Agricultural Uses of Antibiotics 9780841209961, 9780841211575, 0-8412-0996-0

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Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.fw001

Agricultural Uses of Antibiotics

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.fw001

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

320

ACS SYMPOSIUM SERIES

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.fw001

Agricultural Uses of Antibiotics William A . Moats, U.S. Department

of

EDITOR

Agriculture

Developed from a symposium sponsored by the Division of Agricultural and F o o d Chemistry at the 190th Meeting of the American Chemical Society, Chicago, Illinois, September 8-13, 1985

American Chemical Society, Washington, DC 1986

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Library of Congress Cataloging-in-Publication D a t a

Agricultural uses of antibiotics. ( A C S symposium series, I S S N 0097-6156; 320)

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.fw001

"Developed from a symposium sponsored by the Division of Agriculture and Food Chemistry at the 190th Meeting of the American Chemical Society, Chicago, Illinois, September 8-13, 1985." Includes bibliographies and indexes. 1. Antibiotics in veterinary medicine—Congresses. 2. Antibiotics in agriculture—Congresses. 3. Antibiotics in animal nutrition—Congresses. 4. Antibiotic residues—Hygienic aspects—Congresses. 5. Food contamination—Congresses. I. Moats, William A . (William Alden),1928II. American Chemical Society. Division of Agricultural and Food Chemistry. III. American Chemical Society. Meeting (190th: 1985: Chicago, Ill.) IV. Series. [ D N L M : 1. Agriculture—congresses. 2. Antibiotics—congresses. 3. Food Contamination— congresses. 4. Veterinary Medicine—congresses. Q V 350 A278 1985] SF918.A5A37 1986 I S B N 0-8412-0996-0

663.1'929

86-20614

Copyright © 1986 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each chapter in this volume indicates the copyright owner's consent that reprographic copies of the chapter may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc., 27 Congress Street, Salem, M A 01970, for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating a new collective work, for resale, or for information storage and retrieval systems. The copying fee for each chapter is indicated in the code at the bottom of the first page of the chapter. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by A C S of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN THE UNITED STATES OF AMERICA

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

ACS Symposium Series

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.fw001

M . Joan Comstock, Series Editor Advisory

Board

Harvey W. Blanch

Donald E. Moreland

University of California—Berkeley

USDA,

A g r i c u l t u r a l R e s e a r c h Service

Alan Elzerman

W. H. Norton

Clemson University

J. T. B a k e r C h e m i c a l C o m p a n y

John W. Finley

James C. Randall

N a b i s c o B r a n d s , Inc.

Exxon

Chemical Company

Marye Anne Fox

W. D. Shults

The University of T e x a s — A u s t i n

O a k Ridge National Laboratory

Martin L. Gorbaty Exxon

Research and Engineering

Geoffrey K. Smith Co.

Rohm

& Haas C o .

Roland F. Hirsch

Charles S.Tuesday

U.S. Department of Energy

General M o t o r s Research Laboratory

Rudolph J . Marcus

Douglas B. Walters

Consultant, Computers & Chemistry Research

N a t i o n a l Institute o f Environmental Health

Vincent D. McGinniss

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Battelle C o l u m b u s L a b o r a t o r i e s

IBM

Research Department

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

FOREWORD Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.fw001

The A C S S Y M P O S I U M

S E R I E S was f o u n d e d i n 1974 to provide a

m e d i u m for p u b l i s h i n g s y m p o s i a q u i c k l y i n b o o k f o r m . T h e format o f the Series parallels that o f the c o n t i n u i n g A D V A N C E S I N C H E M I S T R Y S E R I E S except that, i n order to save time, the papers are not typeset but are reproduced as they are submitted by the authors i n camera-ready f o r m . Papers are reviewed under the supervision o f the E d i t o r s w i t h the assistance o f the Series A d v i s o r y B o a r d a n d are selected to m a i n t a i n the integrity o f the s y m p o s i a ; however, v e r b a t i m reproductions o f previously p u b lished papers are not accepted. B o t h reviews a n d reports o f research are acceptable, because s y m p o s i a m a y embrace b o t h types o f presentation.

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

PREFACE A N T I B I O T I C S O R D I N A R I L Y A R E D E F I N E D as antibacterial or antiparasitic c o m p o u n d s derived f r o m m i c r o o r g a n i s m s . In this b o o k sulfonamides also are included because, while they are not derived f r o m microorganisms, they

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.pr001

are used i n the same manner as antibiotics. Since the discovery of p e n i c i l l i n , a n e n o r m o u s n u m b e r of a n t i b i o t i c c o m p o u n d s have been isolated. They have f o u n d uses b o t h i n treatment of h u m a n disease a n d i n various aspects of agriculture, i n c l u d i n g treatment of a n i m a l a n d plant diseases, a n d as feed additives to p r o m o t e

growth

animals. S o m e antibiotics such as tylosin were developed specifically

of for

agricultural use. T h i s b o o k was developed to provide a current perspective o n a g r i c u l tural use of antibiotics. Topics include some major

uses of antibiotics,

problems associated w i t h their use f r o m a regulatory standpoint, residues i n f o o d i n c l u d i n g methods of detection, risks to h u m a n health f r o m use i n feeds, trends i n use, a n d overall risks and benefits. The scope, therefore, is m u c h broader t h a n i n several other recent s y m p o s i a that have

focused

mainly o n the controversy regarding the use of antibiotics as feed additives. M a n y of the topics included i n the present volume have not been discussed under one cover before. T h e practice of i n c o r p o r a t i n g low levels of antibiotics i n livestock feeds to promote growth has been particularly controversial. It is feared that this practice w i l l result i n development of resistant bacteria i n animals, w h i c h w i l l in t u r n be passed o n to h u m a n s , thus d i m i n i s h i n g the effectiveness antibiotics i n treatment

of h u m a n disease. A

of

petition f r o m the N a t u r a l

Resources Defense C o u n c i l to b a n such uses of penicillin a n d tetracyclines recently was denied by the Secretary of H e a l t h a n d H u m a n Services. T h e controversy therefore is likely to continue. O p i n i o n o n the subject is quite polarized, a n d several points of view are presented i n this b o o k . T h a n k s are due to participants i n the s y m p o s i u m for their support and diligence i n p r e p a r i n g the material for p u b l i c a t i o n a n d to the A C S B o o k s Department for their unfailing support and guidance. WILLIAM A. MOATS

Meat Science Research Laboratory Agricultural Research Service U.S. Department of Agriculture Beltsville, MD 20705

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

1

A n t i b i o t i c s U s e in A g r i c u l t u r e : An O v e r v i e w

Richard H. Gustafson

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch001

Agricultural Research Division, American Cyanamid Company, Princeton, NJ 08540 Antimicrobial agents and antibiotics in particular have been utilized in livestock rearing for more than thirty years. This has allowed the production of meat, eggs, and milk at levels of efficiency significantly higher than that seen in the pre-antibiotic era. In terms of current kilogram amounts in the U.S., the feed additive uses far surpass all other uses in agriculture and approach the total amount utilized in human medicine. Starting in the 1960's, the use of antibiotic feed additives has been questioned by some as a potential threat to human health. This long standing controversy has been a difficult one for regulatory officials, scientific advisory groups, and legislators who must decide whether directed changes in current agricultural uses are justified. The use of antibiotics in agriculture is a subject of wide variety and complexities. It is also a subject of considerable controversy. It was in the years surrounding 1950 that Dr. Jukes and his colleagues demonstrated that chlortetracycline at low levels, i.e. 20 ppm and lower, could improve performance in livestock (J.). By performance we mean the rate of gain and the amount of feed per unit of gain. Antibiotics, particularly penicillin, had been used in animals prior to that period, but only as an injectable in sick livestock or as mammary infusions in lactating dairy animals suffering from mastitis. After Stokstad and 3ukes opened the door and many agricultural researchers walked in, the development of antimicrobials as feed additives developed at a rapid pace (2). Registration and Commercialization Today, in the United States, the F D A is responsible for examining safety and efficacy data before an antibiotic or synthetic chemical may be commercialized for livestock use. This includes studies on formulations, product stability, conventional and genetic toxicity, environmental safety, metabolism, residue studies in target animals, studies on antibiotic resistance in gut microflora and on salmonella shedding in target animals. Similar requirements are part of registering these products in overseas markets. In general, after a product is discovered in the laboratory, many 0097-6156/86/0320-0001$06.00/ 0 © 1986 American Chemical Society

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch001

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A G R I C U L T U R A L USES O F ANTIBIOTICS

years and millions of dollars are required to bring it to commercialization. Extending registrations of existing products to other livestock species is also a time-consuming and expensive proposition. The big three meat species are poultry, swine, and cattle. The potential markets for these groups of animals may support the required industrial research if the probability of registration and commercial success are high. On the other hand, the minor species, goats, sheep, turkeys, cultured fish often do not support large expensive programs. The dairy industry is also large and antibiotics for the control of mastitis must be considered a significant market. Antibiotics to be used as feed additives must also be inexpensive and fermentation yields often need to be improved before commercialization is possible. Some feed additives are used as biomass products. They are marketed this way because the high costs of extensive purification would make any other course impractical. A biomass product contains the inert spent products of fermentation, remnants of the producing organism, media, precipitation products, etc. The following demonstrates the number of registered feed additives according to category. Table I. The Number of Registered Feed Additives According to Category Antibiotic

Synthetic 13 5 8 9

Anticoccidials Antibacterials Histomonastats Anthelmintics Miscellaneous

3 1

Anticoccidials Antibacterials Anthelmintic

Antibiotic is defined here as a chemical produced in whole or in part by a microorganism in large scale fermentation. Definitions after that are somewhat grey. For example, two of the three ionophore anticoccidials currently used by the poultry industry are also used to promote feed efficiency in cattle, and that effect is certainly a consequence of antibacterial activity in the rumen. Many of the narrow spectrum gram positive antibiotics are poorly absorbed from the gut and registration claims are confined to growth promotion and feed efficiency, particularly at the commonly used levels. Other antibiotics are well absorbed and provide a significant measure of protection against bacterial disease, in addition to promoting growth and feed efficiency. It should be noted that although the summary table provides information on the variety of products which are available, the list doesn't necessarily reflect current uses. For example, synthetic chemicals dominated the anticoccidial market during the 1950's and 60's. With the introduction of monensin, the first ionophore antibiotic used for this purpose the anticoccidial market shifted away from synthetic chemicals and toward antibiotics, where it exists today. Nevertheless, the older synthetic products are still registered and are still part of the armamentarium of anticoccidials. There are more than twice as many synthetic chemicals as antibiotics registered for feed additive use. Table II lists antibacterial drugs, fermentation products and synthesized chemicals approved for use in food animals.

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

1.

GUSTAFSON

Antibiotics

Use in Agriculture:

An

Overview

3

Table II. Antibacterial Feed Additives Antibiotic

Synthetic

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch001

Carbodox Furazolidone Nitrofurazone Sulfamethazine Sulfathiazole

Bacitracin Methylene Disalicylate Bacitracin Zinc Bambermycins Chlortetracycline Erythromycin Lincomycin iNeomycin Novobiocin Nystatin Oxytetracycline Penicillin Streptomycin Tylosin Virginiamycin

The nitrofurans and sulfa drugs are antibacterial feed additives that occupy a continuing important position in the prophylaxis and treatment of livestock disease. These are products which are well absorbed and have good activity against a variety of respiratory pathogens. Carbadox, another synthetic feed additive, has been used extensively in the swine industry for performance improvement and the control of swine dysentery. Uses of Antibiotics in Animal Agriculture The antibiotic feed additives era started with the tetracyclines and this class continues to dominate the field. Chlortetracycline and oxytetracycline were initially used at fairly low levels as promoters of growth and feed efficiency. As the market for these products increased rapidly, production efficicency and fermentation yields also improved. Eventually costs came down and it was economically feasible to use them at higher levels in poultry, swine and cattle, i.e. prophylactic levels which controlled endemic bacterial diseases. A combination for swine was introduced in the 1960's, consisting of chlortetracycline at 100 g/ton, sulfamethazine at 100 g/ton, and penicillin G at 50 g/ton and it was the rapid acceptance of this product by swine producers which was partially responsible for an upsurge of antibiotic use in livestock production. At the same time tetracyclines became increasingly useful in protecting feedlot cattle from the bacterial component of respiratory diseases, as well as reducing the prevalence of liver abscesses caused by Fusobacterium. Anaplasmosis is controlled by tetracyclines in those areas where this disease is endemic in cattle. The poultry industry has also used tetracyclines at higher levels to protect flocks against respiratory diseases as well as controlling certain enteric infections. These prophylactic uses are used particularly at times and in animals in which the risk of bacterial disease is highest. This high risk situation tends to operate in young animals, or when livestock, particularly cattle, are exposed to the stresses of shipment or severe weather. It should be noted that injectable antibiotics are also used extensively in feedlot cattle with a penicillin-streptomycin combination and injectable oxytetracycline used rather extensively for respiratory disease.

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch001

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A G R I C U L T U R A L USES O F ANTIBIOTICS

The current uses of tetracyclines in the poultry industry are almost entirely at the higher levels for the control of bacterial disease. This includes both feed additive and drinking water formulations. Tylosin and erythromycin are commonly used in the livestock industry. These macrolides show wide spectrum activity against gram positive organisms with particularly useful activity against mycoplasma in poultry, swine, and cattle. The combination of tylosin and sulfamethazine is used in swine feed. Erythromycin, the first commercialized macrolide and long the flagship of this group in human medicine, is also registered for use in poultry, swine, and cattle but is used far less than tylosin. Both erythromycin and tylosin are also used therapeutically as injectables and in drinking water for treatment of a variety of infections. The only beta-lactam antibiotics used in food producing animals are penicillin G, ampicillin, amoxicillin, hetacillin, cloxacillin, and cephapiron. Of these, only penicillin G is used as a feed additive, most of it as a combination product in the swine industry. Penicillin feed additives are also registered for use in poultry, but not in cattle. Although penicillin has negligible activity against most gram negative organisms, later generations of beta-lactams have had a much broader spectrum. This group of antibiotics has been the subject of a great deal of pharmaceutical company research, primarily because of the existence in nature of a wide variety of beta-lactamases and cephalosporinases, enzymes which break down various members of this class. Beta-lactam antibiotics continue to be in the forefront of human medicine and as mammary infusion products in the dairy industry. Aminoglycosides registered for feed additive use include streptomycin and neomycin although these antibiotics currently are not being used extensively. Gentamicin and kanamycin are not registered for feed use but are approved as injectables. Gentamicin is also used in an egg dip solution to control specific pathogens in the turkey industry. The general group of antibiotics characterized by gram positive activity and poor absorption from the gut are used principally for growth promotion and feed efficiency in the swine and poultry industry. These include bacitracin, the bambermycins, and virginiamycin. In addition, two products of this type used freqently in Europe and the Far East are avoparcin and nitrovin. The growth promoters major claims are for improved rate of gain and feed efficiency, although occasionally claims at high levels for disease control have been allowed. For example, virginiamycin is registered at 25 to 100 grams/ton for control of swine dysentery in the United States. In the European Economic Community, these growth promoters are used free sale, strictly for performance purposes and disease claims are not made. Therapeutic and prophylactic feed additives for the control of bacterial infections are used by veterinary order. Although the feed additive uses of antibiotics have been emphasized, it should be noted that the uses as injectables for therapy, mammary infusions for mastitis, boluses, pills, capsules, medicated blocks, and drinking water formulations include a wider variety of antibiotics than are added to feeds. Many of these are currently used at the discretion of the meat producer or dairyman, others must be used under the direction of a veterinarian. For example, chloramphenicol is an antibiotic which the veterinarian has access to, but which the F D A has indicated should not be used in livestock destined for human consumption, primarily because of the

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

1.

GUSTAFSON

Antibiotics

Use in Agriculture:

An

Overview

5

Agency's concern about the potential for toxic residues in humans. This antibiotic is used in European livestock.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch001

Extent of Antibiotic Use A question which should be considered here is the extent of antibiotic use in meat production. It's difficult to obtain market figures on individual antibiotic products since the industry considers this information proprietary. It is also unclear how to best express the extent of agricultural uses. Should we be talking about dollars or kilograms? There is no doubt that in terms of kilograms, feed additive use is by far the most significant. The National Academy of Sciences in their 1980 report on feed antibiotics (3) cited figures obtained from the U.S. International Trade Commission, showing non-medical uses to be between 5 and 6 million kilograms in 1978. Unfortunately reporting by weight tends to blur the distinction between growth promoters which are used at 3 or 4 grams per ton such as the bambermycins, and coccidiostats such as monensin, which are used at 100 g/ton. The differences in feed consumption by chickens, swine, and cattle also compound the difficulty of examining antibiotic use. According to figures released by the Animal Health Institute, 1983 sales by American companies of pharmaceuticals, biologicals, and feed additives for animal agriculture exceeded 2 billion dollars (4). These figures represent sales at the manufacturers level. The total feed additive market for 1983 was about half of that total. Pharmaceutical antibacterials sold for $210 million and animal feed antibacterials at $271 million that year. In terms of value to the consumer, the Council of Agricultural Science and Technology reviewed six economic studies and concluded that feed additives save the U.S. consumer approximately $3.5 billion per year in meat prices (5). Antibiotic use accounts for most of this. Although specific figures are proprietary, the tetracyclines have dominated the product lists in terms of total use but tylosin, sulfa drugs, nitrofurans, the gram positive growth promoters and the ionophore antibiotics are also highly significant. The Public Health Question The long controversy surrounding antibiotic feed additives is principally concerned with selection of antibiotic resistant bacteria in livestock. The public health significance under consideration may be reduced to the following questions: "Do the uses of antibiotics in meat animals interfere with the continued efficacy of antibiotics in human medicine?" and secondly, if there is a connection, "would stricter controls on certain feed additive uses help maintain antibiotic effectiveness?" In the United States, the controversy has currently settled on feed additive uses of tetracyclines and penicillin. In response to similar concerns in England in the 1960's, the Swann Committee recommended that feed additives used only for growth promotion and feed efficiency continue to be permitted free sale but that antibiotics used for prevention or control of disease be used only on veterinary prescription. These regulations were implemented in England in 1971 and a few years later, by several other European countries. Most observers agree that these regulations have not altered antibiotic resistance levels in livestock and that antibiotics having disease claims continue to be widely used in these countries, even though veterinary prescriptions are required. The veal calf industry overseas uses antibiotics in milk replacer in

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

A G R I C U L T U R A L USES O F ANTIBIOTICS

6

order to protect the health of these young animals which are particularly susceptible to stress and bacterial infections. The F D A and industry have both been embroiled in the antibiotic resistance controversy for more than 15 years and a resolution of the dispute remains elusive. Part of the reason is that the subject is highly politicized with pressures on the F D A from a variety of sources anxious to introduce new restrictions. The communications media, TV, newspapers, periodicals have also turned their attention to this subject in recent years. Agricultural groups, industry manufacturers, and congressmen with agricultural constituencies have tended to remind the F D A that restrictions are not justified so pressure on the Agency comes from both sides of the issue. It s my opinion that most microbiologists, infectious disease experts, and epidemiologists believe that further government restrictions on the agricultural use of antibiotics would accomplish nothing in the way of public health benefits. This overview has presented an introduction to the subject of antibiotics in animal agriculture and provides a general view of the extent of antibiotic use. The manuscripts to follow will offer more details and provide additional food for thought. Certainly the current efficient production of meat and dairy products is dependent on a wide variety of antibiotics and this will continue to be true in most Western countries in which livestock production is an important part of the economy.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch001

f

Literature Cited 1. 2.

3.

4. 5.

Stokstad, E . L. R.; Jukes, T. H., Proc. Soc. Exptl. Biol. Med., 73; 523528. Gustafson, R. H.; Kiser, J . S., In "The Tetracycline"; Hlavka, J . J.; Boothe, J. H., Eds; HANDBOOK EXP. PHARM.; Springer-Verlag: Heidelberg, 1985; pp. 405-446. "The Effects on Human Health of Subtherapeutic Use of Antimicrobials in Animal Feeds". National Academy of Sciences, National Research Council; Washington, March 1980. Animal Health Institute; 119 Oronoco St., Alexandria, VA. "Antibiotics in Animal Feeds"; Council for Agricultural Science and Technology, Report No. 88, March 1981.

RECEIVED February 19, 1986

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

2

T h e r a p e u t i c U s e of A n t i b i o t i c s

in

Farm

Animals

G. Ziv

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch002

Ministry of Agriculture, Kimron Veterinary Institute, P.O. Box 12, Bet Dagan, Israel The prevention and therapeutic management of disease conditions caused by infectious agents are daily events in the practice of farm animal medicine and surgery. Antimicrobial drugs represent one third of a l l chemicals used in veterinary medicine. The use of antimicrobials by practitioners involves therapy of bovine species, 75%, compared with 25% for the treatment of infections in horses, pigs and other farm animals. Antimicrobials are used to treat mastitis, ent e r i t i s , peritonitis, metritis, pneumonia, septicemia, and localized infections. The successful use of these agents for each indication depends on the same basic principles that apply to a l l microbial infections: (i) identifying the incriminating pathogen, ( i i ) determining the in vitro sensitivity of the pathogen to the antibacterial drug, ( i i i ) attaining and maintaining therapeutic drug concentrations at the infection site, (iv) minimizing local and systemic side-effects of therapy, and (v) the administration of supportive, non-antimicrobial, therapy when indicated. Pharmacokinetic and pharmaceutical properties, cost, and duration of drug residues are also important criteria for drug selection. Antimicrobial therapy is generally applied on a herd basis in order that animals returned to full health and productivity at the earliest opportunity, that the excretion of pathogenic organisms from sick animals be curtailed, and that epidemics of infectious diseases be prevented from developing. A n t i b i o t i c s are used for many purposes i n agriculture but perhaps the most important of these uses i s that for the maintenance of health i n our food animal population. E s s e n t i a l l y , antimicrobial agents are used to treat disease, to prevent the spread of i n f e c t i o n , and to check the m u l t i p l i c a t i o n of tissue-associated i n f e c t i v e agents. A n t i b i o t i c s are very complex therapeutic tools and their correct use requires a wide knowledge of physiology, pathology, pharmacology, diagnostics and l e g i s l a t i o n . A proper acquaintance 0097-6156/86/0320-0008$06.00/0 © 1986 A m e r i c a n C h e m i c a l Society

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch002

2.

ζιν

Therapeutic

Use of Antibiotics

in Farm

Animals

9

with these d i s c i p l i n e s enables the veterinary practitioner to apply a n t i b i o t i c s i n a manner that w i l l ensure t h e i r continued safe and e f f e c t i v e use for the treatment and prevention of diseases i n farm animals* The treatment of b a c t e r i a l diseases i n man and companion a n i ­ mals i s invariably directed at the individual patient whereas i n food producing animals, e s p e c i a l l y pigs and sheep, although a degree of individual therapy may be undertaken, antimicrobial therapy i s generally applied on a herd or flock basis. Present-day antimicro­ b i a l substances are very sophisticated tools i n the armory of the veterinary practitioner and animal producer. Use of these sub­ stances i n the f i e l d may appear to the novice very simple and free from any attendant hazard, yet this i s only true when these drugs are being used by persons f u l l y conversant with their pharmacologi­ c a l properties and thus able to avoid such problems as tissue r e s i ­ dues and c e r t a i n direct toxic a f f e c t s . Rationale for use of a n t i b i o t i c s Any i n d i v i d u a l , group or population of animals i s always susceptible to outbreaks of c l i n i c a l disease. Many present-day a g r i c u l t u r a l practices such as livestock marketing, movement of very young a n i ­ mals and certain forms of i n t e n s i f i c a t i o n can act as trigger factors for the i n i t i a t i o n and development of c l i n i c a l diseases (1). Gene­ r a l l y speaking, disease w i l l be more prevalent i n large groups of intensively managed animals than i n individual animals kept under extensive conditions. Over the years new animal hybrids have been developed, highly productive strains of livestock have been bred, and imported breeds have been introduced into new l o c a l i t i e s with the sole intention of increasing productivity and quality of animal products. This has resulted i n some cases i n an increase prevalence of disease which has to be constantly treated or prevented. In or­ der to cut down economic losses i t i s necessary that b a c t e r i a l d i ­ sease be treated as soon as possible with an a n t i b a c t e r i a l agent. Present-day economics dictates that animals be returned to f u l l health and productivity at the e a r l i e s t opportunity, that the excre­ tion and dissemination of pathogenic organisms from sick animals be c u r t a i l e d , and that epidemics of infectious diseases be prevented from developing (2). Any delay i n the administration of antibacte­ r i a l s , either to the individual or to a herd, w i l l be counter-pro­ ductive and economically unsound. A n t i b a c t e r i a l drugs were deve­ loped with the sole purpose of helping to treat sick farm animals a f f l i c t e d with b a c t e r i a l disease and i n so doing the dissemination of pathogenic micro-organisms within the common environment of man and animals would be very much reduced and i n some cases t o t a l l y prevented. It would not be d i f f i c u l t to speculate on the problems that would arise i f no a n t i b a c t e r i a l medication were available. Large numbers of farm animals would perish, chronic b a c t e r i a l disease would be commonplace and the consequent losses both of l i f e and pro­ d u c t i v i t y would d r a s t i c a l l y i n f l a t e the cost of milk and meat pro­ duction apart from resulting i n the bankruptcy and disappearance of many livestock producers. Epidemiological experience has shown that the introduction of an infectious disease-causing agent into a large group of suscepti-

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch002

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ble animals i n the same pen w i l l ultimately r e s u l t i n a large proportion of these animals becoming infected (3, 4). The r a t i o n a l therapeutic approach to this problem i s to treat the whole group as an i n d i v i d u a l . In this way one avoids having to continually withdraw and treat i n d i v i d u a l animals which would be very costly i n time and also s t r e s s f u l to the animals due to frequent interference by the human attendants when catching animals for medication. The concept of herd medication i s d i f f i c u l t to accept outside the a g r i c u l tural f i e l d . The experience of veterinary practitioners who treat large herds of animals, however, f u l l y supports the practice of herd medication, e s p e c i a l l y i n those areas of animal management where i t i s e s s e n t i a l to keep a l l the animals at a l e v e l of optimal productiv i t y (2). Extensive experience i n veterinary medicine has c l e a r l y indicated the need for such treatment, e s p e c i a l l y when highly i n f e c tious or contagious diseases are involved. It must be r e a l i z e d that unlike the human family unit, a l l the farm animals i n a group that i s being managed under intensive systems are usually of the same age, usually very immature, and are i n constant contact with their faeces. Another factor which presently produces continuing disease problems i n c a l f and pig enterprises i s the need to transfer young growing animals from breeding units to the grower/finisher u n i t s . This frequently involves prolonged t r a v e l l i n g , a change i n d i e t and a mixing together of animals from d i f f e r e n t sources. Inevitably this can culminate i n a disturbance of the g a s t r o - i n t e s t i n a l f l o r a which i s caused by dietary change, reorganisation of s o c i a l dominance (pecking order), and r e d i s t r i b u t i o n of micro-organisms of many species and types within the newly grouped animals. Such d i s t u r bances frequently r e s u l t i n the production of overt c l i n i c a l d i sease. A similar set of circumstances also may occur at weaning. These events are usually t o t a l l y predictable and experience has shown that, under c e r t a i n circumstances, i f pre-emptive medication i s not applied then serious health problems w i l l ensue. The decision to employ herd medication i s never taken l i g h t l y because of the cost of the drugs to treat a large population of a n i mals and the problems which w i l l ensue with the need to adhere to drug withdrawal times. Herd medication on a very large scale also involves such l o g i s t i c a l problems as getting the correct dose of drug to a l l individuals concerned and the d i f f i c u l t i e s of being able to obtain a very large amount of drug when required and without del a y . Care i s also required i n the choice and use of a n t i b i o t i c s f o r the treatment of zoonotic i n f e c t i o n s , e.g. salmonellosis. Some manifestations of this disease are more amenable to treatment than others and special considerations have to be taken i n order to avoid undue selection of multiple a n t i b i o t i c resistance and destruction of indigenous protective microflora. A further extention of herd medication has often i n the past been referred to as prophylactic treatment or disease prevention. Both terms are p a r t i a l l y correct but the rationale f o r their a p p l i cation requires a basic knowledge of epidemiology. The a p p l i c a t i o n of pre-emptive medication depends upon the knowledge that a p a r t i c u l a r b a c t e r i a l agent has been introduced into a population and i s causing c l i n i c a l disease i n individuals within that population (5). Previous veterinary experience w i l l have indicated that i f medicat i o n i s not applied to the group or herd then there w i l l be a c o n t i -

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nuing sequence of infected individuals, accompanied by a prolonged period of sub-optimal performance i n the affected group of animals. Generally speaking, pre-emptive medication does not apply to the i n ­ d i v i d u a l l y housed animal but only to the group or herd of animals i n which c l i n i c a l disease has broken out i n one or more i n d i v i d u a l s . Rapid curtailment of a herd i n f e c t i o n w i l l bring about a cessation of b a c t e r i a l excretion which w i l l be advantageous to the remainder of the herd and also prevent undue contamination of the farm envi­ ronment. An example of pre-emptive medication i s the use of dry-cow therapy i n which a slow release a n t i b i o t i c preparation i s infused into the cow's udder at the end of a l a c t a t i o n to overcome any r e s i ­ dual i n f e c t i o n and to protect against the establishment of new i n f e c t i o n during the dry period and prior to the commencement of a new l a c t a t i o n cycle (2). Selection of a n t i b i o t i c s The selection and use of a n t i b i o t i c s i n c l i n i c a l practice are depen­ dant upon many factors, not the least of which are the particular drug use habits i n the geographical area (6). Because the majority of b a c t e r i a l pathogens are susceptible to several a n t i b i o t i c s , suc­ cessful therapy should not be unexpected with d i f f e r e n t drug use patterns. There are, however, some important factors which should be given consideration when selecting a n t i b a c t e r i a l agents. Bacte­ r i a l s e n s i t i v i t y i s a prominent decision factor which i s commonly of high p r i o r i t y . However, of nearly equal importance i s the a b i l i t y of the drug to achieve reasonable concentrations at the s i t e of i n ­ fection. Additionally, the age and health state of the animal should also be considered along with dosage preparations available, cost, t o x i c i t y , etc. Therapy s h a l l f a i l with the use of a very po­ tent oral a n t i b i o t i c which does not penetrate into the s i t e of i n ­ fection or which i s degraded by ruminai microflora before i t can be absorbed. The l i p i d s o l u b i l i t y and degree of i o n i z a t i o n at physio­ l o g i c a l pH of the drug are important determinants of tissue penetra­ tion. Generally, the more l i p i d - s o l u b l e drugs which are l i t t l e ionized at physiological pH are more widely distributed i n the body and are most l i k e l y to achieve reasonable concentrations i n d i f f i cult-to-penetrate peripheral tissues such as brain and reproductive t r a c t . The pH of the tissue i s also important since tissues with a pH lower than that of blood (7.4) w i l l trap basic a n t i b i o t i c s i n them by causing increased ionization of the a n t i b i o t i c . An example of t h i s i s the mammary gland with a pH of 6.8 to 7.0. In t h i s case the basic macrolide a n t i b i o t i c s which are l i p i d - s o l u b l e and are l i t ­ t l e ionized i n blood w i l l be found i n higher levels i n milk than some less l i p i d - s o l u b l e drugs because they are trapped i n the a c i d i c milk and move from blood into milk more readily than from milk to blood. Conversely, a c i d i c a n t i b i o t i c s may be found i n lower than expected concentrations i n tissues with a pH lower than blood. As a general rule, these relationships can be used to determine the need for dosage adjustment for infections involving s p e c i f i c tissues. In situations where an a n t i b i o t i c i s known to penetrate s p e c i f i c t i s ­ sues poorly, the dosage may need to be increased appropriately. After an a n t i b i o t i c was selected, the primary concern should be to optimize the dosage for maximal e f f i c a c y and minimal t o x i c i t y . Other factors such as economics, frequency of animal handling and

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route of administration are important for both dosage determination and drug selection. Often, because of cost or inadequate animal dosage information, the tendency i s to use too low dosages which may be quite s u f f i c i e n t i n some cases but i n others may lead to the erroneous conclusion that the drug i s not e f f e c t i v e . The dose may actually be appropriate but the dosage i n t e r v a l too long to sustain a c t i v i t y , or the opposite situation with dose too low and i n t e r v a l appropriate. A desired serum or tissue concentration can be determined from the i^i v i t r o b a c t e r i a l s e n s i t i v i t y data. I t i s generally desirable to select a dosage schedule which w i l l provide serum or tissue l e vels equal to or exceeding i n jLn v i t r o i n h i b i t o r y concentrations for a substantial portion of the treatment period. For some bacteria low a n t i b a c t e r i a l concentrations may s u f f i c e i f the more sensitive organisms are involved. Yet the next instance of disease encountered due to the same organism may be refractory to a l l but the highest doses of drug. Once a desired serum or tissue l e v e l has been decided, the dosage schedule may be determined. If the dosage schedule f a i l s to provide the appropriate serum l e v e l , adjustments can be made accordingly. In some cases an increase i n dose w i l l provide a proportional increase i n serum concentration. This i s true for intravenous preparations and for intramuscular administration of some water-soluble a n t i b i o t i c s such as the aminoglycosides. Shortening the dosage i n t e r v a l may i n some cases provide s u f f i c i e n t i n crease i n serum concentration. Unfortunately, the relationship between dosage and serum concentration i s not r e a d i l y predictable i n some cases with o r a l or intramuscular administration. This i s part i c u l a r l y true for slow-absorption formulations such as procaine p e n i c i l l i n G, a m p i c i l l i n trihydrate, and some oxytetracycline i n j e c table products. In these cases increasing the dose w i l l more e f f e c t i v e l y increase the duration of action rather than serum concentrations. If the situation demands infrequent drug administration, the dose must be increased too. In most cases this does not present a t o x i c i t y problem because t o x i c i t y i s usually due to cumulative effects of the drug, as with the aminoglycosides and polymyxins. The foregoing comments are based on the ultimate objective of a n t i b a c t e r i a l therapy, attainment of adequate tissue drug levels to either k i l l the pathogen or i n h i b i t i t s growth. Adequate tissue l e vels are usually interpreted as levels equiyalent or higher than the minimal i n h i b i t o r y concentrations (MIC) of the drug as determined by standardized i n v i t r o procedures. The most r e a d i l y available tissue for analysis and one which i s i n equilibrium with most other tissues i s serum. Hence, serum levels of a n t i b i o t i c s are often used as an i n d i c a t i o n of adequacy of dosage. This relationship i s not i n f a l l i ble, however, since other factors may also play important r o l e s . The state of body defense mechanisms may s i g n i f i c a n t l y a l t e r the e f f i c a c y of a given serum or tissue l e v e l . Thus the interpretation of in vivo studies with antimicrobial agents i s complicated by the importance of the host defenses i n producing the f i n a l cure. The host defenses probably have a more dominant role i n the outcome when bact e r i o s t a t i c a n t i b i o t i c s l i k e tetracyclines, chloramphenicol and s u l fonamides are used than with b a c t e r i c i d a l l y acting a n t i b i o t i c s l i k e the beta-lactam and aminoglycoside a n t i b i o t i c s . This i s probably true i n most c l i n i c a l situations. The amount of drug required to k i l l a certain b a c t e r i a l s t r a i n may vary depending on the number of

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organisms i n the inoculum. This has been termed the inoculum e f f e c t , and although i t s c l i n i c a l importance i s not f u l l y understood, the e f f e c t i s known to vary with the drug used and the bacterium being treated. Additionally, i n some instances serum levels may not accurately r e f l e c t tissue l e v e l s , e s p e c i a l l y with a n t i b i o t i c s with high tissue l e v e l s , such as the macrolides (6). With the exception of some highly sensitive b a c t e r i a l isolates and a few sustained-release formulations of antimicrobial drugs, i t i s usually necessary to administer multiple doses of a drug to cont r o l an i n f e c t i o n . However, at the present time, i t i s not established for most infections whether i t i s better to achieve high l e vels of drug i n serum rapidly and thereby achieve high l e v e l s i n tissues, or whether i t i s more desirable to have a drug present f o r a long period, a l b e i t at lower l e v e l s . There are v i r t u a l l y no w e l l controlled comparisons of these d i f f e r e n t situations (7). Concentrations of antimicrobial agents that are below the MIC but produce morphologic or quantitative alternations i n micro-organisms are defined as subinhibitory concentrations. E f f e c t s of subMIC of antimicrobial agents include: ( i ) alternation of structure of micro-organisms, ( i i ) alternation of numbers of micro-organisms, ( i i i ) alternation of adhesiveness to mucosal surfaces, ( i v ) enhancement of phagocytosis or impairment of expression of antiphagocytic material, and (v) induction of beta-lactamase production by the bacterium (8). Additional studies are needed to define the true c l i n i cal importance of the sub-MIC, post-antibiotic e f f e c t , post-antibiot i c leukocyte enhancement, and greatly fluctuating serum drug concentrations. The preponderance of evidence, although much of i t emp i r i c a l , indicates that tissue and/or blood concentrations that are equal to or above the MIC should be attained, especially with bacter i o s t a t i c - t y p e antimicrobial agents or i n animals with refractory type of i n f e c t i o n s . Since the ultimate test of a n t i b i o t i c effectiveness i s the response of the animal, the use of serum levels f o r predicting e f f i c a c y i s only as valuable as i t s a b i l i t y to predict the response of the whole animal (6). Generally, t h i s c o r r e l a t i o n has held, hence the use of serum l e v e l s f o r determination of a n t i b i o t i c usage. The fore-going l i m i t a t i o n s should, however, be kept i n perspective while using such information c l i n i c a l l y . The situation with the sulfonamides i s quite d i f f e r e n t than with many of the a n t i b i o t i c s . In this instance the c o r r e l a t i o n between attainment of i n v i t r o i n h i b i t o r y concentration requirements as ija vivo serum concentrations and drug e f f i c a c y has not been as good as with most of the a n t i b i o t i c s . As a r e s u l t , a general recommendation of 50 ug/ml of drug i n serum has been widely accepted as the desirable concentration for a l l the sulfonamides. Unfortunately l i t t l e e f f o r t has been expended i n veterinary medicine to determine MIC relationships between various bacteria and sulfonamides (6). An important p r i n c i p l e to be emphasized i s that there i s no single optimal dose f o r any given a n t i b i o t i c . There are too many variables such as host resistance, b a c t e r i a l virulence, b a c t e r i a l a n t i b i o t i c s e n s i t i v i t y and s i t e of i n f e c t i o n to allow a single dosage recommendation to cover a l l situations. While many disease problems can be covered by routine dosage l e v e l s , special situations may require marked elevation of dosage or perhaps even allow f o r a reduced dosage schedule.

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Routes of drug administration The dosage form of an antimicrobial preparation determines i t s route of administration whereas formulation influences systemic a v a i l a b i l i t y of the antimicrobial agent from the dosage form ( 9 ) . Antimic r o b i a l preparations are available i n a wide variety of dosage forms that include tablets, capsules, pastes, and suspensions for o r a l and intra-uterine administration; s t e r i l e solutions and suspensions for i n j e c t i o n s ; ointments for ophthalmic use; and intra-mammary preparations for the l o c a l treatment of m a s t i t i s . The amount of drug i n the preparation l i m i t s i t s use to c e r t a i n species of animals, due to the wide range of their body weights. Convenience of administration and cost of the preparation are two other p r a c t i c a l considerations that influence s e l e c t i o n of the dosage form. The ease of adminis t r a t i o n i s often a c r i t i c a l factor governing user compliance with instructions to administer the preparation at the recommended i n t e r vals. Absorption i s the c r i t i c a l factor that determines entry of an antimicrobial agent into the blood stream when an extravascular route of administration, i . e . o r a l , intramuscular (IM), or subcutaneous (SC) i n j e c t i o n i s used. Absorption, the extent of which depends mainly on the physicochemical properties of the antimicrobial agent, i s associated with intra-mammary or intra-uterine therapy. Intravenous (IV) i n j e c t i o n i s often the most satisfactory route of administration for i n i t i a t i n g therapy for animals with acute i n fections. Antimicrobial therapy with agents that produce a bacter i o s t a t i c e f f e c t and have r e l a t i v e l y long h a l f - l i v e s (such as t e t r a cyclines and sulfonamides) can be i n i t i a t e d with an IV priming dose. To avoid adverse systemic effects that may be associated with high i n i t i a l concentration of drug, the parenteral solution must be injected slowly. The option to employ the IV route of administration can be limited by the lack of a v a i l a b i l i t y of parenteral solutions formulated appropriately for i n j e c t i o n by t h i s route. Because the dose i s introduced d i r e c t l y into the blood stream, IV i n j e c t i o n w i l l provide therapeutic serum concentrations for a short duration than w i l l extravascular routes that provide an adequate rate of absorption. Therefore, for maintenance of therapeutic serum concent r a t i o n s , oral dosing or IM/SC i n j e c t i o n of parenteral (prolongrelease) preparations i s more convenient. The most satisfactory technique for maintaining therapeutic serum concentrations at steady-state l e v e l i s to administer the drug by continuous IV i n f u s i o n . The a p p l i c a t i o n of this technique has considerable l i m i t a t i o n s i n farm animals, but on occasions, i t i s employed· The IM and SC routes are by far the most frequently used extravascular parenteral routes of drug administration i n farm animals. The less frequently used parenteral routes have limited a p p l i c a t i o n , i n that they aim at d i r e c t l y placing high concentrations of antimic r o b i a l agent close to the s i t e of i n f e c t i o n . These routes of admin i s t r a t i o n include i n t r a - a r t i c u l a r or subconjuctival i n j e c t i o n and intra-mammary or intra-uterine infusion. These l o c a l routes d i f f e r from the major parenteral routes i n that absorption into the systemic c i r c u l a t i o n i s not a prerequisite for delivery of drug to the s i t e of action. The combined use of systemic and l o c a l delivery of drug to the s i t e of i n f e c t i o n represents the optimum approach to

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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treatment of conditions where the i n f e c t i o n s i t e may be r e l a t i v e l y inaccessible, such as i n a cow with m a s t i t i s ( 9 ) . Absorption of antimicrobial agents from the IM and SC s i t e s of i n j e c t i o n takes place by passive d i f f u s i o n , similar to that from the g a s t r o - i n t e s t i n a l t r a c t , as well as by bulk flow through i n t e r c e l l u ­ l a r pores i n the c a p i l l a r y endothelial membrane. Factors that i n ­ fluence absorption include the physicochemical properties of the drug, that govern i t s passage across the membrane separating the ab­ sorption s i t e from the blood, the pH of the solution at the absorp­ tion s i t e , and the l o c a l blood flow. Because most antimicrobial agents are weak organic acids or bases, the physicochemical proper­ t i e s that a f f e c t their membrane penetrative capacity are the degree of i o n i z a t i o n and l i p i d - s o l u b i l i t y . The less ionized and more l i p i d - s o l u b l e the drug, the greater w i l l be the rate of absorption by passive d i f f u s i o n . In addition to and often overriding the phy­ sicochemical properties of the drug i s the influence of formulation of the preparation (dosage form) on the b i o - a v a i l a b i l i t y . Age or body weight can a f f e c t the systemic a v a i l a b i l i t y of many antimicrobial agents. In the physically smaller animal (sheep and pig) the peak serum concentration of a drug i s usually higher and i s followed by a rapid decline compared with a lower peak and a slower decline of the a n t i b i o t i c i n serum of the larger animal (cow and horse). The limited experimental data appear to indicate that the extent of systemic a v a i l a b i l i t y of IM-administered a n t i b i o t i c s can vary as widely between d i f f e r e n t sites as between IM and SC s i t e s . A c o r o l l a r y to t h i s observation i s that the location of the extra­ vascular i n j e c t i o n s i t e should be well-defined when determining the systemic a v a i l a b i l i t y of parenteral preparations ( 9 ) . A prolonged-release dosage form i s one that not only contains more drug than a conventional dosage form but releases i t s drug con­ tent more slowly than the conventional preparation. The objective of treatment with a prolonged release antimicrobial preparation i s to achieve a s i t u a t i o n i n which the duration of a n t i b a c t e r i a l e f f e c t i s controlled by the rate of drug release from the dosage form r a ­ ther than by the d i s p o s i t i o n k i n e t i c s of the drug. The convenience of a single administration i s an obvious advantage. An important feature i n the design of prolonged-release dosage forms i s that the rate of release be adequate to maintain e f f e c t i v e serum drug concen­ t r a t i o n s . Another requirement i s that the formulation of parenteral preparations be such that their IM i n j e c t i o n does not cause tissue damage with persistence of residual concentrations at the i n j e c t i o n site. Sporadic c l i n i c a l reports, without the support of data from controlled studies, repeatedly indicate the effectiveness of i n t r a ­ tracheal administration of parenteral antimicrobial preparations i n the treatment of tracheobronchitis and pneumonia i n c a t t l e . The ex­ pectation when using this route of administration i s that a greater therapeutic e f f e c t w i l l be achieved when the drug i s placed as close to the i n f e c t i o n s i t e as possible, rather than r e l y i n g on the syste­ mic c i r c u l a t i o n for drug delivery. The majority of o r a l preparations are s o l i d dosage forms. These include tablets and capsules f o r administration to small farm animals, pastes f o r horses, and a variety of prolonged-release pro­ ducts f o r administration to c a t t l e . The drug i n s o l i d dosage form must dissolve before i t can be absorbed. The d i s s o l u t i o n rate de-

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pends i n part on the physicochemical properties of the drug and partly on the manufacturing process. The inert ingredients of the dosage form can have a profound effect on d i s s o l u t i o n of the active ingredient and thereby control i t s rate of absorption. Dissolution i s often the major underlying source of v a r i a t i o n i n the absorption of a drug from d i f f e r e n t oral preparations, and this process can even influence the effectiveness of therapy. Even though a drug may have the combination of physicochemical properties that are favourable for absorption, i t may s t i l l have low systemic a v a i l a b i l i t y when administered o r a l l y . The drug may be unstable i n g a s t r o - i n t e s t i n a l f l u i d s (such as p e n i c i l l i n G) or be metabolized. Metabolism can be mediated by i n t e s t i n a l microflora or e p i t h e l i a l enzymes or can occur i n the l i v e r preceding entry of the drug into the systemic c i r c u l a t i o n . The importance of the f i r s t pass effect does not appear to have been determined for antimicrob i a l agents i n farm animals, but presumably would apply only to l i pophilic agents that are extensively metabolized (such as chloramphenicol, clindamycin, metronidazole, trimethoprim, and the antibact e r i a l quinolones). The ruminai microflora can hydrolize esters and have been shown to inactivate chloramphenicol by reductive reactions. In calves less than one-week o l d , chloramphenicol i s well absorbed when administered as an oral solution. The systemic a v a i l a b i l i t y of the ant i b i o t i c decreases with ruminai development. Similar observations were made after o r a l a m p i c i l l i n , amoxycillin and cephalexin therapy i n young calves. Trimethoprim i s extensively metabolized i n the l i ver (oxydation followed by conjugation reactions) and may undergo some metabolism i n the rumen. The higher systemic a v a i l a b i l i t y of trimethoprim i n the newborn c a l f and kid can be attributed to lower metabolic a c t i v i t y with lesser f i r s t - p a s s e f f e c t i n the neonatal animal. Although some antimicrobial agents can be metabolized i n the rumen, prolonged o r a l dosing with these or other agents has the potential to disturb a c t i v i t y of the ruminai microflora ( 9 ) . Thus, knowledge of the b i o - a v a i l a b i l i t y and d i s p o s i t i o n kinetics of the antimicrobial agent i s required for optimal dosage with the preparation selected. This information can only be obtained from w e l l designed pharmacokinetic studies i n the target species of farm animals. Types of antimicrobial agents Penicillins. This group includes p e n i c i l l i n G ( b e n z y l - p e n i c i l l i n ) , p e n i c i l l i n VK (phenoxymethyl-penicillin), the isoxazolyl p e n i c i l l i n s o x a c i l l i n , c l o x a c i l l i n , d i c l o x a c i l l i n and n a f c i l l i n , the amino-penic i l l i n s a m p i c i l l i n , h e t a c i l l i n and amoxycillin, the carboxy-penicill i n c a r b e n i c i l l i n , and the t h i e n y l - p e n i c i l l i n t i c a r c i l l i n . These a n t i b i o t i c s are b a c t e r i c i d a l v i a i n h i b i t i o n of synthesis of c e l l wall material necessary for maintenance of c e l l u l a r i n t e g r i ty. The spectrum of action i s quite broad for the group as a whole; however, s p e c i f i c p e n i c i l l i n s may have a limited range of e f f i c a c y . P e n i c i l l i n G i s not only one of the oldest a n t i b i o t i c s , but remains one of the most useful. I t i s limited by i t s poor tissue d i s t r i b u tion and i t s low s e n s i t i v i t y for Gram-negative bacteria. These l i mitations can often be overcome, however, by increasing the dosage since both cost and t o x i c i t y are low. Increasing the dose to i n -

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch002

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crease tissue l e v e l s i s best accomplished using either the watersoluble Na and Κ s a l t s IV or the Na salt IM. By these routes of ad­ ministration the increases i n serum or tissue concentrations are nearly proportional to increases i n dose. Dosage of procaine peni­ c i l l i n G must be increased markedly to produce a useful increase i n serum or tissue l e v e l while benzathine p e n i c i l l i n G can only be used to provide prolonged low levels regardless of the dose. The isoxaz o l y l p e n i c i l l i n s are used primarily for the intra-mammary treatment of mastitis due to penicillinase-producing staphylococci. The amphoteric amino-penicillins have become popular because of their e f f i c a c y against many Gram-negative pathogens associated with neonatal infections, such as e n t e r i t i s and pneumonia. Although am­ p i c i l l i n i s a very e f f e t i v e drug, i t e r r a t i c oral absorption i n preruminants i s an important l i m i t i n g factor. With oral use, the do­ sage should be increased and administered several hours prior to or after feeding. H e t a c i l l i n i s not appreciably better than a m p i c i l l i n in the above respects. Amoxycillin, a very similar drug to ampicil­ l i n , does not share these r e s t r i c t i o n s as i t i s well absorbed i n the presence of food. Sodium s a l t preparations of a m p i c i l l i n are avai­ lable for short duration, high serum l e v e l s while the trihydrate forms are analogous to procaine p e n i c i l l i n G i n producing sustained low serum and tissue l e v e l s . P e n i c i l l i n s available with s p e c i f i c e f f i c a c y against Pseudomonas spp. include c a r b e n i c i l l i n and t i c a r c i l l i n . Both are very e f ­ f e c t i v e but require high dosage (50 mg/kg). They can also be used concurrently with aminoglycosides but should be injected separate­ l y . Cost i s a l i m i t i n g factor i n the use of these drugs. Cephalosporins. These beta-lactam a n t i b i o t i c s share many features with the p e n i c i l l i n s including mechanism, spectrum of action, d i s ­ t r i b u t i o n ans t o x i c i t y potential. At the present time, the cephalo­ sporins are c l a s s i f i e d into three groups, designated as generations. First-generation cephalosporins, introduced into human medicine i n the I960 s and 1970 s, are b a s i c a l l y similar i n a n t i b a c t e r i a l ac­ t i v i t y and d i f f e r mainly i n their pharmacokinetic properties. These include a l l of the currently available o r a l l y active cephalosporins, and are r e l a t i v e l y susceptible to beta-lactamase, active against most Gram-positive bacteria and have a limited spectrum of a c t i v i t y against the Gram-negative organisms. Second-generation cephalosporins, i n i t i a l l y introduced i n the late 1970's, tend to be more resistant to beta-lactamase and more active against a broader spectrum of Gram-negative bacteria. A l ­ though their a c t i v i t y against Gram-positive bacteria i s often thought to be less than the first-generation compounds, this i s usually i n reference to the p e n i c i l l i n - r e s i s t a n t staphylococci. Third-generation cephalosporins were introduced i n the early 1980's. They are more active against many of the Gram-negative or­ ganisms, including Pseudomonas spp., often at the expense of dimi­ nished a c t i v i t y against Gram-positive bacteria, p a r t i c u l a r l y S. aureus. Some third generation cephalosporins have long h a l f - l i v e s as well as good penetration of CSF and peritoneal f l u i d . The cephalosporins are not widely used i n veterinary medical practice due to the a v a i l a b i l i t y of other a n t i b i o t i c s that are ef­ f e c t i v e against the common animal pathogens, less expensive on a treatment regimen basis, and approved for use i n animals. At the 1

f

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A G R I C U L T U R A L USES O F ANTIBIOTICS

present time, only a single first-generation cephalosporin, cephapir i n , i s approved for use i n food animals i n the USA. Available as an intra-mammary infusion preparation, cephapirin i s used i n the treatment of m a s t i t i s i n lactating and non-lactating cows. The first-generation cephalosporins cephalexin, cephoxazole, cephalonium, cephacetrile and cefuroxime are approved i n several European countries as intra-mammary infusion products f o r the treatment of mastitis i n cows. Cephalexin i s also used parenterally for the treatment of neonatal infections i n calves and f o r bovine m a s t i t i s . In most cases, the cephalosporins are not the preferred drug, but rather used i n infections caused by organisms resistant to other ant i b i o t i c s . However, there i s a trend to use the cephalosporins f o r presurgical prophylaxis, especially i n certain orthopedic procedures. The cephalosporin a n t i b i o t i c s are among several classes of compounds currently being examined i n a search f o r new therapeutic antibacterials for use i n veterinary medicine, with primary emphasis on products for food animals (10). Aminoglycosides (aminocyclitols)» These a n t i b i o t i c s are valuable therapeutic agents and among the oldest known a n t i b a c t e r i a l agents for use i n farm animals. They include streptomycin, neomycin, kanamycin, gentamycin, spectinomycin, and the recently introduced amikac i n and apramycin. A l l are rapidly b a c t e r i c i d a l and their a c t i v i t y involves uptake of the a n t i b i o t i c by bacteria followed by binding to b a c t e r i a l ribosomes and i n h i b i t i o n of protein synthesis. Their spectrum of a c t i v i t y covers most Gram-negative bacteria, as well as staphylococci but they have r e l a t i v e l y poor a c t i v i t y against streptococci and no useful a c t i v i t y against anaerobic bacteria or fungi. Bacteria acquire resistance to aminoglycosides by ( i ) mutation of the organism leading to altered ribosomes that no longer bind the drug, ( i i ) by reduced permeability of the bacterium to the drug, or ( i i i ) by b a c t e r i a l enzymes that inactivate the drug. A l l aminoglycoside a n t i b i o t i c s are small, basic water-soluble molecules that form stable s a l t s . None i s absorbed well from the alimentary tract or when applied t o p i c a l l y , and therefore, must be administered parenterally f o r systemic use. In human beings, there i s a higher r i s k of toxicosis when aminoglycoside agents are administered by IV bolus i n j e c t i o n or continuous IV infusion. The margin between therapeutic and toxic concentrations for a l l members of t h i s group i s not as great as that with the p e n i c i l l i n s or macrolides. The two problems occurring less frequently but of great concern are neuromuscular blockage and cardiovascular depression. Ototoxicity i s manifested by damage to the 8th c r a n i a l nerve which include auditory and vestibular dysfunction. Studies of the acoustical e f f e c t s of aminoglycoside a n t i b i o t i c s i n domestic animals have been limited because of d i f f i c u l t i e s i n determining hearing loss i n animals. Nephrotoxicity i s of great potential importance because approximatel y 90% of drug i s eliminated by renal f i l t r a t i o n . Any f a i l u r e i n r e nal f i l t r a t i o n w i l l result i n an excessively high serum concentration of aminoglycoside which i n turn w i l l result i n further renal injury. Aminoglycosides accumulate i n renal parenchyma, mainly i n the cortex, i n concentrations considerably greaters than those i n serum. Streptomycin i s widely used generally i n combination with penic i l l i n for treatment of c a t t l e with shipping fever, m a s t i t i s or a f ter surgery and trauma.

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch002

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Neomycin i s commonly used i n combination with other drugs. Pa­ r e n t e r a l l y , neomycin i s quite nephrotoxic. It i s most often used t o p i c a l l y i n animals with infectious diseases of the eye and exter­ nal ear or contaminated wounds. Neomycin i s also available alone or i n combination with other drugs for the treatment of enteric i n f e c ­ tions and for the intra-mammary treatment of mastitis i n cows. Kanamycin i s unapproved i n the USA for use i n food animals but i n many other countries i t i s used for the treatment of c a t t l e with respiratory tract diseases, m a s t i t i s , and other infectious diseases. Gentamicin i s indicated for control of b a c t e r i a l infections of the uterus i n horses and c a t t l e and as an aid to improving concep­ tion. Treatment i s given by intra-uterine infusion of 2 g mixed with 200 ml s t e r i l e saline d a i l y for 3 days. Gentamicin i s also i n ­ dicated for the treatment of pigs with c o l i b a c i l l o s i s or swine dysentry IM or o r a l l y as well as drinking water administration. A l ­ though unapproved i n the USA, gentamicin has been used i n c a t t l e by intra-mammary infusion for treatment of m a s t i t i s , by intra-uterine infusion for treatment of m e t r i t i s , and parenterally f o r treatment of respiratory tract infections (11). Amikacin i s indicated for the treatment of genital tract i n f e c ­ tions i n the mare by intra-uterine infusion. Apramycin i s unapproved for use i n the USA f o r food animals but i n many European countries i t i s widely used for the same indica­ tions as neomycin. Apramycin i s e f f e c t i v e in v i t r o against neomy­ c i n - and streptomycin-resistant Gram-negative bacteria associated with diseases of new born calves. The future development of aminoglycosides for use i n veterinary medicine w i l l depend on two main factors. The f i r s t i s the cost of producing them as the synthetic process i s expensive. The second i s depdendent on discovering an aminoglycoside that does not accumulate and remain i n kidney tissue for prolonged periods, resulting i n a shorter withdrawal period for food producing animals (11)· Macrolides. This group includes erythromycin, t y l o s i n , oleandomycin and spiramycin. These a n t i b i o t i c s are b a c t e r i o s t a t i c and are ac­ tive against Gram-positive bacteria, including p e n i c i l l i n G-resistant staphylococci, and mycoplasma. They are l i p o p h i l l i c weak bases and can achieve excellent tissue penetration and r e l a t i v e l y long tissue l i f e . In such tissues as lung and mammary gland, drug con­ centrations may be 3 to 4 times serum l e v e l s . Oral absorption i s variable with erythromycin and may be reduced by the presence of feed for some preparations (6). Absorption from IM i n j e c t i o n s i t e i s slow and because of the wide tissue d i s t r i b u t i o n high serum l e ­ vels are d i f f i c u l t to a t t a i n . They are used parenterally and o r a l l y for the treatment of respiratory infections, p a r t i c u l a r l y those as­ sociated with Mycoplasma spp. and for the intra-mammary treatment of bovine m a s t i t i s . Tetracyclines. Oxytetracycline i s widely used i n c l i n i c a l practice against a broad array of pathogens although i t s e f f i c a c y against some Gram-negative bacteria has declined i n recent years. The ex­ tensive tissue d i s t r i b u t i o n of this group i s of particular value i n the treatment of respiratory tract infections. Although a l l the te­ tracyclines are well distributed to respiratory tissues, including

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Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch002

the sinuses, doxycycline appears to accumulate i n bronchial f l u i d s and may have p a r t i c u l a r value i n chronic bronchial infections. Oral absorption i s good i n monogastrics, but questionable i n ruminants. Although systemic e f f e c t s are attainable by oral administration i n ruminants, high serum levels are best achieved by IV administrat i o n . The recently introduced long-acting (LA) formulations of oxytetracycline have gained great popularity. They are used at a r e l a t i v e l y high dose (20 mg/kg) and serum levels s u f f i c i e n t to i n h i b i t the growth of susceptible pathogens can be maintained for 3 days. These products are extensively used i n the treatment and control of several tick-borne infections such as anaplasmosis. Chloramphenicol. This potent b a c t e r i o s t a t i c a n t i b i o t i c remains one of the drugs of choice against many Gram-negative pathogens, a l though i t i s not approved i n the USA for food animal use. I t i s a l so e f f e c t i v e against anaerobes. Chloramphenicol i s most e f f e c t i v e when given IV. Administration by the IM route i s accompanied by err a t i c absorption i n many species. Oral administration i n ruminants i s not e f f e c t i v e beyond the age of 2 to 3 weeks because of ruminai degradation. The h a l f - l i f e of the drug i n the body i s somewhat var i a b l e between species, but i s generally of short duration. The drug i s extensively metabolized i n many species to metabolites which are inactive against bacteria. In most species frequency of adminis t r a t i o n must be 2 or 3 times d a i l y . The drug i s well distributed i n the body, achieving serum concentrations or higher i n many t i s sues. Concentrations i n brain are somewhat lower than i n serum but s t i l l i n the e f f e c t i v e range. This i s a neutral drug so ionization i s not a consideration as regards e f f i c a c y . The drug i s extensively used outside the USA for the treatment of infections due to Gramnegative pathogens associated with pneumonia, p e r i t o n i t i s , gastroe n t e r i t i s , a r t h r i t i s and m a s t i t i s . Trimethoprim. This b a c t e r i o s t a t i c metabolic i n h i b i t o r of folate reductase system i n bacteria i s available as o r a l and parenteral combination with sulfanumides, p a r t i c u l a r l y sulfadiazine and s u l f a doxine. The combination i s very often b a c t e r i c i d a l to a wide range of pathogens otherwise resistant to the sulfonamides. I t i s well distributed throughout the body including the reproductive organs. The need for using a r e l a t i v e l y low dosage of the combination greatl y reduces the likelyhood of toxic reactions. As of yet, t h i s combination i s not approved f o r use i n food animals i n the USA but has attained widespread acceptance elsewhere. The o r a l preparations are probably e f f e c t i v e only i n the preruminant c a l f , f o r others i t must be given parenterally. The combination i s used for the treatment of infections due to Gram-negative and Gram-positive pathogens associated with pneumonia, p e r i t o n i t i s , gastro-enteritis and m a s t i t i s . Some r e s t r i c t i v e tissues (reproductive) may l i m i t penetration of the sulfonamide component with the resultant underdosing of the trimethoprim alone which may account for some therapeutic f a i l u r e s (6). Sulfonamides. This group of well recognized a n t i b a c t e r i a l drugs has long been used i n veterinary medicine although i n recent year their use has declined perhaps due to interest i n a n t i b i o t i c s of more recent development. As a group, the sulfonamides have a bacterios t a t i c , broad spectrum of action and good tissue d i s t r i b u t i o n

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

2. ζιν

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throughout the body. The more l i p i d - s o l u b l e sulfonamides achieve the best tissue l e v e l s , but a l l are generally low i n the mammary gland due to their acidic properties. Since the h a l f - l i v e s of most of the sulfonamides are long and o r a l absorption i s slow, the d a i l y dosage can usually be reduced to two-thirds or one-half of the i n i ­ t i a l dose. Of recent development are dosage forms with reduced ab­ sorption rates to prolong serum and tissue l e v e l s .

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch002

A n t i b i o t i c combinations Any discussion of a n t i b i o t i c s must eventually broach the topic of combination therapy. This i s a much discussed but l i t t l e appre­ ciated area because of the lack of good basic information available (12, 13). The e f f e c t s of a n t i b i o t i c combinations are quite s p e c i f i c for individual b a c t e r i a l species and they may have quite diverse ef­ fects ranging between synergism and antagonism of one another when u t i l i z e d against d i f f e r e n t bacteria. To f u l l y appreciate the value of any combination, i t would require testing on each b a c t e r i a l spe­ cies using a wide range of combination r a t i o s . Because of t h i s va­ r i a b i l i t y i t i s d i f f i c u l t to develop general guidelines. Several combinations may be quite helpful for serious i l l n e s s i n the weak and debilated patient p a r t i c u l a r l y when a mixed b a c t e r i a l population i s involved. The combination may also reduce the development of re­ sistance. In many cases, the disadvantage of increased r i s k of ad­ verse drug reaction, potential antagonism between a n t i b i o t i c s and increased expense may be more important than the advantage of the combination. In many therapeutic situations the drug combinations are com­ p l e t e l y misused (12). Adding another drug to a combination does not reduce the need for sound c l i n i c a l judgement i n therapy. Although there are many disease situations i n which the use of more than one a n t i b a c t e r i a l agent may be j u s t i f i e d , generalizations about various combinations of b a c t e r i c i d a l or bacteriostatic a n t i b i o t i c s or ad­ mixtures have not proven v a l i d . B a s i c a l l y , a n t i b i o t i c combinations should be avoided as a common practice unless they have shown a clear increase i n effectiveness as reported i n the l i t e r a t u r e . Literature cited 1. 2. 3. 4.

5. 6. 7. 8. 9.

Halpin, B. "Patterns of Animal Disease"; Bailiere Tindall: Lon­ don, 1975. Walton, J.R. Zbl. Vet. Med. A, 1983, 30, 81-92. Brander, G.C.; Ellis, P.R. "Animal and Human Health : The Con­ trol of Disease"; Baillier Tindall: London, 1976. Wilson, G.S.; Miles, A. In "Toply and Wilson's Principles of Bacteriology, Virology and Immunity" 6th Edn.; Arnold: London, 1975. Schwabe, C.W.; Riemann, H.P.; Franti, C.E. "Epidemiology in Ve­ terinary Practice"; Lea Febiger: Philadelphia, 1977. Burrows, G.E. Bovine Practitioner 1980, 15, 103-110. Koritz, G.D. J . Amer. Vet. Med. Assoc. 1984, 185, 1072-5. Powers, T . E . ; Varma, K . J . ; Powers, J.D. J . Amer. Vet. Med. Assoc. 1984, 185, 1062-7. Baggot, J.D. J . Amer. Vet. Med. Assoc. 1984, 185, 1076-82.

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

22 10. 11. 12. 13.

AGRICULTURAL USES OF ANTIBIOTICS

Thomson, T.D.; Quay, J.F.; Webber, J.A. J . Amer. Vet. Med. Assoc. 1984, 185, 1109-14. Benitz, A.M. J . Amer. Vet. Med. Assoc. 1984, 185, 1118-23. Burrows, G.E. Bovine Practitioner 1980, 15, 99-102. Stowe, C.M. J . Amer. Vet. Med. Assoc. 1985, 185, 1137-44.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch002

RECEIVED March 3, 1986

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

3

A n t i b i o t i c s in T r e a t m e n t of M a s t i t i s W. D. Schultze

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch003

Milk Secretion and Mastitis Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705 Antibiotics have failed early expectations of chemo-sterilization of the mammary gland. Treatment before bacterial identification necessitates use of broad-spectrum drugs. Cure rates vary with the pathogen: low for Staphylococcus aureus, high for Streptococcus agalactiae. The most effective antibiotics against Gram-negative b a c i l l i are not approved for U.S. use. During lactation, therapy is usually limited to c l i n i cal cases, eliminating clinical signs in 90% of cases but achieving many fewer bacteriologic cures. Mass in-tramammary treatment at the end of lactation is common. Higher cure rates due to higher drug concentrations and long retention in the gland, plus avoidance of milk discard are advantages. Some degree of prophylaxis is afforded against the high new infection rate in the early nonlactating period. Evidence conflicts as to increased resistance to antibiotics as a result of mastitis treatment, but has been reported for populations of staphylococci, streptococci and coliform bacteria.

M a s t i t i s , which i s defined as inflammation of the mammary gland, i s the single most costly disease i n American agriculture Direct losses to the dairyman average $182 per cow, or i n excess of $2 b i l l i o n annually to the dairy industry, nearly 70% of these the r e s u l t of l o s t milk production. In addition to the d i r e c t losses, mastitis causes s i g n i f i c a n t reduction i n milk quality, for both f l u i d consumption and f o r processing, and i n n u t r i t i o n a l value. I t leads to a n t i b i o t i c resistance problems i n milk, meat and the environment, to premature c u l l i n g of c a t t l e , and to reduced sale value of young dairy stock. In mastitis problem herds, annual losses due to masti t i s may exceed $300 per cow. Mastitis i s nearly always caused by b a c t e r i a l infection. The introduction of benzyl p e n i c i l l i n for the treatment of intramammary infections (IMI) caused by Gram positive bacteria, followed by products containing other a n t i m i c r o b i a l agents, was a major advance i n T h i s chapter not subject to U . S . copyright. Published 1986, A m e r i c a n C h e m i c a l Society

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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A G R I C U L T U R A L USES O F ANTIBIOTICS

m a s t i t i s control. I t made possible f o r the f i r s t time a major reduc­ tion of the losses caused by c l i n i c a l m a s t i t i s (2). I t provided a p r a c t i c a l method of eliminating Streptococcus agalactiae, the predom­ inant pathogen a t that time. Hopes soared even to the extreme of forseeing that we might be able to accomplish chemosterilization of the mammary gland, and thus eradicate bovine mastitis. In a c t u a l i t y , t h i s has been f a r from the case. A n t i b i o t i c ther­ apy, although an important component of m a s t i t i s control strategy, i s much less e f f e c t i v e than could be desired. Farm advisors uniformly stress the p r i n c i p l e that treatment must not be r e l i e d upon to r e ­ dress the disease promoting e f f e c t s of bad animal husbandry, unsani­ tary milking practices and defective milking machinery. M a s t i t i s i s a complex of infections, caused by a variety of microorganisms with inherent differences i n s e n s i t i v i t y to antimi­ c r o b i a l agents. Furthermore, s e n s i t i v i t y i n v i t r o does not assure e f f i c a c y i n vivo. Additionally, pathogens have the capacity to gain resistance to a n t i b i o t i c s , p a r t i c u l a r l y under conditions of heavy and poorly controlled use. S e n s i t i v i t y of M a s t i t i s Pathogens to A n t i b i o t i c s A senior B r i t i s h government veterinarian stated i n 1962 (3), "When p e n i c i l l i n was f i r s t used i n treating mastitis only 2% of the strains of staphylococci recovered from cases of mastitis were re­ s i s t a n t to p e n i c i l l i n . Today the figure i s over 70%." Between 1958 and 1961, resistance to p e n i c i l l i n (PEN) increased from 62.0% to 70.6%. Resistance to streptomycin (STR), tetracycline and chloram­ phenicol also increased (4). A n t i b i o t i c resistance increased f o r i s o l a t e s of both mastitis staphylococci and streptococci i n Canada between 1960 and 1967 (5). In Belgium (6), Staphylococcus aureus strains i s o l a t e d from cases of bovine m a s t i t i s showed increase i n PEN resistance from 38% i n 1971 to 78% i n 1974, but then no further i n ­ crease to 1980. The resistance s i t u a t i o n was reported to remain stable i n the Federal Republic of Germany between 1962 and 1975 (7), as also i n A u s t r a l i a between 1974 and 1979 (8) and Denmark, a t a very low l e v e l , for the period 1963 to 1978 (9). Inasmuch as s e l e c t i o n pressure i s considered responsible f o r de­ velopment of a n t i b i o t i c resistance, l o c a l differences i n drug usage may explain the widely varying resistance situations (10). Great d i s p a r i t i e s have been reported within nations as well as between them, f o r example, i n A u s t r a l i a (8) and Switzerland (11 ). In the United States, i t i s the common opinion (based on meagre and geographically r e s t r i c t e d data) that increasing a n t i b i o t i c re­ sistance among pathogens of bovine m a s t i t i s i s not a problem (12.13)» with the exception of resistance to PEN (14). A scan of reports ac­ cumulated over some 15 years (Table l ) supports the view, but also suggests a dramatic increase i n resistance to STR among staphylo­ cocci. I t i s noteworthy that none of the drugs to which Escherichia c o l i i s nearly always sensitive, namely gentamicin, polymyxin Β or chloramphenicol, are approved f o r use i n mastitis therapy i n the U.S. In the use of dry cow therapy (DCT) f o r m a s t i t i s control, we have a model s i t u a t i o n where a n t i b i o t i c s are introduced into the milk compartment a t high concentration and undergo slow d i s s i p a t i o n f o r up to 3 weeks (21,22,23) · In mass dry cow therapy (DCT), a l l cows i n a herd are infused i n a l l mammary quarters with an a n t i b i o t i c

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

b/

MD^

NY CA,IA,MD 92 95

—

23 37 40 11 23

100 100 53 31 30 58 55

42 90 92

^ One research herd of about 180 milking cows

Ρ0Β polymyxin Β

STR dihydrostreptomycin,

1973 1975 1977 1982 1983

15 8

48 24

—

1969 1973 1980

100 100 90

0 0 4

5 19

0

1976 1976 1983

9 30 39 31 61

2 3 46). Z i v (23) has summarized the desirable k i n e t i c and other properties of such a product. The f o l ­ lowing a n t i b i o t i c formulations are presently approved by the U.S. Center for Veterinary Medicine, FDA f o r infusion into the dry mammary gland: erythromycin (300 mg), oxytetracycline-HCl (426 mg), benza­ thine c l o x a c i l l i n (500 mg), cephapirin benzathine (300 mg), novobio­ c i n (400 mg), p e n i c i l l i n (200,000 IU) & novobiocin (400 mg), p e n i c i l ­ l i n (1 χ 10 IU) & dihydrostreptomycin (1 g), p e n i c i l l i n (200,000 IU) & dihydrostreptomycin (300 mg), and procaine p e n i c i l l i n G (100,000 IU) (26). Varying estimates of therapeutic success from use of dry cow formulations have been reported (Table I I ) . Correction for spontane­ ous cure rate i n an appropriate control group i s necessary to pre­ clude serious overestimation of drug e f f i c a c y . Mass DCT i s a popular and commonly recommended strategy i n the U.S., the U.K., A u s t r a l i a , Ireland, the Federal Republic of Germany, France, the Netherlands, South A f r i c a and I s r a e l (54). Antimicrobial treatment at drying off i s r a r e l y practiced i n Austria, Czechoslovak­ i a , Hungary, Spain, Japan, Norway and Poland. New Zealand and most of the Scandinavian countries favor s e l e c t i v e DCT, i n which only those cows receive treatment who have either a history of c l i n i c a l mastitis during the preceding l a c t a t i o n or current signs of i n f e c t i o n (54). I t has been suggested that as m a s t i t i s control using the strategies common among the English-speaking countries reduces d i s ­ ease prevalence we must rethink the question of mass versus s e l e c t i v e therapy (55). b

Selective Dry Cow Therapy. Two c r i t e r i a are c r i t i c a l to the success of a regimen for s e l e c t i v e DCT: the a b i l i t y to detect and thus

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986. 8&

67^

7

Streptococcus uberis only

Percentage computed by correcting f o r author's reported spontaneous cure rate

Percent of infected quarters rendered noninfected

34

b/

61

Procaine p e n i c i l l i n G (200,000 IU); novobiocin (0.4 g)

(0.4 g)

35*/

Procaine p e n i c i l l i n G (500,000 IU); novobiocin (0.6 g)

Novobiocin

lOO^

90^

77-

Neomycin-SO, (0.5 g); benzathine p e n i c i l l i n V (325,000 IU)

45^

75

65

62

Neomycin-SO^ (0.5 g)

59^

90

5 6 ^ '

b/

combined 97

τ τ

Streptococcus : other

7&

9 1

97

Streptococcus agalactiae

Benzathine c l o x a c i l l i n (0.5 g)

Benzathine c l o x a c i l l i n (0.5 g)

84

76

Benzathine p e n i c i l l i n G (200,000 IU); dihydrostreptomycin-SO^ (0.4 g)

Benzathine c l o x a c i l l i n (1 g)

87

Procaine p e n i c i l l i n G (1 χ 10°IU); dihydrostreptoraycin-SO^ (1 g)

Staphylococcus aureus

E f f i c a c y of Dry Cow Therapy Against Intramammary Infections

A n t i b i o t i c s i n formulation

Table I I .

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch003

b/

31-b/

89

n

Coliform bacteria

52

51

50

49

48

49

48

47

46

41

References

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch003

30

A G R I C U L T U R A L USES O F ANTIBIOTICS

select for treatment a high proportion of cows with IMI present at drying o f f , and achievement of a degree of prophylaxis not greatly i n f e r i o r to that afforded by mass DCT. Studies of e f f i c i e n c y of detection have not been encouraging. Selection for treatment of i n d i v i d u a l quarters according to t h e i r strong reaction i n the Whiteside test (a rough measure of inflammation of the gland) (56) detected only 39% of infected quarters and predictably would have permitted 80% of a l l staphylococcal infections to persist into the next l a c t a t i o n . Treating a l l quarters of cows which had had c l i n i c a l mastitis during the previous l a c t a t i o n (57) would have missed 34% of quarters infected at drying off and 39% of those which subsequently became i n fected during the dry period. A comparison of s e l e c t i o n c r i t e r i a i n cluding mastitis history, somatic c e l l count history and current C a l i f o r n i a mastitis test score, alone and i n combination (58), showed predictions ranging from 50% to 92% of infected cows, but included from 49% to 80% of the noninfected cows. Selection for DCT of cows drying o f f with a history of mastitis treatment during l a c t a t i o n or with one or more quarters p o s i t i v e to bactériologie culturing at drying o f f (17) was successful, of course, i n clearing most previously infected glands. The o v e r a l l r e s u l t , however, was a net gain of 1.4% i n infected quarters at the next calving, occasioned by the f a i l u r e to control new dry period IMI among untreated cows and peripartum IMI among both groups. Prophylactic E f f i c a c y of Dry Cow Therapy. Prophylactic efficacy might be expected to go hand i n hand with e f f i c i e n c y of selecting i n fected animals, f o r the new i n f e c t i o n r a t e i n the dry period tends to be higher among cows entering the period with at least one quarter infected (17,59). Some a u t h o r i t i e s , however, question the very existence of prophylaxis as an aspect of DCT (60). Nonetheless, one of the e a r l i e s t uses of a n t i b i o t i c s against bovine mastitis was the prevention of "summer m a s t i t i s " (a special form of the disease not seen i n the U.S.) by treating every cow at drying o f f (61). F i e l d t r i a l s have demonstrated better than 90% control of t h i s disease entity through DCT (62,63,64). Experimental design of most studies has been inadequate to provide convincing evidence of prophylactic e f f i c a c y . Contributing to the problem, designs i n which new IMI at the beginning of the dry period are lumped with new IMI at calving obscure the effects of prophylaxis. They imply an u n r e a l i s t i c expectation of DCT, for peripartum i n f e c t i o n must be treated as a separate problem. I t cannot be attacked merely by stretching the persistence i n the gland of longacting products for DCT. After attempting to correct for biases i n selection of animals for treatment (51), prophylactic efficacy of a p e n i c i l l i n - n o v o b i o c i n formulation was estimated at nearly 50%. Some studies i n which evidence of prophylaxis against new IMI i n the early dry period has been adduced have noted great differences i n t h i s r e gard among a n t i b i o t i c formulations (65) or among species of pathogen (53). I t seems reasonable to expect prophylaxis as a benefit of dry cow therapy but currently there i s i n s u f f i c i e n t documentation to permit a good estimate of i t s magnitude. In addition to DCT, recommended mastitis control i n the U.S. i n cludes the dipping of the cow's teats i n a germicide immediately after each milking. This strategy commonly results i n a lowered prevalence of IMI i n a dairy herd, but also a s h i f t i n pathogen

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

3.

SCHULTZE

Antibiotics

in Treatment of

Mastitis

31

d i s t r i b u t i o n . E f f i c a c y of current mastitis control i s greatest against S^. aureus and Str. agalactiae, for which species cow-to-cow transmission seems of primary importance. We see an increasing number of dairy herds i n which the mastitis problem stems c h i e f l y from exposure of teat ends between milkings, to pathogens o r i g i n a t i n g i n the cow's environment: streptococci other than Str. agalactiae and Gram-negative b a c i l l i members of the coliform group (66). For such herds, r e s t r i c t e d application of DCT would seem reasonable i f the s a c r i f i c e of p o t e n t i a l prophylaxis i n the early dry period were not too great.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch003

A n t i b i o t i c A c t i v i t y and Phagocytosis S e n s i t i v i t y of m a s t i t i s pathogens to an a n t i b i o t i c i n v i t r o merely indicates p o t e n t i a l therapeutic e f f i c a c y . The data from c l i n i c a l t r i a l s r e f l e c t a l e s s encouraging r e a l i t y , i n which both pathogen and host c h a r a c t e r i s t i c s influence the outcome (67). Some pathogens are highly tissue-invasive. Once sequestered and metabolically i n a c t i v e within i n f e c t i o n f o c i they are unaffected by a n t i b i o t i c s that act by disruption of c e l l wall synthesis, such as p e n i c i l l i n s and cephalosporins . The aim of antimicrobial therapy i s to k i l l or temporarily i n activate a s u f f i c i e n t proportion of the population of invading bact e r i a to permit host defense mechanisms to accomplish s t e r i l i z a t i o n of the affected t i s s u e (67). Phagocytosis of c e l l s of the pathogen by several classes of blood-derived leukocytes i s a c r i t i c a l element i n the process. However, i n t r a c e l l u l a r s u r v i v a l within phagocytic c e l l s can be a s i g n i f i c a n t contributor to f a i l u r e of a n t i b i o t i c therapy (68). At least i n the case of Staphylococcus aureus, phagocytos i s i s not always followed by k i l l i n g , and can indeed protect the engulfed bacteria from exposure to c l o x a c i l l i n for up to 4 days. Furthermore, a n t i b i o t i c s and formulation vehicles used i n i n t r a mammary infusion therapy against mastitis can have a deleterious effect on the v i a b i l i t y and phagocytic a c t i v i t y of neutrophilic leukocytes i s o l a t e d from bovine milk. In an i n v i t r o assay for phagocytos i s of 32p_i b ;L d is, aureus, the percentage of phagocytosis was s i g n i f i c a n t l y reduced by addition to the incubation mixture of tiamulin, nitrofurantoin, rifampin, chloramphenicol or amikacin i n quantities r e f l e c t i v e of t h e i r concentration i n milk 6 h after i n j e c t i o n into a mammary gland (69). Also, gentamicin, t e t r a c y c l i n e , and novobiocinp e n i c i l l i n were i n h i b i t o r y at a concentration similar to that i n milk immediately a f t e r i n j e c t i o n . Incubation with chloramphenicol, novob i o c i n - p e n i c i l l i n or tiamulin also a f f e c t s o v e r a l l neutrophil v i a b i l i t y , as measured by exclusion of trypan blue dye from the c e l l (7Q). Disruption of the morphology and function of bovine milk neutrophils was produced i n vivo by intramammary infusion of t e t r a c y c l i n e , gentamicin or chloramphenicol (71). a

e

e

Coda Nowadays, one stated objective of much of the more imaginative masti t i s research i s the reduction i n our dependence on a n t i b i o t i c s and other exogenous chemicals to control bovine m a s t i t i s . Achievement of t h i s goal i s nowhere i n sight. And so, we are l e f t dependent upon antimicrobial therapy, despite i t s many l i m i t a t i o n s , as a major element i n control strategy for bovine m a s t i t i s .

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

32

AGRICULTURAL USES OF ANTIBIOTICS

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Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch003

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Jasper, D. E.; McDonald, J. S.; Mochrie, R. D.; Philpot, W. N.; Farnsworth, R. J.; Spencer, S. B. Proc. 21st Annu. Mtg. Nat. Mastitis Coun., 1982, pp. 182-4. 2. IDF Group of Experts on Mastitis. "Principles of Mastitis Control"; International Dairy Federation Doc. 76: Brussels, 1973; p. 67. 3. Wilson, C. D. J . Soc. Dairy Technol. 1962, 15, 21-7. 4. Wilson, C. D. Vet. Rec. 1961, 73, 1019-24. 5. Greenfield, J.; Bankier, J. C. Can. J. comp. Med. 1969, 33, 3943. 6. Devriese, L. A. Ann. Rech. Vet. 1980, 11, 399-408. 7. Weigt, U . ; Bleckmann, E. Dtsch. Tierärztl. Wschr. 1977, 84, 234-5. 8. Frost, A. J.; O'Boyle, D. Aust. vet. J . 1981, 57, 262-7. 9. Madsen, J. A. Nord. Vet-Med. 1978, 30, 434-6. 10. Finland, M. J. Inf. Pis. 1970, 122, 419-31. 11. Schifferli, D.; Schällibaum, M.; Nicolet, J . Schweiz. Arch. Tierheilk. 1984, 126, 83-90. 12. Davidson, J . N. Proc. 19th Annu. Mtg. Nat. Mastitis Coun., 1980, pp. 181-5. 13. Natzke, R. P. Proc. 21st Annu. Mtg. Nat. Mastitis Coun., 1982, pp. 125-33. 14. McDonald, J. S.; Anderson, A. J. Cornell Vet. 1981, 71, 391-6. 15. Philpot, W. N. J . Dairy Sci. 1969,52, 708-13. 16. Schultze, W. D. Proc. 26th Annu. Mtg. Northeastern Mastitis Conf. 1973, pp. 67-74. 17. Schultze, W. D. J . Dairy Sci. 1983, 66, 892-903. 18. McDonald, J. S.; McDonald, T. J.; Stark, D.R. Am. J. Vet. Res. 1976, 37, 1185-8. 19. McDonald, J. S.; McDonald, T. J.; Anderson, A. J. Am. J. Vet. Res. 1977, 38, 1503-7. 20. Davidson, J. N. J . Am. Vet. Med. Assoc. 1982, 180, 153-5. 21. Ziv, G . ; Saran-Rosenzuaig, A . ; Risenberg, R. Zbl. Vet. Med. Β 1973, 20, 415-24. 22. Ziv, G . ; Gordin, S.; Bechar, G . ; Bernstein, S. Br. vet. J. 1976, 132, 318-22. 23. Ziv, G. Vet.Med./S.A.C. 1980, 75, 657-70. 24. Rollins, L . D . ; Pocurull, D. W.; Mercer, H. D.; Natzke, R. P.; Postle, D. S. J . Dairy Sci. 1974, 57,944-50. 25. Berghash, S. R.; Davidson, J . N.; Armstrong, J. C.; Dunny, G. M. Antimicrobial Agents Chemotherapy 1983, 24, 771-6. 26. Anonymous. "National Drug Withdrawal Guide for Dairymen"; National Milk Producers Federation: Arlington, 1982. 27. Griffin, T. K . ; Dodd, F. H . ; Bramley, A. J. Proc. Br. Cattle Vet. Assoc. 1981-82, 1982, pp. 137-52. 28. Bramley, A. J.; Dodd, F. H. J . Dairy Res. 1984, 51, 481-512. 29. Nicolet, J.; Schällibaum, M.; Schifferli, D.; Schweizer, R. Schweiz. Arch. Tierheilk. 1983, 125, 31-43. 30. Olsen, S. J. In "Proc. IDF Seminar on Mastitis Control 1975"; International Dairy Federation Doc. 85: Brussels, 1975; pp. 41041.

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

3. SCHULTZE

Antibiotics in Treatment of Mastitis

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch003

31.

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Klastrup, O. In "Proc. Symposium on Mastitis Therapy, Copenhag­ en"; Novo Industri A/S: Bagsvaerd, Copenhagen, 1982; pp. 1-7. 32. Senze, Α.; Jakubowski, S. Zeszyt. nauk. wyzsz. Szkol. roln., Wroclaw 1957, No. 10 (Weterynaria III), 135-49 (summary in Ger.) 33. Morris, R. S. Aust. vet. J . 1973, 49, 153-6. 34. Plommet, M.; Le Loudec, C. In "Proc. IDF Seminar on Mastitis Control 1975"; International Dairy Federation Doc. 85: Brussels, 1975; pp. 265-81. 35. McDermott, M. P.; Erb, H. N . ; Natzke, R. P.; Barnes, F. D.; Bray, D. J . Dairy Sci. 1983, 66, 1198-1203. 36. Bakken, G. Acta Agric. Scand. 1981, 31, 273-86. 37. Bakken, G.; Gudding, R. Acta Agric. Scand. 1982, 32, 17-22. 38. Woolford, M.W.;Williamson, J . H . ; Copeman, P. J. Α.; Napper, A. R.; Phillips, D. S. M.; Uljee, E. Proc. 35th Ruakura Farm­ ers' Conf., Hamilton, N.Z., 1983, pp. 115-9. 39. Smith, K. L.; Todhunter, D. A. Proc. 21st Annu. Mtg. Nat. Mast­ i t i s Coun., 1982, pp. 87-100. 40. Eberhart, R. J. Proc. 21st Annu. Mtg. Nat. Mastitis Coun., 1982, pp. 101-11. 41. Dodd, F. H . ; Westgarth, D. R.; Neave, F. K . ; Kingwill, R. G. J . Dairy Sci. 1969, 52, 689-95. 42. Dodd, F. H . ; Griffin, T. K. In "Proc. IDF Seminar on Mastitis Control 1975"; International Dairy Federation Doc. 85: Brussels, 1975; pp. 282-302. 43. Roberts, S. J.; Meek, Α. M.; Natzke, R. P.; Guthrie, R. Proc. 19th World Vet. Cong., Mexico City, 1971, 3, 935-9. 44. Funke, H. Acta Vet. Scand. 1961, 2 (Suppl. 1), 1-88. 45. Smith, Α.; Neave, F. K . ; Dodd, F. H . ; Jones, Α.; Gore, D. N. J . Dairy Res. 1967, 34, 47-57. 46. Ziv, G . ; Saran-Rosenzuaig, Α.; Gluckmann, E. Zbl. Vet. Med. Β 1973, 20, 425-34. 47. Bäckström, G.; Johansson, Α.; Petersson, O.; Winsö, S. Svensk Veterinärtid. 1980, 32, 83-5. 48. Smith, Α.; Westgarth, D. R.; Jones, M. R.; Neave, F. K . ; Dodd, F. H . ; Brander, G. C. Vet. Rec. 1967, 81, 504-10. 49. Postle, D. S.; Natzke, R. P. Vet.Med./S.A.C. 1974, 69, 1535-9. 50. Pankey, J . W.; Barker, R. M.; Twomey, Α.; Duirs, G. N.Z. Vet. J. 1982, 30, 50-2. 51. Schultze, W. D.; Mercer, H. D. Am. J. Vet. Res. 1976, 37, 12759. 52. Pankey, J . W.; Barker, R. M.; Twomey, Α.; Duirs, G. N.Z. Vet. J. 1982, 30, 13-5. 53. Swenson, G. H. Can. J. comp. Med. 1979, 43, 440-7. 54. IDF Group A.2 (Bovine Mastitis) "International Progress in Mastitis Control 1983"; International Dairy Federation Bull. 187: Brussels, 1985; 20 pp. 55. Schultze, W. D. Proc. 14th Annu. Mtg. Nat. Mastitis Coun., 1975, pp. 41-53. 56. Natzke, R. P. J . Dairy Sci. 1971, 54, 1895-1901. 57. Schultze, W. D.; Casman, Ε. Α.; Lillie, J . H. J . Dairy Sci. 1974, 57, 643 (Abstr.). 58. Rindsig, R. B.; Rodewald, R. G . ; Smith, A. R.; Thomsen, Ν. K . ; Spahr, S. L. J . Dairy Sci. 1979, 62, 1335-9. 59. Neave, F. K . ; Dodd, F. H . ; Henriques, E. J . Dairy Res. 1950, 17, 37-49.

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34 60. 61. 62. 63. 64. 65.

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66. 67. 68. 69. 70. 71.

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Ziv, G. In "Progress in Control of Bovine Mastitis: IDF Semin­ ar, Kiel"; Kieler Milchwirtschaftl. Forschungsber. 1986 (In Press). Wilson, C. D. In "Mastitis Control and Herd Management"; Bramley, A. J.; Dodd, F. H . ; Griffin, T. Κ., eds.; N.I.R.D.: Reading, 1981; pp. 113-27. Pearson, J . K. L. Vet. Rec. 1950, 62, 166-8. Pearson, J . K. L . Vet. Rec. 1951, 63, 215-20. Franke, V . ; Tolle, Α.; Reichmuth, J.; Beimgraben, J . In "Heifer Mastitis Seminar, Stockholm-Helsinki"; Svensk Husdjursskötsel: Eskilstuna, 1983; Chap. 2. Natzke, R. P.; Everett, R. W.; Bray, D. R. J . Dairy Sci. 1975, 58, 1828-35. Smith, K. L.; Todhunter, D. Α.; Schoenberger, P. S. J . Dairy Sci. 1985, 68, 1531-53. Craven, N . ; Williams, M. R.; Anderson, J . C. Proc. 4th Int. Sympos. Antibiotics in Agric., 1984, pp. 175-92. Craven, N . ; Anderson, J. C. J . Dairy Res. 1984, 51, 513-23. Ziv, G.; Paape, M. J.; Dulin, A. M. Am. J. Vet. Res. 1983, 44, 385-8. Nickerson, S. C.; Paape, M. J.; Dulin, A. M. Am. J. Vet. Res. (In Press). Nickerson, S. C.; Paape, M. J.; Dulin, A. M. J . Dairy Sci. 1984, 67 (Suppl. 1), 85 (Abstr.).

RECEIVED February 18, 1986

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

4

A n t i b i o t i c s in B e e k e e p i n g Robert J. Argauer

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch004

Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705

A vital role insects play in the pollination of many plants, including some of our most important agricultural crops, was described in 1976 by McGregor, an apiculturist, in a handbook published by the Agricultural Research Service, United States Department of Agriculture (1). According to USDA estimations the value of crops in 1980 requiring bee pollination for seed or fruit in the United States approached $20 billion (2). Honey and beeswax produced was valued at $140 million. In this paper we present a brief history of the use of sulfathiazole, Terramycin®, and Fumidil-B® as antimicrobials in beekeeping. Included are some results of our published research, as well as some of our new research in which we show why the precautions - stated explicitly on the current Terramycin® label to assure that honey intended for human consumption is free of trace amounts of drug residues - also implicitly apply to medicated colonies from which pollen may be collected for human consumption. The normal honey bee colony i s considered by many beekeepers as a superorganism made up of between 10,000 and 60,000 bees. A s t r i c t system of sanitation has been created i n the colony i n order to minimize the spread of diseases that are contagious to honey bees, Apis m e l l i f e r a L. To augment this natural system, and to insure strong and healthy colonies, a p i c u l t u r i s t s , soon a f t e r antimicrobials came into general use i n the 1940's, began the feeding of drugs as a preventive measure to control the spread of American foulbrood disease, caused by Bacillus larvae, and European foulbrood disease, caused by Melissococcus pluton, i n honey bees. The s u s c e p t i b i l i t y of honey bee larvae to American foulbrood was described by Woodrow i n 1941 (7). Farrar (8) reported i n 1956 that one Bacillus larvae spore that gains entrance to a bee larva of the proper age under the right conditions may be multiplied two to three b i l l i o n times i n eight or nine days. He recommended that an occasional colony infected with American foulbrood should be burned. As a preventive measure, a l l remaining colonies should be sprayed with medicated sugar sprays or dusted with medicated powdered sugar dusts. T h i s chapter not subject to U . S . copyright. Published 1986, A m e r i c a n C h e m i c a l Society

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

A G R I C U L T U R A L USES O F ANTIBIOTICS

36

In the United States almost a l l the states have laws and regulations r e l a t i n g to honey bees and beekeeping that are designed primarily to control the spread of bee diseases. The beekeeper consults with his state apiary inspector for state recommendations.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch004

Sulfathiazole Sodium s u l f a t h i a z o l e , though not an a n t i b i o t i c , was one of the early antimicrobial drugs found e f f e c t i v e for the control of American foulbrood. It i s not e f f e c t i v e for the control of European foulbrood. These findings were based on the research of Haseman i n 1946, Johnson i n 1947, Reinhardt i n 1947, and Eckert i n 1948 (3_6). Eckert used a colorimeter to measure s u l f athiazole i n a honey bee colony before and after the medicated sugar syrups were fed to and processed by the bees. He stated, "Due to the dangers of introducing even small quantitites of s u l f a t h i a z o l e i n marketable honey the general use of this drug as a preventive measure i n the control of American foulbrood i s not j u s t i f i e d at the present time." Using microbiological assay i n a series of papers i n the 1950*s, Landerkin and Katznelson (_9) confirmed that s u l f a drugs remained stable for three years at 34°C i n honey and sugar syrup. He found the order of s t a b i l i t y for several drugs i n sugar syrup and honey were as follows: s u l f a drugs > streptomycin > t e t r a c y c l i n e > chlortetracycline > erythromycin > oxytetracycline. Sulfathiazole i s not registered for use i n the United States at the present time. H i s t o r i c a l l y s u l f a t h i a z o l e has been used for f a l l feeding. If a l l stored food were consumed by spring the danger of contaminated harvested honey appeared remote. In 1982 (19) we developed the a n a l y t i c a l chemical methodology based on normal phase HPLC needed to detect small amounts of s u l f a t h i a z o l e (Figures 1, 2) i n honey and to measure the amount of s u l f a t h i a z o l e that may be transferred to stored honey when honey bee colonies were fed medicated sugar solutions. Figure 1 compares the separation of four sulfonamide drugs on a cyano-amino polar phase. Our ultimate goal was to determine i f sodium s u l f a t h i a z o l e can be used i n a manner that would not contaminate honey intended for human consumption. Figure 2 compares the chromatograms obtained for a s u l f a t h i a z o l e standard and f o r an extract of honey f o r t i f i e d with s u l f a t h i a z o l e . We were able to detect s u l f a t h i a z o l e i n the brood nest honey, but not i n the surplus honey (honey stored above the brood nest and available for harvest). The l i m i t of s e n s i t i v i t y was 0.2 ppm. In 1983, Barry and MacEachern (20), using reverse phase HPLC, reported that of nineteen commercial honeys collected by Agriculture Canada inspectors, 8 samples contained s u l f a t h i a z o l e residue at levels ranging from 0.10 to 0.56 ppm. Oxyt etracycline Registration for Use by Beekeepers. Terramycin® (oxytetracycline hydrochloride) i s the only drug that i s registered by the United States Food and Drug Administration for the feeding of medicated sugar syrups and powdered sugar dusts to honey bee colonies as an aid i n the prevention and control of American and European foulbrood diseases. Based on approved use for feeding, given by the dusting

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch004

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Time (minutes) Figure 1. HPLC separation of four sulfonamide drugs on a CN-N^ bonded polar phase (1) Sulfadiazine; (2) sulfapyridine; (3) sulfanilamide; (4) s u l f a t h i a z o l e . Mobile phase: 95% methylene chloride-5% methanol; flow rate 1 ml/min. Detection: 254 nm. Amount injected: 0.2 ]ig each (as the free acids obtained from Sigma Chemical Co.).

Time (minutes) Figure 2. HPLC chromatograms obtained f o r sulfathiazole standard compared with a honey control and honey f o r t i f i e d at 1.0 ppm (80% recovery). Mobile phase: 95% methylene chloride5% methanol; flow rate 1.5 ml/min; 0.2 Pg of s u l f a t h i a z o l e injected as standard; extract injected equivalent to 40 mg of honey.

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and syrup directions printed on the label (Table I ) , each colony receives 200 mg active ingredient per ounce of powdered sugar sprinkled on the ends of the frames, or 50 micrograms active ingredient i n each m i l l i l i t e r of sugar syrup fed either by using feeders or by f i l l i n g the brood combs to cause gourging. To avoid contamination of marketable honey by trace amounts of oxytetracycline, a l l medicated sugar dusts and syrup treatments of honey bee colonies that occur i n the spring and/or f a l l are terminated by the a p i c u l t u r i s t at least 4 weeks before the main honey flow begins. In addition a l l medicated honey or syrup stored during periods of medication i n combs reserved f o r surplus honey i s removed.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch004

TABLE I

Part of Label 60-7000-00-9 f o r the Use of Terramycin® Soluble Powder Distributed by P f i z e r , Inc. Revised Aug. 1976 BEES Terramycin i s recommended as an aid i n the prevention and control of American f o u l brood and European f o u l brood i n bees. Use Terramycin as directed below. DUSTING DIRECTIONS: Use 1 l e v e l teaspoonful (200 mg) of Terramycin Soluble Powder (TSP®) per ounce of powdered sugar per colony, or 1 l b . TM-10® (Terramycin) per 2 l b s . powdered sugar, applying 1 ounce of this mixture per colony. Apply the dust on the outer parts or ends of the frames. Usually 3 dustings at 4-5 day intervals are required i n the spring and/or f a l l at least 4 weeks before the main honey flow to prevent contamination of marketable honey. SYRUP DIRECTIONS: Use 1 l e v e l teapoonful (200 mg) of Terramycin Soluble Powder (TSP®) per 5 l b . j a r containing 1:1 sugar syrup per colony. Dissolve Terramycin Soluble Powder i n a small quantity of water before adding to syrup. Bulk feed the syrup using feeder p a i l s or d i v i s i o n board feeders or by f i l l i n g the combs. Usually 3 applications at 4-5 day intervals are required i n the spring and/or f a l l at least 4 weeks before the main honey flow to prevent contamination of marketable honey. WARNING: A l l Terramycin medicated supplements should be fed early i n the spring or f a l l and consumed by the bee before main honey flow begins to avoid contamination of production honey. Honey or syrup stored during medication periods i n combs for surplus honey should be removed following f i n a l medication of the bee colony and must not be used for human food. Honey from bee colonies l i k e l y to be infected with f o u l brood should not be used f o r preparation of medicated syrup supplements since i t may be contaminated with spores of foul brood and may result i n spreading the disease.

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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Safe Use of Oxytetracyline. To assure honey i s free from even the smallest trace of drug residue by the time i t reaches the market place, researchers have developed methodologies to measure and follow the degradation of oxytetracycline by microbiological and chemical means. Recommendations for use made on the l a b e l have been based p r i n c i p a l l y upon data obtained by microbiological assay that depend on the i n h i b i t i o n of growth of an indicator bacterium by oxytetracycline (10-12, 21). Naturally occurring antimicrobial substances that are found i n honey and i n bees also may give zones of i n h i b i t i o n which may be mistaken for a c t i v i t y of oxytetracycline, e s p e c i a l l y at trace residue l e v e l s . Wilson (22) i n 1974, based on the residue results obtained by D. W. Clarke, A g r i c u l t u r a l D i v i s i o n , P f i z e r , Inc. using the Microbiological Plate Diffusion Method, P f i z e r , Inc. reported that the background i n h i b i t i o n due to honey and/or pollen was about 0.25 ppm. In the early 1970 s we developed (14) a chemical means, based upon the fluorescence of calciumoxytetracycline complex described by Kohn (13) i n 1961, to monitor the d i s t r i b u t i o n of oxytetracycline i n medicated colonies. We now were able to support e a r l i e r observations that were based on microbiological assay, and were able to monitor the s t a b i l i t y of oxytetracycline i n medicated diets registered for use and i n several experimental medicated d i e t s . In b r i e f , the procedure developed involves the extraction of oxytetracycline from a t r i c h l o r o a c e t i c acid solution into ethylacetate-ethylacetoacetate, and addition of calcium chloride and ammonium hydroxide to remove the i n t e r f e r r i n g fluorescent phenols and acids while the oxytetracycline remains i n the organic phase as the fluorescent calcium complex. The s t a b i l i t y of oxytetracycline i n non-acidified water at brood nest temperature (34°C) i s given i n Figure 3. The h a l f - l i f e was determined to be about two days. Temperature i s a v a r i a b l e . The s t a b i l i t y of oxytetracycline i n sugar syrup bee diets (Table II) i s similar to the loss rate i n water at 34°C. Oxytetracycline appears r e l a t i v e l y stable at low temperature, and encased i n experimental bee diet formulations that contain pollen, sugar, or f a t (Tables I I I and IV).

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch004

f

TABLE I I . Relative S t a b i l i t y of Oxytetracycline i n Sugar Syrup Bee Diets (% Recovered) Time (weeks)

0 1 2 3 7 11

(-9°C) Freezer

92 90 87 90 94 90

(4°C) (25°C) (34°C) Refrigerator Room Brood nest

92 92 90 92 86 72

89 70 47 28 8 3

90 34 14 4 3 1

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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A G R I C U L T U R A L USES OF ANTIBIOTICS

TABLE I I I . Relative S t a b i l i t y of Oxytetracycline i n Pollen Patty Bee Diets (% Recovered)

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch004

Time (weeks)

1 2 3 7 11

(-9°C) Freezer

82 79 86 84 90

(4°C) (25°C) (34°C) Refrigerator Room Brood nest

87 83 86 90 86

82 86 85 78 82

75 75 73 55 68

TABLE IV. Relative S t a b i l i t y of Oxytetracycline i n Extender Patty Bee Diets (% Recovered) Time (weeks)

1 2 3 7 11

(-9°C) Freezer

100 96 98 97 100

(4°C) (25°C) (34°C) Refrigerator Room Brood nest

98 92 97 91 94

95 91 98 95 91

98 96 100 94 97

We next applied the method to follow the degradation of oxytetracycline i n syrups packed i n comb c e l l s . 700-1400 bees i n small cages were medicated under controlled feeding conditions (16). Data i n Table V have been "adjusted" to correct for "background fluorescence" observed i n non-medicated control colonies. The amount of oxytetracycline remaining i n the combs approaches the l i m i t s of s e n s i t i v i t y of the method 4-5 weeks after medication ends. In these experiments the highest levels i n stored syrup and honey were recorded at the end of the period during which i t was fed. Oxytetracycline then degrades at a rapid rate s i m i l a r to that for unpacked aqueous sugar syrups. In a subsequent study we repeated the experiment using twelve i s o l a t e d bee colonies maintained i n large polyethylene greenhouse enclosures and fed medicated honey and medicated syrup under controlled environmental conditions (17). The rate of loss observed was similar to data i n Table V down to the l i m i t of s e n s i t i v i t y of the fluorescence method.

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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in Beekeeping

TABLE V. Relative S t a b i l i t y of Oxytetracycline i n Packed Comb C e l l s (micrograms OTC/ml)

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch004

Weeks after start of treatment

1 2 3 4 5

Caged Bees fed medicated syrup One week feeding Two week feeding

124.0 30.3 0.0

113.5 2.5 0.2

71.7 1.4

0.0

6

0.0 Caged Bees fed medicated honey

1 2 3 4 5 6

165.0 30.3 2.7 -

177.8 9.1 2.6 1.4 0.0 0.0

182.5 5.9 1.6 0.2 0.0

Commercial beekeepers prefer preparations that are quick and easy to prepare and use under f i e l d conditions. We therefore compared (18) the residues i n both broodnest and surplus honey after medication of outdoor f r e e - f l y i n g colonies with medicated sugar dusts, and medicated syrup sprays that cause engorging of the nurse bees as described by Farrar (_8). Figure 4 compares the amounts of oxytetracycline residues for 3 colonies, averaged f o r ease of symposium presentation, with a non-medicated control colony. Fluorescence readings f o r the control colony have been converted to oxytetracycline residue values, and have not been subtracted from the values obtained for the treated colonies. To prepare the medicated sugar dusts one teaspoon of animal soluble powder which contains about 200 mg of oxytetracycline was mixed with 28g of powdered sugar per colony per treatment. The dust was applied on the ends of the frames of the brood nest between the two broodcontaining hive bodies of each of three colonies. Ten treatments were given at 4 to 5 day i n t e r v a l s . Medication ended after 6 weeks. Two ml of brood nest honey and 2 ml surplus honey were analyzed. The rate of loss of oxytetracycline i n brood nest honey i s s i m i l a r to data presented e a r l i e r i n Table II and Figure 3. Within 2-3 weeks after treatment oxytetracycline residues f e l l to levels approaching those found i n the non-medicated colony. The residues found i n surplus honey are r e l a t i v e l y much lower when compared to levels i n brood nest honey, and also decreased to background l e v e l s . Figure 5 compares results obtained for medicated sugar syrup sprays (18). Data for 3 colonies have been averaged for presentation. Medicated sprays sugar syrup contained 3.8g of animal soluble powder (200 mg oxytetracycline) i n 1.5 l i t e r s of 50% (w/v) sucrose syrup. The combs of each of 3 colonies were sprayed with

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

A G R I C U L T U R A L USES O F ANTIBIOTICS

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42

TIME

(WEEKS)

Figure 3. Degradation of oxytetracycline i n water at brood nest temperature (34°C). t V -2 days. 2

TREATED CONTROL

TREATMENT ENDED AT WEEK 8 Figure 4. Oxytetracycline i n brood nest honey from honey bee colonies treated with medicated sugar dusts.

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch004

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750 ml of the medicated sugar syrup using a hand-held garden sprayer. Ten treatments were given at 4 to 5 day i n t e r v a l s . The data show no cumulative buildup of oxytetracycline residues. After the treatment ends at 6 weeks, the residues f a l l to levels observed i n the control untreated colony. It i s clear from Figures 4 and 5 that the chemist i s at the mercy of the f r e e - f l y i n g honey bee who i s free to synthesize the nectar of the gods using whatever flower i t so chooses. The background fluorescence started r i s i n g on the fourth week into the experiment completely wiping out the s e n s i t i v i t y of the fluorescence method 3 weeks after the time the medication had ended. We suspect the increased i n t e r f e r i n g fluorescence i n this experiment was caused by a flavanoid extracted from honey made from collected nectar obtained from tamarisk or athel (Tamarix aphylla (L.) Karst) i n bloom i n Arizona near the end of July. This interference was not eliminated by the extraction methodologies that we had developed earlier. Oxytetracycline i n Honey Bee Collected Pollen for Human Consumption In recent years pollen collected i n traps by beekeepers has been made available as a health food for human consumption. Commercial pollen traps are manufactured to f i t i n s i d e , above or below the brood chamber, or at the entrance to the hive. Bottom traps presumably are never used to c o l l e c t pollen intended for human consumption, as these traps c o l l e c t dead bees and insect parts, notwithstanding the fact that medicated sugar dusts, i f used, may f a l l from the treated frames of the brood nest chamber and possibly cause contamination. We already have demonstrated the s t a b i l i t y of oxytetracycline when incorporated into supplemental bee diets that contain pollen (15). Present Label I m p l i c i t l y Applies to Harvested Pollen. For beekeepers who use oxytetracycline for medication, the present l a b e l (Table I) i s e x p l i c i t i n defining the proper use and precautions that need to be followed when honey i s to be harvested and marketed for human consumption. Presumably the label i m p l i c i t l y applies to pollen collected for human consumption as w e l l . This does, however, pose an i n t e r e s t i n g question - i f fresh pollens were collected i n pollen traps placed at the hive entrance of medicated colonies before the 4 week r e s t r i c t i o n elapsed, as stated on the use l a b e l for c o l l e c t i n g marketable honey, would the oxytetracycline be transferred by the honey bee to the pollen. To answer the question f i e l d colonies were medicated by feeding freshly prepared solutions of medicated sugar syrup for several weeks at recommended and twice recommended l e v e l s . Immediately at the end of medication, and every 3 to 4 days thereafter, pollen traps were sampled and emptied to trap samples of pollen f r e s h l y collected by the foraging bees. The data i n Figure 6 c l e a r l y show that oxytetracycline can be transferred by the bee i n the f i e l d to pollen. As the pollen i s being c o l l e c t e d , the bee cements the hundreds of pollen grains together to form a pollen p e l l e t which i s returned to the hive. The amount transferred to the pollen p e l l e t i s a function of the amount of oxytetracycline that remains i n the stored syrups i n the colony

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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TREATED CONTROL

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch004

200-J

1

2

S

4

5

8

7

8

8

10

11

12

13

14

15

TREATMENT ENDED AT MEEK 8

Figure 5 . Oxytetracycline i n brood nest honey from honey bee colonies treated with medicated sugar syrup sprays.

Figure 6. Oxytetracycline i n bee collected pollen. Bee colonies fed medicated sucrose syrups at recommended (XI) and twice recommended levels (X2).

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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and on which the foraging bees feed. These data show that pollens intended for human consumption can become contaminated with trace amounts of oxytetracycline residues i f precautions are not followed.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch004

Safe and E f f i c i e n t Use of Oxytetracycline - Present and Future In t h i s symposium paper we have attempted to provide a synopsis of some of the research that has been performed by industry and government and have emphasized some of our own published research and included new findings concerning marketable pollen, that not only supports but may help to extend the label recommendations for proper use of oxytetracycline i n bee colonies. These research e f f o r t s and the work of state apiary inspectors help combat the spread of bee diseases i n economically important bee colonies while helping to prevent contamination of marketable honey and p o l l e n . Federal Regulations. Present Federal regulations (25) l i m i t residues of tetracyclines i n edible animal tissues to tolerance l i m i t s ranging from 0.1 to 4.0 ppm (mg/kg) (26). Since tolerance levels have not been established for oxytetracycline i n marketable honey or pollen, trace amounts are not permitted. Honeys and pollens are chemically complex and highly variable i n their minor chemical composition, the minor chemicals being a function of the s p e c i f i c species of flowers the bee v i s i t s . It i s precisely this freedom to forage, and the p o s s i b l i t y of variable backgrounds that may cause a false positive reading to be recorded when trace amounts of oxytetracycline are determined at or near the low l i m i t of detection by either microbiological or fluorescence assay. Several methods based upon reversed phase HPLC have been proposed by Jurgens i n Germany (22) and by Takeba and coworkers i n Japan (23), at s e n s i t i v i t y levels between 0.1 and 1.0 ppm. Moats (24) has recently proposed the use of a polymeric reverse phase column to determine tetracyclines i n tissues and blood serum of cattle and swine by HPLC. We expect i n the near future, i n collaborative work with Moats, to explore this advance i n methodology i n order to increase further the s e n s i t i v i t y for detecting oxytetracycline i n honey and pollen with a high degree of confidence. Fumidil-B Fumidil-B® manufactured by Abbott Laboratories i s the water soluble bicyclohexylammonium s a l t of the a n t i b i o t i c fumagillin produced by the fermentation of A s p e r g i l l u s fumigatus and i s used world-wide for the prevention and control of Nosema apis, a disease i n adult honey bees. The drug attacks the a c t i v e l y multiplying disease producing protozoan parasites i n the gut of the adult bee. Katznelson and Jamieson (21) f i r s t demonstrated the effectiveness of fumagillin (the a n t i b i o t i c was dissolved i n methanol and diluted with water) i n preventing the development of nosema i n caged bees. The drug's usefulness was substantiated by others (28>22)· Fumagillin dissolved i n ethanol solution i s readily destroyed by l i g h t (30). C r y s t a l l i n e fumagillin exposed to l i g h t and a i r for one year l o s t 90% of i t s absorptivity at 351 nm (31). However when Fumidil-B i s used as the source of fumagillin

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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considerable residual nosemastatic a c t i v i t y i s retained i n stored medicated sugar syrups to permit the effective control of nosema disease (32). To protect over-wintered colonies, Fumidil-B i n medicated sugar syrup i s commonly fed i n the f a l l . Colonies established from packages are fed medicated syrup as soon as they are established. Any chance of trace residues of fumagillin appearing i n marketable honey from these treatments i s remote since medicated syrups are not fed during the honey flow or immediately before the honey flow. Fumidil-B i s inactive against most bacteria, fungi, and viruses.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch004

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McGregor, S. E. 1977. "Insect pollination of cultivated crop plants." U.S. Department of Agriculture Handbook No. 496, 411 pp.

2.

Levin, M. D. Agriculture.

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Haseman, L . 1946. Sulfa Drugs to Control American Foulbrood. J . Econ. Entomol. 39(1): 5-7.

4.

Johnson, J. P. 1947. Sulfathiazole for American foulbrood disease of honey-bees; Second Report. J . Econ. Entomol. 40: 338-43.

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Reinhardt, J. F. 1947. The sulfathiazole cure of American foulbrood; an explanatory theory. J . Econ. Entomol. 40(1): 458.

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Eckert, J. E. 1948. The use of sodium sulfathiazole in the treatment of American foulbrood disease of honey bees. J . Econ. Entomol. 41: 491-4.

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Woodrow, A. W. 1941. Susceptibility of honeybee larvae to American foulbrood. Glean. Bee Cult. 69: 148-151, 190.

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Farrar, C. L . 1956. 84: 207-11, 218.

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Landerkin, G. B. and Katznelson, H. 1957. Stability of antibiotics in honey and sugar syrup as affected by temperature. Appl. Microbiol. 5: 152-154.

1983. Value of Bee Pollination to U.S. Bull. Ent. Soc. Am. 27(4): 50-51.

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Corner, J . and Gochnauer, T. A. 1971. The persistence of tetracycline activity in medicated syrup stored by wintering honeybee colonies. J . Apic. Res. 10: 67-71.

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Gochnauer, T. A. and Bland, S. E. 1974. Persistence of oxytetracycline activity in medicated syrup stored in honeybee colonies in late spring. J . Apic Res. 13: 153-159.

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch004

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Rousseau, M. and Tabarly, O. 1962. L'emploi des antibiotiques en apiculture: leur action dans l a ruche, leur presence dans l e miel. B u l l . Apic. Doc. S c i . Tech. Inf. 5: 155-176.

13.

Kohn, K. W. 1961. Determination of tetracyclines by extraction of fluorescent complexes. Anal. Chem. 33: 862-866.

14.

Argauer, R. J . and M. G i l l i a m . 1974. A fluorometric method for determining oxytetracycline i n treated colonies of the honey bee, Apis m e l l i f e r a . J . Invertebr. Pathol. 23: 51-4.

15.

G i l l i a m , M. and Argauer, R. J . 1975. S t a b i l i t y of oxytetracycline i n diets fed to honeybee colonies for disease control. J . Invert. Path. 26: 383-386.

16.

G i l l i a m , M., Taber, S., I I I , and Argauer, R. J . 1978. Degradation of oxytetracycline i n medicated sucrose and honey stored by caged honey bees, Apis m e l l i f e r a . J . Invert. Path. 31: 128-130.

17.

Gilliam, Μ., Taber I I I , S., and Argauer, R. J. 1979. Degradation of oxytetracycline i n sugar syrup and honey stored by honeybee colonies. J . Apic. Res. 18: 208-11.

18.

G i l l i a m , M. and Argauer, R. J. 1981. Oxytetracycline Residues i n Surplus Honey, Brood Nest Honey, and Larvae After Medication of Colonies of Honey Bees, Apis m e l l i f e r a , with A n t i b i o t i c Extender Patties, sugar Dust, and syrup Sprays. Environmental Entomology 10: 479-82.

19.

Argauer, R. J . , Shimanuki, Η., and Knox, D. A. 1982. Determination of Sulfathiazole i n Honey from Medicated Honey Bee Colonies by High-Performance Liquid Chromatography on a Cyano-Amino Polar Phase. Environmental Entomology 11: 820-23.

20.

Barry, C. P., MacEachern, G. M. 1983. Reverse Phase Liquid Chromatographic Determination of Sulfathiazole Residues i n Honey. J . Assoc. Off. Anal. Chem. 66: 4-7.

21.

Juergens, U. 1981. High-pressure l i q u i d chromatographic analysis of residues of drugs i n honey. I. Tetracycline. Juergens, Uwe. Z. Lebensm.-Unters. Forsch., 173(5): 356-8.

22.

Wilson, W. T. 1974. Residues of Oxytetracycline i n Honey Stored by Apis m e l l i f e r a . Environmental Entomology 3: 674-676.

23.

Takeba, Κ., Kanzaki, Μ., Murakami, F., Matsumoto, M. 1984. Simplified a n a l y t i c a l method for tetracycline residues in honey by high performance l i q u i d chromatography. Kenkyu Nenpo Tokyo-toritsu E i s e i Kenkyusho. 35: 187-91.

24.

Moats, W. A. 198 . Determination of Tetracycline A n t i b i o t i c s i n Tissues and Blood Serum of Cattle and Swine by High Performance Liquid Chromatography. J . Chromatog. (In Press)

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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25.

Code of Federal Regulations (CFR), Title 21. 1979. Chlortetracycline 556.150, oxytetracycline 556.500, Tetracycline 556.720.

26.

Ashworth, R. B. 1985. Assay of Tetracyclines HPLC. J . Assoc. Off. Anal. Chem. 68: 1013-8.

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Katznelson, H . , Jamieson, C. A. 1952. Disease of Honey Bees with Fumagillin.

28.

Gochnauer, T. A. 1953. Chemical Control of American Foulbrood and Nosema Diseases. Amer. Bee J. 93: 410-411.

29.

Farrar, C. L . 1954. Fumagillin for Nosema Control in Package Bees. Amer. Bee J. 94: 52.

30.

Eble, Τ. Ε . , Garrett, E. R. 1954. Studies on the Stability of Fumagillin. II. J . Am. Pharm. Assoc. 43: 536-538.

31.

Garrett, E. R. 1954. Studies on the Stability of Fumagillin. III. J . Am. Pharm. Assoc. 43: 539-543.

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Furgala, Β . , Gochnauer, T. A. 1969. Disease. Amer. Bee J. 109: 218-219.

in Tissues by

Control of Nosema Science 115: 70-71.

Chemotherapy of Nosema

RECEIVED March 25, 1986

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

5

A n t i b i o t i c s as

Crop

Protectants

1

Arun K. Misra

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch005

Department of Biological Sciences, Morris Brown College, Atlanta University, Atlanta, GA 30314 Antibiotics, the miracle drugs, have a long history of being useful in agriculture. There has been increased interest in recent years in the use of antibiotics for the control of plant diseases. Some antibiotics that are toxic for use in the treatment of human or animal diseases may be used on plants. Antibiotics have been found useful for the control of bacterial, fungal, viral, and mycoplasmal diseases of a variety of crops and ornamental plants. Drug laws of various countries differ regarding use of antibiotics as plant protectants. Concern regarding uncontrolled use of antibiotics is appropriate, but more information is needed about the effectiveness and safety of the use of antibiotics for control of plant diseases. A n t i b i o t i c s are chemicals a n t a g o n i s t i c to l i f e . These are g e n e r a l l y produced by microorganisms and may be very e f f e c t i v e against m i c r o b i a l pathogens(1). Using a n t i b i o t i c s other than i n c o n t r o l l i n g diseases of humans has been c a l l e d non-medical or "non-pharmaceutical". The use of a n t i b i o t i c s i n food and a g r i c u l t u r e i s multifaceted(2-_5) aspect of t h e i r use with plants and animals, covered i n several i n t e r n a t i o n a l conferences, proceedings of which have been published(()-9 ) . The subject of t h i s review w i l l be the use of a n t i b i o t i c s as plant p r o t e c t a n t s . ff

M

1On leave of absence from Botany Department, L. N. Mithila University, C. M. Science College, Darbahnga, Bihar, 846004, India. 0097-6156/ 86/ 0320-0049506.00/ 0 © 1986 American Chemical Society

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Crop P r o t e c t i o n The use of a n t i b i o t i c s i n plant pathology, e s p e c i a l l y f o r the c o n t r o l of plant diseases i s a subiect of i n c r e a s i n g i n t e r e s t ( 1 0 ) . Nearly four decades ago, i n the f o r t i e s , use of a n t i b i o t i c s f o r plant p r o t e c t i o n was l i t t l e recognized. A n t i b i o t i c s were used against plant diseases only when they were found u n s u i t a b l e i n human medicine(11). Later, i t was found that many plant diseases p a r t i c u l a r l y those caused by fungi and b a c t e r i a were e f f e c t i v e l y c o n t r o l l e d using a n t i b i o t i c s (12). However, r e c e n t l y doubts have been expressed concerning the growing use of a n t i b i o t i c s ( 1 3 ) , e s p e c i a l l y because of p o s s i b l e residues i n vegetable products. There i s , however, l i t t l e evidence as to the d e l e t e r i o u s e f f e c t s of spraying a n t i b i o t i c s on crop p l a n t s . G l i o t o x i n , i s o l a t e d by Winding i n 1932 from Gliocladium fimbriaturn was found a n a t a g o n i s t i c to Rhizoctonia s o l a n i , thus h e l p f u l against the r o o t - r o t of potato and tomato. Brown and Boyle i n 1954(14-15) noted that p e n i c i l l i n was a c t i v e against the crown-gaTT bacterium. Zaumeyer (16)found that spraying with streptomycin was e f f e c t i v e against h a l o b l i g h t of beans caused by Pseudomonas p h a s e o l i c o l e . Zalaback(17) and Ark(18) used streptomycin to c o n t r o l bean b l i g h t , caused bv Erwinia amylovora, under f i e l d c o n d i t i o n s . Aureofungin was developed as a plant protectant against the fungal diseases of r i c e i n India(,19). S i m i l a r l y , b l a s t i c i d i n has been used i n r i c e c u l t i v a t i o n i n Japan f o r very long time(20). THe a n t i b i o t i c s used i n plant p r o t e c t i o n have been more s u c c e s s f u l i n c o n t r o l l i n g fungi than other types of plant pathogens. Aureofungin, cycloheximide, g r i s e o f u l v i n , ohyamycin and a host of others(see Table 1) have been used. Extensive reviews i n t h i s field(21.-26) are a v a i l a b l e . Aureofungin i s a heptaene a n t i b i o t i c anïï~~is extracted from Streptomyces cinnamoseous var. t e r r i c o l a . I t belongs to a new a n t i b i o t i c group among the heptaenes(27). I t i s a broad spectrum f u n g i c i d e , e f f e c t i v e against a wicfe v a r i e t y of f u n g i , and i s systemic i n a c t i v i t y . A golden yellow powder, i t i s unstable i n the presence of moisture and l i g h t , and needs to be stored dry and i n darkness. A host of fungal diseases have been c o n t r o l l e d by aureofungin(28-29). C i t r u s gummosis caused by Phytophthora c i t r o p h t h o r a may be cured by 20 mg/ml spray. Control of the D i p l o d i a r o t of mangoes and the A l t e r n a r i a r o t of tomatoes, by t h i s a n t i b i o t i c , e s p e c i a l l y during t r a n s i t and storage of f r u i t s i s noteworthy. Dipping mango f r u i t s i n 100-500 ppm of aurefungin prevents r o t t i n g f o r 11-20 days. Untreated f r u i t s r o t i n 2-3 days. In developing countries where r e f r i g e r a t i o n i s not common, t h i s method i s u s e f u l i n control l i n g post-harvest l o s s . The b l a s t disease of r i c e caused by P i r i c u l a r i a oryzae has been shown to be c o n t r o l l e d by a 7.5 g/hectare spray of aureofungin, four times at 12 days interval(30).Powdery mildew of apples caused by Podosphaera leucotricïïa can be e f f e c t i v e l y handled with aureofungin. Seed-borne i n f e c t i o n

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Table I : Some Common A n t i b i o t i c s used f o r Plant P r o t e c t i o n Antibacterial : A n t i b i o t i c C6, C e l l o c i d i n , Chloramphenicol, C i t r i n i n , Erythromycin, Gramicidin, Kanamycin, Novobiocin, P e n i c i l l i n , P h t o b a c t e r i omycin,Polymycin, Polymyxin, Rhizopin, Streptomycin, Agrimycin, Phytostrep, T e t r a c y c l i n e , and Vancomycin.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch005

Antifungal : A n t i b i o t i c P3, A n t i b i o t i c P9, Antimycin, Antimvcin, Aureofungin, B l a s t i c i d i n , Bulboformin, C a n d i c i d i n , C R R I - a n t i b i o t i c , Cycloheximide, Foliomvcin, N y s t a t i n , Oligomvcin, G r i s e o f u l v i n , Phytoactin, Polyoxin, T e t r i n , T r i c h o t h e c i n , B e n t u r i c i d i n , and Venturomycin. Antimycoplasmal

:

T e t r a c y c l i n e , Erythromycin, and Methacycline. Antiviral : Actinomycin D, A n t i b i o t i c 205-2B, B l a s t i c i d i n , Cycloheximide(actidione), Daunomycin DPB, Mithramycin, Mitomycin C, Pentaene G8, and T u b e r c i d i n .

by Helminthosporium oryzae(30) may be s i g n i f i c a n t l y reduce -d by overnight soaking of paddy seeds i n aureofungin s o l ution. B l a s t i c i d i n s are produced by Streptomyces grieseochro -mogens and i n h i b i t s e v e r a l species of b a c t e r i a and fungi (31). Pseudomonas i s p a r t i c u l a r l y vulnerable to b l a s t i c i d i n S. P i r i c u l a r i a oryzae causing the b l a s t disease of r i c e i s widely c o n t r o l l e d with b l a s t i c i d i n S i n Japan. I t i s appl i e d to the r i c e p l a n t s a f t e r i n f e c t i o n by the fungus has already ocurred(32), since the a n t i b i o t i c a f f e c t s the myce - l i a i phase more than the spore phase. I t would be d e s i r a ble to search f o r spore k i l l i n g a n t i b i o t i c s to c o n t r o l s o i l - i n h a b i t i n g microbes and to destroy the inoculum before i t i n f e c t s the crop. Numerous cases of the use of a n t i b i o t i c s ( e s p e c i a l l y : cycloheximide, ohyamycin, streptomycin, t e t r a c y c l i n e s , p e n i c i l l i n , g r i s e o f u l v i n , and polymyxin) against s e v e r a l b a c t e r i a l and fungal diseases are now known(33-35). In the United States of America, Merck s e l l s preparations of streptomycin and Upjohn s e l l s that of cycloheximide f o r the c o n t r o l of the diseases of ornamental plants(R.Burg,

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personal communication). Interest i n the use of aureofungin i s s t i l l continuing i n some l a b o r a t o r i e s (36^) . A c t i d i o n e has r e c e n t l y been reported to be e f f e c t i v e against s e v e r a l s o i l f u n g i ( 3 7 ) . I t has a l s o been noted that some a n t i b i o t i -cs may supress V e r t i c i l l i u m d a h l i a e and protect peppers against i n f e c t i o n and a l s o stimulate seed germination and growth of the plants(38-39). Bacillomycin(40) has been found e f f e c t i v e against Helminthosporium turcium(41), which i n f e c t s s e v e r a l c e r e a l crops. Antimycin A i s e f f e c t i v e against A l t e r n a r i a s o l a n i spores(42), karumin i s e f f e c t i v e against Rhizoctonia s o l a n i ( 4 3 ) . Nikkomycin i s being used to cure trees of Dutch elm disease i n Hamden, Connecticut(44). I t i n h i b i t s the formation of c h i t i n and stops the myceTia of Ceratocys t i s ulmi from growing normally. There i s a long l i s t of a n t i b i o t i c s that have been t r i e d against the b a c t e r i a l pathogens of plants(45-47). Pseudomonas, Xanthomonas, Agrobacterium, Aplanobacterium and other genera have been found to be i n a c t i v a t e d by streptomycin, t e t r a c y c l i n e , o x y t e t r a c y c l i n e , p e n i c i l l i n , agromvcin, and vancomycin; e s p e c i a l l y i n the crops of cherry, maize, bean, poplar, cotton, r i c e , c i t r u s , apple, plums and s e v e r a l plants of h o r t i c u l t u r a l importance such as geraniums(48-51) Tomato canker caused by Xanthomonas can be c o n t r o l l e d by the a p p l i c a t i o n of t e t r a c y c l i n e ( 5 2 - 5 3 ) . Streptomycin r e s i s t a n t s t r a i n s of b a c t e r i a have Been found on peach, tomato and peppers(54), and the mixture of two a n t i b i o t i c s has helped to stop tïïe build-up of r e s i s t a n c e i n the patho -gens i n some cases(55-56). The s i l v e r y disease of sugarbeet caused by CorynëTSacterium i s i n s u f f i c i e n t l y c o n t r o l l e d by m e r c u r i a l compounds, but i s completely e l i m i n a t e d when seeds are dipped f o r s e v e r a l hours i n a s o l u t i o n of s t r e p tomycin. Most of the work with a n t i b a c t e r i a l a n t i b i o t i c s seem confined t o Europe, India, Japan and New Zealand. Treatment of tomato seeds with a n t i b i o t i c s c o n t r o l s b a c t e r i a l pathogens(57). Xanthobacidin has been s a i d to be a c t i v e against Xanthomonas and other s p e c i e s . Seed-borne b a c t e r i a l tumors i n tobacco may a l s o be t r e a t e d with chloramphenicol and t e t r a c y c l i n e ( 5 8 ) . Bacterium f o r crown-gall(with T i plasmids) can be i n h i b i t e d by spraying t e t r a c y c l i n e s on plants that are i n f e c t e d with Agrobacterium. Screening of the e f f i c a c y of a n t i b i o t i c s against b a c t e r i a l plant pathogens i s continuing(59-60). The involvement of plasmids i n c o n t r o l l i n g r e s i s t a n c e of plant pathogens to a n t i b i o t i c s has now been w e l l studied(58). Caution i n using a n t i b i o t i c s against b a c t e r i a l plant patïïogens i s very important, to avoid r e s i s t a n c e build-up i n the environment. The use of a n t i b i o t i c s f o r the c o n t r o l of plant v i r u s diseases (6^1 ) i s of i n t e r e s t . Several a n t i b i o t i c s have been t e s t e d f o r i n h i b i t i o n of r e p l i c a t i o n of v i r a l n u c l e i c a c i d and/or p r o t e i n synthesis w i t h i n the host c e l l . Chloramphen i c o l , cycloheximide, actinomycin D and others are the most used a n t i b i o t i c s ; and the disease caused by tobacco mosaic

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virus(TMV) the most t r e a t e d . In most of the cases(62)the work i s s t i l l at the t h e o r e t i c a l or experimental l e v e l and the p r a c t i c a l use of a n t i b i o t i c s f o r c o n t r o l of v i r a l d i s eases has not been e s t a b l i s h e d . P r i n c i p l e s i n s e l e c t i n g a p a r t i c u l a r a n t i b i o t i c have not been d e f i n e d . Mostly a broad spectrum, r e l a t i v e l y inexpensive, non-phytotoxic one should be s e l e c t e d . Those with growth s t i m u l a t i n g a c t i v i t y f o r the host plant are p r e f e r r e d ( ^ 3 ) . Much work i s needed i n t h i s area, but mention may be made that a n t i b i o t i c s are known that cure the l e a f - c u r l disease of tomato, and a l s o r e s u l t i n l a r g e r s i z e and number of tomato f r u i t s ( 6 0 ) . C e r t a i n v i r a l diseases of tobacco, potato, cucumber, tomato and other crops have been t r e a t e d with a n t i b i o t i c s , but s t i l l many of the plant pathogens(especially v i r u s e s ) causing widespread and severe damage have not been c o n t r o l l e d successfully. Viruses such as bromegrass mosaic, broadbean mottle, c h i l i mosaic, cowpea yellow mosaic, cucumber mosaic, pea streak, potato v i r u s X, soybean pod mottle, tobacco mosaic, tobacco n e c r o s i s , tobacco tumor, tomato l e a f - c u r l and tomato spotted w i l t have been t r e a t e d with a n t i b i o t i c s such as actinomycin D, b l a s t i c i d i n S, a c t i d i o n e ( c y c l o h e x i m i d e ) , miharamvcin A, ohvamycin, polyoxin A, pentaene G8, chloramphenicol, c i t r i n i n , daunomycin, dextromycin, formvcin, kan^ amycin, mitomycin C, mithramvcin, r i b a v i r i n and t u b e r c i d i n (ICI, 64). An a n t i b i o t i c that i s r e l a t e d to r i b a v i r i n (known i n animal v i r o l o g y ) , c a l l e d t a i z o f u r i n has r e c e n t l y been t r i e d i n Sao Paulo, B r a z i l , against tomato spotted w i l t virus(TSWV) and i s s a i d to be an e f f i c i e n t a n t i - v i r a l drug (65). S i m i l a r l y i n India, DPB(code name f o r an a n t i b i o t i c ) i s u s e f u l i n c o n t r o l l i n g the tomato l e a f - c u r l v i r u s , and a l s o increases the s i z e of the tomato f r u i t s ( 6 6 ) . C y t o v i r i n i s a wide spectrum a n t i b i o t i c against plant v i r u s e s , and has proved e f f e c t i v e against the v i r u s diseases of the crops l i k e r i c e , c i t r u s and sugarcane(Table 2). Mycoplasma-like organisms(MLOs) and r i c k e t t s i a - l i k e organisms(RLOs) are i n a c t i v a t e d more e a s i l y than v i r u s e s by a n t i b i o t i c s , since they have membranes l i k e b a c t e r i a and are a f f e c t e d by a n t i b i o t i c s more d i r e c t l y during membrane biogenesis(62). T e t r a c y c l i n e treatment has been very e f f e c t i v e against s e v e r a l MLO-diseases, e s p e c i a l l y i n egg p l a n t , sandal, mulberry dwarf, sugarcane stunt, and grassy shoot. Grapevine n e c r o s i s , hop c r i n k l e and beet yellows are s a i d to be caused by RLOs. C i t r u s greening and other s i m i l a r diseases may be c o n t r o l l e d with t e t r a c y c l i n e , p e n i c i l l i n and aureofungin(68). It i s o f t e n s a i d that when a suspected v i r u s disease may be c o n t r o l l e d by a n t i b i o t i c s ( T a b l e 3), the cause of the diseases must be mycoplasma and never a v i r u s . This a r b i t r ary statement segregating v i r u s e s from mycoplasma has many times been h e l d v a l i d , but there remain s e v e r a l other i n s tances where a p p l i c a t i o n of a n t i b i o t i c s to the host plant has reduced the pathogenesis of v i r u s e s to a considerable degree(69-70).

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Table II :

A n t i b i o t i c s Used against Plant Viruses

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch005

Viruses

Antibiotics

Bromegrass mosaic

Actinomycin, B l a s t i c i d i n

Broadbean mosaic

Actidione/Cycloheximide

C h i l i mosaic

DPB (chemical name unknown)

Cowpea yellow mosaic

Actinomycin

Cucumber mosaic

Blasticidin

Egg plant mosaic

Actidione

Pea streak

Actidione

Potato v i r u s X

Actinomycin, B l a s t i c i d i n , Miharamycin, Ohvamycin, Polyoxin A

Soybean pod mottle

Actinomycin

Sunhemp mosaic

Pentaene G

Tobacco mosaic

Actidione,Actinomycin D, B l a s t i c i d i n , Chloramphenicol, C i t r i n i n , Daunomycin, Dextromycin, F e r r i m y c i d i n , Formycin, Imanin, Kanamycin, L a u r i s i n , Miharamycin, Mitomycin C, Naramycin, Ohyamycin, Pentaene G8, Polyoxin A, Puromycin, Streptomycin.

Tobacco n e c r o s i s

A c t i d i o n e , Chloramphenicol

Tobacco tumor

Chiorampheni c o l , Daunomyc i n , Mithramycin, T u b e r c i d i n

Tomato l e a f - c u r l

DPB (chemical name unknown)

Tomato spotted w i l t

Taizofurin

A n t i b i o t i c s from higher forms of L i f e I s o l a t i o n and c h a r a c t e r i z a t i o n of a n t i b i o t i c s from microorganisms has been attempted f o r several decades. There has r e c e n t l y been i n c r e a s i n g i n t e r e s t i n e x t r a c t i n g a n t i m i c r o -

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Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch005

Table I I I : Therapeutic drugs against Mycoplasmal Diseases of Plants Disease

Drug

Application

Host-Plant

Dwarf

Tetracycline, c h l o r - , oxy-, dimethyl-,and other d e r i v a tives

root immersion f o l i a r spray girdling

mulberry carrot tomato potato rice

Yellows

Methacycline, dipping, hydroChlorampheni- ponics, spray, c o l , Tetracyc- i n f i l t r a t i o n line

Stunt

Tetracycline

root immersion

corn

Phyllody

Doxycline

f o l i a g e dip

aster

Little-leaf

Tetracycline

foliar

legumes tomato

Greening

Tetracycline

sprays

citrus

Decline

Oxytetracyc-line

transfusion

pear trees

Spike

Tetracycline

girdling

sandal

Yellowing

Tetracycline

trunk i n j e c t i o n

coconut

spray

aster chrysanthemum celery tobacco

b i a l compounds from higher plants(71-73). Lichens, algae, angiosperms and various types of marine organisms are being used as sources of a n t i m i c r o b i a l compounds(74). The emphasi s i s obviously on o b t a i n i n g a n t i b i o t i c s usëTul i n human medicine but searches may a l s o be c a r r i e d out f o r chemical compounds e f f e c t i v e against plant diseases. There are r e p o r t s that plant v i r u s i n h i b i t o r s occur n a t u r a l l y i n p l a n t s , and thev could be p r o t e i n s , glycoprote - i n s , polysaccharides, phenols etc(7_5). E x t r a c t s of mosses, e s p e c i a l l y Sphagnum(76), algae(77_) and Cassia of the family Leguminosae(78) are e f f e c t i v e i n i n h i b i t i n g tobacco mosaic virus(TMV), But much more work i s needed to develop v i r i c i des that may be sprayed s a f e l y and economically on crop plants i n the f i e l d . Often medicinal plants known from f o l k - l o r e are picked up and t h e i r e x t r a c t s t e s t e d against known plant v i r u s e s by mixing them with the inoculum and doing h a l f - l e a f experiments. Each h a l f of the l e a f i s rubbed with v i r u s suspension,

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one h a l f r e c e i v e s untreated v i r u s , while on the other h a l f v i r u s i s mixed with plant e x t r a c t . Numerous plant e x t r a c t s are being screened and u s e f u l compounds have been i s o l a t e d . However, v a l i d a p p l i c a t i o n i n plant p r o t e c t i o n has not been e s t a b l i s h e d , although several drugs f o r use i n human medicine have already been developed(78). Some angiosperms l i k e Acalypha i n d i c a has been shown toHSe a c t i v e against plant pathogenic fungi l i k e A l t e r n a r i a ( 7 9 ) . The biomedical potent i a l of the sea has a l s o r e c e n t l y a t t r a c t e d considerable attention(80-81) and searches f o r plant p r o t e c t i n g a n t i b i o t i c s are underway i n the marine environment.

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Legislation Almost every country has c e n t r a l i z e d drug r e g u l a t i o n s with regard to p e s t i c i d e s and drugs, i n c l u d i n g a n t i b i o t i c s , i n plants and animals. Discussions about the unwarranted use of a n t i b i o t i c s i n human medicine and plant p r o t e c t i o n stem many times from discrepancies i n the drug laws of d i f f e r e nt nations, a thorough account of which has been presented i n several works(TO). A very s i m p l i e f i e d account of the present s i t u a t i o n regarding the use of a n t i b i o t i c s i n plant p r o t e c t i o n reveals that there are extremes of 'no' ( l i k e i n USA and Western Germany) through l i b e r a l y e s ( a s i n Japan and I n d i a ) . Most other countries have adopted a middle path, where a n t i b i o t i c s are allowed i n animal feed, but not i n plant p r o t e c t i o n , or v i c e versa. Reasons f o r t h i s are probably n o n - s c i e n t i f i c . Much of the fear of the unwanted use of a n t i b i o t i c s may be removed a f t e r we have f u r t h e r information i n the f i e l d . The USSR and West Germany r e g u l a r l y p u b l i s h l i s t s of chemicals that are allowed to be used as p e s t i c i d e s and f o r spraying on plants(8283), but a n t i b i o t i c s do not appear i n them. The a g r i c u l t u r a l uses of t e t r a c y c l i n e s have r e c e n t l y been discussed. In the UK i t has now been r e a l i z e d that a f r e s h look i s needed at the problem of using a n t i b i o t i c s i n a g r i c u l t u r e (84). It i s thus necessary to t e s t more and v a r i e d a n t i b i o t i c s against plant pathogens, under c o n t r o l l e d experime n t a l c o n d i t i o n s , before reaching a f i n a l opinion i n the matter. f

f

Growth Promotion In a d d i t i o n to being used as cure against plant diseases, a n t i b i o t i c s may a l s o be used as agents to stop preharvest f r u i t drop, or as a b s c i s s i o n agents to c o l l e c t f r u i t s i n c i t r u s crops(85). Some a n t i b i o t i c s help enhance growth of the crop plants i n a d d i t i o n to c o n t r o l l i n g t h e i r diseases (4). Hence, an i d e a l a n t i b i o t i c that may act against pathogens, increase crop p r o d u c t i v i t y and o f f e r other d e s i r a ble properties without l e a v i n g longterm residues should e a s i l y f i n d approval with drug l e g i s l a t i n g agencies(86).

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Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch005

Conclusion A n t i b i o t i c s are commonly used as drugs f o r humans and a n i mals p a r t i c u l a r l y against b a c t e r i a l i n f e c t i o n s . There has been i n c r e a s i n g i n t e r e s t i n the use of a n t i b i o t i c s f o r the c o n t r o l of plant diseases since these compounds may o f f e r more e f f e c t i v e and/or safer a l t e r n a t i v e s to chemicals p r e s e n t l y used to c o n t r o l plant diseases. The debate over the p o t e n t i a l biomedical consequences of a n t i b i o t i c s and the need to impose some kind of r e s t r a i n t on t h e i r usage i n a g r i c u l t u r e has become very intense l a t e l y . Caution i s appropriate but the present concerns may be unfounded and excessive. The use of a n t i b i o t i c s has been i n c r e a s i n g i n a g r i c u l t u r e , because of t h e i r obvious b e n e f i t s . Their use seems to pose no obvious harm to the environment. I t i s thus b e t t e r to r e f r a i n from making i l l - f o u n d e d arguments, and to put more e f f o r t i n determining what sort of a n t i b i o - t i c s can be s a f e l y and e f f e c t i v e l y used i n crop p r o t e c t i on. Acknowledgment s Dr. Luther S. Williams and Dr. Joe Johnson provided necess -ary encouragements and f a c i l i t i e s to acomplish the task. Dr. Nipen K. Bose helped with pertinent l i t e r a t u r e on the subject. Mr. S i e b r i n Simon helped with improving the l a n guage of the manuscript. Literature

Cited

1. Lancini,G.; Parenti,F. " A n t i b i o t i c s , An Integrated View "; Springer-Verlag : New York, 1982; p. 247. 2. Misato,T.; Yoneyama, K. In "Advances i n A g r i c u l t u r a l Mi -crobiology"; Rao,N.S.S., Ed.; Butterworths : London, 1982; p. 465. 3. Misato,T.; Ko, K.; Yamaguchi,I. Adv. Applied M i c r o b i o l . 1977, 21, 53-88. 4. Thirumalachar, M.J. In "Seed Pathology, problems and progress"; Y r i n o r i , J.T.; S i n c l a i r , J . B . ; Mehta, Y.R.; Mohan, S.K., Ed.; IAPAR, Panama, B r a z i l , 1979; p. 274. 5. Navashin, S.M.; Sazykin, Yu. O. Biologischeskaya 1978 1, 19-43. 6. Anonymous; "Proc. F i r s t Int. Congr. on the Use of A n t i b i o t i c s i n A g r i c u l t u r e " ; N a t l . Acad. Sciences USA, Washington, D.C., 1956; #397. 7. Woodbine, M., Ed.; " A n t i b i o t i c s i n A g r i c u l t u r e " ; Butterworths : London, 1962; p. 345. 8. Woodbine,M., Ed.; " A n t i b i o t i c s and A n t i b i o s i s i n A g r i c u l t u r e , with s p e c i a l reference to Synergism"; Butterworths : London, 1977; p. 386. 9. Woodbine,M. Ed.; " A n t i b i o t i c s i n A g r i c u l t u r e , Benefits and M a l e f i t s " ; Butterworths : London, 1982, p. 367. 10. Misra,A. " A g r i c u l t u r a l A n t i b i o t i c s " ; Associated P u b l i s hing Co. : New D e l h i , 1980; p. 174.

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11. Thirumalachar, M. J . Adv. Applied M i c r o b i o l . 1968,10,313 -337. 12. Zaumeyer, M.J. Annu Rev. M i c r o b i o l . 1956, 12, 415-440. 13. Wen-Chieh, R.C. In "New Trends i n A n t i b i o t i c s , Research and Therapy"; G r a s s i , G.G.; Sabath,L.D., Ed.; E l s e v i e r : Amsterdam, 1981; p. 223. 14.Woodcock, D. Chem. B r i t . 1971, 7, 414-423. 15.Brown,J.C.; Boyte,A.M. Science 1954, 100, 528. 16.Zaumeyer, M.J. In "Medical Encyclopedia"; Anonymous,Ed. ; L i t e r a r y Books : New York, 1957; p. 251. 17. Zalaback, V. P r e s l i a 1966, 38, 112. 18.Ark, P.A. Plant Dis. Reptr., 1958, 42, 1937-1938. 19.Thirumalachar, M.J.; Whitehead, M.D. B u l l . Georgia Acad. S c i . 1971, 29, 253. 20.Heitefuss, R. "Pflanzenschutz"; Georg-Thieme : Stuttga­ 1975; p. 119. 21.Shigaeva,M.; Tulemisova, K.A. R e f e r a t i v n y i Zhurnal 1977,27, 172. 22.Kurylowicz, W.; Kowszyk-Gindifer, Z. Hindustan A n t i b i o t . B u l l . 1979, 21, 115-124. 23.Dekker,J. Annu. Rev. M i c r o b i o l . 1964, 6, 91-117. 24.Babika, J . P r e s l i a 1966, 38, 112-116. 25.Goldberg,H.S7 Adv. Applied M i c r o b i o l . 1964,6, 91-117. 26.Goodman,R.N. "Use of a n t i b i o t i c s i n plant pathology and p r o t e c t i o n " ; Amer. Soc. M i c r o b i o l . : Washington, D.C., 1967; p. 747. 27.Rahalkar, P.W.; Neergaard,P. Hindustan A n t i b i o t . B u l l . 1969, 11, 163-165. 28.Nene, Y.; T h a p a l i y a l , P.W. "Fungicides in Plant Disea­ ses"; Oxford & IBH : New D e l h i , 1979; p. 321. 29.Agrawal, R.K.; Thirumalachar, M.J. In "Plant Disease Problems"; Raychaudhuri, S.P., Ed.; IPS : New D e l h i , 1970; p. 449. 30.Thirumalachar, M.J. Indian Phytopath. 1967, 20,270-279. 31.Yonehara, H. In "Biotechnology of I n d u s t r i a l A n t i b i o t i ­ cs"; Vandamme, E.J., Ed.; Marcel Dekker : New York, 1984; p. 832. 32.Misato,T. Japan Pest. I n f . 1969, 15, 18. 33.Jha, V. " A n t i b i o t i c s f o r Plant Disease Control"; M i t h i ­ l a Univ. : Darbhanga, India, 1978; p. 99. 34.Vandamme, E.J. "Biotechnology of I n d u s t r i a l Antibiotic­ s"; Marcel Dekker : New York, 1984; p. 13. 35.Blakeman, J.P.; S i t e j n i b e r g , A. Trans. B r i t . Mycol. Soc. 1974; p. 62, 537. 36.Singh,K.P.; Chauhan, V.B.; Edward, J.C. Hindustan A n t i ­ b i o t i c B u l l . 1976, 18, 96-98. 37.Dwivedi,R.; Dwivedi, R.S. Proc. Ind. N a t l . Acad. S c i . 1976; 42, 205. 38.Seredinskaya, A. F.; Sabelmikova, V.I.; Danilova,A.T.; Brun, G.Α.; Osipova, R.A. R e f e r a t i v n y i Zhurnal, 1979, 6, 35-38. 39.Utkhade, R.S.; Gaunce, A.P. Canad. J . Botany 1983, 61, 3343-3348. 40.Esterhnizen, B.; Merwe, K.J. Mycologia 1977, 69, 975979.

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Antibiotics

as Crop

Protectants

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41.Subramaniyan, V. " A n t i b i o t i c s , a symposium"; CSIR : New D e l h i , 1958; p. 292. 42.Waggoner, P.E.; Parlange, J.Y. Phytopath. 1977, 67,1007 -1011. 43.Kanniayan, S.; Prasad, N.N. Madras A g r i c u l t u r a l J . 1982, 69,488. 44.Lowe,D.A.; Elander, R.P. Mycologia 1983, 75, 361-373. 45.Misra,A.; Jha,V.; Jha, S.; Sharma, B.P. In "Proc. Vth I n t e r n a t i o n a l Congress on Plant Pathogenic B a c t e r i a " ; Lozano, J.C., Ed.; CIAT : C a l i , Colombia, 1982; p. 210 - 212. 46.Pyke,N.B. ; Milne, K.S.; Neilson, H.F. N.Z. J . Exp. Agr­ ic. 1984, 12, 161-164. 4 7 . E l l i s , J.G.; Kerr, Α.; Montagu,M.van P h y s i o l . P l . Path­ ol. 1979, 15, 311-319. 48.Anonymous B u l l . Kyowa Fermentation Industry, Japan 1983 14, 493. 49.Fredriq, P. Protoplasmologia 1958, 4, 1-14. 50.Glasby, J.S. "Encyclopedia of A n t i b i o t i c s " ; John Wiley : Sussex, 1979; p. 484. 51.Knosel, D.Z. PflKrankh. P f l S c h u t z . 1965, 72, 577-584. 52.Hardy, K. "Bacterial Plasmids"; ASM : Washington, D.C., 1981; p. 104. 53.Kruger,W. S. A f r . J . A g r i c . S c i . 1960, 3, 409-418. 54.Vournakis, J.N.; Elander, R.P. Science 1983, 219, 703 -709. 55.Hotta, K.; Okami, Y.; Umezawa, H. J . A n t i b i o t . 1977, 30, 1146. 56.Demain,A.L. Science 1983, 219, 709-714. 57.Trinci,A. B u l l . B r i t . Mycol. Soc. 1977, 11, 136-144. 58.Depicker, Α.; Montagu, M. van; S c h e l l , J . In "Genetic Engineering of P l a n t s " Kosuge, T.; Meredith, C.P.; H o l l ­ aender, A. Ed.; Plenum Press : New York, 1983; p. 143. 59.Das, C.R.; P a l , A. Indian Phytopath. 1974, 27, 33-36. 60.Misra,A. Z. PflKrankh. P f l S c h u t z . 1977, 84, 244-252. 61.Misra,A.; Nienhaus,F. Phytopath Z. 1977, 89, 76-81. 62.Amelia, J.C.M.; Alexandre, V.; Vincente, M. A n t i v i r a l Res. 1984, 4, 325-327. 63.Russell, A.D.; "Quensel A n t i b i o t i c s : Assessment of An­ tibiotic A c t i v i t y " ; Academic Press : New York, 1983; p . 2. 64.Kluge,S.; Marcinka, K. Acta Virol. 1979, 23, 148-152. 65.De Fazio, G.; Caner, J . ; Vincente, M. Arch. Virol. 1978 58, 153-156. 66.Raychaudhuri, S.P. "Plant Disease Problems"; Indian Phytopathological S o c i e t y : New D e l h i , 1970; p. 489. 67.Raychaudhuri,S.P. ; N a r i a n i , T.K. "Virus and Mycoplasma Diseases of Plants in India"; Oxford & IBH : New D e l h i , 1979; p. 49. 68.Raychaudhuri, S.P. Eur. J . Forest Pathol. 1977, 7,1-5. 69.Martinez, A.L. P h i l l i p i n e Phytopath. 1975, 11, 58-61. 70.Liao,C.H.; Chen, T.A. Phytopath. 1981, 71, 442-445. 71.Mandava,B. "CRC Handbook of Natural P e s t i c i d e s " ; CRC Press : Boca Raton, 1985; p . 552.

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A G R I C U L T U R A L USES O F ANTIBIOTICS

72.Berdy,J.; Aszalos, Α.; Bostian, M.; McNitt, K. "CRC Handbook of A n t i b i o t i c Compounds"; CRC Press : Boca Ra­ ton, 1982; p. 448. 73.Lewis, W.H.; Elvin-Lewis, M.P.F. "Medical Botany"; John Wiley : New York, 1977; p . 37. 74. Misra, Α.; Sinha, R. In "Islamic Medicine"; Al-Awadi, A.R.A., Ed. M i n i s t r y of P u b l i c Health : Kuwait, 1981; p. 390. 75. Misra,A. Z. PflKrankh. P f l S c h u t z . 1977,84, 334-341. 76. Misra,A.; Sinha, R. In "Marine Algae i n Pharmaceutical Sciences"; Hoppe, H. A. Ed.; Walter de Gruyter : New York, 1979; p. 237. 77. Misra, Α.; Sinha, R.; Rani, P.; Sinha, M. Med. Fac. Landbouww., 1978 43, 1043. 78.Satyavati, G.V.; Raina, Μ.Κ.; Sharma, M. "Medicinal Plants of India"; ICMR : New D e l h i , 1976; p.196. 79. Bhowmick, B.N.; Choudhary, B.K. Ind. Bot. Reptr. 1982, 1, 164-165. 80. Kaul, P.N.; Sinderman, C.J. "Drugs and Food from the Sea"; Univ. Oklahoma Press : Norman, 1978; p. 123. 81. C o l w e l l , R.R. Science 1983, 222, 19-24. 82. Anonymous "Plant P r o t e c t i o n Regulation i n Germany"; B i o l o g i s c h e Bundesanstalt : Braunschweig, 1982; p.177. 83. Anonymous "Chemical Agents f o r Plant P r o t e c t i o n Regis­ tered i n the USSR", 1978; R e f e r a t i v e v Zhurnal : Moscow; p. 8. 84. Gustafson, R.; K i e s e r , P. In "The T e t r a c y c l i n e s " ; Hlavka, J . ; Boothe, H. Ed.; Springer-Verlag : New York, 1985; p. 405. 85. Iyenger, M.R.S. Indian Phytopath. 1979, 32, 343-351. 86. V e t t o r a z z i , G. " I n t e r n a t i o n a l Regulatory Aspects of P e s t i c i d e Chemicals"; CRC Press : Boca Raton, 1982; p. 256.

RECEIVED May 16, 1986

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

6

T r e n d s in the U s e o f F e r m e n t a t i o n P r o d u c t s in A g r i c u l t u r e R. W. Burg

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch006

R50G-121, Merck Institute for Therapeutic Research, Rahway, NJ 07065 By far the largest agricultural market for antibiotics is for feed additives. The bulk of this market is taken by antibiotics that are also used in human medicine. However, mounting concern over the hazards of increased resistance to antibiotics has encouraged the search for new types of antibiotics for this use. Some of these newer products are already taking an increasing share of the market. The discovery of the anticoccidial activity of monensin opened an entirely new field for the use of antibiotics in agriculture. The avermectins, a family of compounds with potent anthelmintic, insecticidal and acaricidal activity, have vividly demonstrated that fermentation products can have entirely unanticipated activities. Besides their u t i l i t y in animals, they show great promise for the control of insect pests of plants. Although antibiotics have found only a limited role in the control of plant diseases, the desire to find environmentally acceptable alternatives to the chemicals currently used has prompted new research efforts to discover fermentation products for use as pesticides. There has been a gradual evolution i n the types of fermentation products that have been developed for use i n a g r i c u l t u r e . This evolution has been punctuated by several major discoveries that have served to influence future work. The history begins with the accidental discovery of a new use for an a n t i b i o t i c that was already playing a major role i n the treatment of human diseases. There follows a deliberate search for new a n t i b i o t i c s unrelated to those used i n humans, the detection of a new a c t i v i t y for what had appeared to be a useless a n t i b i o t i c , and, f i n a l l y , the discovery of a family of compounds that has opened up an e n t i r e l y new area for the use of fermentation products i n agriculture and may well play a major role i n the control of both plant and animal diseases. 0097-6156/ 86/ 0320-0061 $06.00/0 © 1986 A m e r i c a n C h e m i c a l Society

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Animal Health The market for animal health products i s estimated to be over $2 b i l l i o n i n the U.S. and nearly as much i n Western Europe. A n t i b i o t i c s dominate the animal health market, and feed additives account for about 50% of that market. A c l a s s i f i c a t i o n of the compounds used for animal health i s shown i n Table I. A n t i b i o t i c s can be used therapeutically to treat b a c t e r i a l , fungal and p a r a s i t i c i n f e c t i o n s . For this purpose, they can be given i n the feed, or administered o r a l l y , parenterally or t o p i c a l l y . A n t i b i o t i c s that are fed at subtherapeutic levels to improve the rate of growth and the feed e f f i c i e n c y are called "growth permittants". They act i n d i r e c t l y by a s t i l l unknown mechanism, although i t seems reasonable that i t i s their antibact e r i a l a c t i v i t y that i s important, and that they must act on a subpopulation of the i n t e s t i n a l f l o r a . Growth promotants act d i r e c t l y , through a physiological mechanism, to enhance growth; and they usually have estrogenic a c t i v i t y . They are administered parenterally, often i n the form of an implant. Table I.

C l a s s i f i c a t i o n of Agents Used for Animals A.

B. C.

Therapeutic Agents 1. A n t i b a c t e r i a l 2. Antifungal 3. A n t i p a r a s i t i c a. Endoparasiticides (1) A n t i c o c c i d i a l s (2) Anthelmintics b. Ectoparasiticides (1) Insecticides (2) Acaricides Growth Permittants Growth Promotants

Table II l i s t s the fermentation products licensed i n the U.S. for parenteral or t o p i c a l administration to animals. Most of these are also used to treat human i n f e c t i o n s . As important as these are for animal health, of far greater economic importance are the a n t i b i o t i c s that are incorporated into animal feeds. Feed Additives. Some a n t i b i o t i c s are also administered i n the feed for the treatment of disease. These are l i s t e d i n Table I I I . For the most part, they are used for the treatment of b a c t e r i a l i n f e c tions and are the same as those l i s t e d i n Table I I . Although these a n t i b i o t i c s are incorporated into the feed, their use d i f f e r s from what has become known as "feed additive a n t i b i o t i c s " or growth permittants. The era of feed additive a n t i b i o t i c s had i t s beginning i n the l a t e 1940's i n a c l a s s i c example of serendipity. Investigators at the Lederle Laboratories were searching for a more convenient source of "animal protein factor", a substance found i n l i v e r and other animal proteins that stimulated the growth of chicks fed a vegetable diet (1). [It had already been demonstrated by workers

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6.

BURG

Fermentation

Table I I .

Products

Agriculture

63

Fermentation Products Administered T o p i c a l l y or Parenterally

Name

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in

EN

Type

Amikacin Ampicillin Bacitracin* Cephapirin Chlortetracycline Cloxacillin Dihydrostreptomycin* Erythromycin Gentamicin Hetacillin Kanamycin Lincomycin Neomycin Novobiocin Oxytetracycline Penicillin G Polymyxin B* Spectinomycin Tetracycline Tylosin Griseofulvin

Semisyn. aminocyc. Semisyn. p e n i c i l l i n Peptide Semisyn. cephalosp. Tetracycline Semisyn. p e n i c i l l i n Aminocyclitoi Macrolide Aminocyclitol Semisyn. p e n i c i l l i n Aminocyclitol

Ivermectin

Semisyn. avermectin

Zeranol

Semisyn.

Aminocyclitol Tetracycline Natural p e n i c i l l i n Peptide Aminocyclitol Tetracycline Macrolide Grisan

zearalenone

RE

Use UG

MA

OP

+ + +

+ + +

+ + + + + + + +

+ + + + + + +

+ +

+ +

+

+ + +

+

+ + +

+ + +

+ +

+

+ + +

+ +

+

+

Dermatophytic i n f e c ­ tions Nematodes and arthropods Growth promotant

*Used only i n combinations EN = Enteric

Table compiled

RE = Respiratory tract UG • Uro-genital tract MA = M a s t i t i s OP = Ophthalmic from information obtained from (20).

at Merck and Co. that p u r i f i e d vitamin Βχ2 could replace the pro­ t e i n factor ( 2 ) ] . One of the materials that was tested was the dried fermentation mash of Streptomyces aureofaciens, the producer of c h l o r t e t r a c y c l i n e . The chicks grew faster and to a greater f i n a l weight than those fed a diet supplemented with l i v e r extract, and the growth was greater than could be accounted for by the con­ tent of vitamin Ή\2· The component of the fermentation mash responsible for the stimulation of growth was i d e n t i f i e d as chlor­ t e t r a c y c l i n e (3), and this a b i l i t y to enhance growth was quickly confirmed i n turkeys and swine. The era of feed additive a n t i ­ b i o t i c s was launched. Oxytetracycline, b a c i t r a c i n and p e n i c i l l i n were soon added to the l i s t of a n t i b i o t i c s that could enhance growth and improve feed

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AGRICULTURAL USES OF ANTIBIOTICS Table I I I .

Fermentation Products Used as Feed Additives f o r the Treatment of Disease For Use In:

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch006

Name Bacitracin Chlortetracycline Erythromycin Hygromycin Β Lasalocid Lincomycin Monensin Neomycin Novobiocin Nystatin Oxytetracycline Penicillin G Salinomycin Streptomycin* Tylosin Virginiamycin

Poultry

Swine

Β Β Β Η C Β C Β Β F Β Β C Β Β

Β Β

Cattle

Sheep

Use Level (g/ton) 50- 500 50- 400 92- 185 812 68- 113 40- 100 90- 110 70- 140 200- 350 50 50- 500 50- 100 40- 60 75 100-1000 25- 100

Η Β Β

*Used only i n combination Β - antibacterial F • antifungal

C = Anticoccidial Η = Anthelmintic

Table compiled from information provided i n (21). e f f i c i e n c y . Table IV l i s t s the a n t i b i o t i c s used as growth permit­ tants i n the U.S. The levels at which these a n t i b i o t i c s are fed to increase the rate of gain and to improve feed e f f i c i e n c y are lower by a factor of 5 to 10 ( c f . Table I I I ) . There has been great concern that the feeding of low levels of a n t i b i o t i c s that are also used i n human medicine could lead to serious human health problems. There i s no question that bacteria develop resistance to these a n t i b i o t i c s , and that they can transfer t h e i r resistance to other bacteria, even to other species. There i s also no question that a n t i b i o t i c resistance has become a serious problem i n human medicine. However, the extent to which the feed­ ing of a n t i b i o t i c s to animals has contributed to the human health problem i s s t i l l unclear and a source of great controversy. In addition to the risks to human health, one must also con­ sider the benefits i n terms of cheaper meat and the saving of grain. In 1981, the Council for A g r i c u l t u r a l Science and Tech­ nology estimated that i t would cost consumers an additional $3.5 b i l l i o n per year i f the use of tetracyclines and p e n i c i l l i n were c u r t a i l e d (4). This estimate did not consider the p o s s i b i l i t y that these a n t i b i o t i c s might be replaced by others o f f e r i n g less r i s k .

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Fermentation

Table IV.

Products

in

65

Agriculture

Fermentation Products Used as Growth Permittants i n the U.S. For Use In:

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Name Bacitracin Bambermycins Chlortetracycline Erythromycin Lincomycin Oxytetracycline Penicillin G Streptomycin* Tylosin Virginiamycin Lasalocid// Monensin// Salinomycin#

Sheep

Poultry

Swine

Cattle

+ + + + + + + + + +

+ + + +

+ + +

+

+ + + + +

+

+

Use Level g/ton

+ + +

4 -50 1 - 4 10 -50 5 -18 2-4 5 -50 2.4-50 12 -19 4 -50 5 -15 10 -30 5 -30

* Used only i n combination # Rumen additives + Increased rate of gain and improved feed e f f i c i e n c y Table compiled

from information provided i n (21).

Because of the desire to reduce the nontherapeutic use i n a n i mals of a n t i b i o t i c s that are also used i n human medicine, pharmaceutical companies have been searching for new types of a n t i b i o t i c s to be used exclusively as feed additives. B a c i t r a c i n , one of the f i r s t a n t i b i o t i c s to be used as a feed additive would f i t t h i s category. Two newer a n t i b i o t i c s , bambermycins and virginiamyc i n , are licensed for use i n poultry and swine (Table IV). These a n t i b i o t i c s are unrelated to any used i n human medicine and, along with lincomycin and t y l o s i n , are taking an increasing share of the market. Other a n t i b i o t i c s , including enramycin F, sold i n Japan, and avoparcin and tiamulin, sold i n the U.K., also f a l l into this category (Table V). There are some a n t i b i o t i c s s t i l l i n development i n the U.S. Merck i s hard at work on efrotomycin, and L i l l y has avilamycin and actaplanin (Table V). The l a t t e r i s being studied not only as a growth permittant but as a means of improving milk production i n dairy c a t t l e . Unfortunately, progress has been slow and development costs are high because of the stringent requirements of the Food and Drug Administration. Growth Promotants. D i e t h y l s t i l b e s t r o l was the major growth promotant i n use for many years. It was very e f f e c t i v e , increasing weight gain i n steers by 15 to 19% and feed e f f i c i e n c y by up to 12%. However, i t has now been banned i n most countries because of i t s reported carcinogenicity. The discovery of the one fermentation product that i s used as a growth promotant i s an interesting study i n epidemiology (5). In

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AGRICULTURAL USES OF ANTIBIOTICS Table V·

Name

Activity

Avoparcin Enramycin F Tiamulin Actaplanin* Avilamycin* Efrotomycin* Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch006

Feed Additives Marketed Outside the or Under Development

Growth permittant Growth permittant Growth permittant Growth permittant, improved milk prod. Growth permittant Growth permittant

U.S.

Used i n

Poultry, C a t t l e , Swine Poultry, Swine Swine Cattle Poultry, Swine Poultry, Swine

*Under development

the midwestern U.S., there were reports of estrogenic effects i n swine that had been fed moldy corn. The fungus Gibberella zeae was i s o l a t e d from the corn, and extracts were shown to have estrogenic a c t i v i t y . A r e s o r c y l i c acid lactone, zearalenone, was isolated and shown to be responsible for the estrogenic e f f e c t s . The compound selected for commercial development was a reduction product, zearalanol or zeranol. Zeranol does not appear to have carcinogenic a c t i v i t y (5). It i s licensed for use as an implant p e l l e t i n beef c a t t l e and lambs (Table II) where i t has about 30 to 50 percent of the a c t i v i t y of d i e t h y l s t i b e s t r o l (6). A n t i c o c c i d i a l s . A new a n t i b i o t i c , monensin, discovered at the L i l l y Laboratories had an uninteresting gram-positive a n t i b a c t e r i a l spectrum. However, shortly after i t s discovery, i t was found to be cytotoxic to tumor c e l l s i n culture, and was isolated on the basis of that a c t i v i t y . As i s often the practice i n pharmaceutical research, i t was submitted to other assays and was found to have a n t i c o c c i d i a l a c t i v i t y i n a chick assay. It was shown to control infections by the s i x economically important species of Eimeria that infect chickens (7). This was an exciting discovery, and there were extensive discussions between representatives of marketing and research concerning the economic f e a s i b i l i t y of such a product. Fortunately, a dramatic increase i n the fermentation y i e l d was attained, and monensin became the dominant a n t i c o c c i d i a l i n the world. Although i t has a small therapeutic index, i t enjoys the unusual advantage of not succumbing to the development of resistance. Monensin belongs to the family of polyether ionophores. These compounds consist of a series of tetrahydrofuran and tetrahydropyran rings and have a carboxyl group that forms neutral s a l t s with a l k a l i metal cations. Their three-dimensional structure presents a l i p o p h i l i c hydrocarbon exterior with the cation encircled i n the oxygen-rich i n t e r i o r . They probably act by transporting cations through the l i p i d bi-layer of c e l l membranes, thereby preventing the concentration of potassium by the c e l l s . Evidence for this i s

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that high concentrations of potassium incorporated into the medium reverse the a c t i v i t y of ionophores again gram-positive bacteria. After the marketing of monensin began, there was a rush to discover more ionophores. The second ionophore to be licensed as a coccidiostat i n the U.S. was X-537A, f i r s t reported by investigators at the Nutley, NJ laboratory of Hoffmann-LaRoche i n 1951 (8), 16 years prior to the announced discovery of monensin. I t was t h e i r misfortune not to have tested t h e i r compounds against cocc i d i a . X-537A, now named l a s a l o c i d , d i f f e r s from most of the other ionophores i n i t s a b i l i t y to complex with divalent cations. Because of i t s smaller s i z e , two molecules can surround one divalent cation. Salinomycin i s also licensed i n the U.S. (Table I I I ) . At least two other ionophores, narasin and maduramicin, have been introduced elsewhere i n the world. Narasin i s a homologue of s a l i nomycin, and maduramicin i s noteworthy because i t i s e f f e c t i v e at a l e v e l of 5 g/ton, only 5 to 10% of the l e v e l required for the other ionophores· The discovery of the coccidiostat a c t i v i t y of monensin marks the second milestone i n the history of the use of fermentation products i n agriculture. U n t i l this discovery, the emphasis had been on the search for a n t i b i o t i c s with a n t i b a c t e r i a l a c t i v i t y . I t was now evident that fermentation products could be used for the control of p a r a s i t i c i n f e c t i o n s . Rumen Additives. The ionophores were found to possess a second remarkable u t i l i t y . Ruminants are walking fermentation vesseles that are able to convert r e l a t i v e l y useless, high cellulose vegetat i o n such as grass into protein. Although this i s a wonderful a b i l i t y , researchers, who seem never to be s a t i s f i e d with nature, have long sought to improve this fermentation. One product of the rumen fermentation, methane, i s of no value to the ruminant. The major fermentation products used by the ruminant are the short-chain f a t t y acids, acetate, butyrate and propionate. Acetate and butyrate can be used for energy, but propionate i s most useful for the synthesis of protein. I f the fermentation could be shifted to reduce methane, acetate and butyrate production and to increase the propionate, the feed e f f i c i e n c y and growth rate could improved. Monensin was tested i n a rumen fermentation assay at the L i l l y Laboratories, and i t was found to produce the desired s h i f t i n the fermentation (9). Monensin has been licensed i n the U.S. for use i n beef c a t t l e for improved feed e f f i c i e n c y , where i t i s administered at 5 to 30 g/ton i n a complete feed. In this application, the rate of growth i s not increased, but the c a t t l e consume about 10% less food. It i s also licensed for increased rate of weight gain i n c a t t l e weighing more than 400 l b . and on pasture, where i t i s fed i n a supplement at a rate of 50 to 200 mg per head per day. Lasalocid and salinomycin have also been licensed for use i n cattle. There have been a number of reports i n the l a s t four years of studies on salinomycin as a growth permittant i n swine. I t has been administered at a l e v e l of 25 to 100 g/ton of feed where i t gave a s i g n i f i c a n t increase i n weight gain and feed e f f i c i e n c y , quite comparable to t y l o s i n (10) or virginiamycin (11). If these studies lead to the development of salinomyin as a growth permit-

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tant for non-ruminants, the ionophores could eventually dominate the entire feed additive market. Anthelmintic Agents. One a n t i b i o t i c has been used as an anthelmin­ t i c agent for many years. Hygromycin Β was isolated at the L i l l y laboratories because of i t s a n t i b a c t e r i a l a c t i v i t y . Although i t i s active against both gram-positive and gram-negative bacteria, i t s a c t i v i t y was too weak to be therapeutically useful. It was tested i n a variety of other assays and was found to be active jLn vivo against the pinworms, A s p i c u l a r i s tetraptera and Syphacia obvelata. The anthelmintic a c t i v i t y was confirmed i n pigs (12). It i s licensed for the control of Ascaris g a l l i , Heterakis gallinae and C a p i l l a r i a obsignata i n chickens when fed at the l e v e l of 8 to 12 g/ton an for the control of Ascaris suum, Oesophagostomum dentatum and T r i c h u r i s suis i n swine, where i t i s fed at 12 g/ton. A number of other fermentation products have been reported to have anthelmintic a c t i v i t y . Among these are the aminoglycoside, G-418, the destomycins, paromomycin, anthelvencin, aspiculamycin, anthelmycin, and the axenomycins. However, none of these has seen commercial use. The third milestone i n the history of the use of fermentation products i n agriculture was the discovery of the avermectins. They were f i r s t detected i n an anthelmintic assay using mice infected with nematospiroides dubius (13). This i s one of the few assays i n which they could have been detected since they lack a n t i b a c t e r i a l and antifungal a c t i v i t y . Further experience has demonstrated that i t was not solely the choice of assay but the great good fortune to have received a group of cultures from the Kitasato Institute and to have made the deci­ sion to screen these cultures i n the N. dubius assay. One of these cultures, 0S-3153, was active. The screening of several tens of thousands of s o i l isolates i n this assay has f a i l e d to detect any remotely similar anthelmintic a c t i v i t y . Of the fermentation pro­ ducts discussed, this i s the only one where the a c t i v i t y for which the product was eventually marketed was found by direct screening. (Zeranol might be considered to be another example, but i t was not discovered by screening.) Tests using helminth infections i n a variety of laboratory animals soon revealed that the avermectins had a c t i v i t y against a variety of nematodes but lacked a c t i v i t y toward cestodes and trematodes. During the course of testing i n a number of other assays, they were found active against the f l o u r beetle, Tribolium confusum (14). This a c t i v i t y against arthropods was confirmed i n mice infected with larvae of the bot f l y , Cuterebra f o n t i n e l l a . The avermectins are active against a wide variety of insects and other arthropods, including mites, ticks and l i c e . Moreover, they are active against nematode, insect and acarine infections of animals when administered i n a single dose given o r a l l y or paren­ t e r a l l y . Equally as exciting as their spectrum i s their extreme potency. For example, avermectin B i exhibits greater than 95% e f f i c a c y against Haemonchus contortus, Ostertagia circumcincta, Trichostrongylus axei, T. colubriformis, Cooperia oncophora and Oesophagostomum columbianum when administered to sheep i n a single o r a l dose of 100 pg/kg (15) · I t i s even more potent against Ancylostoma caninum i n dogs, where i t i s 83 to 100% e f f e c t i v e when given as a single o r a l dose of 3 to 5 yg/kg (15). Undoubtedly the a

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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most sensitive ectoparasite i s the larva of the common c a t t l e grub, Hypoderma lineatum, where a single subcutaneous i n j e c t i o n of 0.2 yg/kg gives 100% control (16). This dual a c t i v i t y against both nematode and arthropod para­ s i t e s of animals was an unexpected bonus from a screen for anthel­ mintic agents. The reason for this broad a c t i v i t y l i e s i n their mode of action. They act by i n t e r f e r i n g with γ-aminobutyric acid (GABA) mediated neurotransmission. When treated with avermectin, the nematode Ascaris suum becomes paralyzed although i t retains normal muscle tone (17). Picrotoxin, an antagonist of GABA, can reverse the effect of avermectin on neurotransmission i n v i t r o . The absence of GABA-mediated neurotransmission i n cestodes and trema-todes explains the lack of a c t i v i t y of the avermectins against these organisms. The compound ultimately chosen for development was a semi­ synthetic derivative of the Bj series i n which the 22,23 double bond i s reduced (18). The mixture consisting of at least 80% 22,23-dihydroavermectin B and not more than 20% 22,23-dihydroavermectin B has been named ivermectin. Its use l e v e l i s 200 yg/kg i n horses, c a t t l e and sheep and 300 yg/kg i n swine. It i s injected subcutaneously i n c a t t l e and swine, and there are oral formulations for use i n horses and sheep. l a

l b

Plant Diseases Fermentation products have played a rather minor role i n the control of plant diseases. Table VI gives a c l a s s i f i c a t i o n of agents used on plants. These are divided into pesticides and growth modulators. The pesticides are c l a s s i f i e d as bactericides, fungicides, i n s e c t i c i d e s , miticides, nematicides and herbicides. There are fermentation products i n each of these categories, and these are l i s t e d i n Table VII. Table VI.

C l a s s i f i c a t i o n of Agents Used on Plants

A.

B.

Pesticides 1· Bactericides 2· Fungicides 3. Herbicides 4. Insecticides 5. Miticides 6. Nematicides Growth Modulators

It should be emphasized that although the t o t a l worldwide market for a g r i c u l t u r a l pesticides i s huge (over $10 b i l l i o n ) , the share held by fermentation products i s quite small. Most of the fungicides l i s t e d i n Table VII. are used i n Japan, often for r i c e b l a s t . There are several reasons for this small market share, but the most s i g n i f i c a n t reason i s probably economic. Although the fermentation products that have found commercial application are often much more active than chemical pesticides, this factor i s not often s u f f i c i e n t to compensate for the higher cost of producing them.

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch006

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In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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There are a few companies that are hoping to change t h i s . Several chemical companies that already have a large share of the chemical pesticide market are a c t i v e l y screening fermentation broths. A major motivation for this probably comes from the present concern about our environment. There i s a perception that "natural" pesticides w i l l have a much less serious environmental impact than "chemicals". Whether this advantage i s r e a l or only psychological remains to be seen. Another incentive to this screening may have arisen from the discovery of two remarkably potent families of fermentation products with i n s e c t i c i d a l and a c a r i c i d a l a c t i v i t y , the milbemycins and the avermectins. Abamectin (a mixture of not less than 80% avermectin B i and not more than 20% avermectin B ^ ) i s already seeing limited use i n F l o r i d a for the protection of ornamentals, and there i s a considerable e f f o r t being made to develop the avermectins for use against a wide variety of insect and mite pests. Two examples of the remarkable potency of avermectin B^ are i t s LD9Q of 0.02 to 0.03 ppm against the two-spotted spider mite, Tetranychus u r t i c a e , when applied to bean plants as a f o l i a r spray; and i t s control of the red imported f i r e ant, Solenopsis i n v i c t a , when applied as a bait at a l e v e l as low as 25 to 50 mg per acre (19). To express this extreme potency i n another way, the spray for mites contains 4.5 mg of abamectin per l i t e r whereas malathion spray, also used as a miticide, contains 3,700 mg per l i t e r . This i s over 800 times as much compound to produce the same e f f e c t .

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch006

a

a

Conclusion Microorganisms are extremely v e r s a t i l e chemists. The wide variety of structures among the r e l a t i v e l y few compounds discussed here i s testimony to that. There are several theories to explain the evolutionary advantage conferred by the synthesis of secondary metabolites. ( I t often seems that they serve primarily to enrich pharmaceutical companies.) U n t i l recently, the idea that they conferred a competitive advantage upon the producing organism seemed reasonable, since most of the products that had been detected had a n t i b i o t i c a c t i v i t y . For many years, screening programs were directed toward the discovery of a n t i b a c t e r i a l and antifungal a n t i b i o t i c s . Now the screening of microorganisms has shifted toward the search for other types of a c t i v i t i e s . Perhaps the products that had been found are more the result of the assays employed than of their synthetic c a p a b i l i t i e s . The a b i l i t y to discover new types of fermentation products may be limited only by the ingenuity i n developing new and sensitive assays along with a certain luck i n selecting the proper microorganisms to t e s t . The future for the use of fermentation products i n agriculture holds much promise. Acknowledgment I wish to thank Dr. Robert Hamill of L i l l y Research Laborat o r i e s f o r providing unpublished information about the discovery and development of monensin.

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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L i t e r a t u r e Cited 1. 2. 3. 4. 5.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch006

6.

7.

8. 9. 10. 11. 12. 13.

14. 15.

16. 17. 18.

19.

20.

21.

Stokstad, E. L. R.; Jukes, T. H.; Pierce, J . ; Page, A. C., J r . ; Franklin, A. L. J . B i o l . Chem. 1949, 180, 647-654. Ott, W. H.; Rickes, E. L.; Wood, T. L. J . B i o l . Chem. 1948, 174, 1047-1048. Stokstad, E. L. R.; Jukes, T. H. Proc. Soc. E x p t l . B i o l . Med. 1950, 73, 523-528. Council for A g r i c u l t u r a l Science and Technology. Report No. 88, Ames, Iowa 1981. Hidy, P. H.; Baldwin, R. S.; Greasham, R. L.; Keith, C. L.; McMullen, J . R. Adv. Appl. M i c r o b i o l . 1977, 22, 59-82. U. S. Office of Technology Assessment. "Drugs i n Livestock Feed", V o l . I, Technical Report, U. S. Government Printing O f f i c e , Washington, D.C. 1979, 37. Shumard, R. F; Callender, M. E. In: Hobby, G. L. (Ed.) A n t i ­ microbial Agents and Chemotherapy - 1967, American Society for Microbiology, Ann Arbor, Mich. 1968, 369-377. Berger, J . ; Rachlin; A. I.; Scott, W. E.; Sternbach, L. H; Goldberg, M. W. J . Am. Chem. Soc. 1951, 73, 5295-5298. Richardson, L. F.; Raun, A P.; Potter, E. L.; Cooley, C. O.; Rathmacher, R. P. J . Anim. S c i . 1974, 39, 250. Leeson, S.; Hacker, R. H.; Wey, D. Can. J . Anim. S c i . 1981, 61, 1063-1065. de Wilde, R. O. Deutsche T i e r a r z t l . Wochenschr. 1984, 91, 22-24. Goldsby, A. I.; Todd, A. C. North Amer. Vet. 1957, 38, 140144. Burg, R. W.; M i l l e r , Β. M; Baker, Ε. E.; Birnbaum, J . ; Currie, S. Α.; Hartman, R.; Kong, Y-L.; Monaghan, R. L.; Olson, G.; Putter, I.; Tunac, J . B.; Wallick, H.; Stapley, E. O.; Oiwa, R; Omura, S. Antimicrob. Agents Chemother. 1979, 15, 361-367. Ostlind, D. Α.; Cifelli, S; Long, R. Vet. Rec. 1979, 105, 168. Egerton, J . R.; Ostlind, D. Α.; B l a i r , L. S.; Eary, C. H.; Suhayda, D.; Cifelli, S.; Riek, R. F; Campbell, W. C. A n t i ­ microb. Agents Chemother. 1979, 15, 372-378. Drummond, R. O. J . Econ. Entomol 1984,, 77, 402-406. Kass, I. W.; Wang, C. C.; Walrond, J . P; Stretton, A. O. W. Proc. Nat. Acad. S c i . U.S. 1980, 77, 6211-6215. Chabala, J . C.; Mrozik, H; Tolman, R. L.; Eskola, P.; L u s i , Α.; Peterson, L. H.; Woods, M. F.; Fisher, M. H.; Campbell, W. C.; Egerton, J . R.; Ostlind, D. A. J . Med. Chem. 1980, 23, 1134-1136. Campbell, W. C.; Burg, R. W.; Fisher, M. H; Dybas, R. A. i n Magee, P. S.; Kohn, G. K; Menn, J . J . (Eds.) Pesticide Syn­ thesis Through Rational Approaches, A.C.S. Symposium Series 255, American Chemical Society, Washington, D.C. 1984, 5-20. Aronson, C. E. (Ed.) Veterinary Pharmaeuticals & B i o l o g i c a l s 1982/1983, Veterinary Medicine Publishing Co., Edwardsville, Kansas, 1983. Leidahl, R. (Ed.) 1985 Feed Additive Compendium, The M i l l e r Publishing Company, Minneapolis, Minnesota, 1984.

RECEIVED February 18, 1986

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B e n e f i t s and Risks of A n t i b i o t i c s U s e in A g r i c u l t u r e Virgil W. Hays

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch007

Department of Animal Sciences, University of Kentucky, Lexington, KY 40546-0215 The a d v i s a b i l i t y of using c e r t a i n a n t i b i o t i c s , p a r t i c u l a r l y p e n i c i l l i n and tetracycline, in animal feeds has been questioned because of their use in human medicine. Any use of an antibiotic that i s prescribed for humans presents some risks to human health, whether the use is for humans, animals or for other purposes; but. the uses also have benefits. Otherwise, they would not persist. Antibiotics are used in animal feeds to increase animal weight, increase efficiency of feed u t i l i z a t i o n , increase reproductive efficiency and decrease morbidity and mortality. These benefits to animals and animal producers are reflected in decreases in food costs to humans. There are also benefits to human health from use of antibiotics in food animals. By reducing the incidence of animal h e a l t h problems, use of antibiotics in food animals reduce the transference of animal infections to humans. The contention that the effectiveness of p e n i c i l l i n and tetracycline for use in human medicine is rapidly diminishing as a result of the proliferation of resistant bacteria caused by subtherapeutic use of antibiotics in animal production is not supported by experimental data. Rather, the evidence suggests that a f a i r l y stable l e v e l of resistance of the i n t e s t i n a l bacteria in humans has long since been e s t a b l i s h e d to penicillin and tetracycline as i t has been in animals. F o r t h e p a s t 35 y e a r s . U.S. l i v e s t o c k a n d p o u l t r y p r o d u c e r s h a v e used antibiotics (products of microbial s y n t h e s i s ) and chemotherapeutics ( c h e m i c a l l y s y n t h e s i z e d p r o d u c t s ) . The drugs a r e a d m i n i s t e r e d i n r e l a t i v e l y l a r g e dosages t o t r e a t s i c k a n i m a l s ( t h e r a p e u t i c a l l y ) and i n lower dosages t o p r e v e n t d i s e a s e i n exposed a n i m a l s ( p r o p h y l a c t i c a 1 1 y)· More commonly, s m a l l amounts ( s u b t h e r a p e u t i c ) o f a n t i b i o t i c s are added t o a n i m a l feeds t o p r e v e n t or reduce d i s e a s e s and t o improve f e e d e f f i c i e n c y and growth. 0097-6156/ 86/ 0320-0074$06.00/ 0 © 1986 American Chemical Society

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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Benefits and Risks of Antibiotics

Use in

Agriculture

75

A p p r o x i m a t e l y 80% o f t h e p o u l t r y . 75% o f t h e swine. 60% o f t h e beef c a t t l e and 7 5 % o f t h e d a i r y c a l v e s m a r k e t e d a r e e s t i m a t e d t o h a v e r e c e i v e d a n t i b i o t i c s a t some t i m e i n t h e i r l i f e (CAST. 2.)· Of t h e a n t i b i o t i c s p r o d u c e d e a c h y e a r i n t h e U.S.. 45 t o 5 5 % a r e administered to animals. Three broad g r o u p i n g s , o f the a n t i b i o t i c substances p r e s e n t l y used i n a n i m a l p r o d u c t i o n , i n c l u d e : (a) broad-spectrum a n t i b i o t i c s , i n c l u d i n g p e n i c i l l i n s and t e t r a c y c l i n e s , which a r e e f f e c t i v e a g a i n s t a w i d e v a r i e t y o f p a t h o g e n i c and n o n - p a t h o g e n i c bacteria; (b) s e v e r a l narrow-spectrum a n t i b i o t i c s t h a t a r e not used i n human m e d i c i n e ; and. (c) t h e i o n o p h o r e a n t i b i o t i c s , monensin. l a s a l o c i d and s a l i n o m y c i n . M o n e n s i n and l a s a l o c i d a r e u s e d as rumen f e r m e n t a t i o n r e g u l a t o r s i n beef c a t t l e , and t h e t h r e e ionophores a r e used as c o c c i d i o s t a t s i n p o u l t r y p r o d u c t i o n . The i o n o p h o r e s . which a r e not used i n human m e d i c i n e , were f i r s t i n t r o d u c e d i n the 1970 s and account f o r most o f t h e i n c r e a s e i n a n t i b i o t i c usage i n a n i m a l p r o d u c t i o n s i n c e t h e 1960 s.

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f

f

Why

Are

A n t i b i o t i c s Used In A n i m a l P r o d u c t i o n ?

The e f f i c a c y o f a n t i b i o t i c s i n i m p r o v i n g r a t e and e f f i c i e n c y o f growth has been w e l l documented by many r e s e a r c h e r s . To i l l u s t r a t e t h e r e s p o n s e s i n a n i m a l s , a c o m p r e h e n s i v e summary i n v o l v i n g 937 experiments and more t h a n 20.000 p i g s i s p r e s e n t e d i n t a b l e 1. Note

T a b l e 1.

. . . 1 Response o f P i g s t o S u b t h e r a p e u t i c L e v e l s o f A n t i b i o t i c s Control

Antibiotic

Improvement» %

S t a r t e r phase (15-57 l b ) Average d a i l y g a i n , l b Feed/gain

.86 2.32

1.01 2.16

16 7

Grower phase (37-108 l b ) Average d a i l y g a i n , l b Feed/gain

1.30 2.91

1.45 2.78

11 5

G r o w e r - f i n i s h e r phase (44-189 l b ) Average d a i l y g a i n , l b Feed/gain

1.50 3.37

1.56 3.30

4 2

Item

\ ) a t a from 378, 280 and 279 e x p e r i m e n t s , i n v o l v i n g 10.023, 4.783 and 5.666 p i g s f o r the t h r e e phases, r e s p e c t i v e l y (Hays. 5.).

t h a t the magnitude of the response i s g r e a t e r f o r the younger a n i m a l s and d e c l i n e s as t h e a n i m a l matures. Similar results could be p r e s e n t e d f o r c h i c k s , t u r k e y s and c a t t l e . For the most p a r t , t h e summary p r e s e n t e d i s b a s e d on d a t a f r o m E x p e r i m e n t S t a t i o n o r Industry research units. We h a v e r e l a t i v e l y few p u b l i s h e d

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experiments carried out i n a production environment. Such o v e r a l l summaries as the one i n t a b l e 1 markedly underestimate the t o t a l benefits derived from a n t i b i o t i c s for three major reasons: (1) the data f o r the more as w e l l as the l e s s e f f e c t i v e a n t i b i o t i c s are pooled; (2) growth rate and feed conversion are accounted f o r , but reduction i n m o r t a l i t y and improved reproductive performance are not appropriately considered; and^ (3) the magnitude of the responses i s s m a l l e r i n the experiment s t a t i o n environment than i n commercial p r o d u c t i o n u n i t environments. T h i s l a t t e r effect on estimates of the average b e n e f i t s i s i l l u s t r a t e d by the data presented i n table 2. By pooling a large number of observations as was done for this table, to overcome at least p a r t i a l l y the b i o l o g i c a l v a r i a t i o n

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Table 2.

Comparison of Response to A n t i b a c t e r i a l Agents by Pigs in Experiment Station and Production Unit Environments*

Location

No. Experiments

Experiment station research units Production units Weight avg.

% Improvement from Antibiotics D a i l y gain Feed/gain 2

128

16.9

7.0

32

28.4

14.5

160

19.2

8.5

Data on 12,000 pigs from 15 to 57 l b . (Hays,

1).

2 Chlortetracycline-penicillin-sulfamethazine, tylosin-sulfamethazine, tetracyclines and carbadox. a s s o c i a t e d with s m a l l experiments, one can i l l u s t r a t e that the responses to a n t i b i o t i c s by young p i g s i n p r o d u c t i o n u n i t s are nearly twice that observed i n Experiment Station units. There are s e v e r a l reasons why the Experiment S t a t i o n or Industry r e s e a r c h u n i t data are l i k e l y to underestimate the r e a l benefits from a n t i b i o t i c s . These include: (1) Animals are selected for uniformity, and any poor-doing or unhealthy animals are not used u n l e s s the experiment i s s p e c i f i c a l l y designed f o r that purpose. The producer must treat i f necessary, a l l o w the unhealthy animals to r e c o v e r , and, to the extent p r a c t i c a l , f i n i s h a l l a n i m a l s . Ample data are a v a i l a b l e to i l l u s t r a t e that the response to a n t i b i o t i c s i s much g r e a t e r i n p o o r l y - d o i n g a n i m a l s . (2) The environment of the animals i s less conducive to stress conditions i n most experimental situations than may be p r a c t i c a l for commercial operations. Animals are grouped in smaller numbers and frequently the space allowance i s e x c e s s i v e . (3) S a n i t a t i o n i s u s u a l l y b e t t e r i n the e x p e r i m e n t a l situation, p a r t i c u l a r l y for pigs and poultry, i n that buildings are u s u a l l y emptied, c l e a n e d and d i s i n f e c t e d between experiments. (4) R a t i o n - b a l a n c i n g and f e e d i n g procedures are g e n e r a l l y more

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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7.

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Benefits and Risks of Antibiotics

Use in

Agriculture

11

p r e c i s e and i n g r e d i e n t q u a l i t y i s c l o s e l y monitored i n most experimental situations. Some c r i t i c s of current commercial production methods suggest that a n t i b i o t i c s are necessary only because of the s t r e s s f u l rearing c o n d i t i o n s and that the r e t u r n to more e x t e n s i v e r e a r i n g systems would obviate the need for a n t i b i o t i c s . Returning to the extensive animal r e a r i n g systems would r e s u l t i n exposure to g r e a t e r e n v i r o n m e n t a l extremes and i n c r e a s e the exposure to i n t e r n a l parasites and the associated s u s c e p t i b i l i t y to diseases, hence would increase rather than decrease the response to a n t i b a c t e r i a l agents. Improved performance of the animals and reduced m o r t a l i t y are d e f i n i t e benefits. The t o t a l aggregate of these benefits to a l l of animal a g r i c u l t u r e i s very substantial and has been estimated to be as much as a $3.5 b i l l i o n per year r e d u c t i o n i n food c o s t s to the U.S. consuming p u b l i c (CAST, 1). Whv the Concern About Using A n t i b i o t i c a In Animal Production? Since 1977, the Food and Drug A d m i n s t r a t i o n (FDA) has been considering a ban on the subtherapeutic use of procaine p e n i c i l l i n and t e t r a c y c l i n e s i n animal feeds. These a n t i b i o t i c s are used i n both humans and a n i m a l s , and any use of an a n t i b i o t i c that i s p r e s c r i b e d f o r humans p r e s e n t s some r i s k to human h e a l t h , whether the use i s for humans, animals, or other purposes. The r i s k i s that pathogenic or d i s e a s e - c a u s i n g b a c t e r i a may d e v e l o p a s t r a i n that r e s i s t s that a n t i b i o t i c . The r e s i s t a n t s t r a i n of the pathogen then may cause human disease that cannot be treated by this a n t i b i o t i c . The r i s k that e x i s t s from the use of a n t i b i o t i c s i n animals arises through a complicated series of events. When an a n t i b i o t i c i s f e d to an a n i m a l , i t comes i n contact with the v a s t and complex b a c t e r i a l p o p u l a t i o n i n the d i g e s t i v e t r a c t . I f present i n b i o l o g i c a l l y e f f e c t i v e amounts, i t a f f e c t s the s e n s i t i v e bacteria, which may i n c l u d e pathogens; but, the t o t a l number o f l i v i n g bacteria remains about the same. The s e n s i t i v e bacteria destroyed or i n h i b i t e d , and the r e s i s t a n t b a c t e r i a m u l t i p l y to take t h e i r p l a c e . These r e s i s t a n t b a c t e r i a may contaminate animal products used by humans as food. Two types of b a c t e r i a l r e s i s t a n c e to a n t i b i o t i c s are known: (a) r e s i s t a n c e due to genes t r a n s f e r r e d to the progeny v i a the chromosome--the r e g u l a r , r e l a t i v e l y s t a b l e g e n e t i c m a t e r i a l and (b) r e s i s t a n c e due to t r a n s f e r of genes on *R plasmids", which are b i t s of g e n e t i c m a t e r i a l s m a l l e r than chromosomes that e x i s t and r e p l i c a t e autonomously i n the c e l l cytoplasm. The t r a n s f e r of r e s i s t a n c e due R p l a s m i d s i s not n e c e s s a r i l y l i m i t e d to other bacteria of the same genus or species, and R plasmids may a l s o carry other genetic factors that increase or decrease the v i r u l e n c e of the organisms to which they are t r a n s f e r r e d (Fagerberg et a l . , 4.)· Another problem r e s u l t s from the f a c t that r e s i s t a n c e to one a n t i b i o t i c may be g e n e t i c a l l y l i n k e d i n some i n s t a n c e s to resistance to one or more other a n t i b i o t i c s . Both types of r e s i s t a n c e i n animal b a c t e r i a can a f f e c t human health. B a c t e r i a of animal o r i g i n that are r e s i s t a n t to a p a r t i c u l a r a n t i b i o t i c may make t h i s a n t i b i o t i c i n e f f e c t i v e f o r c o n t r o l l i n g human i n f e c t i o n s with pathogens b e a r i n g the k i n d o f

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r e s i s t a n c e c a r r i e d by these b a c t e r i a as a consequence of (a) the pathogenic p r o p e r t i e s of the animal b a c t e r i a as such, or (b) the transference of the resistance to other bacteria, which may be human pathogens. The transfer may occur i n either animals or humans, and i f c o n d i t i o n s are r i g h t f o r b a c t e r i a l growth, the t r a n s f e r c o u l d take place i n a prepared food.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch007

A r e A n t i b i o t i c R e s i d u e s A Concern? Another frequently cited concern to humans from use of a n t i b i o t i c s in animal feeds i s that the residues i n the edible animal products may i n c r e a s e the human i n t a k e of a n t i b i o t i c s , and thus cause development of a n t i b i o t i c - r e s i s t a n t pathogenic b a c t e r i a i n humans. To avoid this p o s s i b i l i t y , FDA establishes maximum l e v e l s that can be used i n animal feeds and a minimum time i n t e r v a l between the l a s t use of feed containing a n t i b i o t i c s and the slaughter of the animals. This allows for elimination of a n t i b i o t i c s from the animals before slaughter. A r e v i e w of the U.S. Department of A g r i c u l t u r e ' s m o n i t o r i n g r e c o r d s confirms that a n t i b i o t i c r e s i d u e s i n animal products are not a s i g n i f i c a n t problem. I f a n t i b i o t i c s did remain in the tissues, they would be inactivated by cooking. Certain of the chemotherapeutics, for example the s u l f a drugs, have been of g r e a t e r concern from the p o i n t of view of t i s s u e residues. Though there i s no evidence that the s u l f a residues found in pork l i v e r s or kidneys has or would cause human health problems, they are v i o l a t i v e by our present standards. Therapeutic use (high dosages) of a n t i b i o t i c s are more l i k e l y to r e s u l t i n residues than are feed a d d i t i v e uses, but i t i s important that o n l y approved l e v e l s and required withdrawal periods be adhered to for a l l drugs. Is Subtherapeutic A n t i b i o t i c Use A C o n t r i b u t o r To Human Health Problems? The c u r r e n t concern about s u b t h e r a p e u t i c doses of a n t i b i o t i c s i n animal feeds gained renewed impetus from two s c i e n t i f i c papers (Holmberg et a l . , Z-.£)* by r e s e a r c h e r s at t h e C e n t e r s f o r Disease C o n t r o l (CDC) i n A t l a n t a and a r e l a t e d e d i t o r i a l (Levy, 11) and news a r t i c l e s (Sun, 16-7) i n s c i e n t i f i c journals about them. In e a r l y 1983, Dr. S c o t t Holmberg (a CDC e p i d e m i o l o g i s t ) and his colleagues i d e n t i f i e d 18 persons i n four midwestern states who were affected by a severe diarrhea caused by a s t r a i n of S a l m o n e l l a newport that was resistant to t e t r a c y c l i n e , as w e l l as a m p i c i l l i n and c a r b e n i c i l l i n (chemically synthesized r e l a t i v e s of p e n i c i l l i n ) . Of these 18 persons, 13 had consumed hamburger s u p p l i e d d i r e c t l y from a p a r t i c u l a r herd of c a t t l e , or they had purchased hamburger from markets thought to be s u p p l i e d with meat from the herd. The c a t t l e i n question were produced i n a feedlot i n South Dakota, where t h e y had r e c e i v e d f e e d c o n t a i n i n g c h l o r t e t r a c y c l i n e a t a subtherapeutic l e v e l . Some s u p p o r t e r s o f t h e p r o p o s e d FDA ban on t h e use o f s u b t h e r a p e u t i c l e v e l s of p e n i c i l l i n and t e t r a c y c l i n e s i n animal feeds h a i l e d the r e s u l t s of the CDC study as a c l e a r l i n k between s u b t h e r a p e u t i c a n t i b i o t i c use i n food animal p r o d u c t i o n and a n t i b i o t i c - r e s i s t a n t d i s e a s e s i n humans. Some opponents, on the

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch007

7.

HAYS

Benefits and Risks of Antibiotics

Use in

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79

other hand* p o i n t e d to the l a c k of e v i d e n c e needed to c o n s t i t u t e adequate s c i e n t i f i c proof that the r e s i s t a n t organism came from hamburger» that the hamburger was contaminated with the r e s i s t a n t s t r a i n o f S.a.lmo.llfî.llâ. X L £ . K £ Q X £ . » t h a t i f the hamburger was contaminated* the source of contamination was the herd of c a t t l e implicated* that the herd i t s e l f harbored the resistant organism* or that subtherapeutic usage of c h l o r t e t r a c y c l i n e had any connection to the a n t i b i o t i c resistance of the organism or i t s p r o l i f e r a t i o n . The r e a l l y important p o i n t i s not whether t h i s p a r t i c u l a r CDC investigation did or did not demonstrate the l i n k * but rather, are there problems; and, i f so. how important are the problems r e s u l t i n g from use of a n t i b i o t i c s at subtherapeutic l e v e l s i n animal feeds? In other words, would discontinuing the subtherapeutic use of t e t r a c y c l i n e and p e n i c i l l i n have a s i g n i f i c a n t e f f e c t on a n t i b i o t i c resistance i n consumers of animal products? Long Term Experiments On Impact nf A n t i b i o t i c Restrictions In addition to our work on the animal responses to a n t i b i o t i c s , we have been very much i n v o l v e d i n the impact of a n t i b a c t e r i a l agents on the microflora of the animals and their environment. Since 1972 we have monitored the l e v e l and p a t t e r n s of r e s i s t a n c e i n two separate herds of p i g s and have used p i g s from these herds i n experiments conducted at a t h i r d location. In the control herd, we have used a n t i b i o t i c s much as a swine producer would except that a portion of the herd has been continuously exposed to t e t r a c y c l i n e a n t i b i o t i c s at a l e v e l of 50 to 100 grams per ton of feed. The other herd, a Specific Pathogen Free herd located at our Princeton. K e n t u c k y E x p e r i m e n t S t a t i o n , has had no e x p o s u r e to any a n t i b a c t e r i a l agent for therapeutic or subtherapeutic purposes since the p r o j e c t was s t a r t e d (May. 1972). T h i s herd ( a n t i b i o t i c - f r e e herd) i s c o m p l e t e l y i s o l a t e d from other p i g s . Any new g e n e t i c material i s introduced into the herd by a r t i f i c i a l insemination of selected females to provide the required number of males. A l l diets have been prepared on the research farm and no a n t i b i o t i c containing feeds have been processed i n that m i l l . No animal products are i n c l u d e d i n the d i e t i n order to l i m i t d i e t as an e n t r y source f o r resistant organisms. These two herds have p r o v i d e d i n f o r m a t i o n on development, p e r s i s t e n c y and t r a n s f e r o f a n t i b a c t e r i a l r e s i s t a n c e ; and. furthermore, they have provided information regarding the impact the previously proposed r e s t r i c t i o n s (Fed. Reg. 42:43770 and 42:52645, 1977) of a n t i b i o t i c usage would have on a n t i b i o t i c r e s i s t a n t bacteria of animal o r i g i n as a health hazard to humans. Following a n t i b i o t i c withdrawal, the performance of pigs i n the a n t i b i o t i c - f r e e herd was markedly reduced ( L a n g l o i s et a l . , 9-10). Conception rate on f i r s t service declined (91% vs. 84%), sows farrowed fewer p i g s (10.9 v s . 10.1), fewer p i g s s u r v i v e d to weaning (8.9 v s . 7.5), and the p i g s were s m a l l e r at three-week weaning (12.1 l b . vs. 11.5 lb.). We r e a l i z e that such before- and after-withdrawal comparisons alone are not v a l i d estimates of the average benefits of feed a d d i t i v e usage of a n t i b i o t i c s . However, the d i f f e r e n c e s observed are remarkably s i m i l a r to the average responses from c o n t r o l l e d experiments.

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Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch007

Numerous other problems have been encountered since a n t i b i o t i c s were discontinued. A higher proportion of the pigs do poorly (runt pigs) r e s u l t i n g in more v a r i a b l e as w e l l as an o v e r a l l increase in l e n g t h of time to reach market weight. There has been an i n c r e a s e in the incidence of mastitis* m e t r i t i s and a g a l a c t i a . 10% (before) v s . 66% ( a f t e r ) of sows a f f e c t e d . There has been a g r e a t e r i n c i d e n c e of lameness and s k e l e t a l j o i n t problems. On s e v e r a l o c c a s i o n s , the pigs have had severe s k i n l e s i o n s a t t r i b u t e d to s t a p h y l o c o c c a l i n f e c t i o n s . Two recent outbreaks of diarrhea were diagnosed as p o r c i n e p r o l i f e r a t e d e n t e r i t i s with Campylobacter diagnosed as the c a u s a t i v e organism. C o n v e r s e l y , we have not encountered s i m i l a r or other major health problems in the control (continuous-antibiotic) herd. A n t i b i o t i c Use and B a c t e r i a l R e s i s t a n c e It may conceivably be argued that no one has proposed a complete ban on a n t i b i o t i c usage, and that these studies would not be u s e f u l in evaluating the effects of r e s t r i c t e d a n t i b i o t i c use. However, the impact any r e s t r i c t i o n s would have on a n t i b i o t i c resistance must be considered i n e v a l u a t i n g the e f f e c t i v e n e s s of a l t e r n a t i v e s other than a complete ban. We have monitored the l e v e l and p a t t e r n of a n t i b i o t i c r e s i s t a n c e i n e n t e r i c b a c t e r i a at p e r i o d i c i n t e r v a l s during the past 13 years. I n i t i a l l y , the e n t e r i c b a c t e r i a of the two herds did not d i f f e r g r e a t l y i n l e v e l or pattern of resistance. Though there had been no s p e c i f i c a l l y planned use of a n t i b i o t i c s in t h e s e r e s e a r c h h e r d s and no one a n t i b i o t i c had been used continuously at e i t h e r l o c a t i o n . a n t i b i o t i c s were used experimentally or as a swine producer would use them. As one would expect, after 13 years of continuous exposure, the l e v e l s of a n t i b i o t i c resistance and l e v e l s of m u l t i p l e resistance are higher i n the c o n t r o l herd. In that herd, t e t r a c y c l i n e r e s i s t a n c e i n the f e c a l c o l i f o r m p o p u l a t i o n s has remained high, a v e r a g i n g more than 90%. T e t r a c y c l i n e r e s i s t a n c e of the f e c a l coliforms in the a n t i b i o t i c - f r e e herd has declined from the i n i t i a l high l e v e l of above 90%; but, the resistance l e v e l s t i l l fluctuates between 20 and 55% even though these animals have had no exposure to a n t i b i o t i c s f o r 13 years. During that time more than 5 complete g e n e r a t i o n t u r n - o v e r s of that herd have occurred. We have monitored the resistance to 14 antimicrobial agents; and, the f e c a l c o l i f o r m s , on the average, are r e s i s t a n t to 1.5 to 2.0 of those agents (table 3). Factors A f f e c t i n g Resistance

to A n t i b i o t i c s

We f i n d that the l e v e l of a n t i b i o t i c r e s i s t a n c e i s r e l a t e d to or i n f l u e n c e d by a number of f a c t o r s i n c l u d i n g age of a n i m a l , with organisms from younger pigs having a higher l e v e l of resistance than that of more mature a n i m a l s . A f t e r 11 years of no a n t i b i o t i c exposure, approximately 55% of the f e c a l coliforms from pigs less than s i x months of age were r e s i s t a n t to t e t r a c y c l i n e ; and, on the average were r e s i s t a n t to 2.9 of the 14 agents t e s t e d . In more mature pigs (6 months or older) the l e v e l of t e t r a c y c l i n e resistance was about 25%; and, on the average, the f e c a l c o l i f o r m s were resistant to about 1.5 agents.

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Table 3· Tetracycline Resistance i n Fecal Coliforms of Pigs i n A n t i b i o t i c - f r e e Herd (No A n t i b i o t i c s Since 1972)

Age of p i g . months

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A l l i s o l a t e s (1972-1982) 2-6 7-11 12-23 23

No. isolates

Tetracycline resistance %

Multiple ^ resistance no ·

977 358 701 370

73 26 35 30

2.5 1.1 1.5 1.5

161 51 47 21

55 24 26 24

2.9 1.6 1.4 1.7

f

Samples. March 83 2-6 7-11 12-23 23

1 Average number of a n t i b a c t e r i a l s to which f e c a l coliforms were resistant· We have found that s t r e s s i n g p i g s by t r a n s p o r t i n g them to another location w i l l increase the l e v e l of resistance and w i l l a l s o increase shedding of l a c t o s e - n e g a t i v e coliforms (including SAlmojifî.lla.). These e f f e c t s are v e r y obvious when the t r a v e l i s q u i t e d i s t a n t ; but. s i m i l a r , though s m a l l e r i n magnitude, e f f e c t s are noted when animals are t r a n s p o r t e d l e s s than 10 m i l e s . For example, pigs were sampled p r i o r to and after being transported 200 miles. The l e v e l of t e t r a c y c l i n e resistance increased from 40% to a l e v e l of 80% as a r e s u l t of the stress of moving. Several days were required f o r the l e v e l s of resistance to return to the p r e v i o u s l y lower l e v e l . We have a l s o noted that stressing pigs by forcing them to walk f o r about 0.5 m i l e s w i l l i n c r e a s e the percentage of f e c a l coliforms resistant to t e t r a c y c l i n e s . Short Term Therapeutic

Use and B a c t e r i a l

Resistance

Exposure of animals to t h e r a p e u t i c l e v e l s of a n t i b i o t i c s w i l l markedly raise the l e v e l of resistance, and a long period of time i s required f o r the resistance l e v e l s to return to the pre-treatment level. I n an e x p e r i m e n t replicated five times ( L a n g l o i s et a l . 1Q.), we found that exposure of animals to subtherapeutic l e v e l s of g r a m - p o s i t i v e spectrum a n t i b i o t i c s a l s o resulted i n increased l e v e l s of t e t r a c y c l i n e resistance. During the course of our study we have sampled pigs on two separate occasions (1974 and 1985) from another research herd i n which a n t i b i o t i c s have never been used as feed a d d i t i v e s , but o n l y f o r t h e r a p e u t i c purposes. Of the 100 p i g s sampled i n 1974, o n l y one had been

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treated and that pig was treated more than a year prior to sampling. A n t i b i o t i c resistance l e v e l s i n that herd were between our two herds with 72% of the f e c a l c o l i f o r m s i n the f i r s t sampling being r e s i s t a n t (by the d i s c p l a t e technique) to t e t r a c y c l i n e (Hays and Muir, &). In 1985» 76% of the f e c a l coliforms were resistant (using the S e n s i t i t r e MIC/ID System, 8 mcg/ml). The r e s u l t s from our s t u d i e s make i t v e r y c l e a r that the p r e v i o u s p r o p o s a l [Fed. Reg. 42:43770 (Aug. 30, 1977) and Fed. Reg. 42:52645 (Oct. 21, 1977), as endorsed i n the p e t i t i o n from the N a t u r a l Resources Defense C o u n c i l (NRDC, Fed. Reg. 49:49645, December 21, 1984)] f o r r e s t r i c t i n g f e e d a d d i t i v e use o f subtherapeutic l e v e l s of p e n i c i l l i n and t e t r a c y c l i n e , would have very l i t t l e , i f any, impact on the l e v e l s or patterns of a n t i b i o t i c resistance i n food-producing animals and the r e s u l t i n g exposure of humans to resistant bacteria of animal o r i g i n . For l i v e s t o c k producers to approach the care we have exercised in i n avoiding a n t i b i o t i c exposure would require a complete ban of a l l a n t i b i o t i c s and other a n t i b a c t e r i a l agents. Periodic exposure to prophylactic or therapeutic l e v e l s of a n t i b a c t e r i a l s would r e s u l t in high l e v e l s of resistance and, i n a production environment, that resistance would be very slow i n d e c l i n i n g . Human H e a l t h

Implications

The r e s u l t s of the Seattle-King County (Nolan et a l . 12.) study are v e r y s u p p o r t i v e of t h e c o n c l u s i o n s we h a v e drawn f r o m our experiments. I n c i d e n c e o f fia 1mone Ί Ί a and Campylobacter contamination and a n t i b i o t i c r e s i s t a n c e l e v e l s of these b a c t e r i a associated with processing plants and r e t a i l meats were as high or higher i n p o u l t r y products than f o r other meats. Of the s t r a i n s i s o l a t e d from r e t a i l p o u l t r y , 33% of the Campylobacter jejuni s t r a i n s and 31% of the Salmone1 l a s t r a i n s showed r e s i s t a n c e to tetracyclines. Since the introduction of the ionophore a n t i b i o t i c s to c o n t r o l c o c c i d i o s i s (1971), there has been l i t t l e , i f any, s u b t h e r a p e u t i c use of t e t r a c y c l i n e s or p e n i c i l l i n i n b r o i l e r production. Furthermore, b r o i l e r production and processing and the manufacturing of b r o i l e r feeds are l a r g e l y separated from any aspect of swine and beef p r o d u c t i o n and p r o c e s s i n g . Thus, i n essence, we have experienced a 15 year, l a r g e s c a l e t e s t on the impact of the proposed actions and found i t to be n i l . In 1971, Great B r i t a i n implemented a ban on subtherapeutic use of t e t r a c y c l i n e s i n animal production. This action was taken after c o n s i d e r a b l e debate and was g r e a t l y i n f l u e n c e d by a n t i b i o t i c r e s i s t a n t Sa.lmOJifi.Hfl. i n f e c t i o n s (S.. t y p h i m u r i u m phage type 29) i n humans i n the mid-1960 s» which appeared to be r e l a t e d to s i m i l a r infections i n calves (Anderson, 2)· A n t i b i o t i c s were not approved as feed a d d i t i v e s f o r c a l v e s at that time nor p r e v i o u s l y . Thus, e a r l i e r implementation of the Swann Committee (1&) recommendations would have had no apparent impact on that p a r t i c u l a r epidemic, nor did i t prevent a s i m i l a r epidemic l a t e r (Rowe et a l . , ϋ ) . Dr. Β. Rowe, Director of the Central Public Health Laboratory, London, participated i n the J u l y 19-20, 1984 International Symposium on Sa lmone 1 l a . i n New Orleans. He was quoted i n Food Chemical News (Aug. 6, 1984, page 17) as f o l l o w s : "the B r i t i s h increase i n cases of s a l m o n e l l o s i s caused by m u l t i p l e - d r u g - r e s i s t a n t forms of S J . f

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typhimurium i s t o t a l l y due to i n j u d i c i o u s use of a n t i b i o t i c s i n a n i m a l husbandry*. I f one accepted t h i s sweeping but unsubstantiated conclusion, then one would l o g i c a l l y conclude that the p r o h i b i t i o n o f s u b t h e r a p e u t i c use i n a n i m a l s of t h o s e a n t i b i o t i c s used i n human medicine has had no impact on the situation in Great B r i t a i n . Smith reported a s l i g h t decrease i n t e t r a c y c l i n e - r e s i s t a n t Escherichia c o l i . but the number of pigs excreting these organisms did not change during the 4-year period a f t e r implementation of the Swann Report recommendations by the B r i t i s h Government. Some have attributed a lack of change i n resistance l e v e l s among the organisms from animals i n Great B r i t a i n to i n j u d i c i o u s use of a n t i b i o t i c s through producers and veterinarians finding ways to circumvent the r e g u l a t i o n s . Our r e s e a r c h would i n d i c a t e that on d i s c o n t i n u i n g subtherapeutic use of a n t i b i o t i c s , i n c i d e n c e of disease problems would increase, thus necessitating an increased need for therapeutic use. Furthermore, p e r i o d i c t h e r a p e u t i c use would r e s u l t i n continued excretion of a n t i b i o t i c resistant organisms. Though our r e s e a r c h experiences are s u p p o r t i v e of a l a c k of impact on a n t i b i o t i c r e s i s t a n c e i n Great B r i t a i n from the withdrawal of subtherapeutic use i n animals, we c e r t a i n l y could not agree with the statement attributed to Rowe that multi-drug-resistant Salmonel l a i n humans i s t o t a l l y due to drug use in animals as Rowe concluded. The recent r e p o r t (Holmberg et a l . , 1) on the ^lmojl£Lllâ. n e w p o r t epidemic i n Minnesota and South Dakota has been h i g h l y p u b l i c i z e d and proclaimed as the "direct link'' or "smoking gun" that t i e s feed a d d i t i v e use of t e t r a c y c l i n e s i n beef c a t t l e to s e r i o u s human health problems. Much speculation was required to l i n k the i n f e c t i v e S a l m o n e l l a to that p a r t i c u l a r beef farm and even further to conclude that subtherapeutic use of a n t i b i o t i c s i n the c a t t l e e i t h e r r e s u l t e d i n the development of the r e s i s t a n c e p a t t e r n or r e s u l t e d i n s e l e c t i n g f o r the organism. The authors c o r r e c t l y acknowledged that the organism was not found on that beef farm nor i n ground beef. They f u r t h e r acknowledged that the r e s i s t a n c e p a t t e r n ( t e t r a c y c 1 ine-ampici 11 i n - c a r b e n i c i l l i n ) of the S- newport organism most l i k e l y d i d not d e v e l o p on that farm. However, the t h e s i s presented to the p u b l i c by the media i s as f o l l o w s : the organism entered that group of beef c a t t l e by some means, possibly a s t r a y c a l f from a neighboring farm, and the use of a t e t r a c y c l i n e gave the S a l m o n e l 1 ^ organism the s e l e c t i v e advantage f o r r a p i d proliferation. In r e a l i t y , i f t e t r a c y c l i n e was used with any r e g u l a r i t y on that farm, the t e t r a c y c l i n e resistant S a l m o n e l l a would have had l i t t l e , i f any, s e l e c t i v e advantage because of i t s t e t r a c y c l i n e r e s i s t a n c e , as the m a j o r i t y of the r e s i d e n t gramnegative organisms would have been r e s i s t a n t to t e t r a c y c l i n e . No f a c t u a l evidence was presented to demonstrate that the resistant S a l m o n e l l a newport organism did gain entrance to that beef herd. I f , however, the organism d i d enter the herd, a s e l e c t i v e advantage could have been provided e q u a l l y as or more l i k e l y by the use of a gram-positive spectrum a n t i b a c t e r i a l agent, a change i n the d i e t of the a n i m a l s , a d i s r u p t i o n of feed intake t r i g g e r e d by inclement weather, or the s t r e s s of t r a n s p o r t i n g those animals to these are mere speculations; but, we have research data to support each of them as factors capable of triggering t h e ' p r o l i f e r a t i o n of a previously asymptomatic i n f e c t i o n .

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The authors (Holmberg et a l . Ζ ) did not r u l e out sources other than that beef herd, f o r the SJ. newport b a c t e r i a . I f one assumes that i n f e c t e d hamburger d i d come through that brokerage f i r m and from that Nebraska p r o c e s s o r , the t o t a l c a r c a s s weight from 5 9 c a t t l e p r o b a b l y would not be as much as 4 1 , 0 0 0 l b s . ( 6 2 % y i e l d on 1 1 0 0 l b . c a t t l e ) . Considerably less than that would have gone into boxed beef and/or hamburger. There was probably no more than 2 9 , 0 0 0 pounds a f t e r f a t , bone and waste t r i m and o n l y about 7 0 0 0 l b . of t h a t , at the most, would be hamburger. Where d i d the other 4 1 , 0 0 0 to 6 3 , 0 0 0 l b s . ( 7 0 , 0 0 0 - 2 9 , 0 0 0 or 7 0 0 0 ) come from? Why wasn't there any human i l l n e s s that c o u l d be l i n k e d to the 4 6 c a t t l e that went through other processors? Could i t be that the o r i g i n a l source of those 5 9 c a t t l e had n o t h i n g to do with the i l l n e s s i n c i d e n t s ? Granted that a l l people infected with Salmonel l a do not become i l l and/or are not i d e n t i f i e d ; but, i t seems h i g h l y u n l i k e l y that one can d i v i d e a group of i n f e c t e d c a t t l e n e a r l y i n h a l f and send one h a l f through one chain and r e s u l t i n 1 2 i d e n t i f i e d cases of i l l n e s s and the o t h e r h a l f t h r o u g h a n o t h e r r o u t e and r e s u l t i n no i d e n t i f i a b l e cases. Wouldn't i t be equally or more l i k e l y that the Salmonel l a originated in the beef that was mixed with that of the 5 9 carcasses? Furthermore, the authors d i d not demonstrate that s u b t h e r a p e u t i c use of t e t r a c y c l i n e was r e l a t e d i n any way to the i l l n e s s of the patients. I f one makes a t h i r d assumption that the i n f e c t i v e Salmonel l a did originate i n the hamburger from those 5 9 carcasses from that South Dakota Farm, the use of a n t i b i o t i c s could have been unrelated to the chain of events. The Salmonella newport s t r a i n i s o l a t e d from i l l p a t i e n t s and a d a i r y c a l f that died on a farm adjacent to the beef farm was r e s i s t a n t to t e t r a c y c l i n e , a m p i c i l l i n and c a r b e n i c i l l i n . A m p i c i l l i n and c a r b e n i c i l l i n are not used i n beef c a t t l e except p o s s i b l y on a prescription basis and not l i k e l y then. The authors did not report i l l n e s s in the beef animals and f u r t h e r they s t a t e : "Thus, the beef herd was p r o b a b l y not the o r i g i n a l source of the R p l a s m i d , but the use of s u b t h e r a p e u t i c t e t r a c y c l i n e i n the herd's feed throughout 1 9 8 2 provided a s e l e c t i v e pressure for persistence of the antimicrobial resistant organism"*. One can r e a d i l y see that the c o n c l u s i o n s of the authors are b a s e d on numerous a s s u m p t i o n s r a t h e r than on f a c t u a l data. Unfortunately, t h i s i s the nature of retrospective epidemiological studies. One cannot r e a l l y f a u l t the authors for their sequence of theories leading them to a possible source of the infections. Such e f f o r t s are necessary f o r s o r t i n g out the p o t e n t i a l source of infections; and, t h r o u g h f o l l o w - u p s t u d i e s and d e s i g n e d experiments, we can determine sources of infections and methods for prevention. They e v i d e n t l y did omit some observations on the meat samples they did check and observations on the adjacent dairy herd; and, they did not take samples from the cattle's environment on the beef farm. I f the beef farm was thought to be the source, there should have been more e f f o r t to find the organism on that farm. A n t i b i o t i c Resistance and Virulence The N a t u r a l R e s o u r c e s D e f e n s e C o u n c i l p e t i t i o n i n f e r s from c a l c u l a t i o n s based on a v e r y s m a l l number of o b s e r v a t i o n s that a n t i b i o t i c resistant Salmonel l a are more v i r u l e n t , hence r e s u l t in

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more deaths than do a n t i b i o t i c - s e n s i t i v e Salmonel l a (Ahmed et al.» 1). C o n t r o l l e d experiments with c h i c k s on the e f f e c t s of the resistance carrying plasmids on v i r u l e n c e of Salmonel l a led Smith (JJL) to the f o l l o w i n g c o n c l u s i o n : "The m o r t a l i t y r a t e s from infection with the R+ (resistant) strains were s i m i l a r to. s l i g h t l y lower t h a n , or much lower t h a n t h o s e from i n f e c t i o n s w i t h corresponding R- ( s e n s i t i v e ) s t r a i n s " . Numerous r e p o r t s support Smith's c o n c l u s i o n s . In the more recent Chicago outbreak of Salmonellosis, which has been attributed to milk contaminated with a t e t r a c y c l i n e - r e s i s t a n t s t r a i n of S a l m o n e l l a typhimurium. there were two deaths v e r i f i e d as r e s u l t i n g from infections with the resistant s t r a i n of Salmonel l a i n 16,284 confirmed cases. If one pools these cases with those cited in the NRDC p e t i t i o n , then the i n c i d e n c e of m o r t a l i t y (0.09%) i s s i m i l a r or than that of persons affected by a n t i b i o t i c - s e n s i t i v e Salmonel l a (0.21%) a l s o c i t e d by NRDC. Thus, the s i m i l a r i t y of r i s k s of human i n f e c t i o n s with r e s i s t a n t and s e n s i t i v e s t r a i n s of Salmone11a agrees with r e s e a r c h data obtained i n c o n t r o l l e d experiments (Smith 15). Reducing

Human Exposure

I do not wish to l e a v e the impression that I'm unaware of the importance of b a c t e r i a l contamination of animal products. There i s no question about the significance of Salmonel l a and Campylobacter jejuni being a problem in human health, and food producing animals serve along with pets, humans, w i l d l i f e and other foods as a part of the t o t a l Salmone1 l a - Campylobacter r e s e r v o i r . There are v e r y serious questions, however, regarding the impact of a n t i b i o t i c usage i n animals on a n t i b i o t i c r e s i s t a n c e i n humans. There i s a great deal of research evidence to indicate that the action proposed, to r e s t r i c t certain uses of t e t r a c y c l i n e s and p e n i c i l l i n , would have e s s e n t i a l l y no impact on human exposure to a n t i b i o t i c r e s i s t a n t organisms. Implementing t h e p r o p o s e d a c t i o n w o u l d h a v e an adverse impact on the production costs of animal products. We do need means f o r markedly r e d u c i n g or c o m p l e t e l y e l i m i n a t i n g b a c t e r i a l contamination. An e a r l i e r CDC r e p o r t (Holmberg et a l . , 1984b) indicates there are added r i s k s associated with consumption of raw meats and milk. Thus, using pasteurized milk only and proper h a n d l i n g and cooking of meats are important p r e c a u t i o n s f o r preventing food borne i l l n e s s e s . Other useful means for reducing the health r i s k s from b a c t e r i a l contaminants of animal products and other foods include p u b l i c education on personal hygiene of a l l food h a n d l e r s and a p p r o p r i a t e cooking and h a n d l i n g techniques f o r a l l foods·

Literature Cited 1. Ahmed, A. K., S. Chasis and B. McBarnette. "Petition of the Natural Resources Defense Council, Inc. to the Secretary of Health and Human Services requesting immediate suspension of approval of the subtherapeutic use of penicillin and tetracyclines in animal feeds"; Nov. 20. NRDC, New York, NY. 1984.

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2. Anderson, E.S. "Observations on e c o l o g i c a l e f f e c t s of antibacterial drugs"; in Dunlop, R. H. and Moon, H. W., Eds.; Resistance to Infectious Disease, Saskatoon Modern Press, Saskatoon, Saskatchewan, 1970, pp 157-172. 3.

CAST. "Antibiotics in Animal Feeds"; Council for Agricultural Science and Technology. Ames, IA. Rpt. No. 88, 1981.

4.

F a g e r b e r g , D. J., Q u a r l e s , C. L.; M c K i n l e y , G. A. Antibiotic resistance and its transfer ; Feed Management June, 1979, pp 32, 34 and 37.

5. Hays, V. W. " E f f e c t i v e n e s s of Feed A d d i t i v e Usage of Antibacterial Agents in Swine and Poultry Production"; Rachel le Lab., Long Beach, CA. 1977. 6.

Hays, V. W.; Muir, W. M. Efficacy and safety of feed additive use of antibacterial drugs in animal production. Can. J. Animal Sci., 1978, 59,447.

7. Holmberg, S. D.; Osterholm, M. T.; Senger, Κ. Α.; Cohen, M. L. Drug resistant Salmone11a from animals fed antimicrobials. New England Journal of Medicine. 1984, 311,617. 8.

Holmberg, S. D.; Wells, J. G.; Cohen, M. L. Animal-to-man t r a n s m i s s i o n of antimicrobial-resistant Salmonella: Investigations of U. S. Outbreaks, 1971-1983. Science 1984, 225 ,833.

9.

Langlois, Β. E.; Cromwell, G. L.; Hays, V. W. Influence of chlortetracycline in swine feed on reproductive performance and on incidence and persistence of antibiotic resistant enteric bacteria. J. Animal Sci., 1978, 46, 1369.

10. Langlois, B. E.; Cromwell, G. L.; Hays, V. W. Influence of type of antibiotic and length of antibiotic feeding period on performance and persistence of antibiotic resistant enteric bacteria in growing finishing swine. J. Animal Sci., 1978, 46, 1383. 11. Levy, S. B. Playing antibiotic pool: Time to t a l l y the score. New England Journal of Medicine. 1984, 311, 663. 12. Nolan, C. M.; Harris, Ν. V.; Canova, P. M.; Skillman, S. M.; Cain-Nelson, A. K.; Tenover, F. C.; Coyle, M. B.; Plorde, J. J.; Weiss, N. S.; Martin, D. C. Surveillance of the Flow of Salmonel la and Campylobacter in a Community. Prepared for the U. S. Department of Health and Human Services, Public Health Service, Food and Drug Administration, Bureau of Veterinary Medicine. Contract No. 223-81-7041. Communicable Disease Control Section, Seattle-King County (Washington) Department of Public Health. 1984.

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13· Rowe, B.; Threlfall, E. J.; Ward, L. R.; A s h l e y , A. S. International spread of multiresistant strains of Salmonella typhimurium phage types 204 and 193 from Britain to Europe. Vet Record. 1979, 105. 468. 14. Smith, H. W. "Antibiotic resistant bacteria and associated problems before and after the 1969 Swann Report". in Woodbine, M. Ed. Antibiotics and Antibiosis in Agriculture. Butterworths, London. 1977.

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15. Smith, H. W. The effect on virulence of transferring R factors to Salmonella typhimur ium i n vivo._J. Med. Microbiol. 1972, 5, 451. 16. Sun, M. In search of Salmonella's smoking gun. Science 1984, 226, 30. 17. Sun, M. Use of antibiotics in animal feed challenged. Science. 1984, 226, 144. 18. Swann, M. M. Report of Joint Committee on the Use of Antibiotics in Animal Husbandry and Veterinary Medicine. Cmnd. 4198, Her Majesty's Stationery Office, London. 1969. RECEIVED May 5, 1986

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8

Significance of Antibiotics in Foods and Feeds

Khem M. Shahani and Paul J. Whalen

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch008

Department of Food Science and Technology, University of Nebraska, Lincoln, NE 68583-0919

The use of a n t i b i o t i c s in livestock production for increased feed e f f i c i e n c y i s widespread. Such use may i n d i r e c t l y access human food i n the form of residuals i n products such as meat and milk. For years, scient i s t s and health o f f i c i a l s have warned of the risk of developing resistant pathogens from feeding a n t i b i o t i c s to livestock. Recently, this concern was fueled by a f a t a l case of salmonellosis caused by an a n t i b i o t i c resistant s t r a i n linked to meat. F a i l u r e to withhold milk produced after treating mastitic or otherwise diseased animals i s the primary cause of a n t i b i o t i c s i n milk. L a c t i c cultures produce inherent a n t i b i o t i c s active against a wide range of pathogenic and nonpathogenic bacteria. The benefits of the naturally occurring a n t i b i o t i c s produced i n fermented milk products are being investigated for use i n treating E. c o l i mediated diarrhea and Salmonella/Shig e l l a dysentery i n children. Although the economic advantages of incorporating a n t i b i o t i c s i n animal feed for livestock production are massive, the r i s k to the consumer must be weighed against such advantages.

The advent of a n t i b i o t i c s began a new era i n the treatment of human and animal disease. I n i t i a l l y , a n t i b i o t i c s were used i n a therapeutic mode only. However, i n the early 1950 s, i t was discovered that the residual mash from chlortetracycline production produced a growth promoting effect on chicks which was l a t e r attributed to low levels of the a n t i b i o t i c present i n the mash. Use of a n t i b i o t i c s i n feeds for animal production has grown to account for about half of over 35 m i l l i o n lbs produced annually i n the U.S. (1). Concern that t h i s extensive use may be compromising human therapeutic use i s mounting due to increasing prevalence of multiply a n t i b i o t i c resistant i n t e s t i n a l bacteria, not only i n the f l o r a of the treated animal, but i n the producer personnel as well. Of p r i n c i p a l concern are the complications presented by pathogens such as salmonella whose DNA containing plasmids (R factor) allow acquisition of mul0097-6156/ 86/ 0320-0088506.00/ 0 © 1986 American Chemical Society

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t i p l e resistance and present serious, p o t e n t i a l l y f a t a l , upon transmission to humans.

illness

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A n t i b i o t i c s i n Foods and Feeds A n t i b i o t i c residuals i n food products are considered additives by the FDA. Therefore, such products are considered adulterated and r e s t r i c t e d from interstate commerce. In addition, widespread use of a n t i b i o t i c s for food production may pose the r i s k of toxic or a l l e r g i c reactions i n sensitized individuals. Cultures employed i n fermented dairy products such as cheese or yogurt are extremely susceptible to low levels of a n t i b i o t i c s i n the milk supply where production schedules may be thrown off and product losses incurred due to the presence of a n t i b i o t i c s . A l t e r n a t i v e l y , some l a c t i c cultures synthesize natural a n t i b a c t e r i a l , a n t i b i o t i c - l i k e compounds active against a wide range of pathogenic and nonpathogenic bact e r i a . In view of some adverse effects of a n t i b i o t i c feeding, use of these probiotics i s being promoted. Several countries other than the U.S. permit the use of n i s i n i n dairy products as a bacteriocin which i s not therapeutically employed i n man or animals. A n t i b i o t i c resistant microorganisms. Livestock production employs a n t i b i o t i c s i n 3 basic manners (2): 1) Therapeutic - treatment of disease and i n f e c t i o n 2) Prophylaxis - disease prevention i n healthy animals 3) Growth promotion - continual subtherapeutic or low doses of a n t i b i o t i c s i n normal, healthy animals for improved production (growth rate and/or feed e f f i c i e n c y ) . Therapeutic levels of a n t i b i o t i c s range from 200 - 500 g/ton while prophylactic applications range from 50 - 200 g/ton and subtherapeutic levels i n feed range from 1 - 5 0 g/ton for poultry, swine and calves (3^). I t i s the long terra or continual incorporation of a n t i b i o t i c s i n feeds which concerns many s c i e n t i s t s i n that many of the a n t i b i o t i c s used for production purposes are also used for therapy i n man. As w i l l be discussed, the resistant organisms which result from a n t i b i o t i c s i n feeds can subvert, potenti a l l y , therpeutic application of these drugs should the resistant organisms present a pathogenic s i t u a t i o n i n man. The presence of a n t i b i o t i c resistant i n t e s t i n a l organisms res u l t i n g from the use of a n t i b i o t i c s i n feeds i s well established (4_, 5_, 6). Wanatabe (7) reported on the t r a n s f e r a b i l i t y of this t r a i t and Anderson and Lewis (8) showed the transfer of a n t i b i o t i c resistance between species involving Salmonella typhimurium. Siegel et a l . (5) showed the effect of a n t i b i o t i c feeds on the number of a n t i b i o t i c resistant IS. c o l i i n swine, poultry and c a t t l e as compared to range c a t t l e . As shown i n Table I, extremely high percentiles of resistant cultures were found i n the animals on feed versus very low levels i n the range c a t t l e . The spread of these resistant f e c a l f l o r a to man has been demonstrated by Levy et a l . (6). In this study, the farm personnel acquired a s i g n i f i c a n t l y higher population of tetracycline resistant coliforms (mainly 15. c o l i ) as compared to neighboring participants not employing a n t i b i o t i c supplemented feed. Multiple resistance i n the bacteria

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Table I, E f f e c t Consuming of A n t i b i o t i c Supplemented Feeds on the Incidence of Resistant E. c o l i % Resistant Antibacterials

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Oxytetracycline Dihydrostreptomycin Ampicillin Neomycin Sulfamerazine

Illinois Swine 89.8 93.2 52.5 20.5 82.9

Illinois Poultry 59 72 17 0 21

Illinois Beef 49.1 50.0 13.2 12.3 29.2

Montana Range Cattle* 0 0.6 1.3 0 0.6

*Range c a t t l e , minimally exposed to a n t i b i o t i c s , served as the control. was noted for both human and animal subjects and linked with the tetracycline plasmid. The animal to human link has been reported i n a number of studies. Holmberg et a l . (9) summarized 52 outbreaks of salmonella investigated by the Centers for Disease Control from 1971 to 1983 of which food animals were linked with 69% of the outbreaks involving a n t i b i o t i c resistant salmonella. For 38 outbreaks of confirmed mode or source (Table I I ) , multiply resistant salmonella were involved i n 33% of the community based outbreaks, 40% of the nosocomial, and 75% of the outbreaks i n volving both community and hospital. In addition, i t has been estimated that each culture-documented case among human beings may represent as many as 100 undocumented cases (10). Agency part i c i p a t i o n for these cases was made at the request of the l o c a l health o f f i c i a l s and therefore the survey was not random. However, the resistant strains displayed a f a t a l i t y rate 21 times that of a n t i b i o t i c sensitive strains. O'Brien et a l . (10) found that characterization of plasmid DNA from a n t i b i o t i c resistant salmonella suggests extensive commingling of human and animal bacteria. Table I I . Outbreaks of Salmonellosis Between 1971 and 1983 Reported by CDC

Source Food Animals or Products Food Services Other Sources Person to Person

Number of Outbreaks* Community Nosocomial Both 7/12 3/4 1/2 1/10 0/2 0

0/1 1/2 2/5

0 0 0

Resistant Strains 61% 9% 25% 40% 39.5%

Mean % Resistant Strains 33% 40% 75% Adapted from: Holmberg et a l . (9) *Resistant s t r a i n s / t o t a l outbreak for cases of s p e c i f i c source or mode of transmission.

Evidence for the demonstration of the link of subtherapeutic use of a n t i b i o t i c s i n animal feeds to severe disease i n humans was

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch008

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Antibiotics in Foods and Feeds

proposed i n the highly publicized Minnesota outbreak involving a multiply resistant Salmonella nevport (11). The provision of evidence linking the subtherapeutic use of a n t i b i o t i c s i n feeds to human disease was not conclusive and the proof of o r i g i n as well as meat samples confirming the source for the j>« newport were not clearly established. However, this study c l e a r l y points out the risks involved with establishing multiply resistant enteric pathogens which access human food products. This study combines the l i b e r a l manner i n which a n t i b i o t i c s are used (prescribed or not) by the general public for cold-type symptoms with the onset of a severe disease state i n an otherwise benign and common i l l n e s s . Of the 18 cases investigated, 11 were hospital i z e d f o r an average of 8 days and 10 of these were taking a n t i b i o t i c s prior to the onset of salmonellosis. For the single f a t a l i t y among these cases, the multiply resistant salmonella compromised a n t i b i o t i c therapy where a systemic salmonella i n f e c t i o n occurred. Salmonella i s considered an inherent defect i n raw meats and therefore i s not considered an adulterant since the ultimate use by the comsumer involves cooking which destroys the organism. Concern that salmonella and other common food borne pathogens may be presenting an increased r i s k to human health v i a a n t i b i o t i c resistance prompted contracted studies recommended by the National Research Council i n 1980. Results of these studies (12) showed widespread multiply resistant Salmonella, Campylobacter, Staphylococcus species and other pathogens among livestock on farms, i n slaughter houses and on the meat i n the U.S. Their transmission to man appears equally evident. Whether the organisms are more virulent has not been shown but certainly they have an advantage when a n t i b i o t i c s are employed to which they are resistant. As has been discussed, the use of a n t i b i o t i c s selects f o r resistant organisms i n the intestine and while these organisms may not be pathogenic ( i . e . - IS. c o l i ) they act as reservoirs of resistant plasmids which can be transferred to pathogenic or nonpathogenic organisms a l i k e . Prior to the a n t i b i o t i c era, was a n t i b i o t i c resistance as prevalent? Some l i g h t has been shed on this question by the work of Hughes and Datta (13). These authors studied the Murray c o l l e c t i o n of Enterobacteriaceae strains collected from 1917 to 1954. They tested 692 strains from 433 i s o l a t e s of which almost a l l were from human infections. Nineteen percent of the strains contained conjugative plasmids but none of the plasmids showed a n t i b i o t i c resistance. This work strongly indicates that the mass emergence of resistant strains i s a product of the a n t i b i o t i c era. Residual a n t i b i o t i c s . With the widespread use of a n t i b i o t i c s i n feeds the occurrence of residuals i n milk, meat and eggs becomes inevitable. These residuals result primarily from f a i l u r e by the producer to adhere to adequate withdrawal periods following the use of the a n t i b i o t i c s . In a review by Katz C3), residual a n t i b i o t i c s were found i n a l l animal species marketed i n 1976 - 1978.

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The lowest incidence of v i o l a t i v e residuals were found i n poultry and c a t t l e , with swine and calves having the highest. A n t i b i o t i c s i n milk result primarily from f a i l u r e to withhold milk from the market after therapeutic treatment of mastitic or otherwise diseased animals. The exposure to a n t i b i o t i c residuals i n food products i s an uncontrolled and involuntary s i t u a t i o n . Ingestion of these admittedly low dose levels has been of concern due to potential for s e n s i t i z a t i o n and/or a l l e r g i c reaction to the a n t i b i o t i c (14). About 10% of the general population exhibit an adverse reaction to p e n i c i l l i n (15). The p o s s i b i l i t y of primary s e n s i t i z a t i o n to a n t i b i o t i c s v i a food products, s p e c i f i c a l l y from p e n i c i l l i n i n milk, was e s s e n t i a l l y ruled out by Dewdney and Edwards (16). Their conclusion rests on the improbability of the low levels encountered i n milk (0.01 microgram/ml) as being capable of s e n s i t i z i n g an i n d i v i d u a l . However, i n view of evidence that low dose immunization can favor antibody production (17), these residual levels could be capable of s e n s i t i z a t i o n (30. Upon s e n s i t i z a t i o n the dose required to e l i c i t an a l l e r g i c reaction i s highly dependent upon the individual but may be as l i t t l e as 40 IU (0.024 mg) i n a highly sensitized person. Dewdney and Edwards (16) c i t e the paucity of cases i n the l i t e r a t u r e to warrant the concern given to p e n i c i l l i n residues i n milk and question the v a l i d i t y of those that are. However, given the widespread use and misuse of p e n i c i l l i n and the potential for reaction i n the above estimated 10% of the population, continued concern appears warranted. A n t i b i o t i c s i n milk can affect dramatically the production of fermented dairy products such as cheese, yogurt, buttermilk and sour cream. Routine application of a n t i b i o t i c test k i t s such as the Delvo k i t are required to avoid major losses on the l i n e . Many of the organisms employed are extremely sensitive to a n t i b i o t i c residuals i n the milk. As shown i n Table I I I , as l i t t l e as 0.05 to 1.0 IU/ml of p e n i c i l l i n and 0.05 to 10.0 microgram/ml of aureomycin inhibited the growth of 19 cheese starter cultures (18). Lower levels are capable of affecting the flavor and texture properties of the product (14, 19) as well as promoting the growth of undesirable a n t i b i o t i c resistant coliforms (14, 20). Benefits of A n t i b i o t i c s Modern methods of livestock production are intensive and the environmental conditions stress the animals. The use of a n t i b i o t i c s promotes growth and protects the animals from otherwise certain i n f e c t i o n under these conditions. A n t i b i o t i c - l i k e compounds formed i n l a c t i c acid fermentations prevent p r o l i f e r a t i o n of spoilage and pathogenic microorganisms and increase the shelf l i f e of the products. N i s i n i s a antimicrobial produced by a l a c t i c acid bacterium and i s used i n some countries as a food preservative. Some l a c t i c acid bacteria are capable of favorably influencing the f e c a l f l o r a i n man and animals. Livestock production. The growth promoting attributes of a n t i b i o t i c s i n feeds reside i n their a n t i b a c t e r i a l a c t i v i t y i n the

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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Table I I I .

WHALEN

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Minimum Inhibitory Levels of P e n i c i l l i n and Aureomycin for Common Dairy Starter Cultures

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch008

1

Aureomycin Penicillin yg/ml U/ml Cultures 1.0 0.05 Lactobacillus l a c t i s A 1.0 0.05 Lactobacillus l a c t i s B 3.0 0.05 Lactobacillus l a c t i s VI04 0.5 0.30 Lactobacillus l a c t i s 431 1.0 0.30 Lactobacillus l a c t i s kw 0.3 0.05 Lactobacillus l a c t i s V109 1.0 0.05 Lactobacillus l a c t i s , myc 3.0 0.10 Lactobacillus bulgaricus 488 5.0 0.10 Lactobacillus bulgaricus 444 3.0 0.30 Lactobacillus bulgaricus R 2.0 0.20 Lactobacillus bulgaricus V71 0.3 0.05 Lactobacillus bulgaricus VI2 10.0 1.00 S and R 0.3 0.05 Streptococcus thermophilus H 0.3 0.05 Streptococcus thermophilus T 0.05 0.05 Streptococcus l a c t i s 9 0.05 0.05 Lactobacillus casei 0.2 0.10 Streptococcus durans 0.05 0.05 Micrococcus 8406 •••Adapted from: Shahani and Harper (18). ^Commercial Mixed Culture Containing L. l a c t i s and L. bulgaricus. 2

i n t e s t i n e . This i s corroborated by the lack of improvement i n growth of germ-free chicks fed a n t i b i o t i c supplemented feed (21). The improvement i n growth i s probably a combined effect of the a n t i b i o t i c on the f l o r a and the small i n t e s t i n e . A n t i b i o t i c fed chicks showed h i s t o l o g i c a l changes i n the intestine similar to those for germ-free chicks as well as shorter and thinner walled i n t e s t i n e s . Nutrient uptake i s believed to be enhanced by these changes (3). In ruminants, growth promotion i s due to increased propionate producing bacteria by selective i n h i b i t i o n of competing f l o r a , decreased microbial protein, decreased rumen solids and d i l u t i o n rate and increased metabolizability (not d i g e s t i b i l i t y ) (2). Other contributing factors may be suppression of mild but unrecognized infections, reduced microbial destruction of essent i a l nutrients, reduced microbial toxins and synthesis of v i t a mins or other growth promoting factors which become available subsequently to the animal. Naturally occurring a n t i b i o t i c s i n foods. L a c t i c acid producing bacteria have been selected h i s t o r i c a l l y for food preservation i n fermented foods such as sausage, cheese, yogurt, sauerkraut etc. Their p r i n c i p l e mode of a n t i b i o s i s i s through the rapid production of organic acid (mainly l a c t i c ) and the associated lowering of the pH. Other metabolites such as hydrogen peroxide also havebeen recognized as factors i n the prevention of spoilage of f e r mented products. The consistent use of high numbers of l a c t i c acid bacteria i n fermented meats provides for a rapid fermentation which precludes the p r o l i f e r a t i o n of pathogens such as

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Staphylococcus aureus, Salmonella spp. and Clostridium botulinum (22). In the case of the l a t t e r , the combination of l a c t i c s t a r t e r with either sucrose or dextrose has proven e f f e c t i v e i n preventing toxin production even without n i t r i t e C23, 240 • Table IV i l l u strates the i n h i b i t o r y effect of l a c t i c cultures on the growth and production of enterotoxin by staphylococci i n dry sausage (25).

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch008

Table IV.

I n h i b i t i o n of Pathogenic Staphylococci L a c t i c Cultures i n Sausage

Storage, 3 da Sausage Toxin Log CFU pH formulation + 8.84 5.9 Without l a c t i c starter With l a c t i c 6.78 5.6 starter Adapted from: Niskanen and Nurmi (25)

-

by

Storage, 7 da pH Toxin CFU

Log

8.88

5.7

+

7.53

5.3

-

The provision of l a c t o b a c i l l i as therapy for acute diarrhea brought on by enteropathogenic IS. c o l i (EEC) has been demonstrated i n v i t r o and i n vivo (26). G i l l i l a n d and Speck (27) found a high degree of i n h i b i t i o n of Salmonella typhimurium, Staphylococcus aureus and EEC when grown i n associative culture with Lactobacillus acidophilus (Table V); they attributed the i n h i b i tory action to l a c t i c acid, hydrogen peroxide and other i n h i b i t o r y compounds. The i n h i b i t o r y compounds have been studied further and isolated (28 -_38). They are l i s t e d i n Table VI. The product i o n of these a n t i b i o t i c s generally required s p e c i f i c media and growth conditions. Recent work i n our laboratory investigated the effect of L. acidophilus on Staphylococcus aureus under yogurt production conditons (39). We found the i n h i b i t i o n due to the acid. Hydrogen peroxide production did not account for the t o t a l i n h i b i t i o n observed i n the yogurt. Table V.

Test Culture

s. aureus s.

typhimurium

E. c o l i

Adapted from:

I n h i b i t i o n of Pathogens by L. acidophilus i n Associative Culture Treatment

Pathogen (CFU/ml), 1.5 X T o 2.7 X 1.7 X 10 c 2.3 X 3.3 X 4.3 X 10 7

Control L. acidophilus Control L. acidophilus Control L. acidophilus G i l l i l a n d and Speck

Inhibition (%) 98.2

6

86.5 87.0

6

(27)

Shahani and coworkers (29, ^4, 40, 41_) studied a c i d o p h i l i n and bulgarican from s p e c i f i c strains of L. acidophilus and L. bulgaricus, respectively. These compounds were of low molecular weight and demonstrated a wide range of a c t i v i t y against gram negative and gram positive organisms. Table VII shows the results

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95

Table VI. Natural A n t i b i o t i c s Produced by L a c t i c Cultures

Culture Lactobacillus acidophilus

Lactobacillus brevis

Compound Acidolin Acidophilin Lactocidin Lactobacillin (H 0 ) Lactobrevin Bulgarican Lactolin Diplococcin Nisin 2

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch008

Lactobacillus Lactobacillus Streptococcus Streptococcus

Table VII.

bulgaricus plantarum cremoris lactis

Reference (28) (29) (30)

2

(31-32) (33) (34) (35) (36) (37-38)

In V i t r o A n t i b a c t e r i a l Spectrum of A c i d o p h i l i n

1

2

IC (ug/ml) Strain Test Organism 30 ATCC 6633 Bacillus subtilis 29 Difco 902072 B a c i l l u s cereus 43 ATCC 7954 B a c i l l u s stearothermophilus 45 ATCC 8043 Streptococcus f a e c a l i s 42 ATCC 4532 Streptococcus f a e c a l i s var. liquefaciens 30 NUC Streptococcus l a c t i s 6 40 LY-3 France 7 Lactobacillus l a c t i s 42 ATCC 7469 Lactobacillus casei 8 60 ATCC 8014 Lactobacillus plantarum 9 59 ATCC 7830 Lactobacillus leichmannii 10 30 ATCC 9341 11 Sarcina lutea 29 NU 12 Serratia marcescens 32 NU Proteus vulgaris 13 32 NU 14 Escherchia c o l i 30 ATCC 167 15 Salmonella typhosa 30 ATCC 417 16 Salmonella schottmulleri 30 ATCC 934 17 Shigella dysenteriae NU (coagulase + ve) 50 18 Staphylococcus aureus 60 Phage 80/81 19 Staphylococcus aureus 60 ATCC 9997 20 K l e b s i e l l a pneumoniae 30 ATCC 9459 21 Vibrio comma Adapted from: K i l a r a and Shahani (41). 2lC - concentration i n h i b i t i n g 50% ofgrowth. 5 0

No. 1 2 3 4 5

x

5 0

of i n v i t r o a n t i b a c t e r i a l a c t i v i t y f o r a c i d o p h i l i n against pathogenie and nonpathogenic organisms (41). Beyond the potential product s t a b i l i t y against pathogens imparted by these compounds, the possible health benefits to the consumer have not been shown. N i s i n i s an antimicrobial elaborated by Streptococcus l a c t i s N. Due to the fact that n i s i n i s not used f o r human or animal therapy or as a feed additive and growth promotor, i t s use i n food i s per-

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mitted i n over 35 countries (42). L i t t l e or no d e f i n i t i v e i n f o r mation i s available concerning the probability of the development of cross-resistance between n i s i n and other medically important a n t i b i o t i c s . However, use of n i s i n i s not permitted for food i n the U.S. or Canada. N i s i n i s not inhibitory to yeasts, fungi, or gram negative organisms but i s active against several gram positive streptococci, l a c t o b a c i l l i , C l o s t r i d i a , staphylococci and b a c i l l i (43, 44). For successful application of n i s i n to a food product, Hurst (42) suggests that the food be a c i d i c and that the spoilage organims of concern be gram positive. N i s i n was f i r s t used e f f e c t i v e l y to prevent gas defects caused by Clostridium butyricum i n Swiss cheese (45). The use of n i s i n as a potential adjunct to or replacement of n i t r i t e i n simulated hams was suggested by Rayman et a l . (46). These investigators found that due to an additive e f f e c t , the n i t r i t e l e v e l could be reduced from 150 ppm to 40 ppm while retaining color s t a b i l i t y and preservation q u a l i t i e s . However, they reported also the i n s t a b i l i t y of the n i s i n after storage at low temperatures followed by temperature abuse. The higher levels of n i s i n needed to compensate for these factors may make this use uneconomical according to Hurst (42). Natamycin (Pimaricin) has been approved for use on the surface of cheese and cheese s l i c e s for mold i n h i b i t i o n (47). Shahani (48) found that natamycin treated cheeses inoculated with toxigenic molds e f f e c t i v e l y prolonged the shelf l i f e . Summary The widespread use of a n t i b i o t i c s i n current livestock production methods offers clear cut advantages i n terms of growth and e f f i ciency. However, i n view of the increased incidence of multiple a n t i b i o t i c resistant organisms i n the food products, the risks imparted by resistant food borne pathogens i s of concern to human health. The routes of transfer are varied and may result from routine farm contact with animals and feed, direct transfer from animal to man, or be as complex as transfer from manure to plant f l o r a to man or animal. Evidence of this last mentioned route has been presented by Levy (49). The sheer volume of largely unmanaged or untreated f e c a l material generated from livestock production v i r t u a l l y assures transfer to humans. By comparison, human use of a n t i b i o t i c s i s estimated at less than 1% of animal use, with animals producing manure at a rate up to 400 times that of man (1_)• Nonetheless, r e s t r i c t i o n s on the subtherapeutic use of a n t i b i o t i c s i n feeds should be met with an equal scrutiny of human therapeutic abuse. L i b e r a l prescribing of these valuable drugs, largely to prevent secondary b a c t e r i a l infections following v i r a l cold-type infections, i s a useless remedy and serves to exacerbate the human health question concerning infections involving resistant organisms. That resistant pathogens may complicate human therapeutic application i s a fact. These resistant organisms threaten application of a n t i b i o t i c s deemed v i t a l i n human treatment. The course that industry and government set must balance food production objectives with long term public health considerations. More prudent use of a n t i b i o t i c s i n the feed industry

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

8.

SHAHANI AND

WHALEN

97

Antibiotics in Foods and Feeds

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as well as i n human medicinal practice i s required i n order to e f f e c t i v e l y reserve a n t i b i o t i c s for the treatment of serious microbial disease i n humans. The human microflora can be influenced e f f e c t i v e l y by ingestion of l a c t o b a c i l l i . Work i n our laboratory has shown a severe reduction of coliforms i n the human stool by l a c t o b a c i l l i . These organisms present a highly corapetative environment for salmonella and other pathogens. L a c t o b a c i l l i have a s t a b i l i z i n g effect on the i n t e s t i n a l f l o r a and, i f used with proper consideration, could act to prevent s h i f t s i n the i n t e s t i n a l microflora that favor disease from resistant pathogens. This application could find a role i n c o n t r o l l i n g the buildup of a n t i b i o t i c resistant organisms i n farm personnel who are routinely i n contact with a n t i b i o t i c supplemented feeds. Research on a n t i b i o t i c s which do not produce multiple resistance i s , of course, desirable. N i s i n appears to present just such a situation although i t s applications are narrow. Some feed additives are capable of "curing" multiple resistance i n enterics (50). This area deserves more research and emphasis.

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Armstrong, D. G. In "Antimicrobials and Agriculture"; Woodbine, M., Ed.; Butterworths: London, 1984; p. 31.

3.

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Siegel, D.; Huber, W. G.; Enloe, F. Antimicrob. Agents Chemother. 1974, 6(6), 697-701.

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Holmberg, S. D.; Wells, J . G.; Cohen, M. L. Science 1984, 225, 833-835.

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O'Brien, T. F.; Hopkins, J . D.; Gilleece, E. S.; Medeiros, A. A.; Kent, R. L.; Blackburn, B. O.; Holmes, M. B.; Reardon, J . P.; Vergeront, J . M.; Schell, W. L.; Christenson, E.; Bisset, M. L.; Morse, E. V. N. Engl. J . Med. 1982, 307(1), 1-6.

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Holmberg, S. D.; Osterholm, M. T.; Senger, K. A.; Cohen, M. L. N. Engl. J . Med. 1984, 311(10), 617-622.

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12.

"Report on the Status of the Food and Drug Administration s Proposed Withdrawal of Approvals for Low Level uses of P e n i c i l l i n and Tetracyclines i n Animal Feeds," Subcommittee on Agriculture, Rural Development and Related Agencies, 1985.

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Atkinson, N. F. In "The Effects on Human Health of Subtherapeutic Use of Antimicrobials i n Animal Feeds," National Academy of Sciences, Washington, D.C., 1980.

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Shahani, K. M.; Harper, W. J . 53-54.

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K a s t l i , P.

21.

Coates, M. E. In "Growth i n Animals"; Lawrence, T. L. J . , Ed.; Butterworths: London, 1980; p. 175.

22.

Dacus, J . N.; Brown, W. L.

23.

Christiansen, L. N.; Tompkin, R. B.; Shaparis, A. B.; Johnston, R. W.; Kautler, D. A. J . Food S c i . 1975, 40, 488-490.

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Tanaka, N.; Traisman, E.; Lee, M. H.; Cassens, R. G.; Foster, E. M. J . Food Protection 1980, 43, 450-457.

25.

Niskanen, A.; Nurmi, E.

26.

Sandine, W. E.; Muralidhara, K. S.; E l l i k e r , P. R.; England, D. C. J . Milk Food Technol. 1972, 35(12), 691-702.

27.

G i l l i l a n d , S. E.; Speck, M. L. 820-823.

28.

Hamdan, I. Y.; Mikolajcik, E. M. 631-636.

29.

Shahani, K. M.; V a k i l , J . R.; K i l a r a , A. J . 1977, 11(4), 14-17.

Nature 1983, 302, 725-726.

" A n t i b i o t i c s i n Milk", A. A. Balkema:

Rotterdam,

New

Milk Prod. J . 1958, 49, 15-16,

J . Dairy Res.

1949, 16, 235-241.

Schweiz. Arch. T i e r h e i l k .

1948, 90, 685-695.

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Appl. Microbiol.

1981, 35, 74-78.

1976, 34, 11-20.

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Cult. Dairy Prod.

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33.

Kavasnikov, E. I.; Sodenko, V. I. Mikrobiol. Zh. Kyviv. 1967, 29, 146; Dairy Science Ab. 1967, 29, 3972.

34.

Reddy, G. V.; Shahani, K. M.; Friend, B. A.; Chandan, R. C. Cult. Dairy Prod. J . 1983, 18(2), 15-19.

35.

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37.

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Attaie, R.; Whalen, P. J . ; Shahani, K. M.; Amer, M. A. "Presented at the 80th Annual Meeting of the American Dairy Science Association", Urbana, IL, 1985.

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Shahani, K. M.; V a k i l , J . R.; K i l a r a , A. J . 1977, 12(2), 8-11.

41.

K i l a r a , A.; Shahani, K. M. J . Dairy S c i .

42.

Hurst, A. In "Antimicrobials i n Foods"; Branen, A. L.; Davidson, P. M., Eds.; Marcel Dekker, Inc.: New York, 1983; p. 327.

43.

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1962, 45, 827-832.

R E C E I V E D May 28, 1986

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

9

Risks

to

Human

Health

from

the

Use

of

Antibiotics

in Animal Feeds

Philip J. Frappaolo

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch009

Center for Veterinary Medicine, U.S. Food and Drug Administration, Rockville, MD 20857

Since 1969, the Food and Drug Administration's Center for Veterinary Medicine (formerly the Bureau of Veterinary Medicine) has had cause for concern that the subtherapeutic use of a n t i b i o t i c s i n animal feeds may cause bacteria i n animals to become resistant to a n t i b i o t i c s . This resistance to a n t i b i o t i c s i s said by many knowledgeable s c i e n t i s t s to be transferred to bacteria i n humans, thus making these a n t i b i o t i c s i n e f f e c t i v e i n treating human b a c t e r i a l infections due to compromise of therapy. For this reason, FDA proposed i n 1977 to withdraw the use of p e n i c i l l i n i n animal feed and r e s t r i c t the use of the tetracyclines (chlortetracycline and oxytetracycline) to certain uses i n animal feed. This talk w i l l focus on FDA's e f f o r t s to f i n a l i z e i t s review of the issue and present an update on the current status of the 1977 proposals.

In a l e t t e r to Science i n 1980 (1), U.S. Representative John Dingell (D-MICH) stated with respect to the debate concerning the subtherapeutic use of a n t i b i o t i c s i n animal feeds: "The science of this issue i s well i n hand, but we cannot c a l l upon i t to do the impossible. Twenty years of s c i e n t i f i c investigation have i d e n t i f i e d but not quantified the r i s k to human health. We now face a fork i n the road where prudent policy decision and not further study w i l l be the pathfinder." There are several ways i n which the feeding of a n t i b i o t i c s of animals may pose a potential health hazard to humans and other animals. F i r s t , pathogenic organisms such as Salmonella, existing i n the GI tract of animals, can become resistant to the a n t i b i o t i c ( s ) fed to the host animal at subtherapeutic levels and over time be passed into the environment and/or food to humans. This chapter not subject to U.S. copyright. Published 1986, American Chemical Society

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9. F R A P P A O L O

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101

Since the organisms are a n t i b i o t i c resistant, i f they produce c l i n i c a l i n f e c t i o n i n humans or other animals, then the same a n t i b i o t i c would be an i n e f f e c t i v e treatment. Secondly, resistance that develops i n non-pathogenic bacteria, for example, jE. c o l i may be transferred to pathogenic bacteria either i n animals or humans which may i n turn cause a drug resistant i n f e c t i o n . There i s also concern that a n t i b i o t i c s i n animal feeds may increase the prevalence or prolong the shedding of Salmonella organisms i n animals, thus increasing the r i s k of disease i n animals and humans.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch009

H i s t o r i c a l Perspective The health concerns over the practice of feeding animals a n t i b i o t i c s came to the forefront i n 1965 when i n England there was an epidemic of drug resistant Salmonella typhimurium i n dairy calves that subsequently spread to humans. Thousands of animals as well as seven humans died as a result of the epidemic which lasted for several years. The offending s t r a i n of Salmonella was believed to have originated on a c a l f dealer's premises from which infected calves were sold to many parts of England. The use of a n t i b a c t e r i a l s i n the calves was thought to have caused the development of the resistant s t r a i n of Salmonella. Spread of the organism and treatment of diseased animals with various a n t i b i o t i c s led to the s t r a i n acquiring resistance to eight different drugs by the time the epidemic had run i t s course. This incident and concerns that resistance to a n t i b i o t i c s was increasing led to the formation of the Swann Committee, which examined the use of a n t i b a c t e r i a l s i n feeds i n England. In 1969, the Committee issued i t s report (2) on the use of a n t i b i o t i c s i n veterinary medicine and animal husbandry. I t recommended that a n t i b i o t i c s and other a n t i b a c t e r i a l s be divided into a "feed" class and a "therapeutic" class which would be used only by issuance of a veterinary prescription. The B r i t i s h government accepted the Swann Commmttee recommendations i n 1971. FDA's concerns regarding a n t i b i o t i c resistance and the implications for human and animal health span some 30 years during which symposia, consultations with outside experts and task force reviews were held. Most notable among these actions was the establishment of the FDA Task Force on the Use of A n t i b i o t i c s i n Animal Feeds. Established i n 1970 at the recommendation of FDA's Science Advisory Committee, the Task Force was asked to undertake a comprehensive review of the use of a n t i b i o t i c s i n animal feeds. In i t s report (3) issued i n 1972, the Task Force acknowledged the potential human and animal health hazard of drug resistant bacteria and made a number of recommendations. In addition to basic research to better understand the nature of the problem, the Task Force recommended that r e s t r i c t i o n s be placed on the use of a n t i b a c t e r i a l agents i n feeds which f a i l to meet guidelines established by the Task Force i n regard to safety and/or e f f i c a c y . Agents that do not meet these standards would be prohibited from growth promotion and any subtherapeutic use i n animals but could continue to be used at therapeutic levels for short-term treatment on the order of licensed veterinarians.

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Shortly a f t e r the Task Force made i t s recommendation, FDA i n 1973 established a regulation (4) that specified that a n t i b i o t i c s to be used i n animal feeds for more than two weeks must meet the Task Force's c r i t e r i a for safety i n order to gain approval or to remain on the market. A few years a f t e r the issuance of the Task Force Report, the Commissioner of the Food and Drug Administration (FDA) ordered additional review of the data and the issues involved by the Agency's National Advisory Food and Drug Committee. This review involved public meetings and comments from a l l interested p a r t i e s . After this review and taking into consideration the recommendations of the Advisory Committee, former Commissioner Donald Kennedy directed the Bureau of Veterinary Medicine [now the Center for Veterinary Medicine (CVM)] to publish a Notice of Opportunity for Hearing on a proposal to withdraw approval of New Animal Drug Applications for use of p e n i c i l l i n i n animal feeds. This Notice 05) published i n the FEDERAL REGISTER on August 30, 1977, and was followed on October 21, 1977, by a s i m i l a r Notice (6) which proposed withdrawal of certain subtherapeutic uses of the tetracyclines, s p e c i f i c a l l y chlortetracycline and oxytetracycline i n animal feeds. The manufacturers of the a n t i b i o t i c s requested a hearing. Because of disagreement among some s c i e n t i s t s as to whether the subtherapeutic use of these a n t i b i o t i c s results i n s i g n i f i c a n t health r i s k s , Congress intervened and i n 1979 directed FDA to contract with the National Academy of Sciences (NAS) to study the issues involved and earmarked $250,000 f o r that purpose. Congress also mandated that FDA hold i n abeyance any implementation of i t s proposed actions pending f i n a l results of these studies. Nevertheless, FDA announced i t s intention to hold a formal evidentiary hearing i n response to the drug sponsors' request, but the start of the hearing would be delayed u n t i l release of the NAS report. In addition to the NAS report, other Congressionally mandated studies included the Office of Technology Assessment report 07) on "Drugs i n Livestock Feed" i n June of 1979, and the USDA's report (8) on the "Economic Effects of a Prohibition on the Use of Selected Animal Drugs" i n December of 1978. These reports e s s e n t i a l l y supported the views held by FDA. For example, the OTA report concluded that the health r i s k s from the use of low-level a n t i b i o t i c s are of greater concern than the risks of cancer from DES and furazolidone as used i n l i v e s t o c k p r a c t i c e . They also concluded that the drugs FDA proposed r e s t r i c t i n g could be replaced with alternative drugs. The USDA economic study concluded that while farm and food prices would increase i n i t i a l l y , the economic system would generally "be quite resistant to a more r e s t r i c t i v e policy on animal drug use." This conclusion was reached even though the USDA study was based on the erroneous assumption that a l l feed additive a n t i b i o t i c s would be banned. In March of 1980, the NAS submitted i t s report (9) e n t i t l e d "The Effects on Human Health of Subtherapeutic Use of Antimicrobials i n Animal Feeds." The report stated "the postulations concerning the hazards to human health that might result from the addition of subtherapeutic antimicrobials to feeds

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9.

FRAPPAOLO

Risks to Human Health from Antibiotics in Animal Feeds

103

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch009

have been neither proven nor disproven. The lack of data l i n k i n g human i l l n e s s with subtherapeutic levels of antimicrobials must not be equated with proof that the proposed hazards do not e x i s t . The research necessary to establish and measure a d e f i n i t e r i s k has not been conducted and, indeed, may not be possible." The NAS committee further concluded that i t i s not technically feasible to conduct a single comprehensive epidemiological study that w i l l s e t t l e the issues. They offered suggestions for several less comprehensive, but more f e a s i b l e , studies with the caveat that these studies had potential for c l a r i f y i n g certain points, but would not s e t t l e the issues. They would, i n essence, better define the links i n the chain of events that i s believed to exist from the feeding of subtherapeutic levels of a n t i b i o t i c s i n animals to the development of drug resistant disease i n humans. Recent

Events

In view of the NAS report, Congress, through the appropriations process for f i s c a l 1981, instructed FDA to conduct additional studies to generate new epidemiologic information consistent with the NAS suggestions and hold i n abeyance any proposed actions u n t i l the studies are concluded. In response to the 1981 mandate of Congress to generate additional data, FDA awarded a contract (10) to the Seattle-King County Department of Public Health to conduct an epidemiologic study of Salmonella and Campylobacter i n commercial meat products i n the community and their association with human disease. In August of 1984, CVM received the f i n a l study report and although i t has been accepted as having met contractual obligations, the study i s currently undergoing s c i e n t i f i c review. The study used a dual surveillance approach, one monitoring cases of human i l l n e s s and the other involving the sampling of food for contamination. For human case surveillance, a l l cases of Salmonella and Campylobacter jejuni e n t e r i t i s diagnosed i n enrollees at Group Health Cooperative of Puget Sound, a 320,000 member health maintenance organization (HMO), were investigated over an 18-20 month period. A case/control study was also conducted. In addition, household environmental samples were taken from family members of index cases, household pets, and i n some cases, foods were sampled i n an e f f o r t to i d e n t i f y reservoirs of Campylobacter. Food surveillance was integrated into the health departments meat inspection program and thus provided access to a l l r e t a i l purveyors of meat products i n King County, Washington. Added to the r e t a i l meat surveillance system was a s p e c i f i c a t i o n for culturing poultry products at a large independent poultry processor i n Seattle. The food surveillance system was designed to provide for the culture of 2,000 specimens of food products of animal o r i g i n for Salmonella and Campylobacter during a 20 month-period. In order to evaluate relationships among individual Campylobacter and Salmonella i s o l a t e s , a n t i b i o t i c s u s c e p t i b i l i t y testing was conducted along with serotyping and several types of plasmid analyses. The predominant finding reported by the contractor i n the food surveillance system was s i g n i f i c a n t contamination of r e t a i l poultry

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by Campylobacter j e j u n i ; 22.3% of specimens cultured jejuni while only 3.5% cultured Salmonella. The research contractor concluded that e n t e r i t i s due to Campylobacter jejuni i s more common than that due to Salmonella and that £. jejuni appears to flow from chickens to man v i a consumption of poultry products. Considerable public attention has been focused on the a n t i b i o t i c s i n animal feed issue as of late as a r e s u l t of two recent reports from investigators at the Centers for Disease Control. One report (11) i n the August 24, 1984, issue of Science was a retrospective analysis of a l l CDC investigated Salmonella outbreaks during the 13 year period between 1971 and 1983. They discovered that i n over two-thirds of U.S. outbreaks of miltiple-drug-resistant Salmonella infections that had a defined source, such bacteria came from food animal populations. Animal origins were discovered more commonly i n outbreaks involving antimicrobial-resistant Salmonella than i n outbreaks involving antimicrobial-sensitive s t r a i n s . In addition, the case f a t a l i t y rate for patients with multiple resistant Salmonella infections was found to be 21 times higher than the case f a t a l i t y rate associated with antimicrobial-sensitive Salmonella i n f e c t i o n s . Their assessment was that antimicrobial-resistant bacteria frequently a r i s e from food animals and can cause serious infections i n humans. One of the major c r i t i c i s m s of FDA's s c i e n t i f i c basis for wanting to r e s t r i c t the use of a n t i b i o t i c s i n animal feeds has been that i t has not provided any s p e c i f i c instances of human i l l n e s s due to drug-resistant pathogens that resulted from the subtherapeutic feeding of a n t i b i o t i c s to animals. However, individual events i n the complicated sequence have been documented. Another report (12) by Dr. Scott Holmberg and others at CDC which appeared i n the September 6, 1984, issue of the New England Journal of Medicine purportedly linked, for the f i r s t time, the use of subtherapeutic a n t i b i o t i c s i n livestock feed to the development of serious drug resistant infections i n humans. The a r t i c l e described the investigation of an outbreak of Salmonella newport involving 18 persons i n the Midwest. The epidemic s t r a i n was resistant to a m p i c i l l i n , c a r b e n i c i l l i n , and t e t r a c y c l i n e . Twelve of the patients had been taking p e n i c i l l i n derivatives for other medical problems. Eleven required h o s p i t a l i z a t i o n and there was one death. Through epidemiologic techniques ground beef was implicated as the common food source of the i n f e c t i o n and the meat was traced to c a t t l e presumably from a farm i n South Dakota. The c a t t l e had presumably been fed subtherapeutic levels of c h l o r t e t r a c y c l i n e for growth promotion and disease prevention. A major finding i n this investigation was the i d e n t i f i c a t i o n of a segment of the population, i . e , those receiving a n t i b i o t i c s , that may be at higher r i s k of contracting a severe i l l n e s s due to a resistant Salmonella i n f e c t i o n . Presumably, the use of antimicrobials to which a pathogen i s resistant would constitute s e l e c t i v e pressure permitting the organism to f l o u r i s h . There have also been a number of other studies contracted for by FDA since the 1977 notices on the p e n i c i l l i n s and the t e t r a c y c l i n e s . These studies were designed to provide more information on s p e c i f i c segments i n the postulated chain of events

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linking the subtherapeutic use of a n t i b i o t i c s i n animals to the development of serious disease i n humans. Notable among them was the work by Thomas O'Brien and collegues the results of which were published i n the New England Journal of Medicine i n 1982 (13). This study provided for methodology developments that allowed one to determine whether or not plasmids from d i f f e r e n t sources (man and animal) were i d e n t i c a l or s i m i l a r . The Center's b e l i e f that the continued unrestricted subtherapeutic use of these a n t i b i o t i c s presents risks to human and animal health i s based upon consideration of a number of factors:

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—

—

—

—

Long-term, low-level feeding of p e n i c i l l i n and the tetracyclines promotes, by natural selection from the pool of normal i n t e s t i n a l f l o r a , those enteric (gut) bacteria that contain R-plasmids. R-plasmids, also known as R-factors, are extrachromosomal genetic material which confer a n t i b i o t i c resistance to host bacteria. These plamids can be transferred between various kinds of bacteria through c e l l - t o - c e l l contact (conjugation). Simultaneous resistance to several unrelated a n t i b i o t i c s i s commonly carried on a single plasmid and therefore i s simultaneously transferred from one bacterium to another. IS. C o l i strains bearing R-plasmids can be transferred from animal to man. Under the proper circumstances, organisms of animal o r i g i n can colonize i n the human gut. However, colonization i s not considered necessary for transfer of drug resistance to strains that inhabit the human gut. Use of p e n i c i l l i n and the tetracyclines also causes selection for pathogenicity factors, that i s , disease-causing factors. These factors and drug resistance have been shown to be linked on the same plasmid. Pathogenicity and a n t i b i o t i c resistance can therefore be transferred simultaneously to other organisms. R-plasmids can be transferred from normally nonpathogenic .E. c o l i to certain pathogenic strains of bacteria with which they may come i n contact i n man or animals. Since R-plasmids carry drug resistance, this transfer can result in the creation of pathogenic strains of bacteria which are resistant to a n t i b i o t i c therapy.

Continued unrestricted subtherapeutic use of a n t i b i o t i c s i n animal feed increases the pool of drug-resistant bacteria i n our environment. Moreover, the prospect of pathogens becoming drug resistant i s , as FDA believes, a r e a l threat to human health. In a speech before the Congress i n 1978, former Commissioner Donald Kennedy stated: "the evidence indicates that enteric microorganisms i n animals and man, t h e i r R-plasmids, and human pathogens form a linked ecosystem of their own i n which action at any one point can a f f e c t every other." If the v u l n e r a b i l i t y of microorganisms to a n t i b i o t i c s i s reduced by the use of a n t i b i o t i c s for nonmedical purposes i n animals, the effectiveness of medical treatment w i l l be diminished i n man. Potential risks to animal health also e x i s t , and while the linkage to human health i s i n d i r e c t , animal agriculture faces the r i s k d i r e c t l y . The

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development of resistant s t r a i n s , which i s enhanced by subtherapeutic drug use, reduces the e f f i c a c y of those same drugs for the treatment of animal diseases. The o v e r a l l implications are addressed by Marc Lappe i n his book (14), "Germs That Won't Die." He states: "Organisms almost t o t a l l y resistant to the major a n t i b i o t i c s now run rampant i n h o s p i t a l quarters, nurseries, and animal stockyards a l i k e , creating unprecedented problems for infectious disease control s p e c i a l i s t s and public health o f f i c i a l s . In spite of the awesome nature and speed of this spread of resistant organisms, many American agencies l i k e the Center for Disease Control i n Atlanta, Georgia, have only recently recognized the f u l l implications of this problem. Hospitals and physicians (and veterinarians I might add) s t i l l only grudgingly admit a problem e x i s t s , even as new a n t i b i o t i c s appear to p r o l i f e r a t e as fast as the old ones are outstripped by resistant organisms." NRDC Analysis of Risks to Human Health On November 20, 1984, Secretary Heckler received from the Natural Resources Defense Council (NRDC) a p e t i t i o n to declare the subtherapeutic uses of p e n i c i l l i n and the tetracyclines i n animal feeds an imminent hazard to the public health. NRDC argues that, on the basis of three recently published s c i e n t i f i c s t u d i e s — t h e O'Brien and the two Holmberg studies discussed e a r l i e r — F D A i s l i k e l y to eventually withdraw approval of the subtherapeutic uses of p e n i c i l l i n and the t e t r a c y c l i n e s i n animal feeds. NRDC argues, based on these studies, that these uses meet the c r i t e r i a for imminent hazard under the law. The p e t i t i o n and i t s impact were discussed before Congress, i n hearings before the Committee on Science and Technology i n December of 1984 (15). Before making any recommendation to FDA Commissioner Young and then to Secretary Heckler, the Center for Veterinary Medicine had to evaluate a l l available information, not just the three studies c i t e d , before deciding on the p e t i t i o n . To assist i n i d e n t i f y i n g pertinent available data and information, FDA decided to hold a l e g i s l a t i v e - t y p e hearing on January 25, 1985, on the NIH Campus i n which interested persons were invited to present their views. Some 35 individuals representing industry, academia, government, consumers, a g r i c u l t u r e , pharmaceutical manufacturers, producers of red meat and poultry, and even a member of Congress spoke either for or against the NRDC imminent hazard proposal. F i n a l comments were due i n by February 11, 1985, and an o f f i c i a l transcript was prepared. The c r i t e r i a used to evaluate the p e t i t i o n were the following: — — — — —

The l i k e l i h o o d that FDA w i l l eventually withdraw approval; The severity of harm pending withdrawal of approval; The l i k e l i h o o d of harm pending withdrawal of approval; The r i s k to treated animals from suspended marketing; and Other approaches to protect the public health.

The NRDC p e t i t i o n was unique i n that i t involved an indirect e f f e c t , that i s , the effect from the use of subtherapeutic levels of p e n i c i l l i n and the tetracyclines i n animal feeds on the health

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of man. Previously submitted "imminent hazard" petitions dealt with direct effects as i n the effect of a drug on a treated i n d i v i d u a l . Because of the i n d i r e c t e f f e c t , demonstration of the harm to man i s decidedly more d i f f i c u l t to measure. Quantitation indeed has been one of the major issues since the subtherapeutic use of a n t i b i o t i c s i n feeds question arose i n the 1950 s. NRDC estimated that between 100 and 300 deaths each year (depending on which of the provided estimates were used) may be attributable to the subtherapeutic use of p e n i c i l l i n and the tetracyclines i n animal feeds. In addition, some 270,000 non-fatal cases of salmonellosis may also be due to the subtherapeutic use of a n t i b i o t i c s ( p e n i c i l l i n and the tetracyclines) i n animal feeds. NRDC u t i l i z e d two key rate estimates from the Holmberg paper published i n Science during 1984. Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch009

f

A summary of these estimates i s as follows: 1.

2.

SUMMARY OF THE FIRST ESTIMATE OF MORTALITY RATE: a. Approximately 40,000 cases of salmonellosis are reported each year (CDC data base). b. 20% to 30% of Salmonella isolated from humans are resistant to one or more a n t i b i o t i c s (CDC data base). 40,000 cases times 20% due to resistant Salmonella equals 8,000 cases each year caused by resistant Salmonella. c. 4.2% death rate associated with resistant Salmonella (from Holmberg, et a l . ) . 8,000 cases from resistant Salmonella times a 4.2% death rate from resistant Salmonella equals 336 deaths each year from resistant Salmonella. d. 69% of reported Salmonella outbreaks due to resistant Salmonella are traceable to animal sources (from Holmberg, et a l . ) . 336 deaths from resistant Salmonella times 69% traceable to animal sources equals 232 deaths each year from resistant Salmonella associated with animal sources. e. 50% of the resistant strains of Salmonella from animal sources result from subtherapeutic use of p e n i c i l l i n and the tetracyclines i n animal feeds (NRDC estimate). 232 deaths from resistant Salmonella from animal sources times 50% of resistant Salmonella from animals due to subtherapeutic use of p e n i c i l l i n and the tetracyclines equals 116 deaths each year attributed to subtherapeutic use of p e n i c i l l i n and the tetracyclines i n animal feeds. SUMMARY OF THE SECOND ESTIMATE OF MORTALITY RATE: a. 1,000 to 1,500 deaths each year are associated with Salmonella outbreaks (from private communication with CDC). b. 76.5% of f a t a l cases of Salmonella infections are associated with resistant Salmonella (calculated by NRDC from information contained i n Holmberg, et a l . ) . 1,000 deaths from Salmonella times 76.5% of f a t a l Salmonella infections associated with resistant Salmonella equals 765 deaths each year from resistant Salmonella.

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c.

69% of resistant Salmonella outbreaks are traceable to animal sources (from Holmberg, et a l . ) . 765 deaths from resistant Salmonella times 69% traceable to animal sources equals 528 deaths each year from resistant Salmonella from animal sources. d. 50% of resistant Salmonella from animals result from subtherapeutic use of p e n i c i l l i n and the tetracyclines i n animal feeds (NRDC estimate). 528 deaths from resistant Salmonella from animal sources times 50% of the resistance i n Salmonella from animal sources resulting from subtherapeutic use of p e n i c i l l i n and the tetracyclines i n animal feeds equals 264 deaths each year attributed to subtherapeutic use of p e n i c i l l i n and the tetracyclines i n animal feeds. 3. SUMMARY OF THE MORBIDITY ESTIMATE: a. 40,000 cases of Salmonella infections reported each year (CDC data base). b. 20% of these cases are caused by resistant Salmonella (CDC data base). 40,000 cases times 20% equals 8,000 cases reported each year caused by resistant Salmonella. c. 69% of resistant Salmonella outbreaks traceable to animal sources (from Holmberg, et a l . ) . 8,000 cases from resistant Salmonella times 69% from animal sources equals 5,520 cases reported each year caused by resistant Salmonella attributable to animal sources• d. 50% of resistant Salmonella from animal sources result from subtherapeutic use of p e n i c i l l i n and the tetracyclines i n animal feeds (NRDC estimate). 5,520 cases times 50% equals 2,760 cases reported each year attributed to use of p e n i c i l l i n and the tetracyclines in animal feeds. e. 1% of a l l cases of Salmonella infections are reported (from private communication with CDC). 2,760 cases times 100 equals 276,000 cases of non-fatal salmonellosis each year that are associated with the subtherapeutic use of p e n i c i l l i n and the tetracyclines i n animal feeds. NRDC used Salmonella infections as the model to make their estimates of mortality and morbidity rates. They pointed out that these are conservative estimates (underestimates) because resistance also occurs i n other pathogenic bacteria that cause human diseases. Some of the resistance i n these other pathogens results from the pool of resistant bacteria i n animals, which i s ultimately due i n large part to subtherapeutic use of p e n i c i l l i n and the tetracyclines i n animal feeds. NRDC concluded that there w i l l be no s i g n i f i c a n t negative e f f e c t on animal health from banning subtherapeutic uses of p e n i c i l l i n and the tetracyclines i n animal feeds. They indicated that the use of these drugs for purposes of improving feed e f f i c i e n c y and weight gain i s for economic reasons only and no

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health risks to animals w i l l result i f these uses are discontinued. The only potential animal health r i s k involves the use of these drugs for prevention of animal diseases. Since the p e t i t i o n i s for suspending uses of p e n i c i l l i n and the tetracyclines, there are other a n t i b i o t i c s that can be used to prevent these diseases. Also, there are e f f e c t i v e alternatives to a n t i b i o t i c s , such as vaccines, to prevent diseases. NRDC also advocated changing certain farm management practices, such as reducing the crowding of animals i n feedlots, which should reduce stress and transmission of diseases. Both of those actions i t was said should reduce the need for disease prevention. NRDC pointed out that i t i s not advocating a ban of p e n c i l l i n and the tetracyclines used at therapeutic levels to treat diseases. NRDC also noted that the frequency of a n t i b i o t i c resistance i n bacteria that cause disease increases when animals are fed subtherapeutic levels of these drugs. Thus, when animals become i l l with one of these resistant organisms, treatment with therapeutic levels of the a n t i b i o t i c of choice may not be effective• NRDC contended that the suspension of these subtherapeutic uses of p e n i c i l l i n and the tetracyclines i n animal feeds poses no human health problem. No potential human health problem has been i d e n t i f i e d i n the l i t e r a t u r e . Any r i s k of eating meat from an animal that becomes i l l , because p e n i c i l l i n and the tetracyclines were not available, could be a l l e v i a t e d by using substitute a n t i b i o t i c s and better farming practices to prevent or reduce the incidence of disease. Moreover, there would be an increased probability of e f f e c t i v e l y treating the diseases with therapeutic levels of a n t i b i o t i c s i f they were not used at subtherapeutic levels. According to NRDC, the only possible impact of a ban on humans would be economic. A higher price for meat would be temporary. I t was proposed that the average c i t i z e n consumes almost three times more meat per year than the U.S. Department of Agriculture considers necessary to meet n u t r i t i o n a l needs. Thus, the consumption of a few pounds less meat per person per year because of economic reasons would not have any human health e f f e c t according to NRDC. If the Agency decides to proceed with withdrawal, a formal evidentiary public hearing before an administrative law judge (ALJ) would be required. Under our law, such a hearing would be needed in this case even i f the drug uses i n question were to be found to be an imminent hazard. Granting an imminent hazard p e t i t i o n does not avoid formal proceedings. Rather, granting a p e t i t i o n suspends the marketing of a drug immediately—before the completion of the formal evidentiary public hearing, the ALJ's i n i t i a l decision, and the Commissioner's f i n a l decision. Under the ordinary withdrawal procedures, i n which a drug does not meet statutory requirements but does not present an imminent hazard, the drug may be marketed u n t i l the completion of a l l of these steps. CONCLUSION In an a r t i c l e (16) written for the American Journal of Epidemiology, Reuel Stallones, Chairman of the NAS Committee to

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Study the Human Health Effects of Subtherapeutic A n t i b i o t i c Use i n Animal Feeds stated: "Public policy and the actions stemming from i t cannot always await the accumulation of s c i e n t i f i c evidence and the development of prevailing views among s c i e n t i s t s . " At the time of our o r i g i n a l proposal to ban the subtherapeutic uses of p e n i c i l l i n and the tetracyclines i n animal feeds, the contention was advanced that there were gaps i n the s c i e n t i f i c position to supporting the chain of events l i n k i n g low l e v e l a n t i b i o t i c feedings to disease i n humans. Since then, newly generated data have been useful i n f i l l i n g these gaps i n our knowledge. After FDA reviews and evaluates these new data, the Agency w i l l know whether to proceed with the proposed ban. In sum, two decisions must be made i n the near future. F i r s t , whether the hazards to human health are of such significance as to c a l l for an immediate ban of low l e v e l uses of p e n i c i l l i n and the tetracyclines i n animal feeds, and (2) whether to move forward with the withdrawal proceedings. CVM i s currently engaged i n an active review of a l l available research and other information, p a r t i c u l a r l y that generated since 1977, to assess the impact of this complex s c i e n t i f i c issue on the subtherapeutic feeding of a n t i b i o t i c s to animals. Thank you for allowing me to share these views with you and I am available for any questions that you may have. Literature Cited 1.

Dingell, J . , 1980. "Animal Feeds: Effect of A n t i b i o t i c s " ( l e t t e r ) , Science. 208: 1069. 2. Report of the Joint Committee on the Use of A n t i b i o t i c s i n Animal Husbandry and Veterinary Medicine, 1969. Her Majesty's Stationary Office, London. 3. FDA Task Force, 1972. Report to the Commissioner of the Food and Drug Administration on the Use of A n t i b i o t i c s i n Animal Feeds. FDA #72-6008, 23 pp. 4. Code of Federal Regulations, 1985. A n t i b i o t i c , Nitrofuran, and Sulfonamide Drugs i n the Feed of Animals. Section 558.15: 484-497. 5. FEDERAL REGISTER, 1977. P e n i c i l l i n : Use i n Animal Feed. V o l . 42, No. 168: 43770-43793. 6. FEDERAL REGISTER, 1977. Tetracycline i n Animal Feeds and Tetracycline Containing Premixes. V o l . 42, No. 204: 56254-56289. 7. Office of Technology Assessment, 1979. Drugs i n Livestock Feed. OTA-F-91, 67 pp. 8. U.S. Department of Agriculture Economics, S t a t i s t i c s , and Cooperatives Service, 1978. Economic Effects of a Prohibition on the Use of Selected Animal Drugs. A g r i c u l t u r a l Economic Report #414. IV + 68 pp. 9. Committee to Study the Human Health Effects of Subtherapeutic A n t i b i o t i c Use i n Animal Feeds, 1980. The Effects on Human Health of Subtherapeutic Use of Antimicrobials i n Animal Feeds. ISBN 0-309-03044-7, XVI + 376 pp. 10. FDA Contract #223-81-7041, 1981. Surveillance of the Flow of Salmonella and Campylobacter i n a Community.

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11. Holmberg, S. D., J . G. Wells, M. L. Cohen, 1984. Animal-to-Man Transmission of Antimicrobial-Resistant Salmonella: Investigations of U.S. Outbreaks 1971-1983. Science 225-833-835. 12. Holmberg, S. D., M. T. Osterholm, K. A. Senger, ET AL., 1984, Drug-Resistant Salmonella From Animals Fed Antimicrobials. New England Journal of Medicine, 311: 617-622. 13. O'Brien, T. F., J . D. Hopkins, E. S. Gilleece, ET AL., 1982. Molecular Epidemiology of A n t i b i o t i c Resistance i n Salmonella from Animals and Human Beings i n the United States. New England Journal of Medicine. 307:1-6. 14. Lappe, M., 1982. Germs That Won't Die: Medical Consequences of the Misuse of A n t i b i o t i c s . Anchor Press, Garden City, NY, 246 pp. 15. Committee on Science and Technology: U.S. House of Representatives, 1984. A n t i b i o t i c Resistance. 98th Congress, Second Session. Report No. 150, 139-188. 16. Stallones, R. A., Epidemiology and Public Policy: Pro-And-Anti-Biotic, 1982. American Journal of Epidemiology. Vol. 115, No. 4: 485-491. R E C E I V E D April 1, 1986

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

10 Effects of Low Levels of Antibiotics in Livestock Feeds Thomas H . Jukes

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch010

Department of Biophysics and Medical Physics, University of California, Berkeley, CA 94720 Young vertebrates usually l i v e i n a "below-par" condition of subtle ill health caused by unidentified harmful i n t e s t i n a l microorganisms. This i s shown by the a n t i b i o t i c growth effect, i n which the unidentified microorganisms are suppressed and by the enhanced growth rate of germ-free chicks and rats, i n which the unidentified microorganisms are excluded. When a n t i b i o t i c s are added to the diet of animals, large numbers of resistant enterobacteria become present i n t h e i r i n t e s t i n e s . Resistance is also enhanced by administering antimicrobial drugs to human beings, or to animals by veterinary prescription. The outbreak of Salmonella foodborne i l l n e s s i n I l l i n o i s i n A p r i l 1985 was attributed to a tetracycline-resistant s t r a i n of Salmonella typhimurium. It evidently had no connection with feeding a n t i b i o t i c s in livestock. The resistant s t r a i n was of lower virulence than the average sensitive s t r a i n . A n t i b i o t i c s i n livestock feeds continue to be e f f e c t i v e i n promoting growth and suppressing certain diseases of farm animals after more than 33 years of use. Since about 1952, the American public has been amply supplied with meat produced largely from animals that received feed containing a n t i b i o t i c s . These and other chemicals, including sulfonamides and a n t i p a r a s i t i c drugs such as anthelmintics and coccidiostats added to feed, have saved labor, feed and space, thus revolutionizing animal agriculture. The record of safety of a n t i b i o t i c s i n animal feed i n the US has been excellent, including safety to producers and meat handlers as well as to consumers. A n t i b i o t i c s are commonly added to many livestock feeds at "subtherapeutic" l e v e l s , defined usually as up to 200 parts per m i l l i o n , commonly expressed as 200 grams per ton. This increases growth and suppresses bacteria that cause certain diseases, some of them subacute. The increase i n growth results from an antibacterial effect. 0097-6156/ 86/ 0320-0112506.00/ 0 © 1986 American Chemical Society

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My association with a n t i b i o t i c s i n livestock feeds started i n 1949 when Dr. Robert Stokstad and I found that aureomycin mash increased the growth of young chickens that received a complete d i e t . , The growth-promoting effect of aureomycin (chlortetracycline) was announced at the American Chemical Society meetings i n Philadelphia, A p r i l 1950. The announcement was widely quoted in the press. For example, the Daily Telegraph, London, England, headlined the story, "Drug Speeds Growth 50 p . c ; Effect on Animals," and said that "the American Chemical Society has announced i n Philadelphia that the drug aureomycin, hitherto known for i t s a n t i - i n f e c t i o n properties i s also one of the greatest growth-promoting substances ever discovered...and has increased the rate of growth of hogs by as much as 50%." The a r t i c l e also stated that tests were being made with undersized and undernourished children. So-called normal young animals are i n r e a l i t y s l i g h t l y sick, and this slows their growth. Their growth i s increased by feeding low levels of a n t i b i o t i c s . The response i s produced by several different a n t i b i o t i c s that have no s i m i l a r i t y either i n chemistry or mechanism of action, and whose only common property i s that of i n h i b i t i n g the growth of bacteria. The response may occur i n chickens fed a n t i b i o t i c s at levels as low as one gram per ton of feed. Table 1 l i s t s some characteristics of the a n t i b i o t i c growth effect.

Table I .

The A n t i b i o t i c

Growth

Effect

1.

Low levels of a n t i b i o t i c s increase growth i n healthy animals on n u t r i t i o n a l l y complete diets.

2.

No growth increase by a n t i b i o t i c s in germ-free animals.

3.

Duplication by a n t i b i o t i c s of certain effects seen i n germ-free animals.

4.

Sparing action on nutrient incomplete diets.

5.

Prolonged use of a n t i b i o t i c s on some premises often results i n improved growth i n unsupplemented animals.

physiological

requirement in animals on

The effect has been observed i n animals kept i n c a r e f u l l y cleaned surroundings. Sometimes the effect disappears under such conditions, and at other times i t p e r s i s t s . For example, the growth effect has been obtained i n pigs taken by Caesarean section under aseptic conditions and reared under thoroughly clean, but not s t e r i l e , conditions (1) • A n t i b i o t i c s do not promote growth of s t e r i l e , so-called germ-free animals or of chick embryos. This shows that the growth effect i s not produced by direct action of a n t i b i o t i c s on animals

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but results from a n t i b a c t e r i a l action. Of equal importance i s the fact that germ-free animals grow faster than non-germ-free controls. Table 2 shows results by Forbes and co-workers that i l l u s t r a t e these points (2).

Table I I .

E f f e c t o f A n t i b i o t i c s on Growth o f Germ-Free and " C o n v e n t i o n a l " T u r k e y P o u l t s *

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Status of Turkey Poults Germ-free Conventional Germ-free Conventional

No Supplement No. of Birds Weight 23 33 27 37

202 170 201 170

Antibiotics No. of Birds Weight

g g g g

20 34 25 36

207 212 199 207

g g g g

*After Forbes et a l . , 1958, Ref. 2 Six experiments summarized; 14-day weights. P e n i c i l l i n , 45 ppm, Oleandomycin, 30 ppm Conclusions: Growth increase approximately 20% by excluding contamination, or by feeding a n t i b i o t i c . No e f f e c t of a n t i b i o t i c i n absence of contamination.

The effect on growth i s highly persistent, and has continued for periods of up to 30 years or more i n the same animal colonies, such as at Washington State University, the American Cyanamid Company (Table 3) and the University of Wisconsin. Growth promotion was s t i l l obtained with chicks i n 1984 at Wisconsin by oxytetracycline and p e n i c i l l i n just as markedly as i n 1951 (3). The growth effect occurs i n the presence of resistant i n t e s t i n a l bacteria. One must conclude that i n the i n t e s t i n a l tract there are susceptible deleterious bacteria that are inhibited or eliminated, and also there are harmless i n t e s t i n a l bacteria that become resistant. Upon prolonged use of a n t i b i o t i c s i n the same animal colony, i t has sometimes been found that the control animals grow more rapidly as time goes by i n successive experiments, so that the quantitative growth response becomes l e s s , even though i t p e r s i s t s . In other cases (3), the response has remained about the same. The growth response to a n t i b i o t i c s depends on the "disease l e v e l , " because various subacute diseases i n farm animals are controlled by feeding a n t i b i o t i c s . Under these conditions, the growth response i s increased, because growth i s depressed by such diseases. As a r e s u l t , subtherapeutic levels of a n t i b i o t i c s are

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Table I I I .

Growth Responses ( A l l i n t h e Same Room) t o P e n i c i l l i n (200 ppm) i n t h e D i e t o f C h i c k s 1964-1980*

Average Gains

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Year

Experiments

1964 1965 1966 1967 1968 1969 1970 1971 1972 1973 1974 1975 1976 1977 1978 1979 1980

11 23 43 23 38 39 30 12 24 24 44 44 36 42 42 38 48

Length of experiments: 1964-•1971 1972- •1980

Controls 250 289 291 267 262 266 271 316 193 183 189 188 190 235 230 255 245 — —

Penicillin 294 324 330 311 301 310 310 357 211 203 207 202 206 253 249 275 273

19 to 20 days 13 to 15 days

Each experiment contained two replicates of 10 birds each (5 males, 5 females)• * J . Pensack, personal communication.

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added to animal feeds as a preventive measure i n the control of certain subacute animal diseases. In the decade of the 1950s, the use of a n t i b i o t i c s i n animal feeds led to improvements i n animal health and animal production. This contributed to the r i s e of large units for maintaining meat animals and poultry. These f i r s t 10 years should have given ample time for resistant pathogens to have become widespread. Ten years of this spread of resistance ought to have made a n t i b i o t i c s i n animal feed useless or deleterious so that their commercial use would cease. Yet this has not happened, even after 35 years. The f a i l u r e of such a series of events to take place i s an unexplained riddle. One guess i s that anaerobic i n t e s t i n a l microorganisms, as yet unidentified, have retained their s u s c e p t i b i l i t y to a n t i b i o t i c s and also, perhaps, that a large reservoir of sensitive wild microorganisms exists as a sort of pool that continually reinfects farm animals and depresses their growth, unless a n t i b i o t i c s are added to the d i e t . Most of the research on a n t i b i o t i c s i n feeds was from 1950 to 1960, and this led to many interesting findings that have largely been forgotten (4, 5) . The diseased conditions that existed on farms before a n t i b i o t i c feeding was introduced to stop them have not reappeared so that most people today are not f a m i l i a r with them. A n t i b i o t i c feeding for the control of chronic respiratory disease i n poultry was pioneered by White-Stevens and co-workers who used levels of 100 to 200 grams per ton (6) • These higher levels became used for treatment of various other infections of poultry, and also for other animals. In pigs, feeds containing 50 to 200 grams per ton are used to prevent or treat b a c t e r i a l e n t e r i t i s , leptospirosis and other infections, and i n beef c a t t l e against shipping fever and l i v e r abscesses. Obviously, these higher levels include production of the growth e f f e c t . Another increase i n a n t i b i o t i c usage came i n the 1960s, when a mixture of chlortetracycline, sulfamethazine and p e n i c i l l i n was introduced for addition to pig feeds. A n t i b i o t i c s Used at Low Levels i n Livestock Feeds The use of a n t i b i o t i c s at any l e v e l i n animal feed i s s t r i c t l y regulated by the Center for Veterinary Medicine of the Food and Drug Administration, acting under the US Food, Drug and Cosmetic Act of 1938 as amended i n 1958 and 1963 (7). Twelve different a n t i b i o t i c s are approved for use i n livestock feeds: Bacitracin Bambermycins Erythromycin Lincomycin Neomycin Novobiocin

Oleandomycin Penicillin Chlortetracycline Oxytetracycline Tylosin Virginiamycin.

A l e v e l of a n t i b i o t i c i n edible tissues which i s judged

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safe for human consumption has been set for each a n t i b i o t i c approved for such use. This tolerance i s based on the results of extensive tests for t o x i c i t y , b i r t h defects and carcinogenicity. A method of analysis for the drug i n animal tissues must be developed by the sponsor of the drug and approved by FDA. Tolerances are measured i n uncooked, edible tissues. A withdrawal time i s the time from the last a v a i l a b i l i t y of a medicated feed to an animal u n t i l i t s slaughter. This time i s set so that the l e v e l of residues drops below the lower l e v e l of d e t e c t a b i l i t y of the a n t i b i o t i c and i s based on a tissue residue study i n which animals are dosed with the highest l e v e l of drug i n the feed for the longest time permitted. The method of analysis must be s u f f i c i e n t l y sensitive to detect fractions of a microgram per gram i n tissue. There i s an additional protection against residues, because a n t i b i o t i c s i n meat tend to be destroyed by cooking., For example, Broquist and Kohler found that chicken breast muscle containing 12 parts per m i l l i o n of c h l o r t e t r a c y c l i n e had 0.14 parts per m i l l i o n after roasting at 230° C for 15 minutes and no detectable amounts after half an hour. The o r i g i n a l l e v e l of 12 ppm was about 60 times as high as would be produced by 400 ppm i n the animal feed, without a withdrawal period (8) . The UK Swann Committee reported that the only possible effect of residues on consumers arose from p e n i c i l l i n i n milk from cows treated for udder infections i n which the withdrawal time for the a n t i b i o t i c had not been observed. Cases of skin rashes were reported from the consumption of such milk by sensitive patients. The Committee commented that "there are no known instances i n which harmful effects i n human beings have resulted from a n t i b i o t i c residues i n food other than milk" (9) . The question of a n t i b i o t i c s i n meat and other edible products was reviewed at length by Katz (10). The USA Inspection and Sampling Program (1973 results) indicated that 5.3% of 529 carcass samples examined for residues of streptomycin, tetracycline, erythromycin, neomycin, oxytetracycline and chlortetracycline were positive; only 17 of 5,301 samples, or 0.32%, were p o s i t i v e for p e n i c i l l i n . The levels of residues that can be expected from feeding subtherapeutic quantities of a n t i b i o t i c s vary with the degree of absorption from the i n t e s t i n a l t r a c t . In chickens, the continuous feeding of 50 to 200 grams of c h l o r t e t r a c y c l i n e per ton of feed resulted i n residue levels ranging from .036 to 0.11 micrograms per gram of muscle tissue, and from .058 to .199 per gram of l i v e r tissue. These residues disappeared after one day of withdrawal from supplemented feed. The residues were also destroyed by cooking, which was found to destroy a l l residues of both oxytetracycline and c h l o r t e t r a c y c l i n e i n the muscle of poultry. The only residues surviving cooking were found i n the l i v e r . No p e n i c i l l i n a c t i v i t y was found i n the blood, muscle, l i v e r and kidney tissues of b r o i l e r chickens or i n the eggs of hens fed 100 grams of procaine p e n i c i l l i n per ton. Approximately 98% of the p e n i c i l l i n a c t i v i t y was destroyed i n the upper portion of the i n t e s t i n a l tract, and l i t t l e or no a c t i v i t y reached the small i n t e s t i n e . Katz comments that residues of the tetracyclines

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in the muscle tissue of animals w i l l not survive normal food preparation procedures, and that "no residues w i l l enter the diet of humans unless the muscle tissue i s eaten raw or very rare." Cooking degrades chlortetracycline to i s o c h l o r t e t r a c y c l i n e (11) , and oxytetracycline i s thought to be converted to alpha and beta apo-oxytetracyclines (10)• Katz comments that "the l i t e r a t u r e contains no data to indicate that either of these compounds has any b i o l o g i c a l s i g n i f i c a n c e . " The widespread occurrence of p e n i c i l l i n s e n s i t i v i t y , and the survival of p e n i c i l l i n residues i n meat following cooking, led Katz to point out that "since up to 10% of the population i s potentially sensitive to p e n i c i l l i n and i t s breakdown products, the r i s k i s too great to be ignored," and to warn against injections unless these are c a r e f u l l y controlled. The use of withdrawal procedures should protect consumers against possible s e n s i t i v i t y reactions from p e n i c i l l i n residues. Resistance Much of the debate concerning the use of a n t i b i o t i c s i n livestock feeds has centered on b a c t e r i a l resistance. One of the f i r s t observations made early i n the 1950s, was that the b a c t e r i a l count in animal feces increased after a temporary decrease when a n t i b i o t i c s , such as tetracyclines, were fed (12)• This was i n contrast to the effect of sulfonamides, which reduce the count. Obviously, resistance had occurred because the i n t e s t i n a l bacteria were t h r i v i n g i n the presence of a n t i b i o t i c s . Simultaneously, the growth of the animals was increased. Therefore the resistance i n i t s e l f was not harmful. The i n t e s t i n e of a warm-blooded vertebrate contains 21 t r i l l i o n bacteria, many of which have not been i d e n t i f i e d or grown i n test tubes. Many investigators i n the 1950s t r i e d to find out the nature of the changes i n i n t e s t i n a l bacteria that were produced by feeding a n t i b i o t i c s . The results were variable and often c o n f l i c t i n g (4) • Some reports have pointed to a decrease i n C l o s t r i d i a , but others have not supported these findings. I t i s certain that the growth response can persist for years i n the same animal colony, therefore there must be some type or types of deleterious i n t e s t i n a l microorganisms that do not acquire resistance. Salmonella The most serious association of a n t i b i o t i c s with salmonellosis was the 1965 outbreak i n England of phage type 29 Salmonella typhimurium, resistant to tetracyclines. Six human deaths were attributed to this epidemic. It was traced to "shotgun" treatment of young calves with a n t i b i o t i c s followed by wide dispersal of the calves (5) • Although this epidemic did not involve the use of livestock feeds containing a n t i b i o t i c s , the seriousness of the outbreak led to an inquiry i n the UK and a report by the Swann Committee, 1969, into this use. The report of the committee called for a stop to the use of certain common a n t i b i o t i c s i n animal feeds i n the United Kingdom.

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The Swann Committee report was followed by demands f o r discontinuing the feed use of p e n i c i l l i n and tetracyclines i n the US. These demands were largely based on the claim that transferable resistance was produced by feeding a n t i b i o t i c s , so that genes f o r resistance i n common nonpathogenic organisms, such as Escherichia c o l i , passed through c e l l walls to other bacteria, including pathogens. This transfer of genes f o r resistance can e a s i l y be demonstrated i n test tube experiments, but transfer evidently occurs less frequently i n l i v i n g animals. The Animal Health I n s t i t u t e has commented that the presence of other materials, such as b i l e salts and f a t t y acids, coupled with a very low population of donors i n the i n t e s t i n a l tract compared to the t r i l l i o n s of normally present bacteria, minimize the opportunities for conjugation (13) • It was postulated that farm animals that were fed a n t i b i o t i c s could serve as " f a c t o r i e s " that produced resistant i n t e s t i n a l bacteria and that the genes f o r resistance would be spread throughout the environment so that resistant disease would steadily increase. This would make certain common a n t i b i o t i c s useless i n treating human diseases. Accordingly, FDA proposed i n A p r i l 1977 to remove p e n i c i l l i n and tetracyclines from animal feed use and to place them s o l e l y on veterinary prescription "for the shortest time necessary to achieve the desired r e s u l t . " FDA said "The t h e o r e t i c a l p o s s i b i l i t y that drug-resistant pathogens can be produced by a n t i b i o t i c selection has become a r e a l threat with the emergence of human disease (typhoid and childhood meningitis) caused by a m p i c i l l i n - and chloramphenicol-resistant Salmonella and Haemophilus. The point i s that known routes of transfer exist by which a n t i b i o t i c use i n animals contributes to such threats." (Emphasis i n original.) These examples were inappropriate. Overuse of a m p i c i l l i n i n medical practice was discussed by Wescoe on p. 27 of the FDA's own National Advisory Food and Drug Committee Report, on January 24, 1977. Wescoe said (speaking of a n t i b i o t i c s i n animal feeds), "I r e a l l y f i n d i t d i f f i c u l t to understand how you believe a hazard exists f o r instance, r e l a t i v e to Neisseria gonorrheae, where the disease i s p r a c t i c a l l y a l l human, where i t has been treated worldwide f o r many years by a m p i c i l l i n ... and then s t r a i n to say that maybe that i s i n part due to subtherapeutic doses of the a n t i b i o t i c i n feed." Dr. Wescoe chaired the committee. Typhoid i s treated with chloramphenicol, an a n t i b i o t i c that i s not used i n animal feeds. The two i l l u s t r a t i o n s are examples of the fact that resistant human pathogens can result from medical practice. As a result of FDA's proposals and the need f o r more information, a committee was appointed by the National Academy of Sciences. I t s report (376 pp) was published i n 1980 as "The Effects on Human Health of Subtherapeutic Use of A n t i b i o t i c s i n Animal Feeds" (7). The report noted that the use of antimicrobials i n animal husbandry has steadily increased since 1950, as has animal production. Antimicrobials are perceived as especially b e n e f i c i a l when animals are being reared under

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intensive conditions or are being shipped. The committee pointed out that a number of investigators have asserted that low-level feeding of a n t i b i o t i c s to livestock increases the t o t a l numbers of bacteria containing resistant plasmids above that r e s u l t i n g from therapeutic veterinary prescribed use and both therapeutic and prophylactic uses i n human beings. If this i s true, said the committee, and i f these resistant bacteria reach consumers of meat, there would be an increased r i s k of i n f e c t i o n by resistant pathogens, or there would be an increased l i k e l i h o o d of acquiring a nonpathogenic resistant organism that could transmit infectious resistance to pathogens. "Infectious resistance" refers to the transfer of resistant genes between b a c t e r i a l c e l l s by means of plasmids or episomes. The committee concluded that not enough information was avialable on these issues to determine the effects on human health. The committee recommended a comparison of subtherapeutic with therapeutic use of a n t i b i o t i c s on the prevalence of resistant transfer factors i n meat animals. Also recommended was a study comparing the enteric f l o r a of vegetarians and meat-eaters. A t h i r d study would involve workers i n abattoirs and their contacts. These studies are i n progress under the d i r e c t i o n of Dr. Edward Kass at Harvard University and investigators at the Loraa Linda Medical School. The committee also recommended further research on the mechanisms of the a n t i b i o t i c growth e f f e c t . The report (7) said there i s l i t t l e indication that sale of a n t i b i o t i c s , including p e n i c i l l i n and t e t r a c y c l i n e s , for feed and veterinary use, "has decreased as a result of the Swann Report." The report (7) summarized work by Richmond and Linton i n England who found that 3% of a l l human prescriptions i n a county studied were for tetracyclines, and that sewage from hospitals contained more resistant organisms than did domestic sewage. They concluded that the main selective pressure for tetracycline-resistant organisms was from medical rather than veterinary use. Richmond stated that "no reduction had occurred in the incidence of a n t i b i o t i c - r e s i s t a n t E^_ c o l i i n Europe following the implementation of regulations recommended i n the Swann Report" (7)• I conclude that the results i n Great B r i t a i n and other European countries show that banning the use of p e n i c i l l i n and tetracyclines i n animal feeds has had no measurable effect on the prevalence of a n t i b i o t i c resistance, presumably because of the continued use of these a n t i b i o t i c s i n human and veterinary practice. A n t i b i o t i c s and Salmonella Foodborne I l l n e s s Salmonella are a frequent cause of foodborne i l l n e s s , commonly termed "food poisoning," going back long before the use of a n t i b i o t i c s . Salmonellosis i s of unusual interest and importance to inhabitants of Chicago because of the outbreak s t a r t i n g i n March of 1985, caused by a resistant s t r a i n of Salmonella typhimurium. As a result of recommendations made by the National Academy

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of Sciences committee that studies be made of the transmission of normal enteric microflora between animals and human beings, a study was undertaken for FDA by the Seattle-King County (SKC) Department of Public Health. FDA decided to fund such a study of the pathogens Salmonella and Campylobacter. The report was submitted and released i n August 1984 (14). i±* -1 J ^- caused i l l n e s s ( e n t e r i t i s ) at a rate of 100 per 100,000 persons, 2.5 times as often as Salmonella, and poultry products were contaminated by f j . j e j u n i four times as often as by Salmonella. Food of animal o r i g i n from r e t a i l outlets was systematically cultured for 20 months, during which time the incidence of enteric i l l n e s s was monitored among 320,000 members of a l o c a l health-maintenance group. Major sources of campylobacteriosis were i d e n t i f i e d , estimating that almost half the infections came from eating chicken, p a r t i c u l a r l y raw or undercooked chicken. Raw milk, t r a v e l to underdeveloped nations, fresh mushrooms, and one outbreak from a single goat dairy were also i d e n t i f i e d . The SKC investigators found that beef, pork and turkey were not s i g n i f i c a n t sources of Campylobacter f o r human beings. A main finding was the detection i n r e t a i l poultry of C, j e j uni cultured i n 192 of 862 specimens examined, as compared with 30 for Salmonella. Other types of r e t a i l meat had "negligible contamination by either bacterium." S i m i l a r l y , 48% of the C_. j ej uni e n t e r i t i s cases were estimated to originate i n poultry and none i n beef or pork. Only a few of the cases were hospitalized. There were no deaths. The King County surveillance does not show a connection between the use of a n t i b i o t i c s i n animal feed and either campylobacteriosis or salmonellosis. In September 1984, Dr. S. Holmberg and co-workers of the Centers for Disease Control (CDC) reported an outbreak of salmonellosis i n 18 patients, 13 of whom had consumed hamburger (15). The patients carried multiply resistant Salmonella newport. Twelve of them had received treatment with a m o x i c i l l i n or p e n i c i l l i n . The authors suggest that this "use of antimicrobials to which the S i . newport was resistant contributed selective pressure that allowed growth of the organism." A seemingly i d e n t i c a l s t r a i n of S^. newport, as judged by plasmid c h a r a c t e r i s t i c s , was found following autopsy i n tissues of a dairy c a l f that had died on the dairy farm of one of the patients. The dairy farm was near a beef c a t t l e farm from which hamburger was obtained. Thirteen of the patients had eaten hamburger "from the suspected herd or purchased from markets thought to be supplied with meat from the herd." The authors were informed by the beef farmer that he had added chlortetracycline to the feed for h i s c a t t l e by hand. One patient died, and the authors describe this case by saying, newport of animal o r i g i n apparently contaminated a sigmoidoscope which may have been inadequately disinfected and eventually resulted i n a f a t a l case of nosocomial salmonellosis."

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e

un

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This patient had severe abdominal i n j u r i e s following a traumatic accident, following which his spleen was removed. There are a number of "missing l i n k s " i n the account. The authors said that "suspect hamburger was not available f o r culture." Later i t was disclosed that nine samples were obtained by Holmberg and were received from South Dakota on A p r i l 11, 1983 by CDC. They were examined for the presence of Salmonella. No Salmonella were recovered from any of the specimens, which consisted of s i x samples of ground beef, two of beef l i v e r and one of swiss steak. These results were obtained by use of the Freedom of Information Act, and were made public i n Food Chemical News, June 10, 1985, p. 45. The c a t t l e feed was not analyzed f o r chlortetracycline. Publication of the a r t i c l e by Holmberg and co-workers (15) was followed promptly by sensational p u b l i c i t y i n the media, especially i n USA Today and on t e l e v i s i o n programs. An advertisement by American Broadcasting Company i n the New York Daily News and the New York Post i n v i t e d readers to "watch Burt Wolfe of the Channel 7 Eyewitness News Team as he reveals the frightening side effects we could suffer from the meat we eat," and to tune i n November 7 and 8 at 5:00 p.m. to "find out i f there's much more i n meat—beside f a t and c h o l e s t e r o l — t h a t could k i l l you." As a follow-up to these threats of death by eating meat, ABC, on the next evening, November 9 at 7:00 p.m., aired a coast-to-coast broadcast on the same topic, i n which Dr. Scott Holmberg said, i n reference to the use of a n t i b i o t i c s i n animal feeds, "We are looking at hundreds of thousands of a n t i b i o t i c - r e s i s t a n t b a c t e r i a l infections and hundreds of thousands more drug-resistant b a c t e r i a l infections." The source of these t e r r i f y i n g large s t a t i s t i c s was not revealed. Consumer Reports, March 1985, warned against " l i c k i n g your fingers while eating raw meat" and said that the findings by Holmberg "appear to p u l l the rug out from under" those who had claimed there was no l i n k between a n t i b i o t i c s i n feed and human disease. The "hundreds of thousands of cases" are not v i s i b l e i n a review of nontyphoidal Salmonella outbreaks between January 1971 and December 1983 (16). F i f t y - f i v e Salmonella outbreaks were investigated by CDC i n the 12-year period, and, of these, summary reports were available for 52, which affected 3,653 persons, an average of 281 per year. Of these 52 outbreaks, food-producing animals were implicated i n 18, and foods such as raw milk and eggs are included, as well as beef, among sources of infection. The outbreaks described are confined to those investigated by CDC at the request of state health departments and therefore "are not a random sample of a l l Salmonella oubreaks." However, i t i s of interest to compare the number of affected persons, 281 per year i n the outbreaks studied, with the annual production of 4.2 b i l l i o n pounds of hamburger i n the US, 1982 and 1983. Much of this beef was produced with the aid of a n t i b i o t i c s .

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The I l l i n o i s Outbreak, 1985 When the Salmonella outbreak occurred i n Chicago i n March and A p r i l 1985, Dr. Holmberg was quoted as saying, "We r e a l l y only understand two things. The outbreak i s causing severe human i l l n e s s and the Salmonella i s a drug resistant variety coming from the general animal population." He also said that he thought the bacteria most l i k e l y originated from a dairy herd, and that "the public must now consider the issue of a n t i b i o t i c s i n animal feed." On May 25, Wallace's Farmer reported that the most recent count i n this epidemic, which was the largest i n US history, totaled over 14,000 confirmed cases and two deaths linked to Salmonella poisoning (17) . This i s a mortality rate of 0.014%. According to Holmberg's summary of salmonellosis published i n 1984 and covering the years 1971 to 1983, 17 outbreaks involved resistant organisms and affected 312 persons, 13 of whom (4.2%) died from salmonellosis. Nineteen outbreaks caused by nonresistant organisms resulted i n only 4 (0.2%) f a t a l i t i e s i n 1,912 i l l persons. These percentages have been widely publicized. A f a t a l i t y of 0.26% was reported i n 1972 i n a report to FDA by a task force (18). Holmberg's mortality rate of 0.2% f o r sensitive Salmonella would have produced 28 deaths i n the I l l i n o i s outbreak of 14,000 cases. His mortality rate of 4.2% f o r resistant Salmonella would have led to 588 deaths i n the I l l i n o i s outbreak, 1985. Clearly the resistant s t r a i n i n the I l l i n o i s outbreak was less virulent than the average sensitive s t r a i n . Clearly, we can breathe more freely about a n t i b i o t i c s i n livestock feeds, i n spite of the "media b l i t z . " The I l l i n o i s outbreak i n 1985 involved 16,284 cases with two deaths v e r i f i e d as infections from the tetracycline-resistant s t r a i n (0.012% mortality) (Final Task Force Report, Salmonellosis outbreak, H i l l f a r m Dairy, Melrose Park, IL, September 1985) . Using the "CDC rates," there should have been 684 deaths from a n t i b i o t i c - r e s i s t a n t infections, and 34 deaths from infections with sensitive s t r a i n s . Is Resistance Increasing? It has repeatedly been shown that p e n i c i l l i n and tetracyclines retain their growth-promoting a c t i v i t y when used i n the same a g r i c u l t u r a l surroundings for periods of 30 years or longer. Furthermore, tetracyclines continue to be e f f e c t i v e i n the treatment of both human and animal diseases. Atkinson and Lorian (19) found that c o l i , Staphylococcus aureus, K l e b s i e l l a pneumoniae, and Staph, epidermidis showed " v i r t u a l l y the same s u s c e p t i b i l i t i e s " to tetracycline i n 242 US hospitals, 1971 to 1982. They examined the proposal that b a c t e r i a l resistance to antimicrobials i s increasing worldwide at an alarming pace. They obtained data that included over 43 m i l l i o n individual tests. The study (19) , showed that the resistance of most bacteria to most a n t i b i o t i c s had not changed during the past 12 years. Lorian

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concluded that any general increase of b a c t e r i a l resistance was a myth. Many i n d i v i d u a l cases of resistance are reported i n the s c i e n t i f i c l i t e r a t u r e , and this a t t r a c t s attention, but these cases do not represent a general trend. The opponents of a n t i b i o t i c s i n feeds tended to question L o r i a n s findings rather than adjust t h e i r own conclusions to revealed facts. 1

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch010

Animal Welfare and A n t i b i o t i c s i n Livestock Feeds It has been claimed by some members of the animal rights movement that a n t i b i o t i c s should be banned from use i n feeds. One statement was: "In the early 1900s, farm animals were raised on extensive farms, where there was plenty of land, fresh a i r , and room for animals to respond to t h e i r own b i o l o g i c a l needs. Not only were the farm animals healthy, but the farms themselves were healthy as v i t a l enterprises." (20). This author continued by alleging that the use of a n t i b i o t i c s was not i n the best i n t e r e s t s of the animals because "agri-business farmers must increasingly rely upon a n t i b i o t i c s [which creates] ...unnatural conditions." I doubt whether she was aware of the actual conditions among animals i n "the early 1900s." The land was often contaminated by parasites that caused animal diseases. The fresh a i r was often so fresh that the animals froze to death. However, I was present when a n t i b i o t i c s were introduced into feeds on farms. It was at a time, i n 1950, when bloody diarrhea caused obvious s u f f e r i n g and death i n young pigs, when chickens died i n thousands, suffocated by air-sac disease, and baby calves perished from scours. These various forms of acute d i s t r e s s were r a p i d l y a l l e v i a t e d by a n t i b i o t i c s . The diseases preceded the use of a n t i biotics. The Swann Committee noted that "disease i s one of the p r i n c i p a l causes of suffering i n animals, and i n a l l types of animals the use of a n t i b i o t i c s to control i n f e c t i o n reduced the suffering and makes an important contribution to animal welfare" (9). I t i s indeed i r o n i c a l that the American Humane Society wants to stop animals from being protected against disease and suffering. My only i n t e r p r e t a t i o n i s that the animal rights protagonists don't know anything about farming. This i s the most charitable explanation. Effects on Children Although the topic i s not included i n the t i t l e of this paper, mention should be made of the e f f e c t s of low l e v e l feeding of a n t i b i o t i c s to infants and children. This was investigated extensively i n the 1950s, e s p e c i a l l y i n disadvantaged children i n developing countries where diarrhea and fecalism are common, just as i n young farm animals. The effects were predominantly b e n e f i c i a l and no problems with resistance were reported (5). Recently the use of o r a l rehydration s a l t s with p e n i c i l l i n has been described by UNICEF (21). Discussion There are three main arguments or theories against the use of low levels of a n t i b i o t i c s i n livestock feeds. The f i r s t theory says

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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that this practice turns farm animals into producers of a n t i b i o t i c resistant genes that spread throughout the environment and convert sensitive pathogens to resistance. I have challenged this theory on the basis of the continued effectiveness of a n t i b i o t i c s i n livestock feeds for more than 34 years. The actual results of hospital tests, as reported by Lorian and his co-workers, are also contrary to this theory because these results do not show a general increase i n resistance. The second argument states that resistant salmonellae are more v i r u l e n t than sensitive salmonellae, and that i n consequence the use of a n t i b i o t i c s i n animal feeds increases the danger of Salmonella to public health. This theory was given a test i n Chicago l a s t spring. The resistant s t r a i n of Salmonella was of outstandingly low virulence, less v i r u l e n t than the average sensitive s t r a i n . Lorian has stated that p r a c t i c a l l y a l l the published work on b a c t e r i a l virulence and a n t i b i o t i c s "points to the fact that i n experiments i n animals and the experience i n c l i n i c a l medicine, bacteria that are resistant to one or multiple a n t i b i o t i c s are either equally or less virulent than the nonresistant sensitive organs." Jarolraen and Kemp found that smooth v i r u l e n t strains of Salmonella acquired resistance much less readily than rough strains that were less virulent for mice (22). It has been argued (15) that the virulence of i n f e c t i o n with resistant Salmonella i s heightened i f the infected individuals are simultaneously being dosed for colds, etc. with a n t i b i o t i c s , because the a n t i b i o t i c s destroy sensitive nonpathogenic bacteria in the i n t e s t i n e , thus providing more " l i v i n g space" for resistant Salmonella. But Aserkoff and Bennett (23) found that a course of a m p i c i l l i n or chloramphenicol prolonged salmonellosis regardless of s e n s i t i v i t y or resistance. The third argument i s that a n t i b i o t i c s i n animal feeds, i n veterinary prescriptions, and i n human prescriptions, a l l contribute to resistance, and that only the f i r s t of these three uses should be discontinued. This argument i s challenged by results i n Europe. There was no decrease i n resistance i n E_^ c o l i following the ban on p e n i c i l l i n and tetracycline i n animal feeds as enacted following the Swann report. Salmonellosis remains a major public health problem that i s reduced by sanitary procedures and adequate cooking. I t i s not tied to the use of low levels of a n t i b i o t i c s i n livestock feeds and i t w i l l continue to erupt regardless of a n t i b i o t i c use.

Literature Cited 1. H i l l , E. G.; Larson, N. L. J . Anim. S c i . 1955, 14, 395. 2. Forbes, M.; Supplee, W. C.; Combs, G. R. Proc. Soc. E x p t l . B i o l . Med. 1958, 99, 110. 3. Dafwang, I . I.; Bird, H. R.; Sunde, M. L. Poultry S c i . 1984, 63, 1027. 4. Jukes, T. H. "Antibiotics i n Nutrition"; Medical Encyclopedia Inc.: New York, 1955, 128 pp.

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5. 6. 7.

8. 9.

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10.

11.

12. 13.

14.

15. 16. 17. 18. 19. 20.

21. 22. 23.

Jukes, T. H. Adv. Appl. Microbiol. 1973, 16, 1. White-Stevens, R. H.; Zeibel, H. G.; Walker, N. E. Cereal S c i . 1956, 1, 101. "The Effects on Human Health of Subtherapeutic Use of Antimicrobials i n Animal Feeds"; National Academy of Sciences: Washington, D.C., 1980, Office of Publications, NAS, 2102 Constitution Ave., N.W., Washington, D.C., 20418. Broquist, H. P. Kohler, A. R. A n t i b i o t i c s Annu. 1953:4, 409. Swann, M. M. (Chairman) "Report of Joint Committee on the Use of A n t i b i o t i c s i n Animal Husbandry and Veterinary Medicine"; Her Majesty's Stationery Office: London, 1969. Katz, S. E. "Appendix E: i n "The Effects on Human Health of Subtherapeutic Use of Antimicrobials i n Animal Feeds"; National Academy of Sciences: Washington, D.C., 1980, Office of Publications, NAS, 2102 Constitution Ave., N.W., Washington, D.C., 20418, pp. 158-181. Shirk, R. J . ; Whitehall, A. R.; Hines, L. R. A n t i b i o t i c s Annual, 1956-1957, Medical Encyclopedia, Inc., N.Y., pp. 843-848, 1957. Johansson, K. R.; Peterson, G. E.; Dick, E.C. J. Nutr. 1953, 49, 135. Issue B r i e f i n g Book: "Subtherapeutic Use of A n t i b i o t i c s i n Animal Feeds"; Animal Health I n s t i t u t e : Alexandria, VA 22313, 1985, 30 pp. "Surveillance of the Flow of Salmonella and Campylobacter i n a Community"; Communicable Disease Control Section, SeattleKing County Department of Public Health, Seattle, August, 1984. Holmberg, S. D.; Osterholm, M. T.; Senger, K. A.; Cohen, M. L. N. Engl. J . Med. 1984, 311, 617-622. Holmberg, S. D.; Wells, J. G.; Cohen, M. L. Science 1984, 225, 833-835. Wyant, S. Wallaces Farmer 25 May 1985. FDA Task Force "Report to the Commissioner of the Food and Drug Administration: B e l t s v i l l e , MD." Atkinson, B. A.; Lorian, V. J. C l i n . Microbiol. 1984, 20, 791-796. Knopke, M. " P e n i c i l l i n and Tetracycline Used i n Animal Feeds"; Public Hearing 25 January 1985, Food and Drug Administration, pp. 367-374. DeJong, D. UNICEF News 1981, 107, 26. Jarolmen, H.; Kemp, G. A. J. B a c t e r i o l . 1969, 97, 962. Aserkoff, B.; Bennett, J.V. N. Engl. J. Med. 1969, 281, 636-640.

R E C E I V E D May

28,

1986

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11

Antibiotic Residues in Food: Regulatory Aspects

Robert C. Livingston

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch011

Center for Veterinary Medicine, U.S. Food and Drug Administration, Rockville, MD 20857

The Food and Drug Administration has the r e s p o n s i b i l i t y of ensuring that residues of drugs i n animal-derived food are safe for human consumption. The permitted residue levels and the conditions of use of each i n d i v i d u a l drug are determined by t o x i c o l o g i c a l and chemic a l studies. The studies required for a n t i b i o t i c s vary according to the drug and the proposed application. T h i s paper d i s c u s s e s the f o l l o w i n g p o i n t s : (1) the requirements for approval of a new a n t i b i o t i c as well as those for a new use of an approved a n t i b i o t i c , (2) the e f f e c t s of recent changes i n the requirements on the amount of r e s i d u e s permitted i n food, and (3) d e f i c i e n c i e s i n current methods for determining a n t i b i o t i c residues i n food.

The Food & Drug Administration has the r e s p o n s i b i l i t y for the premarket clearance of a l l animal drugs. The 1958 food additive amendment to the Federal Food, Drug & Cosmetic Act requires sponsors to demonstrate the safety of their products. The Kefauver-Harris amendment of 1962 requires the sponsors to demonstrate, i n addition to safety, the e f f i c a c y of t h e i r drugs. Safety implies safety to the animal as well as to the consumers of animal products. The role of the Center f o r Veterinary Medicine i n the premarket approval process i s to e s t a b l i s h conditions of drug use and to e s t a b l i s h the allowable tolerances for drug residues i n animal-derived food products. The two major questions concerning the use of a n t i b i o t i c s i n agriculture are the safety of the residues i n the animal-derived food and the a n t i b i o t i c resistance that may develop from the use of these drugs i n animals. I w i l l not talk about a n t i b i o t i c resistance as Mr. Frappaolo discusses this issue i n a separate paper. The residue issue can be further divided into the t o x i c i t y and the a l l e r g i c reaction to the drug residues. There i s s u f f i c i e n t concern f o r the a l l e r g i c reaction to p e n i c i l l i n that i t s tolerance i s based upon this concern; however, the rest of the a n t i b i o t i c s have tolerances based on t o x i c i t y other than the a l l e r g i c reaction. This chapter not subject to U.S. copyright. Published 1986, American Chemical Society

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Drugs are used i n agriculture to promote growth, improve feed e f f i c i e n c y and to control disease. Modern methods of producing animal-derived food depend heavily on the use of a n t i b a c t e r i a l substances. In discussing the regulatory concerns for a n t i b i o t i c s i n agriculture, one needs to review how tolerances have been established for drug residues i n animal products. Table 1 gives the

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch011

TABLE I.

DEFINITIONS OF TOLERANCES AND TOXICITY TESTS FOR REQUESTED TOLERANCE WITH SAFETY FACTORS

Toxicity test required

Tolerance

Definition

Negligible tolerance*

Toxicologically insignificant residue

90-Day subacute study i n rat and dog (preferably i n utero for rat)

Finite tolerance

Measureable amount of residue

Lifetime studies i n rat and mouse; 6-month study i n dog; 3-generation reproduction study with teratologic phase

Safety factors 2,000

100+

- Residue must be < 0.1 ppm i n meat and < 10 ppb i n milk and eggs. + - If teratogenic a c t i v i t y i s demonstrated, the safety factor i s 1,000; may also be < 100 when human exposure data are available or when a sensitive measurement i s used to set a no-effect concentration.

d e f i n i t i o n of two types of tolerances that have been used i n regulating animal drugs since 1966. A n e g l i g i b l e tolerance has a value of 0.1 part per m i l l i o n (ppm) i n meat. Negligible tolerances were obtained by drug sponsors by conducting two ninety-day subacute studies generally one i n the rat and one i n the dog. A safety factor of 2000 was used to calculate tolerances based on these two studies. If the calculated tolerance exceeded 0.1 ppm, the t o l e r ance was a r b i t r a r i l y set at 0.1 ppm; consequently, most a n t i b i o t i c s have tolerances of 0.1 ppm. If a sponsor desired a higher tolerance than 0.1 ppm, additional t o x i c o l o g i c a l studies were required. To obtain a f i n i t e tolerance, that i s a tolerance above 0.1 ppm, l i f e time studies i n the rat and mouse were required; i n addition, a s i x month study i n the dog and a three generation reproduction study with a t e r a t o l o g i c a l phase were also required. Because of the chronic nature of these studies, the safety factor was reduced from 2000 to 100. The equation for c a l c u l a t i n g a tolerance based upon the toxi c i t y studies i s presented i n Table 2. The various t o x i c i t y studies are examined to determine the lowest no-effect l e v e l i n each of the species. The no-effect l e v e l i n the most sensitive species i s used to determine the tolerance. The tolerance i s equal to the no-effect

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TABLE I I .

CALCULATION OF TOLERANCE FOR A DRUG RESIDUE

TOLERANCE

NEL X 60 KG (SF) (FF) (0.5 KG/DAY)

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NEL - NO-EFFECT LEVEL IN THE MOST SENSITIVE TEST SPECIES SF

- SAFETY FACTOR

FF

= FOOD FACTOR

l e v e l times the average weight of a person (60 kg) divided by the safety factor, a consumption factor and 0.5 kg, the estimated consumption of meat per day. The consumption factor i s an acknowledgement that organ meats, such as l i v e r and kidney, are not consumed to the same extent as muscle tissue. The consumption factors f o r the various edible products of the different species are given i n Table 3. For example, the consumption factor for muscle i n a l l species i s

TABLE III.

RELATIVE FACTORS FOR ASSIGNING NEGLIGIBLE TOLERANCES MAJOR SPECIES CATEGORIES Tissue

Beef

Pork

Muscle Liver Kidney Skin Fat

1 2 3

1 3 4 4 4

* Not used f o r human

-* 4

Sheep 1 5 5

-* 5

Poultry 1 3

-* 2 2

food.

1. The consumption factor for beef l i v e r i s 2. Because of this doubl i n g of the consumption factor, the tolerance i n l i v e r can be twice the value of the tolerance f o r the drug i n muscle. The consumption factor f o r pork l i v e r i s 3, indicating that pork l i v e r i s consumed less than beef l i v e r . Because of these consumption factors, the tolerances i n the Code of Federal Regulations (CFR) d i f f e r depending on what edible tissue i s being described. However, some of the older tolerances i n the CFR give the same value f o r a l l edible t i s sues. These drugs were regulated before the use of consumption factors were developed. The v i o l a t i o n rate f o r a n t i b i o t i c s , as determined by USDA, also needs to be examined i n order to discuss the regulatory concerns for a n t i b i o t i c residues. Table 4 l i s t s the v i o l a t i v e residue rates f o r a n t i b a c t e r i a l s i n several species f o r the years 1979 through 1983.

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TABLE IV.

Antibiotic Residues in Food

VIOLATIVE RESIDUE RATES FOR ANTIBACTERIALS

1979 MATURE CATTLE CHICKENS TURKEYS BOB VEAL SWINE

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131

2.2 nil 2.4 7.8 10

1980

3.9 6.8

1981

7.3 8.7

(%)

1982

6.1 7

1983 .2 nil .01 7.7 9.2

The residue v i o l a t i o n rate i n mature c a t t l e , chickens, and turkeys, i s very low, 0.2% or less. In f a c t , chickens have almost a zero v i o l a t i o n rate. This i s due to the highly integrated chickenproducing operations i n this country, whereas turkeys are raised more by independent producers. However, the v i o l a t i o n rate for t u r keys i s s t i l l very low. The v i o l a t i o n rate i s not low for a l l species. Bob veal through 1979 to 1983 has had a very large v i o l a t i o n rate r e l a t i v e to the other species. This i s due to the fact that bob veal are given drugs to keep them a l i v e u n t i l they are marketed. As bob veal are marketed as young as 10 days of age, the l i k e l i h o o d of withdrawal periods being followed for bob veal i s not high. Some of the drugs used i n bob veal require more than 10 days to deplete to below their established tolerances. The v i o l a t i o n rate i n swine i s also r e l a t i v e l y high. This i s primarily due to residues of sulfamethazine. The high v i o l a t i o n rate for sulfamethazine i n swine i s due to several factors. Powdered sulfamethazine i s e l e c t r o s t a t i c and tends to adhere to mixing equipment. This effect leads to contamination of nonmedicated feed. Studies indicate that average levels of contamination as high as 3 ppm can occur. These levels i n the withdrawal feed of swine can cause v i o l a t i v e residue l e v e l s . Another problem i s i n the husbandry of swine. Pigs are coprophagic and as l i t t l e as 2-3 ppm of s u l f a methazine i n the feces w i l l also result i n v i o l a t i v e residues. Compounding the problem has been the r e f u s a l of some producers to f o l low the withdrawal period. A recent publication of USDA indicates that half of the v i o l a t i o n s of sulfamethazine i n swine are the result of producers not following the withdrawal period (1). Based on the conservative nature of the n e g l i g i b l e tolerance concept and the low v i o l a t i o n rate of a n t i b a c t e r i a l s , the regulatory concern for a n t i b i o t i c s i s not large. The Center for Veterinary Medicine i s no longer using the concept of a n e g l i g i b l e tolerance i n i t s approval process. The current procedures for c a l c u l a t i n g tolerances for drug residues have the potential of further reducing our regulatory concern for many of the approved animal drugs. Table 5 l i s t s the minimal t o x i c o l o g i c a l t e s t ing for an animal drug by today's standards. These tests e s s e n t i a l l y replace the studies required to obtain a n e g l i g i b l e tolerance, i . e . , the two ninety-day feeding studies. The f i r s t thing that i s required i s a battery of genetic t o x i c i t y tests to help assess potential carcinogenicity of an animal drug. The second requirement i s the

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TABLE V.

0 0

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0

MINIMUM TOXICOLOGICAL TESTING FOR AN ANIMAL DRUG

A BATTERY OF GENETIC TOXICITY TESTS A 90-DAY FEEDING STUDY BOTH IN A RODENT SPECIES (USUALLY THE RAT) AND IN A NON-RODENT MAMMALIAN SPECIES (USUALLY THE DOG). A TWO-GENERATION REPRODUCTION STUDY WITH A TERATOLOGY COMPONENT IN RATS.

ninety-day feeding studies i n both a rodent species, usually the rat and i n a non-rodent mammalian species, usually the dog. The t h i r d requirement i s a two-generation reproduction study with a teratology component i n rats. Although the minimum t o x i c o l o g i c a l studies required by today's standards are more extensive, the 0.1 ppm cap for the tolerance has been raised to 1.0 ppm i n the t o t a l d a i l y diet of an i n d i v i d u a l . Assuming that one-third of the d a i l y diet i s composed of meat products, the 1 ppm i n the diet means that a tolerance of up to 3 ppm i n the meat can be obtained based on these studies. The 3 ppm i s the tolerance i n muscle t i s s u e . Using the consumption factors previously discussed, the t o l e r ance i n kidney, l i v e r , and skin/fat can be several multiples higher than 3 ppm. Most drugs that we see i n the program today would not require a tolerance higher than 3 ppm because their residue levels are usually much less than 3 ppm i n muscle t i s s u e . In fact, several drugs have tissue residues i n the ppb range at zero withdrawal. I f a drug requires an assigned tolerance greater than 3 ppm to obtain approval, i f the residues bioaccumulate, or i f i t i s a suspect carcinogen, additional t o x i c o l o g i c a l tests are required. Table 6 l i s t s

TABLE VI.

TOXICOLOGICAL TEST REQUIRED WHEN RESIDUE LEVELS EXCEED 3PPM,THE DRUG IS A SUSPECT CARCINOGEN, OR THE DRUG IS EXPECTED TO BIOACCUMULATE

0

Chronic bioassays for oncogenicity/chronic t o x i c i t y i n each of two rodent species.

0

A chronic bioassay (one year) i n a non-rodent mammalian species (usually the dog).

0

A teratology study i n a second species.

0

Other specialized t e s t i n g i f necessary.

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the t o x i c o l o g i c a l studies required under these conditions. In addition to the subchronic studies, chronic studies are required i n two rodent species and a non-rodent species, usually the dog. Also, a teratology study i s required i n a second species and depending on s p e c i f i c concerns other specialized testing may be required. A l i b e r a l i z i n g aspect of the new t o x i c o l o g i c a l requirements i s that the safety factor for subchronic studies has been reduced from 2000 to 1000. The recent change i n the method of calculating tolerances within the animal drug program i n CVM w i l l have quite a dramatic effect on the permitted tolerances supported by subchronic studies. Table 7 l i s t s a few representative drugs that are currently regulated i n food-producing animals. The f i r s t column i s the no-effect

TABLE VII.

SELECTED ANTIBIOTICS APPROVED FOR USE IN FOOD PRODUCING ANIMALS

Drug Apramycin Bacitracin (Zn, MD) Erythromycin Gentamicin Oleandomycin Oxytetracycline Tylosin

NEL (mg/kg) 25 >50 25 60 200 365 40

CFR T o l . (ppm) 0.1 0.5 0.1 0.1 0.15 1.0 0.2

Possible T o l . (ppm) 3.0 6.0 3.0 7.2 24.0 438.0 48.0

l e v e l for the drug that was used to determine the current tolerance as l i s t e d i n the CFR. The second column l i s t s the current tolerance. The t h i r d column indicates the possible tolerance based upon the formula given i n Table 2. Apramycin, for example, would have a poss i b l e tolerance of 3 ppm. This i s asubstantial increase over the present tolerance of 0.1 ppm. S i m i l a r i l y , the current tolerance of 0.5 ppm for b a c i t r a c i n could possibly be j u s t i f i e d as 6 ppm based upon the no-effect l e v e l alone. However, current policy would l i m i t the tolerance i n muscle to 3 ppm. The tolerance for erythromycin would increase from 0.1 up to 3 ppm. Gentamicin could possibly jump from 0.1 to 7.2, but again would be limited to 3.0 based upon our p o l i c y . The no-effect l e v e l of the l a s t three drugs l i s t e d i n the table, oleandomycin, oxytetracycline, and t y l o s i n , indicate the safety of these compounds. These drugs would a l l be candidates f o r a revised tolerance of 3 ppm based on conventional t o x i c i t y . These tolerances are not a l l being automatically revised i n the CFR f o r several reasons: (1) the subchronic studies used to calculate the current tolerances may not meet todays standards, (2) most of these drugs were regulated on the basis of residues of parent drug only, (3) the o f f i c i a l methods for monitoring the residues may not meet present standards, and (4) some of the drugs have safety concerns that are not s a t i s f i e d by subchronic studies.

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch011

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Present standards require that drugs be regulated on the basis of t o t a l residues. Total residues r e s u l t i n g from drug administration to an animal consist of the parent drug and a l l compounds derived from i t , i . e . , metabolites, conjugates, and residues bound to b i o l o g i c a l macromolecules. Total residues are t y p i c a l l y determined i n a l l edible tissues by dosing the animal under proposed use conditions with a radiolabeled drug. Several animals are usually employed i n such a study to permit their s e r i a l s a c r i f i c e after the drug has last been administered. From such an experiment, the depletion of t o t a l residues i n each of the tissues can be followed. Figure 1 represents a t y p i c a l depletion curve for t o t a l residues of a drug. The withdrawal period i s approximated by the point i n time where the t o t a l residue curve intersects the safe concentration l e v e l , previously referred to as the tolerance, as determined by the toxicol o g i c a l studies and the formula i n Table 2. To ensure compliance with the withdrawal period, an assay i s needed to monitor t o t a l residues i n the edible tissues. Because i t i s impractical to develop assays for each residue i n each of the edible tissues, the concept of a marker residue and a target tissue i s introduced. The marker residue i s a selected analyte whose l e v e l i n a p a r t i c u l a r tissue has a known relationship to the l e v e l of the t o t a l residue of t o x i c o l o g i c a l concern i n a l l edible tissues. Therefore, i t can be taken as a measure of the t o t a l residue of interest i n the target animal. The information obtained from studies of the depletion of the radiolabeled t o t a l residue can be used to calculate a l e v e l of the marker residue that must not be exceeded i n a selected tissue (the target tissue) i f the t o t a l residue of toxicol o g i c a l concern i n the edible tissues of the target animal i s not to exceed i t s safe concentration. In the example depicted i n Figure 1, the safe concentration i s 2.0 ppm. The marker residue i s at a l e v e l of 1.0 ppm when the safe concentration i s 2.0 ppm. The method i s developed f o r the marker residue at 1.0 ppm and the tolerance for the drug i s 1.0 ppm of the marker residue. The o v e r a l l effect of regulating on t o t a l residues as opposed to the parent drug i s a lowering of the tolerance. The amount by which the tolerance decreases depends on the proportion of the parent drug to the t o t a l residues. For some a n t i b i o t i c s the parent drug i s a good approximation of t o t a l residues because they are not metabolized. For other drugs the parent drug i s a vanishingly small f r a c t i o n of the t o t a l residue and the parent drug would not serve as a marker residue for the t o t a l residue. In the l a t t e r case, the tolerances would be greatly reduced based upon the low percentage of parent drug. A few of the currently regulated a n t i b i otics would not require t o t a l residue studies to support requests for new uses. The tetracyclines are not s i g n i f i c a n t l y metabolized and the parent drug i s a good approximation of the t o t a l residues. We do recognize that degradation to the epi form may occur to a small extent. The aminoglycosides undergo limited metabolism and t h e i r absorption from the GI tract i s low. Residue studies on gentamicin using microbiological assays, radiotracers and RIA techniques a l l gave the same r e s u l t s . The lack of absorption also has been demonstrated with the polypeptides b a c i t r a c i n and bambermycins. Another reason that the tolerance i s not automatically raised i n accordance with our new policy, i s the question of adequate

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

11.

LIVINGSTON

Antibiotic Residues in Food

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch011

SAFE CONCENTRATION = 2.0 PPM TOLERANCE = 1.0 PPM

TIME (DAYS)

Figure 1. Typical depletion curve f o r t o t a l residues and a marker residue i n an edible tissue.

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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methods to monitor residues. Most of the a n t i b i o t i c s have microbiol o g i c a l assays as methods for monitoring residues. These methods are not s p e c i f i c and are limited to measuring b i o l o g i c a l l y active residues. The adequacy of the extraction procedures for these methods has recently been questioned. If the method does not measure a l l of the drug residue, the withdrawal periods w i l l be too short. The need for chemical methods for many of the a n t i b i o t i c s was the subject of a recent Association of O f f i c i a l A n a l y t i c a l Chemists symposium. Sulfamethazine i s an example of a drug where the tolerance would not be raised based only on subchronic studies because of i t s possible carcinogenicity. FDA i s presently conducting chronic studies i n both rats and mice as well as a t o t a l residue study at the National Center for Toxicological Research. These studies are to be completed next year. As the Director of CVM stated i n a speech at the Food Editors Conference i n Dallas l a s t June, these studies " w i l l either exonerate sulfamethazine or w i l l incriminate i t to a point incompatible with continued approval." In either case, the v i o l a t i o n problem disappears. In summary, the regulatory concerns for residues of regulated a n t i b i o t i c s i s not large. This i s due to the conservative procedures for s e t t i n g most tolerances and the low v i o l a t i o n rates. Another reason f o r the lack of concern of residues of regulated a n t i b i o t i c s i s the number of new a n t i b i o t i c s that q u a l i f y for zero withdrawal periods. The fact that t h e i r r e l a t i v e safety enables them to obtain zero withdrawal periods places competitive pressure on sponsors to also develop safer drugs. There are some s p e c i f i c problems but they are being addressed and the future v i o l a t i o n rates should even be lower than present l e v e l s . Literature Cited 1.

Fed. Regis. 50:

20796 (May 20,

1985)

R E C E I V E D June 10, 1986

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

12

The

U.S.

Department

of

Agriculture

Meat

and

Poultry

Antibiotic Residue Testing Program

1

2

Bernard Schwab and Jeffrey Brown 1

U.S. Department of Agriculture, Food Safety and Inspection Service, Beltsville, MD 20705 U.S. Department of Agriculture, Food Safety and Inspection Service, Washington, DC 20250

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch012

2

The USA monitoring and surveillance programs for detecting a n t i b i o t i c residues i n the domestic and imported meat supply are described. An overview of the f i e l d / l a b o r a t o r y tests currently i n use i s also provided.

A n t i b i o t i c s are used extensively i n r a i s i n g meat animals and poultry i n the United States (USA) and other nations. The antimicrobials are used as feed additives or medicants; they allow for faster weight gain and more concentrated rearing practices, and protect the maturing animals against the various diseases that may occur on the farm. The Food Safety and Inspection Service (FSIS) of the United States Department of Agriculture (USDA) i s responsible for providing meat and poultry products to the consumer that are safe, wholesome, and unadulterated. Before marketing, meat animals and poultry must be properly withdrawn from a n t i b i o t i c s to ensure that the levels of a n t i b i o t i c s i n edible tissues at slaughter are at or below the tolerances established by the Food and Drug Administration (FDA). FDA i s responsible for the approval and regulation of animal drugs used i n animal husbandry i n the USA. The FDA i s also responsible for establishing tolerances for any a n t i b i o t i c s that may adulterate food products and animal feed. National Residue Program Since 1967 FSIS has conducted the National Residue Program to help prevent the marketing of animals and poultry containing i l l e g a l residues of a n t i b i o t i c s , drugs, and other chemicals. The National Residue Program operates i n three basic modes: monitoring, s u r v e i l l a n c e , and exploratory. Monitoring i s the random sampling of healthy-appearing animals at slaughter. The data gained from analysis of these samples are This chapter not subject to U.S. copyright. Published 1986, American Chemical Society

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

AGRICULTURAL USES OF ANTIBIOTICS

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch012

138

used to define the p r o f i l e or residues over time and to i d e n t i f y problems• Surveillance i s biased sampling directed at p a r t i c u l a r carcasses or products. Surveillance comes into play when the Program receives information from monitoring or other sources, e.g., from slaughter inspection, indicating that adulterating residues may be present. Product may be held u n t i l laboratory tests determine the appropriate regulatory action. Exploratory sampling i s done generally to gain information about possible residues of concern. A l l exploratory projects have i n common the negative c h a r a c t e r i s t i c that t h e i r design i s not suitable f o r immediate regulatory action. However important to the Program, they are b a s i c a l l y f o r informational purposes. Meat and Poultry products exported to the USA are also checked f o r a n t i b i o t i c residues. Imported meat must meet the same residue standards as those established for domestic production. Monitoring, s u r v e i l l a n c e , and exploratory subprograms as defined above are carried out on foreign production marketed i n the USA. Compound Evaluation and Selection It i s , of course, not f e a s i b l e to monitor residues of a l l chemicals that t h e o r e t i c a l l y could contaminate meat and poultry, nor i s t h i s necessary to adequately protect public health. It i s important, however, to monitor those chemicals that are most l i k e l y to present the greatest r i s k . FSIS i s currently implementing a new prototype system f o r more refined categorization of residues as to their potential impact on public health. FSIS believes that this Compound Evaluation System (CES) w i l l be s u f f i c i e n t l y f l e x i b l e to permit rapid response to new information that may affect previous rankings and to allow f o r the use of s c i e n t i f i c or expert judgement. As such, the CES should serve as a useful guide i n the planning and a l l o c a t i o n of FSIS program resources f o r those residues considered to have the greatest potential effect on public health. Methods and Testing Program The e f f o r t to reduce the incidence of a n t i b i o t i c residues i n the meat supply involves not only FSIS and FDA, but also the farmers, t h e i r trade associations, feed manufacturers, and veterinarians. FSIS has expended considerable resources recently investigating several a n t i b i o t i c residue problems, such as sulfonamides and a n t i b i o t i c residues i n bob veal calves, and sulfonamides and chloramphenicol i n pigs. Agency representatives apprise the industry and other involved parties of the problem and provide resources such as educational materials, f i e l d t e s t s , and other assistance to resolve the residue problems on the farm before the animals are sent to market. When these e f f o r t s do not produce the desired r e s u l t s , the Agency implements intensive in-plant testing programs to detect the residues i n the meat at slaughter and takes corresponding regulatory action against the offending producers.

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

12.

SCHWAB AND BROWN

USDA Antibiotic Residue Testing Program

139

Tests f o r A n t i b i o t i c Residues FSIS currently uses a variety of tests for detecting a n t i b i o t i c residues i n meat; among these are f i e l d , in-plant, and laboratory screen tests, bioassays, immunoassays, and related biochemical techniques.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch012

F i e l d , In-Plant, and Laboratory

Tests

FSIS has developed a series of overnight, inexpensive, easy to perform swab bioassay tests f o r screening tissues, body f l u i d s , or feed extracts f o r a n t i b i o t i c residues. The swab tests are used on the farm, i n the slaughter plant, or i n the laboratory for t h e i r designated purpose. Swab test results indicate whether antimicrobial a c t i v i t y i s present i n the sample at or above allowable levels or absent. Further testing with more sophisticated tests i s required to i d e n t i f y and quantify the a n t i b i o t i c s producing the antimicrobial a c t i v i t y . These are usually done i n a laboratory as required. B a s i c a l l y , a l l swab tests are performed i n the same manner. The analyst (farmer, v e t e r i n a r i a n , laboratory s c i e n t i s t , or any other user) saturates a cotton tipped swab with sample tissue f l u i d s , serum, urine, or feed extract. He then firmly places the saturated cotton swab on the surface of the appropriate growth medium previously surface streaked with the working d i l u t i o n of the appropriate susceptible test organism. The test i s then incubated at the proper temperature overnight and observed the next day for antimicrobial a c t i v i t y . If there i s a zone of i n h i b i t i o n (no growth of the test organism) around the sample swab, the test i s p o s i t i v e ; no i n h i b i t i o n indicates that antimicrobials are absent or below detectable levels i n the sample tested. There are currently five swab tests i n use: o o o o o

Live Animal Swab Test (LAST) Residue Avoidance Feed Test (RAFT) Swab Test on Premises (STOP) Calve Antibiotic/Sulfonamide Test (CAST) STOP I I

LAST i s used by farmers, veterinarians, and other interested parties to screen urine from c u l l dairy cows for a n t i b i o t i c residues before marketing. I f the LAST test i s p o s i t i v e , the animal i s retained for several days and retested before s a l e . A negative LAST test allows the farmer to market h i s c u l l cow with a high degree of confidence that the edible meat, l i v e r , kidney, e t c . , at slaughter w i l l be a n t i b i o t i c residue free or below established tolerances. RAFT allows feed m i l l operators, farmers, e t c . , to test feed or feed constituents f o r antimicrobial a c t i v i t y . A feed containing an a n t i b i o t i c added i n t e n t i o n a l l y (medicated feed) or unintentionally (contaminated feed) and detectable by RAFT w i l l result i n a p o s i t i v e RAFT t e s t . STOP i s used by Federal Meat inspection personnel in-plant to check tissues from slaughtered animals for a n t i b i o t i c residues.

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch012

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AGRICULTURAL USES OF ANTIBIOTICS

Edible tissues from STOP-positive animals are retained u n t i l tested further by FSIS laboratories. If the laboratory report indicates a n t i b i o t i c at or above tolerance l e v e l s , the v i s c e r a and/or the carcass are condemned. If a n t i b i o t i c levels are below tolerance levels upon laboratory t e s t i n g , the tissues are released into the food supply. In-plant STOP-negative animals are released without delay into the food chain. STOP may be used to test a l l food animals and poultry f o r a n t i b i o t i c residues. Since STOP began i n 1979, the incidence of a n t i b i o t i c residues i n the bovine meat supply has been reduced to approximately one percent. CAST allows FSIS meat inspectors to test young veal calves at slaughter for a n t i b i o t i c and sulfonamide residues. This category of veal animal has a lengthy past history of a n t i b i o t i c misuse at slaughter. A positive CAST finding r e s u l t s i n the animal's condemnation or requires further testing at an FSIS laboratory. A CAST-negative animal i s released into the food chain without delay. The FSIS CAST program, started i n June 1985, has been successful i n reducing the incidence of a n t i b i o t i c s and sulfonamides i n the veal supply. STOP II currently i s used exclusively by FSIS laboratories to screen import and domestic monitoring samples for t y l o s i n , novobiocin, virginiamycin, and lincomycin. This test detects these compounds at or above established tolerance l e v e l s . Positive findings indicate that these drugs may be present and were not properly withdrawn before the animals was sold for slaughter. Other tests used by FSIS to detect, i d e n t i f y , and/or quantify a n t i b i o t i c residues i n meat are primarily designed for laboratory use. The conventional bioassays based on methodology developed by FDA and expanded by FSIS use four extractant buffers, five test organisms, five growth media, two incubation temperatures, and p e n i c i l l i n a s e to detect, i d e n t i f y , and/or quantify a n t i b i o t i c s such as the p e n i c i l l i n s , streptomycins, t e t r a c y c l i n e s , neomycins, erythromycin, t y l o s i n , e t c . Bioassay laboratory r e s u l t s are used by FSIS to take regulatory action and by FDA to prosecute farmers with h i s t o r i e s of improperly withdrawing a n t i b i o t i c s before marketing t h e i r herds or f l o c k s . Certain drugs such as chloramphenicol require additional tests f o r t h e i r detection and q u a n t i f i c a t i o n i n meat t i s s u e s . The Competitive Enzyme Labeled Immunoassay for Chloramphenicol (CELIA) was developed and i s used by FSIS laboratories to detect and quantify this drug i n the meat supply; chloramphenicol i s not approved for use i n food animals. CELIA detects 5 ppb chloramphenicol i n tissue extracts. The a n t i b i o t i c i d e n t i f i c a t i o n c a p a b i l i t i e s of FSIS laboratories have rapidly expanded during the past year. Commercially-produced ELISA-type immunoassays, such as the E-Z Screen, are being rapidly adapted by FSIS laboratories for use i n testing meat extracts and body f l u i d s for various a n t i b i o t i c s . These tests are r e l a t i v e l y inexpensive, s p e c i f i c , sensitive to appropriate l e v e l s , and provide results on the same day. Within the next several years, FSIS laboratories w i l l be able to screen f o r and confirm the presence of at least 22 d i f f e r e n t a n t i b i o t i c s .

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch012

12.

SCHWAB AND

BROWN

USDA Antibiotic Residue Testing Program

141

Biochemical sophisticated separation techniques are also used when necessary to confirm and quantify immunoassay test r e s u l t s . FSIS laboratories also use chemical techniques and instrumentation to i d e n t i f y select a n t i b i o t i c residues. The tetracyclines of interest are i d e n t i f i e d by t h i n layer chromatography. Sulfonamides are detected and quantified by fluorescence t h i n lay chromatography and confirmed by gas chromatography/mass spectrometry. Amoxicillin and gentamycin are i d e n t i f i e d and/or quantified by high pressure l i q u i d chromatography. Similar techniques are used to i d e n t i f y ionophores and other antimicrobials of i n t e r e s t . In conclusion, FSIS i s making a determined e f f o r t to reduce a n t i b i o t i c and other man-incurred residues i n the meat supply. The Agency i s providing resources such as educational materials and inexpensive screen tests to industry for preventing antimicrobial residues i n meat animals and poultry before marketing. Screen tests such as STOP and LAST are used in-plant by inspectors to check meat and poultry at slaughter. Additional in-plant screen tests are planned for introduction soon. Laboratory c a p a b i l i t i e s are also being rapidly expanded by improving the bioassays and by introducing rapid, s e n s i t i v e , inexpensive ELISA-type immunoassays. Sophisticated biochemical/physical techniques are also in-place or under active evaluation. R E C E I V E D May

2, 1986

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

13

Microbiological Assay Procedures for Antibiotic Residues

Stanley E. Katz

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch013

Department of Biochemistry and Microbiology, Rutgers University—The State University of New Jersey, New Brunswick, NJ 08903

The c l a s s i c a l microbial assay approaches to measuring a n t i b i o t i c residues, d i f f u s i o n , turbidimetric and acid production were described and the advantages and l i m i tations reviewed. Other systems so discussed and r e viewed were the affinity or receptor methods and the immunological approach using ELISA or EMIT assay techniques. The c l a s s i c a l systems, i n general, could measure a n t i b i o t i c residues at the f r a c t i o n a l ppm to the ppb l e v e l s . The potentials of the receptor and immunological assay system were discussed.

The appearance o f a n t i b i o t i c r e s i d u e s i n f o o d p r o d u c t s o f a n i m a l o r i g i n a r e f o r t h e most p a r t t h e r e s u l t o f improper and c a r e l e s s usage, from d e l i b e r a t e and i n t e n t i o n a l misusage, from t h e improper f o r m u l a t i o n o f a n i m a l f e e d i n g m a t e r i a l s and from t h e i g n o r i n g o f proper withdrawal times. The paths o f misuse o f a n t i b i o t i c s i n a n i m a l a g r i c u l t u r e c a n be a s v a r i e d as t h e i m a g i n a t i o n o f man can d e v i s e ; a l l however, a r e based upon economic needs o r p e r c e i v e d needs t o p r e v e n t d i s e a s e and/or t o t r e a t r e c a l c i t r a n t i n f e c t i o n s . The a n a l y s i s f o r a n t i b i o t i c r e s i d u e s i n e d i b l e a n i m a l t i s s u e , eggs, m i l k , t r a d i t i o n a l l y , h a s been performed u s i n g m i c r o b i o l o g i c a l a s s a y t e c h n i q u e s . P r i m a r i l y , t h e s e a s s a y p r o c e d u r e s were i n h i b i t i o n a s s a y s u t i l i z i n g t h e a g a r d i f f u s i o n systems (1_). M i c r o b i o l o g i c a l a s s a y p r o c e d u r e s measure a c t i v e s p e c i e s , i . e . , t h o s e t h a t c a n i n h i b i t t h e growth o f m i c r o o r g a n i s m s . Metabolic products that are n o t i n h i b i t o r y , a r e n o t measured; c o n j u g a t e d a n t i b i o t i c s , t y p i c a l l y Phase I I m e t a b o l i t e s , u s u a l l y w i l l n o t be d e t e c t e d . D e t e c t i o n o f such m e t a b o l i c p r o d u c t s r e q u i r e s h y d r o l y s i s o f t h e c o n j u g a t e p r i o r to the assay. O v e r a l l , m i c r o b i o l o g i c a l a s s a y methods have been t h e most s e n s i t i v e o f a l l a s s a y systems and t h e a b i l i t y t o measure r e s i d u e s i n t h e ppb t o ppm range i s common and h a s been f o r over 20 y e a r s ( 1 ) . Howe v e r , most o f t h e r e s i d u e a s s a y systems l a c k s p e c i f i c i t y and r e q u i r e c o n f i r m a t i o n by s p e c t r a l systems f o r a p r o p e r i d e n t i f i c a t i o n o f t h e i n d i v i d u a l a n t i b i o t i c or the a n t i b i o t i c family. There a r e many advantages t o t h e u s e o f m i c r o b i a l a s s a y 0097-6156/ 86/ 0320-0142$06.00/ 0 © 1986 American Chemical Society

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch013

13.

KATZ

Microbiological Assay Procedures for Antibiotic Residues

143

methods. With rare exception, these procedures are simple to perform, possess a f a i r degree of precision and accuracy f o r the species they measure and require simple equipment to perform. Unfortunately, microbial systems are usually slow labor-intensive and require overnight incubations and multiple platings and measurements to achieve the ± 25% to ± 35% precision at the ppm to ppb concentration. There are several ways to subdivide the a n a l y t i c a l systems encompassing the area defined as microbial assay procedures. These are: Diffusion Systems cylinder-plate well-plate pad-plate Turbidimetric Systems Competitive Receptor Assays Immunological Systems Diffusion Systems Diffusion systems are based upon the a b i l i t y of the a n t i b i o t i c to d i f f u s e through agar and cause the i n h i b i t i o n of the sensitive assay s t r a i n s . Since the substrate to be assayed i s applied i n a "point source," d i f f u s i o n occurs r a d i a l l y . A c i r c u l a r zone of i n h i b i t i o n forms and the size of the zone i s a function of the concentration. This function i s expressed as a linear relationship between the size of the zone of i n h i b i t i o n and the logarithm of the concentration. By comparing the measurable zone with a standard response l i n e , the concentration of the d i l u t i o n can be determined and the potency of the sample may be calculated. For a complete discussion of the mechanics of d i f f u s i o n , the formation of the zone edge, and the relationships between concentration and zone size, the reader should r e f e r to Kavanagh s c l a s s i c text 02). f

The Cylinder-Plate Procedure. In this procedure the substance being assayed diffuses from cylinders placed upon a uniform thickness of seeded agar, f i l l e d or charged with a fixed volume of the analyte, or reference standards or a series of standard solutions. The p e t r i dishes are incubated at a predetermined temperature and the zones of i n h i b i t i o n measured to the nearest 0.1 mm. The Cup-Plate or Well Procedure. This procedure i s similar to the cylinder-plate system except that wells are cut into the agar with cutters capable of cutting uniform, completely c i r c u l a r wells. As with the cylinder-plate assays, the wells are f i l l e d . Zones are measured after incubation and the concentration determined u t i l i z i n g a comparison with a standard response l i n e . The Pad-Plate Procedure. The pad-plate approach u t i l i z e s f i l t e r paper discs saturated with solution of the analyte as the reservoir. In a l l other respects, the system i s i d e n t i c a l to the other d i f f u s i o n systems. The advantages of the d i f f u s i o n system are: ( i ) variations are adaptable to provide reasonably sensitive assays, ( i i ) the approach i s adaptable to assay most i f not a l l a n t i b i o t i c s , and ( i i i ) the

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analyte solution need not be s t e r i l e or treated s p e c i a l l y . The disadvantages are: ( i ) f i l l i n g cylinder or wells or saturating and placing pads on agar pads i s labor intensive, slow and tedious, ( i i ) most assays require overnight incubation and hence any assay covers a two-day period, and ( i i i ) the pad-plate v a r i a t i o n i s the least sensitive usually capable of measuring ug/mL quantities; i n comparison, the cylinder or well v a r i a t i o n can measure ng/mL l e v e l s , suspended materials interfere i n the cylinder-plate system by plugging the bottoms of the cylinder and l i m i t i n g d i f f u s i o n ; i n contrast the well system i s unaffected since the analyte solution diffuses only horizontally rather than v e r t i c a l l y and horizontally. Several factors affect the d i f f u s i o n assay and must be cont r o l l e d c a r e f u l l y . The depth of the agar i n the cylinder-plate system must be minimal, as thin as possible and as uniform as possible to maximize d i f f u s i o n of the analyte. The temperature of incubators must be uniform throughout and should not vary more than ± 0.2°C. The vegetative assay organism must be sensitive to the analyte, be stable (resistant to spontaneous change), be i n the logarithmic growth phase (for uniformity of response), and be e a s i l y cultured, maintained and standardized. Spores suspension have similar c r i t e r i a except that the spores must be capable of germinating with reasonable synchrony. U t i l i z i n g the d i f f u s i o n assay systems, primarily the cylinderplate procedure, the following l i m i t s of detection and measurement are r e a l i s t i c . Table I. Detection and Measurement Levels of A n t i b i o t i c Residues i n Products of Animal Origin Using Diffusion Assays ReferAnimal Dairy Muscle ences Eggs Products or units/g or mL Penicillins 0.005-0.01 0.01-0.02 0.01-0.02 0.025-0.03 (1) (313) Streptomycins 0.06 -0.10 0.20-0.40 0.20-0.40 0.30 -0.50 (1) (14) (15) Chlortetracycline 0.005-0.10 0.02-0.03 0.02-0.03 0.02 -0.04(1X16-20) Oxytetracycline 0.025-0.03 0.08-0.10 0.08-0.10 0.08 -0.10 U ) (2122) Chloramphenicol 0.025-0.05 0.10-0.20 0.025-0.05 (1) (23) Neomycin 0.05-0.10 0.25-0.50 0.20 -0.30 (1) (2425) Erythromycin 0.025-0.05 0.10-0.20 0.10-0.20 0.10 -0.20 (1) (26Antibiotic

Milk

-17) Some other a n t i b i o t i c s commonly used i n animal production such as the b a c i t r a c i n s , bambermycins and virginiamycins as well as the streptomycins are poorly absorbed from the i n t e s t i n a l tract and residues usually do not occur from feeding. Chloramphenicol i s used i l l e g a l l y i n the United States i n many species; i t i s used l e g a l l y i n Europe, Canada and other parts of the world. The maximum s e n s i t i v i t y (the lower l i m i t of detection and measurement) that can be achieved for any d i f f u s i o n procedure i s a function of the response of the test organism to the a n t i b i o t i c being assayed. In order to increase the s e n s i t i v i t y of such

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procedures, an extraction system must be devised to concentrate the a n t i b i o t i c . Solvent extraction and concentration, and subsequent p a r t i t i o n i n g into a suitable buffer has not achieved any large degree of success simply because of complications from co-extraction of interferences. Use of column concentration/clean-up techniques also has not been exploited since there appears to be l i t t l e advantage to i t , at present. Interferences have been handled, t r a d i t i o n a l l y , by the use of a matrix compensation response curve. B a s i c a l l y , the system i s a series of standard additions to samples of a matrix and the use of these supplementations as the standards i n a response curve. Thus, the recoveries of a n t i b i o t i c s , affected p o s i t i v e l y or negatively, can be corrected for matrix e f f e c t s over a wide range of concentrations. Absolute recoveries are, of course, determined against standards i n buffer. Extractions t r a d i t i o n a l l y have been performed using buffers (jL); the same used to obtain the maximum response i n standard curves. Unfortunately t h i s has been a major f a i l i n g of the plate d i f f u s i o n assay systems. I t i s rare that the pH can be adjusted to the optimum necessary for greatest response simply by blending a matrix with buffer. As much as a 30 to 40% loss of a c t i v i t y can occur by not adjusting the pH properly; analysis f o r residues of the streptomycins and erythromycin, f o r example, can y i e l d results 20% lower by having the pH of the analyte 0.2 units below 8.0; i f the pH i s 0.5 units below 8.0, the loss of potency approaches 50% (14-15). The conventional assay systems (1) include d i l u t i o n s of 1:2 to 1:5, as part of the extraction. Hence, the levels of detection are limited. The use of minimum amounts of extractant coupled with the physical removal of s o l i d s can improve the l i m i t s of detection and measurement. Again, i t i s important to r e i t e r a t e one important fact, only free a n t i b i o t i c i s measured. Bound residues are rarely measured d i r e c t l y using these assays. Another problem with a l l such assays i s the supplementation system. The assumption that the simple addition of drug to a matrix followed by analysis was r e f l e c t i v e of the problems of assaying for a n t i b i o t i c residues i s s i m p l i s t i c and does not address the o v e r a l l problem of assaying f o r a n t i b i o t i c residues. Turbidimetric Systems The methodology i s based upon the relationship between the increasing concentrations of an a n t i b i o t i c and the resulting i n h i b i t i o n of the growth of a microorganism as measured by the development of t u r b i d i t y . The presence of increasing amounts of a n t i b i o t i c i n the assay medium result i n an increasing i n h i b i t i o n of growth. By comparing the response of the assay organism exposed to an unknown quantity of a n t i b i o t i c with the response found from known concent r a t i o n s , the potency of the a n t i b i o t i c i n the sample can be determined (absorbance vs concentration). The procedure requires that the standards and the samples be assayed under exactly the same cond i t i o n s . The most general method u t i l i z e d i s based upon the growth rate. This involves a short (usually 3-4 h) incubation period after which the incubation i s terminated and the absorbance (turbidity)

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measured i n a suitable spectrophotometer, using a flow-through c e l l system. Required for this assay are medium control, uniform test organism seeding, incubation temperature control, and a quenching system to cause the cessation of growth 02). This approach has certain advantages over the d i f f u s i o n system; i t i s more sensitive to low concentrations and the assay i s rapid. However, the l i m i t a t i o n s precluded t h i s approach from widespread a p p l i c a t i o n . Extracts of tissue or body f l u i d s are often turbid or have i n t e r f e r i n g colors and can cause errors. Solvents can i n t e r fere much more i n the turbidimetric systems than i n d i f f u s i o n systems. Surprisingly, s t e r i l i t y i s not a s i g n i f i c a n t problem unless the samples contain very large numbers of organisms. If one inoculates the assay medium to y i e l d a density of 1 x 10 organisms/ mL, i n 4 h assuming no lag phase; the organism concentration would be 4 x 10® organisms/mL (assuming no i n h i b i t o r y material and an organism generation time of 20 min). At 10 organisms/mL there i s minimal measurable t u r b i d i t y . Only i f a rapid growing organism i s present i n large numbers i n the sample extract would an interference be noted.

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5

5

Competitive Receptor Assays This assay, commonly referred to as the Charm Test, i s based upon the a f f i n i t y of a n t i b i o t i c s for s p e c i f i c s i t e s on the c e l l wall of microorganisms and the i r r e v e r s i b l e binding of the a n t i b i o t i c to these s i t e s . By adding C - l a b e l l e d or H - l a b e l l e d a n t i b i o t i c to a sample of milk, urine or the aqueous extract of tissues together followed by microbial binding s i t e s and measuring the quantity of the l a b e l l e d a n t i b i o t i c that binds to the microbial s i t e s , the a n t i b i o t i c residue can be measured. The competition f o r receptor s i t e s prevents the radiolabelled a n t i b i o t i c from binding. Thus the more radiolabelled a n t i b i o t i c bound, the less a n t i b i o t i c i n the sample. The Charm Test was i n i t i a l l y applied to the analysis of 3lactam residues i n milk although i t s application to the analysis of body, f l u i d s , meat extracts, and fermentation broths was indicated. There appears to be no rationale why t h i s basic procedure cannot be applied to a l l types of matrices (water, s o i l , animal feeds, premixes). The primary application of the procedure i s the determination of the presence or absence of 3-lactam (7) residues i n milk and secondarily to measure the levels quantitatively. The receptor assay system has now been expanded to q u a l i t a t i v e l y detect residues of t e t r a c y c l i n e , erythromycin, streptomycin, chloramphenicol, novob i o c i n , and sulfamethazine i n milk, serum and urine (Table II) (30). B a s i c a l l y the procedure to detect 3-lactam residues i n milk i s remarkably simple. A 5 mL sample of milk i s used. To this i s added the C - l a b e l l e d 3-lactam and the b a c t e r i a l receptor s i t e s . The mixture i s incubated f o r 4 min at 85°C to complete the competition f o r receptor s i t e s and centrifuged. The supernatant i s discarded, the p e l l e t i s washed gently so as not to disturb the p e l l e t . The p e l l e t i s resuspended i n water and s c i n t i l l a t i o n f l u i d i s added. For quantitative work, the sample i s counted for 5 min, for screening purposes 1 min. The l a b e l l e d a n t i b i o t i c s contain either a H- or C - l a b e l . ll+

3

1I+

3

ll+

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An entire a n t i b i o t i c screen can be carried out using 4 samples with the assay time being 15 min to 1 h depending upon whether the q u a l i tative or quantitative mode i s desired. Table I I . Limits of Detection and Measurement of A n t i b i o t i c s

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Milk A n t i b i o t i c Family Penicillins Tetracyclines Macrolides Aminoglycosides Chloramphenicol Sulfonamides Novobiocin

0.0025 0.25 0.05 0.025 0.020 0.025 0.010

Serum ugs or units/mL 0.0025 0.25 0.05 0.10 0.50 0.25 —

Urine 0.0025 — 0.05 0.10 — 0.25 —

From these data the p o t e n t i a l of the receptor assay i s evident. Comparison with the microbial d i f f u s i o n assay system, Table I, i n d i cates that the levels of detection and measurement are reasonably s i m i l a r . The receptor assay has the added v i r t u e of allowing for the completion by the analysis within an hour, generally, rather than several hours or the next day. Miscellaneous Assays f o r Residues of A n t i b i o t i c s i n Milk In conjunction with the discussion of the receptor assay system, i t i s l o g i c a l to discuss the variations of the plate assay systems and/or growth systems using colorimetric indicators of i n h i b i t i o n of metabolism or growth. Disc Assay - This i s the simplist of the procedures and involves the placing of a standard 1/2" disc saturated with milk onto the surface of IS. stearothermophilus seeded agar plate and co-incubating with suitable control discs at 55° or 64°C u n t i l w e l l defined zones of i n h i b i t i o n are obtained, usually 3-4 h. Confirmation using p e n i c i l l i n a s e - t r e a t e d milk i s required. Zones 14.0 mm are p o s i t i v e . The lower l i m i t of detection i s 0.008 units penicillin/mL. This type of assay i s simple, reasonably rapid and reasonably sensitive. Quantitation i s possible by using graded concentrations of p e n i c i l l i n i n the control milk. The technique i s limited, however, to 3-lactam a n t i b i o t i c s , primarily p e n i c i l l i n (8). A v a r i a t i o n of the disc assay i s the quantitative estimate using a central point. Each p e t r i dish contains three reference discs which contain 0.016 units penicillin/mL and three discs saturated with the unknown milk. A p e n i c i l l i n a s e disc i s placed i n the center of each plate to help confirm the presence of p e n i c i l l i n . Three plates are used for each milk sample; 13. stearothermophilus i s the assay organism. After incubation for 2-4 h, zones are measured and compared to the diameters of the reference concentration. V a l i d i t y of the difference between zone size of the reference and sample i s determined s t a t i s t i c a l l y . This procedure i s less sensitive and attempts to set the qualitative presence of 3-lactams at 0.016 units/mL rather than at lower levels (3).

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148

Colorimetric Assay f o r 3-Lactams The system discussed, commercially known as the Delvotest (9-11), u t i l i z e s a s t r a i n of B^. stearothermophilus which grows at a very rapid rate and produces acid from the nutrients i n the medium i n the absence of i n h i b i t o r y substances. When i n h i b i t o r y substances such as the 3-lactam a n t i b i o t i c s are present, acid production i s i n h i b i t e d By incorporating bromcresol purple i n the medium, i t i s easy to observe acid production, a change from purple to yellow. Incubation i s performed at 65°C f o r approximately 3 h. Table I I I shows the actual combination of colors that can be obtained and t h e i r interpretation.

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Table I I I . Interpretation of Delvotest Results

Sample Color Yellow Purple or Purple Yellow Purple Purple/Yellow Purple

Heated Confirm —

Penicillinase Treated —

Interpretation -

Yellow

Yellow

+

Purple Purple/Yellow Purple

Purple Purple/Yellow Yellow

4-

The levels of detection are quite good and are shown f o r a number of dairy products i n Table IV. Table IV. Limits of Detection of 3-Lactams i n Milk Using the Delvotest System Milk Type Raw Skim Low fat 1-2% Homogenized Half and Half

Units Penicillin/mL 0.004-0.005 0.006 0.004-0.005 0.004-0.006 0.007

Other miscellaneous assays f o r p e n i c i l l i n or other 3-lactams in milk i s the Penzyme Test which uses c e l l wall enzymes inhibited by 3-lactam drugs i n a k i n e t i c assay. This test system i s purported to be able to detect 0.005 units penicillin/mL and requires approximately 30 min to complete. I t , l i k e many other assays, detects 3-lactam a n t i b i o t i c s only. Application of Delvotest or the disc assay systems to detecting other a n t i b i o t i c s i n milk has not been successful. Only the receptor assay system appears to be v e r s a t i l e and p o t e n t i a l l y applicable to determine the presence of d i f f e r e n t a n t i b i o t i c residues i n d i f f e r e n t matrices. Immunological Systems M i c r o b i o l o g i c a l l y based assay systems invariably measure the active a n t i b i o t i c ( s ) or forms of the a n t i b i o t i c that can be i n h i b i t o r y to microorganisms. Immunological assays can measure both the active a n t i b i o t i c as well as m i c r o b i o l o g i c a l l y inactive species.

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Immunological assays measure those moieties that can cause an a n t i genic response. For the most part, immunological assays should not be interfered with by a n t i b i o t i c s from the other a n t i b i o t i c families, the s p e c i f i c i t y of the antibodies being vaguely similar to the s p e c i f i c i t y of enzyme systems. The basic p r i n c i p l e governing immunoassays for a n t i b i o t i c s i s indicated i n the following reaction:

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Ag Antigen

+

Ab Antibody

• *c

=*» Ag:Ab » Antigen:Antibody Complex

This reaction i s an equilibrium reaction and w i l l continue u n t i l the concentration of antigen i n both the free and complexed form becomes a constant (30). Applications of the immunological systems are limited by the a b i l i t y to develop suitable antibodies. Most a n t i b i o t i c s are r e l a t i v e l y small molecules having molecular weights under 500. Hence these molecules must be complexed with some c a r r i e r protein to create a molecule that can evoke the immune response and the development of antibodies. An antibody i s produced when the antigen carrying a number of antigenic determinants i s introduced into an animal s body. Lines of B c e l l s mature into plasma c e l l s and each produces an immunoglobulin molecule that f i t s a single determinant or a segment of the determinant. In a conventional sense, antibodies are polyclonal proteins because they can be directed against several other components rather than against the antigen alone. Separation of the d i f f e r e n t antibodies i n a polyclonal mixture i s extremely d i f f i c u l t i f not impossible. In contrast, monoclonal antibodies are directed against a s p e c i f i c antigen or a s p e c i f i c segment of the antigenic molecule. Kohler and M i l s t e i n (31) revolutionized immunology by demons t r a t i n g that antibody producing c e l l s (spleen c e l l s ) when fused with malignant mouse myeloma c e l l s produced hybrid c e l l lines whose c e l l s produced only a single antibody. These hybrid c e l l s , known as hybridomas,were e s s e n t i a l l y immortal meaning that they could be grown i n c e l l culture. A complete discussion of the production of antibody by hybridoma i s given by Goding (32). T

Immunological Techniques f o r Analyzing A n t i b i o t i c s Over the l a s t 8-10 years, monoclonal as well as conventional a n t i body techniques have gained popularity f o r the analysis of hormones, various drugs, proteins, bacteria, viruses and parasites. Application of immunological systems f o r the analysis of drug and a n t i b i o t i c residues has lagged because of the general lack of f a m i l i a r i t y with the p r i n c i p l e s of immunology, the d i f f i c u l t i e s i n producing stable reagents, and the d i f f i c u l t i e s i n developing methods using the crude material i n i t i a l l y available. Agglutination. The agglutination assay or the passive hemagglutination i n h i b i t i o n assay i s based upon the least amount of soluble antigen necessary to i n h i b i t agglutination or the clumping of c e l l s that occurs following the union of antigen and antibody.

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A n a l y t i c a l l y t h i s i s the amount of antigen i n the l a s t tube of a d i l u t i o n series that w i l l give a wide ring agglutination pattern. Tubes containing less antigen than this tube allow agglutination to occur. I t i s quite common to use a two-fold d i l u t i o n sequence obviously the greater the i n t e r v a l between concentration, the greater the inaccuracy. The converse i s also evident, the narrower the range, the greater the accuracy. Application of this technique i s very limited for assaying a n t i b i o t i c residues. Steiner (33) developed such a model system u t i l i z i n g gentamicin as the p i l o t a n t i b i o t i c i n matrices such as urine, blood serum, milk and animal feeds. The procedure was r e l a t i v e l y simple. A measured volume of a gentamicin-treated red c e l l suspension was added to the previously mentioned gentamicin supplemented matrices. A fixed volume of gentamicin antibody was added and the mixture incubated at room temperature for 30 to 60 min. The hemagglutination reactions were observed and the concentration of a n t i b i o t i c determined. The l i m i t s of this system for gentamicin were 0.4 ppm f o r chicken serum, burnine, 1.9 ug/mL for milk and 20 g/ton for feeds. Although these l e v e l s are not e s p e c i a l l y s e n s i t i v e , the hemagglutination offers two d i s t i n c t advantages, speed of analysis and s p e c i f i c i t y . The t o t a l assay usually can be completed within 2 h with no observable i n t e r ferences from other a n t i b i o t i c s . Radioimmunoassay. Radioimmunoassay (RIA) was f i r s t described by Berson and Yalow (34) and Luft and Yalow (35). The assay i s based upon the competition for an antibody between a radiolabelled antigen and i t s unlabelled counterpart. The greater the amount of unlabelled antigen i n the test sample, the less radiolabelled antigen bound. The concentration of antigen i n a test sample can be determined from comparisons with standard curves. The primary application of RIA for a n t i b i o t i c s has been i n the medical area, and primarily for a n t i b i o t i c s not used i n agriculture. Assays have been developed f o r gentamicin tobramycin, sisomicin, n e t i l m i c i n and for hygromycin B, an a n t i b i o t i c used primarily i n agriculture (36-37, 22-24). Gentamycin could be measured as low as 80 pg, tobramycin 280 pg, n e t i l m i c i n 300 pg mL. The RIA has d e f i n i t e advantages, small samples, sizes, speed, accuracy, precision, s p e c i f i c i t y . There are s i g n i f i c a n t disadvantages also. The labelled reactant i s unstable ( I ) and costs are r e l a t i v e l y high. The great s e n s i t i v i t y requires considerable d i l u t i o n s ; antibody-bound fractions must be separate from free fractions i n order to obtain accurate counts. In a sense, the receptor assay system i s a RIA-type technique that has been applied to a n t i b i o t i c residue analysis. 1 2 5

Nonisotopic Immunoassays. Nonisotopic immunoassays d i f f e r from the isotopic assays only i n the type of label used, the end-point measurement, and the separation of bound and free fractions (41-43). Fluoroimmunoassays. This assay requires the drug being assayed to be l a b e l l e d with umbelliferyl-B-D-galactoside. The enzyme 3-galactosidase i s added and the fluorescent products are released from the l a b e l l e d a n t i b i o t i c . The antibody i n the bound f r a c t i o n

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i n h i b i t s the enzymatic hydrolysis. The d i f f e r e n t i a l i n fluorescence i s proportional to the a n t i b i o t i c concentration. This technique could o f f e r an excellent approach i f endogenous fluorophores can be removed or minimized. Enzyme M u l t i p l i e d Immunoassay Technique (EMIT). This technique employs enzyme-labelled a n t i b i o t i c s which react analogously to the fluroimmunoassay i n that a reduction of enzyme a c t i v i t y i s a t t r i buted to antibody binding. Higher concentrations of unlabelled drug i n the sample result i n less enzyme-labelled drug bound to the antibody. To perform the EMIT assay i s rather simple and straightforward: The sample i s added to a p l a s t i c tube. [Substrate-antibody reagents could be 3-NAD (B-nicotinamidoadenine dinucleotide and 1-malic acid) 3-NAD and glucose-6-phosphate] The enzyme reagent i s added, malic dehydrogenase glucose-6-dehydrogenase The reaction i s stopped a f t e r 10 min with sodium borate. Measure the i n t e n s i t y of the r e s u l t i n g color. The range of a n t i b i o t i c that can be measured i s usually 0.01 to 1.00 ug antibiotic/mL and has been used f o r gentamicin, c a r b e n i c i l l i n , t i c a r c i l l i n and amikacin (44-46). The use of the EMIT system, to-date, has been i n the c l i n i c a l area and unrelated to measuring residues of a n t i b i o t i c s . The procedure has p o t e n t i a l for residue analysis i f interferences by non-specific factors can be overcome. Enzyme-Linked Immunosorbent Assays (ELISA). Three methods are commonly used: d i r e c t competition, double antibody sandwish and antibody i n h i b i t i o n . Direct competition. The s o l i d phase (a m i c r o t i t e r plate) i s coated with an antibody s p e c i f i c for the antigen being assayed. The sample and enzyme-labelled antigen ( a n t i b i o t i c ) are added. There i s a competition for the antibody between the l a b e l l e d and unlabelled antigen ( a n t i b i o t i c ) . Substrate i s added and the color produced by the enzymatic hydrolyse i s inversely proportioned to the concentration of antigen i n the sample (48>Double-antibody sandwich. Antibody i s coated on or adsorbed to the p l a s t i c plate. The sample to be assayed containing the antigen ( a n t i b i o t i c ) i s added followed by a second antibody that i s conjugated to the enzyme (horseradish peroxidase, a l k a l i n e phosphatase, or 3-galactosidase). The substrate i s added and the intensity of the color produced i s d i r e c t l y proportional to the antigen i n the test sample. Antibody i n h i b i t i o n . Antibody i s preincubated with the sample being assayed. I f any antigen i s present i n the sample i t w i l l bind with antibody. When the assay mixture i s added to a microtiter plate coated with antigen, there i s a decrease i n the i n t e n s i t y of the color produced. The aforementioned procedures (techniques) have s i g n i f i c a n t potential and as assay problems are worked out could provide rapid, s e n s i t i v e , s p e c i f i c and precise methods for the analysis of low l e v e l s of a n t i b i o t i c s i n food and feed products.

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Acknowledgment s New Jersey A g r i c u l t u r a l Experiment Station Publication Number F-01112-01-86 supported by State and U. S. Hatch Act Funds. Literature Cited 1.

2.

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3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Kramer, J . ; Carter, G. G.; Arret, B.; Wilner, J.; Wright, W. W.; Kirshbaum, A. Methods, Reports, and Protocols. Food and Drug Administration, Washington, D.C., 1968. Kavanagh, F. "Analytical Microbiology"; Academic: New York, 1971; Vol. I I . Ginn, R. E.; Case, R. A.; Packard, V. S.; T a t i n i , S. R. J. Assoc. Off. Anal. Chem. 1982, 1407-12. Katz, S. E.; Fassbender, C. A.; Hackett, A. J . ; M i t c h e l l , R. G. J. Assoc. Off. Anal. Chem. 1974, 57, 819-22. Katz, S. E.; Fassbender, C. A. J . Assoc. Off. Anal. Chem. 1978, 61, 918-22. Kelley, W. N. J . Assoc. Off. Anal. Chem. 1982, 65, 1193-1207. Messer, J. W.; L e s l i e , J . E.; Houghtby, G. A.; Peeler, J . T.; Barnett, J . E. J. Assoc, Off. Anal. Chem. 1982, 65, 1208-14. Ouderkirk, L. A. J . Assoc. Off. Anal. Chem. 1979, 62, 985-88. Packard, V. S.; T a t i n i , S.; Ginn, R. E. J . Milk Food Technol. 1975, 38, 601-03. Van Os, J . L.; Beukers, R. J . Food Protection. 1980, 43, 510-11. Van Os, J . L.; Lameris, S. A.; Doodeward, T.; Oostendorp, J . G. Netherlands Milk Dairy J. 1975, 29, 16-34. Katz, S. E.; Fassbender, C. A.; Dinnerstein, P. S.; Dowling, Jr., J. J . J . Assoc. Off. Anal. Chem. 1974, 57, 522-26. Katz, S. E.; Fassbender, C. A.; DePaolis, A. M.; Rosen, J . D. J. Assoc. Off. Anal. Chem. 1978, 61, 564-68. I n g l i s , J . M.; Katz, S. E. J . Assoc. Off. Anal. Chem. 1978, 61, 1098-1102. I n g l i s , J . M.; Katz, S. E. Appl. Environ. Microbiol. 1978, 35, 517-20. Katz, S. E.; Fassbender, C. A. J . Assoc. Off. Anal. Chem. 1970, 53, 968-72. Katz, S. E.; Fassbender, C. A. J . Agr. Food Chem. 1970, 18, 1164-67. Katz, S. E.; Fassbender, C. A.; Dorfman, D. B u l l . Environ. Contam. Toxicol. 1971, 6, 110-16. Katz, S. E.; Fassbender, C. A.; Dowling, J . J . J . Assoc. Off. Anal. Chem. 1972, 55, 123-33. Katz, S. E.; Fassbender, C. A.; Dorfman, D.; Dowling, J r . , J . J . J. Assoc. Off. Anal. Chem. 1972, 55, 134-38. Katz, S. E.; Fassbender, C. A. B u l l . Environ. Contam. Toxicol. 1972, 7, 229-36. Katz, S. E.; Fassbender, C. A.; Dowling, J r . , J . J . J . Assoc. Off. Anal. Chem. 1973, 56, 77-81. Singer, C. J . ; Katz, S. E. J . Assoc. Off. Anal. Chem. 1985, 1037-41. Katz, S. E.; Levine, P. R. J . Assoc. Off. Anal. Chem. 1978, 61, 1103-06.

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13.

KATZ

Microbiological

Assay Procedures for Antibiotic Residues

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25.

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Levine, P. R. Ph.D. Thesis, Rutgers University, New Brunswick, N. J . , 1980. 26. Harpster, C. P.; Katz, S. E. J . Assoc. Off. Anal. Chem. 1980, 63, 1144-48. 26a. Harpster, C. A. Ph.D. Thesis, Rutgers University, New Brunswick, N. J . , 1979. 27. Charm, S. E. U.S. Patents 5 238 521; 4 239 745; 4 239 852, 1980. 28. Charm, S. E. Cultured Dairy Products J . 1979, 14, 24-26. 29. Charm, S. E.; Chi, R. K. J . Assoc. Off. Anal. Chem. 1982, 65, 1186-92. 30. S e l l , S. "Immunology, Immunopathology and Immunity"; Harper and Row: Hagerstown, 1980; 3rd Ed. 31. Kohler, G.; M i l s t e i n , C. Nature. 1975, 256, 495-97. 32. Goding, J . W. J . Immunol. Meth. 1980, 39, 285-308. 33. Steiner, S. J . Ph.D. Thesis, Rutgers University, New Brunswick, N. J . , 1981. 34. Berson, S. A.; Yalow, R. S. In "Radioimmunoassay: A Status Report i n Immunology"; Good, R. A.; Fisher, D. W., Eds.; Sinauer: Stanford, 1971. 35. Luft, R.; Yallow, R. S. "Radioimmunoassay Methodology and Applications i n Physiology and i n C l i n i c a l Studies"; George Thieme Verlag: Stuttgard, 1974. 36. Broughton, A.; Strong, J . E.; Pickering, L. K.; Bodey, G. P. Antimicrob. Agents Chemother. 1976, 10, 652-56. 37. Lewis, J . E.; Nelson, J . C.; Elder, H. A. Nature New B i o l . 1972, 239, 214-16. 38. Watson, R. A. A.; Wenk, M. In "Current Chemotherapy"; Siegenthaler, W.; Lathy, R., Eds.; American Society for Microbiology: Washington, D.C., 1978; Vol. 2. 39. Watson, R. A. A.; Shaw, E. J . ; Edwards, G. R. W. In "Chemotherapy"; Williams, J . D.; Goeddes, A. M., Eds.; Plenum: New York, 1976; Vol. 2. 40. Foglesong, M. A.; LeFeber, D. S. J . Assoc. Off. Anal. Chem. 1982, 65, 48-51. 41. Shaw, E. J . ; Watson, R. A. A.; Landon, J . ; Smith, D. S. J . C l i n . Pathol. 1977, 30, 526-31. 42. Shaw, E. J . ; Watson, R. A. A.; Smith, D. A. C l i n . Chem. 1979, 25, 322-24. 43. Bund, J . ; Wong, R. C.; Feeney, J . E.; Carrico, R. J . ; Boguslaski, R. C. C l i n . Chem. 1977, 23, 1402-08. 44. O'Leary, T. D.; R a t c l i f f , R. M.; Geary, T. D. Antimicrob. Agents Chemother. 1980, 17, 776-78. 45. Erwin, J . R.; Bullock, W. E.; N a t t a l l , C. E. Antimicrob. Agents Chemother. 1976, 9, 1004-11. 46. Hindler, J . Abstracts, Annual American Society for Microbiology Meeting, 1983. 47. V o l l e r , A.; Bidwell, D. E.; B a r t l e t t , A. "The Enzyme-linked Immunosorbent Assay (ELISA), A Guide with Abstracts of MicroPlate Applications"; Dynatech Publication, Nuffield Laboratories of Comparative Medicine; Zoological Soc. of London: Reagents Park. 48. Campbell, G. S.; Mageau, R. P.; Schwab, B.; Johnston, R. W. Antimicrob. Agents Chemother. 1984, 25, 205-11. RECEIVED May 8, 1986

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14

Physicochemical Methods for Identifying Antibiotic Residues in Foods

William A. Moats

Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch014

Meat Science Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705 The physicochemical methods are needed f o r i d e n t i f i c a t i o n and quantitation of a n t i b i o t i c residues i n milk and tissues of animals. Methods successfully employed include high voltage electrophoresis with detection by bioautography and chromatographic procedures. Gas-liquid (GLC), thin-layer (TLC) and high performance l i q u i d chromatography (HPLC) have all been used f o r residue a n a l y s i s . A number of chromatographic methods have been described f o r chloramphenicol and the sulfonamides using all three chromatographic modes. Less work has been reported with residues of other a n t i b i o t i c s . S a t i s f a c t o r y physicochemical confirmatory tests are not available for some compounds. The work on residue monitoring has been divided into microbiological methods covered i n the preceding chapter and physicochemical methods which i s the topic of t h i s chapter. The d i v i s i o n between the two approaches i s somewhat a r b i t r a r y since many methods include elements of both approaches. Physicochemical methods are commonly used f o r i d e n t i f i c a t i o n and/or quantitation of residues detected by various types of screening methods, although they can be used for d i r e c t testing f o r residues. Successful methods mainly employ either high voltage electrophoresis or chromatography f o r separation of compounds and I w i l l discuss a p p l i c a t i o n of these two approaches to residues i n food substrates. For the present discussion, sulfonamides are also included, since they are used i n a s i m i l a r manner to a n t i b i o t i c s . Electrophoresis High voltage electrophoresis (HVE) i n agar gel with detection by bioautography has been used with considerable success i n some laboratories f o r i d e n t i f i c a t i o n of residues (1-6). This procedure has the advantage that a l l a n t i b i o t i c substances detectable by bioautography can be c l a s s i f i e d on the basis of electrophoretic mobility. Further t e s t i n g may be required f o r quantification and to This chapter not subject to U.S. copyright. Published 1986, American Chemical Society

In Agricultural Uses of Antibiotics; Moats, W.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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14.

MOATS

Physicochemical Methods for Identifying Antibiotic Residues

155

distinguish compounds with s i m i l a r electrophoretic m o b i l i t i e s , especially i f only one buffer i s used (7,8). Natural microbial i n h i b i t o r s found i n some animal tissues form a streak unlike any a n t i b i o t i c compound. Smither et a l (9) examined 5442 UK-produced meat samples using the four plate test (FPT) of the European community. Of these, 34 were i n i t i a l l y p o s i t i v e . However, electrophor e s i s demonstrated that only two of the p o s i t i v e s were recognizable a n t i b i o t i c s . On r e t e s t i n g , 20 of the samples o r i g i n a l l y p o s i t i v e were negative and 12 samples were found to contain natural microbial i n h i b i t o r s . Van Schothorst and Van Leusden (5) reported good agreement between electrophoresis and bioassays f o r confirmation of residues found i n kidney. Engel et a l (10) found that of residues detected i n kidney and muscle by the European four-plate test (FPT), only 50% and 37%, respectively, could be confirmed by HVE. They concluded that HVE i s l e s s s e n s i t i v e than the FPT. Chromatographic Methods Enough chromatographic methods f o r a n t i b i o t i c s have been described to warrant a book on the subject (11). These are, however, mainly f o r formulations and c l i n i c a l applications and a p p l i c a t i o n to residue analysis has been rather l i m i t e d . Residue analysis requires greater s e n s i t i v i t y and i s o l a t i o n from more complex substrates than i s the case with other a p p l i c a t i o n s . However, considerable progress has been reported i n recent years, e s p e c i a l l y with chloramphenicol and the sulfonamides. Thin layer chromatography (TLC), high performance l i q u i d chromatography (HPLC), and gas l i q u i d chromatography (GLC) have a l l been used. The applications of GLC f o r analysis of drug residues i n tissues were recently reviewed by Petz (12). Chromatographic methods are frequently suitable f o r determination of residues of a number of compounds i n a single procedure. They also have the potential to detect metabolites. Further confirmation by spectrophotometry and/or mass spectrometry i s possible. A discussion of the a p p l i c a t i o n to s p e c i f i c a n t i b i o t i c residues follows. Sulfonamides Rapid progress has been reported i n the development of methods f o r sulfonamide residues i n t i s s u e s , milk, and eggs since the subject was reviewed by Horwitz (13) i n 1981. The colorimetric method of T i s h l e r et a l (14) has i n the past been used to detect v i o l a t i v e l e v e l s of sulfonamide residues i n animal t i s s u e s . The lack of s p e c i f i c i t y and the variable background l e v e l s produced by t h i s method have been discussed by Horwitz (13), Matusik et a l (15), and Lloyd et a l (16). Recently, a number of s p e c i f i c chromatograpKic methods have been described f o r determination of residues of a v a r i e t y of sulfonamides. These are summarized i n Table I and suggest that HPLC i s emerging as the method of choice followed by GLC and TLC methods. The methods l i s t e d do not include a number described f o r blood and/or urine only. The HPLC methods mainly use UV detectors, but one uses amperometric (18) and one uses fluorescent detection (25). Fluorescent detection a f t e r d e r i v a t i z a t i o n with fluorescamine i s the method most commonly used f o r detection on TLC plates. V i l i m (24) used TLC to

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Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch014

Table I,

Chromatographic Methods for Determination of Sulfonamide Residues i n Tissue, Milk, and Eggs

Method

Substrate

Compounds

Detection

Sensi- Refert i v i t y ence (ppb)

HPLC

Chicken, tissue eggs

SFX, SMMf/ SDM, SQX

UV

5

(17)

Liver, kidney muscle

Several

Amperometric

10

(18)

Chicken tissue

SQX

UV

10

(19)

Chicken tissue

SMM, SDN, SQX

UV

10-30

(20)

Swine l i v e r

Glycopyranosyl SMZ

UV

10

(21)

Chicken tissue, eggs

SQX

UV

10-30

(22)

Beef tissue

SMZ

UV

100

(23)

Pork tissue

SMZ

UV

50

(24)

Chicken tissue, eggs

SMM, BDM, SQX

Fluorescamine Derivative

TLC

—

(25)

Eggs, meat, milk SMR, SDZ SDD, SMX, SQX

UV

100

(26)

Swine tissue

SMZ

UV

50

(27)

Swine tissue

5

UV

50

(28)

Pork l i v e r

SMZ, STH

Colorimetric

100

(29)

Liver, muscle

SMZ, SDM STH, SQX SBM

Fluorescamine Quantity