Experimental studies on trichinosis in Swiss mice

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Experimental studies on trichinosis in Swiss mice

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EXPBEIMSKm STUDIES ON TRICHINOSIS IN SWISS MICE

by Richard Bean Sjbcmer

A dissertation submitted in partial fulfillment of the requirements for th© degree of Doctor of Philosophy, in the Department of Zoology in th© Graduate College of th© State University of Iowa August, 195>0

ProQuest Number: 10583790

All rights reserved INFORMATION TO ALL USERS The quality o f this reproduction is d e p e n d e n t upon th e quality o f th e co p y subm itted. In th e unlikely ev e n t th a t th e author did not send a c o m p le te manuscript an d there are missing pages, these will b e no ted . Also, if m aterial h a d to b e re m o v e d , a n o te will indicate th e deletion.

uest ProQuest 10583790 Published by ProQuest LLC (2017). Copyright o f th e Dissertation is held by th e Author. All rights reserved. This work is p ro tected against unauthorized copying under Title 17, United States C o d e Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346

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Th© writer wishes to express his appreciation to Dr* L* 0* Nolf of the Department of Zoology and to Dr* Albert P* McKee of the Department of Bacteriology for their helpful advice and criticism during th© progress of this study. My sincere thanks are extended to Dr* John R* Porter, Head, of the Department of Bacteriology and to Dr* William M* Hale, formerly Head of th© Department of Bacteriology for their assistance and suggestions in the investigation of this problem. This work was aided by a grant from the National Institutes of Health administered by Dr. Joseph H. Bodine*

The writer is deeply

indebted to Dr. Bodine for his generous assistance.

ii

TABLE OF CONTENTS

page INTRODUCTION............. ..................................

1

MATERIALS AND METHODS

3

.........

EFFECTS OF GRADED DOSES OF TBICHINELLA SPIRALIS IN SWISS MICE . Results and Discussion . . . . . . . . . . ............ .

L L

FACTORS ASSOCIATED WITH A 100 LARVAE PER GRAS INFECTION OF TRICHI NELLA SPIRALIS AND THE EFFECTS OP A DIET SUPPLEMENT UPON THE CONDITION OFTHE H O S T .......................... Experiment I . . . . . . . . . . . . . . . . . ........ • Results and Discussion .......... . . . . . . . . . . Experiment 2 . . . . . . . . . . . . . . . . . . . . . . . Results and Discussion. . . . . . . .....................

S 3 9 10 1L

SECONDARY BACTERIAL INFECTION AND THE EFFECTS OF ANTIETOUCS UPON SURVIVAL TIME AND THE BLOOD PICTURE OF SWISS MICE INFECTED WITH TRICHXRELLA SPIRALIS , Experiment 1 . . . . . . . . . . . . . . . . . Experiment 2 . . . . . . . . . . . . . . . . . . . . . . . Experiment 3 * . . . * ........ . . . . . . . . . . . . . Experiment L . . . . . . . . . . . . . . . Experiment £ . . . ........ . . . . . ... . . Results and Discussion . . .....

22 23 23 2U 26 27 31

SUJBIART

........................

B I B U O O R A F H T .............................

iii

LI

TABUS OF FIGURES Figure 1

2

page Showing the Effects of Graded Boses of T* Spiralis in Survival Tim© and Per Cent Survival for Each Group Consisting of Fifty Swiss Mice * * * * • » * • • • Showing Average Amount of nPurina, laboratory Chow1* Consumed Bally per Animal Through Twenty-four Days of Observation

$

1$

3

Showing Average Amount of Water Consumed Daily per Animal Through Twenty-four Bays of Observation * * # . * 16

h

Showing Average Bectsl Temperature for Ten Mice

5 6 7

8

«

* * * 17

Showing Average Body Weight Daily per Animal Through Twenty-four Days of Observation * • • • » • * « • « *

18

Showing Survival Time and Per Cent Survival for Six Groups of Mice Used in th© Diet Supplement Experiment. •

21

Showing the Effects of Xntraperitoneal Injection of Penicillin and Streptomycin Upon Survival Tim® and Per Cent Survival of Mice Infected with a 100 Ipg Dose of T. Spiralis

28

• •

Showing the Effect© of Subcutaneous Injection of Crysticillim and Pihydro© treptomycin upon Survival Time and Per Gent Survival of Mice Infected with a 100 Ipg Dose of T* Spiralis • • » * • • • • • » » • •

39

TABLE OF T A ® »

Table 1 II HI

TV

V

VI

VII

VIII

page Composition of Diet Supplement................. Diet Regimen for Supplement Experiment

12 13

Results of Cultures Obtained from Ventricular Blood and from the Peritoneal Cavity of Twenty Mice Through Twenty-four Days of a 100 Ipg Infection of T» Spiralis • • • • • • • • . .......... . .........

25

The Figures Represent the Mean Erythrocyte Count for Ten Mice Through Twenty Days of a 100 Ipg Infection of T. Spiralis .........

32

The Figures Represent th© Mean Hemoglobin Value for Ten Mice Through Twenty Days of a 100 Ipg Infection of T. Spiral!© • • • • • • • • • • . . , •

33

The Figures Represent the Mean leucocyte Count for Ten Mice Through Twenty Bay® of a 100 Ipg Infection of T. Spiralis • • • * • • • • » ........

3b

The Figures Represent th© Mean lymphocyte and Monocyte Count for Ten Mice Through Twenty Bays of a 100 Ipg Infection of T. Spiralis . . . . * • • •

35

Th© Figures Represent the Mean Neutrophil and Eosinophil Count for Ten Mic© Through Twenty Days of a 100 Ipg Infection of T. Spiralis • . . . . . .

36

v

1

INTRODUCTION There have been several references in the literature concerning the wide range of susceptibility found in single host species of laboratory animals to experimental infections with Trichinella spiralis.

Roth (1939) reviewed the varied results re­

ported by different investigators using the guinea pig as the experimental host.

A considerable variation in the susceptibility

of the laboratory rat to Trichinella Infection may be noted from the work of McCoy (1931) and Trawinski (1935)•

Inspection of the

literature reveals similar inconsistencies in the susceptibility of the laboratory mouse to infections of T. spiralis (Glaser, 1920; Culbertson and Kaplan, 1938; Rappaport, 19U3). The question of a difference in virulence of different strains of T. spiralis as a contributing factor in the variability encountered was investigated by Rappaport (19U3) • Three strains of the parasite from different sources were studied with the albino mouse as the experimental host.

From this work there appeared to

be little or no variation in virulence of the three strains of T. spiralis as determined by the lethal dose of larvae and survival time of the mice. That the laboratory mouse is subject to numerous infectious diseases is well known, consequently the outbreak and manifestations of a latent infection accompanying an experimental Trichinella infection may be misleading in the interpretation of experimental

2

data*

Th© use of th© Swiss strain of the albino mouse as an experi­

mental host for fm spiralis has not been reported*

Th© first part

of this investigation was undertaken to determine the susceptibility of Swiss mice to graded doses of Trichinelia larvae.

The work pre­

sented in th© second part is an attempt to establish several factors associated with an infective dose of 100 larvae per gram of body weight.

The third part deals with secondary infections and the

effects of several antibiotics on the blood picture and survival time of the host.

5

MATERIALS AND METHODS The Swiss strain of albino mice used in this study has been maintained as an inbred colony by the Department of Bacteriology of the State University of Iowa.

The strain was obtained originally

from the U. S* Department of Agriculture in 1933.

These animals

were normally resistant to Salmonella infections and were free from lymphocytic choriomeningitis and mouse pneumonitis.

Breeder stock

and experimental animals, unless otherwise indicated, were fed a diet of ”Purina Laboratory Chow,” water ad libitum and lettuce twice weekly#

All the mice used in experimental work were ten weeks of

age ~ one week at the time of infection. The Trichinella larvae used for infective purposes were isolated from a wild rat and have been maintained in hooded rats for several year®.

All larvae used as infective doses were obtained

from stock rats with infections of at least six weeks duration. Th© larvae were isolated by the pepsin digest method and known doses were determined by the dilution count method of McCoy (1931). Infective doses of larvae were concentrated in conical centrifuge tubes*

Using a rubber bulb, the larvae were drawn with about 1 cc.

of tap water into a small bore tapered pipette*

By etherization

of the mice it was possible to pass the pipette through the esophagus and introduce the larvae directly into the stomach.

The specific

materials and procedures employed are described with respective experiments.

4

EFFECTS OF GRADED DOSES OF TRICHINELLA SPIRALIS IN SWISS MICE Th© data presented here were taken from several experiments in which mice were infected with graded doses of 5* 25, 50, 75, 100, 200, I4OO and ?00 Trichinella larvae per gram of body weight* Larvae per gram of body weight will hereafter b© referred to as (Ipg)*

Fifty animals, consisting of an equal number of males and

females, were used in each of the dosage levels tested*

The average

weight of the mice at th© time of infection was 23*5 grams, with a standard deviation of 1*52 grams*

An equal number of controls

was carried in the experiments * All animals that survived th® infection were observed for a period of ninety days from th© date of infection* Results and Discussion

The results of th©s© experiments are shown in figure 1. It is indicated from the data that Swiss mice tolerate infective doses of 5 and 25 Ipg with a group survival of 100 and 96 per cent respectively*

Rappaport (19U3) in a comparative study of three

strains of T. spiralis, reported 100 per cent survival for infective doses of 10 and 20 Ipg.

In the same study 6k and 65 per cent survival

was reported for respective doses of 30 and 36-1*5 Ipg* dose of 1*6-55 Ipg resulted in a 1*1* per cent survival#

An infective As shown in

LARVAL D O S E P ER G R A M B O D Y W E I G H T 0

D AY S

O F

%

I N F E C T I O T vl

S U R ­ 0 ■

i

4 i

i

8 i

i

12 i

16

2 0

2 4

2 8

32

36

(CONTROL)

VIVED

100

5

100

25

96 *



5 0

86



75

* • •

• • • • •

• • • • • • •

100



• • • * • • •





• • • » • • • • • • • • • • • • • • • » • • •

• • • • • • • • • • • * • • • • • • • • • • «

• • • * •

14

0 • •



0

2 0 0 • • • • • •

• • m • • • • • •

4 0 0

5 0 0



4 0

• • • • • • • • • • • •

• • * • m

0 • m • • •

m • • • •

• • • • • * • *

• • • • • • • • • • • •

• • • • • • • * • • • • • • • •

REPRESENTS

Figure !•

m • • • • • • • • • m m m • • • •

0

DEATH

OE

ONE

A N IM A L

Sbowing the effects of graded doses of ?• spiralis in survival tine and per cent survival for each groun consisting of fifty Swiss mice.

6

figure X, 86 per cent of the Swiss mice survived a 50 Ipg dose of t* spiralis# Glaser (1920) reported a total dose of 600 Trichinella larvae as fatal in thlrty-on© days, a total dose of 1,200 larvae was fatal in seven days#

In a study of passive Immunity by

Culbertson and Kaplan (1933), infected control mice exhibited a 33*1 per cent survival with a total infective dose of 300 larvae# Thirteen out of twenty-on® animals died within thirteen days of infection* It may be noted from the data shown in figure 1, that an increase in larval dosage from 50 to 75 Ipg results in a sharp decrease from Bh to lit per cent survival#

The deaths occuring with

a 50 Ipg dos© were widely scattered from the second week through th© sixth week of infection#

With a 75 Ipg dose the deaths appear

to be grouped into two areas, an initial death period in th© second week and another death period in the fourth and fifth week of infection#

A 100 Ipg parasitism demonstrated more clearly an initial

death period from the sixth to the twelfth day and a second death period beginning in the third week and extending through th© fourth week of infection*

There were no survivors with 100 Ipg infections

of Trichinella larvae# Rappaport (19U3) reported that all mice receiving an overwhelming dose of 130-280 Ipg died within two and four days after infection#

With graded doses of 200 and 1x00 Ipg all the

7

Swiss mice died between the second and twelfth day of infection. An infective dose of £00 Ipg resulted in the death of all the mice within one and three days#

Severe intestinal damage and marked

hemorrhage were noted with infections of 100, 200, U00, and £00 Ipg, With a 100 Ipg infective dose the damage to the intestinal mucosa was largely repaired by the third week of infection, Rappaport (19U3) found little or no difference in the virulence of three strains of T* spiralis as determined by the lethal dose of larva© and survival time of the mice.

Therefore, the wide

range in the susceptibility of mice to Trichinella infections may b© due in part to secondary processes such as the outbreak of a latent bacterial or virus infection.

The mice used in th© present

study were normally resistant to the more common epizootics found in mouse colonies, Th© data at hand indicates that in regard to per cent survival Swiss mice were less susceptible to comparable infective doses of T, spiralis than were the mice used by the investigators previously cited.

Moreover, Swiss mice exhibited a longer period

of survival with even larger infections.

8

FACTORS ASSOCIATED WITH A 100 LARVAE PER GRAM INFECTION OF TRICHINELLA SPIRALIS AND THE EFFECTS OF A DIET SUPPLEMENT UPON THE CONDITION OF THE HOST The appearance of two death periods with an infective dose of 100 Ipg served as the basis for the following experiments*

The

purpose of this study was to establish several factors associated with the two death periods, such factors to be considered are food and water consumed, rectal temperature and body weight of the host* Data are presented on the effects of a parent©rally administered diet supplement upon th© morbidity and mortality observed with a 100 Ipg parasitism of T* spiralis* Several characteristic symptoms were noted in Swiss mice during the course of a 100 Ipg infection*

By the second day the

animals appeared uneasy and the fur was ruffled*

Diarrhea began in

the third day and persisted for about five days.

During this period

the mice were markedly slow in movement and huddled in a corner of th© cage*

From about day ten to day seventeen the animals were more

active and appeared to be convalescent.

The succeeding and terminal

days of Infection were characterised by muscle weakness, loss in weight, evidence of malnutrition and stupor leading to a moribund condition. Experiment 1 An experiment was undertaken in which daily food and water consumed and body weight were recorded for two groups of mice through

9

twenty days of infection*

Ten female mice were infected as previously-

described, an equal number of females served served as a control* All mice in this and later experiments were housed in metal ca-es with a solid floor, sawdust was used as litter material*

lfPurina

Laboratory Chow” was available at all time in a metal feeder with a one-half inch wire mesh bottom*

The amount of food consumed by each

group was measured by direct weight comparison of the feeder and contents daily*

Water bottles equipped with one-quarter inch inside

diameter glass tubes were inserted through the roof of the cage* Th© length of the tub© was regulated so that th© mice had to raise off the floor to drink*

The volume of water consumed daily was

determined by direct weight comparison of the water bottle, drinking tub© and contents*

It should be noted here, that the above methods

are an attempt to arrive at an approximation of the amount of food and water actually consumed* Results and Discussion

Over the twenty day period of observation th© control mice consumed an average of U *1 grams of chow per day per animal* Infected mice consumed 1*2* £ per cent less chow with a daily average of 2*U grams per animal• Water intake for control mice averaged 7*2 cc* per day per animal*

Infected mice consumed 3U*2 per cent

less water with a daily average of U*7 cc* per animal*

Infected

mice suffered an average weight loss of £,9 grams with a standard deviation of 1*36 grams*

The control group made an average gain

10 of 1*7 grams with a standard deviation of 0.81 grains*

The day to

day observations are not reported here* since data for similar groups , serving as controls, are presented in the following experiment. See group I (normal control) and group IV (infected control) in figures 2, 3* U, and 5* In consideration of a 26*86 per cent protein content of **Purina Laboratory Chow,” a U2*£ per cent reduction in food consumed results in an average daily loss to infected mice of approximately U68 mg* of protein.

In regard to carbohydrate content of the chow,

the infected animals facedan of carbohydrate daily*

average loss of approximately 819 mg#

In addition to the marked reduction in food

and water consumed, severe intestinal damage by th© parasite may interfere with absorption of digested food.

Frothingham (1909)

and Gursch (19U9) studied the intestinal phase of T* spiralis in rats and reported extensive damage of th® villi and mucosa*

More­

over, th© metabolic pool and food stores of the host are subjected to th© nutritional demands of the parasite. Experiment 2 In consideration

ofth© observed anorexia, loss in weight

and debility of the host} the question was raised as to the effects of a diet supplement upon the morbidity and mortality associated with a 100 Ipg infection of T. spiralis.

An experiment was carried

on in which a diet supplement was administered parenterally by subcutaneous injection of the mice*

11

Th© composition of the supplement and the total amount of each constituent injected daily are shown in Table I. supplement was given xn

2 cc * closo3 at

The ^nt^rv^a"Is#

Each dose consisted of 1*1* cc* Amigon (10 per cent solution)* a nonpyrogenic solution of a pancreatic hydrolysate of casein, containing amino acids and polypeptides ; 0*5> cc* of Bacto—Dextrose (£0 per cent solution of d-glucose) and 0*1 cc# of a vitamin mixture*

Purified

diet studies by Woolley (19U1), Gerecedo and Mirone (1$?U7) served as the basis for the composition of the vitamin portion of the supplement* All solutions were made up with pyrogen free 0*8£ per cent saline and rendered sterile by filtration through filter, porosity sise P—3*

a Coops

porcelain

The solutions were stored separately

in amber glass bottles at ii°C and combined daily for injection; sodium bicarbonate was used to adjust the pH of the supplement to the alkaline side of neutrality*

Prior to subcutaneous injection of the

mice the supplement was warmed to about 37°C* A total of 120 mice were used in this experiment, the animals were separated into six groups, composed of ten males and ten females# The description and diet regimen for each group are shown in Table II. Hale and female mice were housed in separate cages under the condi­ tions previously described*

Using th© methods detailed in the previous

experiment, a daily record was kept of the food and water consumed by each group*

The average body weight was recorded daily by weighing

12

Table I Composition of Diet Supplement

Constituent

Total Administered Daily

Aralgen (Mead Johnson and Co.)

U20 milligrams

Bacto**Dextros (Bifeo)

75>0 milligrams

Thiamin chloride

60 micrograms

Riboflavine

60 micrograms

Pyridoxine hydrochloride

150 micrograms

Nicotinamide

600 mierograms

Ascoihic acid

600 micrograms

Inositol

3,000 mierograms

Choline chloride

3,000 micrograms

Calcium pantothenate (dextrorotatory) p-Aminobenzoic acid Folic acid

600 micrograms 3,000 mierograms 30 micrograms

15

Table II Piet Regimen for Supplement Experiment

Group

No* of Mice

Description

Diet Regimen

I

20

Control

*Stock diet

II

20

Control

tfStock diet plus supplement

III

20

Control

IV

20

^Infected control

*Stock diet

V

20

*HfrInfected experimental

#Stock diet plus supplement

VI

20

•^Infected control

*

Supplement (only)

Supplement (only)

Stock diet consisted of "Purina Laboratory Chow/’ lettuce twice weekly and water ad libitum* Infected mice received 100 Ipg dose of T* spiralis*

14

the animals from each cage as a unit*

All the mice were weighed

prior to the injection of the supplement. Xn preliminary experiments a lowered body temperature was noted in handling infected mice. were

Therefore, rectal temperatures

recordedonce daily from 2s00 to lj.:00 p.m. for five males and

fivefemales drawn

at random from each group.

After the method of

Congdon (1912), rectal temperatures were measured with a clinical thermometer.

The small bulb of the thermometer was lubricated with

vaseline and inserted into the rectum to a depth of one-half inch in all cases.

Congdon (1912) recorded the changes in rectal tem­

perature of mice held at different room temperatures*

At a room

temperature of 2£°C the average rectal temperature was 3£.3°C. At $°C room temperature the rectal temperature was 31.2°C.

In the

present study room temperature varied from 21.1°C to 26.1°C.

The

average rectal temperature for ten normal Swiss mice over a period of ten days was 3?.9°C with a range of 36*7°G to 38*8°C and a standard deviation of G.Ul. Results and Discussion

The data obtained over a period of twenty-four days of infection for food and water consumed, rectal temperature and body weight are shown in figures 2, 3, U, and 5 respectively.

All

observations are plotted as the average per animal per day.

In

regard to food and water consumed, rectal temperature and body weight,

FOOD

IN

GRAMS

15

GRO UP X GRO UP I GROUP

-----•— •+■»■

GROUP E GROUP 3C GROUP

20 DAYS

Figure 2.

OF

22

24

IN FEC TIO N

Shewing average amount of nPurina Laboratory Chow” consumed daily per animal through twenty-four days of observation. Each group consisted of twenty mice. Group Group Group Group Group

I (control* stock diet). II (control* stock diet plus supplement)• H I (control, supplement only), (not given food). IV (infected control, stock diet). V (infected experimental, stock diet plus supplement). Group VI (infected control, supplement only), (not given food). Groups IV, V and VI received a 100 Ipg infective dose of T. spiralis.

WATER

IN

(cc.)

16

GROUP X GROUP I G RO UP H

GROUP GROUP 3 t GROUP 3 1

20 DAYS

Figure 3.

OF

22

IN F E C T IO N

Showing average amount of water consumed daily per animal through twenty-four days of observation. Each group consisted of twenty mice. Group Group Group Group Group

I (control, stock diet). IX (control, stock diet plus supplement). I H (control, supplement only). IV (infected control, stock diet). V (infected experimental, stock diet plus supplement). Group VI (infected control, supplement only). Groups IV, V and VI received a 100 Ipg infective dose of T. spiralis.

24

17

RECTAL

TEMP.

DEG.

CENTIGRADE

38.6

38.0

3 7.4

36.8

3 6,2

35.6

3 5.0 GROUP X GROUP I GROUP

3 4 .4

-------------------

GROUP H GROUP 3C GROUP 5 1

20 DAYS

Figure U*

OF

22

24

IN FEC TIO N

Showing average rectal temperature for ten mice drawn at random from each group consisting of twenty mice* Group Group Group Group Group

I (control, stock diet)• II (control, stock diet plus supplement}* III (control, supplement only)• IV (infected control, stock diet)* V (infected experimental, stock diet plus supplement). Group VI (infected control, supplement only) *

Groups XV, V and VI received a 100 Ipg infective dose of T* spiralis*

18

28

26

22

20

GROUP X ---------GROUP I ---------GROUP U ■++++■

14

GROUP H «GROUP V * GROUP VT

r2 20

DAYS

Figure

OF

22

24

IN F E C T IO N

Showing average body weight daily per animal through twenty-four days of observation* Each group consisted of twenty mice* Group Group Group Group Group

I (control, stock diet)* II (control, stock diet plus supplement)* III (control, supplement only)* IV (infected control, stock diet)* V (infected experimental, stock diet plus supplement)* Group VI (infected control, supplement only)*

Groups IV, V and VI received a 100 Ipg infective dose of T* spiralis*

19

It may be noted that there were only minor differences between group IV (infected control, stock diet) and group V (infected experimental, stock diet plus supplement).

Groups IV and V demon­

strate a sharp decrease in the amount of food and water consumed and rectal temperature during the first week of infection.

This

was followed by a period of recovery for food and water intake and an increase in rectal temperature. Through the twenty-four day period of observation, group IV suffered an average weight loss of 6,1* grams per animal; during the corresponding period group V lost an average of £.6 grams per animal.

Thus subcutaneous injection of the supplement had little

effect upon loss in weight of the mice given a 100 Ipg infection of Trichinella larvae.

The terminal days of the infection resulted

in a gradual decline in food and water consumed, rectal temperature and body weight. Group II (control, stock diet plus supplement) consumed less food and water and exhibited a lower rectal temperature through­ out the period of observation than group I (control, stock diet). The mice of group II made an average gain in weight of 1.7 grams per animal, on the other hand, group I gained an average of 3.0 grams. In preliminary experiments ten mice were kept on a starvation diet (without food and water) and all the animals died within two and three days; with access to water only, another group of ten mice died within three and four days.

The survival time

20

and per cent survival for the six groups of mice are shown in figure 6. It is indicated from the period of mortality for group III (control, supplement only) and group IV that starvation may be a factor in the death of mice given a 100 lpg infection of T. spiralis.

That the

mice in group III were able to utilize a portion of the supplement is evidenced in an extended period of survival when compared with the survival time of mice kept on a starvation diet* In group IV (infected control, stock diet) eight out of twenty, i.e*,

per cent of the mice died during the initial mortality

period of six to ten days*

As determined by the survival time of

the infected mice in group V, subcutaneous injection of the supplement was detrimental in that fifteen out of twenty, i.e., 75 por cent of the animals died during the corresponding period of infection* From these experiments, it is evident that a sharp decrease in food and water consumed, loss in body weight and lowered rectal temperature are associated with the initial mortality period observed with a 100 lpg infection of T. spiralis.

A recovery in the amount

of food and water consumed and an increase in rectal temperature was noted from about day ten to day seventeen.

During this period

the infected mice appeared to be convalescent.

The terminal days

of infection and second period of mortality are characterized by further loss in weight, a decline in food and water consumed and lowered rectal temperature*

A parenterally administered diet supplement

resulted in an Increase in the number of deaths occurring in the initial mortality period*

21

GROUP T

01

DAYS 1 81

1 41

OF 1 12 i

°/o

1N F E C T I O N 16 20

SUR­

24

VIVED

CONTROL STOCK

__

100

DIET

CONTROL

100

S T O C K + SUPPL.

• •• • •• ••• •• •• •• ••

CONTROL

in

SUPPL.(ONLY)

INFECTED CONTROL

w

STOCK

0

• • •• • •• •

DIET

INFECTED EXPERIMENTAL STOCK + SUPPL.

• •

IN FE C TE D • • • •

CONTROL

Figure 6#

REPRESENTS



• • • • •



• • • • •

0

• •• ••

• 0

*

• •





0

• • • m m • • • • • •

S U P P L . ( ONLY)



• • • • • •

• • *• ••

DEATH

OF

ONE

AN IM A L

Showing survival time and per cent survival for six groups of mice used in the diet supplement experiment. Each group consisted 01 twenty mice* Groups IV, V and VI received a 100 lpg infective dose of T* spiralis#

22

SECONDARY BACTERIAL INFECTION AND THE EFFECTS OF ANTIBIOTICS UPON SURVIVAL TB'E AND THE BLOOD PICTURE OF SWISS WIGS INFECTED WITH TBXCBINELLA SPIRALIS The hypothesis that Trichinella infections favored bacterial infections was advanced by Pi ana in 1877, cited from Romanovitch (1912)#

Frothingham (1909) observed in rats the penetration and

destruction of intestinal mucosa and villi by T. spiralis, thus presenting an opportunity for entrance of bacterial organisms to the lymph channels and blood stream of the host*

Extensive damage

to the mucosa and villi was reported by Gursch (19U9) in a study of the distribution of maturing and adult trichinae in the digestive tract of rats*

Th© purpose of this study was to determine the role

of secondary bacterial infection in 100 lpg infections of T. spiralis in Swiss mice* became evident*

As the experimental work proceeded, new problems Data are presented on the effects of several anti­

biotics on the blood picture and survival time of the host* Contradictory evidence has appeared in the literature as to the role of secondary bacterial infection in trichinosis.

Herrick

(1915) reviewed the case histories of trichinosis diagnosed in th© Roosevelt and St* Lukes Hospitals of New York during a period of five years*

Of ten cases in which a blood culture was taken no bacteria

were demonstrated*

It should be noted here that blood cultures are

seldom determined in cases of human trichinosis.

Cummins and Carson

(1916) made blood cultures in seven out of fifteen cases and all

23

were negative*

In a severe case of trichinosis Staubli (1909)

reported streptococci in pure culture were isolated from the blood and urine*

In a second case an infection of staphylococci was observed

in deep intramuscular abscesses*

Staubli (1909) cultured the heart

blood and peritoneal exudate from two guinea pigs dying from experi­ mental trichinosis*

Bacteria were demonstrated in both animals, a

diplococcus in one and a streptococcus in the second*

Romanov!tch

(1912) examined th© blood of trichinosed rats, twenty-six out of thirty-six animals exhibited bacteremia* Experiment 1 This experiment was arranged to determine if bacterial organisms could be demonstrated in the peritoneal cavity and blood of twenty normal Swiss mice*

The animals were placed under ether

anesthesia and aseptic operating techniques were used to lay open the abdominal wall*

A hypodermic syringe was used to inject 2 cc*

of sterile saline into the peritoneal cavity, a 0*2 cc* sample was withdrawn and inoculated into a deep meat tube medium*

The peri­

cardial cavity was opened aseptic ally and 0*2 cc* of ventricular blood withdrawn for subsequent culture* at 37*5 C for forty-eight hours*

All cultures were incubated

In all cases examined the cultures

were negative* Experiment 2 In this experiment twenty mice were infected with a 100

24

lpg dog© of T. spiralis*

Cultures were obtained from ventricular

blood and from the peritoneal cavity as described in the previous experiment* in Table III*

Th© results from the culture determinations are shown In thirteen out of seventeen animals examined, i.e.,

76*h per cent, bacteria were found in one or both of the regions from which the cultures were obtained.

The bacteria were isolated

in pure culture and organisms were identified from th© following groupss

Streptococcus pyogenes, alpha hemolytic streptococci,

staphylococci and various members of the Enterobacteriaceae, none of which belonged to the genera Salmonella and Shigella*

In pre­

liminary experiments, bacterial organisms belonging to th© above groups were isolated from th© intestinal tract of ten normal mice* It may be noted here, that these organisms are usually harmless in the intestinal tract but are considered as opportunists when not confined to their natural environment* Experiment 3 The purpose of this experiment was to determine the effects of a bacteria free infective dose of T* spiralis upon the mortality found with a 100 lpg infection in mice*

In previous experiments,

bacterial sterility tests were determined on the larval doses used for infective purposes*

Bacterial organisms were found in the

larval doses in all cases examined# The Trichinella used In this experiment were Isolated as previously described*

The experimental group of infective doses

25

Table III

Results of Cultures Obtained from Ventricular Blood and from th© Peritoneal Cavity of Twenty Mice Through Twenty-four Days of a 100 lpg Infection of T. Spiralis

Mouse Ho*

Day of Infection Culture Obtained

1 2 3 h $ 6 7 8 9 10 11 12 13 Hi 15 16 17 18 19 20

k h 6 6 8 8 6 9 10 10 10 13 13 16 16 16 20 20 2h 2k

- negative 4» positive

Blood Culture

Peritoneal Culture

f

mm

44*

* 4-

4-

4*

¥

4 (died, -

mm

mm

not

examined) 4» mm

4-

4-





(died,

not

44*

(died, *

examined) mm

4-

not

examined) — *»

26

were washed ten times in $0 cc. of sterile tap water, the larvae were allowed to settle out by gravity in conical centrifuge tubes. Ten thousand units of Penicillin

G Sodium (Squibb) and five

thousand units of Streptomycin Hydrochloride (Squibb) were added to each of the final washed suspensions of larvae.

The control larval

doses along with the experimental doses were held at room temperature about seven hours before the mice were infected.

Prior to infection

of the mice the experimental larval doses were washed three times in $0 cc. of sterile tap water to dilute out the antibiotics* Subsequent culture on blood agar plates revealed the experimental doses to be bacteria free*

Bacterial organisms were demonstrated

in all control larval doses which were prepared in the usual manner. A sterile glass pipette was used to administer the infective doses as detailed earlier in the paper.

Ten mice were infected with th©

bacteria free Trichine 11a and an equal number were infected with the control doses. The results of this experiment demonstrated that there was no difference in the per cent survival and time of survival of th© two groups of mice.

There were no survivors in either group

and the survival time was similar to that demonstrated for a 100 lpg infective dose as shown in figure 1. Experiment U In consideration of the bacteremia noted in the second exoeriment, this experiment was carried on to determine if the

27

control of th© bacteremia with antibiotics would alter the mortality and time of survival associated with a 100 lpg infection of Trichinella in Swiss mice*

A total of sixty mice were employed in the study,

th© animals were divided into th© following group®; ten mice, group I (control); ten mice, group XX (antibiotic control); twenty mice, group XXI (infected control) and twenty mice, group IV (infected experimental) • Group XX and group XV were given a dally total dose of three thousand units of Penicillin G. Sodium (Squibb) and ?£0 units of Streptomycin Hydrochloride (Squibb) in three intraperitoneal injections at eight hour interval®. From the results shown in figure 7, it is evident that intraperitoneal injection of Penicillin and Streptomycin had no effect upon the mortality and time of survival of mice infected with a 100 lpg dose of T. spiralis.

Preliminary larval recovery

experiments demonstrated that th© antibiotics did not reduce the larval population harbored by the host.

It is thus indicated that

either th© secondary bacterial infection was not controlled or that bacteremia is not a factor in the mortality observed with a 100 lpg infection. Experiment 5 This experiment was arranged to determine; (1) the blood picture of Swiss mic© Infected with a 100 lpg dose of T. spiralis; (2) the effects of the antibiotics, Ciysticillin and

28

D A Y S

O F

%

I N F E C T I O N

G R O U P

SUR­ 0

X

4

8

12

16

2 0

2 4

2 8

VIVED

3 2

CONTROL

- n-

I O O

ANTIBIOTIC 1 0 0

CONTROL INFECTED CONTROL

TV





• • •

• *



• •



: •

• •

o

m







INFECTED • •

EXPERIMENTAL



REPRESENTS

i?i 'uro ? .

DEATH

OF

S v ie Poo f o l l i l n

• •.:

o n e

irl/rY y o rilo o o o l

oP -5.co L’ - -

TdJ

•• : '

. .



ANIMAL

;3 n:.l 3

,r• I ond

o



m

o llc ctn :. o f

.a3

^ Oi. 3 I

Ty-iy.

• •

*

in je c tio n

®

IVU.;- II C1^’.:01O''\0V O . 1,01 "i’ v i ; 0

IV

D.I

• v v . I \ . :.di I . c vnd :n i c e tod ' L I ; s. 1 •' ‘ I ' l l

C0 - 0 IS l e i

O l

•-O'»

T.’. EO;:

' ‘."IOC®

29

Dihydrostreptomycin, upon th© blood picture of Trichinella infected mice and (3) the effects of the antibiotics upon the mortality and survival time of trichinosed mice, th© observation of ©osinophilia as an aid in the diagnosis of trichinosis was reported by Brown (1897)*

Howard (1899) first

reported the absence of eosinophilia in fatal trichinosis,

Aldridge

(1931) found in twenty-nine cases of trichinosis that an ©osinophilia was present in twenty-seven patients who recovered and absent in two patients in whom the infection was fatal. It was demonstrated by Opi© (190U) and confirmed by Spink (193U) that introduced secondary bacterial infections caused a reduction and gradual disappearance of eosinophils from th© peripheral blood of Trichinella infected guinea pigs.

In view of th© bacteremia

observed in the second experiment with 100 lpg infections in mice, the question arises as to the effects of secondary invasion of enteric bacterial organisms upon the blood picture of trichinosed mice.

If th© secondary bacterial infection could b© controlled

with antibiotics, the results of this control might be evident in the early appearance of an ©osinophilia*

Data aie presented on th©

effects of Crysticillin and Dihydrostreptomycin upon the blood picture and survival time of Swiss mice given 100 lpg infections of T* spiralis, Four groups of mice were employed in this experiment, each of the following groups consisted of twenty female mice; group I

30

(control)| group II (antibiotic control)\ group III (infected control) and group IV (Infected experimental)*

The animals of group II and

group IV were given 1,500 units of Crysticillin (Squibb) and 2,000 units of Dihydrostreptomycin Sulfate (Merck) once daily by subcutaneous injection* groups

The following hematologic data were determined for each

(1)

total erythrocyte count per cu. mm*, (2)

count per cu* mm*, (3) and (U)

total leucocyte

hemoglobin determination in grams per 100 ml*

leucocyte differential*

The observations were made at five

day intervals through twenty days of infection*

Ten mice were drawn

at random and examined from each group, the hematologic findings are reported as the mean for ten animals* All blood samples were obtained from the tail*

A Haden-

Hausser Hemoglobinoiaeter (clinical model) was used in the determination of hemoglobin*

Blood films were stained with Wright*s stain*

The

classification of leucocytes in the differential counts was based upon a blood morphology study of normal white mice by Petri (1933)* In the present study leucocytes are divided into four groups? lymphocytic, monocytic, neutrophilic and eosinophilic*

Pre-

neutrophilic myelocytes are recorded as neutrophils and pre-eosinic myelocytes are reported as eosinophils*

A total of 100 leucocytes

were counted from each blood film, the observations are recorded In per cent*

31

Results and Discussion

It is shown from the results in Table IV, that there was no change in the total erythrocyte count through the twenty day period of observation.

A slight decrease in hemoglobin may be noted

from Table V on day fifteen and day twenty for group H I (infected control) and group IV (infected experimental).

Calculation of

the significance of the difference between the two mean revealed that the observed differences may have arisen due to chance. The data obtained from the total leucocyte counts are shown in Table VI*

A slight increase in th© total leucocyte count was

found for group III and group IV on the tenth day of infection. The observed increase was not significant. Th© results of the leucocyte differential counts are presented in Table VII and Table VIII*

A neutrophilia associated

with a lymphopenia may be noted in group i n and group IV beginning by th© fifth day of infection.

The neutrophils of group III reached

a maximum of U8.3 per cent by the tenth day of infection, a 37*9 per cent neutrophil count was observed for group IV on the correspond­ ing day.

The UQ*3 per cent neutrophilia in group III is significant,

however, the 37*9 per cent neutrophil count in group IV is not significant. Lawrence and Josoy (1932) followed the changes in leucocytes of th© peripheral blood of guinea pigs given intraperitoneal

52

Table IV Th© Figures Represent the Mean Erythrocyte Count for Ten Mice Through Twenty Days of a 100 lpg Infection of T. Spiralis

Mean Erythrocyte Count (Number per cu. mm., Peripheral Blood)

Group Day of Observation

I . . (Control)

II (Antibiotic Control)

III (Infected Gontrol)

IV (Infected Experimental)

0 5 10 IS 20

9 ,7 7 0 ,0 0 0 9 ,7 8 6 ,0 0 0 9 ,8 6 5 ,0 0 0 9 , 8U0, 000 9 ,7 1 9 ,0 0 0

9,66 0,00 0 9 ,7 62 ,00 0 9 ,79 5,0 00 9 ,7 9 9 ,0 0 0 9 ,8 2 0 ,0 0 0

10,31*0,000 10, 115,000 io,U 85,ooo 9,1*80,000 *9 ,7 0 7 ,0 0 0

10,066,000 9, 781*, 000 9 ,85 0,0 0 0 9,315,000 9,75 5,0 00

Bang.

(3 ,6 0 0 ,0 0 0 11, 150, 000)

( 8, 300, 00011, 500, 000)

(8 ,3 5 0 ,0 0 0 1 2,1 0 0,0 0 0 )

(8,1*1*0,00011, 350, 000)

S .D .

i.E. of Mean

680,000

755,000

860,000

710,000

96,181

106,789

100,1*21*

122,857

* Mean for nine animals

53

Table V The Figures Represent the Mean Hemoglobin Value for Ten Mice Through Twenty Bays of a 100 lpg Infection of T. Spiralis

Mean Hemoglobin Value (Grams per 100 ml., Peripheral Blood) Group

(Control)

II (Antibiotic Control)

III (Infected Control)

15* k 15.U 15.3 15.3 15.5

15.3 15.U 15.U I5.lt 15.3

15.lt 15.U 15.2 H i. 1 *lit.U

I Day of Observation

0 5 10 15 20

Range S.D. S.iS. of Mean

*

(1 2 .0 -1 6 .5 )

IV (Infected Sxperimental)

15.U 15.5 15.1 llt.O llt.lt (1 2 .5 -1 6 .0 )

(iit.o~l6.o)

(1U.0-16.0)

Q.U3

G.U2

0.81*

0 .3 5

0.06

0.059

0.119

0.119

Mean for nine animals

34

Table VI The Figures Represent the Mean Leucocyte Count for Ten Mice Through Twenty Days of a 100 lpg Infection of T* Spiralis

Mean Leucocyte Count (Number per cu. mm., Peripheral Blood)

Group I Day of Observation

0 5 10 15 20 Range S.D. S.E. of Mean

(Control)

11 (Antibiotic Control)

III (Infected Control)

IV (Infected Experimental)

12,280 11,900 15,350 13,030 13,150

12,700 12,370 12,770 12,31*0 12,175

11,835 11,550 12,335 12,010 12,205

(8,250** 19,200)

(7 ,1 0 0 18,1*00)

(5 ,o 5 o 19,000)

(8,1*5022,1*00)

2,1*20

2,570

3,lt*o

3,080

3U2

363

1*1*8

1*35

* Mean for nine animals

11,565 11,315 H i, 51*5 12,150 *1 0 ,5 5 5

35

Table VII Th© Figures Represent th© Mean Lymphocyte and Monocyte Count for Ten Mice Through Twenty Days of a 100 lpg Infection of T. Spiralis

Mean Lymphocyte Count In Per Cent (Peripheral Blood) Group I Day of Observation

0 5 10 15 20 Range S.D. S.E. of Mean

(Control)

68.9 65.7 69.1* 66.6 67.7

II (Antibiotic Control)

68.U 68.9 70.0 69.8 68.0

III (Infected Control)

66.7 55.0 lil*5 U8.1 *38.2

IV (Infected Exp© rimental)

71.2 59.2 U9.0 U9.3 37.9

(55-80)

(20-710

(21—81

lt.61*

5.8

llt.6

1U.5

0.66

0.82

(57-76)

2.06

2.05

Mean Monocyte Count in Per Cent (Peripheral Blood) 0 5 10 15 20 Range S.D. S.E. of Mean

9.0 12.6 9.2 10.3 *11.7

7.6

9.6 10.9 10.0 8.6 8.9

9.2 9.6 9.8 1Q.U 8.3

(k-15)

(U-20)

U.58

3.05

3.7

2.62

0.6U

0.U3

0.53

0.37

* Mean for nine animals

(2-18)

10.5 8.8 8.6 8.1* (1KL7)

56

Table VIII Th© Figures Represent th© Mean Neutrophil and Eosinophil Count for Ten Mice Through Twenty Bays of a 100 lpg Infection of T. Spiralis

Mean Neutrophil Count in Per Cent (Peripheral Blood) Group I Bay of Observation

0 5 10 15 20 Range S.D# S.E. of Mean

(Control)

II (Antibiotic Control)

III (Infected Control)

XV (Infected Experimental)

20.? 22.5 19.5 23.9 22.1

21.2 20.3 19.5 18.8 22.2

22.9 31.7 U8.3 39.8 *U6.0

20.0 29.3 37.9 37*k 1*6.9

(12-36)

(12-36)

7.3

li.9

1.1

0.69

(17-67) 13.6

1.9U

(12-65) 12.5 1.71

Mean Eosinophil Count in Per Cent (Peripheral Blood) 0

5 10

15 20 Range S.D. S.E. of Mean

1.1*

1.0 1.0

0.8 0.9 1.1 0.9 1.2

1.2 1.2 0.7 1.0 1.5

(0-lt)

(o-U)

(0-11)

(0-18)

1.1

l.XU

2.1

3.23

0.15

0.16

0.3

o.l*5

* Mean for nine animals

0.7 1.0 1.8 -H-ii.l

U.3 1*.7 6.8

37

bacterial Infections*

They reported a marked increase in the number

of neutrophils, a diminution of lymphocytes and eosinophils and no change in th© number of monocytes*

Similar changes in the leucocyte

differentials were noted for intraperitoneal injection of killed bacteria and diptheria toxin* The marked increase in the number of neutrophils in group III (infected control) is indicative of a bacterial infection*

A

lymphopenia in group III and group IV reached a minimum of 36*2 and 37*9 per cent respectively on day twenty of infection*

Ho

change in the monocyte count wag noted during the period of observa­ tion* Hunter and Group© (1939) infected albino mice with varying total doses of £0 to 300 Trichinella cysts and studied the blood picture over a period of seven weeks*

These investigators reported

no change’in th© total erythrocyte and total leucocyte count*

Th©

neutrophils fluctuated slightly, a moderate lymphopenia was associated with a slight but distinct eosinophila of 25-33 per cent within th© first two to three weeks of infection* Slight ©osinophilia of 1**3 per cent was observed for group IV (infected experimental) on th© tenth day of infection* On the corresponding day a 1*0 per cent eosinophil count was recorded for group III (infected control)*

The significance of the difference

between th© two means on day ten were calculated for group III and group IV*

The slight ©osinophilia in group IV was found to be

58

significant#

It 1© shown from the data that subcutaneous injection

of Crysticillin and Bihydrostreptomyein resulted in a slight but significant increase in circulating eosinophils by the tenth day of infection*

A significant increase in eosinophils in group III

(infected control) did not appear until day twenty of infection* The eosinophil count on day twenty for group III and group IV was Ii*l per cent and 6*8 per cent respectively.

The significance of

the difference between the two means was calculated, the observed difference at this time may be due to chance* Th© per cent survival and survival time of th© four groups of mice used in this experiment are shown in figure 3* no survivors in group III and group IV.

There were

In regard to survival

time, group H I (infected control) eleven out of twenty, i.e., 5>5> per cent of the mic© died in th© initial mortality period.

With

group IV (infected experimental) two out of twenty, i.e., 10 per cent of the mice died during the corresponding period* The data at hand indicate

that subcutaneous injection

of Crysticillin and Dihydrostreptomycin reduced the number of deaths that occur during the initial death period with 100 lpg infections of T. spiralis in Swiss mice.

With the exception of

the hematologic work, this experiment was repeated with an equal number of mice in each of the four groups.

The results found were

essentially the same as that reported in experiment $•

That

secondary bacterial infection of enteric organisms is a contributing

59

GROUP

I

DAYS

0*

4i

i 8i

C>F

2 i 1i

°fa

INFECTION

16

20

24

28

SUR­

32

VIVED

CONTROL

100

A N TIB IO TIC

n

100

CONTROL

nr

INFECTED CONTROL

•• • *• • • • • •

•• ••



• •

••



INFECTED



EXPERIMENTAL



REPRESENTS

Fi:'aire 3.

DEATH

OF

ONE



•• • •• • •• ••• • •• •• ••

ANIMAL

Showing the effects of subcutaneous injection of Crysticillin and Dihydros treptomycin upon survival time and per cent survival of mice infected with a 100 1 o d o s e of P* spiralis* Each proup consisted of twenty mice*

0 0

40

factor in the death of mice during the first period of mortality is further substantiated by the appearance of a slight eosinophila in group I? in the tenth day of infection#

41

Sm&iAHT Swiss mice were infected with graded doses of 5* 25* 50* 75* 100* 200, U00 and 500 Trichinella larvae per gram of body weight* As determined by survival time and per cent survival, the mice were less susceptible to comparable infective doses than were the mice used by other investigators (Glaser, 1920) Culbertson and Kaplan, 1938; Rappaport, 19U3}•

That the Swiss strain of albipo

mice was normally resistant to the more common epizootics of laboratory mice may account for the higher per cent survival and longer period of survival with even larger infective doses than were employed by other workers* With 100 larvae per gram infection® there were no survivors and the majority of the mice died within about the sixth day to the fourth Y/eek of infection*

The deaths of the trichinosed mice

were grouped into an initial mortality period from about the sixth to the twelfth day and a second period of mortality beginning in the third week and extending through the fourth week* A decrease in the amount of food and water consumed, lowered rectal temperature and loss in body weight were found to be associated with th® two mortality periods.

The morbidity and

mortality associated with a 100 larvae per gram infection were not lessened by a parent®rally administered diet supplement*

42

Cultures were obtained from ventricular blood and th© peritoneal cavity of mice given a 100 larva© per gram infective dose of Trichinella*

A bacteremia was demonstrated in 76.14 per cent

of the cases examined*

Thus, secondary bacterial infection of

enteric organisms was found to accompany a severe infection of f * spiralis in mice* The blood picture of Swiss mice given 100 Trichinella larvae per gram was studied through twenty days of infection.

Mo

change was evident in the mean total erythrocyte counts, a slight decrease in hemoglobin was noted by day fifteen in trichinosed mice*

The mean total leucocyte count fluctuated slightly, a

neutrophilia associated with a lymphopenia began by th© fifth day of infection* fection.

The neutrophilia is indicative of a bacterial in­

A slight ©osinophilia of U.l per cent was observed by day

twenty of infection# Subcutaneous injection of the antibiotics, Crysticillin and Dihydrostreptomycin, resulted in a reduction in th® number of deaths occurring in the initial mortality period*

That secondary

bacterial infection is a contributing factor in th© death of Trichinella infected mice during th© initial period of mortality is further substantiated by the appearance of a slight ©osinophilia by th© tenth day of infection in mice treated with Crysticillin and Dihydroatreptomycin.

BIBLIOGRAPHY Aldridge, F. 0* 1931 "An Outbreak of Trichinosis in Pennsylvania Amer* J. Med. Sci., 181: 312-323. Brown, T. R. 1897 "Studies on Trichinosis." Hosp., 8s 79-81.

Bull. Johns Hopkins --- ------------

Cerecedo, L. R. and Miron©, L. 19U7 "The Beneficial Effect of Folic Acid (Lactobacillus easel Factor) on Lactation in Mice Maintained on Highly Purified Diets." Arch. Biochem., 12s 15U—155. Congdon, E. D. 1912 "The Surroundings of the Germ Plasm* III. Th© Internal Temperature of Warm Blooded Animals (Mus decumanus M. musculus, Myoxus glis) in Artificial Climates." Arch. Sntw. mechan. der Organismen., 33s 703-715* Culbertson, J. T. and Kaplan, S. $. 1933 Immunity in Experimental Trichinosis."

"A Study Upon Passive Parasit., 30s 156-166.

Cummins, W. T. and Carson, G. R. 1916 "Trichinosis. A Study of Fifteen Cases." J. Amer. Med. Assoc., 671 806-808. Frothingham, C., Jr. 1909 "Th© Intestinal Lesions Caused by Trichinella Spiralis in Rats." Arch. Int. Med., p p 505-516.

.

Glaser, H. 1920 "Die Empfindlichkeit von Ratte und Maus gegen Trichineninfektion." Arb. a. d. Reichsgesundhei tsamte• Berlin 52s 573-595. Gursch, 0. F. 19U9 "Intestinal Phase of Trichinella Spiralis (Oiren, 1835) Railliet, 1895." J. Faraslt., 35s 19-26. Herrick, W. W. 1915 "Review of Recent Studies in Trichinosis." J. Amer. Med. Assoc., 65i 1870-1872. Howard, W. T., Jr. 1099 "Report of a Fatal Case of Trichinosis Without Eosinophilia but With Large Numbers of Eosinophilic Cells in the Muscle Lesions; With Remarks on the Origin of Eosinophilic Cells in Trichinosis." Phila. Med. J*, Us 1035-1087. Hunter, G. W., Ill, and Groupe, V. 1939 "The Blood Picture of White Mice Infected with Trichinella Spiralis." J. Faraslt., 25 (Suppl.)j 33.

44

Lawrence, J. S., and Josey, A. I* 1932 "Studies in the Physiology of the Eosinophil* XI. The Effects of subcutaneous and Intra­ peri toneal Injections of B. Coli on the WhiteBlood Cells of the Guinea Pig." Folia haemat., IS: 313-322. McCoy, 0. R* 1931 "Immunity of Rats to Reinfection with Trich­ inella Spiralis." Amer. J. Hyg., 11: 131-191. Opie, E. L. 1901 "The Relation of Cells with Eosinophile Granula­ tion to Bacterial Infection." Amer. J. Med. Sci., 12?: 968-1010. Petri, S. 1933 "Morphologic und Sahl der Blutkorperchen bei 7 ca. 30 g. schweren normalen weissen Laboriumsmaussen." Acta. path, et microbiol., 10: 159-238. Rappaport, I. 1913 "A Comparison of Three Strains of Trichinella Spiralis. I. Pathogenicity and Extent of Larval Development in the Musculature." Amer. J. Trap. Med., 23: 313-350. Romano vitch. 1912 "Recherche® sur la trichinose." Pasteur., 26: 351-370.

Ann. de 1* Inst*

Roth, H. 1939 "Experimental Studies on the Course of Trichina Infections in Guinea Pigs. H . Natural Susceptibility of the Guinea Pig to Experimental Trichina Infection." Amer. J. Byg., 29: 69-101. Spink, W* W* 1931 "Effects of Vaccines and Bacterial and Parasitic Infections on Eosinophilia in Trichinous Animals." Arch. Int* Med*, 51: 805-817. Staubli, C.

1909

Trichinosis.

J. F. Bergmann, Wiesbaden, 295 PP*

Trawinski, A. 1935 "Studien uber Immunitat. bei Trichinose." Zentralbl. f* Bakt. Prig. (Abt. I), 131: 115-119. Woolley, D. W. 1911 "Relationship of Pantothenic Acid and Inositol to Alopecia in Mice." Proc. Soc. Exp. Biol, and Med., 16: 565-569*