ASPECTS OF INFECTION AND IMMUNITY IN RELAPSING FEVER

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'ft. 3 X 4 4 5 LD3907 J ,G7 Stein, George Jay, 19091942 Aspects of infection and immunity .S7 in relapsing fever... cNew York. 1942. 2p.l.,73,6, d 3 typewritten leaves, tables,diagr. 29cm. Thesis (Ph.D.) - Mew York university. Graduate school, 1942. Bibliography: 6p. at end.

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1,1T'RARY OF1 NEW :-G!.;v L‘HTVERSTTY UNIVERSITY HEIGHTS

ASPECTS OP INFECTION AND IMMUNITY IN RELAPSING FEVER

George J. r*'Stein lift*

April, 1942

"A dissertation in the Department of Bacteriology submitted to the Faculty of the Graduate School of Arts and Science in partial fulfillment of the re* quirements for the Degree of Doctor of Philosophy.”

TABLE OF CONTENTS

Page I. II.

INTRODUCTION

1

PRESENT KNOWLEDGE OF RELAPSING FEVER A.

General Features of the Disease

4

B.

Bacteriology

5

C.

Immunology

7

D.

Epidemiology and Geographical Distribution

E.

Diagnosis, Prognosis, Prophylaxis, and Therapy

III.

14.

HISTORY AND BRIEF SURVEY OF THE LITERATURE

IV.

11

16

EXPERIMENTAL FINDINGS INFECTION A.

Cultivation Attempts

21

B.

Strains

26

C.

Preservation of Spirochetes

28

D.

Routes of Infection

29

E.

Distribution of Spirochetes in Infected Mice

31

F.

Sedimentation Test

33

C.

Minimum Infective Dose of Spirochetes

35

TABLE OF CONTENTS

Page IMMUNITY A.. Separation of Spirochetes from Infected Blood

36

B.

Preparation of Antisera

40

C.

The Use of Immunological Methods as a Means of Classifying Relapsing Fever Spirochetes

D.

41

Immunological Studies with Spiro­ chetes Separated from Infected Blood by the Use of Saponin

E.

53

The Use of Spirochetes Separated from Infected Blood by Means of Saponin as an Antigen for the Differential Diag­ nosis of Relapsing Fever

V. VI.

57

DISCUSSION

65

SUMMARY AND CONCLUSIONS

73

BIBLIOGRAPHY

1:1^ ■jptl.a ASPECTS OF INFECTION AHD IMMUNITY IN RELAPSING FEVER

I. IHTRQDUCTIOH Spirochetes axe only slightly less ubiquitous than ordinaxy baoteria.

They thrive in virtually axy natural environment which is

capable of sustaining aicrobial life.

They may exist either in a free-

living state, as commensals, or as strict pathogens.

Studies on one of

the latter group of microorganisms from the standpoint of infection and immunity form the subject of this dissertation. During the formative period, of bacteriology, careful and sustained investigations of the spf»h«+•«> were largely neglected be­ cause of their position in the ill-defined zone between bacteria and protozoa.

Over the last forty years* sporadic reports of the discovery

of pathogenic spirochetes have appeared in the literature.

Certain of

these organisms have been found in lesions of Vincent's angina, bronch­ ial spirochetosis, ulcerations, and. other fuso-spirochetal conditions. Others have been demonstrated to possess a definite causal relationship in syphilis, yaws, spirochetal jaundice, and the relapsing fevers of mas and animals. Knowledge of the relapsing fever spirochetes is still very in­ complete, although the literature is replete with a bewildering nomencla­ ture and reports of studies, the latter burdened with highly controversial conqparisons of the various "species1' in this grotq? of organisms. This confused state may be attributed to the fact that the spiro­ chetes may be found in the blood of vertebrates in most parts of the world and are implicated in the production of modified forms of relapsing fever. The type of fever, the character of the symptoms, the mode of transmission,

and the serological qualities present nueerons variations, with the result that the causative agent from a'particular locality has often bean given a specific designation. is ever increasing.

Thus the nuaber of naued species

Investigators in almost every country throughout

the world have established different species naans, each inferring specificity*

Hence classification is either descriptive of a spiro­

chete indigenous to a particular geographical area or tends to denote such singular characteristics as were described by the original ob­ servers. It eay wall be that all these nuearous relapsing fever "species" are merely local varieties of one widely distributed species, which has become adapted to different naans of transmission in various localities. With this in mind, it would be of interest to determine whether or not differentiation of relapsing fever spirochetes into many distinct species is justifiable.

Information pertinent to this problem might be

obtained with the aid of sero-iemunological procedures. Relapsing fever and other infectious diseases are generally characterised by periods of fever which on occasions may be intermittent in nature.

Attempts to recover the etiological agent concerned are fre­

quently unsuccessful, Moreover, symptoms typical of the disease may be v

lacking, as may be effective serodiagnostic measures.

In such cases

there is a real problem of ready and accurate diagnosis; it is then difficult to establish which of many infections is responsible for the pyretic condition.

Of the nmeerous infectious diseases other than relaps­

ing fever which present this difficulty, tuberculosis, tularemia, rat-bite fever, brucellosis, the spotted fevers, typhus fever, Well's disease, the

enteric diseases, syphilis, pneumonia, rheusatic fever, malaria, and trypanosomiasis nay be mentioned.

For this reason the devel­

opment of a reliable method tp distinguish rolapsing fever from other Infections would be valuable. Attempts to accomplish this will be described.

II.

PRESENT KNOU P OB OF RELAPSING FEVER

JL. general Features of the Disease The spirochetes of relapsing fever are exclusively para** sitic and are present primarily in the blood of their hosts. They are transmitted through the agency of blood-sucking arthropods, especially ticks and lice. may persist ixdefinitely.

In these ectoparasites the spirochetes The microorganisms generally produce a

relapsing type of fever which affects man throughout the world. The disease may be described as an acute infectious process, the causative agent being characteristically present in the circulating blood during the height of the infection. To some extent it is olinically distinguishable from the majority of other febrile infections because of the occurrence of paroxysms which alternate with intervals of apparent recovery. However, subsequent pyretic intervals do not always occur, or if they do, an adequate record of their appearance may be lacking. Clinical distinction then is haphazard• In relapsing fever the internal organs commonly exhibit evidences of toxemia} they may be more or less deeply stained with bile. Degenerative changes of the arteries may give rise to focal softening of the brain and infarcts of the kidney and spleen. Char­ acteristically, the liver and spleen especially are enlarged, and spirochetal emboli of the cerebral vessels may be observed. On oc­ casion a macular rash, not uhlike rose spots, may occur and persist

for several days. The average incubation period of about 2 to 9 days is followed in the human by the usual prodromal symptoms.

During the

primary attack chills and fever of 104° F. or more are common.

The

liver is generally enlarged and tender, and the spleen may be readily palpated.

The initial seizure lasts approximately 5 days, when

defervescence occurs and the temperature rapidly returns to normal with apparently complete restoration of health.

However, this picture

merely represents the end of the first paroxysm and rushers in the beginning of an afebrile interval. After about 1 week of seeming normalcy, the primary relapse sets in.

Here the symptoms tend to duplicate those present in the

initial attack, but they are generally less severe and of shorter duration.

A second afebrile period, usually lasting longer th«n the

first, succeeds the primary relapse.

As the disease proceeds on its

course, second, third, and even more relapses may take place, each progressively less prolonged and each interrupted by increasingly extended apyretic intervals.

A case showing as many as eleven relapses

has been recorded.

B. Bacteriology The spirochete of relapsing fever may be described as a spiral, loosely wound, flexible, actively motile microorganism, measuring about 14/u.long and O.^u.thick; it is composed typically

of fire to seven primary waves, several of which aay straighten out «rtnM>ritAt»iiy to form large secondary undulations.

The spirochete

is best observed in the living state by darkfield illumination, exhibiting double refraction and three distinctive forms of motion: rotation, translation, and flexion.

It may be demonstrated by means

of ordinary aniline dyes, being less difficult to stain than most spirochetes.

Involution forma and granules are seen at the stage during

which the spirochetes tend to disappear from the blood.

These granules

may be either degenerative forms or a phase in the development of the organism.

The spirochetes are able to traverse uninjured mucous

membranes, the cornea of the eye, and the normal placental barrier. They have been observed in the cerebrospinal fluid as well as in the blood. Contrary to reports in the literature, authoritative workers in the field contend that a reliable in vitro cultivation technique has not yet been developed.

The prevailing method of maintaining

strains in the laboratory requires employment of constant animal passage.

Monkeys, mice, rats, squirrels, chipmunks, and, occasionally,

guinea pigs and rabbits may be infected.

The disease produced in

experimental animals simulates the infection in man. Spirochetes are most numerous in the blood during the initial febrile attack.

With each successive relapse their numbers

diminish. On the other hand, during the inter-relapse periods they are rarely observed.

Although the blood is microscopically negative

at this stage, it may still be infective for experimental animals

T.

and ectoparasites. Hence it m y be concluded that either the organisms are preeent in extremely scanty numbers or they exist in an unrecognised form* Each febrile attack is terminated by crisis, when the temperature returns to normal and the spirochetes tend to assume entangled and rosette-like aggregations. Agglutination, fragmentation, and granular disintegration ensue. Within a few hours the circulating blood, which may have contained virtually as many organisms as red blood cells, no longer reveals spirochetes. The disappearance of the spirochetes from the blood has been attributed to the rapid development of specific antibodies. However, some antibody-resistant spirochetes may escape destruction and looalise elsewhere in the body.

Studies in animals indicate

that such organisms may persist in the brain for months after apparent recovery.

It has been postulated that the brain and other tissues which

harbor these antibody-resistant spirochetes serve as foci for the institution of relapses.

C.

Immunology Attempts to deteradne the antigenic nature of relapsing

fever spirochetes by chemical analysis are at present not practicable. Furthermore, immunological studies have uncovered little exact information. Host in vitro serological investigations have made use of mixtures of infected blood and antisarum, since isolation of the

microorganism in piura culture has not been achieved.

Actually much

of the accepted evidence concerning immunity in relapsing fever has been established through in vivo observations. An attack of the disease confers an immunity of short dura­ tion.

Consequently, reinfection in man is not an infrequent event and

has been recorded as early as 2 months after recovexy from a previous attack.

Another departure from the Immunological picture which

characterizes most other infectious diseases is the occurrence of relapses after primary infection.

These unconventional features of

immunity in relapsing fever stimulated various investigations. It was early determined that agglutinins, lysins, and spirocheticidins are rapidly evoked toy infection with relapsing fever.

These

antibodies are presumably highly specific and impart complete protection only against the same strain or, at times, against a variant of the same strain of spirochetes.

(The term "variant" refers to those organisms of

a strain which are responsible for the production of a relapse.

It is

believed that when several relapses occur in a single host, each results from invasion of the blood by 811 antigenically distinct variant.) At this point consideration of the relapse phenomenon appears to be appropriate.* During the course of the initial paroxysm, despite * a complete discussion of the theories, mechanisms, and complexities of the relapse phenomenon occupies a position separate in itself and is beyond the scope of this paper. Hence, no experimental studies or critical analyses of this subject have been ventured. References to it will be largely descriptive and will deal in part with the most rational explanation of its mechanism thus far propounded.

the abundant present of spirechatas in the circulating blood, specific agglutinins are likewise demonstrable• As the paroxysm terminates, the spirochetal population diminishes and the antibody concentration rises* This may be readily observed by microscopic examination of a drop of infected blood which reveals the organisms in varying degrees of agglutination and degeneration* By virtue of continued antibody production the blood infection is soon overcome and the spirochetes are abolished from the blood stream* This, however, does not signify complete sterilisation, for some organisms remain latent in the brain and other tissues* Then the spirochetes are presumed to change antigenically, become resistant to the initial anti­ bodies, multiply, and invade the general circulation, thus reinstituting the disease and signalizing a relapse or second onset* The spirochetes of the new paroxysm stimulate the production of homologous antibodies which eradicate all but a few of the second-attack organisms*

These

few remain latent in the tissues, again become altered antigenically so that they escape the action of the initial attack and first-relapse antibodies, proliferate, and establish a third onset or second relapse* Such a chain of events may be repeated ssvsral times* If the spirochete populations and antibody curves are charted over a period of time, severed features become conspicuous* Gradually the severity of the relapses dwindles, the interval between relapses is more prolonged, and finally no additional paroxysms occurs a so-called cure results*

(Figure 1.)

IS

Tho cur# may be more apparent than real* since in animals the spirochetes of relapsing fever may frequently be found in the brain

other tissues long after the cassation of clinical symptoms.

During this period a unique type of immunity exists - one which is termed "premunition" or "infection-immunity." A somewhat similar state has been described as occurring in certain of the virus and protozoan diseases and has also been associated with immunity to superinfection in syphilis. The host* while possessing humoral immunity to relapsing fever* nevertheless harbors a quiescent infection of the tissues* which* under certain infrequently encountered conditions* is capable of breaking down the existing barrier of immunity and producing a frank infection.

These circumstances may account for the delayed

recrudescent attacks sometimes seen in laboratory animals.

Essentially*

premunition may be regarded as a state which reflects a delicate host-parasite equilibrium and which is susceptible to disturbance. It is possible that the latency of the infectious agent may account for the absence of recognizable disease through the promption of a continual production of antibodies.

Thus the threshold level

necessary to prevent reinfection would be constantly maintained. Besides the complexities attendant upon the relapse phenomenon* results of epidemiological observations have further complicated the picture.

A given strain from one host species

passed through another species reveals an alteration in antigenic

specificily.

For example* a patient recovering from relapsing fever

brought on by the bite of an infected tick* may be susceptible to the same strain after it has been passed through a mouse.

Moreover*

individuals infected with one variety of relapsing fever* such as the European type* may* after recovery* become infected with the African* the American* or the Indian variety. In summary* the antigenic constitution of the relapsing fever spirochete appears to be extremely unstable.

The problem is

rendered more complex by the lack of a suitable isolation technique. The difficulty in classifying these organisms into distinct species* groups* and types by current methods may* therefore* be readily understood.

D.

T^oidamlology and Geographical Distribution Relapsing fever is now recognized as occurring far more

commonly than had been hitherto suspected.

It is not uncommon in

the United States* and its manifestations are more protean than one might surmise from the descriptions given in medical texts.

As far

as is known* the disease is almost always transmitted in Nature by ectoparasites.

The two most common vectors are the louse

(Pedlculus huaanus) and various species of the tick (Omithodoros). The mechanism of transmission of the disease to mammals by these arthropods is in a state of dispute. are made:

The following claims

The infected tick transmits the spirochetes while feeding

1) by direct contamination of the bite wound with secretions from

the salivary glands; 2) by fluid from the coxal glands which bathes the area of the bite; 5) by spirochete-laden feces which are deposited on the pVin by the tick and rubbed into the wound. Transmission by the infected louse is effected either by means of 1) the salivary glands* 2) contamination with infected dejecta* or 3) crushing of the louse and introduction of the contaminated body contents into the abraded skin through scratching. Infection in both ticks and lice can be transferred to the eggs through several generations* a point of considerable epidemi­ ological significance. Louse-borne relapsing fever is endemic* becoming epidemic at certain times* particularly during periods of war* famine* economic distress* and social upheaval.

Epidemics have occurred in Russia*

Boland* Germany* Ireland* the Balkan countries, India* China* and Northwest Africa.

In 1869 relapsing fever was conveyed to New York

and Fhilac.alphia from Ireland.

Several small epidemics which emanated

from these foci took place in the United States. Epidemics of relapsing fever and typhus fever are often coexistent and are both prone to occur during periods of stress.

Overcrowding* neglect of

personal hygiene* and the dissemination of vermin* inevitable at such times* excite the development of serious epidemics from endemic areas. In severe outbreaks the mortality rate may be as high as 30 per cent. Tick-home relapsing fever is more prevalent in Africa than elsewhere. America.

However* foci exist also in Asia and North and South

The United States Public Health Service* in the last few

years* has discovered reservoirs of infection in ticks and wild rodents

throughout this covntiy. The distribution of endemic relapsing fever conforms closely to the dispersion of the chief vector* the tick. A possible source of infection is the bedbug. The causative agent is capable of living in this parasite for long periods* but its epidemiological importance is obscure. Mites have also been implicated as reservoirs of the infective organism.

In recent years

relapsing fever has come to be regarded as a disease primarily of small animals which function as reservoirs* with man in the role of an occasional and accidental host. In the absence of general agreement on classification* the varieties of human relapsing fever have been identified in a purely arbitrary manner. The following is only a partial list of the "species” that have been described in various parts of the world. Vector

Classification

Etiological Agent

Louse

European relapsing fever

Tick

Spanish

"

"

"

hispanica

Louse

Indian

”

”

"

carteri

Tick

Persian

"

**

"

persica

Louse

Chinese

"

"

Various species described

"

Algerian

"

"

Borrelia berbera

"

Egyptian and Sudanese relapsing fever

"

egyptica

Tunisian relapsing fever

"

normandl

"

Ethiopian

"

"

savignri

"

West African "

"

duttoni

Tick

Borrelia obermeieri

.

14 Classification

Vector Tick

East African relapsing fever North American

Louse

Etiological Agent

*

Borrelia kochi

*

"

novyi (presumably louse-borne

Relapsing fever in United States Canada and Uexico

Many tick strains of spirochetes; in process or being named

Central and South American relapsing fever

( (

Relapsing fever in Brazil and Peru

Not named

Relapsing fever in West Didies

Not named

Borrelia neotropicalis " venesaaloails

Unclassified tick-borne relapsing fever of North America

E* Diagnosis. Prognosis, Prophylaxis, and Therapy Diagnosis*- Diagnosis can be made by direct microscopic examination under optimal conditions*

During paroxysms Borrelia may

be demonstrated in the blood by darkfield illumination or by thin or thick smear techniques*

Indirectly, injection of blood from a febrile

patient into rats and mice may be employed to initiate a frank blood stream invasion*

This method necessitates a daily search for spirochetes

in the rodents' blood* Differential diagnosis clinically is often difficult*

It is

especially troublesome in such cases as Weil's disease, trypanosomiasis, rat-bite fever, malaria, typhus, and other conditions which may simulate the relapse characteristics of relapsing fever*

In the

absence of positive laboratory findings, considerable uncertainty

sonstimes exists. Prognosis.-

Relapsing fever tends to be self-limiting.

Each relapse is usually less severe than the preceding one.

In

robust individuals the outcome need not be viewed with alarm.

However, in weak, poorly nourished victims and in very young children, or in the aged, it may prove fatal. Symptoms which may be regarded as unfavorable are syncope, acute cardiac dilatation, incontinence, convulsions, coma, and severe toxemia.

Ifader such

circumstances a mortality rate of 30 to 40 per cent has been recorded. fF9Pty 1'*v*s (Vaccine).- Some positive claims have been made with regard to the efficacy of vaccines in promoting resistance to infection.

In view of the practical difficulties encountered in

preparing vaccines effective against agents of such recognized antigenic multiplicity, these assertions might well be viewed with caution. Serum Therapy.—

Serum therapy possesses the same limita­

tions ascribed to vaccine prophylaxis. Chemotherapy.—

Organic arsenicals readily cut short the

period of infection with relapsing fever.

Sometimes a single dose is

sufficient to prevent relapses and "cure" the disease.

The dosage

tolerated is less than that used for other diseases, because relapsing fever lowers a patient's resistance to the toxic properties of the drug.

However, arsenic-fast variants may be encountered just as are

antiserum-fast variants. of value.

In such cases bismuth compounds may prove

III. HISTOBT AMD BRTCT smnng OF THE LITERATURE

Has first reference to relapsing fever dates back to 1741, whan Rutty observed a dlsoaaa of this description associated with typhus fever in Dublin* In 1857, Livingston reported that in South Africa a peculiar relapsing fever often followed the bite of a tick. Oberaaier, in 1868, was the first to observe the spirochete of relaps­ ing fever in the blood of a patient. Four years later, he confined this observation while studying an epidemic in Germany; he fond the infective agent in the blood of easy cases. The following year, 1875, he published his discovery (Oberaaier, 1875). In 1882, Carter, working In India, proved, by naans of in­ oculation expertwants, that the spirochete was pathogenic and produced a relapsing type of fever.

During subsequent decades the above find­

ings inaugurated a flow of publications which not only substantiated the causal relationship of the spirochete but also indicated that the distribution of the disease was world-wide. Thus the fact was unequiv­ ocally established that the etiological agent of relapsing fever is a spirochete of the genus Borrelia. Perhaps excusably, but nevertheless unfortunately, different species naaes were applied to organisns of this genus in various parts of the world.

Host observers believed that the several species could

be distinguished fron one another eorphologically.

This, however, is

rarely the ease. The variations in any single "species" are quite as narked as those which have been pms e s d to indicate differences between i*

«f

spscies. As early as 1807, Laiaheaa.stated, that ha could find no mor­ phological distinctions between the European, Central African, and TnrH««

strains. Macfie and fork* (1917) reemphasized this point.

It was

soon adduced that other presumably distinguishing features, such as the difference in the type of disease, the number of relapses, sero­ logical and cross-iaaunity tests, and the variations in susceptibility of laboratory animals, could not be employed as reliable methods of differentiating species, unless very carefully controlled. In 1906, Carlisle reviewed the early history of relapsing fever in Horth America and reported the first authenticated case seen in tbs tfaited States. The morphologic and pathogenic characteristics of the strain recovered from the patient, as well as attempts at cul­ tivation, were described by Horris, Pappenheieer, and Flournoy (1906). They named the spirochete Snlrillum obermeieri. Working with this strain and related organisms, Hovy and F”

later (1906) conducted

comprehensive biological studies and Included in the publication of their results an account of pathogenesis and immunity in relapsing fever. Numerous investigations on sera of nan and animals conva­ lescent from relapsing fever were carried out ly Manteufel (1908), ty Novy and Knapp (1906), and by subsequent workers.

A& these studies

progressed, it was observed that animals which had recovered from in­ fection with one strain might sometimes be Infected with another.

I^nce

a tendency arose to segregate relapsing fever spirochetes into separate species.

Despite this dubious assumption, nevertheless, immunological

studies performed at this time uncovered valuable information concerning agglutinating, lytie, and spirocheticldal antibodies in relapsing fever. Disparities in the agglutinating titer of ■ a»x© Q o

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rabbit serum.

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it may contain email amounts of oell stroma, as well as traces of mouse serum proteins.

Under these circumstances an extraneous immune reaotion

can occur and may complicate evaluation of the spirochete-antispirochetal reaction.

Prom Tables VIII and IX it is evident that this factor may be

eliminated by diluting the antiserum. 2.

Agglutinin titers during and after infection.

Since it appeared

that a stable antigen was now available, a study of antibody titers during anl after infection was undertaken.

Estimations of the relation between

the severity of infection and antibody titer were also made. Materials and Methods. Mice were injected with the MOb strain and at varying intervals during and subsequent to infection were sacrificed and bled for serum.* Each serum, appropriately diluted, was added to 0.2 cc. of antigen. Readings were taken after incubation for 2 hours at 57° 0. and following refrigeration overnight.

Table X shows the results of these tests.

The data presented in Table X indicate that agglutinins may be demonstrated in the serum as early as 2 days after inoculation of in­ fected blood and may persist for at least 20 days. Antibodies are present in highest concentration between the 4th and 8th days. lapsing fever infectious agent.

As expeoted, re­

bodies coexisted in the blood stream along with the It may also be concluded that the antibody titer need

*In mice the primary attack lasted approximately 4 days.

TABLE X

Agglutinin Titers During and After Infection SaponinTime* Extent** treated of of Antigen (0.2 cc.) collection infection

0*s. 1 day 2+

Antisera - 0.2 cc. Dilutions **** 1:10 1:40 1:100 1:160 1:200 1:400 1:800 mrn tm mm mm mm mm — -

1+

mm c days

2+ 2+ 3+ 2+

3 days

3+ 4+

4 days

MOb

5 days

—*

2+

1+ + 2+

?+ 3+ V* 3+ 3+ ?+

3+ 3+ 3+ 4+ k *

2+ 2+ 2+

11 days

< 1+

■n*

mm

1+ 1+ 1+ 1+

+

-

mm

mi

mm

4*

2+

2+

Ml IM

2+ 2+ 2+

3 *

2+

—

—

3 *

1+

2+

8 days

—

—

3+ V* 3+ 4+ 3*

3+ 4+ 2+ 2+ 3+

3+ 3+

+ + 2+ 2+ 2+

2+ 3+

2+

3*

2+ 3+ 3+ 3+ 2+ 3+ 2+ 2+ 2+ 2+ 1+

•*

1+ 2+ 2+ m.

1+ -

1+ 1* 1+ -

mm

M

1+ 4+ 12 days 3+ 1+ 3+ 4+ 3+ 1+ 13 days 2+ 2+ 3+ 1+ 20 days 1+ 3* 1+ 2+ 36 days*** 1+ ±1+ 3+ Indicates time serum was collected subsequent to injection of infected blood. Indicates extent of infectjon^jn mouse from which serum was obtained. Indicates 0.1 cc. of antigeA intradermally 7 days before mouse was bled for serum. * For each dilution there were two readingsj the first indicates that made 2 hrs. after incubation at 37^ C., the second after refrigeration overnight. -

mm

-

*m

-

Mi

mm

mm

M

—

*****

mm

•m

-

-

2+

* ** ***

mm

not necessarily parallel the severity of the infection. 5.

Slide agglutination tests with homologons and heterologoua

antiaera. Results of a previous experiment suggested that treatment of spirochetes with saponin abolished the sharp serological distinctions between strains but further evidence was considered necessary to sub­ stantiate this observation.

Accordingly, slide agglutination tests

were carried out. Materials and Methods. Sera from rats, mice, and guinea pigs recovered from infection with various strains of relapsing fever were diluted 1:10 in normal saline.

Similar dilutions of sera from animals immunised with MOb anti­

gen were made; normal serum controls were included. A drop of each re­ spective serum was mixed on a slide with a drop of MOb antigen and darkfield examination of each preparation followed without delay.

The re­

sults of these tests areecorded in Table XI. Table XI shows that antisera from three species of animals, each of which had been infected with different strains of spirochetes, agglutinated MOb antigen.

Sera from rats, mice, and rabbits immui-ized

against MOb antigen reacted similarly.

All controls proved negative.

8inee the above test was intended merely for preliminary study, quantitative considerations were deferred for tube agglutination teste. 4. Tube agglutination tests with homologous and heterologous antisera. Materials and Methods. Dilutions of sera from rats, mice, and guinea pigs recovered

TABLE XI

Slide Agglutination Test with Homologous and Heterologous Antiserum

SaponinAntiserum diluted 1:10 treated Period Extent Antigen Derived from after of (0,2cc.) Animal Strain infection* infection** (days) 2D 6