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1981 Characterization of immunity to multocida Robert Morris Nathanson Iowa State University

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NATHANSON, ROBERT MORRIS

CHARACTERIZATION OF IMMUNITY TO PASTEURELLA MULTOCIDA

Iowa State University PH.D. 1981

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University Microfilms International Characterization of immunity to Pasteurella multocida

by

Robert Morris Nathanson

A Dissertation Submitted to the

Graduate Faculty in Partial Fulfillment of the

Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Major: Immunobiology

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Signature was redacted for privacy. In Charge of M,

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For the Major Department

Signature was redacted for privacy. For the Graduate College

Iowa State University Ames, Iowa 1981 ii

TABLE OF CONTENTS

Page INTRODUCTION 1

LITERATURE REVIEW 4

PART I: PASTEURELLA MULTOCIDA: IMMUNOLOGIC H STATUS OF AND TURKEYS GIVEN INACTIVATED VACCINE SUMMARY 12

INTRODUCTION 13

MATERIALS AND METHODS 15

RESULTS 18

DISCUSSION 20

REFERENCES, CITED 26

PART II: PASTEURELLA MULTOCIDA: ANTIBODY MEDIATED 27 RESISTANCE TO VIRULENT CHALLENGE EXPOSURE IN VACCINATED TURKEYS

SUMMARY 28

INTRODUCTION 29

MATERIALS AND METHODS 30

RESULTS 34

DISCUSSION 35

ADDENDUM 38

REFERENCES CITED 42

PART III: CYCLOPHOSPHAMIDE-INDUCED IMMUNOSUPPRESSION 43 DEMONSTRATED IN PASTEURELLA MULTOCIDA VACCINATED CHICKENS

SUMMARY 44

INTRODUCTION 45 iii

MATERIALS AND METHODS

RESULTS

DISCUSSION AND CONCLUSIONS

REFERENCES CITED

PART IV: IN VIVO TESTS FOR DELAYED TYPE HYPERSENSITIVITY TO PASTEURELLA MULTOCIDA IN VACCINATED CHICKENS AND TURKEYS

SUMMARY

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

REFERENCES CITED

PART V: TRANSFORMATION OF LYMPHOCYTES OPTIMAL CULTURE CONDITIONS

SUMMARY

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

REFERENCES CITED iv

Page

PART VI: STUDIES OF CELL-MEDIATED IMMUNITY 96 TO PASTEURELLA MULTOCIDA: A COMPARISON TO MY COBACTE RIUM TUB ERCULOSIS

SUMMARY 97

INTRODUCTION 98

MATERIALS AND METHODS 99

RESULTS 104

DISCUSSION 106

LITERATURE CITED 124

SUMMARY AND CONCLUSIONS 126

BIBLIOGRAPHY 134

ACKNOWLEDGMENTS 140

APPENDIX 141 1

INTRODUCTION

Fowl is an infectious disease of poultry and wild birds

which is caused by a small Gram-negative bacterium, Pasteurella

multoeida. The greatest mortality is usually observed in turkeys that

are older than 16 weeks of age. £. multocida can produce either chronic

or acute . In the chronic form, P^. multocida produces

localized infections in wattles, sinuses, joints, and footpads. Chronic

infections are seen more often in birds that have a reduced suscepti­ bility to P^. multocida. Occasionally, chronic infections can develop

into acute infections. Most acute infections have a very rapid onset;

as little as twelve hours may elapse between the appearance of symptoms

and death. A very rapid and overwhelming septicemia develops, with

symptoms being typical of endotoxin shock. Body temperature is elevated,

nasal secretions become profuse and thickened, and petechial and ecchymotic hemmorhages often occur. Torticollis, cyanosis, diarrhea and anorexia are common. An acute infection may kill thousands of birds in a single day (1). The disease is believed to be spread between birds by excretions from the mouth that contaminate feed and drinking water

(2). A number of wild and domestic animals can act as carriers of avian strains of P. multocida (3).

Two procedures are used currently to vaccinate against fowl cholera: a live, low virulence strain, developed at Clemson University, can be used to vaccinate turkeys through their drinking water (4,5). 2

Alternatively, turkeys or chickens are vaccinated subcutaneously with

formalin-killed P^. multocida suspended in Freund's incomplete adjuvant

(6,7). Neither vaccine provides complete protection against the

disease.

In order to produce more effective vaccines against multocida

infections of poultry, the immunologic responses to vaccination need

to be characterized. Humoral and cell-mediated immunologic responses

have not been completely defined. Certain areas are not well-under­

stood; particularly the role of complement, the relationship of

resistance to antibody titer, the effector activity of T-lymphocytes,

and .

This dissertation reports on vaccine induced immunity to P^. multocida in chickens and turkeys. The principal objective of the

research was to determine: (A) the protective role of antibodies,

(B) whether in vitro and in vivo correlates of cell-mediated immunity

could be demonstrated, (C) whether cell-mediated imune responses to P^. multocida vaccination were of similar magnitude as responses to

Mycobacterium tuberculosis vaccination. Vaccine-induced immunity was studied by various methods, including passive protection, surgical bursectomy, drug induced immunosuppression, lymphocyte transformation, migration inhibition reaction, granuloma formation, and skin testing.

In this research project, a formalin-inactivated multocida bacterin was used to vaccinate chickens and turkeys. The decision to use a killed bacterin instead of a live vaccine was based on a number of 3

salient observations.

(A) Lymphocytes from turkeys that received formalin-inactivated

adjuvanted bacterin underwent blast transformation after

exposure to 2» multocida antigens (8,9).

(B) Leukocyte migration inhibition was observed when formalin-

inactivated adjuvanted bacterin was used (10).

(C) The Clemson University strain, a live vaccine, does not induce

an immunity that is as effective or as durable as a formalin-

inactivated adjuvanted bacterin (9).

(D) Live vaccines occasionally induce significant mortality

(5,11).

(E) Live attenuated P^. miiltocida vaccines are usually given through

the drinking water and are difficult to administer quantita­

tively.

This dissertation represents an effort to characterize the immunity induced by a fowl cholera vaccine. It is hoped that these studies advance our understanding of the role of humoral and cell-mediated immunity in resistance to P. multocida infection. 4

LITERATURE REVIEW

Characteristics of P. multocida Fowl cholera is produced by Pasteurella multocida, a small Gram- negative rod, of which biochemical and physiological characteristics have been described previously (3). A thick capsule composed of hyaluronic acid is sometimes present on recently isolated cultures. The Pasteurellae are aerobic, but are capable of surviving as facultative anaerobes.

P^. multocida possesses a free endotoxin, a substance that is loosely attached to the bacterium. It can be readily washed off in a cold-formalinized saline solution (12,13,14,15,16). The endotoxin is immunogenic and produces symptoms of toxicity when given parenterally

(12,13,15,16). The endotoxin can be concentrated by dialysis and ultra- centrifugation (12,13,14,15,16). It has been analyzed chemically and has been found to contain various , lipids and proteins (14).

Treatment with phenol destroys immunogenicity. Some portion of the immunogenicity is apparently associated with the protein (14).

The bacterium expresses different antigens when the culture conditions are varied. P^. multocida grown in vivo can produce cross- protection against other strains of P^. multocida whereas agar grown cells produce only homologous protection (17,18). P^. multocida grown in vivo are susceptible to freeze-thaw lysis, whereas agar grown cells are not

(19). P^. multocida isolated directly from tissues are immunogenically and possibly antigenically different than those grown on agar. These 5

differences may be a source of variability in in vitro studies dealing with opsonization and phagocytosis.

multocida are often difficult to phagocytize. The presence of the capsule might act to inhibit phagocytosis (20). The Pasteurellae are not considered to be intracellular pathogens.

Serotypes of P. multocida

Several methods of serotyping have been developed (21,22,23).

The two most commonly used are (A) the system of Carter, and (B) the system of Heddleston and Rebers. There are four type strains in the passive hemagglutination system of Carter. The method of Heddleston and Rebers is based on the Ouchterlony gel-diffusion test. Sixteen have been identified by this method.

Different diseases are produced by different serotypes of P^. multocida. Fowl are most commonly infected with P-1059 (type 3

Heddleston system), or X-73 (type 1, Heddleston system). Both are highly pathogenic and produce fowl cholera. The strain X-73 is notoriously virulent. It has a LD-100 of one organism for mice (24).

Infections in humans most commonly result from a cat bite or scratch.

Respiratory infections have been reported to occur in humans (25). 6

Host-range of P. muUocida

2» multocida produces disease in a wide range of species.

Birds fowl cholera Bovine shipping fever, mastitis Sheep mastitis Rabbit snuffles Pig Human cat scratch infection, respiratory infections

£. multocida is often present in apparently healthy farm animals

(26).

Immunity induced by fowl cholera vaccination

The resistance to infection induced by P^. multocida vaccination is

not entirely understood. Some investigators have been unable to show a

correlation between serum antibody titers of vaccinated birds and

resistance to infection (7,27,28), whereas others have indicated that a

correlation may exist (5,9). Passive protection studies have demon­

strated that immunoglobulin G isolated from the serum of vaccinated

chickens can induce protection in nonvaccinated chickens (29).

Recent studies have also indicated that cell-mediated immunity may function in resistance (8,10). Maheswaran reported that peripheral blood leukocytes from immunized turkeys underwent transformation in the presence of various IP. multocida antigens (8). Baba reported that surgical or hormonal bursectomy had no effect on mortality in vaccinated chickens (30). Significant leukocyte migration inhibition was reported in one publication, but not in a second (10,30). 7

It is not known whether humoral or cell-mediated responses

provide protection in vaccinated birds. In addition, the interaction

of humoral and cellular response has not been studied.

Techniques used to analyze the immune response of P. multocida vaccinated

poultry: selective suppression of antibody responses

The avian immune system is different from that of mammals. A

unique structure, the Bursa of Fabricius, acts as a primary lymphoid organ. If the organ is removed immediately after hatching, the bird will develop a severe antibody deficiency. Bursectomized birds are unable to produce a significant antibody response (31,32). In addition, levels of circulating immunoglobulins are often reduced.

The bursectomy procedure produces a suppression of antibody response without suppressing cell-mediated immunity. Therefore, it is possible to study resistance in vaccinated antibody-deficient birds. Thymus dependent functions remain intact and their role in protection can be evaluated (32).

An immunosuppressive tumoricidal drug, cyclophosphamide, can be used in a manner analogous to surgical bursectomy. Treatment of newly hatched chickens with cyclophosphamide causes a prolonged suppression of antibody production and a temporary suppression of cell-mediated immunity (33,34,35,36). Antibody responses are abrogated for at least ten weeks, whereas cell-mediated responses are reduced for less than three weeks. Antibody response and cell-mediated response can be studied as separate components when the birds are vaccinated and their 8

resistance is challenged during the fourth through the tenth week after

treatment.

Techniques used to analyze the immune response of P.. multocida vaccinated

poultry; selective suppression of cell-mediated responses

Niridazole, an antischistosomal drug has been shown to act as a

suppressant of cell-mediated inmunity in various species, including

chickens (37,38,39,40,41,42). Niridazole is metabolized, and a product of its degradation is bound to serum proteins (43). The serum bound metabolite produces T cell suppression. A number of eel 1-mediated responses are suppressed including graft-versus-host reactions, delayed type hypersensitivity, granuloma formation, lymphocyte transformation, and production of migration inhibition factor. Suppression can be maintained for up to three weeks after completion of treatment (37).

It should, therefore, be possible to study resistance in T cell immuno- deficient chickens by administering niridazole prior to vaccination.

In vivo tests for delayed hypersensitivity

Skin testing is a classical means for measuring delayed hyper­ sensitivity (44,45). If a small amount of antigen is placed intradermally into a previously sensitized animal, a localized erythema and induration may develop. The reaction usually persists for several weeks, and upon histological examination an extensive mononuclear infiltrate is seen.

The presence of such a reaction is used as an indication that delayed 9

type hypersensitivity has developed.

Granuloma formation is a phenomenon that is closely related to

the development of skin test reactions. When Mycobacterium tuberculosis,

Corynebacterium parvum, or certain other antigens are incorporated into

an adjuvant and injected parenterally, granulomatous lesions develop

(44). The lesion has the appearance of a hard nodule and can persist for several months. Histologically, an extensive mononuclear cell

infiltrate is seen. Granuloma formation is often indicative of

delayed hypersensitivity. Antigens can be tested by this method to

determine if delayed hypersensitivity is produced.

In vitro tests for delayed-type hypersensitivity

The leukocyte migration inhibition test is an in vitro correlate

of delayed hypersensitivity (46). Lymphocytes isolated from the

peripheral blood of animals may produce leukocyte migration inhibition

factor (LMIF) after re-exposure to antigen. The LMIF acts to inhibit

the random migration of polymorphonuclear . A number of

different migration inhibition assays have been described for use with avian leukocytes (47,48,49,50,51). The migration inhibition test often

correlates with skin test reactions.

The lymphocyte transformation assay is used as an in vitro correlate of delayed hypersensitivity (52,53,54,55). Lymphocytes from antigen sensitized animals may undergo blast transformation upon re-exposure to the antigen. Blast transformation is measured semi quantitatively. 10

and is used as an index of cell-mediated immunity.

Cell-mediated immune responses

Cell-mediated immunity is discussed extensively in this dissertation.

Therefore it is appropriate to elaborate on this topic.

There are a number "of bacterial pathogens that induce cell-mediated

immunity. These include Mycobacterium tuberculosis. abortus,

and Listeria monocytogenes. The viruses, various fungi, and some

protozoa also induce cell-mediated immunity (44). Certain hapten-

carrier conjugates such as dinitrophenyl-bovine gamma globulin (DNP-BGG)

also have this capacity (56,57).

Macrophages process and present antigen to lymphocytes (58). A small

number of T lymphocytes, probably less than two percent, are capable of

undergoing blast transformation in response to antigenic stimulation and will form clones derived from individual lymphocytes (59). These sensitized T lymphocytes may secrete various lymphokines, and recruit and activate new macrophages. Sensitized T lymphocytes also secrete lymphotoxins and will kill some tumors in vitro (60).

Cell-mediated immune responses generally fall into two categories;

(A) those that involve direct participation of intact lymphocytes in the effector mechanism of the reaction (cytotoxic reactions), and (B) those that involve participation by lymphokines (delayed hypersensitivity and associated phenomena) (45). n

PART I: PASTEURELLA MULTOCIDA: IMMUNOLOGIC STATUS OF CHICKENS AND TURKEYS GIVEN INACTIVATED VACCINE 12

SUMMARY

Turkeys were shown to be more susceptible than chickens to

Pasteurella multocida challenge infection. A formal in-inactivated

Pj. multocida bacterin produced a significant level of protective immunity in chickens and turkeys.

In turkeys, vaccination induced an antibody response that rapidly increased until the 10th day after vaccination. The antibody titers then remained relatively unchanged. A second vaccination induced a four-fold increase in antibody titers.

The results indicate that it is feasible to use the formalin inactivated bacterin in studies of the immunity produced by vaccination. 13

INTRODUCTION

Fowl cholera is recognized as a potentially severe poultry disease.

Antibiotics are often added to the feed ration to act prophylactically

against various diseases including fowl cholera. Some poultry management

programs use a combination of antibiotics and vaccination to prevent

cholera.

Certain species of birds are more susceptible to fowl cholera

infection than others. Turkeys are more commonly infected than chickens.

Vaccination provides a degree of protection in both chickens and

turkeys (1).

There are two types of commercial vaccines: (A) live attenuated

£.• multocida or (B) formalin inactivated P^. multocida suspended in

Freund's incomplete adjuvant.

Poultry growers are sometimes reticent to use live fowl cholera vaccines in areas where the disease is not endemic (2). Occasional outbreaks of the disease have been attributed to the use of attenuated vaccine strains.

To develop more effective vaccines, the resistance produced by iP. multocida vaccination needs to be more thoroughly characterized.

This paper reports the results of a preliminary study that was made to determine the feasibility of using the formalin-inactivated bacterin in a study of the immunity produced by vaccination. Several factors were analyzed including (A) the susceptibility of nonvaccinated chickens 14

and turkeys to challenge exposure, (B) the protective effect of vaccination, (C) the development of antibody response following vaccination, and (D) the effect of revaccination on antibody response. 15

MATERIALS AND METHODS

Preparation of vaccine

multocida, strain 1059 was grown on tryptose agar in Roux bottles for 18 hours at 37 C. The cells were washed off the agar surfaces with sterile saline and put into a sterile flask. Bacterial counts were determined by preparing serial dilutions in saline, followed by plating on agar. The concentration was adjusted to 10^ cells/ml of saline. Formalin was then added to the flask to a final concentration of 0.3% of the total volume, and the flask was kept at room temperature for 18 hours to kill the . A sample of the formalinized bacterial suspension was tested by plating on tryptose agar to determine sterility. No colony growth was detected after the formalin treatment. The bacterial suspension was combined in a ratio of 1:1 v/v with Freund's complete adjuvant (Difco) and was used as vaccine.

Tube agglutination test

Antigen was prepared by growing and harvesting the bacteria in the same manner as described for the preparation of the vaccine. Strain 1059 has a capsule which was removed by acid treatment. After addition of formalin, bacteria were centrifuged at 21,000 x G for 15 minutes in a

Sorval RC-2B centrifuge. The pellet was suspended in 150 ml of 1 N 16

HCl acid-saline solution and incubated for 18 hours at 37 C. The cells

were then washed and centrifuged twice with phosphate buffered saline

plus 0.3% formalin, pH 5.8. The cell concentration was adjusted to

equal an optical density of 0.34 as determined in a Spectronic 20

spectrophotometer at a wavelength of 600 nm. Phosphate buffered

saline plus 0.3% formalin was used as the diluent. For the agglutina­

tion test, a twofold dilution of the antisera being tested was used

in each tube. Antigen (1 ml) and diluted antiserum (1 ml) were

combined in each tube and were incubated at 37 C for 18 hours. The

agglutination end point was then determined and recorded as the

agglutination titer (reciprocal of the end point dilution).

Part I - Susceptibility studies: protection studies

One group of eight three-week-old chickens received a single

subcutaneous injection of 1 ml of the iP. multocida vaccine. A second

group of six chickens was used as the control and was not vaccinated.

The birds were challenge exposed at six weeks of age with 10,000

viable P^. multocida by intramuscular injection into the thigh.

One group of eleven three-week-old turkeys also received a single

subcutaneous injection of 1 ml of the P^. multocida vaccine. A second

group of eight turkeys was not vaccinated and was used as control.

The birds were then challenge exposed in the same manner as the chickens. 17

Part II - Development of antibody titers following vaccination and revaccination

Three twelve-week-old turkeys received a subcutaneous injection of 1 ml of the multocida vaccine. Agglutination antibody titers were measured at three-day intervals. A second vaccination was given

26 days after the initial vaccination. Three ml of blood was drawn from the wing vein of each bird on every third day. The antisera were tested using the agglutination procedure described above. 18

RESULTS

Differences in susceptibility to challenge exposure between nonvaccinated

chickens and turkeys

Nonvaccinated turkeys were more susceptible to challenge infection than nonvaccinated chickens. All of the turkeys were dead within 48 hours after challenge exposure. Chickens did not start dying until four days after challenge exposure. Mortality reached 66 percent (Fig. 1).

Other experiments by this author have demonstrated that in groups of nonvaccinated chickens mortality may reach 100% when a larger challenge exposure is given (data not shown). However, the development of clinical signs and mortality is always delayed in chickens in comparison to turkeys.

Protective effect of vaccination

Vaccination of three-week-old birds reduced mortality significantly when challenge exposure was given at six weeks of age. In turkeys, mortality was reduced from 66% to 12% (Fig. 1). Vaccinated birds that de­ veloped fowl cholera succombed at least three days later than nonvaccinated birds (Fig. 1). Other experiments completed by this author have demonstrated that mortality may reach 25% in vaccinated chickens when a larger challenge dose is given (data not shown). 19

Development of antibody titers following vaccination

The geometric mean agglutination titer {G.M.T., geometric mean of the reciprocal of the end point dilution) rapidly increased following vaccination. By day 10 following vaccination a G.M.T. of 56 was recorded. The G.M.T. then remained relatively unchanged until the turkeys were revaccinated (Fig. 2).

Effect of revaccination on agglutination titers

Revaccination on day 26 produced a transitory drop in G.M.T. followed by a dramatic rise, A 4.5-fold increase in G.M.T. was seen.

The G.M.T. increased to 224 following the revaccination (Fig. 2). 20

DISCUSSION

A number of factors are important when evaluating P^. multocida vaccines. These factors include the level and duration of protection and need for revaccination. In addition, a successful £. multocida vaccine must be able to induce cross-protection against different

£. multocida serotypes. Cross-protection is at least partially dependent on the method used to grow P^. multocida (3).

It is evident that the development of P^. multocida vaccines will be facilitated by a deeper understanding of the immunity induced by vaccination. The immunologic mechanisms mediating resistance are not well-understood. Certain aspects need to be more clearly elucidated, particularly the relationship of antibody titer to protection (4,5) and the role of cell-mediated immunity in protection (6,7,8,9).

These results confirm that a formalin inactivated bacterin provides a substantial level of protection in chickens and turkeys.

Revaccination increased agglutination titers significantly. The results demonstrate that there is a distinct difference between chickens and turkeys in their susceptibility to challenge infection and in their response to vaccination (Fig. 1). There is no known reason for this phenomenon. 21

The results from this study suggest that the immunity produced by inactivated adjuvanted bacterins is of sufficient duration and magnitude so that this method of vaccination could be used in further studies where a more detailed analysis of the immune response will be made. Figure 1. Mortality of vaccinated and nonvaccinated chickens and turkeys after challenge exposure to strain P-1059

• nonvaccinated turkeys (n=8).

• vaccinated turkeys (n=ll).

A nonvaccinated chickens (n=6).

• vaccinated chickens (n=8). 23

100 n

90-

80-

0 70-

0 60-

(S 50-

40-

•H 30-

2 20-

10-

0 2 4 6 8 10 12 Days post-challenge Figure 2. Development of agglutination antibody titers following vaccination of turkeys with P-1059 bacterin (n=3). Geometric mean titer is expressed as the geometric mean of the reciprocal of the end point dilution.

A indicates revaccination on day 26. 25

250

200

150

50

5 10 15 20 30 35 40

DAYS POST-VACCINATION 26

REFERENCES CITED

1. Heddleston, K. L., and K. R. Rhoades. 1978. Avian . Pages 181-199 in M. S. Hofstad, ed. Diseases of Poultry 7th edition. The Iowa State University Press, Ames, Iowa.

2. Matsumoto, M., and D. H. Heifer. 1977. A bacterin against fowl cholera in turkeys: Protective quality of various preparations originated from broth cultures. Avian Dis. 21:382-392.

3. Rimler, R. B., P. A. Rebers, and K. R. Rhoades. 1979. Modulation of cross-protection factor(s) of avian Pasteurella multocida. Avian Dis. 24:989-998.

4. Coates, S. R., M. M. Jensen, and E. D. Brown. 1977. The response of turkeys to varying doses of live oral Pasteurella multocida vaccine. Poult. Sci. 56:273-276.

5. Dua, S. K., and S. K. Maheswaran. 1978. Studies on Pasteurella multocida. VI. Nature of systemic immunity and analysis of the correlation between levels of immunity induced by various fowl cholera vaccines and protection against challenge. Avian Dis. 22:748-764.

6. Yamaguchi, Y., and T. Baba. 1974. Demonstration in tissue culture of cellular immunity to fowl cholera. Pages 14-18 in T. Hasegawa ed. Proceedings of the First Intersectional Congress of the lAMS. Vol. 4. Science Council of Japan, Tokyo.

7. Baba, T., T. Ando, and M. Nukena. 1978. Effect of bursectomy and thymectomy on Pasteurella multocida infection in chickens. J. Med. Microbiol. 11:281-288.

8. Maheswaran, S. K., E. S. Thies, and S. K. Dua. 1976. Studies on Pasteurella multocida. III. In vitro assay for cell-mediated immunity. Avian Dis. 20:332-341.

9. Maheswaran, S. K., S. K. Dua, and E. S. Thies. 1980. Studies on Pasteurella multocida. IX. Levamisole-induced augmentation of immune responses to a live fowl cholera vaccine. Avian. Dis. 24:71-81. 27

PART II: PASTEURELLA MULTOCIDA: ANTIBODY MEDIATED RESISTANCE TO VIRULENT CHALLENGE EXPOSURE IN VACCINATED TURKEYS

This paper was published in the American Journal of Veterinary Research 41:1285-1287 (1980) 28

SUMMARY

Aspects of antibody mediated resistance to Pasteurella muTtocida infection in vaccinated turkeys were investigated. multocida immune serum obtained from vaccinated turkeys was shown to confer temporary protection to nonvaccinated turkey poults. Recipients given immune serum were free of clinical signs of disease for at least 8 days after intramuscular (IM) challenge exposure with virulent multocida

1059. All turkeys given normal serum died within 36 hours of challenge exposure.

Vaccinated bursectomized turkeys were more susceptible to IM challenge exposure than were vaccinated nonbursectomized turkeys. In two of three trials, mortality also occurred earlier in the bursectomized turkeys. The presence of specific antibody may be an important determ­ inant in resistance to P. multocida infection. 29

INTRODUCTION

Many domestic and wild animals are susceptible to infection with

Pasteurella multocida. In poultry, P. multocida produces fowl cholera and often fatal septicemia. The mechanisms of resistance to fowl cholera stimulated by vaccination remain controversial. Some investi­ gators have not been able to show a correlation between serum antibody titers in vaccinated turkeys and resistance (1,2), whereas others have indicated that a correlation exists (3). There is also some evidence that indicates that cell-mediated immunity may function in resistance to 2" multocida in vaccinated turkeys (4). To develop more effective vaccines, the mechanisms of resistance to P^. multocida need to be better understood.

The purpose of the present report is to indicate the importance of antibody response in resistance to IM P^. multocida challenge exposure.

Two methods were used to evaluate in vivo antibody activity: (A) the induction of passive protection by transfer of immune sera from vaccinated turkeys, (B) the determination of susceptibility of vaccinated bursectomized turkeys to virulent JP. multocida 1059 challenge exposure. 30

MATERIALS AND METHODS

Preparation of vaccine

Pasteurella multocida strain 1059 was grown on tryptose agar in Roux bottles for 18 hours at 37 C. The bacteria were washed from the agar surfaces with sterile saline solution and were pooled in a sterile flask. Bacterial counts were obtained by making serial dilutions in saline solution followed by plating on agar. The concentration of

£. multocida was adjusted to 10^ cells/ml of saline solution. Formalin was then added to the flask to a final concentration of 0.3% of the total volume and the flask was kept at room temperature for 18 hours to kill the bacteria. A sample of the formalinized bacterial suspension was tested by plating on tryptose agar to determine sterility. The saline suspension of £. multocida was combined in a ratio of 1/1 v/v with

Freund's complete adjuvant and was used as vaccine.

Production of antisera

Antisera against P^. multocida strain 1059 were produced in three

Broad Breasted White turkeys that were given two subcutaneous injections each of 0.1 ml of the vaccine and two injections each of 1 ml given intraperitoneally. Injections were administered at three-week intervals.

Blood samples were collected 2 weeks after the final injection, and the serum was separated and heated at 56 C for 30 minutes and then pooled. 31

Normal serum samples were obtained from several nonvaccinated

turkeys, heat treated, and pooled.

Approximately 100 ml of serum was obtained from each of the groups.

Passive protection - (Experiment I)

Three-week old nonvaccinated turkey poults were used as recipients

of antiserum against P^. multocida or normal serum. Eight poults were

each injected IV with 8 to 9 ml of antiserum. Seven poults were each

injected IV with 8 to 9 ml of normal serum. Signs of distress were not observed after the large transfusions. Twenty-four hours after passive serum transfers, each poult was inoculated IM with 2000 viable P^. multocida organisms in the thigh.

Studies with bursectomized turkeys - (Experiment II)

Removal of the Bursa of Fabricius in the chicken diminishes or eliminates antibody response but leaves cell-mediated responses essentially intact (5). Turkeys were used instead of chickens in the present study because of their greater susceptibility to P^. multocida infection.

Fertile turkey eggs were purchased from a local hatchery and were incubated. Within 24 hours of hatching, a group of birds was bur­ sectomized and another group was left intact and used as controls. The area immediately dorsal to the cloaca was cleared of feathery down and was wiped with an alcohol swab. A small perforation was made and widened with the tips of sharp forceps. Mild pressure was applied to the abdomen 32

to move the bursa into proximity to the incision. Sterile forceps

were inserted through the incision and the suspensory tissue located

just dorsal to the bursa was teased away. The forceps were then moved

in a cranial direction over the surface of the bursa. The apex of the

bursa was grasped and pulled through the incision. The bursa was then

cut at the stalk. Nitrofurazone antibacterial powder^ was applied to the

wound sitsv Healing normally occurred within 2 or 3 days. All poults

used in the experiment were kept together in a heated disinfected room

and were given autoclaved feed.

Bursectomized and nonbursectomized poults were vaccinated at 3 weeks of age with a single subcutaneous injection of 1 ml of vaccine. The poults were challenge exposed at 6 weeks of age, with 2,000 viable JP. multocida organisms by IM inoculation into the thigh. The IM challenge route was chosen because it is quantitative and reproducible.

Experiment II wa,> conducted as three separate trials (Table 1).

Tube agglutination test

£o multocida strain 1059 has a capsule that must be removed by acidic or enzymatic treatment for agglutination to occur.

Agglutination antigen was prepared by growing and harvesting the bacteria in the same manner as described for the preparation of the vaccine. 2 Formalinized bacteria were centrifuged at 21,000 x g for 15 minutes.

^Furacin soluble powder, Easton Veterinary Laboratories, Morton Norwich Products Inc., Norwich, NY.

^Sorvall RC 2-B, Ivan Sorvall Inc., Newtown, CT. 33

The pellet was then suspended in 150 ml of 1 N HCl saline solution and was incubated at 37 C for 18 hours. Cells were centrifuged again and were washed twice with formalinized PBSS (pH 5.8). The cell concentra­ tion was then adjusted to equal an optical density of 0.648 on a spectrophotometer^ at a wave length of 600 nm. Formalinized PBSS

(pH 5.8) was used as the diluent. A two-fold dilution of the antisera being tested was used in each tube. Antigen (1 ml) and antiserum

(1 ml) were combined in each tube and were incubated at 37 C for 18 hours.

^Bausch and Lomb, Rochester, NY. 34

RESULTS

Passive protection - (Experiment I)

Passive protection was indicated when poults survived challenge

exposure with £. multocida 1059. The poults given normal serum showed

clinical signs of disease 24 hours after challenge exposure, and 100%

died within 36 hours. Poults given antiserum from vaccinated turkeys

developed a temporary resistance as indicated by delayed mortality

from day 8 to 13 after challenge exposure (Fig. 1). Of eight poults,

two given antiserum survived the challenge exposure.

Effect of bursectomy on resistance in vaccinated turkeys - (Experiment II)

Poults that were bursectomized, vaccinated, and challenge exposed, were less resistant than were nonbursectomized vaccinated controls

(Table 1). Mortality was significantly higher (P<0.05, test) in the bursectomized group than in the controls. Deaths began to occur earlier in two of three trials, in the bursectomized group (Fig. 2). Bursectomized poults which had been vaccinated also produced some detectable antibody as demonstrated by the agglutination of IP. multocida. However, antibody levels were considerably depressed in the sera of bursectomized poults.

Generally, the antibody titers correlated with observed resistance (Table 2). 35

DISCUSSION

The present results confirm and extend the evidence for antibody mediated protection against _P. multocida infection in vaccinated turkeys.

The data obtained confirm the results of Rebers and Heddleston who demonstrated that passively transferred antiserum can protect against challenge exposure (6). The data also indicate that vaccinated bur- sectomized turkeys were more susceptible to IM challenge exposure with

P^. multocida than were vaccinated turkeys that were not bursectomized.

Turkeys given antiserum survived IP. multocida challenge exposure for

8 days whereas the control birds died within 36 hours after challenge exposure. These observations indicate that antibody against P^. multocida has the capacity to suppress clinical signs of disease and mortality.

Recent studies by Hofing et al. are interesting in view of these results

(7). They found that rabbit antiserum against P^. multocida was not bactericidal and did not enhance phagocytosis when compared with normal serum in in vitro tests. This apparent discrepancy between their in vitro results and the present in vivo results may be due to a modifica­ tion of 2' multocida organisms under different growth conditions. Changes in the organisms can be inferred from certain observations. When P^. multocida is isolated from the blood or tissues of turkeys and is used as a formalini zed bacterin, it is capable of inducing cross-protection in turkeys against challenge exposure by heterologous strains.

However, P. multocida from agar-grown cultures protects only 36

against challenge exposure by the homologous strain (8). In addition,

multocida isolated directly from infected tissues are lysed by

freeze-thaw cycles whereas agar-grown cells are not (9). Conceivably, the aforementioned cited differences between in vivo and in vitro grown

multocida organisms may be extended to differences in susceptibility to bactericidal and opsonizing activities of antibodies.

The role of immunoglobulin (Ig)G in protection against 2- multocida challenge exposure has been demonstrated in passive protection studies by Rebers and Heddleston (6). They have established that IgG antibody from turkeys immunized with P^. multocida infected tissues, could passively cross-protect chickens against agar-grown multocida almost as well as whole antiserum.

The vaccinated bursectomized turkey poults used in this study produced less P^. multocida antibody than did vaccinated nonbursectomized poults. The bursectomized group also had less resistance to challenge exposure. The bursectotny experiments indicate that there is a re­ quirement for humoral response in the development of resistance to £. multocida.

The bursectorny results obtained with turkeys do not agree with results reported by Baba et al^. (10). These authors did not observe antibody dependent protection in vaccinated chickens that had been bursectomized.

The initial site of infection by P^. multocida under field conditions is usually the respiratory system. However, conclusive evidence has not 37

been obtained which indicates that local respiratory immunity is

induced by P^. multocida infection or vaccination. There is no known

selective advantage in giving IM challenge exposure vs. aerosol challenge

exposure in demonstrating the relevance of circulating antibody to

protection. The IM route was chosen for challenge exposure to

deliver uniform and quantitative doses. It is possible that antibodies

which suppress systemic challenge exposure infections also may function

in local respiratory immunity. Further studies are necessary to clarify the role of antibodies in local respiratory immunity.

The present results suggest that presence of antibody may be an important determinant in resistance of turkeys against IM challenge exposure infection with £. multocida. 38

ADDENDUM

At the time this manuscript was being prepared it was found, in preliminary results, that treatment of newly hatched chickens with cyclophosphamide, a potent suppressor of antibody production, diminished the effectiveness of vaccination against challenge exposure. This observation further suggests that antibodies have a role in development of resistance. 39

Table 1. (Experiment II) Effect of bursectomy on resistance to 2- multocida challenge exposure in turkeys. Ratios shown represent (Number dead/number challenge exposed). Bx = bursectomized. Vacc = vaccinated.

Trial Bx + Vacc Vacc only No Bx, No Vacc

Trial 1 7/8 3/7 3/3

Trial 2 9/12 4/11 3/3

Trial 3 12/13 8/13 3/3

Cumulative % 85% 47% 100%

Table 2. (Experiment II) Effect of bursectomy on antibody titers (Reciprocal of dilution) against P. multocida in vaccinated turkeys and relationship to challenge exposure mortality. Results are from turkeys randomly selected in trial two at one day before challenge. Bx = bursectomized. Vacc =

vaccinated. '

Group Bird Titer Status

Bx + Vacc 374 160 Dead 381 10 Dead 382 80 Dead 389 80 Dead 391 5 Dead 392 10 Dead 393 40 A1i ve

Vacc only 485 320 A1i ve 495 320 A1i ve 497 1280 A1i ve 501 80 A1i ve 504 40 Dead Figure 1. (Experiment I) Mortality rate of IP. multocida challenge exposed turkeys given 8 to 9 ml of turkey serum 24 hours before challenge exposure

Normal serum (n=7).

Immune serum (n=8).

Figure 2. (Experiment II) Mortality rate of JP. multocida challenge exposed turkeys

0 Bursectomized and vaccinated (n=12).

# Vaccinated only (n=ll).

This pattern seen in two of three trials. Percent Cumulative Mortality Bercent Cumulative Mortality

O) o o o o o

ro< O O)'

=r »• i®.

fO' 42

REFERENCES CITED

1. Bhasin, J. L., and E. L. Biberstein. 1968. Fowl cholera in turkeys - the efficacy of adjuvant bacterins. Avian Dis. 12:159-168.

2. Alexander, A. M., and M. A. Soltys. 1973. Relationship of serum agglutinins to protective immunity produced in turkeys immunized against fowl cholera. J. Comp. Pathol. 83:191-198.

3. Coates, S. R., M. M. Jensen, and E. D. Brown. 1977. The response of turkeys to varying doses of live Pasteurella multocida vaccine. Poult. Sci. 56:273-276.

4. Maheswaran, S. K., and E. S. Thies. 1979. Pasteurella multocida antigen induced i£ vitro lymphocyte stimulation, using whole blood from cattle and turkeys. Res. in Vet. Sci. 26:25-31.

5. Warner, N. L., A. Ovary, and F. S. Kantor. 1971. Delayed hyper­ sensitivity reactions in normal and bursectomized birds. Int. Arch. Allergy 40:719-728.

6. Rebers, P. A., K. L. Heddleston, B. Wright, and K. Gillette. 1975. Fowl cholera: Cross protective turkey antisera and IgG antibodies induced with Pasteurella multocida infected tissue bacterins. Carbohydr. Res. 40:99-100.

7. Hofing, G. L., H. G. Rush, A. R. Petkus, and J. C. Glorioso. 1979. In vitro killing of Pasteurella multocida: The effect of rabbit granulocyte and specific antibody source. Am. J. Vet. Res. 40:679-683.

8. Heddleston, K. L., and P. A. Rebers. 1972. Fowl cholera: Cross- immunity induced in turkeys with formalin-killed in vivo propagated Pasteurella multocida. Avian Dis. 16:578-586.

9. Rimler, R. B., P. A. Rebers, and K. R. Rhoades. 1979. Fowl cholera: Cross protection induced by Pasteurella multocida separated from infected turkey blood. Avian Dis. 23:730-741.

10. Baba, T., T. Ando, and M. Nukina. 1978. Effect of bursectomy and thymectomy on Pasteurella multocida infection in chickens. J. Med. Microbiol. 11:281-288. 43

PART III: CYCLOPHOSPHAMIDE-INDUCED IMMUNOSUPPRESSION DEMONSTRATED IN PASTEURELLA MULTOCIDA VACCINATED CHICKENS

This paper has been accepted for publication in Avian Diseases 44

SUMMARY

Vaccinated cyclophosphamide-treated chickens were more susceptible to challenge infection than vaccinated untreated chickens. Antibody titers were greatly reduced in cyclophosphamide-treated chickens. The results suggest that the presence of specific antibody is an important determinant in the ability of chickens to resist £. multocida infection. 45

INTRODUCTION

In poultry, Pasteurella tiiultocida produces fowl cholera, which

often results in high mortality. The type of immunity stimulated by

vaccination is still controversial. Some investigations have indicated

that cell-mediated immunity plays a role in resistance to challenge

infection (1,2). Other work suggests that antibody-mediated immunity

is a determinant of challenge resistance (3,4,5,6).

In this study, a tumoricidal agent, cyclophosphamide, was used to

diminish or eliminate antibody response in chickens, and to evaluate the

relative importance of antibody to resistance of vaccinated chickens to

P^. multocida challenge.

Treatment of newly hatched chickens with cyclophosphamide causes suppression of antibody production for at least 10 weeks. Cell-mediated

immunity is suppressed for less than one month (7,8,9,10). Histological studies provide evidence for this differential suppression (8,9).

Cyclophosphamide treatment causes severe and often permanent depletion of bursal cells and chronic degenerative changes in the bursal structure.

Spleens show an absence of germinal centers and plasma cells. Initially, the lymphoid population of the thymus decreases, but it is rapidly repopulated within two or three weeks after treatment is completed.

Cellular immunological functions of birds treated in the newly hatched period appear to recover by three to four weeks of age (7,8,9,10).

Allograft rejection and graft-versus-host reactivity are restored by this age. 46

As a result of this differential suppression, one may evaluate the protective components of the immune response in vivo against challenge infection. 47

MATERIALS AND METHODS

Fertile chicken eggs were incubated and hatched. Within 24 hours

of hatching one group of chickens received intramuscular injections of

4 mg cyclophosphamide.^ The chickens were given daily injections for

four days. A second group, not treated with cyclophosphamide, served

as controls. The birds were kept in heated batteries and fed ad

libitum.

2. multocida strain 1059 was grown on tryptose agar in Roux

bottles for 18 hours at 37 C. Bacteria were harvested in sterile saline

and placed into a sterile flask. Bacterial counts were obtained by

serial dilution in saline and plating on tryptose agar. The concentration of P^. multocida was adjusted to 10^ cells/ml saline. Formalin was then

added to the flask to a final concentration of 0.3% of the total volume

and held at room temperature for 18 hours to kill the bacteria. No

viability was detected after the formalin treatment. The bacterial suspension was combined in a ratio of 1:1 v/v with Freund's complete adjuvant (Difco) and used as vaccine.

Both groups of chickens were vaccinated 26 days after the last administration of cyclophosphamide. Each bird was given a single subcutaneous injection of 1 ml of the vaccine.

Antibody titers were determined by a bacterial agglutination test.

Strain 1059 has a capsule, which was removed by acid treatment. An

^Cytoxan, Mead Johnson Laboratories, Evansville, IN. 48

agglutination antigen was prepared by growing and harvesting the

bacteria as described above. Formalinized bacteria were centrifuged at

21,000 X G in a Sorvall RC2-B centrifuge for 15 minutes. The pellet

was then suspended in 150 ml of 1 N HCl saline solution and incubated

at 37 C for 18 hours. The cells were washed and centrifuged twice

with formalinized phosphate-buffered saline, pH 5.8. The cell con­

centration was adjusted to an optical density of 0.34 as determined in

a Spectronic 20 spectrophotometer^ at a wave length of 600 nm.

Formalinized phosphate-buffered saline, pH 5.8, was used as the diluent.

Antiserum was obtained from each chicken one day before challenge

exposure. In the agglutination test, twofold dilutions of antiserum in

1 ml of diluent were mixed with 1 ml of the antigen and incubated at 37 C

for 18 hours. The agglutination end point was then determined and

recorded as the titer (reciprocal of dilution).

The chickens were challenged 21 days after vaccination with either

2 X 10® (trial 1) or 2 x 10® (trial 2) P.. multocida given by intra­ muscular inoculation into the thigh. The chickens were observed for 21

days after the challenge and mortality was recorded.

^Bausch and Lomb, Rochester, NY. 49

RESULTS

Vaccinated cyclophosphamide-treated chickens were more susceptible

to challenge infection than were the vaccinated untreated chickens

(Table 1). All of the cyclophosphamide-treated chickens died from the

challenge infection. Mortality was significantly higher (p<0.01,

test) in the cyclophosphamide-treated group than in the control group.

Three of 17 treated chickens produced agglutinating antibody against

IP. multocida. All untreated birds produced agglutinating antibody

(Table 1).

Five of 19 vaccinated chickens died, but they survived longer than most of the vaccinated cyclophosphamide-treated chickens (Fig. 1). 50

DISCUSSION AND CONCLUSIONS

The results suggest that there is a correlation between the presence of antibody and of the ability of vaccinated chickens to resist

multocida. This agrees with previous experiments that demonstrated that vaccinated surgically bursectomized turkeys were more susceptible to challenge with K multocida (11). Cell-mediated immunity may not be the primary means of protection with this method of vaccination.

Clearly, it would be an asset to confirm that the cyclophosphamide treatment did not suppress cell-mediated responses to £. multocida.

Unfortunately, this laboratory has not successfully established an assay for cell-mediated immunity to £. multocida. Other studies with cyclophosphamide suggest that cell-mediated immunological functions are restored by the fourth week post-treatment (7,8,9,10). In these experiments, approximately seven weeks elapsed between completion of cyclophosphamide treatment and time of challenge. 51

Table 1. Suppressive effect of cyclophosphamide. Reduction of agglutinating antibody titers of chickens vaccinated with P. tnultocida and relationship to mortality after challenge. Antiserum was obtained from each chicken one day before challenge exposure

Trial Treatment Antibody titer® Mortality^

1 Cyclophosphamide 0-5 9/9

None 40-320 3/9

2 Cyclophosphamide 0-10 8/8

None 40-1280 2/10

Reciprocal of dilution, range of titer of group. One treated bird produced detectable titer in Trial 1. Two treated birds produced detectable titers in Trial 2.

^Number dead/number challenged. Figure 1. Mortality of vaccinated chickens to P^. multocida challenge infection

Data are pooled from trials 1 and 2.

Cyclophosphamide-treated (n=17).

4 Untreated (n=19). Percent Cumulative Mortality fO o o

to- i

"u en

(Q o 54

REFERENCES CITED

1. Baba, T., T. Ando, and M. Nukina. 1978. Effect of bursectomy and thymectomy on Pasteurella multôcida infection in chickens. J. Med. Microbiol. 11:281-288.

2. Maheswaran, S. K., E. S. Thies, and S. K. Dua. 1976. Studies on Pasteurella multôcida. III. In vitro assay for cell-mediated immunity. Avian Dis. 20:332-347.

3. Bierer, B. W., and W. T. Derieux. 1975. The ability of blood plasma from drinking water vaccinated turkeys to protect against a lethal challenge of Pasteurella multôcida. Poult. Sci. 54:2091-2093.

4. Coates, S. R., M. M. Jensen, and E. D. Brown. 1977. The response of turkeys to varying doses of live oral Pasteuralla multôcida vaccine. Poult. Sci. 56:273-276.

5. Dua, S. K., and S. K. Maheswaran. 1978. Studies on Pasteurella multôcida. VI. Nature of the systemic immunity and analysis of the correlation between levels of immunity induced by various fowl cholera vaccines and protection against challenge. Avian Dis. 22:748-764.

6. Heddleston, K. L., P. A. Rebers, and G. Wessman. 1975. Fowl cholera: Immunologic and serologic response in turkeys to live Pasteurella multôcida vaccine administered in the drinking water. Poult. Sci. 54:217-221.

7. Lerman, S. P., and W. P. Weidanz. 1970. The effect of cyclo­ phosphamide on the ontogeny of the humoral immune response in chickens. J. Immunol. 105-614-619.

8. Linna, T. J., D. Frommel, and R. A. Good. 1972. Effect of early cyclophosphamide treatment on the development of lymphoid organs and immunological functions in the chicken. Int. Arch. Allergy 42:20-39.

9. Rouse, B. T., and A. Szenberg. 1974. Functional and morphological observations on the effect of cyclophosphamide on the immune response of the chicken. Aust. J. Expt. Biol. Med. Sci. 52:873-885. 55

10. Sharma, J. M., and L. F. Lee. 1977. Suppressive effect of cyclophosphamide on the T-Cell system in chickens. Infect. Immun. 17:227-230.

11. Nathanson, R. M., M. S. Hofstad, and E. L. Jeska. 1980. Pasteurella multocida antibody-mediated resistance to virulent challenge exposure in vaccinated turkeys. Am. J. Vet. Res. 41: 1285-1287. 56

PART IV: IN VIVO TESTS FOR DELAYED TYPE HYPERSENSITIVITY W PTRTEURELLA MULTOCIDA IN VACCINATED CHICKENS AND TURKEYS 57

SUMMARY

In vivo tests were developed in order to determine whether delayed type hypersensitivity is induced by Pasteurella multocida vaccination.

Chickens and turkeys were vaccinated with a formalin inactivated

multocida bacterin. The birds were studied by skin testing, evaluation of granuloma formation, and treatment with niridazole, an immunosuppressive drug.

Vaccinated chickens and turkeys did not appear to develop delayed type hypersensitivity in these studies. 58

INTRODUCTION

The immune response to Pasteurelia multocida vaccination is not well-understoodo In vaccinated poultry both humoral and cell-mediated immunity may be important in resistance to challenge infection (1).

Certain in vitro tests have indicated that cell-mediated immunity may be induced by vaccination (2,3). There have not been any reports of in vivo tests that were used to evaluate cell-mediated immunity and delayed hypersensitivity to IP. multocida. This report presents the results of a preliminary study in which in vivo tests were developed.

Delayed type hypersensitivity: the phenomenon

Cell-mediated immune responses fall into two general categories,

(A) those that involve direct participation of intact lymphocytes in the effector mechanism of the reaction (cytotoxic reactions), (B) those that involve participation by lymphokines (delayed type hypersensitivity and associated phenomena) (4).

Delayed type hypersensitivity reactions (hereafter called DTH reactions) are inflammatory in nature, and are characterized by the infiltration of mononuclear cells into areas of antigen deposition (5).

The macrophages present in DTH infiltrates are activated.

Various intracellular bacteria, protozoa, fungi, and viruses induce macrophage activation. However, not all antigens possess this capacity. 59

Activated macrophages process and present antigen to lymphocytes.

About two percent of the total lymphocyte population is capable of

undergoing blast transformation in response to antigenic stimulation and

will form clones derived from individual lymphocytes (6). A greatly

expanded population of lymphocytes develops that is capable of responding

to the antigen. T lymphocyte blasts can produce lymphokines and various effector substances when they are exposed to antigen. The production of lymphokines by T lymphocytes is considered to be a hallmark of DTH and cell-mediated immunity.

The purpose of this study was to determine whether DTH has a role in resistance to £. multocida infection. DTH responses were analyzed by skin testing, evaluation of granuloma formation, and drug induced immunosuppression.

In vivo tests for DTH

Skin test reactions and granuloma formation result from the same pathogenetic mechanism (5). In both cases cellular reactions localize around foci of antigen. The skin test reaction is more diffuse than are granulomatous reactions (5).

Immunosuppressive drugs can be used to evaluate DTH. Niridazole, an antischistosomal drug, has been shown to suppress DTH in man, chickens, mice, and rats (7,8,9,10,11). The drug has no apparent suppressive effect on humoral immunity (12). Some studies have shown enhancement of antibody response (7). Niridazole has low toxicity when used in doses 60

of less than 100 mg/kg body weight (7,13). When niridazole is

ingested orally, it is metabolized and becomes bound to serum proteins

(14). The metabolite remains bound for at least three weeks, and

immunosuppression exists throughout this period.

Chickens were treated with niridazole to produce a suppression of cell-mediated immunity. Later the chickens were challenge exposed and the requirement for a protective cellular response was evaluated. 61

MATERIALS AND METHODS

Preparation of vaccine

IP. multocida, strain 1059, was grown on tryptose agar in Roux bottles for 18 hours at 37 C. Bacterial counts were obtained by making serial dilutions in saline followed by plating on agar. The Q concentration of P^. multocida was adjusted to 10 cells/ml of saline.

Formalin was then added to the flask to a final concentration of 0.3% of the total volume. Eighteen hours later a sample of the formalinized bacterial suspension was tested by plating on tryptose agar to determine sterility. The bacterial suspension was combined in a ratio of 1:1 v/v with Freund's complete adjuvant and used as vaccine.

Skin Tests

Preparation of skin test reagents

Two skin test reagents were used: (A) formalin inactivated multocida, strain 1059, and (B) formalin inactivated sonicated P^. multocida, strain 1059.

Reagent A consisted of formalin inactivated P^. multocida at a concentration of TO® bacteria/ml formalin-saline. Reagent B consisted of formalin inactivated sonicated P^. multocida at a concentration of 10^ bacteria/ml formalin-saline. The preparation was sonicated in a

Bronwill Biosonik IV sonicator for eight thirty-second pulses with a high intensity probe.^

^VWR Scientific, San Francisco, CA. 62

Skin test with reagent A

Two groups of turkeys were used. One group of four turkeys was

given two subcutaneous injections each of 0.1 ml of the vaccine and two injections each of 1 ml of the vaccine given intraperitoneally. A second group of three turkeys was not vaccinated and was used as control.

Two weeks after the second vaccination the birds in both groups received an intradermal injection of 0.1 ml of the reagent A. Skin test reactions were measured with a ruler after 48 hours had elapsed.

Skin test with reagent B

Two groups of turkeys were used. The turkeys were three weeks old, and were housed together. In one group of nine turkeys, each bird received 1 ml of the vaccine intraperitoneally. Three weeks later the vaccination was repeated. A second group of nine turkeys was not vaccinated and was used as a control group. Two weeks after the second vaccination the birds in both groups received an intradermal injection of 0.1 ml of reagent B. Skin test reactions were measured with a ruler after 48 hours had elapsed. 63

Evaluation of Granuloma Formation

Preparation of materials

(A) Lyophilized and killed Mycobacterium tuberculosis strain R37a^

was combined with a 1:1 v/v mixture of Freund's incomplete

adjuvantrsaline. The concentration of M. tuberculosis was

adjusted to 5 mg/ml of emulsion.

(B) 2" multocida, strain 1059, was harvested and decapsulated

by a method described previously and then lyophilized (15).

The lyophilized killed P^. multocida was combined with a 1:1

v/v mixture of Freund's incomplete adjuvant:saline. The

concentration of P^. multocida was adjusted to 5 mg/ml of

emulsion.

(C) A negative control consisted of a 1:1 v/v mixture of Freund's

incomplete adjuvant;saline.

Chickens

Three groups of nine chickens were used. The chickens were six weeks old, and were housed together. In one group, each chicken received 1 ml of the M. tuberculosis emulsion subcutaneously. In a second group, each chicken received 1 ml of the P^. multocida emulsion.

The chickens in the third group each received the negative control consisting of the adjuvant emulsion alone. The chickens were examined for granulomas by palpation.

^Difco, Detroit, MI. 64

Drug Induced Immunosuppression

Niridazole

Niridazole^ was suspended in distilled water. Dosage was calculated on the basis of body weight. The mean body weight of the chickens was 762 + 97 grams (six birds were weighed). Niridazole was suspended in distilled water so that one ml provided a dosage of

75 mg/kg body weight. The drug was given orally by gavage using a 2 1/2 inch hypodermic needle with a ball tip.

Chickens

Three groups of chickens were used. The chickens were six weeks old and were housed together. One group of six chickens received one ml of niridazole suspension daily, for a period of five days. On the second day of treatment the chickens were vaccinated subcutaneously with 1 ml of the vaccine described above. A group of five chickens was used as the vaccine control group. The control group was vaccinated on the same day as the group receiving niridazole. The control group was not treated with the drug. Another group of four chickens was left untreated and unvaccinated.

Challenge infection

Blood was drawn from the wing vein of each chicken on the day preceding challenge infection. Agglutination titers were determined

^Ciba-Geigy Corp., Summit, NJ. 65

by a method described previously (15).

The birds were challenged one day after the blood samples were taken. Each bird received 2000 £. multocida by intramuscular injection into the thigh. 66

RESULTS

Skin test reactions

Vaccinated turkeys developed a swelling at the site of injection

24 hours after skin testing with reagent A (Table I). The swelling had

the appearance of a small lump. The lump remained for at least five

weeks. One nonvaccinated turkey developed a small lump.

Four out of nine vaccinated turkeys developed a small white welt at

the site of injection 24 hours after skin testing with reagent B. Each

welt gradually disappeared over a ten-day period. None of the

nonvaccinated turkeys showed any reaction.

Granuloma formation

Granulomas developed in all nine chickens injected with Freund's

incomplete adjuvant plus M. tuberculosis (Table III). The granulomas were present for six weeks, the duration of the experiment. Some of the chickens that were injected with Freund's incomplete adjuvant plus 2" multocida developed a temporary thickening at the site of injection. However, this reaction was probably not a true granuloma because it disappeared within four weeks after the injection. The chickens that were injected with Freund's incomplete adjuvant showed no evidence of granuloma. 67

Drug induced immunosuppression

Niridazole-treated vaccinated chickens had lower mortality than untreated vaccinated chickens (Table IV). The niridazole-treated chickens had a slightly enhanced agglutination titer. All of the chickens that were not vaccinated died from challenge exposure. 68

DISCUSSION

Vaccinated turkeys developed a skin test reaction to both reagents.

The reaction to reagent A (formalin inactivated £. multocida) appeared

to be greater than the reaction to reagent B (formalin inactivated sonicated multocida).

It was not possible to compare the results of the two different skin tests because the vaccination protocols and skin test reagent concentrations were dissimilar. Clearly, only limited conclusions can be drawn from the skin test experiments. Vaccinated turkeys appear to have the capacity to develop DTH-like skin test reactions under con­ ditions that are not well-defined. The author has attempted to elicit skin test reactions in £. multocida vaccinated chickens without success.

There is no obvious explanation for this discrepancy.

The decapsulated £, multocida incomplete adjuvant emulsion did not produce granulomas in chickens. Decapsulated cells were used because the capsule is believed to inhibit phagocytosis (16). The macrophages present in granulomas are highly phagocytic. The data indicate that decapsulated P^. multocida do not induce DTH reactions in chickens. Niridazole-treated vaccinated chickens had lower mortality and higher antibody titers than untreated vaccinated chickens. These data suggest two possibilities: (A) the niridazole was ineffective in producing suppression, or (B) the T lymphocyte functions suppressed by 69

niridazole are not required for challenge protection.

The first alternative seems unlikely because previous reports have

shown that the dosage used in these experiments was effective in

inducing suppression in chickens (7,8). Niridazole treatment has been

reported to enhance antibody response (7). In these experiments, the

treated chickens had an elevated antibody titer as compared to the

untreated chickens* The second alternative appears to be more reasonable.

Suppression of T lymphocyte functions would not significantly affect mortality if they are not important in protection. If antibody mediated responses are important, then their potentiation could enhance protection.

The results indicate that DTH is not a major component of protection in chickens. The skin test data indicate that there is a possibility that DTH might be elicited in vaccinated turkeys.

Previous reports of cell-mediated irrenunity to multocida have all dealt with turkeys. It is possible that chickens respond differently to £. multocida vaccination than turkeys. 70

Table 1» Skin test reactions of JP, multocida vaccinated turkeys to whole cell preparation (Reagent A)®

Vaccinated (n=4) 1 2 3 4

Reaction^ 10 3 4 6

Nonvaccinated (n=3) 1 2 3

Reaction 0 2 0

^Turkeys were skin tested with 10^ formalin killed Pc multocida two weeks after final vaccination.

^Diameter of intradermal swelling in mm at 48 hours after skin testing. 71

Table 2. Skin test reactivity of P^. multocida vaccinated turkeys to sonicated preparation (Reagent B)9

Vaccinated (n=9) 1 2 3 4 5 6 7 8 9

Reaction^ 0 2 2 0 0 0 0 0 2

Nonvaccinated (n=9) 1 2 3 4 5 6 7 8 9

Reaction 0 0 0 0 0 0 0 0 0

^Turkeys were skin tested with 0.1 cc of 10® sonicated P. multocida two weeks after final vaccination.

^Diameter of intradermal swelling in mm at 48 hours after skin testing (9 turkeys). 72

Table 3. Presence of localized granuloma in injected chickens

FIA® 0/9 FIA-PM^ 0/9 FIA-TB^ 9/9

Ratios = Number of chickens with granuloma/number of chickens injected.

These results were obtained five weeks after chickens were Injected.

®1 ml of a 1:1, Freund's incomplete adjuvant:saline emulsion.

^1 ml of a 1:1, Freund's incomplete adjuvant:saline emulsion containing 5 mg decapsulated killed P-1059 P^. multocida.

^1 ml of a 1:1, Freund's incomplete adjuvant:saline emulsion containing 5 mg killed R37a: M. tuberculosis. 73

Table 4. Effect of niridazole treatment on immunity in chickens

Vacc + ni rid® 1 2 3 4 5 6

Titer'' 640 320 640 80 320 640

Mortali ty^ + +

Vacc alone^ 1 2 3 4 5

Titer 640 40 160 40 160

Mortality + + + +

Control® 1 2 3 4

Titer 0 0 0 5

Mortality + + + +

^Vaccinated and niridazole treated (n=6).

^Titer: Agglutination end point expressed as reciprocal of dilution.

^Mortality: Chickens dead from challenge infection +.

^Vaccinated alone: No niridazole treatment (n=5).

^Control: No vaccination. No niridazole (n=4). 74

REFERENCES CITED

1. Dua, S. K., and S. K. Maheswaran. 1978. Studies on Pasteurella mùitôcida. VI. Nature of systemic irnnunity and analysis of the correlation between levels of immunity induced by various fowl cholera vaccines and protection against challenge. Avian Dis. 22:748-764.

2. Maheswaran, S. K., E. S. Thies, and S. K. Dua. 1976. Studies on Pasteurella multocida. III. In vitro assay for cell-mediated immunity. Avian Dis. 20:332-3^7.

3. Yamaguchi, Y., and T. Baba. 1974. Demonstration in tissue culture of cellular immunity to fowl cholera. In, T. Hasegawa Ed. Proceedings of the first intersectional Congress of the lAMS. Science Council of Japan 4:14-18.

4. Cohen, S. 1977. The role of cell-mediated immunity in inflammatory responses. Am. J. Pathol. 88:502-528.

5. Bennacerraf, B., and E. R. Unanue. 1979. Cellular immunity and delayed hypersensitivity. Chapter 6 in^ Textbook of Immunology. Williams and Wilkins, Baltimore MD.

6. Marshall, W. H., F. T. Valentine, and H. S. Lawrence. 1969. Cellular immunity in vitro. Clonal proliferation of antigen- stimulated lymphocytes! J. Expt. Med. 130:327-343.

7. Donahoe, J. P., J. Giambrone, 0. J. Fletcher, and S. H. Kleven. 1975. In vivo function tests of the effect of tilorane and niridazole on cell-mediated immunity in chickens. Am. J. Vet. Res. 38:2013-2017.

8. Walser, M. M. 1978. Evaluation of niridazole as a suppressant of cellular immunity in chickens. Am. J. Vet. Res. 1858-1859.

9. Mahmoud, A. A. F., and K. S. Warren. 1974. Anti-inflammatory effects of tartar emetic and niridazole: Suppression of schistosome egg granuloma. J. Immunol. 112:222-228.

10. Mahmoud, A. A. F., M. A. Mandel, K. S. Warren, and L. T. Webster. 1975. Niridazole II. A potent long acting suppressant of cellular hypersensitivity. J. Immunol. 114:279-283. 75

n. Mattironi, V. D., V. H. Rudolfsky, R. V. Swift, and L. L. Esposito. 1978. Niridazole inhibition of local graft-versus-host reaction in rat muscle. Clin. Immunol. Immunopathol. 10:158-167.

12. Pelley, R. P., R. J. Pelley, A. B. Stavitsky, A. A. F. Mahmoud, and K. S. Warren. 1975. Niridazole, a potent long-acting suppressant of cellular hypersensitivity. III. Minimal suppression of antibody responses. J. Immunol. 115:1477-1482.

13. Paterson, P. Y., J. M. Harvey, and L. T. Webster, Jr. 1977. Niridazole suppression of experimental allergic encephalo- n\yelitis in Lewis rats. J. Immunol. 118:2151-2154.

14. Faigle, J. W., and H. Keberle. 1969. of niridazole in various species, including man. Ann. N.Y. Acad. Sci. 160:544-557.

15. Nathanson, R. M., M. S. Hofstad, and E. L. Jeska. 1980. Pasteurella multocida: Antibody-mediated resistance to virulent challenge exposure in vaccinated turkeys. Am. J. Vet. Res. 41:1285-1287.

16. Maheswaran, S. K., and E. S. Thies. 1979. Influence of encapsulation on phagocytosis of Pasteurella multocida by bovine neutrophils. Infect. Immun. 26:76-81. 76

PART V: TRANSFORMATION OF CHICKEN LYMPHOCYTES: OPTIMAL CULTURE CONDITIONS

This paper will be submitted to

Veterinary Immunology and Immunopathology for publication 77

SUMMARY

The culture conditions for obtaining the maximum transformation of chicken lymphocytes are different from those of mammalian lymphocytes.

Three differences were demonstrated: (A) the optimal cell concentration is much higher, (B) the optimal concentration of Concanavalin A is much higher, (C) ^H-thymidine should be added earlier.

These results apply specifically to cells grown in microti ter tissue cultures. 78

The mitogen Concanavalin A is used extensively to study the activities of lymphocytes. Concanavalin A (Con A) induces T lymphocytes to undergo blast transformation (1). Mitogen stimulated T lymphocytes produce various lymphokines and factors that inhibit cell growth and are immunosuppressive (2). Many investigators have used mitogens to develop models of immunologic phenomena. Previous studies that have dealt with standardization of avian lymphocyte transformation assays have yielded variable results (3,4,5,6).

The purpose of this report is to describe the culture conditions for obtaining the maximum transformation of chicken lymphocytes in microtiter tissue culture plates. These conditions are different from those of mammalian cultures. Three characteristic differences are described in this report: (a) the optimal cell number, (B) the optimal concentration of Con A, (C) the optimal time for addition of H-thymidine

(^H-TdR). 79

MATERIALS AND METHODS

Isolation of leukocytes

Normal chickens aged 10 to 15 weeks were used. Blood was drawn

by venepuncture and was collected into sodium citrate anticoagulant.

One-fourth ml of 6% dextran was added to tubes containing 4 ml of blood. The tubes were centrifuged for 8 minutes at 80 x G and the buffy coat was removed by a Pasteur pipette. The buffy coat leukocytes were washed twice with RPMI-1640 medium by centrifugation.^ Viability was determined by the trypan blue dye exclusion test. Usually at least 95% of the cells were viable. Samples containing more than 5% erythrocytes were discarded.

Cultures

Leukocytes were cultured in Linbro 0.25 ml round bottom microti ter p tissue culture plates. RPMI-1640 tissue culture medium was supple­ mented with a 2 mMolar L-glutamine plus 100 units and 100 yg streptomycin per ml. Fetal bovine serum (10%) was added to the media in all the experiments with the exception of the experiment that was designed to evaluate serum requirements. The same lot of serum was used throughout the study.

^Grand Island Biological Company, Grand Island, NY.

^Linbro Scientific Inc., Hamden, CT. 80

Determination of serum requirement

Two million leukocytes were cultured in media containing 4 mg

Con A and 10% inactivated fetal bovine serum in each 0.25 ml microti ter

culture.^ Controls consisted of cultures lacking either Con A or serum.

All of the cultures were done in triplicate. The cultures were incubated

at 41 C for 56 hours with 5% COg. Lymphocyte transformation was measured

by adding 1 AjCi of ^H-methyl-thymidine (^H-TdR, specific activity 6.7 O Ci/mM) 18 hours prior to termination. Cells were harvested with a

Skatron cell harvester onto glass fiber filters and the radioactivity

was counted in a beta scintillation counter.

Determination of the optimal cell number

Leukocytes were isolated from peripheral blood as described above.

Cell concentrations varied from 5 x 10^ to 2.5 x 10® cells per culture in

increments of 5 x 10^ cells. Each 0.25 ml well contained 4 mg Con A

in RPMI-1640 media. All of the cultures were done in triplicate. The

cultures were incubated at 41 C for 56 hours with 5% COg. Cells were O labeled with H-TdR as described above.

Determination of the optimal mitogen concentration

Two million leukocytes were cultured in each 0.25 ml culture well.

The concentration of Con A was varied in increments of 2 yg from 0 yg

^Con A, Sigma Chemical Co., St. Louis, MO.

2 New England Nuclear, Boston, MA. 81

to 8 pg per culture. Cultures were incubated, labeled with ^H-TdR and

harvested as described above.

Determination of labeling kinetics

Leukocytes were added to tissue culture wells as described

above. Each culture contained 4 yg Con A. All of the cultures were

done in triplicate. Serum and supplements were added as described

above. O H-TdR incorporation was measured over four time intervals.

Four microti ter plates were used for each experiment. The cultures

were incubated at 41 C with 5% COg and labeled with ^H-TdR at different

time intervals. The first plate was labeled for the initial 18

hours of incubation and was then terminated by freezing. The second

plate was labeled at the 18th hour of incubation and terminated at

the 36th. The third plate was labeled at the 36th hour of incubation

through the 54th hour and was then frozen. The final plate was labeled

at the 54th hour and was terminated at the 72nd. Radioactivity was measured as described above. 82

RESULTS

Serum requirements

Serum was required for the proliferation of lymphocytes in Con A stimulated cultures. Cultures that lacked serum incorporated o approximately the same amount of H-TDR as cultures that lacked Con A

(Fig. 1). The findings do not agree with those of Maheswaran et al. who obtained lymphocyte proliferation in the absence of serum (6).

Optimal cell number O The greatest incorporation of H-TdR occurred when the cell number was adjusted to 2 x 10^ cells/culture well. When the cell number was reduced below this optimum, incorporation diminished significantly

(Fig. 2).

Optimal mitogen concentration 3 The greatest incorporation of H-TdR occurred when the concentration of Con A was adjusted to 4 yg/culture well. Concentrations deviating from this optimum resulted in diminished incorporation (Fig. 3).

Labeling kinetics

Cultures that were labeled during the initial 18 hours of incubation o did not incorporate much H-TdR (Fig. 4). When cultures were labeled at the 18th through the 36th hour of incubation the most incorporation occurred. Incorporation diminished slightly when cultures were labeled after the 36th hour. 83

Appearance of cultures

All experiments were monitored by observation with an inverted tissue culture microscope. In all cases the development of focal O areas of proliferation correlated with the incorporation of H-TdR. 84

DISCUSSION

The results demonstrate that the culture conditions for obtaining

the maximum transformation of chicken lymphocytes are different from those of mammals. Three fundamental differences were demonstrated:

(A) the optimal cell concentration is much higher, (B) the optimal concentration of Con A is higher, (C) ^H-TdR should be added earlier.

In humans, mice, cows, rats, and pigs, the cell number is usually adjusted to within 2 x 10^ to 4 x 10^ cells/microtiter culture

(7,8,9,10). For chickens, the optimum is 2 x 10^ cells/microtiter culture. When leukocytes are isolated from chicken spleens by ficoll gradient centrifugation, a similar number of cells are required to produce optimum transformation (unpublished observations). The results obtained in this study agree with the results of Meyers et al. who showed that 2 x 10® cells/microtiter culture was optimum for obtaining transformation of chicken lymphocytes stimulated by phytohemagglutinin

(11).

In this system the optimal concentration of Con A was found to be

4 yg/microtiter culture (20 yg/ml). When human lymphocytes are cultured, Con A is used at a concentration of 1 yg/microtiter culture

(5 yg/ml), or less (7). It is not apparent why these differences exist.

Chicken lymphocytes proliferated very rapidly. The maximum incorporation of H-TdR occurred between 18 and 36 hours of incubation.

A study of chicken cells in macroculture yielded similar results (4). 85

Most lymphocyte transformation assay protocols for mammals specify that

the cells should be labeled only after a minimum of 50 hours of incubation have elapsed. Clearly, this would not be the optimal time to label chicken cultures. The unusually rapid proliferation was probably not caused by the 41 C incubation temperature. Cultures incubated at 37 C showed similar proliferation rates.

These results demonstrate that chicken leukocyte cultures respond differently to Con A than cultures of mammalian origin. These observations are important because when chicken cells are used, the assay should be modified accordingly.

These findings also apply to chicken leukocytes cultured with antigen. The maximum transformation produced by stimulation with the purified protein derivative of Mycobacterium tuberculosis was obtained with a 2 X 10^ cells/microtiter culture (unpublished observations). o The optimum incorporation of H-TdR occurred between 18 and 36 hours of incubation. Figure 1. Serum requirement

Typical response pattern of Con A stimulated chicken lymphocytes cultured in the presence or absence of 10% fetal bovine serum. 87

100 2 80 S 60 0£ 2 40 a: O 30 o 5 20

CM P 10 8 0. 6 o 4 3

media media media media + + Con A serum Con A Figure 2. Optimal cell number

Typical response pattern of varying numbers of Con A stimulated buffy coat leukocytes.

Typical pattern of varying numbers of unstimulated buffy coat leukocytes. 89

2000

1000 800 600 400 i 300

200 O H g 100 O 80- OC 60 p W 40 30 — OiI O 20-

O. 10" o 8- 6- " 1 î 4- 3- 2-

"T" T- T" T- 0.5 1.0 1.5 2.0 2.5 CELLS/CULTURE x 10 Figure 3. Optimal concentration of Con A

Typical response pattern of buffy coat leukocytes stimulated by different concentrations of Con A.

Individual responses of leukocytes from 4 chickens are shown. 91

800- 600-

400 300

200-

100 80 60 40- 30-

20-

10 8 6 4 3

0 2 4 8 jig CON A/CULTURE Figure 4. Labeling kinetics

Typical response of buffy coat leukocytes labeled with ^H-TdR at different time intervals. 93

200-

0 18 18 -O 36 36 4» 54 54 4* 72

HOURS 94

REFERENCES CITED

1. Greaves, M., G. Janassy, and M. Doenhoff. 1974. Selective triggering of human T and B lymphocytes in vitro by polyclonal mitogens. J. Exp. Med. 140:1-18.

2. Rich, R. R., and C. W. Pierce. 1974. Biological expressions of lymphocyte activation. III. Suppression of plaque forming cell responses in vitro by supernatant fluids from concanavalin A-activated spleen cell cultures. J. Immunol. 112:1360-1368.

3. Lee, L. F. 1974. In vitro assay of mitogen stimulation of avian peripheral lymphocytes. Avian Dis. 18:602-604.

4. Aim, G. V. 1970. In vitro studies of chicken lymphoid cells. Acta Path. Microb. Scand. Section B 78:632-640.

5. Tessler, J., and L. A. Page. 1978. Optimal macroculture method for studying mitogenic stimulation of turkey lymphocytes. Can. J. Comp. Med. 42:249-252.

6. Maheswaran, S. K., and E. S. Thies. 1975. Development of a micro- culture system for stimulation of chicken peripheral blood lymphocytes with concanavalin A. Am. J. Vet. Res. 36: 1053-1055.

7. Oppenheim, J. J., and B. Schecter. 1976. Lymphocyte transformation. Chapter 9 N. Rose and H. Freidman eds. Manual of Clinical Microbiology, Washington, D. C.

8. Brownlie, J., and E. J. Scott. 1979. The response of bovine lymphocytes from lymph and blood to phytohaemagglutinin. Vet. Immunol, and Immunopathol. 1:5-13

9. Mitchell, R. N., and W. E. Bowers. 1980. Cell surface glycoproteins of rat lymphocytes. II. Protease-sensitive glycoproteins associated with mitogenic stimulation by periodate or neuraminidase plus galactose oxidase. J. Immunol. 124: 2632-2640.

10. Kaneene, 0. M., R. K. Anderson, D. W. Johnson, R. D. Angus, C. C. Muscoplat, D. E. Pietz, L. C. Vanderwagon, and E. E. Sloane. 1978. Cell-mediated immune responses in swine from a herd infected with Brucella suis. Am. J. Vet. Res. 39: 1607-1611. 95

n. Meyers, P., G. D. Ritts, and D. R. Johnson. 1976. Phyto- hemagglutinin-induced leukocyte blastogenesis in normal and avian leukosis virus infected chickens. Cell Immunol. 27:140-146. 96

PART VI: STUDIES OF CELL-MEDIATED IMMUNITY TO PASTEURELLA MULTOCIDA: A COMPARISON TO MYCOBACTERIUM TUBERCULOSIS The immune response induced by Pasteurella multocida vaccination

was compared to the response induced by Mycobacterium tuberculosis

vaccination. Two in vitro correlates of cell-mediated immunity,

the lymphocyte transformation test, and the leukocyte migration

inhibition test, were used. M. tuberculosis was shown to induce an intense cell-mediated response whereas IP. multocida evoked at most only a very minimal cell-mediated response. 98

INTRODUCTION

The itimune response to Pasteurella multocida vaccination is not well-understood. Vaccination can induce significant levels of circu­ lating antibody, yet it is not clear to what extent cell-mediated immunity is induced. Some studies have indicated that turkeys vaccinated with P^. multocida bacterins may develop cell-mediated immunity (1,2,3). In contrast, the immunity to Mycobacterium tuberculosis has been much more thoroughly studied (4). When either live or dead

M. tuberculosis are inoculated into an animal, a very strong cellular response occurs (5).

The purpose of this study was to determine whether K multocida vaccinated chickens developed: (A) cell-mediated immune responses to iP. multocida antigens and (B) a cellular response similar in magnitude to Mycobacterium tuberculosis vaccinated chickens.

Two methods were used to assay for cell-mediated immunity:

(A) the lymphocyte transformation test and (B) the leukocyte migration inhibition test. Both assays can be used to measure in vitro correlates of cell-mediated immunity (6,7). 99

MATERIALS AND METHODS

Preparation of f « "lultocida antigens

Two JP. miiltocida test antigens were prepared. Antigen A consisted of a sonicate soluble fraction of P^. multocida. Strain 1059 was grown on tryptose agar in Roux bottles for 18 hours at 37 C. The bacteria were removed from the agar surfaces with sterile saline. Cells were washed and centrifuged twice with sterile saline. The suspension was placed in a sonic oscillator chamber and sonicated with a 250 watt

10 kc generator for 7 minutes. A sample of the suspension was plated on tryptose agar to check for contaminants. The sonicated multocida were then centrifuged at 21,000 x G for 15 minutes in a Sorvall RC2-B refrigerated centrifuge.^ A dense pellet and clear supernatant fluid remained. The supernatant fluid was drawn off by a Pasteur pipette and was lyophilized.

Antigen B consisted of a heat stable capsular extract of £. multocida. Strain P-1059 was harvested from tryptose agar with sterile saline. A sample of the suspension was plated on tryptose agar to check for contaminants. The suspension was heated at 56 C for 30 minutes. The heat treated £. multocida were centrifuged at 21,000 x G and the supernatant was used as antigen. The supernatant was frozen prior to storage. Both antigens reacted against antiserum to £. multocida in gel diffusion tests.

^Ivan Sorvall Inc., Newtown, CT. 100

Vaccination of chickens

Killed 2' multocida strain P-1059 was emulsified in Freund's q complete adjuvant. The bacterin contained 10 cells/ml. Chickens,

aged 9 to 12 weeks were vaccinated with 1 ml of the bacterin three

times at three-week intervals.

An additional group of chickens was vaccinated with M. tuberculosis.

Killed M. tuberculosis strain R37a was emulsified in Freund's complete adjuvant. Each chicken received a single subcutaneous injection of 10 mg M. tuberculosis in one ml of emulsified adjuvant.

Blood and spleen samples were taken from birds four to eight weeks after vaccination.

Preparation of peripheral blood leukocytes for the transformation test

Blood was drawn by venepuncture and collected into sodium citrate anticoagulant. One-fourth ml of 6% dextran was added to tubes containing

4 ml of blood. The tubes were centrifuged at low speed for 8 minutes at 80 X G and the buffy coat was removed by aspiration with a Pasteur pipette. The buffy coat leukocytes were washed twice with RPMI-1640 media by centrifugation.^ Viability was determined by the trypan blue dye exclusion test. Usually, at least 95% of the cells were viable.

Samples containing more than 5% erythrocytes were discarded. Leukocyte concentrations were adjusted so that 2 x 10® cells were incubated in

^Grand Island Biological Co., Grand Island, NY. 101

each 0.25 ml round bottom well of a microti ter tissue culture plate.

Heat inactivated fetal bovine sera (10%) was added to the cultures; the same lot of serum was used in each experiment. Media was supple­ mented with 2 mMolar L-glutamine plus 100 units penicillin and 100

Vig of streptomycin per ml.

Isolation of leukocytes from spleens

Spleens were removed from IP- multocida vaccinated chickens. The capsular material surrounding the spleen was discarded. Each spleen was minced in RPMI-1640 medium. The minced suspension was centrifuged at low speed to remove large clumps of tissue. The spleen suspension was layered over ficoll-pague and centrifuged to isolate leukocytes.

The leukocytes were drawn off from the interface with a Pasteur pipette.

The cells were washed and cultured in microti ter plates as described above.

Lymphocyte transformation test

£. multocida antigens were added to leukocyte cultures prior to incubation. Sonicate soluble fraction was used at a concentration of

100 yg/culture, as described by Maheswaran et al. (1). Capsular extract was used at a concentration of 6 yg/culture. The extract was found to produce optimal stimulation at this concentration. Capsular protein was estimated by the method of Lowry et al. (8). 102

Mycobacterial antigen (purified protein derivative of M. tuberculosis, without preservative) was used at a concentration of either 50 yg/culture or 25 yg/cultureJ Cell cultures were labeled with ^H-methyl thymidine (^H-TdR, sp. act. 6.7 Ci/mmole), at either 2 zero hours, 18 hours, or 36 hours of incubation. Cultures were terminated by freezing 18 hours after labeling. Cells were harvested with a Skatron cell harvester onto glass fiber filters and the radio­ activity was counted in a beta scintillation counter. The stimulation index was calculated by the formula:

stimulation index (SI) = z::::i::: i::s%3iagen

Leukocyte migration inhibition test

Leukocytes were isolated from peripheral blood as described above.

Capillary tubes (75 mm without heparin) were filled with the leukocyte O suspension. One end of each capillary tube was closed with capillary tube sealant. Tubes were centrifuged for 5 minutes or until the leukocytes were tightly packed at the bottom of each capillary tube.

The capillary tubes were cut just below the cell boundary and then

^Parke-Davis Tuberculin PPD, Detroit, MI. p New England Nuclear, Boston, MA.

^Fisher Scientific, Pittsburgh, PA. 103

anchored to the bottom of tissue culture plates with sterile stopcock grease. Two migration inhibition antigens were prepared: (A) sonicate soluble fraction was added to RPMI-1640 medium (500 yg/ml) containing

10% fetal bovine sera; (B) heat-killed P.. multoeida (10^ cells/ml) were added to RPMI-1640 containing 10% fetal bovine sera. One ml of media supplemented with antigen was added to tissue culture wells that contained capillary tubes. Three sets of control cultures were made:

(A) sensitized leukocytes without antigen, (B) unsensitized leukocytes with antigen, and (C) unsensitized leukocytes without antigen. The cultures were incubated for 4 hours at 37 C. Cell migration was magnified lOX with a microscope slide projector. Migration distance was measured with a ruler. The percentage of migration-inhibition was calculated with the following formula.

Percentage migration inhibition = ,.0 - ::: :i:L:r:Sgen

X 100 104

Optimal culture conditions for obtaining maximum proliferative response

to P. multocida and M. tuberculosis antigens

The number of cells in culture was a critical factor in trans­

formation assays. It was advantageous to use 2 x 10® rather than 10®

leukocytes per culture. Cultures that contained 2 x 10® cells

incorporated the greatest amount of H-TdR (Fig. 1). These results

agree with the observations by Meyers et al. (9).

Cell cultures that were labeled between 18 and 39 hours of incubation 3 incorporated more H-TdR than cultures labeled at other time intervals

(Figs. 2,3). Cellular proliferation was greatest during this period.

Lymphocyte transformation: response to P.. multocida antigens

Leukocytes from vaccinated chickens had slightly higher stimulation indices than did leukocytes from nonvaccinated chickens (Fig. 4).

Peripheral blood leukocytes and spleen leukocytes responded similarly.

Capsular material and sonicate soluble fraction induced similar responses (Fig. 5).

Lymphocyte transformation: response to purified protein derivative of

M. tuberculosis

Peripheral blood leukocytes from M. tuberculosis vaccinated chickens had much higher stimulation indices than did nonvaccinated chickens 105

(Fig. 6). A concentration of 50 yg purified protein derivative (PPD) per culture produced greater responses than 25 yg. Leukocytes from M. tuberculosis vaccinated chickens had much higher stimulation indices than did leukocytes obtained from multocida vaccinated chickens

(Figs. 4,5,6).

Migration inhibition test: response to £. jnultocida antigens

Significant levels of leukocyte migration inhibition were not demonstrated. Generally, over 30% inhibition must exist before the presence of migration inhibition factor can be assumed (10). When cells from vaccinated chickens were cultured with sonicate soluble fraction, inhibition was 0.14 + .08. Cells from nonvaccinated chickens had an average inhibition of 0.08 + .10 (Fig. 7). It was not clear why leukocytes from nonvaccinated chickens showed some migration inhibition.

In a similar experiment in which leukocytes from vaccinated turkeys were incubated with heat killed £. multocida, significant levels of leukocyte migration inhibition were not demonstrated (Fig. 8). 106

DISCUSSION

Antigen stimulated microti ter cultures that contained 2 x 10®

leukocytes incorporated more H-TdR than did microti ter cultures containing

10® leukocytes (Fig. 1). Mammalian systems require between 2 x 10®

and 5 x 10® cells per microti ter culture. The results agree with the

findings of Maheswaran and Thies (11), and Meyers et al. (9).

Antigen stimulated microtiter cultures that were labeled between

the 18th and 39th hour of incubation incorporated ^H-TdR (Figs. 2,3).

These data agree with the finding of Aim, who observed that mitogen 3 stimulated lymphocytes incorporated the greatest amount of H-TdR

during this period (12).

Leukocytes from 2- multocida vaccinated chickens had slightly

higher stimulation indices than did leukocytes from nonvaccinated

chickens (Figs. 4,5). In contrast, leukocytes from M. tuberculosis

vaccinated chickens had much higher stimulation indices than did non-

vaccinated chickens (Fig. 6). Several factors might account for this

difference: (A) M. tuberculosis may act as a more potent activator of

cell-mediated immunity as compared to P^. multocida, (B) the P^. multocida antigens used in the lymphocyte transformation and leukocyte migration inhibition tests were highly heterogeneous, whereas the M. tuberculosis antigen (PPD) was highly purified. Inhibitory substances may have been present in the P^. multocida sonicate soluble fraction and the capsular material, (C) the £. multocida vaccination protocol might have favored the 107

production of suppressor T cells.

Some consideration should be directed toward a greater understanding of the multocida fractions used in this study. The sonicate soluble fraction and capsular material have not been well-characterized.

Previous studies have demonstrated the extensive heterogeneity of antigens in various £. multocida extracts (13). It is possible that sonicate soluble fraction and capsular material possess similar antigens and may actually be similar or contain identical substances.

A number of other procedures have been used to obtain P^. multocida antigens. These procedures include phenol, trichloroacetic acid, and potassium thiocyanate extractions (14,15,16). In a study of various extracts, Maheswaran found that a sonicate soluble fraction of multocida induced the greatest lymphocyte transformation response (1).

Before in vitro correlates of cell-mediated imunity can be accurately evaluated, the various P^. multocida antigens will have to be more adequately and thoroughly characterized. Figure 1. Optimal cell number

Response pattern of various numbers of leukocytes to stimulation with £» multocida sonicate soluble fraction.

nv=nonvaccinated chicken, Vi=2. multocida vaccinated chicken #1. V2=Po multocida vaccinated chicken #2.

Results expressed as stimulation index + S^D. 109

4-

3 S 2

nv v-| V2 nv v-j V2

2 X10® 1 X10®

CELLS/CULTURE Figure 2. Kinetics of proliferative response to multocida sonicate soluble fraction

nv=nonvaccinated chicken. Vi=_P. multocida vaccinated chicken #1. V2=£. multocida vaccinated chicken #2.

Results expressed as stimulation index S.C. STIMULATION INDEX M CO ^ ai o> Figure 3. Kinetics of proliferative response to purified protein derivative of M. tuberculosis

nv=nonvaccinated chicken. vi=M. tuberculosis vaccinated chicken #1. V2=M. tuberculosis vaccinated chicken #2.

Results expressed as stimulation index + S.D. STIMULATION INDEX Figure 4. Proliferative response to sonicate soluble fraction of P^. multocida: Comparison of the response of leukocytes isolated from the blood and spleen

vacc=£. multocida vaccinated. not vacc=not vaccinated.

There were four chickens tested (n=4) in each group.

Results expressed as stimulation index + S.D. 115

I 5-

vacc

BLOOD SPLEEN Figure 5. Proliferative response to capsular material of 2" multocida

vacc=£. multocida vaccinated. not vacc=not vaccinated.

There were three chickens tested (n=3) in each group.

Results expressed as stimulation index + S.D. STIMULATION INDEX

CO U1 Figure 6. Proliferative response to purified protein derivative of M. tuberculosis

vacc=M. tuberculosis vaccinated. not vacc=not vaccinated.

There were four chickens tested (n=4) in each group.

Results expressed as stimulation index + S.D. 119

20-

50 ug 25 ug 50 ug NOT VACC VACC Figure 7. Leukocyte migration inhibition test: sonicate soluble fraction was used as antigen

vacc=£. multocida vaccinated, not vacc-not vaccinated.

There were six chickens tested (n=6) in each group.

Results expressed as percent migration inhibition + S.D. PERCENT MIGRATION INHIBITION

I ro 00 o» o o o o Figure 8. Leukocyte migration inhibition test (turkey)

Heat killed bacteria (10^/ml) were used as antigen.

vacc=£. multocida vaccinated, not vacc=not vaccinated.

There were six turkeys tested (n=6) in each group.

Results expressed as percent migration inhibition + S.D. PERCENT MIGRATION INHIBITION 124

LITERATURE CITED

1. Maheswaran, S. K., E. S. Thies, and S. K. Dua. 1976. Studies on Pasteurella multocida. III. In vitro assay for cell- mediated immunity. Avian Dis. 20:332-341.

2. Yamaguchi, Y., and T. Baba. 1974. Demonstration in tissue culture of cellular immunity to fowl cholera. Pages 14-18 in T. Hasegawa ed. Proceedings of the First Intersectional Congress of the lAMS. Vol. 4. Science Council of Japan, Tokyo.

3. Baba, T., T. Ando, and M. Nukina. 1978. Effect of bursectomy and thymectomy on Pasteurella multocida infection in chickens. J. Med. Microbiol. 11:281-288.

4. Mackaness, G. B. 1968. The immunology of antituberculosis immunity. Amer. Rev. Resp. Dis. 97:337-344.

5. Bennacerraf, B., and E. R. Unanue. 1979. Cellular immunity in delayed hypersensitivity. Chapter 6 in Textbook of Iimunology. Williams and Wilkins, Baltimore, MD.

6. Oppenbeim, J. J., and B. Schecter. 1976. Lymphocyte transformation. Chapter 9 in N. Rose and H. Freidman eds. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D. C.

7. Vlaovic, M. S., G. M. Buening, and R. W. Loan. 1975. Capillary tube leukocyte migration inhibition as a correlate of cell- mediated immunity in the chicken. Cell. Immunol. 17:335-341.

8. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Fol in phenol reagent. J. Biol. Chem. 193:265-275.

9. Meyers, P., G. D. Ritts, and D. R. Johnson. 1976. Phytohemagglutina­ tion-induced leukocyte blastogenesis in normal and avian leukosis virus-infected chickens. Cell. Immunol. 27:140-146.

10. Watanuki, M., and S. Haga. 1977. The statistical distribution of macrophage migration distance and its application to MI F test. J. Immunol. Methods 15:331-341. 125

11. Maheswaran, S. K., and E. S. Thies. 1975. Development of a microtiter system for stimulation of chicken peripheral blood lymphocytes with concanavalin A. Am. J. Vet. Res. 36:1053-1055.

12. Aim, G. V. 1970. In vitro studies of chicken lymphoid cells. Acta Path. Microb. Scand. Section B. 78:632-640.

13. Bhasin, J. L., and L. Lapointe-Shau. 1980. Antigenic analysis of Pasteurella multocida ( 1) by crossed immuno- electrophoresis: characterization of cytoplasmic and cell envelope associated antigens. Can. J. Microb. 26:676-689.

14. Rebers, P. A., and K. L. Heddleston. 1974. Immunologic comparison of Westphal-type 1ipopolysaccharides and free endotoxins from an encapsulated and nonencapsulated avian strain of Pasteurella multocida. Am. J. Vet. Res. 35:555-560.

15. Pirosky, I. 1938. C. R. Soc. Biol. 127:98-100.

16. Moffat, G. G., T. K. S. Mukkur. 1977. Fowl cholera: Immunization of chickens with potassium thiocyanate (KSCN) extract of Pasteurella multocida serotype 3. Avian Dis. 21:543-548. 126-127

SUMMARY AND CONCLUSIONS

This dissertation consists of a series of five papers that deal with the immune response produced by Pasteurella multocida vaccination.

A sixth paper (Section V) describes a study designed to find the optimal culture conditions for growing chicken lymphocytes. The study was a necessary prerequisite to the in vitro studies reported in Section VI.

The objective of this research was to determine the extent that humoral and cell-mediated responses are elicited by vaccination. The experiments were designed to evaluate: (A) the protective role of antibody response,

(B) whether in vitro and in vivo correlates of cell-mediated immunity can be demonstrated, and (C) whether cell-mediated responses to P^. multocida are of similar magnitude as responses to M. tuberculosis vaccination.

Summary of results

A single vaccination with a formalin inactivated P^. multocida bacterin produced significant antibody titers in chickens and turkeys.

Revaccination produced four-fold increases in titers. The bacterin was capable of inducing a significant level of immunity as demonstrated by resistance to challenge (Section I). Passive protection studies indicated that antiserum was capable of inducing temporary protection.

Antibody titers appeared to correlate with protection (Section II).

Newly hatched chickens were treated with cyclophosphamide for a period of four days and later vaccinated (Section III). The treatment was 128

designed to suppress antibody production but leave cell-mediated immunity

intact. Chickens treated in this manner were very susceptible to challenge infection. Vaccinated bursectomized turkeys were also susceptible to

challenge infection (Section II). If cell-mediated immunity were a major component of protection, it seems likely that groups of vaccinated cyclophosphamide treated birds, and groups of vaccinated bursectomized birds, would not be expected to have appreciable mortality following challenge infection. However, mortality was very high, which suggests

that cell-mediated responses may not be as important as antibody response.

The results indicated that there is a correlation between the presence of agglutinating antibody and resistance to challenge infection.

In vivo tests did not clearly indicate the presence of delayed hypersensitivity to multocida (Section IV). Chickens that were injected with decapsulated £. multocida in Freund's incomplete adjuvant did not develop granulomatous lesions indicative of delayed hyper­ sensitivity. In contrast, chickens that were injected with killed M. tuberculosis in Freund's adjuvant developed intense granulomatous lesions that lasted in excess of ten weeks (Appendix).

JP. multocida vaccinated chickens did not develop skin test reactions

(Section IV). In contrast, a small group of turkeys tested under similar conditions developed skin test reactions that may have been representative of delayed hypersensitivity. Vaccinated chickens that were fed niridazole, a T cell immunosuppressant, did not show increased challenge mortality.

This finding suggests that cell-mediated response is not a major factor in protection (Section IV). However, it was not confirmed that the 129

niridazole produced immunosuppression, so this particular experiment

must be interpreted with caution.

Lymphocytes from vaccinated chickens underwent blast transformation

after exposure to P^. multocida antigens (Section VI). Stimulation

indices rarely exceeded five. When lymphocytes from M. tuberculosis

vaccinated chickens were incubated in the presence of PPD of M.

tuberculosis, stimulation indices in excess of 20 were recorded. M.

tuberculosis appears to act as a much more potent activator of cell-

mediated responses than does P^. multocida. The leukocyte migration

inhibition test, an established in vitro correlate of cell-mediated

immunity, failed to indicate that £. multocida vaccination induced

delayed type hypersensitivity (Section VI).

The lymphocyte transformation response was minimal. This response,

in itself, does not necessarily indicate that cell-mediated immunity is

the primary means of protection. In addition, it is unclear which population of lymphocytes (T or B) were actually proliferating in response

to the £. multocida antigens. However, a role for T lymphocytes cannot be excluded. T lymphocytes may act as helper cells in antibody formation.

Significance of this work

The results of this research are significant for two reasons: (A) they may facilitate the further development of more effective vaccines, and (B) they further our understanding of the immune response to £. multocida. 130

The results demonstrate that in poultry the protective immune response that is induced by P^. multocida adjuvant bacterins is primarily antibody mediated. In contrast, cell-mediated responses do not appear to be as important. It is apparent that P^. multocida vaccines should be produced and administered so as to induce a prolonged antibody response. The selection of adjuvant and immunizing schedules should be adjusted so that continuous antigen stimulation is provided.

The results also demonstrate that the immunity induced by multocida vaccination is similar to that induced by vaccination with other

Gram negative non-intracellular bacteria because the immunity is primarily antibody mediated. Previous studies, particularly those by Baba, and others by Dua and Maheswaran, indicated that P^. multocida vaccination might be an exception and might induce a strong cell-mediated response

(30,9). This discrepancy appears to have been resolved. The studies reported here suggest that while cell-mediated responses may exist, they probably play only a minor role in protective immunity. Cell-mediated immunity is usually demonstrated against intracellular organisms, in­ cluding certain bacteria, parasites, fungi, and most viruses. Clearly, this is not the case with £. multocida. There is no evidence suggesting that multocida is an obligate intracellular pathogen. 131

Inactivated vaccine vs live vaccine

In all of the experiments in this study, a formalin inactivated

multocida bacterin was used for vaccination. It is essential to

recognize that an inactivated bacterin may induce the formation of

immune responses substantially different from a live vaccine. However, previous studies have shown that IP. multocida adjuvanted bacterins induce the most substantial and lasting immunity (9). Maheswaran has reported that turkeys vaccinated with a P. multocida adjuvanted bacterin produced a stronger cell-mediated immune response than turkeys vaccinated with a live vaccine administered in drinking water (9).

The use of inactivated vaccines does not necessarily preclude the development of cell-mediated immune responses. It has been known for many years that Freund's complete adjuvant (incomplete adjuvant plus killed Mycobacteria) induces a state of cellular immunity. Similarly, dinitrophenyl-bovine gamma globulin and a series of a, N-dinitrophenyl-

L-lysines have been shown to activate cell-mediated immune responses

(44,57).

Activated macrophages play an important role in cell-mediated immunity. Materials that induce macrophage activation are often capable of producing cell-mediated immunity. It has not been demonstrated that P^. multocida can induce macrophage activation. Some studies have shown that phagocytized Pasteurellae produce the opposite effect: £. hemolytica are cytocidal for neutrophils and macrophages (61,62). 132

The following conclusions can be made from this study:

(A) Vaccination with a formalin-inactivated adjuvant bacterin

provides a significant level of protective immunity.

(B) Vaccination produces detectable levels of antibody in

chickens and turkeys.

(C) The antibody produced by vaccination is protective.

(D) Various indicators of cell-mediated immunity suggest that

cell-mediated immunity is probably only a minor component

of protection to £. multocida challenge.

(E) The immunity produced by P^. multocida vaccination is different from the immunity produced by M. tuberculosis

vaccination.

Some areas for further study can be clearly identified:

(A) The immune response to purified homogenous antigens should

be investigated. K multocida possesses a wide spectrum of

antigens. A study should be initiated to determine which

antigens are involved in inducing protection, and how they

induce protection. The capsular and sonicate soluble fraction

used in this study was very heterogeneous and could have

possessed substances which interfered with in vitro and in

vivo tests for cell-mediated immunity.

(B) Live attenuated £. multocida vaccines must be further perfected.

The recent development of the MSG £. multocida vaccine in Israel is an important step (63). The MSG vaccine appears to 133

be safer and offer more protection than the CIemson strain.

The Immunity induced by this vaccine should be studied in

order to determine the importance of humoral and cellular

response in protection.

(C) It is not known why turkeys are more susceptible than chickens

to challenge infection. There may be inherent differences in

the immunologic capabilities of chickens and turkeys.

(D) Cross-protection needs to be more thoroughly understood

(see the second article in this series for discussion). Cross-

protection is observed after a £. multocida bacterin has been

grown in vivo and is not observed after growth in vitro. This

phenomenon is important in the development of vaccines that

provide protective immunity against different serotypes of P^.

multocida.

This project has been interesting and productive. The results should be useful in the further development of riiultocida vaccines. 134

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ACKNOWLEDGMENTS

I must express my sincere appreciation to all those who have helped me throughout my graduate studies at Iowa State University. In particular, I wish to express try gratitude to my Major Professor, Dr.

M. S. Hofstad. He was encouraging, patient, and tolerant under circumstances which were not always easy.

My other graduate committee members. Dr. D. L. Harris, Dr. J. G.

Holt, Dr. E. L. Jeska, Dr. P. A. Rebers, and Dr. R. B. Rimler were always available when I needed assistance.

Special thanks are also due to Dr. H. J. Barnes for technical assistance. The conscientious efforts of Marge Davis, my dissertation typist, are appreciated.

My fellow graduate students at the Veterinary Medical Research

Institute are an unusually outstanding group. We have had many enjoyable conversations dealing with immunology, science, and life in general. I have been enriched by these friendships.

My parents have provided love, endless encouragement, and a real interest in my studies. I carry a deep sadness because shortly before the completion of this dissertation niy father died.

Finally, I want to thank my very special friend Elaine, who always stands by me. 141

APPENDIX Figure 1. Reaction of tnuUocida capsular material material (2) and P^. multocida sonicate soluble fraction (3) against antisera obtained from a P. multocida vaccinated chicken (1)

Figure 2. Buffy coat leukocytes isolated from turkey blood by dextran sedimentation. Wrights stain. Magnification 400 X 143 i S CL' Figure 3. Histologic appearance of granulomatous lesion from chicken inoculated with inactivated M. tuberculosis. Intense mononuclear infiltrate is present. H ànd E stain. Magnification 400 X 145