Veterinary Immunology and Immunopathology, 33 (1992) 155-162 Elsevier Science Publishers B.V., Amsterdam

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Use of rats to compare atrophic rhinitis vaccines for protection against effects of heat-labile protein toxin produced by Pasteurella multocida serogroup D J.R. T h u r s t o n a, R.B. R i m l e r ~, M . R . A c k e r m a n n a a n d N.F. Cheville b aAvian Diseases Research Unit and bBrucellosis Research Unit, US Department of Agriculture, Agricultural Research Service, National Animal Disease Center, PO Box 70, Ames, IA 50010, USA (Accepted 13 September 1991 )

ABSTRACT Thurston, J.R., Rimler, R.B., Ackermann, M.R. and Cheville, N.F., 1992. Use of rats to compare atrophic rhinitis vaccines for protection against effects of heat-labile protein toxin produced by Pasteurella multocida serogroup D. Vet. Immunol. Immunopathol., 33:155-162. Four bacterin-toxoid and three bacterin commercial vaccines against atrophic rhinitis were tested in rats for their capacity to immunize against the lethal and systemic effects of purified heat-labile protein toxin (D-toxin) produced by Pasteurella multocida serogroup D. Only one bacterin-toxoid vaccine stimulated sufficient immunity to prevent the death of all rats challenged with D-toxin. None of the vaccines prevented weight loss, leukocytosis or increases in serum complement titers in rats challenged with D-toxin. Rats provide an inexpensive animal model for testing the capacity of vaccines to generate antitoxic immunity against the lethal and systemic effects of D-toxin.

ABBREVIATIONS EDTA, ethylenediamine tetraacetic acid. INTRODUCTION There atrophic cida a n d ditional

are numerous commercial vaccines for the prevention or control of r h i n i t i s . M o s t c o n t a i n c o m b i n a t i o n s o f t o x i g e n i c Pasteurella rnultoBordetella bronchiseptica. S o m e i n c l u d e t o x o i d a n d , f r e q u e n t l y , a d a n t i g e n s s u c h as ' e r y s i p e l a s ' o r E s c h e r i c h i a coli. T h e a p p e a r a n c e o n

Correspondence to: J.R. Thurston, Avian Diseases Research Unit, US Department of Agriculture, Agricultural Research Service, National Animal Disease Center, PO Box 70, Ames, IA 50010, USA.

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J.R. T H U R S T O N E T A L .

the market of modifications of existing atrophic rhinitis vaccines is evidence that a completely effective vaccine is not presently available to hog producers. Current published research on vaccine development emphasizes the importance of protection against heat-labile protein toxin produced by toxigenic P. multocida. Pedersen and Barford ( 1982 ) found that immunization of pregnant gilts with a laboratory-prepared vaccine containing P. multocida toxin (non-detoxified) provided significant protection of offspring against nasal lesions caused by combined experimental infection with B. bronchiseptica and toxin-producing P. multocida. Kobisch and Pennings ( 1989 ) found that immunization of sows with an experimental vaccine containing formalin-inactivated purified dermonecrotic toxin from a toxigenic strain of P. multocida type D reduced clinical signs of atrophic rhinitis in offspring. The vaccine also prevented lung lesions and turbinate atrophy. Offspring of gilts immunized with toxoid (no bacterin included) prepared from toxigenic P. multocida were better protected against severe deviations of the nasal septum than were pigs born to non-vaccinated sows (Foged et al., 1989). Recently, we vaccinated rats with a toxoid prepared from purified heatlabile toxin (D-toxin) produced by a toxigenic P. multocida serogroup D (Thurston et al., 1990). Vaccinated rats challenged with a lethal dose of Dtoxin remained in good condition and had normal hematology, complement titers and weight gain. All these factors were abnormal in non-vaccinated rats given sublethal injections of D-toxin. We have now compared several commercially available atrophic rhinitis vaccines for immunization against the effects of D-toxin in rats. Immunity was assessed in terms of survival and effects on weight, hematology and serum complement titers. MATERIALS AND METHODS

D-Toxin

Purified heat-labile D-toxin was prepared from a non-capsulated serogroup D, P. multocida (strain P-4533 ) originally isolated from the nasal cavity of a pig. Details of the preparation and purification of D-toxin by column chromatography have been described previously (Cheville and Rimler, 1989). DToxin was stored in small aliquots at - 70 ° C and was thawed only once. Rats

White, male, Holtzman rats (Holtzman Laboratory Animals, Madison, WI ) were individually housed on corn cob bedding, and had unlimited access to feed and water.

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Vaccines and diluents

Vaccines listed and described in Table 1 were either purchased or supplied to us by the manufacturers from existing stocks. All vaccines, except Rhinogen P (Oxford Veterinary Laboratories, Worthington, MN), were licensed by the US Department of Agriculture (USDA). We classified the vaccines acTABLEI Vaccines tested Vaccine

Composition and classification

Manufacturer

Anchor True-Vac-2

Bordetella bronchiseptica, Pasteurella multocida toxigenic type D. BACTERIN-TOXOID

Boehringer-Ingelheim Animal Health, Inc., St. Joseph, MO 64502

Sanofi Rhi-CO-Pig D

BordeteUa bronchiseptica, Escherichia coli serotypes K88, K99, 987P and F41, and Pasteurella multocida type D toxoid. BACTERIN-TOXOID

Sanofi Laboratories, Overland Park, KS 66210

Oxford Rhinogen P

Bordetella bronchiseptica, Pasteurella types A and D toxoid. BACTERINTOXOID

Oxford Veterinary Laboratories, Inc., Worthington, MN 56187

Nobl Toxivac AD

Cell-free and non-toxic antigens of Pasteurella rnultocida and Bordetella bronchiseptica. BACTERIN-TOXOID

Nobl Laboratories, Inc., Sioux Center, IA 51250

Solvay RhinitisBac-3 way + D

Bordetella bronchiseptica, Erysipelothrix rhusiopathiae, Pasteurella multocida serotype A, toxigenic D. BACTERIN

Solvay Animal Health, Inc., Mendota Heights, MN 55120-1139

Pitman-Moore Bordegen P/D

Bordetella bronchiseptica, Pasteurella multocida. BACTERIN

Pitman-Moore, Inc., Washington Crossing, NJ 08560

Ambico AR-P+D

Bordetella bronchiseptica, Pasteurella multocida types A and toxigenic D. BACTERIN

Ambico, Inc., Dallas Center, IA 50063

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cording to the USDA, Animal and Plant Health Inspection Service, Science and Technology definitions for inactivated bacterial products (Standard requirements for bacterins, toxoids, bacterin-toxoids, and bacterial extracts. Federal Register 39:16862 ( 10 May) 1974, as amended in 49:22625 (3 May) 1984 ). Briefly, a bacterin is defined as "an antigenic suspension of organisms, representing a whole culture or a concentrate thereof, with or without the unevaluated growth products; a toxoid is an antigenic filtrate resulting from the growth of bacterial organisms which have had the bacterial cells removed; and a bacterin-toxoid is a whole culture or a concentrate thereof which includes the inactivated toxic growth products which retain antigenic value as measured by suitable tests, or it is a combination product containing a bacterin, a toxoid, or a bacterin-toxoid." In this study, there were four bacterintoxoids and three bacterins. Vaccine manufacturers supplied the diluents used to prepare the vaccines and they were used to sham vaccinatecontrol rats. The composition of vaccines or diluents was not made known to us. No diluent was supplied by Solvay, Pitman-Moore or Ambico.

Experimental plan Vaccination of rats and challenge with D-toxin were performed using the same injection schedule, amounts of vaccines and D-toxin as previously described (Thurston et al., 1990). On Days 0 and 10, rats were given a 0.25 ml s.c. injection of either vaccine or diluent. On Day 24, all rats were given s.c. injections of 0.1 ml of D-toxin in a concentration equivalent to 0.8/tg k g - 1 of average body weight. Each vaccine and diluent was given to ten rats, with groups matched for average weight and distribution of weight. Experiments were terminated on Day 27 when surviving rats were anesthetized with a mixture of air and at least 10% CO2 before blood samples were taken. Rats were then killed by overdose with CO2. All rats were weighed daily Monday-Friday. Data accumulated from the testing of normal rats (n = 42 ) are included in Table 2 for comparative purposes.

Hematology When blood samples were taken from rats surviving on Day 27, smears were made for differential leukocyte counts and blood was placed in tubes containing ethylenediamine tetraacetic acid (EDTA) for the determination of total leukocyte counts (WBC) by electronic counting (Nova Celltrak, Waltham, MA).

Serum complement Serum collected on Day 27 was tested for complement activity using sheep erythrocytes sensitized with rabbit anti-sheep hemolysin. C o m p l e m e n t titers

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TABLE 2 Survival, hematology and serum complement titers on Day 27 of rats injected with either vaccines or diluent on Days 0 and 10, and challenged on Day 24 with D-toxin

Injection

Surviving rats per

Average

Average %

Average %

WBC 2

neutrophils

L+ M3

Average complement loglo titer

group I on Day 27 Anchor-BT Diluent

10 3

26 25

28 36

71 60

1.85 2.08

Sanofi-BT

4 1

33 20

42 33

57 67

1.97 2.05

5 0

41 .

66

2.05

Diluent

1 2

14 34

16 28

83 70

1.66 2.05

Solvay-B4

1

55

48

52

2.05

Pitman-Moore-B4

1

51

35

63

2.15

Ambico-B4

0

.

Normal rats s

-

88 77-95

1.74 1.60-1.90

Diluent Oxford-BT

Diluent Nobl-BT

Range

32 .

.

13 6.5-20.8

.

. 11 11-22

.

.

tTen rats per group. 2Count × 106 m l - ~. 3Combined percentage of lymphocytes and monocytes. 4Diluents were not available from the vaccine suppliers. Sn=42.

were expressed as log~o of the 50% hemolytic endpoint (Garvey et al., 1977 ). An aliquot of pooled normal rat serum was included as a standard in each set of complement titrations. RESULTS

Three of the vaccines classified as containing toxoids showed evidence of protecting rats against the lethal effects of D-toxin (Table 2 ). Immunization with Anchor vaccine was effective in preventing death during the interim between the injection of rats with D-toxin on Day 24 and termination on Day 27. Four rats survived after vaccination with the Sanofi vaccine and five after vaccination with the Oxford vaccine. The other vaccines were no more effec-

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tive in protecting rats against the lethal effects of D-toxin than was sham vaccination with their diluents. The most rats surviving from Day 24 to Day 27 among any sham-vaccinated group was three. The clearest demonstration of the effect of D-toxin on the hematology and serum complement titers of vaccinated and sham-vaccinated rats was to compare the data accumulated from testing normal rats (n = 42 ) with the values found for surviving rats (Table 2 ). Vaccinated or sham-vaccinated rats surviving challenge with D-toxin had individual or average values either at the limits or outside the range of values determined for normal rats. All groups of rats had weight gains averaging between 8 and 9 g d a y through Day 24 (Table 3 ). For comparative purposes, weights on Day 24 of rats surviving on Day 27 are shown in a separate column. Individual surviving rats lost weight. The smallest average weight loss following challenge with D-toxin occurred in Anchor-vaccinated rats. An exception to the above observations was one rat immunized with Nobl vaccine that may not have been challenged with D-toxin. TABLE 3 Average weight of rats vaccinated or sham vaccinated with diluent on Days 0 and 10, and challenged with D-toxin on Day 24 Vaccine or diluent

Day 0

10

24

241

27

Anchor Diluent

125 125

219 222

328 (8.5) 2 329 (8.8)

328 (10) 336 (3)

325 312

Sanofi Diluent

125 124

218 217

328 (8.5) 328 (8.5)

329 (4) 327 (1)

303 293

Oxford Diluent

136 136

220 228

332 (8.2) 339 (8.5)

343 (5) 0 (0)

322 -

Nobl Diluent

120 122

206 209

320 (8.5) 328 (8.6)

286 (1) 337 (2)

297 306

Solvay

136

224

330 (8.1)

220 (1)

306

Pitman-Moore

121

207

317 (8.2)

300 (1)

278

Ambico

122

208

317 (8.1)

317 (0)

-

~Average weight on Day 24 of rats surviving (number in parentheses) on Day 27. 2Average daily weight gain for days 0-24.

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161

DISCUSSION For atrophic rhinitis to occur, a minimum requirement apparently is the localization of B. bronchiseptica in the turbinates (Pedersen and Barford, 1981 ). For severe atrophic rhinitis to occur, a site, either in the turbinates or elsewhere, appears to be necessary for toxigenic P. multocida of serogroup D to become established and produce toxin (Pedersen and Barford, 198 l, 1982 ). Purified D-toxin, when sprayed into the nasal cavities of pigs, caused lesions of atrophic rhinitis (Dominick and Rimler, 1986). Vaccines, to be protective, should either minimize colonization of B. bronchiseptica and P. multocida or limit toxin production by the stimulation of antibody against toxins of either organism. Foged et al. ( 1989 ) demonstrated good protection against experimental infection with a combination of B. bronchiseptica and a toxigenic P. multocida by immunization of gilts with a toxoid prepared from purified toxin produced by a serogroup D P. multocida. Their study suggested that antitoxic immunity alone may prevent severe atrophic rhinitis in pigs. None of the vaccines we tested for antitoxic activity protected rats against changes in hematology, serum complement and weight loss caused by injection of D-toxin. Vaccines classified as bacterins were not expected to provide antitoxic immunity and did not do so. Obviously, we could have used different amounts of vaccines, routes and schedules of vaccination; possibly with different results. The study was limited in scope, and the results should not be extrapolated to mean that the tested commercial bacterin or bacterin-toxoid vaccines will be ineffective when used to immunize swine against naturally occurring atrophic rhinitis. The results of this study, and a previous study (Thurston et al., 1990) with an experimental toxoid vaccine that provided protection against lethal and systemic effects of D-toxin, do suggest that rats may be useful in evaluating vaccines intended to generate antitoxic immunity against the lethal and systemic effects of D-toxin. ACKNOWLEDGMENTS The authors thank K. Driftmier for excellent technical assistance, Dr. T.O. Bunn (USDA, Animal and Plant Health Inspection Service, Science and Technology, National Veterinary Services Laboratories) for critical appraisal of the manuscript and the vaccine manufacturers for their total cooperation.

REFERENCES Cheville,N.F. and Rimler,R.B., 1989.A protein toxin from Pasteurella multocida type D causes acute and chronicliver toxicityin rats. Vet. Pathol., 26:148-157. Dominick, M.A. and Rimler, R.B., 1986. Turbinate atrophy in gnotobiotic pigs intranasaUy

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inoculated with protein toxin isolated from type D Pasteurella multocida. Am. J. Vet. Res.. 47: 1532-1536. Foged, N.T., Nielsen, J.P. and Jorsal, S.E., 1989. Protection against progressive atrophic rhinitis by vaccination with Pasteurella multocida toxin purified by monoclonal antibodies. Vet. Rec., 125:7-11. Garvey, J.S., Cremer, N.E. and Sussdorf, D.H., 1977. In: Methods in Immunology. 3rd Edn. W.A. Benjamin, Reading, MS, pp. 382-387. Kobisch, M. and Pennings, A., 1989. An evaluation in pigs ofNobi-Vac AR and an experimental atrophic rhinitis vaccine containing P. multocida DNT-toxoid and B. bronchiseptica. Vet. Rec., 124: 57-61. Pedersen, K.B. and Barford, K., 1981. The aetiological significance ofBordetella bronchiseptica and Pasteurella multocida in atropic rhinitis of swine. Nord. Veterinaermed., 33:513-522. Pedersen, K.B. and Barford, K., 1982. Effect on the incidence of atrophic rhinitis of vaccination of sows with a vaccine containing Pasteurella multocida toxin. Nord. Veterinaermed., 34: 293-302. Thurston, J.R., Rimler, R.B., Ackermann, M.R., Cheville, N.F. and Sacks, J.M., 1990. Immunity induced in rats vaccinated with toxoid prepared from heat-labile toxin produced by Pasteurella multocida serogroup D. Vet. Microbiol., 27:169-174.