Serological Tests as Indicators of Immunity Against Pasteurella multocida Infection in Sheep S. K. Dua and C. C. PandurangaRao*
ABSTRACT
I NTRODUCTION
Five serological tests, i.e. single tube agglutination, doubling dilution tube agglutination, agar agglutination, passive hemagglutination and passive mouse protection tests were evaluated for their efficacy in predicting
Reliable serological test(s) for detection of natural immunity in unvaccinated sheep and for determination of the immune status of vaccinated sheep against Pasteurella multocida are essential not only for elimthe fate of vaccinated and unvaccinated sheep inating naturally acquired immune animals on challenge with an ovine strain of Pasteu- from experiments on the testing of Pasteurella multocida. The passive hemagglutina- rella vaccines but also for judging the tion test predicted the fate of unvaccinated immune status of vaccinated animals under sheep while the agar agglutination test in- field conditions where it is not possible to dicated the immune status of vaccinated sheep. ascertain the same by direct challenge. Dhanda et al (6) reported that the slide or plate agglutination test was the most reliable indicator of immunity while Bain (1, 2) noted agar agglutination and passive mouse protection tests were the best for predicting the fate. These studies were RkSUMA carried out in cattle. Palit and PandurangaRao (12), working with sheep, did not get encouraging results with the plate aggluAfin de pouvoir determiner d'avance le sort tination test. de moutons vaccines et temoins, soumis a une In the present study five serological tests, infection experimentale avec une souche ovine de Pasteurella multocida, les auteurs ont uti- i.e. single tube agglutination, doubling lise les cinq epreuves serologiques suivantes: dilution tube agglutination, passive hemagglutination, agar agglutination and passive l'agglutination lente et simple, l'agglutination mouse protection tests were evaluated both lente a doubles dilutions, I'agglutination sur g'lose, l'hemagglutination passive et l"preuve in nonvaccinated and vaccinated sheep for de protection passive de la souris. L'epreuve their efficacy in predicting the fate on d'hemagglutination passive se revela la meil- challenge. leure pour predire le sort des moutons temoins, tandis que l'epreuve d'agglutination sur gelose donna la meilleure image du statut immunitaire des moutons vaccines.
MATERIALS AND METHODS
*Department of Veterinary Pathobiology, College of Veterinary Medicine, St. Paul, Minnesota 55108 (Dua) and Division of Bacteriology and Virology, Indian Veterinary Research Institute, Wukteswar, Kumaon Izatnagar U.P. India (PandurangaRao). Submitted May 17, 1977.
Volume 42 - October, 1978
Two strains, R, and R4, of Pasteurella multocida were employed in the present study. Both the strains belong to Carter's type B and were isolated from pneumonic lungs of sheep which died of Pasteurella multocida infection in India (8, 9). Each of the strains was passaged at monthly intervals in sheep to maintain virulence
489
and keep the organisms in phase I on blood agar slants and stored in the refrigerator at 4C.
SEROLOGICAL TESTS 1) Single tube agglutination test: The test was done according to the method of Bain (1) with slight modification. Ten drops of fresh serum were added in a tube to two drops of the antigen matching opacity four times of the tube no. 6 of Burroughs Wellcome opacity set. The tube was kept in a water bath at 37°C for 30 minutes. It was then taken out, shaken vigorously and left at room temperature overnight. The results were recorded in the morning. An antigen control was set up with each test. The interpretation was recorded in the following manner: Complete clearing and complete sedimentation + + + + Definite clearing but not complete with a substantial deposit + + + Fifty percent clearing and agglutination + +-+The clearing not obvious but with deposit + + or + depending on the amount of deposit No clearing and no deposit Sheep whose sera gave + + + or more were considered to be immune, + + ± probably immune and the remainder nonimmune.
2) Serial dilution tube agglutination test: Live antigen, adjusted to match opacity tube no. 4 of Burroughs Wellcome opacity set and doubling dilution of the different serum samples. Sheep whose sera gave titre of 1:40 or more were regarded as immune and expected to withstand the challenge.
3) Agar agglutination test : This test was carried out according to the method of Bain (1, 2). The medium used for this test was as follows: Sodium chloride 5 g Difco agar 15 g Distilled water 1000 ml The ingredients were dissolved in water and the following were then added: Difco tryptose Z0 g Difco tryptone 2.5 g Glucose 1.0 g 1.0 g Difcoyeast extract The pH was adjusted to 7.4 and the medium was then filtered. It was distributed
490
in tubes in 15 ml amounts and these tubes were sterilized. One ml of test serum was added to each melted tube and poured in a plate. When the medium solidified, 0.1 ml of 18 hour old broth culture of the strain was spread over the surface of the medium and the plate was incubated for 18 hours at 37°C. Finally the growth was washed with 2.5 ml of sterile normal saline. The washings were letf on the plate and examined with the help of a magnifying lens. The growth was firmly adherent to the surface of the medium in the case of strongly immune sera and with weak sera the bacterial growth came off into the saline with slight agitation. Positive and negative control sera were used while performing the test. The interpretation of this test was as follows: The growth could not be freed from the agar by washing and the saline remained clear +++ The degrees of agglutination of the organism in the saline wash were ++ and ± The growth washed smoothly and easily with little gentle agitation The serum with + or more was considered to be protective (immune)
4) Passive hemagglutination test for measuring the anti-polysaccharide antibodies: The method of Baxi and Crumpton et al (3, 5) with slight modification was followed. Blood from healthy sheep was collected aseptically in sterile Alsever's solution and was centrifuged and the red blood cells were washed three times with 0.1 M phosphate buffer saline (pH 7.2). The packed cells thus finally obtained were used for antigen sensitization. The strain of P. m'ultocida was cultivated on a blood agar plate and colonies were transferred in brain heart infusion broth for 16 hours at 370C. Such suspension of Pasteurella multocida adjusted to opacity 8 (Burroughs Wellcome) in 0.1 M phosphate buffer saline pH 7.2 and was autoclaved at 15 lbs pressure for 30 minutes and the cells were removed by centrifugation at 400 x g for half an hour. The clear supernatant was collected and used for sensitization of erythrocytes. Molisch test (Alpha napthol reaction) 0.5 ml of antigen was added to 4.5 ml of sulphuric acid (89 % by volume) in a test tube. The tube was transferred to tap
Can. J. comp. Med.
water, then to boiling bath for three minutes and allowed to cool. When cooled, 0.2 ml of freshly prepared alcoholic solution of alpha napthol (2%) was added slowly. The contents turned red which was indicative of the presence of polysaccharide in the antigen. Autoclave extract was titrated for the optimum sensitization of erythrocytes. The packed sheep cells were suspended in 0.1 M phosphate buffer saline pH 7.2 to make a 2.5 % suspension. The autoclave extract was diluted 1:4 in phosphate buffer saline and an equal volume of 2.5% sheep cells were added in a flask. The flask was kept in a water bath at 37°C for three hours. Then the sensitized cells were washed with phosphate buffer saline. The cells were finally packed by centrifugation at 500 x g for 20 minutes. A 0.3% suspension of the sensitized cells was made in the phosphate buffer saline and used as antigen. Doubling dilutions of the test serum ranging from 1:10 to 1:1280 were made in 0.5 ml quantities in diluent in perspex hemagglutination plate. An equal (0.5 ml) volume of the 0.3 % of suspension of sensitized cells was added to each of the dilutions of the serum. Two controls, namely sensitized cells plus the diluent and unsensitized cells plus test serum were included in each set. The plate was left at room temperature overnight and results were recorded in the morning. Sheep whose sera gave a titre of 1:40 or more were expected to survive
challenge. 5) Passive mouse protection test: Sets of two to four Swiss albino mice were inoculated with 0.5 ml of test serum subcutaneously and an equal number of control mice which received sterile normal saline instead of serum. All the mice were challenged subcutaneously with 10 mouse MLD of the desired strain of Pasteurella multocida 24 hours after the serum inoculation. The challenge dose was prepared by suitably diluting an eighteen-hour old broth culture of the strain, having approximately 150 live organisms. Survival of even a single mouse in the test group indicated immunity. IMMUNIZATION
The vaccine was prepared according to
Volume 42
-
October, 1978
the method of Dhanda et al (7). The mass production of Pasteurella multocida was accomplished by cultivation of organisms on yeast extract agar-containing Roux Flasks. The growth was harvested by adding sterile physiological saline and bacterial cells were centrifuged to form a pellet which was suspenled in physiological saline so as to adjust approximate 7 x 10' organisms/ml. Finally, the bacterial suspension was treated with 0.3% formalin and tested for its sterility on artificial media. The formalin-killed bacterial suspension thus obtained was emulsified with oil adjuvant (liquid paraffin ten parts, lanolin one part and bacterial suspension 15 parts). The bacterin in 3 ml amounts was administered subcutaneously in the neck region of each sheep. Sheep were inoculated subcutaneously with 1 ml of 18 hour old broth culture of Pasteurella multocida containing approximately 1 x 109 live Pasteurella multocida organisms. The experiment was discontinued after two weeks of postchallenge. All the 42 vaccinated sheep were challenged after three weeks postvaccination.
RESULTS Results of serological tests and challenge in respect of 65 unvaccinated and 42 vaccinated sheep are presented in Tables I and II. On the basis of the results presented in these two tables the accuracy of prediction of the fate by each of the serological tests was worked out and these particulars are presented in Table III. The results show that none of the five sheep (#249, 256, 185, 410, 441) expected to succumb to challenge on the basis of all the five serological tests survived (Table I and II). On the other hand, three sheep (#267, 198 and 460) which were declared immune by four out of five tests (excepting single tube agglutination test where no prediction could be made because of +++± reaction) died on challenge. All these three sheep belonged to the vaccinated group. Subsequently, 52 animals in all, gave a + + + reaction (Tables I, II and III) in the single tube agglutination test, of which 28 survived challenge while the remaining 24 succumbed.
491
TABLE I. Results of Different Serological Tests and Fate on Challenge in Respect of Unvaccinated Sheep Results with i Serological Tests Passive Mouse Serial Protection Result of Passive Agar Dilution Single Direct AgglutiHaemaggluti- Survived Tube Tube Si. Sheep Inoculated Challenge tination nation No. No. Agglutination Agglutination D 0/2 Neg. Neg. Neg. Neg. 1 264 D 0/3 Neg. Neg. Neg. Neg. 2 275 D 0/2 Neg. Neg. Neg. 3 107 + D 0/2 Neg. Neg. Neg. Neg. 4 308 D 0/2 1:10 Neg. Neg. 5 359 N.D. D N.D. Neg. Neg. Neg. 6 197 N.D. D 0/2 Neg. Neg. Neg. 7 181 D Neg. 0/2 1:10 Neg. 8 232 D 0/2 1:10 1:20 Neg. 9 179 1/2 S 1:20 Neg. 10 170 + D 0/2 1:40 1:40 11 175 + D 0/3 Neg. Neg. Neg. Neg. 12 165 D 0/4 Neg. Neg. Neg. Neg. 13 180 D 0/2 Neg. Neg. Neg. 14 166 + D 0/2 1:10 Neg. Neg. Neg. 15 168 N.D. D Neg. Neg. Neg. Neg. 16 196 D 0/2 Neg. Neg. Neg. 17 174 D 0/2 Neg. Neg. 1:40 18 163 D 0/2 Neg. Neg. 1:20 19 167 D 1/2 1:10 Neg. Neg. 20 401 D N.D. Neg. N.D. N.D. 21 447 + + D N.D. 0/2 Neg. Neg. 22 334 D 0/2 Neg. Neg. Neg. 23 324 +++ N.D. D Neg. N.D. Neg. 24 24 ++ D 0/2 Neg. Neg. Neg. 25 69 D 1/4 1:10 Neg. 1:40 26 23 D 1:20 1/4 1:40 27 64 D N.D. 2/4 Neg. Neg. 28 104 + + N.D. D 0/2 N.D. 1:20 Neg. 29 109 S 1:40 0/3 1:20 ++ Neg. 30 110 D 0/2 Neg. Neg. 1:40 31 208 Neg. D 1:10 0/2 Neg. Neg. 32 238 Neg. Neg. D 1:10 0/3 N.D. Neg. 33 239 Neg. D 1:10 0/4 Neg. 1:40 34 242 Neg. D 0/4 Neg. Neg. Neg. 35 247 Neg. S 1:80 3/4 1:40 ++ 36 249 Neg. D 1:10 0/2 Neg. 1:20 37 262 Neg. Neg. D 0/2 Neg. Neg. Neg. 38 480 Neg. D 0/2 1:10 Neg. Neg. 39 432 Neg. D 0/2 Neg. Neg. Neg. 40 265 Neg. D N.D. Neg. Neg. Neg. 41 288 Neg. D 0/2 Neg. N.D. Neg. 42 236 Neg. D N.D. N.D. Neg. Neg. 43 117 D N.D. 1:10 Neg. 44 189 D N.D. Neg. Neg. Neg. 45 161 D N.D. 1:10 N.D. Neg. 46 435 Neg. D N.D. 1:10 N.D. Neg. 47 466 Neg. Neg. D N.D. Neg. Neg. 1:20 48 292 Neg. Neg. D 0/2 Neg. Neg. Neg. 49 274 D N.D. Neg. N.D. 1:20 50 304 D 1:20 0/2 N.D. Neg. 51 306 D 0/2 Neg. N.D. Neg. 52 310 Neg. D 1:10 0/3 N.D. Neg. 53 314 D 0/2 Neg. N.D. Neg. Neg. 54 395 Neg. D 0/2 Neg. N.D. Neg. Neg. 55 398 D 0/2 Neg. N.D. Neg. 56 404 D N.D. Neg. N.D. Neg. 57 452 D 1:10 0/2 Neg. Neg. 93 58 D 1:80 0/2 Neg. 1:40 94 59 D 1:10 0/3 Neg. Neg. 96 60 D 0/4 Neg. Neg. Neg. 61 105 D 1:10 0/3 Neg. 1:40 62 108 S 1:40 2/3 ++ 1:40 63 256 D 0/3 Neg. Neg. Neg. 64 558 D Neg. 0/3 Neg. Neg. 65 564 D = Died, S = Survived, N.D. = Not determined
TABLE II. Results of Different Serological Tests and Fate on Challenge in Respect of Vaccinated Sheep
Results with Serological Tests
Serial Agar Single Dilution Tube AgglutiS. Sheep Tube No. No. Agglutination Agglutination nation 1 164 1:20 ++4 Neg. Neg. 2 169 Neg. ++ 1:40 3 198 ++4 4 200 ++ 1:40 ++ ++ 5 172 1:80 6 199 1:40 ++4 +++ 7 156 1:40 ++4 ++4 8 158 1:80 ++ 9 123 1:80 ++ | 10 264 1:80 ++ ++ 11 283 1:80 ++4 +++ ++i 12 151 1:80 + 13 286 1:40 ++i ++ 14 263 ++4 1:160 ++ 15 381 1:80 ++ ++4 16 462 1:160 ++ ++4 1:40 17 267 +++ 18 427 ++4 1:20 ++ 19 66 1:80 ++4 ++ 20 67 1:80 ++4 ++ 21 403 ++4 1:160 ++ 22 457 1:80 ++4 ++ 23 187 1:20 ++4 ++ 24 366 1:20 N.D 25 194 1:40 ++4 ++ ++4 1:40 26 284 ++ 1:80 27 185 +++ +.D 1:160 28 303 ++4 ++ 1:20 29 469 +++ ++ 1:40 30 460 ++4 ++ 451 1:40 31 ++ ++ 1:40 32 454 ++4 ++ 1:80 33 410 +++ ±+ 1:160 34 441 +++ ++ ++t 1:80 35 259 ++ 1:160 ++ 36 261 1:80 ++ 37 412 + 1:20 38 417 ++4 ++ 1:40 39 267 + ++ 1:40 + 40 289 ++ 1:80 41 559 ++4 ++ 1:40 42 483 + + 4S = Survived, D = Died, N.D. = Not determined
±+
The efficacy of the five tests in predicting the immune status of unvaccinated and vaccinated sheep is shown in Table III. Passive hemagglutination test was found to be the best for indicating the immune status of unvaccinated sheep. In case of vaccinatel sheep, the agar agglutination test was found to be most reliable in predicting the fate on challenge.
DISCUSSION The results in the present investigation Volume 42
October, 1978
Passive Mouse Protection Result of Passive Direct Haemaggluti- Survived tination Inoculated Challenge S 1:40 2/4 D 0/4 1:10 D 2/4 1:40 S 3/4 1:80 S 1:80 2/4 S 1:80 2/4 S 4/4 1:320 S 3/4 1:320 S 1:320 3/4 S 4/4 1:160 S 1:20 3/4 S 3/4 1:160 S 1:20 2/4 S 1:320 3/4 S 1:160 4/4 S 1:80 3/4 D 1:80 3/4 S 1:160 3/4
1:80 1:80 1:80 1:160 1:80 1:80 1:160 1:160 1:160 1:160 1:80 1:80 1:80 1:20 1:160 1:80 1:80 1:60 1:40 1:40 1:40 1:40 1:160 1:80
2/2 2/2 2/4 1/2 3/4 N.D. 2/4 3/4 3/4 2/4 4/4 3/4 N.D. N.D. 2/2 1/2 2/4 2/4 2/4 3/4 0/4 1/4 2/4 2/2
S S S S D D S D S S S D S S S S S S S S S S S S
lend support to the contention that immune status of an animal against P. multocida infection is, by and large, related to the level of circulating antibodies. All the six sheep considered immune on the basis of the results of all these serological tests, withstood the challenge. Similar observations were reported by Dhanda et al (6) in cattle. However, three sheep which were declared immune by four out of five tests (excepting single tube agglutination test where no prediction could be made because of +++± reaction) died on challenge. Similar findings were also reported by Dhanda 493
TABLE III. Evaluation of Serological Tests as Indicators of Immunity
Unvaccinacted Sheep Total No. Total No. Percentage Subjected in which of Accuracy of to Indi- Fate was rect Tests Predicted Prediction
Name of Indirect Test Single tube
agglutination agglutination Agar agglutination... .
Serial dilution tube
.....
Passive hemagglutination ............ Passive mouse
Vaccinated Sheep Total No. Total No. Percentage Subjected in which of Accuracy of Fate was to Indirect Tests Predicted Prediction
45a
42
93.33
JOb
6
64 45
55 41
85.93 91.11
42 41
34 36
60
80.95 87.80
34 80.95 42 96.87 84.61 33 52 46 88.46 39 protection &In addition to these 45, sera of 20 more sheep were also tested and were found to give + + = reaction, the fate of which could not be predicted bIn addition to these ten, sera of 32 vaccinated sheep were also tested and were found to give + + ± reaction, the fate of which could not be predicted. 64
62
........
et al (6). They explained it by stating that "in animals, subjected to physical or physiological stress or in low level of nutrition, the dynamic cellular phenomenon of phagocytosis and other clearance mechanisms might not be as efficient as otherwise, even in the presence of circulating
antibodies". Our findings also showed that 52 sheep in all, gave a ++-+ reaction in single tube agglutination test, of which 28 survived challenge while the remaining 24 succumbed. So, nothing can be predicted about the immune status of animals giving such a reaction in this test. Similar observations were also reported by Bain (1, 2). The efficacy of the five tests in predicting the immunity status of nonvaccinated sheep is shown in Table III. Passive hemagglutination test was found to be the best for indicating the immunity status of nonvaccinated sheep. Statistical examination of the remaining four tests revealed that any one of them was as good as the other in predicting the fate on challenge. For judging the immunity status of vaccinated sheep, agar agglutination test was found to be the best while the single tube agglutination test was the least useful. Statistical examination of the results of the remaining three tests revealed that any one of them was as good as the other. The mechanism of immunity against P. multocida infection is still not definitely known in other species. On the basis of information available, it appears that though circulating antibodies are related to immunity, there is sufficient evidence
494
to believe that cellular defences also play an important role in protection. Regarding the mode of origin of naturally occurring immunity, Dhanda et al (6) attributed it to infection with naturally occurring strains of low virulence while Bain (1, 2) considered that it would develop from arrested infection with virulent P. multocida strains. It is possible that both the mechanisms may be operating in nature. However, the role of local humoral and cell mediated immunity (CMI) in the respiratory tract of the animal should not be ignored (4, 10, 11, 13, 14). Further work is required to elucidate the importance of CMI against P. multocida infection
ACKNOWLEDGMENTS The senior author is grateful to the Indian Council of Agricultural Research, New Delhi for the award of a Research Fellowship. The authors are thankful to Dr. C. M. Singh, Director, Indian Veterinary Research Institute, MukteswarKumaon for providing the necessary facilities.
REFERENCES 1. BAIN, R. V. S. Studies on haemorrhagic septicemia of cattle. II. The detection of naturally acquired immunity. Br. vet. J. 110: 519-524. 1954.
Can. J. comp. Med.
2. BAIN, R. V. S. Studies on haemorrhagic septicemia of cattle. V. Tests for immunity in vaccinated cattle. Br. vet. J. 111: 511-518. 1955. 3. BAXI, K. K., H. BLOBEL und W. SCHARMANN. Untersuchungen Uberdie Antigenstruktur von Pasteurella multocida. Zentbl. Vet. Med. 17B: 441-452. 1970. 4. COLLINS, F. M. Vaccines and cell-mediated immunity. Bact. Rev. 38: 371-402. 1974. 5. CRUMPTON, M. J., D.A.L. DAVIES and A. M. HUTCHISON. The serological specificities of Pasteurella pseudotuberulosis somatic antigens. J. gen. Microbiol. 18: 129-139. 1958. 6. DHANDA, M. R. J.M. LALL, R. N. SETH and P. CHANDERSEKARIAH. Immunological studies on Pasteurella septica. III. Indirect tests as indicators of immunity in haemorrhagic septicemia. Indian J. vet. Sci. 29: 30-46. 1959. 7. DHANDA, M.R., P. C. SEKARIAH, J. M. LALL and P. PRAKASH. Immunological studies on Pasteurella septica. V. Further observations on the immunizing efficacy of adjuvant vaccine. Indian vet. J. 37: 597-606. 1960. 8. DUA, S. K. and C. C. PANDURANGARAO. Immunological studies on Pasteurella multocida of
Volume 42 - October, 1978
ovine origin. Indian J. Anim. Sci. 41: 986-991. 1971. 9. DUA, S.K., C.C. PANDURANGARAO and S. S. KHERA. Studies on the use of bovine hemorrhagic septicemia vaccine in sheep and goats. Indian vet. J. 54: 421-424. 1977. 10. MAHESWARAN, S.K., J.R. McDOWELL and B. S. POMEROY. Studies on Pasteurella multocida. I. Efficacy of an avirulent mutant as a live vaccine in turkeys. Avian. Dis. 17: 396-405. 1973. 11. MAHESWARAN, S.K., E. THEIS and S. K. DUA. Studies on Pasteurella multocida III. In vitro assay for cell-mediated immunity. Avian Dis. 20: 332-341. 1976. 12. PALIT, N. and C. C. PANDURANGARAO. Studies on Bovine Pasteurellosis. M.V.Se. thesis, p. 132, Agra University. 1966. 13. WALDMAN, R.H., C.S. SPENCER and J.E. JOHNSON. III. Respiratory and systemic cellular and humoral immune response to inflenza virus vaccine administered parenterally or nose drops. Cell. Immunol. 3: 294-300. 1972. 14. WOOLCOCK, J.B. Resistance to microbial infecfection Vaccine in theory and practice. Aust. vet. J. 49: 307-317. 1973.
495
ABSTRACT
I NTRODUCTION
Five serological tests, i.e. single tube agglutination, doubling dilution tube agglutination, agar agglutination, passive hemagglutination and passive mouse protection tests were evaluated for their efficacy in predicting
Reliable serological test(s) for detection of natural immunity in unvaccinated sheep and for determination of the immune status of vaccinated sheep against Pasteurella multocida are essential not only for elimthe fate of vaccinated and unvaccinated sheep inating naturally acquired immune animals on challenge with an ovine strain of Pasteu- from experiments on the testing of Pasteurella multocida. The passive hemagglutina- rella vaccines but also for judging the tion test predicted the fate of unvaccinated immune status of vaccinated animals under sheep while the agar agglutination test in- field conditions where it is not possible to dicated the immune status of vaccinated sheep. ascertain the same by direct challenge. Dhanda et al (6) reported that the slide or plate agglutination test was the most reliable indicator of immunity while Bain (1, 2) noted agar agglutination and passive mouse protection tests were the best for predicting the fate. These studies were RkSUMA carried out in cattle. Palit and PandurangaRao (12), working with sheep, did not get encouraging results with the plate aggluAfin de pouvoir determiner d'avance le sort tination test. de moutons vaccines et temoins, soumis a une In the present study five serological tests, infection experimentale avec une souche ovine de Pasteurella multocida, les auteurs ont uti- i.e. single tube agglutination, doubling lise les cinq epreuves serologiques suivantes: dilution tube agglutination, passive hemagglutination, agar agglutination and passive l'agglutination lente et simple, l'agglutination mouse protection tests were evaluated both lente a doubles dilutions, I'agglutination sur g'lose, l'hemagglutination passive et l"preuve in nonvaccinated and vaccinated sheep for de protection passive de la souris. L'epreuve their efficacy in predicting the fate on d'hemagglutination passive se revela la meil- challenge. leure pour predire le sort des moutons temoins, tandis que l'epreuve d'agglutination sur gelose donna la meilleure image du statut immunitaire des moutons vaccines.
MATERIALS AND METHODS
*Department of Veterinary Pathobiology, College of Veterinary Medicine, St. Paul, Minnesota 55108 (Dua) and Division of Bacteriology and Virology, Indian Veterinary Research Institute, Wukteswar, Kumaon Izatnagar U.P. India (PandurangaRao). Submitted May 17, 1977.
Volume 42 - October, 1978
Two strains, R, and R4, of Pasteurella multocida were employed in the present study. Both the strains belong to Carter's type B and were isolated from pneumonic lungs of sheep which died of Pasteurella multocida infection in India (8, 9). Each of the strains was passaged at monthly intervals in sheep to maintain virulence
489
and keep the organisms in phase I on blood agar slants and stored in the refrigerator at 4C.
SEROLOGICAL TESTS 1) Single tube agglutination test: The test was done according to the method of Bain (1) with slight modification. Ten drops of fresh serum were added in a tube to two drops of the antigen matching opacity four times of the tube no. 6 of Burroughs Wellcome opacity set. The tube was kept in a water bath at 37°C for 30 minutes. It was then taken out, shaken vigorously and left at room temperature overnight. The results were recorded in the morning. An antigen control was set up with each test. The interpretation was recorded in the following manner: Complete clearing and complete sedimentation + + + + Definite clearing but not complete with a substantial deposit + + + Fifty percent clearing and agglutination + +-+The clearing not obvious but with deposit + + or + depending on the amount of deposit No clearing and no deposit Sheep whose sera gave + + + or more were considered to be immune, + + ± probably immune and the remainder nonimmune.
2) Serial dilution tube agglutination test: Live antigen, adjusted to match opacity tube no. 4 of Burroughs Wellcome opacity set and doubling dilution of the different serum samples. Sheep whose sera gave titre of 1:40 or more were regarded as immune and expected to withstand the challenge.
3) Agar agglutination test : This test was carried out according to the method of Bain (1, 2). The medium used for this test was as follows: Sodium chloride 5 g Difco agar 15 g Distilled water 1000 ml The ingredients were dissolved in water and the following were then added: Difco tryptose Z0 g Difco tryptone 2.5 g Glucose 1.0 g 1.0 g Difcoyeast extract The pH was adjusted to 7.4 and the medium was then filtered. It was distributed
490
in tubes in 15 ml amounts and these tubes were sterilized. One ml of test serum was added to each melted tube and poured in a plate. When the medium solidified, 0.1 ml of 18 hour old broth culture of the strain was spread over the surface of the medium and the plate was incubated for 18 hours at 37°C. Finally the growth was washed with 2.5 ml of sterile normal saline. The washings were letf on the plate and examined with the help of a magnifying lens. The growth was firmly adherent to the surface of the medium in the case of strongly immune sera and with weak sera the bacterial growth came off into the saline with slight agitation. Positive and negative control sera were used while performing the test. The interpretation of this test was as follows: The growth could not be freed from the agar by washing and the saline remained clear +++ The degrees of agglutination of the organism in the saline wash were ++ and ± The growth washed smoothly and easily with little gentle agitation The serum with + or more was considered to be protective (immune)
4) Passive hemagglutination test for measuring the anti-polysaccharide antibodies: The method of Baxi and Crumpton et al (3, 5) with slight modification was followed. Blood from healthy sheep was collected aseptically in sterile Alsever's solution and was centrifuged and the red blood cells were washed three times with 0.1 M phosphate buffer saline (pH 7.2). The packed cells thus finally obtained were used for antigen sensitization. The strain of P. m'ultocida was cultivated on a blood agar plate and colonies were transferred in brain heart infusion broth for 16 hours at 370C. Such suspension of Pasteurella multocida adjusted to opacity 8 (Burroughs Wellcome) in 0.1 M phosphate buffer saline pH 7.2 and was autoclaved at 15 lbs pressure for 30 minutes and the cells were removed by centrifugation at 400 x g for half an hour. The clear supernatant was collected and used for sensitization of erythrocytes. Molisch test (Alpha napthol reaction) 0.5 ml of antigen was added to 4.5 ml of sulphuric acid (89 % by volume) in a test tube. The tube was transferred to tap
Can. J. comp. Med.
water, then to boiling bath for three minutes and allowed to cool. When cooled, 0.2 ml of freshly prepared alcoholic solution of alpha napthol (2%) was added slowly. The contents turned red which was indicative of the presence of polysaccharide in the antigen. Autoclave extract was titrated for the optimum sensitization of erythrocytes. The packed sheep cells were suspended in 0.1 M phosphate buffer saline pH 7.2 to make a 2.5 % suspension. The autoclave extract was diluted 1:4 in phosphate buffer saline and an equal volume of 2.5% sheep cells were added in a flask. The flask was kept in a water bath at 37°C for three hours. Then the sensitized cells were washed with phosphate buffer saline. The cells were finally packed by centrifugation at 500 x g for 20 minutes. A 0.3% suspension of the sensitized cells was made in the phosphate buffer saline and used as antigen. Doubling dilutions of the test serum ranging from 1:10 to 1:1280 were made in 0.5 ml quantities in diluent in perspex hemagglutination plate. An equal (0.5 ml) volume of the 0.3 % of suspension of sensitized cells was added to each of the dilutions of the serum. Two controls, namely sensitized cells plus the diluent and unsensitized cells plus test serum were included in each set. The plate was left at room temperature overnight and results were recorded in the morning. Sheep whose sera gave a titre of 1:40 or more were expected to survive
challenge. 5) Passive mouse protection test: Sets of two to four Swiss albino mice were inoculated with 0.5 ml of test serum subcutaneously and an equal number of control mice which received sterile normal saline instead of serum. All the mice were challenged subcutaneously with 10 mouse MLD of the desired strain of Pasteurella multocida 24 hours after the serum inoculation. The challenge dose was prepared by suitably diluting an eighteen-hour old broth culture of the strain, having approximately 150 live organisms. Survival of even a single mouse in the test group indicated immunity. IMMUNIZATION
The vaccine was prepared according to
Volume 42
-
October, 1978
the method of Dhanda et al (7). The mass production of Pasteurella multocida was accomplished by cultivation of organisms on yeast extract agar-containing Roux Flasks. The growth was harvested by adding sterile physiological saline and bacterial cells were centrifuged to form a pellet which was suspenled in physiological saline so as to adjust approximate 7 x 10' organisms/ml. Finally, the bacterial suspension was treated with 0.3% formalin and tested for its sterility on artificial media. The formalin-killed bacterial suspension thus obtained was emulsified with oil adjuvant (liquid paraffin ten parts, lanolin one part and bacterial suspension 15 parts). The bacterin in 3 ml amounts was administered subcutaneously in the neck region of each sheep. Sheep were inoculated subcutaneously with 1 ml of 18 hour old broth culture of Pasteurella multocida containing approximately 1 x 109 live Pasteurella multocida organisms. The experiment was discontinued after two weeks of postchallenge. All the 42 vaccinated sheep were challenged after three weeks postvaccination.
RESULTS Results of serological tests and challenge in respect of 65 unvaccinated and 42 vaccinated sheep are presented in Tables I and II. On the basis of the results presented in these two tables the accuracy of prediction of the fate by each of the serological tests was worked out and these particulars are presented in Table III. The results show that none of the five sheep (#249, 256, 185, 410, 441) expected to succumb to challenge on the basis of all the five serological tests survived (Table I and II). On the other hand, three sheep (#267, 198 and 460) which were declared immune by four out of five tests (excepting single tube agglutination test where no prediction could be made because of +++± reaction) died on challenge. All these three sheep belonged to the vaccinated group. Subsequently, 52 animals in all, gave a + + + reaction (Tables I, II and III) in the single tube agglutination test, of which 28 survived challenge while the remaining 24 succumbed.
491
TABLE I. Results of Different Serological Tests and Fate on Challenge in Respect of Unvaccinated Sheep Results with i Serological Tests Passive Mouse Serial Protection Result of Passive Agar Dilution Single Direct AgglutiHaemaggluti- Survived Tube Tube Si. Sheep Inoculated Challenge tination nation No. No. Agglutination Agglutination D 0/2 Neg. Neg. Neg. Neg. 1 264 D 0/3 Neg. Neg. Neg. Neg. 2 275 D 0/2 Neg. Neg. Neg. 3 107 + D 0/2 Neg. Neg. Neg. Neg. 4 308 D 0/2 1:10 Neg. Neg. 5 359 N.D. D N.D. Neg. Neg. Neg. 6 197 N.D. D 0/2 Neg. Neg. Neg. 7 181 D Neg. 0/2 1:10 Neg. 8 232 D 0/2 1:10 1:20 Neg. 9 179 1/2 S 1:20 Neg. 10 170 + D 0/2 1:40 1:40 11 175 + D 0/3 Neg. Neg. Neg. Neg. 12 165 D 0/4 Neg. Neg. Neg. Neg. 13 180 D 0/2 Neg. Neg. Neg. 14 166 + D 0/2 1:10 Neg. Neg. Neg. 15 168 N.D. D Neg. Neg. Neg. Neg. 16 196 D 0/2 Neg. Neg. Neg. 17 174 D 0/2 Neg. Neg. 1:40 18 163 D 0/2 Neg. Neg. 1:20 19 167 D 1/2 1:10 Neg. Neg. 20 401 D N.D. Neg. N.D. N.D. 21 447 + + D N.D. 0/2 Neg. Neg. 22 334 D 0/2 Neg. Neg. Neg. 23 324 +++ N.D. D Neg. N.D. Neg. 24 24 ++ D 0/2 Neg. Neg. Neg. 25 69 D 1/4 1:10 Neg. 1:40 26 23 D 1:20 1/4 1:40 27 64 D N.D. 2/4 Neg. Neg. 28 104 + + N.D. D 0/2 N.D. 1:20 Neg. 29 109 S 1:40 0/3 1:20 ++ Neg. 30 110 D 0/2 Neg. Neg. 1:40 31 208 Neg. D 1:10 0/2 Neg. Neg. 32 238 Neg. Neg. D 1:10 0/3 N.D. Neg. 33 239 Neg. D 1:10 0/4 Neg. 1:40 34 242 Neg. D 0/4 Neg. Neg. Neg. 35 247 Neg. S 1:80 3/4 1:40 ++ 36 249 Neg. D 1:10 0/2 Neg. 1:20 37 262 Neg. Neg. D 0/2 Neg. Neg. Neg. 38 480 Neg. D 0/2 1:10 Neg. Neg. 39 432 Neg. D 0/2 Neg. Neg. Neg. 40 265 Neg. D N.D. Neg. Neg. Neg. 41 288 Neg. D 0/2 Neg. N.D. Neg. 42 236 Neg. D N.D. N.D. Neg. Neg. 43 117 D N.D. 1:10 Neg. 44 189 D N.D. Neg. Neg. Neg. 45 161 D N.D. 1:10 N.D. Neg. 46 435 Neg. D N.D. 1:10 N.D. Neg. 47 466 Neg. Neg. D N.D. Neg. Neg. 1:20 48 292 Neg. Neg. D 0/2 Neg. Neg. Neg. 49 274 D N.D. Neg. N.D. 1:20 50 304 D 1:20 0/2 N.D. Neg. 51 306 D 0/2 Neg. N.D. Neg. 52 310 Neg. D 1:10 0/3 N.D. Neg. 53 314 D 0/2 Neg. N.D. Neg. Neg. 54 395 Neg. D 0/2 Neg. N.D. Neg. Neg. 55 398 D 0/2 Neg. N.D. Neg. 56 404 D N.D. Neg. N.D. Neg. 57 452 D 1:10 0/2 Neg. Neg. 93 58 D 1:80 0/2 Neg. 1:40 94 59 D 1:10 0/3 Neg. Neg. 96 60 D 0/4 Neg. Neg. Neg. 61 105 D 1:10 0/3 Neg. 1:40 62 108 S 1:40 2/3 ++ 1:40 63 256 D 0/3 Neg. Neg. Neg. 64 558 D Neg. 0/3 Neg. Neg. 65 564 D = Died, S = Survived, N.D. = Not determined
TABLE II. Results of Different Serological Tests and Fate on Challenge in Respect of Vaccinated Sheep
Results with Serological Tests
Serial Agar Single Dilution Tube AgglutiS. Sheep Tube No. No. Agglutination Agglutination nation 1 164 1:20 ++4 Neg. Neg. 2 169 Neg. ++ 1:40 3 198 ++4 4 200 ++ 1:40 ++ ++ 5 172 1:80 6 199 1:40 ++4 +++ 7 156 1:40 ++4 ++4 8 158 1:80 ++ 9 123 1:80 ++ | 10 264 1:80 ++ ++ 11 283 1:80 ++4 +++ ++i 12 151 1:80 + 13 286 1:40 ++i ++ 14 263 ++4 1:160 ++ 15 381 1:80 ++ ++4 16 462 1:160 ++ ++4 1:40 17 267 +++ 18 427 ++4 1:20 ++ 19 66 1:80 ++4 ++ 20 67 1:80 ++4 ++ 21 403 ++4 1:160 ++ 22 457 1:80 ++4 ++ 23 187 1:20 ++4 ++ 24 366 1:20 N.D 25 194 1:40 ++4 ++ ++4 1:40 26 284 ++ 1:80 27 185 +++ +.D 1:160 28 303 ++4 ++ 1:20 29 469 +++ ++ 1:40 30 460 ++4 ++ 451 1:40 31 ++ ++ 1:40 32 454 ++4 ++ 1:80 33 410 +++ ±+ 1:160 34 441 +++ ++ ++t 1:80 35 259 ++ 1:160 ++ 36 261 1:80 ++ 37 412 + 1:20 38 417 ++4 ++ 1:40 39 267 + ++ 1:40 + 40 289 ++ 1:80 41 559 ++4 ++ 1:40 42 483 + + 4S = Survived, D = Died, N.D. = Not determined
±+
The efficacy of the five tests in predicting the immune status of unvaccinated and vaccinated sheep is shown in Table III. Passive hemagglutination test was found to be the best for indicating the immune status of unvaccinated sheep. In case of vaccinatel sheep, the agar agglutination test was found to be most reliable in predicting the fate on challenge.
DISCUSSION The results in the present investigation Volume 42
October, 1978
Passive Mouse Protection Result of Passive Direct Haemaggluti- Survived tination Inoculated Challenge S 1:40 2/4 D 0/4 1:10 D 2/4 1:40 S 3/4 1:80 S 1:80 2/4 S 1:80 2/4 S 4/4 1:320 S 3/4 1:320 S 1:320 3/4 S 4/4 1:160 S 1:20 3/4 S 3/4 1:160 S 1:20 2/4 S 1:320 3/4 S 1:160 4/4 S 1:80 3/4 D 1:80 3/4 S 1:160 3/4
1:80 1:80 1:80 1:160 1:80 1:80 1:160 1:160 1:160 1:160 1:80 1:80 1:80 1:20 1:160 1:80 1:80 1:60 1:40 1:40 1:40 1:40 1:160 1:80
2/2 2/2 2/4 1/2 3/4 N.D. 2/4 3/4 3/4 2/4 4/4 3/4 N.D. N.D. 2/2 1/2 2/4 2/4 2/4 3/4 0/4 1/4 2/4 2/2
S S S S D D S D S S S D S S S S S S S S S S S S
lend support to the contention that immune status of an animal against P. multocida infection is, by and large, related to the level of circulating antibodies. All the six sheep considered immune on the basis of the results of all these serological tests, withstood the challenge. Similar observations were reported by Dhanda et al (6) in cattle. However, three sheep which were declared immune by four out of five tests (excepting single tube agglutination test where no prediction could be made because of +++± reaction) died on challenge. Similar findings were also reported by Dhanda 493
TABLE III. Evaluation of Serological Tests as Indicators of Immunity
Unvaccinacted Sheep Total No. Total No. Percentage Subjected in which of Accuracy of to Indi- Fate was rect Tests Predicted Prediction
Name of Indirect Test Single tube
agglutination agglutination Agar agglutination... .
Serial dilution tube
.....
Passive hemagglutination ............ Passive mouse
Vaccinated Sheep Total No. Total No. Percentage Subjected in which of Accuracy of Fate was to Indirect Tests Predicted Prediction
45a
42
93.33
JOb
6
64 45
55 41
85.93 91.11
42 41
34 36
60
80.95 87.80
34 80.95 42 96.87 84.61 33 52 46 88.46 39 protection &In addition to these 45, sera of 20 more sheep were also tested and were found to give + + = reaction, the fate of which could not be predicted bIn addition to these ten, sera of 32 vaccinated sheep were also tested and were found to give + + ± reaction, the fate of which could not be predicted. 64
62
........
et al (6). They explained it by stating that "in animals, subjected to physical or physiological stress or in low level of nutrition, the dynamic cellular phenomenon of phagocytosis and other clearance mechanisms might not be as efficient as otherwise, even in the presence of circulating
antibodies". Our findings also showed that 52 sheep in all, gave a ++-+ reaction in single tube agglutination test, of which 28 survived challenge while the remaining 24 succumbed. So, nothing can be predicted about the immune status of animals giving such a reaction in this test. Similar observations were also reported by Bain (1, 2). The efficacy of the five tests in predicting the immunity status of nonvaccinated sheep is shown in Table III. Passive hemagglutination test was found to be the best for indicating the immunity status of nonvaccinated sheep. Statistical examination of the remaining four tests revealed that any one of them was as good as the other in predicting the fate on challenge. For judging the immunity status of vaccinated sheep, agar agglutination test was found to be the best while the single tube agglutination test was the least useful. Statistical examination of the results of the remaining three tests revealed that any one of them was as good as the other. The mechanism of immunity against P. multocida infection is still not definitely known in other species. On the basis of information available, it appears that though circulating antibodies are related to immunity, there is sufficient evidence
494
to believe that cellular defences also play an important role in protection. Regarding the mode of origin of naturally occurring immunity, Dhanda et al (6) attributed it to infection with naturally occurring strains of low virulence while Bain (1, 2) considered that it would develop from arrested infection with virulent P. multocida strains. It is possible that both the mechanisms may be operating in nature. However, the role of local humoral and cell mediated immunity (CMI) in the respiratory tract of the animal should not be ignored (4, 10, 11, 13, 14). Further work is required to elucidate the importance of CMI against P. multocida infection
ACKNOWLEDGMENTS The senior author is grateful to the Indian Council of Agricultural Research, New Delhi for the award of a Research Fellowship. The authors are thankful to Dr. C. M. Singh, Director, Indian Veterinary Research Institute, MukteswarKumaon for providing the necessary facilities.
REFERENCES 1. BAIN, R. V. S. Studies on haemorrhagic septicemia of cattle. II. The detection of naturally acquired immunity. Br. vet. J. 110: 519-524. 1954.
Can. J. comp. Med.
2. BAIN, R. V. S. Studies on haemorrhagic septicemia of cattle. V. Tests for immunity in vaccinated cattle. Br. vet. J. 111: 511-518. 1955. 3. BAXI, K. K., H. BLOBEL und W. SCHARMANN. Untersuchungen Uberdie Antigenstruktur von Pasteurella multocida. Zentbl. Vet. Med. 17B: 441-452. 1970. 4. COLLINS, F. M. Vaccines and cell-mediated immunity. Bact. Rev. 38: 371-402. 1974. 5. CRUMPTON, M. J., D.A.L. DAVIES and A. M. HUTCHISON. The serological specificities of Pasteurella pseudotuberulosis somatic antigens. J. gen. Microbiol. 18: 129-139. 1958. 6. DHANDA, M. R. J.M. LALL, R. N. SETH and P. CHANDERSEKARIAH. Immunological studies on Pasteurella septica. III. Indirect tests as indicators of immunity in haemorrhagic septicemia. Indian J. vet. Sci. 29: 30-46. 1959. 7. DHANDA, M.R., P. C. SEKARIAH, J. M. LALL and P. PRAKASH. Immunological studies on Pasteurella septica. V. Further observations on the immunizing efficacy of adjuvant vaccine. Indian vet. J. 37: 597-606. 1960. 8. DUA, S. K. and C. C. PANDURANGARAO. Immunological studies on Pasteurella multocida of
Volume 42 - October, 1978
ovine origin. Indian J. Anim. Sci. 41: 986-991. 1971. 9. DUA, S.K., C.C. PANDURANGARAO and S. S. KHERA. Studies on the use of bovine hemorrhagic septicemia vaccine in sheep and goats. Indian vet. J. 54: 421-424. 1977. 10. MAHESWARAN, S.K., J.R. McDOWELL and B. S. POMEROY. Studies on Pasteurella multocida. I. Efficacy of an avirulent mutant as a live vaccine in turkeys. Avian. Dis. 17: 396-405. 1973. 11. MAHESWARAN, S.K., E. THEIS and S. K. DUA. Studies on Pasteurella multocida III. In vitro assay for cell-mediated immunity. Avian Dis. 20: 332-341. 1976. 12. PALIT, N. and C. C. PANDURANGARAO. Studies on Bovine Pasteurellosis. M.V.Se. thesis, p. 132, Agra University. 1966. 13. WALDMAN, R.H., C.S. SPENCER and J.E. JOHNSON. III. Respiratory and systemic cellular and humoral immune response to inflenza virus vaccine administered parenterally or nose drops. Cell. Immunol. 3: 294-300. 1972. 14. WOOLCOCK, J.B. Resistance to microbial infecfection Vaccine in theory and practice. Aust. vet. J. 49: 307-317. 1973.
495
