The Detection of Transmissible Gastroenteritis Viral Antibodies by Immunodiffusion J. Bohoc and J. B. Derbyshire*
ABSTRACT
RASUME
Precipitating antibodies against transmissible gastroenteritis viral antigens were detected by the immunodiffusion test in two transmissible gastroenteritis viral hyperimmune antisera and in antiserum prepared against haemagglutinating encephalomyelitis virus but not in sera from several species of normal animals, in antisera prepared against a variety of other viruses and bacteria or sera from swine with bacterial enteritis. When the immunodiffusion test was compared with the virus neutralization test for the detection of transmissible gastroenteritis viral antibodies in 20 swine sera certain samples which contained high titres of virus neutralizing antibodies failed to produce precipitation while other sera were positive in the immunodiffusion test although their virus neutralizing antibody titres were relatively low. Precipitating antibodies were also detected by immunodiffusion in several samples of milk whey from a sow which had been vaccinated with inactivated transmissible gastroenteritis virus.
Les auteurs ont decele des anticorps precipitant les antigenes viraux de la gastro-enterite transmissible, a l'aide de l'immunodiffusion, dans deux echantillons de serum hyperimmun specifique 'a ce virus, ainsi que dans un immun-serum prepare avec le virus hemagglutinant de l'encephalomyilite porcine; ils obtinrent cependant des resultats negatifs avec le serum d'animaux sains de differentes especes, avec des serums hyperimmuns prepar6s a l'aide d'une variete d'autres virus et bacteries, ainsi qu'avec le serum de porcs atteints d'enterite bacterienne. La comparaison de l'epreuve d'immunodiffusion avec celle de la sero-neutralisation, relativement a la recherche d'anticorps specifiques au virus de la gastro-enterite transmissible, dans le serum de 20 porcs, revela que certains de ces echantillons, riches en anticorps neutralisants, ne produisaient pas de precipitation en milieu gelifie, contrairement a d'autres echantillons qui contenaient moins de ces anticorps. L'immunodiffusion permit aussi de deceler des anticorps precipitants dans plusieurs echantillons du lactoserum d'une truie ayant regu un vaccin inactive contre la gastro-enterite transmissible.
of Veterinary Microbiology and Immunology, University of Guelph, Guelph, Ontario NlG 2W1. Dr. Bohac's present address is Animal Pathology Division, Health of Animals Branch, Agriculture Canada, Animal Diseases Research Institute (W), P.O. Box 640, Lethbridge, Alberta T1J 3Z4. This study forms part of a thesis presented by the senior author to the Faculty of Graduate Studies of the Univeraity of Guelph in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Reprint requests should be addressed to the junior author. Submitted June 13, 1975.
*Department
Volume 40
-
April, 1976
INTRODUCTION In a previous publication (1) we described the detection of precipitating antigens in various materials from piglets infected with transmissible gastroenteritis (TGE) virus by means of the immunodif-
161
fusion reaction. The same technique was used subsequently for the detection of TGE viral antibodies in serum and milk samples from swine infected or vaccinated with TGE virus and the results obtained are reported in this paper. The initial experiments were designed to check further the specificity of the immunodiffusion reaction and involved the use of sera from several species of normal animals, antisera prepared against a variety of bacterial and viral agents and sera from swine with bacterial enteritis. Next, a comparison was made between the reaction obtained in the immunodiffusion test and the neutralizing antibody titres of sera from pigs which had been vaccinated or challenged with TGE virus in the field. Finally, precipitating antibodies were demonstrated in serum and milk samples from a sow which had been vaccinated with an inactivated TGE viral vaccine.
MATERIALS AND METHODS IMMUNODIFFUSION TEST ANTIGENS
The antigens used in the immunodiffusion tests were either alkaline intestinal extracts prepared from piglets infected with V-52 or DL strain of TGE virus or the supernatant obtained after precipitation with 60% ammonium sulphate (ASS60) of alkaline intestinal extract prepared from a piglet infected with the 67-1 strain of virus. The preparation of these antigens was detailed in a previous paper (1). Intestinal extract and ASS60 prepared from a normal piglet were used for control purposes. SERA Two positive TGE hyperimmune antisera, designated S-4497 and S-54, were described earlier (1) and preimmunization samples from the same animals were also used. Additional negative control sera were obtained from two normal piglets and normal horse, sheep, calf, rabbit and guinea pig sera were also tested. The antiviral sera which were used were as follows. Sera from swine immunized with the T80 and
162
Ti strains of swine enterovirus were obtained from Dr. J. Thorsen, University of Guelph and a serum sample from a pig immunized with haemagglutinating encephalomyelitis virus (HEV-21) was supplied by Dr. A. S. Greig, Animal Diseases Research Institute, P.O. Box 11300, Station "H", Ottawa, Ontario K2H 8P9. A bovine anti-infectious bovine rhinotracheitis virus serum and a guinea pig antiparainfluenza type 3 virus serum were obtained from Dr. M. Savan, University of Guelph, and a guinea pig anti-rabies serum was obtained from Dr. R. Ramsden, University of Guelph. Antisera prepared in rabbits against Salmonella typhi and S. choleraesuis were supplied by Dr. P. J. Quinn, University of Guelph and sera from swine immunized with two strains of Escherichia coli were obtained from Dr. C. L. Gyles, University of Guelph. Convalescent serum samples from five piglets diagnosed clinically to be suffering from bacterial enteritis by Dr. A. C. Brandenburg, University of Guelph, were also tested. Serum samples were obtained from ten sows and ten piglets, aged between 14 and 16 weeks, in a herd in which a field trial of a TGE vaccine was in progress. The samples were submitted by the Veterinary Services Laboratory, Ridgetown, Ontario. Five of the sows had been vaccinated late in pregnancy with the DL strain of virus and five of the piglets were from litters born to these sows. The remaining sows had not been vaccinated but all had been exposed to field virus. A series of serum samples was supplied by Dr. J. Thorsen, University of Guelph, from four sows which had been vaccinated with either two or three doses of beta propiolactone inactivated strain V-52 virus by either the intramuscular or intramammary route as described by Thorsen and Djurickovic (7). Sera were collected from these sows on the day of farrowing and at intervals of three days up to the fifteenth day of lactation. All the sera were inactivated at 56°C for 30 minutes and stored at -20'C. MILK
Milk samples were obtained from the above four sows which had been vaccinated with inactivated V-52 virus at the same times that the sera were collected. The milk was centrifuged at 15,000 x g for
Can. J. comp. Med.
TABLE I. Immunodiffusion and Virus Neutralization Tests on Swine Sera with the DL Strain of Transmissible Gastroenteritis Virus Source of Sera
Pig No.
IDa Test VNb Titre
Source of Sera
Pig
No.
ID Test
VN Titre
+ -
512 512 512 32 512
+ + -
< 16'
Unvaccinated Sows
1 2 3 4 5
+ + + +
5 8 16 20 20
Vaccinated Sows
11 12 13 14 15
Piglets from unvaccinated sows
6 7 8 9 10
+ + +
128 8 32 < 16c 16
Piglets from vaccinated sows
16 17 18 19 20
4 16 16 16
"Immunodiffusion test: + indicates formation of precipitin line; - no reaction bVirus neutralizing titres expressed as reciprocals of 50% neutralization end points *Sera cytotoxic at high concentration, but titres < 16.
20 minutes to remove fat and then at 40,000 x g for two hours to remove casein. Lipoprotein was precipitated by dextran sulphate and calcium chloride (2). The milk which was obtained was inactivated at 56C for 30 minutes and stored at -20°C.
IMMUNODIFFUSION TESTS
These tests were conducted as described previously (1). The agar gel was made up in tris-HCl buffer and the diluent for the antigens was borate saline solution at pH 9.0. All the sera described above were subjected to an immunodiffusion test. Details of the antigens and of the dilutions of antigen and sera used in particular tests are given under Results. VIRUS NEUTRALIZATION TESTS
Virus neutralization tests were done by the microcolor procedure described by Witte (9) in stable swine testis (SST) cells with the DL strain of virus. Only the 20 field sera obtained from the TGE vaccination trial were tested for virus neutralization, since only in this experiment was a comparison made between the virus neutralizing titre and the immunodiffusion reaction. The SST cell line was obtained from Dr. C. J. Welter, Diamond Laboratories, and the cells were cultivated in Eagle's minimal essential medium supplemented with 5% foetal calf serum.
Volume 40
-
April, 1976
RESU LTS IMMUNODIFFUSION TESTS WITH VARIOUS SERA With the exception of the 20 samples from the DL strain vaccine trial and those obtained from the sows vaccinated with irnactivated TGE virus, all the sera described above were tested by immunodiffusion with V-52 alkaline intestinal extract antigen. Positive reactions in the form of a single precipitin line were obtained with the TGE antisera S-54 and S-4997 which had been used in previous studies (1) and with the HEV antiserum. The preimmunization sera, those from normal animals and from swine with bacterial enteritis and the various bacterial and other viral antisera were all negative. The control intestinal extract prepared from a normal piglet failed to precipitate with any serum. The immunodiffusion reaction between TGE virus and HEV antiserum was confirmed with strain DL alkaline intestinal extract as antigen and the single precipitin line which developed was identical with the reaction between the DL antigen and TGE antiserum S-54. IMMUNODIFFUSION AND VIRUS NEUTRALIZATION TESTS ON SWINE SERA
Immunodiffusion and virus nieutralization tests were performed on the 20 serum
163
samples obtained from sows and piglets in a field investigation of TGE vaccination. DL strain TGE virus was used in both serological tests. In the immunodiffusion reaction, the sera were tested undiluted, and at dilutions of 1:2 and 1:4, against dilutions of alkaline intestinal extract from 1:1 to 1:16, in checker-board fashion, in order to avoid negative reactions due to prozone phenomena. The results obtained are given in Table 1, from which it will be seen that there were marked discrepancies between the results in the two tests. Several sera with high virus neutralizing titres were negative in the immunodiffusion test, while certain sera which were positive in the immunodiffusion test were found to have relatively low virus neutralizing antibody titres. While the virus neutralizing titres of sera from the vaccinated sows were all higher than those from the unvaccinated group, only one of the former sera was positive in the immunodiffusion test. The reactivity of the sera from the two groups of piglets was similar, irrespective of the vaccination status of their dams and there was no clear relationship between virus neutralizing and precipitating antibody content of these sera. The control alkaline intestinal extract prepared from a normal piglet did not reacvt in the immunodiffusion test with any of the 20 sera. IMMUNODIFFUSION TESTS ON MILK SAMPLES
Samples of serum and milk whey from the four sows vaccinated with beta propiolactone (BPL) inactivated TGE virus collected at intervals after farrowing were tested by immunodiffusion against ASS60
prepared from strain 67-1. The ASS60 used at a dilution of 1:4, which contained four precipitating units of antigen, because this dilution was shown in a preliminary checker-board titration with serum S-54 to react with the highest dilutions of serum. It will be seen from Table II that negative results were obtained in tests with the milk samples from those sows which received only two doses of vaccine and with milk from the sow which was given three intramuscular doses. Milk from Sow 4 which received three intramammary doses of vaccine was positive in the immunodiffusion test from the ninth day after farrowing and serum from this sow was positive at every test. Isolated positive results were also obtained on sera from Sows 1 and 2. The control ASS60 prepared from an intestinal extract from a normal piglet did not react with any of the serum or milk samples. was
DISCUSSION In this study, the immunodiffusion test appeared to be specific for the detection of TGE viral antibodies, except for the cross reactivity of the antigen with HEV antiserum, which was obtained with V-52 and DL viruses. An antigenic relationship between TGE virus and HEV was demonstrated previously by immunodiffusion (5) although no details of the reactants were given. Among other members of the coronavirus group, antigenic relationships have been described only between mouse hapatitis virus and certain human coro-
TABLE II. Immunodiffusion Tests on Serum and Milk Whey from Sows Vaccinated with TGE Virus Inactivatedwith Beta Propiolactone
Immunodiffusion Test Reactions' Days after Farrowing 0 3 6 9 12 Sow Route of No. of doses Sb Wc S W S W S W No. Vaccination of vaccine S W 2 1 Intramuscular ++2 Intramam y 2 - +3 3 Intramuscular 3 4 Intramammary + + + + +++a + indicates formation of precipitin line in immunodiffusion test; - no reaction bSrum oMilk whey
164
15 S W +-
+ +
Can. J. comp. Med.
naviruses (3) although animal strains have not been exhaustively studied in this regard. The nature of the relationship between TGE virus and HEV requires further study by means of reciprocal tests with various antisera and strains of the two viruses. We were able to detect precipitating antibodies in milk whey from a sow vaccinated with inactivated TGE virus and in sera from sows and piglets either vaccinated or infected with the virus. The variation in the results obtained in the immunodiffusion and virus neutralization tests on 20 sera from the field suggested that the two tests were detecting different antibodies. Care was taken to conduct the immunodiffusion tests with a wide range of dilutions of serum and antigen in order to avoid false negative reactions due to prozone phenomena. Failure of precipitating antibodies to neutralize infectivity has been reported for several other viruses (4, 6, 8) but the fact that certain sera with high neutralizing antibody titres failed to precipitate with TGE antigen would limit the diagnostic application of the immunodiffusion test and in the light of present knowledge virus neutralization would seem to be preferable to immunodiffusion for the serological diagnosis of TGE. A similar situation exists in relation to the laboratory diagnosis of infectious bronchitis, in which the precipitating antibody response was shown (10) to be relatively transient, so that sera which neutralized the virus were frequently negative in the immunodiffusion test. It would be of interest to study the dynamics of the neutralizing and precipitating antibody responses in TGE and to relate the different antibodies to immunoglobulin class and to protection.
Volwne 40- April, 1976
ACKNOWLEDGMENTS We wish to thank the following for their kindness in supplying us with the samples of serum and milk which made this study possible: Dr. A. C. Brandenburg, Dr. A. S. Greig, Dr. C. L. Gyles, Dr. P. J. Quinn, Dr. R. Ramsden, Dr. M. Savan, Dr. J. Thorsen and the staff of the Veterinary Services Laboratory, Ridgetown, Ontario. The work was supported financially by the National Research Council of Canada, the Ontario Ministry of Agriculture and Food and Diamond Laboratories, Inc.
REFERENCES 1. BOHAC, J. and J. B. DERBYSHIRE. The detection of transmissible gastroenteritis viral antigens by im^ munodiffusion. Can. J. comp. Med. 39: 67-75. 1975. 2. BOURNE, F. J. IgA immunoglobulin from porcine milk. Biochim. biophys. Acta 181: 485487. 1969. 3. BRADBURNE, A. F. Antigenic relationships amongst coronaviruses. Arch. ges. Virusforsch. 31: 352-364. 1970. 4. KLONTZ, G. W., S. E. SVEHAG and J. R. GORHAM. A study by the agar diffusion technique of precipitating antibody directed against bluetongue virus and its relation to homotypic neutralizing antibody. Arch. ges. Virusforsch. 12: 259-268. 1962. 5. PHILLIP, J. I. H., S. F. CARTWRIGHT and A. C. SCOTT. The size and morphology of TGIE and vomiting and wasting disease viruses of pigs. Vet. Rec. 88: 311-312. 1971. 6. SCHMIDT, N. J. and E. H. LENNETTE. Gel double diffusion studies with group B and group A, type 9 coxsackie viruses. I. The technique and reactions obtained with hyperimmune animal sera and human sera. J. Immun. 89: 85-95. 1962. 7. THORSEN, J. and S. DJURICKOVIC. Experimental immunization of sows with inactivated transmissible gastroenteritis (TGE) virus. Can. J. comp. Med. 35: 99-102. 1971. 8. WHITE, G. and G. R. SCOTT. An indirect gel diffusion precipitation test for the detection of rinderpest antibody in convalescent cattle. Res. vet. Sci. 1: 226-229. 1960. 9. WITTE, K. H. Micro-color test for assay of transmissible gastroenteritis virus neutralizing antibodies. Arch. ges. Virusforsch. 33: 171-176. 1971. 10. WITTER, R. L. The diagnosis of infectious bronchitis of chickens by the agar gel precipitin test. Avian Dis. 6: 478492. 1962.
165
ABSTRACT
RASUME
Precipitating antibodies against transmissible gastroenteritis viral antigens were detected by the immunodiffusion test in two transmissible gastroenteritis viral hyperimmune antisera and in antiserum prepared against haemagglutinating encephalomyelitis virus but not in sera from several species of normal animals, in antisera prepared against a variety of other viruses and bacteria or sera from swine with bacterial enteritis. When the immunodiffusion test was compared with the virus neutralization test for the detection of transmissible gastroenteritis viral antibodies in 20 swine sera certain samples which contained high titres of virus neutralizing antibodies failed to produce precipitation while other sera were positive in the immunodiffusion test although their virus neutralizing antibody titres were relatively low. Precipitating antibodies were also detected by immunodiffusion in several samples of milk whey from a sow which had been vaccinated with inactivated transmissible gastroenteritis virus.
Les auteurs ont decele des anticorps precipitant les antigenes viraux de la gastro-enterite transmissible, a l'aide de l'immunodiffusion, dans deux echantillons de serum hyperimmun specifique 'a ce virus, ainsi que dans un immun-serum prepare avec le virus hemagglutinant de l'encephalomyilite porcine; ils obtinrent cependant des resultats negatifs avec le serum d'animaux sains de differentes especes, avec des serums hyperimmuns prepar6s a l'aide d'une variete d'autres virus et bacteries, ainsi qu'avec le serum de porcs atteints d'enterite bacterienne. La comparaison de l'epreuve d'immunodiffusion avec celle de la sero-neutralisation, relativement a la recherche d'anticorps specifiques au virus de la gastro-enterite transmissible, dans le serum de 20 porcs, revela que certains de ces echantillons, riches en anticorps neutralisants, ne produisaient pas de precipitation en milieu gelifie, contrairement a d'autres echantillons qui contenaient moins de ces anticorps. L'immunodiffusion permit aussi de deceler des anticorps precipitants dans plusieurs echantillons du lactoserum d'une truie ayant regu un vaccin inactive contre la gastro-enterite transmissible.
of Veterinary Microbiology and Immunology, University of Guelph, Guelph, Ontario NlG 2W1. Dr. Bohac's present address is Animal Pathology Division, Health of Animals Branch, Agriculture Canada, Animal Diseases Research Institute (W), P.O. Box 640, Lethbridge, Alberta T1J 3Z4. This study forms part of a thesis presented by the senior author to the Faculty of Graduate Studies of the Univeraity of Guelph in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Reprint requests should be addressed to the junior author. Submitted June 13, 1975.
*Department
Volume 40
-
April, 1976
INTRODUCTION In a previous publication (1) we described the detection of precipitating antigens in various materials from piglets infected with transmissible gastroenteritis (TGE) virus by means of the immunodif-
161
fusion reaction. The same technique was used subsequently for the detection of TGE viral antibodies in serum and milk samples from swine infected or vaccinated with TGE virus and the results obtained are reported in this paper. The initial experiments were designed to check further the specificity of the immunodiffusion reaction and involved the use of sera from several species of normal animals, antisera prepared against a variety of bacterial and viral agents and sera from swine with bacterial enteritis. Next, a comparison was made between the reaction obtained in the immunodiffusion test and the neutralizing antibody titres of sera from pigs which had been vaccinated or challenged with TGE virus in the field. Finally, precipitating antibodies were demonstrated in serum and milk samples from a sow which had been vaccinated with an inactivated TGE viral vaccine.
MATERIALS AND METHODS IMMUNODIFFUSION TEST ANTIGENS
The antigens used in the immunodiffusion tests were either alkaline intestinal extracts prepared from piglets infected with V-52 or DL strain of TGE virus or the supernatant obtained after precipitation with 60% ammonium sulphate (ASS60) of alkaline intestinal extract prepared from a piglet infected with the 67-1 strain of virus. The preparation of these antigens was detailed in a previous paper (1). Intestinal extract and ASS60 prepared from a normal piglet were used for control purposes. SERA Two positive TGE hyperimmune antisera, designated S-4497 and S-54, were described earlier (1) and preimmunization samples from the same animals were also used. Additional negative control sera were obtained from two normal piglets and normal horse, sheep, calf, rabbit and guinea pig sera were also tested. The antiviral sera which were used were as follows. Sera from swine immunized with the T80 and
162
Ti strains of swine enterovirus were obtained from Dr. J. Thorsen, University of Guelph and a serum sample from a pig immunized with haemagglutinating encephalomyelitis virus (HEV-21) was supplied by Dr. A. S. Greig, Animal Diseases Research Institute, P.O. Box 11300, Station "H", Ottawa, Ontario K2H 8P9. A bovine anti-infectious bovine rhinotracheitis virus serum and a guinea pig antiparainfluenza type 3 virus serum were obtained from Dr. M. Savan, University of Guelph, and a guinea pig anti-rabies serum was obtained from Dr. R. Ramsden, University of Guelph. Antisera prepared in rabbits against Salmonella typhi and S. choleraesuis were supplied by Dr. P. J. Quinn, University of Guelph and sera from swine immunized with two strains of Escherichia coli were obtained from Dr. C. L. Gyles, University of Guelph. Convalescent serum samples from five piglets diagnosed clinically to be suffering from bacterial enteritis by Dr. A. C. Brandenburg, University of Guelph, were also tested. Serum samples were obtained from ten sows and ten piglets, aged between 14 and 16 weeks, in a herd in which a field trial of a TGE vaccine was in progress. The samples were submitted by the Veterinary Services Laboratory, Ridgetown, Ontario. Five of the sows had been vaccinated late in pregnancy with the DL strain of virus and five of the piglets were from litters born to these sows. The remaining sows had not been vaccinated but all had been exposed to field virus. A series of serum samples was supplied by Dr. J. Thorsen, University of Guelph, from four sows which had been vaccinated with either two or three doses of beta propiolactone inactivated strain V-52 virus by either the intramuscular or intramammary route as described by Thorsen and Djurickovic (7). Sera were collected from these sows on the day of farrowing and at intervals of three days up to the fifteenth day of lactation. All the sera were inactivated at 56°C for 30 minutes and stored at -20'C. MILK
Milk samples were obtained from the above four sows which had been vaccinated with inactivated V-52 virus at the same times that the sera were collected. The milk was centrifuged at 15,000 x g for
Can. J. comp. Med.
TABLE I. Immunodiffusion and Virus Neutralization Tests on Swine Sera with the DL Strain of Transmissible Gastroenteritis Virus Source of Sera
Pig No.
IDa Test VNb Titre
Source of Sera
Pig
No.
ID Test
VN Titre
+ -
512 512 512 32 512
+ + -
< 16'
Unvaccinated Sows
1 2 3 4 5
+ + + +
5 8 16 20 20
Vaccinated Sows
11 12 13 14 15
Piglets from unvaccinated sows
6 7 8 9 10
+ + +
128 8 32 < 16c 16
Piglets from vaccinated sows
16 17 18 19 20
4 16 16 16
"Immunodiffusion test: + indicates formation of precipitin line; - no reaction bVirus neutralizing titres expressed as reciprocals of 50% neutralization end points *Sera cytotoxic at high concentration, but titres < 16.
20 minutes to remove fat and then at 40,000 x g for two hours to remove casein. Lipoprotein was precipitated by dextran sulphate and calcium chloride (2). The milk which was obtained was inactivated at 56C for 30 minutes and stored at -20°C.
IMMUNODIFFUSION TESTS
These tests were conducted as described previously (1). The agar gel was made up in tris-HCl buffer and the diluent for the antigens was borate saline solution at pH 9.0. All the sera described above were subjected to an immunodiffusion test. Details of the antigens and of the dilutions of antigen and sera used in particular tests are given under Results. VIRUS NEUTRALIZATION TESTS
Virus neutralization tests were done by the microcolor procedure described by Witte (9) in stable swine testis (SST) cells with the DL strain of virus. Only the 20 field sera obtained from the TGE vaccination trial were tested for virus neutralization, since only in this experiment was a comparison made between the virus neutralizing titre and the immunodiffusion reaction. The SST cell line was obtained from Dr. C. J. Welter, Diamond Laboratories, and the cells were cultivated in Eagle's minimal essential medium supplemented with 5% foetal calf serum.
Volume 40
-
April, 1976
RESU LTS IMMUNODIFFUSION TESTS WITH VARIOUS SERA With the exception of the 20 samples from the DL strain vaccine trial and those obtained from the sows vaccinated with irnactivated TGE virus, all the sera described above were tested by immunodiffusion with V-52 alkaline intestinal extract antigen. Positive reactions in the form of a single precipitin line were obtained with the TGE antisera S-54 and S-4997 which had been used in previous studies (1) and with the HEV antiserum. The preimmunization sera, those from normal animals and from swine with bacterial enteritis and the various bacterial and other viral antisera were all negative. The control intestinal extract prepared from a normal piglet failed to precipitate with any serum. The immunodiffusion reaction between TGE virus and HEV antiserum was confirmed with strain DL alkaline intestinal extract as antigen and the single precipitin line which developed was identical with the reaction between the DL antigen and TGE antiserum S-54. IMMUNODIFFUSION AND VIRUS NEUTRALIZATION TESTS ON SWINE SERA
Immunodiffusion and virus nieutralization tests were performed on the 20 serum
163
samples obtained from sows and piglets in a field investigation of TGE vaccination. DL strain TGE virus was used in both serological tests. In the immunodiffusion reaction, the sera were tested undiluted, and at dilutions of 1:2 and 1:4, against dilutions of alkaline intestinal extract from 1:1 to 1:16, in checker-board fashion, in order to avoid negative reactions due to prozone phenomena. The results obtained are given in Table 1, from which it will be seen that there were marked discrepancies between the results in the two tests. Several sera with high virus neutralizing titres were negative in the immunodiffusion test, while certain sera which were positive in the immunodiffusion test were found to have relatively low virus neutralizing antibody titres. While the virus neutralizing titres of sera from the vaccinated sows were all higher than those from the unvaccinated group, only one of the former sera was positive in the immunodiffusion test. The reactivity of the sera from the two groups of piglets was similar, irrespective of the vaccination status of their dams and there was no clear relationship between virus neutralizing and precipitating antibody content of these sera. The control alkaline intestinal extract prepared from a normal piglet did not reacvt in the immunodiffusion test with any of the 20 sera. IMMUNODIFFUSION TESTS ON MILK SAMPLES
Samples of serum and milk whey from the four sows vaccinated with beta propiolactone (BPL) inactivated TGE virus collected at intervals after farrowing were tested by immunodiffusion against ASS60
prepared from strain 67-1. The ASS60 used at a dilution of 1:4, which contained four precipitating units of antigen, because this dilution was shown in a preliminary checker-board titration with serum S-54 to react with the highest dilutions of serum. It will be seen from Table II that negative results were obtained in tests with the milk samples from those sows which received only two doses of vaccine and with milk from the sow which was given three intramuscular doses. Milk from Sow 4 which received three intramammary doses of vaccine was positive in the immunodiffusion test from the ninth day after farrowing and serum from this sow was positive at every test. Isolated positive results were also obtained on sera from Sows 1 and 2. The control ASS60 prepared from an intestinal extract from a normal piglet did not react with any of the serum or milk samples. was
DISCUSSION In this study, the immunodiffusion test appeared to be specific for the detection of TGE viral antibodies, except for the cross reactivity of the antigen with HEV antiserum, which was obtained with V-52 and DL viruses. An antigenic relationship between TGE virus and HEV was demonstrated previously by immunodiffusion (5) although no details of the reactants were given. Among other members of the coronavirus group, antigenic relationships have been described only between mouse hapatitis virus and certain human coro-
TABLE II. Immunodiffusion Tests on Serum and Milk Whey from Sows Vaccinated with TGE Virus Inactivatedwith Beta Propiolactone
Immunodiffusion Test Reactions' Days after Farrowing 0 3 6 9 12 Sow Route of No. of doses Sb Wc S W S W S W No. Vaccination of vaccine S W 2 1 Intramuscular ++2 Intramam y 2 - +3 3 Intramuscular 3 4 Intramammary + + + + +++a + indicates formation of precipitin line in immunodiffusion test; - no reaction bSrum oMilk whey
164
15 S W +-
+ +
Can. J. comp. Med.
naviruses (3) although animal strains have not been exhaustively studied in this regard. The nature of the relationship between TGE virus and HEV requires further study by means of reciprocal tests with various antisera and strains of the two viruses. We were able to detect precipitating antibodies in milk whey from a sow vaccinated with inactivated TGE virus and in sera from sows and piglets either vaccinated or infected with the virus. The variation in the results obtained in the immunodiffusion and virus neutralization tests on 20 sera from the field suggested that the two tests were detecting different antibodies. Care was taken to conduct the immunodiffusion tests with a wide range of dilutions of serum and antigen in order to avoid false negative reactions due to prozone phenomena. Failure of precipitating antibodies to neutralize infectivity has been reported for several other viruses (4, 6, 8) but the fact that certain sera with high neutralizing antibody titres failed to precipitate with TGE antigen would limit the diagnostic application of the immunodiffusion test and in the light of present knowledge virus neutralization would seem to be preferable to immunodiffusion for the serological diagnosis of TGE. A similar situation exists in relation to the laboratory diagnosis of infectious bronchitis, in which the precipitating antibody response was shown (10) to be relatively transient, so that sera which neutralized the virus were frequently negative in the immunodiffusion test. It would be of interest to study the dynamics of the neutralizing and precipitating antibody responses in TGE and to relate the different antibodies to immunoglobulin class and to protection.
Volwne 40- April, 1976
ACKNOWLEDGMENTS We wish to thank the following for their kindness in supplying us with the samples of serum and milk which made this study possible: Dr. A. C. Brandenburg, Dr. A. S. Greig, Dr. C. L. Gyles, Dr. P. J. Quinn, Dr. R. Ramsden, Dr. M. Savan, Dr. J. Thorsen and the staff of the Veterinary Services Laboratory, Ridgetown, Ontario. The work was supported financially by the National Research Council of Canada, the Ontario Ministry of Agriculture and Food and Diamond Laboratories, Inc.
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