An Indirect Fluorescent Antibody Test for Antibodies to Transmissible Gastroenteritis of Swine D. A. Benfield, E.
0.
Haelterman and T. Burnstein*
ABSTRACT The indirect fluorescent antibody test was modified to provide a rapid technique for the detection, screening and titration of antibodies to transmissible gastroenteritis of pigs. Large numbers of slides containing transntissible gastroenteritis antigen were prepared by planting mixtures of infected and uninfected swine testicular cells onto multiwelled teflon-coated slides. After overnight incubation, about one-half of the cells in each well were infected which provided contrast to aid in detecting specific fluorescence in the presence of varying degrees of background staining. Following fixation, antigen slides were stored at -20°C until used. The indirect fluorescent antibody test was compared to the virus neutralization test in both the screening and titration of swine sera containing transmissible gastroenteritis antibodies. The test was found to be sensitive and reliable and to offer certain advact'Ltges over the virus neutralization test.
et titrer les anticorps specifiques au virus de la gastro-enterite transmissible porcine. On prepara a cette fin plusieurs lames infectees, en versant un melange de cellules testiculaires porcines infectees et saines dans les nombreux petits puits dont etaient pourvues les lames enduites de teflon qu'on utilisa. Apres l'incubation de ces lames, durant une nuit, environ 50% des cellules de chacun des puits etaient infectees; ce phenomene assura un contraste qui aida a identifier la fluorescence specifique, en depit de la presence d'une intensite variable de la fluorescence non specifique. Apres la fixation, on entreposa les lames porteuses d'antigene, a -20°C, jusqu'a leur utilisation. On compara aussi les resultats de l'epreuve indirecte d'immunofluorescence a ceux de l'epreuve de la neutralisation du virus, relativement au filtrage et a titrage du serum de porcs porteur d'anticorps specifiques au virus de la iastro-enterite transmissible. La premiere epreuve se revela sensible et fiable; elle pre sentait en plus certains avantages sur la seconde.
RESUMA Cette experience visait a modifier l'epreuve indirecte d'immunofluorescence, dans le but d'obtenir une methode rapide de deceler, filtrer
*Department of Veterinary Microbiology, Pathology and Public Health, School of Veterinary Medicine, Purdue University, West Lafayette, Indiana 47907. Present address of D. Benfield: Department of Microbiology, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, Missouri 65201. Supported in part by U.S. Department of Agriculture Cooperative Agreement No. 12-14-3001-274. Submitted as Journal Paper No. 6857 of the Purdue University Agricultural Experiment Station. Submitted September 12, 1977.
478
INTRODUCTION Serological tests for transmissib. gastroenteritis (TGE) are needed for surveys to determine the prevalence of infection and to aid in diagnosis of the disease. Where baby pigs are affected, a rapid and accurate diagnosis can be m.ade on the basis of the clinical picture, thxe lesions present and by the. use of dire, t immunofluorescence on gut samples (4, 6), When only older animals are involved, clinical signs are equivocal and serological testing may be necessary. At the present
Can. J. comp. Med.
time, neutralization tests (1, 2, 3) are commonly used. Although reliable and simple in principle, they require several days for completion and, with some serum samples, there are problems of toxicity and sterility. For example, we found that 527 of 1517 swine sera originally submitted for brucellosis testing were toxic for cell cultures when used at screening dilutions of 1:8 or 1:16 in a virus neutralization test. This report describes the adaptation of an indirect fluorescent antibody test for serology of TGE and some factors influencing the sensitivity and specificity of the reaction. The results are compared to the sensitivity, specificity and reliability of the neutralization test.
MATERIALS AND METHODS SERA
Sera from several sources were used: a) experimental pigs with known history from a TGE carrier state study, b) swine from two, commercial herds and c) sera submitted to the Purdue Animal Disease Diagnostic Laboratory for brucellosis accreditation. VIRUS
The Purdue strain of TGE virus in the 113th cell culture passage was used in both the indirect fluorescent antibody test (IFAT) and virus neutralization tests (VNT). Virus pools were prepared in a swine testicle (ST) cell line developed by McClurkin (5) and had infectivity titers of 105 to 106.2 TCID50/ml. FLUORESCENT CONJUGATE
A commercial antiporcine IgG prepared in rabbits and conjugated with fluorescin isothiocyanatel was used. Its staining titer
'Miles Laboratories, Inc.,
Volume 42
-
Kankakee, Illinois.
October, 1978
was determined to be 1:80 and a working dilution of 1:20 or 1:30 was used. NEUTRALIZATION TESTS A constant virus-varying serum dilution method was used. Sera were inactivated at 56°C for 30 minutes and diluted in 96well microtiter plates2 in Eagle's minimum essential medium (MEM). Two types of tests were conducted. Some groups of serums were screened at dilutions of 1:8 and 1:16 while others were titrated, using twofold or fourfold dilutions ranging from 1:2 to 1:1280. Volumes of a virus suspension containing approximately 100 TCID50 were added to wells containing equal amounts of diluted serum and the mixtures were allowed to react for one hour at room temperature. Approximately 1.2 x 104 ST cells were then added to each well. Serum controls, known positive and negative sera, and virus and cell controls were included in each test series. Endpoints were based on cytopathic effects determined after five to six days incubation at 370C in an atmosphere of 5% CO2 in air. PREPARATION OF ANTIGEN SLIDES
Antigen slides having mixtures of infected and uninfected cells to provide selfcontained controls were prepared as follows. Teflon-coated slides with twelve 6 mm wells were placed on rubber mesh mats in Petri dishes and autoclaved. Each Petri dish held five slides and served as a moist chamber during incubation. Swine testicle (ST) cells were grown in eight ounce prescription bottles. After five to six days of growth, one half of the cultures were inoculated with TGE virus using an input multiplicity of about one TCID50 of virus per cell. Unadsorbed virus was removed after one hour at room temperature. Cells from both infected and uninfected cultures warp then dispersed with trypsin, susnended in MEM containing 10% fetal bovine serurand pooled to produce a mixture of about 1:1 of cells from infected and uninfected cultures. A viable cell count was made using trypan-blue dye exclusion and the cell sus2Linbro Chemical Co., Inc., New Haven, Connecticut. 3Shandon Southern Instruments, Inc., Sweickly, Pennsylvania.
479
pension was adjusted to about 1.7 x 105 cells/ml. A volume of 0.05 ml of this suspension of the cell mixture was seeded onto each well of the multiwell slides which were then incubated in 5% C02 in air for 16 to 18 hours. After incubation the slides were then rinsed twice with phosphate buffered saline (PBS) and once in distilled water to remove the salt and fixed in acetone for ten minutes at room temperature. The slides were dried and if not used immediately, stored at -20°C.
contrast since they did not fluoresce or had low levels of background staining, which varied with different samples of serum and the dilutions at which they were used. It can also be observed in Fig. 1 that the cell sheets were not confluent. A light cell sheet was used since it was found that confluent or heavy cell sheets appeared to increase nonspecific retention of serum and conjugate. INTERPRETATION OF IFAT
STAINING AND EXAMINATION OF ANTIGEN SLIDES
Early trials indicated that there were variations in the intensity of the staining reactions and that the subjectivity of inDiluted sera were placed on the wells of terpretation might be a source of error. the antigen slides in 0.05 ml amounts and Two experiments were therefore carried incubated for 30 minutes at 37°C in a out to compare individual interpretations humidified incubator. The slides were then of the same tests. In the- first, 20 sera rinsed for 30 min in two changes of PBS (seven negative and 13 positive) from (pH 7.2), once in distilled water and dried. swine used in carrier state experiments and Each well was covered with the rabbit tested with the VNT were coded and antiporcine IgG conjugate and the slides diluted in twofold increments from 1:2 to were again incubated for 30 min and rinsed. 1:32, resulting, in effect, in 100 samples. Coverslips were then mounted onto the The completed IFAT tests were coded, ranstained slides with buffered glycerol (2 domized and read by four readers as either parts glycerol: 1 part PBS). They were positive or negative. None of the readers examined with a Leitz-Orthoplan micro- interpreted any of the known positive scope equipped with a dark field condenser, samples as negative but some of the known 'high pressure mercury vapor lamp, a negative samples were interpreted as posiKP490 excitation and K 530 barrier filter. tive by each of the readers as is shown in Known, positive and negtive control sera Table I. The overall correlation with the were included in each series of tests. Anti- VN test and known history of the samples body titers were expressed as the highest was 96.25%. A second group of 50 sera, collected from dilution of serum which had a fading but definitive fluorescence. If slides could not experimental pigs at various times between be read immediately they were stored at one week and 16 months after exposure to TGE virus, were tested at only the 1:4 -200C. dilution, coded and randomized, and read by the same four readers. In this trial there was 100 % correlation between the VN test, known hist-ory and the IFAT.
RESULTS COMPARISON OF IFAT WITH VNT STAINING PATTERN OF THE ANTIGEN SLIDES By using the procedure of mixing equal quantities of infected and uninfected cells, specific immunofluorescent staining was obtained in approximately 50% of the cells in each well as shown in Fig. 1. This staining pattern was observed consistently in virtually every microscopic field examined and specific fluorescence was limited to the cytoplasm. Uninfected cells provided
480
A comparison of antibody titers as detected by the VNT and IFAT was made using a group of 150 serum samples collected at various times during a carrier state experiment. The sera were diluted in fourfold increments for the IFAT in an attempt to increase the sharpness of endpoints. Twofold dilutions, starting at 1:8 were used in the VN test. The results are shown in Table II. All but one of 44 samples with IFAT titers of less than 1:4
Can. J. comp. Med.
Fig. 1. Immunofluorescent preparation showing transmissible gastroenteritis antigen in swine testicular cells. X125.
TABLE I. Correlation of the IFAT with Known History and VNT as Evaluated by Four Readers" No. False Positivesb at Serum Dilutions
% Correlation with Known History and 1/32 Neutralization Test 1/4 1/8 1/16 Reader No. 1/2 1 ................ 0 1 0 0 0 99 2 ................ 4 3 0 0 0 93 2 2 3 ................ 0 0 0 96 4 ................ 1 1 1 0 0 97 aTwenty sera, 13 positive and seven negatives, were diluted to produce 100 samples; coded, randomized and read by four persons bNone of the positive samples was read as negative
were negative at 1:8 in the VNT. The titers obtained with the IFAT were generally higher: 32 of the 105 positive samples had the same titer as with the VNT, 52 had higher and 22 had lower titers compared to the VNT.
SCREENING FIELD SAMPLES
Serum samples submitted for brucellosis evaluation were tested at dilutions of 1:8
Volume 42
-
October, 1978
and 1:16 with the neutralization test and subsequently tested at a dilution of 1:8 with the IFAT. Of 183 samples, 90 (49%) were positive with the neutralization test, 22 (12%) were negative and the remaining 71 (39%) were toxic. The results of the IFAT agreed completely with those of the VNT with respect to the positive and negative samples. Of the 71 samples found toxic in the neutralization test, 42 were positive with the IFAT and 29 were negative, indicating that whatever caused
481
TABLE II. Comparison of TGE Antibody Titers of 150 Sera Tested With the IFAT and VNT
4
0.
Haelterman and T. Burnstein*
ABSTRACT The indirect fluorescent antibody test was modified to provide a rapid technique for the detection, screening and titration of antibodies to transmissible gastroenteritis of pigs. Large numbers of slides containing transntissible gastroenteritis antigen were prepared by planting mixtures of infected and uninfected swine testicular cells onto multiwelled teflon-coated slides. After overnight incubation, about one-half of the cells in each well were infected which provided contrast to aid in detecting specific fluorescence in the presence of varying degrees of background staining. Following fixation, antigen slides were stored at -20°C until used. The indirect fluorescent antibody test was compared to the virus neutralization test in both the screening and titration of swine sera containing transmissible gastroenteritis antibodies. The test was found to be sensitive and reliable and to offer certain advact'Ltges over the virus neutralization test.
et titrer les anticorps specifiques au virus de la gastro-enterite transmissible porcine. On prepara a cette fin plusieurs lames infectees, en versant un melange de cellules testiculaires porcines infectees et saines dans les nombreux petits puits dont etaient pourvues les lames enduites de teflon qu'on utilisa. Apres l'incubation de ces lames, durant une nuit, environ 50% des cellules de chacun des puits etaient infectees; ce phenomene assura un contraste qui aida a identifier la fluorescence specifique, en depit de la presence d'une intensite variable de la fluorescence non specifique. Apres la fixation, on entreposa les lames porteuses d'antigene, a -20°C, jusqu'a leur utilisation. On compara aussi les resultats de l'epreuve indirecte d'immunofluorescence a ceux de l'epreuve de la neutralisation du virus, relativement au filtrage et a titrage du serum de porcs porteur d'anticorps specifiques au virus de la iastro-enterite transmissible. La premiere epreuve se revela sensible et fiable; elle pre sentait en plus certains avantages sur la seconde.
RESUMA Cette experience visait a modifier l'epreuve indirecte d'immunofluorescence, dans le but d'obtenir une methode rapide de deceler, filtrer
*Department of Veterinary Microbiology, Pathology and Public Health, School of Veterinary Medicine, Purdue University, West Lafayette, Indiana 47907. Present address of D. Benfield: Department of Microbiology, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, Missouri 65201. Supported in part by U.S. Department of Agriculture Cooperative Agreement No. 12-14-3001-274. Submitted as Journal Paper No. 6857 of the Purdue University Agricultural Experiment Station. Submitted September 12, 1977.
478
INTRODUCTION Serological tests for transmissib. gastroenteritis (TGE) are needed for surveys to determine the prevalence of infection and to aid in diagnosis of the disease. Where baby pigs are affected, a rapid and accurate diagnosis can be m.ade on the basis of the clinical picture, thxe lesions present and by the. use of dire, t immunofluorescence on gut samples (4, 6), When only older animals are involved, clinical signs are equivocal and serological testing may be necessary. At the present
Can. J. comp. Med.
time, neutralization tests (1, 2, 3) are commonly used. Although reliable and simple in principle, they require several days for completion and, with some serum samples, there are problems of toxicity and sterility. For example, we found that 527 of 1517 swine sera originally submitted for brucellosis testing were toxic for cell cultures when used at screening dilutions of 1:8 or 1:16 in a virus neutralization test. This report describes the adaptation of an indirect fluorescent antibody test for serology of TGE and some factors influencing the sensitivity and specificity of the reaction. The results are compared to the sensitivity, specificity and reliability of the neutralization test.
MATERIALS AND METHODS SERA
Sera from several sources were used: a) experimental pigs with known history from a TGE carrier state study, b) swine from two, commercial herds and c) sera submitted to the Purdue Animal Disease Diagnostic Laboratory for brucellosis accreditation. VIRUS
The Purdue strain of TGE virus in the 113th cell culture passage was used in both the indirect fluorescent antibody test (IFAT) and virus neutralization tests (VNT). Virus pools were prepared in a swine testicle (ST) cell line developed by McClurkin (5) and had infectivity titers of 105 to 106.2 TCID50/ml. FLUORESCENT CONJUGATE
A commercial antiporcine IgG prepared in rabbits and conjugated with fluorescin isothiocyanatel was used. Its staining titer
'Miles Laboratories, Inc.,
Volume 42
-
Kankakee, Illinois.
October, 1978
was determined to be 1:80 and a working dilution of 1:20 or 1:30 was used. NEUTRALIZATION TESTS A constant virus-varying serum dilution method was used. Sera were inactivated at 56°C for 30 minutes and diluted in 96well microtiter plates2 in Eagle's minimum essential medium (MEM). Two types of tests were conducted. Some groups of serums were screened at dilutions of 1:8 and 1:16 while others were titrated, using twofold or fourfold dilutions ranging from 1:2 to 1:1280. Volumes of a virus suspension containing approximately 100 TCID50 were added to wells containing equal amounts of diluted serum and the mixtures were allowed to react for one hour at room temperature. Approximately 1.2 x 104 ST cells were then added to each well. Serum controls, known positive and negative sera, and virus and cell controls were included in each test series. Endpoints were based on cytopathic effects determined after five to six days incubation at 370C in an atmosphere of 5% CO2 in air. PREPARATION OF ANTIGEN SLIDES
Antigen slides having mixtures of infected and uninfected cells to provide selfcontained controls were prepared as follows. Teflon-coated slides with twelve 6 mm wells were placed on rubber mesh mats in Petri dishes and autoclaved. Each Petri dish held five slides and served as a moist chamber during incubation. Swine testicle (ST) cells were grown in eight ounce prescription bottles. After five to six days of growth, one half of the cultures were inoculated with TGE virus using an input multiplicity of about one TCID50 of virus per cell. Unadsorbed virus was removed after one hour at room temperature. Cells from both infected and uninfected cultures warp then dispersed with trypsin, susnended in MEM containing 10% fetal bovine serurand pooled to produce a mixture of about 1:1 of cells from infected and uninfected cultures. A viable cell count was made using trypan-blue dye exclusion and the cell sus2Linbro Chemical Co., Inc., New Haven, Connecticut. 3Shandon Southern Instruments, Inc., Sweickly, Pennsylvania.
479
pension was adjusted to about 1.7 x 105 cells/ml. A volume of 0.05 ml of this suspension of the cell mixture was seeded onto each well of the multiwell slides which were then incubated in 5% C02 in air for 16 to 18 hours. After incubation the slides were then rinsed twice with phosphate buffered saline (PBS) and once in distilled water to remove the salt and fixed in acetone for ten minutes at room temperature. The slides were dried and if not used immediately, stored at -20°C.
contrast since they did not fluoresce or had low levels of background staining, which varied with different samples of serum and the dilutions at which they were used. It can also be observed in Fig. 1 that the cell sheets were not confluent. A light cell sheet was used since it was found that confluent or heavy cell sheets appeared to increase nonspecific retention of serum and conjugate. INTERPRETATION OF IFAT
STAINING AND EXAMINATION OF ANTIGEN SLIDES
Early trials indicated that there were variations in the intensity of the staining reactions and that the subjectivity of inDiluted sera were placed on the wells of terpretation might be a source of error. the antigen slides in 0.05 ml amounts and Two experiments were therefore carried incubated for 30 minutes at 37°C in a out to compare individual interpretations humidified incubator. The slides were then of the same tests. In the- first, 20 sera rinsed for 30 min in two changes of PBS (seven negative and 13 positive) from (pH 7.2), once in distilled water and dried. swine used in carrier state experiments and Each well was covered with the rabbit tested with the VNT were coded and antiporcine IgG conjugate and the slides diluted in twofold increments from 1:2 to were again incubated for 30 min and rinsed. 1:32, resulting, in effect, in 100 samples. Coverslips were then mounted onto the The completed IFAT tests were coded, ranstained slides with buffered glycerol (2 domized and read by four readers as either parts glycerol: 1 part PBS). They were positive or negative. None of the readers examined with a Leitz-Orthoplan micro- interpreted any of the known positive scope equipped with a dark field condenser, samples as negative but some of the known 'high pressure mercury vapor lamp, a negative samples were interpreted as posiKP490 excitation and K 530 barrier filter. tive by each of the readers as is shown in Known, positive and negtive control sera Table I. The overall correlation with the were included in each series of tests. Anti- VN test and known history of the samples body titers were expressed as the highest was 96.25%. A second group of 50 sera, collected from dilution of serum which had a fading but definitive fluorescence. If slides could not experimental pigs at various times between be read immediately they were stored at one week and 16 months after exposure to TGE virus, were tested at only the 1:4 -200C. dilution, coded and randomized, and read by the same four readers. In this trial there was 100 % correlation between the VN test, known hist-ory and the IFAT.
RESULTS COMPARISON OF IFAT WITH VNT STAINING PATTERN OF THE ANTIGEN SLIDES By using the procedure of mixing equal quantities of infected and uninfected cells, specific immunofluorescent staining was obtained in approximately 50% of the cells in each well as shown in Fig. 1. This staining pattern was observed consistently in virtually every microscopic field examined and specific fluorescence was limited to the cytoplasm. Uninfected cells provided
480
A comparison of antibody titers as detected by the VNT and IFAT was made using a group of 150 serum samples collected at various times during a carrier state experiment. The sera were diluted in fourfold increments for the IFAT in an attempt to increase the sharpness of endpoints. Twofold dilutions, starting at 1:8 were used in the VN test. The results are shown in Table II. All but one of 44 samples with IFAT titers of less than 1:4
Can. J. comp. Med.
Fig. 1. Immunofluorescent preparation showing transmissible gastroenteritis antigen in swine testicular cells. X125.
TABLE I. Correlation of the IFAT with Known History and VNT as Evaluated by Four Readers" No. False Positivesb at Serum Dilutions
% Correlation with Known History and 1/32 Neutralization Test 1/4 1/8 1/16 Reader No. 1/2 1 ................ 0 1 0 0 0 99 2 ................ 4 3 0 0 0 93 2 2 3 ................ 0 0 0 96 4 ................ 1 1 1 0 0 97 aTwenty sera, 13 positive and seven negatives, were diluted to produce 100 samples; coded, randomized and read by four persons bNone of the positive samples was read as negative
were negative at 1:8 in the VNT. The titers obtained with the IFAT were generally higher: 32 of the 105 positive samples had the same titer as with the VNT, 52 had higher and 22 had lower titers compared to the VNT.
SCREENING FIELD SAMPLES
Serum samples submitted for brucellosis evaluation were tested at dilutions of 1:8
Volume 42
-
October, 1978
and 1:16 with the neutralization test and subsequently tested at a dilution of 1:8 with the IFAT. Of 183 samples, 90 (49%) were positive with the neutralization test, 22 (12%) were negative and the remaining 71 (39%) were toxic. The results of the IFAT agreed completely with those of the VNT with respect to the positive and negative samples. Of the 71 samples found toxic in the neutralization test, 42 were positive with the IFAT and 29 were negative, indicating that whatever caused
481
TABLE II. Comparison of TGE Antibody Titers of 150 Sera Tested With the IFAT and VNT
4
