The Indirect Fluorescent Antibody Technique as a Method for Detecting Antibodies in Aborted Fetuses R. B. Miller and B. N. Wilkie* ABSTRACT
In this investigation the indirect fluorescent antibody technique was used to titrate antibodies in bovine sera to parainfluenza 3, infectious bovine rhinotracheitis virus and bovine viral diarrhea virus. These results were compared to those determined on the same samples by hemagglutination inhibition for parainfluenza 3 virus and serum neutralization for bovine virus diarrhea and infectious bovine rhinotracheitis virus. The results of the serological methods agreed closely. The indirect fluorescent antibody technique is a rapid and sensitive method for detecting antibodies and the procedure lends itself to use in diagnostic laboratories. In addition to the above viruses the presence or absence of antibodies to bovine coronavirus and bovine adenovirus 3 were determined by the indirect fluorescent antibody technique in thoracic fluids from 100 aborted fetuses and 50 nonaborted fetuses. Results on these samples were not compared to hemagglutination inhibition or serum neutralization as the condition of fluid samples from aborted fetuses renders interpretation of such tests unreliable. Antibodies to one or more viruses were detected in 30 of the 100 aborted fetuses and in seven of the 50 nonaborted fetuses. Antibodies to more than one agent were detected in eleven of the 100 aborted and in one of the 50 nonaborted fetuses. Reasons for this occurrence and application of the test in determination of causes of abortion are discussed.
RESUME
Cette experience visait 'a utiliser l'immunofluorescence indirecte pour determiner la te*Department of Pathology (Miller) and Department of Veterinary Microbiology and Immunology (Wilkie), Ontario Veterinary College, University of Guelph, Guelph, Ontario NiG 2W1. Submitted July 3, 1978.
Volume 43 - July, 1979
neur du serum de bovins en anticorps, 'a 1'endroit des virus suivants: para-influenza 3, rhino-tracheite infectieuse et diarrhee 'a virus bovines. On compara ensuite ces resultats 'a ceux qu'on avait dej'a obtenus avec les memes echantillons, en utilisant l'inhibition de l'hemagglutination, pour le virus para-influenza 3, et la seroneutralisation, pour ceux de la rhinotrach6ite infectieuse et de la diarrhee 'a virus bovines. Toutes ces methodes serologiques donnerent des resultats 'a peu pries identiques. L'immunofluorescence indirecte represente une mithode rapide et efficace pour la recherche des anticorps; elle se prete bien 'a un usage dans les laboratoires de diagnostic. On utilisa aussi l'immunofluorescence indirecte pour rechercher la presence d'anticorps 'a T'endroit du coronavirus et de l'adenovirus du type 3 bovins, dans le liquide pleural de 100 avortons et de 50 foetus normaux. On ne compara pas les resultats ainsi obtenus, 'a ceux des epreuves de l'inhibition de l'hemagglutination et de la seroneutralisation, parce que la condition des echantillons fluides rend l'interpretation de ces epreuves erratique. On decela des anticorps ia l'endroit d'un ou de plusieurs virus chez 30 des 100 avortons et chez sept des 50 foetus normaux, ainsi qu'a l'endroit de plus d'un virus chez 11 des avortons et chez un des 50 foetus normaux. On commente les raisons de ces resultats et l'application de l'immunofluorescence indirecte 'a la recherche des causes de l'avortement.
INTRODUCTION The bovine fetus is capable of immune response to several antigens (13) and the detection of fetal antibodies in correlation with other findings may be useful in determining cause of abortion (12). The contribution of fetal serology to understanding the epidemiology of infectious abortion may also be considerable. Viral serology using fetal fluids is frequently complicated by their content of bacteria and hemolysed
255
red blood cells which make the results of hemagglutination inhibition tests difficult to interpret and frequently interfere with cell growth in virus neutralization tests. Nevertheless, a semiquantitative and sensitive qualitative test for specific viral antibody has application in diagnosis and when correlated with specific lesions may help in deciding on a presumptive cause for abortion. In addition, a quantitative determination of total fetal serum immunoglobulins is useful in detecting chronic fetal infections (12). The purpose of this investigation was to adapt the indirect fluorescent antibody technique (IFAT) for detection in fetal fluids of antibodies to parainfluenza-3 virus (PI3), bovine viral diarrhea virus (BVD) and the virus of infectious bovine rhinotracheitis (IBR) and to compare these results to those determined by hemagglutination inhibition (HI) for PI3 and serum neutralization (SN) for BVD and IBR. Fetal fluids were also examined by IFAT for antibodies to bovine adenovirus-3 and bovine coronavirus but these results were not compared with those of other tests.
layer cultures on coverslips in Leighton tubes with one of the following viruses: IBR virus isolated from an aborted bovine fetus3, PI3 virus, tenth passage of a field isolate3, BVD virus, tenth passage of the Oregon C24V strain (6),' bovine adenovirus type III, strain WBRI (4)4 and bovine coronavirus, 35th passage on a fetal bovine kidney cell line culture3. With the IBR, bovine adenovirus and BVD virus as soon as CPE was observed (usually about one to two days with bovine adenovirus and IBR viruses and three to four days with BVD virus) the coverslips were removed, air dried and stored on glass slides in sealed containers at -70°C until use. With coronavirus the coverslips were removed and stored as above after three days. As bovine coronavirus is noncytopathogenic on MDBK cells viral antigen was detected using the direct fluorescent antibody technique. The presence of PI3 viral antigen in cells was determined by hemadsorption with 0.3% bovine red blood cells (7). By this test, virus was usually present on about the third day.
9
FLUORESCEIN ISOTHIOCYANATE CONJUGATED RABBIT ANTI-BOVINE IMMUNOGLOBULIN
MATERIALS AND METHODS Twenty-two heat-inactivated bovine sera with virus neutralizing (VN) antibody to IBR, 22 with VN antibody to BVD and 22 found positive on the HI test for PI3 antibodies were titrated for viral antibody by IFAT. One hundred samples of thoracic fluid from aborted and 50 from nonaborted fetuses, the latter described previously (11), were tested by IFAT to detect antibodies to these viruses and to bovine coronavirus and adenovirus 3. Stable line Madin-Darby' bovine kidney (MDBK) cells previously found to be negative for bovine corona, IBR, BVD and PI3 viruses by the direct fluorescent antibody test2 and to be negative for bovine adenovirus by the IFAT were infected as mono-
iMadin-Darby
bovine kidney cells were originally obtained from the American Type Culture Collection.
2The fluorescein isothiocyanate conjugated goat antisera to IBR virus was obtained from Armand Frappier Ii.stitute, Laval-des-Rapides, Quebec, to BVD and P13 viruses from the Colorado Serum Co. and the coronavirus from Norden Laboratories, 493 Champagne Drive, Downsview, Ontario.
256
Anti-bovine immunoglobulin (Ig) was prepared in rabbits by immunization with immunoelectrophoretically pure Ig precipitated from pooled bovine serum by 1/3 saturation with ammonium sulphate. The resulting anti-Ig, which reacted strongly with the major classes of bovine Ig in immunoelectrophoresis, was conjugated with fluorescein isothiocyanate (FITC) by previously reported methods (14). Unconjugated FITC was removed by gel filtration chromatography and conjugated protein having a molar fluorescein to protein ratio of 2:1 was obtained by elution from DEAE-cellulose in 0.1 M Tris-HCl, pH 8.6 with 0.1 M added sodium chloride. FITCanti-bovine Ig was used at a dilution determined to optimize specific staining using known positive and negative bovine sera. 3Dr. M. Savan, Department of Veterinary Microbiology and Immunology, Ontario Veterinary College, University of Guelph, Guelph, Ontario. 4Dr. Y. Elazhary, Department of Veterinary Microbiology
and Immunology, Ontario Veterinary College, University of Guelph, Guelph, Ontario. 5Dr. C. A. Mebus, Department of Veterinary Science, College of Agriculture, University of Nebraska, Lincoln, Nebraska 68503.
Can. J. comp. Med.
POSITIVE AND NEGATIVE CONTROL SERA
Field bovine sera positive for neutralizing antibodies against BVD, IBR, adeno and corona virus, and for inhibiting antibodies against PI3, were used as positive controls. Field sera negative for such antibodies against the particular viruses were used as negative controls. Serum samples were titrated by making serial twofold dilutions in saline and the IFAT titres compared to those obtained in the other serological tests appropriate for each virus (SN or HI).
serum dilution which still reacted with infected cells and conjugate, emitting specific and bright fluorescence. Control, negative cultures did not fluoresce when treated with the same test serum dilution and con-
jugate. RESULTS
COMPARISON OF SEROLOGICAL METHODS Positive and negative sera were easily distinguished, however, precise end points were difficult to determine with IFAT SLIDE PREPARATION since the presence of background fluorescence with many negative sera made Virus inoculated coverslips stored as de- estimation of true negativity in titrations scribed above were divided into eight to of positive sera somewhat subjective. ten squares using a Model K Manostat The titres as determined by the IFAT Marktex Tech Pen6. Positive and negative are compared to the results of SN in Table control sera were placed in squares on op- I for IBR, in Table II for BVD and composite ends of each slide. Dilutions of un- pared to the results of the HI test for PI3 known test sera were placed in the remain- in Table III. Of 22 samples with IBR antiing squares. Coverslips were incubated in body the results coincided exactly in nine. an open humidity chamber at room tempe- The IFAT result was one dilution above rature for 30 minutes, washed once for 15 SN in four and one dilution below in eight. minutes in fluorescent antibody (FA) buf- The IFAT result was two dilutions below fer7 and then, after application of the in one sample. FITC-anti-bovine Ig were incubated for an Of 22 samples with PI3 antibody the additional 30 minutes. Preparations were titres agreed exactly in 13. The IFAT recounterstained with 0.2% w/v aqueous sult was one dilution above the HI test in Evans blue-albumin applied for 30-60 sec- two and one dilution below in seven. In no onds and the slides were then washed once case was the IFAT result two dilutions or for 15 minutes in distilled water. Evans more below the HI test. blue-albumin was prepared by dissolving Of 22 samples with BVD antibody, the 1.0 g of Evans blue in 100 cc of distilled results agreed exactly in six. The IFAT water and diluting with four parts of a result was one dilution above in four and 1.25% distilled water solution of bovine al- one dilution below in ten. The IFAT result bumin8. Coverslips were mounted on micro- was two dilutions below in two samples. scope slides using a 50:50 preparation of FA buffer (as above) and glycerine. Samples were immediately examined for THE IFAT AS APPLIED TO FETAL FLUIDS fluorescence using a Leitz Orthoplan microscope with an HBO 200 W superpressure The presence of IFAT antibodies in fetal mercury lamp, BG38 and KP690 primary fluids of aborted fetuses is summarized in filters, a K510 barrier filter and a dry dark Table IV. IFAT antibodies to one or more ground condenser (Leitz D 0.80). The slides of the viruses were detected in 30 of the were stored at 5°C for 24 hours and re- 100 aborted fetuses. Thirteen had antiexamined. Titration results by SN or HI bodies to adenovirus, thirteen to bovine corowere unknown by the evaluator at the time navirus, eight to PI3, six to IBR and five of comparison by the IFAT. The IFAT titre to BVD. Nineteen had antibodies to only of the test sera was defined as the highest one agent, of these six reacted with adenovirus, five with coronavirus, three with 6Canadian Laboratory Supplies (Canlab), 80 Jutland BVD, three with PI3 and two with IBR. Eight fetuses had antibodies to two agents. Road, Toronto, Ontario M8Z 2H4. 7F. A. Buffer - Difco Laboratories, Detroit, Michigan. Five of these were positive with both 8Bovine albumin fraction V #10017, ICN Pharmaceucorona and adenovirus. ticals Inc., Life Science Group, 1956 Bourbon St., Montreal, Quebec H4M lVl. Six of the 19 aborted fetuses having anti-
Volume 43 - July, 1979
257
TABLE I. A Comparison of Titres to IBR Virus in 22 Bovine Serum Samples as Determined by Serum Neutralization (SN) and the Indirect Fluorescent Antibody Test (IFAT) Number of Samples per Titre 4 2 2 3 4 2 5
IBR SN Titre 128 64 48 32 16 8 4
Total
IFAT 1 Dilution Above SN 0 0 0 1 1 1 1
IFAT Equals SN 2 2 0 0 2 0 3
IFAT 1 Dilution Below SN 2 0 2 2 1 0 1
IFAT 2 Dilutions Below SN 0 0 0 0 0 1 0
22
TABLE II. Comparison of Titres to BVD Virus in 22 Bovine Serum Samples as Determined by Serum Neutralization (SN) and the Indirect Fluorescent Antibody Test (IFAT)
Number of Samples per Titre 3 2 7 1 2 1 1
BVD SN Titre 2048 1280 640 320 256 160 80 64 32
4 1
Total
IFAT 1 Dilution Above SN 0 0 3 0 0 0 1 0 0
IFAT Equals SN 2 1 1 0 1 1 0 0 0
IFAT 1 Dilution Below 1 1 3 1 1 0 0 2 1
IFAT 2 Dilution Below 0 0 0 0 0 0 0 0 0
22
TABLE III. A Comparison of Antibody Titres to PI3 Virus in 22 Samples as Determined by the Hemagglutination Inhibition (HI) Test and the Indirect Fluorescent Antibody Test (IFAT)
P13
Number of Samples per Titre 1 2 3 15 1
HI Titre
128 64 32 16 4
Total
IFAT 1 Dilution Above HI 0 0 0 1 0
IFAT 1 Dilution Below HI 1 0 1 5 0
IFAT 2 Dilution Below HI 0 0 0 0 0
22
body to a single agent were found to have been aborted for other reasons (Table V). In only one of these 19 aborted fetuses did agent isolation correlate with antibody demonstration (IBR). In seven of the 19, although morphological diagnosis was made, etiology was not definitively determined as the lesions could not be associated with the agent inducing the demonstrated antibody. The results of the IFAT as applied to fluids from the nonaborted fetuses are presented in Table VI. Seven of the 50 nonaborted fetuses had viral-specific antibodies and one of these reacted with both PI3 and 258
IFAT Equals HI 0 2 2 8 1
coronavirus. Four had antibodies to PI3, three to IBR and one to coronavirus. Antibodies to BVD or adenovirus were not demonstrated in the nonaborted fetuses.
DISCUSSION The IFAT is a rapid and sensitive method for detecting antibody. In the present experiments titres determined by IFAT on test sera agreed closely with those derived from the same samples by commonly used
Can. J. comp. Med.
TABLE IV. Reactivity of Fetal Fluids from Aborted Fetuses Against Various Viral Antigens as Demonstrated by IFAT& Fetus 1 2 3 4 5
IBR +
BVD +
PI3
Adeno
+
Corona
+
+ 6 7 + 8 + 9 + 10 11 + 12 13 + 14 + 15 + 16 + + 17 18 + 19 + 20 21 + 22 + + 23 24 25 26 + 27 28 29 30 Totals 30 6 5 8 13 aThirty of 100 fluids tested contained antibody to at least one of the viral antigens
+
+ +
13
TABLE V. A Comparison of Organisms Isolated, Antibody Detected and Morphological Diagnoses in 19 Aborted Bovine Fetusesa
Specificity of
Fetus 8 ..................... 27 ..................... 29 ..................... 30 .....................
Organism Antibody Demonstrated Detected none none none none
BVD virus adenovirus adenovirus adenovirus
Morphological Diagnosis not made not made not made not made
2 ..................... none adenovirus skeletal muscle degeneration 19 ..................... none coronavirus peribronchial cuffing 3 ..................... none partial anencephaly PI3 virus 24 ..................... none coronavirus aspiration of meconium 11 ..................... none PI3 virus hepatic necrosis 13 ..................... none IBR virus interstitial pneumonia 14 ..................... none PI3 virus bronchopneumonia 17 ..................... none adenovirus cardiomyopathy 5 ..................... IBR virus IBR virus focal necrosis in intestine 23 ..................... A. fumigatus adenovirus placentitis 6 ..................... IBR virus coronavirus focal hepatic necrosis 10 ..................... IBR virus BVD virus focal hepatic necrosis 12 ..................... coronavirus interstitial nephritis, Sarcocystis encephalitis 15 ..................... A. fumigatus BVD virus placentitis 28 ..................... Bacillus (sp.) coronavirus placentitis &In these 19 aborted fetuses antibody was demonstrated to only one virus
Volume 43
-
July, 1979
259
TABLE VI. Reactivity of Fetal Fluidsa from Nonaborted Fetuses Against Various Viral Antigens as Demonstrated by IFATa
Fetus 2NA 5NA lONA 13NA 24NA 29NA
4ONA
IBR + + +
BVD
P13 + +
Adeno Corona +
+ +
3 0 4 0 1 ,Seven of 50 fluids tested contained antibody to at least one of the viral antigens
techniques of viral serology. The IFAT results did not appear to have been adversely influenced by hemolysis in fetal fluids. However, the condition of thoracic fluids from aborted fetuses (hemolysed and autolyzed) precludes the use of comparison testing by SN or HI. The results on the aborted fetal samples therefore stand on their own. The IFAT is easy to apply to a wide range of viruses simply by growing the virus on coverslip cultures which may then be stored until needed. This procedure therefore lends itself to use in diagnostic laboratories where elaborate facilities for virology or immunology may not be available. Antibodies to one or more viruses were detected in 30 of the 10 aborted fetuses and in seven of the 50 nonaborted fetuses. Eleven of the 10 aborted and one of the 50 nonaborted fetuses had antibodies to more than one agent. These antibodies may have been produced by the fetus. However, while antibodies do not normally cross the bovine placenta (1) breakdown of normal placental structure in impending abortion conceivably could permit maternal antibody to reach the fetus. This hypothesis has not however been tested. A reported comparison of maternal and fetal anti-BVD antibody did not reveal a significant correlation (6). Maternal antibodies were not examined in the cases presented here. It would be expected that if maternal antibodies passed to the fetus through a compromised placenta antibodies to enzootic agents would predominate in the fetus. In this regard four of the 11 aborted fetuses with more than one antibody did have antibodies to IBR virus, a ubiquitous pathogen. In balance, while it seems likely that fetal antibody detected here was endogenously produced proof of this is presently not available. 260
Fetal infections may occur during gestation without leading to abortion and these infections may be expected to stimulate antibody production (8). Our results indicate however that multiple infections are much more common in fetuses that do abort. The relatively large number of aborted fetuses (11 out of 100) with antibodies to more than one agent versus the small number of nonaborted fetuses (one out of 50) with antibodies to more than one agent supports Dunne's (5) suggestion of a multiple etiology for some abortions. The isolation of one agent and the presence of antibody to another agent in six of 100 aborted fetuses may also indicate multiple fetal infection. Therefore, by these criteria 17 of the 100 abortions had a multiple etiology. The results do not exclude the possibility of other undetected infections in these fetuses. As multiple infections in bovine fetuses appear to be common, the usefulness of fetal antibody determination in the diagnosis of abortion may be limited. When multiple abortions occur in one premise repetition of antibody occurrence in different fetuses may facilitate etiological diagnosis. When (or if) a specific lesion is observed in the aborted fetus, antibody detection may corroborate this evidence strengthening conclusions on cause of lesion and/or abortion. For exemple the presence of fetal antibodies to BVD virus and occurrence of cerebellar hypoplasia in the same bovine fetus is characteristic of in utero bovine viral diarrhea infection under certain circumstances (2, 9). When the lesions caused by an agent are less specific than in the above example, etiological correlation may be inappropriate. Kirkbride et al (10), Sawyer et al (12) and Miller and Quinn (11) suggest that IgG is present more frequently in aborted fetuses than in nonaborted fetuses and since immunoglobulins do not normally cross the bovine placenta, their presence in the fetus suggests an infectious cause of abortion. Kirkbride et al (10) using radial immunodiffusion report that 59.1% of 164 aborted bovine fetuses had levels of IgG, IgM or both greater than 20 mgm/ 100lm while only 5.1% of 59 abattoir fetuses exceeded this level. Detection of specific antibody may contribute to etiological diagnosis of abortion and provide useful information in epidemiological investigation of congenital infections. The IFAT using viral infected monolayers on coverslips apparent-
Can. J. comp. Med.
ly provides a simple and satisfactory serological technique for studies of this type.
ACKNOWLEDGMENTS This study was supported by the Ontario Ministry of Agriculture and Food and by grant number 5528134 from the Alberta Agricultural Research Trust and the Alberta Cattle Commission. The authors gratefully acknowledge the technical assistance of Mary Anne Waring, Bev Dahmer, Val Jaspers-Fayer and Judy Little who prepared the reagents and performed the fluorescent antibody techniques. The cheerful cooperation of the staff of the Alberta Provincial Veterinary Laboratory and especially Dr. G. Klavano is appreciated.
REFERENCES 1. BRAMBELL, F. W. R. The passive immunity of the young mammal. Biol. Rev. 33: 488-531. 1958. 2. BRAUN, R. K., B. I. OSBURN and J. W. KENDRICK. Immunologic response of bovine fetus to
Volume 43- July, 1979
bovine viral diarrhea virus. Am. J. vet. Res. 34: 1127-1132. 1973. 3. BROWN, C. L., E. W. JENNEY and L. R. LEE. Fluorescent antibody procedures for bovine viruses. Proc. 72nd a. Meet. U.S. Livestock Sanit. Ass. 1965. pp. 470-477. 1966. 4. DARBYSHIRE, J. H., P. S. DAWSON, P. H. LAMONT, D. C. OSTLER and H. G. PEREIRA. A new adenovirus serotype of bovine origin. J. comp. Path. 75: 327-330. 1965. 5. DUNNE, H. W., S. M. AJINKA, G. R. BUBASH and L. C. GRIEL, Jr. Parainfluenza-3 and bovine entelroviruses as possible important causative factors in bovine abortion. Am. J. vet. Res. 34: 1121-1126. 1973. 6. GILLESPIE, J. H., J. A. BAKER and K. McENTEE. A cytopathogenic strain of virus diarrhea virus. Cornell Vet. 51: 155-159. 1961. 7. HIERHOLZER, J. C. and M. T. SUGGS. Standairdized viral hemagglutination and hemagglutinationinhibition tests. I. Standardization of erythrocyte suspensions. Appl. Microbiol. 18: 816-823. 1969. 8. HUBBERT, W. T. Biology of bovine fetal infection. Proceedings: XX World Vet. Congress 3: 1939-1941. 1975. 9. KAHRS, R. F., F. W. SCOTT and A. de LAHUNTA. Congenital cerebeller hypoplasia and ocular defects in calves following bovine viral diarrheal-mucosal disease infection in pregnant cattle. J. Am. vet. med. Ass. 156: 1443-1450. 1970. 10. KIRKBRIDE, C. A., D. MARTINOVICH and D. A. WOODHOUSE. Immunoglobulins in aborted bovine fetuses. N.Z. vet. J. 25: 180-187. 1977. 11. MILLER, R. B. and P. J. QUINN. Observations on abortion in cattle: A comparison of pathological, microbiological and immunological findings in abor ted fetuses and fetuses collected at abattoirs. Can. J. comp. Med. 39: 270-290. 1975. 12. SAWYER, M., B. I. OSBURN, H. D. KNIGHT and J. W. KENDRICK. A quantitative serologic assay for diagnosing congenital infections of cattle. Am. J. vet. Res. 34: 1281-1284. 1973. 13. SCHULTZ, R. D., H. W. DUNNE and C. E. HEIST. Ontogeny of the bovine immune response. Infection & Immunity 7: 981-991. 1973. 14. WILKIE, B. N., M. GYGAX and B. PAULI. Immunofluorescent studies of bovine hypersensitivity pneumonitis. Can. J. comp. Med. 38: 475-479. 1974.
261
In this investigation the indirect fluorescent antibody technique was used to titrate antibodies in bovine sera to parainfluenza 3, infectious bovine rhinotracheitis virus and bovine viral diarrhea virus. These results were compared to those determined on the same samples by hemagglutination inhibition for parainfluenza 3 virus and serum neutralization for bovine virus diarrhea and infectious bovine rhinotracheitis virus. The results of the serological methods agreed closely. The indirect fluorescent antibody technique is a rapid and sensitive method for detecting antibodies and the procedure lends itself to use in diagnostic laboratories. In addition to the above viruses the presence or absence of antibodies to bovine coronavirus and bovine adenovirus 3 were determined by the indirect fluorescent antibody technique in thoracic fluids from 100 aborted fetuses and 50 nonaborted fetuses. Results on these samples were not compared to hemagglutination inhibition or serum neutralization as the condition of fluid samples from aborted fetuses renders interpretation of such tests unreliable. Antibodies to one or more viruses were detected in 30 of the 100 aborted fetuses and in seven of the 50 nonaborted fetuses. Antibodies to more than one agent were detected in eleven of the 100 aborted and in one of the 50 nonaborted fetuses. Reasons for this occurrence and application of the test in determination of causes of abortion are discussed.
RESUME
Cette experience visait 'a utiliser l'immunofluorescence indirecte pour determiner la te*Department of Pathology (Miller) and Department of Veterinary Microbiology and Immunology (Wilkie), Ontario Veterinary College, University of Guelph, Guelph, Ontario NiG 2W1. Submitted July 3, 1978.
Volume 43 - July, 1979
neur du serum de bovins en anticorps, 'a 1'endroit des virus suivants: para-influenza 3, rhino-tracheite infectieuse et diarrhee 'a virus bovines. On compara ensuite ces resultats 'a ceux qu'on avait dej'a obtenus avec les memes echantillons, en utilisant l'inhibition de l'hemagglutination, pour le virus para-influenza 3, et la seroneutralisation, pour ceux de la rhinotrach6ite infectieuse et de la diarrhee 'a virus bovines. Toutes ces methodes serologiques donnerent des resultats 'a peu pries identiques. L'immunofluorescence indirecte represente une mithode rapide et efficace pour la recherche des anticorps; elle se prete bien 'a un usage dans les laboratoires de diagnostic. On utilisa aussi l'immunofluorescence indirecte pour rechercher la presence d'anticorps 'a T'endroit du coronavirus et de l'adenovirus du type 3 bovins, dans le liquide pleural de 100 avortons et de 50 foetus normaux. On ne compara pas les resultats ainsi obtenus, 'a ceux des epreuves de l'inhibition de l'hemagglutination et de la seroneutralisation, parce que la condition des echantillons fluides rend l'interpretation de ces epreuves erratique. On decela des anticorps ia l'endroit d'un ou de plusieurs virus chez 30 des 100 avortons et chez sept des 50 foetus normaux, ainsi qu'a l'endroit de plus d'un virus chez 11 des avortons et chez un des 50 foetus normaux. On commente les raisons de ces resultats et l'application de l'immunofluorescence indirecte 'a la recherche des causes de l'avortement.
INTRODUCTION The bovine fetus is capable of immune response to several antigens (13) and the detection of fetal antibodies in correlation with other findings may be useful in determining cause of abortion (12). The contribution of fetal serology to understanding the epidemiology of infectious abortion may also be considerable. Viral serology using fetal fluids is frequently complicated by their content of bacteria and hemolysed
255
red blood cells which make the results of hemagglutination inhibition tests difficult to interpret and frequently interfere with cell growth in virus neutralization tests. Nevertheless, a semiquantitative and sensitive qualitative test for specific viral antibody has application in diagnosis and when correlated with specific lesions may help in deciding on a presumptive cause for abortion. In addition, a quantitative determination of total fetal serum immunoglobulins is useful in detecting chronic fetal infections (12). The purpose of this investigation was to adapt the indirect fluorescent antibody technique (IFAT) for detection in fetal fluids of antibodies to parainfluenza-3 virus (PI3), bovine viral diarrhea virus (BVD) and the virus of infectious bovine rhinotracheitis (IBR) and to compare these results to those determined by hemagglutination inhibition (HI) for PI3 and serum neutralization (SN) for BVD and IBR. Fetal fluids were also examined by IFAT for antibodies to bovine adenovirus-3 and bovine coronavirus but these results were not compared with those of other tests.
layer cultures on coverslips in Leighton tubes with one of the following viruses: IBR virus isolated from an aborted bovine fetus3, PI3 virus, tenth passage of a field isolate3, BVD virus, tenth passage of the Oregon C24V strain (6),' bovine adenovirus type III, strain WBRI (4)4 and bovine coronavirus, 35th passage on a fetal bovine kidney cell line culture3. With the IBR, bovine adenovirus and BVD virus as soon as CPE was observed (usually about one to two days with bovine adenovirus and IBR viruses and three to four days with BVD virus) the coverslips were removed, air dried and stored on glass slides in sealed containers at -70°C until use. With coronavirus the coverslips were removed and stored as above after three days. As bovine coronavirus is noncytopathogenic on MDBK cells viral antigen was detected using the direct fluorescent antibody technique. The presence of PI3 viral antigen in cells was determined by hemadsorption with 0.3% bovine red blood cells (7). By this test, virus was usually present on about the third day.
9
FLUORESCEIN ISOTHIOCYANATE CONJUGATED RABBIT ANTI-BOVINE IMMUNOGLOBULIN
MATERIALS AND METHODS Twenty-two heat-inactivated bovine sera with virus neutralizing (VN) antibody to IBR, 22 with VN antibody to BVD and 22 found positive on the HI test for PI3 antibodies were titrated for viral antibody by IFAT. One hundred samples of thoracic fluid from aborted and 50 from nonaborted fetuses, the latter described previously (11), were tested by IFAT to detect antibodies to these viruses and to bovine coronavirus and adenovirus 3. Stable line Madin-Darby' bovine kidney (MDBK) cells previously found to be negative for bovine corona, IBR, BVD and PI3 viruses by the direct fluorescent antibody test2 and to be negative for bovine adenovirus by the IFAT were infected as mono-
iMadin-Darby
bovine kidney cells were originally obtained from the American Type Culture Collection.
2The fluorescein isothiocyanate conjugated goat antisera to IBR virus was obtained from Armand Frappier Ii.stitute, Laval-des-Rapides, Quebec, to BVD and P13 viruses from the Colorado Serum Co. and the coronavirus from Norden Laboratories, 493 Champagne Drive, Downsview, Ontario.
256
Anti-bovine immunoglobulin (Ig) was prepared in rabbits by immunization with immunoelectrophoretically pure Ig precipitated from pooled bovine serum by 1/3 saturation with ammonium sulphate. The resulting anti-Ig, which reacted strongly with the major classes of bovine Ig in immunoelectrophoresis, was conjugated with fluorescein isothiocyanate (FITC) by previously reported methods (14). Unconjugated FITC was removed by gel filtration chromatography and conjugated protein having a molar fluorescein to protein ratio of 2:1 was obtained by elution from DEAE-cellulose in 0.1 M Tris-HCl, pH 8.6 with 0.1 M added sodium chloride. FITCanti-bovine Ig was used at a dilution determined to optimize specific staining using known positive and negative bovine sera. 3Dr. M. Savan, Department of Veterinary Microbiology and Immunology, Ontario Veterinary College, University of Guelph, Guelph, Ontario. 4Dr. Y. Elazhary, Department of Veterinary Microbiology
and Immunology, Ontario Veterinary College, University of Guelph, Guelph, Ontario. 5Dr. C. A. Mebus, Department of Veterinary Science, College of Agriculture, University of Nebraska, Lincoln, Nebraska 68503.
Can. J. comp. Med.
POSITIVE AND NEGATIVE CONTROL SERA
Field bovine sera positive for neutralizing antibodies against BVD, IBR, adeno and corona virus, and for inhibiting antibodies against PI3, were used as positive controls. Field sera negative for such antibodies against the particular viruses were used as negative controls. Serum samples were titrated by making serial twofold dilutions in saline and the IFAT titres compared to those obtained in the other serological tests appropriate for each virus (SN or HI).
serum dilution which still reacted with infected cells and conjugate, emitting specific and bright fluorescence. Control, negative cultures did not fluoresce when treated with the same test serum dilution and con-
jugate. RESULTS
COMPARISON OF SEROLOGICAL METHODS Positive and negative sera were easily distinguished, however, precise end points were difficult to determine with IFAT SLIDE PREPARATION since the presence of background fluorescence with many negative sera made Virus inoculated coverslips stored as de- estimation of true negativity in titrations scribed above were divided into eight to of positive sera somewhat subjective. ten squares using a Model K Manostat The titres as determined by the IFAT Marktex Tech Pen6. Positive and negative are compared to the results of SN in Table control sera were placed in squares on op- I for IBR, in Table II for BVD and composite ends of each slide. Dilutions of un- pared to the results of the HI test for PI3 known test sera were placed in the remain- in Table III. Of 22 samples with IBR antiing squares. Coverslips were incubated in body the results coincided exactly in nine. an open humidity chamber at room tempe- The IFAT result was one dilution above rature for 30 minutes, washed once for 15 SN in four and one dilution below in eight. minutes in fluorescent antibody (FA) buf- The IFAT result was two dilutions below fer7 and then, after application of the in one sample. FITC-anti-bovine Ig were incubated for an Of 22 samples with PI3 antibody the additional 30 minutes. Preparations were titres agreed exactly in 13. The IFAT recounterstained with 0.2% w/v aqueous sult was one dilution above the HI test in Evans blue-albumin applied for 30-60 sec- two and one dilution below in seven. In no onds and the slides were then washed once case was the IFAT result two dilutions or for 15 minutes in distilled water. Evans more below the HI test. blue-albumin was prepared by dissolving Of 22 samples with BVD antibody, the 1.0 g of Evans blue in 100 cc of distilled results agreed exactly in six. The IFAT water and diluting with four parts of a result was one dilution above in four and 1.25% distilled water solution of bovine al- one dilution below in ten. The IFAT result bumin8. Coverslips were mounted on micro- was two dilutions below in two samples. scope slides using a 50:50 preparation of FA buffer (as above) and glycerine. Samples were immediately examined for THE IFAT AS APPLIED TO FETAL FLUIDS fluorescence using a Leitz Orthoplan microscope with an HBO 200 W superpressure The presence of IFAT antibodies in fetal mercury lamp, BG38 and KP690 primary fluids of aborted fetuses is summarized in filters, a K510 barrier filter and a dry dark Table IV. IFAT antibodies to one or more ground condenser (Leitz D 0.80). The slides of the viruses were detected in 30 of the were stored at 5°C for 24 hours and re- 100 aborted fetuses. Thirteen had antiexamined. Titration results by SN or HI bodies to adenovirus, thirteen to bovine corowere unknown by the evaluator at the time navirus, eight to PI3, six to IBR and five of comparison by the IFAT. The IFAT titre to BVD. Nineteen had antibodies to only of the test sera was defined as the highest one agent, of these six reacted with adenovirus, five with coronavirus, three with 6Canadian Laboratory Supplies (Canlab), 80 Jutland BVD, three with PI3 and two with IBR. Eight fetuses had antibodies to two agents. Road, Toronto, Ontario M8Z 2H4. 7F. A. Buffer - Difco Laboratories, Detroit, Michigan. Five of these were positive with both 8Bovine albumin fraction V #10017, ICN Pharmaceucorona and adenovirus. ticals Inc., Life Science Group, 1956 Bourbon St., Montreal, Quebec H4M lVl. Six of the 19 aborted fetuses having anti-
Volume 43 - July, 1979
257
TABLE I. A Comparison of Titres to IBR Virus in 22 Bovine Serum Samples as Determined by Serum Neutralization (SN) and the Indirect Fluorescent Antibody Test (IFAT) Number of Samples per Titre 4 2 2 3 4 2 5
IBR SN Titre 128 64 48 32 16 8 4
Total
IFAT 1 Dilution Above SN 0 0 0 1 1 1 1
IFAT Equals SN 2 2 0 0 2 0 3
IFAT 1 Dilution Below SN 2 0 2 2 1 0 1
IFAT 2 Dilutions Below SN 0 0 0 0 0 1 0
22
TABLE II. Comparison of Titres to BVD Virus in 22 Bovine Serum Samples as Determined by Serum Neutralization (SN) and the Indirect Fluorescent Antibody Test (IFAT)
Number of Samples per Titre 3 2 7 1 2 1 1
BVD SN Titre 2048 1280 640 320 256 160 80 64 32
4 1
Total
IFAT 1 Dilution Above SN 0 0 3 0 0 0 1 0 0
IFAT Equals SN 2 1 1 0 1 1 0 0 0
IFAT 1 Dilution Below 1 1 3 1 1 0 0 2 1
IFAT 2 Dilution Below 0 0 0 0 0 0 0 0 0
22
TABLE III. A Comparison of Antibody Titres to PI3 Virus in 22 Samples as Determined by the Hemagglutination Inhibition (HI) Test and the Indirect Fluorescent Antibody Test (IFAT)
P13
Number of Samples per Titre 1 2 3 15 1
HI Titre
128 64 32 16 4
Total
IFAT 1 Dilution Above HI 0 0 0 1 0
IFAT 1 Dilution Below HI 1 0 1 5 0
IFAT 2 Dilution Below HI 0 0 0 0 0
22
body to a single agent were found to have been aborted for other reasons (Table V). In only one of these 19 aborted fetuses did agent isolation correlate with antibody demonstration (IBR). In seven of the 19, although morphological diagnosis was made, etiology was not definitively determined as the lesions could not be associated with the agent inducing the demonstrated antibody. The results of the IFAT as applied to fluids from the nonaborted fetuses are presented in Table VI. Seven of the 50 nonaborted fetuses had viral-specific antibodies and one of these reacted with both PI3 and 258
IFAT Equals HI 0 2 2 8 1
coronavirus. Four had antibodies to PI3, three to IBR and one to coronavirus. Antibodies to BVD or adenovirus were not demonstrated in the nonaborted fetuses.
DISCUSSION The IFAT is a rapid and sensitive method for detecting antibody. In the present experiments titres determined by IFAT on test sera agreed closely with those derived from the same samples by commonly used
Can. J. comp. Med.
TABLE IV. Reactivity of Fetal Fluids from Aborted Fetuses Against Various Viral Antigens as Demonstrated by IFAT& Fetus 1 2 3 4 5
IBR +
BVD +
PI3
Adeno
+
Corona
+
+ 6 7 + 8 + 9 + 10 11 + 12 13 + 14 + 15 + 16 + + 17 18 + 19 + 20 21 + 22 + + 23 24 25 26 + 27 28 29 30 Totals 30 6 5 8 13 aThirty of 100 fluids tested contained antibody to at least one of the viral antigens
+
+ +
13
TABLE V. A Comparison of Organisms Isolated, Antibody Detected and Morphological Diagnoses in 19 Aborted Bovine Fetusesa
Specificity of
Fetus 8 ..................... 27 ..................... 29 ..................... 30 .....................
Organism Antibody Demonstrated Detected none none none none
BVD virus adenovirus adenovirus adenovirus
Morphological Diagnosis not made not made not made not made
2 ..................... none adenovirus skeletal muscle degeneration 19 ..................... none coronavirus peribronchial cuffing 3 ..................... none partial anencephaly PI3 virus 24 ..................... none coronavirus aspiration of meconium 11 ..................... none PI3 virus hepatic necrosis 13 ..................... none IBR virus interstitial pneumonia 14 ..................... none PI3 virus bronchopneumonia 17 ..................... none adenovirus cardiomyopathy 5 ..................... IBR virus IBR virus focal necrosis in intestine 23 ..................... A. fumigatus adenovirus placentitis 6 ..................... IBR virus coronavirus focal hepatic necrosis 10 ..................... IBR virus BVD virus focal hepatic necrosis 12 ..................... coronavirus interstitial nephritis, Sarcocystis encephalitis 15 ..................... A. fumigatus BVD virus placentitis 28 ..................... Bacillus (sp.) coronavirus placentitis &In these 19 aborted fetuses antibody was demonstrated to only one virus
Volume 43
-
July, 1979
259
TABLE VI. Reactivity of Fetal Fluidsa from Nonaborted Fetuses Against Various Viral Antigens as Demonstrated by IFATa
Fetus 2NA 5NA lONA 13NA 24NA 29NA
4ONA
IBR + + +
BVD
P13 + +
Adeno Corona +
+ +
3 0 4 0 1 ,Seven of 50 fluids tested contained antibody to at least one of the viral antigens
techniques of viral serology. The IFAT results did not appear to have been adversely influenced by hemolysis in fetal fluids. However, the condition of thoracic fluids from aborted fetuses (hemolysed and autolyzed) precludes the use of comparison testing by SN or HI. The results on the aborted fetal samples therefore stand on their own. The IFAT is easy to apply to a wide range of viruses simply by growing the virus on coverslip cultures which may then be stored until needed. This procedure therefore lends itself to use in diagnostic laboratories where elaborate facilities for virology or immunology may not be available. Antibodies to one or more viruses were detected in 30 of the 10 aborted fetuses and in seven of the 50 nonaborted fetuses. Eleven of the 10 aborted and one of the 50 nonaborted fetuses had antibodies to more than one agent. These antibodies may have been produced by the fetus. However, while antibodies do not normally cross the bovine placenta (1) breakdown of normal placental structure in impending abortion conceivably could permit maternal antibody to reach the fetus. This hypothesis has not however been tested. A reported comparison of maternal and fetal anti-BVD antibody did not reveal a significant correlation (6). Maternal antibodies were not examined in the cases presented here. It would be expected that if maternal antibodies passed to the fetus through a compromised placenta antibodies to enzootic agents would predominate in the fetus. In this regard four of the 11 aborted fetuses with more than one antibody did have antibodies to IBR virus, a ubiquitous pathogen. In balance, while it seems likely that fetal antibody detected here was endogenously produced proof of this is presently not available. 260
Fetal infections may occur during gestation without leading to abortion and these infections may be expected to stimulate antibody production (8). Our results indicate however that multiple infections are much more common in fetuses that do abort. The relatively large number of aborted fetuses (11 out of 100) with antibodies to more than one agent versus the small number of nonaborted fetuses (one out of 50) with antibodies to more than one agent supports Dunne's (5) suggestion of a multiple etiology for some abortions. The isolation of one agent and the presence of antibody to another agent in six of 100 aborted fetuses may also indicate multiple fetal infection. Therefore, by these criteria 17 of the 100 abortions had a multiple etiology. The results do not exclude the possibility of other undetected infections in these fetuses. As multiple infections in bovine fetuses appear to be common, the usefulness of fetal antibody determination in the diagnosis of abortion may be limited. When multiple abortions occur in one premise repetition of antibody occurrence in different fetuses may facilitate etiological diagnosis. When (or if) a specific lesion is observed in the aborted fetus, antibody detection may corroborate this evidence strengthening conclusions on cause of lesion and/or abortion. For exemple the presence of fetal antibodies to BVD virus and occurrence of cerebellar hypoplasia in the same bovine fetus is characteristic of in utero bovine viral diarrhea infection under certain circumstances (2, 9). When the lesions caused by an agent are less specific than in the above example, etiological correlation may be inappropriate. Kirkbride et al (10), Sawyer et al (12) and Miller and Quinn (11) suggest that IgG is present more frequently in aborted fetuses than in nonaborted fetuses and since immunoglobulins do not normally cross the bovine placenta, their presence in the fetus suggests an infectious cause of abortion. Kirkbride et al (10) using radial immunodiffusion report that 59.1% of 164 aborted bovine fetuses had levels of IgG, IgM or both greater than 20 mgm/ 100lm while only 5.1% of 59 abattoir fetuses exceeded this level. Detection of specific antibody may contribute to etiological diagnosis of abortion and provide useful information in epidemiological investigation of congenital infections. The IFAT using viral infected monolayers on coverslips apparent-
Can. J. comp. Med.
ly provides a simple and satisfactory serological technique for studies of this type.
ACKNOWLEDGMENTS This study was supported by the Ontario Ministry of Agriculture and Food and by grant number 5528134 from the Alberta Agricultural Research Trust and the Alberta Cattle Commission. The authors gratefully acknowledge the technical assistance of Mary Anne Waring, Bev Dahmer, Val Jaspers-Fayer and Judy Little who prepared the reagents and performed the fluorescent antibody techniques. The cheerful cooperation of the staff of the Alberta Provincial Veterinary Laboratory and especially Dr. G. Klavano is appreciated.
REFERENCES 1. BRAMBELL, F. W. R. The passive immunity of the young mammal. Biol. Rev. 33: 488-531. 1958. 2. BRAUN, R. K., B. I. OSBURN and J. W. KENDRICK. Immunologic response of bovine fetus to
Volume 43- July, 1979
bovine viral diarrhea virus. Am. J. vet. Res. 34: 1127-1132. 1973. 3. BROWN, C. L., E. W. JENNEY and L. R. LEE. Fluorescent antibody procedures for bovine viruses. Proc. 72nd a. Meet. U.S. Livestock Sanit. Ass. 1965. pp. 470-477. 1966. 4. DARBYSHIRE, J. H., P. S. DAWSON, P. H. LAMONT, D. C. OSTLER and H. G. PEREIRA. A new adenovirus serotype of bovine origin. J. comp. Path. 75: 327-330. 1965. 5. DUNNE, H. W., S. M. AJINKA, G. R. BUBASH and L. C. GRIEL, Jr. Parainfluenza-3 and bovine entelroviruses as possible important causative factors in bovine abortion. Am. J. vet. Res. 34: 1121-1126. 1973. 6. GILLESPIE, J. H., J. A. BAKER and K. McENTEE. A cytopathogenic strain of virus diarrhea virus. Cornell Vet. 51: 155-159. 1961. 7. HIERHOLZER, J. C. and M. T. SUGGS. Standairdized viral hemagglutination and hemagglutinationinhibition tests. I. Standardization of erythrocyte suspensions. Appl. Microbiol. 18: 816-823. 1969. 8. HUBBERT, W. T. Biology of bovine fetal infection. Proceedings: XX World Vet. Congress 3: 1939-1941. 1975. 9. KAHRS, R. F., F. W. SCOTT and A. de LAHUNTA. Congenital cerebeller hypoplasia and ocular defects in calves following bovine viral diarrheal-mucosal disease infection in pregnant cattle. J. Am. vet. med. Ass. 156: 1443-1450. 1970. 10. KIRKBRIDE, C. A., D. MARTINOVICH and D. A. WOODHOUSE. Immunoglobulins in aborted bovine fetuses. N.Z. vet. J. 25: 180-187. 1977. 11. MILLER, R. B. and P. J. QUINN. Observations on abortion in cattle: A comparison of pathological, microbiological and immunological findings in abor ted fetuses and fetuses collected at abattoirs. Can. J. comp. Med. 39: 270-290. 1975. 12. SAWYER, M., B. I. OSBURN, H. D. KNIGHT and J. W. KENDRICK. A quantitative serologic assay for diagnosing congenital infections of cattle. Am. J. vet. Res. 34: 1281-1284. 1973. 13. SCHULTZ, R. D., H. W. DUNNE and C. E. HEIST. Ontogeny of the bovine immune response. Infection & Immunity 7: 981-991. 1973. 14. WILKIE, B. N., M. GYGAX and B. PAULI. Immunofluorescent studies of bovine hypersensitivity pneumonitis. Can. J. comp. Med. 38: 475-479. 1974.
261
