Comp. Imraun. Microbiol. infect. Dis. Vol. 15, No. 3, pp. 213-219, 1992 Printed in Great Britain
HOG
CHOLERA
DIAGNOSTIC
0147-9571/92 $5.00 + 0.00 Pergamon Press Ltd
TECHNIQUES
J. E. PEARSON USDA, APHIS, S&T, National Veterinary Services Laboratories, Diagnostic Virology Laboratory, Ames, Iowa, U.S.A. A~traet~Clinical signs and lesions can sometimes provide the basis for a presumptive diagnosis of hog cholera (HC). However, an accurate diagnosis requires laboratory testing. The usual procedure for the detection of viral antigen is the examination of cryostat sections stained with fluorescein-conjugated HC antiserum. A more definitive technique is isolation of the virus in PK-15 cell cultures and identification of the viral antigen in cells using an HC fluorescent antibody conjugate. As bovine viral diarrhea (BVD) virus will cross-react with HC virus, isolation must be confirmed by the comparison of BVD and HC staining or, preferably, by the use of monoclonal antibodies that can differentiate between HC and BVD viruses. Hog cholera surveillance must rely on serology. The fluorescent antibody virus neutralization (FAVN) test is the classical technique, and HC and BVD antibody can usually be differentiated if HC-positive serum samples are tested against both viruses. Recently the enzyme-linked immunosorbent assay (ELISA) and peroxidase-labeled antibody tests have become the commonly used techniques. Key words: Hog cholera, diagnosis, fluorescent antibody, virus neutralization, tissue section, enzyme-linked immunosorbent assay, peroxidase-linked assay, virus isolation, bovine viral diarrhea.
TECHNIQUES DE DIAGNOSTIC DU CHOLI~RA DU PORC R6sum6--Signes cliniques et 16sions peuvent quelquefois fournir la base pour un diagnostic probable de chol6ra du porc (CP). Cependent, un diagnostic pr6cis doit 6tre bas6 sur des tests de laboratoires. La proc6dure normale pour la d6tection d'un antig6ne viral est bas6e sur l'examen de sections de cryostat tach6es d'antiserum CP conjugu6 de fluorescine. Une technique plus complete consiste en l'isolation du virus dans des cultures de cellules PK-15, et en l'identification de l'antig~ne viral dans les cellules utilisant un anticorp conjug~ fluorescent CP. A mesure que le virus de la diarrh6e virale bovine (DVB) r6agit avec le virus CP, l'isolation doit &re confirm6e par la comparaison des taches DVB et CP ou, de pr6f6rence, par l'utilisation d'anticorps monoclonaux grace auxquels on peut diff~rencier entre les virus DVB et CP. La surveillance du chol6ra du porc doit se faire sur la base de la s6rologie. La technique classique utilise le test de neutralisation de l'anticorp fluorescent du virus (NAFV). De plus, les anticorps CP et DVB peuvent 6tre g6n6ralement distingu6s si les 6chantillons de s6rum CP-positif sont compar6s aux deux virus. R6cemment l'analyse immunosobante li6e aux enzymes (AILE) et les tests d'anticorps marqu6s de p+roxidase sont devenus les techniques les plus couramment utilis6es. Mots-clefs: Chol6ra du porc, diagnostic, anticorp fluorescent, neutralisation de virus, section de tissu, analyse immunosorbante li6e aux enzymes, analyse li6e aux p6roxidases, isolation de virus, diarrh6e virale bovine.
INTRODUCTION
Historically, hog cholera (HC) (classical swine fever) was diagnosed on the basis of clinical signs and lesions. The variability of the disease syndrome made these methods unreliable. The current methods rely on detection of antigen in cryostat tissue sections using the direct immunofluorescent test, isolation of the virus in cell culture, or pig inoculation and serological techniques which include the fluorescent antibody virus neutralization (FAVN) 213
214
J.E. PE~,~,sor,r
test, enzyme-linked immunosorbent assay (ELISA), and the peroxidase-linked assay (PLA). There have been previous reviews of these procedures [1, 2]. In recent years, improved methods have been developed using monoclonal antibodies to differentiate between HC and bovine viral diarrhea (BVD) virus infections. There have also been reports on the development of molecular techniques that will enhance HC diagnosis. The threat of introducing persistently infected, clinically normal pigs or infected meat scraps has increased the importance of laboratory diagnosis. C L I N I C A L S I G N S A N D POST M O R T E M L E S I O N S
A presumptive diagnosis of the acute and peracute forms of the disease based on signs and lesions are described in detail elsewhere [3, 4]. The disease spreads rapidly among pigs of all ages; the affected pigs are depressed, anorexic, and develop a fever of 42°C or higher, and have leukopenia, conjunctivitis, and constipation, that may be followed by diarrhea. The pigs will huddle together as if chilled and may vomit. Convulsions may occur, which are usually followed by death. The pigs that survive longer will become gaunt and develop a staggering gait. The latter condition is related to weakness in the hindquarters that progresses into posterior paralysis. In the terminal stages of the disease, a purplish discoloration is observed on the abdomen, snout, ears, and medial sides of the legs. Pigs with the acute form of HC die between 10 and 20 days postinfection (DPI). Lesions are often absent in the peracute form of HC. In the acute form, infarction of the spleen is considered almost pathognomonic. The lymph nodes are swollen and hemorrhagic. Petechial and ecchymotic hemorrhages are seen in the cortex of the kidney and less often in the medullary pyramids and hilus. They are also often observed in the urinary bladder, larynx, epiglottis, heart, serous and mucous membranes, and skin. A catarrhal to necrotic enteritis and infarcts in the lungs may be seen. Microscopic examination of the brain often reveals an encephalitis and perivascular cuffing. There are no pathognomic microscopic lesions. In the chronic or subacute forms, which are caused by less virulent HC strains, the clinical signs may be similar to those seen in the acute form but are much less pronounced. The signs may be too mild to be detected. Pigs infected in u t e r o may have a lifelong high level of viremia with few, or no, clinical signs. Some pigs may have intermittent diarrhea, anorexia, and retarded growth, and the mortality may be above normal. In u t e r o infection with HC may cause losses of piglets, abortions, stillbirths, and fetal anomalies. This variability in clinical signs requires that any suspicious case be confirmed by laboratory investigations. Diseases such as African swine fever and salmonellosis may be confused with HC. Also, BVD can produce similar clinical signs following in u t e r o infection [5]. DETECTION OF HC VIRUS ANTIGEN The fluorescent antibody tissue section technique (FATST) has become the classical method for detection of hog cholera antigen. The tissue of choice is the tonsil, as this is the first to become infected [6], but 4 cm 2 pieces of spleen, kidney, mandibular lymph node, and distal portion of the ileum should also be submitted. The ileum should be examined as it frequently contains viral antigen in the subacute and chronic forms of the disease and may, in some cases, be the only tissue that gives a positive reaction [1]. Tissues should be collected from several pigs and sent to the laboratory refrigerated. Tonsils can be collected
Hog cholera diagnostic techniques
215
from live pigs using human rectal biopsy forceps. In the laboratory, tissue sections are cut at 8 # m on a cryostat, fixed with acetone, and stained with anti-HC directly conjugated antiserum. For controls, tissue sections should also be stained with HC conjugate diluted 1:2 with HC-negative serum and HC conjugate diluted 1:2 with HC-positive serum. Another control that can be used to detect non-specific reactions is staining with a conjugate prepared against serum from a specific pathogen-free pig. Hyperimmune serum for HC conjugate can be prepared by vaccinating pigs with an attenuated strain of H C virus, then inoculating them with virulent virus 18 days later. The pigs are then challenged with a massive intravenous inoculation of hog cholera virus 60-90 days later. The challenge virus is blood from an infected pig collected at the peak of the viremia, and the dosage is 10 ml/kg body wt. The resulting antiserum will usually have a high antibody titer. However, serum from pigs with high antibody titers may not be suitable for producing satisfactory conjugates. The sections are examined by fluorescence microscopy. The cytoplasm of cells infected with HC virus stains bright green; nuclei do not fluoresce. Unirifected cells appear dark green or brown unless the cells are necrotic, in which case they will stain with both the normal and H C conjugate. The cells in the germinal center of the tonsil and in lymphoid tissue of the spleen and intestine may also non-specifically fluoresce. The fluorescence of the epithelial cells of the tonsil is usually specific and, consequently, is the most significant. In some cases, viral antigen cannot be detected in all infected pigs; therefore, tissues from several pigs should be collected for the FATST, and virus isolation should also be attempted on tissues from all suspected cases. Most of the polyclonal HC conjugates will also stain BVD virus, resulting in a false-positive HC diagnosis. To differentiate between BVD and HC on FATST, the tissues that are positive with polyclonal HC conjugate are stained with a monoclonal antibody (MAb) conjugated to horseradish peroxidase (HRP). This technique has been used to discriminate between BVD and HC infected pigs [7, 8]. A more definitive method is to use MAb to differentiate cell culture isolates of HC and BVD. Another method is to perform differential endpoint HC and BVD neutralization tests with the serum from FATST positive pigs. Modified live HC vaccine virus replicates in vaccinated pigs and tissues from these animals may produce positive FATST reactions for 2 weeks after vaccination [9]. The vaccine strain of rabbit origin and field strains can be differentiated by inoculation of rabbits with cell culture isolates. VIRUS ISOLATION IN CELL CULTURE Isolation of HC virus in cell culture is the most definitive diagnostic method it is more sensitive but slower than the FATST. The initial case of HC that will result in regulatory action should be confirmed by virus isolation. The tissues that are collected for the FATST are also suitable for virus isolation. The tonsil is the tissue of choice. A 20% tissue suspension is prepared in cell culture medium. The suspension is inoculated onto confluent monolayers of PK-15 cells on coverslips in Leighton tubes. Alternatively, one part of the suspension is mixed with nine parts of trypsinized PK-15 cells; this mixture is used to inoculate Leighton tubes. If confluent cells are used, the suspension is allowed to adsorb for 1 hr at 37°C, then removed; cell monolayers are washed, and fresh medium is added. Uninoculated cell monolayers are
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used as negative controls. The fetal bovine serum used in all HC diagnostic tests must be free of BVD virus and BVD antibody. Viral replication can be detected as early as 8 hr after inoculation; however, the usual method is to examine coverslips 1 and 2 DPI. The coverslips are washed twice with cell culture medium, fixed in cold acetone, and stained with the direct anti-HC conjugate used in the FATST. The stained cells on coverslips are washed three times with phosphate buffered saline (PBS) and are mounted on microscope slides with buffered glycerine. The cells are examined for cytoplasmic fluorescence. If there is positive fluorescence, the cells must be stained with BVD conjugate. If fluorescence is observed with both HC and BVD conjugates, the isolates can be differentiated using MAb. This has proven to be a reliable method to differentiate HC and BVD viruses [7, 8, 10-13]. Monoclonal antibodies have also been used to identify the C-strain of HC vaccine virus. Suspensions of cells are mixed with 103-105 TCIDs0/ 0.1 ml of suspected HC virus, seeded in microtiter plates or on coverslips in Leighton tubes, and incubated at 37°C in a CO2 incubator. The cells are rinsed with cell culture medium, fixed with cold acetone, and stained with MAbs conjugated with HRP. If microtiter plates are used, cells are fixed with 20% acetone as described for the PLA procedure [14]. The cells are examined with an inverted microscope for plaques that stain dark red-brown in color. In our laboratory, cells infected with five HC isolates from the U.S.A. and 15 BVD virus strains were stained with MAbs. Twenty-four MAbs were obtained from the Central Veterinary Laboratory (CVL), Weybridge, England (courtesy of S. Edwards), and two from the Central Veterinary Institute (CVI), Lelystad, The Netherlands (courtesy of G. Wensvoort). Eleven of the BVD isolates were supplied by Steve Bolin, National Animal Disease Center, Ames, Iowa. The BVD virus isolates were atypical in that they were isolated from swine or wildlife or they produced unusual patterns when examined by radioimmunoprecipitation with standard BVD antiserum. Nine of the CVL MAbs and one of the CVI MAbs stained only the HC isolates. However, two BVD isolates could only be differentiated from HC by use of the CVL MAbs and one of the CVI MAbs (J. B. Katz, National Veterinary Services Laboratories, Ames, Iowa, unpublished data). A time-consuming and expensive but definitive method to differentiate BVD and HC is to inoculate a pig and calf with the isolate and evaluate the antibody response for each virus. Modified live vaccine strains of HC virus can also be isolated in cell culture. Some of the strains will replicate slower than field virus strains and some can be identified by MAb. The strain that has been adapted to rabbits will produce a febrile reaction in rabbits inoculated by the intravenous route, and the rabbits will produce anti-HC-antibody [9]. Pig inoculation can be used to evaluate pathogenicity of HC isolates [2]. Even avirulent HCV may produce persistent infection if pigs are infected in utero; consequently, if a country wants to be H C free, these strains must be eradicated. VIRUS ISOLATION BY PIG INOCULATION The most sensitive method for HCV detection is pig inoculation. It is a time consuming and expensive procedure, but it is about 10-fold more sensitive than cell culture [2]. Infection is confirmed by reisolation of the virus in cell culture inoculated with blood collected 1-4 DPI or by FATST. If the pig survives, infection can be confirmed by the immunologic response of the inoculated pig.
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SEROLOGIC TESTS Serology is the method of choice for surveillance of HC in an apparently disease-free area or for insuring that there are no residual foci of infection during the HC eradication program. However, antibody may not be detected for 4 weeks after infection.
Fluorescent antibody virus neutralization test This test is more time consuming and difficult to perform than the other tests but is still considered in many countries as the standard method to detect HC antibody. A 1:8 dilution of serum and suitable dilution of HC virus that will produce 10 plaques per microscope field are inoculated onto 50% confluent monolayers of PK-15 cells (24 hr after seeding). Four section chamber slides or coverslips in Leighton tubes may be used. After 24 hr incubation, the coverslips or chamber slides are washed with PBS, fixed with acetone, stained with HC conjugate, and examined by a fluorescence microscope. An alternate procedure is to perform the test in 96-well microtiter plates as described for the PLA, except the wells are stained with HC conjugate and examined under an inverted fluorescence microscope [14]. The titer is the highest dilution of serum in which there is a 90% reduction in plaques. Serum samples positive for HC antibody at a 1:16 screening dilution should be tested in parallel titrations to an end point using both HC and BVD viruses. I f the titers are similar, additional samples should be collected to establish whether the pigs have been exposed to HCV.
Enzyme-linked immunosorbent assay procedures Three ELISA test procedures have recently been described for detection of antibody against HCV [11, 15, 16]. The first test is an indirect ELISA with the plate coated with HC MAb, followed by the addition of HC antigen, the test sera, and the anti-swine-IgG serum conjugated to H R P [11]. The second test is a complex-trapping-blocking ELISA using two different MAbs against HCV [15]. A complex-trapping-ELISA kit is commercially available (CVI, Department of Virology, Lelystad, The Netherlands). The third test is a blocking ELISA using polyvalent antibody prepared in rabbits [16]. The sensitivity and specificity of the ELISA tests have been reported to be greater than the FAVN test and the PLA [11, 15, 16]. All ELISA tests can be performed in < 2 4 h r .
Peroxidase-linked assay Two different PLA procedures have been described [17, 18]. The PLA test uses acetone-fixed HCV infected cells. The neutralizing PLA (NPLA) is similar to the FAVN except that the cells are stained with H R P conjugate. In the PLA test, a cell suspension is added to 96-well microplates, and incubated at 37°C for 24 hr in a CO2 incubator. Then 100 TCIDs0 per well of HCV is added, and the plates are covered and incubated for an additional 24 hr. The medium is removed and the cells are fixed with 20% acetone in PBS with 0.05% Tween 80 (PBST) for 10 min, the plates are then drained, and allowed to dry at room temperature. An alternate method to fix the cells is to wash the plates with 0.15 M NaCI and dry for 1 hr at 80°C. The plates with fixed cells are stored in a plastic bag at - 2 0 ° C until needed. Before use, PBST is added to the wells for 10 min; after the PBST is poured off, the test sera are added to the wells at a 1:10 screening dilution, incubated for 30min, and washed three times with PBST.
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Anti-swine-IgG conjugated to H R P is added, plates are incubated for 20 rain, washed three times, and the substrate, 3-amino-9-ethylcarbazole, is added. The test is read using an inverted light microscope. In the neutralization PLA, a 1 : 10 dilution of test sera and virus (100 TCID 5° per 50/~1) are added to a microtiter plate which is incubated for 1 hr at 37°C. PK-15 cell suspension is added, and the plate is sealed and incubated at 37°C with 5% CO2 for 4 days. The plates are then fixed and stained as described for the PLA. The N P L A is reported to be more specific than the PLA. Both the NPLA and PLA have been reported to have sensitivity and specificity equal to or greater than the FAVN test and are much easier to perform [18, 19]. MOLECULAR DIAGNOSTIC TECHNIQUES FOR THE DIAGNOSIS OF HOG CHOLERA Recently the nucleotide sequence of the HC virus has been described [20]. This will provide the basis for developing nucleic acid probes to identify HC virus. A procedure has been described for identification of BVD virus in tissue samples by amplification using the polymerase chain reaction and identification of the virus with a nucleus acid probe [21]. The nucleotide sequence of the two viruses are very similar [20]; consequently, the same procedure should be applicable to HC. Recently a genetically engineered HC vaccine using a live attenuated pseudorabies virus as a vector was described [22]. These techniques will allow the development of serologic tests to differentiate between animals exposed to HC vaccine virus and field strains of HC. However, at this time these molecular techniques are not used for HC diagnosis. CONCLUSION
The acute form of HC can be diagnosed with reasonable accuracy by clinical signs and lesions. If a control or eradication program is to be carried out, laboratory diagnostic techniques must be used to detect mild strains of HCV and diagnose the persistent infection in pigs. Initially the F A T S T and one of the serologic procedures could be used, but eventually virus isolation and characterization using MAb will be needed to increase sensitivity and eliminate the false positive reaction to BVD. REFERENCES 1. Terpstra C. Hog cholera (classical swine fever). In Manual of Recommended Diagnostic Techniques and Requirements for Biological Product, Vol. 2, pp. I-15. Office International des Epizootic, Paris (1989). 2. Carbrey E. A. Diagnostic procedures. In Classical Swine Fever and Related Viral Infections (Edited by Liess B.), pp. 94-114. Nijhoff, Boston (1988). 3. Van Oirschot J. T. Hog cholera. In Disease o f Swine (Edited by Lemon A. D.), pp. 289-300. Iowa State University Press, Ames, Iowa (1986). 4. Van Oirschot J. T. and Terpstra C. Hog cholera virus. In Virus Infections of Vertebrates, Vol. 2, Virus Infections o f Porcines (Edited by Pensaert M. B.), pp. 113-130. Elsevier, Amsterdam (1989). 5. Terpstra C. and Wensvoort G. Natural infections of pigs with bovine viral diarrhoea virus associated with signs resembling swine fever. Res. vet. Sci. 45, 137 142 (1988). 6. Ressang A. A. Studies on the pathogenesis of hog cholera. Zbl. vet. med. B20, 256-271 (1973). 7. Wensvoort G., Terpstra C., Boonstra J., Bloemraad M. and Van Zaane D. Production of monoclonal antibodies against swine fever virus and their use in laboratory diagnosis. Vet. Microbiol. 12, 101-108 (1986). 8. Wensvoort G., Terpstra C., De Kluijver E. P., Kragten C. and Warnaar J. C. Antigenic differentiation of pestivirus strains with monoclonal antibodies against hog cholera virus. Vet. Microbiol. 21, 9-20 (1989). 9. Terpstra C. Detection of C-strain virus in pigs following vaccination against swine fever. Tijdschr. Diergeneesk 103, 678-684 (1978).
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10. Edwards S. and Sands J. J. Antigenic comparisons of hog cholera virus isolates from Europe, America and Asia using monoclonal antibodies. Dtsch. tierarztl. Wschr. 97, 79-81 (1990). I1. Moennig V., Schagemann G., Dahle J., Greiser-Wilke I. and Leder L. A new approach for the diagnosis of hog cholera. Dtsch. tierarztl. Wschr. 97, 91-93 (1990). 12. Zhou Y., Moennig V., Coulibaly C. O. Z., Dahle J. and Liess B. Differentiation of hog cholera and bovine virus diarrhoea viruses in pigs using monoclonal antibodies. J. vet. Med. 36, 76-80 (1989). 13. Hess R. G., Coulibaly C. O. Z., Greiser-Wilke I., Moennig V. and Liess B. Identification of hog cholera viral isolates by use of monoclonal antibodies to pestiviruses. Vet. Microbiol. 16, 315-321 (1988). 14. Buonavoglia C., Falcone E., Pestalozza S., Trani L. D. and D'Amore E. A rapid serum neutralization test in microplates for the detection of antibodies to hog cholera virus. J. vir. Meth. 23, 77-79 (1989). 15. Wensvoort G., Bloemraad M. and Terpstra C. An enzyme immunoassay employing monoclonal antibodies and detecting specifically antibodies to classical swine fever virus. Vet. Microbiol. 17, 129-140 (1988). 16. Leforban Y., Edwards S., Ibata G. and Vannier P. A blocking ELISA to differentiate hog cholera virus antibodies in pig sera from those due to other pestiviruses. Ann. rech. Vet. 21, 119-129 (1990). 17. Jensen M. H. Detection of antibodies against hog cholera virus and bovine viral diarrhea virus in porcine serum. Acta vet. Scand. 22, 85-98 (1981). 18. Afshar A., Dulac G. C. and Bouffard A. Application of peroxidase labelled antibody assays for detection of porcine IgG antibodies to hog cholera and bovine viral diarrhea viruses. J. vir. Meth. 23, 253-262 (1989). 19. Jutting D. R. New test for hog cholera. USDA Foreign Animal Disease Report, Vol. 18, pp. 8-10. Animal and Plant Health Inspection Service, USDA Hyattsville, Md (1990). 20. Meyers G., Rumenapf T. and Thiel H. Molecular cloning and nucleotide sequence of the genome of hog cholera virus. Virology 171, 555-567 (1989). 21. Lopez O. J., Osorio F. A. and Donis R. O. Rapid detection of bovine viral diarrhea virus by polymerase chain reaction. J. clin. Micro. 29, 578-582 (1991). 22. Van Zijl M., Wensvoort G., De Kluyver E., Hulst M.,Van Der Gulden H., Gielkens A., Berns A. and Moormann R. Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus protects swine against both pseudorabies and hog cholera. J. Virol. 65, 2761-2765 (1991).
HOG
CHOLERA
DIAGNOSTIC
0147-9571/92 $5.00 + 0.00 Pergamon Press Ltd
TECHNIQUES
J. E. PEARSON USDA, APHIS, S&T, National Veterinary Services Laboratories, Diagnostic Virology Laboratory, Ames, Iowa, U.S.A. A~traet~Clinical signs and lesions can sometimes provide the basis for a presumptive diagnosis of hog cholera (HC). However, an accurate diagnosis requires laboratory testing. The usual procedure for the detection of viral antigen is the examination of cryostat sections stained with fluorescein-conjugated HC antiserum. A more definitive technique is isolation of the virus in PK-15 cell cultures and identification of the viral antigen in cells using an HC fluorescent antibody conjugate. As bovine viral diarrhea (BVD) virus will cross-react with HC virus, isolation must be confirmed by the comparison of BVD and HC staining or, preferably, by the use of monoclonal antibodies that can differentiate between HC and BVD viruses. Hog cholera surveillance must rely on serology. The fluorescent antibody virus neutralization (FAVN) test is the classical technique, and HC and BVD antibody can usually be differentiated if HC-positive serum samples are tested against both viruses. Recently the enzyme-linked immunosorbent assay (ELISA) and peroxidase-labeled antibody tests have become the commonly used techniques. Key words: Hog cholera, diagnosis, fluorescent antibody, virus neutralization, tissue section, enzyme-linked immunosorbent assay, peroxidase-linked assay, virus isolation, bovine viral diarrhea.
TECHNIQUES DE DIAGNOSTIC DU CHOLI~RA DU PORC R6sum6--Signes cliniques et 16sions peuvent quelquefois fournir la base pour un diagnostic probable de chol6ra du porc (CP). Cependent, un diagnostic pr6cis doit 6tre bas6 sur des tests de laboratoires. La proc6dure normale pour la d6tection d'un antig6ne viral est bas6e sur l'examen de sections de cryostat tach6es d'antiserum CP conjugu6 de fluorescine. Une technique plus complete consiste en l'isolation du virus dans des cultures de cellules PK-15, et en l'identification de l'antig~ne viral dans les cellules utilisant un anticorp conjug~ fluorescent CP. A mesure que le virus de la diarrh6e virale bovine (DVB) r6agit avec le virus CP, l'isolation doit &re confirm6e par la comparaison des taches DVB et CP ou, de pr6f6rence, par l'utilisation d'anticorps monoclonaux grace auxquels on peut diff~rencier entre les virus DVB et CP. La surveillance du chol6ra du porc doit se faire sur la base de la s6rologie. La technique classique utilise le test de neutralisation de l'anticorp fluorescent du virus (NAFV). De plus, les anticorps CP et DVB peuvent 6tre g6n6ralement distingu6s si les 6chantillons de s6rum CP-positif sont compar6s aux deux virus. R6cemment l'analyse immunosobante li6e aux enzymes (AILE) et les tests d'anticorps marqu6s de p+roxidase sont devenus les techniques les plus couramment utilis6es. Mots-clefs: Chol6ra du porc, diagnostic, anticorp fluorescent, neutralisation de virus, section de tissu, analyse immunosorbante li6e aux enzymes, analyse li6e aux p6roxidases, isolation de virus, diarrh6e virale bovine.
INTRODUCTION
Historically, hog cholera (HC) (classical swine fever) was diagnosed on the basis of clinical signs and lesions. The variability of the disease syndrome made these methods unreliable. The current methods rely on detection of antigen in cryostat tissue sections using the direct immunofluorescent test, isolation of the virus in cell culture, or pig inoculation and serological techniques which include the fluorescent antibody virus neutralization (FAVN) 213
214
J.E. PE~,~,sor,r
test, enzyme-linked immunosorbent assay (ELISA), and the peroxidase-linked assay (PLA). There have been previous reviews of these procedures [1, 2]. In recent years, improved methods have been developed using monoclonal antibodies to differentiate between HC and bovine viral diarrhea (BVD) virus infections. There have also been reports on the development of molecular techniques that will enhance HC diagnosis. The threat of introducing persistently infected, clinically normal pigs or infected meat scraps has increased the importance of laboratory diagnosis. C L I N I C A L S I G N S A N D POST M O R T E M L E S I O N S
A presumptive diagnosis of the acute and peracute forms of the disease based on signs and lesions are described in detail elsewhere [3, 4]. The disease spreads rapidly among pigs of all ages; the affected pigs are depressed, anorexic, and develop a fever of 42°C or higher, and have leukopenia, conjunctivitis, and constipation, that may be followed by diarrhea. The pigs will huddle together as if chilled and may vomit. Convulsions may occur, which are usually followed by death. The pigs that survive longer will become gaunt and develop a staggering gait. The latter condition is related to weakness in the hindquarters that progresses into posterior paralysis. In the terminal stages of the disease, a purplish discoloration is observed on the abdomen, snout, ears, and medial sides of the legs. Pigs with the acute form of HC die between 10 and 20 days postinfection (DPI). Lesions are often absent in the peracute form of HC. In the acute form, infarction of the spleen is considered almost pathognomonic. The lymph nodes are swollen and hemorrhagic. Petechial and ecchymotic hemorrhages are seen in the cortex of the kidney and less often in the medullary pyramids and hilus. They are also often observed in the urinary bladder, larynx, epiglottis, heart, serous and mucous membranes, and skin. A catarrhal to necrotic enteritis and infarcts in the lungs may be seen. Microscopic examination of the brain often reveals an encephalitis and perivascular cuffing. There are no pathognomic microscopic lesions. In the chronic or subacute forms, which are caused by less virulent HC strains, the clinical signs may be similar to those seen in the acute form but are much less pronounced. The signs may be too mild to be detected. Pigs infected in u t e r o may have a lifelong high level of viremia with few, or no, clinical signs. Some pigs may have intermittent diarrhea, anorexia, and retarded growth, and the mortality may be above normal. In u t e r o infection with HC may cause losses of piglets, abortions, stillbirths, and fetal anomalies. This variability in clinical signs requires that any suspicious case be confirmed by laboratory investigations. Diseases such as African swine fever and salmonellosis may be confused with HC. Also, BVD can produce similar clinical signs following in u t e r o infection [5]. DETECTION OF HC VIRUS ANTIGEN The fluorescent antibody tissue section technique (FATST) has become the classical method for detection of hog cholera antigen. The tissue of choice is the tonsil, as this is the first to become infected [6], but 4 cm 2 pieces of spleen, kidney, mandibular lymph node, and distal portion of the ileum should also be submitted. The ileum should be examined as it frequently contains viral antigen in the subacute and chronic forms of the disease and may, in some cases, be the only tissue that gives a positive reaction [1]. Tissues should be collected from several pigs and sent to the laboratory refrigerated. Tonsils can be collected
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from live pigs using human rectal biopsy forceps. In the laboratory, tissue sections are cut at 8 # m on a cryostat, fixed with acetone, and stained with anti-HC directly conjugated antiserum. For controls, tissue sections should also be stained with HC conjugate diluted 1:2 with HC-negative serum and HC conjugate diluted 1:2 with HC-positive serum. Another control that can be used to detect non-specific reactions is staining with a conjugate prepared against serum from a specific pathogen-free pig. Hyperimmune serum for HC conjugate can be prepared by vaccinating pigs with an attenuated strain of H C virus, then inoculating them with virulent virus 18 days later. The pigs are then challenged with a massive intravenous inoculation of hog cholera virus 60-90 days later. The challenge virus is blood from an infected pig collected at the peak of the viremia, and the dosage is 10 ml/kg body wt. The resulting antiserum will usually have a high antibody titer. However, serum from pigs with high antibody titers may not be suitable for producing satisfactory conjugates. The sections are examined by fluorescence microscopy. The cytoplasm of cells infected with HC virus stains bright green; nuclei do not fluoresce. Unirifected cells appear dark green or brown unless the cells are necrotic, in which case they will stain with both the normal and H C conjugate. The cells in the germinal center of the tonsil and in lymphoid tissue of the spleen and intestine may also non-specifically fluoresce. The fluorescence of the epithelial cells of the tonsil is usually specific and, consequently, is the most significant. In some cases, viral antigen cannot be detected in all infected pigs; therefore, tissues from several pigs should be collected for the FATST, and virus isolation should also be attempted on tissues from all suspected cases. Most of the polyclonal HC conjugates will also stain BVD virus, resulting in a false-positive HC diagnosis. To differentiate between BVD and HC on FATST, the tissues that are positive with polyclonal HC conjugate are stained with a monoclonal antibody (MAb) conjugated to horseradish peroxidase (HRP). This technique has been used to discriminate between BVD and HC infected pigs [7, 8]. A more definitive method is to use MAb to differentiate cell culture isolates of HC and BVD. Another method is to perform differential endpoint HC and BVD neutralization tests with the serum from FATST positive pigs. Modified live HC vaccine virus replicates in vaccinated pigs and tissues from these animals may produce positive FATST reactions for 2 weeks after vaccination [9]. The vaccine strain of rabbit origin and field strains can be differentiated by inoculation of rabbits with cell culture isolates. VIRUS ISOLATION IN CELL CULTURE Isolation of HC virus in cell culture is the most definitive diagnostic method it is more sensitive but slower than the FATST. The initial case of HC that will result in regulatory action should be confirmed by virus isolation. The tissues that are collected for the FATST are also suitable for virus isolation. The tonsil is the tissue of choice. A 20% tissue suspension is prepared in cell culture medium. The suspension is inoculated onto confluent monolayers of PK-15 cells on coverslips in Leighton tubes. Alternatively, one part of the suspension is mixed with nine parts of trypsinized PK-15 cells; this mixture is used to inoculate Leighton tubes. If confluent cells are used, the suspension is allowed to adsorb for 1 hr at 37°C, then removed; cell monolayers are washed, and fresh medium is added. Uninoculated cell monolayers are
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used as negative controls. The fetal bovine serum used in all HC diagnostic tests must be free of BVD virus and BVD antibody. Viral replication can be detected as early as 8 hr after inoculation; however, the usual method is to examine coverslips 1 and 2 DPI. The coverslips are washed twice with cell culture medium, fixed in cold acetone, and stained with the direct anti-HC conjugate used in the FATST. The stained cells on coverslips are washed three times with phosphate buffered saline (PBS) and are mounted on microscope slides with buffered glycerine. The cells are examined for cytoplasmic fluorescence. If there is positive fluorescence, the cells must be stained with BVD conjugate. If fluorescence is observed with both HC and BVD conjugates, the isolates can be differentiated using MAb. This has proven to be a reliable method to differentiate HC and BVD viruses [7, 8, 10-13]. Monoclonal antibodies have also been used to identify the C-strain of HC vaccine virus. Suspensions of cells are mixed with 103-105 TCIDs0/ 0.1 ml of suspected HC virus, seeded in microtiter plates or on coverslips in Leighton tubes, and incubated at 37°C in a CO2 incubator. The cells are rinsed with cell culture medium, fixed with cold acetone, and stained with MAbs conjugated with HRP. If microtiter plates are used, cells are fixed with 20% acetone as described for the PLA procedure [14]. The cells are examined with an inverted microscope for plaques that stain dark red-brown in color. In our laboratory, cells infected with five HC isolates from the U.S.A. and 15 BVD virus strains were stained with MAbs. Twenty-four MAbs were obtained from the Central Veterinary Laboratory (CVL), Weybridge, England (courtesy of S. Edwards), and two from the Central Veterinary Institute (CVI), Lelystad, The Netherlands (courtesy of G. Wensvoort). Eleven of the BVD isolates were supplied by Steve Bolin, National Animal Disease Center, Ames, Iowa. The BVD virus isolates were atypical in that they were isolated from swine or wildlife or they produced unusual patterns when examined by radioimmunoprecipitation with standard BVD antiserum. Nine of the CVL MAbs and one of the CVI MAbs stained only the HC isolates. However, two BVD isolates could only be differentiated from HC by use of the CVL MAbs and one of the CVI MAbs (J. B. Katz, National Veterinary Services Laboratories, Ames, Iowa, unpublished data). A time-consuming and expensive but definitive method to differentiate BVD and HC is to inoculate a pig and calf with the isolate and evaluate the antibody response for each virus. Modified live vaccine strains of HC virus can also be isolated in cell culture. Some of the strains will replicate slower than field virus strains and some can be identified by MAb. The strain that has been adapted to rabbits will produce a febrile reaction in rabbits inoculated by the intravenous route, and the rabbits will produce anti-HC-antibody [9]. Pig inoculation can be used to evaluate pathogenicity of HC isolates [2]. Even avirulent HCV may produce persistent infection if pigs are infected in utero; consequently, if a country wants to be H C free, these strains must be eradicated. VIRUS ISOLATION BY PIG INOCULATION The most sensitive method for HCV detection is pig inoculation. It is a time consuming and expensive procedure, but it is about 10-fold more sensitive than cell culture [2]. Infection is confirmed by reisolation of the virus in cell culture inoculated with blood collected 1-4 DPI or by FATST. If the pig survives, infection can be confirmed by the immunologic response of the inoculated pig.
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SEROLOGIC TESTS Serology is the method of choice for surveillance of HC in an apparently disease-free area or for insuring that there are no residual foci of infection during the HC eradication program. However, antibody may not be detected for 4 weeks after infection.
Fluorescent antibody virus neutralization test This test is more time consuming and difficult to perform than the other tests but is still considered in many countries as the standard method to detect HC antibody. A 1:8 dilution of serum and suitable dilution of HC virus that will produce 10 plaques per microscope field are inoculated onto 50% confluent monolayers of PK-15 cells (24 hr after seeding). Four section chamber slides or coverslips in Leighton tubes may be used. After 24 hr incubation, the coverslips or chamber slides are washed with PBS, fixed with acetone, stained with HC conjugate, and examined by a fluorescence microscope. An alternate procedure is to perform the test in 96-well microtiter plates as described for the PLA, except the wells are stained with HC conjugate and examined under an inverted fluorescence microscope [14]. The titer is the highest dilution of serum in which there is a 90% reduction in plaques. Serum samples positive for HC antibody at a 1:16 screening dilution should be tested in parallel titrations to an end point using both HC and BVD viruses. I f the titers are similar, additional samples should be collected to establish whether the pigs have been exposed to HCV.
Enzyme-linked immunosorbent assay procedures Three ELISA test procedures have recently been described for detection of antibody against HCV [11, 15, 16]. The first test is an indirect ELISA with the plate coated with HC MAb, followed by the addition of HC antigen, the test sera, and the anti-swine-IgG serum conjugated to H R P [11]. The second test is a complex-trapping-blocking ELISA using two different MAbs against HCV [15]. A complex-trapping-ELISA kit is commercially available (CVI, Department of Virology, Lelystad, The Netherlands). The third test is a blocking ELISA using polyvalent antibody prepared in rabbits [16]. The sensitivity and specificity of the ELISA tests have been reported to be greater than the FAVN test and the PLA [11, 15, 16]. All ELISA tests can be performed in < 2 4 h r .
Peroxidase-linked assay Two different PLA procedures have been described [17, 18]. The PLA test uses acetone-fixed HCV infected cells. The neutralizing PLA (NPLA) is similar to the FAVN except that the cells are stained with H R P conjugate. In the PLA test, a cell suspension is added to 96-well microplates, and incubated at 37°C for 24 hr in a CO2 incubator. Then 100 TCIDs0 per well of HCV is added, and the plates are covered and incubated for an additional 24 hr. The medium is removed and the cells are fixed with 20% acetone in PBS with 0.05% Tween 80 (PBST) for 10 min, the plates are then drained, and allowed to dry at room temperature. An alternate method to fix the cells is to wash the plates with 0.15 M NaCI and dry for 1 hr at 80°C. The plates with fixed cells are stored in a plastic bag at - 2 0 ° C until needed. Before use, PBST is added to the wells for 10 min; after the PBST is poured off, the test sera are added to the wells at a 1:10 screening dilution, incubated for 30min, and washed three times with PBST.
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Anti-swine-IgG conjugated to H R P is added, plates are incubated for 20 rain, washed three times, and the substrate, 3-amino-9-ethylcarbazole, is added. The test is read using an inverted light microscope. In the neutralization PLA, a 1 : 10 dilution of test sera and virus (100 TCID 5° per 50/~1) are added to a microtiter plate which is incubated for 1 hr at 37°C. PK-15 cell suspension is added, and the plate is sealed and incubated at 37°C with 5% CO2 for 4 days. The plates are then fixed and stained as described for the PLA. The N P L A is reported to be more specific than the PLA. Both the NPLA and PLA have been reported to have sensitivity and specificity equal to or greater than the FAVN test and are much easier to perform [18, 19]. MOLECULAR DIAGNOSTIC TECHNIQUES FOR THE DIAGNOSIS OF HOG CHOLERA Recently the nucleotide sequence of the HC virus has been described [20]. This will provide the basis for developing nucleic acid probes to identify HC virus. A procedure has been described for identification of BVD virus in tissue samples by amplification using the polymerase chain reaction and identification of the virus with a nucleus acid probe [21]. The nucleotide sequence of the two viruses are very similar [20]; consequently, the same procedure should be applicable to HC. Recently a genetically engineered HC vaccine using a live attenuated pseudorabies virus as a vector was described [22]. These techniques will allow the development of serologic tests to differentiate between animals exposed to HC vaccine virus and field strains of HC. However, at this time these molecular techniques are not used for HC diagnosis. CONCLUSION
The acute form of HC can be diagnosed with reasonable accuracy by clinical signs and lesions. If a control or eradication program is to be carried out, laboratory diagnostic techniques must be used to detect mild strains of HCV and diagnose the persistent infection in pigs. Initially the F A T S T and one of the serologic procedures could be used, but eventually virus isolation and characterization using MAb will be needed to increase sensitivity and eliminate the false positive reaction to BVD. REFERENCES 1. Terpstra C. Hog cholera (classical swine fever). In Manual of Recommended Diagnostic Techniques and Requirements for Biological Product, Vol. 2, pp. I-15. Office International des Epizootic, Paris (1989). 2. Carbrey E. A. Diagnostic procedures. In Classical Swine Fever and Related Viral Infections (Edited by Liess B.), pp. 94-114. Nijhoff, Boston (1988). 3. Van Oirschot J. T. Hog cholera. In Disease o f Swine (Edited by Lemon A. D.), pp. 289-300. Iowa State University Press, Ames, Iowa (1986). 4. Van Oirschot J. T. and Terpstra C. Hog cholera virus. In Virus Infections of Vertebrates, Vol. 2, Virus Infections o f Porcines (Edited by Pensaert M. B.), pp. 113-130. Elsevier, Amsterdam (1989). 5. Terpstra C. and Wensvoort G. Natural infections of pigs with bovine viral diarrhoea virus associated with signs resembling swine fever. Res. vet. Sci. 45, 137 142 (1988). 6. Ressang A. A. Studies on the pathogenesis of hog cholera. Zbl. vet. med. B20, 256-271 (1973). 7. Wensvoort G., Terpstra C., Boonstra J., Bloemraad M. and Van Zaane D. Production of monoclonal antibodies against swine fever virus and their use in laboratory diagnosis. Vet. Microbiol. 12, 101-108 (1986). 8. Wensvoort G., Terpstra C., De Kluijver E. P., Kragten C. and Warnaar J. C. Antigenic differentiation of pestivirus strains with monoclonal antibodies against hog cholera virus. Vet. Microbiol. 21, 9-20 (1989). 9. Terpstra C. Detection of C-strain virus in pigs following vaccination against swine fever. Tijdschr. Diergeneesk 103, 678-684 (1978).
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10. Edwards S. and Sands J. J. Antigenic comparisons of hog cholera virus isolates from Europe, America and Asia using monoclonal antibodies. Dtsch. tierarztl. Wschr. 97, 79-81 (1990). I1. Moennig V., Schagemann G., Dahle J., Greiser-Wilke I. and Leder L. A new approach for the diagnosis of hog cholera. Dtsch. tierarztl. Wschr. 97, 91-93 (1990). 12. Zhou Y., Moennig V., Coulibaly C. O. Z., Dahle J. and Liess B. Differentiation of hog cholera and bovine virus diarrhoea viruses in pigs using monoclonal antibodies. J. vet. Med. 36, 76-80 (1989). 13. Hess R. G., Coulibaly C. O. Z., Greiser-Wilke I., Moennig V. and Liess B. Identification of hog cholera viral isolates by use of monoclonal antibodies to pestiviruses. Vet. Microbiol. 16, 315-321 (1988). 14. Buonavoglia C., Falcone E., Pestalozza S., Trani L. D. and D'Amore E. A rapid serum neutralization test in microplates for the detection of antibodies to hog cholera virus. J. vir. Meth. 23, 77-79 (1989). 15. Wensvoort G., Bloemraad M. and Terpstra C. An enzyme immunoassay employing monoclonal antibodies and detecting specifically antibodies to classical swine fever virus. Vet. Microbiol. 17, 129-140 (1988). 16. Leforban Y., Edwards S., Ibata G. and Vannier P. A blocking ELISA to differentiate hog cholera virus antibodies in pig sera from those due to other pestiviruses. Ann. rech. Vet. 21, 119-129 (1990). 17. Jensen M. H. Detection of antibodies against hog cholera virus and bovine viral diarrhea virus in porcine serum. Acta vet. Scand. 22, 85-98 (1981). 18. Afshar A., Dulac G. C. and Bouffard A. Application of peroxidase labelled antibody assays for detection of porcine IgG antibodies to hog cholera and bovine viral diarrhea viruses. J. vir. Meth. 23, 253-262 (1989). 19. Jutting D. R. New test for hog cholera. USDA Foreign Animal Disease Report, Vol. 18, pp. 8-10. Animal and Plant Health Inspection Service, USDA Hyattsville, Md (1990). 20. Meyers G., Rumenapf T. and Thiel H. Molecular cloning and nucleotide sequence of the genome of hog cholera virus. Virology 171, 555-567 (1989). 21. Lopez O. J., Osorio F. A. and Donis R. O. Rapid detection of bovine viral diarrhea virus by polymerase chain reaction. J. clin. Micro. 29, 578-582 (1991). 22. Van Zijl M., Wensvoort G., De Kluyver E., Hulst M.,Van Der Gulden H., Gielkens A., Berns A. and Moormann R. Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus protects swine against both pseudorabies and hog cholera. J. Virol. 65, 2761-2765 (1991).
