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Laboratory diagnostic investigations for bovine viral diarrhoea virus infections in cattle
Veterinary Microbiology 64 (1999) 123±134
Laboratory diagnostic investigations for bovine viral diarrhoea virus infections in cattle Torstein Sandvik*,1 Department of Virology and Serodiagnostics, National Veterinary Institute, Oslo, Norway
Abstract There are no pathognomonic clinical signs of infection with bovine viral diarrhoea virus (BVDV) in cattle. Diagnostic investigations therefore rely on laboratory-based detection of the virus, or of virus-induced antigens or antibodies in submitted samples. In unvaccinated dairy herds, serological testing of bulk milk is a convenient method for BVDV prevalence screening. Alternatively, serological testing of young stock may indicate if BVDV is present in a herd. In BVDV positive herds, animals persistently infected (PI) with BVDV can be identified by combined use of serological and virological tests for examination of blood samples. ELISAs have been used for rapid detection of both BVDV antibodies and antigens in blood, but should preferably be backed up by other methods such as virus neutralization, virus isolation in cell cultures or amplification of viral nucleic acid. Detailed knowledge of the performance of the diagnostic tests in use, as well as of the epidemiology of bovine virus diarrhoea is essential for identification of viremic animals in affected herds. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Bovine viral diarrhoea virus; BVDV; Cattle; Diagnosis; Pestivirus
1. Introduction If the success of a virus is measured by its ability to spread, cause disease and still persist within a population without being discovered, pestiviruses are perhaps the most successful of all bovine viruses. In most countries, large parts of the bovine population have been infected with one or more of them, which usually have been referred to as bovine viral diarrhoea virus (BVDV) (Baker, 1987). * Corresponding author. Tel.: +47 2296 4775; fax: +47 2296 4818; e-mail: [email protected] 1 Present address: Department of Pharmacology, Food hygiene and Microbiology, Norwegian College of Veterinary Medicine, Oslo, Norway. 0378-1135/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII S 0 3 7 8 - 1 1 3 5 ( 9 8 ) 0 0 2 6 4 - 8
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During the last decade, research in two fields resulted in major improvements of our knowledge of the biology of pestiviruses. The production of pestivirus-specific monoclonal antibodies (Mabs) provided important information on the nature of viral proteins and on the antigenic diversity of these viruses (Peters et al., 1986; Bolin et al., 1988; Donis et al., 1988; Edwards et al., 1988), but Mabs were also excellent tools for the development of diagnostic tests. Similarly, nucleotide sequencing of complete virus genomes improved our basic understanding of the biology of the viruses (Collett et al., 1988; Deng and Brock, 1992; Ridpath and Bolin, 1995), and allowed the application of powerful molecular genetic tests for detection and classification of different types of ruminant pestiviruses. Recent antigenic and genetic studies of bovine pestivirus isolates have shown that cattle may be infected with at least two different types of BVDV (Ridpath et al., 1994; Paton et al., 1995), and possibly also the ovine border disease virus (Edwards et al., 1988; Moussa et al., 1997). Most of the bovine pestivirus isolates appear to be BVDV type 1, but even within this type considerable antigenic differences have been demonstrated (Edwards, 1997). BVDV type 2 strains were initially recognized by their virulence (Pellerin et al., 1994), but isolates of lower virulence have also been assigned to this type (Wolfmeyer et al., 1997). Most field isolates of BVDV are non-cytopathogenic (ncp), but there are also cytopathogenic (cp) biotypes of the virus, which have been shown to be the cause of mucosal disease (Brownlie et al., 1984). Since most of the ruminant pestiviruses share the basic properties essential in the pathogenesis and epidemiology of bovine virus diarrhoea (BVD), diagnostic methods should be designed to recognize the whole range of viruses isolated from cattle. Together with the better understanding of the disease complex induced by BVDV, the currently available diagnostic tests have made disease control projects based on detection and removal of viremic animals from unvaccinated populations as realistic options to the more conservative vaccination and non-intervention strategies for control of BVD. 2. Properties of diagnostic tests Several terms are used to describe the performance of diagnostic tests (Martin et al., 1987). The diagnostic or epidemiological sensitivity of a test is defined as the percentage of true positives which test positively with the test. The epidemiological specificity is the percentage of true negatives recognized as such by the test. These two terms should not be mixed up with the analytical sensitivity and specificity, which refers to the amount of substance that can be detected with the test, and lack of false positive signals due to cross reactions, respectively. In both senses, the values for sensitivity and specificity should be as high as possible, but in practice they will be inversely related. Among other desired qualities are high repeatability and reproducibility, which ensure similar test results of aliquots repeatedly tested within and at separate laboratories. The predictive value of a test result, which describes the probability that animals which are diagnosed as positive or negative by a test actually have or not have the disease, is not a quality of the test only, but depends on both the epidemiological sensitivity and specificity of the test as well as the prevalence of the disease in the tested population.
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Newly introduced tests should preferably be evaluated against the established reference test methods (`gold standards'). For BVDV diagnostic work, these are virus isolation, and titration of virus neutralizing antibodies, both in cell cultures (Edwards, 1990). These tests are biological assays, which may be influenced e.g. by the susceptibility to infection of the cell cultures, the cell culture medium, the test protocol and format as well as the immunological labelling. Since the reference methods require considerable experience and efforts to provide reliable results, less complicated test methods such as enzyme immunoassays have become attractive alternatives for BVD testing of series of samples. 3. What is to be diagnosed? The pathogenesis of BVDV infections is complex, and results in a wide spectrum of conditions ranging in severity from asymptomatic to lethal (Baker, 1990). In most cases, the clinical signs are vague, and not sufficient for an aetiological diagnosis. One exception to this rule is classical cases of mucosal disease, which may require euthanasia before the diagnosis can be verified by laboratory tests. For most other cases, as well as for general information on the prevalence of BVD, laboratory examination of clinical samples is necessary to establish a diagnosis. In the current Scandinavian BVD control programmes, the aim of the organized diagnostic work is to identify and remove the animals that are sources of infection, which primarily are animals immunotolerant to and persistently infected (PI) with BVDV (Bitsch and Rùnsholt, 1995). To achieve this, identification of several categories of animals is necessary (Table 1). Clinically healthy PI animals older than 2 months are nearly always negative for antibodies to, and have a constant and high level of BVDV in the blood, most other tissues and in excreta such as saliva, feces, urine and milk. It is difficult to estimate the role other categories of BVDV carrying animals (Table 1; b, h and i) play in transmission of the virus, but their existence should be recognized, for both differential diagnostic reasons vs. PI animals and since they may be of importance in Table 1 Categories of animals in an unvaccinated bovine population where BVDV is prevalent, and usual results of testing for antibodies and virus in serum
a. b. c. d. e. f. g. h. i.
Category
Antibody
Virus
Uninfected, naive animals Acutely infected animals Immune animals after acute infection Passively immunized calves PI animals PI calves of immune dams Mucosal disease cases Pregnant cows carrying PI calf Immune bulls
ÿ ÿ ÿ ÿ/ /
ÿ /ÿ ÿ ÿ ÿ/ /ÿ ÿ ÿ
PI ± Persistently infected.
Comments Brief and low virus titre in blood Antibodies detectable for 5±9 months Antibodies detectable for 4±10 weeks Neutralizing antibodies High antibody titre in late pregnancy Semen may be virus positive
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maintenance of the virus in certain herds (Moerman et al., 1993). Also, reliable identification of uninfected herds and individual animals immune to infection is important to exclude these from unnecessary testing, and thereby improving the predictive value of the test result for the herds undergoing full testing. Since all diagnostic investigations for BVD should be conducted with the herd as the epidemiological unit of interest, the first task is to identify herds with PI animals. For dairy herds not vaccinated against BVDV, bulk milk testing for antibodies has proven to be a very useful and cost-effective way of tracing herds likely to house PI animals (Niskanen, 1993). Alternatively, antibody testing of blood samples from selected age groups of a herd will also give information on the time of infection of the herd (Bitsch and Rùnsholt, 1995), but such samples are not as easy to collect as bulk milk. In vitro gene amplification techniques have also been used for the detection of viral nucleic acid in bulk milk (Radwan et al., 1995; Drew et al., 1999). The advantage of this approach is that it may be used in vaccinated herds, but since not all PI animals are lactating this approach of screening is not optimal, and may serve as an example of a test with an excellent analytic, but intermediate epidemiological sensitivity. For further testing of herds likely to have PI animals, individual blood samples remain as the best sample material for laboratory diagnostic work. Since different test protocols may require clotted or anticoagulated blood, the diagnostic laboratory should be consulted before samples are collected. Depending on the performance of the applied diagnostic tests, all samples should be tested for both antibodies to and for BVDV; only for BVDV, or screened for antibodies before samples without high antibody levels are tested for BVDV. Usually, the criteria for a diagnosis of persistent infection with BVDV have been repeated isolation of BVDV from blood without seroconversion in samples drawn 3±4 weeks apart. With additional information on the status of infection of all animals in a herd, sampled once at the same time, there is circumstantial evidence that a given viremic, antibody negative individual is PI with BVDV when all other animals in contact have high levels of antibodies to the virus. In animals acutely infected with BVDV, the virus titres in blood are usually low and of short duration, and BVDV is only rarely isolated if blood samples are drawn at random. If acute infection with BVDV is suspected, paired blood samples drawn at least 3±4 weeks apart should be examined for antibodies to the virus, and the diagnosis should be based on seroconversion. To verify that all PI animals have been removed from a herd, young stock between 8± 12-months old should be tested for BVDV antibodies. This group of animals are normally free from maternal antibodies, and have been at risk for acute infection with BVDV for some time. For many years, laboratory diagnostic investigations for BVDV have been complicated by false positives due to contamination of cell cultures with ncp BVDV, usually from the fetal calf serum used to supplement cell culture media (Bolin et al., 1991). Similarly, BVDV neutralizing antibodies in fetal calf serum may also inhibit replication of virus and conceal the presence of BVDV in sample material. Monitoring to ensure freedom from contamination with BVDV, as well as to ascertain satisfactory susceptibility to infection of the cell cultures are therefore essential quality control measures for all BVDV diagnostic work.
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4. Detection of immune responses against BVDV Immune responses against an infectious agent are classified as cellular or humoral, and immunity may be acquired actively by infection or passively by transfer of colostral antibodies. Cellular immunity, measured as proliferation of peripheral blood mononuclear cells after in vitro exposure to BVDV, has been demonstrated for seropositive cows, but not for a calf believed to be passively immunised with colostral antibodies (Larsson and Fossum, 1992). However, most studies on detection of immune responses against BVDV have concentrated on demonstration of antibodies to the virus, which can be detected in serum from 3 weeks, and thereafter for years after acute infection of immunocompetent animals (Howard, 1990). BVDV specific antibodies can be classified in two functional groups. Antibodies to viral glycoproteins (primarily E2 or gp53) may block the infectivity of or neutralize the virus. These antibodies cross react with other strains of BVDV, but if sera raised against one virus strain are tested against other challenge viruses, different antibody titres may be obtained (Brock, 1995). Conversely, the highly immunogenic nonstructural viral protein NS2-3 (p125), which is essential for the intracellular replication of the virus, is antigenically conserved between all pestiviruses (Collett, 1992). Antibodies to NS2-3 do not neutralize BVDV, but since they can be readily detected by other serological tests, this antigen is important in BVDV serology. If effective recombinant sub-unit vaccines against BVDV become available, simultaneous vaccination and anti-NS2-3 serology for monitoring of natural infection with BVDV should be possible. 4.1. Virus neutralization test When properly calibrated, virus neutralization (VN) assays are sensitive and specific assays for detection of antibodies to BVDV, and have long been recognized as the reference test for BVDV serology. Today, the test is usually performed in 96-well microtitre plates, where serially diluted test sera are incubated with a cytopathogenic challenge virus before susceptible bovine cells are added to and incubated with the neutralized virus for 4 days (Edwards, 1990). Because of the high specificity of the test, it is essential to use a challenge virus antigenically similar to the field viruses in the population to be tested. The VN assay requires substantial investments in selection and monitoring of cell cultures and media to give satisfactory test results, and is therefore, not suitable for examination of few or sporadic samples. 4.2. Enzyme immunoassays Enzyme-linked immunosorbent assays (ELISAs) are versatile diagnostic methods which can be designed to detect almost any immunoreactive molecule. For BVDV serology they have become popular for several reasons; they are independent of cell cultures, they can easily be applied for mass screening, test results can be read in few hours and reliable results can be obtained with milk as test material (Niskanen et al., 1989). Two principally different protocols are possible, activity amplification (AA) or activity modulation (AM) systems (Tijssen, 1985). Among commonly used AA systems
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are indirect ELISAs, where antigen is immobilized and used to trap specific antibodies, which subsequently are detected by enzyme-conjugated species-specific antiglobulins and a chromogenic substrate. The specificity of such systems is determined by the choice of viral antigen, which may be purified virus particles (Chu et al., 1985), detergent extracts of cultured cells inoculated with BVDV (Howard et al., 1985), single viral antigens immobilized with Mabs (Moennig et al., 1991) or recombinant virus proteins produced in bacteria (Kwang et al., 1995). In AM systems, virus-specific antibodies in the test sample compete with or block the binding of conjugated virus-specific antibodies, resulting in a lowered signal for positive samples (Paton et al., 1991). When the performance of such ELISAs are compared to that of a VN assay, only those using purified virus particles as antigen can be expected to give equal results, but still, acceptable agreements have usually been found for anti NS2-3 ELISAs (Moennig et al., 1991). If ELISAs for BVDV serology are to be developed in-house, a convenient source of antigen is cell cultures inoculated with BVDV, e.g. as described by Edwards (1990). ELISA kits are also available commercially. Among problems observed with AA ELISAs for BVDV serology are high and variable background signals caused by contaminations in the test antigen, or cross reactivity of the enzyme-conjugated antiglobulins resulting in low signal-to-noise ratios. AM ELISAs based on blocking of enzyme-conjugated Mabs can be designed to have a high analytical specificity, but it is essential that the employed Mabs recognize all virus strains in order to ensure a sufficient epidemiological sensitivity. 4.3. Other serological tests Many serological tests have been adopted for BVDV serology, including immunodiffusion in agar gels, complement fixation, indirect immunofluorescence, and western blotting. Agar gel immunodiffusion detects primarily antibodies to the NS2-3 antigen, but since the analytical sensitivity is low, it performs best as a simple screening assay (Edwards, 1990). Similarly, the analytical sensitivity of the complement fixation test is not very high, but since the antibodies detected with this assay appear and decline earlier than neutralizing or precipitating antibodies it may be used for the early detection of antibodies in seroconverting animals (Gutekunst and Malmquist, 1964). The indirect immunofluorescence test, in which BVDV-infected cells fixed on microscope slides are used as antigen, is similar to indirect AA ELISAs, but since it has to be read manually, the specificity added by recognition of a typical pattern of infected cell cultures is outweighed by the time consumed by examination of many samples. However, it is an useful alternative test method for sera cytotoxic to cell cultures or giving high background signals in ELISAs. Technically, western blotting is also similar to some ELISAs, but provides information on the molecular specificity of the antibodies. 5. Detection of the virus or viral components BVDV is not among the most fragile viruses, but still, reliable results from virological testing will only be obtained with suitable sample material. If clinical cases are to be
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investigated for BVDV, the choice and handling of samples may influence the success of assays for BVDV. Nasal secretions and faeces are important sources of infection for other animals, but since they are contaminated with other microbes and substances toxic to cell cultures, blood should instead be collected from live animals. From carcasses, samples of spleen, liver, lung or kidneys are suitable for isolation of BVDV in cell cultures. For virological verification of cases of mucosal disease, cp BVDV can be isolated from lesions in the small intestines or from ileal Peyer's patches. Organ samples should be collected aseptically and transported rapidly at refrigerator temperatures to the laboratory. BVDV is not destroyed by freezing, but if samples are frozen they should not be allowed to thaw during the transport. In principle, three different methods for detection of BVDV can be distinguished. The virus may be cultivated for one or more passages in cell cultures, which subsequently are fixed and stained with virus-specific antibodies for visualization of ncp BVDV. Alternatively, BVDV antigens can be detected with specific antibodies directly in organ samples without propagation of the virus. Using molecular biological methods, viral RNA can also be detected in a variety of biological samples. 5.1. Virus isolation in cell cultures BVDV can be replicated in many different types of cell cultures. Compared to many other viruses, the virus titres obtained in cell cultures are low, and may also vary considerably for different virus isolates. Therefore, optimization of the cell culture system is important if the popular micro-titre multi-well format for testing of series of samples is used. Among the most sensitive cell cultures are low-passage cultures of primary bovine kidney, turbinate and testis cells (Edwards, 1990). Fetal calf serum used to supplement the cell culture medium should be free from both BVDV and BVDVspecific antibodies. The sensitivity of a given cell culture system will primarily depend on the volume of the inoculum and the period of incubation. For detection of ncp BVDV in blood from PI animals, cells cultured in 96-well microtitre plates and inoculated with 10±50 ml serum for 4 days may give satisfactory results (Meyling, 1984; Sandvik and Krogsrud, 1995). If attempts to isolate BVDV from acutely infected animals are planned, blood leucocytes should be isolated and cocultivated with cells in larger culture bottles for two or more passages. Since cells infected with ncp BVDVs cannot be distinguished from uninfected controls, cultured cells are usually fixed and incubated with fluorochrome or enzyme labelled BVDV-specific antibodies. For assay of cp BVDV, cells cultured in flasks should be inoculated and examined daily for cytopathic effect. If present in low titres, foci of lysed cells can be seen by phase contrast microscopy, and if the cells are fixed at that stage of infection, viral antigen can be seen at the periphery of the foci. Otherwise, a cytopathogenic virus can be identified as BVDV e.g. by neutralization with BVDV-specific antibodies. Cross neutralization tests using sera raised against different virus strains may be used for demonstration of antigenic differences among isolated viruses. Further antigenic differentiation between BVDV types can be done with panels of Mabs (Paton et al., 1995).
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5.2. Detection of viral antigens The basic principle of this diagnostic method is identical to the cell culture propagation/immunostaining procedure referred to above, but instead of cultured cells for production of antigen, preformed antigen is either detected inside or extracted from cells taken from the host animal. By morphological recognition of cytoplasmatically located BVDV antigens in infected cells, immunohistochemical (IHC) examination of tissue sections provides a more specific diagnosis than detection of extracted antigens in a solution. Test results from IHC examination of skin biopsies has correlated well to findings from blood testing (ThuÈr et al., 1996), but tissue samples are more difficult to obtain from live animals than blood. IHC staining of tissue sections from brain cortex have also given reliable test results for calves PI with BVDV (Hewicker et al., 1990). These methods are most promising for postmortem diagnosis of persistent infection with BVDV, but should be first evaluated on samples from acutely infected cattle. Since blood is readily available and is also used for serology, many studies on BVDV antigen testing of blood have been performed. A study on flow cytometric screening of mononuclear leucocytes in peripheral blood showed that on average 11.2% of these cells were positive for BVDV antigens (Qvist et al., 1990). When this assay was applied to BVDV diagnostic work, it was more sensitive than the cell culture assay used as reference method (Qvist et al., 1991). For rapid and cell culture independent testing of large series of samples this test method could be remunerative, but the introduction of less costintensive ELISA systems has apparently discouraged wide use of the method. After BVDV specific Mabs became available, several antigen capture ELISAs (agELISA) were developed for rapid detection of BVDV antigens extracted from tissues or blood leucocytes (Fenton et al., 1991; Mignon et al., 1991; Shannon et al., 1991; Gottschalk et al., 1992). These agELISAs were based on either Mabs specific for one or more viral antigens for both capture and detection of antigen, or combinations of antisera and Mabs. Since they all recognize the antigenically conserved non-structural protein NS2-3 they should be able to detect most, if not all BVDV strains. The diagnostic performance, independence from cell culture facilities and speed of performance has made such agELISAs invaluable for testing of series of samples in organized control programmes. Initially, such an agELISA appeared to diagnose persistent infection with BVDV selectively (Greiser-Wilke et al., 1993), but later reports have concluded that blood from cattle acutely infected with BVDV also test positively (Brinkhof et al., 1996). Sandvik et al. (1997a) found low and transient antigen levels in five of 24 animals undergoing acute infection with BVDV, and that 99% of animals with high levels of BVDV antigen in blood leucocytes were PI with BVDV (Sandvik, 1997). However, conclusions towards status of infection on basis the of the performance of one agELISA can not be generally applied to other tests, or on test results in different populations of cattle. 5.3. Detection of viral nucleic acid The nucleotide sequencing of several pestivirus genomes has served as basis for a rapid progress in our understanding of the biology of these viruses
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(Donis, 1995). This field of pestivirology is still subject to intense research, but enough data for development of sensitive and specific diagnostic tests have been available for some years. The first reports on diagnostic detection of BVDV nucleic acid used radioactive probes for filter hybridization (Brock et al., 1988; Roberts et al., 1991) but there were no major improvements compared to established diagnostic methods. Later, numerous reports on use of the reverse transcription-polymerase chain reaction (RT-PCR) technique for amplification of BVDV cDNA have been published (reviewed by BelaÂk and Ballagi-PordaÂny, 1993; Sandvik et al., 1997b). Due to the more conserved nucleotide sequences in the 50 untranslated region and the NS3 gene of BVDV, RT-PCR assays using primers specific for these regions have offered the best epidemiological sensitivity. The high analytical sensitivity of double or nested RT-PCR assays is usually not required for routine testing of individual blood samples for BVDV. However, for some sample categories RT-PCR assays are very useful. These include blood from PI calves where the virus is neutralized by maternal antibodies, blood from acutely infected animals, as well as organ samples toxic to cell cultures. Another useful diagnostic application of the RT-PCR method is monitoring of cell cultures and fetal calf serum used as cell culture medium supplement, both of which may be contaminated with ncp BVDV (Bolin et al., 1991, 1994). Nucleotide sequencing of cDNA amplified e.g. by RT-PCR has been used for virological classification of closely related ruminant pestiviruses (Ridpath et al., 1994). These results coincide mostly with data obtained from Mab binding studies, but allow an even finer discrimination between the viruses examined. 6. Concluding remarks As contrasted to sporadic diagnostic investigations of disease conditions which may have been caused by infection with BVDV, control programmes are designed to obtain complete sets of information for herds suspected to be BVDV positive. To make most of the information obtained from laboratory diagnostic investigations, a thorough understanding of both the epidemiology of BVD as well as the performance of diagnostic tests is essential. For example, typical values for the sensitivity and specificity of an excellent agELISA for BVDV may be 97% and 99%, respectively, which means that 3% of the PI animals in a population are not detected. If combined and simultaneous antibody and antigen testing detect a non-viremic antibody negative animal together with 30 non-viremic antibody positive animals and one viremic antibody negative animal, it should be recognized as a clear candidate of the 3% group of missing PI animals, and retested, preferably also by cell culture inoculation or RT-PCR. Similarly, even without suspicion of false test results all investigated herds should be followed up by serological testing to verify that young stock remain seronegative after decline of maternal antibodies. If attempts to eliminate BVDV from individual herds fail, it should preferably be regarded as a failure of understanding of the nature of both diagnostic tests and the biology of BVDV rather than a failure of the employed tests.
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Radwan, G.S., Brock, K.V., Hogan, J.S., Smith, K.L., 1995. Development of a PCR amplification assay as a screening test using bulk milk samples for identifying dairy herds infected with bovine viral diarrhea virus. Vet. Microbiol. 44, 77±92. Ridpath, J.F., Bolin, S.R., Dubovi, E.J., 1994. Segregation of bovine viral diarrhea virus into genotypes. Virology 205, 66±74. Ridpath, J.F., Bolin, S.R., 1995. The genomic sequence of a virulent bovine viral diarrhea virus (BVDV) from the type 2 genotype. Detection of a large genomic insertion in a noncytopathic BVDV. Virology 212, 39± 46. Roberts, K.L., Collins, J.K., Carman, J., Blair, C.D., 1991. Detection of cattle infected with bovine viral diarrhea virus using nucleic acid hybridisation. J. Vet. Diagn. Invest. 3, 10±15. Sandvik, T., Krogsrud, J., 1995. Evaluation of an antigen capture ELISA for detection of bovine viral diarrhea virus in cattle blood samples. J. Vet. Diagn. Invest. 7, 65±71. Sandvik, T., 1997. Studies on diagnosis of pestivirus infections in cattle. Thesis, Norwegian College of Veterinary Medicine, Oslo. Sandvik, T., Fredriksen, B., Lùken, T., 1997a. Level of viral antigen in blood leucocytes from cattle acutely infected with bovine viral diarrhoea virus. J. Vet. Med. B 44, 583±590. Sandvik, T., Paton, D.J., Lowings, P.J., 1997b. Detection and identification of ruminant and porcine pestiviruses by nested amplification of 50 untranslated cDNA regions. J. Virol. Methods 64, 43±56. Shannon, A.D., Richards, S.G., Kirkland, P.D., Moyle, A., 1991. An antigen-capture ELISA detects pestivirus antigens in blood and tissues of immunotolerant carrier cattle. J. Virol. Methods 34, 1±12. ThuÈr, B., Zlinszky, K., Ehrensperger, F., 1996. Immunohistochemical detection of bovine viral diarrhea virus in skin biopsies: a reliable and fast diagnostic tool. J. Vet. Med. B 43, 163±166. Tijssen, P., 1985. Practice and theory of enzyme immunoassays. Elsevier, Amsterdam. Wolfmeyer, A., Wolf, G., Beer, M., Strube, W., Hehnen, H., Schmeer, N., Kaaden, O.-R., 1997. Antigenic and genetic diversity (50 untranslated region) of German and American bovine viral diarrhoea virus isolates. In: Edwards, S., Paton, D., Wensvoort, G. (Eds.), Proc. 3rd Symp. on Pestivirus Infections, Lelystad, September 1996, ESVV/CVL, Weybridge, UK, pp. 99±103.
Laboratory diagnostic investigations for bovine viral diarrhoea virus infections in cattle Torstein Sandvik*,1 Department of Virology and Serodiagnostics, National Veterinary Institute, Oslo, Norway
Abstract There are no pathognomonic clinical signs of infection with bovine viral diarrhoea virus (BVDV) in cattle. Diagnostic investigations therefore rely on laboratory-based detection of the virus, or of virus-induced antigens or antibodies in submitted samples. In unvaccinated dairy herds, serological testing of bulk milk is a convenient method for BVDV prevalence screening. Alternatively, serological testing of young stock may indicate if BVDV is present in a herd. In BVDV positive herds, animals persistently infected (PI) with BVDV can be identified by combined use of serological and virological tests for examination of blood samples. ELISAs have been used for rapid detection of both BVDV antibodies and antigens in blood, but should preferably be backed up by other methods such as virus neutralization, virus isolation in cell cultures or amplification of viral nucleic acid. Detailed knowledge of the performance of the diagnostic tests in use, as well as of the epidemiology of bovine virus diarrhoea is essential for identification of viremic animals in affected herds. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Bovine viral diarrhoea virus; BVDV; Cattle; Diagnosis; Pestivirus
1. Introduction If the success of a virus is measured by its ability to spread, cause disease and still persist within a population without being discovered, pestiviruses are perhaps the most successful of all bovine viruses. In most countries, large parts of the bovine population have been infected with one or more of them, which usually have been referred to as bovine viral diarrhoea virus (BVDV) (Baker, 1987). * Corresponding author. Tel.: +47 2296 4775; fax: +47 2296 4818; e-mail: [email protected] 1 Present address: Department of Pharmacology, Food hygiene and Microbiology, Norwegian College of Veterinary Medicine, Oslo, Norway. 0378-1135/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII S 0 3 7 8 - 1 1 3 5 ( 9 8 ) 0 0 2 6 4 - 8
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During the last decade, research in two fields resulted in major improvements of our knowledge of the biology of pestiviruses. The production of pestivirus-specific monoclonal antibodies (Mabs) provided important information on the nature of viral proteins and on the antigenic diversity of these viruses (Peters et al., 1986; Bolin et al., 1988; Donis et al., 1988; Edwards et al., 1988), but Mabs were also excellent tools for the development of diagnostic tests. Similarly, nucleotide sequencing of complete virus genomes improved our basic understanding of the biology of the viruses (Collett et al., 1988; Deng and Brock, 1992; Ridpath and Bolin, 1995), and allowed the application of powerful molecular genetic tests for detection and classification of different types of ruminant pestiviruses. Recent antigenic and genetic studies of bovine pestivirus isolates have shown that cattle may be infected with at least two different types of BVDV (Ridpath et al., 1994; Paton et al., 1995), and possibly also the ovine border disease virus (Edwards et al., 1988; Moussa et al., 1997). Most of the bovine pestivirus isolates appear to be BVDV type 1, but even within this type considerable antigenic differences have been demonstrated (Edwards, 1997). BVDV type 2 strains were initially recognized by their virulence (Pellerin et al., 1994), but isolates of lower virulence have also been assigned to this type (Wolfmeyer et al., 1997). Most field isolates of BVDV are non-cytopathogenic (ncp), but there are also cytopathogenic (cp) biotypes of the virus, which have been shown to be the cause of mucosal disease (Brownlie et al., 1984). Since most of the ruminant pestiviruses share the basic properties essential in the pathogenesis and epidemiology of bovine virus diarrhoea (BVD), diagnostic methods should be designed to recognize the whole range of viruses isolated from cattle. Together with the better understanding of the disease complex induced by BVDV, the currently available diagnostic tests have made disease control projects based on detection and removal of viremic animals from unvaccinated populations as realistic options to the more conservative vaccination and non-intervention strategies for control of BVD. 2. Properties of diagnostic tests Several terms are used to describe the performance of diagnostic tests (Martin et al., 1987). The diagnostic or epidemiological sensitivity of a test is defined as the percentage of true positives which test positively with the test. The epidemiological specificity is the percentage of true negatives recognized as such by the test. These two terms should not be mixed up with the analytical sensitivity and specificity, which refers to the amount of substance that can be detected with the test, and lack of false positive signals due to cross reactions, respectively. In both senses, the values for sensitivity and specificity should be as high as possible, but in practice they will be inversely related. Among other desired qualities are high repeatability and reproducibility, which ensure similar test results of aliquots repeatedly tested within and at separate laboratories. The predictive value of a test result, which describes the probability that animals which are diagnosed as positive or negative by a test actually have or not have the disease, is not a quality of the test only, but depends on both the epidemiological sensitivity and specificity of the test as well as the prevalence of the disease in the tested population.
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Newly introduced tests should preferably be evaluated against the established reference test methods (`gold standards'). For BVDV diagnostic work, these are virus isolation, and titration of virus neutralizing antibodies, both in cell cultures (Edwards, 1990). These tests are biological assays, which may be influenced e.g. by the susceptibility to infection of the cell cultures, the cell culture medium, the test protocol and format as well as the immunological labelling. Since the reference methods require considerable experience and efforts to provide reliable results, less complicated test methods such as enzyme immunoassays have become attractive alternatives for BVD testing of series of samples. 3. What is to be diagnosed? The pathogenesis of BVDV infections is complex, and results in a wide spectrum of conditions ranging in severity from asymptomatic to lethal (Baker, 1990). In most cases, the clinical signs are vague, and not sufficient for an aetiological diagnosis. One exception to this rule is classical cases of mucosal disease, which may require euthanasia before the diagnosis can be verified by laboratory tests. For most other cases, as well as for general information on the prevalence of BVD, laboratory examination of clinical samples is necessary to establish a diagnosis. In the current Scandinavian BVD control programmes, the aim of the organized diagnostic work is to identify and remove the animals that are sources of infection, which primarily are animals immunotolerant to and persistently infected (PI) with BVDV (Bitsch and Rùnsholt, 1995). To achieve this, identification of several categories of animals is necessary (Table 1). Clinically healthy PI animals older than 2 months are nearly always negative for antibodies to, and have a constant and high level of BVDV in the blood, most other tissues and in excreta such as saliva, feces, urine and milk. It is difficult to estimate the role other categories of BVDV carrying animals (Table 1; b, h and i) play in transmission of the virus, but their existence should be recognized, for both differential diagnostic reasons vs. PI animals and since they may be of importance in Table 1 Categories of animals in an unvaccinated bovine population where BVDV is prevalent, and usual results of testing for antibodies and virus in serum
a. b. c. d. e. f. g. h. i.
Category
Antibody
Virus
Uninfected, naive animals Acutely infected animals Immune animals after acute infection Passively immunized calves PI animals PI calves of immune dams Mucosal disease cases Pregnant cows carrying PI calf Immune bulls
ÿ ÿ ÿ ÿ/ /
ÿ /ÿ ÿ ÿ ÿ/ /ÿ ÿ ÿ
PI ± Persistently infected.
Comments Brief and low virus titre in blood Antibodies detectable for 5±9 months Antibodies detectable for 4±10 weeks Neutralizing antibodies High antibody titre in late pregnancy Semen may be virus positive
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maintenance of the virus in certain herds (Moerman et al., 1993). Also, reliable identification of uninfected herds and individual animals immune to infection is important to exclude these from unnecessary testing, and thereby improving the predictive value of the test result for the herds undergoing full testing. Since all diagnostic investigations for BVD should be conducted with the herd as the epidemiological unit of interest, the first task is to identify herds with PI animals. For dairy herds not vaccinated against BVDV, bulk milk testing for antibodies has proven to be a very useful and cost-effective way of tracing herds likely to house PI animals (Niskanen, 1993). Alternatively, antibody testing of blood samples from selected age groups of a herd will also give information on the time of infection of the herd (Bitsch and Rùnsholt, 1995), but such samples are not as easy to collect as bulk milk. In vitro gene amplification techniques have also been used for the detection of viral nucleic acid in bulk milk (Radwan et al., 1995; Drew et al., 1999). The advantage of this approach is that it may be used in vaccinated herds, but since not all PI animals are lactating this approach of screening is not optimal, and may serve as an example of a test with an excellent analytic, but intermediate epidemiological sensitivity. For further testing of herds likely to have PI animals, individual blood samples remain as the best sample material for laboratory diagnostic work. Since different test protocols may require clotted or anticoagulated blood, the diagnostic laboratory should be consulted before samples are collected. Depending on the performance of the applied diagnostic tests, all samples should be tested for both antibodies to and for BVDV; only for BVDV, or screened for antibodies before samples without high antibody levels are tested for BVDV. Usually, the criteria for a diagnosis of persistent infection with BVDV have been repeated isolation of BVDV from blood without seroconversion in samples drawn 3±4 weeks apart. With additional information on the status of infection of all animals in a herd, sampled once at the same time, there is circumstantial evidence that a given viremic, antibody negative individual is PI with BVDV when all other animals in contact have high levels of antibodies to the virus. In animals acutely infected with BVDV, the virus titres in blood are usually low and of short duration, and BVDV is only rarely isolated if blood samples are drawn at random. If acute infection with BVDV is suspected, paired blood samples drawn at least 3±4 weeks apart should be examined for antibodies to the virus, and the diagnosis should be based on seroconversion. To verify that all PI animals have been removed from a herd, young stock between 8± 12-months old should be tested for BVDV antibodies. This group of animals are normally free from maternal antibodies, and have been at risk for acute infection with BVDV for some time. For many years, laboratory diagnostic investigations for BVDV have been complicated by false positives due to contamination of cell cultures with ncp BVDV, usually from the fetal calf serum used to supplement cell culture media (Bolin et al., 1991). Similarly, BVDV neutralizing antibodies in fetal calf serum may also inhibit replication of virus and conceal the presence of BVDV in sample material. Monitoring to ensure freedom from contamination with BVDV, as well as to ascertain satisfactory susceptibility to infection of the cell cultures are therefore essential quality control measures for all BVDV diagnostic work.
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4. Detection of immune responses against BVDV Immune responses against an infectious agent are classified as cellular or humoral, and immunity may be acquired actively by infection or passively by transfer of colostral antibodies. Cellular immunity, measured as proliferation of peripheral blood mononuclear cells after in vitro exposure to BVDV, has been demonstrated for seropositive cows, but not for a calf believed to be passively immunised with colostral antibodies (Larsson and Fossum, 1992). However, most studies on detection of immune responses against BVDV have concentrated on demonstration of antibodies to the virus, which can be detected in serum from 3 weeks, and thereafter for years after acute infection of immunocompetent animals (Howard, 1990). BVDV specific antibodies can be classified in two functional groups. Antibodies to viral glycoproteins (primarily E2 or gp53) may block the infectivity of or neutralize the virus. These antibodies cross react with other strains of BVDV, but if sera raised against one virus strain are tested against other challenge viruses, different antibody titres may be obtained (Brock, 1995). Conversely, the highly immunogenic nonstructural viral protein NS2-3 (p125), which is essential for the intracellular replication of the virus, is antigenically conserved between all pestiviruses (Collett, 1992). Antibodies to NS2-3 do not neutralize BVDV, but since they can be readily detected by other serological tests, this antigen is important in BVDV serology. If effective recombinant sub-unit vaccines against BVDV become available, simultaneous vaccination and anti-NS2-3 serology for monitoring of natural infection with BVDV should be possible. 4.1. Virus neutralization test When properly calibrated, virus neutralization (VN) assays are sensitive and specific assays for detection of antibodies to BVDV, and have long been recognized as the reference test for BVDV serology. Today, the test is usually performed in 96-well microtitre plates, where serially diluted test sera are incubated with a cytopathogenic challenge virus before susceptible bovine cells are added to and incubated with the neutralized virus for 4 days (Edwards, 1990). Because of the high specificity of the test, it is essential to use a challenge virus antigenically similar to the field viruses in the population to be tested. The VN assay requires substantial investments in selection and monitoring of cell cultures and media to give satisfactory test results, and is therefore, not suitable for examination of few or sporadic samples. 4.2. Enzyme immunoassays Enzyme-linked immunosorbent assays (ELISAs) are versatile diagnostic methods which can be designed to detect almost any immunoreactive molecule. For BVDV serology they have become popular for several reasons; they are independent of cell cultures, they can easily be applied for mass screening, test results can be read in few hours and reliable results can be obtained with milk as test material (Niskanen et al., 1989). Two principally different protocols are possible, activity amplification (AA) or activity modulation (AM) systems (Tijssen, 1985). Among commonly used AA systems
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are indirect ELISAs, where antigen is immobilized and used to trap specific antibodies, which subsequently are detected by enzyme-conjugated species-specific antiglobulins and a chromogenic substrate. The specificity of such systems is determined by the choice of viral antigen, which may be purified virus particles (Chu et al., 1985), detergent extracts of cultured cells inoculated with BVDV (Howard et al., 1985), single viral antigens immobilized with Mabs (Moennig et al., 1991) or recombinant virus proteins produced in bacteria (Kwang et al., 1995). In AM systems, virus-specific antibodies in the test sample compete with or block the binding of conjugated virus-specific antibodies, resulting in a lowered signal for positive samples (Paton et al., 1991). When the performance of such ELISAs are compared to that of a VN assay, only those using purified virus particles as antigen can be expected to give equal results, but still, acceptable agreements have usually been found for anti NS2-3 ELISAs (Moennig et al., 1991). If ELISAs for BVDV serology are to be developed in-house, a convenient source of antigen is cell cultures inoculated with BVDV, e.g. as described by Edwards (1990). ELISA kits are also available commercially. Among problems observed with AA ELISAs for BVDV serology are high and variable background signals caused by contaminations in the test antigen, or cross reactivity of the enzyme-conjugated antiglobulins resulting in low signal-to-noise ratios. AM ELISAs based on blocking of enzyme-conjugated Mabs can be designed to have a high analytical specificity, but it is essential that the employed Mabs recognize all virus strains in order to ensure a sufficient epidemiological sensitivity. 4.3. Other serological tests Many serological tests have been adopted for BVDV serology, including immunodiffusion in agar gels, complement fixation, indirect immunofluorescence, and western blotting. Agar gel immunodiffusion detects primarily antibodies to the NS2-3 antigen, but since the analytical sensitivity is low, it performs best as a simple screening assay (Edwards, 1990). Similarly, the analytical sensitivity of the complement fixation test is not very high, but since the antibodies detected with this assay appear and decline earlier than neutralizing or precipitating antibodies it may be used for the early detection of antibodies in seroconverting animals (Gutekunst and Malmquist, 1964). The indirect immunofluorescence test, in which BVDV-infected cells fixed on microscope slides are used as antigen, is similar to indirect AA ELISAs, but since it has to be read manually, the specificity added by recognition of a typical pattern of infected cell cultures is outweighed by the time consumed by examination of many samples. However, it is an useful alternative test method for sera cytotoxic to cell cultures or giving high background signals in ELISAs. Technically, western blotting is also similar to some ELISAs, but provides information on the molecular specificity of the antibodies. 5. Detection of the virus or viral components BVDV is not among the most fragile viruses, but still, reliable results from virological testing will only be obtained with suitable sample material. If clinical cases are to be
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investigated for BVDV, the choice and handling of samples may influence the success of assays for BVDV. Nasal secretions and faeces are important sources of infection for other animals, but since they are contaminated with other microbes and substances toxic to cell cultures, blood should instead be collected from live animals. From carcasses, samples of spleen, liver, lung or kidneys are suitable for isolation of BVDV in cell cultures. For virological verification of cases of mucosal disease, cp BVDV can be isolated from lesions in the small intestines or from ileal Peyer's patches. Organ samples should be collected aseptically and transported rapidly at refrigerator temperatures to the laboratory. BVDV is not destroyed by freezing, but if samples are frozen they should not be allowed to thaw during the transport. In principle, three different methods for detection of BVDV can be distinguished. The virus may be cultivated for one or more passages in cell cultures, which subsequently are fixed and stained with virus-specific antibodies for visualization of ncp BVDV. Alternatively, BVDV antigens can be detected with specific antibodies directly in organ samples without propagation of the virus. Using molecular biological methods, viral RNA can also be detected in a variety of biological samples. 5.1. Virus isolation in cell cultures BVDV can be replicated in many different types of cell cultures. Compared to many other viruses, the virus titres obtained in cell cultures are low, and may also vary considerably for different virus isolates. Therefore, optimization of the cell culture system is important if the popular micro-titre multi-well format for testing of series of samples is used. Among the most sensitive cell cultures are low-passage cultures of primary bovine kidney, turbinate and testis cells (Edwards, 1990). Fetal calf serum used to supplement the cell culture medium should be free from both BVDV and BVDVspecific antibodies. The sensitivity of a given cell culture system will primarily depend on the volume of the inoculum and the period of incubation. For detection of ncp BVDV in blood from PI animals, cells cultured in 96-well microtitre plates and inoculated with 10±50 ml serum for 4 days may give satisfactory results (Meyling, 1984; Sandvik and Krogsrud, 1995). If attempts to isolate BVDV from acutely infected animals are planned, blood leucocytes should be isolated and cocultivated with cells in larger culture bottles for two or more passages. Since cells infected with ncp BVDVs cannot be distinguished from uninfected controls, cultured cells are usually fixed and incubated with fluorochrome or enzyme labelled BVDV-specific antibodies. For assay of cp BVDV, cells cultured in flasks should be inoculated and examined daily for cytopathic effect. If present in low titres, foci of lysed cells can be seen by phase contrast microscopy, and if the cells are fixed at that stage of infection, viral antigen can be seen at the periphery of the foci. Otherwise, a cytopathogenic virus can be identified as BVDV e.g. by neutralization with BVDV-specific antibodies. Cross neutralization tests using sera raised against different virus strains may be used for demonstration of antigenic differences among isolated viruses. Further antigenic differentiation between BVDV types can be done with panels of Mabs (Paton et al., 1995).
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5.2. Detection of viral antigens The basic principle of this diagnostic method is identical to the cell culture propagation/immunostaining procedure referred to above, but instead of cultured cells for production of antigen, preformed antigen is either detected inside or extracted from cells taken from the host animal. By morphological recognition of cytoplasmatically located BVDV antigens in infected cells, immunohistochemical (IHC) examination of tissue sections provides a more specific diagnosis than detection of extracted antigens in a solution. Test results from IHC examination of skin biopsies has correlated well to findings from blood testing (ThuÈr et al., 1996), but tissue samples are more difficult to obtain from live animals than blood. IHC staining of tissue sections from brain cortex have also given reliable test results for calves PI with BVDV (Hewicker et al., 1990). These methods are most promising for postmortem diagnosis of persistent infection with BVDV, but should be first evaluated on samples from acutely infected cattle. Since blood is readily available and is also used for serology, many studies on BVDV antigen testing of blood have been performed. A study on flow cytometric screening of mononuclear leucocytes in peripheral blood showed that on average 11.2% of these cells were positive for BVDV antigens (Qvist et al., 1990). When this assay was applied to BVDV diagnostic work, it was more sensitive than the cell culture assay used as reference method (Qvist et al., 1991). For rapid and cell culture independent testing of large series of samples this test method could be remunerative, but the introduction of less costintensive ELISA systems has apparently discouraged wide use of the method. After BVDV specific Mabs became available, several antigen capture ELISAs (agELISA) were developed for rapid detection of BVDV antigens extracted from tissues or blood leucocytes (Fenton et al., 1991; Mignon et al., 1991; Shannon et al., 1991; Gottschalk et al., 1992). These agELISAs were based on either Mabs specific for one or more viral antigens for both capture and detection of antigen, or combinations of antisera and Mabs. Since they all recognize the antigenically conserved non-structural protein NS2-3 they should be able to detect most, if not all BVDV strains. The diagnostic performance, independence from cell culture facilities and speed of performance has made such agELISAs invaluable for testing of series of samples in organized control programmes. Initially, such an agELISA appeared to diagnose persistent infection with BVDV selectively (Greiser-Wilke et al., 1993), but later reports have concluded that blood from cattle acutely infected with BVDV also test positively (Brinkhof et al., 1996). Sandvik et al. (1997a) found low and transient antigen levels in five of 24 animals undergoing acute infection with BVDV, and that 99% of animals with high levels of BVDV antigen in blood leucocytes were PI with BVDV (Sandvik, 1997). However, conclusions towards status of infection on basis the of the performance of one agELISA can not be generally applied to other tests, or on test results in different populations of cattle. 5.3. Detection of viral nucleic acid The nucleotide sequencing of several pestivirus genomes has served as basis for a rapid progress in our understanding of the biology of these viruses
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(Donis, 1995). This field of pestivirology is still subject to intense research, but enough data for development of sensitive and specific diagnostic tests have been available for some years. The first reports on diagnostic detection of BVDV nucleic acid used radioactive probes for filter hybridization (Brock et al., 1988; Roberts et al., 1991) but there were no major improvements compared to established diagnostic methods. Later, numerous reports on use of the reverse transcription-polymerase chain reaction (RT-PCR) technique for amplification of BVDV cDNA have been published (reviewed by BelaÂk and Ballagi-PordaÂny, 1993; Sandvik et al., 1997b). Due to the more conserved nucleotide sequences in the 50 untranslated region and the NS3 gene of BVDV, RT-PCR assays using primers specific for these regions have offered the best epidemiological sensitivity. The high analytical sensitivity of double or nested RT-PCR assays is usually not required for routine testing of individual blood samples for BVDV. However, for some sample categories RT-PCR assays are very useful. These include blood from PI calves where the virus is neutralized by maternal antibodies, blood from acutely infected animals, as well as organ samples toxic to cell cultures. Another useful diagnostic application of the RT-PCR method is monitoring of cell cultures and fetal calf serum used as cell culture medium supplement, both of which may be contaminated with ncp BVDV (Bolin et al., 1991, 1994). Nucleotide sequencing of cDNA amplified e.g. by RT-PCR has been used for virological classification of closely related ruminant pestiviruses (Ridpath et al., 1994). These results coincide mostly with data obtained from Mab binding studies, but allow an even finer discrimination between the viruses examined. 6. Concluding remarks As contrasted to sporadic diagnostic investigations of disease conditions which may have been caused by infection with BVDV, control programmes are designed to obtain complete sets of information for herds suspected to be BVDV positive. To make most of the information obtained from laboratory diagnostic investigations, a thorough understanding of both the epidemiology of BVD as well as the performance of diagnostic tests is essential. For example, typical values for the sensitivity and specificity of an excellent agELISA for BVDV may be 97% and 99%, respectively, which means that 3% of the PI animals in a population are not detected. If combined and simultaneous antibody and antigen testing detect a non-viremic antibody negative animal together with 30 non-viremic antibody positive animals and one viremic antibody negative animal, it should be recognized as a clear candidate of the 3% group of missing PI animals, and retested, preferably also by cell culture inoculation or RT-PCR. Similarly, even without suspicion of false test results all investigated herds should be followed up by serological testing to verify that young stock remain seronegative after decline of maternal antibodies. If attempts to eliminate BVDV from individual herds fail, it should preferably be regarded as a failure of understanding of the nature of both diagnostic tests and the biology of BVDV rather than a failure of the employed tests.
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