Aspects of the epidemiology, research, and control of lentiviral infections of small ruminants and their relevance to Dutch sheep and goat farming.

In 1862, the veterinarian Loman reported the first sheep in The Netherlands with symptoms associated with lentiviral infection, although at the time the symptoms were ascribed to ovine progressive pneumonia. In the following century, similar cases were reported by South African, French, American, and Icelandic researchers. Extensive research into the pathology, aetiology, and epidemiology of this slowly progressive and ultimately fatal disease was initiated in several countries, including the Netherlands. Studies of the causative agents--maedi visna virus (MVV) in sheep and caprine arthritis encephalitis virus (CAEV) in goats, comprising the heterogeneous group of the small ruminant lentiviruses (SRLV)--prompted the development of diagnostic methods and the initiation of disease control programmes in many European countries including the Netherlands, as a pioneer in 1982, and in the U.S.A. and Canada.

Diagnostic performance of ELISA and PCR in identifying SRLV-infected sheep and goats using serum, plasma and milk samples and in early detection of infection in dairy flocks through bulk milk testing.

The objective of the study was to evaluate the diagnostic performances of the ELITEST-MVV ELISA for detection of antibodies against small ruminant lentiviruses and of two recently published PCRs for the detection of proviral DNA of SRLV in blood and corresponding individual milk samples. In addition, the feasibility of bulk milk testing was investigated by titrating ELISA positive pooled milk samples in negative milk, and by investigating bulk milk samples by ELISA and PCR in relation to the SRLV-status of the flocks. The results show that plasma and milk are suitable replacements for serum. For sheep, both PCRs showed a better diagnostic performance than for goats. ELISA results for bulk milk samples were promising with a putative detection limit of <3% within-herd prevalence using 1/10 pre-diluted samples and even <1% within-herd prevalence when samples were tested undiluted. In a panel of 249 bulk milk samples, all samples from SRLV free flocks (n=138) tested negative in the ELISA, while 50% of the samples from flocks with an unknown SRLV-status (n=111) were positive. For a subset of 59 bulk milk samples, agreement between ELISA results and leader-gag PCR results was almost 100%. These results demonstrate the potential of bulk milk testing as a cost effective tool for early detection of infection in dairy flocks, which is essential for SRLV-monitoring programs.

Use of serology and polymerase chain reaction for the rapid eradication of small ruminant lentivirus infections from a sheep flock: a case report.

A SRLV-free sheep flock incurred infection which led to an SRLV infection rate of over 50% of the ewes (34/64) within a 30 months period, indicating that environmental conditions were favourable to transmission. An intensive regimen of sampling at short intervals and testing for SRLV antibodies and proviral DNA combined with strict management was implemented for the entire flock, lambs and yearlings included. This resulted in eradication of the infection within two testing and culling rounds with a 3 months interval. The additional value of the proviral DNA detection by PCR in identifying infected animals was clear in that nine infected animals were found that would have been missed if tested by serology alone. PCR also saved two lambs from being culled; they were sero-positive probably due to maternal antibodies, but not infected.

Presence of pro-lentiviral DNA in male sexual organs and ejaculates of small ruminants.

To be able to predict sexual transmissibility of small ruminant lenti viruses (SRLV), it is necessary to know whether or not the virus is excreted in the semen, and under what circumstances. Thus, this research focussed on establishing the presence of proviral DNA of SRLV in semen and in the male genital tract of small ruminants. After initial results established the presence of SRLV in serum, the emergence of proviral DNA of SRLV in semen and presence in blood in a group of naturally SRLV-infected individuals (13 rams and 4 bucks), was followed temporally using real-time polymerase chain reaction (PCR). The same animals were also systematically serologically monitored by enzyme-linked immuno sorbent assay (ELISA) during the breeding season (August-February). A triple monocyte-macrophage count was performed on both blood and semen using a specific monoclonal antibody in conjunction with flow cytometry. The finding that epididymal semen and tissue samples of the testes, epididymides, ampullary, vesicular, prostate and bulbo-urethral glands all tested positive for the presence of proviral DNA indicates that various male sexual organs may contribute directly to shedding of proviral SRLV DNA in ejaculated semen. Our results suggest that small ruminants show intermittent shedding of proviral SRLV DNA into epididymal as well as ejaculated semen. They also demonstrate that a single PCR-negative semen sample cannot be used as a diagnostic tool to predict that subsequent ejaculates will be SRLV-free. No significant relationship was found between numbers of monocytes and/or macrophages in blood or semen and the detection of proviral SRLV in ejaculates.

Specific detection of small ruminant lentiviral nucleic acid sequences located in the proviral long terminal repeat and leader-gag regions using real-time polymerase chain reaction.

Real-time polymerase chain reaction (RT-PCR) detection of proviral nucleic acid sequences of small ruminant lentiviruses (SRLV) in blood samples was developed and evaluated. Priming oligonucleotides were designed on the highly conserved 5' untranslated leader-gag region while those on the long terminal repeat (LTR) assay were derived from literature. DNA was extracted from the buffycoat interlayer of centrifuged blood samples. Real-time PCR was performed by means of LightCycler technology (Roche Applied Science) using melting temperature analysis (SYBR Green I) for detection. Results were compared with those of serology using samples from Dutch sheep and goat flocks with known SRLV statuses, with sequential samples from a natural transmission experiment and samples from different regions in Norway, France, Spain and Italy. Real-time PCR testing, especially the application of oligonucleotides for priming the leader-gag region appeared promising in detecting SRLV specific proviral DNA in blood samples from both sheep and goats.

Evaluation of five enzyme-linked immunosorbent assays and an agar gel immunodiffusion test for detection of antibodies to small ruminant lentiviruses.

In the framework of the Dutch control program for small ruminant lentiviral (SRLV) infections, too many drawbacks were encountered with respect to serological testing. To improve the quality of testing, five enzyme-linked immunosorbent assays (ELISAs) and an agar gel immunodiffusion test (AGIDT) were evaluated. The focus was on the sensitivity, specificity, and variances of the commercially available tests. Clear differences were found among the tests in analytical and diagnostic sensitivity and overall diagnostic performance, whereas no significant differences in specificity were found. For serodiagnosis of sheep with clinical symptoms of maedi-visna virus (MVV) (histopathologically confirmed), one ELISA was significantly more sensitive than the other ELISAs and than the AGIDT, while for asymptomatic sheep originating from infected flocks, three ELISAs and the AGIDT demonstrated similar performance. The diagnostic performance appeared to be related to animal species and virus infection (MVV or caprine arthritis encephalitis virus [CAEV]) as well as the phase of infection/progression of disease. Receiver operating characteristic analysis, demonstrating the diagnostic potential of tests irrespective of defined cutoffs, again revealed clear differences between tests with respect to diagnostic performance for detection of antibodies against CAEV or MVV. An indirect ELISA, of which the solid phase is sensitized with a combination of the core protein p27 of MVV produced in Escherichia coli and a peptide derived from the transmembrane protein gp46, appeared to be the test of choice for serodiagnosis of SRLV infections in sheep and goats.

The effect of maternal antibodies on the detection of bovine virus diarrhoea virus in peripheral blood samples.

Persistently infected animals (PI animals), that is those animals born after an intrauterine infection of the dam during the first 120 days of gestation, are the main source of bovine virus diarrhoea virus (BVD virus) in a cattle population. The success of any BVD virus eradication programme depends on the ability to detect all PI animals at a young age. The purpose of this study was to evaluate the use of the antigen ELISA test and the reverse transcriptase-polymerase chain reaction (RT-PCR) test for the diagnosis of PI animals in the presence of maternal antibodies, and to compare them with the classical virus isolation test. In this experiment, 25 calves born after an experimental infection with a mixture of BVD virus field strains were used. All calves were found to be positive for BVD virus using the virus isolation test, both before the ingestion of colostrum and again at 10 weeks of age. Both the virus isolation test and the antigen ELISA test were shown to be unreliable indicators for the diagnosis of persistent infections with BVD virus, when used in the presence of high levels of maternal antibodies. However, the RT-PCR test gave positive results even in the presence of high maternal antibody titres, indicating the suitability of the RT-PCR test for use in eradication programmes.

Failure of foetal protection after vaccination against an experimental infection with bovine virus diarrhea virus.

A protocol is described to measure the protection of the bovine fetus against an experimental bovine virus diarrhea virus (BVDV) infection after vaccination. Two inactivated experimental vaccines were applied twice with a 3 week interval. A mixture of three different Dutch field strains was used as challenge on mainly the 82nd day of gestation to vaccinated and unvaccinated control animals. The challenge was applied 5 months after completion of the two-fold vaccinations. All calves born from unvaccinated control animals were persistently infected. The calves born from dams vaccinated with the two different inactivated BVDV vaccines were persistently infected in 78 and 60%, respectively.

A comparison of four serological tests for the diagnosis of caseous lymphadenitis in sheep and goats.

A double antibody sandwich ELISA (ELISA A) developed for the detection of Corynebacterium pseudotuberculosis infection in sheep and goats was modified to improve its sensitivity. To establish the sensitivity and specificity of this modified ELISA (ELISA B), sera from 183 sheep and 186 goats were tested using ELISAs A and B. Comparison was also made with two further ELISAs (C and D) developed in Australia that, respectively, detect antibodies to cell wall antigens or toxin.ELISA B had the best performance of the four tests. Its specificity was 98+/-1% for goats and 99+/-1% sheep. Its sensitivity was 94+/-3% for goats and 79+/-5% for sheep. ELISA B will now be tested for use in caseous lymphadenitis eradication and control programmes in The Netherlands. It will also be used in experimental studies of CL in Scotland.

An equine herpesvirus 1 (EHV1) abortion storm at a riding school.

An outbreak of EHV1 abortions occurred at a riding school in The Netherlands in 1991. Seven of twelve pregnant mares aborted, and another foal died at 8 days of age. Six abortions occurred within 12 days in March after an initial abortion on 8 February. Four mares delivered live foals. Virological examination of four aborted foals revealed an EHV1 infection. Serological results for paired sera from 17 horses suggested, that the initial abortion on 8 February was the index case, and probably caused the other six abortions. The index case could well have been caused by reactivation of latent virus induced by transport stress. The laboratory results are discussed in the light of the present knowledge of the pathogenesis and epidemiology of EHV1 abortion.

Differentiation and genomic and antigenic variation among fetal, respiratory, and neurological isolates from EHV1 and EHV4 infections in The Netherlands.

Ten monoclonal antibodies (MAbs) were produced against equine herpes virus type 1 (EHV1). Two appeared type-specific, while the other eight were directed against epitopes common to both EHV1 and EHV4. Two MAbs directed against the glycoprotein gp2 recognized linear epitopes, as demonstrated by Western blotting. With pools of type-specific MAbs, 282 field isolates were typed in an immunoperoxidase monolayer assay (IPMA). From a total of 254 fetal or neonatal isolates, 244 (96%) were typed as EHV1, whereas 14 out of 15 (93%) respiratory tract isolates were typed as EHV4. Surprisingly, 3 out of 13 isolates (23%) originating from horses with neurological disease were typed as EHV4. No antigenic differences were found among 75 randomly selected EHV1 field isolates, using the panel of ten MAbs and six additional MAbs, directed against gp2, gB, or gC. Typing by restriction endonuclease analysis with BamHI corresponded completely with that of MAb analysis. There was a remarkable degree of uniformity in BamHI restriction patterns, with 90% of the investigated EHV1 isolates belonging to the 1P electropherotype. Among 30 randomly selected EHV1 isolates we could not identify the EHV1.1B electropherotype, which has been the predominant electropherotype in Kentucky since 1982. Mobility differences were seen in fragments originating from the repeat regions. These differences were not caused by heterologous cell passage, since all viruses were passaged in equine cell systems.

Seroepidemiological evidence for a relationship between Neospora caninum infections in dogs and cattle.

Dogs from dairy farms with a known prevalence of Neospora caninum antibodies in the cattle were examined for the presence of N. caninum antibodies using an ELISA. Data of farm dogs were compared with those of dogs examined at a university clinic, which originated mainly in urban areas. Of the 152 farm dogs, 36 (23.6%) were seropositive to N. caninum, which was significantly higher than the proportion of seropositives in the clinic dog population (19 of 344, 5.5%). Seroprevalence was significantly higher (P = 0.01) in female dogs than in male dogs. Seroprevalence in dogs increased with age, indicating postnatal infection. Seropositivity to N. caninum in farm dogs was strongly correlated with a high prevalence of N. caninum antibodies in the cattle. At farms where no dogs were present, the seroprevalence to N. caninum in the cattle was significantly lower (P = 0.0002) than in farms where dogs were present. These findings suggest that there is a relationship between N. caninum infection of farm dogs and cattle. Since dogs have been shown to be definitive hosts of N. caninum, cattle may be infected by exposure to canine oocysts. Further research is needed to find out whether and how dogs may acquire the infection from cattle.

A simple, rapid and reliable enzyme-linked immunosorbent assay for the detection of bovine virus diarrhoea virus (BVDV) specific antibodies in cattle serum, plasma and bulk milk.

To detect Bovine Virus Diarrhoea Virus (BVDV)-specific antibodies in cattle serum, plasma and bulk milk, a simple, reliable and rapid blocking ELISA ("Ceditest") has been developed using two monoclonal antibodies ("WB112" and "WB103") directed to different highly conserved epitopes on the non-structural peptide NS3 of pestiviruses. The test can be performed at high reproducibility using undiluted samples. In testing 1000 field serum samples, the ELISA showed a specificity and a sensitivity relative to the virus neutralization test of 99% and 98%, respectively. The blocking ELISA is able to detect specific antibodies in serum obtained 12 days after an acute infection and in serum of vaccinated and challenged animals. A frequency distribution diagram, obtained by testing almost 1800 random Dutch field serum samples, showed a clear separation between a negative population (maximum frequency of the % inhibition at -5%) and a positive population (maximum frequency of the % inhibition at 95%). Based on these data, the prevalence of seropositive animals was 65% (95% confidence interval: 63%-67%). By exchanging plasma- and bulk milk samples between two laboratories (The Netherlands and Denmark), a good overall agreement was found between results obtained with the Ceditest and those obtained with the Danish blocking ELISA as used in the Danish BVDV eradication programme. The results indicate that BVDV infections can reliably be diagnosed by the Ceditest ELISA and that the test is suitable for use in large scale screening and eradication programmes.

Serodiagnosis of neosporosis in individual cows and dairy herds: A comparative study of three enzyme-linked immunosorbent assays.

The performance of three enzyme-linked immunosorbent assays (ELISA) for detection of antibodies to Neospora caninum in bovine sera was evaluated by using various categories of sera. Two commercial ELISA methods, one based on chemically fixed intact tachyzoites and one based on a sonicate lysate of whole tachyzoites, were compared with an in-house ELISA based on a detergent lysate of whole tachyzoites. A brief description of the development of the latter ELISA is also given. There was good agreement among all three tests with regard to postabortion sera. By using acute-phase abortion sera from cows with confirmed N. caninum-induced and non-N. caninum-induced abortions, satisfactory levels of sensitivity and specificity were calculated for all tests. In addition, similar test results were obtained with postpartum samples from dams and calves. However, considerable differences were found between test results of sequential samples and cross-sectional and total-herd samples. Apparently, these discrepancies were due to different sensitivities of the tests for detection of low antibody levels in chronically infected animals. It is suggested that these differences were primarily due to the use of different antigens and different test sample dilutions. It is concluded that all tests are applicable as an additional diagnostic tool in cases of abortion in cattle and for monitoring of congenitally infected calves. For herd screening, the lysate-based ELISAs appear to be more adequate because of their higher sensitivities.

A comparative study of serological tests for use in the bovine herpesvirus 1 eradication programme in The Netherlands.

We compared a gB-ELISA, a gE-ELISA and a Danish test system (consisting of a blocking and an indirect ELISA) for their specificity and sensitivity to detect antibodies against BHV1. The Danish test system showed the highest sensitivity and the gE-ELISA the lowest; the gB-ELISA showed an intermediate sensitivity. If the doubtful zone (25-50% blocking) of the gB-ELISA was considered as positive (gB-ELISA+), the sensitivity almost reached that of the Danish test system. The specificity of all tests appeared to be very high, 99.7, 96.7, 100, 99.7% for the gB-ELISA, gB-ELISA+, gE-ELISA and the Danish test system, respectively. Seroconversion was detected in the gE-ELISA up to 3 weeks later than in the gB-ELISA and the Danish test system. It is concluded that the combination of a gB-ELISA (for screening) and the Danish test (for confirmation) system used in the BHV1 eradication programme in the Netherlands, provides for very high sensitivity (> 99.0%) (Kramps et al., 1994) and a very high specificity (> 99.9%).

Comparative study on four enzyme-linked immunosorbent assays and a cocultivation assay for the detection of antigens associated with the bovine viral diarrhoea virus in persistently infected cattle.

Four commercially available ELISAs for detection of antigens associated with the bovine viral diarrhoea virus in persistently infected cattle have been compared. The tests are equally specific (100%) and the sensitivity of three ELISAs is comparable with that of a conventional cocultivation assay. Performing ELISA on samples from young animals that received colostrum may yield false negative results because of interference of maternal antibodies in the tests. It is concluded that ELISA can be a valuable tool in eradications programs when large numbers of cattle are to be monitored.

Activation of complement by bovine respiratory syncytial virus-infected cells.

Because complement activation is probably involved in the pathogenesis of as well as in recovery from the disease induced by bovine respiratory syncytial virus (BRSV), we studied the activation of complement by BRSV-infected cells in vitro in a homologous system. Binding of C3 on the surface of infected cells was measured in a biotin-streptavidin amplified ELISA, and complement-mediated lysis was measured in a 51Cr release assay. Without antibody, infected cells activated and bound more C3 than uninfected cells. C3 activation that occurred in the absence of antibody was largely mediated by the classical pathway and induced lysis inefficiently. BRSV-specific antibody enhanced complement activation as measured by both C3 ELISA and cytotoxicity assay. In the presence of antibody, C3 activation was largely dependent on the alternative pathway and efficiently induced lysis. Both IgG1 and IgM antibodies enhanced C3 activation, but IgG2 and IgA did not enhance C3 activation in our experiments. Preincubating cells with IgA or IgG2 did not inhibit C3 activation enhanced by IgG1 or IgM. Murine monoclonal IgG1 antibodies against epitopes on the Fusion protein of the virus also enhanced C3 binding, but differed in their capacity to induce complement-mediated lysis.

Analysis of the antibody response to bovine respiratory syncytial virus proteins in calves.

The antibody response in calves to natural infections with bovine respiratory syncytial virus (BRSV) was analysed by radioimmunoprecipitation assays. Antibodies to virus proteins of Mr 200K (L), 87K (G), 46K (F1), 41K (N), 35K (P), 28K and 24K (F2), 27K (M), 22K and less than 14K could be identified. Recovery of 6- to 7-month-old calves from severe BRSV-associated disease was accompanied by the development of an antibody response to the virus, which was directed mainly o the F and N proteins. Calves of 2 to 3 weeks of age possessed moderate levels of maternal antibodies to BRSV particularly directed to the F and N proteins but became seriously ill after infection. The antibody response in these calves was severely suppressed. In the sera of 4- to 9-month-old calves that died in the course of infection, high antibody levels to the virus were found, which were directed at least to the F and N proteins. The presence or development of antibodies to the F and N proteins appears insufficient for protection against or recovery from BRSV infections.

An enzyme-linked immunosorbent assay for detection of antibodies to porcine parvovirus.

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of antibodies to porcine parvovirus (PPV). Antisera to PPV were raised in pigs, for which PPV grown on PK15 cells was used for primary intranasal inoculations, and PPV cultured on autologous kidney cells for booster immunisations. A competition ELISA was developed, based on the principle of a double antibody sandwich assay, using immunoglobulin fractions prepared from these sera. The ELISA was compared with a haemagglutination inhibition (HI) test. The tests were equally sensitive for detecting antibodies early after infection and for detecting a significant increase in antibody titre between paired sera. A high correlation was found between antibody titres of field sera measured by the two tests (r = 0.91). We conclude that ELISA is preferable to the HI test, because it is labour-saving and can be standardised better and automated.

Comparison of a newly developed enzyme-linked immunosorbent assay with complement fixation and neutralisation tests for serology of bovine respiratory syncytial virus infections.

An indirect double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection and titration of serum antibodies to bovine respiratory syncytial virus (BRSV). The ELISA was compared with a complement fixation (CF) test and a test for virus neutralising antibody in serum (virus neutralisation [VN] test). Testing sera collected in dairy herds revealed the closest correlation between the results of the ELISA and the CF test with respect to BRSV antibody titres. The VN test detected BRSV antibodies in a higher percentage of acute phase sera compared to the other two tests in field samples and in early bleedings of experimentally infected calves. However, the VN test was less effective in making a diagnosis of BRSV infections on the basis of a significant titre increase in paired sera. For this purpose the ELISA was found to be the most sensitive test.