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.

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.

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.

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.

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.

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.

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.

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.

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.

Gel electrophoretic analysis of polypeptides from nucleocapsids of Marek's disease virus strains and herpesvirus of turkey.

The polypeptides of nucleocapsids of Marek's disease virus (MDV) strains with different biological properties and of antigenically related herpesvirus of turkey (HVT) strains were analysed by one- and two-dimensional (1D and 2D, respectively) gel electrophoresis. Based on small differences in migration behaviour (size and charge) of a number of corresponding nucleocapsid polypeptides, the virus strains could be differentiated into three groups. The polypeptide pattern of group I, comprising the virulent MDV-strain K and the attenuated strains, HPRS-16/att and CVI988 37th passage, was composed of four major polypeptides (i.e. 140K, 50K, 40K and 33K daltons) and at least four minor polypeptides. The pattern of group II, comprising the naturally occurring non-oncogenic MDV-strains SB-1 and HPRS-24, contained one additional major polypeptide of 39K daltons. The nucleocapsid-specific 2D polypeptide patterns of the HVT strains HVT-Fc 126 and PB-THV1, comprising group III, were distinguishable from each other on the basis of a small difference in size of one major 50K polypeptide. Results were further substantiated by coelectrophoresis experiments.

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.