The Dutch Brucella abortus monitoring programme for cattle: the impact of false-positive serological reactions and comparison of serological tests

The Dutch national Brucella abortus eradication programme for cattle started in 1959. Sporadic cases occurred yearly until 1995; the last infected herd was culled in 1996. In August 1999 the Netherlands was declared officially free of bovine brucellosis by the European Union. Before 1999, the programme to monitor the official Brucella-free status of bovine herds was primarily based on periodical testing of dairy herds with the milk ring test (MRT) and serological testing of all animals older than 1 year of age from non-dairy herds, using the micro-agglutination test (MAT) as screening test. In addition, serum samples of cattle that aborted were tested with the MAT. The high number of false positive reactions in both tests and the serum agglutination test (SAT) and complement fixation test (CFT) used for confirmation seemed to result in unnecessary blockade of herds, subsequent testing and slaughter of animals. For this reason, a validation study was performed in which three indirect enzyme-linked immunosorbent assays (ELISAs), the CFT and the SAT were compared using a panel of sera from brucellosis-free cattle, sera from experimentally infected cattle, and sera from cattle experimentally infected with bacteria which are known to induce cross-reactive antibodies (Pasteurella, Salmonella, Yersinia, and Escherichia). Moreover, four ELISAs and the MRT were compared using a panel of 1000 bulk milk samples from Brucella-free herds and 12 milk samples from Brucella abortus- infected cattle. It is concluded that the ELISA obtained from ID-Lelystad is the most suitable test to monitor the brucelosis free status of herds because it gives rise to fewer false-positive reactions than the SAT.     

Capture ELISA systems for the detection of bovine coronavirus-specific IgA and IgM antibodies in milk and serum

Isotype-capture ELISAs for BCV-specific IgA and IgM were developed and tested on milk and serum samples from Swedish cattle. The capture ELISAs showed higher sensitivity than indirect ELISAs for detection of BCV-specific IgA and IgM. In the capture ELISAs the agreement between detection in milk and serum samples was 94% for IgA and 86% for IgM. The correlation between log(10) titres in milk and serum was r=0.82 (P<0.001) for IgA and 0.84 (P<0.001) for IgM. Milk seemed a better target than serum for diagnosing specific IgA at low levels. There was no variation in the isotype-specific BCV antibody titres between healthy quarters of the same udder, but subclinical mastitis was associated with higher levels of IgA antibodies and weak false IgM positive reactions in undiluted milk. Bovine IgA and IgM antibodies in milk and serum showed high stability towards freezing and thawing and storage at room temperature.The antibody responses to BCV were followed in milk and serum from six dairy cows and in serum from four calves for a period of 1 year after an outbreak of winter dysentery (WD). In this outbreak some animals became reinfected with BCV. The IgA and IgM capture ELISAs differentiated between primarily BCV infected and reinfected animals. In the primarily infected cattle, IgM antibodies were first detected in milk and serum four to nine days after the first WD symptoms observed, and were subsequently detected for at least 2-3 weeks. IgM was also detected in the reinfected cows, but mostly at lower levels and for a shorter period of time than in the primarily infected animals. In milk, however, the IgM response of the reinfected cows was detected for a longer period of time than in serum. Six months after the outbreak, IgA was still detected in both serum and milk of all six cows and also in serum of one calf. The reinfected cows showed higher and more long-lasting peak levels of IgA in milk and serum than the primarily infected cows, indicating boosting of the IgA response.     

Antibodies against bovine herpesvirus (BHV) 5 may be differentiated from antibodies against BHV1 in a BHV1 glycoprotein E blocking ELISA

We examined whether antibodies against bovine herpesvirus (BHV) 5 cross-react with BHV1 antigens and whether they could interfere with BHV1 eradication programmes. Six calves were experimentally infected with different doses of BHV5 strain N569; homologous antibodies were first detectable on day 11 post infection; they cross-reacted in a BHV1 virus neutralisation test, in a BHV1-glycoprotein (g)-B blocking ELISA and in a BHV1-gE ELISA, but not in a BHV1-gE blocking ELISA. This study indicates that, in ongoing BHV1 eradication programmes, based on vaccines that lack gE, BHV5 infections may not lead to false-positive serological reactions in case cattle are tested for BHV1-gE antibodies by the BHV1-gE blocking ELISA; antibodies against BHV5 may be differentiated from antibodies against BHV1. The BHV1-gE blocking ELISA may, therefore, offer opportunities for the serological differentiation between BHV1 and BHV5 infections.     

Reactivation of latent bovine herpesvirus 1 in cattle seronegative to glycoproteins gB and gE

Six heifers were vaccinated intranasally with the live bovine herpesvirus 1 (BHV1) temperature-sensitive (ts) vaccine strain RBL106 within 3 weeks of birth. These calves most likely still had maternal antibodies against BHV1. Thereafter, these heifers were vaccinated several times with an experimental BHV1 glycoprotein-D (gD) subunit vaccine. At the age of 3 years these 6 heifers were seronegative in the BHV1 gB and gE blocking ELISAs, but had neutralizing antibodies against BHV1, probably induced by the vaccinations with the gD subunit vaccine. Five of these 6 heifers excreted BHV1 after treatment with dexamethasone. Restriction enzyme analysis of the genome of the excreted viruses revealed that all 5 isolates had a BHV1.1 genotype and that isolates of 3 heifers were not obviously different from the ts-vaccine strain. The restriction enzyme fragment pattern of the isolate of 1 heifer was clearly different from the pattern of the ts-vaccine strain. It is concluded that cattle can be seronegative against BHV1 gB and gE but can still carry BHV1 in a latent form. This finding strongly suggests that there are completely BHV1 seronegative cattle that are latently infected with BHV1. The impact of this finding on BHV1 eradication programmes is discussed.     

Surveys on Coxiella burnetii infections in Swedish cattle,sheep, goats and moose

Background

Q fever is a zoonotic disease caused by the bacterium Coxiella burnetii. Prevalence data in ruminant species are important to support risk assessments regarding public and animal health. The aim was to investigate the presence of or exposure to C. burnetii in cattle, sheep, goats and moose, and to compare two enzyme-linked immunosorbent assays (ELISAs). National surveys of antibodies against C. burnetii were performed for dairy cattle (n=1537), dairy goats (n=58) and sheep (n=518). Bovine samples consisted of bulk milk, caprine of pooled milk, and ovine of pooled serum. Antibodies were investigated in moose samples (n=99) from three regions. A one-year regional cattle bulk milk survey was performed on the Isle of Gotland (n=119, four occasions). Cattle, sheep and goat samples were analysed with indirect ELISA and moose samples with complement fixation test. For the sheep, goat, and parts of the cattle survey, samples were run in parallel by ELISAs based on antigens from infected ruminants and ticks. Bulk milk samples from the regional cattle survey and vaginal swabs from a subset of the sheep herds (n=80) were analysed for the agent by polymerase chain reaction. Spatial clustering was investigated in the national cattle survey.

Results

The prevalence of antibodies in dairy herds was 8.2% with large regional differences. High risk clusters were identified in the southern regions. The prevalence among dairy herds on the Isle of Gotland varied from 55.9% to 64.6% and 46.4% to 58.9.0% for antibodies and agent, respectively, overall agreement between agent and antibodies was 85.2%. The prevalence of antibodies in sheep was 0.6%, the agent was not detected the vaginal swabs. Antibodies were not detected in goats or moose, although parts of the moose samples were collected in an area with high prevalence in cattle. The overall agreement between the two ELISAs was 90.4%.

Conclusions

The prevalence of antibodies against C. burnetii in dairy cattle in Sweden shows large regional differences. The results suggest that C. burnetii is a rare pathogen among Swedish moose, dairy goat and sheep. ELISAs based on ruminant and tick antigen performed in a similar manner under Swedish conditions.     

Evaluation of a commercially available enzyme-linked immunosorbent assay for detecting antibodies to Fasciola hepatica and Fasciola gigantica in cattle, sheep and buffaloes in Australia

A commercially available ELISA for detecting antibodies to liver fluke was evaluated for use in Australia. Milk and serum samples from cattle and sheep in which infection with Fasciola hepatica was confirmed by detection of eggs in faeces were used to estimate sensitivity. Similar samples collected from cattle and sheep outside the F. hepatica-endemic area were used to estimate specificity. The ELISA was also evaluated for detecting antibodies to F. hepatica in milk from sheep and antibodies to Fasciola gigantica in sera from cattle and buffaloes, but with small numbers of samples. In cattle, the sensitivity and specificity of the ELISA were 98.2% and 98.3% using serum and 97.7% and 99.3% using milk. In infected herds, 41.4% and 41.5% of animals were positive in the serum and milk ELISAs, respectively, whereas F. hepatica eggs were found in faecal samples from 26.5% of animals. In sheep, the sensitivity of the ELISA was 96.9% and the specificity was 99.4%. In infected flocks, 60.2% of animals were positive in the serum ELISA and F. hepatica eggs were found in faecal samples 52.2% of animals. There was perfect agreement in the ELISA between paired serum and milk samples collected from ewes. The assay detected antibodies in sera from cattle and buffaloes with natural and experimental F. gigantica infections. In the experimentally infected animals, antibodies were detected 2 weeks post-infection. We conclude that the ELISA will be a valuable tool for diagnosing F. hepatica infections in cattle and sheep. The assay may also be useful for diagnosing F. gigantica infections but further studies are required to establish sensitivity and specificity.     

Prevalence of Neospora caninum infection in Australian (NSW) dairy cattle estimated by a newly validated ELISA for milk

AIM: To determine the performance characteristics of an Institut Pourquier (IP) enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Neospora caninum in bovine milk and subsequent determination of the prevalence of N. caninum infection in New South Wales (NSW) dairy cattle. METHODS: Matching serum and milk samples from 93 cattle were assayed in two commercially available ELISAs for the detection of anti-N. caninum antibodies. Serum test results of one ELISA (IDEXX) were used to determine the N. caninum infection status of the cattle. Optimised cut-off values for the IP ELISA using milk samples were determined by two-graph receiver operating characteristic (TG-ROC) analysis and then applied to a representative sample of 398 milk samples from dairy herds around NSW. RESULTS: When this ELISA was applied to a representative collection of 398 milk samples from dairy cattle across NSW it demonstrated a 21.1% prevalence of N. caninum infection in those cattle. From the TG-ROC analysis an IP ELISA protocol was derived which suggested a cut-off threshold that would allow milk testing with 97% sensitivity and specificity, respectively, relative to serum testing. CONCLUSIONS: The prevalence of N. caninum in NSW dairy cattle was higher than previously believed. When used on individual milk samples this ELISA demonstrated high sensitivity and specificity and so could be used to accurately identify N. caninum infection. TG-ROC analysis of the IP ELISA optimised the protocol and prescribed cut-off values enabling the ELISA to be used for the screening of N. caninum antibodies in the milk of dairy cattle.     

A serological survey for antibodies against foot-and-mouth disease virus (FMDV) in domestic pigs during outbreaks in Kenya

Foot-and-mouth disease (FMD) is endemic in Kenya and has been well studied in cattle, but not in pigs, yet the role of pigs is recognised in FMD-free areas. This study investigated the presence of antibodies against FMD virus (FMDV) in pigs sampled during a countrywide random survey for FMD in cattle coinciding with SAT 1 FMDV outbreaks in cattle. A total of 191 serum samples were collected from clinically healthy pigs in 17 districts. Forty-two of the 191 sera were from pigs vaccinated against serotypes O/A/SAT 2 FMDV. Antibodies against FMDV non-structural proteins were found in sera from 30 vaccinated and 71 non-vaccinated pigs, altogether 101/191 sera (53 %), and 91 % of these (92/101) also had antibodies measurable by serotype-specific ELISAs, predominantly directed against SAT 1 with titres of 10–320. However, only five high titres against SAT 1 in vaccinated pigs were confirmed by virus neutralisation test (VNT). Due to high degree of agreement between the two ELISAs, it was concluded that positive pigs had been infected with FMDV. Implications of these results for the role of pigs in the epidemiology of FMD in Kenya are discussed, and in-depth studies are recommended.     

Airborne transmission of bovine herpesvirus 1 infections in calves under field conditions

A small scale transmission experiment was performed with bovine herpesvirus 1 (BHV1) in a cattle population under field conditions. 10 calves were housed under strict hygienic conditions, with a distance of 4m between each calf. Five calves were experimentally infected with BHV1, two calves with strain Harberink and three with strain Lam, respectively. Experimentally infected calves were placed at 4 m distance from five susceptible sentinel calves. Airborne transmission to sentinel calves was detected using virus isolation and BHV1 specific polymerase chain reactions in samples of nasal fluids, and BHV1 specific antibodies in serum samples. Strain Harberink was hardly transmitted to sentinel calves, whereas strain Lam was transmitted to all sentinels. Estimating the rate of transmission per day, the total number of calves infected by one (strain Lam) infected calf was 1.18. Comparing this estimated transmission ratio between cattle at a distance of 4 m to the estimated transmission ratio R of BHV1 in susceptible commingled cattle reported before, the effect of the factor distance on the transmission ratio could be calculated. Extrapolating these results, a distance of 4.4 m between cattle populations would be necessary to reduce transmission for this strain to R<1.     

An evaluation of five serological tests for the detection of antibody to bovine herpesvirus 1 in vaccinated and experimentally infected cattle

More than 300 bovine sera from a previously reported vaccination and challenge trial were tested for antibodies to bovine herpesvirus 1 (BHV1) by five serological assays: enzyme-linked immunosorbent assay (ELISA) for IgM and IgG, passive haemagglutination (PHA), and two methods of virus neutralisation (VN). In a statistical comparison of ELISA (IgG), PHA and VN results, the assays showed highly significant correlations (P less than 0.01). The sensitivities of ELISA and 24-hour neutralisation tests were similar, in contrast to passive haemagglutination and one hour neutralisation which failed to detect BHV1 antibodies in some low titre sera.     

Comparison of serological methods for the diagnosis of Neospora caninum infection in cattle

The aims of this study were to evaluate the performance and agreement of various commercial and in-house Neospora caninum antibody assays used in dairy cattle in North America, and to investigate reproducibility of two assays performed in different laboratories. From 1998 to 2005, three enzyme linked immunosorbent assays (ELISAs, a competitive ELISA-VMRD Inc., an indirect ELISA-Biovet Inc., and another indirect ELISA-Herdchek IDEXX Corp.), two indirect fluorescent antibody tests (IFATs, VMRD Inc., and in-house USDA) and one N. caninum agglutination test (NAT, in-house USDA) were utilized to test 397 randomly selected dairy cattle serum samples from 34 herds in eastern Canada for antibodies to N. caninum. The manufacturers' recommended cut-off values were used to evaluate test performance and agreement between tests. One IFAT (VMRD Inc.) performed well (sensitivity and specificity: 0.97 and 0.97, respectively) using reference sera (n = 452), therefore, results from this IFAT on the 397 samples could subsequently be used as the reference standard to calculate test characteristics for the other assays. Only 11% of the 397 sera were found to be N. caninum-positive with the IFAT. Prevalence-adjusted bias-adjusted kappa (PABAK) ranged from 0.06 to 0.99. Positive agreement was moderate to very good (P(pos) = 0.25-0.96). Negative agreement was very good for all assays (P(neg) > 0.94) except NAT (P(neg) = 0.66). Sensitivity was > or =0.89 for all assays except the NAT, which had a significantly lower sensitivity (0.66). Specificity was high (>0.94) for all assays except for one indirect ELISA (specificity = 0.52). This indirect ELISA did not perform satisfactorily when used in 1998, but an improved version of the ELISA performed as one of the best assays in 2004. Reproducibility of the competitive ELISA was excellent, but the reproducibility of the indirect ELISA that was improved was low (concordance correlation coefficient = 0.90 and 0.36, respectively). The performance characteristics observed for most assays in this study make them useful for screening antibodies to N. caninum in cattle.     

Induction of humoral antibodies by a BHV-1 intradermal test]

An intradermal test for the diagnosis of BHV 1 (Intrakutantest Behringwerke AG) was applied to 53 nonvaccinated BHV 1-seronegative cattle aged 7 months to 8 years. Serologic blood testing performed subsequently using ELISA (Enzygnost-IBR/IPV, Behringwerke AG) and SNT revealed seroconversion in 24 of 45 animals without previous maternal antibodies. A second application of the intradermal test after these BHV 1-antibodies had declined, lead to a 'booster-effect' while a control group remained negative. Six of nine animals not affected by the first intradermal application of BHV 1-antigen, developed BHV 1-antibodies following the second intradermal test. Eight animals possessing maternal antibodies showed no serological response to the intradermal test at all. Present results strongly suggest an induction of humoral BHV 1-antibodies by the intradermal application of inactivated BHV 1. Consequently, the indirect control of BHV 1 infections, especially in cattle breeding farms, should be done exclusively by serological examination.     

Evaluation of enzyme-linked immunosorbent assays performed on milk and serum samples for detection of paratuberculosis in lactating dairy cows

OBJECTIVE: To determine whether results obtained for milk and serum samples with ELISAs intended for diagnosis of paratuberculosis in dairy cows were comparable to results obtained by means of mycobacterial culture of fecal samples. DESIGN: Cross-sectional study. ANIMALS: 689 lactating dairy cows in 9 Ontario herds. PROCEDURE: Milk, serum, and fecal samples were obtained from all cows. Fecal samples were submitted for mycobacterial culture. Serum samples were tested with a commercially available ELISA for antibodies against Mycobacterium paratuberculosis, and preserved milk samples were tested with an indirect ELISA for antibodies against M paratuberculosis. RESULTS: Results were positive for 130 of the 689 (18.9%) serum samples, 77 of the 689 (11.1%) milk samples, and 72 of the 689 (10.4%) fecal samples. The level of agreement between results for milk and serum samples was only moderate. Proportions of positive results for serum and fecal samples were significantly different, but proportions of positive results for milk and fecal samples were not significantly different. In addition, results for milk samples had a higher level of agreement with results of mycobacterial culture than did results for serum samples. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the indirect ELISA used on milk samples may be a convenient method of detecting paratuberculosis in dairy herds.     

Antibody responses of naive cattle to two inactivated bovine viral diarrhoea virus vaccines,measured by indirect and blocking ELISAS and virus neutralisation

Two groups of naive heifers were given primary courses of two inactivated bovine viral diarrhoea (BVD) virus vaccines licensed for use in the UK. Their humoral responses in serum and milk were assayed by means of an indirect ELISA detecting antibodies to structural viral glycoproteins, a blocking ELISA specific for antibodies to the non-structural protein NS2-3 and the virus neutralisation test (VNT). For each assay, the numbers of serum or milk samples testing positive at each sample point and the mean values were determined. In both vaccine groups, serum antibody responses were detected by the indirect ELISA and the VNT, with both the numbers of seropositive animals and mean values peaking five weeks after the second vaccination. In the 23 heifers vaccinated with Bovilis BVD, the mean NS2-3-specific ELISA values remained low throughout the trial, with no serum or milk samples testing positive. In the 24 heifers vaccinated with Bovidec, the mean NS2-3 responses peaked below the level of positivity five weeks after the second vaccination, before declining again; NS2-3-specific antibodies were detected in one serum sample and one milk sample from two heifers in this group. A pooled milk sample from each vaccine group tested negative by both ELISAS 12 weeks after the second vaccination.     

Improvement of serological discrimination between herpesvirus-infected animals and animals vaccinated with marker vaccines

Control/eradication plans of bovine herpesvirus 1 (BHV1) and suid herpesvirus 1 (SHV1) infections involve vaccination with inactivated or attenuated gE-deleted marker vaccines and associated companion serological tests to discriminate naturally infected from vaccinated animals. Blocking or competitive enzyme-linked immunosorbent assays (ELISAs) have been designed for the detection of specific antibodies against BHV1 or SHV1 gE glycoprotein. The antigen source usually consists of a crude viral preparation in which gE is associated with other envelope glycoproteins. Such assays suffer from a lack of specificity which is not due to serological cross-reactions with other pathogens. Interestingly, false-positive results occur with sera collected from multivaccinated cattle or pigs. After multivaccination with a marker vaccine, the binding of the conjugated monoclonal antibody used as a tracer, could be hampered by antibodies directed against the other viral glycoproteins.In order to validate the steric hindrance hypothesis, a simple preadsorption of such samples was carried out with a preparation of antigen devoid of gE, prior to the blocking ELISA itself. The decrease in antibody concentrations against the major glycoproteins, clearly leads to a better discrimination between positive and negative samples; that is between infected and multivaccinated animals, without significant loss of sensitivity. This experiment confirms the steric hindrance hypothesis, therefore serum preadsorption could be an easy way to improve the specificity of currently available diagnostic tests.     

Advances in BHV1 (IBR) research.

Bovine Herpesvirus Type 1 (BHV1) is the aetiological agent of a number of diseases and not only of IBR, namely infectious pustular vulvovaginitis (IPV), infectious balanoposthitis (IBP), conjunctivitis, encephalomyelitis, mastitis, abortion, enteritis, and lesions in the interdigital space. The serological identical strains differ, however, in some aspects. Typical genital strains usually cause a mild illness, sometimes not even detected clinically, but serologically. They hamper eradication programmes and do not cause IBR when inoculated intranasally. The other--modern--strains are, however, always able to induce a severe disease in the genital tracts. But infection of field or vaccine virus leads to the development of humoral and cell-mediated immunity. The latter is, however, not transmitted to neonates via colostrum. BHV1 antibodies can be found in bovines in all continents, and in many wild species. Prevalences vary greatly depending on herd size and management. Because seronegative cattle play a role in international trade a number of European countries have eradicated BHV1, with very high costs involved. Marker and conventional vaccines can prevent disease but not infection followed by the state of latency. The genomes of several strains, including the marker strains can remain latent in the same animal and be reactivated after stress or injection of corticosteroids. For the detection of humoral antibodies the ELISA is widely used. It is useful for testing bulk milk samples for antibodies derived from field virus and conventional vaccines but not from gE-deleted marker vaccines. Importing countries should consider only vaccinated animals for import. They should require that the animals are seronegative prior to vaccination.     

Isotype-specific ELISAs for the detection of antibodies to bovine respiratory syncytial virus

Isotype-specific ELISAs for the detection of antibodies to bovine respiratory syncytial virus (BRSV) are described. BRSV-specific IgG1 and IgG2 were determined in indirect double antibody sandwich assays. For IgA and IgM antibody capture assays were used. The isotype specificity of the assays was confirmed by the observation that samples with a high titre of BRSV-specific antibodies of particular isotype were negative in the assays for the other isotypes and vice versa. Comparison of the results obtained in the ELISAs and in the virus neutralisation test showed that acute phase antibodies were more efficiently detected in the latter. It also showed that the presence of BRSV-specific IgA was not correlated with neutralising activity in vitro. The serum antibody response of BRSV-infected seronegative calves from the field consisted of a nearly simultaneous increase of IgM, IgA and IgG1-antibodies in the acute phase of the disease, while the IgG2-response followed at various intervals thereafter. In young animals with maternal antibodies a different pattern was found. There was no increase in IgG1 and IgG2, but six of eight animals showed a weak IgM response and two of these six calves also showed a weak and short lasting IgA response. Because maternal antibodies are insufficiently effective in protecting calves against BRSV, the presence of such antibodies at mucosal surfaces was investigated. Maternal immunity was found to be restricted to IgG1 antibodies in serum. This agrees with the failure of maternal antibodies to protect mucosal surfaces against BRSV infection.     

Use of milk samples and monoclonal antibodies directed against BLV-p24 to identify cattle infected with bovine leukemia virus (BLV)

An indirect double-antibody sandwich (IDAS) enzyme-linked immunosorbent assay (ELISA) using milk samples was developed to identify cows infected with bovine leukemia virus (BLV). Two monoclonal antibodies (McAbs) were used. One, which was directed against BLV core protein p24, was used to coat ELISA plates; the other was used to prepare a horseradish peroxidase (HRP) conjugate directed against bovine immunoglobulin. The IDAS-ELISA detected antibodies directed against BLV-p24 in 97% of the milk samples collected from known seropositive cows identified by the agar gel precipitation test (AGTP). Even when milk samples were diluted 1:50, 93% of the seropositive cows were identified. Only 0.43% of the 4000 milk samples collected in The Netherlands reacted nonspecifically. Nonspecific binding disappeared, however, when these samples were diluted 50 times in BLV-negative milk. In a comparative evaluation of BLV test-kits in various European laboratories, our IDAS-ELISA using McAb directed against p24 was one of the most sensitive.     

Bluetongue virus: European Community inter-laboratory comparison tests to evaluate ELISA and RT-PCR detection methods

European Community national reference laboratories participated in two inter-laboratory comparison tests in 2006 to evaluate the sensitivity and specificity of their 'in-house' ELISA and RT-PCR assays for the detection of bluetongue virus (BTV) antibodies and RNA. The first ring trial determined the ability of laboratories to detect antibodies to all 24 serotypes of BTV. The second ring trial, which included both antisera and EDTA blood samples from animals experimentally infected with the northern European strain of BTV-8, determined the ability of laboratories to detect BTV-8 antibodies and RNA, as well as the diagnostic sensitivity of the assays. A total of six C-ELISAs, six real-time RT-PCR and three conventional RT-PCR assays were used. All C-ELISAs were capable of detecting the BTV serotypes currently circulating in Europe (BTV-1, 2, 4, 8, 9 and 16), however some assays displayed inconsistencies in the detection of other serotypes, particularly BTV-19. All C-ELISAs detected BTV-8 antibodies in cattle and sheep by 21 dpi, while the majority of assays detected antibodies by 9 dpi in cattle and 8 dpi in sheep. All the RT-PCR assays were able to detect BTV-8, although the real-time assays were more sensitive compared to the conventional assays. The majority of real-time RT-PCR assays detected BTV RNA as early as 2 dpi in cattle and 3 dpi in sheep. These two ring trails provide evidence that national reference laboratories within the EC are capable of detecting BTV antibodies and RNA and provide specificity and sensitivity information on the detection methods currently available.     

An international comparison of different laboratory tests for the diagnosis of bovine leukosis: suggestions for international standardization

A collaborative study was conducted to compare the detection limit of different laboratory tests for antibodies against bovine leukemia virus (BLV). Serum and milk samples were tested in agar gel immunodiffusion (AGID), different modifications of indirect ELISA, blocking ELISA and ELISA procedures using monoclonal antibodies to BLV gp51 or BLV p24. The detection limit of reference serum E4 diluted 2-fold in negative serum gave a median value of 1:16 in AGID, indirect ELISA, and monoclonal ELISA p24, 1:128 in monoclonal ELISA gp51, and 1:1024 in blocking ELISA. The detection limit of a 4% immunoglobulin preparation of E4 diluted in negative milk showed median values of 1:800 in indirect ELISA, 1:1000 in monoclonal ELISA, and 1:2400 in blocking ELISA. None of the ELISA procedures could detect all the positive individual milk samples diluted 1:50. The AGID test is the official reference test for detection of antibodies against BLV. Reference serum E4 diluted 1:10 in negative serum must be scored positive in the AGID test. It is suggested that an international reference serum standard be established rather than an official recommendation of a particular ELISA test.