Challenges for bovine viral diarrhoea virus antibody detection in bulk milk by antibody enzyme-linked immunosorbent assays due to changes in milk production levels

Background

Bovine viral diarrhoea (BVD) is considered eradicated from Denmark. Currently, very few (if any) Danish cattle herds could be infected with BVD virus (BVDV). The Danish antibody blocking enzyme-linked immunosorbent assay (ELISA) has been successfully used during the Danish BVD eradication program, initiated in 1994. During the last decade, the cattle herd size has increased while the prevalence of BVDV has decreased. In this study, we investigated how these changes could affect the performance of the Danish blocking ELISA and of the SVANOVIR^®BVDV-Ab indirect ELISA. The latter has successfully been used to eradicate BVD in Sweden.Data (2003–2010) on changes in median herd size and milk production levels, occurrence of viremic animals and bulk milk surveillance were analysed. Additionally, the Danish blocking ELISA and the SVANOVIR ELISA were compared analyzing milk and serum samples. The prevalence of antibody positive milking cows that could be detected by each test was estimated, by diluting positive individual milk samples and making artificial milk pools.

Results

During the study period, the median herd size increased from 74 (2003) to 127 cows (2010), while the prevalence of BVDV infected herds decreased from 0.51 to 0.02 %. The daily milk yield contribution of a single seropositive cow to the entire daily bulk milk was reduced from 1.61 % in 2003 to 0.95 % in 2010 due to the increased herd size. It was observed that antibody levels in bulk milk decreased at national level. Moreover, we found that when testing bulk milk, the SVANOVIR^®BVDV-Ab can detect a lower prevalence of seropositive lactating cows, compared to the Danish blocking ELISA (0.78 % vs. 50 %). Values in the SVANOVIR^®BVDV-Ab better relate to low concentrations of antibody positive milk (R² = 94-98 %), than values in the blocking ELISA (R² = 23–75 %). For sera, the two ELISAs performed equally well.

Conclusions

The SVANOVIR ELISA is recommended for analysis of bulk milk samples in the current Danish situation, since infected dairy herds e.g. due to import of infected cattle can be detected shortly after BVDV introduction, when only few lactating cows have seroconverted. In sera, the two ELISAs can be used interchangeably.     

Assessing the within-herd prevalence of cows antibody-positive to bovine viral diarrhoea virus with a blocking ELISA on bulk tank milk

Milk samples from 135 herds in Brittany were tested by a blocking ELISA for antibodies to bovine viral diarrhoea virus (BVDV) and used to assess the relationship between the bulk milk result and the within-herd prevalence of antibody-positive lactating cows. This relationship was first quantified by using a general linear model and controlling for the number of cows contributing milk to the bulk tank, for the percentage of primiparous cows in the herds and for the number of milkings contributing to the bulk tank. Receiver operating characteristic (ROC) analysis was then used to define classes of percentage inhibition in the bulk milk associated with minimum intraclass and maximum between-class variances of the within-herd prevalence. Only the percentage inhibition of bulk milk had a significant positive effect on within-herd prevalence (R2 = 0.85). The ROC analysis provided three classes of bulk milk results corresponding to different expected levels of within-herd prevalence. Herds with bulk milk percentage inhibitions of 0 to 35 per cent, 35 to 60 per cent and 60 to 100 per cent had mean (sd) observed prevalences of 4.8 (5.7) per cent, 21.6 (14.6) per cent and 66.0 (29.3) per cent, respectively. Herds with a bulk milk inhibition of 0 to 35 per cent were expected to be BVDV-free. A herd with two consecutive bulk milk results four months apart of 60 per cent or more was likely to have a very high prevalence (median of 93 per cent) and could be suspected of harbouring an active infection.     

Antibody titer against bovine viral diarrhea virus (BVDV) in bulk milk samples from Bavaria

A total of 5204 bulk milk samples were tested for antibodies against bovine viral diarrhea virus (BVDV) classified according to the scheme after Alenius. Forty-five percent of the samples from 2002 were classified as class 0 and class 1, 55% as class 2 and 3. 6420 bulk milk samples from 1997 were classified in an independent study in 65.6% class 0 and 1 and 34.4% in class 2 and 3. In class 0 and class 1 farms only very rarely persistent viremic animals have been found, whereas in class 2 and 3 their presence is highly likely. Our studies with non-selected sera defined the serological screening of bulk milk samples as a promising tool for a possible BVDV eradication program in Bavaria.     

Prevalence of bovine viral diarrhoea virus antibodies in bulk tank milk of industrial dairy cattle herds in suburb of Mashhad-Iran

Bulk milk for the presence of antibodies against bovine viral diarrhea virus (BVDV) from 38 industrial dairy cattle herds complexes with 250-3000 Holstein dairy cows in suburb of Mashhad-Iran was tested. None of the herds were vaccinated against BVDV. Commercial indirect ELISA-kit for the detection of specific antibodies was used. The result could be read visually where the optical density (OD) was measured at 450 nm. The percent positivity (PP) values >or=7 and <7 interpreted positive and negative, respectively. According to this study the apparent and the true prevalence of BVDV antibody-positive herds was 89.47 and 93.98%, respectively. The range of PP was 1.59-107.66 among the herds. The OD in 52.63% bulk milk of the herds was very high. It is concluded that exposure to BVD virus was widely distributed in the dairy cattle herds in suburb of Mashhad-Iran.     

Validation of a bulk tank milk antibody ELISA to detect dairy herds likely infected with bovine viral diarrhoea virus in New Zealand

AIMS: To assess the sensitivity and specificity of a bulk tank milk (BTM) antibody enzyme-linked immunosorbent assay (ELISA) to detect likely infection of a dairy herd with bovine vi- ral diarrhoea virus (BVDV). The ELISA was subsequently used to estimate the prevalence of likely infected herds in parts of the North Island of New Zealand.

METHODS: BTM samples from 724 randomly selected dairy herds in the Waikato, Bay of Plenty and Northland regions of New Zealand were tested for BVDV antibodies. From this group, 20 herds were again randomly selected from each of the quartiles of the ELISA percentage inhibition (%INH) result. From each participant herd, serum from 15 randomly selected calves aged 6–18 months and 15 cows was collected and tested using an indirect blocking ELISA for BVDV antibodies.

RESULTS: Among serum results from calves from 50 herds available for analysis, 34 (68%) herds were classified as likely non-infected (0-3 seropositive among 15 calves) and 16 (32%) as likely infected (5–15 seropositive among 15 calves). Receiver- operator characteristic (ROC) analysis identified an optimal cut-off for BTM of 80%INH associated with 81% sensitivity and 91% specificity for likely herd infection. The prevalence of BVDV antibodies in cows within herds and %INH for BVDV in bulk milk were positively correlated (p<0.01). The association between bulk milk %INH and the prevalence of BVDV antibodies in calves was stronger than the same association in cows. Based on the threshold of 80%INH, the 95% confidence interval (CI) for prevalence of likely infection in the 724 herds in the Waikato, Bay of Plenty and Northland regions of New Zealand was 12–17%. Vaccination against BVDV was not significantly associated with the likely infection status of the herd based on prevalence of BVDV antibodies among calves.

CONCLUSION: An ELISA test result for BVDV antibodies in BTM ≥80%INH can be used as a threshold to indicate the presence of likely infection with BVDV in dairy herds in New Zealand, with 81% sensitivity and 91% specificity.     

Evaluation of a blocking ELISA for the detection of bovine viral diarrhoea virus (BVDV) antibodies in serum and milk

The performance characteristics of a blocking ELISA test applied to serum and individual milk for the detection of antibodies to bovine viral diarrhoea virus (BVDV) were assessed using 1189 matched milk/serum samples collected from cows of 42 dairy herds located in Brittany (west of France). This test was based on a monoclonal antibody directed against non-structural protein NS2-3 of pestiviruses. All tests were performed blind. For each type of sample, negative/positive cut-off values were determined using receiver operating characteristic (ROC) analysis. Sensitivity and specificity were estimated using the virus neutralisation test as a reference. For sera, the ROC analysis provided a negative/positive inhibition percentage cut-off value of 50% giving a sensitivity and a specificity of 96.9 and 97.8%. For individual milk samples, the cut-off was fixed at 30%, leading to a sensitivity and a specificity of 96.9 and 97.3%. Using this test, a good overall agreement was found between results obtained on matched milk/serum samples (Kappavalue=0.95). The present results indicate that this blocking ELISA test is reliable enough for use in a mass screening and control scheme on BVDV.     

Validation of a bulk tank milk antibody ELISA to detect dairy herds likely infected with bovine viral diarrhoea virus in New Zealand

AIMS: To assess the sensitivity and specificity of a bulk tank milk (BTM) antibody enzyme-linked immunosorbent assay (ELISA) to detect likely infection of a dairy herd with bovine viral diarrhoea virus (BVDV). The ELISA was subsequently used to estimate the prevalence of likely infected herds in parts of the North Island of New Zealand. METHODS: BTM samples from 724 randomly selected dairy herds in the Waikato, Bay of Plenty and Northland regions of New Zealand were tested for BVDV antibodies. From this group, 20 herds were again randomly selected from each of the quartiles of the ELISA percentage inhibition (%INH) result. From each participant herd, serum from 15 randomly selected calves aged 6-18 months and 15 cows was collected and tested using an indirect blocking ELISA for BVDV antibodies. RESULTS: Among serum results from calves from 50 herds available for analysis, 34 (68%) herds were classified as likely non-infected (0-3 seropositive among 15 calves) and 16 (32%) as likely infected (5-15 seropositive among 15 calves). Receiver-operator characteristic (ROC) analysis identified an optimal cut-off for BTM of 80%INH associated with 81% sensitivity and 91% specificity for likely herd infection. The prevalence of BVDV antibodies in cows within herds and %INH for BVDV in bulk milk were positively correlated (p<0.01). The association between bulk milk %INH and the prevalence of BVDV antibodies in calves was stronger than the same association in cows. Based on the threshold of 80%INH, the 95% confidence interval (CI) for prevalence of likely infection in the 724 herds in the Waikato, Bay of Plenty and Northland regions of New Zealand was 12-17%. Vaccination against BVDV was not significantly associated with the likely infection status of the herd based on prevalence of BVDV antibodies among calves. CONCLUSION: An ELISA test result for BVDV antibodies in BTM >/=80%INH can be used as a threshold to indicate the presence of likely infection with BVDV in dairy herds in New Zealand, with 81% sensitivity and 91% specificity.     

Establishing a pilot bovine viral diarrhoea virus eradication scheme in Somerset

Beginning in April 2006, 41 farms were recruited onto a pilot Bovine viral diarrhoea virus (BVDV) eradication programme across the south of England with the majority of study herds concentrated in Somerset. Each herd was assessed and where relevant cleared of persistently infected (PI) animals. Seven farms dropped out before whole herd screening could be performed. Of the remaining 34 farms, 20 (59 per cent) were classified as infected although two of these were initially misclassified as BVDV-free. Over the course of three years, 61 PIs were identified across 16 of the 20 infected farms. 72 per cent of PIs indentified on the first herd test were below two years of age. PI prevalence ranged from 0.2 to 3.1 per cent of infected herds and was highest in herds that did not vaccinate. By the end of 2009, 24/34 (71 per cent) of study farms were BVDV-free while 10 (29 per cent) remained infected.     

进口奶牛病毒性腹泻病毒快速检测鉴定

采用ELISA方法对3 073头奶牛全血进行牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)检测,结果发现编号a889的血样呈阳性反应,另外有3份血样(a126、a803、b1277)呈可疑反应;进一步的白细胞抽提物ELISA反应表明,a889血样呈阳性反应,其余呈阴性反应。对a889血样进行RT-PCR扩增,得到1条310 bp大小的特异性条带,与预期片段大小吻合;经序列同源性比较分析,确定a889血样中存在牛病毒性腹泻病毒。