Evaluation of three enzyme-linked immunosorbent assays for detection of antibodies to Neospora caninum in bulk milk

Three ELISAs for the detection of antibodies against Neospora caninum in bulk milk were evaluated in 162 Dutch dairy herds. The first ELISA was the Dutch Animal Health Service (AHS) in-house ELISA, developed from the routine in-house serum ELISA. The other two ELISAs were commercial milk ELISAs from IDEXX and LSI. Blood samples of all lactating cows in 162 dairy herds were tested using the AHS in-house serum ELISA. Based on previous studies in the Netherlands a within-herd N. caninum seroprevalence of 15% was associated with increased risk for reproductive losses. This percentage was therefore used as positive seroprevalence cut-off value. Repeatability of the ELISAs was evaluated by testing on three different days. The AHS in-house ELISA lacked specificity, probably due to use of a different batch of antigen on the second and third test-day. Cut-off values were determined using misclassification costs term calculations. At cut-off values 0.6 for the IDEXX and 0.2 for the LSI, a herd sensitivity of 61% (95% CI: 49--73%) and 47% (95% CI: 35--60%) was estimated. Herd specificity at these cut-off values was 92% (95% CI: 87--98%) for the IDEXX and 94% (95% CI: 90--99%) for the LSI ELISA. The positive and negative predictive values were 84% (95% CI: 68--100%) and 86% (95% CI: 79--94%) for the IDEXX ELISA, and 85% (95% CI: 67--100%) and 82% (95% CI: 74--90%) for the LSI ELISA. The agreement between all possible combinations of test-days was expressed by kappa values. These were found to be slightly higher for the IDEXX than for the LSI ELISA. It is concluded that both commercial ELISAs performed satisfactorily to detect a within-herd seroprevalence of N. caninum in lactating cows of at least 15%.     

The suitability of ELISA for the detection of antibodies against Mycobacterium avium ssp. paratuberculosis in bulk milk samples from Rhineland-Palatinate

Paratuberculosis or Johne's Disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a notifiable disease in Germany which produces enormous economical losses in dairy farms. At present,there is no confirmed data about the actual number of infected livestock herds in Germany. A countrywide monitoring program to evaluate the prevalence in dairy herds would only be economically feasible on the basis of bulk milk testing. In this study, we evaluated two ELISA test kits (SVANOVIR Ptb-ELISA, IDEXX-M.pt. Milk test kit) for the detection of antibodies against MAP in bulk milk. First, the Paratuberculosis-status of the herd derived from the history of the farm was used as a gold standard. Paratuberculosis-negative farms were tested negative with each test, but paratuberculosis-positive or Paratuberculosis-serologically-positive farms were detected only in one case (Svanovir) or three cases (IDEXX), respectively. Even if inconclusive results are counted as positive, 82.9 % (Svanovir) or 80 % (IDEXX) of the paratuberculosis-positive or serologically paratuberculosis positive farms were not detected. Nevertheless, a re-validation of both ELISAs by means of ROC and TG-ROC analyses was attempted by searching for ideal cut-offs, optimised for bulk milk. If a high specificity was selected, no acceptable sensitivity could be reached.The best results were obtained using a sensitivity of 32.3 % at a specificity of 100 % (Svanovir). With a small change of the cut-off value, the sensitivity increased to still 57 %, but this reduced the specificity to 67 %. Similar results were obtained with the IDEXX-ELISA. We then evaluated the Svanovir-ELISA for the detection of bulk milk samples on the basis of the current paratuberculosis prevalence within 69 dairy herds from Rhineland-Palatinate using individual milk samples.When the bulk milk samples were tested in two different laboratories using the same ELISA, considerable differences in the results became evident. Nearly all samples were tested with a higher relative test result in one laboratory, which often led to differences in the classification of the prevalence levels.The estimated within-herd seroprevalences ranged between 0 % and 37 %.There was little agreement between the historical paratuberculosis herd status and the within-herd prevalence in milk serum, as reflected in a kappa-index of 0.146.To determine the sensitivity and specificity of the bulk milk ELISA by ROC and TG-ROC analysis, 116 bulk milk samples were used that had been obtained from the 69 dairy herds participating in the study. The optimal ratio of sensitivity (81 %) and specificity (77 %) relative to a "gold standard" was obtained when the cut-off was set at the 10 % level. These values for sensitivity and specificity were better than those obtained in an evaluation of the same ELISA in which the historical Paratuberculosis herd-status was used as a "gold standard." The results of this study question the suitability of the available ELISAs for bulk milk testing.Taking into account that the Svanovir-ELISA for individual milk samples has a sensitivity of 60 96% relative to the blood serum variant of the test, and that the latter has also a limited sensitivity due to the pathogenesis of paratuberculosis, the available test systems examined in this Study do not seem to be suitable for herd diagnosis by using bulk milk samples.     

Evaluation of an iscom ELISA used for detection of antibodies to Neospora caninum in bulk milk

The intracellular parasite Neospora caninum is increasingly recognized as an important cause of abortion and stillbirth in cattle. Presence of specific antibodies indicates infection, and the immunostimulating complex (iscom) enzyme-linked immunoassay (ELISA) has previously been evaluated for use on individual milk and sera. In the present study, this test is investigated for use on bulk milk. In this study, 124 herds were used to analyse the relationship between within-herd prevalences based on individual sera and bulk milk optical densities. The individual test results were translated into a herd-level result, which enabled comparison of the bulk milk test result to the aggregate of individual serum results. The relative contribution of milk from cows with different milk yield and antibody status to the tank, i.e. its composition, was expected to influence the outcome of the bulk milk test. Therefore, sensitivity and specificity were calculated at different cut-off levels, not only using a standard cross-tabulation technique, but also a logistic regression model. By using the latter method, the sensitivity and specificity could be estimated adjusting for milk yield covariates. Specificity was estimated to be high ( approximately 98%) at the 0.20 cut-off, which can be used as a decision threshold to rule in infection. With more equal emphasis on sensitivity and specificity, a lower cut-off should be used. Although infection cannot be completely ruled out, herds with test results below 0.05 are highly likely to be non-infected. The within-herd prevalence of false negative herds is probably less than 10-15% at this level. From what is known about test performance at the individual level and the prevalence of infection, the estimate of the specificity of the bulk milk test should be quite accurate while the sensitivity is likely to be underestimated. We confirmed that the performance of the bulk milk test depends on the milk tank composition. In particular the milk yield of cows with high antibody levels affects the probability of a positive outcome of the bulk milk test.     

A retrospective evaluation of a Bovine Herpesvirus-1 (BHV-1) antibody ELISA on bulk-tank milk samples for classification of the BHV-1 status of Danish dairy herds

Bulk-tank milk samples analysed in a Bovine Herpesvirus-1 (BHV-1) blocking ELISA are still in use in the Danish BHV-1 programme as a tool to classify dairy herds as BHV-1 infected or BHV-1 free herds. In this retrospective study, we used data from the Danish BHV-1 eradication campaign to evaluate performance characteristics of the BHV-1 blocking ELISA in 1039 BHV-1-seropositive and 502 repeatedly BHV-1-negative dairy herds using the results of blood testing of the individual animals as the true infection status. At a cut-off value of 30% blocking reaction, the herd-level relative sensitivity and relative specificity were 82 and 100%, respectively. The herd-level relative sensitivity depended on the within-herd prevalence of seropositive cows and the cut-off value in the assay, but not on the time interval (up to 90 days) between the collection of the bulk-tank milk sample and the individual serum samples. The BHV-1 blocking ELISA on bulk-tank milk could detect seropositive herds (few), with prevalence proportions as low as one seropositive cow out of 70 cows.     

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.     

Use of an enzyme-linked immunosorbent assay in bulk milk to estimate the prevalence of Neospora caninum on dairy farms in Prince Edward Island, Canada

This study evaluated the use of bulk milk as a diagnostic tool for estimation of herd-level Neospora caninum exposure in Atlantic Canada; it was used to estimate the prevalence of dairy farms with a within-herd N. caninum-seroprevalence > or = 15% in Prince Edward Island (PEI). The variation over time of N. caninum antibodies in bulk milk is also reported. Skimmed bulk milk and individual serum samples were analyzed for N. caninum antibodies by using an enzyme-linked immunosorbent assay (ELISA). Bulk milk samples were collected in May 2004 (n = 235), May 2005 (n = 189), and June 2005 (n = 235). The prevalence of dairy farms with a within-herd seroprevalence > or = 15% on PEI was 6.4% in May 2004. In May and June 2005, respectively, 10.1% and 10.2% of farms had a > or = 15% within-herd seroprevalence. In 11 farms that were considered positive based on bulk milk samples, blood samples were collected from all adult cows in September 2005, in conjunction with a 4th bulk milk sample on the same day. The correlation coefficient between serology and bulk milk ELISA was 0.87. The results of this study demonstrate that the prevalence of N. caninum in dairy farms can be estimated by using a bulk milk ELISA.     

Validation of a commercial ELISA for the detection of bluetongue virus (BTV)-specific antibodies in individual milk samples of Dutch dairy cows

A recently developed indirect ELISA for the detection of bluetongue virus (BTV)-specific antibodies in bovine milk samples was compared to that of the routinely used competitive ELISA on serum samples. During the bluetongue outbreak in the Netherlands in 2006, caused by BTV serotype 8, coupled serum and milk samples were obtained from 470 individual cows from 10 BTV-infected farms with an average seroprevalence of 57%. In addition, bulk milk samples of the same farms, and historically BT-negative samples were tested. Compared to the ELISA for sera, the relative specificity and sensitivity of the ELISA for milk samples is 96.5% and 98.9%, respectively when using a S/P% cut-off value of 50% as advised by the manufacturer. The optimal cut-off value was found at S/P% of 90% revealing an optimal specificity (99.0%) combined with an optimal sensitivity (98.1%). Titres in positive individual milk samples ranged from 1 to 2048 with a peak titre of 128. Bulk milk samples contained antibodies with titres ranging from 64 to 512. The ELISA for milk samples was found to be a reliable and robust test. This diagnostic tool is very useful, and may replace the ELISA for serum samples as first choice in order to get insight into the status of lactating individual animals and therewith of the entire herd with respect to BTV infection.     

Testing of bulk tank milk for Salmonella Dublin infection in Danish dairy herds.

The usefulness of enzyme-linked immunosorbent assay (ELISA) was investigated as a simple method to screen for Salmonella Dublin infection in dairy herds, examining bulk tank milk samples for lipopolysaccharide (O:1,9,12) antibodies. The cut-off value for the ELISA on bulk tank milk was established based on individual milk samples (n = 2887) and bulk tank milk from 52 herds. Bulk tank milk samples (n = 5108) were collected from 1464 dairy herds located in 19 different areas. About 10% of the dairy herds in Denmark participated in the study. The percentage of herds changing from test-negative to test-positive in each area was correlated with the incidence of S. Dublin outbreaks in the corresponding county (r = 0.48, n = 19; P < 0.025). The mean level of the OD values obtained in the first and third test rounds was not constant (Pr /t/ = 0.0001). The study demonstrated that the probability of being test-negative in the third test round was 0.926 for a herd with 2 previous test-negative results. It was concluded that the investigated ELISA method was in general accordance with the cases of clinical S. Dublin infection recorded, and that the method has a potential for national screening purposes.     

A longitudinal study of seroprevalence and seroconversion of Neospora caninum infection in dairy cattle in northeast Thailand

A long-term study was carried out in 11 dairy herds in the Khon Kaen province of northeast Thailand between August 2001 and November 2004. The objective was to investigate seroprevalence dynamics of Neospora caninum infection in the herds and to demonstrate patterns of seroconversion in individual cattle. Each herd was visited once a year, in total four times, and sera from cattle > 3 months of age and farm dogs as well as a sample from the bulk milk were collected. All samples were analysed for presence of specific antibodies by an N. caninum iscom ELISA. The overall percentage of antibody-positive cattle was constant and varied only between 10 and 13% over the 4 years, but the variation in within-herd seroprevalence between herds was substantial. Two herds had > or = 20% seropositive animals at all samplings and consistently high bulk milk OD, whereas two herds had no seropositive animal at the last two samplings and low bulk milk OD. Five herds had a decreasing trend of within-herd seroprevalence, whereas the remaining six herds had a higher portion of test-positive individuals at the end of the study. A total of 424 individuals were sampled more than once; 344 (81%) and 32 (8%) were consistently antibody-negative and antibody-positive, respectively. The proportions of animals that changed from being seronegative to seropositive and from being seropositive to seronegative between the years were 3.9-4.6% and 19-39%, respectively. Apparent vertical and horizontal transmission rates were 58% (95% CI; 44-71%) and 5% (95% CI; 3-7%), respectively. In conclusion, the overall percentage of N. caninum antibody-positive cattle was constant over the years, but the within-herd seroprevalence varied substantially between the herds. Seroconversions were likely to occur in individual cattle although most animals had consistent serological status throughout the study.     

Use of bulk milk for detection of Neospora caninum infection in dairy herds in Thailand

The relationship between the level of Neospora caninum antibodies in bulk milk and the seroprevalence in lactating cows was investigated. Bulk milk was also used to estimate the prevalence of N. caninum infection in dairy herds in the northeast and north Thailand. Bulk milk and individual serum from all lactating cows in 11 herds as well as 220 bulk milk samples from nine milk collection centres were analysed for presence of N. caninum antibodies using an iscom ELISA. In the 11 herds the bulk milk absorbances ranged between 0.04 and 0.89 and the seroprevalences varied between 0 and 46%. Five herds had milk absorbances below 0.20, among those were the two herds housing only seronegative lactating cows. In the remaining three herds with such low bulk milk absorbances one or two cows (5-14%) were seropositive. Six of the investigated herds had bulk milk absorbances above 0.20. In the two herds with the highest bulk milk absorbances more than 30% of the cows were seropositive. Using an absorbance of 0.20 to discriminate between negative and positive herds, 102 (46%) of 220 bulk milk samples were judged positive. There was no significant difference in mean bulk milk absorbance between the milk collection centres within each region. However, the proportion of herds with bulk milk absorbances > or =0.50 in the north was statistically (P < 0.01) higher than that in the northeast. It was concluded that bulk milk antibody testing can be used to identify N. caninum-infected herds and that N. caninum is a common infection in dairy herds in Thailand.     

Serosurveillance of Schmallenberg virus in Switzerland using bulk tank milk samples

Infections with Schmallenberg virus (SBV), a novel Orthobunyavirus transmitted by biting midges, can cause abortions and malformations of newborns and severe symptoms in adults of domestic and wild ruminants. Understanding the temporal and spatial distribution of the virus in a certain territory is important for the control and prevention of the disease. In this study, seroprevalence of antibodies against SBV and the spatial spread of the virus was investigated in Swiss dairy cattle applying a milk serology technique on bulk milk samples. The seroprevalence in cattle herds was significantly higher in December 2012 (99.5%) compared to July 2012 (19.7%). This high between-herd seroprevalence in cattle herds was observed shortly after the first detection of viral infections. Milk samples originating from farms with seropositive animals taken in December 2012 (n = 209; mean 160%) revealed significantly higher S/P% ratios than samples collected in July 2012 (n = 48; mean 103.6%). This finding suggests a high within-herd seroprevalence in infected herds which makes testing of bulk tank milk samples for the identification farms with past exposures to SBV a sensitive method. It suggests also that within-herd transmission followed by seroconversion still occurred between July and December. In July 2012, positive bulk tank milk samples were mainly restricted to the western part of Switzerland whereas in December 2012, all samples except one were positive. A spatial analysis revealed a separation of regions with and without positive farms in July 2012 and no spatial clustering within the regions with positive farms. In contrast to the spatial dispersion of bluetongue virus, a virus that is also transmitted by Culicoides midges, in 2008 in Switzerland, the spread of SBV occurred from the western to the eastern part of the country. The dispersed incursion of SBV took place in the western part of Switzerland and the virus spread rapidly to the remaining territory. This spatial pattern is consistent with the hypothesis that transmission by Culicoides midges was the main way of spreading.     

Trichinella spiralis infection induces β-actin co-localized with thymosin β4

A serological survey for Neospora caninum and Besnoitia besnoiti was carried out in beef and dairy cattle in South Australia. Serum samples of dairy cattle (n=133) from 9 properties and tank milk samples from a further 122 dairy herds were tested. An additional 810 sera from beef cattle from 51 properties were also tested. Testing at the individual animal level by IDEXX NEOSPORA X2 Ab test ELISA revealed a low prevalence of N. caninum antibodies of only 2.7% (95% CI; 1.6-3.7%) sera positive, as did the milk testing that showed 2.5% (95% CI; 1.4-3.6%) of tank milks being positive. At the herd level, 29.4% (95% CI; 16.9-41.9%) of beef, and 44.4% (95% CI; 12.0-76.9%) of dairy cattle herds showed serum antibodies. The highest within-herd prevalence in beef was 20% and 25%in dairy, which explains the low herd prevalence in dairy detected by bulk milk testing. Testing for B. besnoiti antibodies by PrioCHECK(?) Besnoitia Ab 2.0 ELISA initially identified 18.4% (95% CI: 15.8-21.0%) of 869 individual cattle sera as positive by ELISA at the manufacturer's suggested cut-off threshold (15 PP). Additional tests by immunoblot and IFAT, however, could not confirm any of the ELISA results. The use of a higher (40 PP) threshold in the ELISA is suggested to improve specificity. There is thus no evidence of B. besnoiti infection in South Australian cattle.     

Diagnosis of bovine brucellosis by enzyme immunoassay of milk

Enzyme-immunoassays using lipopolysaccharide as antigen were developed for the detection of bovine IgG1, IgG2 or IgA Brucella antibodies (Ab) in milk. The results of these tests were compared with those of the milk ring test (MRT) by analyzing 3212 bulk milk samples from farms located in regions where brucellosis is prevalent. Among the 105 herds detected by ELISA and/or MRT, 29 infected herds were detected by ELISA only. The 40 MRT-positive herds were also ELISA positive. Five herds became infected during the study and were detected by ELISA 15 days to 6 months prior to detection by MRT. The ELISA IgG1 titration (IgG1 ELISA) detected 92.8% of the herds found positive in the three ELISA assays. The concomitant use of IgA ELISA raised the sensitivity to 100% but slightly decreased the specificity. The IgG2 ELISA did not improve the diagnosis. The sensitivity of MRT and IgG1 ELISA was compared by testing successive dilutions in negative milk of 110 individual MRT positive milks samples. On average, IgG1 ELISA was 22 times more sensitive than MRT.     

Considerations concerning diagnostic certainties and cut-off values for a bulk milk ELISA for Mycobacterium avium ssp. paratuberculosis

Serological tests for the examination of individual samples from single animals are evaluated based on their ability to detect true positives above a defined threshold value. If results are obtained not from an individual but from a bulk sample this concept usually is adopted such that the threshold is set to allow the detection of a single positive sample within the pool. In conjunction with the development of a diagnostic paratuberculosis ELISA for the examination of bulk milk samples it is discussed which interpolations of this concept are justified when defining the true status of a herd based on the test parameters and the seroprevalence within the herd. Here, bulk milk from up to 50 animals each and the corresponding individual samples of 4241 dairy cows from 28 herds in the state of Brandenburg are investigated, and results are subjected to different evaluation approaches. Based on epidemiological considerations and test parameters a "critical prevalence" is defined which then serves as basis for the deduction of a cut-off value to be used for bulk milk samples. Finally, the practical relevance of this approach is demonstrated by suggesting an initial scheme for paratuberculosis classification of dairy herds with respect to possible control measures.     

Udder health in dairy cattle infected with Neospora caninum

Blood samples were collected from 3449 cows on 57 representative Ontario dairy herds during the summer of 1998 and analysed for antibody to Neospora caninum using an ELISA. Forty-eight herds (2742 cattle) contained at least one N. caninum-seropositive animal. Two composite milk samples were collected from all cattle: the first on the day of blood collection and the second 68 to 365 days later. All milk samples were submitted for bacteriological culture. Ontario Dairy Herd Improvement Corporation (DHI) data were available for 3162 cattle in the 57 herds at the time of bleeding. Furthermore, complete DHI data were available for 1658 cattle that were culled between 12 and 24 months following blood collection. Using a standardised ELISA sample-to-positive (S/P) cut-off of ≥0.45, the corrected seroprevalence was 8.2% overall and 10.1% within seropositive herds. At blood collection the odds of N. caninum-seropositive cows having a high linear score (≥4.0; equivalent to a somatic cell count ≥200,000 cells/ml) was 27% less than for seronegative animals. Similarly, at the time of culling, the odds of having a high linear score was 22% less in N. caninum-seropositive cattle. Overall, linear score was lower in N. caninum-seropositive cattle at culling. After controlling for herd, parity, days in milk, and the interval between collection of milk samples, the odds of N. caninum-seropositive cattle testing positive for an environmental pathogen (i.e. environmental Streptococcus species and coliforms) on the second milk sample was 56% less than for seronegative animals. The odds were 83% less at a higher ELISA S/P cut-off of ≥0.70. Finally, the odds of N. caninum-seropositive cattle developing a new infection with a major pathogen (environmental or contagious) were 60% less than seronegative cows using the higher ELISA S/P cut-off.     

Anti-Neospora caninum antibodies in milk in relation to production losses in dairy cattle

A comprehensive field study was carried out with the following objectives: (a) to assess the usefulness of individual and bulk tank milk analysis for determining Neospora caninum serostatus in individual cows and herds, and (b) to study the associations between N. caninum infection status (based on milk testing), and several productive and reproductive parameters in the animals. Antibodies were detected with a commercially available ELISA test (Bio K 192/5). Analysis of paired serum and milk samples from 1134 lactating cows on 38 farms revealed that 97.6% of the ELISA results were coincident, irrespective of whether serum or milk samples were used. Moreover, multiple linear regression analysis revealed that 86.0% of the variations in ELISA values in milk were due to variations in the serum. The measurement of antibodies in bulk tank milk was a good estimator of the herd level status of N. caninum infection, and enabled detection of infection in 94.7% herds with ≥10.0% seropositive cows and/or in all herds with >4% highly seropositive cows. The odds ratio for abortion in seropositive animals was 9.1 times higher than in seronegative animals. The infection serostatus was also a significant risk factor, as the odds ratio for abortion was even higher (12.0 times) in cows categorized as highly seropositive. ELISA values for the bulk milk from 387 randomly selected herds were negatively associated with average milk production. Moreover, milk production losses mainly occurred on farms categorized as highly positive (i.e. herds with ≥20.0% seropositive cows).     

Comparison of an indirect ELISA with the Brucella milk ring test for detection of antibodies to Brucella abortus in bulk milk samples.

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of Brucella abortus antibodies in bovine bulk milk samples was evaluated. About 31 individual milk samples from B. abortus infected cows were diluted into bulk milk from a brucellosis free herd. Individual milk samples obtained from 96 negative or positive herds to ELISA or Brucella ring test (BRT), were tested by ELISA. All positive cows were bled and serum samples were tested by the complement-fixation test (CFT) which was considered the definitive test. A herd was considered infected if at least, one cow was positive in the CFT. Four samples were negative in the BRT at the dilution 1:10 but positive in the ELISA. For samples positive in both tests, BRT titers ranged from 1:10 to 1:480 while ELISA titers ranged from 1:10 to 1:3200.Using bulk milk samples, the sensitivity of the ELISA (98.1%) was higher than the BRT (72.2%) but the specificity of BRT (90.5%) was not statistically different (P=1.0) from the ELISA (88.1%). The implications of the results for brucellosis control are discussed.     

Bulk-tank milk ELISA antibodies for estimating the prevalence of paratuberculosis in Danish dairy herds

Paratuberculosis (Johne's disease) has been widespread in Danish dairy herds for a long time but the herd-level prevalence has never been determined precisely. To evaluate the prevalence of paratuberculosis in Danish dairy herds in various regions, an ELISA based on a commercially available antigen was adapted for testing bulk-tank milk for the presence of antibodies to Mycobacterium avium subsp. paratuberculosis. Bulk-tank milk samples were collected from six milk-collecting centres from six different areas of the country. Samples from 900 herds (about 7.5% of all Danish dairy herds) were examined, and 70% were positive at the statistically optimal cut-off (sensitivity 97.1%; specificity 83.3%). The technical performance of the ELISA was not sufficient to provide a tool for surveillance because even slight changes in optical density for the samples would change the classification of some samples. The infection is more widespread than previous investigations have shown.     

Bovine viral diarrhoea virus seroprevalence and vaccination usage in dairy and beef herds in the Republic of Ireland

Background

Bovine viral diarrhoea (BVD) is an infectious disease of cattle with a worldwide distribution. Herd-level prevalence varies among European Union (EU) member states, and prevalence information facilitates decision-making and monitoring of progress in control and eradication programmes. The primary objective of the present study was to address significant knowledge gaps regarding herd BVD seroprevalence (based on pooled sera) and control on Irish farms, including vaccine usage.

Methods

Preliminary validation of an indirect BVD antibody ELISA test (Svanova, Biotech AB, Uppsala, Sweden) using pooled sera was a novel and important aspect of the present study. Serum pools were constructed from serum samples of known seropositivity and pools were analysed using the same test in laboratory replicates. The output from this indirect ELISA was expressed as a percentage positivity (PP) value. Results were used to guide selection of a proposed cut-off (PCO) PP. This indirect ELISA was applied to randomly constructed within-herd serum pools, in a cross-sectional study of a stratified random sample of 1,171 Irish dairy and beef cow herds in 2009, for which vaccination status was determined by telephone survey. The herd-level prevalence of BVD in Ireland (percentage positive herds) was estimated in non-vaccinating herds, where herds were classified positive when herd pool result exceeded PCO PP. Vaccinated herds were excluded because of the potential impact of vaccination on herd classification status. Comparison of herd-level classification was conducted in a subset of 111 non-vaccinating dairy herds using the same ELISA on bulk milk tank (BMT) samples. Associations between possible risk factors (herd size (quartiles)) and herd-level prevalence were determined using chi-squared analysis.

Results

Receiver Operating Characteristics Analysis of replicate results in the preliminary validation study yielded an optimal cut-off PP (Proposed Cut-off percentage positivity - PCO PP) of 7.58%. This PCO PP gave a relative sensitivity (Se) and specificity (Sp) of 98.57% and 100% respectively, relative to the use of the ELISA on individual sera, and was chosen as the optimal cut-off since it resulted in maximization of the prevalence independent Youden’s Index.The herd-level BVD prevalence in non-vaccinating herds was 98.7% (95% CI - 98.3-99.5%) in the cross-sectional study with no significant difference between dairy and beef herds (98.3% vs 98.8%, respectively, p = 0.595).An agreement of 95.4% was found on Kappa analysis of herd serological classification when bulk milk and serum pool results were compared in non-vaccinating herds. 19.2 percent of farmers used BVDV vaccine; 81% of vaccinated herds were dairy. A significant association was found between seroprevalence (quartiles) and herd size (quartiles) (p < 0.01), though no association was found between herd size (quartiles) and herd-level classification based on PCO (p = 0.548).

Conclusions

The results from this study indicate that the true herd-level seroprevalence to Bovine Virus Diarrhoea (BVD) virus in Ireland is approaching 100%. The results of the present study will assist with national policy development, particularly with respect to the national BVD eradication programme which commenced recently.     

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.