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.     

Diagnostic performance of the Pourquier ELISA for detection of antibodies against Mycobacterium avium subspecies paratuberculosis in individual milk and bulk milk samples of dairy herds

The objective of the study was to determine the diagnostic performance of the Pourquier ELISA for detection of antibodies against Mycobacterium avium subsp. paratuberculosis (Map) in individual milk samples and in bulk milk samples. For individual milk samples the specificity of the Pourquier ELISA was estimated by testing a panel of individual milk samples from certified Map-free herds. The relative sensitivity of the assay in individual milk samples and agreement of the results with those of serum samples was estimated by testing panels of paired serum-milk samples from seropositive cattle, whole-herd investigations, and moderate or heavy shedders. The specificity of the ELISA for individual milk samples was still 99.8% at a cut-off of 20% sample to positive (S/P) value, clearly lower than the cut-off defined by the manufacturer (30% S/P). The relative sensitivity for individual milk samples as compared with positive serum samples was 87% for a cut-off of 20% S/P, and 80% for a cut-off of 30% S/P. The sensitivity of this ELISA for detection of high shedders was >90% both for individual milk and serum samples, also agreement was very good (kappa=0.91 for all paired samples). The specificity of the Pourquier ELISA in bulk milk samples was investigated by testing bulk milk samples from certified Map-free herds. Feasibility of bulk milk testing was investigated by titrating ELISA positive individual milk samples in negative milk. In addition, 383 bulk milk samples from herds with a known within-herd seroprevalence were tested. The specificity of the ELISA for bulk milk samples was 100% at a cut-off of 12.5% S/P. At the cut-off recommended by the manufacturer (30% S/P) performance of the bulk milk ELISA related to herd status (> or =2 seropositive cows) was rather poor, corresponding with a sensitivity of 24% and a specificity of 99% relative to serology. However, at the revised cut-off for bulk milk of 12.5% S/P and a within-herd seroprevalence of > or =3%, sensitivity and specificity relative to serology were 85% and 96%, respectively. Given the current herd-level seroprevalence in The Netherlands, these test characteristics corresponded with positive and negative predictive values for bulk milk of 67% and 94%, respectively. In conclusion, the diagnostic performance of the Pourquier ELISA for individual milk samples creates opportunities for a cheaper and more feasible testing scheme, while the diagnostic performance for bulk milk samples warrants further consideration.     

BVDV and BHV-1 infections in dairy herds in northern and northeastern Thailand

Bulk milk samples from 220 dairy herds were collected at 9 public milk collection centres in the northeastern and northern Thailand, and a subset of 11 herds was selected for individual testing. The samples were tested for presence of antibodies to BVDV and BHV-1 using an indirect ELISA. The results from the bulk milk testing demonstrated a moderate level of exposure to BVDV and BHV-1 (73% and 67%, respectively). However, the low proportion of herds with high BVDV antibody-levels (13%) and the low within-herd seroprevalence of BVDV and BHV-1 in the 11 herds (24% and 5%, respectively), particularly among the young stock (15% and 0%, respectively), demonstrated a low prevalence of active BVDV infection and a low rate of reactivation of latent BHV-1. The presence of a self-clearance process was also indicated by the results from the individual testing. Moreover, a surprisingly low prevalence of BVDV and BHV-1 antibody-positive herds at one of the milk centres was found. This centre was established 5-10 years before the others. Our impression is that this reflects the self-clearance process, where consecutive replacement of imported infected animals without further spread has resulted in a nearly total elimination of the infections. Based on our experiences and on these results we are convinced that this process can continue if there is awareness of herd biosecurity. This is especially important in the context of a future intensification of the dairy production.     

Aspects of bovine herpesvirus-1 infection in dairy and beef herds in the Republic of Ireland

Background

Infection with bovine herpesvirus-1 (BHV-1) causes a wide range of disease manifestations, including respiratory disease and abortion, with world-wide distribution. The primary objective of the present study was to describe aspects of BHV-1 infection and control on Irish farms, including herd-level seroprevalence (based on pooled sera) and vaccine usage.

Methods

The characteristics of a diagnostic indirect BHV-1 antibody ELISA test when used on serum pools were evaluated using laboratory replicates for use in the seroprevalence study. The output from this indirect ELISA was expressed as a percentage positivity (PP) value. A proposed cut off (PCO) PP was applied in a cross-sectional study of a stratified random sample of 1,175 Irish dairy and beef cattle herds in 2009, using serum pools, to estimate herd seroprevalence. The study was observational, based primarily on the analysis of existing samples, and only aggregated results were reported. For these reasons, ethical approval was not required. Bulk milk samples from a subset of 111 dairy herds were analysed using the same ELISA. Information regarding vaccine usage was determined in a telephone survey.

Results

A PCO PP of 7.88% was determined to give 97.1% sensitivity and 100% specificity relative to the use of the ELISA on individual sera giving maximization of the prevalence independent Youden''s index, on receiver operating characteristics analysis of replicate results. The herd-level BHV-1 seroprevalence was 74.9% (95% CI - 69.9%-79.8%), with no significant difference between dairy and beef herds. 95.5% agreement in herd classification was found between bulk milk and serum pools. Only 1.8 percent of farmers used BHV-1 marker vaccine, 80% of which was live while 75% of vaccinated herds were dairy.A significant association was found between herd size (quartiles) and seroprevalence (quartiles).

Conclusions

The results from this study indicate BHV-1 infection is endemic, although BHV-1 vaccines are rarely used, in the cattle population in Ireland.     

Prevalence of Fasciola hepatica in dairy herds in England and Wales measured with an ELISA applied to bulk-tank milk

An ELISA developed to diagnose Fasciola hepatica infection in cattle by detecting serum antibodies was adapted and validated for use with samples of bulk-tank milk. The prevalence of the infection in 61 dairy herds was established by using serum antibody levels or faecal egg counts measured in a proportion of the cows in each herd. The correlation between the results of the ELISA and the herd seroprevalence was 0.83. Using a cut-off value of 27 per cent positive, the bulk-tank ELISA identified herds in which more than 25 per cent of the cows were infected with a diagnostic sensitivity of 96 per cent (95 per cent confidence interval 89 to 100 per cent) and a diagnostic specificity of 80 per cent (95 per cent confidence interval 66 to 94 per cent). By applying the ELISA to 623 herds in England and 445 herds in Wales, the prevalence of F hepatica infection in England was estimated to be 48 per cent (95 per cent confidence interval 46 to 54 per cent), and in Wales 86 per cent (95 per cent confidence interval 84 to 90 per cent).     

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.     

Seroprevalence of Mycobacterium avium subsp paratuberculosis infection among dairy cows in Colorado and herd-level risk factors for seropositivity

OBJECTIVE: To estimate seroprevalence of Mycobacterium avium subsp paratuberculosis (MAP) infection among adult dairy cows in Colorado and determine herd-level factors associated with the risk that individual cows would be seropositive. DESIGN: Cross-sectional observational study. ANIMALS: 10,280 adult (> or = 2 years old) dairy cows in 15 herds in Colorado. PROCEDURE: Serum samples were tested with a commercial ELISA. A herd was considered to be infected with MAP if results of mycobacterial culture of > or = 1 individual cow fecal sample were positive or if > or = 1 culled cow had histologic evidence of MAP infection. RESULTS: 424 of the 10,280 (4.12%) cows were seropositive. Within-herd prevalence of seropositive cows ranged from 0% to 7.82% (mean, 2.6%). Infection was confirmed in 11 dairies. Cows in herds that had imported > or = 8% of their current herd size annually during the preceding 5 years were 3.28 times as likely to be seropositive as were cows in herds that imported < 8%. Cows in herds with > or = 600 lactating cows were 3.12 times as likely to be seropositive as were cows in herds with < 600 lactating cows. Cows in herds with a history of clinical signs of MAP infection were 2.27 times as likely to be seropositive as were cows in herds without clinical signs. CONCLUSIONS AND CLINICAL RELEVANCE: Annual importation rate, herd size, and whether cows in the herd had clinical signs typical of MAP infection were associated with the risk that individual cows would be seropositive for MAP infection.     

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.     

Comparison of milk and serum enzyme-linked immunosorbent assays for diagnosis of Mycobacterium avium subspecies paratuberculosis infection in dairy cattle.

Milk and serum samples from 35 dairy herds in 17 states were evaluated for cow- and herd-level Mycobacterium avium subspecies paratuberculosis (MAP) antibody test agreement. Evaluation of 6,349 samples suggested moderate agreement between milk and serum enzyme-linked immunosorbent assay (ELISA) results, with a kappa value of 0.50. Cow-level sensitivity (Se) for 18 dairy operations with 1,921 animals was evaluated relative to fecal culture results. At the cow level, the milk ELISA relative Se was not significantly different from that of the serum ELISA (21.2 and 23.5%, respectively). Logistic regression models revealed a positive association between lactation number and milk ELISA status. Non-Holstein cows were more likely to test milk ELISA positive than Holstein cows. Cows in the first 2 weeks of lactation and after week 45 of lactation were more likely to test milk ELISA positive than cows between 3 and 12 weeks of lactation. Milk production > 80% of herd average was negatively associated with testing milk ELISA positive. Animals in the West and Midwest regions were less likely than animals in the Southeast region to test ELISA positive by either test. Estimates for herd-level sensitivity for the milk and serum ELISA, relative to fecal culture results, ranged from 56 to 83%. At the cow and herd levels, milk ELISA performed equivalent to serum ELISA using fecal culture as a reference for MAP infection and has the advantage of decreased labor costs on farms that use Dairy Herd Improvement Association testing.     

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.     

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.     

Evaluation of serum and milk ELISAs for paratuberculosis in Danish dairy cattle

A milk and a serum ELISA for detection of antibodies against Mycobacterium avium ssp. paratuberculosis (MAP) were evaluated against the complement-fixation test (CFT) and culture of faecal samples from 580 cows collected between August 1996 and December 1996. Milk and serum were obtained concurrently from six dairy herds infected with MAP and from two dairy herds without history of infection with MAP.

A cut-off value of 7 OD% was used in the ELISAs. At this cut-off value, all six culture-positive herds were positive in the serum ELISA but one was negative in the milk ELISA. All six culture-positive herds were positive in the CFT. In the two culture-negative herds, the serum and the milk ELISA deemed all serum samples negative at this cut-off value, whereas four serum samples from one of these herds were positive in the CFT. The highest cut-off value enabling the milk ELISA to record all six culture-positive herds as positive was 4 OD%. The highest cut-off value enabling the serum ELISA to record all six culture-positive herds as positive was 17 OD%. Individual-sample relative sensitivities of the ELISAs ranged from 49 to 64% and relative specificities were 80–96% at the cut-off values of 4, 7 and 17 OD%.     

Validation of a Dictyocaulus viviparus MSP-ELISA and cut-off adjustment in a one-year longitudinal field study in dairy cattle herds

A one-year field study analysing lungworm seropositivity by use of the MSP-ELISA was performed (1) to investigate the antibody dynamics in individual milk samples following field (re-)infections of dairy cows with the bovine lungworm Dictyocaulus viviparus, (2) to investigate the correlation between individual and bulk tank milk (BTM) antibody titres and (3) to review the current individual as well as BTM cut-off value, which was extrapolated from dilution experiments (Fiedor et al., 2009). Over a one-year period individual and BTM samples were collected monthly on 15 dairy farms. Following a critical review of previous cut-off values, individual and BTM samples were subjected to different cut-off thresholds. Following Receiver-Operating-Characteristics (ROC) analysis, individual milk samples were assessed with the cut-off value 0.573, previously shown to be associated with each 100% sensitivity and specificity. In addition, the present study enabled BTM cut-off adjustment based on field data. To ensure reliable detection of herds with an in-herd prevalence of ≥20% the BTM cut-off was lowered from 0.493 to 0.410, corresponding to 100% sensitivity and 97.32% specificity. Regression analysis showed that the percentage of seropositive animals related to the corresponding BTM ODR correlated moderately (r=0.581, P<0.001), whereas a strong correlation (r=0.764, P<0.001) was found between mean individual and BTM ODR per herd and sampling month. Seasonal antibody pattern became obvious in a single-peaked antibody curve in late summer/early autumn for individual milk whilst BTM showed a two-peaked distribution with an additional spring peak besides the late summer/early autumn peak. This leads to the conclusion that the BTM-ELISA could be a useful tool to detect and control pasture contamination in the spring, following sexual maturation of hypobiotic lungworm larvae harboured by clinically asymptomatic carrier animals. In addition to the knowledge gained on antibody patterns in dairy herds and the relationship of individual and BTM, the present study enabled sensitivity and specificity calculations for the obsolete BTM cut-off value 0.493 to be performed.     

The Mycobacterium avium subsp. paratuberculosis ELISA response by parity and stage of lactation

Two cross-sectional studies were carried out to determine the enzyme-linked immunosorbent assay (ELISA) response to Mycobacterium avium subsp. paratuberculosis (Map) by cow characteristics and stage of lactation. One of the studies (referred to as "milk-group") used milk samples from all lactating cows (n=7994) in 108 Danish dairy herds. The other study (referred to as "serum-group") used serum samples collected from all cows (n=5323) in a subset of 72 herds from the 108 herds. These samples were analysed using a similar ELISA for detection of antibodies.The results from the ELISAs were interpreted with two cut-off values as the optimal cut-off value is not known, and as several levels are recommended to be used in practice. The results showed that the probability of being ELISA-positive was two to three times lower for cows in parity 1 relative to cows in other parities using both milk and serum ELISA. At the beginning of the lactation the probability of being positive was highest in the milk ELISA. In the serum ELISA the odds of being positive was highest at the end of lactation. The findings are important in the interpretation of ELISA results at cow level with a subsequent tentative diagnosis and correction for parity and stage of lactation should be considered when providing a diagnosis of paratuberculosis. Some issues related to the pathogenesis are also discussed.     

Simulation model estimates of test accuracy and predictive values for the Danish Salmonella surveillance program in dairy herds

The Danish government and cattle industry instituted a Salmonella surveillance program in October 2002 to help reduce Salmonella enterica subsp. enterica serotype Dublin (S. Dublin) infections. All dairy herds are tested by measuring antibodies in bulk tank milk at 3-month intervals. The program is based on a well-established ELISA, but the overall test program accuracy and misclassification was not previously investigated. We developed a model to simulate repeated bulk tank milk antibody measurements for dairy herds conditional on true infection status. The distributions of bulk tank milk antibody measurements for infected and noninfected herds were determined from field study data. Herd infection was defined as having either >or=1 Salmonella culture-positive fecal sample or >or=5% within-herd prevalence based on antibody measurements in serum or milk from individual animals. No distinction was made between Dublin and other Salmonella serotypes which cross-react in the ELISA. The simulation model was used to estimate the accuracy of herd classification for true herd-level prevalence values ranging from 0.02 to 0.5. Test program sensitivity was 0.95 across the range of prevalence values evaluated. Specificity was inversely related to prevalence and ranged from 0.83 to 0.98. For a true herd-level infection prevalence of 15%, the estimate for specificity (Sp) was 0.96. Also at the 15% herd-level prevalence, approximately 99% of herds classified as negative in the program would be truly noninfected and 80% of herds classified as positive would be infected. The predictive values were consistent with the primary goal of the surveillance program which was to have confidence that herds classified negative would be free of Salmonella infection.     

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.     

Bovine herpesvirus type 1 (BHV-1) and bovine viral diarrhoea virus (BVDV) infections in dairy herds: self clearance and the detection of seroconversions against a new atypical pestivirus

The epidemiology of bovine herpesvirus type 1 (BHV-1) and bovine viral diarrhoea virus (BVDV) was studied in a population of small dairy herds that had not been vaccinated. Bulk tank milk samples of 186 herds in Thailand were collected four times between 2002 and 2004. Serum samples from individual animals in 11 herds were also taken on three occasions. The prevalence of BHV-1 in the 186 herds was 61% in 2002, decreasing to 48% in 2004 and for BVDV was 91% in 2002, decreasing to 72% in 2004. A BVDV antigen-positive calf was found in one of the 11 herds, and animals in this herd and three other herds seroconverted to a recently described atypical BVDV strain (HoBi). This study showed a significantly decreasing prevalence for both BHV-1 and BVDV due to a self-clearance process. Further studies are needed to find out how the atypical BVDV strain entered the cattle population.     

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.     

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.     

Bulk milk testing for antibody seroprevalences to BVDV and BHV-1 in a rural region of Peru

Bulk milk from 60 herds of dairy cattle in a rural region in the central highlands of Peru was tested for antibodies to bovine viral-diarrhoea virus (BVDV) and bovine herpesvirus type 1 (BHV-1). None of the herds had been vaccinated against BVDV or BHV-1. Commercially available indirect ELISA-kits were used for antibody detection. True prevalences of BVDV and BHV-1 antibody-positive herds were 96 and 51%, respectively. A relatively low proportion of strongly positive herds suggests, however, a low prevalence of active BVDV infection. BVDV optical densities (ODs) in bulk milk increased with herd size--indicating a higher within-herd prevalence in the larger herds (probably, in part a consequence of a higher rate of animal movement into these herds). For BHV-1, this pattern was not found; a relatively high proportion of the herds was free from BHV-1 infection in each size category. This could indicate a low rate of reactivation of latent BHV-1 infection.