Evaluation of an O antigen enzyme-linked immunosorbent assay for screening of milk samples for Salmonella dublin infection in dairy herds.

Levels of antibodies to the O antigens (O:1,9,12) of Salmonella dublin were tested in 1355 serum, 1143 cow milk and 160 bulk milk samples from dairy herds using an enzyme-linked immunosorbent assay (ELISA). In order to define the background reaction, milk samples from all lactating cows and serum samples from 9 animals were collected in each of 20 salmonellosis-free herds located on the island of Bornholm, where cattle salmonellosis has not been reported. Similar samples were collected from all stalled animals in 10 herds with recent (< 6 months) outbreaks of salmonellosis located in Jutland, where salmonella infection is enzootic. Using herd history of salmonellosis, herd location and clinical status of the herds as criteria, the optimal cutoff in the milk ELISA was determined as being at least 5% of the samples having optical density > 0.5, resulting in herd sensitivity of 1.0 and herd specificity of 0.95. While none of the sera in the herds from Bornholm was ELISA positive, 2 herds had a few reactors in the milk ELISA. Using the same cutoff, all but 1 bulk milk sample from 150 herds on Bornholm was ELISA-negative, and all 10 salmonellosis-positive herds from Jutland were ELISA-positive. A significant correlation was found between ELISA reactions in milk and in serum of cows (34% and 32% respectively, rs = 0.69, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

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).     

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%.     

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.     

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.     

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

Serum and milk samples from 1229 cows on 22 Ontario dairy farms were individually tested for antibodies specific for bovine leukosis virus (BLV) and Neospora caninum by enzyme-linked immunosorbent assay (ELISA). Antibodies against BLV were present in 361 serum samples (29.4%) and 369 milk samples (30.0%). Comparing the 2 tests, agreement was almost perfect (k = 0.86; 95% CI = 0.83 to 0.90) and the proportions of samples positive were not significantly different (P = 0.56). Both tests identified the same 3 herds free of bovine leukosis virus. Antibodies against N. caninum were detected in 138 serum samples (11.2%), and 111 milk samples (9.0%). Agreement between the 2 tests was moderate (k = 0.52; 95% CI = 0.43 to 0.59). Four herds were free of neosporosis by the serum test, while 10 herds were negative by the milk test. The ELISA on milk samples facilitates sample collection to classify herds free of BLV; the milk N. caninum ELISA was less reliable in predicting herd-level infection.     

Occurrence of Coxiella burnetii in Polish dairy cattle herds based on serological and PCR tests

The aim of the research was to assess the prevalence of antibodies to Coxiella burnetii in dairy cattle herds in Poland and to compare the results of real-time PCR and ELISA tests performed on bulk tank milk (BTM) samples. In total, 2635 serum samples collected from 969 dairy cattle herds from all provinces were tested using ELISA. Additionally, BTM specimens from 101 herds were analysed by ELISA and real-time PCR targeting IS1111 element. Presence of anti-C. burnetii antibodies was confirmed in 25.39% of serum samples in 237 herds (24.46%) and the herd-level seroprevalence in Voivodeships varied from 2.5% to 61.4%. Moreover, 46 (45.5%) of analysed bulk tank milk samples gave postive result in ELISA and microbial DNA was detected in 40 (39.6%) of tested herds. The comparative analysis of ELISA and real-time PCR results obtained for BTM samples using the chi-square test showed statistically significant relationship between results of both methods.     

Application of an ELISA Milk Pregnancy Test in Beef Cows

Pregnancy‐associated glycoproteins (PAG) are secreted by the binucleate giant cells of the ruminant placenta and enter maternal circulation at the time of placental attachment. The IDEXX Milk Pregnancy Test (IDEXX, Westbrook, ME) detects a subset of PAG in milk. Although designed as a management tool for dairy cows, there is potential for using the milk PAG test in beef cows. Our objective was to compare the performance of the milk PAG ELISA with a gold standard method for pregnancy diagnosis and determine the agreement between milk and serum PAG analysis in lactating beef cows. Angus and Angus‐crossed cows (n = 332) from two Michigan beef herds were enrolled in this study. Cows were subjected either to timed artificial insemination followed by exposure to a bull or exclusively exposed to a bull. The bulls and cows were separated 30 days prior to examination. Serum and milk samples were collected and submitted within 24 h of collection to a commercial laboratory for PAG analysis using the IDEXX Milk Pregnancy Assay (milk) and the IDEXX Bovine Pregnancy Assay (serum). Concurrently with milk and serum collection, each cow was examined transrectally by palpation or ultrasonography. When compared to transrectal examination, the performance (and 95% confidence intervals) of the milk PAG ELISA was sensitivity of 99.7% (99.0–100.0%) and specificity of 80.8% (65.6–95.9%). The lower specificity is likely due to the low prevalence (9.9%) of open cows (n = 30) in the herds examined. Of the 332 cows examined, 1.8% (n = 6) were classified as rechecks using the milk PAG ELISA. Results of the milk and serum PAG ELISA were in high agreement (kappa coefficient = 0.91). The milk PAG ELISA was accurate in predicting pregnancy status using milk collected from beef cattle between days 37 and 125 post‐insemination and may be useful for aiding management decisions in beef herds.     

Control measures in officially acknowledged brucellosis-free and leukosis unsuspected dairy herds on the basis of bulk milk samples in combination with ELISA tests

1. EC- and National Regulations. Since 1988 the EC-regulations accept in addition to the on Agar Gel Immunodiffusion test (AGIDT) based blood serum testing of cattle herds that are filed as "free from Enzootic Bovine Leucosis" the use of ELISA for this purpose. The regular testings in dairy cattle herds can be done alternatively with single or pooled milk samples, in other herds with pooled blood sera using ELISA. General condition is only a minimal sensitivity of the test to detect the European EBL Antibody Standard ("E4") in a dilution of 1:10 in negative serum or 1:250 in negative milk. Adequate national regulations are in preparation. The present limitation of pool sizes, blood maximum 50 animals without preparation steps 20, and milk after concentration treatment 50 cows is neutralized by proceedings in development of higher sensitive ELISA tests. This limitation should be canceled. Herd bulk milk samples without size limitations are accepted to be tested with "Milk Ring Test" by EC for the regular testings in filed "Brucellosis Free Dairy Cattle Herds". The alternative use of more sensitive (and more specific) ELISA tests for this purpose including the technical conditions is in a final discussion. 2. Scientific-Technical Base for Using the Chances of the Proceeding in the EC-Regulations. The realisation of the EC accepted or final discussed ELISA based bulk milk testing to control filed "EBL- and/or Brucellosis Free Herds" depends on some basic conditions like sensitivity, specificity, and variability of the ELISA systems. Field trials of more than 20,000 bulk milk samples in case of Brucellosis and more than 2,000 in case of EBL show the feasibilities and the limits of the ELISA systems in defining the status of the herds. The Brucellosis respectively the EBL situations of the dairy cattle herds tested in this trail were well known by history and by investigation of single animal blood samples using conventional tests. Special test run variations of pretested assays demonstrated the possibilities to define the EBL status of dairy cattle herds up to 50 lactating cows without preparation of the bulk milk sample and up 100 after concentration of the antibodies by the rennet-ammonium sulfate method. The concentration limit for detection of Brucellosis antibodies is 100 lactating cows. The bulk milk of smaller herds can be tested without concentration. On principle the evaluation of the test values bases on defined relations to a "weak positive" reference.(ABSTRACT TRUNCATED AT 400 WORDS)     

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 enzyme-linked immunosorbent assay using milk samples as a potential screening test of bovine tuberculosis of dairy cows in Korea

An enzyme-linked immunosorbent assay (ELISA) using bulk tank milk samples was evaluated as a screening test for bovine tuberculosis (TB), a contagious chronic disease of cattle. An ELISA with MPB70, a major antigen of Mycobacterium bovis was performed using paired sets of milk and sera samples from 33 tuberculin-positive and 43 tuberculin-negative cattle. Anti-MPB70 antibodies were detected in milk samples and there was a significant correlation between seroreactivities of milk and sera samples (R² = 0.83). Using the tuberculin skin test as the reference test, the sensitivities of ELISA using milk and sera samples were 87.8% and 81.8%, respectively, and the specificities were 97.7% and 100%, respectively.In the screening test using bulk tank milk samples from 931 dairy herds in Whasung, Gyeonggi-do, Korea, the positive rate for anti-MPB70 antibody was 4.5% (42/931) and the tuberculin-positive rate was 2.8% (26/931). Individual milk samples (n = 253) were collected from randomly selected 8 problematic and 3 negative herds (positive and negative in the screening test by MPB70 ELISA using bulk tank milk samples, respectively) and tested by MPB70 milk ELISA. In the problematic herds, positive rates were 10.5% (20/190) for anti-MPB70 antibodies in milk ELISA and 2.1% (4/190) in the tuberculin skin test. More than one dairy cows were positive by milk ELISA among the problematic herds, and all tuberculin-positive dairy cows were positive in the milk ELISA. Further, no positive cows were detected in negative herds both by milk ELISA and tuberculin skin test. These results suggest that an ELISA, using bulk tank milk samples, might be a potential efficient screening test for bovine TB of dairy cows.     

Associations between Neospora caninum specific antibodies in serum and milk in two dairy herds in Scotland

The study evaluated the use of the Mastazyme ELISA for quantification of Neospora caninum (N. caninum) specific IgG in bovine milk and examined the relationship between serum and milk antibodies in two dairy herds. The serum and milk antibodies both had bimodal distributions in each herd. This was mainly due to between cow variation: in both herds, approximately two thirds of cows were either clearly and consistently seropositive or seronegative for N. caninum with one third consistently near the threshold. Milk and serum N. caninum IgG were strongly related. This relationship was modelled using a linear mixed model including a polynomial term for serum, the effect of herd, and between and within cow variance components. The latter gave a significantly better fit to the data than a model that allowed for a different relationship for the positive and negative (according to the serum test) groups of observations. The sensitivity and specificity (based on serum percentage positivity (pp)) of the milk antibody was determined for different milk pp thresholds. In spite of the differences between the relationship of milk to serum seen for the two herds, for those estimates with sufficient precision, sensitivity and specificity greater than 0.73 for both herds were obtained using single thresholds of 14 and 15.5 for milk pp in both herds based on, as our gold standard, serum antibody pp thresholds of 22.5 and 25, respectively. If milk antibody is to be used for detecting persistently infected cows, the higher threshold of 15.5 may be suitable while for epidemiological screening 14 would be preferable. Further validation in a greater number of herds is required, but our results suggest that this test may prove to be a useful adjunct to serum N. caninum IgG assays in the monitoring of N. caninum infection as part of herd health programmes and epidemiological studies.     

Diagnosis of paratuberculosis by fecal culture and ELISA on milk and serum samples in two types of Chilean dairy goat herds.

Fecal culture has been the primary method used to diagnose paratuberculosis in goats. It is laborious, slow, and expensive. Validation of enzyme-linked immunosorbent assays (ELISAs) on milk samples could make paratuberculosis testing more widely available for goat farmers. The aim of this study was to determine the accuracy of serum and milk ELISAs for paratuberculosis, relative to fecal culture, in Chilean dairy goats. Eight dairy goat herds were selected. Feces, blood, and milk samples were collected from all female goats >2 years old. Fecal samples were cultured using Herrold egg yolk medium with mycobactin J and antibiotics. Serum and milk samples were tested using a commercial ELISA kit for Mycobacterium avium subsp. paratuberculosis antibody detection. A total of 383 goats were tested by ELISA and fecal culture. The sensitivity of ELISA on serum and milk relative to fecal culture was 74.3% (95% CI: 59.8-88.8) and 60% (95% CI: 43.8-76.2), respectively. The corresponding values for ELISA specificity based on the percentage of non- M. avium subsp. paratuberculosis-infected goats testing ELISA-negative were 98.6% (95% CI: 96.6-100) and 99.3% (95% CI: 97.9-100) on serum and milk, respectively. Proportions of positive results for serum and fecal samples were significantly different, whereas the proportions of positive results for milk and fecal samples were not significantly different. The milk ELISA had a moderate level of agreement with fecal culture results (Kappa = 0.57). The paratuberculosis ELISA on goat milk samples may be a cost-effective, accurate alternative to fecal culture.     

Prevalence of bovine herpesvirus 1 (BHV-1) infection in Hungarian cattle herds.

Hungarian cattle herds were surveyed for bovine herpesvirus 1 (BHV-1) infection by ELISA of milk and serum samples. In 1993, 75% of the large cattle herds (consisting of more than 50 cattle) and all small herds (small-scale producers' stocks), while in 1997 90% of the small herds were included in the survey. In the case of large herds, 79.3% of the herds and 64.1% of the samples tested were found to be positive. Of the small herds, 13.5% and 15.7% tested positive in 1993 and 1997, respectively. The majority of large herds were Holstein-Friesian dairy stocks. Small herds with an infection rate markedly exceeding the average were found in those counties where the small herds had been in close contact with the large-scale farms, or where new herds were established by using animals of uncontrolled infectious bovine rhinotracheitis (IBR) status originating from large farms. Attention is called to the importance of maintaining the IBR-free status of small herds that constitute one-third of the Hungarian cattle population.     

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.     

Informative Value of an Indirect Enzyme‐Linked Immunosorbent Assay (ELISA) for the Detection of Bovine Viral Diarrhoea Virus (BVDV) Antibodies in Milk

Bulk and individual milk samples from 117 herds located in Brittany (west France) were used to assess: (i) the performance characteristics of an indirect enzyme‐linked immunosorbent assay (ELISA) applied to individual milk for the detection of antibodies to bovine viral diarrhoea virus (BVDV); and (ii) the relationship between the bulk milk result obtained from this test and the within‐herd prevalence of antibody‐positive lactating cows. This ELISA test was based on a monoclonal antibody directed against non‐structural protein NS2‐3 of pestiviruses. At the individual level, based on 1113 matched milk/serum samples, the sensitivity and specificity of this test applied to milk, compared with the virus neutralization test on serum, were 95.0 and 97.7%, respectively. At the herd level, the relationship between the optical density percentage (OD%) of bulk milk and the within‐herd prevalence of antibody‐positive lactating cows was assessed using the receiver operating characteristics (ROC) analysis. Classes of OD% of bulk milk were determined so that they were associated with minimum intraclass and maximum between‐class variances of within‐herd prevalence of antibody‐positive cows. The ROC analysis resulted in two classes of bulk milk results corresponding to different expected levels of within‐herd prevalence. Herds with an OD% of bulk milk <75% and ≥75% had a mean observed prevalence of antibody‐positive cows of 8.9 and 60.6%, respectively. Herds with a bulk milk result <75% were expected to be BVDV free, whereas large variations in prevalence of antibody‐positive cows existed in the herds with OD% ≥75%. The test described in this study is suitable to identify herds likely to have a low prevalence of BVDV antibody‐positive cows.     

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.     

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.     

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.     

Development and evaluation of a highly sensitive and specific blocking enzyme-linked immunosorbent assay and polymerase chain reaction assay for diagnosis of bovine leukemia virus infection in cattle

OBJECTIVE: To develop a blocking ELISA for detection of bovine leukemia virus (BLV) antibodies that is comparable to a radioimmunoprecipitation (RIP) assay, to evaluate use of this ELISA for identification of BLV-infected herds, and to develop a polymerase chain reaction (PCR) assay for direct diagnosis of infection with BLV. SAMPLE POPULATION: Serum samples and pooled bulk-tank milk samples from cattle. PROCEDURE: The blocking ELISA was developed, using BLV gp51 as antigen, captured by a selected bovine polyclonal serum. A nested PCR was conducted with primers specific for a segment of the pol region of the BLV genome. RESULTS: Sensitivity and specificity of the ELISA were comparable to those of the RIP assay. Use of the ELISA on pooled milk samples allowed identification of herds in which prevalence of BLV infection among lactating cows was as low as 2.5%. Pooled milk samples from BLV-free herds did not react in the ELISA. All cattle that had positive results for the nested PCR had BLV antibodies, but cattle with consistantly low antibody titers required examination of sequential DNA samples to detect viral sequences. None of the 63 antibody-negative cattle had positive results for the PCR. CONCLUSIONS AND CLINICAL RELEVANCE: This ELISA is a highly specific and sensitive assay for the detection of BLV antibodies in serum and milk samples of cattle. Examination of pooled milk samples with the ELISA provides a reliable, practical, and economic procedure for identification of BLV-infected herds. The nested PCR also constitutes a specific procedure for direct diagnosis of infection with BLV.