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 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%.     

Prevalence of antibodies to Leptospira interrogans serovar hardjoin bulk tank milk from unvaccinated irish dairy herds

: Bulk tank milk samples, collected from 347 herds throughout the Republic of Ireland using a sampling frame based on seven milk-recording organisations, were tested by ELISA for antibodies to Leptospira interrogans serovar hardjo. These herds, which had not been vaccinated against leptospirosis within the previous five years, were categorised according to their province, milk-recording organisation and size. Two-hundred-and-seventy-three herds (79%) had a positive ELISA titre. Both the probability of a herd being seropositive and the antibody level in the herd milk sample were affected by the province (P < 0.05 and P < 0.01, respectively) and the herd size category (P < 0.05 and P < 0.01, respectively). Larger herds were significantly more likely to have positive reactions and higher mean concentrations of antibody. It was concluded that a high proportion of unvaccinated Irish dairy herds have been exposed to infection with Leptospira hardjo.     

Risk factors for Coxiella burnetii antibodies in bulk tank milk from Danish dairy herds

The aim was to identify risk factors associated with Coxiella burnetii antibody positivity in bulk tank milk (BTM) samples from 100 randomly selected Danish dairy cattle herds. Antibody levels were measured by an enzyme-linked immuno-sorbent assay. Before testing the herds, the farm managers were interviewed about hired labour, biosecurity, housing and herd health during the 12 months prior to the study. Variables considered important for C. burnetii antibody positivity in multivariable logistic regression analysis included the sharing of machines between farms (OR = 3.6), human contacts (OR = 4.2), artificial insemination by other people than artificial insemination technicians (OR = 7.7), routine herd health contract with the veterinarian (OR = 4.3) and hygiene precautions taken by veterinarians (OR = 5). In addition, herd size, hired labour, trading of cattle between farms, quarantine and use of calving and disease pens also showed significant association in univariable analysis. This study demonstrates that strict biosecurity is important for the prevention of infections with C. burnetii.     

Evaluation of indirect and avidin-biotin enzyme linked immunosorbent assays for detection of anti-listeriolysin O antibodies in bovine milk samples

Listeria monocytogenes is a foodborne pathogen that causes a wide spectrum of diseases in humans and animals. Enzyme linked immunosorbent assays (ELISA) [indirect and avidin-biotin (A-B)] for detecting L. monocytogenes antibodies in bovine milk samples (n = 2060) were standardized and evaluated by comparison with bacteriological examination. The tests were standardized by checker board titration. Highly purified listeriolysin O (LLO) was used as an antigen. Receiver operating characteristic (ROC) analysis was performed to decide the cut-off values. The ROC analysis revealed the sensitivities of indirect and A-B ELISA as 100% and specificities as 97.1 and 99.9% respectively. Listeria monocytogenes was isolated from 105 (5.1%) milk samples collected from 52 farms. Anti-LLO IgG antibodies were detected from 137 and 112 milk samples when tested by indirect and A-B ELISA respectively. Of the 52 farms screened, 28 (53.8%) yielded one or more isolates of L. monocytogenes and 33 (63.5%) of the farms had one or more animals simultaneously positive by one or both the assays for anti-LLO antibodies.     

Bulk tank milk ELISA for detection of antibodies to Mycobacterium avium subsp. paratuberculosis: Correlation between repeated tests and within-herd antibody-prevalence

Detection of bulk tank milk (BTM) antibodies using ELISA (BTM-ELISA) may constitute an inexpensive test for surveillance of Mycobacterium avium subsp. paratuberculosis (MAP) infection in dairy cattle herds provided that the test is accurate and consistent. The objectives of this study were to determine: (a) the correlation between repeated BTM reactions; and (b) the association between the BTM antibody ELISA-level and the within-herd prevalence of antibody-positive cows.     

A survey of Salmonella spp and Campylobacter spp in dairy goat faeces and bulk tank milk in the Murcia region of Spain

This study was designed to investigate the occurrence of Salmonella spp and Campylobacter spp in faeces samples from 222 healthy Murciano-Granadina dairy goats reared on 12 farms in Spain and in samples of bulk tank milk from 11 of those herds. Neither Salmonella spp nor Campylobacter spp were isolated from any of the samples. Our results suggest that, under the management practices applied to this breed in Spain, Murciano-Granadina goats are not likely to be a significant reservoir for these food-borne pathogens.     

Evaluation of three commercial enzyme-linked immunosorbent assays for the detection of antibodies against Salmonella spp. in meat juice from finishing pigs in Spain

The control of animal salmonellosis is considered as a major objective in Europe and indirect ELISAs will be important tools for the implementation of control programs for this infection in pigs. We analyse the results yielded by three commercial ELISAs (Herdcheck Swine Salmonella, SALMOTYPE Pig Screen, and PrioCHECK Salmonella) on meat juice samples from a population of slaughter pigs of Aragon, NW Spain, to assess their efficacy using traditional and latent-class approaches. Overall, the Herdcheck Swine Salmonella detected more Salmonella-infected pigs than the other two tests, but its relative sensitivity was low (65.9%). A similar result was observed when only serotypes detectable by this test were considered (69.1%). When a Bayesian approach was used the Herdcheck Swine Salmonella showed also the highest overall accuracy (sensitivity = 88% and specificity = 74%). Our results suggest that a relatively small proportion of the observed prevalence in herds would be explained by using these ELISAs. Also, this study points out that when different ELISA tests are used within the same herd, results may differ substantially. Thus, caution is advised if it is decided to use these assays for herd health classification in Spanish Salmonella control programs.     

Evaluation of an enzyme-linked immunosorbent assay kit to detect Babesia bovis antibodies in cattle

The sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) to detect antibodies; to Babesia bovis was evaluated in 1000 sera from Holstein heifers. Five hundred of them were from cattle naturally or experimentally infected with B. bovis and 500 from uninfected heifers born and raised in a region free of the vector of cattle babesiosis. Additionally, the ELISA was evaluated and compared with an indirect immunofluorescent antibody (IFA) test in 374 heifers inoculated with different kinds of B. bovis antigens in four trials. The cross-reaction was also evaluated in 50 heifers infected with Babesia bigemina and 50 heifers infected with Anaplasma marginale. The mean percentage positivity of negative sera in relation to the ELISA strong positive sera was 8%. The seropositive/seronegative cutoff point was set as twice the mean percentage positivity of negative cattle sera ( = 16%). The sensitivity of the ELISA was 98% with a 95% confidence interval (CI) of 96–99%. The specificity was 95% (CI 93–97%). The agreement was 97% and the kappa value was 0.93. The predictive values of positive and negative results were 95% and 98% respectively. ELISA showed a similar sensitivity to that of the IFA test to detect antibodies to different B. bovis antigens. Its sensitivity ranged from 97.1% to 100% (CI 89–100%), while the sensitivity of the IFA test ranged from 92.8% to 100% (CI 83–100%). ELISA cross-reacted in 8% and 6% of the sera carrying B. bigemina and A. marginale antibodies, respectively, while the IFA showed 4% cross-reaction in each situation. The ELISA evaluated has the advantages of a proper sensitivity, objectivity and capacity to be adapted to test large number of samples in a short period of time. The results indicate that the ELISA is a suitable replacement for the IFA test to detect B. bovis antibodies in cattle sera, especially in epidemiological studies.     

A multiplex real-time PCR for differential detection and quantification of Salmonella spp., Salmonella enterica serovar Typhimurium and Enteritidis in meats

Salmonella (S.) Typhimurium and S. Enteritidis are the major causative agents of food-borne illnesses worldwide. Currently, a rapid detection system using multiplex real-time polymerase chain reaction (PCR) has been applied for other food-borne pathogens such as Escherichia coli, Staphylococcus aureus and Streptococcus spp. A multiplex real-time PCR was developed for the simultaneous detection of Salmonella spp., especially S. Typhimurium and S. Enteritidis, in beef and pork. For the specific and sensitive multiplex real-time PCR, three representative primers and probes were designed based on sequence data from Genbank. Among the three DNA extraction methods (boiling, alkaline lysis, and QIAamp DNA Mini Kit), the QIAamp DNA Mini Kit was the most sensitive in this study. The optimized multiplex real-time PCR was applied to artificially inoculated beef or pork. The detection sensitivity of the multiplex real-time PCR was increased. The specificity of the multiplex real-time PCR assay, using 128 pure-cultured bacteria including 110 Salmonella isolates and 18 non-Salmonella isolates, was 100%, 100% and 99.1% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The sensitivity was 100%, 100% and 91.7% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The multiplex real-time PCR assay developed in this study could detect up to 0.54 ± 0.09 and 0.65 ± 0.07 log₁₀ CFU/ml for S. Typhimurium and S. Enteritidis for beef, 1.45 ± 0.21 and 1.65 ± 0.07 log₁₀ CFU/ml for S. Typhimurium and S. Enteritidis for pork, respectively, with all conditions optimized. Our results indicated that the multiplex real-time PCR assay developed in this study could sensitively detect Salmonella spp. and specifically differentiate S. Typhimurium from S. Enteritidis in meats.     

Association between antibodies to Coxiella burnetii in bulk tank milk and perinatal mortality of Danish dairy calves

Background

Coxiella burnetii is a well-known cause of placentitis and subsequent abortion in ruminants, but there are no reports on the relationship with perinatal mortality. The study was performed to determine the influence of level and change of bulk tank milk (BTM) antibodies to C. burnetii on two outcomes associated with parturition in cattle: a) stillbirth; and b) stillbirth and neonatal mortality combined (perinatal death).

Methods

Twenty-four Danish dairy herds were tested repeatedly for antibodies to C. burnetii in BTM using a commercial ELISA. Samples were collected monthly from July 2008 to July 2009. Information on the 2,362 calvings occurring in the study period was obtained from the Danish Cattle Database. Two multilevel logistic regression models were created for the two outcomes stillbirth and perinatal mortality. One model included the level of BTM antibodies in a specified period before or after the outcome had occurred. The other model included the change in antibodies over time. These predictors were included both at herd and animal level. Furthermore, all models included parity and breed.

Results

The individual monthly BTM antibody levels were highly correlated within herds. Consequently, changes in BTM antibody levels were not found to be associated with neither risk of stillbirth nor the risk of perinatal mortality. However, the risk of stillborn calves and perinatal death was higher with high level of BTM antibodies 8 to 9 months after the incident, but not outside this period.

Conclusion

We conclude that the level of antibodies to C. burnetii in BTM may be associated with perinatal mortality, but the association was not persistent and should be investigated further.     

An enzyme-linked immunosorbent assay for detection of antibodies against bovine pestivirus

A competitive enzyme-linked immunosorbent assay was developed and compared with the serum neutralisation test for bovine pestivirus using 508 cattle sera and serial serum samples from a goat hyperimmunized with five bovine pestivirus isolates. There was 96.7% agreement between the two tests. The relative sensitivity of the enzyme-linked immunosorbent assay compared to the serum neutralisation test was 95.2% and the relative specificity was 99.4%. The titres of individual animals in the assay did not show a close correlation with serum neutralisation test titres. This may be because the antibodies measured in the two tests are directed against different viral proteins. The enzyme-linked immunosorbent assay has the advantage of being quicker and cheaper than the serum neutralisation test. The configuration used in the ELISA means sera from all species can be tested for pestivirus antibody using the same set of reagents.     

Blocking enzyme-linked immunosorbent assay for detection of antibodies against bovine enterovirus

A blocking enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against bovine enterovirus (BEV) in bovine sera. In this ELISA, bovine serum samples were allowed to react with captured viral antigens (by specific chicken IgG), before the addition of specific mouse IgG for measuring non-occupied viral epitopes. The ELISA was slightly more sensitive and required a shorter time period than traditional serum neutralisation (SN). Among the 871 bovine serum samples tested so far, the titres produced by this assay had a significant correlation with those recorded by SN. The ELISA could be used as an alternative assay for SN in a large-scale BEV antibody investigation.     

A longitudinal survey of anti-Ostertagia ostertagi antibody levels in individual and bulk tank milk in two dairy herds in Normandy

The Ostertagia-specific antibody levels in milk were monitored in 2 dairy herds to investigate seasonal variations and the relationship between individual and bulk tank milk antibody levels. Bulk tank and individual milk samples from all lactating animals were collected over a 1-year period at weekly and monthly intervals, respectively. The Ostertagia-specific antibody levels were measured with an indirect ELISA and the test results were expressed as optical density ratios (ODR). A clear seasonal pattern that followed the expected intake of infectious larvae was observed in the individual and bulk tank milk antibody levels of both herds. Within each herd, there was a large variation in the individual ODRs. This variation remained large when the distribution of individual ODRs was plotted according to high and low bulk tank milk ODR categories. The results suggest that the effect of seasonal variations on cut-off levels that predict production responses after anthelmintic control, needs to be assessed.     

检测牛结核抗体新型ELISA方法的建立及应用

利用分子克隆和Splicing by overlapping extension(SOE)技术,表达了重组蛋白rM70-83-E6,Western blot分析rM70-83-E6具有很好的特异性。以蛋白rM70-83-E6作为诊断抗原建立了间接ELISA方法,ELISA诊断方法的判定标准,即S/P≥0.5为阳性,S/P<0.4为阴性,0.5>S/P≥0.4为可疑。ELISA方法的特异性为96.0%、敏感性为69.4%。对67份PPD皮试阳性和50份PPD皮试阴性(117份)血清样品进行了检测,并与PPD皮试比较。67份PPD皮试阳性血清样品中有46份为ELISA检测阳性,阳性符合率为68.7%,50份PPD皮试阴性牛血清都为阴性,阴性符合率为100%,本ELISA方法与PPD皮试诊断方法总复合率为82.1%。研究表明,ELISA方法具有很好的开发和应用前景。     

Evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to bovine virus diarrhoea virus in milk

The present study shows that milk is an appropriate source for detection of seroreactors to bovine virus diarrhoea virus (BVDV). There was close agreement between antibody titres in serum and in skim milk, as determined by an indirect enzyme-linked immunosorbent assay. The antibody titres were usually lower in skim milk than in serum, but all seropositive cows (n = 84) were also skim milk-positive and all but one seronegative cow (n = 55) proved negative in skim milk. During lactation, the level of antibodies to BVDV in milk showed an inverse relationship to the amount of milk produced. However, there was a sufficient level of antibodies in milk throughout lactation to permit an adequate determination of BVDV antibody status in dairy cows. There was a mutual good agreement between milk antibody titre in the four mammary quarters, irrespective of milk cell count. Milk can be used to detect seroreactors to BVDV. Milk is preferable to blood in large-scale epidemiological studies, since the sampling procedure is much simpler.     

Evaluation of PCR inhibitory effect of enrichment broths and comparison of DNA extraction methods for detection of Salmonella Enteritidis using real-time PCR assay

The best enrichment broth and DNA extraction scheme was determined for rapid and sensitive detection of Salmonella Enteritidis in steamed pork using real-time PCR. The inhibitory effect of commonly used Salmonella enrichment broths, Rappaport-Vassiliadis (RV) and Muller-Kauffmann tetrathionate with novobiocin (MKTTn), on real-time PCR was confirmed. The inhibition of PCR was statistically significant (p < 0.05) in RV and MKTTn, as compared with buffered peptone water (BPW) or phosphate-buffered saline. The inhibitory effect of the selective enrichment media was successfully removed by using a modified DNA extraction, PrepMan Ultra Reagent with an additional washing step or the DNeasy Tissue Kit. In three experiments, when applied to detection of Salmonella Enteritidis in steamed pork, the real-time PCR coupled with single 24 h enrichment with BPW performed better than double 48 h enrichment with BPW plus RV or MKTTn. The simple real-time PCR assay using BPW proved to be a rapid and sensitive test for detection of low concentrations of Salmonella Enteritidis in steamed pork samples as compared with the conventional culture method.     

New enzyme-linked immunoassay for the detection of specific antibodies against multiple Leptospira serogroups in bovine sera

Leptospirosis is a zoonotic disease with worldwide endemicity in Argentina, it is a significant public health problem in low-income populations. Bovine leptospirosis is a serious economic problem for cattle production, causing abortions, reduced milk yield, mortality in calves and decreased daily weight gain. We developed an enzyme-linked immunosorbent assay (ELISA) with sonicated Leptospira interrogans serogroup Icterohaemorrhagiae serovar Copenhageni M 20. We evaluated its performance for the detection of specific antibodies against multiple Leptospira serogroups in bovine. The microscopic agglutination test (MAT) was used as the gold standard. The performance of this ELISA was evaluated with a panel of sera (118 MAT confirmed positive and 97 MAT negative). The overall sensitivity was close to 85.6 % and the specificity was 83.5 %, according to the MAT reference method. Analytical specificity of the IgG-ELISA was evaluated using 50 bovine serum samples from animals showing serum antibodies against other pathogens that cause abortion in bovine, such as Brucella sp., Neospora sp. and Bovine Viral Diarrhea (BVD), and no cross-reaction was observed. This IgG-ELISA can be an alternative to the MAT for diagnosis of leptospiral infection in bovine.     

Development of an indirect enzyme-linked immunosorbent assay for detecting equine serum antibodies to the lipopolysaccharide of Salmonella abortusequi

An indirect enzyme-linked immunosorbent assay (IELISA) was developed for the detection of equine serum antibodies to lipopolysaccharide of Salmonella enterica subsp. enterica serovar Abortusequi (LPS), a causative organism of Equine Paratyphoid. The data presented demonstrates that horses immunized with S. abortusequi LPS developed antibodies detectable by the IELISA. By comparison, the tube agglutination test (TAT) did not detect antibody to S. abortusequi LPS as consistently as the IELISA. The data suggests that the IELISA may be a more suitable test for the detection of serum antibodies to S. abortusequi than the TAT.