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%.     

Performance characteristics and optimisation of cut-off values of two enzyme-linked immunosorbent assays for the detection of antibodies to Neospora caninum in the serum of cattle

AIM: To determine the performance characteristics of two enzyme-linked immunosorbent assays (ELISAs) manufactured by Institut Pourquier (IP) for the detection of antibodies against Neospora caninum in bovine sera. METHODS: Sera from 526 cattle were assayed in two ELISAs (IP) for the detection of anti-N. caninum antibodies. Results from a further ELISA (IDEXX) were used to provide the "gold standard"N. caninum infection status of the cattle and the ELISA results assessed by two-graph receiver operating characteristic (TG-ROC) analysis. RESULTS: TG-ROC analysis suggested changes to one of the IP ELISA protocols, arriving at a cut-off threshold that was different to the one recommended by the manufacturer. With that change, both of the ELISAs performed with high sensitivity and specificity (in excess of 98%) for bovine sera. CONCLUSIONS: The analysis of the two IP ELISAs when used on individual bovine sera demonstrated high sensitivity and specificity. TG-ROC analyses optimised the cut-off point suggested by the manufacturer for one of these commercial diagnostic assays and found agreement with the manufacturer's cut-off regarding the other assay. This will help with the accurate identification of infected animals and thereby contributing to the control of neosporosis.     

Herd-level risk factors for bovine viral diarrhea infection in cattle of Tamil Nadu

A cross-sectional study was carried out to identify risk factors for bovine viral diarrhea virus (BVDV) infection in 62 randomly selected dairy herds which were tested for BVD serum antibodies by using an indirect ELISA kit (IDEXX). Results from the chi-square test analysis were interpreted by analyzing by chi-square test. A sum of 500 sera samples were screened and 66 animals (13.20%) showed positive for BVDV antibody. Within herd, BVD seroprevalence was 12–65%. This study concluded that epidemiological risk factors like location, herd size, housing patterns like, tail to tail system, roofing pattern, distance between the manure pit and farm, and distance between farms were significantly associated with BVDV serological status (P?<?0.05).     

Evaluation of the specificity of three enzyme-linked immunosorbent assays for detection of antibodies against Salmonella in bovine bulk milk

Background

The Swedish Salmonella control program has been running for decades and has resulted in a low prevalence of Salmonella in Swedish food producing animals. Routine bacteriology is used to detect Salmonella, however, bacteriology is time consuming, costly and has a low sensitivity. Different enzyme-linked immunosorbent assays (ELISAs) have been developed for detection of antibodies against Salmonella Dublin and S. Typhimurium in bovine bulk milk, individual milk samples as well as in sera. Screening bulk milk for antibodies against Salmonella spp. could improve the cost-effectiveness of the surveillance in Swedish dairy cattle, but as characteristics of tests may vary in different populations, tests should always be evaluated in the specific population where they will be used. Hence, the aim of this study was to evaluate the specificities of three bovine ELISAs when used to analyse bulk milk samples from Swedish dairy cattle. A second aim was to compare the performance of the two Dublin ELISAs tested.

Methods

Bulk milk samples for analysis were randomly selected from samples collected within the Swedish bulk milk sampling scheme and analyzed with the three ELISAs; a Danish in-house Dublin ELISA, PrioCHECK® Salmonella Ab bovine Dublin ELISA and PrioCHECK® Salmonella Ab bovine ELISA (hereafter named mixed ELISA). The specificities of the ELISAs were calculated assuming a disease-free status in Sweden i.e. that all test positive samples were assumed to be false positive results. This assumption can be used when a disease is known to be infrequent.

Results

The calculated specificities of the two Dublin ELISAs and the mixed ELISA, when using the producer’s recommended cut-off value of the corrected optic-density percent (ODC%) were 99.4% (95% Confidence Interval (CI): 98.8% -99.8%), 99.4% (95% CI: 98.8% -99.8%) and 97.9% (95% CI: 96.8% -98.7%), respectively. The correlation between the ODC% values of the two Dublin ELISAs was 0.83.

Conclusions

We conclude that the evaluated ELISAs have sufficiently high specificities to be used as supplement to bacteriological examinations in the Swedish Salmonella control program in cattle as well as a primary screening test in routine surveillance for S. Dublin.     

Evaluation of four commercial enzyme-linked immunosorbent assays for the diagnosis of bovine paratuberculosis in Chilean dairy herds.

The accuracy of 4 commercial enzyme-linked immunosorbent assays (ELISAs) for diagnosis of bovine paratuberculosis was compared using sera from 53 Mycobacterium avium subsp. paratuberculosis (MAP) fecal culture-positive dairy cows (cases) and sera from 345 dairy cattle resident in 11 fecal culture-negative herds on 2 consecutive occasions 1 year apart (controls). The specificity of all 4 ELISA kits was >99%, and their diagnostic sensitivity ranged from 30.2% to 41.5%. Pairwise comparison of ELISAs found no significant differences (McNemar's chi-square test > 0.05), and assay agreement for categorical assay interpretation (positive or negative) was high (>98%) with kappa values ranging from 0.84 to 0.95. Receiver operating characteristic (ROC) curve analysis and the corresponding area under the ROC curves indicate that kit B had the highest overall accuracy. Thus, all 4 ELISA kits for bovine paratuberculosis had comparable accuracy when tested on Chilean dairy cattle, with kit B having a slight statistical advantage based on ROC area under the curve analysis. This suggests that any of the 4 kits could be appropriate for herd certification and for paratuberculosis control programs on Chilean dairy cattle.     

Evaluation of two commercial PRRSV antibody ELISA kits with samples of known status and singleton reactors

Two commercial PRRSV ELISA kits (IDEXX and Bionote) were evaluated for their sensitivity and specificity using 476 PRRS-positive serum samples collected from 7 animal challenge experiments and 1,000 PRRS-negative sera. Both ELISA kits exhibited 100% sensitivity with sera collected 14 to 42 days post-infection, and the results from the kits were highly correlated (R²=0.9207). The specificity of IDEXX or Bionote kit was 99.9% or 99.7%, respectively. In addition, the Bionote ELISA kit was used to examine 100 sera that were determined to be falsely positive either by IDEXX 2XR or 3XR ELISA, and only 7 of these samples were found to be positive. These results indicate that both ELISA kits exhibited similar levels of sensitivity and specificity and would complement one another for the verification of false-positive samples.     

Evaluation of cellular and serological diagnostic tests for the detection of Mycobacterium bovis-infected goats

We have evaluated the comparative intradermal skin (CID) test, the interferon gamma (IFN-γ) assay, two ELISAs with bovine PPD antigen, the standard and the anamnestic using sera obtained, respectively, at the time of the tuberculin injection and 15 days later, for the diagnosis of caprine tuberculosis (TB). The sensitivity and specificity results were high for the CID test (83.7%, 100%), the IFN-γ assay (83.7%, 96%) and the anamnestic ELISA (88.6%, 95.8%). In contrast, they were comparatively low for the standard ELISA (54.9%, 88%). However, test results with the standard ELISA were positive in a group of goats with cavitating TB (100%). A combination of the CID test and the IFN-γ assay offered the highest sensitivity, 95.8%, and also high specificity, 96%. In spite of this, the evidence that the serological tests were most sensitive for the detection of goats with severe lesions (100% positivity) suggested that a combination of CID test and anamnestic ELISA may be most useful as part of an eradication campaign against caprine TB.     

Sensitivity and specificity of an enzyme-linked immunosorbent assay for the detection of bovine viral diarrhea virus antibody in cattle.

A reliable bovine viral diarrhea (BVD) viral antigen was prepared from BVD virus grown on Madin Darby bovine kidney (MDBK) cells by solubilizing the virus with detergent MEGA-10 (decanoyl-N-methylglucamide) followed by removal of hydrophobic proteins with Triton X-100 treatment. By these treatments, problems of high background associated with BVD viral antigen in the enzyme-linked immunosorbent assay (ELISA) were eliminated. With this new antigen, an ELISA was adapted to detect bovine serum antibody against BVD virus. The diagnostic specificity of the assay in 403 bovine sera collected from a BVD virus-free herd was 100%; in 296 bovine sera with serum neutralizing antibody titers of greater than or equal to 1:2, 289 sera were ELISA positive (relative sensitivity of 97.6%), two sera gave false negative reactions (0.7%) and five sera gave suspicious reactions (1.7%). These interpretations were based on positive/negative (P/N) ratio readings, i.e. a P/N ratio of less than 1.50, 1.50-1.99 and greater than or equal to 2.00 were interpreted as negative, suspicious and positive reactions, respectively. The ELISA results gave excellent agreement with serum neutralization in detecting both seropositive and seronegative animals (Kappa = 0.994). The ELISA assay was considered to be technically superior to the serum neutralization test for the routine detection of BVD viral antibody in bovine sera.     

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.     

The Dutch Brucella abortus monitoring programme for cattle: the impact of false-positive serological reactions and comparison of serological tests

The Dutch national Brucella abortus eradication programme for cattle started in 1959. Sporadic cases occurred yearly until 1995; the last infected herd was culled in 1996. In August 1999 the Netherlands was declared officially free of bovine brucellosis by the European Union. Before 1999, the programme to monitor the official Brucella-free status of bovine herds was primarily based on periodical testing of dairy herds with the milk ring test (MRT) and serological testing of all animals older than 1 year of age from non-dairy herds, using the micro-agglutination test (MAT) as screening test. In addition, serum samples of cattle that aborted were tested with the MAT. The high number of false positive reactions in both tests and the serum agglutination test (SAT) and complement fixation test (CFT) used for confirmation seemed to result in unnecessary blockade of herds, subsequent testing and slaughter of animals. For this reason, a validation study was performed in which three indirect enzyme-linked immunosorbent assays (ELISAs), the CFT and the SAT were compared using a panel of sera from brucellosis-free cattle, sera from experimentally infected cattle, and sera from cattle experimentally infected with bacteria which are known to induce cross-reactive antibodies (Pasteurella, Salmonella, Yersinia, and Escherichia). Moreover, four ELISAs and the MRT were compared using a panel of 1000 bulk milk samples from Brucella-free herds and 12 milk samples from Brucella abortus- infected cattle. It is concluded that the ELISA obtained from ID-Lelystad is the most suitable test to monitor the brucelosis free status of herds because it gives rise to fewer false-positive reactions than the SAT.     

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

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

Development of ELISAs based on recombinant antigens for the detection of Toxoplasma gondii-specific antibodies in sheep and cats.

ELISAs using recombinant parasite polypeptides as antigens were developed to measure Toxoplasma gondii-specific antibodies in the sera of sheep and cats. Compared with an ELISA based on traditional parasite antigen, the ELISA for sheep sera had a sensitivity of 79% and a negative predictive value of 80%, and the ELISA for cat sera had a sensitivity of 100% and a negative predictive value of 100%. Both ELISAs had specificities of 100% and positive predictive values of 100%. These ELISAs appear to be a useful cost-effective alternative to ELISAs based on traditional parasite antigen for the measurement of T. gondii-specific antibodies in the sera of sheep and cats.     

Development of a monoclonal antibody-based competitive enzyme-linked immunosorbent assay for the detection of Leptospira borgpetersenii serovar hardjo type hardjobovis antibodies in bovine sera.

A murine monoclonal antibody (designated M553) that binds to an epitope on whole cell antigens prepared from Leptospira borgpetersenii serovar hardjo type hardjobovis and Leptospira interrogans serovar hardjo type hardjoprajitno, was produced and incorporated into a competitive enzyme-linked immunosorbent assay for the detection of bovine antibodies to serovar hardjo. The epitope recognized by M553 was susceptible to periodate oxidation. The M553 antibody was characterized by western blot with hardjobovis whole cell antigen. This antibody does not cross-react with whole cell antigens prepared from 11 other pathogenic Leptospira serovars, or, Leptospira biflexa serovar patoc. The sensitivity estimate of the competitive ELISA was 100% with field sera (n = 165) with serovar hardjo microscopic agglutination test (MAT) titres of > or = 100. The specificity estimate was 100% with sera (n = 128) obtained from a specific pathogen free herd of cattle that were negative in the MAT at a dilution of 1:100 for serovars hardjo, pomona, sejroe, copenhageni, canicola, and grippotyphosa. The specificity estimate with field sera (n = 301) with serovar hardjo MAT titres of < 100, was 98% (95% confidence interval = +/- 1.58%). There was no cross-reactivity with field sera (n = 306) with serovar pomona titres > or = 100 and serovar hardjo titres < 100. The specificity estimate with the combined populations of sera with serovar hardjo MAT titres of < 100 (n = 735) was 99.18% (95% confidence interval = +/- 0.65%). There was a high level of agreement (kappa = 0.977) between the results of the competitive ELISA and those of the MAT.     

Comparative studies with three different bovine blood sample BHV-1 ELISA tests: indirect ELISA and bG-blocking-ELISA

A total of 2754 bovine blood samples were examined for BHV1 antibodies in three different BHV-1 ELISA tests. The tests used were the CHEKIT Trachitest 2nd. Gen. "Best?tigungstest" Serum, the CHEKIT-BHV-1-gB-ELISA (Bommeli-Diagnostics/Intervet), and the BHV-1-gB Antibody Test Kit (IDEXX). A first testing identified 111 (4.0%) of the samples as positive and 2501 (90.8%) samples negative in all three tests. Onehundredand-fortytwo (5.2%) of the samples showed a not negative result in at least one of the two gB-ELISAs. The testing was repeated for 139 of the 142 samples and still 89 (3.2%) samples were found with discrepant results. The remaining 50 samples showed a negative reaction in the three ELISA tests. After Western blot analysis antibodies against glycoprotein B (gB) of BHV-1 were only detected in 11 of the 89 samples with a not negative reaction in at least one of the gB-ELISAs. Three of the 50 ELISA-negative samples showed a positive reaction in the Western blot. The high number of positive results in the gB-ELISAs which were negative in the Western blot assay leads to the conclusion that gB-ELISAs may have a lower specificity as the indirect ELISA. Introducing the gB-ELISA as the standard test for BHV1 serology in Bavaria would result to an increase of the number of BHV-1 positive farms by 8% (gB IDEXX) or 20% (gB Bommeli) 20%.     

Accuracy assessment and screening of a dairy herd with paratuberculosis by three different ELISAs

Although the culture of Mycobacterium avium subsp. paratuberculosisis is the gold standard for the diagnosis of paratuberculosis, this bacterium is difficult to grow. In contrast, serological tests like ELISAs are inexpensive, rapid, and easy to perform. The aims of this study were to evaluate the accuracy of three different ELISAs: one with the commercial antigen PPA-3, another one with L5P (a recently described lipopentapeptide), and a third one with an in-house antigen whole cell lysates (WCL) of M. avium (MAA) strain D4ER (Study 1), and to compare them with other tests for paratuberculosis (PTB) diagnosis (Study 2). In Study 1, the sensitivities of the three ELISAs tested were 74.1%, 37% and 74.1%, respectively, whereas their specificities were 98.9%, 100% and 100%, respectively. In Study 2, we compared the three above-mentioned ELISAs with the intradermal reaction test using Avian PPD (PPDa) and fecal culture associated with Ziehl-Neelsen stain and PCR tests, in a dairy herd with 4.6% of cows with clinical signs of PTB. The results showed that fecal samples from 14 cows (16%) were culture-positive and that fecal samples from nine cows (10%) were PPDa-positive. Most of these animals (culture-positive and PPDa-positive) were detected as positive with any of the three ELISAs tested. Serological results showed that 31% of the animals were positive to ELISA-PPA-3, 17% to ELISA-L5P and 42.5% to ELISA-WCL. The combination of these three ELISAs identified 50.6% of the animals as positive in the infected herd. In particular, the results show that the locally developed ELISA seems to be useful for identifying many infected animals in a herd.     

Specificity of four serologic assays for Mycobacterium avium ss paratuberculosis in llamas and alpacas: a single herd study.

An investigation was conducted for Mycobacterium avium ss paratuberculosis infections in a research herd of llamas and alpacas. Herd culture-negative status was established over a 23-month period by screening any individuals with any signs compatible with paratuberculosis (n = 1), high serology values (n = 8), or other health and research related reasons (n = 24). There were no M. avium ss paratuberculosis isolates from radiometric cultures of multiple tissue and fecal samples from these individuals and no known sources of exposure. Paratuberculosis is uncommon in North American llamas and alpacas: only 5 cases were identified after an extensive search of the Veterinary Medical Data Base, diagnostic laboratory records, publication databases, and personal communications. Therefore, serum samples from llamas (n = 84) and alpacas (n = 16) in the culture-negative herd were used to obtain preliminary estimates of test specificity for 3 enzyme-linked immunoassays (ELISAs) and an agar gel immunodiffusion (AGID) assay kit for detecting serum antibodies to M. avium ss paratuberculosis in South American camelids. The ELISAs were modifications of established bovine assays for antibody detection. With provisional cutoffs, ELISA-A had 52 false positives (specificity 48%), ELISA-B had 8 false positives (specificity 92%), ELISA-C had two false positives (specificity 98%), and the AGID had 0 false positives (specificity 100%). The range of ELISA values for culture-positive llamas and alpacas (n = 10) from other herds overlapped the range of values for culture-negative llamas and alpacas. The accuracy of the ELISAs may be improved by using age- and sex-specific cutoffs because uninfected male llamas and alpacas that were older than 1 year had higher values for some tests. These tests can be used for either llamas or alpacas; the protein-G conjugate ELISA (ELISA-B) may be useful for multispecies applications. These assays are best used for rapid presumptive diagnoses of llamas and alpacas with diarrhea and weight loss and as a screening tool for herds known to be exposed to infection. All seropositive results should be confirmed with culture.     

Rapid immunofiltration assay based on colloidal gold–protein G conjugate as an alternative screening test for bovine and ovine brucellosis

A non-enzymatic rapid immunofiltration assay (NERIFA) was developed as an alternative field test for rapid detection of anti-Brucella antibody in bovine and ovine sera. The assay was based on Brucella abortus lipopolysaccharide as diagnostic antigen and colloidal gold particle–protein G conjugate as detection reagent. Its diagnostic performance was evaluated using undiluted well-defined positive and negative serum samples in comparison with Rose Bengal test (RBT), complement fixation test (CFT) and a commercial and an in-house indirect enzyme-linked immunosorbent assay (ELISA). A perfect test agreement was found between NERIFA and ELISAs by kappa statistics. In addition, McNemar’s analysis of the results showed that the RBT for bovine sera and the CFT for ovine sera were found significantly less performant than indirect ELISAs and NERIFA. The results of the present study indicated that the NERIFA could be considered as a simple, rapid, and accurate field test for screening of ovine and bovine brucellosis. Therefore, this test constitutes a high potential to be used as an alternative model particularly in brucellosis prevalent tropical and subtropical geographical areas.     

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.     

Sensitivity and specificity of an enzyme-linked immunosorbent assay for the detection of infectious bovine rhinotracheitis viral antibody in cattle.

An enzyme-linked immunosorbent assay was developed to detect bovine serum antibody to infectious bovine rhinotracheitis virus. The specificity of this assay in 304 bovine sera, collected from an infectious bovine rhinotracheitis virus-free herd, was 100%; in sera from 62 cattle inoculated with an intranasal vaccine, its diagnostic sensitivity was 27.4% at one month and 100% at six months, postvaccination. In 303 bovine sera with standard serum neutralizing antibody titers of greater than or equal to 1:2 it showed 100% sensitivity; and in 463 random diagnostic samples, comparative tests indicated that enzyme-linked immunosorbent assay detected more seropositive animals (61.6%) than the standard serum neutralizing test (49.9%). The enzyme-linked immunosorbent assay method was considered to be technically superior as a routine diagnostic test for the detection of infectious bovine rhinotracheitis viral antibody in bovine sera.     

Comparison of three enzyme-linked immunosorbent assays for serologic diagnosis of contagious agalactia in sheep.

Serologic diagnosis of ovine contagious agalactia (Mycoplasma agalactiae) with the enzyme-linked immunosorbent assay (ELISA) developed by Agence Fran?aise de Sécurité Sanitaire des Aliments (AFSSA) may produce a few false-positive (FP) and false-negative (FN) results. When the prevalence of disease is low, these erroneous results may generate problems for eradication schemes. To prevent this, 2 commercial ELISAs were compared with the AFSSA ELISA. Flocks of known status were selected and classified into 4 categories: true positive (TP), FP, true negative (TN), and FN; 20 sheep per flock were submitted for blood sampling. A flock was considered positive when at least 1 out of 20 sera was positive or 2 sera were doubtful. In the flock, the diagnostic sensitivity of the 3 kits was very good (100%), and the diagnostic specificity showed an improvement from 46% (AFSSA test) to 88% and 92% (commercial tests). Considering individual animals, very few positive ewes were detected within TN or FP flocks; the proportion of positive ewes varied greatly from one kit to another (48% to 82%) within TP flocks. The kinetics of antibody response in sheep experimentally infected with various field strains of M. agalactiae were quite similar with all 3 ELISAs. The agreement between the 3 tests, assessed using the kappa value, varied from moderate to good (respective values of 0.56, 0.61, and 0.86). The 2 commercial ELISAs showed better performances, probably because of a superior analytical sensitivity, and are a good alternative for the serodiagnosis of contagious agalactia in sheep.