Evaluation of surveillance strategies for bovine tuberculosis (Mycobacterium bovis) using an individual based epidemiological model

The Netherlands holds the bovine tuberculosis-free (BTB-free) status according to European Union standards, but in recent years small outbreaks of the infection have occurred. After the last outbreak in 1999 with 10 infected herds the question raised if the current surveillance system, visual inspection of carcasses at the slaughterhouse, is efficient enough to detect infected cattle in time and to maintain the official BTB-free status.

Through epidemiological modelling, the risk of a major outbreak is quantified, using one of six surveillance strategies. These are the currently used visual inspection of carcasses at the slaughterhouse (SL), the ELISA test on blood samples of carcasses at the slaughterhouse (ELISA-B), the γ-interferon test on blood samples of carcasses at the slaughterhouse (GAMMA-B), comparative tuberculination of the herd (CT), the combined method of single and comparative tuberculination of the herd (ST + CT) and the ELISA test on samples of bulk milk (ELISA-M). Test frequency of the last three methods was varied as well.

A stochastic individual based model (IBM) was developed to simulate a chain of infected herds, where each individual animal is followed in time. The model mimics the nation-wide situation after the introduction of one infected animal into one herd. BTB-transmission is simulated with an S-E₁-E₂-I state transition model. Output is time until detection of the infection, prevalence in the detected herd and the number of infected herds at the time of detection. For the assessment 500 simulations were used, representing 500 BTB-introductions. Model robustness to parameter values was analysed with Monte Carlo elasticity analysis, for which 1000 simulations were used.

Results of median time until detection and median number of infected farms at detection for SL (302 weeks and seven farms) were in agreement with estimates from an outbreak in the Netherlands in 1999. ELISA-B and GAMMA-B performed better than SL with a much lower median time until detection (189 and 97 weeks, respectively). The results for the tuberculination methods (ST + CT and CT) and ELISA-M depended heavily on the frequency in which the tests were performed. The tuberculination methods ST + CT and CT yield comparable results and detect the infection sooner than SL, also at the lowest tested frequency of once in 5 years. ELISA-M is comparable with SL at frequencies of once in 4 or 5 years, and this test works well at frequencies of once a year or higher. Our study results are used for an economical optimisation analysis of the six surveillance strategies.     

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.     

Evaluation of surveillance and sample collection methods to document freedom from infectious bovine rhinotracheitis in cattle populations

OBJECTIVES: To assess the sensitivity of the current surveillance program used in Denmark for detecting outbreaks of infectious bovine rhinotracheitis (IBR) at the herd level and to evaluate the impact of alternative sample collection strategies on the sensitivity of the system in an acceptable time frame. SAMPLE POPULATION: Data from the Danish Central Husbandry Register on cattle of 24,355 and 25,233 beef herds and on 13,034 and 12,003 dairy herds in the years 2000 and 2001, respectively. PROCEDURES: Surveillance programs were evaluated under current sample collection conditions and under 3 alternative scenarios by use of simulation modeling. Data from the current detection component of the surveillance system were used as input, taking into consideration the sensitivity and specificity of bulktank milk and serologic testing. RESULTS:The current system identifies infected dairy herds within a 3-month period with desired accuracy largely because of the test characteristics and number of bulk-tank milk samples. The system is less likely to detect infected beef herds in a timely manner because surveillance in beef herds depends solely on serologic testing at the time of slaughter. The efficiency of surveillance in dairy cattle herds was not decreased substantially when the slaughter-surveillance component was omitted. CONCLUSIONS AND CLINICAL RELEVANCE:Geographically targeted sample collection during the high-risk season (winter) was predicted to increase the probability of rapid detection of IBR infection in cattle. This approach can be used for assessing other surveillance systems to determine the best strategies for detection of infected herds.     

Predicting milk-production responses after an autumn treatment of pastured dairy herds with eprinomectin

The objectives of this study were (1) to determine the effect of a treatment with eprinomectin in autumn of pastured dairy herds on the anti-Ostertagia ostertagi bulk-tank milk antibody level, (2) to determine the overall effect of this treatment on three milk-production parameters (milk yield, protein % and fat %) and (3) to investigate the value of the pre-treatment Ostertagia-specific bulk-tank milk antibody level to predict the production response after anthelmintic treatment. One hundred and nineteen herds in Flanders (Belgium) were randomly assigned to a treatment with eprinomectin or a placebo in October 2004. Bulk-tank milk samples were collected monthly from August 2004 until April 2005, and the antibody levels against O. ostertagi were determined as optical density ratios (ODRs) with an ELISA. The treatment effect over the 4 months following treatment on three production parameters (milk yield, milk-protein %, milk-fat %) was estimated by mixed models with herd as a random effect. The treatment effect on milk yield was also investigated within six categories of the pre-treatment ODR. The ODR values were lower in the eprinomectin group than in the control group at each time point after treatment. The overall effect on milk yield was estimated at 1.2 kg/cow/day, whereas no effect on the milk-protein % and milk-fat % was observed. Herds in the highest pre-treatment ODR category (>0.84) had a positive milk-yield response of 4.0 kg/cow/day (95%-confidence interval: 1.0; 7.0), while the 95%-confidence intervals of the milk-yield responses in the other categories all included zero. This study demonstrates that treatment with eprinomectin of pastured dairy cows in autumn will lower the Ostertagia-specific bulk-tank milk antibody level during the stabling period and can result in a consistent increase in milk yield. The results indicate that an O. ostertagi bulk-tank milk ELISA can be used to identify the herds where the greatest milk-yield response after an anthelmintic treatment is expected.     

Association between measures of milk quality and risk of violative antimicrobial residues in grade-A raw milk

OBJECTIVE: To determine whether bulk-tank standard plate counts or plate loop counts and bulk-tank somatic cell counts (SCC) were associated with detection of violative antimicrobial residues in milk from dairy cattle. DESIGN: Longitudinal study. PROCEDURE: Information for 1994 through 1997 was obtained from a large milk marketing cooperative that operated in multiple states throughout the northeastern and midwestern United States (16,831 herd-years of information from 6,546 farms) and from the Ohio Department of Agriculture Grade-A Milk Certification Program (12,042 herd-years of information from 4,022 farms). Data were analyzed by use of multivariate logistic regression. RESULTS: For both data sets, odds that a violative antibiotic residue would be detected increased as mean SCC for the herd-year increased. Standard plate counts and plate loop counts were not associated with odds that a violative antibiotic residue would be detected. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this study suggested that the odds that a violative antibiotic residue would be found in bulk-tank milk increased as mean SCC for the herd-year increased. This suggests that management practices that would be expected to influence SCC may also influence the risk of antibiotic residue violations.     

Establishing a cost-effective national surveillance system for Bluetongue using scenario tree modelling

Vector-borne diseases pose a special challenge to veterinary authorities due to complex and time-consuming surveillance programs taking into account vector habitat. Using stochastic scenario tree modelling, each possible surveillance activity of a future surveillance system can be evaluated with regard to its sensitivity and the expected cost. The overall sensitivity of various potential surveillance systems, composed of different combinations of surveillance activities, is calculated and the proposed surveillance system is optimized with respect to the considered surveillance activities, the sensitivity and the cost. The objective of this project was to use stochastic scenario tree modelling in combination with a simple cost analysis in order to develop the national surveillance system for Bluetongue in Switzerland. This surveillance system was established due to the emerging outbreak of Bluetongue virus serotype 8 (BTV-8) in Northern Europe in 2006. Based on the modelling results, it was decided to implement an improved passive clinical surveillance in cattle and sheep through campaigns in order to increase disease awareness alongside a targeted bulk milk testing strategy in 200 dairy cattle herds located in high-risk areas. The estimated median probability of detection of cases (i.e. sensitivity) of the surveillance system in this combined approach was 96.4%. The evaluation of the prospective national surveillance system predicted that passive clinical surveillance in cattle would provide the highest probability to detect BTV-8 infected animals, followed by passive clinical surveillance in sheep and bulk milk testing of 200 dairy cattle farms in high-risk areas. This approach is also applicable in other countries and to other epidemic diseases.     

Evidence of Schmallenberg virus circulation in ruminants in Greece

During March 2013, we investigated the presence and the levels of Schmallenberg virus (SBV) circulation in three dairy cow herds and three sheep flocks in Central Macedonia, Greece. In two cow herds, a high number of abortions had been observed during the winter. Six bulk-tank milk samples and 147 individual sera were screened for SBV-specific antibodies by ELISA. Positive reactions were obtained from 5 out of 6 bulk-tank milk samples, 58 out of 90 sera from the 3 cow herds, and 2 sera from 2 of the 3 sheep flocks. Twenty-two ELISA-positive sera were tested by serum neutralization test (SNT). SNT confirmed the presence of neutralizing antibodies against SBV in all samples tested, with titers ranging between 1:32 and ≥1:256. No neutralizing antibodies against Akabane virus (AKAV) or Shamonda virus (SHAV) were detected, indicating that neutralizing antibodies against SBV do not cross react with AKAV or SHAV in SNT. ELISA testing of bulk-tank milk samples proved to be convenient and reliable. None of the tested sera was found positive for SBV by real-time RT-PCR, indicating that the sampling was conducted past the viremia stage. This is the first report of SBV circulation in Greece.     

Evaluation of anti-Ostertagia ostertagi antibodies in individual milk samples as decision parameter for selective anthelmintic treatment in dairy cows

The aim of this study was to explore whether anti-Ostertagia ostertagi antibody levels measured by ELISA in individual milk samples hold promise as a decision parameter for either herd-level decisions or selective anthelmintic treatments by investigating (1) the relationship between individual and bulk-tank milk ELISA results; (2) the relationships of individual milk ELISA results with non-parasitic cow factors and (3) the relationship between individual milk ELISA results and the milk production response after anthelmintic treatment. Twelve farms were randomly allocated to a whole-herd treatment with eprinomectin or a placebo in October 2004 and individual milk samples and a bulk-tank milk sample were collected 1 month before and 1 month after treatment. Linear mixed models were used to investigate the associations of ELISA results with (a) breed, actual milk production, lactation stage, somatic cell count, age and sampling month and (b) the milk production response after anthelmintic treatment. There was a reasonable correlation between the mean individual and bulk-tank milk ELISA results (r = 0.72). Individual cow ELISA results increased with higher lactation number and were lower in November than September. The associations with the other non-parasitic factors were weak and not significant. Milk yield responses to anthelmintic treatment were greater when treatment was given in early lactation and increased with the pre-treatment ELISA result and cow age. However, these latter two interaction terms were not significant when they were put in the model together. We conclude that (1) O. ostertagi ELISA results from individual milk samples may provide more information on the herd's parasitic status than a single bulk-tank milk result; (2) lactation number should be taken into account when interpreting ELISA results from individual milk samples and (3) the value of the O. ostertagi antibody level in individual cow milk samples to predict individual production responses after anthelmintic treatment remains equivocal.     

Bovine tuberculosis surveillance alternatives in Belgium

Belgium obtained the bovine tuberculosis (bTB) officially free status in 2003 (EC Decision 2003/467/EC). This study was carried out to evaluate the components of the current bTB surveillance program in Belgium and to determine the sensitivity of this program. Secondly, alternatives to optimize the bTB surveillance in accordance with European legislation (Council Directive 64/432/EEC) were evaluated. Separate scenario trees were designed for each active surveillance component of the bTB surveillance program. Data from 2005 to 2009 regarding cattle population, movement and surveillance were collected to feed the stochastic scenario tree simulation model. A total of 7,403,826 cattle movement history records were obtained for the 2,678,020 cattle from 36,059 cattle herds still active in 2009. The current surveillance program sensitivity as well as the impact of alternative surveillance protocols was simulated in a stochastic model using 10,000 iterations per simulation. The median (50% percentile) of the component sensitivities across 10,000 iterations was 0.83, 0.85, 0.99, 0.99, respectively, for (i) testing the cattle only during the winter screening, (ii) testing only imported cattle, (iii) testing only purchased cattle and (iv) testing only all slaughtered cattle. The sensitivity analysis showed that the most influential input parameter explaining the variability around the output came from the uncertainty distribution around the sensitivity of the diagnostic tests used within the bTB surveillance. Providing all animals are inspected and post mortem inspection is highly sensitive, slaughterhouse surveillance was the most effective surveillance component. If these conditions were not met, the uncertainty around the mean sensitivity of this component was important. Using an antibody ELISA at purchase and an interferon gamma test during winter screening and at import would increase greatly the sensitivity and the confidence level of Belgium's freedom from bTB infection status.     

Genetic evaluation of IS900 partial sequence of Mycobacterium avium subsp. paratuberculosis Brazilian isolates from bovine milk

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis. Insertion sequence IS900 is used for the identification of MAP. The objective of this study was to verify the genetic conservation of IS900 sequences in raw milk samples. To evaluate genetic conservation, 206 quarter milk samples and 16 bulk-tank milk samples were collected. DNA extraction and IS900 PCR were performed in all samples. Six samples amplified the expected fragment. To confirm the identity of the amplified fragments, PCR products were cloned and sequenced. The resulting sequences were compared with other MAP sequences from GenBank, and it was possible to identify eight polymorphic regions and to form five distinct haplotypes. The number of mutations in each haplotype was verified. IS900 sequence is a very well-conserved sequence that could be used as tool for the molecular detection of this agent and epidemiological purposes. The results showed the first genetic analysis on Brazilian isolates of MAP.     

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.     

A hierarchical trend model for bovine virus diarrhoea virus (BVDV) sero-conversion in Norwegian dairy herds from 1993 through 1997

The Norwegian bovine virus diarrhoea (BVD) control-and-eradication program performed annual testing of the national cattle population since its first screening in January 1993. A bulk-tank milk ELISA antibody test was used for the initial screening of dairy herds. Based on the annual bulk-tank milk-test result, a binary variable denoting herd sero-conversion (a surrogate measure for incidence) was created.The count of herds with sero-conversion in each community was regressed on year and the initial herd-sero-prevalence for each community in a Poisson hierarchical trend model - modelling the risk of sero-conversion. By using this modelling approach, estimates of trend specific for each hierarchical level of organisation were included in the trend model (community, veterinary district and county) could be estimated.The main BVD trend showed a steadily declining sero-conversion risk. The communities in the highest herd-sero-prevalence quartile in 1993 continued to have the highest sero-conversion risk throughout the study period--decreasing from an average predicted sero-conversion risk in 1993 of 0.12 (95% CI; 0.10, 0.13) to 0.02 (0.007, 0.04) in 1997. There was an expressed variation in the level of sero-conversion for all the three hierarchical levels, but the trend only varied at the lowest level (community).     

现代化规模猪场猪伪狂犬病防控探讨

猪伪狂犬病是目前规模猪场主要防控的病毒病之一,2018年12月华北地区某现代化规模猪场,妊娠母猪出现流产现象。通过发病背景、临床症状及死胎剖解、现场情况、实验室血清学及病原学检测,此次妊娠母猪流产是由伪狂犬病毒野毒感染所致。采用优质伪狂犬基因缺失疫苗进行紧急接种、改善通风、消除贼风引起的应激措施后,得到了有效的控制。该措施从猪场的饲养管理角度并结合疫苗防控来控制疫病,以期为现代化规模猪场伪狂犬病的防控提供参考。     

Milk quality assurance for paratuberculosis: simulation of within-herd infection dynamics and economics

A bulk milk quality assurance programme for Mycobacterium avium subsp. paratuberculosis (Map) in dairy herds was simulated with a stochastic simulation model (JohneSSim). The aim of this study was to evaluate the epidemiological and economic effects of preventive management measures and various test schemes in a simulated population of closed Dutch dairy herds over a 20-year period. Herds were certified as ;low-Map bulk milk' if, with a certain probability, the concentration of Map in bulk milk did not exceed a maximum acceptable concentration of 10(3) Map organisms per litre (based on pasteurisation studies). The programme started with an initial assessment; test-negative herds entered a surveillance procedure and test-positive herds a control procedure. The simulations showed that herd examinations by ELISA for the initial assessment, surveillance and control procedures effectively ensure the quality of ;low-Map bulk milk': > 75% of simulated herds were certified and > 96% of certified herds produced bulk milk with < 10(3) Map/L if the initial herd-level prevalence was 30%. Preventive management measures only had a minor effect on bulk milk quality of certified herds. Culling based on biennial faecal culture was more effective than culling based on annual ELISA. Average total discounted costs for 20-year participation in a programme consisting of initial assessment by ELISA, surveillance by biennial ELISA and control by biennial faecal culture were 16 Euro x 10(3) per herd. In conclusion, this study shows that a bulk milk quality assurance programme for closed Dutch dairy herds is feasible and provides information on the cost-effectiveness of different programmes. The concepts of this study equally apply to other countries because mechanisms of paratuberculosis infection, disease, and testing are comparable in other dairy cattle populations.     

The Danish pig health scheme: Nation-wide computer-based abattoir surveillance and follow-up at the herd level

In 1978 an agreement was made among the Danish authorities, the slaughterhouse association and the pig produces to develop and operate a national swine herd health scheme based on data from the routine slaughter inspection.This review of the health scheme first presents available evidence of the diagnostic validity of slaughter inspection data in terms of correlation with clinical history and with detailed post-mortem findings of internal organs. The epidemiological basis for the program further includes the use of slaughter inspection findings in estimating the poor beneficial effects of medical treatment procedures in common, multifactorial disorders, their considerable variation within and among herds of different sizes, their correlation with environmental and managerial herd factors, and some obvious associations among different clinical pictures of these economically important herd health problems.Finally, an overview is presented of the current program, its design and operation, and preliminary results from its first few years of operations are given.     

Detection of Trichinella infection in food animals

The first part of this review article deals with classical methods used for the detection of Trichinella larvae in muscle samples of those animal species which are recognized as traditional sources of trichinellosis for human beings, as well as those species which are important for epidemiological reasons. Special consideration is given to the main applications of these methods (routine slaughter inspection, and epidemiological studies in reservoir animals), and to the major factors that may influence detection methods (sampling site, sample size). Historical, current and future aspects concerning national and EU legislation for Trichinella inspection are also presented. The latter part of this review is directed at serodiagnostic methods for the detection of Trichinella-specific antibodies in different animal species. Classical methods of serodiagnosis such as the complement fixation test and immunofluorescence antibody test are reviewed and the characteristics and performance of the ELISA are discussed. Factors dependent upon the animal species being tested or on components of the ELISA test system are considered. This paper also reviews systematic development of the ELISA in relation to improvements in test specificity and sensitivity. Additionally, remarks are made on implementing this test for surveillance and control programs in domestic pigs and wildlife.     

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.     

Geographical and temporal aspects of an outbreak of off-flavours in bulk-tank milk in Prince Edward Island, Canada

The geographical and temporal dynamics of the outbreak of off-flavours in bulk-tank milk that occurred between September 2000 and June 2002 in Prince Edward Island (PEI) dairy herds were described using spatial, temporal and space-time scan statistics. Results suggested clustering both in time and space. We found one primary cluster (six case herds, feed off-flavour, October 2000 to January 2001) and two secondary clusters formed of feed (5 case herds, October 2000 to January 2001)) and rancid (five case herds, December 2000 to March 2001) off-flavours, respectively. The relative risk (RR) for producing off-flavoured milk was similar for these three clusters (RR=6.4).     

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

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

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.