An estimated prevalence of Johne's disease in a subpopulation of Alabama beef cattle.

The objective of this study was to estimate the overall prevalence of animals that were infected with Mycobacterium avium ssp. paratuberculosis in a subpopulation of Alabama beef cattle. This was determined using a commercial enzyme-linked immunosorbent assay (ELISA) for the detection of M. avium ssp. paratuberculosis-specific antibodies in serum. Serum was collected from 79 herds that were participating in the Alabama Brucellosis Certification program. A total of 2,073 beef cattle were randomly tested by selecting 30 animals per herd in herds greater than 30 and selecting all animals in herds 30 and less for testing. It has been estimated that the commercial ELISA test used has a 60% sensitivity and a 97% specificity. Of the 79 herds tested, 29 herds were seronegative, 24 herds had 1-2 positive animals, and 26 herds had 3 or more seropositive animals. The average number of infected animals per positive herd was 3.3. In addition, a calculated minimum of 53.5% of the herds were identified as Johne's positive herds with a 95% confidence level. Of the total number of animals tested, 8.0% (166/2,073) of them were positive by the ELISA. After adjustments for test sensitivity and specificity and the proportion of animals sampled per herd, the true prevalence was calculated to be 8.75%. These data suggest that approximately 50% of the herds are infected with M. avium ssp. Paratuberculosis, and the overall prevalence of infection in Alabama beef cattle is approximately 8%, which correlates with other previously published regional estimates.     

Babesia gibsoni infection among dogs in the southeastern United States

OBJECTIVE: To identify subclinical Babesia gibsoni infection in American Pit Bull Terriers from the southeastern United States and to determine the genetic sequence of parasite DNA isolated from these dogs. DESIGN: Case series. ANIMALS: 33 American Pit Bull Terriers and 87 dogs of various other breeds. PROCEDURE: Blood smears were examined for microscopic evidence of the parasite, and DNA was extracted from blood samples and used in a polymerase chain reaction (PCR) assay designed to amplify the small subunit ribosomal RNA gene sequence of B. gibsoni. Amplification products of the expected size were sequenced, and sequences were compared with published sequences for B. gibsoni isolates. Hematocrit, platelet count, mean platelet volume, WBC count, and eosinophil count were compared between dogs with positive PCR assay results and dogs with negative results. RESULTS: Results of the PCR assay were positive for 18 of the 33 (55%) American Pit Bull Terriers, including all 10 dogs with microscopic evidence of parasitemia. Only 1 of these dogs was clinically ill at the time blood samples were collected. Results of microscopic evaluation of blood smears and of the PCR assay were negative for the 87 other dogs. Hematocrit and platelet count were significantly lower in dogs with positive PCR assay results than in dogs with negative results. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that American Pit Bull Terriers in the southeastern United States may be subclinically infected with B. gibsoni. However, subclinical infection was not identified in dogs of other breeds from the same geographic area.     

牛尿苷酸合酶缺乏症PCR-RFLP检测方法的建立

提取牛血液和精液DNA,进行PCR扩增、RFLP分析,建立尿苷酸合酶缺乏症(DUMPS)检测方法。对济南市6个奶牛场224头荷斯坦奶牛及53头种公牛进行了DUMPS的PCR-RFLP检测,共检测出2头杂合母牛(携带者),杂合子占检测母牛群的0.89%,在荷斯坦公牛中只检测到一种基因型,没有发现隐性突变基因的携带者。实验证明,该方法重复性好、灵敏性高、稳定性可靠。     

Survey for antibodies to leukemia (C-type) virus in cattle.

Serums from 4,394 dairy cattle in 100 herds and from 2,794 beef cattle in 50 herds were tested for antibody to the bovine (C-type) leukemia virus (BLV), using the agar gel immunodiffusion test. Reactors were found in 66% of the dairy herds (10.2% of the cattle) and in 14% of the beef herds (1.2% of the cattle). The prevalence of reactors was examined with respect to age, herd size, and sex. Few of the reactors were less than 2 years old. There was a high percentage of reactors in small dairy herds (less than 50 cattle). In 22 dairy herds (1,354 cows and 96 bulls), the rate of infection in cows was compared with that in bulls. In those herds, 13.5% of the cows and 10.4% of the bulls were reactors.     

Investigation of the index case herd and identification of the genotypes of Theileria orientalis associated with outbreaks of bovine anaemia in New Zealand in 2012

CASE HISTORY AND CLINICAL FINDINGS: On 7 September 2012 the Ministry for Primary Industries was notified of a dairy cow with regenerative anaemia (haematocrit (HCT) 0.08?L/L) in a herd of 465 Jersey-Friesian cross cows (index case herd) in the Northland region of New Zealand. Organisms consistent with Theileria spp. were present in red blood cells on a blood smear. No other causes of anaemia were detected following examination of affected cows. Blood samples collected from 29 randomly selected cows on 26 September 2012 showed that 24 (83%) were anaemic (HCT≤0.24 L/L) and therefore fitted the case definition for bovine anaemia associated with Theileria orientalis infection.

LABORATORY FINDINGS: Using a T. orientalis type-specific PCR assay that targeted the single subunit rRNA gene, all of six animals tested were positive for T. orientalis type Ikeda. Blood samples collected from clinically affected cattle in 11 subsequent outbreaks from throughout the North Island showed that T. orientalis Ikeda type was a common finding, but mixed infections with Chitose type were also identified. In addition, using a PCR assay that targeted the major piroplasm surface gene, T. orientalis type 5 was detected in one cow from the Waikato region.

DIAGNOSIS: The presence of T. orientalis type Ikeda, as well as type 5, was confirmed in cattle from outbreaks of bovine anaemia in herds throughout the North Island of New Zealand.

CLINICAL RELEVANCE: Two new types of T. orientalis were identified in this investigation, that were associated with a sudden rise in cases of bovine anaemia. The body of evidence showed that the Ikeda type was implicated as the cause of disease observed in this epidemic.     

Developing high throughput quantitative PCR assays for diagnosing Ikeda and other Theileria orientalis types common to New Zealand in bovine blood samples

AIMS: To develop rapid, quantitative PCR (qPCR) assays using high resolution melt (HRM) analysis and type-specific TaqMan assays for identifying the prevalent types of Theileria orientalis found in New Zealand cattle; and to evaluate their analytical and diagnostic characteristics compared with other assays for T. orientalis.

METHODS: Nucleotide sequences aligned with T. orientalis Buffeli, Chitose and Ikeda types, obtained from DNA extracted from blood samples from infected cattle, were used to design HRM and type-specific probe-based qPCR assays. The three type-specific assays were also incorporated into a single-tube multiplex qPCR assay. These assays were validated using DNA extracted from blood samples from cattle in herds with or without clinical signs of T. orientalis infection, other veterinary laboratory samples, as well as plasmids containing T. orientalis type-specific sequences. Diagnostic specificity (DSp) and sensitivity (DSe) estimates for the qPCR assays were compared to blood smear piroplasm results, and other PCR assays for T. orientalis. Copy number estimates of Ikeda DNA in blood were determined from cattle exhibiting anaemia using the Ikeda-specific qPCR assay.

RESULTS: The T. orientalis type-specific and the HRM qPCR assays displayed 100% analytical specificity. The Ikeda-specific qPCR assay exhibited linearity (R²?=?0.997) with an efficiency of 94.3%. Intra-assay CV were ≤0.08 and inter-assay CV were ≤0.095. For blood samples from cows with signs of infection with T. orientalis, the DSp and DSe of the multiplex probe qPCR assay were 93 and 96%, respectively compared with blood smears, and 97 and 100%, respectively compared with conventional PCR assays. For the Ikeda-specific qPCR assay, the number of positive samples (n=66) was slightly higher than a conventional PCR assay (n=64). The concentration of Ikeda genomes in blood samples from 41 dairy cows with signs of infection with T. orientalis ranged between 5.6?×?10⁴ and 3.3?×?10⁶ genomes per µL of blood.

CONCLUSIONS: All qPCR assays had improved specificity and sensitivity over existing conventional PCR assays for diagnosis of T. orientalis Ikeda. The burden of Ikeda DNA in blood was demonstrated using an Ikeda-specific qPCR assay with titrated synthetic gene target.

CLINICAL RELEVANCE: Adoption of high-throughput DNA extraction and qPCR reduced T. orientalis and Ikeda diagnosis times. The Ikeda-specific qPCR assay provides a specific diagnosis for Ikeda in animals with signs of infection with T. orientalis and can be used to monitor the parasite load of Ikeda in blood.     

Association of fecal shedding of mycobacteria with high ELISA-determined seroprevalence for paratuberculosis in beef herds

OBJECTIVE: To evaluate the seroprevalence of paratuberculosis by use of 2 commercial ELISAs in association with prevalence of fecal shedding of mycobacteria within beef cattle herds. DESIGN: Cross-sectional field study. ANIMALS: Six beef herds (affected herds; 522 cattle) with and 3 geographically matched herds (181 cattle) without high seroprevalence of paratuberculosis. PROCEDURES: Blood and fecal samples were collected from adult cattle and assessed for serum anti-Mycobacterium avium subsp paratuberculosis (MAP) antibodies with 2 commercial ELISA kits and submitted for bacterial culture for MAP and environmental bacteria (termed environmental mycobacteria) via a radiometric method, respectively. Species of mycobacterial isolates were identified, and sensitivities and specificities of the 2 ELISAs were compared. RESULTS: Compared with comparison cattle, cattle from affected herds were 9.4 times as likely to have environmental mycobacteria isolated from feces. Among the 6 affected and 3 comparison herds, the proportions of cattle shedding environmental mycobacteria were 0.225 (range, 0.1 to 0.72) and 0.04 (range, 0 to 0.06), respectively. Although relative MAP- detection specificities (compared with bacterial culture of feces) were different between the 2 ELISAs, sensitivities were not. Nine environmental mycobacterial species were identified from participating herds. All affected herds apparently had > or = 1 bovid infected with MAP, although MAP was not isolated from any cattle in comparison herds. CONCLUSIONS AND CLINICAL RELEVANCE: In beef herds with persistently high rates of false- positive ELISA results, which may be associated with recovery of environmental myco- bacteria from feces, organism detection via bacterial culture of feces or PCR assay should direct paratuberculosis control measures.     

Bovine viral diarrhoea (BVD) infections--control and eradication programme in breeding herds in Slovenia

A Slovenian BVD control and eradication programme was initiated in 1994, and the results from testing of bovine herds for antigen and antibodies in 1996 are presented. Samples originating from breeding herds, breeding herds for young bulls, and insemination stations were tested by antigen or antibody ELISA, or by PCR. Out of 7968 samples from 354 herds we found 18% of the animals antibody-positive. In one region situated in the north-east of Slovenia we found the herds to be almost nearly free of BVDV infections (5% prevalence). No positive antigen ELISA findings were done in 374 blood samples from recruitment herds for young bulls, whereas two out of 206 sera were investigated by PCR-reacted positive. The differences in seroprevalence found between regions is thought to be caused by differences in summer pasturing and husbandry practices.     

荷斯坦牛脊椎畸形综合征分子诊断方法的建立与应用

脊椎畸形综合征(Complex Vertebral Malformation,CVM)是由常染色体上SLC35A3基因单碱基突变(G→T)引起的隐性遗传疾病,该基因隐性纯合(CV/CV)时奶牛致死,但CVM携带者表现正常,所以CVM携带者公牛可以通过人工授精技术传播CVM缺陷基因。本研究自行设计检测CVM特异性PCR引物,扩增长度为173bp,然后利用PCR-SSCP方法对186个公牛样品和140个母牛样品进行了检测分析,该方法简便快捷、准确率高,使用样品宽泛,适合大样本筛选。研究结果表明,在所检测的样本中,荷斯坦种公牛和母牛CVM携带率分别为11.3%和12.1%,并通过系谱追踪发现,CVM遗传缺陷的共同祖先是美国名牛"Penstate Ivanhoe Star"(USA.1441440,CV)。通过剔除CVM携带者公牛可以有效地控制CVM遗传缺陷,但是,我国许多CVM携带者公牛冻精依然在商业化使用,所以,有效地监控CVM携带者在奶牛群中的状况对CVM防控计划是有益的。     

Prevalence of Coxiella burnetii infection in Dutch dairy herds based on testing bulk tank milk and individual samples by PCR and ELISA

A study using an ELISA and a real-time PCR assay based on the detection of the repetitive transposon-like gene of Coxiella burnetii revealed that infection with the bacterium was widespread among Dutch dairy herds, with antibodies detected in bulk tank milk (BTM) from 268 of 341 herds (78.6 per cent) and bacterial DNA detected in 193 of 341 herds (56.6 per cent). The BTM samples were taken in November and December 2007. Serological and molecular studies in young and adult cattle selected from 100 herds showed that antibodies were present in the blood of 470 of 2936 (16.0 per cent) lactating cows but only in 19 of 1831 (1.0 per cent) young animals. Bacterial DNA was detected in the milk of 254 of 2925 (8.7 per cent) lactating cows; bacterial DNA was not detected in any of the faecal samples obtained from youngstock. The blood and milk samples were taken from the cattle in the period January to April 2008.     

Seroprevalence to bovine immunodeficiency virus and lack of association with leukocyte counts in Italian dairy cattle

We report herein on the first serological detection of antibodies to bovine immunodeficiency virus (BIV) in Italy. According to criteria of a stratified-random sampling of dairy cattle reared in the Parma area (a province in the Po Valley, Northern Italy), sera from 3166 cows belonging to 272 herds were collected. In addition, sera of 138 bulls from eight artificial-insemination (AI) centres were sampled. Seventy-eight cows (2.5%) from 16 herds (5.8%) and seven bulls (5.1%) from two AI centres were positive for BIV-R29 antibodies in the IFA-test. IFA-positive sera assayed by Western blot had reaction to different viral proteins: 81 out of 85 sera showed antibody to p26 (considered the BIV major internal core protein); four sera reacted to other viral proteins but not to p26. Peripheral blood leukocytes of 60 seropositive and 60 seronegative animals, belonging to eight BIV-infected herds, were enumerated to assess any effect of BIV infection on white-blood cells. No significant differences were detected between the two groups. These data indicate that BIV infection is present in Italian dairy cattle – but the role of BIV in inducing disease remains unclear.     

Theileria orientalis MPSP types in Australian cattle herds associated with outbreaks of clinical disease and their association with clinical pathology findings

Between September 2010 and November 2011, 350 EDTA blood samples were received from 73 Australian cattle herds, as cases suspected to be infected with Theileria orientalis. Beef cattle were predominantly affected, with Angus and Angus-crossbred cattle representing 48% of smear positive samples examined. DNA extracts were tested in conventional polymerase chain reaction (PCR) assays for genes encoding the p32, Ikeda, Chitose and Buffeli major piroplasm surface proteins (MPSP). PCR findings were compared with results of clinical pathology examinations of stained blood smears for parasitaemia and packed cell volume (PCV). PCR testing was much more sensitive than clinical pathology examinations in detecting T. orientalis infections, and concurrent testing of neat and diluted extracts gave significantly more PCR positive results than testing of neat extract alone. Significant associations and correlations were shown between PCR results of p32 and Ikeda assays with PCV levels indicative of anaemia, and with the level of parasitaemia estimated by smears. A high proportion of samples had concurrent Ikeda and Chitose infection, and significantly more clinical cases of theileriosis were associated with the Ikeda MPSP type as the sole infection, compared with sole infection with types Chitose or Buffeli. The findings indicate Ikeda type organisms were significantly associated with clinical parameters of theileriosis in cattle herds in eastern Australia, and that this type is most likely to be responsible for outbreaks of theileriosis experienced in affected Australian herds. In New South Wales, 11 of 14 regulatory districts yielded Ikeda positive samples, with five (Mid-Coast, Cumberland, Central North, Hume and Lachlan) containing 234/307 (76%) of the Ikeda positive samples.     

Surveillance for persistent bovine viral diarrhea virus infection in four artificial insemination centers

Four large bovine artificial insemination centers implemented a program of surveillance of resident and newly acquired bulls for persistent bovine viral diarrhea virus infection. Infection was identified in 12 of 1,538 bulls. Several clinical abnormalities, including acute and chronic mucosal disease, were evident among the persistently infected bulls. Semen produced by such bulls consistently contained bovine viral diarrhea virus, and such contamination was not always accompanied by diminished seminal quality. Infected bulls were detected by means of virus isolation tests performed on blood specimens, but not by use of serologic testing. Ten of the 12 persistently infected bulls were results of embryo transfer. Virologic surveillance of breeding herds, artificial insemination and embryo transfer centers, and the cattle trade is necessary to prevent spread of this virus by modern cattle breeding practices. Attention is also necessary to prevent contamination by this virus of the fluids used for recovery, in vitro manipulation, and transfer of bovine embryos.     

Molecular and serological evidence of Anaplasma phagocytophilum infection of farm animals in the Black Sea Region of Turkey

This study was designed to determine the presence and the prevalence of Anaplasma phagocytophilum infection in sheep and cattle in the Middle and Eastern Black Sea Regions of Turkey in which the potential vector, Ixodes ricinus, is widespread. Blood samples were collected from 720 sheep and 720 cattle from 6 provinces of the region, and used for detecting antibodies to A. phagocytophilum by indirect immunofluorescent antibody test (IFAT) and specific nucleic acids by a nested polymerase chain reaction (PCR). Blood smears were also prepared and examined microscopically for the presence of A. phagocytophilum-like organisms in polymorphonuclear cells. Of the animals examined, antibodies were detected in 110 (15.27%) cattle and 107 (14.86%) sheep and A. phagocytophilum-like organisms were detected in the blood of 73 (10.13%) cattle and 71 (9.86%) sheep. In addition, specific DNA was detected in the blood of 27 (14.75%) cattle and 22 (12.35%) sheep. The results obtained constitute the first molecular and serological evidence of A. phagocytophilum infection in sheep and cattle in the Black Sea Region of Turkey.     

Influence of three types of farm management on the seroprevalence of Q fever as assessed by an indirect immunofluorescence assay.

The influence of three types of farm management on the occurrence of Q fever in cattle was investigated by means of a serological study carried out in Irpinia in southern Italy. Twenty-one herds were permanently housed, 26 were housed in winter and turned out to graze in the spring, and six herds were kept outdoors throughout the year. Blood samples were taken from 1188 cattle and tested by an indirect immunofluorescence assay. The overall seroprevalence of Q fever was 14.4 per cent. The herds which were housed in winter and turned out in the spring had the highest seroprevalence of 19.6 per cent, followed by the permanently housed animals with a seroprevalence of 13.2 per cent and the unhoused cattle had a much lower seroprevalence of 1.9 per cent.     

High prevalence of Tritrichomonas foetus infection in Asturiana de la Montaña beef cattle kept in extensive conditions in Northern Spain

Bovine trichomonosis (BT) and bovine genital campylobacteriosis (BGC) are sexually transmitted diseases that can be important infectious causes of reproductive failure in extensively managed beef cattle where natural mating is a common practice. However, their prevalence in Europe was thought to be insignificant or very low. The purpose of this study was to investigate the prevalence and risk factors associated with BT and BCG in a representative beef cattle breed, Asturiana de la Monta?a (AM), which is usually managed extensively in the mountain areas of Northern Spain and putative risk factors associated with the two diseases are present on most farms holding AM cattle. Preputial smegma samples were collected from 103 bulls belonging to 65 herds. Pathogen detection was undertaken using culture and PCR. Two scraping methods for sample collection (AI pipette and plastic scraper), as well as different culture media and DNA extraction methods were evaluated on field samples. Campylobacter fetus veneralis infection was not detected in any animal in any herd. However, Tritrichomonas foetus infection was demonstrated in 32% (33/103) and 41.5% (27/65) of bulls and herds tested, respectively. AM bulls older than 3 years (39.7%) were more likely to be infected than young bulls (16%) (OR=3.45, CI=1.07-11.19). An increase in repeat breeder cows was reported in herds from which T. foetus was detected (OR=5.2, CI=1.5-17.18). These findings highlight the re-emergence of this disease in extensively managed beef cattle in Spain. For routine diagnosis, the use of a culture technique and PCR in combination is advisable for testing smegma samples under field conditions.     

Use of dried blood smears for detection of feline hemoplasmas using real-time polymerase chain reaction

The objective of the current study was to determine the sensitivity and specificity of real-time polymerase chain reaction (real-time PCR) for feline hemoplasmas when applied to DNA extracted from dried whole-blood smears in comparison to that for DNA extracted from liquid whole blood. Blood samples were collected into ethylenediamine tetra-acetic acid tubes from 305 cats with possible or suspected hemoplasmosis, and dried blood smears from each sample were prepared. DNA was extracted from blood smears and a 160-microl aliquot of each liquid blood sample by using a robotic extractor and was subjected to real-time PCR for feline glyceraldehyde-3-phosphate dehydrogenase (liquid blood), 18S ribosomal RNA (dried blood), and "Candidatus Mycoplasma haemominutum", Mycoplasma haemofelis, and "Candidatus Mycoplasma turicensis" DNA. When using the results for liquid whole blood as the gold standard, the sensitivity of each assay for "Ca. M. haemominutum", M. haemofelis, and "Ca. M. turicensis" was 49 of 66 (74%), 11 of 13 (85%), and 11 of 20 (55%), respectively. The specificity of each assay was 224 of 234 (96%), 287 of 287 (100%), and 280 of 280 (100%), respectively. When possible, liquid blood samples should be submitted for detection of feline hemoplasmas by using real-time PCR. The improved sensitivity of real-time PCR on blood smears for M. haemofelis compared with that of the other hemoplasma species may reflect the higher organism burdens associated with infection with this species.     

Population structure of Reyna Creole cattle in Nicaragua

Reyna Creole cattle originated from Bos taurus cattle brought to Latin America during the Spanish colonization in the fifteenth century and are the only remaining local breed in Nicaragua. However, the current genetic status of this breed is unknown. Therefore, the population structure of three recorded Reyna Creole herds in Nicaragua was studied to estimate their level of inbreeding, effective population size, and generation intervals. Data from 2,609 animals born between 1958 and 2007 were analyzed. A pedigree completeness index higher than 0.8 was required to obtain reliable estimates of the level of inbreeding, and this criterion was met for 367 animals (14%) in two herds. The average level of inbreeding was 13.0%, with values ranging from 0% to 43.8% for individual animals. One of the herds had an average inbreeding level of 21.6%, primarily due to long periods in which the same bulls were used for mating, leading to excessive frequencies of matings between closely related animals. The effective population size differed between years and ranged from 28 to 46 animals, showing that the Reyna Creole cattle breed is endangered, close to critical status. The average generation interval was 6.9 years with values as high as 19.1 years for some sires that were used for artificial insemination over a long period of time. Due to the high level of inbreeding and small population size, urgent actions are required for the development of a breeding program to protect the breed and support its sustainable utilization.     

Economic costs associated with two testing strategies for screening feeder calves for persistent infection with bovine viral diarrhea virus

OBJECTIVE: To develop partial budgets of the economic costs of 2 test strategies for screening cattle for persistent infection with bovine viral diarrhea virus (BVDV). DESIGN: Partial budget analysis. ANIMALS: 938 calves arriving at 2 stocker operations. PROCEDURE: Calves were tested to determine prevalence of persistent BVDV infection. Test strategies that were evaluated included a single-test strategy consisting of immunohistochemical staining of skin biopsy specimens from all animals and a 2-test strategy consisting of polymerase chain reaction (PCR) assaying of pooled blood samples followed by immunohistochemical staining of skin biopsy specimens from animals in pools for which assay results were positive. Break-even costs (i.e., cost of persistent BVDV infection per animal necessary to justify whole-herd diagnostic testing) associated with each test strategy were calculated as a function of disease prevalence and test cost. RESULTS: Apparent prevalence of persistent BVDV infection was 0.32%. Sensitivity and specificity of the PCR assay for pooled samples were 100% and 89.7%, respectively. Regardless of the prevalence of persistent BVDV infection, the break-even cost for the 2-test strategy was lower than the break-even cost for the single-test strategy. However, the economic advantage was greatest when prevalence was low. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that using a 2-test strategy to screen cattle for persistent BVDV infection, whereby the first test involves PCR assaying of pooled samples and the second involves immunohistochemical testing only of those animals represented in pooled samples with positive assay results, will reduce the cost of screening incoming feedlot cattle, compared with immunohistochemical testing of all animals.     

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.