Efficiency of inspection procedures for the detection of tuberculous lesions in cattle

The sensitivity of the abattoir inspection procedure introduced for Australian export beef in 1976 was compared to a detailed necropsy procedure for the detection of tuberculous lesions in cattle. In a sample of cattle that were reactors to the tuberculin test, abattoir inspection failed to detect an estimated 47% of cattle with lesions. The detailed necropsy examination of cattle with lesions of tuberculosis identified 21 sites of infection compared with 13 to 18 in cattle examined by routine meat inspection procedures. Of the lesions detected during detailed necropsy, 15.9% did not involve the thoracic cavity or the medial retropharyngeal lymph nodes. The failure to detect lesions during abattoir inspection has its greatest significance in an animal with a single lesion. If the 245 cattle found with single lesions during detailed necropsy had been examined by abattoir inspection using the 1976 or the 1986 procedures, 0.8 and 8.9%, respectively, of these animals would not have been detected because the diseased tissues would not have been examined. If meat inspection is to provide an effective means of monitoring the level of bovine tuberculosis during the final stages of eradication, a procedure no less sensitive than that introduced in 1976 should be used.     

牛皮蝇蛆病间接ELISA诊断方法的建立

以纹皮蝇Ⅰ期幼虫粗提蛋白为包被抗原,通过方阵试验确定了血清最佳稀释倍数为80倍,抗原最佳包被浓度为26.64μg/mL,并对其特异性、敏感性和重复性进行了试验,建立了牛皮蝇蛆病间接ELSIA诊断方法。再以建立的ELISA诊断方法对采自内蒙古地区的233份牛血清进行了检测。结果表明,所建立的牛皮蝇蛆病间接ELISA诊断方法具有较好的特异性、敏感性和可重复性,可用于牛皮蝇蛆病的血清学检测。     

Single dilution ELISA for detection of serum antibody to foot-and-mouth disease virus in cattle

A single dilution blocking ELISA was developed and evaluated for measuring serum antibody to foot-and-mouth disease virus (FMDV). Basic parameters of the assay were established and a positive-negative threshold determined from testing 176 specific antibody negative sera from Australian cattle. Sera collected from immunised animals in Thailand were tested by ELISA and virus-neutralisation (VN) tests and the results compared. A positive correlation between ELISA and VN titres was recorded for each of the 3 FMDV serotypes endemic in Thailand, with the overall correlation coefficient being r = 0.8990. A positive correlation for each of the serotypes was also found between ELISA titre and the degree of blocking (percentage inhibition) of each test serum at a dilution of 1:16, with the overall correlation being r = 0.8704. This simplified ELISA was sensitive, specific and gave reproducible results, and had the potential to test quickly and efficiently a considerable number of sera.     

Competitive ELISA for the detection of antibodies to Rift Valley fever virus in goats and cattle

Rift Valley fever virus (RVFV) is one of the important emerging viral diseases of serious impact in public health and animal hygiene both in human and animal industries. In this study, we developed a monoclonal antibody-based competitive ELISA for the detection of antibodies to RVFV in goats and cattle. The recombinant N protein of RVFV was expressed in E. coli with a six-histidine tag, and the purified N protein was used for detecting antigen with a competitive monoclonal antibody against RVFV antibodies. The competitive ELISA (C-ELISA) could detect antibodies at 9-11 days after inoculation in goats and cattle with a sensitivity of 94.7% (virus neutralization titer >32) and specificity of 99.7%, respectively. In addition, the C-ELISA did not show any cross-reactivity with positive sera against arboviruses such as Akabane, Aino, Chuzan, Ibaraki and bovine ephemeral fever virus, which are prevalent viral agents in ruminant animals throughout Southeast Asia. The results of the present study indicate that the C-ELISA is a simple, rapid and convenient serodiagnostic method for RVFV in goats and cattle.     

3种口蹄疫O型抗体检测试剂盒对疫苗免疫牛血清抗体检测的比较试验

为了提供检测结果准确且操作便捷的试剂盒,本试验应用两种商品化试剂盒和本实验室研制的一种基于合成肽包被抗原的间接ELISA试剂盒同时对口蹄疫疫苗免疫后14、21和28 d的150份免疫牛血清进行口蹄疫O型抗体水平的检测,并对3种试剂盒测定结果的符合程度、重复性和便捷性进行了比较。试验结果表明3种试剂盒测得的抗体水平检测结果之间高度符合,且3种试剂盒各自的检测结果重复性良好,基于合成肽包被抗原的间接ELISA试剂盒在操作便捷性方面优势明显。     

Development of an ELISA system for detection of anti-Anaplasma marginale antibodies in cattle in Brazil

An ELISA test was developed for detecting antibodies against Anaplasma marginale in bovine sera. Four antigenic preparations were produced from infected red blood cells. Some aliquots of this preparation were stored at -70 degrees C with 30% DMSO in phosphate-buffered saline (PBS) and others were lysed with 0.9% NH4Cl and stored at -20 degrees C. Typical anaplasmal structures were seen by electron microscopy in the antigenic preparations containing the erythrocytes that had been stored with DMSO. The performance of the ELISA test was evaluated by testing 298 positive serum samples collected from immunized cattle, 39 negative serum samples collected from cattle imported from areas free of A. marginale and 50 samples collected from cattle naturally infected in the field. The test gave a specificity of 94.87% and a sensitivity of 100%.     

牛口蹄疫病毒VP2结构蛋白抗体间接ELISA方法的建立

为建立牛口蹄疫(FMD)抗体的检测方法,本研究将口蹄疫病毒(FMDV)的VP2基因,通过pPROEXTM HTb表达载体在大肠杆菌DH5α中表达,获得大小为35ku的重组VP2蛋白(rVP2),western blot证实rVP2可与FMDV5种血清型的牛阳性血清发生特异性反应。以纯化复性的rVP2为抗原建立了FMDVrVP2间接ELISA方法。重复性试验证实批内、批间变异系数均小于10%。特异性交叉试验表明,该抗原不与常见的其他7种牛病阳性血清发生交叉反应。检测非免疫无口蹄疫国家牛阴性血清的特异性为100%;检测感染血清敏感性为97.3%;检测O-AsiaⅠ的二价苗免疫牛血清,与4种商品化试剂盒比较,其符合率分别为69.0%、95.0%、90.4%和86.8%。实验结果表明建立的ELISA方法可以用于口蹄疫感染和免疫抗体检测。     

Evaluation of an antigen-capture ELISA for the detection of bovine herpesvirus type 1 shedding from feedlot cattle

A total of 457 nasal swab specimens from cases of respiratory disease in 2 feed lots were evaluated for the detection of bovine herpesvirus Type 1 (BHV-1) by ELISA. Thirty-three were found to be positive for BHV-1 by the recovery of infectious virus and 21 of these were positive by ELISA, yielding a sensitivity of 64%. Fifteen other virus isolations were made and included bovine viral diarrhea viruses, rhinoviruses and parainfluenza Type 3 viruses; none of these cases were positive with the BHV-1 ELISA. Specificity of the ELISA was 100%. Eighty percent of the specimens with BHV-1 titers greater than 10(5) TCID50 were detected by ELISA; the median amount of virus in positive specimens that were detected by ELISA was 7 X 10(5) TCID50 and the median amount of virus in specimens not detected was 1.5 X 10(4) TCID50. BHV-1 infection was most frequently diagnosed in feedlot cattle that had been in the feedlot for 40-80 days. Approximately half of the infected cattle were carrying virus-neutralizing antibodies in their serum.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bovine viral diarrhoea virus (BVDV) in cattle sera

A microtitre ELISA has been established for the quantitation of antibodies to bovine viral diarrhoea virus (BVDV). Single dilutions of sera were assayed and units of antibody were calculated from a standard curve. In order to detect the maximum number of responding animals both IgG1 and IgG2 antibody should be assayed, although detection of IgG1 alone was nearly as effective. The ELISA was as sensitive as the virus neutralization test for detection of antibody; comparison of an ELISA that detected IgG1 plus IgG2 antibody to BVDV with the virus neutralization test gave a correlation coefficient (r) of 0.89 (P less than 0.001 for 95 compared sera). Although similar amounts of IgG1 and IgG2 antibodies were present in sera from both experimentally- and naturally-infected cattle, antibody to BVDV in colostrum and in the sera from young calves was predominantly IgG1. The number of adult cows with antibody was 40 out of 41 while 36 of 44 calves reared in a beef unit were found to have produced antibody by the time they were 31.5 weeks old, an indication of the high prevalence of BVDV in the cattle population.     

Evaluation of the ELISA test for the antibody detection in cattle naturally and experimentally infected with Cysticerccus bovis

The ELISA test was evaluated for the diagnosis of bovine cysticercosis using heterologous antigens from the larvae of T. solium and T. crassiceps, by using different types of positive and negative control sera, to allow a broader analysis of the results. The ELISA test showed low sensitivity under natural conditions of bovine cysticercosis manifestation, but high rates (up to 90%) under experimental conditions. The high specificity of the test (81-100%) made evident its capacity to differentiate cysticercosis from other bovine diseases. No difference in performance was found among the antigens studied. It was concluded that the ELISA test has deficiencies in detecting anti-cysticercosis antibodies of animals at slaughterhouse. However, it can be useful in detecting experimentally infected animals and differentiating cysticercosis from other bovine diseases.     

An estimate of specificity for a Johne's disease absorbed ELISA in northern Australian cattle

Objective To estimate the specificity of an absorbed enzyme-linked immuno-sorbent assay kit^d for Johne's disease (JD) when used in mature cattle populations resident in northern Australia.
Design Blood samples were collected from beef cattle in northern Queensland, the Northern Territory and northern Western Australia, and from dairy cattle in northern Queensland. The specificity of a serological test for JD was estimated by testing the blood samples with an absorbed ELISA kit. Further samples were collected from cattle with positive ELISA results to determine the presence or absence of infection with Mycobacterium avium subsp paratuberculosis .
Procedure During 1995 and 1996, blood, tissue and gut contents were collected from beef cattle at abattoirs in Queensland and the Northern Territory; and blood and faecal samples were collected from dairy cattle in herds assessed to be most at risk for JD in northern Queensland. The blood samples were tested using an absorbed ELISA kit. Tissues and gut contents from beef cattle that had positive ELISA results were cultured for M avium subsp paratuberculosis , and tissues were examined histo-logically. Faecal samples from dairy cattle with positive ELISA results were cultured for M avium subsp paratuberculosis .
Results Estimates of specificity for this absorbed ELISA in mature northern Australian cattle were 98.0% (97.0 to 98.8%, 95% CI) in beef cattle, and 98.3% (96.7 to 99.3%, 95% CI) in dairy cattle.
Conclusion Estimates of specificity in this study were lower for beef cattle from the Northern Territory and northern Western Australia and for dairy cattle from northern Queensland than those quoted from studies on cattle in southern Western Australia. This should be considered when serological testing using the JD ELISA is carried out on northern Australian cattle.     

An estimate of specificity for a Johne's disease absorbed ELISA in northern Australian cattle

OBJECTIVE: To estimate the specificity of an absorbed enzyme-linked immunosorbent assay kit for Johne's disease (JD) when used in mature cattle populations resident in northern Australia. DESIGN: Blood samples were collected from beef cattle in northern Queensland, the Northern Territory and northern Western Australia, and from dairy cattle in northern Queensland. The specificity of a serological test for JD was estimated by testing the blood samples with an absorbed ELISA kit. Further samples were collected from cattle with positive ELISA results to determine the presence or absence of infection with Mycobacterium avium subsp paratuberculosis. PROCEDURE: During 1995 and 1996, blood, tissue and gut contents were collected from beef cattle at abattoirs in Queensland and the Northern Territory; and blood and faecal samples were collected from dairy cattle in herds assessed to be most at risk for JD in northern Queensland. The blood samples were tested using an absorbed ELISA kit. Tissues and gut contents from beef cattle that had positive ELISA results were cultured for M. avium subsp paratuberculosis, and tissues were examined histologically. Faecal samples from dairy cattle with positive ELISA results were cultured for M. avium subsp paratuberculosis. RESULTS: Estimates of specificity for this absorbed ELISA in mature northern Australian cattle were 98.0% (97.0 to 98.8%, 95% CI) in beef cattle, and 98.3% (96.7 to 99.3%, 95% CI) in dairy cattle. CONCLUSION: Estimates of specificity in this study were lower for beef cattle from the Northern Territory and northern Western Australia and for dairy cattle from northern Queensland than those quoted from studies on cattle in southern Western Australia. This should be considered when serological testing using the JD ELISA is carried out on northern Australian cattle.     

Comparison of blocking dot ELISA and competitive ELISA, using a monoclonal antibody for detection of bluetongue virus antibodies in cattle.

A blocking (B) dot enzyme-linked immunosorbent assay (ELISA), using a monoclonal antibody (mAb) against a group specific antigen of bluetongue virus (BTV) is described for the detection of BTV antibodies to BTV in cattle sera. Dots of BTV antigens were adsorbed to nitrocellulose (NC) strips and/or NC mounted in the windows of dipsticks. After blocking the remaining sites of the NC paper with milk powder solution and immersion in the test sample, the NC strips and dipsticks were exposed to mAb. Bound mAb was detected with peroxidase conjugated anti-mouse IgG (H and L). In the absence of anti-BTV antibody in the test sample, BTV antigen sites were reactive with mAb as indicated by a brown colored dot in the presence of the enzyme substrate, hydrogen peroxide and diaminobenzidine. In the presence of sufficient anti-BTV antibodies no color reaction was observed. The performance of these assays in detecting anti-BTV antibody in field blood eluate samples, prepared from whole blood dried on filter paper, from 395 bluetongue-free cattle in Canada and 635 sentinel cattle in Florida, USA, was evaluated and compared with the standard competitive (C) ELISA. The specificity of the dipstick B-dot ELISA was identical to that of the C-ELISA in testing of BT-free Canadian cattle but not in the testing of samples from the sentinel cattle in Florida, resulting in values of 100% diagnostic and 88.9% relative specificity, respectively. Based on the C-ELISA, the specificity of the NC strip B-dot ELISA was low and in the same order as that of the dipstick assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Foot-and-mouth disease: detection of antibodies in cattle sera by blocking ELISA.

A blocking ELISA was developed for the detection of antibodies to foot-and-mouth disease virus SAT1, SAT2 and SAT3 and for the quantification of antibodies on a single dilution of serum. The avidin-biotin system was used. The test was compared with the liquid-phase ELISA executed at the World Reference Laboratory for foot-and-mouth disease. It was found to have favourable logistics and combined high specificity with high sensitivity. The quantitative test using a single dilution of serum was resource saving and proved to be a reliable and precise method for the assessment of antibody levels.     

An ELISA optimized for porcine epidemic diarrhoea virus detection in faeces

Monoclonal antibodies to porcine epidemic diarrhoea virus (PEDV) membrane protein M were prepared and used for the comparative assessment of three blocking ELISA variants to detect PEDV. The competitive blocking ELISA (CB-ELISA) format showed the highest sensitivity, allowing detection of 10(2.5) plaque-forming units of PEDV/ml in culture medium. Its specificity was verified by inclusion of control samples containing transmissible gastroenteritis virus (TGEV) and rotavirus A in each analysis. Eighty porcine field samples of faeces obtained from 38 herds affected with diarrhoea were examined, and PEDV was found in 15 (19%) samples from 6 (16%) herds. The suitability of the CB-ELISA for the screening herds in epizootiologic situations is discussed.     

Development of a monoclonal blocking ELISA for the detection of antibody to Mycoplasma bovis in dairy cattle and comparison to detection by PCR

A monoclonal antibody blocking enzyme-linked immunosorbent assay (B-ELISA) was developed to detect antibodies to Mycoplasma bovis in cattle sera. The assay was highly specific and sensitive and there was no cross-reaction detected. This method revealed a high prevalence of antibodies (60%) to M. bovis in dairy cattle in North Queensland. The diagnostic potential of this B-ELISA for the detection of antibody to M. bovis was compared with its detection by PCR. There was a strong positive correlation between PCR and B-ELISA titers. Thus, the B-ELISA appears to be a valuable and reproducible tool in the serodiagnosis of M. bovis infection in cattle.     

新疆牛双芽巴贝斯虫病的流行病学调查

本研究使用牛双芽巴贝斯虫HSP20（exon）-iELISA检测方法,对2006-2008年新疆14个地州市的牛双芽巴贝斯虫病流行病学进行了调查。结果显示：（1）新疆存在着牛双芽巴贝斯虫病,且比牛巴贝斯虫病严重。在2006年采集的278份牛血清样品中,阳性血清11份,感染率为5.40%。2007年的532份牛血清样品中检出阳性血清25份,感染率为4.70%。在2008年的530份牛血清中检出阳性血清53份,感染率为7.17%;（2）2008年,发病疫区内牛双芽巴贝斯虫感染率高达30%;（3）牛双芽巴贝斯虫感染的地州市由2006年的8个扩大到2008年的13个;（4）新疆牛双芽巴贝斯虫病的感染率逐年上升,疫区面积不断扩大,流行区内感染率激增。这是新疆首次利用血清学方法对全疆范围内牛双芽巴贝斯虫病进行大规模的流行病学调查。