Bovine leucosis virus contamination of a vaccine produced in vivo against bovine babesiosis and anaplasmosis

Contamination of a batch of tick fever (babesiosis and anaplasmosis) vaccine with bovine leucosis virus (BLV) was detected when a herd, in the final stages of an enzootic bovine leucosis (EBL) accreditation program, developed a large number of seropositive cattle following use of tick fever vaccine. Investigations incriminated a single calf used to produce Anaplasma centrale vaccine from which 13,959 doses were distributed. The failure of this calf to give a positive agar gel immunodiffusion (AGID) test before use was not fully explained. A total of 22,627 cattle from 111 herds receiving contaminated vaccine was tested to validate claims for compensation. Results showed infection rates of 62% and 51.8% in vaccinated dairy and beef cattle, respectively, compared with 6.1% and 1.5% in non-vaccinated cattle in the same herds. The results also indicated that infection did not spread from vaccinated to non-vaccinated in-contact cattle. Heavy reliance is now placed on purchase of calves for vaccine production from EBL accredited-free herds and on transmission tests from the calves to sheep to prevent a recurrence of contamination. The need for a BLV antigen detection test, with the sensitivity of the sheep transmission test but simpler and faster to perform, is evident.     

The suitability of ELISA for the detection of antibodies against Mycobacterium avium ssp. paratuberculosis in bulk milk samples from Rhineland-Palatinate

Paratuberculosis or Johne's Disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a notifiable disease in Germany which produces enormous economical losses in dairy farms. At present,there is no confirmed data about the actual number of infected livestock herds in Germany. A countrywide monitoring program to evaluate the prevalence in dairy herds would only be economically feasible on the basis of bulk milk testing. In this study, we evaluated two ELISA test kits (SVANOVIR Ptb-ELISA, IDEXX-M.pt. Milk test kit) for the detection of antibodies against MAP in bulk milk. First, the Paratuberculosis-status of the herd derived from the history of the farm was used as a gold standard. Paratuberculosis-negative farms were tested negative with each test, but paratuberculosis-positive or Paratuberculosis-serologically-positive farms were detected only in one case (Svanovir) or three cases (IDEXX), respectively. Even if inconclusive results are counted as positive, 82.9 % (Svanovir) or 80 % (IDEXX) of the paratuberculosis-positive or serologically paratuberculosis positive farms were not detected. Nevertheless, a re-validation of both ELISAs by means of ROC and TG-ROC analyses was attempted by searching for ideal cut-offs, optimised for bulk milk. If a high specificity was selected, no acceptable sensitivity could be reached.The best results were obtained using a sensitivity of 32.3 % at a specificity of 100 % (Svanovir). With a small change of the cut-off value, the sensitivity increased to still 57 %, but this reduced the specificity to 67 %. Similar results were obtained with the IDEXX-ELISA. We then evaluated the Svanovir-ELISA for the detection of bulk milk samples on the basis of the current paratuberculosis prevalence within 69 dairy herds from Rhineland-Palatinate using individual milk samples.When the bulk milk samples were tested in two different laboratories using the same ELISA, considerable differences in the results became evident. Nearly all samples were tested with a higher relative test result in one laboratory, which often led to differences in the classification of the prevalence levels.The estimated within-herd seroprevalences ranged between 0 % and 37 %.There was little agreement between the historical paratuberculosis herd status and the within-herd prevalence in milk serum, as reflected in a kappa-index of 0.146.To determine the sensitivity and specificity of the bulk milk ELISA by ROC and TG-ROC analysis, 116 bulk milk samples were used that had been obtained from the 69 dairy herds participating in the study. The optimal ratio of sensitivity (81 %) and specificity (77 %) relative to a "gold standard" was obtained when the cut-off was set at the 10 % level. These values for sensitivity and specificity were better than those obtained in an evaluation of the same ELISA in which the historical Paratuberculosis herd-status was used as a "gold standard." The results of this study question the suitability of the available ELISAs for bulk milk testing.Taking into account that the Svanovir-ELISA for individual milk samples has a sensitivity of 60 96% relative to the blood serum variant of the test, and that the latter has also a limited sensitivity due to the pathogenesis of paratuberculosis, the available test systems examined in this Study do not seem to be suitable for herd diagnosis by using bulk milk samples.     

The diagnosis of enzootic bovine leukosis

This paper reviews the clinical and virological diagnostic procedures for enzootic bovine leukosis (EBL). The clinical diagnosis must be always confirmed by a specific laboratory test for Bovine Leukaemia Virus (BLV). Many virological tests were proposed. The sensitivity of all the diagnostic methods is sufficient to do an early detection of a BLV infection on an individual base. Advantages of the highly sensitive methods like RIA and ELISA appear when the samples to be tested have naturally very low antibody titers (individual milk, bulk milk, pooled sera).     

Experimental and natural infection with the enzootic leukosis virus of cattle

A trial was performed with heifers at the age of six to seven months. The animals were experimentally infected with the lymphocytes of a virus-productive donor. Infection was produced in all the nine cases, as demonstrated by means of the positive syncytial test. As indicated by the results of the trial, the antibodies to the enzootic bovine leucosis virus (BLV) were produced soon after experimental infection. A high sensitivity of the serum-neutralization test and the ELISA method was demonstrated in this connection: by these methods, the antibodies were identified already two to three weeks after experimental infection whereas by the immunodiffusion test they could be detected only after five weeks. Twenty-four animals were exposed to natural contact infection. Within 270 days of the trial, the disease after contact was recorded only in one heifer out of the four that were in close contact with the experimentally infected animals. In this case, as compared with experimental infection, the antibodies were produced much later--after 85 to 93 days. Leucosis was recorded in none of the remaining animals. The reasons why such a favourable result was obtained were the thorough disinfection of the stables after blood collections and the strict observance of the aseptic conditions. The results of experimental infection in three cows were identical with those obtained in young cattle. In the experimentally infected dairy cows, antibodies in milk were determined by the ELISA method. As found, in milk the antibodies to BLV appear two to three weeks later than they do in serum. The ELISA method of BLV antibody detection can be used for the identification of infected animals in herds where enzootic bovine leucosis occurs.     

Cost-effectiveness of bulk-tank milk testing for surveys to demonstrate freedom from infectious bovine rhinotracheitis and bovine enzootic leucosis in Switzerland

In Switzerland, annual surveys to substantiate freedom from infectious bovine rhinotracheitis (IBR) and enzootic bovine leucosis (EBL) are implemented by a random allocation of farms to the respective survey as well as blood sampling of individual animals at farm level. Contrary to many other European countries, bulk-tank milk (BTM) samples have not been used for active cattle disease surveillance for several years in Switzerland. The aim of this project was to provide a financial comparison between the current surveillance programme consisting of blood sampling only and a modified surveillance programme including BTM sampling. A financial spreadsheet model was used for cost comparison. Various surveillance scenarios were tested with different sample sizes and sampling frequencies for BTM samples. The costs could be halved without compromising the power to substantiate the freedom from IBR and EBL through the surveillance programme. Alternatively, the sensitivity could be markedly increased when keeping the costs at the actual level and doubling the sample size. The risk-based sample size of the actual programme results in a confidence of 94,18 % that the farm level prevalence is below 0,2 %. Which the doubled sample size, the confidence is 99,69 % respectively.     

Control and eradication programme of enzootic bovine leucosis (EBL) from selected dairy herds in Turkey

Serum samples of 15,909 cattle from 31 dairy herds located in various regions of Turkey were tested for the presence of antibodies against bovine leucosis virus (BLV) using Agar Gel Immuno-diffusion technique (AGID). 48.3% (15/31) of the herds had seropositive animals and positivity rates were detected from 0.5-34.4% in these herds. In an EBL control/eradication programme all seropositive animals were culled in the infected herds. Thereafter, a total of 74,347 sera were tested for the presence of BLV specific antibodies. The serological results and detail of EBL control/eradication programme were shown in this paper.     

Evaluation of alternative methods for the detection of bovine leukaemia virus in cattle

AIM: To evaluate commercially available enzyme-linked immunosorbent assays (ELISAs) and the polymerase chain reaction (PCR) for their ability to detect antibodies against or nucleic acid of the bovine leukaemia virus (BLV), the causal agent of enzootic bovine leukosis (EBL), and to assess their usefulness in a national eradication programme. METHODS: Eighty-two well-defined sera (including 18 from an OIE reference laboratory) and 399 field sera from New Zealand cattle were tested in five ELISAs and the results compared with the agar gel immunodiffusion (AGID) test and electrophoretic immunoblotting (EIB) results. A polymerase chain reaction-based technique, which could detect BLV-RNA and proviral-DNA, was also evaluated on a subsample of the field cases. RESULTS: Two commercial ELISAs classified 99% of the defined sera correctly, with the other three ranging in their correct classification between 88% and 95%. The ELISAs agreed in their general classification on the majority of the 399 blood samples (91.7%), and with the AGID for more than 95 % of the sera. In a dilution series of the international reference serum E4, the highest dilution with a positive (or suspicious) result ranged from 1:80 to 1:5120. A dilution series of 202 field positive samples tested in the preferred ELISA detected 98% of positive sera at a 15 and 1: 10 dilution, reducing to 78% at a 1:80 dilution of the sera. Agreement between serological tests and PCR was poor, mainly due to failure of the PCR to detect a number of serologically positive animals. CONCLUSION: ELISA tests detected about 10% more reactors than the AGID and the EIB combined. Some ELISA-positive animals were not detected by PCR, raising doubts about the usefulness of PCR-based technology in EBL eradication programmes.     

The eradication experience of enzootic bovine leukosis from Lithuania

Before 1985 the situation regarding enzootic bovine leukosis (EBL) in Lithuanian cattle was described only haphazardly. In 1986 serological investigations were initiated together with an eradication programme. The EBL bovine leukosis virus (BLV) situation was monitored by the Institute of Immunology Vilnius University, national and regional veterinary laboratories. Starting in 1986 all EBL-positive cattle were separated from negative cattle into BLV-infected and BLV-free herds. To create the latter, calves were fed pasteurized milk. The seroprevalence in 1990 was 7.29%, but it steadily declined to 0.32% in 2006.     

Diagnosis of bovine brucellosis by enzyme immunoassay of milk

Enzyme-immunoassays using lipopolysaccharide as antigen were developed for the detection of bovine IgG1, IgG2 or IgA Brucella antibodies (Ab) in milk. The results of these tests were compared with those of the milk ring test (MRT) by analyzing 3212 bulk milk samples from farms located in regions where brucellosis is prevalent. Among the 105 herds detected by ELISA and/or MRT, 29 infected herds were detected by ELISA only. The 40 MRT-positive herds were also ELISA positive. Five herds became infected during the study and were detected by ELISA 15 days to 6 months prior to detection by MRT. The ELISA IgG1 titration (IgG1 ELISA) detected 92.8% of the herds found positive in the three ELISA assays. The concomitant use of IgA ELISA raised the sensitivity to 100% but slightly decreased the specificity. The IgG2 ELISA did not improve the diagnosis. The sensitivity of MRT and IgG1 ELISA was compared by testing successive dilutions in negative milk of 110 individual MRT positive milks samples. On average, IgG1 ELISA was 22 times more sensitive than MRT.     

Feasibility of serological bulk milk testing as a method for BVD surveillance

A commercial ELISA detecting antibodies against bovine viral diarrhoea Virus (BVDV) was analysed for its applicability for bulk-milk screening. Detection limits were analysed using native and concentrated milk samples (milk treated with rennet and ammonium sulfate precipitated) from 10 cows whose sera showed different reactivity levels in the ELISA and from two cows which gave birth to persistently infected calves during the last year. Further this and a second commercial ELISA were used to screen 591 randomly selected bulk-milk samples. To clarify discrepancies thirty-nine herds were included in a follow-up study. A second bulk-milk sample and serum samples from 10 young cattle of 6 to 28 month of age per herd were analysed for antibodies against BVDV. The results of this second testing and the detection of viremic animals in 4 herds confirmed the results from initial bulk-milk testing with both tests. The analysed test is suitable for bulk-milk testing although its application is limited by vaccination.     

Detection of Brucella species in the milk of infected cattle,sheep, goats and camels by PCR

One hundred and three milk samples were collected from 52 cows, 21 ewes, 18 goats and 12 camels. The animals tested positive to at least one of the following: (1) standard tube agglutination test (SAT); (2) Rose Bengal plate test (RBPT); (3) milk ring test (MRT). All milk samples were examined by culture and single-step polymerase chain reaction (PCR) techniques for detection of Brucella species. The PCR assay amplified Brucella-DNA from 29 bovine milk samples, 10 from sheep, 13 from goats and one from a camel. The direct culture method detected Brucella organisms from 24 samples of cows' milk, 12 from sheep, 10 from goats and failed to detect any Brucella organisms from camels' milk. PCR detected up to 100 colony forming units (CFU) of B. abortus per millilitre of milk in 100% of diluted milk samples, and 1000 CFU of B. melitensis from 70% of milk samples. Although the overall sensitivity of the PCR was higher than the culture method, it should be possible to increase the sensitivity to detect lower numbers of Brucella organisms in field samples. The speed and sensitivity of the PCR assay suggest that this technique could be useful for detection of Brucella organisms in bovine milk, as well as in sheep, goat, and camels milk.     

Use of pooled serum or milk samples for the epidemiological surveillance of bovine hypodermosis

An enzyme-linked immunosorbent assay (ELISA) was used on pooled serum and milk samples to determine whether hypodermosis could be detected where a larger sero-epidemiological survey was required. This study was undertaken to assess the potential of this assay for testing sera on milk samples, pooled from 10 cows, and determining the period of the year when detection was optimal. The sensitivity of the assay was determined by increasingly diluting a positive serum with pooled negative sera, from 1:10 to 1:100. The diagnostic lower limit of the assay requires at least two serological reactors within a herd of 100. The kinetic development and depletion of anti-Hypoderma antibody of individual and pooled sera or milk from 30 cows was evaluated from November to July. Anti-Hypoderma antibody levels of two groups of 8 calves, one control and one teated with ivermectin (Ivomec), were tested from October to June. These preliminary results indicate that an ELISA assay on serum or milk samples pooled from 10 cows can be used between February and April to evaluate the prevalence of hypodermosis within cattle herds in France, demonstrating the feasibility of using pooled serum already collected for bovine leucosis testing.     

虎红平板凝集试验与试管凝集试验检测奶牛布鲁菌病的比较

布鲁菌病主要采用凝集试验来检测,本试验用虎红平板凝集试验和试管凝集试验对江苏海安、江都、无锡和兴化地区的660头奶牛的血清样品进行平行检测,虎红平板凝集试验的阳性检出率为9.7%,试验管凝集试验的阳性检出率为5.5%;与后者相比,虎红平板凝集试验的敏感性为100%,特异性为95.3%,符合率为95.6%。这些数据表明,江苏地区某些奶牛场存在布鲁菌感染,虎红平板凝集试验可以用于布鲁菌病初步检测。     

‘Nano-immuno test’ for the detection of live <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subspecies <Emphasis Type="Italic">paratuberculosis</Emphasis> bacilli in the milk samples using magnetic nano-particles and chromogen

Early rapid detection of Mycobacterium avium subspecies paratuberculosis (MAP) bacilli in milk samples is the major challenge since traditional culture method is time consuming and laboratory dependent. We report a simple, sensitive and specific nano-technology based ‘Nano-immuno test’ capable of detecting viable MAP bacilli in the milk samples within 10 h. Viable MAP bacilli were captured by MAP specific antibody-conjugated magnetic nano-particles using resazurin dye as chromogen. Test was optimized using true culture positive (10-bovine and 12-goats) and true culture negative (16-bovine and 25-goats) raw milk samples. Domestic livestock species in India are endemically infected with MAP. After successful optimization, sensitivity and specificity of the ‘nano-immuno test’ in goats with respect to milk culture was 91.7% and 96.0%, respectively. Whereas, it was 90.0% (sensitivity) and 92.6% (specificity) with respect to IS900 PCR. In bovine milk samples, sensitivity and specificity of ‘nano-immuno test’ with respect to milk culture was 90.0% and 93.7%, respectively. However, with respect to IS900 PCR, the sensitivity and specificity was 88.9% and 94.1%, respectively. Test was validated with field raw milk samples (goats-258 and bovine-138) collected from domestic livestock species to detect live/viable MAP bacilli. Of 138 bovine raw milk samples screened by six diagnostic tests, 81 (58.7%) milk samples were positive for MAP infection in one or more than one diagnostic tests. Of 81 (58.7%) positive bovine raw milk samples, only 24 (17.4%) samples were detected positive for the presence of viable MAP bacilli. Of 258 goats raw milk samples screened by six diagnostic tests, 141 (54.6%) were positive for MAP infection in one or more than one test. Of 141 (54.6%) positive raw milk samples from goats, only 48 (34.0%) were detected positive for live MAP bacilli. Simplicity and efficiency of this novel ‘nano-immuno test’ makes it suitable for wide-scale screening of milk samples in the field. Standardization, validation and re-usability of functionalized nano-particles and the test was successfully achieved in field samples. Test was highly specific, simple to perform and easy to read by naked eyes and does not require laboratory support in the performance of test. Test has potential to be used as screening test to estimate bio-load of MAP in milk samples at National level.     

胶体金免疫层析检测牛结核γ干扰素方法的建立

牛结核病是一种重要的人畜共患病,尽管许多国家已经实施了牛群净化,但其仍然威胁人类的公共卫生健康。本研究利用抗牛γ干扰素特异性单克隆抗体,通过免疫金标记技术,建立了胶体金免疫层析试纸条检测牛γ干扰素的方法。研充结果证明,该方法对牛γ干扰素具有高度特异性,与其它动物干扰素没有交叉反应,检测牛γ干扰素的灵敏度与进口ELISA试剂盒相当。试纸条在室温保存6个月、4℃保存12个月,其特异性及灵敏度没有明显变化。对田间送检的23份全血经结核特异性刺激后进行检测,结果与进口结核ELISA试剂盒符合率为91．3％。研究表明,本研究建立的牛γ干扰素免疫层析检测方法具有特异、敏感、稳定、操作简单快捷等特点,适合用于牛结核的快速特异检测。     

Cost-benefit analysis of the control and cure of enzootic bovine leukosis, described in the example of a selected territory

Severe economic damage had been inflicted upon numerous industrialised dairy farms by enzootic bovine leucosis in a selected region with predominance of stepwise production. Initial contamination at the beginning of control action and sanitation had been as high as 98 percent. The annual damage, in monetary terms, had been 3,205,041. - Marks, according to calculations. Now, after eleven years of control and sanitation, eradication of enzootic bovine leucosis has come into sight. Even during these eleven years, control action yielded a benefit of 19,005,451. - Marks, that is 1,727,768.20 Marks per annum or 69. - Marks per cow and annum. It will be possible in forthcoming years to bring the cost factor down and, consequently, to further increase the benefit.     

An enzyme-linked immunosorbent assay for enzootic bovine leukosis virus antibodies

An enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to enzootic bovine leukosis (EBL) virus is described and its sensitivity compared with that of the agar gel immunodiffusion test (AGIDT) using 198 sera collected in Great Britain. There was 95 per cent agreement between the ELISA and AGIDT, when sera with positive/negative ratio (P/N) values of 1 . 5 or greater were considered positive. A total of 259 out of 264 sera (98 per cent) collected in Northern Ireland had P/N values of less than 1 . 5, the remaining sera having P/N values of 1 . 5 and 1 . 6. As Northern Ireland is clinically and serologically free of EBL infection it is proposed that sera with P/N values of 1 . 5 and 1 . 6, which account for approximately 3.5 per cent of the total sera tested, are considered doubtful and should be tested by another serological test.     

Lack of sequence variation in sporadic bovine leucosis in regions of tumour suppressor genes p53 and p16

Regions of the promoter and exons 5-8 of the tumour suppressor gene p53 were analysed in 25 cases of sporadic bovine leucosis. The study included 17 cases of juvenile leucosis, five cases of adult leucosis and three cases of skin leucosis. Exon 2 of tumour suppressor gene p16 was also investigated in the same samples. No sequence variations were present in the analysed areas of the genes. In p53, this fact represents a clear difference in comparison with enzootic bovine leucosis. In p16, no comparative data are available.     

Multiplex PCR for the diagnostic detection of Coxiella burnetii in cow's milk

A multiplex PCR based assay was developed for the highly sensitive and specific detection of Coxiella (C.) burnetii in cow's milk. The assay simultaneously amplifies a diagnostic target within the C. burnetii IS1111 sequence and a control target within the bovine CD18 gene. The internal PCR amplification control allows the discrimination of false negative results (single tube reaction failures) from negative results due to true absence of target sequences. In order to maximize the sensitivity of the assay, a sample preparation method including a centrifugation step to concentrate the bacterium was developed. In milk samples artificially contaminated with serial dilutions of C. burnetii, about four particles per ml could reproducibly be detected. The sensitivities of both assays, multiplex PCR and PCR with only a single pair of primers ('simplex' PCR), were observed to be similar.