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1.
A serologic subgroup of bovine leukemia virus (BLV) has not been identified, whereas genetic diversity among BLVs has been reported by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). To investigate the distribution of BLV provirus variants, 42 isolates from Argentina and Japan were examined by nested PCR for a segment of the env gene, followed by DNA sequencing. The nucleotide sequences were compared with other previously characterized BLV variants from different geographical areas (Belgium, France, Italy, North America, Australia, Japan and Argentina). The majority of analyzed segments had a tendency for nucleotide substitution without changing the amino acid. The constructed phylogenetic tree showed the relations and differences between proviruses and within each one. Most of the samples in Argentina formed one cluster. The samples in Japan, except one, also formed one cluster and some of them showed high homology with the isolates from Australia and the USA. Considering the sequence analysis of env PCR products of all Japanese and Argentine samples and comparing them with the other previously isolated sequences, the variation was up to 3.5% and was characterized geographically in each area.  相似文献   

2.
Analysis of the partial bovine leukaemia virus (BLV) env gp51 gene sequences obtained from three BLV strains isolated in three different regions of Brazil was carried out. The Brazilian BLV env gp51 sequences were compared with seven other corresponding sequences of BLV strains isolated in different countries and with consensus sequence as well. The obtained data point on qualitative and quantitative differences among the analysed strains as far as the occurrence of single point mutations is concerned. Two Brazilian strains show significantly higher mutation rate than other analysed strains. Amino acid analysis did not show, however, any substantial changes of the primary protein structure coded by well conserved region of BLV env gp51 gene. Based on the obtained data, the putative dendogram image of possible phylogenetic relations among the studied BLV strains is presented as well.  相似文献   

3.
Enzootic bovine leukaemia (EBL) which is caused by the bovine leukaemia virus (BLV) still plays a remarkable role despite a significant success in sanitation programmes. In the Federal Republic of Germany it was not possible to eradicate the disease until today. Sporadically during slaughter or necropsy of cattle neoplastic lesions of the lymphatic tissues are observed that need to be clarified with regard to BLV as etiological agent. Due to the fact that in most instances no serological data are available from the respective animals and blood drawings from the original holdings are not easy to obtain the polymerase chain reaction (PCR) opens new avenues as supplementary diagnostic tool to test unfixed lymphatic tissues for the presence of BLV proviral DNA. Lymph node tissues from 10 naturally or experimentally BLV-infected cattle, which have been monitored virologically and serologically, and tissues from 4 negative animals were processed, DNA was extracted and subjected to PCR to amplify BLV env gene specific sequences. The results show that in cattle with BLV-induced leukosis as well as in cattle, which were clinically healthy and unsuspicious at slaughter or at post-mortem, either with persistent lymphocytosis (PL) or without, BLV proviral DNA could be detected easily in samples of lymphatic tissues and in high concordance with serological data. In this article data from the National and OIE reference laboratory for EBL at the Friedrich-Loeffler-Institut (FLI, Germany) are presented. Elaborated laboratory protocols for processing of tissue samples and performing of BLV-PCR are recommended.  相似文献   

4.
With the aim of achieve a better understanding of the epidemiology and distribution of bovine leukaemia virus (BLV) infection in Chile, we assessed the suitability of using DNA isolated from the leukocyte fraction of bulk milk samples to carry out PCR-RFLP and DNA sequence analysis. The env fragment of BLV was successfully amplified from 33 serologically positive bulk milk samples collected from different geographical areas in the south of Chile. Restriction analysis allowed to classify 17 isolates within the Australian subgroup and 16 within the Belgium subgroup. DNA sequence and multiple alignment analysis of eight Chilean isolates showed a significantly higher frequency of single and double nucleotide substitutions. Most of these mutations were non-silent, resulting in changes at the protein level in several important epitopes of gp51. The Chilean sequences and 59 BLV env sequences available at GenBank, were subjected to a phylogenetic analysis, resulting in four different clusters. The groups identified were not related to those previously defined by restriction analysis. Chilean isolates were included in two different clusters and were genetically not related to isolates collected from neighbouring countries. Considering our results we can conclude: (i) bulk milk samples are suitable to identify the presence of BLV allowing epidemiological and genetic studies to be conducted on large geographical areas; (ii) at least four different genetic groups of BLV were identified by phylogenetic analysis, with Chilean isolates included in two different sub clusters.  相似文献   

5.
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.  相似文献   

6.
Polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) is commonly used for genotyping bovine leukemia virus (BLV) in slaughterhouses. However, unclassified BLV genotypes have been sporadically reported. To assess the current status of BLV genetic characterization in cattle, PCR-RFLP was performed on blood samples of 170 cattle (84 Japanese Black, 60 Japanese Black x Holstein, and 26 Holstein) from 17 farms (5 prefectures) at a slaughterhouse in Aichi Prefecture in 2019. A total of 65 samples (38.2%) were BLV positive, and genotype 1 was the most predominant (56/65 samples), followed by genotypes 3 (6 samples) and 5 (1 sample), and two unclassified samples. No relationship between the genotypes and breeds was observed. Sequence and phylogenetic analyses demonstrated that unclassified BLV genotypes clustered with genotype 1 sequences were, therefore, not new genotypes.  相似文献   

7.
OBJECTIVE: To determine the subtypes of feline immunodeficiency virus (FIV) present in the domestic cat population in Melbourne. METHODS: Blood samples were collected from 42 cats that had serum antibodies against FIV. DNA was extracted and subjected to polymerase chain reaction (PCR) to amplify variable regions of the envelope (env) and group specific antigen (gag) genes of FIV. PCR products were directly sequenced or sequenced after cloning when direct sequencing yielded ambiguous results. Phylogenetic analysis was performed and comparisons made with representative sequences of different subtypes. RESULTS: The variable region of the env gene was successfully amplified by PCR from 41 of the 42 cats. All 41 were found to cluster with subtype A env sequences. The variable region of the gag gene was successfully amplified by PCR from all 42 cats. Forty-one were found to cluster with subtype A gag genes and one was found to cluster with subtype B sequences, suggesting that it may be derived from a recombinant env A/gag B virus. CONCLUSIONS: Subtype A is the predominant FIV type in Melbourne, although a subtype A/B recombinant was identified in the population of FIV positive cats. These results of env gene analysis were similar to those in a previous Australian study, suggesting that subtype A predominates in Australia. The results of the gag gene analysis show the importance of analysing multiple areas of the FIV genome when assigning FIV subtypes. Comparison with other major urban centres may provide useful information about the phylogenic diversity of FIV in Australia.  相似文献   

8.
A polymerase chain reaction (PCR)-based detection system was established to identify the presence of bovine leukosis virus (BLV) DNA in bovine semen. Seventy-nine bulls were included in the study. Serum, peripheral blood leukocytes, and semen were collected from each of the 79 bulls. The BLV-specific antibody was detected in serum by agar gel immunodiffusion and viral DNA in blood and semen by PCR. Serologically, 29 of the 79 bulls were BLV positive. Twenty-seven of the 29 seropositive bulls and 1 of the seronegative bulls had BLV DNA in peripheral blood leukocytes. All 79 bulls tested PCR negative for the presence of BLV in semen. This data is strong evidence that properly collected semen from BLV seropositive bulls will not contribute to dissemination of this viral infection.  相似文献   

9.
Concerns about retroviruses in livestock and products derived from them have necessitated the development of tests to detect the bovine leukemia virus (BLV) in blood and milk from cattle. Dairy cattle (n = 101) from 5 different geographical areas were used for this study. A nested polymerase chain reaction (PCR) identified 98% of BLV seropositive cattle (n = 80) from blood and 65% from milk, whereas real-time PCR detected 94% of BLV seropositive cattle from blood and 59% from milk. Bovine leukemia virus was also detected by PCR in approximately 10% of seronegative cattle (n = 21), most likely because of early detection before seroconversion.  相似文献   

10.
Twenty-seven cattle with lymphoma and 46 cows from a known bovine leukemia virus (BLV)-infected herd were tested for anti-BLV antibody by the agar gel immunodiffusion (AGID) test and an enzyme-linked immunosorbent assay (ELISA). The polymerase chain reaction (PCR) and Southern hybridization were used to detect BLV provirus in the tumor DNA of the 27 cattle with lymphoma. The PCR was used to detect BLV provirus in the peripheral blood mononuclear cell DNA of the 46 normal known-exposed cattle. Two presumed false negative AGID test results compared to ELISA were found. Of ten cattle three years of age or less with "sporadic" forms of lymphoma, four had BLV provirus in tumor DNA, detectable by PCR. In two of these four, BLV provirus was clonally integrated based on digestion of tumor DNA with restriction enzymes followed by Southern hybridization. The BLV provirus was not detected by PCR in 5 of 17 cattle with "enzootic" lymphoma and two of these five were seronegative. Among normal BLV-exposed cows, 6.5% (3 of 46) were serologically positive and PCR negative; serologically negative and PCR positive cows occurred with the same frequency. Serological and PCR test results, when considered in all cattle (n = 73), had a concordance rate of 83.6%. Discordant test results occurred with approximately equal frequency between serologically positive and PCR negative (7 of 73, 9.6%) and serologically negative and PCR positive (5 of 73, 6.8%) groups. These data suggest that the role of BLV in some "sporadic" bovine lymphomas, previously unassociated with BLV, should be reexamined. The BLV provirus was not demonstrable in the tumor DNA from five adult cattle with lymphoma, suggesting that BLV may not be the etiological agent in all adult bovine lymphomas. The findings of persistently seronegative PCR positive and seropositive PCR negative cattle indicate that further work is needed to more fully understand the host-virus interaction. Present serological screening methods may not have sufficient sensitivity for determining BLV status in some circumstances.  相似文献   

11.
OBJECTIVE: To evaluate the use of a polymerase chain reaction (PCR) assay in detecting bovine leukosis virus (BLV) in adult dairy cows. DESIGN: Prospective study. ANIMALS: 223 adult dairy cows. PROCEDURE: Cows were tested for BLV status by use of an ELISA and a PCR assay. Sensitivity, specificity, predictive values of positive and negative tests, and the percentage of cows correctly classified by PCR assay were calculated. Ninety-five percent confidence intervals were calculated for sensitivity and specificity. RESULTS: Sensitivity and specificity were 0.672 and 1.00, respectively. Prevalence of BLV in this herd was 0.807. Predictive value of a positive test was 1.00, and predictive value of a negative test was 0.421. The percentage of cows correctly classified by PCR assay was 73.5%. CONCLUSIONS AND CLINICAL RELEVANCE: A positive PCR assay result provided definitive evidence that a cow was infected with BLV. Sensitivity and negative predictive value for PCR assay were low. Consequently, PCR assay alone is unreliable for routine detection of BLV in herds with high prevalence of the disease.  相似文献   

12.
Nine percent of xenogeneic hybridomas originating from a bovine leukemia virus (BLV)-infected cow secreted monoclonal IgM antibodies with multispecific reactivity. Similar reactivity was evident in some antibodies with an unusually long (> 50 amino acids) third complementarity-determining region of the heavy chain. Electron microscopy of hybridomas demonstrated the presence of c-type virus particles consistent with polymerase chain reaction detection of BLV env gene. Some hybridomas contained dilated rough endoplasmic reticulum and cisternae filled with moderately electron-dense granular substance compatible with plasma cells at presecretory stage. The number of chromosomes in xenogeneic hybridomas corresponded to the sum total of mouse and bovine chromosomes. None of the hybridomas showed polyploidy. The immunochemical and genetic analysis of stable bovine immunoglobulin-secreting xenogeneic hybridomas confirms that BLV infection causes polyclonal B cell activation regardless of antigen specificity. Presence of c-type particles in hybridomas suggests that T cell-derived cytokines are not required for sustained BLV expression.  相似文献   

13.
本试验通过PCR技术获得了安徽省地方品种五华鸡禽白血病病毒J亚群(avian leukosis virus subgroup J,ALV-J)env部分基因序列ALV-J-env1,并将该序列与GenBank数据库中登录的8条env基因相应序列进行了比对分析。结果表明,五华鸡已经感染了J亚群禽白血病,且部分鸡个体已经发病。通过分析可见,ALV-J-env1与所比较的基因序列同源性介于94.2%~96.6%之间,说明不同毒株之间的env基因有一定变异。但与ALV-J-env1同源性最高的是来自于中国ALV-J毒株的HQ425636和HM235665基因序列,且在进化树中聚集为一组,暗示它们之间亲缘关系较近,由共同的毒株进化而来。与ALV-J-env1同源性最低的是来源于马来西亚毒株的AY312965基因序列,遗传进化分析也进一步证实,两者之间亲缘关系较远。  相似文献   

14.
A total of 59 monoclonal antibodies (mAbs) specific against the bovine leukaemia virus (BLV) using different antigen preparations was produced. The five antigen preparations for immunizing BALB/c mice were: live cells (CEL), sonicated and ultracentrifuged cells (SOC), cell lysates (LYS), semi-purified BLV (PV), and formalin-treated cells (FOR) from two cell lines permanently infected with BLV (FLK-BLV and BLV-bat2). These viral component presentations were selected to obtain mAbs against specific BLV proteins: located on the cell surface (FOR and CEL), in free virus particles (PV) and intracellular viral proteins (SOC and LYS). Two antigen preparations (SOC and LYS) were lethal to the mice following the intravenous and intrasplenic routes. Six fusions were performed in this study that rendered specific antibodies against BLV. The highest number of hybridomas was produced with SOC; however, the majority of the hybridomas produced (> 90%) were against cellular proteins. Even though immunization with PV gave the lowest number of hybridomas, the majority of them were specific against BLV. Based on the reactivity of the mAbs in Western blot (WB), we classified the mAbs into five groups, namely anti-gp51SU (39 mAbs), anti-gp30TM (six mAbs), anti-Pr72env (nine mAbs), anti-Pr66gag-pro (one mAb) and anti-Prgag (four mAbs). A very high percentage of the mAbs produced (48 of 59) reacted with gp51SU, suggesting that this is the most immunogenic and accessible BLV protein presented in the different antigen preparations. The majority of our mAbs recognized more than one band in WB, suggesting that, aside from reacting with mature proteins, the mAbs also recognized viral precursors.  相似文献   

15.
We examined whether Bovine leukemia virus (BLV) was transmitted by rectal palpation using a common sleeve between a BLV-infected cow and BLV-negative steers. Three of four steers developed antibodies against BLV as determined by agar-gel immunodiffusion (AGID) test between 7 to 10 weeks after the first rectal palpation using common sleeves from BLV-infected cow. In the steers, BLV proviral DNA were detected by PCR 1 to 5 weeks earlier than detection of the antibodies by the AGID test. Our experiments demonstrated that rectal palpation is a potential cause of BLV spread in herds and that detection of BLV proviral DNA in cattle by PCR is useful screening test for early diagnosis of BLV infection.  相似文献   

16.
A double polymerase chain reaction (PCR) assay has been devised for the direct detection of bovine leukemia virus (BLV). The assay was directly performed on blood leukocytes, avoiding the DNA-purification procedures. The PCR products were identified by gel-electrophoresis and the specificity of the test was confirmed by hybridization with a biotinylated oligonucleotide probe. When testing the sensitivity of PCR, less than eight genome copies of the provirus were detected in the background of two million negative lymphocytes. In a BLV infected herd 22 animals of various age groups were examined by the indirect (serological) diagnostic tests of agar-gel immunodiffusion and indirect ELISA as well as by the direct detection method of PCR. The tests were repeated at monthly intervals on five occasions. When examining the specimens from cows and heifers, a close agreement was found between the results of the various methods. The newborn calves, which were the offspring of BLV infected mothers, were consequently negative in PCR throughout the experimental period. However, in the indirect tests the calves were positive during the first samplings and became negative only around four months of age. Since the indirect tests can not discriminate infection from colostral immunity, PCR proved to be a useful complementary assay for the safe diagnosis of BLV infection in young calves.  相似文献   

17.
[目的]了解大庆市某牧场牛白血病病毒（BLV）感染情况。[方法]随机选取大庆市某规模化牧场泌乳期奶牛107头,采用ELISA方法进行BLV血清流行病学调查,并采用荧光定量PCR方法加以验证。[结果]ELISA检测结果阳性率为31.78%（34/107）,荧光定量PCR检测结果阳性率为37.38%（40/107）,两种检验方法的阳性重合率为85.00%;BLV高载量牛占总数的26.17%,低载量牛占总数的11.21%。[结论]调查结果表明,该牧场奶牛存在一定程度的BLV感染,在牛群中传播的风险较高。  相似文献   

18.
237 cattle of a dairy herd in Syria were tested for anti-BLV antibody by the ELISA. 194 animals were additionally examined by the agar gel immunodiffusions test (AGID) on BLV antibodies and 100 by polymerase chain reaction (PCR) for BLV provirus. BLV specific antibodies were determined by means of AGID and ELISA at 62.9% and 69.2% of the examined animals, respectively. Using the PCR method the BLV provirus was detected in 89% of the investigated cattle. Only one ELISA seropositive animal was negative for BLV provirus. The results show the high BLV contamination of this herd and lead to the presumption of wide spread enzootic bovine leukosis in Syria. In the case of the diagnosis of BLV-infection, the PCR-technique compared to the serological tests proved to be much more sensitive. By the detection of BLV antibody, the ELISA showed a higher sensitivity than the AGID and in this way, is advisable as a method of choice for screening investigations. Restriction enzyme and sequence analysis of PCR-amplificates demonstrate that different BLV provirus variants (A, B and C) in the examined herd occur, where the variant C which a high similarity to an Australian BLV provirus isolates showed, occurred most frequently at 92.5%.  相似文献   

19.
Analysis of the partial bovine leukaemia virus (BLV) gp51 gene sequences obtained from five BLV strains isolated in different regions of Iran and BLV-FLK strain was carried out. The Iranian BLV gp51 sequences were compared with seven other corresponding sequences of BLV strains isolated in different countries. Nucleotide sequence analysis showed a variability of 0.003–5.1% and the phylogenetic tree constructed revealed three clusters. The first cluster included French, German and FLK-BLV samples; the second cluster included four Iranian samples; and the third cluster included Australian, Korean, Japanese, Brazilian and Belgian reference strains and one Iranian sample. Iranian samples were had significantly similarity to European and Australian samples.  相似文献   

20.
BACKGROUND: The relation between calf bovine leukosis virus (BLV) infection status and colostrum ingestion is unclear. Two conclusions have been drawn from previous studies. One suggests that colostrum ingestion transmits BLV to neonatal calves. The second suggests that colostral antibodies are protective. HYPOTHESIS: Colostrum from BLV-positive cattle is protective in naturally exposed calves. ANIMALS: Twelve colostrum-deprived Holstein calves and 20 colostrum-fed Holstein calves born to BLV-infected cows. METHODS: Prospective study. Colostrum-deprived calves were tested weekly by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) tests for BLV antibody and provirus for 12 weeks or until the animal became positive for BLV infection. Colostrum-fed calves were fed colostrum derived from BLV-positive cows. Thereafter, ELISA and PCR tests for BLV antibody and provirus were performed every other week until 2 consecutive negative ELISA tests or 1 positive PCR test was achieved. The proportion of calves that converted to BLV-positive status was calculated for each group and compared between groups by using the Fisher exact test. RESULTS: Four of 12 colostrum-deprived calves (33%) became BLV positive, whereas 0 of 20 colostrum-fed calves (0%) became BLV positive. The proportion of calves that became infected was significantly higher in the colostrum-deprived group (P = .014). CONCLUSIONS AND CLINICAL RELEVANCE: Calves born to BLV-positive cows are exposed during parturition, and a proportion of these calves will become infected with BLV. Administration of colostrum from BLV-positive cows greatly decreases the risk of infection.  相似文献   

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