Time course changes in peripheral B-cell clonality in a Japanese Black bull with enzootic bovine leukosis.

A 38-month-old Japanese Black bull presenting with anorexia was given supportive treatment without improvement. Findings including bovine leukemia virus positivity and monoclonal B-cell proliferation strongly suggested the onset of enzootic bovine leukosis (EBL). Pathological findings confirmed the diagnosis of EBL. B-cell clonality were analyzed over time using pre-onset preserved genomic DNA at ages 6 months, 16 months, and 30 months. In the B-cell clonality analysis, two minor peaks at 140 and 220 bp were observed before onset, but another large peak at 175 bp appeared at the time of EBL diagnosis. Although the reason for the proliferation of an independent clone is unknown, detection of clonality abnormalities may lead to the detection of cattle at high risk of developing EBL.

Bovine Leukaemia Virus: Current Epidemiological Circumstance and Future Prospective.

Bovine leukaemia virus (BLV) is a deltaretrovirus that is closely related to human T-cell leukaemia virus types 1 and 2 (HTLV-1 and -2). It causes enzootic bovine leukosis (EBL), which is the most important neoplastic disease in cattle. Most BLV-infected cattle are asymptomatic, which potentiates extremely high shedding rates of the virus in many cattle populations. Approximately 30% of them show persistent lymphocytosis that has various clinical outcomes; only a small proportion of animals (less than 5%) exhibit signs of EBL. BLV causes major economic losses in the cattle industry, especially in dairy farms. Direct costs are due to a decrease in animal productivity and in cow longevity; indirect costs are caused by restrictions that are placed on the import of animals and animal products from infected areas. Most European regions have implemented an efficient eradication programme, yet BLV prevalence remains high worldwide. Control of the disease is not feasible because there is no effective vaccine against it. Therefore, detection and early diagnosis of the disease are essential in order to diminish its spreading and the economic losses it causes. This review comprises an overview of bovine leukosis, which highlights the epidemiology of the disease, diagnostic tests that are used and effective control strategies.

Cytokine TNF-α and its receptors TNFRI and TNFRII play a key role in the in vitro proliferative response of BLV infected animals.

Bovine leukemia virus (BLV) main host cells are B lymphocytes. Infected animals can be classified into high or low proviral load (HPL or LPL respectively), regarding the number of proviral copies infected lymphocytes they carry. After infection, there is an overexpression of several cytokines, particularly TNF-α, which has a delicate regulation mediated by receptors TNFRI and TNFRII; the first one involved with apoptosis, while the other stimulates cell proliferation. The study aimed to quantify TNF-α and its receptors mRNA expression, and in which extent in vitro proliferation was affected, in peripheral blood mononuclear cells (PBMC) from BLV-infected animals with different proviral loads, after the addition or not of synthetic TNF-α (rTNF-α) for 48 h. PBMC from BLV-infected animals showed spontaneous proliferation after 48 h in culture but did not show changes in proliferation rates after 48 h incubation in the presence of the rTNF-α. TNF-α mRNA expression after 48 h culture without exogenous stimulation was significantly lower, regardless of the proviral load of the donor, compared to non-infected animals. In the LPL animals, the expression of TNF-α mRNA was significantly lower with respect to the control group while the expression of TNFRI mRNA was significantly increased. The HPL animals showed a significant decrease in the expression of TNF-α and TNFRII mRNA respect to the control group. After 48 h incubation with rTNF-α, PBMC from infected animals had different responses: TNF-α and TNFRI mRNA expression was reduced in PBMC from the LPL group compared to the BLV negative group, but no differences were observed in PBMC from the HPL group. TNFRII mRNA expression showed no differences between HPL, LPL, and BLV negative groups, though HPL animals expressed 10.35 times more TNFRI mRNA than LPL. These results support the hypothesis that LPL animals, when faced with viral reactivation, present a pro-apoptotic and anti-proliferative state. However, complementary studies are needed to explain the influence of TNFRII on the development of the HLP profile. On the other hand, exogenous stimulation studies reinforce the hypothesis that BLV infection compromises the immune response of the animals.

Novel single nucleotide polymorphisms in the bovine leukemia virus genome are associated with proviral load and affect the expression profile of viral non-coding transcripts.

Bovine leukemia virus (BLV) infects bovine B-cells and causes malignant lymphoma, resulting in severe economic losses in the livestock industry. To control the spread of BLV, several studies have attempted to clarify the molecular mechanisms of BLV pathogenesis, but the details of the mechanism are still enigmatic. Currently, viral non-coding RNAs are attracting attention as a novel player for BLV pathogenesis because these transcripts can evade the host immune response and are persistently expressed in latent infection. One of the viral non-coding RNA, AS1, is encoded in the antisense strand of the BLV genome and consists of two isoforms, AS1-L and AS1-S. Although the function of the AS1 is still unknown, the AS1 RNA might also have some roles because it keeps expressing in tumor tissues. In the present study, we identified novel single nucleotide polymorphisms (SNPs) located in the AS1 coding region and indicated that individuals infected with BLV with minor SNPs showed low proviral load. To evaluate the effect of identified SNPs, we constructed infectious clones with these SNPs and found that their introduction affected the expression profile of AS1 RNA; the amount of AS1-L isoform increased compared with the wild type, although the total amount of AS1 RNA remained unchanged. Prediction analysis also suggested that the introduction of SNPs changed the secondary structure of AS1 RNA. These results explain part of the relationship between BLV expansion in vivo and the expression profile of AS1, although further analysis is required.

L233P mutation in the bovine leukemia virus Tax protein has impact on annexin A3 and type I collagen secretion by host cells.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL) and can be classified into two types based on the amino acid at position 233 in Tax protein, which probably plays crucial roles in leukemogenesis. We previously revealed that L233-Tax-expressing cells secreted chemoattractants for endothelial cells and formed significantly larger tumors accompanying neovascularization than P233-Tax-expressing cells in athymic mice. In the present study, comparative proteomic analysis of the culture medium of Tax-expressing cells revealed that annexin A3 and probably extracellular matrix protein 1 served as chemoattractants. Conversely, L233-Tax-expressing cells were impaired in the secretion of collagen alpha-1 (I) chain precursor, which participates in tissue tension homeostasis, leading to tumor mass development. The analysis also demonstrated that both L233-Tax- and P233-Tax-expressing cells had deficits in the secretion of potentially antiangiogenic molecules, including pigment epithelium-derived factor and collagen alpha-1 (VIII) chain, and they produced complement component 3, which might participate in tumor cell proliferation, metastasis, and immune evasion. These findings provided novel insights into prognostication of EBL and the function of Tax in leukemogenesis induced by BLV.

A target enrichment high throughput sequencing system for characterization of BLV whole genome sequence, integration sites, clonality and host SNP.

Bovine leukemia virus (BLV) is an oncogenic retrovirus which induces malignant lymphoma termed enzootic bovine leukosis (EBL) after a long incubation period. Insertion sites of the BLV proviral genome as well as the associations between disease progression and polymorphisms of the virus and host genome are not fully understood. To characterize the biological coherence between virus and host, we developed a DNA-capture-seq approach, in which DNA probes were used to efficiently enrich target sequence reads from the next-generation sequencing (NGS) library. In addition, enriched reads can also be analyzed for detection of proviral integration sites and clonal expansion of infected cells since the reads include chimeric reads of the host and proviral genomes. To validate this DNA-capture-seq approach, a persistently BLV-infected fetal lamb kidney cell line (FLK-BLV), four EBL tumor samples and four non-EBL blood samples were analyzed to identify BLV integration sites. The results showed efficient enrichment of target sequence reads and oligoclonal integrations of the BLV proviral genome in the FLK-BLV cell line. Moreover, three out of four EBL tumor samples displayed multiple integration sites of the BLV proviral genome, while one sample displayed a single integration site. In this study, we found the evidence for the first time that the integrated provirus defective at the 5' end was present in the persistent lymphocytosis cattle. The efficient and sensitive identification of BLV variability, integration sites and clonal expansion described in this study provide support for use of this innovative tool for understanding the detailed mechanisms of BLV infection during the course of disease progression.

Proteomic profiling of milk small extracellular vesicles from bovine leukemia virus-infected cattle.

Milk small extracellular vesicles (sEV) contain proteins that provide potential information of host physiology and immunology. Bovine leukemia virus (BLV) is an oncogenic virus that causes progressive B-cell lymphosarcoma in cattle. In this study, we aimed to explore the proteomic profile of milk sEV from BLV-infected cattle compared with those from uninfected cattle. Milk sEV were isolated from three BLV-infected and three uninfected cattle. Proteomic analysis was performed by using a comprehensive nanoLC-MS/MS method. Furthermore, gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were used to evaluate the candidates for uniquely or differentially expressed proteins in milk sEV from BLV-infected cattle. Proteomic analysis revealed a total of 1330 common proteins in milk sEV among BLV-infected cattle, whereas 118 proteins were uniquely expressed compared with those from uninfected cattle. Twenty-six proteins in milk sEV were differentially expressed proteins more than two-fold significant difference (p < 0.05) in BLV-infected cattle. GO and KEGG analyses indicated that the candidates for uniquely or differentially expressed proteins in milk sEV had been involved in diverse biological activities including metabolic processes, cellular processes, respond to stimulus, binding, catalytic activities, cancer pathways, focal adhesion, and so on. Taken together, the present findings provided a novel insight into the proteomes of milk sEV from BLV-infected cattle.

Establishment of a simplified inverse polymerase chain reaction method for diagnosis of enzootic bovine leukosis.

Enzootic bovine leukosis (EBL) is a malignant B-cell lymphoma of cattle caused by infection with bovine leukemia virus (BLV). It is defined by clonal and neoplastic expansion of BLV-infected B cells. Currently, multiple examinations are able to comprehensively diagnose this condition. Inverse polymerase chain reaction (PCR) is a useful method to determine retrovirus integration sites. Here, we established a simplified inverse PCR method, involving the evaluation of clonality and similarity of BLV integration sites, to clinically diagnose EBL, and we also assessed its reliability. We found that the novel BLV inverse PCR could detect clonal expansion of infected cells even if they constituted only 5% of the total number of cells, while not amplifying any fragments from BLV-uninfected cells, thus confirming its sufficient sensitivity and specificity for use in EBL diagnosis. Furthermore, 50 clinical cases of bovine leukemia were analyzed using BLV inverse PCR and other PCR-based methods, wherein our method most efficiently determined virus-dependent bovine leukemia, including unidentified clinical cases observed in a previous report. Following further clinical investigations to enhance its reliability, the proposed BLV inverse PCR method has the potential to be applied to EBL diagnosis.

Expression-based analysis of genes related to single nucleotide polymorphism hits associated with bovine leukemia virus proviral load in Argentinean dairy cattle.

In dairy cattle infected with bovine leukemia virus (BLV), the proviral load (PVL) level is directly related to the viral transmission from infected animals to their healthy herdmates. Two contrasting phenotypic groups can be identified when assessing PVL in peripheral blood of infected cows. A large number of reports point to bovine genetic variants (single nucleotide polymorphisms) as one of the key determinants underlying PVL level. However, biological mechanisms driving BLV PVL profiles and infection progression in cattle have not yet been elucidated. In this study, we evaluated whether a set of candidate genes affecting BLV PVL level according to whole genome association studies are differentially expressed in peripheral blood mononuclear cells derived from phenotypically contrasting groups of BLV-infected cows. During a 10-mo-long sampling scheme, 129 Holstein cows were phenotyped measuring anti-BLV antibody levels, PVL quantification, and white blood cell subpopulation counts. Finally, the expression of 8 genes (BOLA-DRB3, PRRC2A, ABT1, TNF, BAG6, BOLA-A, LY6G5B, and IER3) located within the bovine major histocompatibility complex region harboring whole genome association SNP hits was evaluated in 2 phenotypic groups: high PVL (n = 7) and low PVL (n = 8). The log2 initial fluorescence value (N0) transformed mean expression values for the ABT1 transcription factor were statistically different in high- and low-PVL groups, showing a higher expression of the ABT1 gene in low-PVL cows. The PRRC2A and IER3 genes had a significant positive (correlation coefficient = 0.61) and negative (correlation coefficient = -0.45) correlation with the lymphocyte counts, respectively. Additionally, the relationships between gene expression values and lymphocyte counts were modeled using linear regressions. Lymphocyte levels in infected cows were better explained (coefficient of determination = 0.56) when fitted a multiple linear regression model using both PRRC2A and IER3 expression values as independent variables. The present study showed evidence of differential gene expression between contrasting BLV infection phenotypes. These genes have not been previously related to BLV pathobiology. This valuable information represents a step forward in understanding the BLV biology and the immune response of naturally infected cows under a commercial milk production system. Efforts to elucidate biological mechanisms leading to BLV infection progression in cows are valuable for BLV control programs. Further studies integrating genotypic data, global transcriptome analysis, and BLV progression phenotypes are needed to better understand the BLV-host interaction.

Effectiveness of on-farm continuous flow high-temperature short-time pasteurization for inactivation of bovine leukemia virus in milk.

The effectiveness of on-farm continuous flow high-temperature short-time (HTST) pasteurization (i.e., 72°C for 15 s) for the inactivation of bovine leukemia virus (BLV) in milk was investigated with a sheep bioassay. Four sheep that had been inoculated with completely pasteurized milk containing approximately 3.4 × 107 BLV-infected peripheral blood mononuclear cells (PBMC) and treated by either HTST pasteurization or laboratory-scale low-temperature long-time (LTLT) pasteurization (i.e., 60°C for 30 min), remained negative for BLV for at least 17 weeks after inoculation. In contrast, all sheep inoculated with unpasteurized or inadequately pasteurized milk containing the same number of BLV-infected PBMC were tested positive for BLV and anti-BLV antibodies within 3 weeks after inoculation. These results suggest that on-farm continuous flow HTST pasteurization was equivalent value with inactivated BLV on the LTLT procedure and can effectively inactivate BLV in the milk. Therefore, on-farm HTST pasteurization of the pooled colostrum or milk used in automated feeding systems is likely to protect group-housed preweaned calves from BLV infection, thereby improving animal health on dairy farms.

Genetic variation in Japanese Holstein cattle for EBL development.

BACKGROUND: Infection with bovine leukemia virus (BLV), the causative agent for enzootic bovine leukosis (EBL), is increasing in dairy farms of Japan. The tendency of tumor development following BLV infection in certain cow families and bull lines has previously been described. We therefore hypothesized the existence of a genetic component which differentiates cattle susceptibility to the disease. RESULTS: We analyzed routinely collected large-scale data including postmortem inspection data, which were combined with pedigree information and epidemiological data of BLV infection. A total of 6,022 postmortem inspection records of Holstein cattle, raised on 226 farms served by a regional abattoir over 10 years from 2004 to 2015, were analyzed for associations between sire information and EBL development. We then identified statistically the relative susceptibility to EBL development for the progeny of specific sires and paternal grandsires (PGSs). The heritability of EBL development was calculated as 0.19. Similarly, proviral loads (PVLs) of progeny from identified sires and PGSs were analyzed, but no significant differences were found. CONCLUSIONS: These observations suggest that because EBL development in our Holstein population is, at least in part, influenced by genetic factors independent of PVL levels, genetic improvement for lower incidence of EBL development in cattle notwithstanding BLV infection is possible.

High positivity values for bovine leukemia virus in human breast cancer cases from Minas Gerais, Brazil.

Bovine leukemia virus (BLV) is a retrovirus that causes lymphoma in cattle worldwide and has also been associated with breast cancer in humans. The mechanism of BLV infection in humans and its implication as a primary cause of cancer in women are not known yet. BLV infection in humans may be caused by the consumption of milk and milk-products or meat from infected animals. Breast cancer incidence rates in Brazil are high, corresponding to 29.5% a year of cancer cases among women. In 2020, an estimated 66,280 new cases of breast cancer are expected, whereas in 2018 breast cancer has led to 17,572 deaths, the highest incidence and lethality among cancers in women in this country that year. BLV infection occurrence ranges from 60 to 95% in dairy herds. In addition, there are some regions, such as the Minas Gerais State, southeastern Brazil, where the population traditionally consume unpasteurized dairy products. Taken together, this study aimed to verify if there is a higher association between breast cancer and the presence of BLV genome in breast tissue samples within this population that consumes raw milk from animals with high rates of BLV infection. A molecular study of two BLV genes was carried out in 88 breast parenchyma samples, between tumors and controls. The amplified fragment was subjected to BLV proviral sequencing and its identity was confirmed using GenBank. BLV proviral genes were amplified from tumor breast parenchyma samples and healthy tissue control samples from women, revealing a 95.9% (47/49) and 59% (23/39) positivity, respectively. Our results show the highest correlation of BLV and human breast cancer found in the world to date within the population of Minas Gerais, Brazil.

Bovine leukemia virus detection and dynamics following experimental inoculation.

Bovine leukemia virus (BLV) infects more than 40% of the United States cattle population and impacts animal health and production. Control programs aiming to reduce disease prevalence and incidence depend on the ability to detect the BLV provirus, anti-BLV antibodies, and differences in blood lymphocyte counts following infection. These disease parameters also can be indicative of long-term disease progression. The objectives of this study were to determine the timing and to describe early fluctuations of BLV-detection by qPCR, ELISA, and lymphocyte counts. Fifteen Holstein steers were experimentally inoculated with 100 µL of a blood saline inoculum. Three steers served as in-pen negative controls and were housed with the experimentally infected steers to observe the potential for contract transmission. Five additional negative controls were housed separately. Steers were followed for 147 days post-inoculation (DPI). Infections were detected in experimentally infected steers by qPCR and ELISA an average of 24- and 36 DPI, respectively. Significant differences in lymphocyte counts between experimentally infected and control steers were observed from 30 to 45 DPI. Furthermore, a wide variation in peak proviral load and establishment was observed between experimentally infected steers. The results of this study can be used to inform control programs focused on the detection and removal of infectious cattle.

Development of an in situ hybridization assay using an AS1 probe for detection of bovine leukemia virus in BLV-induced lymphoma tissues.

Enzootic bovine leukosis (EBL) is a malignant B cell lymphoma caused by infection with bovine leukemia virus (BLV). Histopathological examination is commonly used for diagnosis of the disease, but observation of lymphoma alone does not confirm EBL because cattle may be affected by sporadic forms of lymphoma that are not associated with BLV. Detection of BLV in tumor cells can be definitive evidence of EBL, but currently, there is no technique available for such a purpose. In this study, we focused on a viral non-coding RNA, AS1, and developed a novel in situ hybridization assay for the detection of BLV from formalin-fixed paraffin-embedded (FFPE) tissues. RNA-seq analysis revealed that all examined B lymphocytes derived from clinical EBL abundantly expressed AS1 RNA, indicating a possible target for detection. The in situ hybridization assay using an AS1 probe clearly detected AS1 RNA in fetal lamb kidney cells persistently infected with BLV. The utility of this assay in clinical samples was assessed using three EBL-derived lymph node specimens and one BLV-negative specimen, and AS1 RNA was detected specifically in the EBL-derived tissues. These results suggest that AS1 RNA is a useful target for the detection of BLV from FFPE specimens of tumor tissues. This technique is expected to become a powerful tool for EBL diagnosis.

Development of a microchip electrophoresis-based, high-throughput PCR-RFLP method to type Tax 233 variants of bovine leukemia virus in Japan.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL). We used microchip electrophoresis in combination with automatic image analysis to develop a novel high-throughput PCR-RFLP to type the gene sequences that encode BLV Tax 233. This method revealed that 233L-Tax is more prevalent than 233P-Tax in cattle in Japan. The proportion infected with BLV carrying the gene encoding 233L-Tax was significantly higher in Holstein cattle than in Japanese Black cattle. Holsteins infected with BLV encoding 233L-Tax had higher proviral loads than did Holsteins infected with BLV encoding 233P-Tax and Japanese Blacks infected with BLV encoding 233L-Tax or 233P-Tax. The novel method developed in this study will be a useful tool for identifying cattle harboring BLV with a higher risk of EBL and viral transmission.

Changes in bovine leukemia virus serological status and lymphocyte count between dry-off and early lactation in Michigan dairy cows.

This study addresses how the serological status of bovine leukemia virus (BLV) and lymphocyte count fluctuate from dry-off to early lactation in dairy cattle. Very few studies have investigated how BLV antibody status and lymphocyte count of cows changes longitudinally during the lactation cycle. Blood samples were collected from dairy cattle (n = 149) on 5 commercial dairy herds in Michigan at dry-off, close-up, and 7 to 10 d after calving. Plasma was analyzed for anti-BLV antibodies using a BLV-ELISA and whole blood was analyzed for lymphocyte counts. We found that BLV seroprevalence increased from dry-off (38.9%) to close-up (43.6%), then slightly decreased from close-up to 7 to 10 d after calving (43.0%). However, the change in seroprevalence was only significant from dry-off to close-up. Cows of third or higher parity were more likely to seroconvert than cows of lower parity and had the highest ELISA-negative prevalence of BLV. Lymphocyte counts were significantly higher in ELISA-positive animals, but only among second and third or greater parity animals. These results indicate that the use of lymphocyte counts as a disease severity monitoring tool for BLV should differ by parity group. Future studies should investigate if changes in seroprevalence are due to new infections or natural changes in antibody concentrations as the cow prepares for colostrum production. More accurate lymphocyte guidelines to be used for monitoring the progression of BLV should be created that consider parity and lactation stage.

Phylogenetic Analysis of South African Bovine Leukaemia Virus (BLV) Isolates.

Bovine leukaemia virus (BLV) causes chronic lymphoproliferative disorder and fatal lymphosarcoma in cattle, leading to significant economic losses in the beef and dairy industries. BLV is endemic globally and eleven genotypes have been identified. To date, only Zambian isolates have been genotyped from Africa. Although high BLV prevalence has been reported in South Africa, there has been no molecular characterisation of South African BLV isolates. To characterise BLV isolates in South Africa for the first time, we investigated the phylogenetic relationships and compared the genetic variability of eight South African BLV isolates with BLV isolates representing the eleven known genotypes from different geographical regions worldwide. Phylogenetic analyses based on full-length and partial env sequences as well as full-length gag sequences revealed that at least two genotypes, genotypes 1 (G1) and 4 (G4), are present in cattle in South Africa, which is consistent with studies from Zambia. However, our analysis revealed that the G1 South African isolate is more similar to other G1 isolates than the G1 Zambian isolates whereas, the G4 South African isolates are more divergent from other G4 isolates but closely related to the G4 Zambian isolate. Lastly, amino acid sequence alignment identified genotype-specific as well as novel amino acid substitutions in the South African isolates. The detection of two genotypes (G1 and G4) in southern Africa highlights the urgent need for disease management and the development of an efficacious vaccine against local strains.

Herd management practices associated with bovine leukemia virus incidence rate in Michigan dairy farms.

The objective of this study was to identify associations between herd management practices and the incidence rate of bovine leukemia virus (BLV) infections in Michigan dairy herds. Previous management risk factor studies were of antibody prevalence rather than the rate of recent infections. Milk samples were collected from cohorts of cows on 112 Michigan dairy herds and tested for BLV using an antibody capture ELISA (n = 3849 cows). Cows were subsequently followed for an average of 21 months. Cows negative for anti-BLV antibodies and still present in their respective herds were retested by the same antibody capture ELISA to estimate within-herd incidence rates. The overall crude incidence rate was 1.46 infections per 100 cow-months at risk for the 1314 retested cows in 107 herds. The average within-herd incidence rate was 2.28 infections per 100 cow-months (range: 0 to 9.76 infections per 100 cow-months). A negative binomial regression model was used to identify herd management practices associated with the within-herd incidence rate. Results of the final multivariable model identified higher herd prevalence, milking frequency, needle reuse, as well as housing post-parturient cows separately, to be associated with increased incidence rate. Utilization of sand bedding for the lactating herd was found to be associated with decreased incidence rates. Results of this study suggest potential routes of BLV transmission which should be further investigated as disease control targets in ongoing control programs.

Detection of genotype 1 bovine leukemia virus from a C.schultzei pool: Do Culicoides spp. have a role on the transmission of bovine leukemia virus?

Bovine leukemia virus (BLV) is known as the etiological agent of Enzootic bovine leukosis (EBL), which is the most common neoplastic disease of cattle. While the major route of virus transmission is believed to be iatrogenic, BLV proviral DNA has been identified in biological materials, including nasal secretions, saliva, milk, colostrum, and semen, and in several insect species, including horses flies. However, insects' role in the natural transmission of BLV has not been clearly demonstrated. This study assessed the possible role of midges - Culicoides spp. - in BLV transmission. BLVs were genetically characterized and BLV infection seroprevelance was determined in 224 cattle sampled from 27 different small family herds in five different districts in Hatay province, southern Turkey. Out of the 25 Culicoides spp. pools, one (4.0%; 1/25) was a C.schultzei pool while 2.67% (6/224) of the sampled cattle were positive for BLV nucleic acid. The seroprevalance rates for the sampled herds and all sampled cattle were 7.40% (2/27) and 1.33% (3/224), respectively. According to the phylogenetic analysis, the sequences of the BLVs from the cattle (n = 6) and the one BLV-positive C.schultzei pool clustered on genotype 1 (G1) BLVs. Although these results do not reveal the exact role of Culicoides spp. or other midges flies in BLV transmission, the simultaneous presence of same substitions in BLVs from both cattle and a C.schultzei pool is noteworthy. Further studies on the env gene and other BLV gene regions detected from cattle and C.schultzei pools are ongoing to understand the possible epidemiological relationship between cattle and flies.

Dairy cattle with bovine leukaemia virus RNA show significantly increased leukocyte counts.

Infection with bovine leukaemia virus (BLV), a retrovirus, causes dysfunction of the immune system and can have a marked economic impact on dairy industries due to decreased milk production and reduced lifespan in affected dairy cattle. The presence of proviral DNA has been the major diagnostic indicator of BLV infection. However in the course of BLV infection, the viral genome can be dormant, without detectable gene expression, resulting in limited impact on infected animals. At present, there is limited knowledge regarding haematological indices in dairy cattle that could indicate activation of the BLV genome and suggest reactivated BLV infection. In this study, BLV infection and BLV genome reactivation were evaluated based on the presence of BLV DNA and BLV env gene transcripts, respectively. BLV RNA transcription was confirmed. Among 93 whole blood samples obtained from asymptomatic dairy cattle, the prevalence of BLV proviral DNA and transcripts was 93.5% (n = 87/93) and 83.9% (n = 78/93), respectively. Between groups with and without BLV, the mean counts of white blood cells and lymphocytes in whole blood were significantly associated with the presence of BLV RNA (P < 0.05), but not with BLV proviral DNA. These results shed light on the activation status of the BLV genome and should be taken into account when evaluating the possible impact of BLV on cattle.