Bovine Leukemia Virus Infected Animals Research Articles

Removing bovine leukemia virus–infected animals with high proviral load leads to lower within-herd prevalence and new case reduction

Bovine leukosis is prevalent in the North American dairy industry and its effect on animal health and production is widely documented. However, not all bovine leukemia virus (BLV) infected animals transmit the virus equally. Animals with high BLV proviral loads (HPL) are associated with higher transmission risks and therefore, their removal may reduce transmission and eventually within-herd prevalence. We aimed to evaluate the impact of selectively removing HPL cows on the within-herd BLV prevalence and incidence rate of BLV infection in 10 dairy herds. Annual blood and/or milk samples were collected from adult cows over 3 years. Positivity with BLV were determined by ELISA tests and proviral loads in blood of BLV-positive animals were estimated with BLV SS1 quantitative PCR assays. Herd managers were encouraged to consider the proviral load when making culling decisions and implement BLV control practices. High proviral load cows had the highest relative risk of removal indicating the farmers prioritized HPL cows for culling. The within-herd BLV prevalence decreased significantly in 4 herds whereas BLV incidence rate decreased in 9 herds. Over the 3 years, the proviral load demonstrated a relatively stable level, suggesting a single proviral load test in an adult cow may suffice to make culling decisions.

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 48h. PBMC from BLV-infected animals showed spontaneous proliferation after 48h in culture but did not show changes in proliferation rates after 48h incubation in the presence of the rTNF-α. TNF-α mRNA expression after 48h 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 48h 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.

Impact of bovine leukemia virus infection on beef cow longevity

Bovine leukosis is a chronic lymphoproliferative disorder caused by bovine leukemia virus (BLV). Previous studies estimate that 38 % of cow-calf beef herds and 10.3 % of individual beef cows in the US are BLV seropositive. About 70 % of BLV infected animals are asymptomatic carriers of the virus, while less than 5% develop lymphosarcoma, the leading reason for carcass condemnation at the US slaughterhouses. Studies provide evidence that BLV infection leads to decreased immune function making animals more vulnerable to other diseases, which could shorten their productive lifespan and increase economic losses in the cattle industry. BLV seropositive dairy cows are reportedly more likely to be culled sooner compared with their uninfected herd mates. Beyond simple prevalence studies, little is known about the impact of BLV infection in beef cattle production or specifically on beef cow longevity. Our objective was to determine the association between BLV infection and cow longevity in beef cow-calf operations. Twenty-seven cow-calf herds from the Upper Midwest volunteered to participate in this study. Female beef cattle (n = 3146) were tested for serum BLV antibodies by ELISA. A subsample of 648 cows were also tested for BLV proviral load (PVL). Culling data was collected for the subsequent 24 months. Twenty-one herds (77.7 %) had at least one BLV-infected animal, and 29.2 % (930/3146) of tested animals were BLV seropositive. Of the BLV-positive cows, 33.7 % (318/943) were culled compared with 32.1 % (541/1682) of the seronegative cows. BLV status did not affect cows' longevity within herds (P = 0.062). However, cows with high BLV PVL had decreased survival within the herd compared with ELISA- negative cows (P = 0.01). Overall, infection with BLV did not impact beef cow longevity unless the disease had progressed to a point of high BLV PVL.

Changes in blood chemistry values of the cattle with associative course of leukemia and brucellosis

This paper describes the results obtained when the characteristics of metabolic process changes in leukemia, including brucellosis-complicated leukemia, were studied. To do it, 50 blood serum samples were taken from cows with specific antibodies against bovine leukemia virus (BLV) under the results of immunological diffusion reactions (IDR), indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA). All samples were serologically tested to detect a number of bacterial infections resulted in a possibility to establish that the BLV carrier state in most animals is combined with a bacterial infection, especially brucellosis (46%), chlamydia (20%), paratuberculosis (12%) and campylobacteriosis (8 %). At the next stage, 3 groups of 10 animals each were formed to study the metabolic process level, i.e. clinical healthy animals with no specific antibodies revealed during diagnostic tests for leukemia and other infections (Group 1); BLV carriers (Group 2); brucellosisand virus carrier animals (Group 3). Analysis of the blood chemistry values obtained for the experimental groups showed an uneven path of changes, especially for the protein and fat metabolism parameters. The difference was in albumin and cholesterol concentrations reduced in BLV infected animals, while their level was significantly increased in animals with leukemia associated with brucellosis, on the contrary.

Bulls as a source of bovine leukemia virus during natural breeding

Bovine leukosis is a chronic lymphoproliferative disorder in cattle caused by the deltaretrovirus, bovine leukemia virus (BLV). Most BLV infected animals remain asymptomatic and act as carriers of the virus; 30 to 40% of infected cattle develop a persistent lymphocytosis, while less than 5% progress to lymphosarcoma. The major route of virus transmission is believed to be iatrogenic through procedures that permit the transfer of blood between cattle. Proviral DNA has been identified in nasal secretions, saliva, semen, and smegma. Natural transmission through these secretions has not been clearly demonstrated. Our research group has identified the use of bulls in dairy herds as a risk factor for BLV infection at the herd level, and has also shown a high prevalence ( 45%) of infected beef bulls in Michigan. Natural service is still used in half of dairy operations across the US, and is the most commonly used breeding method (approximately 90%) in beef cattle herds in the US. Given these factors, there may be risk of BLV transmission during natural breeding via smegma or blood transfer following trauma to the penis, vulva, and vagina. The overall goal of our research team is to develop cost effective strategies to reduce the transmission of BLV in cattle herds. The objective of this study is to assess the risk of BLV transmission by breeding bulls during natural breeding.

Breeding beef bulls as a source of BLV transmission

Bovine leukosis is a chronic lymphoproliferative disorder caused by bovine leukemia virus (BLV) that leads to economic losses in the beef and dairy industries. The USDA NAHMS Beef 1997 study estimated that 38% of cow-calf beef herds and 10.3% of individual adult cows in the US are BLV seropositive. Most BLV infected animals are asymptomatic carriers of the virus while a proportion of infected cattle develop lymphocytosis (30-40%) and lymphosarcoma (5-10%). The major route of virus transmission is iatrogenic through the transfer of blood from infected cattle. BLV proviral DNA has also been identified in nasal secretions, saliva, milk, colostrum, and semen, which could serve as potential sources for virus transmission. In the US, natural service accounts for 90% of the breeding in beef cow-calf operations. During natural breeding, minor genital tract trauma resulting from copulation may lead to blood transfer and transmission of BLV. Alternatively, BLV transmission may occur via transfer of infected secretions including semen and smegma during copulation. Little is known about the prevalence of BLV in breeding bulls and few studies have evaluated semen or smegma as a potential route of BLV transmission. The objectives of this study were to 1) determine the prevalence of BLV in breeding bulls presented for breeding soundness exams (BSE) at Michigan State University (MSU), 2) compare lymphocyte counts in BLV positive and negative bulls, and 3) evaluate the presence of BLV proviral DNA in bull smegma and semen.

Co-infection with Mycobacterium bovis does not alter the response to bovine leukemia virus in BoLA DRB3*0902, genetically resistant cattle

High proviral load (HPL) profile in bovine leukemia virus infected animals poses increased risk of transmission, and development of HPL or low proviral load (LPL) profile may be attributed to host genetics. Genetic resistance and susceptibility has been mapped to the Major Histocompatibility Complex class II DRB3 gene (BoLA DRB3). The aim of this work was to determine the effect of Mycobacterium bovis infection on certain virological and host immunological parameters of BLV experimental infection. Twenty-six Argentinian Holstein calves carrying the resistance-associated marker allele BoLA DRB3*0902, susceptibility-associated marker allele BoLA DRB3*1501, or neutral BoLA DRB3 alleles, exposed to M. bovis were used. Twenty calves were inoculated with BLV, three were naturally infected and other three were BLV-negative. Seven from twenty six (27%) of the animals resulted positive to the PPD test. The proviral load, absolute leukocyte and lymphocyte counts, time to seroconversion, antibody titer against BLV, and viral antigen expression in vitro at various times post inoculation were determined and compared between PPD+ and PPD− animals. From a total of 23 BLV positive animals (naturally and experimentally infected), 13 (56.5%) developed HPL, and 10 (43.5%) developed LPL. None of the investigated parameters were affected by infection with M. bovis. We concluded that the ability of cattle carrying resistance-associated marker to control BLV and to progress towards a LPL phenotype was not altered by M. bovis co-infection.

Toll-like receptors, IFN-γ and IL-12 expression in bovine leukemia virus-infected animals with low or high proviral load

Bovine leukemia virus (BLV) infection is widespread mainly in dairy cattle and 5–10% of infected animals will die due to lymphosarcoma; most cattle remain asymptomatic but 30% develop persistent lymphocytosis (PL). BLV transmission depends on infected cell exchange and thus, proviral load is determinant. Understanding the mechanisms which govern the control of viral dissemination will be desirable for the design of effective therapeutic or preventive strategies for BLV. The development of high proviral load (HPL) or low proviral load (LPL) might be associated to genetic factors and humoral immune responses, however cellular responses are not fully described. We aimed to characterize cytokines and toll-like receptors (TLR) expression related to the proviral load profiles. IFN-γ and IL-12 mRNA expression level was significantly higher in PBMC from infected cattle (LPL n=6 and HPL n=7) compared to uninfected animals (n=5). While no significant differences were observed in IL-12 expression between LPL and HPL group, IFN-γ expression was significantly higher in LPL animals. Infected cattle exhibited higher expression levels of TLR3, 7–9. Animals with HPL had significantly higher expression of TLR7/8 than uninfected cattle. TLR8 and TLR9 were up-regulated in HPL group, and TLR3 was up-regulated in LPL group. This is the first report related to TLR gene expression in BLV infected cattle and represents evidence of the involvement of these receptors in BLV recognition. Further studies on different subpopulations of immune cells may help clarify their role in response to BLV and its consequences on viral dissemination.

Bovine immunodeficiency virus and bovine leukemia virus and their mixed infection in Iranian Holstein cattle

Bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV) have worldwide distributions, but their prevalences in Iran are unknown. We investigated the presence of infections in Iranian Holstein cattle and determined changes in hematological values for infected animals. Nested PCR was used on blood samples from 143 animals Holstein cattle to detect proviral BIV and BLV gag sequences. Flow cytometric analysis was performed using monoclonal antibodies (mAbs) against CD4, CD8, and CD21 bovine T lymphocyte subsets. Proviral BIV and BLV gag sequences were detected in 20.3% and 17% of the animals, respectively. BIV-BLV confection was also detected in 4.2% of the study population but this was not statistically significant. Flow cytometric analysis showed that both BIV-infected cows and non-infected ones had CD4/CD8 ratios of 2.45 and 1.43, respectively, and this difference was significant. BLV infected and non-infected animals had no significant differences in their CD4/CD8 ratio. In comparison to non-infected cattle, those with both BIV and BLV had a significant decrease in their CD4/CD8 ratios (1.5 % vs. 2.3; P = 0.01). This is the first report of BIV and BLV infections in Iran. We found no evidence that infection with one agent predisposed an animal to infection with the other. BIV infection may have a role in decreasing T CD8 counts, but this may depend on the genetics of the cattle and virus strains involved.

Cellular immune response cytokine expression during the initial stage of bovine leukemia virus (BLV) infection determines the disease progression to persistent lymphocytosis

We have established experimental models of bovine leukemia virus (BLV) infection followed by progression to persistent lymphocytosis (PL) positive (BLV +PL +) or PL negative (BLV +PL −) stages of infection. Two out of six BLV infected animals developed PL + 4 weeks after BLV infection. One other animal became PL + late in the course of infection and three infected animals stayed PL −. These animals (PL −) exhibited transient lymphocytosis 3–4 weeks after infection and sustained PL − lymphocyte counts up to 24 weeks after infection. Competitive RT-PCR analysis of IFN- γ mRNA expression revealed that peripheral blood mononuclear cells (PBMC) of animals with PL + status developed by 4 weeks after infection had augmented IFN- γ mRNA expression 3–4 weeks after BLV infection. However PBMC of animals that sustained a long-termed PL − lymphocyte count had elevated IFN- γ mRNA expression 1–24 weeks after infection. Competitive RT-PCR analysis of IL-2 mRNA expression showed an increase in the levels of IL-2 mRNA in PL − animals. Interleukin-10 (IL-10) mRNAs expression were elevated both in PL + and PL − animals from 3 and 12 weeks after infection respectively. We suggest that early and extended expression of cellular response cytokines may delay the progression to PL + in enzootic bovine leukemia.

Interleukin-12 p40 mRNA expression in bovine leukemia virus-infected animals: increase in alymphocytosis but decrease in persistent lymphocytosis.

Interleukin-12 (IL-12), a key cytokine in immune regulation, has an important role in activating the cell-mediated immune response in infectious diseases. Recently, a dichotomy between IL-12 and IL-10 regarding progression of a variety diseases has emerged. IL-12 activates type 1 cytokine production and has an antagonistic effect on type 2 cytokines. Here, by using quantitative competitive PCR, we show that peripheral blood mononuclear cells from bovine leukemia virus-infected animals in the alymphocytotic stage of disease express an increased amount of IL-12 p40 mRNA. In contrast, IL-12 p40 mRNA expression by cells from animals with late-stage disease, termed persistent lymphocytosis, was significantly decreased compared to that by normal and alymphocytotic animals. Interestingly, IL-12 p40 mRNA was also detected in tumor-bearing animals. IL-12 p40 expression occurred only in monocytes/macrophages, not B or T lymphocytes. The present study combined with previous findings suggest that IL-12 in bovine leukemia virus-infected animals may regulate production of other cytokines such as gamma interferon and IL-10 and the progression of bovine leukosis in animals that develop more advanced disease such as a persistent lymphocytosis of B cells or B-cell lymphosarcoma.

Provirus Variants of the Bovine Leukemia Virus and Their Relation to the Serological Status of Naturally Infected Cattle

Infection of cattle with the bovine leukemia virus (BLV) results in a strong permanent antibody response to the BLV antigens some weeks after infection. However, cattle may carry provirus and not have detectable antibody titers. To prove the occurrence of different BLV provirus variants in German cattle and to study the influence of special BLV variants on the immunoreaction, a 444-bp fragment of theenvgene of 35 naturally BLV infected animals was analyzed. Seven different groups of BLV provirus variants were found on the basis of restriction fragment length polymorphism. Three BLV provirus variant groups and five additionally sequenced BLV isolates showed a high similarity to BLV provirus isolates from other geographical areas. The variation in nucleotide sequence of the five BLV isolates compared with nine previously sequenced BLV isolates ranged up to 5.3%. While BLV provirus variant groups A, C, D, E, F, and G were clearly related to agar-gel immunodiffusion test (AGID)- and enzyme-linked immunosorbent assay (ELISA)-positive animals, BLV provirus variant group B was solely found in permanent AGID- and ELISA-negative or in transient ELISA-positive animals. Altogether, these results indicate that special BLV provirus variants may be responsible for atypical forms of BLV infection in cattle.

Optimization of lymphocyte proliferation assay for cells with high spontaneous proliferation in vitro: CD4 + T cell proliferation in bovine leukemia virus infected animals with persistent lymphocytosis

Evaluation of antigen specific lymphocyte proliferation is extremely difficult with cells displaying high spontaneous proliferation in vitro, such as bovine leukemia virus (BLV) and T cell leukemia virus type I (HTLV-I) infected cells. We introduced modifications to a standard proliferation protocol, and for the first time report antigen specific proliferation in BLV infected animals with persistent lymphocytosis (a benign proliferation of B lymphocytes). Serum (autologous, heterologous or fetal) but not medium was a major factor contributing to spontaneous proliferation. Spontaneous proliferation was strongly serum concentration dependent and reducing serum to 0.5–1% resulted in decreased background proliferation and development of a functional assay. Addition of indomethacin (3–6 μg/ml) further increased antigen specific responses not only from persistently lymphocytotic but also from lymphosarcoma animals. Thus, this modified proliferation protocol can be used in infections where background proliferation hinders evaluation of antigen-specific CD4 + T cells.

Increased interleukin-10 mRNA expression in tumor-bearing or persistently lymphocytotic animals infected with bovine leukemia virus.

Interleukin-10 (IL-10), produced by Th2 helper T cells, B cells, and macrophages, can inhibit cytokine production by Th1 cells and enhance B-cell proliferation and differentiation. Here, we show that peripheral blood mononuclear cells (PBMCs) from bovine leukemia virus-infected animals with late-stage disease express considerably more IL-10 mRNA than animals that are not infected or that are in the early stages of disease. In contrast, the quantities of type 1 cytokines, IL-2 and gamma interferon, decrease with disease progression. In addition, we observed that IL-10 is expressed principally by monocytes/macrophages, not B lymphocytes, in persistently lymphocytotic animals. This observation supports a role for monocytes/macrophages in progression of bovine leukemia virus infection and, of importance, indicates that proliferating B cells are not the source of IL-10 expression. These findings suggest that IL-10 produced by monocytes/macrophages may influence the progression of bovine leukosis in animals that develop persistent lymphocytosis of B cells or B-cell lymphosarcoma.

Monoclonal antibody specific for bovine CD 5 antigen which enhances mitogen-induced blastogenesis and IL-2 production

This study reports on the functional characteristics of a bovine T-cell differentiation antigen recognized by the monoclonal antibody (mAb) 8C11. This mAb has previously been found to react with a 67-kD molecule shared by thymocytes and peripheral blood T cells and to be undetectable on the B cells of healthy animals. This antigen is also largely expressed on the B cells from bovine leukemia virus-infected animals. Molecules with a similar cell distribution have been described in other species (mouse, human, rat and sheep), and were termed CD5 molecules. In order to confirm the CD5-like nature of the target molecule recognized by 8C11, functional T-cell assays were carried out. We report here that this mAb, like its human and murine homologues, enhances the proliferative responses of T cells to mitogens or alloantigens but does not directly stimulate T-cell division. Moreover, we have shown an enhancing effect of this 8C11 mAb on bovine interleukin-2 production by concanavalin A-stimulated bovine peripheral blood mononuclear cells.