Recombinants from a proviral bovine leukemia virus genome corresponding to the 3' region transactivate viral LTR in NIH3T3 and non-infected FLK cells

Bovine leukemia virus (BLV), like human T-cell leukemia viruses, Types I and II, contains three open reading frames at the 3' end of its genome. The longest open reading frame encodes a transactivator protein which is generated by a doubly-spliced mRNA. A series of co-transfection experiments, using proviral BLV pX expression plasmids under the control of the Moloney leukemia virus LTR and the indicator plasmid containing the assayable lac Z gene under the control of BLV LTR, revealed that both NIH3T3 cells and non-infected fetal lamb kidney cells are able to express an active transactivator protein.     

Bovine leukaemia proviral DNA detection in cattle using the polymerase chain reaction

Bovine leukaemia virus (BLV) is the causative agent in enzootic bovine leukosis a disease occurring worldwide. This virus is normally detected by the agar gel immunodiffusion or ELISA assays which rely on the appearance of antibodies to a major surface protein of the virus, gp51, present in the serum of infected cattle. We have used the polymerase chain reaction, which depends on the amplification of specific DNA sequences as a sensitive assay for the detection of BLV. It was possible to detect proviral DNA in 100 pg of tumour DNA from an infected host using agarose gel electrophoresis followed by ethidium bromide staining. The sensitivity of the assay was increased by two log orders when hybridization analysis, using a BLV proviral DNA probe, was used in combination with amplification of the DNA. Proviral DNA was detected in both lymphocytic and tumour DNA and at all stages of infection in cattle.     

In vitro expression of bovine leukemia virus in isolated B-lymphocytes of cattle and sheep

The purpose of this study was to determine the effect of T-lymphocytes and phytohemagglutinin (PHA), a T-cell mitogen, on the expression of bovine leukemia virus (BLV) in cultured B-lymphocytes from BLV-infected cattle and sheep. Bovine B-lymphocytes were isolated by negative selection via complement-mediated lysis of T-lymphocytes. Additionally, bovine and ovine B-lymphocytes were positively selected using fluorescence activated cell sorting. Expression of BLV in cultured bovine and ovine B-lymphocytes occurred in the absence of T-lymphocytes and without PHA stimulation. The results of this study demonstrate that BLV replication in cultured B-lymphocytes is T-cell independent. This finding may have implications for the mechanism of viral latency within infected B-lymphocytes.     

Cross-reactive antibodies to BLV and HTLV in bovine and human hosts with retrovirus infection

Sera of 22 cattle naturally infected with bovine leukemia virus (BLV), of 2 calves vaccinated with BLV, and of 22 patients with human T cell leukemia virus type I (HTLV-I) infection were tested to BLV and HTLV-I by enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB). Sera of 22 healthy cattle and from 22 healthy persons, and mouse monoclonal antibody to BLV-gp51, to HTLV-I-p24, or to HTLV-I-p19 were also tested. Sera of virus-infected hosts gave significantly higher ELISA values than sera of healthy donors to both BLV and HTLV-I. The correlation between ELISA values of bovine sera to BLV and those to HTLV-I was r = 0.76, whereas that of human sera was r = 0.35. By WB and competitive WB assays, bovine sera that were ELISA-positive to BLV reacted with one or more of p12, p15, and p24 of BLV, and with only p24 of HTLV-I. Human sera that were ELISA-positive to HTLV-I reacted with p12 and p24 of BLV, and with one or more of p12, p15, p19, and p24 of HTLV-I. These results demonstrate that BLV and HTLV-I are capable of evoking cross-reactive immune response in at least some hosts under natural infection as well as by virus vaccination.     

Lymphoid leukosis viruses,their recognition as ‘persistent’ viruses and comparisons with certain other retroviruses of veterinary importance

Diseases caused by lymphoid leukosis virus (LLV), a retrovirus, take a long time after infection to develop and have a wide variety of pathological manifestations. This long latent period is characteristic of persistent virus infections. Disease produced by LLV infection and its underlying mechanisms is compared with persistent infections caused by other retroviruses in birds and mammals of veterinary importance. The diseases considered for comparison are those caused by reticuloendotheliosis, feline leukaemia, bovine leukosis and equine infectious anaemia viruses. There are significant changes in the immunological status in all diseases caused by these viruses. LLV infections follow this trend with, in manifestations of neoplastic disease, a perturbation of the normal switch that occurs from IgM to IgG synthesis. There are also indications of other immunological disturbances.Factors other than immunological disturbances may contribute to the length of time after infection required for the many forms of LLV infection to appear. Such additional factors may include the operation of biological clocks, such as the arrival of sexual maturity, and also the very nature of retroviruses. These factors, like the immunological changes, play major roles in the maintenance and progression of persistent retrovirus infections.Abbreviations ACTH adrenocorticotropic hormone - AEV avian erythroblastosis virus - AMV avian myeloblastosis virus - BLV bovine leukaemia virus - CAV chicken anaemia virus - EBL enzootic bovine leukaemia - EIAV equine infectious anaemia virus - env envelope gene - FeLV feline leukaemia virus - FeSV feline sarcoma virus - FOCMA feline oncovirus membrane-associated antigen; gag, group antigen gene - HTLV human T-cell leukaemia virus - LLV lymphoid leukosis virus - L/S leukosis/sarcoma - LTR long terminal repeat - MAV myeloblastosis-associated virus - MDV Marek disease virus - MuLV mouse leukaemia virus - ORF open reading frame; pol, polymerase gene - REV reticuloendotheliosis virus - RIF resistance-inducing factor - RSV Rous sarcoma virus     

Detection of multiple retroviral infections in cattle and cross-reactivity of bovine immunodeficiency-like virus and human immunodeficiency virus type 1 proteins using bovine and human sera in a western blot assay.

Bovine antibovine immunodeficiency-like virus (BIV) antibodies were detected by Western blot analysis (WBA) using a chemiluminescence protocol. Bovine sera with anti-BIV activity, obtained from cows in two dairy herds, had antibodies directed against a variety of BIV-specific antigens indicating chronic infections. These sera were also tested for serological reactivity against bovine leukemia virus (BLV) and bovine syncytial virus (BSV). Cows most commonly had anti-BSV antibodies (12 of 39). Evidence for infection with BSV and BIV or BSV and BLV occurred with almost equal frequency (5 of 39 and 4 of 39, respectively) while only one instance of BIV and BLV coseropositivity was detected. The high prevalence of BSV seropositivity is consistent with a relatively infectious virus, which, as is known, may be transferred congenitally. Similar rates of coseropositivity of BIV or BLV with BSV in this population suggest that BIV is no more infectious than BLV and probably requires prolonged close contact for transmission. Seven of nine cows with anti-BIV antibodies detected primarily human immunodeficiency virus type 1 (HIV-1) p51 and p63 antigens by WBA using an alkaline phosphatase detection system, suggesting that HIV-1 proteins have potential usefulness in screening cattle for BIV seropositivity. Six human sera that showed strong reactivity against multiple HIV-1 proteins and the serum from one of three patients considered to be an "indeterminate" HIV-1 reactor, cross-reacted primarily with BIV p26. This is the first report of human sera with antibody to BIV-specific proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serological diagnosis of infection with the putative bovine leukemia virus.

We report on an accurate, rapid and inexpensive test for the identification of animals infected with the Bovine C-type virus (BLV). The test involves the detection of serum antibodies to BLV using the immunofluorescent (IF) technique on acetone-fixed, infected cells. The specificity of the test was demonstrated by the fact that virus was found by electron microscopy in 90% of cattle showing positive reactions. In contrast, virus was not found despite extensive examination in antibody negative animals. Thus, the presence of IF antibody is an accurate indicator of current rather than past BLV infection. In order for the IF test to be specific it is of critical importance that the target cells used are infected only with BLV. BLV antibodies can also be detected by the immunoprecipitation (Ouchterlony) technique. However, a significant proportion of BLV infected animals showing positive reactions in the IF test failed to show precipitin antibodies to the virus. Likewise, BLV infection was demonstrated by both the IF test and electron microscopy in many animals with persistently normal levels of blood lymphocytes. Thus, neither the precipitin test nor the blood lymphocyte count (Bendixen's key) can be used to rule out BLV infection.     

Changes in B cell and T cell subsets in bovine leukaemia virus-infected cattle

Direct immunofluorescence and fluorescence-activated cell sorter techniques were used for the detection of surface immunoglobulin positive (SIg+) cells in peripheral blood lymphocytes (PBL's) of bovine leukaemia virus (BLV) infected cattle with or without persistent lymphocytosis (PL+, PL-) and in BLV-free cattle. The percentage of SIg+ cells was more than twice as high in BLV+PL+ cattle than in BLV-free and BLV+PL- cattle. Bovine T cells, and T cell subsets were identified indirectly by the same techniques using three monoclonal antibodies (MAb's) specific for all T cells (IL-A43), T helper (BoT4) cells (IL-A12) and T cytotoxic (BoT8) cells (IL-A17). The major histocompatibility complex (MHC) determinants of both class II (BoT4) and class I (BoT8) as well as all T cells were significantly reduced in BLV+PL+ compared to BLV-free cattle. The actual decrease in the BoT8 cell subset or the dilution effect that would change effector:target cell ratio suggests that a resultant decrease in cytotoxic activity in BLV+PL+ cattle may play an important role in the progress of BLV infection in cattle.     

Antioxidant status and biomarkers of oxidative stress in bovine leukemia virus-infected dairy cows

Bovine leukemia virus (BLV) is among the most widespread livestock pathogens in many countries. Despite advances in understanding the pathogenesis of this disease, little is known about the involvement of oxidative stress. Therefore, this study examined the antioxidant status and the markers of oxidative stress in BLV-infected dairy cows. BLV infection was associated with an increase in triacylglycerol levels, a decrease in glutathione peroxidase (GSH-Px) activity and a tendency toward lower superoxide dismutase activity in the infected animals. No significant difference was observed in other markers of oxidative stress (i.e., conjugated dienes, hydroperoxides and malondialdehyde) in the infected animals compared to controls. A novel method for the analysis of oxidative stress, Z-scan based on the measurement of the mean-value of θ in low density lipoprotein indicated that the infected animals had low-density lipoprotein particles that were slightly less modified than those from the healthy group. Thus, we conclude that BLV infection is associated with a selective decrease in GSH-Px activity without any alteration in the common plasma markers of oxidative stress.     

Bovine leukemia virus: A major silent threat to proper immune responses in cattle

Bovine leukemia virus (BLV) infection is widespread in the US dairy industry and the majority of producers do not actively try to manage or reduce BLV incidence within their herds. However, BLV is estimated to cost the dairy industry hundreds of millions of dollars annually and this is likely a conservative estimate. BLV is not thought to cause animal distress or serious pathology unless infection progresses to leukemia or lymphoma. However, a wealth of research supports the notion that BLV infection causes widespread abnormal immune function. BLV infection can impact cells of both the innate and adaptive immune system and alter proper functioning of uninfected cells. Despite strong evidence of abnormal immune signaling and functioning, little research has investigated the large-scale effects of BLV infection on host immunity and resistance to other infectious diseases. This review focuses on mechanisms of immune suppression associated with BLV infection, specifically aberrant signaling, proliferation and apoptosis, and the implications of switching from BLV latency to activation. In addition, this review will highlight underdeveloped areas of research relating to BLV infection and how it causes immune suppression.     

Purified bovine plasma blocking factor decreases Bovine leukemia virus p24 expression while increasing protein synthesis and transcriptional activity of peripheral blood mononuclear cells in short-term culture

Bovine leukemia virus (BLV) induces a persistent infection in the B-cells causing polyclonal expansion of B-cells in one-third of infected cattle and lymphosarcoma in less than 5% of infected cattle. While BLV is difficult to detect in vivo, it is readily produced by cultured lymphocytes and is diminished when supplemented by bovine plasma. This phenomenon is attributed to a poorly characterized plasma blocking factor (PBF). We assessed the effects of bovine plasma on cell viability and BLV p24 expression, and the effects of purified PBF on protein synthesis and gene expression of short-term cultures of bovine lymphocytes. The addition of 25% plasma or semi-purified PBF to cultures had no significant effect on cell viability but caused significant decreases in BLV p24 production and significantly increased de novo protein synthesis. Utilizing a human microarray, the RNA messages of 83 genes involved in cell division, cell metabolism, and gene regulation were up-regulated.     

Seroprevalence of Mycobacterium avium subspecies paratuberculosis, Neospora caninum, Bovine leukemia virus, and Bovine viral diarrhea virus infection among dairy cattle and herds in Alberta and agroecological risk factors associated with seropositivity

A province-wide, cross-sectional seroprevalence and agroecological risk factor study of Mycobacterium avium subspecies paratuberculosis (MAP), Neospora caninum (NC), Bovine leukemia virus (BLV), and Bovine viral diarrhea virus (BVDv) genotypes 1 and 2 (BVDv1 and BVDv2) infection in dairy cattle herds in Alberta was conducted. Among adults, the seroprevalence of MAP, NC, and BLV was 9.1%, 18.5%, and 26.9%, respectively. For MAP, based on a herd test cutpoint of 2 or more seropositive cows, 58.8% of herds were infected. Herd-level seroprevalence for NC and BLV was 98.7% and 86.7%, respectively, based on a herd-test cutpoint of 1 seropositive cow. Among unvaccinated dairy heifers, the seroprevalence for BVDv1 and BVDv2 infection was 28.4% and 8.9%, respectively, while herd-level infection was 53.4% and 19.7%. Seroprevalence for MAP varied moderately by agroecological region, whereas that for NC, BLV, and BVDv1 and BVDv2 did not. For MAP, aridity and soil pH (correlated features of the region) were also important.     

Viral infections and bovine mastitis: a review

This review deals with the role of viruses in the aetiology of bovine mastitis. Bovine herpesvirus 1, bovine herpesvirus 4, foot-and-mouth disease virus, and parainfluenza 3 virus have been isolated from milk from cows with clinical mastitis. Intramammary inoculations of bovine herpesvirus 1 or parainfluenza 3 virus-induced clinical mastitis, while an intramammary inoculation of foot-and-mouth disease virus resulted in necrosis of the mammary gland. Subclinical mastitis has been induced after a simultaneous intramammary and intranasal inoculation of lactating cows with bovine herpesvirus 4. Bovine leukaemia virus has been detected in mammary tissue of cows with subclinical mastitis, but whether this virus was able to induce bovine mastitis has not been reported. Bovine herpesvirus 2, vaccinia, cowpox, pseudocowpox, vesicular stomatitis, foot-and-mouth disease viruses, and bovine papillomaviruses can play an indirect role in the aetiology of bovine mastitis. These viruses can induce teat lesions, for instance in the ductus papillaris, which result in a reduction of the natural defence mechanisms of the udder and indirectly in bovine mastitis due to bacterial pathogens. Bovine herpesvirus 1, bovine viral diarrhoea virus, bovine immunodeficiency virus, and bovine leukaemia virus infections may play an indirect role in bovine mastitis, due to their immunosuppressive properties. But, more research is warranted to underline their indirect role in bovine mastitis. We conclude that viral infections can play a direct or indirect role in the aetiology of bovine mastitis; therefore, their importance in the aetiology of bovine mastitis and their economical impact needs further attention.     

A rapid method for the large scale preparation of bovine leukemia virus antigen

Bovine leukemia virus (BLV) is the etiologic agent responsible for enzootic bovine leukosis. Detection of cattle seropositive for BLV and laboratory studies of BLV require the preparation of large quantities of BLV antigen. A convenient and economical method for concentrating virus from large volumes of cell culture medium involves the precipitation of the virus in the cold by polyethylene glycol (PEG). The present work demonstrates the feasibility of rapidly precipitating BLV with 10% PEG and subsequently separating the resuspended virus from the residual PEG on a disposable desalting column.     

Comparison of four tests to evaluate the reactivity of rabbit sera against envelope or Gag-related proteins of bovine leukemia virus (BLV)

Bovine leukemia virus (BLV) has a long latency period during which animals are inapparently infected, may spread the disease, and are only detected by serological techniques or by the most cumbersome molecular biology techniques. We have compared techniques for detecting either total antibodies (ELISA), anti-p24 and Gag-related proteins (Western blot), or anti-gp51 (agar gel immunodiffusion, AGID, and syncytia inhibition, SI) in rabbits inoculated experimentally with inocula of variable immunogenicity. The two tests to detect antibodies to gp51 correlated well in sera clearly positive or clearly negative by either one, but correlation was poor in the intermediate groups. All sera positive by AGID were also positive by ELISA, but results did not agree in sera negative by AGID, ELISA proving to be more sensitive. Western blot was a good technique for detecting antibodies against Gag-related proteins. However, no band was identified to clearly correspond to anti-Env-related proteins. As for other retroviruses, testing of animals for infection with BLV should include the detection of antibodies anti-Gag and anti-Env proteins.     

To compare the incubation period following intratracheal and subcutaneous inoculation of bovine leukosis virus infected lymphocytes and to study their clearance from the circulation

The migration of fluorescein isothiocyanate labelled lymphocytes through the tracheobronchial mucosa has been studied in cattle. Following intratracheal inoculation of labelled non-infected autologous lymphocytes and bovine leukosis virus (BLV) infected heterologous (presumed allogeneic) lymphocytes, the labelled lymphocytes appeared in the blood circulation between 4 and 7 days post inoculation. Following intravenous inoculation of labelled autologous lymphocytes, the cells could be detected in the circulation for 10 days post inoculation whereas BLV infected and non-infected heterologous lymphocytes could be detected for only 2 days. The migration of BLV-infected heterologous lymphocytes through the tracheobronchial mucosa caused a delay in the appearance of labelled lymphocytes in the circulation and a corresponding delay in the appearance of BLV antibodies. Comparison was made of the effect of two different routes of inoculation, subcutaneous and intratracheal on the incubation period as indicated by the detection of antibody. Subcutaneous inoculation of 1 X 10(4), 5 X 10(3), 1 X 10(3) of lymphocytes from a BLV infected cow caused seroconversion whereas 5 X 10(2) cells did not. Intratracheal inoculation of 5 X 10(3) cells caused sero-conversion. One animal did not develop BLV antibody until 30 weeks after inoculation although BLV could be isolated from the blood at 24 and 26 weeks post inoculation.     

Seroprevalence of bovine leukemia virus in dairy cattle in Argentina: comparison of sensitivity and specificity of different detection methods

Bovine leukemia virus (BLV) is a retrovirus that induces a chronic infection in cattle, which develop in three possible pathological forms: asymptomatic course, persistent lymphocytosis (PL) and lymphosarcoma. Once infected, cattle remain virus carriers for life and start to show a serological reaction within a few weeks after infection. Eradication and control of the disease is based on early diagnostic and segregation of the carriers. The agar gel immunodiffusion (AGID) test has been the serological test of choice for routine diagnosis of serum samples. Nevertheless, in more recent years, the enzyme-linked immunosorbent assay (ELISA) has replaced the AGID for large scale testing. Although Argentina has over 60 million cattle population, no nationwide studies have been conducted yet to determine the prevalence of the infection. To estimate the rate of BLV infection in dairy cattle in Argentina, a survey for specific antibodies in >10,000 serum samples from animals over 18 months old, belonging to 363 different herds from the largest dairy production areas of the country, was carried out in our laboratory, along 1999. For this purpose, we developed an ELISA to detect serum antibodies against the BLV virus. The cut-off of the ELISA was established over 339 serum samples, using polymerase chain reaction and southern blot (PCR-SB) as confirmatory test. The sensitivity and specificity of the ELISA was of 97.2 and 97.5%, respectively, while the local official AGID test showed a sensitivity of 79.7% and specificity of 99.0%. To know the seroprevalence of BLV on dairy herds, and also the incidence of the infection within the herd, the serological survey was based on individual serum samples. The results show that the prevalence of infected individuals is of 32.85%, while the percentage of infected herds, harboring one or more infected animals, is of 84%. These results indicate a medium level of seropositive animals when taken individually, but a high prevalence of infected farms, which has been notoriously increased in the last 15 years as shown when compared with previous data from particular geographic areas, indicating that BLV constitutes a serious sanitary problem for dairy producers in Argentina. They also indicate the poor sensitivity of the official AGID test used in the country.     

Detection of bovine leukosis virus in bronchoalveolar lung washings and nasal secretions

Cattle and sheep persistently infected with bovine leukosis virus (BLV) were studied for the presence of the virus in bronchoalveolar lung washings and nasal secretions. The virus was demonstrated in the cellular fraction of the lung washings in six out of nine cattle and in one out of six sheep. In no instance was bovine leukosis isolated from the cell-free bronchoalveolar lung washings. The virus was isolated from the nasal secretion of only one of six naturally infected milking cows despite frequent sampling; the virus-infected nasal secretion was from a sick 10-year-old cow. Bovine leukosis virus was not isolated from cellular fractions of nasal secretions.