Serum immunoglobulin concentrations in cattle naturally infected with bovine leukemia virus

In order to elucidate whether natural infection of BLV in cattle might induce humoral immunological responses, changes in IgG1, IgG2, and IgM concentrations in the sera of infected cattle were determined. Twelve BLV-infected cattle were used. Cattle of different breeds were classified serologically and haematologically into BLV + PL+, BLV + PL- and BLV-free groups. Ig concentrations in the sera of the three groups were quantitated using a commercial single radial immunodiffusion assay. The findings were compared to those of BLV-free cattle. The serum IgM concentrations were significantly lower in cattle with PL (P less than 0.001) than in BVL + PL- and BLV-free cattle. The IgM concentrations tended to be lower in BLV+ PL- than those of BVL-free cattle. There were no significant differences in IgG1 and IgG2 serum concentrations between the three experimental cattle groups. IgG1 was the predominant subtype in the sera of all cattle groups.     

Molecular studies of T-lymphocytes from cattle infected with bovine leukemia virus

Although bovine leukemia virus (BLV) is mainly associated with infections of B-lymphocytes, we have previously reported the statistically significant increase in the T-lymphocytes obtained from BLV-infected asymptomatic aleukemic (AL) cattle. In this report the presence of BLV provirus in the DNA of immunoaffinity purified T-lymphocytes from AL animals was assessed using a highly specific radiolabelled (32P) BLV-DNA provirus probe and solid phase DNA hybridization. The BLV provirus was found in the DNA of the peripheral blood mononuclear cells of all AL animals tested and three of the four purified T-lymphocyte preparations from these animals. The purified T-lymphocyte preparations used in this study contained less than 4% detectable B-lymphocytes. One animal had no detectable B-lymphocytes in the purified T-lymphocyte preparation and the DNA from these cells also gave positive hybridization results. The lymphocyte blastogenesis assay was then used as an indicator of the functional ability of lymphocytes from these BLV-infected AL cattle to respond to mitogenic stimuli. The responsiveness of lymphocytes from these animals to the mitogens concanavalin A (Con A), phytohemagglutinin (PHA), and pokeweek mitogen (PWM) was comparable to that of lymphocytes from BLV-negative animals when changes in 3H-thymidine uptake (c.p.m.) were used as measurement of mitogenic-induced blastogenesis. This indicated that infection of the T-lymphocytes by BLV does not appear to alter the overall response of the lymphocyte populations to mitogenic stimuli. High levels of spontaneous blastogenesis in the absence of mitogenic stimulation were observed for lymphocyte preparations of AL animals. The reason for this proliferation of lymphocytes is unclear; however, sera from these AL animals were found to contain a blastogenesis-augmenting factor(s) when added to lymphocytes from BLV-negative control animals in the presence of Con A, PHA and PWM.     

Lesions in clinically healthy cattle persistently infected with the virus of bovine viral diarrhea--glomerulonephritis and encephalitis

Four clinically healthy cattle persistently infected with the virus of bovine viral diarrhea were examined for viral antigen and lesions. Antigen was seen by direct immunofluorescence in cytoplasm of the neurons of the brain and cervical part of the spinal cord, cells and basement membrane of renal glomeruli, reticular cells of lymph nodes and spleen, epithelial cells of small intestinal crypts and renal and testicular tubules, and endothelial cells of blood vessels. Infected neurons were pyknotic and surrounded by astrocytes and macrophages. A few blood vessels in the brains were cuffed with mononuclear cells. Basement membranes of renal glomeruli were irregularly thick with eosinophilic material, and mesangial cells in the glomeruli were plentiful. The virus had a direct effect on some tissues, but was restricted in its cytopathogenicity and was not eliminated by defense mechanisms of the host. Renal glomerular lesions were believed to have an immunologic basis.     

Antibody titers against bovine coronavirus and shedding of the virus via the respiratory tract in feedlot cattle

OBJECTIVE: To describe patterns of seroconversion to bovine coronavirus (BCV) and shedding of BCV from the respiratory tract in feedlot cattle. ANIMALS: 1,074 calves in feedlots in Ohio, Texas, and Nebraska. PROCEDURE: Nasal swab specimens were obtained at time of arrival (day 0) and at various times during the initial 28 days after arrival at feedlots. Specimens were tested for BCV, using an antigen-capture ELISA. Serum samples were obtained at time of arrival and again 28 days after arrival; sera were analyzed for antibodies to BCV, using an antibody-detection ELISA. RESULTS: Samples from 12 groups of cattle entering 7 feedlots during a 3-year period revealed that 78 of 1,074 (7.3%) cattle were shedding BCV (range, 0 to 35.9% within specific groups). At time of arrival, 508 of 814 (62.4%) cattle had low (< 50) or undetectable BCV antibody titers. Seroconversion to BCV during the initial 28 days after arrival was detected in 473 of 814 (58%) cattle tested (range, 20.3 to 84.1 % within specific groups). In cattle shedding BCV from the nasal passages, 49 of 68 (72.1 %) seroconverted, and 472 of 746 (63.3%) cattle that were not shedding the virus seroconverted. CONCLUSIONS AND CLINICAL RELEVANCE: Bovine coronavirus can be detected in populations of feedlot cattle in the form of viral shedding as well as seroconversion to the virus. Although only a few cattle were shedding the virus at the time of arrival at a feedlot, most of the cattle seroconverted to BCV by 28 days after arrival.     

Upper-normal prediction limits of lymphocyte counts for cattle not infected with bovine leukemia virus

A study was conducted to develop valid estimates of lymphocyte count (LC; cells per microliter) of individual, clinically normal dairy cattle. Estimated weighted regression was used on repeated measures of individual LC to examine 6 models predicting LC as a function of age in cattle not infected with bovine leukemia virus. The generalized growth curve model of analysis of variance was used to estimate intercepts, slopes, and prediction limits for the models and to compare the LC-to-age relationship between Holstein and Guernsey breeds. The best-fitting model (P = 0.0001) with the narrowest prediction interval was LC = 4,414.4 - 84.6X, where X = (age -48) if age less than or equal to 48 months, and X = 0 if age greater than 48 months, and 163.6 and 8.1 are the SE of the estimates, respectively. Upper one-sided 95%-predicted normal LC tended to be higher than estimates derived from traditional hematologic keys that use confidence limits of mean LC. Difference was not found in the LC-to-age relationship between the Holstein and Guernsey cattle (P = 0.67). Results of this study provided estimates of normal LC that are more specific in diagnosing lymphocytosis in individual cattle.     

Provirus variants of bovine leukemia virus in naturally infected cattle from Argentina and Japan

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.     

S-phase evaluation with bromodeoxyuridine in lymphocytes from cattle infected with bovine leukemia virus (BLV)

Bromodeoxyuridine (BrdUrd), an analogue of thymidine, can be detected by means of monoclonal antibodies and utilized as a marker of the S-phase. In this paper a determination of the S-phase in BLV+ cattle with lymphocytosis has been performed by incorporating bromodeoxyuridine in the DNA. This evaluation was compared to the DNA content, demonstrating that i) bromodeoxyuridine incorporation is a reliable marker of S-phase in BLV+ cattle with lymphocytosis and ii) cytofluorimetry is the method of choice, together with immunocytochemistry, to demonstrate bromodeoxyuridine incorporation.     

Response of cattle persistently infected with bovine virus diarrhoea virus to bovine leukosis virus

Six cattle persistently infected with bovine virus diarrhoea virus (BVDV) and seronegative, and two control, virus negative seropositive cattle were inoculated with lymphocytes infected with bovine leukosis virus (BLV). The two controls produced a normal immune response to BLV, developing antibodies at four and five weeks after inoculation. Two of the six cattle persistently infected with BVDV developed a strong antibody response by six weeks after inoculation with BLV. Four developed a depressed response to BLV, characterised in three by a 'hooking' reaction in the immunodiffusion test which persisted in successive bleedings but was interspersed occasionally by a weak positive reaction. In one of these animals, a series of 'hooking' reactions was followed by a number of negative results. The fourth animal remained serologically negative until 16 weeks after inoculation when a 'hooking' reaction was observed followed by a series of negative results. BLV was isolated from all the cattle persistently infected with BVDV at 42 or 58 weeks after inoculation regardless of whether the serum samples gave negative, 'hooking', weak positive or positive reactions in the immunodiffusion test. BLV was consistently isolated from the nasal secretions of a steer which was BVDV negative but seropositive. The possibility of decreased immune responsiveness to BLV in animals persistently infected with BVDV should be considered when formulating regulations governing the testing of animals for freedom from BLV.     

Ki-67 antigen expression in lymphocytes of cattle infected with bovine leukemia virus (BLV).

The Ki-67 monoclonal antibody, which recognizes an antigen present on the nuclear membrane surface of mammalian cells in the replication phase, has been used for the determination of the cellular cycle of peripheral blood lymphocytes on a group of cattle positive for bovine leukemia virus (BLV) and with blood values showing a persistent lymphocytosis. The results obtained have shown that: 1. Both of the techniques used (immunofluorescence and immunoperoxidase) are easily applicable and give uniform results; 2. Cattle with a persistent lymphocytosis show an absolute number of cells in cycle significantly more elevated compared with cattle positive for BLV with normal blood values.     

The role of neighboring infected cattle in bovine leukemia virus transmission risk

A cohort study was conducted to evaluate the risk of bovine leukemia virus (BLV) transmission to uninfected cattle by adjacent infected cattle in 6 dairy farms. Animals were initially tested in 2010–2011 using a commercial ELISA kit. Uninfected cattle were repeatedly tested every 4 to 6 months until fall of 2012. The Cox proportional hazard model with frailty showed that uninfected cattle neighboring to infected cattle (n=53) had a significant higher risk of seroconversion than those without any infected neighbors (n=81) (hazard ratio: 12.4, P=0.001), implying that neighboring infected cattle were a significant risk factor for BLV transmission. This finding provides scientific support for animal health authorities and farmers to segregate infected cattle on farms to prevent spread of BLV.     

Neutrophil function in sheep experimentally infected with bovine leukemia virus

In vitro neutrophil adherence, random migration, chemotaxis, resting and phagocytosis-associated oxygen consumption and bactericidal responses were assessed in sheep experimentally infected with bovine leukemia virus (BLV). Neutrophil function was examined in two groups of 9 control and 9 BLV-infected sheep at 0, 1, 2, 3, 5, 7, 11 and 15 weeks post-infection. Enhanced neutrophil adherence, chemotaxis and resting oxygen consumption responses were found in the infected group at 2, 11 and 15 weeks respectively. Significant alterations between groups were not demonstrated during the other time intervals.     

Immunocompetence of sheep experimentally infected with bovine leukemia virus

The humoral and cellular immunological reactivity of sheep were studied throughout the first 32 weeks following experimental infection with bovine leukemia virus (BLV). Seroconversion of BLV-inoculated sheep occurred within 4 weeks, but infection was not transmitted to contact control sheep. Despite the persistence of the viral infection, no differences were demonstrated in leukograms, serum IgG concentrations, humoral response to immunization with an irrelevant antigen (rabbit red blood cells), phytomitogen (Concanavalin A and Pokeweek mitogen)-induced lymphocyte blastogenesis, or chemical (1-chloro, 2-4 dinitrobenzene) skin contact hypersensitivity, between BLV-infected and uninfected contact control sheep. These results demonstrate the absence of a nonspecific immunosuppressive effect of BLV and further negate the influence of a generalized immunological deficit on the development of clinical disease in BLV-infected animals.     

Cytotoxic T-lymphocyte responses in cattle infected with bovine viral diarrhea virus

A system for a reproducible in vitro restimulation of bovine viral diarrhea virus (BVDV)-specific cytotoxic T-cells (CTL) was developed. Lymphocyte cultures of BVDV-immunised cattle were stimulated with infectious BVDV isolate PT810 and recombinant bovine interleukin-2 for 12 to 25 days. A specific lysis of Concanavalin A-stimulated BVDV-infected autologous target cells was observed, whereas allogeneic BVDV-infected target cells were only marginally lysed as detected by flow cytometry. BVDV-specific lymphocyte transformation was further characterised by the expression of bovine lymphocyte activation antigens and bovine MHC class-II molecules. Secondary stimulation of CTL was influenced by in vitro production of BVDV-specific neutralising antibodies, which were secreted exclusively in BVDV-inoculated lymphocyte cultures of immunised cattle. These results demonstrate the presence of CTL in peripheral blood mononuclear cells (PBMC) of immunised cattle which can kill autologous BVDV-infected antigen-presenting cells after in vitro restimulation.     

Seroprevalence of bovine immunodeficiency virus and bovine leukemia virus in dairy and beef cattle in hokkaido

Serological survey of bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV) infection was conducted in dairy cattle from 10 different regions of Hokkaido, Japan. Among 390 cattle, 11.0% of cattle were BIV-seropositive and 3.3% were BLV-seropositive. Moreover, in two dairy farms, where bovine leukosis has been reported, prevalence of BIV infections were 6.4 and 9.1%, respectively. In contrast, among 150 beef cattle, 16.6% were BIV-seropositive while none was BLV-seropositive. Dual infections with BLV and BIV in dairy cattle were tested by using 107 BLV-seropositive sera, and 20 sera were found BIV-positive (18.7%). These results indicate that BIV infection was widespread in Hokkaido.