Histopathology and distribution of cells harboring bovine leukemia virus (BLV) proviral sequences in ovine lymphosarcoma induced by BLV inoculation.

Six sheep with lymphosarcoma induced by hypodermic inoculation of bovine leukemia virus (BLV) materials were examined to elucidate the relation between pathologic lesions and integration of BLV provirus in cellular DNAs. Antibodies to BLV gp-antigens had been detected since the 3rd week after the inoculation, and BLV was positive when checked 3 months later. Lymphosarcomas followed the leukemic phase in 4 sheep. The other 2 sheep showed initial lesions of lymphosarcoma and were aleukemic clinically. Five animals were killed by enthanasia and autopsied at 2.5 to 3.5 years postinoculation (pi) because of their diseased condition. One animal died 10 years pi following the 4th leukemic episode. Sarcomatous lesions were confirmed grossly and histologically, and the proliferating neoplastic cells were classified into lymphocytic, prolymphocytic, lymphoblastic and histiocytic types. Integration of BLV provirus in cellular DNAs of the peripheral blood lymphocytes (PBL) and neoplastic cells of sarcomatous lesions was examined by Southern blotting technique. BLV provirus was demonstrated in the PBL of all infected animals and in most of the sarcomatous lesions of the spleen, kidney and lymph nodes except 4 lymph nodes showing slight neoplastic infiltration. The results indicated that ovine lymphosarcoma could be caused by BLV and the cells carrying proviral information seemed to be disseminated and proliferate in the lesions.     

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.     

Humoral and cellular responses in calves experimentally infected with bovine leukemia virus (BLV)

In order to elucidate whether infection by Bovine Leukemia Virus (BLV) might induce an immunodeficient state, we inoculated sixteen calves with BLV. The calves were followed up for two years and were tested for humoral and cellular responses using various parameters, namely the appearance of antibodies to the BLV antigens, the changes in the numbers of lymphocytes involved, and the ratio between the two main populations of lymphocytes. Antibodies to the BLV antigens were of both the IgG and the IgM classes of immunoglobulins. The levels of antibodies of the IgM class were higher than those of IgG. There was a temporary decrease of reactive antibodies to the BLV antigens, to below detectable levels, during the 14-24 weeks post infection. A significant decrease in the level of plasma IgM was found in all BLV infected calves exhibiting lymphocytosis, while the level of IgG in the plasma of all experimental calves did not diverge significantly from the initial values, throughout the experiment. BLV infection was followed by lymphocytosis of B-cells in most infected calves, which persisted for the whole course of the experiment, while a decrease in the population of T-cells in peripheral blood was observed for a period of several months in all infected calves.     

Protective effects of vaccination with bovine leukemia virus (BLV) Tax DNA against BLV infection in sheep

A DNA vaccination trial was performed on sheep to determine whether vaccination with bovine leukemia virus (BLV) transactivator Tax DNA is effective against BLV infection. Immunization was carried out with cationic liposomes containing the Tax-expressing plasmid DNA and subsequently all sheep were challenged with BLV. BLV titers in peripheral blood mononuclear cell (PBMC) determined by syncytium formation assay and BLV provirus load detected by genomic PCR analysis showed higher levels of virus titers in control sheep than those in Tax-vaccinated sheep. Higher levels of IFN-gamma mRNA expression have been demonstrated in vaccinated sheep after the challenge. These results suggested that Th1 type immune response induced by Tax DNA vaccine inhibited BLV propagation in vaccinated sheep at the early phase of infection.     

Bovine leukosis virus in sheep,lymphocyte modification and surface-immunoglobulin-bearing cell numbers

Lymphocytes from sheep experimentally infected with bovine leukosis virus (BLV) and from non-infected normal sheep were examined for the presence of surface Ig by an immunofluorescence test. Surface Ig-bearing lymphocytes in blood from BLV-infected sheep increased when lymphocyte counts of blood were elevated in comparison with normal animals. The mitogen stimulation of cultured lymphocytes from BLV-infected sheep and from non-infected normal sheep was determined by measuring ³H-thymidine incorporation. Peripheral blood lymphocytes (PBL) from BLV-infected leukemic sheep with elevated PBL counts responded poorly to phytohemagglutinin M and concanavalin A but responded well to lipopolysaccharide compared with lymphocytes from uninfected animals. In BLV-infected preleukemic sheep with low PBL counts, stimulation indices of mitogen responses of lymphocytes with phytohemagglutinin M, concanavalin A, and pokeweed mitogen were low compared with those of lymphocytes from uninfected animals. The results indicated that B cells were affected by BLV infection in sheep as suggested by the increased number of surface Ig-bearing lymphocytes and that significant alteration of mitogen stimulation of lymphocytes occured in sheep with BLV infection.     

T lymphocyte subset alterations following bluetongue virus infection in sheep and cattle

To determine potential mechanisms of differential disease expression in ruminants infected with bluetongue virus (BTV), clinically normal, BTV-seronegative, yearling sheep and cattle were infected subcutaneously with a standardized insect-source inoculum of BTV serotype 17 (BTV-17) (three infected and one contact control each) or animal adapted BTV serotype 10 (BTV-10) (three sheep only). BTV was isolated from peripheral blood cell components of infected sheep and cattle and all infected animals showed evidence of seroconversion by 14 days post infection (PI). Sheep infected with both serotypes of BTV developed pyrexia, oral lesions, and leukopenia which were most severe on days 7-8 PI. Analysis of peripheral blood mononuclear leukocytes with specific monoclonal antibodies and flow cytometry revealed panlymphocytopenia on day 7 PI. This response was further characterized by an increase in the CD4/CD8 ratio (greater than 3) resultant from a greater decrease in absolute numbers of circulating SBU-T8(CD8+) ("cytotoxic/suppressor") lymphocytes compared to SBU-T4 (CD4)+ ("helper") lymphocytes. SBU-T19+ lymphocytes were also decreased below baseline values on days 5-14 post infection. On day 14 PI there were increased CD8+ lymphocytes and decreased CD4/CD8 ratios (approximately 0.6) in these sheep. Clinical and hematologic changes in cattle infected with BTV-17 were minimal and consisted of mild pyrexia (rectal temperature 103 degrees F) on day 9 PI in two of three infected animals and mild leukopenia on several days PI in one animal. This leukopenia was the result of a pan T lymphocytopenia with CD4/CD8 ratios in the expected range (1-2). Similar to infected sheep, infected cattle did have a shift (decrease, approximately 0.8) in the peripheral CD4/CD8 ratio associated with an increase in circulating BoT8 (CD8)+ lymphocytes on day 14 post infection. Lymphocytes in the peripheral blood of all sheep and cattle infected with BTV-17 proliferated in vitro in response to purified BTV-17. These results confirm and extend those of previous studies that indicate species differences in the hematologic response to an equivalent BTV infection in domestic ruminants.     

Decreased expression of L‐selectin (CD62L) on lymphocytes in enzootic bovine leukaemia

Expression of L‐selectin was determined by single‐ and two‐colour immunofluorescence on granulocytes, peripheral blood mononuclear cells (PBMC) and blasts of bovine origin by means of a monoclonal antibody IVA94 which recognizes bovine L‐selectin (CD62L). Cells were separated from peripheral blood of healthy cattle and colleagues infected with bovine leukaemia virus (BLV). BLV‐infected animals comprised lymphocytotic and non‐lymphocytotic cows. L‐selectin was expressed on 90–98 % of granulocytes in all tested animals. The percentage of PBMC expressing L‐selectin was lower in cattle with persistent lymphocytosis than in non‐lymphocytotic or BLV‐free cattle, and inversely correlated with lymphocyte counts. The ratio of B lymphocytes stained for L‐selectin was significantly decreased from 60.2 ± 1.9 % in BLV‐free cattle to 43.8 ± 3.6 and 22.5 ± 5.7 % in non‐lymphocytotic and lymphocytotic cattle, respectively. B‐lymphocytes stained for L‐selectin exhibited about 50 % reduction in L‐selectin expression in BLV‐infected cattle compared with BLV‐free cattle, as judged by the mean fluorescence intensity (MFI). The percentage of L‐selectin‐positive PBMC not bearing surface immunoglobulin M (predominantly T lymphocytes) was comparable in BLV‐free and BLV‐infected cattle. However, L‐selectin expression on T lymphocytes was reduced (about 50 %) in BLV‐infected cattle, as judged by the MFI. We suppose that BLV infection results in a decreased L‐selectin expression on lymphocytes, and accordingly, it may contribute to deregulation of the host immune system.     

T and B lymphocytes in horses persistently infected with equine infectious anaemia virus

The percentage of T and B lymphocytes in the peripheral blood of horses chronically infected with equine infectious anaemia (EIA) virus was determined and the results were compared with the percentage of these cells in healthy uninfected horses. Cells with membrane receptors for sheep erythrocytes (T and active T lymphocytes) were determined by E and A rosette techniques, while cells with receptors for the C3b component of complement and those with receptors for mouse erythrocytes (B lymphocytes), were determined by the EAC rosette method. The percentage of Fc positive cells was assayed by the EA rosette test.The majority of peripheral blood lymphocytes (PBL) from both uninfected and EIA-infected horses formed rosettes of each kind with only three erythrocytes indicating a low density of the corresponding receptors on the cell membrane under the condition of the assays used. The percentage of T lymphocytes in the peripheral blood of diseased horses (52.4±1.6%), as detected by E rosettes, was significantly (p<0.01) higher than in control animals (42.4±3.5%). In clinically healthy horses 8.9±1.1% of PBL were identified by A rosettes as active T cells, whereas animals with a chronic form of EIA had a much lower (p<0.001) percentage of these cells (4.7±0.7%). In the B lymphocyte subpopulations the percentages of cells bearing Fc and C3b receptors were markedly elevated (p<0.001) in EIA-infected horses (24.7±0.8% and 42.8±2.2% respectively) as compared to uninfected animals (15.1±1.4% and 29.6±1.2% respectively). Receptors for mouse erythrocytes, as yet undescribed on equine PBL, were demonstrated in approximately equal proportions on lymphocytes from EIA-infected (24.8±1.5%) and uninfected horses (24.3±2.1%).     

Alterations in lymphocyte subpopulations in peripheral blood of sheep persistently infected with border disease virus.

Fourteen sheep persistently infected with border disease virus were investigated to examine the effects of persistent viraemia on lymphocyte subpopulations in peripheral blood and their responses to the mitogen phytohaemagglutinin (PHA) in vitro. Persistently infected sheep had significantly more CD8+ (cytotoxic-suppressor) T-lymphocytes than uninfected sheep of the same age (P less than 0.001). The total number of CD4+ (helper) T-lymphocytes were not significantly different but there were more T-lymphocytes (CD5+) which were CD4- and CD8- in normal sheep than persistently infected sheep (P less than 0.001). Peripheral lymphocytes obtained from persistently infected sheep showed significantly reduced blastogenesis induced by PHA than those obtained from normal sheep (P less than 0.001).     

Lymphosarcoma development in sheep experimentally infected with bovine leukaemia virus

Twelve sheep were experimentally infected with a phytohemagglutinin (PHA) treated short term culture of lymphocytes from a cow naturally infected with BLV at the PL stage. Five of 12 (42%) BLV infected sheep had histologically confirmed lymphosarcoma 10-16 months after infection. The PBL's were increased to leukemic levels 3-21 weeks before death due to lymphoblastic leukemia. Lymphocyte proliferation and appearance of immature lymphocytes and lymphoblastic cells in the blood were a characteristic feature of tumour development following inoculation with an Australian strain of BLV. In contrast to a number of previous studies the peripheral lymph nodes of all infected sheep were clinically normal throughout the experimental period but at death gross tumours were evident in the mesentric lymph nodes and the heart in all cases. All the other lymph nodes, liver, spleen, kidney and lung were histologically infiltrated with lymphoid tumour cells. Gross tumours were present in the abomasum (1 out of 5) in the urinary tract (2 out of 5) and in the uterus (1 out of 2). The majority of the tumour cells isolated from the various tissues were centroblastic demonstrating that the malignant leukemia in experimentally infected sheep was of a multicentric centroblastic type. The central nervous system was not involved in any case.     

Breed susceptibility to ovine progressive pneumonia (maedi/visna) virus

In this retrospective study of breed differences in susceptibility to disease caused by ovine progressive pneumonia (OPP) virus, 29 Border Leicester sheep were compared with 46 Columbia sheep. As judged by frequency and severity of clinical signs and lesions attributable to the infection, Border Leicester sheep were markedly more susceptible than Columbia sheep and experimentally infected sheep were slightly more susceptible than naturally infected sheep. Differences in susceptibility to infection by the virus were not determined.     

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.     

Ultrastructure and frequency of mastitis caused by ovine progressive pneumonia virus infection in sheep

Twenty-five sheep, experimentally (n = 15) or naturally (n = 6) infected with ovine progressive pneumonia virus and noninfected controls (n = 4), were evaluated for histological and ultrastructural lesions of mastitis. Histologically, nine of 15 experimentally infected sheep and all six naturally infected sheep had lympho-plasmacytic mastitis. Severity of the lesion increased with length of time after infection. Periductal lymphatic nodules were seen in five sheep experimentally infected for 2.8 years or longer and in five naturally infected sheep that were 3.7 years old or older. Ultrastructurally, responses to ovine progressive pneumonia virus were diffuse lympho-plasmacytic infiltrates in glandular interstitium, lymphocytic and occasional plasmacytic infiltrates in ductal walls and lumens, lymphoblasts surrounded by small lymphocytes in glandular interstitium, and degeneration of epithelium releasing cells and cellular debris into the lumen. Based on the prevalence of lesions, the mammary tissue was more susceptible to ovine progressive pneumonia virus than other target organs: lung, brain, and synovium. Lesions did not differ between breeds of sheep. Ovine progressive pneumonia virus was not seen in the mammary tissue but was isolated from 15 of 17 mammary glands.     

Bovine herpesvirus-1 (BHV-1), bovine leukemia virus (BLV) and bovine viral diarrhea virus (BVDV) infections in Algerian dromedary camels (<Emphasis Type="Italic">Camelus dromaderius</Emphasis>)

This study was performed to investigate the presence of bovine herpesvirus-1 (BHV-1), bovine leukemia virus (BLV) and bovine viral diarrhea virus (BVDV) infections in dromedary camels (Camelus dromaderius) kept in mixed herds with sheep and goats in Algeria, since the prevalence of BHV-1, BVDV, and BLV infections among dromedary camels in Algeria is unknown. Totally, 111 camel sera were collected from two provinces (Laghouat and Ghardaia) in Algeria. The sera were analyzed for BHV-1 specific antibodies, BVDV specific antibodies and BVDV antigen using the ELISA, and BLV nucleic acid using PCR. The seropositivity rate was 9.0% for BVDV-specific antibody, although 41.4% of camels tested were positive for BVDV antigen. Moreover, there was no evidence of BHV-1 and BLV infections. The results indicated that camels might represent an important source for BVDV infection in all ruminants, including cattle, sheep, and goats bred in mixed herds in Algeria, since they had a higher BVDV prevalence rates. Therefore, the prevention and control measures for BVDV infection should put in place in camel populations to limit the spread of BVDV infection to ruminant populations in Algeria.     

两种方法测定羊外周血淋巴细胞增殖的研究

本试验比较~3H胸腺嘧啶核苷(~3H-TdR)渗入法和BrdU-ELISA方法测定羊外周血淋巴细胞增殖。分离健康羊外周血淋巴细胞,与不同浓度的非特异刺激因子刀豆素A(concanavalinA,1.0,0.5,0.25,0.125,0.0625,0.0313,0.0156,0.0078,0.0039μg/孔)培养,分别用~3H胸腺嘧啶核苷(~3H-TdR)和5-溴-2’-脱氧尿苷(BrdU)标记细胞,测定刺激细胞和对照细胞的每分钟脉冲数cpm或光吸收值OD,计算刺激指数SI和光吸收值差△OD,统计学方法分析SI和△OD的相关程度。同样的方法测定羊试验感染肝片吸虫(Fasciola hepatica)后,在感染早期阶段(PIWO-PIW4)其外周血淋巴细胞在特异性抗原-片形吸虫分泌排泄产物(ESP,5μg/孔)刺激下的增殖。结果表明,刺激指数SI和光吸收值差△OD在两个试验中的相关系数分别为0.946和0.924,SI和OD用均呈强正线性相关。BrdU-ELISA方法无同位素放射物,且敏感、快速、方便,和~3H-TdR渗入法一样可以用于测定淋巴细胞增殖。     

Infectivity of bovine leukaemia virus infected cattle: an ELISA for detecting antigens expressed in in vitro cultured lymphocytes.

A simple ELISA is described for quantifying expression of bovine leukaemia virus (BLV) antigens in short-term cultures of peripheral blood lymphocytes (PBL) isolated from infected cattle. The PBL-ELISA demonstrated that antigen expression levels in infected cattle could vary by more than 50-fold. Inoculation of sheep with dilutions of lymphocytes from two BLV-infected cattle, differentiated in the PBL-ELISA by 50 to 100-fold, suggested that antigen expression levels were correlated with infectivity. Haematological data indicated that increased antigen expression in PBL cultures was associated with an increased number of circulating B-lymphocytes, irrespective of whether or not an animal had lymphocytosis. This supported the hypothesis that BLV-infected cattle that are PBL-ELISA positive are more infectious and may present a greater risk of transmitting the disease. The applicability of the PBL-ELISA to a field situation was assessed with 98 BLV-infected cattle from three commercial dairy herds with infection prevalences of 11%, 23% and 47%. Similar percentages (49%, 50% and 52%) of PBL-ELISA positive cattle were identified among those infected cattle available for testing in the three herds. An additional 22 infected cattle from an experimental herd were tested to assess the stability of antigen expression levels over an 8 month period. Fewer (27%) of these cattle were identified as PBL-ELISA positive and antigen expression levels were generally lower than those observed in the commercial herds. Antigen expression levels in the experimental herd remained stable over the period of the study. The potential of the PBL-ELISA to assist in BLV eradication programs by identifying those seropositive cattle with the greatest potential to transmit infection is discussed.     

Detection of duck plague virus by reverse passive hemagglutination test

A reverse passive hemagglutination (RPHA) test was developed to detect duck plague virus (DPV). The technique used sheep erythrocytes stabilized with formaldehyde and pyruvaldehyde and coated with immunoglobulin G (IgG) containing anti-DPV antibody prepared from antiserum produced in sheep. Optimum coating of stabilized erythrocytes occurred at 25 C and pH 4.0 with a concentration of IgG of 20-40 micrograms/ml and a 90-min incubation period. The coated cells were stable for 40 days when stored at 4 C or for at least 4 months (the longest period tested) when frozen at -70 C or -196 C. The RPHA test was conducted at 25 C and read after 3 hours. The high specificity of the test is indicated by the absence of cross-reactions with heterologous virus strains, with specimens prepared from normal duck livers, and with normal chicken embryo chorioallantoic fluid, as well as by the inhibition of hemagglutination only with DPV antiserum. The RPHA test detected six strains of DPV in all virus-containing specimens as well as the immunofluorescence (IF) test did; however, conventional plaque assays (PA) failed to detect virus in five specimens that contained three non-plaque-forming strains of DPV. The mean quantity of DPV that could be detected in the RPHA test was 25 plaque-forming units or 65 fluorescent units per ml. Although the RPHA test was less sensitive than either the PA or the IF test, there was a positive correlation in the titers of DPV antigens between all three tests. The RPHA test is a rapid, simple procedure that is sufficiently sensitive for diagnostic detection of DPV in acute infections, especially in tissues of ducks dying of the disease.     

Ovine and murine T cell epitopes from the non-structural protein 1 (NS1) of bluetongue virus serotype 8 (BTV-8) are shared among viral serotypes

Bluetongue virus (BTV) is a non-enveloped dsRNA virus that causes a haemorrhagic disease mainly in sheep. It is an economically important Orbivirus of the Reoviridae family. In order to estimate the importance of T cell responses during BTV infection, it is essential to identify the epitopes targeted by the immune system. In the present work, we selected potential T cell epitopes (3 MHC-class II-binding and 8 MHC-class I binding peptides) for the C57BL/6 mouse strain from the BTV-8 non-structural protein NS1, using H2^b-binding predictive algorithms. Peptide binding assays confirmed all MHC-class I predicted peptides bound MHC-class I molecules. The immunogenicity of these 11 predicted peptides was then determined using splenocytes from BTV-8-inoculated C57BL/6 mice. Four MHC-class I binding peptides elicited specific IFN-γ production and generated cytotoxic T lymphocytes (CTL) in BTV-8 infected mice. CTL specific for 2 of these peptides were also able to recognise target cells infected with different BTV serotypes. Similarly, using a combination of IFN-γ ELISPOT, intracellular cytokine staining and proliferation assays, two MHC-class II peptides were identified as CD4⁺ T cell epitopes in BTV-8 infected mice. Importantly, two peptides were also consistently immunogenic in sheep infected with BTV-8 using IFN-γ ELISPOT assays. Both of these peptides stimulated CD4⁺ T cells that cross-reacted with other BTV serotypes. The characterisation of these T cell epitopes can help develop vaccines protecting against a broad spectrum of BTV serotypes and differentiate infected from vaccinated animals.