Existence of cytotoxic activity against BLV-transformed cells in lymphocytes from normal cattle and sheep

Peripheral blood lymphocytes (PBL) from normal cattle and sheep were tested for their cytotoxic activity against several target cells using a 20-hour 51Cr release assay. The following characteristics of the effector cells were observed; 1) PBL from animals showed cytotoxic activity against two sheep cell lines (FLK and SF-28) that were transformed with bovine leukemia virus. However, normal sheep and bovine cells and Molony leukemia virus-induced mouse lymphoma cell line (YAC-1) were not killed by these cells. 2) A time course study showed that the activity was first observed at 4 to 8 hours and reached a maximum at 20 to 30 hours after incubation. 3) Cytotoxic activity was observed in both adherent and nonadherent cell fractions when PBL were passed through a nylon-wool column. This indicated that the effector cells showed some degree of adherence. 4) Treatment of PBL with carrageenan did not change the cytotoxic activity against target cells, indicating that phagocytic capability is not perhaps necessary for cytotoxicity to take place. These results indicate that the effector cells participating in the cytotoxic reaction resembled natural killer cells or natural cytotoxic cells which are present in murine and human systems. However, analysis of the cell surface markers of the effector cells is yet to be done in future studies.     

Spontaneous and mitogen-induced RNA synthesis by blood lymphocytes from bovine leukemia virus-infected and normal cows

Peripheral blood lymphocytes (PBL) from normal and bovine leukemia virus (BLV)-infected cattle were prepared by density gradient technique and incubated with and without phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). RNA synthesis was determined at different periods of incubation by 3H-uridine incorporation. PBL from BLV-infected cows with persistent lymphocytosis (PL) showed the highest spontaneous RNA synthesis. PBL from BLV-infected cows with normal lymphocyte counts synthesized more RNA than cells from normal animals. Decreased mitogen responses were observed in PBL from infected cows with PL in comparison to normal and BLV-infected cattle without PL. PHA and PWM did not show significant differences in their degree of stimulation of RNA synthesis.     

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.     

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.     

Investigation of the possible role of the tuberculin intradermal test in the spread of enzootic bovine leukosis

The single intradermal comparative test was used with both avian and bovine tuberculin. Three cattle infected with bovine leukosis virus (BLV) were used as a source of infection. BLV-positive and susceptible animals were tuberculin tested alternately. Fifteen susceptible calves and 15 susceptible sheep were tested. A further three calves and three sheep were used as controls; the needles of the tuberculin syringes were deliberately contaminated with blood from the BLV-infected cattle, before being used in the test. Whereas all three calves and the three sheep inoculated intradermally with contaminated needles developed BLV infections, all of the other 30 animals have remained serologically negative to BLV for 10 months. Transmission of BLV with needles contaminated with BLV-infected blood was prevented by wiping the needles with absorbent cotton wool.     

Development and serial passage of persistent lymphocytosis associated with bovine leukemia virus infection in cattle

Two calves each were inoculated with 1.5 x 10(8) or 5 x 10(9) lymphocytes collected from each one cow which had persistent lymphocytosis (PL) and antibodies to bovine leukemia virus (BLV). A sudden increase in the number of peripheral blood lymphocytes (PBL) was observed 14 and 23 days, respectively, after inoculation and the maximum number reached 29,000 and 52,000/microliters 72 and 57 days after inoculation. Although the degree of PL decreased gradually in these cattle, it continued until 14 and 44 months after inoculation when one animal was sacrificed and the other died of lymphosarcoma. The PL was passaged in cattle by inoculation of a large number of PBL obtained from cattle at the stage of PL (PLL). The degree of PL was severer in cattle inoculated with a larger number of PLL. PL was not caused by inoculation of PBL obtained from either BLV-infected non-PL cattle or cattle free of BLV. The PL was also caused by inoculation of PLL into BLV-infected non-PL cattle. On the other hand, it was not observed after inoculation of a large amount of cell-free virus obtained from short-term cultures of PLL. Antibodies to BLV developed earlier and to higher levels in cattle inoculated with PLL than in those inoculated with cell-free virus. These facts show that infection with BLV was established more effectively by PLL than by cell-free virus, the infection may occur by lymphocyte to lymphocyte interaction and the actual number of infected BLV may have an important role in development of PL.     

Further phenotypic characterization of target cells for bovine leukemia virus experimental infection in sheep

To determine the phenotype of target cells for bovine leukemia virus (BLV) infection in sheep, we analyzed blood lymphocytes from BLV-infected clinically healthy and leukemic sheep by use of monoclonal antibodies. In clinically healthy and leukemic sheep that were BLV-infected, the blood concentration of T lymphocytes was within normal values, but the number of B lymphocytes was increased in several cases. In addition, the number of blood lymphocytes expressing the BLV antigen correlated well with that of B lymphocytes. Double immunofluorescence staining demonstrated that lymphocytes expressing BLV antigens bore B-cell but not T-cell surface markers. Moreover, neoplastic cells in the lymph nodes of leukemic sheep were stained immunohistochemically with an anti-B monoclonal antibody but not with any of anti-T monoclonal antibody tested, indicating that tumor cells are of B-lymphocyte origin. Collectively, these results show that BLV antigen-positive cells obtained from BLV-infected sheep that have no clinical signs and BLV-induced lymphosarcoma cells belong to the B-lymphocyte lineage.     

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.     

Circulating immune complexes in bovine leukemia virus (BLV)-infected cattle.

Circulating immune complexes (ICs) were detected in the sera of bovine leukemia virus (BLV)-seropositive cattle. Immune complexes were precipitated in 2.5% polyethylene glycol (PEG) and further dissociated. Bovine leukemia virus antigens, IgG and IgM molecules were detected after solubilization in the presence of sodium dodecyl sulphate, and quantitated by enzyme-linked immunosorbent assay (ELISA) assays. Mean values of IgG and IgM in BLV-containing ICs did not significantly differ from those obtained from ICs originating from BLV-seronegative animals. However, differences were found in the composition of ICs from older BLV-positive animals as compared to those obtained from young animals. The ratio of IgG/IgM was 5.02 in animals aged 5-10 years, while this ratio was 11.66 in animals of less than 5 years of age and 10.19 in controls. This might indicate a possible increase in the contribution of IgM molecules to the structural composition of ICs in BLV-infected cattle as related to age or stage of infection.     

Experimental infection of sheep with bovine leukemia virus: infectivity of blood, nasal and saliva secretions

This study was designed to determine the relative infectivity of lymphocytes and secretions from BLV-infected cattle with and without persistent lymphocytosis (BLV+PL+ and BLV+PL-). Ninety-seven sheep of mixed sex and age were assembled into 21 experimental groups. The recipient sheep were inoculated intravenously with serial dilutions of whole blood, saliva or nasal secretions from BLV+PL+ and BLV+PL- donor cows. Between 200 to 20,000 cells from single and mixed BLV+PL+ or single and mixed BLV+PL- donor cattle were used for inoculation. A very small number of BLV-infected lymphocytes (200 cells) was sufficient to induce BLV infection in sheep inoculated with diluted whole blood from BLV+PL+ cattle. The inoculation of whole blood (containing up to 20,000 lymphocyte cells) from BLV+PL- cattle did not induce BLV infection in recipient sheep. Saliva and nasal secretions also failed to bring about BLV transmission.     

Tumor-associated antigen on bovine leukemia virus-induced bovine lymphosarcoma

Specific tumor-associated antigen (TAA) was detected on enzootic bovine leukosis (EBL) cells by monoclonal antibodies against TAA. One of the monoclonal antibodies, c143, reacted with all EBL tumor cells tested but not with bovine leukemia virus (BLV) antigens. c143 reacted slightly with bovine fetal thymus and mitogen-stimulated lymphocytes from BLV-free cows but not with normal bovine lymphoid cells. TAA may be a good tumor marker of EBL tumor cells. We sacrificed eight TAA-positive but clinically normal animals and examined them in order to elucidate whether or not they had gross or histological tumors. At necropsy, four animals had tumors macroscopically. Three animals had no tumors histologically but had initial lesions showing follicular hyperplasia and the TAA on affected lymph nodes. The one remaining showed medullary hyperplasia in the spleen but there were no findings of tumors. Thus, c143 is a useful tool not only for diagnosing EBL, but also for screening of BLV-infected cattle with potential to develop tumors in the future.     

Suppression of immunological responses in rabbits experimentally infected with bovine leukemia virus

Ten 2- to 4-month-old rabbits were inoculated subcutaneously with bovine leukemia virus (BLV)-infected bovine or sheep cells. By 6 weeks after inoculation all ten rabbits had converted to BLV antibody-positive, and BLV or BLV antigen was detected in lymphocytes from most of the rabbits tested, although there were few antigen-producing cells. Three rabbits showed continuous respiratory symptoms after infection and one died with pneumonia. Humoral immune responses against mouse serum were significantly suppressed in BLV-infected rabbits compared with non-infected control rabbits. The lymphocyte blastogenesis response was also suppressed in BLV-infected rabbits. At the time of necropsy, six rabbits showed pulmonary lesions; however, none of the BLV-infected rabbits had tumors during an observation period of over 1 year.     

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.     

Non-specific natural cytotoxic factor released from bovine peripheral blood lymphocytes.

Natural cytotoxicity against bovine leukemia cells (PC-3 cells) was found in bovine peripheral blood lymphocytes (PBL), and in non-adherent cells but not in adherent cells to nylon-wool column. Natural cytotoxic cells (NCC), which have natural cytotoxic activity, are found in T cell-rich fraction. When NCC were cocultured with PC-3 cells, natural cytotoxic factor (NCF) was released rapidly from NCC, and dose-response curve for NCF was almost linear induction. Cytotoxicity against PC-3 cells by NCC or NCF was increased with an increment of incubation period. Cytotoxicity against K562 cells, CL-1 cells, M1 cells or EL-4 cells by NCF was almost the same level as that against PC-3 cells, but that against those cell lines by NCC was not found. NCF activity in culture fluid from NCC cocultured with K562 cells or CL-1 cells was lower than that from NCC cocultured with PC-3 cells.     

Antibody seroprevalences against peste des petits ruminants (PPR) virus in camels, cattle, goats and sheep in Ethiopia

A questionnaire-survey data indicated that 26% of 276 farmers reported the presence of respiratory disease in their herds in 2001. The incidence was perceived as "high" in small ruminants and camels, but as "low" in cattle. Simultaneously, 2815 serum samples from camels (n=628), cattle (n=910), goats (n=442) and sheep (n=835) were tested. The peste des petits ruminants (PPR) antibody seroprevalence was 3% in camels, 9% in cattle, 9% in goats and 13% in sheep. The highest locality-specific seroprevalences were: camels 10%, cattle 16%, goats 22% and sheep 23%. The animals had not been vaccinated against rinderpest or PPR. Antibody seroprevalences detected in camels, cattle, goats and sheep confirmed natural transmission of PPR virus under field conditions.     

Seroepidemiological study of Rift Valley fever (RVF) in animals in Saudi Arabia

Serological prevalence of IgG antibodies against Rift Valley fever (RVFV) virus was investigated in 22 major localities in five different regions of Saudi Arabia where vaccination against RVF virus (RVFV) is not practiced. The study excludes the southwestern region where a major outbreak of RVF occurred in 2000 and where annual vaccination of ruminants is practiced. Sheep and goat IgG-sandwich ELISA were used to test serum samples from sheep and goats, and bovine IgG-sandwich ELISA was used to test cattle sera. A nonspecies-specific, nonantibody isotype-specific ELISA was used to test camel sera. A total of 3,480 sheep, goats, cattle and camels with no previous history of vaccination against RVFV were randomly tested. All tested animals were negative for IgG class antibodies against the virus except four out of 1,508 sheep and three out of 913 goats, which tested positive. All animals were clinically normal and no evidence was found of virus activity in the studied areas. It is, therefore, most likely that those rare positive cases, which constituted 0.002% of the total animals tested, were either false positives or vaccinates smuggled from the outbreak zone. The need for regular monitoring of animals both within the outbreak zone of 2000 and other parts of the kingdom is strongly emphasized.     

Comparative activities of semicarbazide-sensitive amine oxidase (SSAO) in five domestic species

Semicarbazide-sensitive amine oxidase (SSAO) activity was measured spectrophotometrically using benzylamine as a substrate, in the serum of healthy males and females of horses, camels, cattle, sheep and goats. The animals were born and raised in the same area, and the blood collection was made on the same day to avoid variations. Also the concentrations of protein and copper were measured in the same samples. There were no significant gender-related differences in SSAO activity between the tested animals regardless of species. Activities of SSAO in either male or female of horse were significantly different (p < 0.05) from the remaining tested animals. The highest activities (expressed as micromole of benzaldehyde production/mg protein/hr) were found in horses (9.592 and 9.458), followed by camels (3.226 and 2.407), cattle (1.014 and 1.648), goat (0.750 and 0.572) and sheep (0.435 and 0.244). Insignificant higher activities of SSAO were noted in all males of the tested animals compared to that in females except in cattle. The results suggest that horses are endowed with a very high activity of this enzyme amounting to 3-21 times higher than that found in large and small ruminants. There were no significant differences between the levels of protein and copper in either sex of all the tested species.     

Detection of B and T cells, with lectins or antibodies, in healthy and bovine leukemia virus-infected cattle

Lectins, polyclonal antibodies and monoclonal antibodies (MAbs) were evaluated as markers for bovine lymphocytes obtained from healthy animals and from cattle infected with bovine leukemia virus (BLV). In the blood from healthy cattle the proportion of cells identified as T lymphocytes with the lectin Helix pomatia (HP) (67.8 +/- 6.2%) using the indirect immunofluorescence technique was similar to the proportion of cells identified by the MAbs P5 (66.1 +/- 3.8%) and BLT-1 (59.8 +/- 7.1%). The proportion of B cells in blood from healthy animals identified with a polyclonal antibody to bovine IgM (18.0%) was similar to that identified with a MAb to bovine IgM (16.2%). However, greater variation between individual values was detected with the MAb (SD = 8.2) than with the polyclonal antibody (SD = 4.0). In the blood from BLV-infected cattle with persistent lymphocytosis, both the polyclonal and the MAb revealed a threefold increase of B cells. A proportion of the B cells had an increased amount of immunoglobulin molecules in their plasma membrane as indicated by flow cytometry. The proportion of T lymphocytes, identified by the MAb P5, was reduced to one-third of that in non-infected cattle. The indirect HP labelling gave inconsistent results and seems not to detect solely T lymphocytes among blood lymphocytes from BLV-infected cattle.