Misao Onuma

Activation of bovine peripheral blood macrophages in Theileria sergenti-infected calves

Macrophage activation in Theileria sergenti-infected calves was studied by testing the production of oxygen metabolites in macrophages following specific and non-specific stimulation with T sergenti merozoites or zymosan, respectively. Six calves were inoculated with merozoites and three calves with sporozoites. All showed significant macrophage activation within one month after inoculation (P less than 0.05). Activation of macrophages appeared earlier than parasitaemia or the peak of antibody titre against T sergenti. The highest chemiluminescence response, indicative of macrophage activation, was observed when the merozoites were opsonised with immune sera.

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.

Immunohistological demonstration of virus and tumor associated antigens in tissues in experimental and spontaneous Bovine leukemia virus (BLV) infection

Expression of bovine leukemia virus (BLV) antigens in vivo has not been shown. After BLV infection, however, production of antibodies directed towards BLV proteins (e.g. gp51) can be easily demonstrated. Thus, production of BLV proteins has to take place somewhere in infected cattle. Tissues and organs of experimentally infected cattle were fixed in acetone and embedded in paraffin. Monoclonal antibodies directed to gp51 were used to demonstrate BLV expression immunohistologically by the peroxidase-antiperoxidase (PAP) method. The same samples were also used to demonstrate a tumor associated antigen (TAA) employing a monoclonal antibody. Our results indicate that very few cells, found in the intestinal mucosa, produce gp51 in vivo. The expression of TAA, however, increases significantly shortly after infection with BLV and remains high throughout life.

Detection of Cross‐Reactive Antibody to BLV p24 in Sera of Human Patients Infected with HTLV

For detection of antibody to bovine leukemia virus (BLV) major core protein of p24 and cross‐reactive antibody in human patients infected with human T cell leukemia virus type I (HTLV‐I), monoclonal antibody, D432 against BLV p24 was used by competitive binding enzyme‐linked immunoadsorbed assay (ELISA). In sera from cattle with enzootic bovine leukosis (EBL) which were positive for BLV antibodies by immunodiffusion test, 109 out of 112 (97.3%) were positive for BLV p24 antibody by competitive binding ELISA. By using the same procedures, 21 samples from adult T cell leukemia (ATL) patients and healthy carriers with HTLV‐I were tested for cross‐reactive antibody to BLV p24. All 21 samples were positive for HTLV‐I antibodies by immunofluorescence test and/or ELISA. By competitive binding ELISA using non‐treated BLV antigens, none of these 21 samples inhibited the binding of the D432. When the BLV antigen was treated by several different denaturation procedures, several HTLV‐I positive samples showed the inhibition of the D432 binding and the most effective treatment was by 2‐mercaptoethanol (2‐ME). Sixteen out of 21 samples showed the presence of cross‐reactive antibody against 2‐ME‐treated BLV antigens. The cross‐reactivity of human sample to BLV p24 antigen was further confirmed by Western blotting of the 2‐ME‐treated BLV antigens. None of the 28 samples from leukemia patients other than ATL which were negative for HTLV‐I antibodies showed inhibition of the D432 by the competitive binding ELISA.

Effect of platelet-derived factor on expression of bovine leukemia virus genome.

SummaryPlasma of cattle infected with bovine leukemia virus (BLV) contains a factor, plasma blocking factor (PBF), that inhibits the expression of viral genome in cultured lymphocytes from BLV infected cattle. When platelet lysate was added to this culture, BLV antigen became detectable in the culture and there are some factors (PDF) in platelet lysate which have inhibitory activity against PBF. The PDF was present in platelet from BLV-free cattle as well as BLV-infected cattle at relatively high titer. The effect of platelet lysate against PBF on the expression of BLV genome seemed to be irreversible.

The Screening of Cattle with Potential for Developing Leukemia by Using Monoclonal Antibody against Bovine Leukemia Cells

Tumor cells from cattle with enzootic bovine lymphosarcoma (EBL) have a tumor‐associated antigen (TAA) which is distinct from bovine leukemia virus (BLV)‐induced antigens. We were able to sacrifice 8 TAA‐positive cattle with no clinical signs of EBL and to examine whether or not they had gross or histological tumors. At necropsy, 4 animals had tumors macroscopically. Three animals had no tumors histologically but had initial lesions showing follicular hyperplasia and had the TAA on affected lymph nodes. The remaining one showed medullary hyperplasia in the spleen but there were no findings of tumors. These results suggest that most BLV‐infected cattle which are TAA‐positive but have no clinical signs of EBL, do have tumors and have a higher potential for developing EBL in the future when compared to BLV‐infected but TAA‐negative cattle.

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.

Regression of bovine lymphosarcoma by treatment with cell-wall skeleton of Nocardia rubra

Five bovine-leukaemia-virus-positive cattle with enlarged subcutaneous lymphatic nodules and having tumour-associated antigens (TAA) in their peripheral blood lymphocytes (PBL) were treated by injection of the cell-wall skeleton of Nocardia rubra (N-CWS) into the nodules. All treated animals received two or three injections of N-CWS (each 0.5-4 mg per nodule) at 2 or 4-week intervals. The effect of the treatment was evaluated by the size of the nodules. Complete regression of nodules was observed in seven out of ten nodules treated in five animals. Decrease of TAA-positive cells was also observed in their peripheral blood lymphocytes for all five treated animals. In one cow, the TAA-positive cells remained low for at least 280 days after treatment.

The Effect of GD3 Ganglioside Obtained from Bovine Lymphosarcoma on Bovine Normal Mononuclear Cell

The effect of immunological function of GD3 on normal bovine lymphocyte was examined with various in vitro assay systems. The GD3 level in sera from enzootic bovine leukemia (EBL) cattle was significantly increased compared with that of normal cattle (EBL: 0.62±0.24 μg/ml; normal cattle: 0.33±0.09 μg/ml, P<0.05). Lymphocyte blastogenesis elicited by concanavalin A was inhibited by addition of a 50 μg/ml concentration, or more, of GD3. Inhibitory effect of GD3 in IL‐2‐dependent T cell line and EBL tumor cell line was hardly observed compared with normal peripheral blood mononuclear cells. GD3 also inhibited mixed lymphocyte reaction and allo cytotoxic T lymphocyte reaction.

Chemotherapy and immunotherapy of Bovine leukosis

To prevent the progression of the disease, we treated leukemic or preleukemic cows with (1) adriamycin (ADM) entrapped in liposomes conjugated with monoclonal antibody, c143, against tumor-associated antigens (TAA) of bovine leukemic cells and (2) an immunotherapy using an immunopotentiator consisting of the cell wall skeleton of Nocardia rubra (N-CWS). Five leukemic or preleukemic cows with TAA-positive peripheral blood lymphocytes (PBL) received four injections of ADM alone (0.4 mg/kg body weight) or c143-conjugated liposomes containing the same dose of ADM (L-ADM-c143) through the jugular vein at about 4-day intervals. In three animals treated with L-ADM-c143, the TAA-positive cells gradually decreased with treatment and finally two animals became TAA-negative during a 6-week period and a 14-week period after treatment, respectively. About 6 weeks later, however, TAA-positive cells gradually increased. In the control, two animals treated with ADM alone showed no decrease of TAA-positive cells. Five TAA-positive animals with enlarged subcutaneous lymphatic nodules, each nodule estimated to be from 1 to 4 cm3 in size, were treated by injection of N-CWS into the tumors. Complete regression of tumor was observed in seven out of ten tumors treated in five animals. Decrease of TAA-positive cells was also observed in PBL for all five treated animals. In one animal, the TAA-positive cells remained low for at least 280 days after treatment. This study documents that ADM treatment and intralesionally administered N-CWS are effective in the treatment of bovine leukosis.