Yoshimi Kawakami

Detection and identification of equine herpesvirus-1 and -4 by polymerase chain reaction

A rapid method for detection and identification of equine herpesvirus-1 and -4 (EHV-1 and EHV-4) was developed using polymerase chain reaction (PCR). Primers for PCR were designed from aligned nucleotide sequences of glycoprotein B genes of EHV-1 and EHV-4 to amplify specific regions for EHV-1 or EHV-4 or a common region of both viruses. By using type specific primer mixture, amplified fragments were identified as EHV-1 or EHV-4 in a one-step reaction. We have applied this technique on specimens from aborted fetuses. The samples contained only EHV-1 and there was complete accordance between the results of PCR and virus isolation. Our PCR system could differentiate the two virus types rapidly in a one-step reaction.

The genomic diversity among equine herpesvirus-1 strains isolated in Japan

SummaryThe DNAs from nine Japanese field isolates of equine herpesvirus-1 (EHV-1) were analyzed by digestion with the restriction endonuclease Bam HI and Southern hybridization. Comparing restriction profiles among the EHV-1 strains, there was no considerable difference between isolates before and after vaccine application, but some minor variations in the mobility ofBam HI fragments were observed. To identify these variable fragments, all genomic DNA sequences of the Japanese prototype of EHV-1 have been cloned asBam HI restriction fragments into the plasmid pUC-18. Physical maps of the virus DNA were constructed by a combination of Southern blot analysis and double enzyme digestion of the cloned fragments. By using these cloned fragments as probes in Southern blot analysis, the areas of heterogeneity observed among the field EHV-1 isolates were located in both terminals of UL, the center of UL, IR, US and TR regions of the genome.

Changes in the hybridization patterns of populations of Theileria sergenti during infection.

Restriction fragment length polymorphisms (RFLPs) of Theileria sergenti DNA were analysed using probes of a genomic DNA fragment (pTs 2) and a cDNA corresponding to this genomic probe (C-Ts 2). Each of the probes detected RFLPs in DNA from different stocks of Theileria sergenti. Additionally, using these probes, alterations in hybridization patterns were observed in samples of the parasites harvested at different times after individual calves had been infected with Theileria sergenti. This result suggests that the Theileria sergenti stocks used were mixed parasite populations.

Genomic analysis of Theileria sergenti stocks in Japan with DNA probes

Restriction fragment length polymorphisms of Theileria sergenti DNA from 18 different infections of cattle in 14 locations in Japan were analyzed by Southern blotting using T. sergenti genomic DNA fragments as probes. Probe pTs 2 hybridized with four fragments in BamHI digested piroplasm DNA, at 8.0, 7.3, 6.0 and 3.4 kb. Probe pTs 11-D1 hybridized with multiple fragments. With each probe, polymorphisms were observed among stocks from different locations. However, there was no correlation between the patterns of hybridization bands and the locations where parasites were collected. Analysis of the hybridization patterns of stocks obtained from individual cattle in the same grazing areas showed an almost identical pattern.

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.

Establishment of B-cell lines from tumor of enzootic bovine leukosis.

Two lymphoid cell lines were established from enzootic bovine leukosis tumor cells. Suspension cell cultures of these cell lines have been maintained in vitro for over 2 yr. The cell grew as floating cells without attaching to the glass surface. These 2 cell lines have B-cell surface marker, tumor-associated antigen on the cell surface and bovine leukemia provirus in the genomes.

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.

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.

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.

Responses of peripheral blood mononuclear cells in calves infected with Theileria sergenti

The role of peripheral blood mononuclear cell (PBMC) in Theileria sergenti-infected calves was studied by various in vitro assay systems. Proliferation of T cells in mixed lymphocyte protozoa culture (MLPC) increased with parasitemia, and the addition of monoclonal antibodies against T. sergenti merozoites in this MLPC enhanced the response. However, the addition of antibody-positive autologous serum resulted in the suppression of the response. Cell-mediated cytotoxicity of PBMC increased after peak parasitemia. This cytotoxicity increased on co-cultivation of PBMC with T. sergenti merozoites, but the addition of autologous serum suppressed the response.