Takane Matsui

Isolation of Neospora caninum from the brain of a pregnant sheep.

Neospora caninum was isolated from the brain of a naturally infected pregnant sheep by inoculation of immunodeficient mice with a homogenate of the brain tissue. The ewe showed no clinical signs. Tachyzoites were observed in the tissues of the nu/nu mice injected with the brain tissue homogenate and the diagnosis was confirmed by immunohistochemical staining with anti-N. caninum antibodies and by detecting N. caninum-specific DNA by polymerase chain reaction.

Integration of region X of hepatitis B virus genome in human primary hepatocellular carcinomas propagated in nude mice.

Tissues of human primary hepatocellular carcinoma (PHC) from six patients infected with hepatitis B virus (HBV) were propagated in nude mice, as well as a strain of hepatitis B surface antigen-positive PHC (PLC/PRF/5). Integration of viral DNA into chromosomal DNA of tumour cells was evaluated by the capacity to hybridize with radiolabelled DNA probes, each representing fundamental parts of the HBV genome, that is S and C genes and regions pre-S and X. All PHC cells possessed region X integrated in their chromosomes. However, integration of the S gene, C gene and region pre-S was found in only six of the seven PHCs. Based on these findings, the integration of region X seems to be most closely associated with carcinogenesis in HBV infection.

Unexpectedly high incidence of visceral AA-amyloidosis in slaughtered cattle in Japan.

Experimental mouse AA amyloidosis can be transmissible by dietary ingestion of amyloid fibrils and it is well known that AA amyloidosis occasionally develops in aged cattle. Bovine liver and intestine have conventionally been used in Oriental foods, and the incidence of visceral AA amyloidosis in slaughtered cattle was evaluated. Renal tissues from 302 aged cattle older than 4 years were obtained from a local abattoir. Amyloid deposition was microscopically examined and amyloid protein was immunochemically determined. Renal amyloid deposition was seen in 15 out of 302 cattle with no previous history of diseas, an incidence of 5.0%. Amyloid protein in these cattle was AA and they had pathological findings in their visceral organs on gross examination. The incidence of visceral AA amyloidosis in slaughtered cattle in this study was disturbingly high compared with those (0.4–2.7%) previously reported from Japan and other foreign countries. AA amyloidosis is a life-threatening complication in patients with chronic inflammatory diseases and these patients at risk should avoid ingesting food that may possibly contain AA amyloid fibrils. More detailed information on cattle amyloidosis is required to guarantee the safety of our food.

Prevention of Escherichia coli O157:H7 infection in gnotobiotic mice associated with Bifidobacterium strains

Previous reports have shown that Escherichia coli O157:H7 infection is strongly modified by intestinal microbes. In this paper, we examined whether bifidobacteria protect against E. coli O157:H7 infections using gnotobiotic mice di-associated with Bifidobacterium strains (6 species, 9 strains) and E. coli O157:H7. Seven days after oral administration of each Bifidobacterium strain, the mice were orally infected with E. coli O157:H7 and their mortality was examined. Bifidobacterium longum subsp. infantis 157F-4-1 (B. infantis 157F) and B. longum subsp. longum NCC2705 (B. longum NS) protected against the lethal infection, while mice associated with all other Bifidobacterium strains, including type strains of B. longum subsp. infantis and B. longum subsp. longum, died. There were no significant differences in the numbers of E. coli O157:H7 in the faeces among the Bifidobacterium-associated mouse groups. However, the Shiga toxin concentrations in the cecal contents and sera of the GB mice associated with B. infantis 157F and B. longum NS were significantly lower than those of the other groups. However, there were no significant differences in the volatile fatty acid concentrations and histopathological lesions between these two groups. These data suggest that some strains of B. longum subsp. longum/infantis can protect against the lethal infections of E. coli O157:H7 by preventing Shiga toxin production in the cecum and/or Shiga toxin transfer from the intestinal lumen to the bloodstream.

Differentiation analysis of bovine T-lymphosarcoma☆

The tumor cells from three different types (calf, skin and thymic) of bovine lymphosarcoma identified as sporadic bovine leukosis (SBL) were transplanted to nude mice and established as lymphoid tumor lines. To clarify the T-cell lineage, clonality and differentiation stage, these cell lines were examined for the immunophenotypic profile, the expression and the gene rearrangement of T-cell receptor (TCR) genes. The tumor cells from all three types showed alpha-naphthyl acetate esterase-positive staining and the absence of surface immunoglobulin (sIg). Moreover, the tumor cells appeared to be all BoCD5+ BoCD6+ BoCD4- BoCD8- cells in the FACS analysis with mAbs. Rearrangement of TCR-beta and -delta, but not of TCR-alpha and -gamma, genes was found to be unique for each tumor line showing T-cell clonality. Mature or truncated TCR-beta, -gamma and -delta transcripts were detected in the tumor cells, but all of them were found to be non-functional in cDNA cloning analysis. These results suggested that tumor cells from SBL are immature double-negative T-cells expressing non-functional TCR gamma delta transcripts to somewhat differentiated degrees.

Histopathological and immunohistochemical studies of intracranial nervous-system tumours in four cattle

Intracranial nervous-system tumours were diagnosed in three of 1092 bovine necropsy specimens submitted to the Department of Veterinary Pathology, Obihiro University between April 1983 and March 1996. A fourth case was a referral from the Department of Veterinary Pathology, Rakuno Gakuen University. Histopathological examination revealed four types of tumour: intracranial malignant peripheral nerve sheath tumour (MPNST), choroid plexus papilloma, differentiated fibrillary astrocytoma and anaplastic (malignant) astrocytoma. Immunohistochemically, the intracranial MPNST was strongly positive for S-100 protein and vimentin, and in places weakly positive for glial fibrillary acid protein (GFAP). The choroid plexus papilloma was strongly positive for epithelial membrane antigen (EMA), keratin, S-100 protein and vimentin, and positive for GFAP in places. The cytoplasm and fibrous component in the differentiated fibrillary astrocytoma were strongly positive for S-100 protein and GFAP. The anaplastic (malignant) astrocytoma was strongly positive for vimentin, S-100 protein and keratin in the cytoplasm and fibrous processes, and weakly positive for GFAP and EMA in places. Myelin basic protein (MBP) and synaptophysin showed a weak positive reaction in the marginal areas of the tumour.

p53 mutation as a potential cellular factor for tumor development in enzootic bovine leukosis

Mutations of p53 in the lymphocytes from peripheral blood and from tumoral lymph nodes in six naturally occurring bovine leukemia virus (BLV)-infected cows were examined. A point mutation of the p53 gene was found in three of six (50%) BLV-infected cows. These p53 gene mutations resulted in amino acid substitutions of codons 144, 167 and 241. The BLV-infected cow in the tumor stage had abnormally proliferating monoclonal B-lymphocytes having the p53 mutation. However, the mutation was not found in somatic cells, except for tumor cells. These results show that p53 mutation plays an important role in the pathogenesis of BLV-induced neoplasms, and that the B-lymphocyte bearing p53 mutations may be a target cell for tumor formation of enzootic bovine leukosis.

Bovine herpesvirus meningoencephalitis association with infectious bovine rhinotracheitis (IBR) vaccine

During 1992, on a farm in the Tokachi district of Hokkaido, Japan, approximately 20 Holstein-Friesian calves showed neuroparalysis and died within 7–10 days after routine vaccination. Six male calves, aged about 1.5 months, were submitted to our laboratory for pathological examination and diagnosed as acute or subacute necrotizing meningoencephalitis due to bovine herpes virus (BHV) infection. The main necropsy findings included a few hemorrhages or clots, and malacic lesions localized in the cortical to subcortical area of the cerebrum. Histopathological brain lesions were characterized by laminar or focal necrosis of neurons, accompanying macrophages, polymorphonuclear cell infiltration, severe astrogliosis, and perivascular cuffing in all six calves. Nuclear basophilic inclusion bodies, which showed positive reaction with immunocytochemical staining of BHV antigen, were observed in the necrotic neurons, astroglia and oligodendroglia in five affected calves. BHV antigens were also seen in the cell bodies and cell processes of the necrotic neurons, which was indicative of cell-to-cell propagation of infection. There was a general tendency for more severe lesions to be located at the cortex to subcortex of the cerebrum. Milder lesions were observed in the cerebellum and brain stem. These findings suggest that the infectious route to the cerebrum in the present cases was through the olfactory bulbs and/or along the meninges beginning from the ethmoid bone, rather than through the trigeminal ganglia route as had been emphasized in studies dealing with experimental infection.

Putative bovine B cell lineage tumor in sporadic bovine leukosis

Two calves (C924 and C928) clinically identified as suffering from the calf type of sporadic bovine leukosis (SBL) were examined for immunophenotypical distribution of their peripheral blood lymphocytes (PBL) with T cell or B cell specific monoclonal antibodies (mAbs). Flow cytometric profile analysis indicated that both calves possessed a high population of MHC class II positive cells and a low population carrying T cell associated markers. More than 93% of the PBL of C928 were positive for MHC class II and BoCD5, but were not IgM positive, indicating that clonal tumor cells (MHC-II+, BoCD5+ and IgM-) accounted for most of the PBL of C928. A tumor cell line, BLC3, established from calf C924 in nude mice was examined for immunophenotype, DNA rearrangement of immunoglobulin (Ig) genes and expression of cellular oncogenes c-abl, c-myc and c-myb. BLC3 showed a characteristic profile, being MHC class I and MHC class II positive, but without T cell associated markers. Although the rearranged configuration in the Ig loci was not clearly found in Southern blot analysis using sheep Ig probes, steady-state levels of c-myb transcripts were detected in tumor cell line BLC3 by Northern blot hybridization. Taken together, our data show that the calf type of SBL found in these two calves is induced by immature pre-B cells, including tumor cell line BLC3 established in nude mice.

Clinical experience of using multidetector-row CT for the diagnosis of disorders in cattle

To determine the clinical usefulness of multidetectorrow CT for the diagnosis of disorders in cattle, images were obtained from 27 cattle, which were then subjected to postmortem and histopathological examinations. The cattle were divided into three categories of disorder: neurological (18 cases), skeletal (four cases) and other (five cases). In five cattle, which were suspected to have brain diseases, no abnormalities were identified by either CT or histopathological examination. Eight types of lesions were detected by CT in the cattle with neurological and vestibular disorders. The diseases diagnosed included hydrocephalus (three cases), intracranial arachnoid cysts (three cases), otitis media (five cases), cerebral abscess (one case), meningoencephalocele (one case), porencephaly (one case), bicephalus (one case) and rupture of the spinal cord (one case). Lesions were identified in all the cattle with skeletal disorders, including luxation (two cases), fracture (two cases), spondylosis (one case) and congenital disorders of the skeletal system (one case). Morphological disorders in the eyes (one case), nasal cavity (two cases), frontal sinuses (one case), thyroid glands (two cases), lung fields (two cases) and abdominal organs (two cases) were diagnosed by CT.