Masanobu Goryo

Fatal necrotic enteritis associated with Clostridium perfringens in wild crows (Corvus macrorhynchos)

Sporadic outbreaks of fatal enteritis occurred among free-living wild crows (‘large billed’ or ‘wok’ crow; Corvus macrorhynchos) in an open-air park in Japan in 2002. Eight crows were found dead during February, followed by two more in September, and five of the eight were examined histopathologically. At necropsy, all cases showed a markedly dilated small intestine, especially the jejunum and ileum, with large amounts of gas, and dark red to greenish–brown soft content. The necrotic intestinal wall was markedly thickened with multifocal haemorrhages. All cases had multifocal white foci in the liver, and four cases showed marked splenomegaly. Histologically, there was severe necrotic enteritis characterized by extensive mucosal necrosis and multifocal haemorrhages, as well as inflammatory cell infiltrations. A prominent pseudo-membrane formation was noted in the affected intestine. Severe adhesive peritonitis was also observed in three cases. Gram-positive bacilli were present in large numbers in the lumen, and in and around necrotic lesions in the affected intestine. The bacilli were positive for Clostridium perfringens enterotoxin type A by immunohistochemistry, and were also positive for C. perfringens type A using the immunofluorescence method. C. perfringens was isolated by anaerobic culture from the intestinal contents. The present enteritis was thought to be induced by proliferated C. perfringens in the intestine, and to be the cause of death.

Experimental induction and oral transmission of avian AA amyloidosis in vaccinated white hens

Abstract Avian AA amyloidosis is commonly observed in adult birds afflicted with bacterial infections or chronic inflammatory disorders. Experimental AA amyloidosis in birds can be induced by repeated inflammatory stimulation, such as injection with casein or vaccination with oil-emulsified bacterins. However, the transmission of amyloidosis among avian species has not been studied well to date. In the present study, we confirm the potential induction of avian AA amyloidosis by inoculation of Salmonella enteritidis (SE) vaccine or Mycoplasma gallisepticum vaccine. To determine the transmission of chicken AA amyloidosis among white hens, we induced experimental AA amyloidosis in vaccinated chickens by intravenous or oral administration of chicken AA fibrils. Amyloid deposits were observed in chickens injected with SE and inoculated with chicken AA fibrils intravenously (21/26: 81%) and orally (8/12: 67%). These results suggest that chicken AA amyloidosis can be induced by vaccinations, and may be transmitted among like species by oral administration.

An immunohistochemical study of an equine B-cell lymphoma

The tissues of an 8-year-old thoroughbred castrated male horse with equine lymphoma were examined immunohistochemically. Neoplastic masses were observed in the mediastinum, mesenteric lymph nodes, gastric mucosa and serosa, liver capsule, and spleen capsule with associated lymph nodes. Histopathologically, the neoplastic cells were seen to consist predominantly of a mixture of well differentiated small and large types. Immunohistochemically, the small lymphoid cells were MHC class IIlow+ and PanT- and the large lymphoid cells were MHC class IIhigh+ and PanT-. These findings revealed that the neoplastic cells were of B-lymphocyte lineage.

Pathomorphologc Findings of Lead Poisoning in White-fronted Geese (Anser albifrons)

Nineteen lead-poisoned white-fronted geese (Anser albifrons), including nine immature birds, were examined pathologically. Subacute lead poisoning due to ingestion of spent lead shots was diagnosed pathologically and confirmed by demonstrating high lead concentration in the liver. The liver lead concentration ranged from 6.9 to 67.7 mg/kg wet weight. The most suggestive gross lesions were mottled bile-stained liver in eight geese and proventricular impaction and/or the presence of lead pellets in the gizzard. Histologic lesions of the liver consisted of Kupffer cell hemosiderosis, large bile plugs in dilated canaliculi, bile pigmentation in hepatocytes, and bile extravasation and associated hepatic necrosis. Seven geese of the remaining 11 birds also had hepatic necrosis in the liver, the greenish discoloration of which was obscure macroscopically. The liver discoloration was considered a jaundice due to both rapid overproduction of bile from increased breakdown of erythrocytes and intrahepatic impaired excretion of bile. The severity of lesions was not correlated to the liver lead concentrations. All examined geese had hemosiderosis of mononuclear phagocytic system cells in the spleen and hypoplasia or edema of the bone marrow with increased numbers of polychromatic erythroblasts. These prominent changes probably resulted from excess breakdown of erythrocytes, hypercholia followed by intrahepatic cholestasis, and disrupted crythropoiesis in bone marrow caused by lead.

B-1a, B-1b and conventional B cell lymphoma from enzootic bovine leukosis

In order to characterize the phenotypes of tumor cells and to clarify from which B cell lineage the lymphomas were derived, ten cows with enzootic bovine leukosis were examined by means of immunohistologic staining and flow cytometry. The tumor cells expressed mainly major histocompatibility complex (MHC) class II+ (10/10), BoCD11b+ (9/10), IgG1+ (8/10), B-B2+ (8/10) BoCD5+ (7/10), and lambda light chain+ (7/10). Tumor cells from only one animal expressed sIgM+ (1/10). Tumor cells from all ten animals were negative for IgG2, BoCD3, BoCD4, BoCD8, WC1-N2, and IL-2R alpha. The phenotypes of these tumor cells were all slightly different, suggesting that bovine leukemia virus (BLV)-induced lymphoma expresses phenotypic diversity. Moreover, tumor cells from seven cattle coexpressed BoCD5 and BoCD11b (B-1a cells). On the other hand, tumor cells from two of them only expressed BoCD11b (B-1b cells), and those from one were negative for both BoCD5 and BoCD11b (conventional B cells). Therefore, we concluded that BLV-induced lymphoma cells can be derived from B-1a, B-1b and conventional B cells.

Phenotypic Analysis of Neoplastic Cells from Calf, Thymic, and Intermediate Forms of Bovine Leukosis

Immunohistochemistry and flow cytometric analysis were used with monoclonal antibodies to examine the phenotype of neoplastic cells from cattle with sporadic bovine leukosis (three cases of calf form, two cases of thymic form, and three cases of intermediate form). Three cases of calf form and two cases of intermediate form were positive for B cell lineage in immunohistologic examination and in flow cytometric analysis for B-B2+, sIgM+, and major histocompatibility class II+. Two cases of thymic form and one case of intermediate form were CD2+, CD5+, CD6+, and CD8+ in immunohistologic examination and in flow cytometric analysis. The results show that neoplastic cells develop from B and T cell lineages in sporadic bovine leukosis.

Pathological and immunohistochemical study of chickens with co-infection of Marek's disease virus and chicken anaemia virus

Chicken anaemia virus (CAV) is the most important confounding pathogen in Mareks disease virus (MDV) infection. The effect of CAV co-infection at 4 weeks of age after inoculation of virulent MDV (vMDV, KS strain) or very virulent MDV (vvMDV, Md/5 strain) in 1-day-old chicks was investigated by pathological and immunohistochemical studies. CAV increased the mortality rates induced by vMDV or vvMDV. The packed cell volume was reduced significantly in vMDV–CAV infection; however, no reduction or non-significant reduction was observed in vMDV infection. Bone marrow hypoplasia was related to CAV co-infection and none of the birds inoculated with vMDV or vvMDV had hypoplasia. Severe atrophy of the thymus and bursa of Fabricius was observed in the vvMDV–CAV and vvMDV groups. Complete regeneration of the thymus cortex and bursa of Fabricius in the vMDV group was noted and was in contrast to sequential lymphoid depletion after CAV inoculation in the vMDV–CAV group. The spleen was either regenerated, lymphoid depleted or had lymphoproliferative lesions. Lymphoid depletion in the spleen was not detected in the vMDV group; however, it was prominent in the vMDV–CAV and vvMDV–CAV groups during the first 2 weeks after CAV inoculation. CAV inclusions and antigens were detected in the thymus cortex and spleen of vMDV–CAV and vvMDV–CAV groups during the experiment. Severe depletion of CD8+ T cells was observed in depleted spleen and thymus. The neoplastic foci appeared around splenic arterioles and venules, and stained mainly by CD4 antibody; however, CD8+ T cells were singly dispersed or were present in clusters. It could be concluded that CAV was responsible for bone marrow hypoplasia, severe anaemia and hindrance of lymphoid organ regeneration in MDV–CAV co-infection.

A recombinant avian leukosis virus associated with fowl glioma in layer chickens in Japan.

Fowl glioma is characterized by multiple nodular growth of astrocytes, and fowl glioma-inducing virus belonging to avian leukosis virus has been isolated from Japanese bantam as a causal agent. Subcutaneous neoplasms of the head and neck have been reported in layer chickens since 2003 in Japan, and fowl glioma concurred in these affected layers. In the present study, the histopathology of 240 layers, including 18 layers with subcutaneous neoplasms and 222 layers kept with the affected layers, was performed to clarify the characteristics of fowl glioma in layers. Microscopically, 103 layers showed non-suppurative encephalitis, and four layers had locally extensive proliferation or multiple nodules of astrocytes. Gliomas concurred in 11 layers with subcutaneous neoplasms and occurred independently in three layers. In addition, two layers had locally extensive proliferation of small, round cells in the cerebrum. The fowl glioma-inducing virus genome was not detected in the affected brains by nested polymerase chain reaction. Ten isolates were obtained from the affected brains. By nucleotide sequencing of the env gene, SU coding regions of these isolates were most closely related to myeloblastosis-associated virus-like viruses, but TM regions showed the highest similarity to endogenous viral (ev) loci. The genome of one isolate mainly consisted of ev loci and contained several parts of other avian leukosis/sarcoma viruses. These results show that the causal avian leukosis virus of fowl glioma is not just fowl glioma-inducing virus and that different avian leukosis virus strains having oncogenicity in the central nervous system by recombination are spread in layers in Japan.

Superoxide dismutase activity as a measure of hepatic oxidative stress in cattle following ethionine administration

The goal of this study was to assess if oxidative stress, as measured by alterations in the concentrations of antioxidant enzymes in the liver and erythrocytes of cattle, could be induced following dl-ethionine administration. Whole blood, serum and liver biopsy samples were collected 0, 4, 7 and 10 days after intra-peritoneal ethionine administration to five cows. The activities of the antioxidant enzymes copper zinc superoxide dismutase (Cu, Zn SOD) and catalase were assessed in the liver biopsies which were also examined histopathologically. Significant increases in hepatic Cu, Zn SOD concentrations (P<0.01) were noted on days 7 and 10 post-treatment. Hepatic catalase activity decreased significantly (P<0.01) on days 4, 7 and 10 post-treatment and erythrocyte Cu, Zn SOD activity was significantly increased on day 10. Serum biochemical analysis revealed a significant increase (P<0.01) in non-esterified fatty acid concentrations on day 4 and significant decreases in total cholesterol and phospholipid levels on days 4 (P<0.05), 7 (P<0.01) and 10 (P<0.01). In this model system, dl-ethionine administration was effective in inducing oxidative stress particularly reflected in the liver.

Quantification of tumour‐induced angiogenesis by image analysis

Quantitative techniques for in vivo and in vitro angiogenesis were developed using an image analyser. In the in vivo study, a Millipore chamber filled with mouse sarcoma 180 (S180) cells was transplanted subcutaneously to the dorsal side of a mouse, and the area of neovascularization induced by the tumour cells was quantified by image analysis. Images of vascular networks with poor contrast had their contrast improved by Laplacean transformation. The area of vascular network was 16.9 mm2 in the control group without tumour cells and 44.2 mm2 in the group with tumour cells, demonstrating a significant increase in neovascularized area by tumour cells. In the in vitro study, migration of vascular endothelial cells was induced with conditioned media of S180 cells. Image analysis was used to count automatically the nuclei of migrated endothelial cells, which were stained violet with Giemsas solution. This automated measurement by image analyser is expected to save labour and time. Checkerboard analysis revealed that the endothelial cell migration induced by S180‐conditioned medium was due to chemotaxis. The quantitation method using an automated image analyser is valuable in evaluating the induction of neovascularization by tumours and the effect of pharmacological agents on tumour angiogenesis in vivo and in vitro