Persistence of inclusions and antigens of chicken anemia virus in Marek's disease lymphoma.

The pathogenesis of the co-infection of CAV to MDV is complicated. In order to investigate the impact of CAV on the transformation phase of MD, MDV and, subsequently, CAV, were inoculated at 1day and 4weeks of age, respectively. Chickens were divided into six groups; vvMDV, vvMDV-CAV, vMDV, vMDV-CAV, CAV and a control group. The CAV inclusions and antigens were continuously detected in MD lymphomas in the vMDV-CAV and vvMDV-CAV groups in large bizarre-shape (presumably CD4+ T cells) and small MD lymphoid cells (presumably CD8+ T cells). The MD lymphomas were composed primarily of CD4+ T cells, but CD8+ T cells were infiltrated singly or in clusters. CAV enhanced the MDV-induced brain lesions in the vMDV-CAV group. The lymphoproliferative lesion (LP) in the vvMDV-CAV and vMDV-CAV groups was non-significantly higher than those in vvMDV and vMDV groups, respectively. CAV significantly increased the LP lesion in sciatic nerves. In conclusion, MD lymphomas enabled CAV replication and dissemination. The depletion of CTLs by CAV did not significantly affect progression of MD lymphoma, although they are essential for possible transition of lymphomatous to inflammatory lesion.

Gliomatosis cerebelli in a Saint Bernard dog.

A 6-year-old, neutered male Saint Bernard dog was presented with a 1-month history of ataxia, hypermetria and head tilt. High-field magnetic resonance imaging revealed a mass in the cerebellar vermis. During necropsy examination, a cream-coloured irregular area was observed in the cerebellar white matter. Microscopically, the mass comprised a diffuse neoplastic proliferation of spindle cells with oval pleomorphic nuclei in the white and grey matter of the cerebellum and pons and in the subpial area. Neoplastic infiltration was not found in the cerebrum. Immunohistochemistry revealed that the neoplastic cells were positive for vimentin and partially positive for glial fibrillary acidic protein. Based on these findings, the neoplastic lesion was diagnosed as gliomatosis cerebelli, without involvement of the cerebrum.

Early pathophysiologic feature of arthropathy in juvenile dogs induced by ofloxacin, a quinolone antimicrobial agent.

Arthropathy in dogs induced by ofloxacin, a quinolone antimicrobial agent, was pathophysiologically investigated. In the in vivo studies, ofloxacin was administered orally once or twice at 20 mg/kg/day to male juvenile (3-month-old, n=3) or adult (36-month-old, n=2) dogs, and the humeral and femoral heads were examined pathologically. Unlike adult dogs, fluid-filled vesicles were macroscopically observed on the articular surfaces of one juvenile dog 24 hours after a single treatment with ofloxacin. These lesions were seen in all juvenile dogs by twice dosing. Microscopically, fissures or cavity formations in the middle zone of the articular cartilage were noted only in juvenile dogs. Furthermore, the cartilage matrix from the abnormal area to the articular surface showed a decreased safranin-O staining intensity, suggesting proteoglycan depletion. Ultrastructurally, chondrocytes in the middle zone of juvenile dogs displayed dilatation of the cisternae in the rough endoplasmic reticulum as an initial hallmark. In the in vitro studies, chondrocytes isolated from the articular cartilage of naive juvenile dogs were exposed to ofloxacin at 6.3-100 microg/ml for 24 hours. Although no changes were noted in the deoxyribonucleic acid synthesis, protein synthesis, or proteoglycan release at concentrations of up to 100 microg/ml, the proteoglycan synthesis was evidently decreased in a dose-dependent manner from 12.5 microg/ml. The results obtained suggest that the inhibitory action of ofloxacin on proteoglycan syntheses in the chondrocytes may largely contribute to the early morphologic features in the articular cartilage of the juvenile dog.

A non-arthropathic dose and its disposition following repeated oral administration of ofloxacin, a new quinolone antimicrobial agent, to juvenile dogs.

A non-arthropathic dose and disposition of ofloxacin, a potent new quinolone antimicrobial agent, were assessed in male juvenile (3-month-old) dogs, when administered orally at 5, 10 and 20 mg/kg/day once daily for 8 consecutive days. Ofloxacin concentrations in sera and articular cartilages were analyzed by high-performance liquid chromatography (HPLC). Macroscopically, arthropathy characterized by fluid-filled vesicles in articular surface of the humerus and femur was observed in animals receiving 10 and 20 mg/kg/day of ofloxacin, but not in those given 5 mg/kg/day. At 20 mg/kg/day, arthropathy of comparable severity also occurred on day 2. Microscopically, the cavity formation in the middle zone of the articular cartilage was first identified and then necrotic chondrocytes were found numerous around the cavity, followed by appearance of chondrocyte clusters. In pharmacokinetics, peak serum concentration (Cmax) and area under the concentrations (AUC0-24) were increased in a dose-dependent manner. However, no remarkable differences in these two parameters were noted between a single and repeated treatments, suggesting no accumulation of the drug. The articular ofloxacin concentration 2 hr after treatment was approximately 1.8 (day 2) to 2.0 times (day 8) higher than the serum concentration. Based on these results, a non-arthropathic dose of ofloxacin in male juvenile dogs following an 8-day treatment is considered to be 5 mg/kg/ day, and its Cmax, AUC0-24 and articular cartilage concentrations 2 hr after treatment were 3.4 microg/ml, 35.1 microg-hr/m/ and 7.0 microg/g, respectively, under these experimental conditions. Thus, arthropathy due to ofloxacin may be predicted by monitoring serum drug concentration.

Pituitary chromophobe carcinoma with a low level of serum gonadotropin and an aspermatogenesis in a dog.

A 5-year-old male Shiba dog with progressive neurologic signs was examined by computed tomography (CT). A CT image of the brain disclosed a large, spherical high-density lesion in the thalamus and diencephalon. Serum LH, FSH and testosterone levels were all low. Macroscopically the large mass was connected with the sella turcia, and it was histopathologically diagnosed as a pituitary chromophobe carcinoma. An aspermatogenesis was observed in the testes. Therefore, it was suggested that the low levels of gonadotropin secretion from the pituitary gland due to the pituitary tumor resulted in the failure of maturation of spermatozoa and spermatids.

Serum parathyroid hormone and calcitonin levels in racehorses with fracture.

Serum parathyroid hormone (PTH) and calcitonin (CT) levels in fractured racehorses were measured by radioimmunoassay. Racehorses with fracture of large bone such as the radius, third metacarpus, third carpus, digital bone or tibia, showed normal PTH level and elevated CT level in the serum. Serum PTH level was slightly higher in racehorses with sesamoid bone fracture compared to that of healthy racehorses, but not statistically significant. Moreover, serum CT level of racehorses with sesamoid bone fracture was significantly higher than that of healthy racehorses. Racehorses with sesamoid bone fracture and large bone fracture might be in different conditions of calcium regulation.

Apoptosis of proliferative cortical tubular epithelia in chronic progressive nephrosis of rats.

To investigate the role of apoptosis in the pathogenesis of rat chronic progressive nephrosis (CPN), the kidney of male F344/DuCrj rats, 19, 59, and 111 weeks of age, was examined histologically. In situ analysis for DNA fragmentation and proliferating cell nuclear antigen (PCNA) was performed simultaneously by TdT-mediated dUTP-biotin nick end labeling (TUNEL) and immunohistochemistry, respectively. CPN was seen in all the kidneys of 59-week-old (n=6) and 111-week-old rats (n=16), correlating significantly (p<0.01) with age. There were apoptotic bodies (ABs) in the single-layered epithelia of dilated tubules (SLD) and the multilayered epithelia (ML) of the cortical tubules. There were no ABs in any of the kidneys of the 19-week-old (n=5) or 59-week-old rats (n=6). Proliferative activity might have been enhanced in the single-layered and flattened epithelia, SLD, and ML of the cortical tubules in the kidneys of the 59-week-old rats (n=6) compared with that in 111-week-old rats (n=8). The correlations between the TUNEL-positive ratio and number of PCNA-positive cells, and age and the CPN grade were significant (p<0.01) exclusively in the ML. Thus, the results suggest that apoptosis occurs in the proliferative ML of rat CPN, and the pathological significance might be the removal of abnormal or excess cells.

Apoptosis and cell proliferation in rat hepatocytes induced by barbiturates.

To examine the effect on cell population in hepatocytes of phenobarbital (PB) and other barbiturates, PB, allobarbital (ALB), barbital sodium (BS) and barbituric acid (BA) were given orally to male rats for 7 consecutive days. Although there was no apparent change in non-promoting BA, hepatomegaly was induced by PB, BS and ALB, which are promoters of hepatocarcinogenesis. In PB- and BS-treated livers, hepatomegaly was attributable to hepatocyte proliferation and enzyme induction. In ALB-treated liver, it was attributable to enzyme induction. The level of cell proliferation was reduced to less than the control values following withdrawal of PB, ALB and BS. It seemed that the degree of suppression of cell proliferation following withdrawal of these compounds correlated to the degree of cell proliferation (PB>BS>ALB) during treatment. In PB-treated liver, apoptosis was induced during treatment, serving to eliminate the excess of hepatocytes. This suggests that short-term administration of PB neither induced suppression of apoptosis nor disturbed homeostasis of hepatocyte populations.

Cholangiohepatitis in broiler chickens in Japan: histopathological, immunohistochemical and microbiological studies of spontaneous disease.

Forty-five broiler carcasses from 6 different flocks were condemned due to liver lesions at processing meat inspection, and collected for pathological and bacterial examinations. All affected chickens showed liver enlargement with discolouration and an apparent acinar pattern. The enlarged gallbladder and the extrahepatic bile ducts contained yellow inspissated cream-coloured material. Histopathologically, extensive proliferation of bile ductules with fibrosis was observed in interlobular connective tissue, and it spread to form bridges with adjoining triads. Destruction and obstruction of portal bile ducts with multiple granulomas due to bacterial infection and outflow of the bile were frequently observed. Many Gram-positive bacilli were seen in the lesions, and they were identified as Clostridium perfringens by indirect immunofluorescence staining technique. Clostridium perfringens was isolated from affected livers. These findings are consistent with cholangiohepatitis. Therefore, it is suggested that C. perfringens might be important in the pathogenesis of cholangiohepatitis in broiler chickens.

Cholangiohepatitis in chickens induced by bile duct ligations and inoculation of Clostridium perfringens.

The association of bile duct ligation with the development of cholangiohepatitis due to Clostridium perfringens was examined. Chickens with only the cystoenteric duct ligated had no significant liver lesions even if C. perfringens was inoculated into the cystoenteric duct. Chickens with both the cystoenteric and hepatoenteric ducts ligated had enlargement of the liver with an apparent acinar pattern. Histopathologically, proliferation of bile ductules was seen. Bile ductules proliferated extensively, and fibrosis formed bridges between triads. The gall bladder and extrahepatic bile ducts were thickened and distended with yellow inspissated material in chickens that had both ducts ligated and had C. perfringens inoculated into the cystoenteric duct. Multiple granulomas and intrahepatic cholangitis were frequently observed. Infiltration of lymphocytes and plasma cells with germinal centres, and heterophilic extramedullary haematopoiesis were seen in the portal areas. Thus, cholangiohepatitis similar to field cases can only be induced by both ligation of bile ducts and inoculation of C. perfringens.

Distribution of T-lymphocyte subpopulation in blood and spleen of normal cattle and cattle with enzootic bovine leukosis.

Immunocytochemical and flow cytometry techniques were used to examine T-lymphocyte subpopulations in peripheral blood lymphocytes (PBLs) and spleen from cases of enzootic bovine leukosis (EBL) in adult cattle, and from normal cattle (adult and young), with a panel of monoclonal antibodies against bovine leucocyte differentiation molecules. Both in PBLs and spleen, the percentages of T-lymphocyte subpopulations (CD3+, CD4+, CD8+, and WC1 + gamma delta T lymphocytes) of EBL-affected and normal adult cattle were significantly lower than those of normal young cattle. The percentages of these T-lymphocyte subpopulations in the PBLs of adult cattle with EBL were lower than those of normal adult cattle, but the converse was true in the spleen. It is suggested that tumour immunity occurred in the spleen. Histological examination revealed no follicular hyperplasia in the spleen, and the proliferation of neoplastic cells began in the red pulp. It is concluded that the spleen is not the organ initially responsible for the transformation of EBL lymphoma and that neoplastic cells migrating from peripheral blood are metastatic.

Hemorrhagic enteritis associated with Clostridium perfringens type A in a dog.

A female Shetland sheep dog died suddenly with hemorrhagic diarrhea and vomitting, and was examined pathologically and microbiologically. Gross pathological change was restricted to the intestinal tract. The intestine contained watery, blood-stained fluid. Histopathologically, the principal intestinal lesion was superficial mucosal hemorrhagic necrosis at the jejunoileum. Many Gram-positive bacilli were found adhering to the necrotic mucosal surface in parts of the intestinal tract. Clostridium perfringens in pure culture were isolated from jejunal contents by anaerobic culture. These results suggested that the typical lesion of this case coincided with canine hemorrhagic enteritis and enterotoxemia due to C. perfringens infection could be the cause of sudden death.

Spontaneous pinealoma in a male Crj:CD (SD) IGS rat.

A pinealoma (benign) was found in a 61-week-old male Crj:CD (SD) IGS rat. The neoplasm was located between the cerebral hemispheres and the cerebellum. Histologically, the tumor cells consisted of two cell types: large, pale-staining cells and small dark-staining cells. A fibrovascular stroma divided the tumor cells into incomplete lobules or nest structures. Relatively numerous mitoses were noted in the tumor cells. Ultrastructurally, the tumor cells contained dense-cored vesicles, approximately 120 nm in diameter.

Drug resistance and P-glycoprotein expression in endothelial cells of newly formed capillaries induced by tumors.

The inhibitory effects of vincristine (VCR) with or without verapamil (VER) on angiogenesis in mouse subcutaneous fascia induced by mouse sarcoma 180 cells were assessed using the dorsal air sac method. VCR combined with VER had inhibitory effects on tumor-induced angiogenesis, but VCR alone did not inhibit capillarization. The chemosensitivity of human umbilical vein endothelial cells (HUVEC) and tumor-derived endothelial cells from rat KMT-17 fibrosarcoma (TEC) was examined using the microculture tetrazolium assay. VCR and taxol had strong anti-proliferative activity against HUVEC, but only weakly inhibited the proliferation of TEC. In combination with VER, VCR and Taxol inhibited the proliferation of TEC. Cisplatin, mitomycin C, and 5-fluorouracil had weakly anti-proliferative activity against both HUVEC and TEC. Expression of P-glycoprotein (P-gp) was found in TEC, but not in HUVEC using western blot analysis. These findings indicate that drug resistance and P-gp expression appeared on newly formed capillaries induced by rodents tumors.

Tumor-associated antigen in lymph nodes during the progression of enzootic bovine leukosis.

A monoclonal antibody, (MAb) c143, that recognizes a tumor-associated antigen (TAA) that is upregulated on neoplastic B cells in cattle with enzootic bovine leukosis (EBL), was used as a marker to study disease progression. Immunohistochemical examination of neoplastic tissue and lymph nodes from animals with EBL, revealed three morphologically definable stages of change in the architecture of lymph nodes associated with infiltration and proliferation of neoplastic cells: 1) presence of c143 positive cells at the marginal sinus with no apparent changes in lymph node architecture at the earliest stage of neoplastic cell accumulation, 2) presence of positive cells extending into and distorting the architecture of the lymph node with clear evidence of proliferation, and 3) presence of positive cells throughout the lymph node with total disruption of lymph node architecture. The results indicated that, at the earliest stages of EBL, neoplastic cells in peripheral blood may accumulate at the marginal sinus area and subsequently proliferate and infiltrate pressing follicles leading to the development of clinical signs of lymphosarcoma.

Further analysis of the phenotype and distribution of tumor cells in sporadic B-cell and T-cell lymphomas in the lymph node and spleen of cattle.

Immunohistologic studies were performed to identify the phenotype and distribution of neoplastic lymphocytes in the spleens of BLV-negative animals examined by PCR and diagnosed as having sporadic bovine leukosis. Tumor cells from three cases of sporadic bovine leukosis were identified as of B-cell lineage. Tumor cells from three additional cattle were identified as CD3+ CD4- CD8+, CD3+ CD4- CD8-, and CD3+ CD4- WC1+, respectively. The last case was diagnosed as a gamma/delta T-cell lymphoma. Differences in morphology proliferative characteristics were recognized between B- and T-cell type lymphomas. The tumor cells in B-cell type lymphoma were characterized as follows: medium or large in size, round or polymorphic nucleus with rough chromatin with some tumor cells containing a convoluted nucleus. These tumor cells of B-cell type lymphoma were present in the red pulp and periarteriolar lymphoid sheath. Tumor cells of the T-cell type lymphoma were uniformly smaller than B-cell type and present around arteries or replaced red pulp of the spleen.

Phenotype analysis of lymphoid cells in Marek's disease of CD4(+) or CD8(+) T-cell-deficient chickens: Occurrence of double negative T-cell tumour.

Chickens rendered either CD4(+) or CD8(+) T-cell-deficient by thymectomy and subsequent inoculation of anti-CD4 or anti-CD8 monoclonal antibody (mAb), were infected with Marek's disease herpesvirus (MDV). All the non-treated control (4/4) and CD8-deficient chickens (5/5) formed gross tumours. However, one of three CD4(+) T-cell-deficient chickens developed gross tumours. Histologically, all chickens had tumourous lesions in the liver and nerve fibres. In CD4(+) T-cell-deficient chickens, the phenotypes were CD3(+)CD4(-)CD8(-)Vss1-alphassTCR(+) T-cells and CD3(-)CD4(-)CD8(-) cells. These findings indicate that the double negative T-cells (CD3(+) CD4(-) CD8(-)) and CD3(-) CD4(-) CD8(-) cells are transformed morphologically in addition to CD3(+) CD4(+) CD8(-) T-cells by MDV infection.

The proto-oncogene c-myb is expressed in sporadic bovine lymphoma, but not in enzootic bovine leukosis.

We examined bovine c-myb gene expression in six samples of sporadic bovine lymphomas (two calf, three thymic and one intermediate) and five of enzootic bovine leukosis. Tumor cells of the sporadic bovine lymphomas were of immature cell lineage (one B lymphoma and five T lymphomas). The c-myb mRNA was expressed in almost all the sporadic bovine lymphomas (except for one thymic form) including a BoCD8 single positive T lymphoma. On the contrary, c-myb was not expressed in mature B lymphomas of enzootic bovine leukosis. The results suggest that c-myb expression is closely associated with tumor cell differentiation of bovine lymphomas.

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.

Changes in the distribution of cells expressing tumour-associated antigen in lymph nodes during the progression of enzootic bovine leukosis.

A monoclonal antibody (MAb), c143, that recognizes a tumour-associated antigen that is "upregulated" on neoplastic B cells in cattle with enzootic bovine leukosis (EBL), was used as a marker to study disease progression. An immunohistochemical examination of neoplastic tissue and superficial cervical lymph nodes from 14 animals with EBL revealed three morphologically definable stages of change in the structure of lymph nodes, associated with the distribution of c143-positive cells: (1) the presence of c143-positive cells at the marginal sinus with no apparent changes in lymph node structure; (2) the presence of positive cells extending into and distorting the architecture of the lymph node, with clear evidence of proliferation before overt changes (enlargement of lymph nodes) were evident; and (3) the presence of positive cells throughout the lymph node with total disruption of lymph node structure when clinical signs of lymph node enlargement were evident. The results indicated that the bovine leukaemia virus-transformed lymphocytes or neoplastic cells in peripheral blood accumulate in the marginal sinus area at the earliest stages, and subsequently proliferate and infiltrate into follicles, leading to the development of clinical signs of lymphosarcoma.