Kazuaki Yamazoe

Expression of Myogenic Regulating Factors, Myogenin and MyoD, in Two Canine Botryoid Rhabdomyosarcomas

Myogenin and MyoD regulate the development of skeletal muscle, and their expressions are specific to the stages of myogenesis. Therefore, these myogenic regulatory proteins could be considered as sensitive and specific markers for rhabdomyosarcoma. In this report we investigated the immunohistochemical reactivities of myogenin and MyoD in two canine bladder botryoid rhabdomyosarcomas that were different in the degree of differentiation. MyoD was stained in the Ki-67 antigen-positive undifferentiated mesenchymal cells, which had proliferative activity similar to myoblasts differentiated from mesoblasts. In contrast, multinucleated neoplastic cells were positive for myogenin and α-sarcomeric actin but not for Ki-67 antigen, similar to the myotubes differentiated from myoblastic cells. The expressions of myogenin and MyoD were closely correlated to the histologic features of myogenic neoplastic cells.

Odontoameloblastoma in a Japanese Monkey (Macaca fuscata)

Only a few odontogenic tumors have been reported in domestic animals and nonhuman p r i m a t e ~ . * . ~ , ~ , ~ J ~ Odontoameloblastoma is rarely observed in dogs and In nonhuman primates, a few cases have been described, including only two in macaques.14J3 This report describes a necropsy case of odontoameloblastoma in the mandible of a 14-year-old female Japanese monkey (Mucucafuscata) that was referred to a veterinary clinic at the Japanese Monkey Center in Inuyama in May 1992 because of diffuse swelling of the left mandible. She had no past or familial history of any diseases of the head and neck region. Swelling of her mandibular left molar region continued to enlarge gradually. Regional lymph nodes appeared not to be involved. Serum creatine phosphokinase level was remarkably elevated (294 U/liter). The animal was sent to Gifu University and euthanatized after physical examination in November 1992. She had been kept in either a family or single-caged housing and had received a standard diet of monkey chow with fresh fruits. Physical examination revealed diffuse swelling of the left mandible, which was bony and tender on palpation. Radiographic examination showed an irregular, multilocular, wellcircumscribed radiolucent lesion with scalloping of the margins (Fig. l). A complete necropsy was done immediately after the monkey was euthanatized. The mandible and other organs were fixed in 10% buffered formalin. After fixation, tissue blocks were dehydrated and embedded in paraffin in the usual manner, sectioned at 5 pm thickness, and stained with hematoxylin and eosin (HE). Selected sections of the mandible were also stained with periodic acid-Schiff and alcian blue. For immunohistochemical studies, the labeled streptavidin-biotin (LSAB) method was applied using commercial kits (Dako Corp., Santa Barbara, CA). The primary antibodies used were anti-keratin, anti-carcinoembryonic antigen (CEA), anti& 100 protein, anti-glial fibrillary acidic protein (GFAP) (polyclonal, Dako Corp.), anti-epithelial membrane antigen (EMA), anti-neuron specific enolase (NSE) (monoclonal, Dako Corp.) and anti-cytokeratin AE 1 and AE3 (monoclonal, Signet Labs, Inc., Dedham, MA). To analyze cell proliferation, bromodeoxyuridine (BrdU) (Sigma Chemical Co., St. Louis, MO) was administered intraperitoneally at a dose of 100 mg/kg 30 minutes prior to necropsy. Parts of the mandible and intestine were dissected and fixed in 10% formalin or 70% ethanol and embedded in paraffin. Proliferating cells at the S-phase were identified by immunohistochemical techniques using the LSAB method with anti-BrdU (monoclonal, Immunotech S.A., Marseilles, France). For electron microscopic examination, tissues from the tumor mass were fixed with 2% phosphate-buffered glutaraldehyde and 1 O/o osmium tetroxide and routinely processed. Ultrathin sections were cut and then stained with uranyl acetate and lead citrate and examined with a Hitachi H-800 electron microscope at 75 kV. Grossly, the tumor was 4.5 x 3.5 x 2.5 cm, and the buccal cortical bone was expanded by the growth of tumor, resulting in an asymmetrical jaw. A transverse cut of the tumor revealed extensive destruction of the mandible and replacement by a faintly lobulated gelatinous substance (Fig. 2). Microscopically, the tumor contained epithelial components resembling those of enamel organ. These components were well developed, consisting of irregular cords of epithelial follicular structure. There were columnar ameloblast-like cells arranged in palisades in the peripheral area and a loose stel-

HISTOPATHOLOGIC FEATURES OF NATURALLY OCCURRING HEPATOZOONOSIS IN WILD MARTENS (MARTES MELAMPUS) IN JAPAN

Nodular lesions containing Hepatozoon sp. schizonts or merozoite gametocytes were found in the tissues of 67 (96%) of 70 wild caught martens (Martes melampus) examined in Gifu, Japan, 1991 and 1992. The heart was the most commonly parasitized organ (96%), followed by the perirenal adipose tissue (36%); the diaphragm, mesentery, tongue, omentum and perisplenic adipose tissue generally had a prevalence of 10 to 15%. In the heart, two types of nodular lesions were differentiated based on developmental stages: nodules containing schizonts and nodules consisting of an accumulation of phagocytes containing merozoites or gamonts. Under electron microscopy, mature schizonts contained membrane-bound merozoites with a single nucleus and small scattered electrondense cytoplasmic granules in the schizont nodules; the merozoites and gamonts were engulfed in a phagosome-like vacuole of phagocytes with the nucleus compressed to one side due to the parasite in the merozoite-gamont nodule.

Basal Cell Tumor in a Japanese Macaque (Macaca fuscata)

A rare case of basal cell tumor of the skin in a wild-caught female Japanese macaque (Macaca fuscata) was studied. Tumor growth, which was composed of cells resembling basal cells, was observed in the dermis and subcutis and showed a mixture of ribbon, solid, glandular, and cystic patterns. The tumor was separated from surrounding normal tissue by a compressed zone of fibrous connective tissue. The nuclei were round and hyperchromatic and possessed a single centrally located nucleolus. Mitotic figures were common. Immunohistochemically, a positive reaction for epithelial membrane antigen, keratin, and cytokeratin AE3 was evident. Electron microscopic examination revealed a small number of tonofilaments in the cytoplasm.

Spontaneous vascular mineralization in the brains of cows

The frequency of occurrence and the characteristics of vascular mineralization in the brains of cows were studied. Mineralized lesions were found in the pallidal arteries in eight of 13 cows. They appeared as basophilic deposits in the arterial walls, were sheath-like or tubular in shape, and were accompanied by intimal oedema or fibrous thickening. The deposits were positive for periodic acid-Schiff, von Kossas, alizarin red and Berlin blue stains. Elemental analysis revealed the presence of large amounts of phosphorus, calcium and zinc, as well as small amounts of iron, copper and sodium.

Hypertrophic Pulmonary Osteoarthropathy in a Raccoon Dog (Nyctereutes procyonoides) with Chronic Pulmonary Inflammatory Lesions

A female raccoon dog (Nyctereutes procyonoides) captured September 1992 in Nara prefecture, Japan, suffering from swollen joints of all four limbs was necropsied. Radiographically, there was a shadow in the right pulmonary caudal lobe and periosteal bone formation around several joints. Microscopically, there was extensive edema, along with one large and many small pyogranulomatous nodules and purulent bronchitis in the right lobe and left caudal lobe of the lungs. There were intermingled branching filamentous bacteria in the lesion, which stained positively with Warthin-Starry, Grocott, and Gram stains, and partially with periodic acid Schiff and Ziehl-Neelsen stains. Extensive exostosis with coralloid trabeculation had developed bilaterally at the carpal, stifle and hock joints in the surrounding well vascularized loose connective tissue. Degeneration of articular cartilage and villous proliferation of synovial membranes were apparent. This is the first report of hypertrophic pulmonary osteoarthropathy in the raccoon dog.

Identification of a CD4 variant in Microminipigs not detectable with available anti-CD4 monoclonal antibodies

The Microminipig is an extra-small sized novel miniature pig developed in Japan. In the process of peripheral blood mononuclear cells analysis by flow cytometry, CD4+ cells could not be detected in some pigs with an anti-pig CD4 antibody (clone 74-12-4), or in some pigs with two other antibodies from different clones (MIL17 and PT90A). In a herd of 178 Microminipigs, 87 pigs (48.9%) were reactive with the anti-CD4 antibody (designated as CD4.A), and 91 pigs (51.1%) were non-reactive (designated as CD4.B). The CD4 types of piglets delivered from parents with CD4.A were CD4.A or CD4.B, and piglets delivered from parents with CD4.B were only CD4.B. This implies that the CD4.A pigs were homozygous for CD4.A or heterozygous for CD4.A and CD4.B, and the CD4.B pigs were homozygous for CD4.B. The CD4.B trait might be recessive. Significant differences could not be found in the percentage of CD3+ and CD8+ cells in whole lymphocytes between CD4.A and CD4.B animals. In the profile of CD4.B pigs, CD4+CD8+ T cells appeared to be detected in the CD4-CD8+ T cell region because the CD8 dull T cell population was observed. Thus, we considered that the CD4 molecules may be expressed on helper T cells, but the CD4 expressing cells could not be detected with the three anti-pig CD4 antibodies. Clinical abnormalities have not been observed in CD4.B pigs. Significant differences were not observed in immunoglobulin concentrations between CD4.A and CD4.B, though lower tendency was observed in plasma IgM concentrations from CD4.B pigs >36-months-old. These results imply that the CD4.B does not affect basic humoral immunity in vivo.

Body and major organ sizes of young mature microminipigs determined by computed tomography

To understand the anatomical characteristics of microminipigs, one of the smallest miniature pigs, as a large animal model, we measured the body and organ sizes of four-, five-, six-, and seven-month-old microminipigs (n = 4, females) using computed tomography. In addition, the results were compared with those of young mature beagles (10 months old, two males and three females), which have been widely used as a large animal model. The microminipigs at 4–6 months of age were much smaller than the beagles. However, when the microminipigs reached seven months of age, their overall size was similar to that of the beagles. The thoracic cavity volume of the seven-month-old microminipigs was less than half that of the beagles, and the cavity was largely filled by the heart. The liver size of the seven-month-old microminipigs was approximately half of that of the beagles. Moreover, the spleen of the seven-month-old microminipigs was different in morphology, but not different in size from that of the beagles. In addition, although their volumes were the same, the kidneys of the seven-month-old microminipigs, unlike those of the beagles, were flattened in shape. Collectively, the major abdominal organs of the seven-month-old microminipigs were either the same size or smaller than those of the beagles, but the abdominal cavity volume of the seven-month-old microminipigs was larger than that of the beagles. Thus, the abdominal cavity of microminipigs is assumed to be filled with the gastrointestinal tract. The anatomical characteristics of the young mature microminipigs revealed in our study suggest that microminipigs could have great potential as a large animal model for biomedical research.

Subclinical renal oxalosis in wild-caught Japanese macaques (Macaca fuscata)

Various degrees of crystal deposition were found in the kidneys of 12 out of 59 Japanese macaques (Macaca fuscata) caught in the wild in Gifu, Japan. The needle- or rod-shaped crystals, which were radially arranged and occurred in the lumen and epithelium of the renal (mainly the proximal) tubules, were birefringent under polarized light. They stained with alizarin red S at a pH of 7.0 but not 4.2, and were identified as calcium oxalate. The morphological features of the renal lesions were similar to those previously reported in oxalate poisoning, and it was believed that the macaques ingested the oxalate in plants.

Arteriosclerosis associated with a natural Marek's disease infection in a Japanese bantam (Gallus gallus).

A severe case of arteriosclerosis was observed in a female Japanese bantam (Gallus gallus) associated with Mareks disease (MD) infection. The hen, showing clinical, macroscopic and microscopic evidence of MD infection, harbored arteriosclerosis in the aorta and large arteries characterized by an extremely thickened intima and marked luminal occlusion. Proliferating cell nuclear antigen (PCNA) immunostain-ing showed many labeled nuclei in the thickened intima, and less frequent positivity in the media. This is the first report on avian arteriosclerosis in natural MD infection.