Toshifumi Oyamada

Intracellular Ca2+ storage sites in the carp heart: comparison with the rat heart.

The Ca(2+)-releasing mechanisms of the sarcoplasmic reticulum responsible for cardiac muscle contraction in carp were examined and compared with these mechanisms in rats. Morphologically, the ventricular muscles of the carp heart are composed of an outer compact and an inner spongy layer. In the present study, ventricular muscle preparations were obtained from the compact layer of the carp heart, because the spongy layer does not contribute significantly to the overall force of contraction. Electron microscopic observations showed that the sarcoplasmic reticulum in the carp ventricular muscle, compared to that in the rat ventricular muscle, was poorly developed. Consistent with this finding, specific [3H]ryanodine binding to partially purified sarcoplasmic reticulum preparations obtained from carp ventricular muscle as compared with the preparations isolated from the rat ventricular muscle showed a lower affinity and a smaller number of binding sites. Additionally, a higher Ca2+ concentration was required to cause a half maximal stimulation of [3H]ryanodine binding in the carp heart. In skinned ventricular muscle fibers isolated from carp hearts, the caffeine-induced contracture was significantly weaker than that observed in rat hearts. These results suggest that, in carp hearts, the sarcoplasmic reticulum has an important role as a supply source of Ca2+ for muscle contraction, though the storage capacity and/or amount of Ca2+ release in carp was significantly smaller than that in rats.

Histopathological development of equine cutaneous papillomas

The histopathological development of equine cutaneous papillomas was studied in 78 warts naturally occurring in 50 one to 3-year-old Thoroughbred or Arab horses and in 54 warts experimentally induced in three 2-year-old Thoroughbreds. Lesions in the natural cases were categorized into three phases, growth, development and regression. Main lesions of the growing phase were marked hyperplasia of the basal cells and mild to moderate acanthosis, hyper- and parakeratosis with a few intranuclear inclusion bodies (IIB) which were positive with anti-bovine papillomavirus serum. In the developing phase, there was prominent acanthosis with cellular swelling and fusion, and marked hyper- and parakeratosis. Many IIB were also present in swollen or degenerative prickle cells and granular cells, with a high degree of parakeratosis in keratinocytes. In the regressing phase, epidermal layers were almost normal with only slight hyperplastic change. However, there was rete peg proliferation downward into the dermis with moderate proliferation of fibroblasts and collagen fibres. In addition, in 10 spontaneous and one experimental wart, the lesions were fibropapillomas and this has never been described in horses previously. It was concluded that papillomas were initiated by basal cell hyperplasia without viral antigen production, with formation of acanthosis and hyper- and parakeratosis with IIB production. These findings were confirmed by examination of the experimental cases on the basis of the gross diameter of the warts.

Malignant Ameloblastic Fibro-odontoma in a Dog

An 11-year-old male Collie was presented with a swelling of the face caused by tumor masses arising from the gingiva. Postmortem examination revealed metastases to the lymph nodes, lung, liver, and orbital cavity. Histologically, the tumor represented a combination of fibrosarcomatous proliferation, pulpal mesenchyme, and undifferentiated odontogenic epithelium, with a follicular or plexiform growth pattern. In addition, the follicular areas of the tumor showed a biphasic character, and there were numerous apoptotic cells in plexiform areas. Furthermore, acidophilic material resembling dysplastic dentine or enamel matrix was observed in the metastatic lesion in the lung. Based on the histological characters, the present case was diagnosed as malignant ameloblastic fibro-odontoma. This study is the first known description of a possible malignant ameloblastic fibro-odontoma in a dog with metastasis to distant organs.

Age-related changes in the testes of horses.

Atrophy of seminiferous tubules and interstitial fibrosis are frequently observed in aged horses. Samples from 8 male Thoroughbreds, age 4-24 years, were subjected to histological, electron microscopical and immunohistochemical examination and statistical analysis. There were statistically significant increases in collagen fibres in the lamina propria of seminiferous tubules and testicular interstitium in 3 horses age 23 and 24 years compared with 5 horses age 4-20 years (P<0.001). Lamina propria surrounding atrophic tubules was thickened by an increase in collagen type IV and elastic fibres and by proliferation of bizarre myoid cells. Basal lamina was also thickened but had decreased reactivity for collagen type IV. Some myoid cells changed morphologically to a swollen and irregular shape and contained abundant cytoplasmic organelles. Laser scanning microscopy revealed that cytoplasmic actin filaments were decreased; the remaining filaments were positive for alpha-smooth muscle actin and vimentin, and matrix metalloproteinase-2 was secreted. These myoid cells transformed into myofibroblasts. The changes are interpreted as evidence of injured structure and function of the lamina propria and basal lamina and may explain the functional decline of the blood-testis barrier. Myoid cells may play an important role in the progression of testicular fibrosis.

Cutaneous Toxoplasmosis in a Female Japanese Cat

A 16-year-old female Japanese cat was presented with a single mammary-gland nodule approximately 3 cm in diameter. Histologically, the nodule consisted of necrotizing granulomatous panniculitis, vasculitis, and mastitis, and contained free and clustered protozoal organisms. The organism was present in the cytoplasm of macrophages, fibroblasts, endothelial cells, and mammary-gland epithelia. The organism was positive for anti-Toxoplasma gondii and anti-Neospora caninum antibodies. Electron microscopy showed single and grouped tachyzoites, with morphologic features similar to those of T. gondii. Polymerase chain reaction and deoxyribonucleic acid sequence analysis was consistent with T. gondii infection. This is the first report of cutaneous toxoplasmosis in a Japanese cat.

Analysis of a Growth-Promoting Factor for Babesia caballi Cultivation

Serum-free media were examined to culture Babesia caballi. Daigos T (DT) basal medium supplemented with Daigos GF21 (GF21) or GIT medium, which already contains GF21, supported the parasite propagation at 37 C in a humidified atmosphere under 5% CO2 in air. Growth of B. caballi was dependent of the suitable concentration (10–20%) of GF21. Therefore, GF21 was suggested as the growth-promoting factor for B. caballi. However, GIT medium did not support the growth of parasites from cryopreserved stabilates, and serum supplementation was essential for the retrieval of parasites.

Congenital Hepatic Fibrosis in a Newborn Calf

Congenital hepatic fibrosis was observed in a newborn calf. Light microscopy revealed that periportal areas were linked via connective tissue to the central vein regions and to other periportal areas. Hyperplastic fibers were positive for type I collagen. A remarkable increase in the number of myofibroblasts that were positive for α-smooth muscle actin and vimentin was observed in the inner wall of the sinusoids, indicating the occurrence of various fibrogenesis. Ultrastractually, foci of cells resembling cholangiole epithelium cells were observed within the sinusoids, thereby suggesting either ductal plate dysplasia or a bile duct anomaly.

Expression of insulin-like factor 3 hormone-receptor system in the reproductive organs of male goats.

Relaxin-like factor (RLF), generally known as insulin-like factor 3 (INSL3), is essential for testis descent during fetal development. However, its role in adult males is not fully understood. We investigate the function of INSL3 in male Saanen goats by identifying cell types expressing its receptor, relaxin/insulin-like family peptide receptor (RXFP)2 and by characterizing the developmental expression pattern of INSL3 and RXFP2 and the binding of INSL3 to target cells in the male reproductive system. A highly specific RXFP2 antibody that co-localizes with an anti-FLAG antibody in HEK-293 cells recognizes RXFP2-transcript-expressing cells in the testis. INSL3 and RXFP2 mRNA expression is upregulated in the testis, starting from puberty. INSL3 mRNA and protein expression has been detected in Leydig cells, whereas RXFP2 mRNA and protein localize to Leydig cells, to meiotic and post-meiotic germ cells and to the epithelium and smooth muscle of the cauda epididymis and vas deferens. INSL3 binds to all of these tissues and cell types, with the exception of Leydig cells, in a hormone-specific and saturable manner. These results provide evidence for a functional intra- and extra-testicular INSL3 ligand-receptor system in adult male goats.

Serosurveillance for Francisella tularensis Among Wild Animals in Japan Using a Newly Developed Competitive Enzyme-Linked Immunosorbent Assay

Tularemia, a highly infectious zoonotic disease caused by Francisella tularensis, occurs sporadically in Japan. However, little is known about the prevalence of the disease in wild animals. A total of 632 samples obtained from 150 Japanese black bears, 142 Japanese hares, 120 small rodents, 97 rats, 53 raptors, 26 Japanese monkeys, 21 Japanese raccoon dogs, 20 masked palm civets, and three Japanese red foxes between 2002 and 2010 were investigated for the presence of antibodies to F. tularensis by competitive enzyme-linked immunosorbent assay (cELISA) and the commonly used microagglutination (MA) test. Seropositive cELISA and MA results were obtained in 23 and 18 Japanese black bears, three and two Japanese raccoon dogs, and two and one small rodents, respectively. All MA-positive samples (n=21) were also positive by cELISA. Six of seven samples that were only positive by cELISA were confirmed to be antibody-positive by western blot analysis. These findings suggest that cELISA is a highly sensitive and useful test for serosurveillance of tularemia among various species of wild animals. Because this is the first study to detect F. tularensis-seropositive Japanese raccoon dogs, these could join Japanese black bears as sentinel animals for tularemia in the wild in Japan. Further continuous serosurveillance for F. tularensis in various species of wild animals using appropriate methods such as cELISA is important to assess the risks of human exposure and to improve our understanding of the ecology of F. tularensis in the wild.

Time-related Pathological Changes in Horses Experimentally Inoculated with Equine Influenza A Virus

To investigate the pathology of equine influenza, necropsy of 7 horses experimentally infected with equine influenza A virus (EIV) subtype H3N8 was conducted on post-infection days (PID) 2, 3, 7, and 14. Histopathologically, rhinitis or tracheitis including epithelial degeneration or necrosis with loss of ciliated epithelia and a reduction in goblet cell numbers, was observed in the respiratory tracts on PIDs 2 and 3. Epithelial hyperplasia or squamous metaplasia and suppurative bronchopneumonia with proliferation of type II pneumocytes were observed on PIDs 7 and 14. Viral antigen was detected immunohistochemically in the epithelia of the nasal mucosa, trachea, and bronchi on PIDs 2 and 3. The sodA gene of Streptococcus equi subsp. zooepidemicus, a suspected cause of suppurative bronchopneumonia, was detected in paraffin-embedded lung tissue sections, but only on PIDs 7 and 14. These findings suggest that damage caused to ciliated epithelia and goblet cells by EIV infection results in secondary bacterial bronchopneumonia due to a reduction in mucociliary clearance.