Skulls and canines of 460 sea otters from Lopatka Cape, Kamchatka, were examined to assess development patterns, individual variation and sexual differences. An allometric formula was applied to morphometrical data, and the relative growth of each character to total length of skull was analyzed. In both sexes, most morphometrical characters ceased growth at about 2 years of age. Canine root length increased rapidly during the first year of life, while crown length decreased due to remarkable wear. There was large individual variation in the feeding and breathing/sniffing apparatus, while there was little variation in braincase size. There were sexual differences in most characteristics, although males and females showed similar growth patterns. The coronoid process of the mandible showed positive allometry in both sexes, and we attributed this finding to feeding habits. The fact that only male mastoids showed positive allometry may be due to the need for male otters to maintain a passing territory.

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It has been hitherto considered that mature erythroblasts migrate toward the sinusoid along the cytoplasmic processes of macrophages of erythroblastic islands in bone marrow. Our previous report, however, has demonstrated the morphological features of a mature erythroblastic island passing through the sinusoidal endothelium. In this study, the possibility of migration of erythroblastic islands toward the sinusoid was examined in rat bone marrow by light microscopical histoplanimetry. As a result, the more mature erythroblasts were not regularly arranged in the peripheral direction of the erythroblastic islands. Immature erythroblasts were populated more in the erythroblastic islands away from the sinusoid than in those islands neighboring the sinusoid, whereas mature erythroblasts were more in erythroblastic islands neighboring the sinusoid. These findings suggest that the formation of erythroblastic islands occurs in a region away from the sinusoid, and that erythroblastic islands migrate towards the sinusoids as erythroid maturation proceeds.

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The adverse effect of estrogenic chemicals on luteinizing hormone-immunoreactive (LH-ir) cells in the adenohypophysis of common carp (Cyprinus carpio) was examined using immunocytochemical and morphometric methods. Adult male fish were collected from two contaminated sites (Ishizu and Wada Rivers) and from a control pond at Agriculture, Food and Environmental Sciences, Research Center of Osaka Prefecture. The concentration of nonylphenol, bisphenol A and 17&Εαχυτε;β-estradiol in the Ishizu River was 3-4 times higher than that in the Wada River. The proportion and size of LH-ir cells were evaluated using the point-counting method by optical microscopy. In control carp, the proportion of LH-ir cells in the breeding season was significantly lower than in the pre- and post-breeding seasons. The same tendency was also found in Ishizu and Wada River carp, but without statistical significance. The proportion of LH-ir cells in Ishizu River carp was significantly lower than that of the control and the Wada River in all seasons. The LH-ir cells in control carp increased in size in the breeding season. LH-ir cells in Ishizu River carp were significantly (p<0.05) smaller than those in control fish, but not different from Wada River carp. A disturbance in the secretory function of LH-ir cells was found in carp from the Ishizu River; granulation and vacuolation were not in synchronization with those of control and Wada River fish. Our data suggest that the estrogenic chemicals in the Ishizu River interfere with functions of LH-ir cells directly or through the testis.

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In the present study, we examined specific markers for taste bud cells in the mouse and the postnatal development of volatile papilla taste bud cells in ddY mice. We examined the immunoreactivity of 4 types of carbonic anhydrase isoenzymes, CA I, CA II, CA III and CA VI, as specific markers for taste bud cells, and K8.13 cytokeratin antibody as a specific marker for the lingual epithelial cells. Of the carbonic anhydrase isoenzymes, only CA III immunoreactivity was clearly detected in the spindle shaped gustatory cells. CA VI immunoreactivity was detectable in suspentacular cells. CA I and CA II antibodies did not recognize any taste bud cell specifically. K8.13 cytokeratin immunoreactivity was detected in the lingual epithelial cells, but not in taste bud cells. At 7 days after birth, the suckling phase, very small taste buds developed from the anaplastic gustatory cells. At 14 days after birth, the taste buds showed larger size than those at 7 days after birth. At 21 days birth, after the weaning phase, taste bud structure approximated the mature structure. These results demonstrate the specificity of anti-CA III and anti-CA VI for gustatory cells and suspentacular cells, respectively. These markers should be useful for an analysis of taste bud development in mice.

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A virus-like cytopathic agent isolated from swine farms with a history of recurrent abortion episodes was investigated. We employed a differential display reverse transcription-polymerase chain reaction (ddRT-PCR) to obtain genetic information of the cytopathic agent. Partial nucleotide sequence (527 bp) obtained from differentially displayed PCR fragments showed 88.7% similarity with the 23S rRNA gene of Mycoplasma hyopneumoniae. Unexpectedly, the 5' portion (1-333 bp) of the sequence shared 96.1% similarity with 5' untranslated region (UTR) of human prostate tumor inducing gene 1 (PTI-1). Cytopathic effects and extranuclear DNA fluorescence were no longer observed when BM-cyclin was added in the culture medium, suggesting that BM-cyclin sensitive mycoplasma-like organisms caused the cell death. Further evidence supporting the cytopathic agent as a mycoplasma-like organism was obtained by the capability of ³H-thymidine and ³H-uridine incorporation, a single peak in buoyant density gradient profile (1.20-1.24 g/ml), and ultrastructural morphology. Unlike M. hyopneumoniae, the organism was not propagated in Friis medium. Nucleotide sequence of 16S rRNA obtained from the cytopathic agent showed 0.8-1.0% divergences with other M. hyorhinis strains, suggesting that the newly isolated cytopathogenic swine mycoplasma was a variant form of M. hyorhinis. Striking homology between a portion of the 23S rRNA gene of M. hyorhinis and 5' UTR of human PTI-1 implicated that M. hyorhinis might potentially be related to the evolution of human PTI-1.

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The concentration of feline serum amyloid A (fSAA) was determined by a direct enzyme-linked immunosorbent assay (ELISA) by using fSAA specific monoclonal antibodies, to evaluate the fSAA as an inflammatory marker in cats. The mean concentration ± standard deviation of fSAA was found to be 0.60 ± 1.06 μg/m l and 33.65 ± 67.59 μg/ml in serum samples from normal cats (n=45) and cats (n=312) with various diseases and disorders, respectively. A significant difference (p<0.001) was found between the two groups. It was also found that the concentration of fSAA begins to increase rapidly at approximately 3-6 hr after spay, and increases up to significantly high levels in some disorders, like injury, renal failure, infectious diseases, etc.

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The aim of this study is to understand host immune responses in immunocompetent and immunocompromised mice against Bartonella henselae infection. BALB/c and nude (BALB/c nu/nu) mice were inoculated intraperitoneally with 10⁸ colony forming units of B. henselae (Houston-1 strain). Blood, brain, liver, spleen, kidney and bone marrow samples were collected 0, 3, 7, 14, 21 and 28 days after infection and submitted to bacteriological, serological and genetical examinations. B. henselae was isolated only from the liver 3 days after infection. DNA of the inoculums was detected by polymerase chain reaction from blood, liver, and spleen samples collected from BALB/c and blood from nude mice 3 and 7 days after infection. No bacterial DNA was detected from both BALB/c and nude mice thereafter during 4 weeks observation periods. These results indicate that the T-cell may not participate in the effective elimination of the organisms from mice. In addition, western blot analysis revealed that the antigens of 27.3- and 31.5-kDa reacted with IgM antibodies from the blood of BALB/c and nude mice after 3 days of infection, suggesting that these antigens were recognized by thymus-independent mechanism. Furthermore the antigens were detected from the culture-supernatants of B. henselae, indicating that these antigens were secreted from the organisms.

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The phenotype and function of peritoneal cavity macrophage-derived dendritic cells (PEC-DC) was previously reported. In this study we have gone further in using our established culture system to generated discrete Peyer's patch dendritic cells (DPP-DC) from murine discrete Peyer's patch macrophages (DPP-Mø), following stimulation with granulocyte macrophage colony stimulating factor (GM-CSF) plus interleukin 4 (IL-4) for 7 days. DPP-Mø from murine small intestines were obtained by mechanical disruption of discrete Peyer's patches (DPP), followed by metrizamide density gradient centrifugation to remove Peyer's patch resident DC and debri, after which an overnight adherent step in tissue culture medium was carried out for macrophage enrichment. Characterization of the generated DPP-DC was carried out using well-established criteria of morphology, expression of membrane antigens and capacity for antigen presentation. Dendritic cells expressed DEC-205, F4/80 and CD34 at high levels, but exhibited very low CD11c levels. They were shown to present soluble protein antigen to CD3⁺ spleen T cells. A comparison of the surface antigen expression in the progenitor DPP-Mø population and the generated DPP-DC showed a significant decrease in MHC class II levels and a marked down regulation of the co-stimulatory molecule CD86 (B7-2). High expression of the haemopoietic progenitor marker CD34 indicates that the generated DC, possess a haemopoietic rather than myeloid origin. Taken together, these results may provide a better understanding of the complex network regulating mucosal immune responses.

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Four sheep were immunized with synthesized peptides, derived from bovine leukemia virus (BLV) envelope glycoprotein, encapsulated in mannan coated liposomes as adjuvant. On the seventh day after the immunization, the sheep were intradermally challenged with BLV antigen, or synthesized peptides. The areas challenged with antigen were increased skin thickness and biopsied sequentially for immunohistological examinations. Strong delayed-type hypersensitivity was induced in sheep immunized and challenged with peptides encapsulated in mannan-coated liposomes. The major phenotype of the infiltrating lymphocytes was CD5⁺. The ratio of CD4⁺ to CD8⁺ cells was about 1:1.

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To clarify the relationship between plasma antioxidant activity and diseases in dogs, plasma samples were collected from 6 healthy dogs and 16 diseased dogs (6 dogs with cancer, 5 dogs with hepatic disease, and 5 dogs with inflammation ), and measured superoxide anion scavenging activities. Antioxidant activities of canine plasma were evaluated by measuring their superoxide anion (O₂^-·) scavenging activities with electron spin response spectroscopy combined with spin trapping reagent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Total O₂^-· scavenging activities in the presence of plasma of diseased dogs tended to be higher than those in healthy controls, especially significant higher activities in the presence of canine plasma of hepatic disease and inflammation were observed. In diseased dogs, KCN-insensitive activities, suggesting the activity of manganese-containing superoxide dismutase (Mn-SOD), were significantly higher than those in healthy controls. Therefore, it seems that there is a possibility of utilizing of plasma O₂^-· scavenging activity as one of clinical indicators for oxidative-related diseases such as cancer, hepatic disease and inflammation in dogs.

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Twelve horses kept at a riding club suffered from pyoderma. All the horses displayed crusting, scaling and alopecia. The lesions were distributed in the chest, back, rump and limbs. Some of the horse patients also showed epilation with an attached crust similar to a `paintbrush lesion' of dermatophilosis, but normal skin flora or opportunistic pathogenic bacteria were only isolated from the lesions. Some patients clearly showed weight loss, anemia and low levels of serum protein and cholesterol. General condition and skin lesions of the patients were improved gradually with improvement of feed and environment after being moved to new stables. Malnutrition under conditions of poor hygiene and poor management due to neglect might be associated with these equine cases of pyoderma in the herd.

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An intact male beagle dog aged 1 year was referred because of shortness of breath, exercise intolerance and cardiac murmur. Based on the results from electrocardiography, thoracic radiography and echocardiography, the dog was diagnosed as Ebstein's anomaly. Although the orally administered digoxin, vasodilators and diuretics partially improved congestive signs, the dog became to be refractory and died 20 months after the diagnosis. Necropsy confirmed malformation and apical displacement of the basal attachment of tricuspid valve leaflets.

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To determine the effects of ozone on the phagocytosis of bovine polymorphonuclear leukocytes (PMNs), ozone gas was administered in vitro on the blood and milk of healthy lactating cows, cows with acute mastitis, and cows with milk fever. In the blood of healthy dairy cattle, although there was no significant effect of ozone gas on the viability of the leukocytes, phagocytosis of PMNs significantly decreased. In contrast, ozone gas administration in vitro significantly increased phagocytosis of PMNs from the blood of cows with acute mastitis and milk fever, and from mastitic milk. These findings showed that ozone administration in vitro has positive and negative effects on bovine PMN phagocytosis, depending on the health status of the animal.

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Larva migrans caused by the common raccoon ascarid, Baylisascaris procyonis, is a zoonotic disease of critical importance in North America. Recently we encountered the first proven outbreak of this disease in Japan in domestic rabbits (Oryctolagus cuniculus) in a small wildlife park. In this park, raccoons (Procyon lotor) had been kept for 9 years, and one raccoon was donated to the park by a pet owner 8 weeks prior to the occurrence of an outbreak in rabbits. Of 12 total raccoons, three raccoons including the donated one shed B. procyonis eggs in the feces, and two of these positive raccoons were kept in metal mesh cages on wooden pedestals, 2 m distant from the rabbit enclosure. Circumstantial evidence indicates that the donated raccoon is the likely source of this outbreak. Treatment of the raccoons with an ascaricide and decontamination by extensive flaming of the cages and the contaminated dirt floor of the park achieved a transient disappearance of ascarids from all 12 enclosed raccoons. Three months after the control measures began, recurrent ascarid infection was detected in three young raccoons of less than 1.5 years of age. The potential risk of serious zoonosis by B. procyonis as well as the difficulty in a clearance of contaminated areas should be considered by pet owners and public health workers in Japan.

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For studying protein trafficking in Babesia-infected erythrocyte, we describe the cloning of a Rab5, one of molecular marker for vesicle trafficking in eukaryotic cells, gene homologue in Babesia gibsoni (BgRab5). The full-length cDNA of BgRab5 is 1,020 bp long with an open reading frame encoding a protein of 220 amino acids. The deduced amino acid sequence of BgRab5 contained the highly conserved GTP-binding consensus sequence and shares about 40% homology with that of Rab5 from Plasmodium falciparum, Toxoplasma gondii, Dog, Lotus japonicusor, Oryza sativa. Northern blot analysis showed that the BgRab5 probe hybridized with a 1kb band in total RNA from parasitized erythrocytes, that was consistent with the size of the BgRab5 full-length cDNA.

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The relationship between deoxyribonucleic acid (DNA) damage and the cell death induced by γ-irradiation was examined in three kinds of cells, Chinese hamster ovary fibroblast CHO-K1, human melanoma HMV-II and mouse leukemia L5178Y. Cell survival was determined by a clonogenic assay. The induction and rejoining of DNA strand breaks induced by radiation were measured by the alkaline and neutral comet assays. L5178Y cells were the most radiosensitive, while CHO-K1 cells and HMV-II cells were radioresistant. There was an inverse relationship between the survival fraction at 2 Gy (SF2) and the yield of initial DNA strand breaks per unit dose under the alkaline condition for the comet assay, and also a relationship between SF2 and the residual DNA strand breaks (for 4 hr after irradiation) under the neutral condition for the comet assay, the latter being generally considered to be relative to cellular radiosensitivity. In the present analysis, it was considered that the alkaline condition for the comet assay was optimal for evaluating the initial DNA strand breaks, while the neutral condition was optimal for evaluating the residual DNA strand breaks. Since the comet assay is simpler and more rapid than other methods for detecting radiation-induced DNA damage, this assay appears to be a useful predictive assay for evaluating cellular clonogenic radiosensitivity of tumor cells.

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A nine months old Japanese domestic cat with a solitary bone cyst, which is a benign tumor-like lesion and is particularly uncommon in feline practice was referred. Radiographic examination revealed an expansive radiolucency in the distal metaphysis of the right ulna and pathologic fracture. The histological examination demonstrated immature osteogenesis consisting of osteoblasts surrounded by connective tissue. We applied drainage and instillation of steroid solution into the cystic cavity. Clinical signs, including lameness and pain, disappeared three weeks after the therapy started. Fourteen months after the therapy, the cystic lesion of bone was remodeling successfully without re-developing. We conclude that our procedure was useful treatment in this case.

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To investigate the viability of timed artificial insemination (TAI) protocols in grazing Japanese Black cattle with long open period, ovarian status and progesterone and estradiol-17 β profiles of the animals during the protocol were monitored. In 1998, prosta-glandin F_2a (PG) was administered to 36 animals seven days after GnRH injection. Three out of the 36 animals were inseminated after detection of estrus and did not receive further treatment. The second GnRH was injected to the remaining 33 animals 48 hr after the PG injection and TAI was performed 24 hr later. In 1999, PG was injected to 25 animals six days after GnRH, the second GnRH was injected to 22 animals 48 hr after PG, and TAI was performed 16 hr later (The other three animals were inseminated before the time of TAI). The percentage of the animals with at least one functional corpus luteum and one follicle equal to or greater than 10 mm in diameter at PG injection was similar between the groups in 1998 and 1999. Likewise, the hormonal profiles were similar between the two groups. Pregnancy rates (PR) after the TAI protocols and natural mating in 1998 and 1999 were 75.0% and 88.0%, respectively. These figures were comparable to the PR obtained by conventional estrus synchronization protocols using PG (in 1995; 69.4%) or CIDR (in 1996; 59.1%). In conclusion, the TAI protocol can be applicable into grazing Japanese Black cattle with long open period.

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Interleukin-6 (IL-6) regulates essential physiological functions such as acute phase reaction, immune response and hematopoiesis. We here studied possible protective effects of IL-6 on skin damage caused by 7,12-dimethylbenz[a]anthracene (DMBA) by using IL-6 null mice. The mice were topically applied with a single dose of DMBA (500 μg /mouse) on the dorsal skin. Osmotic pumps filled with recombinant human IL-6 (rhIL-6) were implanted subcutaneously on the ventral side of the mice. Control mice received PBS instead of rhIL-6 by the pump. Severe skin damage was observed in IL-6-null mice, whereas only epidermal hyperplasia was observed in the wild-type mice. Recombinant hIL-6 treatment to DMBA-treated IL-6-null mice suppressed the occurrence of the skin damage, indicating.

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