The hypothesis that apoptotic factors play some roles in the denucleation of erythroblasts has been confirmed by the immunohistological detection of both phosphatidylserine and thrombospondin as phagocytosis-inducing factors in general apoptotic events. Both phosphatidylserine and thrombospondin were detected on the surface of cell membrane of mature erythroblasts, while thrombospondin was also detected in more immature erythroblasts. The intensities of their immune reactions increased as the erythroids matured. During denucleation, the positivities of both phosphatidylserine and thrombospondin were restricted on the surface of the cell membrane surrounding the protruding nuclei. Thus, the apoptotic process involves denucleation of erythroblasts and phosphatidylserine, and thrombospondin acts as phagocytosis-inducing factors in the denucleation event.

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In this study, we have evaluated our recently developed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for the molecular subtyping of Shiga toxin-producing Escherichia coli (STEC). A total of 200 STEC strains including O157 (n=100), O26 (n=50), O111 (n=10), and non-O26/O111/O157 (n=40) serogroups isolated during 2005-2006 in Japan, which were identified to be clonally different by pulsed-field gel electrophoresis (PFGE) were further analyzed by the PCR-RFLP assay in comparison to PFGE. Ninety-five of O157, 48 of O26, five of O111 and 19 of non-O26/O111/O157 STEC strains yielded one to three amplicons ranging from 6.0 to 15.5 kb in size by the specific primer set targeting region V which is located in the upstream of stx genes. These strains were classified into 41 (O157), 8 (O26), 4 (O111) and 17 (non-O26/O111/O157) groups based on the RFLP patterns obtained by subsequent restriction digestion, respectively. Although the discriminatory power of PCR-RFLP assay was somewhat less than that of PFGE, it is more convenient for molecular subtyping of STEC strains especially for O157, the most important serogroup implicated in human diseases, as well as to identify the outbreak-associated isolates because of its simplicity, rapidity, ease and good reproducibility.

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To identify factors that control coat color in Akita-inu dogs, we sequenced all the exons of the melanocortin 1 receptor (MC1R), β-defensin103 (CBD103) and agouti signaling protein (ASIP) genes of dogs with four distinct coat colors, namely, brindle, sesame, red and white. Then we examined correlations among specific alleles and coat color. In the case of the MC1R gene, all white dogs were homozygous for a nonsense mutation, R306ter, while brindle, sesame, and red dogs had at least one R306 allele. In the case of the CBD103 gene, all brindle dogs were heterozygous for the G23del mutation (deletion of codon 23, encoding glycine), while all sesame and red dogs were homozygous for G23. In the case of the ASIP gene, all dogs, regardless of coat color, had at least one S82 H83 allele. A missense mutation in the ASIP gene, P87L, was identified for the first time in some Akita-inu dogs but was not associated with any specific coloration. Our results indicate that the 2 key mutations, R306ter in the MC1R gene and G23del in the CBD103 gene, are associated with the phenotypic discriminations among brindle, red/sesame, and white coats, while no mutation that might potentially be associated with the discrimination of a sesame coat from a red coat is present in the coding sequences of these three genes.

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Clinical application of genetic clonality analysis by PCR has been available in feline lymphoma. However, it is in under dispute whether the analysis can be applied for cellular lineage determination. To evaluate utility of the analysis for lineage determination, the results between immunohistochemistry and the genetic analysis were compared. Based on the result with immunohistochemistry, sensitivity of the primer for IgH was 89% and specificity was 75%. Sensitivity of the primer for TCRg was 25% and specificity was 100%. Cross-lineage IgH rearrangement was found in 25% of T-cell lymphomas. The present study suggests that the genetic analysis had better be applied as not a definite but an adjunctive tool for lineage determination in feline lymphomas.

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In order to introduce the Canine Behavioral Assessment and Research Questionnaire (C-BARQ), which is a standardized system for evaluating the behavioral traits of dogs, to Japan, where the environment with respect to dog ownership is thought to differ from those of the United States and Europe, we compared demographic information on dogs in the United States and Japan and examined whether similar factors could be extracted from both countries using questionnaire items of the C-BARQ. The C-BARQ was completed by 11,410, and 734 dog owners respectively in the United States and Japan, and some demographic differences were found, such as breed and neuter status. Data from completed questionnaires were subjected to factor analysis, and the resulting factors were tested for reliability. In the United States, factor analysis yielded 11 factors from 63 items that accounted for 52.9% of the common variance. In Japan, 15 factors were extracted, and these accounted for 57.0% of the common variance. The present factors for the United States were almost identical to the factors identified in a previous study, and similar factors were extracted in both countries. Therefore, the C-BARQ can function effectively as a fundamental behavioral evaluation system for dogs in Japan.

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We evaluated the anti-tumor effect of adenoviral vector-mediated p53 gene therapy on the growth of canine osteosarcoma xenografts formed in nude mice. Nude mice were subcutaneously transplanted with cells of 2 P53 mutant canine osteosarcoma cell lines, POS and CHOS. The osteosarcoma xenografts were injected with either an adenoviral vector that expresses canine wild-type P53 (AxCA-cp53) or LacZ (AxCA-LacZ). Tumor growth was significantly inhibited in the xenografts injected with AxCA-cp53 in comparison to those injected with AxCA-LacZ or PBS during the observation period of 27 days. An increase of the amount of p21^WAF1/CDKN1A mRNA, and the number of apoptotic cells was shown in the tumors injected with AxCA-cp53 in comparison to those injected with AxCA-LacZ or PBS. The present study revealed that the adenoviral vector-mediated p53 gene transfer had an anti-tumor effect in canine osteosarcoma xenografts formed in nude mice.

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Changes in inflammatory parameters, leukocyte surface markers, functional responses and cytokine mRNA expression of leukocytes of dairy cows with naturally occurring chronic Staphylococcus aureus (S. aureus) mastitis and healthy cows were determined to elucidate the leukocyte responses to S. aureus infection of the mammary gland. Increased values in inflammatory parameters and matrix metalloproteinase activities in milk revealed the characteristics of cows with chronic mastitis. Expression of L-selectin and CD18 molecules on neutrophils and proportion of CD8 cells in milk from cows with S. aureus mastitis were significantly (P<0.05) increased compared with those found in healthy cows. The FcR-stimulated CL response of blood neutrophils was significantly (P<0.05) decreased in cows with S. aureus mastitis. Significantly (P<0.05) decreased mitogenic responses of lymphocytes were found in cows with S. aureus mastitis; however, the values were not restored to those of healthy cows when stimulated with both mitogens and the cytokine IL-1β. The mRNA expression of TNF-α, IL-1β and IL-8 on milk leukocytes from cows with S. aureus was found to be increased compared with that of healthy cows. The changes of immune responses found in cows with S. aureus mastitis appear to be influenced by the severity and duration of inflammation in infected quarters. The down-regulation of the leukocyte functions found in cows with S. aureus mastitis appears to be associated with the progress of the chronic stage of S. aureus mastitis.

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Many cases of encephalitic disease with unknown etiologies have been reported in specific breeds of small dogs. High mobility group box 1 (HMGB1) in neuronal cells was recently found to be a novel cytokine-like mediator that is a marker of neuronal necrosis and inflammation. The aim of this study was to determine whether HMGB1 levels are elevated in the cerebrospinal fluid (CSF) of dogs suspected of having encephalitis. CSF was obtained from 31 dogs that were diagnosed with an encephalitic disease by clinical examinations and magnetic resonance image (MRI) scanning. The CSF samples were analyzed via western blotting (WB) and an enzyme-linked immunosorbent assay (ELISA) with a polyclonal antibody against HMGB1. The mean HMGB1 concentration was significantly higher in the encephalitic dogs than that in the healthy controls. The concentrations of HMGB1 were correlated with the cell counts and total protein concentrations, which are known CSF indicators of the neuronal inflammation associated with encephalitis. These results suggest that HMGB1 protein in CSF confirms the presence of necrosis and inflammation in most cases of canine encephalitis and that HMGB1 will be a new indicator of encephalitis.

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We examined the refractive value in healthy Beagles by skiascopy. The mean refractive value of 54 eyes of 27 Beagles was 0.08 ± 0.87 (mean ± SD) diopters (D). The numbers of eyes defined as having emmetropia, myopia and hyperopia were 34, 8 and 12, respectively. Anisometropia was detected in 4 dogs. The mean refractive values in the 3-6-year-old and 8-9-year-old groups were 0.26 ± 0.84 and -0.29 ± 0.82 D, respectively, with a significant difference between the two groups (P<0.05).

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A 7-year-old Miniature Dachshund presented with severe chronic jaundice and elevated liver enzymes. Destructive cholangitis was diagnosed according to histopathological findings of remarkable ductopenia with inflammatory infiltrates and fibrosis in the portal areas. Supportive therapy with prednisolone, high-dose ursodeoxycholic acid, human placental extract and antibiotics was tried, and the patient showed recovery of clinical signs 3 months after diagnosis. A second liver biopsy was performed about 1 year after initial diagnosis, and bile duct restoration was confirmed with continuous inflammation around portal areas and inside the lobules. Although we could not determine which treatment was effective in this case, destructive cholangitis in dogs may be recoverable with long-term supportive therapies.

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This study compared the immunosuppressive effects of dexamethasone (DEX), flunixin meglumine (FLU) and meloxicam (MEL) on the peripheral blood mononuclear cells (PBMCs) of seven healthy Holstein calves in vitro. DEX significantly inhibited lymphocyte proliferation and expression of interferon (IFN)-γ, interleukin (IL)-2 and IL-4 messenger RNA (mRNA) in comparison with FLU and MEL. FLU and MEL dose-dependently inhibited lymphocyte proliferation, but did not significantly reduce mRNA expression. Our in vitro study indicates that steroidal anti-inflammatory drugs (SAIDs) as well as nonsteroidal anti-inflammatory drugs (NSAIDs) have immunosuppressive effects on calf PBMCs. These findings are important for assessing the indications and complications of NSAIDs in calves.

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An 8-year-old male Golden Retriever had lameness and claw abnormality in the second digit of the left forelimb. Radiography revealed osteomyelitis in the distal phalanx bone of the affected limb. Microscopic examination of the claw revealed numerous hyphae in the claw matrix. Fungal DNA fragments coding the ribosomal internal transcribed spacer region (ITS) were detected from the claw matrix as well as fungal colonies of the clinical isolates by PCR. Nucleotide sequencing revealed that the amplicons shared > 99% homology with Fusarium sp. Therapy including oral itraconazole resulted in regrowth of a new claw, in which no hyphae were detected. To the authors' knowledge, this is the first case report of canine onychomycosis in which Fusarium sp. was isolated from the affected claw.

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It is known that pica, the consumption of non-nutritive substances such as kaolin, can be induced by administration of toxins or emetic agents in rats. In the present study, we examined the effects of intraperitoneal (i.p.) administration of cyclophosphamide on pica behavior and on the concentration of 5-hydroxyindoleacetic acids (5HIAA) in cerebrospinal fluid (CSF) in the following five strains of adult male rats: Sprague Dawley (SD), Wistar, Fischer 344 (F344), Wistar-Imamichi (WI) and Long Evans (LE). Cyclophosphamide (25 mg or 50 mg/kg) was injected (i.p.) into the rats and kaolin and food intake were measured at 24 hr after injection. The animals were anesthetized with urethane (1 g/kg) at 3 hr after injection of cyclophosphamide, and CSF was collected from the cisterna magna. WI and LE rats clearly showed pica behavior as compared with the other strains. In LE rats, the concentration of 5HIAA in CSF also increased in a dose-dependent manner of cyclophosphamide. The pretreatment with ondansetron (5-HT₃ antagonist) restored both changes (kaolin consumption and 5HIAA levels) induced by cyclophosphamide. These results suggest that the LE rat is sensitive to cyclophosphamide, that pica induced by cyclophosphamide mimics many aspects of emesis including the serotonergic response in the central nervous system and that use of the pica model would be a practical method for evaluating the effects of antiemetic drugs in addition to the mechanism of emesis.

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I have developed a congenic mouse strain for the A^y allele at the agouti locus in an inbred DDD/Sgn strain, DDD.Cg-A^y. DDD.Cg-A^y females are extremely obese and significantly heavier than B6.Cg-A^y females. The objectives of this study were to determine the genetic basis of obesity in DDD.Cg-A^y mice, and to determine whether or not their high body weight was due to the presence of DDD background-specific modifiers. I performed quantitative trait locus (QTL) analyses for body weight and body mass index in two types of F₂ mice [F2 A^y (F₂ mice carrying the A^y allele) and F₂ non-A^y (F2 mice without the A^y allele)] produced by crossing C57BL/6J females and DDD.Cg-A^y males. The results of the QTL analysis of F₂ A^y mice were very similar to those obtained for F₂ non-A^y mice. It was unlikely that the high body weight of DDD.Cg-A^y mice was due to the presence of specific modifiers. When both F₂ datasets were merged and analyzed, four significant body weight QTLs were identified on chromosomes 6, 9, and 17 (2 loci) and four significant obesity QTLs were identified on chromosomes 1, 6, 9, and 17. Although the presence of DDD background-specific modifiers was not confirmed, a multifactorial basis of obesity in DDD.Cg-A^y females was thus revealed.

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The aim of this study was to examine the blood coagulation profiles of ferrets and compare them with those of rats. The ferret activated partial thromboplastin time (aPTT) was slightly longer than the rat aPTT. In contrast, the ferret prothrombin time and thrombin time were profoundly shorter than the corresponding rat values. The fibrinogen level in ferret plasma was 2 times higher than that in rats. Heparin prolonged all blood coagulation times in a concentration-dependent manner in both ferret and rat plasma. A significantly (P<0.01) higher concentration of heparin was required to double the aPTT in ferrets than rats. These blood coagulation data for ferrets will be useful in experimental animal studies.

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The adhesion-preventing effect of trehalose in visceral organs was evaluated in a rabbit model. All rabbits underwent a hysterotomy, during which mild abrasion and desiccation was induced on the surfaces of visceral organs. In the control group, the intestines were kept dry during hysterotomy. In the saline and trehalose groups, saline solution and 7% trehalose solution, respectively, were sprayed on the organ surfaces. Adhesion formation on organ surfaces on which abrasion and desiccation were induced declined significantly in the trehalose group compared with the other groups. In contrast, at the hysterotomy site, there was no significant difference in adhesions between groups, suggesting that trehalose prevents adhesions in organs that have developed minor inflammation by abrasion and desiccation.

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This study evaluated the effect of butylated hydroxytoluene (BHT), a lipid-soluble antioxidant, on dog sperm in chilling storage and cryopreservation. In Experiment 1, 0.2, 0.4, 0.8 and 1.6 mM BHT were added to egg yolk Tris extender (EYT), and sperm were stored at 4°C for 96 hr. Sperm motility, viability, acrosomal integrity and morphological abnormality in the BHT treatment groups were not different from those of the control (0 mM BHT). In Experiment 2, the effect of BHT in EYT containing 0.75% Equex STM paste and 5% glycerol on survivability of cryopreserved sperm was examined after culture at 39°C for 3 hr. Sperm motility, viability and acrosomal integrity in the 0.2 to 0.8 mM BHT treatment groups were not different from those of the control. However, sperm motility, viability and acrosomal integrity decreased when 1.6 mM BHT was added to the extender (P<0.05). In conclusion, supplementation of the extender with 0.2 to 0.8 mM BHT did not affect characteristics of dog sperm in chilling storage and cryopreservation. Supplementation of 1.6 mM BHT did not affect characteristics of chilled sperm but impaired longevity of cryopreserved sperm in the dog.

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This study was performed to evaluate the use of dimethylacetamide (DMA) and dimethyl sulfoxide (DMSO) in boar sperm cryopreservation. Semen from eight boars was cryopreserved following treatment with 3, 5, and 7% DMA and DMSO, and 3% glycerol (control). After thawing, sperm conventional parameters and membrane integrities were evaluated. There were no significant differences among different DMA concentrations in all evaluations. Membrane intactness were higher in 5% and 7% DMSO than 3% DMSO (P<0.05). Sperm motility of 5% DMSO was lower than that of 3% glycerol (P<0.005), and membrane intactness were lower in 5% DMA and DMSO than 3% glycerol (P<0.05). DMA and DMSO didn't improve sperm quality and glycerol remains the most useful for boar sperm cryopreservation.

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In order to understand ovarian toxicity of aryl hydrocarbon receptor (AhR) agonists, in situ gene expression of the AhR was examined during follicle development in immature rats. In situ hybridization on frozen sections of ovaries from 24-day-old Sprague-Dawley rats showed that the AhR mRNA was localized in the granulosa cells and occasionally in the theca cells of the follicles irrespective of the developmental stage. In situ gene quantification on granulosa cell layers collected by laser microdissection further revealed that the granulosa cells expressed less AhR mRNA according to development of belonging follicles, but more β-subunit of inhibin A mRNA, a quality control gene. These results may help to elucidate vulnerable developmental stages of follicles to toxicities of the AhR agonists.

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From April to July 2009, there was a high rate of reproductive losses in a 30-sow, farrow-to-finish swine herd in Yamagata Prefecture, Japan. Histopathological examinations of heart tissue from stillborn and preweaning piglets showed nonsuppurative, necrotizing lesions. Immunohistochemical staining and polymerase chain reaction analysis of myocardial lesions revealed the presence of porcine circovirus type 2 (PCV2) antigens and DNA in these tissues. Indirect immunofluorescence also showed that the PCV2 antibody positive rate in the sows was higher in May 2009 than in December 2008. The results of this study suggest that PCV2 spread to this farm and caused a high rate of reproductive losses.

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