Density and morphologic features of hoof wall tubules in horses’ hooves are described. The sample population consisted of hooves (n=12) from both live and deceased adult horses. Full wall thicknesses of the hoof wall were prepared histologically from sites at the toe, medial quarter and lateral quarter. In defined areas, tubules were counted, and density was calculated across the three histologically defined areas of the stratum externum, stratum medium and stratum internum along with the descriptive morphologic features of the tubules. Morphologically, distinct zones were demarcated by varying tubular cortical and medullary characteristics. Tubule density showed a radially increasing pattern across the wall thickness. Significant regional differences in density were found in the toe, lateral and medial walls. These findings represent a first study of regional differences of tubular densities. Hoof horn clippings from live feet may have potential diagnostic and/or prognostic value.

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To provide anatomical data on the nutrient foramen (NF), the authors observed the number, site and its index (SI), position, diameter and penetrating direction of the nutrient canal (NC) in the femur and tibia of German shepherd dogs. The femur often had more than two NF on the caudal surface with 30.0 to 86.7% of SI. The proximal NF of the femur was regarded to be the major NF, because it was present in most dogs and had the largest diameter. The tibia always had only one NF with 23.2 to 38.3% of SI on the lateral border. Their direction of the NC followed Bérard’s rule with the exception of the proximal NF of the femur. These results describe the anatomical condition of the NF and NC in the dog.

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Clinical efficacies of mutant prevention concentration (MPC) and mutant selection window (MSW) hypotheses have been evaluated for human clinical isolates. We tested the MSW hypothesis by evaluating the relationships between MPCs and mutation frequencies against enrofloxacin for avian pathogenic Escherichia coli (APEC) isolates. Mutation frequencies of strains with MPC:MIC ratios of 8 to 16 were significantly higher than those of strains with an MPC:MIC ratio of 4. Mutation frequencies and MPCs of serogroup O2 strains were lower than those of the other strains; these results may correlate with the absence of fluoroquinolone-resistant O2 strains. Our results support the MSW hypothesis that the range of the MSW is involved in selection of resistant mutants.

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The purpose of the present study was to determine the prevalence of antibiotic-resistant enterococci in dogs and cats subjected to differing antibiotic pressures, and the prevalence of vancomycin resistance genes in isolates from these animals. Enterococci were isolated from fecal samples of 65 healthy dogs and 29 healthy cats brought to animal hospitals, from rectal swabs of 73 puppies and 15 kittens from five breeders and two pet shops, and from fecal samples of 20 dogs and 9 cats that were treated with antibiotics in Nippon Veterinary and Life Science University Animal Medical Center. The rates of resistance to ampicillin among isolates from the kitten–puppy group and healthy dog–cat group were 6.8 and 4.3%, respectively. In contrast, the rates of resistance to ampicillin in enterococci from the treatment group under antibiotic pressure were 37.5%. There was a significant difference between the antibiotic-treated group and the untreated group (P<0.01). Similarly, in the treatment group, the rate of resistance to enrofloxacin was extremely high (75.0%). In comparison, in the healthy group and kitten–puppy group, the rates of resistance to enrofloxacin were 23.4 and 12.1%, respectively. Among these groups, a significant difference was also observed in the apparent resistance rates (P<0.01). Vancomycin-resistant enterococci (VRE) harboring vanA or vanB were not detected in any groups. Therefore, contamination of VRE in dogs and cats is still considered to be minimal in Japan.

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Pulmonary surfactant protein A (SP-A) is used as a biomarker to understand the clinical features of pulmonary diseases and associated prognostic indices in human medicine. This study was conducted to investigate whether or not serum SP-A concentration can be used as a biomarker for identifying pulmonary parenchymal diseases in dogs. Thirty-two dogs with pulmonary parenchymal diseases, 34 with nonrespiratory diseases and 57 healthy dogs were included. Serum SP-A concentration was measured in all dogs using sandwich enzyme linked immunosorbent assay with an anti-dog SP-A polyclonal antibody. Median serum SP-A concentration in healthy dogs was <2.0 ng/ml, whereas that in dogs with aspiration pneumonia (n=11), primary lung tumors (n=9) and blunt traumatic lung injury (BTLI; n=12) was 3.1, 7.2 and 2.6 ng/ml, respectively; these values were significantly higher than those in healthy dogs. The serum SP-A concentration in dogs with nonrespiratory diseases was comparable with that in healthy dogs. No correlation was observed between the serum SP-A and plasma C-reactive protein concentrations in dogs with aspiration pneumonia and BTLI. There was a significant correlation between the serum SP-A concentration and thoracic radiographic changes in dogs with BTLI. These findings suggest that the serum SP-A concentration may be a useful clinical biomarker of alveolar damage that can be used for differential diagnosis of pulmonary parenchymal diseases and nonrespiratory diseases in dogs.

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Lymphoid malignancies, such as leukemia, and many types of lymphoma are common and severe disorders in dogs. Since shortening remission duration caused by resistance to chemotherapy often becomes clinically critical problems, development of novel and effective therapy should be required. The present study investigated the status of NF-κB and effect of its inhibitor, bortezomib, in six canine neoplastic lymphoid cell lines. NF-κB p65 and p50 were detected in the nuclear fraction of GL-1, CLBL-1 and CL-1, suggesting that NF-κB was constitutively activated in the cells. NF-κB p65 was detected in the cytoplasmic fraction of UL-1 and Ema. After incubation with bortezomib, NF-κB p50 and p65 became undetectable in the nuclear fraction of GL-1, CLBL-1 and CL-1, and CLBL-1, respectively, and p65 was clearly degraded in the cytoplasmic fraction of CLBL-1 and CL-1. Bortezomib inhibited the proliferation of all cell lines except Nody-1 in a concentration-dependent manner. The results indicated that constitutive activation of NF-κB could contribute to the proliferation of canine neoplastic lymphoid cells, and bortezomib would have suppressive effects on the NF-κB activation and the proliferation of neoplastic lymphoid cells in dogs.

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The characteristics of surface antigens in canine hepatocellular carcinoma (cHCC) have not been clarified. The objective of this study was to investigate surface antigens, which are considered as stem/progenitor or cancer cell markers, in cHCC cell lines. Expression of various antigens including CD29, CD34, CD44, CD90, CD133 and Dlk-1 was assessed in four cHCC cell lines by flow cytometry. CD44, CD133 and Dlk-1 expression was detectable in all cell lines, and three cell lines expressed CD29. These results indicate that CD29, CD44, CD133 and Dlk-1 have potential as suitable markers in cHCC identification, suggesting that these findings will contribute to the establishment of an early diagnostic tool for the identification of hepatocellular maturation processes.

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Previously, we have demonstrated that the alarm pheromone deteriorates sexual behavior in male rats, which was blocked by pretreatment with a corticotrophin-releasing hormone (CRH) antagonist. Studies have shown that an opioid antagonist blocked the deterioration of male sexual behavior following intracerebroventricular administration of CRH. Therefore, possibly, the pheromone effects could also be mediated by the opioid system. In this study, we pretreated rats with naloxone, an opioid receptor antagonist, and examined the following sexual behavior modulations in male rats that were exposed to the alarm pheromone. Naloxone blocked the deterioration of sexual behavior in a dose-dependent manner. On the basis of these data and the results of the previous study, we conclude that the alarm pheromone activates the CRH system, which then activates the opioid system, to deteriorate male sexual behavior.

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Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of diarrhea in neonatal and postweaning piglets. Fimbrial adhesion of ETEC has been considered an important colonization factor with antigenicity. To safely and effectively deliver the F4 (K88) fimbrial adhesin FaeG to the immune system, we have previously constructed the secretory expression vector pNZ8112-faeG, and FaeG was produced in cytoplasmic form in Lactococcus lactis. In this work, BALB/c mice were immunized with recombinant L. lactis to further determine the immunogenicity of recombinant FaeG (rFaeG) via the subcutaneous or oral route. Subcutaneous immunization in mice with recombinant L. lactis induced a significant increase in the F4-specific serum IgG titer and the number of antibody-secreting cells (ASCs) in the spleen. Oral immunization of mice with recombinant L. lactis induced mucosal and systemic F4-specific immune responses and increased the number of ASCs in the spleen, mesenteric lymph nodes and Peyer’s patches. High-dose (2.8 × 10¹¹ CFU) recombinant strains and adjuvant cholera toxin B subunit enhanced specific mucosal immune responses. The results suggest the feasibility of delivering rFaeG expressed in L. lactis to the immune system in order to induce an F4-specific immune response.

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Of the various classification systems for non-Hodgkin’s lymphoma, the updated Kiel classification is valuable for veterinary practice, because of its utility to classify the subtypes not only by histopathology but also by cytomorphology. However, there are only a few reports of small number of feline lymphomas to apply the updated Kiel classification. In this study, immunogical subtypes and morphology of 76 feline lymphomas were evaluated and classified into subtypes of the updated Kiel classification. Of the cases, 49% were T-cell lymphoma, 25% were B-cell lymphoma and 26% were undetermined immunological subtype. Based on the updated Kiel classification, most subtypes were identified also in feline lymphomas as in dogs and humans. Globule leukocyte lymphoma was specific for cats and relatively common in feline alimentary lymphomas. Of the present cases, 64% were high-grade subtypes, whereas 36% were low-grade subtypes. The present study indicated that feline lymphomas could be morphologically classified by the modified updated Kiel classification.

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The p16, p15 and p14 genes are widely known as tumor suppressor genes in human medicine. Although a large number of genetic and epigenetic aberrations in these genes have been reported in human malignancies, canine malignancies have not been well analyzed on the aberrations of these genes. In this study, the full-length complementary DNA (cDNA) of the canine p16 gene was cloned using the 5’ and 3’ rapid amplification of cDNA ends methods. Based on the sequence data, primers specific for p16, p15 and p14 were designed. Using these primers, the expression of p16, p15 and p14 mRNAs could be individually evaluated by reverse transcriptase polymerase chain reaction. Genomic aberrations were also examined using genomic polymerase chain reaction. Two of the 6 canine lymphoid tumor cell lines did not express detectable levels of p16, p15 and p14 mRNAs, and wide-ranging deletions in the p15-p14-p16 genomic locus were suspected. Wide-ranging deletions were also speculated in 2 of 14 dogs with T-cell lymphoid tumors. On the other hand, similar failure of amplification suggesting wide-ranging deletions were not observed in any of the 14 dogs with B-cell lymphoma. Deletion of the p15-p14-p16 genomic locus could be one of the molecular aberrations in canine lymphoid tumor cells.

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Mast cell tumors (MCTs) are the most common tumors in dogs, accounting for 16–21% of cutaneous tumors. Although several small molecule inhibitors, including imatinib mesylate, have been used for the treatment of MCTs, the response rate remains less than 50%. In this study, the effects of different selective signal inhibitors on MCT cell growth were evaluated using 4 different cell lines derived from dogs. We found that the phosphoinositide 3-kinase (PI3K) signaling pathway is crucial for the proliferation of MCT cells in the presence or absence of c-kit gene mutations. Here, we propose a novel therapeutic strategy to target the PI3K pathway for the treatment of canine MCTs.

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Canine degenerative myelopathy is an adult-onset, progressive neurodegenerative disease that occurs in multiple dog breeds, particularly Pembroke Welsh Corgis. Recently, a degenerative myelopathy-associated mutation of the canine SOD1 gene was identified as c.118G>A (p.E40K). In the present study, genotyping assays using conventional and real-time PCR methods were developed, and a preliminary genotyping survey was performed on 122 randomly selected Pembroke Welsh Corgis without any degenerative myelopathy-related clinical signs to determine the current allele frequency in Japan. Both of the assays provided clear-cut genotyping. The survey demonstrated the frequencies of the G/G wild-type, G/A heterozygote and A/A homozygote to be 9.0, 42.6 and 48.4%, respectively, indicating that the prevalence of the mutant A allele (69.7%) in Pembroke Welsh Corgis is extremely high in Japan.

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In order to investigate the prevalence of tick-borne diseases, equine piroplasmosis, equine granulocytic anaplasmosis and Lyme borreliosis in Central Italy, blood samples from 300 horses were analyzed for the presence of antibodies against Babesia caballi, Theileria equi, Anaplasma phagocytophilum and Borrelia burgdorferi using the IFAT. The blood samples were also subjected to PCR assays in order to detect pathogen DNA. A total of 78 (26.0%) and 123 (41.0%) horses were found to be seropositive for B. caballi and T. equi, respectively, while 41 (13. 4%) and 21 (7.0%) horses were, respectively, seropositive for A. phagocytophilum and B. burgdorferi. Seropositivity for more than one agent was detected in 76 horses using IFAT. The most common association observed was between T. equi and B. caballi (14.7%). In addition, 54 horses (18.0%) were found to be positive for one or more tick-borne pathogens (TBPs) using PCR testing. Among these, 28 (9.3%) harbored single infections, while 26 (8.7%) were found to be co-infected with two or more pathogens. The correlation (K value) between IFAT and PCR results was 0.32 for T. equi, 0.34 for B. caballi, 0.62 for B. burgdorferi and 0.48 for A. phagocytophilum, reflecting an unprecedented degree of multiple exposures to TBPs in horses.

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The ecologies of Lyme disease Borrelia spp. are very specific to location, as they are dependent upon the spirochete species and genotypes, the vectors and the host vertebrates present. In Hokkaido, Japan, where two human pathogenic, Lyme disease Borrelia spp. are present, and human cases are reported annually, the ecologies have been poorly studied. Our goal was to determine whether variation in borrelial infection rates among rodent species sharing an environment, is due to immunological or ecological differences. To this end, we examined the relationships between tick burden and borrelial infection, by including examination of agreement between nested PCR, as a test for infection, and serology, as a test for exposure. We collected 868 rodents, comprised of four species commonly found in Hokkaido, and tested for infection rates with Borrelia spp. using PCR for the borrelial flaB gene, seroprevalence of Borrelia afzelii and Borrelia garinii using ELISA, and attachment of ticks by direct counts. We noted a correlation between differential nymph and larval burdens and the borrelial infection rates found among the four rodent species. Furthermore, there was significant correlation between infection and seroprevalence of B. afzelii and B. garinii (P<0.01), between infection and Ixodes persulcatus nymph burden (P<0.01), and between seroprevalence and I. persulcatus nymph burden (P<0.01). The close agreement among rodent species seroprevalences with infection rates and tick burdens suggest the differences in infection rates of Borrelia spp. may largely be a direct consequence of differential exposure to vectors.

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A case of signet ring cell lymphoma in a 3-year-old mixed-breed sow is described. Macroscopical examination revealed enlargement of superficial, thoracic and abdominal lymph nodes and multiple tumor masses in the liver. The neoplastic tissue was composed of follicle center-like structures, in which neoplastic cells with Russell bodies were conspicuous. The bodies were immunostained for IgM (κ), and corresponded to moderately dense amorphous material within markedly distended cisternae of rough endoplasmic reticulum (RER) at the ultrastructural level. In contrast to typical signet ring cell lymphoma, the component cells of which resemble follicular center B lymphocytes with poorly developed RER, most neoplastic cells had features of plasma cells characterized by a cartwheel arrangement of heterochromatin and development of RER. Signet ring cells frequently had one or a few large Russell bodies occupying the entire cytoplasm, which may have been caused by abundant synthesis and defective secretion of immunoglobulin.

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A 33-year-old red-crowned crane (Grus japonensis) had a diffuse intestinal thickening from the duodenum to colon. Microscopically, neoplastic cells were arranged in sheets and occasionally nests or cords without gland or squamous differentiation. Metastatic tumor cells were found in the lungs, heart, kidneys and adrenal glands. Immunohistochemically, the neoplastic cells were strongly positive for pan-cytokeratin and cytokeratin 8 and 18 and only partly positive for E-cadherin antibodies. Immunostaining for CD3 was positive in normal lymphocytes, and NSE was also positive in normal nerve fibers. But, the neoplastic cells were not immunoreactive to CD3 and NSE. Based on the histological features and the epithelial characteristics in the immunohistochemical stain, the present case was diagnosed as undifferentiated carcinoma originating from the intestine. Interestingly, the neoplastic cells showed a unique growth pattern; they never invaded the submucosa or muscularis throughout the intestine, whereas they spread lymphogenously or hematogenously to other organs.

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Influences of psychotropic drugs, six antipsychotics and three antidepressants, on acetylcholine receptor-operated potassium current (I_K.ACh) were examined by a whole-cell patch clamp method in freshly isolated guinea-pig atrial myocyte. I_K.ACh was induced by a superfusion of carbachol (CCh) or by an intracellular application of guanosine 5’-[thio] triphosphate (GTPγS). To elucidate mechanism for anticholinergic action, IC₅₀ ratio, the ratio of IC₅₀ for GTPγS-activated I_K.ACh to CCh-induced I_K.ACh, was calculated. Antipsychotics and antidepressants inhibited CCh-induced I_K.ACh in a concentration-dependent manner. The IC₅₀ values were as follows; chlorpromazine 0.53 μM, clozapine 0.06 μM, fluphenazine 2.69 μM, haloperidol 2.66 μM, sulpiride 42.3 μM, thioridazine 0.07 μM, amitriptyline 0.03 μM, imipramine 0.22 μM and maprotiline 1.81 μM. The drugs, except for sulpiride, inhibited GTPγS-activated I_K.ACh with following IC₅₀ values; chlorpromazine 1.71 μM, clozapine 14.9 μM, fluphenazine 3.55 μM, haloperidol 2.73 μM, thioridazine 1.90 μM, amitriptyline 7.55 μM, imipramine 7.09 μM and maprotiline 5.93 μM. The IC₅₀ ratio for fluphenazine and haloperidol was close to unity. The IC₅₀ ratio for chlorpromazine, clozapine, thioridazine, amitriptyline, imipramine and maprotiline was much higher than unity. The present findings suggest that the psychotropics studied suppress I_K.ACh. Chlorpromazine, clozapine, thioridazine, amitriptyline, imipramine, maprotiline and sulpiride are preferentially acting on muscarinic receptor. Fluphenazine and haloperidol may act on G protein and/or potassium channel.

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To verify availability of skin conductance (SC) as an indicator for the sympathetic nervous system (SNS) activity in dogs, the changes in SC and blood levels of stress-related hormones induced by drugs were compared. SC and cortisol, adrenaline and noradrenaline levels were measured in 5 dogs on 4 occasions with or without drug-induced sedation at 7-day intervals (no treatment, intramuscular medetomidine 0.01 mg/kg, intramuscular acepromazine 0.1 mg/kg and intravenous fentanyl 0.02 mg/kg). The fentanyl treatment produced significantly higher levels of SC and plasma cortisol and adrenaline compared with the other 3 treatments. The plasma noradrenaline level also tended to be higher following the fentanyl treatment. These results indicate that SC may reflect changes in the SNS activities in dogs.

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A total of 466 rodents were captured in the Republic of Zambia from 2006 to 2010. Based on morphological observations and phylogenetic analyses of mitochondrial gene sequences, rodents were divided into 10 groups consisting of 39 Rattus rodents, 263 multimammate rats, 18 other Murinae rodents, 95 gerbils, 11 pouched mice, 1 giant-pouched rat, 38 fat mice and 1 dormouse. Rodent antibodies except that from Rattus were examined for their cross-reactivity to commercially available antibody detection reagents. Anti-mouse immunoglobulin G (IgG) was most cross-reactive to heterologous antibodies including multimammate rat, gerbil, pouched mouse and fat mouse. Thus, anti-mouse IgG would be a useful detection tool in serological examination of the Zambian rodent population. Preliminary sero-surveillance for plague, leptospirosis and hantavirus infection was performed by ELISA.

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In this study, we aimed to determine the effect of trehalose coating and the optimal dose of basic fibroblast growth factor (bFGF), an osteoinductive protein, loaded onto tailor-made bone implants for implant-induced bone formation in vivo. We fabricated tailor-made α-tricalcium phosphate bone implants (11 mm diameter with 2 parallel cylindrical holes). bFGF 0, 1, 10, 100 or 200 μg/implant was incorporated into implants with and without a trehalose coating, and these were subsequently implanted into dogs to correct temporal bone defects of the same size and shape. Four weeks after implantation, we analyzed the bone implants and surrounding tissues by using micro-computed tomography imaging and histological analyses, as well as gross evaluation. No significant difference in new bone formation was observed between implants with and without a trehalose coating at any of the bFGF doses. Bone implants with 100 and 200 μg bFGF showed significantly more new bone formation at the implant site and within the cylindrical holes of the implants than those without bFGF (P<0.05). However, heterotopic bone formation on the skull near the implant was observed in the group that received 200 μg bFGF. These results suggest that 100 μg bFGF is the optimal dose for this implant in dogs, and that the trehalose coating may not be necessary in vivo, probably due to the presence of blood proteins and electrolytes at the implant site.

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The uterine tissue of three healthy nonpregnant goats was evaluated experimentally as a graft for closure of a bladder defect. Goats were subjected to ovariectomy, and then one detubularized uterine horn was used as a graft to close a large defect in the ventral aspect of the bladder. Follow up included monthly radiographic and ultrasonographic examinations and evaluations of kidney function and electrolytes changes for six months. The goats were euthanized after six months, and both the bladder and the graft were examined macroscopically and histologically. The technique required less invasive procedures than those described with alternative techniques of cystoplasty, achieved a high survival rate without life-threatening complications post surgery, was associated with no significant changes in kidney function and electrolytes levels, showed proper healing of the transplanted graft by regeneration rather than repair without scaring or fibrosis and with complete covering of the graft by a healthy urothelium and was associated with no malignant transformation. Hysterocystoplasty is a non-invasive, non-life-threatening technique due to the close position of the bladder to the uterus, and proper healing of the graft reflects absence of pressure on its blood supply. It is an acceptable alternative technique for closure of a large bladder defect and avoids disadvantages of alternative techniques of cystoplasty. It might be accepted by owners of pets, and further studies in clinical cases of dogs are advised.

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Clothianidin (CTD) is a neonicotinoid developed in the 1990s as an insecticide having selective toxicity, but it was later found to cause reproductive abnormalities in rats through oxidative stress. There is an attempt to preserve endangered animals, including the Japanese crested ibis, in Japan. However, there is a concern that neonicotinoid affects the reproduction of this bird, since it is used in its habitat. CTD toxicity in the birds is poorly understood, so we investigated whether or not the daily oral administration of CTD has any deleterious effects on the reproductive functions of mature male quails as experimental animals. The animals were randomly divided into four groups of 6 or 7 quails each, treated orally with 0, 0.02, 1 or 50 mg CTD/kg body weight (Control, CTD0.02, CTD1 and CTD50). After that the males bred with untreated females to estimate the egg weights, and rates of fertilization and normal development, the testes, liver and spleen were examined histologically. Vacuolization and the number of germ cells having fragmented DNA in seminiferous tubules, and the number and size of vacuoles in hepatocytes increased dose-dependently. There were no significant differences in egg weights and fertilization rates between the groups, but some eggs of the CTD1 and CTD50 groups failed to develop, and embryonic length decreased dose-dependently. Thus, it was found that CTD affected the reproduction of the male quail through the fragmentation of germ cells and the inhibition or delay of embryonic development.

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In this study, we validated three commercial ELISA (NSP-ELISA) kits that detect antibodies to a nonstructural protein of foot-and-mouth disease virus (FMDV) in terms of their specificities and sensitivities. Although the specificities of the NSP-ELISA kits were as high as that of liquid-phase blocking ELISA (LPBE) in non-infected, non-vaccinated animals, the sensitivities of the NSP-ELISA kits were significantly lower than those of the present LPBE and did not agree with the findings of a previous report on infected animals in the field. Therefore, although countries can adopt both a “vaccination-to-kill” policy and a “vaccination-to-live” policy after emergency vaccination during an FMD epidemic, the NSP-ELISA kits do not seem to be suitable for the latter policy in Japan. These results should be useful for choosing appropriate control measures for potential future FMD epidemics in Japan and elsewhere.

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Ovine herpesvirus 2 (OvHV-2) causes sheep-associated malignant catarrhal fever (SA-MCF), and is responsible for economic losses in cattle and other susceptible species around the world. A survey of 154 serum samples from 14 flocks in 3 Japanese prefectures (Hokkaido, Aomori and Iwate) was undertaken between 2007 and 2008 to test for antibodies to OvHV-2. OvHV-2 was present in 56 sheep and 2 goats, with 37.66% of samples having a positive reaction using a serum neutralization test. The immune reaction reported in goats could result from Caprine herpesvirus-2. These results indicate that sheep are reservoirs for OvHV-2 in the field in Japan, and they might transmit the virus to susceptible cattle and wild fauna.

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