The species Erysipelothrixrhusiopathiae displays genetic heterogeneity; however, E. rhusiopathiae serovar 1a strains currently circulating in Japan exhibit remarkably low levels of genetic diversity and group into clonal sublineages of Lineage IVb (IVb-1 and IVb-2). In the present study, based on whole genome sequencing data, we designed primers for a multiplex PCR assay to simultaneously detect and differentiate the sublineages of E. rhusiopathiae strains. Among the one hundred and twenty-seven isolates of various serovar strains, including isolates from a wide range of hosts and geographic origins, the PCR assay could successfully detect and differentiate the serovar 1a strains belonging to the sublineages.

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Rhodococcus equi is a facultative intracellular bacterium that can escape from bactericidal mechanisms associated with phagocytosis. Virulence-associated protein A (VapA), encoded on a virulence-associated plasmid, is essential for intracellular survival in macrophages, but its function is not known. Here, we show that the extracellular addition of recombinant glutathione S-transferase (GST)-VapA fusion protein rescued the intracellular replication defect of a mutant lacking the vapA gene. Furthermore, the virulence-plasmid-cured strain could also multiply to nearly wild-type levels by the addition of GST-VapA. The present data suggest that VapA can alter the intraphagocytic environment, thereby affecting its suitability for the growth of R. equi.

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Cytochrome P450 1A1 (CYP1A1) is a heme-containing mono-oxygenase involved in metabolism of environmental contaminants. Two variants of dog CYP1A1 with a single residue difference were identified and designated Sap1 and Sap2. Compared with Sap1, Sap2 had a Trp50Leu substitution. The biochemical characteristics of the variants were comparatively analyzed using heterologous expression in Escherichia coli. The membrane fraction of E. coli expressing Sap2 exhibited higher CYP holoprotein and heme contents than the Sap1-containing membranes, although the level of total CYP1A1 protein (i.e., apoprotein + holoprotein) was comparable between the groups. As normalized to holo-CYP content, the Sap2-expressing membranes showed lower CYP1A1-specific enzyme activities, such as 7-ethoxyresorufin O-dealkylation (EROD), than the Sap1 group. In single substitution variants of residue 50, proteins with hydrophobic residues having mass similar to Leu exhibited lower EROD activities than those with hydrophobic residues having larger mass than Leu. In addition, variants with polar or charged residues having mass similar to Leu showed activities that were comparable to those of Sap2. Taken together, these findings suggest that the Trp50Leu substitution leads to an enhancement of holo-CYP1A1 formation, but diminishes the enzyme activity because of the small size of Leu compared with Trp.

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Vaccination is the most effective method for controlling the infectious diseases that threaten the poultry industry worldwide. The use of adjuvants or immunostimulants is often necessary to improve vaccine efficacy, particularly for vaccines based on recombinant protein or inactivated pathogens. The adjuvant effects of zymosan A on antigen-specific antibody production were investigated in chickens. First, the optimal adjuvant dose of zymosan A was determined. Chicks were immunized with dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH) at a dosage of 2 mg/kg body weight (BW) with or without zymosan A (at a dosage of 0.5 mg/kg BW) co-administration at 4, 5 and 6 weeks of age. Different routes of immunization (oral, intranasal (i.n.), intraocular (i.o.), subcutaneous (s.c.), intramuscular (i.m.) and intraperitoneal (i.p.) were tested. Anti-DNP IgY and IgA concentrations in serum samples from all chicks were measured by an enzyme-linked immunosorbent assay. The results revealed that co-administration of zymosan A with DNP-KLH significantly increased anti-DNP IgY concentrations in chicks immunized by the oral and s.c. routes of administration when compared with control groups. In addition, co-administration of zymosan A with DNP-KLH significantly increased anti-DNP IgA concentrations in chicks immunized by the oral, i.o. and s.c. routes compared with control groups. In conclusion, zymosan A is a useful immune-potentiator adjuvant in chickens, and its co-administration with vaccine antigens enhances humoral immune responses.

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The present study aimed to determine whether circulating serum concentrations of 25-hydroxyvitamin D [25-(OH) D] differed between healthy dogs and dogs with acute pancreatitis (AP). Twenty-two healthy dogs and twenty client-owned dogs with AP were enrolled in the study. Serum concentrations of 25-(OH) D, blood ionized calcium (iCa), and serum C-reactive protein (CRP) were measured. Concentrations of serum 25-(OH) D and blood iCa in dogs with AP were significantly lower than those of healthy dogs, and serum concentrations of CRP in dogs with AP were significantly higher than those of healthy dogs. A significant difference in 25-(OH) D serum concentrations was observed between survivor and non-survivor dogs with AP. After resolution of clinical signs, concentrations of serum 25-(OH) D, blood iCa, and serum CRP did not differ compared to those before treatment. This study shows that dogs with AP exhibit decreased 25-(OH) D levels, which might be associated with calcium imbalances and mortality rate in canine AP.

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Diabetes is a metabolic disorder that worsens clinical outcome following cerebral ischemia. Protein phosphatase 2A (PP2A) is a conserved, heterotrimeric, serine/threonine phosphatase with various cellular functions. PP2A subunit B is abundant in brain tissue and modulates PP2A function. The aim of this study was to investigate PP2A subunit B protein expression in the cerebral cortex of non-diabetic and diabetic animals with middle cerebral artery occlusion (MCAO) injury. Sprague-Dawley rats were injected with streptozotocin (40 mg/kg, i.p.) to induce diabetic conditions. After 4 weeks of streptozotocin treatment, the rats underwent MCAO to induce focal cerebral ischemia. The cerebral cortex tissue was collected 24 hr after MCAO. Body weight and blood glucose were measured, and Western blot analysis was performed to elucidate the expression of PP2A subunit B. We confirmed decreased body weight and increased blood glucose in diabetic animals. Reverse transcription-PCR and Western blot analyses showed decreased PP2A subunit B expression in the cerebral cortices of MCAO-injured animals. Moreover, diabetic animals with MCAO showed more severe decreases in PP2A subunit B protein levels than non-diabetic animals following MCAO. The decline of PP2A subunit B indicates degradation of neuronal function. These findings suggest that conspicuous decreases in PP2A subunit B may exacerbate cerebral ischemia under diabetic conditions following MCAO.

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Neosporosis is caused by the intracellular protozoan parasite Neospora caninum. This major disease-causing pathogen is responsible for inducing abortion in cattle, and these adverse events occur sporadically all over the world, including Japan. Currently, there are no vaccines on the market against infection with N. caninum. Because live and attenuated vaccines against N. caninum have had safety and effectiveness issues, development of a next-generation vaccine is urgently required. To develop a vaccine against neosporosis, my laboratory has been focused on the following: 1) understanding the host immune responses against Neospora infection, 2) identifying vaccine antigens and 3) developing an effective antigen-delivery system. The research strategy taken in my laboratory will have strong potential to progress current understanding of the pathogenesis of N. caninum infection and promote development of a novel subunit vaccine based on the specific vaccine antigen with an antigen-delivery system for controlling neosporosis.

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In Hokkaido, Japan, wild sika deer are highly infected with Fasciola flukes, suggesting that the flukes complete their life cycle via intermediate host snails and definitive host animals occurring in the natural environment. However, infected snails have been found only in cattle farms contaminated with fasciolosis. This study reports the first Fasciola larva infection in Galba truncatula snails occurring in the Shoro and Atsuma rivers in the natural environment. Molecular analysis revealed that the nad1 haplotype of the larvae was consistent with that of Fasciola adults obtained from sika deer in Hokkaido. These results indicated that Fasciola flukes complete their life cycle via G. truncatula and sika deer occurring in the natural environment.

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Aminopeptidase N (APN) is a member of the highly conserved M1 family of metalloproteases, and is considered to be a valuable target for the treatment of a variety of diseases, e.g., cancer, malaria, and coccidiosis. In this study, we identified an APN gene (TgAPN2) in the Toxoplasma gondii genome, and performed a biochemical characterization of the recombinant TgAPN2 (rTgAPN2) protein. Active rTgAPN2 was first produced and purified in Escherichia coli. The catalytic activity of the enzyme was verified using a specific fluorescent substrate, H-Ala-MCA; the rTgAPN2 was relatively active in the absence of added metal ions. The addition of some metal ions, especially Zn²⁺, inhibited the activity of the recombinant enzyme. The activity of rTgAPN2 was reduced in the presence of the EDTA chelator in the absence of added metal ions. The optimum pH for enzyme activity was 8.0; the enzyme was active in the 3–10 pH range. The substrate preference of rTgAPN2 was evaluated. The enzyme showed a preference for substrates containing N-terminal Ala and Arg residues. Finally, bestatin and amastatin were shown to inhibit the activity of the enzyme. In conclusion, rTgAPN2 shared general characteristics with the M1 family of aminopeptidases but also had some unique characteristics. This provides a basis for the function of aminopeptidases and the study of drug targets.

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Sarcocystis species are heteroxenous cyst-forming coccidian protozoan parasites with a wide host range, including rodents. In this study, Sarcocystis spp. samples were isolated from Bandicota indica, Rattus argentiventer, R. tiomanicus and R. norvegicus across five provinces of Thailand. Two major groups of Sarcocystis cysts were determined in this study: large and small cysts. By sequence comparisons and phylogenetic analyses based on the partial sequences of 28S ribosomal DNA, the large cysts showed the highest identity value (99%) with the S. zamani in GenBank database. While the small cysts could be divided into 2 groups of Sarcocystis: S. singaporensis and presupposed S. zuoi. The further analysis on 18S rDNA supported that the 2 isolates (S2 and B6 no.2) were as identified as S. singaporensis shared a high sequence identity with the S. singaporensis in GenBank database and the unidentified Sarcocystis (4 isolates, i.e., B6 no.10, B6 no.12, B10 no.4 and B10 no.7) showed 96.3–99.5% identity to S. zuoi as well as high distinct identity from others Sarcocystis spp. (≤93%). The result indicated that these four samples should be S. zuoi. In this study, we provided complete sequence of internal transcribed spacer 1 (ITS1), 5.8S rDNA and internal transcribed spacer 2 (ITS2) of these three Sarcocystis species and our new primer set could be useful to study the evolution of Sarcocystis.

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Infection of boar-hunting dogs with Paragonimus westermani was investigated in Western Japan. Blood and rectal feces were collected from 441 dogs in the three districts (205 in Kinki, 131 in Chugoku and 105 in Shikoku District). In a screening ELISA for serum antibody against P. westermani antigen, 195 dogs (44.2%) showed positive reaction. In the 195 dogs, 8 dogs were found excreting P. westermani eggs after molecular analysis of fecal eggs, and additional 7 were identified serologically for the parasite infection because of their stronger reactivity against P. westermani antigen than against antigens of other species of Paragonimus. A spatial analysis showed that all of the P. westermani infections were found in Kinki and Chugoku Districts. In this area, dogs’ experience of being fed with raw boar meat showed high odds ratio (3.35) to the sero-positivity in the screening ELISA, and the frequency of such experiences was significantly higher in sero-positive dogs. While clear relationship was not obtained between predation of boars by dogs during hunting and their sero-positivity. Therefore, it is suggested that human activity of feeding with wild boar meat is the risk factor for P. westermani infection in boar-hunting dogs. Considering that hunting dogs could play as a major definitive host and maintain the present distribution of P. westermani in Western Japan, control measures for the infection in hunting dogs, such as prohibition of raw meat feeding and regular deworming, should be undertaken.

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This study aimed to investigate the neuropathogenesis of equine herpes virus 9 (EHV-9) by studying the effects of a single point mutation introduced in two different EHV-9 genes. The two EHV-9 mutants, 14R and 19R, were generated carrying a point mutation in two separate EHV-9 genes. These mutants, along with the wild-type EHV-9, were used to infect a hamster model. The EHV-9- and 19R-infected groups showed earlier and more severe clinical signs of infection than the 14R-infected group. The white blood cells (WBCs) count was significantly increased in both EHV-9- and 19R-infected groups compared to the 14R-infected group at the 4th day post infection (DPI). Viremia was also detected earlier in both EHV-9- and 19R-infected groups than 14R-infected group. There were differences in the anterograde transmission pattern of both EHV-9 and 19R compared to 14R inside the brain. Serum TNF-α, IL-6 and IFN-γ levels were significantly increased in both EHV-9- and 19R-infected groups compared to the 14R-infected group. Histopathological and immunohistochemical analyses revealed that the mean group scores for the entire brain were significantly higher in both EHV-9- and 19R- infected groups than 14R-infected group. Collectively, these results confirm that the gene product of Open Reading Frame 19 (ORF19) plays an important role in EHV-9 neuropathogenicity and that the mutation in ORF19 is responsible for the attenuation of EHV-9.

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The right third eyelid of an adult female brown bear (Ursus arctos) was swollen and removed. Histopathology revealed a tumor exhibiting proliferation with mild infiltration, consisting of multi-stratified glandular structures of the innermost laminal neoplastic cells and the basaloid neoplastic cells, and with eosinophilic thick basal lamina material around the glandular structures. Both types of neoplastic cells exhibited moderate anisokaryosis, and mitotic figures were observed in the basaloid neoplastic cells. The laminal neoplastic cells were cytokeratin (CK) 8/18-positive. In contrast, the basaloid neoplastic cells were CK14- and p63-positive, but α-smooth muscle actin- and calponin-negative. The case described herein is the first report of basal cell adenocarcinoma in the gland of the third eyelid of a bear.

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A 14-month-old Japanese black beef steer presented with severe chronic diarrhea and emaciation and was euthanized. Postmortem examination showed thickened and corrugated intestinal mucosa and enlarged granulomatous mesenteric lymph nodes with caseating necrosis. Numerous epithelioid cells and multinucleated giant cells infiltrated in the lamina propria and the submucosal tissue of the intestines. These cells were also observed in the systemic organs. Many acid-fast bacilli were detected in the cytoplasm of these cells and were identified as ‘Mycobacterium avium subsp. hominissuis’ (Mah) on the basis of the results of molecular examinations and immunohistochemistry. These findings indicate that Mah can cause systemic mycobacteriosis, and this unique infection needs to be distinguished from Johne’s disease and tuberculosis in cattle.

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DNA methylation is an epigenetic mechanism controlling gene expression without affecting DNA sequences, and aberrant DNA methylation patterns are features of a number of diseases. Notably, epigenetic errors in cancer cells have been intensively studied over the last two decades in humans; however, little is known concerning dogs and cats. To analyze DNA methylation and gene expression changes in feline lymphoma cells, we added the DNA methylation inhibitor 5-aza-2’-deoxycytidine (5-aza) to three cell lines (3281 and FT-1 cells derived from T-cell lymphoma and MS4 cells derived from B-cell lymphoma). Adding 5-aza significantly retarded cell growth in a dose-dependent manner in all cell lines, and there were aberrant gene expression patterns. Transcription factor Sox11 expression in 3281 cells was de-repressed by 5-aza treatment, and subsequent promoter DNA demethylation was analyzed by bisulfite sequencing. Cell cycle analysis suggested that inhibition of cell growth was due to DNA replication arrest, and this supported the result of increased expression of p27kip1 gene which disturbed cells of 3281 and FT-1 entering the S phase. In this study, 5-aza suppressed the growth of feline lymphoma cells, but further experiments with normal lymph cells are necessary to confirm specificity of this drug treatment and to expand it for clinical use.

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The present study was undertaken to clarify the factors that reduce the viable pathogen count in milk collected from the udders of subclinical mastitic cows during preservation. Milk was centrifuged to divide somatic cells (cellular components, precipitates) and antimicrobial peptides (soluble components, supernatants without fat layer); each fraction was cultured with bacteria, and the number of viable bacteria was assessed prior to and after culture. In 28.8% of milk samples, we noted no viable bacteria immediately after collection; this value increased significantly after a 5-hr incubation of milk with cellular components but not with soluble components (48.1 and 28.8%, respectively). After culture with cellular components, the numbers of bacteria (excluding Staphylococcus aureus and Streptococcus uberis) and yeast decreased dramatically, although the differences were not statistically significant. After cultivation with soluble components, only yeasts showed a tendency toward decreased mean viability, whereas the mean bacterial counts of S. uberis and T. pyogenes tended to increase after 5-hr preservation with soluble components. These results suggest that most pathogens in high somatic cell count (SCC) milk decreased during preservation at 15 to 25°C, due to both the cellular components and antimicrobial components in the milk. Particularly, the cellular components more potently reduced bacterial counts during preservation.

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Attenuated derivative rabies virus Ni-CE replicates in muscle cells less efficiently than does the parental pathogenic strain Nishigahara. To examine the mechanism underlying the less efficient replication of Ni-CE, we compared the activities of Ni-CE and Nishigahara phosphoproteins, viral interferon (IFN) antagonists, to suppress IFN-β promoter activity in muscle cells and we demonstrated a defect of Ni-CE phosphoprotein in this ability. Treatment with an IFN-β-neutralizing antibody improved the replication efficiency of Ni-CE in muscle cells, indicating that produced IFN inhibits Ni-CE replication. The results indicate the importance of IFN antagonism of rabies virus phosphoprotein for viral replication in muscle cells.

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Changes in stroke volume variation (SVV) and pulse pressure variation (PPV) in response to fluid infusion were experimentally evaluated during vecuronium infusion and sevoflurane anesthesia in 5 adult, mechanically ventilated, euvolemic, beagle dogs. Sequential increases in central venous pressure (CVP; 3–7[baseline], 8–12, 13–17, 18–22 and 23–27 mmHg) were produced by infusing lactated Ringer’s solution and 6% hydroxyethyl starch solution. Heart rate (beats/min), right atrial pressure (RAP, mmHg), pulmonary arterial pressure (PAP, mmHg), pulmonary capillary wedge pressure (PCWP, mmHg), transpulmonary thermodilution cardiac output (TPTDCO, l/min), stroke volume (SV, ml/beat), arterial blood pressure (ABP, mmHg), extravascular lung water (EVLW, ml), pulmonary vascular permeability index (PVPI, calculated), SVV (%), PPV (%) and systemic vascular resistance (SVR, dynes/sec/cm⁵) were determined at each predetermined CVP range. Heart rate (P=0.019), RAP (P<0.001), PAP (P<0.001), PCWP (P<0.001), TPTDCO (P=0.009) and SV (P=0.04) increased and SVR (P<0.001), SVV (P<0.001) and PPV (P<0.001) decreased associated with each stepwise increase in CVP. Arterial blood pressure, EVLW, PVPI and the arterial partial pressures of oxygen and carbon dioxide did not change. The changes in SVV and PPV directly reflected the fluid load and the minimum threshold values for detecting fluid responsiveness were SVV ≥11% and PPV ≥7% in dogs.

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Propofol is an anesthetic agent suspended in an emulsion system that includes egg yolk lecithin and soybean oil, because of which, there is concern about the use of propofol in patients allergic to these substances. We examined the association between propofol administration and incidence of adverse events in dogs with allergy to egg yolk lecithin and soybean oil. On the basis of the findings of an allergen-specific immunoglobulin E (IgE) test, 14 dogs with high levels (high-IgE group) and 7 dogs with low levels (normal-IgE group) of IgE were selected. Following intravenous administration of propofol, the incidence of anaphylactic reactions and plasma histamine concentrations under general anesthesia maintained with isoflurane throughout surgery were compared between the two groups. The frequency of anaphylactic reactions and plasma histamine concentrations were compared by the chi-square test and Student t-test, respectively. The statistical significance for both tests was set at P<0.05. In the high- and normal-IgE groups, the average frequencies of anaphylactic reactions after propofol administration were 21.4 and 14.3%, and the mean plasma histamine concentrations were 167.9 ± 94.5 nM and 65.7 ± 40.3 nM, respectively. Animals of neither groups experienced shock-like symptoms. These results revealed that propofol might be relatively safe, although careful perioperative anesthesia monitoring and standby protocols are required when using propofol in dogs with a history of allergic diseases or high chicken- or soybean-specific IgE levels.

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A four-year-old dachshund presented with a two-week history of pyrexia, depression, and cough. Four months earlier, the owner observed the dog swallow a whole popsicle stick, but the animal showed no clinical signs at that time. Radiography, ultrasonography, and computed tomography confirmed an intrathoracic linear foreign body and pleural effusion in the right thorax. The pleural fluid was bloody and purulent, and contained inflammatory cells and Escherichia coli. The dog was diagnosed with pyothorax induced by a foreign body, and was treated successfully by surgical removal of the foreign body, partial lung lobectomy, thoracic lavage, and antibiotics. The foreign body was identified as a popsicle stick that the dog had eaten.

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In the present study, bull sperm in the first and second ejaculates were divided into subpopulations based on their motility characteristics using a cluster analysis of data from computer-assisted sperm motility analysis (CASA). Semen samples were collected from 4 Japanese black bulls. Data from 9,228 motile sperm were classified into 4 clusters; 1) very rapid and progressively motile sperm, 2) rapid and circularly motile sperm with widely moving heads, 3) moderately motile sperm with heads moving frequently in a short length, and 4) poorly motile sperm. The percentage of cluster 1 varied between bulls. The first ejaculates had a higher proportion of cluster 2 and lower proportion of cluster 3 than the second ejaculates.

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The pandemic 2009 H1N1 influenza A virus emerged in humans and caused the first influenza pandemic of the 21st century. Mexican isolates, A/Mexico/4108/2009 (H1N1) (Mex4108) and A/Mexico/InDRE4478/2009 (H1N1) (Mex4487) derived from a mild case and from a cluster of severe cases, showed heterogeneity in virulence in a cynomolgus macaque model. To compare the more pathogenic differences, we generated recombinant viruses and compared their virulence in ferrets. Ferrets infected with recombinant Mex4487 displayed a slightly higher rate of viral replication and severe pneumonia in the early stage of infection. In contrast, prolonged lower virus shedding of recombinant Mex4108 than that of recombinant Mex4487 was detected in throat swabs. Thus, Mex4487 induces severe pneumonia in infected individuals, whereas Mex4108 might have wide-spreading potential with mild disease.

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Thirty-two muskrats (Ondatra zibethicus) were captured for surveillance of avian influenza virus in wild waterfowl and mammals near Lake Chany, Western Siberia, Russia. A/muskrat/Russia/63/2014 (H2N2) was isolated from an apparently healthy muskrat using chicken embryos. Based on phylogenetic analysis, the hemagglutinin and neuraminidase genes of this isolate were classified into the Eurasian avian-like influenza virus clade and closely related to low pathogenic avian influenza viruses (LPAIVs) isolated from wild water birds in Italy and Sweden, respectively. Other internal genes were also closely related to LPAIVs isolated from Eurasian wild water birds. Results suggest that interspecies transmission of LPAIVs from wild water birds to semiaquatic mammals occurs, facilitating the spread and evolution of LPAIVs in wetland areas of Western Siberia.

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NOTE
Internal Medicine

The first report of the ante-mortem diagnosis of Ollulanus tricuspis infection in two dogs
Daiki KATO¹⁾, Mariko OISHI¹⁾, Koichi OHNO^1)*, Ko NAKASHIMA¹⁾, Atsuhito WADA¹⁾, Tatsushi MORITA²⁾, Soichi IMAI²⁾, Masaya TSUBOI³⁾, James K. CHAMBERS³⁾, Kazuyuki UCHIDA³⁾ and Hajime TSUJIMOTO^1)
1)Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–8657, Japan
²⁾Division of Veterinary Infectious Disease, Department of Veterinary Pathobiology, Nippon Veterinary and Life Science University, 1–7–1 Kyonanchyo Musashino, Tokyo 180–8602, Japan
³⁾Department of Veterinary Pathology, Graduate School of Agricultural

Should have been
NOTE
Internal Medicine

The first report of the ante-mortem diagnosis of Ollulanus tricuspis infection in two dogs

Daiki KATO¹⁾, Mariko OISHI¹⁾, Koichi OHNO^1)*, Ko NAKASHIMA¹⁾, Atsuhito WADA¹⁾, Shin-ichiro FUKUMOTO²⁾, Tatsushi MORITA³⁾, Soichi IMAI³⁾, Masaya TSUBOI⁴⁾, James K. CHAMBERS⁴⁾, Kazuyuki UCHIDA⁴⁾ and Hajime TSUJIMOTO^1)
1),Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–8657, Japan
²⁾Laboratory of Veterinary Parasitology, Department of Pathobiology, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501, Japan
³⁾Division of Veterinary Infectious Disease, Department of Veterinary Pathobiology, Nippon Veterinary and Life Science University, 1–7–1 Kyonanchyo Musashino, Tokyo 180–8602, Japan
⁴⁾Department of Veterinary Pathology, Graduate School of Agricultural

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