Toll-like receptors (TLRs) play critical roles in innate immunity by recognizing a broad range of microbial components as ligands. The activation of TLRs is an important step not only for the innate immune response, but also for the development of the subsequent antigen-specific adaptive immune response. However, little is known about TLR expression in the female genital mucosa during gestation. In the present study, gene transcriptions of TLRs 1 to 9 were investigated in both the mesometrial side and the anti-mesometrial side of the uterus during gestation in the mouse reproductive organ during the gestation period. In the mesometrial side, gene transcriptions of TLR 1, 3, 4 and 9 were decreased in the late gestation period, whereas an increase of gene transcriptions of TLR 4 and 9 was seen in the early gestation period. In the anti-mesometrial side, gene transcriptions of TLR 1 and 9 were also decreased in the late gestation period, and TLR 9 gene transcription was increased in the early gestation period. On the other hand, gene transcriptions of TLR 3 and 4 were not changed in the late gestation period, but they were increased in the early gestation period. Gene transcriptions of TLR 2, 5, 6, 7 and 8 were not changed statistically in either side during the gestation period. These results suggest that the expressions of particular TLRs may be regulated in the uterus during the gestation period to maintain the pregnant state.

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The mammalian immune system is classified into two categories, innate and adaptive immunity, and innate immunity is an immunological first line of defense for the mucosal immune system. Toll-like receptors (TLRs) play critical roles in innate immunity, as they recognize specific molecular patterns found in microbial pathogens, and the activation of TLRs is an important step not only for the innate immune response, but also for the development of the subsequent antigen-specific adaptive immune response. Despite the importance of TLRs in mucosal immunity, little is known about their expression in the female genital mucosa during gestation. In the present study, gene expressions of TLRs 1 to 9 were investigated together with NF-κB and FoxP3 gene expressions in the vaginae of pregnant mice to understand the immune response of the female genital mucosa during pregnancy. We found that mRNA expressions of TLR4, TLR5, TLR6, TLR7 and TLR9 were significantly decreased during the late gestation period, whereas temporary increases were seen in the middle gestation period. Gene transcriptions of TLR1, TLR2, TLR3 and TLR8 were not changed specifically during the gestation period. The mRNA expression of NF-κB was not changed at any time during the gestation period, while the FoxP3 mRNA expression was increased in the middle gestation period. These results suggest that expressions of particular TLRs would be down-regulated during gestation so as to maintain the pregnant state.

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The main and accessory olfactory bulbs were examined by histological methods and lectin histochemistry in the Japanese striped snake. As the results, the histological properties are similar between the main olfactory bulb and the accessory olfactory bulb. In lectin histochemistry, 21 lectins used in this study showed similar binding patterns in the main olfactory bulb and the accessory olfactory bulb. In detail, 15 lectins stained these olfactory bulbs with similar manner, and 6 lectins did not stain them at all. Two lectins, Lycopersicon esculentum lectin (LEL) and Solanum tuberosum lectin (STL), stained the nerve and glomerular layers and did not stain any other layers in both olfactory bulbs. Four lectins, Soybean agglutinin (SBA), Vicia villosa agglutinin (VVA), Peanut agglutinin (PNA) and Phaseolus vulgaris agglutinin-L (PHA-L) stained the nerve and glomerular layers more intensely than other layers in both olfactory bulbs. In addition, VVA showed the dot-like stainings in the glomeruli of both olfactory bulbs. These findings suggest that the degree of development and the properties of glycoconjugates are similar between the main olfactory bulb and the accessory olfactory bulb in the Japanese striped snake.

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We compared craniodental morphology among 5 populations of the Siberian weasel Mustela sibirica including 2 insular ones (Tsushima and Taiwan). Skulls of the insular individuals tended to be smaller than those of continental ones. Shape differences were also detected, but not so pronounced. Considering these results, the Taiwan population should be regarded as a distinct subspecies M. s. taivana from the mainland ones. The Tsushima population may also possibly be a distinct subspecies from the mainland ones, but more detailed studies using a larger number of specimens are needed for a conclusion. The introduced population in Honshu is also differentiated from the source population. This suggests a high morphological plasticity in M. sibirica.

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Streptococcus suis (S. suis) is an emerging zoonotic pathogen causing significant economic losses in the swine industry. Here, we investigated the antimicrobial susceptibility, associated antibiotic-resistant determinants and sequence type (ST) of S. suis isolates from diseased pigs in China from 2008 to 2010. Serotype 2 was the most frequently observed strain (n=95) among the 106 S. suis strains collected, followed by serotypes 3 (n=3), 5 (n=3), 4 (n=2), 7 (n=1), 11 (n=1) and 28 (n=1). Multilocus sequence typing analysis revealed that ST1 (n=21) and ST7 (n=74) were the predominant STs, and serotype 2 was found to be significantly correlated with ST7 (P=0.017, Fisher’s exact test) and CC1 (P=0.024, Fisher’s exact test). The antimicrobial susceptibility results indicated that the antibiotic resistance rate was highest for tetracycline (99.1%), followed by azithromycin (68.9%), erythromycin (67.9%), clindamycin (67.9%), trimethoprim/sulfamethoxazole (16%), levofloxacin (2.8%), chloramphenicol (1.9%), cefaclor (0.9%) and ceftriaxone (0.9%). Antibiotic-resistant genes tet(M), tet(O), tet(O/W/32/O), tet(O/32/O), tet(S), tet(W), tet(L), tet(40), erm(B), mef(A/E) and msr(D) could be detected, and several tandem organizations of antibiotic resistance genes were also found in this study. In conclusion, S. suis strains isolated from diseased pigs in China were less diverse and multi-drug resistant.

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Mycoplasma haemomuris is a causative organism of infectious anemia or splenomegaly in rodents. Here, we report two distinct genetic groups among M. haemomuris strains detected from rats and mice, respectively, by examining the nucleotide sequences of the 16S-23S rRNA intergenic transcribed spacer region that has been shown to be a stable genetic marker for mycoplasma species. Our results may reveal host-tropism of each cluster of M. haemomuris strains, and suggest an idea to distinguish M. haemomuris into two different genetic clusters.

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Vibrio vulnificus secrets a pore-forming toxin called Vibrio vulnificus hemolysin (VVH). In this study, we showed that methyl-beta-cyclodextrin (MβCD), an oligosaccharide, decreased binding of VVH to Chinese hamster ovary (CHO) cells, resulting in inhibition of its cytotoxicity. When the VVH was incubated with MβCD, cytotoxicity of the toxin was inhibited from 100.3 ± 7.2% to 19.6 ± 5.3%. Binding analysis showed that the amount of VVH on the cells was decreased from 101.4 ± 9.2% to 18.1 ± 8.0% only when MβCD was present in the culture media. Our results indicate that the inhibition of cytotoxicity of VVH by MβCD was due to a decrease in the amount of toxin binding to CHO cells.

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Hepatic stellate cells (HSCs) intracellularly preserve vitamin A in the normal liver. When the liver is damaged, HSCs transform into myofibroblast-like cells, and then proliferate and increase their expression of collagen. Cultured on a plastic plate, HSCs spontaneously activate. To maintain HSCs in a quiescent state with low expression of collagen, coating methods with extracellular matrixes (ECMs) such as Matrigel-coating or laminin-rich coating are commonly used for HSC cultivation. Kishimoto et al. [14] reported that Fragmin®/protamine microparticles (F/P-MPs) have the ability to absorb heparin-binding cytokines like ECMs. Therefore, we examined whether the cultivation on an F/P-MPs-coated plate maintains the quiescent state of RI-T cells (derived from rat HSCs) including the suppression of collagen expression. We found that the mRNA levels of collagen type IαI and TGF-β1 in RI-T cells were significantly suppressed in the cultivation on F/P-MPs-coated plates compared to cultures on noncoated and Matrigel-coated plates. We conclude that the F/P-MPs coating method is useful for maintaining with low expressions of collagen IαI and TGF-β 1 mRNA levels in HSCs.

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Melamine-cyanurate (M-C) crystals, formed by melamine and cyanuric acid, are water-insoluble urolites that can cause renal tubule occlusion, leading to kidney failure. The morphology of the crystals in vivo differs from that in vitro, being rounded in the former case but needle-like in the latter. The reason for this difference remains unknown, but we have previously found that serum proteins, such as bovine serum albumin (BSA), can contribute to in vitro formation of rounded M-C crystals. In the present study, using scanning electron microscopy, we confirmed that, like BSA, polyvinylpyrrolidone (PVP), a synthetic macromolecule, can alter the crystal morphology to a spherical form. The size and surface structure differed between BSA- and PVP-induced M-C crystals.

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Pneumocystis carinii (P. carinii) is an opportunistic fungal pathogen commonly found in many mammalian host species, but rarely in goats. A 3-year-old, female, Tokara-native-goat (Capra hircus domesticus) died of apparent malnutrition caused by multibacillary paratuberculosis. While inflammatory response was slightly observed in the respiratory organs, P. carinii trophozoites and cysts were immunohistochemically observed in the pulmonary alveoli of the infected animal. P. carinii specific DNA was amplified from the formalin fixed and paraffin embedded lung samples. Molecular phylogenetic analyses of the mitochondrial large subunit ribosomal RNA region of P. carinii revealed genetic divergence from previously described P. carinii isolates from other mammalian host species. This is the first description of concurrent infection with P. carinii and the Mycobacterium avium subspecies paratuberculosis in a domestic goat.

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The aim of this study was to determine the antimicrobial resistance of Escherichia coli isolated from the uteri of bitches with pyometra, and 38 E. coli isolates were used. The antimicrobials used were ampicillin (ABPC), amoxicillin/clavulanic acid, gentamicin, minocycline, cefazolin, levofloxacin (LVFX), trimethoprim-sulfamethoxazole (ST) and fosfomycin (FOM). Resistance to ABPC occurred most frequently, followed by LVFX and ST. Multi-drug resistance, defined as resistance against 3 or more classes of antimicrobials, was found in 23.7% of all isolates. Nine out of 13 resistant strains were multi-drug resistant, but no strain was found to be resistant to FOM. This suggests that FOM should be administered for E. coli from pyometra.

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We have developed a rapid and efficient genotyping method for detection of the mouse leptin obese mutation (Lep^ob) using tetra-primer amplification refractory mutation system-polymerase chain reaction (tetra-primer ARMS-PCR). In this method, whole blood collected onto gamma-ray sterilized Flinders Technology Associates (FTA) filter paper is used as PCR template without a DNA purification step. Three genotypes (Lep^ob/Lep^ob, Lep^ob/+ and +/+) differentiated by single-tube PCR and electrophoresis were perfectly consistent with those determined by PCR-restriction fragment length polymorphism (PCR-RFLP). This method can save material costs and operation time, because it does not require restriction enzyme digestion and could be set up in most specific pathogen-free (SPF) barrier facilities.

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Histological and immunohistochemical evaluations of lobular dissecting hepatitis (LDH) were performed in nine American cocker spaniel dogs. Histological examination showed diffuse fibrosis with weak inflammatory reaction of extensive neutrophils, macrophages, lymphocytes and plasma cells. Immunohistochemical examination revealed that the myofibroblastic cells positive for anti-α-smooth muscle actin and anti-vimentin antibodies produced reticular and collagen fibers and brought about the dissection of hepatic cords and diffuse disappearance of hepatocytes. Reticular fibers invading between hepatocytes and the surrounding small group of hepatocytes were strongly positive for anti-collagen type III and anti-collagen type IV antibodies. The positivity to anti-fibronectin and anti-laminin antibodies was frequently continuous on the basement membrane of the sinusoids of the remaining hepatic cords and between the hepatocytes. Positive findings for anti-E-cadherin antibody were not observed between the hepatocytes showing positive findings for anti-collagen type III and anti-collagen type IV antibodies. These results may explain the expression of fibronectin and laminin that occurs prior to the invasion of reticular fibers between hepatocytes. The present study further suggests that expression of an extracellular matrix mainly containing fibronectin and laminin between the hepatocytes and proliferation of collagen fibers and reticular fibers have a major role in the rupture of the hepatic cords and disappearance of hepatocytes.

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A 13-year-old castrated male Labrador retriever dog presented with a mass caudal to the first molar of his left mandible. Although the tumor was excised, a recurrent tumor was detected one month later and resected. Both tumors displayed invasive growth and were composed of neoplastic proliferation arranged in irregular lobules, nests and cords continuous with mucosal epithelium. The most prominent feature of the tumors was the presence of many proliferating spindle cells admixed with palisading basal-like cells, acanthocytes and stellate cells. In immunohistochemical examinations, the spindle cells were found to be positive for vimentin; cytokeratin AE1/AE3, 5/6, 14 and 19; and p63. The other neoplastic cells were positive for all of these markers shown above except vimentin. Based on these findings, the tumors were diagnosed as spindle cell ameloblastic carcinoma.

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A 33 month-old male flying squirrel kept in a zoo developed progressive dyspnea and died. Macroscopically, the liver and lung were enlarged with numerous nodular vesicles. Histologically, these organs were replaced by numerous collapsed vesicles demarcated by fibrous tissues. The cysts lined by a cellular, germinal layer contained numerous brood capsules with abundant production of well-developed protoscolices. Protoscolices were about 80–100 μm in diameter, and had hooks being visible as refractive structures. This zoo locates in the east of Hokkaido where is an endemic area of Echinococcus multilocularis infection. From epidemiology and pathological findings, this animal was diagnosed as E.multilocularis infection. This report describes the pathology of the first case of E. multilocularis infection in a flying squirrel.

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Insulin-like factor 3 (INSL3) is a local regulator in mammalian gonads, but little is known of its function in bovine corpus luteum (CL). Here, we show that RXFP2 protein, the receptor of INSL3, was expressed throughout the estrous cycle and significantly high at the early luteal stage compared to the regressed luteal stage. INSL3 stimulated progesterone secretion, but not prostaglandin F2α and viability in cultured luteal cells. Together, these results suggest that INSL3 plays a luteotropic role as a local regulator in the bovine CL.

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The effect of washing in Vibrio parahaemolyticus contaminated and hygienic seawater on fish, and the frequency and level of natural V. parahaemolyticus contamination in fish were investigated. In the first experiment, live horse mackerel was experimentally kept in seawater artificially contaminated with V. parahaemolyticus. After washing in contaminated and hygienic seawater, the contamination in fish was quantitatively analyzed. Washing fish in the seawater contaminated with V. parahaemolyticus increases the contamination level on the surface and in the gills of the fish. Washing in hygienic seawater was effective in reducing the contamination in fish and cutting board surfaces, but not in the gills or viscera. In the second experiment, natural V. parahaemolyticus contamination in various fish caught by us was analyzed. V. parahaemolyticus was detected in 6 of 28 gill samples and 10 of 28 viscera samples of naturally contaminated fish. The means of V. parahaemolyticus level on gills were 3.3 and 3.9 log cfu/g, and those in viscera were 2.6 and 4.4 log cfu/g by culture method and a real-time PCR assay, respectively. These results indicate that the gills and viscera are able to spread the pathogens to fish meat as well as fish surface contamination by washing in the contaminated seawater. Washing with hygienic seawater and control of contamination from gills and viscera are critically important to prevent V. parahaemolyticus infections.

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Rectal contents of beef cattle and pigs were collected between October 2010 and February 2011 in Japan. Campylobacter jejuni were isolated from 36.0% (90/250) of beef cattle from 88.0% (22/25) of beef farms. C. coli were isolated from 3.6% (9/250) of beef cattle from 16.0% (4/25) of beef farms and from 42.4% (106/250) of pigs from all 25 pig farms. As to enrofloxacin, 40.0% (36/90) of C. jejuni isolates and 66.7% (6/9) of C. coli isolates from beef cattle and 44.3% (47/106) of C. coli isolates from pigs were resistant. Additionally, 15.1% (16/106) of C. coli isolates from pigs were resistant to erythromycin and enrofloxacin. The high prevalence of Campylobacter carriers and significant antimicrobial resistance of the isolates were found.

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Twenty nine oil-soaked birds were collected from around the Coast of Tsushima Island. The contents of eight elements in the livers and kidneys of the birds were investigated. Statistically higher concentrations of vanadium and thallium in the liver and of titanium in the kidney were found in the birds that were found dead compared with those that died after rescued. A significant correlation (r=0.695, P<0.01) was observed only for the molybdenum content between the kidneys and livers from the birds found dead. Although the controls of the eight elements of birds investigated in the present study remain unexplained, some of lower concentration in rescued birds can be blamed on a decrease in food intake of birds. The relation between oil contamination and concentration of elements need to be further explored.

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A serological survey was carried out in the northern prefectures of Hokkaido, Iwate and Aomori in Japan, for the presence of antibodies against Toxoplasma gondii, Chlamydiapsittaci var. ovis, Mycobacterium paratuberculosis, Coxiella burnetii, Brucella spp., Leptospirosis and Orf virus (ORFV). Out of 267 samples tested, highest overall prevalence (28.78%) was found for T. gondii. The 12.59% of tested sheep were positive for C. psittaci var. ovis. A total of 8.67% were found to be seropositive for C. burnetii. Levels of these infections were found in all three prefectures. Seroconversion to ORFV was detected in Hokkaido and Iwate Prefectures (2.57%). Animals were positive only for L. ballum (1.50%), in Hokkaido and Aomori Prefectures. No animals tested positive for Brucella spp. and M. paratuberculosis.

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Tramadol is an atypical opioid analgesic widely used in small animal practice. This study was designed to determine the effect of a single intravenous (IV) dose of tramadol on the minimum alveolar concentration (MAC) of sevoflurane in dogs. Six beagle dogs were anesthetized twice to determine the sevoflurane MAC with or without an administration of tramadol (4 mg/kg, IV) at 7 days interval. The sevoflurane MAC was determined using a tail clamp method in each dog ventilated with positive pressure ventilation. The tramadol administration produced a significant reduction in the sevoflurane MAC by 22.3 ± 12.2% (1.44 ± 0.28% with tramadol versus 1.86 ± 0.30% without tramadol, P=0.010). This MAC reduction had been determined from 122 ± 19 to 180 ± 41 min following the tramadol administration. During this period, the plasma concentrations of tramadol and its metabolite, O-desmethyltramadol (M1), decreased from 429 ± 64 to 332 ± 55 ng/ml and from 136 ± 24 to 114 ± 68 ng/ml, respectively, but these changes were not statistically significant. There was no significant difference in heart rate, mean arterial blood pressure and SpO₂ between the control and tramadol treatment. The dogs that received tramadol treatment sometimes breathed spontaneously. Therefore, their respiratory rates significantly increased, and PETCO₂ decreased during the MAC determination. In conclusion, the single IV dose of tramadol produced a significant reduction in the sevoflurane MAC in dogs.

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The epidural distribution of iohexol (0.2 ml/kg) administered at thoracic vertebrae (Thoracic group) and lumbar vertebrae (Lumbar group) was compared by computed tomographic (CT) epidurography in dogs. The total spread of iohexol was similar between the 2 groups upon reaching a similar cranial level. The maximal CT values were higher at the C7/T1 and T4/T5 levels in Thoracic group, but they were higher at the T13/L1 and L4/L5 levels in Lumbar group (P<0.05). This result suggests that the distribution pattern of the drug administered epidurally at thoracic vertebrae and lumbar vertebrae is different in dogs.

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Many highly pathogenic avian influenza (HPAI) outbreaks occurred in Japan during the 2010–11 winter. H5N1 HPAI viruses were isolated from 63 wild birds including migrating and resident birds, and caused HPAI outbreaks in 24 chicken farms by the end of March. In the present study, all virus strains isolated from wild birds in western Japan together with the viruses in the preceding works were phylogenetically and epidemiologically analyzed. Furthermore, the virus distributions in the raptors that died of H5N1 HPAI virus infection were assessed. The virus isolates in Japan were classified into three groups by phylogenic analysis of their hemagglutinins, supporting the previous report (Sakoda et al., 2012). The viruses in each group were continuously isolated in respective limited areas, indicating that viruses were maintained in local bird populations throughout the outbreak periods. Some viruses were genetically closely related to the Korean isolates around the same periods, suggesting that migratory birds were suspected of contributing to transportation of the viruses across the sea. Viruses were recovered from systemic tissues including digestive organs of the deceased raptors, indicating that they were infected with HPAI viruses by their predatory behavior, eating infected birds or carrion in the environment.

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Widespread outbreaks of highly pathogenic avian influenza (HPAI) caused by H5N1 viruses occurred in wild birds in Japan from 2010–2011. Forty out of 63 deceased wild birds belonged to the order Anseriformes, and mandarin duck was one of the dominant species. To estimate the risk of mandarin ducks as a source of virus infection in the environment, we examined the pathogenicity of a causal H5N1 HPAI virus to mandarin ducks. About half of the mandarin ducks died by inoculation with 10^7.0TCID₅₀ of A/mandarin duck/Miyazaki/22M807-1/2011 (H5N1). Viruses were mainly recovered from the trachea of the ducks sacrificed at three days post inoculation (d.p.i.). Viruses were recovered from the laryngopharyngeal swabs of the observation group until 5 d.p.i. In ducks that died at the late phase of infection, viruses were detected in the systemic organs, such as lung, kidney and colon. Together, these results showed that the H5N1 HPAI viruses, which belonged to clade 2.3.2.1 and are mainly circulating in East Asia, were lethal to mandarin ducks, indicating that mandarin ducks have the potential to disseminate the virus to other bird species. Therefore, wild birds should be kept out of poultry farms to prevent HPAI outbreaks in the future.

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An outbreak of a disease with parapox-like symptoms was reported in South Korea in April 2012. Three of 45 Korean native cattle, age 20–24 months, were affected. Parapoxviruses were detected and identified by electron microscopy and polymerase chain reaction (PCR). To determine the genetic characteristics of the Korean strains, the sequence of the major envelope protein (B2L) was determined and compared with published reference sequences. Phylogenetic analysis revealed that the parapoxvirus strains were closely related to not only isolates from Japan, but also isolates from Germany, Sudan and the United states. This is the first report on an outbreak and the molecular characterization of BPSV in Korea.

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