Recently, dysfunction of antigen-specific T cells is well documented as T-cell exhaustion and has been defined by the loss of effector functions during chronic infections and cancer in human. The exhausted T cells are characterized phenotypically by the surface expression of immunoinhibitory receptors, such as programmed death 1 (PD-1), lymphocyte activation gene 3 (LAG-3), T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) and cytotoxic T-lymphocyte antigen 4 (CTLA-4). However, there is still a fundamental lack of knowledge about the immunoinhibitory receptors in the fields of veterinary medicine. In particular, very little is known about mechanism of T cell dysfunction in chronic infection in cattle. Recent our studies have revealed that immunoinhibitory molecules including PD-1/ programmed death-ligand 1 (PD-L1) play critical roles in immune exhaustion and disease progression in case of bovine leukemia virus (BLV) infection, Johne’s disease and bovine anaplasmosis. This review includes some recent data from us.

Fusarenon-X (FX) is a type B trichothecene mycotoxin that is frequently observed along with deoxynivalenol (DON) and nivalenol (NIV) in agricultural commodities. This review aims to give an overview of the literature concerning the toxicology and toxicokinetics of FX. FX is primarily found in cereals grown in temperate regions, but it can also be found worldwide because of the global transport of products. The major toxicity of FX occurs through inhibition of protein synthesis, followed by the disruption of DNA synthesis. Moreover, FX has also been shown to induce apoptosis in in vitro and in vivo studies. The targets of FX are organs containing actively proliferating cells, such as the thymus, spleen, skin, small intestine, testes and bone marrow. FX causes immunosuppression, intestinal malabsorption, developmental toxicity and genotoxicity. In addition, sufficient evidence of carcinogenicity in experimental animals is currently lacking, and the International Agency for Research on Cancer (IARC) classifies it as a group 3 carcinogen.

A Canis skull, right half of the mandible and part of the left half of the mandible were subjected to three-dimensional (3D) computed tomography (CT) observation and mitochondrial DNA (mtDNA) analysis in order to determine whether the specimens belonged to the extinct Japanese wolf, Canis lupus hodophilax (Temminck, 1839). Osteometric analysis of the skull and right half of the mandible revealed that the material (JW275) was indeed typical of the Japanese wolf. Sequence analysis of a 600-bp mtDNA region revealed that the JW275 belonged to haplotype Group B, which is characterized by an 8-bp deletion in the mtDNA control region. The findings of this study suggest that 3D CT analysis is well suited to examining fragile and valuable biological samples, as it removes the need for destructive sampling.

We attempted the isolation of variant infectious bursal disease (IBD) viruses by using sentinel chickens immunized with inactivated classical-type IBD vaccine. Immunized sentinel chickens with high levels of neutralizing antibodies and non-immunized sentinel chickens were raised together with broiler chickens in a commercial farm. Severe atrophy of the bursa of Fabricius was observed from the second week after cohabitation in non-immunized sentinel chickens. However, in immunized sentinel chickens and broiler chickens, atrophy was observed from the third week after cohabitation. The IBD virus (IBDV) isolated from the bursa of Fabricius of immunized sentinel chickens, designated as strain IBDV TY2, showed severe atrophy of the bursa in infected SPF chickens. Antiserum to the IBDV TY2 strain showed higher neutralizing activity to heterologous IBDV strains than did antiserum to the K strain vaccine virus. Phylogenetic analysis revealed that the nucleotide sequences encoding the hypervariable region of virus protein 2 of the IBDV TY2 strain did not cluster with the classical, variant or very virulent IBDV groups. Based on these results, we suggest that the IBDV TY2 strain may constitute a novel variant type of IBDV.

To characterize the Erysipelothrix rhusiopathiae Met-203 type surface protective antigen (Spa) A strains causing swine erysipelas in Japan, the nucleotide sequence of the hypervariable region of the spaA gene was determined in 80 E. rhusiopathiae (serotype 1a) isolates collected from pigs with chronic and subacute swine erysipelas in 14 prefectures in 2008–2014. In this study, 14 (17.5%) isolates were Met-203 type SpaA strains. We confirmed the pathogenicity of a Met-203 type SpaA strain in specific-pathogen-free pigs. In this experiment, the two challenged pigs displayed arthritis, urticaria and other clinical signs, but recovered within 10 days. Our results reveal the existence of the E. rhusiopathiae Met-203 type strains that have been causing chronic erysipelas in Japan.

The virulent strains of Haemophilus parasuis are the causative agents of Glässer’s disease, which can cause systemic infection and result in polyserositis, meningitis and arthritis. The development of novel, effective vaccines would be beneficial to preventing H. parasuis infections. Here, we report a novel immunogenic protein, glutathione-binding protein A (GbpA), which can elicit a significant humoral antibody response and confer significant protection against challenge with a lethal dose of a highly virulent H. parasuis strain. The H. parasuis strain can be fully eliminated in the immunized mice. The results indicate that GbpA has the potential to be used as an effective component of a new vaccine against H. parasuis.

Understanding the molecular mechanisms of DNA double-strand break (DSB) repair processes, especially nonhomologous DNA-end joining (NHEJ), is critical for developing next-generation radiotherapies and chemotherapeutics for human and animal cancers. The localization, protein-protein interactions and post-translational modifications of core NHEJ factors, such as human Ku70 and Ku80, might play critical roles in controlling NHEJ activity. XRCC4-like factor (XLF) is a core NHEJ factor and plays a key role in the Ku-dependent NHEJ repair process in human cells. Recently, companion animals, such as canines, have been proposed to be a good model for many aspects of cancer research, including the development of chemotherapeutics. However, the localization and regulation of core NHEJ factors in canine cells have not been elucidated. Here, we show that the localization of canine XLF changes dynamically during the cell cycle. EYFP-canine XLF localizes in the nuclei of interphase cells and accumulates immediately at microirradiated DSB sites. The structure of a putative human XLF nuclear localization signal (NLS) and a putative 14-3-3 binding motif are evolutionarily conserved in canine, chimpanzee and mouse XLF. However, the putative β-TRCP-recognizable degron of human XLF is not conserved in canine and mouse. Additionally, some vital human XLF phosphorylation sites, including the ATM major phosphorylation site (S251), are not conserved in canine XLF. Our findings might be useful for the study of the molecular mechanisms of NHEJ in canine cells and for the development of new radiosensitizers that target XLF.

The aim of this study was to evaluate the efficacy of pimobendan with conventional therapies on survival and reocurrence of pulmonary edema in dogs with congestive heart failure (CHF) caused by myxomatous mitral valve disease (MMVD). Records of 197 client-owned dogs from 14 veterinary hospitals were included in this study. Dogs were administered conventional treatments with or without pimobendan. Sixty-four dogs received a standard dose of pimobendan (0.20–0.48 mg/kg every 12 hr (q12hr)), 49 dogs received a low dose of pimobendan (0.05–0.19 mg/kg q12hr), and 84 dogs received conventional therapy alone. Dogs in the standard-dose and low-dose pimobendan groups had significantly longer median survival times than dogs in the conventional group (334, 277 and 136 days, respectively; P<0.001). The reoccurrence rate of pulmonary edema in the standard-dose group was significantly lower than in the low-dose and conventional groups (43%, 59% and 62%, respectively; P<0.05). Combination of pimobendan with a conventional treatment regimen significantly prolonged survival time after an initial episode of pulmonary edema in dogs with CHF caused by MMVD. There was no difference in survival between dogs administered standard and low doses of pimobendan, but pimobendan did prevent the reoccurrence of pulmonary edema in a dose-dependent manner.

Canine pancreatitis is a relatively common disorder, and its mortality rate remains high. However, prognostic factors for pancreatitis based on evidence are limited. Moreover, the relationship between changes in C-reactive protein (CRP) concentration—an important prognostic factor for human patients with acute pancreatitis—and the prognosis of dogs with pancreatitis has not been widely studied. Therefore, we examined prognostic factors for canine pancreatitis during the first medical examination and evaluated the usefulness of serial CRP measurements during hospitalization. Sixty-five dogs met the inclusion criteria, including 22 that were hospitalized and treated. In Study 1, a multivariate analysis revealed that three factors— decreased platelet count and a marked (greater than 1,000 µg/l) elevation of specific canine pancreatic lipase (Spec cPL) concentration at the first medical examination, as well as elevated blood urea nitrogen (BUN) and/or creatinine (CRE) level—were significantly different between the survivors and nonsurvivors. Moreover, CRP concentrations on the third and fourth days were significantly different between the two groups in Study 2. An evaluation of the decreased platelet count, remarkable elevation of Spec cPL concentration at the first medical examination, elevation of BUN and/or CRE as well as serial CRP concentration measurements may be useful for predicting the prognosis of canine pancreatitis.

Reduction in glomerular filtration rate (GFR) is a common complication in advanced stages of heart failure (HF). The convenient and precise assessment for GFR would be useful for early detection of renal impairment in HF dogs. Our hypothesis of this study was the GFR would be reduced in advanced stages of HF from chronic mitral valvular insufficiency (CMVI), as indicated by renal markers including serum cystatin-C (Cys-C) and symmetric dimethylarginine (SDMA) concentrations. Forty-three client-owned dogs consisting of 33 dogs with different stages of HF from CMVI and 10 age-matched healthy dogs were enrolled in this study. Serum Cys-C and SDMA concentrations along with other renal (i.e., urea nitrogen and creatinine) and echocardiographic markers were evaluated in healthy and CMVI dogs. Serum Cys-C concentrations were 1.4 ± 0.4 mg/l in control, 2.1 ± 0.9 mg/l in ISACHC I, 2.9 ± 0.8 mg/l in ISACHC II and 3.6 ± 0.6 mg/l in ISACHC III dogs, whereas serum SDMA concentrations were 8 ± 2 µg/dl in control, 14 ± 3 µg/dl in ISACHC I, 18 ± 6 µg/dl in ISACHC II and 22 ± 7 µg/dl in ISACHC III dogs. There was close correlation of serum Cys-C and SDMA concentrations to serum creatinine, urea nitrogen and the severity of HF. Our study demonstrated that the GFR was decreased in dogs with CMVI having earlier stages of HF.

Mesenchymal stem cells (MSCs) are adult multipotent stem cells that are capable of self-renewal and differentiation into multiple cell lineages. Methods for cell therapy using MSCs have been developed in equine medicine. Recently, human dental pulp stem cells (DPSCs) have drawn much attention owing to their trophic factor producing ability and minimally invasive collection methods. However, there have been no reports on equine dental pulp-derived cells (eDPCs). Therefore, the aim of this study was to isolate and characterize the eDPCs from discarded wolf teeth. Plastic-adherent spindle-shaped cells were isolated from wolf teeth. The doubling time of the isolated eDPCs was approximately 1 day. Differentiation assays using induction medium eDPCs differentiated into osteogenic, chondrogenic and adipogenic lineages. The eDPCs expressed mesenchymal makers (CD11a/18, CD44, CD90 CD105 and MHC class I and II), but did not express hematopoietic markers (CD34 and CD45). Taken together, the results show that eDPCs can be isolated from discarded wolf teeth, and they satisfy the minimal criteria for MSCs. Thus, these eDPCs can be referred to as equine DPSCs (eDPSCs). These eDPSCs may become a new source for cell therapy.

The association between pancreatic disorder and abdominal fat necrosis in cattle remains unclear. The pancreases of 29 slaughtered cattle with or without fat necrosis were collected to investigate pathological changes. Japanese Black (JB) cattle were classified into the FN group (with abdominal fat necrosis; n=9) and N group (without fat necrosis; n=5). The pancreases were also collected from 15 Holstein Friesian (HF) cows. All JB cattle showed high body condition scores. Regarding the pathological findings, fatty pancreas which involves adipocyte infiltration into the pancreas and fat necrosis (saponification) were observed in 25 and 27 cases, respectively. Immunohistochemical staining with anti-Iba-1 antibody showed large numbers of macrophages surrounding the saponified fat in the pancreas. CD3-positive T cells were significantly more common in the pancreas of both the FN and N groups compared with the HF group (P<0.05). Furthermore, fibrosis in the pancreas exhibited a correlative tendency with the formation of necrotic fat mass in the peritoneal cavity (P<0.1). These results indicate that obesity leads to increased severity of pancreatic disorder, including fatty pancreas and pancreatitis. The pathological lesions in the pancreas may play a key role in abdominal fat necrosis through the inflammatory process.

A 10-year-old neutered male Labrador Retriever dog was diagnosed with idiopathic megaesophagus. Despite receiving conventional treatments including elevated feeding, the dog showed repeated regurgitation and aspiration pneumonia, consequently developing weight loss and severe malnutrition. For the purpose of controlling regurgitation, an esophagostomy tube was placed for draining the esophageal fluid. Additionally, an esophagogastric tube was placed for nutritional support. After tube placement, the average frequency of regurgitation was reduced from 2.4 times a day to 0.1 times a day. The nutritional state of the dog improved gradually, and the body weight increased from 18.5 to 27.9 kg. The dog died on day 951, and necropsy revealed a gastric ulcer (2.5 cm in diameter), presumably esophagostomy tube-induced injury. This case report suggests that patients with idiopathic megaesophagus and persistent regurgitation might benefit from esophageal drainage through an esophagostomy tube.

The baboon model of Bordetella pertussis infection is the newest and most clinically accurate model of the human disease to date. However, among the 15 experimentally infected baboons in this study, a subset of baboons did not exhibit the expected high bacterial colonization levels or increase in white blood cell count. Moreover, cultures of nasopharyngeal wash samples from several baboons suggested B. bronchiseptica coinfection. Analysis of serum antibodies recognizing filamentous hemagglutinin, pertussis toxin and B. pertussis lipo-oligosaccharide indicated that several baboons had likely been previously exposed to Bordetella species and that prior exposure correlated with partial protection from B. pertussis infection. Notably, all animals with a baseline Fha titer of 5 IU/ml or below exhibited symptoms typical of the model, suggesting this value can be used as inclusion criteria for animals prior to study enrollment. While B. pertussis infection is endemic to human populations and B. bronchiseptica is common in wild small mammals, this study illustrates that baboons can readily harbor both organisms. Awareness of Bordetella species that share antigens capable of generating protective immune responses and tracking of prior exposure to those species is required for successful use of the baboon model of pertussis.

An imported crossbred Angus beef steer aged eight to twelve months died suddenly on the eighth day of a quarantine period in Japan. Gross examination showed the peritoneum and mesentery consisted of numerous nodules of various sizes. Histological examination revealed chronic suppurative granulomatous peritonitis with eosinophilic rosettes surrounding colonies of Gram-negative bacilli. The bacteria isolated from the nodules were confirmed to be Actinobacillus lignieresii based on the results of 16S rRNA gene sequencing and immunohistochemistry. Antibiotic sensitivity testing showed that the isolate was resistant to penicillin. Thus, a diagnosis of atypical actinobacillosis caused by A. lignieresii was made.

A six-year-old castrated male Maltese dog presented to a private animal clinic with a mass on the dog’s lower lip without any other clinical signs. The mass (3 × 2 × 2 cm) was whitish and grossly well circumscribed, and a histopathological examination revealed that the mass was composed of normal cartilage tissue surrounded by fibrous connective tissues. Based on the gross findings, histopathology and anatomical location of the mass, the first diagnosis of a cartilaginous choristoma in a dog was made.

By simply adding a high concentration of calcium solution to the surface of the bullfrog heart, we reproduced electrocardiogram (ECG) abnormalities representing those observed in hypercalcemia, such as Osborn waves and shortening of the QT interval. The rise in extracellular calcium concentration may have activated the outward potassium currents during phase 3 of the action potential, and thus decreased its duration. In addition to the known decrease in the duration of phase 2, such changes in phase 3 were also likely to contribute to the shortening of the QT interval. The dual recordings of the action potential in cardiomyocytes and the ECG waves enabled us to demonstrate the mechanisms of ECG abnormalities induced by hypercalcemia.

There is concern about the zoonotic potential of rodent-borne hepatitis E virus, designated as HEV-C1. However, epizootiological information about HEV-C1 is limited. To address this issue, serum samples from 443 small mammals captured at 5 sites in Hanoi, Vietnam, were examined for anti-HEV-C1 IgG antibodies. In addition, livers of seropositive animals were examined for viral RNA. Anti-HEV-C1 antibodies were detected in 57 (12.9%) of the 443 serum samples. Seropositive animals were found in all of the sites (4.7% to 22.2%). Anti-HEV-C1 antibodies were detected from 48 (12.3%) of 389 Rattus norvegicus and 9 (19.6%) of 46 R. tanezumi, but were not detected from 8 Suncus murinus. Viral RNAs were detected from 13 (22.8%) of the 57 seropositive rodents. The detection rate of viral RNA in seropositive R. tanezumi (66.7%, 6/9) was significantly higher than that in seropositive R. norvegicus (14.6%, 7/48). The results suggest that R. tanezumi is more susceptible than R. norvegicus to HEV-C1 infection. Phylogenetic analysis revealed that Vietnamese strains were divided into 3 clusters in genetic group 2 of HEV-C1. Multiple clusters of viruses were detected at several sites without species specificity, suggesting that 3 clusters of HEV-C1 co-circulate in Hanoi, Vietnam.

A 3-year-old French bulldog presented for evaluation of recurrent swelling and a fistula on the right cheek after a dog fight. A large volume of serous fluids was identified on the wound immediately after atropine drops. A diagnosis of parotid salivary duct rupture secondary to trauma was made. On surgical exploration, the thickened proximal segment of the severed duct was identified and circumferentially double ligated with 3–0 silk. No evidence of swelling and normal appearance of the parotid salivary gland were identified 4 months postoperative recheck. No further problems were noted 10 months postoperatively phone-call. To the author’s knowledge, this is the first reported case of successful proximal parotid duct ligation of parotid salivary duct rupture secondary to non-iatrogenic trauma.

A 12-year-old, spayed female Schnauzer presented with constipation. A mass was observed in the pelvic cavity, and metastasis was not identified. Mass resection was performed through celiotomy with pubic osteotomy, and hemangiosarcoma was diagnosed. At 10 weeks post-operatively, the patient died of multiple metastasis. Primary intrapelvic hemangiosarcoma is rare in dogs.

Uridine 5’-diphospho-glucuronosyltransferase (UGT) liver activity was measured using estradiol-17β as a substrate in dairy cows with follicular cysts. The activity was significantly lower than that in dairy cows with normal estrous cycles (P<0.01). Liver disorders, such as fatty liver and hepatitis, were observed in half cows with follicular cysts, and liver UGT activity was lower than that in cows with normal estrus cycles. In addition, the liver UGT activity was significantly lower in dairy cows with follicular cysts without liver disorders than in dairy cows with normal estrous cycles. Therefore, the cows were divided into those with low, middle and high liver UGT activities, and liver disorder complication rates were investigated. The complication rate was significantly higher in the low- (78.1%) than in the middle- (22.2%) and high-level (8.3%) groups, suggesting that liver disorders are closely associated with the development of follicular cysts in dairy cows and that steroid hormone metabolism is delayed because of reduced liver UGT activity, resulting in follicular cyst formation. We conclude that reduced estradiol-17β glucuronidation in the liver and liver disorders are associated with follicular cyst occurrence in dairy cows.

Equine and human chorionic gonadotropins were administered to two female Amur leopard cats to induce estrus and ovulation during non-breeding season. Fresh semen collected from male cats was surgically inseminated into the uterine horn of the females. In one animal, two fetal sacs without heartbeats were observed on abdominal ultrasonography 31 days after insemination, which indicated that embryo death had occurred. In the other animal, fetal heartbeats were detected in two fetal sacs 29 days after insemination, which confirmed as pregnancy. This animal delivered two newborns 68 days after insemination; the one of the kittens was assumed to be stillbirth, and the other grew normally. In this study, we successfully obtained a kitten from an Amur leopard cat by artificial breeding for the first time in Japan.

Endometritis is a common puerperal disease in livestock. Thus, a practical clinical test for the establishment of diagnosis is needed. We developed a goat model for subclinical endometritis diagnosis by examining cytological, ultrastructural features and molecular expression in cervical discharge collected by cervical cytobrush method. A suspension of E. coli was infused into the uterine horn of goats, and cervical discharge, peripheral blood and endomertrial biopsy samples were collected before inoculation and at 3, 6, 12, 24, 72, 120 and 168 hr post-inoculation (pi). In experimental goats, total leukocytes increased at 6–12 hr pi in hematological examinations, abundant neutrophils were observed in cervical discharge smears, and some micro-villi shortened or were lost from the epithelium at 3 hr pi, with some inflammatory cells infiltrated into the uterine glands and lamina propria. The mRNA expression levels of the Toll-like receptor 4 (TLR4), cytokines and β-defensin-2 increased significantly within 12 hr pi and returned to pre-inoculation levels at 7 day pi. The results showed that subclinical endometritis was simulated in this bacterial infusion goat model, and the evaluation of cervical cytology was in good agreement with molecular changes of cytokines in cervical discharge, hematology and histology. We thus conclude that the simple cervical cytobrush technique can effectively obtain cervical discharge samples for cytology examination in the early diagnosis of subclinical endometritis in goats.

The cellular isoform of the prion protein (PrP^C) plays critical roles in the development of prion disorders. Although PrP mRNA is ubiquitously present in a tissue-specific manner, the DNA methylation of PrP gene (Prnp) is still unknown. In this study, we demonstrated that the CpG island (CGI, positioned at −218 to +152 bp from the transcriptional start site) including the Prnp core promoter region was completely unmethylated in all tested tissues. On the other hand, CpG methylation in the CGI shore region (positioned at −599 to −238 bp) occurred in various tissue- and site-specific proportions. Interestingly, the correlation analysis between CpG methylation status and PrP mRNA levels showed that one CpG site methylation at −576 was negatively correlated with the PrP mRNA level (Pearson’s r = −0.374, P=0.035). Taken together, our results suggest that Prnp is a typical housekeeping gene and various methylation frequencies of the CGI shore region are likely to affect Prnp expression in a tissue-specific manner.

The sequence at the hemagglutinin (HA) cleavage site (CS) plays a key role in determining the pathogenicity of avian influenza viruses. Three types of HA CS sequences, QREKR/GL, QRKKR/GL and QRRKR/GL, were previously reported in Taiwanese H5N2 viruses that were isolated from chickens from 2003 to 2013. However, no HA CS sequence was reported for viruses isolated after 2013. This article presents the HA CS sequences and pathogenicity of H5N2 viruses that were isolated from chickens in Taiwan during 2013–2015. Two novel HA CS sequences, QKEKR/GL and KREKREKR/GL, were found in the viruses isolated in 2013 and 2014, and pathogenicity tests showed that the viruses with these novel HA CS sequences are low and high pathogenic viruses, respectively. In contrast, the HA CS sequence QREKR/GL was found in all viruses that were isolated in 2015, and all of these viruses were low pathogenic viruses. After 10 passages in embryonated chicken eggs, a virus strain that was isolated in 2003 evolved into a viral quasispecies that contained at least four distinct types of HA CS sequences. These results highlight the potential of Taiwanese H5N2 viruses to change their pathogenicity and HA CS sequences via mutations. Furthermore, viruses with the HA CS sequence QREKR/GL were more prevalent than others in 2015. These findings are useful for understanding the mechanism of sequence changes at the HA CS and for refining H5N2 virus control measures in Taiwan.

Gallid herpesvirus 2 (GaHV-2) causes malignant lymphomas in chickens (Marek’s disease, MD). Although MD is controlled through vaccination efforts, field isolates of GaHV-2 have increased in virulence worldwide and even cause MD in vaccinated chickens. GaHV-2 strains are classified into four categories (mild, virulent, very virulent and very virulent +) based on the virulence exhibited in experimental infection in unvaccinated or MD-vaccinated susceptible chickens. Although MD cases are sporadically reported in Japan, the recent field strains of GaHV-2 in Japan have not been characterized. During isolation of recent field strains by using primary chicken kidney cell cultures, a method classically used for GaHV-2 isolation, vaccine strains were simultaneously isolated. Therefore, it is necessary to separate vaccine strains to characterize the virulence and pathogenicity of the GaHV-2 strains currently distributed in Japan. In this study, we prepared cell suspensions from the spleens of MD-symptomatic chickens, inoculated day-old-chicks and isolated GaHV-2 strains by primary chicken kidney cell cultures at 2−3 weeks post inoculation. The isolated strains were passaged several times on chicken embryo fibroblast cells, and PCR analysis revealed that the isolated strains were not contaminated with vaccine strains. Moreover, the contaminant vaccine strains were completely removed by the purification of plaques observed in chicken kidney cells. These procedures are necessary to isolate GaHV-2 field strains from vaccine strains in order to carry out future studies to characterize these strains and glean insights into GaHV-2 virulence and pathogenicity.

Canine distemper virus (CDV) is one of the most serious pathogens found in many species of carnivores, including domestic dogs. In this study, hemagglutinin (H) genes were detected in five domestic Vietnamese dogs with diarrhea, and two CDVs were successfully isolated from dogs positive for H genes. The complete genome of one isolate, CDV/dog/HCM/33/140816, was determined. Phylogenetic analysis showed that all Vietnamese CDVs belonged to the Asia-1 genotype. In addition, the H proteins of Vietnamese CDV strains were the most homologous to those of Chinese CDVs (98.4% to 99.3% identity). These results indicated that the Asia-1 genotype of CDV was the predominant genotype circulating among the domestic dog population in Vietnam and that transboundary transmission of CDV has occurred between Vietnam and China.

The effectiveness of a vaccine preserved for emergency use in Japan was analyzed under experimental conditions using cows and pigs in order to retrospectively evaluate the effectiveness of the emergency vaccination performed in the 2010 epidemic in Japan. Cows and pigs were administered a vaccine preserved for emergency use in Japan at 3 or 30 days before virus infection (dbv) and were subsequently infected with the foot-and-mouth disease virus (FMDV) O/JPN/2010, which was isolated in the 2010 epidemic in Japan. All animals vaccinated at 30 dbv and one of three pigs vaccinated at 3 dbv showed no vesicular lesions during the experimental period. The virus titers and viral RNA loads obtained from clinical samples were lower in the vaccinated cows than in the non-vaccinated cows. The viral excretion periods were shorter in the vaccinated cows than in the non-vaccinated cows. In contrast, in the vaccinated pigs, the virus titers and viral RNA loads obtained from the samples, except for those obtained from sera, were not decreased significantly, and the viral excretion periods were not sufficiently shortened. These results suggest that the vaccine can protect against clinical signs of infection by the FMDV O/JPN/2010 in animals; however, it should be noted that in vaccinated and infected animals, especially pigs, clinical samples, such as saliva and nasal swabs, may contain excreted viruses, even if no clinical signs were exhibited.

Equine herpesvirus type 4 (EHV-4) is one of the most important pathogens in horses. To clarify the key genes of the EHV-4 genome that cause abortion in female horses, we determined the whole genome sequences of a laboratory strain and 7 Japanese EHV-4 isolates that were isolated from 2 aborted fetuses and nasal swabs of 5 horses with respiratory disease. The full genome sequences and predicted amino acid sequences of each gene of these isolates were compared with of the reference EHV-4 strain NS80567 and Australian isolates that were reported in 2015. The EHV-4 isolates clustered in 2 groups which did not reflect their pathogenicity. A comparison of the predicted amino acid sequences of the genes did not reveal any genes that were associated with EHV-4-induced abortion.

Various techniques for screening and detection of bovine leukemia virus (BLV) were compared to ascertain a rapid and simple technique for routine examination. The performance of real-time PCR, nested PCR and loop-mediated isothermal amplification (LAMP) assays was compared using DNA extracted from whole blood instead of white blood cells (WBCs) of 23 cattle. Real-time PCR, LAMP and nested PCR detected 18, 16 and 11 BLV-positive cattle, respectively. These results suggest that LAMP using DNA from whole blood could enable rapid examination, as isolation of WBCs and electrophoresis is time-consuming and could be useful as a simple and rapid method for routine screening of BLV.

We attempted to prepare a cell line that produces maedi/visna virus (MVV) and is free of contamination by other viruses and mycoplasmas. Three cell lines, which originated from a sheep, goat and bat, were infected with MVV and passaged approximately every 5 days. The cultured cells were then subjected to polymerase chain reaction analysis for MVV provirus. As a result, a cell line persistently infected with MVV was established from ZZ-R cells, which originated from the fetal goat tongue. The 50-fold concentrated culture fluid formed a precipitation line against reference antiserum.

Canine parvovirus type 2 (CPV-2) is the main etiological agent of viral enteritis in dogs. Actually in literature, CPV-2 has been reported with clinical signs that vary from the classical disease, and immunochromatography test and PCR technique have been introduced to veterinary hospitals to confirm CPV-2 diagnosis and other infections. However, the reliability of these techniques has been poorly analyzed. In this study, we evaluated the sensitivity and specificity of veterinary clinical diagnosis, immunochromatography test and PCR technique. Our data indicate that variations in the clinical signs of CPV-2 complicate the gathering of an appropriate diagnosis; and immunochromatography test and PCR technique do not have adequate sensitivity to diagnose positive cases.

Craniodental morphology of the Eurasian otter (Lutra lutra) in the Korean Peninsula, Japanese islands and Kinmen Island (Taiwan) was studied using geometric morphometrics to identify the skull variations between the populations. Forty adult skulls were examined (29 specimens from the Korean Peninsula, six from Shikoku, Honshu and Hokkaido of Japan, and five from Kinmen Island). Images of the dorsal and ventral views of the skull and the right lateral view of the mandible were analyzed. Specimens from the Korean Peninsula were larger than those from the Japanese islands and Kinmen Island. However, no correlation was observed between the shape variations in the three populations and the centroid size of the skull. The Mann-Whitney U-test showed that relative warps (RWs) RW1, RW2 and RW4 of the dorsal view and RW2 of the ventral view of the skull differed significantly between the populations. Some craniodental differences between the populations were seen in the dorsal and ventral views of the skull, mostly at the snout and parietal regions. The MANOVA test revealed significant differences between the specimens from the Japanese islands and Korean Peninsula and between the specimens from the Korean Peninsula and Kinmen Island. RWs plots showed an overlap of all three populations. In conclusion, the comparisons of the three examined populations revealed significant differences in their craniodental morphology.

The objective of this study was to determine the dose-dependent effects of isoflurane on various cardiovascular parameters and the stable range of isoflurane concentrations in Asiatic black bears (Ursus thibetanus). Seven Asiatic black bears were intramuscularly injected with medetomidine, zolazepam and tiletamine (MZT) to induce anesthesia, and anesthesia was maintained by administering isoflurane in 100% oxygen (4 l/min) without mechanical ventilation. Several cardiovascular parameters were measured at five end-tidal isoflurane concentrations (0.5, 1.0, 1.5, 2.0, and 2.5%). Blood was collected from the femoral artery before administration of isoflurane and after each administration for immediate blood gas analysis. Isoflurane produced dose-dependent increases in heart rate, respiratory rate, minute volume, end-tidal carbon dioxide (CO₂) partial pressure and the partial pressure of arterial CO₂, and dose-dependent decreases in non-invasive blood pressure and tidal volume. Rectal temperature, oxygenation and acid-base balance were unaffected by isoflurane. All parameters in this study were in a clinically acceptable range at all times. The data show that the combination of MZT and isoflurane is suitable for general anesthesia in Asiatic black bears with spontaneous breathing during prolonged procedures. End-tidal isoflurane concentrations of 0.5 to 2.5% can be used in Asiatic black bears without adverse side effects.

Anesthesia is an inevitably important component of diagnosis and treatments examining the health condition of wild animals. Not only does anesthesia become an essential tool in minimizing stress of the patients and providing an opportunity to deliver accurate and safe procedures, but it also ensures the safety of the medical crew members. This study was conducted to investigate the dose-response cardiorespiratory effects of isoflurane during spontaneous ventilation in ten cinereous vultures. Each bird was administered isoflurane at initial concentration of 1.0, 1.5, 2.0, 2.5 and 3.0 and then an end-tidal isoflurane concentrations (ETiso) of 1.0% for an equilibration period of 15 min in the given order. At the end of the equilibration period, the direct blood pressure (BP), heart rate (HR), respiratory rate (RR) and end tidal CO₂ partial pressure (P_ETCO₂) were recorded, and blood gas analysis was performed. Increasing isoflurane concentrations during spontaneous ventilation led to dose-dependent increases in HR and P_ETCO₂, with minimal changes in RR, decreased arterial BP and respiratory acidosis. Overall, isoflurane for anesthesia of spontaneously breathing cinereous vultures is a suitable choice for diagnostic or surgical procedures.

To help plan conservation of the endangered Miyako horse, a biological resource of the Miyako Islands in Japan, we characterized the genetics of the breed by genotyping 32 microsatellites and identifying mitochondrial DNA haplotypes. We also calculated genetic distances between individuals based on the proportion of shared alleles and visualized the genetic relationships with a phylogenetic tree. Two important results were obtained. One is that accurate pedigree registration of the horse by using microsatellites is possible, as the exclusion power of parentage testing is 0.999998. Another is that the current genetic diversity of the horses was clarified. The average number of alleles, observed heterozygosity and expected heterozygosity were 4.2, 0.701 and 0.649, respectively, for the 35 analyzed horses. The probability values for bottleneck models (infinite allele model: 0.00000; stepwise mutation model: 0.00026; and two-phase model: 0.00000) suggested that Miyako horses have experienced a recent genetic bottleneck. Only one mitochondrial haplotype was identified. Consequently, genetic diversity within the population is relatively well-maintained despite a very small population size (41 at the time of the study), and the first priority in conservation of the Miyako horse is to increase the population size.

The ranges of pronation/supination of forearms in raccoons, raccoon dogs and red pandas were nondestructively examined. Three carcasses of each species were used for CT analysis, and the left forearms were scanned with a CT scanner in two positions: maximal supination and maximal pronation. Scanning data were reconstructed into three-dimensional images, cross-sectional images were extracted at the position that shows the largest area in the distal part of ulna, and then, the centroids of each cross section of the radius and ulna were detected. CT images of two positions were superimposed, by overlapping the outlines of each ulna, and then, the centroids were connected by lines to measure the angle of rotation, as an index of range of mobility. The measurements in each animal were analyzed, using the Tukey–Kramer method. The average angle of rotation was largest in raccoons and smallest in raccoon dogs, and the difference was significant. In the maximally pronated forearm of all species, the posture was almost equal to the usual grounding position with palms touching the ground. Therefore, the present results demonstrate that the forearms of raccoons can supinate to a greater degree from the grounding position with palms on the ground, as compared with those of raccoon dogs and red pandas.

Two radiated tortoises (Astrochelys radiata) exhibited anorexia and hypokinesia. In both cases, hematological and serum biochemical examinations revealed high alkaline phosphatase levels, moderately high aspartate aminotransferase levels and white blood cell counts approximately within the normal range. Despite being treated, the tortoises died 9 and 43 days after the first clinical examination. Gross pathological examinations revealed that the livers of both animals were extremely swollen and contained pale yellow necrotic tissue. Histopathological assessment revealed that the livers contained a massive area of hepatic necrosis surrounded by migration of macrophages and multinucleated giant cells. In one of the cases, severe fibrosis was observed. The present study provides reference information for similar cases in the future.

A penile tumor (4 × 2.5 × 1 cm) was surgically removed from an African pygmy hedgehog (Atelerix albiventris) aged 3 years and 5 months. The tumor was continuous with the dorsal fascia of the penile head. Histopathologically, tumor cells were pleomorphic (oval-, short spindle- and star-shaped cells) with low cell density. Abundant edematous stroma was weakly positive for Alcian blue staining and positive for colloidal iron reaction. Tumor cells displayed no cellular atypia or karyokinesis. Tumor cell cytoplasm was positive for vimentin antibody, while cytoplasm and nuclei were positive for S-100 protein antibody. Tumor cell ultrastructure matched that of fibroblasts, and the rough endoplasmic reticulum was enlarged. The tumor was diagnosed as myxoma. This represents the first report of myxoma in a hedgehog.