The vomeronasal organ of sheep was examined using lectin histochemistry in order to compare the types and amounts of the glycoconjugates among various components of the vomeronasal sensory and non-sensory epithelia. In the vomeronasal sensory epithelium, Dolichos biflorus agglutinin (DBA) stained particular cells, located at the same level as the vomeronasal receptor cells, while the distribution, shape and number of the stained cells did not correspond to those of the vomeronasal receptor cells. Datura stramonium lectin (DSL), Concanavalin A (Con A), Phaseolus vulgaris agglutinin-E (PHA-E) and Phaseolus vulgaris agglutinin-L (PHA-L) labeled the basal cells of both vomeronasal sensory and non-sensory epithelia. While, Wheat germ agglutinin (WGA), Succinylated-wheat germ agglutinin (s-WGA), Lycopersicon esculentum lectin (LEL), Solanum tuberosum lectin (STL) and Ricinus communis agglutinin-I (RCA-120) labeled the basal cells of the sensory epithelium, and Bandeiraea simplicifolia lectin-I (BSL-I) stained the basal cells of the non-sensory epithelium, respectively. Seventeen lectins labeled the free border of both vomeronasal sensory and non-sensory epithelia, while Sophora japonica agglutinin (SJA), Jacalin and Pisum sativum agglutinin (PSA) labeled neither free border of the sensory nor that of non-sensory epithelia. The expression pattern of glycoconjugate was similar, but not identical, in the free border between the sensory and non-sensory epithelia. These results indicate that there are dissimilar features in the type and amount of glycoconjugates between the vomeronasal sensory and non-sensory epithelia, and at the same time, among the various cell types either in the vomeronasal sensory or non-sensory epithelium.

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Expression of six selective genes in peripheral blood cells was evaluated as diagnostic biomarkers for enzootic bovine leukosis (EBL) by using 10 EBL and 15 clinically healthy cattle. The clinically healthy cattle generally showed lower gene expression levels. Although wide variations of gene expression were found in some clinical cases of EBL, 4 and 5 among 10 EBL cattle showed higher expression of interleukin 2 receptor gene (IL2R) and Wilms’ tumor gene (WT1), respectively. Expression of IL2R in peripheral blood cells in EBL cattle was statistically increased; however, the lower sensitivity and higher variation in the gene expressions among clinical cases of EBL would be problems as diagnostic biomarkers.

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To shed light on the role of central oxytocin (OXT) in regulating social behavior in cattle, the impact of intracerebroventricularly administered OXT agonist, carbetocin (CBT), on the social behavior of a group of familiar steers was investigated. In the first experiment, we determined the dose response of intracerebroventricularly administered CBT (0.5, 5 or 50 nmol) on plasma cortisol level and behavior using 7 steers aged from 6 to 10 months. Five of the steers were assigned to the second experiment. CBT (50 or 200 nmol/200μl) in artificial cerebrospinal fluid (aCSF) or aCSF (200 μl) was injected into the third ventricle. Immediately after the injection, the animal and two peers were taken outside to the adjacent paddock. Thirty minutes later, maintenance and social behaviors of the animal were observed for 2 hr. CBT had no effect either on the basal cortisol level or on the maintenance and the abnormal behavior in steers with their movement restricted by a stanchion stall in the first experiment. However, in the same steers with no movement restrictions in the second experiment, CBT facilitated lying, probably because of its sedative effect via OXT receptor activation, which disturbed some aspects of social behavior. These results suggest that central OXT receptor activation might not affect social behavior itself among “familiar members”, because the stimulation of the central OXT system by intracerebroventricular administration of CBT did not facilitate social behavior between familiar steers.

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This study evaluated the clinical usefulness of serum thymidine kinase (TK) activity for diagnosing bovine leukosis cases for which clinical diagnosis was difficult (‘BL with difficult diagnosis’). Median TK activity values in 24 ‘BL with difficult diagnosis’ and 36 cattle for which BL was clinically confirmed by cytology findings of enlarged superficial lymph nodes (‘clinically confirmed BL’) were 36.8 and 39.4 U/l, respectively (no significant difference). The percentage with positive TK activity (>5.4 U/l) was also similar in both groups (83.3% for ‘BL with difficult diagnosis’ and 97.2% for ‘clinically confirmed BL’). TK activity was significantly higher in cows with ‘BL with difficult diagnosis’ compared to those with other tumors (N=13) and those with inflammatory diseases (N=14). Maximum TK activity in cows with other tumors and inflammatory diseases was not high (<10 U/l). Median TK activities in cows with other tumors and those with inflammatory diseases were 1.8 and 1.4 IU/l, respectively. Positive TK activity was found in a significantly higher percentage of cows with ‘BL with difficult diagnosis’ (83.3%) relative to the percentages of cows with other tumors (15.3%) and inflammatory diseases (21.4%). Thus, TK activity is an appropriate marker for detecting BL onset in cows with ‘BL with difficult diagnosis’ as well as ‘clinically confirmed BL’ group. While the specificity of TK activity required for BL diagnosis is not clear, simultaneous evaluation of serum lactate dehydrogenase activity may assist in the differential diagnoses of other tumors and inflammatory diseases from BL.

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Because the role of bone turnover during early to peak lactation in dairy cows is unclear, plasma levels of osteoclast- or collagen-specific bone resorption and osteoblast-specific bone formation markers from postpartum week 3 (W+3) to W+7 were compared with prepartum W-3 levels in multiparous (n=15) and primiparous (n=13) Holstein cows. The multiparous group showed a consistently low level of osteoclast-specific bone resorption markers, while collagen-specific resorption and osteoblast-specific formation markers decreased at postpartum W+7 and from W+5, respectively. In the primiparous group, the plasma levels of all three markers decreased from postpartum W+3. These results suggest that the calcium demand during the transition from early to peak lactation in dairy cows is less affected by bone turnover.

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It is known that Helicobacter hepaticus or Helicobacter bilis infection causes chronic inflammation of the colon and liver. Chronic active hepatitis was found in radiation exposure experiments using male C3H/HeNrs mice at our institute. Histopathologically, 103 cases among 978 mice (64–91 weeks of age at autopsy) had hepatic lesions regardless of irradiation exposure. Mild lesions showed only focal necrosis and focal inflammation in the liver. Severe cases were accompanied by hepatocytomegaly, bile duct hyperplasia, hypertrophy and activation of Kupffer cells, cholangitis, pleomorphic hepatocytes and/or tumor. Helical-shaped bacteria were detected between hepatocytes by Warthin-Starry silver stain and immunohistochemistry (IHC) with an antibody against Helicobacter pylori. It was suggested that these cases of chronic hepatitis were caused by Helicobacter spp. Although chronic hepatitis occurred frequently in mice exposed high-dose irradiation compared with nonirradiated mice in one lot, it was not concluded that radiation might influence the incidence or degree of hepatitis. Our report suggested that natural Helicobacter spp. infection in mice can occur in an experimental animal facility. Therefore, it is suggested that monitoring of Helicobacter infection is very important for quality control of animal experiments.

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Ovine theileriosis is a tick-borne disease that restricts the development of small ruminant husbandry in northern China. In this study, we report on a molecular epidemiological survey of ovine Theileria spp. in 198 blood samples taken from sheep in northern China. The DNA samples were screened by a nested polymerase chain reaction (PCR) targeting the 18S rRNA gene of ovine Theileria spp. The prevalence of ovine Theileria spp. in Yanji, Nongan, Longjing, Toudao and Jinchang was 80%, 40%, 37%, 24% and 32%, respectively. The sequencing analyses approved the present of the T. orientalis and/or T. luwenshuni in these regions. Taken together, we have demonstrated a high incidence of Theileria spp. in northern China that calls for the need to design effective control programs for ovine theileriosis.

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Water buffalo (WB) is an important domestic animal in Vietnam. This study utilized a card agglutination test to investigate seroprevalence of surra in WB population. Sera were collected from 585 WB from 4 different regions in Cao Bang and Thai Nguyen Provinces. Among them, 131 samples (22.4%) were positive for surra. The highest prevalence (24.6%) was found among 3 to 5 years old WB. Buffaloes less than 3 years old had the lowest prevalence (15.6%). Among 27 abortion cases, 9 WB (33.3%) were surra positive. For treatment of surra, Berenil^® demonstrated a 100% cure rate, while that of Trypamidium^® was only 40%. Our findings suggest that the current control strategy has not succeeded in reducing prevalence of surra in Vietnam.

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A 2-year-old Thoroughbred horse presented with acute onset of colitis, and the intussuscepted jejunum was surgically resected. A transmural mass protruding into the lumen was found at the leading edge of the intussusceptum. Based on histological and immunohistochemical examinations, the mass was diagnosed as diffuse large B-cell lymphoma with metastasis to the mesenteric lymph nodes. Anatomical localization of the mass in the intussusception and absence of other obvious underlying diseases indicated that the intussusception had occurred in association with the mass. To our knowledge, this case is the first report of equine intussusception associated with focal intestinal lymphoma.

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Anoikis is a form of apoptosis triggered by inadequate or inappropriate cell matrix contacts. Anoikis resistance has been utilized to isolate adult stem or progenitor cell populations from various tissues. The aim of this study was to characterize the stem or progenitor cell markers expressed in anoikis-resistant cells isolated from mouse colonic epithelium. Mouse colonic epithelial cells were isolated by Ca²⁺-depletion, and anoikis-resistant cells were obtained by culturing the cells in ultra-low attachment dishes. Flow cytometry analysis showed that anoikis-resistant cells are positive for the epithelial cell marker CD326, but negative for the hematopoietic cell marker CD45, eliminating the possibility of hematopoietic cell contamination. The majority of anoikis-resistant cells were also positive for the stem or progenitor cell markers CD133 and DCLK1 by immunofluorescent analysis. Reverse transcriptase-polymerase chain reaction analysis revealed that anoikis-resistant cells express +4 position cell marker Hopx, but did not express the other reported stem or progenitor cell markers, Lgr5, Musashi1, Bmi1, mTert and Olfm4. CD133 and DCLK1 double positive cells were observed both apical and basal crypts in mouse proximal colonic tissues. Together, anoikis-resistant cells in mouse colon epithelium were shown to be positive for CD133, DCLK1, Hopx and CD326, but negative for CD45, Lgr5, Musashi1, Bmi1, mTert and Olfm4. This study has shown a possible approach to isolating stem cells from the intestinal epithelium.

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Chediak-Higashi syndrome (CHS) is an autosomal recessive hereditary disorder in Japanese Black cattle, caused by a mutation of the Lyst gene. So far, the mutation has been detected by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. However, this method is disadvantaged by its low-throughput performance. Here, we report an alternative method involving real-time PCR with TaqMan minor groove binder probes, which shortens the total assay time by more than 120 min, analyzing 10 samples in a duplicated manner. Using this method, we examined 102 Japanese Black cattle and found that 8.8% of the cattle were CHS-carriers. These data indicate that our technique is useful for routine diagnostic testing for CHS in Japanese Black cattle.

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The aim of this study was to investigate blood coagulation times in genetically obese rats and diet-induced obese (DIO) mice in order to clarify the relationship between visceral obesity and blood coagulation. WBN/Kob-Lepr^fa (fa/fa) rats, a genetically obese model, exhibited a significantly shorter activated partial thromboplastin time (aPTT) and prothrombin time (PT) than age-matched Wistar rats. C57BL/6J mice fed a high-fat diet (60%), a DIO model, exhibited significantly shorter aPTT, PT and thrombin time than lean mice fed a standard diet. Higher body weight, visceral fat weight and insulin resistance were also shared by fa/fa rats and DIO mice. These results suggest that visceral obesity is related to accelerated blood coagulation in addition to disrupted metabolism of glucose and lipids.

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In the following study, we mainly investigate the effects of static magnetic field (SMF) (128 mT, 1 hr/day during 5 consecutive days) on 25-hydroxyvitamin D₃ and calcium homeostasis. Wistar male rats, weighing 50–70 g, were randomly divided into four experimental groups: control, SMF-exposed rat, co-exposed rats (the last day and after exposure rats received a single dose of vitamin D per os) and supplemented with vitamin D group (without exposure to SMF). Exposure to SMF induced a decrease of plasmatic 25-hydroxyvitamin D₃ level (P<0.001). While, calcium and phosphorus levels were not affected (P>0.05). The same treatment failed also to alter body, relative liver and kidney weights. Interestingly, oral supplementation with vitamin D corrected hypovitaminosis D induced by SMF. Likewise, the same treatment failed to alter calcium homeostasis. More studies are needed to evaluate how SMF induces hypovitaminosis D.

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Poultry products have consistently been identified as important sources of Salmonella infection in humans, because vertical transfer of infection from breeding hens to progeny is an important aspect of the epidemiology of Salmonella spp. infection within the poultry industry. The aim of this study was to estimate the prevalence of Salmonella contamination in poultry products from 15 different located geographical areas from among the 50 poultry slaughterhouses authorized to operate in Korea and to characterize all the isolates by genotyping, phage typing and antibiotic resistance pattern. Salmonella was isolated from 10 (66.7%) of the first and 5 (33.3%) of the last chilling waters and from 32 (42.7%) carcasses originating from 9 slaughterhouses. The major prevalent serotypes of Salmonella originating from 2 duck slaughterhouses and 13 chicken slaughterhouses tested were S. Typhimurium and S. Enteritidis, respectively. Regarding the characteristics of their antibiotic resistance, 8 of the 11 ampicillin resistant (Am^R) isolates carried bla_TEM only, two carried bla_TEM and bla_CTX-M-14 and one carried bla_CTX-M-3 and only one Am^R isolate with the bla_CTX-M-3 β-lactamase gene was an ESBL-producing Salmonella strain. Twenty-seven Salmonella isolates showed nalidixic acid resistance with a mutation at amino acid codon Asp87 in gyrA and no mutation in the parC gene. In all the phenotypic and genotypic properties of the 18 S. Enteritidis and 8 S. Typhimurium based on PFGE, phage types and antibiotic resistance pattern, the predominant patterns were XEI/BEI-PT32a-Na^R (n=5) and XTI/BTI-RNDC-no resistant antibiotics (n=6), respectively.

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Lyme disease Borrelia spp. are transmitted by Ixodes ticks, and more than 10 species of borreliae have been identified around the world. Recently, another Borrelia sp. has been reported in Asia (Japan, Korea, China, Taiwan and Thailand) as Borrelia valaisiana-related sp. In the present study, we obtained and genetically characterized 19 B. valaisiana-related sp. strains from mammals and ticks. Genetic analyses showed that the Borrelia strains were distinct from B. valaisiana found in Europe. Multilocus sequence typing revealed that these Borrelia isolates formed a monophyletic group with B. yangtze strains in China. Some of the strains were isolated from the bladders of small mammals, and also two strains were experimentally confirmed to be infectious in C3H/HeN mice. We observed that the Borrelia sp. was maintained in the Ixodes granulatus tick after molting. These results suggested that small mammals and I. granulatus were possible reservoir hosts and the vector tick for the Borrelia sp., respectively. B. valaisiana, originally found in Europe, was transmitted mainly by I. ricinus, and birds were mainly thought to be reservoir hosts. Our results suggested that Japanese isolates of B. yangtze (formerly B. valaisiana-related sp.) were distinguishable from B. valaisiana according to the reservoir host and its vector tick. In this study, we also deposited borrelia strain Okinawa-CW62 into bioresource centers as a reference strain of B. yangtze(=DSM 24625, JCM 17189).

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With the aim of comparing the prevalence of Shiga toxin-producing Escherichia coli (STEC) O157 and O26 between beef and dairy cattle, we collected rectal content samples from 250 beef cattle on 25 beef farms and 250 dairy cows on 25 dairy farms from July through September 2011. STEC O157 was isolated from 16 beef cattle on 7 beef farms, while no STEC O157 was isolated from any dairy farms. This result suggests that the prevalence of STEC O157 is higher in beef cattle than in dairy cattle. STEC O26 was isolated from 1 animal each from beef and dairy cattle herds, and therefore, it was not possible to compare statistically the prevalence of STEC O26 in beef and dairy cattle.

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Colopexy was accomplished in eight healthy mixed-breed dogs by use of a 3-portal laparoscopic technique without major intraoperative and postoperative complications. A permanent adhesion between the colon and the abdominal wall was observed. Concentrations of acute-phase C-reactive protein (CRP) were measured in serum as a marker of systemic inflammation postoperatively, and no relevant increase in CRP concentrations was found.

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Poly-l-lactic acid (PLLA) has been clinically used as a bioabsorbable material and attains a piezoelectric charge upon molecular orientation by the application of a shear force to the C-axis of the crystal line region. Previous studies showed that implanted drawn PLLA films or rods accelerate the ossification due to piezoelectric effect. In this study, we originally designed helically-twisted PLLA fiber to produce piezoelectricity in bioabsorbable suture upon tensile stress. The piezoelectricity of the helical PLLA fibers was evaluated using a lock-in amplifier system in vitro. The ossification induced by helical PLLA fibers was examined by implanting them in the rat patellar ligament supporting a physiological tensile load. We observed that 57° and 45° twisted PLLA fibers generated a higher piezoelectric potential than did 27° twisted fibers. The animal experiment showed that the formation of osseous tissue around helical PLLA fibers was more significant than around non-helical control fibers at 4 weeks after their implantation. These results suggest that helical PLLA fiber may be useful for the surgical suture or artificial ligament, which connects to the bone.

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In healthy male dogs, peripheral plasma testosterone (T), prostaglandin E₂ (PGE₂) and seminal plasma PGE₂ levels were measured before, during and after ejaculation, and semen quality was examined after oral administration of PGE₂. Plasma T and PGE₂ levels did not change during these periods, but the seminal plasma PGE₂ level of combined the first and second fractions was significantly higher than those at 0–5 and 5–10 min after the start of ejaculation of the third fraction. Semen volume but not quality increased after PGE₂ administration. In conclusion, large amounts of PGE₂ are released from the prostate gland during the early part of ejaculation, and PGE₂ plays an essential role in secretion of seminal plasma.

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The objective of this study was to evaluate the possible protection of resveratrol against lung injury induced by arsenic trioxide (As₂O₃). Twenty-four healthy Chinese Dragon Li cats of either sex were randomly divided into four groups: control (1 ml/kg physiological saline), As₂O₃ (1 mg/kg), resveratrol (3 mg/kg) and resveratrol (3 mg/kg) + As₂O₃ (1 mg/kg). The resveratrol + As₂O₃- treated group was given resveratrol 1 hr before As₂O₃ (1 mg/kg) administration. We found that pretreatment with resveratrol in a clinically comparable dose regimen can reversed the changes in morphological and biochemical parameters induced by As₂O₃ in the lung. Resveratrol treatment also upregulated the activities of antioxidant enzymes and attenuated As₂O₃-induced increases in reactive oxygen species, 8-hydroxydeoxyguanosine and malondialdehyde production in the lung. In addition, resveratrol attenuated the As₂O₃-induced reduction in the ratio of reduced glutathione to oxidized glutathione, the content of total glutathione and lung arsenic burden. These findings indicated that resveratrol can provide significant protection against As₂O₃-induced oxidative damage.

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Virus-neutralizing (VN) testing is essential for evaluating virus-specific immunity in equine herpesvirus type-1 (EHV-1) infection. We developed a focus-reduction neutralization test (FRNT) for EHV-1 using 96-well plates for faster large-scale testing with sufficient sensitivity. We used an overlay medium containing Avicel (FMC Biopolymer), a microcrystalline cellulose with lower viscosity than the methylcellulose. The foci were visualized by immuno-staining with anti-EHV-1 gp14 monoclonal antibody. The FRNT successfully detected seroconversion in horses experimentally infected with EHV-1 (n=3) and in those infected naturally (n=16). The FRNT for EHV-1 was high-throughput and time saving. The FRNT can be used in large-scale seroepidemiological studies of EHV-1 and in evaluating vaccine efficacy.

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Equine cells are required for isolation of viruses that infect the horse. However, only a few equine cell lines and cell cultures are available so far. Fetal horse kidney (FHK)-Tcl3.1 cell is a novel cell line established by introducing simian virus 40 (SV40) large T antigen. In the present study, the ability to propagate equine viruses was compared between FHK-Tcl3.1 cells and other equine cells. FHK-Tcl3.1 cells efficiently increased many viruses derived from or having pathogenicity to horses and produced high infective titers in culture fluids. These results indicate that FHK-Tcl3.1 cells would be useful for propagation and serological tests of viruses that affect Equidae.

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Equine coronavirus (ECoV) outbreaks have occurred three times at Obihiro Racecourse in Hokkaido, Japan. The third ECoV outbreak occurred between late February and early April 2012. The main clinical signs of affected horses were anorexia, pyrexia and leucopenia; gastrointestinal disease was observed in about 10% of affected horses. Two ECoV strains were isolated from diarrheal samples. All paired sera (9/9) collected from febrile horses showed seroconversion by neutralization test. Sequence and phylogenetic analysis of the ECoV isolated showed that putative amino acid sequences in S and N genes were highly conserved among ECoV strains. In contrast, sequences of the region coding 4.7kDa non-structural protein (p4.7) differed among the strains. Because of the diversity of the p4.7 region, this region should be useful for epidemiological investigation of ECoV.

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Sexual dimorphism in the craniomandibular traits in the Korean water deer Hydropotes inermis argyropus was examined for the first time. Multivariate analyses using only cranial traits showed a clear separation between sexes. However, the separation was not obvious in the discriminant analysis using only mandibular traits. The most clearly dimorphic trait was in the incisive bone breadth, which was about 12% larger in males. The incisive bone width reflects the characteristically large canines in male. In contrast to this, most of the cranial measurements, except for the incisive breadth, were larger in female, indicating a larger overall skull size. Given that males are generally larger than females, this sexually dimorphic pattern is unique among mammals. We propose that factors, for example, a unique parental care, have influenced the larger skull size in the females of this species.

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The Red-crowned Crane Grus japonensis is an endangered species that has two separate breeding populations, one in the Amur River basin and the other in north and east Hokkaido, Japan. So far, only two (Gj1 and Gj2) and seven (Gj3-Gj9) haplotypes in D-loop of mtDNA were identified in Japan and in the continent, respectively. We obtained feathers from three cranes found in northeast Honshu. The crane in Akita in 2008, which also arrived at west Hokkaido, had a novel haplotype (Gj10). Another crane in Akita in 2009 showed a heteroplasmy (Gj7 and a novel type, Gj12). The third crane in Miyagi in 2010 also showed another type, Gj11. These results suggest that three Red-crowned Cranes appeared in Honshu and west Hokkaido were from the continent.

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