Caspase activation is associated with skeletal muscle differentiation in mouse embryos. We examined the relationship between cardiac myogenesis and cell death using mice hearts at embryonic days (E) 11.5–15.5 and fetal rat heart H9C2 cells. The number of TdT-mediated dUTP nick end labeling (TUNEL)-positive cells increased with fetal age and was much higher than that of single-stranded DNA (ssDNA)- and active caspase-3 (aCasp3)-positive cells. TUNEL and aCasp3 double staining resulted in 3 types of positive cells: TUNEL⁺/aCasp3⁺, TUNEL⁺/aCasp3^- and TUNEL^-/aCasp3⁺. TUNEL⁺/aCasp3^- cells were the most common but lacked morphological features of apoptosis, such as nuclear condensation or fragmentation. The expression of anti-apoptotic factors increased during E11.5–15.5. Furthermore, TUNEL-positive H9C2 cells without nuclear condensation or fragmentation were observed only in myotubes later in the culture period. In this study, the dynamics of TUNEL-positive cardiomyocyte was inconsistent with the activation of apoptosis cascade, and their morphological feature was clearly different from representative apoptosis. From these findings, we concluded that the increased number of TUNEL-positive cardiomyocyte, having the DNA strand breaks, would be associated with the progression of cardiac myogenesis.

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In the olfactory system, G proteins couple to the olfactory receptors, and G proteins expressed in the main olfactory system and vomeronasal system vary according to animal species. In this study, G protein α subunits expressed in the main olfactory system and vomeronasal system of the snake were identified by immunohistochemistry. In the olfactory epithelium, only anti-G_αolf/s antibody labeled the cilia of the receptor cells. In the vomeronasal epithelium, only anti-G_αo antibody labeled the microvilli of the receptor cells. In the accessory olfactory bulb, anti-G_αo antibody stained the whole glomerular layer. These results suggest that the main olfactory system and the vomeronasal system of the snake express G_αolf and G_αo as G proteins coupling to the olfactory receptors, respectively.

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‘Candidatus Mycoplasma haemobos’, sometimes causative of bovine infectious anemia at various extents, has been demonstrated throughout the world. Here, we show two distinct types of ‘Ca. M. haemobos’ are distributed among cattle in Japan, by examining the primary and secondary structures of the 16S-23S rRNA intergenic spacer region that has been shown to be a stable genetic marker for mycoplasma species. Our results may explain differences in severity of anemic condition as well as provide a genetic marker for an epidemiological study of bovine hemoplasma infections.

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This study analyzed molecular-based identification of yeasts that associated with bovine clinical mastitis in Japan. Over 3,200 quarter milk samples from Holstein dairy cows collected in 2011 on Hokkaido and Honshu islands were examined. Yeast isolates were characterized by polymerase chain reaction amplification and sequencing of the D1/D2 region of the 26S rDNA. Molecular characterization confirmed that Candida spp. and Pichia spp. were most frequently isolated species. Our molecular analysis of mastitic milk samples demonstrated the prevalence of Pichia kudriavzevii(22/58) and Candida tropicalis(14/58). In addition, we demonstrated that molecular analysis of the D1/D2 region of the 26S rDNA is a rapid and reliable method for identifying clinically significant yeasts in dairy hygiene, including potentially new or emerging pathogenic species.

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The responses of 64 Holstein cows with milk fever to first treatment with 500 ml of either of 2 intravenous calcium (Ca) solutions, one containing Ca alone (group A, n = 32) or 1 containing Ca, phosphate and magnesium (group B, n = 32), were evaluated by selected clinical signs and serum biochemical data. Based on the cow’s ability to stand, treatment response was categorized into “immediate response” (stood after single treatment), “delayed response” (stood after repeated treatments) and “non-response” (slaughtered despite repeated treatments). No significant differences among the response categories were found between the two groups, suggesting that the solution containing Ca borogluconate alone was sufficient for the first treatment of milk fever.

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GM1 gangliosidosis is a fatal, progressive neurodegenerative lysosomal storage disease caused by mutations in the β-galactosidase (GLB1) gene. In feline GM1 gangliosidosis, a pathogenic mutation (c.1448G>C) in the feline GLB1 gene was identified in Siamese cats in the United States and Japan and in Korat cats in Western countries. The present study found the homozygous c.1448G>C mutation in 2 apparent littermate native kittens in Bangladesh that were exhibiting neurological signs. This is the first identification of GM1 gangliosidosis in native domestic cats in Southeast Asia. This pathogenic mutation seems to have been present in the domestic cat population in the Siamese region and may have been transferred to pure breeds such as Siamese and Korat cats originating in this region.

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Deregulated Wnt signaling pathway is implicated in many hereditary diseases and tumorigenesis including colorectal cancer, hepatocellular carcinoma and gastric cancer. In this study, to assess the relationship between chemically induced gastric tumor and canonical Wnt signaling pathway in genetically intact mice, histopathological and quantitative mRNA analyses were performed in C57BL/6J mice given drinking water containing N-methyl- N-nitrosurea (MNU). 60.5% of gastric adenoma and 27.9% of adenocarcinoma were observed 48 weeks after first administration. Also, in immunohistochemical analysis, aberrant expressions of phospho-GSK-3β, β-catenin, cyclin D1, c-Myc, osteopontin and COX-2 were found. In double immunofluorescent-antibody stains, β-catenin accumulation was colocalized with other proteins. mRNA levels of cyclin D1, c-myc and COX-2 were relatively higher in adenocarcinoma. Altogether, canonical Wnt pathway was highly involved in MNU induced gastric neoplasia of C57BL/6J mice, and it could be a considerably suitable system for the study to examine the linkage between gastric tumorigenesis and the canonical Wnt pathway.

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Feline cases of lung fluke infection were recently reported in the upper basin of the Oi River in Shizuoka Prefecture. The causative species of these cases were not identified, although a field survey conducted about 40 years ago in this area demonstrated the prevalence of Paragonimus miyazakii metacercariae in intermediate host crabs. To clarify the current status of lung fluke metacercarial infection in host crabs, we collected the Japanese freshwater crab or Sawagani, Geothelphusa dehaani, at 14 sampling sites in the Kawane area, which is located in the upper basin of the Oi River. Sawagani harboring Paragonimus metacercariae were collected at all sampling sites in this study with a total prevalence of 42% (281 of 677 crabs examined) and a range of 16% to 92%. The number of metacercariae per infected crab ranged from 1 to 19 with a mean of 2.2. Based on the morphological features of the metacercariae and adult worms recovered from rats that were experimentally infected with the metacercariae, the lung flukes examined were identified as P. miyazakii Kamo, Nishida, Hatsushika and Tomimura, 1961; ITS2 sequence data corroborate this conclusion. P. miyazakii is still widely prevalent in this area, implying that the recently reported feline paragonimiasis cases were likely caused by P. miyazakii infection.

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We used 24 wild boars trapped from December 2009 to January 2010 and a further 65 from July 2010 to August 2010 in Misato Town, Shimane Prefecture, Japan. We collected blood, spleens, skins and ticks from the wild boars, which were examined for rickettsial infections using polymerase chain reaction (PCR) primers for the genes rickettsial 17-kDa antigen and citrate synthase (gltA). We amplified Rickettsia tamurae AT-1 DNA from the tick Amblyomma testudinarium and from wild boar skins where ticks attached. Antibodies against spotted fever group Rickettsia were detected in wild boar sera using immunofluorescence, whereas blood and spleen samples contained no rickettsial DNA. This study suggests that wild boars have a role as an amplifier and a transporter of A. testudinarium, which harbor R. tamurae. One case of R. tamurae infection in humans was reported in Shimane Prefecture. Therefore, R. tamurae infections in humans might increase, if wild boar populations and their habitats expand.

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A robust molecular marker is needed for discrimination of amphistome species, because identification based on morphology alone requires specialized knowledge and techniques. In this study, we performed morphological and molecular characterization of Explanatum explanatum, a species that causes severe liver damage in definitive host species. Fifty-five adult amphistomes were collected from cattle and water buffaloes in Myanmar. Eighteen of the amphistomes, arbitrarily chosen, were morphologically identified as E. explanatum using sagittal sections. All of the 55 amphistome isolates had identical second internal transcribed spacer (ITS2) of ribosomal DNA sequences; these sequences differed at 7 nucleotide sites from those of the closest species, Paramphistomum leydeni. Our data indicate that the ITS2 sequence could be a useful molecular marker for epidemiological studies on E. explanatum.

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We investigated for the first time the prevalence of avian haemosporidia of genera Plasmodium, Haemoproteus and Leucocytozoon among birds and mosquitoes on Tsushima Island of Japan, which is located between Japan and the Korean Peninsula. Of 55 wild birds belonging to 33 species, 16 (29.1%) tested positive for haemosporidia as follows: Plasmodium spp. (11/55; 20.0%); Haemoproteus spp. (2/55; 3.6%); and Leucocytozoon spp. (3/55; 5.5%). A genetic lineage isolated from the Eurasian Sparrowhawk (Accipiter nisus) was identical to that of the known avian malaria parasite P. circumflexum. Several genetic lineages were identical or closely related to the parasite lineages that were previously detected in birds and mosquitoes in Japan and Korea. Another single identical genetic lineage was also detected in both migratory and resident birds. A total of 753 mosquitoes from 12 species were collected; and one fully fed Aedes albopictus was positive for avian Plasmodium(1/753; 0.13%) which is identical to a genetic lineage detected in both mosquitoes in Japan and birds in Korea. Blood-meal identifications of blood-fed mosquitoes showed direct contact between the mosquitoes and 4 species of mammals including humans, cattle, rodents and the endangered Tsushima leopard cat (Prionailurus bengalensis euptilura). Migratory birds use Tsushima Island as a site for wintering, breeding and resting, and our results suggest the transmission of avian haematozoa between resident and migratory birds during their stay on Tsushima Island.

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A dog was suspected of suffering from ectopic Dirofilaria immitis infection, because a large white nematode worm was detected in the anterior chamber of the left eye. A cylinder-shaped fibrin sac in the anterior chamber was found in the eye of the dog by slit lamp microscopy. After successful surgical removal of the worm, the corneal wound produced by the keratotomy healed in a short period. The worm was estimated to be extremely young, 5th-stage-immature male D. immitis, equivalent to a 90–120-day-old worm postinfection, by close morphological measurement and an experimental infection study. Thus, an immature worm can exhibit erratic parasitism in a host’s eye. The fibrin sac was considered to be a trace of the invasion route, and the cornea may have been the port of entry into the anterior chamber of the eye in the erratic migration of D. immitis.

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Canine polyneuropathy is a neurological disorder characterized by a dysfunction of multiple peripheral nerves. The etiology of the disease is diverse; it may occur in cases of infectious, immune-mediated, or hereditary conditions or in association with endocrinopathy, neoplasm, or chemical intoxication. It is often difficult to determine the etiology through clinical symptoms. The aim of this study is to investigate pathological differences among three canine polyneuropathy cases with each presumably having a different etiology. Cases included a 13-month-old female border collie (Dog No.1), a 21-month-old male chihuahua (Dog No.2) and an 11-year-old male beagle (Dog No.3). Clinical examinations revealed hindlimb ataxia and sensory loss in Dog No.1, forelimb paralysis and vertebral pain in Dog No.2, and paddling-gait and hypothyroidism in Dog No.3. Histopathologically, axonal swelling and pale myelin were observed in Dog No.1. Giant axons mimicking giant axonal neuropathy were obvious in Dog No.2. Dog No.3 showed atrophic axons and severe interstitial edema. Distributions of peripheral nerve lesions coincided with respective clinical symptoms. According to their clinical and pathological features, Dogs No.1 and No.2 were suspected of hereditary polyneuropathy, while Dog No.3 seemed to have hypothyroidism-associated polyneuropathy. As each case demonstrated unique pathological features, different pathogeneses of peripheral nerve dysfunction were suggested.

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A 20-year-old Gentoo penguin was found dead with a clinical history of inappetence and dyspnoea. At necropsy, the lungs showed severe congestion/hemorrhage and atelectasis. Histopathologically, fibrosis was observed exclusively around parabronchi with severe collagen deposition. In fibrotic lesions, there were numerous depositions of crystalline structures accompanied by epithelioid cells and multinucleated giant cells (foreign body type). In addition to irregularly lamellar structures as the morphology, the crystals were demonstrated calcium oxalate (CaOx) by the Alizarin red S staining with and without polarized light and von Kossa’s staining. Myocobacteria and fungi were not found by special and immuohistochemical stainings. Pulmonary dystrophic oxalosis is a very rare lesion in Gentoo penguin.

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A 7-year-old spayed female domestic short-haired cat presented with dysuria and hematuria that had been unresponsive to medical therapy. Imaging tests such as ultrasonography, urethrocystography and computed tomography revealed a pelvic mass compressing the urethra. Based on histological examination of the mass following surgical resection, the cat was diagnosed squamous cell carcinoma (SCC) derived from the uterine remnant. After surgery, dysuria was resolved, but on instead, urine and fecal incontinence were observed. Then, about four months after surgery, recurrence of the mass and the symptoms was observed. Consequently, the cat was ultimately euthanized. This is the first report of SCC arising from the uterine remnant in a spayed female cat.

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Proteinase-activated receptor (PAR) is expressed on various cells, and the PAR family consists of PAR1, PAR2, PAR3, and PAR4. Individual PARs are activated during inflammatory conditions in which they regulate inflammatory responses in various diseases. For example, PAR activation is known to induce prostaglandin E₂ (PGE₂) production, and then upregulated PGE₂ suppresses PAR1 expression in a negative feedback loop. Surprisingly, PGE₂ effects on PAR2, which is a well-researched and attractive target for drug development, remain unknown. Therefore, we investigated PAR2 regulation by PGE₂. Using HEK293T cells, we showed that PGE₂ inhibits extracellular signal-regulated kinase (ERK) phosphorylation induced by a PAR2-activating peptide (PAR2-AP). AH-6809 (an inhibitor of PGE₂ receptors 1 [EP1] and 2 [EP2]), but not ONO-AE3-208 (a PGE₂ receptor 4 [EP4] inhibitor), reversed the inhibitory effects of PGE₂ on PAR2-AP-induced ERK phosphorylation. Studies on PAR2 expression revealed that PGE₂ suppressed cell surface expression of PAR2 and induced internalization of PAR2, and not PAR4, in N2a mouse neuroblastoma cells that were transiently transfected with either PAR2 or PAR4. Furthermore, forskolin, an adenylate cyclase activator, induced PAR2 internalization and inhibited PAR2-AP-induced phosphorylation of ERK. Because EP2 (not EP1) also increases intracellular cyclic AMP, we conclude that PGE₂ inhibited PAR2-dependent signal transduction by inducing the internalization of PAR2 through an EP2-dependent increase in intracellular cyclic AMP. This novel regulatory pathway in which PAR2 function is regulated by PGE₂ will broaden our understanding of PAR2-dependent inflammation and could provide novel strategies for drug development.

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Lymphoma is one of the most common malignant tumors in canine. Chemotherapy results in a high rate of remission; however, relapse and clinical drug resistance are usually seen within a year. Protein phosphatase 2A (PP2A) acts as a tumor suppressor and plays a critical role in mammalian cell transformation. Increased protein levels of SET, endogenous PP2A inhibitor, have been reported to correlate with poor prognosis in human leukemia. Here, we test the potential therapeutic role for a SET antagonist in canine lymphoma. We observed SET protein levels increased in multiple canine lymphoma cell lines compared with primary peripheral blood cells. A novel SET antagonist OP449 increased PP2A activity and effectively killed SET high-expressing canine lymphoma cells, but not SET low-expressing cells. Caspase-3 activation and enhanced Annexin V positive staining were observed after OP449 treatment, suggesting apoptotic cell death by OP449. Consistent with this, pan-caspase inhibitor Z-VAD-FMK blocked OP449 induced cell death. These data demonstrated the potential therapeutic application of SET antagonists for canine lymphoma.

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Interaction between acetylcholine receptor-operated potassium channel (K.ACh channel) and GTP binding protein was examined by an immunoprecipitation-Western blotting system in mouse isolated atrium. The carbachol-induced negative inotropic action in indomethacin-pretreated mouse atrium was significantly inhibited by a K.ACh channel blocker, tertiapin or atropine. Kir3.1 K.ACh channel (Kir3.1) was immunoprecipitated with a mouse anti-Kir3.1 antibody. Coprecipitating Gβ with Kir3.1, detected by Western blotting, was significantly augmented by carbachol. Atropine, but not tertiapin, significantly inhibited the carbachol-induced coprecipitating Gβ with Kir3.1. The data indicate that immunoprecipitation with Kir3.1 and Western blotting of Gβ system is a useful method for assessing interaction between K.ACh channel and GTP binding protein in mouse atrium.

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The aim of the study was to determine interrelationships between bone tissue metabolism indices and morphological, biomechanical and densitometric properties of hard dental tissues. First primary maxillary incisor from 6-month-old pigs (N=27) was evaluated in terms of weight and length. Mean volumetric tooth mineral density, total tooth volume, enamel total volume, enamel volumetric mineral density, dentine total volume and dentine volumetric mineral density were estimated with the use of quantitative computed tomography and micro computed tomography techniques. Tooth mineral density and tooth mineral content were evaluated with the use of dual-energy X-ray absorptiometry. Microhardness of enamel was measured using Vicker’s test. Evaluations of total calcium, ionized calcium, magnesium, phosphorus, alkaline phosphatase, bone alkaline phosphatase, osteocalcin, C-terminal telopeptide of type-I collagen (CTX), insulin-like growth factor-1, growth hormone and parathyroid hormone were performed in plasma and serum samples. Pearson’s correlation coefficients were determined between all the investigated variables, and P<0.05 was considered as statistically significant. The obtained results have shown mainly mutual dependences between biochemical indicators of bone metabolism. Evaluation of CTX concentration in serum of pigs has shown the highest predictive value in relation to morphological, densitometric and biomechanical properties of teeth.

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It is well established that growth hormone (GH) is secreted in a pulsatile manner. Although the GH pulse-generating mechanism is not fully understood, we have previously reported that neuropeptide Y (NPY) profiles in the cerebrospinal fluid were negatively correlated with serum GH pulses. In addition, it is known that photic stimulation suppresses GH pulses for a certain period of time. In the present study, to investigate the involvement of NPY in regulating GH pulse generation, NPY gene expression in the arcuate nucleus (ARC) of the hypothalamus in rats was analyzed at around the lights on. First, we confirmed that GH pulses did not occur for around 1.5 hr after the start of the light phase. Then, we analyzed the activity of neurons and expression of NPY mRNA 1 hr before and 0.5 and 2 hr after lights on. Both the activity of neurons, which was evaluated by immunohistochemical detection for phosphorylated-cAMP response element binding protein (pCREB), and NPY mRNA levels in the caudal ARC were higher at 0.5 hr after lights on than the other two time points, while pCREB-positive cell numbers in the rostral ARC remained unchanged throughout the experimental period. In addition, NPY immunoreactivity in the periventricular nucleus (PeVN) was also higher at 0.5 hr after lights on than the other time points. These results suggest that NPY neurons in the caudal ARC projecting to the PeVN play a role in inhibiting GH pulses at the commencement of the light phase.

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In poultry, the benefits of probiotic supplementation on broiler performance and health have been reported. The present study examined the effect of supplemental yeast probiotic preparations on body weight, thyroid hormone metabolism and serum lipid profile in broilers. The experiment was carried out on hybrid Marshall broiler chickens (n=200). Two hundred 1-day-old chicks were randomly selected and distributed into 4 groups of 50 1-day-old chicks each (control, C; E₁0.5%, E₂1.5% and E₃2.0%, experimental groups). The birds were housed in an environmentally controlled poultry house with the floor covered with wood shavings. Chicks were fed commercial broiler starter diet for the first 28 days of age, followed by pelleted finisher diet from 29–42 days. Feed and water were provided ad libitum. Chickens fed 2.0% probiotic had a significantly (P<0.05) higher body weight when compared with the control group. The blood glucose concentration was significantly (P<0.05) different in the E₃1.5% probiotic supplemented group when compared with the control. There was highly significant (P<0.05) difference in T₄ level in the E₃2.0% probiotic group when compared with the control. However, there was a significant difference in cholesterol concentrations observed in the experimental group supplemented with E₁0.5% probiotic. The concentration of triglycerides was not affected by the probiotic supplementation. Significant differences (P<0.01) were observed in HDL concentrations in all experimental groups supplemented with the probiotic when compared with the control group. In conclusion, supplementing broiler feeds with a yeast probiotic increased body weight, enhanced thyroid hormone metabolism, and decreased cholesterol and lipoproteins in broiler chickens.

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Perineal hernia in dogs is very problematic and mostly requires surgical reconstruction of the weak pelvic diaphragm. Tissue or synthetic grafts have been used for the correction after failure of the conventional herniorrhaphy. Aim of this clinical trial was to assess the possible use of the autologous tunica vaginalis communis as a free graft for perineal hernia repair in intact male dogs. Seven unilateral and 2 bilateral perineal hernias in nine intact male dogs free from testicular and scrotal neoplasms were included in this study. The median surgical time for unilateral herniorrhaphy was 75 min. The median follow-up time was 13 months. The success of the autografting, based on no recurrence and comfort of the animals during urination and defecation, was found in ten of 11 hernias; giving a success rate of 90.91%. One hernia (9.09%) recurred 10 days after surgery. Histopathological examination of the apposing area between the graft and the adjacent tissue, taken during the repair of the recurred case at day 20, revealed neovascularization and connective tissue ingrowth. In conclusion, the tunica vaginalis autograft can be used for perineal herniorrhaphy in intact male dogs.

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Equine bone scintigraphy is usually performed with horses in standing position under sedation. However, swaying motion often leads to poor-quality images. To examine the usefulness of motion correction (MC) processing, equine bone scintigrams were evaluated using Scheffé’s method of paired comparisons. A significant difference in evaluation scores was detected by analysis of variance (F test, P<0.01). According to all observers, Yardstick analysis scores were higher for images use of MC processing than for those no use of MC processing, for all parts. Overall scores of 5 observers were as follows: without MC 100% acquisition time (AT, lowest), use of MC with 25% AT, MC 50% AT, MC 75% AT and MC 100% AT (highest). Thus, MC processing shortens AT in equine bone scintigraphy, and it contributes to a reduction in the external radiation exposure of nurses/technicians.

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We evaluated transcervical insemination (TCI) using frozen semen by flexible endoscopy in dogs. Eight female and eight male beagles were used in this study. A flexible endoscope and a washing tube were used for TCI. A tracheal tube was used as an alternative to the penis and was an auxiliary for inserting the flexible endoscope. The mean time required to insert the washing tube into the external os of the uterus after inserting the endoscope into the tracheal tube was 7.5 min. Slight or mild pain was observed in all bitches during TCI. However, TCI could be easily performed with retention in all bitches and without sedation anesthesia. The tracheal tube was useful to ensure the visual field using air sufflation. Clinical signs suspicious of infection were not observed in any bitches from the TCI to the pregnancy diagnosis. The conception rate was 87.5%, and the mean number of fetuses was 6.3. TCI using a flexible endoscope in bitches was performed quickly with minimal invasiveness. We present a new method of TCI in dogs. This method should be studied in small and large breeds to obtain more detailed results.

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Changes in ovarian structures and hormonal profiles in estradiol dipropionate (EDP)-induced pseudopregnant sows following PGF_2α-analogue (PGF_2α-A) administration and practicality of the estrus synchronization protocol using EDP and PGF_2α-A on estrus expression and reproductive performance in commercial conditions were investigated. Pseudopregnancy was defined as absence of estrus maintained for at least 20 days after EDP treatment in this study. When 4 pseudopregnant sows induced by 20 mg EDP were treated with PGF_2α-A as 0.175 mg cloprostenol twice at a 24-hr interval between 20 and 28 days after EDP treatment, plasma progesterone concentrations rapidly decreased after treatment. The luteinizing hormone surge and ovulation were detected in all sows. The number of ovulated follicles was 17.3 ± 1.1 (SEM). On commercial farms, 94.2% of 52 gilts and 95.2% of 21 sows received EDP became pseudopregnant. When these pseudopregnant females (48 gilts and 20 sows) were treated with PGF_2α-A as described above, estrus was detected in all females at 6.1 ± 0.3 days for gilts and 5.5 ± 0.2 days for sows after the first PGF_2α-A treatment. There were no significant differences in farrowing rate (85.0 - 100%), average total litter size (10.0 - 11.4), average born alive litter size (9.4 - 10.3) and average piglet birth weight (1.56 - 1.71 kg) between PGF_2α-A treated pseudopregnant female pigs that were inseminated during synchronized estrus and females inseminated during spontaneous estrus. This study indicates that estrus synchronization programs using EDP and PGF_2α-A are available as practical and convenient procedures for commercial pig farms.

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