Hydroxyapatite (HA) has polarization capability and is able to accumulate electrical storage in bone. Experiments were conducted to measure the polarization capability of rabbit femurs. After preparing and polarizing bone samples using 2% KOH treatment (denoted 2% koh), 2% KOH and baking (2% koh+bake) and decalcification (decalcification) as well as untreated bone (untreated), stored charges were quantitatively determined using thermally stimulated depolarization current (TSDC) measurements. In TSDC spectra, untreated and 2% koh samples showed peaks at 100 and 500°C, while 2% koh+bake showed one peak at 580°C and decalcification one peak around 100°C. These evidences indicated that collagen and inorganic components play a major role in polarization of the bone at different temperature conditions.

View full abstract

In this study, age-dependent histological changes in the kidneys of Brown Norway rat, a strain useful for conducting aging research, were evaluated. Examination was performed at 3, 12, 18, 24 and 30 months of age. Sclerotic and hypertrophic changes of the glomeruli were observed, and quantitative scores of these changes persistently increased with age. A marginal increase in scores was observed for glomerular cystic changes and tubulointerstitial damage. Further, urothelial hyperplasia was observed in the renal papillae, particularly at 30 months of age. In conclusion, the findings of the present study demonstrate that the Brown Norway strain exhibits persistent, but mild progression of age-dependent renal histological changes.

View full abstract

The present study aimed to determine the complete citrate synthase (gltA) and heat-shock protein (groEL) gene sequences of Anaplasma bovis and to infer phylogenetic relationships within the genus Anaplasma. Multiple alignments from single and concatenated sequences of the 16S rRNA, gltA and groEL genes of the genus Anaplasma were subjected to phylogenetic analyses. Percent identities of A. bovis nucleotide sequences were found highest with A. phagocytophilum in gltA (65.4%) and groEL (79.8%). Single gene phylogenetic tree results assumed similar phylogenetic positions within the genus Anaplasma, except for A. bovis. However, consensus and concatenated sequence phylogenetic trees showed similar results, revealing 2 subgroups within the genus.

View full abstract

This study aimed to investigate and compare the antagonistic effects of atipamezole, yohimbine and prazosin on medetomidine-induced diuresis in healthy cats. Five cats were repeatedly used in each of the 9 groups. One group was not medicated. Cats in the other groups received 40 µg/kg medetomidine intramuscularly and saline (as the control), 160 µg/kg prazosin, or 40, 160 or 480 µg/kg atipamezole or yohimbine intravenously 0.5 hr later. Volume, pH and specific gravity of urine; plasma arginine vasopressin (AVP) level; and creatinine, osmolality and electrolyte levels in both urine and plasma were measured. Both atipamezole and yohimbine, but not prazosin, antagonized medetomidine-induced diuresis. The antidiuretic effect of atipamezole was more potent than that of yohimbine, but was not dose dependent, in contrast to the effect of yohimbine at the tested doses. Both atipamezole and yohimbine reversed medetomidine-induced decreases in both urine specific gravity and osmolality and increases in plasma osmolality and free-water clearance. Antidiuresis of either atipamezole or yohimbine was not related to the area under the curve for AVP level, although the highest dose of both atipamezole and yohimbine initially and temporarily increased plasma AVP levels, suggesting that this may partly influence the antidiuretic effects of both agents. The diuretic effect of medetomidine in cats may be mediated by α₂-adrenoceptors, but not α₁-adrenoceptors. Atipamezole and yohimbine can be used as antagonistic agents against medetomidine-induced diuresis in healthy cats.

View full abstract

Eight Holstein calves (10 ± 3 weeks) were used to examine the interaction between a bacteria-based probiotic agent (probiotic) and the function of peripheral blood mononuclear cells (PBMCs). The probiotic, consisting of Lactobacillus plantarum, Enterococcus faecium and Clostridium butyricum, was administered orally at 3.0 g/100 kg body weight to calves once daily for 5 consecutive days. Calves given the vehicle alone with no probiotic served as the control. In the treatment group, increases in numbers of CD282⁺ (TLR2) monocytes, CD3⁺ T cells and CD4⁺, CD8⁺ and WC1⁺ γδ T cell subsets were noted on day 7 post-placement compared to predose day and the control group. Expression of interleukin (IL)-6, interferon-gamma (INF-γ) and tumor necrosis factor-alpha (TNF-α) was elevated in peripheral leukocytes on days 7 and 14. These results suggest that peripheral blood leukocytes in healthy calves may be stimulated via the gastrointestinal microbiota, which was increased by the oral probiotic treatment, with overall stability of the rumen bacterial flora. The 5-day repeated administration of a bacteria-based probiotic may enhance cellular immune function in weaned calves.

View full abstract

Treatments for canine lymphoma often fail, because tumor cells acquire multidrug resistance (MDR). MDR can develop through several mechanisms, among which the overexpression of drug transporters in tumor cells is a well-studied mechanism. ATP-binding cassette sub-family G member 2 (ABCG2) belongs to the ABC-transporters, that are representative drug efflux pumps associated with MDR in human tumor cells. However, the regulation of ABCG2 gene expression in canine tumors is not well understood. The purpose of the present study was to reveal the regulatory mechanism of ABCG2 gene expression in 4 canine lymphoid tumor cell lines, GL-1, CLBL-1, UL-1 and Ema. Treatment with phorbol 12-myristate 13-acetate (PMA), the protein kinase C (PKC) activator, stimulated MAPK/ERK pathway in GL-1, UL-1 and Ema cells and JNK pathway in UL-1 and Ema cells. When GL-1 and UL-1 cells were treated with PMA and the MAPK/ERK kinase inhibitor U0126, ABCG2 gene expression levels were elevated above those in untreated cells. Similarly, ABCG2 gene expression increased above control levels in UL-1 and Ema cells treated with PMA and the JNK inhibitor SP600125. However, ABCG2 gene expression was unaffected by U0126 exposure in CLBL-1 cells, in which activation of MAPK/ERK pathway was observed in non-treated cells. These results suggested that MAPK/ERK and JNK pathways downregulate ABCG2 gene expression, which is upregulated by unidentified but possibly PKC-dependent pathways, in several types of canine lymphoid tumor cells.

View full abstract

Antimalarial drugs, dihydroartemisinin (DHA) and artesunate (ATS), exhibit iron-dependent cytotoxicity in tumor cells. We hypothesized that erythrophagocytic uptake of heme-iron enhances the cytotoxicity of DHA and ATS. Erythrophagocytic (EP) treatment of the canine histiocytic sarcoma cell line DH82 markedly increased the cytotoxicity of DHA and ATS compared to controls. Succinyl acetone, an inhibitor of intracellular heme synthesis, decreased the cytotoxicity of DHA and ATS in normal cells, but this change was not observed in EP cells. These results suggest that exogenous heme derived from erythrocytes can enhance the cytotoxicity of DHA and ATS. Furthermore, our study suggests that heme could be a novel component of tumor treatment in veterinary medicine.

View full abstract

The objectives of this study were to set specific dog breed and sex standards for total cholesterol (T-Cho) and total triglyceride (T-TG) concentrations in dogs and to quantify the associations between dog age and concentrations of both lipids for different breeds. Increased age was associated with higher T-Cho and T-TG concentrations in all five breed groups (P<0.05); T-Cho concentrations increased by 62.5 mg/dl between 9 and 16 years of age, and T-TG concentrations increased by 4.8 mg/dl per year of age (P<0.05). Miniature Schnauzers had the highest T-Cho concentrations of the studied breeds, while Miniature Dachshunds had the lowest concentrations (P<0.05). Veterinarians should consider dog age and breed when they use the lipid concentrations for diagnostic purposes.

View full abstract

GM2 gangliosidosis variant 0 (Sandhoff disease, SD) is a fatal, progressive neurodegenerative lysosomal storage disease caused by mutations of the HEXB gene. In canine SD, a pathogenic mutation (c.283delG) of the canine HEXB gene has been identified in toy poodles. In the present study, a TaqMan probe-based real-time PCR genotyping assay was developed and evaluated for rapid and large-scale genotyping and screening for this mutation. Furthermore, a genotyping survey was carried out in a population of toy poodles in Japan to determine the current mutant allele frequency. The real-time PCR assay clearly showed all genotypes of canine SD. The assay was suitable for large-scale survey as well as diagnosis, because of its high throughput and rapidity. The genotyping survey demonstrated a carrier frequency of 0.2%, suggesting that the current mutant allele frequency is low in Japan. However, there may be population stratification in different places, because of the founder effect by some carriers. Therefore, this new assay will be useful for the prevention and control of SD in toy poodles.

View full abstract

Mycoplasma pulmonis is one of the most prevalent bacterial pathogens that infects laboratory mice and rats. To develop an M. pulmonis-specific antigen for serological diagnosis, we cloned the cDNA of P46-like lipoprotein (P46L), an M. pulmonis putative periplasmic protein. P46L is a homolog of P46, an M. hyopneumoniae antigen. We produced recombinant P46L fused to glutathione S-transferase (GST) in Escherichia coli. Immunoblot analysis revealed that sera from Mycoplasma-infected mice and rats contained anti-P46L antibodies. We developed an ELISA using the recombinant P46L-GST protein as an antigen. Thirteen of the 14 samples from rats naturally infected with M. pulmonis were determined to be positive according to the commercial ELISA (MONILISA Myco) and positive by our ELISA. Furthermore, 18/19 samples from mice experimentally infected with M. pulmonis were positive using our P46L-GST ELISA. In contrast, only 8/19 samples from infected mice were positive by the commercial ELISA. Our results indicate that P46L-GST was an appropriate antigen for developing a serological test to determine M. pulmonis infection in laboratory mice and rats.

View full abstract

Cryptosporidium andersoni and Cryptosporidium muris infections have been found in the mice and/or cattle. The oocysts of C. andersoni and C. muris have been sporadically detected in human feces, but the infectious capacity and features have been unknown, because of the scarcity of reports involving human infections. To assess the infectivity and the clinical and pathological features of C. andersoni and C. muris in primates, an experimental infectious study was conducted using cynomolgus monkeys. The monkeys were orally inoculated with oocysts of two different C. andersoni Kawatabi types and C. muris RN-66 under normal and immunosuppressive conditions. The feces of the monkeys were monitored for about 40 days after the administration of oocysts using the flotation method, but no shedding oocysts were observed under either both normal or immunosuppressive conditions. Gross and histopathological examinations were performed on the immunosuppressive monkeys, but these revealed no evidence of Cryptosporidium infections, even though the monkeys were subjected to immunosuppressive conditions. It is hypothesized that C. andersoni and C. muris pose little danger of infection in primates even under immunosuppressive conditions.

View full abstract

The prevalence of Hypoderma spp. in yaks grazed in the east of Qinghai province was investigated in 2008. In this area, the prevalence in young yaks (1- to 3-year-old) was very high at 82.2–98.7%, whilst in adult yaks (4-year-old and older), the prevalence was 42.4–50.6%. The seasonal development and migration pattern of Hypoderma larvae in yak bodies was found to be similar for different locations in this area. The numbers of first, second and third instar larvae detected in yak bodies peaked in October, December and March, respectively. Different doses of ivermectin (125 to 500 µg/kg body weight) almost completely dewormed the larvae from yaks, suggesting that using a quarter of the prescribed dose (500 µg/kg body weight) was effective. In October of each year between 2009 and 2012, ivermectin (125 µg/kg body weight) was administered to a total of 562,995 yaks grazed in four counties in Qinghai province, and the pevalence of Hypoderma larval infection in yaks was reduced to 0.5–1.0%.

View full abstract

Toxoplasmosis, an important zoonosis, can be transmitted by eating meat or drinking milk of animals infected with Toxoplasma gondii. Samples were collected from 238 donkeys in the year 2010 in Italy, which included 207 females and 31 males of five breeds and crossbreeds with the average age 9 years (1 month−24 years). Sera were tested for T. gondii antibodies using a latex agglutination test and the indirect fluorescent antibody test; 5 and 8% seropositivity were recorded, respectively. We found significant correlation between the presence of T. gondii antibodies and sex, age, grazing and presence of cats on the farms and their access to donkey feed. This is the first detection of T. gondii antibodies in donkeys in Italy.

View full abstract

We describe a case of human Becker muscular dystrophy (BMD)-like myopathy that was characterized by the declined stainability of dystrophin at sarcolemma in a pig and the immunostaining for dystrophin on the formalin-fixed, paraffin-embedded (FFPE) tissue. The present case was found in a meat inspection center. The pig looked appeared healthy at the ante-mortem inspection. Muscular abnormalities were detected after carcass dressing as pale, discolored skeletal muscles with prominent fat infiltrations and considered so-called “fatty muscular dystrophy”. Microscopic examination revealed following characteristics: diffused fat infiltration into the skeletal muscle and degeneration and regeneration of the remaining skeletal muscle fibers. Any lesions that were suspected of neurogenic atrophy, traumatic muscular degeneration, glycogen storage disease or other porcine muscular disorders were not observed. The immunostaining for dystrophin was conducted and confirmed to be applicable on FFPE porcine muscular tissues and revealed diminished stainability of dystrophin at the sarcolemma in the present case. Based on the histological observations and immunostaining results, the present case was diagnosed with BMD-like myopathy associated with dystrophin abnormality in a pig. Although the genetic properties were not clear, the present BMD-like myopathy implied the occurrence of dystrophinopathy in pigs. To the best of our knowledge, this is the first report of a natural case of myopathy associated with dystrophin abnormalities in a pig.

View full abstract

A 6-year-old Thoroughbred gelding was euthanized after a 2-month period of abnormal neurological signs, such as circling left in his pen and hitting his head and body against the wall. After the horse was euthanized on the farm, a half of the brain and whole blood were submitted for diagnostic tests. Histopathological examination of the brain revealed granulomatous and eosinophilic meningoencephalitis with numerous intralesional nematodes, predominantly affecting the cerebrum. Multifocal malacic foci were scattered in the brain parenchyma. The intralesional parasites were identified as Halicephalobus gingivalis by morphological features and PCR testing. To the best of our knowledge, this is the first report of meningoencephalitis caused by H. gingivalis in the horse in Korea.

View full abstract

We have previously suggested that activation of the hypothalamic-pituitary-adrenal (HPA) axis is dependent on cyclooxygenase (COX)-2-related signaling under infectious and restraint stresses, but less dependent on it under hypoglycemic stress. In the present study, we evaluated the neuronal activity in the brain to elucidate the possible mechanisms underlying a stress-specific relevance between COX-2-related signaling and activation of the HPA axis under infectious (lipopolysaccharide, LPS), hypoglycemic (2-deoxy-D-glucose, 2DG) and restraint (1 hr) stress conditions. The number of c-Fos-immunoreactive (IR) cells in several brain regions including the paraventricular nucleus (PVN) and supraoptic nucleus (SON) was increased at 120 min after application of all stress stimuli. The number of c-Fos-IR cells at 30 min was increased only by 2DG in the SON, but not in the PVN. In the PVN, a selective COX-2 inhibitor (NS-398) did not affect the number of c-Fos-IR cells at any time points. On the other hand, in the SON, NS-398 increased c-Fos-IR cells at 30 min after LPS and restraint stresses, but not after 2DG injection. These results suggest that, among the brain regions responding to acute stresses, the PVN and SON are commonly activated under three acute stresses. In addition, it is also suggested that COX-2-related signaling decreases neuronal activity in the SON under infectious and restraint, but not hypoglycemic, stresses, which may be involved in the suppression of the HPA axis.

View full abstract

We investigated biodegradability and new bone formation after implantation of tetrapod-shaped granular artificial bone (Tetrabone^®) or β-tricalcium phosphate granules (β-TCP) in experimental critical-size defects in dogs, which were created through medial and lateral femoral condyles. The defect was packed with Tetrabone^® (Tetrabone group) or β-TCP (β-TCP group) or received no implant (control group). Computed tomography (CT) was performed at 0, 4 and 8 weeks after implantation. Micro-CT and histological analysis were conducted to measure the non-osseous tissue rate and the area and distribution of new bone tissue in the defect at 8 weeks after implantation. On CT, β-TCP was gradually resorbed, while Tetrabone^® showed minimal resorption at 8 weeks after implantation. On micro-CT, non-osseous tissue rate of the control group was significantly higher compared with the β-TCP and Tetrabone groups (P<0.01), and that of the β-TCP group was significantly higher compared with the Tetrabone group (P<0.05). On histology, area of new bone tissue of the β-TCP group was significantly greater than those of the Tetrabone and control groups (P<0.05), and new bone distribution of the Tetrabone group was significantly greater than those of the β-TCP and control groups (P<0.05). These results indicate differences in biodegradability and connectivity of intergranule pore structure between study samples. In conclusion, Tetrabone^® may be superior for the repair of large bone defects in dogs.

View full abstract

The present study evaluated and compared the oxidative stress status of dogs undergoing laparoscopic or open ovariectomy. Twelve healthy female dogs were divided into two groups according to the type of the surgical procedure, laparoscopic or open ovariectomy. Plasma total oxidant status (TOS), total antioxidant status (TAS) and oxidative stress index (OSI) levels for the evaluation of oxidative stress were determined. Increases in plasma TOS and OSI levels and decreases in TAS levels were observed in both groups after surgery. The TOS level was significantly lower in the laparoscopic ovariectomy group compared with the open surgery group. Laparoscopic ovariectomy is a safe and beneficial surgical alternative to traditional ovariectomy with respect to oxidative stress status in dogs.

View full abstract

The purpose of this study was to evaluate the gene expression of growth factors and growth factor receptors of primary hepatic masses, including hepatocellular carcinoma (HCC) and nodular hyperplasia (NH), in dogs. Quantitative real-time reverse transcriptase-polymerase chain reaction was performed to measure the expression of 18 genes in 18 HCCs, 10 NHs, 11 surrounding non-cancerous liver tissues and 4 healthy control liver tissues. Platelet-derived growth factor-B (PDGF-B), transforming growth factor-α, epidermal growth factor receptor, epidermal growth factor and hepatocyte growth factor were found to be differentially expressed in HCC compared with NH and the surrounding non-cancerous and healthy control liver tissues. PDGF-B is suggested to have the potential to become a valuable ancillary target for the treatment of canine HCC.

View full abstract

The extracellular matrix of the cervix that comprises collagen, elastin, proteoglycans and glycosaminoglycans (GAGs) is thought to have an essential role in cervical relaxation. This study investigated the proportion of collagen and smooth muscle as well as the GAGs in cervices obtained from healthy bitches at different stages of the estrous cycle and bitches with open- and closed-cervix pyometra. Cervices were collected after ovariohysterectomy. The proportion of collagen to smooth muscle was determined using Masson’s trichrome staining. Alcian blue staining was used to evaluate the relative distribution of cervical GAGs. The proportion of cervical collagen relative to smooth muscle was higher at estrus compared to anestrus (P≤0.05). It was also higher (P≤0.05) in bitches with open- compared to those with closed-cervix pyometra. Overall, hyaluronan (HA) was the predominant GAG in the canine cervix. In the luminal epithelium, the staining intensity for HA was stronger in estrus than in anestrus (P≤0.05), but not in diestrus (P>0.05). On the contrary, the intensity for the combined keratan sulfate (KS) and heparan sulfate (HS) was stronger in anestrus than in estrus and diestrus (P≤0.05). In bitches with pyometra, the staining intensity of the stroma for KS and HS was weaker in open- compared to closed-cervix pyometra (P≤0.05). Collectively, the different profiles of collagen and GAG suggest that the metabolism of both collagen and GAGs in the canine cervix is associated with hormonal statuses during the estrous cycle and cervical patency of bitches with pathological uterine conditions, such as pyometra.

View full abstract

The objective of the present study was to determine an optimum temperature and extender for short-term transport of canine ejaculated semen. There was no significant difference in the qualities of semen diluted with two kinds of extender, egg yolk Tris-citrate fructose (EYT-FC) or glucose (EYT-GC) extender, between the 2, 8 or 12 and the 4°C control groups during storage for up to 48 hr, while the 16–24°C groups showed decreased sperm motility during storage for 48 hr. However, the 2°C group showed slightly lower sperm motility and slightly higher sperm abnormality than the 4°C group. Therefore, we concluded that semen qualities can be maintained for up to 48 hr when canine semen samples are extended with EYT-FC or EYT-GC and stored at a temperature in the range of 4–12°C.

View full abstract

Maedi/visna (MV) is a lentiviral disease of sheep caused by the maedi/visna virus (MVV). Although MV is prevalent in many countries, it had not been reported in Japan. In 2011, however, three sheep in northern Japan were reported to be seropositive against the MVV antigen, indicating a persistent MVV infection. In the present study, we isolated MVV from one sheep to confirm MVV infection and conducted genomic classification of the virus. The co-culture of leukocytes from a seropositive sheep with fetal goat lung cells resulted in the formation of syncytial cells and the amplification of a long terminal repeat sequence of MVV by polymerase chain reaction. The isolate was confirmed as being MVV, rather than the caprine arthritis-encephalitis virus based on phylogenetic analysis of the gag gene sequence. Although the sheep was asymptomatic, nonpurulent meningitis and demyelination were found in the spinal cord. These were considered to be early lesions associated with pathogenic MVV infection. Therefore, the present study demonstrated that MVV is distributed in Japan.

View full abstract

Here, we used a sheep bioassay to determine the effect of freezing colostrum to prevent the transmission of bovine leukemia virus (BLV) among neonatal calves. Leukocytes were isolated from the colostrum of a BLV-infected Holstein cow and were then either left untreated (control) or freeze-thawed. A sheep inoculated intraperitoneally with the untreated leukocytes was infected with BLV at 3 weeks after inoculation, whereas the sheep inoculated with treated leukocytes did not become infected. The uninfected sheep was inoculated again with leukocytes isolated from the colostrum of another BLV-infected Holstein cow after freezing treatment, and again it did not become infected with BLV. Finally, this sheep was inoculated with the leukocytes isolated from the colostrum of another virus-infected cow without freezing treatment, and it became infected with BLV at 4 weeks after inoculation. The results indicate that colostrum should be frozen as a useful means of inactivating the infectivity of BLV-infected lymphocytes.

View full abstract

In prion diseases, abnormal prion protein (PrP^Sc) is considered as the main component of the infectious agent. Delineation of PrP^Sc conformation is expected to be a critical factor in understanding properties of prions. However, practical methods to differentiate between conformers of PrP^Sc are inadequate. Here, we used two PrP^Sc-specific monoclonal antibodies (mAbs), 3B7 and 3H6, and found that mAb 3H6 detected a limited portion of PrP^Sc in five mice-adapted prion strains. The quantity of mAb 3H6-precipitated PrP^Sc was significantly lesser in 22L compared to other strains. This result provides a direct evidence of the conformational heterogeneity of PrP^Sc within the prion strains. Conformation-specific probes, like these mAbs, have the potential to be powerful tools for investigating conformational variations in PrP^Sc.

View full abstract

Fowl adenovirus (FAdv) serotype 2 causes inclusion body hepatitis (IBH) disease which adversely affects the broiler industry in Thailand. We developed an indirect ELISA based on the recombinant hexon protein produced by E. coli. The recombinant hexon protein was tested with sera, in both infected and noninfected chickens. The recombinant hexon protein was standardized with an antigen concentration of 3.75 µg/ml and test sera. The intra- and inter-assays were repeatable. The cutoff value from TG-ROC curve analysis was 0.106. The specificity and sensitivity were 80 and 80%, respectively. The correlation coefficient (r) of absorbance values from this ELISA compared with the serum neutralization test was 0.76. This ELISA might be helpful for IBH diagnosis and surveillance.

View full abstract

Trunk musculature in Urodela species varies by habitat. In this study, trunk musculature was examined in five species of adult salamanders representing three different habitats: aquatic species, Amphiuma tridactylum and Necturus maculosus; semi-aquatic species, Cynops pyrrhogaster; terrestrial species, Hynobius nigrescens and Ambystoma tigrinum. More terrestrial species have heavier dorsal and ventral trunk muscles than more aquatic forms. By contrast, the lateral hypaxial musculature was stronger in more aquatic species. The number of layers of lateral hypaxial musculature varied among Urodela species and did not clearly correlate with their habitats. The M. rectus abdominis was separated from the lateral hypaxial musculature in both terrestrial and semi-aquatic species. In aquatic species, M. rectus abdominis was not separated from lateral hypaxial musculature. Lateral hypaxial musculature differed in thickness among species and was relatively thinner in terrestrial species. In more terrestrial species, dorsal muscles may be used for stabilization and ventral flexing against gravity. Ventral muscle may be used in preventing dorsally concave curvature of the trunk by dorsal muscles and by weight. The lengthy trunk supported by limbs needs muscular forces along the ventral contour line in more terrestrial species. And, the locomotion on well-developed limbs seems to lead to a decrease of the lateral hypaxial musculature.

View full abstract