An H5N1 highly pathogenic avian influenza virus that invaded Japan through waterfowl migration

In 2010, an H5N1 highly pathogenic avian influenza virus (HPAIV) was isolated from feces of apparently healthy ducks migrating southward in Hokkaido, the northernmost prefecture of Japan. The H5N1 HPAIVs were subsequently detected in domestic and wild birds at multiple sites corresponding to the flyway of the waterfowl having stopovers in the Japanese archipelago. The Hokkaido isolate was genetically nearly identical to H5N1 HPAIVs isolated from swans in the spring of 2009 and 2010 in Mongolia, but less pathogenic in experimentally infected ducks than the 2009 Mongolian isolate. These findings suggest that H5N1 HPAIVs with relatively mild pathogenicity might be selected and harbored in the waterfowl population during the 2009-2010 migration seasons. Our data provide "early warning" signals for preparedness against the unprecedented situation in which the waterfowl reservoirs serve as perpetual sources and disseminators of HPAIVs.     

Trehalose may prevent postsurgical adhesions in a rabbit model of hysterotomy

The adhesion-preventing effect of trehalose in visceral organs was evaluated in a rabbit model. All rabbits underwent a hysterotomy, during which mild abrasion and desiccation was induced on the surfaces of visceral organs. In the control group, the intestines were kept dry during hysterotomy. In the saline and trehalose groups, saline solution and 7% trehalose solution, respectively, were sprayed on the organ surfaces. Adhesion formation on organ surfaces on which abrasion and desiccation were induced declined significantly in the trehalose group compared with the other groups. In contrast, at the hysterotomy site, there was no significant difference in adhesions between groups, suggesting that trehalose prevents adhesions in organs that have developed minor inflammation by abrasion and desiccation.     

Screening of therapeutic targets for canine mast cell tumors from a variety of kinase molecules

Options of systemic treatment for canine MCT have been still limited and most canine cases with MCTs eventually undergo relapses even after achievement of a remission. Thus additional therapies are required to establish for the tumor. To identify the novel candidate therapeutic targets for canine MCT, the mRNA expression and phosphorylation statuses of several receptor or non-receptor kinases as well as the inhibitory effect of 95 specific inhibitors on the growth were assessed in three canine MCT cell lines (HRMC, VIMC1 and CMMC1). Among the 14 targets, the mRNAs of 11, 7 and 7 kinases were amplified in HRMC, VIMC1 and CMMC1, respectively. The mRNAs of VEGFR3, PDGFRα, SRC, YES, LCK and FYN were detected in all cell lines. The phosphorylation of 12, 8 and 7 kinases was observed by using specific antibody arrays in HRMC, VIMC1 and CMMC1, respectively. DTK, EPHB6, AMPKα1, CREB, STAT5a and STAT5b were phosphorylated in all cell lines. The 10, 9 and 17 inhibitors exhibited the biological activity against the growth of HRMC, VIMC1 and CMMC1, respectively. Only three inhibitors such as SB218078 (for Chk1), PDGF RTK inhibitor IV (for PDGFR) and radicicol (for Hsp90) suppressed the growth of all three cell lines. The present study indicated that several kinases, such as Chk1, PDGFR and Hsp90, could be used as therapeutic targets in the treatment for canine MCT. Further studies and clinical trials are warranted to apply the inhibitors for the treatment of the tumor.     

Direct detection of equine herpesvirus type 1 DNA in nasal swabs by loop-mediated isothermal amplification (LAMP)

We evaluated loop-mediated isothermal amplification (LAMP) as a means of detecting equine herpesvirus type 1 (EHV-1) DNA directly from nasal swabs. To increase the sensitivity, we added a step in which the samples were heat-treated to the original LAMP procedure. The detection limit of the LAMP assay with heat treatment was 10 times more sensitive than the original LAMP assay even when the DNA extraction step was omitted. In addition, the LAMP assay with heat treatment was more sensitive than the original LAMP assay and the polymerase chain reaction using clinical samples. The LAMP assay with heat treatment is easy to perform and so should be applicable to the diagnosis of EHV-1 infections in clinical laboratories.     

Effects of Dietary Vitamin A on Juvenile Red Sea Bream Chrysophrys major

A 55-d feeding experiment was conducted to evaluate the effects of dietary vitamin A on growth of juvenile red sea bream Chrysophrys major . Nine semi-purified diets with supplemental retinol palmitate at levels of 0, 300, 600, 1,500, 3,000, 4,500, 6,000, 15,000 and 30,000 retinol eq/kg diet, were fed twice daily to triplicate groups of 10 juveniles per tank with an initial weight of 1.178 ± 0.001 g. Weight gain and specific growth rate increased with the increase of supplemental retinol palmitate in the diet up to a level of 6,000 retinol eq/kg diet, beyond which those indices tended to decrease slightly or to plateau. Significantly lower weight gain and specific growth rate were observed in fish fed with 0 and 300 retinol eq/kg diet. No significant differences were found in survival rates among the different levels of supplementation. The crude protein, lipid, moisture and ash contents in the whole body were not affected by the different vitamin A supplemental levels. Besides the reduced growth, fish fed with low levels of vitamin A supplement showed no other signs of deficiency. No hypervitaminosis A symptoms were observed in fish fed on high levels of supplementation. Fish fed on diets with 0 and 300 retinol eq/kg diet showed a significantly lower content of vitamin A (as a total retinol) in the liver than those in groups fed with higher levels of supplementation. Vitamin A was stored in the liver mainly as retinyl esters. Total retinal content of the eye showed no significant differences among the treatments; however, fish fed with 0 and 300 retinol eq/kg of diet had slightly higher concentrations. According to the results of the mean weight gain, specific growth rate and liver retinol content, the requirement of dietary vitamin A for juvenile red sea bream was estimated to be between 1,500 and 6,000 retinol eq/kg.     

Serum progesterone and estradiol-17beta concentrations, and lapaloscopic observations of the ovary in the cheetah (Acinonyxjubatus) with pregnant mare serum gonadotropin and human chorionic gonadotropin treatments.

In 3 adult female cheetahs, induced-superovulation treatment was conducted, by means of 200 IU of pregnant mare serum gonadotropin (PMSG) and 100 IU of human chorionic gonadotropin (hCG) 80 hr after PMSG. The administration of PMSG created a sharp increase in the estradiol-17beta concentration, resulting in 232 pg/ml 8 hr later in one specimen out of three. The hCG administration showed an increase in the progesterone concentration of 2.29 ng/ml 46 hr later. In addition, after direct observation of the ovary surface by laparoscopy, 5 follicles in the right ovary over 2 mm in diameter, and 7 corpora lutea (5 in the right ovary and 2 in the left) were found. It is assumed that ovulation can be induced with hCG after 80 hr on PMSG during a cheetah's diestrus or proestrus.     

Colitis associated with Clostridium difficile in specific-pathogen-free C3H-scid mice

Soft feces and a decreased delivery rate were observed in a specific-pathogen-free (SPF) C3H-scid mouse breeding colony. Grossly, the ceca were shrunken and edematous in the affected mice. Histopathologically, severe edema in the cecal submucosa as well as infiltration of inflammatory cells in the lamina propria and submucosa of the ceca and colon were observed. No pathogenic microorganisms were detected by the routine microbiological tests. By anaerobic bacterial-examination, Clostridium (C.) difficile with toxin A was isolated from the cecal contents of the affected mice. The mice were diagnosed with C. difficile-associated colitis. This case appears to be the first report of natural infection with C. difficile in SPF mice with clinical signs.     

Efficacy of five commercial disinfectants and one anionic surfactant against equine herpesvirus type 1

We investigated the influences of various reaction conditions on equine herpesvirus type 1 (EHV-1) disinfection by 5 commercial disinfectants (3 quaternary ammonium compounds [QACs] and 2 chlorine-based disinfectants) and 1 anionic surfactant. QACs at their highest recommended concentrations had no virucidal effect on EHV-1 with a 10-min reaction time at 0°C or a 1-min reaction time at room temperature. Chlorine-based disinfectants achieved EHV-1 disinfection with a 10-min reaction time at −10°C or a 30-sec reaction time at room temperature. In the presence of 5% fetal bovine serum, QACs (except for benzalkonium chloride) showed more stable virucidal effects than did chlorine-based disinfectants. The virucidal effect of the anionic surfactant was almost equivalent to that of the QACs.     

Melatonin protects the integrity of granulosa cells by reducing oxidative stress in nuclei,mitochondria, and plasma membranes in mice

Melatonin protects luteinized granulosa cells (GCs) from oxidative stress in the follicle during ovulation. However, it is unclear in which cellular components (e.g., nuclei, mitochondria, or plasma membranes) melatonin works as an antioxidant. GCs from immature (3 wks) ICR mice were incubated with hydrogen peroxide (H2O2; 0.01, 0.1, 1, 10 mM) in the presence or absence of melatonin (100 μg/ml) for 2 h. DNA damage was assessed by fluorescence-based immunocytochemistry using specific antibodies for 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative guanine base damage in DNA, and for histone H2AX phosphorylation (γH2AX), a marker of double-strand breaks of DNA. Mitochondrial function was assessed by the fluorescence intensity of MitoTracker Red probes, which diffuse across the membrane and accumulate in mitochondria with active membrane potentials. Lipid peroxidation of plasma membranes was analyzed by measuring hexanoyl-lysine (HEL), a oxidative stress marker for lipid peroxidation. Apoptosis of GCs was assessed by nuclear fragmentation using DAPI staining, and apoptotic activities were evaluated by caspase-3/7 activities. H2O2 treatment significantly increased the fluorescence intensities of 8-OHdG and γH2AX, reduced the intensity of MitoTracker Red in the mitochondria, increased HEL concentrations in GCs, and enhanced the number of apoptotic cells and caspase-3/7 activities. All these changes were significantly decreased by melatonin treatment. Melatonin reduced oxidative stress-induced DNA damage, mitochondrial dysfunction, lipid peroxidation, and apoptosis in GCs, suggesting that melatonin protects GCs by reducing oxidative stress of cellular components including nuclei, mitochondria, and plasma membranes. Melatonin helps to maintain the integrity of GCs as an antioxidant in the preovulatory follicle.     

Cesium radioactivity in peripheral blood is linearly correlated to that in skeletal muscle: Analyses of cattle within the evacuation zone of the Fukushima Daiichi Nuclear Power Plant

The accident at the Fukushima Daiichi Nuclear Power Plant (FNPP) released a large amount of radioactive substances into the environment. Furthermore, beef contaminated with radioactive cesium above the 500 Bq/kg safety standard was circulated in the food chain in 2011. Japanese consumers remain concerned about the safety of radioactively contaminated food. In our previous study, we detected a linear correlation between radioactive cesium (¹³⁷Cs) activity in blood and muscle around 500 to 2500 Bq/kg in cattle. However, it was unclear whether the correlation was maintained at a lower radioactivity close to the current safety standard of 100 Bq/kg. In this study, we evaluated 17 cattle in the FNPP evacuation zone that had a ¹³⁷Cs blood level less than 10 Bq/kg. The results showed a linear correlation between blood ¹³⁷Cs and muscle ¹³⁷Cs (Y = 28.0X, R² = 0.590) at low radioactivity concentration, indicating that cesium radioactivity in the muscle can be estimated from blood radioactivity. This technique would be useful in detecting high‐risk cattle before they enter the market, and will contribute to food safety.