Genetic characterization and susceptibility on poultry and mammal of H7N6 subtype avian influenza virus isolated in Japan in 2009

From February to March 2009, six strains of H7N6 subtype avian influenza virus were isolated from quails in three farms in Aichi prefecture in Japan. The isolates were shown to be low pathogenic for chicken by the examination performed using the "Manual of Standards for Diagnostic Tests and Vaccines" by World organisation for Animal Health (OIE). The deduced amino acid sequence at the cleavage site was PE (I/Q/L) PKRR (nucleotide sequences were cct gaa (a/c) (t/a) a cc (a/g) aaa aga aga), suggesting persistence in domestic poultry for some time. The direct putative ancestor strain could not be elucidated by phylogenetic analysis of all genome segments of the quail isolates. Diverged date from a putative common ancestor in a non-rooted phylogenetic tree among quail viruses was estimated between March 2002 and July 2004. Three putative N-linked glycosylation sites resided in the vicinity of the receptor binding pocket of HA1 region. They are considered to decrease the reactivity of neutralizing antibody against the virus. Experiments for the infectivity and pathogenicity of a quail strain to poultry indicated that the quail isolate had higher infectivity to quails than chickens and ducks. Direct and dust-borne and/or droplet-borne transmissions among quail were proven in quails with and without direct contact with experimentally infected quails. The virus is seldom transmitted among chickens either directly or indirectly, and indirect transmission from infected quails to chickens was not observed. The pathogenicity of the quail strain for mammalian, pig and mouse was low, although it could replicate in those animals.     

Expressions of protein oxidation markers, dityrosine and advanced oxidation protein products in Cisplatin-induced nephrotoxicity in rats

The purpose of this study was to evaluate whether dityrosine and advanced oxidation protein products (AOPP) reflect the severity of cisplatin-induced nephrotoxicity. Immunoexpression of dityrosine in kidneys and plasma AOPP concentration were examined up to day 4 post-cisplatin injection in rats. Cisplatin injection induced tubular injury on days 2-4 after injection and increased serum creatinine and BUN on days 3 and 4. On days 2-4, dityrosine was immunostained in the cytoplasm of damaged tubular cells, and their immunostaining intensity increased time-dependently. Plasma AOPP levels were significantly increased on days 3 and 4. These results suggest that expressions of dityrosine and AOPP were associated with the severity of renal injury and may be useful markers for the development of cisplatin-induced nephrotoxicity.     

Development and application of West Nile virus subgenomic replicon RNA expressing secreted alkaline phosphatase

We have developed a West Nile virus (WNV) subgenomic replicon harboring the secreted alkaline phosphatase (SEAP) reporter gene instead of viral structural genes (designated repWNV/SEAP). The repWNV/SEAP allowed easy evaluation of viral replication efficiency by direct measurement of SEAP secretion in the cell culture medium in physical containment level 2 facilities. Furthermore, we validated the availability of this system using a known anti-flavivirus gene, mouse oligoadenylate synthetase 1b (Oas1b). The Oas1b-transfected cells were more resistant to repWNV/SEAP replication than the original cells. Thus, this system not only affords a useful tool for identification/evaluation of anti-flavivirus genes/drugs in terms of safety, ease of use and reliability, but should be able to reduce or replace the bioassay using laboratory animals.     

Cloned cows with short telomeres deliver healthy offspring with normal-length telomeres

Dolly, the first mammal cloned from a somatic cell, had shorter telomeres than age-matched controls and died at an early age because of disease. To investigate longevity and lifetime performance in cloned animals, we produced cloned cows with short telomeres using oviductal epithelial cells as donor cells. At 5 years of age, despite the presence of short telomeres, all cloned cows delivered multiple healthy offspring following artificial insemination with conventionally processed spermatozoa from noncloned bulls, and their milk production was comparable to that of donor cows. Moreover, this study revealed that the offspring had normal-length telomeres in their leukocytes and major organs. Thus, cloned animals have normal functional germ lines, and therefore germ line function can completely restore telomere lengths in clone gametes by telomerase activity, resulting in healthy offspring with normal-length telomeres.     

In-straw cryoprotectant dilution for bovine embryos vitrified using Cryotop

The aim of this study was to develop an in-straw dilution method suitable for 1-step bovine embryo transfer of vitrified embryos using the Cryotop vitrification-straw dilution (CVSD) method. The development of embryos vitrified using the CVSD method was compared with those of embryos cryopreserved using in-straw vitrification-dilution (ISVD) and conventional slow freezing, outside dilution of straw (SFODS) methods. In Experiment 1, in vitro-produced (IVP) embryos cryopreserved using the CVSD method were diluted, warmed and exposed to the dilution solution at various times. When vitrified IVP embryos were exposed to the dilution solution for 30 min after warming, the rates of embryos developing to the hatched blastocyst stage after 72 h of culture (62.0-72.5%) were significantly lower (P<0.05) than those of embryos exposed to the solution for 5 and 10 min (82.4-94.3%), irrespective of supplementation with 0.3 M sucrose in the dilution solution. In Experiment 2, the rate of embryos developing to the hatching blastocyst stage after 48 h of culture in IVP embryos cryopreserved using the SFODS method (75.0%) was significantly (P<0.05) lower than those of embryos cryopreserved using the CVSD and ISVD methods (93.2 and 97.3%, respectively). In Experiment 3, when in vivo-produced embryos that had been cryopreserved using the CVSD, ISVD and SFODS methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception and delivery rates among groups. In Experiment 4, when IVP embryos derived from oocytes collected by ovum pick-up that had been cryopreserved using the CVSD and ISVD methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception rates among groups. Our results indicate that this simplified regimen of warming and diluting Cryotop-vitrified embryos may enable 1-step bovine embryo transfer without the requirement of a microscope or other laboratory equipment.     

Isolation of Staphylococcus aureus from raw fish in relation to culture methods

Five hundred and fifty fish samples from various stages in the course of distribution in Hyogo Prefecture (209 retailed in super markets, 173 obtained from fishery cooperatives at a harbor, 91 caught by trawling and 77 caught by rod fishing) were examined for contamination with Staphylococcus aureus (S. aureus). S. aureus was detected in 41 (19.6%) of the retail fish samples and 46 (26.6%) of the samples from the fishery cooperatives. No S. aureus was isolated from the live fish (91 trawled and 77 fished by rod). With regard to the retail fish, the contamination rate of processed fish (26.0%) was significantly higher than that of unprocessed fish (14.2%). For 88 samples, the efficacy of the selective medium was compared using Baird-Parker agar and mannitol salt agar supplemented with egg yolk (MSEY agar) by the direct plate and enrichment culture methods. Using the direct culture method, the S. aureus positive rate with the Baird-Parker agar (30.7%) was significantly higher (P<0.01) than that with the MSEY agar (6.8%). The enrichment culture method remarkably raised the S. aureus detection rate. Seventy-eight (85.7%) of 91 isolates belonged to the human ecovar. Sixty-two (68.1%) of the 91 isolates had some enterotoxin genes, including 44 (48.4%) with the sea gene. These data showed that the fish were contaminated with S. aureus after landing and that Baird-Parker agar had an advantage in detecting S. aureus with a direct plate culture.     

Bovine quantitative trait loci analysis for growth, carcass, and meat quality traits in an F2 population from a cross between Japanese Black and Limousin

A genome-wide scan for QTL affecting economically important traits in beef production was performed using an F(2) resource family from a Japanese Black x Limousin cross, where 186 F(2) animals were measured for growth, carcass, and meat-quality traits. All family members were genotyped for 313 informative microsatellite markers that spanned 2,382 cM of bovine autosomes. The centromeric region of BTA2 contained significant QTL (i.e., exceeding the genome-wide 5% threshold) for 5 carcass grading traits [LM area, beef marbling standards (BMS) number, luster, quality grade, and firmness), 8 computer image analysis (CIA) traits [LM lean area, ratio of fat area (RFA) to LM area, LM area, RFA to musculus (M.) trapezius area, M. trapezius lean area, M. semispinalis lean area, RFA to M. semispinalis area, and RFA to M. semispinalis capitis area], and 5 meat quality traits (contents of CP, crude fat, moisture, C16:1, and C18:2 of LM). A significant QTL for withers height was detected at 80.3 cM on BTA5. We detected significant QTL for the C14:0 content in backfat and C14:0 and C14:1 content in intermuscular fat around the 62.3 to 71.0 cM region on BTA19 and for C14:0, C14:1, C18:1, and C16:0 content and ratio of total unsaturated fatty acid content to total SFA content in intramuscular fat at 2 different regions on BTA19 (41.1 cM for C14:1 and 62.3 cM for the other 4 traits). Overall, we identified 9 significant QTL regions controlling 27 traits with genome-wide significance of 5%; of these, 22 traits exceeded the 1% genome-wide threshold. Some of the QTL affecting meat quality traits detected in this study might be the same QTL as previously reported. The QTL we identified need to be validated in commercial Japanese Black cattle populations.     

Pharmacokinetics of zonisamide and drug interaction with phenobarbital in dogs

The purposes of the present study were to elucidate the pharmacokinetics of zonisamide, determine the presence of a drug interaction with phenobarbital, and evaluate how long any interaction lasted after discontinuation of phenobarbital in dogs. Five dogs received zonisamide (5 mg/kg, p.o. and i.v.) before and during repeated oral administration of phenobarbital (5 mg/kg, bid, for 30–35 days). Zonisamide (5 mg/kg, p.o.) was also administered 8, 10, and 12 weeks after discontinuation of phenobarbital. Blood was sampled until 24 h after each zonisamide administration and serum concentrations of zonisamide were determined. Repeated phenobarbital decreased the maximum serum concentration, area under the serum concentration vs. time curve, apparent elimination half-life, and bioavailability of zonisamide. Total clearance increased. Time to maximum serum concentration and volume distribution were not changed. The maximum serum concentration and area under the serum concentration vs. time curve of zonisamide continued to be low until 10 weeks after the discontinuation of phenobarbital. They were restored to the same serum concentration as before phenobarbital administration 12 weeks after the discontinuation of phenobarbital. These data suggested that repeated administration of a clinical dose of phenobarbital enhanced the clearance of zonisamide and the enhanced clearance lasted at least 10 weeks after the discontinuation of phenobarbital. Caution may be necessary when zonisamide is given with phenobarbital and when antiepileptic therapy is changed from phenobarbital to zonisamide.     

Soil particles inhibit colony formation of Escherichia coli on the surface of agar media

When enumerating Escherichia coli in serial dilution of soil using selective media, soils was found to exert an inhibitory effect on colony formation on the surface of the agar plates. The inhibitory effect did not appear to be due to either soil bacteria or soluble chemicals but to the soil particles themselves. No inhibition was observed when the cells were enumerated by the most probable number (MPN) method. Some alternative plating methods, such as pour-plating or overlaying, were effective in removing the inhibitory effect. Removal of soil particles by simple sedimentation was also effective in enhancing the enumeration efficiency. These observations would be practically important for enumerating bacteria in the soil environment.