Evaluation of a PCR-restriction fragment length polymorphism (PCR-RFLP) assay for molecular epidemiological study of Shiga toxin-producing Escherichia coli

In this study, we have evaluated our recently developed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for the molecular subtyping of Shiga toxin-producing Escherichia coli (STEC). A total of 200 STEC strains including O157 (n=100), O26 (n=50), O111 (n=10), and non-O26/O111/O157 (n=40) serogroups isolated during 2005-2006 in Japan, which were identified to be clonally different by pulsed-field gel electrophoresis (PFGE) were further analyzed by the PCR-RFLP assay in comparison to PFGE. Ninety-five of O157, 48 of O26, five of O111 and 19 of non-O26/O111/O157 STEC strains yielded one to three amplicons ranging from 6.0 to 15.5 kb in size by the specific primer set targeting region V which is located in the upstream of stx genes. These strains were classified into 41 (O157), 8 (O26), 4 (O111) and 17 (non-O26/O111/O157) groups based on the RFLP patterns obtained by subsequent restriction digestion, respectively. Although the discriminatory power of PCR-RFLP assay was somewhat less than that of PFGE, it is more convenient for molecular subtyping of STEC strains especially for O157, the most important serogroup implicated in human diseases, as well as to identify the outbreak-associated isolates because of its simplicity, rapidity, ease and good reproducibility.     

Basal levels and GnRH-induced responses of peripheral testosterone and estrogen in Holstein bulls with poor semen quality

The present study investigated the basal levels and GnRH-induced responses of peripheral testosterone and estrogen in Holstein bulls with poor semen quality. On the basis of semen parameters, bulls (n=5) having poor semen quality were selected as experimental bulls, and good semen quality bulls (n=4) were used as control bulls. Both groups were treated intramuscularly once with GnRH (250 μg of fertirelin acetate). Blood samples were collected at -1 day (d), -30 min and 0 h (treatment) followed by every 30 min for 5 h and 1, 3 and 5 d post-GnRH treatment (PGT), and LH, testosterone and estradiol-17β (E(2)) concentrations were measured. The pretreatment concentrations were used as basal levels. The percentage increments based on the 0-h levels were calculated per bull for each sampling time until 5 h PGT, and differences were compared between the experimental and control groups. The PGT concentrations of testosterone and basal and PGT concentrations of E(2) were significantly lower in the experimental group. The testosterone increment in the experimental group was delayed and significantly lower from 1 to 5 h PGT than those in the control group. It can be suggested that bulls with poor semen quality have delayed and lower GnRH-induced testosterone response and may also have lower estrogen levels.     

Emphysematous cystitis in a chemically-induced diabetic dog

Emphysematous cystitis is a rare disorder caused by bacterial infection and characterized by gas accumulation within the bladder wall with cyst formation. This report describes the histopathological characteristics of emphysematous cystitis found in a diabetic female beagle induced by streptozotocin and alloxan. Macroscopically, multiple cyst-like structures were observed on the cut surface of the urinary mucosa. During fixation, small specimens cut from the mucosa floated on the surface of the fixative solution. Histopathologically, multiple cysts were lined with a single layer of flattened cells found to be immunohistochemically positive for vimentin, partially positive for α-smooth muscle actin or macrophage scavenger receptor, class A, and thought to be myofibroblasts, fibroblasts or macrophages. Multinucleated giant cells were observed around the cysts, and gram-negative short bacilli were observed in the lumen of the urinary bladder. From these findings, this case was diagnosed as emphysematous cystitis.     

Induction of apoptotic lesions in liver and lymphoid tissues and modulation of cytokine mRNA expression by acute exposure to deoxynivalenol in piglets

Six 1-month-old piglets were intravenously injected with deoxynivalenol (DON) at the concentration of 1 mg/kg body weight, with three pigs each necropsied at 6 and 24 h post-injection (PI) for investigation of hepatotoxicity and immunotoxicity with special attention to apoptotic changes and cytokine mRNA expression. Histopathological examination of the DON-injected pigs revealed systemic apoptosis of lymphocytes in lymphoid tissues and hepatocytes. Apoptosis of lymphocytes and hepatocytes was confirmed by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method and immunohistochemical staining against single-stranded DNA and cleaved caspase-3. The number of TUNEL-positive cells in the thymus and Peyer''s patches of the ileum was increased at 24 h PI compared to 6 h PI, but the peak was at 6 h PI in the liver. The mRNA expression of interleukin (IL)-1β, IL-6, IL-18, and tumor necrosis factor (TNF)-α in the spleen, thymus and mesenteric lymph nodes were determined by semi-quantitative RT-PCR, and elevated expression of IL-1β mRNA at 6 h PI and a decrease of IL-18 mRNA at 24 h PI were observed in the spleen. IL-1β and IL-6 mRNA expressions increased significantly at 6 h PI in the thymus, but TNF-α decreased at 6 h PI in the mesenteric lymph nodes. These results show the apoptosis of hepatocytes suggesting the hepatotoxic potential of DON, in addition to an immunotoxic effect on the modulation of proinflammatory cytokine genes in lymphoid organs with extensive apoptosis of lymphocytes induced by acute exposure to DON in pigs.     

Oral administration of L-citrulline changes the concentrations of plasma hormones and biochemical profile in heat-exposed broilers

We examined the effects of oral administration of L-citrulline (L-Cit) on plasma metabolic hormones and biochemical profile in broilers. Food intake, water intake, and body temperature were also analyzed. After dual oral administration (20 mmol/head/administration) of L-Cit, broilers were exposed to a high ambient temperature (HT; 30 ± 1°C) chamber for 120 min. Oral administration of L-Cit reduced (p < .001) rectal temperature in broilers. Food intake was increased (p < .05) by heat stress, but it was reduced (p < .05) by L-Cit. Plasma levels of 3,5,3′-triiodothyronine, which initially increased (p < .0001) due to heat stress, were reduced (p < .01) by oral administration of L-Cit. Plasma insulin levels were increased by heat exposure (p < .01) and oral L-Cit (p < .05). Heat stress caused a decline (p < .05) in plasma thyroxine. Plasma lactic acid (p < .05) and non-esterified fatty acids (p < .01) were increased in L-Cit-treated heat-exposed broilers. In conclusion, our results suggest that oral L-Cit can modulate plasma concentrations of major metabolic hormones and reduces food intake in broilers.     

Relationship between the first ovulation within three weeks postpartum and subsequent ovarian cycles and fertility in high producing dairy cows

The aim of this study was to investigate the relationship between the first ovulation within 3 weeks postpartum and subsequent ovarian cycles and fertility in high producing dairy cattle in Hokkaido, Japan. In Experiment 1, 110 cows (44 primiparous and 66 multiparous) were used to determine the effects of the first ovulation within 3 weeks postpartum on subsequent ovarian cycles. Milk samples were collected twice weekly from 7 to 100 days postpartum. The first ovulation was identified by an increase in milk progesterone (P4) to more than 1 ng/ml within 3 weeks postpartum. The numbers of cows showing ovulation and anovulation within 3 weeks postpartum were 31 (70.5%) and 13 (29.5%) in the primiparous cows and 35 (53.0%) and 31 (47.0%) in the multiparous cows, respectively. The patterns of ovarian resumption after calving were classified into two types (normal ovarian cycles and abnormal ovarian cycles) on the basis of milk P4 concentrations. Initiation of normal ovarian function in cows ovulated within 3 weeks postpartum occurred earlier than in anovulated cows regardless of the number of calvings (primiparous, 27.8 days vs. 44.4 days; multiparous, 30.6 days vs. 55.7 days; P<0.01). Out of the multiparous cows that ovulated within 3 weeks postpartum, initiation of normal ovarian function followed by a normal luteal phase was earlier than when it was followed by an abnormal luteal phase (25.5 days vs. 40.4 days; P<0.05). Milk P4 concentrations after the first ovulation were lower than those after the second ovulation in both the primiparous and multiparous cows (P<0.05). In Experiment 2, 22 multiparous cows were used to determine the effects of the first ovulation within 3 weeks postpartum on subsequent fertility. Blood samples were collected once a week from 0 to 3 weeks postpartum. The interval from parturition to first service in ovulated cows was shorter than in anovulated cows (68.4 days vs. 94.8 days; P<0.05). The conception rate by 100 days after calving tended to be higher in ovulated cows than in anovulated cows (50.0% vs. 16.7%, P=0.09). In conclusion, our data strongly suggests that ovulation within 3 weeks postpartum is a crucial phenomenon for subsequent resumption of ovarian function and conception, and thus it can be used as an index of subsequent reproductive performance.     

A 90-day Feeding Toxicity Study of l-Serine in Male and Female Fischer 344 Rats

A subchronic feeding study of l-serine (l-Ser) was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0, 0.06, 0.5, 1.5 or 5.0% concentrations of l-Ser for 90 days. There were no toxicologically significant, treatment-related changes with regards to body weight, food intake, water intake or urinalysis data. In several of the hematology, serum biochemistry and organ weight parameters, significant changes were observed between some of the treated groups and the controls. All these changes, however, were subtle and lacked any corresponding pathological findings. In addition, the increased or decreased values remained within the range of the historical control values. In fact, histopathological assessment revealed only sporadic and/or spontaneous lesions. In conclusion, the no-observed-adverse-effect-level (NOAEL) for l-Ser was, therefore, determined to be at least a dietary dose of 5.0% (2765.0 mg/kg body weight/day for males and 2905.1 mg/kg body weight/day for females) under the present experimental conditions.     

Effects of light and microsite conditions on tree size of 6-year-old Cryptomeria japonica planted in a group selection opening

We examined the extent to which direct and indirect measures of light and microsite conditions could explain variation in tree height and diameter at the base of 6-year-old Cryptomeria japonica trees planted in a group selection opening of about 0.32 ha on a steep slope at Shiiba, Miyazaki Prefecture, southern Japan. We first used the gap light index (GLI) and soil thickness (ST) as directly measured indices. For an indirect measure of light, we used a between-cohort competition index (BCI) estimated from the position and total height of residual trees. For indirect measures of microsite, we examined topographic indices (slope, plan and profile curvature, average slope gradient, and relative elevation) derived from digital elevation models (DEMs) with different resolutions ranging from 2 to 10 m. The multiple linear regression using GLI and ST explained about 45% of variation in tree size, while simple regression using only GLI explained about 35%. The contribution of ST was about half of GLI. The multiple regressions using BCI and the topographic indices did not explain any more variation than using BCI alone (R ² of about 0.26). We conclude that microsite conditions with shallower soil and steeper slope have negative effects on tree growth in group selection openings, although the relative importance is smaller than light conditions. More comprehensive studies considering several openings with more heterogeneous topography including different species are needed to generalize our growth prediction using the indirect measures, which are useful for practical forest management.     

Annual net production of the five Sargassaceae species in Yoro,western Wakasa Bay,Sea of Japan

ABSTRACT:   Annual net production of the five Sargassaceae species was investigated in Yoro, western Wakasa Bay, Sea of Japan. The Sargassaceae species were collected at a depth of 2.0–2.5 m and subjected to stratified clipping for 13 months. Annual net production was estimated at 2407 g dry wt/m² for Sargassum patens , 2132 g dry wt/m² for Sargassum macrocarpum , 1458 g dry wt/m² for Sargassum siliquastrum , 1197 g dry wt/m² for Myagropsis myagroides , and 1471 g dry wt/m² for Sargassum piluliferum . The ratio of annual net production to maximum biomass (P/B_max) for these species ranged between 1.3 and 1.7, being higher than in previous studies which used the same method. Winter storms with strong wave exposure and relatively calm condition in Yoro were considered to augment P/B_max. The total losses estimated from the different units of stratified clipping were compared. If larger and less segregated units were applied, the total loss would decrease. It was suggested that a 50 cm stratum would be applicable to the larger species (3 m or longer) and a stratum of 20 cm or less would be applicable to mid-sized or smaller species (less than 3 m).