Characterization of a novel nicotinamide adenine dinucleotide-cytochrome b5 reductase mutation associated with canine hereditary methemoglobinemia

Hereditary methemoglobinemia associated with nicotinamide adenine dinucleotide-cytochrome b5 reductase (b5R) deficiency is a rare autosomal recessive disorder in animals. Recently, nonsynonymous b5R gene (CYB5R3) variants have been reported to be associated with canine and feline hereditary methemoglobinemia. However, the underlying molecular mechanisms of canine and feline methemoglobinemia caused by these nonsynonymous variants have not yet been reported. Previously, we reported a Pomeranian dog family with hereditary methemoglobinemia, carrying CYB5R3 mutation of an A>C transition at codon 194 in exon 7, replacing an isoleucine residue with leucine (p.Ile194Leu). In this study, we investigated the enzymatic and structural properties of the soluble form of wild-type and Ile194Leu canine b5Rs to characterize the effects of this missense mutation. Our results showed that the kinetic properties of the mutant enzyme were not affected by this amino acid substitution. The secondary structure of the wild-type and Ile194Leu b5Rs detected by circular dichroism showed a similar pattern. However, the mutant enzyme exhibited decreased heat stability and increased susceptibility to trypsin hydrolysis. Moreover, the thermostability and unfolding measurements indicated that the mutant enzyme was more sensitive to temperature-dependent denaturation than the wild-type b5R. We concluded from these results that unstable mutant enzyme properties with normal enzymatic activity would be associated with hereditary methemoglobinemia in the Pomeranian dog family.     

Anethole inhibits growth of recently emerged multidrug resistant toxigenic Vibrio cholerae O1 El Tor variant strains in vitro

To search natural compounds having inhibitory effect on bacterial growth is important, particularly in view of growing multidrug resistant (MDR) strains of bacterial pathogens. Like other bacterial pathogens, MDR Vibrio cholerae, the causative agent of diarrheal disease cholera, is becoming a great concern. As an approach of searching new antimicrobial agents, here, we show that anethole, a well-studied natural component of sweet fennel and star anise seeds, could potentially inhibit the growth of MDR O1 El Tor biotype, the ongoing 7th cholera pandemic variant strains of toxigenic V. cholerae. The minimum inhibitory concentration (MIC) of anethole against diverse O1 El Tor biotype strains is evaluated as 200 µg/ml. Moreover, the effect of anethole is bactericidal and exerts rapid-killing action on V. cholerae cells. This study is the first report which demonstrates that anethole, purified from natural compound, is a potent inhibitor of growth of toxigenic V. cholerae. Our data suggest that anethole could be a potential antimicrobial drug candidate, particularly against MDR V. cholerae mediated infections.     

Reduction of transport stress of ayu by obligated schooling

We investigated stress responses of ayu Plecoglossus altivelis under the hypothesis that overcrowding during transport can be a stressor and the stress predisposes fish to disease by decline in immunocompetence. The extent of stress response was determined under different fish densities. As inferred from the serum concentration of cortisol 1 day after the treatment, the density at more than 23 kg/m³, which was within the range of normal transporting procedures, acutely evoked intense stress responses. To examine a possible way to reduce the stress response of fish under crowding conditions, we compared two fish groups established in water tanks with circulating (whirling) and turbulent currents at the same density. The cortisol levels 1 day after the treatment were 2.5-fold higher in turbulent current than in whirling current. Fish in whirling current formed a school, while those in turbulent current aggregated with each other. Schooling of fish induced by water flow may reduce their stress responses during transport. A transport system with a directional water flow should keep fish in good condition without reducing the number of fish contained.     

Viability of maternally heat-stressed mouse zygotes in vivo and in vitro

Mammalian preimplantation embryos are susceptible to heat stress. This present study examined how maternal heat stress affects the development of mouse zygotes in vivo and in vitro. In Experiment 1, zygotes collected from female mice that were heat‐stressed for 12 h on day 1 of pregnancy were cultured in vitro. Maternally heat‐stressed zygotes developed normally to the two‐cell stage, but the majority of embryos failed to develop into morulae or blastocysts. In Experiment 2, pregnant mice were heat‐stressed on day 1 or from day 1 to day 3 of pregnancy. The number of living fetuses on day 14 of pregnancy was lower in heat‐stressed mice than in non‐stressed mice, but the difference was significant only in successively heat‐stressed mice. These results demonstrate that maternally heat‐stressed zygotes have reduced in vitro viability, but this phenomenon does not necessarily lead to embryo loss in the maternal environment.     

Prevalence,O-genotype and Shiga toxin (Stx) 2 subtype of Stx-producing Escherichia coli strains isolated from Argentinean beef cattle

The aims of this study were to investigate prevalence, O-genotype, and virulence gene profile including Shiga toxin (Stx) 2 gene-subtype of Stx-producing Escherichia coli (STEC) in beef cattle from the Bahía Blanca in Argentina. Rectal swabs were collected from 283 beef cattle in 2012. stx genes were detected in 90 (32%) out of the 283 rectal swabs by stx gene-specific PCR assay. The positive cases were 13 with stx1, 58 with stx2, and 19 with both stx1 and stx2. Among 90 stx gene-positive samples, 45 STEC strains were isolated, which included 3 stx1, 34 stx2, and eight stx1 and stx2 genes positive isolates. O-genotyping grouped 45 STEC strains into 19 different O-genotypes such as Og8, Og145, Og171, Og185 (4 from each), Og22, Og153, Og157 (3 from each) and others. Various stx2 gene-subtypes were identified in 42 STEC strains: 13 positive cases for stx2a, 11 for stx2c, 3 for stx2g, 10 for stx2a and stx2d, 4 for stx2a and stx2c, and 1 for stx2b, stx2c and stx2g. efaI gene, generally prevalent in clinical strains, was detected in relatively high in the STEC strains. These data suggest that stx2a and stx2c were distributed not only in O145 and O157 but also in minor O-genotypes of STEC in Argentina.     

CONTRAST-ENHANCED ULTRASONOGRAPHY FOR CHARACTERIZATION OF CANINE FOCAL LIVER LESIONS

In six normal beagles and 27 dogs with spontaneous focal or multifocal liver lesions, contrast-enhanced ultrasonography using Sonazoid^® was performed. Sonazoid^® is a newly developed second-generation contrast agent with the ability to be used for real-time contrast imaging along with parenchymal imaging. An appropriate protocol for the evaluation of all three phases (arterial, portal, and parenchymal) was established based on the results for normal beagles. By evaluation of the echogenicity of hepatic nodules during the arterial and parenchymal phases it was possible to differentiate malignant tumors from benign nodules with very high accuracy. In 15 of 16 dogs diagnosed as malignant tumors, nodules were clearly hypoechoic to the surrounding normal liver during the parenchymal phase. Additionally, malignant tumors had different echogenicity compared with the surrounding normal liver during the arterial phase in 14 of 15 dogs. In the portal phase, there were no characteristic findings. Contrast-enhanced ultrasonography with Sonazoid^® appears to improve the characterization of canine focal and multifocal hepatic lesions.     

Prevalence of Shiga toxin-producing Escherichia coli in Yezo sika deer (Cervus nippon yesoensis) in the Tokachi sub-prefecture of Hokkaido,Japan

In food hygiene, the surveillance of foodborne pathogens in wild animals is indispensable because we cannot control hygienic status of them. Yezo sika deer (Cervus nippon yesoensis), which are found only on the island of Hokkaido, Japan, are the most common game animal in the country. In this study, we analyzed the incidence of Shiga toxin-producing Escherichia coli (STEC) in Yezo sika deer hunted in the Tokachi sub-prefecture, which is one of the densest zones for the sub-species. Real-time polymerase chain reaction testing detected STEC in 18.3% of fecal samples (59/323) collected from deer hunted between 2016 and 2017, whereas no Shigella and Salmonella markers were detected. No correlation was found between STEC detection from fecal samples and characteristics of carcasses, such as hunting area, age, and fascioliasis. From 59 STEC-positive fecal samples, we isolated 37 STEC strains, including 34 O- and H-genotyped strains, in which 16 different serogroups were detected. Genetic analysis revealed that our isolates included various stx gene types (stx1⁺/stx2⁻, stx1⁺/stx2⁺, and stx1⁻/stx2⁺) and carried eae. This study demonstrated that STEC strains with various features colonized the Yezo sika deer, similar to other subspecies of sika deer. We conclude that continuous surveillance activity is important to monitor the suitability of game animals as a food source and to assess the validity of the food safety management system for game meat production.