Multiplex PCR method for differentiating highly pathogenic Yersinia enterocolitica and low pathogenic Yersinia enterocolitica,and Yersinia pseudotuberculosis

A multiplex PCR method for rapid and sensitive diagnosis, differentiating three pathogenic Yersinia groups such as the highly pathogenic Y. enterocolitica, including serotype O8, low pathogenic Y. enterocolitica, and Y. pseudotuberculosis, was developed. Four primer pairs were chosen to detect the genes fyuA, ail, inv, and virF, responsible for the virulence in pathogenic Yersinia species. Under the multiplex PCR conditions, the unique band patterns for the highly pathogenic Y. enterocolitica, low pathogenic Y. enterocolitica, and Y. pseudotuberculosis were generated from Yersinia strains. The detection limit of this method was 10¹–10³ CFU per reaction tube. This multiplex PCR method could detect highly pathogenic Y. enterocolitica O8 from the wild rodent fecal samples that were culture-positive. Therefore, the new multiplex PCR method developed in this study is a useful tool for rapid and sensitive diagnosis, distinguishing three pathogenic Yersinia groups.     

Induction of spermatogenesis in Japanese eel by recombinant goldfish gonadotropins

We previously demonstrated the biological activities of single-chain recombinant gonadotropins (scGTHs) of goldfish Carrassius auratus follicle-stimulating hormone (scFSH) and luteinizing hormone (scLH), produced by a baculovirus–silkworm larvae system, by using in vivo bioassays with some fishes including Japanese eel Anguilla japonica. Among the bioassays, we succeeded in induction of spermatogenesis of sexually immature male Japanese eels by both scFSH and scLH, especially resulting in the occurrence of spermatozoa in scLH-administered males. However, those recombinant hormones did not induce enlargement of testes. In order to further confirm the potency of recombinant GTHs for use in aquaculture species, we administered scFSH and scLH to males of Japanese eel at higher dosage and frequency (eight times with 2–5 days interval) than those of the previous study (five or six times with 7 days intervals), including combination of scFSH and scLH administration (scFSH–scLH). Gonadosomatic indices (GSI) of scLH- and scFSH–scLH-administered males were larger than those of initial control males and of control males that were injected with saline. Enlargement of testes was also confirmed by measurement of testicular lobe size in scFSH-, scLH-, and scFSH–scLH-administered males. By histological observation, occurrence of spermatozoa was confirmed in scLH- and scFSH–scLH-administered eels. Although milt production was not induced, higher dosage and frequency of scGTH administration was effective in promoting testicular development of immature eels. Thus, single-chain fish GTHs produced by the baculovirus–silkworm larvae system could be a useful tool for promotion of gonadal maturation in aquaculture fishes.     

A case of juvenile form of dilated cardiomyopathy in a 6-month-old Shiba Inu dog

A 6-month-old Shiba Inu dog was brought to the Veterinary Medical Teaching Hospital because of a cough, exercise intolerance, and pulmonary edema. The dog had a Levine 2/6 systolic murmur. Transthoracic echocardiography revealed left atrial and ventricular dilatation (left atrium to aortic ratio: 2.8), mitral and tricuspid valve regurgitation, and severe left ventricular myocardial hypokinesia (fractional shortening was 11.8%). Bubble contrast echocardiography did not reveal a congenital shunt; therefore, the dog was clinically diagnosed with early onset dilated cardiomyopathy. From the first visit, the dog was treated with pimobendan, taurine, torasemide, and isosorbide dinitrate. After 435 days, echocardiography revealed that systolic function had not improved. On Day 465, atrial fibrillation was confirmed via electrocardiogram, and treatment with diltiazem hydrochloride was initiated. The dog continued to appear clinically stable thereafter, until it died suddenly 1087 days after the initial visit. A postmortem histopathological examination identified severe enlargement of the left atrial and ventricular chambers as well as attenuated wavy fibers in the ventricular myocardium, which confirmed dilated cardiomyopathy in a juvenile. This is the first report of a juvenile form of dilated cardiomyopathy in a Shiba Inu dog. This case report provides evidence that the extended prognosis of this dog differed from that in previously reported cases of dilated cardiomyopathy in young dogs.Key clinical message:This is the first reported case of a juvenile form of dilated cardiomyopathy in a Shiba Inu dog. This report provides evidence that the prognosis of this dog differed from that in previously reported cases of dilated cardiomyopathy in young dogs.     

Genetic characterization of a spontaneous green-type pigmentation mutant of Porphyra yezoensis and the significance of using heterozygous conchocelis in nori farming

ABSTRACT: In order to characterize the spontaneous green-type pigmentation mutant of Porphyra yezoensis , growth and contents of photosynthetic pigments were compared with those of the wild type in gametophytic blades. The growth of the green mutant was slower than that of the wild type. The content of phycoerythrin was markedly lower in the green mutant than in the wild type. For genetic analysis, the green mutant was crossed with the wild type. In the cross, the heterozygous conchocelis (the wild-type color) produced many sectored F ₁ gametophytic blades, which were composed of both parental colors. The segregation ratio by counting the wild-type sectors and the green-type sectors of the F ₁ blades was approximately 1 : 1. This suggests that the green mutant is governed by a single recessive gene. All the monospore germlings from the sectored F ₁ gametophytic blades that originated from the heterozygous conchocelis developed into single-colored blades with either the green color or the wild-type color. The advantages of using heterozygous conchocelis (hybrid conchocelis) and monospores from F ₁ gametophytic blades that originated from the heterozygous conchocelis in commercial farming of Porphyra yezoensis are detailed and discussed.     

Total selenium content of agricultural soils in Japan

To evaluate the selenium (Se) level in agricultural soils in Japan and to investigate its determining factors, 180 soil samples were collected from the surface layer of paddy or upland fields in Japan and their total Se contents were determined. Finely ground soil (50 mg) was wet-digested with HNO₃ and HClO₄ solution and the released Se was reduced to Se(IV). The concentration of Se(IV) was then determined by high-performance liquid chromatography with a fluorescence detector after treatment with 2,3-diaminonaphthalene and extraction with cyclohexane. The total Se content ranged from 0.05 to 2.80 mg kg⁻¹ with geometric and arithmetic means of 0.43 and 0.51 mg kg⁻¹, respectively. The overall data showed a log-normal distribution. In terms of soil type, volcanic soils and peat soils had relatively high Se content and regosols and gray lowland soils had relatively low Se content. In terms of land use, upland soils had significantly higher Se content than paddy soils. Among regions, soils in the Kanto, Tohoku, Hokkaido and Kyushu regions had relatively high content. The total Se content had a significant positive correlation with the organic carbon content ( P  < 0.01) and the equation for the estimation of total Se content with organic carbon suggested that on average approximately 48% (0.24 mg kg⁻¹) of the total Se was in inorganic forms and approximately 52% (0.25 mg kg⁻¹) was in organic forms. Soil pH, on the contrary, did not show a significant relationship with the total Se content. In conclusion, the organic matter content, in combination with volcanic materials, was the main determining factor of the total Se content of agricultural soils in Japan.     

Time-related Pathological Changes in Horses Experimentally Inoculated with Equine Influenza A Virus

To investigate the pathology of equine influenza, necropsy of 7 horses experimentally infected with equine influenza A virus (EIV) subtype H3N8 was conducted on post-infection days (PID) 2, 3, 7, and 14. Histopathologically, rhinitis or tracheitis including epithelial degeneration or necrosis with loss of ciliated epithelia and a reduction in goblet cell numbers, was observed in the respiratory tracts on PIDs 2 and 3. Epithelial hyperplasia or squamous metaplasia and suppurative bronchopneumonia with proliferation of type II pneumocytes were observed on PIDs 7 and 14. Viral antigen was detected immunohistochemically in the epithelia of the nasal mucosa, trachea, and bronchi on PIDs 2 and 3. The sodA gene of Streptococcus equi subsp. zooepidemicus, a suspected cause of suppurative bronchopneumonia, was detected in paraffin-embedded lung tissue sections, but only on PIDs 7 and 14. These findings suggest that damage caused to ciliated epithelia and goblet cells by EIV infection results in secondary bacterial bronchopneumonia due to a reduction in mucociliary clearance.     

The Composition of Volatile Aroma Components, Flavanones, and Polymethoxylated Flavones in Shiikuwasha (Citrus depressa Hayata) Peels of Different Cultivation Lines

Citrus peels are important sources of various pleasant aroma compounds and valuable bioactive substances. To investigate differences in the composition and content of Shiikuwasha (Citrus depressa Hayata) peels from different cultivation lines, the composition of volatile aroma components, flavanones, and polymethoxylated flavones (PMFs) in four Shiikuwasha cultivation lines was examined. The composition of volatile aroma components in cold-pressed extracts of Shiikuwasha peels was analyzed using gas chromatography-flame ionization detection and gas chromatography-mass spectrophotometry. The extracts contained mainly monoterpene hydrocarbons (93.40-97.25%), including limonene (46.52-68.26%) and γ-terpinene (21.48-30.52%). Differences in the composition of volatile aroma compounds in the Shiikuwasha cultivation lines were revealed using principal component analysis. Additionally, the composition of flavanones and PMFs was determined using high-performance liquid chromatography methods. Neohesperidin (96.58%) was the predominant flavanone in 'Izumi kugani' peel, while the other peels had high hesperidin contents (89.26-98.66%). Moreover, the PMFs of Shiikuwasha peels were composed of nobiletin (56.74-64.77%) and tangeretin (23.17-34.70%).