Quadruple or quintuple conversion of hlb, sak, sea (or sep), scn, and chp genes by bacteriophages in non-beta-hemolysin-producing bovine isolates of Staphylococcus aureus

In 13 of 43 non-beta-hemolysin-producing bovine isolates of Staphylococcus aureus, two truncated beta-hemolysin (hlb) genes were demonstrated by PCR and sequencing, and one truncated hlb gene was located beside the integrase (int) gene of phage origin. The staphylokinase (sak) gene was detected in all 13 isolates in which the truncated hlb genes were detected by PCR. Enterotoxin A (sea) and enterotoxin P (sep) genes were also detected in 5 and 2 of the 13 isolates, respectively. Moreover, the scn and chp genes encoding staphylococcal complement inhibitor (SCIN) and chemotaxis inhibitory protein of S. aureus (CHIPS) were detected in 13 and 4 of the 13 isolates, respectively. The bacteriophage induced by mitomycin C treatment was able to lysogenize one beta-hemolysin-producing isolate of S. aureus, and the sak and scn genes were detected from the lysogenized isolate. These results suggest quadruple or quintuple conversion of hlb, sak, sea (or sep), scn, and chp genes by bacteriophages among non-beta-hemolysin-producing bovine isolates of S. aureus.     

Proportions of melanocyte stimulating hormone-immunoreactive cells in the adenohypophysis of Silky fowl and hyperpigmentation-free cockerels

Alpha‐melanocyte stimulating hormone (α‐MSH) is one of the main regulators for melanocytes, and the adenohypophysis is one of the major tissues for the synthesis of this hormone. The Silky fowl is a characteristic breed of chicken with hyperpigmentation throughout the body. The involvement of the adenohypophysis in the hyperpigmentation of this breed is not known. In the present study, the proportion of melanocyte stimulating hormone‐immunopositive cells (MSH cells) in the adenohypophysis was immunocytochemically compared between Silky, Red Cornish × New Hampshire (RN) crossbred and Japanese bantam cockerels at 15 weeks of age. After the body and gland were weighed, the adenohypophyseal cells were enzymatically dispersed and immunostained for α‐MSH, and the immunopositive MSH cells were counted. The weights of the body and adenohypophysis were heaviest in the RN crossbred, followed by the Silky and lightest in the bantam cockerels. In contrast, the ratio between adenohypophysis and bodyweight was much larger in the Silky and the bantam than in the RN crossbreed (P < 0.05). The population of MSH cells in the adenohypophysis was larger in the Silky (14.3%) than in the RN crossbreed (8.0%) and the bantam (8.1%) cockerels (P < 0.05). From these results, it was concluded that prepubertal Silky cockerel have numerous MSH cells in the adenohypophysis suggesting a relationship to hyperpigmentation.     

A molecular epidemiological survey of Babesia, Hepatozoon,Ehrlichia and Anaplasma infections of dogs in Japan

Tick-borne diseases are often encountered in canine clinical practice. In the present study, a molecular epidemiological survey of dogs in Japan was conducted to understand the prevalence and geographical distribution of Babesia spp., Hepatozoon spp., Ehrlichia spp. and Anaplasma spp. Pathogen-derived DNA in blood samples obtained from 722 dogs with a history of exposure to ticks and/or fleas was examined by PCR. The prevalence of Babesia gibsoni, Babesia odocoilei-like species, Hepatozoon canis and Ehrlichia spp./Anaplasma spp. was 2.4% (16/722), 0.1% (1/722), 2.5% (18/722) and 1.5% (11/722), respectively. While B. gibsoni and Ehrlichia spp./Anaplasma spp. were detected in the western part of Japan, H. canis was detected in Tohoku area in addition to western and central parts of Japan.     

Localization of putative pituitary stem/progenitor cells in female dairy cattle

Research on sex-determining region Y-box 2 (SOX2)-positive pituitary stem/progenitor cells, as a source of hormone-producing cells, is progressing rapidly in rodents. However, the stem/progenitor cells supplying hormone-producing cells that are essential for growth, reproduction, and lactation in bovines have not yet been identified. In this study, we characterized SOX2-positive cells in the pituitary gland of dairy cattle (Holstein heifers) after sexual maturity. Immunofluorescence analysis revealed that the localization pattern of SOX2-positive cells in the dairy cattle pituitary gland was similar to that observed in the rodent pituitary gland; the marginal cell layer (MCL), dense cell clusters, and single cells scattered in the parenchyma of the anterior lobe. Furthermore, most of the SOX2-positive cells were positive for the pituitary stem/progenitor cell niche markers E-cadherin and cytokeratin 8+18, which have been reported in rodents. In addition, in the MCL of the anterior lobe, there was a subpopulation of SOX2-positive cells positive for paired-related homeobox 1 and 2, whereas negative for S100β. Moreover, in the parenchyma of the anterior lobe, co-localization of SOX2 and pituitary hormones was infrequent. In summary, this study reveals the localization of putative pituitary stem/progenitor cells positive for SOX2 in dairy cattle. These results provide valuable information to support further investigation of cell supply in the dairy cattle pituitary gland.     

Isolation of chicken taste buds for real‐time Ca2+ imaging

We isolated chicken taste buds and used a real‐time Ca²⁺ imaging technique to investigate the functions of the taste cells. With RT‐PCR, we found that isolated chicken taste bud‐like cell subsets express chicken gustducin messenger RNA. Immunocytochemical techniques revealed that the cell subsets were also immunopositive for chicken gustducin. These results provided strong evidence that the isolated cell subsets contain chicken taste buds. The isolated cell subsets were spindle‐shaped and approximately 61–75 μm wide and 88–98 μm long, and these characteristics are similar to those of sectional chicken taste buds. Using Ca²⁺ imaging, we observed the buds' response to 2 mmol/L quinine hydrochloride (a bitter substance) and their response to a mixture of 25 mmol/L L‐glutamic acid monopotassium salt monohydrate and 1 mmol/L inosine 5′‐monophosphate disodium salt, umami substances. The present study is the first morphological demonstration of isolated chicken taste buds, and our results indicate that the isolated taste buds were intact and functional approaches for examining the taste senses of the chicken using Ca²⁺ imaging can be informative.     

Comparison of collagen content, distribution and architecture among the pectoralis, iliotibialis lateralis and puboischiofemoralis muscles with different myofiber composition in Silky cocks

The total amount of collagen, the relative distributions of types I and III collagens in perimysium and endomysium, and the collagen fiber architecture were compared among the pectoralis (PT), iliotibialis lateralis (ITL) and puboischiofemoralis (PIF) muscles in Silky cocks. All of the myofibers in the PT muscle were type IIB, the myofibers in the ITL muscle were divided into type IIA, 41.7% and IIB, 58.3%, and the PIF muscle was composed of type I, 24.6%; IIA, 64.6%; and transitional, 10.8%. The total amount of collagen differed significantly among the PT (2.92 mg/g), PIF (4.20 mg/g) and ITL (8.06 mg/g) material, where only the PIF was a whole muscle with epimysium. On the image analysis of the immunohistochemical preparations, the percentage area of perimysial collagen to the total area in each type differed significantly among the PIF, PT and ITL muscles, where it was 26.8, 50.0 and 74.4% for the type I collagen and 27.4, 32.9 and 61.7% for the type III collagen, respectively. In the scanning electron micrography of the perimysium in macerated preparations, thick bundles of collagen fibers were observed in the ITL muscle, thinner but broad platelets in the PT muscle, and a coarse tissue of thinner collagen fibers in the PIF muscle. However, the endomysial fabric of collagen fibrils was similar among the muscles. Small, transverse collagen fibers, which branched off from the thicker perimysia, occupied narrow interendomysial spaces and separated the primary myofiber fasciculi. The results indicate that the ITL muscle, localized in the distorted and overextended part of the leg and subject to strong external forces, had highly developed perimysial collagen fiber bundles, but the ITL endomysial collagen architecture was similar to that of the PT and PIF muscles.     

Effects of enzymatic deamidation by protein-glutaminase on structure and functional properties of wheat gluten

Protein-glutaminase (PG) purified from Chryseobacterium proteolyticum was used to investigate its deamidation effects on wheat gluten. Water-insoluble gluten was able to be deamidated to the extent of deamidation degree (DD) 72% in 200 mM sodium phosphate buffer (pH 7) at 40 degrees C for 30 h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis exhibited an upper shift of gluten bands with only deamidation for 1.5-2.0 h (DD 35-45%) compared to the bands of nondeamidated gluten. Results of Fourier transform infrared analysis revealed alterations in secondary structure of gluten by PG deamidation. The assignment within amide I region showed decreases in both inter- (around 1695 cm(-1)) and intramolecular beta-sheets (around 1680 cm(-1)) by deamidation suggesting the deterioration of the aggregation ability of gluten molecules. Solubility and emulsification properties of gluten at pH 7 were improved by deamidation, while both properties at pH 3 were deteriorated by deamidation. Enzyme-linked immunosorbent assay identified that allergenicity of deamidated gluten as compared to the nondeamidated cohorts was decreased remarkably as the deamidation time was prolonged.     

Comparison of behavioral effects of morphine and fentanyl in dogs and cats

Behavioral effects induced by intravenous administration of morphine at 0.3, 0.6, 1.2, and 2.4 μg/kg and fentanyl at 5, 10, 20, and 40 μg/kg were evaluated in dogs and cats. In dogs, fentanyl and morphine depressed activity and level of consciousness in a dose- dependant manner. In cats, higher doses of fentanyl stimulated activity temporarily, but excitement, so-called "opioid mania," was not observed. Morphine induced distinctive behavioral changes characterized by sitting with fixed staring, and "opioid mania" was not observed in cats.     

Genetic and enzymatic analyses of metalloprotease (aureolysin) from Staphylococcus aureus isolated from domestic animals.

The gene (aur) encoding the metalloprotease (aureolysin) of Staphylococcus aureus from domestic animals was analyzed by polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (PCR-RFLP) and sequencing. The aur gene was detected in all 74 isolates from cows, pigs and chickens by PCR amplification and was classified into types I and II by PCR-RFLP patterns. The type II aur gene was contained in 36 (94.7%) of 38 protease-positive isolates as judged by skim milk agar plate culture, while type I was contained in 28 (77.8%) of 36 protease-negative isolates. The deduced amino acid sequences of aureolysins from type I and II isolates were almost identical with those of the published data. Subsequently, the two aureolysins were purified from the culture supernatants of type I and II isolates. The molecular weights of purified type I and II aureolysins were both estimated at 34kDa by SDS-polyacrylamide gel electrophoresis. These aureolysins had maximum proteolytic activity at 30-50 degrees C and pH 7.0-8.0. Their activity was inhibited by metal- and zinc-specific inhibitors, such as EDTA, EGTA and 1,10-phenanthroline. Specific activity (activity/protein) of type II aureolysin was two times higher than that of type I. These results indicated that the aur gene is highly conserved with two allelic forms (types I and II) among bovine, porcine and avian isolates of S. aureus.     

Population density and range use of sika deer,Cervus nippon, on Mt. Ohdaigahara, central Japan

We investigated the population density and range use of sika deer,Cervus nippon, on Mt. Ohdaigahara, central Japan, from July 1996 to July 1997. The deer population density estimated by the block count was high at 17.5–30.9 deer/km². Although the deer remained this area from spring to fall, they moved out in winter, probably because of deep snow. A spotlight count showed that the deer prefer the eastern area as spring-summer range, where the forest consists of coniferous trees over a floor cover ofSasa nipponica, andS. nipponica grassland. The deer also use dense coniferous forest for cover, especially in summer and fall. Part of this study was presented at the 108th Annual Meeting of the Japanese Forestry Society (1997). This study was financially supported by the Environment Agency, Japan.