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Swine edema disease is caused by Shiga toxin (Stx) 2e-producing Escherichia coli (STEC). Addition of highly concentrated zinc formulations to feed has been used to treat and prevent the disease, but the mechanism of the beneficial effect is unknown. The purpose of the present study was to investigate the effects of highly concentrated zinc formulations on bacterial growth, hemolysin production, and an Stx2e release by STEC in vitro. STEC strain MVH269 isolated from a piglet with edema disease was cultured with zinc oxide (ZnO) or with zinc carbonate (ZnCO 3 ), each at up to 3,000 ppm. There was no effect of zinc addition on bacterial growth. Nonetheless, the cytotoxic activity of Stx2e released into the supernatant was significantly attenuated in the zinc-supplemented media compared to that in the control, with the 50% cytotoxic dose values of 163.2 ± 12.7, 211.6 ± 33.1 and 659.9 ± 84.2 after 24 hr of growth in the presence of ZnO, ZnCO 3 , or no supplemental zinc, respectively. The hemolytic zones around colonies grown on sheep blood agar supplemented with zinc were significantly smaller than those of colonies grown on control agar. Similarly, hemoglobin absorbance after exposure to the supernatants of STEC cultures incubated in sheep blood broth supplemented with zinc was significantly lower than that resulting from exposure to the control supernatant. These in vitro findings indicated that zinc formulations directly impair the factors associated with the virulence of STEC, suggesting a mechanism by which zinc supplementation prevents swine edema disease.

Porcine epidemic diarrhea (PED) was detected for the first time in seven years in Japan in October 2013 in Okinawa Prefecture. By December 2013, PED had spread into Miyazaki and Kagoshima Prefectures in the southern part of Kyushu, one of the regions with the highest farm density in the country. The objective of the study here was to assess the spatial dynamics of PED spread during the 8 months of the epidemic in the southern part of Kyushu between December 2013, the month observed first case in the studied region, and July 2014. Information on location and capacity of all farms in the prefectures (n=1269) was obtained from a government database containing demographic information for livestock producers. Additionally, data on PED detection (positive or negative) was obtained from the regional Livestock Hygiene Service Center. The Cuzick-Edwards (CE) test, the Knox test, the directional test, and the permutation model of the scan statistic were used to assess the spatio-temporal distribution of the epidemic. PED cumulative farm level incidence was 19.5% (248/1269) through the study period. The highest density of positive farms was observed in the most farm-populated areas of the prefecture. The CE test revealed an extensive degree of spatial clustering, with clustering of positive sites being significant (P<0.01) up to the 35th level of neighborhood (approximately 5km in the studied data). The observed-to-expected ratio of cases was maximized at short spatio-temporal distances, with values of the observed-to-expected ratio of cases maximized when the thresholds were set at 2km and 10days, respectively. A significant (P<0.01) direction of spread was detected towards the northeastern direction. The permutation model detected five significant (P<0.01) clusters occurring at different stages of the epidemic wave. The strong spatio-temporal clustering of PED-infected farms during the first 6 months of the epidemic in the southern part of Kyushu is consistent with results obtained elsewhere and demonstrates the rapid spread of the virus in naïve populations., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Porcine epidemic diarrhea (PED) is an emerging disease of pigs in several countries. In the present study, individual sow productivity of Berkshire sows exposed to PED virus at different stages of production was compared. On a commercial farrow-to-finish farm in Kagoshima Prefecture, Japan, the clinical presence of PED was observed in the farrowing barn on January 6, 2014, and all gilts and sows were immunized on January 9, except those in the farrowing barn. The sows were categorized into six groups based on the period in which they were exposed to PED virus: between days 0-30 (G1), 31-60 (G2), 61-90 (G3), or after 91 days of pregnancy (G4), during lactation (L), and after weaning (W). The control group was not exposed to PED during the period of PED outbreak. The study was based on 574 production records. The sows of the G4 and L groups had the fewest piglets weaned (4.8±0.4, and 4.0±0.3 pigs, respectively; P<0.05) and the greatest pre-weaning mortality (33.1±4.8%, and 39.7±4.1%, respectively; P<0.05). The number of piglets weaned and pre-weaning mortality, however, did not differ among the G1, G2, G3, and uninfected groups. The G4 and W groups had slightly lesser farrowing rates than the uninfected group (P<0.05), however, similar subsequent piglet litter performance as the uninfected group. In conclusion, the effect of PED on individual sow productivity differed with the production stage in which sows were exposed to PED virus., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Since late 2013, after an absence of seven years, outbreaks of porcine epidemic diarrhea virus (PEDV) infection have reemerged and swept rapidly across Japan, resulting in significant economic losses. In this study, we report the emergence, mixed infection, and genetic characterization of 15 novel field PEDV variants with large genomic deletions. The sizes of deletion varied between 582 nt (194 aa) and 648 nt (216 aa) at positions 28-714 (10-238) on the S gene (protein). Among 17 PEDV samples isolated from individual pigs, all of them contained at least two distinct genotypes with large genomic deletions, and 94.1% of them were found to consist of strains with an intact S gene. These variants were found in eight primary and nine recurrent outbreaks, and they might be associated with persistent PEDV infection in the farms. Full-length S and ORF3 genes of eight variants derived from 2 samples were characterized. This is the first report of mixed infections caused by various genotypes of PEDV and would be important for the studies of viral isolation, pathogenesis, and molecular epidemiology of the disease., Competing Interests: The authors have declared that no competing interests exist.

We improved a loop-mediated isothermal amplification (LAMP) assay permitting sensitive and rapid Mycoplasma bovis detection. A total of 55 bacterial strains were examined in this study, including 33 M. bovis strains, 14 non-M. bovis mycoplasmas and eight non-mycoplasma bacterial strains. M. bovis was successfully detected by the LAMP assay within 60 min without cross-reaction to any other bacteria. Furthermore, a total of 135 nasal swab samples were tested directly using our LAMP assays, the previously reported LAMP assay, conventional PCR assay without pre-culture and comparing standard culture methods. The improved LAMP assay showed sensitivity and specificity of 97.2% and 90.9%, respectively (with a kappa coefficient of 0.8231), and the sensitivity of our revised LAMP assay was increased compared to existing methods.

This study compared the reproductive and growth performances of pigs before and after depopulation and restocking after a foot-and-mouth disease outbreak in Japan. Data for the time period before and after depopulation and restocking were obtained from three farrow-to-finish farms. As a result of depopulation and restocking, hygiene levels were improved, and common infectious diseases, such as porcine reproductive and respiratory syndrome and Aujeszky's disease, remained undetected on the farms. Compared with before depopulation, reproductive and growth performances improved after depopulation; the number of total pigs born was higher, the postweaning mortality rate was lower, and the age at slaughter was lower (P<0.05). In summary, depopulation and restocking improved the reproductive and growth performances of pigs.

Porcine epidemic diarrhea (PED) is an emerging disease of pigs that has recently led to large numbers of piglet deaths in a number of countries of Eastern Asia and The Americas. The objective of the present study was to identify and compare risk factors associated with PED infection in locally and non-locally PED-exposed farms in Japan. A questionnaire was administered to a convenience selection of pig farms located throughout Japan. Questionnaires were administered between November 2013 (when the first case was reported in Japan) and August 2014. PED-positive farms (cases, n=124) were asked to provide information on their status (positive or negative) and select herd management practices for the two weeks prior to onset of PED clinical signs. Negative farms (controls, n=128) were given the same questionnaire and asked herd management practices for the two weeks prior to a given reference date. This date was assigned based on the date of PED occurrence in the town/prefecture in which the farm was located. Case and control farms were categorized as "locally exposed" if they were located within a 5km radius from a PED-infected farm and "non-locally exposed", otherwise. Logistic regression analysis was used to identify factors associated with PED infection. Two separate regressions were done for locally exposed and non-locally exposed farms using PED status (positive/negative) as the dependent variable. PED in locally-exposed farms was associated (P<0.05) with increased farm size (in 100 pig increments), shorter distances to the closest PED-positive farm (less than1,001m), and a disinfectant contact time of less than 20min. In non-locally exposed farms, PED was associated (P<0.05) with increased feed truck visits to the farm, no visit of the veterinarian, and again a disinfectant contact time of less than 20min. These findings suggest that the mechanisms of PED spread in Japan were different for farms closer to case-farms compared to farms that were further away from PED cases. These results will contribute to understanding the epidemiology of the disease in Japan and will ultimately aid in designing and implementing effective prevention and control strategies in Japan and other regions epidemically infected by the PED virus., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A methanol extract of the flowers of Mammea siamensis (Calophyllaceae) was found to inhibit enzymatic activity against aromatase (IC50=16.5 µg/mL). From the extract, two new geranylated coumarins, mammeasins C (1) and D (2), were isolated together with seven coumarins: 8-hydroxy-5-methyl-7-(3,7-dimethyl-octa-2,6-dienyl)-9-(2-methyl-1-oxobutyl)-4,5-dihydropyrano[4,3,2-de]chromen-2-one (9), 8-hydroxy-5-methyl-7-(3,7-dimethyl-octa-2,6-dienyl)-9-(3-methyl-1-oxobutyl)-4,5-dihydropyrano[4,3,2-de]chromen-2-one (10), mammeas A/AA (14), A/AB (15), A/AA cyclo D (18), E/BA (23), and E/BC cyclo D (25). The structures of 1 and 2 were elucidated on the basis of spectroscopic evidence. Among the isolates including 17 previously reported coumarins, 1 (IC50=2.7 µM), 2 (3.6 µM), and mammea B/AB cyclo D (21, 3.1 µM) showed relatively strong inhibitory activities comparable to the activity of the synthetic nonsteroidal aromatase inhibitor aminoglutethimide (2.0 µM).

The fine crystals of an Al-containing layered silicate, whose negative layer charge is generated by an isomorphous substitution in the tetrahedral SiO4 framework, successfully grew on monodisperse amorphous silica microspheres with diameters of 1.0 and 2.6 μm. The fine, plate-like crystals were observed to thoroughly cover the surface of the silica spheres, irrespective of their size, by the hydrothermal reactions of the silica powder in aqueous alkali solution containing Al and Mg ions in a rotating Teflon-lined autoclave. The crystal size increased when the concentration of the precursors was low. The presence of fluorine in the reaction media enlarged the crystalline phase in the direction of the layer stacking while reducing the plate size. The difference in the crystal size affected the kinetics on the hinokitiol desorption in n-hexane from the layered silicates modified with organoammonium ions. The organically modified layered silicate behaved as an exfoliated nanosheet in the nonpolar solvent. The less harmful elements in this hybrid suggest that it can be used in cosmetic and pharmaceutical applications as a drug support, without flaking off the fine layers on the microspherical substrates.

Since late 2013, outbreaks of porcine epidemic diarrhea virus (PEDV) have reemerged in Japan. In the present study, we observed a high detection rate of PEDV, with 72.5 % (148/204) of diarrhea samples (suckling, weaned, and sows) and 88.5 % (77/87) of farms experiencing acute diarrhea found to be positive for PEDV by reverse transcription PCR. Sequencing and phylogenic analyses of the partial spike gene and ORF3 of PEDV demonstrated that all prevailing Japanese PEDV isolates belonged to novel genotypes that differed from previously reported strains and the two PEDV vaccine strains currently being used in Japan. Sequence and phylogenetic analysis revealed prevailing PEDV isolates in Japan had the greatest genetic similarity to US isolates and were not vaccine-related. Unlike vaccine strains, all prevailing field PEDV isolates in Japan were found to have a number of amino acid differences in the neutralizing epitope domain, COE, which may affect antigenicity and vaccine efficacy. The present study indicates recent PEDV isolates may have been introduced into Japan from overseas and highlights the urgent requirement of novel vaccines for controlling PEDV outbreaks in Japan.

Nucleotide sequence determination and analysis of the cps gene involved in the capsular polysaccharide biosynthesis of Actinobacillus pleuropneumoniae serotype 15 revealed the presence of three open reading frames, designated as cps15ABC genes. At the protein level, Cps15A and Cps15B showed considerably high homology to CpsA (67.0 to 68.7%) and CpsB (31.7 to 36.8%), respectively, of A. pleuropneumoniae serotypes 1, 4 and 12, revealing the common genetic organization of the cps among serotypes 1, 4, 12 and 15. However, Cps15C showed no homology to any proteins of A. pleuropneumoniae serotypes, indicating that cps15C may be specific to serotype 15. This study will provide the basic molecular knowledge necessary for the development of diagnostics and a vaccine for A. pleuropneumoniae serotype 15.

We investigated the associations of hind limb claw lesions with claw measures and reproductive performance of sows on a commercial breeding farm. We used a five-point scale (score of 0-4) for each hind limb claw region to generate a claw lesion score (CLS). Of 110 sows, 1.8% had no hind claw lesions, and the remaining 98.2% had at least one. The proportions of sows with a CLS of 2 and 3 were 49.1% and 3.6%, respectively. The junction region had the highest frequency of a CLS of ≥ 2 (P < 0.05). The mean toe length was 45.0 ± 0.4 to 46.4 ± 0.4 mm. Sows with the highest CLS of ≥ 2 in the lateral claw had longer sole dimensions than did sows with the highest CLS of ≤ 1 (P < 0.05). There were no significant differences in claw width or height between sows with the highest CLS of ≥ 2 and ≤ 1. There was also no difference in reproductive performance between sows with the highest CLS of ≥ 2 and ≤ 1. In conclusion, most sows had hind claw lesions. The association between CLS and claw measures indicates that the sole length can be used as an indicator of claw lesions., (© 2014 Japanese Society of Animal Science.)

The objective of this study was to determine the effects of stillbirth and dystocia on subsequent reproductive performance in Japanese Black cattle. Data were collected for 34,763 calvings from 13,186 animals on 826 farms. Stillbirth was associated with a decreased first service conception rate compared to cows with a normal calving (44.4 versus 54.0%, respectively, P < 0.001). Cows with a normal calving required fewer artificial inseminations per conception and had a lower stillbirth rate at subsequent parity than those with stillbirth and dystocia (P < 0.001). Japanese Black cows with stillbirth and dystocia had inferior subsequent reproductive performance compared with those with a normal calving., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Chloroplasts originated from cyanobacteria through endosymbiosis. The original cyanobacterial endosymbiont evolved to adapt to the biochemically rich intracellular environment of the host cell while maintaining its photosynthetic function; however, no such process has been experimentally demonstrated. Here, we show the adaptation of a model cyanobacterium, Synechocystis sp. PCC 6803, to a biochemically rich environment by experimental evolution. Synechocystis sp. PCC 6803 does not grow in a biochemically rich, chemically defined medium because several amino acids are toxic to the cells at approximately 1 mM. We cultured the cyanobacteria in media with the toxic amino acids at 0.1 mM, then serially transferred the culture, gradually increasing the concentration of the toxic amino acids. The cells evolved to show approximately the same specific growth rate in media with 0 and 1 mM of the toxic amino acid in approximately 84 generations and evolved to grow faster in the media with 1 mM than in the media with 0 mM in approximately 181 generations. We did not detect a statistically significant decrease in the autotrophic growth of the evolved strain in an inorganic medium, indicating the maintenance of the photosynthetic function. Whole-genome resequencing revealed changes in the genes related to the cell membrane and the carboxysome. Moreover, we quantitatively analyzed the evolutionary changes by using simple mathematical models, which evaluated the evolution as an increase in the half-maximal inhibitory concentration (IC50) and estimated quantitative characteristics of the evolutionary process. Our results clearly demonstrate not only the potential of a model cyanobacterium to adapt to a biochemically rich environment without a significant decrease in photosynthetic function but also the properties of its evolutionary process, which sheds light of the evolution of chloroplasts at the initial stage.

Specimens of Neoempheria previously considered to be N. ferruginea (Brunetti, 1912) are studied and revised. Eleven new species are described from Japan, Nepal, Philippines, and Thailand; N. biceltisuta sp. n., N. bifurcata sp. n., N. bisecuriata sp. n., N. brevispathulata sp. n., N. carinata sp. n., N. cuneata sp. n., N. dilatata sp. n., N. denticulata sp. n., N. forficulata sp. n., N. latisternata sp. n., and N. muticata sp. n. Neoempheria sakhalinensis Zaitzev, 2001, is recorded from Japan for the first time. Male genitalia of these 12 species are illustrated. A key to the species is provided. A lectotype of Mycomya ferruginea Brunetti, 1912 is designated (deposited in the Zoological Survey of India, Kolkata) and the species redescribed. Morphological similarities and differences among N. ferruginea and allied species are discussed. The usefulness of the male gonocoxite and associated structures, and the gonapophysis of the female 8th and 9th abdominal sternites for taxonomy are discussed.

We investigated the reproductive and lifetime performances of Kagoshima Berkshire gilts and sows. We examined 20 605 parity records of 4419 pigs for 2008 to 2012 on a farrow-to-finish commercial farm. The mean parity (± SD) of all animals was 3.0 ± 2.1. For farrowing performance, the highest numbers of total pigs born and pigs born alive were found in sows with parities 5 and 6 and with parity 3-6, respectively (P < 0.05). Regarding weaning and mating performance, sows with parity 2 had the lowest preweaning mortality (P < 0.05). The longest weaning-to-first-mating interval was found in parity 1 pigs, and the interval decreased as parity increased (P < 0.05). Parities 0 and 1 pigs had the lowest farrowing rate and those with parity 4 had the highest farrowing rate (P < 0.05). The mean parity at culling, total number of pigs born alive in a lifetime, and nonproductive days in a lifetime were 5.5 ± 2.93, 49.2 ± 24.72 pigs, and 132.1 ± 83.34 days, respectively. These animals had a lower litter size and fertility that the F1 crossbred sows mainly used in Japan, but a similar tendency for performance by parity., (© 2013 Japanese Society of Animal Science.)

Morphological features of a Japanese fly that developes in orchids, Orchidophaga gastrodiacola Kato, 2006, assigned to the family Scathophagidae, are reviewed and this species is proposed as a junior synonym of Chyliza vittata Meigen, 1826, of the family Psilidae. Morphological features of specimens from Japan correspond well with those from European. Japanese specimens of this transpalaearctic species utilize buds and stems of the achlorophyllous mycoheterotrophic Gastrodia elata Blume. This association is an exceptional habitat in comparisons to other congeners and European specimens of the same species.

A methanol extract of the flowers of Mammea siamensis (Calophyllaceae) was found to inhibit nitric oxide (NO) production in lipopolysaccharide-activated RAW264.7 cells. From the extract, two new geranylated coumarins, mammeasins A (1) and B (2), were isolated together with 17 known compounds including 15 coumarins. The structures of 1 and 2 were determined on the basis of their spectroscopic properties as well as of their chemical evidence. Among the isolates, 1 (IC(50) = 1.8 μM), 2 (6.4 μM), surangins B (3, 5.0 μM), C (4, 6.8 μM), and D (5, 6.2 μM), kayeassamins E (7, 6.1 μM), F (8, 6.0 μM), and G (9, 0.8 μM), mammea A/AD (11, 1.3 μM), and mammea E/BB (16, 7.9 μM) showed NO production inhibitory activity. Compounds 1, 9, and 11 were found to inhibit induction of inducible nitric oxide synthase (iNOS). With regard to mechanism of action of these active constituents (1, 9, and 11), suppression of STAT1 activation is suggested to be mainly involved in their suppression of iNOS induction., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Bovine coronavirus (BCoV) and group A bovine rotavirus (BRV) are two of major causes for neonatal calf diarrhea. In the present study, a one-step duplex RT-PCR was established to detect and differentiate BCoV and group A BRV from fecal samples. The sensitivity of this method for BCoV and group A BRV was 10 PFU/100 µl and 1 PFU/100 µl, respectively. Twenty-eight diarrhea fecal samples were detected with this method, the result showed that 2 samples were identified as co-infected with BCoV and group A BRV, 26 samples were group A BRV positive, and 2 samples were negative. It proved that this method is sensitive for clinical fecal samples and is worth applying to laboratory diagnosis for BCoV and group A BRV.

Two new species of the genus Cyamops (Diptera: Periscelididae), the first from New Zealand, are described. The two newly described species are: Cyamops alessandrae and Cyamops crosbyi. A key to the genera of the subfamily Stenomicrinae and to the species of Cyamops from the Australasian/Oceanian Region and detailed illustrations of structures of the male terminalia are provided.

The minimum inhibitory concentration(MIC)of a traditional medicine containing 17 components against 9 strains of Trichophyton verrucosum and 13 strains of Malassezia pachydermatis was determined using a method recommended by the Clinical and Laboratory Standards Institute(CLSI). We also measured the MIC of each of the 17 components using the same method, and identified the main antifungal components.In order to evaluate MIC as a parameter of the antifungal effects using the microdilution method, we prepared 10% working solutions from 10% (w/v)medicines. The geometric mean MIC of the medicinal extract against T. verrucosum was 2.51%, and that against M. pachydermatis was 2.25%. The components that exhibited antifungal effects were Rheum palmatum, Glycyrrhiza uralensis, Magnolia obovata, Phellodendron amurense, and Geranium thunbergii., (Copyright © 2011 The Japanese Society for Medical Mycology)

The antimicrobial susceptibilities of 72 Brachyspira hyodysenteriae isolates collected from clinical cases of swine dysentery (SD) in 11 prefectures in Japan between 1985 and 2009 were investigated by an agar dilution method using five antimicrobial agents. There is a tendency of Japanese field isolates of B. hyodysenteriae to acquire resistance to the main antimicrobials used in SD treatment such as tiamulin, valnemulin, and efrotomycin. A responsible approach for selection and use of antimicrobial agents is required for SD treatment.

We examined the susceptibility in vitro of 99 isolates of Mycoplasma (M. bovirhinis, M. bovis, M. alkalescens and M. bovoculi) and one of Acholeplasma laidlawii collected in 2008 and 2009 from Japanese homebred cattle and cows imported from Australia to 13 antimicrobial agents. Mycoplasma species isolated from homebred cattle were susceptible to spectinomycin and fluoroquinolones but resistant to macrolides, tetracyclines, thiamphenicol and flumequine. Kanamycin, lincomycin and chloramphenicol maintained intermediate effectiveness against these species. The isolates from the imported cows maintained sufficient susceptibility to all antimicrobials examined except for erythromycin and thiamphenicol. These results showed that four species of Mycoplasma isolated from Japanese homebred cattle had obviously higher drug resistance compared with previous reports, even within this decade.

Two 60-day-old pigs showing clinical signs of malgrowth and diarrhea were diagnosed as atypical porcine proliferative enteropathy (PPE). The intestinal mucosal lesions in the piglets were characterized by the adenomatous proliferation of the crypt epithelium together with growth of small curved bacteria within the enterocytes. The lesions could be seen in the ileum and other portions of the intestine histologically, although no significant thickening of the gut wall could be observed grossly in the present case. The macroscopic findings are extremely important for the diagnosis of PPE, however, this paper shows that the histopathological and/or immunohistochemical findings were also critical to identify the disease.

During 2004 and 2006, multidrug-resistant Salmonella enterica subspecies enterica serovar Infantis (Salmonella Infantis) isolates (n = 120) were recovered from broiler cecal samples collected from a meat-processing plant, and the isolates were examined. The study was conducted to detect and characterize extended-spectrum beta-lactamase (ESBL)-producing Salmonella Infantis isolates recovered from broiler chickens and determine the mechanisms of transfer of the resistance traits. Extended-spectrum cephalosporins-resistant Salmonella Infantis isolates producing ESBL TEM-52 were detected. The mutant bla(TEM-52) gene and the wild-type bla(TEM-1) gene that mediated resistance to ampicillin (an extended-spectrum penicillin) and cephalothin (a narrow-spectrum cephalosporin) were located on approximately 50-kb conjugative plasmids among beta-lactam-resistant (n = 29) isolates. The bla(TEM) genes did not cotransfer with aadA1, sul1 (both associated with class 1 integrons), tetA, and dfrA5, signifying a chromosomal location of these non-beta-lactam resistance-encoding genes. This is the first report describing TEM-52-producing S. enterica from food-producing animals in Japan. An emergence of TEM-type ESBL is an important concern to public health because this readily transferable resistance mechanism threatens the value of the third-generation cephalosporins and may reduce the clinical utility of this class of antibiotics against pathogenic Gram-negative bacteria.

An epidemiologic study was conducted to investigate the incidence and characterize the antimicrobial resistance determinants, analyzing plasmid profiles, and establishing the genetic relationship among beta-lactam-resistant isolates of Salmonella Infantis from broilers in Southern Japan. A total of 120 isolates were recovered from 56 flocks belonging to 44 holdings during 2004-2006. The percentages of resistance were as follows: ampicillin (24%), cephalothin (23%), cefoxitin (0%), ceftazidime (11%), cefotaxime (11%), chloramphenicol (0%), kanamycin (7.5%), ofloxacin (20%), oxytetracycline, streptomycin and sulfamethoxazole (100%) and trimethoprim (75%). The incidence of bla(TEM)-encoded beta-lactam resistance in 2004-2006 was significantly higher than in 1998-2003 (P<0.001). BlnI-digested PFGE patterns generated two related clusters implicated in the dissemination of beta-lactam resistance. Two types of plasmid profiles were observed and two plasmids of ca. 50 and 180-kb size were carried by beta-lactam-resistant isolates. Streptomycin resistance was conferred by aadA1 (n=116), aadA1-aadA2 (n=1), and aadA1-strA-strB (n=3). Resistances to kanamycin, oxytetracycline, sulfamethoxazole and trimethoprim were conferred by aphA1 (n=9, 100%), tetA (n=120, 100%) sul1 (n=120, 100%) and dfrA5 (n=90, 100%), respectively. Two types of class 1 integrons were detected: 1.0 kb (n=120) and, 1.0/1.5 kb (n=3). Integrons of 1.0/1.5 kb were found in isolates with the aadA1-strA-strB gene combination. For the first time, all S. Infantis isolates showed resistance to at least three classes of antimicrobial agents; and the intestinal tract of healthy poultry was a reservoir of the extended-spectrum cephalosporin-resistant isolates of serovar Infantis.

Although fibrin sealant (FS) has an advantage of high biocompatibility, its adhesive force and sealing effect have been generally considered to be inadequate. In the present study, a high adhesive force and sealing effect were obtained by first rubbing fibrinogen solution into the target tissue, attaching polyglycolic acid (PGA) felt to the treated area, and finally spraying it with FS. This method was compared with three conventional FS application methods and a method using fibrin glue-coated collagen fleece. The adhesive force resulting from the present method was 12 times higher than that for the sequential application method, 4.5 times higher than the spray method, 2.5 times higher than the rubbing and spray method, and 2.2 times higher than the use of fibrin glue-coated collagen fleece. The high adhesive force of FS with PGA felt seemed to be due the high fibrin content of the fibrin gel (FG). Light and electron microscopic observations suggested that the formation of FG in closer contact with the muscle fibers was a factor contributing to this superior adhesive force. Comparison of the sealing effect of the present method with other methods using various biomaterials in combination with FS showed that the sealing effect of FS with PGA felt was 1.4 times higher that of polyglactin 910, 1.8 times that of polytetrafluoroethylene, and 6.7 times that of oxidized regenerated cellulose.

A prototype rotor with two grooves for the multistage centrifugal isotope separation in solid state was developed to test a new idea. This idea is based on the sedimentation of constitutional atoms in solid. In the performance test using indium specimen, it is verified that the developed rotor can receive all injected molten-indium droplets from an automatic raw-material feeding system even at the high rotational speed of 97,000 rpm without the loss of rotational stability, and the received indium specimens can be transferred in/between two grooves through the plastic flow under the influence of strong centrifugal force even in the solid state. The isotope ratio of centrifuged indium specimens was analyzed employing the secondary ion mass spectrometry, and it is confirmed that intended isotope separation by the centrifugation is realized in the solid state. The developed rotor can be used to perform the isotope separation on at least solid metals under the conditions of up to 400 degrees C in specimen temperature and 0.4x10(6)g in centrifugal force field.

We screened extracts of 342 species of marine algae collected from Japanese coastlines for antibacterial activity against Propionibacterium acnes, and found a novel antibacterial compound, which we named Sargafuran, from the MeOH extract of the marine brown alga, Sargassum macrocarpum. Sargafuran has low cytotoxicity, and the MIC against P. acnes was 15 microg ml(-1), showing a broad antibacterial activity against Gram-positive bacteria. A time-kill study showed that Sargafuran was bactericidal and completely killed P. acnes at 4 x MIC by lysing bacterial cells. These results suggest that Sargafuran might be useful as a lead compound to develop new types of anti-P. acnes substances and new skin care cosmetics to prevent or improve acne.

We examined the usefulness of PCR-based restriction fragment length polymorphism (PCR-RFLP) and species-specific PCR combined with a newly devised rapid biochemical test using microplates for identifying weakly beta-hemolytic intestinal spirochetes (WBHIS) isolated from pigs. WBHIS strains showing atypical biochemical characteristics were decisively identified at the species level by PCR-RFLP and species-specific PCR. Identification of WBHIS at the species level in routine diagnostic work will certainly contribute to clarifying the pathogenicity of WBHIS.

Antimicrobial susceptibility and resistance genes of 135 strains of Salmonella enterica serovar Infantis isolated from poultry in Kagoshima were examined. One strain (0.7%) was resistant to ampicillin (A), 97% to streptomycin (S), 95.6% to sulphamethoxazole (Su), 96.3% to oxytetracycline (T), 11.1% to kanamycin (Km) and 36.3% to ofloxacin (O). Multiresistant phenotypes identified were ASSuT-Km, SSuT-Km, SSuT-O and SSuT. Class 1 integrons were detected in 94.8% of isolates. Approximately 89% of oxytetracycline-resistant strains carried the tetA gene and all of the 131 streptomycin-resistant isolates carried the aadA1a gene. Forty-percent of kanamycin-resistant isolates carried the aphA1-Iab gene. All isolates were susceptible to chloramphenicol. Recognition of TEM-type beta-lactamase in a S. Infantis strain from chickens is a recent rare finding in Japan.

To determine the teratogenic potential of Aino virus (AINOV) in cattle, pregnant cows and fetal cattle were infected with a fresh isolate of AINOV. Five pregnant cows were inoculated intravenously with the virus at 122 to 162 days of gestation and allowed to give birth. All of the cows developed neutralizing antibodies to the virus, indicating that the cows had been infected with the virus; however, no clinical abnormalities were seen in their six newborn calves, and no specific antibodies to the virus were detected in the precolostral serum of calves. Five fetuses with fetal ages ranging from 132 to 156 days were inoculated in utero with the virus. One weak newborn and four stillborn calves were delivered at gestation days 256 to 263, i.e., less than the standard gestation term; they had congenital abnormalities including arthrogryposis, hydranencephaly and cerebellar hypoplasia. Antibodies specific to AINOV were detected in their precolostral serum. These results demonstrate that AINOV is a potential etiological agent of congenital malformation of cattle.

Edema disease (ED) of pigs is an enterotoxaemic disease caused by enterotoxaemic Escherichia coli (ETEEC) infection. Antimicrobial therapy for pigs with ED is controversial because it may induce death of sickish piglets. In this study, we investigated the effects in vitro of 7 antimicrobial agents, ampicillin, gentamicin, colistin, bicozamycin, fosfomycin, sulfamethoxazole-trimethoprim and enrofloxacin, on the release and production of shiga toxin (Stx) 2e by ETEEC strains. We found that more Stx 2e accumulated in the bacterial cells than was released into supernatant. Associated with inhibition of cell wall synthesis, the exposure to ampicillin or fosfomycin increased the release of Stx 2e. The production levels of Stx 2e in all antimicrobial-treated cultures were equal to the level in the control or less than in the control. These results suggest that cell wall synthesis inhibitors, such as ampicillin and fosfomycin, may change for the worse in the signs in ETEEC infectious pigs. On the other hand, gentamicin, colistin, bicozamycin and enrofloxacin may be useful for the treatment of pigs with ED.

Using the rapid glass plate method, the Newcastle disease virus strain MET95 showed much weaker haemagglutination (HA) activity for chicken erythrocytes than 69 other Newcastle disease viruses, including 56 field strains isolated from chickens reared in Japan between 1988 and 2001. Using erythrocytes from other avian species, only the MET95 strain failed to show HA activity for erythrocytes from ducks, geese or pigeons. The haemagglutinin-neuraminidase protein of the MET95 strain was shown to have unique substitutions of isoleucine for thereonine and leucine at amino acide residues 216 and 552. It is suggested that these two substitutions might relate to the unique HA activity of the MET95 strain. This HA activity may be a useful marker for this strain.

Fifty-seven Shiga toxin-producing Escherichia coli (STEC) strains isolated from pigs with edema disease (ED) from 1997 to 2001 in Japan were examined for antimicrobial susceptibilities. The susceptibilities were compared with those of E. coli ATCC 23546 isolated from pig with ED in the 1950's. Consequently, the isolated STECs showed high susceptibility to peptides and bicozamycin in a way similar to the reference strain. On the other hand, the STECs showed low susceptibility to beta-lactams, tetracyclines, novobiocin, fosfomycin, trimethoprim, and old quinolones. It became clear that the susceptibilities of the isolated STECs had diminished in regard to antimicrobials.

Seven adult free-ranging sika deer (Cervus nippon yesoensis) were examined by histology, immunohistochemistry and electron microscopy for intestinal spirochetal infection. Histologically epithelial and goblet cell hyperplasia and edema of the lamina propria mucosa with macrophage and lymphocyte infiltration were observed in the cecum and colon in 6 of the 7 deer. Numerous argyrophilic spirochetes were present in the crypts and some had invaded epithelial and goblet cells and caused degeneration. Immunohistochemically the organisms stained positively with polyclonal antisera against Brachyspira (Serpulina) hyodysenteriae and B. pilosicoli. Ultrastructurally they were 6-14 microm long, 0.2-0.3 microm wide and had 4-6 coils and 13 axial filaments per cell; such features were closely similar to those in the Brachyspira species. These results showed that the spirochetes were capable of inducing enteritis in deer and this intestinal spirochete infection might already be prevalent among wild sika deer in Japan. There is a possibility that this spirochetal colitis is a new syndrome in sika deer and that the same and/or similar spirochetes have infected ruminants, including sika deer and cattle.

Fifty-nine calves, aged 11 days to 9 months, from three farms breeding Japanese Black beef cattle in Miyazaki Prefecture, Japan, were examined for the presence of Shiga toxin-producing Escherichia coli (STEC). A high prevalence of STEC was detected among calves, with 45 (76.3%) animals carrying STEC including different serogroups (O26, O74, O111, O114, O119, O127, O153, O157, and ONT) and toxin types. The number of STEC in the feces was estimated by a combined method involving enumeration of colony-forming units by a plate-most-probable-number (plate-MPN) technique and polymerase chain reaction for the detection of Shiga toxin genes. Fecal shedding ranged from 10(1) to 10(10) MPN/g feces. To evaluate the safety and efficacy of bicozamycin (BCM: previously named as bicyclomycin) in eradicating STEC, 30 calves carrying STEC with or without diarrhea were examined. Fifteen calves were treated orally with BCM (10 mg/kg/day) once daily for 5 days, and the other 15 were untreated. Twenty-four hours after the last dose, fecal specimens were collected from both groups to compare the number of coliforms and STEC with those before treatment. BCM-treated animals had a significantly lower number of coliforms and STEC compared to the untreated calves. The STEC eradication rate was 86.6% (13/15) in the BCM-treated group, compared to 0% (0/15) in the control group. The corresponding cure rates for diarrhea were 87.5 (7/8) and 0% (0/3), respectively. No adverse reactions were observed in the calves during treatment. It is concluded that BCM is an effective agent for the eradication of STEC in calves with or without diarrhea.

We examined the preventative effect against enterohemorrhagic Escherichia coli O157:H7 (EHEC) infection in rabbits on administration of probiotics contained in Streptococcus faecalis, Clostridium butyricum and Bacillus mesentericus. The probiotics were administered to 5 days old Japanese white rabbits throughout the experiment. Inoculation of 10(6) colony forming units (CFUs) of EHEC per rabbit was tried 8 days after birth. Three days after EHEC inoculation, in the control group (not administered probiotics), diarrhea was observed in approximately 80% of the rabbits, on the other hand, in the group administered probiotics, rabbits showed diarrhea in 15%. In necropsy, the number of EHEC in the contents of the cecum and the number of attaching and effacing (AE) lesions were fewer in the probiotics group than in the control group. These results suggest that infant rabbits are useful as a colitis model for EHEC, and the probiotics are effective for the prevention of the growth of EHEC in the intestine and for diarrhea.

A comparative study on the adhesion of attaching and effacing Escherichia coli (AEEC) to the enterocytes between the colon of a calf and the jejunum of a piglet showed differences in the developmental process of attaching and effacing (AE) lesions. In the calf, pedestals consisted of fused microvilli, while in the piglet they developed from the apical epithelial cell membranes after effacing microvilli. Microvilli adjacent to the AEEC attachment site were atrophic in the calf, whereas they were elongated in the piglet. The production of AE lesions in the calf may be indicative of a novel developmental process with AEEC infection.

The adherent site of "attaching and effacing Escherichia coli" (AEEC; O103: H-, SK-1 strain) on the intestinal epithelial cells of chicks infected naturally and experimentally was ultrastructurally investigated. The eaeA gene was detected by polymerase chain reaction in the SK-1 strain of E. coli isolated from the intestinal content of a chick infected naturally, however, the bundle-forming pilus (bfp) gene could not be detected. The SK-1 strain (bfp-) of AEEC could attach to the intestinal epithelial cell and induce attaching-effacing lesions in the intestine of chicks. Transmission electron microscopy revealed numerous pilus-like microfilaments in the space between colibacilli and the membranes of the intestinal epithelial cells. The present study suggests that SK-1 strain (eaeA+, bfp-) may attach closely to the intestinal epithelial cells by a novel adhesion different from bfp.

The existence of natural infection with attaching and effacing Escherichia coli (AEEC) in chicks was reported. Numerous Gram-negative bacilli were attached to the enterocyte in association with lesions characterized on a wavy appearance of the mucosal surface in the intestines of six chicks. Immunohistochemically, these bacteria reacted positively with antiserum to Escherichia coli (O 103). By electron microscopy, numerous colibacilli were seen to be closely attached to the surface membranes of the enterocytes. In regions of bacterial attachment, almost all cell microvilli were effaced, and some of the remaining ones were elongated and/or disoriented. Part of the cell membrane formed a cup invagination and pedestal-like protrusion associated with the attached bacteria. A concentration of electron-dense material was seen beneath the adherent organisms. Bacteriologically, numerous E. coli (O 103:H-) were isolated from the jejunal contents of two chicks and the E. coli did not produce verotoxin or enterotoxin. The characteristic lesions could also be induced in the cecal mucosa of young chicks experimentally inoculated with the isolated E. coli. This is the first report of natural infection in chicks with AEEC.

Western blotting was used to analyse the antigens in extracts of adult and infective larvae (SIL) of Strongyloides papillosus with serum of infected rabbits. Several proteins in both the extracts reacted with immunoglobulin G in the serum. A protein of about 16 kDa in the extract of SIL showed an especially strong reaction. No cross-reaction was observed among proteins in the extracts of adult worms of such cattle helminths as Fasciola gigantica, Homalogaster paloniae, Trichuris discolor and Setaria digitata to S. papillosus-infected rabbit serum. The cuticle of the worms and substances in the worm tracks showed positive reaction with the immunoperoxidase test on SIL with the serum.

The radiopacity of zinc phosphate, polycarboxylate and glass ionomer dental luting materials was determined and the values were compared with those of tooth substrates. The radiographs of the 2 mm thick specimens were taken together with tooth slices and pure aluminum step wedges. The radiographic density of the specimens was measured using a densitometer and expressed in terms of the equivalent thickness of aluminum per unit thickness of material. All the zinc phosphate and polycarboxylate cements showed radiopacity values greater than that of enamel (3.5), while one glass ionomer material showed a radiopacity value lower than that of dentin (2.0). A radiopacity value greater than 3.5 may be desirable for the detection of luting materials or for their distinction from decayed tooth structure on the dental radiograph.

The suitability of duck egg shell (DES) for chick embryo culture was investigated. Chick embryos were transferred into DESs with all egg contents after 3 days of normal incubation and cultured. The vessels made of polyethylene cling film were used for shell-less control. Among 35 embryos cultured in DESs, 21 survived until 16 days of incubation (13 days after transfer) and finally 3 newly hatched chicks were obtained at 22 days of incubation. One of them died 4 days later, but remaining two became full-grown cocks showing normal body weight and production of fertile sperms. Among 37 embryos cultured in polyethylene vessels, none survived over the period of 19 days of incubation. It is suggested that DES culture system may be useful for the various experiments using chick embryos.

In young broiler chicks which were inoculated with 10(8) cells of Treponema hyodysenteriae within 24 hr after hatching, numerous treponemes were observed by scanning electron microscopy on the surface of the cecal mucosa 7 and 14 days after the inoculation. However, in the groups inoculated with 10(7) cells, treponemes were not observed on the cecal mucosa 14 days after the inoculation, and the isolation rate from the cecal contents was lower than that from cecal contents of chicks inoculated with 10(8) cells. While the cecal mucosa of noninfected chicks had a smooth surface, that of the chicks infected with treponemes was generally roughened and the epithelium was eroded. Numerous treponemes were also observed within the eroded epithelium.

Observation by scanning electron microscopy served to demonstrate that Treponema hyodysenteriae colonized the cecal mucosa of young chicks on day 3 after inoculation and produced lesions from 9 days after inoculation. Severe and clearly visible lesions were present on the cecal mucosa of all chicks necropsied 18, 21, and 24 days after inoculation. Severe lesions such as roughness of mucosal surface and disappearance of the mucosal foldings were observed at low magnification. The lesions were characterized by a destruction of superficial epithelial cells and dilation of crypt orifices. The dilated orifices of crypts were clearly observed, and the number of the crypts increased as compared with that of the crypts on the cecal surface in non-infected chicks. At high magnification, mucosal epithelial cells were found to be desquamated and the surface of the lamina propria mucosae had appeared. Numerous treponemas became visible on the surface of damaged epithelial cells and on the surface of the lamina propria mucosae.