Relationship between phylogenetic groups of Escherichia coli and Pathogenicity among Isolates from chickens with Colibacillosis and healthy chickens.

Avian pathogenic Escherichia coli (APEC) is closely related to extraintestinal pathogenic E. coli, which are frequently assigned to specific phylogenetic groups (phylogroups). Therefore, we investigated the association between phylogroups of E. coli isolates and those recovered from commercial broiler and layer chickens with colibacillosis. We used 104 E. coli isolates from chickens with colibacillosis (hereafter referred to as "colibacillosis-related isolates"), 56 E. coli isolates obtained from fecal samples of clinically healthy broiler chickens, and 58 isolates obtained from environmental samples of layer chicken housing facilities where clinically healthy layer chickens were reared (hereafter referred to as "healthy chicken-related isolates"). The prevalence of phylogroup F among colibacillosis-related isolates was significantly (P < 0.05) higher than that among healthy chicken-related isolates, while phylogroups A and B1 were more frequently distributed in healthy chicken-related isolates. Fifty-seven (87%) of 65 colibacillosis-related isolates belonging to phylogroup F were defined as APEC based on the presence of virulence-associated genes according to a previously established criterion. In contrast, none of the healthy chicken-related isolates were defined as APEC. As evidenced by the chicken embryo lethality assay, 87 of the 92 healthy chicken-related isolates tested had embryo lethality rates of <30% and were considered avirulent, whereas 59 of the 104 colibacillosis-related isolates were considered virulent. Nonetheless, among isolates exhibiting embryo lethality rates of <30%, the mean lethality rate of embryos inoculated with colibacillosis-related isolates was significantly higher than that of embryos inoculated with healthy chicken-related isolates. These observations suggest that phylogroup F predicts colibacillosis among E. coli strains with virulence-associated genes.

Prevalence of qnrS-positive Escherichia coli from chicken in Thailand and possible co-selection of isolates with plasmids carrying qnrS and trimethoprim-resistance genes under farm use of trimethoprim.

One hundred and twenty chicken samples from feces (n = 80), the carcass surface at slaughter at 2 meat chicken farms (n = 20), and retail chicken meat from 5 markets (n = 20) collected during 2018 and 2019 were examined for the prevalence of plasmid-mediated quinolone resistance (PMQR) in Escherichia coli. We detected qnrS-positive E. coli in a total of 74 samples from feces (n = 59), the carcass surface (n = 7), and retail meat (n = 8). These 74 qnrS-positive isolates were tested for antimicrobial susceptibility to determine the minimum inhibitory concentrations (MICs) of certain antimicrobials and genetically characterized. Ampicillin-resistance accounted for 71 of the 74 isolates (96%), followed by resistance to oxytetracycline (57/74; 77%), enrofloxacin (ERFX) (56/74; 76%), sulfisoxazole (SUL) (56/74; 76%), trimethoprim (TMP) (49/74; 66%), and dihydrostreptomycin (48/74; 65%). All farm-borne SUL- and TMP-resistant isolates except one were obtained from samples from farm A where a combination of sulfadiazine and TMP was administered to the chickens. Concentrations of ERFX at which 50 and 90% of isolates were inhibited were 2 µg/mL and 32 µg/mL, respectively. Diverse pulsed-field gel electrophoresis (PFGE) patterns of XbaI-digested genomic DNA were observed in the qnrS-positive isolates from fecal samples. Several isolates from feces and the carcass surface had identical XbaI-digested PFGE patterns. S1-nuclease PFGE and Southern blot analysis demonstrated that 7 of 11 dfrA13-positive fecal isolates carried both the qnrS and dfrA13 genes on the same plasmid, and 2 of 3 dfrA1-positive isolates similarly carried both qnrS and dfrA1 on the same plasmid, although the PFGE patterns of XbaI-digested genomic DNA of the isolates were different. These results suggest that the qnrS gene is prevalent in chicken farms via horizontal transfer of plasmids and may partly be co-selected under the use of TMP.

Genetic Characterization of CTX-M-2-Producing Klebsiella pneumoniae and Klebsiella oxytoca Associated With Bovine Mastitis in Japan.

CTX-M-2-producing Klebsiella oxytoca (K. oxytoca) has not received much attention in animal husbandry compared with Klebsiella pneumoniae (K. pneumoniae), a major reservoir of extended-spectrum ß-lactamase (ESBL) genes. Bacteriological examinations of 1,466 mastitic milk samples between October 2012 and December 2014 were conducted. Ninety-five K. pneumoniae isolates (total prevalence: 6.5%) and 81 K. oxytoca isolates (total prevalence: 5.5%) were obtained. Seventeen K. pneumoniae isolates obtained from 15 animals reared on 11 farms and 9 K. oxytoca isolates obtained from 9 animals reared on the same farm were phenotypically confirmed to be ESBL producers. All nine ESBL-producing K. oxytoca isolates were obtained from one farm between June and November 2013 and related to a significantly (p < 0.05) higher monthly prevalence of mild mastitis (in June, August, September, October, and November 2013). Pulsed-field gel electrophoresis (PFGE) patterns of ESBL-producing K. pneumoniae isolates were distinguished from each other by more than 6-band differences except for two isolates from two animals, whereas all nine K. oxytoca isolates showed an identical PFGE pattern. Transferability of the bla CTX-M-2 gene was found in 14 K. pneumoniae and 9 K. oxytoca isolates by conjugation analysis. Of these isolates, the bla CTX-M-2 gene was detected on plasmids belonging to the incompatibility (Inc) groups P and N derived from five K. pneumoniae and nine K. oxytoca isolates, respectively, although the plasmids from the remaining nine K. pneumoniae were untypeable. All the transconjugants exhibited elevated minimum inhibitory concentrations of ampicillin, cefotaxime, and ceftiofur compared with those in the wild-type, recipient strain. Restriction fragment length polymorphism analysis demonstrated that the IncN plasmids extracted from eight of nine transconjugants, which received resistance against ß-lactams from K. oxytoca, showed an identical DraI digestion pattern. These results suggest that the CTX-M-2-producing K. oxytoca strain with the above-mentioned characteristics may have clonally spread within a farm, whereas the bla CTX-M-2 gene in K. pneumoniae possibly disseminated among the farms through different plasmids. Thus, monitoring of ESBL genes, including the bla CTX-M-2 gene, among causative agents of bacterial mastitis in cows can help to develop relevant treatments and control practices.

Research Note: Longitudinal monitoring of chicken houses in a commercial layer farm for antimicrobial resistance in Escherichia coli with special reference to plasmid-mediated quinolone resistance.

Plasmid-mediated quinolone resistance (PMQR) genes located on conjugative plasmids can be transferred to other bacteria in the absence of antimicrobial selective pressure. To elucidate the prevalence of resistance, including PMQR in an egg-producing commercial layer farm in western Japan where no antimicrobials were used, minimum inhibitory concentrations (MIC) for a total of 375 Escherichia coli isolates obtained from chicken houses in the farm between 2012 and 2017 were determined using the agar dilution methods. Eighty-seven isolates resistant to oxytetracycline (OTC) accounted for 23.0% of the tested isolates, followed by isolates resistant to dihydrostreptomycin (DSM) (18.4%), sulfisoxazole (18.1%), ampicillin (AMP) (14.4%), trimethoprim (TMP) (14.4%), and nalidixic acid (10.1%). The prevalence rate of multidrug-resistant (MDR) isolates-which are resistant to 3 or more antimicrobial classes, including ß-lactams, aminoglycosides, quinolones, folate pathway inhibitors, tetracyclines, and phenicols-was inversely related to the age of chickens at the time of bacterial examination. Probably, the prevalence of MDR isolates in layer chickens may have decreased with age owing to the absence of selective pressure. Furthermore, 45 isolates exhibiting enrofloxacin MICs of more than 0.25 µg/mL were examined for PMQR genes. The transfer of PMQR genes was tested by conjugation analysis. Southern blot analysis of genomic DNA revealed that the qnrS1 (5 isolates), qnrS2 (1 isolate), and qnrS13 genes (1 isolate) were located on plasmids with sizes ranging from approximately 60 to 120 kpb. In 1 of the 5 qnrS1-positive isolates and in an isolate with qnrS13, the qnrS genes were transferred to recipient strains. The plasmid harboring the qnrS1 gene was typed as IncF by PCR-based replicon typing. On this plasmid, the blaTEM, aadA, tetA, and dfrA1 genes responsible for resistance to AMP, DSM, OTC, and TMP, respectively, were detected. The tetA gene was detected in the plasmid harboring the qnrS13 gene, which was typed as IncI1. These results suggest that despite the low prevalence of quinolone resistance in this farm, various PMQR genes, located on diverse plasmids, exist.

Outbreaks of highly pathogenic avian influenza in zoo birds caused by HA clade 2.3.4.4 H5N6 subtype viruses in Japan in 2016.

In late 2016, two zoos, one in northern Japan and the other in central Japan, experienced highly pathogenic avian influenza (HPAI) outbreaks, in which multiple zoo birds were infected with H5N6 subtype HPAI virus (HPAIV). Here, we report an overview of these HPAI outbreaks. HPAIV infections were confirmed by virus isolation in three black swans (Cygnus atratus) and three snowy owls (Bubo scandiacus) kept in the Omoriyama Zoo hospital. At Higashiyama Zoo and Botanical Gardens, following the death of a black swan at a zoo pond, nine waterfowl, including two black swans, four cackling geese (Branta hutchinsii leucopareia), two mallards (Anas platyrhynchos), and a wigeon (Anas penelope), died after HPAIV infection in isolation facilities. Based on the presence of H5-specific antibodies in their sera, two surviving black swans and a surviving mallard at Higashiyama Zoo appeared to have HPAIV infection, although the virus was not isolated. The detectable levels of antibodies (≥10 HI) were maintained for at least 5-9 months, as determined by haemagglutinin inhibition test. Isolation of two H5N6 subtype HPAIVs from an open-air pond where affected zoo birds were previously housed at Higashiyama Zoo strongly indicates that wild waterfowl associated with aquatic environments brought the virus to the zoo. The phylogenetic relationships of the 18 isolates indicated direct viral transmission among birds within each zoo. In both zoos, containment of suspected birds in isolation facilities might have allowed the virus spread among birds inside the facility. However, maintaining containment measures and strict sanitation procedures could facilitate successful physical containment and clearance of HPAIV in both zoos.

Plasmid-mediated quinolone resistance in Escherichia coli isolates from commercial broiler chickens and selection of fluoroquinolone-resistant mutants.

Plasmid-mediated quinolone resistance (PMQR) is a potential concern for animal husbandry and public health. Escherichia coli isolates from a total of 109 fecal samples collected from 6 commercial broiler farms between 2007 and 2011 were examined for PMQR genes, and transfer of these genes was tested by conjugation analysis to elucidate the prevalence and spread of PMQR in broiler chickens. Two isolates from 2 farms harbored the aac(6')-Ib-cr gene that was not detected in plasmids using Southern blot analysis of S1 nuclease-digested genomic DNA separated by pulsed-field gel electrophoresis. In these 2 isolates, nucleotide mutations in the gyrA and parC genes that result in amino acid substitutions were detected. Additionally, a total of 6 isolates originating from 6 chickens from the 2 farms were positive for the qnrS1 gene. In 2 of the 6 isolates, the qnrS1 gene was transferred to a recipient strain. Two transconjugants harboring the qnrS1 gene were cultured on media supplemented with successively higher concentrations of enrofloxacin (ERFX). After a 5-time subcultivation, the ERFX MICs reached 8 and 16 µg/mL, and no nucleotide mutations were detected in the gyrA, gyrB, parC, and parE genes. Our results suggest that the prevalence of PMQR was relatively low in broiler chickens and that exposure of bacteria carrying PMQR genes to the selective pressure of fluoroquinolones can result in resistance to fluoroquinolone, which is not caused by mutations in genes encoding topoisomerases.

Genotypic and phenotypic characterization of Salmonella enterica subsp. enterica serovar Typhimurium monophasic variants isolated in Thailand and Japan.

Monophasic variants of Salmonella enterica serovar Typhimurium isolated in Thailand and Japan were characterized to elucidate the genetic basis of the monophasic phenotype, genetic relatedness, and antimicrobial resistance. A total of 20 Salmonella isolates agglutinated with anti-O4 and anti-H:i serum and not agglutinated with either anti-H:1 or anti-H:2 serum were identified as monophasic variants of Salmonella serovar Typhimurium because they harbored IS200, specific to this serovar, and lacked the fljB gene. An allele-specific PCR-based genotyping method that detects a clade-specific single nucleotide polymorphism indicated that seven swine isolates and one human isolate from Thailand were grouped into clade 1; five isolates from layer chicken houses and layer chicken feces from Japan were grouped into clade 8, together with two Salmonella serovar Typhimurium isolates from chicken houses in Japan; and five isolates from swine feces from Thailand and two isolates from layer chicken feces from Japan were grouped into clade 9. Multilocus sequencing typing demonstrated that sequence type (ST) 34 isolates were solely grouped into clade 9. Clade 1 and 8 isolates were assigned as ST19. Pulsed-field gel electrophoresis revealed multiple types within each of the clades. The presence of antimicrobial resistance genes and plasmid replicon type, of the clade 1 and 9 isolates were comparable to those reported for epidemic strains of monophasic variants. Our results suggest that monitoring monophasic variants of serovar Typhimurium is important for understanding of the spread of these variants in Thailand and Japan.

Characterization of Aerococcus viridans isolated from milk samples from cows with mastitis and manure samples.

Thirty-eight Aerococcus viridans isolates were obtained from milk from 478 cows with clinical mastitis in a farm during the periods between November 2011 and February 2012, and between December 2012 and March 2013. Additional isolates were obtained from processed manure (a mixture of composted manure, straw and hydrated lime) and bedding materials. The processed manure was later used to cover the floor of the stalls in barns as bedding materials. The temperatures recorded in the composted and processed manure were not as high as those generally observed during satisfactory composting. To reveal the association of A. viridans in manure-related products with intramammary infection in cows, isolates were characterized by their DNA fragment patterns as determined by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. Isolates obtained from milk, processed manure and bedding materials had identical DNA fragment patterns. Antimicrobial susceptibilities were determined for 29 isolates from milk, processed manure and bedding materials. Of these, 26 (89.7%) were resistant to clindamycin, whereas virtually all the isolates were susceptible to 12 other antimicrobials including cefalosporins that have been used to treat bovine mastitis in Japan. In vitro, three A. viridans isolates from milk and an isolate from processed manure survived for 3 hr in Good's buffer (pH 9) at high temperature (50°C). The results suggest that the processed manure and bedding materials in this farm were possible sources of A. viridans that caused infection in the cows with mastitis.

Intestinal carriage and excretion of Campylobacter jejuni in chickens exposed at different ages.

Campylobacter jejuni is usually recovered from chickens in commercial broiler farms after 2 to 3 weeks of age. This study was conducted to clarify whether fecal excretion is associated with the age of exposure to this bacterium. Day-of-hatch broiler chickens were separated from a flock in a local commercial farm, kept in isolation rooms, and esophageally inoculated with C. jejuni (5.5 × 10(7) to 5.4 × 10(8) CFU) at 0, 7, 14, 21, 28, and 35 days of age. The remaining chicks were placed on the farm. Fecal samples obtained from the birds with the experimental infection and those reared on the farm were monitored for C. jejuni. Cecal contents obtained on necropsy were also cultured. In chickens inoculated with C. jejuni at 0 to 14 days of age, fecal excretion of C. jejuni was not observed until 42 days of age, although the organism was recovered from the cecal contents of these birds. When chickens were inoculated at 21 to 35 days of age, C. jejuni was isolated from fecal samples 2 or 3 days after inoculation, and the birds continually shed the organism until they reached 49 days of age, with the maximal numbers of the organism ranging from 1.7 × 10(8) to 1.0 × 10(10) CFU/g. In the commercial broiler farm, C. jejuni was first isolated from fecal samples obtained from two of five chickens at 28 days of age, and the organism was isolated from all five birds tested at 43 days of age. Restriction fragment length polymorphism analysis of the fla gene of C. jejuni isolates revealed that birds on the farm were colonized with C. jejuni after placement of the chickens on the farm. These observations indicate that chickens younger than 2 to 3 weeks old may carry C. jejuni in the ceca if they were exposed to this organism. Our results also suggest that fecal excretion of C. jejuni in commercial broiler chickens older than 3 to 4 weeks of age may be mainly caused by exposure of chickens at this age to this organism.

CTX-M-type extended-spectrum ß-lactamase-producing Klebsiella pneumoniae isolated from cases of bovine mastitis in Japan.

Three Klebsiella pneumoniae isolates producing extended-spectrum beta-lactamase (ESBL) were obtained from three dairy cows with clinical mastitis in two farms in western Japan. Two of the 3 isolates from cows in different farms were able to transfer plasmids carrying the blaCTX-M-2 gene to Escherichia coli recipient. Pulsed-field gel electrophoresis (PFGE) patterns of the 2 isolates were different from each other, although restricted-fragment patterns of the two conjugative plasmids were similar to each other. Additionally, PCR-based replicon typing revealed that both the plasmids belonged to type Inc.T. These results suggest that ESBL-encoding genes can be distributed in bacteria on dairy farms through the plasmids.

The characterization of low pathogenic avian influenza viruses isolated from wild birds in northern Vietnam from 2006 to 2009.

Due to concerns that wild birds could possibly spread H5N1 viruses, surveillance was conducted to monitor the types of avian influenza viruses circulating among the wild birds migrating to or inhabiting in northern Vietnam from 2006 to 2009. An H5N2 virus isolated from a Eurasian woodcock had a close phylogenetic relationship to H5 viruses recently isolated in South Korea and Japan, suggesting that H5N2 has been shared between Vietnam, South Korea, and Japan. An H9N2 virus isolated from a Chinese Hwamei was closely related to two H9N2 viruses that were isolated from humans in Hong Kong in 2009, suggesting that an H9N2 strain relevant to the human isolates had been transmitted to and maintained among the wild bird population in Vietnam and South China. The results support the idea that wild bird species play a significant role in the spread and maintenance of avian influenza and that this also occurs in Vietnam.

Antibody survey on avian influenza viruses using egg yolks of ducks in Hanoi between 2010 and 2012.

In Vietnam, numerous surveillance programs are conducted to monitor the prevalence of avian influenza (AI) viruses. Three serological methods-the agar-gel immunodiffusion test, hemagglutination inhibition (HI) test, and enzyme-linked immunosorbent assay-are well established for detection of AI virus antibodies in poultry sera. Several recent reports have validated egg yolk as an alternative source for detection of AI virus antibodies. In this study, we investigated AI virus antibodies in ducks by HI testing using egg yolk. Ten duck eggs were collected every month from 10 randomly selected markets in Hanoi from April 2010 to March 2012. The HI test was performed using low pathogenic avian influenza (LPAI) viruses (H3, H4, H6, H7, H9, and H11 subtypes) and highly pathogenic avian influenza (HPAI) viruses (H5N1 clade 2.3.4 and 2.3.2.1) as antigens. HI testing for H3, H6, and H9 was 29% positive in November 2010, 50% positive in October and November 2010, and 12% positive in June 2011. These results indicated that several epidemics of LPAI viruses had occurred during the study period. In addition, antibodies against H7 were negative. The results of HI testing for H5N1 showed that the reactivity of the dominant HI antibody shifted from H5N1 clade 2.3.4 to clade 2.3.2.1. In conclusion, egg yolk is useful for long term monitoring of AI virus antibodies and the use of egg-based antibody detection may contribute to improvements in animal welfare.

Isolation and characterization of H6N1 and H9N2 avian influenza viruses from Ducks in Hanoi, Vietnam.

We report the genetic characterization of low pathogenic avian influenza (LPAI) viruses isolated from domestic ducks in northern Vietnam in 2009. In total, 22 influenza A viruses consisting of 21 H6N1 subtypes and one H9N2 subtype were isolated from 1488 ducks collected in February, March, and April 2009, accounting the overall virus isolation rate for 1.5%. No H5N1 strain was isolated in this study. Phylogenetic analysis indicated that all the eight genes of the H6N1 and H9N2 subtypes analyzed in this study were similar to those isolated in Korea, southeast China and northern Japan, and wild birds which migrate along the coastal East Asian Flyway are estimated to transmit these viruses. There was no evidence that the H6N1 and H9N2 subtypes share the gene segments with H5N1 subtypes. However, it is important to monitor the prevalence and genetical backgrounds of LPAI viruses among poultry in an area where several different influenza A subtypes are in circulation.

Reintroduction of H5N1 highly pathogenic avian influenza virus by migratory water birds, causing poultry outbreaks in the 2010-2011 winter season in Japan.

H5N1 highly pathogenic avian influenza virus (HPAIV) was reintroduced and caused outbreaks in chickens in the 2010-2011 winter season in Japan, which had been free from highly pathogenic avian influenza (HPAI) since 2007 when HPAI outbreaks occurred and were controlled. On 14 October 2010 at Lake Ohnuma, Wakkanai, the northernmost part of Hokkaido, Japan, H5N1 HPAIVs were isolated from faecal samples of ducks flying from their nesting lakes in Siberia. Since then, in Japan, H5N1 HPAIVs have been isolated from 63 wild birds in 17 prefectures and caused HPAI outbreaks in 24 chicken farms in nine prefectures by the end of March in 2011. Each of these isolates was genetically closely related to the HPAIV isolates at Lake Ohnuma, and those in China, Mongolia, Russia and Korea, belonging to genetic clade 2.3.2.1. In addition, these isolates were genetically classified into three groups, suggesting that the viruses were transmitted by migratory water birds through at least three different routes from their northern territory to Japan. These isolates were antigenic variants, which is consistent with selection in poultry under the immunological pressure induced by vaccination. To prevent the perpetuation of viruses in the lakes where water birds nest in summer in Siberia, prompt eradication of HPAIVs in poultry is urgently needed in Asian countries where HPAI has not been controlled.

Molecular epidemiology of avian influenza viruses circulating among healthy poultry flocks in farms in northern Vietnam.

Repeated epizootics of highly pathogenic avian influenza (HPAI) virus subtype H5N1 were reported from 2003 to 2005 among poultry in Vietnam. More than 200 million birds were killed to control the spread of the disease. Human cases of H5N1 infection have been sporadically reported in an area where repeated H5N1 outbreaks among birds had occurred. Subtype H5N1 strains are established as endemic among poultry in Vietnam, however, insights into how avian influenza viruses including the H5N1 subtype are maintained in endemic areas is not clear. In order to determine the prevalence of different avian influenza viruses (AIVs), including H5N1 circulating among poultry in northern Vietnam, surveillance was conducted during the years 2006-2009. A subtype H5N1 strain was isolated from an apparently healthy duck reared on a farm in northern Vietnam in 2008 and was identified as an HPAI. Although only one H5N1 virus was isolated, it supports the view that healthy domestic ducks play a pivotal role in maintaining and transmitting H5N1 viruses which cause disease outbreaks in northern Vietnam. In addition, a total of 26 AIVs with low pathogenicity were isolated from poultry and phylogenetic analysis of all the eight gene segments revealed their diverse genetical backgrounds, implying that reassortments have occurred frequently among strains in northern Vietnam. It is, therefore, important to monitor the prevalence of influenza viruses among healthy poultry between epidemics in an area where AIVs are endemic.

Antimicrobial susceptibility of Escherichia coli isolated from feces of wild cranes migrating to Kagoshima, Japan.

Susceptibility to 13 antimicrobial agents was examined for 138 Escherichia coli isolates obtained from 192 fecal samples of wild cranes that migrated for wintering to the Izumi plain, Kagoshima prefecture in Japan. The numbers of isolates that were resistant to the antimicrobials used in this study are as follows: oxytetracycline (OTC), 22 isolates; minocycline, 7 isolates; ampicillin (ABPC), 4 isolates; nalidixic acid, 4 isolates; enrofloxacin, 2 isolates; kanamycin, one isolate. Multidrug resistant isolates exhibiting 2-4 drug resistances were obtained. All of the OTC-resistant isolates carried either the tet (A) or tet(B) gene. The bla(TEM) gene was found in all of the ABPC-resistant isolates.

Antimicrobial resistance in fecal Escherichia coli isolated from growing chickens on commercial broiler farms.

To investigate the effects of rearing practices of commercial broiler chickens on the incidence of antimicrobial resistance in commensal Escherichia coli isolates, fecal E. coli isolates obtained in 4 farms were screened for anitimicrobial resistance. Ten E. coli isolates were recovered from each of the fecal samples collected from 10 birds in the farms at the ages of 2 days, 14-17 days, and 47-50 days. In 2 out of the 4 farms, no antimicrobials were used during the rearing period. In the other two farms, following collection of the fecal samples at 14 and 15 days of age, oxytetracycline (OTC), sulfadimethoxine (SDMX), and tylosin were given to birds on one farm and SDMX was used in the other. Isolates resistant to ampicillin and OTC that were obtained from an untreated flock at different sampling times were closely related to each other by pulsed-field gel electrophoresis patterns (PFGE) of XbaI-digested chromosomal DNA. PFGE analysis together with in vitro conjugation experiments suggested that diversity of resistance phenotypes within a clone may be resulted from the acquisition and loss of R-plasmids in an untreated and a treated flock. The numbers of resistance phenotypes observed among fecal isolates increased during the growth of the chickens in all the farms. The results in the present study suggest that persistence of commensal E. coli strains resistant to antimicrobials even in the absence of antimicrobial administration. It is also hypothesized that horizontal transmission of resistance determinants resulted in the emergence of different resistance phenotypes in those farms.

Susceptibility of two species of wild terrestrial birds to infection with a highly pathogenic avian influenza virus of H5N1 subtype.

The recent epidemic caused by H5N1 highly pathogenic avian influenza (HPAI) viruses has spread over many parts of Asia, Europe and Africa. Wild birds, particularly waterfowl, are considered to play a role in viral dissemination. However, detailed information on whether wild terrestrial birds act as carriers is currently unavailable. To investigate the susceptibility of terrestrial birds to HPAI viruses, two species of wild bird (great reed warbler and pale thrush) that are common in East Asia were infected with H5N1 HPAI virus. The results showed that both species were highly susceptible to the virus. The great reed warbler showed fatal infection with 100% mortality, but the pale thrush survived for longer periods (>8 days) with viral shedding. These findings suggest that there is variation in clinical outcome after infection of wild terrestrial birds, and that some bird species could become subclinical excretors of the H5N1 virus.

Salmonella isolated from the feces of migrating cranes at the Izumi Plain (2002-2008): serotype, antibiotic sensitivity and PFGE type.

From November 2002 to February 2008, 2,251 crane feces were collected at the Izumi Plain in Kagoshima Prefecture. Salmonella enterica was isolated from 359 feces (15.9%), of which 332 (92.5%) were Salmonella Typhimurium (ST), 9 were S. Hvittingfoss/II, 4 were S. Abaetetuba, 3 were S. Enteritidis, 2 were S. Konstanz, 1 was S. Pakistan and 8 were untyped isolates, respectively. Against 12 antimicrobial agents, no resistant strains were found in 154 isolates examined, but one was found to be resistant to ampicillin. By pulsed-field gel electrophoresis (PFGE), all but one of the 68 ST isolates tested showed indistinguishable banding patterns; one had a different pattern. The results suggest that ST strains from the same origin would spread in crane flocks during their stay at Izumi Plain every winter.

Possible circulation of H5N1 avian influenza viruses in healthy ducks on farms in northern Vietnam.

To estimate the prevalence of influenza A subtype H5N1 viruses among domestic ducks in the period between October and November 2006 when H5N1 outbreaks had been absent, 1106 healthy ducks raised in northern Vietnam were collected. Inoculation of all throat and cloacae samples into embryonated eggs resulted in the isolation of subtype H3N8 in 13 ducks, but not H5N1 viruses. Serological analyses demonstrated that five ducks (0.45%) solely developed H5N1 subtype-specific hemagglutinin-inhibiting and neuraminidase-inhibiting antibodies together with anti-non-structural protein 1 antibodies. The results suggested that the ducks were naturally infected with H5N1 viruses when obvious H5N1 outbreaks were absent.