Toshiyuki Murase

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.

Monitoring of layer feed and eggs for Salmonella in eastern Japan between 1993 and 1998.

In order to investigate contamination of chicken farms with Salmonella, feed and eggs were sampled from 16 commercial layer farms in eastern Japan between 1993 and 1998 and cultured for salmonellae. Salmonella enterica subsp. enterica isolates belonging to 19 serovars were obtained from the feed. Six of the 19 serotypes, including Salmonella serovar Enteritidis, were observed in isolates recovered from the eggs. Salmonella serovar Enteritidis strains obtained from a feed sample and egg contents in a layer farm showed pulsed-field gel electrophoresis patterns that were genetically related and belonged to a single phage type, suggesting that the contamination of the farms was linked to the occurrence of salmonellae in feed.

Monitoring of chicken houses and an attached egg-processing facility in a laying farm for Salmonella contamination between 1994 and 1998.

Two chicken houses and an attached egg-processing facility in a laying farm were sampled between 1994 and 1998 to investigate Salmonella contamination. Each of the houses was environmentally controlled and fitted with egg belts that transported eggs from the houses to the egg-processing facility. Four hundred twenty-eight Salmonella isolates were obtained from 904 environmental samples collected from the houses. Two hundred fifty-two of the 428 (58.9%) isolates yielded five serotypes as follows: Salmonella enterica subsp. enterica serovar Livingstone, Salmonella serovar Cerro, Salmonella serovar Montevideo, Salmonella serovar Mbandaka, and Salmonella serovar Corvallis. The remaining (41.1%, 176 of 428) isolates included four other serotypes and isolates that were untypeable. Salmonella isolates obtained from the drain water collected after the washing of the eggs in the egg-processing facility yielded the same serotypes as those found in the chicken houses. Strains having an identical pulsed-field gel electrophoresis (PFGE) pattern were continually recovered from a house for more than 1 year. Several strains of Salmonella Cerro, Salmonella Mbandaka, and Salmonella Montevideo obtained from both the houses and from the egg-processing facility were indistinguishable by PFGE, respectively. These results suggest that Salmonella organisms originating from a single clone colonized the chicken houses and that the egg belts are likely to be one of the means by which Salmonella organisms are spread from one house to the others.

Growth of Salmonella enterica serovar enteritidis in albumen and yolk contents of eggs inoculated with this organism onto the vitelline membrane

By using an in vitro model simulating the potential opportunities for Salmonella enterica serovar Enteritidis (SE) to proliferate within eggs contaminated with this organism following oviposition, we investigated growth of SE in eggs. Seventy to 140 CFU of one of three SE strains originating either from egg contents, chicken meat, or a human infection were experimentally inoculated onto the vitelline membrane of eggs collected from specific-pathogen-free flocks of chickens and incubated at 25 degrees C. SE organisms were detected in 6 of 71 yolk contents of the eggs inoculated with any of the test strains attaining levels ranging from 2.0 x 10(2) to 4.2 x 10(8) CFU/ml by day 6. The organisms were also detected in the albumen from 38 of 55 eggs tested, growing to levels ranging from 1.0 x 10(2) to 4.3 x 10(8) CFU/ml by day 6 after inoculation. An additional three yolk contents and 15 albumen samples were culture positive for SE following enrichment. There was no correlation between the number of the organisms in the yolk contents and that in the albumen from each of the eggs. When 73 to 91 CFU of the egg strain were inoculated into samples of separated albumen obtained from eggs that were stored at 4 degrees C for 1 to 4 weeks or at 25 degrees C for 1 week, slight growth (3.0 x 10(2) to 7.4 x 10(3) CFU/ml) was found in only 3 of the 60 albumen samples by day 6 after inoculation, but the organisms were recovered from 52 samples following enrichment. The results suggest that the environment on or near the vitelline membrane can be conducive to SE proliferation over time.

Evolutionary genetics of highly pathogenic H5N1 avian influenza viruses isolated from whooper swans in northern Japan in 2008

In April and May 2008, highly pathogenic avian influenza viruses subtype H5N1 were isolated from dead or moribund whooper swans in Aomori, Akita and Hokkaido prefectures in northern Japan. To trace the genetic lineage of the isolates, the nucleotide sequences of all eight genes were determined and phylogenetically analyzed. The Japanese strains were nearly identical to chicken viruses isolated in Russia in April 2008 and closely related to viruses isolated from dead wild birds in Hong Kong in 2007–2008. Their HA genes clustered in clade 2.3.2. On the other hand, NA and the other internal genes were closely related to those of clade 2.3.4 viruses (genotype V) whose NP genes originated from an HA clade 2.3.2 virus. In conclusion, the H5N1 viruses isolated in Japan, Russia and Hong Kong were derived from a common ancestor virus belonging to genotype V that was generated from genetic reassortment events between viruses of HA clades 2.3.2 and 2.3.4.

Multiplication and Motility of Salmonella enterica Serovars Enteritidis, Infantis, and Montevideo in In Vitro Contamination Models of Eggs

The invasive ability of Salmonella enterica serovars Enteritidis, Infantis, and Montevideo in eggs was examined. Strains of these serovars originating from egg contents, laying chicken houses, and human patients were experimentally inoculated (0.1-ml dose containing 78 to 178 cells) onto the vitelline membrane of eggs collected from specific-pathogen-free chickens and incubated at 25 degrees C. The test strains were detected in 25 of 138 yolk contents by day 6, indicating the penetration of Salmonella organisms through the vitelline membrane. There were no significant differences in overall rates of penetration between serovars. The organisms were also detected in the albumen from 125 of 138 eggs tested by day 6. Growth to more than 10(6) CFU/ml was observed in 48 of the 125 albumen samples. An inoculum of 1000 Salmonella cells was added to 15 ml of albumen at the edge of a petri plate. A 10-mm-diameter cylindrical well, the bottom of which was sealed with a polycarbonate membrane with 3.0-microm pores, was filled with egg yolk and placed into the albumen at the center of the dish, which was maintained at 25 degrees C. Experiments were performed in triplicate with each strain. Salmonella organisms in all the albumen samples were detected by day 11. However, motility of the organisms toward the yolk was observed in only two dishes inoculated with the Salmonella Enteritidis strain from a human patient and in one dish inoculated with the Salmonella Infantis strain from liquid egg. The albumen samples obtained from the dishes inoculated with the Salmonella Enteritidis strain had high numbers of bacteria (>10(8) CFU/ml). The present study suggests that Salmonella organisms in egg albumen are unlikely to actively move toward the yolk, although depositionon or near the vitelline membrane can be advantageous for proliferation.

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.

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.

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.

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.