Izumi Plain Research Articles

Metagenomic analysis of the gut microbiota of hooded cranes (Grus monacha) on the Izumi plain in Japan.

Recent advances in DNA sequencing technology have dramatically improved our understanding of the gut microbiota of various animal species. However, research on the gut microbiota of birds lags behind that of many other vertebrates, and information about the gut microbiota of wild birds such as migratory waterfowl is particularly lacking. Because the ecology of migratory waterfowl (e.g., lifestyle, diet, physiological characteristics) differs from that of other birds, the gut microbiota of migratory waterfowl likely also differs, but much is still unknown. The hooded crane (Grus monacha) is an important representative migratory waterbird species and is listed as endangered on the International Union for Conservation of Nature and Natural Resources Red List of Threatened Species. In this study, we analyzed the bacterial and viral microbiota in the gut of hooded cranes by using deep sequencing data from fecal samples of hooded cranes that winter on the Izumi plain in Japan, and found that Cetobacterium, Clupeiformes, and Pbunavirus were clearly present in the fecal samples of hooded cranes. These findings advance our understanding of the ecology of hooded cranes.

Isolation and genetic characterization of multiple genotypes of both H5 and H7 avian influenza viruses from environmental water in the Izumi plain, Kagoshima prefecture, Japan during the 2021/22 winter season

In the 2021/22 winter, one H5N1 and nine H5N8 high pathogenicity avian influenza viruses (HPAIVs) of clade 2.3.3.4b were isolated from the water in crane roosts on the Izumi plain, Japan. Additionally, we isolated low pathogenicity avian influenza viruses (LPAIVs) of five subtypes: H1N1, H4N2, H4N6, H7N7, and H10N4. H5N8 HPAIVs belonging to the G2a group were isolated throughout winter, whereas H5N1 HPAIV belonging to the G2b group were isolated only in early winter. These findings suggest co-circulation of both G2a and G2b HPAIVs in early winter. Although two H7N7 LPAIVs were isolated from cranes’ roost water collected on the same day, the gene constellations of the two isolates were clearly different, indicating the contemporary invasion of at least two different genotypes of H7N7 LPAIVs in the Izumi plain. This study underscores the importance of monitoring both HPAIVs and LPAIVs to understand avian influenza virus ecology in migratory waterfowl populations.

Avian paramyxovirus serotype-1 isolation from migratory birds and environmental water in southern Japan: An epidemiological survey during the 2018/19-2021/2022 winter seasons.

Newcastle disease caused by highly pathogenic viruses of avian paramyxovirus serotype-1 (APMV-1) is a highly contagious poultry disease. Although a large-scale epidemic of Newcastle disease had occurred in Japan between the 1950s and the 2000s, there have been no outbreaks anywhere since 2010. In addition, there are no reports of epidemiological surveys of APMV-1 in wild birds in Japan in the last 10 years. We conducted the first epidemiological survey of APMV-1 in the Izumi plain, Kagoshima prefecture of southern Japan from the winter of 2018 to 2022. A total of 15 APMV-1 strains were isolated, and isolation rates from roosting water and duck fecal samples were 2.51% and 0.10%, respectively. These results indicate that the isolation method from environmental water may be useful for efficient surveillance of APMV-1 in wild birds. Furthermore, this is the first report on the success of APMV-1 isolation from environmental water samples. Genetic analysis of the Fusion (F) gene showed that all APMV-1 isolates were closely related to virus strains circulating among waterfowl in Far East Asian countries. All isolates have avirulent motifs in their cleavage site of F genes, all of which were presumed to be low pathogenic viruses in poultry. However, pathogenicity test using embryonated chicken eggs demonstrated that some isolates killed all chicken embryos regardless of viral doses inoculated (102 -106 50% egg infectious dose). These results indicated that APMV-1 strains, which are potentially pathogenic to chickens, are continuously brought into the Izumi plain by migrating wild birds.

Genetic Characterization of Avian Influenza Viruses Isolated from the Izumi Plain, Japan in 2019/20 Winter Season.

The Izumi plain in the Kagoshima Prefecture, Japan, is known as an overwintering site for more than 30,000 migratory waterfowl, including endangered crane species. We previously reported that environmental water samples, from artificial wet paddies created as crane roost sites on the Izumi plain, are useful for avian influenza virus (AIV) surveillance. During the 2019/20 winter season, we collected 238 water samples from the crane roost sites and isolated 22 AIVs of six subtypes: one H1N1, one H3N2, seven H3N8, four H4N6, nine H6N6, and one H11N2 subtypes. Genetic analyses revealed that AIVs of the same subtype isolated from the Izumi plain during a single winter season exhibited multiple genetic constellations. Furthermore, phylogenetic analyses suggested that our H3N2 isolate may be a genetic reassortant between close relatives to our H3N8 and H11N2 isolates. Our study highlighted the importance of monitoring AIV circulation to better understand AIV ecology in migratory waterfowl populations.

Genetically Diverse Highly Pathogenic Avian Influenza A(H5N1/H5N8) Viruses among Wild Waterfowl and Domestic Poultry, Japan, 2021.

Genetic analyses of highly pathogenic avian influenza H5 subtype viruses isolated from the Izumi Plain, Japan, revealed cocirculation of 2 genetic groups of clade 2.3.4.4b viruses among migratory waterfowl. Our findings demonstrate that both continuous surveillance and timely information sharing of avian influenza viruses are valuable for rapid risk assessment.

Prevalence and organ tropism of crane-associated adenovirus 1 in cranes overwintering on the Izumi plain, Japan.

Crane-associated adenovirus 1 (CrAdV-1) is a proposed novel virus in the genus Aviadenovirus, first detected in faecal samples from hooded cranes (a vulnerable crane species) on the Izumi plain, a major overwintering site for migratory cranes in Japan. CrAdV-1 was genetically characterized in that study; however, its virological characteristics remain largely unclear. To investigate the prevalence and organ tropism of CrAdV-1, we collected swab and organ samples from dead or debilitated cranes on the Izumi plain. CrAdV-1 gene was detected in 47% (45/95) of tested cranes, comprising mainly hooded cranes but also white-naped and sandhill cranes. These results indicate that CrAdV-1 shedding is widespread among cranes overwintering on the Izumi plain. Phylogenetic analyses revealed that the 68 nucleotide sequences determined from the positive swabs formed a single cluster, suggesting phylogenetic differences between CrAdV-1 and other aviadenoviruses. CrAdV-1 prevalence showed a significant linear increase with time through the overwintering period (November to February), especially among juveniles. These findings indicate that CrAdV-1 spreads mainly by transmission between juveniles progressively through the overwintering period. The CrAdV-1 gene-positive rate was significantly higher in cloacal swabs than conjunctival or tracheal swabs. Copy numbers for the partial CrAdV-1 gene sequence were markedly high in the colon samples from three of the four cranes investigated for organ tropism. We also detected relatively high copy numbers in the cerebrum, trachea, lung and heart, suggesting that CrAdV-1 mainly targets these four organs and transmitted via the faecal-oral route and airborne transmission. These results contribute to further understanding of the virological characteristics of CrAdV-1.

Global subtype diversity, spatial distribution patterns, and phylogenetic analysis of avian influenza virus in water.

The current COVID-19 pandemic highlights the need for zoonotic infectious disease surveillance. Avian influenza virus (AIV) poses a significant threat to animal and public health due to its pandemic potential. Virus-contaminated water has been suggested as an important AIV spread mechanism among multiple species. Nevertheless, few studies have characterized the global AIV subtype diversity and distribution in environmental water. Therefore, this study aims to provide an updated descriptive and phylogenetic analysis of AIVs isolated in water samples from high risk-sites for influenza outbreaks (i.e. live bird markets, poultry farms, and wild bird habitats) on a global scale. The descriptive analysis evidenced that 21 subtypes were reported from nine countries between 2003 and 2020. Fourteen AIV subtypes were solely reported from Asian countries. Most of the viral sequences were obtained in China and Bangladesh with 47.44% and 23.93%, respectively. Likewise, the greatest global AIV subtype diversity was observed in China with 12 subtypes. Live bird markets represented the main sampling site for AIV detection in water samples (64.1%), mostly from poultry cage water. Nevertheless, the highest subtype diversity was observed in water samples from wild bird habitats, especially from the Izumi plain and the Dongting Lake located in Japan and China, respectively. Water from drinking poultry troughs evidenced the greatest subtype diversity in live bird markets; meanwhile, environmental water used by ducks had the highest number of different subtypes in poultry farms. Maximum-likelihood phylogenetic trees of hemagglutinin (HA) and neuraminidase (NA) genes showed that some sequences were closely related among different poultry/wild bird-related environments from different geographic origins. Therefore, the results suggest that even though the availability of gene sequences in public-access databases varies greatly among countries, environmental AIV surveillance represents a useful tool to elucidate potential viral diversity in wild and domestic bird populations.

Pathogenicity of H5 highly pathogenic avian influenza virus in rooks (Corvus frugilegus)

ABSTRACTRooks (Corvus frugilegus) are considered migratory crows in Japan. Some rooks share a wintering site in the Izumi plain in Kagoshima Prefecture with hooded cranes (Grus monacha) and white-necked cranes (Grus vipio), which are designated as “endangered” in the International Union for Conservation of Nature (IUCN) Red List of Threatened Species. Highly pathogenic avian influenza (HPAI), caused by H5 subtype viruses, has recently been reported in these crane species in Japan, in conjunction with a massive decrease in their population. In the present study, the pathogenicity of HPAI virus was assessed in rooks to evaluate the likelihood that they are a source of infections in other bird species. One of four rooks intranasally inoculated with A/mandarin duck/Miyazaki/22M807-1/2011 (H5N1) died at 10 days post-inoculation (d.p.i.). The other three rooks exhibited seroconversion but no clinical signs. All the rooks had shed virus by the oral route at <103 50% egg infectious dose/ml until 7 d.p.i. Virus was also recovered from multiple tissues of the rook that succumbed to the infection. These results suggest that rooks are susceptible to infection with H5 HPAI viruses, leading to prolonged viral shedding. The rooks shed the virus at low titres however, indicating that they are likely to function as transmission vectors in wintering bird flocks. The rooks exhibited clear antibody responses against the H5 HPAI virus, and thus serological surveillance of them in the field should be helpful for assessing viral pervasion into the habitats of crane species.

Transition in genetic constellations of H3N8 and H4N6 low-pathogenic avian influenza viruses isolated from an overwintering site in Japan throughout different winter seasons.

The Izumi plain in Kagoshima Prefecture, Japan, is an overwintering site for migratory ducks and endangered cranes. We have surveyed avian influenza viruses (AIVs) in this area since 2012 and isolated low-pathogenic AIVs (LPAIVs) of various subtypes every winter season. H3N8 LPAIVs were isolated during the 2012/13 and 2016/17 seasons, and H4N6 LPAIVs were isolated during the 2012/13 and 2013/14 seasons. In the 2017/18 season, one H3N8 and two H4N6 LPAIV strains were isolated from environmental water samples. Genetic and phylogenetic analysis for each gene segment from these H3N8 and H4N6 LPAIVs suggested that our isolates were genetic reassortants generated by intermixing between AIVs circulating not only in Eurasia but also in Africa and/or North America. Comparison of the genetic constellations of our three isolates with their counterparts isolated during previous seasons from the Izumi plain revealed a drastic transition in the genetic constellations of both subtypes. These findings emphasize the importance of continuous surveillance of AIVs on the Izumi plain.

Identification of a distinct lineage of aviadenovirus from crane feces.

Viruses are believed to be ubiquitous; however, the diversity of viruses is largely unknown because of the bias of previous research toward pathogenic viruses. Deep sequencing is a promising and unbiased approach to detect viruses from animal-derived materials. Although cranes are known to be infected by several viruses such as influenza A viruses, previous studies targeted limited species of viruses, and thus viruses that infect cranes have not been extensively studied. In this study, we collected crane fecal samples in the Izumi plain in Japan, which is an overwintering site for cranes, and performed metagenomic shotgun sequencing analyses. We detected aviadenovirus-like sequences in the fecal samples and tentatively named the discovered virus crane-associated adenovirus 1 (CrAdV-1). We determined that our sequence accounted for approximately three-fourths of the estimated CrAdV-1 genome size (33,245bp). The GC content of CrAdV-1 genome is 34.1%, which is considerably lower than that of other aviadenoviruses. Phylogenetic analyses revealed that CrAdV-1 clusters with members of the genus Aviadenovirus, but is distantly related to the previously identified aviadenoviruses. The protein sequence divergence between the DNA polymerase of CrAdV-1 and those of other aviadenoviruses is 45.2-46.8%. Based on these results and the species demarcation for the family Adenoviridae, we propose that CrAdV-1 be classified as a new species in the genus Aviadenovirus. Results of this study contribute to a deeper understanding of the diversity and evolution of viruses and provide additional information on viruses that infect cranes, which might lead to protection of the endangered species of cranes.

Surveillance of antibiotic resistance in Escherichia coli isolated from wild cranes on the Izumi plain in Kagoshima prefecture, Japan.

The prevalence of antibiotic resistance in 376 Escherichia coli (E. coli) isolates from fecal samples of Hooded and White-naped cranes was investigated on the Izumi plain in Kagoshima prefecture, Japan, during winter 2016 and 2017. Resistance to oxytetracycline, ampicillin, and nalidixic acid were observed in 10.9%, 3.1–4.4%, and 2.1–7.7% of isolates, respectively. Since the previous surveillance in 2007, isolation rates of antibiotic-resistant E. coli recovered from wild cranes have remained at significantly low levels compared with those in Japanese livestock. Our results indicate that surveillance of antibiotic-resistant E. coli from wild cranes wintering in the Izumi plain could be a useful strategy to indicate natural environmental pollution by antibiotic-resistant bacteria in the environment.

Phylogenetic variations of highly pathogenic H5N6 avian influenza viruses isolated from wild birds in the Izumi plain, Japan, during the 2016-17 winter season.

During the 2016-2017 winter season, we isolated 33 highly pathogenic avian influenza viruses (HPAIVs) of H5N6 subtype and three low pathogenic avian influenza viruses (LPAIVs) from debilitated or dead wild birds, duck faeces, and environmental water samples collected in the Izumi plain, an overwintering site for migratory birds in Japan. Genetic analyses of the H5N6 HPAIV isolates revealed previously unreported phylogenetic variations in the PB2, PB1, PA, and NS gene segments and allowed us to propose two novel genotypes for the contemporary H5N6 HPAIVs. In addition, analysis of the four gene segments identified close phylogenetic relationships between our three LPAIV isolates and the contemporary H5N6 HPAIV isolates. Our results implied the co-circulation and co-evolution of HPAIVs and LPAIVs within the same wild bird populations, thereby highlighting the importance of avian influenza surveillance targeting not only for HPAIVs but also for LPAIVs.

Genetic characterization of low-pathogenic avian influenza viruses isolated on the Izumi plain in Japan: possible association of dynamic movements of wild birds with AIV evolution.

The Izumi plain in Kagoshima Prefecture, Japan, is an overwintering site of endangered cranes (hooded cranes and white-naped cranes) and of many other migratory birds (including wild ducks) that are considered carriers of avian influenza viruses (AIVs). To assess the risks of a highly pathogenic avian influenza outbreak in the crane populations, we tested various environmental samples for AIVs in this area. In the 2014-2015 winter season, we isolated one AIV of the H6N2 subtype from the cranes' roost water and two AIVs of the H11N9 subtype from a crane fecal sample and a cloacal swab of a dead spot-billed duck. Genetic analysis of these AIV isolates indicated that our H6N2 isolate is genetically close to AIVs isolated from wild birds in Southeast Asian countries, except that the PB1 and NS genes belong to the North American virus lineage. All genes of the two H11N9 isolates are related to AIVs belonging to the Eurasian virus lineage. Notably, in our phylogenetic trees, H11 HA and N9 NA genes showing high sequence similarity to the corresponding genes of isolates from wild birds in South Africa and Spain, respectively, did not cluster in the major groups with recent wild-bird isolates from East Asia. These results suggest that AIVs with viral gene segments derived from various locations and bird species have been brought to the Izumi plain. These findings imply a possible association of dynamic movements of wild birds with AIV evolution.

Characterization of gizzards and grits of wild cranes found dead at Izumi Plain in Japan.

We analyzed the gizzards, and grits retained in the gizzards of 41 cranes that migrated to the Izumi Plain during the winter of 2015/2016 and died there, either due to accident or disease. These included 31 Hooded Cranes (Grus monacha) and 10 White-naped Cranes (G. vipio). We determined body weight, gizzard weight, total grit weight and number per gizzard, and size, shape, and surface roundness of the grits. Average gizzard weights were 92.4 g for Hooded Cranes and 97.1 g for White-naped Cranes, and gizzard weight positively correlated with body weight in both species. Average total grit weights per gizzard were 19.7 g in Hooded Cranes and 25.7 g in White-naped Cranes, and were significantly higher in the latter. Average percentages of body weight to grit weight were 0.8% in Hooded Cranes and 0.5% in White-naped Cranes. Average grit number per gizzard was 693.5 in Hooded Cranes and 924.2 in White-naped Cranes, and were significantly higher in the latter. The average grit size was 2.8 mm in both species. No differences were found in the shape and surface roundness of grits between the two species. To the best of our knowledge, this is the first study on the grits retained in the gizzards of Hooded and White-naped Cranes.

Genetic characterization of an avian H4N6 influenza virus isolated from the Izumi plain, Japan.

An influenza A virus of H4N6 subtype was isolated from the Izumi plain, Japan, in 2013. Genetic analyses revealed that two viral genes (M and NS gene segments) of this isolate were genetically distinct from those of the H4N6 virus isolated from the same place in 2012. Furthermore, three viral genes (PB2, PB1 and M gene segments) of this isolate share high similarity with those of the North American isolates of 2014. These results suggest a high frequency of genetic reassortment of avian influenza viruses in Asian waterfowl and intercontinental movements of avian influenza viruses via migratory waterfowl.

鹿児島県出水平野におけるツル類の死亡個体数の推移（2003～2013）と主な解剖所見

鹿児島県出水平野におけるツル類の死亡個体数の推移（2003～2013）と主な解剖所見

Isolation and characterization of influenza A viruses from environmental water at an overwintering site of migratory birds in Japan

The Izumi plain in Kagoshima prefecture, Japan, is an overwintering site of more than 10,000 cranes. The wet paddy areas are artificially created to provide roosting sites for the cranes every winter. Since wild ducks, known to be a natural reservoir of influenza A viruses, also overwinter in this area, the cranes' roost water likely serves as a source of influenza A virus infection. To assess this potential risk, we collected 126 water samples from the cranes' roost in the 2012/2013 winter season for virus isolation. We isolated six influenza viruses of three subtypes (H3N8, H4N6, and H4N8) from the water samples collected in the months of November and December. Genetic analysis of our isolates indicated that these viruses were genetically similar to the low-pathogenic avian influenza viruses circulating among Eurasian waterfowl. These findings suggest the possibility of the cranes becoming infected with the avian influenza viruses that are present in their roost water.

Antimicrobial Susceptibility of &lt;i&gt;Escherichia coli&lt;/i&gt; 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.

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.

出水平野に飛来するツル糞便からの&lt;I&gt;Salmonella&lt;/I&gt;Typhimuriumの分離および分離株の性状

Between November, 2000, and February, 2002, 342 specimens of crane feces were collected from the Izumi Plain in Kagoshima Prefecture for the isolation of Salmonella spp. AlthoughSalmonellaTyphimurium (ST) was isolated from 64 samples (18.7%), no other serotypes were found. Except for one isolate resistant to gentamicin, tests using minimum inhibitory concentration (MIC) of 12 antibiotics detected no resistant strains among the 64 isolates of this study and among 3 strains isolated in 1997. Pulsed-field gel electrophoresis (PFGE) ofXbaI-digested chromosomal DNA showed indistinguishable banding patterns in all 32 isolates tested. These results suggest that ST strains with the PFGE pattern may spread in crane flocks during their annual winter stay on the Izumi Plain.