Characterizing the temporal patterns of avian influenza virus introduction into Japan by migratory birds.

The objectives of the present study were to observe the temporal pattern of avian influenza virus (AIV) introduction into Japan and to determine which migratory birds play an important role in introducing AIV. In total, 19,407 fecal samples from migratory birds were collected at 52 sites between October 2008 and May 2015. Total nucleic acids extracted from the fecal samples were subjected to reverse transcription loop-mediated isothermal amplification to detect viral RNA. Species identification of host migratory birds was conducted by DNA barcoding for positive fecal samples. The total number of positive samples was 352 (prevalence, 1.8%). The highest prevalence was observed in autumn migration, and a decrease in prevalence was observed. During autumn migration, central to southern Japan showed a prevalence higher than the overall prevalence. Thus, the main AIV entry routes may involve crossing the Sea of Japan and entry through the Korean Peninsula. Species identification was successful in 221 of the 352 positive samples. Two major species sequences were identified: the Mallard/Eastern Spot-billed duck group (115 samples; 52.0%) and the Northern pintail (61 samples; 27.6%). To gain a better understanding of the ecology of AIV in Japan and the introduction pattern of highly pathogenic avian influenza viruses, information regarding AIV prevalence by species, the prevalence of hatch-year migratory birds, migration patterns and viral subtypes in fecal samples using egg inoculation and molecular-based methods in combination is required.

Molecular Analysis of the Cyanobacterial Community in Gastric Contents of Egrets with Symptoms of Steatitis.

Many deaths of wild birds that have drunk water contaminated with hepatotoxic microcystin-producing cyanobacteria have been reported. A mass death of egrets and herons with steatitis were found at the agricultural reservoir occurring cyanobacterial waterblooms. This study aimed to verify a hypothesis that the egrets and herons which died in the reservoir drink microcystin-producing cyanobacteria and microcystin involves in the cause of death as well as the symptoms of steatitis. The cyanobacterial community in gastric contents of egrets and herons that died from steatitis was assessed using cyanobacterial 16S rRNA-based terminal-restriction fragment length polymorphism (T-RFLP) profiling and a cyanobacterial 16S rRNA-based clone library analysis. In addition, PCR amplification of the mcyB-C region and the mcyG gene, involved in microcystin biosynthesis, was examined. The cyanobacterial community in the gastric contents of two birds showed a simplistic composition. A comparison of cyanobacterial T-RFLP profiling and cloned sequences suggested that the genus Microcystis predominated in both samples of egrets died. Although we confirmed that two egrets which died in the reservoir have taken in cyanobacterial waterblooms containing the genus Microcystis, no mcy gene was detected in both samples according to the mcy gene-based PCR analysis. This study is the first to show the profiling and traceability of a cyanobacterial community in the gastric contents of wild birds by molecular analysis. Additionally, we consider causing symptoms of steatitis in the dead egrets.

Reproductive cycle observation of the Okinawa rail (Gallirallus okinawae) in the wild.

The captive breeding program of the Okinawa rail started in 2008. For successful captive breeding, information related to reproduction, such as age at sexual maturity, testicular cycles and ovulatory cycles, is essential to predict when reproduction is possible and when certain reproductive behaviors are most likely to occur. We made gross and histological observations of the reproductive organs of Okinawa rails to gain understanding of sexual maturity, the testicular cycle and the ovulatory cycle. We found that the weight of the testis was smallest in December and largest in March. Changes in the diameter of the seminiferous tubule showed the same pattern. Mature sperm were observed from March to June. The heaviest ovary was observed in April. A single peak of reproduction, from March to April, was observed in males and females. Our observations suggested that the Okinawa rail is a seasonal breeder. Establishing suitable breeding pairs will be critical to ensure success of the Okinawa rail captive breeding program. Our results suggested that pairing must be started before March. If supportive breeding is used, semen should be collected from March to June and artificial insemination conducted in April.

Steatitis in egrets and herons from Japan.

More than 70 egrets and herons were found sick or dead at an agricultural water reservoir in Kanagawa Prefecture, Japan between September and October 2008. The birds showed weakness, lethargy, and inability to fly before death. Postmortem findings included large amounts of firm subcutaneous and cavitary fat comprised of necrotic adipose tissues with infiltrates of heterophils and macrophages. The birds were diagnosed with steatitis on the basis of the gross lesions and histopathology. Egrets with steatitis had low blood levels of vitamin E. High counts of cyanobacteria (Microcystis aeruginosa) were found in the reservoir concurrent with the outbreak of steatitis. No microcystin was detected in the reservoir water or the livers from the egrets. This is the first report of steatitis in wild birds in Japan.

T-cell lymphoma in a wild Okinawa rail (Gallirallus okinawae).

The Okinawa rail (Gallirallus okinawae) is an endangered species that inhabits the northern part of Okinawa Main Island in southern Japan. A wild Okinawa rail was rescued from a road in Kunigami Village in Okinawa in October 2009. The bird subsequently died and underwent necropsy. Tumors were found in the liver, spleen and part of the small intestine. Microscopically, lymphoid neoplasm was confirmed in these tissues. The tumor cells were mainly positive for CD3 and CD8α by immunohistochemistry. No Marek's disease virus genes were detected by PCR of a liver tumor. This is the first report of T-cell lymphoma in the Okinawa rail.