Diversity of Chlamydiales detected in pet birds privately kept in individual homes in Japan.

Chlamydia-related bacteria of the Chlamydiales order have recently been described as emerging pathogens that cause pneumonia and abortion in animals and humans. We investigated the presence of Chlamydiales using real-time polymerase chain reaction (PCR) by targeting the 16S rRNA gene of a broad range of Chlamydiales in 827 fecal samples from pet birds kept in individual homes in Japan. Of the 827 samples, 493 (59.6%) tested positive for the Chlamydiales 16S rRNA gene in the real-time PCR assay. We determined the nucleic acid sequences of PCR products from 17 Chlamydiales strains. A homology search and phylogenetic analysis using these sequences confirmed that the detected Chlamydiales included C. pecorum and a broad range of Chlamydia-related bacteria. To the best of our knowledge, this is the first study to detect a wide range of Chlamydia-related bacteria in birds.

Genome editing by introduction of Cas9/sgRNA into plant cells using temperature-controlled atmospheric pressure plasma.

Previously, we developed a technique to introduce a superfolder green fluorescent protein (sGFP) fusion protein directly into plant cells using atmospheric-pressure plasma. In this study, we attempted genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system using this protein introduction technique. As an experimental system to evaluate genome editing, we utilized transgenic reporter plants carrying the reporter genes L-(I-SceI)-UC and sGFP-waxy-HPT. The L-(I-SceI)-UC system allowed the detection of successful genome editing by measuring the chemiluminescent signal observed upon re-functionalization of the luciferase (LUC) gene following genome editing. Similarly, the sGFP-waxy-HPT system conferred hygromycin resistance caused by hygromycin phosphotransferase (HPT) during genome editing. CRISPR/Cas9 ribonucleoproteins targeting these reporter genes were directly introduced into rice calli or tobacco leaf pieces after treatment with N2 and/or CO2 plasma. Cultivation of the treated rice calli on a suitable medium plate produced the luminescence signal, which was not observed in the negative control. Four types of genome-edited sequences were obtained upon sequencing the reporter genes of genome-edited candidate calli. sGFP-waxy-HPT-carrying tobacco cells exhibited hygromycin resistance during genome editing. After repeated cultivation of the treated tobacco leaf pieces on a regeneration medium plate, the calli were observed with leaf pieces. A green callus that was hygromycin-resistant was harvested, and a genome-edited sequence in the tobacco reporter gene was confirmed. As direct introduction of the Cas9/sgRNA (single guide RNA) complex using plasma enables genome editing in plants without any DNA introduction, this method is expected to be optimized for many plant species and may be widely applied for plant breeding in the future.

Reproductive status of a female white cockatoo (Cacatua alba) based on relationships among urofecal steroid hormone dynamics, molting, and body weight.

The detailed reproductive physiology of cockatoos based on gonadal hormone dynamics is unclear. In this study, we aimed to investigate ovarian activity by monitoring urofecal sex steroid hormone profiles in a captive female white cockatoo (Cacatua alba) and to noninvasively reveal basic reproductive physiology by comparing the hormone profiles with the laying dates, body mass changes, and molt progress. Urofeces were collected regularly for approximately 4 years from one female that frequently laid unfertilized eggs under single-rearing conditions. Urofecal progesterone (P4) and estradiol-17ß (E2) were measured by enzyme immunoassay. In addition, body mass and the number of fallen feathers were measured periodically. The urofecal P4 concentration peaked at an average of 17.7 days after the start of the rise in urofecal E2 concentration, and egg laying began on the day after the peak urofecal P4 concentration. The clutch size was usually two eggs, with an average interval of 4.5 days between eggs in each egg-laying cycle. There was a significant correlation between the dynamics of E2 concentration in urofeces and body mass. The results strongly suggest that E2 and P4 reflect the follicle growth and ovulation status, respectively, and that noninvasive monitoring of hormone dynamics using urofeces can accurately capture ovarian activity in the white cockatoo. Furthermore, changes in body mass can predict follicular growth, and reproduction and molt are antagonistic.

Proventricular dilatation disease associated with avian bornavirus infection in a Citron-crested Cockatoo that was born and hand-reared in Japan.

A 5-month-old female Citron-crested Cockatoo (Cacatua sulphurea citrinocristata) that was born and hand-reared in Japan died with suspected proventricular dilatation disease (PDD). Macroscopic and microscopic examinations of the bird revealed characteristic features of PDD, i.e., distention of the proventriculus and infiltration of lymphocytes and plasma cells in ganglia of various organs and in central and peripheral nerves. A linkage of this PDD case to infection with avian bornavirus (ABV) was documented by RT-PCR amplification of the virus genomes from the affected bird. Phylogenetic analysis revealed that the ABV identified in this study clustered into the genotype 2, which is one of the dominant ABV genotypes worldwide. To the best of our knowledge, this is the first report of a natural case of PDD associated with ABV infection in Japan.

A novel genotype of beak and feather disease virus in budgerigars (Melopsittacus undulatus).

Beak and feather disease virus (BFDV) is a causative agent for psittacine beak and feather disease (PBFD), which shows a characteristic feather disorder in psittacine birds. Nineteen budgerigars, which were clinically suspected to have PBFD, were examined by two polymerase chain reactions (PCR), which target each of open reading frames (ORFs) V1 and C1. All of the 19 samples were detected BFDV by the PCR targeting ORF C1, whereas only two of them were detected by the PCR targeting ORF V1. It was assumed that BFDV derived from budgerigar (budgerigar BFDV) has two genotypes, which are tentatively classified as budgerigar BFDV genotype 1 and genotype 2 by the PCR amplification patterns. Whole genome sequences of six budgerigar BFDVs were determined to reveal the existence of two genotypes. In the phylogenic analysis, six budgerigar BFDV sequences formed a unique group branched from the other 23 published BFDV sequences. The budgerigar BFDV genotype 1 and genotype 2 were also segregated each other, and budgerigar BFDV genotype 2 was particularly distantly related with the other BFDVs. These results suggest budgerigar BFDV is a unique in the known BFDVs and is divided into two genotypes.

Phylogenetic analysis of Newcastle disease virus genotypes isolated in Japan.

We genetically analyzed field isolates of the Newcastle disease (ND) virus isolated in Japan from 1930 to 2001. The coding region of the fusion protein was amplified by reverse transcriptase PCR and directly sequenced. Phylogenetic analysis revealed the presence of viruses belonging to six of the eight known genotypes. It can be concluded from this study that ND outbreaks in Japan have been of multiple etiologies. [All sequences used in this study were sent to DDBJ and assigned accession numbers AB 070382 to AB 074042.]