Establishment of immortalized Egyptian Rousettus bat cell lines.

The Egyptian Rousettus bat (Rousettus aegyptiacus) is a common fruit bat species that is distributed mainly in Africa and the Middle East. Bats serve as reservoir hosts for numerous pathogens. Human activities, such as hunting bats for food, managing vermin, and causing habitat loss, elevate the likelihood of transmission of bat pathogens to humans and other animals. Consequently, bat cell lines play a crucial role as research materials for investigating viral pathogens. However, the inherent limitation of finite cell division in primary cells necessitates the use of immortalized cells derived from various bat tissues. Herein, we successfully established six fibroblast cell lines derived from an infant bat heart and lungs and an elderly bat heart. Three of the six cell lines, called K4DT cells, were transduced by a combination of cell cycle regulators, mutant cyclin-dependent kinase 4, cyclin D1, and human telomerase reverse transcriptase. The other three cell lines, named SV40 cells, were transfected with simian virus 40 large T antigen. Transgene protein expression was detected in the transduced cells. All three K4DT cell lines and one lung-derived SV40 cell line were virtually immortalized and nearly maintained the normal diploid karyotypes. However, the two other heart-derived SV40 cell lines had aberrant karyotypes and the young bat-derived cell line stopped proliferating at approximately 40 population doublings. These bat cell lines are valuable for studying pathogen genomics and biology.

Morphological and molecular identification of Eimeria tetartooimia oocysts from a Japanese green pheasant (Galliformes; Phasianidae; Phasianus versicolor) at a zoo in Japan.

We detected Eimeria oocysts from Japanese green pheasants (Phasianus versicolor) at a zoo in Osaka, Japan. The oocyst isolates were subspherical or ovoidal shaped and measured 17.2 (range 14.7-20.0) µm in length and 14.8 (13.3-16.7) µm in width with a length/width (L/W) ratio of 1.2 (1.0-1.4) and each had one polar granule. The oocysts lacked a residuum and micropyle. Sporocysts measured 9.8 (6.7-13.3) µm in length and 5.9 (4.7-7.3) µm in width, with a L/W ratio of 1.2 (1.1-1.4). Compared to previously published values, this strain shows morphological similarities with an isolate of E. teetartooimia from ring-necked pheasants from other countries. Phylogenetic analysis of the 18S rRNA and mitochondrial cytochrome c oxidase subunit I genes places the isolate in a clade related to chicken Eimeria spp., such as E. acervulina or E. brunetti. Although further analysis is needed, this information can be helpful for the diagnosis and determination of virulence of Eimeria spp. in pheasants.

Molecular phylogeny of Blastocystis isolates from wild rodents captured in Indonesia and Japan.

Blastocystis sp. is a common intestinal protist found worldwide in a variety of animals, including humans. Currently, 17 subtypes (STs) of Blastocystis isolates from mammalian and avian host species have been reported based on the small subunit ribosomal RNA gene (SSU rDNA). Among these, human Blastocystis were only identified among STs 1-9. Except ST9, all other STs comprised isolates from humans and other animal species. Entire sequence data of the SSU rDNA of nine Blastocystis isolates from laboratory rats or guinea pigs previously showed ST4, whereas Blastocystis isolates from wild rodents have not been addressed genetically. In this study, Blastocystis infection in wild rodents was surveyed in Indonesia and Japan, and 11 and 12 rodent Blastocystis parasites were obtained from Rattus exulans and R. novercious, respectively. All new Blastocystis isolates from wild rodents were identified as ST4 based on the SSU rDNA sequences. The best tree inferred with the entire sequences of the SSU rDNA of all ST4 isolates including 17 data registered in GenBank clearly showed monophyletic ST4A and ST4B clades. Although ST4 isolates from laboratory rats were separated into these two clades, all Blastocystis isolates from wild rodents in the present study were positioned into the clade ST4A and further separated into two sub-clusters within the clade ST4A according to the location of the host species. Considering the fact that laboratory rats were susceptible to both ST4A and ST4B, separation of the monophyletic sub-clusters of Blastocystis isolates from Indonesian Polynesian rats and Japanese brown rats may indicate the presence of geographical variations rather than a host-specific separation. In either way, the robust host preference to rodent species of ST4 Blastocystis was also confirmed.

Evidence of infection with Leptospira interrogans and spotted fever group rickettsiae among rodents in an urban area of Osaka City, Japan.

We examined 33 rodents captured in an urban area of Osaka City, Japan for IgG antibodies against Seoul virus, severe fever with thrombocytopenia syndrome virus, hepatitis E virus, Leptospira interrogans, Yersinia pestis, spotted fever, typhus and scrub typhus group rickettsiae. We found that 3 (9.1%) and 1 (3.0%) of the 33 rodents had antibodies against L. interrogans and spotted fever group rickettsiae, respectively. DNAs of leptospires were detected from 2 of the 3 seropositive rodents, but DNA of rickettsia was not detected. Phylogenetic analysis and multiple locus sequence typing revealed that the 2 leptospires were L. interrogans belonging to a novel sequence type. There is a potential risk for acquiring rodent-borne zoonotic pathogens even in cities in developed countries.

Gene duplication and concerted evolution of mitochondrial DNA in crane species.

The gene duplication in mitochondrial DNA (mtDNA) has been reported in diverse bird taxa so far. Although many phylogenetic and population genetic analyses of cranes were carried out based on mtDNA diversity, whether mtDNA contains duplicated regions is unknown. To address the presence or absence of gene duplication in cranes and investigate the molecular evolutionary features of crane mtDNA, we analyzed the gene organization and the molecular phylogeny of mtDNA from 13 crane species. We found that the mtDNA in 13 crane species shared a tandem duplicated region, which consists of duplicated sequence sets including cytochrome b (Cytb), NADH6, control region (CR) and three genes of tRNA. The gene order in the duplicated region was identical among all the 13 crane species, and the nucleotide sequences found within each individual showed high similarities. In addition, phylogenetic trees based on homologous sequences of CR and Cytb indicated the possibility of concerted evolution among the duplicated genes. The results suggested that the duplication event occurred in the common ancestor of crane species or some older ancestors.

Blastocystis phylogeny among various isolates from humans to insects.

Blastocystis is a common unicellular eukaryotic parasite found not only in humans, but also in various kinds of animal species worldwide. Since Blastocystis isolates are morphologically indistinguishable, many molecular biological approaches have been applied to classify these isolates. The complete or partial sequences of the small subunit rRNA gene (SSU rDNA) are mainly used for comparisons and phylogenetic analyses among Blastocystis isolates. However, various lengths of the partial SSU rDNA sequence have been used for phylogenetic inference among genetically different isolates. Based on the complete SSU rDNA sequences, consensus terminology of nine subtypes (STs) of Blastocystis sp. that were supported by phylogenetically monophyletic nine clades was proposed in 2007. Thereafter, eight additional kinds of STs comprising non-human mammalian Blastocystis isolates have been reported based on the phylogeny of SSU rDNA sequences, while STs 11 and 12 were only proposed on the base of partial sequences. Although many sequence data from mammalian and avian Blastocystis are registered in GenBank, only limited data on SSU rDNA are available for poikilotherm-derived Blastocystis isolates. Therefore, the phylogenetic positions of the reptilian/amphibian Blastocystis clades are unstable. The phylogenetic inference of various STs comprising mammalian and/or avian Blastocystis isolates was verified herein based on comparisons between partial and complete SSU rDNA sequences, and the phylogenetic positions of reptilian and amphibian Blastocystis isolates were also investigated using 14 new Blastocystis isolates from reptiles with all known isolates from other reptilians, amphibians, and insects registered in GenBank.

Isolation and characterization of major histocompatibility complex class II B genes in cranes.

In this study, we isolated and characterized the major histocompatibility complex (MHC) class II B genes in cranes. Genomic sequences spanning exons 1 to 4 were amplified and determined in 13 crane species and three other species closely related to cranes. In all, 55 unique sequences were identified, and at least two polymorphic MHC class II B loci were found in most species. An analysis of sequence polymorphisms showed the signature of positive selection and recombination. A phylogenetic reconstruction based on exon 2 sequences indicated that trans-species polymorphism has persisted for at least 10 million years, whereas phylogenetic analyses of the sequences flanking exon 2 revealed a pattern of concerted evolution. These results suggest that both balancing selection and recombination play important roles in the crane MHC evolution.

Molecular evidence of Sarcocystis species in captive snakes in Japan.

Sarcocystis nesbitti, using snakes as the definitive host, is a causative agent of acute human muscular sarcocystosis in Malaysia. Therefore, it is important to explore the distribution and prevalence of S. nesbitti in snakes. Nevertheless, epizootiological information of S. nesbitti in snakes remains insufficient because few surveys have assessed Sarcocystis infection in snakes in endemic countries. In Japan, snakes are popular exotic pet animals that are imported from overseas, but the degree of Sarcocystis infection in them remains unclear. The possibility exists that muscular sarcocystosis by S. nesbitti occurs in contact with captive snakes in non-endemic countries. For a total of 125 snake faecal samples from 67 snake species collected at animal hospitals, pet shops and a zoo, this study investigated the presence of Sarcocystis using polymerase chain reaction (PCR) for the 18S ribosomal RNA gene (18S rDNA). Four (3.2%) faecal samples were positive by PCR. Phylogenetic analysis of the 18S rDNA sequences obtained from four amplification products revealed one isolate from a beauty snake (Elaphe taeniura), Sarcocystis zuoi, which uses rat snakes as the definitive host. The isolate from a Macklot's python (Liasis mackloti) was closely related with unidentified Sarcocystis sp. from reticulated pythons in Malaysia. The remaining two isolates from tree boas (Corallus spp.) were closely related with Sarcocystis lacertae, Sarcocystis gallotiae and unidentified Sarcocystis sp. from smooth snakes, Tenerife lizards and European shrews, respectively. This report is the first of a study examining the distribution of Sarcocystis species in captive snakes in Japan.

Comparative molecular phylogeny and evolution of sex chromosome DNA sequences in the family Canidae (Mammalia: Carnivora).

To investigate the molecular phylogeny and evolution of the family Canidae, nucleotide sequences of the zinc-finger-protein gene on the Y chromosome (ZFY, 924-1146 bp) and its homologous gene on the X chromosome (ZFX, 834-839 bp) for twelve canid species were determined. The phylogenetic relationships among species reconstructed by the paternal ZFY sequences closely agreed with those by mtDNA and autosomal DNA trees in previous reports, and strongly supported the phylogenetic affinity between the wolf-like canids clade and the South American canids clade. However, the branching order of some species differed between phylogenies of ZFY and ZFX genes: Cuon alpinus and Canis mesomelas were included in the wolf-like canid clades in the ZFY tree, whereas both species were clustered in a group of Chrysocyon brachyurus and Speothos venaticus in the ZFX tree. The topology difference between ZFY and ZFX trees may have resulted from the two-times higher substitution rate of the former than the latter, which was clarified in the present study. In addition, two types of transposable element sequence (SINE-I and SINE-II) were found to occur in the ZFY final intron of the twelve canid species examined. Because the SINE-I sequences were shared by all the species, they may have been inserted into the ZFY of the common ancestor before species radiation in Canidae. By contract, SINE-II found in only Canis aureus could have been inserted into ZFY independently after the speciation. The molecular diversity of SINE sequences of Canidae reflects evolutionary history of the species radiation.

Molecular characterization of crane Coccidia, Eimeria gruis and E. reichenowi, found in feces of migratory cranes.

Eimeria gruis and E. reichenowi have lethal pathogenicity to a number of species of cranes. These parasites develop at multiple organs or tissues in infected cranes, thus lacking the specificity of infection sites shown by other Eimeria spp. in spite of morphologic similarity. To date, there have been many reports of crane Eimeria infections, however, genetic examinations of these parasites have never been published. In the present study, we isolated oocysts of E. gruis and E. reichenowi from crane feces at a wintering area in Japan. By phylogenic analysis, we first demonstrated that partial sequences of the isolates formed their own cluster, located separately from other Eimeria spp.

Molecular characterization of a Cryptosporidium isolate from a banded mongoose Mungos mungo.

Cryptosporidium spp. has been found in more than 150 species of mammals, but there has been no report in mongooses. In this study, we report the isolation of Cryptosporidium sp. in a banded mongoose Mungos mungo, which was brought from Tanzania to Japan; the isolate was analyzed genetically to validate the occurrence of a new, host-adapted genotype. Cryptosporidium diagnostic fragments of 18S ribosomal RNA and 70-kDa heat shock protein genes were amplified from this isolate and compared with the other Cryptosporidium species and genotypes reported previously. Analyses showed that the mongoose isolate represents a new genotype, closely related to that of bears.

First record of Cryptosporidium infection in a raccoon dog (Nyctereutes procyonoides viverrinus).

Cryptosporidium species have been found in more than 150 species of mammals, but there has been no report in raccoon dogs. Here we found the Cryptosporidium organism in a raccoon dog, Nyctereutes procyonoides viverrinus, and identified this isolate using PCR-based diagnostic methods. Cryptosporidium diagnostic fragments of the 18S ribosomal RNA, Cryptosporidium oocyst wall protein and 70-kDa heat shock protein genes were amplified from the isolate and sequenced to reveal the phylogenetic relationships between it and other Cryptosporidium species or genotypes reported previously. The results showed that the raccoon dog isolate represented the C. parvum cattle genotype which could be a causative agent in human cryptosporidiosis.

A survey of Blastocystis sp. in livestock, pets, and zoo animals in Japan.

The prevalence of Blastocystis sp. was examined in fecal samples collected from cattle, pigs, dogs, and a variety of zoo animals (primates, carnivores, herbivores, pheasants, and ducks) by direct observation of fresh fecal suspensions or cultured materials, using light microscopy. The cattle and pigs were randomly sampled from 11 and 12 commercial farms, respectively, located in the western region of Japan. The dog material used in this study was obtained from pets housed in an animal shelter in the city of Osaka. Zoo animals were chosen based on housing conditions that minimized the possibility of intra-zoo transmission of the organism. The prevalence rate among the groups varied greatly. A high prevalence of infection was observed in the farm animal group, ranging from 95% (58/61) in pigs to 71% (39/55) in cattle, whereas the dog fecal samples were completely free of the organism. Prevalence of the organism in the zoo animal were 85% (29/34) in primates, 80% (8/10) in pheasants, 56% (9/16) in ducks, and 0% (0/58) in various carnivores and herbivores. Among the zoo animals infected with Blastocystis, eight species of primates, eight species of pheasants, and four species of ducks were confirmed as new hosts. Since Blastocystis organisms isolated from various animals were morphologically indistinguishable from Blastocystis hominis by light microscopy, further genomic studies are required for analysis of the zoonotic potential or etiological significance of these isolates.

The primary structure of cassowary (Casuarius casuarius) goose type lysozyme.

The complete amino acid sequence of cassowary (Casuarius casuarius) goose type lysozyme was analyzed by direct protein sequencing of peptides obtained by cleavage with trypsin, V8 protease, chymotrypsin, lysyl endopeptidase, and cyanogen bromide. The N-terminal residue of the enzyme was deduced to be a pyroglutamate group by analysis with a LC/MS/MS system equipped with the oMALDI ionization source, and then confirmed by a glutamate aminopeptidase enzyme. The blocked N-terminal is the first reported in this enzyme group. The positions of disulfide bonds in this enzyme were chemically identified as Cys4-Cys60 and Cys18-Cys29. Cassowary lysozyme was proved to consist of 185 amino acid residues and had a molecular mass of 20408 Da calculated from the amino acid sequence. The amino acid sequence of cassowary lysozyme compared to that of reported G-type lysozymes had identities of 90%, 83%, and 81%, for ostrich, goose, and black swan lysozymes, respectively. The amino acid substitutions at PyroGlu1, Glu19, Gly40, Asp82, Thr102, Thr156, and Asn167 were newly detected in this enzyme group. The substituted amino acids that might contribute to substrate binding were found at subsite B (Asn122Ser, Phe123Met). The amino acid sequences that formed three alpha-helices and three beta-sheets were completely conserved. The disulfide bond locations and catalytic amino acid were also strictly conserved. The conservation of the three alpha-helices structures and the location of disulfide bonds were considered to be important for the formation of the hydrophobic core structure of the catalytic site and for maintaining a similar three-dimensional structure in this enzyme group.