Vitamin B12 biosynthesis of Cetobacterium ceti isolated from the intestinal content of captive common bottlenose dolphins (Tursiops truncatus).

In comparison with terrestrial mammals, dolphins require a large amount of haemoglobin in blood and myoglobin in muscle to prolong their diving time underwater and increase the depth they can dive. The genus Cetobacterium is a common gastrointestinal bacterium in dolphins and includes two species: C. somerae and C. ceti. Whilst the former produces vitamin B12, which is essential for the biosynthesis of haem, a component of haemoglobin and myoglobin, but not produced by mammals, the production ability of the latter remains unknown. The present study aimed to isolate C. ceti from dolphins and reveal its ability to biosynthesize vitamin B12. Three strains of C. ceti, identified by phylogenetic analyses with 16S rRNA gene and genome-based taxonomy assignment and biochemical features, were isolated from faecal samples collected from two captive common bottlenose dolphins (Tursiops truncatus). A microbioassay using Lactobacillus leichmannii ATCC 7830 showed that the average concentration of vitamin B12 produced by the three strains was 11 (standard deviation: 2) pg ml-1. The biosynthesis pathway of vitamin B12, in particular, adenosylcobalamin, was detected in the draft genome of the three strains using blastKOALA. This is the first study to isolate C. ceti from common bottlenose dolphins and reveal its ability of vitamin B12 biosynthesis, and our findings emphasize the importance of C. ceti in supplying haemoglobin and myoglobin to dolphins.

Prevalence and antimicrobial-resistance profiles of Salmonella spp. isolated from green anoles (Anolis carolinensis) collected on the Haha-jima of the Ogasawara archipelago, Japan.

We investigated the prevalence of Salmonellaenterica and its antimicrobial resistance from 79 green anoles, the invasive alien species inhabits Haha-jima of the Ogasawara archipelago. Samples were collected during the period between 2009 and 2010. The resistance of S. enterica of these samples against 12 common antimicrobial agents was also determined. Salmonella strains, including serovar Oranienburg and Aberdeen, were detected from the large intestines of 30.4% of 79 green anole samples. And 37.5% of which were resistant to Oxytetracycline. This study suggests that green anoles may play an important role of the infection of S. enterica on this island. Attention is needed from the aspect of public and ecological health.

Prevalence of Salmonella in green anoles (Anolis Carolinensis), an invasive alien species in Naha and Tomigusuku Cities, Okinawa Main Island, Japan.

Here, we investigated the prevalence of Salmonella enterica, with and without resistance to 17 common antimicrobial agents, in 706 green anoles (Anolis carolinensis) that were collected in Naha and Tomigusuku Cities, Okinawa Main Island, Japan, between 2009 and 2014. Salmonella strains, including S. enterica Weltevreden and Enteritidis serovars, were identified in the large intestinal content samples extracted from 15 (2.1%) of the analyzed green anoles. No antimicrobial resistance was detected. Thus, the present study demonstrates that although the prevalence of Salmonella and the risk of its transmission from the green anoles to humans or other animals on Okinawa Main Island are relatively low, the green anole population nevertheless represents a potential source of Salmonella infection that could affect human health in this region.

Prevalence of serum antibodies to Toxoplasma gondii in the small Indian mongoose (Herpestes auropunctatus) on Amami-Oshima Island, Japan.

Prevalence of antibodies to Toxoplasm gondii was studied using the latex agglutination (LA) method, followed by sucrose density gradient centrifugation (SDGC) method on the small Indian mongoose (Herpestes auropunctatus), which inhabits Amami-Oshima Island. Of the 362 samples, 38 (10.5%) revealed positive. Single or double peaks in the 7-8 and/or 12-14 fraction to LA titer by SDGC indicated the early stage of T. gondii infection. It is suggested that domestic/feral cats play an important role for spreading this zoonotic pathogen to the mongoose as well as other species that are endemic to this island. Future studies are warranted to prevent the transmission of T. gondii among cats and wild animals in order to maintain the ecosystem health.

Trans-species polymorphism of the Mhc class II DRB-like gene in banded penguins (genus Spheniscus).

The Major Histocompatibility Complex (Mhc) class II DRB locus of vertebrates is highly polymorphic and some alleles may be shared between closely related species as a result of balancing selection in association with resistance to parasites. In this study, we developed a new set of PCR primers to amplify, clone, and sequence overlapping portions of the Mhc class II DRB-like gene from the 5'UTR end to intron 3, including exons 1, 2, and 3 and introns 1 and 2 in four species (20 Humboldt, six African, five Magellanic, and three Galapagos penguins) of penguin from the genus Spheniscus (Sphe). Analysis of gene sequence variation by the neighbor-joining method of 21 Sphe sequences and 20 previously published sequences from four other penguin species revealed overlapping clades within the Sphe species, but species-specific clades for the other penguin species. The overlap of the DRB-like gene sequence variants between the four Sphe species suggests that, despite their allopatric distribution, the Sphe species are closely related and that some shared DRB1 alleles may have undergone a trans-species inheritance because of balancing selection and/or recent rapid speciation. The new primers and PCR assays that we have developed for the identification of the DRB1 DNA and protein sequence variations appear to be useful for the characterization of the molecular evolution of the gene in closely related Penguin species and might be helpful for the assessment of the genetic health and the management of the conservation and captivity of these endangered species.

Prevalence of antimicrobial-resistant Escherichia coli in endangered Okinawa rail (Gallirallus okinawae) inhabiting areas around a livestock farm.

Antimicrobial resistance (AMR) is an important issue for public, animal and environmental health. It has been suggested that livestock farms could be a source origin of AMR, and some wild animals that inhabit this area may play an important role in the spread of AMR in the natural environment. The prevalence of AMR in Escherichia coli was examined from Okinawa rails (Gallirallus okinawae), an endemic bird in Okinawa Main Island, Japan. Forty-eight faecal samples of wild Okinawa rails were collected from around a livestock farm area (LA), near human settlements, in which a population of the Okinawa rail had newly inhabited for feeding, and a forest area (FA), their natural habitat. Among 16 E. coli-positive faecal samples collected around LA, 11/16 (69%) showed antimicrobial resistance and five multiple drug resistance patterns were identified. However, among 15 E. coli-positive faecal samples from FA, 3/15 (20%) showed antimicrobial resistance, and three multiple drug resistance patterns were identified. These results indicate that the endangered Okinawa rail may also play an important role as a potential vector for the spread of AMR in the natural environment. To maintain ecological health, it is imperative that in situ/ex situ conservation projects that include translocation plans for endangered species are aware of these data.

Paternity determination of captive bottlenose dolphins (Tursiops truncatus) using microsatellite DNA analysis.

We applied previously published PCR primer pairs to amplify alleles at three polymorphic microsatellite loci to determine the genetic relationship of 6 bottlenose dolphins (Tursiops truncatus) that were living together in a Japanese aquarium. The three microsatellite loci were sufficient to determine the haplotype relationships of the six dolphins, which represented three different generations. It was confirmed that this genotyping method is simple and economical for assessing, establishing and maintaining genetic diversity in captive populations and will become a very effective technique for ex situ conservation in aquariums and zoos.

Detection of avian Plasmodium spp. DNA sequences from mosquitoes captured in Minami Daito Island of Japan.

Several species of birds in Minami Daito Island, an oceanic island located in the far south from the main islands of Japan, were found to be infected with avian Plasmodium. However, no vector species of the avian malaria in this island have been revealed yet. To speculate potential vectors, we collected mosquitoes there and investigated using a PCR procedure whether the mosquitoes harbor avian malaria or not. Totally 1,264 mosquitoes including 9 species were collected during March 2006 to February 2007. The mosquitoes collected were stored every species, sampled date and location for DNA extraction. Fifteen out of 399 DNA samples showed positive for the partial mtDNA cytb gene of avian Plasmodium. Estimated minimum infection rate among collected mosquitoes was 1.2% in this study. Four species of mosquitoes; Aedes albopictus, Culex quinquefasciatus, Lutzia fuscanus and Mansonia sp. had avian Plasmodium gene sequences. Detected DNA sequences from A. albopictus and L. fuscanus were identical to an avian Plasmodium lineage detected in bull-headed shrike (Lanius bucephalus) captured in the island. Different sequences were detected from C. quinquefasciatus, which were corresponding to an avian Plasmodium from a sparrow (Passer montanus) and Plasmodium gallinaceum. Our results suggest that A. albopictus, Lutzia fuscanus, C. quinquefasciatus, and Mansonia sp. could be potential vectors of avian malaria in Minami Daito Island. This study was the first report of molecular detection of avian Plasmodium from mosquitoes in Japan.

Salmonella infection in green anoles (Anolis carolinensis), an invasive alien species on Chichi Island of the Ogasawara archipelago in Japan.

We investigated the presence of Salmonella in the green anole (Anolis carolinensis), an invasive alien species on Chichi Island, Japan. Samples were also collected from feral goats and public toilets on the island to examine infectious routes. Salmonellae were isolated from 27.1% of 199 samples; 32.6% of 141 cloacal samples from anoles, 62.5% of 8 intestinal samples from anole carcasses, 16.7% of 12 fecal samples from goats and 2.6% of 38 toilet bowl swabs. The serotype of most isolates was Salmonella Oranienburg (94.4% of 54). Although we did not confirm the infection pathways, our results indicated that green anoles are a risk factor as a source of Salmonella for public health. It is important to consider endemic pathogens that may be amplified by alien species within their introduced areas.

Sequence determination of the heavy-chain constant region in four immunoglobulin classes of Mongolian gerbils (Meriones unguiculatus).

We determined partial cDNA sequences of four immunoglobulin (Ig) classes-IgM, IgG1, IgE, and IgA-of Mongolian gerbil (Meriones unguiculatus). Each deduced Ig heavy-chain constant (IGHC) region-Cµ, Cγ1, Cε, and Cα-is structurally similar to its counterparts in the mouse and rat, and phylogenetic analysis suggests that the gerbil Igs are evolutionarily close to their counterparts. In spite of the high sequence homology to the other rodent Cγ sequences, the gerbil Cγ1 sequence differs from our previously reported Cγ2. This result indicates that the gerbil has at least two IgG subclasses. These four gerbil IGHC cDNA sequences will be useful for determining gerbil Ig isotypes and examining the expression of gerbil Ig mRNAs in response to parasitic and bacterial infections.

Prevalence of avian haematozoa in wild birds in a high-altitude forest in Japan.

The infection dynamics of avian haematozoa, which includes the genera Plasmodium, Haemoproteus, and Leucocytozoon, are complicated by a variety of environmental factors and host-parasite interactions. In Japan, the prevalence of haematozoa in wild birds has recently been determined in several local areas. However, no information on the annual prevalence of avian haematozoa in a single study site has been reported. Here, we investigated the long-term infection dynamics of haematozoa in wild birds inhabiting a mountain forest of Japan. Blood samples were collected from 415 wild birds captured in the Chichibu mountains in Saitama Prefecture at an altitude of 1650 m between 2007 and 2010. All obtained samples were examined for haematozoan infection using nested polymerase chain reaction (PCR) of the cytochrome b (cytb) genes of haematozoa. A total of 62 out of 415 (14.9%) forest birds were PCR positive for haematozoa. Relatively high infection rates of Leucocytozoon were found among several bird species (Parus ater, 64.3%; Parus montanus, 81.8%) and may be due to the host preference of vector black flies and host nestling pattern in this forest. Phylogenetic analysis of amplified cytb sequences revealed for the first time that a variety of lineages of avian haematozoa are distributed among wild bird hosts in a high-altitude forest stand in Japan. Notably, significant seasonal changes of the prevalence of avian haematozoa were not observed; however, continuous investigation will likely provide detailed information on host-parasite interactions, including local environmental factors, that influence the dynamics of avian haematozoan infections.

Partial sequence of Mongolian gerbil (Meriones unguiculatus) immunoglobulin gamma heavy chain constant region.

We determined the sequence of the immunoglobulin gamma heavy-chain constant (IGHC) region of the Mongolian gerbil (Meriones unguiculatus). To isolate a part of the IGHC complementary DNA, we designed primers on the basis of highly conserved sequences in mouse, rat and hamster. The deduced IGHC is structurally similar to counterparts in other mammalian species and shows 84.6% identity to the IGHC of hamster IgG, 76.6% to rat IgG1, 83.3% to rat IgG2a, 78.1% to mouse IgG1, 81.8% to mouse IgG2a, 79.1% to mouse IgG2b and 79.2% to mouse IgG3 at the nucleotide level. The results suggest that gerbil IgG is closely related to hamster IgG and rat IgG2a.

HLA alleles are associated with postherpetic neuralgia but not with herpes zoster.

OBJECTIVE: In some herpes zoster (HZ) patients, one symptom is problematic; postherpetic neuralgia (PHN), a persistence of pain such as spontaneous pain and stimulus-evoked pain, allodynia or hyperpathia, for more than 6 months after healing of the vesicular eruptions. In our previous study, we reported the association between HLA alleles, HLA-A*33 and B*44, and PHN patients. In this study, we aim to refine the association of these alleles with PHN or HZ using higher-resolution HLA typing technique with an increased sample size. METHODS: HLA allele frequencies were compared in 70 PHN patients, 80 HZ patients, 52 HZ(-) patients, and 140 Japanese controls using HLA typing kits of SSOP protocols. RESULT AND CONCLUSION: The allele frequencies of HLA-A*3303, B*4403, and DRB1*1302 in PHN(+) patients were significantly higher than those in Japanese controls (P=0.00007, P=0.000002, and P=0.0003, respectively). The frequencies of above alleles in PHN(+) patients were also significantly higher than those in PHN(-) patients (P=0.03, P=0.006 and P=0.03, respectively). However, no association was found in comparison of HZ(+) patients and HZ(-). And the frequency of HLA-B*5101 in HZ(-) patients was significantly higher than those in HZ(+) and PHN(+) patients (P=0.003 and P=0.009, respectively) indicating that HLA-B*5101 functions as a protective allele to HZ.

Analysis of the sequence variations in the Mhc DRB1-like gene of the endangered Humboldt penguin (Spheniscus humboldti).

The Major Histocompatibility Complex (Mhc) genomic region of many vertebrates is known to contain at least one highly polymorphic class II gene that is homologous in sequence to one or other of the human Mhc DRB1 class II genes. The diversity of the avian Mhc class II gene sequences have been extensively studied in chickens, quails, and some songbirds, but have been largely ignored in the oceanic birds, including the flightless penguins. We have previously reported that several penguin species have a high degree of polymorphism on exon 2 of the Mhc class II DRB1-like gene. In this study, we present for the first time the complete nucleotide sequences of exon 2, intron 2, and exon 3 of the DRB1-like gene of 20 Humboldt penguins, a species that is presently vulnerable to the dangers of extinction. The Humboldt DRB1-like nucleotide and amino acid sequences reveal at least eight unique alleles. Phylogenetic analysis of all the available avian DRB-like sequences showed that, of five penguin species and nine other bird species, the sequences of the Humboldt penguins grouped most closely to the Little penguin and the mallard, respectively. The present analysis confirms that the sequence variations of the Mhc class II gene, DRB1, are useful for discriminating among individuals within the same penguin population as well those within different penguin population groups and species.