Phylogenetic analysis of Pomacea canaliculata isolates collected from rice fields in different origins of China by combined mitochondrial 12S and 16S genes.

To study the genetic relationships of Pomacea canaliculata collected from rice fields in China, the mitochondrial (mt) 12S and 16S of 9 P. canaliculata isolates from 5 southern provinces in China were sequenced and analyzed. The intra-specific sequence variations of P. canaliculata were 0-1.1% for 12S and 0--0.6% for 16S, while the inter-specific variations among common Pomacea species in mt 12S and 16S were 3.0-11.7% and 2.3-10.1%, respectively. Phylogenetic analysis based on combined sequences of mt 12S and 16S revealed complex genetic structure of P. canaliculata in China. Two phylogenetic groups of P. canaliculata were indicated in China with one group sistered to P. canaliculata isolates from USA, and two groups were even found in the same province. The phylogenetic relationships of Pomacea spp. also could be effectively inferred by combined sequences of mt 12S and 16S. These findings provided basic information for further study of population genetics and diffusion pattern of P. canaliculata in China as well as in the world.

Molecular identification of Pomacea canaliculata and P. insularum from rice paddy in different origins in China using mitochondrial adenosine triphosphate subunit 6 gene.

To identify Pomacea canaliculata and P. insularum using a molecular approach, the partial sequences of mitochondrial (mt) adenosine triphosphate subunit 6 (patp6) genes of two apple snails species from eight provinces of China were obtained. The intra-specific variations in mt patp6 within P. canaliculata were 0-4.5%, and no sequence difference in this region was observed within P. insularum. However, high inter-specific variations between P. canaliculata and P. insularum were detected, with sequence differences of 8.9-10.1%. Phylogenetic analyses based on sequences of mt patp6 revealed that P. canaliculata and P. insularum were grouped in different clades, but the genetic trees could not reveal geographically genetic relationships of P. canaliculata isolates from different origins. These findings will provide basic information for further study of molecular epidemiology and control of Pomacea invasion in China as well as in the world.

Characterization of the complete mitochondrial genomes of Nematodirus oiratianus and Nematodirus spathiger of small ruminants.

BACKGROUND: Nematodirus spp. are among the most common nematodes of ruminants worldwide. N. oiratianus and N. spathiger are distributed worldwide as highly prevalent gastrointestinal nematodes, which cause emerging health problems and economic losses. Accurate identification of Nematodirus species is essential to develop effective control strategies for Nematodirus infection in ruminants. Mitochondrial DNA (mtDNA) could provide powerful genetic markers for identifying these closely related species and resolving phylogenetic relationships at different taxonomic levels. METHODS: In the present study, the complete mitochondrial (mt) genomes of N. oiratianus and N. spathiger from small ruminants in China were obtained using Long-range PCR and sequencing. RESULTS: The complete mt genomes of N. oiratianus and N. spathiger were 13,765 bp and 13,519 bp in length, respectively. Both mt genomes were circular and consisted of 36 genes, including 12 genes encoding proteins, 2 genes encoding rRNA, and 22 genes encoding tRNA. Phylogenetic analyses based on the concatenated amino acid sequence data of all 12 protein-coding genes by Bayesian inference (BI), Maximum likelihood (ML) and Maximum parsimony (MP) showed that the two Nematodirus species (Molineidae) were closely related to Dictyocaulidae. CONCLUSIONS: The availability of the complete mtDNA sequences of N. oiratianus and N. spathiger not only provides new mtDNA sources for a better understanding of nematode mt genomics and phylogeny, but also provides novel and useful genetic markers for studying diagnosis, population genetics and molecular epidemiology of Nematodirus spp. in small ruminants.

Genetic variability of Baylisascaris schroederi from the Qinling subspecies of the giant panda in China revealed by sequences of three mitochondrial genes.

The present study examined the variations in three mitochondrial (mt) DNA sequences, namely cytochrome b (cytb), cytochrome c oxidase subunit 3 (cox3) and NADH dehydrogenase subunit 5 (nad5), among Baylisascaris schroederi isolates from the Qinling subspecies of the giant panda in Shaanxi province, northwestern China. No differences in length were detected in the three mt fragments from different isolates. The intra-specific sequence variations within all B. schroederi samples were 0-2.6% for pcytb, 0-1.8% for pcox3 and 0-2.1% for pnad5, while the inter-specific sequence differences among members of the genus Baylisascaris were 8.2-15.2%, 6.2-15.9% and 8.4-16.0% for pcytb, pcox3, pnad5, respectively. A phylogenetic analysis of the combined sequences of pcytb, pcox3 and pnad 5 showed that all B. schroederi samples in the present study were located in two large clusters, with one cluster containing samples from giant pandas in Sichuan province. These findings provide basic information for further study of molecular epidemiology and control of B. schroederi infection in the Qinling subspecies of the giant panda and throughout China.

Genetic variability in the three mitochondrial genes among Oesophagostomum asperum isolates from different regions in Shaanxi and Hunan Provinces, China.

The present study examined sequence variations in three mitochondrial DNA (mtDNA) regions, namely, NADH dehydrogenase subunit 5 (nad5), adenosine triphosphate subunit 6 (atp6) and cytochrome c oxidase subunit 3 (cox3), among Oesophagostomum asperum isolates from different regions in Shaanxi and Hunan provinces, China. The lengths for partial sequences of nad5 (pnad5), atp6 (patp6) and cox3 (pcox3) were 427 bp, 381 bp and 337 bp, respectively. The intra-specific sequence variations among all O. asperum samples were 0-2.11%, 0-1.84% and 0-1.48% for pnad5, patp6 and pcox3, respectively, while the inter-specific sequence differences among Oesophagostomum species in pig and small ruminants were 18-21.3% for pnad5, 18.3-24.5% for patp6 and 10.6-13.7% for pcox3. A phylogenetic analysis based on combined sequences of three mtDNA fragments indicated that all O. asperum isolates were grouped in one solid clade, and the Oesophagostomum spp. from pig were located in another clade. However, these mtDNA fragments could not reveal genetic relationships of geographical isolates of O. asperum in China. These results provided valuable information for studying population genetics of Oesophagostomum spp., and for controlling Oesophagostomum infection in animals as well as humans.

Characterization of Haemaphysalis flava (Acari: Ixodidae) from Qingling subspecies of giant panda (Ailuropoda melanoleuca qinlingensis) in Qinling Mountains (Central China) by morphology and molecular markers.

Tick is one of important ectoparasites capable of causing direct damage to their hosts and also acts as vectors of relevant infectious agents. In the present study, the taxa of 10 ticks, collected from Qinling giant pandas (Ailuropoda melanoleuca qinlingensis) in Qinling Mountains of China in April 2010, were determined using morphology and molecular markers (nucleotide ITS2 rDNA and mitochondrial 16S). Microscopic observation demonstrated that the morphological features of these ticks were similar to Haemaphysalis flava. Compared with other Haemaphysalis species, genetic variations between Haemaphysalis collected from A. m. qinlingensis and H. flava were the lowest in ITS2 rDNA and mitochondrial 16S, with sequence differences of 2.06%-2.40% and 1.30%-4.70%, respectively. Phylogenetic relationships showed that all the Haemaphysalis collected from A. m. qinlingensis were grouped with H. flava, further confirmed that the Haemaphysalis sp. is H. flava. This is the first report of ticks in giant panda by combining with morphology and molecular markers. This study also provided evidence that combining morphology and molecular tools provide a valuable and efficient tool for tick identification.

Genotyping Cryptosporidium andersoni in cattle in Shaanxi Province, Northwestern China.

The present study examined the prevalence and genotypes of Cryptosporidium andersoni in cattle in Shaanxi province, China. A total of 2071 fecal samples (847 from Qinchuan cattle and 1224 from dairy cattle) were examined for the presence of Cryptosporidium oocysts, and 70 samples (3.4%) were C. andersoni-positive and those positive samples were identified by PCR amplification of the small subunit ribosomal RNA (SSU rRNA) and the Cryptosporidium oocyst wall protein (COWP) genes. C. andersoni was the only species found in the examined cattle in this province. Fifty-seven C. andersoni isolates were characterized into 5 MLST subtypes using multilocus sequence typing analysis, including a new subtype in the native beef breed Qinchuan cattle. All of these C. andersoni isolates presented a clonal genetic structure. These findings provide new insights into the genetic structure of C. andersoni isolates in Shaanxi province and basic data of Cryptosporidium prevalence status, which in turn have implications for controlling cryptosporidiosis in this province.

Molecular characterization of Cyclospora-like organisms from golden snub-nosed monkeys in Qinling Mountain in Shaanxi province, northwestern China.

Cyclospora spp. have been identified as one of the most important intestinal pathogens causing protracted diarrhea in animals and human beings. To determine the Cyclospora species in the non-human primate Rhinopithecus roxellanae, a total of 71 fecal samples from 19 endangered snub-nosed monkeys in Shaanxi province were collected and examined using Sheater's sugar flotation technique and by sequencing the fragments of 18S rDNA. Only two Cyclospora isolates from 2 golden snub-nosed monkeys (R. roxellanae) were obtained and identified between July 2011 and August of 2012. The sequences of the 18S rDNA for the two Cyclospora isolates were 477 bp, with no nucleotide variation between them. Phylogenetic analysis based on the 18S rDNA sequences revealed that the two Cyclospora isolates were posited into the clade Cyclospora spp. and sistered to C. colobi. These results first showed that Cyclospora infection occurred in R. roxellanae in hot and rainy weather, which would provide useful information for further understanding the molecular epidemiology of Cyclospora spp. and the control of Cyclospora infection in non-human primates as well as in human beings.

Genetic variability among Dicrocoelium dendriticum isolates from different regions in Shaanxi Province, China revealed by sequences of three mitochondrial genes.

The genetic variations in three mitochondrial DNA (mtDNA) regions, namely portion of cytochrome c oxidase subunit 1 (pcox1), NADH dehydrogenase subunit 1 (pnad1) and cytochrome b (pcytb), were examined for Dicrocoelium dendriticum samples isolated from different origins in Shaanxi Province, northwestern China. The intra-specific sequence differences within D. dendriticum samples were 0-0.52% for pcox1, 0-0.73% for pnad1 and 0-0.58% for pcytb. Phylogenetic analyses based on combined sequences of three mtDNA showed that all D. dendriticum samples were clustered together in same clade of Paragonimus westermani. But the phylogenetic trees could not reveal geographically genetic relationships of D. dendriticum isolates in this province. These findings will provide basic information for further study of molecular epidemiology and control of D. dendriticum infection in this province as well as in China.