Wen-Yu Cheng

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.

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.

Pseudanoplocephala crawfordi is a member of genus Hymenolepis based on phylogenetic analysis using ribosomal and mitochondrial DNA sequences.

Abstract Pseudanoplocephala crawfordi is one of the important zoonotic cestodes causing economic significance and public health concern. In the present study, the phylogenetic position of P. crawfordi isolated from pigs was re-inferred using molecular markers of internal transcribed spacer ribosomal DNA (ITS rDNA) and partial NADH dehydrogenase subunit 1 (pnad1) mitochondrial DNA. The lengths of ITS1, ITS2 rDNA and pnad1 were 757 bp, 628 bp and 458 bp, respectively. Sequence differences in the ITS1, ITS2 rDNA and pnad1 between P. crawfordi and Hymenolepis species were smaller than that between cestodes within genus Hymenolepis. Phylogenetic analyses based on three gene fragments showed that P. crawfordi was grouped into cluster of Hymenolepis species. These results suggested that P. crawfordi would be one member of genus Hymenolepis but not in a new genus Pseudanoplocephala.

Prevalence of the intestinal parasite infection in cattle in Shaanxi province, northwestern China

A survey of intestinal parasite infection in cattle was conducted in Shaanxi province, northwestern China from November 2010 to January 2012, including Qinchuan cattle and dairy cattle. A total of 1736 fecal specimens (783 and 953 for Qinchuan and dairy cattle, respectively) in 18 farms (including 7 Qinchuan cattle and 11 dairy cattle farms) were obtained. The prevalences of intestinal parasites in Qinchuan cattle were slightly different from that in dairy cattle but with no statistically difference (P>0.05) except nematodes. Coccidian oocysts were the most common intestinal parasite, which were found in all cattle farms with prevalences ranged from 17.44 to 67.69%. The nematode eggs were found in 17 of 18 farms investigated with prevalences ranged from 0 to 43.75%. Cryptosporidium oocysts and the cestode eggs were only found in 10 and 5 farms, respectively. The present study showed that the prevalences of the intestinal parasites were affected by the related factors of geographical region, breed, age, and season. Therefore, the integrated strategies and measures should be taken to control intestinal parasites infection in cattle in Shaanxi province.

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

Abstract 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.

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

Abstract 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 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.