Ya-Biao Weng

Genetic variability among Schistosoma japonicum isolates from different endemic regions in China revealed by sequences of three mitochondrial DNA genes

The present study examined sequence variation in three mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 3 (cox3), NADH dehydrogenase subunits 4 and 5 (nad4 and nad5), among Schistosoma japonicum isolates from different endemic regions in China, and their phylogenetic relationships were re-constructed. A portion of the cox3 gene (pcox3), a portion of the nad4 and nad5 genes (pnad4 and pnad5) were amplified separately from individual trematodes by polymerase chain reaction (PCR) and the amplicons were subjected to direct sequencing. In the mountainous areas, sequence variations between parasites from Yunnan and those from Sichuan were 0.3% for pcox3, 0.0-0.1% for pnad4, and 0.0-0.2% for pnad5. In the lake/marshland areas, sequence variations between male and female parasites among different geographical locations were 0.0-0.3% for pcox3, 0.0-0.7% for pnad4, and 0.0-1.6% for pnad5. Sequence variations between S. japonicum from mountainous areas and those from lake/marshland areas were 0.0-0.5% for pcox3, 0.0-0.7% for pnad4, and 0.0-1.6% for pnad5. Phylogenetic analyses based on the combined sequences of pcox3, pnad4 and pnad5 revealed that S. japonicum isolates from mountainous areas (Yunnan and Sichuan provinces) clustered together. For isolates from the lake/marshland areas, isolates from Anhui and Jiangsu provinces clustered together and was sister to samples from Jiangxi province, while isolates from Hubei and Zhejiang province clustered together. However, isolates from different geographical locations in Hunan province were in different clades. These findings demonstrated the usefulness and attributes of the three mtDNA sequences for population genetic studies of S. japonicum, and have implications for studying population biology, molecular epidemiology, and genetic structure of S. japonicum, as well as for the effective control of schistosomiasis.

Seroprevalence of Toxoplasma gondii infection in pet dogs in Lanzhou, Northwest China

BackgroundIn recent years, surveys of Toxoplasma gondii infection in dogs have been reported worldwide, including China. However, little is known about the prevalence of T. gondii in pet dogs in Northwest China. In the present study, the prevalence of T. gondii in pet dogs in Lanzhou, China was investigated using the modified agglutination test (MAT).ResultsIn this survey, antibodies to T. gondii were found in 28 of 259 (10.81%) pet dogs, with MAT titers of 1:20 in 14 dogs, 1:40 in nine, 1:80 in four, and 1:160 or higher in one dog. The prevalence ranged from 6.67% to 16.67% among dogs of different ages, with low rates in young pet dogs, and high rates in older pet dogs. The seroprevalence in dogs >3 years old was higher than that in dogs ≤1 years old, but the difference was not statistically significant (P > 0.05). The seroprevalence in male dogs was 12.50% (17 of 136), and in female dogs it was 8.94% (11 of 123), but the difference was not statistically significant (P > 0.05).ConclusionsA high prevalence of T. gondii infection was found in pet dogs in Lanzhou, Northwest China, which has implications for public health in this region. In order to reduce the risk of exposure to T. gondii, further measures and essential control strategies should be carried out rationally in this region.

Characterization of the complete mitochondrial genomes of five Eimeria species from domestic chickens.

Chicken coccidiosis caused by members of the genus Eimeria causes significant economic losses worldwide. In the present study we sequenced the complete mitochondrial DNA (mtDNA) sequences of six Eimeria species and analyzed features of their gene contents and genome organizations. The complete mt genomes of E. acervulina, E. brunetti, E. maxima, E. necatrix, E. tenella and E. praecox were 6179bp, 6148bp, 6169bp, 6214bp, 6213bp and 6174bp in size, respectively. All of the mt genomes consist of 3 genes for proteins (cox1, cox3, and cytb), 12 gene fragments for the large subunit (LSU) rRNA, and 7 gene fragments for the small subunit (SSU) rRNA, but no transfer RNA genes. The organization of the mt genomes is similar to that of Plasmodium, but distinct from Babesia and Theileria. The putative direction of translation for 3 genes (cox1, cox3, and cytb) was the same in all six Eimeria species. The contents of A+T of the mt genomes were 65.35% for E. acervulina, 65.43% for E. brunetti, 64.53% for E. maxima, 65.04% for E. necatrix, 64.98% for E. tenella and 65.59% for E. praecox. The AT bias has a significant effect on both the codon usage pattern and amino acid composition of proteins. Phylogenetic analyses using concatenated nucleotide sequences of the 2 protein-coding genes (cytb and cox1), with three different computational algorithms (Bayesian analysis, maximum parsimony and maximum likelihood), all revealed distinct groups with high statistical support, indicating that the six Eimeria spp. represent six distinct but closely-related species. These data provide novel mtDNA markers for studying the molecular epidemiology and population genetics of the six Eimeria spp., and should have implications for the molecular diagnosis, prevention and control of coccidiosis in domestic chickens.

First report of Toxoplasma gondii seroprevalence in peafowls in Yunnan Province, Southwestern China

BackgroundToxoplasma gondii is an intracellular protozoan parasite infecting almost all warm-blooded animals, including birds, with a worldwide distribution. Surveys of T. gondii infection in wild birds have been reported extensively in the world, but little is known of T. gondii infection in peafowls worldwide. This study was performed to determine the seroprevalence of T. gondii infection in peafowls in Yunnan Province, southwestern China.MethodsSera from 277 peafowls, including 272 blue peafowls (Pavo cristatus) and 5 green peafowls (Pavo muticus) originated from two geographic areas in Yunnan Province were assayed for T. gondii antibodies using the modified agglutination test (MAT).ResultsSpecific T. gondii antibodies were detected in 35 of 277 (12.64%) peafowls (MAT titer ≥ 1:5). Seropositive birds were found in both species, 33 in 272 blue peafowls and 2 in 5 green peafowls. There was no significant difference in T. gondii seroprevalence between the adolescent birds (6.74%) and the adult birds (6.67%) (P > 0.05). The geographical origins of peafowls was found to be highly associated with T. gondii infection in the present study, a statistically significant difference in T. gondii seropositivity was observed between peafowls from Kunming (31.08%) and those from Xishuangbanna Dai Autonomous Prefecture (5.91%) (OR = 10.956, 95% CI = 1.632-73.545, P = 0.014). Statistical analyses showed that there were no significant interactions between ages and geographical origins of peafowls (P > 0.05).ConclusionsThe results of the present survey indicated that infection of peafowls with T. gondii is widespread in Yunnan Province, which has significant public health concerns and implications for prevention and control of toxoplamosis in this province. To our knowledge, this is the first seroprevalence report of T. gondii infection in China’s southwestern Yunnan Province.

Seroprevalence of Toxoplasma gondii infection in household and stray cats in Lanzhou, northwest China.

BackgroundToxoplasma gondii is an important protozoan parasite infecting humans and almost all warm-blooded animals. As the only definitive host, cats play a crucial role in the transmission of T. gondii infection by shedding parasite oocysts in their feces. However, little information on T. gondii infection in cats was available in Lanzhou, northwest China. This study was performed to determine the seroprevalence of T. gondii infection in household and stray cats in Lanzhou, northwest China.ResultsA total of 221 (179 households and 42 strays) blood samples were collected from clinically healthy cats admitted to several pet hospitals located in Lanzhou City, between November 2010 and July 2011 for the serological detection of T. gondii infection. The majority (207) of these cats represented Chinese Lihua cats. 47 of 221 (21.3%) examined cats were seropositive for T. gondii infection using the modified agglutination test (MAT) at the cut-off of 1:25. The seroprevalence in household and stray cats was assessed to be 15.6% and 45.2%, respectively, and the difference was statistically significant (P < 0.05). The seroprevalence ranged from 15.1% to 25.8% among different age groups, but the differences were not statistically significant (P > 0.05). Studies showed that there was no relationship between seroprevalence and the gender (P > 0.05).ConclusionsThe present survey indicated the high seroprevalence of T. gondii in cats in Lanzhou, northwest China, which poses a threat to animal and human health. Therefore, measures should be taken to control and prevent toxoplasmosis of cats in this area.

ISSR, an effective molecular approach for studying genetic variability among Schistosoma japonicum isolates from different provinces in mainland China.

In the present study, inter-simple sequence repeats (ISSRs) markers were used to examine the genetic variability of Schistosoma japonicum isolates from different provinces in mainland China, using S. japonicum from Japan and S. mansoni from Puerto Rico for comparison. Of the 30 primers screened, 4 produced highly reproducible ISSR fragments. Using these primers, 107 discernible DNA fragments were generated with 105 (98.13%) being polymorphic, indicating considerable genetic variation among the examined S. japonicum isolates. The percentage of polymorphic bands among S. japonicum isolates from mainland China and Japan was 82.24%, 43.93% among mountainous type isolates and 64.49% among lake/marshland type isolates from mainland China. UPGMA analysis revealed that all of the S. japonicum samples were grouped into two clades, the first contained isolates from mainland China, and the other one contained samples from Japan. Within the cluster of S. japonicum isolates from mainland China, isolates from mountainous Sichuan and Yunnan provinces grouped together, whereas isolates from lake/marshland regions (Anhui, Jiangsu and Hubei provinces) clustered together. The results of present study demonstrated that the ISSR markers are useful for studying genetic diversity and population structure of S. japonicum isolates from mainland China.

The complete mitochondrial genome sequence of Eimeria mitis (Apicomplexa: Coccidia)

In this study the complete mitochondrial DNA (mtDNA) sequence of Eimeria mitis was sequenced, and its gene contents and genome organizations were compared with that of other Eimeria spp. The complete mt genome sequence of E. mitis is 6407 bp in size. It consists of 3 protein-coding genes (cox1, cox3, and cytb), 12 gene fragments for the large subunit (LSU) rRNA, and 7 gene fragments for the small subunit (SSU) rRNA, but no transfer RNA genes, similar to that of Eimeria spp. The putative direction of translation for three genes (cox1, cox3, and cytb) was the same as those of six other Eimeria spp. The A+T content of the E. mitis mt genome was 67.30%. The E. mitis mt genome sequence provides novel mtDNA marker for studying the molecular epidemiology and population genetics of E. mitis and has implications for the molecular diagnosis of chicken coccidiosis caused by E. mitis.

A specific PCR assay for the identification and differentiation of Schistosoma japonicum geographical isolates in mainland China based on analysis of mitochondrial genome sequences

In the present study, near-complete mt genome sequences for eight representative Schistosoma japonicum samples from seven endemic provinces in mainland China were analyzed. Sequence differences among the eight mt genomes of S. japonicum samples were 0.20-2.51%. Variation in protein-coding genes was greater than that in rRNA genes. The mt DNA sequences of S. japonicum samples from south-western (SW) China were 2 bp [position 11727-11728 within tRNA-Cys, microsatellite (AG) indel] longer than those of the parasites from the lower Yangtze/Zhejiang areas. Representative DNA sequencing confirmed that such (AG) indel could be exploited for identification and differentiation of S. japonicum populations in SW Chinas Yunnan and Sichuan province which have two (AG) repeats from those in all remaining endemic provinces along the Yangtze River below the Three Gorges regions or close to the east coast of China (e.g., Zhejiang) which have only one (AG) repeat. Phylogenetic analyses based on the concatenated amino acids of 12 protein-coding genes also showed that samples from SW China (Sichuan and Yunnan provinces), above the Three Gorges Dam, formed a distinct cluster. Based on this indel polymorphism, a pair of specific primers was designed and used to develop a specific-PCR polyacrylamide gel detection assay. There was an obvious length difference in the amplified PCR products between S. japonicum samples from the two endemic types. The specific-PCR assay allowed the specific identification of S. japonicum, with no amplicons being amplified from other closely related trematodes, and the minimum amount of DNA detectable was 0.05 ng. This approach is inexpensive, easy to perform and the whole detection process can be completed within 4h. Examination of 81 S. japonicum samples from SW Chinas Yunnan and Sichuan provinces, and 264 samples from the lower Yangtze provinces (Hubei, Jiangsu, Jiangxi, Anhui and Hunan) and from Zhejiang validated the value of the specific PCR assay and proved its reliability. These findings indicate that the specific PCR assay would provide a useful tool for the epidemiological surveillance and for tracing the source of S. japonicum infection in humans and animals in China.

Sequence variability in three mitochondrial genes between the two pig nodule worms Oesophagostomum dentatum and O. quadrispinulatum

In this study, sequence variation in three mitochondrial DNA regions, namely cytochrome c oxidase subunit (cox1) and NADH dehydrogenase subunits 1 and 4 (nad1 and nad4), between Oesophagostomum dentatum and O. quadrispinulatum isolated from pigs in different geographical origins in Mainland China was examined, and their phylogenetic relationships were reconstructed. A partial of the cox1 (pcox1), nad1, and nad4 genes (pnad1 and pnad4) were amplified separately from individual nodule worms by PCR and were subjected to direct sequencing in order to define sequence variations. While the intraspecific sequence variations within each of the two species were 0.3–5.2% for pcox1, 0–4.9% for pnad1, and 0–7.1% for pnad4, the interspecific sequence differences were significantly higher, being 10.7–13.4% for pcox1, 11–14.6% for pnad1, and 14.9–18% for pnad4, respectively. There were a number of nucleotide positions in the pcox1, pnad1, and pnad4 sequences with no apparent intraspecific variation but distinct interspecific differences among those samples of Oesophagostomum spp. examined, which may be used as genetic makers for the identification and differentiation of the Oesophagostomum spp. Phylogenetic analyses using three inference methods, namely Bayesian inference, maximum likelihood, and maximum parsimony based on the combined sequences of pcox1, pnad1, and pnad4 revealed that the O. dentatum and O. quadrispinulatum form monophyletic groups, respectively. These findings demonstrated clearly the usefulness of the three mitochondrial sequences for studying systematics, population genetic structures, and the molecular ecology of Oesophagostomum spp.

Genetic diversity and relatedness of Fasciola spp. isolates from different hosts and geographic regions revealed by analysis of mitochondrial DNA sequences.

The present study examined sequence variability in a portion of the mitochondrial cytochrome c oxidase subunit 1 (pcox1) and NADH dehydrogenase subunits 4 and 5 (pnad4 and pnad5) among 39 isolates of Fasciola spp., from different hosts from China, Niger, France, the United States of America, and Spain; and their phylogenetic relationships were re-constructed. Intra-species sequence variations were 0.0-1.1% for pcox1, 0.0-2.7% for pnad4, and 0.0-3.3% for pnad5 for Fasciola hepatica; 0.0-1.8% for pcox1, 0.0-2.5% for pnad4, and 0.0-4.2% for pnad5 for Fasciola gigantica, and 0.0-0.9% for pcox1, 0.0-0.2% for pnad4, and 0.0-1.1% for pnad5 for the intermediate Fasciola form. Whereas, nucleotide differences were 2.1-2.7% for pcox1, 3.1-3.3% for pnad4, and 4.2-4.8% for pnad5 between F. hepatica and F. gigantica; were 1.3-1.5% for pcox1, 2.1-2.9% for pnad4, 3.1-3.4% for pnad5 between F. hepatica and the intermediate form; and were 0.9-1.1% for pcox1, 1.4-1.8% for pnad4, 2.2-2.4% for pnad5 between F. gigantica and the intermediate form. Phylogenetic analysis based on the combined sequences of pcox1, pnad4 and pnad5 revealed distinct groupings of isolates of F. hepatica, F. gigantica, or the intermediate Fasciola form irrespective of their origin, demonstrating the usefulness of the mtDNA sequences for the delineation of Fasciola species, and reinforcing the genetic evidence for the existence of the intermediate Fasciola form.