Feng-Cai Zou

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 Genotyping of Toxoplasma gondii Isolates From Wild Birds in China

Abstract: Toxoplasma gondii is an important cosmopolitan opportunistic protozoan parasite, which threatens the health of human beings and animals. Genetic characterization of isolates from South America has revealed high genetic diversity. In contrast, isolates from North America and Europe were highly clonal, with 3 major lineages known as the Types I, II, and III. However, limited information on T. gondii genotypes has been reported in The Peoples Republic of China. Here we conducted a survey to determine genetic diversity of this parasite in wild birds of China. In total, tissues from breast muscle of 178 wild birds, including 98 common pheasants (Phasianus colchicus), 35 tree sparrows (Passer montanus), 22 house sparrows (Passer domesticus), 20 saxaul sparrows (Passer ammodendri), and 1 cinnamon sparrow (Passer rutilans), were tested for T. gondii infection, 4 of which were found to be positive for the T. gondii B1 gene by PCR amplification. These positive DNA samples were typed at 10 genetic markers, including 9 nuclear loci, i.e., SAG1, 5′- and 3′-SAG2, alternative SAG2, SAG3, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico. Of these, 3 isolates were genotyped with complete data for all loci, and 2 genotypes (Type I and Type II variant) were identified. This is the first report of genetic typing of T. gondii isolates from wild birds from different regions in China. The results suggest that the Type I and II variant strains are circulating in wild birds in China, and these birds are potential reservoirs for T. gondii transmission.

Genetic characterization, species differentiation and detection of Fasciola spp. by molecular approaches

Liver flukes belonging to the genus Fasciola are among the causes of foodborne diseases of parasitic etiology. These parasites cause significant public health problems and substantial economic losses to the livestock industry. Therefore, it is important to definitively characterize the Fasciola species. Current phenotypic techniques fail to reflect the full extent of the diversity of Fasciola spp. In this respect, the use of molecular techniques to identify and differentiate Fasciola spp. offer considerable advantages. The advent of a variety of molecular genetic techniques also provides a powerful method to elucidate many aspects of Fasciola biology, epidemiology, and genetics. However, the discriminatory power of these molecular methods varies, as does the speed and ease of performance and cost. There is a need for the development of new methods to identify the mechanisms underpinning the origin and maintenance of genetic variation within and among Fasciola populations. The increasing application of the current and new methods will yield a much improved understanding of Fasciola epidemiology and evolution as well as more effective means of parasite control. Herein, we provide an overview of the molecular techniques that are being used for the genetic characterization, detection and genotyping of Fasciola spp..

Genomics and molecular genetics of Clonorchis sinensis: Current status and perspectives

Clonorchiasis caused by Clonorchis sinensis is an important foodborne parasitosis of humans and animals, and is predominantly a hepatobiliary disease. Globally, nearly 35 million people were infected with C. sinensis, with approximately 15 million being in China. Patients would chronically present fatigue, jaundice, abdominal discomfort, along with the increased risk of developing into a form of cholangiocarcinoma that is fatal to humans. Treatment of clonorchiasis by praziquantel has been very successful, but this is dependent on early accurate diagnosis and correct species identification. The present article reviews the current status of knowledge in genomics and functional genomics of C. sinensis, and summarizes the main DNA-based techniques for the specific diagnosis of C. sinensis infection and studies of genetic variation in C. sinensis, and provides perspectives for future studies. The advances in genomics and molecular genetics of C. sinensis shed new sight on our understanding of population structure of C. sinensis as well as the prevention and control of clonorchiasis.

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.

Characterization of the Complete Mitochondrial Genome Sequence of Spirometra erinaceieuropaei (Cestoda: Diphyllobothriidae) from China

Sparganosis, caused by the plerocercoid larvae of members of the genus Spirometra, can cause significant public health problem and considerable economic losses. In the present study, the complete mitochondrial DNA (mtDNA) sequence of Spirometra erinaceieuropaei from China was determined, characterized and compared with that of S. erinaceieuropaei from Japan. The gene arrangement in the mt genome sequences of S. erinaceieuropaei from China and Japan is identical. The identity of the mt genomes was 99.1% between S. erinaceieuropaei from China and Japan, and the complete mtDNA sequence of S. erinaceieuropaei from China is slightly shorter (2 bp) than that from Japan. Phylogenetic analysis of S. erinaceieuropaei with other representative cestodes using two different computational algorithms [Bayesian inference (BI) and maximum likelihood (ML)] based on concatenated amino acid sequences of 12 protein-coding genes, revealed that S. erinaceieuropaei is closely related to Diphyllobothrium spp., supporting classification based on morphological features. The present study determined the complete mtDNA sequences of S. erinaceieuropaei from China that provides novel genetic markers for studying the population genetics and molecular epidemiology of S. erinaceieuropaei in humans and animals.

Genetic variability within and among Haemonchus contortus isolates from goats and sheep in China

BackgroundHaemonchus contortus (order Strongylida) is a common parasitic nematode infecting small ruminants and causing significant economic losses worldwide. Knowledge of genetic variation within and among H. contortus populations can provide a foundation for understanding transmission patterns, the spread of drug resistance alleles and might assist in the control of haemonchosis.Methods152 H. contortus individual adult worms were collected from seven different geographical regions in China. The second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA and mitochondrial nicotinamide dehydrogenase subunit 4 gene (nad 4) were amplified by polymerase chain reaction (PCR) and sequenced directly. The sequence variations and population genetic diversities were determined.ResultsNucleotide sequence analyses revealed 18 genotypes (ITS-2) and 142 haplotypes (nad 4) among the 152 worms, with nucleotide diversities of 2.6% and 0.027, respectively, consistent with previous reports from other countries, including Australia, Brazil, Germany, Italy, Malaysia, Sweden, the USA and Yemen. Population genetic analyses revealed that 92.4% of nucleotide variation was partitioned within populations; there was no genetic differentiation but a high gene flow among Chinese populations; some degree of genetic differentiation was inferred between some specimens from China and those from other countries.ConclusionsThis is the first study of genetic variation within H. contortus in China. The results revealed high within-population variations, low genetic differentiation and high gene flow among different populations of H. contortus in China. The present results could have implications for studying the epidemiology and ecology of H. contortus in China.

Contracaecum rudolphii B: gene content, arrangement and composition of its complete mitochondrial genome compared with Anisakis simplex s.l.

In the present study, we sequenced the complete mt genome (14,022 bp) of parasitic nematode Contracaecum rudolphii B and its structure and organization compared with Anisakis simplex s.l. The mt genome of C. rudolphii B is slightly longer than that of A. simplex s.l. (13,916 bp). C. rudolphii B mt genome is circular, and consists of 36 genes, including 12 genes for proteins, 2 genes for rRNA and 22 genes for tRNA. This genome contains a high A+T (70.5%) content. The mt gene order for C. rudolphii B is the same as those for A. simplex s.l., but it is distinctly different from other nematodes compared. The start codons inferred in the mt genome of C. rudolphii B are TTG and ATT. Six protein-coding genes use TAA as a stop codon whereas five genes use T and one genes use TAG as a termination codon. This pattern of codon usage reflects the strong bias for A and T in the mt genome of C. rudolphii B. Phylogenetic analyses using concatenated amino acid sequences of the 12 protein-coding genes, with three different computational algorithms (Bayes, ML and MP), all revealed distinct groups with high statistical support, indicating that C. rudolphii B and A. simplex s.l. is distinct but closely related species. These data provide additional novel mtDNA markers for studying the molecular epidemiology and population genetics of the C. rudolphii B, and should have implications for the molecular diagnosis, prevention and control of anisakidosis in humans and animals.

Oesophagostomum dentatum and Oesophagostomum quadrispinulatum: Characterization of the complete mitochondrial genome sequences of the two pig nodule worms

In the present study, the complete mitochondrial DNA (mtDNA) sequences of the pig nodule worm Oesophagostomum quadrispinulatum were determined for the first time, and the mt genome of Oesophagostomum dentatum from China was also sequenced for comparative analysis of their gene contents and genome organizations. The mtDNA sequences of O. dentatum China isolate and O. quadrispinulatum were 13,752 and 13,681 bp in size, respectively. Each of the two mt genomes comprises 36 genes, including 12 protein-coding genes, two ribosomal RNA and 22 transfer RNA genes, but lacks the ATP synthetase subunit 8 gene. All genes are transcribed in the same direction and have a nucleotide composition high in A and T. The contents of A+T are 75.79% and 77.52% for the mt genomes of O. dentatum and O. quadrispinulatum, respectively. Phylogenetic analyses using concatenated amino acid sequences of the 12 protein-coding genes, with three different computational algorithms (maximum likelihood, maximum parsimony and Bayesian inference), all revealed that O. dentatum and O. quadrispinulatum represent distinct but closely-related species. These data provide novel and useful markers for studying the systematics, population genetics and molecular diagnosis of the two pig nodule worms.

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.