Yongnian Zhang

Characterization of microRNAs in Taenia saginata of zoonotic significance by Solexa deep sequencing and bioinformatics analysis

The beef tapeworm Taenia saginata infects human beings with symptoms ranging from nausea, abdominal discomfort to digestive disturbances and intestinal blockage. In the present study, microRNA (miRNA) expressing profile in adult T. saginata was analyzed using Solexa deep sequencing and bioinformatics analysis. A total of 15.8 million reads was obtained by Solexa sequencing, and 13.3 million clean reads (1.73 million unique sequences) was obtained after removing reads smaller than 18 nt. Ten conserved miRNAs corresponding to 607,382 reads were found when matching the reads against known miRNAs of Schistosoma japonicum in miRBase database. The miR-71 had the most abundant expression in T. saginata, followed by miR-219-5p, but some other common miRNAs such as let-7, miR-40, and miR-103 were not identified in T. saginata. Nucleotide bias analysis found that the known miRNAs showed high bias and the uracil was the dominant nucleotide, particularly at the first and 11th positions which were almost at the beginning and middle of conserved miRNAs. One novel miRNA (Tsa-miR-001) corresponding to ten precursors was identified and confirmed by stem-loop RT-PCR. To our knowledge, this is the first report of miRNA profiles in T. saginata, which will contribute to better understanding of the complex biology of this zoonotic trematode. The reported data of T. saginata miRNAs should provide valuable references for miRNA studies of closed related zoonotic Taenia cestodes such as Taenia solium and Taenia asiatica.

Sensitive and rapid detection of Paragonimus westermani infection in humans and animals by loop-mediated isothermal amplification (LAMP)

In the present study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for the detection of Paragonimus westermani adults, metacercariae, and eggs in human and animal samples. The LAMP amplification can be finished in 45xa0min under isothermal condition at 60°C by employing a set of four species-specific primer mixtures and the results can be checked by naked-eye visualization. No amplification products were detected with deoxyribunucleic acid (DNA) of related trematode species including Fasciola hepatica, Fasciola gigantica, Clonorchis sinensis, Opisthorchis viverrini, Schistosoma mansoni, and Schistosoma japonicum. The method was further validated by examining P. westermani DNA in intermediate hosts including freshwater crabs and crayfish, as well as in sputum and pleural fluid samples from patients of paragonimiasis. These results indicated that the LAMP assay was highly specific, sensitive, and rapid, and it was approximately 100 times more sensitive than conventional specific PCR. The LAMP assay established in this study provides a rapid and sensitive tool for the detection of P. westermani DNA in freshwater crabs, crayfish, sputum, and pleural fluid samples, which has important implications for effective control of human paragonimiasis.

Identification and characterization of microRNAs in Trichinella spiralis by comparison with Brugia malayi and Caenorhabditis elegans.

Trichinella spiralis is an important zoonotic nematode causing trichinellosis which is associated with human diseases such as malaise, anorexia, nausea, vomiting, abdominal pain, fever, diarrhea, and constipation. microRNAs (miRNAs) are endogenous small non-coding RNAs that play important roles in the regulation of gene expression. The objective of the present study was to examine the miRNA expression profile of the larvae of T. spiralis by Solexa deep sequencing combined with stem-loop real-time polymerase chain reaction (PCR) analysis. T. spiralis larvae were collected from the skeletal muscle of naturally infected pigs in Henan province, China, by artificial digestion using pepsin. The specific identity of the T. spiralis larvae was confirmed by PCR amplification and subsequent sequence analysis of the internal transcribed spacer of ribosomal DNA. A total of 17,851,693 reads with 2,773,254 unique reads were obtained. Eleven conserved miRNAs from 115 unique xsmall RNAs (sRNAs) and 12 conserved miRNAs from 130 unique sRNAs were found by BLAST analysis against the known miRNAs of Caenorhabditis elegans (ftp://ftp.ncbi.nih.gov/genomes/Caenorhabditis_elegans) and Brugia malayi dataset (http://www.ncbi.nlm.nih.gov/genomeprj?Db=genomeprj&cmd=ShowDetailView&TermToSearch=9549) in miRBase, respectively. One novel miRNA with 12 precursors were identified and certified using the reference genome of B. malayi, while no novel miRNA was found when using the reference genome of C. elegans. Nucleotide bias analysis showed that the uracil was the prominent nucleotide, particularly at the 1st, 6th, 18th, and 23th positions, which were almost at the beginning, middle, and the end of the conserved miRNAs. The identification and characterization of T. spiralis miRNAs provides a new resource to study regulation of genes and their networks in T. spiralis.

Angiostrongylus cantonensis: identification and characterization of microRNAs in male and female adults.

Angiostrongylus cantonensis causes eosinophilic meningitis and eosinophilic pleocytosis in humans and is of significant socio-economic importance globally. microRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial roles in gene expression regulation, cellular function and defense, homeostasis and pathogenesis. They have been identified in a diverse range of organisms. The objective of this study was to determine and characterize miRNAs of female and male adults of A. cantonensis by Solexa deep sequencing. A total of 8,861,260 and 10,957,957 high quality reads with 20 and 23 conserved miRNAs were obtained in females and males, respectively. No new miRNA sequence was found. Nucleotide bias analysis showed that uracil was the prominent nucleotide, particularly at positions of 1, 10, 14, 17 and 22, approximately at the beginning, middle and the end of the conserved miRNAs. To our knowledge, this is the first report of miRNA profiles in A. cantonensis, which may represent a new platform for studying regulation of genes and their networks in A. cantonensis.

Multi-host Model-Based Identification of Armillifer agkistrodontis (Pentastomida), a New Zoonotic Parasite from China

Background Pentastomiasis is a rare parasitic infection of humans. Pentastomids are dioecious obligate parasites requiring multiple hosts to complete their lifecycle. Despite their worm-like appearance, they are commonly placed into a separate sub-class of the subphylum Crustacea, phylum Arthropoda. However, their systematic position is not uncontested and historically, they have been considered as a separate phylum. Methodology/Principal Findings An appraisal of Armillifer agkistrodontis was performed in terms of morphology and genetic identification after its lifecycle had been established in a multi-host model, i.e., mice and rats as intermediate hosts, and snakes (Agkistrodon acutus and Python molurus) as definitive hosts. Different stages of the parasite, including eggs, larvae and adults, were isolated and examined morphologically using light and electron microscopes. Phylogenetic and cluster analysis were also undertaken, focusing on the 18S rRNA and the Cox1 gene. The time for lifecycle completion was about 14 months, including 4 months for the development of eggs to infectious larvae in the intermediate host and 10 months for infectious larvae to mature in the final host. The main morphological difference between A. armillatus and Linguatula serrata is the number of abdominal annuli. Based on the 18S rRNA sequence, the shortest hereditary distance was found between A. agkistrodontis and Raillietiella spp. The highest degree of homology in the Cox 1 nucleic acid sequences and predicted amino acid sequences was found between A. agkistrodontis and A. armillatus. Conclusion This is the first time that a multi-host model of the entire lifecycle of A. agkistrodontis has been established. Morphologic and genetic analyses supported the notion that pentastomids should be placed into the phylum Arthropoda.

Molecular Detection of Diphyllobothrium nihonkaiense in Humans, China

The cause of diphyllobothriosis in 5 persons in Harbin and Shanghai, China, during 2008–2011, initially attributed to the tapeworm Diphyllobothrium latum, was confirmed as D. nihonkaiense by using molecular analysis of expelled proglottids. The use of morphologic characteristics alone to identify this organism was inadequate and led to misidentification of the species.

A Protein Microarray for the Rapid Screening of Patients Suspected of Infection with Various Food-Borne Helminthiases

Background Food-borne helminthiases (FBHs) have become increasingly important due to frequent occurrence and worldwide distribution. There is increasing demand for developing more sensitive, high-throughput techniques for the simultaneous detection of multiple parasitic diseases due to limitations in differential clinical diagnosis of FBHs with similar symptoms. These infections are difficult to diagnose correctly by conventional diagnostic approaches including serological approaches. Methodology/Principal Findings In this study, antigens obtained from 5 parasite species, namely Cysticercus cellulosae, Angiostrongylus cantonensis, Paragonimus westermani, Trichinella spiralis and Spirometra sp., were semi-purified after immunoblotting. Sera from 365 human cases of helminthiasis and 80 healthy individuals were assayed with semi-purified antigens by both a protein microarray and the enzyme-linked immunosorbent assay (ELISA). The sensitivity, specificity and simplicity of each test for the end-user were evaluated. The specificity of the tests ranged from 97.0% (95% confidence interval (CI): 95.3–98.7%) to 100.0% (95% CI: 100.0%) in the protein microarray and from 97.7% (95% CI: 96.2–99.2%) to 100.0% (95% CI: 100.0%) in ELISA. The sensitivity varied from 85.7% (95% CI: 75.1–96.3%) to 92.1% (95% CI: 83.5–100.0%) in the protein microarray, while the corresponding values for ELISA were 82.0% (95% CI: 71.4–92.6%) to 92.1% (95% CI: 83.5–100.0%). Furthermore, the Youden index spanned from 0.83 to 0.92 in the protein microarray and from 0.80 to 0.92 in ELISA. For each parasite, the Youden index from the protein microarray was often slightly higher than the one from ELISA even though the same antigen was used. Conclusions/Significance The protein microarray platform is a convenient, versatile, high-throughput method that can easily be adapted to massive FBH screening.

Four Human Cases of Diphyllobothrium nihonkaiense (Eucestoda: Diphyllobothriidae) in China with a Brief Review of Chinese Cases

We described 4 human infection cases of zoonotic fish-tapeworm, Diphyllobothrium nihonkaiense, identified with morphological and molecular characters and briefly reviewed Chinese cases in consideration of it as an emerging parasitic disease in China. The scolex and mature and gravid proglottids of some cases were seen, a rosette-shaped uterus was observed in the middle of the mature and gravid proglottids, and the diphyllobothriid eggs were yellowish-brown in color and displayed a small knob or abopercular protuberance on the opposite end of a lid-like opening. The average size of the eggs was recorded as 62–67×42–45 μm. The parasitic materials gathered from 4 human cases were morphologically identified as belonging to the genera Diphyllobothrium and Adenocephalus. The phylogenetic analysis based on the nucleotide sequences of cytochrome c oxidase subunit 1 gene of the etiologic agents confirmed that the 4 cases were D. nihonkaiense infection. The finding of 4 additional D. nihonkaiense cases suggests that D. nihonkaiense might be a major causative species of human diphyllobothriasis in China. A combined morphological and molecular analysis is the main method to confirm D. nihonkaiense infection.