Expansion of Cyclophyllidea Biodiversity in Rodents of Qinghai-Tibet Plateau and the "Out of Qinghai-Tibet Plateau" Hypothesis of Cyclophyllideans.

The Cyclophyllidea comprises the most species-rich order of tapeworms (Platyhelminthes, Cestoda) and includes species with some of the most severe health impact on wildlife, livestock, and humans. We collected seven Cyclophyllidea specimens from rodents in Qinghai-Tibet Plateau (QTP) and its surrounding mountain systems, of which four specimens in QTP were unsequenced, representing "putative new species." Their complete mitochondrial (mt) genomes were sequenced and annotated. Phylogenetic reconstruction of partial 28S rDNA, cox1 and nad1 datasets provided high bootstrap frequency support for the categorization of three "putative new species," assigning each, respectively, to the genera Mesocestoides, Paranoplocephala, and Mosgovoyia, and revealing that some species and families in these three datasets, which contain 291 species from nine families, may require taxonomic revision. The partial 18S rDNA phylogeny of 29 species from Taeniidae provided high bootstrap frequency support for the categorization of the "putative new species" in the genus Hydatigera. Combined with the current investigation, the other three known Taeniidae species found in this study were Taenia caixuepengi, T. crassiceps, and Versteria mustelae and may be widely distributed in western China. Estimates of divergence time based on cox1 + nad1 fragment and mt protein-coding genes (PCGs) showed that the differentiation rate of Cyclophyllidea species was strongly associated with the rate of change in the biogeographic scenarios, likely caused by the uplift of the QTP; i.e., species differentiation of Cyclophyllidea might be driven by host-parasite co-evolution caused by the uplift of QTP. We propose an "out of QTP" hypothesis for the radiation of these cyclophyllidean tapeworms.

Genetic Evolution and Implications of the Mitochondrial Genomes of Two Newly Identified Taenia spp. in Rodents From Qinghai-Tibet Plateau.

The larva of Taeniidae species can infect a wide range of mammals, causing major public health and food safety hazards worldwide. The Qinghai-Tibet Plateau (QTP), a biodiversity hotspot, is home to many species of rodents, which act as the critical intermediate hosts of many Taeniidae species. In this study, we identified two new larvae of Taenia spp., named T. caixuepengi and T. tianguangfui, collected from the plateau pika (Ochotona curzoniae) and the Qinghai vole (Neodon fuscus), respectively, in QTP, and their mitochondrial genomes were sequenced and annotated. Phylogenetic trees based on the mitochondrial genome showed that T. caixuepengi has the closest genetic relationship with T. pisiformis, while T. tianguangfui was contained in a monophyletic group with T. crassiceps, T. twitchelli, and T. martis. Biogeographic scenarios analysis based on split time speculated that the speciation of T. caixuepengi (∼5.49 Mya) is due to host switching caused by the evolution of its intermediate host. Although the reason for T. tianguangfui (∼13.11 Mya) speciation is not clear, the analysis suggests that it should be infective to a variety of other rodents following the evolutionary divergence time of its intermediate host and the range of intermediate hosts of its genetically close species. This study confirms the species diversity of Taeniidae in the QTP, and speculates that the uplift of the QTP has not only a profound impact on the biodiversity of plants and animals, but also that of parasites.

A comparison of loop-mediated isothermal amplification (LAMP) with other surveillance tools for Echinococcus granulosus diagnosis in canine definitive hosts.

BACKGROUND: Cystic echinococcosis is highly prevalent in northwest China. A cost-effective, easy to operate diagnostic tool with high sensitivity and specificity would greatly facilitate the monitoring of Echinococcus infections in canine definitive hosts. METHODS: The primers used in the LAMP assay were based on the mitochondrial nad5 gene of E. granulosus sensu stricto (E. granulosus s.s., or E.g.s.s.) and were designed using Primer Explorer V4 software. The developed LAMP assay was compared with a conventional PCR method, copro-ELISA and microscopy, using the faeces of dogs experimentally infected with E.g.s.s., and field-collected faeces of domestic dogs including 190 from Qinghai province highly endemic for E.g.s.s. and 30 controls from an area in Gansu, where a domestic dog de-worming program was in operation. RESULTS: The positivity rates obtained for the field-collected faecal samples were 12.6%, 1.6% and 2.1% by the LAMP, PCR and copro-ELISA assays, respectively. All samples obtained from the control dogs were negative. Compared with the conventional PCR, the LAMP assay provided 88.8% specificity and 100% sensitivity. The higher sensitivity of the LAMP method was also shown by the fact that it could detect the presence of laboratory challenge dog infections of E. granulsous s.s. four days earlier than the PCR method. Three copro-samples shown positive by the commercial copro-ELISA were all negative by LAMP, PCR and microscopy, which suggests these samples may have originated from another infection rather than E. granulsous s.s., possibly E. shiquicus or E. Canadensis, which is also present in China. CONCLUSIONS: We have developed a potentially useful surveillance tool for determining the prevalence of canine E. granulosus s.s. infections in the field. The LAMP assay may lead to a more cost-effective and practicable way of tracking Echinococcus infections in canids, especially when combined with the copro-ELISA.

Loop-mediated isothermal amplification (LAMP) assay for the identification of Echinococcus multilocularis infections in canine definitive hosts.

BACKGROUND: Alveolar echinococcosis, caused by the metacestode larval stage of Echinococcus multilocularis, is a zoonosis of public health significance and is highly prevalent in northwest China. To effectively monitor its transmission, we developed a new rapid and cheap diagnostic assay, based on loop-mediated isothermal amplification (LAMP), to identify canine definitive hosts infected with E. multilocularis. METHODS: The primers used in the LAMP assay were based on the mitochondrial nad5 gene of E. multilocularis and were designed using Primer Explorer V4 software. The developed LAMP assay was compared with a conventional PCR assay, using DNA extracted from the feces of dogs experimentally infected with E. multilocularis, on 189 dog fecal samples collected from three E. multilocularis-endemic regions in Qinghai province, the People's Republic of China, and 30 negative control copro-samples from dogs from an area in Gansu province that had been subjected to an intensive de-worming program. Light microscopy was also used to examine the experimentally obtained and field collected dog copro-samples for the presence of E. multilocularis eggs. RESULTS: The E. multilocularis-positivity rates obtained for the field-collected fecal samples were 16.4% and 5.3% by the LAMP and PCR assays, respectively, and all samples obtained from the control dogs were negative. The LAMP assay was able to detect E. multilocularis DNA in the feces of experimentally infected dogs at 12 days post-infection, whereas the PCR assay was positive on the 17th day and eggs were first detectable by light microscopy at day 44 post-challenge. CONCLUSION: The earlier specific detection of an E. multilocularis infection in dog copro-samples indicates that the LAMP assay we developed is a realistic alternative method for the field surveillance of canines in echinococcosis-endemic areas.

Differential diagnosis of Moniezia benedeni and M. expansa (Anoplocephalidae) by PCR using markers in small ribosomal DNA (18S rDNA).

Moniezia benedeni and M. expansa are common ruminant tapeworms of worldwide distribution, causing gastrointestinal disorders and even death in sheep and goats. In this study, a polymerase chain reaction- (PCR-) based approach for precise species identification was developed and also applied to faecal DNA diagnosis of the tapeworm infection. Since nuclear ribosomal DNA (rDNA) appears to be a useful target for species and/or strain markers, the 18S regions of the rDNA of M. benedeni and M. expansa were amplified and sequenced. The lengths and GC contents of the regions sequenced were 2476-2487 bp and 51.9-52.1% for M. benedeni and 2473 bp and 51.9-52.0% for M. expansa, respectively. Alignment and comparison of the 18S sequences of the two species showed 92.5-93.3% homology. No matches for the 18S regions of M. benedeni and M. expansa were found with other species by BLAST search, which made the 18S sequences appropriate markers for the design of distinctive primers for the two Moniezia species. Our 18S-based PCR could detect as low levels as 0.5 pg genomic DNA or the DNA extracted from 0.2 g faecal sample collected from the rectum of an M. expansa-infected goat. The results indicate that this PCR approach is a reliable alternative for the differential diagnosis of Moniezia species in faecal samples.

The nuclear 18S ribosomal RNA gene as a source of phylogenetic information in the genus Taenia.

Most species of the genus Taenia are of considerable medical and veterinary significance. In this study, complete nuclear 18S rRNA gene sequences were obtained from seven members of genus Taenia [Taenia multiceps, Taenia saginata, Taenia asiatica, Taenia solium, Taenia pisiformis, Taenia hydatigena, and Taenia taeniaeformis] and a phylogeny inferred using these sequences. Most of the variable sites fall within the variable regions, V1-V5. We show that sequences from the nuclear 18S ribosomal RNA gene have considerable promise as sources of phylogenetic information within the genus Taenia. Furthermore, given that almost all the variable sites lie within defined variable portions of that gene, it will be appropriate and economical to sequence only those regions for additional species of Taenia.

[The signaling systems in Echinococcus multilocularis].

Signaling pathway is the way by which cells receive various stimulation signals, and produce a series of corresponding responses, such as cell proliferation, differentiation and apoptosis. During infection with Echinococcus multilocularis, both parasite and host cells may secrete many cytokines such as insulin, which make stimulating signals transmitted into the cells through their receptors on the surface of cells. As a result, the parasite can grow and proliferate in the host. Study on related signaling pathways and their blocking drugs will play a crucial role in the control of alveolar echinococcosis caused by the larvae of E. multilocularis.

Mitochondrial genes and genomes support a cryptic species of tapeworm within Taenia taeniaeformis.

Taenia taeniaeformis is a globally distributed cestode, which uses felids as definitive and rodents as intermediate hosts. The complete mitochondrial DNA (mtDNA) of T. taeniaeformis from Germany (Tt-GER) was sequenced, and compared with that of another isolate from China (GenBank NC_014768; Tt-CHN), both taken from cats. Analysis of the two mtDNAs indicated that the isolates are significantly different from one another with 12.6% and 9.9% nucleotide and amino acid divergence between them, for concatenated protein-coding genes; overall difference based on a pairwise nucleotide alignment of complete mtDNAs was 11.8%. A phylogenetic analysis based on the 12 protein-coding genes of all available taeniid mtDNAs confirmed the two T. taeniaeformis isolates as sister taxa (likely separate species) and early divergent members of the genus, as suggested previously by morphology. Phylogenetic analysis of published fragments of mt genes rrnS, cox1 and nad1, which represent multiple geographic isolates of T. taeniaeformis also resolve two distinct clades that at present do not seem to be geographically isolated. Mean pairwise (nucleotide) differences between the two clades of T. taeniaeformis were approximately 11%, 10% and 13% in partial rrnS (182bp), cox1 (371bp) and nad1 (459bp) genes, respectively. Differences between entire mtDNAs and partial mt genes of the two T. taeniaeformis isolates are of a similar magnitude between established taeniid sister species. Tt-CHN differs from all other Taenia mtDNAs in lacking a short (∼69bp) non-coding region between trnY and trnL1. Partial mt fragment analysis highlighted likely misidentifications of T. taeniaeformis on GenBank.

[Research advances in interplay of host immune mechanism and Echinococcus multilocularis metacestodes].

Through affecting on host innate and acquired immune responses, Echinococcus multilocularis orchestrates various interplays that are beneficial not only to facilitate its intrahepatic proliferation and maturation during life cycle, but also to limit pathological process in its intermediate host. This review reveals the role of the metacestode's immune-related molecules in modulating host responses and optimizing its own survival.

Genetic variation of the 8-kDa glycoprotein family from Echinococcus granulosus, Taenia multiceps and Taenia hydatigena.

BACKGROUND: Echinococcosis, coenurosis and cysticercosis are debilitating diseases which prevail in China. Immunological diagnosis of metacestodosis is important in disease control. The 8-kDa glycoproteins from taeniid cestodes have successfully been used for diagnosis of human cysticercosis in immunological assays. The aim of the present study was to investigate genetic variations and phylogenetic relationships of the 8-kDa proteins for evaluating the possibility of utilizing these proteins as diagnostic antigens for other metacestode infections. METHODS: The genes and complementary DNAs (cDNAs) encoding the 8-kDa proteins from Echinococcus (E.) granulosus, Taenia (T.) multiceps and T. hydatigena were amplified using PCR method. Their amplicons were cloned into the vector pMD18 and the positive clones were sequenced. Sequence data were analyzed with the SeqMan program, and sequence homology searches were performed using the BLAST program. Alignments were conducted using the ClustalX program, and the phylogenetic analyses were performed with the Protein Sequences Program and the Puzzle Program using the Neighbor-joining method. RESULTS: Fifteen, 18 and 22 different genomic DNA sequences were identified as members of the 8-kDa protein gene family from E. granulosus, T. multiceps and T. hydatigena, respectively. Eight, four and six different cDNA clones respectively from E. granulosus, T. multiceps and T. hydatigena were characterized. Analysis of these sequences revealed 54 unique 8-kDa protein sequences. Phylogenetic trees demonstrated that the taeniid 8-kDa proteins are clustered into eight clades at least: Ts18, Ts14, TsRS1, TsRS2, T8kDa-1, T8kDa-2, T8kDa-3 and T8kDa-4. CONCLUSION: We found that the gene family encoding for the taeniid 8-kDa antigens is comprised of many members with high diversity, which will provide molecular evidence for cross-reaction or specific reaction among metacestode infections and may contribute to the development of promising immunological methods for diagnosis of metacestodosis.

[Application of in vitro cultivation technique for metacestodes in study of Echinococcus spp].

With the development of the in vitro cultivation of Echinococcus metacestodes, the technique is widely applied in research areas such as the pathogenic biological characteristics and the mechanism of infection and pathopoiesis of echinococcus, and development of novel therapeutic agents against echinococcosis. These will help futher understand the disease and its control. This paper reviews the application of the in vitro cultivation technique of Echinococcus spp.