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.

Complete mitochondrial genomes of Taenia multiceps, T. hydatigena and T. pisiformis: additional molecular markers for a tapeworm genus of human and animal health significance.

BACKGROUND: Mitochondrial genomes provide a rich source of molecular variation of proven and widespread utility in molecular ecology, population genetics and evolutionary biology. The tapeworm genus Taenia includes a diversity of tapeworm parasites of significant human and veterinary importance. Here we add complete sequences of the mt genomes of T. multiceps, T. hydatigena and T. pisiformis, to a data set of 4 published mtDNAs in the same genus. Seven complete mt genomes of Taenia species are used to compare and contrast variation within and between genomes in the genus, to estimate a phylogeny for the genus, and to develop novel molecular markers as part of an extended mitochondrial toolkit. RESULTS: The complete circular mtDNAs of T. multiceps, T. hydatigena and T. pisiformis were 13,693, 13,492 and 13,387 bp in size respectively, comprising the usual complement of flatworm genes. Start and stop codons of protein coding genes included those found commonly amongst other platyhelminth mt genomes, but the much rarer initiation codon GTT was inferred for the gene atp6 in T. pisiformis. Phylogenetic analysis of mtDNAs offered novel estimates of the interrelationships of Taenia. Sliding window analyses showed nad6, nad5, atp6, nad3 and nad2 are amongst the most variable of genes per unit length, with the highest peaks in nucleotide diversity found in nad5. New primer pairs capable of amplifying fragments of variable DNA in nad1, rrnS and nad5 genes were designed in silico and tested as possible alternatives to existing mitochondrial markers for Taenia. CONCLUSIONS: With the availability of complete mtDNAs of 7 Taenia species, we have shown that analysis of amino acids provides a robust estimate of phylogeny for the genus that differs markedly from morphological estimates or those using partial genes; with implications for understanding the evolutionary radiation of important Taenia. Full alignment of the nucleotides of Taenia mtDNAs and sliding window analysis suggests numerous alternative gene regions are likely to capture greater nucleotide variation than those currently pursued as molecular markers. New PCR primers developed from a comparative mitogenomic analysis of Taenia species, extend the use of mitochondrial markers for molecular ecology, population genetics and diagnostics.

The first outbreak of Taenia ovis infection in China.

Infection of Taenia ovis metacestodes in sheep or goats causes great economic losses due to condemnation of carcasses. T. ovis infection is not formally recorded in China to date. In October, 2015, T. ovis infection occurred in Jingtai County, China, and 113 of 192 sheep from one farm were infected. Cysts resided in the cardiac and skeletal muscle, and evaginated metacestodes had four suckers and scolex armed with approximately 23 hooks. Using cox1 and nad1 as molecular markers, the sample was further identified and the results showed that the cox1 and nad1 nucleotide sequences of the sample shared 99% identity with that of T. ovis and 75%-91.3% with those of other Taenia species. Taken together, these results confirm the first occurrence of T. ovis in China.

Genetic diversity in Echinococcus shiquicus from the plateau pika (Ochotona curzoniae) in Darlag County, Qinghai, China.

The metacestode of Echinococcus shiquicus has been recorded previously in the lung and liver of its intermediate host, the plateau pika (Ochotona curzoniae), but there is limited information regarding other organ sites. There is also limited evidence of intra-specific genetic variation within E. shiquicus. A PCR-amplified mitochondrial (mt) nad1 gene fragment (approximately 1400bp in size), with unique EcoRI and SspI restriction sites, was used to distinguish cysts or cyst-like lesions of E. shiquicus from E. multilocularis. Then, the complete mt nad1 and cox1 genes for the E. shiquicus isolates were amplified and sequenced. Phylogenetic tree and haplotype network analyses for the isolates were then generated based on a concatenated dataset of the nad1 and cox1 genes using the neighbour-joining (NJ) method and TCS1.21 software. Nineteen of eighty trapped pikas were found to harbor cysts (71 in total) when dissected at the survey site. Seventeen animals had cysts (fertile) present only in the lungs, one animal had fertile cysts in the lungs and spleen, and one individual had an infertile kidney cyst. Restriction endonuclease analysis of a fragment of the nad1 gene indicated all the cysts were due to E. shiquicus. Genetic diversity analysis revealed that the nad1 and cox1 genes varied by 0.1-1.2% and 0.1-1.0%, respectively. Haplotype network analysis of the concatenated nad1 and cox1 sequences of the isolates showed they were classified into at least 6 haplotypes, and different haplotype percentages ranged from 4.2% to 29.6%. Although, high haplotype diversity was evident in the study area, the complete nad1 and cox1 gene sequences obtained indicated that all samples represented isolates of E. shiquicus. The study has also provided a new PCR-restriction endonuclease-based method to rapidly distinguish E. shiquicus from E. multilocularis which provides a useful tool for epidemiological investigations where the two species overlap.

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.

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.