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Large scale screening of digeneans for Neorickettsia endosymbionts using real-time PCR reveals new Neorickettsia genotypes, host associations and geographic records.

ABSTRACT: Digeneans are endoparasitic flatworms with complex life cycles including one or two intermediate hosts (first of which is always a mollusk) and a vertebrate definitive host. Digeneans may harbor intracellular endosymbiotic bacteria belonging to the genus Neorickettsia (order Rickettsiales, family Anaplasmataceae). Some Neorickettsia are able to invade cells of the digenean's vertebrate host and are known to cause diseases of wildlife and humans. In this study we report the results of screening 771 digenean samples for Neorickettsia collected from various vertebrates in terrestrial, freshwater, brackish, and marine habitats in the United States, China and Australia. Neorickettsia were detected using a newly designed real-time PCR protocol targeting a 152 bp fragment of the heat shock protein coding gene, GroEL, and verified with nested PCR and sequencing of a 1371 bp long region of 16S rRNA. Eight isolates of Neorickettsia have been obtained. Sequence comparison and phylogenetic analysis demonstrated that 7 of these isolates, provisionally named Neorickettsia sp. 1-7 (obtained from allocreadiid Crepidostomum affine, haploporids Saccocoelioides beauforti and Saccocoelioides lizae, faustulid Bacciger sprenti, deropegid Deropegus aspina, a lecithodendriid, and a pleurogenid) represent new genotypes and one (obtained from Metagonimoides oregonensis) was identical to a published sequence of Neorickettsia known as SF agent. All digenean species reported in this study represent new host records. Three of the 6 digenean families (Haploporidae, Pleurogenidae, and Faustulidae) are also reported for the first time as hosts of Neorickettsia. We have detected Neorickettsia in digeneans from China and Australia for the first time based on PCR and sequencing evidence. Our findings suggest that further surveys from broader geographic regions and wider selection of digenean taxa are likely to reveal new Neorickettsia lineages as well as new digenean host associations.

SUBMITTER: Greiman SE

PROVIDER: S-EPMC4049572 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Large scale screening of digeneans for Neorickettsia endosymbionts using real-time PCR reveals new Neorickettsia genotypes, host associations and geographic records.

Greiman Stephen E SE Tkach Vasyl V VV Pulis Eric E Fayton Thomas J TJ Curran Stephen S SS

PloS one 20140609 6

Digeneans are endoparasitic flatworms with complex life cycles including one or two intermediate hosts (first of which is always a mollusk) and a vertebrate definitive host. Digeneans may harbor intracellular endosymbiotic bacteria belonging to the genus Neorickettsia (order Rickettsiales, family Anaplasmataceae). Some Neorickettsia are able to invade cells of the digenean's vertebrate host and are known to cause diseases of wildlife and humans. In this study we report the results of screening 7 ...[more]

PMID: 24911315

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Project description:BackgroundThe porcine circovirus type 2 (PCV2) is divided into eight genotypes including the previously described genotypes PCV2a to PCV2f and the two new genotypes PCV2g and PCV2h. PCV2 genotyping has become an important task in molecular epidemiology and to advance research on the prophylaxis and pathogenesis of PCV2 associated diseases. Standard genotyping of PCV2 is based on the sequencing of the viral genome or at least of the open reading frame 2. Although, the circovirus genome is small, classical sequencing is time consuming, expensive, less sensitive and less compatible with mass testing compared with modern real-time PCR assays. Here we report about a new PCV2 genotyping method using qPCR.MethodsBased on the analysis of several hundred PCV2 full genome sequences, we identified PCV2 genotype specific sequences or single-nucleotide polymorphisms. We designed six TaqMan PCR assays that are specific for single genotypes PCV2a to PCV2f and two qPCRs targeting two genotypes simultaneously (PCV2g/PCV2d and PCV2h/PCV2c). To improve specific binding of oligonucleotide primers and TaqMan probes, we used locked nucleic acid technology. We evaluated amplification efficiency, diagnostic sensitivity and tested assay specificity for the respective genotypes.ResultsAll eight PCV2 genotype specific qPCRs demonstrated appropriate amplification efficiencies between 91 and 97%. Testing samples from an epidemiological field study demonstrated a diagnostic sensitivity of the respective genotype specific qPCR that was comparable to a highly sensitive pan-PCV2 qPCR system. Genotype specificity of most qPCRs was excellent. Limited unspecific signals were obtained when a high viral load of PCV2b was tested with qPCRs targeting PCV2d or PCV2g. The same was true for the PCV2a specific qPCR when high copy numbers of PCV2d were tested. The qPCR targeting PCV2h/PCV2c showed some minor cross-reaction with PCV2d, PCV2f and PCV2g.ConclusionGenotyping of PCV2 is important for routine diagnosis as well as for epidemiological studies. The introduced genotyping qPCR system is ideal for mass testing and should be a valuable complement to PCV2 sequencing, especially in the case of simultaneous infections with multiple PCV2 genotypes, subclinically infected animals or research studies that require large sample numbers.

Dataset Information

Large scale screening of digeneans for Neorickettsia endosymbionts using real-time PCR reveals new Neorickettsia genotypes, host associations and geographic records.

Publications

Large scale screening of digeneans for Neorickettsia endosymbionts using real-time PCR reveals new Neorickettsia genotypes, host associations and geographic records.

Similar Datasets

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