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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.

plos one

Large Scale Screening of Digeneans for Neorickettsia Endosymbionts Using Real-Time PCR Reveals New Neorickettsia Genotypes, Host Associations and Geographic Records