Real-time PCR detection and phylogenetic relationships of Neorickettsia spp. in digeneans from Egypt, Philippines, Thailand, Vietnam and the United States

• Eight unique isolates of Neorickettsia discovered in SE Asia, Africa and North America.
• Seven isolates represent new species level genetic lineages.
• This is the first report of Neorickettsia from digeneans in Africa and three SE Asian countries.
• Phylogenetic relationships Neorickettsia has been studied 16S and GroESL genes.
• Host associations of digeneans harboring Neorickettsia are discussed.

Neorickettsia (Rickettsiales, Anaplasmataceae) is a genus of obligate intracellular bacterial endosymbionts of digeneans (Platyhelminthes, Digenea). Some Neorickettsia are able to invade cells of the digenean's vertebrate host and are known to cause diseases of domestic animals, wildlife, and humans. In this study we report the results of screening digenean samples for Neorickettsia collected from bats in Egypt and Mindoro Island, Philippines, snails and fishes from Thailand, and fishes from Vietnam and the USA. Neorickettsia were detected using a 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 1853 bp long region of the GroESL operon and a 1371 bp long region of 16S rRNA. Eight unique genotypes of Neorickettsia were obtained from digenean samples. Neorickettsia sp. 8 obtained from Lecithodendrium sp. from Egypt; Neorickettsia sp. 9 and 10 obtained from two species of Paralecithodendrium from Mindoro, Philippines; Neorickettsia sp. 11 from Lecithodendrium sp. and Neorickettsia sp. 4 (previously identified from Saccocoelioides lizae, from China) from Thailand; Neorickettsia sp. 12 from Dicrogaster sp. Florida, USA; Neorickettsia sp. 13 and SF agent from Vietnam. Sequence comparison and phylogenetic analysis demonstrated that the forms, provisionally named Neorickettsia sp. 8–13, represent new genotypes. We have for the first time detected Neorickettsia in a digenean from Egypt (and the African continent as a whole), the Philippines, Thailand and Vietnam based on PCR and sequencing evidence. Our findings suggest that further surveys from the African continent, SE Asia, and island countries are likely to reveal new Neorickettsia lineages as well as new digenean host associations.

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