Ultrastructure and localization of Neorickettsia in adult digenean trematodes provides novel insights into helminth-endobacteria interaction

Background: Neorickettsia are a group of intracellular α proteobacteria transmitted by digeneans (Platyhelminthes, Trematoda). These endobacteria can also infect vertebrate hosts of the helminths and cause serious diseases in animals and humans. Neorickettsia have been isolated from infected animals and maintained in cell cultures, and their morphology in mammalian cells has been described. However, limited information is available on the morphology and localization of Neorickettsia in the trematode host. Methods: We used a Neorickettsia-infected strain of the model trematode Plagiorchis elegans to infect Syrian Golden hamsters to produce adult worms. Ultrastructure of Neorickettsia was assessed by transmission electron microscopy of high pressure freezing/freeze substitution fixed specimens. A Neorickettsia surface protein from P. elegans (PeNsp-3) was cloned and antibodies against the recombinant protein were used to localize Neorickettsia by immunohistochemistry. Results: Ultrastructural analysis revealed moderate numbers of pleomorphic endobacteria with a median size of 600 × 400 nm and characteristic double membranes in various tissue types. Endobacteria showed tubular membrane invaginations and secretion of polymorphic vesicles. Endobacteria were unevenly localized as single cells, or less frequently as small morula-like clusters in the ovary, Mehlis' gland, vitelline follicles, uterus, intrauterine eggs, testis, cirrus-sac, tegument, intestine and the oral and ventral sucker. Examination of hamster small intestine infected with P. elegans showed many endobacteria at the host-parasite interface such as the oral and ventral sucker, the tegument and the excretory pore. Conclusions: We conclude that adult P. elegans trematodes carry Neorickettsia endobacteria in varying numbers in many tissue types that support vertical transmission, trematode to trematode transmission via seminal fluid, and possibly horizontal transmission from trematodes to vertebrate hosts. These means appear to be novel mechanisms of pathogen transmission by endoparasitic worms.