Detection of new Anaplasmataceae in the digestive tract of fish from southeast Asia

Abstract

The bacteria of the genus Neorickettsia are strict intracellular bacteria of the Anaplasmataceae family. They are transmitted to their definitive host (humans and animals) by ingestion of raw fish, snails or aquatic insects that are parasitised by infected trematodes. Among them, three species (N. sennetsu, N. helminthoeca and N. risticii) are known to cause diseases in mammals (humans and animals) [1] but two have not yet been associated with the disease (SF agent, and Ehrlichia species trout isolate). About 50 million people in the world are infested by food-borne trematodes, predominantly in southeast Asia. Uncooked fish and shellfish consumption is the principal mode of transmission. Fish-borne trematodes in humans are increasing in endemic areas, and are emerging in Europe and America [2]. Globalisation, international travels and new cooking fashions such as eating raw fish ‘Sushi’ and ‘Sashimi’ are likely to lead to new emerging infections caused by Neorickettsia. We studied here the presence of Neorickettsia in fish and fish-based ingredients that are usually consumed uncooked in southeast Asia. A total of 126 fish and 19 ingredients made from uncooked fish were studied. Between January and February 2007, 57 frozen fish and seven ingredients imported from Thailand and Vietnam were bought in the Asian market in Marseille. In March 2007, 69 other fish preserved in 70% ethanol and 12 supplementary ingredients were collected in Cambodia. After dissection of defrosted fish, stomach and intestine were collected for direct examination for parasite by binocular microscope, and then processed for PCR analysis, culture and histology. Found worms were kept in the 70% ethanol for morphological and molecular identification of the 18S gene by using primers P6.F and 18sr2R [3]. Following DNA extraction of the fish digestive tract, DNA of Anaplasmataceae was detected by rrS PCR using primers: Ehr16SD and Ehr16SR [1]. We completed rrS sequences found by using specific primers (Ncs-614F: 5¢-TTG-GTG-TAG-GGG-TGAAAT-CC, Ncs-606R: 5¢-CCC-CTA-CAC-CAA-AAATTC-CA) and non-specific primers (NcsMA-14F: 5¢-GGC-AGA-CGG-GTG-CGT-AAC, Ncs-1321R: 5¢-GAC-GGG-CAG-TGT-GTA-CAA-GA) newly designed respectively by alignment of rrS sequences of three new Anaplasmataceae with Neorickettsia sequences available in GenBank and by alignment of rrS sequence of all Anaplasmataceae available in GenBank. DNA fragment of the citrate synthase gene (gltA) of Anaplasmataceae was identified by using F1 and R1b as previously described [1]. Negative controls consisted of DNA extracted from human colon, uninfected fish and sterile water. DNA of A. phagocytophilum was used as positive control of rrS PCR and N. sennetsu for gltA PCR. We designed new primers CG-442F (5¢-GGC-CCATAC-CCC-ARN-AAT-G) and CG-877R (5¢-GCDAGT-TTT-TGT-CAR-GTN-GA) to confirm the species of positive fishes. The sequence diversity of the rrS gene was used to define a new species when it was at least 3% between bacterial species. The new genus was definite when rrs gene sequence similarities were lower than 97% [4]. We identified 35 worms in six Channa striata imported into Marseille from Thailand. Sequence analysis of the 18S gene showed that all worms were belonging to Neoechinorhynchus pseudemydis species. Three new Anaplasmataceae were detected in three fish by rrS PCR by using Ehr16SD and Ehr16SR primers (Table 1). After completion of the rrS sequences, the Anaplasmataceae from the stomach of Channa striata fish imported from Thailand was identified as a new genus with a Corresponding author and reprint requests: P. Brouqui, URMITE CNRS ⁄ IRD UMR 6236, IFR 48, Universite de la Mediterranee, Faculte de Medecine, 27 Boulevard Jean Moulin, 13385 Marseille cedex 5, France E-mail: philippe.brouqui@univmed.fr