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

2009; Elsevier BV; Volume: 15; Linguagem: Inglês

10.1111/j.1469-0691.2008.02252.x

1469-0691

Piseth Seng, Jean‐Marc Rolain, Didier Raoult, P. Brouqui,

Toxoplasma gondii Research Studies

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) [1Brouqui P Matsumoto K Bacteriology and phylogeny of Anaplasmataceae.in: Raoult D Parola P Rickettsial diseases. Informa Healthcare, New York2007: 179-212Crossref Google Scholar] 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 [2Abdussalam M Kaeferstein FK Mott KE Food safety measures for the control of foodborne trematode infections.Food Control. 1995; 6: 71-79Crossref Scopus (29) Google Scholar]. 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 [3Ando K Tsunemori M Akahane H Tesana S Hasegawa H Chinzei Y Comparative study on DNA sequences of ribosomal DNA and cytochrome c oxidase subunit 1 of mitochondrial DNA among five species of gnathostomes.J Helminthol. 2006; 80: 7-13Crossref PubMed Scopus (24) Google Scholar]. Following DNA extraction of the fish digestive tract, DNA of Anaplasmataceae was detected by rrS PCR using primers: Ehr16SD and Ehr16SR [1Brouqui P Matsumoto K Bacteriology and phylogeny of Anaplasmataceae.in: Raoult D Parola P Rickettsial diseases. Informa Healthcare, New York2007: 179-212Crossref Google Scholar]. We completed rrS sequences found by using specific primers (Ncs-614F: 5′-TTG-GTG-TAG-GGG-TGA-AAT-CC, Ncs-606R: 5′-CCC-CTA-CAC-CAA-AAA-TTC-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 [1Brouqui P Matsumoto K Bacteriology and phylogeny of Anaplasmataceae.in: Raoult D Parola P Rickettsial diseases. Informa Healthcare, New York2007: 179-212Crossref Google Scholar]. 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-CCA-TAC-CCC-ARN-AAT-G) and CG-877R (5′-GCD-AGT-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% [4Stackebrandt E Frederiksen W Garrity GM et al.Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology.Int J Syst Evol Microbiol. 2002; 52: 1043-1047Crossref PubMed Scopus (1280) Google Scholar]. 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 1269 bp sequence homology of 93% with Ehrlichia sp. trout isolate, N. sennetsu and N. risticii. The Anaplasmataceae from the stomach of Mastacembelus armatus fish imported from Vietnam was also identified as a new genus with 1207 bp sequence homology of 95% with N. sennetsu and 94% with N. risticii. This taxonomic position was confirmed by 232 bp gltA gene sequence homology of 67% with N. helminthoeca (Fig. 1). Finally a new species of Neorickettsia was identified with a 1247 bp rrs sequence homology of 97% with Ehrlichia sp. trout isolate and 96% with N. risticii in the intestine of Xenentodon cancila fish collected in Cambodia. No worm was found in the fish PCR-positive for Anaplasmataceae. These four gene sequences identified have been deposited in GenBank under the accession numbers EU780453, EU780452, EU780454 and EU780451.TABLE 1Three new Anaplasmataceae from three fish and no Anaplasmataceae found in 35 worms of negative Channa striata fishNews AnaplasmataceaeSequence size (bp)GeneSimilarity of sequenceTrematodeNeorickettsia sp. Xenentodon cancila isolate1256rrS97% with Ehrlichia sp. trout isolate and 93% with Neorickettsia risticiiNone93% with Neorickettsia risticiiAnaplasmataceae from Channa striata fish126993% with Ehrlichia sp. trout isolate, Neorickettsia risticii and Neorickettsia sennetsuNoneAnaplasmataceae from Mastacembelus armatus fish125695% with Neorickettsia risticii and 94% with Neorickettsia sennetsuNone232gltA67% with Neorickettsia helminthoeca Open table in a new tab All 35 worms found in the Anaplasmataceae rrS PCR-negative fishes were themselves negative for Anaplasmataceae. The main objective of this study was to evaluate the presence of Anaplasmataceae in fish usually consumed raw in southeast Asia. We identified three new Anaplasmataceae in three fishes: Channa striata, Mastacembelus armatus and Xenentodon cancila fish. Based upon a taxonomic note to define a new bacterial species by using the rrS sequence analysis, a new species of Neorickettsia called Neorickettsia sp. Xenentodon cancila isolate and two new genus of Anaplasmataceae from Channa striata and Mastacembelus armatus fish were defined [4Stackebrandt E Frederiksen W Garrity GM et al.Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology.Int J Syst Evol Microbiol. 2002; 52: 1043-1047Crossref PubMed Scopus (1280) Google Scholar]. Bacteria of the Neorickettsia genus were known to be transmitted by digenetic trematodes, such as: Nanophyetus salmincola for N. helminthoeca; trematode of freshwater snails, aquatic insects and bats for N. risticii; and Stellantchasmus falcatus for SF agent [1Brouqui P Matsumoto K Bacteriology and phylogeny of Anaplasmataceae.in: Raoult D Parola P Rickettsial diseases. Informa Healthcare, New York2007: 179-212Crossref Google Scholar]. Recently, Pusterla et al. reported a new Neorickettsia species detected by rrS PCR in major organs of rainbow trout in northern California. Digenetic trematodes, the majority of which were found in gallbladder and intestine, were also positive [5Pusterla N Johnson E Chae J et al.Molecular detection of an Ehrlichia-like agent in rainbow trout (Oncorhynchus mykiss) from Northern California.Vet Parasitol. 2000; 92: 199-207Crossref PubMed Scopus (12) Google Scholar]. In our study, no trematode was found in the two positive fish, Channa striata and Mastacembelus armatus. The diversity of new Anaplasmataceae found in this study lead us to hypothesise that fish are likely to be parasitised by a wide diversity of Anaplasmataceae and that new cooking fashions developing all over the world might be the origin of a new emerging human disease.