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Detection of Anaplasmataceae in ticks collected in Morocco

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

10.1111/j.1469-0691.2008.02251.x

1469-0691

Piseth Seng, M’hammed Sarih, C. Socolovschi, Najma Boudebouch, M. Hassar, Philippe Parola, Didier Raoult, Philippe Brouqui,

Mosquito-borne diseases and control

Bacteria within the Anaplasmataceae include Gram-negative intracellular bacteria that have been known for a long time as the agents of veterinary diseases. However, in the recent years, five members of Anaplasmataceae have been reported to infect humans, including Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis, and E. ewingii, E. canis and Neorickettsia sennetsu [1Brouqui P Matsumoto K Bacteriology and phylogeny of Anaplasmataceae.in: Raoult D Parola P Rickettsial diseases, Informa. Informa Health Care, New York2007: 179-198Crossref Google Scholar]. The abundance of ticks in North Africa represents potential risks for animal and human public health. However, agents within the Anaplasmataceae family have been poorly investigated in North Africa, including in Morocco. In this work, we analysed ticks collected in this country for evidence of infection by Anaplasmataceae. A total of 167 ticks were collected by blanket dragging of the vegetation and by direct removal from dog and cattle in different regions of Morocco, including Kenitra, Marrakech, Taza, Casablanca and Meknès. All the ticks were identified by one of us (MS) and stored in 70% ethanol until they were further processed. DNA was extracted and Anaplasmataceae were detected by rrS PCR using primers Ehr16SD and Ehr16SR, which amplified a 345 bp of DNA fragment [2Sanogo YO Davoust B Inokuma H Camicas JL Parola P Brouqui P First evidence of Anaplasma platys in Rhipicephalus sanguineus (Acari: Ixodida) collected from dogs in Africa.Onderstepoort J Vet Res. 2003; 70: 205-212PubMed Google Scholar] We used sterile water and DNA of uninfected tick as negative controls and DNA of A. phagocytophilum was used as positive control of rrS PCR of Anaplasmataceae. In order to identify the detected Anaplasmataceae, PCR products were amplified and purified, and then the DNA sequenced as previously described [2Sanogo YO Davoust B Inokuma H Camicas JL Parola P Brouqui P First evidence of Anaplasma platys in Rhipicephalus sanguineus (Acari: Ixodida) collected from dogs in Africa.Onderstepoort J Vet Res. 2003; 70: 205-212PubMed Google Scholar]. All obtained sequences were assembled by Chromas Prov.1.34 (Chromas Prov 1.34 Technelysium Pty Ltd., Atlanta, GA, USA) then compared in GenBank as previously described. The phylogenetic tree is produced by software MEGA 4 (The Biodesign Institute, Tempa, FL, USA) after aligning the sequences found with the sequences in GenBank by CLUSTAL W 1.83 (Welcome Trust, Cambridge, UK). Tick species were identified immediately after collection and revealed 125 of Rhipicephalus sanguineus, 25 of Rh. bursa, three of Rh. turanicus and 14 of Ixodes ricinus. After the sequencing, several Anaplasmataceae were identified in 19 ticks tested positive by rrS PCR for Anaplasmataceae allowing sequence analysis. The sequence identified in one I. ricinus specimen collected from vegetation in the Taza region was found to be closely related to the corresponding sequence of 'Candidatus Midichloria mitochondrii' (GenBank accession number AJ566640), with 99.1% (342/345) base pair similarity. A total of 16 ticks were found to harbour Wolbachia spp. The amplified sequences from these ticks showed 97–99% similarity with the corresponding sequence of W. pipientis (GenBank accession number DQ981321). These ticks include 12 Rh. sanguineus (five collected from dogs in Marrakech, five from Kenitra and two from Meknes region), three Rh. bursa (two from Kenitra and one from Meknes) and one Rh. turanicus collected in the Kenitra region. One Rh. sanguineus collected from dogs in the Casablanca region was found to be unambiguously infected with A. platys, (100% similarity with the corresponding sequence of A. platys GenBank accession number AF156784). Finally, the sequence identified in one Rh. bursa collected in Kenitra was 100% similar to those of A. centrale, A. ovis and A. marginale (GenBank accession number, EF520690, EF587237, DQ341370, respectively). For the first time in Morocco, we have identified by molecular tools several bacteria of the Anaplasmataceae family in ticks. A. platys is a parasite of canine platelets and dogs naturally infected with this agent do not necessarily show clinical signs. A. platys has been reported throughout the world, including in Europe, Asia, Australia, America and Africa. In 2003, it had been detected for the first time in Rh. sanguineus from Africa (Democratic Republic of Congo) by Sanogo et al. [2Sanogo YO Davoust B Inokuma H Camicas JL Parola P Brouqui P First evidence of Anaplasma platys in Rhipicephalus sanguineus (Acari: Ixodida) collected from dogs in Africa.Onderstepoort J Vet Res. 2003; 70: 205-212PubMed Google Scholar]. The second bacterium detected here was similar to A. centrale, A. marginale and A. ovis. The size of the fragment that was sequenced was not big enough to differentiate among these three species. All these three species are known as targeting the red blood cells of ruminants, A. marginale being the most pathogenic, A. centrale being less pathogenic in cattle and A. ovis being moderately pathogenic in small ruminants. Although anaplasmosis is more frequently associated with haemolytic anaemia in goats, A. ovis can also cause disease in sheep. In the Old World, A. ovis has been associated with R. bursa [3Friedhoff KT Tick-borne diseases of sheep and goats caused by Babesia, Theileria or Anaplasma spp.Parassitologia. 1997; 39: 99-109PubMed Google Scholar]. However, Fuente et al. have identified A. marginale in the salivary gland of R. bursa removed from Iberian red deer [4Kocan KM de la FJ Blouin EF Garcia-Garcia JC Anaplasma marginale (Rickettsiales: Anaplasmataceae): recent advances in defining host-pathogen adaptations of a tick-borne rickettsia.Parasitology. 2004; 129: S285-S300Crossref PubMed Scopus (206) Google Scholar]. 'Candidatus Midichloria mitochondrii' is an intramitochondrial bacterium that has been detected in Europe in I. ricinus ticks. Its pathogenicity for humans or animals is unknown [5Sassera D Beninati T Bandi C et al."Candidatus Midichloria mitochondrii", an endosymbiont of the tick Ixodes ricinus with a unique intramitochondrial lifestyle.Int J Syst Evol Microbiol. 2006; 56: 2535-2540Crossref PubMed Scopus (130) Google Scholar]. Finally, Wolbachia spp. are a group of bacteria associated with arthropods (insects, ticks, spiders, etc.) as well as filarial nematodes. Basically, a single species has been properly described – W. pipientis [1Brouqui P Matsumoto K Bacteriology and phylogeny of Anaplasmataceae.in: Raoult D Parola P Rickettsial diseases, Informa. Informa Health Care, New York2007: 179-198Crossref Google Scholar]. More work is needed for a precise identification of the Anaplasmataceae identified in this work. These preliminary results, however, increase our knowledge about the prevalence of Anaplasmataceae pathogens in Morocco and may help to implement measures to control transmission to humans and animals in this region.