Multiple host-switching of Haemosporidia parasites in bats

Screened material was obtained in Madagascar and Cambodia. In Madagascar, specimens were obtained at eight different field sites in the provinces of Mahajanga, Toliara and Antananarivo. The fieldwork was conducted between November 2001 and late 2004, in collaboration with the Institut Pasteur de Madagascar and WWF-Madagascar. In Cambodia, two field sites were sampled in the provinces of Kampot and Mondolkiri between December 2004 and May 2006, in collaboration with the Institut Pasteur du Cambodia and Wildlife Conservation Society. The field research was conducted with local and national permits from the Direction des Eaux et Forêts of Madagascar and the Ministry of Agriculture, Forestry and Fisheries in Cambodia. A total of 530 bats, belonging to seven families (Pteropodidae, Rhinolophidae, Hipposideridae, Megadermatidae, Emballonuridae, Vespertilionidae, and Molossidae) were trapped in the wild. A small sample of blood was obtained and the host was released without injury or in some cases retained as a voucher specimen.

Thin blood smears were made for each animal. They were fixed in methanol and stained by incubation with 10% Giemsa for 10 minutes. The smears were examined by microscopy for haemosporidian parasites. Parasites isolated from bats were referred to as Haemosporidia sp. Positive blood smears were catalogued in the Département 'Régulations, Développement et Diversité Moléculaire', Muséum National d'Histoire Naturelle, Paris, France.

DNA was extracted from blood samples using the phenol/chloroform technique [12]. The 709 bp cytochrome b fragments were amplified using PCR and nested-PCR. The PCR reaction was carried out in a total volume of 25 μl under the following condition: 1 μl of each of the primers: PLAS 1 (5'-GAGAATTATGGAGTGGATGGTG-3') and PLAS 2 (5'-GTGGTAATTGACATCCWATCC-3'), 1 mM of each dNTP, 1 U of Taq polymerase (Solis), 3 mM MgCl₂. The PCR conditions were: 5 min at 94°C, 30 sec at 94°C, 30 sec at 55°C and 1 min 30 sec at 72°C for 40 cycles and a final 10 min extension at 72°C. The nested-PCR was carried out using 1 μl of the PCR products and performed with the following primers: PLAS 3 (5'-GGTGTTTYAGATAYATGCAYGC-3') and PLAS 4 (5'-CATCCWATCCATARTAWAGCATAG-3'). The conditions were: 5 min at 94°C, 30 sec at 94°C, 30 sec at 55°C, 1 min 30 sec at 72°C for 40 cycles and a final 10 min extension at 72°C. The PCR products were sequenced using PLAS3 and PLAS4 primers by Macrogen (Korea). All of these primers were designed by the research group. They are specific to haemosporidian parasites and do not amplify others Apicomplexa parasites or host DNA. Relevant sequences were selected for the phylogenetic analysis. There are numerous avian parasite sequences available and some representative sequences were chosen based on geographical localizations.

The nucleotide sequences (709 bp) were translated into amino acid sequences to minimize homoplasy due to saturation of synonymous mutations since some taxa have diverged over hundreds of millions years. The sequences were aligned using CLUSTAL W [13]. Reference sequences of at least 219 amino acids without ambiguous positions were retrieved from GenBank. In the case of identical sequences in amino acids, only one sequence for the analysis was kept. Maximum Likelihood (ML) was performed using Phyml [14] and Parsimony analysis (P) was performed using Phylip package (version 3.62) [15]. Nodal robustness was evaluated by non-parametric bootstrap (100 replicates). Bayesian analyses were conducted with MrBayes V3.1 software [16] using gamma distribution, 1 000 000 generations (average standard deviation of split sequences is below 0.01), with tree sampling every 100 generations and a burn-in of 2500 trees. The aim of the study was to analyse Haemosporidia phylogeny, so Theileria annulata, Babesia gibsoni and Toxoplasma gondii which belong to the phylum Apicomplexa but are non-haemosporidian parasites were choose as out-groups. The phylogenetic study was run using cytochrome b sequences. This is the most abundant marker in GenBank for a variety of haemosporidian parasites and widely used in phylogenetic studies because reference sequences from other genes are lacking.