artonella and Rickettsia in arthropods from the Lao PDR and from orneo, Malaysia

Rickettsioses and bartonelloses are arthropod-borne diseases of mammals with widespread geographical distributions. Yet their occurrence in specific regions, their association with different vectors and hosts and the infection rate of arthropod-vectors with these agents remain poorly studied in South-east Asia. We conducted entomological field surveys in the Lao PDR (Laos) and Borneo, Malaysia by surveying fleas, ticks, and lice from domestic dogs and collected additional samples from domestic cows and pigs in Laos. Rickettsia felis was detected by real-time PCR with similar overall flea infection rate in Laos (76.6%, 69/90) and Borneo (74.4%, 268/360). Both of the encountered flea vectors Ctenocephalides orientis and Ctenocephalides felis felis were infected with R. felis. The degrees of similarity of partial gltA and ompA genes with recognized species indicate the rickettsia detected in two Boophilus spp. ticks collected from a cow in Laos may be a new species. Isolation and further characterization will be necessary to specify it as a new species. Bartonella clarridgeiae was detected oonotic disease transmission potted fever in 3/90 (3.3%) and 2/360 (0.6%) of examined fleas from Laos and Borneo, respectively. Two fleas collected in Laos and one flea collected in Borneo were co-infected with both R. felis and B. clarridgeiae. Further investigations are needed in order to isolate these agents and to determine their epidemiology and aetiological role in unknown fever in patients from these areas. © 2011 Elsevier Ltd. Presented at the 6th International Meeting on Rickettsia and Rickttsial Diseases at Heraklion, Crete, Greece on June 5–7, 2011. ∗ Corresponding author at: Centre for Rickettsioses and other Arthropod Borne ax: +33 0491387772. E-mail address: philippe.parola@univmed.fr (D. Raoult). 147-9571© 2011 Elsevier Ltd. oi:10.1016/j.cimid.2011.10.003 Open access under CC BY license. Open access under CC BY license. Bacterial Diseases, Marseille, France. Tel.: +33 0491324375; icrobiolo 52 T. Kernif et al. / Comparative Immunology, M Since the beginning of the 20th century, ticks (Acarina), lice (Anoplura and Mallophaga) and fleas (Siphonaptera) have been implicated as vectors, reservoirs, and/or amplifiers of agents of human zoonoses, including rickettsioses and bartonelloses [14]. These diseases have been poorly investigated in South-east Asia, including the Lao PDR (Laos) [2,16] and Malaysian Borneo [10]. Among hospitalized patients in Vientiane, Laos, acute rickettsial infection was identified as the cause of fever in115 (27%) of 427 adults [16] and cause of jaundice or hepatitis in 29 (7.3%) from 392 patients admitted in Mahosot Hospital [27]. The organisms identified by serological analysis were Orientia tsutsugamushi, Rickettsia typhi, and spotted fever group Rickettsia (SFGR) (R. helvetica, R. felis, R. conorii subsp. indica, and Rickettsia “AT1”) [16,27]. In addition, Bartonella clarridgeiae and Rickettsia felis were detected in fleas collected in Phu Khao Khoay, near Vientiane [30]. A clinical case of murine typhus (R. typhi) was reported in a traveller from Brunei [9] and R. felis and R. typhi have been detected from flea species associated with small mammals in these area [3]. A recent study on flea-host associations, for example, compiled no more than 15 fleas species described to date to occur on small terrestrial mammals on Borneo Island, highlighting the lack of our understanding of the species diversity of possible vector and their role in transmitting diseases [32]. As part of our recent efforts to better understand the diversity, occurrence and distribution of ectoparasites of medical importance in South-East Asia, we collected ectoparasites from domestic dogs and some additional samples from domestic cows and pigs in Laos. We investigated the collected arthropods for Rickettsia and Bartonella species in order to examine which species of these disease-causing groups are present in northern Laos and Sabah, East Malaysia, on Borneo Island. 1. Materials and methods 1.1. Study sites and sampling In Luang Namtha Province (20◦55′N, 101◦07′E), northwest Laos, ectoparasites were sampled in eight villages, including Tavane, Nikhom, Viengthong, Jamai, Hoitu, Houkhou, Nongbauvieng and Nahom (Fig. 1A and B). On Borneo (6◦02′N, 116◦7′E), in the state of Sabah, Malaysia, arthopods were collected near the capital town Kota Kinabalu and in the districts of Keningau, Penampang, Tambunan, Tamparuli, Tuaran, and Ranau which are near the Crocker Range National Park and Kinabalu National Park (Fig. 1C). We particularly focused on these sites as this study was part of our initial efforts to investigate possible interactions of wildlife and domestic animals and the potential of zoonotic diseases transmission near protected forests. 1.2. Collections and morphological identification of ectoparasites In Luang Namtha Province, northwest Laos, fleas, ticks and lice were collected from domestic dogs, pigs and cows. In Borneo Island, fleas were only collected from domestic dogs. People encountered while travelling through the gy and Infectious Diseases 35 (2012) 51– 57 study area were asked for access to their dogs. We sampled only one dog per household of those consenting; usually, the sampled dog was chosen by the house owner based on availability and tameness. We brushed the dorsal fur of dogs from the neck to the tail for ten minutes with a flea comb (Trixie, Tarp, Germany, art. no. 23762). All ectoparasites were transferred with forceps to a tube containing 70% ethanol for later counting and identification and transported to France. All ectoparasites were morphologically identified to the species level by using morphological criteria within standard taxonomic keys [4,28]. 1.3. Molecular analysis DNAs of ectoparasites were extracted using the BioRobot MDx Workstation (Qiagen, Courtaboeuf, France) with a customized extraction protocol following the manufacturer’s instructions. For DNA extraction, a negative control (one non-infected tick from laboratory colony) was used for each 15 samples. DNA was stored at 4 ◦C until use for further analysis. Real-time quantitative (q)PCR was performed according to the manufacturer’s protocol using a 7900HT Fast RealTime PCR system (Applied Biosystems, Foster City, CA). Positive samples were considered when cycle thresholds were Ct ≤ 35. All samples were screened using SFGRspecific qPCR targeting gltA gene [25] and Bartonella genus-specific real-time PCR with a 21-bp probe targeting the intergenic spacer (ITS) [30]. Positive fleas for Rickettsia DNA were tested subsequently with primers and probe targeting a chromosomal gene specific of R. felis bioB, as described previously [30]. Positive ticks for Rickettsia DNA were tested by regular PCR targeting gltA and OmpA genes [23]. Bartonella positive samples were tested by regular PCR using primers amplifying ITS at fragment [15]. The Bartonella and Rickettsia DNA sequencing reagents were obtained with BigDye Terminator Cycle Sequencing Ready Reaction Kit (ABI PRISM, PE Applied Biosystems, Foster City, CA). The resulting sequences were edited and assembled using Chromas Pro 1.34 (Technelysium Pty. Ltd., Tewantin, Australia). The sequences were then analyzed by Basic Local Alignment Search Tool (BLAST) sequencing and further compared with other sequences available in the GenBank. R. montanensis, R. felis, and B. elizabethae DNA were included as positive control. Two negative controls were used for each test: sterile water and DNA extracted from non-infected ticks taken from a colony at the Unité des Rickettsies, Marseille. We used Fisher’s Exact Test for categorical data as implemented in the software package R [17].