Tick Vector Mapping and Pathogen Characterization Study in Laos 2 (Tick Map 2 Project)

Tick vector mapping and pathogen characterization study in Laos 2 (Tick Map 2 project) Funded by the U.S. Naval Medical Research Center-Asia (NMRC-A) in support of the Department of Defense Global Emerging Infections Surveillance and Response System (DoD-GEIS) Project coordinator: + Dr. Paul Brey, Director, Institut Pasteur du Laos, Vientiane, Lao PDR + Dr. Ian Sutherland, Chief of Entomological Sciences, U.S. Naval Medical + Dr. Jeffrey Hertz, Chief of Entomological Sciences, U.S. Naval Medical Research Center – Asia, Sembawang, Singapore Member of staff: + Khamsing Vongphayloth, Research entomologist, Institut Pasteur du Laos + Khaithong Lakeomany, Technician entomologist, Institut Pasteur du Laos + Nothasine Phommavanh, Technician entomologist, Institut Pasteur du Lao PDR Collaborators: + Marc Eloit , Virologist, Pathogen Discovery Unit, Institut Pasteur Paris + Paul Newton and Matthew Robinson, LOWMRU Wellcome Trust Vientiane Lao PDR + Marc Grandadam, Arbovirology Lab, Institut Pasteur du Laos Background Vector-borne diseases constitute a significant infectious disease risk for for local populations. In Laos, definitive diagnosis is often not available for vector-borne illnesses, so the infectious diseases, which are a threat to rural and urban populations are not well defined. In order to identify common and emerging vector-borne pathogens in Laos, NMRC-A Singapore (SG) has established a study to assess the distribution and infection potential of ticks and tick- borne pathogens. In this study, tick vectors are surveyed to provide biological specimens for pathogen screening. The samples are transported to the Institut du Pasteur (IPL) laboratory in Vientiane, where a wide range of tests can be performed to identify both the arthropod species and the pathogens with which they may be infected. In order to understand the infectious disease threats in a range of environments in Laos, IPL must collect and screen specimens from several sites and provinces throughout Laos. Further characterization and targeted analyses of positive pathogen samples we have already collected from our first tick study has also been carried out. This study is known as the “Tick vector mapping and pathogen characterization study in Laos” (TickMap 2). Nakai District, Khammouan Province, is uniquely positioned to serve as a study site for TickMap 2 and includes a large protected area bordering Vietnam in south-central Laos, a region in which many causes of vector-borne illness are not diagnosed, and from which new infectious diseases may emerge. All local staff is already familiar with the requirements of study specimen identification, sample collection, and the principles of vector biology research. Based on the data and unprecedented success of our prior and ongoing initial phase project lines, including the recording of at least four new species of tick (Vongphayloth et al. 2016), the potential discovery of three new species of rickettsia (Taylor et al. 2016), and a new tick–virus interaction that has never previously been described to science, we are initiating the TickMap 2 project to complement and augment our prior and ongoing tick research efforts in Laos. In this report, we update all our data on field collection missions, arboviral–bacterial screening, and molecular identification of ticks from the study. Objectives + Survey and modern ID of indigenous tick species distribution. + Collection, morphologic identification, extraction of vector DNA for submission, and development of regional repository. This will provide a valuable resource for downstream modern molecular analysis and genotyping. + Building of local capacities and competencies. Methods Field site collection Our first field collection of ticks was carried out between the 12th and 19th January, 2017 in the Nakai Nam Theun National Protected Area (NNT NPA), known as the Watershed Management and Protection Authority area (WMPA), located in Nakai District, Khammouan Province, Laos. Ticks were sampled in two main sites: the Korbong village area (location: 17.879643°N, 105.389993°E), and the Namnoy River area (location: 17.768548°N, 105.381989°E). In the Korbong village area, ticks were collected in secondary forest close to the rice fields of the villagers whereas in the Namnoy River area, ticks were collected inside the primary forest area. The second field collection of ticks was carried out between the 20th and 25th of February 2017 in the Namnoy River area only. Tick sampling procedure A dragging method was used for tick sampling from forest floors and vegetation. These standard dragnets were swept along the forest ground at approximately 1–10 m intervals around natural animal trail ways before being examined for ticks. Ticks were removed from the sheets using forceps and transferred to 1.5 ml labeled cryotubes (using PACS labeling and specimen tracking system Black and Vetch). All live tick samples were then transported to the Nakai field laboratory. In the lab, ticks were placed in the freezer at −20°C for 10 minutes. Then adults, nymphs, and tick larvae were separated, counted, and subsequently transported to IP- Laos in Vientiane Capital and stored at −80°C until processing for further analysis (species identification and pathogen detection). All study sites were data-logged for full GPS parameters. Tick identification Ticks were identified and grouped under microscopes in cooling conditions (on ice packs) by using reference determination from Dr. Richard G. Robbins of the US Armed Forces Pest Management Board (AFPMB), together with related references from Southeast Asia, Japan, Korea, the Ryukyu Islands (Yamaguti, Tipton et al. 1972), L. E. Robinson keys for genus Amblyomma (Nuttall, Cooper et al.), and keys from Thailand (Tanskull and Inlao 1989) for adult Haemaphysalis ticks. As there are no morphological identification keys available for pre-imago forms of Haemaphysalis spp., larval and nymph stages in this genus were grouped by using their main morphological characteristics, especially the capitulum. After tick identification and pooling, all information was registered with the Pathogen Asset Control System (PACS) software and all tick samples were stored at −80°C in IP-Laos, Vientiane Capital, for further analysis. Laboratory procedure for pathogen screening and molecular tick identification Sample preparation and RNA/DNA extraction Tick samples were pooled into groups of one to ten by species or genus, sex, stage of development, collection period, and site. Specimens were placed in a 1.5 ml vial containing 1 ml of 1X cold Phosphate Buffered Saline (PBS) and Lysing Matrix A zirconium beads (MP Biomedicals). Tick pools were homogenized for 10 min at a vibration frequency of 25/s in a TissueLyser II system (Qiagen). After grinding, beads and tissues were spun down by centrifugation for 5 min at 3000 rpm. To obtain total nucleic acid (both DNA and RNA) for bacterial and viral detection by polymerase chain reaction (PCR), 100 μl of each pool was extracted and purified by using NucleoSpin® 8 Virus extraction kit following manufacturer’s protocol. The remaining 400 μl of each pool was stored at –80°C for pathogen isolation. Arboviral viral screening Multiple sets of primers have been selected and tested for screening arthropod specimens for the presence of phlebovirus, flavivirus, and alphavirus sequences by means of conventional nested RT-PCR as previously described (Sanchez-Seco, Rosario et al. 2001; Sanchez-Seco, Rosario et al. 2005). Next generation sequencing on tick samples RT-PCR screening targeting three main arbovirus genera evidenced the presence of genome fragments in some tick samples. Until now, direct sequencing strategies have not allowed fort preliminary identification of the sequence detected as a probable consequence of the use of degenerated primers for both RT-PCR and direct amplicon sequencing. A collaboration with Dr. Marc Eloit (Pathogen discovery and NGS platform at the Institut Pasteur in Paris) has been initiated to solve the sequencing priming problem and increase the chances of identifying new arboviruses by accessing larger or complete genome sequences. This platform was selected as the staff had good experience in the preparation and virome analysis of ticks. Bacterial screening To investigate the occurrence of Rickettsia spp. in ticks in Laos, a molecular screening approach targeting the 17kDa gene was taken (Jiang et al. 2004). The presence ofLeptospira spp., Anaplasma phagocytophilum, Ehrlichia chaffeensis, Coxiella burnetii and other Ehrlichia spp., Neorickettsia spp., and Anaplasma spp. was also investigated. The bacterial screening was carried out in collaboration with the Lao-Oxford University-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), based at Mahosot Hospital, Vientiane. Statistical analysis Infection rate (IR) was calculated using Excel add-in software for mosquito surveillance developed by the CDC. Estimates of the IR are usually presented as the number of infected mosquitoes per 1,000 tested. The simplest estimate, the minimum infection rate (MIR), is calculated: ([number of positive pools / total specimens tested] x 1000), with the data representing a single species or species group collected over a time period and geographic area relevant to the goals of the surveillance program (https://www.cdc.gov/westnile/resourcepages/mosqSurvSoft.html) . Molecular tick identification using the COI – barcode region of the mtDNA For accurate molecular identification of ticks, we tested the primers LCO/HCO (Folmer et al. 1994) and

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