Tick Map 3 project: Mapping of Vectors and Reservoir Hosts in Lao PDR

Project Coordinator:
• Dr. Paul Brey, Director, Institut Pasteur du Laos, Vientiane, Lao PDR
• Dr. Jodi Fiorenzano, Chief of Entomological Sciences, U.S. Naval Medical Research Center – Asia, Sembawang, Singapore
• Dr. Khamsing Vongphayloth, Research entomologist, Institut Pasteur du Laos

Staff members:
• Khaithong Lakeomany, Technician entomologist, Institut Pasteur du Laos
• Nothasine Phommavanh, Technician entomologist, Institut Pasteur du Laos

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)

Summary

• Our field missions were carried out in two provinces of Lao PDR in 2020. A total of 2,206 ticks and other ectoparasites were collected during our course of study, of which 9 were fleas, 658 were mites, and 1,542 were ticks collected from different methods. Additional mosquitoes and sandflies were also collected, but the data was not reported herein (available on request). Three genera of mites and 15 species of ticks belonging to 5 genera were identified.
• Two sequences of Phleboviruses have identified form nymph pools of Haemaphysalis ticks. The sequences were identity to Yongjia Tick Virus 75%, Lesvos Virus from tick 79%, and Dabieshan Tick Virus 92%.
• A total of 100 pools have been screened for the presence of Rickettsia spp., and Anaplasma/Coxiella spp. at LOMWRU. Only one pool (1/100) of Haemaphysalis ticks was positive for Rickettsia spp. (17kDa). None of the tick pools (0/100) were positive for Anaplasma/ Coxiella spp. The team will continue to try to identify the Rickettsia species.
• Our partner at IP-Paris will continue to work on a deep sequencing study on arboviruses from the tick samples that we sent using a new method of RNA transportation.
• We will continue our tick study in Laos in 2021 for both ticks and their pathogens associated, especially Rickettsia, Phleboviruses and Flaviviruses.

Background

Vector-borne diseases constitute a significant infectious disease risk for deployed military personnel and for local populations. In Laos, definitive diagnosis is often not available for vector-borne illnesses, so the infectious diseases which are a threat to military and civilian populations are not well-defined.

In order to identify common and emerging vectorborne pathogens in Laos, NAMRU-2 Singapore (SG) has established a study to assess the distribution and infection potential of vectors (including ticks and associated arthropods). In this study, tick and associated arthropod vectors will be surveyed from the environment and their associated hosts to provide biological specimens for diagnostic purposes. The samples will be transported to the Institut Pasteur du Laos (IPL) laboratory in Vientiane, where a wide range of diagnostic tests can be performed to identify both the vector and 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 2 sites and 2 provinces throughout Laos. In order to identify common and emerging vectorborne pathogens in Laos, NAMRU-2 Singapore (SG) has established a study to assess the distribution and infection potential of vectors (including ticks and associated arthropods). In this study, tick and associated arthropod vectors will be surveyed from the environment and their associated hosts to provide biological specimens for diagnostic purposes. The samples will be transported to the Institut Pasteur du Laos (IPL) laboratory in Vientiane, where a wide range of diagnostic tests can be performed to identify both the vector and 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 2 sites and 2 provinces throughout Laos.

Strategic objective

• Collect approximately 600–1,000 ticks and associated arthropod specimens from study areas by the completion of the study protocol and deliver those samples to the IPL laboratory in Vientiane.

Specific objectives

• Survey and modern ID of indigenous tick and associated arthropod species distribution. • Collection, ID, extraction of vector DNA for submission and development of the regional repository. This will provide a valuable resource for downstream modern molecular analysis and genotyping. • Building of local capacities and competencies.

Major Milestones

• (Q1) Develop a protocol for collection, purchase equipment/supplies required for entomological collections, DNA/RNA extraction, and molecular characterization.
• (Q2/Q4) Collection (6 field missions), identification, extraction, and submission of molecular products to IPL/NMRC-A repository.
• (Q4) Present results and submit a final report.

Methodology

Study site and time Our first field mission of 12 days was conducted during the dry season, between 27/01-07/02/2020, in Xiengkhouang province. Two sites were selected and coded as loc.1: 20.029696, 103.751026 (Sopthang village); and loc.2: 19.734953; 103.555913 (Tha village) located in Kham district. All remaining fields were conducted during the rainy season because of government lockdown according to the pandemic of the SARS CoV-2 situation. The second 12- day field mission was made between 19-30/07/2020 from 2 sites in Luangprabang province coding as loc.3: 20.551757°, 102.627598° (Pha Thok village) located in Ngoy district; and loc.4: 20.594703°, 102.445530° (Far village) located in Nambak district. Six-day field mission was repeated in loc.2 and loc.3 in August and September respectively. See Fig. 1 and Tab. 1 below for more detail.

Figure 1. Fieldwork locations during our study in Xiengkhouang (loc.1 and loc.2) and Luangprabang (loc.3 and loc.4).

Table 1. Time table and coordinates of our field collection

Sample collection procedure

Tick dragging/flagging: Tick dragnets were swept/ dragged along the forest ground at approximately 1–10 m intervals before being examined for ticks. Ticks were removed from the sheets using forceps, then transferred to 1.5 ml labeled cryotubes, and stored -20°C.

Small Mammal Trapping for ectoparasites: In each study site, 45 rodent traps (baited with bananas, or sweet potatoes, or dried fish/meat) were placed in the format of transect according to the topography. All rodents were released after checking for ectoparasites.

Additional tick collection was carried out by examining domestic animals (dogs, buffalos and cows). The animal owners were asked to help to examine their animals. Once ticks attached to animals were found, they were collected by direct hand removal and/or with forceps. Additional tick collection was also made during our traveling by examination of ticks from wild boars selling in a market in Paek district, Xiengkhouang province.

In all sites of collection, an additional collection of mosquitoes and sandflies was taken, but the data is not added in this report (available as request).

All tick samples were stored at −20°C in the field then transported to IP-Laos in Vientiane Capital and stored at −80°C until processing for further analysis (species identification and pathogen detection). Mites collected from rodents were stored in 90% ethanol.

Laboratory work

Sample identification and preparation

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, all larval and nymph stages were grouped into the genus. After tick identification and pooling, all information was registered with the Pathogen Asset Control System (PACS) soft sample identification and preparation 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, all larval and nymph stages were grouped into the genus. 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. ware and all tick samples were stored at −80°C in IP-Laos, Vientiane Capital, for further analysis.

Chigger mites from rodents were mounted on the slide using PVA mounting medium. Mite samples were identified using a compound microscope to genus level by referring to the published taxonomic key of Nadchatram & Dohany 1974.

RNA/DNA extraction

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 future pathogen isolation. 22

Arboviral screening

As we could not conduct our field collection during the lockdown period from March to May 2020, we decided to use reactions for PCR and sequencing from IPL to screen for phleboviruses including severe fever thrombocytopenia syndrome virus (SFTSV). A total of 22 pools were screened by means of conventional nested RT-PCR as previously described by Sa´nchez-Seco et al. 2003.

Bacterial screening

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. To investigate the occurrence of Rickettsia spp. in ticks, a molecular screening approach targeting the 17kDa gene was taken (Jiang et al. 2004). The presence of Anaplasma spp. and Coxiella was also investigated.

Results

Rodent trapping

A total of 17 rodents were collected during our course of study, of which 12 were collected from Xienkhouang province and 5 from Luangprabang province. A total of 655 mites and 3 ticks were collected from the rodents (See Tab. 2 below).

Number, species abundance and composition of ticks and other ectoparasites

A total of 2,206 ticks and other ectoparasites were collected during our course of study, of which 9 were fleas collected from dogs, 658 were mites collected from rodents, and 1,542 were ticks collected from different methods. The most majority of ectoparasites were collected from Xiengkhouang province by dragging method (see Tab. 2 below for more detail).

Table 2.  The number of ticks and other ectoparasites collected from different methods by districts.

So far, two families of mites have been identified as Laelapidae including 1 genus Echinolaelaps spp., and Trombiculidae including 2 genera: Helenicula spp. and Walchia spp. For the ticks, 15 species belonging to 5 genera were identified (Tab. 3 below).

Table 3. Species abundance and composition of ticks and other ectoparasites

Preliminary results on laboratory work on pathogen-associated with ticks

A total of 1,191 tick and mites collected from Xiengkhouang in our first field mission were pooled into 180 pools of one to ten samples by species or genus, sex, stage of development, collection period, and site. In order to assess the distribution and infection potential of tick-borne pathogens (especially rickettsia) in Laos with our limited budget on bacterial screening, we included 12 pools of ticks from our collections in 2019 from Khammouane and Vientiane Provinces (see Tab.4 below for more detail).

Table 4. Total number of ticks and mites pooled so far

Arboviral screening

As we could not conduct our field collection during the lockdown period from March to May 2020, we decided to use reactions for PCR and sequencing from IPL to screen for Pan-phleboviruses including severe fever thrombocytopenia syndrome virus (SFTSV). A total of 22 pools were screened by means of conventional nested RT-PCR as previously described by Sa´nchez- Seco et al. 2003. Two nymph pools of Haemaphysalis ticks were positive for Pan-phleboviruses. The sequencing was performed from these two pools and obtained two sequences of 210 nucleotides. These two sequences were identity 100% to each other. The blast search showed that the sequences were identity to Yongjia Tick Virus 75%, Lesvos Virus from tick 79%, and Dabieshan Tick Virus 92% (Fig. 2 below).

Figure 2. Blast results from phlebovirus sequences obtained from Haemaphysalis ticks

Bacterial screening

So far, a total of 100 pools containing 510 ticks have been screened for the presence of Rickettsia spp., and Anaplasma/ spp. (including Anaplasma phagocytophilum) at Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU). Only one pool (1/100) of Haemaphysalis ticks (a pool of 12 larvae) was positive for Rickettsia spp. (17kDa). None of the tick pools (0/100) were positive for Anaplasma/Coxiella spp. (Tab. 5). The team will continue to try to identify the Rickettsia species.

Table 5. Rickettsial screening from ticks

Next generation Sequencing at IP-Paris

Nucleic acid extracted from 74 pools containing 356 ticks was selected and sent to IP-Paris for a more deep sequencing study on the diversity of arbovirus from ticks in Laos. These samples set included two samples that we obtained the sequences by Pan Phleboviruses (See Tab. 6 below for more detail). Because of the pandemic of COVID-19, it is impossible for us to send the samples in dry ice to our partner as the shipment company has stopped their operation. So, we have selected an alternative method to send our samples using RNA stable tubes. The tube kit allows us to store RNA in dry form and transport it at ambient temperature. However, the shipment takes a long time so we do not have the results in this report.

Table 6. Samples selected to send to IP-Paris for deep sequencing study

Challenges and Follow-up Actions

• The pandemic of COVID-19 is the only challenge that we have faced so far. However, we have finished all of our field collections as plan at the time of the project.
• All samples were stored as -20 – -80 for future pathogen study.
• Our partner, LOMWRU, will continue to try to identify the Rickettsia species.
• And also, our partner at IP-Paris will continue to work on a deep sequencing study on samples that we sent using the new method of RNA transportation.
• We will continue our study in 2021, in Laos, on investigating more ticks and their pathogens associated.

References

Jiang J., Chan T., Temenek J.J., Dasch G.A., Ching W. and Richards A.L. (2004). Development of a Quantitative Real time Polymerase Chain Reaction Assay Specific for Orentia Tsutsugamushi. American Journal of Tropical Medicine and Hygiene, 70(4): 351-356

Nadchatram, M. and A. L. Dohany. 1974. A pictorial key to the subfamilies, genera and subgenera of Southeast Asian chiggers (Acari, Prostigmata, Trombiculidae). Bulletin from the Institute for Medical Research Federation of Malaysia, 16: 1–67.

Nuttall, G. H. F., W. F. Cooper, C. Warburton, L. E. Robinson, and D. R. Arthur. 1926. Ticks: pt. IV. The genus Amblyomma. Cambridge University Press. Sanchez-Seco, M.P., et al., Detection and identification of Toscana and other phleboviruses by RT-nested-PCR assays with degenerated primers. J Med Virol, 2003. 71(1): p. 140-9.

Tanskull, P. and I. Inlao. 1989. Keys to the adult ticks of Haemaphysalis Koch, 1844, in Thailand with notes on changes in taxonomy (Acari: Ixodoidea: Ixodidae). J Med Entomol 26(6): 573–600.

Yamaguti N., V. J. Tipton, H. L. Keegan, and S. Toshioka (1971). Ticks of Japan, Korea, and the Ryukyu islands. Brigham Young University Science Bulletin, Biological Series 15(1):1.