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Identification of Trombiculid Chigger Mites Collected on Rodents from Southern Vietnam and Molecular Detection of Rickettsiaceae Pathogen

Trombiculid “chigger” mites (Acari) are ectoparasites that feed blood on rodents and another animals. A cross-sectional survey was conducted in 7 ecosystems of southern Vietnam from 2015 to 2016. Chigger mites were identified with morphological characteristics and assayed by polymerase chain reaction for detection of rickettsiaceae. Overall chigger infestation among rodents was 23.38%. The chigger index among infested rodents was 19.37 and a mean abundance of 4.61. A total of 2,770 chigger mites were identified belonging to 6 species, 3 genera, and 1 family, and pooled into 141 pools (10–20 chiggers per pool). Two pools (1.4%) of the chiggers were positive for Orientia tsutsugamushi. Ricketsia spp. was not detected in any pools of chiggers. Further studies are needed including a larger number and diverse hosts, and environmental factors to assess scrub typhus.


INTRODUCTION
Trombiculid mites (Acari: Trombiculidae) are ectoparasites that are found in grasses and herbaceous vegetation. The larval mites (chiggers) feed blood on rodents and another animals and birds. More than 3,000 species of chiggers have been identified, but only about 20 species frequently attack humans, and the most relevant species e.g., Eutrombicula alfreddugesi in North and South America, Neotrombicula autumnalis in Europe, and Leptotrombidium spp. in Asia, that are considered to play an important role in transmission of Orientia tsutsugamushi.
The life cycle of Trombiculid mites include 7 stages of active and inactive stages. The larval stage chigger is the only stage of development that parasitizes a wide range of animals, including rats, squirrels, shrews, and birds . Humans are accidentally infected with O. tsutsugamushi through bites of chigger mites in grassy fields, in open and forested areas. The bite sites are on the skin, especially thin and clothing constricts.
Orientia tsutsugamushi is a gram-negative bacteria and causative agent of scrub typhus, is a vector-borne zoonotic disease with the potential of causing life-threatening febrile infection in humans. Orientia tsutsugamushi, is maintained in the Tromboculid mites through transovarial transmission. Scrub typhus is endemic in South and East Asia regions with more than one billion people at risk with approximately one million new cases reported annually.
There was limited surveillance. Surveys on the species composition of chigger mites were conducted at disperse sites in southern as well as throughout Vietnam. Study on pathogens in chigger mites have not been performed in southern Vietnam.
We performed this cross-sectional study to identify a species composition of chigger mites and presence of rickettsiaceae bacteria, especially Orientia tsutsugamushi in the mites parasitized in rodents collected in southern Vietnam.

Site description
Seven ecosystems in southern Vietnam including Bu Gia Map National park (Binh Phuoc province), Can Gio Man-grove Forest (Ho Chi Minh City), Lang Sen Wetland Reserve (Long An province), Lung Ngoc Hoang Nature Reserve (Hau Giang province), Cat Tien National Park (Dong Nai province), U Minh Ha National Park (Ca Mau province), U Minh Thuong National Park (Kien Giang province).

Rodent collection
Rodent were collected from July to October 2015 (rainy season) and March to April 2016 (dry season). A total of 70 Sherman collapsible live capture traps (H. B. Sherman Traps, Inc., Tallahassee, Florida, USA) at each of the sites were set each night (from 16:00 to 05:00 the following day). Traps were baited with fried fish, potatoes or other local food. Rodents were removed from the traps and placed individually in a white cloth bag labeled with the time of collection, date and an unique collection that identified the survey site and other pertinent information, and then transported to laboratory of Ectoparasitology, Department of Parasitology and Entomology, Ho Chi Minh Institute of Malariology.
Rodents were euthanized at the laboratory of Ectoparasitology Department according to the protocol of Ho Chi Minh Institute of Malariology, Parasitology and Entomology with ketamine (100 mg/kg) and xylazine (10 mg/kg) was intraperitoneally injected at dosage of 0.2 ml/100 gm body weight. Rodents were identified to species using morphology and taxonomic keys.
All animals received a good care by well-trained staffs. The study protocols were performed according to the ethical guidelines for care and use of experimental animals approved by the Ethical committee board of Ho Chi Minh Institute of Malariology, Parasitology and Entomology (Permit number 034/2015/ HCMIMPE-IRB).
All personnel were trained on safety measures when conducting investigations, collection, treatment and identification. If participants developed infectious diseases (occupational diseases), they were entitled to benefits and regimes according to current regulations.

Chigger mites collection
Rodents were carefully examined e.g., the groin, leg, anal area, ear, nose, eyelid, and crest by brushing these areas with fine combs. Then chigger mites were collected into plastic vials containing 70% ethanol and labeled individually with an unique number assigned to each of survey sites.

Chigger identification
The chigger mites collected from rodents were identified based on morphologic criteria following taxonomic keys. The chiggers were mounted on glass slides with Hoyer's medium and observed under a binocular microscope at x400 magnification. The key for identification of chigger mite species was based on documents of Van Chau Nguyen. Mites were put on a pool of 10-20 individuals according to their genera and host species. Stored at 4˚C for pathogen identification.

Rickettsiaceae detection
The genomic DNA was extracted from the chigger mite pools using the ISOLATE II Genomic DNA Kit (Cat. BIO-52066, Bioline, UK) according to the manufacturer's instructions. A final elution volume of 50 μl DNA samples were alliquoted into 2 eppendoft tubes and store at -80˚C until used.
A multiplex PCR method was performed for the detection and differential diagnosis of Oriental tsutsugamushi and Rickettsia spp. based on 2 primers sets using groEL gene sequences.
All PCR amplicons were electrophorozed in 2% agarose containing ethidium bromide. Bacteria species were identified based on amplicon size.

Data processing and analysis
The epidemiological parameters such as mean abundance of chiggers (total number of chiggers collected divided by total number of rodents), chigger infestation rate (percentage of rodents infested with chiggers), and chigger index (total number of chiggers divided by total number of infestedrodents ) were calculated using SPSS software, version 16.0 (SPSS, Chicago, Illinois, USA). Map was drawn using ArcView GIS software, version 3.1 (ESRI).

Composition and distribution of Chigger mites
A total of 2,770 chigger mites were collected from 143 rodents with an overall chigger index of 19.37 and a mean abundance of chiggers 4.61.
The results show that R. norvegicus, R. fulvescens, R. edwardsi and R. rattus were the main hosts of 6 chigger mite species, especially Leptotrombidium spp. The remaining rodents were the hosts of some other species. The distribution of chigger mites parasitizing on rodents according to the survey sites were shown in Fig.1.
The geographical distribution showed that L. (L.) deliense was collected from rodents at all surveyed sites, while As. Indica was collected at 6 sites, G. (W.) chinensis and G. (W.) parapacifica at 5 sites. The remaining chigger mite species showed narrow distribution, e.g., G. (W.) lupell was found at 2 survey sites and L. (L.) striatum at 3 sites.

Pathogens in chigger mites
The results showed that 1.4% pools of chigger mites were positive with Orientia tsutsugamushi (Fig. 2). One sample of Ascoschoengastia (L.) indica was infected O. tsutsugamushi (4.35%). One pool of L. (L.) deliense was infected O. tsutsugamushi (1.72%). There were not a positive pool for Rickettsia spp. (Table 3). At 7 survey sites, an O. tsutsugamushi -positive mite pool was found in Bu Gia Map National Park, Binh Phuoc province belonged to As. (L.) indica species (parasitized on R. argentiveter).  The other one was L. (L.) deliense parasitized on R. norvegicus host collected in U Minh Ha National Park, Ca Mau province (Fig. 3).

DISCUSSION
Trombiculid mites' habitat is prevalent in an area of overgrown fields, wooded, grassy and herbaceous vegetation. More than 3,000 species of chiggers are known in the world, about 350 species have been described in Southeast Asia .
Chiggers mites (family Trombiculidae) are considered to be common ectoparasites of rodents collected from the Mekong Delta, in contrast to gamasid mites, fleas, ticks, and liceare infrequently collected. In our study, 5 per 7 survey sites are in Mekong Delta area, and were forested reserves and parks with abudance of trees, high humidity, lower temperature, light intensity, moist soil and covered with vegetation. It is the ideal environment for the living of trombiculid mites. In the other maner, due to the present human activities, the predator mammals as well as raptorial birds are reduced and the numbers of general species such as squirels and wild birds increased. The Central South-Southern Region was the region with the Table 3. Detection rate of rickettsiae in chigger mites by PCR Our result had a lower species composition than other studies that were carried out in different regions of Vietnam such as provinces of Quang Binh and Ha Tinh, 16 species; Con Dao Island, 7 species; Quang Nam and Kon Tum, 6 species. In our study, the chigger mites were only collected from rats, while other studies were retrieved them from rodent host groups (wild birds, rabbits, squirels, raising birds, garbage and animal nests). Of which, 6 species were collected in the rainy season and 5 species were in the dry season. Leptotrombidium (L.) striatum was only collected in the rainy season (July to October) that corresponded to the increasing number of hospitalized scrub typhus patients begun in late August, peaked in October, and then decreased in December.
This The chigger mite infestion rate of O. tsutsugamushi in this study was 1.4% at 7 survey sites and that was lower than 2.7% at 9 survey sites in Korea, and 17.7% in Shandong province, China.
This study provided specific data for the species composition, and distribution of chigger mites infested rodents in southern Vietnam and determined the presence of O. tsutsugamushi infection. It is very helpful in development of monitoring strategy, prevention and control of scrub typhus transmitted through chigger mites infested in the rodents.
Further studies are needed with a larger number of host sample size, diversity of environmental condition to give the overall geographic distribution and assess the full risk of human exposure to Trombiculid "chigger" mites.