Medical and Veterinary Entomology (2021), doi: 10.1111/mve.

12531

High prevalence and low diversity of chigger infestation

in small mammals found in Bangkok Metropolitan parks
S. A. W U L A N D H A R I 1 , Y. P A L A D S I N G 2 , W. S A E S I M 3 ,
V. C H A R O E N N I T I W A T 2 , P. S O N T H A Y A N O N 4 , R. K U M L E R T 5 ,
S. M O R A N D 2,6 , S. S U M R U A Y P H O L 1 and K. C H A I S I R I 2
1
Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, 2 Department of
Helminthology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, 3 Environment Department, Bangkok
Metropolitan Administration, Bangkok, Thailand, 4 Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical
Medicine, Mahidol University, Bangkok, Thailand, 5 Division of Vector-Borne Diseases, Department of Disease Control, Ministry of
Public Health, Nonthaburi, Thailand and 6 CNRS-CIRAD, Faculty of Veterinary Technology, Kasetsart University, Bangkok, Thailand

Abstract. Chiggers are recognized as vectors of scrub typhus disease caused by

the bacteria, Orientia tsutsugamushi. The risk of disease exposure is mainly related
to chigger bites when humans or animals roam into vector-infested habitats. In big
cities, urban public parks could provide areas for the animal–human interface and
zoonotic pathogen transmission. The ecology and epidemiology of urban scrub typhus
are still poorly understood in Thailand. Small mammals were trapped and examined
for chigger infestation in urban public parks across metropolitan Bangkok, Thailand.
We found a high prevalence of infestation (76.8%) with surprisingly low diversity. Two
chigger species, Leptotrombidium deliense and Ascoschoengastia indica, were identified
using morphological characteristics and molecular confirmation. The generalized linear
model identified host intrinsic variables (i.e. body mass index) with host density,
habitat composition and open field as the extrinsic factors explaining the abundance
of chigger infestation. The bacteria O. tsutsugamushi was not detected in chiggers
(90 chigger-pooled samples) and animal host tissues (164 spleen samples). However,
the existence of chigger vectors calls for the Bangkok Metropolitan Administration
and public health authorities to develop a comprehensive scrub typhus monitoring and
prevention strategy in the parks and nearby communities.
Key words. Chiggers, Bangkok, Orientia, public park, small mammals, Thailand,
urban.

Introduction intracellular bacteria Orientia tsutsugamushi (Alphaproteobac-

Chigger is a common term for the tiny larval stages of trom-
biculid mites (Acari: Trombiculidae), also known as harvest
mites, scrub itch mites or redbugs. Chiggers are parasitic only
at the larval stage, feeding on a wide range of vertebrates
(and incidentally on humans). At other stages (nymph and
adult), they prey on soft-bodied arthropods and soil nema-
todes (Elliott et al., 2019). The mites are the main vectors of
teria: Rickettsiaceae), the causative agent of scrub typhus dis-
ease in the Asia Pacific region (Kelly et al., 2009). In Thailand,
scrub typhus is highly prevalent and has been realized as one
of the main causes of acute febrile illness (Wangrangsimakul
et al., 2020). Sporadic human cases have also been reported
recently in other regions, such as the Middle East (i.e. the United
Arab Emirates) and South America (i.e. Chile), caused by two
other bacterial species: Orientia chuto and Candidatus Orientia

Correspondence: K. Chaisiri, Faculty of Tropical Medicine, Mahidol University, 420/6 Ratchavithi Rd., Ratchathewi, Bangkok, Thailand 10400.
E-mail: kittipong.cha@mahidol.ac.th

© 2021 The Royal Entomological Society 1

2 S. A. Wulandhari et al.
chiloensis, respectively (Izzard et al., 2010; Abarca et al., 2020).
These highlight the recent situation of global spread of scrub
typhus outside the endemic region. In addition to the role of
scrub typhus vector, chigger bites also result in intense irri-
tation and dermatitis (trombiculiasis) in humans and animals
(Santibáñez et al., 2015; Elliott et al., 2019).
Trombiculid mites are cosmopolitan, occurring across diverse
landscapes and habitat types, e.g. forest, scrub vegetation,
grassland, plantation, agricultural land, swamp, seacoast and
urban settings (Dohany et al., 1977; Park et al., 2015; Peng
et al., 2018; Chaisiri et al., 2019). There are over 3000 Trom-
biculidae species worldwide, with many described in the Ori-
ental, Australasian and Afrotropical regions compared to other
areas (Elliott et al., 2019). Chiggers in the genus Leptotrom-
bidium are the important vectors transmitting scrub typhus to
humans (Lv et al., 2018; Elliott et al., 2019). Leptotrombidium
akamushi, Leptotrombidium pallidum, Leptotrombidium scutel-
lare and Leptotrombidium deliense are the major vectors in the
Asia Pacific region (Stekolnikov, 2013; Santibáñez et al., 2015;
Elliott et al., 2019). The first three species tend to occupy the
temperate region of the endemic zone, e.g. Northern and East-
ern China, Japan and Korea (Zhang et al., 2013; Roh et al., 2014;
Yoshimoto & Yoshimoto, 2019). The latter species, L. deliense is
responsible for disease transmission in tropical areas represented
by Southern China, South Asia and Southeast Asian countries,
including Thailand (Rodkvamtook et al., 2013; Chakraborty &
Sarma, 2017; Lv et al., 2018).
In Thailand, the geographic distribution of trombiculid mites
mainly follows the information and research on human scrub
typhus’s local epidemiology (Kumlert et al., 2018). Lak-
shana (1973) reported that within 30 provinces of Thailand,
there were two subfamilies of chigger mites, 27 genera and 121
species. Subsequently, Chaisiri et al. (2016) revised the chig-
ger checklist of Thailand, reporting 99 validated species across
the country’s six geographical regions and emphasized lack of
information for some particular regions, e.g. the central plain,
including Bangkok, the capital city.
Small mammals (e.g. rodents, shrews and tree shrews) and
ground-dwelling birds play important roles as the ‘maintenance
hosts’, by either contributing to the abundance of chiggers (pro-
vide feeding meals) or reservoirs of the Orientia bacteria (Elliott
et al., 2019). An individual host was estimated to carry numer-
ous chiggers, ranging from 1000 to 10 000 at one time and could
support up to four generations of chiggers through its lifespan
(Audy, 1961). Chigger infestation status (e.g. prevalence, abun-
dance and species richness) on the maintenance hosts (partic-
ularly small mammals) is influenced by host intrinsic factors,
e.g. host taxonomy, body weight, maturity, behaviour, exploita-
tion habit, home range and site of attachment on the host body
(Frances et al., 1999; Chaisiri et al., 2019; Matthee et al., 2020).
Also, extrinsic factors, such as seasonal conditions, geographical
distribution, latitudinal gradient, landscape complexity, habitat
type, ecotone and degree of human land use, also play signifi-
cant roles in chigger infestation patterns (Frances et al., 1999;
Kuo et al., 2015; Moniuszko & Makol, 2016; Peng et al., 2018;
Chaisiri et al., 2019; Mathee et al., 2020).
People engaged in agriculture and habiting forested areas
are at high risk of infection with scrub typhus disease. In
endemic areas, the risk of infection is mainly associated with
© 2021 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12531
farming and outdoor activities, posing a high disease burden
(Park et al., 2015; Wangrangsimakul et al., 2020). Human scrub
typhus incidence is not only reported from occupational-related
groups such as farmers in the countryside but also occurred
in the others such as the elderly, children, pregnant women,
tourists and even in the urban population (Kim et al., 2006;
Wei et al., 2014; Weitzel et al., 2018). In Bangkok, a rapid
increase in population, capital investment and employment
has expanded the city to surrounding areas. Regarding urban
expansion, recreational green spaces are essential for providing
city dwellers a good quality of life. Big cities worldwide
usually provide open space or public parks for recreation
and rest from the city’s hustle and busyness. Apart from
that, parks are also places for biodiversity hotspots (flora
and fauna). Some synanthropic small mammals (e.g. rats,
squirrels, shrews and tree shrews) are usually present in urban
parks, providing interaction between humans and wildlife.
Zoonotic pathogens, particularly vector-borne diseases, could be
exchanged between humans and animals. A serological survey
in Bangkok suburbs revealed high human exposure to scrub
typhus (21% prevalence) related to contact in orchards and farms
(Strickman et al., 1994). Wei et al. (2014) suggested that people
who had activities in the city park pose a higher risk than those
who did not. Moreover, risk behaviours, e.g. sitting on the lawn,
close contact with wildlife and sitting near rodent burrows,
significantly increased the risk for scrub typhus in a city park
(Wei et al., 2014).
Therefore, this study aims to understand the relationship
among the vector (chiggers), the maintenance host (small mam-
mals) and the causative bacterial agent of scrub typhus at public
parks in the Bangkok Metropolitan area. This study’s specific
objectives are to (1) to investigate the diversity, prevalence and
abundance of chigger infestation in small mammals; (2) to inves-
tigate the vectorial role of chiggers in carrying Orientia bacteria
and (3) to identify potential factors explaining chigger occur-
rence in the public parks of Bangkok.
Materials and methods
Study sites
A cross-sectional study was conducted in Bangkok, Thai-
land, from September to the beginning of December 2018 (wet
season). Small mammals were collected using live-trapping
at the selected seven sites by focusing on the green space
or public parks in the Bangkok Metropolitan area (see Pal-
adsing et al., 2020). These included Suan Luang Rama IX
(SL: lat 13.686583 and long 100.663942), Suan Lumpini (LP:
lat 13.731026 and long 100.541407), Suan Serithai (ST: lat
13.786245 and long 100.672598), Suan Taweevanarom (TW:
lat 13.744377 and long 100.352274), Suan Thonburirom (TB:
lat 13.651955 and long 100.491626), Suan Vareepirom (VP: lat
13.850450 and long 100.774289) and Suan Wachirabenchatad
(WB: lat 13.812098 and long 100.554368), (see Fig. 1A). The
parks share some common characteristics, such as vegetation,
water reservoirs and areas for human activities. However, each
park’s habitat conditions differ depending on their internal archi-
tecture and external characteristics around the parks; some are
 Chiggers from Bangkok public parks 3

(A)

(B)

Fig. 1. Geographical distribution of the study sites for chigger infestation on small mammals in the Bangkok Metropolitan area. (A) The selected seven
public parks as the sampling sites across Bangkok. Pixel colours on the map represent associated land cover labels and RGB codes of the GlobCover2009
Land Cover Map at a resolution of 300 × 300 m (http://due.esrin.esa.int/page_globcover.php). (B) The abundance of chigger infestation at each study
site (size of the red circles indicates chigger abundance on individual hosts), and the pie charts showing the proportional occurrence of Leptotrombidium
deliense (in orange) and Ascoschoengastia indica (in blue) in the seven study sites. [Colour figure can be viewed at wileyonlinelibrary.com].

© 2021 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12531
4 S. A. Wulandhari et al.
surrounded by dense human settlements and buildings, while
others are in the middle of agricultural lands and fields.
The trapping line method was applied following Herbreteau
et al. (2011), i.e. 10 lines (each line comprising 10 traps)
were randomly distributed throughout the area of a particular
park and retained for four nights within the same site. The
traps were checked every morning, and the captured animals
were subsequently assigned the project ID number. Global
Positioning System coordinates were immediately taken at each
positive trap. Trapping success and adjusted-trapping success
(Aplin et al., 2003) were calculated from each trapping site as a
proxy for estimating small mammal density in the parks.
Animal trapping and handling
Animals were transported to the parasitological laboratory
at the Department of Helminthology, Faculty of Tropical
Medicine, Mahidol University, on the same day of capture. They
were euthanized and identified to species level based on either
the morphological characteristics following Aplin et al. (2003)
or the DNA barcoding approach (see Pagès et al., 2010; Pal-
adsing et al., 2020). Information on gender (male or female)
and maturity (adult or juvenile) of the small mammals was also
recorded. We also calculated the animal body mass index (BMI)
with the following formula: weight in gram/(length)2 in millime-
tres. Animals were humanely euthanized using 5% isoflurane
inhalation. Biological specimens (e.g. blood, lung, liver, kid-
ney, spleen, gastrointestinal tract, ears and faeces) were subse-
quently collected in ethanol, RNAlater and fresh frozen for fur-
ther parasite/pathogen investigations (other studies will be pub-
lished elsewhere). The Faculty of Tropical Medicine—Animal
Care and Use Committee, Mahidol University (FTM-ACUC)
approved the protocol for animal trapping and handling under
document number FTM-ACUC 016/2018E. Specifically, the
exemption protocol for the chigger study was also approved
under document number FTM-ACUC 015/2020E.
Investigation of chigger infestation on small mammals
After animal euthanization and blood collection, bodies were
first checked by the naked eye for the presence of chiggers.
Different body parts, e.g. head, pectoral region, axillary line,
abdomen, legs and anogenital area, were carefully checked with
great attention inside the ears. Subsequently, the ears were
cut and examined for chigger infestation under a stereomicro-
scope. The chigger samples collected from the same hosts were
counted to estimate the abundance of infestation and preserved
in 70–95% ethanol. To identify and estimate chigger species
richness, around 10–20% of chiggers from each infested animal
were subsampled by differences in observed sizes and micro-
scopic appearance. Individual mites were put on glass slides and
mounted in Berlese’s fluid (clearing agent). The prepared spec-
imens were examined and measured using the PrimoVert light
microscope supplied with Axiocam 105 through ZEN Lite imag-
ing software (Carl ZEISS, Germany).
Chigger mites were initially identified to subgenus level fol-
lowing the pictorial key of Nadchatram & Dohany (1974),
© 2021 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12531
whereas species identification followed the identification keys
(Fernandes & Kulkarni, 2003; Stekolnikov, 2013). To estimate
chigger infestation status on small mammals, prevalence, mean
abundance and mean intensity were calculated using Quantita-
tive Parasitology 3.0 software (Rozsa et al., 2000).
The remaining chigger specimens from the same hosts, same
species and study sites were pooled in 95% ethanol for further
molecular experiments. Chigger specimens were verified to the
correct species using autofluorescence (ZEISS LSM 700 laser
scanning confocal microscope with fluorescein isothiocyanate
filter mode) and bright-field microscopy described by Kumlert
et al. (2018) before pooling them in the same tube.
Molecular procedures for chigger species confirmation
Genetic material was extracted from individual chigger speci-
mens using Genomic DNA Mini Kit (Geneaid, Taiwan), follow-
ing the manufacturer’s protocol. The mitochondrial cytochrome
oxidase subunit I (mtCOI) was selected as a genetic marker
for the chigger taxonomic study. We used the universal primers
derived from Folmer et al. (1994), which yield product size
around 650–700 bp. Polymerase chain reaction (PCR) was
carried out in 50-μL reaction containing 5 μL of DNA tem-
plate, OnePCR master mix (GeneDireX, Taiwan) and 2 μL of
each primer (at 0.4 μM final concentration). PCR was run for
40 cycles: initial denaturation at 94 ∘ C for 1 min; 5 cycles of
94 ∘ C for 1 min, 45 ∘ C for 90 s, 72 ∘ C for 90 s; 35 cycles of 94 ∘ C
for 1 min, 50 ∘ C for 90 s, 72 ∘ C for 1 min and finishing with a
final extension at 72 ∘ C for 5 min. PCR amplicons were visual-
ized in 1% agarose gel electrophoresis containing SYBR Safe
dye (Invitrogen, US) at 120 V for 40 min. PCR products were
sent for purification and Sanger sequencing at U2Bio (Thailand)
Sequencing Service.
The DNA sequences were quality checked, edited and aligned
using ClustalW multiple alignment, and a phylogenetic tree
was created using MEGA-X software (Kumar et al., 2018).
The phylogenetic tree was constructed using the maximum
likelihood method based on the Hasegawa–Kishino–Yano
model (HKY) with Gamma distributed and invariant sites
(G + I). The mtCOI sequences of trombiculid mites: L.
deliense (GenBank accession no. HQ324977.1), Leptotrom-
bidium imphalum (HQ324949.1), Leptotrombidium palpale
(AB300499.1), L. scutellare (AB300498.1), Leptotrombidium
fletcheri (AB300489.1), Leptotrombidium fuji (AB300496.1)
and Ascoschoengastia indica (KY930732.1) was compared with
chigger specimens from the Bangkok public parks. The DNA
sequence of house dust mite Dermatophagoides pteronyssinus
(KP406749.1) was an outgroup for phylogenetic reconstruction.
Molecular detection of O. tsutsugamushi in chiggers and small
mammal hosts
Similar chigger species were selected for specimen pooling,
based on different study site (public park) per pool. Ten pools of
chiggers (100 individuals from the same species per pool) and
80 pools (30 individuals from the same species within the same

site per pool) were prepared. Increased numbers of chiggers per
pool were applied following previous Orientia detection studies,
from one individual up to >100 chiggers per pool (Takhampunya
et al., 2014; Kuo et al., 2015; Elliott et al. 2019). Here, we
decided to prepare a majority of pool samples with 30 individual
chiggers following Frances et al. (1999) and Shim et al. (2009).
In addition, we prepared pools of 100 chiggers to increase
the amount of starting DNA template, which might improve
the Orientia screening. DNA from each pooled sample was
extracted using the Genomic DNA Mini Kit (Geneaid, Taiwan),
following the manufacturer’s protocol. DNA was extracted from
spleen tissues (n = 164) following the same protocol as chiggers
for small mammals.
Conventional nested PCR was performed to target the two
gene markers: 56 and 47 kDa type-specific antigen (TSA) of Ori-
entia bacteria in the chigger and small mammal samples. Ampli-
fication of 56 kDa TSA gene (around 700 bp) was performed
with the following primers: CG56F (5′ -TTACAATGGATAA
AACGCTTTGAA-3′ ) and CG56R (5′ -AGAAAAACCTAGA
AGTTATAGCGTACA-3′ ) for the first round PCR; and
RTS8F (5′ -AGGATTAGAGTGTGGTCCTT-3′ ) and RTS9R
(5′ -ACAGATGCACTATFAGGCAA-3′ ) for the second round
PCR (Takhampunya et al., 2014). PCR composition and
thermocycler condition were run following the protocol of
Takhampunya et al. (2014). In brief, the first round PCR ampli-
fication was carried out in 25 μL reaction containing 2.5 μL of
DNA template, OnePCR master mix (GeneDireX, Taiwan) and
1 μL of each primer (at 0.4 μM final concentration). The PCR
ran with an initial denaturation at 98 ∘ C for 2 min, followed by
40 cycles of 98 ∘ C for 10 s, 53 ∘ C for 20 s, 72 ∘ C for 1 min and
finishing with a final extension at 72 ∘ C for 5 min. The PCR
product from the first round was diluted to one-tenth of the
volume and used as a template (2.5 μL) in the second round
amplification. PCR ran following the same protocol described
earlier, except that annealing temperature at 55 ∘ C for 15 s
was applied. PCR products were visualized in 1% agarose gel
electrophoresis containing SYBR Safe dye (Invitrogen, US) at
120 V for 40 min.
For 47 kDa TSA gene amplification, we performed nested
PCR using the primers: Ot-145F (5′ -ACAGGCCAAGATATTG
GAAG-3′ ) and Ot-1780R (5′ -AATCGCCTTTAAACTAGATTT
ACTTATTA-3′ ) for the first-round amplification; and Ot-263F
(5′ -GTGCTAAGAAARGATGATACTTC-3′ ) and Ot-1133R
(5′ -ACATTTAACATACCACGACGAAT-3′ ) for the second-
round amplification (Masakhwe et al., 2018). Both the first
and second PCR ran in a 25-μL reaction containing 2 μL of
DNA template, OnePCR master mix (GeneDireX, Taiwan) and
0.7 μL of each primer (at 0.3 μM final concentration). Thermo-
cycler condition was performed similarly on both amplification
round, starting with an initial denaturation at 95 ∘ C for 2 min,
followed by 40 cycles of 94 ∘ C for 30 s, 54 ∘ C for 30 s, 68 ∘ C
for 2 min and completed with a final extension at 72 ∘ C for
7 min. The expected amplicon size was around 870 bp. All
the assays included negative control samples. The positive
control samples, DNA of O. tsutsugamushi (Karp strain)
from Lao Oxford Mahosot Wellcome Trust Research Unit,
Lao People’s Democratic Republic, were also applied in all
assays.
© 2021 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12531
Chiggers from Bangkok public parks 5
Multivariate analysis of independent variables explaining
the abundance of chigger infestation
Parasite and pathogen infections (i.e. diversity and abundance)
in wild rodents are commonly explained by intrinsic factors such
as host attributes (e.g. gender, maturity and body mass) or extrin-
sic factors (e.g. habitat, land use type and other environmental
factors). We hypothesized that such intrinsic and extrinsic fac-
tors might explain chigger infestation status (in terms of chigger
abundance) on small mammals from the Bangkok public parks.
We investigated each public park’s land use characteristics
using Global Land Cover Map (GlobCover2009 V2.3), a raster
in GEOTIFF format containing ID values for different land
cover categories at a resolution of 300 m × 300 m. Land use
boundaries of each study site were created, and proportion
of the land use types, i.e. (1) post-flooding/rain-fed land, (2)
mosaic cropland/vegetation, (3) shrub/grassland, (4) close to a
mixed forest and (5) artificial surface/urban within a certain
park were calculated using ‘PatchStat’ function in ‘SDMTools’
package implemented in R freeware (R Core Team, 2019).
Land use characteristics for each site were verified again
with Google Earth (Google Earth Pro v7.3.2) before using
parameters in further analyses (see more details in Paladsing
et al., 2020). The total area (metre2 ) and boundary perimeter
(metre) of each park were also measured using Google Earth
Pro v7.3.2. All these variables were taken into account for
generating a composite habitat variable of each park through the
principal components analysis (PCA) approach (component 1)
using ‘princomp’ function in ‘stats’ package implemented in R
freeware (R Core Team, 2019). The PCA was used to explore
whether the habitat characteristics of each study site differ from
each other. We also adopted the score of principal component
1 as a composite habitat variable in subsequent multivariate
analysis to understand the abundance of chigger infestation.
Also using Google Earth (Google Earth Pro v7.3.2), we man-
ually measured the distances (meter) from individual captured
small mammals to the four habitat categories in/around the pub-
lic parks. These four habitat categories include (1) water body
(e.g. surface water, water resources, ponds, river, lake and canal);
(2) open field (e.g. grassland, fallow, shrub, cropland and parts of
area that is not covered by shaded trees; (3) shaded tree (e.g. tree
plantation and parts of the full-shaded area that contain peren-
nial trees and (4) building (e.g. park toilet, office, greenhouse,
shelter, apartment, house, mall and other types of human build-
ings).
The generalized linear model (GLM) identified the potential
effects of host attributes and environmental factors on the abun-
dance of chigger infestation. Variables included in the initial
model were as follows: Chigger abundance = BMI + Sex +
Maturity + Host density (proxy) + Habitat composite + Dis-
tance to human building + Distance to water body + Distance
to open field + Distance to shaded trees. Data distribution of
the response variable (Chigger abundance) was tested using
‘descdist’ function in ‘fitdistrplus’ package implemented in R
freeware (R Core Team, 2019). GLM with Akaike’s informa-
tion criterion corrected for sample size (AICc) was performed
with negative binomial distribution using ‘glm.nb’ and ‘glmulti’
functions in ‘gmulti’ and ‘lme4’ packages. We selected the
best model for further discussion based on AICc, ΔAICc and
6 S. A. Wulandhari et al.

Table 1. Prevalence (%), mean abundance (CMA), mean intensity (CMI) and range of chigger infestation on small mammals (n = 164) from public
parks in the Bangkok Metropolitan area

No. of animal Total chiggers % Prevalence Range of

captured number (95%CI) CMA (95%CI) CMI (95%CI) infestation

Host species
Rattus rattus-complex 132 10 991 85.6 (78.4–90.7) 83.27 (64.4–109.7) 97.2 (76.5–125.4) 1–728
Rattus exulans 11 35 9.1 (0.47–40.4) 3.18 (0.00–9.55) 35 (0.0–0.0) 35
Rattus norvegicus 1 20 100 (5–100) 20 (0.0–0.0) 20 (0.0–0.0) 20
Tupaia belangeri 20 132 55 (32–75.5) 6.6 (2.9–14.15) 12 (5.4–22.8) 1–43
Site
WB 55 2455 87.3 (75.6–93.8) 44.6 (30.9–66.7) 51.1 (36.7–75.5) 1–334
SL 38 2789 92.1 (79.2–97.8) 73.4 (48.4–122.7) 79.7 (53.6–132.8) 1–497
LU 4 86 75 (24.8–98.7) 21.5(5.0–33.0) 28.7(20.0–36.3) 20–43
ST 17 213 29.4 (12.3–54-1) 12.5 (1.4–55.5) 42.6 (6.8–113.6) 6–182
VP 23 4522 91.3 (72.2–98.4) 196.6 (125.3–290.8) 215.4 (138.6–307.3) 6–728
TB 12 161 25 (7.2–54.2) 13.4 (0.4–49.7) 53.7 (5.0–140.0) 5–140
TW 15 952 73.3 (46.5–90.3) 63.5 (26.7–150.9) 86.5 (35.2–201.6) 3–406
Sex
Male 74 4959 83.8 (73.7–90.7) 67 (48.1–94.4) 79.9 (58.3–110.6) 1–508
Female 90 6219 71.1 (60.6–79.5) 69.1 (46.7–101) 91.7 (67.7–141.2) 1–728
Maturity
Adult 115 9787 81.7 (73.5–87.9) 85.1 (65.8–114.2) 104.1 (79.9–136.8) 1–728
Juvenile 49 1391 65.3 (51–77.8) 28.3 (15.8–57.2) 43.4 (25.5–84.2) 1–406

Akaike’s weight (W r ) scores (Burnham & Anderson, 2002).
The variance inflation factor was computed using a ‘vif’ func-
tion in the ‘car’ package implemented in R freeware (R Core
Team, 2019) to identify the degree of multicollinearity among
the explicative variables. Also, the quality of the selected model
was evaluated with goodness-of-fit and power analysis tests
using ‘chisq_gof’ and ‘modelPower’ functions in ‘sjstats’ and
‘lmSupport’ packages, respectively.
Results
Prevalence, abundance and species richness of chigger
infestation on small mammals from Bangkok public parks
We trapped 164 small mammals (Muridae: Rattus exulans,
Rattus norvegicus and Rattus rattus-complex and Scadentia:
Tupaia belangeri) in seven public parks around Bangkok. A total
of 11 178 chiggers were collected from the ears of the animal
hosts. Among the mammals examined, 126 individuals were
infested with chiggers, yielding a total prevalence of infestation
at 76.8%. Host species prevalence of infestation varied from
9.1% to 100%, noting the small number of R. norvegicus (n = 1)
and R. exulans (n = 11). The highest prevalence of infestation for
the study sites was found in small mammals from SL (92.11%),
followed by VP (91.3%), WB (87.3%), LP (75%), TW (73.3%)
and ST (29.4%); whereas mammals from TB showed the lowest
prevalence at 25%, no significant difference was observed on
prevalence of infestation between male (83.8%) and female
host (71.1%), (chi-square = 0.3274, P = 0.5672) and between
adult (81.7%) and juvenile (65.3%), (chi-square = 0.5056,
P = 0.4771).
The murid rodents, R. rattus-complex and R. norvegicus had
the highest mean abundance of chiggers (CMA) on host species
of 83.2 and 20, respectively, followed by the tree shrew, T. belan-
geri (CMA = 6.6) and lastly, the Polynesian rat, R. exulans
(CMA = 3.18). Chigger abundance significantly differed among
the public parks (Kruskal-Wallis test = 44.75, P < 0.001). Ani-
mals from VP had the highest mean abundance (CMA = 196.6),
at least two times higher than the other parks, whereas ani-
mals residing in TB and ST had a much lower abundance
(CMA = 13.4 and 12.5, respectively) (Fig. 1B). There was
no significant difference in chigger abundance between male
(CMA = 67) and female hosts (CMA = 69.1). However, adults
significantly had higher chigger abundance (CMA = 85.1) than
the young hosts (CMA =28.3) (Mann–Whitney U test = 1788,
P < 0.001). Table 1 presents more details of chigger infestation
status.
After 10–20% subsampling from each host (intentionally
selected by differences in observed sizes and microscopic
appearance), we prepared 1187 individual chiggers on per-
manent slides for taxonomic identification, only two chigger
species: L. deliense (n = 239; 20.1%) and A. indica (n = 948;
79.9%) were identified (Fig. 2). Leptotrombidium deliense was
present in four public parks: WB (n = 14), LR (n = 51), VP
(n = 94) and TW (n = 80), whereas A. indica occurred in all
study sites. Leptotrombidium deliense was found on R. rat-
tus-complex and T. belangeri, while A. indica occurred widely
on the four host species.
We conducted mtCOI gene amplification of selected 19 indi-
vidual chigger samples (9 L. deliense and 10 A. indica after
morphological identification) from various study sites. Phylo-
genetic reconstruction (see Fig. 3) illustrates the taxonomic
classification and supports the two chigger species morpho-
logical identification. Using nucleotide Basic Local Align-
ment Search Tool (BLASTn), L. deliense specimens from the
present study showed 98.60–98.90% sequence similarity to
L. deliense: accession no. HQ324977.1, whereas A. indica

© 2021 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12531
 Chiggers from Bangkok public parks 7

(A) (B)

(C) (D)

(E) (F)

Fig. 2. Fluorescence and bright-field microscopy of chiggers from rodent hosts in Bangkok public parks, Leptotrombidium deliense (A) and
Ascoschoengastia indica (B). Bright-field microscopy showing scutum and whole body aspect of L. deliense (C and E) and A. indica (D and F).
[Colour figure can be viewed at wileyonlinelibrary.com].

showed 87.79–90.64% sequence similarity to A. indica: acces-
sion no. MG728110.1 and 86.74–88.65% to A. indica: accession
no. KY930732.1. The nucleotide sequences of chiggers estab-
lished in this study were deposited in the nucleotide database
(GenBank) under accession numbers: ADP04 (MW478634),
ADP05 (MW478635), ADP06 (MW478636), ADP07 (MW478
637), ADP08 (MW478638), ADP09 (MW478639), ADP10
(MW478640), ADP17 (MW478641), ADP18 (MW478642),
ADP19 (MW478643), LDP23 (MW475712), LDP24 (MW47
5713), LDP26 (MW475714), LDP27 (MW475715), LDP28
© 2021 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12531
(MW475716), LDP29 (MW475717), LDP30 (MW475718),
LDP33 (MW475719) and LDP34 (MW475720).
Non-detected O. tsutsugamushi in chiggers and small
mammals from Bangkok public parks
Applying the O. tsutsugamushi positive control in all PCR
experiments (56 and 47 kDa TSA genes), we could not detect
any genetic material of the bacteria in both chiggers (90
8 S. A. Wulandhari et al.

Fig. 3. Phylogenetic reconstruction based on mitochondrial COI sequences of individual chiggers on small mammals from public parks of Bangkok
compared with available DNA sequences in the NCBI database: KY930732.1 (Ascoschoengastia indica), MG728110.1 (A. indica), HQ324977.1 (Lep-
totrombidium deliense), AB300489.1 (Leptotrombidium fletcheri), AB300496.1 (Leptotrombidium fuji), HQ324949.1 (Leptotrombidium imphalum),
AB300499.1 (Leptotrombidium palpale), AB300498.1 (Leptotrombidium scutellare) and KP406749.1 (Dermatophagoides pteronyssinus) as the out-
group. Samples labelled with LDPxx and ADPxx are from the present study with abbreviated public park names. The percentage of trees in which
the associated taxa clustered together is shown next to the branches. Tree topology is drawn to scale, with branch lengths indicating the number of
substitutions per site.

chigger-pooled samples) and animal host tissues (164 spleen
samples).
Associated variables explaining the abundance of chigger
infestation on small mammals from Bangkok public parks
composite were the three variables that appeared in all of
those top five models, suggesting that they contribute a strong
influential effect to explain chigger abundance. Using deviance
analysis (type II Wald Chi-square tests), we found significant
associations between chigger abundance and larger body mass
of mammals and living in/closed to open field habitat (Fig. 5).

Regarding physical characteristics of the Bangkok public

parks, PCA revealed that the study sites appeared dissimilar
from each other in habitats and land use types inside and
around the park (Fig. 4). Accordingly, we derived the PCA
score (component 1) of each site as representative of habitat
composite variable in GLM and some other extrinsic factors and
host attributes to explain the abundance of chigger infestation
on small mammals. GLM with AICc selection computed 500
candidate models; among these, the top 5 models are presented
in Table 2. The first model (Chigger abundance − BMI + Host
density (proxy) + Habitat composite + Distance to open field)
was selected as the best-describing factors influencing chigger
abundance (see Table 3). BMI, Host density (proxy) and habitat
Discussion
The study’s findings showed that the prevalence (76.8%) of chig-
ger infestation on small mammal hosts was comparable to pre-
vious reports in other areas of Thailand, e.g. 81.8% of small
mammals from a rural village of an ethnic minority in Chiang-
mai Province (Rodkvamtook et al., 2013), 88.9% of small mam-
mals from military training sites in Chonburi Province (Rod-
kvamtook et al., 2018), and 23.8–95% of small mammals from
the nation-wide surveys (Coleman et al., 2003; Takhampunya
et al., 2018; Chaisiri et al., 2019). The prevalence was higher
than 33% reported in rats from Tak Province (Takhampunya

© 2021 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12531
 Chiggers from Bangkok public parks 9

habitats outside urban area (where biodiversity is limited in

Fig. 4. Principal component analysis (PCA) of habitat characteristics
and their distribution among the selected seven public parks across
Bangkok, with the two first axes of the PCA explaining 61.17% of the
variance. [Colour figure can be viewed at wileyonlinelibrary.com].
et al., 2018) and 44.1% in rats from orchards in Nonthaburi
Province (Frances et al., 1999), suggesting that urban public
parks provide a suitable habitat for chigger species to survive
since they exploit the available hosts to maintain their population
in such a big city. Our findings are corroborated by a previ-
ous study in South Africa, which showed a higher prevalence
of chigger infestation on rodents in urban settings than in agri-
cultural areas and natural habitats (Matthee et al., 2020). In con-
trast, greater chigger diversity was reported in biodiversity-rich
urban), i.e. rodents living in forest, mountainous landscape, and
upland and lowland agricultural areas (Peng et al., 2018; Chaisiri
et al., 2019; Matthee et al., 2020). This trend perfectly sup-
ports our findings of only two chigger species: L. deliense and
A. indica recovered from small mammals in Bangkok’s urban
public parks.
The study revealed that host attributes and extrinsic environ-
mental factors influenced the abundance of chigger infestation in
mammalian hosts found in urban public parks. Adult hosts har-
boured a significantly higher number of infested chiggers than
younger ones; this is because adults live longer and roam fur-
ther, searching for food. With more activity and mobility, they
have a better chance for increased exposure to ectoparasites and
chigger foci in the environment than juvenile animals (Webber
et al., 2015; Matthee et al., 2020). Another consideration is that
animals with a larger body mass or BMI provide a larger sur-
face area for different ectoparasite species to feed on (Fernandes
et al., 2015).
Extrinsic environmental factors could also play an important
role in the abundance of chigger infestation on animal hosts.
Unlike endoparasites that generally live inside their host, and
whose abundance and species composition tend to vary due to
host intrinsic factors (e.g. immunity and competition for nutri-
ents from the host), ectoparasites live outside the fur or skin of
the host’s body. As such, internal host factors and off-host envi-
ronment conditions affect parasite abundance (Morand, 2015).
There was a significant effect of habitat characteristics in the
public parks (i.e. composite habitat variable, including area and
perimeter of each park, and the proportion of land use classi-
fication inside and around the parks) on chigger abundance in
the small mammal hosts. For example, we found the highest
chigger abundance in mammal hosts from VP. The park pos-
sesses the largest area and perimeter, located in Bangkok suburb,
and surrounded by open fields, e.g. cropland and paddy fields.
In contrast, the parks with lower chigger abundance (e.g. ST,

Table 2. The top five generalized linear model (GLM, with negative binomial distribution) testing for intrinsic and extrinsic variables on chigger
abundance in small mammals from the public parks in Bangkok Metropolitan area. The best model (in bold) is selected for further discussion.

Model Dependent variable–independent variables K Log-like AICc ΔAICc Wr

1 Chigger abundance − BMI*** + Host density 4 −747.45 2009.47 0 0.0731

(proxy) + Habitat composite + Distance to open field***
2 Chigger abundance − BMI*** + Host density (proxy) + Habitat 3 −752.99 2009.57 0.1 0.0694
composite
3 Chigger abundance − BMI*** + Host density (proxy) + Habitat 4 −751.96 2010.17 0.7 0.0515
composite + Distance to human building
4 Chigger abundance − BMI** + Host density (proxy) + Habitat 5 −746.02 2010.22 0.75 0.0502
composite + Distance to human building + Distance to open
field***
5 Chigger abundance–Sex + BMI*** + Host density 5 −747.08 2011.53 2.06 0.0261
(proxy) + Habitat composite + Distance to open field***

Akaike’s Information Criterion corrected for sample size (AICc) was used for model selection after 500 computed models. The initial model for AICc
selection was Chigger abundance − BMI + Sex + Maturity + Host density (proxy) + Habitat composite + Distance to human building + Distance to
water body + Distance to open field + Distance to shaded trees. All the selected models were fitted with host taxonomy as a random effect. K, the
number of estimated variables; Log-like, maximized value of the logarithm of the likelihood function; ΔAICc, the difference between AICc value of
a given model and the model with minimum AICc and W r = Akaike weights. Analysis of deviance (type II test) significant level for each variable is
indicated as following: *P = 0.05–0.01; **P = 0.01–0.001 and ***P < 0.001.

© 2021 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12531
10 S. A. Wulandhari et al.

Table 3. The final generalized linear model (GLM) (with negative et al. (2018) that abundance of the low-host specific ectopara-
binomial distribution) showing significant factors for chigger abundance sites (e.g. L. deliense and A. indica chiggers in our case) tends
on small mammals from the public parks across the Bangkok Metropoli- to decrease with an increasing urbanization gradient. Because
tan area (GLM with negative binomial distribution) chiggers are living most of their lifespan off the host in the
environment, their survival and fitness could largely depend
Variance
inflation on the environmental niche. They will not succeed in a higher
Parameters/effects Chi-square P-value factor level of disturbed habitat by urbanization. In addition to these
environmental factors, previous studies revealed that abundance
BMI*** 14.015 <0.001 1.024 and diversity of chiggers are largely influenced by climatic fac-
Host density (proxy) 0.405 0.524 1.011
tors, such as seasonality, temperature and humidity (reviewed
Habitat composite 1.412 0.234 1.066
Distance to open field*** 11.001 <0.001 1.037 by Elliott et al., 2019). Optimal temperature and humidity are
Observation 164 important for chigger activity and development, which also
Log-likelihood −747.45 determines chigger abundance. In tropical and temperate zones,
AICc 2009.47 high chigger diversity tends to be found in warmer or drier con-
Model power (power analysis) 0.876 ditions comparing to wet or cold season (Chaisiri et al., 2019;
The goodness of fit/coefficient of 0.999 Matthee et al., 2020).
determination (R2 )
Screening Orientia spp. bacteria, the causative agent of scrub
Selection of the model used Akaike’s Information Criterion corrected for typhus in vectors and maintenance hosts in the environment, is
sample size (AICc). Analysis of deviance table (type II Wald Chi-square an important strategy to understand disease transmission better
tests), significant level for each variable is indicated as following: and alert the potential risk of exposure (Kuo et al., 2015).
*P = 0.05–0.01; **P = 0.01–0.001 and ***P < 0.001. Although we intensively screened for Orientia spp. in chiggers
and small mammal hosts from Bangkok public parks using
TB and LP) are rather small in size (except the LP) and located standardized molecular methods, the bacteria were not detected
closer to Bangkok’s heart with dense buildings, human accom- from these samples. Chiggers and the mammalian hosts in these
modation and streets. urban parks may not carry Orientia spp., or the population
We also found that animals with high chigger abundance may harbour a very small amount of the pathogen. Previous
were significantly associated with open field habitat (usually publications documented that a higher risk of scrub typhus
peridomestic). This finding could be explained by Maaz exposure is associated with a landscape characterized by forest

Fig. 5. Effect plots of the four explicative variables after best model fitting with generalized linear model to estimate abundance of chigger infestation on
small mammals from public parks across Bangkok. Confidence intervals (95%) are shown in grey. Marks on the X-axis represent individual observations.
[Colour figure can be viewed at wileyonlinelibrary.com].

© 2021 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12531

and peridomestic habitats, e.g. fields, agricultural lands, fallows
in the vicinity of a forested area and high elevated land (Chaisiri
et al., 2017; Wangrangsimakul et al., 2020). Non-occurrence
of Orientia in the present study may be explained by Bangkok
landscape characteristics, itself as the capital city is located in
the large central plain (1–2 m elevation) of the country with
no elevated land or natural forest within the radius of 50 km.
There is a clear difference in landscape prone scrub typhus
compared to the two studies of urban scrub typhus incidences
in Guangzhou, China (Wei et al., 2014) and Seoul, South Korea
(Park et al., 2015). In Guangzhou city, there was a report of a
human scrub typhus outbreak in Haizhu District between 2006
and 2012. The district is a short distance (<10 km) to the south
of Baiyun Mountain (427 m in elevation). In Seoul, urban scrub
typhus cases were reported between 2010 and 2013. The capital
city of South Korea is surrounded by mountain peaks, e.g. the
Bukhan Mountain to the north and the Gwanak Mountain to
the south, within distances of around 8 km and 12 km from
the city centre, respectively. The majority of the human cases
were related to or near the Gwanak Mountain activities, e.g.
trekking, gardening and harvesting agricultural products (Park
et al., 2015).
It can be hypothesized that lack of biodiversity-rich areas
such as natural habitat or preserve forest (particularly on the
mountainous landscape) in Bangkok may limit the abundance
and diversity of chiggers and probably affect Orientia bacte-
ria’s ecological niche and transmission (particular horizontally).
This hypothesis has been postulated by Chaisiri et al. (2019),
with the evidence that (1) chigger diversity in murid rodents
from Thailand gradually decreased towards urbanization and
(2) scrub typhus incidence was found to be positively corre-
lated with chigger species richness. This finding is in line with
the low disease incidence in Bangkok; only 20 and 31 cases of
scrub typhus were documented during 2018 and 2019, respec-
tively (Report 506: Bureau of Epidemiology, Ministry of Pub-
lic Health, Thailand). However, we cannot conclude that pub-
lic parks in Bangkok are safe from scrub typhus because the
important chigger vector species, L. deliense and A. indica have
been recently found in the public parks. The former species, L.
deliense, is the most dominant scrub typhus vector in the tropi-
cal region of Asia and Pacific islands, widely spread in India,
South China, Taiwan, Thailand, Vietnam, Malaysia, Philip-
pines, New Guinea and other countries (Stekolnikov, 2013; Lv
et al., 2018). The latter species is a generalist, broad host range
widely found throughout Thailand and some other countries in
the Asia Pacific region (Fernandes and Kulkarni, 2003; Chaisiri
et al., 2019). Ascoschoengastia indica was one of over 45 chig-
ger species harbouring O. tsutsugamushi, along with the follow-
ing examples: Blankaartia acuscutellaris, Cheladonta ikaoen-
sis, Eutrombicula wichmanni, Gahrliepia saduski, Helenicula
miyagawai, L. akamushi, L. arenicola, L. chiangraiensis, L.
deliense, L. fletcheri, L. fuji, L. gaohuense, L. imphalum, L.
intermedium, L. kitasatoi, L. orientale, L. pallidum, L. palpale,
L. pavlovskyi, L. scutellare, Neotrombicula japonica, Schoen-
gastiella ligula and Walchia pacifica (Frances et al., 1999; San-
tibáñez et al., 2015; Elliott et al., 2019).
The present study was conducted under a cross-sectional
approach but did not cover the seasonal effect. Accordingly, fur-
ther studies should be designed to cover both the wet and dry
© 2021 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12531
Chiggers from Bangkok public parks 11
periods (or a kind of longitudinal study for a couple of years) to
determine the effect of seasonal variation on chigger abundance,
diversity and Orientia infection. Our results show no evidence of
O. tsutsugamushi infection in chiggers and rodent tissues after
conventional nested PCR experiments, other molecular detec-
tion methods may probably offer higher sensitivity, e.g. quan-
titative PCR is suggested for bacterial investigation in future
studies (Jiang et al., 2004). Although the chigger specimens
are very tiny and contain relatively low biomass (i.e. genetic
material), the potential issue of PCR inhibition in O. tsutsuga-
mushi detection cannot be overlooked, particularly when work-
ing with pooled samples. We also suggest that high-throughput
sequencing technology, e.g. amplicon sequencing or metage-
nomics, could be alternatively applied to improve the bacterium
Orientia’s detection and reveal some other potential pathogens
carried by chiggers. In addition, Orientia serological detection
of the maintenance hosts could also help to determine history
of exposure and may help investigating scrub typhus risk in the
study areas.
Our research outcomes provide a basis for future studies in
this neglected field of vector-borne disease in an urban setting.
With our results, the high prevalence of chiggers, as well as the
occurrence of important scrub typhus vector species in public
parks of Bangkok, should provide a message to the Office of
Public Parks, the Bangkok Metropolitan Administration and
other public health sectors to develop a plan to control vectors
and prevent disease outbreaks.
Acknowledgements
We would like to gratefully thank the Environment Department,
Bangkok Metropolitan Administration (Director of the Environ-
ment Department, Mrs. Wullaya Wattanarat and the Director of
Bangkok Public Park Office, Mrs. Arom Wongmaha) for grant-
ing a permission to work in the area of Bangkok public parks. We
would like to deliver special appreciations also to the staffs from
the seven public parks for their kind cooperation and facilitation
during the fieldworks conducted at their properties. Financial
support for the project was granted by the Office of the Higher
Education Commission and the Thailand Research Fund (Grant
no. MRG6180023). Funding for M.Sc. in Tropical Medicine of
Wulandhari from the Malaria Consortium’s Dr. Sylvia Meek
Scholarship for Entomology. Also, we would like to acknowl-
edge the Faculty of Tropical Medicine - Animal Care and Use
Committee, Mahidol University (FTM-ACUC) for an informa-
tive guideline and consultation on development of the animal
research protocols. We thank Warren, R. from ENAGO (https://
university.enago.com/mahidol-university/) for English editing
throughout the manuscript (Reference No. MAHDLW-151).
All authors declare that we have no conflicts of interest
regarding the content in this article.
Author contributions
Conceptualization: Chaisiri K.; Morand S.; Sumruayphol S.;
Sonthayanon P. Supervision: Sumruayphol S.; Chaisiri K.;
Sonthayanon P. Sample and data acquisition: Wulandhari S.A.;
12 S. A. Wulandhari et al.
Paladsing Y.; Saesim W.; Charoennitiwat V.; Kumlert R.,
Chaisiri K. Data curation: Wulandhari S.A.; Charoennitiwat
V.; Chaisiri K. Formal analysis and interpretation: Wulandhari
S.A.; Sonthayanon P.; Morand S.; Sumruayphol S.; Chaisiri K.
Manuscript writing—original draft: Wulandhari S.A.; Chaisiri
K. Manuscript writing—review and edit: Wulandhari S.A.; Pal-
adsing Y.; Saesim W.; Charoennitiwat V.; Sonthayanon P.; Kum-
lert R.; Morand S.; Sumruayphol S.; Chaisiri K.
Data availability statement
All DNA sequence data from the present study will be avail-
able in the nucleotide database on NCBI website following an
embargo from the date of publication. Also, other data that sup-
port the findings of this study are available from the correspond-
ing author upon reasonable request.
References
Abarca, K., Martínez-Valdebenito, C., Angulo, J. et al. (2020) Molecular
description of a novel Orientia species causing scrub typhus in Chile.
Emerging Infectious Diseases, 26, 2148–2156.
Aplin, K., Brown, P., Jacob, J., Krebs, C. & Singleton, G. (2003)
Field Methods for Rodent Studies in Asia and the Indo-Pacific, p.
220. ACIAR Monograph No. 100, Australian Centre for International
Agricultural Research, Canberra.
Audy, J.R. (1961) The ecology of scrub typhus. Studies in Disease
Ecology (ed. by J.M. May), pp. 389–432. Hafner Publishing Company
Inc., New York.
Burnham, K.P. & Anderson, D.R. (2002) Model Selection and Multi-
model Inference: a Practical Information-Theoretic Approach, 2nd
edn, p. 488. Springer International Publishing, New York.
Chaisiri, K., Stekolnikov, A.A., Makepeace, B.L. & Morand, S. (2016)
A revised checklist of chigger mites (Acari: Trombiculidae) from
Thailand, with the description of three new species. Journal of
Medical Entomology, 53, 321–342.
Chaisiri, K., Cosson, J.F. & Morand, S. (2017) Infection of rodents by
Orientia tsutsugamushi, the agent of scrub typhus, in relation to land
use in Thailand. Tropical Medicine and Infectious Diseases, 2, 53.
Chaisiri, K., Gill, C.A., Stekolnikov, A.A. et al. (2019) Ecological
and microbiological diversity of chigger mites, including vectors
of scrub typhus on small mammals across stratified habitats in
Thailand. Animal Microbiome, 1, 18. https://doi.org/10.1186/s42523-
019-0019-x.
Chakraborty, S. & Sarma, N. (2017) Scrub typhus: an emerging threat.
Indian Journal of Dermatology, 62, 478–485.
Coleman, R.E., Monkanna, T., Linthicum, K.J. et al. (2003) Occurrence
of Orientia tsutsugamushi in small mammals from Thailand. Ameri-
can Journal of Tropical Medicine and Hygiene, 69, 519–524.
Core Team, R. (2019) R: a Language and Environment for Statistical
Computing. R Foundation for Statistical Computing, Vienna.
Dohany, A.L., Phang, O.W. & Rapmund, G. (1977) Chigger (Acarina:
Trombiculidae) surveys of the west coast beaches of Sabah and
Sarawak. The Southeast Asian Journal of Tropical Medicine and
Public Health, 8, 200–206.
Elliott, I., Pearson, I., Dahal, P., Thomas, N.V., Roberts, T. & Newton,
P.N. (2019) Scrub typhus ecology: a systematic review of Orientia
in vectors and hosts. Parasites & Vectors, 12, 513. https://doi.org/10
.1186/s13071-019-3751-x.
© 2021 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12531
Fernandes, S. & Kulkarni, S.M. (2003) Studies on the Trombiculid Mite
Fauna of India, p. 539. Zoological Survey of India, Kolkata.
Fernandes, F.R., Cruz, L.D., Linhares, A.X. & Von Zuben, C.J. (2015)
Effect of body size on the abundance of ectoparasitic mites on the wild
rodent Oligoryzomys nigripes. Acta Parasitol, 60, 515–524.
Folmer, O., Black, M., Hoeh, W., Lutz, R. & Vrijenhoek, R. (1994) DNA
primers for amplification of mitochondrial cytochrome c oxidase
subunit I from diverse metazoan invertebrates. Molecular Marine
Biology and Biotechnology, 3, 294–299.
Frances, S.P., Watcharapichat, P., Phulsuksombati, D. & Tanskul, P.
(1999) Occurrence of Orientia tsutsugamushi in chiggers (Acari:
Trombiculidae) and small animals in an orchard near Bangkok,
Thailand. Journal of Medical Entomology, 36, 449–453.
Herbreteau, V., Jittapalapong, S., Rerkamnuaychoke, W., Chaval, Y.,
Cosson, J.F. & Morand, S. (2011) Protocols for Field and Laboratory
Rodent Studies, p. 46. Kasetsart University Press, Bangkok.
Izzard, L., Fuller, A., Blacksell, S.D. et al. (2010) Isolation of a novel
Orientia species (O. chuto sp. nov.) from a patient infected in Dubai.
Journal of Clinical Microbiology, 48, 4404–4409.
Jiang, J., Chan, T.-C., Temenak, J.J., Dasch, G.A., Ching, W.-M. &
Richards, A.L. (2004) Development of a quantitative real-time poly-
merase chain reaction assay specific for Orientia tsutsugamushi. The
American Journal of Tropical Medicine and Hygiene, 70, 351–356.
Kelly, D.J., Fuerst, P.A., Ching, W.M. & Richards, A.L. (2009) Scrub
typhus: the geographic distribution of phenotypic and genotypic
variants of Orientia tsutsugamushi. Clinical Infectious Diseases, 15,
203–230.
Kim, Y.S., Lee, H.J., Chang, M., Son, S.K., Rhee, Y.E. & Shim, S.K.
(2006) Scrub typhus during pregnancy and its treatment: a case series
and review of the literature. American Journal of Tropical Medicine
and Hygiene, 75, 955–959.
Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. (2018)
MEGA X: molecular evolutionary genetics analysis across computing
platforms. Molecular Biology and Evolution, 35, 1547–1549.
Kumlert, R., Chaisiri, K., Anantatat, T. et al. (2018) Autofluorescence
microscopy for paired-matched morphological and molecular identifi-
cation of individual chigger mites (Acari: Trombiculidae), the vectors
of scrub typhus. PLoS One, 13, e0193163. https://doi.org/10.1371/
journal.pone.0193163.
Kuo, C.C., Lee, P.L., Chen, C.H. & Wang, H.C. (2015) Surveillance of
potential hosts and vectors of scrub typhus in Taiwan. Parasites &
Vectors, 8, 611.
Lakshana, P. (1973) A checklist of the trombiculid mites of Thailand
(Prostigmata: Trombiculidae). United States Army Medical Compo-
nent, South East Asia Treaty Organization, Bangkok 44p.
Lv, Y., Guo, X.G. & Jin, D.C. (2018) Research Progress on Leptotrom-
bidium deliense. Korean Journal of Parasitology, 56, 313–324.
Maaz, D., Krücken, J., Blümke, J. et al. (2018) Factors associated with
diversity, quantity and zoonotic potential of ectoparasites on urban
mice and voles. PLoS One, 13, e0199385.
Masakhwe, C., Linsuwanon, P., Kimita, G. et al. (2018) Identification
and characterization of Orientia chuto in trombiculid chigger mites
collected from wild rodents in Kenya. Journal of Clinical Microbiol-
ogy, 56, e01124–e01118.
Matthee, S., Stekolnikov, A.A., Van der Mescht, L., Froeschke, G. &
Morand, S. (2020) The diversity and distribution of chigger mites
associated with rodents in the south African savanna. Parasitology,
147, 1038–1047.
Moniuszko, H. & Makol, J. (2016) Host-parasite association in trombi-
culid mites (Actinotrichida: Trombiculidae) of temperate zone—the

case of Hirsutiella zachvatkini (Schluger, 1948); are we dealing with
prolonged contact with the host? Parasites & Vectors, 9, 61.
Morand, S. (2015) (macro-) evolutionary ecology of parasite diversity:
from determinants of parasite species richness to host diversification.
International Journal for Parasitology: Parasites and Wildlife, 4,
80–87.
Nadchatram, M. & Dohany, A.L. (1974) A Pictorial Key to the
Subfamilies, Genera and Subgenera of Southeast Asian Chiggers
(Acari, Prostigmata, Trombiculidae), p. 67. Institute Penyelidikan
Perubatan, Kuala Lumpur.
Pagès, M., Chaval, Y., Herbreteau, V. et al. (2010) Revisiting the
taxonomy of the Rattini tribe: a phylogeny-based delimitation of
species boundaries. BMC Evolutionary Biology, 10, 184.
Paladsing, Y., Boonsri, K., Saesim, W. et al. (2020) Helminth fauna
of small mammals from public parks and urban areas in Bangkok
metropolitan with emphasis on community ecology of infection in
synanthropic rodents. Parasitology Research, 119, 3675–3690.
Park, S.W., Ha, N.Y., Ryu, B. et al. (2015) Urbanization of scrub
typhus disease in South Korea. PLoS Neglected Tropical Diseases,
9, e0003814. https://doi.org/10.1371/journal.pntd.0003814.
Peng, P.Y., Guo, X.G., Jin, D.C. et al. (2018) Landscapes with differ-
ent biodiversity influence distribution of small mammals and their
ectoparasitic chigger mites: a comparative study from Southwest
China. PLoS One, 13, e0189987. https://doi.org/10.1371/journal.pone
.0189987.
Rodkvamtook, W., Gaywee, J., Kanjanavanit, S. et al. (2013) Scrub
typhus outbreak, northern Thailand, 2006–2007. Emerging Infectious
Diseases, 19, 774–777.
Rodkvamtook, W., Kuttasingkee, N., Linsuwanon, P. et al. (2018)
Scrub typhus outbreak in Chonburi Province, Central Thailand, 2013.
Emerging Infectious Diseases, 24, 361–365.
Roh, J.Y., Song, B.G., Park, W.I. et al. (2014) Coincidence between
geographical distribution of Leptotrombidium scutellare and scrub
typhus incidence in South Korea. PLoS One, 9, e113193. https://doi
.org/10.1371/journal.pone.0113193.
Rozsa, L., Reiczigel, J. & Majoros, G. (2000) Quantifying parasites in
samples of hosts. Journal of Parasitology, 86, 228–232.
Santibáñez, P., Palomar, A.M., Portillo, A., Santibáñez, S. & Oteo, J.A.
(2015) The role of chiggers as human pathogens. In: Samie, A. (Eds),
An Overview of Tropical Diseases. InTechOpen. 173–202. https://doi
.org/10.5772/61978.
© 2021 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12531
Chiggers from Bangkok public parks 13
Shim, S.K., Choi, E.N., Yu, K.O. et al. (2009) Characterisation of
Orientia tsutsugamushi genotypes from wild rodents and chigger
mites in Korea. Clinical Microbiology and Infection, 15, 311–312.
Stekolnikov, A.A. (2013) Leptotrombidium (Acari: Trombiculidae) of
the world. Zootaxa, 3728, 1–173.
Strickman, D., Tanskul, P., Eamsila, C. & Kelly, D.J. (1994) Prevalence
of antibodies to rickettsiae in the human population of suburban
Bangkok. American Journal of Tropical Medicine and Hygiene, 51,
149–153.
Takhampunya, R., Tippayachai, B., Promsathaporn, S. et al. (2014)
Characterization based on the 56-Kda type-specific antigen gene of
Orientia tsutsugamushi genotypes isolated from Leptotrombidium
mites and the rodent host post-infection. American Journal of Tropical
Medicine and Hygiene, 90, 139–146.
Takhampunya, R., Korkusol, A., Promsathaporn, S. et al. (2018) Hetero-
geneity of Orientia tsutsugamushi genotypes in field-collected trom-
biculid mites from wild-caught small mammals in Thailand. PLoS
Neglected Tropical Diseases, 12, e0006632.
Wangrangsimakul, T., Elliott, I., Nedsuwan, S. et al. (2020) The esti-
mated burden of scrub typhus in Thailand from national surveillance
data (2003-2018). PLoS Neglected Tropical Diseases, 14, e0008233.
https://doi.org/10.1371/journal.pntd.0008233.
Webber, Q.M.R., McGuire, L.P., Smith, S.B. & Willis, C.K.R. (2015)
Host behaviour, age and sex correlate with ectoparasite prevalence
and intensity in a colonial mammal, the little brown bat. Behaviour,
152, 83–105.
Wei, Y., Luo, L., Jing, Q. et al. (2014) A city park as a potential
epidemic site of scrub typhus: a case–control study of an outbreak
in Guangzhou, China. Parasites & Vectors, 7, 513.
Weitzel, T., Aylwin, M., Martínez-Valdebenito, C. et al. (2018) Imported
scrub typhus: first case in South America and review of the literature.
Tropical diseases, Travel Medicine and Vaccines, 4, 10. https://doi
.org/10.1186/s40794-018-0070-8.
Yoshimoto, T. & Yoshimoto, T. (2019) Scrub typhus in Japan. American
Journal of Clinical Microbiology and Antimicrobials, 2, 1042.
Zhang, W.Y., Wang, L.Y., Ding, F. et al. (2013) Scrub typhus in mainland
China, 2006-2012: the need for targeted public health interventions.
PLoS Neglected Tropical Diseases, 7, e2493. https://doi.org/10.1371/
journal.pntd.0002493.
Accepted 10 May 2021