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Emerging and re-emerging infectious diseases con- found worldwide. Indonesia has one of the highest
tinue to contribute to morbidity and mortality in de- prevalence levels of antibodies to R. typhi among peo-
veloping nations (Azad et al. 1997). Rickettsial dis- ple in the world (Richards et al. 2002). Studies in Java
eases are endemic in Indonesia, but because of lack of found that people living in urban areas had a higher
diagnosis and agent-speciÞc diagnostic assays, the dis- prevalence of antibodies to R. typhi than less urban
ease burden is unknown across the archipelago (Rich- areas with lower concentrations of people (Richards
ards et al. 1995). These pathogens are zoonotic (the et al. 2002). Flea-borne spotted fever (cat ßea typhus)
vertebrate reservoir is usually rodent species) and are caused by Rickettsia felis has been identiÞed in Cteno-
spread to humans by infected ectoparasites (inverte- cephalides felis found in North America, Europe, Af-
brate hosts). Xenopsylla cheopis, an important plague rica, Asia, Australia, and New Zealand and in X. cheopis
vector, is also a vector of several rickettsial pathogens in Java, Indonesia (Jiang et al. 2006). Scrub typhus
(Traub et al. 1978, Azad and Traub 1989, Jiang et al. (Orientia tsutsugamushi) has been recognized in In-
2006). Murine typhus (also known as endemic typhus donesia since World War II, where it was a major
and ßeaborne typhus), caused by Rickettsia typhi, is nonbattle injury for military forces (GrifÞths 1945).
Richards et al. (2003) found evidence of spotted fever
group (SFG) rickettsiae (SFGR) infection in human
Authors, as employees of the U.S. Government, conducted the work
as part of their ofÞcial duties. Title 17 U.S.C. ¤ 105 provides that
residents of Gag Island, Indonesia, located northwest
“copyright protection under this title is not available for any work of of the island of Irian Jaya in Eastern Indonesia.
the United States Government.” Title 17 U.S.C ¤ 101 deÞnes a U.S. To determine the presence and prevalence of
Government work as a work prepared by an employee of the U.S. rickettsia pathogens in ectoparasites, we surveyed
Government as part of the personÕs ofÞcial duties.
1 United States Naval Medical Research Unit 2, Jakarta, Indonesia. live captured, small mammals trapped in lowland
2 Corresponding author: Navy Environmental Preventive, Medi- and highland villages on four islands in Indonesia. In
cine Unit 2, 1887 Powhatan Street, Norfolk, VA 23511 (e-mail: this study, we report results from a survey of X.
kbarbara@bvwireless.net). cheopis found on small mammals in four islands of
3 National Institute of Health Research and Development, Health
Materials and Methods land (⬎100 m asl) villages on four islands of Indonesia
between April 2007 and May 2008. All aspects of an-
Site Description. Fleas were collected as part of
imal use were conducted using protocols approved by
larger rodent-borne disease serosurvey focusing on
Rickettsia spp. Collection sites are shown in Fig. 1. The the NAMRU-2 Institutional Animal Care and Use
Þrst collection was conducted in Loji village, Sim- Committee and the National Institute of Health Re-
penan subdistrict (lowland: 8 m above sea level [asl]; search and Development, Indonesian Ministry of
07⬚03⬘12”S/106⬚32⬘50”E) and Cihamerang village, Health.
Kabandungan subdistrict (highland: 910 m asl; Small mammals were collected using Sherman
06⬚47⬘31”S/106⬚34⬘35”E), Sukabumi District, West (H. B. Sherman Traps, Tallahassee, FL) and Toma-
Java 21Ð30 April 2007. The second was conducted hawk (Tomahawk Live Trap, Tomahawk, WI) style
15Ð21 May 2007 in Air Manis village, Padang District traps baited with roasted coconut. Tomahawk and
(lowland: 10 m asl; 00⬚59⬘09”S/100⬚21⬘39”E) and Koto Sherman traps were set out in the late afternoon for
Rantang village, Agam District (highland: 890 m asl; three nights at each lowland and highland site for a
00⬚14⬘45”S/100⬚21⬘00”E), West Sumatra. The third total of six trap nights at each location (four lowland,
Þeld trip was conducted in 22Ð28 August 2007 in Ma- four highland). Traps were checked the following
lalayang village, Manado District (lowland: 35 m asl; morning, and those with animals were returned to the
01⬚26.909⬘ N/124⬚49.430⬘ E) and Kinilow village, To- processing site. Trapped animals were killed (or se-
mohon District (highland: 730 m asl; 01⬚21.747⬘ dated) and identiÞed. Ectoparasites were removed
N/124⬚49.941⬘ E), North Sulawesi. The Þnal collection from each animal by vigorously brushing to dislodge.
was conducted in 23Ð29 May 2008 in Bukit Bangkirai, After brushing, each animal was examined for any
Kutai Kartanegara (highland: 110 m asl; 01⬚01.706⬘ remaining ectoparasites by searching through the pel-
S/116⬚51.972⬘ E) and Manggar Baru, Balikpapan Dis- age with Þne forceps. Ectoparasites collected were
trict (lowland: 7 m asl; 01⬚12.939S/116⬚58.630⬘ E), East placed into micro Eppendorf tubes and snap frozen in
Kalimantan. liquid nitrogen until identiÞed and tested by polymer-
Mammal and Flea Collections. Animals and ßeas ase chain reaction (PCR) in the laboratory. Rattus,
were collected from lowland (⬍50 m asl) and high- Suncus, and Mus species were killed at the Þeld sites,
November 2010 BARBARA ET AL.: RICKETSIAL INFECTIONS IN INDONESIAN ECTOPARASITES 1175
and all other species were sedated and then released Ninety-eight percent (98.1%) of ßeas collected were
after sample collection. Fleas were identiÞed as X. X. cheopis. X. cheopis were collected from Rattus tan-
cheopis or Nosopsyllus spp. by using standard taxo- ezumi, Rattus exulans, Rattus norvegicus, and Suncus
nomic keys for ectoparasites of commensal rodents murinus (Table 1). Eight small mammal species (R.
(Mahadevan et al. 1969). Flea load (number of ßeas on exulans, R. norvegicus, R. tanezumi, Rattus timoanicus,
an individual animal) and ßea indexes (average num- Rattus whiteheadi, Leopoldamys sabanus, Maxomys ra-
ber of ßeas per animal) were calculated for each mam- jah, and Mus musculus), one shrew species (S. muri-
mal species. Voucher specimens are deposited at the nus), two squirrels (Sundasciurus lowii and Rhinosciu-
United States Naval Medical Research Unit 2 labora- rus laticaudatus), one civet (Viverra tangalunga), and
Table 1. Mammal species collected, total number of individuals collected, total Xenopsylla cheopis collected with flea indexes, and
range of number of X. cheopis collected during small mammal surveys conducted during 2007–2008 in a) West Sumatra; b) North
Sulawesi; c) East Kalimantan; and d), West Java, Indonesia
Lowland Highland
Mammal species Total no. Total Flea Total no. Total Flea
Range Range
individuals X. cheopis index individuals X. cheopis index
a) West Sumatra
Rattus tanezumi 51 3 0.06 0Ð2 43 87 2.02 0Ð20
Leopoldamys sabanus 0 0 0 0 3 0 0 0
cation. R. tanezumi was the most frequently collected Rickettsia spp. Testing. Thirty-seven pools of X.
mammal at all sites, except the East Kalimantan low- cheopis were tested. Six pools were found positive for
land site, where R. norvegicus was the most frequently Rickettsia spp. (Table 2); four (10.8%) R. typhi, one
collected mammal. Total ßea load (combined data (2.7%) R. felis, and one (2.7%) codetection of R. felis
lowland and highland) was greatest on R. tanezumi, and a SFGR. The detection of a tick-borne rickettsia
except at the Kalimantan site, where the greatest num- was conÞrmed to be a member of the SFG of rickett-
ber of ßeas were collected on R. norvegicus. siae by sequencing regions of the ompB (149 bp) and
Table 2. Rickettsia-positive Xenopsylla cheopis collected during small mammal surveys conducted in 2007–2008 at locations in West
Java, West Sumatra, North Sulawesi, and East Kalimantan, Indonesia; data shown derived from X. cheopis pools (n ⴝ 37)
a
Target gene htrA conserved rickettsia 17-kD antigen gene.
b
Target sequence speciÞc for tick-borne SFGR.
c
Target gene OmpB region speciÞc for R. typhi.
d
Target gene OmpB region speciÞc for R. felis.
e
Primers speciÞc for ampliÞcation of OmpB gene fragment of typhus group (TG).
f
Primers speciÞc for ampliÞcation of OmpB gene fragment of SFG.
g
Visible, but weak band.
November 2010 BARBARA ET AL.: RICKETSIAL INFECTIONS IN INDONESIAN ECTOPARASITES 1177
ompA (1,328 bp) genes. The ampliÞed genes were inant species in the Kalimantan lowland site, with R.
found to be 100% identical with Rickettsia sp. TwKM01 tanezumi and R. exulans predominate in the highland
ompB gene (EF219464), and 100% identical with Rick- site. R. tanezumi and S. murinus appear to be the
ettsia spp. TwKM01 ompA gene (EF219467) from rhi- primary host for R. typhi- and R. felis-infected X. cheo-
picephalus haemaphysaloides. The detection of R. felis pis. All ßeas that tested positive for R. typhi, R. felis, or
was conÞrmed by sequencing regions of the r. felis a SFGR were collected in the lowland sites, except for
ompB gene Rf1396 F/Rf1524R (120 bp). The ampliÞed one R. typhi-infected X. cheopis found at the highland
genes were found to be 97% identical with R. felis site in West Java on R. tanezumi.
ompB gene (AF. 182279). In one pool of X. cheopis collected from East Kali-
Health Surveillance Center. The opinions or assertions ex- real-time polymerase chain reaction assay speciÞc for
pressed herein are the private views of the authors and are Orientia tsutsugamushi. Am. J. Trop. Med. Hyg. 70: 351Ð
not to be construed as representing those of the Department 356.
of Defense or the Department of the Navy. Jiang, J., P. J. Blair, J. G. Olson, E. Stromdahl, and A. L.
Richards. 2005. Development of a duplex quantitative
real-time PCR assay for the detection of tick-borne spot-
References Cited ted fever group rickettsiae and Rickettsia rickettsii. Int.
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Azad, A. F., and R. Traub. 1985. Transmission of murine Bangs, and A. L. Richards. 2006. Rickettsia felis in Xe-