Medical and Veterinary Entomology (2014) 28 (Suppl.

1), 104–108

S H O R T C O M M U N I C AT I O N

Identification of rickettsiae from wild rats and cat fleas

in Malaysia
S. T. T A Y 1 , A. S. M O K H T A R 1 , K. C. L O W 2 , S. N. M O H D Z A I N 3 ,
J. J E F F E R Y 4 , N. A B D U L A Z I Z 4 and K. L. K H O 1
1
Department of Medical Microbiology, Tropical Infectious Diseases Research and Education Centre, Faculty of Medicine, University
of Malaya, Kuala Lumpur, Malaysia, 2 Laboratory Animal Resource Unit, Faculty of Medicine, Universiti Kebangsaan Malaysia,
Kuala Lumpur, Malaysia, 3 Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia and
4
Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia

Abstract. Rickettsioses are emerging zoonotic diseases reported worldwide. In spite

of the serological evidence of spotted fever group rickettsioses in febrile patients
in Malaysia, limited studies have been conducted to identify the animal reservoirs
and vectors of rickettsioses. This study investigated the presence of rickettsiae in the
tissue homogenates of 95 wild rats and 589 animal ectoparasites. Using PCR assays
targeting the citrate synthase gene (gltA), rickettsial DNA was detected in the tissue
homogenates of 13 (13.7%) wild rats. Sequence analysis of the gltA amplicons showed
98.6–100% similarity with those of Rickettsia honei/R. conorii/R. raoultii (Rickettsiales:
Rickettsiaceae). Sequence analysis of outer membrane protein A gene (ompA) identified
Rickettsia sp. TCM1 strain from two rats. No rickettsia was detected from Laelaps
mites, Rhipicephalus sanguineus and Haemaphysalis bispinosa ticks, and Felicola
subrostratus lice in this study. R. felis was identified from 32.2% of 177 Ctenocephalides
felis fleas. Sequence analysis of the gltA amplicons revealed two genotypes of R. felis
(Rf31 and RF2125) in the fleas. As wild rats and cat fleas play an important role in the
enzoonotic maintenance of rickettsiae, control of rodent and flea populations may be
able to reduce transmission of rickettsioses in the local setting.

Key words. Cat fleas, reservoirs, rickettsioses, wild rats, Malaysia.

Rickettsiae are Gram-negative obligate intracellular bacteria
which have been associated with a variety of infections including
Rocky Mountain spotted fever, epidemic typhus, murine typhus,
Mediterranean spotted fever etc. Arthropods such as ticks,
fleas, mites and lice are important vectors for transmission of
rickettsioses. As the organisms are difficult to culture directly
from samples, molecular tools such as PCR and sequence
analysis have been used to facilitate the identification and
characterization of rickettsial species. To date, 26 rickettsial
species with validated and published names have been reported;
Rickettsia sibirica, R. heilongjiangensis, R. japonica, R. conorii,
R. honei, R. aeschlimannii, R. raoultii, R. slovaca, R. helvetica,
R. massiliae and R. monacensis are amongst those tick-borne
rickettsiae that have been reported in the Asia region (Parola
et al., 2013).
Rickettsia felis, the causative agent of flea-borne spotted
fever, has been recently recognized as an emerging global
health threat (Pérez-Osorio et al., 2008). First described in the
Ctenocephalides felis cat flea, R. felis has a wide cosmopolitan
distribution, detected from arthropods in > 20 countries and five
continents (Parola, 2011). As the clinical manifestations of R.
felis infections are non-specific and difficult to differentiate from
other causes of febrile disease such as murine typhus and dengue
(Pérez-Osorio et al., 2008), disease caused by R. felis infection
is potentially underdiagnosed. The importance of R. felis as a
common cause of febrile diseases in sub-Saharan rural Africa
has been recently documented (Mediannikov et al., 2013).
Current data on the prevalence and type of rickettsioses in
Malaysia are scarce. Although evidence is available of nat-
ural infection of spotted fever group rickettsiae in the wild

Correspondence: Sun T. Tay, Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Tel.: 03-79676676; Fax: 03-79676660; E-mail: tayst@um.edu.my

104 © 2014 The Royal Entomological Society


rodents (Rattus and Tupaia species) and Haemaphysalis ticks in
Malaysia (Tay et al., 1996, 1998), attempts to isolate rickettsiae
have not been successful. A high antibody prevalence to R. honei
(TT118 strain) spotted fever group rickettsia has been reported
in febrile patients in rural and urban areas in Malaysia (Tay et al.,
2000, 2003); however, information is lacking concerning the
animal reservoirs and vectors of this rickettsial species. Rick-
ettsia honei has also been reported in patients from Thailand
(Jiang et al., 2005), a neighbouring country of Malaysia. Other
than R. honei, a rickettsial species closely related to R. raoul-
tii has been reported from an Amblyomma tick from Thailand
(Doornbos et al., 2013). Using specific polymerase chain reac-
tion (PCR) assays, R. felis DNA has been detected in fleas in our
previous study (Mokhtar & Tay, 2011).
To identify risk, and emergence of new rickettsial organisms,
mapping of animal hosts and arthropod vectors in a particular
area is essential. Rats are competent reservoir hosts of several
zoonotic pathogens and have been used as a sentinel organism
for rickettsial studies (Psaroulaki et al., 2010; Kuo et al., 2012).
Furthermore rats can bring diseases to humans by increasing
exposure to potentially infected ticks, mites and fleas, and
together, they play important roles as amplifying hosts and
transporters of rickettsiae. Hence, this study was performed with
the aim to assess wild rats and animal ectoparasites as potential
reservoirs for rickettsioses in Malaysia.
A total of 95 wild rats (58 Rattus diardii and 37 R. norvegicus)
caught at the wet markets in Kuala Lumpur and Pulau Pinang
from January 2008 to December 2011 were included in this
investigation. Rodents were caught using live traps, and anaes-
thetized using an ether-charged chamber. Rats were dissected
and organs (kidney, liver, spleen and heart) were harvested asep-
tically and kept at −80 ∘ C prior to processing. The organs were
homogenized using a pestle and mortar in phosphate-buffered
saline. DNA was extracted from 20 mg of animal tissues using a
QIAamp® DNA Mini Kit (Qiagen, Hilden, Germany). Ectopar-
asites were collected from the rats and pooled for screening of
rickettsiae. In addition, ticks and lice collected from cats and
dogs from three sampling sites in Malaysia (Kuala Nerang, Pen-
dang and Ampang) during January to August 2010 were investi-
gated in this study (Table 1). A total of 177 fleas collected from
several locations in Kuala Lumpur and Selangor (Hulu Lan-
gat and Gombak), Malaysia were also included in this study.
The ectoparasites were kept in micro-centrifuge tubes contain-
ing 70% alcohol for identification by entomologists. A protocol
described by Alekseev et al. (2001) was used to prepare a DNA
template from the ectoparasites. Briefly, each ectoparasite was
immersed in 100 𝜇L of 0.7 m ammonium hydroxide and boiled
for 20 min. The extracted DNA was then resuspended in 10 𝜇L
of sterile distilled water prior to amplification.
A PCR assay utilizing primer pair CS-78 and CS-323 was used
to amplify the rickettsial citrate synthase (gltA) gene fragment
(Labruna et al., 2004). The positive samples were further sub-
jected to amplification using (a) primers CS-239 and CS-1069,
targeting a 830-bp fragment of the citrate synthase (gltA-1)
gene fragment (Labruna et al., 2004); (b) primers Rr190_70p
and Rr_602n, targeting a 532-bp fragment of the rickettsial
190-kDa outer membrane protein gene (ompA) (Regnery et al.,
1991) and (c) primers 120-M59f and 120-807, targeting an
approximately 860-bp fragment of the rickettsial 135-kDa outer
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, 28 (Suppl. 1), 104–108
Rickettsiae from wild rats and cat fleas 105
Table 1. Molecular detection of rickettsiae from rats and animal
ectoparasites using gltA PCR assay in this study.
No. (%) rat positive
Sample for rickettsial DNA
Wild rats (n = 95)
Rattus diardii (n = 58) 9 (15.5)
i. PM1 (male, spleen)
ii. PM2 (female, spleen, liver)
iii. BB7 (female, kidney∗, spleen)
iv. DK2 (male, kidney∗, spleen, liver)
v. KL2 (male, liver, spleen)
vi. KL8 (female, spleen)
vii. KL18 (female, kidney)
viii. PP26 (female, heart)
ix. PP34 (female, heart)
Rattus norvegicus (n = 37) 4 (10.8)
i. DK6 (female, kidney, spleen, liver)
ii. PP33 (male, kidney)
iii. PP35 (male, heart)
iv. PP36 (male, heart)
Ectoparasite, animal host (n = 589)
Laelaps spp. mite, rats (n = 63) 0 (0)
Haemaphysalis bispinosa ticks, cats (n = 7) 0 (0)
Felicola subrostratus lice, cats (n = 73) 0 (0)
Rhipicephalus sanguineus ticks, dogs 0 (0)
(n = 165)
Heterodoxus spiniger lice, dogs (n = 104) 0 (0)
Ctenocephalides felis fleas, cats and dogs 57 (32.2)
(n = 177)
∗OmpA was amplified.
membrane protein gene (ompB) (Roux & Raoult, 2000). The
gltA amplicon from R. honei (strain TT118) was cloned into
the PCR2.1-TOPO T/A plasmid vector and used as a positive
control for PCR assays. For detection of R. felis, DNA extracts
from R. felis-positive fleas from a previous study (Mokhtar &
Tay, 2011) were used as positive controls. Purification of ampli-
cons was carried out using a LaboPass PCR Purification Kit
(Cosmos Genetech, Seoul, Korea) as described by the manu-
facturer. Amplicons were sequenced using a BigDye termina-
tor cycle sequencing kit (Applied Biosystems, Foster City, CA,
U.S.A.) on an ABI-3730 Genetic Analyzer (Applied Biosys-
tems, Foster City, CA, U.S.A.) using their respective PCR
primers. Sequences were imported into the BioEdit sequence
alignment program and inspected manually (Hall, 1999). The
neighbour-joining method of MEGA software (version 5.1) was
employed to determine the phylogenetic status of the isolates
(Tamura et al., 2011). The reliability of different phylogenetic
groupings was evaluated using bootstrap tests (1000 bootstrap
replicates). For the phylogenetic study, gltA sequences of the
type strains were retrieved from the Genbank database.
Table 1 shows the detection of rickettsiae in the wild rats
investigated in this study. A total of 13 (13.7%) wild rats were
positive for rickettsiae by gltA PCR assay. Positive specimens
were derived from the tissue homogenates of the livers (n = 4),
hearts (n = 4), kidneys (n = 5) and spleens (n = 7). Sequence
analysis of the gltA gene fragments (265 nucleotides) from five
rats (PM1, PM2, BB7, DK2 and DK6) was performed using
bidirectional sequences. All demonstrated 100% similarity with
106 S. T. Tay et al.

55 Rat specimen PP26/PP33

50 Rat specimen PP34
43
Rat specimen KL18
Rat specimen PP36/KL2/KL8
R. honei RB (AF018074)
R. conorii NIAID Malish 7 (U59730)
13
R. raoultii strain Elanda23/95 (EU036985)
R. raoultii Khabarovsk (DQ365804)
Rat specimen PM1/PM2/DK2/DK6/BB7/PP35 (KF963597-KF963601)
R. massiliae Mtu 1 (U59719)
7
59 R. rhipicephali Burgdorfer 3-7-female 6 (U59721)

48 R. parkeri NIAID maculatum 20 (U59732)

40 R. sibirica 246 (U59734)
R. africae ESF5 (U59733)
10
9 R. raoultii strain Marne (DQ365803)
R. heilongjiangii 054 (AF178034)
41
R. japonica YM (U59724)
47
23 R. slovaca 13B (U59725)

R. montananensis (U74756)
28
66 R. rickettsii R (Bitterroot) (U59729)
R. aeschlimannii MC16 (U59722)
60
R. tamurae strain AT-1 (AF394896)
R. asiatica strain IO1 (AF394901)
57 36 R. helvetica C9P9 (U59723)

83 R. akari MK(Kaplan) (U59717)

R. australis Phillips (U59718)

99 Flea specimen HL2a (KF963602)

97 Rickettsia sp. RF2125 (AF516333)
94
R. felis URRWXCal2 (AF210692)
36
59 Flea specimen HL15c (KF963603)
64 R. felis Rf31 (AF516331)

R. prowazekii Breinl (M17149)

99 R. typhi Wilmington (U59714)
R. bellii 369L421 (U59716)
R. canadensis 2678 (U59713)

0.02

Fig. 1. Unrooted dendrogram showing the phylogenetic position of rickettsiae detected from rat and ectoparasite specimens in this study with other
published Rickettsia species (based on comparison of gltA sequences). Bootstrap values are indicated at nodes.

those of R. honei/R. conorii/R. raoultii (Table 1, GenBank
Accession numbers: KF963597-KF963601). Greater variation
(ranging from 98.4% to 100% compared with those of R.
honei/R. conorii/R. raoultii) was noted for gltA sequences
derived from the remaining eight rats (based on one-directional
sequences). Figure 1 is a dendrogram showing the clustering of
the rickettsiae detected from our rat specimens with published R.
honei/R. conorii/R. raoultii type strains. Further differentiation
of rickettsiae to the species level is not possible owing to
the high sequence similarity shared amongst the taxonomically
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, 28 (Suppl. 1), 104–108
very close related Rickettsia species (as indicated by the low
bootstrapping values in the dendrogram).
None of the gltA-positive rat specimens were positive for
amplification using primers targeting gltA-1 and ompB. OmpA
amplicons were obtained from two kidney homogenates only.
The ompA sequences (BB7 and DK2, GenBank Accession
numbers: KF963604 and KF963605) were identical to Rickettsia
sp. TCM1 strain (GenBank Accession number: AB359459)
which is closely related to R. japonica, the causative agent
of Japanese spotted fever (Mahara, 2006; Takada et al., 2009)

which has recently been isolated from a H. hystricis tick
in Thailand (Takada et al., 2009). At this point, we are not
clear as to why the results of gltA sequence analysis for the
rickettsiae detected from the kidney homogenates of two rats
were not identical with those of ompA sequence analysis. Mixed
rickettsial infections are suspected in these rats. Additionally, in
a multi-template condition as for the tissue homogenates in this
study, the difference in the amplification results can be caused by
PCR bias whereby certain genes are amplified more efficiently
compared with others (Kanagawa, 2003). Probe hybridization
may be useful to confirm the presence of two different rickettsiae
from the same tissue specimen. We have attempted to infect
Vero cells with six rickettsiae-positive rat specimens, however,
no rickettsiae were detected from the infected cells after the first
passage using gltA PCR assays.
Table 1 shows the results of the molecular detection of
rickettsiae from animal ectoparasites in this study. The ectopara-
sites collected from the wild rats in this study were mainly spiny
rat mites (Laelaps spp.), none of which (processed in 26 pools)
were positive for rickettsiae by gltA PCR assay. Rickettsial DNA
was also not detected from the DNA extracts of Rhipicephalus
sanguineus and H. bispinosa ticks, although these ticks have
been considered as potential vectors for rickettsial pathogens
including R. conorii (Lane et al., 2005; Rovery et al., 2008). So
far, no report is available on the importance of H. spiniger and F.
subrostratus lice as vectors in the transmission of rickettsioses.
None of these lice were positive for rickettsial DNA in this study.
Rickettsia felis was detected from 32.2% of 177 C. felis fleas
in this study. This detection rate is higher than that of 2.9%
reported in a previous study in Malaysia (Mokhtar & Tay, 2011),
but similar to the high infection rates in wild cat flea popula-
tions in South America (varying from 13.5 to 90%, Labruna
et al., 2007) and laboratory cat flea colonies in the United States
(varying from 43 to 100%, Reif et al., 2008). Sequence anal-
ysis of the gltA amplicons from our flea specimens demon-
strated two R. felis genotypes. The gltA amplicons from 56 of
57 flea specimens (represented by specimen HL2a, GenBank
Accession number: KF963602) demonstrated 100% sequence
similarity with that of Rickettsia sp. RF2125 (GenBank Acces-
sion number: AF516333), which has been reported in fleas
in a previous study (Mokhtar & Tay, 2011). The gltA ampli-
con from a single flea specimen (HL15c, GenBank Accession
number: KF963603) showed 100% sequence similarity with
that of R. felis Rf31 (GenBank Accession number: AF516331)
which was first described in fleas from the Thai-Myanmar
border (Parola et al., 2003). Amplification of a longer cit-
rate synthase gene (gltA-1) sequences of the flea specimens
HL2a (GenBank Accession number: KF963606) and HL15c
(GenBank Accession number: KF963607) demonstrated 97.7%
(708/725 nt) and 97.2% (705/725 nt) similarity when compared
with that of R. felis type strain URRWXCal2 (GenBank Acces-
sion number: CP000053), respectively. OmpB was only ampli-
fied from the flea specimen HL15c (GenBank Accession num-
ber: KF963608), and the sequence was 95.0% (756/796 nt) sim-
ilar to that of R. felis type strain URRWXCal2.
Figure 1 shows the phylogenetic placement of the R. felis
gltA sequences obtained from our flea specimens with published
R. felis sequences. As R. felis cross-reacts with typhus and
spotted fever group rickettsiae, it may contribute to the high
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, 28 (Suppl. 1), 104–108
Rickettsiae from wild rats and cat fleas 107
seroprevalence of typhus and spotted fever group rickettsiae
observed in our previous serosurveys (Tay et al., 2000, 2003).
The absence of R. typhi (causative agent of murine typhus) in
this study reflects the absence or very low frequency of R. typhi
in fleas from this area.
In conclusion, the findings of this study suggest the presence
of two different spotted fever group rickettsiae (tentatively
identified as ‘R. honei/R. conorii/R. raoultii’ and Rickettsia sp.
TCM1) among wild rats in Malaysia. As the rats were caught
from market areas, it is possible for the infection to spill over
into the human population through the ectoparasite vectors that
feed on both rats and humans. In addition, owing to the frequent
feeding behaviour and mobility of fleas, rapid spreading of
flea-borne R. felis to human populations is possible. Close
association with domestic animals, i.e. dogs and cats and their
ectoparasites, can increase human risk of acquiring rickettsial
infections. Further research to isolate and characterize the
rickettsial organism is important to improve our understanding
about human rickettsioses in Malaysia. In addition, control
of rodent and flea populations may be able to reduce the
transmission of rickettsioses in this part of the world.
Acknowledgements
This study was funded by grants HIR/E000013-20001 (subpro-
gramme 4), PV086/2011B and RP013/2012A from University
of Malaya, Kuala Lumpur, Malaysia. We acknowledge the assis-
tance given by the technical assistance given by S. Samulung,
J. Nagappan, N. H. Mohd Isa, N. Shahimin, B. Douadi and K.
S. Chai.
Author’s declaration of interests
No competing interests have been declared.
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Accepted 22 January 2014