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

104 © 2014 The Royal Entomological Society

 Rickettsiae from wild rats and cat fleas 105

rodents (Rattus and Tupaia species) and Haemaphysalis ticks in Table 1. Molecular detection of rickettsiae from rats and animal
Malaysia (Tay et al., 1996, 1998), attempts to isolate rickettsiae ectoparasites using gltA PCR assay in this study.
have not been successful. A high antibody prevalence to R. honei
(TT118 strain) spotted fever group rickettsia has been reported No. (%) rat positive
Sample for rickettsial DNA
in febrile patients in rural and urban areas in Malaysia (Tay et al.,
2000, 2003); however, information is lacking concerning the Wild rats (n = 95)
animal reservoirs and vectors of this rickettsial species. Rick- Rattus diardii (n = 58) 9 (15.5)
ettsia honei has also been reported in patients from Thailand i. PM1 (male, spleen)
(Jiang et al., 2005), a neighbouring country of Malaysia. Other ii. PM2 (female, spleen, liver)
than R. honei, a rickettsial species closely related to R. raoul- iii. BB7 (female, kidney∗, spleen)
iv. DK2 (male, kidney∗, spleen, liver)
tii has been reported from an Amblyomma tick from Thailand
v. KL2 (male, liver, spleen)
(Doornbos et al., 2013). Using specific polymerase chain reac- vi. KL8 (female, spleen)
tion (PCR) assays, R. felis DNA has been detected in fleas in our vii. KL18 (female, kidney)
previous study (Mokhtar & Tay, 2011). viii. PP26 (female, heart)
To identify risk, and emergence of new rickettsial organisms, ix. PP34 (female, heart)
mapping of animal hosts and arthropod vectors in a particular Rattus norvegicus (n = 37) 4 (10.8)
area is essential. Rats are competent reservoir hosts of several i. DK6 (female, kidney, spleen, liver)
zoonotic pathogens and have been used as a sentinel organism ii. PP33 (male, kidney)
for rickettsial studies (Psaroulaki et al., 2010; Kuo et al., 2012). iii. PP35 (male, heart)
Furthermore rats can bring diseases to humans by increasing iv. PP36 (male, heart)
Ectoparasite, animal host (n = 589)
exposure to potentially infected ticks, mites and fleas, and
Laelaps spp. mite, rats (n = 63) 0 (0)
together, they play important roles as amplifying hosts and Haemaphysalis bispinosa ticks, cats (n = 7) 0 (0)
transporters of rickettsiae. Hence, this study was performed with Felicola subrostratus lice, cats (n = 73) 0 (0)
the aim to assess wild rats and animal ectoparasites as potential Rhipicephalus sanguineus ticks, dogs 0 (0)
reservoirs for rickettsioses in Malaysia. (n = 165)
A total of 95 wild rats (58 Rattus diardii and 37 R. norvegicus) Heterodoxus spiniger lice, dogs (n = 104) 0 (0)
caught at the wet markets in Kuala Lumpur and Pulau Pinang Ctenocephalides felis fleas, cats and dogs 57 (32.2)
from January 2008 to December 2011 were included in this (n = 177)
investigation. Rodents were caught using live traps, and anaes- ∗OmpA was amplified.
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 membrane protein gene (ompB) (Roux & Raoult, 2000). The
homogenized using a pestle and mortar in phosphate-buffered gltA amplicon from R. honei (strain TT118) was cloned into
saline. DNA was extracted from 20 mg of animal tissues using a the PCR2.1-TOPO T/A plasmid vector and used as a positive
QIAamp® DNA Mini Kit (Qiagen, Hilden, Germany). Ectopar- control for PCR assays. For detection of R. felis, DNA extracts
asites were collected from the rats and pooled for screening of from R. felis-positive fleas from a previous study (Mokhtar &
rickettsiae. In addition, ticks and lice collected from cats and Tay, 2011) were used as positive controls. Purification of ampli-
dogs from three sampling sites in Malaysia (Kuala Nerang, Pen- cons was carried out using a LaboPass PCR Purification Kit
dang and Ampang) during January to August 2010 were investi- (Cosmos Genetech, Seoul, Korea) as described by the manu-
gated in this study (Table 1). A total of 177 fleas collected from facturer. Amplicons were sequenced using a BigDye termina-
several locations in Kuala Lumpur and Selangor (Hulu Lan- tor cycle sequencing kit (Applied Biosystems, Foster City, CA,
gat and Gombak), Malaysia were also included in this study. U.S.A.) on an ABI-3730 Genetic Analyzer (Applied Biosys-
The ectoparasites were kept in micro-centrifuge tubes contain- tems, Foster City, CA, U.S.A.) using their respective PCR
ing 70% alcohol for identification by entomologists. A protocol primers. Sequences were imported into the BioEdit sequence
described by Alekseev et al. (2001) was used to prepare a DNA alignment program and inspected manually (Hall, 1999). The
template from the ectoparasites. Briefly, each ectoparasite was neighbour-joining method of MEGA software (version 5.1) was
immersed in 100 𝜇L of 0.7 m ammonium hydroxide and boiled employed to determine the phylogenetic status of the isolates
for 20 min. The extracted DNA was then resuspended in 10 𝜇L (Tamura et al., 2011). The reliability of different phylogenetic
of sterile distilled water prior to amplification. groupings was evaluated using bootstrap tests (1000 bootstrap
A PCR assay utilizing primer pair CS-78 and CS-323 was used replicates). For the phylogenetic study, gltA sequences of the
to amplify the rickettsial citrate synthase (gltA) gene fragment type strains were retrieved from the Genbank database.
(Labruna et al., 2004). The positive samples were further sub- Table 1 shows the detection of rickettsiae in the wild rats
jected to amplification using (a) primers CS-239 and CS-1069, investigated in this study. A total of 13 (13.7%) wild rats were
targeting a 830-bp fragment of the citrate synthase (gltA-1) positive for rickettsiae by gltA PCR assay. Positive specimens
gene fragment (Labruna et al., 2004); (b) primers Rr190_70p were derived from the tissue homogenates of the livers (n = 4),
and Rr_602n, targeting a 532-bp fragment of the rickettsial hearts (n = 4), kidneys (n = 5) and spleens (n = 7). Sequence
190-kDa outer membrane protein gene (ompA) (Regnery et al., analysis of the gltA gene fragments (265 nucleotides) from five
1991) and (c) primers 120-M59f and 120-807, targeting an rats (PM1, PM2, BB7, DK2 and DK6) was performed using
approximately 860-bp fragment of the rickettsial 135-kDa outer bidirectional sequences. All demonstrated 100% similarity with
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, 28 (Suppl. 1), 104–108
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 very close related Rickettsia species (as indicated by the low
Accession numbers: KF963597-KF963601). Greater variation bootstrapping values in the dendrogram).
(ranging from 98.4% to 100% compared with those of R. None of the gltA-positive rat specimens were positive for
honei/R. conorii/R. raoultii) was noted for gltA sequences amplification using primers targeting gltA-1 and ompB. OmpA
derived from the remaining eight rats (based on one-directional amplicons were obtained from two kidney homogenates only.
sequences). Figure 1 is a dendrogram showing the clustering of The ompA sequences (BB7 and DK2, GenBank Accession
the rickettsiae detected from our rat specimens with published R. numbers: KF963604 and KF963605) were identical to Rickettsia
honei/R. conorii/R. raoultii type strains. Further differentiation sp. TCM1 strain (GenBank Accession number: AB359459)
of rickettsiae to the species level is not possible owing to which is closely related to R. japonica, the causative agent
the high sequence similarity shared amongst the taxonomically of Japanese spotted fever (Mahara, 2006; Takada et al., 2009)
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, 28 (Suppl. 1), 104–108
 Rickettsiae from wild rats and cat fleas 107

which has recently been isolated from a H. hystricis tick seroprevalence of typhus and spotted fever group rickettsiae
in Thailand (Takada et al., 2009). At this point, we are not observed in our previous serosurveys (Tay et al., 2000, 2003).
clear as to why the results of gltA sequence analysis for the The absence of R. typhi (causative agent of murine typhus) in
rickettsiae detected from the kidney homogenates of two rats this study reflects the absence or very low frequency of R. typhi
were not identical with those of ompA sequence analysis. Mixed in fleas from this area.
rickettsial infections are suspected in these rats. Additionally, in In conclusion, the findings of this study suggest the presence
a multi-template condition as for the tissue homogenates in this of two different spotted fever group rickettsiae (tentatively
study, the difference in the amplification results can be caused by identified as ‘R. honei/R. conorii/R. raoultii’ and Rickettsia sp.
PCR bias whereby certain genes are amplified more efficiently TCM1) among wild rats in Malaysia. As the rats were caught
compared with others (Kanagawa, 2003). Probe hybridization from market areas, it is possible for the infection to spill over
may be useful to confirm the presence of two different rickettsiae into the human population through the ectoparasite vectors that
from the same tissue specimen. We have attempted to infect feed on both rats and humans. In addition, owing to the frequent
Vero cells with six rickettsiae-positive rat specimens, however, feeding behaviour and mobility of fleas, rapid spreading of
no rickettsiae were detected from the infected cells after the first flea-borne R. felis to human populations is possible. Close
passage using gltA PCR assays. association with domestic animals, i.e. dogs and cats and their
Table 1 shows the results of the molecular detection of ectoparasites, can increase human risk of acquiring rickettsial
rickettsiae from animal ectoparasites in this study. The ectopara- infections. Further research to isolate and characterize the
sites collected from the wild rats in this study were mainly spiny rickettsial organism is important to improve our understanding
rat mites (Laelaps spp.), none of which (processed in 26 pools) about human rickettsioses in Malaysia. In addition, control
were positive for rickettsiae by gltA PCR assay. Rickettsial DNA of rodent and flea populations may be able to reduce the
was also not detected from the DNA extracts of Rhipicephalus transmission of rickettsioses in this part of the world.
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. Acknowledgements
subrostratus lice as vectors in the transmission of rickettsioses.
None of these lice were positive for rickettsial DNA in this study. This study was funded by grants HIR/E000013-20001 (subpro-
Rickettsia felis was detected from 32.2% of 177 C. felis fleas gramme 4), PV086/2011B and RP013/2012A from University
in this study. This detection rate is higher than that of 2.9% of Malaya, Kuala Lumpur, Malaysia. We acknowledge the assis-
reported in a previous study in Malaysia (Mokhtar & Tay, 2011), tance given by the technical assistance given by S. Samulung,
but similar to the high infection rates in wild cat flea popula- J. Nagappan, N. H. Mohd Isa, N. Shahimin, B. Douadi and K.
tions in South America (varying from 13.5 to 90%, Labruna S. Chai.
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- Author’s declaration of interests
strated two R. felis genotypes. The gltA amplicons from 56 of
57 flea specimens (represented by specimen HL2a, GenBank No competing interests have been declared.
Accession number: KF963602) demonstrated 100% sequence
similarity with that of Rickettsia sp. RF2125 (GenBank Acces-
sion number: AF516333), which has been reported in fleas References
in a previous study (Mokhtar & Tay, 2011). The gltA ampli-
Alekseev, A.N., Dubinina, H.V., Van De Pol, I. & Schouls, L.M.
con from a single flea specimen (HL15c, GenBank Accession
(2001) Identification of Ehrlichia spp. and Borrelia burgdorferi in
number: KF963603) showed 100% sequence similarity with
Ixodes ticks in the Baltic Regions of Russia. Journal of Clinical
that of R. felis Rf31 (GenBank Accession number: AF516331)
Microbiology, 39, 2237–2242.
which was first described in fleas from the Thai-Myanmar
Doornbos, K., Sumrandee, C., Ruang-Areerate, T., Baimai, V.,
border (Parola et al., 2003). Amplification of a longer cit-
Trinachartvanit, W. & Ahantarig, A. (2013) Rickettsia sp. closely
rate synthase gene (gltA-1) sequences of the flea specimens related to Rickettsia raoultii (Rickettsiales: Rickettsiaceae) in an
HL2a (GenBank Accession number: KF963606) and HL15c Amblyomma helvolum (Acarina: Ixodidae) tick from a Varanus
(GenBank Accession number: KF963607) demonstrated 97.7% salvator (Squamata: Varanidae) in Thailand. Journal of Medical
(708/725 nt) and 97.2% (705/725 nt) similarity when compared Entomology, 50, 217–220.
with that of R. felis type strain URRWXCal2 (GenBank Acces- Hall, T.A. (1999) BioEdit: a user friendly biological sequence alignment
sion number: CP000053), respectively. OmpB was only ampli- editor and analysis program for Windows 95/98/NT. Nucleic Acids
fied from the flea specimen HL15c (GenBank Accession num- Symposium Series, 41, 95–98.
ber: KF963608), and the sequence was 95.0% (756/796 nt) sim- Jiang, J., Sangkasuwan, V., Lerdthusnee, K., Sukwit, S., Chuenchitra,
ilar to that of R. felis type strain URRWXCal2. T. & Rozmajzl, P.J. (2005) Human infection with Rickettsia honei,
Figure 1 shows the phylogenetic placement of the R. felis Thailand. Emerging Infectious Diseases, 11, 1473–1475.
gltA sequences obtained from our flea specimens with published Kanagawa, T. (2003) Bias and artifacts in multitemplate polymerase
R. felis sequences. As R. felis cross-reacts with typhus and chain reactions (PCR). Journal of Bioscience and Bioengineering, 96,
spotted fever group rickettsiae, it may contribute to the high 317–323.

© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, 28 (Suppl. 1), 104–108
108 S. T. Tay et al.

Kuo, C.C., Huang, J.L., Lin, T.E. & Wang, H.C. (2012) Detec- Regnery, R.L., Spruill, C.L. & Plikaytis, B.D. (1991) Genotypic identi-
tion of Rickettsia spp. and host and habitat associations of fleas fication of rickettsiae and estimation of intraspecies sequence diver-
(Siphonaptera) in eastern Taiwan. Medical and Veterinary Entomol- gence for portions of two rickettsial genes. Journal of Bacteriology,
ogy, 26, 341–350. 173, 1576–1589.
Labruna, M.B., Whitworth, T., Horta, M.C. et al. (2004) Rickettsia Reif, K.E., Stout, R.W., Henry, G.C., Foil, L.D. & Macaluso, K.R.
species infecting Amblyomma cooperi ticks from an endemic area for (2008) Prevalence and infection load dynamics of Rickettsia felis in
Brazilian spotted fever in the state of Sao Paolo, Brazil. Journal of actively feeding cat fleas. PLoS One, 3, e2805.
Clinical Microbiology, 42, 90–98. Roux, V. & Raoult, D. (2000) Phylogenetic analysis of members of
Labruna, M.B., Ogrzewalska, M., Moraes-Filho, J., Lepe, P., Gallegos, the genus Rickettsia using the gene encoding the outer-membrane
J.L. & López, J. (2007) Rickettsia felis in Chile. Emerging Infectious protein rOmpB (ompB). International Journal of Systematic and
Diseases, 13, 1794–1795. Evolutionary Microbiology, 50, 1449–1455.
Lane, A.M., Shaw, M.D., McGraw, E.A. & O’Neill, S.L. (2005) Rovery, C., Brouqui, P. & Raoult, D. (2008) Questions on Mediterranean
Evidence of a spotted fever-like rickettsia and a potential new vector spotted fever a century after its discovery. Emerging Infectious
from northeastern Australia. Journal of Medical Entomology, 42, Diseases, 14, 1360–1367.
918–921. Takada, N., Fujita, H., Kawabata, H., Ando, S., Sakata, A., Takano,
Mahara, F. (2006) Rickettsioses in Japan and the Far East. Annals of the A. & Chaithong, U. (2009) Spotted fever group Rickettsia sp.
New York Academy of Sciences, 1078, 60–73. closely related to Rickettsia japonica, Thailand. Emerging Infectious
Mediannikov, O., Socolovschi, C., Edouard, S. et al. (2013) Common Diseases, 15, 610–611.
epidemiology of Rickettsia felis infection and malaria, Africa. Emerg- Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S.
ing Infectious Diseases, 19, 1775–1783. (2011) MEGA5: molecular evolutionary genetics analysis using max-
Mokhtar, A.S. & Tay, S.T. (2011) Molecular detection of Rickettsia felis, imum likelihood, evolutionary distance, and maximum parsimony
Bartonella henselae, and B. clarridgeiae in fleas from domestic dogs methods. Molecular Biology and Evolution, 28, 2731–2739.
and cats in Malaysia. American Journal of Tropical Medicine and Tay, S.T., Ho, T.M. & Rohani, M.Y. (1996) Use of hemolymph test for
Hygiene, 85, 931–933. detection of rickettsiae in Malaysian ticks. Southeast Asian Journal
Parola, P. (2011) Rickettsia felis: from a rare disease in the USA to a of Tropical Medicine and Public Health, 27, 642–644.
common cause of fever in sub-Saharan Africa. Clinical Microbiology Tay, S.T., Kaewanee, S., Ho, T.M., Rohani, M.Y. & Devi, S. (1998)
and Infection, 17, 996–1000. Serological evidence of natural infection of wild rodents (Rattus spp
Parola, P., Sanogo, O.Y., Lerdthusnee, K. et al. (2003) Identification of and Tupaia glis) with rickettsiae in Malaysia. Southeast Asian Journal
Rickettsia spp. and Bartonella spp. in fleas from the Thai-Myanmar of Tropical Medicine and Public Health, 29, 560–562.
border. Annals of the New York Academy of Sciences, 990, 173–181. Tay, S.T., Ho, T.M., Rohani, M.Y. & Devi, S. (2000) Antibodies to
Parola P, Paddock, C.D., Socolovschi, C. et al. (2013) Update on Orientia tsutsugamushi, Rickettsia typhi and spotted fever group rick-
tick-borne rickettsioses around the world: a geographic approach. ettsiae among febrile patients in rural areas of Malaysia. Transac-
Clinical Microbiology Reviews, 26, 657–702. tions of the Royal Society of Tropical Medicine and Hygiene, 94,
Pérez-Osorio, C.E., Zavala-Velázquez, J.E., Arias León, J.J. & 280–284.
Zavala-Castro, J.E. (2008) Rickettsia felis as emergent global Tay, S.T., Kamalanathan, M. & Rohani, M.Y. (2003) Antibody preva-
threat for humans. Emerging Infectious Diseases, 14, 1019–1023. lence of Orientia tsutsugamushi, Rickettsia typhi and TT118 spotted
Psaroulaki, A., Antoniou, M., Toumazos, P. et al. (2010) Rats as fever group rickettsiae among Malaysian blood donors and febrile
indicators of the presence and dispersal of six zoonotic microbial patients in the urban areas. Southeast Asian Journal of Tropical
agents in Cyprus, an island ecosystem: a seroepidemiological study. Medicine and Public Health, 34, 165–170.
Transactions of the Royal Society of Tropical Medicine and Hygiene,
104, 733–739. Accepted 22 January 2014

© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, 28 (Suppl. 1), 104–108