Bennett, S.

Eremeeva, M.E.

Hayes, E.K.

Hu, R.

Karpathy, S.E.

Kerr, N.

Krueger, L.

Moore, W.

Tilzer, A.

Velten, R.K.

Williams, A.M.

Elsevier - Publisher Connector 

Detection of Rickettsia felis and Rickettsia typhi in an area of California endemic for murine typhus 

10.1111/j.1469-0691.2008.02140.x
Detection of Rickettsia felis and Rickettsia typhi in an area of California
endemic for murine typhus
S. E. Karpathy1, E. K. Hayes1, A. M. Williams1, R. Hu2, L. Krueger3, S. Bennett3, A. Tilzer3,
R. K. Velten3, N. Kerr4, W. Moore5 and M. E. Eremeeva1
1Rickettsial Zoonoses Branch, National Center for Zoonotic, Vector-Borne and Enteric Diseases, Centers
for Disease Control and Prevention, Atlanta, GA, 2California Department of Public Health, Vector-
Borne Disease Section, Ontario, CA, 3Orange County Vector Control District, Garden Grove, CA, USA,
4Long Beach Department of Health and Human Services, Vector Management Program, Long Beach,
CA, USA, and 5Long Beach Department of Health and Human Services, Animal Control Division, Long
Beach, CA, USA
INTRODUCTION
Murine typhus is a mild febrile illness caused by
the Gram-negative bacteriumRickettsia typhi. It has
usually been associated with a transmission cycle
involving rats, Rattus norvegicus and Rattus rattus,
and the oriental rat ﬂea, Xenopsylla cheopis [1]. A
second transmission cycle has been described
involving peridomestic opossums, Didelphis vir-
giniana, and their associated ﬂea, Ctenocephalides
felis [2].Another rickettsial agent,Rickettsia felis, has
also been associated with C. felis [3], and has been
implicated in a murine typhus-like disease of
humans, cat ﬂea rickettsiosis [4]. Current serolog-
ical diagnostic assays are unable to differentiate
between these two infections, partly due to the
limited availability of R. felis antigens, suggesting
that some human cases of cat ﬂea rickettsiosis are
being misdiagnosed as murine typhus. We have
developed a multiplex TaqMan based PCR assay
that facilitates the concurrent detection and differ-
entiation of R. typhi and R. felis DNA for vector
surveillance and for an acute-stage diagnosis of the
disease.
METHODS
The PCR primers gltAF.287 (5¢-GAT TTT TTA GAA GTG
GCA TAT TTG-3¢) and gltAR.552 (5¢-GGK ATY TTA GCW
ATC ATT CTA ATA GC-3¢) were selected to amplify a 265-bp
fragment of the citrate synthase gene, gltA, from all rickettsial
species. The primers were tested using genomic DNA from 14
isolates of Rickettsia prowazekii, eight isolates of R. typhi, and 57
isolates of spotted fever group Rickettsia. A single band of the
expected size was ampliﬁed from all the DNA samples.
Speciﬁc species TaqMan probes were designed to permit
the discrimination of R. typhi and R. felis from all other
Rickettsia. Locked nucleic acid bases, shown in parentheses,
were incorporated into the probes to raise the melting
temperatures and to increase speciﬁcity [5] (R. typhi gltA,
5¢-CalRed 610-TT (T)A (C)TA (C)A (A)AG (A)T (T)G (C)T (C)
A-BHQ2; R. felis gltA, 5¢-Cy-5-CTA (C)GG A(G)A ATT (G)C-
C A-BHQ3). The speciﬁcity of the TaqMan probes was tested
using two isolates of R. prowazekii, two isolates of R. typhi, and
19 spotted fever group rickettsial isolates.
All quantitative PCR reactions were conducted in a 20-lL
ﬁnal reaction volume using the Brilliant qPCR kit (Stratagene,
La Jolla, CA, USA) in an iCycler iQ (BioRad, Hercules, CA,
USA). Each reaction consistedof 2 lLof 10· core reactionbuffer,
4.5 mM MgCl2, 300 nM each primer, 400 nM R. typhi gltA
probe, 600 nM R. felis gltA probe, 800 lM combined dNTPs,
1.25 U of SureStart Taq DNA polymerase, and 4 lL of the DNA
sample. All reactions were run with a non-template control in
which 4 lL of sterilewaterwas substituted for theDNAsample.
The cycle parameters consisted of 10 min of enzyme activation
at 95C followed by 50 cycles of a 30 s denaturation at 95C, 30 s
at 62C to allow the primers and probes to anneal to the target
DNA, and 1 min at 72C for extension. The reaction was
concluded with a ﬁnal extension at 72C for 10 min.
Positive control plasmids were constructed by using prim-
ers gltAF.287 and gltAR.552 to amplify the target fragments
from R. typhi strain Wilmington and R. felis strain LSU. These
fragments were cloned into pCR2.1 using the TA cloning kit
(Invitrogen, Carlsbad, CA, USA), following the manufacturer’s
instructions. The construction of the clone was conﬁrmed via
sequencing.
RESULTS
Using control plasmid DNA, the detection limit
for this assay was veriﬁed using the control
Corresponding author and reprint requests: Marina E. Ereme-
eva, Rickettsial Zoonoses Branch, Mail Stop G-13, National
Center for Zoonotic, Vector-borne and Enteric Diseases,
Centers for Disease Control and Prevention, 1600 Clifton Road
NE, Atlanta, GA 30333, USA
E-mail: meremeeva@cdc.gov
The ﬁndings and conclusions described in this manuscript are
those of the authors and do not necessarily represent the views
of the Centers for Disease Control and Prevention and the
Department of Health and Human Services.
No conﬂicts of interest declared.
 2009 No claim to original government works, CMI, 15 (Suppl. 2), 218–219
plasmids and was determined to be between 1
and 2.5 copies ⁄ reaction for both R. typhi and
R. felis.
This assay was applied to detect the presence of
R. typhi and R. felis DNA in ﬂeas from California
(Table 1). Fleas of the same species and from the
same animal were combined into pools of two
ﬂeas, and minimal infection rates were estab-
lished. In total, 1271 pools were screened; 463
pools (36.4%) were positive for R. felis, 17 pools
(1.3%) were positive for R. typhi, and 21 pools
(1.7%) were positive for both agents. This assay
was also used to screen ﬂeas from Hawaii
(X. cheopis) and from the Democratic Republic of
Congo (Pulex irritans, Xenopsylla brasiliensis, Tunga
penetrans, C. felis, and Echidnophaga gallinacea).
The Taqman assay has also been used to screen
opossum tissues and opossum and rat blood
samples from California. No evidence of rickett-
siemia was obtained in 133 animals tested; how-
ever, R. felis DNA was detected in liver and ⁄ or
kidney samples from three of 16 opossums tested.
Selected amplicons from each collection site were
sequenced to conﬁrm the identity of the DNA
species detected, and respective amplicons were
>98% identical to either R. typhi Wilmington or
R. felis URRWXCal2.
ACKNOWLEDGEMENTS
We wish to thank K. McCaustland for advice concerning
primer and probe design, and G. Dasch for thoughtful
discussions. This research was supported in part by an
appointment of S. Karpathy to the Research Participation
Program at the Centers for Disease Control and Prevention
administered by the Oak Ridge Institute for Science and
Education through an interagency agreement between the US
Department of Energy and CDC.
REFERENCES
1. Azad AF. Epidemiology of murine typhus. Annu Rev
Entomol 1990; 35: 553–569.
2. Williams SG, Sacci JB Jr, Schriefer ME et al. Typhus and
typhuslike rickettsiae associated with opossums and their
ﬂeas in Los Angeles County, California. J Clin Microbiol
1992; 30: 1758–1762.
3. Adams JR, Schmidtmann ET, Azad AF. Infection of
colonized cat ﬂeas, Ctenocephalides felis (Bouche´), with a
rickettsia-like microorganism. Am J Trop Med Hyg 1990; 43:
400–409.
4. Schriefer ME, Sacci JB Jr, Dumler JS, Bullen MG, Azad AF.
Identiﬁcation of a novel rickettsial infection in a patient
diagnosed with murine typhus. J Clin Microbiol 1994; 32:
949–954.
5. Kumar R, Singh SK, Koshkin AA, Rajwanshi VK, Meldg-
aard M, Wengel J. The ﬁrst analogues of lna (locked nucleic
acids): phosphorothioate-lna and 2¢-thio-lna. Bioorg Med
Chem Lett 1998; 8: 2219–2222.
Table 1. PCR detection of Rickettsia in California ﬂea pools
Flea species
Total ﬂeas
tested
Flea pools
tested
Minimal infection
rate (%)
Ctenocephalides felis 2222 1200 21.6
Pulex irritans 106 55 11.3
Xenopsylla cheopis 4 4 50
Diamanus montanus 4 3 50
Echidnophaga gallinacea 15 8 26.7
Leptosylla segnis 1 1 0
Total 2352 1271 21.3
Karpathy et al. Rickettsia felis and R. typhi in California 219
 2009 No claim to original government works, CMI, 15 (Suppl. 2), 218–219


file:///data/core-remote/dit/data/elsevier/pdf/d2e/aHR0cDovL2FwaS5lbHNldmllci5jb20vY29udGVudC9hcnRpY2xlL3BpaS9zMTE5ODc0M3gxNDYzNTUzOQ==.pdf

Detection of Rickettsia felis and Rickettsia typhi in an area of California endemic for murine typhus

Abstract

Similar works

Full text

Available Versions

Elsevier - Publisher Connector

Elsevier - Publisher Connector