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Molecular evidence supports the role of dogs as potential reservoirs for Rickettsia felis

Journal: Vector-Borne and Zoonotic Diseases

Manuscript ID: Draft

Manuscript Type: Original Research

Date Submitted by the Author:

n/a

Complete List of Authors: Hii, Sze Fui; The University of Queensland, School of Veterinary

Science Kopp, Steven; The University of Queensland, School of Veterinary Science Abdad, Mohammad; Australia Rickettsial Reference Laboratory Thompson, Mary; The University of Queensland, School of Veterinary Science O'Leary, Caroline; The University of Queensland, School of Veterinary Science Rees, Robert; Bayer Animal Health Traub, Rebecca; The University of Queensland, School of Veterinary Science

Keyword: Rickettsia felis

Abstract:

Rickettsia felis causes flea-borne spotted fever in humans worldwide. The cat flea, Ctenocephalides felis, serves as vector and reservoir host for this disease agent. To determine the role of dogs as potential reservoir hosts for spotted fever group rickettsiae, we screened blood from 100 pound dogs in Southeast Queensland by using a highly sensitive genus-specific PCR. Nine of the pound dogs were positive for rickettsial DNA and subsequent molecular sequencing confirmed amplification of R. felis. A high prevalence of R. felis in dogs in our study suggests that dogs may act as an important reservoir host for R. felis and as a potential source of human rickettsial infection.

Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801

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The Southeast Queensland region of Australia, showing the location of the major metropolitan area, Brisbane.

135x94mm (94 x 94 DPI)

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Phylogenetic tree obtained from neighbour-joining analysis of the ompB gene of Rickettsia sp.

detected in 9 pound dogs clustered with R. felis GQ385243. 226x170mm (150 x 150 DPI)

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Article title:

Molecular evidence supports the role of dogs as potential reservoirs for Rickettsia felis

Authors:

Sze Fui Hii1, Steven R. Kopp

1, Mohammad Y. Abdad

2, Mary F. Thompson

1, Caroline A.

O’Leary1, Robert L. Rees

3, Rebecca J. Traub

1

Authors affiliations:

1The University of Queensland, School of Veterinary Science, Gatton, Queensland, Australia;

2Australian Rickettsial Reference Laboratory, Geelong, Victoria, Australia;

3Bayer Animal

Health, Brisbane, Queensland, Australia

Disclaimers

The opinions expressed by authors contributing to this journal do not necessarily

reflect the opinions of the institutions with which the authors are affiliated.

Address correspondence to:

Sze Fui Hii

School of Veterinary Science, The University of Queensland, Gatton Campus, Queensland

4343, Australia

Email: sze.hii@uqconnect.edu.au

Running Title:

Rickettsia felis infection in dogs

Word counts

Text – 2960 words; Abstract – 108 words

The number of figures

2 figures

Keyword:

Rickettsia felis, dogs, Ctenocephalides felis, flea-borne spotted fever

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Abstract:

Rickettsia felis causes flea-borne spotted fever in humans worldwide. The cat flea,

Ctenocephalides felis, serves as vector and reservoir host for this disease agent. To determine

the role of dogs as potential reservoir hosts for spotted fever group rickettsiae, we screened

blood from 100 pound dogs in Southeast Queensland by using a highly sensitive genus-

specific PCR. Nine of the pound dogs were positive for rickettsial DNA and subsequent

molecular sequencing confirmed amplification of R. felis. A high prevalence of R. felis in

dogs in our study suggests that dogs may act as an important reservoir host for R. felis and as

a potential source of human rickettsial infection.

Text:

Introduction

Vector-borne diseases are a significant cause of mortality and morbidity in humans

and animals worldwide, and are currently recognised as a priority risk area by the World

Health Organisation. Vector-borne diseases have a devastating impact on health, and

emerging vector-borne diseases are increasingly being recognised. Characterisation of these

emerging diseases, including their vectors, reservoir hosts, prevalence and distribution is

important to allow development of integrated control programmes. One such group of

emerging vector borne disease agents are the Rickettsia, a group of obligate intracellular

bacteria responsible for a number of diseases in humans and animals.

Rickettsia felis has traditionally been grouped as a member of the spotted fever group

(SFG) of rickettsial organisms (Bouyer et al. 2001). Some researchers however, have

classified it as part of the ‘transitional’ group due to the presence of phenotypic and genetic

anomalies in R. felis, which include the presence of plasmids and its ability to serologically

cross-react with both typhus group (TG) and SFG rickettsiae (Gillespie et al. 2007).

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Rickettsia felis was first detected in the cat flea, Ctenocephalides felis (Adams et al. 1990),

and is an emerging human pathogen which causes flea-borne spotted fever or cat flea typhus

throughout the world. The first case of flea-borne spotted fever in humans was reported in

1994 in Texas in the United States of America (USA) (Schriefer et al. 1994). Since then, a

number of human cases have been reported worldwide including Spain (Oteo et al.

2006,Perez-Arellano et al. 2005), Germany (Richter et al. 2002), Mexico (Zavala-Velazquez

et al. 2000), Kenya (Richards et al. 2010), and Taiwan (Tsai et al. 2008). More recently, a

cluster of five patients were diagnosed with R. felis infection in Victoria, Australia (Williams

et al. In press 2010.). Clinical signs of human flea-borne spotted fever include pyrexia,

headache, malaise, myalgia, rash and eschar (Perez-Arellano et al. 2005, Richter et al. 2002).

These signs are very similar to those caused by related Rickettsia species, including R. typhi

(murine typhus) (Schriefer et al. 1994), R. rickettsii (Rocky Mountain spotted fever)(Chen et

al. 2008), R. conorii (Mediterranean spotted fever)(Mert et al. 2006), R. australis

(Queensland tick typhus) and Orientia tsutsugamushi (scrub typhus) (Unsworth et al. 2007).

The first detection of R. felis was from a laboratory flea colony in the USA (Adams et

al. 1990). Since then the distribution of R. felis infection in different flea species collected

from dogs and cats, as determined by molecular methods, has been found to be global , with

infection rates of 15% in New Zealand (Kelly et al. 2004), 31% in Brazil (Horta et al. 2006),

43.6% in Spain (Nogueras et al. 2010), 67.4% in the USA (Hawley et al. 2007) and 81% in

New Caledonia (Mediannikov et al. 2010).

The cat flea, C. felis, has been reported to be both the primary vector and reservoir of

R. felis (Reif et al. 2009). Vertical transmission through transovarial and transstadial

transmission of R. felis in the cat flea has been reported, and plays an important role in the

maintenance of this pathogen in the environment (Azad et al. 1992, Wedincamp et al. 2002).

In spite of this progress in understanding the biology of R. felis, a definitive host has not been

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identified and clinical signs of infection have only been reported in humans. Non-domestic or

wild animals including opossums and feral raccoons have also been shown to harbor R. felis

(Sashika et al. 2010, Schriefer et al. 1994). Although they have been implicated as potential

mammal hosts, their roles as reservoir hosts for human infection still requires further

elucidation.

While the domestic cat has previously been implicated as a potential primary reservoir

for R. felis (Case et al. 2006, Higgins et al. 1996), recent evidence from a number of studies

does not support this hypothesis. A prevalence study using molecular techniques reported

19.8% of flea sets collected from cats in eastern Australia harbored R. felis DNA (Barrs et al.

2010). However, the pathogen was not detected in the blood of these cats, and hence it was

speculated that domestic cats are unlikely to act as the primary vertebrate reservoir (Barrs et

al. 2010). Studies conducted in the USA (Bayliss et al. 2009) and Canada (Kamrani et al.

2008) using gltA and/or ompB gene amplification on high-risk groups of cats did not result in

detection of R. felis.

R. felis DNA has been detected using PCR assays in cats’ blood in an experimental

infection study (Wedincamp et al. 2000), and in skin biopsy and gingival swabs of cats

(Lappin et al. 2009) in the USA. However, natural infection in cats with active rickettsemia

has not been verified by PCR assays. As a reported host for C. felis, dogs could potentially

act as a reservoir for human flea-borne spotted fever. This is supported by a number of

molecular studies, some carried out in Australia, which have demonstrated R. felis in a

variety of fleas and ticks collected from dogs (Kelly et al. 2004, Schloderer et al. 2006). To

date, there are only two reports outlining detection of R. felis DNA in dogs. In Spain, a PCR

assay was used to detect R. felis in the blood of a dog owned by two people diagnosed with

flea-borne spotted fever (Oteo et al. 2006). This dog was afebrile but showed signs of fatigue,

vomiting and diarrhea. A case report from Germany used Western blotting to demonstrate R.

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felis infection in an asymptomatic dog owned by two patients with flea-borne spotted fever

(Richter et al. 2002). Moreover, a seroprevalence study of R. felis in Spain reported that

51.1% of dogs were seropositive to this pathogen (Nogueras et al. 2009).

The present study thus aims to determine whether dogs may serve as a mammalian

reservoir host for R. felis by employing molecular techniques in a targeted prevalence survey

on a high-risk group of poorly-cared-for pound dogs from Southeast Queensland (SE QLD)

in Australia.

Materials and methods

Geographical area

The study was undertaken in SE QLD, Australia (Figure 1). Brisbane, which

represents the major metropolitan area of SE QLD, is the capital city of Queensland. It is

situated in a subtropical region with warm, humid summers and short, mild winters.

Samples

Samples were collected from January 2010 through to June 2010 from pound dogs

sourced from the SE QLD area. Two EDTA-anticoagulated whole blood samples were

collected from 100 pound dogs by venipuncture in the Clinical Studies Centre, School of

Veterinary Science, University of Queensland (UQ). Sex and estimated age were recorded

prior to sample collection. One of the EDTA-anticoagulated whole blood samples from each

dog was sent to either IDEXX Laboratories in Brisbane or the veterinary clinical pathological

laboratory at School of Veterinary Science, UQ for basic hematological evaluation, including

a complete blood count. The remaining blood sample was stored at -20oC until DNA

extraction.

DNA extraction

DNA was extracted from whole blood samples collected from dogs into EDTA tubes.

The DNeasy Blood & Tissue Kits (QIAGEN, Hilden, Germany) were used and the

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manufacturer’s protocol followed with minor modifications. The blood volume used was

increased to 200 µl and the final elution volume was decreased to 50 µl in order to yield more

concentrated target DNA.

Molecular detection

DNA samples were tested using a previously described genus-specific PCR assay

(Paris et al. 2008). The assay was performed using primers ompB-F 5’-

CGACGTTAACGGTTTCTCATTCT-3’ and ompB-R 5’-

ACCGGTTTCTTTGTAGTTTTCGTC-3’ targeting a 252 bp region of the outer membrane

protein B (ompB) gene common to SFG rickettsiae. 2 µl of extracted DNA was added to a 23

µl reaction mixture containing 5x PCR buffer, 200 µmol/L dNTP, 1.0mmol/L MgCl2, 0.5

units of GoTaq polymerase (Promega, Madison, WI, USA), 10 pmol of each forward and

reverse primer and a final volume of nuclease free water (NFW). DNA from R. conorii

israelensis and NFW were used as positive and negative controls respectively.

PCR amplification was performed with the initial activation step at 95oC for 3 min,

followed by 40 cycles of amplification at 95oC for 30 s, 54

oC for 30 s and 72

oC for 30 s, and

a final extension step of 72oC for 7 min. Amplified product was examined on 1.6% agarose

gels stained with SYBR Safe (Invitrogen) run at 100 V for 30 min and visualized using UV

transillumination. Positive PCR products were purified using the QIAquick PCR Purification

Kit (QIAGEN) according to manufacturer’s protocol and submitted to the University of

Queensland Animal Genetics Laboratory (AGL) for DNA sequencing.

Phylogenetic analysis

DNA sequences were analyzed using Finch TV 1.4.0 (Geospiza Inc.) and aligned

using BioEdit version 7.0.5.3 (Hall 1999) with the ompB gene of the following rickettsiae

species, R. felis, R. conorii, R. honei, R. asiatica, R. tasmanensis, R. rickettsii and R. australis

(GenBank accession no. GQ385243, AF123726, AF123711, DQ110870, GQ223393, X16353

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and AF123709 respectively). Neighbor-joining analyses were conducted with Tamura-Nei

parameter distance estimates, and trees constructed using Mega 4.1 software

(www.megasoftware.net). Bootstrap analyses were conducted using 1000 replicates.

Statistical analysis

The prevalence and 95% confidence intervals (CI) were calculated for PCR results

using Win Episcope 2.0. The association between PCR results with host factor (age and

gender), time of sample collection (summer, autumn and winter), blood profile parameters

including packed cell volume (PCV), erythrocyte count, hemoglobin and leukocyte count and

presence of fleas infestation was studied using Chi-Square tests or Fisher’s exact test for

independence. Continuous data (blood parameters) was analyzed using one-way analysis of

variance. Significance was set at p≤ 0.025. Univariate analyses were conducted using SPSS

version 17.0 software (SPSS Inc., Chicago, IL, USA) and Excel 2007 (Microsoft, USA).

Animal Ethics

This project was approved by the University of Queensland Animal Ethics Committee,

approval number SVS/419/09/BAYER/ARC LINKAGE.

Results

Of the 100 pound dogs, 56 were male and 44 female. Fifty three blood samples were

collected during summer (January and February), 32 during autumn (March - May) and 15

during winter (June). The sampled group was represented by a single puppy (<12 weeks), 24

juveniles (12 weeks - 1 year), 68 adults (1 - 10 years) and 7 geriatrics (>10 years). Most dogs

were of mixed breed. All dogs appeared healthy.

Nine (9%) of 100 pound dogs’ blood were positive for Rickettsia by PCR. The

sequences amplified from all nine PCR-positive dogs showed 99.7% similarity to the existing

R. felis ompB sequence present in GenBank (accession no. GQ385243). Phylogenetic analysis

(Figure 2) also closely grouped all 9 sequences with R. felis GQ385243.

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Of these 9 dogs, 2 were juveniles, 6 were adults and 1 was geriatric. Seven dogs were

male and 2 were female.

Blood profile parameters were obtained from all (n=97) except 3 dogs due to clotting

of the blood sample. Two of the infected dogs were found to be mildly anemic (PCV 32%).

However, 31 non-infected dogs were also found to be mildly anemic. There was no

significant age or sex predisposition observed in PCR-positive dogs. There was also no

significant difference in time of sample collection and blood profile parameters between

infected and non-infected dogs.

Discussion

Our study represents the first report of R. felis infection in dogs in Australia. By using

a PCR assay targeting the ompB gene, 9% of dogs in this study were found to carry R. felis

DNA. All amplicons were 99.7% homologous to R. felis (GQ385243) with only one

nucleotide difference, which may suggest degree of genetic variability in different

geographical isolates.

Our report also describes first cluster of canine rickettsemia caused by R. felis in the

world diagnosed by PCR. Since the first recognition of R. felis in C. felis in 1990, household

pets have been implicated as a potential reservoir as they stay in close proximity to humans,

and are frequently infested with C. felis. A high prevalence of R. felis in dogs in our study has

shown that dogs may act as an important reservoir and sentinel host for human infection. Our

findings contrast with those of several groups who have attempted to detect rickettsemia in

cats in several studies, including one conducted in Australia (Assarasakorn et al. 2010, Barrs

et al. 2010, Bayliss et al. 2009, Hawley et al. 2007). This suggests that dogs, and not cats,

may be an important reservoir host for R. felis. Dogs are also known to be ubiquitous, easily

accessible and susceptible to a wide range of emerging human diseases (Cleaveland et al.

2006).

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To our knowledge, this study also describes the presence of Rickettsia spp DNA in

dogs’ blood by PCR for the first time in Australia. Previous surveys of spotted fever

rickettsial diseases in Australia involving dogs were conducted using serological assays

(Izzard et al. 2010, Sexton et al. 1991). Rickettsial infection is difficult to specifically

determine using serological tests alone, due to cross-reaction among SFG Rickettsia spp

(Sexton et al. 1991). Moreover, the early classification of R. felis as part of the TG is because

of its serotypical similarity to the TG rather than the SFG. Only when the presence of the

ompA gene in the R. felis genome was demonstrated was it reclassified into the SFG (Bouyer

et al. 2001). Thus, diagnosis of R. felis infection or exposure via serological methods can be

challenging, and would reinforce the hypothesis that R. typhi cases in the past may have been

misdiagnosed R. felis infections.

The pathogenicity of R. felis infection in dogs is unclear at this time. All the pound

dogs in this study appeared healthy. To date, association of clinical disease and R. felis

infection in animals has not been reported. Previously, in Spain, R. felis was detected by PCR

in two patients and their dog, which showed signs of fatigue, vomiting and diarrhea (Oteo et

al. 2006). The authors did not elaborate on the clinical signs in this dog and no further work-

up was performed.

Australia is endemic with several TG and SFG rickettsial diseases, which includes

epidemic typhus (R. prowazekii), murine typhus (R. typhi), scrub typhus (O. tsutsugumushi),

Q fever (Coxiella burnetii), Queensland tick typhus (R. australis) and Flinders Island spotted

fever (R. honei). This report, as well as three other studies on R. felis (Barrs et al. 2010,

Schloderer et al. 2006, Williams et al. In press 2010.), suggests this pathogen is endemic in

Australia and causes human infections. Following the detection of R. felis in this region, local

health authorities should be aware of the occurrence of this flea-borne spotted fever and its

zoonotic potential.

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Flea infestation in dogs was not evaluated in the present study and a history of flea

infestation in all these dogs could not be confirmed. Nevertheless, flea infestation was

observed in some dogs. Fleas that are commonly found to infest dogs in Australia are C. felis

(Schloderer et al. 2006), which is a non-host specific flea. Other fleas that infest dogs in

Australia are C. canis and Echidnophaga gallinacea (Schloderer et al. 2006), albeit rarely. R.

felis infection in fleas collected from dogs and cats in Australia was first reported in 2006

(Schloderer et al. 2006). Recently, R. felis DNA was detected by PCR in 19.8% of flea sets

collected from cats (Barrs et al. 2010). In the present study, fleas were not collected, thus the

role of fleas as a vector for R. felis infection in dogs in SE QLD remains unclear. Future study

on prevalence of R. felis in fleas and ticks sourced from dogs in this region is required to

evaluate the epidemiology of this flea-borne spotted fever.

R. felis DNA has been detected in a variety of arthropods, including fleas, ticks and

mites, collected from a variety of mammals (Cardoso et al. 2006, Choi et al. 2007, Schloderer

et al. 2006). Although ticks such as Rhipicephalus sanguineus and Amblyomma cajennense

(Cardoso et al. 2006, Oliveira et al. 2008), which infest dogs, were reportedly carrying this

agent, it is possible that these arthropods may just feed on rickettsemic blood, and act as

mechanical or non-competent vectors (Reif et al. 2009).

Horizontal transmission between the cat flea and vertebrates is still poorly understood.

Observation of R. felis in the salivary gland of C. felis under transmission electron

microscope (Macaluso et al. 2008) suggests the potential for infection of vertebrates through

blood feeding. Seroconversion in cats experimentally infected with R. felis through blood

feeding by infected cat fleas (Wedincamp et al. 2000) also suggests that horizontal

transmission is possible. However, in this experimental infection study, rickettsemia in cats

was found to subside rapidly. Horizontal transmission through blood feeding on infected cats

and artificially infected meals, co-feeding between infected and uninfected fleas, and larval

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feeding on flea feces and eggs was not detected in a study by Wedincamp and colleagues

(Wedincamp et al. 2002). Hence, the transmission dynamics of R. felis infection in dogs

remains an important area for future exploration.

In conclusion, detection of R. felis infection in dogs in this study is highly suggestive

that dogs may act as a reservoir host for R. felis. Further studies are required to study the

transmission dynamic and pathogenicity of R. felis in dogs.

Acknowledgment

This study was supported by grants from Bayer Animal Health Australia and the

Centre for Companion Animal Health, School of Veterinary Science, the University of

Queensland.

Disclosure statement

No competing financial interests exist.

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Figure:

Figure 1. The Southeast Queensland region of Australia, showing the location of the major

metropolitan area, Brisbane.

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Figure 2. Phylogenetic tree obtained from neighbour-joining analysis of the ompB gene of

Rickettsia sp. detected in 9 pound dogs clustered with R. felis GQ385243.

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