Figures
Abstract
Bartonellae are emerging vector-borne pathogens infecting erythrocytes and endothelial cells of various domestic and wild mammals. Blood samples were collected from domestic and wild canids in Iraq under the United States Army zoonotic disease surveillance program. Serology was performed using an indirect immunofluorescent antibody test for B. henselae, B. clarridgeiae, B. vinsonii subsp. berkhoffii and B. bovis. Overall seroprevalence was 47.4% in dogs (n = 97), 40.4% in jackals (n = 57) and 12.8% in red foxes (n = 39). Bartonella species DNA was amplified from whole blood and representative strains were sequenced. DNA of a new Bartonella species similar to but distinct from B. bovis, was amplified from 37.1% of the dogs and 12.3% of the jackals. B. vinsonii subsp. berkhoffii was also amplified from one jackal and no Bartonella DNA was amplified from foxes. Adjusting for age, the odds of dogs being Bartonella PCR positive were 11.94 times higher than for wild canids (95% CI: 4.55–31.35), suggesting their role as reservoir for this new Bartonella species. This study reports on the prevalence of Bartonella species in domestic and wild canids of Iraq and provides the first detection of Bartonella in jackals. We propose Candidatus Bartonella merieuxii for this new Bartonella species. Most of the Bartonella species identified in sick dogs are also pathogenic for humans. Therefore, seroprevalence in Iraqi dog owners and bacteremia in Iraqi people with unexplained fever or culture negative endocarditis requires further investigation as well as in United States military personnel who were stationed in Iraq. Finally, it will also be essential to test any dog brought back from Iraq to the USA for presence of Bartonella bacteremia to prevent any accidental introduction of a new Bartonella species to the New World.
Author Summary
Bartonellae are emerging vector-borne pathogens infecting erythrocytes and endothelial cells of various domestic and wild mammals. Blood samples were collected from domestic and wild canids in Iraq under the United States Army zoonotic disease surveillance program. Serology was performed using an indirect immunofluorescent antibody test for B. henselae, B. clarridgeiae, B. vinsonii subsp. berkhoffii and B. bovis. Overall seroprevalence was 47% in dogs (n = 97), 40% in jackals (n = 57) and 13% in red foxes (n = 39). Bartonella species DNA was amplified from whole blood and representative strains were sequenced. DNA of Candidatus Bartonella merieuxii, a new Bartonella species similar to but distinct from B. bovis, was amplified from 37% of the dogs and 12% of the jackals. B. vinsonii subsp. berkhoffii was also amplified from one jackal and no Bartonella DNA was amplified from foxes. Dogs were more likely to be Bartonella PCR positive than wild jackals and foxes, suggesting the role of dogs as reservoir for this new Bartonella species. As most Bartonella species isolated or detected in dogs are also infecting humans, it will be important to test Iraqi people, especially from Baghdad, and American veterans who served in Iraq for the presence of infection by Candidatus Bartonella merieuxii.
Citation: Chomel BB, McMillan-Cole AC, Kasten RW, Stuckey MJ, Sato S, Maruyama S, et al. (2012) Candidatus Bartonella merieuxii, a Potential New Zoonotic Bartonella Species in Canids from Iraq. PLoS Negl Trop Dis 6(9): e1843. https://doi.org/10.1371/journal.pntd.0001843
Editor: Joseph M. Vinetz, University of California San Diego School of Medicine, United States of America
Received: July 25, 2012; Accepted: August 17, 2012; Published: September 27, 2012
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: The project was funded under a US Army contract (USAMRAA/WRAIR Contract Number: W81XWH10P0215). The funders had no role in study design, data collection* and analysis, or preparation of the manuscript. Decision to publish was approved by Public Affairs and Dr. McMillan-Cole supervisors (e-mail of Dr. McMillan-Cole of April 19, 2012). * Sample collection was performed by the military under the supervision of United States Army zoonotic disease surveillance program.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Bartonella are fastidious, hemotropic, gram negative, rods that have an affinity for the erythrocytes, endothelial cells and macrophages of their hosts [1]–[3]. These bacteria cause vasoproliferative lesions by entering endothelial cells resulting in cellular proliferation and migration [4]. The organisms are highly adaptive which enables them to persist within the cells of their mammalian hosts for extended periods [2]. Culturing the bacteria is often frustrating [5], [6], as colonies can take up to 45 days to grow on enriched blood media [2].
These bacteria are primarily arthropod-borne [1]–[3]; the main vectors definitively identified are the sand fly (Lutzomyia verrucarum) for B. bacilliformis, the cat flea (Ctenocephalides felis) for B. henselae, and the human body louse (Pediculus humanis corporis) for B. quintana [5]. Vector-borne transmission of Bartonella to bank voles and gerbils (Gerbillus nanus, G. dasyurus) by Ctenophthalmus nobilis fleas and to jirds (Meriones crassus) by Xenopsylla ramesis has also been established [7], [8]. Bartonella DNA has been detected in ticks and biting flies as well [9], [10], suggesting many more possible arthropod vectors [2], [3], recently supported through experimental demonstration of vector competence for B. birtlesii by Ixodes ricinus [11].
More than 25 species/subspecies of Bartonella and many Candidatus species have been identified in a wide range of domestic and wild animals [10; http://www.bacterio.cict.fr]. Of these, 14 are recognized as zoonotic or potentially zoonotic, including 10 isolated from dogs and cats.
The clinical spectrum of Bartonella infections in humans is quite broad [1], [3], [12]. Worldwide, cat scratch disease (CSD) is the most commonly recognized zoonotic disease caused by Bartonella species [2] that occurs in immunocompetent individuals [1]–[3]. B. henselae and B. quintana cause severe systemic diseases in immunocompromised patients, such as bacillary angiomatosis and bacillary peliosis [1]–[3], [12]. Bartonella infections are an important cause of culture-negative endocarditis in humans [13], comprising an estimated 3–10% of all endocarditis cases [13], [14]. Other manifestations of Bartonella infections include prolonged fever of unknown origin with or without lymphadenopathy [15]. When using an insect based enrichment medium, people with extensive arthropod exposure and animal contact had high infection rates (24–41%) of Bartonella spp. [16].
Several Bartonella species, most of which are zoonotic, have been isolated or detected by PCR amplification and DNA sequencing from dogs, including B. vinsonii subsp. berkhoffii, B. henselae, B. clarridgeiae, B. rochalimae, B. quintana, B. koehlerae, B. elizabethae, B. vinsonii subsp. arupensis, B. taylorii, B. volans-like, B. bovis and B. bovis-like [3], [17]–[20]. Recently, six Italian dogs and one Greek dog were infected with a novel, uncultured Bartonella sp. strain designated as strain HMD [GenBank accession number EF614393], which is phylogenetically similar, but distinct from B. bovis [19]. As the abnormalities observed in dogs infected with Bartonella spp. are very similar to the clinical and pathologic abnormalities seen in humans, the findings in this manuscript may prove to be of comparative medical importance [2], [3]. The role of dogs as a primary reservoir for various Bartonella species is still unclear [2]. Nevertheless, dogs are excellent sentinels as part of a surveillance system for the detection of human infections [21]. In Iraq, limited information is available on zoonotic diseases in canids. A recent study investigated the presence of Q fever antibodies in military working dogs (MWD) deployed in Iraq and free-roaming dogs eliminated through a feral animal control program in locations throughout Iraq [22]. None of the MWD seroconverted versus 5.5% of the indigenous dogs. To our knowledge, presence of Bartonella infection has never been reported in Iraq. Therefore, the objective of the present study was to investigate the presence of Bartonella spp. by PCR amplification and to assess seroprevalence of B. henselae, B. clarridgeiae, B. vinsonii subsp. berkhoffii and B. bovis in domestic and wild canids of Iraq.
Materials and Methods
Subject enrollment, sample collection, and storage
Whole blood samples (1.5–2 ml) were collected between February and December 2008 under the US Army feral animal control and zoonotic disease surveillance program from the saphenous or jugular veins in EDTA tubes from 193 canids, including 97 (50.3%) stray dogs (Canis familiaris), 57 (29.5%) jackals (Canis aureus) and 39 (20.2%) red foxes (Vulpes vulpes) located on several United States military bases throughout Iraq. Categorical age, sex, and location data was also collected with samples, when possible.
Ethics statement
All animals were chemically restrained prior to blood collection and humanely treated prior to euthanasia, in accordance with the rules of the ethic committee from the US Army feral animal control and zoonotic disease surveillance program and by the University of California, Davis (UCD) Animal Use and Care Committee (Protocol # 12668 originally approved on March 8, 2007) in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The blood samples were frozen and shipped to the UCD School of Veterinary Medicine. In a pilot study on 40 dogs, 10 jackals and 35 foxes ((McMillan-Cole, Master of Preventive Veterinary Medicine, Davis, CA; data not shown), either major bacterial contamination or lack of isolation of Bartonella was observed when blood culture were attempted. Therefore, the blood samples analyzed in the present study were only processed by DNA extraction.
Serological analysis
Presence of antibodies against B. henselae H1 (ATCC 49882) and U4 (UC Davis), B. clarridgeiae (ATCC51734), B. vinsonii subsp. berkhoffii genotype I (ATCC 51672) and B. bovis (strain 91-4T: CIP 106692T and CCUG 43828T) were tested on the serum or blood supernatant samples, using an indirect immune-fluorescent-antibody assay (IFA) as previously described [18], [23], [24]. Serum samples were diluted to 1∶32 and 1∶64 and 20 µl of each dilution was added to the test wells. Positive and negative controls were included on each slide. Three conjugate (Cappel fluorescein-conjugate goat anti-dog IgG fraction, MP Biomedicals, Aurora, OH, USA) dilutions were used for the four antigens being tested: 1∶1400 (B. vinsonii subsp. berkhoffii and B. bovis), 1∶2800 (B. henselae H1 and U4) and 1∶3600 (B. clarridgeiae). For each well, 20 µl of the conjugates were added to the respective slides. The intensity of the fluorescence was graded subjectively by two independent masked readers, with sample fluorescence graded ≥2 at the 1∶64 dilution reported as positive.
DNA extraction and PCR-RFPL
The DNA was extracted as previously described [25]. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the gltA gene was then performed for all samples [25]. The primers used for the gltA gene were BhCS.781p 5′- and BhCS.1137n 5′, as previously described [26]. An approximately 400 bp fragment of the gltA gene was amplified and then verified by gel electrophoresis. The amplified product was then digested with restriction endonucleases TaqI (Promega, Madison, WI), HhaI, MseI and DdeI (New England BioLabs, Ipswich, MA, USA). Banding patterns were compared with 19 standard strains of Bartonella, including B. henselae and B. clarridgeiae, B. koehlerae and Bartonella ruminant strains.
Sequencing and phylogenetic analysis
Two representative DNA products from the dog and jackal groups were further analyzed by PCR of the 16S-23S rRNA intergenic spacer region (ITS) [27] and of the rpoB gene [28] and submitted to sequencing. As the gltA gene sequence for the HMD strain had not been previously performed, DNA from strain HMD was submitted by one of the authors (EB), and after PCR testing with the appropriate primers, was submitted for sequencing (Davis Sequencing, Davis, CA, USA). Amplified products from the gltA gene, rpoB gene, and the ITS region were sequenced in both directions and consensus sequences were compared. The sequence alignments obtained in this study were also referenced against those of other known Bartonella species deposited in the GenBank/EMBL/DDBJ database using BLAST [Table 1]. The Clustal W program [29] was used to align each isolate and compare homologous gltA, rpoB and ITS spacer sequences to identify genetic variants. A phylogenetic tree was constructed from concatenated sequences of gltA, rpoB and ITS from representative Bartonella isolates, including ruminant-associated Bartonella species, by using neighbor-joining methods. Nucleotide substitution rates were calculated by Kimura's two-parameter distance model, as previously described [30], using MEGA version 5.0 software [31]. Bootstrap analysis was performed with 1,000 trials.
Statistical analysis
Bartonella prevalence data generated from diagnostic tests and binomial proportion confidence intervals (CI) were calculated using Wald and Fisher's exact methods where appropriate in OpenEpi, version 2.3.1 (Dean AG, Sullivan KM, Soe MM. OpenEpi: Open Source Epidemiologic Statistics for Public Health). All variables were initially screened using univariate logistic regression to check for statistically significant associations with serology and PCR outcomes. Due to quasi-complete separation of PCR data, species and location variables were reclassified in biologically meaningful groups prior to univariate analysis. Using forward stepwise selection methods, categorical variables were retained in separate multivariate logistic regression models for PCR and IFA. Variables that induced changes >10% in the β-coefficients of covariates after inclusion in the model were considered to be potential confounders, and evaluated along with biological reasoning and prior knowledge. Likelihood ratio tests comparing models with and without two-way interaction terms were used to determine presence of effect measure modification. All logistic regression models and resulting adjusted odds ratios with 95% CI were generated in SAS, version 9.3 (SAS Institute Inc., Cary, NC).
Results
Dogs
Most of the dogs (79.38%; 77/97) were captured in Baghdad and surrounding suburbs (Inner Baghdad: 35, North Baghdad: 27, Outer Baghdad: 15). The other dogs were from locations classified as North Iraq (n = 15), West Iraq (n = 3) or South Iraq (n = 2). Forty-three (57.3%) of the 75 dogs for which sex was reported (missing from 22 dogs) were male. Age was not recorded for 27 animals, with 60 (85.7%) of the 70 dogs classified as adults. Thirty-six dogs (37.1%; 95% CI = 27.5%–46.73%) were PCR positive [Table 2]. RFLP analysis of the gltA PCR product from the four enzyme digestions indicated similar banding patterns for all 36 dogs (data not shown). Banding pattern was similar to B. clarridgeiae using TaqI, HhaI and MseI. However, DdeI allowed differentiating these strains from B. clarridgeiae. A representative dog “strain” (F059) was chosen for further genetic analysis. Sequencing of the gltA gene fragment showed that the closest related Bartonella species were respectively B. bovis, B. schoenbuchensis, B. melophagi, B. chomelii and B. capreoli [Table 3]. The partial gltA sequence of the HMD strain was deposited in GenBank under the number JX073031. Sequencing of the 16S-23S ITS spacer region indicated that DNA of “strain” F059 was 100% identical to the previously reported HMD strain [19] and most closely related to ruminant Bartonella species [Table 3]. For the rpoB sequences, DNA of “strain” F059 was also 100% identical to strain HMD and with the closest related species being B. bovis and other ruminant Bartonella species [Table 3]
A phylogenetic tree of the concatenated sequences of the gltA and rpoB genes and the 16S-23S ITS indicted that all strains clustered together and aligned most closely to B. bovis (Figure 1). However, partial sequences were less than 95% similar for all genes, suggesting that these canid strains likely belong to a new Bartonella species. DNA of other Bartonella species, including B. vinsonii subsp. berkhoffii was not PCR amplified from the blood of these dogs. The 36 PCR positive dogs were from either Baghdad (n = 30, 39%) or North Iraq (n = 6, 40%). Within Baghdad and its suburbs, 57.14% (20/35) of the dogs from Inner Baghdad, 40% (6/15) from Outer Baghdad and 14.8% (4/27) from North Baghdad were PCR positive. Of the male dogs, 46.5% (20/43) were PCR positive compared to 46.8% (15/32) of the females. Only 10% (1/10) of the young dogs compared to 53.3% (32/60) of the adults were PCR positive.
The phylogenetic tree was based on concatenated sequences of two housekeeping genes (gltA and rpoB) and the 16S-23S rRNA intergenic spacer region and was constructed from 941 base-pair sequences, inferred using the Neighbor-Joining method, and 1000 replicates in the bootstrap test. Scale bar indicates 10 substitutions per nucleotide position.
Overall, 47.4% of the dogs (46/97; 95% CI: 37.5%–57.4%) were seropositive to at least one Bartonella antigen [Table 2]. Among the 97 dogs, 34% (33/97) were both PCR and serology positive, 3.1% (3/97) were PCR positive and seronegative, 13.4% (13/97) were seropositive and PCR negative, and 49.5% (48/97) were both PCR and seronegative [Table 2]. Of the 36 PCR positive dogs, 66.6% (24/36) were seropositive for all four antigens and five dogs were seropositive for three antigens, with 86.1% (31/36) being seropositive for B. bovis, specifically.
Jackals
Of the 57 jackals sampled, 55.6% (30/54) were collected from North Baghdad, 18.5% (10/54) from Outer Baghdad, 16.7% (9/54) from North Iraq, and 9.3% (5/54) from West Iraq (complete location data was missing for three jackals). The sex ratio of the sampled jackals was 83% (44/53) male and 17% (9/53) female (sex data was absent for four jackals). Age was recorded dichotomously for 51 jackals, with 74.5% (38/51) classified as adults and 25.5% (13/51) as young. Seven jackals (12.3%; 95% CI: 5.08%–23.68%) were PCR positive for Bartonella, including one young male from North Iraq infected with B. vinsonii subsp. berkhoffii [Table 2]. The other six jackals infected with a “strain” identical to strain HMD were also male, four being from North Baghdad and two from North Iraq. Five of these animals were reported as adults and age classification was missing from one sample.
DNA of a representative jackal “strain” (F040) was chosen for genetic analysis. The gltA gene sequence aligned most closely with B. bovis and the other ruminant Bartonella species [Table 4]. The 16S-23S ITS region sequence of DNA from jackal “strain” F040 was 100% identical to the HMD strain, aligning most closely with B. schoenbuchensis and the other ruminant Bartonella species. For the rpoB sequences, DNA amplification from jackal F040 was 100% identical to strain HMD and aligned most closely with B. bovis and the other ruminant Bartonella species [Table 4].
Overall, 40.4% (23/57; 95% CI: 27.6%–53.1%) of the jackals were seropositive for at least one Bartonella antigen [Table 2]. Of the seven PCR positive jackals, only one was seronegative, which was a Bartonella sp. strain HMD adult male from North Baghdad. All six other jackals were IFA positive for the four Bartonella antigens. Of the 17 seropositive jackals that were PCR negative, 70.6% (12/17) were seropositive for the four antigens tested.
Foxes
Blood samples were collected from 39 red foxes, comprised of 21 from West Iraq, 15 from Baghdad (four from Inner and 11 from Outer Baghdad) and three from North Iraq. There were 20 male and 19 female foxes, including 29 adults and 10 young individuals. None of the fox samples were Bartonella PCR positive [Table 2]. Seroprevalence in foxes was 12.8% (5/39; 95% CI: 4.3%–27.4%).
Comparisons among canids
Species, sex, age and location data were collected for most of the canids and submitted to a univariate analysis [Tables 5 and 6). Species and age were then included in the multivariate models for both PCR and serology outcomes [Tables 7 and 8]. Adjusting for age, the odds of dogs being Bartonella PCR positive were 11.94 times higher than of wild canids (95% CI: 4.55–31.35). Adjusting for species, the odds of adult canids being PCR positive were 5.62 times higher than young canids (95% CI: 1.19–26.59). The age-adjusted odds of dogs being Bartonella seropositive were 8.71 times higher than for foxes (95% CI: 3.03–25.01). Similarly, age-adjusted odds of jackals being Bartonella seropositive were 5.64 times higher than of foxes (95% CI: 1.87–17.07).
Discussion
This study reports on the prevalence of Bartonella species in domestic and wild canids of Iraq and provides the first detection of Bartonella in jackals. Dogs and jackals were frequently infected with a new Bartonella species similar to strain HMD, initially described from dogs in southern Italy and Greece [19] and more recently in two dogs from Sri Lanka [32]. The PCR prevalence in Iraqi dogs (45.4%) was similar to the prevalence in the dogs from Basilicata, Italy (42.8%, 6/14).
Based on the criteria suggested by La Scola et al. [33], the low similarity percentages (<95.5%) between the Iraqi, Greek, Italian and Sri Lankan “strains” and B. bovis clearly suggest that the “strains” infecting dogs belong to a new Bartonella species. We propose Candidatus Bartonella merieuxii for this new species. B. vinsonii subsp. berkhoffii, a common Bartonella species isolated from dogs [3], was not detected in the Iraqi dogs and detected only from one jackal. Similarly, B. rochalimae was not detected from any of the canids, which is especially interesting as red foxes have been found to be infected by this Bartonella species in Israel and Europe [30]. Given the statistically significantly higher PCR prevalence of Candidatus B. merieuxii infection in dogs than in wild canids, domestic dogs may represent a major reservoir of this new species in Iraq. Because this new species is genetically related to B. bovis, we sought to investigate the seroprevalence of B. bovis in these canids in addition to the antigens usually used for canids in our laboratory. Most (86%) dogs and jackals that were PCR positive were also seropositive for B. bovis and for the three other antigens. The lowest seroprevalence observed was for B. clarridgeiae and B. vinsonii subsp. berkhoffii, suggesting that these Bartonella species may be found less frequently in Iraqi canids as compared to other parts of the world.
The high seroprevalence in stray dogs and jackals correlates with previously reported studies on seroprevalence of Bartonella species in animals of the tropics, Middle East, and some African nations [1], [2]. A recent study of stray dogs in the tropics [32] reported an overall seroprevalence of 8.3% (41/455), ranging from 0% in dogs from Vietnam to almost 11% in dogs from Bogota, Colombia. These data contrast with reports in domestic pet dogs in the United States and Europe, where overall seroprevalence is <5% [1]. In Thailand, prevalence of Bartonella bacteremia in stray dogs was 31.3% (60/192) [17].
Cross-reactivity is known to occur between B. henselae and B. clarridgeiae, especially in human sera [2], and therefore it is unclear whether the seropositivity for more than one Bartonella species was due to cross-reactions or to co-infection or sequential infection to different Bartonella spp. over an extended time period. Given the relatively small sample sizes of some canid groups and locations, larger studies will be important to strengthen our findings.
To date, no vector has been proven as the source of infection for any Bartonella spp. in dogs. The current hypothesis on the mode of transmission of B. vinsonii subsp. berkhoffii involves ticks, because seropositive dogs were also frequently seropositive for other known tick-borne pathogens [3], [34], [35]. Similarly, although the body louse is considered the primary vector for B. quintana, B. quintana DNA has been amplified from both fleas and ticks, providing support for being possible Bartonella vectors [9], [36]. Furthermore, Candidatus Bartonella merieuxii was detected in Rhipicephalus sanguineus ticks, as DNA was amplified from three tick pools including one pool from salivary glands from female ticks and one gut content pool collected two dogs PCR positive for Candidatus B. merieuxii [19]. Therefore, ticks may be possible candidate vectors for this new Bartonella species. Unfortunately, information on the presence of ectoparasites was not available and no quantitative measure of the fleas and ticks was reported. Given the high prevalence of Bartonella spp. reported in dogs and to a lesser extent in jackals, studies to detect Bartonella spp. DNA in arthropods in Iraq are warranted.
In humans, clinical diagnosis of atypical CSD and bacteremia associated with other emerging clinical manifestations of bartonellosis is difficult [2], [3]. Domestic cats are generally asymptomatic carriers of B. henselae and B. clarridgeiae. Symptomatic bacteremic dogs are often seronegative and frequently manifest similar pathology and disease manifestations as reported in human patients [3], [19], [37]. Furthermore, most of the Bartonella species identified in sick dogs are also pathogenic for humans [2]. Bartonella quintana, for instance, causes a high percentage of endocarditis in people and has been associated with endocarditis in dogs [2], [38], [39]. Therefore, seroprevalence in Iraqi dog owners and bacteremia in Iraqi people with unexplained fever or culture negative endocarditis requires further investigation. This is especially true in Baghdad and the surrounding region, where most of the positive dogs were detected. Similarly, it will be important to investigate pre and post-deployment seroprevalence in United States military personnel who were stationed in Iraq. It would be critical to test for Bartonella bacteremia in veterans with unexplained fever, lymphadenopathy, or culture negative endocarditis. Finally, it will also be essential to test any dog brought back from Iraq to the USA for presence of Bartonella bacteremia to prevent any accidental introduction of a new Bartonella species to the New World.
In the context of zoontic bartonellosis, the dog appears to serve as both a sentinel and a reservoir for human infections. As a sentinel, infection in both dogs and humans (most often cases of endocarditis) has now been documented for eight Bartonella species or subspecies. As a reservoir, persistent infection in dogs has been documented in association with B. vinsonii subsp. berkhoffii, B. henselae and B. koehlerae [3]. As such, there are reports of dog bite transmission of B. henselae to people [40] and two recent publications that implicate needle stick transmission B. vinsonii subsp. berkhoffii or B. henselae from dogs to veterinarians [41], [42]. In the context of diagnostic testing, several of our laboratories are attempting isolation of this novel Bartonella sp. to facilitate additional genetic characterization and the development and validation of Bartonella species specific serologic assays. In addition, attempts at isolation from sick soldiers deployed in Iraq would seem reasonable.
In summary, presence of Bartonella infection was detected in domestic dogs and wild canids in different areas of Iraq. Our findings identify a potential role for dogs as a major reservoir of a new and potentially zoonotic Bartonella species.
Description of Candidatus Bartonella merieuxii sp. nov
Bartonella merieuxii (méri.eu.xii N. L. gen. N. merieuxii of Mérieux, in honor of Charles Mérieux, a French physician, founder of the Mérieux Foundation and former C.E.O. of the Mérieux Institute in Lyon, France. His broad interest in comparative medicine and in one health, within the Pasteur's tradition at its best (“No borders between the two medicines” was his favorite saying), allowed him to build many bridges between physicians and veterinarians in the field of zoonoses. DNA of this species has been amplified and sequenced from dogs and wild canids (jackals) in the old world.
Acknowledgments
We would like to thank all the soldiers of the 43rd Medical Detachment Veterinary Services (MDVS) and the 28th Medical Detachment Veterinary Medicine (MDVM) deployed in Iraq in 2007–2009 who helped collect the blood samples and Dr. Chin-Shang Li for consultation on SAS coding.
Disclaimer
The views expressed in the present article are those of the authors and do not reflect the official policy or position of the Department of the Army, the Department of Defense, or the US Government.
Author Contributions
Conceived and designed the experiments: BBC ACMC. Performed the experiments: BBC ACMC RWK. Analyzed the data: BBC ACMC MJS SS SM. Contributed reagents/materials/analysis tools: RWK SS SM PPVPD EBB. Wrote the paper: BBC ACMC. Contributed to writing of article: MJS SS SM PPVPD EBB.
References
- 1. Boulouis HJ, Chang CC, Henn JB, Kasten RW, Chomel BB (2005) Factors associated with the rapid emergence of zoonotic Bartonella infections. Vet Res 36: 383–410.
- 2. Chomel BB, Boulouis HJ, Maruyama S, Breitschwerdt EB (2006) Bartonella spp. in pets and effect on human health. Emerg Infect Dis 12: 389–94.
- 3. Breitschwerdt EB, Maggi RG, Chomel BB, Lappin MR (2010) Bartonellosis: an emerging infectious disease of zoonotic importance to animals and human beings. J Vet Emerg Crit Care (San Antonio) 20: 8–30.
- 4. Dehio C (2001) Bartonella interactions with endothelial cells and erythrocytes. Trends Microbiol 9(6):279–85.
- 5. Duncan AW, Maggi RG, Breitschwerdt EB (2007) A combined approach for the enhanced detection and isolation of Bartonella species in dog blood samples: pre-enrichment liquid culture followed by PCR and subculture onto agar plates. J Microbiol Methods 69: 273–278.
- 6. Gundi VA, Bourry O, Davoust B, Raoult D, La Scola B (2004) Bartonella clarridgeiae and B. henselae in dogs, Gabon. Emerg Infect Dis 10: 2261–2262.
- 7. Bown KJ, Bennet M, Begon M (2004) Flea-borne Bartonella grahamii and Bartonella taylorii in bank voles. Emerg Infect Dis 10: 684–687.
- 8. Morick D, Krasnov BR, Khokhlova IS, Gottlieb Y, Harrus S (2011) Investigation of Bartonella acquisition and transmission in Xenopsylla ramesis fleas (Siphonaptera: Pulicidae). Mol Ecol 20: 2864–2870.
- 9. Chang CC, Chomel BB, Kasten RW, Romano V, Tietze N (2001) Molecular evidence of Bartonella spp. in questing adult Ixodes pacificus ticks in California. J Clin Microbiol 39: 1221–1226.
- 10. Halos L, Jamal T, Maillard R, Girard B, Guillot J, et al. (2004) Role of Hippoboscidae flies as potential vectors of Bartonella spp. infecting wild and domestic ruminants. Appl Environ Microbiol 70: 6302–6305.
- 11. Reis C, Cote M, Le Rhun D, Lecuelle B, Levin ML, et al. (2011) Vector competence of the tick Ixodes ricinus for transmission of Bartonella birtlesii. PLoS Negl Trop Dis 5(5):e1186.
- 12. Chomel BB, Kasten RW, Sykes JE, Boulouis HJ, Breitschwerdt EB (2003) Clinical impact of persistent Bartonella bacteremia in humans and animals. Ann N Y Acad Sci 990: 267–278.
- 13. Brouqui P, Raoult D (2006) New insight into the diagnosis of fastidious bacterial endocarditis. FEMS Immunol Med Microbiol 47: 1–13.
- 14. Chomel BB, Kasten RW, Williams C, Wey AC, Henn JB, et al. (2009) Bartonella endocarditis: a pathology shared by animal reservoirs and patients. Ann N Y Acad Sci 1166: 120–126.
- 15. Jacobs RF, Schutze GE (1998) Bartonella henselae as a cause of prolonged fever and fever of unknown origin in children. Clin Infect Dis 26: 80–84.
- 16. Maggi RG, Mascarelli PE, Pultorak EL, Hegarty BC, Bradley JM, et al. (2011) Bartonella spp. bacteremia in high-risk immunocompetent patients. Diagn Microbiol Infect Dis 71: 430–437.
- 17. Bai Y, Kosoy MY, Boonmar S, Sawatwong P, Sangmaneedet S, et al. (2010) Enrichment culture and molecular identification of diverse Bartonella species in stray dogs. Vet Microbiol 146: 314–319.
- 18. Henn JB, Gabriel MW, Kasten RW, Brown RN, Koehler JE, et al. (2009) Infective endocarditis in a dog and the phylogenetic relationship of the associated “Bartonella rochalimae” strain with isolates from dogs, gray foxes, and a human. J Clin Microbiol 47: 787–790.
- 19. Diniz PP, Billeter SA, Otranto D, De Caprariis D, Petanides T, et al. (2009) Molecular documentation of Bartonella infection in dogs in Greece and Italy. J Clin Microbiol 47: 1565–1567.
- 20. Pérez C, Maggi RG, Diniz PP, Breitschwerdt EB (2011) Molecular and serological diagnosis of Bartonella infection in 61 dogs from the United States. J Vet Intern Med 25: 805–810.
- 21. Henn JB, Gabriel MW, Kasten RW, Brown RN, Theis JH, et al. (2007) Gray foxes (Urocyon cinereoargenteus) as a potential reservoir of a Bartonella clarridgeiae-like bacterium and domestic dogs as part of a sentinel system for surveillance of zoonotic arthropod-borne pathogens in northern California. J ClinMicrobiol 45: 2411–2418.
- 22. Havas KA, Burkman K (2011) A comparison of the serological evidence of Coxiella burnetii exposure between military working dogs and feral canines in Iraq. Mil Med 176: 1101–1103.
- 23. Chomel BB, Abbott C, Kasten RW, Floyd-Hawkins KA, Kass PH, et al. (1995) Bartonella henselae prevalence in domestic cats in California: Risk factors and association between bacteremia and antibody titers. J Clin Microbiol 33: 2445–2450.
- 24. Chomel BB, Carlos ET, Kasten RW, Yamamoto K, Chang CC (1999) Bartonella henselae and Bartonella clarridgeiae infection in domestic cats from The Philippines. Am J Trop Med Hyg 60: 593–597.
- 25. Chang CC, Kasten RW, Chomel BB, Simpson DC, Hew CM, et al. (2000) Coyotes (Canis latrans) as the reservoir for a human pathogenic Bartonella sp.: molecular epidemiology of Bartonella vinsonii subsp. berkhoffii infection in coyotes from central coastal California. J Clin Microbiol 38: 4193–4200.
- 26. Norman AF, Regnery R, Jameson P, Greene C, Krause DC (1995) Differentiation of Bartonella-like isolates at the species level by PCR-restriction fragment length polymorphism in the citrate synthase gene. J Clin Microbiol 33: 1797–1803.
- 27. Jensen WA, Fall MZ, Rooney J, Kordick DL, Breitschwerdt EB (2000) Rapid identification and differentiation of Bartonella species using a single-step PCR assay. J Clin Microbiol 38: 1717–1722.
- 28. Renesto P, Gouvernet J, Drancourt M, Roux V, Raoult D (2001) Use of rpoB gene analysis for detection and identification of Bartonella species. J Clin Microbiol 39: 430–437.
- 29. Thompson JD, Higgins DG, Gibson TG (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids res 22: 4673–4674.
- 30. Henn JB, Chomel BB, Boulouis HJ, Kasten RW, Murray WJ, et al. (2009) Bartonella rochalimae in raccoons, coyotes, and red foxes. Emerg Infect Dis 15: 1984–1987.
- 31. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, et al. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731–2739.
- 32.
Brenner EC, Chomel BB, Singhasivanon OU, Namekata DY, Kasten RW, et al.. (2012) Bartonella infection in urban and rural dogs from the tropics: Brazil, Colombia, Sri Lanka and Vietnam. Epidemiol Infect 140 [ahead of print] doi:10.1017/S0950268812000519.
- 33. La Scola B, Zeaiter Z, Khamis A, Raoult D (2003) Gene-sequence-based criteria for species definition in bacteriology: the Bartonella paradigm. Trends Microbiol 11: 318–321.
- 34. Billeter SA, Levy MG, Chomel BB, Breitschwerdt EB (2008) Vector transmission of Bartonella species with emphasis on the potential for tick transmission. Med Vet Entomol 22: 1–15.
- 35. Angelakis E, Billeter SA, Breitschwerdt EB, Chomel BB, Raoult D (2010) Potential for tick-borne bartonelloses. Emerg Infect Dis 16: 385–391.
- 36. Rolain JM, Franc M, Davoust B, Raoult D (2003) Molecular detection of Bartonella quintana, B. koehlerae, B. henselae, B. clarridgeiae, Rickettsia felis, and Wolbachia pipientis in cat fleas, France. Emerg Infect Dis 9: 338–342.
- 37. Honadel TE, Chomel BB, Yamamoto K, Chang C, Farver TB (2001) Seroepidemiology of Bartonella vinsonii subsp. berkhoffii exposure among healthy dogs. J Am Vet Med Assoc 219: 480–484.
- 38. Benslimani A, Fenollar F, Lepidi H, Raoult D (2005) Bacterial zoonoses and infective endocarditis, Algeria. Emerg Infect Dis 11: 216–224.
- 39. Znazen A, Rolain JM, Hammami N, Kammoun S, Hammami A, et al. (2005) High prevalence of Bartonella quintana endocarditis in Sfax, Tunisia. Am J Trop Med Hyg 72: 503–507.
- 40. Rolain JM, Boureau-Voultoury A, Raoult D (2009) Serological evidence of Bartonella vinsonii lymphadenopathies in a child bitten by a dog. Clin Microbiol Infect 15 Suppl 2: 122–123.
- 41. Oliveira AM, Maggi RG, Woods CW, Breitschwerdt EB (2010) Suspected needle stick transmission of Bartonella vinsonii subspecies berkhoffii to a veterinarian. J Vet Intern Med 24(5):1229–1232.
- 42. Lin JW, Chen CM, Chang CC (2011) Unknown fever and back pain caused by Bartonella henselae in a veterinarian after a needle puncture: a case report and literature review. Vector Borne Zoonotic Dis 11(5):589–591.