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¹ Department of Vector Biology and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention P.O. Box 5, Changping, Beijing 102206, China
² Yunnan Institute of Endemic Diseases Control and Prevention, Dali City, Yunnan, China
³ Corresponding author (email: Liuqiyong{at}icdc.cn)

	ABSTRACT

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ABSTRACT:   Previous studies have demonstrated a diversity of Bartonella spp. in rodent populations in Yunnan Province, China. Although Bartonella spp. have been isolated from cat fleas and cattle ticks collected from their animal hosts, little is known about Bartonella carried by rodent fleas. In this study, Bartonella DNA was detected by polymerase chain reaction (PCR) in two of five species of rodent fleas. These included Xenopsylla cheopis and Ctenophthalmus lushuiensis, which were collected from Rattus tanezumi flavipectus and from the nests of voles, respectively, during 1997 from two sites in western Yunnan Province, China. Sequence analysis of the Bartonella citrate synthase gene (gltA) amplicons obtained from six of 65 grouped flea samples showed that Bartonella genetic variants were clustered in four groups. One from Xenopsylla cheopis was identical to Bartonella tribocorum, whereas the other three genotypes from Ctenophthalmus lushuiensis were related to the vole-associated Bartonella isolates and cat-associated Bartonella clarridgeiae. This is the first detection of this Bartonella variant from fleas in China. Therefore, further investigations are needed to clarify the distribution of Bartonella in rodents and their ectoparasites in China to define the role of these arthropods in the transmission routes of Bartonella.
  Key words:  Bartonella, Ctenophthalmus lushuiensis, genetic diversity, phylogenetic analysis, rodent flea, Xenopsylla cheopis.

	INTRODUCTION

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The genus Bartonella is an oxidase-negative, fastidious, gram-negative hemotropic bacillus. These diverse species infect a variety of mammalian hosts and arthropod vectors, causing a broad spectrum of diseases. In addition to animal hosts in close contact with humans such as cats, dogs, and cattle, rodents are one of the most important reservoirs. Many Bartonella species have been identified in rodents, and there is an increasing number of human and canine infections reportedly caused by rodent-borne Bartonella (Daly et al., 1993; O’Halloran et al., 1998; Kerkhoff et al., 1999; Welch et al., 1999; Kosoy et al., 2003; Serratrice et al., 2003; Fenollar et al., 2005). Bartonellae are transmitted by variety of bloodsucking arthropods; identified vectors include the sand fly (Lutzomyia verrucarum) as a vector for Bartonella bacilliformis (Alexander, 1995), the body louse (Pediculus humanus) for Bartonella quintana (Maurin and Raoult, 1996), and the cat flea (Ctenocephalides felis) for Bartonella henselae (Chomel et al., 1996). For rodent Bartonellae, two vectors have been suggested: The oriental rat flea (Xenopsylla cheopis) has been demonstrated to be a competent vector of an unidentified Bartonella species that infects bank voles (Clethrionomys glareolus) (Krampitz, 1962), and the vole ear mite (Trombicula microti) has been proposed as the vector of Bartonella vinsonii vinsonii (Baker, 1946). Several researchers have suggested a role for rodent fleas as the vector of Bartonella species based on detection of Bartonella DNA from fleas collected worldwide (Parola et al., 2003; Rolain et al., 2003; Stevenson et al., 2003; Reeves et al., 2005; Loftis et al., 2006; Marie et al., 2006). In experimental transmission studies, Bown et al. (2004) demonstrated that the rodent flea Ctenophthalmus nobilis is a competent vector of at least two Bartonella species: Bartonella grahamii, which has previously been associated with human infection (Kerkhoff et al., 1999; Serratrice et al., 2003), and Bartonella taylorii.

Areas with a high prevalence of Bartonella infections in rodents are often characterized by the presence of a diversity of Bartonella species and strains (Birtles et al., 1994, 2001; Kosoy et al., 1997). During the period 2001–2005, a high prevalence of Bartonella was demonstrated through isolation from rodents (Apodemus species, Eothenomys species, and Rattus species) from the west, northwest, southwest, south, and middle of Yunnan Province, China; isolates typically clustered according to the genus of rodent hosts (Ying et al., 2002; Li et al., 2004a; Bai et al., 2005). Although no evidence of vector involvement was provided in these studies, we have isolated Bartonella from cat fleas (Ctenocephalides felis) collected from dogs, and ticks (Boophilus microplus) collected from cattle in northwestern Yunnan Province (Li et al., 2005). Based on the host range of fleas and the high prevalence of Bartonella spp. in rodents in Yunnan Province, we hypothesized that rodent fleas may represent an important vector of Bartonella in rodent populations. In this study, we report molecular evidence for the presence of a diversity of Bartonella genotypes from the rodent fleas collected in Yunnan Province, China, during 1997.

	MATERIALS AND METHODS

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Flea samples

Fleas were collected from rodents and burrows at five sites (Table 1) in Longling, Longchuan, Yingjiang, Lincang, and Gengma counties of southwestern Yunnan Province, China, during March to November of 1997. Sites were located in residential and farmland mountainous areas around the towns. All fleas were classified to species level by an entomologist, and species pools were stored in 70% ethanol.

Eight to 11 fleas of the same species from the same sites were grouped and washed three times with sterile deionized water for surface decontamination. Each group of fleas was placed in a sterilized mortar and crushed using a sterile pestle in liquid nitrogen. DNA was prepared from the crushed fleas using a QIAamp Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.

PCR amplification

Polymerase chain reaction (PCR) amplification was performed using a MyCycler^TM Thermal Cycler (Bio-Rad, Hercules, CA, USA). Three pairs of primers were used for the amplification of Bartonella DNA. The previously described PCR primers: BhCS.781p-BhCS.1137n (5'-GGGGACCAGCTC ATGGT GG-3', 5'-AATGCAAA AAGAACAGTAAACA-3') described by Norman et al. (1995) targets a 379 base-pair (bp) fragment of the citrate synthase gene (gltA); Bh.311p-Bh.452n (5'-CTCTTTCTTCAGATGATGATCC-3', 5'-AA CCAACTGAGCT ACAAGCCCT-3') described by Jensen et al. (2000) targets the 5'-teminal 200 bp partial sequence of the 16S-23S rRNA intergenic spacer region (ITS); and TIle.455p-TAla.885n (5'-GCTTGTA GCTCAGTTGGT-TAG-3', 5'-TGCTTGCAAAGCAGGTGC TCT-3') described by Li et al. (2004b) amplifies about 200–400 bp of tRNA^Ile-tRNA^Ala ITS in the 16S-23S rRNA ITS. DNA amplification was performed using a 25 µl reaction volume. Each reaction mixture contained 1 µl of 10 pmol/µl of each primer, 1.5 U Taq DNA polymerase (Sino-American Biotechnology Co., Beijing, China), 2 µl of 2.5 mM deoxynucleosides triphosphate mixture, 2.5 µl of 10x PCR buffer, 1.5 µl of 20 mM MgCl₂, and 5 µl of DNA template. Each PCR analysis was performed using a negative control (molecular-grade water) and positive control (DNA from B. henselae strain Houston-1 [ATCC49882]). The extraction, creation of the reaction mixture, sample addition, PCR amplification, and product analysis were performed in different rooms to minimize cross contamination.

DNA sequencing and data analyses

The citrate synthase sequence (gltA) is acknowledged as the phylogenetic marker for Bartonella (Birtles and Raoult, 1996; La Scola et al., 2003). To detect and identify Bartonella DNA, we used PCR assays and sequencing of the amplicons derived from the gltA gene of Bartonella species and compared the Bartonella species from the rodent fleas with the available Bartonella strains in GenBank. The PCR amplicons from the gltA fragments amplified using BhCS.781p-BhCS.1137n primers were purified using the Promega Wizard PCR Preps Kit (Promega, Madison, Wisconsin, USA); the purified amplicons were cloned into pGEM-T Easy vector system (Promega) according to the manufacturer’s protocol; and the white-colored recombinant colonies were selected. Sequencing was performed using a Big-Dye^® Terminator v3.1 Cycle Sequencing Kit and an ABI PRISM 3730xl DNA analyzer (Applied Biosystems, Foster City, California, USA). New sequences were compared with published sequences using the BLAST program from the National Center for Biotechnology Information Website (available from: http://www.ncbi.nlm.nih.gov/BLAST/). Values of similarity between the sample sequences and published Bartonella sequences were calculated using the clustalV analysis with the Megalign module of the DNAStar5 Package (DNASTAR, Inc., Madison, Wisconsin, USA). The sequences were aligned using ClustalX version 1.81 (Thompson et al., 1997). Phylogenetic analysis was performed using MEGA3.1 (Kumar et al., 2004) and the PAUP* 4.0 beta version programs (Sinauer Associates, Inc., Sunderland, Massachusetts, USA).

Nucleotide sequence accession numbers

The gltA gene partial sequences of the Bartonella spp. detected in the different groups of rodent fleas in this study are available in the GenBank database under accession numbers DQ884382 (F12YN), DQ884378 (F13YN), DQ884379 (F14YN), DQ884380 (F15YN), DQ884381 (F16YN), and DQ884377 (F47YN).

	RESULTS

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Flea sampling

A total of 641 rodent fleas comprising five species was found: 317 X. cheopis, 174 Leptopsylla segnis, 49 Ctenophthalmus lushuiensis, 50 Nosopsyllus elongatus, and 51 Palaeopsylla remota (Table 2). Xenopsylla cheopis, L. segnis, and N. elongates were collected from the bodies of Rattus tanezumi flavipectus. Ctenophthalmus lushuiensis specimens were collected in three nests of voles (Eothenomys spp. based on range and habitat). Because all the C. lushuiensis samples were mixed, we have no detailed information to infer about the origin of the four positive samples and if they came from different nests of voles. Palaeopsylla remota specimens were collected from the bodies of Anourosorex squamipes. Fleas were divided into 65 groups as follows: 33 groups of X, 17 groups of L. segnis, five groups of C. lushuiensis, five groups of N. elongatus, five groups of P. remota.

Six of the 65 groups were PCR positive (F12YN, F13YN, F14YN, F15YN, F16YN, and F47YN); PCR products were the expected size, using all three primer pairs. Among the six groups collected from two sites, five groups were C. lushuiensis collected from nests of voles and the other was X. cheopis collected from Rattus tanezumi flavipectus (Table 3). Negative controls failed to yield detectable PCR products, whereas the positive controls gave consistent expected PCR products.

A pair-wise comparison was performed using Bartonella deposited in the Gen-Bank. The gltA sequences of F12YN and F14YN were identical and had 100% sequence similarity to a homologous amplicon of the Bartonella strain Em1712yn (AF391282) of Eothenomys miletus from northwestern Yunnan, China (Ying et al., 2002). There was one nucleotide mutation, GA, at position 206 among the DNA sequences of F13YN, F12YN, and F14YN; however, the deduced amino acid composition was not affected. These gltA amplicon sequences differed from those described for all the known Bartonella variants. F15YN had the highest BLAST sequence similarity (97%) to Bartonella strains detected from rodents from Greece (Tea et al., 2004) and Sweden (Holmberg et al., 2003) and 95% similarity to B. clarridgeiae. Based on the pair-wise divergences of 338 bp gltA sequences, F16YN was most similar (98%) to the Bartonella strain Em1531yn (AF391281) (Ying et al., 2002) of E. miletus from the middle of Yunnan among all available sequences of Bartonella deposited in the GenBank at 97.4–97.6% (with nine nucleotide mutations) and similar to F13YN, F12YN, and F14YN, which had no variation in amino acid sequences. DNA similarity values based on the 379 bp of the gltA gene from F47YN compared to other Bartonella strains show that F47YN was more than 99% similar to Bartonella strains from Rattus norvegicus from Beijing (GenBank accession numbers DQ 884392, DQ884391, DQ884389, DQ 884388, DQ884387, and DQ884383), Bartonella strains from R. tanezumi flavipectus and R. norvegicus, fleas from X. cheopis from Indonesia (GenBank accession numbers AY902189, AY902191, AY902190, and AY902188), and the Bartonella strains from Rattus rattus from Portugal (GenBank accession no. AF 086636). All of these Bartonella are almost identical to Bartonella tribocorum, but there are two different amino acids found at two different positions (IT, IV) in F47YN compared to B. tribocorum and the other Bartonella strains, which had the same amino acid (I) at these two positions based on 109 amino acids.

The gltA-based phylogeny of Bartonella

The sequence alignment of gltA of the Bartonella was 338 base pairs. The phylogenetic tree derived from the gltA data set using distance methods with MEGA3.1 showed four clusters of Bartonella strains identified within the flea samples (Fig. 1). The clusters between the flea sample sequences and published Bartonella sequences derived from interpretations of the parsimony analysis with the PAUP* 4.0 beta version were identical (data not shown). The genotype of F15YN formed a clade with the Grecian (strain af102nev, AY435103) and Swedish (strain af82up, AF391788) Bartonella isolates and was related to Bartonella clarridgeiae. Another two groups were the E. miletus–associated Bartonella isolates originating from different geographic areas of Yunnan Province. One group consisted of F12YN, F13YN, F14YN, and Em1712yn (AF391282) using supporting significant bootstrap values (99%). This genotype was similar to another E. miletus–associated group, which included F16YN and Em1531yn (AF391281). These two vole-associated Bartonella spp. represent a genotype that is related to Bartonella taylorii (from Apodemus spp. or voles). The genotype of F47YN, the five Beijing’s R. norvegicus isolates (DQ884392, DQ884391, DQ 884389, DQ884388, DQ884387, and DQ 884383), the four Indonesian isolates (AY902189, AY902191, AY902190, and AY902188), and the one Portuguese Rattus rattus isolate (AF086636) formed a well-supported cluster (94% bootstrap support) and were closely related to B. tribocorum.

View larger version (25K): [in this window] [in a new window]   FIGURE 1. The phylogenetic tree based on 338 base pairs (bp) of gltA for Bartonella strains drawn using the MEGA3.1 software (Sinauer Associates, Inc., Sunderland, Massachusetts, USA). The distance matrix was calculated using the Kimura-2 parameter. The tree was obtained using the neighbor-joining method. The scale bar represents 5% divergence. The support of each branch was determined using 500 bootstrap samples and is indicated by the value at the node. The sequence of Brucella abortus was chosen as the out-group in the tree. The position of new sequences generated in this study is indicated with a black diamond.

	DISCUSSION

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The Bartonella DNA detected in pools of C. lushuiensis from three nests of voles showed genotypic diversity into three clusters according to the gltA gene phylogenetic analysis: F12YN, F14YN, F13YN, and E. miletus Bartonella strain Em-1712yn (AF391282) from the west of Yunnan were the same genotype and were different from the F16YN genotype and another E. miletus Bartonella strain Em1531yn (AF391281) from the middle of Yunnan. Even though they are branched together, there was no solid evidence to indicate that these Bartonella spp. are B. taylorii based on the similarity value (86.5%) of the gltA gene. Based on La Scola et al. (2003), who proposed that newly encountered Bartonella isolates should be considered new species if a 327 bp gltA fragment shares < 96.0% sequence similarity with validated species, we suspect that these flea-associated Bartonella spp. are new genotypes. Further experiments are needed in order to prove it they are two separate new species or subspecies.

Interestingly, the F15YN genotype is more closely related to cat-associated B. clarridgeiae (Kordick et al., 1997) than the other recognized Bartonella strains. A similar report by Loftis et al. (2006) showed that the new Bartonella genotype from X. cheopis in Egypt was related to B. clarridgeiae based on the groEL gene. Within the B. clarridgeiae clade, there are two Bartonella isolates from Apodemus flavicollis from Greece that are 97.0% similar to the genotype of F15YN. Because B. clarridgeiae is a human pathogen, further studies may be warranted related to pathogenesis, range of host species (including rodents), modes of transmission, and epidemiology of human exposure to this Bartonella variant.

Bartonella organisms cause a persistent, often asymptomatic intra-erythrocytic bacteremia in their mammalian hosts. These arthropod-borne pathogens depend on the ecology of the blood-sucking arthropod vector, and an intra-erythrocytic bacterium can be transmitted among natural reservoir hosts, a cycle that is difficult to interrupt in the natural environment. Various flea vectors play an important role in this infection cycle. Xenopsylla cheopis, which uses R. norvegicus and R. tanezumi flavipectus as its major host, is a notorious vector for transmission of plague (Yersinia pestis) and endemic typhus (Rickettsia typhus). Ctenophthalmus lushuiensis is endemic in Yunnan Province, and it is carried by the common flea of Eothenomys spp. (Xie et al., 2000). Our data demonstrate that Bartonella spp. detected in fleas associated with voles is similar to Bartonella spp. from the voles, and the Bartonella spp. detected in the rat fleas is identical to Bartonella isolates from rats. This correlation between animal hosts and arthropod vectors indirectly reflects the characteristics of host specificity of the fleas and partial host specificity of Bartonella (Breitschwerdt and Kordick, 2000) and suggests that the two species of fleas presumably play a role as the transmission vectors of rodent-associated Bartonella in their natural cycle.

Previous investigations concerning Bartonella infections in rodents, cat fleas, and ticks have shown a high genetic diversity in Bartonella in Yunnan, China (Ying et al., 2002; Bai et al., 2005; Li et al., 2005). Our findings further confirm that there are different Bartonella genotypes in the rodents, and the rodent fleas can carry Bartonella in Yunnan Province. Humans are incidental hosts for the numerous rodent-associated Bartonella species. An increasing number of bacteria are identical or closely related to Bartonella and have been obtained from rodents and are being isolated from human patients. Further studies are needed in China to clarify the characteristics of the new flea-associated Bartonella and the potential vectors and the role of the ectoparasites in the route of transmission.

	ACKNOWLEDGMENTS

 
We thank S. Karpathy (Viral and Rickettsial Zoonoses Branch, Centers for Disease Control and Prevention, Atlanta, Georgia, USA) for the phylogenetic analysis using the PAUP* 4.0 beta version, C. Dehio (Division of Molecular Microbiology, Biozentrum, University of Basel, Basel, Switzerland) for critically reading the manuscript, and. M. Y. Kosoy (Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado, USA) for many helpful discussions. We also thank M. E. Eremeeva (Viral and Rickettsial Zoonoses Branch, Centers for Disease Control and Prevention) for reading and making corrections to the manuscript.

This research was supported by the National Natural Science Foundation of China (NSFC) grant 30371246 and the Project of State Scientific & Technological Development of the 10th Five-Year Plan grant 2003BA712A09-02.

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Received for publication 24 February 2007.

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