• View in gallery

    Phylogenetic relations of 112 sequences of partial gltA of bartonellae isolated from Rattus rats in Thailand. The phylogenetic tree was constructed by N-J method and bootstrap values with 1,000 replicates. Only bootstrap replicates > 70% are noted. The sequences were classified into six groups, each indicated by a rectangle or a circle. The numbers shown in parentheses are Bartonella isolates with identical DNA sequences for the respective Bartonella genetic variants and for the associated host species. RA = Rattus argentiventer; RE = Rattus exulans; RN = Rattus norvegicus; RNI = Rattus nitidus; RR = Rattus rattus; RRE = Rattus remotus.

  • View in gallery

    Phylogenetic relations of 23 sequences of partial gltA of bartonellae isolated from Bandicota spp. and Mus spp. in Thailand. The phylogenetic tree was constructed by N-J method and bootstrap values with 1,000 replicates. Only bootstrap replicates > 70% are noted. The sequences were classified into three groups, each indicated by a rectangle or a circle. The numbers shown in parentheses are Bartonella isolates with identical DNA sequences for the respective Bartonella genetic variants and for the associated host species. BEB = Berylmys berdmorei; BI = Bandicota indica; BS = Bandicota savilei; MC = Mus cervicolor.

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Prevalence and Genetic Heterogeneity of Bartonella Strains Cultured from Rodents from 17 Provinces in Thailand

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  • 1 Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado; Department of Entomology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; International Emerging Infections Program, Thai MOPH-US CDC Collaboration, Bangkok, Thailand

To study the distribution and diversity of Bartonella in rodents from Thailand, 330 rodents belonging to 13 species were tested. The majority (80.6%) of rodents examined belonged to the genus Rattus. Bartonellae were cultured from 41.5% of the rodents with a wide range of prevalence by host species and regions. Sequencing of gltA revealed diverse Bartonella strains. Bartonellae from Rattus spp. belonged to 23 variants and clustered with Bartonella coopersplainensis, Bartonella elizabethae, Bartonella phoceensis, Bartonella rattimassiliensis, Bartonella tribocorum, and an unknown geno-group. Bartonellae from Bandicota spp. belonged to six variants and clustered with B. coopersplainensis, B. rattimassilliensis, and B. tribocorum. Three variants from Mus spp. clustered with B. coopersplainensis or B. rattimassilliensis. The only isolate from a Berylmys berdmorei fell into the B. tribocorum group. The observations highlight the need to study these agents for their role in human febrile illnesses of unknown etiology in Thailand and elsewhere in Asia.

INTRODUCTION

The genus Bartonella includes a variety of genetically and phenotypically related gram-negative bacteria. Infections caused by these microorganisms are widespread in diverse mammalian species, including felines, canines, ruminants, and rodents. In addition to the well-known human pathogens Bartonella bacilliformis, Bartonella quintana, and Bartonella henselae, a growing number of Bartonella species have been associated with emerging diseases with a wide spectrum of manifestations in humans.14 Recently, a new Bartonella strain was isolated from three febrile patients in Thailand, and has been described as Bartonella tamiae.5 Many bartonellae are transmitted by blood-feeding arthropods. Although sandflies and human body lice are known vectors of human Bartonella, fleas, ticks, and other arthropods have been found infected with bartonellae and some have been implicated as potential vectors transmitting bartonellae between animals and from animals to humans.69

Studies conducted in a variety of regions around the world have shown that bartonellae are widely distributed in rodent communities. 10,11 In Asia, Bartonella infections were highly prevalent in rodents from southern China, Bangladesh, and Japan. 1012 Genetic analyses indicated that rodents harbor extremely diverse Bartonella strains. Many novel rodent-associated Bartonella species have been described and at least four—Bartonella elizabethae, Bartonella grahamii, Bartonella vinsonii subsp. arupensis, and Bartonella washoensis—have been implicated as causative agents of human infections, 3,4,13,14 indicating that rodents may serve as reservoirs for Bartonella species that are pathogenic to humans. The degree of host-specificity appears to differ by region and Bartonella species. In Europe, the same Bartonella species were commonly shared in multiple rodent species. 15 In North America and in some Asian countries, particular Bartonella species were restricted to certain rodent species. 3,11,16,17

In Thailand, an early study by Castle and colleagues 16 reported the presence of bartonellae in rodents in the northern province of Chiang Rai. Importantly, a number of isolates obtained from the study were closely related to B. grahamii and B. elizabethae, species that have been associated with human illness. 13,14 The high prevalence of these bacteria in rodents in some communities in Asian countries and the close contact of people with rodents and their ectoparasites increases the likelihood that rodent-borne bartonellae are a significant cause of human morbidity in Thailand. To better understand the distribution and diversity of Bartonella in rodents from Thailand, we analyzed rodent samples collected from 17 provinces across Thailand and characterized the isolates by molecular techniques.

The objectives of this study were to 1) study the prevalence of Bartonella species in rodents from different parts of Thailand by culturing blood samples, 2) evaluate the genetic diversity of recovered Bartonella strains, 3) study host-specificity of Bartonella strains in Thailand, and 4) compare Bartonella strains obtained from rodents in Thailand with those from other countries.

MATERIALS AND METHODS

Study sites, trapping, and processing of small mammals.

Rodents were trapped in 17 provinces from five regions across Thailand in 2004. The regions included the Central Region (Nonthaburi, Phetchaburi, Phra Nakhon Si Ayutthaya, Prachuap Khiri Khan, Sa Kaeo, and Suphan Buri provinces), the Eastern Region (Chon Buri province), the North-eastern Region (Nakhon Ratchasima, Ubon Ratchathani, and Udon Thani provinces), Northern Region (Chiang Mai, Nan, and Tak provinces), and the Southern Region (Chumphon, Nakhon Si Thammarat, Surat Thani, and Yala provinces). Handling of rodents was conducted in compliance with the Animal Welfare Act and other Federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals (National Research Council [NRC] Publication). All procedures involving animals were conducted under animal use protocols approved by the Institutional Animal Care and Use Committees of the Armed Forces Research Institute of Medical Sciences (AFRIMS) (Bangkok, Thailand).

Rodents were captured by live traps baited with bananas or dried fish. Animals were collected from orchards, cultivated rice-fields, grassland areas, edges of dense forest, stream margins, and around houses. Animals were euthanized and identified to species. Blood samples were collected post-mortem by cardiocentesis, stored on dry ice, and transported to the AFRIMS laboratory. Whole blood was stored at −70°C and then shipped on dry ice to the Bartonella Laboratory of the U.S. Centers for Disease Control and Prevention (Fort Collins, CO).

Culturing.

Frozen whole blood was thawed and diluted to 1:4 in brain heart infusion medium (BHI) (BBL, Becton Dickinson Microbiology System, Cockeysville, MD) containing 5% fungizone (Amphotericin B) to reduce the fungal overgrowth. Diluted blood (0.1 mL) was plated on heart infusion agar supplemented with 5% rabbit blood (BBL, Becton Dickinson Microbiology Systems). Agar plates were incubated aerobically at 35°C in 5% CO2 for up to 4 weeks. Plates were monitored for growth at least once per week after initial plating, and every 2–3 days after passaging. Bacterial colonies were tentatively identified as Bartonella species based on colony morphology and microscopic examination. Passages were continued until a pure culture was obtained. Colonies were harvested from initial plates and from subsequent passages by adding 5 mL of BHI plus 10% glycerol to each plate, gently scraping the layer of bacteria from the surface of the agar plate, and storing the material in individual cryovials.

Polymerase chain reaction (PCR) and sequence analyses.

Cultures were verified as bartonellae by PCR amplification of the citrate synthase gene (gltA) using the published primers BhCS781.p and BhCS1137.n 18 followed by sequencing of the amplicon. Crude DNA extracts were obtained by heating a heavy suspension of the organisms at 95°C for 10 minutes. The PCR amplifications were performed in a 50 μL reaction mixture containing 5 μL of 10X PCR buffer, 5 pmol of each primer, 200 μM of each dNTP (Invitrogen, Cergy-Pontoise, France), 2.5 U Taq DNA polymerase (EuroblueTaq, Eurobio, Les Ulis, France), and 2.5 μL DNA. The PCR was carried out in a PTC 200 Peltier thermal cycler (MJ Research, Inc., Watertown, MA) using the following parameters: a 3 minute denaturation at 95°C, followed by 35 cycles of 1 minute denaturation at 95°C, 1 minute annealing at 56°C, and 1 minute elongation at 72°C. Amplification was completed by holding the reaction mixture at 72°C for 10 minutes. The PCR products were visualized for amplicons of the correct size by electrophoresis in a 1.5% agarose gel with ethidium bromide staining. Amplicons of the proper size were purified using a QIAquick PCR Purification Kit (Qiagen, Germantown, MD) and sequenced in both directions using an Applied Biosystems Model 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA). Sequencing reactions were carried out in a PTC 200 Peltier thermal cycler using the same primers for PCR assay at a concentration of 1.6 μM. Cycle parameters for the sequencing reactions were 45 cycles at 96°C for 20 seconds, 50°C for 20 seconds, and 60°C for 4 minutes.

Sequences were analyzed using Lasergene sequence analysis software (version 8, DNASTAR, Madison, WI) to determine the consensus of sequences for the amplified region of the gltA. Unique sequences obtained in this study were submitted to GenBank. The Clustal V program within Megalign module of Lasergene was used to compare homologous Bartonella gltA sequences from the present study and from GenBank. The neighbor-joining (N-J) method by Kimura’s 2-parameter distance method and bootstrap calculation was carried out with 1,000 resamplings. A criterion of ≥ 96% homology to gltA was used to define groups. 19

RESULTS

Rodents.

A total of 330 rodents were sampled in 17 provinces from all five regions of Thailand (Table 1). These rodents represented 13 species of four genera, including the greater bandicoot rat (Bandicota indica) (46), Savile’s bandicoot rat (Bandicota savilei) (7), the small white-toothed rat (Berylmys berdmorei)(1), the rice-field rat (Rattus argentiventer) (3), the Pacific rat (Rattus exulans) (95), the lesser rice-field rat (Rattus losea) (4), the Himalayan field rat (Rattus nitidus) (3), the Norway rat (Rattus norvegicus) (22), the black rat (Rattus rattus) (135), the island rat (Rattus remotus) (2), the red spiny rat (Rattus surifer) (2), the Ryukyu mouse (Mus caroli) (3), and the fawn-colored mouse (Mus cervicolor) (7). The number of rodents sampled from each region varied from 2 (Eastern region) to 105 (Southern region), and the number of rodent species ranged from 2 (Eastern region) to 9 (Northern region). Rattus rattus and R. exulans were the most prevalent rodents in most regions. Rattus norvegicus and B. indica also were common in north-eastern and southern regions, respectively (Table 1).

Prevalence of Bartonella infection by host species and by region.

Bartonella cultures were obtained from blood of 41.5% (137/330) tested rodents. All isolates were confirmed as bartonellae by PCR amplification and sequencing of a portion of the gltA. Bartonellae were detected from 10 rodent species (Table 1). The proportion of culture-positive samples varied by host species, from 3.2% (3/95, R. exulans) to 86.4% (19/22, R. norvegicus) (we only compared four host species [B. indica, R. exulans,R. norvegicus, and R. rattus] with tested samples ≥ 14) (Table 1). Statistical analyses indicated that Bartonella prevalence in the R. exulans was significantly lower than that in the other three species (Fisher’s exact test, P < 0.01), and significantly higher in the R. norvegicus than in the other species (Fisher’s exact test, P < 0.04).

Bartonella prevalence also varied among study regions, ranging from 26.9% (18/67, Central) to 60.0% (63/105, Southern) per region, with the prevalence in the Southern region being significantly higher than in other regions (Fisher’s exact test, P < 0.02) (the Eastern region was excluded from this analysis because of the very small sample size).

Heterogeneity of Rattus rat-associated Bartonella strains.

Bartonella strains associated with Rattus rats were extremely heterogeneous. Twenty-three unique genetic variants with the divergence of 0.3% to 18.6% were identified among 112 sequences obtained from Rattus rats. Of these, 16 were novel and were assigned with GenBank accession nos. FJ655399-FJ655402 and FJ655404-FJ655415 (Table 2). Phylogenetic analyses showed that the 23 variants clustered into six distinct groups (Figure 1). The first group consisted of six variants of 27 sequences obtained from R. exulans (2), R. norvegicus (16), and R. rattus (9). These sequences were similar to Bartonella tribocorum, a bacterium that was originally isolated from R. norvegicus in France. 20 In this group, 18 (64.3%) sequences (R. exulans [1], R. norvegicus [12], R. rattus [5]) were of the same variant (AF075164) obtained from R. norvegicus in New Orleans, Louisiana, USA.2 One variant with three sequences was found only in R. norvegicus and was identical to the strain (AF075163) obtained from R. norvegicus in New Orleans, Louisiana, USA. Two sequences (R. exulans [1], R. rattus [1]) were identical to the strain (AY589566) isolated from R. rattus in Bangladesh. 10 The second group contained three sequences obtained from R. exulans (1) and R. norvegicus (2). Two of them were identical to the other sequences found in R. norvegicus in New Orleans, Louisiana, USA, and Beijing, China (AC nos. AF075166 and DQ884386, respectively). This group clustered with B. elizabethae, a bacterium isolated from a human patient in Massachusetts. 13 The third group, similar to Bartonella rattimassiliensis isolated from R. norvegicus in Marseille, France, 21 was comprised of eight variants of 64 sequences obtained from R. argentiventer (2), R. norvegicus (1), R. nitidus (1), R. rattus (59), and R. remotus (1). In this group, 56 sequences from R. argentiventer (1), R. norvegicus (1), R. nitidus (1), R. rattus (52), and R. remotus (1) were identical to a previously described variant (AF342933) obtained from R. tanazumi in Yunnan, China, 11 and this variant was the most prevalent in this study. The fourth group clustered with Bartonella phoceensis isolated from R. norvegicus in Marseille, France, 21 and included 4 variants of 10 sequences all obtained from R. rattus. The fifth group contained one variant of 7 sequences from R. rattus. This variant, distant from the previously mentioned variants, was identical to an early Bartonella strain (AF363238) isolated from R. norvegicus in Yunnan, China, 11 and belonged to the group of Bartonella coopersplainensis, a new species that was described from R. leucopus in Queensland, Australia (Gundi and others, in press). The last group contained one sequence from R. rattus, and is distant from all other Rattus rat-related Bartonella or any previously described Bartonella species.

Phylogenetic analysis of Bartonella strains associated with rodents other than Rattus rats.

Six variants with divergence of 0.3% to 17.3% were identified among 19 sequences from Bandicota rats. One variant was identical to a previously described Bartonella strain (AY264493) isolated from B. indica from Chiang Rai in Thailand. 16 The other five were novel variants described in GenBank as AC nos. FJ655392-FJ655395 and FJ668633. The variants clustered into three groups (Figure 2). The first group contained three variants of 14 sequences obtained from B. indica (12) and B. savilei (2), and clustered with B. rattimassilliensis. In this group, the variant with AC no. FJ655394 found in two B. indica was identical to a variant found in R. rattus (FJ655409). The second group included two sequences obtained from B. savilei that each represented a unique variant (4.1% divergence, AC nos. FJ655395 and FJ668633) and clustered with B. tribocorum. The two sequences each were identical to sequences of B. tribocorum (AJ005494) and a R. rattus (FJ655400) from the present study. The third group consisted of the last variant of 3 sequences obtained from B. indica, and clustered with B. coopersplainensis. This variant was identical to a previously isolated strain (AY264493) from B. indica from Chiang Rai Province in Thailand. 16

Three sequences obtained from M. cervicolor each represented a unique variant (FJ655390, FJ655391, and FJ668634). The first two were close to each other (98.9% similarity) and belonged to the clade of B. tribocorum. The third sequence (FJ668634) was distant from the previous two sequences (13.6–15.1% divergence) but identical to a variant identified in R. rattus (AF363238) and belonged to the clade of B. coopersplainensis (Figure 2).

A Bartonella strain was isolated from the only Berylmys berdmorei tested in the study. The sequence, assigned with FJ655403, was identical to the sequence of an isolate (FJ655395) obtained from a B. savilei and sequences of B. tribocorum (AJ005494) (Figure 2).

DISCUSSION

The overall prevalence of bartonellae in rodents in Thailand was comparable with reports from other regions, but varied significantly by host species. Within the genus Rattus, the black rat (R. rattus), the Pacific rat (R. exulans), and the Norway rat (R. norvegicus) were the most prevalent species captured. Similar levels of Bartonella infection among these rats were expected as all are commensal and share many characteristics, such as habitat and diet. Interestingly, Bartonella prevalence was very high in the Norway rat (86.4%) and the black rat (65.2%), but low in the Pacific rat (3.16%), suggesting that the Norway rat and the black rat may serve as major reservoirs of Bartonella species in Thailand. An earlier study also reported lower rates of infection with other pathogens (Salmonella species, Vibrio parahaemolyticus, so-called NAG Vibrio, Campylobacter species, Plesiomonas shigelloides, Angiostrongylus cantonensis, Hymenolepis diminuta, Hymenolepis nana, and Raillietina siriraji) in the Pacific rat than in the Norway rat and other mammals. 22 Such results may suggest that the Pacific rat is intrinsically more resistant to these types of infections. Because bartonellae are typically transmitted by arthropods, it is also possible that the ectoparasite loads are lower in Pacific rats compared with black rats and Norway rats. Further studies are required to answer these questions. Bartonellae were not detected in the lesser rice-field rat, the red spiny rat, or the Ryukyu mouse. This may be a reflection of under representation of these species in the collection.

Phylogenetic analyses of the gltA sequences obtained from Bartonella cultures showed that rodents, especially Rattus rats in Thailand, harbor a great number of Bartonella strains representing a variety of distinct species. Six groups/species, including B. elizabethae, B. coopersplainensis, B. phoceensis, B. rattimassiliensis, B. tribocorum, and an unknown genotype were found among Rattus rats of different species, with B. rattimassiliensis and B. tribocorum as the most common species. Moreover, in many cases, the same genetic variant was shared among different Rattus species. Previous studies from different global regions have shown that Rattus rats are reservoirs of B. elizabethae, B. phoceensis, B. rattimassiliensis, and B. tribocorum.2,20,21,23 Our results support these findings and provide new information that Rattus rats can harbor additional Bartonella species.

Testing in rodents other than Rattus rats confirmed a pattern of lacking host-specificity. A majority of Bandicota rodent samples (14/19) carried B. rattimassiliensis, the most prevalent species found in Rattus spp. collected. Bartonella tribocorum, another prevalent species in Rattus spp., was found in rodents of all other three genera as well. Bartonella coopersplainensis was found in both Bandicota and Mus spp. Although identical sequences were found in Rattus and Bandicota spp., and in Rattus and Mus spp., it is unlikely that bartonellae had spillover between rodents of different species 10 because the rodents of different species carrying the same variants were trapped in discrete geographic areas (data not shown).

In summary, our study provides information on the distribution and genetic diversity of Bartonella strains in rodent communities from various habitats in different parts of Thailand. The findings compliment earlier studies conducted in Bangladesh, Japan, southern China, and other regions in Asia, and help elucidate the interaction of bartonellae with their rodent-hosts in natural conditions.

Table 1

Number of rodents captured in 2004 from Thailand tested for Bartonella infection, and Bartonella distribution by host species and region

Table 1
Table 2

Bartonella variants identified in the present study, number of sequences of each variant, and their hosts*

Table 2
Figure 1.
Figure 1.

Phylogenetic relations of 112 sequences of partial gltA of bartonellae isolated from Rattus rats in Thailand. The phylogenetic tree was constructed by N-J method and bootstrap values with 1,000 replicates. Only bootstrap replicates > 70% are noted. The sequences were classified into six groups, each indicated by a rectangle or a circle. The numbers shown in parentheses are Bartonella isolates with identical DNA sequences for the respective Bartonella genetic variants and for the associated host species. RA = Rattus argentiventer; RE = Rattus exulans; RN = Rattus norvegicus; RNI = Rattus nitidus; RR = Rattus rattus; RRE = Rattus remotus.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 5; 10.4269/ajtmh.2009.09-0294

Figure 2.
Figure 2.

Phylogenetic relations of 23 sequences of partial gltA of bartonellae isolated from Bandicota spp. and Mus spp. in Thailand. The phylogenetic tree was constructed by N-J method and bootstrap values with 1,000 replicates. Only bootstrap replicates > 70% are noted. The sequences were classified into three groups, each indicated by a rectangle or a circle. The numbers shown in parentheses are Bartonella isolates with identical DNA sequences for the respective Bartonella genetic variants and for the associated host species. BEB = Berylmys berdmorei; BI = Bandicota indica; BS = Bandicota savilei; MC = Mus cervicolor.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 5; 10.4269/ajtmh.2009.09-0294

*

Address correspondence to Ying Bai, Centers for Disease Control and Prevention, Division of Vector-Borne Infectious Diseases, Fort Collins, CO 80522. E-mail: bby5@cdc.gov

Authors’ addresses: Ying Bai and Michael Y. Kosoy, Centers for Disease Control and Prevention, Division of Vector-Borne Infectious Diseases, Fort Collins, CO 80522, Tel: 1-970-266-3555 and 1-970-266-3522, Fax: 1-970-225-4257, E-mail: bby5@cdc.gov. Kriangkrai Lerdthusnee and Jason H. Richardson, Department of Entomology, Armed Forces Research Institute of Medical Sciences, Bangkok 10400, Thailand, Tel: 66-2-644-4888 and 66-2-644-5777, Fax: 66-2-354-7885. Leonard F. Peruski, International Emerging Infections Program, Thai MOPH-US CDC Collaboration, Bangkok 11000, Thailand, Tel: 66-2-591-4039, Fax: 66-2-591-5753.

Acknowledgments: We wish to express our gratitude to Taweesak Monkanna, Surachai Leepitakrat, Sucheera Insuan, and Weerayut Charoensongserkit from the Department of Entomology, AFRIMS, Bangkok, Thailand, for assistance in trapping and handling rodents.

Disclaimer: Reference to trade names, vendors, proprietary products, or specific equipment is not an endorsement, a guarantee, or a warranty by the Department of the Defense or the U.S. Armed Forces, and does not imply an approval to the exclusion of other products or vendors that also may be suitable. The conclusions and opinions expressed in this document are those of the authors and do not reflect the official position of the U.S. Government, Department of Defense, or the U.S. Army.

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