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ABSTRACT |
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INTRODUCTION |
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The objectives of this study were 1) to investigate the prevalence of Bartonella infections among small mammals in an urban slum in Dhaka; 2) to evaluate the diversity of the Bartonella bacterial community by analyzing partial sequence of citrate synthase gene (gltA); and 3) to compare Bartonella strains obtained from rodents in Bangladesh with the strains obtained in other countries.
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MATERIALS AND METHODS |
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Sampling. Collection of samples from small mammals 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 (NRC Publication). The study protocol was approved by the Animal Experimentation Ethics Committee at the ICDDR, B: Center for Health and Population Research in Dhaka, Bangladesh, and by the Animal Care and Use Committee, Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, CO.
Trapped animals were transported to the laboratory, where they were anesthetized and visually inspected to identify species. Blood samples were collected by cardiocentesis. After collection of blood, the animals were humanely euthanized without regaining consciousness. Whole blood was frozen at –20°C for < 2 days and stored at –70°C before shipment on dry ice to CDC laboratory in Fort Collins, CO.
Culturing. Details of procedures used for isolation of Bartonella bacteria from mammalian blood have been described previously.6 Frozen whole blood was thawed and diluted 1:4 in brain heart infusion media containing 5% fungizone (Amphotericin B). The fungizone was added to diminish fungal overgrowth that can occur on plates before the growth of fastidious Bartonella colonies. Diluted blood (0.1 mL) was pipetted onto heart infusion agar plates containing 5% rabbit blood. Plates were incubated aerobically at 35°C in 5% CO2 for up to 4 weeks. Plates were monitored for bacterial growth at least once per week after initial plating. Bacterial colonies were tentatively identified as Bartonella species based on colony morphology and microscopic examination. Each isolate was collected after at least two passages. Single colonies were harvested from initial plates and from subsequent passages; passages were continued until a pure culture, free from contamination, was obtained. Colonies were harvested 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 into individual polypropylene freezer vials.
Polymerase chain reaction. Bartonella isolates were confirmed by polymerase chain reaction (PCR) amplification of a region of the citrate synthase (gltA) gene followed by sequencing the resulting amplicon. DNA extractions were performed on whole bacterial cells using a Qiagen DNA extraction kit (Qiagen, Chasworth, CA). Each PCR was conducted in a PTC 200 Peltier Thermal cycler (MJ Research, Waltham, MA) using the following program parameters: initial denaturing at 95°C for 5 minutes and 95°C for 1 minute, 56°C for 1 minute, 72°C for 1 minute, and repeat steps 2–4 for an additional 34 cycles. PCR products were analyzed for the presence of amplicons of the correct size by electrophoresis in 1.5% agar gels containing ethidium bromide.
Sequencing. Amplicons of the proper size were purified using a QIAquick PCR Purification Kit (Qiagen) followed by CEQ DTCS-Quick Start kit and Qiagen DyeEx 2.0 Spin Kit. Cycle parameters for the sequencing reactions were 96°C for 30 s, 55°C for 1 minute, 65°C for 2 minutes, and each step was repeated for 39 additional cycles. Sequences were analyzed using Lasergene (DNASTAR, Madison, WI) sequence analysis software to determine consensus sequences for the amplified region of the gltA gene. Unique sequences obtained in this study were submitted to GenBank, and accession numbers were assigned. The Clustal V program within Megalign (DNASTAR) was used to align and compare homologous Bartonella gltA sequences from this study and from the Gen-Bank database. The resulting alignment was analyzed using the PAUP software program for parsimony analysis (Center for Biodiversity, Illinois Natural History Survey, Champaign, IL).
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RESULTS |
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Prevalence of Bartonella infection by species of mammals.
Bartonella-like organisms were cultured from the blood of 42.8% (86/201) tested animals. All isolates were genetically confirmed as Bartonella by amplification of a portion of the citrate synthase gene. Prevalence of Bartonella culture–positive animals by species was as follows: BB, 63.2% (48/76); RR, 32.3% (32/99); SM, 42.9% (6/14); MM, 0 (0/12) (Table 1
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Based on nucleotide and amino acid sequence analyses the 10 identified gltA variants form three distinct clusters (Figure 1). The first cluster combined isolates that are close or identical to Bartonella elizabethae. This group predominantly consisted of sequences of isolates obtained from BB (37), but also contained isolates from RR (4), and all isolates from SM (6). Although there were 5-bp differences among nucleotide sequences within this group, peptide sequences revealed a single amino acid difference or no differences between the isolates. In fact, variants AY589561, AY589562, and EF204537 were identical to B. elizabethae by peptide sequence analysis. Interestingly, all six Bartonella sequences obtained from SM were close to each other by nucleotide analysis and different from all other sequences that were found in Bangladesh or elsewhere. The second cluster comprised isolates that are close or identical to Bartonella tribocorum. A single base pair difference was present between AF075164 and AF086636 and AJ005494, but no differences in amino acids sequences were observed. In this group, 20 sequences were from RR isolates and three sequences were from BB isolates. The third cluster consisted of a group of seven identical sequences (AY589565: four from BB and three from RR) and another group of six identical sequences (AY589563: five from RR and one from BB). Although there were 5-bp differences between the nucleotide sequences, as above, there was no difference between amino acids sequences within the third cluster.
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DISCUSSION |
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Comparative analyses of the citrate synthase gene sequences obtained from Bartonella cultures showed that rodents in Dhaka harbor a diverse assemblage of Bartonella genotypes, some of which seem to represent distinct species. Although we used only a portion of the citrate synthase gene for phylogenetic analysis, this gene has been shown to be a reliable tool for distinguishing between closely related Bartonella genotypes.11,12 By using this gene, we were able to compare the variety of Bartonella genotypes identified in mammals from Dhaka with Bartonella strains found in other geographical areas.
The most intriguing results of the study were the discoveries that many rats in Dhaka were infected with a large number of Bartonella strains that are genetically related or identical to homologous sequences of Bartonella strains that were previously found in France, Portugal, and in the United States.9,10 The human pathogen, B. elizabethae also belongs to this phylogenetic lineage.7 Childs and others13 and Ellis and others9 hypothesized an Old World origin for the group of bacteria that include B. elizabethae. A possible explanation for our findings is that this bacterium could have spread from Asia to other parts of the world through infected rats traveling by ship. Most cultures obtained from B. bengalensis in Dhaka (79%) were genetically related to B. elizabethae. This identifies a potential risk for delivery of this Bartonella species from one continent to another as their vertebrate reservoir has been introduced throughout the world. The results also suggest that Bartonella infections could serve as ecological markers to retrospectively describe the dispersion of rats from their original center.
Findings of a high proportion of domestic rats infected with B. elizabethae–related bacteria in Bangladesh raise potential public health concerns of humans acquiring the Bartonella infection and the need to study whether these agents are responsible for cases of endocarditis and unknown febrile illnesses in this country and in other countries of South Asia.
Received March 13, 2007. Accepted for publication May 14, 2007.
Acknowledgments: The authors thank all people from Safeway Pest Control, Dhaka, Bangladesh, who trapped and processed the animals. Authors’ addresses: Ying Bai, Kelly W. Sheff, and Michael Y. Kosoy, Centers for Disease Control and Prevention, Division of Vector Borne Infectious Diseases, Fort Collins, CO 80522. Susan P. Montgomery, Centers for Disease Control and Prevention, Division of Vector Borne Infectious Diseases, Fort Collins, CO 80522. Current address: Centers for Disease Control and Prevention, Division of Parasitic Diseases, Chamblee, GA. Manjur A. Chowdhury, Safeway Pest Control, Dhaka, Bangladesh. Robert F. Breiman, ICDDR, B: Centre for Health and Population Research, Dhaka, Bangladesh. Current address: International Emerging Infections Program, CDC-KEMRI, Nairobi, Kenya. Hidenori Kabeya, Nihon University, College of Bioresource Sciences, Fujisawa, Japan.
* Address correspondence to Michael Y. Kosoy, CDC, Rampart Road, Fort Collins, CO 80521. E-mail: mck3{at}cdc.gov or mkosoy{at}cdc.gov 
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REFERENCES |
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This article has been cited by other articles:
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  Y. Bai, M. Y. Kosoy, K. Lerdthusnee, L. F. Peruski, and J. H. Richardson Prevalence and Genetic Heterogeneity of Bartonella Strains Cultured from Rodents from 17 Provinces in Thailand Am J Trop Med Hyg, November 1, 2009; 81(5): 811 - 816. [Abstract] [Full Text] [PDF]
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S. Harrus, G. K. Bar-Gal, A. Golan, R. Elazari-Volcani, M. Y. Kosoy, D. Morick, B. Avidor, and G. Baneth Isolation and Genetic Characterization of a Bartonella Strain Closely Related to Bartonella tribocorum and Bartonella elizabethae in Israeli Commensal Rats Am J Trop Med Hyg, July 1, 2009; 81(1): 55 - 58. [Abstract] [Full Text] [PDF]
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