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      Endemic Foci of the Tick-Borne Relapsing Fever Spirochete Borrelia crocidurae in Mali, West Africa, and the Potential for Human Infection

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          Abstract

          Background

          Tick-borne relapsing fever spirochetes are maintained in endemic foci that involve a diversity of small mammals and argasid ticks in the genus Ornithodoros. Most epidemiological studies of tick-borne relapsing fever in West Africa caused by Borrelia crocidurae have been conducted in Senegal. The risk for humans to acquire relapsing fever in Mali is uncertain, as only a few human cases have been identified. Given the high incidence of malaria in Mali, and the potential to confuse the clinical diagnosis of these two diseases, we initiated studies to determine if there were endemic foci of relapsing fever spirochetes that could pose a risk for human infection.

          Methodology/Principal Findings

          We investigated 20 villages across southern Mali for the presence of relapsing fever spirochetes. Small mammals were captured, thin blood smears were examined microscopically for spirochetes, and serum samples were tested for antibodies to relapsing fever spirochetes. Ornithodoros sonrai ticks were collected and examined for spirochetal infection. In total, 11.0% of the 663 rodents and 14.3% of the 63 shrews tested were seropositive and 2.2% of the animals had active spirochete infections when captured. In the Bandiagara region, the prevalence of infection was higher with 35% of the animals seropositive and 10% infected. Here also Ornithodoros sonrai were abundant and 17.3% of 278 individual ticks tested were infected with Borrelia crocidurae. Fifteen isolates of B. crocidurae were established and characterized by multi-locus sequence typing.

          Conclusions/Significance

          The potential for human tick-borne relapsing fever exists in many areas of southern Mali.

          Author Summary

          Tick-borne relapsing fever is a spirochete-caused, recurrent illness acquired by the bite of fast-feeding ticks. In Mali, the potential for people to acquire relapsing fever is unknown although a few human cases have been reported there. Human malaria is also abundant in Mali, and could be complicating the diagnosis of relapsing fever. The relapsing fever spirochete, Borrelia crocidurae, is maintained in natural cycles involving small mammals and its tick vector Ornithodoros sonrai. Therefore, we investigated 20 villages across southern Mali to determine if relapsing fever spirochetes were circulating in small mammals and ticks that lived with people. We found that 11.3% of the 726 mammals tested showed evidence of prior infection, while 2.2% of the animals were actively infected. The tick vector was abundant in two villages we sampled, and overall 17.3% of the individual ticks tested were infected with spirochetes. We also isolated the spirochetes, Borrelia crocidurae, from rodents and ticks and compared their genetic makeup to other species of African spirochetes. We conclude that in some areas of Mali, people are at risk of acquiring tick-borne relapsing fever. Therefore, we recommend that blood smears from acutely ill patients be examined microscopically for spirochetes.

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          Most cited references42

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          Phylogeny of hard- and soft-tick taxa (Acari: Ixodida) based on mitochondrial 16S rDNA sequences.

          Ticks are parasitiform mites that are obligate hematophagous ectoparasites of amphibians, reptiles, birds, and mammals. A phylogeny for tick families, subfamilies, and genera has been described based on morphological characters, life histories, and host associations. To test the existing phylogeny, we sequenced approximately 460 bp from the 3' end of the mitochondrial 16S rRNA gene (rDNA) in 36 hard- and soft-tick species; a mesostigmatid mite, Dermanyssus gallinae, was used as an outgroup. Phylogenies derived using distance, maximum-parsimony, or maximum-likelihood methods were congruent. The existing phylogeny was largely supported with four exceptions. In hard ticks (Ixodidae), members of Haemaphysalinae were monophyletic with the primitive Amblyomminae and members of Hyalomminae grouped within the Rhipicephalinae. In soft ticks (Argasidae), the derived phylogeny failed to support a monophyletic relationship among members of Ornithodorinae and supported placement of Argasinae as basal to the Ixodidae, suggesting that hard ticks may have originated from an Argas-like ancestor. Because most Argas species are obligate bird octoparasites, this result supports earlier suggestions that hard ticks did not evolve until the late Cretaceous.
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            Sequence typing reveals extensive strain diversity of the Lyme borreliosis agents Borrelia burgdorferi in North America and Borrelia afzelii in Europe.

            The genetic polymorphism of Borrelia burgdorferi and Borrelia afzelii, two species that cause Lyme borreliosis, was estimated by sequence typing of four loci: the rrs-rrlA intergenic spacer (IGS) and the outer-membrane-protein gene p66 on the chromosome, and the outer-membrane-protein genes ospA and ospC on plasmids. The major sources of DNA for PCR amplification and sequencing were samples of the B. burgdorferi tick vector Ixodes scapularis, collected at a field site in an endemic region of the north-eastern United States, and the B. afzelii vector Ixodes ricinus, collected at a similar site in southern Sweden. The sequences were compared with those of reference strains and skin biopsy isolates, as well as database sequences. For B. burgdorferi, 10-13 alleles for each of the 4 loci, and a total of 9 distinct clonal lineages with linkage of all 4 loci, were found. For B. afzelii, 2 loci, ospC and IGS, were examined, and 11 IGS genotypes, 12 ospC alleles, and a total of 9 linkage groups were identified. The genetic variants of B. burgdorferi and B. afzelii among samples from the field sites accounted for the greater part of the genetic diversity previously reported from larger areas of the north-eastern United States and central and northern Europe. Although ospC alleles of both species had higher nucleotide diversity than other loci, the ospC locus showed evidence of intragenic recombination and was unsuitable for phylogenetic inference. In contrast, there was no detectable recombination at the IGS locus of B. burgdorferi. Moreover, beyond the signature nucleotides that specified 10 IGS genotypes, there were additional nucleotide polymorphisms that defined a total of 24 subtypes. Maximum-likelihood and parsimony cladograms of B. burgdorferi aligned IGS sequences revealed the subtype sequences to be terminal branches of clades, and the existence of at least three monophyletic lineages within B. burgdorferi. It is concluded that B. burgdorferi and B. afzelii have greater genetic diversity than had previously been estimated, and that the IGS locus alone is sufficient for strain typing and phylogenetic studies.
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              Identification of an uncultivable Borrelia species in the hard tick Amblyomma americanum: possible agent of a Lyme disease-like illness.

              Bites from the hard tick Amblyomma americanum are associated with a Lyme disease-like illness in the southern United States. To identify possible etiologic agents for this disorder, A. americanum ticks were collected in Missouri, Texas, New Jersey, and New York and examined microscopically. Uncultivable spirochetes were present in approximately 2% of the ticks. Borrelia genus-specific oligonucleotides for the flagellin and 16S rRNA genes were used for amplification of DNA. Products were obtained from ticks containing spirochetes by microscopy but not from spirochete-negative ticks. Sequences of partial genes from spirochetes in Texas and New Jersey ticks differed by only 2 of 641 nucleotides for flagellin and 2 of 1336 nucleotides for 16S rRNA. Phylogenetic analysis showed that the spirochete was a Borrelia species distinct from previously characterized members of this genus, including Borrelia burgdorferi. Gene amplification could be used to detect these spirochetes in ticks and possible mammalian hosts.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, USA )
                1935-2727
                1935-2735
                November 2012
                29 November 2012
                : 6
                : 11
                : e1924
                Affiliations
                [1 ]Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Montana, United States of America
                [2 ]Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Twinbrook, Maryland, United States of America
                [3 ]Laboratory of Virology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Montana, United States of America
                [4 ]Malaria Research and Training Center, University of Sciences, Techniques and Technologies of Bamako, Bamako, Mali
                University of California Davis, United States of America
                Author notes

                The authors have declared that no completing interests exist.

                Conceived and designed the experiments: TGS JMA RJF NS OM SFT. Performed the experiments: TGS JMA JEL RJF SJR BNM DS NS OM SFT. Analyzed the data: TGS RJF SJR BNM. Contributed reagents/materials/analysis tools: TGS JMA RJF SJR BNM. Wrote the paper: TGS.

                [¤]

                Current address: Department of Biological Sciences, Mississippi State University, Starkville, Mississippi, United States of America

                Article
                PNTD-D-12-00992
                10.1371/journal.pntd.0001924
                3510061
                23209863
                674911dc-c8da-43de-8911-670715640dcc
                Copyright @ 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.

                History
                : 6 August 2012
                : 12 October 2012
                Page count
                Pages: 13
                Funding
                This study was supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Ecology
                Microbiology
                Bacterial Pathogens
                Host-Pathogen Interaction
                Vector Biology
                Zoology
                Mammalogy
                Parasitology
                Medicine
                Epidemiology
                Global Health
                Public Health

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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