Field Project in Mali, West Africa In 2012 we completed our fieldwork and sampling of the small mammals and ticks for relapsing fever spirochetes in Mali. The following is a summary of our total effort to date. We set traps on 36 nights during the study in 20 villages with a total effort of 2,909 trap-nights. We captured 744 animals for an overall trap success of 26% (744 animals/2,909 trap-nights) that included 14 species of rodents and shrews;however, seven species comprised 96% (717 of 744 total) of the individuals. Mastomys natalensis, Mastomys erythroleucus, and Mastomys huberti together comprised 76% of all captures (565 of 744 total). Mastomys natalensis was captured most frequently and this animal was the most ubiquitous, as we captured these rats at 17 of the 20 villages we sampled. Praomys daltoni and the African Giant Shrew, Crocidura olivieri, were also captured in many of the villages. In contrast, M. huberti, which is restricted to the Inland Delta of the Niger and Bani Rivers, was captured only in and near Belenikegny. In Soromba and the other nearby villages in the southern-most region we worked, M. natalensis was the only species we captured. Sixty skulls were prepared from nine of the species captured and these samples were deposited as voucher specimens in the Smithsonian Institution, National Museum of Natural History, Division of Mammals, Collection of African Mammals, Washington DC. Identifications of 717 of the 744 animals captured were also supported by mitochrondrial cyt-b DNA sequences, and 36 representative sequences for 12 species are deposited in GenBank. Serum samples from 726 animals were tested for antibodies to investigate prior infection with relapsing fever spirochetes. For all locations, 82 animals (11.3%) were seropositive by immunoblot analysis with antibodies binding to multiple proteins in the borrelia whole-cell lysate and to the purified GlpQ. One or more of the animals captured in 14 of the 20 villages were seropositive. Animals that contained antibodies to relapsing fever spirochetes were distributed from Djougounte to Petaka, the most westward and eastward locations, respectively, that we sampled. However, more than half of the seropositive animals were captured in two villages near Bandiagara: Kalibombo and Doucombo. Here, 45 of 128 of the animals (35%) were seropositive, and included M. natalensis, P. daltoni and C. olivieri. These three species that showed evidence of prior infection were captured in houses and lived in close proximity to humans. Thin blood smears were stained and examined for spirochetes from 724 of 744 animals captured. We detected spirochetes in 16 animals (2 shrews and 14 rodents);actively infected animals were captured in five villages. The number of spirochetes observed varied from 1 to 264 spirochetes in the 50 microscopic fields examined. The majority of infected animals were captured in Kalibombo and Doucombo, the same two villages with the highest prevalence of seropositive animals. In these two villages, 13 of 130 animals (10%) had detectable spirochetes when captured, including M. natalensis (11 individuals), P. daltoni (1 individual) and C. olivieri (1 individual). Serum samples from three slide-positive M. natalensis contained live spirochetes and these samples were inoculated into laboratory mice. We isolated spirochetes from two of the mice and characterized them by multi-locus sequence typing. We focused our tick collecting efforts in Doucombo and Kalibombo because of the higher percentage of infected and seropositive animals there compared to other locations. During April 2011, September October 2011, and January 2012, we found O. sonrai ticks in small mammal burrows inside 24 houses in Doucombo and 14 houses in Kalibombo. In total, we collected 734 O. sonrai, which included 501 nymphs, 146 males and 87 females. From this total, the 208 ticks collected during April 2011 were preserved in 70% ethanol and examined individually by PCR for spirochete infection by targeting only the IGS locus. The mitochondrial 16S rDNA sequence was also determined for five of these ticks, which confirmed their identity as O. sonrai. In this group of 208 ticks, borrelia DNA was detected in 37 of them for a prevalence of infection of 17.8%. DNA sequences for the IGS locus were of two types with 99.2% identity. The two sequences were submitted to GenBank for comparisons to other available sequences and both sequences aligned closest to the IGS sequence of the Achema strain of B. crocidurae that originated from O. sonrai ticks collected in Mauritania. Ticks collected during September - October 2011 and January 2012 were kept alive and shipped to our laboratory in Hamilton. At the time of preparing this report, 5 pools of ticks and 70 single ticks (18 nymphs, 28 males, 24 females) have been fed on individual mice. Two tick pools and 11 individual ticks (2 nymphs, 7 males, 2 females) transmitted spirochetes and infected blood from these mice produced 13 isolates of spirochetes in mBSK-c medium. DNA samples from the 15 isolates that originated from ticks and M. natalensis were analyzed by reverse field and 2-dimensional gel electrophoresis and MLST. Six distinct plasmid profiles (I VI) were found among the undigested borrelia DNA samples from the isolates. Like all borreliae, these spirochetes contained numerous linear plasmids (at least ten) that ranged in size from approximately 12.5 to 100 kilobases and circular plasmids of undetermined size. MLST segregated the 15 spirochete isolates into four primary groups. Each group had identical 16S rDNA and flaB sequences that were unique from members of the other groups, while glpQ sequences segregated the spirochetes into the same groups for 13 of the 15 isolates. Four IGS sequence types were found that included the two sequence types we identified in the ethanol-preserved ticks. The IGS sequences varied among the groups but there was not strict congruence. For example, IGS sequence type 1 was shared among all group A and four of the five group D spirochetes. Overall, three of the four groups of spirochetes (A, B, and C) were distinguished by their unique sequences and plasmid profiles. The fourth group (D) was distinct from the others but also displayed some heterogeneity in the glpQ and IGS sequences. The group D isolate DOS-6 was unique from all other isolates by its glpQ and IGS sequences, and its unique plasmid profile. Sixty-two borrelia DNA sequences have been deposited in GenBank. All DNA sequences confirmed the identity of the spirochetes as the relapsing fever spirochete Borrelia crocidurae.
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