Composition and transstadial retention of the salivary glands in Ornithodoros hermsi (Acari: Argasidae).

Following several days of blood feeding by larval and nymphal ixodid (hard) ticks, the salivary glands degenerate and are completely replaced in the next life stage. Yet, what happens during the molt of immature argasid (soft) ticks after their rapid and small bloodmeal has remained a mystery. Multiple studies of nymphal Ornithodoros hermsi Wheeler (Acari: Argasidae) ticks infected with the relapsing fever spirochete Borrelia hermsii suggested the salivary glands in these ticks may not disintegrate after feeding. Therefore, cohorts of second-stage O. hermsi nymphs were fed and examined daily after the bloodmeal by fresh dissections and weekly by histological cross-sections of the entire tick. The composition of the salivary glands was typical for argasid ticks in having agranular (Type I) and granular (Type II) acini, the latter being surrounded by a myo-epithelial sheath. In all 197 ticks examined from 1 to 63 days after feeding, morphologically intact salivary glands were present. During apolysis, 5 ticks had extralimital clusters of granular acini adhering to otherwise intact glands. Our observations demonstrate that the salivary glands of nymphal O. hermsi do not disintegrate after feeding and new acini are produced during the molt for incorporation into the existing glands. Cumulatively, these findings suggest a fundamental difference in the transstadial development of argasid and ixodid ticks.

Borrelia nietonii sp. nov.: Relapsing Fever Spirochetes Transmitted By the Tick Ornithodoros hermsi Designated Previously as Borrelia hermsii Genomic Group II.

Background: The taxonomic status of the relapsing fever spirochete Borrelia hermsii in western North America was established in 1942 and based solely on its specific association with the soft tick vector Ornithodoros hermsi. Multilocus sequence typing (MLST) of the 16S rRNA, flaB, gyrB, glpQ, and 16S-23S rRNA intergenic spacer of B. hermsii isolates collected over many years from various geographic locations and biological sources identified two distinct clades designated previously as B. hermsii Genomic Group I (GGI) and Genomic Group II (GGII). To better assess the taxonomic relationship of these two genomic groups to each other and other species of Borrelia, DNA sequences of the entire linear chromosome were determined. Materials and Methods: Genomic DNA samples were prepared from 11 spirochete isolates grown in Barbour-Stoenner-Kelly-H medium. From these preparations, DNA sequences of the entire linear chromosome of two isolates of B. hermsii belonging to each genomic group and seven additional species were determined. Results: Chromosomal sequences of four isolates of B. hermsii contained 919,212 to 922,307 base pairs. DNA sequence identities between the two genomic groups of B. hermsii were 95.86-95.99%, which were more divergent than chromosomal sequences comparing Borrelia parkeri and Borrelia turicatae (97.13%), Borrelia recurrentis and Borrelia duttonii (97.07%), and Borrelia crocidurae and B. duttonii (97.09%). The 3' end of the chromosome of the two GGII isolates also contained a unique intact oppA gene absent from all other species examined. Conclusion: Previous MLST and the chromosomal sequences presented herein support the division of the B. hermsii species complex into two species, B. hermsii sensu stricto ( = GGI) and Borrelia nietonii sp. nov. ( = GGII). We name this unique relapsing fever spirochete in honor of our late friend and colleague Dr. Nathan Nieto for his outstanding contributions to our understanding of tick-borne relapsing fever.

Transovarial Transmission of Borrelia hermsii by Its Tick Vector and Reservoir Host Ornithodoros hermsi.

Transovarial passage of relapsing fever spirochetes (Borrelia species) by infected female argasid ticks to their progeny is a widespread phenomenon. Yet this form of vertical inheritance has been considered rare for the North American tick Ornithodoros hermsi infected with Borrelia hermsii. A laboratory colony of O. hermsi was established from a single infected female and two infected males that produced a population of ticks with a high prevalence of transovarial transmission based on infection assays of single and pooled ticks feeding on mice and immunofluorescence microscopy of eggs and larvae. Thirty-eight of forty-five (84.4%) larval cohorts (groups of larvae originating from the same egg clutch) transmitted B. hermsii to mice over four and a half years, and one hundred and three single and one hundred and fifty-three pooled nymphal and adult ticks transmitted spirochetes during two hundred and fourteen of two hundred and fifty-six (83.6%) feedings on mice over seven and a half years. The perpetuation of B. hermsii for many years by infected ticks only (without acquisition of spirochetes from vertebrate hosts) demonstrates the reservoir competence of O. hermsi. B. hermsii produced the variable tick protein in eggs and unfed larvae infected by transovarial transmission, leading to speculation of the possible steps in the evolution of borreliae from a tick-borne symbiont to a tick-transmitted parasite of vertebrates.

Vector Specificity of the Relapsing Fever Spirochete Borrelia hermsii (Spirochaetales: Borreliaceae) for the Tick Ornithodoros hermsi (Acari: Argasidae) Involves Persistent Infection of the Salivary Glands.

The relapsing fever spirochetes Borrelia hermsii and Borrelia turicatae are each maintained and transmitted in nature by their specific tick vectors, Ornithodoros hermsi Wheeler (Acari: Argasidae) and Ornithodoros turicata (Duges), respectively. The basis for this spirochete and vector specificity is not known, but persistent colonization of spirochetes in the tick's salivary glands is presumed to be essential for transmission by these long-lived ticks that feed in only minutes on their warm-blooded hosts. To examine this hypothesis further, cohorts of O. hermsi and O. turicata were infected with B. hermsii and examined 7-260 d later for infection in their midgut, salivary glands, and synganglion. While the midgut from all ticks of both species at all time points examined were infected with spirochetes, the salivary glands of only O. hermsi remained persistently infected. The salivary glands of O. turicata were susceptible to an early transient infection. However, no spirochetes were observed in these tissues beyond the first 32 d after acquisition. Ticks of both species were fed on mice 112 d after they acquired spirochetes and only those mice fed upon by O. hermsi became infected. Thus, the vector competency for B. hermsii displayed by O. hermsi but not O. turicata lies, in part, in the persistent infection of the salivary glands of the former but not the latter species of tick. The genetic and biochemical mechanisms supporting this spirochete and vector specificity remain to be identified.

Establishment of a Genetically Confirmed Breeding Colony of Mastomys natalensis from Wild-Caught Founders from West Africa.

Mastomys natalensis are a ubiquitous and often dominant rodent across sub-Saharan Africa. Importantly, they are a natural reservoir for microbial pathogens including Lassa virus (LASV), the etiological agent of Lassa fever in humans. Lassa-infected rodents have been documented across West Africa and coincide with regions where annual outbreaks occur. Zoonotic transmission to humans most often occurs directly from infected rodents. Little is known about LASV infection kinetics and transmissibility in M.natalensis, primarily due to available animals. Here, we describe the establishment of a laboratory breeding colony of genetically confirmed M.natalensis from wild-captured rodents. This colony will provide a convenient source of animals to study LASV and other emerging pathogens that utilize M. natalensis in their enzootic lifecycles.

Transgenic functional complementation with a transmission -associated protein restores spirochete infectivity by tick bite.

The relapsing fever spirochete Borrelia hermsii and the Lyme disease spirochete Borrelia burgdorferi sensu stricto each produces an abundant, orthologous, outer membrane protein, Vtp and OspC, respectively, when transmitted by tick bite. Gene inactivation studies have shown that both proteins are essential for spirochete infectivity when transmitted by their respective tick vectors. Therefore, we transformed a vtp-minus mutant of B. hermsii with ospC from B. burgdorferi and examined the behavior of this transgenic spirochete in its soft tick vector Ornithodoros hermsi. IFA staining indicated up to 97.8 % of the transgenic B. hermsii upregulated OspC in the ticks' salivary glands compared to no more than 12.8 % in the midgut, similar to our previous findings with wild-type B. hermsii producing Vtp. Transformation with ospC also restored B. hermsii infectivity to mice when fed upon by infected ticks. Previous sequence analysis of Vtp for 79 isolates and DNA samples of B. hermsii in our laboratory showed this protein is highly polymorphic with 9 divergent amino acid types, yet strikingly the signal peptide is identical among all samples and the same for all OspC signal peptides for B. burgdorferi and related species examined to date. Searches in multiple genome sequences for other species of relapsing fever spirochetes failed to find the same signal peptide sequence to help identify potential transmission-associated proteins. However, some candidate signal peptides with highly similar sequences were found and worthy of future efforts with other species. While OspC of B. burgdorferi restored infectivity to a Vtp-minus mutant of B. hermsii, the functions of these proteins are not known. Our results should stimulate investigators to search for orthologous transmission-associated proteins in other tick-borne spirochetes to better understand how this group of pathogens has coevolved with diverse tick vectors.

Host Competency of the Multimammate Rat Mastomys natalensis Demonstrated by Prolonged Spirochetemias with the African Relapsing Fever Spirochete Borrelia crocidurae.

African multimammate rats, Mastomys natalensis, are widely distributed in sub-Saharan Africa and live in close association with humans. In West Africa, numerous field studies have shown these animals may be naturally infected with the relapsing fever spirochete Borrelia crocidurae, the primary cause of tick-borne relapsing fever in this region of the continent. However, naturally infected individual rats have never been examined over time; therefore, the true host competency of these rats for this spirochete is unknown. Therefore, using animals from an established laboratory colony of M. natalensis, rats were experimentally infected with B. crocidurae and their blood examined to 28 days postinoculation. These animals were highly susceptible to infection and displayed prolonged and cyclic spirochetemias. Our results demonstrate these peridomestic rodents are likely competent hosts for infecting argasid tick vectors and play a primary role in the enzootic cycle for B. crocidurae in West Africa.

Prevalence and Strains of Colorado Tick Fever Virus in Rocky Mountain Wood Ticks in the Bitterroot Valley, Montana.

The Rocky Mountain wood tick, Dermacentor andersoni, has long been known to transmit human pathogens. Within the Bitterroot Valley, Ravalli County, Montana, these agents include Rickettsia rickettsii, Francisella tularensis, and Colorado tick fever virus (CTFV). Found in the western United States where wood ticks occur, CTFV causes a biphasic, febrile illness in humans and persists in enzootic cycles involving the ticks and small mammals. CTFV belongs to the genus Coltivirus, family Reoviridae, whose genome consists of 12 double-stranded RNA segments. Previous studies revealed the presence of CTFV-infected ticks and rodents in select locations within the valley in the 1960s and 1970s, using animal and cell culture methods for detection. We aimed to determine the range and prevalence of the virus in adult questing ticks throughout the valley using molecular tools and to examine the genomic variation between virus strains. Adult D. andersoni ticks were collected during 2002-2003 and 2009-2013. RNA extractions and reverse transcription-polymerase chain reaction were performed on 921 ticks, of which 61 ticks were positive for CTFV, resulting in a 6.6% prevalence of infection. Four genetic loci, one from each of the segments 9, 10, 11, and 12, within the viral genome were sequenced. Reassortment was detected between CTFV sequence strains within the valley. This study confirmed the prevalence of CTFV in D. andersoni ticks within the Bitterroot Valley, which has remained at levels found in the 1950s and 60s. Additional CTFV sequences were obtained and evidence of reassortment was observed between strains within the valley.

Argasid and ixodid systematics: Implications for soft tick evolution and systematics, with a new argasid species list.

The systematics of the genera and subgenera within the soft tick family Argasidae is not adequately resolved. Different classification schemes, reflecting diverse schools of scientific thought that elevated or downgraded groups to genera or subgenera, have been proposed. In the most recent classification scheme, Argas and Ornithodoros are paraphyletic and the placement of various subgenera remains uncertain because molecular data are lacking. Thus, reclassification of the Argasidae is required. This will enable an understanding of soft tick systematics within an evolutionary context. This study addressed that knowledge gap using mitochondrial genome and nuclear (18S and 28S ribosomal RNA) sequence data for representatives of the subgenera Alectorobius, Argas, Chiropterargas, Ogadenus, Ornamentum, Ornithodoros, Navis (subgen. nov.), Pavlovskyella, Persicargas, Proknekalia, Reticulinasus and Secretargas, from the Afrotropical, Nearctic and Palearctic regions. Hard tick species (Ixodidae) and a new representative of Nuttalliella namaqua (Nuttalliellidae), were also sequenced with a total of 83 whole mitochondrial genomes, 18S rRNA and 28S rRNA genes generated. The study confirmed the utility of next-generation sequencing to retrieve systematic markers. Paraphyly of Argas and Ornithodoros was resolved by systematic analysis and a new species list is proposed. This corresponds broadly with the morphological cladistic analysis of Klompen and Oliver (1993). Estimation of divergence times using molecular dating allowed dissection of phylogeographic patterns for argasid evolution. The discovery of cryptic species in the subgenera Chiropterargas, Ogadenus and Ornithodoros, suggests that cryptic speciation is common within the Argasidae. Cryptic speciation has implications for past biological studies of soft ticks. These are discussed in particular for the Ornithodoros (Ornithodoros) moubata and Ornithodoros (Ornithodoros) savignyi groups.

Colony formation in solid medium by the relapsing fever spirochetes Borrelia hermsii and Borrelia turicatae.

Relapsing fever (RF) in North America is caused primarily by the spirochete Borrelia hermsii and is associated with the bite of its tick vector Ornithodoros hermsi. Although this spirochete was known long before the discovery of the Lyme disease (LD) spirochete, Borrelia burgdorferi, basic methods to facilitate the study of B. hermsii have lagged behind. One important technique to expedite the study of the molecular biology and pathogenesis of B. hermsii would be a reliable method to grow and clone these bacteria in solid medium, which we now describe. We have defined the solidifying agent, plating temperature, oxygen concentration, and pH for the efficient plating of two species of RF spirochetes, B. hermsii and Borrelia turicatae. Importantly, this technique allowed us to successfully isolate virulent, clonal cell lines of spirochetes, and to enumerate and isolate viable B. hermsii from infected mouse blood and tick tissues. Our results also demonstrate the value of testing a range of several environmental variables to increase the efficiency of bacterial isolation, which may be helpful for researchers working on other prokaryotes that are intractable for in vitro growth.

Conspecific hyperparasitism: An alternative route for Borrelia hermsii transmission by the tick Ornithodoros hermsi.

Ixodid and argasid ticks may hyperparasitize other individuals of their own species to acquire a blood meal, however most accounts are based on single observations and the behavior has rarely been studied. While maintaining laboratory colonies of Ornithodoros species, we noticed that unfed ticks occasionally fed on other ticks that were feeding on mice, and unfed ticks parasitized engorged ticks when confined together in tubes. Therefore, we investigated hyperparasitism by Ornithodoros hermsi and the ability of these ticks to acquire and transmit the relapsing fever spirochete Borrelia hermsii when feeding on other ticks. Various combinations of unfed and recently engorged male, female and nymphal ticks were confined for 1-2h as individual pairs or in groups, then examined to determine the number of ticks that acquired blood by feeding on others. Unfed O. hermsi males were far more likely to hyperparasitize other ticks than were females and nymphs, as 78.6% of males (114 of 145 ticks) fed when confined with recently engorged ticks. Unfed females and nymphs also hyperparasitized other ticks but far less frequently (only 6.7% combined; 17 of 254 ticks). Infection experiments demonstrated that unfed males acquired B. hermsii when parasitizing nymphs that had recently engorged on a spirochetemic mouse, and unfed infected males transmitted spirochetes to recently engorged nymphs. Some ticks infected via hyperparasitism subsequently transmitted B. hermsii to mice. Hyperparasitism by O. hermsi occurred more frequently than expected, although possibly influenced by our experimental design. The significance of this behavior as it may influence the horizontal transfer of B. hermsii in nature is not known but worthy of future consideration.

Ecological niche modeling and distribution of Ornithodoros hermsi associated with tick-borne relapsing fever in western North America.

Tick-borne relapsing fever in western North America is a zoonosis caused by the spirochete bacterium, Borrelia hermsii, which is transmitted by the bite of infected Ornithodoros hermsi ticks. The pathogen is maintained in natural cycles involving small rodent hosts such as chipmunks and tree squirrels, as well as the tick vector. In order for these ticks to establish sustained and viable populations, a narrow set of environmental parameters must exist, primarily moderate temperatures and moderate to high amounts of precipitation. Maximum Entropy Species Distribution Modeling (Maxent) was used to predict the species distribution of O. hermsi and B. hermsii through time and space based on current climatic trends and future projected climate changes. From this modeling process, we found that the projected current distributions of both the tick and spirochete align with known endemic foci for the disease. Further, global climate models predict a shift in the distribution of suitable habitat for the tick vector to higher elevations. Our predictions are useful for targeting surveillance efforts in areas of high risk in western North America, increasing the efficiency and accuracy of public health investigations and vector control efforts.

Borrelia lanei sp. nov. extends the diversity of Borrelia species in California.

The diversity of Borrelia species discovered in California appears to be particularly high. A divergent group of Borrelia strains collected from Ixodes ticks in California was described by Postic and co-workers and designated 'genomospecies 2' (Postic D, Garnier M, Baranton G. Int J Med Microbiol 2007;297:263-271; Postic D, Ras NM, Lane RS, Hendson M, Baranton G. J Clin Microbiol 1998;36:3497-3504). We performed multilocus sequence analysis (MLSA) using eight housekeeping loci (clpA, clpX, nifS, pepX, pyrG, recG, rplB and uvrA) on 12 strains of this Borreliagenospecies to confirm that these strains form a distinct group within the Borreliaburgdorferi s. l. complex (Margos G, Hojgaard A, Lane RS, Cornet M, Fingerle V et al.Ticks Tick Borne Dis 2010;1:151-158). Phylogenetic and genetic distance analyses based on sequences of the MLSA housekeeping genes corroborated the distinctness of this group; genetic distances to all other members of the B. burgdorferi s.l. complex were 96 % or lower. We propose the name Borrelia lanei sp. nov. for this genospecies in honor of Professor Robert S. Lane, University of California Berkeley, for his contributions to Borrelia and tick research. The type strain for Borrelia lanei sp. nov., strain CA28-91T, has been deposited to two culture collections (=DSM 17992T=CIP 109135T).

Flavivirus Infection of Ixodes scapularis (Black-Legged Tick) Ex Vivo Organotypic Cultures and Applications for Disease Control.

Ixodes scapularis ticks transmit many infectious agents that cause disease, including tick-borne flaviviruses (TBFVs). TBFV infections cause thousands of human encephalitis cases worldwide annually. In the United States, human TBFV infections with Powassan virus (POWV) are increasing and have a fatality rate of 10 to 30%. Additionally, Langat virus (LGTV) is a TBFV of low neurovirulence and is used as a model TBFV. TBFV replication and dissemination within I. scapularis organs are poorly characterized, and a deeper understanding of virus biology in this vector may inform effective countermeasures to reduce TBFV transmission. Here, we describe short-term, I. scapularis organ culture models of TBFV infection. Ex vivo organs were metabolically active for 9 to 10 days and were permissive to LGTV and POWV replication. Imaging and videography demonstrated replication and spread of green fluorescent protein-expressing LGTV in the organs. Immunohistochemical staining confirmed LGTV envelope and POWV protein synthesis within the infected organs. LGTV- and POWV-infected organs produced infectious LGTV and POWV; thus, the ex vivo cultures were suitable for study of virus replication in individual organs. LGTV- and POWV-infected midgut and salivary glands were subjected to double-stranded RNA (dsRNA) transfection with dsRNA to the LGTV 3' untranslated region (UTR), which reduced infectious LGTV and POWV replication, providing a proof-of-concept use of RNA interference in I. scapularis organ cultures to study the effects on TBFV replication. The results contribute important information on TBFV localization within ex vivo I. scapularis organs and provide a significant translational tool for evaluating recombinant, live vaccine candidates and potential tick transcripts and proteins for possible therapeutic use and vaccine development to reduce TBFV transmission.IMPORTANCE Tick-borne flavivirus (TBFV) infections cause neurological and/or hemorrhagic disease in humans worldwide. There are currently no licensed therapeutics or vaccines against Powassan virus (POWV), the only TBFV known to circulate in North America. Evaluating tick vector targets for antitick vaccines directed at reducing TBFV infection within the arthropod vector is a critical step in identifying efficient approaches to controlling TBFV transmission. This study characterized infection of female Ixodes scapularis tick organ cultures of midgut, salivary glands, and synganglion with the low-neurovirulence Langat virus (LGTV) and the more pathogenic POWV. Cell types of specific organs were susceptible to TBFV infection, and a difference in LGTV and POWV replication was noted in TBFV-infected organs. This tick organ culture model of TBFV infection will be useful for various applications, such as screening of tick endogenous dsRNA corresponding to potential control targets within midgut and salivary glands to confirm restriction of TBFV infection.

Serosurvey of Crimean-Congo Hemorrhagic Fever Virus in Cattle, Mali, West Africa.

AbstractCrimean-Congo hemorrhagic fever is a tick-borne disease caused by the arbovirus Crimean-Congo hemorrhagic fever virus (CCHFV, family Bunyaviridae, genus Nairovirus). CCHFV can cause a severe hemorrhagic fever with high-case fatality rates in humans. CCHFV has a wide geographic range and has been described in around 30 countries in the Middle East, Asia, Europe, and Africa including Mali and neighboring countries. To date, little is known about the prevalence rates of CCHFV in Mali. Here, using banked bovine serum samples from across the country, we describe the results of a seroepidemiological study for CCHFV aimed at identifying regions of circulation in Mali. In total, 1,074 serum samples were tested by a modified in-house CCHFV-IgG-enzyme-linked immunosorbent assay (ELISA) with confirmatory testing by commercial ELISA and immunofluorescence assay. Overall, 66% of samples tested were positive for CCHFV-specific IgG antibodies. Regional seroprevalence rates ranged from 15% to 95% and seemed to correlate with cattle density. Our results demonstrate that CCHFV prevalence is high in many regions in Mali and suggest that CCHFV surveillance should be established.

Host associations and genomic diversity of Borrelia hermsii in an endemic focus of tick-borne relapsing fever in western North America.

BACKGROUND: An unrecognized focus of tick-borne relapsing fever caused by Borrelia hermsii was identified in 2002 when five people became infected on Wild Horse Island in Flathead Lake, Montana. The terrestrial small mammal community on the island is composed primarily of pine squirrels (Tamiasciurus hudsonicus) and deer mice (Peromyscus maniculatus), neither of which was known as a natural host for the spirochete. Thus a 3-year study was performed to identify small mammals as hosts for B. hermsii. METHODS: Small mammals were captured alive on two island and three mainland sites, blood samples were collected and examined for spirochetes, and serological tests performed to detect anti-B. hermsii antibodies. Ornithodoros hermsi ticks were collected and fed on laboratory mice to assess infection. Genomic DNA samples from spirochetes isolated from infected mammals and ticks were analyzed by multilocus sequence typing. RESULTS: Eighteen pine squirrels and one deer mouse had detectable spirochetemias when captured, from which 12 isolates of B. hermsii were established. Most pine squirrels were seropositive, and the five species of sciurids combined had a significantly higher prevalence of seropositive animals than did the other six small mammal species captured. The greater diversity of small mammals on the mainland in contrast to the islands demonstrated that other species in addition to pine squirrels were also involved in the maintenance of B. hermsii at Flathead Lake. Ornithodoros hermsi ticks produced an additional 12 isolates of B. hermsii and multilocus sequence typing identified both genomic groups of B. hermsii described previously, and identified a new genomic subdivision. Experimental infections of deer mice with two strains of B. hermsii demonstrated that these animals were susceptible to infection with spirochetes belonging to Genomic Group II but not Genomic Group I. CONCLUSIONS: Pine squirrels are the primary hosts for the maintenance of B. hermsii on the islands in Flathead Lake, however serological evidence showed that numerous additional species are also involved on the mainland. Future studies testing the susceptibility of several small mammal species to infection with different genetic types of B. hermsii will help define their role as hosts in this and other endemic foci.

Fleas and trypanosomes of peridomestic small mammals in sub-Saharan Mali.

BACKGROUND: Fleas are obligate blood-feeding ectoparasites and vectors of several bacterial zoonotic pathogens as well as trypanosomes that parasitize rodents and other small mammals. During investigations of tick- and rodent-borne diseases in Mali, West Africa, we included fleas and rodent-borne trypanosomes, both of which are poorly known in this country, but are attracting greater public health interest. METHODS: Small mammals were captured in 20 Malian villages from December 2007 to October 2011. Fleas were collected and identified to species, and thin blood smears were prepared, stained and examined microscopically for trypanosomes. RESULTS: We captured 744 small mammals, 68 (9.1 %) of which yielded fleas. Two species of fleas, Xenopsylla cheopis and Xenopsylla nubica, were collected from six species of rodents and one species of shrew. Multimammate rats, Mastomys natalensis, were hosts for 58.5 % of all fleas collected. Xenopsylla cheopis was found in the moister southern savannah while X. nubica was mostly restricted to the drier Sahel. Trypanosomes were found in 3 % of 724 blood smears, although 91 % of parasitemic animals originated from two villages where black rats (Rattus rattus) and M. natalensis were the primary hosts and X. cheopis the dominant flea. The trypanosomes were morphologically consistent with the Trypanosoma (Herpetosoma) lewisi group, flea-borne hemoflagellates that parasitize domestic rats. CONCLUSIONS: Xenopsylla cheopis and trypanosomes parasitize peridomestic rats that commingle with people in southern Mali. Given the increasing awareness of flea-borne trypanosomes as possible human pathogens, we hope our findings will stimulate future investigators to examine the potential public health significance of flea-borne trypanosomosis in West Africa.

Laboratory Response to 2014 Ebola Virus Outbreak in Mali.

Aware of the rapid spread of Ebola virus (EBOV) during the current West African epidemic, Mali took several proactive steps to rapidly identify cases within its borders. Under the Mali International Center for Excellence in Research program, a collaboration between the National Institute of Allergy and Infectious Diseases and the Malian Ministry of Higher Education and Scientific Research established a national EBOV diagnostic site at the University of Sciences, Techniques and Technologies of Bamako in the SEREFO Laboratory. Two separate introductions of EBOV occurred in Mali from neighboring Guinea, but both chains of transmission were quickly halted, and Mali was declared "Ebola free" on 18 January 2015 and has remained so since. The SEREFO Laboratory was instrumental in the success of Mali's Ebola response by providing timely and accurate diagnostics. As of today, the SEREFO Laboratory has tested 103 samples from 88 suspected cases, 10 of which were EBOV positive, since the Ebola diagnostics unit started in April 2014. The establishment of Ebola diagnostics in the SEREFO Laboratory, safety precautions, and diagnostics are described.

Transcriptional Profiling the 150 kb Linear Megaplasmid of Borrelia turicatae Suggests a Role in Vector Colonization and Initiating Mammalian Infection.

Adaptation is key for survival as vector-borne pathogens transmit between the arthropod and vertebrate, and temperature change is an environmental signal inducing alterations in gene expression of tick-borne spirochetes. While plasmids are often associated with adaptation, complex genomes of relapsing fever spirochetes have hindered progress in understanding the mechanisms of vector colonization and transmission. We utilized recent advances in genome sequencing to generate the most complete version of the Borrelia turicatae 150 kb linear megaplasmid (lp150). Additionally, a transcriptional analysis of open reading frames (ORFs) in lp150 was conducted and identified regions that were up-regulated during in vitro cultivation at tick-like growth temperatures (22°C), relative to bacteria grown at 35°C and infected murine blood. Evaluation of the 3' end of lp150 identified a cluster of ORFs that code for putative surface lipoproteins. With a microbe's surface proteome serving important roles in pathogenesis, we confirmed the ORFs expression in vitro and in the tick compared to spirochetes infecting murine blood. Transcriptional evaluation of lp150 indicates the plasmid likely has essential roles in vector colonization and/or initiating mammalian infection. These results also provide a much needed transcriptional framework to delineate the molecular mechanisms utilized by relapsing fever spirochetes during their enzootic cycle.