Tom G. Schwan

Experimental Infection of Ixodes scapularis Larvae (Acari: Ixodidae) by Immersion in Low Passage Cultures of Borrelia burgdorferi

Abstract We describe a procedure for the introduction of Borrelia burgdorferi, the spirochetal agent of Lyme disease, into larvae of the tick vector Ixodes scapularis. Internalized spirochetes were observed in larvae examined after 15 or 45 min immersion at 32°C in liquid culture suspensions of low passage B. burgdorferi strain B31. Larval ticks immersed in low passage strain B31 were able to feed to repletion on white-footed mice. Midguts of larvae contained many spirochetes 1 wk postengorgement, while larvae incubated with high passage strain B31 were free of detectable spirochetes at the same interval. Larvae incubated with low passage strain B31 were competent to transmit the pathogen to mice, as shown by serology, reisolation of B. burgdorferi from mice, and xenodiagnosis. Ticks maintained the infection transstadially to the nymphal stage and transmitted the infection to naive mice, replicating an essential aspect of natural infection. This method requires no special equipment and allows artificial infection of large numbers of ticks at the larval stage. It will facilitate studies of the contribution of specific B. burgdorferi genetic loci to tick colonization.

Variable small protein (Vsp)‐dependent and Vsp‐independent pathways for glycosaminoglycan recognition by relapsing fever spirochaetes

Tick‐borne relapsing fever, caused by pathogenic Borrelia such as B. hermsii and B. turicatae, features recurrent episodes of bacteraemia, each of which is caused by a population of spirochaetes that expresses a different variable major protein. Relapsing fever is also associated with the infection of a variety of tissues, such as the central nervous system. In this study, we show that glycosaminoglycans (GAGs) mediate the attachment of relapsing fever spirochaetes to mammalian cells. B. hermsii strain DAH bound to immobilized heparin, and heparin and dermatan sulphate blocked bacterial binding to host cells. Bacterial binding was diminished by inhibition of host cell GAG synthesis or sulphation, or by the enzymatic removal of GAGs. GAGs mediated the attachment of relapsing fever spirochaetes to potentially relevant target cells, such as endothelial and glial cells. B. hermsii was able to attach to GAGs independently of variable major proteins, because strains expressing the variable major proteins Vsp33, Vlp7 or no variable major protein at all each recognized GAGs. Nevertheless, we found that a variable major protein of B. turicatae directly promoted GAG binding by this relapsing fever spirochaete. B. turicatae strain Oz1 serotype B, which expresses the variable major protein VspB, bound to GAGs more efficiently than did B. turicatae Oz1 serotype A, which expresses VspA. Recombinant VspB, but not VspA, bound to heparin and dermatan sulphate. Previous studies have shown that strain Oz1 serotype B grows to higher concentrations in the blood than does Oz1 serotype A. Thus, relapsing fever spirochaetes have the potential to express Vsp‐dependent and Vsp‐independent GAG‐binding activities and, for one pair of highly related B. turicatae strains, differences in GAG binding correlate with differences in tissue tropism.

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

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.

Platelet activation by a relapsing fever spirochaete results in enhanced bacterium-platelet interaction via integrin alphaIIbbeta3 activation

Borrelia hermsii, a spirochaete responsible for relapsing fever in humans, grows to high density in the bloodstream and causes thrombocytopenia. We show here that B. hermsii binds to human platelets. Extended culture in bacteriological medium resulted in both diminished infectivity in vivo and diminished platelet binding in vitro. Platelet binding was promoted by the platelet integrin αIIbβ3: the bacterium bound to purified integrin αIIbβ3, and bacterial binding to platelets was diminished by αIIbβ3 antagonists or by a genetic defect in this integrin. Integrin αIIbβ3 undergoes a conformational change upon platelet activation, and bacteria bound more efficiently to activated rather than resting platelets. Nevertheless, B. hermsii bound at detectable levels to preparations of resting platelets. The bacterium did not recognize a point mutant of αIIbβ3 that cannot acquire an active conformation. Rather, B. hermsii was capable of triggering platelet and integrin αIIbβ3 activation, as indicated by the expression of the platelet activation marker P‐selectin and integrin αIIbβ3 in its active conformation. The degree of platelet activation varied depending upon bacterial strain and growth conditions. Prostacyclin I2, an inhibitor of platelet activation, diminished bacterial attachment, indicating that activation enhanced bacterial binding. Thus, B. hermsii signals the host cell to activate a critical receptor for the bacterium, thereby promoting high‐level bacterial attachment.

Geographic Distribution and Genetic Characterization of Lassa Virus in Sub-Saharan Mali

Background Lassa fever is an acute viral illness characterized by multi-organ failure and hemorrhagic manifestations. Lassa fever is most frequently diagnosed in Nigeria, Sierra Leone, Liberia, and Guinea, although sporadic cases have been recorded in other West African countries, including Mali. The etiological agent of Lassa fever is Lassa virus (LASV), an Arenavirus which is maintained in nature and frequently transmitted to humans by Mastomys natalensis. The purpose of this study was to better define the geographic distribution of LASV-infected rodents in sub-Saharan Mali. Methodologies/Principal Findings Small mammals were live-trapped at various locations across Mali for the purpose of identifying potential zoonotic pathogens. Serological and molecular assays were employed and determined LASV infected rodents were exclusively found in the southern Mali near the border of Côte dIvoire. Overall, 19.4% of Mastomys natalensis sampled in this region had evidence of LASV infection, with prevalence rates for individual villages ranging from 0 to 52%. Full-length genomic sequences were determined using high throughput sequencing methodologies for LASV isolates generated from tissue samples of rodents collected in four villages and confirmed the phylogenetic clustering of Malian LASV with strain AV. Conclusions/Significance The risk of human infections with LASV is greatest in villages in southern Mali. Lassa fever should be considered in the differential diagnosis for febrile individuals and appropriate diagnostic techniques need to be established to determine the incidence of infection and disease in these regions.

Interaction of Borrelia burgdorferi Hbb with the p66 promoter

Borrelia burgdorferi, an agent of Lyme disease, encodes the β3-chain integrin ligand P66. P66 is expressed by B. burgdorferi in the mammal, in laboratory media, and as the bacteria are acquired or transmitted by the tick, but is not expressed by the bacterium in unfed ticks. Attempts to reveal factors influencing expression revealed that P66 was expressed in all in vitro conditions investigated. Candidate regulators identified in a search of the B. burgdorferi genome for homologs to other bacterial transcription factors were cloned and introduced into E. coli carrying a p66 promoter-signal sequence-phoA (alkaline phosphatase, or AP) fusion. Three candidate transcription factors—two that decreased AP activity (Hbb and BB0527), and one that increased AP activity (BBA23)—were identified. BBA23 and BB0527 did not bind to the p66 promoter at physiologically relevant concentrations. In contrast, several promoter fragments, including p66, were bound by Hbb (BB0232), with slightly different affinities. Consistent with results from other laboratories, Hbb appears to recognize multiple DNA sequences. Changes in the expression of p66 and bb0232 in the tick at various points with respect to feeding on mice, along with the results of the reporter experiment in the surrogate host E. coli, are consistent with Hbb/BB0232 being involved in regulating p66 expression.

Lyme disease agent in Egypt

Abstract : We here report our findings after evaluating individuals from various geographical locations in Egypt for exposure to Borrelia burgdorferi. Our initial screening of sera collected from high-risk individuals with fever or unknown origin (FUO) or meningitis and of low-risk individuals (routine blood bank donors) as controls revealed several reactive samples. To our surprise, all reactive samples were from routine blood bank donors living in an isolated oasis of Egypt. We therefore extended our investigation by assaying 61 individuals from this location, all of whom were either blood bank donors or patients at a clinic for dermatological disorders including sexually transmitted diseases (STD). In Egypt, dermatologists generally care for patients with STDs. A commercially available indirect fluorescent antibody (IFA) assay (Lyme-check, Diagnostic Technology, Hauppauge, New York, USA) for determination of antibodies to B. burgdorferi was performed on the sera from 145 patients with FUO, STD and meningitis and from routine blood bank donors.

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

BackgroundFleas 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.MethodsSmall 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.ResultsWe captured 744 small mammals, 68 (9.1xa0%) 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 58.5xa0% 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 3xa0% of 724 blood smears, although 91xa0% 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.ConclusionsXenopsylla 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.

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.