Ectoparasites (sucking lice, fleas and ticks) of small mammals in southeastern Kenya.

During 1998-2000, at least 14 species (n = 309) of small mammals were live-trapped and examined for ectoparasites in moist forests of the Taita and Shimba Hills and drier savannah habitats of Nguruman, southeastern Kenya. Ectoparasites were recorded from 11 species of mammals. Five species of sucking lice [Hoplopleura inexpectans Johnson, H. intermedia Kellogg & Ferris, Polyplax reclinata (Nitzsch), P. waterstoni Bedford and Schizophthirus graphiuri Ferris], six species of fleas (Ctenophthalmus leptodactylous Hubbard, Dinopsyllus grypurus Jordan & Rothschild, D. lypusus Jordan & Rothschild, Hypsophthalmus campestris Jordan & Rothschild, Listropsylla basilewskyi Smit and Xiphiopsylla lippa Jordan) and at least six species of ticks (Amblyomma sp., Haemaphysalis sp., Ixodes sp., I. alluaudi Neumann, I. cumulatimpunctatus Schulze, I. muniensis Arthur & Burrow and Rhipicephalus sp.) were recorded from these hosts. Four of the five species of sucking lice were host specific whereas P. reclinata was recorded from two different species of white-toothed shrews, Crocidura spp. Although fleas and ticks were less host specific, C. leptodactylous, D. grypurus and I. cumulatimpunctatus were only recorded from the murid rodent Praomys delectorum (Thomas), Amblyomma sp. was only recorded from the nesomyid rodent Beamys hindei Thomas, Rhipicephalus sp. was only recorded from the murid Lemniscomys striatus (L.) and I. muniensis was only recorded from the dormouse Graphiurus microtis (Noack). More species of ectoparasites and significantly greater infestation prevalences were recorded from small mammals in moist habitats compared with those from the savannah habitat. At least one of the fleas recorded, D. lypusus, is a known vector of Yersinia pestis Lehmann & Neumann, the causative agent of plague, which is present in the region.

Temporal regulation of outer surface proteins of the Lyme-disease spirochaete Borrelia burgdorferi.

In the 20 years since the first agent of Lyme disease was discovered, much interest has focused on the possible biological roles of a few outer surface proteins (Osps) in the alternating life cycle that includes ticks and vertebrate hosts. Two major proteins, OspA and OspC, are differentially regulated by the spirochaete Borrelia burgdorferi during the several days when ticks feed. The reciprocal decrease in OspA with the rapid up-regulation of OspC by the spirochaetes when ticks are feeding suggests that OspA aids in spirochaete attachment while OspC assists in the dissemination of spirochaetes from tick to vertebrate. Future experiments in ticks with mutant spirochaetes that lack these proteins should clarify the speculative functions currently given to these proteins.

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 alphaIIbbeta3: the bacterium bound to purified integrin alphaIIbbeta3, and bacterial binding to platelets was diminished by alphaIIbbeta3 antagonists or by a genetic defect in this integrin. Integrin alphaIIbbeta3 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 alphaIIbbeta3 that cannot acquire an active conformation. Rather, B. hermsii was capable of triggering platelet and integrin alphaIIbbeta3 activation, as indicated by the expression of the platelet activation marker P-selectin and integrin alphaIIbbeta3 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.

Serodiagnosis of Louse-Borne relapsing fever with glycerophosphodiester phosphodiesterase (GlpQ) from Borrelia recurrentis.

Human louse-borne relapsing fever occurs in sporadic outbreaks in central and eastern Africa that are characterized by significant morbidity and mortality. Isolates of the causative agent, Borrelia recurrentis, were obtained from the blood of four patients during a recent epidemic of the disease in southern Sudan. The glpQ gene, encoding glycerophosphodiester phosphodiesterase, from these isolates was sequenced and compared with the glpQ sequences obtained from other relapsing-fever spirochetes. Previously we showed that GlpQ of Borrelia hermsii is an immunogenic protein with utility as a serological test antigen for discriminating tick-borne relapsing fever from Lyme disease. In the present work, we cloned and expressed the glpQ gene from B. recurrentis and used recombinant GlpQ in serological tests. Acute- and convalescent-phase serum samples obtained from 42 patients with louse-borne relapsing fever were tested with an indirect immunofluorescence assay (IFA) and an enzyme-linked immunosorbent assay (ELISA) that used whole cells of B. recurrentis and with immunoblotting to whole-cell lysates of the spirochete and Escherichia coli producing recombinant GlpQ. The geometric mean titers of the acute- and convalescent-phase serum samples measured by IFA were 1:83 and 1:575, respectively. The immunoblot analysis identified a high level of reactivity and seroconversion to GlpQ, and the assay was more sensitive than the whole-cell IFA and ELISA using purified, recombinant histidine-tagged GlpQ. Serum antibodies to GlpQ and other antigens persisted for 27 years in one patient. We conclude that assessment of anti-GlpQ antibodies will allow serological confirmation of louse-borne relapsing fever and determination of disease prevalence.

The relapsing fever spirochete Borrelia hermsii contains multiple, antigen-encoding circular plasmids that are homologous to the cp32 plasmids of Lyme disease spirochetes.

Borrelia hermsii, an agent of tick-borne relapsing fever, was found to contain multiple circular plasmids approximately 30 kb in size. Sequencing of a DNA library constructed from circular plasmid fragments enabled assembly of a composite DNA sequence that is homologous to the cp32 plasmid family of the Lyme disease spirochete, B. burgdorferi. Analysis of another relapsing fever bacterium, B. parkeri, indicated that it contains linear homologs of the B. hermsii and B. burgdorferi cp32 plasmids. The B. hermsii cp32 plasmids encode homologs of the B. burgdorferi Mlp and Bdr antigenic proteins and BlyA/BlyB putative hemolysins, but homologs of B. burgdorferi erp genes were absent. Immunoblot analyses demonstrated that relapsing fever patients produced antibodies to Mlp proteins, indicating that those proteins are synthesized by the spirochetes during human infection. Conservation of cp32-encoded genes in different Borrelia species suggests that their protein products serve functions essential to both relapsing fever and Lyme disease spirochetes. Relapsing fever borreliae replicate to high levels in the blood of infected animals, permitting direct detection and possible functional studies of Mlp, Bdr, BlyA/BlyB, and other cp32-encoded proteins in vivo.

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.

Temporal changes in outer surface proteins A and C of the lyme disease-associated spirochete, Borrelia burgdorferi, during the chain of infection in ticks and mice.

The Lyme disease-associated spirochete, Borrelia burgdorferi, is maintained in enzootic cycles involving Ixodes ticks and small mammals. Previous studies demonstrated that B. burgdorferi expresses outer surface protein A (OspA) but not OspC when residing in the midgut of unfed ticks. However, after ticks feed on blood, some spirochetes stop making OspA and express OspC. Our current work examined the timing and frequency of OspA and OspC expression by B. burgdorferi in infected Ixodes scapularis nymphs as they fed on uninfected mice and in uninfected I. scapularis larvae and nymphs as they first acquired spirochetes from infected mice. Smears of midguts from previously infected ticks were prepared at 12- or 24-h intervals following attachment through repletion at 96 h, and spirochetes were stained for immunofluorescence for detection of antibodies to OspA and OspC. As shown previously, prior to feeding spirochetes in nymphs expressed OspA but not OspC. During nymphal feeding, however, the proportion of spirochetes expressing OspA decreased, while spirochetes expressing OspC became detectable. In fact, spirochetes rapidly began to express OspC, with the greatest proportion of spirochetes having this protein at 48 h of attachment and then with the proportion decreasing significantly by the time that the ticks had completed feeding. In vitro cultivation of the spirochete at different temperatures showed OspC to be most abundant when the spirochetes were grown at 37 degrees C. Yet, the synthesis of this protein waned with continuous passage at this temperature. Immunofluorescence staining of spirochetes in smears of midguts from larvae and nymphs still attached or having completed feeding on infected mice demonstrated that OspA but not OspC was produced by these spirochetes recently acquired from mice. Therefore, the temporal synthesis of OspC by spirochetes only in feeding ticks that were infected prior to the blood meal suggests that this surface protein is involved in transmission from tick to mammal but not from mammal to tick.

Effects of environmental pH on membrane proteins in Borrelia burgdorferi.

Borrelia burgdorferi, the causative agent of Lyme disease, alternates between the microenvironments of the tick vector, Ixodes scapularis, and a mammalian host. The environmental conditions the spirochete encounters during its infectious cycle are suspected to differ greatly in many aspects, including available nutrients, temperature, and pH. Here we identify alterations in the membrane protein profile, as determined by immunoblotting and two-dimensional nonequilibrium pH gradient gel electrophoresis (2D-NEPHGE), that occur in virulent B. burgdorferi B31 as the pH of the medium is altered. Initial comparisons of cultures incubated at pHs 6.0, 7.0, and 8.0 yielded alterations in the expression of seven membrane proteins as determined by probing with hyperimmune rabbit serum. Six of these membrane proteins (54, 45, 44, 43, 35, and 24 kDa) were either present in increased amounts in or solely expressed by cultures incubated at pHs 6.0 and 7.0. The 24-kDa protein that decreased in expression at pH 8.0 was identified as outer surface protein C (OspC). In addition, a 42-kDa membrane protein increased in amount in cultures incubated at pH 8.0. Similar changes were observed with serum from a mouse infected by tick bite, with the recognition of two additional bands (48 and 46 kDa) unique to pHs 6. 0 and 7.0. When membrane fractions were analyzed by 2D-NEPHGE, at least 37 changes in the membrane protein profile between cells incubated at pHs 6.0, 7.0, and 8.0 were observed by immunoblotting and silver staining. Environmental cues such as pH may prove important in the regulation of virulence determinants and factors necessary for the adaptation of B. burgdorferi to the tick or mammalian microcosm.

Lethal effect of Rickettsia rickettsii on its tick vector (Dermacentor andersoni).

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, was lethal for the majority of experimentally and transovarially infected Rocky Mountain wood ticks (Dermacentor andersoni). Overall, 94.1% of nymphs infected as larvae by feeding on rickettsemic guinea pigs died during the molt into adults and 88. 3% of adult female ticks infected as nymphs died prior to feeding. In contrast, only 2.8% of uninfected larvae failed to develop into adults over two generations. Infected female ticks incubated at 4 degreesC had a lower mortality (80.9%) than did those held at 21 degreesC (96.8%). Rickettsiae were vertically transmitted to 39.0% of offspring, and significantly fewer larvae developed from infected ticks. The lethal effect of R. rickettsii may explain the low prevalence of infected ticks in nature and affect its enzootic maintenance.

Tick-borne relapsing fever in British Columbia, Canada: first isolation of Borrelia hermsii.

The spirochete that causes tick-borne relapsing fever, Borrelia hermsii, was isolated in pure culture during 1995 and 1996 from three acutely ill human patients infected in southern British Columbia, Canada. The geographic area of exposure is a known focus of this disease dating back to 1930 when the first case was recognized in a human. Analyses of plasmid DNA, protein profiles, and reactivity with a species-specific monoclonal antibody identified the new isolates of spirochetes as B. hermsii, all of which were most similar to an isolate of this spirochete from northern California described previously. These are the first reported isolates of B. hermsii from Canada.

Evaluation of the role of the Yersinia pestis plasminogen activator and other plasmid-encoded factors in temperature-dependent blockage of the flea.

Yersinia pestis, the plague bacillus, has a plasminogen activator (pla) gene on the 9.5-kb plasmid pPla that is hypothesized to play a role in producing the foregut blockage in the flea vector that precedes transmission. In this study, however, Y. pestis that lacked pPla, the 70-kb virulence plasmid, or both plasmids, proved able to block Xenopsylla cheopis fleas normally. Blockage rates decreased with increasing environmental temperature for fleas infected with either wild type or pPla- Y. pestis. Thus, procoagulant ability of the Y. pestis pla gene product does not mediate blockage, nor does its ability to induce fibrinolysis at>28 degreesC account for failure to block at elevated temperatures. A Y. pestis strain that lacked all or part of the third plasmid of 110 kb, however, failed to colonize the flea midgut normally, indicating that one or more genes on the large plasmid may be required for vectorborne transmission.

Bloodstream- versus tick-associated variants of a relapsing fever bacterium.

The relapsing fever spirochete, Borrelia hermsii, alternates infections between a mammal and a tick vector. Whether the spirochete changes phenotypically in the different hosts was examined by allowing the tick vector Ornithodoros hermsi to feed on mice infected with serotype 7 or serotype 8 of B. hermsii. Upon infection of ticks, the spirochetal serotype-specific variable major proteins (Vmps) 7 and 8 became undetectable and were replaced by Vmp33. This switch from a bloodstream- to tick-associated phenotype could be induced in culture by a decrease in temperature. After tick-bite transmission back to mice, the process was reversed and the spirochetes resumed expression of the same Vmp present in the previous infectious blood meal.

Estimation of vector infectivity rates for plague by means of a standard curve-based competitive polymerase chain reaction method to quantify Yersinia pestis in fleas.

The prevalence of infectivity within a vector population is a critical factor in arthropod-borne disease epidemiology but it is difficult to estimate. In the case of bubonic plague, infective flea vectors contain large numbers of Yersinia pestis within a bacterial mass that blocks the flea's foregut, and only such blocked fleas are important for biologic transmission. A bacterial quantitation method could therefore be used to assess the prevalence of plague-infective (blocked) fleas in a population. We developed a standard, curve-based, competitive polymerase chain reaction (PCR) procedure to quantitate Y. pestis in individual fleas. The quantitative PCR (Q-PCR) method equaled a colony count reference method in accuracy and precision when evaluated using mock samples and laboratory-infected fleas. The Q-PCR was more reliable than colony count, however, for field-collected fleas and for blocked fleas collected after their death. In a sample of fleas collected from a prairie dog colony in the aftermath of a plague epizootic, 48% were infected but less than 2% contained numbers of Y. pestis indicative of blockage. The method provides a means to monitor plague epizootics and associated risks of flea-borne transmission to humans, and is applicable to the study of other vector-borne diseases.

Borrelia burgdorferi erp proteins are immunogenic in mammals infected by tick bite, and their synthesis is inducible in cultured bacteria.

Borrelia burgdorferi, the causative agent of Lyme disease, can contain multiple genes encoding different members of the Erp lipoprotein family. Some arthropod-borne bacteria increase the synthesis of proteins required for transmission or mammalian infection when cultures are shifted from cool, ambient air temperature to a warmer, blood temperature. We found that all of the erp genes known to be encoded by infectious isolate B31 were differentially expressed in culture after a change in temperature, with greater amounts of message being produced by bacteria shifted from 23 to 35 degrees C than in those maintained at 23 degrees C. Mice infected with B31 by tick bite produced antibodies that recognized each of the Erp proteins within 4 weeks of infection, suggesting that the Erp proteins are produced by the bacteria during the early stages of mammalian infection and may play roles in transmission from ticks to mammals. Several of the B31 Erp proteins were also recognized by antibodies from patients with Lyme disease and may prove to be useful antigens for diagnostic testing or as components of a protective vaccine.

Population structure of the relapsing fever spirochete Borrelia hermsii as indicated by polymorphism of two multigene families that encode immunogenic outer surface lipoproteins.

The tick-borne relapsing fever spirochete Borrelia hermsii evades the mammalian immune system by periodically switching expression among members of two multigene families that encode immunogenic, antigenically distinct outer surface proteins. The type strain, B. hermsii HS1, has at least 40 complete genes and pseudogenes that participate in this multiphasic antigenic variation. Originally termed vmp (for variable major protein) genes, they have been reclassified as vsp (for variable small protein) and vlp (for variable large protein) genes, based on size and amino acid sequence similarities. To date, antigenic variation in B. hermsii has been studied only in the type strain, HS1. Nucleotide sequence comparisons of 23 B. hermsii HS1 genes revealed five distinct groups, the vsp gene family and four subfamilies of vlp genes. We used PCR with family- and subfamily-specific primers, followed by restriction fragment length polymorphism analysis, to compare the vsp and vlp repertoires of HS1 and seven other B. hermsii isolates from Washington, Idaho, and California. This analysis, together with pulsed-field gel electrophoresis genome profiles, revealed that the eight isolates formed three distinct groups, which likely represent clonal lineages. Members of the three groups coexisted in the same geographic area, but they could also be isolated across large geographical distances. This population structure may result from immune selection by the host, as has been proposed for other pathogens with polymorphic antigens.

Tick-borne relapsing fever in the northwestern United States and southwestern Canada.

Records from 182 cases of tick-borne relapsing fever (TBRF) were reviewed. In confirmed cases, there was febrile illness, and spirochetes were identified on peripheral blood preparations. In probable cases, there were clinical features of TBRF and either the same exposure as a confirmed case or serological (indirect fluorescent antibody test and western blotting [WB]) evidence of infection with Borrelia hermisii. Sera also were tested for antibody to Borrelia burgdorferi. We identified 133 confirmed and 49 probable cases of TBRF. A Jarisch-Herxheimer reaction was reported in 33 (54.1%) of 61 cases for which this information was available. Most patients who had antibodies to B. hermsii were serologically positive for B. burgdorferi, and WB demonstrated false positivity of testing for B. burgdorferi. Thirty-five (21%) of 166 cases were unreported to public health authorities. In 52 cases, there were more than two relapses before the diagnosis. This study demonstrates that TBRF is underrecognized and underreported and may be falsely identified as Lyme disease.

Characterization of an endosymbiont infecting wood ticks, Dermacentor andersoni, as a member of the genus Francisella.

A microorganism (Dermacantor andersoni symbiont [DAS]) infecting Rocky Mountain wood ticks (D. andersoni) collected in the Bitterroot Mountains of western Montana was characterized as an endosymbiont belonging to the genus Francisella. Previously described as Wolbachia like, the organism's DNA was amplified from both naturally infected tick ovarial tissues and Vero cell cultures by PCR assay with primer sets derived from eubacterial 16S ribosomal DNA (rDNA) and Francisella membrane protein genes. The 16S rDNA gene sequence of the DAS was most similar (95.4%) to that of Francisella tularensis subsp. tularensis. Through a combination of Giménez staining, PCR assay, and restriction fragment length polymorphism analysis, 102 of 108 female ticks collected from 1992 to 1996 were infected. Transovarial transmission to female progeny was 95.6%, but we found no evidence of horizontal transmission.

Rickettsia peacockii sp. nov., a new species infecting wood ticks, Dermacentor andersoni, in western Montana.

Rickettsia peacockii, a new species of spotted fever group rickettsiae, was identified from Rocky Mountain wood ticks (Dermacentor andersoni) collected in the Sapphire Mountain Range on the eastern side of Bitterroot Valley, Montana. DNA from R. peacockii SkalkahoT (T = type strain) in naturally infected tick tissue was amplified by a PCR assay with primer sets derived from eubacterial 16S ribosomal DNA (rDNA), rickettsial citrate synthase, and 190-kDa surface antigen (rOmpA) genes. Partial 16S rDNA and rOmpA gene sequences exhibited levels of similarity of 99.7 and 93.2%, respectively, with the sequences of the spotted fever agent Rickettsia rickettsii R. By using Gimenez staining, fluorescent antibody tests, a PCR assay, and a restriction fragment length polymorphism analysis, 76 of 115 female ticks (minimal field infection rate, 66.1%) collected between 1992 and 1995 were found to be infected. The organism is passed transstadially and transovarially (minimal vertical transmission rate, 73.3%), and infections are localized in ovarial tissues. Attempts to cultivate R. peacockii were unsuccessful.