Use of a sentinel host system to study the questing behavior of Ixodes spinipalpis and its role in the transmission of Borrelia bissettii, human granulocytic ehrlichiosis, and Babesia microti.

Ixodes spinipalpis maintains Borrelia bissettii spirochetes in Colorado in a cycle involving wood rats and deer mice. This tick has been described as nidicolous, remaining either attached to its rodent hosts or in the rodent nest. Nidicolous ticks pose little risk of pathogen transmission to humans if they do not actively quest for hosts. To investigate the questing potential of I. spinipalpis, sentinel mice were placed in an area where I. spinipalpis had been commonly found on wood rats and deer mice. Concurrently, wild rodent populations were trapped and analyzed for Lyme disease spirochetes, the agent of human granulocytic ehrlichiosis (aoHGE), and Babesia microti. A total of 122 I. spinipalpis larvae and 10 nymphs were found on 19% of 244 sentinel mice. In addition, 4 sentinel mice became infested with Malaraeus telchinus or Orchopeas neotomae fleas. Questing I. spinipalpis were positively associated with woody shrubs and negatively associated with sunny and grassy areas. Four sentinel mice became infected with aoHGE after having been fed upon only by I. spinipalpis larvae. One sentinel mouse became infected with B. bissettii after having an I. spinipalpis nymph feed on it, and one sentinel mouse became coinfected with aoHGE and B. bissettii after it was fed upon by a single I. spinipalpis nymph. These sentinel mouse conversions suggest the possibility that the aoHGE is transovarially transmitted by I. spinipalpis, and that I. spinipalpis is capable of simultaneously transmitting B. bissettii and the aoHGE. The findings that I. spinipalpis quest away from rodent nests and will attach to and infect sentinel mice may be of public health importance. It suggests the potential transmission of the agents of human granulocytic ehrlichiosis and Lyme disease to other hosts by I. spinipalpis, in regions of the western United States where Ixodes pacificus is not found.

Borrelia lonestari DNA in adult Amblyomma americanum ticks, Alabama.

Polymerase chain reaction analysis of 204 Amblyomma americanum and 28 A. maculatum ticks collected in August 1999 near the homes of patients with southern tick-associated rash illness and in control areas in Choctaw County, Alabama, showed Borrelia lonestari flagellin gene sequence from two adult A. americanum. The presence of B. lonestari in A. americanum ticks from Alabama suggests that this suspected pathogen may be widespread in the southeastern United States.

Infection of Ixodes scapularis (Acari: Ixodidae) with Borrelia burgdorferi using a new artificial feeding technique.

To study interactions between Ixodes scapularis (Say) and Borrelia burgdorferi, an artificial feeding system was refined to allow controlled manipulation of single variables. The feeding system uses a mouse skin mounted on a water-jacketed glass membrane feeder. I. scapularis were infected using either BSK-H-cultured B. burgdorferi spirochetes or a B. burgdorferi-infected mouse skin as the source of spirochetes. Sixty-six percent of nymphs successfully fed to repletion using the artificial feeding systems with at least 75% of nymphs becoming infected with B. burgdorferi. Strain B31 B. burgdorferi spirochetes from passages 2-17 were equally infectious to nymphal ticks. At concentrations of one spirochete per microliter, 12% of nymphs acquired infection and 14 and 100 spirochetes per microliter resulted in 50 and 100% infection rates, respectively. Eighty-nine percent of nymphs fed by artificial feeding molted to the adult stage. When subsequently fed as adults, these I. scapularis successfully transmitted infectious B. burgdorferi spirochetes to mice.

The efficacy of co-feeding as a means of maintaining Borrelia burgdorferi: a North American model system.

Although research on co-feeding as a means of maintaining tick-borne pathogens has focused chiefly on viruses, recent interest has been directed toward the importance of this phenomenon in maintaining the Lyme disease spirochete, Borrelia burgdorferi. In the current study, an experimental model was developed to determine under what conditions immature co-feeding ticks exchange B. burgdorferi using the principal North American vector (Ixodes scapularis) and reservoir (Peromyscus leucopus) species. Experiments conducted with the density of ticks likely to be encountered in nature (8 nymphs & < 40 larvae) demonstrated that no co-feeding larvae became infected; in contrast, horizontal transmission infected 30-64% of test larvae. Only the highest densities of ticks (40 nymphs & > 200 larvae) produced infected larvae (5%) upon co-feeding of larvae and nymphs. An important role for co-feeding in the ecology of Lyme disease in North America has yet to be established.

Babesia microti and Borrelia bissettii transmission by Ixodes spinipalpis ticks among prairie voles, Microtus ochrogaster, in Colorado.

An endemic transmission cycle of Babesia microti was discovered in Colorado in the foothills of the Rocky Mountains. B. microti were found by PCR in 4 of 25 Ixodes spinipalpis tick pools tested (a 3.2 % minimum infection rate) and in 87% (13 of 15) of Microtus ochrogaster (the prairie vole) spleen and blood samples. Using naturally infected I. spinipalpis collected from wild-caught M. ochrogaster as vectors, B. microti and Borrelia bissettii were successfully transmitted to laboratory-born M. ochrogaster. Neither I. spinipalpis, nor M. ochrogaster (the prairie vole) have been previously reported as a vector or a reservoir host of B. microti. Unlike the east coast of the United States where Peromyscus leucopus is an important reservoir for B. microti, evidence for Peromyscus spp. (neither P. maniculatus nor P. difficilis) as B. microti reservoirs was not found in this study.

Isolation of thogoto virus (Orthomyxoviridae) from the banded mongoose, Mongos mungo (Herpestidae), in Uganda.

Small wild vertebrates were trapped during an investigation into possible vertebrate reservoirs of o'nyong-nyong (ONN) fever virus in Uganda in 1997. Antibody neutralization test results and virus isolation attempts were negative for ONN virus, confirming the work of earlier investigators, who also failed to find evidence for a nonhuman ONN virus reservoir. In the course of these ONN virus studies, Thogoto virus was isolated from one of eight banded mongooses (Mongos mungo). This is the first isolation of Thogoto virus from a wild vertebrate. Neutralizing antibodies to Thogoto virus were also found in two of the other mongooses.

Isolation of Borrelia burgdorferi from Neotoma fuscipes, Peromyscus maniculatus, Peromyscus boylii, and Ixodes pacificus in Oregon.

The number of Lyme disease cases in Oregon has increased in recent years despite the fact that the pathogen, Borrelia burgdorferi, has never been isolated in the state. Rodent and tick surveys were undertaken in 1997 to isolate and characterize strains of B. burgdorferi from Oregon and to identify potential reservoirs and vectors of Lyme disease. Borrelia burgdorferi was isolated from Neotoma fuscipes, Peromyscus maniculatus, P. boylii, and Ixodes pacificus. Both N. fuscipes and P. maniculatus were infested with I. pacificus and I. spinipalpis. Although I. pacificus infested P. boylii, I. spinipalpis was not found on this rodent, and only 4% of the P. boylii were infected with B. burgdorferi compared with the 19% and 18% infection rates found in N. fuscipes and P. maniculatus, respectively. Variation in the molecular weights of the outer surface proteins A and B were found in these first confirmed isolates of B. burgdorferi from Oregon, as well as truncated forms of outer surface protein B.

Geographic survey of vector ticks (Ixodes scapularis and Ixodes pacificus) for infection with the Lyme disease spirochete, Borrelia burgdorferi.

Populations of adult Ixodes scapularis and Ixodes pacificus, the two principal vectors of Lyme disease spirochetes in the United States, were collected from 17 sites in 12 states. Female ticks were fed on experimental rabbits; ticks and rabbits were subsequently examined for infection with Borrelia burgdorferi. Fourteen rabbits were exposed to I. scapularis ticks from the northeastern states of Connecticut, New York, New Jersey, and Maryland; all 14 rabbits became infected with B. burgdorferi. A total of 165/226 (73%) of these northeastern ticks was infected. Similarly, ticks from the midwestern states of Michigan, Wisconsin, and Minnesota transmitted infection to all three exposed rabbits; 29/51 (57%) of these midwestern I. scapularis were infected. In marked contrast, none of the 12 rabbits exposed to I. scapularis ticks from the southeastern states of South Carolina, Georgia, Florida, and Mississippi acquired infection with B. burgdorferi, and 0/284 (0%) of these ticks contained spirochetes. Four rabbits were exposed to I. pacificus collected from one location in California; 2/4 of these rabbits acquired infection and 2/57 (4%) of the I. pacificus were infected with B. burgdorferi. The antigenic profiles of all 58 strains tested were consistent with an identity of B. burgdorferi sensu lato. The availability of a human Lyme disease vaccine adds urgency to our efforts to calculate the ecological transmission risk throughout the United States, as an aid to the judicious use of such a vaccine.

Mosquito feeding modulates Th1 and Th2 cytokines in flavivirus susceptible mice: an effect mimicked by injection of sialokinins, but not demonstrated in flavivirus resistant mice.

Culex pipiens and Aedes aegypti mosquitoes were fed on C3H/HeJ mice and systemic cytokine production was quantified from stimulated lymphocytes harvested four to ten days after feeding. Mosquito feeding on C3H/HeJ mice significantly down regulated IFN gamma production seven to ten days post feeding by Cx. pipiens and seven days after Ae aegypti feeding. Th2 cytokines, IL-4 and IL-10, were significantly up regulated 4-7 days after Cx. pipiens and Ae. aegypti feeding. The immunosuppressive effect of Cx. pipiens feeding on systemic cytokine production was not evident in congenic flavivirus resistant (C3H/RV) mice, as systemic IFN gamma and IL-2 were significantly up regulated at days 7 and 10, correlating with a significant decrease in IL-4 10 days after feeding by Cx. pipiens mosquitoes. Inoculation of 5-1000 ng of sialokinin-I into C3H/HeJ mice mimicked the effect of Ae. aegypti feeding by down regulating Th1 cytokines and significantly up regulating Th2 cytokines four days post inoculation. Injections of sialokinin-II resulted in only moderate effects on IFN gamma and IL-4 production seven and ten days after injection. Thus natural feeding by two arbovirus vectors had a profound T cell modulatory effect in vivo in virus susceptible animals which was not demonstrated in the flavivirus resistant host. Moreover, sialokinin-I and sialokinin-II mimicked the effect of mosquito feeding by modulating the host T cell response. These results may lend new insight into specific aspects of the role of the mosquito vector in potentiating virus transmission in the mammalian host.

Duration of immunity to reinfection with tick-transmitted Borrelia burgdorferi in naturally infected mice.

The ability of naturally infected and cured mice to resist reinfection with tick-transmitted Borrelia burgdorferi was tested over a 1-year period. All of the mice were resistant to reinfection when they were challenged at 1.5 months after cure. The majority of animals were resistant to reinfection for up to 10.5 months after cure, but this resistance was lost at 1 year after cure. Both protected and unprotected animals showed a diverse array of antibodies on Western immunoblots. Protection was not associated with the killing of spirochetes in ticks, and naturally infected mice produced no antibodies to outer surface protein A (OSP A). The titers to whole Borrelia sonicate and OSP C, however, remained high throughout the 1-year study period. The levels of borreliacidal antibodies were highest in the 1.5 month-after-cure group. Natural immunity to reinfection with B. burgdorferi is limited in time, is complex, and may involve both humoral and cellular components.

Ability of the Lyme disease spirochete Borrelia burgdorferi to infect rodents and three species of human-biting ticks (blacklegged tick, American dog tick, lone star tick) (Acari:Ixodidae).

The infectivity of a diverse collection of Borrelia burgdorferi strains from North America for mice was determined as a prelude to vector competence experiments with the 3 primary human-biting tick species in the eastern United States (Ixodes scapularis Say, Dermacentor variabilis (Say), Amblyomma americanum (L.)]. Of the 34 B. burgdorferi strains inoculated into mice, 29 were infectious; the exceptions were 5 isolates from Texas. Vector competence experiments were conducted with 2 strains from the southern United States (North Carolina and Georgia). Both strains were extremely infectious to I. scapularis larvae. Moreover, I. scapularis efficiently maintained these spirochetes transstadially and transmitted infection as nymphs. D. variabilis larvae were intermediate in susceptibility but generally did not maintain the infection transstadially. A. americanum larvae were completely refractory to infection with these 2 southern B. burgdorferi strains. Three isolates from Michigan D. variabilis were inoculated into mice, subsequently exposed to I. scapularis and D. variabilis larvae. Larval I. scapularis were 5-fold more susceptible to infection with these strains than were larval D. variabilis. Although nymphal I. scapularis efficiently transmitted a Michigan isolate, nymphal D. variabilis did not. In all these experiments, I. scapularis was the only species that proved to be vector competent for B. burgdorferi.

Duration of tick attachment as a predictor of the risk of Lyme disease in an area in which Lyme disease is endemic.

Animal studies have shown an exponential increase in the risk of Borrelia burgdorferi infection after 48-72 h of deer tick attachment. Persons with tick bites were prospectively studied to determine if those with prolonged tick attachment constitute a high-risk group for infection. Ticks were identified, measured for engorgement, and assayed by polymerase chain reaction (PCR) for B. burgdorferi DNA. Duration of attachment was determined from the scutal index of engorgement. Of 316 submissions, 229 were deer ticks; 14% were positive by PCR. Paired sera and an intact tick for determination of duration of attachment were available for 105 subjects (109 bites). There were 4 human cases (3.7% of bites) of B. burgdorferi infection. The incidence was significantly higher for duration of attachment > or =72 h than for <72 h: 3 (20%) of 15 vs. 1 (1.1%) of 94 (P = .008; odds ratio, 23.3; 95% confidence interval, 2.2-242). PCR was an unreliable predictor of infection. Tick identification and measurement of engorgement can be used to identify a small, high-risk subset of persons who may benefit from antibiotic prophylaxis.

Incomplete protection of hamsters vaccinated with unlipidated OspA from Borrelia burgdorferi infection is associated with low levels of antibody to an epitope defined by mAb LA-2.

Efforts to develop a recombinant vaccine for Lyme disease have focused on using the outer surface protein A (OspA) of Borrelia burgdorferi as an immunogen. We evaluated the effectiveness of an unlipidated recombinant OspA as a vaccine in hamsters. This molecule is soluble and can be produced in high yield in Escherichia coli, characteristics that permit simple and relatively low cost production. Vaccination with unlipidated OspA protected a substantial portion of animals--59-79%, depending on the challenge strain and route--against moderate doses of spirochetes delivered either by injection or by bite of infected nymphal ticks (Ixodes scapularis). The instances of vaccine failure were associated with development of low levels of antibody to a particular OspA epitope, one defined by mAb LA-2. At least 50 ng ml-1 of LA-2 equivalent antibody was necessary for protection of hamsters. Lower LA-2 equivalent antibody concentrations occurred in unprotected animals in the presence of high-titered polyclonal antibody to native OspA. A competitive binding assay to quantitate this serum fraction is described that should be of use in monitoring the quality of the antibody response to OspA in vaccine trials. Concentrations of LA-2 equivalent antibody parallel the ability of the serum specimens to inhibit the growth of B. burgdorferi in culture.

Discovery of an enzootic cycle of Borrelia burgdorferi in Neotoma mexicana and Ixodes spinipalpis from northern Colorado, an area where Lyme disease is nonendemic.

An intensive enzootic cycle of Borrelia burgdorferi was seen in populations of the Mexican wood rat, Neotoma mexicana, and Ixodes spinipalpis ticks in northern Colorado. Cultures of rodent ear tissue and ticks yielded 63 spirochetal isolates: 38 N. mexicana, 2 Peromyscus difficilis, and 23 I. spinipalpis. All 63 isolates were identified as B. burgdorferi sensu lato by polymerase chain reaction; a representative subset was characterized as B. burgdorferi by SDS-PAGE and immunoblotting. A tick-derived spirochete isolate was infectious to laboratory mice and I. scapularis, the principal vector of Lyme disease in endemic areas of the United States. The risk of human contact with infected I. spinipalpis appears to be minimal from this epidemiologically silent focus in northern Colorado, since this tick is restricted to wood rat nests in this semiarid environment.

Amblyomma americanum: a potential vector of human ehrlichiosis.

Polymerase chain reaction primers specific for Ehrlichia chaffeensis were used to amplify DNA from extracts of pooled ticks. Amplification was performed on extracts from 140 pools (1,579 total ticks) consisting of three tick genera collected from five states. The characteristic 389-basepair product was observed after amplification of extracts from seven different pools of adult Amblyomma americanum (117 pools, 1,462 ticks), but not from pools of nymphs. No specific product was observed after amplification of 20 pools (105 ticks) of Dermacentor variabilis and three pools of Ixodes scapularis (12 ticks). Ehrlichia chaffeensis was present in A. americanum at a minimum frequency of > or = 0.48%, suggesting that A. americanum may be a vector of human ehrlichiosis.

Detection of Borrelia burgdorferi in ticks by species-specific amplification of the flagellin gene.

We developed a polymerase chain reaction (PCR) that specifically amplifies a fragment of the flagellin gene (fla) of Borrelia burgdorferi, the causative agent of Lyme disease. This fla target, amplified with nested primers, was conserved among all 80 strains of B. burgdorferi tested. Strains examined included cultures from ticks, humans, and rodents from major B. burgdorferi-endemic regions of the United States and parts of Europe and Asia. Templates from B. hermsii, B. parkeri, B. turicatae, and B. coriaceae were not amplified, nor were eukaryotic DNAs from three tick genera. Several host DNAs potentially present in a tick blood meal also were not amplified. Approximately six B. burgdorferi per PCR reaction could be detected by ethidium bromide staining of amplified DNA. Colony-raised Ixodes dammini were used to evaluate the method. One infected nymph in a pool of 40 ticks was routinely detected. The specificity of the assay for detecting B. burgdorferi-infected ticks in pools was 94% (29 of 31). This protocol should prove useful for assessing infection rates in other putative arthropod vectors.

The hamster immune response to tick-transmitted Borrelia burgdorferi differs from the response to needle-inoculated, cultured organisms.

The human immune response to natural infection with Borrelia burgdorferi appears to differ from that seen in small mammals infected by needle inoculation. In humans, antibody to outer surface proteins A and B (OspA and OspB) is not detectable until late in infection, but small mammals inoculated with B. burgdorferi produce early antibody to OspA and OspB. To investigate this disparity we compared the immune response in hamsters to B. burgdorferi after needle inoculation with cultured organisms or infected tick homogenates with the immune response after tick transmitted (natural) infection. We determined that the antibody response to OspA and OspB after natural infection of hamsters is similar to that seen in humans, and differs from the antibody response after hamster infection by needle inoculation. High titers of antibody to OspA and OspB were undetectable even 42 wk after bite by B. burgdorferi-infected ticks. The failure to produce antibody to OspA and OspB was not dependent on challenge dose, because animals inoculated by needle with low doses (1 x 10(5) to 1 x 10(6) cells) of B. burgdorferi produced antibody to OspA and OspB. A rapid but limited anti-41-kDa response was observed. One possible new Ag, 43 kDa (p43), was identified. The antibody response to p43 was independent of the route of inoculation. Our results suggest that the hamster immune response to tick-transmitted Borrelia burgdorferi differs from the response to needle inoculated, cultured organisms.

Duration of adult female Ixodes dammini attachment and transmission of Borrelia burgdorferi, with description of a needle aspiration isolation method.

The relationship between the attachment duration of adult female Ixodes dammini and the transmission of Borrelia burgdorferi was studied. Sixteen rabbits were exposed to spirochete-infected female ticks for specified intervals. All five rabbits exposed to ticks that fed to repletion (greater than 120 h) became infected, as did two of three exposed for 48 h. In contrast, five rabbits exposed to a cumulative total of 53 infected female I. dammini for 36 h failed to become infected, as did three rabbits exposed for 24 h. A needle aspirate method facilitated the isolation of spirochetes from host skin.

Potential for insect transmission of HIV: experimental exposure of Cimex hemipterus and Toxorhynchites amboinensis to human immunodeficiency virus.

Human immunodeficiency virus (HIV) was detected in bedbugs (Cimex hemipterus) up to 8 d after oral exposure to highly concentrated virus in blood meals, but no virus replication was observed. HIV did not replicate in either intraabdominally inoculated bedbugs or intrathoracically inoculated mosquitoes (Toxorhynchites amboinensis). The virus was not detected in bedbug feces. Mechanical transmission of HIV by bedbugs could not be demonstrated in an in vitro model. The persistence of HIV in an insect or on its mouthparts is one of many factors necessary for mechanical transmission in nature. The risk of insect transmission of HIV appears to be extremely low or nonexistent.

Surveillance of St. Louis encephalitis virus vectors in Grand Junction, Colorado, in 1987.

Grand Junction, Colorado, was the site of a St. Louis encephalitis (SLE) outbreak in 1985. Epidemiologic and ecologic investigations in 1985 and 1986 suggested that Culex tarsalis may not have been the exclusive vector in the outbreak and that Cx. pipiens may have contributed to transmission as an accessory vector. A limited field study in 1987 generally confirmed observations from 1986 that Cx. pipiens was more abundant than Cx. tarsalis in late summer when SLE virus transmission normally occurs. In both years, infection rates in Cx. tarsalis were higher than in Cx. pipiens, but in 1987 the only SLE virus isolate from Cx. pipiens was obtained early in the season. Truck trap collections showed that Cx. pipiens was the principal vector species collected, comprising 86% of the total. Light trap collections underestimated the population of Cx. pipiens; gravid trap collections gave a closer approximation of the relative proportions of Cx. pipiens and Cx. tarsalis in the vector mosquito population after midsummer.