Direct detection of Borrelia burgdorferi in Ixodes scapularis (Acari: Ixodidae) nymphs by hybridization to ribosomal RNA

A method for direct detection of Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner has been developed. Cells are lysed to facilitate release of ribosomal RNA. Lysates are filtered onto nylon membranes that are hybridized with probes specific for sequences in B. burgdorferi 23S rRNA. The technique is rapid and does not require any enzymatic amplification steps. With the use of a cocktail containing five different probes, approximately 1,000 organisms could be detected. The assay was successfully applied to direct detection of B. burgdorferi in Ixodes scapularis Say nymphs.     

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.     

Vector competence of Ixodes muris (Acari: Ixodidae) for Borrelia burgdorferi

The vector competence of Ixodes muris (Bishopp & Smith) was determined for Borrelia burgdorferi, the etiologic agent of Lyme disease. Larval I. muris were fed on ICR outbred mice infected with the B-31 laboratory strain of B. burgdorferi. Replete larvae, at 5 d after feeding, were assayed for infection by culture in Barbour-Stoner-Kelly (BSK-H) media. Infection frequency in I. muris replete larvae was 66%. Resultant nymphs were fed on naive ICR outbred mice to determine the ability of I. muris to transmit infection. Infection frequency in fed nymphs declined to 38% and only 1/5 mice was positive for B. burgdorferi on ear biopsy culture. We demonstrated that I. muris is capable of acquiring and transmitting B. burgdorferi but is a relatively poor vector compared with I. scapularis (Say).     

Experimental infection of Ixodes scapularis larvae (Acari: Ixodidae) by immersion in low passage cultures of Borrelia burgdorferi

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 degrees 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.     

OspB antibody prevents Borrelia burgdorferi colonization of Ixodes scapularis

Borrelia burgdorferi outer surface protein OspB is expressed by spirochetes in the Ixodes scapularis gut. ospB is transcribed from a bicistronic operon with ospA, a known spirochete adhesion gene in the tick gut. Here we examine whether OspB also has a specific function in ticks. OspB specifically binds to a protein or protein complex within the tick gut. We also assessed whether selected nonborreliacidal OspB antibodies or F(ab)(2) fragments interfere with B. burgdorferi-tick attachment in vivo. We examined engorged ticks that fed on B. burgdorferi N40-infected scid mice that had been treated with OspB F(ab)(2) fragments. Control F(ab)(2) fragments did not interfere with B. burgdorferi colonization of the tick gut, whereas OspB F(ab)(2) fragments significantly inhibited the attachment of spirochetes to the tick gut. These studies show that nonbactericidal OspB antibodies interfere with B. burgdorferi colonization of I. scapularis, highlighting a specific role for OspB in spirochete- arthropod interactions and suggesting new antibody-mediated strategies for interfering with B. burgdorferi transmission.     

Contrasts in tick innate immune responses to Borrelia burgdorferi challenge: immunotolerance in Ixodes scapularis versus immunocompetence in Dermacentor variabilis (Acari: Ixodidae)

The blacklegged tick, Ixodes scapularis Say, transmits the Lyme disease spirochete Borrelia burgdorferi, whereas the American dog tick, Dermacentor variabilis (Say), is unable to transmit the bacterium. We compared the innate immune response of these ticks against spirochetes directly inoculated into the hemocoel cavity of ticks. In I. scapularis, some Borrelia were found associated with hemocytes, while numerous other spiral-shaped, intact bacteria remained free in the hemolymph. In contrast, in D. variabilis only remnants of the bacteria were evident in the hemolymph, indicating lysis; intact spirochetes were rare. Spirochetes were observed bound to or within the organs of both tick species, although many more spirochetes were found associated with the I. scapularis organs. The few spirochetes observed with the D. variabilis organs appeared to be dead because D. variabilis tissues rarely contained culturable bacteria, unlike I. scapularis tissues. When spirochetes were incubated with I. scapularis hemolymph plasma in vitro, bacterial survival and motility were not reduced. In contrast, incubation of spirochetes with D. variabilis hemolymph plasma resulted in > 50% of the spirochetes becoming nonmotile by 45 min. The differences in the responses of the two different tick species indicate that I. scapularis is immunotolerant when challenged with B. burgdorferi and dependent on a slow phagocytic response to clear Borrelia from the hemolymph. In contrast, D. variabilis is highly immunocompetent (i.e., innate immunity), using plasma borreliacidal factors and a rapid increase in phagocytic cells to clear the infection and limit tissue invasion.     

Life history of Ixodes (Ixodes) jellisoni (Acari: Ixodidae) and its vector competence for Borrelia burgdorferi sensu lato.

Ixodes (Ixodes) jellisoni Cooley & Kohls, a nonhuman biting and little known tick, is one of 4 members of the I. ricinus complex in the United States. A localized population of I. jellisoni inhabiting a grassland biotope in Mendocino County, CA, was studied from 1993 to 1997. Rodent trapping in all seasons revealed that the only host of both immature and adult I. jellisoni was the heteromyid rodent Dipodomys californicus Merriam. Field investigations suggested that I. jellisoni is nidicolous in habit, and laboratory findings demonstrated that it reproduces parthenogenetically. Known parthenogenetic females (n = 4) produced an average of 530 eggs of which 74% hatched, which was comparable to the fecundity and fertility of wild-caught females (n = 8). After the transstadial molt, 57 F1 or F2 nymphs derived from 2 wild-caught or 4 laboratory-reared, unmated females produced only females. Ixodes jellisoni males were not found on 112 wild-caught D. californicus individuals that were captured an average of 2 times. Collectively, these findings suggest that I. jellisoni may be obligatorily parthenogenetic. Borrelial isolates were obtained from 85% of 58 D. californicus and 33% of 21 I. jellisoni females removed from this rodent. None of the 7 infected female ticks passed borreliae ovarially to its F1 larval progeny. Eight D. californicus and 5 I. jellisoni-derived isolates that were genetically characterized belonged to 2 restriction pattern groups of Borrelia burgdorferi s.l. Neither restriction pattern group has been assigned to a particular genospecies yet. After placement on naturally infected D. californicus, noninfected larval ticks acquired and transstadially passed spirochetes as efficiently as (group 1 borreliae) or 6 times more efficiently (group 2 borreliae) than Ixodes pacificus Cooley & Kohls. As few as 1-4 infected I. jellisoni nymphs were capable of transmitting group 1 or group 2 borreliae to naive D. californicus. We conclude that I. jellisoni is a competent vector of both restriction fragment groups when D. californicus is used as the animal model.     

Isolation of Borrelia burgdorferi from saliva of the tick vector, Ixodes scapularis.

A method for cultivating and isolating Lyme disease spirochetes, Borrelia burgdorferi, from the saliva of vector ticks, Ixodes scapularis (formerly known as Ixodes dammini), is described. Saliva was collected from partially engorged ticks after application of pilocarpine to induce salivation. B. burgdorferi was isolated from 8 of 14 (57%) of the saliva samples derived from ticks infected with the bacteria, as determined by direct immunofluorescent-antibody assay of tick hemolymph. A comparison of the protein profiles of the salivary isolates and a highly passaged strain (B31) showed that the salivary isolates all lacked a 22-kDa protein known to increase with continuous passage, but exhibited larger amounts of the OspA and OspB proteins than did the highly passaged B31 strain.     

Ixodes scapularis (Acari: Ixodidae): abundance and rate of infection with Borrelia burgdorferi in four state parks in Wisconsin

Four state parks located in Lyme disease endemic regions of Wisconsin were surveyed for the presence of Ixodes scapularis Say during May and June of 1998 by drag sampling along hiking trails. Nymphal abundance varied between parks, with the average number of nymphs encountered in 1 h ranging from 6.2 +/- 3.8-47.1 +/- 36.3 (mean +/- SD). Questing nymphs were tested for the presence of Borrelia burgdorferi by culture in BSK medium and 7-12% was found to be infected. The average risk of encountering an infected nymph (entomologic risk index) ranged from 0.5 to 5.2 infected nymphs per hour. The highest entomological risk index was recorded from a small island park in northwestern Wisconsin during the last week in May (8.0 infected nymphs per hour). These results indicate a lower risk for human Lyme disease exposure in Wisconsin state parks in comparison with highly endemic areas of the northeastern United States.     

Isolation of Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) from Ixodes scapularis and Dermacentor albipictus ticks (Acari: Ixodidae) in Oklahoma.

Borrelia burgdorferi was isolated from Ixodes scapularis Say and Dermacentor albipictus Packard that were removed as partially fed adults from white-tailed deer, Odocoileus virginianus Zimmermann, in Oklahoma. Isolation in media was accomplished only after homogenates of pooled field-collected ticks were inoculated into laboratory-reared Peromyscus leucopus and reisolated from the urinary bladder into BSK II media. Both isolates were confirmed by western blot analysis and reactivity with monoclonal antibody H5332. These are the first reported isolates of B. burgdorferi from Oklahoma from these two tick species and are the first isolates from ticks from the south-central United States that were infective for laboratory-reared P. leucopus.     

Ixodes scapularis ticks (Acari: Ixodidae) from Louisiana are competent to transmit Borrelia burgdorferi, the agent of Lyme borreliosis

The principal vector of Borrelia burgdorferi, the Lyme borreliosis spirochete, in the Northeast and Midwestern regions of the United States is the blacklegged tick Ixodes scapularis. Because of a favorable environment, I. scapularis is also plentiful in the South; however, a correlation with Lyme borreliosis cases does not exist in this region of the United States. Concern existed that something intrinsic to ticks found in Louisiana could mitigate their ability to transmit B. burgdorferi. Therefore, we set out to assess the ability of I. scapularis ticks from Louisiana to become infected with and transmit B. burgdorferi using mice as hosts. In the laboratory, mating adult female ticks collected in southeastern Louisiana were fed on the ears of rabbits. After oviposition and egg hatching, the resulting larvae were fed on mice that had been needle-inoculated with two different strains of B. burgdorferi sensu stricto, B31 and JD1. Larvae were found to be positive for spirochetes. Additional fed larvae were allowed to molt into the nymphal stage. Flat nymphs remained infected with B. burgdorferi. Infected nymphs were allowed to feed on na?ve mice, all of which became infected as shown by culture of ear biopsy specimens. Na?ve larvae were then fed on these same mice to assess transmissibility. The resulting engorged larvae harbored spirochetes. We have demonstrated that the I. scapularis ticks found in Louisiana are fully competent to carry and transmit B. burgdorferi infection.     

Infection of Ixodes ricinus (Acari: Ixodidae) by Borrelia burgdorferi sensu lato in North Africa

Free-living adult Ixodes ricinus L, were collected in Amdoun, situated in the Kroumiry mountains in northwestern Tunisia (North Africa). Using direct fluorescence antibody assay, the infection rate of field-collected I. ricinus by Borrelia burgdorferi sensu lato was 30.5% (n = 72). No difference in infection rate was observed between male and female ticks. Spirochetes that had been isolated from I. ricinus from Ain Drahim (Kroumiry Mountains) in 1988 were identified as Borrelia lusitaniae (formerly genospecies PotiB2). This is the first identification of a genospecies of Borrelia burgdorferi sensu lato from the continent of Africa.     

Glass capillary tube feeding: a method for infecting nymphal Ixodes scapularis (Acari: Ixodidae) with the lyme disease spirochete Borrelia burgdorferi

We evaluated an artificial capillary feeding method to infect nymphal Ixodes scapularis (Say) ticks with Borrelia burgdoeferi, the causative agent of Lyme disease. Thirty to 70% of the nymphs were infected after feeding for 2.5 h from glass capillary tubes filled with a solution of spirochetes. Capillary infection was stable and persisted in the nymphs for at least 10 d after feeding. Capillary feeding also maintained natural vector competence patterns because I. scapularis ticks acquired infection unlike Dermacentor variablis (Say), which did not become infected. Capillary infected I. scapularis nymphs were capable of transmitting the infection to naive mice although not as efficiently as naturally infected nymphs. The capillary infection method is convenient and is a better alternative to syringe inoculation as a means of infecting animals with B. burgdorferi.     

Prevalence of Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) in Ixodes scapularis (Acari: Ixodidae) adults in New Jersey, 2000-2001

Using polymerase chain reaction, we analyzed 529 Ixodes scapularis Say adults collected from 16 of New Jersey's 21 counties for the presence of Borrelia burgdorferi, the etiological agent of Lyme disease. Overall, 261 (49.3%) were positive. B. burgdorferi was detected in ticks obtained from each county and from 53 of the 58 (93.1%) municipalities surveyed. The observed statewide prevalence in New Jersey is similar to those reported from other northeastern and mid-Atlantic states.     

Control of immature Ixodes scapularis (Acari: Ixodidae) on rodent reservoirs of Borrelia burgdorferi in a residential community of southeastern Connecticut

A 3-yr community-based study was conducted on residential properties on Mason's Island, Mystic, CT, to determine the efficacy of a rodent-targeted acaricide (fipronil) to control immature Ixodes scapularis (Say) on Peromyscus leucopus. Results indicated that modified commercial bait boxes were effective as an acaricide delivery method for reducing nymphal and larval tick infestations on white-footed mice by 68 and 84%, respectively. Passive application of fipronil significantly reduced the infection rate of Borrelia burgdorferi among white-footed mice by 53%. Moreover, the abundance of questing I. scapularis adults on treated properties was reduced by 77% and fewer were infected with spirochetes (31%) compared with untreated sites (47%) after 3 yr of treatment. Likewise, the abundance of host-seeking nymphs was significantly reduced on treated properties by >50%. Finally, infection rates in flagged nymphal ticks for both B. burgdorferi and Anaplasma phagocytophilum were reduced by 67 and 64%, respectively, after only 2 yr of treatment. Results from this 3-yr trial indicate that the use of fipronil passively applied to reservoir animals by bait boxes is an environmentally acceptable means to control ticks, interrupt the natural disease transmission cycle, and reduce the risk of Lyme disease for residents of treated properties.     

Transmission dynamics of Borrelia burgdorferi s.s. during the key third day of feeding by nymphal Ixodes scapularis (Acari: Ixodidae)

Nymphal Ixodes scapularis Say are the principal vectors of Lyme disease spirochetes (Borrelia burgdorferi sensu stricto) in the eastern United States. Physicians frequently face the decision of whether or not to administer prophylactic antibiotics to human tick bite victims in Lyme disease endemic regions, based on the overall probability that such bites will result in infection with B. burgdorferi s.s. We evaluated the transmission dynamics of B. burgdorferi s.s. during the key third day of nymphal I. scapularis feeding, when the risk of transmission rapidly increases. The cumulative probability that 50% of infected ticks transmitted B. burgdorferi s.s. occurred at 68 h of tick attachment and our overall estimate that a human tick bite would result in transmission of B. burgdorferi s.s. was 2.4%.     

Attempted transmission of Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) (JDI strain) by Ixodes scapularis (Acari: Ixodidae), Dermacentor variabilis, and Amblyomma americanum.

Laboratory-reared Ixodes scapularis Say, Amblyomma americanum (L.), and Dermacentor variabilis (Say) were fed on New Zealand white rabbits experimentally infected with Borrelia burgdorferi (JDI strain). At repletion, spirochetes could be detected by dark-field microscopy only in I. scapularis. Acquisition rates were 18 and 21%. When previously exposed nymphs of each species were fed on susceptible rabbits, I. scapularis was the only tick of the three species that transmitted B. burgdorferi. When a single rabbit was experimentally infected with B. burgdorferi and infested at 7-d intervals with I. scapularis, A. americanum, D. variabilis, and a second time with I. scapularis, B. burgdorferi was detected again only in cultures from the two groups of I. scapularis. When molted nymphs from each tick species were allowed to feed on susceptible rabbits, spirochetes again were isolated only at necropsy from the rabbits on which the two groups of I. scapularis fed.     

Rates of Borrelia burgdorferi infection in Ixodes dammini (Acari: Ixodidae) in southwestern Wisconsin.

Ixodes dammini Spielman, Clifford, Piesman & Corwin collected from a study site in southwestern Wisconsin were examined for Borrelia burgdorferi infection. Adult spirochete infection rates varied seasonally (38.1%, spring 1990; 60.3%, fall 1990; 41.2%, spring 1991) and were similar to or higher than rates reported in various studies from the northeastern United States. Statistical comparison of seasonal infection rates indicated a significant difference between the fall 1990 adult population and the subsequent overwintered population of the same cohort (spring 1991). Additionally, a significant decrease in the intensity of spirochete infection was observed in the overwintered adult sample.     

A technique for longitudinally sampling individual adult Ixodes scapularis (Acari: Ixodidae)

The first technique for repeatedly sampling individual Ixodes scapularis adult ticks was developed and validated. Gut samples from the same individual ticks were removed and analyzed at weekly intervals. Multiple analyses were conducted on each gut sample (e.g., total protein concentration, presence of viable B. burgdorferi spirochetes, and concentration of outer surface protein A [OspA]). Female I. scapularis survived for up to 25 d after gut sampling. Seventy-five percent of females oviposited after the sampling procedure, with 14% of ticks laying >1,500 eggs. No significant differences in either fecundity or length of survival were found between B. burgdorferi-infected and uninfected I. scapularis. This technique will enable longitudinal studies on both tick-pathogen interactions and physiological studies that have hitherto not been attempted.     

Deer density and the abundance of Ixodes scapularis (Acari: Ixodidae)

The abundance of Ixodes scapularis Say (Ixodes dammini Spielman, Clifford, Piesman & Corwin), the vector tick of the Lyme disease spirochete and other human pathogens, is related to the presence of its primary reproductive stage host, white-tailed deer (Odocoileus virginianus Zimmerman). However, this relationship has not been quantified in terms that would guide wildlife management in areas in which the public is, or is likely to become, exposed to infected ticks. In this study, deer density and tick abundance were measured in an emergent area for Lyme disease at three spatial scales using estimation methods appropriate for each. Simple linear regression was used to relate (1) the number of ticks found on deer at tagging stations in southern Maine to harvest-derived estimates of the density of deer within the towns in which they were killed, (2) tick densities estimated from fall flagging counts to deer densities estimated from pellet group counts made within multiple transects distributed through 5.2-km2 study sites, and (3) tick counts to pellet group counts within the individual transects. At the broadest scale, ticks on deer decreased with elevation and distance from the coast and increased with deer density, although deer and tick presence were only weakly related. Among the 5.2-km2 study sites and within individual transects, tick abundance related more strongly to deer pellet group counts. Few ticks were collected at deer densities <7/km2.