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Ixodes scapularis Ticks (Acari: Ixodidae) from Louisiana Are Competent to Transmit Borrelia burgdorferi, the Agent of Lyme Borreliosis

(CC)
Mary B. Jacobs, Jeanette E. Purcell, Mario T. Philipp
DOI: http://dx.doi.org/10.1603/0022-2585-40.6.964 964-967 First published online: 1 November 2003

Abstract

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 naïve mice, all of which became infected as shown by culture of ear biopsy specimens. Naïve 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.

  • Lyme borreliosis
  • Borrelia burgdorferi
  • Ixodes scapularis
  • Louisiana

Lyme borreliosis, the most frequently reported arthropod-borne disease in the United States, is seldom reported in Louisiana. In the year 2001, eight Louisiana cases were reported to the Centers for Disease Control and Prevention (CDC), whereas as many as 4,083 cases of Lyme borreliosis were reported in the state of New York, an area of high endemicity (Center for Disease Control and Prevention 2003).

Effective transmission of Borrelia burgdorferi (Johnson, Schmidt, Hyde, Steigerwalt, and Brenner), the spirochete that causes the disease, requires the concomitant presence of ticks of the Ixodes ricinus complex, a reservoir host (usually a rodent), and deer. In endemic areas of the eastern United States, the tick, rodent, and deer species most often involved in this enzootic transmission cycle are I. scapularis (Say), the blacklegged tick (Oliver 1996); Peromyscus leucopus, the white-footed mouse (Main et al. 1982); and Odocoileus virginianus, the white-tailed deer (Anderson and Magnarelli 1980).

Both the blacklegged tick and the white-tailed deer are abundant in Louisiana (Hesselton and Hesselton 1982, Baker 1984, Dennis et al. 1998), as is the cotton mouse, P. gossypinus, which is sympatric with the white-footed mouse in Louisiana (Barko et al. 2000). Cotton mice have been shown to be hosts for both I. scapularis ticks (Lavender and Oliver 1996) and B. burgdorferi (Oliver et al. 2000) in the southeast.

Because all of the components required for B. burgdorferi transmission are available in Louisiana, the low incidence of Lyme borreliosis in this state is intriguing. Other investigators before us have questioned the apparent sparse overall incidence of this disease in the southeastern United States, and several hypotheses have been put forward to explain this fact. Availability of lizards as hosts for immature tick stages (Durden et al. 2002)—some lizard species may be poor hosts for B. burgdorferi (Lane and Quistad 1998)—and presence of spirochetal species or isolates of relatively lower pathogenicity or infectivity (Barthold et al. 1990, Oliver 1996) have been postulated, among others, as factors that could contribute to the low incidence of Lyme borreliosis in the southeastern United States.

Although I. scapularis is the most likely vector of southeastern isolates of B. burgdorferi (Sanders and Oliver 1995), the competency of I. scapularis from Louisiana to transmit B. burgdorferi sensu stricto has never been verified. For this reason, we evaluated the competency of a local I. scapularis strain as vector for the B31 and JD1 strains of B. burgdorferi sensu stricto. The ability of larvae to acquire spirochetes from mice, transstadial transmission of spirochetes from larvae to nymphs, spirochetal transmission from nymphs to mice, and from mice thus infected back to larvae were all evaluated in the laboratory. Here we report the results of this study.

Materials and Methods

Spirochete Strains.

Borrelia burgdorferi sensu stricto strains JD1, a human isolate (Piesman et al. 1987), and B31, a tick isolate (Burgdorfer et al. 1982), were cultivated at 34°C in complete BSK-H medium (Sigma, St. Louis, MO). Complete medium contained, in addition, 6–10% rabbit serum and the antibiotics rifampicin, 45.4 μg/ml; phosphomycin, 194 μg/ml; and amphotericin B, 0.25 μg/ml (all from Sigma).

Tick Colonies.

Adult I. scapularis used to generate nymphs and larvae for this study were flagged in the parishes of East Baton Rouge, East Feliciana, and St. Tammany, LA. These progenitor adults had been fed on a female New Zealand White rabbit (Oryctolagus cuniculus; Charles River Laboratories, Wilmington, MA). Twenty-five mating pairs were placed under a nylon stocking covered by a cotton bag on each rabbit ear. After 9 d, females had fed to repletion and subsequently were kept in individual jars at 22°C. The female ticks oviposited within 2 wk of feeding, and larvae began hatching 5 wk later. Ticks of all life cycle stages were either held at 22°C, 97% RH with a photoperiod of 16:8 (L:D) h or kept at 4°C for long-term storage.

Mouse Strains and Inoculations.

Six- to- 8-wk-old female mice (Mus musculus) of the CD-1 strain (Crl:CD-1; Charles River Laboratories) were used in this study. Spirochetes grown either to mid-logarithmic (≈2 × 107 spirochetes/ml) or stationary phase (108 spirochetes/ml), as determined by darkfield microscopy at 500× magnification, were used for mouse needle inoculations. The organisms were injected subcutaneously at the back of the mouse neck in a volume of 0.5 ml of complete BSK-H medium. To evaluate transmission by tick bite, 20–25 nymphs were placed on the head and neck of each uninfected mouse and allowed to feed to repletion. Each engorged nymph collected was surface sterilized by immersion in 3% H2O2 for 20 min followed by a 20-min immersion in 70% EtOH. Ticks were allowed to air dry and crushed individually in a microvial in 25-μl sterile phosphate-buffered saline (PBS) and then divided into two parts. One-half was dried on a slide and assessed by direct immunofluorescence as described below. The other half was transferred to 5 ml of complete BSK-H and incubated at 34°C to allow proliferation of spirochetes. One week after tick or needle inoculation, an ear biopsy specimen was obtained from each mouse and cultured at 34°C in complete BSK-H medium. The cultures were monitored under a dark-field microscope to determine presence of spirochetes.

Direct Immunofluorescence.

Engorged larvae and flat or engorged nymphs were crushed in PBS. One-half of the volume was spread on a slide and allowed to dry, acetone-fixed, and incubated at 37°C with 40 μl of a 1:10 dilution of fluorescein-labeled anti-B. burgdorferi or anti-Borrelia species antibody (Kirkegaard & Perry, Gaithersburg, MD). Slides were examined under a fluorescence microscope at 600× magnification.

Infection of Ticks.

Larvae were allowed to feed to repletion on mice at 3–4 wk postinoculation of mice through tick or needle. One week after drop-off, a small portion of the fed larvae (15–31 ticks from each infected group) were crushed and smeared on a slide as described above and examined by direct fluorescence assay (DFA) to determine the infection prevalence. Fed larvae were allowed to molt in a humidified chamber at 22°C. The resultant nymphs were later stored at 4°C for 10 mo and were assayed for transstadial infection by DFA.

Results

Transmission of B. burgdorferi from Mice to Tick Larvae.

Mice were needle inoculated with the JD1 (n = 2) or the B31 (n = 2) strain of B. burgdorferi sensu stricto as described in Materials and Methods. These strains were chosen on the basis that they are pathogenic and could cause Lyme borreliosis if transmitted by local isolates of I. scapularis. Larvae were placed on the mice and allowed to feed to repletion. Between 15 and 31 fed larvae were recovered from each of the mice, and between 74 and 100% of these larvae were infected with B. burgdorferi, as assessed by DFA on tick squashes. These infection yields were obtained regardless of the B. burgdorferi strain (Table 1).

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Table 1

Transstadial Infection of I. scapularis Nymphs.

Larvae were fed on mice that were infected either with the JD1 or B31 strain of B. burgdorferi and allowed to molt. The nymphs were then stored at 4°C for 10 mo. Assaying for infection by DFA showed that 80–100% of the flat nymphs were positive for spirochetes (Table 2). This demonstrates that B. burgdorferi can survive transstadially in the Louisiana local population of I. scapularis.

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Table 2

Transmission of B. burgdorferi from I. scapularis Nymphs to Mice, and Subsequently, to Larvae.

Four naïve mice were used in this experiment. Louisiana I. scapularis nymphs were either uninfected or infected with the JD1 or B31 strain of B. burgdorferi. Groups of uninfected and JD1-infected nymphs were placed on one mouse each, and B31-infected nymphs were placed on the remaining two animals. All ticks were allowed to feed to repletion. Between 3 and 12 engorged nymphs were recovered from each mouse. Between 75 and 100% of the engorged ticks were infected with B. burgdorferi (Table 3). All of the mice exposed to infected nymphs became infected, determined by ear-punch biopsy culture. The mouse exposed to control ticks remained uninfected. Between 10 and 20 Louisiana I. scapularis larvae were then fed to repletion on each of the infected mice. Between 80 and 100% of these larvae became infected with B. burgdorferi (Table 3). This experiment shows that LA I. scapularis nymphs are competent to transmit B. burgdorferi to mice and that mice thus infected can in turn convey the infection to blood-feeding larvae.

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Table 3

Discussion

Ixodes scapularis is considered to be a polytypic species with a widespread geographic distribution exhibiting north-south and east-west morphological clines in eastern North America (Hutcheson et al. 1995, Hutcheson and Oliver 1996, 1998). Southern I. scapularis are genetically more varied than the northern strains (Oliver 1996), and it seemed at least conceivable that a Louisiana strain might not be competent to transmit B. burgdorferi, thus explaining the very low incidence of Lyme borreliosis in this state.

Our experiments demonstrated that a Louisiana local population of I. scapularis is fully competent as a vector of B. burgdorferi in laboratory mice. Tick larvae were able to become infected with the spirochete, convey it transstadially, and transmit it back to mice when in the nymphal stage. Moreover, larvae became readily infected when fed on tick-inoculated mice. Unfed larvae are essentially spirochete-free as transovarial transmission of B. burgdorferi is very inefficient (Schoeler and Lane 1993, Patrican 1997). Although we did not employ local Peromyscus mice in our experiments, our results indicate that local I. scapularis ticks could maintain the spirochetal enzootic cycle. The pertinence of employing locally derived species of hosts, vectors, and parasites when conducting vector-competence studies has been pointed out (Lane et al. 1994). However, in view of the described limited pathogenicity and infectivity of some Southern isolates of B. burgdorferi (Oliver 1996), we preferred to choose for our study B. burgdorferi strains from the Northeast of known pathogenicity, lest such (or similar) strains could be available for transmission in Louisiana.

What are the determinants of the low Lyme borreliosis incidence in Louisiana in particular, and in the southern United States as a whole, compared with the northeast? Possible answers to this question were comprehensively assessed by Oliver in his review on Lyme borreliosis in the South (Oliver 1996). Among them Oliver listed (1) underreporting of disease cases, (2) decreased infectivity and pathogenicity of some B. burgdorferi isolates, (3) differences in density of human population with respect to that in the northeast, (4) diminished synchrony of the tick life cycle, which entails the possibility that infected overwintered nymphs fail to feed on and infect a new cohort of reservoir hosts before larval feeding, (5) availability of lizards for feeding of the immature tick stages-as already mentioned, some, but not all lizard species are incompetent reservoir hosts for B. burgdorferi, because their plasma complement seems to be borreliacidal (Lane and Quistad 1998); and (6) differences in feeding behavior of nymphal I. scapularis, with the suggested possibility that southern nymphal ticks rarely bite humans. In addition, infection with B. burgdorferi in questing southern I. scapularis nymphs is rare or absent, a fact that stands in stark contrast with the >25% infection found in the northern I. scapularis nymphal populations (Piesman 2002). These factors, alone or in concert, may contribute to the low incidence of Lyme borreliosis in Louisiana. Our results exclude as a factor that the local population of I. scapularis is vector incompetent.

Acknowledgements

This work was supported by Centers for Disease Control and Prevention Grant U50/CCU606604 and National Institute of Health Grant RR00164. Technical assistance and care of the animals used in this study by W. Cyprian and N. Garrett is gratefully acknowledged. The authors also thank A. Mackay, Department of Entomology, Louisiana State University Agricultural Center, for advice and help in the collection of adult ticks.

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References Cited

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