Date of Award

1994

Degree Type

Dissertation

First Advisor

Roger Lebrun

Abstract

Ticks in the Ixodes ricinus-species complex have been implicated as vectors of Lyme disease spirochetes (Borrelia burgdorferi). A variety of other ticks, notably American dog ticks (Dermacentor variabilis) and Lone Star ticks (Amblyomma americanum) in the U.S., appear incapable of transmitting Lyme disease spirochetes despite ingesting these bacteria. In this study, I confirmed that deer ticks, but not dog ticks or Lone Star ticks became infected with Lyme disease spirochetes after feeding on infected hosts. In addition, I assessed several potential physiologic factors that could affect the ability of ticks to acquire, maintain, and transmit B. burgdorferi. One factor in particular, found in tick saliva, appears responsible for preventing spirochete killing in I. scapularis but not D. variabilis or A. americanum.

In one study, I assessed anti-microbial activity of the tick's immune system. In particular, I determined whether ticks exhibited measurable phenoloxidase and antimicrobial activity. Phenoloxidase activity in the hemolymph of larvae of the greater wax moth (Galleria me/lone/la L) was compared to phenoloxidase activity in I. scapularis, D. variabilis and A. americanum. Although activity was detected in wax moth hemolymph, no phenoloxidase activity was detected in three species of ixodid ticks. In addition, Enterobacter cloacae was used in an attempt to induce anti-microbial activity in punched cuticle of three species of ixodid ticks. Using this assay, no anti-microbial activity was detected in ticks. Moreover, no anti-microbial substances were found in the midgut of I. scapularis, D. variabilis and A. americanum. Factors associated with the tick's humoral immunity do not appear to play a role in vector competence for Lyme disease spirochetes.

In another study, host-associated borreliacidal factors were examined in vitro.

Spirochetes survived in the presence of pre-immune rabbit serum but were killed in the presence ofimmune rabbit serum. Heat-inactivation (56 °c, 1 hr) destroyed the killing effect of immune serum but the borreliacidal activity of heat-inactivated immune serum could be restored by the addition of pre-immune serum. Using a similar assay, spirochetes survived in tick midgut extract cultures when ticks were removed from preimmune hosts. Spirochetes also survived in I. scapularis midgut extract cultures, but not in D. variabilis or A. americanum midgut extract cultures when ticks were removed from immune hosts. As it did in immune serum, heat-inactivation destroyed the killing effect of D. variabilis and A. americanum midgut extract cultures. An indirect immunofluoresence assay indicated that anti-B. burgdorferi antibody concentrations were similar in both the host and the tick midgut extract. Taken together, these experiments suggest that borreliacidal activity of host immune serum is mediated by the activity of complement. Furthermore, this same activity is found in the midgut of D. variabilis and A. americanum feeding on immune hosts but is not found in I. scapularis. Since vector competence appeared to be related to the activity of host antibody and complement in the tick midgut, I determined whether substances existed in I. scapularis that might inhibit or inactivate antibody or complement. We tested for the presence of an antibody-cleaving enzyme in the gut extract of I. scapularis and were unable to demonstrate any activity in several dilutions of gut extract after 30 minutes at 37oc. A spirochete survival assay was performed in cobra venom, which possesses powerful anticomplement activity, and in the saliva collected from I. scapularis, D. variabilis and A. americanum. Spirochete survival of over 95% was observed in immune serum with cobra venom and I. scapularis saliva but no spirochete survival was found in cultures containing saliva from D. variabilis and A. americanum. We suggest that an anticomplement factor in the saliva of I. scapularis inactivates complement in the midgut of engorged ticks.

Finally, the duration of tick attachment may serve as a useful predictor of risk for acquiring various tick-transmitted infections such as Lyme disease and babesiosis. We measured three tick engorgement indices (El) at known time intervals after tick attachment and used these indices to determine the length of time that ticks were attached to tick-bite victims in selected Rhode Island and Pennsylvania communities where the agents of Lyme disease and human babesiosis occur. Regression equations developed correlate tick engorgement indices with duration of feeding. More than 60%of tick-bite victims removed adult ticks by 36 hours of attachment, but only 10% found and removed the smaller nymphal ticks within the first 24 hours of tick feeding. A table containing specific EI prediction intervals were calculated for both nymphs and adults allowing practitioners or clinical laboratories to use easily-measured tick engorgement indices to predict transmission risk by determining the duration of feeding by individual ticks.

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