The prophylactic potential of a single injection of sustained-release doxycycline hyclate (Atridox) was compared to that of a single oral dose of doxycycline hyclate in a murine model of Lyme borreliosis. Prophylaxis, as measured by the lack of cultivable spirochetes and demonstrable pathology, was noted for 43% of orally treated mice; in contrast, the sustained-release doxycycline hyclate completely protected mice from infection and resultant pathology.Since Lyme borreliosis became a reportable disease in 1982, it has consistently been the most common vector-borne disease reported in the United States (3). Given the lack of an available vaccine, the prophylaxis of tick bite infection consists of personal protective measures directed against ticks in areas where Borrelia burgdorferi is endemic (4). Reports on the efficacy of post-tick exposure antimicrobial prophylaxis in animals and humans demonstrate a high degree of variability, and opinions vary greatly as to the necessity of such prophylactic treatment (5,11,16,17,19). The need for effective prophylaxis is underscored by the increase in cases of Lyme disease and the seriousness of untreatable sequelae in a subset of infected individuals (7,8). This study compares the effectiveness of a single oral dose of doxycycline hyclate, designed to mimic levels in the plasma of humans given 200 mg orally (6,14), to that of a subcutaneous injection of sustained-release doxycycline (Atridox; Atrix Laboratories, Fort Collins, Colo.) in a mouse model of Lyme borreliosis (22).Laboratory-reared nymphal ticks (Ixodes scapularis) that were raised as previously described (13) and that have previously been shown to be free of Anaplasma phagocytophila and Babesia microti (2, 21) were infected with B. burgdorferi strain B31. Five infected ticks were placed on the heads and neck areas of specific-pathogen-free, 7-week-old female C3H/HeJ mice (Jackson Laboratories, Bar Harbor, Maine). Seventy-two hours after tick infestation, the partially engorged ticks were removed from all of the mice. Since three or four infected ticks (average, 3.4) fed on each mouse, the mice were then randomly assigned to receive 2 mg of oral doxycycline hyclate in water, 4.2 mg of a sustained-release doxycycline hyclate copolymer formulation (Atrix Laboratories), or a water or poly (DL-lactide) in N-methyl-2-pyrrolidone copolymer (Atrix Laboratories) control. The oral doxycycline or water vehicle was delivered by gavage in 0.1 ml of tissue-grade water. Sustainedrelease doxycycline hyclate was mixed according to the manufacturer's instructions and transferred to a 1-ml Luerlock syringe (Becton Dickinson, Chicago, Ill.) fitted with a 25-gauge needle for subcutaneous injection. A total of 0.05 ml was delivered to each mouse. At 4 weeks posttreatment, an ear biopsy was obtained and cultured in Barbour-Stoenner-Kelly medium (15) to determine B. burgdorferi infection status (18). The mice were euthanized (by exposure to CO 2 ) 8 weeks after tick infestation, and heart and bladder tissues were placed in culture me...
Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) (1-8). To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick (3,9-13). For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle (14,15). Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva (9,16-19). As a tick feeds the host typically responds with a strong hemostatic and innate immune response (11,13,20-22). Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding (3,11,20,21,23). The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens (3,20,24-27). To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.
bThe impact of the Borrelia burgdorferi surface-localized immunogenic lipoprotein BBA66 on vector and host infection was evaluated by inactivating the encoding gene, bba66, and characterizing the mutant phenotype throughout the natural mouse-tickmouse cycle. The BBA66-deficient mutant isolate, Bb ⌬A66 , remained infectious in mice by needle inoculation of cultured organisms, but differences in spirochete burden and pathology in the tibiotarsal joint were observed relative to the parental wild-type (WT) strain. Ixodes scapularis larvae successfully acquired Bb ⌬A66 following feeding on infected mice, and the organisms persisted in these ticks through the molt to nymphs. A series of tick transmission experiments (n ؍ 7) demonstrated that the ability of Bb ⌬A66 -infected nymphs to infect laboratory mice was significantly impaired compared to that of mice fed upon by WT-infected ticks. trans-complementation of Bb ⌬A66 with an intact copy of bba66 restored the WT infectious phenotype in mice via tick transmission. These results suggest a role for BBA66 in facilitating B. burgdorferi dissemination and transmission from the tick vector to the mammalian host as part of the disease process for Lyme borreliosis.
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