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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Clinical isolates of Borrelia burgdorferi sensu stricto have been categorized into disseminated and nondisseminated groups based on distinct ribosomal spacer restriction fragment length polymorphism genotypes (RSTs). In order to determine whether transmission by tick bite would alter the dissemination dynamics and disease produced by distinct genotypes, disseminated isolates (RST1), nondisseminated isolates (RST3), and a standard laboratory strain (B-31) were established in a murine cycle utilizing infections transmitted by ticks. B-31 spirochetes circulated in the blood of inbred C3H/HeJ mice longer than in the blood of outbred mice. The majority of C3H mice exposed to RST1-infected ticks contained cultivable spirochetes in their blood for up to 17 days; in contrast, mice exposed to RST3 isolates demonstrated a precipitous decline in infection after day 7 postexposure. A quantitative PCR (q-PCR) assay demonstrated that the densities of spirochetes in blood were similar for the RST1 and RST3 isolates, except during the 2nd week postexposure, when the RST1 isolates displayed a markedly higher density in blood. Spirochete load in the heart and bladder of infected mice was measured by q-PCR at 8 weeks postexposure; the numbers of spirochetes in these tissues were similar for mice infected with either disseminated or nondisseminated strains. Similarly, histopathology samples of heart, bladder, and joint tissue obtained at 8 weeks postexposure did not reveal greater pathology in mice infected with the disseminated isolates. We conclude that although the spirochetemia induced by tick-transmitted disseminated isolates was more intense and of longer duration than that induced by nondisseminated isolates, the resultant pathologies produced by these strains were ultimately similar.Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most common vector-borne disease in the United States (3). Lyme disease spirochetes (B. burgdorferi) are transmitted by Ixodes scapularis and Ixodes pacificus ticks in the United States and Ixodes ricinus and Ixodes persulcatus ticks in Europe and Asia (7). Lyme disease usually begins with a characteristic, expanding skin lesion referred to as erythema migrans (EM), which may be accompanied by flu-like symptoms. Some patients may develop more serious sequelae such as arthritis, myocarditis, and lesions of the peripheral and central nervous system (4, 23). Although B. burgdorferi sensu lato is a diverse group of bacteria representing up to 11 named genospecies, only three pathogenic genospecies (B. burgdorferi sensu stricto, Borrelia afzelii, and Borellia garinii) are commonly known to cause human disease (10, 16). All three agents of Lyme disease are found in Europe and are associated with distinct clinical manifestations (24, 25); in contrast, B. burgdorferi sensu stricto is the sole agent associated with human cases of Lyme disease reported from North America (14).B. burgdorferi sensu stricto isolates cultured from Lyme disease patients in Westchester County, N.Y., have been c...

Laboratory testing for the diagnosis of Lyme disease is performed primarily by serologic assays and is accurate for detection beyond the acute stage of the infection. Serodiagnostic assays to detect the early stages of infection, however, are limited in their sensitivity, and improvement is warranted. We analyzed a series of Borrelia burgdorferi proteins known to be induced within feeding ticks and/or during mammalian infection for their utility as serodiagnostic markers against a comprehensive panel of Lyme disease patient serum samples. The antigens were assayed for IgM and IgG reactivity in line immunoblots and separately by enzyme-linked immunosorbent assay (ELISA), with a focus on reactivity against early Lyme disease with erythema migrans (EM), early disseminated Lyme neuroborreliosis, and early Lyme carditis patient serum samples. By IgM immunoblotting, we found that recombinant proteins BBA65, BBA70, and BBA73 reacted with early Lyme EM samples at levels comparable to those of the OspC antigen used in the current IgM blotting criteria. Additionally, these proteins reacted with serum samples from patients with early neuroborreliosis and early carditis, suggesting value in detecting early stages of this disease progression. We also found serological reactivity against recombinant proteins BBA69 and BBA73 with early-Lyme-disease samples using IgG immunoblotting and ELISA. Significantly, some samples that had been scored negative by the Centers for Disease Control and Prevention-recommended 2-tiered testing algorithm demonstrated positive reactivity to one or more of the antigens by IgM/IgG immunoblot and ELISA. These results suggest that incorporating additional in vivo-expressed antigens into the current IgM/IgG immunoblotting tier in a recombinant protein platform assay may improve the performance of early-Lyme-disease serologic testing.A ccurate diagnoses are essential to treat patients with Lyme disease, a tick-borne illness caused by the bacterial agent Borrelia burgdorferi. Diagnosis in the initial stages of Lyme disease can be made by clinical signs such as the onset of flu-like symptoms with the presence of a rash termed erythema migrans (EM) at the site of the tick bite (1, 2). Aiding the diagnostic evaluation, Lyme disease in the United States is endemic and transmitted by the tick vectors Ixodes scapularis in the Northeast and upper Midwest, and Ixodes pacificus in parts of the Pacific Northwest (http://www.cdc .gov/lyme/stats/index.html). However, it is not always apparent that a patient was bitten by an infected tick, and the EM may not appear or may go unnoticed, leading to a disseminated infection with more severe clinical symptoms, including arthritis, carditis, and neuropathy (2). In these instances, diagnosis is performed by serological testing to determine if the patient has been exposed to B. burgdorferi.The standard for serologic Lyme disease testing is a 2-tiered test recommended by the Centers for Disease Control and Prevention whereby the first tier is commonly an enzyme immunoassay ...

Feline heartworm disease, caused by the filarial nematode Dirofilaria immitis, has been diagnosed with increased frequency in areas endemic for canine heartworm infection. The routine methods for determining the infection status of dogs, such as identification of circulating microfilariae in blood or identification of circulating antigen in serum, plasma or blood, have proven inadequate for screening cats. The inadequacies are due to the likelihood of single-sex infections and clinical disease during prepatent infections. Current antibody detection methodologies rely on crude or partially purified worm antigen preparations that may result in poor specificity. This report describes the cloning, expression, and diagnostic utility of the D. immitis homologue (PDi33) of the Onchocerca volvulus aspartyl protease inhibitor (Ov33). PDi33 is present in all stages that occur in the mammalian host (microfilariae, L3, L4, adult males, and females) and is released by adults cultured in vitro. An indirect enzyme-linked immunosorbent assay (ELISA) using antibody to recombinant PDi33 as a diagnostic marker for infection in cats was very sensitive and was useful for identifying prepatent infections. Testing of sera from cats infected with common gastrointestinal parasites also indicated excellent specificity. The same ELISA in dogs, although demonstrating reasonable sensitivity and specificity, appeared to be of less value as compared with the currently accepted antigen detection methodologies.