Lyme disease was reproduced in specific pathogen-free beagle dogs by exposure to Borrelia burgdorferi-infected ticks (Ixodes dammini). Seroconversion and disease frequency were higher after exposure to infected adult ticks than to infected nymphs. Young pups developed clinical disease more readily than older dogs. The incubation period lasted 2-5 months. Acute recurrent lameness with fibrinopurulent arthritis was the dominant clinical sign. Dogs recovered but developed persistent mild polyarthritis. B. burgdorferi persisted in recovered dogs for at least 1 year. Isolation of B. burgdorferi and detection by polymerase chain reaction was most successful from skin biopsies at the site of the tick bite. Antibody to B. burgdorferi antigens was first detected by ELISA and Western blots by 4-6 weeks after exposure. High serum levels persisted during 17 months of observation. In contrast to infection from ticks, inoculation of dogs with cultured B. burgdorferi resulted in seroconversion with a shorter duration of antibody persistence and no clinical disease.

Sixteen specific-pathogen-free beagles were infected with Borrelia burgdorferi. Three groups of 4 dogs were treated with antibiotics for 30 consecutive days starting 120 days after tick exposure; 4 dogs were untreated controls. At day 420 after tick exposure and again before euthanasia, 2 dogs of each group were treated with prednisone for 14 days. All dogs contracted infection and 11 developed acute arthritis 50-120 days after exposure. After day 120, one of 12 antibiotic-treated dogs and 2 of 4 untreated dogs became lame. Antibiotic therapy reduced the frequency of Borrelia-positivity in subsequent skin biopsy samples. After prednisone treatment, both control dogs developed severe polyarthritis. At euthanasia, single tissues of the antibiotic-treated dogs and multiple tissues of all control dogs were Borrelia-positive by polymerase chain reaction. Viable spirochetes were not recovered from antibiotic-treated dogs. Two antibiotic-treated dogs showed histologic evidence of minimal lesions, whereas all control dogs had mild polyarthritis with periarteritis.

Abstract. Seven specific-pathogen-free (SPF) ponies, 1-5 years old, were exposed to Borrelia burgdorferiinfected adult ticks while being treated with dexamethasone over 5 consecutive days. One SPF pony (pony No. 178) was first exposed to laboratory-reared nymphs without B. burgdorferi infection and 3 weeks later was exposed to B. burgdorferi-infected adult ticks with concurrent dexamethasone treatment for 5 consecutive days. Four uninfected ponies treated with dexamethasone, exposed to laboratory-reared ticks without B. burgdorferi infection served as uninfected controls. Clinical signs, bacteriologic culture, polymerase chain reaction (PCR) for bacterial DNA, immunologic responses, and gross lesions and histopathologic changes were investigated during the experiment or at necropsy 9 months after tick exposure. In all of the seven challenged ponies, infection with B. burgdorferi was detected from monthly skin biopsies and various tissues at postmortem examination by culture and by PCR. However, pony No. 178 exposed to laboratory-reared nymphs (without B. burgdorferi infection) and challenged with B. burgdorferi-infected adult ticks 2 months later did not develop a B. burgdorferi infection. All of the infected ponies seroconverted. Control ponies and pony No. 178 were negative by culture, PCR, and serology. Except for skin lesions, we failed to induce any significant histopathologic changes in this study. This is the first report of successful tick-induced experimental infection in ponies by exposure to B. burgdorferi-infected ticks. This Lyme disease model will be very useful to evaluate efficacy of vaccines against the Lyme agent and the effect of antibiotic therapy on horses infected with B. burgdorferi.

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Abstract. Numerous reports have described diagnostic methods based on the polymerase chain reaction (PCR) used to detect Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease. The result of conventional PCR tests has been only qualitative, either positive or negative; it does not present any quantitative information about the number of the agents in the specimen. A quantitative PCR method (IS900 TaqMan) zooepidemicus).Even at high cell numbers (10 5 CFU/reaction), most of the organisms tested negative for the IS900 insertion element except M. marinum and M. scrofulaceum. This finding for M. scrofulaceum was consistent with previous reports that several M. scrofulaceum-like isolates were positive for IS900. Those isolates had 71-79% homology with M. a. paratuberculosis in the region of IS900. When used in conjunction with the new liquid medium-based ESP culture system II for bovine clinical fecal samples, IS900 TaqMan confirmed that the ESP II-positive samples contained 10 5 -10 6 CFU/ml of M. a. paratuberculosis. All of the 222 ESP II-positive and acid-fast bacilli-positive samples tested in this study were positive by IS900 TaqMan. IS900 TaqMan was also useful in the study of growth characteristics of 3 groups of M. a. paratuberculosis strains in bovine fecal samples from 3 shedding levels (heavy, medium, and low) based on cell numbers measured by Herrold egg yolk (HEY) agar culture. When cultured in ESP medium, M. a. paratuberculosis reached 10 5 -10 6 CFU/ml within 2 weeks for heavy shedders, 3-4 weeks for medium, and 6-8 weeks for low shedders. No significant growth was observed after up to 5 weeks of incubation for some of low shedders. No or extremely slow growth characteristic of low shedders might be a possible explanation for frequent false-negative results by HEY. The detection time was dependent on the inoculum size and the growth rate of M. a. paratuberculosis. Generation times were inversely proportional to the shedding level: 1-2 days for medium and heavy shedders and Ͼ4 days for low shedders. IS900 TaqMan could be a useful tool for determining viable cell counts by measuring changes in cell numbers over the incubation period.

Results obtained with use of the new ELISA agreed favorably with those of a previous ELISA. Practitioners must be aware of variability in the sensitivity of the test, which depends on the clinical and shedding status of the cattle, because this may affect interpretation of test results.

Sera collected from dogs experimentally infected withBorrelia burgdorferi by tick inoculation were analyzed for an antibody response to each of the six invariable regions (IRs; i.e., IR1 to IR6) of VlsE, the variable surface antigen of B. burgdorferi. Six synthetic peptides (C1 to C6), which reproduced the six IR sequences were used as peptide-based, enzyme-linked immunosorbent assay (ELISA) antigens. Two IRs, IR2 and IR6, were found to be immunodominant. Studies with serially collected serum samples from experimentally infected dogs revealed that the antibody response to IR6 appears earlier and is stronger than that to IR2. Thus, the IR6 sequence alone appeared to be sufficient for serodiagnosis. When C6 alone was used as antigen, the peptide-based ELISA was positive in 7 of 23 dogs (30%) as early as 3 weeks postinfection. All dogs (n = 33) became strongly positive 1 or 2 weeks later, and this response persisted for the entire study, which lasted for 69 weeks. Of 55 sera submitted by veterinarians from dogs suspected of having Lyme disease, 19 were also positive by the C6 ELISA, compared to 20 positives detected by immunoblot analysis using cultured B. burgdorferi lysates as antigen. The sensitivity of using C2 and C6 together for detecting specific antibody in both experimentally infected and clinically diagnosed dogs was not better than sensitivity with C6 alone, confirming that C6 suffices as a diagnostic probe. Moreover, the C6 ELISA yielded 100% specificity with serum samples collected from 70 healthy dogs, 14 dogs with infections other than B. burgdorferi, and 15 animals vaccinated with either outer surface protein A, whole-spirochete vaccines, or the common puppy-vaccines. Therefore, this C6 ELISA was both sensitive and specific for the serodiagnosis of canine Lyme disease and could be used with vaccinated dogs.