Richard H. Jacobson

Status of Borrelia burgdorferi infection after antibiotic treatment and the effects of corticosteroids: an experimental study.

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.

Development and Application of Quantitative Polymerase Chain Reaction Assay Based on the ABI 7700 System (TaqMan) for Detection and Quantification of Mycobacterium Avium Subsp. Paratuberculosis

Numerous reports have described diagnostic methods based on the polymerase chain reaction (PCR) used to detect Mycobacterium avium subsp. paratuberculosis, the causative agent of Johnes 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) was developed to measure the number of M. a. paratuberculosis organisms present in field and clinical samples. The sensitivity of IS900 TaqMan was 1 colony-forming unit (CFU) for M. a. paratuberculosis ATCC 19698. The specificity of the method was determined by testing 14 mycobacterial species (M abscessus, M. asiaticum, M. avium subsp. avium, M. bovis, M. fortuitum subsp. fortuitum, M. intracellular, M. kansasii, M. marinum, M. phlei, M. scrofulaceum, M. simiae, M. smegmatis, M. terrae, and M. ulcerans) and 9 nonmycobacterial species (Borrelia burgdorferi, Chlamydia psittaci, Ehrlichia canis, E. equi, E. risticii, Escherichia coli, E. coli O157:H7, Streptococcus equi, and S. zooepidemicus). Even at high cell numbers (105 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 105–106 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 105–106 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 >4 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.

Cross-reactivity between B. burgdorferi and other spirochetes affects specificity of serotests for detection of antibodies to the Lyme disease agent in dogs

Abstract Western immunoblots, the kinetics-based enzyme-linked immunosorbent assay (KELA), and the microagglutination test were used to evaluate cross-reactivity among antibodies to serovars of Leptospira interrogans (leptospiral serovars), and B. burgdorferi from naturally infected dogs, and to Serpulina (Treponema) hyodysenteriae from vaccinated rabbits. Whole-cell lysates from Borrelia spp., leptospiral serovars, and Serpulina spp. were used for SDS-PAGE, western blots, and KELA. Cross-reactivity occurred between the antibodies to B. burgdorferi and leptospiral serovars when tested on the heterologous antigens. Antibodies to leptospiral serovars tended to cross-react more strongly with antigens of B. burgdorferi spp. than did antibodies to B. burgdorferi when tested against antigens of leptospiral serovars. The antibodies against B. burgdorferi showed a lesser degree of cross-reactivity to the antigens of S. hyodysenteriae and S. innocens than they did to leptospiral serovars. We conclude that cross-reactivity occurs between B. burgdorferi and leptospiral serovars. Validation and interpretation of ELISA tests for detection of antibody activity to whole cell lysates of the Lyme agent must take this cross-reactivity into consideration. Conversely, dogs infected with the Lyme agent do not show significant cross-reactivity in the microagglutination test for antibody to the leptospiral serovars.

BALB/c mice infected with Brucella abortus express protracted polyclonal responses of both IgG2a and IgG3 isotypes

A polyclonal IgG2a response dependent on the secretion of endogenous IFN-gamma has been demonstrated in BALB/c mice injected with killed whole cells of Brucella abortus [1]. Here we report intense and protracted polyclonal responses of IgG2a and also of IgG3 isotypes in BALB/c mice undergoing primary infections with B. abortus attenuated vaccine strain 19 or virulent strain 2308. Ratios of total serum Ig levels between infected mice and age matched controls were greater than 38 for IgG3 and greater than 12 for IgG2a between weeks 4 and 8 post-infection. Polyclonal increases of IgM and IgG1 that were proportionally much lower (ratios < 2 and < or = 3, respectively) also occurred in infected mice during this time. It is hypothesized that both IgG3 and IgG2a polyclonal responses required IFN-gamma, which was induced by B. abortus primarily in a T cell-independent fashion during the first weeks of infection, and from T cells thereafter.

Vaccination against Lyme Disease with recombinant Borrelia burgdorferi outer-surface protein A (rOspA) in horses

Eight 1-year-old ponies were vaccinated with recombinant OspA (ospA gene derived from B. burgdorferi B31) with adjuvant (aluminium hydroxide). Four ponies were used as non-vaccinated controls with adjuvant. One hundred and twelve days after the first vaccination, the vaccinated and non-vaccinated ponies were challenged by exposure to B. burgdorferi-infected adults tick (Ixodes scapularis) collected from Westchester County, New York (tick infection rate >/=60%). Protection from infection was evaluated by culture for B. burgdorferi from three monthly skin biopsies taken near the site of tick bites. B. burgdorferi was not isolated from any of the vaccinated ponies. In contrast, three of four control ponies challenged by tick exposure were skin culture positive. At the time of tick exposure, vaccinated ponies had antibody to B. burgdorferi demonstrable by KELA (kinetic-ELISA), western blot and a serum growth inhibition assay. Antibodies in the challenge control ponies were only detectable by two to three months after tick exposure and remained at intermediate levels until termination of the study. By western blot analysis, antibodies to OspA first appeared in the sera of vaccinated ponies three weeks after the first vaccination. The absence of additional bands, known to develop when the animal is infected, suggests that infection was blocked after tick exposure of vaccinated ponies. Results from this study show that vaccination with recombinant OspA protected ponies against infection after experimental challenge with B. burgdorferi-infected ticks.

Reproducibility of a Commercial Enzyme-Linked Immunosorbent Assay for Bovine Paratuberculosis among Eight Laboratories

Interlaboratory reproducibility of an absorbed enzyme-linked immunosorbent assay (ELISA) kit for detection of bovine serum antibodies to Mycobacterium paratuberculosis was evaluated. A panel of 30 bovine sera (15 positives and 15 negatives) was tested in triplicate microtiter wells on each of 2 days at 8 different laboratories. One laboratory had invalid results because of positive or negative serum control optical density (OD) readings beyond the acceptable range specified by the kit. The coefficient of variation (CV) for mean OD values was influenced by low ODs on test negative sera at 2 laboratories, thus the CVs on positive sera were considered a more representative measure of kit reproducibility. Between-well CVs averaged 6.7% ± 2.8% (mean ± standard deviation), and between-day CVs averaged 14.5% ± 9.8% among the 7 laboratories with valid assays on the 15 positive sera. The OD values were converted to positive or negative classifications for each assay well, and the results were compared. Among 1,392 assays in 7 laboratories, 98.6% were in agreement. Eleven of 18 discrepant results were due to a sample that consistently gave OD values near the cutoff for a positive test. Exclusion of that serum from the analysis resulted in a 99.8% rate of agreement among laboratories. Results indicated that the absorbed ELISA kit provided reproducible results within and between laboratories.

Feline immunodeficiency virus, feline leukaemia virus, Toxoplasma gondii, and intestinal parasitic infections in Taiwanese cats.

A population consisting of 70 breeder cats, 43 clinical cases, and 16 feral cats was examined for the presence of Toxoplasma gondii, feline immunodeficiency virus (FIV), and feline leukaemia virus (FeLV). No oocysts of T. gondii were observed in 96 faecal samples; faecal samples were not available from the feral cats. Other intestinal parasites identified included Isospora felis (three cats), Isospora rivolta (five), Dipylidium canium (two), Toxocara cati (four), Toxascaris leonina (one), and Ancylostoma sp. (two). Using a kinetics-based enzyme-linked immunosorbent assay on 117 sera including all the feral cats, nine had antibody to T. gondii antigen, three for antigens to FIV, and seven to the p27 antigen of FeLV. Of the nine cats with antibody to T. gondii, only one was also infected with FIV.

Babesia bovis: In vitro phagocytosis promoted by immune serum and by antibodies produced against protective antigens

The in vitro phagocytosis of bothBabesia bovis-infected red cells and of parasites exposed by lysis of infected red blood cells is demonstrated in a phagocytic mouse model. Twenty-fourB. bovis immune sera were tested alone or as a pool as were antibodies (DS antibodies) raised against aB. bovis protective fraction, prepared by dextran sulfate precipitation. All the immune sera failed to promote significant levels of phagocytosis, whereas the other antibodies (DS antibodies) consistently induced phagocytosis of infected cells in all the experiments carried out. This study shows that antibody specificity is critical to the opsonization of infected red cells and parasites during in vitro phagocytosis and suggests that phagocytosis is one of the mechanisms in the in vivo immune response againstBabesia species.

Protection against tick-transmitted Lyme disease in dogs vaccinated with a multiantigenic vaccine.

In an effort to develop a safe and effective vaccine for the prevention of Lyme borreliosis that addresses concerns raised over currently available vaccines, dogs were vaccinated twice with a multiantigenic preparation of Borrelia burgdorferi, strain N40, on days 0 and 20 of the experiment. About 70 and 154 days after the first immunization, dogs were challenged by exposing them to field-collected Ixodes scapularis ticks harboring B. burgdorferi. Vaccinated dogs were completely protected from infection by all criteria utilized to assess infection, developed high-titer anti-B. burgdorferi serum antibodies and growth inhibitory activity which persisted for over 200 days, and did not demonstrate any untoward consequence of vaccination. Serum absorption experiments revealed that borreliacidal and most likely protective antibodies in dogs receiving the multiantigenic preparation were not only elicited against the OspA antigen, but were also produced against additional yet to be determined targets on B. burgdorferi organisms. These data demonstrate that a multiantigenic vaccine is effective in preventing Lyme disease transmitted via the natural vector.

A multilaboratory evaluation of a commercial enzyme-linked immunosorbent assay test for the detection of antibodies against Mycobacterium avium subsp. paratuberculosis in cattle.

Five laboratories participated in a study to evaluate sources of variation in results from an enzyme-linked immunosorbent assay (ELISA) for antibodies against Mycobacterium avium subsp. paratuberculosis. Each laboratory repeatedly tested duplicates of a negative, positive (P), and high-positive (HP) serum sample, which were supplied by the United States Department of Agriculture: Animal and Plant Health Inspection Service: Veterinary Services, National Veterinary Services Laboratories, Ames, IA, on all 96-well microtiter plates when routinely testing other samples for M. avium subsp. paratuberculosis antibodies. These 3 sera were aliquoted and sent to the 5 participating laboratories. This study focused on variation in test results because of assay reagents and laboratory techniques and did not account for biologic variability associated with the time course of infection in cattle. Overall, results from 868 microtiter plates were used in the study. For each sample a sample-to-positive (S/P) ratio was calculated according to the manufacturers directions. The S/P ratio for the P sample ranged from 0.06 to 1.039 (mean = 0.466 and 0.484 for wells 1 and 2, respectively) and those for the HP sample ranged from 2.446 to 8.727 (mean = 4.027 and 3.980 for wells 1 and 2, respectively). The majority of the variation in S/P ratio for the P sample was attributed to kit lot (37.5%), followed by random (unexplained) error (27.0%), laboratory (18.3%), and kit lot by laboratory (11.9%). By eliminating plates in which the separation between negative and positive control ODs was less than 0.4, the proportion of variation attributed to laboratory was reduced markedly. These results confirm that there is variability in M. avium subsp. paratuberculosis ELISA results and that several sources contribute to the observed variability. The study gives a relative estimate of the contribution of various sources to the overall variability observed in the M. avium subsp. paratuberculosis ELISA results with kit lot being a primary contributor. Similar data for other ELISA tests for antibodies to M. avium subsp. paratuberculosis or other antigens also should be developed.