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ABSTRACTMycobacterium aviumsubsp. paratuberculosisis shed into the milk and feces of cows with advanced Johne's disease, allowing the transmission of M. aviumsubsp. paratuberculosisbetween animals. The objective of this study was to formulate an optimized protocol for the isolation of M. aviumsubsp. paratuberculosisin milk. The parameters investigated included chemical decontamination with N-acetyl-l-cysteine–sodium hydroxide (NALC-NaOH), alone and in combination with antibiotics (vancomycin, amphotericin B, and nalidixic acid), and the efficacy of solid (Herrold's egg yolk medium [HEY]) and liquid (Bactec 12B and para-JEM) culture media. For each experiment, raw milk samples from a known noninfected cow were inoculated with 102to 108CFU/ml of live M. aviumsubsp. paratuberculosisorganisms. The results indicate that an increased length of exposure to NALC-NaOH from 5 to 30 min and an increased concentration of NaOH from 0.5 to 2.0% did not affect the viability of M. aviumsubsp. paratuberculosis. Additional treatment of milk samples with the antibiotics following NALC-NaOH treatment decreased the recovery of viable M. aviumsubsp. paratuberculosiscells more than treatment with NALC-NaOH alone. The Bactec 12B medium was the superior medium of the three evaluated for the isolation of M. aviumsubsp. paratuberculosisfrom milk, as it achieved the lowest threshold of detection. The optimal conditions for NALC-NaOH decontamination were determined to be exposure to 1.50% NaOH for 15 min followed by culture in Bactec 12B medium. This study demonstrates that chemical decontamination with NALC-NaOH resulted in a greater recovery of viable M. aviumsubsp. paratuberculosiscells from milk than from samples treated with hexadecylpyridinium chloride (HPC). Therefore, it is important to optimize milk decontamination protocols to ensure that low concentrations of M. aviumsubsp. paratuberculosiscan be detected.

ABSTRACTCD40 and CD40 ligand (CD40L) have costimulatory effects as part of a complex series of events in host immunity. In this study, the expression of CD40 and CD40L on peripheral blood mononuclear cells (PBMCs) isolated from cattle with Johne's disease were measured on freshly isolated PBMCs and on cells cultured for 8, 24, and 72 h in the presence or absence of live Mycobacterium aviumsubsp. paratuberculosisand exogenous gamma interferon, interleukin 10, and transforming growth factor ß. Results demonstrated greater CD40 and CD40L expression on fresh PBMCs obtained from animals in the clinical stage of disease (symptomatic) than those from healthy control animals or cows in the subclinical stage of disease (asymptomatic). A similar expression profile with greater magnitude was noted for cultured PBMCs, with increased CD40 expression after 8 and 24 h of culture and increased CD40L expression between 24 and 72 h on PBMCs obtained from clinically infected animals. The addition of live M. aviumsubsp. paratuberculosisto cell cultures resulted in downregulation of CD40L expression in naturally infected cows, regardless of the disease stage. In contrast, the addition of live M. aviumsubsp. paratuberculosisto cultures resulted in upregulation of CD40 expression on cells obtained from clinically infected animals, while a decrease in expression was noted for healthy and subclinically infected cows. No effects of exogenous cytokines on CD40 or CD40L expression were observed. These results clearly point for the first time to a disparity in the expression of these costimulatory molecules on immune cells from cattle in different stages of Johne's disease and suggest further investigation into their roles in paratuberculosis pathogenesis.

ABSTRACTThe cross-reactivity of mycobacterial antigens in immune-based diagnostic assays has been a major concern and a criticism of the current tests that are used for the detection of paratuberculosis. In the present study, Mycobacterium aviumsubsp. paratuberculosisrecombinant proteins were evaluated for antigenic specificity compared to a whole-cell sonicate preparation (MPS). Measures of cell-mediated immunity to M. aviumsubsp. paratuberculosisantigens were compared in calves inoculated with live M. aviumsubsp. paratuberculosis, M. aviumsubsp. avium(M. avium), Mycobacterium kansasii, or Mycobacterium bovis. Gamma interferon (IFN-?) responses to MPS were observed in all calves that were exposed to mycobacteria compared to control calves at 4 months postinfection. Pooled recombinant M. aviumsubsp. paratuberculosisproteins also elicited nonspecific IFN-? responses in inoculated calves, with the exception of calves infected with M. bovis. M. aviumsubsp. paratuberculosisproteins failed to elicit antigen-specific responses for the majority of immune measures; however, the expression of CD25 and CD26 was upregulated on CD4, CD8, gamma/delta (?d) T, and B cells for the calves that were inoculated with either M. aviumsubsp. paratuberculosisor M. aviumafter antigen stimulation of the cells. Stimulation with MPS also resulted in the increased expression of CD26 on CD45RO+CD25+T cells from calves inoculated with M. aviumsubsp. paratuberculosisand M. avium. Although recombinant proteins failed to elicit specific responses for the calves inoculated with M. aviumsubsp. paratuberculosis, the differences in immune responses to M. aviumsubsp. paratuberculosisantigens were dependent upon mycobacterial exposure. The results demonstrated a close alignment in immune responses between calves inoculated with M. aviumsubsp. paratuberculosisand those inoculated with M. aviumthat were somewhat disparate from the responses in calves infected with M. bovis, suggesting that the biology of mycobacterial infection plays an important role in diagnosis.

ABSTRACTA major drawback of current whole-cell vaccines for Mycobacterium aviumsubsp. paratuberculosisis the interference with diagnostic tests for bovine tuberculosis (TB) and paratuberculosis. The current study was designed to explore the effects of immunization with a heat-killed whole-cell vaccine (Mycopar) on diagnostic test performance and to characterize host immune responses to vaccination over a 12-month period. Neonatal dairy calves were assigned to treatment groups consisting of (i) controls, not vaccinated (n= 5), and (ii) vaccinates, vaccinated with Mycopar vaccine (n= 5). The results from this study demonstrated a rapid initiation of M. aviumsubsp. paratuberculosis-specific gamma interferon (IFN-?) in vaccinated calves by 7 days, with robust responses throughout the study. Vaccinated calves also had responses to M. bovispurified protein derivative tuberculin (BoPPD) but minimal reactivity to ESAT-6/CFP-10, an M. bovisrecombinant fusion protein. The levels of antigen-specific interleukin-4 (IL-4) and IL-10 were markedly decreased in vaccinated calves between days 7 and 90 of the study but thereafter were similar to the levels in controls. Vaccinated calves began to seroconvert at 4 months, with 4/5 calves having detectable M. aviumsubsp. paratuberculosisantibody by 6 months. The responses in test platforms for bovine TB were negligible in the vaccinate group, as only one calf had a response, which was in the suspect range of the comparative cervical skin test. Serum antibody responses to M. bovisantigens ESAT-6, CFP-10, and MPB83 were negative on the Vet TB STAT-PAK, DPP VetTB, and DPP BovidTB tests. These results suggest that the Mycopar vaccine will interfere with diagnostic tools for paratuberculosis but result in low interference with the comparative cervical skin test and emerging serologic tests for M. bovis.

ABSTRACTThe objective of this study was to determine if experimental infection of neonatal calves with Mycobacterium aviumsubsp. paratuberculosiswould invoke changes in the percentages of total B cells in the peripheral blood mononuclear cell population and of subpopulations of B cells as determined by CD5, CD25, and CD45RO markers during a 12-month period. Experimental infection groups included control (noninfected), oral (infected with M. aviumsubsp. paratuberculosisstrain K-10), oral/DXM (pretreatment with dexamethasone before oral inoculation), i.p. (intraperitoneal inoculation), and oral/M (oral inoculation with mucosal scrapings from a cow with clinical disease) groups. Over the course of the study, the percentages of total B cells in nonstimulated and antigen-stimulated cell cultures increased for oral and i.p. group calves, with the highest percentages noted at 3 and 6 months. Oral/M group calves had increased percentages of activated B cells, as determined by CD5dimand CD5brightmarkers, at 9 and 12 months. Experimental infection by all methods resulted in increased expression of CD25+and CD45RO+B cells early in the study, but the most significant results were observed at 12 months for oral/DXM and oral/M group calves. Immunoblot analyses with a whole-cell sonicate of M. aviumsubsp. paratuberculosisdemonstrated the most reactivity with sera from i.p. group calves and the least reactivity with sera from oral group calves. Further evidence of M. aviumsubsp. paratuberculosis-specific antibody responses in the i.p. group calves was demonstrated using the ethanol vortex enzyme-linked immunosorbent assay (EvELISA) method. In summary, an induction of B cell responses was noted after experimental infection with M. aviumsubsp. paratuberculosis, with differences in responses noted according to the method of experimental inoculation.

ABSTRACTThe objective of this study was to observe early markers of cell-mediated immunity in nai¨ve calves infected with Mycobacterium aviumsubsp. paratuberculosisand how expression of these markers evolved over the 12-month period of infection. Groups for experimental infection included control (noninfected), oral (infected orally with M. aviumsubsp. paratuberculosisstrain K-10), oral/DXM (pretreatment with dexamethasone before oral inoculation), intraperitoneal (i.p.) inoculation, and oral/M (oral inoculation with mucosal scrapings from a cow with clinical disease) groups. One of the earliest markers to emerge was antigen-specific gamma interferon (IFN-?). Only i.p. inoculated calves had detectable antigen-specific IFN-? responses at 7 days, with responses of the other infection groups becoming detectable at 90 and 120 days. All infection groups maintained robust IFN-? responses for the remainder of the study. At 1 month, calves in the oral and oral/M groups had higher antigen-stimulated interleukin-10 (IL-10) levels than calves in the other treatment groups, but IL-10 secretion declined by 12 months for all calves. T-cell activation markers such as CD25, CD26, CD45RO, and CD5 were significantly upregulated in infected calves compared to noninfected controls. Oral inoculation of calves resulted in significantly increased antigen-specific lymphocyte proliferation at 9 and 12 months, as well as inducible nitric oxide synthase (iNOS) secretion at 6 and 12 months. These results demonstrate that infection of nai¨ve calves with M. aviumsubsp. paratuberculosisinvoked early immunologic responses characterized by robust antigen-specific IFN-? responses and induction of CD25 and CD45RO expression on T-cell subsets. These were followed by antigen-specific lymphocyte proliferation, iNOS secretion, and expression of CD26 and CD5brightmarkers in the latter part of the 12-month study.

ABSTRACTCurrently, paratuberculosis vaccines are comprised of crude whole-cell preparations of Mycobacterium aviumsubsp. paratuberculosis. Although effective in reducing clinical disease and fecal shedding, these vaccines have severe disadvantages as well, including seroconversion of vaccinated animals and granulomatous lesions at the site of vaccination. DNA vaccines can offer an alternative approach that may be safer and elicit more protective responses. In an effort to identify protective M. aviumsubsp. paratuberculosissequences, a genomic DNA expression library was generated and subdivided into pools of clones (∼1,500 clones/pool). The clone pools were evaluated to determine DNA vaccine efficacy by immunizing mice via gene gun delivery and challenging them with live, virulent M. aviumsubsp. paratuberculosis. Four clone pools resulted in a significant reduction in the amount of M. aviumsubsp. paratuberculosisrecovered from mouse tissues compared to mice immunized with other clone pools and nonvaccinated, infected control mice. One of the protective clone pools was further partitioned into 10 clone arrays of 108 clones each, and four clone arrays provided significant protection from both spleen and mesenteric lymph node colonization by M. aviumsubsp. paratuberculosis. The nucleotide sequence of each clone present in the protective pools was determined, and coding region functions were predicted by computer analysis. Comparison of the protective clone array sequences implicated 26 antigens that may be responsible for protection in mice. This study is the first study to demonstrate protection against M. aviumsubsp. paratuberculosisinfection with expression library immunization.

ABSTRACTThis study describes the development of a nested PCR assay that uses a unique element (ISMap02) for Mycobacterium aviumsubsp. paratuberculosisthat is present at six copies within the genome. In addition, the sensitivity of the assay with this element was compared to the sensitivity of detection of the IS900element in both conventional and real-time PCR assays. The specificity of the ISMap02element was evaluated by PCR of the DNA extracted from isolates of M. aviumsubsp. paratuberculosisand M. aviumsubsp. avium, as well as DNA from M. fortuitum, M. scofulaceum, M. phlei, M. smegmatis, and M. gordonae. Only M. aviumsubsp. paratuberculosisDNA was detectable after amplification with the ISMap02primers. The sensitivity of detection for the ISMap02element in either a conventional or a real-time PCR format was less than 100 fg DNA or 102CFU/ml in serial titration curves with pure bacteria. These results were comparable to those obtained for the IS900element. Experimental spiking of a negative fecal sample followed by M. aviumsubsp. paratuberculosisDNA extraction resulted in detection thresholds of 102CFU/g for the IS900element and 103CFU/g for the ISMap02element by using a real-time PCR format, but this sensitivity dropped 10-fold for both elements in a conventional PCR format. Analyses of fecal samples obtained from naturally infected animals demonstrated a sensitivity for the detection of M. aviumsubsp. paratuberculosisDNA by use of the ISMap02element similar to that achieved by use of the IS900element when it was used in a conventional PCR format. The real-time PCR format improved the levels of detection of both elements, but not to a significant degree. In conclusion, the ISMap02element provides a very sensitive and specific alternative as a diagnostic reagent for use in PCR assays for the detection of paratuberculosis.

ABSTRACTImmunological diagnosis of Mycobacterium bovisinfection of cattle is often confounded by cross-reactive responses resulting from exposure to other mycobacterial species, especially Mycobacterium avium. Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are dominant gamma interferon (IFN-?)-inducing antigens of tuberculous mycobacteria, and they are absent from many environmental nontuberculous mycobacteria. Because M. aviumexposure is the primary confounding factor in the diagnosis of M. bovis-infected animals, in vitro responses to a recombinant ESAT-6:CFP-10 (rESAT-6:CFP-10) fusion protein by blood leukocytes from cattle naturally exposed to M. aviumor experimentally challenged with Mycobacterium aviumsubsp. aviumor Mycobacterium aviumsubsp. paratuberculosiswere compared to responses by M. bovis-infected cattle. Responses to heterogeneous mycobacterial antigens (i.e., purified protein derivatives [PPDs] and whole-cell sonicates [WCSs]) were also evaluated. Tumor necrosis factor alpha (TNF-a), IFN-?, and nitric oxide responses by M. bovis-infected cattle to rESAT-6:CFP-10 exceeded (P< 0.05) the corresponding responses by cattle naturally sensitized to M. avium. Experimental infection with M. bovis, M. avium, or M. aviumsubsp. paratuberculosisinduced significant (P< 0.05) IFN-? and nitric oxide production to WCS and PPD antigens, regardless of the mycobacterial species used for the preparation of the antigen. Responses to homologous crude antigens generally exceeded responses to heterologous antigens. Nitric oxide and IFN-? responses to rESAT-6:CFP-10 by blood leukocytes from M. bovis-infected calves exceeded (P< 0.05) the corresponding responses of noninfected, M. avium-infected, and M. aviumsubsp. paratuberculosis-infected calves. Despite the reported potential for secretion of immunogenic ESAT-6 and CFP-10 proteins by M. aviumand M. aviumsubsp. paratuberculosis, it appears that use of the rESAT-6:CFP-10 fusion protein will be useful for the detection of tuberculous cattle in herds with pre-existing sensitization to M. aviumand/or M. aviumsubsp. paratuberculosis.

Gamma interferon (IFN-gamma) plays a significant role in the control of mycobacterial infections, including Mycobacterium avium subsp. paratuberculosis. However, the contribution of other immunoregulatory cytokines, such as interleukin-10 (IL-10) and transforming growth factor beta (TGF-beta), in Johne's disease has not been investigated as yet. In this study, we examined the effects of in vivo and in vitro infection with M. avium subsp. paratuberculosis on the production of IFN-gamma, IL-10, and TGF-beta by peripheral blood mononuclear cells (PBMC). We also examined the effects of exogenous IFN-gamma, IL-10, and TGF-beta on M. avium subsp. paratuberculosis survival in the cell cultures. PBMC obtained from naturally infected cows, regardless of their disease status, specifically upregulated IL-10 and TGF-beta in culture supernatants in response to stimulation with live M. avium subsp. paratuberculosis. Nonstimulated PBMC recovered from subclinically infected animals secreted the lowest levels of TGF-beta, but after stimulation with live M. avium subsp. paratuberculosis, TGF-beta levels in the culture supernatants increased to levels similar to that produced by PBMC from healthy animals. The numbers of viable M. avium subsp. paratuberculosis recovered from cultures from naturally infected animals were higher than those from healthy cows after in vitro infection with M. avium subsp. paratuberculosis. The addition of exogenous IL-10 and TGF-beta to PBMC isolated from healthy cows inhibited the bactericidal activity of these cells as evidenced by the increased number of viable M. avium subsp. paratuberculosis recovered from these cultures compared to cell cultures containing medium alone. These data suggest important immune regulatory roles for IL-10 and TGF-beta during infection with M. avium subsp. paratuberculosis that may be directly related to their effects on macrophage activation and killing of M. avium subsp. paratuberculosis.

ABSTRACTGamma interferon (IFN-γ) plays a significant role in the control of mycobacterial infections, including Mycobacterium aviumsubsp. paratuberculosis. However, the contribution of other immunoregulatory cytokines, such as interleukin-10 (IL-10) and transforming growth factor β (TGF-β), in Johne's disease has not been investigated as yet. In this study, we examined the effects of in vivo and in vitro infection with M. aviumsubsp. paratuberculosison the production of IFN-γ, IL-10, and TGF-β by peripheral blood mononuclear cells (PBMC). We also examined the effects of exogenous IFN-γ, IL-10, and TGF-β on M. aviumsubsp. paratuberculosissurvival in the cell cultures. PBMC obtained from naturally infected cows, regardless of their disease status, specifically upregulated IL-10 and TGF-β in culture supernatants in response to stimulation with live M. aviumsubsp. paratuberculosis. Nonstimulated PBMC recovered from subclinically infected animals secreted the lowest levels of TGF-β, but after stimulation with live M. aviumsubsp. paratuberculosis, TGF-β levels in the culture supernatants increased to levels similar to that produced by PBMC from healthy animals. The numbers of viable M. aviumsubsp. paratuberculosisrecovered from cultures from naturally infected animals were higher than those from healthy cows after in vitro infection with M. aviumsubsp. paratuberculosis. The addition of exogenous IL-10 and TGF-β to PBMC isolated from healthy cows inhibited the bactericidal activity of these cells as evidenced by the increased number of viable M. aviumsubsp. paratuberculosisrecovered from these cultures compared to cell cultures containing medium alone. These data suggest important immune regulatory roles for IL-10 and TGF-β during infection with M. aviumsubsp. paratuberculosisthat may be directly related to their effects on macrophage activation and killing of M. aviumsubsp. paratuberculosis.

Diagnosis of paratuberculosis (Johne's disease) is stymied by the lack of 1 diagnostic tool that can be used to detect both subclinically and clinically infected animals. At present, fecal culture remains the single diagnostic test that can detect infection in both disease states provided the animals actively shed Mycobacterium paratuberculosisin their feces. Yet, fecal culture has a disadvantage associated with the protracted incubation period of 8–16 weeks before results are available. Detection of nucleic acids specific to M. paratuberculosisin fecal samples is a technique that can circumvent the culture method. This study describes a rapid, simple, and effective method to extract DNA from fecal samples and modification of a polymerase chain reaction assay for optimal sensitivity of detection. An evaluation of 1,000 well-characterized fecal samples was performed by the Colorado Department of Agriculture (Denver, CO) and the National Animal Disease Center (Ames, IA) to determine the sensitivity, specificity, and reproducibility of the new method. Results from this study show that the sensitivity of detection was highly dependent on the load of bacteria in the fecal sample with 81% detection of samples containing >70 colony-forming units (cfu)/g of feces and a 45% detection rate for samples containing less than 1 cfu/g. Similarly, reproducibility of the technique between the 2 laboratories (n= 250 samples) was much higher (75%) for the fecal samples containing high levels of M. paratuberculosisand reduced to 25% for samples with less than 1 cfu/g. An overall specificity of 83% was obtained for known negative samples. The method described here is rapid, simple, and inexpensive compared with other techniques. In addition, this method can detect animals that are shedding less than 1 cfu/g.

Johne's disease (paratuberculosis) of cattle is widespread and causes significant economic losses for producers due to decreased production and poor health of affected animals. The chronic nature of the disease and the lack of a reproducible model of infection hinder research efforts. In the present study, instillation of Mycobacterium avium subsp. paratuberculosis into the tonsillar crypts of neonatal calves resulted in peripheral colonization as detected by antemortem culture of feces and postmortem (320 days postchallenge) culture of intestinal tissues. Antigen-specific blastogenic, gamma interferon (IFN-gamma), and nitric oxide responses by blood mononuclear cells from infected calves exceeded prechallenge responses beginning 194 days postchallenge. Upon in vitro stimulation with paratuberculosis antigens, CD4(+) cells from infected calves proliferated, produced IFN-gamma, and increased expression of CD26 and CD45RO (indicative of an activated memory phenotype). Utilizing a lipoarabinomannan-based enzyme-linked immunosorbent assay, specific serum immunoglobulin was detected as early as 134 days postchallenge and generally increased after this time point. Two antigens of approximately 50 and approximately 60 kDa were particularly immunodominant early in infection, as shown by immunoblot with serum collected within 2 weeks postchallenge. Findings indicate that the intratonsillar inoculation route will prove useful as an experimental model for paratuberculosis infection. Additionally, this study confirms that mycobacteria-specific antibody is detectable early in the course of experimental Johne's disease, even preceding the development of specific cell-mediated responses.