Introduction: Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis, respectively the causative agents of bovine tuberculosis (bTB) and bovine paratuberculosis (PTB), share a high number of antigenic proteins. This characteristics makes the differential diagnosis of the diseases difficult. The interferon gamma (IFN-γ), C-X-C motif chemokine ligand 10 (CXCL10), matrix metallopeptidase 9 (MMP9), interleukin 22 (IL-22) and thrombospondin 1 (THBS1) bovine genes have already been shown to be accurate transcriptional biomarkers of bTB. In order to improve the diagnosis of bTB and PTB, in the present study we evaluated the risk of false positivity of these bTB biomarkers in cattle with PTB. Material and methods: The transcription of these genes was studied in 13 PTB-infected cattle, using Mycobacterium avium subsp. paratuberculosis (MAP)-stimulated peripheral blood mononuclear cells (PBMC). Results: Overall, the levels of IFN-γ, CXCL10, MMP9 and IL-22 transcripts in MAP-stimulated PBMC failed to differentiate animals with PTB from healthy animals. However, as bTB-afflicted cattle do, the MAP-infected group also displayed a lower level of THBS1 transcription than the non-infected animals. Conclusion: The results of this study add new specificity attributes to the levels of transcription of IFN-γ, CXCL10, MMP9 and IL-22 as biomarkers for bTB. | Instituto de Biotecnología | Fil: Klepp, Laura Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina | Fil: Klepp, Laura Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina | Fil: Colombatti Olivieri, Maria Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina | Fil: Colombatti Olivieri, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina | Fil: Moyano, Roberto Damian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina | Fil: Moyano, Roberto Damian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina | Fil: Romano, Maria Isabel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina | Fil: Romano, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina | Fil: Malovrh, Tadej. University of Ljubljana. Veterinary Faculty. Institute for Microbiology and Parasitology; Eslovenia | Fil: Ocepek, Matjaž. University of Ljubljana. Veterinary Faculty. Institute for Microbiology and Parasitology; Eslovenia | Fil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina | Fil: Blanco, Federico Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina | Fil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina | Fil: Bigi, Fabiana. Consejo Nacional de investigaciones Científicas y Tecnológicas; Argentina

Two Korean black goat (approx. 2 and 3 years old) showing diarrhea and chronic weight loss were submitted to Animal and Plant Quarantine Agency. At necropsy, there were thickening of small intestine and enlargement of mesenteric lymph nodes. Microscopically, they had granulomatous enteritis in the small and large intestine and granulomatous lymphadenitis. By polymerase chain reaction (PCR) and acid fast stain, strong positive reaction and acid-fast rod bacteria were detected. According to the result of histopathology and PCR, we confirmed

Objective—To estimate the risk of subclinical Mycobacterium avium subsp paratuberculosis (MAP) infection in cows that ingested MAP DNA–positive raw colostrum as calves, compared with risk in cows that ingested MAP DNA–negative raw colostrum as calves. Animals—205 calves born in 12 commercial dairy herds. Procedures—Each calf was separated from its dam within 30 to 60 minutes after birth and fed raw colostrum. For each calf, samples of the colostrum fed were collected and tested for the presence of MAP DNA by use of a nested PCR assay for the target gene ISMAP02. Calves fed colostrum positive or negative for MAP DNA were classified into exposed (n = 69) and unexposed (136) groups, respectively. Each calf was tested for MAP infection at 30, 42, and 54 months of age by use of a serum ELISA and bacterial culture of feces. Weibull hazard regression models were used to evaluate the association between exposure to MAP DNA–positive colostrum and time to testing positive for MAP infection. Results—Hazard of MAP infection was not different between groups (exposed vs unexposed) when serum ELISA, bacterial culture of feces, or both diagnostic tests (parallel interpretation) were positive. Conclusions and Clinical Relevance—Heifer calves fed MAP DNA–positive colostrum were at no greater risk of MAP infection, compared with heifer calves fed MAP DNA–negative colostrum. This result contradicts findings from other studies and should be interpreted with caution.

Objective-To investigate the epidemiologic and financial impacts of targeted sampling of subpopulations of cows, compared with random sampling of all cows, for classification of dairy herd infection status for paratuberculosis. Animals-All cows from 4 infected herds with a low-to-moderate prevalence of paratuberculosis and from 1 noninfected herd in California. Procedure-The infection status of each cow was classified on the basis of results of an ELISA or combined ELISA and fecal culture results. Thirteen sampling schemes designed to randomly sample cows on the basis of lactation number, stage of lactation, and milk production were evaluated. Sampling without replacement was used to obtain a probability of herd detection of paratuberculosis for each evaluated sampling method and for simulated sample sizes between 30 and 150 cows. Marginal cost-effectiveness analysis was used to determine the cost increase relative to the increase in detection probability. Results-Sampling cows in the third or higher lactation and greater than or equal to 200 days into lactation yielded the highest detection probability in most instances, resulting in a detection probability that was 1.4 to 2.5 times that obtained by sampling 30 cows in the second or higher lactation. Costs of testing via the alternative method with a 95% detection probability were approximately $300 lower in a high-prevalence herd (31 %) and $800 lower in a low-prevalence herd (9%), compared with use of the reference method. Conclusions and Clinical Relevance-Detection of herds with paratuberculosis could be improved, and costs of testing substantially reduced by sampling targeted groups of cows.

Objective—To evaluate the effect of delayed exposure of dairy cattle to Mycobacterium avium subsp paratuberculosis (MAP) on the incidence of those cows testing positive for MAP and developing clinical Johne's disease (CJD). Animals—79 cows not exposed to MAP as calves (unexposed cohort) and 260 cows exposed to MAP as calves (exposed cohort). Procedures—Cows in the unexposed cohort were born into 5 MAP-uninfected herds and introduced at various ages into 5 MAP-infected herds where the exposed cohort cows were born and raised. Beginning when each cow was 24 months old, fecal and serum samples were collected annually from 2003 through 2006. Feces were cultured for MAP, and an ELISA was used to analyze serum samples for antibodies against MAP. Date and reason for culling were obtained from herd records. Incidence of positive culture and ELISA results and CJD was compared between unexposed and exposed cohort cows with Cox regression. Results—Compared with exposed cohort cows, the hazard ratios for unexposed cohort cows having positive culture results, having positive ELISA results, and developing CJD were 0.12, 0.03, and 0.001, respectively, and those ratios increased by 2%, 6%, and 17%, respectively, for each month spent in an MAP-infected herd. Conclusions and Clinical Relevance—Delayed exposure of cows to MAP resulted in lower incidences of positive culture and ELISA results and CJD in those cows, compared with incidences of cows exposed to MAP since birth. The hazard of testing positive for MAP or developing CJD increased with time, regardless of cohort.

Objective: To develop a better system for classification of herd infection status for paratuberculosis (Johne's disease [JD]) in US cattle herds on the basis of the risk of potential transmission of Mycobacterium avium subsp paratubeculosis. Sample: Simulated data for herd size and within-herd prevalence; sensitivity and specificity for test methods obtained from consensus-based estimates. Procedures: Interrelationships among variables influencing interpretation and classification of herd infection status for JD were evaluated by use of simulated data for various herd sizes, true within-herd prevalences, and sampling and testing methods. The probability of finding ≥ 1 infected animal in herds was estimated for various testing methods and sample sizes by use of hypergeometric random sampling. Results: 2 main components were required for the new herd JD classification system: the probability of detection of infection determined on the basis of test results from a sample of animals and the maximum detected number of animals with positive test results. Tables were constructed of the estimated probability of detection of infection, and the maximum number of cattle with positive test results or fecal pools with positive culture results with 95% confidence for classification of herd JD infection status were plotted. Herd risk for JD was categorized on the basis of 95% confidence that the true within-herd prevalence was ≤ 15%, ≤ 10%, ≤ 5%, or ≤ 2%. Conclusions and Clinical Relevance: Analysis of the findings indicated that a scientifically rigorous and transparent herd classification system for JD in cattle is feasible.

Objective-To compare sensitivity of several methods of bacteriologic culture of pooled bovine fecal samples for detection of Mycobacterium paratuberculosis and evaluate homogeneity in number of M paratuberculosisin pooled fecal samples. Sample Population-Feces from 10 dairy cows that shed M paratuberculosis at various concentrations and 1 dairy cow known to be free of infection with M paratuberculosis. Procedure-5 fecal pooling methods, 2 culture methods, and 2 pool sizes were evaluated. Each pooled sample contained 1 infected sample and 4 or 9 uninfected samples. Results-Sensitivity of detection of M paratuberculosis was greater with smaller pool size (5 vs 10 samples/ pool). Detection sensitivity was also associated with concentration of bacteria in the infected sample. Results indicated that, compared with concurrent bacterial culture of individual infected samples, 37 to 44% of pooled samples with low bacterial concentrations yielded positive culture results and 94% of pooled samples with high bacterial concentrations yielded positive results. Conclusions and Clinical Relevance-Bacteriologic culture of pooled fecal samples may provide a valid and cost-effective method of detecting M paratuberculosis infection in cattle herds.

A commercial rapid-absorbed ELISA developed to detect antibodies to Mycobacterium paratuberculosis in bovine serum was modified for use with goat serum. Diagnostic sensitivity was evaluated, using a group of 163 goats from a herd with endemic paratuberculosis. Blood and fecal samples were obtained simultaneously, and prevalence of shedding of M paratuberculosis in the feces was estimated by detection of DNA of the mycobacterial insertion sequence, IS900, using a commercial test kit. Diagnostic specificity was evaluated, using blood samples from a total of 123 goats in 10 herds that were considered clinically free of paratuberculosis. The IS900 DNA was detected in 35 of the 163 goats (21%) from the infected herd. Serum antibody to M paratuberculosis was detected in 19 of the 35 IS900 DNA-positive goats, for apparent sensitivity of 54%. Serum antibody was detected in 18 of the 128 IS900 DNA-negative goats from the infected herd. Negative results for serum antibody to M paratuberculosis were obtained for all 123 goats from the herds that were considered clinically free of paratuberculosis.

Concanavalin A (conA) blast proliferation as a quantitative measure of lymphoblast proliferative capacity by blood mononuclear cell supernatants was measured in cattle naturally infected with Mycobacterium paratuberculosis and in healthy control cattle. Blast cell proliferation was significantly reduced in infected animals, compared with control cattle when blood mononuclear cells were stimulated with conA. Proliferation was significantly greater than media control when M bovis purified protein derivative and johnin were used to stimulate cells from the infected group. After sensitizing control and affected cattle with M paratuberculosis bacterin (live M bovis and keyhole limpet hemocyanin in Freund's incomplete adjuvant), infected animals had no difference in blast cell proliferative capacity with the mycobacterial antigens and cona stimulation, whereas healthy animals had significantly increased blast proliferation in response to all the sensitizing antigens. The blast cell proliferative capacity in infected animals with keyhole limpet hemocyanin stimulation was increased significantly after sensitization; however, it remained significantly less than that in the sensitized control group. These data indicate that cattle naturally infected with M paratuberculosis probably produce suboptimal interleukin-2 (IL-2) activity in response to a potent IL-2 inducer (conA) and fail to optimize IL-2 activity when sensitized with a potent immunogen (keyhole limpet hemocyanin).

To establish whether Mycobacterium paratuberculosis could be cultured from Dulbecco phosphate-buffered saline solution (DPBSS) and to test 3 sampling methods, DPBSS supplemented with 2% fetal bovine serum was inoculated with M paratuberculosis at concentrations of 10(4), 10(3), 10(2), 10(1), and 10(0) colony-forming units/ml. The inoculated media was sampled after mixing, after centrifugation, and after centrifugation and decontamination with 0.75% hexadecylpyridinium chloride. The samples were inoculated onto 3 slants of Herrolds egg yolk medium supplemented with sodium pyruvate and mycobactin J and 1 slant without mycobactin J. Mycobacterium paratuberculosis was isolated following all 3 sampling methods for all concentrations. Treatment with hexadecylpyridinium chloride decreased the number of colonies isolated. To test the efficacy of a 10-step wash procedure for removing M paratuberculosis from bovine ova, washed zona pellucida intact bovine ova were incubated in DPBSS supplemented with 2% fetal bovine serum containing concentrations of 10(4), 10(3), 10(2), 10(1), and 10(0) colony-forming units of M paratuberculosis/ml for 12 hours at 22 C. Ten zona pellucida intact ova were removed from each concentration and washed by passing through 10 changes of DPBSS supplemented with 15% fetal bovine serum. The media from each wash step was inoculated onto slants of Herrolds egg yolk medium. The ova were included with the tenth wash step. Mycobacterium paratuberculosis was isolated from 1 of 10 tenth-wash steps at the 10(4) concentration and 5 of 10 tenth-wash steps at 10(3).