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

Paratuberculosis (Johne’s disease) has devastating outcomes on ruminant health and impacts on national and international trade. The current work assessed the diagnostic value of the VersaTREK automated liquid culture system in isolating Mycobacterium avium subspecies paratuberculosis (MAP) from faecal and intestinal tissue samples from ovine under South African conditions and compared it with the current method of choice, histopathological examination. Intestinal tissue and faecal samples from 111 sheep (including complete set from 104 slaughter sheep from flocks with a history of MAP infection as well as incomplete sample sets from 7 sheep) were analysed using the liquid culture method. One set of tissues was subjected to histopathological examination. Deoxyribonucleic acid (DNA) extracted from culture isolates was subjected to polymerase chain reaction (PCR) amplification using primers that target the IS900 regions of the MAP for species verification. Overall, the VersaTREK automated liquid culture in combination with IS900 PCR showed a comparable level of detection in tissues (12.6%) as histopathology (13.5%), but the detection rate for faecal samples was lower than for tissues (10.8%). A combination of histopathology and faecal culture increased the detection rate from 13.5% (n = 14/104) and 9.6% (n = 10/104), respectively, to 15.4% (n = 16/104). Contribution: Our findings highlight the diagnostic utility of the VersaTREK automated liquid culture system in detecting MAP in ovine samples collected both ante and postmortem. However, an inhibitory effect on the MAP isolation rate observed when the antibiotic cocktail was added to the culture medium warrants further investigation. The outcome of the study is beneficial in guiding the strategic planning of the nationwide control programme.

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 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.

An ELISA containing lipoarabinomannan (LAM) antigen was used to detect antibodies in milk and serum for diagnosis of Mycobacterium paratuberculosis infection in dairy cattle. In experiment 1, milk and serum samples were obtained from 25 cows, and subjected to LAM ELISA testing immediately, and after 1 year of storage at -70 C. Milk samples, with and without a commonly used chemical preservative, were tested. There was no significant difference in LAM ELISA results between fresh and frozen samples or between preserved and unpreserved milk samples. In experiment 2, milk samples were collected daily from 30 cows over a 14-day period. The day-to-day coefficient of variation was 0.19 for milk LAM ELISA and was 0.15 for serum LAM ELISA, with no statistically significant time effect detected. In experiment 3, single milk, serum, and fecal samples were obtained from 764 cows. The fecal samples were cultured for M paratuberculosis to identify infected cows, and the serum and milk samples were subjected to LAM ELISA testing. Results were compared, using the area under the receiver operating characteristic curves. The milk LAM ELISA had specificity (+/- 95% confidence limits) of 87 +/- 8.1% when the cutoff was set at 50% sensitivity, and specificity of 83 +/- 9.1% when sensitivity was set at 60%. The area under the receiver operating characteristic curve was 0.85 +/- 0.03 for the milk ELISA and 0.75 +/- 0.02 for the serum ELISA. In this population of cattle, the milk LAM ELISA had comparable accuracy to serum LAM ELISA, although the milk LAM ELISA was slightly less reproducible (higher coefficient of variation).

The purpose of this study was to document the effect of calfhood vaccination for Mycobacterium paratuberculosis on a serologic ELISA. Fifteen calves vaccinated with a killed paratuberculosis vaccine and 5 unvaccinated control calves were tested from the first through the fifteenth month of life. Age of vaccination ranged from 5 to 40 days. Blood samples were collected prior to vaccination and periodically thereafter. Serum antibody was analyzed by use of the ELISA. All calves were Elisa-negative prior to vaccination. Thirteen of 15 vaccinated calves became ELISA-positive between 2 and 6 months after vaccination. The unvaccinated cohort remained Elisa-negative. Widespread use of vaccine may interfere with diagnosis of paratuberculosis and with control programs that are based on serologic tests that measure humoral antibody.

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).

The performance of the serum complement fixation (CF) test was compared with that of a serum agar gel immunodiffusion (AGID) test on 74 subclinically infected and 154 uninfected cattle in 6 commercial midwestern dairy herds with Mycobacterium paratuberculosis infection and on 30 cattle in a herd that was free of infection. Infection status of cattle within herds was established by performance of a series of 3 or more fecal cultures and of ileocecal lymph node cultures of culled cattle. In cattle with subclinical infection detected by culturing, the sensitivity estimates of the CF and AGID tests were 10.8% (3.6% SE) and 18.9% (4.5% SE), respectively. In the cattle classified as disease free, the specificity estimates of the CF and AGID tests were 97.4% (1.3% SE) and 99.4% (0.6% SE), respectively. Neither set of estimates was significantly different. Negative test results obtained with the use of either test in apparently normal cattle from suspect herds should be interpreted with caution because both tests suffer from low sensitivities in subclinically infected animals. However, the AGID test may be more useful in regulatory situations in which the CF test is currently used because the AGID test is easier to perform and to interpret.

Fecal samples from 131 cattle clinically suspect for paratuberculosis were cultured bacteriologically, using the traditional sedimentation processing method and a processing method that included a centrifugation step. Of 16 samples that were contaminated, 6 were culture-positive on at least 1 medium and by 1 processing method. Ten of 131 (7.6%) fecal samples processed by both methods were lost because of contamination. The number of culture-positive samples (using both processing methods) were 65 of 121 (53.7%) on media without miconazole and 60 of 121 (49.6%) on media with miconazole. Seven of the 121 (5.8%) samples were culture-positive, using centrifugation, after 16 weeks' incubation at 37 C. Thirteen of 60 (21.7%) isolates were obtained only with centrifugation, and 10 of these had low colony counts, suggesting that a centrifugation step may have concentrated microorganisms that would have gone undetected without centrifugation. Six of 60 (10%) isolates positive for M paratuberculosis on the sedimentation method were negative on the centrifugation method. Contamination rates were significantly (P less than 0.001) increased when centrifugation was used. The miconazole significantly (P less 0.001) decreased contamination rates when centrifugation was used.

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.