Bovine tuberculosis is a major problem in many countries; hence, new and better diagnostic tools are urgently needed. In this work, we have tested ESAT6, CFP10, PE13, PE5, MPB70, TB10.4, and TB27.4 for their potentials as diagnostic markers in field animals from Northern Ireland, Mexico, and Argentina, regions with low, medium, and high prevalences of bovine tuberculosis, respectively. At all three sites, ESAT6 and CFP10 were superior diagnostic antigens, while their combination performed even better at the two sites where the combination was tested, providing the best coverage for the detection of diseased populations. The high sensitivity in the skin test reactor groups, combined with the high specificity in the tuberculosis-free groups, indicated that a diagnosis could correctly be made for 85% of the infected animals, based on their responses to these two antigens. Furthermore, TB10.4, PE13, and PE5 have the potential to supplement ESAT6 and CFP10 in a future five-component diagnostic cocktail.
This assessment aimed to elaborate a statistical nationwide model for analyzing the space-time dynamics of bovine tuberculosis in search of potential risk factors that could be used to better target surveillance measures. A database comprising Mycobacterium bovis molecular profiles from all isolates obtained from Belgian outbreaks during the 1995-to-2006 period (n ؍ 415) allowed the identification of a predominant spoligotype (SB0162). Various databases compiling 49 parameters to be tested were queried using a multiple stepwise logistic regression to assess bovine tuberculosis risk factors. Two isolate datasets were analyzed: the first included all Mycobacterium bovis isolates, while the second included only data related to the SB0162 type strain. When all Mycobacterium bovis isolates were included in the model, several risk factors were identified: history of bovine tuberculosis in the herd (P < 0.001), proximity of an outbreak (P < 0.001), cattle density (P < 0.001), and annual amplitude of mean middle-infrared temperature (P < 0.001). The approach restricted to the predominant SB0162 type strain additionally highlighted the proportion of movements from an infected area during the current year as a main risk factor (P ؍ 0.009). This study identified several risk factors for bovine tuberculosis in cattle, highlighted the usefulness of molecular typing in the study of bovine tuberculosis epidemiology, and suggests a difference of behavior for the predominant type strain. It also emphasizes the role of animals' movements in the transmission of the disease and supports the importance of controlling trade movements.
On the basis of DNA-DNA hybridization and numerical taxonomy analysis, the mycobacterial species Mycobacterium avium is subdivided into three subspecies, M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum, which share extensive sequence identity (8). Nevertheless, these subspecies can be differentiated from each other on the basis of host range, mycobactin dependence, and the presence of specific insertion elements (17). M. avium subsp. paratuberculosis causes Johne's disease, a severe gastroenteritis in ruminants, with a significant impact on the agricultural economy, particularly the dairy industry (17). In the Belgian cattle population, paratuberculosis prevalence was determined by a serological survey, conducted from December 1997 to March 1998. This approach resulted in an estimated true herd prevalence of M. avium subsp. paratuberculosis infection of 6% (6). Dairy cattle usually start fecal shedding at 2 years of age and develop clinical symptoms around 4 years of age. Infection with M. avium subsp. paratuberculosis is commonly acquired early in life via the fecal-oral route through the ingestion of contaminated colostrum, milk, water, or feed (46) and possibly through intrauterine transmission (42). M. avium subsp. paratuberculosis is extremely robust, and bacteria were reported to survive up to 250 days in water and feces and on pastures (27).Cell-mediated immune responses seem to control the initial infection for a sustained period of time, and clinical symptoms only appear in cows after a number of years, often after the first or second calving, possibly because of enhanced intracellular multiplication of M. avium subsp. paratuberculosis organisms caused by alterations in the hormonal milieu (15). Decreased cell-mediated responses are likely related to a loss of antigen-specific CD4 ϩ T cells, which is most prominent in the ileum lesions from symptomatic animals (24). Also, Khalifeh et al. demonstrated that transforming growth factor  and interleukin-10 (IL-10) mRNA levels are higher in cows that have progressed to the clinical stage of the disease, compared to subclinically infected or healthy cows (23). It is not clear for the moment, whether this reflects a shift from a Th1-to a Th2-biased immune response or rather the development of a regulatory T-cell circuit (10). Vaccines consisting of whole killed or attenuated live M. avium subsp. paratuberculosis bacilli can provide partial protection by delaying fecal shedding and reducing the number of clinically affected animals, but they do not protect against infection. In the context of bovine tuberculosis (M. bovis) control and eradication programs, it is worth mentioning that animals immunized with these paratuberculosis vaccines develop positive reactions in the tuberculin skin test (the reference bovine tuberculosis detection method), and therefore paratuberculosis vaccination is subject to approval by
Differential delayed-type hypersensitivity skin testing with tuberculin purified protein derivatives from Mycobacterium bovis and M. avium is the standard for diagnosing bovine tuberculosis. However, improved tests based on defined, specific antigens are urgently needed. In the present study, a combination of bioinformatics, molecular biology, and bovine models of infection were used to screen mycobacterial proteins for their potential as diagnostic reagents which could be used in a whole-blood assay for diagnosis of tuberculosis. Initial screening of 28 proteins selected in silico and expressed as recombinants in Escherichia coli indicated that CFP-10, ESAT-6, TB27.4, TB16.2, TB15.8, and TB10.4 induced strong gamma interferon responses in experimentally infected cattle. A more thorough investigation over time in two groups of animals infected with a high (10 6 CFU) and a low (10 4 CFU) dose of M. bovis revealed that, for both groups, the strength of the in vitro response to individual antigens varied greatly over time. However, combining the results for ESAT-6, CFP-10, and TB27.4, possibly supplemented with TB10.4, gave sensitivities at different infection stages close to those obtained with M. bovis purified protein derivative. Importantly, while responsiveness to ESAT-6 and CFP-10 correlated strongly for individual samples, the same was not the case for ESAT-6 and TB27.4 responsiveness. The results suggest that combinations of specific antigens such as these have great potential in development of optimized diagnostic systems for bovine tuberculosis.
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