d. It was previously shown that the ability to develop a well-defined granulomatous response correlated with the severity of mycobacterial infections. When human PBMCs and macrophages were treated with 105 heat-killed M. tuberculosis strain H37Rv they formed aggregates that remained very small and loose. Similarly, human PBMCs infected with avirulent mycobacteria such as M. smegmatis and M. avium formed loose aggregates. We observed that ovine PBMCs infected with Map isolates from sheep and cattle formed well defined aggregates after 10 days of incubation at 37 uC. Map-induced aggregates displayed morphological characteristics similar to natural granulomas, such as threedimensional aggregation of lymphocytes around macrophages In accordance with our results, granulomatous lesions consistent with Map infection have been recently found in tissue sections from lambs experimentally infected with bovine and ovine isolates of Map. The advantages of in vitro models of granuloma include reduced cost, increased control, and that they can provide insights into host-mycobacteria interactions at stages of granuloma formation too early to address with animal models. However, the granuloma constitutes a complex immune microenvironment highly affected by additional physiological signals which are exclusively produced in infected tissues. As consequence, certain aspects of in vivo granulomas may be different or absent in in vitro models, including intra-granulomatous necrosis, accumulation of fibrin and collagen, and presence and distribution of bacilli. Threedimensional in vitro models of granuloma may be very useful to: understand what factors or molecules play a role in granuloma formation and in its continued integrity, evaluate the granuloma-inducing activity of particular antigens or attenuated mutants, and provide a platform for testing vaccine and drug candidates. In order to protect paratuberculosis-free herds, control programs have 
been developed in some countries. The success of these control programs depends on the ability to make decisions regarding on-farm management practices and the movement of animals between regions. For instance, policies regarding mixed farming of cattle and sheep have been based on the apparent host specificity of Map. However, our results suggested that sheep might be order LY341495 susceptible to infection with Map isolates not only from sheep but from cattle, goats, deer, fallow deer and wild boar as well. Therefore, the implementation of measures to prevent the risk of Map transmission from these animal species to sheep in multispecies livestock operations or on farms where sheep share pastures with wildlife animal should be recommended. Because a lack of correlation between genotype and intracellular phenotype of Map isolates in ovine macrophages was observed, destocking polices that aim to eliminate Map should assume equivalence of strains and PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19660899 should not be based on genotype distinction. Conclusion Map isolates from cattle, sheep, goats, fallow deer, deer and wild boar showed successful survival phenotype within ovine macrophages regardless of genotype. This phenotype correlated with stimulation of anti-inflammatory, antiapoptotic and antidestructive responses within ovine macrophages. In addition, we showed that two Map isolates from cattle and sheep with distinct genotypes were able to induce the formation of in vitro granulomas with a well-defined edge and comparable to the granulomas observed in clinical specimens with