tion of the antiapoptotic BCL2-A1 gene was also observed in Map-infected bovine MDMs relative to uninfected cells. Although MDMs can provide insights into Map-host interactions at the very early stages of the infection, this cellular model is unable to mimic later stages of the infection, such as the first stages of granuloma formation. To address this issue, we have developed a three-dimensional in vitro model enabling the formation of Map-induced granulomas by incubating infected ovine PBMCs with an extracellular matrix. Since the ability to develop a well-defined granulomatous response was previously shown to correlate with the severity of mycobacterial infections, the second objective of our study was to assess whether in vitro granuloma formation was induced by two Map isolates from cattle PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19657842 and sheep with distinct genotypes. By comparing the interaction of distinct Map isolates with ovine macrophages at two different stages of the infection, we are seeking to identify differences in host specificity and pathogenicity between Map isolates and to provide scientific data to support effective control management strategies against ovine paratuberculosis. This information might be very useful in situations where veterinarians and producers have to assess risks and introduce effective management strategies to control paratuberculosis in multispecies livestock operations or on farms where livestock share pastures with wildlife animals potentially infected with Map. Materials and Methods Ethics Statement Experimental procedures were performed by clinical veterinarians in strict accordance with the recommendations in the Spanish Ethical Guide for the care of animals used for experimental and other scientific purposes. Blood collection procedure was approved by the Institutional Animal Care and Use Committee of NEIKER-Tecnalia and by the Department of Agriculture, Diputacion Foral de Bizkaia, Spain. Map Isolates, Bacterial Culture and Preparation of Bacterial Suspensions Nine Map isolates from cattle, sheep, goat, red deer, fallow deer, and wild boar species were selected from the collection of isolates at the Mycobacteria laboratory, NEIKERTecnalia, on the basis of varied hosts and genomic profiles as per Sevilla et al., 2007. These isolates of Map were previously recovered from fecal or tissue specimens of domestic or wildlife animal species and maintained as glycerol stocks at 280uC. Aliquots of these glycerol stocks were utilized to directly inoculate all subsequent cultures for use in infection of macrophages. Most of the specimens were collected in several geographic areas of Spain, but three 
isolates from India, Portugal and The United States were also included in the study. Map reference strain K10, a sequenced and laboratory-adapted strain recovered from a clinical case of paratuberculosis, was obtained from the American Type Culture Collection . The 10 isolates of Map selected for our study were grown in T25 tissue culture flasks at 3761uC for up to 3 months in 10 ml of Middlebrook 7H9 broth supplemented with 10% oleic acid-albumin-dextrosecatalase , 0.05% Tween-80 and 2 mg L-1 of mycobactin J. Bacterial cells were harvested by centrifugation at 3,000 x rpm for 20 min in a AEB-071 Beckman Coulter Allegra X-12 centrifuge. Bacterial pellets were resuspended in 2 ml of Hank’s balanced salt solution, and the resultant suspension was passed 20 times through a 27-gauge needle in order to declump cells. The turbidity of the bacterial suspension was