Small numbers of leucocyte clusters (< 0.22 clusters per field) were observed in liver samples from mice inoculated with PBS, though no acid fast bacilli (AFB) were detected in any of these samples. Large differences in the mean ranked density of leucocyte clusters between strains were identified (p<0.001) with the wild type strain JD87/107 having the highest mean ranked densities of clusters (Figure  2b). Strain 2eUK2001 showed evidence of higher mean rank densities than the 316FUK2001 and IIUK2001 strains (p = 0.03). The ranked density of leucocyte clusters with AFB showed highly statistically significant differences between the means of MAP strains Cytoskeletal Signaling (p<0.001), with the JD87/107 strain consistently showing

higher mean densities, with this effect being more pronounced from 8 weeks post infection (Figure  2c). The vaccine strains all tended to exhibit increasingly lower mean ranked densities over the lifetime of the experiment (p=0.002), with consistent patterns of differences between strains (p=0.008): strain IIUK2001 showed the largest mean rank densities, strain 316FUK2001 the lowest, with 2eUK2001 intermediate. The histopathology results show that all strains elicited

a similar inflammation at 4 weeks. Only thereafter some differences between the inflammatory responses to the strains became apparent. In addition, the analysis of mean bacterial counts and AFB positive clusters showed the reduced ability of the vaccine strains to survive and persist within mice. Overall, these Dapagliflozin results provide proof of attenuation of selleck compound the vaccine strains with respect

to a wild type MAP strain. Discussion In this study, we examined genomic and phenotypic characteristics of a panel of MAP vaccine strains obtained from several laboratories around the world including both low and high passage examples of the 316 F lineage. Using a mouse model, we assessed the virulence ofrepresentative clades of three vaccine strains (2e, II, 316 F) with respect to a virulent MAP clinical isolate. The vaccine strains were clearly attenuated with regard to their ability to survive and persist in the mice as evidenced from the reduced numbers of MAP recovered and reduced numbers of leucocyte clusters containing AFB in the livers. This supports previous studies showing decreased persistence of the same 316 F and 2e strains in calves after 8 months [29] and illustrates the utility of the C57BL/6 mouse model for virulence studies. Using a pan-genomic MAP/MAH microarray we demonstrated that the genomes of all but one of the 316 F strains in the test panel contain the same full genome complement as the reference virulent RAD001 clinical trial bovine MAP type II strain MAPK10. One 316 F strain obtained from Norway (316FNOR1960) contained a single deleted region (vGI-19) spanning 21 ORF’s (including 10 MAP specific genes). Two strains not of the 316 F lineage (2eUK2000 and IIUK2000) contained a different deleted region (vGI-20), identical in both strains, spanning 34 ORF’s (including 10 MAP specific genes).

In studies where no genotyping method was used, it was assumed that each isolate represented a strain. BGB324 mouse results and discussion Comparative performance

of the five molecular methods The percentage of correctly identified strains obtained using the five identification methods, and the number of misidentified non-targeted species greatly depended upon the method used (Tables 1 and 2). The percentage of misidentified strains ranged from 16.8% to 67.4% (Table 2). The m-PCR method of Kabeya et al. [15] had the worst performance, and produced unreliable results for all three of its targeted species (Tables 1 CHIR98014 concentration and 2). Although all strains of A. cryaerophilus and A. skirrowii were correctly identified, a further eight and six non-targeted species, respectively, were mistakenly identified as one of these two species (Table 1). Furthermore, only 4.8% of the A. butzleri strains were correctly identified, with six non-targeted species being confused with this species (Tables 1 and 2). Globally, the Kabeya et Luminespib chemical structure al. m-PCR method correctly identified just 32.6% (31/95) of the studied strains. Although this method

was also designed to differentiate subgroups 1A and 1B of A. cryaerophilus, not all strains of these subgroups were correctly identified (Table 2). This correlates with the in silico observations of Douidah et al. [9] who reported that the primer used [15] were not specific enough to provide correct identification of A. cryaerophilus at the subgroup level. Further to this, Debruyne et al.[21] have suggested, that based on results of AFLP and hsp60 analyses, the subgroup nomenclatures 1A and 1B should be abandoned. The second least reliable method analysed was the m-PCR technique described by Houf et al.[14]. This correctly

identified 55.8% (53/95) of the strains (Table 2), including all those belonging RAS p21 protein activator 1 to its targeted species (A. butzleri, A. cryaerophilus, and A. skirrowii; Table 1). This method was 100% reliable for the identification of A. butzleri, and there was no confusion with other species. However, nine of the fourteen non-targeted species generated the typical amplicon of A. cryaerophilus; two that of A. skirrowii; and two simultaneously generated both amplicons (Tables 1 and 2). Only A. cibarius produced no amplification when using this method (Table 2). These results agree with previous studies that showed the existence of misidentifications when using this method [1, 5–7]. A similar number of correctly identified strains (83.2%) were obtained when using the other three evaluated methods (Pentimalli et al.[16]; the combined method of Douidah et al. [9] and De Smet et al.[17]; and Figueras et al.[18]). However, the number of misidentified non-targeted species differed depending upon the method used (Tables 1 and 2). Most misidentification occurred when using the method of Pentimalli et al.[16]. In this case, four non-targeted species were confused with A. butzleri, one with A.