Mycobacterium Avium subsp. paratuberculosis isolates induce in vitro granuloma formation and show successful survival phenotype, common anti-inflammatory and antiapoptotic responses within ovine macrophages regardless of genotype or host of origin.

The analysis of the early macrophage responses, including bacterial growth within macrophages, represents a powerful tool to characterize the virulence of clinical isolates of Mycobcaterium avium susbp. paratuberculosis (Map). The present study represents the first assessment of the intracellular behaviour in ovine monocyte-derived macrophages (MDMs) of Map isolates representing distinct genotypes (C, S and B), and isolated from cattle, sheep, goat, fallow deer, deer, and wild boar. Intracellular growth and survival of the selected isolates in ovine MDMs was assessed by quantification of CFUs inside of the host cells at 2 h p.i. (day 0) and 7 d p. i. using an automatic liquid culture system (Bactec MGIT 960). Variations in bacterial counts over 7 days from the baseline were small, in a range between 1.63 to 1.05-fold. After 7 d of infection, variations in the estimated log10 CFUs between all the tested isolates were not statistically significant. In addition, ovine MDMs exhibited enhanced anti-inflammatory, antiapoptotic and antidestructive responses when infected with two ovine isolates of distinct genotype (C and S) or with two C-type isolates from distinct hosts (cattle and sheep); which correlated with the successful survival of these isolates within ovine MDMs. A second objective was to study, based on an in vitro granuloma model, latter stages of the infection by investigating the capacity of two Map isolates from cattle and sheep to trigger formation of microgranulomas. Upon 10 d p.i., both Map isolates were able to induce the formation of granulomas comparable to the granulomas observed in clinical specimens with respect to the cellular components involved. In summary, our results demonstrated that Map isolates from cattle, sheep, goats, deer, fallow-deer and wild boar were able not only to initiate but also to establish a successful infection in ovine macrophages regardless of genotype

Phase contrast images showing the presence of day-10 granuloma-like aggregates after the infection of ovine PBMCs with bovine and ovine <i>Map</i> isolates.

<p>Ovine PBMCs (5 x 10⁵) seeded on an extracellular matrix were infected with the bovine K-10 reference strain (A, B and C) or with an ovine isolate of <i>Map</i> (2349/06-1) (D, E and F) at MOI (bacteria:cells) of 1:8. Magnification in A and D  =  4X, in B and E  =  20X and in C and F  =  40X. Bars  =  300 µm.</p

Morphological characterization of the cell populations recruited within <i>in vitro-</i> ovine granulomas.

<p>Primary ovine PBMCs (5x10⁵) were seeded on an extracellular matrix and subsequently infected with the K10 reference strain (A, B and C) or with an ovine isolate of <i>Map</i> (2349/06-1) (D, E and F) at MOI (Bacteria:cells) 1:8. At 10 days p.i., the granuloma-like aggregates were harvested, processed for histopathology and stained with HE (A, B, D and E) and ZN stains (C and F). Original magnification in A and D  =  200X and in B, C, E and F  =  1000X. As shown in image B, macrophages (asterisk) and lymphocytes (arrows) were present in the granulomas. In images C and F, acid-fast bacilli (arrows) were observed within macrophages by ZN staining.</p

Genes and primer sequences used in the qRT-PCR assays.

<p>Genes and primer sequences used in the qRT-PCR assays.</p

Expression of cytokines and proteins involved in inhibition of apoptosis or tissue destruction in ovine MDMs infected with bovine and ovine isolates of Map.

<p>Ovine MDMs were infected with (A) C-type Map isolates from cattle (K10 strain) and sheep (p38I isolate), or with (B) two ovine isolates with C- (p38I isolate) or S-genotype (2349/06-1 isolate). At 4, 14 and 24 h p.i. gene expression was assessed by qRT-PCR. Isolates are identified in the figure by their corresponding host of origin and IS1311 PCR-REA type (C or S). Bars represent the average results of two independent infection experiments (± SD). No statistically significant differences in the expression of the indicated genes between the tested isolates were observed.</p

IS<i>1311</i> PCR-REA types, PFGE profiles and estimations of span, K, and plateau for the quantification of each <i>M. avium</i> subsp. <i>paratuberculosis</i> isolate in the Bactec MGIT 960 system.

<p>Growth of all the isolates in the Bactec MGIT 960 system fitted to a one-phase exponential-decay model according to the following equation [log₁₀ inoculum size =  span X <i>e (-K</i> X TTD) + plateau]. Span is the difference between TTD at time zero and the plateau, <i>K</i> is the degree of decay for the log₁₀ CFU, and plateau is the value for log₁₀ CFU curve flattening.</p

Entry and intracellular growth of Map isolates and the reference strain K10 in ovine MDMs.

a<p>Uptake was calculated as the percentage of the inoculated bacteria that was recovered from each cell lysate at day 0.</p>b<p>Values shown are means of three repeated experiments ± standard deviations (SD).</p>c<p>Day 0 = 2 h post infection.</p>d<p>Growth changes (n-fold) were calculated by dividing the number of log10 CFU at day 7 by that at day 0 for each Map isolate.</p>e<p>Indicates a significant change between day 0 and day 7 (P<0.05).</p

Number of microgranulomes generated <i>in vitro</i> 5 and 10 days after infection of ovine PBMCs with (A) the K10 reference strain and with (B) the ovine 2349/06-1 isolate.

<p>Aggregate numbers were estimated under a light microscope. The mean aggregate number estimated by triplicate is shown for each MOI (1:8, 1:16 and 1:33) ± SD.</p