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

Abstract

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.

Granulomatous enteritis in rainbow trout (Oncorhynchus mykiss) associated with soya bean meal regardless of water dissolved oxygen level

“…The aetiology of the disease is Mycobacterium avium spp. paratuberculosis and the bacteria can be detected within granulomas after ZN staining as previously shown (Abendaño et al, 2014).…”

Modelling Bovine Granuloma Formation In Vitro upon Infection with Mycobacterium Avium Subspecies Paratuberculosis

“…Basic Map-infection studies of bovine MDMs have helped elucidate the variety of natural immunological responses to the bacterium [38]. Pro-inflammatory cytokines interleukin (IL)-1 [39][40][41] and tumor necrosis factor (TNF)-α [42][43][44], anti-inflammatory cytokines IL-10 [40,[43][44][45] and IL-6 [40,41,45,46], and chemokine macrophage inflammatory protein (MIP)-1 [45] have all been demonstrated to be differentially regulated upon Map-exposure. However, these assays have been performed at MOIs ranging from 2:1 up to 10:1 Map bacilli per host cell, and it is questionable whether that high an environmental load of mycobacteria represents the start of a natural infection.…”

Increased Lytic Efficiency of Bovine Macrophages Trained with Killed Mycobacteria

“…bovis . Bacterial load associated with each treatment at 2 h and 7 days p. i. was estimated as previously described [6,7]. In the phagocytosis test, bacterial cell uptake did not differ among the groups (S2 Table).…”

“…The phagocytosis assays were performed at 78 and 186 dpt to estimate the bacterial load reduction associated with each treatment as previously described [6,7]. For the first phagocytic assay, peripheral blood was drawn into heparinized Vacutainer tubes, diluted 1:2 in Hanks balanced salt solution (HBSS), layered over 10 ml of Ficoll-Paque gradient (1.084 g/cm 3 ) and centrifuged at 900 x g for 30 min.…”