Comparison of the Virulence and Pathogenicity of MAP Strains

MAP-C, MAP-S and different strains within these lineages appear to differ in virulence and may be associated with different immunopatho- logical forms of paratuberculosis in different host species.

The heparin-binding haemagglutinin ad- hesin mediates the binding of mycobacteria to epithelial cells and fibroblasts early in infection and may play a role in dissemination. Lefranpois et al. (2013) determined that the C-terminal re­gions of the hbhA gene differ between MAP-C and MAP-S strains and that this correlates with their different affinities for heparin and con­sequently their adherence properties. There is some evidence that different MAP strains have different capacities for entry and survival in macrophages, although it is difficult to tease out the effects of bacterial virulence factors from host responses as the interplay of both will de­termine the outcome of infection. Intracellular studies undertaken by Janagama et al. (2006) and Gollnick et al. (2007) showed that MAP-C strains persisted in relatively higher numbers in bovine monocyte-derived macrophages (MDMs) when compared with a MAP-S strain. However, Abendano et al. (2013) showed that MAP-S and C strains from sheep and goats persisted within bovine macrophages in lower numbers than those from cattle, bison, deer and wild boar re­gardless of MAP lineage. A more recent study by Alonso-Hearn et al. (2019) analysed infec­tion data of both bovine and ovine MDMs with genotypically distinct MAP isolates, including MAP-C, MAP-S and Type B strains, from six dif­ferent host species. Overall, MAP isolates from goat and sheep persisted or grew minimally within macrophages, whereas isolates from cat­tle and non-domesticated animals grew to high­er numbers, suggesting that these isolates may be more virulent or better adapted to infect do­mestic ruminants.

Studies have been performed to determine the genetic responses of different MAP strains to macrophages. Zhu et al. (2008) undertook transcriptional analysis of different MAP strains in MDMs using selective capture of transcribed sequences. Despite variations in the genes iden­tified, the different MAP strains responded in a generally similar fashion to oxidative stress, to metabolic and nutritional starvation, in cell survival and in upregulating genes involved in cell wall biosynthesis. However, transcription of MAP_1728 (YfnB), MAP_1738 (MmpL5), MAP_1729c and MAP_1730 (hypothetical proteins with unknown function) was upregu- lated only in MAP-C strains, consistent with their absence from MAP-S strains examined to date (Tables 6.2 and 6.3). Similarly, transcrip­tional and proteomic profiling of MAP-C and MAP-S strains under iron-replete or -depleted conditions revealed that different strains utilize different metabolic pathways under different conditions, likely due to strain-specific polymor­phisms in the promoter of the iron storage gene bfrA (Janagama et al., 2010).

A few studies have presented evidence that different MAP strains may play a role in polar­izing the host immune responses, which may determine the different disease pathologies observed. Janagama et al. (2006) investigated cytokine responses to different MAP strains in a bovine MDM system using real-time PCR as­says, and Motiwala et al. (2006) performed a global-scale transcriptional analysis of human macrophages (THP-1 cells) exposed to differ­ent MAP strains. Both studies reported that MAP-C strains induced anti-inflammatory and anti-apoptotic pathways in the host cells with­out causing major alterations in the transcrip­tion of proinflammatory genes, which would favour bacterial survival and persistence. This pattern of gene expression was found to be the same for bovine, bison and human MAP-C strains, as defined by SSR analysis, although the magnitude of the responses varied.

Protein expression profiling studies have been undertaken to investigate the responses of isolates representing different MAP lineages to conditions mimicking the host environment such as oxidative and nitrosative stress, and the stressors of temperature flux, hypoxia and nutri­ent starvation. These studies have only employed one MAP-S and one MAP-C strain but observed lineage-specific differential expression of a num­ber of proteins: 10 and 9 in response to oxidative and nitrosative stress, respectively (Kawaji et al., 2010); 2 7 to temperature flux (Gumber and Whittington, 2009); 21 and 26 to hypoxia and starvation, respectively (Gumber et al., 2009). Janagama et al. (2010) performed protein pro­filing of the two lineages under iron-depleted and -replete conditions and revealed that un­der iron-replete conditions, ribosomal proteins, bacterioferritin, mycobacterial heme and utili­zation and degrader proteins were upregulated but under iron-depleted conditions aconitase, succinate dehydrogenases and superoxide dis­mutase were downregulated in a MAP-C strain only. A study by Hughes et al. (2012) analysed seven MAP-S (Type I) and 18 MAP-C strains and identified 17 and 16 proteins specific for MAP-S and MAP-C, respectively, which were not de­pendent on growth phase and were also exhib­ited in MAP isolated directly from pathogenic lesions.

Verna et al. (2007) investigated strain­specific differences in the pathology of disease in sheep. Infection with different MAP-C strains resulted in a common pattern, character­ized by focal lesions, mainly in the mesenteric lymph nodes, as well as the presence of fibrous tissue and occasionally necrosis and numer­ous Langhans giant cells in the granulomas. Infection with a MAP-S strain induced more severe lesions, occurring mainly in the intesti­nal lymphoid tissue, and there was a conspicu­ous absence of necrosis, fibrosis and giant cells. Lesions induced by the MAP-S strain were more severe than those induced by MAP-C strains, which suggests that MAP-C strains had a slow, localized development in the early stages of in­fection. The development of giant cells may be linked to MAP strain rather than host determi­nants, since giant cells are a feature of natural cases of bovine paratuberculosis and leporine paratuberculosis (Beard et al., 2001) but not ovine paratuberculosis caused by MAP-S strains.

Experimental infection of lambs with MAP-C and MAP-S strains revealed specific an­tibody and IFN-y production was significantly higher in animals infected with MAP-C strains, whereas no consistent IFN-y responses were ob­served in animals infected with MAP-S strains (Fernandez et al., 2014). Furthermore, sheep infected with passaged MAP-C strains showed more severe and diffuse disease in the small in­testine than those infected with passaged MAP-S strains, though lesions induced by MAP-C strains showed a regressive character, unlike those induced by the MAP-S strains. These re­sults suggest that sheep naturally infected with MAP-C strains may be able to recover from the infection.

Different MAP-C strains have also been found to elicit different immune responses in vitro and in vivo. Differential immune responses in peripheral cells, in the proliferative respons­es and in cell functionality at the local level were found between two different Argentinian MAP-C strains in experimentally infected calves (Colavecchia et al., 2016).

6.5