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This chapter describes the results of the most current studies on the pathogenesis and host immunity after infection with Mycobacterium avium subsp. paratuberculosis and other closely related mycobacterial pathogens.

Supplemental Material, DS1_VET_10.1177_0300985819844823 for Quantification of Macrophages and Mycobacterium avium Subsp. paratuberculosis in Bovine Intestinal Tissue During Different Stages of Johne’s Disease by Caitlin J. Jenvey, Jesse M. Hostetter, Adrienne L. Shircliff, John P. Bannantine and Judith R. Stabel in Veterinary Pathology

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International audience; Mycobacteria have a complex cell wall structure that includes many lipids which are often species specific. Besides giving a phenotypic signature these lipids are often involved in infectious processes of pathogenic mycobacteria by interfering with the host immune system. The biosynthesis pathway of the simplest glycopeptidolipids (GPLs) is relatively well understood and involves more than fifteen genes. Even though M. avium subsp. paratuberculosis (Map) produces a lipopeptide rather than GPL, its genome contains nevertheless a locus highly similar to the GPL biosynthetic pathway of M. avium subsp. avium (Mav). We showed that the module composition of the non-ribosomal protein synthase (Nrp) of Map, the enzyme involved in the synthesis of the peptidyl moiety, is dramatically different from that of other GPL producers such as M. smegmatis (Ms) and Mav. While Map isolates do not produce GPLs, they do produce lipopeptides without the carbohydrate moiety. However, the picture is not as clear regarding the diversity of lipopeptides produced among two lineages classified as type I/III or S-type (ovine) and type II or C-type (bovine) Map strains that have emerged from the common ancestor, M. avium subsp. hominissuis. The S-type isolates are readily distinguishable from C-type isolates based on genome studies and readily discriminated by genotyping methods. In addition to the genotypic distinctions between S- and C-type strains, phenotypic differences have been documented. To provide a genomic basis for the synthesis of the diversity of lipopeptides in Map, its recently published genome sequence was explored using in silico methods and completed by biochemical investigations. Interestingly we discovered a change in the chemical structure of the lipopeptide of the S strains. These findings add new phenotypic evidence that contribute to separate the S type to the C type. Furthermore deciphering the biosynthesis pathway of cell wall lipopeptides should contribute to better understand the determinants of the adaptation of a pathogen to a specific host but also the factors favoring transmission to a new host.

Background The Mycobacterium tuberculosis 19-kDa lipoprotein has been reported to stimulate both T and B cell responses as well as induce a number of Th1 cytokines. In order to evaluate the Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis) 19-kDa lipoprotein as an immunomodulator in cattle with Johne's disease, the gene encoding the 19-kDa protein (MAP0261c) was analyzed. Results MAP0261c is conserved in mycobacteria, showing a 95% amino acid identity in M. avium subspecies avium, 84% in M. intracellulare and 76% in M. bovis and M. tuberculosis. MAP0261c was cloned, expressed, and purified as a fusion protein with the maltose-binding protein (MBP-19 kDa) in Escherichia coli. IFN-γ production was measured from 21 naturally infected and 9 control cattle after peripheral blood mononuclear cells (PBMCs) were stimulated with a whole cell lysate (WCL) of M. avium subsp. paratuberculosis or the recombinant MBP-19 kDa. Overall, the mean response to MBP-19 kDa was not as strong as the mean response to the WCL. By comparison, cells from control, non-infected cattle did not produce IFN-γ after stimulation with either WCL or MBP-19 kDa. To assess the humoral immune response to the 19-kDa protein, sera from cattle with clinical Johne's disease were used in immunoblot analysis. Reactivity to MBP-19 kDa protein, but not MBP alone, was observed in 9 of 14 infected cattle. Antibodies to the 19-kDa protein were not observed in 8 of 9 control cows. Conclusions Collectively, these results demonstrate that while the 19-kDa protein from M. avium subsp. paratuberculosis stimulates a humoral immune response and weak IFN-γ production in infected cattle, the elicited responses are not strong enough to be used in a sensitive diagnostic assay.

Background Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a facultative intracellular pathogen that resides within host macrophages during infection of ruminant animals. We examined survival of M. paratuberculosis infections within cultured macrophages to better understand the interplay between bacterium and host. Results Serial plating of M. paratuberculosis infected macrophage lysates on Herold's egg yolk medium showed that mycobacterial replication takes place between 0 and 24 hours post-infection. This initial growth phase was followed by a steady decline in viability over the next six days. Antibodies against M. paratuberculosis were affinity purified and used in conjunction with transmission electron microscopy to track the development of intracellular bacilli. Immunogold labeling of infected macrophages with antibody against M. paratuberculosis showed degraded intracellular mycobacteria that were unrecognizable by morphology alone. Conversely, when macrophages were heavily infected with M. paratuberculosis, no degraded forms were observed and macrophages were killed. Conclusions We present a general description of M. paratuberculosis survival within cultured macrophages using transmission electron microscopy and viability counts. The results of this study provides further insight surrounding M. paratuberculosis-macrophage infections and have implications in the pathogenesis of M. paratuberculosis, a pathogen known to persist inside cattle for many years.

Periparturient cows are immunosuppressed. Critical neutrophil functions (egress and phagocytic activities) and lymphocyte functions (immunoglobulin and cytokine secretion) are all impaired during this time. Exact causes ofperiparturient immunosuppression are not known but tremendous fluxes in endocrine factors undoubtedly effect changes in immunological competence. Periparturient cows experience large changes in plasma concentrations of vitamins and also are adjusting to the calcium, energy and protein demands necessary for lactation. The end of gestation appears to induce a progressive suppression of immune function. There appears to be a teleological basis for suppression of antigen specific immunity in the periparturient female. Development of immunity against self and paternal antigens released into the cow as a consequence of tissue damage associated with parturition would serve little useful purpose. However, as a result of this suppression, innate immune function is also impaired (perhaps inadvertently) and opportunistic infections such as mastitis and metritis occur with increased frequency in postpartum cows., American Association of Bovine Practitioners Proceedings of the Annual Conference, 1999