Fig 1.
Representative immunohistochemical staining of tissue samples collected from surgically isolated terminal small intestinal segments infected with M. avium subsp. paratuberculosis at one to two months PI.
M. avium subsp. paratuberculosis staining (brown colour and arrowheads) was observed in the dome regions (A, B), and was most abundant within cells of the lymphoid follicles in the submucosa (C, D). FAE, follicle-associated epithelium; D, dome region; V, villus; F, follicle; IF, interfollicular region; Ls, lymphatic sinus; Ca, capsule. Magnification is at x20 (A, C) and x100 (B, D).
Fig 2.
Representative immunohistochemical staining of tissue samples collected from surgically isolated mid-jejunal intestinal segments infected with M. avium subsp. paratuberculosis at one to two months PI.
M. avium subsp. paratuberculosis antigen staining (brown colour and arrowheads) was observed in the dome regions (A, B), and was most abundant within cells of the lymphoid follicles in the submucosa (C, D). M. avium subsp. paratuberculosis staining was not observed in the interfollicular regions. FAE, follicle-associated epithelium; D, dome region; V, villus; F, follicle; IF, interfollicular region; Ls, lymphatic sinus; Ca, capsule. Magnification is at x20 (A, C) and x100 (B, D).
Table 1.
PCR and IHC Detection of M. avium subsp. paratuberculosis in uninfected terminal small intestine segments (IPP-C), adjacent infected segments (IPP-M), and mesenteric lymph nodes (LN).
Table 2.
PCR and IHC Detection of M. avium subsp. paratuberculosis in uninfected mid-jejunal segments (JPP-C), adjacent infected segments (JPP-M), and mesenteric lymph nodes (LN).
Fig 3.
Recombinant Mycobacterium avium subsp. paratuberculosis proteins expressed in E. coli and purified by affinity chromatography.
Asterisks denote the expected migration of each recombinant protein by SDS-PAGE. “Empty vector” and “MBP alone” represent eluate from E. coli cells harbouring pET30a or pMAL-c4e, respectively, with no insert. Molecular weight standards are shown.
Fig 4.
Net IgA-ASC and IgG-ASC responses to recombinant M. avium subsp. paratuberculosis proteins.
Cell suspensions were prepared from PP collected from M. avium subsp. paratuberculosis-infected and uninfected compartments in mid-jejunal segments and assayed for IgA (A) and IgG (B) ASCs specific to recombinant M. avium subsp. paratuberculosis proteins. Cell suspensions were prepared from PP collected from M. avium subsp. paratuberculosis-infected and uninfected compartments in terminal small intestinal segments and assayed for IgG (C) ASCs specific to recombinant M. avium subsp. paratuberculosis proteins. The net number of ASC specific to each M. avium subsp. paratuberculosis protein in infected compartments was calculated using the formula provided in Materials and Methods. M. avium subsp. paratuberculosis proteins for which all calves (n = 3) had detectable net ASC responses are shown with significant (P < 0.05) increases in the frequency of M. avium subsp. paratuberculosis-protein specific ASCs denoted by an asterisk. Data presented are mean values with SEM.
Table 3.
Summary of recombinant M. avium subsp. paratuberculosis proteins and bacterial lysate in which IgA and IgG ASC responses were detected in all three calves.
The net frequency of IgA and IgG plasma cells was determined by comparing ASC frequency for individual proteins in M. avium subsp. paratuberculosis-infected and uninfected compartments of mid-jejunal segments (1) and in the terminal small intestine segments (2).
Fig 5.
Secretory IgA in the intestinal contents of M. avium subsp. paratuberculosis-infected (grey bars) and uninfected (white bars) compartments collected from mid-jejunal (A) and terminal small intestinal (B) segments. Secretory IgA levels were measured using an IgA-specific ELISA with intestinal contents diluted 1:50. Intestinal samples were assayed from calves 6, 7, and 10, for which there was no evidence of M. avium subsp. paratuberculosis infection in the PBS injected compartments. Data presented are mean A490 values and error bars represent SEM.
Fig 6.
Serum IgG reacting with recombinant M. avium subsp. paratuberculosis proteins before and after injecting M. avium subsp. paratuberculosis into intestinal segments of 10 to 14 day old calves.
Specific IgG levels were determined using an IgG-specific ELISA with sera diluted 1:600. For 10 of the 13 proteins, there was significantly (P < 0.05) increased reactivity with serum collected from calves at two month PI (black bars; n = 5) when compared to 1 month PI (grey bars; n = 5) (Groups i and ii). For 4 of these proteins (Group i), serum reactivity was significantly (P < 0.05) increased at 2 months PI when compared to both pre-infection (white bars) and one month PI. For three M. avium subsp. paratuberculosis proteins (Group iii) the level of serum reactivity did not change significantly following M. avium subsp. paratuberculosis infection. Data presented are mean A490 values with SEM.