Marked Differences in Mucosal Immune Responses Induced in Ileal versus Jejunal Peyer’s Patches to <i>Mycobacterium avium</i> subsp. <i>paratuberculosis</i> Secreted Proteins following Targeted Enteric Infection in Young Calves

In cattle, Mycobacterium avium subsp. paratuberculosis infection is primarily mediated through M cells overlying Peyer’s patches (PP) in the ileum. The capacity of M. avium subsp. paratuberculosis to invade ileal PP (IPP) versus discrete PP in the jejunum (JPP) and subsequent differences in mucosal immune responses were investigated. Intestinal segments were surgically prepared in both mid-jejunum, containing two JPPs, and in terminal small intestine containing continuous IPP. M. avium subsp. paratuberculosis (109 CFU) was injected into the lumen of half of each intestinal segment when calves were 10–14 days-old and infection confirmed 1–2 months later by PCR and immunohistochemistry. Thirteen recombinant M. avium subsp. paratuberculosis proteins, previously identified as immunogenic, were used to analyze pathogen-specific B- and T-cell responses in PP and mesenteric lymph nodes. IgA plasma cell responses to 9 of 13 recombinant proteins were detected in JPP but not in IPP. Secretory IgA reacting in ELISA with 9 of the 13 recombinant proteins was detected in luminal contents from both jejunal and ileal segments. These observations support the conclusion that pathogen-specific IgA B cells were induced in JPP but not IPP early after a primary infection. The presence of secretory IgA in intestinal contents is consistent with dissemination of IgA plasma cells from the identified mucosa-associated immune induction sites. This is the first direct evidence for M. avium subsp. paratuberculosis uptake by bovine JPP and for local induction of pathogen-specific IgA plasma cell responses after enteric infection. We also provide evidence that bacterial invasion of IPP, a primary B lymphoid tissue, provides a novel strategy to evade induction of mucosal immune responses. Over 60% of PPs in the newborn calf small intestine is primary lymphoid tissue, which has significant implications when designing oral vaccines or diagnostic tests to detect early M. avium subsp. paratuberculosis infections.在牛体内，禽分枝杆菌副结核亚种（Mycobacterium avium subsp. paratuberculosis）感染主要通过回肠派尔集合淋巴结（Peyer’s patches, PP）上方的M细胞介导。本研究探究了禽分枝杆菌副结核亚种侵袭回肠派尔集合淋巴结（ileal PP, IPP）与空肠离散派尔集合淋巴结（jejunal PP, JPP）的能力差异，以及由此引发的黏膜免疫应答差异。研究人员分别在包含2个JPP的中段空肠，以及带有连续IPP的末端小肠段制备手术模型。在犊牛10~14日龄时，向每段肠腔的半侧注入10^9菌落形成单位（Colony-Forming Unit, CFU）的禽分枝杆菌副结核亚种，并于1~2个月后通过聚合酶链式反应（Polymerase Chain Reaction, PCR）与免疫组织化学法（Immunohistochemistry）验证感染情况。本研究采用13种此前已被鉴定为具有免疫原性的重组禽分枝杆菌副结核亚种蛋白，分析派尔集合淋巴结与肠系膜淋巴结中病原体特异性B细胞与T细胞应答情况。在JPP中可检测到针对13种重组蛋白中9种的IgA浆细胞应答，但IPP中未检测到此类应答。在空肠与回肠肠腔内容物中，均可检测到可通过酶联免疫吸附试验（Enzyme-Linked Immunosorbent Assay, ELISA）与13种重组蛋白中9种发生反应的分泌型IgA。上述结果支持以下结论：原发感染早期，病原体特异性IgA B细胞仅在JPP中被诱导产生。肠内容物中分泌型IgA的存在，与黏膜相关免疫诱导位点产生的IgA浆细胞的播散相一致。本研究首次直接证明了牛JPP可摄取禽分枝杆菌副结核亚种，且肠道感染后可局部诱导产生病原体特异性IgA浆细胞应答。本研究同时证实，细菌侵袭作为初级B淋巴组织的IPP，是一种规避黏膜免疫应答诱导的全新策略。新生犊牛小肠中超过60%的派尔集合淋巴结属于初级淋巴组织，这一发现为设计用于早期检测禽分枝杆菌副结核亚种感染的口服疫苗或诊断试剂提供了重要参考。