Panebra Alfredo, Lee Youngsub, Lillehoj Hyun S
Animal Biosciences and Biotechnology Laboratory, U.S. Department of Agriculture-ARS, Beltsville Agricultural Research Center, Beltsville, MD 20705, USA.
Animal Biosciences and Biotechnology Laboratory, U.S. Department of Agriculture-ARS, Beltsville Agricultural Research Center, Beltsville, MD 20705, USA.
Poult Sci. 2025 Apr 5;104(6):105112. doi: 10.1016/j.psj.2025.105112.
Coccidiosis is caused by apicomplexan protozoa of the genus Eimeria, which invade chicken intestinal epithelial cells, resulting in gut damage and a major poultry welfare problem worldwide. In this study, we developed a Bacillus subtilis (B. subtilis)-based vaccine delivering E. acervulina profilin (3-1E) antigen to induce protective immunity against coccidiosis in the host. A library of pBE-S-3-1E plasmid was constructed by subcloning a 3-1E open reading frame into the shuttle vector pBE-S. This library comprised approximately 900 recombinants, all expressing and secreting 3-1E, but each recombinant contained a distinct signal peptide. Following three rounds of screening using 3-1E-specific monoclonal antibodies (mAbs), 25 higher expressor recombinants were isolated and sequenced to identify the signal peptide driving 3-1E expression. From these 25 candidates, four high-expressing recombinants (#147, #241, #285-2, and #879), along with the empty vector (EV), were selected for further in vitro and in vivo assays. All recombinant clones sporulated, but clone #241 germinated at a higher rate compared to the others. Secretion of 3-1E by all germinated recombinant clones was confirmed by western blot and indirect ELISA, and further visualized by immunofluorescence assay (IFA). The conditioned media of all recombinants induced nitrite release that were neutralized by 3-1E mAb (#320) and induced significant expression of chicken IL-4, IL-6, TNF-α, IL-10 and IFN-γ (p<0.05) in HD11 macrophage cells. In vitro, phagocytosis of 3-1E recombinants by HD11 cells was significantly decreased in the following order #147> #241> #285-2> #879 compared to EV (P<0.0001). Finally, a pilot trial (N=30) was conducted to evaluate humoral and cellular immune responses in broiler chickens which were orally immunized with recombinant spores, as well as to assess spore persistence in chicken ceca in vivo. Chickens immunized with all recombinant spores exhibited significantly higher serum IgY and cecal sIgA levels to recombinant 3-1E protein compared to EV group (P<0.0001). Furthermore, splenocytes from immunized chickens demonstrated significantly increased proliferation when stimulated with recombinant 3-1E protein compared to the EV group. All colonies collected from the ceca of chickens immunized with 3-1E-recombinant spores at 10 days post-immunization were identified as positive by colony PCR.
球虫病由艾美耳属顶复门原生动物引起，这些原生动物侵入鸡的肠道上皮细胞，导致肠道损伤，是全球范围内一个主要的家禽福利问题。在本研究中，我们开发了一种基于枯草芽孢杆菌的疫苗，该疫苗递送堆型艾美耳球虫肌动蛋白结合蛋白（3-1E）抗原以诱导宿主对球虫病产生保护性免疫。通过将3-1E开放阅读框亚克隆到穿梭载体pBE-S中构建了pBE-S-3-1E质粒文库。该文库包含约900个重组体，均表达并分泌3-1E，但每个重组体含有一个独特的信号肽。使用3-1E特异性单克隆抗体（mAb）进行三轮筛选后，分离出25个高表达重组体并进行测序以鉴定驱动3-1E表达的信号肽。从这25个候选物中，选择四个高表达重组体（#147、#241、#285-2和#879）以及空载体（EV）进行进一步的体外和体内试验。所有重组克隆都形成了芽孢，但克隆#241的萌发率高于其他克隆。通过蛋白质免疫印迹和间接酶联免疫吸附测定法确认了所有萌发的重组克隆分泌3-1E，并通过免疫荧光测定法（IFA）进一步可视化。所有重组体的条件培养基诱导亚硝酸盐释放，该释放被3-1E mAb（#320）中和，并在HD11巨噬细胞中诱导鸡白细胞介素-4、白细胞介素-6、肿瘤坏死因子-α、白细胞介素-10和干扰素-γ的显著表达（p<0.05）。在体外，与EV相比，HD11细胞对3-1E重组体的吞噬作用按以下顺序显著降低：#147>#241>#285-2>#879（P<0.0001）。最后，进行了一项初步试验（N=30），以评估用重组芽孢口服免疫的肉鸡的体液和细胞免疫反应，以及评估芽孢在鸡盲肠中的体内持久性。与EV组相比，用所有重组芽孢免疫的鸡对重组3-1E蛋白表现出显著更高的血清IgY和盲肠分泌型IgA水平（P<0.0001）。此外，与EV组相比，用重组3-1E蛋白刺激时，免疫鸡的脾细胞增殖显著增加。在免疫后10天从用3-1E重组芽孢免疫的鸡的盲肠中收集的所有菌落通过菌落PCR鉴定为阳性。
