Zekarias Bereket, Mo Hua, Curtiss Roy
The Biodesign Institute, Center for Infectious Diseases and Vaccinology, Arizona State University, 1001 S. McAllister, Tempe, AZ 85287-5401, USA.
Clin Vaccine Immunol. 2008 May;15(5):805-16. doi: 10.1128/CVI.00457-07. Epub 2008 Mar 12.
Clostridium perfringens-induced necrotic enteritis (NE) is a widespread disease in chickens that causes high mortality and reduced growth performance. Traditionally, NE was controlled by the routine application of antimicrobials in the feed, a practice that currently is unpopular. Consequently, there has been an increase in the occurrence of NE, and it has become a threat to the current objective of antimicrobial-free farming. The pathogenesis of NE is associated with the proliferation of C. perfringens in the small intestine and the secretion of large amounts of alpha toxin, the major virulence factor. Since there is no vaccine for NE, we have developed a candidate live oral recombinant attenuated Salmonella enterica serovar Typhimurium vaccine (RASV) that delivers a nontoxic fragment of alpha toxin. The 3' end of the plc gene, encoding the C-terminal domain of alpha toxin (PlcC), was cloned into plasmids that enable the expression and secretion of PlcC fused to a signal peptide. Plasmids were inserted into Salmonella enterica serovar Typhimurium host strain chi8914, which has attenuating pabA and pabB deletion mutations. Three-day-old broiler chicks were orally immunized with 10(9) CFU of the vaccine strain and developed alpha toxin-neutralizing serum antibodies. When serum from these chickens was added into C. perfringens broth cultures, bacterial growth was suppressed. In addition, immunofluorescent microscopy showed that serum antibodies bind to the bacterial surface. The immunoglobulin G (IgG) and IgA titers in RASV-immunized chickens were low; however, when the chickens were given a parenteral boost injection with a purified recombinant PlcC protein (rPlcC), the RASV-immunized chickens mounted rapid high serum IgG and bile IgA titers exceeding those primed by rPlcC injection. RASV-immunized chickens had reduced intestinal mucosal pathology after challenge with virulent C. perfringens. These results indicate that oral RASV expressing an alpha toxin C-terminal peptide induces protective immunity against NE.
产气荚膜梭菌引起的坏死性肠炎（NE）是鸡群中一种广泛传播的疾病，可导致高死亡率并降低生长性能。传统上，NE通过在饲料中常规使用抗菌药物来控制，但目前这种做法并不受欢迎。因此，NE的发生率有所增加，并且它已成为当前无抗养殖目标的一个威胁。NE的发病机制与产气荚膜梭菌在小肠中的增殖以及大量α毒素（主要毒力因子）的分泌有关。由于尚无针对NE的疫苗，我们开发了一种候选活口服重组减毒鼠伤寒沙门氏菌疫苗（RASV），该疫苗可递送α毒素的无毒片段。编码α毒素C末端结构域（PlcC）的plc基因的3'端被克隆到质粒中，该质粒能够表达并分泌与信号肽融合的PlcC。将质粒插入具有衰减型pabA和pabB缺失突变的鼠伤寒沙门氏菌宿主菌株chi8914中。给3日龄的肉鸡口服10⁹CFU的疫苗菌株，它们产生了α毒素中和血清抗体。当将这些鸡的血清添加到产气荚膜梭菌肉汤培养物中时，细菌生长受到抑制。此外，免疫荧光显微镜检查显示血清抗体与细菌表面结合。RASV免疫鸡的免疫球蛋白G（IgG）和IgA滴度较低；然而，当给鸡注射纯化的重组PlcC蛋白（rPlcC）进行非肠道加强注射时，RASV免疫鸡迅速产生高血清IgG和胆汁IgA滴度，超过了rPlcC注射引发的滴度。用强毒产气荚膜梭菌攻击后，RASV免疫鸡的肠道黏膜病理学变化减轻。这些结果表明，表达α毒素C末端肽的口服RASV可诱导针对NE的保护性免疫。
