College of Veterinary Medicine, Chonbuk National University, Iksan Campus, Gobong-ro 79, Iksan, 54596, Republic of Korea.
Department of Animal Resources Science, Dankook University, Cheonan, 31116, South Korea.
Vet Res. 2018 Jul 5;49(1):57. doi: 10.1186/s13567-018-0552-8.
The obligate intracellular pathogen Lawsonia intracellularis (LI), the etiological agent of proliferative enteropathy (PE), poses a substantial economic loss in the swine industry worldwide. In this study, we genetically engineered an O-antigen-deficient (rough) Salmonella strain secreting four selected immunogenic LI antigens, namely OptA, OptB, LfliC, and Lhly. The genes encoding these antigens were individually inserted in the expression vector plasmid pJHL65, and the resultant plasmids were transformed into the ∆asd ∆lon ∆cpxR ∆rfaL Salmonella Typhimurium (ST) strain JOL1800. The individual expression of the selected LI antigens in JOL1800 was validated by an immunoblotting assay. We observed significant (P < 0.05) induction of systemic IgG and mucosal IgA responses against each LI antigen or Salmonella outer membrane protein in mice immunized once orally with a mixture of four JOL1800-derived strains. Further, mRNA of IL-4 and IFN-γ were highly upregulated in splenic T cells re-stimulated in vitro with individual purified antigens. Subsequently, immunized mice showed significant protection against challenge with 10 TCID LI or 2 × 10 CFU of a virulent ST strain. At day 8 post-challenge, no mice in the immunized groups showed the presence of LI-specific genomic DNA (gDNA) in stool samples, while 50% of non-immunized mice were positive for LI-specific gDNA. Further, all the immunized mice survived the virulent ST challenge, compared to a 20% mortality rate observed in the control mice. Collectively, the constructed rough ST-based LI vaccine candidate efficiently elicited LI and ST-specific humoral and cell-mediated immunity and conferred proper dual protection against PE and salmonellosis.
专性细胞内病原体劳森氏菌（LI）是增生性肠炎（PE）的病原体，给全球养猪业造成了巨大的经济损失。在本研究中，我们对一个缺失 O 抗原（粗糙）的沙门氏菌菌株进行了基因工程改造，使其分泌四种选择的免疫原性 LI 抗原，即 OptA、OptB、LfliC 和 Lhly。这些抗原的编码基因分别被插入表达载体质粒 pJHL65 中，然后将这些质粒转化到 Salmonella Typhimurium（ST）菌株 JOL1800 的 ∆asd ∆lon ∆cpxR ∆rfaL 缺失株中。通过免疫印迹分析，我们验证了这些选择的 LI 抗原在 JOL1800 中的单独表达。我们观察到，单次口服混合了四种 JOL1800 衍生株的混合物后，小鼠体内针对每个 LI 抗原或沙门氏菌外膜蛋白的系统 IgG 和黏膜 IgA 反应显著（P < 0.05）。此外，在用单个纯化抗原体外重新刺激脾 T 细胞时，IL-4 和 IFN-γ 的 mRNA 高度上调。随后，免疫的小鼠在受到 10 TCID LI 或 2×10 CFU 强毒 ST 菌株的攻击时表现出显著的保护作用。在攻毒后第 8 天，免疫组小鼠的粪便样本中未检测到 LI 特异性基因组 DNA（gDNA），而未免疫组小鼠中有 50%呈 LI 特异性 gDNA 阳性。此外，与对照组小鼠 20%的死亡率相比，所有免疫的小鼠都存活下来，成功地抵抗了强毒 ST 的攻击。总之，构建的粗糙 ST 型 LI 疫苗候选物能够有效诱导 LI 和 ST 特异性体液和细胞免疫，并对 PE 和沙门氏菌病提供适当的双重保护。
