Streptococcus pneumoniae capsular polysaccharides (CPSs) are major determinants of bacterial pathogenicity. CPSs of different serotypes form the main components of the pneumococcal vaccines Pneumovax, Prevnar7, and Prevnar13, which substantially reduced the S. pneumoniae disease burden in developed countries. However, the laborious production processes of traditional polysaccharide-based vaccines have raised the cost of the vaccines and limited their impact in developing countries. The aim of this study is to develop a kind of low-cost live vaccine based on using the recombinant attenuated Salmonella vaccine (RASV) system to protect against pneumococcal infections. We cloned genes for seven different serotypes of CPSs to be expressed by the RASV strain. Oral immunization of mice with the RASV-CPS strains elicited robust Th1 biased adaptive immune responses. All the CPS-specific antisera mediated opsonophagocytic killing of the corresponding serotype of S. pneumoniae in vitro. The RASV-CPS2 and RASV-CPS3 strains provided efficient protection of mice against challenge infections with either S. pneumoniae strain D39 or WU2. Synthesis and delivery of S. pneumoniae CPSs using the RASV strains provide an innovative strategy for low-cost pneumococcal vaccine development, production, and use.

SignificancePneumococcal infection-caused diseases are responsible for substantial morbidity and mortality worldwide. Traditional pneumococcal vaccines are developed based on purified capsular polysaccharides (CPS) or CPS conjugated to a protein carrier. Production processes of the traditional vaccines are laborious, and thereby increase the vaccine cost and limit their use in developing nations. A cost-effective pneumococcal vaccine using the recombinant attenuatedSalmonellavaccine (RASV) was developed in this study. We cloned and expressed genes for seven serotypes of CPSs in the RASV strain. The RASV-delivered CPSs induced robust humoral and cell-mediated responses and mediated efficient protection of mice against pneumococcal infection. Our work provides an innovative strategy for mass producing low-cost bioconjugated polysaccharide vaccines for needle-free mucosal delivery against pneumococcal infections.

Shigella flexneri 2a (Sf2a) is one of the most frequently isolated Shigella strains that causes the endemic shigellosis in developing countries. In this study, we used recombinant attenuated Salmonella vaccine (RASV) strains to deliver Sf2a O-antigen and characterized the immune responses induced by the vectored O-antigen. First, we identified genes sufficient for biosynthesis of Sf2a O-antigen. A plasmid containing the identified genes was then introduced into the RASV strains, which were manipulated to produce only the heterologous O-antigen and modified lipid A. After oral immunization of mice, we demonstrated that RASV strains could induce potent humoral immune responses as well as robust CD4+ T-cell responses against Sf2a Lipopolysaccharide (LPS) and protect mice against virulent Sf2a challenge. The induced serum antibodies mediated high levels of Shigella-specific serum bactericidal activity and C3 deposition. Moreover, the IgG+ B220low/int BM cell and T follicular helper (Tfh) cell responses could also be triggered effectively. The live attenuated Salmonella with the modified lipid A delivering Sf2a O-antigen polysaccharide showed the same ability to induce immune responses against Sf2a LPS as the strain with the original lipid A. These findings underscore the potential of RASV delivered Sf2a O-antigen for induction of robust CD4+ T-cell and IgG responses and warrant further studies toward the development of Shigella vaccine candidates with RASV strains.

Shigella flexneri (S. flexneri), a leading cause of bacillary dysentery, is a major public health concern particularly affecting children in developing nations. We have constructed a novel attenuated Salmonella vaccine system based on the regulated delayed antigen synthesis (RDAS) and regulated delayed expression of attenuating phenotype (RDEAP) systems for delivering the S. flexneri 2a (Sf2a) O-antigen. Methods: The new Salmonella vaccine platform was constructed through chromosomal integration of the araC PBADlacI and araC PBADwbaP cassettes, resulting in a gradual depletion of WbaP enzyme. An expression vector, encoding Sf2a O-antigen biosynthesis under the control of the LacI-repressible Ptrc promoter, was maintained in the Salmonella vaccine strain through antibiotic-independent selection. Mice immunized with the vaccine candidates were evaluated for cell-mediate and humoral immune responses. Results: In the presence of exogenous arabinose, the Salmonella vaccine strain synthesized native Salmonella LPS as a consequence of WbaP expression. Moreover, arabinose supported LacI expression, thereby repressing Sf2a O-antigen production. In the absence of arabinose in vivo, native Salmonella LPS synthesis is repressed whilst the synthesis of the Sf2a O-antigen is induced. Murine immunization with the Salmonella vaccine strain elicited robust Sf2a-specific protective immune responses together with long term immunity. Conclusion: These findings demonstrate the protective efficacy of recombinant Sf2a O-antigen delivered by a Salmonella vaccine platform.

为了探究干酪乳杆菌对断奶仔猪回肠黏膜组织发育及免疫功能的影响,将96头14日龄断奶仔猪随机分为空白对照组、乳杆菌对照组、K88攻毒组、预防组,每组4个重复,采用组织化学和免疫组织化学方法观察回肠黏膜组织结构及上皮内淋巴细胞数量的变化.结果显示,饲用干酪乳杆菌进行预防能够显著提高断奶仔猪回肠绒毛高度、V/C值,增加PCNA含量,提高肠上皮内淋巴细胞数量.表明干酪乳杆菌能保护肠道组织,提高肠道免疫力,降低大肠杆菌对肠道的损害.

试验旨在研究中草药饲料添加剂对育肥猪生产性能、肉品质及其主要生理生化特性的影响.试验选取120头体重(100±1.55) kg的“杜×长×大”三元杂交育肥猪,随机分成5个处理组,每个处理6个重复,每个重复4头猪:对照组(A)饲喂玉米-豆粕型基础日粮,试验组复方1组(B)、复方2组(C)、藿香组(D)与五味子组(E)分别饲喂添加不同中草药的试验饲粮,试验期14 d.试验期间,猪舍内平均温度为(34.46±2.14)℃,平均湿度为(49±2.46)％.结果表明:在高温环境中试验C组可显著提高育肥猪的日增重,并显著地降低了料重比(P＜0.05);试验B组和E组显著降低了育肥猪背最长肌的滴水损失及剪切力(P＜0.05);而且不同的中草药添加剂对糖类代谢也表现出显著的调控作用(P＜0.05).由此可见,高温环境中添加上述中草药饲料添加剂可提高育肥猪日增重、饲料转化率及肉品质,缓解高温对猪生产性能的影响.

<span id="ChDivSummary" name="ChDivSummary" class="abstract-text">为研究干酪乳杆菌对断奶仔猪十二指肠黏膜组织结构和功能及发育的影响,采用组织化学和免疫组织化学方法,以168头14日龄断奶仔猪为试验对象,随机分成7组,每组4个重复。研究结果显示:1)干酪乳杆菌预防组（E组）、预防治疗组（F组）的肠绒毛高度分别比大肠杆菌K88攻毒组（D组）明显增高了11.6%（P&lt;0.01）和11.2%（P&lt;0.01）。2)E组、F组的绒毛高度与肠隐窝深度的比值（V/C）分别比D组显著增加了21.3%（P&lt;0.01）和20.5%（P&lt;0.01）。3)E组、F组的肌层厚度分别比D组增加了11.8%（P&lt;0.05）和10.2%（P&lt;0.05）。4)D组、E组、F组和直接治疗组（G组）各组间增殖细胞核抗原（PCNA）含量在统计学上差异不显著,但使用干酪乳杆菌预防和治疗的E组和F组的增殖细胞核抗原（PCNA）含量均高于K88攻毒的D组。以上结果表明,饲用干酪乳杆菌进行预防能够显著提高断奶仔猪小肠绒毛高度,明显增加V/C值、肌层厚度及PCNA含量,提示干酪乳杆菌能有效维护断奶仔猪肠道健康,减轻大肠杆菌对肠黏膜结构的损害,促进肠道的发育。</span>

益生菌是一类定植于动物肠道,可辅助动物消化功能,维护肠道菌群平衡并可影响肠道免疫系统,有益于动物健康的重要调节性菌群.该类菌群与动物肠上皮细胞间互作的分子机制包括菌体表面分子如磷脂壁酸(phosphatidic acid,LTA)、表面层蛋白(S layer protein)等与宿主的粘附相关蛋白分子结合,通过占位效应抑制有害菌群在肠道内的定植;益生菌还可刺激肠道细胞分泌β防御素2、细菌素和有机酸等可抑制甚至杀灭有害菌群;在益生菌作用下,肠道上皮细胞可增强粘液糖蛋白、紧密连接蛋白occludin和ZO-1等分子的表达,加厚并加固肠道黏膜屏障;益生菌相关抗原可通过与抗原递呈细胞表面模式识别受体(TLRs等)分子结合,激活递呈细胞,启动各免疫细胞的交互作用,调节肠道免疫状态.

4-1BB is expressed on activated T cells and other immune and non-immune cells. It plays important roles in human and mouse T cell function. However, the swine 4-1BB sequence remains unknown and its role in swine T cell response has not been studied. In the present study, we for the first time described the cloning of the swine 4-1BB gene and the property of the protein. Two 4-1BB variants were detected in swine. The coding sequences of variant 1 and variant 2 were 768 and 726 nucleotides in length, respectively, and both variants were coded by 7 exons in the swine genome. Comparison of nucleotide and amino acid sequences showed that both swine 4-1BB variants were more closely related to bovine and human sequences than to either the mouse or rat sequence. Prediction analysis showed that swine 4-1BB belonged to the tumor necrosis factor receptor (TNFR) superfamily like human and mouse 4-1BB and the tertiary structures of the swine 4-1BB variants were much more similar to mouse 4-1BB than to human 4-1BB. The 1556bp 5' regulatory sequence cloned by nested PCR efficiently induced green fluorescent protein expression in porcine peripheral blood mononuclear cells (PBMC) post nucleofection. Moreover, 4-1BB protein was widely expressed in pig tissues and both variants of swine 4-1BB protein were transmembrane proteins and expressed on the membrane of porcine PBMCs.

piggyBac, a type II transposon that is useful for efficient transgenesis and insertional mutagenesis, has been used for effective and stable transfection in a wide variety of organisms. In this study we investigate the potential use of the piggyBac transposon system for forward genetics studies in the apicomplexan parasite Eimeria tenella. Using the restriction enzyme-mediated integration (REMI) method, E. tenella sporozoites were electroporated with a donor plasmid containing the enhanced yellow fluorescent protein (EYFP) gene flanked by piggyBac inverted terminal repeats (ITRs), an Asc I-linearized helper plasmid containing the transposase gene and the restriction enzyme Asc I. Subsequently, electroporated sporozoites were inoculated into chickens via the cloacal route and transfected progeny oocysts expressing EYFP were sorted by flow cytometry. A transgenic E. tenella population was selected by successive in vivo passage. Southern-blotting analysis showed that exogenous DNA containing the EYFP gene was integrated into the parasite genome at a limited number of integration sites and that the inserted part of the donor plasmid was the fragment located between the 5' and 3' ITRs as indicated by primer-specific PCR screening. Genome walking revealed that the insertion sites were TTAA-specific, which is consistent with the transposition characteristics of piggyBac.

CD28 is one of the most important co-stimulatory molecules required for effective activation of resting T cells in human and mouse. However, there are few studies on porcine CD28 (pCD28) until now. In the present study, we cloned and characterized the full-length cDNA of CD28 from the miniature pig. The open reading frame (ORF) sequence of pCD28 gene was organized into four exons, which were predicted to be in correspondence with the signal sequence, immunoglobulin variable-like (IgV) domain, transmembrane domain and cytoplasmic tail, respectively. We also identified the putative ligand binding site of CD28 within the IgV domain and the consensus motifs (one “YMNM” motif and two proline-rich motifs) within the cytoplasmic domain. Porcine CD28 was confirmed to be expressed on the cell membrane as indicated by indirect immunofluorescence assay (IFA). The putative promoter region of pCD28 was also cloned by the modified nested PCR and the cloned region could successfully drive the expression of yellow fluorescent protein (YFP) expression in porcine peripheral blood mononuclear cells (PBMCs). The present study is the first report of cloning and characterization of CD28 in porcine. Our work provided fundamental information for further researches on the structure and function of CD28 in porcine.

Eimeria parasites are obligate intracellular apicomplexan protists that can cause coccidiosis, resulting in substantial economic losses in the poultry industry annually. As the component of anticoccidial vaccines, seven Eimeria spp. of chickens are characterized with potent immunogenicity. Whether genetically modified Eimeria spp. maintains this property or not needs to be verified. In this study, two identical transgenic lines of Eimeria tenella were developed by virtue of single sporocyst isolation from a stably transfected population expressing fused protein of M2 ectodomain of avian influenza virus (M2e) and enhanced yellow fluorescent protein (EYFP). The chromosomal integration and expression of M2e-EYFP were confirmed by Southern blot, plasmid rescue and Western blot analysis. We found that the reproduction of transgenic parasites was higher than that of the parental strain. Chickens challenged with wild type E. tenella after immunization with 200 oocysts of transgenic parasites had similar performance compared to those in non-immunized and non-challenged group. In another trial, the performance of transgenic parasite-immunized birds was also comparable to that of the Decoquinate Premix-treated chickens. These results suggest that this transgenic line of E. tenella is capable of inducing potent protection against homologous challenge as a live anticoccidial vaccine. Taking together, our study indicates that transgenic eimerian parasites have the potential to be developed as a vaccine vehicle for animal use in the future.

Transgenic technology is an effective approach to assess the roles of specific genes in the activation and differentiation of T cells and modify T cell qualities. However, porcine T cell transfection is poorly documented. Here, we developed a non-virus-based method for the transfection of resting and ConA-stimulated porcine peripheral blood T cells using "Nucleofection™" gene transfer technology; both plasmid DNA- and mRNA-mediated nucleofection systems were developed. The results demonstrated for the first time that plasmid DNA encoding green fluorescent protein (GFP) and in vitro transcribed GFP mRNA could be delivered efficiently into resting and activated porcine T cells. For both methods, the onset of gene expression was rapid and occurred within 2 h post-nucleofection. Optimised plasmid DNA-mediated nucleofection induced approximately 40% transgene expression with 51% cell viability in resting T cells and approximately 20% transgene expression with 53% cell viability in activated T cells at 24 h post-gene delivery. However, optimised mRNA-based nucleofection resulted in higher transfection efficiencies and cell viability, with more than 50% transgene expression and 62% viability for resting T cells and approximately 40% transgene expression and 59% viability for activated T cells. Finally, we measured the impact of the developed nucleofection systems on T cell function by detecting the mRNA levels of the activation markers CD25, CD69 and the cytokine IFN-γ; cell proliferation of the nucleofected resting peripheral blood mononuclear cells (PBMC) after ConA stimulation was also examined. The nucleofected resting PBMCs proliferated normally and up-regulated CD25, CD69 and IFN-γ mRNA expression levels in a manner comparable to non-nucleofected cells. These results indicate that the developed nucleofection systems have no adverse effects on T cell function and can be utilised in swine immunological research.

Low transfection efficiency has been the major hurdle for gene transfection in Eimeria tenella.In order to overcome this difficulty,the electroporation parameters were optimized for traditional electroporation apparatus of Bio-Rad Gene Pulser and newly developed Amaxa’s Nucleofector I device,respectively.By adjusting series of key parameters,which include the concentration of plasmid DNA,the electric field intensity,the ambient temperature and bult-in programs of Nucleofector I,we found the optimal condition for electroporation of E.tenellasporozoites is at a voltage of 2000Vand a capacitance of 25μF,using 50μg plasmids/107 sporozoites and cuvettes with a 4mm gap,which together resulting in a transfection efficiency of 3.8×10-4.

Recognition of MHC-peptide complexes by T lymphocytes results in robust T cell proliferation,which is regarded as one of the hallmarks of the host cell-mediated immune responses against pathogens.To conveniently detect cell-mediated immune responses of swine upon pathogen infection or vaccine immunization,we developed a method for studying peripheral blood T cell proliferation of swine via MTT assay.Firstly,peripheral blood mononuclear cells(PBMC) were collected through density gradient centrifugation,and then peripheral blood lymphocytes(PBL) were obtained by discarding adherent monocytes after co-culture overnight.Flow cytometry analysis showed that the percentage of T cells in PBL was 80%.Next,the MTT assay was used to detect effects of the concentration of ConA and cell culture density on porcine T cell proliferation.The result of MTT suggested that when 2×106/mL T cells were cultured with 5 μg/mL ConA,the highest level of T cell proliferation was achieved.The MTT assay for studying porcine T cell proliferation could facilitate assessing the T cell mediated immune response against pathogens or vaccines.

Androgens play key roles in sex differentiation, gonadal maturation and reproductive behaviors and their actions are generally mediated through androgen receptor (AR). In the present study, isolation, sequencing and characterization of cDNA encoding AR and its temporal and spatial expression profiles in both sexes of Spinibarbus denticulate were carried out. Androgen receptor of Spinibarbus denticulate (sdAR) was 3172bp in length and encoded a 95.4kDa protein of 865 amino acids. Phylogenetic analysis and multiple amino acids sequence alignment indicated the close relationship and high score similarity of sdAR with ARs of other cyprinid species. A single transcript of approximate 3.2kb was identified in testis, liver and brain. RT-PCR assay characterized that sdAR mRNA was broadly distributed in both central nervous system (CNS) and most of peripheral tissues in male fish, while was confined to olfactory, telencephalon and hypothalamus of CNS and peripheral tissues including liver, spleen, head kidney, heart, and red muscle in females. During the embryonic development, sdAR mRNA was firstly detected at 16-cells stage and mid blastula stage with very weak signal. Little or no signal was detected in mid gastrula and neurula stages. The expression was occurred in the following developmental phases as well as in larvae of 4 days post hatching. During gonadal recrudescence process, liver of both sexes and testis were the most AR mRNA abundant tissues. In male fish, abundance of sdAR mRNA significantly varied in pituitary at fully recrudesced stage and brain at late recrudescing phase, respectively. No significant variation was found throughout the ovary recrudesce in each tissue checked. Our present work provided preliminary evidences that AR mediated androgen action on reproduction and development in both sexes of S. denticulate.

Genetic manipulation of Apicomplexan parasite Eimeria tenella is only in its earliest stages. In the current study, transfection of E. tenella was conducted by electroporating sporozoites along with linear or circular plasmid DNA, and with or without restriction enzyme. Transfection system containing both linear DNA and restriction enzyme resulted in a transfection efficiency of 2.2×10−3 in vitro, which is 200-fold higher than that using circular plasmid DNA alone. In another transfection strategy, PCR amplicons of expression cassette, instead of whole plasmid DNA, were subjected to transfection, and it was also found successful. These results suggest that linear DNA and restriction enzyme together in the transfection system greatly improve the transfection efficiency of E. tenella. The high transfection efficiency makes possible the establishment of stable transfection in vivo; and the success of PCR-based, restriction enzyme-mediated transfection will further simplify the transfection process for E. tenella and other Apicomplexan parasites.