Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is a highly contagious disease with high morbidity and mortality that affects the global swine industry. Despite efforts to control it, there is still a widespread dissemination of PRRSV with obvious genetic variations in swine population, resulting in huge economic losses annually. Consequently, accurate laboratory diagnosis is crucial for the rapid confirmation of PRRSV infections. An immunoperoxidase monolayer assay (IPMA) was developed for the specific and sensitive detection of PRRSV based on a broad-spectrum anti-PRRSV monoclonal antibody (mAb) 28F6. The mAb 28F6-based IPMA could specifically detect PRRSV and possessed no cross-reactions with CSFV, PCV2, and PEDV. Sensitivity analysis showed that the limit of detection of the IPMA reached 10− 2.25 TCID50/100 µL. There was no significant difference in the detection of PRRSV of different passages with different batches of mAb 28F6, indicating that the IPMA had excellent repeatability. Additionally, the IPMA was capable of detecting multiple PRRSV variants, including field strains (e.g., BJ-4, HN07-1, and HNhx) and vaccine strains (e.g., HuN4-F112, JXA1-R, TJM-F92, GDr180, VR2332, CH-1R, and R98). Validation of the IPMA with qRT-PCR showed 100

Background Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a highly contagious disease with high morbidity and mortality that affects the global swine industry. So far, there is still a widespread dissemination of PRRSV with obvious genetic variations in swine population, resulting in huge economic losses annually. Therefore, accurate laboratory diagnosis is needed to quickly confirm PRRSV infection. Results An immunoperoxidase monolayer assay (IPMA) was developed for the specific and sensitive detection of PRRSV based on a broad-spectrum anti-PRRSV monoclonal antibody (mAb) 28F6. The mAb 28F6-based IPMA could specifically detect PRRSV and possessed no cross-reactions with CSFV, PCV2, and PEDV. Sensitivity analysis showed that the limit of detection of the IPMA reached 10− 2.25 TCID50/100 µL. There was no significant difference in the detection of PRRSV of different passages with different batches of mAb 28F6, indicating that the IPMA had good repeatability. In addition, the IPMA could recognize a number of PRRSV variants including field strains such as BJ-4, HN07-1, and NADC30-like strain, as well as vaccine strains like HuN4-F112, JXA1-R, TJM-F92, GDr180, VR2332, CH-1R, and R98. Validation of the IPMA showed that it was in 100% consistency with qRT-PCR on the detection of 108 clinical samples. Conclusions The IPMA could meet the demand for the specific and sensitive detection of PRRSV, which is helpful for accurate monitoring and early warning of PRRSV infections.

Porcine Epidemic Diarrhea Virus (PEDV) poses a significant threat to neonatal piglets, particularly due to the limited efficacy of existing vaccines and the scarcity of efficacious therapeutic drugs. Gegen Qinlian Decoction (GQD) has been employed for over two millennia in treating infectious diarrhea. Nonetheless, further scrutiny is required to improve the drug’s efficacy and elucidate its underlying mechanisms of action. In this study, a modified GQD (MGQD) was developed and demonstrated its capacity to inhibit the replication of PEDV. Animal trials indicated that MGQD effectively alleviated pathological damage in immune tissues and modulated T-lymphocyte subsets. The integration of network analysis with UHPLC-MS/MS facilitated the identification of active ingredients within MGQD and elucidated the molecular mechanisms underlying its therapeutic effects against PEDV infections. In vitro studies revealed that MGQD significantly impeded PEDV proliferation in IPEC-J2 cells, promoting cellular growth via virucidal activity, inhibition of viral attachment, and disruption of viral biosynthesis. Furthermore, MGQD treatment led to increased expression levels of IFN-α, IFN-β, and IFN-λ3, while concurrently decreasing the expression of TNF-α, thereby enhancing resistance to PEDV infection in IPEC-J2 cells. In conclusion, our findings suggest that MGQD holds promise as a novel antiviral agent for the treatment of PEDV infections.

Artemisia argyi (AA) is promising as a potential feed additive. Microbial fermentation is beneficial to the degradation of cell walls and the better release of bioactive compounds of AA. However, there are few reports on the application of fermented AA as a feed additive for broilers. The present study intended to evaluate the application value of fermented AA as a feed additive for broilers by examining the effects of the dietary supplementation of Aspergillus niger-fermented AA and unfermented AA on growth performance, slaughter performance, and meat quality of brokers. A total of 360 newly hatched (1-day-old) broilers with similar body weight were randomly divided into the following 5 groups: basal diet group as control (C) group, basal diet +3% unfermented AA (E1) group, basal diet + 1% fermented AA (E2) group, basal diet + 3% fermented AA (E3) group, basal diet + 5% fermented AA (E4) group. Each group included 6 replicates with 12 broilers per replicate, and the feeding trail lasted for 48 d. Body weight and feed intake were recorded every 2 wk, and the feed gain ratio was calculated to assess growth performance. At 42 d, 6 broilers from each group were slaughtered, and the carcass traits were calculated. The results showed that compared with the control group, Aspergillus Niger could effectively destroy AA fiber, which contributed to better release of AA bioactive compounds. Moreover, dietary supplementation with AA could improve the growth performance of broilers (P < 0.05), and the effect of fermented AA was better than unfermented AA, especially 3% fermented AA. From 28 to 42 d, compared with the control group, the average daily gain of broilers in the group supplementation with 3% fermented AA was significantly increased (P < 0.05), and the feed-to-gain ratio was decreased (P < 0.05). At 42 d, the dressing percentage, half-eviscerated carcass percentage, eviscerated carcass percentage, and breast muscle percentage of broilers in the groups of 1, 3, and 5% fermented AA diets were significantly improved (P < 0.05), and the thigh muscle percentage of broilers in the group with 3% fermented AA diets was significantly improved (P < 0.05). Meanwhile, the meat quality of broilers in the group with fermented AA diets was also significantly improved. Birds in AA groups had higher a* value and lower shear force of breast muscle, especially the group supplementation with 3% fermented AA (P < 0.05). In conclusion, fermented AA has good application value as a potential feed additive for broilers, dietary supplementation of fermented AA can improve the production performance and meat quality of broiler chickens, of which 3% fermented AA is more effective.

猪瘟(classical swine fever,CSF)是危害猪健康的重要传染病之一,具有急性、热性和高度接触性等特性,给世界养猪业造成了巨大的经济损失.PCR-ELISA 检测方法是在PCR和 ELISA 基础上创新的一种新技术,其灵敏性和准确性都高于单纯的 PCR 检测和ELISA检测.根据GenBank中猪瘟石门株序列,设计特异性引物,分别在 5'端标记生物素和地高辛,经过PCR扩增后,加入到包被有链霉亲和素的ELISA反应板上,然后通过DIG-HRP抗体进行显色反应,建立PCR-ELISA检测方法.结果表明:PCR-ELISA 最低检测到 1.81×104 拷贝/μL,普通 PCR 最低检测到 1.81×106 拷贝/μL,因此 PCR-ELISA 灵敏性比普通PCR高约 100 倍;对 27 份未知样品进行检测,普通 PCR 检测出阳性样品 15 份,阳性率为55.5%,PCR-ELISA检出 20 份阳性样品,阴性 7 份,阳性率为 74%,阳性检出率增加 18.5%;用建立的Real-Time PCR进行验证,检测结果与 PCR-ELISA 一致,两者的符合度为 100%;然后用PK15 细胞对普通PCR 未检测出的阳性样品进行病毒分离培养,通过间接免疫荧光(IFA)及普通PCR检测,结果显示该 5 份样品均为阳性.说明本研究所建立的 PCR-ELISA方法具有灵敏、特异、准确、快速、安全等优势,可应用于 CSFV 的快速诊断,为 CSFV 的流行病学监测提供有力保障.

Broiler ascites syndrome (AS), is a nutritional and metabolic disease that occurs in fast-growing commercial broiler chickens. AS can cause poor growth and a significant increase in the rate of broiler deaths, which has resulted in serious economic losses to the poultry industry. The classic traditional Chinese medicine Qiling Jiaogulan Powder (QLJP) has been demonstrated to have a certain therapeutic effect on broiler AS. However, its pharmacological mechanism remains to be elucidated. This study was performed to investigate the multitarget action mechanism of QLJP in the treatment of broiler AS based on network pharmacology analysis using a broiler AS model. First, all chemical components and targets of QLJP were obtained from the Traditional Chinese Medicine System Pharmacology Analysis Platform (TCMSP). Targets related to broiler AS were further obtained through the GeneCards database and the NCBI Gene sub-database. A protein-protein interaction (PPI) network was constructed. Then, enrichment analyses were performed to predict the potential mechanisms of QLJP in the treatment of broiler AS. Finally, the treatment effect of QLJP on AS was verified in a broiler AS model. Network pharmacology analysis generated 49 active ingredients and 167 core targets of QLJP, and a QLJP-single drug-target-disease network was successfully constructed. Gene enrichment analysis indicated that the core targets have played major roles in the Cell cycle, FOXO signaling pathways, etc. We demonstrated that QLJP improved clinical and organ damage symptoms and significantly reduced the ascites heart index in broilers with AS induced by administration of high-energy, high-protein diets and high-sodium drinking water in a low-temperature environment. QLJP may regulate lung oxidative stress, the cell cycle and apoptosis by activating the FOXO3a signaling pathway to interfere with the occurrence and development of AS in broilers. QLJP administration may be a good clinical strategy for the prevention and treatment of broiler AS.

Chinese herbal medicine plays an important role in regulating the nutritional metabolism of poultry and maintaining or improving normal physiological functions and animal health. The present study investigated the effects of dietary supplementation with Qiling Jiaogulan Powder (QLJP) on pulmonary fibrosis and pulmonary arteriole remodeling in low temperature-exposed broilers. Seven-day-old Ross 308 broilers (n = 240) were reared adaptively to 14 days of age. The broilers were randomly divided into six groups: A control group (basal diet and normal feeding temperature); model group (basal diet); low-, medium- and high-dose QLJP groups (basal diet supplemented with 1 g/kg, 2 g/kg, 4 g/kg QLJP); and L-Arg group (basal diet supplemented with 10 g/kg L-arginine). Additionally, all the broilers, except the broilers in the control group, from the age of 14 days old, had a house temperature continuously lowered by 2 °C each day until it reached 12 °C at 21 days of age, and the low temperature was maintained until the end of the experiment. There were four replicates per group and 10 birds per replicate. The results showed that the structure of the lung tissue was clearer and basically intact in the broilers in the QLJP groups, with a small number of collagen fibers formed, and the content of hydroxyproline (HYP) was significantly reduced. QLJP improved pulmonary arteriole lesions, such as tunica media thickening, intimal hyperplasia, arterial wall hypertrophy, and lumen narrowing. QLJP reduced the relative media thickness (%) and relative medial area (%) of the pulmonary arteriole, and significantly decreased the expression level of the alpha-smooth muscle actin (α-SMA) protein in pulmonary arteriole, which alleviated pulmonary arteriole remodeling. The quantitative real-time PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA) results showed that QLJP treatment significantly reduced the gene and protein levels of transforming growth factor-beta l (TGF-β1) and Smad2 in the lung and downregulated the gene and protein levels of collagen type I alpha 1 (COL1A1) and matrix metalloproteinase 2 (MMP2). In conclusion, the results of our study suggested that dietary supplementation with QLJP improved pulmonary fibrosis and pulmonary arteriole remodeling by inhibiting the expression of genes related to the TGF-β1/Smad2 signaling pathway and inhibited the occurrence and development of pulmonary arterial hypertension in low-temperature-exposed broilers.

[目的]利用噬菌体展示技术制备一种可用于检测血清p53抗体的噬菌体.[方法]利用基因工程手段将编码p53蛋白N端20～31位的多肽SD的基因克隆到丝状噬菌体载体fADL-le的pⅢ蛋白基因中,制备展示该外源肽的噬菌体phage-SD并进行了Western blot鉴定.在此基础上,分别以phage-SD和重组p53蛋白为检测抗原,利用ELISA方法对乳腺癌患者血清p53抗体进行检测.[结果]成功制备出能特异性识别血清p53抗体的噬菌体phage-SD.ELISA检测结果表明,60例乳腺癌患者中phage-SD检测到13例p53抗体阳性患者,检出率为21.67％,与重组p53蛋白检测效率(21.67％)一致.[结论]成功制备一种灵敏度高、特异性强的可用于血清p53抗体检测的噬菌体phage-SD,为新型血清p53抗体检测试剂的开发及临床应用奠定基础.

African swine fever (ASF) is a highly contagious disease caused by African swine fever virus (ASFV), affecting domestic and wild boars. The polyprotein pp220 of ASFV is responsible for producing the major structural proteins p150, p37, p14, p34, and p5 via proteolytic processing. The p34 protein is the main component of the ASFV core shell. However, the immunologic properties of the p34 protein in vitro and in vivo remain unclear. The results showed that the recombinant p34 protein expressed in prokaryotes and eukaryotes could react with convalescent swine sera to ASFV, suggesting that p34 is an immunogenic protein. Significantly, anti-p34 antibodies were found to inhibit the replication of ASFV in target cells. Furthermore, rabbits immunized with the recombinant C-strain of classical swine fever virus containing p34 produced both anti-p34 humoral and cellular immune responses. In addition, the p34 protein could induce a cell-mediated immune response, and a T-cell epitope on the p34 protein was identified using immunoinformatics and enzyme-linked immunospot (ELIspot) assay. Our study demonstrates that the p34 protein is a novel antigen of ASFV with protective potential.

本试验旨在筛选和鉴定与猪程序性死亡因子1(PD-1)及其配体(PD-L1)相互作用的表位多肽,为阻断猪PD-1/PD-Ls通路逆转机体的免疫功能提供新策略.根据已解析人与鼠的PD-1与PD-L1相互作用的关键氨基酸位点信息,分析猪PD-1与PD-L1蛋白相互结合的关键氨基酸位点,在关键氨基酸位点处设计系列表位多肽,固相合成法合成表位多肽;分离自然感染猪圆环病毒2型(PCV2)仔猪的外周血单个核细胞(PBMC),检测表位多肽与猪重组蛋白PD-1、PD-L1和PBMC的结合能力,选取能结合猪PD-L1和PBMC的表位多肽pPD-15,检测其对猪PBMC增殖的影响,分析其作为佐剂免疫后对小鼠猪瘟病毒(CSFV)抗体水平的影响,最后,高效液质联用色谱法(HPLC-MS)检测表位多肽pPD-15的纯度和氨基酸序列正确性.结果显示,表位多肽pPD-15能结合猪PD-L1和PBMC;增殖试验显示,pPD-15组M1的平均荧光强度(74.20％)比空白对照组M1的平均荧光强度(4.37％)提高了 69.83％,表明表位多肽pPD-15可促进猪PBMC的增殖;动物免疫试验显示,pPD-15作为佐剂可以提高CSFV抗体水平;HPLC-MS结果显示,合成的表位多肽pPD-15纯度高,氨基酸序列正确.本试验证实,表位多肽pPD-15具有提高体内外免疫力的能力,为研制新型免疫调节佐剂提供了科学依据.

为探讨孕期砷(arsenic,As)暴露对子代雄性小鼠精子质量的影响,本试验检测分析了不同发育阶段子代雄性小鼠的精子活率、畸形率及促卵泡生成素(follicle stimulating hormone,FSH)和促黄体生成素(luteinizing hormone,LH)水平.结果显示,砷暴露组的5周龄和8周龄子代雄性小鼠精子活率与对照组相比显著下降,8周龄的头部畸形率显著升高;5周龄和16周龄的FSH水平均显著低于对照组,8周龄的略下降但无显著性差异;3个发育阶段的LH水平与对照组相比均无显著变化.结果表明,母鼠孕期砷暴露可通过降低子代雄性小鼠的FSH水平、导致精子畸形率升高、精子活率下降,影响精子质量.

为建立一种检测猪圆环病毒3型(porcine circovirus type 3,PCV3)的PCR-ELSIA方法,根据GenBank上报道的PCV3全基因组序列,针对其高度保守区域设计1对特异性引物,上、下游引物的5'端分别标记生物素和地高辛.经PCR扩增获得大量带有标记的目的基因,依靠生物素-链霉亲和素的非共价结合作用固定目的基因于载体上,再通过羊抗DIG-HRP的显色反应,建立PCR-ELISA检测方法.结果表明,该方法与猪繁殖与呼吸综合征病毒、猪瘟病毒、猪细小病毒和猪圆环病毒2型无交叉反应,特异性良好.PCR-ELISA方法最低检测限为5.58×102 copies/μL,敏感性比常规PCR高100倍(常规PCR的检测限为5.58×104 copies/μL).PCR-ELISA方法批内和批间重复性检测的变异系数分别为1.90％～5.20％和3.89％～9.03％.对132份临床样品进行PCV3检测,PCR-ELSIA检出率为9.10％(12/132),高于常规PCR的检出率(4.55％,6/132).综上,该方法具有特异、灵敏、安全高效的优点,可为PCV3的诊断、防控和流行病学调查提供新的技术手段.

本研究对具核梭杆菌(F.nucleatum)感染鼠巨噬细胞进行共同培养之后,分别在10个时段采取上清液和巨噬细胞,用ELISA、qRT-PCR法和扫描电镜检测TNF-a、IL-1α、IL-6和IL-8细胞因子,并观察巨噬细胞的超微形态变化.结果 显示,4种细胞因子均有高水平表达;IL-8在共培养6～72 h呈高水平表达;IL-6和TNF-α表达水平也较高;IL-1从3h表达,12h达最高值,48 h后无表达.qRT-PCR检测结果显示,TNF-α的RNA表达呈波浪式,分别于6,60 h各出现1个高峰值;IL-1a和IL-6呈现正态表达,从3h开始明显表达至60 h达峰值,此后缓慢下降;IL-8从6h逐渐上升,至72 h仍呈高水平表达.超微形态观察显示,在F.nucleatum感染的前24 h,巨噬细胞呈收缩状,其表面和周围有大量发育良好F.nucleatum,并呈团块状黏附,巨噬细胞逐渐伸出伪足.30 h后F.nucleatum逐渐发育不良,小而细,而巨噬细胞表面粗糙,有许多大小不一的结节,伪足变粗大.48 h巨噬细胞表面出现胞吐孔,少量细小的F.nucleatum仍然黏附于巨噬细胞伪足.此后,发现少量巨噬细胞凋亡.结果 表明,F.nucleatum可诱导巨噬细胞产生大量炎性细胞因子,并能长时间与巨噬细胞共生;巨噬细胞能够吞噬F.nucleatum,其胞吐作用可传递F.nuclea-tum抗原信息.

目前,全球因肥胖引发的基础代谢性疾病日益严重,而由肥胖基因编码的瘦素(Lep)是中枢神经系统调节能量代谢进程中的重要能量调节因子,以下丘脑为靶点,通过激活其受体传递抑制摄食、调节脂肪沉积与能量代谢信号;但内源性瘦素升高和瘦素受体(LepR)信号的增加并不能使肥胖者的体重下降;大量研究表明瘦素多效性导致了其功能机制的复杂性.论文主要就瘦素及其受体的分子生物学特性、调控能量代谢的机制、转运缺陷及信号通路调控等及其与肥胖的关系做简要综述,以期为肥胖相关代谢性疾病的研究提供理论依据.

为了研究yibT基因在鼠伤寒沙门氏菌增殖过程中的作用及分子调控机制,采用基因工程方法提取沙门氏菌的RNA,反转录成cDNA,然后利用实时荧光定量PCR技术(RT-qPCR)检测WT、WTΔyibT、WTΔyibT/pyibT的lptA-G和Lpt转运蛋白及csgD、rpoS的信使核糖核酸(mRNA)表达水平,研究yibT基因对沙门氏菌生物膜形成的影响.结果表明,yibT基因缺失后,可不同程度致使转运蛋白lptA-G的mRNA表达水平下调(P<0.05),其中lptB下调最为显著(P<0.01);yibT缺失可导致csgD表达量(P<0.05)和rpoS表达量显著下调(P<0.01);当yibT回补过表达后可致csgD、rpoS的mRNA表达水平均有不同程度的上调(P<0.05);同时,yibT基因的缺失可导致yecF和uspB的mRNA表达水平均不同程度的下降(P<0.05).因此,推测yibT基因可能通过参与介导脂多糖的转运与合成来影响鼠伤寒沙门氏菌的生物膜形成,其可能是一种全局转录调控因子或DNA聚合酶,通过影响菌体DNA的复制而调控鼠伤寒沙门氏菌的多个生物学过程.

结直肠癌(CRC)是全球第三大流行癌症,对人类的健康危害很大.大量研究证明,结直肠局部微生态改变与CRC的发生有关.通过宏基因组学及高通量测序技术运用,发现CRC患者的肿瘤微生态环境和粪便样本富集了大量具核梭杆菌.因此,具核梭杆菌被认为是CRC发生、发展的危险因素之一.迄今为止,具核梭杆菌引起CRC的发生机理还不清楚.论文从具核梭杆菌对大肠及CRC组织固有免疫细胞的抑制作用、具核梭杆菌特异性毒性因子对CRC发生的影响、具核梭杆菌的促癌性miRNA形成和具核梭杆菌促炎性因子对CRC细胞的影响等几个方面阐述了具核梭杆菌引起CRC的发生机理.

新城疫病毒(NDV)作为疫苗载体可以在宿主的呼吸道组织细胞中有效复制,诱导产生针对外来抗原的局部和全身免疫反应.NDV的基因组较大,可以很好地容纳外源基因.作为RNA病毒,与宿主细胞发生DNA重组的可能性较小.由于宿主范围的限制,人群中没有预先存在的NDV抗体,NDV对人类来说相对安全.因此,NDV作为一种疫苗载体颇受关注.论文简要论述了NDV的生物学特征,外源基因的插入方式,重点阐述了重组新城疫病毒载体疫苗的研究进展,以期为设计其他有效的兽用和人用活载体疫苗提供参考.

Chemokine CCL19, together with its receptor CCR7, is one of the most important factors recruiting immune cells into target organ during virus infection. Our previous study has shown that CCL19 played a vital role in the process of T cell trafficking into bursae during bursal disease virus (IBDV) infection. In this study, we hypothesized that CCL19 could exert direct influences on IBDV replication other than recruiting immune cells. A eukaryotic expression vector of pEGFP-N1/CCL19 was successfully constructed and identified by PCR, double enzymes digestion, and sequencing. Different concentrations of pEGFP-N1/CCL19 plasmids were transfected into DF1 cells and CCL19 protein was highly expressed. Then, DF1 cells were infected with IBDV B87 strain post-transfection. Based on PCR and Western blot results, CCL19 could obviously decrease the gene levels of VP1 and VP2 and the protein levels of VP2 and VP3. When CCL19 was knocked down, the gene levels of VP1 and VP2 were significantly upregulated. Moreover, indirect immunostaining revealed that the IBDV content was largely decreased after CCL19 overexpression. Additionally, CCL19 inhibitory effects might rely on activation of the JNK signal pathway. Taken together, chemokine CCL19 directly blocks IBDV replication in DF1 cells, indicating that CCL19 could play crucial functions other than recruiting T cells during the pathogenesis of IBDV.

为探究趋化因子配体19(CCL19)与新城疫病毒La Sota株重组后对新城疫病毒免疫原性的影响,试验利用反向遗传操作系统,将CCL19插入新城疫病毒疫苗株La Sota基因组磷蛋白和基质蛋白之间,经病毒拯救后获得重组病毒LS-CCL19.对LS-CCL19的EID50、MDT、ICPI等生物学特性进行测定,并通过对7日龄雏鸡进行接种测定抗体,检测其与La Sota株诱导抗体的变化.结果显示:拯救病毒LS-CCL19的EID50=101046/mL、MDT＞120 h、ICPI=O,HI抗体效价在二次免疫后7 d达到最高,低于La Sota株.研究表明,重组病毒LS-CCL19与La Sota株具有相似的生物学特性,能够诱导机体产生抗体,但抗体水平有待提升.