以重组PvpA蛋白作为抗原的鸡毒支原体抗体间接ELISA检测方法的建立

为建立鸡毒支原体(MG)种特异性检测的间接ELISA检测方法,本研究以原核表达的PvpA蛋白作为包被抗原,初步建立了检测MG抗体的间接ELISA检测方法.特异性试验结果表明,重组PvpA蛋白与鸡新城疫病毒、鸡传染性支气管炎病毒、滑液支原体、大肠杆菌“O”抗原、禽沙门氏茵“O”抗原阳性血清均不发生交叉反应.该方法的批内变异系数小于8％,批间变异系数小于11％,表明具有较好的重复性.采用该检测方法与进口试剂盒对不同省份送检的鸡血清进行检测比较,两者阳性和阴性符合率均达到90％以上.本研究建立的间接ELISA检测方法为MG抗体检测试剂盒的研制奠定了基础.     

检测犬源狂犬病毒中和抗体ELISA方法的建立

为了有效监测犬免疫狂犬病疫苗后的保护效力,以狂犬病毒(Rabies virus,RV)糖蛋白的主要优势抗原表位区G3蛋白(RV G3)作为包被抗原,建立了一种检测狂犬病毒中和抗体效价的间接ELISA方法.通过优化反应条件,确定抗原最佳包被量为8 mg/L,血清的最佳稀释度为1∶100,酶标二抗的稀释度为1∶3 000.特异性试验表明,该抗原不与犬瘟热病毒、犬腺病毒、犬细小病毒阳性血清发生交叉反应;批内和批间重复性试验的平均变异系数都小于10%;敏感性达1∶1 280.此方法检测134份血清样品的结果与美国SYNBIOTICS试剂盒相比,总符合率达95.6%.     

间接ELISA检测抗传染性法氏囊病病毒抗体方法的建立

采用原核表达的传染性法氏囊病毒(IBDV)核衣壳蛋白VP2作为诊断抗原,建立检测IBD血清抗体的间接ELISA方法.抗原最佳包被量为每孔174.67ng,待检血清最佳稀释度为1:40,待检血清样品的D492nm≥0.43Dpos+0.57Dneg判为阳性,反之判为阴性.对采自安徽省部分地区的979鸡血清样品进行检测,IBDV抗体阳性率为39.73%.结果证实,间接ELISA法用于IBDV血清抗体的监测具有良好的特异性,是一种快速简便的血清学方法,值得在基层推广应用.     

O型口蹄疫疫苗免疫家畜抗体水平的检测

准确监测是防控口蹄疫的重要环节。采集某地区乡镇规模化养殖场及散养户的牛、羊、猪血清,采用正向间接血凝试验进行O型口蹄疫疫苗免疫抗体水平的检测。结果显示,该地区牛、羊的口蹄疫疫苗免疫合格率平均为76%,猪的口蹄疫疫苗免疫合格率平均为60％;规模化养殖场较个体散养户免疫效果好;牛口蹄疫O型灭活苗的免疫效果好于牛口蹄疫Asia I-O型双价灭活疫苗的免疫效果。     

产蛋期蛋鸡不同类型玉米净能研究

本试验利用禽用开放式呼吸测热装置进行能量代谢试验,通过间接测热法结合替代法测定不同类型玉米在产蛋期蛋鸡饲粮中的表观代谢能和净能。选用34周龄产蛋期海兰褐蛋鸡180只,随机分为6组,每组30只。试验选用1种玉米-豆粕型基础饲粮和5种待测饲粮。待测饲粮由5种待测玉米(3种2018年10月收获的正常玉米和2种2016年收获储存3年的陈化玉米),分别以50%比例替代基础饲粮构成。试验鸡在舍内笼养,预试期7 d,正试期27 d,其中正试期分为3期,每期9 d(适应3 d、呼吸测热3 d、绝食测热3 d)。每期试验中,从每组中选择4只试验鸡,称重后分别放入呼吸测热装置的12个代谢室(每个代谢室2只),每2个代谢室对应1种饲粮,测定气体交换和排泄物总量,呼吸测热的同时进行消化代谢试验。结果表明:与基础饲粮相比,5种玉米待测饲粮的表观代谢能显著提高(P<0.05),3种正常玉米待测饲粮的净能显著高于基础饲粮和2种陈化玉米待测饲粮(P<0.05);2种陈化玉米的表观代谢能和净能显著低于3种正常玉米(P<0.05)。本试验中,3种正常玉米的表观代谢能分别为16.19、15.85、16.17 MJ/kg,2种陈化玉米的表观代谢能分别为15.12和15.06 MJ/kg;3种正常玉米的净能分别为12.39、12.57、12.25 MJ/kg,2种陈化玉米的净能分别为11.29和12.05 MJ/kg。     

羊弓形虫病间接ELISA诊断方法的建立

为比较5种弓形虫抗原的诊断效果,建立实用的羊弓形虫病诊断方法,本研究制备了弓形虫速殖子全虫抗原和4个重组蛋白(GRA1、MIC3、MAG、BAG1),正交试验比较其免疫反应性,从中筛选出相对优势抗原并建立羊弓形虫病间接ELISA(iELISA)诊断方法,用于检测弓形虫特异的IgG抗体。研究结果表明：除BAG1外上述4种抗原均具有较好的免疫反应性,综合考虑选择致密颗粒蛋白1(GRA1)作为诊断抗原建立了GRA1-iELISA,其检测弓形虫抗体灵敏度大于1∶100(血清稀释度),且不与羊莫氏巴贝斯虫、吕氏泰勒虫、捻转血毛线虫阳性血清发生交叉反应,特异性良好;运用建立的方法和改良凝集试验分别对采自山羊屠宰场的80份血清样品进行检测,测得的弓形虫感染阳性率分别为28.75%(23/80)和31.25%(25/80),二者总符合率为82.5%。本研究建立的ELISA检测方法对羊弓形虫病的诊断以及商业化试剂盒的开发具有重要参考价值。     

Callose and β‐1,3‐glucanase inhibit Phytophthora cinnamomi in a resistant avocado rootstock

Phytophthora root rot (PRR) of avocado, caused by Phytophthora cinnamomi, is a significant threat to sustainable production wherever the crop is grown. Resistant rootstocks in combination with phosphite applications are the most effective options for managing this disease. Recently, the mechanisms underpinning PRR resistance have been investigated by the avocado community. Here, biochemical assays and confocal and scanning electron microscopy were used to investigate early defence responses in PRR resistant and ‐susceptible avocado rootstocks. Zoospore germination and subsequent hyphal growth for the pathogen were significantly inhibited on the surface of resistant avocado roots. When penetration occurred in the resistant R0.06 rootstock, callose was deposited in the epidermal cells, parenchyma and cortex of roots. In addition, β‐1,3‐glucanase was released early (6 h post‐inoculation, hpi) in response to the pathogen, followed by a significant increase in catalase by 24 hpi. In contrast, susceptible R0.12 roots responded only with the deposition of lignin and phenolic compounds incapable of impeding pathogen colonization. In this study, PRR resistance was attributed to a timely multilayered response to infection by P. cinnamomi.     

Development of a Pseudomonas syringae–Eutrema salsugineum pathosystem to investigate disease resistance in a stress tolerant extremophile model plant

To improve the ability to understand how plants respond to multiple and/or concurrent stresses, disease resistance was investigated in Eutrema salsugineum, an extremophile model plant that is highly tolerant of abiotic stress. Compared to Arabidopsis (Col‐0), both Yukon and Shandong Eutrema accessions exhibit increased resistance to Pseudomonas syringae pv. tomato DC3000 (Pst) and pv. maculicola (Psm), with Shandong Eutrema exhibiting greater resistance to Pst than Yukon Eutrema. RT‐PCR of the EsPR1 (Pathogenesis‐related 1) defence marker gene confirmed RNA‐Seq data that healthy Shandong Eutrema constitutively expresses EsPR1. The data suggests that Shandong Eutrema exists in a highly primed state of defence preparedness, as it displays heightened resistance compared to defence‐primed natural accessions of Arabidopsis (Can‐0, Bur‐0). Pathogen‐triggered PR1 expression was delayed in Yukon Eutrema; however, these plants were resistant to Pst suggesting that Yukon Eutrema employs a PR1‐independent mechanism to resist Pst. This study demonstrates that Eutrema is an excellent model to investigate biotic stress tolerance. The Eutrema–P. syringae pathosystem will facilitate future studies to understand how this extremophile tolerates both abiotic and biotic stress, and will allow exploration of the interplay of these responses to inform efforts to improve stress tolerance in crops.