为调查广东省鸭圆环病毒(DuCV)流行情况,本研究根据已报道的PCR方法,对2018年12月-2020年7月采自广东省9个不同地区的910份临床鸭组织病料进行鸭圆环病毒(DuCV)、大肠杆菌(E.coli)、腺病毒(FAV)、细小病毒(DPV)、禽流感病毒(AIV)和鸭坦布苏病毒(DTUMV)检测.结果表明:DuCV总检出率为23.18％(211/910);2019年第三季度较高,为34.61％;DuCV单重感染为16.24％(32/197),二重感染和三重感染分别为62.43％(132/197)和21.31％(42/197).研究结果表明,DuCV在广东省鸭养殖场中存在不同程度的流行,夏季和冬季为多发,DuCV的感染会造成免疫抑制进而导致其他病原的感染,加重疾病的严重程度和死亡率.本研究调查结果为广东省养殖户了解广东地区DuCV的流行态势提供可支撑的数据.

依据大肠杆菌(Escherichia coli,E.coli)持家基因PhoA PCR检测,从临床病料中分离鉴定出11株鸭致病性E.coli.经O抗原血清型鉴定以及毒力分析,筛选出1株O78血清型鸭E.coli强毒株EC-KP120427.以此菌株为基础,使用单因素法以及正交法,获得O78血清型鸭E.coli强毒株新型高密度发酵培养基,配方中初糖采用甘油替代葡萄糖,无机盐仅添加磷酸氢二钾,同时补充铁、锰、铝3种微量元素,在普通摇床上37℃培养菌液D600nm可达到9.0以上.使用赛多利斯5L发酵罐,对EC-KP120427菌株进行发酵培养试验.使用优化的新型高密度发酵培养基,按1∶10比例接种对数生长期种子菌,菌液D600nm值为1.0、控制温度37℃、pH 7.0、调节空气通气量1.5～5.0 L/min、转速100～400 r/min、发酵至对数生长期以15 mL· h1·L-1流速慢速流加50％葡萄糖溶液,培养菌液D600nm值可高达30.0以上,成功实现了O78血清型鸭E.coli强毒株高密度发酵.本研究为致病性E.coli高密度发酵技术标准的建立奠定基础,同时为水禽E.coli细菌疫苗的改进提供了技术支持.

鸭坦布苏病毒病是由鸭坦布苏病毒(Duck tembusu virus,DTMUV)引起的一种以产蛋量和采食量严重下降为主要特征的传染病.应用RT-PCR方法扩增DTMUV GDHD2014-3毒株的囊膜(E)蛋白全基因序列,并将其克隆至原核表达载体pMAL-c5x,构建重组表达质粒E-pMAL.将其转化E.coli BL21感受态细胞,经诱导表达条件优化,在30℃条件下IPTG诱导,可溶性表达重组的全长E蛋白(rf E),大小约110 ku.用Amylose Resin对目的蛋白进行纯化,并将纯化的rf E蛋白免疫Balb/c小鼠,制备多克隆血清抗体.经Nu-PAGE以及Western blot分析,成功实现rf E蛋白可溶性表达,并能与DTMUV灭活疫苗免疫鸭血清多抗产生特异性反应.间接免疫荧光试验结果表明,rf E蛋白免疫小鼠获取的多克隆血清抗体能与DTMUV反应.实现了rf E蛋白的可溶性表达及纯化,并验证rf E蛋白具有良好的免疫原性,为DT-MUV的亚单位疫苗的研制以及相关诊断试剂的开发奠定了基础.

采集广东省3个城市、4个马场的马厩、训练场、打圈场及过道中的土壤、沙粒样本共计355份,采用优化改良的菌落印记方法对广东省部分马场土壤马红球菌(Rhodococcus equi,R.equi)进行分布调查.结果 显示,马场土壤中普遍存在R.equi,其平均菌量达106 CFU/g;训练场和打圈场的R.equi菌量和分离率都相对较高,而马场和季度之间的R.equi菌量和分离率均无明显差异,表明R.equi在马场土壤中的分布与马匹活动有关,与季节、地理位置等其他因素无明显相关性;因此,马场应注意马匹活动区域的定期清洁和消毒.本研究通过系统的流行病学方法进行调查与分析,为R.equi防控及治疗提供理论基础和依据,并为我国R.equi种质资源库的建立提供基本资源,具有重要的公共卫生学意义.

鸭细小病毒病是由鹅细小病毒(Goose parvovirus,GPV)或番鸭细小病毒(Muscovy duck parvovirus,MDPV)引起的一种传染病.经典MDPV和GPV毒株引起的病鸭主要症状为腹泻、脚软、渗出性肠炎,三周龄内雏鸭感染发病率和死亡率都很高.但是2008年下半年以来,福建省、浙江省、安徽省及江苏省等地的雏半番鸭和樱桃谷鸭陆续出现一种新型细小病毒病,该病发病率10％～30％,病死率低于3％,临床症状主要为软脚、短嘴和生长障碍.通过对该病病原进行全基因组测序及系统发育树分析,结果发现该病原与鹅细小病毒亲缘性很近.本文通过比较新型鹅细小病毒(Novel goose parvovirus,N-GPV)与经典的MDPV和GPV在基因组、感染宿主范围和致病性的区别,为新型鹅细小病毒病的防控提供理论依据.

Since early 2015, mule duck and Cherry Valley duck flocks have been suffering from short beak and dwarfism syndrome. This widely spreading infectious disease is characterized by growth retardation, smaller beak and tarsus with high morbidity and low mortality rate. For better understanding, we identified and characterized virus isolates named AH and GD from diseased Cherry Valley duck and mule duck flocks and investigated the damage caused by novel parvovirus-related virus (NGPV) to tissues and organs, including kidney, brain, pancreas, liver, spleen, bursa of fabricius and myocardial tissues.

Iron is an important growth factor of pathogenic bacterium, the research shows that the iron uptake system regulates Riemerella anatipestifer iron metabolism and is one of the important pathogenic factor of Riemerella anatipestifer. In this study, the relationship between Riemerella anatipestifer ATP dependent outer membrane siderophore receptor protein(SRP) and iron metabolism, bacterial virulence factor was investigated. The mutant of SRP protein was constructed and the growth curve test showed that the growth of the mutant strain was slower about 10 hours than that of wild type strain in the iron limited environment, it indicated that SRP protein was one of the essential factors of Riemerella anatipestifer infection and had a vital role in maintaining Riemerella anatipestifer iron metabolism in vivo . The animal experiment showed that the dead time of duck infected the mutant was later 24 hours than that of the wild type strain. It showed that the growth rate of mutant strain and the speed reaching lethal number was obviously later than the wild type. Thus it indicated that SRP was one of the important virulence factor in Riemerella anatipestifer.

Rhodococcus equi is a Gram positive facultative intracellular pathogen,which mainly causes subacute or chronic abscessating bronchopneumonia of foals in foals up to 6 months.So far,the study of R.equi in China is rarely seen,due to lack of the standard pathogenic survey method.To establish a rapid assay for R.equi detection,a colony blot assay was developed with the Rabbit anti-avirulence R.equi polyclonal antibody and Rabbit anti-virulence R.equi VapA polyclonal antibody.After optimizing the reaction conditions,the colony blot was able to specifically detect R.equi and efficiently distinguished the virulence from avirulence R.equi.Compared with R.equi PCR method,the colony blot test had the advantages of high sensitivity and specificity.The established assay could be applied to investigate the R.equi epidemiology in the environment.

为建立检测马红球菌(R.equi)抗体的方法,本研究对致病性R.equi的标志性抗原毒力相关脂蛋白A(VapA)进行融合表达,并以纯化的VapA重组蛋白作为包被抗原,通过优化反应条件,建立了R.equi血清VapA抗体间接ELISA检测方法.结果表明:该方法仅与马VapA阳性血清发生特异性反应,而与H3N8马流感、马鼻肺炎、马传染性贫血抗血清无交叉反应,具有较强的特异性;并且其批内和批间变异系数均小于15％,具有较好的重复性和稳定性.该检测方法的建立可以应用于我国马群R.equi血清流行病学的调查,为我国马病防控平台提供基础依据和数据支撑.

马鼻肺炎(Equine rhinopneumonitis,ER)是由马疱疹病毒l型(Equineherpesvirus1,EHV-1)和马疱疹病毒4型(Equineherpesvirus4,EHV-4)引起的一种马属动物急性、热性传染病.临床表现为发热、白细胞减少、呼吸道症状、妊娠马流产、胎儿死亡以及脑脊髓炎等症状.自Dimock等[1]于1933年在美国首次报道以来,已有40多个国家或地区相继发现该病,如:日本、土耳其、波兰、阿根廷等[2-4].