本文主要研究内容
作者张益（2019）在《鸭主要病毒性疾病流行病学调查分析》一文中研究指出：随着养鸭业的快速发展,中国鸭饲养量平均每年以10%-15%的速度递增,由于养殖模式改变、养殖数量和饲养密度不断增加以及各省市间频繁的活禽调运,鸭病出现“老病未除,新病又起”的复杂局面,对鸭的危害较大,造成较大经济损失,制约了养鸭业的健康发展。鸭病毒病的相关研究大多集中于对发病鸭或鸭群的零星报道,而对鸭整体病毒病的流行情况不甚了解,尤其对贵州省鸭群的病毒性疾病感染情况更鲜有报道。因此,本文采用文献资料查阅的方法掌握鸭主要病毒病的研究概况;采用血清学方法检测感染抗体、PCR/RT-PCR方法检测病原核酸掌握贵州省鸭病毒性疾病的感染状况;对贵州省新发的病毒性疫病进行了诊断、扩增病毒全基因组进行分子流行病学调查,以期为鸭病毒性疫病的流行感染状况提供基础数据,为进一步制定全面的监测计划及科学防控鸭病毒性疫病提供参考。1.鸭病毒性疫病研究概况文献资料查阅分析以web of science数据库为文献来源(1950-2019年),采用文献计量学方法对鸭病毒性疾病领域的全球发文趋势和文献产出量进行分析;采用VOSviewer软件提取关键词聚类与可视化功能,绘制鸭9种病毒性疾病(AIV、DHV、NDV、DPV、GPV、DRV、DTUMV、MDPV和DuCV)可视化知识图谱;根据中国产业信息网、《中国畜牧兽医年鉴》相关信息,分析贵州省养鸭业基本概况。经文献计量学方法分析鸭流行病学领域和鸭9种病毒性疫病文献,文献产出量整体呈增长趋势,美国和中国发文量显著高于其他国家;《AVIAN DISEASES》是发文量最高的文献来源出版物;传染病学是鸭流行病学最主要的研究方向;鸭坦布苏病毒病是2010年左右新发的病毒病,且主要疫区在中国;H5和H7型高致病禽流感、畜牧分布地图的研究方法、可能与其先天免疫有关的的RIG-1基因是现阶段鸭流行病学领域的研究热点。经VOSviewer软件分别对9种鸭病毒性疾病进行可视化知识图谱分析:鸭禽流感的主要疫区是中国、埃及、朝鲜、日本,常用检测方法是ELISA和RT-PCR;鸭病毒性肝炎主要疫区是中国,RT-PCR是最常用检测方法;鸭新城疫主要疫区是中国和韩国,易与禽流感病毒混合感染,特别是高致病型禽流感H5N1;鸭瘟常用的检测方法是ELISA和PCR;鹅细小病毒病主要疫区是中国和台湾,易和大肠杆菌混合感染,鸭源新型鹅细小病毒正在被广泛关注;鸭坦布苏病毒病和日本脑炎病毒有一定关系,蚊子可能和坦布苏病毒病的传播有关;呼肠孤病毒有MDRV和NDRV两种基因型,目前以MDRV基因型为主;鸭短喙侏儒综合征和番鸭细小病毒病有一定关系,番鸭细小病毒病主要疫区是中国,易和大肠杆菌混合感染;鸭圆环病毒病易和鸭瘟混合感染,和其关系比较近的是鹅圆环病毒。经中国产业网、中国畜牧兽医年鉴相关数据和现场疫情调查:全国31个地区,有种鸭场地区27个,贵州种鸭场存栏量位居全国第27名;贵州省养鸭场仅269家,三穗县是贵州省鸭养殖业的主产区。以上结果为鸭流行病学及病毒性疾病的研究提供了基础数据。2.贵州省鸭主要病毒性疫病现场流行病学调查分析对发病养鸭场(7个养殖场共28230羽发病鸭群)进行现场疫情调查;对三穗县养鸭场及其他发病鸭场抽样采集病(死)鸭血液样本168份,采用血凝与血凝抑制试验检测未免疫鸭血清中H9亚型AIV和NDV抗体,采用ELISA检测试剂盒检测未免疫鸭血清中GPV、DHV、DRV、DTMUV、MDPV和DuCV抗体;抽样采集病(死)鸭内脏组织样本68份,采用建立的GPV-MDPV-DPV多重PCR方法、AIV通用型RT-PCR检测试剂盒和其他病原的单重PCR方法检测鸭9种主要病毒性疫病病原核酸。对7个发病鸭场调查:贵州省养鸭场集约化程度不高,规模不大,发病鸭主要是花边鸭、樱桃谷鸭和三穗麻鸭,养鸭场主要免疫接种了禽流感H5、H7和鸭瘟疫苗。临床上神经、呼吸、食欲、个体大小、眼部、喙部均有出现病理表现;剖解时胸骨、皮下、心脏、肝脏、胰腺、脾脏、胆囊、肺脏、肾脏、脑部、气管、食道、腺胃、肠浆膜面、泄殖腔均有病理变化。血清学方法检测出NDV感染抗体阳性率为47.62%(80/168),DuCV感染抗体阳性率为22.61%(38/168),DTMUV感染抗体阳性率8.93%(15/168),H9型AIV感染抗体阳性率为7.14%(12/168),而DHV、DRV、MDPV和GPV感染抗体检测结果均为阴性。建立了GPV-MDPV-DPV多重PCR方法,优化最佳引物浓度分别为GPV NS 0.3μmol/L、MDPV VP1μmol/L、DPV UL6μmol/L,最佳退火温度为54.1℃,对3种病毒核酸的最小检出量分别为GPV 22.45 pg、MDPV 204.6pg和DPV 237.3 pg;采用GPV-MDPV-DPV多重PCR方法、AIV通用型RT-PCR检测试剂盒和其他病原的单重PCR方法对68份临床样本进行9种病原核酸检测,NDV特异性核酸片段检出率为32.35%(22/68),AIV特异性核酸片段得检出率为20.59%(14/68),GPV VP3基因特异性核酸片段检出率为11.76%(8/68),DuCV特异性核酸片段检出率为7.35%(5/68),DPV特异性核酸片段检出率为5.88%(4/68),而DHV、TMUV、DRV、GPV NS基因和MDPV特异性核酸片段均未被检测出。3.鸭“短喙-侏儒综合征”诊断及新型鹅细小病毒全基因组测序为对导致鸭疑似“短喙-侏儒综合征”的病原进行检测,对患病鸭进行临床症状、解剖大体病理变化观察、细菌分离培养、PCR检测病原核酸,然后基于PCR检测结果进行病毒全基因扩增和测序分析。结果:患病鸭生长缓慢、个体矮小、被毛凌乱、鸭上喙不同程度变短且舌头外露下垂、排绿色或白色稀粪等临床症状;解剖可见胸骨易折、心包积液、出血、肝土黄、脾脏肿大并伴有点状出血、胆囊肿大、肺脏充血和出血、腺胃水肿伴有点状出血、肾脏肿大、充血、出血等大体病理变化;内脏样本中未分离出细菌;对病料进行9种病毒性疫病病原核酸检测,仅检测出GPV VP3基因661bp特异性核苷酸片段,且与NCBI上公布的15株GPV的同源性高于97%;合成鹅细小病毒引物进行分段扩增,经序列拼接获得4988 bp的核苷酸序列;核苷酸同源性比对结果显示,与2015年以来公布的7株新型鹅细小病毒(Novel goose parvovirus,N-GPV)分离株中SDHZ1604株、SDDY1605株和AH1606株的核苷酸的同源性最高,为97.0%,与另外4株SDLC01、JS1603、QH15和M15的同源性分别为96.7%、96.6%、96.3%和93.9%;与2012年经典分离株GPV LH的同源性为93.3%;与2015年以前地方分离株的同源性为90.4%~94.4%;与MDPV的同源性在73.2%~84.1%之间,与2015年MDPV P1分离株的核苷酸同源性为73.1%;遗传进化分析结果显示,与15株GPV毒株均处在同一大的分支,并且与AH1606、JS1603、QH15、SDDY1605、SDHZ1604和SDLC01等6株2015年以来的GPV分离株处于同一小的分支;与5株MDPV毒株均处在不同的进化分支。结论:经文献调查分析,鸭主要病毒性疫病的文献产出量整体呈增长趋势,鸭坦布苏病毒病和鸭源新型鹅细小病毒病是新发病,鸭禽流感是鸭病毒性疾病领域研究热点,鸭的病毒病之间混合感染和与细菌病混合感染的情况比较常见,鸭病毒性疾病最常用的检测技术是PCR/RT-PCR和ELISA。血清学方法检测出NDV、DuCV、DTMUV和H9型AIV感染抗体;PCR/RT-PCR方法检测出NDV、AIV、GPV、DuCV和DPV病原核酸。临床上短喙、长舌、侏儒病鸭内脏样本检测出鹅细小病毒核酸,获得鹅细小病毒全基因组序列,经比对及遗传进化分析,明确该病毒为鸭源新型鹅细小病毒(Novel goose parvovirus,N-GPV)。
Abstract
sui zhao yang ya ye de kuai su fa zhan ,zhong guo ya si yang liang ping jun mei nian yi 10%-15%de su du di zeng ,you yu yang shi mo shi gai bian 、yang shi shu liang he si yang mi du bu duan zeng jia yi ji ge sheng shi jian pin fan de huo qin diao yun ,ya bing chu xian “lao bing wei chu ,xin bing you qi ”de fu za ju mian ,dui ya de wei hai jiao da ,zao cheng jiao da jing ji sun shi ,zhi yao le yang ya ye de jian kang fa zhan 。ya bing du bing de xiang guan yan jiu da duo ji zhong yu dui fa bing ya huo ya qun de ling xing bao dao ,er dui ya zheng ti bing du bing de liu hang qing kuang bu shen le jie ,you ji dui gui zhou sheng ya qun de bing du xing ji bing gan ran qing kuang geng xian you bao dao 。yin ci ,ben wen cai yong wen suo zi liao cha yue de fang fa zhang wo ya zhu yao bing du bing de yan jiu gai kuang ;cai yong xie qing xue fang fa jian ce gan ran kang ti 、PCR/RT-PCRfang fa jian ce bing yuan he suan zhang wo gui zhou sheng ya bing du xing ji bing de gan ran zhuang kuang ;dui gui zhou sheng xin fa de bing du xing yi bing jin hang le zhen duan 、kuo zeng bing du quan ji yin zu jin hang fen zi liu hang bing xue diao cha ,yi ji wei ya bing du xing yi bing de liu hang gan ran zhuang kuang di gong ji chu shu ju ,wei jin yi bu zhi ding quan mian de jian ce ji hua ji ke xue fang kong ya bing du xing yi bing di gong can kao 。1.ya bing du xing yi bing yan jiu gai kuang wen suo zi liao cha yue fen xi yi web of scienceshu ju ku wei wen suo lai yuan (1950-2019nian ),cai yong wen suo ji liang xue fang fa dui ya bing du xing ji bing ling yu de quan qiu fa wen qu shi he wen suo chan chu liang jin hang fen xi ;cai yong VOSviewerruan jian di qu guan jian ci ju lei yu ke shi hua gong neng ,hui zhi ya 9chong bing du xing ji bing (AIV、DHV、NDV、DPV、GPV、DRV、DTUMV、MDPVhe DuCV)ke shi hua zhi shi tu pu ;gen ju zhong guo chan ye xin xi wang 、《zhong guo chu mu shou yi nian jian 》xiang guan xin xi ,fen xi gui zhou sheng yang ya ye ji ben gai kuang 。jing wen suo ji liang xue fang fa fen xi ya liu hang bing xue ling yu he ya 9chong bing du xing yi bing wen suo ,wen suo chan chu liang zheng ti cheng zeng chang qu shi ,mei guo he zhong guo fa wen liang xian zhe gao yu ji ta guo jia ;《AVIAN DISEASES》shi fa wen liang zui gao de wen suo lai yuan chu ban wu ;chuan ran bing xue shi ya liu hang bing xue zui zhu yao de yan jiu fang xiang ;ya tan bu su bing du bing shi 2010nian zuo you xin fa de bing du bing ,ju zhu yao yi ou zai zhong guo ;H5he H7xing gao zhi bing qin liu gan 、chu mu fen bu de tu de yan jiu fang fa 、ke neng yu ji xian tian mian yi you guan de de RIG-1ji yin shi xian jie duan ya liu hang bing xue ling yu de yan jiu re dian 。jing VOSviewerruan jian fen bie dui 9chong ya bing du xing ji bing jin hang ke shi hua zhi shi tu pu fen xi :ya qin liu gan de zhu yao yi ou shi zhong guo 、ai ji 、chao xian 、ri ben ,chang yong jian ce fang fa shi ELISAhe RT-PCR;ya bing du xing gan yan zhu yao yi ou shi zhong guo ,RT-PCRshi zui chang yong jian ce fang fa ;ya xin cheng yi zhu yao yi ou shi zhong guo he han guo ,yi yu qin liu gan bing du hun ge gan ran ,te bie shi gao zhi bing xing qin liu gan H5N1;ya wen chang yong de jian ce fang fa shi ELISAhe PCR;e xi xiao bing du bing zhu yao yi ou shi zhong guo he tai wan ,yi he da chang gan jun hun ge gan ran ,ya yuan xin xing e xi xiao bing du zheng zai bei an fan guan zhu ;ya tan bu su bing du bing he ri ben nao yan bing du you yi ding guan ji ,wen zi ke neng he tan bu su bing du bing de chuan bo you guan ;hu chang gu bing du you MDRVhe NDRVliang chong ji yin xing ,mu qian yi MDRVji yin xing wei zhu ;ya duan hui zhu ru zeng ge zheng he fan ya xi xiao bing du bing you yi ding guan ji ,fan ya xi xiao bing du bing zhu yao yi ou shi zhong guo ,yi he da chang gan jun hun ge gan ran ;ya yuan huan bing du bing yi he ya wen hun ge gan ran ,he ji guan ji bi jiao jin de shi e yuan huan bing du 。jing zhong guo chan ye wang 、zhong guo chu mu shou yi nian jian xiang guan shu ju he xian chang yi qing diao cha :quan guo 31ge de ou ,you chong ya chang de ou 27ge ,gui zhou chong ya chang cun lan liang wei ju quan guo di 27ming ;gui zhou sheng yang ya chang jin 269jia ,san sui xian shi gui zhou sheng ya yang shi ye de zhu chan ou 。yi shang jie guo wei ya liu hang bing xue ji bing du xing ji bing de yan jiu di gong le ji chu shu ju 。2.gui zhou sheng ya zhu yao bing du xing yi bing xian chang liu hang bing xue diao cha fen xi dui fa bing yang ya chang (7ge yang shi chang gong 28230yu fa bing ya qun )jin hang xian chang yi qing diao cha ;dui san sui xian yang ya chang ji ji ta fa bing ya chang chou yang cai ji bing (si )ya xie ye yang ben 168fen ,cai yong xie ning yu xie ning yi zhi shi yan jian ce wei mian yi ya xie qing zhong H9ya xing AIVhe NDVkang ti ,cai yong ELISAjian ce shi ji he jian ce wei mian yi ya xie qing zhong GPV、DHV、DRV、DTMUV、MDPVhe DuCVkang ti ;chou yang cai ji bing (si )ya nei zang zu zhi yang ben 68fen ,cai yong jian li de GPV-MDPV-DPVduo chong PCRfang fa 、AIVtong yong xing RT-PCRjian ce shi ji he he ji ta bing yuan de chan chong PCRfang fa jian ce ya 9chong zhu yao bing du xing yi bing bing yuan he suan 。dui 7ge fa bing ya chang diao cha :gui zhou sheng yang ya chang ji yao hua cheng du bu gao ,gui mo bu da ,fa bing ya zhu yao shi hua bian ya 、ying tao gu ya he san sui ma ya ,yang ya chang zhu yao mian yi jie chong le qin liu gan H5、H7he ya wen yi miao 。lin chuang shang shen jing 、hu xi 、shi yu 、ge ti da xiao 、yan bu 、hui bu jun you chu xian bing li biao xian ;pou jie shi xiong gu 、pi xia 、xin zang 、gan zang 、yi xian 、pi zang 、dan nang 、fei zang 、shen zang 、nao bu 、qi guan 、shi dao 、xian wei 、chang jiang mo mian 、xie shi qiang jun you bing li bian hua 。xie qing xue fang fa jian ce chu NDVgan ran kang ti yang xing lv wei 47.62%(80/168),DuCVgan ran kang ti yang xing lv wei 22.61%(38/168),DTMUVgan ran kang ti yang xing lv 8.93%(15/168),H9xing AIVgan ran kang ti yang xing lv wei 7.14%(12/168),er DHV、DRV、MDPVhe GPVgan ran kang ti jian ce jie guo jun wei yin xing 。jian li le GPV-MDPV-DPVduo chong PCRfang fa ,you hua zui jia yin wu nong du fen bie wei GPV NS 0.3μmol/L、MDPV VP1μmol/L、DPV UL6μmol/L,zui jia tui huo wen du wei 54.1℃,dui 3chong bing du he suan de zui xiao jian chu liang fen bie wei GPV 22.45 pg、MDPV 204.6pghe DPV 237.3 pg;cai yong GPV-MDPV-DPVduo chong PCRfang fa 、AIVtong yong xing RT-PCRjian ce shi ji he he ji ta bing yuan de chan chong PCRfang fa dui 68fen lin chuang yang ben jin hang 9chong bing yuan he suan jian ce ,NDVte yi xing he suan pian duan jian chu lv wei 32.35%(22/68),AIVte yi xing he suan pian duan de jian chu lv wei 20.59%(14/68),GPV VP3ji yin te yi xing he suan pian duan jian chu lv wei 11.76%(8/68),DuCVte yi xing he suan pian duan jian chu lv wei 7.35%(5/68),DPVte yi xing he suan pian duan jian chu lv wei 5.88%(4/68),er DHV、TMUV、DRV、GPV NSji yin he MDPVte yi xing he suan pian duan jun wei bei jian ce chu 。3.ya “duan hui -zhu ru zeng ge zheng ”zhen duan ji xin xing e xi xiao bing du quan ji yin zu ce xu wei dui dao zhi ya yi shi “duan hui -zhu ru zeng ge zheng ”de bing yuan jin hang jian ce ,dui huan bing ya jin hang lin chuang zheng zhuang 、jie pou da ti bing li bian hua guan cha 、xi jun fen li pei yang 、PCRjian ce bing yuan he suan ,ran hou ji yu PCRjian ce jie guo jin hang bing du quan ji yin kuo zeng he ce xu fen xi 。jie guo :huan bing ya sheng chang huan man 、ge ti ai xiao 、bei mao ling luan 、ya shang hui bu tong cheng du bian duan ju she tou wai lou xia chui 、pai lu se huo bai se xi fen deng lin chuang zheng zhuang ;jie pou ke jian xiong gu yi she 、xin bao ji ye 、chu xie 、gan tu huang 、pi zang zhong da bing ban you dian zhuang chu xie 、dan nang zhong da 、fei zang chong xie he chu xie 、xian wei shui zhong ban you dian zhuang chu xie 、shen zang zhong da 、chong xie 、chu xie deng da ti bing li bian hua ;nei zang yang ben zhong wei fen li chu xi jun ;dui bing liao jin hang 9chong bing du xing yi bing bing yuan he suan jian ce ,jin jian ce chu GPV VP3ji yin 661bpte yi xing he gan suan pian duan ,ju yu NCBIshang gong bu de 15zhu GPVde tong yuan xing gao yu 97%;ge cheng e xi xiao bing du yin wu jin hang fen duan kuo zeng ,jing xu lie pin jie huo de 4988 bpde he gan suan xu lie ;he gan suan tong yuan xing bi dui jie guo xian shi ,yu 2015nian yi lai gong bu de 7zhu xin xing e xi xiao bing du (Novel goose parvovirus,N-GPV)fen li zhu zhong SDHZ1604zhu 、SDDY1605zhu he AH1606zhu de he gan suan de tong yuan xing zui gao ,wei 97.0%,yu ling wai 4zhu SDLC01、JS1603、QH15he M15de tong yuan xing fen bie wei 96.7%、96.6%、96.3%he 93.9%;yu 2012nian jing dian fen li zhu GPV LHde tong yuan xing wei 93.3%;yu 2015nian yi qian de fang fen li zhu de tong yuan xing wei 90.4%~94.4%;yu MDPVde tong yuan xing zai 73.2%~84.1%zhi jian ,yu 2015nian MDPV P1fen li zhu de he gan suan tong yuan xing wei 73.1%;wei chuan jin hua fen xi jie guo xian shi ,yu 15zhu GPVdu zhu jun chu zai tong yi da de fen zhi ,bing ju yu AH1606、JS1603、QH15、SDDY1605、SDHZ1604he SDLC01deng 6zhu 2015nian yi lai de GPVfen li zhu chu yu tong yi xiao de fen zhi ;yu 5zhu MDPVdu zhu jun chu zai bu tong de jin hua fen zhi 。jie lun :jing wen suo diao cha fen xi ,ya zhu yao bing du xing yi bing de wen suo chan chu liang zheng ti cheng zeng chang qu shi ,ya tan bu su bing du bing he ya yuan xin xing e xi xiao bing du bing shi xin fa bing ,ya qin liu gan shi ya bing du xing ji bing ling yu yan jiu re dian ,ya de bing du bing zhi jian hun ge gan ran he yu xi jun bing hun ge gan ran de qing kuang bi jiao chang jian ,ya bing du xing ji bing zui chang yong de jian ce ji shu shi PCR/RT-PCRhe ELISA。xie qing xue fang fa jian ce chu NDV、DuCV、DTMUVhe H9xing AIVgan ran kang ti ;PCR/RT-PCRfang fa jian ce chu NDV、AIV、GPV、DuCVhe DPVbing yuan he suan 。lin chuang shang duan hui 、chang she 、zhu ru bing ya nei zang yang ben jian ce chu e xi xiao bing du he suan ,huo de e xi xiao bing du quan ji yin zu xu lie ,jing bi dui ji wei chuan jin hua fen xi ,ming que gai bing du wei ya yuan xin xing e xi xiao bing du (Novel goose parvovirus,N-GPV)。
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