绿头鸭卵黄硒含量与禽流感新城疫病毒抗体滴度关系探讨

绿头鸭(Anas platyrhynchos)是大庆龙凤湿地的优势物种,为研究绿头鸭卵黄硒含量与病毒抗体之间的相关关系,采用血凝抑制试验及原子荧光光谱法(AFS)检测105枚绿头鸭卵黄内硒含量及H1、H5、H9亚型禽流感病毒(AIV)和新城疫病毒(NDV)抗体。结果表明,卵黄硒含量为0.48μg/g±0.15μg/g;卵黄内H1、H5、H9亚型AIV和NDV抗体阳性率分别为:100%、59.05%、71.43%、68.57%;其评价抗体滴度分别为:6.17log2、2.89log2、3.34log2、4.22log2。经相关分析结果表明,卵黄内H1、H5、H9亚型AIV和NDV抗体滴度与卵黄硒含量的相关系数分别为:0.077、0.714、0.690、0.611。由此可见,龙凤湿地绿头鸭种群卵黄硒含量与相关病毒抗体之间呈明显的正相关,说明硒元素对动物机体抗病毒能力起到一定的增强作用。     

鲁北地区商品肉鸭AIV和NDV抗体效价的检测与分析

为了解鲁北地区商品肉鸭中禽流感病毒(AIV)和新城疫病毒(NDV)的母源抗体、免疫抗体消长规律,分析在养殖过程中潜在的疫病风险,选择了三个规模化鸭场,分别采集了不同日龄鸭的血液样品,通过血凝试验和血凝抑制试验检测了AIV H5亚型(Re-6,Re-8)、H9亚型、H7亚型和NDV的抗体情况。结果表明:三个鸭场的AIV H5亚型和NDV抗体效价均处于较高水平,说明AIV H5免疫后的抗体能够产生有效保护,AIV H9亚型母源抗体在肉鸭12日龄左右消失,存在被感染的隐患;仅有两份血清样品检测到AIV H7亚型抗体效价,其余都为0;NDV在鸭群中存在不同程度的隐性感染。建议做好对AIV和NDV的抗体监测工作,并为防控相关疾病做好技术与产品储备。     

重组禽流感病毒(H5+H7)二价灭活疫苗(H5N1 Re-8株+H7N9 H7-Re1株)对商品肉鸡的免疫效力研究

为评估重组禽流感病毒(H5+H7)二价灭活疫苗(H5N1 Re-8株+H7N9 H7-Re1株)对商品肉鸡的免疫保护效果,本研究将该疫苗按照现地免疫程序分别接种10日龄白羽肉鸡和14日龄黄羽肉鸡,疫苗接种前和接种后每周采血,分离血清,检测H5亚型Re-8株和H7亚型H7-Re1株禽流感病毒(AIV)HI抗体滴度,并在初次免疫后3周和出栏时以鼻腔感染方式分别进行攻毒试验。结果显示,免疫前,所有肉鸡血清中H7亚型H7-Re1株AIV HI抗体滴度均低于1log2,H5亚型Re-8株AIV平均HI抗体滴度分别为5.3log2(白羽肉鸡)和4.1log2(黄羽肉鸡);一次免疫的白羽肉鸡在免疫后3周至出栏时,2种亚型AIV平均HI抗体滴度在6.0log2~6.7log2;2次免疫的黄羽肉鸡在初次免疫后3周至出栏时,2种亚型AIV平均HI抗体滴度均在6.0log2以上。在一次免疫后3周时和出栏时,采用高致病性H5、H7亚型AIV攻毒后,对照组白羽肉鸡和黄羽肉鸡均全部死亡,免疫组白羽肉鸡和黄羽肉鸡均不发病、不排毒、不死亡,获得100%的免疫保护。本研究结果表明,重组AIV二价灭活疫苗对商品肉鸡具有良好的免疫效果,为该疫苗的现地应用提供了参考依据。     

禽流感病毒H9亚型灭活油乳剂疫苗的免疫试验

以低致病性禽流感病毒（AIV）H9N2亚型株的鸡胚适应毒经尿囊腔接种10日龄鸡胚，从24h后死亡胚及96h后4℃致冷死亡胚收集尿囊液，尿囊液的血凝价约7log2，每毫长约含10^8.6个ELD50的病毒。将尿囊液经0.1%福马林灭活后，用超滤器将尿囊液浓缩5倍后分别制成双联灭活疫苗。对4周龄SPF鸡肌肉注射0.5ml或0.3ml单联H9亚型AIV灭活苗，在接种后2、3、4、5周后，血清中对H9亚型AIV的血凝抑制（HI）抗体效价分别是5.1、7.2、8.3、10log2或3.8、6.5、6.3、8.31log2。注射0.5ml的H9亚型AIV和NDV二联油乳苗的鸡，在接种后2、3、4、5周对H9亚型AIV及Lasota株NDV的HI滴度分别是4.6、5.6、6.7、8log2及7、7.6、6.7、7log2。显然，二联苗对H9亚型AIV及NDV均产生了理想的免疫效果。     

贵州省HPAIV和NDV免疫抗体检测及其R语言分析

为探究贵州省不同地区、养殖方式、温度和海拔对H5亚型禽流感病毒(Avian influenza virus,AIV)、H7亚型AIV和新城疫病毒(Newcastle disease virus,NDV)免疫抗体阳性率的影响,采用血凝抑制试验对2015—2018年本实验室收集的贵州省各市(州)294个养殖场的鸡血清(共计11 017份)进行上述疫病的免疫抗体检测,并对检测结果使用易侕统计软件(www.empowerstats.com)和R语言进行单因素和多因素分析。统计结果显示,H5,H7亚型AIV和NDV的免疫抗体阳性率分别为84.80%,64.95%和90.48%。单因素分析结果显示,H5,H7亚型AIV免疫抗体阳性率在贵州省的不同地区中差异均极显著(P0.01);不同养殖方式对H5亚型AIV的免疫效果影响差异显著(P0.05);不同温度对H5亚型AIV的免疫效果影响差异极显著(P0.01);不同海拔对H5,H7亚型AIV和NDV的免疫效果影响不显著(P0.05)。多因素分析结果显示,综合考虑不同地区、养殖方式、温度和海拔4个因素对H5,H7亚型AIV和NDV免疫抗体阳性率的影响,只有H5亚型AIV的免疫效果受到显著影响(P0.05)。结果表明,贵州省H5亚型AIV和NDV的免疫抗体阳性率均达到国家农业部动物疫病监测合格要求;贵州省不同地区H5和H7亚型AIV免疫抗体阳性率存在极显著差异;不同养殖方式和温度对H5亚型AIV的免疫效果影响差异均显著;贵州省的平均海拔条件有利于H7亚型AIV和NDV的免疫防控。本研究为贵州省H5,H7亚型AIV和NDV疫苗合理免疫程序的制定提供一定的理论依据。     

H9N2亚型禽流感病毒NS1基因缺失减毒活疫苗候选株对蛋鸡的免疫效力评价

为评价H9N2亚型禽流感病毒(AIV)NS1基因缺失减毒活疫苗候选株r TX-NS1-128对蛋鸡的免疫效力,本研究对蛋鸡H9N2亚型AIV母源抗体半衰期、疫苗适宜免疫时间、抗体免疫保护临界值以及免疫鸡抗体消长规律等进行研究。结果显示,雏鸡H9亚型AIV母源血凝抑制(HI)抗体半衰期为3.29 d。雏鸡母源抗体水平对疫苗的免疫效果有较大的影响,以母源HI抗体滴度约为4 log2时免疫为宜。少数HI抗体滴度为4 log2~6 log2的免疫鸡攻毒后可在5 d或3 d内从咽喉短暂排毒,HI抗体滴度为4 log2、5 log2、6 log2和≥7 log2时攻毒保护率分别为80%、90%、90%和100%,建议该疫苗的抗体免疫保护临界值为5 log2。鸡一次免疫后平均HI抗体滴度在5 log2以上可持续9周左右。本研究为H9N2亚型禽流感新型减毒活疫苗的开发和应用提供依据。     

2008年～2012年山东胶东鸡场NDV IBV AIV(H9)感染情况调查

为了解2008年1月～2012年6月山东胶东青岛、潍坊和烟台三地区新城疫病毒(NDV)、传染性支气管炎病毒(IBV)和H9亚型禽流感病毒(AIV(H9))的感染情况,采用RT-PCR对送检的5 001份患有呼吸道症状的病料进行了检测.结果显示,3地区3种病毒的总阳性率为48.49％,其中AIV(H9)感染率最高,为26.15％.青岛地区NDV和IBV的阳性率均最高,分别为15.94％和11.63％,潍坊地区AIV(H9)阳性率最高(29.23％).4年半间AIV(H9)感染下降明显,但NDV近3年感染明显抬头.寒冷月份3种病毒感染较高,3月份NDV和AIV(H9)阳性率均最高,分别为21.07％和38.17％;10月份IBV阳性率最高为15.40％.3种病毒混合感染阳性率为6.66％,以NDV+ AIV(H9)二重感染最常见(2.98％),NDV+IBV+AIV(H9)三重感染为0.40％.结果表明,山东胶东地区NDV、IBV和AIV(H9)的感染情况较普遍,存在一定程度的混合感染,为有效防控山东省规模鸡场该3种主要疾病提供了依据.     

新疆青格达湖湿地鸟类中H5、H7亚型AIV和NDV流行病学监测

为了解新疆青格达湖湿地野鸟携带禽流感病毒(AIV)和新城疫病毒(NDV)情况,对青格达湖湿地鸟类进行了血样和喉肛棉拭子、组织、粪便样品采集。采用RT-PCR试验对喉肛棉拭子、组织、粪便样品进行了病原学检测,采用血凝(HA)和血凝抑制(HI)试验进行禽流感和新城疫血清抗体的检测,结果均为阴性。结果表明:所检测的样品并未携带AIV(H5、H7亚型)和NDV。     

鸡新城疫、禽流感(H9亚型)二联油乳剂灭活疫苗最小免疫剂量试验

将ND-AI(H9亚型)二联苗分别以1.0、0.5、0.2、0.1、0.05、0.02 mL 6个不同剂量免疫5周龄SPF鸡,免疫后3周采血测NDV及AIV 的HI抗体,并用H9亚型AIV及NDV F48株攻击.结果表明,这6个免疫组鸡的NDV HI抗体几何平均效价分别为2 10、2 8.8、2 8.5、2 7.0、2 6.1、2 5.7;AIV HI抗体水平分别为2 9.3、2 8.4、2 7.7、2 5.7、2 5.1、2 2.8;对照组鸡的NDV及AIV HI抗体均为零.攻毒试验证实,以0.1～1.0 mL剂量免疫鸡获得了100%的抵抗NDV及AIV强毒攻击的保护力;0.02 mL和 0.05 mL的免疫剂量也可使其对NDV与AIV的保护率达到90%～100%.     

山西地区鸡群禽流感和新城疫的免疫状况调查

采用血凝和血凝抑制试验,对山西文水、清徐、太谷、平遥、祁县、榆次、原平、大同、运城、太原等地区的养殖场及散养鸡群,在2007—2012年间的新城疫病毒(NDV)和禽流感病毒(AIV)(H9、H5)抗体监控和流行病学调查。结果表明,在监测的6年间,山西地区鸡的NDV和AIV(H9、H5)水平分别是(4.8-6.7)log2、(3.9-5.4)log2、(6.8-8.3)log2,都呈现缓慢的、波动上升的趋势;对太谷地区的监控,看出养殖人员对ND和AI(H5)的预防越来越重视,为养殖业的发展和疫病防控创造了条件。     

Serological and virological studies of Newcastle disease and avian influenza in slaughter-age ostriches (Struthio camelus) in Japan

Serum samples from 191 ostriches (Struthio camelus) in Japan were tested for antibodies to Newcastle disease virus (NDV) and avian influenza virus (AIV). Twenty-two (12%) contained NDV-specific neutralizing antibodies by a virus-neutralization (VN) test without vaccination. Antibodies to AIV were not detected in the any sera by an agar gel precipitation test. Seven serum samples that had vaccinated with live NDV by eye drop were all positive by the VN test at 1 month post vaccination. A haemagglutination inhibition (HI) test for NDV seemed not to be suitable for ostriches because of non-specific agglutination of chicken red blood cells. No haemagglutinating viruses were isolated. This is the first report on detection of antibodies against NDV in ostriches in Japan.     

Serologic survey of slaughter-age ostriches (Struthio camelus) for antibodies to selected avian pathogens

Serum samples from 163 slaughter-age ostriches (Struthio camelus) in Ohio and Indiana were tested for antibodies to avian influenza virus (AIV), Newcastle disease virus (NDV), paramyxovirus (PMV) 2, PMV3, PMV7, infectious bursal disease virus (IBDV), Bordetella avium, Mycoplasma synoviae, Mycoplasma gallisepticum, Ornithobacterium rhinotracheale, Salmonella pullorum, Salmonella gallinarum, and Salmonella typhimurium. One ostrich had antibodies to AIV H5N9, 57% of the ostriches had antibodies to NDV, four ostriches had antibodies to both NDV and PMV2, and one ostrich had antibodies to NDV, PMV2, PMV3, and PMV7. None of the ostriches had antibodies to IBDV, B. avium, M. synoviae, M. gallisepticum, O. rhinotracheale, S. pullorum, S. gallinarum, and S. typhimurium. This is the first report of antibodies to avian influenza and PMV7 in ostriches in the United States.     

鸡新城疫-禽流感二联灭活疫苗免疫不同日龄商品肉鸡的效果比较

使用鸡新城疫-禽流感（H9N2 HP株）二联灭活疫苗分别免疫3、7、14日龄三组商品肉鸡各40羽,同时设一组空白对照组。各免疫组及对照组于3、7、14、21、28、35、42日龄采血检测新城疫、禽流感抗体,于21、28、35日龄进行禽流感病毒攻毒,对比不同日龄免疫组的抗体消涨情况及不同日龄禽流感攻毒结果。发现对照组随鸡日龄增加,新城疫与禽流感抗体逐渐下降,在42日龄时下降至0,而不同免疫组新城疫与禽流感抗体均先下降,21日龄左右开始上升,至35日龄新城疫与禽流感抗体升至6log2以上。3日龄免疫组的禽流感免疫保护效果最好,21、28、35日龄时禽流感强毒攻毒保护率均达100%;7日龄免疫组在21、35日龄时禽流感强毒攻毒保护率均达100%,28日龄时禽流感强毒攻毒保护率达70%;14日龄免疫组在28、35日龄时禽流感强毒攻毒保护率均达100%,21日龄时禽流感强毒攻毒保护率只达30%。试验表明,商品肉鸡选择3日龄免疫鸡新城疫-禽流感（H9N2 HP株）二联灭活疫苗时禽流感免疫保护效果最好,采用3日龄免疫程序可以提高新城疫与禽流感的免疫保护效果,减少养殖业的经济损失。     

不同剂型新城疫-禽流感二联灭活疫苗免疫SPF鸡后抗体水平对比试验

不同剂型的佐剂对疫苗的免疫效果有较大的影响,本试验旨在对比不同剂型佐剂的新城疫-禽流感(简称"新-流")二联灭活疫苗的免疫效果,从而为生产上选择最佳剂型佐剂应用于新城疫-禽流流感二联灭活疫苗提供依据。分别将油包水型、水包油型、水包油包水型佐剂与新城疫及禽流感灭活抗原乳化成不同剂型的新城疫-禽流感(H9亚型,HP株)二联灭活疫苗,将这三种不同剂型的新城疫-禽流感(H9亚型,HP株)二联灭活疫苗分别免疫一组7日龄SPF鸡。每羽颈部皮下注射0.2 m L,同时设一组未免7日龄SPF鸡作为对照组,各免疫组与对照组SPF鸡于免疫组免后6、10、15、21、28、35 d进行采血,检测新城疫与禽流感抗体水平。结果表明:免疫油包水型疫苗组SPF鸡免疫后新城疫与禽流感抗体上升最快,在免后10、15、21、28、35 d的新城疫与禽流感抗体也最高,其次是免疫水包油包水型的,而免疫水包油型疫苗组的SPF鸡新城疫与禽流感抗体上升最慢,而且在免后10、15、21、28、35 d的新城疫与禽流感抗体也最低。此外,从各免疫组可以看出,在免后6 d时新城疫抗体已开始产生,在1log2以下,而禽流感抗体仍未产生。可见免疫新-流二联苗后,新城疫抗体比禽流感抗体更早产生,油包水型新-流二联苗的免后抗体高于水包油包水及水包油型,而且能持续刺激机体产生高水平抗体,更具优势。     

Evaluation of different methods of inactivation of Newcastle disease virus and avian influenza virus in egg fluids and serum

Viruses conveyed in shipments of eggs, viral diagnostic reagents, or avian serum samples are a potential hazard for susceptible poultry. Different methods of treatment of those materials to eliminate the hazard of virulent and avirulent strains of Newcastle disease virus (NDV) or avian influenza virus (AIV) were evaluated. The NDV strains tested were more thermostable than the AIV strains. The results suggest that standard pasteurization methods would not reliably inactivate the concentrations of NDV used. beta-Propiolactone (BPL) (greater than or equal to 0.025%) inactivated NDV or AIV in allantoic fluid, but higher concentrations were needed to inactivate virus diluted in serum. Hemagglutination (HA) of NDV and AIV and hemolysis (HL) activity of NDV were reduced or eliminated by 0.4% BPL. Formalin (greater than or equal to 0.04%) inactivated either virus but adversely affected HA and HL activity. NDV or AIV was inactivated by binary ethylenimine (BEI) (0.01 M) with no adverse effect on HA or HL. Heat (56 C) or BEI (0.01 M) had no apparent effect on hemagglutination-inhibition (HI) titers of NDV and AIV antisera, the effect of formalin (0.1%) was variable, and BPL (greater than or equal to 0.25%) depressed the HI titers of both antisera. The optimum method should achieve virus inactivation without harming the treated material.     

Newcastle disease and avian influenza A virus in wild waterfowl in South Africa

In an intensive ostrich farming area in South Africa with a history of ostrich influenza outbreaks, we conducted a survey of avian influenza virus (AIV) and Newcastle disease virus (NDV) in wild aquatic birds. During late autumn and winter 1998, the time of year when outbreaks in ostriches typically start to occur, 262 aquatic birds comprising 14 species were sampled and tested for both virus infections. From eight samples, AIV, serotype H10N9, could be isolated. All isolates were apathogenic as determined by the intravenous pathogenicity index (0.00). Conversely, none of 33 sera of these wild birds showed antibodies against H10. However, one bird was found serologically positive for H6 AIV. This AIV serotype was later isolated from ostriches during an avian influenza outbreak in this area. No NDV was isolated although 34 of 46 serum samples contained NDV-specific antibodies. This is the first H10N9 isolate to be reported from Africa. In addition, our data support the notion that wild aquatic birds may function as a reservoir for AIV and NDV in South Africa.     

禽肉产品中禽流感与新城疫多重PCR-DHPLC检测方法的建立

应用多重RT-PCR反应(mRT-PCR)结合变性高效液相色谱(DHPLC)技术建立禽产品中禽流感病毒和新城疫病毒的快速检测方法。以禽流感病毒NP基因和新城疫病毒F基因各设计1对引物,经优化单相RT-PCR(sRT-PCR)反应体系,建立多重RT-PCR(mRT-PCR)同时检测禽流感和新城疫,其PCR扩增产物经DHPLC技术进行快速检测分析后,检测极限分别达10-0.5ELD50/0.1mL和10-1.5ELD50/0.1mL。与普通凝胶电泳比较,敏感性高2个数量级。特异性试验表明,mRT-PCR-DHPLC仅检测到禽流感病毒与新城疫病毒,而对其他5种禽类病原体和SPF鸡胚尿囊液均未检测到阳性吸收峰。所建立的方法检测不同亚型禽流感病毒29株、不同来源及不同毒力的新城疫病毒16株,结果与实时荧光定量PCR检测结果完全一致;与病原分离法同时检测人工感染鸡的组织脏器,两种方法检测禽流感的符合率为94.4%(34/36),检测新城疫的符合率为95.4%(21/22)。病原分离法的检出率虽高于DHPLC方法,但经统计学分析两者差异不显著,两种方法同时对27份进口鸡胗样品与90份棉拭子样品进行检测,阴性符合率为100%,可适用于进出口禽肉产品的检测。     

禽流感病毒和新城疫病毒二联RT-PCR检测方法的建立

参照国内外已发表的禽流感病毒(AIV)和新城疫病毒(NDV)的基因序列及其相关的RT-PCR检测方法,根据禽流感病毒M蛋白基因和新城疫病毒NP基因各设计一套特异性通用引物,扩增目的带分别为600bp和340 bp。通过对相关病毒检测,建立了AIV和NDV通用型二联RT-PCR检测方法。该方法具有快速、敏感、特异等优点,可为AIV和NDV的检测、流行病学调查及疫苗使用等奠定基础。     

Maternal transfer of antibodies specific for avian influenza viruses in captive whooper swans (Cygnus cygnus)

The transfer of maternal antibodies to offspring can effectively protect against avian influenza virus (AIV) infection during early life in chickens and can prevent AIV spread by decreasing the overall percentage of the avian population susceptible to this pathogen. Herein, we evaluated maternal antibody transfer dynamics in whooper swans (Cygnus cygnus), which represent an important AIV host species. In total, 57 eggs from 19 nests were collected to study the relationship between egg yolk AIV-specific antibody concentrations and factors including egg size, laying order, maternal serum AIV antibody titer, and maternal body condition. Overall, we found that AIV-specific antibodies were present in the serum of 63.2 % of surveyed female swans and were transferred to 50.8 % of analyzed eggs. We found maternal AIV-specific antibody concentration and body weight to be positively correlated with egg yolk AIV antibody concentration, whereas egg laying order was negatively correlated with yolk antibody titer. Overall, these findings maternal transfer of AIV-specific antibodies may function as a key mechanism governing the dynamics of AIV infection in swan populations.