2014—2015年深圳野生鸟类禽流感流行病学监测

为调查深圳野生鸟类的禽流感感染情况,于2014年1月~2015年4月采集了678只野鸟的569份粪便,62份肛门拭子,26份咽肛拭子和21份血样.采用病原分离鉴定法、AIV-Ag(通用型)抗原速测卡胶体金法、荧光RT-PCR试验对样品拭子进行病原学检测.采用血凝抑制实验(HI)对血清样品进行H5N1(Re-4)、H5N1(Re-6)、H5N1(Re-7)、H7、H9等5种血清型AIV抗体检测.结果显示:26份咽肛拭子分离出1株H9N2亚型禽流感病毒,阳性率为3.85%;粪便和拭子样品的抗原检测全部阴性;血清样品中H5(Re-4)阳性数1份,阳性率为9.09%;H9阳性数7份,阳性率为63.64%;未检出H5(Re-6)、H5(Re-7)及H7阳性抗体.     

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。     

贵州省部分地区H7亚型禽流感监测与分析

为了解贵州省H7亚型禽流感感染情况,采用HI试验对全省9个地区家禽血清样本进行血清抗体检测,PT-PCR方法对家禽咽-肛拭子、养禽场环境样本和野鸟粪便进行病原核酸检测。结果显示:在1570份家禽血清样本中,H7亚型禽流感血清抗体阳性率为0.45%(7/1570);在350份家禽咽-肛拭子和310份养殖场环境样本中,均未检测出H7亚型禽流感病毒核酸;在160份野鸟粪便中,有1份为H7亚型禽流感病毒核酸阳性,阳性率为0.63%(1/160)。试验提示,在贵州省养禽场中不存在H7亚型禽流感感染,但迁徙野禽中可能存在H7亚型禽流感隐性感染,应当引起重视。     

深圳地区救护野鸟中禽流感的流行病学监测

为调查深圳地区救护野鸟中禽流感感染状况,于2015年4月至2015年5月从深圳市野生动物救护中心放养区采集了野鸟肛拭子77份、野鸟血清11份,利用RT-PCR方法对拭子样品进行了AIV抗原检测;利用血凝抑制实验(HI)对血清样品进行H5N1(Re-4)、H5N1(Re-6)、H5N1(Re-7)、H7、H9等5种血清型AIV抗体检测。结果显示:拭子样品的AIV-Ag抗原检测全部阴性;而血清样品中H5(Re-4)阳性数1份,阳性率为1/11;H9阳性数1份,阳性率为1/11;未检出H5(Re-6)、H5(Re-7)及H7阳性抗体。     

安徽省猪群H5亚型禽流感的血清学调查

对安徽省16个地区规模猪场商品猪、散养猪和定点屠宰生猪进行随机抽样,采集血清样品及相应的鼻腔棉拭样品共2588份。首先采用微量血凝抑制试验对血清样品进行H5亚型禽流感抗体检测,然后使用H5亚型禽流感病毒抗原快速检测试剂盒对抗体阳性的鼻腔棉拭样品进行抗原检测。结果显示:(1)在所监测范围内,散养猪和屠宰生猪均未检测到H5亚型禽流感抗体。(2)64个规模猪场1616份血清样品中有11个猪场67份样品检测出H5亚型禽流感抗体,阳性场占17.19%(11/64),各阳性场抗体检出率从10%～50%不等,样品阳性率为4.15%(67/1616);不同地区猪场的抗体检出率分别为:芜湖地区15%(12/80)、安庆地区18%(9/50)、淮北地区15%(9/60)、宣城地区7.14%(10/140)、合肥地区4.62%(18/390)、巢湖地区4%(4/100)、马鞍山地区3.75%(3/80)和黄山地区1.03%(2/194)。(3)从抗体阳性的鼻腔棉拭子样品中均未检测到病毒抗原。表明安徽省多个规模猪场均有H5亚型禽流感抗体阳性猪存在,应当引起高度重视。     

西藏林芝地区市售家禽禽流感免疫状况调查

为了解林芝地区市售家禽禽流感免疫状况,于2008年10～12月间采集了5种家禽的249份血清样本,采用HA和HI试验进行了禽流感血清抗体的检测,发现在5种家禽的249份血清中检出了179份AIV阳性抗体,其中H5阳性数32份,阳性率为12.9%;H9阳性数43份,阳性率为17.3%;H1阳性数19份,阳性率为7.6%;H3阳性数85份,阳性率为34.7%。表明林芝地区市售家禽禽流感免疫状况仍需进一步改进。     

贵州省H7亚型禽流感病毒感染的血清学和病原学分析

为了解贵州省近几年H7亚型禽流感病毒感染情况,分别采集鸡血清3 031份、卵黄962份、咽-肛拭子1 607份、环境土壤拭子394份及野鸟粪便122份样本共计6 116份,采用血凝抑制试验(HI)和实时荧光定量RT-PCR方法进行H7亚型禽流感病毒特异性抗体和核酸检测。结果显示,在鸡血清和卵黄样本中,H7亚型禽流感表达特异性抗体的阳性率分别为1.2%(37/3 031)和2.3%(22/962);在2 123份鸡咽-肛拭子、环境样本和野鸟粪便中,均未检测出H7亚型禽流感病毒核酸。这些结果表明贵州省家禽、野鸟和环境土壤中尚未发现H7亚型禽流感病毒感染。     

2009年～2011年新疆雁形目野生鸟类禽流感血清流行病学调查与分析

本研究采集了新疆野鸟血清样品260份,进行了禽流感H1～H16亚型的血清学监测,结果显示,野鸭类血清样品2009年检出5个血清亚型,其中H8检出率高达15.38%,H13为9.62%;2010年检出11个血清亚型,H5亚型的阳性率最高,达14.29%;2011年检出12个血清亚型,其中H9检出率最高,达15.79%。不同品种野鸭的禽流感阳性检出率分析表明,绿头鸭禽流感抗体阳性率58.82%,赤麻鸭为68.66%,赤嘴浅鸭为34.09%,凤头浅鸭为44.44%。灰雁的血清样品检测结果表明,H13亚型的阳性率最高,为40.91%。1份天鹅血清样品检测出H13抗体阳性。对乌鸦、斑鸫、野鸽和红隼等野鸟血清样品检测,全部为阴性。本次在覆盖新疆边境的野生鸟类栖息地开展的禽流感血清学调查,掌握了新疆地区野生鸟类禽流感血清学监测数据,为新疆地区乃至全国的禽流感预警预测提供了科学依据。     

野生鸟类禽流感病毒感染情况的调查

为了解野生鸟类禽流感病毒(AIV)的携带感染情况,2006年～2010年,本研究在湖南省主要候鸟迁徙地收集115只野鸟组织或拭子样品、75份野鸟的新鲜粪便样品和72份血清样品。组织或拭子样品采用RT-PCR方法检测和鸡胚接种病毒分离鉴定,血清样品分别进行H5(含Re-5和Re-4)、H6、H7、H9、H10和H11抗体检测。结果表明,从斑鸠和绿头鸭组织中分别分离到H5N1亚型和H3N2亚型AIV;72份血清中有17份抗体为阳性,其中H5(Re-5)亚型5份、H5(Re-4)亚型1份、H6亚型1份、H7亚型2份和H9亚型8份,阳性率分别为6.94%、1.39%、1.39%、2.78%和11.11%。H10和H11亚型未检测到抗体阳性。     

盘锦地区候鸟禽流感调查研究

候鸟是禽流感病毒的自然宿主,对流感病毒的储存和蔓延起重要作用。本研究对辽河口湿地的迁徙候鸟进行了禽流感流行病学分析,在2012~2017年间,从双台子河口自然保护区内采集了各种野生水鸟,共采集野鸟样品棉拭384份、野鸟尸体脏器39份、环境水样500份、野鸟粪便3 355份,通过荧光定量RT-PCR方法进行检测(通用型、H5、H7和H9亚型),结果在多种候鸟粪便样品中检测到H5、H9亚型流感病毒。     

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.     

Evaluation of a competitive ELISA for antibody detection against avian influenza virus

Active serologic surveillance is necessary to control the spread of the avian influenza virus (AIV). In this study, we evaluated a commercially-available cELISA in terms of its ability to detect AIV antibodies in the sera of 3,358 animals from twelve species. cELISA detected antibodies against reference H1- through H15-subtype AIV strains without cross reactivity. Furthermore, the cELISA was able to detect antibodies produced following a challenge of the AIV H9N2 subtype in chickens, or following vaccination of the AIV H9 or H5 subtypes in chickens, ducks and geese. Next, we tested the sensitivity and specificity of the cELISA with sera from twelve different animal species, and compared these results with those obtained by the hemagglutination-inhibition (HI) test, the "gold standard" in AIV sera surveillance, a second commercially-available cELISA (IZS ELISA), or the agar gel precipitation (AGP) test. Compared with the HI test, the sensitivities and specificities of cELISA were 95% and 96% in chicken, 86% and 88% in duck, 97% and 100% in turkey, 100% and 87% in goose, and 91% and 97% in swine, respectively. The sensitivities and specificities of the cELISA in this study were higher than those of IZS ELISA for the duck, turkey, goose, and grey partridge sera samples. The results of AGP test against duck and turkey sera also showed significant correlation with the results of cELISA (R-value >0.9). In terms of flock sensitivity, the cELISA correlated better with the HI test than with commercially-available indirect ELISAs, with 100% flock sensitivity.     

供港猪群流感病毒的分离鉴定及流行病学调查

为了对供港猪群中的猪流感流行情况进行分析,从华南地区供港猪群中用无菌棉拭子采集鼻腔粘液样品,采用鸡胚接种方法,从供港猪群中分离出了2株不同亚型的猪流感病毒株,经国家流感中心鉴定分别为H1N1和H3N2亚型。本研究设计了猪流感常见亚型的HA和NA分型特异性引物,建立了猪流感型特异性RT-PCR检测方法;对分离鉴定的2株猪流感病毒和禽流感H5N1 HI检测抗原进行了RT-PCR检测,并对其部分HA和NA基因进行克隆测序分析。对供港猪群的血清检测结果表明:供港猪群中H1N1和H3N2亚型抗体阳性率分别为26.87%、38.26%,禽流感H5N1和H9N2亚型抗体阳性率均为0%。     

Influenza virus infections in migrating waterfowl: results of a two year study in Germany

In order to determine the actual prevalence of avian influenza viruses (AIV) in wild birds in Germany, extensive surveillance studies were carried out between March 2003 and January 2005. More than 3.000 samples of 79 different species of wild birds (migratory and resident birds) were taken and 1.151 established pools investigated. Samples came from 80 different regions of Germany. Forty AIV isolates representing 14 combinations of eight different hemagglutinin and eight neuraminidase subtypes, among them H5 and H7, were identified. All H5 and H7 isolates were found to be of low pathogenicity. The overall incidence of the investigated pools based on virus isolation was 3,5 % for AIV, with considerable variability noted among species, season and location. All AIV were isolated from birds sampled in autumn. Most of the AIV isolates came from the resting or wintering areas of mallards breeding far north. This study adds to the understanding of the ecology of influenza viruses in wild birds and empahsizes the constant need for surveillance in times of an ongoing and expanding epidemic of highly pathogenic AI.     

An enzyme-linked immunosorbent assay for detection of avian influenza virus subtypes H5 and H7 antibodies

Background

Avian influenza virus (AIV) subtypes H5 and H7 attracts particular attention because of the risk of their potential pathogenicity in poultry. The haemagglutination inhibition (HI) test is widely used as subtype specific test for serological diagnostics despite the laborious nature of this method. However, enzyme-linked immunosorbent assays (ELISAs) are being explored as an alternative test method.H5 and H7 specific monoclonal antibodies were experimentally raised and used in the development of inhibition ELISAs for detection of serological response specifically directed against AIV subtypes H5 and H7. The ELISAs were evaluated with polyclonal chicken anti-AIV antibodies against AIV subtypes: H1N2, H5N2, H5N7, H7N1, H7N7, H9N9, H10N4 and H16N3.

Results

Both the H5 and H7 ELISA proved to have a high sensitivity and specificity and the ELISAs detected H5 and H7 antibodies earlier during experimental infection than the HI test did. The reproducibility of the ELISA’s performed at different times was high with Pearson correlation coefficients of 0.96-0.98.

Conclusions

The ELISAs are a potential alternative to the HI test for screening of large amounts of avian sera, although only experimental sera were tested in this study.     

Avian influenza A viruses in birds --an ecological, ornithological and virological view

Avian influenza A viruses (AIV) are the causative agents of the presently most important poultry disease. Ten countries in Asia and several other countries in Eastern Europe suffer high losses from the lethal effects of these viruses of the H5N1 subtype. AIV of other subtypes cause in additional countries severe losses. The threat to health and well-being of the avifauna, domestic poultry and possibly mammals including humans are worldwide of major concern. The European Union reacted with a complete import ban on untreated meat, eggs, poultry products as well as free-living and pet birds. Extensive surveillance of free-living birds and domestic poultry that is maintained in free-range and close to open waters were initiated in an attempt to gather information on the current status of infection with these viruses and to target appropriate countermeasures for the protection of domestic poultry (in-house keeping) and to safeguard food production for humans. Since the monitoring of free-living birds is labour-intensive, costly, and time-consuming, only birds should be included in the monitoring programme that harboured in the past most if not all influenza A viruses. The birds of the order Anatiformes, family Anatidae, subfamilies Anserinae and Anatinae, provided 65.9 % of all avian AIV isolates. The cosmopolitan Common Mallard (Anas platyrhynchos) is the dominant species with the highest rate of isolations among all bird species. Second in frequency is the North-American Blue-winged Teal (Spatula discors). Consequently, free-living anatiform birds of the genera Anas and Spatula should comprise the main focus for the collection of cloacal and pharyngeal swabs. With the likely exception of the most recent H5N1 viruses, signs of disease were not recorded in AIV infected anatiform birds. AIV isolations were definitely less frequently obtained from birds of the orders Phasianiformes (including domestic chickens and turkeys), Charadriiformes (plovers and lapwings), Lariformes (gulls), Columbiformes (pigeons) and Psittaciformes (psittacines) and need less attention in sampling efforts. This review presents also data on taxonomy and most suitable means for isolation and typing of haemagglutinating viruses. The different frequencies of the detection of 16 haemagglutinin (HA) subtypes and 9 subtypes of neuraminidase (NA) surface antigens are composed on the basis of extensive literature retrievals. Both antigens occure in isolates at different frequencies. Only 103 of all 144 possible HA x NA combinations were described so far. The AIV that contain the HA subtypes H3, H4, H6 are most frequently isolated whereas the AIV of the subtypes H5 and H7 were less frequently encountered. All other HAs are rather rare. AIV that possess the NA of the subtypes N2, N1, N8 and N3 are frequent and all other NAs are rarely detected.     

天津地区猪流感血清学调查

2002年-2005年期间,采用血凝抑制试验对天津市10个区县44个养猪场进行了猪流感病毒抗体检测,结果75%的被调查猪场抗体检测结果有阳性,检测的648份血清样品中,69.6%为阳性。流感病毒抗体亚型调查结果显示该地区流行的猪流感病毒主要为H1和H3亚型,抗体阳性率分别为55.4%和39.4%。部分猪群中存在抗H9亚型流感病毒抗体,阳性率为5.4%,但未发现H5亚型流感病毒抗体。此外,部分猪群中同时存在2种或3种亚型(H1,H3和H9)流感病毒的抗体,表明这些猪曾经同时被2种或3种不同亚型的流感病毒感染。     

禽流感病毒N4亚型神经氨酸酶基因的克隆和序列分析

应用无特定病原体 (SPF)鸡胚增殖禽流感病毒 A/ Turkey/ Ontario/ 6 118/ 6 8(H8N4 )毒株 ,Tri Zol L S Reagent提取病毒 RNA,RT- PCR扩增神经氨酸酶 (NA)基因全片段 ,克隆到 p MD18- T载体上 ,并进行了鉴定和序列测定。所获得的 NA基因片段长 14 4 1bp,编码 4 90个氨基酸残基。根据推导的氨基酸序列进行预测 ,有 9个潜在的糖基化位点和2 0个半胱氨酸残基