Evaluation of effectiveness and efficiency of wild bird surveillance for avian influenza

This study aimed to assess which method of wild waterbird surveillance had the greatest probability of detecting highly pathogenic avian influenza (HPAI) H5N1 during a period of surveillance activity, the cost of each method was also considered. Lake Constance is a major wintering centre for migratory waterbirds and in 2006 it was the site of an HPAI H5N1 epidemic in wild birds. Avian influenza surveillance was conducted using harmonised approaches in the three countries around the lake, Austria, Germany and Switzerland, from 2006–2009. The surveillance consisted of testing birds sampled by the following methods: live birds caught in traps, birds killed by hunters, birds caught in fishing nets, dead birds found by the public and catching live Mute Swans (Cygnus olor); sentinel flocks of Mallards (Anas platyrhynchos) were also used. Scenario tree analysis was performed including sensitivity analysis, followed by assessment of cost-effectiveness. Results indicated that if HPAI H5N1 was present at 1% prevalence and assuming HPAI resulted in bird mortality, sampling dead birds found by the public and sentinel surveillance were the most sensitive approaches despite residual uncertainty over some parameters. The uncertainty over the mortality of infected birds was an influential factor. Sampling birds found dead was most cost-effective, but strongly dependent on mortality and awareness of the public. Trapping live birds was least cost-effective. Based on our results, we recommend that future HPAI H5N1 surveillance around Lake Constance should prioritise sentinel surveillance and, if high mortality is expected, the testing of birds found dead.     

新疆艾比湖野鸟H1N2亚型禽流感病毒的分离鉴定及全基因组序列分析

旨在调查野鸟中存在的禽流感病毒,并为禽流感的防制提供依据。采集新疆艾比湖自然保护区野鸟的粪便棉拭子进行禽流感病毒的分离与鉴定,将PCR产物进行纯化后用T载体连接并转化到DH5α细胞中;菌液PCR鉴定后将目的菌液送至上海生工测序并比对测序结果,在NCBI上进行基因序列分析。结果显示,从新疆艾比湖自然保护区野鸟的粪便棉拭子分离得到1株H1N2亚型禽流感病毒;经序列分析得知,该毒株为1株低致病性禽流感病毒,且存在与多种亚型病毒的重配现象。     

Australian surveillance for avian influenza viruses in wild birds between July 2005 and June 2007

Objective   To identify and gain an understanding of the influenza viruses circulating in wild birds in Australia.
Design   A total of 16,303 swabs and 3782 blood samples were collected and analysed for avian influenza (AI) viruses from 16,420 wild birds in Australia between July 2005 and June 2007. Anseriformes and Charadriiformes were primarily targeted.
Procedures   Cloacal, oropharyngeal and faecal (environmental) swabs were tested using polymerase chain reaction (PCR) for the AI type A matrix gene. Positive samples underwent virus culture and subtyping. Serum samples were analysed using a blocking enzyme-linked immunosorbent assay for influenza A virus nucleoprotein.
Results   No highly pathogenic AI viruses were identified. However, 164 PCR tests were positive for the AI type A matrix gene, 46 of which were identified to subtype. A total of five viruses were isolated, three of which had a corresponding positive PCR and subtype identification (H3N8, H4N6, H7N6). Low pathogenic AI H5 and/or H7 was present in wild birds in New South Wales, Tasmania, Victoria and Western Australia. Antibodies to influenza A were also detected in 15.0% of the birds sampled.
Conclusions   Although low pathogenic AI virus subtypes are currently circulating in Australia, their prevalence is low (1.0% positive PCR). Surveillance activities for AI in wild birds should be continued to provide further epidemiological information about circulating viruses and to identify any changes in subtype prevalence.     

野鸟源H5N8禽流感病毒遗传变异分析与致病性评估

本试验在野鸟禽流感病毒紧急疫情检测过程中鉴定并分离到1株H5N8高致病性禽流感病毒,利用病毒全基因组测序、系统发育及关键氨基酸位点分析解析了该野鸟源H5N8禽流感病毒分离株遗传进化情况,通过体外复制动力学试验及小鼠感染试验,评价了该野鸟源H5N8禽流感病毒分离株对哺乳动物致病性。进化分析显示,该病毒株属于Clade 2.3.4.4,可以不经适应直接感染小鼠并在呼吸系统内复制,表现出有限的组织嗜性,对小鼠呈低致病性。其在体内外复制能力较低。结果表明,本试验加深了对野生鸟携带H5N8禽流感病毒的认识和理解、对野鸟源H5N8禽流感病毒生物学特性的评价,为预测野鸟源H5N8禽流感病毒遗传进化趋势及其生物安全风险评估提供借鉴和参考。     

1株野鸭源H6N2亚型AIV对SPF鸭的致病性

本研究选取2周龄SPF鸭（绍兴麻鸭）自天然孔感染1株野鸭源H6N2亚型LPAIV,评价其对幼龄鸭的致病性。结果显示,试验感染鸭临床症状较轻微,病毒在鸭消化道复制能力较呼吸道内更强,且具有水平传播的能力。仅能在盲肠扁桃体和法氏囊2个组织器官中检测到病毒,但可对法氏囊等多个组织器官造成不同程度的损伤。此外感染鸭可产生HI抗体,并在第21天达到高峰。结果表明,该株H6N2亚型LPAIV对幼龄鸭的致病力较低,在AIV病毒传播中幼龄鸭起到了一定作用,为研究H6N2亚型LPAIV的致病机制及AIV发病机理提供了理论数据。     

一株野鸟源H9N2亚型禽流感病毒的全基因组序列分析及致病性研究

为进一步了解2014年分离自我国南方野鸟粪便中的一株H9N2亚型禽流感病毒(AIV)Wide Bird/Hu N/SC1400/2014(H9N2)(WB/400/14)的生物学特性,本研究对其进行全基因组序列测定、进化分析及SPF鸡、SPF鸭和BALB/c小鼠的感染性试验。序列分析显示:该分离株的HA裂解位点基序为333PAASDR↓GL340,其中不存在多个连续的碱性氨基酸,符合低致病性禽流感病毒(LPAIV)氨基酸序列特征。该分离株不同基因片段来源较复杂,分别与H9、H6、H4、H1、H11、H10、H3等多种亚型的LPAIV同源性较高,呈现明显的多样性。感染性试验显示,WB/400/14不能够在SPF鸡和小鼠体内有效复制,但病毒感染SPF鸭后能够在部分脏器中检测到病毒的存在,并且感染鸭能通够过咽喉和泄殖腔同时向外排毒,而同居感染鸭仅通过泄殖腔向外排毒,表明分离株在SPF鸭群中具有良好的水平传播能力。本研究为AIV的监测和防控提供实验依据。     

野鸟禽流感的研究进展及西藏的预防和控制对策

高致病性禽流感是由A型流感病毒引起的一种传染病,不仅给世界养禽业造成重大损失,也具有重要的公共卫生学意义。作者综述了高致病性禽流感的研究进展和流行特点,以及野鸟感染禽流感的概况,并结合实际情况提出西藏预防和控制禽流感的建议。     

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。     

野生鸟类高致病性禽流感抗体水平检测

高致病性禽流感对禽类及人类危害十分严重.高致病性禽流感流行病学特点之一是随野生鸟类的迁徙,该病在全球广泛扩散和蔓延.野生鸟类在紧急免疫后的血清抗体水平直接关系到疫情的控制、养禽业的发展以及人类的健康.我国GB/T 18936高致病性禽流感诊断技术对火鸡、鸭、鹅(包括野生鸟类)等禽类接种禽流感疫苗免疫效力评价缺乏足够的血清学依据,国际国内对野生鸟类血清抗体水平检测资料比较欠缺.因此对野生鸟类高致病性禽流感血清抗体水平进行检测,就显得十分重要和必要.     

Surveillance for highly pathogenic avian influenza in wild birds in the USA

As part of the USA's National Strategy for Pandemic Influenza, an Interagency Strategic Plan for the Early Detection of Highly Pathogenic H5N1 Avian Influenza in Wild Migratory Birds was developed and implemented. From 1 April 2006 through 31 March 2009, 261 946 samples from wild birds and 101 457 wild bird fecal samples were collected in the USA; no highly pathogenic avian influenza was detected. The United States Department of Agriculture, and state and tribal cooperators accounted for 213 115 (81%) of the wild bird samples collected; 31, 27, 21 and 21% of the samples were collected from the Atlantic, Pacific, Central and Mississippi flyways, respectively. More than 250 species of wild birds in all 50 states were sampled. The majority of wild birds (86%) were dabbling ducks, geese, swans and shorebirds. The apparent prevalence of low pathogenic avian influenza viruses during biological years 2007 and 2008 was 9.7 and 11.0%, respectively. The apparent prevalence of H5 and H7 subtypes across all species sampled were 0.5 and 0.06%, respectively. The pooled fecal samples (n= 101 539) positive for low pathogenic avian influenza were 4.0, 6.7 and 4.7% for biological years 2006, 2007 and 2008, respectively. The highly pathogenic early detection system for wild birds developed and implemented in the USA represents the largest coordinated wildlife disease surveillance system ever conducted. This effort provided evidence that wild birds in the USA were free of highly pathogenic avian influenza virus (given the expected minimum prevalence of 0.001%) at the 99.9% confidence level during the surveillance period.     

野鸟在禽流感流行病学中的作用研究进展

作者对中国特别是辽宁省野鸟存在和迁移情况进行了详细总结;同时结合野鸟的迁移,携带禽流感病毒情况,深入分析、探讨野鸟在禽流感传播中的作用,明确预防控制野鸟禽流感的重要性,为野鸟禽流感的防控提供参考。     

禽流感病毒神经氨酸酶基因在重组杆状病毒中的表达

根据已知H5N1亚型禽流感病毒(AIV)神经氨酸酶(NA)基因序列设计并合成引物。从H5N1亚型病毒感染的鸡胚尿囊液中提取总RNA,反转录后采用高保真DNA聚合酶扩增NA基因,构建转移载体pFastBacHTA-NA,并与大肠杆菌DH10Bac的Bacmid质粒重组,构建重组转座质粒rBacmid-NA。在脂质体介导下将rBacmid-NA转染sf9昆虫细胞获得重组杆状病毒。在sf9昆虫细胞中表达NA蛋白,通过SDS-PAGE、Western blot和激光共聚焦检测蛋白。结果表明:表达的NA蛋白分子量约为53 ku,该蛋白能与H5N1亚型AIV血清发生特异性反应,证明NA蛋白表达正确,具有良好的免疫反应性。     

Recent developments in avian influenza research: epidemiology and immunoprophylaxis

Influenza A viruses have been isolated from humans, from several other mammalian species and a wide variety of avian species, among which, wild aquatic birds represent the natural hosts of influenza viruses. The majority of the possible combinations of the 15 haemagglutinin (HA) and nine neuraminidase (NA) subtypes recognized have been identified in isolates from domestic and wild birds. Infection of birds can cause a wide range of clinical signs, which may vary according to the host, the virus strain, the host's immune status, the presence of any secondary exacerbating microorganisms and environmental factors. Most infections are inapparent, especially in waterfowl and other wild birds. In contrast, infections caused by viruses of H5 and H7 subtypes can be responsible for devastating epidemics in poultry. Despite the warnings to the poultry industry about these viruses, in 1997 an avian H5N1 influenza virus was directly transmitted from birds to humans in Hong Kong and resulted in 18 confirmed infections, thus strengthening the pandemic threat posed by avian influenza (AI). Indeed, reassortant viruses, harbouring a combination of avian and human viral genomes, have been responsible for major pandemics of human influenza. These considerations warrant the need to continue and broaden efforts in the surveillance of AI. Control programmes have varied from no intervention, as in the case of the occurrence of low pathogenic (LP) AI (LPAI) viruses, to extreme, expensive total quarantine-slaughter programmes carried out to eradicate highly pathogenic (HP) AI (HPAI) viruses. The adoption of a vaccination policy, targeted either to control or to prevent infection in poultry, is generally banned or discouraged. Nevertheless, the need to boost eradication efforts in order to limit further spread of infection and avoid heavy economic losses, and advances in modern vaccine technologies, have prompted a re-evaluation of the potential use of vaccination in poultry as an additional tool in comprehensive disease control strategies. This review presents a synthesis of the most recent research on AI that has contributed to a better understanding of the ecology of the virus and to the development of safe and efficacious vaccines for poultry.     

广东省部分地区规模化猪场猪流感病毒感染的血清学调查与分析

为了解广东省规模化猪场猪流感病毒(SIV)感染情况,本研究采用ELISA方法和血凝抑制试验(HI),对2011年～2012年采集的1 050份血清样品进行SIV血清学检测.两种检测方法结果显示1 050份血清样品中SIV抗体阳性率分别为50.4％(ELISA)和50.2％(HI).其中珠三角地区和粤东地区的感染率高于粤西地区.SIV亚型调查结果显示该地区流行的SIV主要为H1和H3亚型,抗体阳性率分别为39.2％和18.2％(ELISA).部分猪场存在H9亚型SIV抗体.部分猪群中同时存在H1和H3两种亚型SIV抗体,表明猪群中存在不同亚型SIV混合感染.本研究为广东省猪流感的预防提供参考依据.     

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.     

H9亚型禽流感病毒SD96株HA蛋白特异性单抗的制备及鉴定

本研究用H9N2亚型禽流感病毒分离株A／Chicken／Shandong／6／1996（CK／SD／6／96）免疫6周龄BALB／c雌性小鼠,取免疫后小鼠脾细胞与骨髓瘤细胞SP2／0融合。采用血凝抑制（HI）和ELISA方法筛选阳性细胞克隆,连续多次克隆培养后获得3株能稳定分泌H9亚型HA特异性单抗的细胞株1F4C4、9F12D1、2C5H12。3株阳性杂交瘤细胞的培养上清及腹水对CK／SD／6／96的HI效价分别为2^6、2^4、2^1和2^12、2^10、2^7。特异性试验表明该3株单抗均不与其他主要禽类病毒和其他14个HA亚型的禽流感病毒发生交叉反应。1F4C4和2C5H12抗体亚类为IgG2b,9F12D1为IgG2a。这3株单抗对H9亚型不同抗原群流感毒株有不同程度的中和作用,表现出单抗识别抗原住点的差异性,为进一步研究H9亚型流感病毒的抗原性差异以及抗原识别提供了物质基础和条件。     

不同H9N2亚型禽流感病毒株致病力的比较试验

参照NDV评价毒力的方法对1998—2008年间收集的25株H9N2AIV的致病性进行了比较研究。结果显示,25株H9N2AIV不同毒株间致病性有较大差异,大致分为3类：致病性偏强、致病性中等和致病性较弱。从中选出致病力有差异的8个毒株进行了1日龄雏鸡脑内接种指数（ICPI）、6周龄鸡静脉接种指数（IVPI）以及8周龄SPF鸡人工感染排毒试验,结果证明大部分毒株ICPI和IVPI基本上为0,排毒散毒的高峰期在攻毒后第5天到第6天。在25个毒株中,3#和12#表现出了比较高的致病性,不仅其EID50值最高（分别为10-8.8/0.2mL和10-8.8/0.2mL）、ELD50值最高（分别为10-7.9/0.2mL和10-8.7/0.2mL）,而且鸡胚平均死亡时间也最短（分别为66.5,69h）。3#、12#还出现了1日龄雏鸡脑内接种指数（分别为0.238,0.437）和6周鸡静脉内接种指数（分别为0.34,0.51）。在8周龄SPF鸡人工感染排毒试验中3#和12#毒株的排毒量大,排毒时间也明显长于其他毒株（第2～9天）。以上试验结果表明我国H9N2AIV不同毒株致病性有明显差异,呈多态性,致病性偏强的毒株造成鸡群较高的死亡率。