【作者】 李晓霞；

【导师】 柴同杰；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2009， 博士

【摘要】 畜禽传染病如新城疫(Newcastle disease,ND)、禽流感(Avian Influenza,AI)、口蹄疫(Foot-and-Mouth disease,FMD)等疾病的病原都可以通过空气传播,造成传染病的流行。2002-2003年SARS和近几年来高致病性禽流感(Highly pathogenic avian influenza,HPAI)的暴发流行,警示人们应重视病毒气溶胶及其气源性传染规律的研究。ND是危害最严重的家禽传染病之一,世界动物卫生组织(World Organisation for Animal Health, OIE)将其定为法定报告类疫病。然而,ND传播途径研究没能得到应有的重视。Gloster (1983)通过对两起ND爆发的分析,认为ND流行主要是由气载新城疫病毒(Newcastle disease virus, NDV)引起的;气源传播在新城疫流行中起着重要作用。这种传播方式在1970–1971年的英格兰ND爆发和1973年北爱尔兰ND流行中起了重要作用,并已经有证据证明NDV可发生远距离的传播。以上学者的观点尽管确认了ND的气源性传染途径,然而,NDV气溶胶的发生和传染机制不详,需要实验证明。因此,开展本研究。本课题建立了NDV气溶胶发生及传播感染的实验模型,动态检测了人工感染鸡NDV气溶胶排出的时间,浓度;验证NDV F48E9株可以通过气源传播感染健康鸡群。并进一步研究了感染NDV的鸡群排出的NDV气溶胶向周围环境扩散并感染10m、20m外的鸡群;对NDV气溶胶不同感染途径的感染剂量进行对比研究;在此基础上,收集生产鸡舍中的空气,对其中的NDV进行分子检测及分离鉴定,对气载NDV分离株的部分分子特性进行了研究;应用ERIC-PCR动态监测了感染NDV气溶胶SPF鸡肠道菌群的变化。本研究不仅对深入认识ND的传染机制非常必要,而且对揭示像SARs、禽流感等气源性传染病的传播机制带来有益的启示。1新城疫病毒气溶胶发生及其传染的实验研究本实验建立一个NDV气溶胶发生、传播及感染的实验模型,进行实验1(T1)和实验2(T2)。隔离器A中的SPF鸡(G1)点眼、滴鼻接种NDV后用AGI-30收集A中的气体,检测气体样品中的NDV,用蚀斑计数法测定NDV气溶胶浓度;隔离器B中的SPF鸡(G2)接受气源性被动感染。定期收集实验鸡的口咽和泄殖腔拭子,检测拭子中的NDV,确定排毒情况;每7天收集实验鸡血液样品,应用HI-T检测抗体。试验结果发现:T1和T2分别在攻毒后第二天和第三天检测到NDV气溶胶,其浓度分别在第13天和第11天达到高峰,为1.69×104 PFU/m3空气和9.14×103 PFU/m3空气,到第40天NDV气溶胶浓度仍分别为6.98×103 PFU /m3空气和3.78×103 PFU/m3空气;T1G1和T2G1组鸡均在攻毒后第二天检测到排毒;T1G2组和T2G2组接受气源被动感染7天后检测分别有80%和60%的鸡排毒,抗体分别在感染后第7天和第14天呈阳性,为4.07和7.40,一直到第四十天均为阳性。结果表明SPF鸡在感染NDV后三天内排出NDV,并能够很快形成气溶胶;NDV气溶胶能够感染健康鸡只。2新城疫病毒气溶胶向周围环境扩散感染的实验研究共进行两组实验,一组是新城疫病毒气溶胶向周围环境扩散的实验研究;一组是10m,20m气源传播感染实验。扩散试验时设一组SPF鸡经点眼、滴鼻接种NDV后用AGI - 30收集接种鸡处(S0)距离接种感染鸡10m (S10)、20m(S20)处中的气体,检测并定量气体样品中的NDV,揭示动物舍NDV气溶胶向周围环境的传播情形。结果表明,S0处气体样品中从接种后第3天开始检测到NDV,浓度在11天达到高峰,一直到第30天RT-PCR检测结果仍为阳性;S10处及S20处气体样品中均从接种后第5天开始检测到NDV,浓度在11-13天达到最大值,一直到第25天RT-PCR检测结果仍为阳性。感染试验时设三组试验鸡,分别为人工接种鸡(G0),点眼、滴鼻接种NDV,在距离接种鸡10m (G10)、20m (G20)处SPF鸡接受气源感染的哨兵组鸡,通过检测哨兵组鸡的排毒及抗体来确定是否发生气源感染,具体检测方法同试验一。气源扩散感染试验结果表明G10及G20持续气源被动感染7后开始检测,均检测到排毒直到第28天仍然排毒。结果表明SPF鸡在感染NDV后三天内排出NDV,并能够很快形成气溶胶,传播扩散到20m;NDV气溶胶能够感染10m、20m处健康鸡只。3 SPF鸡经不同途径的感染剂量及致死剂量实验研究本实验分别通过呼吸道(通过人工发生定量NDV气溶胶感染SPF鸡)、消化道(喂服)及肌肉注射途径定量感染NDV,通过检测鸡的特异性抗体判定鸡是否发生感染,ID50、LD50计算方法采用的加权直线回归法(Bliss法)。结果表明NDV气溶胶半数感染吸入剂量为30.53 PFU,95%置信区间为9.46 - 98.54 (PFU);半数致死剂量为117.05 PFU,95%置信区间为23.71 - 577.92 PFU。NDV通过消化道半数感染灌服剂量为4419.78 PFU,95%置信区间为1095.34 -17834.14 (PFU);半数致死剂量为44743.91PFU,95%置信区间为10161.62 - 197017.62 (PFU)。NDV经肌肉注射途径半数感染注射剂量为16.63 PFU,95%置信区间为4.85 - 57.00 (PFU);半数致死剂量为69.60 PFU,95%置信区间为14.24 - 340.27 (PFU)。结果表明不同感染途径的致病性有差异,肌肉注射致病性最强,气溶胶次之,消化道最弱。4鸡舍中气载鸡新城疫病毒的检测及分离鉴定本研究采用AGI-30气体收集器在五个大型鸡舍舍内收集气体(15份/舍),同时采集鸡的气管和泄殖腔拭子,采用兼并引物RT-PCR检测气体样品和拭子中的新城疫病毒(NDV),并鉴别NDV的毒力;同时分离、纯化气体样品中的NDV,用常规毒力学实验鉴别分离株的毒力。结果五个鸡舍中,在鸡舍A中,2份气体样品和4份口咽及泄殖腔拭子中检测到弱毒,均没检测到强毒。在鸡舍B中,7份气体样品和5份口咽及泄殖腔拭子中检测到弱毒,而仅在5份气体样品中检测到强毒;在鸡舍C中,6份气体样品和5份口咽及泄殖腔拭子中检测到弱毒,而仅在2份气体样品中检测到强毒;在鸡舍E中,7份气体样品和1份口咽及泄殖腔拭子中检测到弱毒,而在所有样品中均未检测到强毒。分离纯化后总共得到7株NDV,其中3株为强毒,4株为弱毒。在鸡舍A中得到一株弱毒YTCX08,在鸡舍B中得到一株强毒DZ07和一株弱毒DZ0702,在鸡舍C中得到2株强毒MH07和MH0702和1株弱毒MH0703,在鸡舍E中得到一株弱毒NY06。结果表明兼并引物RT-PCR可快速、直接对气体样品进行检测及鉴别诊断,有助于ND的防治。5气载新城疫病毒野毒株F基因重要片段的克隆及遗传变异分析根据已发表文献针对新城疫病毒(NDV) F基因序列设计了1对引物,对从气体中分离的7株NDV毒株的F基因重要功能区片段扩增进行了扩增和序列测定,并与多株已报道的NDV参考株相应片段进行序列比较,经遗传基因进行化树分析。结果表明,七株气载新城疫病毒分离株之间的同源性为79.3% - 100%;氨基酸的同源性为77.4% - 100%,分离到的四株野毒NY06、MH0703、YTCX08与DZ0702均属于基因II型,在裂解位点的氨基酸序列(111GGRQGRL117)与NDV弱毒株特征相符合;MH07属于基因IX型;DZ07、MH0702均属于基因VII型,这三株在裂解位点的氨基酸序列( 111GRRQK RF117 )与NDV强毒株特征相符合。6新城疫病毒气溶胶感染SPF鸡肠道菌群结构变化的研究SPF鸡定量感染NDV气溶胶后,定期采集试验鸡的粪便,检测其中大肠杆菌的浓度变化,同时应用ERIC-PCR对肠道菌群结构进行了动态检测;同时对健康鸡进行同样研究作对比。结果表明,通过与对照组肠道大肠杆菌的检测比较,结果大肠杆菌在感染初期高于对照组(P<0.01),而严重感染开始出现死亡时开始降低,明显低于对照组(P<0.01),恢复期大肠杆菌总数开始回升,与对照组差异不显著(P>0.05)。ERIC-PCR结果比较发现NDV气溶胶感染组鸡只与同日龄对照组鸡肠道菌群的图谱间有一定的差异。在大多数鸡群出现临床症状(2-10天)菌群结构也发生了改变,在恢复期(10-15天)菌群重新建立,并有一个重新选择的过程,到完全恢复后菌群结构基本稳定。结果表明感染NDV气溶胶后鸡群肠道菌群发生了改变,采用ERCI-PCR指纹图谱技术可以动态监测动物肠道菌群结构变化。更多还原

【Abstract】 Airborne and droplet transmission are one of the major routes for spreading animal viral diseases, such as Newcastle disease, Avian Influenza and foot-and-mouth disease. Recent emergence of respiratory viral pathogens such as SARS and avian influenza viruses have attracted public attention and are believed to be transmitted via the airborne route, raising various scientific and public health issues related to airborne transmission and control of infectious agents. Newcastle disease (ND) is included in the World Organization (OIE) for Animal Health list of notifiable diseases (former list A). Airborne transmission was also one of the important routes for spreading of Newcastle disease, which was considered a significant factor in the 1970-1971 ND outbreaks in England and the 1973 ND epidemic in Northern Ireland. Airborne transport over long distances was known to have occurred. Despite the recognition of airborne as a spread route for NDV, the mechanism of aerosol generation, transmission is still unclear. The notion of airborne spread needs experiment authentication. Therefore, we experimentally investigated airborne transmission of ND in a model system. Using this model system, we monitored occurrence and transmission of NDV aerosol. We have not only established the time of aerosol occurrence but also determined the aerosol concentrations, dynamic changes and their relationship with virus shedding. This experiment also confirmed the airborne transmission of the F48E9 strain. NDV aerosol originating from infected chickens could spread to their surrounding air (10m, 20m away) from the infected chickens and could infect the healthy chickens at that point. The infectious and lethal doses of NDV aerosol were compared by other infectious routs. The difference of intestinal flora of NDV aerosol infected group and healthy group by ERIC-PCR based fringerprint on molecular ecology. Airborne Newcastle disease (ND) viruses in the air of five chicken houses were detected and differentiated by reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate primers. Airborne ND viruses were also isolated and virulence identified by in-vivo tests. Part of the fusion gene of isolate s were amplified by RT- PCR and sequenced.Not only is this result important in better understanding the mechanism of ND transmission, it also has implications in controlling avian influenza and other contagious diseases.The study consists of six parts:1. Occurrence and transmission of Newcastle Disease Virus aerosol originating from infected chickens under experimental conditions.One model was established to better study emission, transmission and infection mechanism of NDV (Newcastle Disease Virus, NDV) aerosol. Two trials (T1) and (T2) were performed. SPF chickens (G1) were housed in the isolator A and were inoculated with NDV strain F48E9 by means of dripping noses and eyes. After G1 was inoculated, the air in the isolator A was sampled by AGI-30 and airborne NDV was detected and quantified (Plaque per m3 air). SPF chickens were housed in the isolator B, which were used as aerosol challenged group (G2). Oropharyngeal and cloacal swabs were collected from each chicken, which used for NDV detection and determined virus shedding of chickens. The antibody response to NDV infection of Serum samples was assessed by the hemagglutination inhibition (HI) test. In T1 and T2, NDV aerosol could be detected at 2 dpi (days post inoculation) and 3 dpi respectively, at 13 dpi and 11 dpi the concentration of NDV aerosol respective was 1.69×104 PFU/m3 air and 9.14×103 PFU/m3 air which reached a peak, at 40 dpi the concentration still was 6.98×103 PFU/m3 air and 3.78×103 PFU/m3 air; at 2 dpi the shedding NDV in the swaps of chickens in T1G1 and T2G1 were confirmed; 7 days post infected by NDV aerosol, the presence of NDV in the swaps of 80% chickens in T1G2 and 60% chickens in T2G2 could be detected, The chickens of T1G2 and T2G2 had antibody titers for NDV reaching 4.07 and 7.40 at 7 dpi and 14 dpi respectively, positive results according to OIE standards, the antibody was consistently detected positive up to 40 dpi.The results of two trials showed that infected SPF chickens could emission NDV within 3 days post inoculation and come into NDV aerosol and then infected healthy chickens.2. Spread and infection of Newcastle disease virus aerosol under field conditions.In this study, two trials were conducted, I: Study on spread of NDV aerosol to their surrounding air; II: Infection of NDV aerosol. I: After SPF chickens were inoculated with NDV and were housed in one cage , the NDV aerosol originating from inoculated chickens (S0) and10m (S10)、20m (S20) away were collected with All Glass Impinger-30 (AGI-30) to studied the occurrence and concentration of NDV aerosol.The spreading of NDV aerosol from the infected chickens to the ambient air was characterized. Th results showed that NDV aerosol was initially detectabl at day 3 post inoculation (dpi) at the S0 Aand at day5 post inoculation (dpi) at S10 and S20. The aerosol concentration peaked at 11dpi at S0 and was consistently detectable up to 30 dpi. , at 11-13 dpi at S10 and S20 and was consistently detectable up to 25 dpi. II: 3 groups (infected group, 10m aerosol infected group and 20 m aerosol infected group as sentinel chickens). Aerosol infection did occur, as shown by NDV shedding and seroconversion to NDV in sentinel chickens. The detailed methods were in accordance with the methods in Trial I. Th results showed that after 7 days of aerosol exposed infection, all the sentinel chickens were tested positive. Up to 28 dpi, some sentinel chickens were still positive.The results indicated that viruses shed from infected chickens readily aerosolized and aerosol could spread into the ambient air (20m away) and the chickens there were infected by the NDV aerosol.3. Studies to quantify the infectious and lethal dose of Newcastle disease virus for chickens through defferent routs.In this study, SPF chickens were ramdomly divided into 3 groups: aerosol infectedgroup, intramuscular injection, gastrointestinal tract infected group. Each chicken was challenged with the same volume of different dilution of NDV. Infection was determined by the specific antibody to NDV. Computation Method for ID50 and LD50 Based on Bliss. The results showed that aerosol routs: ID50 was 30.53 PFU, 95% confidence interval was 9.46 - 98.54 (PFU); LD50 was 117.05 PFU,95% confidence interval was 23.71 - 577.92 (PFU). Gastrointestinal tract infected rout: ID50 was 4419.78 PFU, 95% confidence interval was 1095.34 -17834.14 (PFU); LD50 was 44743.91 PFU,95% confidence interval was 10161.62 - 197017.62 (PFU). Intramuscular injection rout: ID50 was 16.63 PFU, 95% confidence interval was 14.85 - 57.00 (PFU); LD50 was 69.60 PFU,95% confidence interval was 14.24 - 340.27 (PFU).The results indicated that it was stronger with Intramuscular injection than aerosol infection. That with Gastrointestinal tract infection was weakest.4. Molecular detection and isolation of airborne chicken Newcastle disease virus in chicken houses Airborne Newcastle disease (ND) viruses in the air of five chicken houses were detected and differentiated by reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate primers. Fifteen air samples were collected with All Glass Impinger-30 (AGI-30) air samplers in each house. Airborne ND viruses were also isolated and virulence identified by in-vivo tests. Only avirulent viruses were detected in two air samples and four swab samples in house A. Avirulent viruses were detected in seven air samples and five swab samples and virulent viruses were detected only in five air samples in house B. Avirulent viruses were detected in six air samples and five swab samples and virulent viruses were detected only in two air samples in house C. Avirulent viruses were detected in seven air samples and one swab samples in house E. Seven NDV strains were obtained. Three were virulent strains and four were avirulent strains. One strain avirulent (YTCX08) was isolated in house A; One virulent strain (DZ07) and one avirulent strain (DZ0702) were isolated in house B; Two virulent strains (MH07, MH0702) and one avirulent strain (MH0703) were isolated in house C; one avirulent strain (NY06) was isolated in house The results showed that it was feasible to detect and differentiate NDV in the air samples using degenerate primers based RT-PCR. This technique could decrease the time it required identify NDV infected flocks while distinguishing between virulent and avirulent viruses. It will help effectively to control Newcastle disease.5. Nucleotide Sequence Analysis of Main Function Domain of F Gene of airborne Newcastle disease virusOne pair of specific primers was designed and synthesized according to fusion ( F) protein gene sequences of Newcastle disease virus (NDV), Part of the fusion gene of seven isolates were amplified by RT- PCR and sequenced. A phylogenetic tree based on obtained sequences of reference NDV strains was constructed and the deduced amino acid sequences showed that the homologies of t he nucleotide are 79.3% - 100 % and the homologies of the deduced amino acid sequences are77.4% - 100%. NY06、MH0703、YTCX08 and DZ0702 belong to the genotype II and their amino acid sequences of the cleavage site region residues (111GG RQGRL117 ) matched to the characteristics of avirulent strains; MH07 belongs to the genotype IX and DZ07、MH0702 belong to genotype VII, and their amino acid sequences of the cleavage site region residues (111GRRQK RF117 ) matched to the characteristics of virulent strains.6. Analysis of the structure features of Intestinal communities of chickens infected with NDV aerosolThe article compare the difference of intestinal E. coli floras between healthy chickens and the chickens infected with NDV aerosol. The results showed that Significant difference in E. coli floras in the feces of the group infected with NDV aerosol increased (P<0.01) in Early infection and also was detected in the later phase (P<0.01). The difference of intestinal flora of NDV aerosol infected group and healthy group by ERIC-PCR based fringerprint on molecular ecology. Some difference was detected in the ERIC-PCR fingerprints between infected group and healthy group. The intestinal microbial population was changed when the chickens exhibited clinical symptoms (2-10 dpi), and was rebuilt in the recovery stage (10-15 dpi), and then was stable after fully recovery.The results showed that the intestinal flora of NDV aerosol infected chickens was changed and ERIC–PCR based fingerprints can analyze the dynamic monitoring of animal intestinal flora.更多还原