【作者】 姚美玲；

【导师】 柴同杰；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2010， 博士

【摘要】 人类所认识的气源性传播的病毒性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)、猪繁殖障碍与呼吸综合征(Porcine reproductive and respiratory syndrome, PRRS)、鸡传染性支气管炎(Infectious bronchitis)等达到20多种。AI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。人们普遍认为AI可以经过直接接触和包括气溶胶(或大液滴)或暴露于病毒污染物的间接接触两种途径传播。然而,禽流感病毒(Avian Influenza Virus, AIV)气溶胶的发生、传播与感染机制不详。因此,本课题建立了AIV气溶胶发生及传染的实验模型,动态检测了实验鸡AIV气溶胶的形成时间、浓度及气溶胶对健康鸡只的感染情况；对AIV经不同途径对SPF鸡的感染剂量进行了对比研究；进一步对AIV气溶胶感染SPF鸡后,在实验鸡体内的分布进行了动态观察,并分析了AIV感染对SPF鸡免疫功能的影响；在此基础上,收集生产鸡舍中的空气,应用荧光RT-PCR对AIV气溶胶进行了检测。本研究阐明了禽流感病毒气溶胶(AIV-Aerosol)的发生、传染过程与机制,揭示了AIV气溶胶的传染规律,对于认识像ND、FMD等病毒性气源性呼吸道传染病的传播感染机制,带来有益的启示。本研究分为4部分：1气载H9N2亚型禽流感病毒气溶胶的发生及其传播特性本研究建立了H9N2亚型禽流感病毒(AIV)气溶胶的传染模型,通过观察实验鸡对该病毒经直接接触及气溶胶传染的过程,以探讨AIV气溶胶的发生与传染机制,以及实验动物机体免疫应答状况。实验共进行两个重复：实验1(T1)和实验2(T2)。SPF鸡随机分为3组,每组15只：攻毒组(G1)、接触感染组(G2)和气溶胶感染组(G3),其中G1、G2饲养在隔离器A中,G3饲养在隔离器B中。G1的SPF鸡经点眼、滴鼻接种AIV,G2、G3的SPF鸡不接种病毒,分别经直接接触或气溶胶暴露感染AIV。定期用AGI-30收集隔离器A中的气体样品,测定其中的AIV气溶胶浓度；收集实验鸡的口咽和泄殖腔棉拭子样品,检测棉拭子中的AIV,确定排毒情况；收集实验鸡血液样品,检测抗体滴度。结果显示,T1和T2分别在攻毒后第3天和第2天检测到AIV气溶胶,其浓度在第7天达到高峰,分别为4800 PFU/m3空气和7200 PFU/m3空气；T1G1和T2G1组鸡分别在攻毒后第3天、第2天检测到排毒；G2组最早可在第4天检测到排毒,组内所有实验鸡均可测到排毒；G3组最早可在第7天检测到排毒,仅有部分实验鸡可测到排毒(T1G3：87%,T2G3：80%)；G1组分别在攻毒后第5天和第4天开始检测到抗体,并在第21天和第14天达到高峰,峰值分别为7.07、7.20。这表明SPF鸡人工接种感染AIV后,可通过气管和泄殖腔排出病毒；进而引起饲料和饮水的污染,传播病毒；感染鸡排出的病毒能形成气溶胶,并随着空气流通传播。因此看出,除了经由污染物引起的直接接触传染外,即使彼此间没有直接接触,同一鸡舍或者相邻鸡舍的鸡只也可以通过气溶胶互相传染禽流感。2 H9N2亚型禽流感病毒不同感染途径感染剂量的测量本实验对AIV经不同途径对SPF鸡的感染剂量进行了对比研究。经呼吸道(分别通过人工定量发生AIV气溶胶和滴鼻感染SPF鸡)及消化道(喂服)定量感染AIV,通过检测鸡的特异性抗体判定鸡是否发生感染,计算半数感染剂量(ID50)。结果表明AIV气溶胶感染的ID50为212 TCID50,滴鼻感染ID50为398 TCID50,消化道感染的ID50为23988 TCID50。这说明了AIV对SPF鸡的感染力因感染途径的不同而有差异,气溶胶感染力最强,滴鼻途径次之,消化道感染最弱。3 H9N2亚型禽流感病毒气溶胶感染对SPF鸡免疫功能的影响及组织病理学观察用H9N2 AIV经气溶胶途径感染4周龄SPF鸡,对多个器官的病理变化进行了动态观察,并测量了免疫器官指数及体重的变化。结果发现,胸腺、法氏囊、脾脏的器官指数均出现一个上升、下降、再上升的过程。并且,从第5天开始,胸腺指数显著低于对照组(p<0.05),其他器官指数与对照组相比差异不显著(p>0.05)。剖检可见自第2天开始心、肝、肺、肾、胰腺、胸腺、法氏囊、脾等器官即出现不同程度的充血、肿胀；第5天前后开始萎缩,有的出现坏死灶,肾小管充满白色尿酸盐沉淀；之后逐渐恢复。病理切片显示心、肺、胰腺等出现炎性细胞浸润,肺、胰腺、胸腺、法氏囊、脾脏、肝脏等有出血、淋巴细胞减少、坏死、崩解等变化。ND和IBD疫苗免疫后,抗体水平较低,与对照组相比差异极显著(p<0.01)。这说明H9N2亚型AIV气溶胶感染可导致多器官尤其是免疫器官的损害,进而在一定程度上导致免疫抑制。4鸡舍环境H9亚型AIV气溶胶的检测用AGI-30空气微生物取样器收集山东省6个鸡场的舍内空气样品,同时采集鸡的气管和泄殖腔棉拭子,应用荧光RT-PCR对空气样品及棉拭子中的AIV进行了检测。6个被检鸡舍中,A、C、D和E4个鸡舍检测到气载H9亚型AIV,鸡舍B和F中未检查到病毒。所有被检鸡只的咽喉和泄殖腔棉拭子样品中均未检出该病毒。结果表明,在流行过H9亚型AIV的鸡舍内存在该病毒的气溶胶,这将对人和动物的健康造成危害,应该引起足够的重视。更多还原

【Abstract】 Animal airborne transmission viral diseases ever known are more than 20, such as Newcastle disease (ND), Avian Influenza (AI) and Foot-and-mouth disease (FMD), Porcine reproductive and respiratory syndrome (PRRS) and so on. AI is included in the World Organization (OIE) for Animal Health list of notifiable diseases. Direct contact and indirect contact—including aerosols or droplets and contaminants—are two important routes through which AI spread. However, the occurrence and transmission mechanisms of AIV aerosol remained unknown, and lack of experimental testify. In this study, an infection model was established to determine the process of direct contact and aerosol infection of specific pathogen free (SPF) chickens after inoculation. We have not only established the time of aerosol occurrence but also determined the aerosol concentrations, dynamic changes and their relationship with virus shedding. Infection doses of AIV through different routes for SPF chickens were quantified and compared. Lesions of SPF chickens infected with AIV aerosol and effection on immune function were monitored dynamically. Furthermore, air samples from chicken farms were collected and airborne AIV were detected by fluorescent RT-PCR.Not only were these results important in better understanding the transmission mechanism of AI, but also had implications in controlling ND, FMD and other contagious diseases.This study consists of four parts:1 The occurrence and transmission characteristics of airborne H9N2 avian influenza virusTo better understand the transmission route of H9N2 avian influenza virus (AIV), an infection model of H9N2 subtype AIV was established and two duplicate trials (T1 and T2) were conducted to observe the process of aerosol infection and direct contact in SPF chickens. Fifteen chickens (inoculation group, G1) were inoculated with H9N2 AIV and housed together with another 15 chickens (direct contact group, G2) in the same positive-negative-pressure isolator (A). Fifteen chickens (aerosol challenged group, G3) were bred in another isolator (B) which was connected with A. The SPF chickens of G1 were inoculated with AIV ocularly and nasally, and the SPF chickens of G2 and G3 were not inoculated with virus, but infected through direct contact and aerosols, respectively. Air samples in isolator A were collected with AGI-30 for the detection of aerosol forming. The seroconversion was assessed by the hemagglutination inhibition (HI) test and viral shedding was detected by RT-PCR and HI-Test. AIV aerosols were initially detected in chickens of T1 and T2 at day 3 and 2 post inoculation (dpi), respectively, reaching their peak concentrations of 7,200 PFU/m3 air and 4,800 PFU/m3 air at 7 dpi, respectively. AIV shedding was detected in chickens of T1G1 and T2G1 at 3 and 2 dpi, respectively. Virus shedding was detected in all chickens of G2, but only in 80-87% chickens of G3. Antibodies were initially detected at 5 and 4 dpi in chickens of T1G1 and T2G1, respectively, reaching their peak levels of 7.07 and 7.20 at 21 and 14 dpi, respectively. The results showed that after being infected with AIV, SPF chickens could excrete viruses via trachea and cloaca, thus leading to contamination of feedstuff and drinking water, as well as viral transmission; infected chickens may excrete viruses to form aerosols which may be transmitted by air flow. Therefore, it is obvious that in addition to direct contact infection due to contaminants, chickens in the same or adjacent henhouses could also be infected mutually with AI, despite no direct contact.2 Quantification and comparison of infection doses of H9N2 subtype avian influenza virus through different routes for SPF chickensIn this study, infection doses of H9N2 subtype avian influenza virus through different routes for SPF chickens were quantified and compared. SPF chickens were randomly divided into 3 groups:aerosol infection group, intranasal infection group and gastrointestinal tract infection group. Each chicken was inoculated with the same volume of different dilution of AIV. Infection was determined by detecting of the specific antibody to AIV. Infection dose 50 percent (ID50) were determined by the method of Reed-Muench. The results showed that ID50 of aerosol infection was 212 TCID50, of intranasal route was 398 TCID50, and of gastrointestinal tract infection was 23988 TCID50. It indicated that the efficiency was different when SPF chickens infected with AIV through different routes, aerosol infection was stronger than that of intranasal route, and the gastrointestinal tract infection was the weakest.3 Impact on immunologic function of SPF chicken experimentally infected with H9N2 AIV aerosolThe pathological changes of SPF chickens infected with H9N2 AIV aerosol were observed dynamically, and immune organ index and weight were measured. The results showed that the thymus index was significantly lower than the control group (p<0.05) since 5 dpi, but the bursa and spleen index showed no significant (p>0.05). At 2 dpi, histopathological changes of heart, liver, lung, kidney, pancreas, thymus, bursa and spleen showed hyperemia, hemorrhage and tumefaction. Atrophy of the thymus, bursa and spleen was detected at 5 dpi, and renal tubular filled with white urate deposition, then gradually recover. Pathology showed heart, lung and pancreas appeared inflammatory cell infiltration, and lung, pancreas, thymus, bursa, spleen, liver showed hemorrhage, lymphocytes decreased, necrosis and/or collapse. Antibodies reactions to ND and IBD vaccines were significantly lower compared with the control group (p<0.01). This showed that the H9N2 subtype AIV infection could cause multiple organs damages, in particular the immune organs, and thus led to immunosuppression to some extent.4 Detection of airborne H9 subtype avian influenza virus in chicken housesAir samples from 6 chicken farms of Shandong Province were collected by AGI-30 air sampler, and oropharyngeal and cloacal swabs were sampled simultaneously. Fifteen air samples were collected in each chicken house. Fluorescent RT-PCR was used for the detection of AIV in air and swab samples. Airborne H9 AIV was detected in four (A, B, C and E) out of 6 farms, and no AIV was detected in swabs. The results showed that airborne H9 AIV was detectable in farms after an outbreak. The AIV aerosol would do great harm to human and animal health and should be paid more attention to.更多还原