【作者】 杨萍萍；

【导师】 朱瑞良；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2012， 博士

【摘要】 禽波氏杆菌（Bordetella avium，B. avium）最早于1967年分离自火鸡，主要引起禽类的鼻炎、眼炎等呼吸道症状。近年来不断从出现孵化率降低和弱雏率增高的种鸡孵化场鸡胚中分离到该菌，为探明其致病性的变化，本研究对2001-2011年间出现疫情的规模化种鸡孵化场的致病病原进行了分析，然后对分离到的B. avium菌株进行了强毒株的筛选以及基因分型，对筛选到的强毒株进行了定植规律检测，并研究了其在免疫抑制状态下致病性的变化。本研究共分为五个部分：1.规模化种鸡孵化场胚胎性疫病病原分析本试验对2001~2011年间全国8省市共计72个种鸡孵化场送检或采集的将出壳死亡鸡胚或死亡的刚出壳雏鸡进行了病原的检测和分析。经多重PCR和RT-PCR扩增以及细菌、支原体的分离培养，在33个孵化场检测到了鸡传染性贫血病毒（CIAV）、禽网状内皮增殖症病毒（REV）和鸡白血病病毒（ALV）三种病毒的单纯感染和多重感染，占45.8%；有6个孵化场存在有支原体感染，占检测孵化场总数的8.33%；在52个孵化场检测到了细菌的单纯感染或多重感染，占到了72.2%。共分离到禽波氏杆菌、葡萄球菌、大肠杆菌、沙门氏菌和绿脓杆菌5种细菌，共计105株。在检测的47种药物中5类细菌耐受药物的数量在15-32种，耐药性总体呈上升趋势，其中以葡萄球菌的耐药最为严重。孵化率下降、弱雏率增加等状况主要由细菌的多重感染（47.22%）和病毒与细菌的共感染（19.44%）引起，单纯的病毒、细菌和支原体的感染仅占了极少的比例，分别为6.94%、9.72%和4.71%。2. B.avium鸡胚分离株的生物学特性及致病性研究1991-2010年间分离到的22株B. avium菌株的培养特性及生化特征基本一致，且均具有不同程度的耐药性。在所检测的47种药物中，不同分离株耐受药物数量为8-30种。对近年来的12株鸡胚分离株进行11日龄鸡胚ELD50和1日龄雏鸡LD50的测定，结果发现ELD50在6.75-27.95CFU之间，LD50在9.2×105-3.4×108CFU之间，从中筛选出3株毒力较强的菌株Ba15、Ba17和Ba21。3株B.avium经鼻腔感染1日龄雏鸡后发现Ba15对雏鸡的生长抑制和ND油苗免疫后的抗体水平增长的抑制作用最强，在肺脏和气管中的感染率为100%，在各内脏中引起感染的鸡只比例在30%-40%。对Ba15以不同方式感染雏鸡发现腹腔感染的危害最严重，生长抑制和免疫抑制作用最强，内脏的感染比例最高（100%），比较接近胚胎期感染，经鼻腔和经眼两种方式感染的比较发现鼻腔感染的危害略强于经眼感染，B.avium入侵内脏的鸡只比例分别为30-40%和30%。3.禽波氏杆菌鼻腔攻毒雏鸡的定植规律研究用筛选到的强毒株Ba15经鼻腔感染1d雏鸡，采用组织匀浆涂布细菌计数法、接种环划线法、抹片染色法以及建立的细胞内抗原原位检测的间接免疫酶和间接免疫荧光组化法进行细菌感染后在组织中的实时和定位检测，在感染后1h即可在气管和肺脏中检测到少量的B.avium，攻毒后12h内肺脏中细菌的数量急剧上升，5d时入侵肝脏、心脏和脾脏，7d时发现其在肾脏的感染。内脏感染后的荷菌数量高峰期出现在14-28日龄，35日龄后在内脏中的感染比例逐渐下降，和B.avium感染后抗体水平的增长以及抗病能力的增强直接相关。经过间接免疫酶和间接免疫荧光组化检测B.avium感染后在组织细胞中的定植部位主要是位于胞浆和细胞间质中，且呈弥散性分布。4.禽波氏杆菌RAPD基因分型研究用细菌基因组抽提试剂盒对22株禽波氏杆菌的DNA进行提取，获得了高纯度和高浓度的细菌基因组DNA，并建立了稳定性和重复性好的RAPD扩增程序。经过对一系列随机引物的筛选，有6条引物能够产生清晰且多态性好的条带。运用筛选的6条随机引物对22株B.avium进行扩增，获得了RAPD指纹图谱。运用分析软件对指纹图谱进行统计分析，计算出各菌株间的遗传距离并进行聚类分析，绘制亲缘关系树状图，最终以遗传距离0.402为界将22株B.avium分成了3个聚类群，其中第一聚类群又可以分为3个亚群，第三聚类群可以分为两个亚群。其中第一聚类群包括了2000年之前的10个分离株（Ba1-Ba10）和2001年之后分离到的Ba12、Ba13、Ba14、Ba20，第二聚类群和第三聚类群中均为2001年之后的分离株，其中Ba15、Ba17、Ba18、Ba22属于第二聚类群，Ba11、Ba16、Ba19和Ba21属于第三聚类群。5. REV与禽波氏杆菌共感染的致病作用研究B. avium单纯感染鸡胚致病力较弱，鸡胚死亡时间较晚，主要危害是产生大量弱雏，且孵出的雏鸡长期带菌，生长缓慢，产蛋日龄延后。REV与B. avium共感染鸡胚比B.avium单纯感染时的鸡胚死亡时间提前，孵化率仅为30%，死胚率、弱雏率增高，且弱雏的最终成活率为0。B. avium单纯感染和与REV的共感染对不同日龄雏鸡的LD50的检测表明，雏鸡的感染日龄至关重要，不管是单纯感染组还是共感染组，高日龄的LD50值明显高于低日龄感染组。胚胎期及1日龄REV感染均显著降低了B. avium对不同日龄雏鸡LD50，尤其是胚胎期REV后B. avium对1日龄雏鸡的LD50值最低。REV作为一种免疫抑制性病毒，感染越早对B. avium的LD50影响越大。B. avium单纯鼻腔感染对雏鸡的生长抑制和免疫抑制作用均较弱，但胚胎期REV感染的雏鸡再经鼻腔感染B. avium后，对生长抑制和免疫抑制均具有协同作用，尤其是免疫抑制方面，尽管是以REV引起为主，但与B. avium共感染后加快了免疫器官细胞的凋亡、坏死，并且使抗体水平的持续时间缩短。另外共感染还提高了鸡群中“僵鸡”的比例，使B. avium在内脏中的检出时间提前、感染比例增大，并且在内脏中的持续感染时间延长，同时还发现在共感染组中继发其他细菌感染的病例数增多。B. avium单纯于1、7、14、和21日龄经鼻腔感染雏鸡时发现，感染日龄至关重要，1日龄和7日龄的雏鸡比较敏感，21日龄感染组症状上仅表现为一过性感染。不同日龄感染组之间相比，感染日龄越低对雏鸡的生长抑制和免疫抑制越严重，B. avium入侵内脏的病例数也越多。1日龄雏鸡感染REV后再分别于1、7、14、和21日龄经鼻腔感染B. avium发现，在对雏鸡的生长抑制和免疫抑制上具有一定的协同作用，并且各日龄共感染组与单纯感染组相比内脏的感染比例均有不同程度的增加。在共感染组中低日龄时雏鸡对B. avium的易感性仍强于高日龄，但感染日龄之间的差异在显著缩小。总之，本研究结果表明我国种鸡孵化场的胚胎性疫病多病原共感染的现象非常普遍，用RAPD方法将十年来的B. avium流行株分成了三个聚类群，并从分离到的B. avium鸡胚分离株中筛选出一株强毒株，对其进行了定植规律和与REV共感染的致病研究，发现在免疫抑制状态下，B. avium入侵内脏的时间提前，感染比例增大，病理损伤更严重，且对生长抑制和免疫抑制具有明显的协同作用，继发细菌感染的病例增多，大大增加了B. avium在鸡群中的危害。更多还原

【Abstract】 Bordetella avium (B. avium) was first isolated from young turkeys in1967. It mainlycauses respiratory diseases, including ophthalmia and rhinitis, in poultries. However, in recentyears our research group constantly isolated B. avium strains from dead embryos in somebreeder hatcheries with reduced hatchability rate and increased hatched-weak chicken rate. Toanalyze the pathogenicity of B. avium isolates from chick embryos, the study conducted ananalysis of the causatives in scale hatcheries of2001-2011. B. avium strains isolated fromembryos were gene typed by RAPD and screened for virulen strain whose colonizationpattern would be detected and pathogenicity changes under immune inhibition would bestudied.This study is divided into five parts:1Pathogen analysis of embryonal diseases in scale hatcheriesIn this experiment, pathogen detection and analysis were conducted on dead chickembryos or newly hatched chicks from72chicken hatcheries in8provinces of China from2001to2011. Multiplex PCR and RT-PCR amplification, as well as bacterial andmycoplasma isolation were done about the samples. Single infection and multiple infectionsof CIAV, REV and ALV were detected from33hatcheries, accounting for45.8%.Mycoplasma infection lied in6hatcheries, with a rate of8.33%. Single infection and multipleinfections of bacteria were detected from52hatcheries, accounting for72.2%. Five kinds ofbacteria were isolated with a total number of105, including Staphylococcus aureus,Escherichia coli, Bordetella avium, Salmonella and Pseudomonas aeruginosa. Number oftolerated drugs of5kinds of bacteria was15-32from47tested drugs. General antibioticresistance is on the rise, with the most serious drug-resistance of Staphylococcus. The declinein hatchability and increase of weak chick rate are primarily caused by multiple bacterialinfection (47.22%) and viral and bacterial co-infection (19.44%). Single virus, bacteria andmycoplasma infections accounted for only a very small proportion, with infection rates of6.94%,9.72%and4.71%respectively.2Biological characteristic and pathogenicity of B.avium isolates from chickembryos Culture characteristics and biochemical characteristics of22B. avium isolates in1991-2010are basically the same, and all of them have resistance to drugs. Tolerated drugnumbers of different isolates vary from8to30in47detected drugs. ELD50to11-day-oldchick embryo and LD50to1-day-old chicks were detected about12isolates from chickembryo in recent years. Results showed that their ELD50were between6.75-27.95CFU andLD50between9.2×105-3.4×108CFU. Accordingly three virulent strains Ba15, Ba17, andBa21were screened from them. Ba15had strongest inhibition of growth and immuneresponse to NDV among the3isolates after nasal infection in1-day-old chicks, with a rate of100%infection in the lungs and trachea, and30%-40%infection in various internal organs. Itwas discovered by challenging chicks with Ba15in different ways that intra-abdominalinfection with highest proportion of visceral infections (100%), close to fetal infections, hadstrongest growth inhibition and immune suppression. Nasal infection with a infection rate of30-40%in viscera organs was more serious than ocular infection (30%).3Colonization pattern of B. avium in nasal challenged SPF chickensBacterial counting of the homogenate, inoculating loop line method, smear stainingmethod, indirect immunoenzyme histochemistry, as well as indirect immunofluorescencehistochemistry were used to detect B. avium infection in different tissues after intranasalinfection of Ba15. Results showed that the bacteria were isolated from tracheas and lungs at1h post-infection. As time prolonged, B. avium continually increased in lungs within12hpost-infection. Afterwards, they colonized livers, hearts and spleens at5d and then infectedkidneys at7d. The peak of bacteria count appeared between14-28days of age. After35daysof age, the proportion of infections in the viscera organs gradually decreased. It was directlyrelated with antibody levels of B. avium and enhanced disease resistance. Visceral organswere colonized extensively by B. avium which were distributed densely in intercellular spaceand cytoplasm by detecting with indirect immunoenzyme histochemistry and indirectimmunofluorescence histochemistry.4Gene typing of B. avium isolates by RAPD analysisBacterial genomic DNAs of high purity and high concentration were obtained by using acommercially available kit. RAPD amplification procedure which had good stability andreproducibility were established. Accordingly, a set of20commercially available primers wasscreened out to identify suitable primers for RAPD analysis of B. avium isolates. At last6primers R1, R2, R4, R6, R8and R10resulted in clear fingerprints of well polymorphism andwere used to evaluate the B. avium isolates. Based on their RAPD patterns, a dendrogram allowed the separation of the B. avium isolates into3genetic similarity clusters. The firstcluster could be divided into3subsets, and the third cluster could be divided into2subsets.The first cluster included10isolates before2000and isolate Ba12, Ba13, Ba14and Ba20after2001. The isolates in the second cluster and the third cluster were all after2001. Ba15,Ba17, Ba18and Ba22were in the second cluster and Ba11, Ba16, Ba19and Ba21were in thethird cluster.5Synergistic effects of co-infection of REV and B. aviumB. avium of single infection in chick embryo had weak virulence with late death time ofchick embryos. The main harm is to generate a large number of weak chicks, and the chickswould be carriers for a long time, grow slowly and egg laying days delay. The death of chickembryos in co-infection of REV and B. avium were ahead of time with a hatching rate of only30%. Dead embryo rate and weak young chick rate increased, and the ultimate survival rate ofweak young chicks was0. Results of LD50detection of single B. avium infection andco-infection of REV and B. avium at different ages showed that infection age is very essential.Whether in single infection or co-infection, LD50value of high age was significantly higherthan that of low age. REV infection both at the embryonic period and1day old significantlyreduced the LD50values of B. avium, especially REV infection at the embryonic period. REV,as an immunosuppressive virus, earlier infects in chicks, greater effect to pathogenicity of B.avium.Effects of B. avium single nasal infection on growth inhibition and immune inhibition areweak. REV infection to embryos then nasal infection of B. avium to chicks had synergy ongrowth inhibition and immunosuppressive. Immunosuppression, although mainly caused byREV, B. avium infection accelerated the immune organ cell apoptosis, necrosis, and shortenedthe duration of antibody levels. In addition, co-infection improved the proportion of stiffchicken number. The detection time of B. avium in the visceral organs were ahead of time, theinfection rate increased, infection time extended in the viscera, and secondary infectionnumber also increased.It was discovered that infection age is crucial in single infection of B. avium at1,7,14,and21ages. At the age of1and7, chicks are more sensitive. Symptoms showed onlytransient infection when challenged at21ages. Compared between infections at different ages,the lower infection age was, the more serious effect on growth inhibition and immunesuppression, as well as the more numbers of cases of B. avium invaded into visceral organs.When B. avium was challenged at1,7,14, and21ages after REV infection at1age, it was showed that a certain degree of synergy appeared on growth inhibition and immunesuppression. Compared to single infection of B. avium, the proportion of viscera invaded by B.avium in co-infection increased. The chicks of low ages were still more susceptible than thoseof high ages, however, the difference between the infections at different ages reduced.In short, the study results show that multi-pathogen infection was widespread in chickenhatcheries of our country. B. avium isolates in nearly ten years were divided into3clusters.One virulent strain was screened and its colonization pattern in chickens and synergisticpathogenicity with REV were studied. Under state of immune suppression B. avium invadedthe visceral ahead of time, the infection ratio increased and pathological changes were moreserious. At the same time co-infection of REV and B. avium had synergistic effect on growthsuppression and immune suppression. Immunosuppressive virus infection significantlyincreased the hazard of B. avium in the chickens.更多还原