鸡包涵体肝炎过程中免疫细胞变化规律的研究
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摘要
本研究在鸡包涵体肝炎（Inclusion Body Hepatitis，IBH）过程中，通过检测单核巨噬细胞和T、B淋巴细胞在外周血、各淋巴器官和肝脏中的消长规律及其凋亡规律与其表达黏附分子CD18的变化，从而揭示其发病过程中的分子免疫机理。本研究采用禽腺病毒Ⅷ型毒株（FAV-8）1：50倍稀释，0.3ml/只口服接种200只1日龄SPF雏鸡，分别在攻毒后1，3，5，7，9，12，15，20，25，30天迫杀攻毒组10只，对照组10只，制备外周血、各淋巴器官的淋巴细胞悬液，用流式细胞仪检测了血液和淋巴器官中T淋巴细胞及其亚群的变化、B淋巴细胞的变化规律，血液和脾脏中单核巨噬细胞的变化，血液中的单核巨噬细胞与淋巴细胞的凋亡规律及其表面CD18黏附分子的变化规律等。并同时采集肝组织和各淋巴组织等，制备石蜡切片和肝脏冰冻切片，做H.E.染色和免疫组化染色，观察病理变化和肝脏中淋巴细胞及其亚群以单核巨噬细胞的变化。结果显示：（1）IBH病毒对SPF雏鸡主侵肝脏，眼观病变呈以肝脏为主的出血性坏死,同时免疫器官有不同程度的出血，反复发病，呈不完全恢复。（2）血液Ia+ 活化单核细胞的损伤最明显，并在第5天时达到高峰，但血液和脾脏中K1+单核巨噬细胞一直处于积极状态。（3）早期凋亡单核细胞第5天就达到高峰，晚期凋亡单核细胞均显著高于对照；则早期凋亡淋巴细胞第7天显著高于对照，之后有下降趋势。（4）法氏囊Bu-1a+B淋巴细胞的流失最为严重，脾脏和血液中B细胞在病变高峰期显著升高，对本病的免疫起着积极作用。（5）CD8+ T细胞受损最重，CD3+ T和CD4+ T细胞虽有损伤，但显著或不显著高于对照的多些。在血液和脾脏中的TCRγδ+ T细胞在病变严重时期有过显著升高，但在病毒攻击的初期和强病变之后均显著降低。（6）单核细胞表面CD18第15到20天显著高于对照，之后逐渐下降，而淋巴细胞表面CD18第20天显著高于对照，之后下降。（7）肝脏中第3天开始出现K1+ 单核巨噬细胞，到第9天时达到高峰，之后明显下降；第3天开始出现少量CD3+T细胞，到第12天阳性细胞逐渐增多，之后数量减少，到第25天再升高；CD4+T细胞第12天时阳性细胞的数量较多；CD8+T细胞的数量比CD4+T多；TCRγδ+ T细胞的数量少。脾脏对照呈阳性，空白对照和健康鸡对照均呈阴性。以上结果表明，IBH患病鸡的免疫功能抑制和反复发病并呈现不完全恢复是因为由淋巴细胞和单核细胞的大量损伤和凋亡所导致。（8）本研究首次采用流式细胞仪检测了实验性IBH过程中SPF雏鸡血液、胸腺、法氏囊和脾脏的淋巴细胞及其亚群和单核巨噬细胞的变化规律及其在血液中凋亡规律；首次应用免疫组化法检测了实验性IBH过程中肝脏中免疫细胞的类型及其变化规律。
This study revealed molecular immunity mechanism of Avian Inclusion Body Hepatitis（IBH）by detecting the growth and decay or apoptosis regulation of macrophages and T、B lymphocytes in blood and immunity organ or liver，and also detecting the regulation of cells that express the adhesion molecules CD18. Two hundreds of one-day-old SPF chickens were inoculated FAV-8 mouth injection at a dose of 0.3ml per chicken，consisting of 1：50（w/v）tissuer virus suspension.And 10 of experimental group and 10 of control group were killed at 1,3,5,7,9,12,15,20,25 and 30 days postinfection respectively.All the lymphocyte suspension of peripheral blood,thymus,Fabricius bursa and spleen,were collected.And detected the growth and decay or apoptosis regulation of macrophage and T、B lymphocytes in blood and in variety immunity organ，and also detected the regulation of its adhesion molecules CD18 with flow cytometry. At the same time, liver and every immunity organ were fixed in 10% formalin for preparing waxy embedded sections and liver ice embedded sections，and to reveal the pathological changes and the regulation of lymphocytes or its subpopulations and macrophages in liver by the staining method of H.E. and immunohistochemistry . The research results that：(1) The IBH virus violates the liver of chicken primarily, showing the the view pathological changes of hemorrhagic and necrotic hepatitis mainly, and bleeds with the immunity organ.The disease develops again and again,and never completely recovered. (2)The Ia+ macrophage in blood was damaged mostly and to highest level at 5 day postinfection ,but the K1+ macrophages were in a positive situation all the time in blood or spleen. (3) The early apoptosis macrophages had arrived the highest level at 5 day postinfection, and the late apoptosis macrophages were significantly higher than control continuously; The early apoptosis lymphocytes were higher than control at 7 day postinfection, and then they were decreasing. (4)The Bu-1a+B lymphocytes in Fabricius bursa were cleared seriously,but it goes up significantly at the period of strong pathological changes in spleen and in blood . (5)Whether in thymus, spleen or in blood, the CD8+ T Lymphocytes were damaged mostly.Although the CD3+T and CD4+ T Lymphocytes had been harmed, the most of them were higher than control. TCRγδ+ T Lymphocytes in the blood and spleen had gone up significantly at the period of serious pathological changes, but they had greater drop in yield after the initial stage of virus attack and the strong pathological changes. (6)The adhesion molecule CD18 of macrophage was
higher than control from 15 day postinfection to 20 day postinfection ,and then decreasing; the adhesion molecule CD18 of lymphocyte was higher than control at 20 day postinfection.(7) K1+ macrophages appeared at 3 day postinfection in liver, they arrived at the highest level at 9 day postinfection, and then they were decreasing; CD3+T lymphocytes also appeared at 3 day postinfection in liver, and they were increasing at 12 and 25 day postinfection respectively;CD4+T lymphocyte, the amount of them was increasing at 12 day postinfection;CD8+T lymphocytes, the amount of them was more than the CD4+T lymphocytes，;A little of TCRγδ+ T lymphocytes were appeared in liver.(8) This research examined the regulation of macrophages in blood or in spleen and its apoptosis in blood in the process of experimental IBH by flow cytometry for the first time; Examined the regulation of lymphocytes of experimental IBH in liver by the method of immunohistochemistry for the first time.
This study revealed molecular immunity mechanism of Avian Inclusion Body Hepatitis（IBH）by detecting the growth and decay or apoptosis regulation of macrophages and T、B lymphocytes in blood and immunity organ or liver，and also detecting the regulation of cells that express the adhesion molecules CD18. Two hundreds of one-day-old SPF chickens were inoculated FAV-8 mouth injection at a dose of 0.3ml per chicken，consisting of 1：50（w/v）tissuer virus suspension.And 10 of experimental group and 10 of control group were killed at 1,3,5,7,9,12,15,20,25 and 30 days postinfection respectively.All the lymphocyte suspension of peripheral blood,thymus,Fabricius bursa and spleen,were collected.And detected the growth and decay or apoptosis regulation of macrophage and T、B lymphocytes in blood and in variety immunity organ，and also detected the regulation of its adhesion molecules CD18 with flow cytometry. At the same time, liver and every immunity organ were fixed in 10% formalin for preparing waxy embedded sections and liver ice embedded sections，and to reveal the pathological changes and the regulation of lymphocytes or its subpopulations and macrophages in liver by the staining method of H.E. and immunohistochemistry . The research results that：(1) The IBH virus violates the liver of chicken primarily, showing the the view pathological changes of hemorrhagic and necrotic hepatitis mainly, and bleeds with the immunity organ.The disease develops again and again,and never completely recovered. (2)The Ia+ macrophage in blood was damaged mostly and to highest level at 5 day postinfection ,but the K1+ macrophages were in a positive situation all the time in blood or spleen. (3) The early apoptosis macrophages had arrived the highest level at 5 day postinfection, and the late apoptosis macrophages were significantly higher than control continuously; The early apoptosis lymphocytes were higher than control at 7 day postinfection, and then they were decreasing. (4)The Bu-1a+B lymphocytes in Fabricius bursa were cleared seriously,but it goes up significantly at the period of strong pathological changes in spleen and in blood . (5)Whether in thymus, spleen or in blood, the CD8+ T Lymphocytes were damaged mostly.Although the CD3+T and CD4+ T Lymphocytes had been harmed, the most of them were higher than control. TCRγδ+ T Lymphocytes in the blood and spleen had gone up significantly at the period of serious pathological changes, but they had greater drop in yield after the initial stage of virus attack and the strong pathological changes. (6)The adhesion molecule CD18 of macrophage was
higher than control from 15 day postinfection to 20 day postinfection ,and then decreasing; the adhesion molecule CD18 of lymphocyte was higher than control at 20 day postinfection.(7) K1+ macrophages appeared at 3 day postinfection in liver, they arrived at the highest level at 9 day postinfection, and then they were decreasing; CD3+T lymphocytes also appeared at 3 day postinfection in liver, and they were increasing at 12 and 25 day postinfection respectively;CD4+T lymphocyte, the amount of them was increasing at 12 day postinfection;CD8+T lymphocytes, the amount of them was more than the CD4+T lymphocytes，;A little of TCRγδ+ T lymphocytes were appeared in liver.(8) This research examined the regulation of macrophages in blood or in spleen and its apoptosis in blood in the process of experimental IBH by flow cytometry for the first time; Examined the regulation of lymphocytes of experimental IBH in liver by the method of immunohistochemistry for the first time.
引文
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3 Reece.R. L et al. Observation on naturally occuring inclusion body hepatitis in Victorian chikens. Australia Veterinary Journal ,1986,63(6):201—202
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8 Howell， J. Inclusion Body Hepatitis in chickens. Canada Vererinary Journal， 1970, 11(5)
9 Christensen N.H and Md Saifuddin .A Primary Epidemic of Inclusion Body Hepatitis in Broilers .Avian Diseases, 1989,33：622-630
10 Pilkinton et al.Adenovirus - Induced Inclusion Body Hepatitis in Four—day—old Broiler Breeders. Avion Diseases , 1997,41:472-747
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25 Kefford. B. Serological identification of aian aenoviruses iolated From a cases of Inclusion Body Hepatitis in a cockeral. Avian Diseases， 1985,24(4)
26 James. Inclusion Body Hepatitis in I—day—old turkeys. Avian Disease， 1988, 32(12)
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3 Reece.R. L et al. Observation on naturally occuring inclusion body hepatitis in Victorian chikens. Australia Veterinary Journal ,1986,63(6):201—202
4 Jack. S. W. Further Characterization of an Avian Adenovirus Associated with Inclusion Body Hepatitis in Bobwhite Quails .Avian Diseases , 1990,34:526-530
5 Young J.A.Inclusion Body Hepatitis Outbreak in Broiler Flocks .The Veterinary Record January, 1972,15
6 Reece.R.L.An Unusual Case of Inclusion Body Hepatitis in a Cockerel .Avion Diseases ,30(1)
7 缪延武译.对马来西亚肉用鸡的包涵体肝炎病鸡中禽腺病毒的分离和鉴定.国外兽医学——畜禽传染病，1985，5（1）：38-39
8 Howell， J. Inclusion Body Hepatitis in chickens. Canada Vererinary Journal， 1970, 11(5)
9 Christensen N.H and Md Saifuddin .A Primary Epidemic of Inclusion Body Hepatitis in Broilers .Avian Diseases, 1989,33：622-630
10 Pilkinton et al.Adenovirus - Induced Inclusion Body Hepatitis in Four—day—old Broiler Breeders. Avion Diseases , 1997,41:472-747
11 阴天榜，刘兴友等.家禽免疫学[M].北京:中国农业科技出版社,1999.10~25
12 朱广柱、林钧安、王大庸等.鸡包涵体肝炎病理形态学观察.中国兽医杂志，1987,12
13 Scott. P. C.Inclusion body hepatitis associated with adenovirus-like particles in a cockatiel .Australian Veterinary Journal, 1986, l.63(10)
14 刁有祥,李久芹等.鸡包涵体肝炎的诊断.中国畜禽传染病,1996,1:40~41
15 王立钧.一起肉鸡包涵体肝炎的诊断.Animal Husbandry＆Veterinary Medicine,2000,32(4):26
16 Wells. R. J. H. A fatal adenovirus infeetion of broiller chickens: inclusion body hepatitis .The Veterinary Record， 1974, 25(5)
17 殷震、刘景华主编.动物病毒学. 科学出版社 , 1995
18 Saifuddin. MD. Reproduction of Inclusion Body Hepatitis in conventionally raised chickens inoculated with a New Zealand Isolate of avian adenovirus. New Zesland veterinany Journal， 1990,6:62~65
19 Saifluddin. Md， Wilks. C. R.Pathogenesis of an acute Viral hepatitis:inclusion body hepatitis in the chicken. Archives of virology, 1991,116(1—4):33—43
20 Hourells. R. J. A fatal adenovirus infection of oroiler chickens Inclusion Body Hepatitis. The Veter inary Record， 1974, 25(5)
21 Howell. J. H. Inclusion Body Hepatitis in chickens canada. Veternary Journal， 1970, 11(5)
22 Macpherson I. Inclusion Body Hepatitis in a broiler integration. The Veterinary Record， 1974,34
23 Young. J. A.. Inclusion Body Hepatitis break out in broilerflooks. The Veterinary Recorcl Journal， 1972,15
24 Hoffman， R:.Lesions in chickens with spontaneous or experimental infectious hepecto—myelopojeetic
disease in Germany .Avian Disease， 1974,19(2):225-237
25 Kefford. B. Serological identification of aian aenoviruses iolated From a cases of Inclusion Body Hepatitis in a cockeral. Avian Diseases， 1985,24(4)
26 James. Inclusion Body Hepatitis in I—day—old turkeys. Avian Disease， 1988, 32(12)
27 Shrivastava. P. K. Prevalence of Inclusion Body Hepatitis in chicen in Uttar Pradesh， a Pathomorphological study. Inclion Veterinary Joural， 1990, 67(12)
28 王纯洁.实验性鸡包涵体肝炎病毒抗原定位及发病机理的研究.硕士学位论文.1998
29 刘月焕.实验性鸡包涵体肝炎的病理学研究（内蒙古农农学院硕士学位论文）
30 Pettit.J. R. Inclusion Body Hepatitis in broiler chickens. Avian Diseases， 1972,16
31 Fadly A. M,Role of the Bursa of Fabricius in the Pathogenicity of Inclusion Body Hepatitis and Infeetious Bursa Disease Viruses .Avian Diseases , 20(3)
32 王百合,王安忠等.肉仔鸡包涵体肝炎诊断初报.现代化农业,1995,9:19
33 刘聚祥,杨润德等.鸡包涵体肝炎油乳剂灭活苗的研制(简报).河北农业大学学报,2000,23(3):86~87
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