低致病性禽流感病毒与禽源大肠杆菌的协同致病作用
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摘要
目前，低致病性禽流感病毒(mildly pathogenic avian influenza virus，MPAIV)在我国分布较广，对鸡群的产蛋率、肉鸡的增重有明显影响，尤其是和大肠杆菌混合感染时可产生一定的病死率，在肉鸡可招致较高致死率，给养禽业造成了严重的经济损失。迄今，有关MPAIV与大肠杆菌(Escherichia coli,E.coli)协同致病作用机理的研究很少。针对MPAIV H9亚型在我国分布较广，MPAIV与禽大肠杆菌病常并发、继发等实际情况，在SPF鸡以不同方式人工感染MPAIV和E.coli O26株后，对试验鸡的致死率、对细菌在血液及各脏器的动态分布、病理变化、MPAIV的排毒规律等作了深入研究，以探索两者之间可能的协同致病作用，为揭示这两种疾病并发、继发的原因，最终控制其流行提供理论依据。
1．实验鸡经不同顺序接种H9亚型禽流感病毒、禽源大肠杆菌后死亡率比较试验
将10日龄SPF鸡随机分组，每组25羽，隔离饲养，气管内接种。A组10日龄接种MPAIV；B组10日龄同时接种大肠杆菌和MPAIV；C组10日龄接种大肠杆菌，48h后再接种MPAIV；D组10日龄接种MPAIV，48h后再接种大肠杆菌；E组10日龄接种大肠杆菌；F组作为健康对照组。在各组攻毒结束后，观察死亡情况，结果如下：
A、B、C、D组死亡率分别为12％、32％、16％、24％，E组没有死亡，进行卡方检验后，A、B、C、D与E组、B与A组死亡率差异显著。
结果表明：与单独接种MPAIV或E.coli组相比，先MPAIV后大肠杆菌接种组的死亡率有了明显的提高，同时接种组的死亡率有显著的提高，而先接种大肠杆菌再接种MPAIV，死亡率的变化不明显，表明协同致病程度与2种病原接种顺序的先后有关。
2．实验鸡单独或混合感染禽源大肠杆菌后细菌的动态分布
将10日龄SPF鸡随机分组，每组27羽，隔离饲养。A组10日龄气管内接种大肠杆菌；B组10日龄气管内同时接种大肠杆菌与MPAIV；C组为健康对照组。各组在接种后1-96定时分批扑杀：观察它们的临诊症状，并进行细菌学检查。其结果如下：
细菌学检查发现各组的气管、肺、血液、脾脏、肝脏和肾脏中细菌数量与接种时间有关：①A组接种后12-72h出现菌血症，B组在接种后3-96h出现菌血症。细菌数后者恒高于前者；②各试验组肺中的检菌率明显高与其它脏器。比较B、A两组发现，B组肺中细菌明显减少时间延长12h，在这延长时间内细菌数目差异极显
一
著；③A、B组气管接种后 lh出现细菌的增殖，但 B组气管中细菌增殖比。迅速，
细菌数目也比A组多，l2 细菌数目差异显著，在3h后差异不显著。④各组脾脏。
肾脏和肝脏在接种后偶尔可以分离到细菌，而且数量很少。
上述结果表明：试验鸡经气管接种病原性大肠杆菌后，细菌主要在肺中定居并
大量繁殖，进入血液，引起全身反应；联合感染大肠杆菌和MPAIV后，可使大肠
杆菌在肺中定居时间延长。
3．实验鸡人工感染Hg亚型禽流感病毒、禽源大肠杆菌后呼吸器官的超微动态病理
变化
将10日龄SPF鸡随机分组，接种组30羽、对照组20羽，隔离饲养。A组10
日龄气管内接种大肠杆菌；B组IO日龄气管内同时接种大肠杆菌和＊PA w：C组
10日龄气管内接种 MPAIV；D组为健康对照。在接种后定时分批扑杀，采集气管、
肺等进行组织学的超微病理切片和常规病理切片观察。其结果如下：
组织病理学观察可见，B组气管和肺的炎症发生时间早于其它组，恢复也慢，
肺在接种大肠杆菌和 MPAIV后第 lh开始出现炎症一直持续至试验结束，气管粘膜
纤毛严重脱落。
超微结构观察结果为：B组在接种大肠杆菌和 MPAIV后 4－48h，气管纤毛全部脱
落，整个粘膜层均可见MPAIV粒子；肺在接种大肠杆菌和 MPAIV后 6h，肺11型
上皮细胞全部溶解成空泡，只留下I型上皮细胞，严重病变持续到接种后48h，仍
可见＊型上皮细胞细胞器空泡化。
E COlf O26株可引起气管纤毛细胞的损伤，而 MPAIV不仅能破坏气管粘膜的
上皮细胞，而且还能损害肺组织中的＊型上皮细胞。当 E COn与MPAIV混合感染
时，气管、肺的病变更为严重。
4．实验鸡单独或混合感染Hg亚型禽流感病毒后气管、肺及泄殖腔样品的病毒分离
10日龄叩F鸡随机分组，接种组30羽。对照组15羽，隔离饲养。A组12日
龄气管内接种＊PA w：B组12日龄气管内同时接种大肠杆菌与MPA IV：C组IO日
龄气管内接种大肠杆菌dh后再气管内接种MPA W：D组u 日龄气管内接种
MPAIV，48h后再气管内接种大肠杆菌；E组作为健康对照组。在MPAIV接种后的
第7－17天内，定时分批扑杀，采其气管和肺，研磨成组织匀浆液，同时采其泄殖腔
棉拭样，进行病毒的分离。
结果显示：两种病原联合感染时，对病毒在气管、肺中的滞留时间无明显影响；
但联合感染组试验鸡泄殖腔排毒时间略长于MPAIV单独接种组。
Mildly pathogenic avian influenza virus H9N2 (MPAIV) is widely distributed and
recently has resulted in considerable economic losses to poultry industry in China. Clinically, mildly pathogenic avian influenza virus infection is often complicated with avian colibacciilosis. But there are few reports about the synergism between these two agents currently. We investigated their synergistic pathogenesis from the following four aspects in the synergism model: (1) the mortality rate in the synergism; (2) the dynamic distribution of Escherichia coli (E.coli) in the synergism; (3) the pathologic abnormalities in the synergism; (4) MPAIV isolation in the synergism.
10-day-old specific pathogen free (SPF) chickens were inoculated intratracheally with virulent E.coli (026) and /or mildly pathogenic avian influenza virus (MPAIV). The mortality rate of chickens inoculated with 10 8 colony-forming units (CPU) E.coli and 2× 104.3 ELD50 MPAIV simultaneously was 32%. Chickens inoculated first with 2 × 1043 ELD50 MPAIV and then 10 8 CPU E.coli at an interval of 48 hours experienced the mortality rate of 24%, whereas those inoculated with 10 8 CPU E.coli and 2× 10 4.3 ELD50 MPAIV at the same interval exhibited the mortality rate of 16%. the group
infected with MPAIV alone experienced the mortality rate of 12% while the group infected with E.coli alone had no death.
10-day-old SPF chickens inoculated intratracheally with 108 CPU virulent E.coli (026) and /or 2 × 1043 ELD50 mildly pathogenic avian influenza virus (MPAIV), respectively, were euthanatized at different times from 1 to 96 h post-inoculation and examined for bacterial counts, macroscopic and microscopic lesions, and ultrastructural pathologic abnormalities. The challenging strain E.coli could be reisolated from trachea, lung of the chickens during the experiments. The distribution of E.coli isolated from blood and the other tissues correlated with the time of postinoculation. Chickens inoculated with E.coli and MPAIV had more severe and persistent respiratory lesions than those inoculated with E. coll or MPAIV alone.
the group inoculated with MPAIV and E.coli was more severe inflammation of the trachea, lung, and air sac and dispersion necrosis of parenchyma organs than that of groups inoculated with MPAIV or E.coli alone. Chickens inoculated with MPAIV and E.coli simultaneously had the most severe and persistent lesions than the other experimental groups. The most severe pathological changes that ciliated cells were deciliated totally and the type II alveolar cells were vacuolated completely were taken place in trachea 4-48h and in lung 6h postinoculation. There were moderate pathological changes in lung and trachea at 6h post inoculation with E.coli alone. The pathological changes that ciliated cells were deciliated partly and the lamellar bodies were vanished in the type II alveolar cell appeared in trachea 3-48h and in lung 12h postinoculation with MPAIV alone.
Samples of tracheas, lungs and cloacal swabs were taken from 3 chickens each group at different intervals postinoculation for isolating challenged MPAIV in 9-day-old embryonating chicken eggs. The results showed that there was no difference for the persistence of MPAIV in the tracheas and lungs between the group infected with MPAIV and E.coli and the group infected with MPAIV alone, while the duration of
virus shedding from intestine for the group infected with both MPAIV and E.coli seemed to be slightly longer than that infected with MPAIV alone.
These results suggest that E.coli colonized mainly the trachea and lung, MPAIV might maintain the time that E.coli colonized the trachea and lung of chickens, and it might facilitate E.coli invasion into the respiratory tract of the chickens resulting in more severe pathologic abnormalities. There was a pathogenic synergism between E. coli and MPAIV.
1．实验鸡经不同顺序接种H9亚型禽流感病毒、禽源大肠杆菌后死亡率比较试验
将10日龄SPF鸡随机分组，每组25羽，隔离饲养，气管内接种。A组10日龄接种MPAIV；B组10日龄同时接种大肠杆菌和MPAIV；C组10日龄接种大肠杆菌，48h后再接种MPAIV；D组10日龄接种MPAIV，48h后再接种大肠杆菌；E组10日龄接种大肠杆菌；F组作为健康对照组。在各组攻毒结束后，观察死亡情况，结果如下：
A、B、C、D组死亡率分别为12％、32％、16％、24％，E组没有死亡，进行卡方检验后，A、B、C、D与E组、B与A组死亡率差异显著。
结果表明：与单独接种MPAIV或E.coli组相比，先MPAIV后大肠杆菌接种组的死亡率有了明显的提高，同时接种组的死亡率有显著的提高，而先接种大肠杆菌再接种MPAIV，死亡率的变化不明显，表明协同致病程度与2种病原接种顺序的先后有关。
2．实验鸡单独或混合感染禽源大肠杆菌后细菌的动态分布
将10日龄SPF鸡随机分组，每组27羽，隔离饲养。A组10日龄气管内接种大肠杆菌；B组10日龄气管内同时接种大肠杆菌与MPAIV；C组为健康对照组。各组在接种后1-96定时分批扑杀：观察它们的临诊症状，并进行细菌学检查。其结果如下：
细菌学检查发现各组的气管、肺、血液、脾脏、肝脏和肾脏中细菌数量与接种时间有关：①A组接种后12-72h出现菌血症，B组在接种后3-96h出现菌血症。细菌数后者恒高于前者；②各试验组肺中的检菌率明显高与其它脏器。比较B、A两组发现，B组肺中细菌明显减少时间延长12h，在这延长时间内细菌数目差异极显
一
著；③A、B组气管接种后 lh出现细菌的增殖，但 B组气管中细菌增殖比。迅速，
细菌数目也比A组多，l2 细菌数目差异显著，在3h后差异不显著。④各组脾脏。
肾脏和肝脏在接种后偶尔可以分离到细菌，而且数量很少。
上述结果表明：试验鸡经气管接种病原性大肠杆菌后，细菌主要在肺中定居并
大量繁殖，进入血液，引起全身反应；联合感染大肠杆菌和MPAIV后，可使大肠
杆菌在肺中定居时间延长。
3．实验鸡人工感染Hg亚型禽流感病毒、禽源大肠杆菌后呼吸器官的超微动态病理
变化
将10日龄SPF鸡随机分组，接种组30羽、对照组20羽，隔离饲养。A组10
日龄气管内接种大肠杆菌；B组IO日龄气管内同时接种大肠杆菌和＊PA w：C组
10日龄气管内接种 MPAIV；D组为健康对照。在接种后定时分批扑杀，采集气管、
肺等进行组织学的超微病理切片和常规病理切片观察。其结果如下：
组织病理学观察可见，B组气管和肺的炎症发生时间早于其它组，恢复也慢，
肺在接种大肠杆菌和 MPAIV后第 lh开始出现炎症一直持续至试验结束，气管粘膜
纤毛严重脱落。
超微结构观察结果为：B组在接种大肠杆菌和 MPAIV后 4－48h，气管纤毛全部脱
落，整个粘膜层均可见MPAIV粒子；肺在接种大肠杆菌和 MPAIV后 6h，肺11型
上皮细胞全部溶解成空泡，只留下I型上皮细胞，严重病变持续到接种后48h，仍
可见＊型上皮细胞细胞器空泡化。
E COlf O26株可引起气管纤毛细胞的损伤，而 MPAIV不仅能破坏气管粘膜的
上皮细胞，而且还能损害肺组织中的＊型上皮细胞。当 E COn与MPAIV混合感染
时，气管、肺的病变更为严重。
4．实验鸡单独或混合感染Hg亚型禽流感病毒后气管、肺及泄殖腔样品的病毒分离
10日龄叩F鸡随机分组，接种组30羽。对照组15羽，隔离饲养。A组12日
龄气管内接种＊PA w：B组12日龄气管内同时接种大肠杆菌与MPA IV：C组IO日
龄气管内接种大肠杆菌dh后再气管内接种MPA W：D组u 日龄气管内接种
MPAIV，48h后再气管内接种大肠杆菌；E组作为健康对照组。在MPAIV接种后的
第7－17天内，定时分批扑杀，采其气管和肺，研磨成组织匀浆液，同时采其泄殖腔
棉拭样，进行病毒的分离。
结果显示：两种病原联合感染时，对病毒在气管、肺中的滞留时间无明显影响；
但联合感染组试验鸡泄殖腔排毒时间略长于MPAIV单独接种组。
Mildly pathogenic avian influenza virus H9N2 (MPAIV) is widely distributed and
recently has resulted in considerable economic losses to poultry industry in China. Clinically, mildly pathogenic avian influenza virus infection is often complicated with avian colibacciilosis. But there are few reports about the synergism between these two agents currently. We investigated their synergistic pathogenesis from the following four aspects in the synergism model: (1) the mortality rate in the synergism; (2) the dynamic distribution of Escherichia coli (E.coli) in the synergism; (3) the pathologic abnormalities in the synergism; (4) MPAIV isolation in the synergism.
10-day-old specific pathogen free (SPF) chickens were inoculated intratracheally with virulent E.coli (026) and /or mildly pathogenic avian influenza virus (MPAIV). The mortality rate of chickens inoculated with 10 8 colony-forming units (CPU) E.coli and 2× 104.3 ELD50 MPAIV simultaneously was 32%. Chickens inoculated first with 2 × 1043 ELD50 MPAIV and then 10 8 CPU E.coli at an interval of 48 hours experienced the mortality rate of 24%, whereas those inoculated with 10 8 CPU E.coli and 2× 10 4.3 ELD50 MPAIV at the same interval exhibited the mortality rate of 16%. the group
infected with MPAIV alone experienced the mortality rate of 12% while the group infected with E.coli alone had no death.
10-day-old SPF chickens inoculated intratracheally with 108 CPU virulent E.coli (026) and /or 2 × 1043 ELD50 mildly pathogenic avian influenza virus (MPAIV), respectively, were euthanatized at different times from 1 to 96 h post-inoculation and examined for bacterial counts, macroscopic and microscopic lesions, and ultrastructural pathologic abnormalities. The challenging strain E.coli could be reisolated from trachea, lung of the chickens during the experiments. The distribution of E.coli isolated from blood and the other tissues correlated with the time of postinoculation. Chickens inoculated with E.coli and MPAIV had more severe and persistent respiratory lesions than those inoculated with E. coll or MPAIV alone.
the group inoculated with MPAIV and E.coli was more severe inflammation of the trachea, lung, and air sac and dispersion necrosis of parenchyma organs than that of groups inoculated with MPAIV or E.coli alone. Chickens inoculated with MPAIV and E.coli simultaneously had the most severe and persistent lesions than the other experimental groups. The most severe pathological changes that ciliated cells were deciliated totally and the type II alveolar cells were vacuolated completely were taken place in trachea 4-48h and in lung 6h postinoculation. There were moderate pathological changes in lung and trachea at 6h post inoculation with E.coli alone. The pathological changes that ciliated cells were deciliated partly and the lamellar bodies were vanished in the type II alveolar cell appeared in trachea 3-48h and in lung 12h postinoculation with MPAIV alone.
Samples of tracheas, lungs and cloacal swabs were taken from 3 chickens each group at different intervals postinoculation for isolating challenged MPAIV in 9-day-old embryonating chicken eggs. The results showed that there was no difference for the persistence of MPAIV in the tracheas and lungs between the group infected with MPAIV and E.coli and the group infected with MPAIV alone, while the duration of
virus shedding from intestine for the group infected with both MPAIV and E.coli seemed to be slightly longer than that infected with MPAIV alone.
These results suggest that E.coli colonized mainly the trachea and lung, MPAIV might maintain the time that E.coli colonized the trachea and lung of chickens, and it might facilitate E.coli invasion into the respiratory tract of the chickens resulting in more severe pathologic abnormalities. There was a pathogenic synergism between E. coli and MPAIV.
引文
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25 Larsen C T, Domermuth C H, Sponenberg D P, et al. Colobacillosis of turkeys exacerbated by hemorrhagic enteritis virus. Laboratory studies. Avian Dis. 1985,29:729-732
26. Newberry L A, Skeeles J K, Kreider D L, et al. Use of virulent hemorrhagic enteritis virus for the induction of colibacillosis in turkeys. Avian Dis. 1993, 37: 1-5
27 Smit W H, Goren E, Litjens J B, et al. "Vaccination reaction": syndrome of broilers after vaccination against Newcastle disease and infectious bronchitis. Tijdschr. Diergeneeskd. 1976, 101: 645-657
28. Gross W B. Factors affecting the development of respiratory disease complex in chickens. Avian Dis. 1990, 34: 607-610
39 Nakamura K, Ueda H, Tanimura T, et al. Effect of mixed live vaccine (Newcastle disease and infectious bronchitis); Mycoplasma gallisepticum on the chicken respiratory tract and on Eicherichia coli infection. J.Comp. Pathol. 1994, 111:33-42
30. Pierson F W, Larsen C T, Domermuth C H. Newcastle disease virus or Bordetella avium, aviruent hemorrhagic enteritis virus, and Escherichia coli. Avian Dis. 1996. 40: 837-840
31．高崧，彭大新，甘军纪等．MPAIV、NDV Lasota株分别与低致病性禽源E．coli的联合感染试验．动物医学进展，2001，22(4)：72-74
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22．石火英，高崧，王保安等．实验性鸡大肠杆菌病病理学动态变化。畜牧兽医学报，2002，33(5)：508-512
23 Nivas S C, Peterson A C, York M D, et al. Epizootiological investigations of colibacillosis in turkeys. Avian Dis. 1977,21:514-530
24. Sponenberg D P, Domermuth C H, Larsen C T. Fied outbreaks of colibacilloisis of turkeys associated with hemorrhagic enteritis virus. Avian Dis 1985,29:828-842.
25 Larsen C T, Domermuth C H, Sponenberg D P, et al. Colobacillosis of turkeys exacerbated by hemorrhagic enteritis virus. Laboratory studies. Avian Dis. 1985,29:729-732
26. Newberry L A, Skeeles J K, Kreider D L, et al. Use of virulent hemorrhagic enteritis virus for the induction of colibacillosis in turkeys. Avian Dis. 1993, 37: 1-5
27 Smit W H, Goren E, Litjens J B, et al. "Vaccination reaction": syndrome of broilers after vaccination against Newcastle disease and infectious bronchitis. Tijdschr. Diergeneeskd. 1976, 101: 645-657
28. Gross W B. Factors affecting the development of respiratory disease complex in chickens. Avian Dis. 1990, 34: 607-610
39 Nakamura K, Ueda H, Tanimura T, et al. Effect of mixed live vaccine (Newcastle disease and infectious bronchitis); Mycoplasma gallisepticum on the chicken respiratory tract and on Eicherichia coli infection. J.Comp. Pathol. 1994, 111:33-42
30. Pierson F W, Larsen C T, Domermuth C H. Newcastle disease virus or Bordetella avium, aviruent hemorrhagic enteritis virus, and Escherichia coli. Avian Dis. 1996. 40: 837-840
31．高崧，彭大新，甘军纪等．MPAIV、NDV Lasota株分别与低致病性禽源E．coli的联合感染试验．动物医学进展，2001，22(4)：72-74
32．甘孟候主编．禽流感．北京：北京农业出版社，第1版，1995