麻杏石甘粉防治人工感染鸡传染性支气管炎的试验研究
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摘要
本试验主要研究麻杏石甘粉对人工感染传染性支气管炎病毒(IBV)雏鸡的保护作用,并初步探讨其可能的作用机制,为研究和开发中药防治动物疾病提供新的思路和方法。
方法:IBV-M41标准毒株经SPF鸡胚复壮传代5次后测定其半数感染量,以100EID50为参照值,通过滴鼻、点眼的方法对15日龄雏鸡进行人工感染试验。接毒24h后,观察鸡的临床表现并进行实验室诊断。然后将240只15日龄雏鸡随机分为8组:正常组、模型组、麻杏石甘粉高、中、低剂量组及麻杏石甘散高、中、低剂量组。除健康组外其余7组接种IBV尿囊液。两种药物治疗组于攻毒48h后分别以0.5%、1%、1.5%的麻杏石甘粉和0.12%、0.24%、0.36%的麻杏石甘散药物浓度进行干预,连续给药3d,于雏鸡18、21、24、27、30日龄时,每组随机取6只鸡,无菌心脏采血,分离血清。ELISA方法测定血清中IBV抗体滴度,然后将雏鸡处死,取肺、支气管等组织做病理切片,免疫组织化学方法测支气管单位面积IgA分泌细胞的变化;计算脾脏、法氏囊的器官指数。为评价麻杏石甘粉的毒性,以半数致死量(LD50)作为评价急性毒性强度的指标将40只小白鼠随机分为4组,每组10只,并且保证雌雄各半,给药途径为口服,试验期为7d,观察动物临床表现和死亡情况。
结果:(1)给每只供试雏鸡滴鼻0.2mL100 EID50供试毒液,同时对雏鸡进行冷刺激(频繁室内洒水,关闭鸡舍内的取暖设施),从攻毒后的鸡只临床症状、死亡鸡只的剖检病变症状说明,本试验成功建立了鸡传染性支气管炎模型;(2)麻杏石甘可溶性粉治疗IB总有效率达90%-93.3% ,治愈率为86.7%-90%,证实雏鸡群应用麻杏石甘粉治疗此病效果显著高于麻杏石甘散,饮服本药的最适治疗浓度为0.1%-0.15%;(3)与模型组相比,麻杏石甘粉组通过促进机体细胞活化,导致组织迅速再生,改善IBV模型肺与支气管组织的病理学变化并且效果优于麻杏石甘散组;(4)抗体测定结果显示给IBV感染雏鸡药物干预后,血清IBV抗体的滴度升高明显,麻杏石甘粉高、中剂量组均为抗体阳性表现,显著高于麻杏石甘散组;(5)攻毒后3d,在IBV的刺激下,各组IgA分泌细胞的数量较正常组都有不同程度的增多,但到攻毒后9d,模型组及麻杏石甘散组IgA分泌细胞的增加幅度已不是很明显,而麻杏石甘粉组仍然保持着较好的上升趋势,尤其在机体的康复期仍能高水平的分泌抗体,提高了雏鸡抵御强毒攻击的能力;(6)急性毒性试验结果表明,LD50>60000mg/kg,说明麻杏石甘可溶性粉无毒,可安全服用。
结论:麻杏石甘粉对IBV感染的雏鸡具有良好的保护作用,对IBV模型的气管及肺组织的病理损伤有修复作用,并能调节机体免疫功能,促进雏鸡产生抗IBV抗体,提高机体的细胞免疫和体液免疫,从而杀灭侵入机体的病原微生物并提高雏鸡抗病能力,达到抗病毒的作用;并且有效增强鸡体的黏膜免疫功能,提高了雏鸡抵御强毒攻击的能力。以上效果均显著高于传统的散剂剂型并且无毒副作用,能有效治疗雏鸡的传染性支气管炎。
The study was carried out to evaluate the protective effect of the new dosage forms of Chinese herbal medicine Maxing Shigan powder on Infectious Bronchitis Virus (IBV) through artificial infection in chickens,and initially discuss its possible function mechanism. The result of this study will provided a novel thought and method for studying Chinese herbal medicine to prevent and treat Animal diseases.
Method: Determine the EID50 of IBV-M41 strain after rejuvenation on embryo at 5 generation, then 15-day-old chicks were inoculated intranasally with IBV-M41 refer to 100 EID50, the clinical symptom were observed and chicks were diagnosed by empirical method at 24h post infection. Two hundred and forty 15-day-old chicks were randomly divided into eight groups of 30 each: blank control group, model group,low, middle and high dose of Maxingshigan soluble powder groups and Maxingshigan coarse powder groups. Except for blank control group, chickens in other groups were inoculated intranasally with 0.2 ml inoculum of IBV-M41 respectively. Birds in the two kinds of drug therapeutic groups were intervened with 0.5%, 1% and 1.5% dose of Maxingshigan soluble powder and 0.12%, 0.24% and 0.36% dose of Maxingshigan coarse powder respectively at 48h post infection, successive administration for 3 days. Clinical signs were recorded daily before or after drug usage and serum was collected at 18,21,24,27,30 days of age with six chicks from each group randomly; the IBV antibody titers in serum were determined by ELISA; pathological sections of lung and bronchus were observed; the changes of IgA-secreting cells per unit area of bronchus were measured by immunohistochemistry; the weights of whole body, spleen and bursa of Fabricius were weighed for the calculation of immune organ index. For evaluating the toxicity of Maxingshigan soluble powder, the index of acute toxicity intensity was evaluated by LD50, forty mouses were randomly divided into four groups of 10 each, the route of administration was orally. The test period was 7d, clinical manifestation and death was observed.
Result: (1)IBV infected model could be replicated successfully by inoculating with 0.2 ml inoculum of IBV-M41 and under the physical environment of low temperature and high moisturization. It could be confirmed by the clinical symptoms and signs, the pathological appearances of lung and bronchus tissue; (2)The total efficiency of Maxingshigan soluble powder on IBV infection was 90%-93.5%, the cure rate was 86.7%-90%, it was confirmed that the therapeutic effect of Maxingshigan soluble powder on IBV infection was significantly better than Maxingshigan coarse powder and the optimum therapeutic concentration was 0.1%-0.15%; (3)Compared with model group, Maxingshigan soluble powder groups could rapidly induce tissue regeneration through promotion of cell activation, and could improve the pathological changes of the bronchus and lung tissues of the IBV infected model which was better than Maxingshigan coarse powder groups; (4)After administrated with Chinese herb medicine, the serum level of IBV antibody rised obviously, high and middle dose of Maxingshigan soluble powder groups showed positive which was significantly higher than Maxingshigan coarse powder groups; (5) at 3 days postinfection, the areas of IgA secreting cells in the groups challenged with virus continued to increase, but at 9 days postinfection, the increase extent of IgA secreting cells in model group and Maxingshigan coarse powder groups was not obviously, but in high and middle dose of Maxingshigan soluble powder groups,it still maintained upward trend,especially in the rehabilitative period of the body,it still secreted antibody at a high level,therefore enhanced the chicken’s ability of resisting the virus; (6)The result of acute toxicity test was LD50>60000mg/kg, explained that Maxingshigan soluble powder is non-toxic, which was safe to take.
Conclusion: Maxingshigan soluble powder had significant therapeutic effect on IBV infection, meanwhile reparation effect on pathology injury caused by IBV infection, and immune regulation. It could increase serum antibody against IBV, improve the cellular immunity and humoral immunity, which would eventually kill the pathogenic microorganism and speed up the chick’s recovery from IBV infection; it also had the ability of enhancing the function of Mucosal immunity, enhanced the chicken’s ability of resisting the virus. The effects aboved of Maxingshigan soluble powder were significantly higher than the traditional coarse powder, moreover it without toxic and side-effect, thereby protect the chickens with IBV infection.
方法:IBV-M41标准毒株经SPF鸡胚复壮传代5次后测定其半数感染量,以100EID50为参照值,通过滴鼻、点眼的方法对15日龄雏鸡进行人工感染试验。接毒24h后,观察鸡的临床表现并进行实验室诊断。然后将240只15日龄雏鸡随机分为8组:正常组、模型组、麻杏石甘粉高、中、低剂量组及麻杏石甘散高、中、低剂量组。除健康组外其余7组接种IBV尿囊液。两种药物治疗组于攻毒48h后分别以0.5%、1%、1.5%的麻杏石甘粉和0.12%、0.24%、0.36%的麻杏石甘散药物浓度进行干预,连续给药3d,于雏鸡18、21、24、27、30日龄时,每组随机取6只鸡,无菌心脏采血,分离血清。ELISA方法测定血清中IBV抗体滴度,然后将雏鸡处死,取肺、支气管等组织做病理切片,免疫组织化学方法测支气管单位面积IgA分泌细胞的变化;计算脾脏、法氏囊的器官指数。为评价麻杏石甘粉的毒性,以半数致死量(LD50)作为评价急性毒性强度的指标将40只小白鼠随机分为4组,每组10只,并且保证雌雄各半,给药途径为口服,试验期为7d,观察动物临床表现和死亡情况。
结果:(1)给每只供试雏鸡滴鼻0.2mL100 EID50供试毒液,同时对雏鸡进行冷刺激(频繁室内洒水,关闭鸡舍内的取暖设施),从攻毒后的鸡只临床症状、死亡鸡只的剖检病变症状说明,本试验成功建立了鸡传染性支气管炎模型;(2)麻杏石甘可溶性粉治疗IB总有效率达90%-93.3% ,治愈率为86.7%-90%,证实雏鸡群应用麻杏石甘粉治疗此病效果显著高于麻杏石甘散,饮服本药的最适治疗浓度为0.1%-0.15%;(3)与模型组相比,麻杏石甘粉组通过促进机体细胞活化,导致组织迅速再生,改善IBV模型肺与支气管组织的病理学变化并且效果优于麻杏石甘散组;(4)抗体测定结果显示给IBV感染雏鸡药物干预后,血清IBV抗体的滴度升高明显,麻杏石甘粉高、中剂量组均为抗体阳性表现,显著高于麻杏石甘散组;(5)攻毒后3d,在IBV的刺激下,各组IgA分泌细胞的数量较正常组都有不同程度的增多,但到攻毒后9d,模型组及麻杏石甘散组IgA分泌细胞的增加幅度已不是很明显,而麻杏石甘粉组仍然保持着较好的上升趋势,尤其在机体的康复期仍能高水平的分泌抗体,提高了雏鸡抵御强毒攻击的能力;(6)急性毒性试验结果表明,LD50>60000mg/kg,说明麻杏石甘可溶性粉无毒,可安全服用。
结论:麻杏石甘粉对IBV感染的雏鸡具有良好的保护作用,对IBV模型的气管及肺组织的病理损伤有修复作用,并能调节机体免疫功能,促进雏鸡产生抗IBV抗体,提高机体的细胞免疫和体液免疫,从而杀灭侵入机体的病原微生物并提高雏鸡抗病能力,达到抗病毒的作用;并且有效增强鸡体的黏膜免疫功能,提高了雏鸡抵御强毒攻击的能力。以上效果均显著高于传统的散剂剂型并且无毒副作用,能有效治疗雏鸡的传染性支气管炎。
The study was carried out to evaluate the protective effect of the new dosage forms of Chinese herbal medicine Maxing Shigan powder on Infectious Bronchitis Virus (IBV) through artificial infection in chickens,and initially discuss its possible function mechanism. The result of this study will provided a novel thought and method for studying Chinese herbal medicine to prevent and treat Animal diseases.
Method: Determine the EID50 of IBV-M41 strain after rejuvenation on embryo at 5 generation, then 15-day-old chicks were inoculated intranasally with IBV-M41 refer to 100 EID50, the clinical symptom were observed and chicks were diagnosed by empirical method at 24h post infection. Two hundred and forty 15-day-old chicks were randomly divided into eight groups of 30 each: blank control group, model group,low, middle and high dose of Maxingshigan soluble powder groups and Maxingshigan coarse powder groups. Except for blank control group, chickens in other groups were inoculated intranasally with 0.2 ml inoculum of IBV-M41 respectively. Birds in the two kinds of drug therapeutic groups were intervened with 0.5%, 1% and 1.5% dose of Maxingshigan soluble powder and 0.12%, 0.24% and 0.36% dose of Maxingshigan coarse powder respectively at 48h post infection, successive administration for 3 days. Clinical signs were recorded daily before or after drug usage and serum was collected at 18,21,24,27,30 days of age with six chicks from each group randomly; the IBV antibody titers in serum were determined by ELISA; pathological sections of lung and bronchus were observed; the changes of IgA-secreting cells per unit area of bronchus were measured by immunohistochemistry; the weights of whole body, spleen and bursa of Fabricius were weighed for the calculation of immune organ index. For evaluating the toxicity of Maxingshigan soluble powder, the index of acute toxicity intensity was evaluated by LD50, forty mouses were randomly divided into four groups of 10 each, the route of administration was orally. The test period was 7d, clinical manifestation and death was observed.
Result: (1)IBV infected model could be replicated successfully by inoculating with 0.2 ml inoculum of IBV-M41 and under the physical environment of low temperature and high moisturization. It could be confirmed by the clinical symptoms and signs, the pathological appearances of lung and bronchus tissue; (2)The total efficiency of Maxingshigan soluble powder on IBV infection was 90%-93.5%, the cure rate was 86.7%-90%, it was confirmed that the therapeutic effect of Maxingshigan soluble powder on IBV infection was significantly better than Maxingshigan coarse powder and the optimum therapeutic concentration was 0.1%-0.15%; (3)Compared with model group, Maxingshigan soluble powder groups could rapidly induce tissue regeneration through promotion of cell activation, and could improve the pathological changes of the bronchus and lung tissues of the IBV infected model which was better than Maxingshigan coarse powder groups; (4)After administrated with Chinese herb medicine, the serum level of IBV antibody rised obviously, high and middle dose of Maxingshigan soluble powder groups showed positive which was significantly higher than Maxingshigan coarse powder groups; (5) at 3 days postinfection, the areas of IgA secreting cells in the groups challenged with virus continued to increase, but at 9 days postinfection, the increase extent of IgA secreting cells in model group and Maxingshigan coarse powder groups was not obviously, but in high and middle dose of Maxingshigan soluble powder groups,it still maintained upward trend,especially in the rehabilitative period of the body,it still secreted antibody at a high level,therefore enhanced the chicken’s ability of resisting the virus; (6)The result of acute toxicity test was LD50>60000mg/kg, explained that Maxingshigan soluble powder is non-toxic, which was safe to take.
Conclusion: Maxingshigan soluble powder had significant therapeutic effect on IBV infection, meanwhile reparation effect on pathology injury caused by IBV infection, and immune regulation. It could increase serum antibody against IBV, improve the cellular immunity and humoral immunity, which would eventually kill the pathogenic microorganism and speed up the chick’s recovery from IBV infection; it also had the ability of enhancing the function of Mucosal immunity, enhanced the chicken’s ability of resisting the virus. The effects aboved of Maxingshigan soluble powder were significantly higher than the traditional coarse powder, moreover it without toxic and side-effect, thereby protect the chickens with IBV infection.
引文
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