肉鸡呼吸道菌群分子生物学分析及芽孢杆菌Pab02喷雾剂的初步应用研究
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摘要
本文采用三种分子生物学方法(ERIC-PCR-AGE、ERIC-PCR-DGGE和V3-PCR-DGGE)联合活菌计数法分析肉鸡呼吸道菌群结构的多样性,枯草芽孢杆菌Pab02喷雾剂对肉鸡生长性能和呼吸道菌群的影响以及Pab02预防和治疗金黄色葡萄球菌感染的效果,结果如下：
1.选取48只7日龄健康肉公鸡,随机分为2组,每组24只,CK组为对照组,P组为喷雾芽孢杆菌Pab02组。P组动物房内每日8时喷雾1ml/m3的Pab02制剂(108cfu/ml),28日龄时对肉鸡称重。结果,与CK组相比,P组净增重提高了9.40g,平均日增重提高了2.68g,全期耗料降低125g,平均日耗料降低5.59g,料肉比降低0.02,但差异性均不显著(P>0.05)。结果表明通过喷雾枯草芽孢杆菌Pab02对肉鸡的采食量、日增重、料肉比等生长性能有所提高,但无明显作用。
2.采用活菌计数法和三种分子生物学分析法对28和49日龄肉鸡呼吸道细菌结构及多样性进行检测,结果,49日龄时呼吸道细菌总数略高于28日龄时；喉部的细菌总数比气管高一个数量级,喉部细菌总数为106cfu/g,气管细菌总数为105cfu/g；喷雾Pab02后,喉部细菌数显著增高(P<0.05),气管细菌数量略有增多(P>0.05)；49日龄时呼吸道各段的条带数量显著多于28日龄(P<0.05),两个日龄段之间呼吸道各段菌群相似性为59%-84%；喉部的条带数量显著多于气管(P<0.05),喉与气管的菌群相似性为60%-84%。说明,肉鸡在49日龄时,呼吸道菌群多样性均显著高于28日龄时,,但两日龄之间呼吸道菌群相似性仍然很高；喉部的菌群多样性显著高于气管,但两者之间的菌群相似性仍然很高。喷雾Pab02后,喉部的条带数显著增多(P<0.05),相似性提高了6-16个百分点；气管的条带数量变化不显著(P>0.05),相似性提高了11-16个百分点。说明喷雾Pab02能增加呼吸道细菌数量和多样性,并能提高菌群的相似性,但是对不同部位的调节能力不同,对肉鸡喉部的调节作用较大,对气管的调节作用较弱。
3.利用金色葡萄球菌G1分别通过滴鼻感染两组肉鸡,然后采用活菌计数和分子生物学方法检测菌群结构,结果,在感染G1前和后均喷雾Pab02不能显著改变喉部的细菌总数(P>0.05),但能显著减少气管中的细菌总数和整个呼吸道(喉部和气管)中的葡萄球菌数(P<0.05)；在感染前喷雾Pab02或在感染后喷雾Pab02均不能显著改变呼吸道中的细菌总数(P>0.05),但能显著减少呼吸道中的葡萄球菌数(P<0.05)；在感染G1前和后均喷雾Pab02或者仅在感染G1前喷雾Pab02能够恢复呼吸道菌群多样性和相似性,而在感染G1后再喷雾Pab02不能恢复呼吸道菌群多样性和相似性。
4.通过组织学方法观察喷雾枯草芽孢杆菌Pab02以及滴鼻感染金色葡萄球菌G1后对肉鸡肺组织的影响。结果,Pab02喷雾组肺泡正常,未见破裂和融合,未见肺间质增生,未见炎性渗出物,但淋巴小结增多；感染G1后,可见肺泡破裂融合,出血严重,炎性细胞大量增多,有炎性渗出物和严重化生灶；在感染前后均喷雾Pab02能使肺脏中淋巴小结增多,而且未见肺损伤；在感染前喷雾Pab02,能使肺内淋巴小结增多,炎性细胞增多,可见肺泡扩张现象；感染后喷雾Pab02,肺脏中可见肺泡破裂融合,炎性细胞大量增多,可见炎性渗出物和轻微化生灶。结果表明Pab02喷雾剂对肺组织不产生损伤,具有刺激肺脏免疫功能的作用；对呼吸系统的感染有预防和治疗作用,但是预防作用大于治疗作用。
The paper was focused on the combination three molecular biology methods (ERIC-PCR-AGE, ERIC-PCR-DGGE and V3-PCR-DGGE) and viable total count to analyse multiplicity of microorganism community in respiratory tract of broilers; the growth performance, microorganism community structure in respiratory tract, threatment and prevention Staphylococcus aureus infection of broilers were effected by the spraying agent of Bacillus subtilis PabO2. The results are reported as follows.
1.48 7-day-old Avian broilers were randomly divided into four groups. Group CK was the control group, Group P was sprayed lml/m3 of the spraying agent of B. subtilis PabO2 (108cfu/ml) in the room. At the age of 28-day-old, the broilers were weighted. The results were showed that net gain of Group P increased 9.4g, average daily gain increase 2.68g, total feed consumption decreased 125g, average daily feed decreased 5.59g, F/G decreased 0.02, compared with Group CK, the differences of all were not significant (P>0.05).The results indicated that the spraying agent of B. subtilis PabO2 could improve growth performances of feed intake, daily gain and F/G to spray PabO2, but it could not play a significant role.
2. Bacteria in respiratory tract of broilers at of age 28 days and 49 days were detected by viable total count and three molecular biology models.The results showed total bacterial number in respiratory tract of broilers at age of 49 days was little larger than that of broilers at age of 28 days; Total bacterial number of throat was one order higher than that of trachea, Total bacterial number of throat was 106cfu/g,that of trachea was 105 cfu/g; After spraying PabO2,Total bacterial number of throat increased significantly (P<0.05), total bacterial number of trachea increased,but not significantly(P>0.05). Bands of respiratory tract of broilers at age of 49 days were more than that of broilers at age of 28 days significantly(P<0.05), the similarity of microorganism community in respiratory tract was 59%～84%;bands of throat were more than that of trachea significantly(P<0.05),the similarity of microorganism community between throat and trachea was 60%～84%. The finding indicated that multiplicity of microorganism community in respiratory tract of broilers at age of 49 days was more than broilers at age of 28 days,multiplicity of microorganism community in throat was more than in trachea.After spraying PabO2,bands of throat increased significantly(P<0.05),similarity increased by 6～16 percentage points; bands of trachea did not increased significantly(P>0.05), similarity increased by 11～16 percentage points. The results indicated spraying PabO2 could increase number and multiplicity of bacteria in respiratory tract,however,there were different modulatory effects on different locations, modulatory effects on throat was larger than on trachea.
3.Broilers of two groups were infected by Staphylococcus aureus G1,then microorganism community structure of respiratoty tract was tested by viable total count and molecular biology models.The results showed that spraying PabO2 before and after infected by G1 could not significantly change total bacterial number of throat(P>0.05),but could significantly reduce total bacterial number of trachea and Staphylococcus number of respiratory tract(P<0.05);spraying PabO2 before or after infected by G1 could not significantly change total bacterial number of respiratory tract(P>0.05),but could significantly reduce Staphylococcus number of respiratory tract(P<0.05). Spraying PabO2 before and after infected by Glor spraying PabO2 only before infected by G1 could renew the multiplicity and similarity of microorganism community,however, spraying PabO2 after infected by G1 could not do.
4. The histological sections of lung were observed by optical microscope, lung tissue of broilers after spraying PabO2 was normal, alveolar did not rupture or fuse, lung interstitium did not hyperplasia, inflammatory exudation did not infiltrate,moreover,the number of lymphonodulis increased; There were ruptured and fused alveolar, inflammatory exudation and more inflammatory cells, severe haemorrhage and metaplasia in lung of the broilers infected by G1; There were more lymphonodulis instead of lung injury in the lung of broilers sprayed PabO2 before and after infected by G1; There were expanded alveolar and more inflammatory cells, but number of lymphonodulis increased in lung of broilers sprayed before infected by G1;There were ruptured and fused alveolar,inflammatory exudation,more inflammatory cells,and slight metaplasia in the lung of broilers sprayed after infected by G1. Finding indicated spraying agent of PabO2 did not injury lung tissue, on the contrary could stimulate immune function; spraying agent of PabO2 could prevent and cure respiratory disease caused by Staphylococcus aureus,and the preventive effect is better than the therapeutic effect.
1.选取48只7日龄健康肉公鸡,随机分为2组,每组24只,CK组为对照组,P组为喷雾芽孢杆菌Pab02组。P组动物房内每日8时喷雾1ml/m3的Pab02制剂(108cfu/ml),28日龄时对肉鸡称重。结果,与CK组相比,P组净增重提高了9.40g,平均日增重提高了2.68g,全期耗料降低125g,平均日耗料降低5.59g,料肉比降低0.02,但差异性均不显著(P>0.05)。结果表明通过喷雾枯草芽孢杆菌Pab02对肉鸡的采食量、日增重、料肉比等生长性能有所提高,但无明显作用。
2.采用活菌计数法和三种分子生物学分析法对28和49日龄肉鸡呼吸道细菌结构及多样性进行检测,结果,49日龄时呼吸道细菌总数略高于28日龄时；喉部的细菌总数比气管高一个数量级,喉部细菌总数为106cfu/g,气管细菌总数为105cfu/g；喷雾Pab02后,喉部细菌数显著增高(P<0.05),气管细菌数量略有增多(P>0.05)；49日龄时呼吸道各段的条带数量显著多于28日龄(P<0.05),两个日龄段之间呼吸道各段菌群相似性为59%-84%；喉部的条带数量显著多于气管(P<0.05),喉与气管的菌群相似性为60%-84%。说明,肉鸡在49日龄时,呼吸道菌群多样性均显著高于28日龄时,,但两日龄之间呼吸道菌群相似性仍然很高；喉部的菌群多样性显著高于气管,但两者之间的菌群相似性仍然很高。喷雾Pab02后,喉部的条带数显著增多(P<0.05),相似性提高了6-16个百分点；气管的条带数量变化不显著(P>0.05),相似性提高了11-16个百分点。说明喷雾Pab02能增加呼吸道细菌数量和多样性,并能提高菌群的相似性,但是对不同部位的调节能力不同,对肉鸡喉部的调节作用较大,对气管的调节作用较弱。
3.利用金色葡萄球菌G1分别通过滴鼻感染两组肉鸡,然后采用活菌计数和分子生物学方法检测菌群结构,结果,在感染G1前和后均喷雾Pab02不能显著改变喉部的细菌总数(P>0.05),但能显著减少气管中的细菌总数和整个呼吸道(喉部和气管)中的葡萄球菌数(P<0.05)；在感染前喷雾Pab02或在感染后喷雾Pab02均不能显著改变呼吸道中的细菌总数(P>0.05),但能显著减少呼吸道中的葡萄球菌数(P<0.05)；在感染G1前和后均喷雾Pab02或者仅在感染G1前喷雾Pab02能够恢复呼吸道菌群多样性和相似性,而在感染G1后再喷雾Pab02不能恢复呼吸道菌群多样性和相似性。
4.通过组织学方法观察喷雾枯草芽孢杆菌Pab02以及滴鼻感染金色葡萄球菌G1后对肉鸡肺组织的影响。结果,Pab02喷雾组肺泡正常,未见破裂和融合,未见肺间质增生,未见炎性渗出物,但淋巴小结增多；感染G1后,可见肺泡破裂融合,出血严重,炎性细胞大量增多,有炎性渗出物和严重化生灶；在感染前后均喷雾Pab02能使肺脏中淋巴小结增多,而且未见肺损伤；在感染前喷雾Pab02,能使肺内淋巴小结增多,炎性细胞增多,可见肺泡扩张现象；感染后喷雾Pab02,肺脏中可见肺泡破裂融合,炎性细胞大量增多,可见炎性渗出物和轻微化生灶。结果表明Pab02喷雾剂对肺组织不产生损伤,具有刺激肺脏免疫功能的作用；对呼吸系统的感染有预防和治疗作用,但是预防作用大于治疗作用。
The paper was focused on the combination three molecular biology methods (ERIC-PCR-AGE, ERIC-PCR-DGGE and V3-PCR-DGGE) and viable total count to analyse multiplicity of microorganism community in respiratory tract of broilers; the growth performance, microorganism community structure in respiratory tract, threatment and prevention Staphylococcus aureus infection of broilers were effected by the spraying agent of Bacillus subtilis PabO2. The results are reported as follows.
1.48 7-day-old Avian broilers were randomly divided into four groups. Group CK was the control group, Group P was sprayed lml/m3 of the spraying agent of B. subtilis PabO2 (108cfu/ml) in the room. At the age of 28-day-old, the broilers were weighted. The results were showed that net gain of Group P increased 9.4g, average daily gain increase 2.68g, total feed consumption decreased 125g, average daily feed decreased 5.59g, F/G decreased 0.02, compared with Group CK, the differences of all were not significant (P>0.05).The results indicated that the spraying agent of B. subtilis PabO2 could improve growth performances of feed intake, daily gain and F/G to spray PabO2, but it could not play a significant role.
2. Bacteria in respiratory tract of broilers at of age 28 days and 49 days were detected by viable total count and three molecular biology models.The results showed total bacterial number in respiratory tract of broilers at age of 49 days was little larger than that of broilers at age of 28 days; Total bacterial number of throat was one order higher than that of trachea, Total bacterial number of throat was 106cfu/g,that of trachea was 105 cfu/g; After spraying PabO2,Total bacterial number of throat increased significantly (P<0.05), total bacterial number of trachea increased,but not significantly(P>0.05). Bands of respiratory tract of broilers at age of 49 days were more than that of broilers at age of 28 days significantly(P<0.05), the similarity of microorganism community in respiratory tract was 59%～84%;bands of throat were more than that of trachea significantly(P<0.05),the similarity of microorganism community between throat and trachea was 60%～84%. The finding indicated that multiplicity of microorganism community in respiratory tract of broilers at age of 49 days was more than broilers at age of 28 days,multiplicity of microorganism community in throat was more than in trachea.After spraying PabO2,bands of throat increased significantly(P<0.05),similarity increased by 6～16 percentage points; bands of trachea did not increased significantly(P>0.05), similarity increased by 11～16 percentage points. The results indicated spraying PabO2 could increase number and multiplicity of bacteria in respiratory tract,however,there were different modulatory effects on different locations, modulatory effects on throat was larger than on trachea.
3.Broilers of two groups were infected by Staphylococcus aureus G1,then microorganism community structure of respiratoty tract was tested by viable total count and molecular biology models.The results showed that spraying PabO2 before and after infected by G1 could not significantly change total bacterial number of throat(P>0.05),but could significantly reduce total bacterial number of trachea and Staphylococcus number of respiratory tract(P<0.05);spraying PabO2 before or after infected by G1 could not significantly change total bacterial number of respiratory tract(P>0.05),but could significantly reduce Staphylococcus number of respiratory tract(P<0.05). Spraying PabO2 before and after infected by Glor spraying PabO2 only before infected by G1 could renew the multiplicity and similarity of microorganism community,however, spraying PabO2 after infected by G1 could not do.
4. The histological sections of lung were observed by optical microscope, lung tissue of broilers after spraying PabO2 was normal, alveolar did not rupture or fuse, lung interstitium did not hyperplasia, inflammatory exudation did not infiltrate,moreover,the number of lymphonodulis increased; There were ruptured and fused alveolar, inflammatory exudation and more inflammatory cells, severe haemorrhage and metaplasia in lung of the broilers infected by G1; There were more lymphonodulis instead of lung injury in the lung of broilers sprayed PabO2 before and after infected by G1; There were expanded alveolar and more inflammatory cells, but number of lymphonodulis increased in lung of broilers sprayed before infected by G1;There were ruptured and fused alveolar,inflammatory exudation,more inflammatory cells,and slight metaplasia in the lung of broilers sprayed after infected by G1. Finding indicated spraying agent of PabO2 did not injury lung tissue, on the contrary could stimulate immune function; spraying agent of PabO2 could prevent and cure respiratory disease caused by Staphylococcus aureus,and the preventive effect is better than the therapeutic effect.
引文
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