犬源益生芽孢杆菌的筛选及其对攻毒幼犬的保护作用
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摘要
微生态制剂作为生物兽药和饲料添加剂已广泛用于治疗和预防仔猪、犊牛、羔羊、家禽等动物肠炎、腹泻病。但是犬用微生态制剂研究应用较少,目前专门用于宠物犬的国产微生态制剂还没有问世。开发犬用微生态制剂,不仅对生态养犬有重要意义,而且给生物兽药业和饲料工业带来巨大的经济效益。而要开发高质量的微生态制剂,菌种的选择是关键。因此,本研究从不同品种宠物犬的粪样中分离和纯化出可作为犬用微生态制剂的备用菌株,并对筛选出的菌株从安全性、分子生物学鉴定和对攻毒幼犬的保护作用等方面进行了系统评价,其主要内容和结果如下:
1、用营养琼脂平板从不同品种宠物犬的粪样中分离、筛选出13株芽孢杆菌,经耐酸和耐胆盐试验初筛到5株抗酸性强和胆盐耐受力强的芽孢杆菌。采用双层琼脂平板法,用5株芽孢杆菌对5株常见的条件性病原微生物进行抑菌实验,筛选到芽孢杆菌Y_(10)具有较好的抑菌效果。药敏实验表明,芽孢杆菌Y_(10)对常见的7种抗生素敏感,可作为犬用微生态制剂的备用菌株。
2、采用固体发酵法制备芽孢杆菌Y_(10)菌粉,以浓度梯度为10~9cfu/g、10~8cfu/g和10~7cfu/g的浓度添加于饲料中,对40只昆明系小白鼠进行慢性攻毒实验。方差分析得出各处理组和对照组小鼠体重、免疫器官指数、血液生化指标、盲肠优势菌群数量和结肠短链脂肪酸含量差异不显著,表明芽孢杆菌Y_(10)对小白鼠是安全的,无毒副作用。
3、利用分子生物学技术,从16S rDNA序列同源性和系统发育角度鉴定芽孢杆菌Y_(10)为Bacillus licheniformis。
4、选取健康幼龄犬16只,研究了日粮中添加芽孢杆菌Y_(10)对攻毒幼犬腹泻的影响,并且应用PCR-DGGE技术分析实验不同阶段幼犬肠道菌群结构及多样性。攻毒实验结果显示:未饲喂芽孢杆菌Y_(10)的试验组4中犬都出现了腹泻,饲喂了芽孢杆菌Y_(10)的试验组3只有一只出现了腹泻现象,说明芽孢杆菌能显著降低幼犬的腹泻率(P＜0.05)。PCR-DGGE分析肠道菌群结果:①实验第0天各组间DGGE图谱条带相似性指数都在56%以上(试验组中的一个体除外)。多样性分析显示图谱条带组内和各组间都没有显著性差异(P＞0.05),实验用的仔犬肠道菌群组成相对稳定。②实验第9天各处理组肠道菌群组间相似性在58%以上。阳性对照组组内个体图谱间相似性指数在69%～95%,阴性对照组组内个体图谱间相似性指数为58%～71%。各处理组的多样性从平均11条上升到14条,阳性对照组3个重复的平均条带数为14.7,阴性对照组的3个重复的平均条代数为12.7,说明饲喂芽孢杆菌能够提高犬肠道微生物的多态性。试验组3攻毒后的平均条带数为13.3,间于阳性和阴性对照组,表明饲喂芽孢杆菌后,当幼犬受到外源微生物时侵染时,机体肠道内微生物多态性降低。试验组4攻毒后发生了腹泻现象,平均条带数为16.6,高于其他各处理组,表明当幼犬受到外源微生物感染时,其机体肠道内菌群失调,大量微生物被排出了体外。
综上所述,安全性实验评估和对攻毒幼犬的保护作用的检测结果均表明犬源益生芽孢杆菌Y_(10)(Bacillus licheniformis)可作为开发犬用微生态制剂的备用菌株,为犬源微生态制剂的发展提供了可行的备用菌种。而且,犬源益生芽孢杆菌Y_(10)能较显著改善犬肠道环境,促进微生态平衡,从而降低腹泻率,为预防犬肠道细菌性疾病提供了新的思路。
Probiotics, as a biological veterinary drugs and feed additives, has been widely used in the treatment and prevention animal enteritis and diarrhea of piglet, calf, lamb, poultry. However, the research and application of probiotics in dogs devoted to the domestic pet has not yet come out. It is not only having important ecological significance for the Kennel, but also bringing the huge enormous bebefit to biology of veterinary drugs and feed industry to develop canine microbiologic preparation. And to develop high-quality probiotics, selection of microbe species is the key. Therefore, this study was to separate and purificate a bacillus sp used as backup with probiotics strains from different varieties of pet dogs, and made a systematic evaluation of the selected strains in the security, identification of molecular biology and the protection of challenged puppies. The main contents and results were as follows:
1. 13 strains of Bacillus were isolated and purificated from different varieties of healthy pet dogs feces by using nutritional broth medium, in which five strains demonstrated a good survival ability in the presence of bile salts and at low pH. Taking five common conditions for the instructions of bacteria pathogenic microorganisms and using double-agar plate method of five strains to bacteria inhibition test, Bacillus Y_(10) was found to have a very good bacteriostatic effect. Sensitivity experiments showed that Bacillus Y_(10) were sensitive to seven common antibiotics as a backup strain of canine probiotics.
2. Prepared using solid-state fermentation of Bacillus Y_(10) powder to the concentration gradient of 10~9 cfu / g, 10~8 cfu / g and 10~7 cfu / g added to the feed of 40 Kunming mice to chronic experimental infection. Analysis of variance of the treatment group and control group of mice body weight size, immune indices, blood biochemical indicators, the number of cecal and colonic flora advantage short-chain fatty acids, there were no significant differences, indicating that Bacillus Y_(10) to mice were safe, the role of non-toxic negative.
3. Using molecular biology techniques, Bacillus Y_(10) was identified for Bacillus licheniformis, from the 16S rRNA sequence homology and phylogenetic.
4. Selected 16 healthy puppies to study the effects of dietary Bacillus Y_(10) in diarrhea and applied PCR-DGGE to analysis puppies at different stages of experimental intestinal flora structure and diversity. The experimental results showed that groups of not fed Bacillus Y_(10) had emerged diarrhea, compared in only one of the fed Bacillus Y_(10) groups, which indicated that Bacillus could significantly reduce the the rate of diarrhea (P <0.05). The DGGE results indicated that:①DGGE band pattern similarity indices of all groups in the first experimental day were at or above 56%. Diversity analysis showed that stripe pattern group and groups were not significantly different, indicating that the intestinal flora of puppies used were relatively stable.②The similarity indices of the experimental treatment group of intestinal flora in ninth day were at more than 58 percent. Positive control group between the individual patterns of similarity indices was at 69%～95% and the negative control group was at 58%～71%. The intestinal Microflora diversity of two groups was improves gradually from 11 to 14. The results of bands diversity showed that feeding Bacillus bacteria can enhance intestinal microbial polymorphism in dogs. The average band number of test group 1 after infection was at 13.3, between positive and negative in the control group, indicating that intestinal microbial polymorphism of puppies was reduced when infected after feeding Bacillus later. When puppies of test group 2 happened diarrhea, the average number of bands was at 16.6, higher than the other treatment groups, indicating that when infected by exogenous microbial, the puppies organism occurred in the intestinal flora imbalance, which resulted in a large number of micro-organisms discharged in vitro.
To sum up, the safety of assessment experiment and the protective effect in challenged puppies of drug testing results showed that Bacillus Y_(10) (Bacillus licheniformis) could be used as the probiotics strains, which provided a viable and valuable species for the development of canine probiotics. Moreover, the source dogs of Bacillus Y_(10) prebiotics could significantly improve the intestinal environment and the promotion of micro-ecological balance, thereby reducing the rate of diarrhea and providing a new way for the prevention of bacterial disease in dogs.
1、用营养琼脂平板从不同品种宠物犬的粪样中分离、筛选出13株芽孢杆菌,经耐酸和耐胆盐试验初筛到5株抗酸性强和胆盐耐受力强的芽孢杆菌。采用双层琼脂平板法,用5株芽孢杆菌对5株常见的条件性病原微生物进行抑菌实验,筛选到芽孢杆菌Y_(10)具有较好的抑菌效果。药敏实验表明,芽孢杆菌Y_(10)对常见的7种抗生素敏感,可作为犬用微生态制剂的备用菌株。
2、采用固体发酵法制备芽孢杆菌Y_(10)菌粉,以浓度梯度为10~9cfu/g、10~8cfu/g和10~7cfu/g的浓度添加于饲料中,对40只昆明系小白鼠进行慢性攻毒实验。方差分析得出各处理组和对照组小鼠体重、免疫器官指数、血液生化指标、盲肠优势菌群数量和结肠短链脂肪酸含量差异不显著,表明芽孢杆菌Y_(10)对小白鼠是安全的,无毒副作用。
3、利用分子生物学技术,从16S rDNA序列同源性和系统发育角度鉴定芽孢杆菌Y_(10)为Bacillus licheniformis。
4、选取健康幼龄犬16只,研究了日粮中添加芽孢杆菌Y_(10)对攻毒幼犬腹泻的影响,并且应用PCR-DGGE技术分析实验不同阶段幼犬肠道菌群结构及多样性。攻毒实验结果显示:未饲喂芽孢杆菌Y_(10)的试验组4中犬都出现了腹泻,饲喂了芽孢杆菌Y_(10)的试验组3只有一只出现了腹泻现象,说明芽孢杆菌能显著降低幼犬的腹泻率(P＜0.05)。PCR-DGGE分析肠道菌群结果:①实验第0天各组间DGGE图谱条带相似性指数都在56%以上(试验组中的一个体除外)。多样性分析显示图谱条带组内和各组间都没有显著性差异(P＞0.05),实验用的仔犬肠道菌群组成相对稳定。②实验第9天各处理组肠道菌群组间相似性在58%以上。阳性对照组组内个体图谱间相似性指数在69%～95%,阴性对照组组内个体图谱间相似性指数为58%～71%。各处理组的多样性从平均11条上升到14条,阳性对照组3个重复的平均条带数为14.7,阴性对照组的3个重复的平均条代数为12.7,说明饲喂芽孢杆菌能够提高犬肠道微生物的多态性。试验组3攻毒后的平均条带数为13.3,间于阳性和阴性对照组,表明饲喂芽孢杆菌后,当幼犬受到外源微生物时侵染时,机体肠道内微生物多态性降低。试验组4攻毒后发生了腹泻现象,平均条带数为16.6,高于其他各处理组,表明当幼犬受到外源微生物感染时,其机体肠道内菌群失调,大量微生物被排出了体外。
综上所述,安全性实验评估和对攻毒幼犬的保护作用的检测结果均表明犬源益生芽孢杆菌Y_(10)(Bacillus licheniformis)可作为开发犬用微生态制剂的备用菌株,为犬源微生态制剂的发展提供了可行的备用菌种。而且,犬源益生芽孢杆菌Y_(10)能较显著改善犬肠道环境,促进微生态平衡,从而降低腹泻率,为预防犬肠道细菌性疾病提供了新的思路。
Probiotics, as a biological veterinary drugs and feed additives, has been widely used in the treatment and prevention animal enteritis and diarrhea of piglet, calf, lamb, poultry. However, the research and application of probiotics in dogs devoted to the domestic pet has not yet come out. It is not only having important ecological significance for the Kennel, but also bringing the huge enormous bebefit to biology of veterinary drugs and feed industry to develop canine microbiologic preparation. And to develop high-quality probiotics, selection of microbe species is the key. Therefore, this study was to separate and purificate a bacillus sp used as backup with probiotics strains from different varieties of pet dogs, and made a systematic evaluation of the selected strains in the security, identification of molecular biology and the protection of challenged puppies. The main contents and results were as follows:
1. 13 strains of Bacillus were isolated and purificated from different varieties of healthy pet dogs feces by using nutritional broth medium, in which five strains demonstrated a good survival ability in the presence of bile salts and at low pH. Taking five common conditions for the instructions of bacteria pathogenic microorganisms and using double-agar plate method of five strains to bacteria inhibition test, Bacillus Y_(10) was found to have a very good bacteriostatic effect. Sensitivity experiments showed that Bacillus Y_(10) were sensitive to seven common antibiotics as a backup strain of canine probiotics.
2. Prepared using solid-state fermentation of Bacillus Y_(10) powder to the concentration gradient of 10~9 cfu / g, 10~8 cfu / g and 10~7 cfu / g added to the feed of 40 Kunming mice to chronic experimental infection. Analysis of variance of the treatment group and control group of mice body weight size, immune indices, blood biochemical indicators, the number of cecal and colonic flora advantage short-chain fatty acids, there were no significant differences, indicating that Bacillus Y_(10) to mice were safe, the role of non-toxic negative.
3. Using molecular biology techniques, Bacillus Y_(10) was identified for Bacillus licheniformis, from the 16S rRNA sequence homology and phylogenetic.
4. Selected 16 healthy puppies to study the effects of dietary Bacillus Y_(10) in diarrhea and applied PCR-DGGE to analysis puppies at different stages of experimental intestinal flora structure and diversity. The experimental results showed that groups of not fed Bacillus Y_(10) had emerged diarrhea, compared in only one of the fed Bacillus Y_(10) groups, which indicated that Bacillus could significantly reduce the the rate of diarrhea (P <0.05). The DGGE results indicated that:①DGGE band pattern similarity indices of all groups in the first experimental day were at or above 56%. Diversity analysis showed that stripe pattern group and groups were not significantly different, indicating that the intestinal flora of puppies used were relatively stable.②The similarity indices of the experimental treatment group of intestinal flora in ninth day were at more than 58 percent. Positive control group between the individual patterns of similarity indices was at 69%～95% and the negative control group was at 58%～71%. The intestinal Microflora diversity of two groups was improves gradually from 11 to 14. The results of bands diversity showed that feeding Bacillus bacteria can enhance intestinal microbial polymorphism in dogs. The average band number of test group 1 after infection was at 13.3, between positive and negative in the control group, indicating that intestinal microbial polymorphism of puppies was reduced when infected after feeding Bacillus later. When puppies of test group 2 happened diarrhea, the average number of bands was at 16.6, higher than the other treatment groups, indicating that when infected by exogenous microbial, the puppies organism occurred in the intestinal flora imbalance, which resulted in a large number of micro-organisms discharged in vitro.
To sum up, the safety of assessment experiment and the protective effect in challenged puppies of drug testing results showed that Bacillus Y_(10) (Bacillus licheniformis) could be used as the probiotics strains, which provided a viable and valuable species for the development of canine probiotics. Moreover, the source dogs of Bacillus Y_(10) prebiotics could significantly improve the intestinal environment and the promotion of micro-ecological balance, thereby reducing the rate of diarrhea and providing a new way for the prevention of bacterial disease in dogs.
引文
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