PCR-DGGE实验技术在畜牧科学研究中的应用

本文综述了以变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)为主要手段结合聚合酶链式反应(polymerase chain reaction,PCR)实验技术的基本原理、主要实验流程,优缺点以及它在畜禽肠道等微生物的群落结构和多样性研究的最新动态。     

应用PCR-DGGE分析鸭场土壤细菌群落结构

为了解贵州省三穗县8个养鸭场环境土壤中细菌群落结构多样性概况,本试验采用试剂盒提取土壤样品细菌总DNA,PCR扩增细菌16S rDNA V3可变区,并通过变性梯度凝胶电泳(DGGE)技术对扩增产物进行凝胶分离,回收测序共得到43条特异性条带,其中7、8号养鸭场条带数相对较少.DGGE图谱经Quantity One软件分析,结果显示,同一鸭场样品相似性存在差异,绝大部分鸭场样品间相似性均高于50%,所测序列与GenBank中相应原核生物16S rDNA序列经同源性比对可见相似性在90%~100%之间,共包括5个大纲的细菌种类:变形菌门(Proteobacteria)的α,β类群、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)、酸杆菌门(Acidobacteria)和厚壁菌门(Firmicutes).     

Foal-Heat Diarrhea Is Not Caused by the Presence of Yeasts in Gastrointestinal Tract of Foals

The aim of this study was to evaluate whether a normal yeast flora is present in the intestine of healthy foals and whether establishing yeast flora or yeast overgrowth might be associated with foal-heat diarrhea. Among the sampled foals, 49% showed foal-heat diarrhea and 28.3% were positive for yeasts. Candida famata was isolated 9 times; Geotrichum sp., 6; Saccharomyces cerevisiae, 5; Cryptococcus laurentii, 4; Rhodotorula sp., 3; Zygosaccharomyces sp., 2; and Candida colliculosa, Candida globosa, and Candida pelliculosa 1 time, respectively. The colony-forming unit ranged from 1 to 10 in all positive cultures. Fifteen of 92 (16.3%) foals with heat diarrhea and 11 of 92 (11.9%) asymptomatic animals yielded positive samples for yeasts. None of the animals shed the same yeast more than 2 times consecutively, and yeasts were found in pure or mixed culture. Equine gastrointestinal tract did not appear to be colonized by yeasts during the first 15 days of life, and yeasts are not involved in foal-heat diarrhea.     

苏云金芽胞杆菌在小鼠肠道的消长及对小鼠肠道菌群的影响

利用选择性培养平板计数法,研究了苏云金芽胞杆菌(Bt)营养体和芽胞在小鼠肠道的数量消长及对小鼠肠道生理性有益菌群(大肠杆菌、双歧杆菌和乳酸杆菌)数量的影响,同时利用基于16S rDNA作为分子标记的变性梯度凝胶电泳(DGGE)研究了Bt对小鼠肠道微生物多样性的影响。结果表明:Bt营养体和芽胞在小鼠肠道内很快死亡,也未对小鼠肠道有益菌群数量和细菌多样性产生不良影响。说明苏云金芽胞杆菌(Bt)营养体及芽胞对小鼠是安全的。     

木聚糖酶对肉仔鸡后肠道微生物的影响

主要研究了小麦基础口粮添加木聚糖酶对肉仔鸡后肠道微生物的影响.将120只7日龄肉仔鸡分成2组,日粮分别添加0,0.1%木聚糖酶,饲喂至21日龄,研究木聚糖酶对肉仔鸡后肠道菌群数量变化的影响.结果表明,加酶未使同肠和盲肠乳酸杆菌、双歧杆菌和大肠杆菌数量发生显著变化.利用变性梯度凝胶电泳技术研究了木聚糖酶对肉仔鸡后肠细菌群体的影响.结果表明,加酶组回肠和盲肠的图谱比对照组条带相对较多,但差异不显著;组内个体间的图谱相似性比组问的相似性相对较大.木聚糖酶影响肉仔鸡后肠微生物数量和种群的作用效果不明显.     

Ammonia‐oxidizing Archaea in laboratory‐scale activated sludge systems for wastewater of low‐ or high‐ammonium concentration

Ammonia‐oxidizing bacteria (AOB) is known as ammonia‐oxidizer in wastewater treatment systems. However, ammonia‐oxidizing Archaea (AOA) is found from various environments, including wastewater treatment systems. In this study, to investigate the relationships between AOA population and ammonia concentration, AOA was monitored in two laboratory‐scale reactors treating artificial wastewater of different ammonium concentrations by denaturing gradient gel electrophoresis targeting ammonia monooxygenase genes. At day 60 of the operation, AOA populations dominant in each reactor differed, suggesting the importance of influent ammonia concentration in dominant AOA selection.     

Influences of quorum‐quenching probiotic bacteria on the gut microbial community and immune function in weaning pigs

The aim of this study is to investigate the dynamic gut microbial diversity in weaning swine after administering feed supplemented with probiotic bacteria that specifically inhibit the activity of quorum molecules. Initially, the universal quorum molecule autoinducer‐2 (AI‐2) bioassay results indicated that AI‐2 activity was profoundly inhibited in enterohemorrhagic Escherichia coli (EHEC) O157:H7 in the presence of Lactobacillus acidophilus strain 30SC cell extract, although the growth of EHEC was not affected. Based on plate counting results, bacterial community analysis revealed a specific reduction in coliforms compared to the control, whereas the population of lactobacilli increased in weaning swine in in vivo trials. Supplementation with L. acidophilus strain 30SC did not affect the counts of other communities, such as total aerobes and yeast/mold. In addition, PCR‐denaturing gradient gel electrophoresis analysis showed a significant difference in the 16S rRNA gene products after administering L. acidophilus strain 30SC. Selected bands were sequenced, and most of them were identified as uncultured bacterium clones or a Lactobacillus‐ and Bifidobacterium‐specific community. Therefore, our results indicate that quorum‐quenching probiotic bacteria can significantly modulate the gut microbiota of swine and these beneficial effects can contribute to the improvement of performance and health in the gastrointestinal tract of weaning pigs.     

莱芜黑山羊胃肠道血管活性肠肽免疫反应阳性细胞的研究

比较观察血管活性肠肽(VIP)在莱芜黑山羊胃肠道内的分布,用免疫组织化学方法显示胃肠壁各层及各肠段的分布情况。结果表明,血管活性肠肽以贲门部数量最多,结肠、空肠、幽门、盲肠、直肠、胃底次之,十二指肠、回肠、食管中未发现VIP细胞。VIP细胞形态多样,呈椭圆形和锥体形,常有突起伸至其他细胞附近,也有突起直达上皮表面。     

Distribution of 5-hydroxytryptamine,Ghrelin or Glucagon-like Peptide-1 Immunopositive Cells in the Gastrointestinal Tract of Meihua Pig

In order to analyze the distributions and densities of endocrine cells in the gastrointestinal tract of Meihua pig (an elite native breed of Guangdong province, China), 6 healthy piglets weaned at 30 days of age were fed with the same diet till 65 days of age. At 65 days of age, different sections of the gastrointestinal tract of each piglet were collected, and the features and densities of 5-hydroxytryptamine (5-HT), ghrelin and glucagon-like peptide-1 (GLP-1) immunopositive cells were detected by immunohistochemistry. The results showed that the 5-HT positive cells distributed at the epithelial layer throughout the intestinal tract (duodenum, jejunum, ileum and colon) with a variety of features (e.g. cone, round, spindle), and they could be classified into either open-shape or closed-shape type, the density of 5-HT positive cells in duodenum was highest; The ghrelin positive cells were located along the gastrointestinal tract (gastric glands, pylorus, duodenum and jejunum) with both open-shape and closed shape, and its density decreased along the tract with most cells in gastric glands; The GLP-1 positive cells were observed along the intestinal tract, but its density increased from duodenum to ileum and then decreased in colon. This study might provide supporting data for further study of the gastrointestinal physiology of Meihua pig.     

梅花猪胃肠道5-羟色胺、胃饥饿素和胰高血糖素样肽-1免疫阳性细胞分布特征研究

为了解中国优良地方猪种梅花猪胃肠道内分泌细胞的分布与形态特征,本试验选取6头30日龄断奶健康梅花仔猪,在相同条件下饲养至65日龄,所有仔猪饲喂相同日粮,自由采食饮水。试验结束时,收集仔猪胃肠道各段组织样品,应用免疫组织化学方法观察3种胃肠道内分泌激素5-羟色胺（5-HT）、胃饥饿素（ghrelin）和胰高血糖素样肽-1（GLP-1）免疫阳性细胞的分布及形态特征,并对阳性细胞密度进行分析。结果显示,梅花猪的肠道不同部位（十二指肠、空肠、回肠和结肠）均存在5-HT阳性细胞,主要位于肠上皮组织中,呈锥形、圆形、梭形等形态,可分为开放型和闭合型,其中十二指阳性细胞肠密度显著高于其他肠段（P<0.05）;梅花猪的胃肠道不同部位均有Ghrelin阳性细胞,阳性细胞密度也是随胃肠道走向递减,胃腺体部密度最高;梅花猪的肠道不同部位均有GLP-1阳性细胞,阳性细胞密度沿肠道走向呈抛物线分布,其中回肠数量最多。本试验结果为进一步探究梅花猪胃肠道生理奠定了基础。     

Presence of a Temperature Gradient Among Genital Tract Portions and the Thermal Changes Within These Portions Over the Estrous Cycle in Beef Cows

The aim of the present study was to describe the temperature of the different portions of the female genital tract and their relation to rectal temperature and to investigate the effect of steroid hormones profiles on these variables over the estrous cycle in cattle. Four nonpregnant Japanese Black cows were investigated daily over two successive estrous cycles using a digital thermometer with a long probe and rounded-end sensor to record the temperature of the rectum (RT), vagina (VT), cervix (CT), uterine body (UBT) and uterine horns (UHT). Blood samples were collected immediately before temperature recording to assay peripheral levels of progesterone (P₄) and estradiol-17β (E₂). Moreover, transrectal ultrasonography was carried out after temperature recording to monitor the ovulatory follicle and track ovulation. During the experiment, the ambient temperature and relative humidity were recorded for further calculation of the temperature humidity index (THI). The temperature within the genital tracts in these cows progressively increased towards the uterine horns from the vagina. The VT, CT, UBT and UHTs were significantly higher in association with peripheral P₄ concentrations greater than 4 ng/ml (mid-luteal phase) when compared with lower peripheral P₄ concentrations. The VT was more significantly (P<0.01) correlated to the CT, UBT and UHTs than RT. In conclusion, a temperature gradient was present among the vagina, cervix and uterus over the estrous cycle, and changes in peripheral P₄ concentrations were associated with the thermal variations within these portions. The VT could be more beneficial than RT in monitoring temperature of deeper portions of the female genital tract in bovine.     

Evaluation of the immune response induced by intradermal vaccination by using a needle-less system in comparison with the intramuscular route in conventional pigs

The immune response induced by intradermal vaccination using a needle-less device was evaluated in conventional pigs in comparison with the more conventional intramuscular vaccination; to this purpose, vaccination against Aujeszky’s Disease (AD) was used as a model of antiviral immunity. Two groups of pigs (n = 10 each) were vaccinated 4 weeks apart respectively by the intramuscular (IM group) and intradermal route (ID group; needle-less I.D.A.L.® vaccinator) with an AD modified live virus. Ten pigs injected with the vaccine adjuvant only were kept as sham-vaccinated controls (C group).On blood samples collected at 0, 2, 4, 5, 6 and 7 weeks post-vaccination (PV) ADV-specific virus neutralizing (VN) antibodies, IFN-γ secreting cells (SC), lymphocyte subsets and IFN-γ gene expression in PBMC were evaluated.VN antibodies increased after the 1st vaccination and peaked after the 2nd vaccination in both vaccinated groups. Also IFN-γ SC reached maximum levels in both groups after administration of the booster dose. Pigs in the control group remained negative for both parameters throughout the study. Flow cytometry showed persistently higher levels of CD3−CD8α+ Natural Killer cells in both vaccinated pigs. The ID group showed an earlier and regulated activation characterized by an increase of cytotoxic CD8β+ T lymphocytes and CD25+ cells after the boosting dose. No statistically significant differences between treated and control groups were detected for memory CD4+CD8α+^low T cells. Upregulation of IFN-γ gene expression in PBMC was detected in ID and IM pigs after both vaccine administrations, although at a different extent. Overall, the results showed that the intradermal vaccine delivery by a needle-less device can prime a strong humoral and cellular immune response comparable to that obtained by the intramuscular vaccination.