目的 运用高通量测序分析一只临床死亡的阿拉伯狒狒胃肠道菌群的组成及多样性,结合其临床症状和检测结果进一步分析该动物的死亡原因,为野生动物的饲养管理和物种健康提供基础资料.方法 以2017年12月20日成都动物园一只死亡的幼龄雌性阿拉伯狒狒为研究对象,收集其胃、十二指肠、空肠、回肠、盲肠和结肠的内容物及粪便,利用高通量测序技术对细菌16SrRNA基因的V4区进行测序,分析细菌菌群的组成和多样性差异.结果 十二指肠的菌群有效序列数较高,而盲肠和粪便菌群的alpha多样性较高;胃肠道菌群丰度不同,但都主要来自变形菌门、厚壁菌门和拟杆菌门,而大肠具有较多的梭杆菌门;海尔曼螺杆菌和大肠埃希菌分别在胃和小肠富集,嗜肺巴氏杆菌和具核梭杆菌在结肠富集,并且结肠具有较多来自变形菌门的特有细菌;菌群的功能预测提示胃和小肠具有腹泻和胃肠炎风险,结肠具有菌群失调风险.结论 阿拉伯狒狒的胃肠道细菌菌群的高通量测序分析和临床诊断及器官组织病理检测结果表明该动物患有的间质性肺炎与小肠富集的大肠埃希菌关系密切,其直接死亡可能与嗜肺巴氏杆菌有关,这些症状进一步导致其结肠菌群失调而加剧了死亡,提示饲养管理中应加强对动物园阿拉伯狒狒等野生动物肠道大肠埃希菌和嗜肺巴氏杆菌的监测,提高野生动物的饲养管理和物种健康.

运用实时荧光定量PCR技术,分析1只临床死亡小熊猫胃肠道13种菌群的差异性.结果显示:检测的13种菌群在小熊猫不同肠段的数量有差异,肠杆菌科(Enterobacteriaceae)在各肠段的菌群丰度差异显著(P<0.05),其菌群丰度在空肠高达9.35;白色瘤胃球菌(Ruminococcus albus)和黄色瘤胃球菌(Ruminococcus flavefaciens)的数量在直肠最多,菌群丰度分别达5.29和3.91;其他菌群的数量均在结肠最多,其中溶纤维丁酸弧菌(Butyrivibrio fibrisolvens)、埃氏巨球形菌(Megasphaera elsdenii)和梭菌类群IV(Clostridium cluster IV)的数量在结肠中较高,菌群丰度分别为9.03、6.06和9.13;与胃相比,菌群丰度在小熊猫空肠、结肠和直肠差异显著(P<0.05).综合分析,小熊猫的空肠、结肠和直肠对其健康生长可能起着重要的作用,肠杆菌科的细菌数量最多且在不同肠段差异显著,是今后小熊猫胃肠道菌群研究值得关注的菌群之一.

The aim of this experiment was to evaluate the bacteria diversity in gastrointestinal tract (GIT) of a dead wild Rhinopithecus roxellanae,and to analyze the phylogenetic tree of cloning sequencing bands.The GIT contents of a dead wild Rhinopithecus roxellanae were collected,and the polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) technology with the cloning sequencing,cluster analysis and principal component analysis (PCA) of bands were used to detect the bacterial diversity and to build the phylogenetic tree.The results showed as follows:1) numerous bacteria were obtained from the GIT of wild Rhinopithecus roxellanae.Samples from the stomach,duodenum,jejunum and ileum clustering together,samples from the cecum,colon and rectum clustering together,and samples from the faeces single clustering together.2) The 18 identified DGGE bands were belong to five phylum,they were Proteobacteria (38.89％),Firmicutes (22.22％),Bacteroidetes (5.56％),Actinobacteria (5.56％),Verrucomicrobia (5.56％) and uncultured bacterium (22.22％).The Proteobacteria and Firmicutes were detected along the GIT.3) The phylogenetic tree analysis showed that only one of uncultured bacterium was similar to the identified classification of Enterococcus faecalis,while the other three uncultured bacteria had a significant difference with the known branch of the bacteria.This indicated that a large number of flora information was unkowned in the GIT of the wild Rhinopithecus roxellanae.The results suggest that Proteobacteria is predominant in the GIT of a dead wild Rhinopithecus roxellanae,and the bacteria diversity shows a tendency of high-low-high according to the GIT from front to back.

This study aimed to evaluate the bacterial diversity in the gastrointestinal tract (GIT) of Ailurus fulgens.The polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) technology combined with the cloning sequencing,cluster analysis and principal component analysis (PCA) of bands were used to detect the bacterial composition and diversity.The results showed as follows: 1) numerous bacteria were obtained from the GIT of Ailurus fulgens by using the PCR-DGGE profiles.There was a difference in each intestinal segment.However,there were some similarities between the adjacent intestinal segment.Samples from the colon and faeces had the higher bacterial diversity,the secondly for samples from stomach and rectum,and the samples from the jejunum and ileum had the lower bacterial diversity.2) The sequencing results of PCR-DGGE profiles of bacteria from gastrointestinal tract of Ailurus fulgens showed that most of the bacteria belonged to Firmicutes,Bacteroides,Proteobacteria and Verrucomicrobia.The common bands were mainly inclued uncultured Bacteroidetes bacterium,Enterococcus faecalis,uncultured Clostridium sp.,Lactococcus lactis and Weissella cibaria,and Firmicutes was the dominant bacterial.The specific bands were Comamonas sp.,Clostridium sp.and Akkermansia.In summary,a large amounts of bacteria exist in the GIT of Ailurus fulgens,and the diversity of bacteria shows a tendency of high-low-high according to the GIT from front to back.

为了解成都地区犬布氏杆菌病的流行情况并建立一种快速、可靠、特异、灵敏的布氏杆菌的检测方法,对来自宠物医院、狗场等的860份犬血清样品进行了血清学检测,并针对布氏杆菌属保守基因设计1对特异性引物并建立了一种基于SYBR Green的荧光定量PCR检测方法.血清学检测结果显示,成都地区存在犬布氏杆菌病的总阳性率为1.51％,其中流浪狗阳性率最高,其次是狗场,宠物医院犬布氏杆菌病的阳性率最低.本研究建立的荧光定量PCR具有较高的特异性,标准曲线相关系数为0.996,扩增效率为91％,能够用于未知样品的定量检测.用建立的荧光定量PCR检测了33份虎红平板凝集试验(RBPT)阳性血清,结果,其中10份呈阳性;检测了30份随机抽取的阴性血清,其中2份呈阳性.结论:本研究建立的荧光定量PCR与RBPT的检测结果有差异,两种方法均检测到成都地区犬布氏杆菌病.

The aim of this study was to analyze the composition and function of ileum microbial community of healthy chickens. The samples of ileum contents of 15 chickens were investigated by high?throughput sequen?cing. Operational taxonomic units ( OTUs) were picked with a 97% similarity threshold and the sequences less than two in all samples were removed. Than the results were analyzed by bioinformation software and PICRUSt functional profiles. The results showed as follows:1) a total of 424 062 tags were assigned to 1 002 non?sin?gleton OTUs from all samples. Each sample had (28 271±8 855) tags and (261±82) OTUs on average. 2) In total, 20 phyla and 160 genera were detected, and 4 shared core phyla were contained in all samples. They were Firmicutes (85.35%), Proteobacteria (6.09%), Cyanobacteria (2.15%) and Actinobacteria (0.16%). Nineteen shared core genera were contained in all samples, which consisted 93. 75% of the overall bacteria community. 3) Three hundred and twenty?eight functions were detected in all ileum samples. Heat?map of the first 20 functions showed that there were some differences in efficiency of each sample. Therefore, our research reveals that the chicken ileum is complex for the phylogenetic diversity of the microbial communities, but the main microbial communities are relatively stable. The findings of this study will provide basic information and lay the foundation of the research in composition and function of the microbiota of chicken ileum.

<span id="ChDivSummary" name="ChDivSummary" class="abstract-text">为对肉鸡盲肠微生物的多样性进行探索,选取15只饲养在相同条件下的科宝-500肉鸡,利用Illumina MiSeq测序平台与Qiime等16srRNA序列分析工具在22日龄时采集盲肠内容物进行研究。结果显示:1)测序共获得359 158条有效序列与3 210个OTU,并且由稀释曲线可以证明此次测序结果比较全面的覆盖了肉鸡盲肠微生物群落。2)各样品菌群之间存在差异,得到序列的数量以及菌群多样性差别较大。3)通过核心菌群分析可知,4个共享菌群在门水平上分别为:拟杆菌门Bacteroidetes（70.0%±12.1%）、厚壁菌门Firmicutes（25.4%±11.2%）、变形菌门Proteobacteria（4.3%±3.7%）和蓝菌门Cyanobacteria（0.3%±0.5%）。4)有益菌群如乳酸杆菌属Lactobacillus、双歧杆菌属Bifidobacterium虽然存在于盲肠微生物中,但相对丰度不高。5)PICRUSt分析盲肠菌群对应的基因功能发现:功能转运、嘌呤代谢、DNA修复重组蛋白、氧化磷酸化和甲烷代谢等基因功能丰度最高。由此可见,个体差异虽然对肉鸡盲肠微生物结构影响较大,但是仍然存在不少共性。</span>

为探明植物乳杆菌BS22对黄曲霉毒素的解毒作用及对肉鸡肠道菌群的影响,设饲料添加不同量的BS22试验(不饲喂)和饲料添加BS22饲喂肉鸡试验.在含有50μg黄曲霉毒素B1(AFB1)的1 kg饲料中加入106、107、108 CFU/g植物乳杆菌BS22,于加入0、15、30d检测AFB1含量.检测结果:加入BS22后,各组饲料中的AFB1含量均显著下降;同一添加量下,随饲料贮存时间的增加,AFB1含量均有所提高.肉鸡饲喂设3个组:Ⅰ组(对照组),饲喂基础日粮;Ⅱ组,饲喂基础日粮+50 μg/kg黄曲霉毒素B1;Ⅲ组,饲喂基础日粮+50 μg/kg黄曲霉毒素B1+1.0× 108 CFU/g植物乳杆菌BS22.试验期为28 d.采用RCR-DGGE比较4周龄肉鸡嗉囊、腺胃、十二指肠、空肠、回肠及盲肠内容物及黏膜细菌群落的结构.对消化道内容物而言,除回肠外,消化道其他部位的内容物的细菌多样性指数、均匀度和丰富度均是Ⅲ组的高于Ⅱ组和对照组;聚类图显示,除腺胃和盲肠外,其他部位均是Ⅱ组与对照组聚在一起,而Ⅲ组单独存在.对消化道黏膜而言,除空肠和盲肠外,其他部位的黏膜的细菌多样性指数和丰富度均是Ⅱ组的均高于Ⅲ组和对照组,且Ⅲ组和对照组的细菌多样性指数相近或相同;除回肠和盲肠外,其他部位Ⅲ组和对照组聚在一起,而试验Ⅱ组单独存在.综合分析,黄曲霉毒素B1对各肠段内容物菌群的作用不明显,对黏膜菌群有较大影响,尤其是对十二指肠的黏膜菌群影响更明显,植物乳杆菌BS22能调节由于黄曲霉毒素B1引起的肠道黏膜菌群失衡.

This experiment was conducted to compare bacteria community diversity of liquid and solid phases of ruminal contents of Mongolian sheep. The samples of liquid and solid phases of ruminal contents from 5 Mongolian sheep were investigated using 16S rRNA amplicon high-throughput sequencing technique by Illumi-na MiSeq platform, and the results were analyzed by UPARSE-operational taxonomic units ( OTU) and un-weighted pair group method with arithmetic mean ( UPGMA) . The results showed that a total of 91 971 se-quences and 1 871 OTUs across all samples were observed, and the average number of sequence and OTU was 13 138 and 267 per sample, respectively. Compared between the samples of liquid and solid phases: at phyla level, dominant bacteria with significant differences were Bacteroidetes and Firmicutes, and the percentage of Bacteroidetes of the samples of liquid and solid phases was 67% and 45%, respectively;at genus level, domi-nant bacteria with significant differences were Prevotella and an unknown genus, dominant bacterium of liquid phase was Prevotella whose percentage reached 53%, and that of solid phase was the unknown genus whose percentage reached 32%; at species level, dominant bacteria with significant differences were Prevotella ru-minicola, and the percentage of liquid and solid phases reached 41% and 11%, respectively. The alpha diversi-ty of solid phase was higher compared with that of liquid phase, and the samples from the same animal clus-tered together by principal component analysis ( PCA) and principal coordinate analysis ( PCoA) . In conclu-sion, both the contents of liquid and solid phases have high diversity of bacteria, and the diversity of solid phase is higher compared with that of liquid phase.

<span id="ChDivSummary" name="ChDivSummary" class="abstract-text">为探讨蒙古羊瘤胃液相和固相细菌的多样性,采用变性梯度凝胶电泳技术结合聚合酶链式反应（PCRDGGE）及条带的克隆测序,比较瘤胃液相和固相细菌的差异,同时采用聚类分析和主成分分析（PCA）方法分析瘤胃液相和固相细菌的多样性。结果表明:1)瘤胃液相和固相样品均含有物种丰富的细菌菌群,但液相比固相具有更高的平均条带数,分别为30条和24条;2)瘤胃液相样品比固相样品具有更高的多样性指数、均匀度和丰富度,其数值分别为3.45、0.90和30.20及3.23、0.84和24.62;3)同一动物个体瘤胃液相样品和固相样品聚类在一起,相似性系数均高达0.81;4)共性条带测序结果表明瘤胃的优势细菌主要是Uncultured rumen bacterium和Uncultured Bacteroidetes bacterium,而特异性条带主要是Alcaligenes sp.。不同动物个体的瘤胃液相和固相均含有丰富的细菌菌群,且物种丰富度均较高;同一动物个体的液相与固相相比具有更高的细菌多样性。</span>

This experiment aimed at analyzing the structure of gut microbiome in young, adult and old Rhi?nopithecus roxellana, and analyzing the change trend of the gut microbiome with the age increasing. Performed the polymerase chain reaction?denaturing gradient gel electrophoresis ( PCR?DGGE) with the total DNA extrac?ted from the faeces of young, adult and old Rhinopithecus roxellana, and the results were analyzed by UPGMA cluster and principal component, and the biological diversity of Rhinopithecus roxellana in different ages was compared. The number of eubacteria, Lactobacillus spp. , Enterobacteriaceae, Bifidobacterium spp. in gut mi?crobiome was detected by fluorescence quantitative PCR technology. The results showed as follows: not only the UPGMA cluster analysis, but also the principal component analysis and the biological diversity comparison indicated that the diversity of gut microbiome of Rhinopithecus roxellana in different ages existed obvious differences. The advantages of the band of the young and adult Rhinopithecus roxellana were more than the old Rhinopithecus roxellana in the fingerprint. The number of eubacteria in the gut microbiome of Rhinopithecus roxellana in different ages was all in the level 1011. The number of Enterobacteriaceae in the gut microbiome of the young Rhinopithecus roxellana was significantly less than the adult’ s and the old’ s ( P<0.05) . The num?ber of Lactobacillus spp. in the gut microbiome of the adult Rhinopithecus roxellana was significantly more than the young’s and the old’s (P<0.01). The number of Bifidobacterium spp. in the gut microbiome of the old Rhinopithecus roxellana was significantly less than the adult’ s and the young’ s ( P<0.01) . In conclusion, a?long with the age increasing, the diversity of the gut microbiome of Rhinopithecus roxellana shows an increas?ing trend from childhood to adulthood and shows a reducing trend from adulthood to senectitude. And, the number of advantage bacterium group is reduced in the senile period. Moreover, the number of gut microbiome of Rhinopithecus roxellana has great differences in different ages.

In order to compare the bacterial flora diversity of rumen, reticulum, omasum and abomasum from sheep, polymerase chain reaction-denaturing gradient gel electrophoresis ( PCR-DGGE) analysis compounded with cloning and sequencing of bands in DGGE profiles were used, and cluster analysis and principal constitu-ent analysis ( PCA) of bacterial flora were carried out. The results showed as follows: the average number of DGGE bands of contents in rumen, reticulum, omasum and abomasum was 18, 13, 16 and 15, respectively;Shannon diversity index, evenness and richness of bacteria in rumen and omasum were higher, which were 2.83, 0.79 and 17.00, and 2.82, 0.79 and 16.80, respectively, however, those of reticulum and abomasum were lower, which were 2.52, 0.70 and 12.60, and 2.73, 0.76 and 15.40, respectively; clustering analysis results suggested that there were some differences in bacterial flora among the contents in different stomachs, but the similarity coefficients from the same segment of stomach of different animals were all above 0.63;most of the sequenced bands belonged to Bacteroidetes and Firmicutes, and Bacteroidetes, Firmicutes, uncultured rumen bacterium, uncultured bacterium and Veillonellaceae were predominant in stomach, while the special bacteria were Acinetobacter sp. and Ruminococcaceae bacteria. In conclusion, there are rich kinds and large a-mount of bacteria in the four stomachs of sheep. With rearward moving of position of intestinal tract, the diver-sity increased firstly and then decreased and finally increased.

The trial was conducted to investigate the effect of moisture content on pH,lactic acid content,the number of lactobacillus and dry matter recovery(DMR)in lactobacillus solid-state fermentation feed.There were five groups in the test,group A,B,C,D and E,each group included 3 replications.The moisture content of these treatments were 25%,30%,35%,40% and 45%,respectively.The results showed that:the pH reduced most fast in group D with the largest number of lactobacillus;group C had the highest content of lactic acid;there was a negative correlation between DMR and moisture content;the optimum moisture content of fermentation feed was 35% ～ 40%.

This experiment was conducted to study the diversity of rumen microflora under different cellulose enrichment conditions by polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE).Carboxymethyl cellulose(CMC),peptone-cellulose(PCS),J and K mediums were used in this study,and rumen contents were incubated at 37 and 50 ℃,respectively.The diversity of rumen mircroflora was analyzed by PCR-DGGE,cloning and sequencing of common and specific bands in DGGE profiles,cluster analysis and principal component analysis.The results showed as follows: there were differences among DGGE profiles of different mediums at 37℃,the similarity coefficient between J and PCS mediums was 0.77,and that between J and CMC mediums was 0.76.The DGGE profiles of different mediums were similar to each other at 50℃,the similarity coefficient between J and PCS mediums was 0.78,and that between CMC and PCS mediums reached 0.84.The common bands were Streptococcus gallolyticus and Ureibacillus themosphaericus,and were predominant in rumen contents;the specific bands were Streptococcus gallolyticus,Pseudomonas mosselii,Alcaligenes and uncultured Lachnospiraceae bacterium clone.In conclusion,the diversity of rumen microflora can be affected by different cellulose enrichment mediums and culture temperatures.

Two kinds of hyperimmune sera,porcine-anti-rotavirus IgG and rabbit-anti-rotavirus IgG,were prepared by inoculating respectively piglets and rabbits with porcine rotavirus(RV) purified by diffe-rential centrifugation,and purified by affinity chromatography.A double antibody sandwich ELISA for detection of RV was developed based on the two kinds of IgG.The optimal coating concentration of porcine-anti-rotavirus IgG was 4 μg/mL,the optimal working concentration of rabbit-anti-rotavirus IgG was 3.5 μg/mL,the reaction time of sample was 90 min,and the optimal working dilution of HRP-labelled goat-anti-rabbit IgG was 1∶8 000.The positive standard value was 0.161(D 450 nm).The coefficient of variation of reproducibility was less than 10%,and at least 1.25 μg/mL antigen could be detectable.The ELISA had no cross-reaction with classical swine fever virus,porcine pseudorabies virus,porcine transmissible gastroenteritis virus,porcine epidemic diarrhea virus,Escherichia coli and Salmonella.The prepared plates and reagents could preserved at least four months at room temperature and 4 ℃.Seventy clinical fecal samples were detected by the ELISA and the colloidal gold card,and the positive ratio was 22.9% and 20.0% respectively.The results revealed that the ELISA possessed good specificity and reproducibility,and higher sensitivity,indicating a suitable method for rapid detection of RV.