Quantitative comparisons of select cultured and uncultured microbial populations in the rumen of cattle fed different diets

ABSTRACT: BACKGROUND: The number and diversity of uncultured ruminal bacterial and archaeal species revealed by 16S rRNA gene (rrs) sequences greatly exceeds that of cultured bacteria and archaea. However, the significance of uncultured microbes remains undetermined. The objective of this study was to assess the numeric importance of select uncultured bacteria and cultured bacteria and the impact of diets and microenvironments within cow rumen in a comparative manner. RESULTS: Liquid and adherent fractions were obtained from the rumen of Jersey cattle fed hay alone and Holstein cattle fed hay plus grain. The populations of cultured and uncultured bacteria present in each fraction were quantified using specific real-time PCR assays. The population of total bacteria was similar between fractions or diets, while total archaea was numerically higher in the hay-fed Jersey cattle than in the hay-grain-fed Holstein cattle. The population of the genus Prevotella was about one log smaller than that of total bacteria. The populations of Fibrobacter succinogenes, Ruminococcus flavefaciens, the genus Butyrivibrio, and R. albus was at least one log smaller than that of genus Prevotella. Four of the six uncultured bacteria quantified were as abundant as F. succinogenes, R. flavefaciens and the genus Butyrivibrio. In addition, the populations of several uncultured bacteria were significantly higher in the adherent fractions than in the liquid fractions. These uncultured bacteria may be associated with fiber degradation. CONCLUSIONS: Some uncultured bacteria are as abundant as those of major cultured bacteria in the rumen. Uncultured bacteria may have important contribution to ruminal fermentation. Population dynamic studies of uncultured bacteria in a comparative manner can help reveal their ecological features and importance to rumen functions.     

Effect of Saccharomyces cerevisiae on alfalfa nutrient degradation characteristics and rumen microbial populations of steers fed diets with different concentrate-to-forage ratios

Live yeast（Saccharomyces cerevisiae） constitutes an effective additive for animal production;its probiotic effect may be related to the concentrate-to-forage ratio（CTFR）.The objective of this study was to assess the effects of S.cerevisiae（SC） on fiber degradation and rumen microbial populations in steers fed diets with different levels of dietary concentrate.Ten Simmental × Local crossbred steers（450 ± 50 kg BW） were assigned to a control group or an SC group.Both groups were fed the same basal diet but the SC group received SC supplementation（8 × 10^9 cfu/h/d through the ruminal fistula）following a two-period crossover design.Each period consisted of four phases,each of which lasted 17 d：10 d for dietary adaptation,6 d for degradation study,and 1 d for rumen sample collection.From the 1~（st） to the 4~（th） phase,steers were fed in a stepwise fashion with increasing CTFRs,i.e.,30：70,50：50,70：30,and 90：10.The kinetics of dry matter and fiber degradation of alfalfa pellets were evaluated;the rumen microbial populations were detected using real-time PCR.The results revealed no significant（P〉 0.05） interactions between dietary CTFR and SC for most parameters.Dietary CTFR had a significant effect（P〈 0.01） on degradation characteristics of alfalfa pellets and the copies of rumen microorganism;the increasing concentrate level resulted in linear,quadratic or cubic variation trend for these parameters.SC supplementation significantly（P〈 0.05） affected dry matter（DM） and neutral detergent fiber（NDF）degradation rates（c_（DM）,c_（NDF）） and NDF effective degradability（ED_（NDF））.Compared with the control group,there was an increasing trend of rumen fungi and protozoa in SC group（P 〈 0.1）;copies of total bacteria in SC group were significantly higher（P〈 0.05）.Additionally,percentage of Ruminobacter amylophilus was significantly lower（P〈 0.05）but percentage of Selenomonas ruminantium was significantly higher（P〈 0.05） in t     

Effect of Saccharomyces cerevisiae on alfalfa nutrient degradation characteristics and rumen microbial populations of steers fed diets with different concentrate-to-forage ratios

Live yeast (Saccharomyces cerevisiae) constitutes an effective additive for animal production; its probiotic effect may be related to the concentrate-to-forage ratio (CTFR). The objective of this study was to assess the effects of S. cerevisiae (SC) on fiber degradation and rumen microbial populations in steers fed diets with different levels of dietary concentrate. Ten Simmental × Local crossbred steers (450 ± 50 kg BW) were assigned to a control group or an SC group. Both groups were fed the same basal diet but the SC group received SC supplementation (8 × 10⁹ cfu/h/d through the ruminal fistula) following a two-period crossover design. Each period consisted of four phases, each of which lasted 17 d: 10 d for dietary adaptation, 6 d for degradation study, and 1 d for rumen sample collection. From the 1^st to the 4^th phase, steers were fed in a stepwise fashion with increasing CTFRs, i.e., 30:70, 50:50, 70:30, and 90:10. The kinetics of dry matter and fiber degradation of alfalfa pellets were evaluated; the rumen microbial populations were detected using real-time PCR. The results revealed no significant (P > 0.05) interactions between dietary CTFR and SC for most parameters. Dietary CTFR had a significant effect (P < 0.01) on degradation characteristics of alfalfa pellets and the copies of rumen microorganism; the increasing concentrate level resulted in linear, quadratic or cubic variation trend for these parameters. SC supplementation significantly (P < 0.05) affected dry matter (DM) and neutral detergent fiber (NDF) degradation rates (c_DM, c_NDF) and NDF effective degradability (ED_NDF). Compared with the control group, there was an increasing trend of rumen fungi and protozoa in SC group (P < 0.1); copies of total bacteria in SC group were significantly higher (P < 0.05). Additionally, percentage of Ruminobacter amylophilus was significantly lower (P < 0.05) but percentage of Selenomonas ruminantium was significantly higher (P < 0.05) in the SC group. In a word, dietary CTFR had a significant effect on degradation characteristics of forage and rumen microbial population. S. cerevisiae had positive effects on DM and NDF degradation rate or effective degradability of forage; S. cerevisiae increased rumen total bacteria, fungi, protozoa, and lactate-utilizing bacteria but reduced starch-degrading and lactate-producing bacteria.     

Ecological characterization of three different phylogenetic groups belonging to the cellulolytic bacterial species Fibrobacter succinogenes in the rumen

To study the group‐dependent ecology of Fibrobacter succinogenes in the rumen, real‐time polymerase chain reaction assays for two phylogenetic groups (groups 2 and 3) of F. succinogenes were newly established and applied to rumen samples. Both the assays targeting the bacterial 16S rDNA were sensitive and accurate, showing wide quantifiable ranges (10⁴?10⁹ and 10²?10⁹ copies of 16S rDNA) and high recoveries of known amounts of added DNA (96.9 and 98.0%). The quantity of group 1 was confirmed to be numerable by subtracting assay values of groups 2 and 3 from that of F. succinogenes species (groups 1–3). By using the developed assays and the above subtractive calculation, the quantities of all three groups were evaluated in solid and liquid fractions of the rumen content and also on hay stems. In the solid fraction, groups 1 and 2 were abundantly present, compared with group 3 (P < 0.05). On untreated hay stems, group 1 was dominant throughout 48 h. In addition, group 1 showed growth even on the cellulase‐treated hay stems, unlike the other two groups. These results suggest that F. succinogenes group 1 greatly contributes to rumen fiber digestion, even for less degradable materials.     

Validation and application of real-time polymerase chain reaction assays for representative rumen bacteria

Real‐time polymerase chain reaction (PCR) assays for 11 representative rumen bacterial species were validated. The sensitivity was tested by using the serially diluted target 16S rDNA from respective bacterial species. The recovery of the target DNA and the assay reproducibility were determined using DNA from rumen fluid spiked with different quantities of the target. Minimum detection levels for the target were 10–100 copies in pure culture. The recovery of the added target ranged from 82.4 to 116.6%. The intra‐ and inter‐assay variations of each assay were <9.4 and <12.6%, respectively. Therefore, the real‐time PCR assays evaluated in the present study are considered to be sufficiently reliable for monitoring all 11 bacterial species in the rumen. The assays were then applied to the monitoring of the bacterial species attached to ruminally incubated rice straw. Among the monitored fibrolytic species, Fibrobacter succinogenes was found to be the most dominant, accounting for 2.61% of total bacteria after 24 h incubation. Selenomonas ruminantium and Streptococcus bovis, non‐fibrolytics, were detected on the rice straw at 8.96% and 1.16% of total bacteria, respectively. Such high levels of non‐fibrolytics on the plant fiber suggest a synergistic relationship between fibrolytics and non‐fibrolytics.     

酵母发酵饲料对瘤胃菌群数量及多样性的影响

本试验研究日粮中添加酵母发酵饲料对蒙古绵羊瘤胃菌群数量及多样性的影响.选用10只安装永久性瘤胃瘘管且体重约40 kg的14月龄蒙古羯羊,分为对照组和试验组,每组各5只.对照组饲喂基础日粮,试验组饲喂基础日粮+酵母发酵饲料(13.37％).预试期15 d,正试期5 d.于正试期晨饲后0、3、6、9、12 h依次采集瘤胃液...     

The mcrA gene and 16S rRNA gene in the phylogenetic analysis of methanogens in the rumen of faunated and unfaunated cattle

The influence of rumen protozoa on the composition of rumen methanogens was studied by using seven growing Holstein cattle divided into two groups: four faunated and three unfaunated. 16S ribosomal RNA gene (rDNA) and methyl coenzyme‐M reductase (MCR) α subunit (mcrA) gene clonal libraries were constructed. The results of each analysis showed that Methanobacteriales was dominant in the rumen of both groups. By mcrA gene analysis, 22.1% of unfaunated clones were classified into unfaunated group 1, which was not detected from faunated cattle. The 16S rRNA gene analysis showed that the number of operational taxonomic units was higher in unfaunated than faunated cattle, suggesting the diversity of methanogens tended to be higher by the removal of protozoa. The results of the LIBSHUFF program indicated that the 16S rRNA gene and mcrA gene clone libraries for the faunated group differed from those for the unfaunated group (P = 0.001). It was suggested that the presence of protozoa strongly affected the composition of rumen methanogens.     

Quantitative analysis of ruminal methanogenic microbial populations in beef cattle divergent in phenotypic residual feed intake (RFI) offered contrasting diets

Background

Methane (CH₄) emissions in cattle are an undesirable end product of rumen methanogenic fermentative activity as they are associated not only with negative environmental impacts but also with reduced host feed efficiency. The aim of this study was to quantify total and specific rumen microbial methanogenic populations in beef cattle divergently selected for residual feed intake (RFI) while offered (i) a low energy high forage (HF) diet followed by (ii) a high energy low forage (LF) diet. Ruminal fluid was collected from 14 high (H) and 14 low (L) RFI animals across both dietary periods. Quantitative real time PCR (qRT-PCR) analysis was conducted to quantify the abundance of total and specific rumen methanogenic microbes. Spearman correlation analysis was used to investigate the association between the relative abundance of methanogens and animal performance, rumen fermentation variables and diet digestibility.

Results

Abundance of methanogens, did not differ between RFI phenotypes. However, relative abundance of total and specific methanogen species was affected (P < 0.05) by diet type, with greater abundance observed while animals were offered the LF compared to the HF diet.

Conclusions

These findings suggest that differences in abundance of specific rumen methanogen species may not contribute to variation in CH₄ emissions between efficient and inefficient animals, however dietary manipulation can influence the abundance of total and specific methanogen species.     

First report of Wolbachia from field populations of Culex mosquitoes in south-western Nigeria

Recent reports on finding Wolbachia-strain infections in field mosquito species in some West African countries and the potential for developing these as disease vector biocontrol tools have prompted a search for Wolbachia in mosquitoes within the study area. Using a completely randomised design, mosquito traps were set at different locations in a rural and an urbanised community. One hundred and eighty (180) mosquitoes were trapped and pooled on the basis of genus, sex and site of collection, because there have been no earlier reports of Wolbachia isolated from Nigeria. Twenty pools, made up of not more than ten mosquitoes per pool, were homogenised and analysed for Wolbachia-specific DNA. Mosquitoes were trapped within Ede (urbanised community) and Akoda (rural community). Genomic DNA was extracted from trapped mosquito samples and used as a template in a PCR reaction. The Wolbachia sp. specific 16S rRNA gene was amplified, sequence analysis of PCR products was performed and a chromatogram of the sequence was subjected to Basic Local Alignment Search Tool analysis to identify the Wolbachia sp. This sequence was subsequently submitted to GenBank with accession number MK127541. The first evidence of the presence of the endosymbiont, Wolbachia in field-caught mosquitoes is hereby documented. The homology of this strain of Wolbachia bears similarities to those reported recently from other parts of West Africa and forms a single clade with a Wolbachia sp. from Mali, with a strong bootstrap support of 99%. This finding of a Wolbachia strain in mosquitoes at Ede could form the basis for more searches for diverse strains of Wolbachia in Nigeria.     

荷斯坦奶牛瘤胃微生物分离鉴定及多样性分析

研究为分离获得荷斯坦奶牛瘤胃内容物中的细菌,建立系统进化树,获取有益菌种。采用培养组学技术和16S rDNA分子鉴定方法相结合,对3头健康荷斯坦奶牛瘤胃内容物中细菌进行分离培养。共分离得到105株细菌,包括肠球菌属(Enterococcus)共15株14.29%,芽孢杆菌属(bacillus)共11株10.48%,不动杆菌属(Acinetobacter)共14株13.33%,葡萄球菌属(Staphylococcus)共22株20.95%,梭菌属(Clostridium)共1株0.95%,狭义梭菌属(Clostridium sensu stricto)共2株1.90%,短杆菌科(Brevibacteriaceae)共2株1.90%,链球菌属(Streptococcus)共11株10.48%,气球菌属(Aerococcus)共4株3.81%,柠檬酸杆菌属(Citrobacter)共14株13.33%,杆菌属(Brachybacterium)共1株0.95%,克雷伯氏菌属(Klebsiella)共1株0.95%,普罗维登斯菌属(Providencia)共3株2.86%,沙雷氏菌属(Serratia)共4株3.81%。结果中所占比例最高的菌属是葡萄球菌属;系统进化树分析和GenBank中的同源性比对结果发现,从细菌门、纲、目、科、属、种分析,分支明确;26个菌种同源性都在95.01%~100%之间。分离纯化出11株芽孢杆菌属(Bacillus)细菌具有潜在益生菌活性。可作为饲料添加剂饲喂荷斯坦奶牛。     

基于16S rRNA高通量测序技术分析草原红牛瘤胃微生物多样性和功能预测的研究

为系统探讨草原红牛瘤胃内的微生物多样性及其功能,本试验利用16S rRNA基因高通量测序技术检测分析草原红牛(20月龄左右,均重为577.5 kg)瘤胃液样本菌群结构并进行PICRUSt功能预测。结果显示:通过Illumina Miseq测序平台共获得35 848条优质序列,聚类分析得到387个操作分类单元(OTU),经分类学鉴定分属15个门、20个纲、25个目、41个科及110个属;厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes)为优势菌群,所占比例分别为50.09%和41.11%;基于属的组成,依次为普雷沃菌属(Prevotella)15.20%、未知属f型拟杆菌目(norankfBacteroidales)BS11菌群8.66%、瘤胃菌科(Ruminococcaceae)NK4A214菌群6.96%、理研菌科(Rikenellaceae)RC9菌群5.56%、未知属(Christensenellaceae)R-7菌群4.01%、瘤胃球菌属2(Ruminococcus2)3.33%等;16S rRNA基因组的PICRUSt功能预测结果显示,瘤胃内菌群功能主要集中在碳水化合物转运及代谢,表面草原红牛体内含有大量的纤维素和木质素降解酶基因。综上,基于16S rRNA基因的高通量测序技术全面揭示了草原红牛瘤胃菌群的多样性,且预测其含有丰富的蛋白质分解、木质纤维素降解酶系,为探索草原红牛瘤胃微生物的认知提供了基础,也为挖掘其他重要营养生理功能相关的瘤胃微生物功能基因提供了参考。     

Detection and identification of rumen bacteria constituting a fibrolytic consortium dominated by Fibrobacter succinogenes

A fibrolytic consortium, dominated by the rumen cellulolytic bacterium Fibrobacter succinogenes, was artificially constructed on hay stems to detect and identify rumen bacteria that can potentially interact with F. succinogenes . Consortium-bacterial members were determined by DGGE and sequencing analysis targeted bacterial 16S rDNA. An artificial consortium was formed in a 2-step incubation of hay stems; the first step with group 1, 2 or 3 F. succinogenes strains, the second step with rumen fluid. After consortium formation, morphologically different bacteria were observed in association with F. succinogenes . DGGE exhibited more than 30 bands, the pattern of which depended on the F. succinogenes group. Sequencing suggested that Butyrivibrio fibrisolvens, Pseudobutyrivibrio ruminis , Clostridium sp., F. succinogenes group 2, Prevotella ruminicola and unclassified Bacteroides were prominent in the group 1 consortium and that Treponema bryantii , B. fibrisolvens , Acinetobacter sp, and Wolinella succinogenes were prominent in the group 2 consortium. However, in the group 3 consortium, F. succinogenes -like bacteria were microscopically undetectable, whereas cellulolytic Ruminococcus albus and F. succinogenes group 1 were prominent, suggesting that the group 3 cannot be a core member of this consortium. This study is the first attempt to identify bacterial members of a fibrolytic consortium dominated by a specific bacterium.     

牛无浆体16S rRNA基因的克隆测序及分析

从自然感染无浆体的重庆黄牛无菌采集血液,提取全血基因组,用血营养菌16S rRNA基因的通用引物进行PCR扩增,得到长约1500 bp的扩增片段,将其克隆到pMD18-T载体后进行测序,并与5条边缘无浆体、4条中央无浆体、4条牛无浆体、4条羊无浆体和3条嗜吞噬细胞无浆体16S rRNA基因序列进行系统发育分析.结果表明所克隆的基因片段长度为1412 bp,GenBank登录号为FJ169957.序列比较结果显示,所获得的序列与Kawa-hara公布的牛无浆体日本株(AB211163)同源性最高,达到99.0%,系统发育分析发现,该序列被聚类到牛无浆体群,并与嗜吞噬细胞无浆体群聚类到一个大的分支.本文从分子水平证实重庆地区存在牛无浆体,牛无浆体与嗜吞噬细胞无浆体的亲缘关系比其他3种无浆体更近.     

Change in body condition and carcass characteristics of cull ewes fed diets supplemented with rumen bypass fat

Thirty Malpura ewes (>6 years age) distributed into three groups of 10 each were maintained on concentrate supplemented with rumen protected fat at 0 (T₁), 20 (T₂) and 40 (T₃) g kg⁻¹ and chick pea straw for a period of three months. Towards the end of feeding experiment a metabolism trial was conducted on five representative ewes from each treatment. Blood and rumen liquor samples were analyzed at 0 and 90 days of feeding for blood biochemical and rumen metabolites. Five representative ewes were slaughtered at the initiation of the study and all the experimental ewes were slaughtered after termination of the experiment. The gain in weight (kg) and final body condition score was higher (P<0.05) in T₂ and T₃ as compared to T₁. The concentrate intake increased (P<0.05) with bypass fat (RBF) supplementation. The serum glucose and population of spirotrichs and total protozoa in rumen liquour sample increased (P<0.05) with concentrate as well as concentrate with RBF supplementation. Pre-slaughter weight, hot carcass weight, dressing percent, loin eye area, bone percent and carcass fat improved (P<0.05) with RBF supplementation. Composition of Longissimus dorsi muscle also revealed improvement when compared with 0 day composition. The feeding protocol also revealed higher returns by RBF supplementation. It is therefore concluded that RBF supplementation is advantageous in improving body conditions of cull ewes.     

用于肠道中的乳酸菌和双歧杆菌研究的分子生物学技术

综述了利用16S rRNA基因为靶基因的各种分子生物学技术来研究肠道中乳酸菌和双歧杆菌的方法，指出了肠道中乳酸菌和双歧杆菌的组成及其菌数。     

鸭疫里氏杆菌的分离与16 S rRNA的鉴定

从疑似患有鸭疫里氏杆菌病的病死鸭群中采取的肝脏、心脏、脑等病料中分离病原,进行生化鉴定,自6只病死鸭的12份病料中分离鉴定出6株鸭疫里氏杆菌。根据鸭疫里氏杆菌16 S rRNA基因的保守序列设计特异引物,对6株鸭疫里氏杆菌进行PCR扩增,均能扩增出680 bp的特异条带;从凝胶中回收DNA目的条带并测序,测序结果与GenBank中鸭疫里氏杆菌相应序列相似率达到99.99%。     

禽大肠杆菌的分离与16S rRNA的鉴定

从疑似患有大肠杆菌病的病死鸡群中采取粪便样品,分离病原进行生化鉴定,从8份样品中分离鉴定出6株大肠杆菌。根据细菌16S rRNA 基因的高度保守性,设计合成大肠杆菌的共同引物,对随机选取的1株细菌进行PCR扩增,并与GenBank中的E.coli 16S rRNA进行序列比对,确定这株细菌与大肠杆菌的同源性达99%以上。本方法特异性好,为实验室鉴定大肠杆菌提供了一种简单、容易操作的手段。     

慢性瘤胃酸中毒状态下奶山羊瘤胃细菌内几种相关细菌数量变化的研究

研究慢性瘤胃酸中毒（subacute rumen acidosis,SARA）状态下,相关瘤胃微生物数量的变化。将6只体重相近,体况良好,并安装有永久性瘤胃瘘管的泌乳期关中奶山羊作为试验对象,以逐渐增加精料的方式诱导动物发生SARA。试验分4期进行,4期日粮的精粗比分别为5∶5、6∶4、7∶3和8∶2。分别采用16S rRNA 探针杂交法和传统培养法对SARA状态下瘤胃内微生物数量变化进行测定。随着日粮精料比例的增加,牛链球菌、乳酸杆菌、反刍兽新月单胞菌、埃氏巨型球菌和淀粉分解菌的数量出现不同程度的增加,而3种主要纤维分解菌的数量有所下降,其中淀粉分解菌的数量极显著增加（P<0.01）。在SARA状态下瘤胃细菌总数、乳酸产生菌和乳酸利用菌数量均有所增长,同时3种主要纤维分解菌的数量明显下降。     

竹叶提取物对肉牛生产性能、瘤胃发酵特征及微生物数量的影响

试验研究竹叶提取物对肉牛生产性能、瘤胃发酵特征及微生物数量的影响.选取24头6月龄、体重(259.30±12.17) kg的西门塔尔杂交公牛,随机分为4组,每组6个重复.各组肉牛日粮中分别添加0(对照组)、40、80、120 g/d竹叶提取物,试验期180 d.结果 显示,80 g/d组肉牛的平均日增重和平均干物质采食...     

饲粮组成对育肥羔羊肠道微生物菌群结构的影响

为研究饲粮组成改变对育肥羔羊肠道菌群结构及多样性的影响,选择生长发育良好、体重相近的2月龄蒙系公羔羊76只,随机分成2组,每组38只,分别饲喂不同的饲粮,对照组为精料+苜蓿饲粮,处理组为精料+全株玉米青贮+苜蓿+花生秧的全混合饲粮,试验期122 d,其中预试期20 d.试验结束后,每组随机选取4只羔羊屠宰并采集小肠及盲...