牛瘤胃产蛋氨酸热带假丝酵母菌的分离鉴定及发酵条件优化

试验旨在从牛瘤胃微生物中筛选出能够合成并分泌蛋氨酸的菌株.试验以草原红牛瘤胃液为样品,使用蛋氨酸缺陷培养基分离纯化,通过革兰氏染色、游离蛋氨酸含量测定、16S rDNA测序等鉴定菌株.采用单因素设计和正交试验对培养基和发酵条件进行优化,通过体外模拟瘤胃发酵试验测试菌株.结果 显示,分离的菌株为热带假丝酵母菌(Candi...     

牛瘤胃乳酸片球菌的分离鉴定与生物学特性研究

为了获得生物学特性良好的乳酸菌,试验利用厌养菌分离技术从成年牛瘤胃中分离出一株牛瘤胃乳酸菌,命名为Y-A-2,并对该菌进行了形态分析、生化试验、16S rRNA序列同源性分析和生物学特性研究。结果表明:Y-A-2菌株为乳酸片球菌,可发酵纤维二糖、水杨苷、菊糖;最适生长温度为26℃,最适生长温度下pH值随着时间的延长逐渐降低,具有一定的耐盐能力,不耐强酸强碱。说明该菌具有良好的生物学活性,有助于牛瘤胃微生态制剂的研制和控制牛瘤胃的微生物发酵进程。     

山羊瘤胃内产乳酸菌的分离鉴定及其产D-、L-乳酸特性的研究

为阐明瘤胃中产乳酸菌的种类及其产乳酸特性,本试验以饲喂精粗比例为6:4的健康山羊瘤胃液为接种物,利用厌氧分离技术,经形态学和16s rRNA序列分析法,从山羊瘤胃中分离获得6株细菌(分别命名为L2、L3、L5、L8、L10和L12).菌株主要为3类:L2、L8、L10为牛链球菌,L5为马链球菌,L3和L12为食窦魏斯氏...     

绵羊瘤胃内纤维降解菌的分离鉴定

试验从蒙古绵羊瘤胃中分离出3株厌氧细菌,通过细菌纤维降解性试验发现3株菌皆具有纤维降解能力;通过革兰氏染色试验发现3株菌均为革兰氏阳性菌;通过生理生化试验和16S rDNA基因序列测定及同源性分析,最终确定分离的3株菌分别为牛链球菌（Streptococcus bovis）、婴儿链球菌（Streptococcus infantarius）和粪肠球菌（Enterococcus faecium）,编号为E1、E2和E3。     

一株枯草芽孢杆菌YB-6的筛选、生物学特性及发酵工艺的研究

为了研究从牛的瘤胃内容物中经过分离、筛选、鉴定得到一株枯草芽孢杆菌YB-6,试验对该菌株的生理生化特性、16S rDNA基因序列同源性、产酶特性、耐受能力、抗生素敏感性及对致病菌的抑菌能力和发酵工艺进行了研究。结果表明:枯草芽孢杆菌YB-6是一株生物学特性良好、抗逆性强的益生菌株,同时通过工艺优化,发酵活菌数与原有工艺比较提高了142.8%。     

10株对多种抗生素耐药的禽链球菌的分离鉴定

为有效预防和治疗禽链球菌病,2012—2013期间,笔者从前来就诊的病死禽脏器中分离得到10株疑似禽源链球菌的分离株,经过染色镜检、培养特性、生化试验及生化编码查询等检测表明：10种致病菌都属于链球菌属。各菌株生化反应特性不同,但大多数能发酵蔗糖,5株细菌吡咯芳氨酸酶（PYR）试验阳性。生化编码册查询结果显示：10株分离菌有9株属于D群链球菌、1株是C群兽疫链球菌属。药敏试验结果表明：10株分离菌对萘啶酸、罗美沙星完全耐药;对丁胺卡那霉素、多黏菌素、洛美沙星、新生霉素、万古霉素和美洛西林高敏,对其他多种抗生素都有一定的耐药性。     

基于PFGE技术的酸乳中乳酸菌的基因分型

采用选择性培养基分离酸乳中的保加利亚乳杆菌和嗜热链球菌,并利用生理生化和糖发酵实验结合16S rDNA同源性分析对分离所得菌株进行鉴定,获得保加利亚乳杆菌和嗜热链球菌分离菌株。利用脉冲场凝胶电泳（pulsed field gel electrophoresis,PFGE）分别对保加利亚乳杆菌和嗜热链球菌分离株进行基因分型。结果表明：所检测的酸乳样品中均含有保加利亚乳杆菌和嗜热链球菌;仅有2 种酸乳的嗜热链球菌为同一菌株,其余酸乳中的保加利亚乳杆菌及嗜热链球菌均为不同菌株。PFGE可以对保加利亚乳杆菌和嗜热     

10株对多种抗生素耐药的禽链球菌的分离鉴定

为有效预防和治疗禽链球菌病,2012—2013期间,笔者从前来就诊的病死禽脏器中分离得到10株疑似禽源链球菌的分离株,经过染色镜检、培养特性、生化试验及生化编码查询等检测表明:10种致病菌都属于链球菌属。各菌株生化反应特性不同,但大多数能发酵蔗糖,5株细菌吡咯芳氨酸酶(PYR)试验阳性。生化编码册查询结果显示:10株分离菌有9株属于D群链球菌、1株是C群兽疫链球菌属。药敏试验结果表明:10株分离菌对萘啶酸、罗美沙星完全耐药;对丁胺卡那霉素、多黏菌素、洛美沙星、新生霉素、万古霉素和美洛西林高敏,对其他多种抗生素都有一定的耐药性。     

奶牛瘤胃中反刍兽月形单胞菌的分离鉴定

采用厌氧分离技术从奶牛瘤胃中分离出1株细菌,通过对其形态、培养特性、生理生化特性、16SrRNA基因序列测定与同源性分析等研究,确定分离菌株为反刍兽月形单胞菌（Selenomonas ruminantium）,为进一步研究其对瘤胃发酵的影响奠定了基础。     

牛瘤胃乳酸菌的分离与鉴定

为分离筛选用于牛活菌制剂的乳酸菌菌株,试验采用API 50CHL对从牛瘤胃中分离的60株乳酸菌进行生化鉴定,对鉴定出的菌株进行耐酸耐碱、生长温度、耐氧、耐药和抑菌性测定。结果显示,经生化鉴定出的3株菌分别为嗜酸乳杆菌（L.acidophilus）、发酵乳杆菌（L.fermentum）和乳明串珠菌（L.holzapfelii）,分别命名为L1、L2和L3。其中,L1耐酸性最强,L2耐碱性最强,L1在37～40 ℃之间均生长良好且兼性厌氧;L1、L2、L3耐药率为60%～100%;3株乳酸菌对大肠杆菌和金黄色葡萄球菌均有不同程度的抑制作用,其中L1抑菌效果最好,抑菌圈直径分别为18.30和16.40 mm。提示,L1、L2可作为抗性乳酸菌活菌制剂的候选菌株。     

Effects of feeding polyclonal antibody preparations on rumen fermentation patterns, performance, and carcass characteristics of feedlot steers

In a previous study, preparations of polyclonal antibodies (PAP) against Fusobacterium necrophorum (PAP-Fn) or Streptococcus bovis (PAP-Sb) were successful in decreasing ruminal counts of target bacteria and increasing ruminal pH in steers fed high-grain diets. The objective of this study was to evaluate the effects of feeding PAP-Fn or PAP-Sb on performance, carcass characteristics, and ruminal fermentation variables of feedlot steers. In Exp. 1, during 2 consecutive years, 226 or 192 Angus and Angus crossbred steers were fed a high-grain diet containing either PAP-Sb or PAP-Fn, or both. When measured on a BW basis, steers fed only PAP-Sb had a greater G:F (P < 0.05) than those fed no PAP. Nevertheless, when both PAP were fed, feed efficiency was similar (P > 0.10) to steers fed no PAP or only PAP-Sb. Steers receiving PAP-Fn (alone or in combination with PAP-Sb) had a decreased (P < 0.05) dressing percentage. Steers receiving PAP-Fn (alone or in combination with PAP-Sb) had a decreased severity of liver abscess (P < 0.05). No differences (P > 0.10) were observed in any other carcass characteristics. In Exp. 2, sixteen ruminally cannulated Angus crossbred steers (BW = 665 +/- 86 kg) were fed a high-grain diet containing either PAP-Sb or PAP-Fn, or both. Feeding only PAP-Fn or PAP-Sb for 19 d decreased (P < 0.05) ruminal counts of S. bovis when compared with steers fed both or no PAP. The ruminal counts of F. necrophorum in steers fed PAP-Fn alone or in combination with PAP-Sb were decreased by 98% (P < 0.05) after 19 d, when compared with the counts in control steers. Mean daily ruminal pH was greater (P < 0.05) in steers fed both PAP when compared with feeding either or no PAP. Ruminal pH in the first 4 h after feeding was greater (P < 0.05) for steers receiving PAP-Fn alone or in combination with PAP-Sb. Steers receiving either PAP alone or in combination had less (P < 0.05) ruminal NH(3)-N concentrations in the first 4 h after feeding when compared with those of control steers. Polyclonal antibody preparations against S. bovis were effective in enhancing G:F of steers fed high-grain diets, but dressing percentage was decreased. Mechanisms of enhancement of G:F remain unknown but may be related to changes in ruminal counts of target bacteria and associated effects on ruminal fermentation products.     

Effects of feeding polyclonal antibody preparations on ruminal bacterial populations and ruminal pH of steers fed high-grain diets

Three experiments with factorial arrangements of treatments were designed to test the efficacy of avian-derived polyclonal antibody preparations (PAP) against Streptococcus bovis (PAP-Sb) or Fusobacterium necrophorum (PAP-Fn) in reducing ruminal counts of target bacteria in beef steers supplemented or not with feed additives (300 mg of monensin/d and 90 mg of tylosin/d; MT). Feeding increasing doses of PAP-Sb in Exp. 1 or a single dose in Exp. 2 reduced S. bovis counts in a cubic fashion (P = 0.014). In Exp. 1 and 2, inclusion of MT in the diet had no effect (P > 0.05) on ruminal S. bovis counts. In Exp. 2, ruminal pH was increased (P < 0.05) by feeding PAP-Sb, MT, and PAP-Sb plus MT. Ruminal F. necrophorum counts were reduced by feeding PAP-Fn (P = 0.002) and MT (P < 0.001). Reduction in ruminal F. necrophorum counts was greater (P = 0.008) when feeding MT alone than when feeding PAP-Fn and MT together. In Exp. 3, ruminal S. bovis counts were not affected (P = 0.64) by PAP-Fn. Ruminal pH was not affected (P = 0.61) by feeding PAP-Fn, and the total anaerobic bacterial count was not affected (P > 0.05) by either PAP-Sb or PAP-Fn in Exp. 1 or Exp. 3. In conclusion, PAP of avian origin and against S. bovis or F. necrophorum were effective in reducing target ruminal bacterial populations. These PAP could be effective in preventing the deleterious effects associated with these bacteria, and possibly in enhancing animal performance.     

Preliminary observations of interaction between bacteriophages and Streptococcus bovis bacteria on ruminal epithelium primoculture.

Five Streptococcus bovis strains (47/3, 59/2, 4/1, 46/2 and 44/9) isolated from calf ruminal fluid samples were examined for the adherence to cultured ruminal epithelium cells. Four strains (47/3, 59/2, 4/1 and 46/2) were able to attach to the cultured epithelial cells. However, S. bovis 47/3 strain attached to the target cells in significantly greater numbers than the other strains. Strain 44/9 did not adhere to cells of ruminal epithelium. The adherent bacteria were observed on the surface of differentiated (mainly keratinized) cells of ruminal epithelium primoculture only. The different effect of F4, F5 and F6 bacteriophages was ascertained on S. bovis bacteria adhering to rumen epithelial primoculture. A significant decrease in the number of adherent bacteria was shown after cultivation of strains 47/3 and 4/1 with F6 bacteriophage and of 47/3 strain with F4 phage. The F5 bacteriophage had no significant effect on these bacteria.     

Effects of various adjuvants on efficacy of a vaccine against Streptococcus bovis and Lactobacillus spp in cattle

OBJECTIVE: To determine efficacy of vaccines incorporating QuilA, alum, dextran combined with mineral oil, or Freund adjuvant for immunization of feedlot cattle against Streptococcus bovis and Lactobacillus spp. ANIMALS: 24 steers housed under feedlot conditions. PROCEDURE: Steers were randomly assigned to 4 experimental groups and a control group. Animals in experimental groups were inoculated on days 0 and 26 with vaccines containing Freund adjuvant (FCA), QuilA, dextran combined with mineral oil (Dex), or alum as adjuvant. Serum anti-S bovis and anti-Lactobacillus IgG concentrations were measured, along with fecal pH, ruminal fluid pH, and number of S bovis and Lactobacillus spp in ruminal fluid. RESULTS: Throughout the study, serum anti-S bovis and anti-Lactobacillus IgG concentrations for animals in the Dex, QuilA, and alum groups were similar to or significantly higher than concentrations for animals in the FCA group. Serum anti-S bovis and anti-Lactobacillus IgG concentrations were significantly increased on days 26 through 75 in all 4 experimental groups, and there was a linear relationship between anti-S bovis and anti-Lactobacillus IgG concentrations. For animals in the QuilA and Dex groups, mean pH of feces throughout the period of experiment were significantly higher and numbers of S bovis and Lactobacillus spp in ruminal fluid on day 47 were significantly lower than values for control cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that immunization of feedlot steers against S bovis and Lactobacillus spp with vaccines incorporating Freund adjuvant, QuilA, dextran, or alum as an adjuvant effectively induced high, long-lasting serum anti-S bovis and anti-Lactobacillus IgG concentrations. Of the adjuvants tested, dextran may be the most effective.     

牛链球菌在瘤胃中产酸的代谢机制及调控

牛链球菌(S.bovis)是瘤胃主要的乳酸产生菌,在饲喂高精料饲粮导致瘤胃乳酸中毒进程中扮演重要角色。已有研究证实S.bovis利用碳水化合物代谢产酸主要受葡萄糖转运方式、酵解产酸途径中酶和中间代谢物调控。另外,研究也发现环境p H、增殖生长阶段及分解代谢控制蛋白(Ccp A)等对其产酸速率和模式也有显著影响。本文对近年来有关S.bovis利用饲料中碳水化合物发酵产酸代谢途径及影响因素研究加以综述,为从微生物代谢角度解析瘤胃乳酸中毒机制提供参考。     

In vitro bacterial growth and in vivo ruminal microbiota populations associated with bloat in steers grazing wheat forage

The role of ruminal bacteria in the frothy bloat complex common to cattle grazing winter wheat has not been previously determined. Two experiments, one in vitro and another in vivo, were designed to elucidate the effects of fresh wheat forage on bacterial growth, biofilm complexes, rumen fermentation end products, rumen bacterial diversity, and bloat potential. In Exp. 1, 6 strains of ruminal bacteria (Streptococcus bovis strain 26, Prevotella ruminicola strain 23, Eubacterium ruminantium B1C23, Ruminococcus albus SY3, Fibrobacter succinogenes ssp. S85, and Ruminococcus flavefaciens C94) were used in vitro to determine the effect of soluble plant protein from winter wheat forage on specific bacterial growth rate, biofilm complexes, VFA, and ruminal H2 and CH4 in mono or coculture with Methanobrevibacter smithii. The specific growth rate in plant protein medium containing soluble plant protein (3.27% nitrogen) was measured during a 24-h incubation at 39 degrees C in Hungate tubes under a CO2 gas phase. A monoculture of M. smithii was grown similarly, except under H2:CO2 (1:1), in a basal methanogen growth medium supplemented likewise with soluble plant protein. In Exp. 2, 6 ruminally cannulated steers grazing wheat forage were used to evaluate the influence of bloat on the production of biofilm complexes, ruminal microbial biodiversity patterns, and ruminal fluid protein fractions. In Exp. 1, cultures of R. albus (P < 0.01) and R. flavefaciens (P < 0.05) produced the most H2 among strains and resulted in greater (P < 0.01) CH4 production when cocultured with M. smithii than other coculture combinations. Cultures of S. bovis and E. ruminantium + M. smithii produced the most biofilm mass among strains. In Exp. 2, when diets changed from bermudagrass hay to wheat forage, biofilm production increased (P < 0.01). Biofilm production, concentrations of whole ruminal content (P < 0.01), and cheesecloth filtrate protein fractions (P < 0.05) in the ruminal fluid were greater on d 50 for bloated than for nonbloated steers when grazing wheat forage. The molecular analysis of the 16S rDNA showed that 2 different ruminal microbiota populations developed between bloated and nonbloated animals grazing wheat forage. Bloat in cattle grazing wheat pastures may be caused by increased production of biofilm, resulting from a diet-influenced switch in the rumen bacterial population.     

Effect of virginiamycin on ruminal fermentation in cattle during adaptation to a high concentrate diet and during an induced acidosis.

The objective of Exp. 1 was to compare the effects of virginiamycin (VM; 0, 175, or 250 mg x animal(-1) x d(-1)) and monensin/tylosin (MT; 250/ 90 mg x animal(-1) x d(-1)) on ruminal fermentation products and microbial populations in cattle during adaptation to an all-concentrate diet. Four ruminally cannulated, Holstein steers were used in a 4x4 Williams square design with 21-d periods. Steers were stepped up to an all-concentrate diet fed at 2.5% of BW once daily. Ruminal pH, protozoal counts, and NH3-N and VFA concentrations generally were unaffected by VM or MT. Mean counts of Lactobacillus and Streptococcus bovis were lower (P<.05) for VM-treated compared with control or MT-treated steers. Both VM and MT prevented the increase in Fusobacterium necrophorum counts associated with increasing intake of the high-concentrate diet observed in the control. The objective of Exp. 2 was to compare the effects of VM and MT on ruminal pH, L(+) lactate and VFA concentrations, and F. necrophorum numbers during carbohydrate overload. Six ruminally cannulated Holstein steers were assigned randomly to either the control, VM (175 mg/d), or MT (250 + 90 mg/d) treatments. Acidosis was induced with intraruminal administration of a slurry of ground corn and corn starch. The VM and MT premixes were added directly to the slurry before administration. Carbohydrate challenge induced acute ruminal acidosis (pH was 4.36 and L (+) lactate was 19.4 mM) in controls by 36 h. Compared with the controls, steers receiving VM or MT had higher (P<.05) ruminal pH, and the VM group had a lower (P<.05) L (+) lactate concentration. Fusobacterium necrophorum numbers initially increased in VM- and MT-administered steers. In the control steers, F. necrophorum was undetectable by 36 h. Virginiamycin seemed to control the growth of ruminal lactic acid-producing bacteria and, therefore, has the potential to moderate ruminal fermentation in situations that could lead to rapid production of lactic acid.     

Effects of adding polyclonal antibody preparations on ruminal fermentation patterns and digestibility of cows fed different energy sources

Nine ruminally cannulated cows fed different energy sources were used to evaluate an avian-derived polyclonal antibody preparation (PAP-MV) against the specific ruminal bacteria Streptococcus bovis, Fusobacterium necrophorum, Clostridium aminophilum, Peptostreptococcus anaerobius, and Clostridium sticklandii and monensin (MON) on ruminal fermentation patterns and in vivo digestibility. The experimental design was three 3 × 3 Latin squares distinguished by the main energy source in the diet [dry-ground corn grain (CG), high-moisture corn silage (HMCS), or citrus pulp (CiPu)]. Inside each Latin square, animals received one of the feed additives per period [none (CON), MON, or PAP-MV]. Dry matter intake and ruminal fermentation variables such as pH, total short-chain fatty acids (tSCFA), which included acetate, propionate, and butyrate, as well as lactic acid and NH(3)-N concentration were analyzed in this trial. Total tract DM apparent digestibility and its fractions were estimated using chromic oxide as an external marker. Each experimental period lasted 21 d. Ruminal fluid sampling was carried out on the last day of the period at 0, 2, 4, 6, 8, 10, and 12 h after the morning meal. Ruminal pH was higher (P = 0.006) 4 h postfeeding in MON and PAP-MV groups when compared with CON. Acetate:propionate ratio was greater in PAP-MV compared with MON across sampling times. Polyclonal antibodies did not alter (P > 0.05) tSCFA, molar proportion of acetate and butyrate, or lactic acid and NH(3)-N concentration. Ruminal pH was higher (P = 0.01), 4 h postfeeding in CiPu diets compared with CG and HMCS. There was no interaction between feed additive and energy source (P > 0.05) for any of the digestibility coefficients analyzed. Starch digestibility was less (P = 0.008) in PAP-MV when compared with CON and MON. In relation to energy sources, NDF digestibility was greater (P = 0.007) in CG and CiPu vs. the HMCS diet. The digestibility of ADF was greater (P = 0.002) in CiPu diets followed by CG and HMCS. Feeding PAP-MV or monensin altered ruminal fermentation patterns and digestive function in cows; however, those changes were independent of the main energy source of the diet.     

Isolation,Identification and Biological Characteristics Research of Streptococcus bovis from Yak

The objective of this study was to isolate and identify Streptococcus bovis from yak, and detect their hemolytic, pathogenicity and antimicrobial susceptibility. 45 fecal samples collected from the diarrheal yaks in Aba state in Northwest Sichuan province were used to isolate Streptococcus bovis with sheep blood culture media under 37℃ and 5% CO₂ condition cultured for 24 h. The isolated bacteria were identified by 16S rRNA amplification and sequencing. Total 14 Streptococcus bovis were identified, among which, 8 Streptococcus lutetiensis strains and 6 Streptococcus gallolyticus strains. 9 Streptococcus bovis strains showed α hemolytic, and 5 Streptococcus bovis showed β hemolytic. Both Streptococcus lutetiensis and Streptococcus gallolyticus caused the experimental mice mild diarrhea. The isolated 14 Streptococcus bovis were sensitive to penicillin, cefotaxime, vacomycin, acetylspiramycin, ciprofloxacin, rifampicin, teicoplanin, ampicillin and gentamicin, and highly tolerant to streptomycin, kanamycin, lincomycin, erythrocin, tetracycline and clindamycin.This study illustrated the biological characteristics of the isolated Streptococcus bovis from yak, which made the basis for the prevention and control of diseases caused by Streptococcus bovis.