表达猪乳铁蛋白肽重组屎肠球菌对断奶仔猪生长性能的影响及其抗ETEC感染效果研究

本试验以表达猪乳铁蛋白肽的重组屎肠球菌（pNZ8112-PLFcin/Ef）饲喂断奶仔猪，研究其对仔猪生长性能的影响和抗产肠毒素大肠杆菌（enterotoxigenic Escherichia coli，ETEC）感染的效果。选取28日龄体重相近的健康断奶仔猪36头，随机分为3组（重组屎肠球菌组、空载体组和培养基组），每组3个重复，每个重复4头仔猪。重组屎肠球菌组和空载体组分别饲喂添加pNZ8112-PLFcin/Ef （6×10¹² CFU/kg）和pNZ8112/Ef （6×10¹² CFU/kg）的基础日粮，而培养基组饲喂含有相同体积的GM17液体培养基的基础日粮。试验期26 d。结果显示，与培养基组相比，重组屎肠球菌组断奶仔猪的平均日增重极显著提高（P<0.01）；料重比显著降低（P<0.05）；腹泻率明显降低，肠道菌群的均匀度和多样性指数均下降。为进一步探究表达猪乳铁蛋白肽的重组屎肠球菌对断奶仔猪抵抗ETEC感染的保护作用，在连续饲喂21 d后，每个重复中随机挑选1头体况相近的断奶仔猪灌服ETEC。结果发现，与培养基组相比，攻菌后重组屎肠球菌组断奶仔猪血清白细胞介素-2（IL-2）、免疫球蛋白G （IgG）含量、肠黏液中分泌型免疫球蛋白A （sIgA）水平均显著升高（P<0.05）；脾脏指数显著提高（P<0.05），但胸腺指数、肠段长度及重量则均无显著差异（P>0.05）。综上所述，表达猪乳铁蛋白肽的重组屎肠球菌能够起到促进断奶仔猪生长及抗ETEC感染的保护作用。     

粪肠球菌生长曲线的测定及其对小鼠脑组织的影响

为测定致羔羊脑炎粪肠球菌（Enterococcus faecalis）的生长曲线，寻求一种快速而准确的方法测定不同生长时期粪肠球菌数量，并客观评价其毒性强弱及其对小鼠脑组织的影响，试验采用平板菌落计数法和OD-Monitor振荡比浊法（D_λ值法）测定粪肠球菌的生长曲线，探究该菌在合适时间段内的吸光值（D_{600 nm}）与平板菌落计数法测定的活菌数（CFU）的关系。用粪肠球菌感染小鼠，观察记录小鼠的死亡情况，最后采用Karber法计算粪肠球菌感染小鼠的半数致死量（LD₅₀）。用LD₅₀的剂量感染小鼠，及时采集死亡小鼠脑组织，未死亡的小鼠72 h后全部剖杀取脑组织，一部分做涂片染色，制作病理切片，观察病理变化；一部分进行培养，用于PCR方法进行细菌的回收鉴定。结果显示，用两种方法测定此株粪肠球菌的生长曲线基本一致，在2~8 h生长迅速，为对数生长期，8~14 h生长缓慢，为稳定期，14 h之后死亡数增加，进入衰亡期；对12 h粪肠球菌D_{600 nm}与CFU的关系进行探讨，成功建立回归方程：y=20.769x-1.3422，R²=0.997；其感染小鼠的LD₅₀为7.77×10¹¹个活菌。以此剂量感染小鼠，脑组织涂片染色和培养染色，均能看到革兰氏阳性球菌；PCR结果显示，均出现了大小为112 bp的条带。对脑组织进行病理学观察发现该菌可导致脑组织充血、出血、形成微血栓，脑膜充血。通过生长曲线和其D_{600 nm}与CFU关系的建立，可实时监测粪肠球菌数量，为后期更深入研究粪肠球菌穿越血脑屏障的机制奠定重要的理论基础。     

粪肠球菌替代抗生素对断奶仔猪生长性能、腹泻率、血液生化指标和免疫器官的影响

本试验在断奶仔猪饲粮中添加不同水平的粪肠球菌,研究其替代抗生素对断奶仔猪生长性能、腹泻指数和腹泻率、血液生化指标以及免疫器官的影响。选取120头(28±2)日龄平均体重为(7.60±0.81)kg的"杜×长×大"断奶仔猪,随机分为5组,每组6个重复(公母各占1/2),每个重复4头,每个重复为1圈。对照组饲喂基础饲粮,抗生素组在基础饲粮中添加100mg/kg硫酸黏菌素和400 mg/kg杆菌肽锌,3个粪肠球菌组分别在基础饲粮中添加40、200和1 000 mg/kg粪肠球菌,试验期31 d。试验结果显示:1)在1~14 d,饲粮中添加抗生素和粪肠球菌对断奶仔猪的平均日采食量(ADFI)、平均日增重(ADG)以及料重比(F/G)无显著影响(P0.05)。在15~31 d,与对照组和抗生素组相比,200和1 000 mg/kg组可极显著提高ADFI和ADG(P0.01)。在1~31 d,与对照组相比,200和1 000 mg/kg组的ADFI均极显著提高(P0.01),而ADG分别显著和极显著增加(P0.05和P0.01);与抗生素组相比,1 000 mg/kg组可极显著提高ADFI(P0.01),而200和1 000 mg/kg组的ADG分别显著和极显著增加(P0.05和P0.01)。2)在1~14 d,与对照组相比,40 mg/kg组的腹泻指数和腹泻率显著降低(P0.05)。在15~31 d和1~31 d,与对照组和抗生素组相比,40和1 000 mg/kg组均可在一定程度上降低腹泻率(P0.05)。3)与对照组相比,粪肠球菌可以显著提高血液中总蛋白含量(P0.05),其中200和1 000 mg/kg组达到极显著水平(P0.01),且这2组分别可以显著和极显著提高球蛋白含量(P0.05和P0.01)。与抗生素组相比,200和1 000 mg/kg组可以显著提高总蛋白含量(P0.05)。与对照组相比,抗生素组和1 000 mg/kg组的白球比显著降低(P0.05),另外,抗生素组血液中谷丙转氨酶和谷草转氨酶活性显著降低(P0.05),200 mg/kg组的谷丙转氨酶活性显著降低(P0.05)。4)与对照组相比,1 000 mg/kg组的肝脏重量和脾脏指数有一定程度的提高(P0.05),与抗生素组相比,200和1 000 mg/kg组可显著提高肝脏重量(P0.05)。结果表明,饲粮中添加粪肠球菌可改善断奶仔猪的生长性能(饲喂15~31 d),降低腹泻率,提高仔猪免疫力,其中添加量为1 000 mg/kg时效果最好。     

Isolation,Identification and Multidrug-resistant Analysis of the New Sequence Type ST989 Strain of Enterococcus faecium

Gram-positive cocci were isolated from half a month diseased lambs with a fatal infection in a sheep farm in Minhe county, Qinghai province.The clinical symptoms observed were drooping, reduced feed intake, emaciated and fever.The infected lambs died several hours after the disease been observed.Necropsy of two dying lambs revealed that for one lamb, the liver was enlarged and capsule of the liver dropped, and other organs had no obvious changes;For the other one, the organs had no obvious changes.After gram staining, gram-positive cocci were observed from all the samples.Partial Tuf gene sequences of these isolates were amplified and sequenced for species identification, BLASTn conparison result revealed that they had the highest sequence similarity with that of Enterococcus faecium (E.faecium) AUS0085 (99.8%), we thus primarily confirmed that these strains belonged to E.faecium.Antimicrobial susceptibility test indicated that these strains could be divided into two classes.Class 1 was resistant to β-lactams, aminoglycosides, macrolides, tetracyclines, quinolones, rifamycins, lincomycin and clindamycin;Susceptible to chloromycetin and vancomycin.Class 2 was resistant to β-lactams, aminoglycosides, tetracyclines, quinolones, rifamycins;Intermediate resistance to erythromycin and roxithromycin;But susceptible to clarithromycin, azithromycin, clindamycin, lincomycin, chloromycetin and vancomycin.Multilocus sequence typing (MLST) based on seven housekeeping genes for E.faecium revealed that E.faecium lby1 strain in class 1 and E.faecium lbg3 strain in class 2 were defined as a new sequence type ST989.Phylogenetic tree analysis revealed the representative strain of these isolates, E.faecium lbg3, was clustered together with E.faecium ST468.The pathogenicity test revealed that E.faecium lby1 and lbg3 strains both had pathogenicity.     

Cloning,Expression and Bioinformatics Analysis of Enterococcus faecalis from Northeast Black Bear

In order to detect Enterococcus faecalis endocarditis antigen (EfaA) of bear that was used to disclose the infected Northeast Black bear immediately, a pair of PCR primers was synthesized by EfaA gene of Enterococcus faecalis that was recorded in GenBank (accession number:U03756.1) and the target fragment was 689 bp. The PCR product was cloned by pMD19-T vector, then was transferred to Escherichia coli DH5α competent cells and the positive clones were filtered. Recombinant plasmid (pMD19-T-EfaA) was extracted. Identification of PCR and digestion, sequencing, the structure prediction of proteins were operated. The results showed that EfaA gene was cloned successfully, and the gene homology with ATCC 29212 was 100.0%. pMD19-T-EfaA was constructed successfully, structure of proteins was predicted. This study provided theoretical basis for diagnostic methods of Enterococcus faecalis and laid basis for prevention and control of Northeast Black bear disease.     

基质辅助激光解吸电离飞行时间质谱在动物源细菌耐药性监测中的研究应用

细菌鉴定是细菌耐药性监测过程中的重要工作环节之一,基质辅助激光解析电离飞行时间质谱(Matrix-assisted laser desorption ionization time-off flight mass spectrometry, MALDI-TOF MS)能够高效鉴定细菌。为了快速监测五家养殖场来源的大肠杆菌和肠球菌的临床耐药特征,本研究利用MALDI-TOF MS和微量肉汤稀释法,快速鉴定临床分离的大肠杆菌和肠球菌,并对其进行耐药表型检测。结果显示,MALDI-TOF MS实现了对临床分离菌株(31株大肠杆菌和34株肠球菌)的快速鉴定;鸡源大肠杆菌和肠球菌的耐药情况最为严重,其次为羊和牛。其中,鸡源的大肠杆菌均对四环素(100%)和氨苄西林(91.67%)耐药率最高,肠球菌对苯唑西林(62.07%)耐药率较高。研究结果表明,不同动物源细菌临床耐药性表型严重程度有所不同,与此同时,MALDI-TOF MS技术可以同时实现对动物源大肠杆菌和肠球菌的快速鉴定,值得在动物源细菌耐药性检测领域推广应用。     

Positive effects of dietary supplementation of three probiotics on milk yield,milk composition and intestinal flora in Sannan dairy goats varied in kind of probiotics

In this study, we investigated the effects of Saccharomyces cerevisiae (SC), Bacillus subtilis (BS) and Enterococcus faecalis (EF), singly and in combination, on the dry matter intake (DMI), milk production and composition, and faecal microflora of Saanen dairy goats. Fifty goats were randomly divided into five groups: (a) basal diet (control); (b) basal diet + SC; (c) basal diet + BS; (d) basal diet + EF; and (e) basal diet + mixed probiotics. Each treated animal received 5 g/d of probiotics for a total administration of 5 × 1,011 CFU/goat per day. The inclusion of B. subtilis and E. faecalis in the diet of lactating Saanen goats increased DMI (p < .05). Enhanced milk yield was observed with BS and EF. Milk fat percentage was significantly increased by feeding mixed probiotics compared with the control (p < .05); supplying SC, BS and mixed probiotics enhanced the protein percentage (p < .05). The milk lactose percentage in the SC and BS groups was higher than in the control (p < .05). The amount of milk total solids was higher after feeding EF or mixed probiotics than in the control group (p < .05). Non-fat solids showed no notable differences among groups (p > .05). There was no significant influence on gut bacterial abundance and diversity from adding these three probiotics, singly or in combination. Bacteroidales, Escherichia–Shigella and Christensenellaceae abundances were decreased by supplying these probiotics but Succinivibrionaceae increased. In conclusion, there were positive influences of probiotic feed supplementation on intake, milk performance and intestinal microecology.     

产enterocin E5细菌素粪肠球菌发酵条件的优化

采用琼脂扩散法测定粪肠球菌发酵上清液对大肠埃希菌K88的抑菌活性,从而确定产enterocin E5细菌素粪肠球菌的发酵条件。结果表明,粪肠球菌E5产细菌素的发酵培养基为MRS培养基,最适温度为37℃,最适起始pH值6.5,最佳接种量2%,种龄14 h,发酵时间16 h,最佳培养基组分氮源为1%胰蛋白胨、0.5%酵母浸粉,最佳碳源为1%葡萄糖、0.5%蔗糖,0.1%Tween-80有利于enterocin E5的产生。这是分离自北京优良商品猪黑六产enterocin E5粪肠球菌的首次报道。     

屎肠球菌对AA肉鸡血清和肝脏抗氧化功能的影响

试验旨在研究日粮中添加屎肠球菌对爱拔益加（Arbor Acres,AA）肉鸡血清和肝脏抗氧化功能的影响。选取1日龄AA肉鸡600只（公鸡）,随机分为5组,每组6个重复。对照组（CON组）饲喂基础日粮;抗生素组（Ant组）在基础日粮中添加0.1%金霉素;低（LEF组）、中（MEF组）、高（HEF组）剂量屎肠球菌组分别在基础日粮中添加50、100、200 mg/kg屎肠球菌。结果表明：①21日龄时,HEF组肉鸡血清中总抗氧化能力（T-AOC）、谷胱甘肽过氧化物酶（GSH-Px）活性均显著高于CON组（P<0.05）,Ant组T-AOC显著高于CON组（P<0.05）;42日龄时,MEF组和HEF组血清中丙二醛（MDA）含量显著低于CON组（P<0.05）,LEF组血清中超氧化物歧化酶（SOD）活性显著高于CON组和Ant组（P<0.05）。②21日龄时,MEF组和HEF组肉鸡肝脏中T-AOC、GSH-Px活性均显著高于CON组（P<0.05）,且HEF组肉鸡肝脏中T-AOC显著高于Ant组（P<0.05）;42日龄时,HEF组肉鸡肝脏中过氧化氢酶（CAT）活性显著高于CON组（P<0.05）。结果提示,屎肠球菌能显著提高21与42日龄血清和肝脏中T-AOC和GSH-Px活性,降低血清中MDA含量,增加抗氧化酶活性,改善肉鸡抗氧化应激能力,且200 mg/kg屎肠球菌添加量在提高血清和肝脏抗氧化能力上与抗生素效果相当。     

Enterococcus faecium NCIMB 10415 supplementation improves the meat quality and antioxidant capacity of muscle of broilers

The present study was conducted to investigate the effects of Enterococcus faecium (E. faecium) on the meat quality and antioxidant capacity of muscle in broilers. A total of 600 Arbor Acre broiler chickens (1‐day‐old, male) were randomly divided into five treatments with six replicates (20 chickens per replicate) for each treatment. The five treatments were the control treatment (CON, basal diet), antibiotic treatment (ANT, basal diet supplemented with 0.1% chlortetracycline) and E. faecium‐supplemented treatments (LEF, MEF and HEF, basal diet supplemented with 50, 100 and 200 mg/kg of E. faecium respectively). The experiment lasted 42 days in two periods of 21 days. Results showed that there were no differences in breast meat quality among different treatments (p > 0.05). Compared with the CON and ANT treatments, the yellowness of thigh meat in E. faecium‐supplemented treatments was significantly increased (p < 0.05); the shear force of thigh meat in the LEF and MEF treatments was lower than that of the CON treatment (p < 0.05). In addition, the concentration of the inosine monophosphate (IMP) in the breast and thigh meat of the MEF treatment was significantly higher than that of the other treatments (p < 0.05). At 21 days, the total antioxidant capability (T‐AOC) level and glutathione peroxidase (GSH‐Px) activity of breast meat and superoxide dismutase (SOD) in the thigh meat of the MEF treatment were greatly increased (p < 0.05). At 42 days of age, the catalase (CAT), GSH‐Px and T‐AOC activities in the breast meat of the MEF treatment were increased (p < 0.05) and the CAT activity of thigh meat in the LEF and MEF treatments was increased (p < 0.05). In conclusion, E. faecium supplementation increased the meat quality of the thigh muscle, increased the IMP content and the activities of CAT, SOD, T‐AOC, and GSH‐Px of muscle in broilers. Supplementation with 100 mg/kg E. faecium had the greatest effects.