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1.
Streptococcus equi ssp. zooepidemicus(SEZ) is a pathogen associated with a wild range of animal species. Frequent outbreaks have occurred in recent years in pigs, horses, goats and dogs which is liable to infect humans. There is a lack of efficient vaccines against this disease and the occurrence of antibiotic resistance may render drug therapies ineffective. In this study, gene deletion mutant(ΔSEZ) in pathogenicity islands SeseCisland_4 was constructed. The mutant ΔSEZ had a 52-fold decrease in 50% lethal dose(LD_(50)) and had less capacity to adhere epithelial cells. Importantly, immunization of mice with attenuated vaccine ΔSEZ at the dose of 10~2 colony-forming units(CFU) mL~(–1) elicited a significant humoral antibody response, with an antibody titer of 1:12 800. Therefore, 10~2 CFU mL~(–1) might be used as the appropriate immune dose for the attenuated vaccine ΔSEZ, which provided mice with efficient protection against virulent SEZ. In addition, the hyperimmune sera against 10~2 CFU m L–1 attenuated vaccine ΔSEZ could confer significant protection against virulent SEZ infection in the passive immunization experiment and exhibited efficient bactericidal activity in the whole blood assay. Meanwhile, no viable bacteria was detected in blood when mice were immunized with ΔSEZ at the dose of 10~2 CFU mL~(–1) via hypodermic injection. Thereafter, the mutant ΔSEZ at the dose of 10~2 CFU mL~(–1) could confer significant protection in mice and had less negative effects on host, which could be an effective attenuated vaccine candidate for the prevention of SEZ.  相似文献   

2.
Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide. The pathogen is seed-transmitted, so seed detection to prevent distribution of contaminated seed is crucial in disease management. In this study, we adapted a quantitative real-time PCR (qPCR) assay to droplet digital PCR (ddPCR) format for A. citrulli detection by optimizing reaction conditions. The performance of ddPCR in detecting A. citrulli pure culture, DNA, infested watermelon/melon seed and commercial seed samples were compared with multiplex PCR, qPCR, and dilution plating method. The lowest concentrations detected (LCD) by ddPCR reached up to 2 fg DNA, and 102 CFU mL−1 bacterial cells, which were ten times more sensitive than those of the qPCR. When testing artificially infested watermelon and melon seed, 0.1% infestation level was detectable using ddPCR and dilution plating method. The 26 positive samples were identified in 201 commercial seed samples through ddPCR, which was the highest positive number among all the methods. High detection sensitivity achieved by ddPCR demonstrated a promising technique for improving seed-transmitted pathogen detection threshold in the future.  相似文献   

3.
Pseudomonas aeruginosa(PA) is an opportunistic pathogen of humans and animals and a common source of nosocomial infections especially of the respiratory tract. Pseudomonas aeruginosa is also a major bacterial disease of poultry and in particular, eggs and newly hatched chicks. In this study, we developed a simple, accurate and rapid molecular detection method using cross priming amplification(CPA) with a nucleic acid test strip to detect P. aeruginosa. The assay efficiently amplified the target gene within 45 min at 62°C only using a simple water bath. The detection limit of the method was 1.18×10~2 copies μL~(–1) for plasmid DNA and 4.4 CFU mL~(–1) for bacteria in pure culture, and was 100 times more sensitive than conventional PCR. We screened 83 clinical samples from yellow-feather broiler breeder chickens and hospitalized/treated dogs and cats using CPA, PCR and traditional culture methods. The positive-sample ratios were 15.3%(13/83) by CPA, 13.3%(11/83) by PCR and 12.1%(10/83) by the culture method. The established CPA method has significant advantages for detecting P. aeruginosa. The method is easy to use and possesses high specificity and sensitivity without the requirements of complicated experimental equipment. The PA-CPA assay is especially fit for outdoor and primary medical units and is an ideal system for the rapid detection and monitoring of P. aeruginosa.  相似文献   

4.
TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in...  相似文献   

5.
Citrus Huanglongbing(HLB, yellow shoot disease) is one of the most serious citrus diseases worldwide. To better improve the detection sensitivity, a droplet digital PCR(ddPCR) assay was developed for the rapid detection of ‘Candidatus Liberibacter asiaticus'(Las), the putative causal agent of HLB. The detection of sensitivity comparison using positive plasmid indicated that dd PCR was superior to quantitative PCR(qPCR) for detecting and quantifying Las at low concentrations. The Las detection of 40 field samples also showed that six of 13 asymptomatic samples(46.15%) with high Ct value(35) were positive by dd PCR. This methodology showed great potential for early HLB infection diagnosis.  相似文献   

6.
肌注氟苯尼考在鸭疫里氏杆菌感染鸭体内的药动学特征   总被引：1，自引：0，他引：1  
【目的】研究并比较肌注氟苯尼考在2周龄健康和鸭疫里氏杆菌感染鸭体内的药代动力学特征。【方法】240只鸭随机分为2组,各120只鸭,其中一组用鸭疫里氏杆菌腿部感染,分别以30 mg·kg~(-1)体重单剂量肌内注射氟苯尼考,采血点为给药前和给药后5、10、15、20、30、45 min和1、1.5、2、4、8、10、14 h。采用高效液相色谱法(HPLC)测定血浆氟苯尼考的浓度,采用紫外检测器检测,检测波长为223 nm,流动相为乙腈和水,其体积比为26和74,流速为1 m L·min-1,色谱柱为Agilent C18(4.6 mm×150 mm,5μm),进样量为20μL。药动学分析软件Win Nonlin 5.2.1以非房室模型拟合处理血药浓度-时间数据,计算相关的药动学参数,并用统计学软件SPSS17.0对药动学参数进行t检验统计学分析。【结果】氟苯尼考在健康鸭体内的峰浓度(Cmax)为(22.88±3.11)μg·m L-1,表观分布容积(Vd/F)为(2.39±0.81)L·kg~(-1),体清除率(ClB/F)为(0.64±0.11)L·h-1·kg~(-1),消除半衰期(t1/2β)为(2.57±0.51)h和药时曲线下面积(AUC)为(47.28±7.87)μg·m L-1·h;氟苯尼考在感染鸭体内的峰浓度(Cmax)为(19.77±1.82)μg·m L-1,表观分布容积(Vd/F)为(2.44±0.46)L·kg~(-1),体清除率(ClB/F)为(0.63±0.08)L·h-1·kg~(-1),消除半衰期(t1/2β)为(2.74±0.54)h和药时曲线下面积(AUC)为(48.11±6.62)μg·m L-1·h。统计分析氟苯尼考在健康鸭和感染鸭的药动学参数,结果表明,除了Cmax差异显著(P0.05),其他参数差异均不显著(P0.05)。【结论】两者的主要药动学参数相比较,感染鸭体内的Cmax显著低于(P0.05)健康鸭体内的C_(max),其他参数无显著性差异。氟苯尼考以30 mg·kg~(-1)体重肌内注射在健康和感染鸭体内具有吸收迅速,峰浓度高,体内分布广泛,消除较快的特点。  相似文献   

7.
为提高葡萄球菌冻干菌粉的活菌数,制备高活性肉用葡萄球菌发酵剂。以前期研究获得的1株优良肉用发酵剂菌株松鼠葡萄球菌SL4为研究对象,对其菌体收集条件、冷冻干燥保护剂及初始菌悬液活菌数进行优化。结果表明:松鼠葡萄球菌SL4最适离心收集条件为离心转速6 000r/min,离心时间10min;冷冻干燥保护剂配方为脱脂奶粉100g/L,甘油23.56mL/L,甘露醇50g/L,海藻糖51.68g/L;初始菌悬液活菌数为109 CFU/mL。经以上条件优化后松鼠葡萄球菌SL4冻干存活率可达95%。  相似文献   

8.
Is it possible that Giant Panda (Ailuropoda melanoleuca) post-thawed sperm fertilization is improved with pentoxifylline (PF), platelet activated factor (PAF) and prostaglandin F 2-alpha (PGF2α)? In our study Giant Panda post-thawed sperm was incubated in Ham’s F-10 medium with different concentration of PF, PAF and PGF2α under 37°C. The effects of PF, PAF and PGF2α on Giant Panda post-thawed sperm fertility were evaluated through sperm motility, survival time, sperm membrane integrity, acrosome state and heterospecific egg penetration. The results were that PF, PAF and PGF2α all can affect Giant Panda post-thawed sperm in vitro fertilizing capability. In the experiment: 1 mg·mL?1 PF was most suitable for improving Giant Panda post-thawed sperm in vitro fertilizing capability. In the 1 mg·mL?1 PF group, sperm survival time was (15.33±4.73) h, the heterospecific egg penetration was 51.44% after incubating for 4 hours, the heterospecific egg penetration was 7.49% after incubating for 6 hours. The results of the 1 mg·mL?1 PF group were significantly higher than those of the control group (P<0.01). The results were also higher than those of the other treatment groups; Treated Giant Panda post-thawed sperm with 50 ng·mL?1 PAF had a better effect than 100 ng·mL?1 PAF, but the sperm fertilizing capability was damaged when incubation time exceeded 2 hours; 50 ng·mL?1 PGF2α had no significant effect on Giant Panda postthawed sperm, but when the PGF2α treated concentration was increased, sperm in vitro fertilizing capability decreased because of the damaged motility and declined acrosomal reaction rate. The conclusions suggest that it is possible to improve post-thawed Giant Panda sperm fertility with 1 mg·mL?1 PF.  相似文献   

9.
我国东北部分地区原料奶中蜡样芽孢杆菌污染情况调查   总被引：1，自引：0，他引：1  
对中国东北3个地区的原料奶中微生物污染情况进行调查与鉴定,主要检测原料奶中蜡样芽孢杆菌数,以及菌落总数和大肠菌群数,确定原料奶中微生物的污染状况与蜡样芽孢杆菌污染的关系.结果表明,3个地区的蜡样芽孢杆菌数平均值均≤105 CFU/mL.而细菌总数平均值则为106～107 CFU/mL,都属于超标奶.大肠菌群平均值,地区1为1 349 MPN/100mL.地区2为2 574 MPN/100 mL,地区3为2 032 MPN/100 mL.综合分析得出,蜡样芽孢杆菌数与原料奶的微生物污染状况无相关性.  相似文献   

10.
采用正交法建立了由栅藻(Scencdesmus obliquus)、小球藻(Chlorella vulgans)、亚硝化细菌(Nitritebacteria)、硝化细菌(Nitrate bacteria)组成的复合藻-菌净化系统去除氨态氮和亚硝酸态氮的最优化模型,确定了单胞藻与细菌的最优化数量配比关系,即栅藻∶小球藻∶亚硝酸化细菌∶硝化细菌=2.13∶1∶2.38∶3.73。利用该系统模型与单藻、单菌去除池塘老化水体中的氨态氮、亚硝酸态氮显示:去除氨态氮和亚硝酸态氮的效率远远高于单藻、单菌,其去除率分别为97.3%和68.8%。同时该系统模型还具有增加养殖水体溶解氧的作用,可使水体中的溶解氧在试验设定的参数中短时间达到9.7 mg.L-1,并可为水产动物提供1.6×106CFU.mL-1的天然藻类饵料。  相似文献   

11.
CB-4, a bacterial strain with highly effective herbicidal activity, was isolated from infected corn leaves. Through morphology, physiological and biochemical tests, and 16 S ribosomal DNA gene sequencing methods, CB-4 was identified as Pseudomonas aeruginosa. We conducted activity-evaluation experiments in the laboratory to assess the herbicidal potential of metabolites produced by strain CB-4. Crude extracts of strain CB-4 have high inhibition activity on Digitaria sanguinalis. In general, the root and shoot growth parameters of D. sanguinalis were significantly reduced by metabolites of strain CB-4. The IC50 of the culture filtrate extracts for the radicula and coleoptile of D. sanguinalis were 0.299 and 0.210 mg mL-1, respectively. Component 2 of the herbicidal activity of the crude toxin from strain CB-4 was successfully purified for the first time by using high-speed counter current chromatography with a two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-water（4：5：4：5, v/v） and high-performance liquid chromatography. We concluded that the metabolites of strain CB-4 have the potential to be developed as a microbe-based herbicide.  相似文献   

12.
Chemical potassium(K) fertilizer is commonly used in apple(Malus domestica L. Borkh) production but K is easily fixed by soil, resulting in reduced K fertilizer utilization and wasted resources. K-solubilizing bacteria(KSB) can cost-effectively increase the soluble K content in rhizosphere soil. Therefore, the objectives were to select high-efficiency KSB from apple orchards under various soil management models and evaluate their effects on apple seedling growth. Maize(Zea mays L.) straw mulching(MSM) increased the total carbon(TC), total nitrogen(TN) and available potassium(AK) in the rhizosphere and improved fruit quality. The number of KSB in the rhizosphere soil of MSM was 9.5×104 CFU g–1 soil, which was considerably higher than that in the other mulching models. Fourteen KSB strains were isolated with relative K solubilizing ability ranging from 17 to 30%, and five strains increased the dry weight per apple seedling. The most efficient strain was identified as Paenibacillus mucilaginosus through morphological observation and sequence analysis of 16 S rDNA, named JGK. After inoculation, the colonization of JGK in soil decreased from 4.0 to 1.5×10~9 CFU g~(–1) soil within 28 d. The growth of the apple seedlings and the K accumulation in apple plants were promoted by irrigation with 50 mL JGK bacterial solution(1×10~9 CFU mL~(–1)), but there was no significant increase in the AK content of rhizosphere soil. High-performance liquid phase analysis(HPLC) data showed that the JGK metabolites contained phytohormones and organic acids. Hence, the JGK strain promoted the growth of two-month-old apple seedlings by stimulating function of the produced phytohormones and enhanced K solubility by acidification for apple seedling uptake. This study enriches the understanding of KSB and provides an effective means to increase the K utilization efficiency of apple production.  相似文献   

13.
【目的】通过分析tlr4基因对羊肠道主要细菌数量的影响,评价转基因羊tlr4的生物安全性。【方法】收集15只转基因羊tlr4和15只非转基因羊的粪便样本,采用传统平板培养法和实时荧光定量PCR法(quantitative Real-Time PCR,qPCR)分析转基因羊tlr4和非转基因羊肠道中主要细菌数量的变化。【结果】传统培养法的结果表明,转tlr4基因对羊肠道中大肠杆菌(E.coli)、沙门氏菌(Salmonella)、双歧杆菌(Bifidobacterium)、粪肠球菌(Enterococcus)和金黄色葡萄球菌(Staphylococcus)的生长均有一定程度的抑制作用,在数量上转基因羊tlr4和非转基因羊的差异不显著(P0.05);qPCR法的结果表明,转tlr4基因对羊肠道中大肠杆菌、双歧杆菌、拟杆菌(Bacteroides)和粪肠球菌的数量亦无显著性影响(P0.05)。【结论】显示抗病基因tlr4处理对羊肠道主要细菌基本无显著性影响。  相似文献   

14.
摘要：该文通过水质理化因子监测、虾体和水体中可培养细菌和弧菌检测及高通量测序方法,系统解析过硫酸氢钾（PMS）干预下对虾养殖池塘水体环境指标和菌群结构的变化情况。结果表明：施用0.2 g/L PMS对对虾养殖水体中低含量氨氮也具有一定改善作用,泼洒PMS 24h和72h后的水温、溶解氧、pH、盐度、亚硝酸盐无显著差异。PMS泼洒后对虾肝胰腺内可培养细菌和弧菌数量分别由3.13×106 CFU/g和1.98×106 CFU/g降低至4.30×105 CFU/g和1.09×105 CFU/g,弧菌占比由63.36%降低至25.35%;水体中可培养细菌和弧菌数量分别由2.70×104 CFU/mL和6.00×103 CFU/m降低至8.50×103 CFU/mL和1.20×103 CFU/mL,弧菌占比由22.22%降低至14.11%,PMS可显著降低虾体和水体中可培养细菌数量及弧菌占比。对水体菌群结构进行高通量测序分析表明,放线菌门、拟杆菌门、蓝藻门和变形菌门为主要优势菌门,泼洒PMS前后（PB1/PA1）,腈基降解菌科、PeM15、DS001、Llumatobacteraceae、微杆菌科、红细菌科相对丰度显著升高（P<0.05）,巴纽尔斯菌科、腐螺菌科、Stappiaceae相对丰度显著降低（P<0.05）,对比泼洒PMS前后3天的变化趋势表明,泼洒PMS后池塘优势菌相对丰度发生显著变化且菌群结构PCoA指数偏离度较低。相关研究结果为评判PMS在水产养殖中的防控作用及其科学使用提供数据支撑。  相似文献   

15.
Reversed-phase high-performance liquid chromatography (RP-HPLC) method with C4 column and C18 column for analyzing β-lactoglobulin and α-lactalbumin in bovine milk was developed and the performance and...  相似文献   

16.
To reveal the insecticidal mechanism of terpinen-4-ol, the activity of Na＋,K＋-ATPase in insects tested were determined in vivo and in vitro. The results showed that terpinen-4-ol and its ester derivatives had strong contact activity to housefly and the contact toxicities of its derivatives except Z3 were all superior or equivalent to terpinen-4-ol. All the 7 compounds had strong inhibition towards activity of Na＋,K＋-ATPase. With poisoning symptom exacerbating, the inhibition rates were gradually increased. In vitro, the IC50 of terpinen-4-ol, Z1, Z2, Z4, Z5, and Z6 was 155.89, 197.98, 96.02, 121.36, 124.85, and 153.74 μg mL% respectively. There was well correlation between the LDs0 of terpinen-4-ol derivatives to housefly and the IC50 of terpinen-4-ol derivatives to Na＋,K＋-ATPase in housefly. In conclusion, Na＋,K＋-ATPase was likely the target of terpinen-4-ol against insects.  相似文献   

17.
Laccase(EC 1.10.3.2)is known to oxidize various aromatic and nonaromatic compounds via a radical-catalyzed reaction,which generally includes two types of laccase,Lac1 and Lac2.Lac1 oxidizes toxic compounds in the diet,and Lac2 is known to play an important role in melanizing the insect exoskeleton.In this study,we cloned and sequenced the cDNA of the diamondback moth,Plutella xylostella Lac2(PxLac2),from the third instar larvae using polymerase chain reaction(PCR)and rapid amplification of cDNA ends techniques.The results showed that the full-length PxLac2 cDNA was 1 944 bp long and had an open reading frame of 1 794 bp.PxLac2 encoded a protein with 597 amino acids and had a molecular weight of 66.09 kDa.Moreover,we determined the expression levels of PxLac2 in different stages by quantitative PCR(qPCR).The results indicated that PxLac2 was expressed differently in different stages.We observed the highest expression level in pupae and the lowest expression level in fourth instar larvae.We also investigated the enzymatic properties of laccase,which had optimal activity at pH 3.0 and at 35°C.Under these optimal conditions,laccase had a Michaelis constant(K_m)of 0.97 mmol L~(-1),maximal reaction speed(V_m)of 56.82 U mL~(-1),and activation energy(E_a)of 17.36 kJ mol~(-1) to oxidize2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt).Type II copper enhanced laccase activity below 0.8mmol L~(-1) and reduced enzyme activity above 0.8 mmol L~(-1) with an IC_(50) concentration of 1.26 mmol L~(-1).This study provides insights into the biological function of laccase.  相似文献   

18.
The objective of this study was to investigate the effect of lactic acid bacteria(LAB) inoculums on fermentation quality and in vitro digestibility of corn stover silage. Corn stover was ensiled without(control) or with Lactobacillus plantarum(LP), Enterococcus faecalis(EF), and Enterococcus mundtii(EM) for 45 days. The fermentation characteristics were assessed, and subsequent in vitro dry matter digestibility(DM-D), neutral detergent fiber digestibility(NDF-D), volatile fatty acids(VFA), methane(CH_4) production, cellulolytic bacteria proportions and their activities per corn stover silage were also determined. There was no significant difference(P0.05) among the silage pH, lactic acid, crude protein(CP), water soluble carbohydrates(WSC) and lignocelluloses contents of different treatments. The relative proportions of Ruminococcus flavefaciens and Fibrobacter succinogenes, carboxymethyl-ocellulose and β-glycosidase activities, DM-D, NDF-D, and VFA production of in vitro incubation was higher(P0.05) for silages inoculated with LP and EF than those of the control silage. Silage inoculated with LP showed the lowest(P0.05) CH_4 production per unit yield of VFA, which was positively corresponded to the lowest(P0.05) ratio of acetate to propionate. In summary, the ensiling fermentation quality and subsequent utilization of corn stover silage were efficiently improved by inoculated with L. plantarum.  相似文献   

19.
With a view to closing the nutrient loop between sanitation and fish culture, advanced fry of different species of fish (rohu, bata, mrigal, common carp, tilapia, punti) and larvae of freshwater prawn were reared in twelve experimental tanks in a mixed culture system for 120 days using three treatments (fresh urine, stored urine and mix of fresh and stored human urine) and control in triplicate. The total weight for all the species of fish and prawn in the stored urine (420.0 g) was 18 and 27% higher compared to fresh (356.0 g) and mixed urine (332.0 g) treatments, respectively. Likewise, primary productivity of phytoplankton and heterotrophic bacterial load related to phosphate level of water were also highest and lowest in the stored urine (GPP—508 ± 39.87 mg C m–2 h–1; heterotrophic bacteria—38.38 cfu × 103 mL–1) and control system (GPP—214 ± 38.09 mg C m–2 h–1; heterotrophic bacteria—13.53 cfu × 103 mL–1), respectively. The mean count of E. coli, on the other hand, did not differ between urine fed treatment and control. As a possible mechanism, it is suggested that stored urine upon transfer to aquaculture pond underwent degradation and mineralization that induced the autotrophic and heterotrophic food webs conducive to fish growth.  相似文献   

20.
Three goats fitted with cannula were used to provide rumen liquor to investigate the effects of limiting amino acids on rumen fermentation and microbial community in vitro. The removal method was used in the current experiments. Treatments are total essential amino acid （TEAA）, His-removal, Lys-removal, Met-removal, and branch chain amino acid （BCAA）- removal. Results indicated that, pH-value ranged between 5.9 and 6.8, with the highest mean value for the group with BCAA-removal （6.54） in the culture. Concentration of NH3-N ranged between 10.99 to 30.51 mg 100 mL^-1, with the group of TEAA recording the highest average NH3-N concentration （17.85 mg 100 mL^-1）. Yields of microbial protein and limiting degree on microbial growth varied with treatments （P 〈 0.01）, and the lowest accrued in treatment with BCAA-removal （0.1389, 0.1772, and 0.3161 mg mL^-1 for bacteria, protozoa, and mixed microbes, respectively）, compared to the group with TEAA, microbial production of mixed microbes decreased by 44.52%. As for micro-flora, protozoa to bacteria ratio was the lowest for the group with Lys-removal （89.12%）, while the highest for the group with BCAA-removal （127.60%） （P 〈 0.01）. Furthermore, PCR-SSCP analysis revealed that, microbial profile subjected to substrates within bacteria and protozoa groups. It was therefore concluded that, dietary amino acid influenced both rumen fermentation and microbial characteristics.  相似文献   

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