Use of phage for the identification of Brucella canis and Brucella ovis cultures

The brucella phage strains R, R/O and R/C standardised at routine test dilution on their propagating strains were effective in identifying cultures of Brucella ovis and B canis and in differentiating these from other non-smooth brucella isolates.     

猪霍乱沙门菌抗噬菌体菌株筛选及受体基因突变位点分析

【目的】 筛选抗噬菌体菌株并分析其受体基因突变位点,为解析猪霍乱沙门菌噬菌体抗性突变的产生机制提供理论依据。【方法】 以猪霍乱沙门菌CICC 21501及其裂解性噬菌体vB_SenS_S528（噬菌体S528）为研究对象,通过波动实验筛选自发突变的抗噬菌体菌株,观察菌落形态,并测定其对噬菌体的敏感性、吸附特性、生长曲线及对温度和pH的敏感性。通过全基因组重测序结合PCR验证定位耐受基因突变位点。【结果】 成功筛选出1株抗噬菌体S528的突变菌株,命名为B6-2。B6-2菌落边缘粗糙,与野生菌相比,生长曲线无显著差异,对pH表现出敏感性,在40、50 ℃时表现出温度耐受性。噬菌体S528对抗噬菌体菌株B6-2的吸附率减少60%（对野生菌吸附率为93%）。通过全基因组重测序及比对分析得知,B6-2菌株有3个位点发生了突变,分别为PROKKA_04510基因中2个位点和rfbC基因中的1个位点发生突变。突变基因片段的PCR产物电泳及测序结果均表明,PROKKA_04510基因上的2个位点并未发生突变,而真正发生突变的位点位于rfbC基因的350 bp处,碱基由C突变为T。【结论】 筛选到1株与受体改变有关的抗噬菌体菌株B6-2,其通过rfbC基因上的碱基突变来阻止噬菌体吸附。     

Experimental infection of goat fetuses in utero with a stable, rough mutant of Brucella abortus.

Fetuses of goats in their last trimester of pregnancy were experimentally infected with Brucella abortus strain RB51, a stable rough mutant deficient in the perosamine O-chain content of its lipopolysaccharide. RB51 maintained its rough phenotype in vivo and did not induce abortion. Infection with RB51 resulted in the production of significant levels of IgG type antibodies specific for B abortus cellular antigens distinct from the perosamine O-chain. These findings suggest that strain RB51 will be useful in the pregnant goat for studying the role of brucella antigens other than the lipopolysaccharide O-chain in the immune response to brucellosis.     

The identity, distribution and epizootiological significance of brucella isolates in Australia, 1981 to 1985

Results listing the identification of brucella isolates received by the National Brucellosis Reference Centre, National Biological Standards Laboratory, Canberra from 1981 to 1985 are presented. The distribution of brucella species and biotypes is shown on a host and state basis. Cultures isolated in Australia were identified as Brucella abortus biotypes 1, 2 and 4, and Strain 19; B. suis biotype 1, and B. ovis. B. melitensis biotype 3 was recovered from man infected in the Mediterranean area. B. abortus biotype 1 was the most frequent isolate. Atypical cultures isolated from cattle included B. suis biotype 1, and erythritol-utilising mutants of Strain 19. The epizootiological implications of these findings are discussed in relation to their impact on the national campaign to eradicate bovine brucellosis.     

Properties and partial genetic characterization of Nepean phage and other lytic phages of Brucella species.

Nepean (Np), a new brucellaphage, was associated with atypical Brucella abortus strains from Ontario cattle. Carriage of Np was associated with loss of smooth lipopolysaccharide, changes in some protein bands in acrylamide gel electrophoresis profiles, increased susceptibility to colistin, and increased resistance to ultraviolet killing. Nepean (Np) was compared with brucellaphages Tb, Fi, Wb, Iz and R/C. All were morphologically identical, with icosahedral capsids (50-65 nm diameter) and short tails (15-25 nm long), but Np had a more restricted host range, replicating only in smooth strains of B. abortus. All six brucellaphages were generally similar in resistance to chemical and physical agents. Brucellaphage DNA was double stranded and unmethylated; its molecular size was 38 kilobase pairs. The DNAs of Tb, Fi, Wb, Iz and R/C could not be differentiated by restriction endonuclease digest profiles produced by BgII, EcoRI, HindIII or PvuII. Nepean (Np) DNA was very similar to that of the other brucellaphages, but with every enzyme used its profile differed in the number and/or position of at least one fragment. However, there was complete cross-hybridization of Tb and Np DNAs. Hybridization techniques failed to detect Brucella DNA in Dp or Tb phages, or phage DNA in Brucella cells. Extrachromosomal plasmid DNA was not detected.     

病绵羊体内分离的拟似绵羊附睾种布氏菌的首次系统鉴定与分类

我们对新疆地方病防治研究所近年来从患附睾炎的公绵羊精液中分离出１５株拟似布氏菌，除按常规检定外，还采用了Ｓ群及Ｒ群噬菌体裂解试验、绵羊附睾种血清的试管凝集反应，并对有的菌株做了ＤＮＡ Ｇ＋Ｃｍｏｌ％含量测定，最后判定：在这十五株菌中有７株为绵羊附睾种布氏菌。     

Isolation and identification of anaerobic bacteria from ovine foot rot in Spain

A microbiological study was made of 125 Merino sheep showing clinical signs of foot rot. A total of 435 strictly anaerobic strains were isolated, belonging to the following genera: Bacteroides, Peptostreptococcus, Tissierella, Fusobacterium, Megasphaera, Eubacterium, Acidaminococcus, Clostridium, Peptococcus and Propionibacterium. Of the 35 species obtained, the following were found in more than 10 per cent of animals sampled: Bacteroides nodosus, B putredinis, B buccae, B ruminicola subspecies brevis, Tissierella praeacuta, Peptostreptococcus anaerobius and Megasphaera elsdenii. Six culture media were used for isolation. Agar brucella and agar brucella enriched with G-N anaerobe supplement proved to be the most efficient for isolating anaerobic bacteria.     

布鲁氏菌omp28基因的克隆、表达及反应原性分析

本研究克隆了羊种布鲁氏菌16M株、羊种布鲁氏菌M28株、犬种布鲁氏菌、绵羊附睾种布鲁氏菌、牛种布鲁氏菌A19株、猪种布鲁氏菌S2株的omp28基因并对以上不同种菌株的omp28基因序列及编码的氨基酸序列进行了比对,结果显示不同种布鲁氏菌omp28基因之间仅6个碱基不同,而且只有2个氨基酸不同,亲水性分析结果显示两处氨基酸的差异对蛋白亲水性不造成影响.将羊种布鲁氏菌16M的omp28基因亚克隆到pET32a中表达,OMP28在低温下诱导以可溶性形式高效表达.Westem-blot结果显示OMP28反应原性良好,是布鲁氏菌病诊断抗原的可能选择.     

鼠伤寒沙门菌烈性噬菌体的分离鉴定与生物学特性

旨在分离鼠伤寒沙门菌烈性噬菌体,为控制该病原菌感染或污染提供生物制剂。以1株从市售散装牛奶中分离到的多重耐药性鼠伤寒沙门菌为宿主菌,采用人工诱导方法,连续1周每日给3只试验鸡饲喂宿主菌悬液,7 d后,采用双层琼脂平板法从试验鸡粪便中分离培养噬菌体,并对其效价、核酸类型、宿主谱、热稳定性与酸碱耐受性以及对鼠伤寒沙门菌感染小鼠的治疗效果进行分析。结果表明：分离到1株鼠伤寒沙门菌噬菌体,命名为KM104。该病毒在体外高效裂解同源宿主菌株及另1株受试鼠伤寒沙门菌,形成清晰透明、直径约1 mm圆形噬菌斑,核酸类型为DNA,基因组约为28 kb,最佳感染比（MOI）为1∶10,对宿主菌感染的潜伏期约为6 min,70 min时效价最高,达4.6×10⁸ PFU·mL^-1,在20~60℃、pH2.0~8.0范围内保持较高的生物学活性,并有效减轻鼠伤寒沙门菌引起的小鼠十二指肠组织损伤。结果提示,噬菌体KM104能在体内外高效裂解鼠伤寒沙门菌,且增殖迅速,对环境适应性强,其耐酸特性利于抵御胃酸的分解,具有应用于生物防治的潜力。     

Effect of Bacteriophage in Enterotoxigenic Escherichia coli (ETEC) Infected Pigs

We evaluated effect of enterotoxigenic Escherichia coli (ETEC) specific lytic phage CJ12 in ETEC infected pigs. Phage was mixed with feed at a ratio of 1:1,000 (0.1%). One week after initially providing phage mixed feed, pigs were challenged orally with 10(11) CFU of ETEC and body weight, diarrhea score, bacterial CFU and phage PFU in the feces were measured. Pigs of phage treated groups C (10(6) PFU/g) and D (10(8) PFU/g) showed more resistance to diarrhea due to ETEC infection compared to positive control group B on the third day after the initial challenge. Moreover, during the quantitation of ETEC in feces, both groups C and D showed approximately 63.92 and 60.73% reduced ETEC compared to positive control group B. Phages were successfully isolated from feces in both groups C and D during the experiment without any adverse effects, suggesting the possibility of using CJ12 as a feed additive.     

The role of the differential complement fixation test, using rough and smooth brucella antigens, in the anamnestic test

To assess the ability of the differential complement fixation test to distinguish vaccinal reactors from infected cattle, approximately 1,000 heifers were tested by the complement fixation test (CFT) using rough and smooth brucella antigens, before the injection of 45/20 vaccine and at 3 and 6 or 10 weeks after vaccination. Before vaccination 91.5% of heifers were negative to the rough antigen but 0.6% were positive with high titre (greater than or equal to 128). By 10 weeks after injection of 45/20 vaccine 97.6% of heifers were positive to the rough CF antigen, at greater than or equal to 8, a majority reaching greater than or equal to 128. Nineteen pre-vaccinal reactors to the standard CFT were killed and Brucella abortus was isolated from the tissues of 14. Twenty-six post-vaccinal reactors were killed and B. abortus was isolated from the tissues of 8. In the 22 B. abortus infected animals the differential CFT classified 9 correctly as infected, 5 incorrectly as vaccination reactions and 8 as inconclusive. The differential CF was ineffective in distinguishing titres resulting from vaccination with 45/20 vaccine from those due to infection.     

1株C3样奇异变形杆菌烈性噬菌体的生物学特性及基因组分析

从仔猪粪样中分离鉴定奇异变形杆菌（Proteus mirabilis）的烈性噬菌体,并对其进行生物学特性分析和基因组分析。以Proteus mirabilis为宿主菌,用双层平板法从仔猪粪样中分离噬菌体。利用电子显微镜观察噬菌体大小和形态。提取噬菌体DNA,并使用Illumina Hiseq进行高通量测序,对基因组进行注释和分析。结果显示：分离到的奇异变形杆菌噬菌体命名为vB_PmiP_P59（Proteus mirabilis phage vB_PmiP_P59）。P59属于短尾噬菌体科,形态为C3形：头部为扁平的椭圆形,长152 nm,宽54 nm;尾部长约40 nm,宽12 nm。P59的最佳感染复数为0.001;潜伏期为30 min,爆发期为105 min,裂解量为2 754 PFU·cell^-1;P59在不高于50℃,pH 5~10时稳定。P59噬菌体基因组全长90 187 bp,（G+C）%含量为34.65%,共编码136个ORF和4个tRNA,不携带抗生素抗性基因和毒力基因。蛋白质组分析表明,P59与C3样噬菌体phiEco32有30.47%的一致性。奇异变形杆菌噬菌体vB_PmiP_P59属于C3样烈性噬菌体,并对奇异变形杆菌有显著的抑菌效果。     

1株宽噬菌谱鸡白痢沙门氏菌噬菌体的分离及生物学特性分析

本研究采用双层琼脂平板法从现代化肉鸡养殖场采集的粪便、污水和垫料样品中分离宽噬菌谱鸡白痢沙门氏菌烈性噬菌体,并通过透射电子显微镜观察、噬菌谱分析、热稳定性、pH稳定性、最佳感染复数及一步生长曲线等对分离纯化的噬菌体进行生物学特性分析。结果显示,试验成功分离纯化出一株鸡白痢沙门氏菌噬菌体,命名STP98-a。该噬菌体噬菌斑直径4~5 mm,圆形透明,无晕圈,其头部偏椭圆形,长径约61 nm,横径约67 nm,尾长约112 nm,直径约10 nm,属于长尾噬菌体科;能裂解不同血清型鸡白痢沙门氏菌和肠炎沙门氏菌肠亚种,噬菌谱宽;在30~60℃保持高活性;在pH 3.0~12.0稳定;最佳感染复数为0.001;一步生长曲线结果显示,其感染宿主菌潜伏期为5 min,裂解期为135 min,裂解量为93。综上所述,STP98-a为一株宽噬菌谱鸡白痢沙门氏菌烈性噬菌体,可用于鸡白痢沙门氏菌噬菌体制剂的研发。     

猪致病性大肠杆菌O9菌蜕的制备及免疫保护力

通过PCR方法克隆噬菌体PhiXl74的融菌基因E,将其与温控表达载体pBV220连接,成功构建温控融菌表达盒,设计扩增温控表达盒的2对引物,使其5′端分别与lacZ基因敲除基因的首尾50bp基因同源,使用Red系统同源重组,使用麦康凯平板筛选、PCR鉴定白色菌落,温控诱导融菌蛋白的表达,制备大肠杆菌O9的菌影,并对其融菌效率及免疫保护力进行了初步研究。结果显示,成功构建了温控融菌表达盒,经同源重组成功获得猪致病性大肠杆菌O9温控融菌株,温控表达溶菌蛋白能够成功抑制细菌的增殖,并使活菌数下降3个指数,对小鼠安全性好,皮下免疫组攻毒保护率达到71.43%。     

The activity of chosen bacteriophages on Yersinia enterocolitica strains

The aim of the present study was to evaluate the lytic activity of three bacteriophages on Yersinia enterocolitica strains isolated from humans and pigs. The Y. enterocolitica strains tested belonged to 0:3, 0:9 and 0:2 serogroups. The ZD5 phage was obtained from a water sample, but remaining phages were obtained from the lysogenic Y. frederiksenii 7291 and Y. enterocolitica 8684 strains. All the Y. enterocolitica strains tested which belonged to 0:9 serogroup did not show any susceptibility to the bacteriophages used. The bacteriophages tested showed different lytic activity on the Y. enterocolitica 0:3 strains investigated. The phage susceptibility of Y. enterocolitica 0:3 strains revealed 9 different phage patterns. ZD5 phage showed the highest lytic activity, because it produced confluent lysis of the most Y. enterocolitica 0:3 strains tested. The Y. enterocolitica 0:2 strains isolated from pigs showed the similar phage susceptibility. The Y. kristensenii and Y. pseudotuberculosis strains tested were not sensitive to the bacteriophages used.     

奶牛乳房炎金黄色葡萄球菌裂解性噬菌体裂解酶LysIMEP5基因的克隆及序列分析

为了克隆奶牛乳房炎金黄色(葡萄球菌裂解性噬菌体裂解酶Lys IMEP5基因,分析其生物信息学特性,以本实验室分离的奶牛乳房炎金黄色葡萄球菌裂解性噬菌体v B_Sau S_IMEP5为材料,根据其全基因组学信息,获取裂解酶基因序列,应用Primer 5.0设计特异性引物。采用PCR方法扩增并克隆Lys IMEP5基因,通过BLAST进行序列比对分析,利用在线软件对蛋白结构进行预测。成功克隆到裂解酶Lys IMEP5基因,测序结果通过DNAMAN比对与原序列完全匹配,无任何基因突变,同源性分析显示,与已报道的金黄色葡萄球菌噬菌体裂解酶相似性最高为83.1%;裂解酶Lys IMEP5基因序列全长1371 bp,共编码456个氨基酸,为亲水性蛋白,分子质量为51.717 k Da,理论等电点为9.70;Lys IMEP5同时存在CHAP片段和Amidase-3片段;该蛋白无跨膜区,无信号肽,以无规则卷曲为主。从奶牛乳房炎金黄色葡萄球菌裂解性噬菌体中成功克隆出Lys IMEP5基因,通过对该蛋白的结构预测分析为后续克隆表达及开发新型绿色抑菌剂奠定了基础。     

Isolation,Identification and Characterization Analysis of Broad-spectrum Escherichia coli O157∶H7 CVCC4050 Phage vB_EcoM_GXBP08

【Objective】 This study was aimed to explore the biological characteristics and genome characteristics of broad-spectrum Escherichia coli O157∶H7 phage.【Method】 The broad-spectrum Escherichia coli O157∶H7 lytic phage was seperated from a swine farm sewage in Nanning of Guangxi using the double-layer agar culture method.The host range and the corresponding titers of phage were determined through the methods of spot tests and plaque tests.The methods of transmission electron microscope observation,determination of the best multiplicity of infection in the plural,one-step growth curve drawing,thermal sensitivity and pH stability evaluation,sterilization experiment and the whole genome sequencing were used to analyze the morphological features,biological characteristics and the whole genomic characteristics of phage.【Result】 A broad-spectrum lytic phage against Escherichia coli O157∶H7 was successfully isolated and purified which was named as vB_EcoM_GXBP08.Phage vB_EcoM_GXBP08 could lyse 14 Escherichia coli strains with high efficiency,and the phage titers could reach 10⁹ to 10¹⁰ PFU/mL.Using Escherichia coli O157∶H7 CVCC4050 as host bacteria,the optimal multiplicity of infection (MOI) of phage vB_EcoM_GXBP08 was 1.One-step growth curve indicated that the latent period of vB_EcoM_GXBP08 was 20 min,the outbreak period was 50 min,and the outbreak quantity was 154 PFU/cell. The tolerable temperature range of phage vB_EcoM_GXBP08 was 30 to 70 ℃,and it could maintain activity at pH 4.0 to pH 10.0.The results of bactericidal experiment against CVCC4050 showed that phage vB_EcoM_GXBP08 had good germicidal efficacy when MOI was 1.According to transmission electron microscope observation and the whole genome analysis,phage vB_EcoM_GXBP08 belonged to Caudovirales order,Myoviridae family,T4-like Phagus,which had a genome consisting of 108 114 bp with a GC content of 36.23%.Phage vB_EcoM_GXBP08 genome harbored 8 CDS associated with lysis proteins and lacked virulence genes and drug resistance genes associated with antibiotic resistance,toxins and virulence factors.【Conclusion】 Phage vB_EcoM_GXBP08 had a broad host range,high titer,good thermal stability and acid-base stability,and a strong bactericidal effect in liquid medium.The results provided reference for the development of broad-spectrum phage and its application in the prevention and treatment of Escherichia coli O157∶H7 infection in food industry and breeding industry.     

噬菌体降低铜绿假单胞菌所致腐蛋的效果

铜绿假单胞菌是孵化过程中造成腐蛋的主要病原菌,拟尝试用裂解性噬菌体防治。选用腐蛋中筛选的铜绿假单胞菌PA-MH12,以此为宿主从河水中筛选到1株裂解性噬菌体MH12-Q,对其进行了生物学特性研究,体外进行了对铜绿假单胞菌所致腐蛋的防控效果试验。结果显示：噬菌体MH12-Q属于肌尾噬菌体科（Myoviridae）,效价可达到10¹³pfu·mL^-1,完全裂解效价（CLC）为10¹⁰pfu·mL^-1,最适生长温度为37℃,pH稳定性好,最适pH为6~8,且其在pH4~10的效价都能维持在较高的水平。最佳感染复数（OMOI）为0.001,感染宿主的潜伏期为30 min,爆发期为70 min。噬菌体MH12-Q可使高浓度铜绿假单胞菌的感染率从70%降低到40%。铜绿假单胞菌噬菌体MH12-Q的生物学性质适合生产应用,对孵化场腐蛋具有较好的防控作用,能够提高感染铜绿假单胞菌的鸡胚成活率。     

奶牛乳腺炎源金黄色葡萄球菌噬菌体P82的分离及特性研究

【目的】 为开发噬菌体"鸡尾酒"制剂防治奶牛乳腺炎提供生物学材料。【方法】 以实验室分离的奶牛乳腺炎源金黄色葡萄球菌82为宿主菌,通过点滴法和双层平板法在奶牛场的污水、弃奶和粪便混合物中分离并纯化其噬菌体,通过噬菌斑及透射电子显微镜对该株噬菌体的形态特征进行观察。利用核酸酶处理分析该噬菌体核酸类型,并对其裂解谱、最佳感染复数、一步生长曲线、热稳定性、pH稳定性进行研究。【结果】 分离筛选出金黄色葡萄球菌82的裂解性噬菌体P82,电镜下观察其头部为二十面体,大小约为120 nm×83 nm,有一条带可伸缩尾鞘的长尾,长约126 nm,属于有尾噬菌体目,肌尾噬菌体科。通过琼脂糖凝胶电泳鉴定其核酸类型为双链DNA （dsDNA）。噬菌体P82的最佳感染复数为0.001,对奶牛乳腺炎源金黄色葡萄球菌裂解率为36.51%（23/63）,宿主谱较广;其生长潜伏期约为25 min,爆发期约为45 min,裂解量约为74 PFU/cell;在pH 4.0~12.0时活性稳定,在pH 2.0和13.0时则完全失去活性。噬菌体P82热稳定性较高,在70 ℃作用60 min后仍有裂解能力,在80 ℃作用40 min时则失去裂解能力。【结论】 本研究分离的噬菌体P82效价较高、裂解能力强、增殖速度快、噬菌谱广,对不同温度和pH均有较好的耐受度,能作为专一性裂解奶牛乳腺炎源金黄色葡萄球菌的噬菌体候选株,可与其他噬菌体混合制成噬菌体"鸡尾酒"制剂用于防治奶牛乳腺炎,为奶牛乳腺炎的噬菌体疗法提供材料。     

Urease activity of Brucella species

Examination of the urease activity of 604 brucella strains showed a limited correlation with species. Most strains of B canis, B neotomae and B suis gave a positive urease reaction within 15 minutes, although some exceptions were noted. A substantial proportion of strains of B abortus and B melitensis also hydrolysed urea as rapidly as most B suis strains. Although most B ovis strains were negative to the urease test, 28.9 per cent of those examined gave positive reactions.