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1.
为分离纯化奶牛腐蹄病坏死杆菌,分析其与其他菌株的亲缘关系,本研究利用坏死杆菌白细胞毒素特异性引物,对奶牛腐蹄病病牛蹄部拭子样品进行了PCR检测,利用厌氧培养基对PCR检测阳性样品进行了坏死杆菌的分离培养,以分离的坏死杆菌基因组DNA为模板,对白细胞毒素基因进行了克隆和序列分析。结果显示,9份奶牛腐蹄病病牛蹄部拭子样品PCR检测结果均为阳性,对其中一份样品中的坏死杆菌进行分离培养,获得了纯培养物,命名为bFR13-1。坏死杆菌bFR13-1菌株白细胞毒素基因测序结果显示,与GenBank已发表的H05、A25和B35菌株的白细胞毒素基因在核苷酸水平的同源性分别为98.40%、98.35%和90.79%,推导氨基酸的同源性分别为97.7%、97.6%和89.0%。进化树分析结果显示,坏死杆菌bFR13-1菌株白细胞毒素与H05菌株的同源性最高,bFR13-1菌株与H05菌株和A25菌株呈较近亲缘关系。结果表明,不同坏死杆菌分离株的白细胞毒素呈现一定的变异性,这种变化是否与坏死杆菌致病性相关,值得深入研究。 相似文献
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Jin J Xu D Narongwanichgarn W Goto Y Haga T Shinjo T 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2002,64(3):273-276
The 16S-23S rRNA intergenic spacer regions (ISRs) of Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme were characterized. Products of two sizes, about 360 bp (small) and 530 bp (large), were generated by PCR amplification from the 16S-23S rRNA ISR of all the strains tested. The large and small 16S-23S rRNA ISRs of F. necrophorum exhibited a level of sequence similarity of 93.9% to 99.7% and 94.2% to 98.6% homologies within the species, respectively. Only the large spacer regions in these bacteria contained one or two tRNA genes. F. necrophorum subsp. necrophorum contains the isoleucine and alanine tRNA gene, whereas F. necrophorum subsp. funduliforme contains the isoleucine tRNA gene. 相似文献
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Fusobacterium necrophorum, a gram-negative, non-spore-forming anaerobe, is a normal inhabitant of the alimentary tract of animals and humans. Two types of F. necrophorum, subspecies necrophorum (biotype A) and funduliforme (biotype B), have been recognized, which differ morphologically, biochemically and biologically. The organism is an opportunistic pathogen that causes numerous necrotic conditions (necrobacillosis) such as bovine hepatic abscesses and ruminant foot abscesses. Subspecies necrophorum strains are considered to be more virulent for cattle and have been shown to produce greater amounts of leukotoxin than subspecies funduliforme strains. The leukotoxin operon of F. necrophorum consists of three genes (lktBAC) of which the leukotoxin structural gene (lktA) is the second gene in the operon. In this study, the promoter regions of the leukotoxin operons from the two subspecies were identified and their nucleotide sequence compared. The promoter regions were found to differ in sequence, in length of the sequence between the upstream determinant (oppF) and the first gene of the leukotoxin operon (lktB), and in promoter strength as assayed in Escherichia coli host cells. 相似文献
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Narongwanichgarn W Misawa N Jin JH Amoako KK Kawaguchi E Shinjo T Haga T Goto Y 《Veterinary microbiology》2003,91(2-3):183-195
The phylogenic relationships of two subspecies of Fusobacterium necrophorum were investigated by randomly amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR). With each of the 12 random primers, the DNA fingerprints generated were subjected to cluster analysis for dendrograms. The analysis indicated that twelve strains were organized into two major clusters, and that all strains of each subspecies were confined to one cluster. Furthermore, two of the random primers examined each generated a unique band in F. n. necrophorum strains. We cloned these specific bands and determined the nucleotide sequences. A search for amino acid sequence homologies revealed that the two specific fragments had significant homology to the rpoB gene of Lactococcus lactis subsp. lactis and the hemagglutinin-related protein gene of Ralstonia solanacearum, respectively. New specific primers designed for the rpoB gene were able to amplify 900bp fragments from both subspecies. However, the specific primers designed for the hemagglutinin-related protein gene amplified only a 250bp fragment of the genome of the F. n. necrophorum strains, suggesting that this gene is unique to F. n. necrophorum. These results were further confirmed by dot blot hybridization. Finally, a one-step duplex PCR technique in a single tube for the rapid detection and differentiation of the F. necrophorum subspecies was developed. 相似文献
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坏死梭杆菌是动物和人的各种坏死化脓感染的条件性致病菌.坏死梭杆菌的白细胞毒素是一种高度不稳定性分泌蛋白,被认为是主要的毒力因子.坏死梭杆菌白细胞毒素基因的开放阅读框(lktAORF)包括9 726 bp,编码3 241个氨基酸,总分子质量为336 ku的蛋白,且与其他细菌的细胞毒素没有任何相似的序列.覆盖在整个坏死梭杆菌lktA ORF上的5个短的重叠的多肽分别是BSBSE,SX,GAS,SH和FINAL,将它们在大肠埃希菌中表达,所有的多肽都有免疫原性,但GAS引起最小的抗体反应,BSBSE和SH对坏死梭杆菌攻击诱导产生了很强的保护力,比坏死梭杆菌的培养上清内全长活性lkt或无活性上清的保护性要好得多. 相似文献
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Tadepalli S Stewart GC Nagaraja TG Jang SS Narayanan SK 《Veterinary microbiology》2008,127(1-2):89-96
Fusobacterium equinum, a gram negative, rod-shaped and an obligate anaerobic bacterium is a newly described species. The organism is associated with necrotic infections of the respiratory tract in horses that include necrotizing pneumonia, pleuritis and paraoral infections. The species is closely related to F. necrophorum that causes liver abscesses in cattle and sheep, calf-diphtheria in cattle, and foot-rot in sheep and cattle. Leukotoxin, an exotoxin, is an important virulence factor in bovine strains of F. necrophorum. Our objective was to examine strains (n=10) of F. equinum for leukotoxin (lktA) gene and its toxic effects on equine leukocytes. Southern hybridization and partial DNA sequencing revealed that all the 10 strains had the lktA gene with greater similarities to F. necrophorum subsp. necrophorum. The secreted leukotoxin was detected in the culture supernatant and its biological activity was determined by viability assays with equine polymorphonuclear cells (PMNs) using flow cytometry. While culture supernatants of four strains (E1, E7, E9, and E10) were highly toxic to equine PMNs; strain E5 was moderately toxic and the remaining strains (E2, E3, E4, E6, and E8) were only mildly toxic. Our data indicated that F. equinum isolates had lktA gene and its product was toxic to equine leukocytes. Therefore, leukotoxin may be an important virulence factor in F. equinum infections. 相似文献
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为了对1例貉源犬瘟热(CD)进行病毒检测并分析其血凝素H基因变异情况,本研究从1只疑似犬瘟热病死的貉采病料进行研磨,利用RT-PCR方法扩增犬瘟热病毒H基因,对扩增出的H基因片段进行克隆测序,并对得到的H基因序列进行分析。结果表明,该貉感染犬瘟热病毒,得到的H基因核苷酸和氨基酸序列与CDV野毒株同源性较高,分别为89.1%~98.0%和85.4%~97.5%。遗传进化分析表明其属于Asia-1型野毒株,N连接糖基化位点分析结果表明,该毒株在542aa处比参考野毒株多了1个潜在的N糖基化位点,在525aa-550aa间多出了1个抗原表位。 相似文献
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Immunogenicity and protective effects of truncated recombinant leukotoxin proteins of Fusobacterium necrophorum in mice 总被引：4，自引：0，他引：4
Fusobacterium necrophorum, a gram-negative, anaerobic and rod-shaped bacterium, is generally an opportunistic pathogen and causes a wide variety of necrotic infections in animals and humans. Leukotoxin, a secreted protein, is a major virulence factor. The gene encoding the leukotoxin (lktA) in F. necrophorum has been cloned, sequenced and expressed in Escherichia coli. Because of low expression levels, problems associated with purifying full-length recombinant protein, and of the physical instability of the protein, five overlapping leukotoxin gene truncations were constructed. The recombinant polypeptides (BSBSE, SX, GAS, SH, and FINAL) were expressed in E. coli and purified by nickel-affinity chromatography. The objectives were to investigate the effectiveness of the purified truncated polypeptides to induce protective immunity in mice challenged with F. necrophorum. The polypeptides, individually or in combination, and inactivated native leukotoxin or culture supernatant of F. necrophorum were homogenized with an adjuvant and injected into mice on days 0 and 21. Blood samples were collected to measure serum anti-leukotoxin antibody titers on days 0, 21 and 42 and on day 42, mice were experimentally challenged with F. necrophorum. All polypeptides were immunogenic, with GAS polypeptide eliciting the least antibody response. Two polypeptides (BSBSE and SH) induced significant protection in mice against F. necrophorum infection. Protection was better than the full-length native leukotoxin or inactivated supernatant.The study demonstrated that the leukotoxin of F. necrophorum carries epitopes that induce protective immunity against experimental fusobacterial infection, thus providing further evidence to the importance of leukotoxin as a major virulence factor. 相似文献
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D. B. Sun R. Wu G. L. Li J. S. Zheng X. P. Liu Y. C. Lin D. H. Guo 《Veterinary research communications》2009,33(7):749-755
To analyze immunodominant regions of leukotoxin protein of Fusobacterium necrophorum strain H05, a series of truncated forms of leukotoxin gene were expressed in Escherichia coli using the vector pGEX-6p-1 or pPROEX HTa. The results of SDS-PAGE showed the truncated forms PL1, PL2, PL4, and PL5 were
expressed in Escherichia coli using the vector pGEX-6p-1, and the truncated forms PL3 was expressed in Escherichia coli using the vector pPROEX HTa. These recombinant proteins were able to react with antisera against Fusobacterium necrophorum strain A25. In five recombinant proteins, the recombinant proteins PL1, PL3 and PL4 as vaccine were able to elicit formation
of the better protective effects on mice against infection of Fusobacterium necrophorum strain A25. 相似文献
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The effect of cultural conditions on the production of leukotoxin by biotypes A and B of F. necrophorum was investigated. Biotypes A and B were grown in prereduced, anaerobically sterilized, brain-heart infusion (BHI) broth. The average leukotoxin titer of culture supernatant was 18 times higher from biotype A strains than from biotype B strains. Leukotoxin activity peaked during the late-log and early-stationary phases of growth, then declined precipitously in both biotypes. F. necrophorum biotype A was grown in different media (BHI, liver infusion, and Eugon broths), at various pH (6.6, 7.3, 7.7, and 8.2), incubation temperatures (30, 35, 39, and 43 degrees C), redox potentials (-352 to +375 mV), and iron concentrations (less than 0.2, 4.2, 42.1, and 361.4 microM). Anaerobic BHI broth with pH from 6.6 to 7.7 at 39 degrees C incubation temperature supported maximal F. necrophorum growth and leukotoxin production. The optimum redox potential for F. necrophorum growth was in the range of -230 to -280 mV. However, the presence of titanium III citrate or dithiothreitol (7.78 mM) in the medium decreased (P less than 0.05) the leukotoxicity of F. necrophorum. Low iron concentration (less than 0.2 microM) decreased (P less than 0.05) growth rate but not leukotoxin activity of F. necrophorum, whereas high iron concentration inhibited the leukotoxin activity. 相似文献
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Bicalho ML Machado VS Oikonomou G Gilbert RO Bicalho RC 《Veterinary microbiology》2012,157(1-2):125-131
The objective of this study was to evaluate the relationship between bacterial species-specific virulence factors (VFs) present in the uterus at 3 different stages of lactation (1-3, 8-10, and 34-36 days in milk (DIM)) and the incidence of metritis and clinical endometritis in dairy cows. The following VF genes were investigated: plo (pyolysin), cbpA (collagen-binding protein), and fimA (fimbriae expression) which are Arcanobacterium pyogenes specific; fimH (a type 1 pilus component), Escherichia coli specific; and lktA (leukotoxin), Fusobacterium necrophorum specific. Uterine swabs were collected from 111 postpartum dairy cows. PCR was used to detect the presence of plo, cbpA, fimA, fimH, and lktA genes. A. pyogenes cbpA was detected in only 5 samples and therefore was not subjected to further analysis. E. coli (fimH) was significantly associated with metritis and endometritis when detected at 1-3 DIM; F. necrophorum (lktA) was significantly associated with metritis when detected at 1-3 and 8-12 DIM and with endometritis when detected at 34-36 DIM; and A. pyogenes (fimA and plo) was associated with metritis (fimA) when detected at 1-3 DIM and endometritis (fimA and plo) when detected at 8-10 and 34-36 DIM. 相似文献
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H5N1亚型禽流感病毒新疆株NS基因的克隆和序列分析 总被引：1，自引：0，他引：1
根据已发表的H5N1禽流感病毒NS基因全序列设计了1对引物，对4株禽流感病毒新疆株的NS基因进行了RT—PCR扩增，并将其克隆到pMD18-T载体上，分别获得了全长为817、851、854、854bp的全基因序列。序列分析结果表明，新疆毒株间的核苷酸同源性为97．8％～98．9%，氨基酸同源性为96．5％～98．7％，与广东、香港和东南亚等不同地区的11个毒株比较，同源性为90．0％～99．4％；与来自野禽、猪等不同种类的11个流感毒株比较，同源性为81．4％～99．3％。系统进化树分析表明，新疆株独立分支。 相似文献
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为了解凉山州腹泻仔猪猪圆环病毒2型(PCV2)的感染情况,采用PCR方法对采自四川省凉山州西昌市和喜德县7个猪场85份腹泻仔猪粪便、病变组织等样品进行PCV2检测,并将扩增到的PCV2 ORF2基因片段进行测序和序列分析。结果显示,PCV2阳性样品有22份,阳性率为25.9%,阳性猪场占71.4%(5/7)。22个ORF2基因片段长度均为459 bp,核苷酸同源性为92.8%～100%,氨基酸同源性为93.5%～100%。构建的系统进化树显示22个测序序列分别处于2个分支:PCV2b和PCV2d,但未形成明显的地理分支。结果表明凉山州猪群中PCV2感染较为普遍,且以PCV2d亚型为主,并存在一定程度的遗传变异。 相似文献
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G. Bennett J. Hickford H. Zhou J. Laporte J. Gibbs 《Research in veterinary science》2009,87(3):413-415
Lameness in the dairy industry in New Zealand causes a problem in lost production, animal welfare and associated costs. To understand what bacteria may be present on the hooves of lame dairy cattle in this grass-fed system, samples were scraped from lame dairy cows and examined for the presence of Fusobacterium necrophorum (F. necrophorum) and Dichelobacter nodosus (D. nodosus) using the polymerase chain reaction (PCR). The PCR primers were designed to detect the presence of the lktA gene, which encodes a leukotoxin unique to F. necrophorum, and the fimA gene of D. nodosus. A total of 148 hoof scrapings were collected by farm staff over the period September 2005 to May 2006. F. necrophorum was detected in 79/148 of the samples, while D. nodosus was detected in 7/148 of the samples. The frequent finding of F. necrophorum within dairy herds in New Zealand is noteworthy and the occasional finding of D. nodosus on some dairy cattle suggests a possible role in both ovine and bovine hoof pathology. 相似文献
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通过克隆鹅细小病毒(Goose parvovirus,GPV)分离株VP1-V3非重叠区基因,并对其进行序列分析,为鹅细小病毒感染与疫苗免疫的鉴别诊断奠定理论基础.进行鹅胚病毒增殖,收集尿囊液,提取基因组,参考发表的B株序列,设计合成一对引物,经PCR扩增,克隆VP1-VP3基因,筛选阳性克隆,对其进行序列测定及同源性分析.结果表明,克隆的基因片段为901 bp,VP1-VP3基因共594 bp,编码198个氨基酸;弱毒株之间亲缘关系很近,核苷酸同源性99.5％～100％,氨基酸同源性98.5％～100％;强毒株与B株亲缘关系较近,核苷酸同源性96.6％,氨基酸同源性97.5％;弱毒株与强毒株亲缘关系较远,核苷酸同源性92％～93％,氨基酸同源性96％～97.5％;弱毒株与B株亲缘关系最远,核苷酸同源性92.5％～93％,氨基酸同源性93.9％～95.5％.说明强毒株与弱毒株之间核苷酸序列存在差异. 相似文献
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Four pairs of primers containing BamHⅠ and XhoⅠ sites were designed for amplification of 43 ku outer membrane protein （43K OMP） of bovine Fusobacterium necrophorum strain H05 according to the GenBank. The PCR products of the truncated 43K OMP genes were digested with BamHⅠ and XhoⅠrestriction endonuclease, and then the digested products were ligated to the pET-32a vector with His tag. The positive plasmids of four truncated 43K OMP genes were transformed into E.coli BL21(DE3). Protein expression of four truncated 43K OMP genes were induced using 1.0 mmol/L IPTG. The result indicated that the four truncated 43K OMP genes were successfully expressed in E.coli, and molecular weights of the expressed proteins were all about 30 ku. This study would provide some basis for further research of immunogenicity of the outer membrane protein of Fusobacterium necrophorum. 相似文献
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为调查广东地区鸡传染性支气管炎病毒(IBV)的流行及其遗传变异情况,本研究通过病料SPF鸡胚接种和鸡胚尿囊液的RT-PCR鉴定,于2013年从广东湛江地区不同发病鸡场分离到两株IBV,分别命名为CK/CH/GD/ZJ10/2013和CK/CH/GD/ZJ11/2013,并对这两株IBV的S1基因进行序列分析。结果显示,CK/CH/GD/ZJ10/2013株S1基因全长1 626 bp,编码542个氨基酸,其裂解位点为NRFRR,属于基因型Ⅲ,并推测其为基因型Ⅲ毒株(CK/CH/GX/NN11-3)与基因型Ⅰ毒株(GX-NN-6)在S1基因处发生重组而产生的新毒株;CK/CH/GD/ZJ11/2013株S1基因全长1620 bp,编码540个氨基酸,其裂解位点为HRRRR,属于基因型Ⅰ(类QX型);两株IBV的S1基因间核苷酸序列及其推导的氨基酸序列同源性较低,与位于同一基因型的参考毒株间同源性较高,而与中国使用的Mass型常规疫苗H120和H52之间的同源性最低,仅为75.7%~76.3%和77.1%~77.9%。本研究可为广东省IBV的流行病学调查和分子生物学研究提供参考。 相似文献
20.
SHANG Hui-qin LI Lin CHEN Rui-ai LIU Hong-bo LIANG Mei-lan HE Dong-sheng 《中国畜牧兽医》2015,42(8):1963-1972
In order to investigate the epidemiology and genetic variation of avian infectious bronchitis virus (IBV) in Guangdong province, two strains of IBV were isolated by inoculation of embryo and RT-PCR detection from diseased chickens at different farms in Zhanjiang, Guangdong province in 2013.We denoted these two strains of IBV as CK/CH/GD/ZJ10/2013 and CK/CH/GD/ZJ11/2013, respectively.Analysis of the S1 gene sequences from these two isolated strains showed that the S1 gene of CK/CH/GD/ZJ10/2013 was 1 626 bp, which encoded 542 amino acids with a cleavage site sequence of NRFRR, while the S1 gene of CK/CH/GD/ZJ11/2013 was 1620 bp, encoded 540 amino acids with a cleavage site sequence of HRRRR.Phylogenetic analysis revealed that these two isolated strains were clustered into genetic groups Ⅲ and Ⅰ(QX-like type), respectively.Homology analysis demonstrated that nucleotide and deduced amino acid sequence homologies between these two isolated strains were lower, while the homologies were higher among the same genotypes, however, the homologies were lower comparing with current vaccine strains such as H120 and H52, with which the nucleotide homologies ranged from 75.7% to 76.3% and the amino acid homologies ranged from 77.1% to 77.9%.Further analysis showed that the CK/CH/GD/ZJ10/2013 strain was formed from a recombination event between CK/CH/GX/NN11-3 and GX-NN-6.Taken together, this study provided valuable insight into prevention and control of IBV infection in Guangdong province. 相似文献