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1.
Mai YAMAMOTO Takashige KASHIMOTO Ping TONG Jianbo XIAO Michiko SUGIYAMA Miyuki INOUE Rie MATSUNAGA Kohei HOSOHARA Kazue NAKATA Kenji YOKOTA Keiji OGUMA Koichiro YAMAMOTO 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2015,77(7):823-828
Vibrio vulnificus is the causative agent of primary septicemia, wound
infection and gastroenteritis in immunocompromised people. In this study, signature-tagged
mutagenesis (STM) was applied to identify the virulence genes of V.
vulnificus. Using STM, 6,480 mutants in total were constructed and divided into
81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool
was intraperitoneally injected into iron-overloaded mice, and in vivo
surviving mutants were collected from blood samples from the heart (OUTPUT pools). From
the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the
tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated
mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes,
were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than
1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000.
Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase,
UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine
cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized
with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a
transposon, they were protected against V. vulnificus infection. In this
study, we demonstrated that the STM method can be used to search for the virulence genes
of V. vulnificus. 相似文献
2.
为明确天津市某猪场保育猪疑似发生猪链球菌病的病原菌及其致病力等生物学特性,将猪场一头发病猪扑杀,无菌采集病料于哥伦比亚血琼脂平板上划线培养,对分离的疑似菌株进行形态学观察、生化试验、16S rDNA序列分析和血清型鉴定,确定分离菌株为猪链球菌8型,命名为TJS31。药敏试验结果显示,TJS31株对8种受试药物耐药,4种受试药物中介,1种受试药物敏感。致病性试验结果显示,TJS31株经腹腔接种昆明小鼠,24 h内可以引起小鼠出现精神萎靡、扎堆、呼吸急促、发抖、运动迟缓、死亡等,LD50为1.8×108 CFU/mL。毒力基因检测发现TJS31的毒力基因型为sly+/gdh+/orf2+/gapdh+,不携带mrp、epf和fbps基因。研究结果丰富了我国猪链球菌病病原的生物学资料,为猪链球菌8型的防控提供了依据,也为深入开展猪链球菌8型的致病机制和耐药机理奠定了基础。 相似文献
3.
从江苏省某屠宰场猪的扁桃体中分离到1株细菌,通过培养特性、菌体形态、菌落形态、染色特性、生化试验以及荚膜多糖(cps)基因的PCR检测,确定为猪链球菌2型,命名为HA0609。本试验针对猪链球菌7种主要毒力因子——谷氨酸脱氢酶(gdh)、溶菌酶释放蛋白(mrp)、胞外因子(epf)、溶血素(sly)、纤连蛋白/血纤蛋白原结合蛋白(fpbs)、次黄嘌呤核苷酸脱氢酶(impdh)及毒力相关序列orf2,进行PCR检测。与已知强毒株比较,该菌株2种主要毒力因子sly和epf均为阴性。动物试验显示HA0609对猪、兔和Balb/c鼠均无致病性。 相似文献
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5.
Vanier G Sekizaki T Domínguez-Punaro MC Esgleas M Osaki M Takamatsu D Segura M Gottschalk M 《Veterinary microbiology》2008,127(3-4):417-424
Streptococcus suis, a major pathogen of swine, is an emerging zoonotic agent which causes meningitis and septic shock. In this study, we investigated the ability of S. suis mutant strain (SRTDeltaA) lacking the sortase A gene (srtA) to interact with host cells and extracellular matrix (ECM) proteins, as well as its virulence in a mouse infection model. We demonstrated that mutant SRTDeltaA had reduced capacity to adhere to and invade porcine brain microvascular endothelial cells compared to the wild-type strain. In addition, mutant SRTDeltaA also showed significantly less adherence to plasma fibronectin, cellular fibronectin and collagen type I. However, disruption of srtA had little effect on the virulence of S. suis in a mouse intraperitoneal model of infection. These results indicate that surface proteins anchored by sortase A are required for a normal level of bacterial binding. However, other factors may also be important for S. suis virulence and interaction with host tissues. 相似文献
6.
现有的布鲁菌减毒活疫苗存在一定毒力,且野强毒株和减毒活疫苗株间缺少可供鉴别的抗原,导致在血清学检测上自然感染与疫苗接种很难区分,限制了现有的减毒活疫苗的广泛应用.本文拟对布鲁菌的减毒活疫苗株S2进行遗传改造,克服上述缺陷.本研究利用同源重组的方法,得到了布鲁菌S2株omp10基因缺失株.分别用基因缺失株和疫苗株感染小鼠,比较基因缺失株小鼠体内的存活能力.结果成功构建了布鲁菌S2株omp10基因缺失株,动物试验结果表明,基因缺失株仍能在小鼠体内存活,具备作为减毒活疫苗的特性.与原始S2株比较,基因缺失株的感染力进一步减弱.表明omp10基因在布鲁菌的毒力及体内生存方面发挥了作用,为基因标记疫苗的研制奠定了基础. 相似文献
7.
从全国部分猪场采集疑似猪链球菌感染病例脑样品20份进行细菌分离,成功分离到10株细菌,通过猪链球菌及血清类型鉴定,最终确定其中一株为2型猪链球菌。应用猪链球菌7种主要毒力因子特异性基因扩增检测方法检测所分离到的2型猪链球菌的毒力因子分布情况,并应用小鼠攻毒试验对其致病性进行观察研究。结果表明:分离的2型猪链球菌具备7种毒力因子;动物试验表明SS2能引起小鼠的急性败血症及脑膜炎;细菌回归试验结果表明,试验组死亡小鼠的脑、心脏、肝脏、脾脏、肺脏、肾脏均有细菌定植,且能分离出攻毒菌。此分离菌株的研究为研制2型猪链球菌病疫苗奠定了基础。 相似文献
8.
为了解血清2型猪链球菌(S.suis2)河南分离株的毒力基因型及cps2j全基因突变情况,本研究扩增S.suis2毒力基因gdh、cps2j、mrp、ef 、sly、orf2和fbps,并对s.suis2的cps2j进行全基因序列测定和同源性分析.结果表明,gdh、cps2j、mrp、ef、sly、orf2和fbps基因在8个S.suis2分离株中的检出率分别为100％(8/8)、100％(8/8)、62.5％(5/8)、75％(6/8)、87.5％(7/8)、100％(8/8)和100％(8/8);其中cps2j全基因核苷酸及推导的氨基酸序列与来源不同的S.suis2分离株同源性分别达98％和97％以上,以该基因构建的系统发育树表明8株S.suis2河南分离株与国内外不同参考株同源性高,亲缘关系密切. 相似文献
9.
Kerro Dego O Prado ME Chen X Luther DA Almeida RA Oliver SP 《Veterinary microbiology》2011,151(3-4):379-385
Streptococcus uberis is an important mastitis pathogen that affects dairy cows worldwide. In spite of the economic impact caused by the high prevalence of S. uberis intramammary infections (IMI) in many well-managed dairy herds, pathogenic strategies and associated virulence factors of S. uberis are not well understood. It has been shown that S. uberis attaches to and internalizes into mammary epithelial cells and can survive inside cells for extended periods of time. We hypothesize that early attachment to and internalization into mammary epithelial cells is a critical step for the establishment of intramammary infection. The aim of this study is to identify and characterize chromosomally encoded virulence factors of S. uberis that allow early bacterial attachment to and internalization into mammary epithelial cells. A common approach used to identify virulence factors is by generating random insertion mutants that are defective in adherence to and internalization into mammary epithelial cells using pGh9:ISS1 mutagenesis system. A random insertion mutant library of S. uberis strain UT888 was created using a thermo-sensitive plasmid pGh9:ISS1 carrying ISS1 insertion sequence. Integration of the insertion sequence into the chromosome of these mutant clones was confirmed by PCR and Southern blot. Southern blot analysis of mutant clones also showed that insertional integration was random. Of 1000 random chromosomal insertion mutants of S. uberis strain UT888 screened, 32 had significantly reduced ability to adhere to and internalize into mammary epithelial cells. Chromosomal mapping of insertion sequence integration sites in some of these defective mutants showed integration into penicillin binding protein 2A (pbp2A), sensor histidine kinase, tetR family regulatory protein, phosphoribosylaminoimidazole carboxylase catalytic subunit (purE), lactose phosphotransferase, phosphoribosylamine glycine ligase (purD), and other genes involved in metabolic activities. These proteins may have a significant role in early bacterial colonization of the mammary gland during infection. 相似文献
10.
Nuttapone SANGKANJANAVANICH Tsutomu KAKUDA Yasunori SUZUKI Yukako SASAKI Shinji TAKAI 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2021,83(8):1182
Rhodococcus equi is a Gram-positive facultative intracellular bacterium that causes pyogranulomatous pneumonia in foals and immunocompromised people. In the present study, signature-tagged transposon mutagenesis was applied for the negative selection of R. equi mutants that cannot survive in vivo. Twenty-five distinguishable plasmid-transposon (plasposon) vectors by polymerase chain reaction (PCR), each containing a unique oligonucleotide tag, were constructed and used to select the transposon mutants that have in vivo fitness defects using a mouse systemic infection model. Of the 4,560 transposon mutants, 102 mutants were isolated via a real-time PCR-based screening as the mutants were unable to survive in the mouse model. Finally, 50 single transposon insertion sites were determined via the self-cloning strategy. The insertion of the transposon was seen on the virulence plasmid in 15 of the 50 mutants, whereas the remaining 35 mutants had the insertion of transposon on the chromosome. The chromosomal mutants contained transposon insertions in genes involved in cellular metabolism, DNA repair and recombination, gene regulation, non-ribosomal peptide synthesis, and unknown functions. Additionally, seven of the chromosomal mutants showed a reduced ability to multiply in the macrophages in vitro. In this study, we have identified several biosynthetic pathways as fitness factors associated with the growth within macrophages and survival in mice. 相似文献
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12.
Yanlong Pei Valeria Parreira Vivian M. Nicholson John F. Prescott 《Canadian journal of veterinary research》2007,71(1):1-7
Rhodococcus equi can cause severe or fatal pneumonia in foals as well as in immunocompromised animals and humans. Its ability to persist in macrophages is fundamental to how it causes disease, but the basis of this is poorly understood. To examine further the general application of a recently developed system of targeted gene mutation and to assess the importance of different genes in resistance to innate immune defenses, we disrupted the genes encoding high-temperature requirement A (htrA), nitrate reductase (narG), peptidase D (pepD), phosphoribosylaminoimidazole-succinocarboxamide synthase (purC), and superoxide dismutase (sodC) in strain 103 of R. equi using a double-crossover homologous recombination approach. Virulence testing by clearance after intravenous injection in mice showed that the htrA and narG mutants were fully attenuated, the purC and sodC mutants were unchanged, and the pepD mutant was slightly attenuated. Complementation with the pREM shuttle plasmid restored the virulence of the htrA and pepD mutants but not that of the narG mutant. A single-crossover mutation approach was simpler and faster than the double-crossover homologous recombination technique and was used to obtain mutations in 6 other genes potentially involved in virulence (clpB, fadD8, fbpB, glnA1, regX3, and sigF). These mutants were not attenuated in the mouse clearance assay. We were not able to obtain mutants for genesfurA, galE, and sigE using the single-crossover mutation approach. In summary, the targeted-mutation system had general applicability but was not always completely successful, perhaps because some genes are essential under the growth conditions used or because the success of mutation depends on the target genes. 相似文献
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Bonifait L Vaillancourt K Gottschalk M Frenette M Grenier D 《Veterinary microbiology》2011,148(2-4):333-340
Streptococcus suis is a major swine pathogen that is responsible for severe infections such as meningitis, endocarditis, and septicemia. S. suis is also recognized as a zoonotic agent and expresses several virulence factors. The recently identified subtilisin-like protease (SspA) of S. suis plays an important role in the pathogenicity of this bacterium in animal models. The objective of the present study was to clone, purify, and characterize the SspA of serotype 2 S. suis P1/7. The SSU0757 gene encoding SspA was amplified and a 4798-bp DNA fragment was obtained. It was cloned into the expression plasmid pBAD/HisB and then inserted into Escherichia coli to overproduce the protein. The recombinant protease was purified by chromatography procedures and showed a molecular weight of 170 kDa by SDS-PAGE. Its activity was optimal at pH 7 and at temperatures ranging from 25°C to 37°C. It had a high specificity for the chromogenic substrate succinyl-Ala-Ala-Pro-Phe-pNa while specific inhibitors of serine proteases inhibited its activity. In addition to degrading gelatin, the protease hydrolyzed the Aα chain of fibrinogen, which prevented fibrin formation by thrombin. The recombinant subtilisin-like protease also showed toxicity towards brain microvascular endothelial cells. Lastly, sera from pigs infected with S. suis reacted with the recombinant SspA, indicating that it is produced during infections. In conclusion, the SspA of S. suis shared similarities with subtilisin-like proteases produced by other pathogenic streptococci and may contribute to the pathogenic process of S. suis infections. 相似文献
15.
Vermoote M Vandekerckhove TT Flahou B Pasmans F Smet A De Groote D Van Criekinge W Ducatelle R Haesebrouck F 《Veterinary research》2011,42(1):51
ABSTRACT: Helicobacter (H.) suis has been associated with chronic gastritis and ulcers of the pars oesophagea in pigs, and with gastritis, peptic ulcer disease and gastric mucosa-associated lymphoid tissue lymphoma in humans. In order to obtain better insight into the genes involved in pathogenicity and in the specific adaptation to the gastric environment of H. suis, a genome analysis was performed of two H. suis strains isolated from the gastric mucosa of swine. Homologs of the vast majority of genes shown to be important for gastric colonization of the human pathogen H. pylori were detected in the H. suis genome. H. suis encodes several putative outer membrane proteins, of which two similar to the H. pylori adhesins HpaA and HorB. H. suis harbours an almost complete comB type IV secretion system and members of the type IV secretion system 3, but lacks most of the genes present in the cag pathogenicity island of H. pylori. Homologs of genes encoding the H. pylori neutrophil-activating protein and γ-glutamyl transpeptidase were identified in H. suis. H. suis also possesses several other presumptive virulence-associated genes, including homologs for mviN, the H. pylori flavodoxin gene, and a homolog of the H. pylori vacuolating cytotoxin A gene. It was concluded that although genes coding for some important virulence factors in H. pylori, such as the cytotoxin-associated protein (CagA), are not detected in the H. suis genome, homologs of other genes associated with colonization and virulence of H. pylori and other bacteria are present. 相似文献
16.
Domínguez-Bernal G Tierrez A Bartolomé A Martínez-Pulgarín S Salguero FJ Antonio Orden J de la Fuente R 《Veterinary microbiology》2008,130(3-4):298-311
Live attenuated Salmonella enterica strains have been extensively studied as potential vectors for the oral delivery of heterologous antigens. Due to its ability to target immune cells, its specific mechanism for crossing the intestinal barrier, and its swine-restricted tropism, S. enterica subspecies enterica serovar Choleraesuis (S. Choleraesuis) has attracted a great deal of interest for the production of bacterial-based oral carriers specifically adapted to swine. In this study, two mutants of S. Choleraesuis were constructed and their attenuation and intracellular fate analysed with the purpose of engineering new attenuated live strains with improved properties as oral vaccine carriers. Those strains harboured a specific deletion either within the phoP or rpoS genes, which encode virulence-related regulators in S. Typhimurium. In comparison to the wild-type parental S. Choleraesuis, the mutant strains, especially DeltaphoP, were extremely low in virulence in the murine model and in the natural host, the pig. Moreover, when compared with a commercial live vaccine strain, SC-54, the two mutants showed a higher level of attenuation in mice and DeltaphoP also in pigs. In addition, DeltarpoS and DeltaphoP presented a proliferation and survival phenotype within swine intestinal primary fibroblast and macrophage cell cultures, respectively. Collectively, the present results indicate that the DeltarpoS and DeltaphoP strains of S. Choleraesuis gather adequate features to be potential candidates for vaccine vectors for the specific delivery of heterologous antigens adapted to pigs. 相似文献
17.
Slater JD Allen AG May JP Bolitho S Lindsay H Maskell DJ 《Veterinary microbiology》2003,93(3):197-206
Genetic tools for studying streptococci are much less sophisticated than those that are available for many other bacterial genera. In this paper, we describe the development of a transposon mutagenesis system that we have used to mutate two important veterinary streptococci, Streptococcus equi and Streptococcus suis. The system uses a temperature-sensitive suicide vector to deliver Tn917 via electroporation, transposing Tn917 into the chromosomal DNA of the two streptococci. The transposon insertions can be rescued from the streptococcal chromosomes by plasmid rescue and selection in E. coli, with subsequent insertion site analysis by DNA sequencing. Transposition appeared to have occurred in an essentially random fashion when chromosomal DNA of S. suis and S. equi mutants was analysed by Southern blotting. However, when analysis of 60 S. equi mutants was carried out using the S. equi genome sequence database, 60% of transposon insertions had occurred within a 15 kb region of the genome whereas the other insertions appeared to have occurred essentially randomly. This finding suggests that Southern blot analysis for assessing the randomness of transposon libraries may need to be interpreted with caution. However, this observation notwithstanding, the Tn917 based system described in this paper will facilitate the study of S. suis and S. equi. 相似文献
18.
Shizhong Geng Xinan Jiao Paul Barrow Zhiming Pan Xiang Chen 《Veterinary microbiology》2014,168(2-4):388-394
Salmonella Gallinarum biovar Pullorum (S. Gallinarum biovar Pullorum) is the causative agent of pullorum disease (PD) in chickens which results in considerable economic losses to the poultry industries in developing countries. PCR-Signature Tagged Mutagenesis was used to identify virulence determinants of S. Gallinarum biovar Pullorum and novel attenuated live vaccine candidates for use against this disease. A library of 1800 signature-tagged S. Gallinarum biovar Pullorum mutants was constructed and screened for virulence-associated genes in chickens. The attenuation of 10 mutants was confirmed by in vivo and in vitro competitive index (CI) studies. The transposons were found to be located in SPI-1 (2/10 mutants), SPI-2 (3/10), the virulence plasmid (1/10) and non-SPI genes (4/10). One highly attenuated spiC mutant persisted in spleen and liver for less than 10 days and induced high levels of circulating antibody and protective immunity against oral challenge in young broiler chickens. The spiC mutant is a potential new vaccine candidate for use with chickens against this disease. 相似文献
19.
Experimental infection of specific pathogen free piglets with French strains of Streptococcus suis capsular type 2. 总被引：3，自引：0，他引：3
F Berthelot-Hérault R Cariolet A Labbé M Gottschalk J Y Cardinal M Kobisch 《Canadian journal of veterinary research》2001,65(3):196-200
A standardized model of Streptococcus suis type 2 infection in specific-pathogen-free piglets, housed in high-security barns, was used to compare the virulence of 3 French field strains of S. suis serotype 2 isolated from tonsils of a healthy pig (strain 65) or from diseased pigs (meningitis, strain 166', or septicemia, strain 24). In one of the 2 trials, 7-week-old pigs, in 3 groups of 8, were inoculated intravenously with 2 x 10(8) colony-forming units of S. suis type 2. In each group, 1 uninfected animal was a sentinel. Eight animals were also used as negative control group. The experiment was repeated under similar conditions with strains 65 and 166'. Virulence differed markedly among these S. suis strains when clinical signs, zootechnical performances, lesions, and bacteriological data were analyzed. Strain 65 did not induce clinical signs in inoculated pigs. In contrast, pigs infected with the other 2 strains exhibited clinical signs and typical lesions of S. suis type 2 infections. Differences in virulence were also observed between the 2 virulent strains. Sentinel animals exhibited the same manifestations as those recorded in inoculated piglets. Results were similar in the second trial, indicating that under the present experimental conditions, results were reproducible. The standardized conditions described in this study could be a useful tool to further study about the S. suis infection. 相似文献
20.
Streptococcus Suis: Past and Present 总被引：109，自引：0，他引：109
Staats J.J. Feder I. Okwumabua O. Chengappa M.M. 《Veterinary research communications》1997,21(6):381-407
Staats, J.J., Feder, I., Okwumabua, O. and Chengappa, M.M., 1997. Streptococcus suis: past and present. Veterinary Research Communications, 21 (6), 381-407Steptococcus suis is a Gram-positive, facultatively anaerobic coccus that has been implicated as the cause of a wide range of clinical disease syndromes in swine and other domestic animals. In swine, the disease has spread worldwide but is more prevalent in countries with intensive swine management practices. The disease syndromes caused by S. suis in swine include arthritis, meningitis, pneumonia, septicaemia, endocarditis, polyserositis, abortions and abscesses. S. suis has also been implicated in disease in humans, especially among abattoir workers and swine and pork handlers. In humans, S. suis type 2 can cause meningitis, which may result in permanent hearing loss, septicaemia, endocarditis and death. The pathogenic mechanism of S. suis is not well defined. Several virulence factors have been identified, but their roles in pathogenesis and disease have not been well elucidated. Much work is in progress on characterization of virulence factors and mechanisms, with emphasis on the control of the disease. Because of the non-availability of suitable immunoprophylaxis, control of S. suis infection has depended mainly on the use of antimicrobials. 相似文献