选择性捕获鸡白痢沙门菌在巨噬细胞内的转录序列
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摘要
鸡白痢沙门菌呈世界范围分布。目前,在西方部分发达国家鸡白痢已经被消灭或基本消灭,而在国内该病的危害仍严重。鸡白痢沙门菌多造成2-3周龄雏鸡死亡,成年鸡则可带菌而无临床症状。感染后存活的鸡常表现生殖系统病变,引起蛋鸡产蛋量下降,并污染种蛋,造成巨大的经济损失。该病既可垂直传播,又可水平传播,造成严重的危害。探究鸡白痢沙门菌潜在的致病基因,了解其致病机理,对于寻求新的有效防治措施具有重要的意义。
随着各种分子生物学和免疫学研究手段的出现,人们对病原体在感染宿主过程中转录的基因序列的研究也逐渐深入。选择性捕获转录序列(selective capture of transcribed sequences,SCOTS)技术在研究病原体体内转录序列方面有着独特的优势。本研究比较了鸡白痢沙门菌在鸡体内和体外巨噬细胞系中的存活消亡趋势,以鸡巨噬细胞系HD-11为侵染模型,用SCOTS技术捕获该菌在巨噬细胞中的转录序列,并初步分析了其中一个序列在侵染细胞过程中的转录水平。
1鸡白痢沙门菌体内外侵染实验
为了探究鸡白痢沙门菌S06004在与鸡体单核吞噬细胞系统和体外巨噬细胞系的相互作用的过程中,细菌定殖、复制的变化趋势,进行了细菌的体内侵染实验和体外巨噬细胞侵染实验。体内侵染实验中,以口服和肌注两种方式感染9日龄非免疫海兰白蛋鸡,感染后第三天吞噬细胞中的细菌量都达到最高点,第九天下降到每克组织中带菌量不足500 cfu,且这种状态能够持续至少5天。在体内侵染实验中,细菌的定殖趋势为先发生复制增殖、后消减至很低水平,并且低水平的带菌状态会维持一段时间。同时在体外侵染实验中,不论给予细菌1h还是2.5h的作用时间,进入巨噬细胞内的细菌量在增殖3h左右时都达到最高点,消减到较低的水平后,能够维持一段时间。在体外实验中胞内的细菌同样具有先增殖后消减,并维持一段低水平细菌携带率的平台期的存活趋势。鸡白痢沙门菌S06004株对鸡巨噬细胞系HD-11的体外侵染实验结果和体内吞噬细胞侵染实验结果基本吻合,故采用鸡白痢沙门菌侵染鸡巨噬细胞系HD-11可以在一定程度上模拟细菌体内感染时与内脏器官吞噬细胞系统的相互作用。
2选择性捕获鸡白痢沙门菌在巨噬细胞内的转录序列
应用SCOTS方法鉴定鸡白痢沙门菌S06004株在感染鸡巨噬细胞系HD-11过程中细菌表达的基因。根据细菌体内和体外侵染实验的研究结果,选用鸡白痢沙门菌S06004侵染鸡巨噬细胞系HD-11,以模拟细菌体内侵染吞噬细胞过程,通过SCOTS技术对转录序列cDNAs的选择性捕获,检测了细菌在与巨噬细胞相互作用过程中毒力基因以及相关调控基因的表达,共得到16个序列。除了一个未知序列,这些体内转录序列包括毒力相关的III型分泌系统中分泌素蛋白和Ⅰ型分泌系统中外膜蛋白的编码基因、代谢相关的敏感激酶和核苷水解酶的编码基因、运输功能基因、一些质粒编码基因以及环境适应性功能基因等。
3实时荧光定量PCR动态分析鸡白痢沙门菌体内转录序列7-6在侵染过程中的转录水平
根据SCOTS技术捕获得到的体内转录序列7-6,设计了针对其特异性检测的一对引物,同时设计鸡白痢沙门菌16sRNA基因的引物作为内参。提取鸡白痢沙门菌S06004株在侵染细胞过程中各个阶段的总RNA,反转录成cDNAs,利用荧光定量PCR比较基因在不同阶段的表达水平的差异。结果表明:捕获序列7-6在体外培养时不表达;在侵染初期表达量最高;在细菌胞内增殖阶段,基因的表达量先上升后下降,在增殖3h时表达量最高,在增殖5h后,7-6序列的表达下降到很低的水平。基因的转录变化与细菌的巨噬细胞侵染过程密切关联。
Salmonella pullorum is distributed in worldwide. Nowadays, the pullorum disease has been eliminated or partially eliminated in the developed countries, however, there it is an important avain disease causing serious losses in the poultry industry in China. Salmonella pullorum mainly causes the death of 2-3 weeks old chickens, and the adult chickens appear as bacteria carrier without clinical symptoms. Most of the survival chickens show the reproductive disorders, including decreased egg production, production of contaminated eggs, which cause severe economic losses. The disease can spread vertically, as well as transmit horizontally, which is the main reason for the reproductive disorders. It is important to explore the novel potential virulence-related gene and develop new strategies for the control of the pullorum disease.
With the development of various molecular biological and immunological research methods, the gene expression of pathogens in infection process can be evaluated. It is notable that identification of transcribed sequences of pathogens in vivo is very important for exploring the pathogenetic mechanism. SCOTS is applicable to many kinds of bacteria from which transcribed sequences can be isolated. In this study, it was carried out an invasion test both in chicken model and macrophage cell line to compare the growth property of Salmonella pullorum in vivo and in vitro. In avian macrophage cell line HD-11, the bacterial transcribed sequences of S. pullorum was captured using SCOTS and the expression develop captured sequence 7-6 was analyzed by real-time quantitative PCR.
1 Comparison of invasion process in vivo and in vitro of Salmonella pullorum
In order to investigate the specific infection of Salmonella pullorum in chicken model and in the avian macrophages cell line HD-11 in vitro, 9-days-old chickens were inoculated orally or intramuscularly with S. pullorum S06004. The bacteria within macrophages of spleen and liver can reach the highest point at the third day after inocularion, and would be less than 500 cfu/g at the ninth day, which can be persist more than 5 days. The result of invasion in vivo indicatied that bacteria in macrophages were proliferated at first, then reduced to a lower level which can last some time. Meanwhile bacteria infected the macrophage in vitro, intracellular bacteria reached the highest point in 3h at proliferation stage, then reduced to a lower level which also lasted a few hours. So the in vitro invasion of HD-11 cells by S. pullorum can simulate the invasion in vivo in some extent.
2 Identification of Salmonella pullorum genes expressed within macrophages by selective capture of transcribed sequences
The selective capture of transcribed sequences(SCOTS) was used to identify Salmonella pullorum S06004 genes expressed during the process of infection of avian macrophage cell line HD-11. According to bacterial invasion in vivo and in vitro, the invasion of Salmonella pullorum S06004 in avian macrophage cell line HD-11 can simulate in vivo. The transcribed sequences were captured, and identified the virulence genes and related regulation genes expressed within macrophages. 16 sequences were obtained. Except an unknown sequence, these sequences included the coding genes of virulence-related secreted proteins in type III secretion system, outer membrane protein coding genes of typeⅠsecretion system, sensitive kinase related to metabolism and nucleoside hydrolase coding genes, transport genes, and some plasmid genes, environmental adaptability functional gene.
3 Kinetic detection of the expression level of a captured sequence 7-6 during the infection process by real-time quantitative PCR
It was designed a pair of primers of the captured sequence 7-6 of S. pullorum for the specific detection and the primers of 16sRNA gene were used as internal control. The total RNA of infected cells at different time was extracted, and reverse transcribed into cDNAs, and then the gene expression levels of 7-6 was monitored using real-time PCR. The results showed: the captured sequence 7-6 was not expressed in vitro, and then there are the highest expression in early invasion, at the intracellular bacteria proliferation phase, the expression level reached to the highest at 3h proliferation, and then decreased to a very low level after 5h proliferation. The changes in transcriptional level of 7-6 were related to the infection process of macrophages.
随着各种分子生物学和免疫学研究手段的出现,人们对病原体在感染宿主过程中转录的基因序列的研究也逐渐深入。选择性捕获转录序列(selective capture of transcribed sequences,SCOTS)技术在研究病原体体内转录序列方面有着独特的优势。本研究比较了鸡白痢沙门菌在鸡体内和体外巨噬细胞系中的存活消亡趋势,以鸡巨噬细胞系HD-11为侵染模型,用SCOTS技术捕获该菌在巨噬细胞中的转录序列,并初步分析了其中一个序列在侵染细胞过程中的转录水平。
1鸡白痢沙门菌体内外侵染实验
为了探究鸡白痢沙门菌S06004在与鸡体单核吞噬细胞系统和体外巨噬细胞系的相互作用的过程中,细菌定殖、复制的变化趋势,进行了细菌的体内侵染实验和体外巨噬细胞侵染实验。体内侵染实验中,以口服和肌注两种方式感染9日龄非免疫海兰白蛋鸡,感染后第三天吞噬细胞中的细菌量都达到最高点,第九天下降到每克组织中带菌量不足500 cfu,且这种状态能够持续至少5天。在体内侵染实验中,细菌的定殖趋势为先发生复制增殖、后消减至很低水平,并且低水平的带菌状态会维持一段时间。同时在体外侵染实验中,不论给予细菌1h还是2.5h的作用时间,进入巨噬细胞内的细菌量在增殖3h左右时都达到最高点,消减到较低的水平后,能够维持一段时间。在体外实验中胞内的细菌同样具有先增殖后消减,并维持一段低水平细菌携带率的平台期的存活趋势。鸡白痢沙门菌S06004株对鸡巨噬细胞系HD-11的体外侵染实验结果和体内吞噬细胞侵染实验结果基本吻合,故采用鸡白痢沙门菌侵染鸡巨噬细胞系HD-11可以在一定程度上模拟细菌体内感染时与内脏器官吞噬细胞系统的相互作用。
2选择性捕获鸡白痢沙门菌在巨噬细胞内的转录序列
应用SCOTS方法鉴定鸡白痢沙门菌S06004株在感染鸡巨噬细胞系HD-11过程中细菌表达的基因。根据细菌体内和体外侵染实验的研究结果,选用鸡白痢沙门菌S06004侵染鸡巨噬细胞系HD-11,以模拟细菌体内侵染吞噬细胞过程,通过SCOTS技术对转录序列cDNAs的选择性捕获,检测了细菌在与巨噬细胞相互作用过程中毒力基因以及相关调控基因的表达,共得到16个序列。除了一个未知序列,这些体内转录序列包括毒力相关的III型分泌系统中分泌素蛋白和Ⅰ型分泌系统中外膜蛋白的编码基因、代谢相关的敏感激酶和核苷水解酶的编码基因、运输功能基因、一些质粒编码基因以及环境适应性功能基因等。
3实时荧光定量PCR动态分析鸡白痢沙门菌体内转录序列7-6在侵染过程中的转录水平
根据SCOTS技术捕获得到的体内转录序列7-6,设计了针对其特异性检测的一对引物,同时设计鸡白痢沙门菌16sRNA基因的引物作为内参。提取鸡白痢沙门菌S06004株在侵染细胞过程中各个阶段的总RNA,反转录成cDNAs,利用荧光定量PCR比较基因在不同阶段的表达水平的差异。结果表明:捕获序列7-6在体外培养时不表达;在侵染初期表达量最高;在细菌胞内增殖阶段,基因的表达量先上升后下降,在增殖3h时表达量最高,在增殖5h后,7-6序列的表达下降到很低的水平。基因的转录变化与细菌的巨噬细胞侵染过程密切关联。
Salmonella pullorum is distributed in worldwide. Nowadays, the pullorum disease has been eliminated or partially eliminated in the developed countries, however, there it is an important avain disease causing serious losses in the poultry industry in China. Salmonella pullorum mainly causes the death of 2-3 weeks old chickens, and the adult chickens appear as bacteria carrier without clinical symptoms. Most of the survival chickens show the reproductive disorders, including decreased egg production, production of contaminated eggs, which cause severe economic losses. The disease can spread vertically, as well as transmit horizontally, which is the main reason for the reproductive disorders. It is important to explore the novel potential virulence-related gene and develop new strategies for the control of the pullorum disease.
With the development of various molecular biological and immunological research methods, the gene expression of pathogens in infection process can be evaluated. It is notable that identification of transcribed sequences of pathogens in vivo is very important for exploring the pathogenetic mechanism. SCOTS is applicable to many kinds of bacteria from which transcribed sequences can be isolated. In this study, it was carried out an invasion test both in chicken model and macrophage cell line to compare the growth property of Salmonella pullorum in vivo and in vitro. In avian macrophage cell line HD-11, the bacterial transcribed sequences of S. pullorum was captured using SCOTS and the expression develop captured sequence 7-6 was analyzed by real-time quantitative PCR.
1 Comparison of invasion process in vivo and in vitro of Salmonella pullorum
In order to investigate the specific infection of Salmonella pullorum in chicken model and in the avian macrophages cell line HD-11 in vitro, 9-days-old chickens were inoculated orally or intramuscularly with S. pullorum S06004. The bacteria within macrophages of spleen and liver can reach the highest point at the third day after inocularion, and would be less than 500 cfu/g at the ninth day, which can be persist more than 5 days. The result of invasion in vivo indicatied that bacteria in macrophages were proliferated at first, then reduced to a lower level which can last some time. Meanwhile bacteria infected the macrophage in vitro, intracellular bacteria reached the highest point in 3h at proliferation stage, then reduced to a lower level which also lasted a few hours. So the in vitro invasion of HD-11 cells by S. pullorum can simulate the invasion in vivo in some extent.
2 Identification of Salmonella pullorum genes expressed within macrophages by selective capture of transcribed sequences
The selective capture of transcribed sequences(SCOTS) was used to identify Salmonella pullorum S06004 genes expressed during the process of infection of avian macrophage cell line HD-11. According to bacterial invasion in vivo and in vitro, the invasion of Salmonella pullorum S06004 in avian macrophage cell line HD-11 can simulate in vivo. The transcribed sequences were captured, and identified the virulence genes and related regulation genes expressed within macrophages. 16 sequences were obtained. Except an unknown sequence, these sequences included the coding genes of virulence-related secreted proteins in type III secretion system, outer membrane protein coding genes of typeⅠsecretion system, sensitive kinase related to metabolism and nucleoside hydrolase coding genes, transport genes, and some plasmid genes, environmental adaptability functional gene.
3 Kinetic detection of the expression level of a captured sequence 7-6 during the infection process by real-time quantitative PCR
It was designed a pair of primers of the captured sequence 7-6 of S. pullorum for the specific detection and the primers of 16sRNA gene were used as internal control. The total RNA of infected cells at different time was extracted, and reverse transcribed into cDNAs, and then the gene expression levels of 7-6 was monitored using real-time PCR. The results showed: the captured sequence 7-6 was not expressed in vitro, and then there are the highest expression in early invasion, at the intracellular bacteria proliferation phase, the expression level reached to the highest at 3h proliferation, and then decreased to a very low level after 5h proliferation. The changes in transcriptional level of 7-6 were related to the infection process of macrophages.
引文
[1]陆承平.兽医微生物(第三版).北京,中国农业出版社, 2002.
[2] Altmeyer RM, J K McNern, J C Bossio, I Rosenshine, B B Finlay, and J E Galan. Cloning and molecular characterization of a gene involved in Salmonella adherence and invasion of cultured epithelial cells. Mol Microbiol, 1993, 7: 89-98.
[3] Barrow PA. The paratyphoid salmonella. Rev. Sci. Tech, 2000, 19: 351-375.
[4] Shivaprasad HL. Fowl typhoid and pullorum disease. Rev. Sci. Tech, 2000, 19: 405-424.
[5] Jones MA, Wigley P, Page KL, Hulme SD, Barrow PA. Salmonella enterica serovar Gallinarum requires the Salmonella pathogenicity island 2 type III secretion system but not the Salmonella pathogenicity island 1 type III secretion system for virulence in chickens. Infect. Immun, 2001, 69: 5471-5476.
[6] Cobb SP, McVicar CM, Davies RH, Ainsworth H. Fowl typhoid in caged layer birds. Vet. Rec, 2005, 157: 268.
[7] Parmar D, Davies R. Fowl typhoid in a small backyard laying flock. Vet. Rec, 2007, 160: 348.
[8] Wigley P, Berchieri Jr A, Page KL, Smith AL, Barrow PA. Salmonella enterica serovar Pullorum persists in splenic macrophages and in the reproductive tract during persistent, disease-free carriage in chickens. Infect Immun, 2001, 69: 7873-7879.
[9] Gewirtz AT, Rao AS, Simon Jr PO, Merlin D, Carnes D, Madara JL, Neish AS. Salmonella typhimurium induces epithelial IL-8 expression via Ca (2+)-mediated activation of the NF-kappaB pathway. J. Clin. Invest, 2000, 105: 79-92.
[10] Gewirtz AT, Simon Jr PO, Schmitt CK, Taylor LJ, Hagedorn CH, O’Brien AD, Neish AS, Madara JL. Salmonella typhimurium translocates flagellin across intestinal epithelia, inducing a proinflammatory response. J. Clin. Invest, 2001, 107: 99-109.
[11] Wallis TS, Galyov EE, et al. Molecular basis of Salmonella-induced enteritis. Mol Microbiol, 2000, 36: 997-1005.
[12] Wallis TS, Wood M, Watson P, Paulin S, Jones M, Galyov E. Sips, Sops, and SPIs but not stn influence Salmonella enteropathogenesis. Adv. Exp. Med. Biol, 1999, 473: 275-280.
[13] Zeng H, Carlson AQ, Guo Y, Yu Y, Collier-Hyams L S, Madara JL, Gewirtz AT, Neish AS. Flagellin is the major proinflammatory determinant of enteropathogenic Salmonella. J. Immunol, 2003, 171: 3668-3674.
[14] Smith AL and Beal R. The avian enteric immune system in health and disease. In: Davison F, Kaspers B, Schat KA (Eds), Avian Immunology. Academic Press, London, pp, 2008, 243-271.
[15] Smith AL, Beal R. The avian enteric immune system in health and disease. In: Davison F,Kaspers B, Schat KA (Eds), Avian Immunology. Academic Press, London, pp, 243-271.
[16] Withanage GS, Wigley P, Kaiser P, Mastroeni P, Brooks H, Powers C, Beal R, Barrow P, Maskell D, McConnell I. Cytokine and chemokine responses associated with clearance of a primary Salmonella enterica serovar Typhimurium infection in the chicken and in protective immunity to rechallenge. Infect Immun, 2005, 73: 5173-5182.
[17] Henderson SC, Bounous DI, Lee MD, et al. Early events in the pathogenesis of avian salmonellosis. Infect Immun, 1999, 67: 3580-3586.
[18] Kaiser P, Rothwell L, Galyov EE, Barrow PA, Burnside J, Wigley P. Differential cytokine expression in avian cells in response to invasion by Salmonella typhimurium, Salmonella enteritidis and Salmonella gallinarum. Microbiology, 2000, 146 (12): 3217-3226.
[19] Withanage GS, Kaiser P, Wigley P, Powers C, Mastroeni P, Brooks H, Barrow P, Smith A, Maskell D, McConnell I. Rapid expression of chemokines and proinflammatory cytokines in newly hatched chickens infected with Salmonella enterica serovar typhimurium. Infect Immun, 2004, 72: 2152-2159.
[20] Iqbal M, Philbin VJ, Withanage GS, Wigley P, Beal RK, Goodchild MJ, Barrow P, McConnell I, Maskell DJ, Young J, Bumstead N, Boyd Y, Smith AL. Identification and functional characterization of chicken toll-like receptor 5 reveals a fundamental role in the biology of infection with Salmonella enterica serovar typhimurium. Infect Immun, 2005, 73: 2344-2350.
[21] Withanage GS, Wigley P, Scott D, et al. Infection of the Reproductive Tract and Eggs with Salmonella enterica serovar Pullorum in the Chicken Is Associated with Suppression of Cellular Immunity at Sexual Maturity. Infect Immun, 2005, 73(5): 1443-1450.
[22] Berchieri A, Wigley P, Page K, Murphy CK, Barrow PA. Further studies on vertical transmission and persistence of Salmonella enterica serovar Enteritidis phage type 4 in chickens. Avian Pathol, 2001, 30: 297-310.
[23] Chan K, Baker S, Kim CC, Detweiler CS, Dougan G, Falkow S. Genomic comparison of Salmonella enterica serovars and Salmonella bongori by use of an S. enterica serovar typhimurium DNA microarray. J Bacteriol, 2003, 185: 553-563.
[24] Hughes S, Poh TY, Bumstead N, Kaiser P. Re-evaluation of the chicken MIP family of chemokines and their receptors suggests that CCL5 is the prototypic MIP family chemokine, and that different species have developed different repertoires of both the CC chemokines and their receptors. Dev Comp Immunol, 2007, 31: 72-86.
[25] Henderson SC, Bounous DI, Lee MD, et al. Early events in the pathogenesis of avian salmonellosis. Infect Immun, 1999, 67: 3580-3586.
[26] Wigley P, Jones MA, Barrow PA. Salmonella enterica serovar Pullorum requires the Salmonella pathogenicity island 2 type III secretion system for virulence and carriage in the chicken. Avian Pathol, 2002b, 31: 501-506.
[27] Collier-Hyams LS, Zeng H, Sun J, Tomlinson AD, Bao ZQ, Chen H, Madara JL, Orth K., Neish AS. Cutting edge: Salmonella AvrA effector inhibits the key proinflammatory, anti-apoptotic NF-kappa B pathway. J Immunol, 2002, 169: 2846-2850.
[28] Mastroeni P, Menager N. Development of acquired immunity to Salmonella. J. Med Microbiol, 2003, 52: 453-459.
[29] Barrow PA, Huggins MB, Lovell MA. Host specificity of Salmonella infection in chickens andmice is expressed in vivo primarily at the level of the reticuloendothelial system. Infect Immun, 1994, 62: 4602-4610.
[30] Hensel M. Salmonella pathogenicity island 2. Mol Microbiol, 2000, 36; 1015–1023.
[31] Cheminay C, Mohlenbrink A, Hensel M. Intracellular Salmonella inhibit antigen presentation by dendritic cells. J Immunol, 2005, 174: 2892-2899.
[32] Jones MA, Hulme SD, Barrow PA, Wigley P. The Salmonella pathogenicity island 1 and Salmonella pathogenicity island 2 type III secretion systems play a major role in pathogenesis of systemic disease and gastrointestinal tract colonization of Salmonella enterica serovar Typhimurium in the chicken. Avian Pathol, 2007, 36: 199-203.
[33] Jones MA, Wigley P, Page KL, Hulme SD, Barrow PA. Salmonella enterica serovar Gallinarum requires the Salmonella pathogenicity island 2 type III secretion system but not the Salmonella pathogenicity island 1 type III secretion system for virulence in chickens. Infect Immun, 2001, 69: 5471-5476.
[34] Kogut MH, Tellez GI, McGruder ED, Hargis BM, Williams JD, Corrier DE, DeLoach JR. Heterophils are decisive components in the early responses of chickens to Salmonella enteritidis infections. Microb Pathog, 1994, 16: 141-151.
[35] Wigley P, Hulme S, Rothwell L, Bumstead N, Kaiser P, Barrow P. Macrophages isolated from chickens genetically resistant or susceptible to systemic salmonellosis show magnitudinal and temporal differential expression of cytokines and chemokines following Salmonella enterica challenge. Infect Immun, 2006, 74: 1425-1430.
[36] Wigley P, Hulme SD, Bumstead N, Barrow PA. In vivo and in vitro studies of genetic resistance to systemic salmonellosis in the chicken encoded by the SAL1 locus. Microbes Infect, 2002a, 4: 1111-1120.
[37] Withanage GS, Kaiser P, Wigley P, Powers C, Mastroeni P, Brooks H, Barrow P, Smith A,Maskell D, McConnell I. Rapid expression of chemokines and proinflammatory cytokines in newly hatched chickens infected with Salmonella enterica serovar typhimurium. Infect Immun, 2004, 72: 2152-2159.
[38] Mariani P, Barrow PA, Cheng HH, Groenen MM, Negrini R, Bumstead N. Localization to chicken chromosome 5 of a novel locus determining salmonellosis resistance. Immunogenetics, 2001, 53: 786-791.
[39] Wigley P, Hulme S, Rothwell L, Bumstead N, Kaiser P, Barrow P. Macrophages isolated from chickens genetically resistant or susceptible to systemic salmonellosis show magnitudinal and temporal differential expression of cytokines and chemokines following Salmonella enterica challenge. Infect Immun, 2006, 74: 1425-1430.
[40] Shivaprasad HL. Fowl typhoid and pullorum disease. Rev Sci Tech, 2000, 19: 405-424.
[41] Babu U, Dalloul RA, Okamura M, Lillehoj HS, Xie H, Raybourne RB, Gaines D, Heckert RA. Salmonella enteritidis clearance and immune responses in chickens following Salmonella vaccination and challenge. Vet. Immunol Immunopathol, 2004, 101: 251-257.
[42] Babu U, Scott M, Myers MJ, Okamura M, Gaines D, Yancy HF, Lillehoj H, Heckert RA, Raybourne RB. Effects of live attenuated and killed Salmonella vaccine on T-lymphocyte mediated immunity in laying hens. Vet. Immunol Immunopathol, 2003, 91: 39-44.
[43] Beal RK, Powers C, Wigley P, Barrow PA, Smith AL. Temporal dynamics of the cellular, humoral and cytokine responses in chickens during primary and secondary infection with Salmonella enterica serovar Typhimurium. Avian Pathol, 2004a, 33: 25-33.
[44] Withanage GS, Wigley P, Kaiser P, Mastroeni P, Brooks H, Powers C, Beal R, Barrow P, Maskell D, McConnell I. Cytokine and chemokine responses associated with clearance of a primary Salmonella enterica serovar Typhimurium infection in the chicken and in protective immunity to rechallenge. Infect Immun, 2005, 73: 5173-5182.
[45] Beal RK, Powers C, Davison TF, Barrow PA, Smith AL. Clearance of enteric Salmonella enterica serovar Typhimurium in chickens is independent of B-cell function. Infect Immun, 2006, 74: 1442-1444.
[46] Desmidt M, Ducatelle R, Mast J, Goddeeris BM, Kaspers B, Haesebrouck F. Role of the humoral immune system in Salmonella enteritidis phage type four infection in chickens. Vet. Immunol Immunopathol, 1998, 63: 355-367.
[47] Lucy C, Peter K, Paul B. The immunobiology of avian systemic salmonellosis. Veterinary Immunology and Immunopathology, 2009, 128: 53-59.
[48] Woodward MJ, Gettinby G, Breslin MF, Corkish JD, Houghton S. The efficacy of Salenvac, aSalmonella enterica subsp. Enterica serotype Enteritidis iron-restricted bacterin vaccine, in laying chickens. Avian Pathol, 2002, 31: 383-392.
[49] Wigley P, Berchieri Jr A, Page KL, Smith AL, Barrow PA. Salmonella enterica serovar Pullorum persists in splenic macrophages and in the reproductive tract during persistent, disease-free carriage in chickens. Infect Immun, 2001, 69: 7873-7879.
[50] Wigley P, Hulme SD, Powers C, Beal RK, Berchieri Jr A, Smith A, Barrow P. Infection of the reproductive tract and eggs with Salmonella enterica serovar pullorum in the chicken is associated with suppression of cellular immunity at sexual maturity. Infect Immun, 2005b, 73: 2986-2990.
[51] Qimron U, Madar N, Mittrucker HW, Zilka A, Yosef I, Bloushtain N, Kaufmann SH, Rosenshine I, Apte RN, Porgador A. Identification of Salmonella typhimurium genes responsible for interference with peptide presentation on MHC class I molecules: Deltayej Salmonella mutants induce superior CD8+ T-cell responses. Cell Microbiol, 2004, 6: 1057-1070.
[52] Uchiya K, Groisman EA, Nikai T. Involvement of Salmonella pathogenicity island 2 in the up-regulation of interleukin-10 expression in macrophages: role of protein kinase A signal pathway. Infect Immun, 2004, 72: 1964-1973
[53] Henderson SC, Bounous DI, Lee MD. Early events in the pathogenesis of avian salmonellosis. Infect Immun, 1999, 67: 3580-3586.
[54] Hassan JO, and CurtissⅢR . Virulent Salmonella typhimurium induced lymphocyte depletion and immunosuppression in chickens. Infect Immun, 1994, 62: 2027-2036.
[55] Carter PB, and Collins FM. The route of enteric infection in normal mice. J Exp Med, 1974, 139: 1189-1203.
[56] Carter PB, and Collins FM. Peyer’s patch responsiveness to Salmonella in mice. J Reticuloendothel Soc, 1974, 17: 38-46.
[57] Liebler EM, McPress CL, Landsverk T. Lymphocyte subpopulations in jejunal and ileal Peyer’s patchs of calves with experimental Salmonella dublin infection. J Vet Med, 1994, 41: 113-125.
[58] Kimbrough TG, Miller SI. Assembly of the typeⅢsecretion needle complex of Salmonella typhimurium. Microbes Infect, 2002, 4: 75-82.
[59] Kubori T, Matsushima Y, Nakamura D, et al. Supramolecular structure of the Salmonella typhimurium typeⅢprotein secretion system. Science, 1998, 280: 602-605.
[60] Kubori T, Sukhan A, Aizawa SI, et al. Molecular characterization and assembly of the needle complex of the Salmonella typhimurium type III protein secretion system. PROC NATL ACAD SCI USA, 2000, 97: 10225-10230.
[61] Pugsley AP, The complete general secretory pathway in gram-negative bacteria. Microbiol Rev, 1993, 57: 50-108.
[62] Daefler S, Russel M. The Salmonella typhimurium InvH protein is an outer membrane lipoprotein for the proper localization of InvG. Mol Microbiol, 1998, 28: 1367-1380.
[63] Behlau L, Miller SI. A PhoP-repressed gene promotes Salmonella typhimurium invasion of epithelial cells. J Bacteriol, 1993, 175: 4475-4484.
[64] Pegues DA, Hantman MJ, Behlau I, et al. PhoP/PhoQ transcriptional repression of Salmonella typhimurium invasion genes: evidence for a role in protein secretion. Mol Mirobiol, 1995, 17: 169-181.
[65] Kimbrough TG, Millerl SI. Contribution of Salmonella typhimurium type III secretion components to needle complex formation. Proc Natl Acad Sci USA, 2000, 97: 11008-11013.
[66] Groisman EA, Ochman H. Cognate gene clusters govern invasion of host epithelial cells by Salmonella typhimurium and Shigella flexneri. EMBO J, 1993, 12: 3779-3787.
[67] Sasakawa C, Komatsu K, Tobe T, et al. Eight genes in region 5 that form an operon essential for invasion of epithelial cells by Shigella flexneri 2a. J Bacterial, 1993, 175: 2334-2346.
[68] Collazo CM, Galan JE. Requirement for exported proteins in secretion through the invasion-associated type III system of Salmonella typhimurium. Infect Immun, 1996, 64: 3524-3531.
[69] Darwin KH, Miller VL. Molecular basis of the interaction of Salmonella with the intestinal mucosa. Clin Microbiol Rev, 1999, 12: 405-428.
[70] Gincocchio CC, Galan JE. Functional conservation among members of the Salmonella typhimurium InvA family of proteins. Infect Immun, 1995, 63: 729-732.
[71] Li J, Ochman H, Groisman EA, et al. Relationship between evolutionary rate and cellular location among the Inv/Spa invasion proteins of Salmonella enterica. ProC Natl Acad Sci USA, 1995, 82: 7252-7256.
[72] Eichelberg K, Ginocchio CC, Galan JE. Molecular and functional characterization of the invasion genes invB and invC: homology of InvC to the F0F1 ATPase family of proteins. J Bacteriol, 1994, 176: 4501-4510.
[73] Monack DM, Raupach B, Hromockyj AE, et al. Salmonella typhimurium invasion induces apoptosis in infected macrophages. Proc Natl Acad Sci USA, 1996, 93: 9833-9838.
[74] Hersh D, Monack DM, Smith MR, et al. The salmonella invasin SipB induces macrophage apoptosis by binding to caspase-1. Proc Natl Acad Sci USA, 1999, 96: 2396-2401.
[75] Santos RL, Tsolis RM, B?umler AJ, et al. Salmonella enterica serovar Typhimurium inducescell death in bovine monocyte-derived macrophages by early sipB-dependent and delayed sipB independent mechanisms. Infect Immun, 2001, 69: 2293-2301.
[76] Watson PR, Gautier AV, Paulin SM, et al. Salmonella enterica serovars Typhimurium and Dublin can lyse macrophages by a mechanism distinct from apoptosis. Infect Immun, 2000, 68: 3744-3747.
[77] Hueck CJ, Hantman MJ, Bajaj V, et al. Salmonella typhimurium secreted invasion determinants are homologous to Shigella Ipa proteins. Mol Microbiol, 1995, 18: 479-490.
[78] Kaniga K, Tucker S, Trollinger D, et al. Homologs of the Shigella IpaB and IpaC invasins are required for Salmonella typhimurium entry into cultured epithelial cells. J Bacteriol, 1995, 177: 3965-3971.
[79] Collazo CM, Galan JE. The invasion-associated type III system of Salmonella typhimurium directs the translocation of the Sip proteins into the host cell. Mol Microbiol, 1997, 24: 747-756.
[80] Hayward RD, Koronakis V. Direct nucleation and bundling of actin by the SipC protein of invasive Salmonella. EMBO J, 1999, 18: 4926-4934.
[81] Hardt WD, Chen LM, Schuebel KE, et al. S. typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells. Cell, 1998, 93:815-826.
[82] Zhou D, Moosekar M, Galan JE. Role of the S. typhimurium actin-binding protein SipA in bacterial internalization. Science, 1999, 283: 2092-2096.
[83] Shea JE, Hensel M, Gleeson C, et al. Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium. Proc Natl Acad Sci USA, 1996, 93: 2593-2597.
[84] Coombes BK, Brown NF, Valdez Y, et al. Expression and secretion of Salmonella pathogenicity Island-2 virulence genes in response to acidification exhibit differential requirements of a functional type III secretion apparatus and SsaL. J Biol Chem, 2004, 279: 49804-49815.
[85] Daniel V, Zurawski, Stein MA. The SPI2-encoded SseA chaperone has discrete domains required for SseB stabilization and export, and binds within the C-terminus of SseB and SseD. Microbiol, 2004, 150: 2055-2068.
[86] Cirillo DM, Valdivia RH, Monack DM, et al. Macrophage-dependent induction of the Salmonella pathogenicity island 2 type III secretion system and its role in intracellular survival. Mol. Microbiol, 1998, 30: 175-188.
[87] Medina E, Paglia P, Nikolaus T, et al. Pathogenicity island 2 mutants of Salmonella enterica serovar Typhimurium are efficient carriers for heterologous antigens and enable modulation of immune responses. Infect Immun,1999, 67: 1093-1099.
[88] Nikolaus T, Deiwick J, Rappl C. SseBCD proteins are secreted by the type III secretion systemof Salmonella pathogenicity island 2 and function as a translocon. J Bacteriol, 2001, 183: 6036-6045.
[89] Beuzon CR, Banks G, Deiwick J. PH-dependent secretion of SseB, a product of the SPI2 type III secretion system of Salmonella typhimurium. Mol Microbiol, 1999, 33: 806-816.
[90] Klein JR, Jones BD. Salmonella pathogenicity island 2-encoded proteins SseC and SseD are essential for virulence and are substrates of the type III secretion system. Infect Immun, 2001, 69: 737-743.
[91] Vazquez TA, Xu Y, Jones CJ, et al. Salmonella pathogenicity island 2-dependent evasion of the phagocyte NADPH oxidase. Science, 2000, 287: 1655-1658.
[92] Kuhle V, Hensel M. SseF and SseG are translocated effectors of the type III secretion system of Salmonella pathogenicity island 2 that modulate aggregation of endosomal compartments. Cell Microbiol, 2002, 4: 813-824.
[93] Hansen-Wester I, Stecher B, Hensel M. Type III secretion of Salmonella enterica serovar Typhimurium translocated effectors and SseFG. Infect Immun, 2002, 70: 1403-1409.
[94] Boucrot E, Henry T, Borg JP, et al. The intracellular fate of Salmonella depends on the recruitment of kinesin. Science, 2005, 308:1174-1178.
[95] Salcedo SP, Holden DW. SseG, a virulence protein that targets Salmonella to the Golgi network. EMBO J, 2003, 22: 5003-5014.
[96] Deiwick J, Salcedo SP, Boucrot E, et al. The translocated Salmonella effector proteins SseF and SseG interact and are required to establish an intracellular replication niche. Infect Immun, 2006, 74: 6965-6972.
[1]陆承平.兽医微生物(第三版).北京,中国农业出版社, 2002.
[2] Popoff MY, Bockemul J, Gheesling LL, et al. Sulllement 2001 (no. 45) to the Kauffmann-White scheme. Res Microbiol, 2003, 154: 173-4.
[3] (美)塞弗(Saif,Y. M.),主编苏敬良,主译高福,索勋.禽病学.中国农业出版社, 2005.
[4]蔡宝祥.家畜传染病学(第四版).北京,中国农业出版社, 2006.
[5] Mark S, Chadfield Derek, J Browm, et al. Comparison of intestinal invasion and macrophage response of Salmonella Gallinarum and other host-adapted Salmonella enterica serovars in the avian host. Veterinary Microbiology, 2003, 92: 46-64.
[6] France Daigle, James E Graham. Identification of Salmonella typhi genes expressed within macrophages by selective capture of transcribed sequences(SCOTS). Molecular Microbiology, 2001, 41(5): 1211-1222.
[7] Wang Xueqin, Zhou Daoguo. Salmonella invasion. Journal of Microbes and Infection, 2009, 4(2): 112-123.
[8] Miha Lavric, Michele NM, Travis WB, et al. Gene expression modulation in chicken macrophages exposed to Mycoplasma synoviae or Escherichia coli. Veterinary Microbiology, 2008, 126: 111-121.
[9] Rafael Antonio Casarin Penha Filho, Jacqueline Boldrin de Paiva, Mariana Dias da Silva, et al. Control of Salmonella Enteritidis and Salmonella Gallinarum in birds by using live vaccine candidate containing attenuated Salmonella Gallinarum mutant strain. Vaccine, 2010, 10: 1016-1023.
[10] Lotte Bohez, Inne Gantois, Richard Ducatelle, et al. The Salmonella Pathogenicity Island 2 regulator ssrA promotes reproductive tract but not intestinal colonization in chickens. Veterinary Microbiology, 2008, 126: 216-224.
[1] Diatchenko L, Lau YFC, Campbell AP, et al. Suppression subtractive hybridization: A method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA, 1996, 93: 6025-6030.
[2] J E Graham and Josephine E Clark-Curtiss. Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS). Proc Natl Acad Sci USA, 1999, 96: 11554-11559.
[3] M Hensel JE, Shea C, Gleason MD, Jones E Dalton and D W Holden, Science, 269, 400(1995).
[4] M J Mahan, J M Slauch and J J Mekalanos, Science, 259, 686(1993).
[5] R H Valdivia and S Falkow, Science, 277, 2007(1993).
[6] Handfield M, Brady LJ, Progulske-Fox A, et al. IVIAT: a novel method to identify microbial genes expressed specially during human infection. Trends Microbiol, 2000, 8(7): 336-339.
[7] Dozois CM, Daigle F, Curtiss R III. Identification of pathogen specific and conserved genes expressed in vivo by an avian pathogenic Escherichia coli strain. Proc Natl Acad Sci USA, 2003, 100(1): 247-252.
[8] Graham JE, Clark-Curtiss JE. Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS). Proc Natl Acad Sci USA, 1999, 96: 11554-11559.
[9] Daigle F, Graham JE, Curtiss R III. Identification of Salmonella typhi genes expressed within macrophages by selective capture of transcribed sequences (SCOTS). Mol Microb, 2001, 41(5): 1211-1222.
[10] Graham JE, Peek RM, Krishna U, et al. Global analysis of Helicobacter pylori gene expression in human gastric mucosa. Gastroenterology, 2002, 123(5): 1637-1648.
[11] Hou JY, Graham JE, Clark-Curtiss JE. Mycobacterium avium Genes Expressed during Growthin Human Macrophages Detected by Selective Capture of Transcribed Sequences (SCOTS). Infect Immun, 2002, 70(7): 3714-3726.
[12] Liu S, Graham JE, Bigelow L, et al. Identification of Listeria monocytogenes Genes Expressed in Response to Growth at Low Temperature. Appl Environ Microb, 2002, 68(4): 1697-1705.
[13] Baltes N, Gerlach GF. Identification of genes transcribed by Actinobacillus pleuropneumoniae in necrotic porcine lung tissue by using selective capture of transcribed sequences. Infect Immun, 2004, 72(11): 6711-6716.
[14]陈祥.禽病原性大肠杆菌的生物学特性及其可能毒力相关基因的鉴定[D].扬州大学博士论文, 2006.
[15] France Daigle, James E Graham. Identification of Salmonella typhi genes expressed within macrophages by selective capture of transcribed sequences(SCOTS). Molecular Microbiology, 2001, 41(5): 1211-1222.
[16]耿士忠.鸡白痢沙门氏菌毒力相关基因鉴定及其突变株特性研究[D].扬州大学博士论文, 2008.
[17]李求春.鸡白痢沙门氏菌抑制差减杂交文库的构建及ipaJ、mobA基因的克隆与表达[D].扬州大学硕士论文, 2007.
[1] Wigley P, Hulme SD, Powers C, et al. Infection of the reproductive tract and eggs with Salmonella enteria serovar Pullorum in the chicken is associated with suppression of cellular immunity at sexual maturity. Infect Immun, 2005, 73(5): 2986-2990.
[2] Heid CA, Stevens J, Livak KJ, et al. Real time quantitativa PCR Genome Res, 1996, 6: 986-994.
[3] Winer J, Jung CK, Shackel I, et al. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem, 1999, 270: 41-9.
[4]金鑫,李妍,陈代杰. PCR扩增rRNA基因在细菌菌种鉴定中的应用.国外医药抗生素分册, 2003, 24(5): 223-225.
[5]马莉,谢秀兰,岳华.鸡β-actin基因实时荧光定量PCR方法的建立.中国畜牧兽医, 2007, 34(2): 73-75.
[6]袁亚男,刘文忠.实时荧光定量PCR技术的类型、特点与应用.中国畜牧兽医, 2008, 35(3): 27-30.
[7]张贺,李波,周虚,谭建华.实时荧光定量PCR技术研究进展及应用.动物医学进展, 2006, 27(增): 5-12.
[8]李文涛,王俊东,杨利峰,王宏伟,韩少杰.实时荧光定量PCR技术及其应用.生物技术通讯, 2006, 17(1): 112-114.
[9]胡晓红,刘昕,黄正根,彭惠民,袁科,程英,高云.应用于PCR及实时荧光定量PCR的细菌DNA提取方法.第三军医大学学报, 2006, 28(7): 734-735.
[10]洪云,李津,汪和睦,赵铠.实时荧光定量PCR技术进展.国际流行病学传染病学杂志, 2006, 33(3): 161-163.
[2] Altmeyer RM, J K McNern, J C Bossio, I Rosenshine, B B Finlay, and J E Galan. Cloning and molecular characterization of a gene involved in Salmonella adherence and invasion of cultured epithelial cells. Mol Microbiol, 1993, 7: 89-98.
[3] Barrow PA. The paratyphoid salmonella. Rev. Sci. Tech, 2000, 19: 351-375.
[4] Shivaprasad HL. Fowl typhoid and pullorum disease. Rev. Sci. Tech, 2000, 19: 405-424.
[5] Jones MA, Wigley P, Page KL, Hulme SD, Barrow PA. Salmonella enterica serovar Gallinarum requires the Salmonella pathogenicity island 2 type III secretion system but not the Salmonella pathogenicity island 1 type III secretion system for virulence in chickens. Infect. Immun, 2001, 69: 5471-5476.
[6] Cobb SP, McVicar CM, Davies RH, Ainsworth H. Fowl typhoid in caged layer birds. Vet. Rec, 2005, 157: 268.
[7] Parmar D, Davies R. Fowl typhoid in a small backyard laying flock. Vet. Rec, 2007, 160: 348.
[8] Wigley P, Berchieri Jr A, Page KL, Smith AL, Barrow PA. Salmonella enterica serovar Pullorum persists in splenic macrophages and in the reproductive tract during persistent, disease-free carriage in chickens. Infect Immun, 2001, 69: 7873-7879.
[9] Gewirtz AT, Rao AS, Simon Jr PO, Merlin D, Carnes D, Madara JL, Neish AS. Salmonella typhimurium induces epithelial IL-8 expression via Ca (2+)-mediated activation of the NF-kappaB pathway. J. Clin. Invest, 2000, 105: 79-92.
[10] Gewirtz AT, Simon Jr PO, Schmitt CK, Taylor LJ, Hagedorn CH, O’Brien AD, Neish AS, Madara JL. Salmonella typhimurium translocates flagellin across intestinal epithelia, inducing a proinflammatory response. J. Clin. Invest, 2001, 107: 99-109.
[11] Wallis TS, Galyov EE, et al. Molecular basis of Salmonella-induced enteritis. Mol Microbiol, 2000, 36: 997-1005.
[12] Wallis TS, Wood M, Watson P, Paulin S, Jones M, Galyov E. Sips, Sops, and SPIs but not stn influence Salmonella enteropathogenesis. Adv. Exp. Med. Biol, 1999, 473: 275-280.
[13] Zeng H, Carlson AQ, Guo Y, Yu Y, Collier-Hyams L S, Madara JL, Gewirtz AT, Neish AS. Flagellin is the major proinflammatory determinant of enteropathogenic Salmonella. J. Immunol, 2003, 171: 3668-3674.
[14] Smith AL and Beal R. The avian enteric immune system in health and disease. In: Davison F, Kaspers B, Schat KA (Eds), Avian Immunology. Academic Press, London, pp, 2008, 243-271.
[15] Smith AL, Beal R. The avian enteric immune system in health and disease. In: Davison F,Kaspers B, Schat KA (Eds), Avian Immunology. Academic Press, London, pp, 243-271.
[16] Withanage GS, Wigley P, Kaiser P, Mastroeni P, Brooks H, Powers C, Beal R, Barrow P, Maskell D, McConnell I. Cytokine and chemokine responses associated with clearance of a primary Salmonella enterica serovar Typhimurium infection in the chicken and in protective immunity to rechallenge. Infect Immun, 2005, 73: 5173-5182.
[17] Henderson SC, Bounous DI, Lee MD, et al. Early events in the pathogenesis of avian salmonellosis. Infect Immun, 1999, 67: 3580-3586.
[18] Kaiser P, Rothwell L, Galyov EE, Barrow PA, Burnside J, Wigley P. Differential cytokine expression in avian cells in response to invasion by Salmonella typhimurium, Salmonella enteritidis and Salmonella gallinarum. Microbiology, 2000, 146 (12): 3217-3226.
[19] Withanage GS, Kaiser P, Wigley P, Powers C, Mastroeni P, Brooks H, Barrow P, Smith A, Maskell D, McConnell I. Rapid expression of chemokines and proinflammatory cytokines in newly hatched chickens infected with Salmonella enterica serovar typhimurium. Infect Immun, 2004, 72: 2152-2159.
[20] Iqbal M, Philbin VJ, Withanage GS, Wigley P, Beal RK, Goodchild MJ, Barrow P, McConnell I, Maskell DJ, Young J, Bumstead N, Boyd Y, Smith AL. Identification and functional characterization of chicken toll-like receptor 5 reveals a fundamental role in the biology of infection with Salmonella enterica serovar typhimurium. Infect Immun, 2005, 73: 2344-2350.
[21] Withanage GS, Wigley P, Scott D, et al. Infection of the Reproductive Tract and Eggs with Salmonella enterica serovar Pullorum in the Chicken Is Associated with Suppression of Cellular Immunity at Sexual Maturity. Infect Immun, 2005, 73(5): 1443-1450.
[22] Berchieri A, Wigley P, Page K, Murphy CK, Barrow PA. Further studies on vertical transmission and persistence of Salmonella enterica serovar Enteritidis phage type 4 in chickens. Avian Pathol, 2001, 30: 297-310.
[23] Chan K, Baker S, Kim CC, Detweiler CS, Dougan G, Falkow S. Genomic comparison of Salmonella enterica serovars and Salmonella bongori by use of an S. enterica serovar typhimurium DNA microarray. J Bacteriol, 2003, 185: 553-563.
[24] Hughes S, Poh TY, Bumstead N, Kaiser P. Re-evaluation of the chicken MIP family of chemokines and their receptors suggests that CCL5 is the prototypic MIP family chemokine, and that different species have developed different repertoires of both the CC chemokines and their receptors. Dev Comp Immunol, 2007, 31: 72-86.
[25] Henderson SC, Bounous DI, Lee MD, et al. Early events in the pathogenesis of avian salmonellosis. Infect Immun, 1999, 67: 3580-3586.
[26] Wigley P, Jones MA, Barrow PA. Salmonella enterica serovar Pullorum requires the Salmonella pathogenicity island 2 type III secretion system for virulence and carriage in the chicken. Avian Pathol, 2002b, 31: 501-506.
[27] Collier-Hyams LS, Zeng H, Sun J, Tomlinson AD, Bao ZQ, Chen H, Madara JL, Orth K., Neish AS. Cutting edge: Salmonella AvrA effector inhibits the key proinflammatory, anti-apoptotic NF-kappa B pathway. J Immunol, 2002, 169: 2846-2850.
[28] Mastroeni P, Menager N. Development of acquired immunity to Salmonella. J. Med Microbiol, 2003, 52: 453-459.
[29] Barrow PA, Huggins MB, Lovell MA. Host specificity of Salmonella infection in chickens andmice is expressed in vivo primarily at the level of the reticuloendothelial system. Infect Immun, 1994, 62: 4602-4610.
[30] Hensel M. Salmonella pathogenicity island 2. Mol Microbiol, 2000, 36; 1015–1023.
[31] Cheminay C, Mohlenbrink A, Hensel M. Intracellular Salmonella inhibit antigen presentation by dendritic cells. J Immunol, 2005, 174: 2892-2899.
[32] Jones MA, Hulme SD, Barrow PA, Wigley P. The Salmonella pathogenicity island 1 and Salmonella pathogenicity island 2 type III secretion systems play a major role in pathogenesis of systemic disease and gastrointestinal tract colonization of Salmonella enterica serovar Typhimurium in the chicken. Avian Pathol, 2007, 36: 199-203.
[33] Jones MA, Wigley P, Page KL, Hulme SD, Barrow PA. Salmonella enterica serovar Gallinarum requires the Salmonella pathogenicity island 2 type III secretion system but not the Salmonella pathogenicity island 1 type III secretion system for virulence in chickens. Infect Immun, 2001, 69: 5471-5476.
[34] Kogut MH, Tellez GI, McGruder ED, Hargis BM, Williams JD, Corrier DE, DeLoach JR. Heterophils are decisive components in the early responses of chickens to Salmonella enteritidis infections. Microb Pathog, 1994, 16: 141-151.
[35] Wigley P, Hulme S, Rothwell L, Bumstead N, Kaiser P, Barrow P. Macrophages isolated from chickens genetically resistant or susceptible to systemic salmonellosis show magnitudinal and temporal differential expression of cytokines and chemokines following Salmonella enterica challenge. Infect Immun, 2006, 74: 1425-1430.
[36] Wigley P, Hulme SD, Bumstead N, Barrow PA. In vivo and in vitro studies of genetic resistance to systemic salmonellosis in the chicken encoded by the SAL1 locus. Microbes Infect, 2002a, 4: 1111-1120.
[37] Withanage GS, Kaiser P, Wigley P, Powers C, Mastroeni P, Brooks H, Barrow P, Smith A,Maskell D, McConnell I. Rapid expression of chemokines and proinflammatory cytokines in newly hatched chickens infected with Salmonella enterica serovar typhimurium. Infect Immun, 2004, 72: 2152-2159.
[38] Mariani P, Barrow PA, Cheng HH, Groenen MM, Negrini R, Bumstead N. Localization to chicken chromosome 5 of a novel locus determining salmonellosis resistance. Immunogenetics, 2001, 53: 786-791.
[39] Wigley P, Hulme S, Rothwell L, Bumstead N, Kaiser P, Barrow P. Macrophages isolated from chickens genetically resistant or susceptible to systemic salmonellosis show magnitudinal and temporal differential expression of cytokines and chemokines following Salmonella enterica challenge. Infect Immun, 2006, 74: 1425-1430.
[40] Shivaprasad HL. Fowl typhoid and pullorum disease. Rev Sci Tech, 2000, 19: 405-424.
[41] Babu U, Dalloul RA, Okamura M, Lillehoj HS, Xie H, Raybourne RB, Gaines D, Heckert RA. Salmonella enteritidis clearance and immune responses in chickens following Salmonella vaccination and challenge. Vet. Immunol Immunopathol, 2004, 101: 251-257.
[42] Babu U, Scott M, Myers MJ, Okamura M, Gaines D, Yancy HF, Lillehoj H, Heckert RA, Raybourne RB. Effects of live attenuated and killed Salmonella vaccine on T-lymphocyte mediated immunity in laying hens. Vet. Immunol Immunopathol, 2003, 91: 39-44.
[43] Beal RK, Powers C, Wigley P, Barrow PA, Smith AL. Temporal dynamics of the cellular, humoral and cytokine responses in chickens during primary and secondary infection with Salmonella enterica serovar Typhimurium. Avian Pathol, 2004a, 33: 25-33.
[44] Withanage GS, Wigley P, Kaiser P, Mastroeni P, Brooks H, Powers C, Beal R, Barrow P, Maskell D, McConnell I. Cytokine and chemokine responses associated with clearance of a primary Salmonella enterica serovar Typhimurium infection in the chicken and in protective immunity to rechallenge. Infect Immun, 2005, 73: 5173-5182.
[45] Beal RK, Powers C, Davison TF, Barrow PA, Smith AL. Clearance of enteric Salmonella enterica serovar Typhimurium in chickens is independent of B-cell function. Infect Immun, 2006, 74: 1442-1444.
[46] Desmidt M, Ducatelle R, Mast J, Goddeeris BM, Kaspers B, Haesebrouck F. Role of the humoral immune system in Salmonella enteritidis phage type four infection in chickens. Vet. Immunol Immunopathol, 1998, 63: 355-367.
[47] Lucy C, Peter K, Paul B. The immunobiology of avian systemic salmonellosis. Veterinary Immunology and Immunopathology, 2009, 128: 53-59.
[48] Woodward MJ, Gettinby G, Breslin MF, Corkish JD, Houghton S. The efficacy of Salenvac, aSalmonella enterica subsp. Enterica serotype Enteritidis iron-restricted bacterin vaccine, in laying chickens. Avian Pathol, 2002, 31: 383-392.
[49] Wigley P, Berchieri Jr A, Page KL, Smith AL, Barrow PA. Salmonella enterica serovar Pullorum persists in splenic macrophages and in the reproductive tract during persistent, disease-free carriage in chickens. Infect Immun, 2001, 69: 7873-7879.
[50] Wigley P, Hulme SD, Powers C, Beal RK, Berchieri Jr A, Smith A, Barrow P. Infection of the reproductive tract and eggs with Salmonella enterica serovar pullorum in the chicken is associated with suppression of cellular immunity at sexual maturity. Infect Immun, 2005b, 73: 2986-2990.
[51] Qimron U, Madar N, Mittrucker HW, Zilka A, Yosef I, Bloushtain N, Kaufmann SH, Rosenshine I, Apte RN, Porgador A. Identification of Salmonella typhimurium genes responsible for interference with peptide presentation on MHC class I molecules: Deltayej Salmonella mutants induce superior CD8+ T-cell responses. Cell Microbiol, 2004, 6: 1057-1070.
[52] Uchiya K, Groisman EA, Nikai T. Involvement of Salmonella pathogenicity island 2 in the up-regulation of interleukin-10 expression in macrophages: role of protein kinase A signal pathway. Infect Immun, 2004, 72: 1964-1973
[53] Henderson SC, Bounous DI, Lee MD. Early events in the pathogenesis of avian salmonellosis. Infect Immun, 1999, 67: 3580-3586.
[54] Hassan JO, and CurtissⅢR . Virulent Salmonella typhimurium induced lymphocyte depletion and immunosuppression in chickens. Infect Immun, 1994, 62: 2027-2036.
[55] Carter PB, and Collins FM. The route of enteric infection in normal mice. J Exp Med, 1974, 139: 1189-1203.
[56] Carter PB, and Collins FM. Peyer’s patch responsiveness to Salmonella in mice. J Reticuloendothel Soc, 1974, 17: 38-46.
[57] Liebler EM, McPress CL, Landsverk T. Lymphocyte subpopulations in jejunal and ileal Peyer’s patchs of calves with experimental Salmonella dublin infection. J Vet Med, 1994, 41: 113-125.
[58] Kimbrough TG, Miller SI. Assembly of the typeⅢsecretion needle complex of Salmonella typhimurium. Microbes Infect, 2002, 4: 75-82.
[59] Kubori T, Matsushima Y, Nakamura D, et al. Supramolecular structure of the Salmonella typhimurium typeⅢprotein secretion system. Science, 1998, 280: 602-605.
[60] Kubori T, Sukhan A, Aizawa SI, et al. Molecular characterization and assembly of the needle complex of the Salmonella typhimurium type III protein secretion system. PROC NATL ACAD SCI USA, 2000, 97: 10225-10230.
[61] Pugsley AP, The complete general secretory pathway in gram-negative bacteria. Microbiol Rev, 1993, 57: 50-108.
[62] Daefler S, Russel M. The Salmonella typhimurium InvH protein is an outer membrane lipoprotein for the proper localization of InvG. Mol Microbiol, 1998, 28: 1367-1380.
[63] Behlau L, Miller SI. A PhoP-repressed gene promotes Salmonella typhimurium invasion of epithelial cells. J Bacteriol, 1993, 175: 4475-4484.
[64] Pegues DA, Hantman MJ, Behlau I, et al. PhoP/PhoQ transcriptional repression of Salmonella typhimurium invasion genes: evidence for a role in protein secretion. Mol Mirobiol, 1995, 17: 169-181.
[65] Kimbrough TG, Millerl SI. Contribution of Salmonella typhimurium type III secretion components to needle complex formation. Proc Natl Acad Sci USA, 2000, 97: 11008-11013.
[66] Groisman EA, Ochman H. Cognate gene clusters govern invasion of host epithelial cells by Salmonella typhimurium and Shigella flexneri. EMBO J, 1993, 12: 3779-3787.
[67] Sasakawa C, Komatsu K, Tobe T, et al. Eight genes in region 5 that form an operon essential for invasion of epithelial cells by Shigella flexneri 2a. J Bacterial, 1993, 175: 2334-2346.
[68] Collazo CM, Galan JE. Requirement for exported proteins in secretion through the invasion-associated type III system of Salmonella typhimurium. Infect Immun, 1996, 64: 3524-3531.
[69] Darwin KH, Miller VL. Molecular basis of the interaction of Salmonella with the intestinal mucosa. Clin Microbiol Rev, 1999, 12: 405-428.
[70] Gincocchio CC, Galan JE. Functional conservation among members of the Salmonella typhimurium InvA family of proteins. Infect Immun, 1995, 63: 729-732.
[71] Li J, Ochman H, Groisman EA, et al. Relationship between evolutionary rate and cellular location among the Inv/Spa invasion proteins of Salmonella enterica. ProC Natl Acad Sci USA, 1995, 82: 7252-7256.
[72] Eichelberg K, Ginocchio CC, Galan JE. Molecular and functional characterization of the invasion genes invB and invC: homology of InvC to the F0F1 ATPase family of proteins. J Bacteriol, 1994, 176: 4501-4510.
[73] Monack DM, Raupach B, Hromockyj AE, et al. Salmonella typhimurium invasion induces apoptosis in infected macrophages. Proc Natl Acad Sci USA, 1996, 93: 9833-9838.
[74] Hersh D, Monack DM, Smith MR, et al. The salmonella invasin SipB induces macrophage apoptosis by binding to caspase-1. Proc Natl Acad Sci USA, 1999, 96: 2396-2401.
[75] Santos RL, Tsolis RM, B?umler AJ, et al. Salmonella enterica serovar Typhimurium inducescell death in bovine monocyte-derived macrophages by early sipB-dependent and delayed sipB independent mechanisms. Infect Immun, 2001, 69: 2293-2301.
[76] Watson PR, Gautier AV, Paulin SM, et al. Salmonella enterica serovars Typhimurium and Dublin can lyse macrophages by a mechanism distinct from apoptosis. Infect Immun, 2000, 68: 3744-3747.
[77] Hueck CJ, Hantman MJ, Bajaj V, et al. Salmonella typhimurium secreted invasion determinants are homologous to Shigella Ipa proteins. Mol Microbiol, 1995, 18: 479-490.
[78] Kaniga K, Tucker S, Trollinger D, et al. Homologs of the Shigella IpaB and IpaC invasins are required for Salmonella typhimurium entry into cultured epithelial cells. J Bacteriol, 1995, 177: 3965-3971.
[79] Collazo CM, Galan JE. The invasion-associated type III system of Salmonella typhimurium directs the translocation of the Sip proteins into the host cell. Mol Microbiol, 1997, 24: 747-756.
[80] Hayward RD, Koronakis V. Direct nucleation and bundling of actin by the SipC protein of invasive Salmonella. EMBO J, 1999, 18: 4926-4934.
[81] Hardt WD, Chen LM, Schuebel KE, et al. S. typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells. Cell, 1998, 93:815-826.
[82] Zhou D, Moosekar M, Galan JE. Role of the S. typhimurium actin-binding protein SipA in bacterial internalization. Science, 1999, 283: 2092-2096.
[83] Shea JE, Hensel M, Gleeson C, et al. Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium. Proc Natl Acad Sci USA, 1996, 93: 2593-2597.
[84] Coombes BK, Brown NF, Valdez Y, et al. Expression and secretion of Salmonella pathogenicity Island-2 virulence genes in response to acidification exhibit differential requirements of a functional type III secretion apparatus and SsaL. J Biol Chem, 2004, 279: 49804-49815.
[85] Daniel V, Zurawski, Stein MA. The SPI2-encoded SseA chaperone has discrete domains required for SseB stabilization and export, and binds within the C-terminus of SseB and SseD. Microbiol, 2004, 150: 2055-2068.
[86] Cirillo DM, Valdivia RH, Monack DM, et al. Macrophage-dependent induction of the Salmonella pathogenicity island 2 type III secretion system and its role in intracellular survival. Mol. Microbiol, 1998, 30: 175-188.
[87] Medina E, Paglia P, Nikolaus T, et al. Pathogenicity island 2 mutants of Salmonella enterica serovar Typhimurium are efficient carriers for heterologous antigens and enable modulation of immune responses. Infect Immun,1999, 67: 1093-1099.
[88] Nikolaus T, Deiwick J, Rappl C. SseBCD proteins are secreted by the type III secretion systemof Salmonella pathogenicity island 2 and function as a translocon. J Bacteriol, 2001, 183: 6036-6045.
[89] Beuzon CR, Banks G, Deiwick J. PH-dependent secretion of SseB, a product of the SPI2 type III secretion system of Salmonella typhimurium. Mol Microbiol, 1999, 33: 806-816.
[90] Klein JR, Jones BD. Salmonella pathogenicity island 2-encoded proteins SseC and SseD are essential for virulence and are substrates of the type III secretion system. Infect Immun, 2001, 69: 737-743.
[91] Vazquez TA, Xu Y, Jones CJ, et al. Salmonella pathogenicity island 2-dependent evasion of the phagocyte NADPH oxidase. Science, 2000, 287: 1655-1658.
[92] Kuhle V, Hensel M. SseF and SseG are translocated effectors of the type III secretion system of Salmonella pathogenicity island 2 that modulate aggregation of endosomal compartments. Cell Microbiol, 2002, 4: 813-824.
[93] Hansen-Wester I, Stecher B, Hensel M. Type III secretion of Salmonella enterica serovar Typhimurium translocated effectors and SseFG. Infect Immun, 2002, 70: 1403-1409.
[94] Boucrot E, Henry T, Borg JP, et al. The intracellular fate of Salmonella depends on the recruitment of kinesin. Science, 2005, 308:1174-1178.
[95] Salcedo SP, Holden DW. SseG, a virulence protein that targets Salmonella to the Golgi network. EMBO J, 2003, 22: 5003-5014.
[96] Deiwick J, Salcedo SP, Boucrot E, et al. The translocated Salmonella effector proteins SseF and SseG interact and are required to establish an intracellular replication niche. Infect Immun, 2006, 74: 6965-6972.
[1]陆承平.兽医微生物(第三版).北京,中国农业出版社, 2002.
[2] Popoff MY, Bockemul J, Gheesling LL, et al. Sulllement 2001 (no. 45) to the Kauffmann-White scheme. Res Microbiol, 2003, 154: 173-4.
[3] (美)塞弗(Saif,Y. M.),主编苏敬良,主译高福,索勋.禽病学.中国农业出版社, 2005.
[4]蔡宝祥.家畜传染病学(第四版).北京,中国农业出版社, 2006.
[5] Mark S, Chadfield Derek, J Browm, et al. Comparison of intestinal invasion and macrophage response of Salmonella Gallinarum and other host-adapted Salmonella enterica serovars in the avian host. Veterinary Microbiology, 2003, 92: 46-64.
[6] France Daigle, James E Graham. Identification of Salmonella typhi genes expressed within macrophages by selective capture of transcribed sequences(SCOTS). Molecular Microbiology, 2001, 41(5): 1211-1222.
[7] Wang Xueqin, Zhou Daoguo. Salmonella invasion. Journal of Microbes and Infection, 2009, 4(2): 112-123.
[8] Miha Lavric, Michele NM, Travis WB, et al. Gene expression modulation in chicken macrophages exposed to Mycoplasma synoviae or Escherichia coli. Veterinary Microbiology, 2008, 126: 111-121.
[9] Rafael Antonio Casarin Penha Filho, Jacqueline Boldrin de Paiva, Mariana Dias da Silva, et al. Control of Salmonella Enteritidis and Salmonella Gallinarum in birds by using live vaccine candidate containing attenuated Salmonella Gallinarum mutant strain. Vaccine, 2010, 10: 1016-1023.
[10] Lotte Bohez, Inne Gantois, Richard Ducatelle, et al. The Salmonella Pathogenicity Island 2 regulator ssrA promotes reproductive tract but not intestinal colonization in chickens. Veterinary Microbiology, 2008, 126: 216-224.
[1] Diatchenko L, Lau YFC, Campbell AP, et al. Suppression subtractive hybridization: A method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA, 1996, 93: 6025-6030.
[2] J E Graham and Josephine E Clark-Curtiss. Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS). Proc Natl Acad Sci USA, 1999, 96: 11554-11559.
[3] M Hensel JE, Shea C, Gleason MD, Jones E Dalton and D W Holden, Science, 269, 400(1995).
[4] M J Mahan, J M Slauch and J J Mekalanos, Science, 259, 686(1993).
[5] R H Valdivia and S Falkow, Science, 277, 2007(1993).
[6] Handfield M, Brady LJ, Progulske-Fox A, et al. IVIAT: a novel method to identify microbial genes expressed specially during human infection. Trends Microbiol, 2000, 8(7): 336-339.
[7] Dozois CM, Daigle F, Curtiss R III. Identification of pathogen specific and conserved genes expressed in vivo by an avian pathogenic Escherichia coli strain. Proc Natl Acad Sci USA, 2003, 100(1): 247-252.
[8] Graham JE, Clark-Curtiss JE. Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS). Proc Natl Acad Sci USA, 1999, 96: 11554-11559.
[9] Daigle F, Graham JE, Curtiss R III. Identification of Salmonella typhi genes expressed within macrophages by selective capture of transcribed sequences (SCOTS). Mol Microb, 2001, 41(5): 1211-1222.
[10] Graham JE, Peek RM, Krishna U, et al. Global analysis of Helicobacter pylori gene expression in human gastric mucosa. Gastroenterology, 2002, 123(5): 1637-1648.
[11] Hou JY, Graham JE, Clark-Curtiss JE. Mycobacterium avium Genes Expressed during Growthin Human Macrophages Detected by Selective Capture of Transcribed Sequences (SCOTS). Infect Immun, 2002, 70(7): 3714-3726.
[12] Liu S, Graham JE, Bigelow L, et al. Identification of Listeria monocytogenes Genes Expressed in Response to Growth at Low Temperature. Appl Environ Microb, 2002, 68(4): 1697-1705.
[13] Baltes N, Gerlach GF. Identification of genes transcribed by Actinobacillus pleuropneumoniae in necrotic porcine lung tissue by using selective capture of transcribed sequences. Infect Immun, 2004, 72(11): 6711-6716.
[14]陈祥.禽病原性大肠杆菌的生物学特性及其可能毒力相关基因的鉴定[D].扬州大学博士论文, 2006.
[15] France Daigle, James E Graham. Identification of Salmonella typhi genes expressed within macrophages by selective capture of transcribed sequences(SCOTS). Molecular Microbiology, 2001, 41(5): 1211-1222.
[16]耿士忠.鸡白痢沙门氏菌毒力相关基因鉴定及其突变株特性研究[D].扬州大学博士论文, 2008.
[17]李求春.鸡白痢沙门氏菌抑制差减杂交文库的构建及ipaJ、mobA基因的克隆与表达[D].扬州大学硕士论文, 2007.
[1] Wigley P, Hulme SD, Powers C, et al. Infection of the reproductive tract and eggs with Salmonella enteria serovar Pullorum in the chicken is associated with suppression of cellular immunity at sexual maturity. Infect Immun, 2005, 73(5): 2986-2990.
[2] Heid CA, Stevens J, Livak KJ, et al. Real time quantitativa PCR Genome Res, 1996, 6: 986-994.
[3] Winer J, Jung CK, Shackel I, et al. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem, 1999, 270: 41-9.
[4]金鑫,李妍,陈代杰. PCR扩增rRNA基因在细菌菌种鉴定中的应用.国外医药抗生素分册, 2003, 24(5): 223-225.
[5]马莉,谢秀兰,岳华.鸡β-actin基因实时荧光定量PCR方法的建立.中国畜牧兽医, 2007, 34(2): 73-75.
[6]袁亚男,刘文忠.实时荧光定量PCR技术的类型、特点与应用.中国畜牧兽医, 2008, 35(3): 27-30.
[7]张贺,李波,周虚,谭建华.实时荧光定量PCR技术研究进展及应用.动物医学进展, 2006, 27(增): 5-12.
[8]李文涛,王俊东,杨利峰,王宏伟,韩少杰.实时荧光定量PCR技术及其应用.生物技术通讯, 2006, 17(1): 112-114.
[9]胡晓红,刘昕,黄正根,彭惠民,袁科,程英,高云.应用于PCR及实时荧光定量PCR的细菌DNA提取方法.第三军医大学学报, 2006, 28(7): 734-735.
[10]洪云,李津,汪和睦,赵铠.实时荧光定量PCR技术进展.国际流行病学传染病学杂志, 2006, 33(3): 161-163.