人布鲁氏菌病标准化PCR诊断方法及BP26基因标记疫苗研究
摘要
布鲁氏菌病为动物源性疾病,是由细胞内寄生的布鲁氏菌引起的传染-变态反应性人兽共患传染病,在世界范围内广泛流行,对畜牧养殖业的发展和人类健康造成严重威胁,已成为重要的公共卫生问题。布鲁氏菌病的诊断方面存在两个主要的问题:一是快速准确诊断,二是现有减毒活疫苗的鉴别诊断。
PCR检测技术避免操作活菌,并能检测到标本中较低丰度的病原,适合布鲁氏菌这种高危险性病原的检测。用PCR方法对布鲁氏菌病早期、复发期以及伴随多种并发症的患者及药物治疗后患者进行诊断,具有较好的应用前景,我国目前尚无应用PCR检测方法在临床诊断上跟踪随访的研究报道。本研究针对布鲁氏菌的保守基因bp26设计了属的引物,针对插入性序列IS711设计了牛、羊、猪种引物,建立了单重和多重PCR检测方法,在临床上对免疫学诊断为患者、可疑患者和非患者人群进行检测,并结合流行病学个案数据库资料拟定了进一步跟踪随访检测的对象,为建立人布鲁氏菌病标准化PCR诊断方法提供研究依据。
目前应用血清学方法不能区分布鲁氏菌减毒疫苗株的反应与自然感染,发展基因标记疫苗可解决这一问题。M5是我国广泛应用的减毒活疫苗株,本研究应用基因同源重组方法,用卡那抗性基因替换部分bp26基因,构建了bp26突变株,并根据布鲁氏菌的保守基因建立了能够区分bp26突变株与疫苗株的PCR鉴别方法。bp26突变株在胞内外的生存能力显示,bp26突变株在小鼠体内的免疫应答和免疫保护效果均低于M5疫苗株,表明bp26基因在M5疫苗株的免疫保护作用中发挥了作用。考虑到bp26基因在免疫原性和保护性方面的重要性,我们对BP26蛋白进行了表达及纯化,并致力于开发有效的布鲁氏菌亚单位疫苗和作为人布鲁氏菌病血清学的特异性诊断抗原用于临床诊断研究。
Brucellosis is a kind of typical zoonosis that is caused by cytozoic Brucella, which spreads worldwide and becomes a major public health problem with the threat of the livestock industry and human being health.There are two major problems in the diagnosis of brucellosis,the one is the lack of rapid and accurate diagnostic method,the other is the lack of identifying method of diagnosis for present attenuated live vaccine.The clinical diagnostic criterion of human brucellosis is based on clinical manifestations,history exposed to Brucella and laboratory diagnosis.The diagnosis of brucellosis always requires laboratory confirmation because of its various clinical manifestations without specific clinical signs.Present laboratory testing methods is the isolation of pathogen or demonstration of high titres of specific antibodies,but all these mehthods have serious shortcomings. Although the isolation of Brucella is the best evidence of confirmation of brucellosis, it is rarely applied to clinical diagnosis because the method is a time-consuming one, and with low positive rate of isolation of patients with long-term clinical courses or with focal complications,and harm to laboratory workers' health.As a simple method,serological testing method has been used to acquire main evidence of diagnosis of brucellosis,but it lacks specificity in spreading areas and in those persons exposed professionally to Brucella.Moreover,cross-reaction with other bacteria can also occur.Although we have developed some methods for clinical diagnosis,such as different kinds of serological methods and automated culture system,it still has some restrictions to apply.PCR assays by targeting to nucleic acids have been considered as the best way to the replacement of Brucella isolation and culture,because of its simple,rapid,sensitive,specific and no requirement of operation on live bacteria.Recent years,foreign scholars carried out studies on clinical diagnosis for brucellosis in acute stage and follow-up studies after treatment. They believed that PCR detection was useful tool in discovering patients who were in early acute stage or relapse.The standardization of detection methods,However, have not been set,the method of PCR detection is not applied to clinical diagnosis of brucellosis widely.Domestic research found results various.Most of them showed the method of PCR detection low in sensitivity.Some studies reported false-positive, all of them lack systematic and in-depth study and evaluation.As to now,there is no report concerning researching on the method of PCR detection that is used to evaluate the treatment effectiveness by follow-up patients after treatment and discover new patients who are in the early acute stage.So in our study,we are trying to do those things 1) build a standardized PCR diagnostic methods which is suitable for China's national condition and grass roots medical staff in CDC to evaluate the treatment effectiveness by following-up patients after treatment and discover new patients who are in the early acute stage;2) apply it to isolate and culture Brucella from blood of whom are with acute febrile in clinical;3) apply the blood with bacteria to optimize the template processing method;4) evaluate its applications in clinical diagnosis and following-up patients,by combining with epidemiological information on cases,so that it could offer a tool for pharmaceutical research and the effectiveness evaluation.The prevention of Brucellosis is mainly relied on inoculating animals.Attenuated live vaccine is used to prevent Brucellosis in China now.But it is hard to distinguish difference between attenuated live vaccine and natural infection,so the efforts of inoculating animals by plan is curbed,which reveals that rebuilding marker vaccine could be the direction of future research.
Objective:
1.To build a standardized PCR diagnostic methods which is suitable for China's national condition and grass roots medical staff in CDC to evaluate the treatment effectiveness by following-up patients after treatment and discover new patients who are in the early acute stage.
2.To isolate Brucella from patients blood and to identify its phenotype so as to offer bacteriological evidence for PCR diagnosis research.
3.To establish two database which contain epidemiological information on cases and sample information separately.
4.To build safe and effective vaccine strains with genetic markers so that infection could be distinguished between the attenuated live vaccine and wild strains.
Methods:
1.To establish database which contains epidemiological information on cases collecting at CDC Brucellosis outpatient service,such as personal information, clinical manifestation,laboratory diagnosis.At the same time,the blood and serum samples of out-patients would be collected and preserved at -20℃which are used to extract genome and test PCR.
2.To establish a standardized PCR diagnostic methods.Primers were designed separately to optimize single-PCR and multi-PCR detection and reaction conditions, which were specific to BP26 encoding gene and IS711 gene sequence,so that they could be applied to identifying Brucella spp.
3.To establish a boiling method in order to extract Brucella genome from patients blood.M5 strains and BP26 primers were used to amplify Brucella DNA by extracting lysate,simulate and experimental animals at different time points,so as to determine a method fit for clinical application.
4.To isolate and culture Brucella from patients' blood,and BP26 encoding gene and primers were used to identify bacterial strains phenotype,so as to verify specificity of amplified target sequence.
5.Preparations of templates from blood isolated Brucella.spp.then tested with different primers by PCR detection,so as to find most sensitive method for PCR detection applying to patients,no-patients and suspect patients who immunology diagnosed.Furthermore,we would use it to track target people in three,six and twelve months separately combining with epidemiological information of cases and to analyze and evaluate the reliability of PCR diagnosis and evaluation of clinical application.
6.In the method of homologous recombination,a genetic marked vaccine strain was rebuilt by replacing attenuated live Brucella vaccine strain of the bp26 gene M5 with kanamycin resistance gene.Two pairs of primers which were towards deleted sequences of bp26 gene and various types of highly conserved sequence were designed to rebuild PCR detection so as to identify vaccine and mutant strains.Wild Brucella strains were used to attack mice immune system so as to explore mice immune protection from marked vaccine strain.
Results:
1.Database of Brucellosis epidemiological information on cases was built in Songyuan CDC.Information on demography,clinical manifestation,history exposed to Brucella spp.,results of laboratory diagnosis,treatment protocols,samples of whole blood and serum blood were collected.
2.Methods of single-PCR and multi-PCR detection were established.Primers for diagnosing Brucella BP26 encoding gene were designed.And primers used to diagnose Brucella insert sequences IS711 and to identify B.mellitensis,B.abortus, and B.suis were designed too.The specificity was verified among genomes of standard Brucella spp.and vaccine strains.
3.Ways of Brucella DNA extraction from blood were established.With the PCR detection of mice blood among lystae,simulate and experimental animals at different time points,results showed that the sensitivity of pyrolysis method was better than inhibitor stimulation,and the sensitivity of PCR assay was better than isolated culture.
4.Ten strains of Brucella were identified as B.melitensis which were isolated cultured from patients' blood samples.In the positive isolates and other isolates in PCR identification,BP26,primers for B.melitensis were amplified the target bands in the ten bacteria strains,and other isolates were not amplified the target bands.
5.In the templates of blood which were positive isolated and cultured Brucella, the sensitivity of B4/B5 was the highest in accordance with the results of isolated culture.The sensitivity of BP26,primers of B.melitensis and 26A/26B were lower, with the coincidence rate of 67%.
6.Serological diagnosis was performed to test patients' blood samples.The results showed that the sensitivity of BP26 primer and primer of B.melitensis were lower than that tested by serological detection methods.The sensitivity of B4/B5 primer was high with high rate of coincidence.Furthermore,positive amplification was shown among suspected cases and non-patients,but was not shown among healthy people.
7.Analysis of clinical data showed that there were seven cases who suffered Brucellosis(SAT＞1:100),two cases who were confirmed as suspected cases (PAT0.04),one case confirmed as non-patient among the ten Brucella sufferers who were proved as existing Brucella in the blood samples.Positive predictive value of PCR detection was 67%～100%.
8.Positive rate of PCR detection were 82.9%,80.0%and 60.0%separately among those who were diagnosed as patients(34 cases),non-patients(25 cases) and suspected cases(16 cases) by immunology.
9.By constructing mutative box,which means the recombinant plasmid of pUC19K-NC,and under the principle of gene homologous recombination,part of bp26 gene in the M5 vaccine strain was deleted,M5△bp26 tagged by molecular was constructed.Two pairs of primers were designed specific to deletion sequences of bp26 gene and every highly conserved sequences,respectively.Double-PCR detection methods were established and optimized which could be used to identify vaccine strains and mutant strains.
10.Bacterial strains marked by M5△bp26 could live in macrophages and mice body,but the viability of it was lower than mild strain.Marked strains and vaccine strains M5 could both induce specific antibody in the body of Balb/c mice,but humoral immune response induced by marked strains was weaker and had shorter duration than that induced by M5.Immune protective effect of marked strains was dissatisfactory when it was attacked by B.mellitensis 16M.
Conclusions:
1.Primers could be used to identify the strains of Brucella,which were designed to target to BP26 coding gene of Brucella diagnostic antigen and parenthetical sequences of IS711 designed to identify B.melitensis,B.abortus and B.suis separately.The sensitivity of boiling method was better than inhibitor stimulation,so it could be performed on clinical research of PCR detection.
2.When being used to detect blood infected by Brucella or came from Brucellosis patients,it showed that the sensitivities of PCR detection using BP26 primers or primers of B.melitensis were low,but specificities were high;the sensitivities of PCR detection using B4 primers and B5 primers were high,and high coincidence rate with indicators of isolated culture and indicators of immunology. Positive amplification was shown among suspected cases and non-patients,but was not shown among healthy people.The method of PCR detection could be used to follow up Brucellosis patients after treatment.
3.Database of Brucellosis epidemiological information on cases which were collected at Songyuan CDC was built.
4.Molecular marked strains M5△bp26 which deleted part of bp26 gene were built,and dual-PCR detection methods were established to identify vaccine strains and mutant strains.Bacterial strains marked by M5△bp26 could live in macrophages and mice body,but the its viability was lower than mild strain.Marked strains and vaccine strains M5 could both induce specific antibody in the body of Balb/c mice,but humoral immune response induced by marked strains was weaker and had shorter duration than that induced by M5.
5.Objectives remaining to study are:
(1)To follow up those people whose blood are proved positive when being cultured isolated or detected positive with PCR assay or who are proved to be Brucellosis patients diagnosed by immunological detection after their treatment in three,six,twelve months separately.To evaluate status and future of PCR techniques for the diagnosis and follow-up of human brucellosis used to discover relapses patients.
(2) Following up those people who are diagnosed as Brucella positive by PCR detection but non-patients when diagnosed by immunology detection,so as to make sense of the PCR detection method when it is used to discover missed diagnosis.
(3) Immune protective effect of marked strains was dissatisfactory,which were deleted of bp26 gene.But BP26 antigen had been expressed and purified,which could be used as subunit vaccine to be continued to research on.
PCR检测技术避免操作活菌,并能检测到标本中较低丰度的病原,适合布鲁氏菌这种高危险性病原的检测。用PCR方法对布鲁氏菌病早期、复发期以及伴随多种并发症的患者及药物治疗后患者进行诊断,具有较好的应用前景,我国目前尚无应用PCR检测方法在临床诊断上跟踪随访的研究报道。本研究针对布鲁氏菌的保守基因bp26设计了属的引物,针对插入性序列IS711设计了牛、羊、猪种引物,建立了单重和多重PCR检测方法,在临床上对免疫学诊断为患者、可疑患者和非患者人群进行检测,并结合流行病学个案数据库资料拟定了进一步跟踪随访检测的对象,为建立人布鲁氏菌病标准化PCR诊断方法提供研究依据。
目前应用血清学方法不能区分布鲁氏菌减毒疫苗株的反应与自然感染,发展基因标记疫苗可解决这一问题。M5是我国广泛应用的减毒活疫苗株,本研究应用基因同源重组方法,用卡那抗性基因替换部分bp26基因,构建了bp26突变株,并根据布鲁氏菌的保守基因建立了能够区分bp26突变株与疫苗株的PCR鉴别方法。bp26突变株在胞内外的生存能力显示,bp26突变株在小鼠体内的免疫应答和免疫保护效果均低于M5疫苗株,表明bp26基因在M5疫苗株的免疫保护作用中发挥了作用。考虑到bp26基因在免疫原性和保护性方面的重要性,我们对BP26蛋白进行了表达及纯化,并致力于开发有效的布鲁氏菌亚单位疫苗和作为人布鲁氏菌病血清学的特异性诊断抗原用于临床诊断研究。
Brucellosis is a kind of typical zoonosis that is caused by cytozoic Brucella, which spreads worldwide and becomes a major public health problem with the threat of the livestock industry and human being health.There are two major problems in the diagnosis of brucellosis,the one is the lack of rapid and accurate diagnostic method,the other is the lack of identifying method of diagnosis for present attenuated live vaccine.The clinical diagnostic criterion of human brucellosis is based on clinical manifestations,history exposed to Brucella and laboratory diagnosis.The diagnosis of brucellosis always requires laboratory confirmation because of its various clinical manifestations without specific clinical signs.Present laboratory testing methods is the isolation of pathogen or demonstration of high titres of specific antibodies,but all these mehthods have serious shortcomings. Although the isolation of Brucella is the best evidence of confirmation of brucellosis, it is rarely applied to clinical diagnosis because the method is a time-consuming one, and with low positive rate of isolation of patients with long-term clinical courses or with focal complications,and harm to laboratory workers' health.As a simple method,serological testing method has been used to acquire main evidence of diagnosis of brucellosis,but it lacks specificity in spreading areas and in those persons exposed professionally to Brucella.Moreover,cross-reaction with other bacteria can also occur.Although we have developed some methods for clinical diagnosis,such as different kinds of serological methods and automated culture system,it still has some restrictions to apply.PCR assays by targeting to nucleic acids have been considered as the best way to the replacement of Brucella isolation and culture,because of its simple,rapid,sensitive,specific and no requirement of operation on live bacteria.Recent years,foreign scholars carried out studies on clinical diagnosis for brucellosis in acute stage and follow-up studies after treatment. They believed that PCR detection was useful tool in discovering patients who were in early acute stage or relapse.The standardization of detection methods,However, have not been set,the method of PCR detection is not applied to clinical diagnosis of brucellosis widely.Domestic research found results various.Most of them showed the method of PCR detection low in sensitivity.Some studies reported false-positive, all of them lack systematic and in-depth study and evaluation.As to now,there is no report concerning researching on the method of PCR detection that is used to evaluate the treatment effectiveness by follow-up patients after treatment and discover new patients who are in the early acute stage.So in our study,we are trying to do those things 1) build a standardized PCR diagnostic methods which is suitable for China's national condition and grass roots medical staff in CDC to evaluate the treatment effectiveness by following-up patients after treatment and discover new patients who are in the early acute stage;2) apply it to isolate and culture Brucella from blood of whom are with acute febrile in clinical;3) apply the blood with bacteria to optimize the template processing method;4) evaluate its applications in clinical diagnosis and following-up patients,by combining with epidemiological information on cases,so that it could offer a tool for pharmaceutical research and the effectiveness evaluation.The prevention of Brucellosis is mainly relied on inoculating animals.Attenuated live vaccine is used to prevent Brucellosis in China now.But it is hard to distinguish difference between attenuated live vaccine and natural infection,so the efforts of inoculating animals by plan is curbed,which reveals that rebuilding marker vaccine could be the direction of future research.
Objective:
1.To build a standardized PCR diagnostic methods which is suitable for China's national condition and grass roots medical staff in CDC to evaluate the treatment effectiveness by following-up patients after treatment and discover new patients who are in the early acute stage.
2.To isolate Brucella from patients blood and to identify its phenotype so as to offer bacteriological evidence for PCR diagnosis research.
3.To establish two database which contain epidemiological information on cases and sample information separately.
4.To build safe and effective vaccine strains with genetic markers so that infection could be distinguished between the attenuated live vaccine and wild strains.
Methods:
1.To establish database which contains epidemiological information on cases collecting at CDC Brucellosis outpatient service,such as personal information, clinical manifestation,laboratory diagnosis.At the same time,the blood and serum samples of out-patients would be collected and preserved at -20℃which are used to extract genome and test PCR.
2.To establish a standardized PCR diagnostic methods.Primers were designed separately to optimize single-PCR and multi-PCR detection and reaction conditions, which were specific to BP26 encoding gene and IS711 gene sequence,so that they could be applied to identifying Brucella spp.
3.To establish a boiling method in order to extract Brucella genome from patients blood.M5 strains and BP26 primers were used to amplify Brucella DNA by extracting lysate,simulate and experimental animals at different time points,so as to determine a method fit for clinical application.
4.To isolate and culture Brucella from patients' blood,and BP26 encoding gene and primers were used to identify bacterial strains phenotype,so as to verify specificity of amplified target sequence.
5.Preparations of templates from blood isolated Brucella.spp.then tested with different primers by PCR detection,so as to find most sensitive method for PCR detection applying to patients,no-patients and suspect patients who immunology diagnosed.Furthermore,we would use it to track target people in three,six and twelve months separately combining with epidemiological information of cases and to analyze and evaluate the reliability of PCR diagnosis and evaluation of clinical application.
6.In the method of homologous recombination,a genetic marked vaccine strain was rebuilt by replacing attenuated live Brucella vaccine strain of the bp26 gene M5 with kanamycin resistance gene.Two pairs of primers which were towards deleted sequences of bp26 gene and various types of highly conserved sequence were designed to rebuild PCR detection so as to identify vaccine and mutant strains.Wild Brucella strains were used to attack mice immune system so as to explore mice immune protection from marked vaccine strain.
Results:
1.Database of Brucellosis epidemiological information on cases was built in Songyuan CDC.Information on demography,clinical manifestation,history exposed to Brucella spp.,results of laboratory diagnosis,treatment protocols,samples of whole blood and serum blood were collected.
2.Methods of single-PCR and multi-PCR detection were established.Primers for diagnosing Brucella BP26 encoding gene were designed.And primers used to diagnose Brucella insert sequences IS711 and to identify B.mellitensis,B.abortus, and B.suis were designed too.The specificity was verified among genomes of standard Brucella spp.and vaccine strains.
3.Ways of Brucella DNA extraction from blood were established.With the PCR detection of mice blood among lystae,simulate and experimental animals at different time points,results showed that the sensitivity of pyrolysis method was better than inhibitor stimulation,and the sensitivity of PCR assay was better than isolated culture.
4.Ten strains of Brucella were identified as B.melitensis which were isolated cultured from patients' blood samples.In the positive isolates and other isolates in PCR identification,BP26,primers for B.melitensis were amplified the target bands in the ten bacteria strains,and other isolates were not amplified the target bands.
5.In the templates of blood which were positive isolated and cultured Brucella, the sensitivity of B4/B5 was the highest in accordance with the results of isolated culture.The sensitivity of BP26,primers of B.melitensis and 26A/26B were lower, with the coincidence rate of 67%.
6.Serological diagnosis was performed to test patients' blood samples.The results showed that the sensitivity of BP26 primer and primer of B.melitensis were lower than that tested by serological detection methods.The sensitivity of B4/B5 primer was high with high rate of coincidence.Furthermore,positive amplification was shown among suspected cases and non-patients,but was not shown among healthy people.
7.Analysis of clinical data showed that there were seven cases who suffered Brucellosis(SAT＞1:100),two cases who were confirmed as suspected cases (PAT0.04),one case confirmed as non-patient among the ten Brucella sufferers who were proved as existing Brucella in the blood samples.Positive predictive value of PCR detection was 67%～100%.
8.Positive rate of PCR detection were 82.9%,80.0%and 60.0%separately among those who were diagnosed as patients(34 cases),non-patients(25 cases) and suspected cases(16 cases) by immunology.
9.By constructing mutative box,which means the recombinant plasmid of pUC19K-NC,and under the principle of gene homologous recombination,part of bp26 gene in the M5 vaccine strain was deleted,M5△bp26 tagged by molecular was constructed.Two pairs of primers were designed specific to deletion sequences of bp26 gene and every highly conserved sequences,respectively.Double-PCR detection methods were established and optimized which could be used to identify vaccine strains and mutant strains.
10.Bacterial strains marked by M5△bp26 could live in macrophages and mice body,but the viability of it was lower than mild strain.Marked strains and vaccine strains M5 could both induce specific antibody in the body of Balb/c mice,but humoral immune response induced by marked strains was weaker and had shorter duration than that induced by M5.Immune protective effect of marked strains was dissatisfactory when it was attacked by B.mellitensis 16M.
Conclusions:
1.Primers could be used to identify the strains of Brucella,which were designed to target to BP26 coding gene of Brucella diagnostic antigen and parenthetical sequences of IS711 designed to identify B.melitensis,B.abortus and B.suis separately.The sensitivity of boiling method was better than inhibitor stimulation,so it could be performed on clinical research of PCR detection.
2.When being used to detect blood infected by Brucella or came from Brucellosis patients,it showed that the sensitivities of PCR detection using BP26 primers or primers of B.melitensis were low,but specificities were high;the sensitivities of PCR detection using B4 primers and B5 primers were high,and high coincidence rate with indicators of isolated culture and indicators of immunology. Positive amplification was shown among suspected cases and non-patients,but was not shown among healthy people.The method of PCR detection could be used to follow up Brucellosis patients after treatment.
3.Database of Brucellosis epidemiological information on cases which were collected at Songyuan CDC was built.
4.Molecular marked strains M5△bp26 which deleted part of bp26 gene were built,and dual-PCR detection methods were established to identify vaccine strains and mutant strains.Bacterial strains marked by M5△bp26 could live in macrophages and mice body,but the its viability was lower than mild strain.Marked strains and vaccine strains M5 could both induce specific antibody in the body of Balb/c mice,but humoral immune response induced by marked strains was weaker and had shorter duration than that induced by M5.
5.Objectives remaining to study are:
(1)To follow up those people whose blood are proved positive when being cultured isolated or detected positive with PCR assay or who are proved to be Brucellosis patients diagnosed by immunological detection after their treatment in three,six,twelve months separately.To evaluate status and future of PCR techniques for the diagnosis and follow-up of human brucellosis used to discover relapses patients.
(2) Following up those people who are diagnosed as Brucella positive by PCR detection but non-patients when diagnosed by immunology detection,so as to make sense of the PCR detection method when it is used to discover missed diagnosis.
(3) Immune protective effect of marked strains was dissatisfactory,which were deleted of bp26 gene.But BP26 antigen had been expressed and purified,which could be used as subunit vaccine to be continued to research on.
引文
[1]卫生部疾病控制司编著.布鲁氏菌病手册[M].北京:人民卫生出版社,2008
[2]Yagupsky P,Baron EJ.Laboratory exposures to brucellae and implications for bioterrorism[J].Emerging InfectiousDiseases,2005,11(8):1180-1185
[3]Memish ZA,Balkhy HH.Brucellosis and international traval[J].J Travel Med,2004,11(1):49-55
[4]Ross HM,Foster G,Reid RJ,et al.Brucella species infection in sea-mammals [J].Vet Rec,1994,134(14):359
[5]Foster G,Jahans KL,Reid RJ,et al.Isolation of Brucella species from cetaceans,seals and an otter[J].Vet Rec,1996,138(24):583-6
[6]Cloeckaert A,Verger JM,Grayon M,et al.Classification of Brucella spp.Isolated from marine mammals by DNA polymorphism at the omp2 locus[J].Microbes Infect,2001,3(9):729-738
[7]Jahans KL,Foster G,Broughton ES.The characterization of Brucella strains isolated from marine mammals[J].Vet Microbiol,1997,57(54):373-382
[8]Clavareau C,Wellemans V,Walravens K,et al.Phenotypic and molecular characterization of a Brucella strain isolated from a minke whale[J].Microbiology,1998,144(Pt 12):3267-3273
[9]Verger JM,Grayon M,Cloeckaert A,et al.Classification of Brucella strains isolated from marine mammals using DNA-DNA hybridization and ribotyping [J].Res Microbiol,2000,151(9):797-799
[10]吉林省地方病第一防治研究所.2000年-2008年吉林省布病疫情及分析[C].全省布病防治工作会议资料,2008.
[11]Sarafidis K,Agakidis C,Diamanti E,et al.Congenital brucellosis:a rare cause of respiratory distress in neonates[J].Am J Perinatol,2007,24(7):409-12
[12]Arroyo Carrera I,Lopez Rodriguez MJ,Sapina AM,et al.Probable transmission of brucellosis by breast milk[J].J Trop Pediatr,2006,52(5):380-1
[13]Palanduz A,Palanduz S,G(u|")ler K,et al.Brucellosis in a mother and her young infant:probable transmission by breast milk[J].Int J Infect Dis,2000,4(1):55-6
[14]李元凯,邱海燕.布氏菌L型最适培养基的优选及L型布氏菌生长特性的研究[J].中国地方病杂志,2001,(3):56-58
[15]Ariza J,Corrediora J,Pallares R,et al.Characteristics of and risk factors for a relapse of brucellosis in humans[J].Clin Infect Dis,1995,20(5):1241-1249
[16]Solera J,Martinez-Alfaro E,Espinosa A,et al.Multivariate model for predicting relapses in human brucellosis[J].J Infect,1998,36(1):85-92
[17]Bowden RA,Estein SM,Zygrnunt MS,et al.Identification of protective outer membrane antigens of Brucella ovis by passive immunization of mice with monoclonal antibodies[J].Microbes Infect,2000,2(5):481-488
[18]Ratushna VG,Sturgill DM,Ramamoorthy S,et al.Molecular targets for rapid identification of Brucella spp[J].BMC Microbiology,2006,6:13
[19]Vizcaino N,Cloeckaert A,Verger J,et al.DNA polymorphism in the genus Brucella[J].Microbes Infect,2000,2(9):1089-1100
[20]Halling SM,Tatum FM,Bricker BJ.Sequence and characterization of an insertion sequence,IS711,from Brucella ovis[J].Gene,1993,133(1):123-127
[21]Quahrani S,Michaux S,Sriwidada,et al.Identification and sequence analysis of IS6501,an insertion sequence in Brucella spp.relationship between genomic structure and the number of IS6501 copies[J].Gen Microbiol,1993,139(12):3265-3273
[22]Halling SM,Zehr ES.Polymorphism in in Brueella spp.due to highly repeated DNA[J].Bacteriol,1990,172(12):6637-6640
[23]Halling SM,Bricker BJ.Characterization and occurrence of two repeated palindromic DNA elements of Brucella spp:Bru-RS1 and Bru-RS2[J].Mol Microbiol,1994,14(4):681-689
[24]Allen CA,Adams LG,Ficht TA.Transposon-derived Brucella abortus rough mutants are attenuated and exhibit reduced intracellular survival [J]. Infect Immun, 1998, 66(3): 1008-1016
[25] Godfroid F, Taminiau B, Danese I, et al. Identification of the perosamine synthetase gene of Brucella melitensis 16M and involvement of lipopolysaccharide O side chain in Brucella survival in mice and in macrophages[J]. Infect Immun, 1998, 66(11): 5485-5493
[26] Cloeckaert A, Grayon M, Verger JM, et al. Conservation of seven genes involved in the biosynthesis of the lipopolysaccharide O-side chain in Brucella spp. [J]. Res Mccrobiol, 2000, 151(3): 209-216
[27] Cloeckaert A, Vizcaino N, Paquet JY, et al. Major outer member proteins of Brucella spp: past, present and future [J]. Vet.Microbiol, 2002, 90(1-4): 229-247
[28] Ficht TA, Bearden SW, Sowa BA, et al. A 36-kilodalton Brucella abortus cell envelope protein is encoded by repeated sequences closely linked in the genomic DNA [J]. Infect Immun. 1988, 56(8): 2036-2046
[29] Ficht TA, Bearden SW, Sowa BA, et al. DNA sequence and expression of the 36-kilodalton out membrane protein gene of Brucella abortus [J]. Infect Immun, 1989, 57(11): 3281-3291
[30] Cloeckaert A, Verger JM, Grayon M, et al. Restriction site polymorphism of the genes encoding the major 25kDa and 36 kDa outer-membrane proteins of Brucella [J]. Microbiology, 1995, 141(Pt 9): 2111-2121
[31] Vizcaino N, Verger JM, Grayon M, et al. DNA polymorphism at the omp-31 locus of Brucella spp: evidence for a large deletion in Brucella abortus, and other species-specific markers [J]. Microbiology, 1997, 143(Pt 9): 2913-2921
[32] Vizcaino N, Cloeckaert A, Zygmunt MS, et al. Cloning, nucleotide sequence and expression of the Brucella melitensis omp31 gene coding for an immunogenic major outer membrane protein [J]. Infect Immune, 1996, 64(9): 3744-3751
[33]Vizcaino N,Kittelberger R,Cloeckaert A,et al.Minor nucleotide substitutions in the omp31 gene of Brucella ovis result in antigenic differences in the major outer membrane protein that it encodes compared to those of the other Brucella species[J].Infect Immune,2001,69(11):7020-7028
[34]Cloeckaert A,Verger J M,Grayon M,et al.Polymorphism at the dnaK locus of Brucella species and identification of a Brucella melitensis species-specific marker[J].Med Microbilo,1996,45(3):200-205
[35]Yang X,Walters N,Robison A,et al.Nasal immunization with recombinant Brucella melitensis bp26 and trigger factor with cholera toxin reduces B.melitensis colonization[J].Vaccine,2007,25(12):2261-2268
[36]Ahvonen P,Jansson E,Aho K.Marked cross-agglutination between brucellae and a subtype of Yersinia enterocolitica[J].Acta Pathol Microbiol Scand,1969,75(2):291-295
[37]Corbel MJ.The relationship between the protective and cross reacting antigenes of Brucella spp.,Yersinia enterocolitica 0:9 and Salmonella serotypes of Kauffmann-White group N.Contr[J].Microbiol Immunol,1979,5:50-63
[38]Kittelberger R,Reichel MP,Joyce MA,et al.Serological crossreactivity between Brucella abortus and Yersinia enterocolitica O:9.Ⅲ.Specificity of the in vitro antigen-specific gamma interferon test for bovine brucellosis diagnosis in experimentall Yersinia enterocolitica O:9-infected cattle[J].Vet Microbiol,1997,57(4):361-371
[39]陈伟业,王淑杰,王永,等.重组布氏杆菌BP26 和 OMP31蛋白作为间接ELISA诊断抗原的研究[J].中国人兽共患病学报,2006,22(6):518-582
[40]Al Dahouk S,Tomaso H,Nockler K,et al.Laboratory-based diagnosis of brucellosis-a review of the literature.Part Ⅱ:serological tests for brucellosis.Clin Lab 2003,49(11-12):577-589
[41]Al Dahouk S,Tomaso H,Nockler K,et al.The detection of Brucella spp.using PCR-ELISA and real-time PCR assays[J].Clin Lab,2004,50(7-8):387-394
[42] Kiel FW, Khan MY. Analysis of 506 consecutive positive serologic tests for brucellosis in Saudi Arabia [J]. J Clin Microbiol, 1987, 25(8): 1384-1387
[43] Irmak H, Buzgan T, Evirgen O, et al. Use of a new, simple and rapid diagnostic test, the Brucella-IgM and IgG flow assays in the serodiagnosis of human brucellosis inan endemic area in Eastern Turkey [J]. Am J Trop Med Hyg 2004, 70: 688-694.
[44] Lucero NE, Escobar GI, Ayala SM, et al. Fluorescence polarization assay for diagnosis of human brucellosis [J]. J Med Microbiol, 2003, 52(Pt 10): 883-887
[45] Maichomo MW, McDermott JJ, Arimi SM, et al. Assessment of the Rose-Bengal plate test for the diagnosis of humanbrucellosis in health facilities in Narok district, Kenya [J]. East Afr Med J, 1998, 75: 219-222.
[46] Orduna A, Almaraz A, Prado A, et al. Evaluation of an immunocapture -agglutination test (Brucellacapt) for serodiagnosis of human brucellosis [J]. J Clin Microbiol, 2000, 38(11): 4000-4005
[47] Al-Attas RA, Al-Khalifa M, Al-Qurashi AR, et al. Evaluation of PCR, culture and serology for the diagnosis of acute human brucellosis [J]. Ann Saudi Med, 2000, 20(3-4): 224-8
[48] Mitka S, Anetakis C, Souliou E, et al. Evaluation of Different PCR Assays for Early Detection of Acute and Relapsing Brucellosis in Humans in Comparison with Conventional Methods [J]. Journal of Clinical Microbiology, 2007, 45 (4) : 1211-1218
[49] Maha Almuneef, Ziad A. Memish Letters to the Editor: Persistence of Brucella Antibodies after Successful Treatment of Acute Brucellosis in an Area of Endemicity [J]. Clin Microbiol, 2002,40(6): 2313
[50] Yagupsky P, Peled N, Press J, et al. Rapid detection of Brucella melitensis from blood cultures by a commercial system [J]. Eur J Clin Microbiol Infect Dis, 1997, 16(8): 605-607.
[51] Yagupsky P, Peled N, Press J, et al. Comparison of BACTEC 9240 Peds Plus medium and Isolator 1.5 microbial tube for detection ofBrucella melitensis from blood cultures[J].J Clin Microbiol,1997,35(6):1382-1384
[52]Colmenero JD,Reguera JM,Martos F,et al.Complications associated with Brucella melitensis infection:A study of 530 cases[J].Medicine,1996,75(4):195-211
[53]Navarro E,Fernandez JA,Escribano J,et al.PCR Assay for Diagnosis of Human Brucellosis[J].Clin Microbiol,1999,37(5):1654-1655
[54]唐浏英,邱海燕,李元凯,等.多聚酶链反应用于布鲁氏菌病病人诊断的流行病学评价[J].中华流行病学杂志,1997,18(3):153-155
[55]尚德秋,唐浏英,邱海燕,等.布氏菌病新诊断技术的研究[J].中国地方病防治杂志1999,14(3):141-144
[56]李铁锋,何玉萍,孙海波.分子生物学方法在布氏菌病病原学诊断中的应用[J].中国地方病防治杂志,1999,14(6):345-347
[57]王季秋,李铁锋,张桂珍,等.全血聚合酶链反应试验在诊断布氏菌病中的应用效果观察[J].中国地方病防治杂志,2003,18(1):10-12
[58]王丽,马国柱.PCR技术用于布鲁氏菌病的诊断研究[J].中国地方病防治杂志,2004,19(2):65-67
[59]赵智香.布鲁氏菌病诊断方法的对比研究[D].内蒙古农业大学,2007
[60]李铁锋,王季秋,森林,等.PCR检测布氏菌DNA的试验研究[J].中国地方病防治杂志,2002,17(4):211-213
[61]Baddour MM,Alkhalifa DH.Evaluation of three polymerase chain reaction techniques for detection of Brucella DNA in peripheral human blood[J].Can J Microbiol,2008,54(5):352-7
[62]Romero C,Gamazo C,Pardo M,et al.Specific detection of Brucella DNA by PCR[J].J Clin Microbiol,1995,33(3):615-617
[63]Da Costa M,Guillou JP,Garinp-Bastuji,et al.Specificity of six gene sequences for the dectection of the genus Brucella by DNA amplification[J].Appl Bacteriol,1996,81:267-275
[64]Gallien P,Dorn C,Alban G,et al.Detection of Brucella species in organs of naturally infected cattle by polymerase chain reaction[J].Vet Rec,1998,142(19):512-514
[65]Bricker BJ.PCR as a diagnositic tool for brucellosis[J].Vet Microbiol,2002,90(1-4):435-446
[66]Navarro E,Escribano J,Fernandez J,et al.Comparison of three different PCR methods for detection of Brucella spp in human brucellosis[J].FEMS Immunol Med Microbiol,2002,34(2):147-151
[67]Mayfield JE,Bricker BJ,Godfrey H,et al.The cloning and nucleotide sequence of a gene coding for an immunogenic Brucella abortus protein[J].Gene,1988,63(1):1-9
[68]Bricker BJ,SM Halling.Enhancement of the Brucella AMOS PCR assay for differentiation of Brucella abortus vaccine strains S19 and RB51[J].J Clin Microbiol,1995,33(6):1640-1642
[69]Zerva L,Bourantas K,Mitka S,et al.Serum is the preferred clinical specimen for diagnosis of human brucellosis by PCR[J].J Clin Microbiol,2001,39(4),1661-1664
[70]Leal-Klevezas DS,Martinez-Vazquez IO,Lopez-Merino A,et al.Single-Step PCR for Detection of Brucella spp.from Blood and Milk of Infected Animals[J].Journal of Clinical Microbiology,1995,33(12):3087-3090
[71]Navarro E,Fernandez JA,Escribano J,et al.PCR Assay for Diagnosis of Human Brucellosis[J].Clin Microbiol,1999,37(5):1654-1655
[72]Morata P,Queipo-Ortuno MI,de Dios Colmenero J.Strategy for Optimizing DNA Amplification in a Peripheral Blood PCR Assay Used for Diagnosis of Human Brucellosis[J].Journal of Clinical Microbiology,1998,36(9):2443-2446
[73]杜昕颖,陈泽良,王玉飞,等.多重PCR特异检测布鲁氏菌方法的建立[J].解放军预防医学杂志,2008,26(5):341-343
[74]Matar GM,Khneisser IA,Abdelnoor AM.Rapid laboratory confirmation of human brucellosis by PCR analysis of a target sequence on the 31-Kilodalton Brucella DNA[J].J Clin Microbiol,1996,34(2):477-478
[75]Navarro-Martinez A,Navarro E,Castano MJ,et al.Rapid diagnosis of human brucellosis by Quantitative Real-Time Polymerase Chain Reaction:A case report of brucellar spondylitis[J].Clin Microbiol,2008,46(1 ):385-387
[76]Nielsen K,JR Duncan.Antibody isotype response in adult cattle vaccinated with Brucella abortus S19[J].Vet Immunol Immunopathol,1988,19(3-4):205-214
[77]Nielsen KH,Duncan JR,Stemshorn BW.Non-specific reactions to the Brucella abortus SAT[J].Vet Rec,1985,116(20):550-552
[78]Ko J,Splitter GA.Molecular host-pathogen interation in Brucellosis:current understanding and future approaches to vaccine development for mice and humans[J].Clin Microbiol Rev,2003,16(1):65-78
[79]Gerhardt G,Schurig NA,Michael J.Brucellosis vaccines:past,present and future[J].Vet Microbiol,1990,20(20):479-496
[80]Cerone SI,AE Parma,RA Bowden,et al.Kinetic analysis of cattle anti-Brucella abortus S45/20 non-agglutinating antibodies in antibody-dependent cell-mediated cytotoxicity[J].Vet Microbiol,1987,13(3):273-279
[81]尚得秋.布氏杆菌病(M)//唐家琪.自然医源性疾病.北京:科学出版社,2003,p.869-883
[82]Zygmunt MS,Debbarh HS,Cloeckaert A,et al.Antibody response to Brucella melitensis outer membrane antigens in naturally infected and Revl vaccinated sheep[J].Veterinary microbiology,1994,39(1-2):33-46
[83]Verger JM,Grayon M,Zundel E,et al.Comparison of the efficacy of Brucella suis strain 2 and Brucella melitensis Rev.1 live vaccines against a Brucella melitensis experimental infection in pregnant ewes[J].Vaccine,1995,13(2):191-196
[84]Stevens MG,Olsen SC,Cheville NF.Lymphocyte proliferation in response to immunodominant antigens of Brucella abortus 2308 and RB51 in strain 2308-infected cattle[J].Infect Immun,1994,62(10):4646-4649
[85]Stevens MG,SC Olsen,Palmer MV,et al.Immune responses and resistance to brucellosis in mice vaccinated orally with Brucella abortus RB51[J].Infect Immun,1996,64(11 ):4534-4541
[86]Stevens MG,Olsen SC,Pugh GW,et al.Comparison of immune responses and resistance to brucellosis in mice vaccinated with Brucella abortus 19 or RB51[J].Infect Immun,1995,63(1):264-270
[87]Vemulapalli R,He Y,Buccolo LS,et al.Complementation of Brucella abortus RB51 with a functional wboA gene results in O-antigen synthesis and enhanced vaccine efficacy but no change in rough phenotype and attenuation [J].Infect Immun,2000,68(7):3927-3932
[88]Olsen SC,Stofregen WS.Essential role of vaccines in brucellosis control and eradication programs for livestock[J].Expert Rev Vaccines,2005,4(6):915-925
[89]Cassataro J,Velikovsky CA,de la Barrera S,et al.A DNA Vaccine Coding for the Brucella Outer Membrane Protein 31 Confers Protection against B.melitensis and B.ovis Infection by Eliciting a Specific Cytotoxic Response [J].Infection and Immunity,2005,73(10):6537-6546
[90]Seco-Mediavilla P,Verger JM,Grayon M,et al.Epitope mapping of the Brucella melitensis BP26 immunogenic protein:usefulness for diagnosis of sheep brucellosis[J].Clin Diagn Lab Immunol,2003,10:647-651
[91]胡森,步志高.布氏杆菌病概况及其研究进展[J].畜牧兽医科技信息,2003,(12):9-11
[92]布鲁氏菌病诊断标准及处理原则(GB 15988-1995)
[93]WS布鲁氏菌病诊断方法,北京:中华人民共和国卫生部,2007
[94]Gotuzzo E,Carrillo C,Guerra J,et al.An evaluation of diagnostic methods for brucellosis-the value of blood marrow culture[J]. J Infect Dis, 1986, 153(1): 122-125
[95] Morata P, Queipo-Ortuno MI, de Dios Colmenero J. Strategy for Optimizing DNA Amplification in a Peripheral Blood PCR Assay Used for Diagnosis of Human Brucellosis [J]. Journal of Clinical Microbiology, 1998, 36(9): 2443-2446
[96] Morata P, Queipo-Ortuno MI, Reguera JM et al. Post treatment follow-up of brucellosis by PCR assay [J]. Clin Microbiol, 1999, 37: 4163-4166.
[97] Nimri LF. Diagnosis of recent and relapsed cases of human brucellosis by PCR assay[J]. BMC Infect Dis, 2003, 28: 3-5
[98] Morata P, Queipo-Ortuno MI, Reguera JM, et al. Diagnostic yield of a PCR assay in focal complications of brucellosis [J]. J Clin Microbiol, 2001, 39(10): 3743-6
[99] Gazapo E, Gonzalez Lahoz J, Subiza JL, et al. Changes in IgM and IgG antibodies concentrations in brucellosis over time: importance for diagnosis and follow-up. J Infect. Dis. 1989.159(2):219-225
[100]Cloeckaert A, Debbarh HS, Vizcaino N, et al. Cloning, nucleotide sequence, and expression of the Brucella melitensis bp26 gene coding for a protein immunogenic in infected sheep [J]. FEMS Microbiol left, 1996, 140(2-3): 139-144
[101]Lindler LE, Hadfield TL, Tall BD, et al. Cloning of a Brucella melitensis group 3 antigen gene encoding Omp28, a protein recognized by the humoral immune response during human brucellosis [J]. Infect Immun, 1996, 64(7): 2490-2499
[102]Rossetti OL, Arese AI, Boschiroli ML, et al. Cloning of Brucella abortus gene and characterization of expressed 26-kilodalton periplasmic protein: potential use for diagnosis [J]. J Clin Microbiol, 1996, 34(1): 165-169
[103]Salih-Alj Debbarh H, Cloeckaert A, Cloeckaert A, et al. Enzyme-linked immunosorbent assay with partially purified cytosoluble 28-kilodalton protein for serological differentiation between Brucella melitensis-infected and B. melitensis Rev. 1-vaccinated sheep[J]. Clin Diagn Lab Immunol, 1996,3(3): 305-308
[104]Debbarh HS, Zygmunt MS, Dubray G, et al. Competitive enzyme-linked immunosorbent assay using monoclonal antibodiesto the Brucella melitensis BP26 protein to evaluate antibody responses in infected and B. melitensis Rev.1 vaccinated sheep. Vet Microbiol, 1996, 53(3-4): 325-337
[105]Boschiroli ML, Cravero SL, Arese AI, et al . Protection against infection in mice vaccinated with a Brucella abortus mutant[J]. Infect Immun, 1997, 65(2): 798-800
[106] Cloeckaet A, Jacques I, Grillo MJ, et al. Development and evaluation as vaccines in mice of Brucella melitensis Rev.l single and double deletion mutants of the bp26 and omp31 genes coding for antigens of diagnostic significance in ovine brucellosis. Vaccine, 2004, 22(21-22): 2827-2835
[107] Jacques I, Verger JM, Laroucau K, et al. Immunological responses and protective efficacy against Brucella melitensis induced by bp26 and omp31 B. melitensis Rev.l deletion mutants in sheep. Vaccine, 2007, 25(5): 794-805
[108] Campos E, Cravero L, Delgui L, et al. Brucella abortus INTA2, a novel strain 19 (Delta)bp26::luc (Delta)bmp18 double mutant lacking drug resistance markers [J]. Vet Microbiol, 2002, 87(1): 1-13
[109] Roop RM, Gee JM, Robertson GT, et al. Brucella stationary-phase gene expression and virulence[J]. Annu Rev Microbiol, 2003, 57: 57-76
[110] Ficht TA. Intracellular survival of Brucella: defining the link with persistence[J]. Vet Microbiol, 2003, 92(3): 213-223
[111]Guilloteau LA, Laroucau K, Olivier M, et al. Residual virulence and immunogenicity of CGV26 and CGV2631 B. melitensis Rev. 1 deletion mutant strains in sheep after subcutaneous or conjunctival vaccination[J]. Vaccine. 2006, 24(17): 3461-3468
[112]Rajashekara G, DA Glover, M Banai, et al. Attenuated bioluminescent Brucella melitensis mutants GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091) confer protection in mice [J]. Infect Immun, 2006, 74(5): 2925-2936
[113]Mosmann TR, Sad S. The expanding universe of T-cell subset:Thl,Th2 and more[J]. Immunol Today, 1996, 17(3): 138-146
[114] Murphy EA, Sathiyaseelan J, Parent MA, et al. Interferon-gamma is crucial for surviving a Brucella abortus infection in both resistant C57BL/6 and susceptible Balb/c mice[J]. Immunology, 2001, 103(4): 511-8
[115] Jiang X, Baldwin CL. Iron augments macrophage-mediated killing of Brucella abortus alone and in conjunction with interferon-gamma [J]. Cell Immunol, 1993, 148(2): 397-407
[2]Yagupsky P,Baron EJ.Laboratory exposures to brucellae and implications for bioterrorism[J].Emerging InfectiousDiseases,2005,11(8):1180-1185
[3]Memish ZA,Balkhy HH.Brucellosis and international traval[J].J Travel Med,2004,11(1):49-55
[4]Ross HM,Foster G,Reid RJ,et al.Brucella species infection in sea-mammals [J].Vet Rec,1994,134(14):359
[5]Foster G,Jahans KL,Reid RJ,et al.Isolation of Brucella species from cetaceans,seals and an otter[J].Vet Rec,1996,138(24):583-6
[6]Cloeckaert A,Verger JM,Grayon M,et al.Classification of Brucella spp.Isolated from marine mammals by DNA polymorphism at the omp2 locus[J].Microbes Infect,2001,3(9):729-738
[7]Jahans KL,Foster G,Broughton ES.The characterization of Brucella strains isolated from marine mammals[J].Vet Microbiol,1997,57(54):373-382
[8]Clavareau C,Wellemans V,Walravens K,et al.Phenotypic and molecular characterization of a Brucella strain isolated from a minke whale[J].Microbiology,1998,144(Pt 12):3267-3273
[9]Verger JM,Grayon M,Cloeckaert A,et al.Classification of Brucella strains isolated from marine mammals using DNA-DNA hybridization and ribotyping [J].Res Microbiol,2000,151(9):797-799
[10]吉林省地方病第一防治研究所.2000年-2008年吉林省布病疫情及分析[C].全省布病防治工作会议资料,2008.
[11]Sarafidis K,Agakidis C,Diamanti E,et al.Congenital brucellosis:a rare cause of respiratory distress in neonates[J].Am J Perinatol,2007,24(7):409-12
[12]Arroyo Carrera I,Lopez Rodriguez MJ,Sapina AM,et al.Probable transmission of brucellosis by breast milk[J].J Trop Pediatr,2006,52(5):380-1
[13]Palanduz A,Palanduz S,G(u|")ler K,et al.Brucellosis in a mother and her young infant:probable transmission by breast milk[J].Int J Infect Dis,2000,4(1):55-6
[14]李元凯,邱海燕.布氏菌L型最适培养基的优选及L型布氏菌生长特性的研究[J].中国地方病杂志,2001,(3):56-58
[15]Ariza J,Corrediora J,Pallares R,et al.Characteristics of and risk factors for a relapse of brucellosis in humans[J].Clin Infect Dis,1995,20(5):1241-1249
[16]Solera J,Martinez-Alfaro E,Espinosa A,et al.Multivariate model for predicting relapses in human brucellosis[J].J Infect,1998,36(1):85-92
[17]Bowden RA,Estein SM,Zygrnunt MS,et al.Identification of protective outer membrane antigens of Brucella ovis by passive immunization of mice with monoclonal antibodies[J].Microbes Infect,2000,2(5):481-488
[18]Ratushna VG,Sturgill DM,Ramamoorthy S,et al.Molecular targets for rapid identification of Brucella spp[J].BMC Microbiology,2006,6:13
[19]Vizcaino N,Cloeckaert A,Verger J,et al.DNA polymorphism in the genus Brucella[J].Microbes Infect,2000,2(9):1089-1100
[20]Halling SM,Tatum FM,Bricker BJ.Sequence and characterization of an insertion sequence,IS711,from Brucella ovis[J].Gene,1993,133(1):123-127
[21]Quahrani S,Michaux S,Sriwidada,et al.Identification and sequence analysis of IS6501,an insertion sequence in Brucella spp.relationship between genomic structure and the number of IS6501 copies[J].Gen Microbiol,1993,139(12):3265-3273
[22]Halling SM,Zehr ES.Polymorphism in in Brueella spp.due to highly repeated DNA[J].Bacteriol,1990,172(12):6637-6640
[23]Halling SM,Bricker BJ.Characterization and occurrence of two repeated palindromic DNA elements of Brucella spp:Bru-RS1 and Bru-RS2[J].Mol Microbiol,1994,14(4):681-689
[24]Allen CA,Adams LG,Ficht TA.Transposon-derived Brucella abortus rough mutants are attenuated and exhibit reduced intracellular survival [J]. Infect Immun, 1998, 66(3): 1008-1016
[25] Godfroid F, Taminiau B, Danese I, et al. Identification of the perosamine synthetase gene of Brucella melitensis 16M and involvement of lipopolysaccharide O side chain in Brucella survival in mice and in macrophages[J]. Infect Immun, 1998, 66(11): 5485-5493
[26] Cloeckaert A, Grayon M, Verger JM, et al. Conservation of seven genes involved in the biosynthesis of the lipopolysaccharide O-side chain in Brucella spp. [J]. Res Mccrobiol, 2000, 151(3): 209-216
[27] Cloeckaert A, Vizcaino N, Paquet JY, et al. Major outer member proteins of Brucella spp: past, present and future [J]. Vet.Microbiol, 2002, 90(1-4): 229-247
[28] Ficht TA, Bearden SW, Sowa BA, et al. A 36-kilodalton Brucella abortus cell envelope protein is encoded by repeated sequences closely linked in the genomic DNA [J]. Infect Immun. 1988, 56(8): 2036-2046
[29] Ficht TA, Bearden SW, Sowa BA, et al. DNA sequence and expression of the 36-kilodalton out membrane protein gene of Brucella abortus [J]. Infect Immun, 1989, 57(11): 3281-3291
[30] Cloeckaert A, Verger JM, Grayon M, et al. Restriction site polymorphism of the genes encoding the major 25kDa and 36 kDa outer-membrane proteins of Brucella [J]. Microbiology, 1995, 141(Pt 9): 2111-2121
[31] Vizcaino N, Verger JM, Grayon M, et al. DNA polymorphism at the omp-31 locus of Brucella spp: evidence for a large deletion in Brucella abortus, and other species-specific markers [J]. Microbiology, 1997, 143(Pt 9): 2913-2921
[32] Vizcaino N, Cloeckaert A, Zygmunt MS, et al. Cloning, nucleotide sequence and expression of the Brucella melitensis omp31 gene coding for an immunogenic major outer membrane protein [J]. Infect Immune, 1996, 64(9): 3744-3751
[33]Vizcaino N,Kittelberger R,Cloeckaert A,et al.Minor nucleotide substitutions in the omp31 gene of Brucella ovis result in antigenic differences in the major outer membrane protein that it encodes compared to those of the other Brucella species[J].Infect Immune,2001,69(11):7020-7028
[34]Cloeckaert A,Verger J M,Grayon M,et al.Polymorphism at the dnaK locus of Brucella species and identification of a Brucella melitensis species-specific marker[J].Med Microbilo,1996,45(3):200-205
[35]Yang X,Walters N,Robison A,et al.Nasal immunization with recombinant Brucella melitensis bp26 and trigger factor with cholera toxin reduces B.melitensis colonization[J].Vaccine,2007,25(12):2261-2268
[36]Ahvonen P,Jansson E,Aho K.Marked cross-agglutination between brucellae and a subtype of Yersinia enterocolitica[J].Acta Pathol Microbiol Scand,1969,75(2):291-295
[37]Corbel MJ.The relationship between the protective and cross reacting antigenes of Brucella spp.,Yersinia enterocolitica 0:9 and Salmonella serotypes of Kauffmann-White group N.Contr[J].Microbiol Immunol,1979,5:50-63
[38]Kittelberger R,Reichel MP,Joyce MA,et al.Serological crossreactivity between Brucella abortus and Yersinia enterocolitica O:9.Ⅲ.Specificity of the in vitro antigen-specific gamma interferon test for bovine brucellosis diagnosis in experimentall Yersinia enterocolitica O:9-infected cattle[J].Vet Microbiol,1997,57(4):361-371
[39]陈伟业,王淑杰,王永,等.重组布氏杆菌BP26 和 OMP31蛋白作为间接ELISA诊断抗原的研究[J].中国人兽共患病学报,2006,22(6):518-582
[40]Al Dahouk S,Tomaso H,Nockler K,et al.Laboratory-based diagnosis of brucellosis-a review of the literature.Part Ⅱ:serological tests for brucellosis.Clin Lab 2003,49(11-12):577-589
[41]Al Dahouk S,Tomaso H,Nockler K,et al.The detection of Brucella spp.using PCR-ELISA and real-time PCR assays[J].Clin Lab,2004,50(7-8):387-394
[42] Kiel FW, Khan MY. Analysis of 506 consecutive positive serologic tests for brucellosis in Saudi Arabia [J]. J Clin Microbiol, 1987, 25(8): 1384-1387
[43] Irmak H, Buzgan T, Evirgen O, et al. Use of a new, simple and rapid diagnostic test, the Brucella-IgM and IgG flow assays in the serodiagnosis of human brucellosis inan endemic area in Eastern Turkey [J]. Am J Trop Med Hyg 2004, 70: 688-694.
[44] Lucero NE, Escobar GI, Ayala SM, et al. Fluorescence polarization assay for diagnosis of human brucellosis [J]. J Med Microbiol, 2003, 52(Pt 10): 883-887
[45] Maichomo MW, McDermott JJ, Arimi SM, et al. Assessment of the Rose-Bengal plate test for the diagnosis of humanbrucellosis in health facilities in Narok district, Kenya [J]. East Afr Med J, 1998, 75: 219-222.
[46] Orduna A, Almaraz A, Prado A, et al. Evaluation of an immunocapture -agglutination test (Brucellacapt) for serodiagnosis of human brucellosis [J]. J Clin Microbiol, 2000, 38(11): 4000-4005
[47] Al-Attas RA, Al-Khalifa M, Al-Qurashi AR, et al. Evaluation of PCR, culture and serology for the diagnosis of acute human brucellosis [J]. Ann Saudi Med, 2000, 20(3-4): 224-8
[48] Mitka S, Anetakis C, Souliou E, et al. Evaluation of Different PCR Assays for Early Detection of Acute and Relapsing Brucellosis in Humans in Comparison with Conventional Methods [J]. Journal of Clinical Microbiology, 2007, 45 (4) : 1211-1218
[49] Maha Almuneef, Ziad A. Memish Letters to the Editor: Persistence of Brucella Antibodies after Successful Treatment of Acute Brucellosis in an Area of Endemicity [J]. Clin Microbiol, 2002,40(6): 2313
[50] Yagupsky P, Peled N, Press J, et al. Rapid detection of Brucella melitensis from blood cultures by a commercial system [J]. Eur J Clin Microbiol Infect Dis, 1997, 16(8): 605-607.
[51] Yagupsky P, Peled N, Press J, et al. Comparison of BACTEC 9240 Peds Plus medium and Isolator 1.5 microbial tube for detection ofBrucella melitensis from blood cultures[J].J Clin Microbiol,1997,35(6):1382-1384
[52]Colmenero JD,Reguera JM,Martos F,et al.Complications associated with Brucella melitensis infection:A study of 530 cases[J].Medicine,1996,75(4):195-211
[53]Navarro E,Fernandez JA,Escribano J,et al.PCR Assay for Diagnosis of Human Brucellosis[J].Clin Microbiol,1999,37(5):1654-1655
[54]唐浏英,邱海燕,李元凯,等.多聚酶链反应用于布鲁氏菌病病人诊断的流行病学评价[J].中华流行病学杂志,1997,18(3):153-155
[55]尚德秋,唐浏英,邱海燕,等.布氏菌病新诊断技术的研究[J].中国地方病防治杂志1999,14(3):141-144
[56]李铁锋,何玉萍,孙海波.分子生物学方法在布氏菌病病原学诊断中的应用[J].中国地方病防治杂志,1999,14(6):345-347
[57]王季秋,李铁锋,张桂珍,等.全血聚合酶链反应试验在诊断布氏菌病中的应用效果观察[J].中国地方病防治杂志,2003,18(1):10-12
[58]王丽,马国柱.PCR技术用于布鲁氏菌病的诊断研究[J].中国地方病防治杂志,2004,19(2):65-67
[59]赵智香.布鲁氏菌病诊断方法的对比研究[D].内蒙古农业大学,2007
[60]李铁锋,王季秋,森林,等.PCR检测布氏菌DNA的试验研究[J].中国地方病防治杂志,2002,17(4):211-213
[61]Baddour MM,Alkhalifa DH.Evaluation of three polymerase chain reaction techniques for detection of Brucella DNA in peripheral human blood[J].Can J Microbiol,2008,54(5):352-7
[62]Romero C,Gamazo C,Pardo M,et al.Specific detection of Brucella DNA by PCR[J].J Clin Microbiol,1995,33(3):615-617
[63]Da Costa M,Guillou JP,Garinp-Bastuji,et al.Specificity of six gene sequences for the dectection of the genus Brucella by DNA amplification[J].Appl Bacteriol,1996,81:267-275
[64]Gallien P,Dorn C,Alban G,et al.Detection of Brucella species in organs of naturally infected cattle by polymerase chain reaction[J].Vet Rec,1998,142(19):512-514
[65]Bricker BJ.PCR as a diagnositic tool for brucellosis[J].Vet Microbiol,2002,90(1-4):435-446
[66]Navarro E,Escribano J,Fernandez J,et al.Comparison of three different PCR methods for detection of Brucella spp in human brucellosis[J].FEMS Immunol Med Microbiol,2002,34(2):147-151
[67]Mayfield JE,Bricker BJ,Godfrey H,et al.The cloning and nucleotide sequence of a gene coding for an immunogenic Brucella abortus protein[J].Gene,1988,63(1):1-9
[68]Bricker BJ,SM Halling.Enhancement of the Brucella AMOS PCR assay for differentiation of Brucella abortus vaccine strains S19 and RB51[J].J Clin Microbiol,1995,33(6):1640-1642
[69]Zerva L,Bourantas K,Mitka S,et al.Serum is the preferred clinical specimen for diagnosis of human brucellosis by PCR[J].J Clin Microbiol,2001,39(4),1661-1664
[70]Leal-Klevezas DS,Martinez-Vazquez IO,Lopez-Merino A,et al.Single-Step PCR for Detection of Brucella spp.from Blood and Milk of Infected Animals[J].Journal of Clinical Microbiology,1995,33(12):3087-3090
[71]Navarro E,Fernandez JA,Escribano J,et al.PCR Assay for Diagnosis of Human Brucellosis[J].Clin Microbiol,1999,37(5):1654-1655
[72]Morata P,Queipo-Ortuno MI,de Dios Colmenero J.Strategy for Optimizing DNA Amplification in a Peripheral Blood PCR Assay Used for Diagnosis of Human Brucellosis[J].Journal of Clinical Microbiology,1998,36(9):2443-2446
[73]杜昕颖,陈泽良,王玉飞,等.多重PCR特异检测布鲁氏菌方法的建立[J].解放军预防医学杂志,2008,26(5):341-343
[74]Matar GM,Khneisser IA,Abdelnoor AM.Rapid laboratory confirmation of human brucellosis by PCR analysis of a target sequence on the 31-Kilodalton Brucella DNA[J].J Clin Microbiol,1996,34(2):477-478
[75]Navarro-Martinez A,Navarro E,Castano MJ,et al.Rapid diagnosis of human brucellosis by Quantitative Real-Time Polymerase Chain Reaction:A case report of brucellar spondylitis[J].Clin Microbiol,2008,46(1 ):385-387
[76]Nielsen K,JR Duncan.Antibody isotype response in adult cattle vaccinated with Brucella abortus S19[J].Vet Immunol Immunopathol,1988,19(3-4):205-214
[77]Nielsen KH,Duncan JR,Stemshorn BW.Non-specific reactions to the Brucella abortus SAT[J].Vet Rec,1985,116(20):550-552
[78]Ko J,Splitter GA.Molecular host-pathogen interation in Brucellosis:current understanding and future approaches to vaccine development for mice and humans[J].Clin Microbiol Rev,2003,16(1):65-78
[79]Gerhardt G,Schurig NA,Michael J.Brucellosis vaccines:past,present and future[J].Vet Microbiol,1990,20(20):479-496
[80]Cerone SI,AE Parma,RA Bowden,et al.Kinetic analysis of cattle anti-Brucella abortus S45/20 non-agglutinating antibodies in antibody-dependent cell-mediated cytotoxicity[J].Vet Microbiol,1987,13(3):273-279
[81]尚得秋.布氏杆菌病(M)//唐家琪.自然医源性疾病.北京:科学出版社,2003,p.869-883
[82]Zygmunt MS,Debbarh HS,Cloeckaert A,et al.Antibody response to Brucella melitensis outer membrane antigens in naturally infected and Revl vaccinated sheep[J].Veterinary microbiology,1994,39(1-2):33-46
[83]Verger JM,Grayon M,Zundel E,et al.Comparison of the efficacy of Brucella suis strain 2 and Brucella melitensis Rev.1 live vaccines against a Brucella melitensis experimental infection in pregnant ewes[J].Vaccine,1995,13(2):191-196
[84]Stevens MG,Olsen SC,Cheville NF.Lymphocyte proliferation in response to immunodominant antigens of Brucella abortus 2308 and RB51 in strain 2308-infected cattle[J].Infect Immun,1994,62(10):4646-4649
[85]Stevens MG,SC Olsen,Palmer MV,et al.Immune responses and resistance to brucellosis in mice vaccinated orally with Brucella abortus RB51[J].Infect Immun,1996,64(11 ):4534-4541
[86]Stevens MG,Olsen SC,Pugh GW,et al.Comparison of immune responses and resistance to brucellosis in mice vaccinated with Brucella abortus 19 or RB51[J].Infect Immun,1995,63(1):264-270
[87]Vemulapalli R,He Y,Buccolo LS,et al.Complementation of Brucella abortus RB51 with a functional wboA gene results in O-antigen synthesis and enhanced vaccine efficacy but no change in rough phenotype and attenuation [J].Infect Immun,2000,68(7):3927-3932
[88]Olsen SC,Stofregen WS.Essential role of vaccines in brucellosis control and eradication programs for livestock[J].Expert Rev Vaccines,2005,4(6):915-925
[89]Cassataro J,Velikovsky CA,de la Barrera S,et al.A DNA Vaccine Coding for the Brucella Outer Membrane Protein 31 Confers Protection against B.melitensis and B.ovis Infection by Eliciting a Specific Cytotoxic Response [J].Infection and Immunity,2005,73(10):6537-6546
[90]Seco-Mediavilla P,Verger JM,Grayon M,et al.Epitope mapping of the Brucella melitensis BP26 immunogenic protein:usefulness for diagnosis of sheep brucellosis[J].Clin Diagn Lab Immunol,2003,10:647-651
[91]胡森,步志高.布氏杆菌病概况及其研究进展[J].畜牧兽医科技信息,2003,(12):9-11
[92]布鲁氏菌病诊断标准及处理原则(GB 15988-1995)
[93]WS布鲁氏菌病诊断方法,北京:中华人民共和国卫生部,2007
[94]Gotuzzo E,Carrillo C,Guerra J,et al.An evaluation of diagnostic methods for brucellosis-the value of blood marrow culture[J]. J Infect Dis, 1986, 153(1): 122-125
[95] Morata P, Queipo-Ortuno MI, de Dios Colmenero J. Strategy for Optimizing DNA Amplification in a Peripheral Blood PCR Assay Used for Diagnosis of Human Brucellosis [J]. Journal of Clinical Microbiology, 1998, 36(9): 2443-2446
[96] Morata P, Queipo-Ortuno MI, Reguera JM et al. Post treatment follow-up of brucellosis by PCR assay [J]. Clin Microbiol, 1999, 37: 4163-4166.
[97] Nimri LF. Diagnosis of recent and relapsed cases of human brucellosis by PCR assay[J]. BMC Infect Dis, 2003, 28: 3-5
[98] Morata P, Queipo-Ortuno MI, Reguera JM, et al. Diagnostic yield of a PCR assay in focal complications of brucellosis [J]. J Clin Microbiol, 2001, 39(10): 3743-6
[99] Gazapo E, Gonzalez Lahoz J, Subiza JL, et al. Changes in IgM and IgG antibodies concentrations in brucellosis over time: importance for diagnosis and follow-up. J Infect. Dis. 1989.159(2):219-225
[100]Cloeckaert A, Debbarh HS, Vizcaino N, et al. Cloning, nucleotide sequence, and expression of the Brucella melitensis bp26 gene coding for a protein immunogenic in infected sheep [J]. FEMS Microbiol left, 1996, 140(2-3): 139-144
[101]Lindler LE, Hadfield TL, Tall BD, et al. Cloning of a Brucella melitensis group 3 antigen gene encoding Omp28, a protein recognized by the humoral immune response during human brucellosis [J]. Infect Immun, 1996, 64(7): 2490-2499
[102]Rossetti OL, Arese AI, Boschiroli ML, et al. Cloning of Brucella abortus gene and characterization of expressed 26-kilodalton periplasmic protein: potential use for diagnosis [J]. J Clin Microbiol, 1996, 34(1): 165-169
[103]Salih-Alj Debbarh H, Cloeckaert A, Cloeckaert A, et al. Enzyme-linked immunosorbent assay with partially purified cytosoluble 28-kilodalton protein for serological differentiation between Brucella melitensis-infected and B. melitensis Rev. 1-vaccinated sheep[J]. Clin Diagn Lab Immunol, 1996,3(3): 305-308
[104]Debbarh HS, Zygmunt MS, Dubray G, et al. Competitive enzyme-linked immunosorbent assay using monoclonal antibodiesto the Brucella melitensis BP26 protein to evaluate antibody responses in infected and B. melitensis Rev.1 vaccinated sheep. Vet Microbiol, 1996, 53(3-4): 325-337
[105]Boschiroli ML, Cravero SL, Arese AI, et al . Protection against infection in mice vaccinated with a Brucella abortus mutant[J]. Infect Immun, 1997, 65(2): 798-800
[106] Cloeckaet A, Jacques I, Grillo MJ, et al. Development and evaluation as vaccines in mice of Brucella melitensis Rev.l single and double deletion mutants of the bp26 and omp31 genes coding for antigens of diagnostic significance in ovine brucellosis. Vaccine, 2004, 22(21-22): 2827-2835
[107] Jacques I, Verger JM, Laroucau K, et al. Immunological responses and protective efficacy against Brucella melitensis induced by bp26 and omp31 B. melitensis Rev.l deletion mutants in sheep. Vaccine, 2007, 25(5): 794-805
[108] Campos E, Cravero L, Delgui L, et al. Brucella abortus INTA2, a novel strain 19 (Delta)bp26::luc (Delta)bmp18 double mutant lacking drug resistance markers [J]. Vet Microbiol, 2002, 87(1): 1-13
[109] Roop RM, Gee JM, Robertson GT, et al. Brucella stationary-phase gene expression and virulence[J]. Annu Rev Microbiol, 2003, 57: 57-76
[110] Ficht TA. Intracellular survival of Brucella: defining the link with persistence[J]. Vet Microbiol, 2003, 92(3): 213-223
[111]Guilloteau LA, Laroucau K, Olivier M, et al. Residual virulence and immunogenicity of CGV26 and CGV2631 B. melitensis Rev. 1 deletion mutant strains in sheep after subcutaneous or conjunctival vaccination[J]. Vaccine. 2006, 24(17): 3461-3468
[112]Rajashekara G, DA Glover, M Banai, et al. Attenuated bioluminescent Brucella melitensis mutants GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091) confer protection in mice [J]. Infect Immun, 2006, 74(5): 2925-2936
[113]Mosmann TR, Sad S. The expanding universe of T-cell subset:Thl,Th2 and more[J]. Immunol Today, 1996, 17(3): 138-146
[114] Murphy EA, Sathiyaseelan J, Parent MA, et al. Interferon-gamma is crucial for surviving a Brucella abortus infection in both resistant C57BL/6 and susceptible Balb/c mice[J]. Immunology, 2001, 103(4): 511-8
[115] Jiang X, Baldwin CL. Iron augments macrophage-mediated killing of Brucella abortus alone and in conjunction with interferon-gamma [J]. Cell Immunol, 1993, 148(2): 397-407
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |