弓形虫病分子诊断方法及弓形虫分离株耐药性的研究
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摘要
弓形虫病(Toxoplasmosis)是由刚地弓形虫(Toxoplasma gondii)引起的以孕妇(畜)流产、弱胎、畸胎、死胎和儿童(幼畜)生长受阻、死亡等为特征的人畜共患病。该病广泛存在于世界各地,宿主范围十分广泛,人及大多数动物感染率都较高,是人类优生的大敌,是当今新生儿致畸的四大病因之一。弓形虫病在猪场感染率高,严重影响着养猪业的发展。建立准确快速的诊断方法和研制安全高效疫苗是预防和控制弓形虫病的重要措施。MIC3是弓形虫在速殖子、缓殖子及子孢子期都表达的粘附蛋白,与弓形虫入侵宿主细胞密切相关,已证明其具有良好的免疫原性。本研究利用原核表达的弓形虫微线体蛋白3(MIC3)建立了SPA-ELISA和AG-ELISA检测方法,利用细胞培养方法从临床血清学阳性的病料中分离得到了10株弓形虫虫株,并对虫株的耐药性进行了研究。主要研究成果如下:
本研究利用本室构建的pGEX-KG-MIC3质粒转化感受态细胞,确定了pGEX-KG-MIC3在E.coli BL21-CodonPlus菌株中高效表达的最佳条件,SDS-PAGE实验表明表达的融合蛋白分子量为66KD,表达产物经Western blot检测证实具有较强的免疫反应原性。
利用纯化的重组蛋白rMIC3替代传统的虫体成份作为诊断抗原建立了SPA-ELISA和AG-ELISA诊断方法。这两种方法检测猪其它常见病原体阳性血清,结果全为阴性;SPA-ELISA和AG-ELISA分别能检测到1:6400和1:12800稀释血清中的抗T.gondii特异性抗体;诊断试剂的批内(间)重复试验,ELISA变异系数＜10%;在4℃贮存下诊断试剂的保质期均超过8个月。结果表明,本研究建立的两种诊断方法均具有良好的特异性、敏感性、稳定性及较长的保质期。
为验证和比较SPA-ELISA和AG-ELISA的广适性,在临床上,检测了四种不同动物血清,共332份,并与MAT方法进行了比较,结果表明,除山羊血清外,SPA-ELISA对猪、犬、猫血清的阳性检出率均高于MAT方法,二者符合率超过90%。利用AG-ELISA检测四种动物血清,共304份,并与MAT方法进行比较,结果显示,AG-ELISA对四种动物血清的阳性检出率均高于MAT方法。说明了AG-ELISA具有更广泛的宿主适用范围。
为进一步验证AG-ELISA的实际应用效果,利用已建立的AG-ELISA监测了人工感染弓形虫猪体内抗体水平的动态变化,同时用MAT方法及ELISAkit平行测定。结果表明,首次感染第10d,8头人工感染猪的AG-ELISA抗体均为阳性,第35d,AG-ELISA抗体水平达到高峰,随后缓慢下降,在第57d时仍可检测到ELISA抗体。Kappa一致性试验结果表明,AG-ELISA与MAT方法及ELISAkit符合良好。
为提高弓形虫细胞分离的阳性率,对细胞培养条件进行了优化,结果显示,细胞培养的最佳条件为选用DMEM高糖培养基,加入1%血清和10%SRC。采集猪的抗凝全血,利用细胞培养方法进行了弓形虫虫株的分离。利用上述培养条件,对19份弓形虫AG-ELISA抗体阳性的抗凝血进行了弓形虫虫株的分离培养,经显微镜观察、PCR检测以及动物接种试验证明,成功地分离得到10株弓形虫分离株。耐药性试验初步显示其中的一株对磺胺类药物具有一定的抗药性。
本研究建立的SPA-ELISA和AG-ELISA诊断方法,解决了目前弓形虫病不易快速、准确、简便诊断的难题,对多种动物弓形虫病的临床诊断、疾病控制等均具有重要意义。猪源弓形虫虫株的分离及磺胺耐药现象的初步证实,为弓形虫病的有效防治及合理用药奠定了基础。
Toxoplasmosis causes reproductive failure in pregnant women and pregnant animals, manifested as puny,and abnormal embryo,abortion and stillbirths.The Toxoplasma gondii is ubiquitous among people and animals throughout the world and is high infection in most herds.It is the enemy of human prepotency and one of the four factors leading to abnormal embryo in pregnant women.Because of above reasons,establishing quick diagnostic methods and isolating wild strains are very important to prevent and control toxoplasmosis.In this research,MIC3 of Toxoplasrna gondii was highly expressed in E.coli;rMIC3-SPA-ELISA and rMIC3-AG-ELISA diagnostic methods for swine toxoplasmosis were established.This research will be useful for vaccination,diagnosis, prevention and control of swine toxoplasmosis.The results of the research are as following:
1.Expression of MIC3 in E.coli
MIC3 of Toxoplasma gondii are considered as an important candidate antigen of diagnostic methods and DNA vaccine.The MIC3 are expressed during tachyzoite, bradyzoite and sporozoite period.The recombinant plasmid pGEX-KG-MIC3 was respectively introduced into E.coli BL21-CodonPlus.After induction by IPTG,high expressions were found.An average molecular weight of 66 KD was determined by SDS-PAGE.The expressed products of MIC3 could specifically bind to the antibody against Toxoplasma gondii by Western blot.
2.Development of SPA-ELISA and AG-ELISA
The quick diagnostic methods of SPA-ELISA and AG-ELISA for swine toxoplasmosis were established by rMIC3.Both methods did not react with positive sera of other pathogen and could detect positive serum at a maximum dilution of 1:6400. OD_(630) values of positive and negative sera remained constant under different storage time from 1 month to 8 months in 4℃.All the results indicated that SPA-ELISA and AG-ELISA had high specificity,good sensitivity and longer shelf life of products.
A total of 304 serum samples from four species of animals(pigs,goats,dogs and cats) were detected by AG-ELISA and MAT methods.AG-ELISA showed a higher prevalence than MAT in pig sera and goat sera,and obtained the same prevalence in dog sera and cat sera.Kappa results were estimated and a good agreement was observed in AG-ELISA×MAT(κ=0.88,P＜0.001).
In order to validate the effect of AG-ELISA,sera from experimentally infected pigs were detected by AG-ELISA,ELISA kit and MAT,and the time course of anti-T.gondii antibody responses for each of these tests was determined.AG-ELISA was able to detect all animals as positive at day 10,and MAT detected them starting at day 14.The peak of antibody level was detected at day 35 in AG-ELISA and MAT.The antibody level has remained high at the 57th day after inoculation in all the methods.Kappa results were estimated and a good agreement was observed both in AG-ELISAxMAT(κ=0.86, P＜0.001) and AG-ELISA×ELISA kit(κ=0.83,P＜0.001).
3.Isolating for T.gondii wild strains and studying on its drug resistance
Anticoagulating blood samples collected from serum positive farms were used to isolate T.gondii parasites.The optimizing test indicated that the best culture medium was DMEM medium with high glucose,1%FBS and 10%SRC.19 blood samples collected from the pigs of T.gondii antibody positive farms were cultured with PK-15 cell line.Ten T.gondii strains were isolated,and the validity of the strains was confirmed by specific PCR and ELISA methods.These results demonstrated that the method of cell culture is an effective way to isolate T.gondii strain from animals.One of the ten isolates was proved to be resistant for sulfonamides.
In conclusion,the rapid,accurate and convenient diagnostic methods of SPA-ELISA and AG-ELISA of animal toxoplasmosis have been established.They could be used widely for serological detection of T.gondii antibody in many species of animals.The isolation of pig-derived wild strains and the confirmation for sulfonamides resistance have laid the foundation for control of toxoplasmasis.
本研究利用本室构建的pGEX-KG-MIC3质粒转化感受态细胞,确定了pGEX-KG-MIC3在E.coli BL21-CodonPlus菌株中高效表达的最佳条件,SDS-PAGE实验表明表达的融合蛋白分子量为66KD,表达产物经Western blot检测证实具有较强的免疫反应原性。
利用纯化的重组蛋白rMIC3替代传统的虫体成份作为诊断抗原建立了SPA-ELISA和AG-ELISA诊断方法。这两种方法检测猪其它常见病原体阳性血清,结果全为阴性;SPA-ELISA和AG-ELISA分别能检测到1:6400和1:12800稀释血清中的抗T.gondii特异性抗体;诊断试剂的批内(间)重复试验,ELISA变异系数＜10%;在4℃贮存下诊断试剂的保质期均超过8个月。结果表明,本研究建立的两种诊断方法均具有良好的特异性、敏感性、稳定性及较长的保质期。
为验证和比较SPA-ELISA和AG-ELISA的广适性,在临床上,检测了四种不同动物血清,共332份,并与MAT方法进行了比较,结果表明,除山羊血清外,SPA-ELISA对猪、犬、猫血清的阳性检出率均高于MAT方法,二者符合率超过90%。利用AG-ELISA检测四种动物血清,共304份,并与MAT方法进行比较,结果显示,AG-ELISA对四种动物血清的阳性检出率均高于MAT方法。说明了AG-ELISA具有更广泛的宿主适用范围。
为进一步验证AG-ELISA的实际应用效果,利用已建立的AG-ELISA监测了人工感染弓形虫猪体内抗体水平的动态变化,同时用MAT方法及ELISAkit平行测定。结果表明,首次感染第10d,8头人工感染猪的AG-ELISA抗体均为阳性,第35d,AG-ELISA抗体水平达到高峰,随后缓慢下降,在第57d时仍可检测到ELISA抗体。Kappa一致性试验结果表明,AG-ELISA与MAT方法及ELISAkit符合良好。
为提高弓形虫细胞分离的阳性率,对细胞培养条件进行了优化,结果显示,细胞培养的最佳条件为选用DMEM高糖培养基,加入1%血清和10%SRC。采集猪的抗凝全血,利用细胞培养方法进行了弓形虫虫株的分离。利用上述培养条件,对19份弓形虫AG-ELISA抗体阳性的抗凝血进行了弓形虫虫株的分离培养,经显微镜观察、PCR检测以及动物接种试验证明,成功地分离得到10株弓形虫分离株。耐药性试验初步显示其中的一株对磺胺类药物具有一定的抗药性。
本研究建立的SPA-ELISA和AG-ELISA诊断方法,解决了目前弓形虫病不易快速、准确、简便诊断的难题,对多种动物弓形虫病的临床诊断、疾病控制等均具有重要意义。猪源弓形虫虫株的分离及磺胺耐药现象的初步证实,为弓形虫病的有效防治及合理用药奠定了基础。
Toxoplasmosis causes reproductive failure in pregnant women and pregnant animals, manifested as puny,and abnormal embryo,abortion and stillbirths.The Toxoplasma gondii is ubiquitous among people and animals throughout the world and is high infection in most herds.It is the enemy of human prepotency and one of the four factors leading to abnormal embryo in pregnant women.Because of above reasons,establishing quick diagnostic methods and isolating wild strains are very important to prevent and control toxoplasmosis.In this research,MIC3 of Toxoplasrna gondii was highly expressed in E.coli;rMIC3-SPA-ELISA and rMIC3-AG-ELISA diagnostic methods for swine toxoplasmosis were established.This research will be useful for vaccination,diagnosis, prevention and control of swine toxoplasmosis.The results of the research are as following:
1.Expression of MIC3 in E.coli
MIC3 of Toxoplasma gondii are considered as an important candidate antigen of diagnostic methods and DNA vaccine.The MIC3 are expressed during tachyzoite, bradyzoite and sporozoite period.The recombinant plasmid pGEX-KG-MIC3 was respectively introduced into E.coli BL21-CodonPlus.After induction by IPTG,high expressions were found.An average molecular weight of 66 KD was determined by SDS-PAGE.The expressed products of MIC3 could specifically bind to the antibody against Toxoplasma gondii by Western blot.
2.Development of SPA-ELISA and AG-ELISA
The quick diagnostic methods of SPA-ELISA and AG-ELISA for swine toxoplasmosis were established by rMIC3.Both methods did not react with positive sera of other pathogen and could detect positive serum at a maximum dilution of 1:6400. OD_(630) values of positive and negative sera remained constant under different storage time from 1 month to 8 months in 4℃.All the results indicated that SPA-ELISA and AG-ELISA had high specificity,good sensitivity and longer shelf life of products.
A total of 304 serum samples from four species of animals(pigs,goats,dogs and cats) were detected by AG-ELISA and MAT methods.AG-ELISA showed a higher prevalence than MAT in pig sera and goat sera,and obtained the same prevalence in dog sera and cat sera.Kappa results were estimated and a good agreement was observed in AG-ELISA×MAT(κ=0.88,P＜0.001).
In order to validate the effect of AG-ELISA,sera from experimentally infected pigs were detected by AG-ELISA,ELISA kit and MAT,and the time course of anti-T.gondii antibody responses for each of these tests was determined.AG-ELISA was able to detect all animals as positive at day 10,and MAT detected them starting at day 14.The peak of antibody level was detected at day 35 in AG-ELISA and MAT.The antibody level has remained high at the 57th day after inoculation in all the methods.Kappa results were estimated and a good agreement was observed both in AG-ELISAxMAT(κ=0.86, P＜0.001) and AG-ELISA×ELISA kit(κ=0.83,P＜0.001).
3.Isolating for T.gondii wild strains and studying on its drug resistance
Anticoagulating blood samples collected from serum positive farms were used to isolate T.gondii parasites.The optimizing test indicated that the best culture medium was DMEM medium with high glucose,1%FBS and 10%SRC.19 blood samples collected from the pigs of T.gondii antibody positive farms were cultured with PK-15 cell line.Ten T.gondii strains were isolated,and the validity of the strains was confirmed by specific PCR and ELISA methods.These results demonstrated that the method of cell culture is an effective way to isolate T.gondii strain from animals.One of the ten isolates was proved to be resistant for sulfonamides.
In conclusion,the rapid,accurate and convenient diagnostic methods of SPA-ELISA and AG-ELISA of animal toxoplasmosis have been established.They could be used widely for serological detection of T.gondii antibody in many species of animals.The isolation of pig-derived wild strains and the confirmation for sulfonamides resistance have laid the foundation for control of toxoplasmasis.
引文
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