ME 49株弓形虫速殖子对昆明小鼠的致病性
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摘要
【目的】通过ME 49株弓形虫速殖子对小鼠致病性的研究,了解该虫株速殖子的毒力特点,为深入研究弓形虫弱毒株的致病特征提供研究基础。【方法】以ME 49株弓形虫速殖子为研究对象,用血球计数板将速殖子梯度浓度稀释为<10~0,10~0-10~6个/mL,腹腔注射昆明小鼠,死亡小鼠的组织直接涂片检测肺脏及肠系膜淋巴结速殖子或大脑组织中弓形虫包囊数量,肺脏常规石蜡切片进行H.E及IHC染色,统计涂片和MAT方法的结果分析弓形虫的感染情况,并统计小鼠的感染率和生存率。【结果】0-60 DPI,死亡小鼠组织涂片显微镜检查和MAT法检测小鼠血清中弓形虫IgG抗体结果显示,<10~0、10~0、10~1及≥10~2速殖子浓度组小鼠的弓形虫感染率分别为0、20%、60%及100%。10~4速殖子浓度组小鼠有3只分别在12、16、19 DPI死亡;10~5速殖子浓度组小鼠有2只在11 DPI死亡,1只在8 DPI死亡;10~6速殖子浓度组小鼠有2只在9 DPI死亡,在7、8、10 DPI各死亡1只,感染弓形虫的小鼠生存率为62.07%(18/29)。对死亡小鼠的肺脏和肠系膜淋巴结直接涂片,显微镜镜检可见弓形虫速殖子;H.E及IHC染色,肺脏可以观察到弓形虫包囊及弓形虫抗原。对<10~0、10~0、10~1及10~2处死小鼠的大脑组织研磨镜检,结果显示<10~0和10~0感染组阳性小鼠大脑中弓形虫包囊数均为0;10~1感染组阳性小鼠大脑中弓形虫包囊数分别为60、100和0;10~2感染组阳性小鼠大脑中弓形虫包囊数量分别为20、40、40、60和0。60-600 DPI,10~3速殖子浓度组小鼠在184、435和569 DPI各有1只死亡,大脑中未检测到弓形虫包囊;10~5速殖子浓度组小鼠1只在188 DPI死亡,大脑包囊数量为20,其余小鼠存活时间为590 DPI。【结论】本研究探讨了ME 49株弓形虫速殖子对昆明小鼠的致病性,≥10~2速殖子浓度可引起小鼠100%感染,10~6速殖子浓度可引起小鼠100%死亡,小鼠死亡的时间为7-19 DPI,感染小鼠生存率为62.07%(18/29),大脑包囊成囊率为53.85%(7/13),平均包囊量为0-53个包囊/小鼠大脑,最长存活时间590 DPI。ME 49株弓形虫速殖子对小鼠致病性较弱,大脑可见弓形虫包囊,高浓度速殖子可引起小鼠死亡。
【Objective】In order to provide a basis for study the pathogenic characteristic of Toxoplasma gondii(T.gondii),ME49 strain of T.gondii tachyzoites were explored to understand its virulence.【Method】In this study,the tachyzoites of ME 49 strain T.gondii were diluted with the concentration of<10~0,10~0-10~6 and inoculated to Kunming mice by intraperitoneal injection.The tachyzoites in lung and mesentery lymph nodes of mice were checked,and the cysts of T.gondii in brain were numbered.Besides,the paraffin sections in the lungs were stained with HE and IHC.The results of smear and MAT were used to analyze the infection rate and survival rate of mice.【Result】0 DPI-60 DPI,the result of T.gondii IgG antibody by MAT showed that the seroprevalence of mice with<10~0,10~0,10~1 and ≥10~2 were 0,20%,60% and 100%,respectively.Three mice with 10~4 tachyzoites died at 12 DPI,16 DPI and 19 DPI,respectively.Two mice died at 11 DPI and one mouse died at 8 DPI with 10~5 tachyzoites.Two mice with 10~6 tachyzoites died at 9 DPI,three mice died at 7 DPI,8 DPI and 10 DPI,respectively,and the survival rate of mice infected with T.gondii was 62.07%(18/29).The tachyzoites of lungs and mesentery lymph nodes of dead mice were found by microscopy,and the cysts and antigen of T.gondii were observed in the lung by HE and IHC.The cysts in brain of T.gondii infection-positive mice with<10~0,and 10~0 tachyzoites were not found,the cysts in brain of T.gondii positive mice with 10~1 tachyzoite were 60,100 and0,respectively,10~2 tachyzoites of that were 20,40,60,and 0,respectively,60 DPI-600 DPI,three mice died with 10~3 tachyzoites at 184 DPI,435 DPI and 569 DPI,respectively,but no cysts of T.gondii were detected in the brains of mice.One mouse died at 188 DPI with the 10~5 tachyzoites and the cysts was 20,while the survived time of remaining mice was 590 DPI.【Conclusion】In this study,the pathogenicity of ME 49 strain of T.gondii tachyzoites in Kunming mice was explored.100% infection rate for mice was≥10~2 T.gondii tachyzoites,the 100% lethal dose was 10~6 tachyzoites,the time of death in infected mice was 7 DPI-19 DPI,the brain cysts was 0-53 per mouse,and the longest survival time was 590 DPI.The T.gondii tachyzoites of ME 49 strain had low pathogenicity to Kunming mice,and the cysts could be formed in brain,and only cause death of mice at high concentration of tachyzoites.
【Objective】In order to provide a basis for study the pathogenic characteristic of Toxoplasma gondii(T.gondii),ME49 strain of T.gondii tachyzoites were explored to understand its virulence.【Method】In this study,the tachyzoites of ME 49 strain T.gondii were diluted with the concentration of<10~0,10~0-10~6 and inoculated to Kunming mice by intraperitoneal injection.The tachyzoites in lung and mesentery lymph nodes of mice were checked,and the cysts of T.gondii in brain were numbered.Besides,the paraffin sections in the lungs were stained with HE and IHC.The results of smear and MAT were used to analyze the infection rate and survival rate of mice.【Result】0 DPI-60 DPI,the result of T.gondii IgG antibody by MAT showed that the seroprevalence of mice with<10~0,10~0,10~1 and ≥10~2 were 0,20%,60% and 100%,respectively.Three mice with 10~4 tachyzoites died at 12 DPI,16 DPI and 19 DPI,respectively.Two mice died at 11 DPI and one mouse died at 8 DPI with 10~5 tachyzoites.Two mice with 10~6 tachyzoites died at 9 DPI,three mice died at 7 DPI,8 DPI and 10 DPI,respectively,and the survival rate of mice infected with T.gondii was 62.07%(18/29).The tachyzoites of lungs and mesentery lymph nodes of dead mice were found by microscopy,and the cysts and antigen of T.gondii were observed in the lung by HE and IHC.The cysts in brain of T.gondii infection-positive mice with<10~0,and 10~0 tachyzoites were not found,the cysts in brain of T.gondii positive mice with 10~1 tachyzoite were 60,100 and0,respectively,10~2 tachyzoites of that were 20,40,60,and 0,respectively,60 DPI-600 DPI,three mice died with 10~3 tachyzoites at 184 DPI,435 DPI and 569 DPI,respectively,but no cysts of T.gondii were detected in the brains of mice.One mouse died at 188 DPI with the 10~5 tachyzoites and the cysts was 20,while the survived time of remaining mice was 590 DPI.【Conclusion】In this study,the pathogenicity of ME 49 strain of T.gondii tachyzoites in Kunming mice was explored.100% infection rate for mice was≥10~2 T.gondii tachyzoites,the 100% lethal dose was 10~6 tachyzoites,the time of death in infected mice was 7 DPI-19 DPI,the brain cysts was 0-53 per mouse,and the longest survival time was 590 DPI.The T.gondii tachyzoites of ME 49 strain had low pathogenicity to Kunming mice,and the cysts could be formed in brain,and only cause death of mice at high concentration of tachyzoites.
引文
[1]DUBEY J P.Toxoplasmosis of Animals and Humans.Boca Raton,Florida,USA:CRC Press,Taylor&Francis Group,2010,1-313.
[2]DUBEY J P.The history of Toxoplasma gondii-the first 100 years.Journal of Eukaryotic Microbiology,2008,55(6):467-475.
[3]汪涛,汤自豪.弓形虫病:速殖子、包囊和卵囊的传播.中国人兽共患病学报,2012,28(11):1133-1136.WANG T,TANG Z H.Toxoplasmosis:The spread of tachyzoites,cysts and oocysts.Chinese Journal of Zoonoses,2012,28(11):1133-1136.(in Chinese)
[4]DORNY P,PRAET N,DECKERS N,GABRIEL S.Emerging food-born parasites.Veterinary Parasitology,2009,163(3):196-206.
[5]孔阳光,王凯,杨玉荣.弓形虫在猫科动物小肠上皮细胞内发育过程的研究进展.中国兽医学报,2016,36(10):1809-1812.KONG Y G,WANG K,YANG Y R.Progress in research on developing Toxoplasma gondii in small intestine epithelial cell of felids.Chinese Journal of Veterinary Science,2016,36(10):1809-1812.(in Chinese)
[6]LOPES W D Z,COSTA A J D,SANTANA L F,SANTOS R S D,ROSSANESE W M,LOPES W C Z,COSTA G H N.Aspects of toxoplasma infection on the reproductive system of experimentally infected rams(Ovis aries).Journal of Parasitology Research,2009,2:910-911.
[7]DUBEY J P,FERREIRA L R,MARTINS J,MCLEOD R.Oral oocyst induced mouse model of toxoplasmosis:effect of infection with Toxoplasma gondii strains of different genotypes,dose,and mouse strains(transgenic,out-bred,in-bred)on pathogenesis and mortality.Parasitology,2012,139(1):1-13.
[8]DUBEY J P.Bradyzoite-induced murine toxoplasmosis:stage conversion,pathogenesis,and tissue cyst formation in mice fed bradyzoites of different strains of Toxoplasma gondii.The Journal of Eukaryotic Microbiology,1997,44(6):592-602.
[9]付晓莹,冯永杰,梁宏德,杨玉荣.中国弓形虫分离株基因型及致病性的研究进展.中国人兽共患病学报,2015,31(7):669-673.FU X Y,FENG Y J,LIANG H D,YANG Y R.Genotypes and pathogenesis of Toxoplasma gondii isolates in China.Chinese Journal of Zoonoses,2015,31(7):669-673.(in Chinese)
[10]DUBEY J P,DESMONTS G.Serological responses of equids fed Toxoplasma gondii oocysts.Equine Veterinary Journal,1987,19(4):337-339.
[11]KIJLSTRA A,JONGERT E.Toxoplasma-safe meat:close to reality.Trends in Parasitology,2009,25(1):18-22.
[12]COOK A J C,GILBERT R E,BUFFOLANO W,ZUFFEREY J,PETERSEN E,JENUM P A,FOULON W,SEMPRINI A E,DUNND T.Sources of toxoplasma infection in pregnant women:European multicentre case-control study.BMJ,2000,321(7254):142-147.
[13]FALLAH M,RABIEE S,MATINI M,TAHERKHANI H.Seroepidemiology of toxoplasmosis in primigravida women in Hamadan,Islamic Republic of Iran,2004.Eastern Mediterranean Health Journal,2008,14(1):163-171.
[14]HAN K,SHIN D W,LEE T Y,LEE Y H.Seroprevalence of Toxoplasma gondii infection and risk factors associated with seropositivity of pregnant women in Korea.The Journal of Parasitology,2008,94(4):963-965.
[15]LOPES W D Z,RODRIGUEZ J D,SOUZA F A,SANTOS T R D,SANTOS R S D,ROSANESE W M,LOPES W R Z,SAKAMOTO CA,COSTA A J D.Sexual transmission of Toxoplasma gondii in sheep.Veterinary Parasitology,2013,195(1/2):47-56.
[16]DUBEY J P.Toxoplasma gondii oocyst survival under defined temperatures.The Journal of Parasitology,1998,84(4):862-865.
[17]JOHNSON A M.Speculation on possible life cycles for the clonal lineages in the genus toxoplasma.Parasitology Today.1997,13(10):393-397.
[18]RIEMANN H P,MEYER M E,THEIS J H,KELSO G,BEHYMER DE.Toxoplasmosis in an infant fed unpasteurized goat milk.The Journal of Pediatrics.1975,87(4):573-576.
[19]SACKS J J,ROBERTO R R,BROOKS N F.Toxoplasmosis infection associated with raw goat's milk.Journal of the American Medical Association,1982,248(14):1728-1732.
[20]LI M,MO X W,WANG L,CHEN H,LUO Q L,WEN H Q,WEI W,ZHANG A M,DU J,LU F L,LUN Z R,SHEN J L.Phylogeny and virulence divergency analyses of Toxoplasma gondii isolates from China.Parasites&Vectors,2014,7(1):133-144.
[21]陆瑶瑶,冯永杰,董辉,杨玉荣.VEG株弓形虫卵囊对昆明小鼠的毒力研究.中国人兽共患病学报,2017,33(7):624-627.LU Y Y,FENG Y J,DONG H,YANG Y R.Virulence of VEG strain Toxoplasma gondii oocysts against Kunming mice.Chinese Journal of Zoonoses,2017,33(7):624-627.(in Chinese)
[22]SARAF P,SHWAB E K,DUBEY J P,SU C.On the determination of Toxoplasma gondii virulence in mice.Experimental parasitology,2017,174:25-30.
[23]ZHOU P,NIE H,ZHANG L X,WANG H Y,YIN C C,SU C,ZHUX Q,ZHAO J L.Genetic characterization of Toxoplasma gondii isolates from pigs in China.The Journal of Parasitology,2010,96(5):1027-1029.
[24]ZHOU P,SUN X T,YIN C C,YANG J F,YUAN Z G,YAN H K,ZHU X Q,ZOU F C.Genetic characterization of Toxoplasma gondii isolates from pigs in southwestern China.The Journal of Parasitology,2011,97(6):1193-1195.
[25]CHAICHAN P,MERCIER A,GALAL L,MAHITTIKORN A,ARIEY F,MORAND S,BOUMEDIENE F,UDONSOM R,HAMIDOVIC A,MURAT J B,SUKTHANA Y,DARDE M L.Geographical distribution of Toxoplasma gondii genotypes in Asia:Alink with neighboring continents.Infection Genetics Evolution,2017,53:227-238.
[26]CHENG W S,LIU F,LI M,HU X D,CHEN H,PAPPOE F,LUO QL,WEN H Q,XING T,XU Y F,SHEN J L.Variation detection based on next-generation sequencing of type Chinese 1 strains of Toxoplasma gondii with different virulence from China.BMCGenomics,2015,16:888.
[27]YANG Y R,FENG Y J,YAO Q X,WANG Y H,LU Y Y,LIANG HD,ZHU X Q,ZHANG L X.Seroprevalence,isolation,genotyping,and pathogenicity of Toxoplasma gondii strains from sheep in China.Frontiers in Microbiology,2017,8:136.
[28]YANG Y R,FENG Y J,LU Y Y,DONG H,LI T Y,JIANG Y B,ZHU X Q,ZHANG L X.Antibody detection,isolation,genotyping,and virulence of Toxoplasma gondii in captive felids from China.Frontiers in Microbiology,2017,8:1414.
[29]YANG Y R,YING Y Q,VERMA S K,CASSINELLI A B M,KWOK O C H,LIANG H D,PRADHAN A K,ZHU X Q,SU C,DUBEY J P.Isolation and genetic characterization of viable Toxoplasma gondii from tissues and feces of cats from the central region of China.Veterinary Parasitology,2015,211:283-288.
[30]王凯,冯永杰,付晓莹,陆瑶瑶,梁宏德,杨玉荣.ToxoDB#17型弓形虫卵囊对昆明小鼠致病性的研究.中国人兽共患病学报,2017,33(1):49-52.WANG K,FENG Y J,FU X Y,LU Y Y,LIANG H D,YANG Y R.Virulence and pathogenicity of Toxoplasma gondii ToxoDB#17 strain oocysts in Kunming mice.Chinese Journal of Zoonoses,2017,33(1):49-52.(in Chinese)
[31]LU Y Y,DONG H,FENG Y J,WANG K,JIANG Y B,ZHANG L X,YANG Y R.Avirulence and lysozyme secretion in Paneth cells after infection of BALB/c mice with oocysts of Toxoplasma gondii strains TgCatCHn2(ToxoDB#17)and TgCatCHn4(ToxoDB#9).Veterinary Parasitology,2018,252:1-8.
[2]DUBEY J P.The history of Toxoplasma gondii-the first 100 years.Journal of Eukaryotic Microbiology,2008,55(6):467-475.
[3]汪涛,汤自豪.弓形虫病:速殖子、包囊和卵囊的传播.中国人兽共患病学报,2012,28(11):1133-1136.WANG T,TANG Z H.Toxoplasmosis:The spread of tachyzoites,cysts and oocysts.Chinese Journal of Zoonoses,2012,28(11):1133-1136.(in Chinese)
[4]DORNY P,PRAET N,DECKERS N,GABRIEL S.Emerging food-born parasites.Veterinary Parasitology,2009,163(3):196-206.
[5]孔阳光,王凯,杨玉荣.弓形虫在猫科动物小肠上皮细胞内发育过程的研究进展.中国兽医学报,2016,36(10):1809-1812.KONG Y G,WANG K,YANG Y R.Progress in research on developing Toxoplasma gondii in small intestine epithelial cell of felids.Chinese Journal of Veterinary Science,2016,36(10):1809-1812.(in Chinese)
[6]LOPES W D Z,COSTA A J D,SANTANA L F,SANTOS R S D,ROSSANESE W M,LOPES W C Z,COSTA G H N.Aspects of toxoplasma infection on the reproductive system of experimentally infected rams(Ovis aries).Journal of Parasitology Research,2009,2:910-911.
[7]DUBEY J P,FERREIRA L R,MARTINS J,MCLEOD R.Oral oocyst induced mouse model of toxoplasmosis:effect of infection with Toxoplasma gondii strains of different genotypes,dose,and mouse strains(transgenic,out-bred,in-bred)on pathogenesis and mortality.Parasitology,2012,139(1):1-13.
[8]DUBEY J P.Bradyzoite-induced murine toxoplasmosis:stage conversion,pathogenesis,and tissue cyst formation in mice fed bradyzoites of different strains of Toxoplasma gondii.The Journal of Eukaryotic Microbiology,1997,44(6):592-602.
[9]付晓莹,冯永杰,梁宏德,杨玉荣.中国弓形虫分离株基因型及致病性的研究进展.中国人兽共患病学报,2015,31(7):669-673.FU X Y,FENG Y J,LIANG H D,YANG Y R.Genotypes and pathogenesis of Toxoplasma gondii isolates in China.Chinese Journal of Zoonoses,2015,31(7):669-673.(in Chinese)
[10]DUBEY J P,DESMONTS G.Serological responses of equids fed Toxoplasma gondii oocysts.Equine Veterinary Journal,1987,19(4):337-339.
[11]KIJLSTRA A,JONGERT E.Toxoplasma-safe meat:close to reality.Trends in Parasitology,2009,25(1):18-22.
[12]COOK A J C,GILBERT R E,BUFFOLANO W,ZUFFEREY J,PETERSEN E,JENUM P A,FOULON W,SEMPRINI A E,DUNND T.Sources of toxoplasma infection in pregnant women:European multicentre case-control study.BMJ,2000,321(7254):142-147.
[13]FALLAH M,RABIEE S,MATINI M,TAHERKHANI H.Seroepidemiology of toxoplasmosis in primigravida women in Hamadan,Islamic Republic of Iran,2004.Eastern Mediterranean Health Journal,2008,14(1):163-171.
[14]HAN K,SHIN D W,LEE T Y,LEE Y H.Seroprevalence of Toxoplasma gondii infection and risk factors associated with seropositivity of pregnant women in Korea.The Journal of Parasitology,2008,94(4):963-965.
[15]LOPES W D Z,RODRIGUEZ J D,SOUZA F A,SANTOS T R D,SANTOS R S D,ROSANESE W M,LOPES W R Z,SAKAMOTO CA,COSTA A J D.Sexual transmission of Toxoplasma gondii in sheep.Veterinary Parasitology,2013,195(1/2):47-56.
[16]DUBEY J P.Toxoplasma gondii oocyst survival under defined temperatures.The Journal of Parasitology,1998,84(4):862-865.
[17]JOHNSON A M.Speculation on possible life cycles for the clonal lineages in the genus toxoplasma.Parasitology Today.1997,13(10):393-397.
[18]RIEMANN H P,MEYER M E,THEIS J H,KELSO G,BEHYMER DE.Toxoplasmosis in an infant fed unpasteurized goat milk.The Journal of Pediatrics.1975,87(4):573-576.
[19]SACKS J J,ROBERTO R R,BROOKS N F.Toxoplasmosis infection associated with raw goat's milk.Journal of the American Medical Association,1982,248(14):1728-1732.
[20]LI M,MO X W,WANG L,CHEN H,LUO Q L,WEN H Q,WEI W,ZHANG A M,DU J,LU F L,LUN Z R,SHEN J L.Phylogeny and virulence divergency analyses of Toxoplasma gondii isolates from China.Parasites&Vectors,2014,7(1):133-144.
[21]陆瑶瑶,冯永杰,董辉,杨玉荣.VEG株弓形虫卵囊对昆明小鼠的毒力研究.中国人兽共患病学报,2017,33(7):624-627.LU Y Y,FENG Y J,DONG H,YANG Y R.Virulence of VEG strain Toxoplasma gondii oocysts against Kunming mice.Chinese Journal of Zoonoses,2017,33(7):624-627.(in Chinese)
[22]SARAF P,SHWAB E K,DUBEY J P,SU C.On the determination of Toxoplasma gondii virulence in mice.Experimental parasitology,2017,174:25-30.
[23]ZHOU P,NIE H,ZHANG L X,WANG H Y,YIN C C,SU C,ZHUX Q,ZHAO J L.Genetic characterization of Toxoplasma gondii isolates from pigs in China.The Journal of Parasitology,2010,96(5):1027-1029.
[24]ZHOU P,SUN X T,YIN C C,YANG J F,YUAN Z G,YAN H K,ZHU X Q,ZOU F C.Genetic characterization of Toxoplasma gondii isolates from pigs in southwestern China.The Journal of Parasitology,2011,97(6):1193-1195.
[25]CHAICHAN P,MERCIER A,GALAL L,MAHITTIKORN A,ARIEY F,MORAND S,BOUMEDIENE F,UDONSOM R,HAMIDOVIC A,MURAT J B,SUKTHANA Y,DARDE M L.Geographical distribution of Toxoplasma gondii genotypes in Asia:Alink with neighboring continents.Infection Genetics Evolution,2017,53:227-238.
[26]CHENG W S,LIU F,LI M,HU X D,CHEN H,PAPPOE F,LUO QL,WEN H Q,XING T,XU Y F,SHEN J L.Variation detection based on next-generation sequencing of type Chinese 1 strains of Toxoplasma gondii with different virulence from China.BMCGenomics,2015,16:888.
[27]YANG Y R,FENG Y J,YAO Q X,WANG Y H,LU Y Y,LIANG HD,ZHU X Q,ZHANG L X.Seroprevalence,isolation,genotyping,and pathogenicity of Toxoplasma gondii strains from sheep in China.Frontiers in Microbiology,2017,8:136.
[28]YANG Y R,FENG Y J,LU Y Y,DONG H,LI T Y,JIANG Y B,ZHU X Q,ZHANG L X.Antibody detection,isolation,genotyping,and virulence of Toxoplasma gondii in captive felids from China.Frontiers in Microbiology,2017,8:1414.
[29]YANG Y R,YING Y Q,VERMA S K,CASSINELLI A B M,KWOK O C H,LIANG H D,PRADHAN A K,ZHU X Q,SU C,DUBEY J P.Isolation and genetic characterization of viable Toxoplasma gondii from tissues and feces of cats from the central region of China.Veterinary Parasitology,2015,211:283-288.
[30]王凯,冯永杰,付晓莹,陆瑶瑶,梁宏德,杨玉荣.ToxoDB#17型弓形虫卵囊对昆明小鼠致病性的研究.中国人兽共患病学报,2017,33(1):49-52.WANG K,FENG Y J,FU X Y,LU Y Y,LIANG H D,YANG Y R.Virulence and pathogenicity of Toxoplasma gondii ToxoDB#17 strain oocysts in Kunming mice.Chinese Journal of Zoonoses,2017,33(1):49-52.(in Chinese)
[31]LU Y Y,DONG H,FENG Y J,WANG K,JIANG Y B,ZHANG L X,YANG Y R.Avirulence and lysozyme secretion in Paneth cells after infection of BALB/c mice with oocysts of Toxoplasma gondii strains TgCatCHn2(ToxoDB#17)and TgCatCHn4(ToxoDB#9).Veterinary Parasitology,2018,252:1-8.