人芽囊原虫感染大鼠的方式改良及病理切片观察
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摘要
目的建立人芽囊原虫感染大鼠动物模型。方法将30只雄性SD大鼠随机分为对照组和4个感染组(10~5、 10~6、 10~7和10~8),每组6只。4个感染组分别灌服人芽囊原虫滋养体10~5、 10~6、 10~7和10~8个/鼠,对照组灌服等量PBS。感染后第3天开始收集粪样,于洛克氏-鸡蛋-血清(LES)培养基中培养72 h后镜下观察,PCR和测序确认感染前后虫株的基因型。感染后第3～18天连续收集5次大鼠粪样培养观察,记录阳性鼠感染情况。感染后第21天将大鼠全部无痛处死,分别收集十二指肠、空肠、回肠、结肠和盲肠等肠段的内容物培养72 h后观察,计算其最终感染数和感染密度;并取不同部位的组织进行切片,苏木精-伊红(HE)染色后观察肠道内是否存在虫体。采用皮尔森相关性分析比较各肠段寄生数目的相关关系。结果 10~5、 10~6、 10~7和10~8感染组中确定感染人芽囊原虫的大鼠数目分别为2、 3、 5和6只。其中10~5感染组感染后第6天检出阳性结果,其余感染组第3天检出阳性,对照组均为阴性。PCR及序列比对证实,感染大鼠分离的人芽囊原虫与感染所用虫株均为ST7型。肠道内容物培养结果显示,回肠、盲肠和结肠的内容物均成功培养出人芽囊原虫,且盲肠与结肠内的虫数之间呈正相关,盲肠与结肠内容物培养获得的人芽囊原虫数呈正相关性(r=0.541, P <0.01),其余肠段未发现人芽囊原虫。HE染色结果显示,盲肠有人芽囊原虫寄生并存在黏蛋白,未发现虫体入侵、炎细胞浸润、黏膜糜烂等病理损伤;其余肠段未发现人芽囊原虫寄生。结论采用人芽囊原虫滋养体可成功建立大鼠感染模型。灌服10~5个人芽囊原虫滋养体可成功感染大鼠, 10~8个滋养体的感染效果稳定,各组随着感染人芽囊原虫数量的增加,感染的大鼠数也增加。
Objective To establish a rat model for Blastocystis hominis infection and lay a foundation for the further study of B. hominis. Methods Total 30 male Sprague-Dawley rats were randomly divided into four infection groups and one control group with 6 in each group. In the infection groups, the rats were infected with 10~5, 10~6, 10~7 and 10~8 of B. hominis trophozoites, respectively, by oral gavage. In the control group rats were given the same volume of PBS only. Three days after infection fecal samples were collected until 18 days after infection with once every three days. Total 5 fecal samples were collected for each rat. The fecal samples were cultured in LES medium for 72 hours and B. hominis in the culture were observed under microscope and confirmed by PCR.The genotype of B. hominis before and after infection was identified by DNA sequencing of small subunit r DNA(SSU r DNA). The number of infected rats was calculated and clinical sign was observed. On the 21 st day after infection, all the rats were euthanized and the contents of the intestines including duodenum, jejunum, ileum, colon and cecum were collected and cultured in LES for 72 hours before microscopic examination of B. hominis in the culture. The final infection rate and infection intensity were calculated. The collected intestinal tissues were sectioned and stained by Hematoxylin-Eosin(HE) to observer the infection and pathological changes. Pearson correlation was used for the statistic analysis. Results Total 2, 3, 5 and 6 rates were confirmed to be infected with B. hominis in the groups infected with 10~5, 10~6, 10~7 and 10~8 B. hominis trophozoites, respectively. All nfected rats had been identified to be infected with B. hominis on the 3 rd day after infection except for the rats in group infected with105 trophozoites that in which the parasites were identified on the 6 th day post infection. No rats got infected in the control group. Sequence analysis of SSU rDNA amplified from the infected parasites confirmed that the subtype of the infected B. hominis was ST7. There was no mutation within the amplified SSU rNDA for the parasite before and after infection in the rats. In vitro culture of the intestinal contents showed that B. hominis parasites were found in the ileum, cecum and colon. The number of B. hominis identified in cecum and the cultured parasite in cecum was correlated with those identified in the colons( r = 0.541, P < 0.05). No parasite was found in other parts of intestine. HE staining of intestinal sections showed that B. hominis and mucin were found in the section of cecum, however, there was no invasion of parasite into mucosa, or lesion or erosion or inflammatory cell infiltration in the mucosa identified. Conclusion A rat model was successfully established for infection of B. hominis in our lab by oral gavage with as less as 10~5 trophozoites, however it is more stable to establish infection in rats using 108 of trophozoites. The more the trophozoites used for the infection, the higher infection rate can be obtained.
Objective To establish a rat model for Blastocystis hominis infection and lay a foundation for the further study of B. hominis. Methods Total 30 male Sprague-Dawley rats were randomly divided into four infection groups and one control group with 6 in each group. In the infection groups, the rats were infected with 10~5, 10~6, 10~7 and 10~8 of B. hominis trophozoites, respectively, by oral gavage. In the control group rats were given the same volume of PBS only. Three days after infection fecal samples were collected until 18 days after infection with once every three days. Total 5 fecal samples were collected for each rat. The fecal samples were cultured in LES medium for 72 hours and B. hominis in the culture were observed under microscope and confirmed by PCR.The genotype of B. hominis before and after infection was identified by DNA sequencing of small subunit r DNA(SSU r DNA). The number of infected rats was calculated and clinical sign was observed. On the 21 st day after infection, all the rats were euthanized and the contents of the intestines including duodenum, jejunum, ileum, colon and cecum were collected and cultured in LES for 72 hours before microscopic examination of B. hominis in the culture. The final infection rate and infection intensity were calculated. The collected intestinal tissues were sectioned and stained by Hematoxylin-Eosin(HE) to observer the infection and pathological changes. Pearson correlation was used for the statistic analysis. Results Total 2, 3, 5 and 6 rates were confirmed to be infected with B. hominis in the groups infected with 10~5, 10~6, 10~7 and 10~8 B. hominis trophozoites, respectively. All nfected rats had been identified to be infected with B. hominis on the 3 rd day after infection except for the rats in group infected with105 trophozoites that in which the parasites were identified on the 6 th day post infection. No rats got infected in the control group. Sequence analysis of SSU rDNA amplified from the infected parasites confirmed that the subtype of the infected B. hominis was ST7. There was no mutation within the amplified SSU rNDA for the parasite before and after infection in the rats. In vitro culture of the intestinal contents showed that B. hominis parasites were found in the ileum, cecum and colon. The number of B. hominis identified in cecum and the cultured parasite in cecum was correlated with those identified in the colons( r = 0.541, P < 0.05). No parasite was found in other parts of intestine. HE staining of intestinal sections showed that B. hominis and mucin were found in the section of cecum, however, there was no invasion of parasite into mucosa, or lesion or erosion or inflammatory cell infiltration in the mucosa identified. Conclusion A rat model was successfully established for infection of B. hominis in our lab by oral gavage with as less as 10~5 trophozoites, however it is more stable to establish infection in rats using 108 of trophozoites. The more the trophozoites used for the infection, the higher infection rate can be obtained.
引文
[1]Tan KS.Blastocystis in humans and animals:new insights using modern methodologies[J].Vet Parasitol,2004,126(1/2):121-144.
[2]Duda A,Kosikbogacka D,Lanochaarendarczyk N,et al.The prevalence of Blastocystis hominis and other protozoan parasites in soldiers returning from peacekeeping missions[J].Am J Trop Med Hyg,2015,92(4):805-806.
[3]Ocana-Losada C,Cuenca-Gomez JA,Cabezas-Fernandez MT,et al.Clinical and epidemiological characteristics of intestinal parasite infection by Blastocystis hominis[J].Rev Clin Esp,2018,218(3):115-120.
[4]辛致炜,廖振捷,廖德君,等.人芽囊原虫体外纯培养法的改良及形态观察[J].中国寄生虫学与寄生虫病杂志,2017,35(6):623-625.
[5]Zhou XB,Zhang X,Qiao JY,et al.Encystation:survival of Blastocystis hominis in immunocompetent mice abdomen cavity[J].Parasitol Res,2010,106(6):1315-1320.
[6]Moe KT,Singh M,Gopalakrishnakone P,et al.Cytopathic effect of Blastocystis hominis after intramuscular inoculation into laboratory mice[J].Parasitol Res,1998,84(6):450-454.
[7]姚繁荣,乔继英,赵晏,等.人芽囊原虫感染小鼠试验[J].中国寄生虫学与寄生虫病杂志,2005,23(6):444-448.
[8]Ajjampur SS,Png CW,Chia WN,et al.Ex vivo and in vivo mice models to study Blastocystis spp.adhesion,colonization and pathology:closer to proving koch’s postulates[J].PLoSOne,2016,11(8):e0160458.
[9]Yoshikawa H,Yoshida K,Nakajima A,et al.Fecal-oral transmission of the cyst form of Blastocystis hominis in rats[J].Parasitol Res,2004,94(6):391-396.
[10]李娟,邓婷,李小花,等.人芽囊原虫感染大鼠模型的建立[J].赣南医学院学报,2013,33(1):4-7.
[11]王东,薛长贵.人芽囊原虫不同感染途径对免疫抑制小鼠肠黏膜的影响[J].现代预防医学,2012,39(18):4813-4815.
[12]Elwakil HS,Hewedi IH.Pathogenic potential of Blastocystis hominis in laboratory mice[J].Parasitol Res,2010,107(3):685-689.
[13]Moe KT,Singh M,Howe J,et al.Experimental Blastocystis hominis infection in laboratory mice[J].Parasitol Res,1997,83(4):319-325.
[14]张红卫,李文,颜秋叶,等.人芽囊原虫对实验感染昆明小鼠肠黏膜超微结构的影响[J].中国寄生虫学与寄生虫病杂志,2006,24(3):187-191.
[15]Stensvold CR,Suresh GK,Tan KS,et al.Terminology for Blastocystis subtypes:a consensus[J].Trends Parasitol,2007,23(3):93-96.
[16]Yason JA,Ajjampur SSR,Tan KSW.Blastocystis isolate Bexhibits multiple modes of resistance against antimicrobial peptide LL-37[J].Infect Immun,2016,84(8):2220-2232.
[17]孙煦苧,廖德君,刘静,等.m LES法培养人芽囊原虫的实验观察[J].中国病原生物学杂志,2018,13(3):259-262,266.
[18]Mohamed RT,El-Bali MA,Mohamed AA,et al.Subtyping of Blastocystis sp.isolated from symptomatic and asymptomatic individuals in Makkah,Saudi Arabia[J].Parasit Vectors,2017,10(1):174.
[19]Windsor JJ,Macfarlane L,Hughes-Thapa G,et al.Incidence of Blastocystis hominis in faecal samples submitted for routine microbiological analysis[J].Br J Biomed Sci,2002,59(3):154-157.
[20]Hirata T,Nakamura H,Kinjo N,et al.Prevalence of Blastocystis hominis and Strongyloides stercoralis infection in Okinawa,Japan[J].Parasitol Res,2007,101(6):1717-1719.
[21]Wong KH,Ng GC,Lin RT,et al.Predominance of subtype3 among Blastocystis isolates from a major hospital in Singapore[J].Parasitol Res,2008,102(4):663-670.
[22]Zhang WZ,Ren GX,Zhao W,et al.Genotyping of enterocytozoon bieneusi and subtyping of Blastocystis in cancer patients:relationship to diarrhea and assessment of zoonotic transmission[J].Front Microbiol,2017,8:1835.
[23]Zhang SX,Yang CL,Gu WP,et al.Case-control study of diarrheal disease etiology in individuals over 5 years in southwest China[J].Gut Pathog,2016,8:58.
[24]Tian LG,Wang TP,Lv S,et al.HIV and intestinal parasite co-infections among a Chinese population:an immunological profile[J].Infect Dis Poverty,2013,2:18.
[25]胡缨,李艳文,刘晓泉,等.慢性病患者合并人芽囊原虫感染的情况调查[J].中国卫生检验杂志,2017,27(17):2558-2560.
[26]Liu DY,Lu ZC,Liu XQ,et al.Comparative analysis of intestinal parasitic infections of outpatients in Guangxi medical university affiliated hospital in 2005 and 2013[J].Int J Clin Exp Med,2015,8(9):16640-16645.
[27]何姗姗,伍玲园,刘晓泉,等.巴马瑶族自治县人芽囊原虫感染情况调查[J].中国寄生虫学与寄生虫病杂志,2013,31(1):76-77.
[28]Defaye M,Nourrisson C,Baudu E,et al.Efficient and reproducible experimental infections of rats with Blastocystis spp.[J].PLoS One,2018,13(11):e0207669.
[29]Chandramathi S,Suresh KG,Mahmood AA,et al.Urinary hyaluronidase activity in rats infected with Blastocystis hominis:evidence for invasion?[J].Parasitol Res,2010,106(6):1459-1463.
[30]Chandramathi S,Suresh K,Sivanandam S,et al.Stress exacerbates infectivity and pathogenicity of Blastocystis hominis:in vitro and in vivo evidences[J].PLoS One,2014,9(5):e94567.
[31]Suresh K,Venilla GD,Tan TC,et al.In vivo encystation of Blastocystis hominis[J].Parasitol Res,2009,104(6):1373-1380.
[32]Ragavan ND,Govind SK,Chye TT,et al.Factors that influence the shedding of Blastocystis cysts in an irritable bowel syndrome(IBS)patient:an evidence-based case study[J].Parasitol Res,2015,114(8):2999-3005.
[33]苗雅娟,吴秀萍,王光明,等.黏蛋白在肠道寄生虫感染中的作用[J].湖北农业科学,2012,51(12):2409-2411,2415.
[2]Duda A,Kosikbogacka D,Lanochaarendarczyk N,et al.The prevalence of Blastocystis hominis and other protozoan parasites in soldiers returning from peacekeeping missions[J].Am J Trop Med Hyg,2015,92(4):805-806.
[3]Ocana-Losada C,Cuenca-Gomez JA,Cabezas-Fernandez MT,et al.Clinical and epidemiological characteristics of intestinal parasite infection by Blastocystis hominis[J].Rev Clin Esp,2018,218(3):115-120.
[4]辛致炜,廖振捷,廖德君,等.人芽囊原虫体外纯培养法的改良及形态观察[J].中国寄生虫学与寄生虫病杂志,2017,35(6):623-625.
[5]Zhou XB,Zhang X,Qiao JY,et al.Encystation:survival of Blastocystis hominis in immunocompetent mice abdomen cavity[J].Parasitol Res,2010,106(6):1315-1320.
[6]Moe KT,Singh M,Gopalakrishnakone P,et al.Cytopathic effect of Blastocystis hominis after intramuscular inoculation into laboratory mice[J].Parasitol Res,1998,84(6):450-454.
[7]姚繁荣,乔继英,赵晏,等.人芽囊原虫感染小鼠试验[J].中国寄生虫学与寄生虫病杂志,2005,23(6):444-448.
[8]Ajjampur SS,Png CW,Chia WN,et al.Ex vivo and in vivo mice models to study Blastocystis spp.adhesion,colonization and pathology:closer to proving koch’s postulates[J].PLoSOne,2016,11(8):e0160458.
[9]Yoshikawa H,Yoshida K,Nakajima A,et al.Fecal-oral transmission of the cyst form of Blastocystis hominis in rats[J].Parasitol Res,2004,94(6):391-396.
[10]李娟,邓婷,李小花,等.人芽囊原虫感染大鼠模型的建立[J].赣南医学院学报,2013,33(1):4-7.
[11]王东,薛长贵.人芽囊原虫不同感染途径对免疫抑制小鼠肠黏膜的影响[J].现代预防医学,2012,39(18):4813-4815.
[12]Elwakil HS,Hewedi IH.Pathogenic potential of Blastocystis hominis in laboratory mice[J].Parasitol Res,2010,107(3):685-689.
[13]Moe KT,Singh M,Howe J,et al.Experimental Blastocystis hominis infection in laboratory mice[J].Parasitol Res,1997,83(4):319-325.
[14]张红卫,李文,颜秋叶,等.人芽囊原虫对实验感染昆明小鼠肠黏膜超微结构的影响[J].中国寄生虫学与寄生虫病杂志,2006,24(3):187-191.
[15]Stensvold CR,Suresh GK,Tan KS,et al.Terminology for Blastocystis subtypes:a consensus[J].Trends Parasitol,2007,23(3):93-96.
[16]Yason JA,Ajjampur SSR,Tan KSW.Blastocystis isolate Bexhibits multiple modes of resistance against antimicrobial peptide LL-37[J].Infect Immun,2016,84(8):2220-2232.
[17]孙煦苧,廖德君,刘静,等.m LES法培养人芽囊原虫的实验观察[J].中国病原生物学杂志,2018,13(3):259-262,266.
[18]Mohamed RT,El-Bali MA,Mohamed AA,et al.Subtyping of Blastocystis sp.isolated from symptomatic and asymptomatic individuals in Makkah,Saudi Arabia[J].Parasit Vectors,2017,10(1):174.
[19]Windsor JJ,Macfarlane L,Hughes-Thapa G,et al.Incidence of Blastocystis hominis in faecal samples submitted for routine microbiological analysis[J].Br J Biomed Sci,2002,59(3):154-157.
[20]Hirata T,Nakamura H,Kinjo N,et al.Prevalence of Blastocystis hominis and Strongyloides stercoralis infection in Okinawa,Japan[J].Parasitol Res,2007,101(6):1717-1719.
[21]Wong KH,Ng GC,Lin RT,et al.Predominance of subtype3 among Blastocystis isolates from a major hospital in Singapore[J].Parasitol Res,2008,102(4):663-670.
[22]Zhang WZ,Ren GX,Zhao W,et al.Genotyping of enterocytozoon bieneusi and subtyping of Blastocystis in cancer patients:relationship to diarrhea and assessment of zoonotic transmission[J].Front Microbiol,2017,8:1835.
[23]Zhang SX,Yang CL,Gu WP,et al.Case-control study of diarrheal disease etiology in individuals over 5 years in southwest China[J].Gut Pathog,2016,8:58.
[24]Tian LG,Wang TP,Lv S,et al.HIV and intestinal parasite co-infections among a Chinese population:an immunological profile[J].Infect Dis Poverty,2013,2:18.
[25]胡缨,李艳文,刘晓泉,等.慢性病患者合并人芽囊原虫感染的情况调查[J].中国卫生检验杂志,2017,27(17):2558-2560.
[26]Liu DY,Lu ZC,Liu XQ,et al.Comparative analysis of intestinal parasitic infections of outpatients in Guangxi medical university affiliated hospital in 2005 and 2013[J].Int J Clin Exp Med,2015,8(9):16640-16645.
[27]何姗姗,伍玲园,刘晓泉,等.巴马瑶族自治县人芽囊原虫感染情况调查[J].中国寄生虫学与寄生虫病杂志,2013,31(1):76-77.
[28]Defaye M,Nourrisson C,Baudu E,et al.Efficient and reproducible experimental infections of rats with Blastocystis spp.[J].PLoS One,2018,13(11):e0207669.
[29]Chandramathi S,Suresh KG,Mahmood AA,et al.Urinary hyaluronidase activity in rats infected with Blastocystis hominis:evidence for invasion?[J].Parasitol Res,2010,106(6):1459-1463.
[30]Chandramathi S,Suresh K,Sivanandam S,et al.Stress exacerbates infectivity and pathogenicity of Blastocystis hominis:in vitro and in vivo evidences[J].PLoS One,2014,9(5):e94567.
[31]Suresh K,Venilla GD,Tan TC,et al.In vivo encystation of Blastocystis hominis[J].Parasitol Res,2009,104(6):1373-1380.
[32]Ragavan ND,Govind SK,Chye TT,et al.Factors that influence the shedding of Blastocystis cysts in an irritable bowel syndrome(IBS)patient:an evidence-based case study[J].Parasitol Res,2015,114(8):2999-3005.
[33]苗雅娟,吴秀萍,王光明,等.黏蛋白在肠道寄生虫感染中的作用[J].湖北农业科学,2012,51(12):2409-2411,2415.