Skip to content
Licensed Unlicensed Requires Authentication Published by De Gruyter April 13, 2018

Primary culture of cat intestinal epithelial cells in vitro and the cDNA library construction

  • Gui hua Zhao , Ye Liu , Yun tang Cheng , Qing song Zhao , Xiao Qiu , Chao Xu , Ting Xiao , Song Zhu , Gong zhen Liu and Kun Yin EMAIL logo
From the journal Acta Parasitologica

Abstract

Felids are the only definitive hosts of Toxoplasma gondii. To lay a foundation for screening the T. gondii-felids interaction factors, we have developed a reproducible primary culture method for cat intestinal epithelial cells (IECs). The primary IECs were isolated from a new born cat’s small intestine jejunum region without food ingress, and respectively in vitro cultured by tissue cultivation and combined digestion method with collagenase XI and dispase I, then purified by trypsinization. After identification, the ds cDNA of cat IECs was synthesized for constructing pGADT7 homogenization three-frame plasmid, and transformed into the yeast Y187 for generating the cDNA library. Our results indicated that cultivation of primary cat IECs relays on combined digestion to form polarized and confluent monolayers within 3 days with typical features of normal epithelial cells. The purified cells cultured by digestion method were identified to be nature intestinal epithelial cells using immunohistochemical analysis and were able to maintain viability for at least 15 passages. The homogenizable ds cDNA, which is synthesized from the total RNA extracted from our cultured IECs, distributed among 0.5–2.0 kb, and generated satisfying three-frame cDNA library with the capacity of 1.2 × 106 and the titer of 5.2 × 107 pfu/mL. Our results established an optimal method for the culturing and passage of cat IECs model in vitro, and laid a cDNA library foundation for the subsequent interaction factors screening by yeast two-hybrid.

  1. Ethics considerations: The study protocol was subject to approval by the Ethical Review Committee of the Shandong Academy of Medical Sciences, Shandong Institute of Parasitical Disease.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (81702026, 31300617, 31502057); the National Science Foundation of Shandong Province (ZR2014YL039, BS2013SW015, ZR201709180311); the Medicine and Health Science Technology Development Plan of Shandong Province (2017WS103); and the Innovation Project of Shandong Academy of Medical Sciences.

References

Alvarado-Esquivel C., Torres-Castorena A., Liesenfeld O., García-López C., Estrada-Martínez S., Sifuentes-Álvarez A., et al. 2009. Seroepidemiology of Toxoplasma gondii infection in pregnant women in rural Durango. Journal of Parasitology, 95, 271–274. 10.1645/GE-1829.1Search in Google Scholar

Boyle J.P., Radke J.R. 2009. A history of studies that examine the interactions of Toxoplasma with its host cell: Emphasis on in vitro models. International Journal for Parasitology, 39, 903–91410.1016/j.ijpara.2009.01.008Search in Google Scholar PubMed

Chatterton J.M., Evans R., Ashburn D., Joss A.W., Ho-Yen D.O. 2002. Toxoplasma gondii in vitro culture for experimentation. Journal of Microbiological Methods, 51, 331–33510.1016/S0167-7012(02)00101-XSearch in Google Scholar PubMed

Commodaro A.G., Chiasson M., Sundar N., Rizzo L.V., Belfort R. Jr., Grigg M.E. 2016. Elevated Toxoplasma gondii Infection Rates for Retinas from Eye Banks, Southern Brazil. Emerging Infectious Diseases, 22, 691–693. 10.3201/eid2204.141819Search in Google Scholar PubMed

Daryani A., Sarvi S., Aarabi M., Mizani A., Ahmadpour E., Shokri A., et al. 2014. Seroprevalence of Toxoplasma gondii in the Iranian general population: a systematic review and meta-analysis. Acta Trop, 137, 185–194. 10.1016/j.actatropica.Search in Google Scholar PubMed

Dubey J.P. 2009. History of the discovery of the life cycle of Toxoplasma gondii. International Journal for Parasitology, 39, 877–88210.1016/j.ijpara.2009.01.005Search in Google Scholar PubMed

Dubey J.P., Beattie C.P. 2010. Toxoplasmosis of animals and humans. CRC Press, Second edition, pp. 313Search in Google Scholar

Dumètre A., Dardé M.L. 2003. How to detect Toxoplasma gondii oocystsin environmental samples? FEMS Microbiology Reviews, 27, 651–66110.1016/S0168-6445(03)00071-8Search in Google Scholar PubMed

Ferguson D.J. 2004. Use of molecular and ultrastructural markers to evaluate stage conversion of Toxoplasma gondii in both the intermediate and definitive host. International Journal for Parasitology, 34, 347–46010.1016/j.ijpara.2003.11.024Search in Google Scholar PubMed

Grabinger T., Luks L., Kostadinova F., Zimberlin C., Medema J.P., Leist M., Brunner T. 2014. Ex vivo culture of intestinal crypt organoids as a model system for assessing cell death induction in intestinal epithelial cells and enteropathy. Cell Death & Disease, 5, e1228. 10.1038/cddis.2014.183Search in Google Scholar PubMed PubMed Central

Hunter C.A., Sibley L.D. 2012. Modulation of innate immunity by Toxoplasma gondii virulence effectors. Nature Reviews Microbiology, 10, 766–778. 10.1038/nrmicro2858Search in Google Scholar PubMed PubMed Central

Jones J.L., Dubey J.P. 2012. Food borne toxoplasmosis. Clinical Infectious Diseases, 55, 845–851. 10.1093/cid/cis508Search in Google Scholar PubMed

Kuratnik A., Giardina C. 2013. Intestinal organoids as tissue surrogates for toxicological and pharmacological studies. Biochemical Pharmacology, 85, 1721–1726. 10.1016/j.bcp.2013.04.016Search in Google Scholar PubMed

Meireles L.R., Ekman C.C., Andrade jR. HF., Luna E.J. 2015. Human toxoplasmosis outbreaks and the agent infecting from findings from a systematic review. Revista do Instituto de Medicina Tropical de São Paulo, 57, 369–376. 10.1590/S0036-46652015000500001Search in Google Scholar PubMed PubMed Central

Ogoina D., Onyemelukwe G.C., Musa B.O., Obiako R.O. 2013. Seroprevalence of IgM and IgG antibodies to Toxoplasma infection in healthy and HIV-positive adults from Northern Nigeria. Journal of Infection in Developing Countries, 7, 398–403. 10.3855/jidc.2797Search in Google Scholar PubMed

Sibley L.D. 2011. Invasion and a cellular survival by protozoan parasites. Immunological Reviews, 240, 72–91. 10.1111/j.1600-065X.2010.00990.xSearch in Google Scholar PubMed PubMed Central

Speer C.A., Dubey J.P. 1998. Ultrastructure of early stages of infections in mice fed Toxoplasma gondii oocysts. Parasitology, 116, 35–4210.1017/S0031182097001959Search in Google Scholar PubMed

Swierzy I.J., Muhammad M., Kroll J., Abelmann A., Tenter A.M., Luder C.G. 2014. Toxoplasma gondii within skeletal muscle cells: a critical interplay for food-borne parasite transmission. International Journal for Parasitology, 44, 91–98. 10.1016/j.ijpara.2013.10.001Search in Google Scholar PubMed

Torrey E.F., Yolken R.H. 2013. Toxoplasma oocysts as a public health problem. Trends in Parasitology, 29, 380–384. 10.1016/j.pt.2013.06.001Search in Google Scholar PubMed

Wu L., Chen S.X., Jiang X.G., Fu X.L., Shen Y.J., Cao J.P. 2012. Separation and purification of Toxoplasma gondii tachyzoites from in vitro and in vivo culture systems. Experimental Parasitology, 130, 91–94. 10.1016/j.exppara.2011.10.006Search in Google Scholar PubMed

Zhan K., Jiang M., Sui Y., Yan K., Lin M., Zhao G. 2017. Establishment of immortalized mouse intestinal epithelial cells line and study of effects of Arg-Arg on inflammatory response. In Vitro Cellular & Developmental Biology-animal, 53, 538–546. 10.1007/s11626-017-0143-4Search in Google Scholar PubMed

Zhou P., Chen Z., Li H.L., Zheng H., He S., Lin R.Q., Zhu X.Q. 2011. Toxoplasma gondii infection in humans in China. Parasites & Vectors, 4, 1–9. 10.1186/1756-3305-4-165Search in Google Scholar PubMed PubMed Central

Received: 2017-8-2
Revised: 2018-2-8
Accepted: 2018-2-13
Published Online: 2018-4-13
Published in Print: 2018-6-26

© 2018 W. Stefański Institute of Parasitology, PAS

Downloaded on 29.3.2024 from https://www.degruyter.com/document/doi/10.1515/ap-2018-0041/html
Scroll to top button