This study describes a simple method for the large-scale isolation of pure Toxoplasma
gondii tachyzoites and bradyzoites. T. gondii tachyzoites were obtained from infected
human foreskin fibroblasts (HFFs) and peritoneal exudates of mice, while tissue cysts
containing bradyzoites were collected from chronically infected mice. Harvested cells
and brain tissues were incubated in Hanks balanced salt solution (HBSS), containing
0.25% trypsin and 0.5% taurodeoxycholic acid (TDC) for 5 min. Subsequent washes
in phosphate buffered saline (PBS) were conducted, and the cell viability of the preparations
was good, as determined by flow cytometry and ability to reinfect HFF cells
and propagate in mice. The purification procedure allowed for a rapid preparation of pure T. gondii tachyzoites and bradyzoites in sufficient quantity that can be used for
downstream procedures. The advantage of the new method is that it is convenient
and inexpensive.The National Natural Science Foundation of China (No. 31502071), Youth Innovative Talents Project of Guangdong province Education Department (No. 2017KQNCX212), Guangdong province (2017GDK07), Start-up Research Grant Program provided by Foshan University, Foshan city, Guangdong province for distinguished researchers, Guangdong Science and Technology Plan Project (Grant No: 1244060045607389XC), and School of Life Science and Engineering fund (Grant No: KLPREAD201801-02).http://www.wileyonlinelibrary.com/journal/vms3am2021Paraclinical Science

Aboelhadid, Shawky M.

Abouhajer, Fathi

Al Nasr, Ibrahim

Alajmi, Reem A.

Cenci-Goga, Beniamino T.

El Wakil, Abeer

El-Ashram, Saeed

El-Khadragy, Manal F.

Hamady, Gamal Ali Abdelhafez

Hargis, Billy M.

Ji, Yongsheng

Karama, Musafiri

Marufu, Munyaradzi C.

Mei, Kun

Metwally, Dina M.

Salama, Dina

Shujian, Huang

Suo, Xun

Tellez-Isaias, Guillermo

Zhang, Haoji

Zhang, Yu

Zhili, Li

Marufu, Munyaradzi Christopher

UPSpace at the University of Pretoria

Vet. Med. Sci. 2021;7:357–361.    |  357wileyonlinelibrary.com/journal/vms3 DOI: 10.1002/vms3.364  O R I G I N A L  A R T I C L EA rapid and simple single-step method for the purification of Toxoplasma gondii tachyzoites and bradyzoitesSaeed El-Ashram1,2  |   Yu Zhang1 |   Yongsheng Ji3 |   Dina Salama4,5 |   Kun Mei1 |   Li Zhili1 |   Huang Shujian1 |   Haoji Zhang1 |   Shawky M. Aboelhadid5 |   Reem A. Alajmi6 |   Dina M. Metwally6 |   Manal F. El-Khadragy7 |   Billy M. Hargis8 |   Guillermo Tellez-Isaias8 |   Beniamino T. Cenci-Goga9 |   Musafiri Karama10 |   Munyaradzi C. Marufu11 |   Fathi Abouhajer12 |   Gamal Ali Abdelhafez Hamady13 |   Abeer El Wakil14 |    Ibrahim Al Nasr15,16 |   Xun Suo171College of Life Science and Engineering, Foshan University, Foshan, Guangdong Province, China2Faculty of Science, Kafrelsheikh University, Kafr el-Sheikh, Egypt3School of Basic Medical Sciences, Division of Life Science and Medicine, University of Science and Technology of China, Hefei, China4Department of Parasitology and Animal Disease, National Research Centre, Dokki, Giza, Egypt5Parasitology Department, Faculty of Veterinary Medicine, Beni Suef University, Beni-Suef, Egypt6Department of Zoology, Faculty of Science, King Saud University, Riyadh, Saudi Arabia7Department of Zoology and Entomology, Faculty of Science, Helwan University, Cairo, Egypt8Department of Poultry Science, University of Arkansas, Fayetteville, AR, USA9Dipartimento di Medicina Veterinaria, Laboratorio di Ispezione degli Alimenti di Origine Animale, University of Perugia, Perugia, Italy10Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, South Africa11Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, South Africa12Faculty of Education, Asmarya University for Islamic Sciences, Zliten, Libya13Department of Animal Production, Faculty of Agriculture, Al-Azhar University, Nasr City, Cairo, Egypt14Department of Biological & Geological Sciences, Alexandria University, Alexandria, Egypt15College of Science and Arts in Unaizah, Qassim University, Unaizah, Saudi Arabia16College of Applied Health Sciences in Ar Rass, Qassim University, Ar Rass, Saudi Arabia17National Animal Protozoa Laboratory & College of Veterinary Medicine, China Agricultural University, Beijing, ChinaThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.© 2020 The Authors. Veterinary Medicine and Science published by John Wiley & Sons LtdCorrespondenceSaeed El-Ashram, College of Life Science and Engineering, Foshan University, 18 Jiangwan street, Foshan 528231, Guangdong Province, China.Email: saeed_elashram@yahoo.comXun Suo, National Animal Protozoa Laboratory & College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China.Email: suoxun@cau.edu.cn.Funding informationStart-up Research Grant Program provided by Foshan University, Foshan city, AbstractThis study describes a simple method for the large-scale isolation of pure Toxoplasma gondii tachyzoites and bradyzoites. T. gondii tachyzoites were obtained from infected human foreskin fibroblasts (HFFs) and peritoneal exudates of mice, while tissue cysts containing bradyzoites were collected from chronically infected mice. Harvested cells and brain tissues were incubated in Hanks balanced salt solution (HBSS), containing 0.25% trypsin and 0.5% taurodeoxycholic acid (TDC) for 5 min. Subsequent washes in phosphate buffered saline (PBS) were conducted, and the cell viability of the prepa-rations was good, as determined by flow cytometry and ability to reinfect HFF cells and propagate in mice. The purification procedure allowed for a rapid preparation of 358  |     EL-ASHRAM Et AL.1  | INTRODUC TIONToxoplasma gondii is an intracellular protozoan parasite. Felids are the definitive hosts and a wide range of warm-blooded animals, in-cluding humans, rodents, birds, livestock and marine mammals are considered intermediate hosts. Sexual reproduction only occurs in felids. Sporozoites attach and penetrate the felid enterocytes after oocyst excystation in the intestinal lumen. Sporozoites undergo multiplication by endodyogeny to form tachyzoites, while brady-zoites are able to invade the intestinal cells, entering into the enteric phase of the sexual cycle. Bradyzoites switch to tachyzoites and go through several rounds of asexual division. After the sporulated oo-cysts are being ingested by intermediate hosts, sporulated oocysts will be excysted into the intestinal lumen. Sporozoites will find their way into extraintestinal sites to develop into tissue cysts in visceral organs, muscular and neural tissues (El-Ashram et al., 2015). A sub-stantial amount of research on separation of eimerian oocysts and nematode eggs from faecal materials has been published over the last decade. Techniques for the concentration and purification of oocysts and eggs from faecal samples include saturated salt solution floatation, sucrose density, zinc sulphate and Percoll discontinuous density gradient centrifugation (Kawazoe et al., 1992). For example, sucrose gradient ultracentrifugation has been exploited to separate subcellular fractions of Eimeria tenella sporozoites. Recently, atten-tion has focused on the isolation of endocytic organelles from mac-rophages by sucrose gradient (Lamberti et al., 2015). Furthermore, discontinuous sucrose gradients and isopycnic Percoll gradients have been used to isolate Cryptosporidium spp. oocysts and sporo-zoites (Arrowood & Sterling, 1987). Discontinuous sucrose gradient has been reported to be a simple and rapid procedure to obtain via-ble Cryptosporidium spp. oocysts (Bautista et al., 1999). In addition, T. gondii tachyzoites, which were obtained by two consecutive discon-tinuous sucrose gradient separations maintain their biological activ-ity (Garberi et al., 1990). However, the aforementioned procedures are cumbersome, and tachyzoite yield is low. Although, it is techni-cally possible to obtain pure zoites from oocysts collected from cat faeces using the sucrose gradient method; however, the method re-mains complicated. Currently, in vivo research on Toxoplasma using cats is very expensive because cats are an expensive species that are difficult to breed as their yields are very low. Therefore, most research on T. gondii is carried out using tachyzoites, which can be harvested in significant quantities from experimentally infected human foreskin fibroblasts (HFFs), the peritoneal cavity/exudates of experimentally infected mice, brain tissues and HFF cells. However, to obtain T. gondii tachyzoites from purification of murine peritoneal exudates, brain tissues and HFF cells remains a serious challenge. In this short communication, we report a very simple approach for the isolation of highly purified tachyzoites and bradyzoites from infected human foreskin fibroblasts (HFFs) and peritoneal exudates of mice.2  | MATERIAL S AND METHODSSix- to eight- week-old specific pathogen free (SPF) female BALB/c mice were purchased from the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences (Beijing, China). Two genotypes of T. gondii were used in this study: the avirulent type II T. gondii Prugniaud (Pru) strain and the virulent T. gondii RH strain. The avirulent type II T. gondii Prugniaud (Pru) strain maintained by passage every 4–5 weeks in mice by oral infection with five cysts where tachyzoites of T. gondii RH strain were kept stored in liquid nitrogen. The experimental infection was carried out by inoculating tachyzoites of the T. gondii RH strain to mice intraperitoneally. Successful T. gondii infection was achieved within 4–5 days after intraperitoneal infection, fol-lowed by recovery of the parasites from the peritoneal exudate. Three days after infection, mice were sacrificed by cervical dislo-cation. Furthermore, HFF cells were incubated in DMEM medium (HyClone) supplemented with 8% fetal bovine serum (HyClone) and infected with the T. gondii RH strain tachyzoites (1x107 para-sites) for 72 hr. Tachyzoites and tachyzoites were collected from the peritoneal cavity and brain tissues, respectively in 5 ml phos-phate buffered saline (PBS). Peritoneal cells and brain tissues Guangdong province for distinguished researchers, Guangdong Science and Technology Plan Project (Grant No:1244 0600 4560 7389XC) and School of Life Science and Engineering fund (Grant No: KLPREAD201801-02).pure T. gondii tachyzoites and bradyzoites in sufficient quantity that can be used for downstream procedures. The advantage of the new method is that it is convenient and inexpensive.K E Y W O R D Sflow cytometry, purification, T. gondii tachyzoites and bradyzoites, trypsin, taurodeoxycholic acidHighlights• Purification of Toxoplasma gondii tachyzoites and bradyzoites• Harvested cells and tissues were incubated in digestive solution• Preparation viability was assessed by flow cytometry and re-infection• The new method is convenient and inexpensive.     |  359EL-ASHRAM Et AL.were washed three times in PBS, placed in 5 volumes of digestive medium (i.e. 5 volumes of digestive medium/ 1 volume of cell- or tissue-containing zoites)—Hanks balanced salt solution (HBSS) containing 0.25% trypsin and 0.5% taurodeoxycholic acid (TDC). After their washing thrice with PBS, 5 ml of the trypsin-TDC me-dium was added to a T-75 flask containing Toxoplasma-infected HFF cells. The mixture was incubated at 40°C for 5 min with in-termittent shaking and free zoites were monitored by the light microscopy. The zoite-containing solution was diluted 10 times in HBSS solution at 1:10 (v:v). Tachyzoites or bradyzoites were collected by centrifugation at 4,000 × g for 5 min, and super-natant was discarded (Figure 1). Tachyzoites were washed three times with PBS, centrifuged at 4,000 × g for 5 min and propa-gated in mice by intraperitoneal inoculation. HFFs with 80%–90% confluence were infected with tachyzoites. The viability of the purified zoites was detected by a flow cytometer (FCM) as fol-lows: the isolated tachyzoites and bradyzoites were added to Eppendorf tubes containing 5 µl of Annexin V-FITC (fluorescein isothiocyanate conjugated), 5 µl propidium iodide (PI) or 1.5 ml PBS. Furthermore, Annexin+/PI-(early apoptosis) and Annexin+/PI+(late apoptosis or already dead) parasites were sorted by fluoresence-activated cell sorter (FACS)(Demchenko, 2013; Yin, et al., 2015; Yin et al., 2015).3  | RESULTS AND DISCUSSIONHFF and murine peritoneal exudates of T. gondii RH strain-infected HFF cells (Figure 2a,b) and mice (Figure 2c,d) are shown below be-fore and after purification, respectively. Additionally, the purified bradyzoites are displayed in Figure 3. The viability of tachyzoites and bradyzoites was established by flow cytometry and re-infectivity. As shown in Figure 4, more than 99% of collected parasites were Annexin−/PI−, indicating the high viability of parasites.There are several published reports on purification of Toxoplasma tachyzoites by passing the parasite through a 25-gauge needle, filtration through a 3-μm pore size, sucrose gradient density centrifugation (Garberi et al., 1990; Khan & Grigg, 2017). Toxoplasma tachyzoite prurification can also be achieved by sonic vibration and trypsin digestion (Tsunematsu, 1960) or by a sin-tered glass filter. These procedures are often complex and pose scale-up challenges. Additionally, recovery rates can be low, and some methods have failed to produce pure tachyzoite prepara-tions. In this study, we developed a rapid and simple method for purifying tachyzoites and bradyzoites with optimal yield and pu-rity. The new proposed method has the following advantages: (a) zoites can be obtained quickly and easily; (b) the technique can be performed in low resource settings and capable of handling F I G U R E  1   Flow chart summarizing the purification steps of T. gondii tachyzoites and bradyzoites360  |     EL-ASHRAM Et AL.larger numbers of cells and tissues; (c) zoites obtained in this way are free of cellular debris; and (d) the viability and infectivity of tachyzoites and bradyzoites are maintained. A more detailed study is required to explore the effect of trypsin on surface anti-gens of parasites.ACKNOWLEDG EMENTSThis work was supported by the National Natural Science Foundation of China (No. 31502071), Youth Innovative Talents Project of Guangdong province Education Department (No. 2017KQNCX212), Guangdong province (2017GDK07), Start-up Research Grant Program F I G U R E  3   Purified bradyzoites from infected murine brainF I G U R E  4   Viability of T. gondii tachyzoites by flow cytometryF I G U R E  2   Tachyzoite-infected human foreskin fibroblast (HFF) cell line (a) and purified tachyzoites from HFF cell line (b1 and b2); and tachyzoite-infected peritoneal cells (c) and purified tachyzoites from HFF cell line (d)(a) (b)1 (b)2(c) (d)     |  361EL-ASHRAM Et AL.provided by Foshan University, Foshan city, Guangdong province for distinguished researchers, Guangdong Science and Technology Plan Project (Grant No: 1244060045607389XC), and School of Life Science and Engineering fund (Grant No: KLPREAD201801-02).CONFLIC TS OF INTERE S TThe author declares no conflicts of interest.AUTHOR CONTRIBUTIONSSaeed El-Ashram: Conceptualization, Investigation and Methodology.Saeed El-Ashram, Yu Zhang, Yongsheng Ji, Dina Salama, Kun Mei, Li Zhili, Huang Shujian, Haoji Zhang, Shawky M. Aboelhadid, Reem A. Alajmi, Dina M. Metwally, Manal F. El-Khadragy, Billy M. Hargis, Guillermo Tellez-Isaias, Beniamino T. Cenci-Goga, Musafiri Karama, Munyaradzi C. Marufu, Fathi Abouhajer, Gamal Ali Abdelhafez Hamady, Abeer El Wakil, Ibrahim Al Nasr and Xun Suo: Writing-original draft, Writing-review and editing.AUTHOR CONTRIBUTIONSaeed El-Ashram: Conceptualization; Investigation; Methodology; Writing-original draft; Writing-review & editing. Yu Zhang: Writing-original draft; Writing-review & editing. Yongsheng Ji: Writing-original draft; Writing-review & editing. Dina Salama: Writing-original draft; Writing-review & editing. Kun Mei: Writing-original draft; Writing-review & editing. Li Zhili: Writing-original draft; Writing-review & editing. Huang Shujian: Writing-original draft; Writing-review & editing. Haoji Zhang: Writing-original draft; Writing-review & edit-ing. Shawky M Aboelhadid: Writing-original draft; Writing-review & editing. Reem Reem Alajmi: Writing-original draft; Writing-review & editing. dina metwally: Writing-original draft; Writing-review & editing. Manal El-Khadragy: Writing-original draft; Writing-review & editing. Billy Hargis: Writing-original draft; Writing-review & edit-ing. Guillermo Tellez: Writing-original draft; Writing-review & edit-ing. Beniamino Cenci-Goga: Writing-original draft; Writing-review & editing. Musafiri Karama: Writing-original draft; Writing-review & editing. Munyaradzi Marufu: Writing-original draft; Writing-review & editing. Fathi Abouhajer: Writing-original draft; Writing-review & editing. gamal ali abdelhafez hamady: Writing-original draft; Writing-review & editing. Abeer El Wakil: Writing-original draft; Writing-review & editing. Xun Suo: Writing-original draft; Writing-review & editing.E THIC AL S TATEMENTAll experiments were conducted according to the ethical standards and protocols approved by the Committee of Animal Experimentation of College of Life Science and Engineering, Foshan University, China (permission number 2019- FOSU- 04).PEER RE VIE WThe peer review history for this article is available at https://publo ns.com/publo n/10.1002/vms3.364.ORCIDSaeed El-Ashram  https://orcid.org/0000-0003-0389-1980 R E FE R E N C E SArrowood, M. J., & Sterling, C. R. (1987). Isolation of Cryptosporidium oocysts and sporozoites using discontinuous sucrose and isopycnic Percoll gradients. Journal of Parasitology, 73, 314–319. https://doi.org/10.2307/3282084Bautista, E., Pezzani, B. C., Cordoba, A., De Luca, M. M., & Basualdo, J. A. (1999). Relationship between discontinuous sucrose gradient and viability of Cryptosporidium sp. oocysts. Revista Argentina De Microbiologia, 31, 188–192.Demchenko, A. P. (2013). Beyond annexin V: Fluorescence response of cellular membranes to apoptosis. Cytotechnology, 65, 157–172. https://doi.org/10.1007/s1061 6-012-9481-yEl-Ashram, S., Yin, Q., Barta, J. R., Khan, J., Liu, X., & Suo, X. (2015). Immunoproteomic technology offers an extraordinary diagnostic approach for Toxoplasma gondii infection. Journal of Microbiological Methods, 119, 18–30. https://doi.org/10.1016/j.mimet.2015.09.011Garberi, J. C., Blanco, J. C., Angel, S. O., Pzsenny, V., Arakelian, M. C., & Pastini, A. (1990). Toxoplasma gondii: A rapid method for the iso-lation of pure tachyzoites. Preliminary characterization of its ge-nome. Memorias do Instituto Oswaldo Cruz, 85, 429–434. https://doi.org/10.1590/S0074 -02761 99000 0400007Kawazoe, U., Tomley, F. M., & Frazier, J. A. (1992). Fractionation and an-tigenic characterization of organelles of Eimeria tenella sporozoites. Parasitology, 104(Pt 1), 1–9.Khan, A., & Grigg, M. E. (2017). Toxoplasma gondii: Laboratory Maintenance and Growth. Curr Protoc Microbiol, 44, 20C.21.21-20C.21.17.Lamberti, G., de Araujo, M. E., & Huber, L. A. (2015). Isolation of mac-rophage early and late endosomes by latex bead internalization and density gradient centrifugation. Cold Spring Harbor Protocols, 2015(12), pdb.prot083451.Tsunematsu, Y. (1960). Purification of toxoplasma by means of sonic vibra-tion and tryptic digestion. The American Journal of Tropical Medicine and Hygiene, 9, 556–561. https://doi.org/10.4269/ajtmh.1960.9.556Yin, Q., El-Ashram, S., Liu, H., Sun, X., Zhao, X., Liu, X., & Suo, X. (2015). Interferon-gamma release assay: An effective tool to detect early toxoplasma gondii infection in mice. PLoS One, 10, e0137808. https://doi.org/10.1371/journ al.pone.0137808Yin, Q., El-Ashram, S., Liu, X. Y., & Suo, X. (2015). Early detection of Toxoplasma gondii-infected cats by interferon-gamma release assay. Experimental Parasitology, 157, 145–149. https://doi.org/10.1016/j.exppa ra.2015.08.015How to cite this article: El-Ashram S, Zhang Y, Ji Y, et al. A rapid and simple single-step method for the purification of Toxoplasma gondii tachyzoites and bradyzoites. Vet. Med. Sci. 2021;7:357–361. https://doi.org/10.1002/vms3.364

A rapid and simple single-step method for the purification of Toxoplasma gondii tachyzoites and bradyzoites

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A rapid and simple single-step method for the purification of Toxoplasma gondii tachyzoites and bradyzoites

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