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Transfection of live, tick derived sporozoites of the protozoan Apicomplexan parasite Theileria parva
Veterinary Parasitology 208 (2015) 238–241
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short Communication
Transfection of live, tick derived sporozoites of the protozoan Apicomplexan parasite Theileria parva Ine De Goeyse a,∗ , Famke Jansen a , Maxime Madder a , Kyoko Hayashida b , Dirk Berkvens a , Dirk Dobbelaere c,1 , Dirk Geysen a a
Institute of Tropical Medicine, Department of Biomedical Sciences, Nationalestraat 155, 2000 Antwerp, Belgium Division of Collaboration and Education, Research Center for Zoonosis Control, Hokkaido University, Sapporo-shi, Hokkaido 001-0020, Japan c Division of Molecular Pathobiology, Vetsuisse Faculty, University of Bern, Länggassstrasse 122, CH-3012 Bern, Switzerland b
a r t i c l e
i n f o
Article history: Received 28 May 2014 Received in revised form 12 January 2015 Accepted 17 January 2015 Keywords: Theileria parva Transfection Nucleofection Sporozoites
a b s t r a c t Theileria parva is an important veterinary protozoan causing the tick-borne disease East Coast fever. Transfection of Theileria parasites will facilitate the investigation of many aspects of this apicomplexan infection and its unique host–parasite interaction. The pathogen has the extraordinary capacity of transforming B and T cells into clonally dividing cancerous cell lines in a reversible way. Sequence data of the entire T. parva genome are available and in vitro infected cell lines can easily be generated, thereby eliminating the use of animals in the evaluation of the evolution of the transfected sporozoites. Here we report, for the first time, on experiments towards successful transient transfection of T. parva sporozoites, making use of a new generation transfection device. Plasmid DNA containing the strong EF-1␣ promoter and an Azami Green reporter gene were integrated by nucleofection into freshly purified T. parva sporozoites. Expression of Azami Green was detected with a fluorescence microscope and confirmed by counter staining with a monoclonal directed against a sporozoite protein. Despite the fact that transfection efficiencies are still low, this is the first step towards a stable infection method of T. parva parasites. In the long run, transfected parasites might become an alternative way to induce immunity without clinical signs. © 2015 Elsevier B.V. All rights reserved.
Theileria parva, an important protozoan belonging to the Apicomplexa family, causes the fatal disease East Coast fever in cattle, restricted to the East Coast and the central part of Southern Africa. This disease, together with tropical theileriosis (Theileria annulata), causes major economic losses to the farming community in developing countries. Infective sporozoites, transmitted through the saliva of the brown ear tick Rhipicephalus appendiculatus,
∗ Corresponding author. Tel.: +32 3 2470721; fax: +32 3 2476268. E-mail address: [email protected] (I. De Goeyse). 1 Deceased. http://dx.doi.org/10.1016/j.vetpar.2015.01.013 0304-4017/© 2015 Elsevier B.V. All rights reserved.
enter host’s lymphocytes and mature into intracellular schizonts. Theileria is unique among parasites in its ability to stimulate the uncontrolled proliferation of the blood lymphocytes it infects (Dobbelaere and Küenzi, 2004). This pathogen has the extraordinary capacity of transforming peripheral blood lymphocytes (PBMCs) in vitro into clonally dividing cancerous cell lines in a reversible way. Once the intracellular parasite dies off, the Theileria containing cell will revert to its normal phenotype. Moreover, the genome of T. parva is sequenced (Gardner et al., 2005) and a database to support vaccine development is available (Visendi et al., 2011).
I. De Goeyse et al. / Veterinary Parasitology 208 (2015) 238–241
Genetic manipulation of parasites can help to improve the understanding of the fundamental biology of parasites. Transfection is a widely used technique to study gene regulation and function. The possibility to transfect members of the Apicomplexa has already been proven for many of them, such as Plasmodium spp., Babesia bovis, Toxoplasma gondii, Eimeria tenella and Neospora caninum (Donald and Roos, 1993; Soldati and Boothroyd, 1993; Van Wye et al., 1996; Beckers et al., 1997; Ménard and Janse, 1997; Kelleher and Tomley, 1998; Sultan et al., 1999). Regarding Theileria, only one report of a transient transfection event has been recorded for T. annulata (Adamson et al., 2001). Until now, transfection of Theileria using infected cell cultures has failed probably due to the fact that the transfecting plasmids had to cross two membranes (host lymphocyte, and parasite membrane). Despite the more time-consuming purification of sporozoites from salivary glands, this is the only way to transfect the parasite for the moment. We report here on experiments resulting for the first time in transient transfection of T. parva sporozoites using a 4D-NucleofectionTM System (Lonza), based on the momentary formation of small pores in cell membranes by applying an electrical pulse. A novel foreign protein expression vector pMotif-EF1␣-100 was derived from pRNAi-GL (Takara, Japan), a conventional mammalian protein expression vector. The original CMV promoter gene was replaced with the elongation factor 1 alpha (EF-1˛) gene promoter region of T. parva, as used successfully in transfection of Babesia and Plasmodium (Fernandez-Becerra et al., 2003; Suarez and McElwain, 2009). The minus 263 to 169 position of the 5 region of the EF-1˛ orf (TP01 0726) was amplified from the T. parva Muguga strain genomic DNA. The promoter sequence is preceded by a ‘motif’ sequence, consisting of three tandem putative transcription factor binding sites of T. parva (Guo and Silva, 2008). Further, a transmembrane Azami Green sequence, preceded by a secretion signal sequence was integrated to visualize transfected parasites (Fig. 1). Azami Green is a green-emitting fluorescent protein from a stony coral, Galaxeidae, which absorbs light maximally at 492 nm and emits the green light at 505 nm. The protein is stable and fluorescence is brighter in cultured cells than EGFP (Karasawa et al., 2003). Infection can be confirmed in the early stages by staining fixed cells with monoclonal antibodies against the Polymorphic Immunodominant Molecule (PIM), present in rhoptries of T. parva sporozoites or at the surface of the schizont (Toye et al., 1996). T. parva (Muguga stock) infected R. appendiculatus ticks were obtained from either the International Livestock Research Institute (Nairobi, Kenya) or the Centre for Ticks and Tick-Borne Diseases (Lilongwe, Malawi). Sporozoites were prepared from salivary glands of infected adult ticks pre-fed on rabbit ears for 4 days (Shaw et al., 1999). All experiments were performed in accordance with the procedures approved by the Animal Ethics Committee (BM2013-04). Ticks were dissected and both salivary glands removed, stored on ice in gland diluent (RPMI with antibiotics) until crushing in 2 ml tissue grinders
239
(type Potter-Elvehjem, VWR, Belgium). After centrifugation at 1000 × g for 10 min, the supernatant, containing the liberated sporozoites, was taken off and centrifuged at 10,000 × g for 10 min. Pellets were resuspended in transfection buffer (Lonza) and mixed with 2 g of plasmid DNA. PBMC were purified from bovine blood samples collected in Alsever’s solution (Sigma) using a Nycoprep gradient as described previously (Goddeeris and Morrison, 1988). Harvested cells were counted and resuspended in culture medium (RPMI 1640 containing 25 mM Hepes & l-glutamin (Gibco® ) supplemented with 15% Foetal calf serum (FCS, Gibco® ), 10% l-glutamin (Gibco® ), 0.05 mg/ml gentamycin (Gibco® ) and 0.025 mM 2-mercaptoethanol (ICN Biochemicals) at a concentration of 4 × 106 PBMC/ml. For the actual transfection experiments, 107 purified sporozoites were suspended in 20 l SF transfection buffer (Lonza), together with 2 g plasmid DNA. Different transfection protocols (CA107, CA150, EO101, 4D-NucleofectionTM System Lonza) were tested. After transfection, sporozoite diluent (RPMI-1640 containing BSA and antibiotics) was added and sporozoites were transferred, mixed with 107 purified bovine PBMCs and divided over 5 wells on a 24-well plate. Transfection events were checked by spotting PBMCs on cytospin slides, 24 h, 48 h and 72 h post infection. Cells were fixated with 4% paraformaldehyde. A fluorescence microscope (Zeiss, Axio Imager M1 with Z-drive, camera, and AxioVision software with co-localisation module) was used for monitoring transfected parasites. The DAPI filterset was used for DNA visualization and the GFP filter for Azami Green expression. In order to confirm possible transfection events, a counterstaining with monoclonal anti-PIM antibody was developed. Cytospin slides were stained with IL-S36.5 antiPIM (Toye et al., 1996) as first antibody and Alexa fluor® 555 Goat anti-mouse IgG (Invitrogen) as secondary antibody. PBMCs infected with sporozoites were examined daily and the first schizonts became visible after three days. All transfection events were seen between 24 h and 46 h post transfection. After 72 h multinucleated schizonts were visible, but no fluorescent schizonts were detected. For the first time, transfection events were observed in T. parva parasites (Figs. 2 and 3, non-transfected sporozoite Fig. 4). The fluorescence with the GFP-filter corresponded with DAPI staining and PIM staining. Screening for in vitro infection is time-consuming. An alternative approach to increase sensitivity of detection would be the use of a luciferase construct as successfully shown by some groups (Wu et al., 1995, 1996; de Koning-Ward et al., 1999), followed by flow cytometry analysis. However, high transfection efficiencies are required for the use of flow cytometry systems. Although we used the latest nucleofection technique, transfection efficiencies were low. Our results are comparable with the 0.3–0.9 % transfection results of T. annulata, as reported by Adamson et al. in 2001. The main obstacle is the development of an optimal purification method to obtain sufficient live sporozoites for transfection in a less cumbersome way. There are indications in our experiments of a much lower amount of viable purified sporozoites than expected. Although, best transfection results so far were seen with sporozoites freed
240
I. De Goeyse et al. / Veterinary Parasitology 208 (2015) 238–241
Fig. 1. Transfection construct for Theileria parva: sequence details. A transmembrane Azami Green sequence, used as a selectable marker, is cloned after a strong gene promoter region of T. parva (EF-1˛).
Fig. 2. Cells spotted on cytospin slide 24 h post transfection. Images are sections of a multiple z-stack. Magnification 100× (Axio Imager): (a) DAPI staining; (b) expression of Azami Green; (c) combination of the two images.
Fig. 3. Cells spotted on cytospin slide 45 h post transfection. Images are sections of a multiple z-stack. Magnification 100× (Axio Imager): (a) DAPI staining; (b) expression of Azami Green; (c) PIM staining; (d) combination of the three images.
from infected salivary glands dissected out of living ticks. In a first approach we took sporozoites from cryopreserved T. parva stabilates, but no positive transfection event was obtained, probably due to traces of glycerol having a known negative effect on transfection. However purification of sporozoites from salivary glands is very demanding pre-feeding of ticks is required and a considerable number of adult ticks need to be dissected depending on
infection rate and age. Dissection of living ticks is very labour-intensive and time-consuming and free extracellular sporozoites have a limited lifespan. Although equal numbers of sporozoites and PBMCs (theoretically calculated 2 × 106 sporozoites and purified PBMCs) were added in one well of a 24 well plate, only 5–10 infections were detected with or without pulse. Most likely the liberation of sporozoites by crushing salivary glands was the
Fig. 4. Cells spotted on cytospin slide 24 h post transfection. Sporozoites stained with anti-PIM monoclonal antibody (c), but no expression of Azami Green (b). Images are sections of a multiple z-stack. Magnification 100× (Axio Imager): (a) DAPI staining; (b) expression of Azami Green; (c) PIM staining; (d) combination of the three images.
I. De Goeyse et al. / Veterinary Parasitology 208 (2015) 238–241
most important limiting factor here. Salivary glands are quite sticky and clumping of sporozoites with residues of salivary gland cells could explain the low infection rate. Crushing higher numbers of salivary glands could increase the amount of free sporozoites. In summary, we present the first successful transient transfection of T. parva sporozoites. Transient transfection of T. annulata sporozoites has already been reported in 2001 (Adamson et al., 2001) using sporozoites from cryopreserved stabilates of ground up infected tick material. We used freshly purified live sporozoites and used a different promoter and fluorochrome. Optimization of the protocol to increase transfection efficiencies is needed for stable transfection tests. This study describes the first steps towards transfection of T. parva parasites, but protocol optimization for higher numbers of free-living sporozoites will improve transfection efficiencies. Further research on several aspects of the unique host–parasite interaction and even the generation of attenuated parasites would be possible after the successful establishment of stable transfection, which could lead to a breakthrough in the vaccination research. Acknowledgements The authors like to thank Dr. M. Janssens for the initial steps in the development of the methodology, Dr. R. De Deken, A. Van Hul, K. De Witte, G. De Deken, L. Verhelst and M. Van den Bogaerde for their technical support. Infected ticks were received from ILRI, Kenya and CTTBD, Malawi. This project was partly funded by SOFI-B funds, Belgian Ministry of Science. References Adamson, R., Lyons, K., Sharrard, M., Kinnaird, J., Swan, D., Graham, S., Shiels, B., Hall, R., 2001. Transient transfection of Theileria annulata. Mol. Biochem. Parasitol. 114 (1), 53–61. Beckers, C.J., Wakefield, T., Joiner, K.A., 1997. The expression of Toxoplasma proteins in Neospora caninum and the identification of a gene encoding a novel rhoptry protein. Mol. Biochem. Parasitol. 89 (2), 209–223. de Koning-Ward, T.F., Speranc¸a, M.A., Waters, A.P., Janse, C.J., 1999. Analysis of stage specificity of promoters in Plasmodium berghei using luciferase as a reporter. Mol. Biochem. Parasitol. 100 (1), 141–146. Dobbelaere, D.A., Küenzi, P., 2004. The strategies of the Theileria parasite: a new twist in host–pathogen interactions. Curr. Opin. Immunol. 16 (4), 524–530. Donald, R.G., Roos, D.S., 1993. Stable molecular transformation of Toxoplasma gondii: a selectable diahydrofolate reductase-thymidylate
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synthase marker based on drug-resistance mutations in malaria. Proc. Natl. Acad. Sci. U. S. A. 90 (24), 11703–11707. Fernandez-Becerra, C., de Azevedo, M.F., Yamamoto, M.M., del Portillo, H.A., 2003. Plasmodium falciparum: new vector with bi-directional promoter activity to stably express transgenes. Exp. Parasitol. 103 (1–2), 88–91. Gardner, M.J., Bishop, R., Shah, T., de Villiers, E.P., Carlton, J.M., Hall, N., Ren, Q., Paulsen, I.T., Pain, A., Berriman, M., Wilson, R.J.M., Sato, S., Ralph, S.A., Mann, D.J., Xiong, Z., Shallom, S.J., Weidman, J., Jiang, L., Lynn, J., Weaver, B., Shoaibi, A., Wasawo, D., Crabtree, J., Wortman, J.R., Haas, B., Angiuoli, S., Creasy, T., Lu, C., Suh, B., Silva, J., Utterbacl, T., Feldblyum, T., Pertea, M., Allen, J., Taracha, E., Salzberg, S.L., White, O., Fitzhugh, H.A., Morzaria, S., Venter, J.C., Fraser, C.M., Nene, V., 2005. Genome sequence of Theileria parva, a bovine pathogen that transforms lymphocytes. Science 309, 134–137. Goddeeris, B.M., Morrison, W.I., 1988. Techniques for the generation, cloning, and characterization of bovine cytotoxic T cells specific for the protozoan Theileria parva. J. Tissue Cult. Methods 11, 101–110. Guo, X., Silva, J.C., 2008. Properties of non-coding DNA and identification of putative cis-regulatory elements in Theileria parva. BMC Genomics 9, p582. Karasawa, S., Araki, T., Yamamoto-Hino, M., Miyawaki, A., 2003. A greenemitting fluorescent protein from Galaxeidae coral and its monomeric version for use in fluorescent labeling. J. Biol. Chem. 278 (36), 34167–34171. Kelleher, M., Tomley, F.M., 1998. Transient expression of betagalactosidase in differentiating sporozoites of Eimeria tenella. Mol. Biochem. Parasitol. 97 (1–2), 21–31. Ménard, R., Janse, C.J., 1997. Gene targeting in malaria parasites. Methods 13, 148–157. Shaw, M.K., Tilney, L.G., Musoke, A.J., 1999. The entry of Theileria parva sporozoites into bovine lymphocytes: evidence for MHC class I involvement. J. Cell Biol. 113, 87. Soldati, D., Boothroyd, J.C., 1993. Transient transfection and expression in the obligate intracellular parasite Toxoplasma gondii. Science 260 (5106), 349–352. Suarez, C.E., McElwain, T.F., 2009. Stable expression of a GFP-BSD fusion protein in Babesia bovis merozoites. Int. J. Parasitol. 39 (3), 289–297. Sultan, A.A., Thathy, V., Nussenzweig, V., Ménard, R., 1999. Green fluorescent protein as a marker in Plasmodium berghei transformation. Infect. Immun. 67 (5), 2602–2606. Toye, P., Nyanjui, J., Goddeeris, B., Musoke, A.J., 1996. Identification of neutralization and diagnostic epitopes on PIM, the polymorphic immunodominant molecule of Theileria parva. Infect. Immun. 64 (5), 1832–1838. Van Wye, J., Ghori, N., Webster, P., Mitschler, R.R., Elmendorf, H.G., Haldar, K., 1996. Identification and localization of rab6, separation of rab6 from ERD2 and implications for an ‘unstacked’ Golgi, in Plasmodium falciparum. Mol. Biochem. Parasitol. 83 (1), 107–120. Visendi, P., Ng’ang’a, W., Bulimo, W., Bishop, R., Ochanda, J., de Villiers, E.P., 2011. TparvaDB: a database to support Theileria parva vaccine development. In: Database (Oxford), bar015. Wu, Y., Sifri, C.D., Lei, H.H., Su, X.Z., Wellems, T.E., 1995. Transfection of Plasmodium falciparum within human red blood cells. Proc. Natl. Acad. Sci. U. S. A. 92, 973–977. Wu, Y., Kirkman, L.A., Wellems, T.E., 1996. Transformation of Plasmodium falciparum malaria parasites by homologous integration of plasmids that confer resistance to pyrimethamine. Proc. Natl. Acad. Sci. U. S. A. 93, 1130–1134.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short Communication
Transfection of live, tick derived sporozoites of the protozoan Apicomplexan parasite Theileria parva Ine De Goeyse a,∗ , Famke Jansen a , Maxime Madder a , Kyoko Hayashida b , Dirk Berkvens a , Dirk Dobbelaere c,1 , Dirk Geysen a a
Institute of Tropical Medicine, Department of Biomedical Sciences, Nationalestraat 155, 2000 Antwerp, Belgium Division of Collaboration and Education, Research Center for Zoonosis Control, Hokkaido University, Sapporo-shi, Hokkaido 001-0020, Japan c Division of Molecular Pathobiology, Vetsuisse Faculty, University of Bern, Länggassstrasse 122, CH-3012 Bern, Switzerland b
a r t i c l e
i n f o
Article history: Received 28 May 2014 Received in revised form 12 January 2015 Accepted 17 January 2015 Keywords: Theileria parva Transfection Nucleofection Sporozoites
a b s t r a c t Theileria parva is an important veterinary protozoan causing the tick-borne disease East Coast fever. Transfection of Theileria parasites will facilitate the investigation of many aspects of this apicomplexan infection and its unique host–parasite interaction. The pathogen has the extraordinary capacity of transforming B and T cells into clonally dividing cancerous cell lines in a reversible way. Sequence data of the entire T. parva genome are available and in vitro infected cell lines can easily be generated, thereby eliminating the use of animals in the evaluation of the evolution of the transfected sporozoites. Here we report, for the first time, on experiments towards successful transient transfection of T. parva sporozoites, making use of a new generation transfection device. Plasmid DNA containing the strong EF-1␣ promoter and an Azami Green reporter gene were integrated by nucleofection into freshly purified T. parva sporozoites. Expression of Azami Green was detected with a fluorescence microscope and confirmed by counter staining with a monoclonal directed against a sporozoite protein. Despite the fact that transfection efficiencies are still low, this is the first step towards a stable infection method of T. parva parasites. In the long run, transfected parasites might become an alternative way to induce immunity without clinical signs. © 2015 Elsevier B.V. All rights reserved.
Theileria parva, an important protozoan belonging to the Apicomplexa family, causes the fatal disease East Coast fever in cattle, restricted to the East Coast and the central part of Southern Africa. This disease, together with tropical theileriosis (Theileria annulata), causes major economic losses to the farming community in developing countries. Infective sporozoites, transmitted through the saliva of the brown ear tick Rhipicephalus appendiculatus,
∗ Corresponding author. Tel.: +32 3 2470721; fax: +32 3 2476268. E-mail address: [email protected] (I. De Goeyse). 1 Deceased. http://dx.doi.org/10.1016/j.vetpar.2015.01.013 0304-4017/© 2015 Elsevier B.V. All rights reserved.
enter host’s lymphocytes and mature into intracellular schizonts. Theileria is unique among parasites in its ability to stimulate the uncontrolled proliferation of the blood lymphocytes it infects (Dobbelaere and Küenzi, 2004). This pathogen has the extraordinary capacity of transforming peripheral blood lymphocytes (PBMCs) in vitro into clonally dividing cancerous cell lines in a reversible way. Once the intracellular parasite dies off, the Theileria containing cell will revert to its normal phenotype. Moreover, the genome of T. parva is sequenced (Gardner et al., 2005) and a database to support vaccine development is available (Visendi et al., 2011).
I. De Goeyse et al. / Veterinary Parasitology 208 (2015) 238–241
Genetic manipulation of parasites can help to improve the understanding of the fundamental biology of parasites. Transfection is a widely used technique to study gene regulation and function. The possibility to transfect members of the Apicomplexa has already been proven for many of them, such as Plasmodium spp., Babesia bovis, Toxoplasma gondii, Eimeria tenella and Neospora caninum (Donald and Roos, 1993; Soldati and Boothroyd, 1993; Van Wye et al., 1996; Beckers et al., 1997; Ménard and Janse, 1997; Kelleher and Tomley, 1998; Sultan et al., 1999). Regarding Theileria, only one report of a transient transfection event has been recorded for T. annulata (Adamson et al., 2001). Until now, transfection of Theileria using infected cell cultures has failed probably due to the fact that the transfecting plasmids had to cross two membranes (host lymphocyte, and parasite membrane). Despite the more time-consuming purification of sporozoites from salivary glands, this is the only way to transfect the parasite for the moment. We report here on experiments resulting for the first time in transient transfection of T. parva sporozoites using a 4D-NucleofectionTM System (Lonza), based on the momentary formation of small pores in cell membranes by applying an electrical pulse. A novel foreign protein expression vector pMotif-EF1␣-100 was derived from pRNAi-GL (Takara, Japan), a conventional mammalian protein expression vector. The original CMV promoter gene was replaced with the elongation factor 1 alpha (EF-1˛) gene promoter region of T. parva, as used successfully in transfection of Babesia and Plasmodium (Fernandez-Becerra et al., 2003; Suarez and McElwain, 2009). The minus 263 to 169 position of the 5 region of the EF-1˛ orf (TP01 0726) was amplified from the T. parva Muguga strain genomic DNA. The promoter sequence is preceded by a ‘motif’ sequence, consisting of three tandem putative transcription factor binding sites of T. parva (Guo and Silva, 2008). Further, a transmembrane Azami Green sequence, preceded by a secretion signal sequence was integrated to visualize transfected parasites (Fig. 1). Azami Green is a green-emitting fluorescent protein from a stony coral, Galaxeidae, which absorbs light maximally at 492 nm and emits the green light at 505 nm. The protein is stable and fluorescence is brighter in cultured cells than EGFP (Karasawa et al., 2003). Infection can be confirmed in the early stages by staining fixed cells with monoclonal antibodies against the Polymorphic Immunodominant Molecule (PIM), present in rhoptries of T. parva sporozoites or at the surface of the schizont (Toye et al., 1996). T. parva (Muguga stock) infected R. appendiculatus ticks were obtained from either the International Livestock Research Institute (Nairobi, Kenya) or the Centre for Ticks and Tick-Borne Diseases (Lilongwe, Malawi). Sporozoites were prepared from salivary glands of infected adult ticks pre-fed on rabbit ears for 4 days (Shaw et al., 1999). All experiments were performed in accordance with the procedures approved by the Animal Ethics Committee (BM2013-04). Ticks were dissected and both salivary glands removed, stored on ice in gland diluent (RPMI with antibiotics) until crushing in 2 ml tissue grinders
239
(type Potter-Elvehjem, VWR, Belgium). After centrifugation at 1000 × g for 10 min, the supernatant, containing the liberated sporozoites, was taken off and centrifuged at 10,000 × g for 10 min. Pellets were resuspended in transfection buffer (Lonza) and mixed with 2 g of plasmid DNA. PBMC were purified from bovine blood samples collected in Alsever’s solution (Sigma) using a Nycoprep gradient as described previously (Goddeeris and Morrison, 1988). Harvested cells were counted and resuspended in culture medium (RPMI 1640 containing 25 mM Hepes & l-glutamin (Gibco® ) supplemented with 15% Foetal calf serum (FCS, Gibco® ), 10% l-glutamin (Gibco® ), 0.05 mg/ml gentamycin (Gibco® ) and 0.025 mM 2-mercaptoethanol (ICN Biochemicals) at a concentration of 4 × 106 PBMC/ml. For the actual transfection experiments, 107 purified sporozoites were suspended in 20 l SF transfection buffer (Lonza), together with 2 g plasmid DNA. Different transfection protocols (CA107, CA150, EO101, 4D-NucleofectionTM System Lonza) were tested. After transfection, sporozoite diluent (RPMI-1640 containing BSA and antibiotics) was added and sporozoites were transferred, mixed with 107 purified bovine PBMCs and divided over 5 wells on a 24-well plate. Transfection events were checked by spotting PBMCs on cytospin slides, 24 h, 48 h and 72 h post infection. Cells were fixated with 4% paraformaldehyde. A fluorescence microscope (Zeiss, Axio Imager M1 with Z-drive, camera, and AxioVision software with co-localisation module) was used for monitoring transfected parasites. The DAPI filterset was used for DNA visualization and the GFP filter for Azami Green expression. In order to confirm possible transfection events, a counterstaining with monoclonal anti-PIM antibody was developed. Cytospin slides were stained with IL-S36.5 antiPIM (Toye et al., 1996) as first antibody and Alexa fluor® 555 Goat anti-mouse IgG (Invitrogen) as secondary antibody. PBMCs infected with sporozoites were examined daily and the first schizonts became visible after three days. All transfection events were seen between 24 h and 46 h post transfection. After 72 h multinucleated schizonts were visible, but no fluorescent schizonts were detected. For the first time, transfection events were observed in T. parva parasites (Figs. 2 and 3, non-transfected sporozoite Fig. 4). The fluorescence with the GFP-filter corresponded with DAPI staining and PIM staining. Screening for in vitro infection is time-consuming. An alternative approach to increase sensitivity of detection would be the use of a luciferase construct as successfully shown by some groups (Wu et al., 1995, 1996; de Koning-Ward et al., 1999), followed by flow cytometry analysis. However, high transfection efficiencies are required for the use of flow cytometry systems. Although we used the latest nucleofection technique, transfection efficiencies were low. Our results are comparable with the 0.3–0.9 % transfection results of T. annulata, as reported by Adamson et al. in 2001. The main obstacle is the development of an optimal purification method to obtain sufficient live sporozoites for transfection in a less cumbersome way. There are indications in our experiments of a much lower amount of viable purified sporozoites than expected. Although, best transfection results so far were seen with sporozoites freed
240
I. De Goeyse et al. / Veterinary Parasitology 208 (2015) 238–241
Fig. 1. Transfection construct for Theileria parva: sequence details. A transmembrane Azami Green sequence, used as a selectable marker, is cloned after a strong gene promoter region of T. parva (EF-1˛).
Fig. 2. Cells spotted on cytospin slide 24 h post transfection. Images are sections of a multiple z-stack. Magnification 100× (Axio Imager): (a) DAPI staining; (b) expression of Azami Green; (c) combination of the two images.
Fig. 3. Cells spotted on cytospin slide 45 h post transfection. Images are sections of a multiple z-stack. Magnification 100× (Axio Imager): (a) DAPI staining; (b) expression of Azami Green; (c) PIM staining; (d) combination of the three images.
from infected salivary glands dissected out of living ticks. In a first approach we took sporozoites from cryopreserved T. parva stabilates, but no positive transfection event was obtained, probably due to traces of glycerol having a known negative effect on transfection. However purification of sporozoites from salivary glands is very demanding pre-feeding of ticks is required and a considerable number of adult ticks need to be dissected depending on
infection rate and age. Dissection of living ticks is very labour-intensive and time-consuming and free extracellular sporozoites have a limited lifespan. Although equal numbers of sporozoites and PBMCs (theoretically calculated 2 × 106 sporozoites and purified PBMCs) were added in one well of a 24 well plate, only 5–10 infections were detected with or without pulse. Most likely the liberation of sporozoites by crushing salivary glands was the
Fig. 4. Cells spotted on cytospin slide 24 h post transfection. Sporozoites stained with anti-PIM monoclonal antibody (c), but no expression of Azami Green (b). Images are sections of a multiple z-stack. Magnification 100× (Axio Imager): (a) DAPI staining; (b) expression of Azami Green; (c) PIM staining; (d) combination of the three images.
I. De Goeyse et al. / Veterinary Parasitology 208 (2015) 238–241
most important limiting factor here. Salivary glands are quite sticky and clumping of sporozoites with residues of salivary gland cells could explain the low infection rate. Crushing higher numbers of salivary glands could increase the amount of free sporozoites. In summary, we present the first successful transient transfection of T. parva sporozoites. Transient transfection of T. annulata sporozoites has already been reported in 2001 (Adamson et al., 2001) using sporozoites from cryopreserved stabilates of ground up infected tick material. We used freshly purified live sporozoites and used a different promoter and fluorochrome. Optimization of the protocol to increase transfection efficiencies is needed for stable transfection tests. This study describes the first steps towards transfection of T. parva parasites, but protocol optimization for higher numbers of free-living sporozoites will improve transfection efficiencies. Further research on several aspects of the unique host–parasite interaction and even the generation of attenuated parasites would be possible after the successful establishment of stable transfection, which could lead to a breakthrough in the vaccination research. Acknowledgements The authors like to thank Dr. M. Janssens for the initial steps in the development of the methodology, Dr. R. De Deken, A. Van Hul, K. De Witte, G. De Deken, L. Verhelst and M. Van den Bogaerde for their technical support. Infected ticks were received from ILRI, Kenya and CTTBD, Malawi. This project was partly funded by SOFI-B funds, Belgian Ministry of Science. References Adamson, R., Lyons, K., Sharrard, M., Kinnaird, J., Swan, D., Graham, S., Shiels, B., Hall, R., 2001. Transient transfection of Theileria annulata. Mol. Biochem. Parasitol. 114 (1), 53–61. Beckers, C.J., Wakefield, T., Joiner, K.A., 1997. The expression of Toxoplasma proteins in Neospora caninum and the identification of a gene encoding a novel rhoptry protein. Mol. Biochem. Parasitol. 89 (2), 209–223. de Koning-Ward, T.F., Speranc¸a, M.A., Waters, A.P., Janse, C.J., 1999. Analysis of stage specificity of promoters in Plasmodium berghei using luciferase as a reporter. Mol. Biochem. Parasitol. 100 (1), 141–146. Dobbelaere, D.A., Küenzi, P., 2004. The strategies of the Theileria parasite: a new twist in host–pathogen interactions. Curr. Opin. Immunol. 16 (4), 524–530. Donald, R.G., Roos, D.S., 1993. Stable molecular transformation of Toxoplasma gondii: a selectable diahydrofolate reductase-thymidylate
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