Cryopreservation of Trypanosoma evansi after DEAE-cellulose purification: Evaluation of infective parameters Kaio César Simano Tavares a , Aleksandro Schafer Da Silva b , Patricia Wolkmer b , Silvia Gonzalez Monteiro b , Luiz Claudio Miletti a, * a Universidade do Estado de Santa Catarina, Laboratório de Bioquímica de Hemoparasitas e Vetores – LABHEV, Avenida Luiz de Camões, n° 2090, Bairro Conta Dinheiro Lages 88520-000, SC, Brazil b Departamento de Microbiologia e Parasitologia da Universidade Federal de Santa Maria, Bairro Camobi – Prédio 20, Sala 4232, 97105-900 Santa Maria – RS, Brazil article info Article history: Received 10 March 2010 Accepted 17 May 2010 Keywords: Trypanosoma evansi Cryopreservation DEAE-cellulose abstract Cryopreservation is a method of keeping parasites alive in a laboratory. However, this technique may also damage the parasite. Alternatively, parasites may be maintained by in vitro culture. Unfortunately, for Trypanosoma evansi no effective medium that is able to maintain the parasite for more than 4 months has been described. In this study, we examined the effect of purifying trypomastigote through DEAE-cel- lulose chromatography before and after cryopreservation, by analyzing the pre-patent period, longevity, parasitemia, and count of viable parasites. Our results showed a three-times increase in the concentration of viable trypomastigote in DEAE-purified cryopreserved parasites as compared to non-DEAE-purified cryopreserved parasites. This indicates that DEAE-cellulose chromatography followed by cryopreserva- tion is an effective method for the storage and preservation of T. evansi, with the advantage that the stocked parasites will be ready to use in molecular biology procedures. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Trypanosoma evansi is the pathogenic trypanosomatid with largest distribution worldwide (Losos, 1980). The pathogen causes a disease in horses called ‘‘Surra” in Africa and ‘‘Mal das Cadeiras” in South America. Currently, T. evansi causes great economic losses in many countries including Australia (Reid et al., 2001), the Phil- ippines (Dargantes et al., 2009), and Egypt (Abdel-Rady, 2008). Be- sides horses, the pathogen causes disease in a variety of animals, including capybaras, coatis, small rodents, dogs, cattle, and pigs (Herrera et al., 2004). A few years ago the first case of T. evansi infection in humans was described in India (Joshi et al., 2005). Currently, the cryopreservation of trypanosomes is achieved by using infected blood samples (Dar et al., 1972). The benefit of iso- lating other trypanosomatids using DEAE-cellulose and glycerol- frozen samples for future cultivation has been demonstrated (Truc et al., 2004). Maintenance of a T. evansi strain has also been done by inoculation of rodents with blood or by cryopreservation, since it is difficult to achieve a long-term maintenance in culture (>4 months) (Baltz et al., 1985). The use of animals for mainte- nance purposes of trypanosomes requires the sacrifice of many individuals, as some strains are highly virulent. Cryopreservation is a viable alternative because it avoids the use of animals while it maintains some natural characteristics of the parasite (Miyake et al., 2004). Diethylaminoethyl-cellulose ion-exchange chromatography has been used for the purification of T. evansi trypomastigotes. It has been shown to be effective for the isolation of parasites for future use in molecular studies and for diagnostic purposes (Reid et al., 2001; Holland et al., 2001; Gutierrez et al., 2004). In this study we show that the DEAE-cellulose cryopreservation of purified parasites from experimentally infected rats can be used to keep T. evansi viable and pathogenic. The cryopreserved para- sites can be used in molecular biology procedures that require purified parasites, including nucleic acid and protein extraction, immunofluorescence, and culture medium. 2. Materials and methods 2.1. Isolation of T. evansi from naturally infected blood The T. evansi isolate was derived from a naturally infected dog previously described (Colpo et al., 2005). The parasite was kept in vivo culture by successive passages through Wistar rats, and 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.05.021 * Corresponding author. Address: Departamento de Produção Animal e Alimen- tos, Centro de Ciências Agroveterinárias, Universidade do Estado de Santa Catarina, Av. Luiz de Camões, 2090, Bairro Conta Dinheiro Lages 88520-000, SC, Brazil. Tel.: +55 49 2101 9175. E-mail address: miletti@cav.udesc.br (L.C. Miletti). Research in Veterinary Science 90 (2011) 257–259 Contents lists available at ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc