Trypanosoma vivax: a simplified protocol forin vivogrowth, isolation and cryopreservation

Abstract

S u m m a ry :A rodent ad apte d clone o f Trypanosoma vivax w as used to infect cyclophospham ide treated mice and rats. Fresh blood containing trypanosomes, w as centrifuged in a density gradient of three Percoll solutions, 1 .0 7 , 1 .0 6 , 1 .0 5 g /m l, respectively, carefully layered on top o f each other. The yields o f this simple procedure for trypanosome purification w ere about six times higher than those obtained w ith the conventional anion-exchange columns. C ryopreservation o f trypanosomes using glycerol yielded 9 0 % via b le parasites, whereas using dimethylsulfoxide, a more com m only used cryoprotectant, the v iability w as only 3 5 %.KEY WORDS : Trypanosoma vivax, cyclophosphamide, isolation, cryopre servation, Percoll, rodents. Résumé : T r y p a n o s o m a v i v a x : u n p r o t o c o l e sim ple p o u r l a CULTURE IN VIVO, L'ISOLEMENT ET LA CRYOCONSERVATION Un clone de Trypanosoma vivax a d a p té aux rongeurs a été utilisé p o ur infecter des souris et des rats traités au cyclophospham ide. Du sang frais contenant des trypanosomes a été centrifugé dans un g radien t de densité d e trois solutions d e

Trypanosoma vivax Infections: Pushing Ahead with Mouse Models for the Study of Nagana. I. Parasitological, Hematological and Pathological Parameters

“…IL 1392 was chosen for its stable expression of VSG ILDat 1.2, characteristic of rodent-adapted West African T. vivax isolates that together with closely related South American stocks pertain phylogenetically to the same clade [66]. Initially, our studies showed that the IL 1392 isolate, retained its infective characteristics and mouse infection profile after a long cryopreservation period (see Material and Methods) [11], [22]. In addition, the experimental mouse models used in this work proved easy to handle on infection and reflected the general characteristic features observed in livestock, namely the remodeling of secondary lymphoid organs, cardinal severe anemia, genetically-related differences in resistance to the parasite (but not to death) and the development of multifocal tissue hemorrhages, necrosis and consequent systemic pathologies.…”

“…T. vivax - specific forward and reverse ribosomal promoter primers were also deduced from the Zaria Y486 chromosome 3: TvrDNAF (5' CTGATTTCGCCACTGCTATTATTTGC 3') and TvrDNAR (5' CGCTTCACTTGATGATCGTTTCG 3'), respectively. Parasites were maintained by weekly passages in mice and new stabilates were appropriately and regularly frozen in polysoma buffer/glycerol, as previously described [11]. Blood smears were prepared from infected mouse blood, air dried, fixed in methanol for 5 minutes and further stained with 5% Giemsa for 20 minutes.…”

“…Several pieces of evidence point to the importance of host genetic factors in determining individual susceptibility and/or resistance to this infection [3], [7], [8], [9], [10], [11]. Trypanotolerance is defined as the ability demonstrated by cattle of different genetic backgrounds to control trypanosomosis [12], [13].…”

“…Several early studies were conducted in more affordable mouse or rat experimental models of infection in attempts to throw light on trypanotolerance, antigenic variation, the pathogenesis of intravascular coagulation, and T. vivax immunobiology and dynamics [7], [11], [19], [22], [23], [24], [25]. However, these studies used a variety of more or less virulent isolates from cattle, goats, sheep, horses and donkeys to explore the ability of T. vivax stocks to infect several intact or immunosuppressed mouse strains.…”

Trypanosoma (Duttonella) vivax: its biology, epidemiology, pathogenesis, and introduction in the New World - a review

“…It is thus necessary to produce the antigens by cultivation in sheep or cattle, host species in which separation of parasites and blood cells on DEAE-cellulose is quite difficult and produces inconsistent results. For this reason, some authors proposed a technique based on the use of a Percoll gradient (Ndao et al 2004) or even the combined used of a gradient and DEAE-cellulose (Gonzalez et al 2005). However, a rodent adapted strain of T. vivax from French Guiana is now available at the Centre International de Recherche Développement de l'Elevage en Zone subhumide, Bobo Dioulasso, Burkina Faso (M Desquesnes unpublished observations).…”