Complete In Vitro Life Cycle of Trypanosoma congolense: Development of Genetic Tools

Abstract

BackgroundAnimal African trypanosomosis, a disease mainly caused by the protozoan parasite Trypanosoma congolense, is a major constraint to livestock productivity and has a significant impact in the developing countries of Africa. RNA interference (RNAi) has been used to study gene function and identify drug and vaccine targets in a variety of organisms including trypanosomes. However, trypanosome RNAi studies have mainly been conducted in T. brucei, as a model for human infection, largely ignoring livestock parasites of economical importance such as T. congolense, which displays different pathogenesis profiles. The whole T. congolense life cycle can be completed in vitro, but this attractive model displayed important limitations: (i) genetic tools were currently limited to insect forms and production of modified infectious BSF through differentiation was never achieved, (ii) in vitro differentiation techniques lasted several months, (iii) absence of long-term bloodstream forms (BSF) in vitro culture prevented genomic analyses.Methodology/Principal FindingsWe optimized culture conditions for each developmental stage and secured the differentiation steps. Specifically, we devised a medium adapted for the strenuous development of stable long-term BSF culture. Using Amaxa nucleofection technology, we greatly improved the transfection rate of the insect form and designed an inducible transgene expression system using the IL3000 reference strain. We tested it by expression of reporter genes and through RNAi. Subsequently, we achieved the complete in vitro life cycle with dramatically shortened time requirements for various wild type and transgenic strains. Finally, we established the use of modified strains for experimental infections and underlined a host adaptation phase requirement.Conclusions/SignificanceWe devised an improved T. congolense model, which offers the opportunity to perform functional genomics analyses throughout the whole life cycle. It represents a very useful tool to understand pathogenesis mechanisms and to study potential therapeutic targets either in vitro or in vivo using a mouse model.

Importance of Nonenteric Protozoan Infections in Immunocompromised People

“…However, T. b. brucei, T. b. evansi, T. congolense, and T. lewisi infections have been identified in several cases of human disease (138,285,299,305,311,495,521,560,573). These trypanosomes infect a range of domestic, feral, and wild animals (124,261,262,371) and probably come into frequent contact with humans.…”

“…Trypanosoma congolense is a pathogen of livestock animals in sub-Saharan Africa, causing weight loss, anemia, and immunosuppression (124). Like T. brucei, T. congolense has a tsetse fly vector, and so its range is restricted to that of the tsetse fly (124).…”

Prolyl oligopeptidase-like deficient Trypanosoma evansi parasites are associated with reduced interleukin-10 concentrations in vivo and in vitro

“…(http://bio.lonza.com/fileadmin/groups/marketing/Downloads/ Protocols/Generated/Optimized_Protocol_322.pdf) Briefly, for each reaction, a pellet of 10 6 parasites was resuspended in 20 µL of solutions P1-P5 along with 1 µg of NotIlinearized pLEW20-green fluorescent protein (GFP), a plasmid that was previously used for GFP expression in T. brucei (Wirtz et al 1999;Coustou et al 2010), and transfected using one of the programs recommended by the manufacturer. Transfected parasites were first cultured for 24 h in the supplemented IMDM medium (Hirumi and Hirumi, 1989) before addition of 2 µg mL −1 of phleomycin for the selection of viable clones.…”

Identification and Characterization of a <i>Trypanosoma congolense</i> 46 kDa Protein as a Candidate Serodiagnostic Antigen

“…[5]. The disease can affect various species of mammals, and it is particularly important in cattle from the economic viewpoint [15].…”