In vitro production of Trypanosoma equiperdum antigen and its evaluation for use in serodiagnosis of dourine.

A modified Baltz's in vitro cultivation system for the propagation of Trypanosoma equiperdum strain OVI was established to develop a replacement for the conventional production procedure of dourine diagnostic antigen in rats. To increase trypanosome yields we designed an optimized culture medium by addition of supplemental compounds. Trypanosomes were adapted to this medium by two succeeding cultivation steps which led to a substantial proliferation rate and an increased cell density tolerance, respectively. As a result, adapted parasites could be propagated to maximum cell densities of >2×10(6) cells/ml, facilitating in vitro antigen production in preparative quantities comparable to the conventional method. A panel of 180 horse field sera, previously sent for testing to the German National Reference Laboratory for Dourine, was tested by complement fixation test using culture-derived as well as conventionally produced dourine antigen. Cohen's kappa values for results obtained with two batches of culture-derived antigen as compared to conventional antigen were 0.91 (95% confidence interval [CI]: 82.2-99.7) and 0.83 (95% CI: 70.3-95.3), respectively. Performance of antigens for diagnostic purposes was characterized in an inter-laboratory comparative study deploying 14 sera from horses with defined dourine statuses. Complement fixation test results from 15 participating European laboratories showed a diagnostic sensitivity of 94.1% (95% CI: 89.4-98.7) and a diagnostic specificity of 96.2% (95% CI: 92.5-99.9) for conventional antigen and a slightly higher diagnostic sensitivity of 96.0% (95% CI: 92.2-99.8) and a diagnostic specificity of 97.1% (95% CI: 94.0-100) for culture-derived antigen. We conclude that our novel approach for dourine antigen production from in vitro-grown trypanosomes described and evaluated herein meets the requirements for the prospective purpose in quantitative and qualitative terms and should be considered by the competent authorities as an alternative for the animal experiment currently prescribed by international standards.

Functional characterization of three aquaglyceroporins from Trypanosoma brucei in osmoregulation and glycerol transport.

Previous studies using bloodstream form Trypanosoma brucei have shown that glycerol transport in this parasite occurs via specific membrane proteins, namely a glycerol transporter and glycerol channels [1]. Later, we cloned, expressed and characterized the transport properties of all three aquaglyceroporins (AQP1-3) [2], which were found permeable for water, glycerol and other small uncharged solutes like dihydroxyacetone [3]. Here, we report on the cellular localization of TbAQP1 and TbAQP3 in bloodstream form trypanosomes. Indirect immunofluorescence analysis showed that TbAQP1 is exclusively localized in the flagellar membrane, whereas TbAQP3 was found in the plasma membrane.In addition, we analyzed the functions of all 3 AQPs, using an inducible inheritable double-stranded RNA interference methodology. All AQP knockdown cell lines were still able to survive hypo-osmotic stress conditions, except AQP2 knockdown parasites. Depleted TbAQP2 negatively impacted cell growth and the regulatory volume recovery, whereas AQP1 und 3 knockdown trypanosomes displayed phenotypes consistent with their localization in external membranes. A simultaneous knockdown of all 3 AQPs showed that the cells were able to substitute the missing glycerol uptake capability through a putative glycerol transporter.