Isothermal microcalorimetry - A quantitative method to monitor Trypanosoma congolense growth and growth inhibition by trypanocidal drugs in real time.

Trypanosoma congolense is a protozoan parasite that is transmitted by tsetse flies, causing African Animal Trypanosomiasis, also known as Nagana, in sub-Saharan Africa. Nagana is a fatal disease of livestock that causes severe economic losses. Two drugs are available, diminazene and isometamidium, yet successful treatment is jeopardized by drug resistant T. congolense. Isothermal microcalorimetry is a highly sensitive tool that can be used to study growth of the extracellular T. congolense parasites or to study parasite growth inhibition after the addition of antitrypanosomal drugs. Time of drug action and time to kill can be quantified in a simple way by real time heat flow measurements. We established a robust protocol for the microcalorimetric studies of T. congolense and developed mathematical computations in R to calculate different parameters related to growth and the kinetics of drug action. We demonstrate the feasibility and benefit of the method exemplary with the two standard drugs, diminazene aceturate and isometamidium chloride. The method and the mathematical approach can be translated to study other pathogenic or non-pathogenic cells if they are metabolically active and grow under axenic conditions.

Recombinant expression of trypanosome surface glycoproteins in Pichia pastoris for the diagnosis of Trypanosoma evansi infection.

Serodiagnosis of surra, which causes vast economic losses in livestock, is still based on native antigens purified from bloodstream form Trypanosoma (T.) evansi grown in rodents. To avoid the use of laboratory rodents in antigen preparation we expressed fragments of the invariant surface glycoprotein (ISG) 75, cloned from T. brucei gambiense cDNA, and the variant surface glycoprotein (VSG) RoTat 1.2, cloned from T. evansi gDNA, recombinantly in Pichia (P.) pastoris. The M5 strain of this yeast has an engineered N-glycosylation pathway resulting in homogenous Man5GlcNAc2 N-glycosylation which resembles the predominant Man9-5GlcNAc2 oligomannose structures in T. brucei. The secreted recombinant antigens were affinity purified with yields of up to 10mg and 20mg per liter cell culture of rISG 7529-465-E and rRoTat 1.223-385-H respectively. In ELISA, both recombinant proteins discriminated between pre-immune and immune serum samples of 25 goats experimentally infected with T. evansi. The diagnostic potential of rRoTat 1.223-385-H but not of rISG 7529-465-E was confirmed with sera of naturally infected and control dromedary camels. The results suggest that rRoTat 1.223-385-H expressed in P. pastoris requires further evaluation before it could replace native RoTat 1.2 VSG for serodiagnosis of surra, thus eliminating the use of laboratory animals for antigen production.

Prevalence of mixed Trypanosoma congolense infections in livestock and tsetse in KwaZulu-Natal, South Africa.

Trypanosoma congolense causes the most economically important animal trypanosomosis in Africa. In South Africa, a rinderpest pandemic of the 1890s removed many host animals, resulting in the near-eradication of most tsetse species. Further suppression was achieved through spraying with dichlorodiphenyltrichloroethane (DDT); however, residual populations of Glossina austeni and G. brevipalpis remained in isolated pockets. A total of 506 of these tsetse flies were captured in the Hluhluwe-iMfolozi Park, the St Lucia Wetland Park and Boomerang commercial farm. The polymerase chain reaction (PCR) was used to determine the infection rate and frequency of mixed infections of these flies. Additionally, 473 blood samples were collected from cattle at communal diptanks and a commercial farm in the area and each one examined by the haematocrit centrifugation technique (HCT). Furthermore, buffy coats from these blood samples were spotted onto FTA Elute cards and the DNA extracted from each one tested using 3 separate PCRs. The HCT revealed the presence of trypanosomes in only 6.6% of the blood samples; by contrast, species-specific PCR detected trypanosome DNA in 50% of the samples. The species-specific PCR detected trypanosome DNA in 17% of the tsetse flies, compared with the nested PCR targeting rDNA which detected trypanosome DNA in only 14% of the samples. Over time, the transmission of Savannah-type T. congolense and Kilifi-type T. congolense as mixed infections could have an impact on disease manifestation in different hosts in the area.

Establishment of a panel of reference Trypanosoma evansi and Trypanosoma equiperdum strains for drug screening.

The animal pathogenic protozoan, Trypanosoma evansi, leads to a wasting disease in equines, cattle and camels, commonly known as Surra. It is extensively distributed geographically with a wide range of mammalian hosts and causes great economical loss. Trypanosoma equiperdum causes a venereal disease called Dourine in horses and donkeys. Chemotherapy appears to be the most effective form of control for T. evansi, whereas infections caused by T. equiperdum are considered incurable. Due to emerging drug resistance, efficient control of T. evansi is severely threatened, emphasising the urgent need to find new alternative drugs. A drug profile for a panel of T. evansi and T. equiperdum strains has been established for the four standard drugs currently used in treatment. The (3)H-hypoxanthine incorporation assay was used to obtain 50% inhibitory concentration (IC(50)) values for each standard drug against the various strains. The results indicate the presence (and in some cases, the emergence) of drug resistance in several strains. This panel of characterised strains with known drug sensitivities and resistances will be of great value for the screening of new active compounds, in comparison with the four standard drugs currently available.

Deltamethrin-impregnated bednets reduce human landing rates of sandfly vector Lutzomyia longipalpis in Amazon households.

The entomological efficacy of using 25% deltamethrin EC insecticide-treated bednets (ITNs) was evaluated against the sandfly Lutzomyia longipalpis Lutz and Neiva (Diptera: Psychodidae), the principal vector of zoonotic visceral leishmaniasis (ZVL) in Latin America. A crossover field study in Amazon Brazil (Marajó Island, Pará State) demonstrated that, compared with untreated nets, the insecticide increased the barrier effect of nets by 39% (95% confidence interval [CI] 34-44%), reduced human landing rates by 80% (95% CI 62-90%) and increased the 24-h mortality rate from 0% to 98% (95% CI 93-99%) inside ITNs. The presence of an ITN also reduced the human landing rate on unprotected persons outside the net in the same room by 56% (95% CI 52-59%), and increased 24-h mortality to 68% (95% CI 62-73%) compared to 0.4% (0.1-2.0%) in untreated houses. The reduction in human landing rates in ITN rooms was associated with a doubling in the proportion of sandflies alighting on walls compared with that in untreated rooms, which was attributed to insecticide-induced excito-repellency. There was no evidence that sandflies were diverted onto unprotected hosts. Human landing catches inside houses peaked between 19.00 hours and 23.00 hours and declined steadily to zero at 02.00 hours and thereafter. House-to-house questionnaires established that only 34% of households owned at least one net (median two, range 1-8), only 20% of the population slept under a net (33% of 0-5-year-old children), and the majority (73%) of the population slept in hammocks. Combined data pertaining to sleeping times for children and sandfly activity period indicate that > 50% of sandfly bites inside houses, and substantially more outside houses, were taken before a third of children were potentially protected by a net. This study demonstrates the clear entomological efficacy of ITNs against Lu. longipalpis in this endemic region. The effectiveness of ITNs at preventing ZVL infection and disease has still to be evaluated.