Midgut lectin activity and sugar specificity in teneral and fed tsetse

Abstract. . Midgut infection rates of Trypanosoma congolense in Glossina palpalis palpalis and of Trypanosoma brucei rhodesiense in Glossina pallidipes are potentiated by the addition of D+ glucosamine to the infective feed, but not to the levels of super-infection reported for G. m. morsitans. G. p. palpalis and G.pallidipes are shown to possess two trypanocidal molecules: a glucosyl lectin which can be inhibited by D+ glucosamine and a galactosyl molecule inhibited by D+ galactose. Addition of both D+ glucosamine and D+ galactose to the teneral infective feed promotes super-infection of the midguts of G.p.palpalis. The glucosyl lectin is specific for rabbit erythrocytes and is present in guts of fed G.m.morsitans and G.p.palpalis , titres of lectin activity do not increase substantially after the second bloodmeal. The galactosyl specific molecule does not show any erythrocyte specificity, although haemolytic activity is observed only in G.p.palpalis and not in G.m.morsitans. The presence of two trypanocidal molecules in some species of tsetse may account for the innate refractoriness of these flies to trypanosome infection.
As D+ glucosamine also inhibits the killing of procyclic trypanosomes taken as an infective feed, it is suggested that the midgut lectin is normally responsible for the agglutination of trypanosomes in the fly midgut by binding to the pro-cyclic surface coat, prior to establishment in the ecto-peritrophic space.     

Haemolymph lectin and the maturation of trypanosome infections in tsetse

The tsetse immune system has recently been shown to be involved in trypanosome maturation; lectin secreted in the midgut, normally responsible for preventing the establishment of midgut infections, induces established midgut trypanosomes to mature. We now show that a second lectin, present in tsetse haemolymph, is essential to complete the maturation process. Interactions between tsetse lectins and parasite surface coats probably determine trypanosome transmissibility and may be partly responsible for the distribution of trypanosomiasis in Africa.     

Vector competence of Glossina palpalis gambiensis for Trypanosoma brucei s.l. and genetic diversity of the symbiont Sodalis glossinidius

Tsetse flies transmit African trypanosomes, responsible for sleeping sickness in humans and nagana in animals. This disease affects many people with considerable impact on public health and economy in sub-Saharan Africa, whereas trypanosomes' resistance to drugs is rising. The symbiont Sodalis glossinidius is considered to play a role in the ability of the fly to acquire trypanosomes. Different species of Glossina were shown to harbor genetically distinct populations of S. glossinidius. We therefore investigated whether vector competence for a given trypanosome species could be linked to the presence of specific genotypes of S. glossinidius. Glossina palpalis gambiensis individuals were fed on blood infected either with Trypanosoma brucei gambiense or Trypanosoma brucei brucei. The genetic diversity of S. glossinidius strains isolated from infected and noninfected dissected flies was investigated using amplified fragment length polymorphism markers. Correspondence between occurrence of these markers and parasite establishment was analyzed using multivariate analysis. Sodalis glossinidius strains isolated from T. brucei gambiense-infected flies clustered differently than that isolated from T. brucei brucei-infected individuals. The ability of T. brucei gambiense and T. brucei brucei to establish in G. palpalis gambiensis insect midgut is statistically linked to the presence of specific genotypes of S. glossinidius. This could explain variations in Glossina vector competence in the wild. Then, assessment of the prevalence of specific S. glossinidius genotypes could lead to novel risk management strategies.     

Control of tsetse and trypanosomiasis transmission in Uganda by applications of lambda-cyhalothrin

The pyrethroid insecticide lambda-cyhalothrin was evaluated in field trials against Glossina f.fuscipes and sleeping sickness transmission in Iyolwa sub-county, Tororo District, Uganda. The insecticide was applied selectively to the resting-sites of tsetse, by bush-spraying, using 10% wettable powder (10WP) formulation at an application rate of 11.6 g a.i./ha over an area of 28 km2, or by a 2% Electrodyn formulation (2ED) applied at 0.9 g a.i./ha over 30 km2. In a third trial area of 32 km2, 215 pyramidal traps treated with lambda-cyhalothrin 100 mg/m2 were set. The best impact was obtained with 10WP lambda-cyhalothrin which eliminated tsetse within 1-2 months, whereas G.f.fuscipes persisted at very low density in part of the area treated with 2ED lambda-cyhalothrin. In both treated areas the numbers of human sleeping sickness cases fell to no more than one per month, compared with four to twelve per month previously. The overall rate of cattle trypanosomiasis (T.brucei and T.vivax) was also reduced slightly. Insecticide-treated traps remained fully effective for at least 6 months under field conditions and catches were reduced 20-90-fold. These results in the control of tsetse and trypanosomiasis transmission lead us to recommend lambda-cyhalothrin for tsetse control operations.     

Rate of trypanosome killing by lectins in midguts of different species and strains of Glossina

The activity of lectins in different species of tsetse was compared in vivo by the time taken to remove all trypanosomes from the midgut following an infective feed and in vitro by agglutination tests. Teneral male Glossina pallidipes Austen, G. austeni Newstead and G. p. palpalis R-D. removed 50% of all Trypanosoma brucei rhodesiense Stephens & Fantham infections within 60 h. A 'refractory' line of G. m. morsitans Westwood took 170 h to kill 50% infections while a 'susceptible' line of the same species failed to kill 50%. Agglutination tests with midgut homogenates showed differences between fly stocks which accorded with differences in rate of trypanosome killing in vivo. Flies fed before an infective feed were able to remove trypanosomes from their midguts more quickly than flies infected as tenerals. Increasing the period of starvation before infection increased the susceptibility to trypanosome infection of non-teneral flies. Teneral flies showed little agglutinating activity in vitro, suggesting that lectin is produced in response to the bloodmeal. Feeding flies before infection also abolished the differences in rate of trypanosome killing found between teneral 'susceptible' and 'refractory' G. m. morsitans, suggesting that maternally inherited susceptibility to trypanosome infection is a phenomenon limited to teneral flies. Electron micrographs of midguts of G. m. morsitans suggest that procyclic trypanosomes are killed by cell lysis, presumably the result of membrane damage caused by lectin action.     

A new transmission risk index for human African trypanosomiasis and its application in the identification of sites of high transmission of sleeping sickness in the Fontem focus of southwest Cameroon

A new index for the risk for transmission of human African trypanosomiasis was developed from an earlier index by adding terms for the proportion of tsetse infected with Trypanosoma brucei gambiense group 1 and the contribution of animals to tsetse diet. The validity of the new index was then assessed in the Fontem focus of southwest Cameroon. Averages of 0.66 and 4.85 Glossina palpalis palpalis (Diptera: Glossinidae) were caught per trap/day at the end of one rainy season (November) and the start of the next (April), respectively. Of 1596 tsetse flies examined, 4.7% were positive for Trypanosoma brucei s.l. midgut infections and 0.6% for T. b. gambiense group 1. Among 184 bloodmeals identified, 55.1% were from pigs, 25.2% from humans, 17.6% from wild animals and 1.2% from goats. Of the meals taken from humans, 81.5% were taken at sites distant from pigsties. At the end of the rainy season, catches were low and similar between biotopes distant from and close to pigsties, but the risk for transmission was greatest at sites distant from the sties, suggesting that the presence of pigs reduced the risk to humans. At the beginning of the rainy season, catches of tsetse and risk for transmission were greatest close to the sties. In all seasons, there was a strong correlation between the old and new indices, suggesting that both can be used to estimate the level of transmission, but as the new index is the more comprehensive, it may be more accurate.     

Trypanosoma brucei s.l.: Microsatellite markers revealed high level of multiple genotypes in the mid-guts of wild tsetse flies of the Fontem sleeping sickness focus of Cameroon

To identify Trypanosoma brucei genotypes which are potentially transmitted in a sleeping sickness focus, microsatellite markers were used to characterize T. brucei found in the mid-guts of wild tsetse flies of the Fontem sleeping sickness focus in Cameroon. For this study, two entomological surveys were performed during which 2685 tsetse flies were collected and 1596 (59.2%) were dissected. Microscopic examination revealed 1.19% (19/1596) mid-gut infections with trypanosomes; the PCR method identified 4.7% (75/1596) infections with T. brucei in the mid-guts. Of these 75 trypanosomes identified in the mid-guts, Trypanosoma brucei gambiense represented 0.81% (13/1596) of them, confirming the circulation of human infective parasite in the Fontem focus. Genetic characterization of the 75 T. brucei samples using five microsatellite markers revealed not only multiple T. brucei genotypes (47%), but also single genotypes (53%) in the mid-guts of the wild tsetse flies. These results show that there is a wide range of trypanosome genotypes circulating in the mid-guts of wild tsetse flies from the Fontem sleeping sickness focus. They open new avenues to undertake investigations on the maturation of multiple infections observed in the tsetse fly mid-guts. Such investigations may allow to understand how the multiple infections evolve from the tsetse flies mid-guts to the salivary glands and also to understand the consequence of these evolutions on the dynamic (which genotype is transmitted to mammals) of trypanosomes transmission.     

Diversity of response to Trypanosoma brucei gambiense infections in the Forecariah mangrove focus (Guinea): perspectives for a better control of sleeping sickness

At a time when human African trypanosomiasis (HAT) elimination again seems a reachable goal in many parts of sub-Saharan Africa, it is becoming increasingly important to characterise the factors involved in disease resurgence or maintenance to develop sustainable control strategies. In this study conducted in the Forecariah mangrove focus in Guinea, HAT patients and serological suspects (SERO) were identified through mass screening of the population with the Card Agglutination Test for Trypanosomiasis (CATT) and were followed up for up to 2 years. Analysis of the samples collected during the follow-up of HAT patients and SERO was performed with PCR (TBR1/TBR2) and the trypanolysis serological test (TL) in order to clarify the role played by these individuals in the epidemiology of HAT. PCR positivity was higher in TL⁺ than in SERO TL⁻ (50% vs. 18%, respectively). Whereas CATT plasma titres decreased both in treated HAT patients and SERO TL⁻, SERO TL⁺ maintained high CATT titres. Four out of 17 SERO TL⁺ developed HAT during the study. These results strongly suggest that SERO TL⁺ individuals are asymptomatic carriers. In the context where disease prevalence is sufficiently low, treating SERO TL⁺ individual may thus be of crucial importance in order to cut transmission.     

An agent-based model of sleeping sickness: simulation trials of a forest focus in southern Cameroon

An agent-based model (AMB) used to simulate the spread of Human African Trypanosomiasis is presented together with the results of simulations of a focus of the disease. This model is a completely spatialized approach taking into account a series of often overlooked parameters such as human behaviour (activity-related movements), the density and mobility of the disease vectors--tsetse flies (Glossina spp.)--and the influence of other tsetse feeding hosts (livestock and wild animal populations). The agents that represent humans and tsetse flies move in a spatially structured environment managed by specialized location agents. Existing compartmental mathematical models governed by differential equations fail to incorporate the spatial dimension of the disease transmission. Furthermore, on a small scale, transmission is unrealistically represented by entities less than one. This ABM was tested with data from one village of the Bipindi sleeping sickness focus (southern Cameroon) and with obtained realistic simulations of stable transmission involving an animal reservoir. In varying different spatial configurations, we observe that the stability of spread is linked to the spatial complexity (number of heterogeneous locations). The prevalence is very sensitive to the human densities and to the number of tsetse flies initially infected in a given location. A relatively low and durable prevalence is obtained with shortening the phase I. In addition, we discuss some upgrading possibilities, in particular the linkage to a Geographical Information System (GIS). The agent-based approach offers new ways to understanding the spread of the disease and a tool to evaluate risk and test control strategies.     

Prevalence of enlarged salivary glands in wild populations of Glossina pallidipes in Kenya,with a note on the ultrastructure of the affected organ

Enlarged salivary gland was found to be widespread among wild populations of Glossina pallidipes in Kenya. The incidence of this abnormality varied from 0.9% in Meru National Park in Central Kenya to 5.4% in the Shimba hills area on the Kenya coast. Ultrastructurally, the enlarged glands were multinucleated with lumen reduced substantially in size. A large number of viruses filled both the lumen and the broken pieces of epithelial cytoplasm. In some cases Trypanosoma brucei trypanosomes were seen in the lumen of the enlarged glands. The epithelial cytoplasm was heavily vacuolated. Comment is made on the suitability of the diseased flies as transmitters of T. brucei.