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.     

The effect of starvation on the susceptibility of teneral and non-teneral tsetse flies to trypanosome infection

Transmission of vector-borne diseases depends largely on the ability of the insect vector to become infected with the parasite. In tsetse flies, newly emerged or teneral flies are considered the most likely to develop a mature, infective trypanosome infection. This was confirmed during experimental infections where laboratory-reared Glossina morsitans morsitans Westwood (Diptera: Glossinidae) were infected with Trypanosoma congolense or T. brucei brucei. The ability of mature adult tsetse flies to become infected with trypanosomes was significantly lower than that of newly emerged flies for both parasites. However, the nutritional status of the tsetse at the time of the infective bloodmeal affected its ability to acquire either a T. congolense or T. b. brucei infection. Indeed, an extreme period of starvation (3-4 days for teneral flies, 7 days for adult flies) lowers the developmental barrier for a trypanosome infection, especially at the midgut level of the tsetse fly. Adult G. m. morsitans became at least as susceptible as newly emerged flies to infection with T. congolense. Moreover, the susceptibility of adult flies, starved for 7 days, to an infection with T. b. brucei was also significantly increased, but only at the level of maturation of an established midgut infection to a salivary gland infection. The outcome of these experimental infections clearly suggests that, under natural conditions, nutritional stress in adult tsetse flies could contribute substantially to the epidemiology of tsetse-transmitted trypanosomiasis.     

A study on the maturation of procyclic Trypanosoma brucei brucei in Glossina morsitans centralis and G. brevipalpis

Abstract. Teneral Glossina morsitans centralis and G. brevipalpis were fed in vitro upon medium containing procyclic Trypanosoma brucei brucei derived from the midguts of G. m. centralis or G. brevipalpis which had immature trypanosome infections. The tsetse were then maintained on rabbits and, on day 31, were dissected to determine the infection rates. In G. m. centralis the midgut and salivary gland infection rates by T. b. brucei were 46.0% and 27.0% with procyclic trypanosomes from G. m. centralis, and 45.4% and 24.7% with procyclic trypanosomes from G. brevipalpis, respectively. In G. brevipalpis the rates were 20.2% and 0.0% with procyclic trypanosomes from G. m. centralis, and 28.0% and 0.0% with procyclic trypanosomes from G. brevipalpis, respectively. Teneral G. m. centralis and G. brevipalpis were also fed similarly upon procyclic T. b. brucei derived from G.m.centralis or G. brevipalpis on day 31 of infection, the former tsetse species had mature infections while the latter were without infections in the salivary glands. In G.m.centralis the infection rates in the midgut and salivary glands were 48.9% and 17.0%, and 38.0% and 17.0% when fed on procyclic trypanosomes from G.m.centralis and G. brevipalpis, respectively. In G. brevipalpis the rates were 21.5% and 0.0%, and 10.7% and 0.0% with procyclic trypanosomes of G.m.centralis and G. brevipalpis origin, respectively. Thus, procyclic T. b. brucei from susceptible G.m.centralis could not complete cyclical development in refractory G. brevipalpis, whereas those from G. brevipalpis developed to metatrypanosomes in the salivary glands of G.m.centralis. Teneral and 15-day-old non-teneral G.m.centralis were fed in vitro upon heparinized goat's blood containing T. b. brucei bloodstream trypomastigotes, or upon medium containing procyclic T. b. brucei derived from G.m.centralis with mature infections. On day 31 their infection rates were determined. The infection rates by T. b. brucei in the midgut and salivary glands of G.m.centralis fed on the infected blood were 70.4% and 40.4% when fed as teneral tsetse, as against 15.3% and 4.0% when fed as non-teneral tsetse. Those tsetse which were fed on the medium containing procyclic trypanosomes showed rates of 50.0% and 25.6%, as against 11.6% and 2.5%, respectively. It would appear, therefore, that maturation of T. b. brucei in tsetse is probably not determined simply by an interaction between lectin and procyclic trypanosomes in the midgut of non-teneral tsetse, but it is the result of a complex interaction between many interrelated physiological factors of both the trypanosome and the tsetse vector.     

Post eclosion age predicts the prevalence of midgut trypanosome infections in Glossina

The teneral phenomenon, as observed in Glossina sp., refers to the increased susceptibility of the fly to trypanosome infection when the first bloodmeal taken is trypanosome-infected. In recent years, the term teneral has gradually become synonymous with unfed, and thus fails to consider the age of the newly emerged fly at the time the first bloodmeal is taken. Furthermore, conflicting evidence exists of the effect of the age of the teneral fly post eclosion when it is given the infected first bloodmeal in determining the infection prevalence. This study demonstrates that it is not the feeding history of the fly but rather the age (hours after eclosion of the fly from the puparium) of the fly when it takes the first (infective) bloodmeal that determines the level of fly susceptibility to trypanosome infection. We examine this phenomenon in male and female flies from two distinct tsetse clades (Glossina morsitans morsitans and Glossina palpalis palpalis) infected with two salivarian trypanosome species, Trypanosoma (Trypanozoon) brucei brucei and Trypanosoma (Nannomonas) congolense using Fisher's exact test to examine differences in infection rates. Teneral tsetse aged less than 24 hours post-eclosion (h.p.e.) are twice as susceptible to trypanosome infection as flies aged 48 h.p.e. This trend is conserved across sex, vector clade and parasite species. The life cycle stage of the parasite fed to the fly (mammalian versus insect form trypanosomes) does not alter this age-related bias in infection. Reducing the numbers of parasites fed to 48 h.p.e., but not to 24 h.p.e. flies, increases teneral refractoriness. The importance of this phenomenon in disease biology in the field as well as the necessity of employing flies of consistent age in laboratory-based infection studies is discussed.     

The influence of sex and fly species on the development of trypanosomes in tsetse flies

Unlike other dipteran disease vectors, tsetse flies of both sexes feed on blood and transmit pathogenic African trypanosomes. During transmission, Trypanosoma brucei undergoes a complex cycle of proliferation and development inside the tsetse vector, culminating in production of infective forms in the saliva. The insect manifests robust immune defences throughout the alimentary tract, which eliminate many trypanosome infections. Previous work has shown that fly sex influences susceptibility to trypanosome infection as males show higher rates of salivary gland (SG) infection with T. brucei than females. To investigate sex-linked differences in the progression of infection, we compared midgut (MG), proventriculus, foregut and SG infections in male and female Glossina morsitans morsitans. Initially, infections developed in the same way in both sexes: no difference was observed in numbers of MG or proventriculus infections, or in the number and type of developmental forms produced. Female flies tended to produce foregut migratory forms later than males, but this had no detectable impact on the number of SG infections. The sex difference was not apparent until the final stage of SG invasion and colonisation, showing that the SG environment differs between male and female flies. Comparison of G. m. morsitans with G. pallidipes showed a similar, though less pronounced, sex difference in susceptibility, but additionally revealed very different levels of trypanosome resistance in the MG and SG. While G. pallidipes was more refractory to MG infection, a very high proportion of MG infections led to SG infection in both sexes. It appears that the two fly species use different strategies to block trypanosome infection: G. pallidipes heavily defends against initial establishment in the MG, while G. m. morsitans has additional measures to prevent trypanosomes colonising the SG, particularly in female flies. We conclude that the tsetse-trypanosome interface works differently in G. m. morsitans and G. pallidipes.     

Comparison of the susceptibility of different Glossina species to simple and mixed infections with Trypanosoma (Nannomonas) congolense savannah and riverine forest types

Abstract Teneral Glossina morsitans mositans, G.m.submorsitans, G.palpalis gambiensis and G.tachinoides were allowed to feed on rabbits infected with Trypanosoma congolense savannah type or on mice infected with T.congolense riverine-forest type. The four tsetse species and subspecies were also infected simultaneously in vitro on the blood of mice infected with the two clones of T.congolense via a silicone membrane. The infected tsetse were maintained on rabbits and from the day 25 after the infective feed, the surviving tsetse were dissected in order to determine the infection rates.
Results showed higher mature infection rates in morsitans-gwup tsetse flies than in palpalis-group tsetse flies when infected with the savannah type of T.congolense. In contrast, infection rates with the riverine-forest type of T.congolense were lower, and fewer flies showed full development cycle. The intrinsec vectorial capacity of G.m.submorsitans for the two T.congolense types was the highest, whereas the intrinsic vectorial capacity of G.p.gambiensis for the Savannah type and G.m.morsitans for the riverine-forest type were the lowest. Among all tsetse which were infected simultaneously with the two types of T.congolense , the polymerase chain reaction detected only five flies which had both trypanosome taxa in the midgut and the proboscis. All the other infections were attributable to the savannah type.
The differences in the gut of different Glossina species and subspecies allowing these two sub-groups of T.congolense to survive better and undergo the complete developmental cycle more readily in some species than other are discussed.     

Lectin signalling of maturation of T.congolense infections in tsetse

The process of maturation of Trypanosoma congolense Broden in tsetse has been shown to be initiated by lectin secreted in the fly midgut. In the present study the duration of lectin signal required to induce maturation was determined by the sequential addition or removal of a specific lectin inhibitor (D+glucosamine) to the diet of infected male Glossina morsitans Westwood. An established midgut infection of T.congolense was found to require, at most, 72 h exposure to midgut lectin to begin the process of maturation. Longer exposure to midgut lectin increased the frequency of maturation, suggesting clonal variation in response to lectin stimulation occurs within trypanosome stocks. It is suggested that this variation corresponds to differences in lectin binding sites on the trypanosome surface. Midgut trypanosomes retained their ability to mature throughout their life in the fly; when lectin activity in the midgut was inhibited, the trypanosomes remained as procyclic forms but when this inhibition was removed maturation was able to proceed. This indicates that the process of maturation is dependent upon a signal from the fly and is not predetermined by the trypanosomes undergoing a fixed number of division cycles. The possible role of lectins in the maturation of trypanosomes in vitro is discussed.     

Increased expression of unusual EP repeat-containing proteins in the midgut of the tsetse fly (Glossina) after bacterial challenge

Proteins containing a glutamic acid-proline (EP) repeat epitope were immunologically detected in midguts from eight species of Glossina (tsetse flies). The molecular masses of the tsetse EP proteins differed among species groups. The amino acid sequence of one of these proteins, from Glossina palpalis palpalis, was determined and compared to the sequence of a homologue, the tsetse midgut EP protein of Glossina m. morsitans. The extended EP repeat domains comprised between 36% (G. m. morsitans) and 46% (G. p. palpalis) of the amino acid residues, but otherwise the two polypeptide chains shared most of their sequences and predicted functional domains. The levels of expression of tsetse EP protein in adult teneral midguts were markedly higher than in midguts from larvae. The EP protein was detected by immunoblotting in the fat body, proventriculus and midgut, the known major immune tissues of tsetse and is likely secreted as it was also detected in hemolymph. The EP protein was not produced by the bacterial symbionts of tsetse midguts as determined by genome analysis of Wigglesworthia glossinidia and immunoblot analysis of Sodalis glossinidius. Bacterial challenge of G. m. morsitans, by injection of live E. coli, induced augmented expression of the tsetse EP protein. The presence of EP proteins in a wide variety of tsetse, their constitutive expression in adult fat body and midguts and their upregulation after immunogen challenge suggest they play an important role as a component of the immune system in tsetse.     

Lectin and peritrophic membrane development in the gut of Glossina m.morsitans and a discussion of their role in protecting the fly against trypanosome infection

Newly emerged Glossina m.morsitans Westwood tsetse flies lack a peritrophic membrane which develops to fully line the midgut after c. 80-90 h. Midgut lectins are mainly associated with the peritrophic membrane. Lectin levels in the blood-free gut of adult flies rise slowly up to 8 days and then rapidly to at least 14 days post-eclosion (when the last of our recordings was made). Despite starving flies for 4 days prior to the agglutination assay, gut lectin levels in older flies are 100-200 times more than those in newly ecloded flies. This is inconsistent with the idea that there is a simple relationship between lectins and the protection of tsetse flies against trypanosome infection. Various theories put forward to account for age-dependent variation in the ability of tsetse to become infected with trypanosomes are discussed in the light of these findings.     

A comparison of African buffalo, N''Dama and Boran cattle as reservoirs of Trypanosoma congolense for different Glossina species

Teneral Glossina morsitans centralis Machado were fed on the flanks of the African buffalo (Syncerus caffer Sparrman), N'Dama (Bos taurus L.) or Boran (Bos indicus L.) cattle infected with Trypanosoma congolense Broden. The infected tsetse were maintained on rabbits and on day 30 after the infected feed, the surviving tsetse were dissected to determine the infection rates. The mean infection rates (% +/- SE) in the midgut of tsetse fed on buffalo, N'Damas and Borans were 23.5 +/- 3.3, 31.6 +/- 2.7 and 33.7 +/- 4.6, respectively. The differences were not significant. However, the mean mature infection rate in tsetse fed on the buffalo (13.2 +/- 2.1%) was significantly lower compared to the rates in tsetse fed on the N'Dama (20.4 +/- 1.4) or the Boran cattle (21.4 +/- 1.1). When groups of teneral G.m.centralis, G.pallidipes Austen, G.p.gambiensis Vanderplank, G.f.fuscipes Newstead, G.brevipalpis Newstead and G.longipennis Corti were fed simultaneously on either an infected buffalo, an N'Dama or a Boran steer, the mature infection rates ranged from 0 to 16.1%. Irrespective of the host species used, the T.congolense infection rate was highest in G.m.centralis, lowest in the palpalis and fusca group tsetse, with G.pallidipes being intermediate. Nevertheless, the trypanoresistant African buffalo and N'Dama may serve as reservoirs of T.congolense as can trypanosusceptible Boran cattle.     

Identification of midgut proteins that are differentially expressed in trypanosome-susceptible and normal tsetse flies (Glossina morsitans morsitans)

Molecules in the midgut of tsetse flies (Diptera: Glossinidiae) are thought to play important roles in the life cycle of African trypanosomes by influencing initial parasite establishment and subsequent differentiation events that ultimately lead to maturation of mammal-infective trypanosomes. The molecular composition of the tsetse midgut is, therefore, of critical importance to disease transmission by these medically important vectors. In this study we compared protein expression profiles of midguts of the salmon mutant and wild type Glossina morsitans morsitans Westwood that display marked differences in their susceptibility to infection by African trypanosomes. Isotope coded affinity tag (ICAT) technology was used to identify 207 proteins including 17 that were up regulated and nine that were down regulated in the salmon mutants. Several of the up regulated molecules were previously described as tsetse midgut or salivary gland proteins. Of particular interest was the up regulation in the salmon flies of tsetse midgut EP protein, a recently described molecule with lectin-like activity that was also found to be induced in tsetse by bacterial challenge. The up regulation of the EP protein in midguts of salmon mutants was confirmed by two-dimensional gel electrophoresis and tandem mass spectrometry.     

An Investigation into the Protein Composition of the Teneral Glossina morsitans morsitans Peritrophic Matrix

Background

Tsetse flies serve as biological vectors for several species of African trypanosomes. In order to survive, proliferate and establish a midgut infection, trypanosomes must cross the tsetse fly peritrophic matrix (PM), which is an acellular gut lining surrounding the blood meal. Crossing of this multi-layered structure occurs at least twice during parasite migration and development, but the mechanism of how trypanosomes do so is not understood. In order to better comprehend the molecular events surrounding trypanosome penetration of the tsetse PM, a mass spectrometry-based approach was applied to investigate the PM protein composition using Glossina morsitans morsitans as a model organism.

Methods

PMs from male teneral (young, unfed) flies were dissected, solubilised in urea/SDS buffer and the proteins precipitated with cold acetone/TCA. The PM proteins were either subjected to an in-solution tryptic digestion or fractionated on 1D SDS-PAGE, and the resulting bands digested using trypsin. The tryptic fragments from both preparations were purified and analysed by LC-MS/MS.

Results

Overall, nearly 300 proteins were identified from both analyses, several of those containing signature Chitin Binding Domains (CBD), including novel peritrophins and peritrophin-like glycoproteins, which are essential in maintaining PM architecture and may act as trypanosome adhesins. Furthermore, 27 proteins from the tsetse secondary endosymbiont, Sodalis glossinidius, were also identified, suggesting this bacterium is probably in close association with the tsetse PM.

Conclusion

To our knowledge this is the first report on the protein composition of teneral G. m. morsitans, an important vector of African trypanosomes. Further functional analyses of these proteins will lead to a better understanding of the tsetse physiology and may help identify potential molecular targets to block trypanosome development within the tsetse.     

Ultrastructural studies of certain aspects of the development of Trypanosoma congolense in Glossina morsitans morsitans

The course of Trypanosoma congolense infections in Glossina morsitans morsitans was followed by electron-microscopic examination of ultrathin sections of the guts and proboscises of infected flies. Guts dissected from flies 7 days after infection with culture procyclic forms of T. congolense had heavy trypanosome infections in the midgut involving both the endo- and ectoperitrophic spaces. Trypanosomes were also seen in the process of penetrating the fully formed peritrophic membrane in the central region of the midgut. By post infection day 21, trypanosomes had reached the proboscis of the fly and were found as clumps of epimastigote forms attached to the labrum by hemidesmosomes between their flagella and the chitinous lining of the food canal. Desmosome connections were observed between the flagella of adjacent epimastigotes. Flies examined at postinfection days 28 and 42 had, in addition to the attached forms in the labrum, free forms in the hypopharynx.     

Inhibition of the DNA amplification of trypanosomes present in tsetse flies midguts: implications for the identification of trypanosome species in wild tsetse flies

The present study was carried out in order to investigate if there was really a failure of PCR in identifying parasitologically positive tsetse flies in the field. Tsetse flies (Glossina palpalis gambiensis and Glossina morsitans morsitans) were therefore experimentally infected with two different species of Trypanosoma (Trypanosoma brucei gambiense or Trypanosoma congolense). A total of 152 tsetse flies were dissected, and organs of each fly (midgut, proboscis or salivary glands) were examined. The positive organs were then analysed using PCR. Results showed that, regardless of the trypanosome species, PCR failed to amplify 40% of the parasitologically positive midguts. This failure, which does not occur with diluted samples, is likely to be caused by an inhibition of the amplification reaction. This finding has important implications for the detection and the identification of trypanosome species in wild tsetse flies.     

Cleavage of trypanosome surface glycoproteins by alkaline trypsin-like enzyme(s) in the midgut of Glossina morsitans

EP and GPEET procyclin, the major surface glycoproteins of procyclic forms of Trypanosoma brucei, are truncated by proteases in the midgut of the tsetse fly Glossina morsitans morsitans. We show that soluble extracts from the midguts of teneral flies contain trypsin-like enzymes that cleave the N-terminal domains from living culture-derived parasites. The same extract shows little activity against a variant surface glycoprotein on living bloodstream form T. brucei (MITat 1.2) and none against glutamic acid/alanine-rich protein, a major surface glycoprotein of Trypanosoma congolense insect forms although both these proteins contain potential trypsin cleavage sites. Gel filtration of tsetse midgut extract revealed three peaks of tryptic activity against procyclins. Trypsin alone would be sufficient to account for the cleavage of GPEET at a single arginine residue in the fly. In contrast, the processing of EP at multiple sites would require additional enzymes that might only be induced or activated during feeding or infection. Unexpectedly, the pH optima for both the procyclin cleavage reaction and digestion of the trypsin-specific synthetic substrate Chromozym-TRY were extremely alkaline (pH 10). Direct measurements were made of the pH within different compartments of the tsetse digestive tract. We conclude that the gut pH of teneral flies, from the proventriculus to the hindgut, is alkaline, in contradiction to previous measurements indicating that it was mildly acidic. When tsetse flies were analysed 48 h after their first bloodmeal, a pH gradient from the proventriculus (pH 10.6+/-0.6) to the posterior midgut (pH 7.9+/-0.4) was observed.     

Molecular characterization of a tsetse fly midgut proteolytic lectin that mediates differentiation of African trypanosomes

Differentiation of bloodstream-form trypanosomes into procyclic (midgut) forms is an important first step in the establishment of an infection within the tsetse fly. This complex process is mediated by a wide variety of factors, including those associated with the vector itself, the trypanosomes and the bloodmeal. As part of an on-going project in our laboratory, we recently isolated and characterized a bloodmeal-induced molecule with both lectin and trypsin activities from midguts of the tsetse fly, Glossina longipennis [Osir, E.O., Abubakar, L., Imbuga, M.O., 1995. Purification and characterization of a midgut lectin-trypsin complex from the tsetse fly, Glossina longipennis. Parasitol. Res. 81, 276-281]. The protein (lectin-trypsin complex) was found to be capable of stimulating differentiation of bloodstream trypanosomes in vitro. Using polyclonal antibodies to the complex, we screened a G. fuscipes fuscipes cDNA midgut expression library and identified a putative proteolytic lectin gene. The cDNA encodes a putative mature polypeptide with 274 amino acids (designated Glossina proteolytic lectin, Gpl). The deduced amino acid sequence includes a hydrophobic signal peptide and a highly conserved N-terminal sequence motif. The typical features of serine protease trypsin family of proteins found in the sequence include the His/Asp/Ser active site triad with the conserved residues surrounding it, three pairs of cysteine residues for disulfide bridges and an aspartate residue at the specificity pocket. Expression of the gene in a bacterial expression system yielded a protein (M(r) approximately 32,500). The recombinant protein (Gpl) bound d(+) glucosamine and agglutinated bloodstream-form trypanosomes and rabbit red blood cells. In addition, the protein was found to be capable of inducing transformation of bloodstream-form trypanosomes into procyclic forms in vitro. Antibodies raised against the recombinant protein showed cross-reactivity with the alpha subunit of the lectin-trypsin complex. These results support our earlier hypothesis that this molecule is involved in the establishment of trypanosome infections in tsetse flies.     

An antimicrobial peptide with trypanocidal activity characterized from Glossina morsitans morsitans

Tsetse flies (Diptera:Glossinidae) are vectors of African trypanosomes, the protozoan agents of devastating diseases in humans and animals. Prior studies in trypanosome infected Glossina morsitans morsitans have shown induced expression and synthesis of several antimicrobial peptides in fat body tissue. Here, we have expressed one of these peptides, Attacin (GmAttA1) in Drosophila (S2) cells in vitro. We show that the purified recombinant protein (recGmAttA1) has strong antimicrobial activity against Escherichia coli-K12, but not against the enteric gram-negative symbiont of tsetse, Sodalis glossinidius. The recGmAttA1 also demonstrated inhibitory effects against both the mammalian bloodstream form and the insect stage Trypanosoma brucei in vitro (minimal inhibitory concentration MIC50 0.075 microM). When blood meals were supplemented with purified recGmAttA1 during the course of parasite infection, the prevalence of trypanosome infections in tsetse midgut was significantly reduced. Feeding fertile females GmAttA1 did not affect the fecundity or the longevity of mothers, nor did it affect the hatchability of their offspring. We discuss a paratransgenic strategy, which involves the expression of trypanocidal molecules such as recGmAttA1 in the midgut symbiont Sodalis in vivo to reduce trypanosome transmission.     

Glossina dynamics in and around the sleeping sickness endemic Serengeti ecosystem of northwestern Tanzania

We investigated the dynamics of Glossina spp. and their role in the transmission of trypanosomiasis in the sleeping sickness endemic Serengeti ecosystem, northwestern Tanzania. The study investigated Glossina species composition, trap density, trypanosome infection rates, and the diversity of trypanosomes infecting the species. Tsetse were trapped using monopyramidal traps in the mornings between 06:00 to 11:00 and transported to the veterinary laboratory in Serengeti National Park where they were sorted into species and sex, and dissected microscopically to determine trypanosome infection rates. Age estimation of dissected flies was also conducted concurrently. Tsetse samples positive for trypanosomes were subjected to PCR to determine the identity of the detected trypanosomes. Out of 2,519 tsetse trapped, 1,522 (60.42%) were G. swynnertoni, 993 (39.42%) were G. pallidipes, three (0.12%) were G. m. morsitans, and one (0.04%) was G. brevipalpis. The trap density for G. swynnertoni was between 1.40 and 14.17 while that of G. pallidipes was between 0.23 and 9.70. Out of 677 dissected G. swynnertoni, 63 flies (9.3%) were infected, of which 62 (98.4%) were females. A total of 199 G. pallidipes was also dissected but none was infected. There was no significant difference between the apparent densities of G. swynnertoni compared to that of G. pallidipes (t = 1.42, p = 0.18). Molecular characterization of the 63 infected G. swynnertoni midguts showed that 19 (30.2%) were trypanosomes associated with suid animals while nine (14.3%) were trypanosomes associated with bovid animals and five samples (7.9%) had T. brucei s.l genomic DNA. Thirty (47.6%) tsetse samples could not be identified. Subsequent PCR to differentiate between T. b. brucei and T. b. rhodesiense showed that all five samples that contained the T. brucei s.l genomic DNA were positive for the SRA molecular marker indicating that they were T. b. rhodesiense. These results indicate that G. swynnertoni plays a major role in the transmission of trypaniosomiasis in the area and that deliberate and sustainable control measures should be initiated and scaled up.     

Infectivity of Trypanosoma brucei cultivated at 28 C with tsetse fly salivary glands

When transformed procyclic noninfective trypanosomes of several unrelated stocks of Trypanosoma brucei were cultivated in T-30 Falcon flasks at 28 C in a liquid medium containing head-salivary gland explants of Glossina morsitans morsitans some of the organisms developed into forms infective for mice. Infective trypanosomes were detected 7 to 14 days after the cultures were prepared and they persisted for varying periods of up to 88 days when the cultures were terminated. A few of the salivary glands became invaded with parasites about the time infective organisms appeared in the cultures. Using T. brucei TREU 929, it was shown that trypanosomes grown with between 2m and 50 explants were capable of producing infections consistently for prolonged periods. On the other hand, trypanosomes cultivated with 25 or fewer explants rarely infected mice. Infectivity titrations on trypanosome suspensions from cultures of stocks TREU 1275 and TREU 929 revealed that the maximum number of infective organisms was present 26 to 50 days after initiation of the cultures. Control cultures of trypanosomes grown in medium alone were generally not infective but 2 of the 6 stocks gave rise to a few sporadic infections. A few epimastigote-like and metacyclic-like trypanosomes were seen in stained preparations of infective inocula.