Cell Adhesion in Trypanosoma: In Vitro Studies of the Interaction of Trypanosoma vivax with Immobilized Organic Dyes1

Certain bloodstream forms of Trypanosoma vivax have been shown to attach to Amicon Matrex? Gel Green A dye beads in a manner similar to the in vivo binding of T. vivax to the inner surface of the tsetse fly proboscis. We now report an in vitro assay for trypanosome-bead attachment and show that only the 9,10-anthraquinone portion of the dye molecule is involved in the binding of trypanosomes to beads and that bead-bound dyes with similar structures also support binding to differing degrees. The binding is dependent upon the amount of dye on the beads and this, and other evidence, suggests that an array of dye molecules, rather than individual molecules, may be the actual recognition site. Various external effectors, including temperature, soluble protein-dye complexes, and serum of mice with chronic T. vivax infections, reduce trypanosome binding, indicating that at least one immunogenic trypanosome macromolecule is involved. The trypanosome-bead interaction mimics the in vivo binding to tsetse proboscis and warrants closer examination as a model of trypanosome cell adhesion in the tsetse fly.     

Trypanosoma vivax: mechanical transmission in cattle by one of the most common African tabanids,Atylotus agrestis

The role of mechanical vectors in the transmission of African livestock trypanosomes has always been controversial relative to tsetse flies, their cyclical vectors. An experiment was carried out in Burkina Faso to demonstrate mechanical transmission of Trypanosoma vivax by one of the most common tabanids in Africa: Atylotus agrestis. Eight heifers (crossbred zebuxBaoulé), free of trypanosome infection, were kept in a corral covered by a mosquito net, together with two heifers infected experimentally with a local stock of T. vivax. On average, 324 A. agrestis, freshly captured with Nzi traps, were introduced daily over 20 days. Parasitological, PCR and serological examinations were carried out regularly to assess infections and levels of parasitaemia. Microscopic examination of buffy-coats indicated that five of the eight receiver-heifers were infected on days 8, 13, 32, 41, and 48. PCR results indicated that these five heifers were already infected by day 13. Mechanical transmission of T. vivax by A. agrestis was demonstrated unequivocally, at a high rate (63% in 13-20 days). Conditions of transmission in this experiment are discussed in terms of natural rates of challenge. The importance of tabanids as mechanical vectors of T. vivax should be re-considered, in light of these results. Creation of tsetse free zones in Africa will generally lead to the disappearance of T. congolense, T. brucei, and most often T. vivax as well; however, in areas where T. vivax can be mechanically transmitted, clearance of tsetse may not be sufficient to eradicate livestock trypanosomosis.     

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.     

The Mode of Attachment of Trypanosoma vivax in the Proboscis of the Tsetse Fly Glossina fuscipes: an Ultrastructural Study of the Epimastigote Stage of the Trypanosome*

SYNOPSIS. The ultrastructure of attached Trypanosoma vivax epimastigote clusters in the proboscis of the tsetse fly Glossina fuscipes is described from electron micrographs of thin sections. Some flagellates are attached directly to the lining of the insect's labrum by their flagella, most of which are aligned along the long axis of the proboscis. Other trypanosomes are attached indirectly, their flagella adhering to those of flagellates which are directly attached. Junctional complexes similar to those described from metazoan epithelia are found on the flagellar membrane. A long zonular hemidesmosome attaches the flagellum to the proboscis wall and a series of closely set macular desmosomes link the flagellar membranes of adjacent flagellates. Unlike the trypomastigote stages of T. vivax, more than one row of macular desmosomes may be present along the flagellum-body junction of the trypanosome. It is suggested that all these Junctional complexes serve to buttress the flagellate's attachment to its insect host and so maintain anchorage of the parasite during the fly's blood meals. The ability of the flagellum of trypanosomatids to form Junctional complexes may be a factor contributing to their success as parasites, this adaptation enabling them to multiply while attached to host surfaces.     

Molecular cloning and characterization of Trypanosoma vivax alternative oxidase (AOX) gene, a target of the trypanocide ascofuranone

Trypanosoma vivax causes nagana disease in cattle. Since T. vivax is transmitted not only by tsetse flies but also by other biting flies (non-cyclic transmission), the parasite has been distributed to and has had a significant economic impact on wide geographical areas, including Africa and South America. Our previous study on Trypanosoma brucei brucei showed that the trypanosome alternative oxidase (TAO, TbAOX) is a promising target of chemotherapy. For this reason, we also have cloned the T vivax AOX (TvAOX) gene and characterized the recombinant enzyme. The deduced amino acid sequence (328 a.a.) of TvAOX shares 76% identity with TbAOX and contains the diiron-coordination motifs (-E-, -EXXH-) that are conserved among AOXs. The Km of recombinant TvAOX (rTvAOX) expressed in Escherichia coli for ubiquinol (87.0 +/- 0.54 microM) was significantly lower than the value for recombinant TbAOX (rTbAOX) (714 +/- 4.5 microM). Ascofuranone, the most potent inhibitor of TbAOX, was a competitive inhibitor of rTvAOX with a Ki value (0.40 +/- 0.00 nM) significantly lower than that for rTbAOX (1.29 +/- 0.00 nM). The non-cyclic transmission ability of T. vivax and the in vivo chemotherapeutic efficacy of ascofuranone against T. vivax and T. b. brucei infection are discussed in terms of these Km and Ki values.     

Partial structure of glutamic acid and alanine-rich protein,a major surface glycoprotein of the insect stages of Trypanosoma congolense

The tsetse fly transmitted salivarian trypanosome, Trypanosoma congolense of the subgenus Nanomonas, is the most significant of the trypanosomes with respect to the pathology of livestock in sub-Saharan Africa. Unlike the related trypanosome Trypanosoma brucei of the subgenus Trypanozoon, the major surface molecules of the insect stages of T. congolense are poorly characterized. Here, we describe the purification and structural characterization of the glutamic acid and alanine-rich protein, one of the major surface glycoproteins of T. congolense procyclic and epimastigote forms. The glycoprotein is a glycosylphosphatidylinositol-anchored molecule with a galactosylated glycosylphosphatidylinositol anchor containing an sn-1-stearoyl-2-l-3-HPO(4)-1-(2-O-acyl)-d-myo-inositol phospholipid moiety. The 21.6-kDa polypeptide component carries two large mannose- and galactose-containing oligosaccharides linked to threonine residues via phosphodiester linkages. Mass spectrometric analyses of tryptic digests suggest that several or all of the closely related glutamic acid and alanine-rich protein genes are expressed simultaneously in a T. congolense population growing in vitro.     

New epidemiological features on animal trypanosomiasis by molecular analysis in the pastoral zone of Sideradougou, Burkina Faso

A multidisciplinary work was undertaken in the agropastoral zone of Sidéradougou, Burkina Faso to try to elucidate the key factors determining the presence of tsetse flies. In this study the PCR was used to characterize trypanosomes infecting the vector ( Glossina tachinoides and Glossina palpalis gambiensis ) and the host, i.e. cattle. A 2-year survey involved dissecting 2211 tsetse of the two Glossina species. A total of 298 parasitologically infected tsetse were analysed by PCR. Trypanosoma vivax was the most frequently identified trypanosome followed by the savannah type of T. congolense and, to a lesser extent, the riverine forest type of T. congolense , and by T. brucei . No cases of T. simiae were found. From the 107 identified infections in cattle, the taxa were the same, but T. congolense savannah type was more frequent, whereas T. vivax and T. congolense riverine forest types were found less frequently. A correlation was found between midgut infection rates of tsetse, nonidentified infections and reptile bloodmeals. These rates were higher in G.p. gambiensis , and in the western part of the study area. T. vivax infections were related to cattle bloodmeals, and were more frequent in G. tachinoides and in the eastern study area. The PCR results combined with bloodmeal analysis helped us to establish the relationships between the vector and the host, to assess the trypanosome challenge in the two parts of the area, to elucidate the differences between the two types of T. congolense , and to suspect that most midgut infections were originating from reptilian trypanosomes.     

Sensitive detection of trypanosomes in tsetse flies by DNA amplification.

African trypanosome species were identified using the Polymerase Chain Reaction (PCR) by targeting repetitive DNA for amplification. Using oligonucleotide primers designed to anneal specifically to the satellite DNA monomer of each species/subgroup, we were able to accurately identify Trypanosoma simiae, three subgroups of T. congolense, T. brucei and T. vivax. The assay was sensitive and specific, detecting one trypanosome unequivocally and showing no reaction with non-target trypanosome DNA or a huge excess of host DNA. The assay was used to identify developmental stage trypanosomes in the tsetse fly. The use of radioisotopes was not necessary and mixed infections could be detected easily by incorporating more than one set of primers in a single reaction. The use of crude preparations of template made the process very rapid. The methodology should be suitable for large-scale epidemiological studies.     

Trypanosoma brucei: a membrane-associated protein in coated endocytotic vesicles

Membrane proteins were isolated from purified Trypanosoma brucei coated endocytotic vesicles by phase separation with Triton X-114. The largest abundant membrane protein was a doublet band with a molecular mass of about 77 kDa. A specific antiserum was prepared against this protein by immunization with antigen bands excised from sodium dodecyl sulfate-polyacrylamide gels. Immunoblot analyses with this antiserum showed that the 77-kDa protein was present in other T. brucei, in T. congolense, and in T. vivax bloodstream-stage parasites but absent from procyclic (tsetse fly midgut)-stage trypanosomes. Antigenically related molecules of 58, 300, and 15.5 kDa were also detected. The 300- and 15.5-kDa molecules were not in purified coated vesicles; they were detected in whole bloodstream- and procyclic-form T. brucei organisms. Immunofluorescent studies localized the antigen to the region between the flagellar pocket and the nucleus of bloodstream-form parasites. Ultrastructurally, the antigen was detected on membranes of endosomes and lysosome-like structures that contained endocytosed markers.     

Seasonal variation of tsetse fly species abundance and prevalence of trypanosomes in the Maasai Steppe,Tanzania

Tsetse flies, the vectors of trypanosomiasis, represent a threat to public health and economy in sub‐Saharan Africa. Despite these concerns, information on temporal and spatial dynamics of tsetse and trypanosomes remain limited and may be a reason that control strategies are less effective. The current study assessed the temporal variation of the relative abundance of tsetse fly species and trypanosome prevalence in relation to climate in the Maasai Steppe of Tanzania in 2014–2015. Tsetse flies were captured using odor‐baited Epsilon traps deployed in ten sites selected through random subsampling of the major vegetation types in the area. Fly species were identified morphologically and trypanosome species classified using PCR. The climate dataset was acquired from the African Flood and Drought Monitor repository. Three species of tsetse flies were identified: G. swynnertoni (70.8%), G. m. morsitans (23.4%), and G.pallidipes (5.8%). All species showed monthly changes in abundance with most of the flies collected in July. The relative abundance of G. m. morsitans and G. swynnertoni was negatively correlated with maximum and minimum temperature, respectively. Three trypanosome species were recorded: T. vivax (82.1%), T. brucei (8.93%), and T. congolense (3.57%). The peak of trypanosome infections in the flies was found in October and was three months after the tsetse abundance peak; prevalence was negatively correlated with tsetse abundance. A strong positive relationship was found between trypanosome prevalence and temperature. In conclusion, we find that trypanosome prevalence is dependent on fly availability, and temperature drives both tsetse fly relative abundance and trypanosome prevalence.     

Trypanosoma (Duttonefla) vivax

Although Trypanosoma vivax was first discovered in 1905 (Ref. 1), the fact that most stocks of this parasite are restricted to ruminant hosts has retarded investigation of this species compared with the experimentally more amenable T. brucei and T. congolense. The veterinary importance of T. vivax (Box 1) and a recent report suggesting that T. vivax may have an even more extended range than previously thought (Box 2) prompts an evaluation of the current knowledge of the biology of this trypanosome.     

Factors affecting trypanosome maturation in tsetse flies

Trypanosoma brucei brucei infections which establish successfully in the tsetse fly midgut may subsequently mature into mammalian infective trypanosomes in the salivary glands. This maturation is not automatic and the control of these events is complex. Utilising direct in vivo feeding experiments, we report maturation of T. b. brucei infections in tsetse is regulated by antioxidants as well as environmental stimuli. Dissection of the maturation process provides opportunities to develop transmission blocking vaccines for trypanosomiasis. The present work suggests L-cysteine and/or nitric oxide are necessary for the differentiation of trypanosome midgut infections in tsetse.     

N-acetyl D-glucosamine stimulates growth in procyclic forms of Trypanosoma brucei by inducing a metabolic shift

SUMMARYThe lectin-inhibitory sugars D-glucosamine (GlcN) and N-acetyl D-glucosamine (GlcNAc) are known to enhance susceptibility of the tsetse fly vector to infection with Trypanosoma brucei. GlcNAc also stimulates trypanosome growth in vitro in the absence of any factor derived from the fly. Here, we show that GlcNAc cannot be used as a direct energy source, nor is it internalized by trypanosomes. It does, however, inhibit glucose uptake by binding to the hexose transporter. Deprivation of D-glucose leads to a switch from a metabolism based predominantly on substrate level phosphorylation of D-glucose to a more efficient one based mainly on oxidative phosphorylation using L-proline. Procyclic form trypanosomes grow faster and to higher density in D-glucose-depleted medium than in D-glucose-rich medium. The ability of trypanosomes to use L-proline as an energy source can be regulated depending upon the availability of D-glucose and here we show that this regulation is a graded response to D-glucose availability and determined by the overall metabolic state of the cell. It appears, therefore, that the growth stimulatory effect of GlcNAc in vitro relates to the switch from D-glucose to L-proline metabolism. In tsetse flies, however, it seems probable that the effect of GlcNAc is independent of this switch as pre-adaptation to growth in proline had no effect on tsetse infection rate.     

Feeding behaviour of Glossina pallidipes and g. morsitans centralis on Boran cattle infected with trypanosoma congolense or T. vivax under laboratory conditions

In field studies, tsetse flies (Diptera: Glossinidae) feed more successfully on cattle infected with Trypanosoma congolense Broden (Kinetoplastida: Trypanosomatidae) than on cattle infected with T. vivax Ziemann or uninfected cattle. Here we describe the first laboratory investigation of this phenomenon. In the first experiment, caged Glossina pallidipes Austen were fed for 1 and 5 min on a Boran steer infected with T. congolense clone IL 1180 and on an uninfected steer. Feeding success was recorded in this way five times over several weeks. The same protocol was subsequently used in three additional experiments with the following combinations: G. pallidipes and a steer infected with T. vivax stock IL 3913, G. morsitans centralis Machado and a steer infected with T. congolense, and G. morsitans centralis and a steer infected with T. vivax. The four experiments were replicated once, making eight experiments in total. In three experiments there was increased tsetse feeding success, measured at 1 min, after a steer became infected (T. congolense, two experiments and T. vivax, one experiment). Analysis of all data combined found no significant differences in tsetse feeding success on the different groups of cattle prior to infection, but after infection tsetse feeding success was significantly greater on the infected cattle (P< 0.001). Trypanosoma congolense infection led to a greater increase in tsetse feeding success than T. vivax infection. The increase in feeding success was not related to changes in the level of anaemia, skin surface temperature or parasitaemia. A possible explanation is the effects of trypanosome infection on cutaneous vasodilation and/or blood clotting in infected cattle. When allowed to feed for 5 min, nearly all tsetse engorged successfully and effects of cattle infection on feeding success were not found.     

Dynamics of infection and competition between two strains of <Emphasis Type="Italic">Trypanosoma brucei brucei</Emphasis>in the tsetse fly observed using fluorescent markers

Background  

Genetic exchange occurs between Trypanosoma brucei strains during the complex developmental cycle in the tsetse vector, probably within the salivary glands. Successful mating will depend on the dynamics of co-infection with multiple strains, particularly if intraspecific competition occurs. We have previously used T. brucei expressing green fluorescent protein to study parasite development in the vector, enabling even one trypanosome to be visualized. Here we have used two different trypanosome strains transfected with either green or red fluorescent proteins to study the dynamics of co-infection directly in the tsetse fly.     

Tsetse-trypanosome interactions: rites of passage.

Trypanosomes that cause sleeping sickness (Trypanosoma brucei rhodesiense and T. b. gambiense) are entirely dependent on tsetse for their transmission between hosts, but the flies are not easily infected. This situation has not arisen by chance - the tsetse has evolved an efficient defence system against trypanosome invasion. In this review, Susan Welburn and Ian Maudlin chart the progress of trypanosomes through the fly and identify some of the hazards faced by both parasite and fly that affect vector competence of tsetse.     

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.