Evolutionary history of trypanosomes from South American caiman (Caiman yacare) and African crocodiles inferred by phylogenetic analyses using SSU rDNA and gGAPDH genes

In this study, using a combined data set of SSU rDNA and gGAPDH gene sequences, we provide phylogenetic evidence that supports clustering of crocodilian trypanosomes from the Brazilian Caiman yacare (Alligatoridae) and Trypanosoma grayi, a species that circulates between African crocodiles (Crocodilydae) and tsetse flies. In a survey of trypanosomes in Caiman yacare from the Brazilian Pantanal, the prevalence of trypanosome infection was 35% as determined by microhaematocrit and haemoculture, and 9 cultures were obtained. The morphology of trypomastigotes from caiman blood and tissue imprints was compared with those described for other crocodilian trypanosomes. Differences in morphology and growth behaviour of caiman trypanosomes were corroborated by molecular polymorphism that revealed 2 genotypes. Eight isolates were ascribed to genotype Cay01 and 1 to genotype Cay02. Phylogenetic inferences based on concatenated SSU rDNA and gGAPDH sequences showed that caiman isolates are closely related to T. grayi, constituting a well-supported monophyletic assemblage (clade T. grayi). Divergence time estimates based on clade composition, and biogeographical and geological events were used to discuss the relationships between the evolutionary histories of crocodilian trypanosomes and their hosts.     

Purification of major variable-surface glycoproteins from African trypanosomes by reverse-phase high-performance liquid chromatography

Reverse-phase high-performance liquid chromatography (RP-HPLC) was used in a one-step procedure to purify and analyze several different major variable-surface glycoproteins (VSGs) from lysates of African trypanosomes. RP-HPLC was used to fractionate lysates of trypanosomes and the VSG localized to the major peak of the elution profile using a rabbit antiserum to the cross-reacting determinant of the VSG. Polyacrylamide gel electrophoresis of HPLC fractions showed that the purity of isolated VSGs was equivalent to or better than that attained using conventional purification procedures. The elution positions of purified VSGs from a variety of cloned trypanosomes were identical, indicating the presence of a common hydrophobic feature on the surface of these highly polymorphic antigens. Preliminary experiments have shown that purification of VSG from trypanosome lysates may be scaled up to preparative levels. The results show that RP-HPLC is a useful procedure for rapid preparation of highly purified trypanosome VSGs and that analysis of their various molecular forms will be facilitated by the application of HPLC methods.     

Trypanosoma rhodesiense: protection in mice by inoculations of homologous parasite products

Mice were immunized against Trypanosoma rhodesiense (Wellcome strain) with whole lyophilized trypanosomes, with antigens produced by disrupting lyophilized trypanosomes under pressure, and with excretions and secretions of the living parasites. The survival rate in groups of 40 mice inoculated with disrupted trypansomes and challenged with the homologous strain was 48% with a soluble fraction, and 70% with a particulate fraction of the parasites. There was 95% survival after challenge in a group immunized with lyophilized trypanosomes; none of the controls survived. Results were essentially the same whether or not an aluminum hydroxide adjuvant was used. In subsequent experiments, complete protection was obtained with either crude excretion-secretion (ES) antigens or the particulate fraction of the ES antigen, while 40% of the mice survived challenge after inoculations of ES supernatant fluid. Mice immunized with crude ES antigen failed to survive challenge with a heterologous strain, although their mean survival time was prolonged several days beyond that of the controls.     

Sensitivity of an antigen detection enzyme immunoassay for diagnosis of Trypanosoma congolense infections in goats and cattle

The sensitivity of a monoclonal antibody-based antigen-detection enzyme immunoassay (antigen-ELISA) for the diagnosis of Trypanosoma congolense was evaluated using sera from experimentally infected goats and cattle. Ten goats (Galla x East African Masai) and 7 steers (Bos indicus) were infected with different clones of T. congolense and left to run a chronic course for 46 and 24 mo, respectively. During this period, monthly blood samples were collected and analyzed for the presence of trypanosomes and antigens in peripheral blood. Of 383 caprine blood samples, 361 (94.3%) were positive for circulating antigens whereas only 42 (10.9%) had demonstrable trypanosomes as revealed by the microhematocrit centrifugation technique. In cattle, 570 (82.5%) of 691 blood samples were antigen-ELISA positive compared to 136 (19.7%) samples with detectable trypanosomes. In an analysis of serum samples from goats in an area known to be endemic for trypanosomiasis, 106 (80.9%) of 131 were positive for T. congolense antigens whereas none of the corresponding blood samples had detectable trypanosomes. Control sera from 24 goats in a trypanosomiasis-free region were all antigen-ELISA negative. Hence, the antigen-ELISA was at least 4 times more sensitive than the microhematocrit centrifugation technique in monitoring T. congolense infections in goats and cattle.     

Two genotypic groups of morphologically similar fish trypanosomes from the Okavango Delta, Botswana

Blood smears and blood lysate samples from freshwater fishes captured in the Okavango Delta, Botswana, were examined to determine whether their trypanosomes were all Trypanosoma mukasai, a species of supposed broad host specificity and widespread existence across Africa. Trypanosomes and/or babesiosomes occurred in 20/32 blood smears, and morphometric analysis of trypanosomes from 13/32 smears showed features suggestive of T. mukasai, including nuclear indices consistently >1. In 16/32 blood lysate samples from which DNA was extracted, trypanosome DNA was detected in 12/16 by PCR (polymerase chain reaction), using trypanosome-specific ssu rRNA gene primers. Two samples positive for trypanosomes in blood smears yielded no amplifiable trypanosome DNA, but 4 samples with no detectable infection in blood smears were positive for trypanosome DNA, suggesting an overall trypanosome prevalence rate of 17/32 (53%) among fishes and demonstrating the value of PCR in trypanosome recognition. Cloning and sequencing of the 12 amplified fragments revealed 2 genotypic groups among these fish trypanosomes. Group 1 trypanosomes were from cichlids and 3 families of catfishes, Group 2 from 2 types of catfishes. Sequence comparison showed that the consensus Group 1 sequence was most similar to that of Trypanosoma cobitis, representing European fish trypanosomes of the carassii type, while the consensus Group 2 sequence showed similarity with a trypanosome sequence from another African catfish, Clarias angolensis. It was concluded that the identification of T. mukasai remains a problem, but at least 2 genotypic groups of trypanosomes occur in Okavango Delta fishes, and catfishes in this region appear to contain both types.     

Trypanosoma congolense: mechanical removal of the surface coat in vitro

By shaking suspensions of Trypanosoma congolense in isotonic buffer the surface coat could be separated from the cell body. The release of radioactivity from trypanosomes, selectively labeled in the surface coat by diazoniobenzenesulfonate, was used to follow the kinetics of coat detachment. The proteins in the supernatants of shaken trypanosomes were analyzed by sodium dodecyl sulfate—polyacrylamide gel electrophoresis. The shaking conditions had to be carefully controlled to avoid complete rupture of trypanosomes. Otherwise the coat protein was rapidly degraded by endogenous proteases. The influence of several parameters on the yield of coat release and the degree of degradation of the coat protein was investigated, including the ratio of trypanosome suspension volume to shaking vessel volume, vessel surface, temperature, shaking frequency, and preincubation of the trypanosomes at 0 C. By combining these parameters an optimal scheme was developed which allowed the separation of more than 90% of the coat protein from T. congolense, the detached protein showing no degradation at all. These results could be confirmed by electron microscopy of shaken and unshaken trypanosomes.     

Trypanosoma brucei: Antigenic analysis of bloodstream,vector, and culture stages by the quantitative fluorescent antibody methods

Quantitative direct fluorescent antibody methods were used in antigenic analysis of developmental stages of Trypanosoma brucei brucei strains, most of them having the same variant antigen B, which were derived from a cyclically transmissible stabilate. Antigen-B trypanosomes were used for initiation of cultures in modified Tobie's (Tm) medium and in Glossina morsitans morsitans organ cultures, and for the infective feed of G. m. morsitans. Antisera against antigen-B bloodstream forms and against Tm-grown culture forms were developed in rabbits by inoculations of disrupted organisms mixed (1:1) with complete Freund's adjuvant. The globulin fractions of the antisera were conjugated with fluorescein isothiocyanate, and processed on Sephadex G-25 and DEAE-cellulose columns. The DEAE fractions with 2.0 and 4.7 or 4.8 molar fluorescein:protein ratios were pooled and concentrated twofold.Examination of 109 flies at 30 or 31 days after the infective feed revealed about 18.3% midgut, about 10.1% proventricular, and about 3.7% salivary-gland infections. A salivary gland suspension from one of the infected flies gave rise to a parasitemia in a mouse, and trypanosomes from the first parasitemia were transferred by two 3-day syringe passages into another mouse. Smears were prepared of trypanosomes (antigens B-164, B-167) from the first parasitemias from these two mice, of intact B-antigen trypanosomes, of culture forms (CT) from Tm medium, and of procyclics (CG) from Glossina cultures as well as of midgut (GM), proventricular (GP), and salivary-gland (GS) forms from tsetse flies. All these forms were fixed by one or more of the three following methods: complete fixation (CoFix) by the formalin-NH₄OH-Tween 80 procedure; fixation before affixation to slides (F+); fixation after affixation to slides (F?). The best results with regard to fluorescence intensity and specificity were obtained by using the CoFix technique.Statistical analyses of the fluorescence means of the antigens subjected to direct and inhibition staining gave the following results: (1) CT, CG, GM, and GP forms were antigenically the same. (2) GM and GP trypanosomes from different flies were antigenically indistinguishable. (3) The surface antigen of the variant-B bloodstream trypanosomes was different from these antigens of culture, midgut, and proventricular forms. It differed also from those of metacyclics from two flies and of B-164 and B-167 bloodstream forms. (4) No antigenic differences were found, in preparations fixed by the F? method, between B-164 and B-167 bloodstream trypanosomes and the metacyclics from two flies, one of which served as the source of the salivary-gland trypomastigotes (GS-98) that gave rise to these two bloodstream form antigens. (5) Closer antigenic relationships were noted between B forms and B-164 and B-167 trypanosomes than between B and CT organisms in smears fixed by the F+ technique, but no such differences were discernible in preparations fixed by the F? procedure.     

A function for a specific zinc metalloprotease of African trypanosomes

The Trypanosoma brucei genome encodes three groups of zinc metalloproteases, each of which contains approximately 30% amino acid identity with the major surface protease (MSP, also called GP63) of Leishmania. One of these proteases, TbMSP-B, is encoded by four nearly identical, tandem genes transcribed in both bloodstream and procyclic trypanosomes. Earlier work showed that RNA interference against TbMSP-B prevents release of a recombinant variant surface glycoprotein (VSG) from procyclic trypanosomes. Here, we used gene deletions to show that TbMSP-B and a phospholipase C (GPI-PLC) act in concert to remove native VSG during differentiation of bloodstream trypanosomes to procyclic form. When the four tandem TbMSP-B genes were deleted from both chromosomal alleles, bloodstream B (-/-) trypanosomes could still differentiate to procyclic form, but VSG was removed more slowly and in a non-truncated form compared to differentiation of wild-type organisms. Similarly, when both alleles of the single-copy GPI-PLC gene were deleted, bloodstream PLC (-/-) cells could still differentiate. However, when all the genes for both TbMSP-B and GPI-PLC were deleted from the diploid genome, the bloodstream B (-/-) PLC (-/-) trypanosomes did not proliferate in the differentiation medium, and 60% of the VSG remained on the cell surface. Inhibitors of cysteine proteases did not affect this result. These findings demonstrate that removal of 60% of the VSG during differentiation from bloodstream to procyclic form is due to the synergistic activities of GPI-PLC and TbMSP-B.     

Trypanosoma (Megatrypanum) melophagium in the sheep ked Melophagus ovinus from organic farms in Croatia: phylogenetic inferences support restriction to sheep and sheep keds and close relationship with trypanosomes from other ruminant species

Trypanosoma (Megatrypanum) melophagium is a parasite of sheep transmitted by sheep keds, the sheep-restricted ectoparasite Melophagus ovinus (Diptera: Hippoboscidae). Sheep keds were 100% prevalent in sheep from five organic farms in Croatia, Southeastern Europe, whereas trypanosomes morphologically compatible with T. melophagium were 86% prevalent in the guts of the sheep keds. Multilocus phylogenetic analyses using sequences of small subunit rRNA, glycosomal glyceraldehyde-3-phosphate dehydrogenase, spliced leader, and internal transcribed spacer 1 of the rDNA distinguished T. melophagium from all allied trypanosomes from other ruminant species and placed the trypanosome in the subgenus Megatrypanum. Trypanosomes from sheep keds from Croatia and Scotland, the only available isolates for comparison, shared identical sequences. All biologic and phylogenetic inferences support the restriction of T. melophagium to sheep and, especially, to the sheep keds. The comparison of trypanosomes from sheep, cattle, and deer from the same country, which was never achieved before this work, strongly supported the host-restricted specificity of trypanosomes of the subgenus Megatrypanum. Our findings indicate that with the expansion of organic farms, both sheep keds and T. melophagium may re-emerge as parasitic infections of sheep.     

The inadvertent introduction into Australia of Trypanosoma nabiasi, the trypanosome of the European rabbit (Oryctolagus cuniculus), and its potential for biocontrol

Wild rabbits (Oryctolagus cuniculus) in Australia are the descendents of 24 animals from England released in 1859. We surveyed rabbits and rabbit fleas (Spilopsyllus cuniculi) in Australia for the presence of trypanosomes using parasitological and PCR-based methods. Trypanosomes were detected in blood from the European rabbits by microscopy, and PCR using trypanosome-specific small subunit ribosomal RNA (SSU rRNA) gene primers and those in rabbit fleas by PCR. This is the first record of trypanosomes from rabbits in Australia. We identified these Australian rabbit trypanosomes as Trypanosoma nabiasi, the trypanosome of the European rabbit, by comparison of morphology and SSU rRNA gene sequences of Australian and European rabbit trypanosomes. Phylogenetic analysis places T. nabiasi in a clade with rodent trypanosomes in the subgenus Herpetosoma and their common link appears to be transmission by fleas. Despite the strict host specificity of trypanosomes in this clade, phylogenies presented here suggest that they have not strictly cospeciated with their vertebrate hosts. We suggest that T. nabiasi was inadvertently introduced into Australia in the 1960s in its flea vector Spilopsyllus cuniculi, which was deliberately introduced as a potential vector of the myxoma virus. In view of the environmental and economic damage caused by rabbits in Australia and other islands, the development of a virulent or genetically modified T. nabiasi should be considered to control rabbits.     

Development of Trypanosoma (M.) theileri in Tabanids

Thus far the life cycle of Trypanosoma (Megatrypanum) theileri has not been studied. We collected tabanids during the mass hatching, when only few tabanids are infected with trypanosomes. Tabanids were caught immediately after attacking a bait cow to serve as controls or after they had been allowed to engorge on the Trypanosoma (M.) theileri-infected cow. Tabanids were kept in the laboratory and used to study the developmental cycle of T. (M.) theileri in the tabanid gut. From day 1 to day 10 the presumably unfed controls and the engorged tabanids were dissected and cytological smears made from the mid- and hindgut. In total 2.6% (1/38) of the controls and 39% (23/59) of the engorged tabanids were positive for trypanosomes in the 1991 season. From day 1 to day 4 after engorgement trypanosomes were found in the midgut. Epimastigotes with a length of 29 μm on day 1 after infection multiplied by inequal division to form smaller epimastigotes of 26 μm on day 3. On day 4 morphologically indistinguishable trypanosomes of 21 μm total length were found in both mid- and hindgut. From day 5 to day 10 trypanosomes were found only in the hindgut in which the transformation to metacyclics was demonstrated, i.e. epimastigotes transformed to amastigote stages of 5 μm in total length.     

Phylogenetic analysis of Bolivian bat trypanosomes of the subgenus schizotrypanum based on cytochrome B sequence and minicircle analyses

The aim of this study was to establish the phylogenetic relationships of trypanosomes present in blood samples of Bolivian Carollia bats. Eighteen cloned stocks were isolated from 115 bats belonging to Carollia perspicillata (Phyllostomidae) from three Amazonian areas of the Chapare Province of Bolivia and studied by xenodiagnosis using the vectors Rhodnius robustus and Triatoma infestans (Trypanosoma cruzi marenkellei) or haemoculture (Trypanosoma dionisii). The PCR DNA amplified was analyzed by nucleotide sequences of maxicircles encoding cytochrome b and by means of the molecular size of hyper variable regions of minicircles. Ten samples were classified as Trypanosoma cruzi marinkellei and 8 samples as Trypanosoma dionisii. The two species have a different molecular size profile with respect to the amplified regions of minicircles and also with respect to Trypanosoma cruzi and Trypanosoma rangeli used for comparative purpose. We conclude the presence of two species of bat trypanosomes in these samples, which can clearly be identified by the methods used in this study. The presence of these trypanosomes in Amazonian bats is discussed.     

Molecular evidence for trypanosomatids in Culex mosquitoes collected during a West Nile virus survey

Adult mosquitoes were previously collected and tested for West Nile virus during an intense WNV outbreak in 2003-2004 along the Cache la Poudre River in Colorado, USA. A subset of these mosquitoes was also tested for infection with trypanosomatids using nested PCR to amplify 18S rRNA. Of the 69 pools of Culex pipiens that were screened for both pathogens, 4.3% were positive for WNV and 11.6% tested positive for trypanosomes; no pools were found to be co-infected with both pathogens. One hundred and forty-three pools of Culex tarsalis, considered to be the principal WNV vector in this area, were tested in the same manner. 7.7% were positive for WNV and 20.3% of these pools tested positive for trypanosomes. Five pools of C. tarsalis were found to be co-infected with both pathogens, which was approximately 2.2 times more frequent than would be expected if these pathogens are independent of each other. Sequencing and maximum parsimony analysis of 18S rRNA revealed that four of the isolates arise in or near clades of described avian trypanosomes, likely indicating that these are vectored pathogens between birds and mosquitoes. Unexpectedly, the majority (24/28, 86%) of our positive samples form their own separate clade within the order Trypanosomatida with 100% bootstrap support. We have identified a potential new clade of trypanosomatids that exist within important mosquito vectors and discuss the potential ecological connections between these trypanosomes, arboviruses and mosquitoes.     

The Biology of Trypanosoma diemyctyli, Tobey. III. Factors influencing the cycle of Trypanosoma diemyctyli in the vertebrate host Triturus v. viridescens.

SYNOPSIS. The effects of some environmental influences on the cycle of Trypanosoma diemyctyli in Triturus v. viridescens are described. Bleeding of the host produced a reduction in the number of trypanosomes but did not affect their growth rate. The temperature at which the host was maintained affected the cycle of the trypanosomes. The length of the post-inoculation latent stage increased from 24 hours at 25°C. to an indefinitely long time at 5°C. The trypanosomes were found to be dimorphic. Adult parasites of the short form had a range of 45–75 μ and those of the long form of 76–116 μ. Growth rate of the trypanosomes was inhibited or greatly retarded at temperatures of 10°C. or lower and was greatest at 25°C. The size attained by the parasites and the number of parasites were greatest at 15°C. At this temperature the infection was pathogenic and the dimorphic parasites were in their long form. At the higher temperatures (20–25°C.) the infection was non-pathogenic with the trypanosomes in their short form.
The infection is primarily one of adult newts. Experiments indicated that the larvae were resistant to the trypanosomes at all temperatures while the red efts were not. The latter are usually free from the trypanosomes because they are not exposed to them. Attempts to infect other newts and to locate any cryptic stages by the injection of blood and tissues from infected newts gave negative results.
Starvation, sodium salicylate, and treatments used to control fungus infection of the newts had no detectable effects on the trypanosomes.     

The Utilization of Glucose and Proline by Culture Forms of Trypanosoma brucei*

Exponentially dividing culture forms of Trypanosoma brucei did not utilize glucose provided in the culture medium. The inclusion of 2-deoxyglucose in the medium had no effect on the growth of the trypanosomes. Glucose could be replaced by proline in the liquid phase of biphasic medium without affecting the doubling time of the organisms. Proline added to the culture medium in this way disappeared during the log phase of growth. Glucose in the culture medium was used by the trypanosomes only when the stationary growth phase had been reached. Lipid accumulated in stationary phase trypanosomes grown in glucose-containing medium, but there was no lipid accumulation in log phase organisms or in those which had been grown in proline-containing medium. Bloodstream trypanosomes transferred to liquid medium rapidly utilized glucose over the first 12 hr of culture, and this was accompanied by an accumulation of free pyruvate in the medium. The rate of glucose utilization fell off over the next 36 hr; this was accompanied by a lowering of free pyruvate in the medium and a rise in the proline oxidase activity of the trypanosomes. The possible biologic significance of proline to trypanosomes developing in the midgut of the tsetse vector is discussed.     

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.     

Phylogenetic position of the giant anuran trypanosomes Trypanosoma chattoni,Trypanosoma fallisi,Trypanosoma mega,Trypanosoma neveulemairei,and Trypanosoma ranarum inferred from 18S rRNA gene sequences

Phylogenetic relationships within the kinetoplastid flagellates were inferred from comparisons of small-subunit ribosomal RNA gene sequences. These included 5 new gene sequences, Trypanosoma fallisi (2,239 bp), Trypanosoma chattoni (2,180 bp), Trypanosoma mega (2,211 bp), Trypanosoma neveulemairei (2,197 bp), and Trypanosoma ranarum (2,203 bp). Trees produced using maximum-parsimony and distance-matrix methods (least-squares, neighbor-joining, and maximum-likelihood), supported by strong bootstrap and quartet-puzzle analyses, indicated that the trypanosomes are a monophyletic group that divides into 2 major lineages, the salivarian trypanosomes and the nonsalivarian trypanosomes. The nonsalivarian trypanosomes further divide into 2 lineages, 1 containing trypanosomes of birds, mammals, and reptiles and the other containing trypanosomes of fish, reptiles, and anurans. Among the giant trypanosomes, T. chattoni is clearly shown to be distantly related to all the other anuran trypanosome species. Trypanosoma mega is closely associated with T. fallisi and T. ranarum, whereas T. neveulemairei and Trypanosoma rotatorium are sister taxa. The branching order of the anuran trypanosomes suggests that some toad trypanosomes may have evolved by host switching from frogs to toads.     

The use of experimental artefacts in African trypanosome research

When trypanosomes are removed from the field and maintained in laboratory conditions, phenotypic changes commonly occur such that the lines used by many investigators in routine work show several differences from the populations that affect humans and cattle in Africa. Whether these differences are important or irrelevant of course depends on the purpose of each particular experiment, but an awareness of what the differences are can be a useful aid in the interpretation of results. Furthermore, trypanosomes can be manipulated in the laboratory to possess particular characteristics that aid in the testing of hypotheses that are difficult to test using 'wild-type' trypanosomes. In this article, Mike Turner describes how some defined trypanosome lines have been created, how they differ from one another and several of their uses.     

Expression and function of the Trypanosoma brucei major surface protease (GP63) genes

The genome of the African trypanosome Trypanosoma brucei (Tb) contains at least three gene families (TbMSP-A, -B, and -C) encoding homologues of the abundant major surface protease (MSP, previously called GP63), which is found in all Leishmania species. TbMSP-B mRNA occurs in both procyclic and bloodstream trypanosomes, whereas TbMSP-A and -C mRNAs are detected only in bloodstream organisms. RNA interference (RNAi)-mediated gene silencing was used to investigate the function of TbMSP-B protein. RNAi directed against TbMSP-B but not TbMSP-A ablated the steady state TbMSP-B mRNA levels in both procyclic and bloodstream cells but had no effect on the kinetics of cultured trypanosome growth in either stage. Procyclic trypanosomes have been shown previously to have an uncharacterized cell surface metalloprotease activity that can release ectopically expressed surface proteins. To determine whether TbMSP-B is responsible for this release, transgenic variant surface glycoprotein 117 (VSG117) was expressed constitutively in T. brucei procyclic TbMSP-RNAi cell lines, and the amount of surface VSG117 was determined using a surface biotinylation assay. Ablation of TbMSP-B but not TbMSP-A mRNA resulted in a marked decrease in VSG release with a concomitant increase in steady state cell-associated VSG117, indicating that TbMSP-B mediates the surface protease activity of procyclic trypanosomes. This finding is consistent with previous pharmacological studies showing that peptidomimetic collagenase inhibitors block release of transgenic VSG from procyclic trypanosomes and are toxic for bloodstream but not procyclic organisms.     

Genetic characterization of Trypanosoma brucei circulating in domestic animals of the Fontem sleeping sickness of Cameroon

To improve our knowledge on the transmission dynamics of trypanosomes, Trypanosoma brucei was identified in domestic animals of the Fontem sleeping sickness focus of Cameroon and their genetic characterizations were performed using seven polymorphic microsatellite markers. About 397 domestic animals including 225 pigs, 87 goats, 65 sheep and 20 dogs were sampled. The card agglutination test for trypanosomiasis was positive for 254 (63.98%) animals while the parasitological examinations (thin blood film and capillary tube centrifugation) revealed 86 (21.66%) trypanosome infections. The PCR based method revealed 140 (35.26%) infections of trypanosomes of the subgenus Trypanozoon. The genetic characterization of these 140 positive samples revealed 89 different alleles: 82 in pigs, 72 in goat, 60 in sheep and 48 in dog. Whatever the microsatellite marker used, most of positive samples were amplified. However, the sensitivity (percentage of samples amplified for each marker) of these markers varies significantly between them (χ(2) = 120.32; P < 0.0001). This study showed a high level (80.00%) of mixed genotypes as well as a wide range of T. brucei genotypes circulating in domestic animals of the Fontem sleeping sickness focus of Cameroon. This indicates that several T. brucei genotypes can naturally be transmitted simultaneously to tsetse flies during a single blood meal.