Conserved sequence of the TgsGP gene in Group 1 Trypanosoma brucei gambiense

The trypanosome responsible for the majority of cases of human trypanosomiasis in Africa is Group 1 Trypanosoma brucei gambiense. Currently the most reliable test for the parasite is based on a single gene, which encodes a 47 kDa receptor-like T. b. gambiense-specific glycoprotein, TgsGP, expressed in the flagellar pocket of bloodstream forms. Although TgsGP has been demonstrated in T. b. gambiense throughout its geographic range, similar genes have been demonstrated in other T. brucei sspp. isolates, and there are no data on the extent of sequence variation in TgsGP. Here we have carried out a comparison of TgsGP sequences in a range of Group 1 T. b. gambiense isolates and compared the gene to homologues in other T. brucei sspp. in order to provide information to support the use of this gene as the key identification target for Group 1 T. b. gambiense. We demonstrate that the sequence of TgsGP is well conserved in Group 1 T. b. gambiense across the endemic range of gambian human trypanosomiasis and confirm that this gene is a suitable target for specific detection of this parasite. The TgsGp-like genes in some isolates of T. b. brucei, T. b. rhodesiense and Group 2 T. b. gambiense are closely similar to VSG Tb10.v4.0178, which may be the ancestral gene from which TgsGP was derived.     

Population genetic structure of Central African Trypanosoma brucei gambiense isolates using microsatellite DNA markers

Genetic variation of microsatellite loci is a widely used method for the analysis of population genetic structure of microorganisms. Seven microsatellite markers were used here to characterize Trypanosoma brucei gambiense isolates from Central Africa sub-region in order to improve knowledge on the population genetic structure of this subspecies. These markers confirmed the low genetic polymorphism within Central African T. b. gambiense isolates from the same focus and strong differentiation between different foci. The presence of many multilocus genotypes of T. b. gambiense and the excess of heterozygotes found in this study play in favour of a clonal reproduction of this parasite. But some data may be indicative of a unique recombination event in one subsample. The high F_ST value indicates low migration rates between T. b. gambiense subpopulations (foci). Very negative F_IS suggests fairly small clonal population sizes of this pathogen in the different human trypanosomosis foci of Central Africa.     

The susceptibility of chickens to Trypanosoma brucei subspecies

Chickens were susceptible to infection with three different stocks of the subgenus Trypanozoon: two of presumptive Trypanosoma b. brucei and one of T. b. rhodesiense. Two groups of chickens were used: the first hatched following inoculation with either T. b. brucei or T. b. rhodesiense during embryonic development, and the second were infected as adult birds. In both experimental groups, parasitaemia persisted for prolonged periods, but was mostly subpatent and detectable only by subinoculation of blood into mice. In chickens infected as embryos, parasitaemias were patent for five weeks after hatching, but subpatent thereafter (to weeks 13 to 17). Quantitative estimations of the parasitaemias of seven of the birds hatched from embryos inoculated with T. b. brucei revealed fluctuations in the number of circulating trypanosomes, with an initial peak between days 2 to 9 after hatching. Between weeks 13 to 17 after hatching the chickens appeared to have recovered spontaneously from the trypanosome infections. Homologous challenge at week 20 failed to produce a recrudescence of parasitaemia, indicative of a possible acquired immunity.The infections of ten chickens inoculated with either T. b. brucei or T. b. rhodesiense as adult birds were microscopically subpatent throughout the observation period of six weeks, but subinoculation of blood into mice showed the chickens were parasitaemic from week one and thereafter. Different aspects of infection of avian hosts by the Trypanozoon subspecies are discussed.     

APOL1 expression is induced by Trypanosoma brucei gambiense infection but is not associated with differential susceptibility to sleeping sickness

Most African trypanosome species are sensitive to trypanolytic factors (TLFs) present in human serum. Trypanosome lysis was demonstrated to be associated with apolipoprotein L-I (APOL1). Trypanosoma brucei (T. b.) gambiense and Trypanosoma brucei rhodesiense, the two human infective trypanosome species, have both developed distinct resistance mechanisms to APOL1 mediated lysis. Whereas T. b. rhodesiense resistance is linked with the expression of the serum resistance associated (SRA) protein that interacts with APOL1 inside the parasite lysosome, inhibiting its lytic action; T. b. gambiense resistance is rather controlled by a reduced expression of the parasite HpHb receptor, limiting APOL1 absorption by trypanosomes. Based on this last observation we hypothesised that variation in the host APOL1 environment could significantly alter T. b. gambiense growth and thus resistance/susceptibility to sleeping sickness. To test this hypothesis, we have measured blood APOL1 relative expression in HAT patients, uninfected endemic controls and serologically positive subjects (SERO TL⁺) that are suspected to control infection to parasitological levels that are undetectable by the available test used in the field. All RNA samples were obtained from medical surveys led in the HAT mangrove foci of Coastal Guinea. Results indicate that APOL1 expression is a complex trait dependant on a variety of factors that need to be taken into account in the analysis. Nevertheless, multivariate analysis showed that APOL1 expression levels were significantly higher in both HAT and SERO TL⁺ subject as compared to endemic controls (p = 0.006). This result suggests that APOL1 expression is likely induced by T. b. gambiense, but is not related to resistance/susceptibility in its human host.     

Monitoring the pleomorphism of Trypanosoma brucei gambiense isolates in mouse: Impact on its transmissibility to Glossina palpalis gambiensis

Substantial differences have been observed between the cyclical transmission of three Trypanosoma brucei gambiense field isolates in Glossina palpalis gambiensis (Ravel et al., 2006). Differences in the pleomorphism of these isolates in rodent used to provide the infective feed to Glossina, could explain such results, since stumpy forms are preadapted for differentiation to procyclic forms when taken up in a tsetse bloodmeal. To assess this possibility, mice were immunosuppressed and inoculated intraperitoneally with the three isolates (six mice for each trypanosome isolate); then parasitaemia and pleomorphism were determined daily for each mouse. The three T. b. gambiense isolates induced different infection patterns in mouse. The parasitaemia peak was rapidly reached for all the isolates and maintained until mice death for two isolates, while the third isolate rapidly showed a falling phase followed by a second parasitaemia plateau. The proportion of the stumpy forms varied from 15% to 70% over the duration of the experiment and according to the isolate. One isolate, which displayed the highest proportion of stumpy forms and reached the stumpy peak at the onset of the falling phase of parasitaemia, was used to study the relationship between the proportion of stumpy forms and transmissibility to tsetse fly. The results indicated that the transmissibility of trypanosomes was not correlated to the proportion of non-dividing stumpy forms.     

Aspartate aminotransferase in Leishmania is a broad-spectrum transaminase

The substrate specificity of aspartate aminotransferase (ASAT, E.C. 2.6.1.1.) from Leishmania was examined following observations of artefacts on gels stained for alanine aminotransferase (ALAT, E.C. 2.6.1.2.) after thin-layer starch-gel electrophoresis. Leishmanial ASAT acted on L-aspartate, L-alanine, L-tryptophan and L-tyrosine. Interpretation of ALAT zymograms must thus take into account the presence of interfering ASAT bands, and the need is emphasized for rigorous controls in isoenzyme electrophoresis.     

Leishmaniasis in Brazil: XV. Biochemical distinction of Leishmania mexicana amazonensis,L. braziliensis braziliensis and L. braziliensis guyanensis—aetiological agents of cutaneous leishmaniasis in the Amazon Basin of Brazil

Enzymic profiles of the three known agents of human cutaneous leishmaniasis in the lower Amazon region are compared. Of 14 enzymes, 10 (ASAT, ALAT, GPI, G6PD, MDH, ACON, PEP, HK, MPI and ACP) differentiate Leishmania mexicana amazonensis from L. braziliensis braziliensis or L. braziliensis guyanensis: this supports their taxonomic status as distinct species. In contrast, only slight mobility differences of four enzymes (ASAT, ALAT, PGM, MPI) separate L. b. braziliensis and L. b. guyanensis, which are distinguished biochemically for the first time: this indicates that they are closely related.Four stocks of L. b. panamensis correspond with L. b. guyanensis on mobilities of 10 enzymes (ASAT, ALAT, PGM, GPI, G6PD, MDH, PK, HK, MPI, ACP), although these two subspecies are known to be separable by kinetoplast DNA buoyancies and the enzyme 6PGDH.The generation of practical, regional biochemical keys to the medically important leishmanias is discussed.     

Differentiation of Trypanosoma brucei, T. rhodesiense, and T. gambiense by the indirect fluorescent antibody test

Epidemiological studies, if they are to lead to appropriate preventive procedures, require knowledge of the host distribution of the parasite. Progress in the epidemiology of African trypanosomiasis is restricted by the lack of a reliable and simple method of differentiating Trypanosoma brucei, T. rhodesiense, and T. gambiense. The recently introduced blood inoculation infectivity test promises to fulfil this need by distinguishing T. brucei from T. rhodesiense, but it would not be suitable for separating T. brucei from T. gambiense, since rats and mice are frequently refractory to infection by fresh isolates of T. gambiense. Previous studies had indicated that the indirect fluorescent antibody test might differentiate not only the subgenera of the salivarian trypanosome species but also members of the subgenus Trypanozoon. A method of performing the test is described that enables T. brucei, T. rhodesiense, and T. gambiense to be differentiated by the titre of the sera. The method might be used in conjunction with the blood inoculation infectivity test to distinguish between new isolates of the subgenus Trypanozoon in East Africa, and also to search for possible animal reservoirs of T. gambiense in West Africa.     

Characterization of Leishmania spp. from Kuwait by isoenzyme electrophoresis

Leishmania stocks isolated from skin lesions of 26 patients in Kuwait were compared among themselves and with Leishmania stocks collected from other parts of the Old and New Worlds on the basis of their isoenzyme patterns for seven enzymes by means of thin layer starch gel electrophoresis. The enzymes examined were: alanine aminotransferase (ALAT) E.C.2.6.1.2; aspartate aminotransferase (ASAT) E.C.2.6.1.1; glucose-phosphate isomerase (GPI) E.C.5.3.1.9; glucose-6-phosphate dehydrogenase (G6PD) E.C.1.1.1.49; malate dehydrogenase (MDH) E.C.1.1.1.37; malic enzyme (ME) E.C.1.1.1.40 and phosphoglucomutase (PGM) E.C.2.7.5.1.The isoenzyme patterns obtained fell clearly into 11 groups for the stocks of Leishmania tested. The Kuwaiti stocks separated into six groups. The patterns obtained with 15 Kuwaiti stocks were identical with those obtained with Leishmania tropica major (identified on clinical and geographical characteristics); seven stocks were identical with L. tropica minor similarly identified; three stocks had isoenzyme patterns different from each other and from all other leishmanias examined in this study; and one stock gave isoenzyme patterns identical with those of an L. donovani isolated in the Sudan.The isoenzyme patterns were the same in three stocks classified as L. tropica minor, L. aethiopica and a Leishmania isolated in Baghdad which caused a visceral disease in rats.     

The composition of the Trypanosoma brucei subgroup in nonhuman reservoirs in the Lambwe Valley, Kenya, with particular reference to the distribution of T. rhodesiense

Identification by means of the blood incubation infectivity test (BIIT) of 159 Trypanosoma brucei subgroup strains recently isolated from non-human hosts in the Lambwe Valley, Kenya, has defined the distribution in these hosts of both T. brucei and T. rhodesiense in an endemic sleeping sickness area. The presence of a small third group strongly suggestive of a population intermediate between these two species has also been revealed for the first time.     

Estimating the burden of rhodesiense sleeping sickness during an outbreak in Serere,eastern Uganda

Background  

Zoonotic sleeping sickness, or HAT (Human African Trypanosomiasis), caused by infection with Trypanosoma brucei rhodesiense, is an under-reported and neglected tropical disease. Previous assessments of the disease burden expressed as Disability-Adjusted Life Years (DALYs) for this infection have not distinguished T.b. rhodesiense from infection with the related, but clinically distinct Trypanosoma brucei gambiense form. T.b. rhodesiense occurs focally, and it is important to assess the burden at the scale at which resource-allocation decisions are made.     

Further enzymic characters of Trypanosoma cruzi and their evaluation for strain identification

Starch-gel electrophoresis of 38 enzymes was attempted with extracts of Trypanosoma cruzi culture forms. 18 of the enzymes that gave discrete electrophoretic bands were selected for routine characterization of T. cruzi stocks; the enzymes were: aspartate aminotransferase (E.C. 2.6.1.1, ASAT); alanine aminotransferase (E.C.2.6.1.2, ALAT); phosphoglucomutase (E.C.2.7.5.1, PGM); glucosephosphate isomerase (E.C.5.3.1.9, GPI); malate dehydrogenase (oxaloacetate decarboxylating) (NADP⁺) (E.C.1.1.1.40, ME); glucose 6-phosphate dehydrogenase (E.C.1. 1.1.49, G6PD); malate dehydrogenase (E.C.1.1.1.37, MDH); aconitate hydratase (E.C.4.2.1.3, ACON); isocitrate dehydrogenase (NADP⁺) (E.C.1.1.1.42, ICD); alcohol dehydrogenase (NADP⁺) (E.C.1.1.1.2, ADH); lactate dehydrogenase (E.C.1.1.1.27, LDH); aminopeptidase (cytosol) (E.C.3.4.11.1, PEP); pyruvate kinase (E.C.2.7.1.40, PK); phosphoglycerate kinase (E.C.2.7.2.3, PGK); enolase (E.C.4.2.1.11, ENO); hexokinase (E.C.2.7.1.1, HK); mannosephosphate isomerase (E.C.5.3.1.8, MPI); and glutamate dehydrogenase (E.C. 1.4.1.2, GD). ADH (NADP⁺) in the genus Trypanosoma, and PGK, MPI and ENO, in T. cruzi, were apparently demonstrated for the first time.Between six and 18 enzymes were used to characterize more than 250 T. cruzi stocks, newly isolated from a wide range of sources in northern and central Brazil. All stocks were identified as belonging to T. cruzi zymodemes 1, 2 or 3, as originally defined—that is, by combination of electrophoretic patterns of ASAT, ALAT, PGM, GPI, ME and G6PD. The composite range of results with all enzymes confirmed the presence of three principal T. cruzi zymodemes, but some enzymic characters overlapped between zymodemes and others suggested subgroups within individual zymodemes. Seven (MDH, ACON, LDH, PK, PGK, ENO, HK) of the 18 enzymes did not distinguish the three zymodemes; five (ASAT, PGM, GPI, ICD, PEP) distinguished all three zymodemes; 10 (ASAT, ALAT, PGM, GPI, ME, G6PD, ICD, ADH, PEP, GD) distinguished zymodemes 1 and 2, of which seven plus MPI and eight plus MPI separated zymodemes 1 from 3 and 2 from 3 respectively. T. cruzi stocks were taken from a small area of the natural species distribution; the full range of enzymic characters within the species T. cruzi is expected to be far more complex.The epidemiological distribution of the zymodemes continued to accord with local transmission cycles and supported the hypothesis that distinct T. cruzi strains might be responsible for the enigmatic distribution of chronic Chagas's disease.Some of the difficulties in the empirical selection of new electrophoretic methods and the interpretation of results were presented, and the present and prospective significance of T. cruzi enzymic characters was discussed.Until the stability and genetic basis of T. cruzi enzymic characters are better understood it is recommended that isoenzymic profiles be confirmed routinely, both before and after stocks are used experimentally, as representative of a given zymodeme. A multiple biochemical approach to T. cruzi strain identification is recommended, using characters suitable for a numerical taxonomy.     

Sleeping sickness survey in Musoma district,Tanzania: Further study on the vector role of Glossina

In a follow-up survey of sleeping sickness in the Ikoma-Serengeti region of Tanzania, carried out in May and June 1972, 11,060 G. swynnertoni and 95 G. pallidipes were collected, triturated mainly in batches of 50, and the supernate of each batch was inoculated into cyclophosphamide-treated mice. Nine strains of brucei-subgroup were isolated from G. swynnertoni, which indicated the vector rô1e of the local tsetses in the transmission cycle of infection in this endemic region.All the 9 brucei-subgroup isolates gave consistently negative results by the Blood Incubation Infectivity Test (BIIT) whereas the respective controls gave positive results. However, T. (T.) brucei and particularly T. (T.) rhodesiense standards which were tested simultaneously with the test strains gave inconsistent results. It would seem therefore that the BII test in its present form is not reliable for differentiating between the above two trypanosome species, and so the 9 brucei-subgroup isolates could not be classified further.In the Ikoma-Serengeti region there are no restricted foci of Rhodesian sleeping sickness but rather the various components comprising zoonosis interact sporadically throughout this part of Tanzania. The biocenosis of infection favours intense circulation of the parasites, mainly via the tsetse vectors, among game animals some of which function as natural reservoirs; in this ecological zone man is only an incidental host.     

The testing of proven Trypanosoma brucei and T. rhodesiense strains by the blood incubation infectivity test

The authors describe a simple test (the blood incubation infectivity test) by which Trypanosoma brucei (sensu stricto) may be differentiated from T. rhodesiense without recourse to human volunteers. The method consists in incubating the strain of trypanosome under test for 5 hours at 37°C in vitro in human blood, followed by observation of the effect of this procedure on the strain''s infectivity to rats.     

The use of culture-derived metacyclic trypanosomes in studies on the serological relationships of stocks of Trypanosoma brucei gambiense

Metacyclic trypanosomes of five stocks of Trypanosoma brucei gambiense were produced in vitro in tsetse head-salivary gland explant cultures and used to infect rabbits. Sera were collected from the rabbits and monitored by agglutination tests for antibody production to nine serotype antigens of T. b. gambiense. In the case of a Nigerian stock of T. b. gambiense the sequences of antibody production were found to be similar in animals infected with the stock transmitted by tsetse flies and from culture. Many similarities were also found between the patterns of antibody production in rabbits infected with stocks of T. b. gambiense from Senegal, Nigeria, Zaire and Uganda. The occurrence of similar serotypes in geographically different stocks of T. b. gambiense provides further support for continuing efforts to develop improved serodiagnostic tests for sleeping sickness based on variable trypanosome antigens and to find techniques for immunoprophylaxis.     

A comparison of electrophoretic methods for isoenzyme characterization of trypanosomatids. I: Standard stocks of Trypanosoma cruzi zymodemes from northeast Brazil

An investigation of the relative merits of cellulose acetate electophoresis (CAE) and starch-gel electrophoresis (SGE) was made for 18 enzymes of T. cruzi using standard stocks of zymodemes Z1, Z2 and Z3. The 18 enzymes were those shown previously to be the most suited to routine screening of T. cruzi on starch-gel, namely, aspartate aminotransferase (E.C.2.6.1.1. ASAT); alanine aminotransferase (E.C.2.6.1.2. ALAT); phosphoglucomutase (E.C.-2.7.5.1. PGM); glucosephosphate isomerase (E.C.-5.3.1.9. GPI); malate dehydrogenase (oxaloacetate decarboxylating) (NADP⁺) (E.C. 1.1.1.40. ME); glucose-6-phosphate dehydrogenase (E.C.1.1.1.49 G6PD); malate dehydrogenase (E.C. 1.1.1.37. MDH); aconitate hydratase (E.C.4.2.1.3. ACON); isocitrate dehydrogenase (NADP⁺) (E.C.1.1.1.42. ICD); alcohol dehydrogenase (NADP⁺) (E.C.-1.1.1.2. ADH); lactate dehydrogenase (E.C.1.1.1.27. LDH); aminopeptidase (cytosol) (E.G.3.4.11.1. PEP); pyruvate kinase (E.C.2.7.1.40. PK); phosphoglycerate kinase (E.C.2.7.2.3. PGK); enolase (E.C.4.2.1.11. ENO); hexokinase (E.C.2.7.1.1. HK); mannose phosphate isomerase (E.C.5.3.1.8. MPI); and glutamate dehydrogenase (E.C.1.4.1.2. GD). Of these MDH and PEP failed to give satisfactory patterns on CAE. The cellulose acetate zymograms of the other 16 enzymes were as good as, and in some cases better than, those of starch. Increased CAE resolution for ME and G6PD enabled all three zymodemes to be distinguished. Single CAE bands replaced double SGE bands in some cases, and vice versa, without affecting the zymodeme classification. It was concluded that CAE and SGE were both suitable for isoenzyme characterization and were complementary to each other. CAE characterization of T. cruzi was recommended for use in field work and simple laboratories because of its simplicity, transportability, low maintenance requirements and low capital expenditure. Isoelectric focusing (IEF) of ASAT, ALAT, GPI and PGM on Ampholine PAG plates gave poor results, in our hands, and was considered impracticable for routine characterization of T. cruzi.     

A second note on a high rate of infection of the salivary glands of Glossina morsitans after feeding on a reedbuck infected with Trypanosoma rhodesiense

Another instance of a high rate of infection of the salivary glands of G. morsitans, after feeding on an infected reedbuck, is recorded; and it is suggested that the species of animal may be a determining factor in the rate of infection of the flies. Some other figures are given for comparison. Robertson's views on an endogenous cycle of T. gambiense in monkeys are referred to and the opinion is expressed that they are not applicable to infections with T. rhodesiense. It is suggested that surveys of animals in sleeping sickness areas are needed and also further experiments with single animals and with groups of animals.