Brucei Stocks Research Articles

Genetic Diversity of Trypanosoma evansi in Buffalo based on Internal Transcribed Spacer (ITS) Regions

The nucleotide sequences of 18S rDNA and internal transcribed spacer (ITS) regions were used for studying the relationships of Trypanosoma evansi isolate from a buffalo. The sequences were analyzed and compared to 18S rDNA and the ITS regions of the other Trypanosoma spp. Maximum likelihood phylogenetic trees were constructed using Leishmania major as the outgroup. The tree of 18S rDNA indicated that T. evansi (buffalo B18) isolate was closely related to those of Taiwan and T. brucei stock. The ITS tree showed the genetic diversity among 32 clones of T. evansi (B18) within a single host. This data will be useful for epidemiological and dynamic studies for designing the rational control programs of the disease.

Molecular profiles of Trypanosoma brucei, T. evansi and T. equiperdum stocks revealed by the random amplified polymorphic DNA method.

A total of 20 random primers (10-mers) were used to amplify RAPD markers from the genomic DNA of four Trypanosoma brucei stocks from East and West Africa, four T. evansi stocks from Africa, Asia and South America and one T. equiperdum stock from Asia. Between 65 and 88 reproducible fragments ranging from 0.25 to 2.15 kb were generated from these stocks depending on the stock/primer combination. The similarity coefficient (SC) among the stocks of T. brucei from Kenya, Nigeria, Tanzania and Zambia ranged from 62.9% to 74.0% (average: 67.6%). The SC among the stocks of T. evansi from Kenya, China and Brazil was 76.4%-95.5% (average: 86.4%), while the SC between T. evansi stock from China and Brazil was 95.5%. For T. evansi and T. equiperdum, the SC among the stocks ranged from 81.2% to 94.4% (average: 87.6%). As for the SC among the stocks of T. brucei and T. evansi, it was found to be from 54.7% to 80.3% (average: 68.0%) and the SC among stocks of T. brucei and T. equiperdum was from 59.4% to 76.9% (average: 68.1%). Our results indicate that the stocks of T. evansi from China and from Brazil are more closely related to the stock of T. equiperdum from China than to the stocks of T. evansi isolated from Kenya and to the stocks of T. brucei. In addition, our results further support the hypothesis that T. evansi stocks from China and Brazil could have arisen from a single lineage. The possible evolution of T. evansi and T. equiperdum is also discussed.

Response of a T. b. rhodesiense stock with reduced drug susceptibility in vitro to treatment in mice and cattle

In vivo drug susceptibility tests involving treatment of infected mice and cattle were performed on two trypanosome stocks, a T. brucei brucei and a T. b. rhodesiense, isolated in South Eastern Uganda. The T. b. rhodesiense stock had expressed reduced susceptibility to diminazene aceturate and isometamidium chloride in vitro, while the other, a T. b. brucei stock was susceptible. Diminazene aceturate at 14 mg/kg was not sufficient to cure all T. b. rhodesiense infected mice. Similarly, in the case of isometamidium chloride, 33% of infected mice treated with 2.0 mg/kg drug were not cured. In contrast, mice infected with the susceptible T. b. brucei and treated similarly with either drug were all cured. However, when cattle infected with the T. b. rhodesiense stock, or the susceptible T. b. brucei stock, or a 1:1 mixture of the two were treated with 7 mg/kg diminazene aceturate, they were all cured. Use of diagnostic PCR employing T. brucei specific primers confirmed that although the cattle had acquired infection pre-treatment, no trypanosome DNA amplification signal was demonstrated in the samples collected 60 days post-treatment. The reduced susceptibility of this T. b. rhodesiense, which could be demonstrated in mice as well as in culture, may indicate the existing potential for evolution of resistance in South Eastern Uganda.

Population Structure of Trypanosoma brucei s. l. in Cote D'Ivoire Assayed by Multilocus Enzyme Electrophoresis: Epidemiological and Taxonomical Considerations

Fifty-two Trypanosoma brucei stocks isolated in Côte d'Ivoire from sympatric locations were analyzed by cellulose acetate electrophoresis of isoenzymes. Of 13 genetic loci surveyed, 5 appeared as variable, which made it possible to delimit 12 different zymodemes. The most abundant zymodeme involved stocks isolated from both humans and pigs, which is consistent with the hypothesis that pig is a reservoir of human African trypanosomiasis in Côte d'Ivoire, as already proposed by other authors. Population genetic analysis of the isozyme data indicated a strong linkage disequilibrium, which suggests that genetic recombination is severely restricted in this sample and favors the hypothesis that the trypanosome populations surveyed are basically clonal. Nevertheless, additional studies are required to better estimate the long-term stability of these clones and the possible interference of gene exchange at an evolutionary scale. The results corroborate the hypothesis that a majority of human T. brucei stocks from West Africa correspond to a fairly homogeneous cluster of genotypes (T. brucei gambiense 'Group I', Gibson, 1986).

Genetic diversity and population structure of Trypanosoma brucei: clonality versus sexuality

Genomic fingerprinting by arbitrarily primed PCR was used to analyze the genetic variability among 59 Trypanosoma brucei stocks representing the three T. brucei subspecies isolated from various hosts and different countries in Africa. 14 oligonucleotide primers revealed 355 polymorphic binary characters which were used for phenetic and phylogenetic analysis and to perform recombination tests exploring the linkage disequilibrium in the sample. These was good concordance between arbitrarily primed PCR polymorphisms and isoenzyme data previously collected for many of the same strains [1]. However, the arbitrarily primed PCR typing was more discerning than multilocus enzyme electrophoresis typing. Phenetic and phylogenetic analysis using arbitrarily primed PCR markers did not confirm T. brucei brucei and T. brucei rhodesiense as separate subspecies, but T. brucei gambiense group I was monophyletic, confirming this group as suitable for the subspecies status. With this exception, there were no clear lineages among the sample, other than clustering of East African stocks and clustering of West African stocks. Some features of the phylogenetic analysis suggested that the population structure was not strictly clonal though recombination tests showed linkage disequilibrium, even in the absence of repeated genotypes. While genotypes appear stable enough for tracking in applied studies, sexuality will impact at the evolutionary time scale, and may be more frequent under some ecological conditions. The arbitrarily primed PCR approach should be an effective and simple approach to follow epidemics and to quantify the role of sexuality in T. brucei populations.

Identification of Trypanosoma brucei gambiense group I by a specific kinetoplast DNA probe.

A set of 26 Trypanosoma brucei stocks from various African countries, previously characterized by multilocus enzyme electrophoresis (MLEE) for 18 polymorphic loci, have been selected to be representative of the three T. brucei classic subspecies. The kinetoplast DNA minicircle variable regions from these stocks have been amplified using the polymerase chain reaction (PCR) technique, and hybridized with the amplified variable regions of three T. brucei reference stocks, previously identified as T. brucei brucei, T. brucei gambiense, and T. brucei rhodesiense, respectively. Both T. b. brucei and T. b. rhodesiense probes hybridized only with their own stocks, but the T. b. gambiense probe specifically hybridized with a group of 12 stocks that represented most of the human stocks from West and Central Africa in our sample. These stocks, which appeared as a clearly separable cluster based on previous MLEE analysis, probably correspond to T. brucei gambiense group I. No other stock hybridized with this amplified fragment. Since the T. b. gambiense probe obtained is specific for many isolates that are pathogenic for humans in Central and West Africa, it appears to be a promising tool for epidemiologic and medical surveys.

Identification of Trypanosoma brucei gambiense by PCR amplification of variant surface glycoprotein genes

We have developed a sensitive and specific method to identify Trypanosoma brucei gambiense using the polymerase chain reaction (PCR) to amplify the gene encoding variant surface glycoprotein (VSG) Antat 11.17. The test was capable of distinguishing T. b. gambiense from T. b. brucei in most foci of gambian sleeping sickness and gave positive results with previously well-characterised Type I T. b. gambiense stocks from Ivory Coast, Nigeria, Cameroon, Congo, Zaire and Sudan. The test gave negative results with T. b. rhodesiense from Zambia, Kenya and Uganda, virulent or Type II T. b. gambiense from Ivory Coast and T. b. brucei stocks from East and West Africa. The test was modified for colorimetric detection in dot blot format by using nested biotinylated primers in a two-step reaction. Comparison of DNA sequences of VSG genes from T. b. gambiense and other T. brucei ssp. stocks showed a high level of homology, suggesting recent gene flow.

Experimental immunity against trypanosomiasis in rabbits

Rabbits were immunized with specially prepared subcellular fractions of a cloned Trypanosoma brucei brucei stock 427. One fraction was found to be highly immunoprophylactic in rabbits exposed to the vector fly genus Glossina m. morsitans (tsetse flies) infected with EATRO Trypanosoma brucei brucei 1969. The most immunoprotective fraction IPF A from T. b. brucei 427 gave 75% protection to animals challenged by EATRO T. b. brucei 1969. IPF A also showed the highest IgG antibody titre against the heterologous and homologous strains. In in vitro studies, the sera from these immunized rabbits killed different strains of parasites, T. b. brucei strains 1969 and 999 which were grown in continuous culture of salivary gland cells from G. m. morsitans.

Sequential infection of tsetse flies with Trypanosoma congolense and Trypanosoma brucei

The question whether tsetse flies can be experimentally infected with more than one trypanosome species or strain by sequential feeding was investigated using DNA probe technology to identify directly the small numbers of trypanosomes in the fly gut. Bloodstream form trypanosomes of Trypanosoma congolense or T. brucei ssp. were used for initial infection, followed by sequential feeds using either T. congolense or T. brucei ssp. Midgut trypanosome populations were subsequently analysed by hybridising dot blots with species-specific DNA probes. Two different T. brucei stocks were also fed in succession and the midgut trypanosome populations analysed by molecular karyotype. Contrary to expectations from previous reports, it was comparatively easy to superinfect flies with a second trypanosome species or stock, although the presence of trypanosomes already in the gut did not aid establishment of those incoming. Thus, to develop a mixed infection, a prerequisite for trypanosome mating, flies do not necessarily have to pick up both parental trypanosomes on their first feed.

Multilocus isozyme identification of Trypanosoma brucei stocks isolated in Central Africa: evidence for an animal reservoir of sleeping sickness in Congo

Six Congolese and 3 Zairian Trypanosoma brucei stocks were studied by isozyme cellulose acetate electrophoresis. Twenty isozyme systems were used, of which only 5 showed variability. These 5 polymorphic systems made it possible to identify 5 different zymodemes. Zymodemes isolated from man were recorded both from pig and sheep too, which confirms the results of previous authors. This favors the existence of an animal reservoir of human African trypanosomiasis in the Congo, which could play a role in the transmission of the disease, at least by the maintenance of residual foci.

Electrophoretic analysis of Trypanosoma brucei sub-group stocks from cattle, tsetse and patients from Lambwe Valley, Western Kenya

Trypanosoma brucei stocks (43) isolated from man, cattle and tsetse, all coming from a small area of Lambwe Valley, in Kenya were characterized using isoenzyme analysis. The tsetse isolates revealed the largest number of zymodemes, indicating a lot of heterogeneity among these isolates. Cluster analysis of all the trypanosomes characterized showed that the organisms could be grouped into three distinct trypanosome types; human, cattle and tsetse. The human type was represented in all the three groups. This was not the case either with the cattle nor the tsetse T. brucei type. T. brucei zymodeme which was found to be predominant during the 1980 outbreak of sleeping sickness was not observed this time round. It is suggested that this zymodeme was either unstable and had changed or it had been eliminated during the insecticides ground spraying operations against G. pallidipes. It is concluded that the heterogeneity observed among the fly isolates is an adaptive mechanism for survival of trypanosomes.

Trypanosoma brucei brucei: Expression of drug resistance in vitro

The susceptibility of five Trypanosoma brucei brucei stocks to diminazene aceturate (Berenil) and isometamidium chloride (Samorin) was determined in vivo and in vitro. Two stocks were resistant in mice to both drugs. Differences in drug-induced growth inhibition occurred between drug-resistant and drug-sensitive stocks when trypanosome bloodstream forms were incubated for 24 hr at 37 °C in 4% CO 2 in air in a feeder layer-free culture system. Diminazene concentrations which inhibited growth of trypanosomes by 50% (EC 50) differed more than 50-fold between drug-resistant and drug-sensitive stocks, but there were only minor differences in EC 50 values for isometamidium. However, differences in growth inhibition due to isometamidium occurred when trypanosomes were incubated with drug concentrations (0.001–0.05 μg/ml) below the EC 50 values. Trypanosome stocks which were propagated in vitro in the presence of drugs for a period of 10 days greatly differed in their viability. In contrast to drug-sensitive trypanosomes, drug-resistant stocks could be maintained without loss of viability in the presence of 0.01 μg/ml diminazene aceturate or 0.0001–0.001 μg/ml isometamidium chloride.

Feeder layer-free in vitro assay for screening antitrypanosomal compounds against Trypanosoma brucei brucei and T. b. evansi

A drug-susceptible Trypanosoma brucei brucei stock, a multidrug-resistant T. b. brucei stock, and a T. b. evansi stock resistant to two commercial trypanocides were adapted to a feeder layer-free culture system. Bloodstream forms were grown continuously in a liquid medium at 37 degrees C in 4% CO2 in air. Samples of trypanosome populations in the logarithmic growth phase were incubated with various concentrations of commercial and experimental compounds. Growth inhibition was monitored after a 24-h incubation and quantified by comparing the number of generations between control and drug-treated cultures. Some of the experimental compounds [taxol, formicin B, thioridazine, Ro 15-0216, and DL-alpha-(difluoromethyl)ornithine hydrochloride monohydrate] showed activity against both drug-susceptible and drug-resistant trypanosomes. Other compounds [sinefungin, 1,3,5-triacetylbenzene tris(guanylhydrazone)trimethanesulfonate hydrate, and 9-deazainosine] which inhibited the growth of drug-susceptible trypanosomes showed little or no effect upon drug-resistant parasites. Gossypol, however, had no antitrypanosomal effect on either trypanosome stock. The results obtained in this study correlate with observations obtained from drug screening in mice. The main advantages of the described in vitro screening assay are as follows: (i) lower amounts of drugs are required, (ii) results are obtained more rapidly, (iii) animals are not necessary, and (iv) the method is less labor intensive. These advantages result in an economical and rapid assay for primary drug screening.

A simple and rapid method to initiate Trypanosoma brucei brucei and T. brucei evansi bloodstream form cultures

Initiation of cultures of bloodstream forms of trypanosomes from the Trypanozoon subgenus is an established laboratory procedure. The trypanosomes are usually separated from the blood cells of the donor animal by centrifugation on a density gradient (Hirumi et al., 1977) or by differential centrifugation (Hill et al., 1978; Brun et al., 1981; Zweygarth et al., 1982; Baltz et al., 1985). Isolation on an anion exchange column (Lanham and Godfrey, 1970) has been little used for this purpose. We describe a simple and rapid method for the initiation of trypanosome bloodstream form cultures from infected host blood, avoiding centrifugation and anion exchange column procedures. Two T.b. brucei stocks, CP 271 and CP 547, and two T.b. evansi stocks, CP 1134 and CP 2087, were used. Stock CP 271 was isolated in 1980 from a goat in Matuga/Kenya, stock CP 547 was isolated in 1985 from a naturally infected bovine in Jilib/Somalia. The two T.b. evansi stocks were both isolated from camels, stock CP 1134 in 1979 in Kulal/Kenya, stock CP 2087 in 1980 in the Sudan.

Effect of In vitro Cultivation on the Stability of Resistance of Trypanosoma brucei brucei to Diminazene, Isometamidium, Quinapyramine, and Mel B

A Trypanosoma brucei brucei stock resistant to diminazene aceturate, isometamidium chloride, quinapyramine sulfate, and Mel B was grown in vitro and its response to these drugs compared to that of a drug-sensitive trypanosome stock. There was little if any change of drug sensitivity after in vitro propagation as bloodstream forms for 120, 177, and 275 days and after in vitro transformation of bloodstream forms into procyclic, epimastigote, and finally metacyclic forms. Drug resistance was stable during in vitro maintenance in the absence of drugs in both culture systems. The response of resistant and sensitive T. b. brucei to diminazene in vitro correlated with their sensitivity pattern in vivo. Thus, in vitro techniques can be used to study drug resistance in trypanosomes.

Efficacy of experimental trypanocidal compounds against a multiple drug-resistant Trypanosoma brucei brucei stock in mice.

Several experimental trypanocidal compounds, 6-amidino-2-(4-amidinophenyl)indole dilactate (DAPI), DL-alpha-difluoromethylornithine (DFMO), 2-(dimethylamino)-4'-[(1-methyl-2-nitroimidazole-5-yl) methoxy] aceto-anilide (Ro 15-0216), sinefungin, and triacetylbenzene-tris(guanylhydrazone)trimethanesulfonate hydrate (TBG-MS) were tested to evaluate their ability to cure mouse infections with a multiple drug-resistant Trypanosoma brucei brucei stock (CP 547). This stock proved to be drug-resistant against diminazene aceturate, homidium chloride, isometamidium, quinapyramine sulfate, Mel B, and pentamidine isethionate but fully sensitive to suramin. Compared with the sensitive stock CP 462, the drug-resistant stock CP 547 was completely resistant to 16-fold the curative dose of sinefungin and partially resistant to 4-fold the curative dose of DAPI and to 13-fold the curative dose of TBG-MS, a dose that killed 25% of the animals due to its toxicity. Ro 15-0216 cured all mice when 18 times the usual curative dose level was given. DFMO was equally effective against both stocks.

Characterization of the genes for fructose-bisphosphate aldolase in Trypanosoma brucei

In Trypanosoma brucei stock 427 the glycolytic enzyme fructose-bisphosphate aldolase is encoded by two tandemly linked genes of identical sequence. Such a tandem arrangement of aldolase genes is also present in other T. brucei stocks of unrelated origin. In stock 427 one of the allelic genes is a pseudogene, as a result of a one-nucleotide deletion. The genes code for a polypeptide of 371 amino acids, with a calculated molecular weight of 40 940. The protein that is predicted from the gene sequence has 45–48% positional identity with known aldolase sequences of other organisms. The trypanosomal protein is, however, unique in having a 10 amino-acid insertion near its N-terminus and high number of basic residues, a feature it shares with other glycolytic enzymes of T. brucei. These glycolytic enzymes have in common that they are located in microbody-like organelles, the glycosomes. We have previously proposed that the positively charged residues may be involved in the import of the proteins into the organelles.

Characterization of genes coding for two major metacyclic surface antigens in Trypanosoma brucei.

In African trypanosomes, only a very small fraction of the total repertoire of variable antigen types (VATs) is expressed by the metacyclic form. In Trypanosoma brucei stock EATRO 1125, the VATs AnTat 1.30 and 1.45 are reproducibly present in about 15% and 4% of the metacyclic population, respectively. The genes encoding the corresponding antigens or variant surface glycoproteins (VSGs) are in telomeres of large chromosomes, as are some non-metacyclic VSG genes from the same stock. Their activation mechanism has been studied in seven independent clones, 3 of which, referred to as 'first wave' metacyclic VATs (M-VATs), have been cloned from the first wave of parasitemia after cyclic transmission. In all these clones, activation of the antigen gene was linked to the transposition of an expression linked copy (ELC) of the gene to a telomeric expression site. For first wave M-VATs, this site seems variable, although restricted to large chromosomes, and it can be re-used for VSG gene expression in the bloodstream form. In 'late bloodstream' M-VATs, isolated from established chronic infections, the active expression site, at the end of a 200 kb chromosome, is the one preferred for the expression of late antigen types. It can be concluded that no characteristic feature in the genomic location and expression mechanism can distinguish metacyclic antigen genes from those expressed in the bloodstream forms, although the control of their expression must clearly be different.

Nonvariant antigens limited to bloodstream forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense.

The presence of nonvariant antigens (NVAs) limited to bloodstream forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense was demonstrated for the first time by immunodiffusion and immunoelectrophoresis. Noncloned and cloned populations were employed in preparation of polyclonal antisera in rabbits and of antigens to be used in the immunologic reactions. The NVAs could be shown best in systems in which hyperimmune rabbit sera (adsorbed with procyclic forms to eliminate antibodies against antigens common to bloodstream form and procyclic stages) were reacted with trypanosomes characterized by heterologous variant-specific antigens (VSAs). The NVAs demonstrated in this study are very likely different from the common parts of VSAs. As has been suggested by experiments with living trypanosomes, at least a part of the NVAs appears to be located on the surface of the bloodstream forms. In these experiments involving the quantitative indirect fluorescent antibody test, the amount of fluorescence recorded for the heterologous system, i.e. ETat 5 trypanosomes incubated with anti-AmTat 1.1 serum, equalled approximately 3.0% of the fluorescence emitted by the AmTat 1.1 bloodstream forms treated with their homologous antiserum. Evidently, only small amounts of NVAs are present on the surfaces of T. brucei bloodstream forms. In addition to the NVAs, the electrophoresis results suggested the presence of antigenic differences between procyclic stages belonging to different T. brucei stocks.

Enzyme variation in Trypanosoma brucei spp. I. Evidence for the sub-speciation of Trypanosoma brucei gambiense.

Three groups of stocks of Trypanosoma brucei ssp, defined by the criteria of host, human serum resistance and place of isolation as T. b. gambiense, T. b. brucei (Nigeria) and T. b. brucei/rhodesiense (non-gambiense, Uganda) were screened for electrophoretic variation at 20 enzyme loci. One enzyme (Peptidase C) was found to differentiate all T. b. gambiense stocks from the other T. brucei stocks and, taken together with specific variants of 5 other enzymes, could be used to unambiguously define T. b. gambiense stocks. Using a population genetics approach, the frequencies of the different variants in the three groups of stocks were estimated and from them the average similarity and difference between groups were measured using the statistics of genetic identity (I) and genetic distance (D). These results show firstly, that T. b. gambiense is more different from the other two groups than they are from each other and, secondly, that the values of I and D obtained are consistent with T. b. gambiense constituting a sub- or sibling species of T. brucei. Three domestic animal isolates from Zaire and Cameroun were also screened for enzyme variation and two of these identified as T. b. gambiense, thereby establishing the existence of an animal reservoir host. In parallel, these stocks were tested for human serum resistance resulting in the same identification. Studies of the antigen repertoires and antigen gene structure were carried out by other workers on all the T. b. gambiense stocks reported here and the same conclusions reached as to the identification and ability to discriminate this subspecies from other groups of T. brucei stocks. The results presented here are discussed in relation to other published data on enzyme variation in T. brucei.