Kinetoplast DNA from Trypanosoma vivax and T. congolense

We have analysed kinetoplast DNA (kDNA) of the African trypanosomes Trypanosoma vivax and T. congolense. The maxi-circles from these organisms resemble those of T. brucei in size, but only to a limited extent in sequence as judged from restriction enzyme digests and DNA X DNA hybridization. The kDNA networks of T. vivax have three distinguishing features: they contain the highest maxi-circle concentration of any kDNA (at least twice that of T. brucei); they contain the smallest mini-circles (465 bp) yet found thus far and the width of the kDNA nucleoid in thin sections is correspondingly small (55 nm against 91 nm for T. brucei); they contain a substantial fraction of mini-circle dimers.     

TSIDER1, a short and non-autonomous Salivarian trypanosome-specific retroposon related to the ingi6 subclade

Retroposons of the ingi clade are the most abundant transposable elements identified in the trypanosomatid genomes. Some are long autonomous elements (ingi, L1Tc) while others, such as RIME and NARTc, are short non-coding elements that parasitize the retrotransposition machinery of the active autonomous ones for their own mobilization. Here, we identified a new family of short non-autonomous retroposons of the ingi clade, called TSIDER1, which are present in the genome of Salivarian (African) trypanosomes, Trypanosoma brucei, T. congolense and T. vivax, but absent in the T. cruzi and Leishmania spp. genomes and, as such, TSIDER1 is the only retroposon subfamily conserved at the nucleotide level between African trypanosome species. We identified three TvSIDER1 families within the genome of T. vivax and the high level of sequence conservation within the TvSIDER1a and TvSIDER1b groups suggests that they are still active. We propose that TvSIDER1a/b elements are using the Tvingi retrotransposition machinery, as they are preceded by the same conserved pattern characteristic of the ingi6 subclade, which corresponds to the retroposon-encoded endonuclease binding site. In contrast, TcoSIDER1, TbSIDER1 and TvSIDER1c are too divergent to be considered as active retroposons. The relatively low number of SIDER elements identified in the T. congolense (70 copies), T. vivax (32 copies) and T. brucei (22 copies) genomes confirms that trypanosomes have not expanded short transposable elements, which is in contrast to Leishmania spp. (～2000 copies), where SIDER play a role in the regulation of gene expression.     

Régime alimentaire des glossines et diversité des espèces de trypanosomes dans un foyer actif de trypanosomose humaine africaine au Gabon

Feeding host is an important factor upon which depend the Glossina infection rate and the proportion of different species of trypanosome. Glossina feed both upon animals and humans. In order to identify species of trypanosomes present in the Komo-Mondah focus and to verify whether there is any relationship between the prevalence of sleeping sickness and the feeding habits of Glossina, we have carried out an entomological survey in this focus of Gabon. Flies were dissected and organs were analysed by PCR, while the origin of blood meals was determined by ELISA. Three species of trypanosomes were found: Trypanosoma congolense “forest type” (14/104; 13.46%), Trypanosoma vivax (11/104; 10.58%) and Trypanosoma brucei s.l. (65/104; 62.5%) with 13.46% (14/104) of mixed infections of T. brucei s.l. and T. congolense Glossina palpalis palpalis was caught in all biotopes investigated (91.85%) and was infected by all these species of trypanosomes. Glossina caliginea was not infected and Glossina fuscipes fuscipes was infected by T. brucei s.l. Tsetse flies feed more on animals than on humans in almost all villages, but there was no significant difference between the number of blood meals taken upon these two groups of vertebrates (Chi 2 = 7.43; p > 0.05). A negative correlation was found between the zoophylic/anthropophylic index and the prevalence of HAT. This result is insufficient to conclude that this index can be used as an indicator of the degree of prevalence of HAT. In contrary, the trypanosomian risk seems to be an appropriate indicator of the prevalence of HAT in an area. The identification of the reservoir hosts in this focus would be useful for a good understanding of the HAT epidemiology.     

Recombinant tumor necrosis factor alpha does not inhibit the growth of African trypanosomes in axenic cultures

Mice whose tumor necrosis factor alpha (TNF-alpha) genes were disrupted developed higher levels of parasitemia than wild-type mice following infection with Trypanosoma congolense IL1180 or T. brucei brucei GUTat3.1, confirming the results of earlier studies. To determine whether TNF-alpha directly affects the growth of these and other bloodstream forms of African trypanosomes, we studied the effects of recombinant mouse, human, and bovine TNF-alpha on the growth of two isolates of T. congolense, IL1180 and IL3338, and two isolates of T. brucei brucei, GUTat3.1 and ILTat1.1, under axenic culture conditions. The preparations of recombinant TNF-alpha used were biologically active as determined by their capacity to kill L929 cells. Of five recombinant TNF-alpha lots tested, one lot of mouse TNF-alpha inhibited the growth of both isolates of T. brucei brucei and one lot of bovine TNF-alpha inhibited the growth of T. brucei brucei ILTat1.1 but only at very high concentrations and without causing detectable killing of the parasites. The other lots of mouse recombinant TNF-alpha, as well as human TNF-alpha, did not affect the growth of any of the test trypanosomes even at maximal concentrations that could be attained in the culture systems (3,000 to 15,000 U of TNF-alpha/ml of medium). These results suggest that exogenously added recombinant TNF-alpha generally does not inhibit the growth of African trypanosomes under the culture conditions we used. The impact of TNF-alpha on trypanosome parasitemia may be indirect, at least with respect to the four strains of trypanosomes reported here.     

Physical and transcriptional organization of the ribosomal RNA genes of the savannah-type Trypanosoma congolense

Ribosomal RNA genes have been cloned from the major species of African trypanosomes. Complete nucleotide sequence composition of the small subunit (SSU) and portions of the large subunit (LSU) ribosomal RNA genes was determined for each of these trypanosome species. In contrast to the situation in Trypanosoma brucei, in savannah-type T. congolense the LSU ribosomal RNA is cleaved twice, to generate two additional prominent fragments. This leads to the different profiles observed when the rRNA molecules from these two trypanosome species are resolved in agarose gels. From the nucleotide sequences of the 18S RNA, a phylogenetic tree was derived depicting the relationships among the T. congolense complex of trypanosomes and the other species of trypanosomes.     

Hybridisation with a repetitive DNA probe reveals the presence of small chromosomes in Trypanosoma vivax

We have isolated and cloned a tandemly repeating element from trypanosoma vivax for use as a species-specific DNA probe. The repeat hybridises only with DNA from T. vivax, not with DNA from other Salivarian trypanosome species (T. brucei spp., T. congolense, T. simiae). The monomer of the repeat is approximately 180 bp long and is 64% GC rich. Hybridisation of the cloned fragment with size-fractionated large DNA molecules of 3 T. vivax stocks revealed a band in the position expected for minichromosomes, although these were believed absent in T. vivax. This band migrated to the 100-250 kb area of the gel at 4 different pulse frequencies and also hybridised with a telomeric repeat probe from T. brucei. The band is unlikely to be simply degraded material, since it failed to hybridise with another highly repetitive sequence from T. vivax and was consistently present in different trypanosome preparations. We conclude that T. vivax does possess mini-chromosomes, although possibly only 1 or 2 per cell.     

Ability of trypanosome-infected tsetse flies (Diptera: Glossinidae) to acquire an infection with a second trypanosome species

The epidemiology of human and animal trypanosomiasis is determined to a large extent by the number of infected tsetse flies in a specific area. In the field, a substantial proportion of infected flies carry mixed trypanosome infections. The way in which these tsetse flies acquire a mixed infection is not fully understood. In particular, the susceptibility of tsetse flies to sequential infection with trypanosomes is not well understood. Accordingly, laboratory studies were made of the effects of age and prior infection on the probability of Glossina morsitans morsitans (Westwood) developing an infection of Trypanosoma congolense and Trypanosoma brucei brucei after feeding on infected mice. Results of these experiments clearly showed that 20-30-d-old G. m. morsitans can still pick up and develop a mature infection in the mouthparts/hypopharynx for T. congolense or in the salivary glands for T. b. brucei. However, their ability to acquire infection was significantly lower compared with teneral flies. Furthermore, 20-30-d-old flies that already carry a mature T. congolense or T. b. brucei infection remained at least as susceptible to a secondary trypanosome infection compared with noninfected flies of the same age. The immunological and epidemiological repercussions of those findings are discussed.     

Loop-mediated isothermal amplification for detection of African trypanosomes

While PCR is a method of choice for the detection of African trypanosomes in both humans and animals, the expense of this method negates its use as a diagnostic method for the detection of endemic trypanosomiasis in African countries. The loop-mediated isothermal amplification (LAMP) reaction is a method that amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions with only simple incubators. An added advantage of LAMP over PCR-based methods is that DNA amplification can be monitored spectrophotometrically and/or with the naked eye without the use of dyes. Here we report our conditions for a highly sensitive, specific, and easy diagnostic assay based on LAMP technology for the detection of parasites in the Trypanosoma brucei group (including T. brucei brucei, T. brucei gambiense, T. brucei rhodesiense, and T. evansi) and T. congolense. We show that the sensitivity of the LAMP-based method for detection of trypanosomes in vitro is up to 100 times higher than that of PCR-based methods. In vivo studies in mice infected with human-infective T. brucei gambiense further highlight the potential clinical importance of LAMP as a diagnostic tool for the identification of African trypanosomiasis.     

Effect of isometamidium chloride treatment on susceptibility of tsetse flies (Diptera: Glossinidae) to trypanosome infections

Experiments were conducted to determine the effect of a single isometamidium chloride treatment of teneral tsetse flies, Glossina morsitans morsitans Westwood (Diptera: Glossinidae), on the subsequent susceptibility to an infection with Trypanosoma congolense or Trypanosoma brucei brucei. Flies were offered a first bloodmeal on sterile gamma-irradiated defibrinated bovine blood that contained either 10 or 100 microg ofisometamidium chloride/ml. Treated flies were subsequently infected with T. congolense IL 1180 or T. b. brucei AnTAR1 on day 3, 5, 10, or 20 posttreatment. To determine the effect of a single treatment with isometamidium chloride at 10 microg/ml on the fly's susceptibility to infection with isometamidium chloride-resistant trypanosome strains, treated flies were infected with one of two resistant isogenic T. congolense IL 1180 strains 3 d after the first feed. Results showed that a single isometamidium chloride treatment at 10 microg/ml blood sufficed to reduce significantly the fly's subsequent susceptibility to infection. Only 6.8% of the flies that were treated with isometamidium chloride developed a mature infection with T. congolense in the mouthparts compared with 34.3% of the control group. None of the flies that were administered isometamidium chloride and subsequently infected on day 3 or 6 with T. b. brucei developed a metacyclic infection in the salivary glands compared with 22.7% of the control flies. Likewise for the resistant T. congolense strains, a single treatment with isometamidium chloride significantly reduced the subsequent susceptibility to infection (6.5 and 33.5% of flies with metacyclic infections for treated and untreated flies, respectively). In practice and with respect to the release of sterile male flies to eradicate an isolated tsetse fly population, our results show that administering isometamidium chloride during the first bloodmeal (and before release) would significantly reduce the ability of these released males to transmit trypanosomes.     

Cloning and analysis of Trypanosoma (Nannomonas) congolense ILNat 2.1 VSG gene

Genes encoding various Trypanosoma (Trypanozoon) brucei variable surface glycoproteins (VSGs) show considerable conservation among different members of this species, known as isotypes. The occurrence of isotypes in other salivarian trypanosomes has not been well documented. We have cloned sequences encoding Trypanosoma (Nannomonas) congolense ILNat 2.1 VSG, and used it in DNA blot hybridization analyses of this and other T. congolense clones originating from geographically separate regions of East Africa. The data indicate that the expression of ILNat 2.1 VSG gene proceeds by duplicative transposition resulting in the presence of an extra expression-linked copy in the expressing clones examined. Furthermore the ILNat 2.1 VSG gene sequence is absent or has greatly diverged, in all other T. (N.) congolense clones that belong to different serodemes. This suggests that some T. (N.) congolense VSGs may be limited to their respective antigen repertoires. The data are discussed in the light of their implications for antigenic variation in T. (N.) congolense, and parasite epidemiology.     

Glycosyl-sn-1,2-dimyristylphosphatidylinositol is the membrane anchor for Trypanosoma equiperdum and T. (Nannomonas) congolense variant surface glycoproteins

We have analysed the structures of the Trypanosoma (Nannomonas) congolense and T. equiperdum variant surface glycoprotein (VSG) membrane anchors. Myristic acid uptake, phospholipase treatment, and nitrous acid deamination showed that, for each species, the anchor is glycosyl-sn-1,2-dimyristylphosphatidylinositol, as has been previously described for T. brucei. Osmotic lysis of these trypanosomes resulted in the release of soluble VSG, lacking fatty acid. In both species and in T. evansi, an endogenous phospholipase C, which cleaved diacylglycerol from membrane form VSG, was identified.     

Immune depression in African trypanosomiasis: the role of antigenic competition.

The capacity of trypanosome-infected cattle to mount an immune response to a simultaneous or subsequent challenge with other trypanosomes was investigated using various clones of Trypanosoma congolense and T. brucei. In animals infected simultaneously with equal numbers of trypanosomes of two different clones, the variant-specific antibody response to one clone was severely depressed while the response to the other was not affected. In cattle infected with one clone and then subsequently challenged severely depressed depending on the time interval between the two inoculations. These observations were consistent regardless of whether the clones of trypanosomes used were derived from the same or different species. The characteristics of these responses would suggest that the inability of trypanosome-infected cattle to respond well to a simultaneous or subsequent challenge with other trypanosomes or other antigens may be due to antigenic competition.     

Resistance of cattle to tsetse-transmitted challenge with Trypanosoma brucei or Trypanosoma congolense after spontaneous recovery from syringe-passaged infections

Groups of cattle were inoculated intravenously with cloned populations of bloodstream forms of Trypanosoma brucei or Trypanosoma congolense. All five steers infected with T. brucei ILTat 2.1 and six of the eight steers infected with T. congolense IL 13-E14 became aparasitemic within 16 and 32 weeks postinfection, respectively. Examination of sera from animals infected with T. brucei by indirect immunofluorescence and neutralization assays revealed the presence of antibodies against all the metacyclic variable antigen types (VATs) of the infecting clone. The neutralizing capacity of the sera increased with the course of infection from 1:10 at 2 months to 1:100 at 3 to 4 months postinfection. The recovered animals were completely immune to challenge by Glossina morsitans subsp. centralis infected with clone IL Tat 2.1, which had initiated the infection, as well as with another clone (IL Tat 2.2) belonging to the same serodeme, but they were susceptible to a tsetse-transmitted heterologous challenge with isolate STIB 367-H. Similar results were obtained with sera from T. congolense IL 13-E14-infected steers. The six steers infected with a different T. congolense ILNat 3.1 clone did not recover spontaneously; however, 2 months postinfection, sera from five of them also contained neutralizing antibodies against ILNat 3.1 metacyclic VATs. These results indicate that some of the bloodstream VATs that arise during the course of a chronic infection possess surface epitopes in their variable surface glycoproteins that are identical to those of the metacyclic VATs. It is suggested that in chronic infection, the infecting trypanosomes could exhaust their VAT repertoire, including those that cross-react with metacyclics, thereby leading to both "self-cure" and subsequent immunity to homologous cyclically transmitted challenge.     

Surface carbohydrates of procyclic forms of African trypanosomes studied using fluorescence activated cell sorter analysis and agglutination with lectins

Living culture form procyclics of Trypanosoma brucei brucei, T.b. rhodesiense, T.b. gambiense, T. congolense and T. simiae were tested for binding of eight different lectins. The binding of fluorescein isothiocyanate (FITC)-conjugated lectins was measured using a fluorescence activated cell sorter (FACS) and by agglutination with unlabelled lectins. Five of the lectins failed to bind to any of the procyclic organisms in both tests. All parasites bound concanavalin A (Con A) and all T.b. brucei, T.b. rhodesiense and T. congolense procyclics bound Ricinus communis agglutinin 120 (RCA) and wheat germ agglutinin (WGA). Trypanosoma b. gambiense procyclics failed to bind RCA and thus could be easily discriminated from other subspecies of T. brucei. Similarly, T. simiae did not bind WGA, unlike T. congolense, the other species of the genus Nannomonas. All positive reactions were inhibited by 0.2 M concentrations of the relevant sugars. The results indicate that all species and subspecies of the procyclic culture forms tested have surface-exposed structures resembling alpha-D-mannose moieties and that T.b. brucei, T.b. rhodesiense and T. congolense have surface-exposed molecules resembling D-galactose and N-acetyl D-glucosamine (or sialic acid) moieties. Molecules resembling D-galactose and N-acetyl D-glucosamine residues are absent or inaccessible in T.b. gambiense and T. simiae respectively. A group of T. congolense clones of parasite stocks isolated at Kilifi on the Kenyan coast showed quantitatively different binding of RCA when compared to the other T. congolense clones tested indicating that these organisms differ in surface carbohydrate structure.     

Antigenicity and protective effects of type 3 pneumococcal polysaccharide in rats.

Total hemolytic complement and C3 levels were found to drop to 6.25% and 50% of preinfection levels, respectively, during trypanosome infections. Chemotherapeutic elimination of the trypanosomes with Berenil led to recovery of preinfection levels within 7 days and 11 days when cattle infected with Trypanosome congolense and Trypanosoma vivax, respectively, were treated 37 days after onset of infection. Recovery was slower in T. vivax-infected cattle treated on day 50. Berenil treatment had no effect on complement levels in control animals. The possible causes and implications of these low complement levels in bovine trypanosomiasis are discussed.     

Glycosylphosphatidylinositol-anchored surface molecules of Trypanosoma congolense insect forms are developmentally regulated in the tsetse fly.

Procyclic culture forms of Trypanosoma congolense have been shown to express a glutamic acid/alanine-rich protein (GARP) on their surface. By labelling T. congolense procyclic culture forms with glycosylphosphatidylinositol (GPI) precursors, we show that GARP is bound to the membrane by a GPI anchor and demonstrate the presence of two additional GPI-anchored surface molecules of 24-34 and 58 kDa that are abundantly expressed. The 24-34 kDa molecule, which is recognised by monoclonal antibodies that bind to the surface of living trypanosomes, is resistant to proteolysis, suggesting that it consists (predominantly) of non-proteinaceous material. We have therefore named it protease-resistant surface molecule (PRS). In common with the EP and GPEET procyclins of Trypanosoma brucei, the relative expression of the T. congolense GPI-anchored molecules changes during parasite development in the tsetse fly. PRS is abundantly expressed by procyclic trypanosomes in the midgut shortly after infection, but is downregulated in established midgut forms and completely absent from the epimastigote form in the proboscis. In contrast, GARP is downregulated in parasites in the tsetse fly midgut, but upregulated in the epimastigote form. Unexpectedly, 14 days post-infection, procyclic forms frequently are negative for both PRS and GARP, suggesting that they might be expressing another stage-specific surface antigen at this point in the life cycle.     

Variant surface glycoproteins of Trypanosoma congolense bloodstream and metacyclic forms are anchored by a glycolipid tail

The variant surface glycoproteins (VSGs) of both metacyclic and bloodstream forms of Trypanosoma congolense are shown to be anchored to the plasma membrane through a glycolipid similar to that found in Trypanosoma brucei. Release of soluble VSG from both metacyclic and bloodstream forms is associated with the exposure of an antigenic determinant homologous to the cross-reacting determinant of T. brucei VSGs. Release of soluble VSG of T. congolense can be achieved by lysates of both bloodstream and metacyclic forms of T. congolense, by lysates of T. brucei bloodstream forms, but not by lysates of procyclic forms.     

Detection of Trypanosoma congolense and T. brucei subspecies in cattle in Zambia by polymerase chain reaction from blood collected on a filter paper

To facilitate epidemiology studies of African trypanosomiasis in cattle in Zambia, we adapted a polymerase chain reaction (PCR) method using blood spotted on filter papers. For easy preparation of template DNA from the dried blood, we adapted a simple DNA extraction method using Chelex-100, an anion-exchange resin. Using primers directed for repetitive nuclear DNA sequences, species-specific DNA amplifications were detected from the blood of rats infected with Zambian isolates of T. congolense and T. brucei subspecies. The method was sensitive enough to detect a single trypanosome for both species. In the Eastern Province of Zambia, 240 cattle were examined for motile flagellates in the buffy coat by the microhematocrit method, and 100 of them were positive for the test. These 100 animals were further examined by thin blood smears and PCR for species identification. The thin blood smear revealed 62 and 14 animals with T. congolense and T. brucei subspecies infection, respectively, whereas the PCR detected 73 of the former and 38 of the latter species. These results indicate that dried blood spots on filter papers are a useful source of DNA for detection of African trypanosomes by PCR.     

Suppression of antibody response to Leptospira biflexa and Brucella abortus and recovery from immunosuppression after Berenil treatment.

Zebu cattle infected with either Trypanosoma congolense EATRO 1800 or Trypanosoma vivax EATRO 1721 had suppressed humoral immune responses to Leptospira biflexa injected intravenously and to attenuated Brucella abortus injected subcutaneously. T. congolense infections were more suppressive than T. vivax infections. In cattle infected with T. vivax, the suppression of immune responses to both bacterial immunogens was abrogated when the animals were treated with Berenil at the time of antigen administration. In cattle infected with T. congolense, simultaneous Berenil treatment at the time of vaccination abolished the suppression of immune response to L. biflexa, and lessened but did not abrogate the suppression of immune response to B. abortus.     

Trypanosoma congolense infections: antibody-mediated phagocytosis by Kupffer cells

Immunohistochemical double-label technique was used to detect trypanosomal antigen in macrophages. Immunoglobulin (Ig)M as well as IgG2a monoclonal antibodies (mAb) specific for the variant surface glycoprotein (VSG) mediated phagocytosis of Trypanosoma congolense variant antigenic type (VAT) TC13 by macrophages [bone marrow-derived macrophage cell line from BALB/c (BALB.BM)] in vitro. Administration of these IgM or IgG2a antibodies to BALB/c mice 30 min after injection of 3 x 10(8) T. congolense mediated phagocytosis of trypanosomes by Kupffer cells of the liver within 1 h. Plasma levels of the monokines interleukin (IL)-1beta, IL-10, and IL-12p40 were significantly increased 6-48 h after phagocytosis. In BALB/c mice infected with 10(3) T. congolense, a small degree of phagocytosis of trypanosomes by Kupffer cells, mediated by actively synthesized antibodies, was detected as early as 5 days after infection. Phagocytosis of trypanosomes was dramatically enhanced on day 6. Concomitantly, the Kupffer cells trippled in size. In BALB/c mice infected for 6 days, treatment with IgM or IgG2a mAb specific for T. congolense VSG led to clearance of VAT TC13 parasitemia but did not prevent death at the second parasitemia of a different VAT. We conclude that IgM as well as IgG antibody mediate phagocytosis of trypanosomes by Kupffer cells.