Congopain from Trypanosoma congolense: drug target and vaccine candidate

Trypanosomes are the etiological agents of human sleeping sickness and livestock trypanosomosis (nagana), which are major diseases in Africa. Their cysteine proteases (CPs), which are members of the papain family, are expressed during the infective stages of the parasites' life cycle. They are suspected to act as pathogenic factors in the mammalian host, where they also trigger prominent immune responses. Trypanosoma congolense, a major pathogenic species in livestock, possesses at least two families of closely related CPs, named CP1 and CP2. Congopain, a CP2-type of enzyme, shares structural and functional resemblances with cruzipain from T. cruzi and with mammalian cathepsin L. Like CPs from other Trypanosomatids, congopain might be an attractive target for trypanocidal drugs. Here we summarise the current knowledge in the two main areas of research on congopain: first, the biochemical properties of congopain were characterised and its substrate specificity was determined, as a first step towards drug design; second, the possibility was being explored that inhibition of congopain by host-specific antibodies may mitigate the pathology associated with trypanosome infection.     

Bovine trypanotolerance: A natural ability to prevent severe anaemia and haemophagocytic syndrome?

Trypanotolerance is the capacity of certain West-African, taurine breeds of cattle to remain productive and gain weight after trypanosome infection. Laboratory studies, comparing Trypanosoma congolense infections in trypanotolerant N'Dama cattle (Bos taurus) and in more susceptible Boran cattle (Bos indicus), confirmed the field observations. Experiments using haemopoietic chimeric twins, composed of a tolerant and a susceptible co-twin, and T cell depletion studies suggested that trypanotolerance is composed of two independent traits. The first is a better capacity to control parasitaemia and is not mediated by haemopoietic cells, T lymphocytes or antibodies. The second is a better capacity to limit anaemia development and is mediated by haemopoietic cells, but not by T lymphocytes or antibodies. Weight gain was linked to the latter mechanism, implying that anaemia control is more important for survival and productivity than parasite control. Anemia is a marker for a more complex pathology which resembles human haemophagocytic syndrome: hepatosplenomegaly, pancytopenia and a large number of hyperactivated phagocytosing macrophages in bone marrow, liver and other tissues. Thus, mortality and morbidity in trypanosome-infected cattle are primarily due to self-inflicted damage by disproportionate immune and/or innate responses. These features of bovine trypanotolerance differ greatly from those in murine models. In mice, resistance is a matter of trypanosome control dependent on acquired immunity. However, a model of anaemia development can be established using C57BL/6J mice. As in cattle, the induction of anaemia was independent of T cells but its development differed with different trypanosome strains. Identification of the molecular pathways that lead to anaemia and haemophagocytosis should allow us to design new strategies to control disease.     

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

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

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

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

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

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

Factors associated with acquisition of human infective and animal infective trypanosome infections in domestic livestock in Western Kenya

Background

Trypanosomiasis is regarded as a constraint on livestock production in Western Kenya where the responsibility for tsetse and trypanosomiasis control has increasingly shifted from the state to the individual livestock owner. To assess the sustainability of these localised control efforts, this study investigates biological and management risk factors associated with trypanosome infections detected by polymerase chain reaction (PCR), in a range of domestic livestock at the local scale in Busia, Kenya. Busia District also remains endemic for human sleeping sickness with sporadic cases of sleeping sickness reported.

Results

In total, trypanosome infections were detected in 11.9% (329) out of the 2773 livestock sampled in Busia District. Multivariable logistic regression revealed that host species and cattle age affected overall trypanosome infection, with significantly increased odds of infection for cattle older than 18 months, and significantly lower odds of infection in pigs and small ruminants. Different grazing and watering management practices did not affect the odds of trypanosome infection, adjusted by host species. Neither anaemia nor condition score significantly affected the odds of trypanosome infection in cattle. Human infective Trypanosoma brucei rhodesiense were detected in 21.5% of animals infected with T. brucei s.l. (29/135) amounting to 1% (29/2773) of all sampled livestock, with significantly higher odds of T. brucei rhodesiense infections in T. brucei s.l. infected pigs (OR = 4.3, 95%CI 1.5-12.0) than in T. brucei s.l. infected cattle or small ruminants.

Conclusions

Although cattle are the dominant reservoir of trypanosome infection it is unlikely that targeted treatment of only visibly diseased cattle will achieve sustainable interruption of transmission for either animal infective or zoonotic human infective trypanosomiasis, since most infections were detected in cattle that did not exhibit classical clinical signs of trypanosomiasis. Pigs were also found to be reservoirs of infection for T. b. rhodesiense and present a risk to local communities.     

The effect of starvation on the susceptibility of teneral and non-teneral tsetse flies to trypanosome infection

Transmission of vector-borne diseases depends largely on the ability of the insect vector to become infected with the parasite. In tsetse flies, newly emerged or teneral flies are considered the most likely to develop a mature, infective trypanosome infection. This was confirmed during experimental infections where laboratory-reared Glossina morsitans morsitans Westwood (Diptera: Glossinidae) were infected with Trypanosoma congolense or T. brucei brucei. The ability of mature adult tsetse flies to become infected with trypanosomes was significantly lower than that of newly emerged flies for both parasites. However, the nutritional status of the tsetse at the time of the infective bloodmeal affected its ability to acquire either a T. congolense or T. b. brucei infection. Indeed, an extreme period of starvation (3-4 days for teneral flies, 7 days for adult flies) lowers the developmental barrier for a trypanosome infection, especially at the midgut level of the tsetse fly. Adult G. m. morsitans became at least as susceptible as newly emerged flies to infection with T. congolense. Moreover, the susceptibility of adult flies, starved for 7 days, to an infection with T. b. brucei was also significantly increased, but only at the level of maturation of an established midgut infection to a salivary gland infection. The outcome of these experimental infections clearly suggests that, under natural conditions, nutritional stress in adult tsetse flies could contribute substantially to the epidemiology of tsetse-transmitted trypanosomiasis.     

The effect of trypanosomosis on pregnancy in trypanotolerant Orma Boran cattle

A study was undertaken to investigate the influence of trypanosomosis on the outcome of pregnancy in trypanotolerant Orma Boran (Bos indicus) exposed to natural tsetse challenge in an area of Kenya infested predominantly with Glossina pallidipes. Of 73 pregnant Orma heifers, 58 (79.5%) produced live calves at term, 13 (17.8%) aborted and 2 (2.7%) died of trypanosomosis. Of the 71 surviving animals, 22 (31%) were infected with Trypanosoma vivax , 21 (29.6%) T. congolense, and 26 (36.6%) had mixed infections with T. vivax and T. congolense. These results suggest that in areas of high trypanosomosis risk reproductive function is affected even in trypanotolerant cattle, and that both T. vivax and T. congolense can be responsible for the abortions observed in the field. It is suggested that maintenance of pregnancy in the face of trypanosome challenge was dependent on individual variation among the Orma cattle, but as challenge increased beyond the limits of effectiveness of trypanotolerance, disruption of pregnancy occurred.     

Serological characterizations of tandem repeat proteins for detection of African trypanosome infection in cattle

Serological diagnosis is a useful method to detect African trypanosome infection in livestock animals. Currently available serological tests utilize whole parasites or crude antigens, and recombinant antigens may improve reproducibility/standardization and reduce production costs. With a goal of identifying such recombinant proteins, we computationally identified proteins with tandem repeat (TR) domain from the parasite proteomes and evaluated their potential for serological diagnosis of African trypanosome infections in cattle. Among those tested, Tbg4 demonstrated the best performance with 92% sensitivity, followed by TbbGM6 (85%), TcoGM6 (85%), Tbg2 (65%) and Tbg5 (65%). Although further evaluations such as investigating cross-reactivity to other infections are needed, our data indicate the potential of these antigens for detection of African trypanosome infection in cattle.     

G protein-coupled receptor/arrestin3 modulation of the endocytic machinery

Nonvisual arrestins (arr) modulate G protein-coupled receptor (GPCR) desensitization and internalization and bind to both clathrin (CL) and AP-2 components of the endocytic coated pit (CP). This raises the possibility that endocytosis of some GPCRs may be a consequence of arr-induced de novo CP formation. To directly test this hypothesis, we examined the behavior of green fluorescent protein (GFP)-arr3 in live cells expressing beta2-adrenergic receptors and fluorescent CL. After agonist stimulation, the diffuse GFP-arr3 signal rapidly became punctate and colocalized virtually completely with preexisting CP spots, demonstrating that activated complexes accumulate in previously formed CPs rather than nucleating new CP formation. After arr3 recruitment, CP appeared larger: electron microscopy analysis revealed an increase in both CP number and in the occurrence of clustered CPs. Mutant arr3 proteins with impaired binding to CL or AP-2 displayed reduced recruitment to CPs, but were still capable of inducing CP clustering. In contrast, though constitutively present in CPs, the COOH-terminal moiety of arr3, which contains CP binding sites but lacks receptor binding, did not induce CP clustering. Together, these results indicate that recruitment of functional arr3-GPCR complexes to CP is necessary to induce clustering. Latrunculin B or 16 degrees C blocked CP rearrangements without affecting arr3 recruitment to CP. These results and earlier studies suggest that discrete CP zones exist on cell surfaces, each capable of supporting adjacent CPs, and that the cortical actin membrane skeleton is intimately involved with both the maintenance of existing CPs and the generation of new structures.     

Control of Glossina longipennis (Diptera: Glossinidae) by insecticide-treated targets at Galana ranch, Kenya, and confirmation of the role of G. longipennis as a vector of cattle trypanosomiasis

Glossina longipennis Corti was studied in Galana Ranch, Kenya over a four year period, in two areas (Tank E and Lali) where the species was abundant and other species were absent or scarce. There was active transmission of trypanosomiasis to cattle in both areas, the parasite species being Trypanosoma vivax Ziemann and T. congolense Broden. Mean infection rates of the G. longipennis were 1.1% and 0. 55% for T. vivax and T. congolense respectively at Tank E, and 0.88% and 0.15% at Lali. Experimental transmission studies showed that cattle in fly-proof enclosures challenged with wild G. longipennis collected from Galana became infected with both trypanosome species. A tsetse control operation in one area (Tank E) using targets impregnated with deltamethrin in an oil formulation reduced the population of G. longipennis by 98% over one year, despite evidence of re-invasion. Populations of G. longipennis in the other area (Lali) were relatively stable over the whole study period. The effect of tsetse control on the incidence of cattle trypanosomiasis at Tank E was less clear than that on tsetse numbers, probably due to the lack of a sustained reduction in tsetse numbers. However, a significant relationship was demonstrated between fortnightly incidence measurements and electric net catches of G. longipennis at Tank E. A further significant predictor of incidence was rainfall in the previous four to seven weeks. This study confirms the importance of G. longipennis as a vector of bovine trypanosomiasis in areas where it is the predominant tsetse present.     

Comparison of the susceptibility of different Glossina species to simple and mixed infections with Trypanosoma (Nannomonas) congolense savannah and riverine forest types

Abstract Teneral Glossina morsitans mositans, G.m.submorsitans, G.palpalis gambiensis and G.tachinoides were allowed to feed on rabbits infected with Trypanosoma congolense savannah type or on mice infected with T.congolense riverine-forest type. The four tsetse species and subspecies were also infected simultaneously in vitro on the blood of mice infected with the two clones of T.congolense via a silicone membrane. The infected tsetse were maintained on rabbits and from the day 25 after the infective feed, the surviving tsetse were dissected in order to determine the infection rates.
Results showed higher mature infection rates in morsitans-gwup tsetse flies than in palpalis-group tsetse flies when infected with the savannah type of T.congolense. In contrast, infection rates with the riverine-forest type of T.congolense were lower, and fewer flies showed full development cycle. The intrinsec vectorial capacity of G.m.submorsitans for the two T.congolense types was the highest, whereas the intrinsic vectorial capacity of G.p.gambiensis for the Savannah type and G.m.morsitans for the riverine-forest type were the lowest. Among all tsetse which were infected simultaneously with the two types of T.congolense , the polymerase chain reaction detected only five flies which had both trypanosome taxa in the midgut and the proboscis. All the other infections were attributable to the savannah type.
The differences in the gut of different Glossina species and subspecies allowing these two sub-groups of T.congolense to survive better and undergo the complete developmental cycle more readily in some species than other are discussed.     

Erythrophagocytosis of desialylated red blood cells is responsible for anaemia during Trypanosoma vivax infection

Trypanosomal infection‐induced anaemia is a devastating scourge for cattle in widespread regions. Although Trypanosoma vivax is considered as one of the most important parasites regarding economic impact in Africa and South America, very few in‐depth studies have been conducted due to the difficulty of manipulating this parasite. Several hypotheses were proposed to explain trypanosome induced‐anaemia but mechanisms have not yet been elucidated. Here, we characterized a multigenic family of trans‐sialidases in T. vivax, some of which are released into the host serum during infection. These enzymes are able to trigger erythrophagocytosis by desialylating the major surface erythrocytes sialoglycoproteins, the glycophorins. Using an ex vivo assay to quantify erythrophagocytosis throughout infection, we showed that erythrocyte desialylation alone results in significant levels of anaemia during the acute phase of the disease. Characterization of virulence factors such as the trans‐sialidases is vital to develop a control strategy against the disease or parasite.     

Trying to predict and explain the presence of African trypanosomes in tsetse flies.

Trypanosome infections identified by polymerase chain reaction on field-caught tsetse flies from various locations were analyzed with respect to factors intrinsic and extrinsic to the trypanosome-tsetse association. These factors were then simultaneously analyzed using artificial neural networks (ANNs) and the important factors were identified to predict and explain the presence of trypanosomes in tsetse. Among 4 trypanosome subgroups (Trypanosoma brucei s.l., T. congolense of the 'savannah' and of the 'riverine-forest' types, and T. simiae), the presence of the 2 types of T. congolense was predictable in more than 80% of cases, suggesting that the model incorporated some of the key variables. These 2 types of T. congolense were significantly associated in tsetse. Among all the examined factors, it was the presence of T. congolense savannah type that best explained the presence of T. congolense riverine forest type. One possible biological mechanism would be 'hitchhiking,' as previously suspected for other parasites. The model could be improved by adding other important variables to the trypanosome tsetse associations.     

Tsetse immunity and the transmission of trypanosomiasis

Cyclical transmission of African trypanosomes - Trypanosoma congolense and subspecies of T. brucei - depends on their uptake by and development within their tsetse fly vectors. Tsetse susceptibility to such trypanosome infection seems to be controlled by maternally inherited rickettsia-like organisms (RLOs) (Fig. 1) and it now seems that the RLOs may exert this effect by controlling midgut lectins in the fly. Ian Maudlin and Susan Welburn explain the latest findings.     

A comparison of African buffalo, N''Dama and Boran cattle as reservoirs of Trypanosoma congolense for different Glossina species

Teneral Glossina morsitans centralis Machado were fed on the flanks of the African buffalo (Syncerus caffer Sparrman), N'Dama (Bos taurus L.) or Boran (Bos indicus L.) cattle infected with Trypanosoma congolense Broden. The infected tsetse were maintained on rabbits and on day 30 after the infected feed, the surviving tsetse were dissected to determine the infection rates. The mean infection rates (% +/- SE) in the midgut of tsetse fed on buffalo, N'Damas and Borans were 23.5 +/- 3.3, 31.6 +/- 2.7 and 33.7 +/- 4.6, respectively. The differences were not significant. However, the mean mature infection rate in tsetse fed on the buffalo (13.2 +/- 2.1%) was significantly lower compared to the rates in tsetse fed on the N'Dama (20.4 +/- 1.4) or the Boran cattle (21.4 +/- 1.1). When groups of teneral G.m.centralis, G.pallidipes Austen, G.p.gambiensis Vanderplank, G.f.fuscipes Newstead, G.brevipalpis Newstead and G.longipennis Corti were fed simultaneously on either an infected buffalo, an N'Dama or a Boran steer, the mature infection rates ranged from 0 to 16.1%. Irrespective of the host species used, the T.congolense infection rate was highest in G.m.centralis, lowest in the palpalis and fusca group tsetse, with G.pallidipes being intermediate. Nevertheless, the trypanoresistant African buffalo and N'Dama may serve as reservoirs of T.congolense as can trypanosusceptible Boran cattle.     

Transition models to assess risk factors for new and persistent trypanosome infections in cattle-analysis of longitudinal data from the Ghibe Valley, southwest Ethiopia

The objective of this study was to apply transition models to distinguish between factors associated with both incident and persistent trypanosome infections. Data collected from 1561 cattle were analyzed from a long-term study involving 8 herds in which both trypanosome infections (a total of 56,931 cattle sampling-months) and tsetse (Glossina spp.) challenge were monitored monthly from March 1986 to March 1998. Both pour-on and insecticide-target tsetse control programs and mass treatment with diminazene aceturate before tsetse control were associated with significant decreases in both incidence and persistence of trypanosome infection relative to noncontrol periods, as were seasonal and sex effects. The magnitudes of the effects were, however, often different for new and persistent infections. For persistence of infection, there were 2 trends. In general, the duration of infection increased during the study, despite the regular treatment with diminazene aceturate. The transition model had 2 major benefits. The first was to identify an increasing duration of infections with time, taking into account other factors associated with increasing infection risk. The second was to highlight different patterns in the effects of certain factors on new and persistent trypanosome infections.     

Nutritional stress affects the tsetse fly's immune gene expression

Tsetse-transmitted trypanosomiasis poses a serious threat to human and animal health in sub-Saharan Africa. The majority of tsetse flies ( Glossina spp.) in a natural population will not develop a mature infection of either Trypanosoma congolense or Trypanosoma brucei sp. because of refractoriness, a phenomenon that is affected by different factors, including the tsetse fly's immune defence. Starvation of tsetse flies significantly increases their susceptibility to the establishment of a trypanosome infection. This paper reports the effects of nutritional stress (starvation) on (a) uninduced baseline levels of gene expression of the antimicrobial peptides attacin, defensin and cecropin in the tsetse fly, and (b) levels of expression induced in response to bacterial ( Escherichia coli ) or trypanosomal challenge. In newly emerged, unfed tsetse flies, starvation significantly lowers baseline levels of antimicrobial peptide gene expression, especially for attacin and cecropin. In response to trypanosome challenge, only non-starved older flies showed a significant increase in antimicrobial peptide gene expression within 5 days of ingestion of a trypanosome-containing bloodmeal, especially with T. brucei bloodstream forms. These data suggest that a decreased expression of immune genes in newly hatched flies or a lack of immune responsiveness to trypanosomes in older flies, both occurring as a result of fly starvation, may be among the factors contributing to the increased susceptibility of nutritionally stressed tsetse flies to trypanosome infection.     

Trypanosoma congolense: paraoxonase 1 prolongs survival of infected mice

In vitro studies have suggested that a fraction of human high density lipoprotein (HDL), termed trypanosome lysis factor (TLF), can protect against trypanosome infection. We examined the involvement of two proteins located in the TLF fraction, apolipoprotein A-II (apoA-II) and paraoxonase 1 (PON1), against trypanosome infection. To test whether PON1 is involved in trypanosome resistance, we infected human PON1 transgenic mice, PON1 knockout mice, and wild-type mice with Trypanosoma congolense. When challenged with the same dosage of trypanosomes, mice overexpressing PON1 lived significantly longer than wild-type mice, and mice deficient in PON1 lived significantly shorter. In contrast, mice overexpressing another HDL associated protein, apoA-II, had the same survival as wild-type mice. Together, these data suggest that PON1 provides protection against trypanosome infection. In vitro studies using T. brucei brucei indicated that HDL particles containing PON1 and those depleted of PON1 did not differ in their lysis ability, suggesting that protection by PON1 is indirect. Our data are consistent with an in vivo role of HDL protection against trypanosome infection.