Congopain, the major cysteine protease from Trypanosoma congolense, is synthesized as an inactive zymogen, and further converted into its active form after removal of the proregion, most probably via an autocatalytic mechanism. Processing of recombinant procongopain occurs via an apparent one-step or a multistep mechanism depending on the ionic strength. The auto-activation is pH-dependent, with an optimum at pH 4.0, and no activation observed at pH 6.0. After addition of dextran sulfate (10 microg/ml), an approx. 20-fold increase of processing (expressed as enzymatic activity) is observed. Furthermore, in the presence of dextran sulfate, procongopain can be processed at pH 8.0, an unusual feature among papain-like enzymes. Detection of procongopain and trypanosomal enzymatic activity in the plasma of T. congolense-infected cattle, together with the capacity of procongopain to be activated at weakly basic pH, suggest that procongopain may be extracellularly processed in the presence of blood vessel glycosaminoglycans, supporting the hypothesis that congopain acts as a pathogenic factor in host-parasite relationships.

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 hostspecific antibodies may mitigate the pathology associated with trypanosome infection.

A 69-kDa immunodominant protein of Trypanosoma congolense was identified as a member of the hsp70 family that is homologous to mammalian BiP. We report here the expression of the gene encoding the T. congolense BiP in a bacterial system. Dot blotting of the truncated recombinant proteins confirmed that BiP antigenicity is mainly located in the C-terminal third of the molecule. A recombinant fragment corresponding to this region was used as an antigen in an indirect ELISA and an initial evaluation of its diagnostic potential for bovine trypanosomosis was performed. The test showed limited sensitivity for detection of primary-infected cattle but was capable of accurately detecting secondary infections. As BiP and its derivatives may be produced at low cost under stable forms allowing standardization of the tests, they warrant further evaluation as antigens fro serodiagnosis of bovine trypanosomosis.

The catalytic domains of two closely related cysteine proteinases (CP1 and CP2) from Trypanosoma congolense, referred to as C1 and C2, were expressed as proforms in Escherichia coli (C1) and in the baculovirus system (C1 and C2). While the bacterial expression system did not allow recovery of active C1, the baculovirus system led to secretion of inactive zymogens which could be processed at acidic pH into mature enzymes. Active C1 and C2 were purified from serum-free culture supernatants by anion-exchange chromatography and characterised. Their kinetic parameters and pH activity profiles confirmed the relatedness between C2 and native CP2 (congopain). These properties also underline major functional differences between C1 and C2, that appear to relate to discrete but essential sequence differences. It is likely that these two enzymes perform distinct roles in vivo, in the parasite and/or in the host-parasite relationships.

In order to test the hypothesis that trypanosome cysteine proteinases (CPs) contribute to pathology of trypanosomosis, cattle were immunised with CP1 and/or CP2, the major CPs of Trypanosoma congolense, and subsequently challenged with T. congolense. Immunisation had no effect on the establishment of infection and the development of acute anaemia. However, immunised cattle, unlike control cattle, maintained or gained weight during infection. Their haematocrit and leukocyte counts showed a tendency to recovery after 2-3 months of infection. Cattle immunised with CP2 mounted early and prominent IgG responses to CPs and to the variable surface glycoprotein following challenge. Thus trypanosome CPs may play a role in anaemia and immunosuppression; conversely, anti-CP antibody may modulate the trypanosome-induced pathology.

The S2 subsite of mammalian cysteine proteinases of the papain family is essential for specificity. Among natural amino acids, all these enzymes prefer bulky hydrophobic residues such as phenylalanine at P2. This holds true for their trypanosomal counterparts: cruzain from Trypanosoma cruzi and congopain from T. congolense. A detailed analysis of the S2 specificity of parasitic proteases was performed to gain information that might be of interest for the design of more selective pseudopeptidyl inhibitors. Nonproteogenic phenylalanyl analogs (Xaa) have been introduced into position P2 of fluorogenic substrates dansyl-Xaa-Arg-Ala-Pro-Trp, and their kinetic constants (Km, kcat/Km) have been determined with congopain and cruzain, and related host cathepsins B and L. Trypanosomal cysteine proteases are poorly stereoselective towards D/L-Phe, the inversion of chirality modifying the efficiency of the reaction but not the Km. Congopain binds cyclohexylalanine better than aromatic Phe derivatives. Another characteristic feature of congopain compared to cruzain and cathepsins B and L was that it could accomodate a phenylglycyl residue (kcat/Km = 1300 mM-1.s-1), while lengthening of the side chain by a methylene group only slightly impaired the specificity constant towards trypanosomal cysteine proteases. Mono- and di-halogenation or nitration of Phe did not affect Km for cathepsin L-like enzymes, but the presence of constrained Phe derivatives prevented a correct fitting into the S2 subsite. A model of congopain has been built to study the fit of Phe analogs within the S2 pocket. Phe analogs adopted a positioning within the S2 pocket similar to that of the Tyr of the cruzain/Z-Tyr-Ala-fluoromethylketone complex. However, cyclohexylalanine has an energetically favorable chair-like conformation and can penetrate deeper into the subsite. Fitting of modeled Phe analogs were in good agreement with kinetic parameters. Furthermore, a linear relationship could be established with logP, supporting the suggestion that fitting into the S2 pocket of trypanosomal cysteine proteases depends on the hydrophobicity of Phe analogs.

The ability of the prodomains of trypanosomal cysteine proteinases to inhibit their active form was studied using a set of 23 overlapping 15-mer peptides covering the whole prosequence of congopain, the major cysteine proteinase of Trypanosoma congolense. Three consecutive peptides with a common 5-mer sequence YHNGA were competitive inhibitors of congopain. A shorter synthetic peptide consisting of this 5-mer sequence flanked by two Ala residues (AYHNGAA) also inhibited purified congopain. No residue critical for inhibition was identified in this sequence, but a significant improvement in Ki value was obtained upon N-terminal elongation. Procongopain-derived peptides did not inhibit lysosomal cathepsins B and L but did inhibit native cruzipain (from Dm28c clone epimastigotes), the major cysteine proteinase of Trypanosoma cruzi, the proregion of which also contains the sequence YHNGA. The positioning of the YHNGA inhibitory sequence within the prosegment of trypanosomal proteinases is similar to that covering the active site in the prosegment of cysteine proteinases, the three-dimensional structure of which has been resolved. This strongly suggests that trypanosomal proteinases, despite their long C-terminal extension, have a prosegment that folds similarly to that in related mammal and plant cysteine proteinases, resulting in reverse binding within the active site. Such reverse binding could also occur for short procongopain-derived inhibitory peptides, based on their resistance to proteolysis and their ability to retain inhibitory activity after prolonged incubation. In contrast, homologous peptides in related cysteine proteinases did not inhibit trypanosomal proteinases and were rapidly cleaved by these enzymes.

Trypanosomosis is one of the major constraints on animal production in areas of Africa which have the greatest potential for significant increases in domestic livestock populations and livestock productivity. While the eradication of trypanosomosis from the entire continent is an unrealistic goal, considerable effort has been invested in the control of this disease through the use of trypanocidal drugs, management of the vector and exploitation of the genetic resistance exhibited by indigenous breeds. There is little hope that a conventional, anti-infection vaccine will be produced in the near future. Drug resistance is developing faster than generally thought. The control of the tsetse fly has been attempted over many decades. The decreasing efficacy of available trypanocidal drugs and the difficulties of sustaining tsetse control increase the imperative need to enhance trypanotolerance through selective breeding, either within breeds or through cross-breeding. Trypanotolerance has been defined as the relative capacity of an animal to control the development of the parasites and to limit their pathological effects, the most prominent of which is anaemia. A major constraint on selection for trypanotolerance in cattle, for both within-breed and cross-breeding programmes, has been the absence of practical reliable markers of resistance or susceptibility. Distinct humoral immune response to trypanosome infection is the major feature of bovine trypanotolerance. The role that these responses play in the control of infection or disease is being addressed by ongoing research, but remains a matter of speculation at present. Results in recent years have shown that packed cell volume (PCV) in particular and parasitaemia, the two principal indicators of trypanotolerance, are strongly correlated to animal performance. However, although direct effects of trypanosome infections on PCV and growth are obvious, more sensitive diagnostic methods for reflecting parasite control are required so that individual animals can be categorised reliably for their parasite control capability. One key finding is the major contribution made by each of the indicators evaluated to the overall trypanotolerance variance. Preliminary genetic parameters for PCV provide evidence that trypanotolerance is not only a breed characteristic but is also a heritable trait within the N'Dama population; this brings new opportunities for improved productivity through selection for trypanotolerance. More reliable estimation of genetic parameters of the indicators may well show that these parameters must be handled simultaneously for optimal progress. This would require diagnostics for assessing parasite control capability that identify trypanosome species more accurately, especially in mixed infections. A major advantage of trypanotolerant livestock, particularly N'Dama cattle, is the resistance or adaptation of this breed to many of the important pathogenes which prevail in the sub-humid and humid tropics. Research on practical indicators of resistance to these conditions will be required to establish relevant integrated strategies based on disease-resistant livestock. Selective breeding will require the integration of the traits that farmers hold important for their production systems.

Congopain and cruzipain, the major cysteine proteinases from Trypanosoma congolense and Trypanosoma cruzi, were compared for their activities towards a series of new, sensitive fluorogenic substrates of the papain family of cysteine proteinases and for their sensitivity to inhibition by cystatins and related biotinylated peptidyl diazomethanes. Low Ki values, in the 10 pM range, were found for the interaction of both proteinases with natural cystatin inhibitors. The kinetic constants for the hydrolysis of cystatin-derived substrates, and the inhibition by related diazomethanes were essentially identical. Unlike cathepsins B and L, the related mammal papain family proteinases, congopain and cruzipain accomodate a prolyl residue in P2'. Substrates having the sequence VGGP from P2 to P2' were hydrolysed by both congopain and cruzipain with a k(cat)/Km greater than 4.10(3) mM(-1) s(-1). Irreversible diazomethane inhibitors, deduced from the unprime sequence of cystatin-derived substrates, inhibited the two parasite proteinases. N-terminal labelling of diazomethanes with a biotin group did not alter the rate of inhibition significantly, which provides a useful tool for examining the distribution of these enzymes in the parasite and in the host. Despite their similar activities on cystatin-derived substrates, congopain and cruzipain had significantly different pH-activity profiles when assayed with a cystatin-derived substrate. They were correlated with structural differences, especially at the presumed S2 subsites.

As part of a study on livestock productivity under trypanosomosis risk in the region of Boundiali, northern Côte d'Ivoire, 21 herds of cattle (N'Dama, Baoulé and Zebu crosses) and 20 flocks of Djallonké and Djallonké × Sahel sheep were monitored monthly for body weight, packed red cell volume and trypanosomal parasitaemia over various periods between January 1984 and December 1992. A tsetse control campaign using biconical traps impregnated with alphacypermethrin started in December 1987. Tsetse control reduced the relative tsetse density by over 95% between 1988 and 1992, and this was associated with reductions in the prevalence of Trypanosoma congolense over the same period of over 90% both in sheep and cattle. Average reductions in the prevalence of T. vivax were lower, on average 68% in adults and 85% in young animals. Attempts were made in the design of the study to allow comparisons between controlled and uncontrolled areas; however, there were too many confounding and uncontrollable factors to allow such comparisons to be made. It was necessary, therefore, to compare data collected from all herds and flocks before and after the intervention, with the consequential difficulties in accounting for uncontrollable year-to-year variations in factors affecting trypanosome prevalence in livestock.

A comparison of T-cell-mediated immune responses in trypanotolerant N'Dama and susceptible Boran cattle during primary infection with tsetse-transmitted Trypanosoma congolense was conducted to assess whether different patterns of T-cell activation occurred during trypanosome infection. Proliferation and IFN-gamma synthesis in response to trypanosome antigens and to the mitogen Con A were measured in LNC before infection and 10 and 35 days postinfection. Phenotypic analysis of LNC was also carried out. No significant differences in the in vitro proliferation of LNC to VSG, to hsp70/BiP, or to Con A were detected between the breeds. In contrast, IFN-gamma production in response to Con A was higher in Boran cattle at 35 days p.i. A reduction in the number of CD2+ and CD4+ T-cells and an increase in the percentage of B-cells, CD8+ T-cells, and gamma delta T-cells during infection in both N'Dama and Boran was revealed by cytofluorimetric analysis of lymph node cells.

Trypanosomiasis is a serious constraint to livestock production in sub-Saharan Africa. Some breeds of cattle are genetically more resistant to the pathogenic effects of trypanosome infection. We measured B-cell activation and the quantity and isotype of antibody produced at the cellular level in six trypanotolerant N'Dama and five trypanosusceptible Boran cattle. The frequencies of spleen cells secreting total and parasite-specific IgM and IgG were measured prior to and 16, 28, and 35 days after a primary challenge with Trypanosoma congolense. Boran cattle had higher frequencies of splenic cells secreting IgM specific for trypanosome-derived variable surface glycoprotein (VSG), cysteine protease (congopain, CP), and heat shock protein (hsp 70/BiP) and the nonparasite antigen, ovalbumin, than did N'Dama cattle. In contrast, the number of VSG-specific IgG-secreting cells was significantly greater in N'Dama than in Boran cattle. During infection, low titers of anti-VSG IgM were detected transiently in the serum of all animals. However, N'Dama had significantly more VSG-specific IgG in blood than Boran during infection. The peripheral blood mononuclear cell population of N'Dama cattle contained a higher percentage of surface IgM-positive B-cells prior to and throughout infection than were found in the blood of Boran. In addition, during infection N'Dama cattle had more circulating lymphocytes that could be activated in vitro to undergo differentiation into IgM- and IgG-secreting cells. These findings demonstrate differences in the frequency of trypanosome-specific antibody-secreting cells in the spleen and in the activation state of B-cells in the blood between N'Dama and Boran cattle during a primary infection with T. congolense.

Memory T- and B-cell responses to trypanosome antigens were measured in peripheral blood mononuclear cells, spleen and lymph node cells obtained from four trypanotolerant N'Dama cattle which had been exposed to six experimental infections with Trypanosoma congolense. These cattle were treated with trypanocidal drugs following each infection and had remained aparasitemic for 3 years prior to this study. The antigens used were whole trypanosome lysate, variable surface glycoprotein, a 33-kDa cysteine protease (congopain) and a 70-kDa heat-shock protein. As parameters of T-cell-mediated immunity, we measured T-cell proliferation and IFN-gamma production. Lymph node cells, spleen cells and peripheral blood mononuclear cells all proliferated to a mitogenic stimulus (concanavalin A) but only lymph node cells responded to trypanosome antigens. Similarly, IFN-gamma was produced by both lymph node and spleen cells stimulated with concanavalin A but only by lymph node cells stimulated with variable surface glycoprotein and whole trypanosome lysate. T. congolense-specific antibodies were detected in sera and in supernatants of cultured lymph node and spleen cells after in vitro stimulation with lipopolysaccharide and recombinant bovine interleukin-2. In conclusion, we have demonstrated that memory T- and B-cell responses are detectable in various lymphoid organs in cattle 3 years following infection and treatment with T. congolense.

T-cell-mediated immune responses to defined antigens of Trypanosoma congolense were measured in cattle undergoing primary infection. The antigens used were the variable surface glycoprotein and two invariant antigens, a 33-kDa cysteine protease (congopain) and a recombinant form of a 69-kDa heat-shock protein. Proliferative responses were highest during the second week postinfection and were detected in cells obtained from the lymph node draining the site of infection but not in peripheral blood mononuclear cells. Production of IL-2 and IFN-gamma was measured in supernatants from antigen-stimulated lymph node cell cultures. Expression of IL-2, IL-4, and IFN-gamma mRNA was detected in antigen-stimulated lymph node cells by reverse transcription-polymerase chain amplification.

Approximately 390 East African Zebu calves from birth to 3 years of age and their darns were monitored monthly from 1986 to 1992 in nine village herds in an area of high trypanosomiasis risk in southwest Ethiopia where there was resistance to all available trypanocidal drugs. Cattle were individually treated with diminazene aceturate when they were detected to be parasitaemic and their packed red cell volume decreased below 26%, or when they showed clinical signs of trypanosomiasis. The average monthly trypanosome prevalence among cattle between 6 and 36 months of age was 18%. Within this environment, animals achieved average body weights of 79 +/- 14 (SD) kg, 134 +/- 21 kg and 183 +/- 22 kg at 12, 24 and 36 months of age respectively. Annual mortalities ranged from 8 to 24%, from 6 to 15% and from 4 to 16% in the age ranges 0-12, 13-24 and 25-36 months respectively. Calves parasitaemic in any one month in 1988, when early rains failed, had a higher average mortality in that month (3.1%) than those that were aparasitaemic (1.4%). Live weight gains of calves born to the dams detected as parasitaemic on more than half the occasions during the first 6 months postpartum were 14% lower than those of calves from dams not detected as parasitaemic over this period. An effect of parasitaemia in the calf on weight gain to 12 months could not be demonstrated, but animals detected as parasitaemic on more than six of the 12 monthly samples between 13 and 24 or 25 and 36 months of age had growth rates on average 22% lower than those of animals not detected as parasitaemic. All these effects of trypanosomiasis on productivity, however, were temporary and animals later compensated for periods of poor growth. Regular trypanocidal chemotherapy in a situation of high levels of drug resistance may have helped to maintain the health and productivity of these young cattle.

Bovine trypanosomiasis is prevalent in 66,000 km 2 of southwest Ethiopia infested with tsetse flies (Fordet al., 1976; Krug, 1971). Since 1986, East African zebu cattle raised under trypanosomiasis risk in the Ghibe valley of southwest Ethiopia have been studied to investigate the effects of trypanosomiasis on health and productivity. As the project progressed it became apparent that many animals were repeatedly being detected parasitaemic despite treatment with diminazene aceturate at a dose of 3.5 mg/kg bodyweight (Rowlandset al.,1993). A model to distinguish between new and recurrent infections was developed (Rowlands et al.,1991) in order to determine if the high infection rate following treatment ofTrypanosoma congolense infections, the predominant species, was due solely to the high tsetse challenge or instead to relapses of infections following treatment. Rowlandset al. (1993) showed that changes in T. congolenseprevalence matched corresponding changes in tsetse challenge. However, the number of detected parasitaemias attributable to new infections accounted for only about two thirds of all parasitaemias, implying that a proportion of the detected parasitaemias was due to recurrent infections. These results pointed to the existence of drug resistant trypanosomes and this was confirmed by Codjiaet al. (1993). Twelve stabilates obtained from cattle at Ghibe were inoculated into individual Boran calves and characterized for their sensitivity to diminazene aceturate, isometamidium chloride and homidium chloride. All 12 stabilates produced infections resistant to treatment with diminazene aceturate, while 11 produced infections resistant to isometamidium chloride and homidium chloride. With this evidence of very high levels of drug resistance under laboratory conditions it is perhaps surprising that a higher prevalence of parasitaemia was not being detected in the field. The prevalence of T. congolenseat the time that isolates were collected was 37%. It would appear, therefore, that, whilst treatment may not have been eliminating infections, it may have

Approximately 320 East African Zebu cows over 36 months of age were monitored monthly from 1986 to 1992 in nine village herds in an area of high trypanosomiasis risk in southwest Ethiopia where there was resistance to all available trypanocidal drugs. Cows were individually treated with diminazene aceturate when their packed cell volume either decreased below 26% and were detected parasitaemic, or when they showed clinical signs of trypanosomiasis. The average annual monthly trypanosome prevalence was 25% (range 18–39%). Average cow body weight was 196 kg but was 16 kg lower on average during 1988, a year when early rains failed. The median calving interval was 463 days ranging from 379 to 620 days for different years and seasons. Cows detected parasitaemic in more than half of the monthly samples taken over the period 1–150 days post partum had an average calving interval 39 ± 18 (SE) days longer than those not detected parasitaemic. Calving interval was also inversely related to both post partum body weight and to the change in body weight between 1 and 150 days post partum. The median age at first calving was 40.5 months. Heifers detected parasitaemic at least once between 19 and 30 months of age calved at an average age 2.9 ± 1.2 (SE) months older than those not detected parasitaemic. Over 8% of calvings resulted in abortions or still births and there was a significant increase from 7 to 10% in the rate of abortion associated with cases of parasitaemia detected during the last 3 months of pregnancy. Annual abortion rate and annual trypanosome prevalence also appeared to be correlated. Except for the high incidence of abortions the effects of trypanosomiasis on reproduction appeared generally to be small.