Department of Parasitology

The overall objective of the department is to improve the control of three major parasitic diseases transmitted by vectors in tropical countries: malaria, trypanosomiasis and leishmaniasis. Within the department three laboratories, protozoology, entomology and serology, each with its own expertise and infrastructure, collaborate closely on topics related to the parasite, the vector, the host and their mutual interactions. Links with control programmes have been developed, complementary to bench and field research, training and technical assistance.

 

 

 

Coosemans

Marc Coosemans

 

Malaria

Research activities

Research on malaria focuses on

1.Immune response against exoerythrocytic schizonts (M. Wéry)

Supported by European Union and Vlaamse Gemeenschap. Partners: Institut Pasteur (Paris), Imperial College (London) Katholieke Universiteit Nijmegen, Universidad del Valle (Cali, Colombia)

1.1. Immunogenicity of CS and LSA-1 based peptides in the P. berghei model

The availability of synthetic peptides representing defined epitopes of malaria antigens has contributed to the research of immune responses to the EE stages. Moreover, if the amino acid sequences are conserved between the human malaria parasite P. falciparum and the rodent parasite P. berghei, then peptides representing falciparum EE stage antigens can be screened in the berghei model and the role of conserved antigens in malaria immunity can be studied. This approach has been used to study peptides based on invariant regions of the P. falciparum CS protein for functional activity and protection in the P. berghei model. Peptides representing predicted T helper cell epitopes of another malaria antigen of P. falciparum hepatic stages - LSA-1, have been screened for immunogenicity in the P. berghei model. The following experiments are being conducted:

Provisional conclusions

Our studies suggest that synthetic peptides representing conserved sequences of malaria antigens could form the basis of a peptide vaccine, and that an effective pre-erythrocytic vaccine would require the incorporation of epitopes based on both sporozoite and liver stage antigens.

1.2. Immune humoral responses to P. berghei pre-erythrocytic forms in vitro.

Sera antibodies generated in vivo by immunisation of C57BL6 mice with irradiated sporozoites were assayed in IFAT and in tests measuring in vitro the inhibition of sporozoite invasion (ISI) and the reduction of EE forms in the hepatoma cell line Hep G2 cells.

Serum antibodies generated in groups of mice immunised with 18, 15, 12 & 10 krad irradiated sporozoites showed reactivity in immunofluorescence to sporozoites and maturing EE forms in Hep G2 cells, up to serum dilutions of 1/160. No reactivity to blood stage antigens was observed.

A low inhibition of 27% of sporozoite invasion (ISI) was recorded in the presence of immune sera from mice immunised with 18 krad irradiated sporozoites. Moreover, antibodies caused a significant reduction of EE forms when added to infected Hep G2 cells after sporozoite invasion. Antibodies from mice immunised with 12 - 16 krad irradiated sporozoites provided almost 80 % reduction in the number of developing EE forms. These in vitro experiments indicate specific humoral immune responses to the EE forms in the immunised mice.

Isotypes present in sera of immunised mice, before and after challenge

Titration of different IgG classes were performed in Balb/c and C57 black mice (a) after immunisation with irradiated sporozoites and (b) after challenge with live sporozoites. Antibodies are present in immunised mice. The results also show that the challenge produces a sharp increase of the titres obtained for immunoglobulins G2a and G2b isotypes. These isotypes show a protective activity on sporozoites invasion in hepatoma cells and schizont development in vitro.

See Graphic.

2.Genetic characterisation of vector populations (M. Coosemans)

Ssupported by: Compagnie Maritime Belge and Belgian Administration for Development Co-operation.
Partners: Centre Muraz (Burkina Faso), Institute of Malariology (Vietnam), ORSTOM (France)

All known malaria vectors are groups and complexes, rather than single species. Differences in vectorial capacity and behaviour determine the role of individual species in the epidemiology of malaria transmission, and have to be considered when designing effective vector control programmes. Recognizing sibling species of Anopheles will allow discrimination between vectors and non-vectors. Sibling species often exhibit major differences in behaviour, spatial and seasonal distribution and responses to different control measures. Preventive measures, such as vector control activities, play a major role in national malaria control programmes and it is therefore crucial to know precisely which species should be targeted for control.

A cellulose acetate electropheresis system was developed and used to study the enzyme polymorphisms in the Anopheles gambiae complex related to feeding and resting behaviour (in Burundi) and vectorial competence (in Burkina Faso). In Burkina Faso, a two fold difference in P. falciparum infection rate was found for well defined genotypes. However, infected specimens were found in all the genotypes and consequently no refractory mechanism occurs in these natural populations.

In the framework of the Vietnam project, a study is now analysing the species of the Minimus group. Two species have been identified by isoenzyme analysis. Further work is now performed using PCR-RFLP techniques. A new network has been set up for the identification and characterisation of malaria vectors in Southeast Asia, consisting of:

3.Malaria control projects (M. Coosemans, M. Wéry)

The aim is to improve and to optimise malaria control activities by using control tools already available, i.e. preventive and curative measures. This requires a structural approach (institutional strengthening and training) but also a good knowledge of all aspects of the malaria situation (health care, drug resistance, diagnosis, epidemiology, parasitology, socio-economic aspects, ecology, entomology). All these aspects are taken into consideration in our overseas projects. Specific questions generated by field applications are translated into research projects. Such projects were previously undertaken in Burundi (1981-1994) and in Sri Lanka (1991-1993). Currently, a project is carried out in Southeast Asia (Vietnam) and another one is planned in Mali.

3.1. Support to the national malaria control programme in the province of Hoa Binh (Vietnam)

Supported by the Belgian Administration for Development Co-operation (BADC) (1995-1998)
Partners:The project is carried out in close collaboration with the Ministry of Health, the Institute of Malariology in Hanoi, the Provincial Health Authorities.

Objective

In this project research is focused on the integration of the control activities within the Primary Health System. The project will have a positive impact not only on the province but also at the national and regional level.

Operational research:

Population:

Vector:

Diagnosis:

Treatment:

Prevention:

Epidemics:

Health system:

3.2. Support to the national malaria control programme in Mali

A fact-finding mission was done in April. An objective-oriented intervention plan has been submitted to BADC and the Ministry of Health in Mali.

Funding: Belgian Co-operation (BADC) (1997-2001)

Objectives:

Research:

Assistance and training activities

1.Programme for malaria control in Southeast Asia

Submitted for funding by the E.U., DG I

A sub regional malaria control programme in three Southeast Asian Countries (Laos, Cambodia en Vietnam) will be supported by the European Union (29 million ECU). Applied and operational research will be closely linked to the field activities. A Technical Support Group of European Institutes has been created to offer their expertise to this project: London School of Hygiene and Tropical Medicine, the Liverpool School of Tropical Medicine, the Department of Infectious Diseases and Tropical Medicine of the University of Munich, ORSTOM (Paris) and ITM Antwerp.

2.International Course of Malariology and Control of Malaria

Supported by the Belgian Co-operation (BADC)

Three international courses have previously been organised in Bujumbura (Burundi) in collaboration with WHO (1986, 1991, 1993) where more then 50 participants of 16 different African countries have been trained. These courses are organised every two years. The next course has been prepared and will be organised in Cameroon in Collaboration with the French Co-operation and WHO (September - December 1996).

Objectives:

TrypanosomiasIs

Research activities

Research on trypanosomiasis focuses on issues

1.Vector related research programmes

1.1.Rearing of tsetse flies (P. Elsen)

In 1996 the number of colonies has been drastically reduced to one Glossina morsitans morsitans strain and one G. palpalis gambiensis strain. For further rationalisation a colony of G.m.morsitans is developed with in vitro feeding. Evaluation of this method of feeding on performance in cyclical transmission will be evaluated. If no difference occurs, the in vivo feeding shall be replaced by the in vitro one.

The production of flies for research and training lies between 400 and 600 per week for each line. A total capacity of 7,000 flies is currently maintained. Higher capacities have been reached for short periods of three months.

1.2. Genetics of the fly (P. Elsen)

In a previous work on two colonies of Glossina palpalis gambiensis, hexokinase has been shown to be probably sex-linked. We analysed this enzyme in the progeny of eighty couples and confirmed its linkage to the sexual X chromosome. However, two heterozygous males were observed, the offspring of whom allowed us to propose an alternative hypothesis.

Regarding the results obtained on Triatomine bugs we have analysed the peptide profile by means of SDS-PAGE of the saliva of our G. m. morsitans colony. The polymorphism observed between specimens of a same population incite us to add this tool in our investigations on the genetic of the vectorial capacity. Two projects have been prepared and submitted for that purpose.

1.3. Trypanosome development in tsetse flies ( M. Coosemans, D. Le Ray)

Supported by: EC-DG XII SCIENCE project 1992-1995 SCI*-CT92-0761, Compagnie Maritime Belge, NFWO project, approved (1997-2000), IUAP project (1997-2001), EC-TMR network project, in preparation.
partners: ULB (Belgium), Liverpool School of Tropical Medicine, V.U.B (Belgium), the Tsetse Research Laboratory (Bristol, UK), ORSTOM (Montpellier, France), UCL-ICP (Belgium)

Salivaria trypanosomiases are sustained by a remarkable biological relationship between the vector and the parasite. Almost a century after Bruce’s report of tsetse flies as vectors, there is still a huge gap in our knowledge on the biological fundamentals of their relationship. Until now, experimental work has mainly focused on the colonisation of the tsetse midgut by procyclic trypanosomes. Independently of this initial stage, midgut trypanosomes have to ‘mature’ to the final, infective metacyclic stage in the salivary glands. Experimental data on tsetse-trypanosome interactions in the course of this maturation process are scanty.

Objective:

To characterise the molecular mechanisms which determine transmission of T. brucei by tsetse flies.

In this field, the Unit of Entomology carried out research on biocommunication at the protein level between the tsetse fly and the trypanosome. For the first time, evidence was given for an active interaction of the trypanosome with a specific protein component of the tsetse fly. This protein activates the signal transduction mechanisms of the parasite. Moreover, the dynamics and morphogenic events of trypanosome development within the tsetse fly were described. As a result, an excellent transmitting tsetse-trypanosome model was optimised (G. m. morsitans ITMA - T.b. brucei EATRO 1125). Only by the presence of a unique infrastructure and expertise at the Institute can this model be maintained and kept available for research purposes such as the study on tsetse-trypanosome ligand-receptor interactions. The multidisciplinary study of the fundamentals of tsetse-trypanosome interaction will allow to develop new methods to estimate the vectorial potency of a natural tsetse population. These data are a prerequisite for a better understanding of the epidemiology of sleeping sickness and will result in an improvement of existing disease control activities. Future research will focus on the characterisation of protein ligands of the tsetse fly which bind to a trypanosome receptor, activating the signal transduction mechanism of the parasite. cDNA libraries, expressed in COS-cells, derived from proventriculus / oesophagus tissue and from salivary glands will be established.

2.Host related research programmes (N. Van Meirvenne & P. Büscher)

2.1. Human Salivaria trypanosomiases

The unit is active on the domain of diagnosis (for epidemiological as well as for clinical purpose), of treatment and of control of parasite development in the hosts. Diagnostic research is focused on the study into parasitological, serological, genetic and bioclinical parameters and their implementation in primary diagnosis and stage determination (operational and applied research). Newly developed tests such as alternative CATT versions, LATEX/T.b. gambiense and LATEX/IgM are under evaluation. A study into the use of recombinant antigens and synthetic peptides in antibody detection tests has been started. Research on treatment focuses on alternative treatment protocols with existing drugs and on the follow-up of patients after treatment (operational and applied research). Basic research is conducted to gain a better insight in the pathogenesis and related neuropathogenesis of sleeping sickness. Particular interest is paid to the interaction between the trypanosome and the host with respect to cytokines and their receptors, on the occurrence of auto-antibodies and on the protein composition of cerebrospinal fluid.

2.2. Animal Salivaria trypanosomiases

The unit is active on the development and evaluation of diagnostic tests for T.b. brucei, T. vivax, T. congolense, T. evansi infections in small ruminants, bovine, camels and buffaloes. Antibody, antigen and DNA/RNA detection systems: IFAT, CATT, LATEX, ELISA, PCR.

Collaborative research with the Veterinary Department is conducted on the role of complement in trypanotolerance.

2.3. Ongoing research projects

Collaborative research between ILRI and the Belgian Universities

CCGIAR-ILRI project (1979 - 1997), financed by the Belgian Administration for Development Cooperation (BADC)

Diagnosis of trypanosomiases in small ruminants

CCGIAR-ITC project (1995 - 1997), financed by the BADC; in collaboration with the Veterinary Department and the International Trypanotolerance Centre, Banjul, The Gambia.

Central Serum Bank for Sleeping Sickness

financed by WHO/TDR (1981 - 1997).

Longitudinal study of seropositive persons

IPR, Bouaké, Ivory Coast (1996 -1998), financed by FAC / OCEAC

Alternative treatment of early second stage trypanosomiasis patients

In collaboration with EPICENTRE, Paris and MSF France, Adjumani, Uganda (1995 - 1997). Suspended due to rebellion activities in Northern Uganda; financed by FAC/OCEAC

Reduction of treatment risks associated with Melarsoprol®

In collaboration with EPICENTRE, Paris and MSF France, Adjumani, Uganda (1995 - 1997);.financed by FAC/OCEAC

Approved research projects

Evaluation of diagnostic tests

Yaoundé, Cameroun (1997 - 1998), financed by: FAC/OCEAC

Development and evaluation of new dipstick test

Yaoundé, Cameroun et République Centrafricaine (1997 - 1998), supported by FAC/OCEAC

Therapeutical trial and research on stage determination

In collaboration with: Bureau Central de la Trypanosomiase (ZR), Centre de Développement Intégral-Bwamanda (ZR), Projet de Recherches Cliniques sur la Trypanosomiase (CI), Medische Missie Samenwerking (BE) and the Department of Clinical Research (1996 - 1999), supported by BADC.

Assistance and training activities

1.Production and distribution of reagents and materials

Diagnostic reagents, cryostabilates, sera, cerebrospinal fluid, antisera, trypanosome preparations have been provided to internal and external research groups.

2.Analyses on request

For control programs, for the Clinical Laboratory of ITM and for external research groups.

3.Support of projects involved in the control of Salivaria trypanosomiases

Technical and scientific services are provided to national and international programmes in most of the countries where human and animal trypanosomiases occur.

4.Training facilities

The Unit has accepted the training of numerous people involved in control and research programmes at various levels (technicians, clinicians, co-ordinators, students and graduated researchers).

5.Co-ordination

The Unit has initiated the setting up of the Belgian Forum for Trypanosomiasis (BELFORT) consisting of workgroups covering the following topics: bioresearch, chemotherapy, general control strategy, vector control)

LeishmaniasIs

(D. Le Ray)
RESEARCH and training ACTIVITIES

Leishmaniases of the New World

Two projects were run in parallel :

1.Project “Medicina Tropical y Salud”

The project continued to evolve in a very positive manner. The meeting of the Management Committee in November 1995 was preceded by an evaluation coordinated by Dr Chr. Darras (BADC/OPS La Paz) in collaboration with the Bolivian partners of the Faculty of Medicine and of the Health Secretariat of Cochabamba, with the participation of three advizers from the Antwerp Institute (Dr. V. Tellier, Nutrition; Pr P. Van der Stuyft, Epidemiology and Pr D. Le Ray, Protozoology) and of the BADC/ Brussels. The meeting of the Management Committee was chaired by the Under Secretary of Health, who stressed the national importance of the project developed at Cochabamba from a normative viewpoint (essential drugs, reference laboratories), as well as within the framework of the new laws on local community participation in the identification of primary needs and in the management of health care.

Mutual concertation visits between Bolivian coordinators and Belgian partners covered the four elements of the project: public health, nutrition, Chagas and leishmaniases. Two complementary projects have strengthened the current project : on the one hand a VLIR/RUG/ITMA project for a post-graduate course in Tropical Medicine in Cochabamba, aiming to train the local doctors in health problems of the tropical zone of Chapare-Carrasco; on the other hand, the renewed selection by the INCO.DC (EC, DG XII) programme of the “Leishbolpe” research project on Leishmaniases, aimed primarily at identifying the epidemiology and the control of the disease in the Chapare-Carrasco region, and at developing the parasitology and molecular biology capacities in the Research Centre (IIBISMED, CUMETROP) of the Faculty of Medicine of Cochabamba. The identification of the conditions of extension 1997-2001 of the BADC project was started during the summer of 1996. The extension agreement was signed in September 1996 during the visit to Bolivia of the Secretary of State for Cooperation, in agreement with the recommendations put forward in 1995 by the Belgo-Bolivian Commission. This extension comprises two additional interventions: tuberculosis and malaria, as well as an expansion of the activities along the Amazonian fluvial axis of the Ichilo and Mamoré rivers around the harbour of Puerto Villaroel.

2.Project “Leishbolpe”

This project aims to study the cutaneo-mucosal Leishmaniases of the New World, in various aspects :

2.1.Molecular epidemiology in Peru and Bolivia

The allopatric sampling in the Peruvian Andes and the forest regions of Peru and Bolivia was continued: at present more than 200 parasite isolates have been transferred to the ITMA, of which 180 were characterised by molecular karyotyping, electrophoresis of the isoenzymes and RAPD (Random Amplification of Polymorphic DNA). In Peru, the following species were encountered in the indicated proportions in forest regions (L. (V.) braziliensis, 74.5%; L.(V.) guyanensis, 9 %; L.(V.) lainsoni, 9 %; hybrids between L.(V.) braziliensis and L.(V.) peruviana, 5 %, L.(L.) amazonensis, 1 % and non identified species, 1.5 %) and in the Andes (L.(V.) peruviana, 90 % and L.(V.) guyanensis, 10 %). In Bolivia, the epidemiological complexity seems less important, within the limits of the achieved sampling, and probably because of a lesser ecological complexity in Bolivia : only two species were found (L.(V.) braziliensis, 80 % and L. (L.) amazonensis, 20 %.

2.2.Parasitic markers for clinical risk

The sympatric sampling from Peruvian (Pilcopata, Madre de Dios) and Bolivian (Chapare, Cochabamba) foci was continued. Our objective is to measure the genomic/genetic variability in order to find clinical risk markers (mucosal metastases or not).

So far, 88 and 79 cutaneous and muco-cutaneous stocks have been isolated from Peruvian and Bolivian patients respectively. From these, 122 stocks were transferred to Antwerp for cryopreservation and characterisation.

Up to now, karyotypic analysis (4 chromosomes) has been done on 66 stocks from the two sources (26 mucous, M and 40 cutaneous, C). Phenetic analysis has confirmed the results previously obtained on a smaller number of stocks : (i) certain genotypes are preferentially associated with M or C lesions, and (ii) within the genotypes M and C, a heterogeneity is observed. Among the studied chromosomes, the one containing the gp63 genes (a major surface glycoprotein considered to be an important factor of virulence, see below) shows the best clinical association: the probability that a Leishmania is found associated with a mucosal lesion decreases with the size of its gp63 chromosome. These results have led us to propose the hypothesis that the variation in chromosome size could involve certain essential genes, and therefore be associated to certain phenotype changes. In this context, a detailed analysis was undertaken of the contents of the other chromosomes studied (see below).

2.3.Sexual recombination among parasites

Validation of the clinical risk markers must be done through genetic study of the population, so that the frequency of sexual recombination can be assessed. Indeed, in case of sexual recombination, only the direct markers (e.g. the responsible genes) would remain valid, whereas the other markers would be rapidly dissociated from the phenotype by recombination.

Population studies conducted with Dr Tibayrenc’s team (ORSTOM, Montpellier) suggested a clonal structure of the leishmanias. We have however also shown the existence of rare hybrids (Dujardin et al., 1995a), as well as the occurrence of pseudo-sexuality (Dujardin, 1995b; Bañuls et al., submitted for publication). In order to understand the impact of these phenomena, two experimental lines of research were followed.

First, in order to study the phenotype characteristics of the hybrids, we compared their behaviour in in vitro culture to that of their presumed parents, L.(V.) braziliensis (rapid growth) and L.(V.) peruviana (slow growth). Our preliminary findings suggest a dominance of the peruviana character in the hybrids. These results should be confirmed and expanded by the study of other phenotype characters.

Second, in order to study the stability of the hybrid genotypes (existence of mendelian segregation?) and to assess their propagation potential in the natural populations (fitness), we have started a long term maintenance in vitro for several strains of hybrids. The caryotype of these different strains is regularly checked: until now, no difference has been observed. In parallel, the ploidy is also analyzed (in collaboration with Mr J. Van Den Abeele, Entomology and Dr Van Boxtael, UIA, Antwerp). This work is part of the M.Sc thesis of Miss Maiza Campos (VUB, Brussels)

2.4.Characterization of chromosomes of epidemiological interest

During the past few years, our epidemiological studies by karyotype analysis of leishmanias have highlighted 3 particularly interesting chromosomes, in as much as the reduction in their size is correlated to the reduction in gravity of the lesions seen on the affected patient. These observations were made in three epidemiological situations: (i) L. braziliensis, severe lesions + mucosal metastasis vs L. peruviana, benign lesions without metastasis (chromosome gp63), (ii) mucosal L. braziliensis vs cutaneous L. braziliensis (chromosomes gp63 and 149), and (iii) L. peruviana, large lesions vs small lesions (chromosome 22). In order to test the hypothesis that the reduction in size could be linked to the deletion of essential genes, the study of the genomic organisation of these 3 chromosomes was started.

In the case of chromosome gp63, we had already shown (Victoir et al.,1995) a reduction in number of the copies of the gp63 genes (major surface glycoprotein, implicated in the virulence) between L. braziliensis and L. peruviana, as well as the existence of several families of that gene (associated by other authors to different functions of the protein). PCR analysis showed that this deletion could concern one of these families (Victoir et al., submitted for publication). Our future efforts will therefore be concentrated on verification of this result and analysis of possible functional consequences. This will be done within the framework of a new project supported by the NFWO.

In the case of chromosome 22, we have shown that size polymorphism was partially due to a change in the number of copies of the genes coding for the ribosomal (rDNA). We proved by physical cartography that these genes are regrouped in clusters at the extremity of the chromosome, which could explain their high rate of rearrangement (subtelomeric regions are unstable). An unexpected effect of the analysis of this chromosome was the discovery by sequencing of a new kinasis protein of Leishmania.

Analysis of chromosome 149 was started within the framework of a MSc thesis (Mr Amha Kebede, VUB) and has shown that size polymorphism is partially due to variation in the number of copies of the genes coding for the mini-exon (essential sequence to the functionality of all the RNA carriers of Trypanosomatids). Cartography of this chromosome is in progress.

For the 3 chromosomes under study, our hypothesis seems therefore to be confirmed: essential genes are implicated in the polymorphism of the studied chromosomes. In the next stage of our research, we will study the further functional effects of these rearrangements by qualitative and quantitative analysis of the corresponding RNA messengers.

2.5.Development of new characterisation tools for diagnosis and epidemiology

Our research into the parasitic genome, and especially the description of a complex and variable organisation of the gp63 genes, have enabled us to develop interesting new tools for the characterisation of leishmanias. After having finalized an RFLP method for the characterization of the leishmanias of the New World, Miss Kathleen Victoir (VUB) has developed a second generation of tools within her doctorate thesis.

This method consists of an amplification of the genes of the gp63, followed by treatment with restriction enzyme. Her evaluation on an important number of stocks of the New World has shown that this method made it possible to distinguish all the species under observation. This result is essential for the future development of the diagnosis and epidemiology. This method enables us to characterize small quantities of parasites and therefore the simultaneous treatment of a large amount of data. Moreover, as this method uses primers specific for Leishmania, it should permit direct analysis of human and insect tissue.

Visceral leishmaniasis

1.Recombinant antigens with diagnostic relevance

We have pursued the molecular analysis of the specific antigen 24 (Ag24) of the leishmanias with the purpose of selecting recombinant DNA clones (within the framework of the PhD thesis of Mr Bernard Couvreur, ULB, Nivelles).

Initially, we sought to isolate the DNA sequences coding for this antigen by immunological screening of a bank of complementary DNA (cDNA) with a specific rabbit antiserum. The difficulty in getting rid of the antibodies anti-E.coli present in the serum made this approach disappointing.

For this reason, we followed an alternative approach based on the fractioning of the RNA messengers. Two fractions enabled us, after in vitro translation and immuno-precipitation, to reproduce the profile associated to the Ag24 entirely or partially. These two fractions have then been used to prepare banks of cDNA by RT-PCR. The recombinant plasmids were analyzed as pools, RNA corresponding to the inserts was produced and translated in vitro, and corresponding proteins were immunoprecipitated with an antiserum specific to the Ag24. After subdivision of the positive pools, we obtained a first positive clone for which the expression product was recognized specifically by the serum anti-Ag24. The analysis of the coding sequence revealed a significant homology with the peptidic sequence of one of the derived units of the proteasome complex of the Eucaryotes and the Archeobacteria. In parallel, amino-terminal sequencing of the proteic bands obtained by direct analysis of immuno-electrophoresis gel is presently running in collaboration with Dr Rudy Wattiez of the University of Mons-Hainaut (Biological Chemistry Laboratory). The first of the sequenced constituants also presents a significant homology with the derived units of the proteasome complex. Moreover this sequence is different from the one of the clone that resulted from the screening of the cDNA banks.

The hypothesis of an identity between Ag24 and the proteasome of Leishmania does not therefore sound unreasonable. The next stage of the project will be designed to (i) complete the analysis of the cDNA clones as well as the amino-terminal sequencing, and (ii) verify our hypothesis.

2.Project DAT/WHO-TDR

Multicenter evaluation of the direct agglutination test (DAT) and of its field kit nears completion. Two missions in Sudan and Kenya were carried out by Dr M. Boelaert (Epidemiology, Public Health) and Mrs D. Jacquet (Protozoology) in order to check, complete and process the data collected by the local investigators. On the other hand, the WHO-TDR and MSF-Holland have kept up a continuous demand for production of DAT antigen for various African and Asian countries. These two organisations have co-financed the purchase by Antwerp of a cell incubator-shaker in order to increase our production capacity. Lastly, a new contract was signed with WHO (CTD) for quality control of DAT antigen and serology recently developed by the National Centre of Dacca, Bangladesh.

American trypanosomiasis

A collaboration was set up last year with the University of Santiago, Chili (Department of Biochemistry, Dr A. Solari) and the University of Uppsala, Sweden (Department of Medical Genetics, Dr J. Henriksson), for the interpretation of caryotype data of T.cruzi, with the aid of algorhithms specially designed by our laboratory.

This study showed a strong correlation between karyotype and isoenzymate variability. Comparison with our studies on Leishmania showed that karyotype variability was significantly more important within T.cruzi than within each of the Leishmania species. This suggests the existence of different mechanisms of chromosomic rearrangement among the American trypanosomes, and highlights the relativity of species status among protozoa. We are pursuing the interpretation of our results in order to find out whether a correlation exists between the size of certain chromosomes and the clinical origin of the samples.

Assistance Activities

1.Diagnosis

This year, serodiagnosis of visceral leishmaniasis with DAT was transferred to the ITMA Clinical Biology Laboratory. As for parasitological diagnosis, 23 requests were received (against 12 last year), of which 9 turned out to be positive. We have also developed the diagnosis of leishmaniasis with PCR. Until now, this has been applied only to the subspecies of the subgenus Viannia, because of the specificity of the available samples: 3 cases of infection were diagnosed. From next year onwards, we will investigate the possibilities of translating research on the genes gp63 into a method of species diagnosis.

One request for xenodiagnosis of Chagas disease was received. This proved to be negative.

2.Reference Cryobank

In the course of this year, we received 55 stocks of Leishmania from Peru and Bolivia, and sent out 94 stocks of Leishmania from our Reference Cryobank on request to foreign laboratories.

Publications

Bakhiet M, Büscher P, Kristensson K, Wigzel H, Olsson T. Different trypanozoon species possess CD8 dependent lymphocyte triggering factor-like activity. Immunol Lett 1996; 50: 71-80.

Bakhiet M, Jansson L, Büscher P, Holmdahl R, Kristensson K, Olsson T. Control of parasitemia and survival during Trypanosoma brucei brucei infection is related to strain dependent ability to produce interleukin-4. J Immunol 1996; 157: 3518-26.

Bakhiet M, Olsson T, Mhlanga J, Büscher P, Lycke N, Van der Meide PH, Kristensson K. Human and rodent interferon-gamma as a growth factor for Trypanosoma brucei. Eur J Immunol 1996; 26: 1359-64.

Baptista JL, Vervoort T, Van der Stuyft P, Wéry M. Variations dans les taux de lipides plasmatiques en fonction de l'infection à Plasmodium falciparum à São Tomé. Parasite 1996; 4: 335-40.

Binh LN, Lieu TT, Verlé P, La PT, Tuy TQ, Yen PT. ParaSight®-F test prospects in hypo-endemic and epidemic prone regions of Vietnam. J Contr Malaria Parasit Dis 1996; 1: 34-8 (in Vietnamese).

Bringmann G, Götz R, François G. Synthesis of pindikamine A, a michellamine-related dimer of a non-natural, “skew” naphtylisoquinoline. Tetrahedron 1996; 52: 13419-26.

Bringmann G, Holenz J, Aké Assi L, Steenackers T, François G. Eine potentielle neue Generation von pflanzlicher Wirkstoffe gegen Malaria. In: Blick-Forschung, Lehre, Dienstleistung. Würzburg: Bayerische Julius-Maximilians-Universität, 1996; 2: 85-7.

Bringmann G, Ledermann A, François G. Dimeric murrayfoline A, a potential bis-carbazole-alkaloid: “biomimetic” synthesis, atropoisomer separation, and antimalarial activity. Heterocycles 1995; 40: 293-300.

Bringmann G, Koppler D, Wiesen B, François G, Sankara Narayanan AS, Almeida MR, Schneider H, Zimmermann U. Ancistroheynine A, the first 7, 8'-coupled naphtylisoquinoline alkaloid from Ancistrocladus heyneanus. Phytochemistry 1996; 43: 1405-10.

Bringmann G, Saeb W, Koppler D, François G. Jozimine A (“dimeric” dioncophylline A), a non-natural michellamine analog with high antimalarial activity. Tetrahydron 1996; 52: 13409-18.

Cham MK, D'Alessandro U, Todd J, Bennett S, Fegan G, Cham BA, Greenwood BM. Implementation of a National Malaria Control Programme using insecticide impregnated bednets in The Gambia, West Africa. Health Pol Plann 1996; 11: 292-8.

Chatterjee S, François G, Druilhe P, Timperman G, Wéry M. Immunity to Plasmodium berghei exoerythrocytic forms derived from irradiated sporozoites. Parasitol Res 1996; 82: 297-303.

Coosemans M, Verlé P. Sustainability and integration prospects of malaria vaccines in comparison with other malaria control activities. J Contr Malaria Parasit Dis 1995; 3: 8-10 (in Vietnamese).

D'Alessandro U. An efficacy trial of a malaria vaccine in Gambian infants and comparison with insecticide-treated bednets. Ann Trop Med Parasitol 1996; 90: 373-8.

D'Alessandro U, Langerock P, Bennett S, Francis N, Cham K, Greenwood BM. The impact of a national impregnated bed net programme on the outcome of pregnancy in primigravidae in The Gambia. Trans Roy Soc Trop Med Hyg 1996; 90: 487-92.

Doko A, Verhulst A, Pandey VS, Büscher P, Lejon V. Détection d'antigènes circulants au cours d'une infection expérimentale à Trypanosoma brucei brucei chez des bovins Borgou, Lagunaire et zébus Bororo blancs. Rev Elev Méd Vét Pays Trop 1996; 49: 207-11.

François G, Aké Assi L, Holenz J, Bringmann G. Constituents of Picralima nitida display pronounced inhibitory activities against asexual erythrocytic forms of Plasmodium falciparum in vitro. J Ethnopharmacol 1996; 54: 113-17.

François G, Passreiter CM, Woerdenbag HJ, Van Looveren M. Antiplasmodial activities and cytotoxic effects of aqueous extracts and sesquiterpene lactones from Neurolaena lobata. Planta Med 1996; 62: 126-9.

François G, Timperman G, Holenz J, Aké Assi L, Geuder T, Maes L, Dubois J, Hanocq M, Bringmann G. Naphthylisoquinoline alkaloids exhibit strong growth inhibiting activities against Plasmodium falciparum and P. berghei in vitro. Structure-activity relationship of dioncophylline C. Ann Trop Med Parasitol 1996; 90: 115-23.

François G, Van Looveren M, Timperman G, Chimanuka B, Aké Assi L, Holenz G, Bringmann G. Larvicidal activity of the naphthylisoquinoline alkaloid dioncophylline A against the malaria vector Anopheles stephensi. J Ethnopharmacol 1996; 54: 125-30.

Horsmanheimo L, Harvima IT, Harvima RJ, Brummer-Korvenkontio H, François G, Reunala T. Histamine and leukotriene C4 release in cutaneous mosquito-bite reactions. J Allergy Clin Immunol 1996; 98: 408-11.

Lieu TTT, Verlé P, Tuy TQ, Hung LX, Kieu DV. Can we use the patients feeling as base for fever detection. J Contr Malaria Parasit Dis 1996; 3: 6-8 (in Vietnamese).

Smits A, Coosemans M, Van Bortel W, Barutwanayo M, Delacollette C. Readjustment of the malaria vector control strategy in the Rusizi Valley, Burundi. Bull Entomol Res 1995; 85: 541-48.

Smits A, Roelants P, Van Bortel W, Coosemans M. Enzyme polymorphism in the Anopheles gambiae (Diptera: Culicidae) complex related to feeding and resting behavior in the Imbo valley, Burundi. J Med Entomol 1996; 33: 545-53.

Soenens J, François G, Van den Eeckhout E, Herdewijn P. Synthesis of 3'-amino-3'-deoxyadenosine derivatives as potential drugs for the treatment of malaria. Nucleosid Nucleotid 1995; 14: 409-11.

Stevens WJ, Van den Abbeele J, Bridts CH. Anaphylactic reaction after bites by Glossina morsitans (tsetse fly) in a laboratory worker. J Allergy Clin Immunol 1996; 98: 700-1.

Thomson MC, Connor S, Bennett S, D'Alessandro U, Milligan P, Aikins M, Langerock P, Jawara M, Greenwood BM. Geographical perspectives on bednet use and malaria transmission in The Gambia, West Africa. Soc Sci Med 1996; 43: 101-12.

Trung HD, Manh ND, Hinh TD, Roelants P, Van Bortel W, Smits A, Verlé P, Coosemans M. Preliminary results of electrophoresis on cellulose acetate gel in the research of A. minimus in Vietnam. J Contr Malaria Parasit Dis 1996; 4: 40-6 (in Vietnamese).

Van Bortel W, Barutwanayo M, Delacollette C, Coosemans M. Motivation à l'utilisation des moustiquaires imprégnées dans une zone à paludisme stable au Burundi. Trop Med Int Health 1996; 1: 71-80.

Van Bortel W, Delacollette C, Barutwanayo M, Coosemans M. Deltamethrin-impregnated bednets as an operational tool for malaria control in a hyper-endemic region of Burundi: impact on vector population and malaria morbidity. Trop Med Int Health 1996; 1: 824-35.

Van den Abbeele J, Van Driessche E, Claes Y, Le Ray D, Coosemans M. Trypanosome-binding proteins in the tsetse flies Glossina palpalis gambiensis and G. morsitans morsitans. Int J Parasitol 1995; 26: 113-6.

Verlé P, Binh LN, Lieu TT, Yen PT, Coosemans M. ParaSight®-F test to diagnose malaria in hypo-endemic and epidemic prone regions of Vietnam. Trop Med Int Health 1996; 1: 794-6.

Verlé P, Binh LN, Lieu TT, Yen PT, Tuy TT, Cong LD. Can we still use chloroquine and sulphadoxine-pyrimethamin systematically in northern Vietnam?  J Contr Malaria Parasit Dis 1996; 4: 8-10 (in Vietnamese).

Verlé P, Dulieu P. Project voor malariabestrijding in Vietnam. Dimensie 3, 1996; 4: 10-3.

Verlé P, Dulieu P. Un projet de lutte contre la malaria au Vietnam. Dimension 3, 1996; 4: 10-3.

Abstracts

Bañuls AL, Sidibé I, Brisse S, Arevalo J, Le Ray D, Tibayrenc M. Multiple primer RAPD analysis for studying genetic diversity and molecular taxonomy of Leishmania spp. In: International Workshop on Molecular Epidemiology and Evolutionary Genetics of Pathogenic Microorganisms, CDC Atlanta, 17-19 June 1996. [s.l.]: [s.n.], [1996]: 54.

Bañuls AL, Sidibé I, Brisse S, Arevalo J, Le Ray D, Tibayrenc M. Multiple primer RAPD analysis for studying genetic diversity and molecular taxonomy of Leishmania spp. Mem Inst Oswaldo Cruz 1996; 91(Suppl.): 184, Abstract 248.

Barutwanayo M, Bisore S, Butoyi G, Coosemans M. Malaria vector control during the political unrest in Burundi. Parassitologia 1996; 38: 252.

Chamekh M, Chimfwembe E, Van Hamme L, Van den Abbeele J, Cornelis P, Le Ray D, Pays E, Hamers R. The molecular basis of human serum resistance in Trypanosoma brucei rhodesiense: role of serum resistant associated gene. In: Joint Meeting of Parasitology, Brussels 13-15 May 1996, Book of abstracts. [s.l.]: [s.n.], [1996].

Chimanuka B, François G, Timperman G, Hamers R. Chronobiology of Plasmodium chabaudi (IP-PC1): the importance of detailed recording of the total parasitaemia and the differential distribution of malaria parasites. In: 28ème Congrès du Groupe d'Etudes des Rythmes Biologiques (GERB), 20-22 mai 1996, Saint-Etienne, France. [s.l.]: [s.n.], [1996]: 9.

Coosemans M. Old and new tools for malaria control. In: Second Indonesian-Dutch-Flemish Meeting on Infectious Diseases and Immunology, 29 May - 1 June 1996, Noordwijk, The Netherlands. [s.l.]: [s.n.], [1996].

Coosemans M. Typologie du paludisme en Afrique tropicale. Méd Trop 1996; 55(Suppl.): 113.

Coosemans M, Smits A, Roelants P. Enzyme polymorphism of Anopheles gambiae s.s. in relation to habitat, behaviour and malaria transmission in South Western Burkina Faso. Parassitologia 1996; 38: 253.

Dujardin JC. Chromosomal size polymorphism in Leishmania of the braziliensis complex: involvement of genes coding for key functions. In: Joint Meeting of Parasitology, Brussels, 13-15 May 1996, Book of abstracts. [s.l.]: [s.n.], [1996]: O35.

Dujardin JC. Use and limits of molecular karyotyping for genetic stuies of microorganisms: the example of Leishmania. In: International Workshop on Molecular Epidemiology and Evolutionary Genetics of Pathogenic Microorganisms, CDC Atlanta, 17-19 June 1996. [s.l.]: [s.n.], [1996]: 27.

Dujardin JC, Bañuls AL, Victoir K, Arevalo J, Llanos-Cuentas A, Tibayrenc M, Le Ray D. Eco-genetics of L. (V.) braziliensis and L. (V.) peruviana: from population to genome. Ann Trop Med Parasitol 1995; 89: 119-20.

François G, Bringmann G, Boyd MR, Holenz J, Timperman G, Steenackers T, van Looveren M, Assi LA. New and highly efficient drugs against malaria: the naphthylisoquinoline alkaloids. Trop Med Int Health 1996; 1: A29.

François G, Bringmann G, Boyd MR, Timperman G, Holenz J. Naphthylisoquinoline alkaloids: a new type of antimalarial drugs from plants. In: “Aktuelle Entwicklungen in der Naturstofforschung”, 7. Irseer Naturstofftage der DECHAMA e.V., 22-24 Februar 1995, Irsee, Deutschland. [s.l.]: [s.n.], [1995]: 21.

Lejon V, Büscher P, Magnus E, Van Meirvenne N. Rationalization of stage determination in sleeping sickness. Scand J Immunol 1996; 43: 711.

Passreiter CM, François G, Woerdenbag HJ. In vitro activities of germacranolides and furanoheliangolides from Neurolaena lobata against two tumour cell lines and Plasmodium falciparum. In: 44th Annual Congress of the Society for Medicinal Plant Research, 3-7 September 1996, Prague, Czech Republic. [s.l.]: [s.n.], [1996]: 23.

Tuy TQ, Verlé P, Coosemans M, Wéry M, Cong LD. Hypo-endemic malaria in Vietnam: approach to integrated control. Parassitologia 1996; 38: 252.

Van den Abbeele J, Le Ray D, Coosemans M. Development of Trypanosoma brucei in the tsetse. Joint Meeting of Parasitology, Brussels, 13-15 May 1996, Book of abstracts.  [s.l.]: [s.n.], [1996].

Victoir K, Dujardin JC, Bañuls AL, De Doncker S, Arevalo J, Hamers R, Le Ray D, Tibayrenc M. Plasticity of the gp63 genes in Leishmania (Viannia) peruviana and Leishmania (Viannia) braziliensis. In: International Workshop on Molecular Epidemiology and Evolutionary Genetics of Pathogenic Microorganisms, CDC Atlanta, 17-19 June 1996. [s.l.]: [s.n.], [1996]: 51.

CATT- T.b.gambiense