THE DEVELOPMENT OF A NEW PLANT-BASED CULTURE MEDIUM FOR PLASMODIUM FALCIPARUM, IN VITRO STUDIES ON THE ANTIMALARIAL ACTIVITIES OF FOUR COMMONLY USED MEDICINAL PLANTS IN NIGERIAN AND SOME ASPECTS OF THE IMMUNOLOGICAL IMPLICATIONS OF THE USE OF INSECTICIDES TREATED CURTAINS FOR THE PREVENTION OF MALARIA IN CHILDREN.
BY
ADEROUNMU ADEOLA OMOTAYO
THE DEVELOPMENT OF A NEW PLANT-BASED CULTURE MEDIUM FOR PLASMODIUM FALCIPARUM, IN VITRO STUDIES ON THE ANTIMALARIAL ACTIVITIES OF FOUR COMMONLY USED MEDICINAL PLANTS IN NIGERIAN AND ASPECTS OF THE IMMUNOLOGICAL IMPLICATIONS OF THE USE OF INSECTICIDES TREATED CURTAINS FOR THE PREVENTION OF MALARIA IN CHILDREN.
A thesis submitted (but NEVER defended) in partial fulfilment of the requirement for the degree of Doctor of Philosophy (Ph. D) in Medical Parasitology of the University of Lagos
By ADEROUNMU Adeola Omotayo
B.Sc., M.Sc. UNILAG
SEPTEMBER 2003
DECLARATION
I hereby declare that this thesis titled:
"The development of a new plant-based culture medium for plasmodium falciparum, in vitro studies on the antimalarial activities of four commonly used medicinal plants in nigerian and aspects of the immunological implications of the use of insecticides treated curtains for the prevention of malaria in children,"
is an original research carried out by me, Aderounmu Adeola Omotayo in the Department of Medical Microbiology and Parasitology, College of Medicine of the University of Lagos, Lagos, Nigeria and the Department of Immunology, Stockholm University, Sweden.
DEDICATION
"This project is dedicated to the African children suffering morbidity and mortality due to malaria and other preventable childhood diseases"
ACKNOWLEDGEMENT
The University of Lagos, School of Post-Graduate Studies granted the permission for this research to be carried out at Stockholm University.
UNESCO-American Society of Microbiologists and the International Centre for Culture and Science (ISC) in Switzerland provided the scholarship that facilitated my stay in Stockholm.
I am grateful to Professor A.F Fagbenro-Beyioku for her valuable guidance and support right from the days of my MSc programme at idiaraba and during the time away from home.
My sincere gratitude to Professor Klavs Berzins of the Department of Immunology, Stockholm University, (SU), for being a very kind man and at the same time for being my host and supervisor. I will never forget you. I appreciate the tutelage of Dr. Ahmed Bolad during all the time spent in and out of the laboratory.
I am grateful to Dr. Bolaji Thomas for providing a useful lead and for wonderful words of encouragements.
Special thanks to:
Professor Marita Troye-Blomberg, the Head of my department at Stockholm University
Margareta Hagstedt for her assistance in the Laboratory
All my seniors and friends in CMUL, Ninan, Bola, Amisu, Sola, Gbenga, Emeka
All my seniors and friends at SU, Jacob, Ben, Sallah, Nina-Maria, Gawa, Masashi, Manijeh, Shiva,
I remain indebted to my parents, Reverend and Mrs S. A. Aderounmu and to my siblings, Tosin, Wale, Ayoola, Yemi and Ayodele.
TABLE OF CONTENTS
Declaration
Certification
Acknowledgement
Table of contents
Chapter 1
Introduction and Literature review
Chapter 2
Material and methods
Chapter 3
Results
Chapter 4
Discussion
Chapter 5
Summary
REFERENCES
ABSTRACT
Malaria infection has been a major worldwide cause of death for centuries. An estimated 300-500 cases each year cause 1.5 to 2.7 million deaths, more than 90% in children under 5 years of age in Africa. The laboratory cultivation of malaria parasites is an important tool for the understanding of parasite biology, biochemistry, molecular biology, immunology and pharmacology. The requirements for human serum and the general high cost of existing approaches used for the cultivation of malaria parasites pose technical limitations to the application of these methods in many poor countries where malaria is endemic. In the first part of this study, short-term cultivation of Plasmodium falciparum, F32 strian, was achieved in a medium containing plant exudate and mice liver extract following earlier cultivation of the parasites in medium containing human serum. The parasitaemia increased from 4.0% to 7.6% on day 4 and the addition of hypoxanthine (0.02-0.04µM) to the new medium enhanced parasite growth as 9% parasitaemina was observed at 48 hours in a separate culture well containing the new medium and hypoxanthine. All the asexual stages of the erythrocytic phase of the parasite life cycle were seen throughout the duration of the cultivation. The results obtained in this study probably represents the first successful attempt to cultivate P. falciparum in vitro in a plant-based media supplemented with animal extract. In addition, the essence of the ongoing research is to develop an inexpensive malaria culture system that will enhance malaria research in poorer countries mostly afflicted with the scourge of the infection of P. falciparum. It is also known that the spread of multidrug resistance to P. falciparum, which is on the increase, has contributed to malaria specific mortality. This has highlighted the irrepressible need to develop new antimalarial drugs from natural products. Such drugs need be not only available but also affordable, safe to use and effective for developing countries where malaria undoubtedly remains one of the worst scourges contributing to mortality. In this study, the crude organic and aqueous extracts of four commonly used herbal plants from Nigeria have been evaluated for antimalarial activity against P.falciparum in vitro. These plants which include Cymbopogon giganteus, Azadirachta indica, Enantia chlorantha and Morinda lucida inhibited parasite growth efficiently in terms of reduced number of infected ring stages of the parasite. It is shown here that E.chlorantha-known to contain alkaloids, lignin, saponins and tannin-with crude extract inhibition of 68.9% and 54.0% at 500µg/ml as organic and aqueous respectively, is a promising antimalarial plant. When combined, lower concentrations of these extracts gave higher efficacy. The results justify the ethnomedicinal use of these four plants for the treatment of fever and malaria. Apart from chemotherapy, another malaria control strategy is currently being monitored closely is the use insecticides treated nets (ITNs) and curtains. These chemical barriers form a promising preventive measure against the deadly Plasmodium malaria parasite but concerns have been raised that the use of ITN may place young children at an increased risk of developing severe malaria at an older age by delaying the development of acquired immunity to malaria. Exposure to infection is regarded as a prerequisite for the natural development of immunity to malaria and protection against the blood stages of the parasite seems to rely on immunoglobulins (Ig)G, specifically on IgG1 and IgG3 antibodies. Interventions to reduce or eliminate malaria in children living in high transmission areas could lead to loss of these malaria antibodies resulting to rebound mortality in later childhood. This study has been conducted to compare the immunological responses in children from 2 villages in the vicinity of Ouagadougou in Burkina Faso. Enzyme-linked immunosorbent assay, (ELISA) analyses of plasma samples from asymptomatic children, aged 3-7 years, living in villages with and without insecticides treated curtains, (ITC) show no clear difference in the protective immunoglobulin levels in the two groups of children. In addition, PCR analyses of the DNA from these children show no distinction in the multiplicity of infection. These results probably indicate that the use of ITC does not interfere with the acquisition of antimalarial immunity associated with childhood in malaria endemic areas.
CHAPTER 1
INTRODUCTION
Plasmodium falciparum infection annually causes an estimated 500 million infections with 1.5-2.7 million deaths, more than 90% in children under 5 years of age in Africa (Good, 2001; Sach and Malaney, 2002). Efforts to prevent and control the disease have been hampered by the development of resistance to insecticides in the mosquito vector and to therapeutic agents in the parasite. Antimalarial regimens are difficult and there is yet no effective vaccine against malaria (Rozmajzl et al., 2001). Malaria is endemic in Nigeria and the population at highest risk includes children, pregnant women and the non-immune. Not less than 25% of infant deaths and 20% of maternal mortality cases in Nigeria are attributed to malaria. In addition, more than 65% of Nigeria's population of 100-120 million people experiences at least one attack of malaria each year (Aderounmu, 1999, M.Sc thesis).
Cultivation of malaria parasites is an important tool for the understanding of parasite biology, biochemistry, molecular biology, immunology and pharmacology (Ringwald et al., 1999). Since Trager and Jensen establised the in vitro culture of P.falciparum in 1976, the medium used has been complemented by the addition of 10% human serum. The requirements for human serum pose technical limitations to the application of this method (Asahi and Kanazawa, 1994: Asahi et al., 1996). For many reasons, it would be advantageous to replace human serum in the culture medium. In malaria-endemic regions, local sera may contain antimalarial drugs or immune factors that render them useless for culturing the parasites (Siddiqui and Palmer, 1980; Divo and Jensen, 1982a; Jensen et al.,1982). A few reports however have shown that African donors can support the growth of laboratory adapted strains of parasites and fresh isolates and that acute phase homologous serum may be useful for the continuous in vitro culture of reference strain (Oduola et al., 1992; Binh et al., 1997 and Ringwald et al., 1999).
It has generally been accepted that nonimmune human serum is required for optimal parasite growth. However the requirement for a regular supply of nonimmune human serum entails difficulties in conducting research in most of the African continent, where malaria transmission occurs at a high level throughout the year. Nonimmune human type AB-positive serum is relatively scarce and expensive in countries where malaria is not endemic (Divo and Jensen, 1982a; Ringwald et al., 1999).
Furthermore, serum from donors living in malaria-free areas differ considerably in their ability to support parasite growth and therefore, it is recommended that several units of serum from different donors be pooled together to reduce the batch-to-batch differences in the support of parasite growth (Divo and Jensen, 1982b; Jensen, 1988). It cannot also be excluded that drugs not directed against the malaria parasites may nevertheless influence the development of the parasites. More problems include blood type compatibility and risks associated with the handling of infectious agents (Lingnau et al., 1994). In addition, any widely used vaccine should not be grown in human serum when there is a real possibility of contamination with infectious agents (Divo and Jensen, 1982a).
A number of successful attempts to replace the human serum components of the medium used for the in vitro cultivation has been reported (Siddiqui and Richmond-Crum,1977; Ifediba and Vanderberg, 1980; Sax and Rieckamn 1980; Divo and Jensen, 1982b; Willet and Canfield 1984; Asahi and Kanazawa 1994; Oduola et al., 1985; Lingnau et al., 1993, 1994; Ofulla et al., 1993,1994). All outlined the obvious disadvantages of drawing experimental data from parasites grown in serum supplemented medium due to batch-to-batch invariabilities problems with availability and cost in some places and the probability of contracting viral infections. Parallel comparisons of studies from different laboratories can therefore be easily standardised once this important variable has been excluded. These investigators have used both commercial and defined formulations to mimic the growth requirements supplied by the human serum though no thorough identification has been carried out as to which factor(s) of human serum are necessary for growth (Ofulla et al., 1993; Asahi et al., 1996; Binh et al.,1997 and Flores et al.,1997). The use of various animal sera and serum factions as substitutes for human sera have had only limited success hence the attempts to find a suitable sustitute for human serum are continuing.
Importance of the culture system to malaria research
The methods for cultivation of the erythrocytic stages of P.falciparum reported by Trager and Jensen (1976) have been usefully applied in nearly all aspects of research on malaria: chemotherapy, drug resistance, immunology and vaccine development, pathogenesis, gametocytogenesis and mosquito transmission, genetics, the basis for resistance of certain red cells, cellular and molecular biology and biochemistry of the parasites and their relationship with their host erythrocytes (Trager and Jensen, 1997).
Chemotherapy
It was obvious that cultures of P. falciparum could be used for the direct testing of blood schizonticides against the parasites and hence to screen new antimalarial drugs (Trager, 1987). Malaria cultures provide accessible forms of the target organism itself, developing within its natural host cell, the erythrocyte, but apart from the intact host. It is possible to expose the parasite to any desired concentration of drug for any desired time and access the extent of inhibition. Particularly important in this context has been the development of a rapid measurement of the antimalarial activity of a large number of compounds using microdilution method. In view of the striking success of artemisinin, natural products, especially extracts of medicinal plants provide a logical starting point (Kirby, 1996). In vitro studies are particularly useful for attempts to discover the mode of antimalarial compounds.
Drug resistance
This can be easily demonstrated and quantitated in cultures. Moreover, a short-term in vitro test can be applied to a large number of clinical isolates to determine the prevalence of drug-resistant malaria (Ringwald et al., 1996). Cultures have been useful in attempts to determine the genetic and biochemical basis for drug resistance.
Vaccine development
A prerequisite for the ultimate goal of a vaccine against malaria is a method for in vitro propagation of the parasites (WHO, 1975). The main use of cultures in relation to development of malaria vaccines has been the identification of target antigens for both the asexual erythrocytic stages and for those of the sexual stages (Kaslow et al., 1992; Feng et al., 1993). They have also been used for the in vitro assessment of immunity, especially to test for the antibodies that inhibit merozoite invasion. In addition, the cultures have been used to provide gametocytes to infect mosquitoes. The mosquitoes in turn are used for both studies on transmission blocking immunity and to infect volunteers in clinical vaccination trials. The cultures have proved useful for tests of vaccines against sporozoites and pre-erythrocytic stages as well as for those against the asexual erythrocytic stages.
Pathogenesis, protein export and take-over of erythrocytes
The phenomenon of sequestration of late-stage parasites in the capillaries of deep organs is responsible for the severe manifestations of falciparum malaria including, in particular, cerebral malaria (Turner, 1997). Sequestration is mediated by attachment of trophozoite- and schizont-infected red cells via the knobs to endothelial cells of small blood vessels. The formation of knobs requires the production by the parasite of at least 3 proteins that are exported from the parasite to reach the host erythrocyte plasma. When the cultures made all stages of P.falciparum readily available, Langreth et al., (1978) found that the knobs appeared on the infected cells at a time corresponding to the time of sequestration.