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Genetics of Drug Resistance in Rodent Malaria Rose GENETICS OF DRUG RESISTANCE IN RODENT MALARIA ROSE ANN PADUA, B.Sc. Submitted to the University of Edinburgh as a Thesis in Fulfilment of the Requirement for a degree of Doctor of Philosophy Institute of Animal Genetics, University of Edinburgh February, 1980. C18It4 I. CONTENTS Page ACKNOWLEDGEMENTS V ABSTRACT VI SU?1ARY vii INTRODUCTION 1 1.1 The Malaria Problem 1 1.2 Life Cycle 3 1.3 Antimalarial Drugs 4 1.4 Drug Resistance in Human Malaria 5 1.5 Chloroquine- resistance in the Laboratory 7 1.6 Chlorauine-Mode of Action and Mechanism of Resistance 10 1.7 Mefloquine - Mode of Action 14 1.8 Genetics 16 1.8.1 Cytogenetics 16 1.8.2 Hybridization Between Strains 18 1.9 Aims of this Investigation 20 2 MATERIALS AND METHODS 22 2.1 Lines of P. chabaudi 22 2.1.1 Origins 22 2.1.2 Life Cycle 22 2.1.3 Definition of Terms 22 2.1.4 Parasite Lines 23 2.2 Maintenance of Parasites in the Laboratory 25 2.2.1 Mammalian Host 25 2.2.2 Blood passage of Parasites 25 2.2.3 Mosquito Transmission 26 2.2.4 Liquid Nitrogen Preservation 26 2.3 Parasitaemja Estimations 27 II. Page 2.4 Preparation of Standard Inocula 27 6 2.4.1 Inocula Containing 10 Parasites 27 2.4.2 Inocula Containing 0.5 Parasite for Cloning 27 2.5 Starch Gel Electrophoresis 28 2.6 Source, Preparation and Administration of Drugs 29 2.6.1 Chloroquine (X) 29 2.6,2 Pyrimethamine (PYR) 29 2.6.3 Mefloquine (IlF) 30 2.6.4 Mepacrine (MP) 30 2.6.5 Quinine (QN) 30 2.7 Standard Drug Tests 30 2.7.1 chioroquine 31 2.7.2 Pyrimethamine 31 2.7.3 Mefloquine 32 27.4 Mepacrine 32 2.7.5 Quinine 32 2.8 Chioroquine-resistance 32 2.8.1 Selection of a High Level of chioroquine- 32 Resistance 2.8.2 Tests for Different Levels of Chioroquine- Resistance 33 2.8.3 In vitro Drug Tests 33 2.8.4 Genetic Studies 34 2.8.5 Reconstruction and Competition Experiments 36 2.9 Mefloquine 37 2.9.1 Selection for Mefloquine-Resistance 37 2.9.2 Stability of Mefloquine-Resistance 38 2.9.3 Cross-Resistance to Quinine 38 III. Page 3 RESULT 39 3.1 Selection of Chloroquine-Resistant Lines 39 3.2 Response of Selected Lines to Chloroquine 39 3.2.1 4-Day Versus 6-Day Tests 39 3.2.2 Infectivity During Drug Treatment 42 3.2.3 Drug Delay Tests 43 3.2.4 Response to Different Doses of Chloroquine 43 3.3 Stability of Resistance of Selected Lines 45 3.3.1 Response of AS(15CQ) Line 45 3.3.2 Response of AS(20Q) Line 45 3.3.3 Response of AS(30Q) Line 45 3.4 Response of Selected Lines to Other Drugs 49 3.4.1 Response to Mefloquine 49 3.4.2 Response to Mepacrine 49 3.4.3 Response to Quinine 52 3.5 Genetic Analyses 52 3.5.1 Crosses 1 & 2: AJ(Sens) x AS(30CQ) 52 3.5.2 Tests on Uncloned Progeny 55 3.5.3 Control Studies 55 3.5.4 Characterization of Clones Derived from - Cross 1 57 3.5.5 Characterization of Clones from Cross 2 63 3.6 Reconstruction and Competition Experiments 63 3.6.1 Reconstruction 63 3.6.2 Competition 70 3.7 In vitro Chloroquine Drug Tests 75 3.8 Selection of Mefloquine-Resistance 78 3.9 Response of Selected Lines to Chloroquine 81 3.9.1 Response of AS(12MF) Line 81 3.9.2 Response of AS(30MF) Line 81 Iv. Page 3.10 Stability of Resistance of Selected Lines 81 3.10.1 Response of AS(lMF) Line 81 3.10.2 Response of AS(30MF) Line 81 3.11 Cross-Resistance to Quinine 84 4 DISCUSSION 87 4.1 General Mechanism of Drug Resistance 87 4.2 Variation Due to Host Effects 90 4.3 Chioroquine-Resistance in AS 91 4.4 Genetic Analyses 93 4.4.1 Cross 1 94 4.4.2 Cross 2 96 4.5 Reconstruction and Competition Experiments 97 4.6 In vitro Drug Tests 101 4.7 Mefloquine-Resistance 103 4.8 Cross-Resistance to Quinine 106 4.9 General Discussion 107 REFERENCES 112 APPENDIX 139 PUBLICATIONS 148 V. ACKNOWLEDGEMENTS I would like to thank my supervisors, Professor Geoffrey Beale and Dr David Walliker for their encouragement, useful advice and critical appraisal of the manuscript. In addition, I would like to thank my colleagues, Dr Virgilio Rosario and Dr Andrew Tait, for many useful suggestions, and Robin Gray for his help in the production of this thesis. I thank Richard Fawcett for his technical assistance in carrying out some of the electrophoresis gel runs reported in this thesis and Edie Joyce for the maintenance of the animal house. I certify that, apart from the assistance mentioned above, all of the experiments presented in this thesis were planned and carried out by myself. This work has been supported by the Medical Research Council. February, 1980. VI. ABSTRACT Chioroquine-resistance was studied in the rodent malaria parasite, Plasmodium. chabaudi. Parasite lines exhibiting different degrees of resistance were obtained by submitting a line already resistant to a low level of the drug to a gradual increase of drug pressure. The chloroquine-resi st ant lines were found to be stable after mosquito transmission and after multiple blood passage in the absence of the drug. The inheritance of the high level of chloroquine-resistance 1 (resistant to 30 mg kg- chloroquine for 6 days) was examined by crossing this resistant line with a drug sensitive (sensitive to 3 mg 1 kg- chloroquine for 6 days) line. The progeny of the cross showed intermediate levels of chloroquine-resistance suggesting that the high level of resistance was due to more than one mutation. Competition studies between different lines of P. chabaudi revealed that the highly resistant line was at a selective advantage over the sensitive forms and that cross products were favoured over parental resistant lines. A mefloquine-resistant line was established by a gradual increase in drug pressure. This resistance was variable in its stability after mosquito transmission and after blood passage without drug pressure. A clone was obtained which was stable on blood passage in the absence of the drug (to 15mg kg- 1 Mefloquine for 4 days). VII. SUMMARY Chioroquine-resistance has been studied in the rodent malaria parasite Plasmodium chabaudi. Parasite lines exhibiting different degrees of resistance were obtained by submitting a line already resistant to a low level of the drug to a gradual increase of drug pressure. Lines showing different levels of chloroquine-resistance were produced; low (3CQ) intermediate (15CQ) and high (30CQ) These levels could be distinguished by drug tests using varying doses of chioroquine and by using other drugs to which chloro- quine appeared to be cross-resistant; these included mefloquine, mepacrine and quinine. Tests using chloroquine administered for four and six days were compared. These experiments indicated that the duration of drug treatment as well as the dose administered was important for antimalarial activity. Chloroquine administered at a high dose -1 (30 mg Kg ) for four days did not eliminate chloroquine-sensitive parasites whereas lower doses administered for six days did eradicate chloroquine-sensitive parasites. The chloroquine-resistant lines selected were found to be stable after mosquito transmission and after multiple blood passage in the absence of the drug. The inheritance of the high level of chioroquine resistance (30Q) was examined by crossing this resistant line with a drug sensitive line. These two lines were polymorphic for two enzyme markers VIII. and differed in their response to pyrimethamine. The progeny of the cross were cloned and each clone was examined for drug response and enzyme types. Clones which were classified as 1 sensitive (to 3 mg Kg_ for 6 days), intermediate (3Q) or 15 CQ) or highly resistant (30cQ) were detected. The presence of clones showing intermediate levels of chloroquine resistance suggested that the high level (30Q) was due to more than one mutation. Blood mixtures of the highly resistant (30CQ). and the drug sensitive line did not generate parasites with inter- mediate levels of chioroquine-resistance. In vitro chioroquine drug tests were conducted using a chloroquine- sensitive and the highly resistant (30cQ) lines. In the presence of chloroquine, the resistant parasites formed vacuoles and their development appeared to be arrested at a specific stage whereas sensitive parasites continued to develop in the presence of the drug forming parasites which appeared abnormal. Ino- culations from cultures into uninfected mice revealed that the resistant parasites remained viable at all but the highest of the doses of chloroquine used whereas the sensitive parasites showed a reduced viability after exposure to the drug. Competition studies were carried out in which known proportions of two lines differing in specific characters were injected into mice. After varying periods of time the infections were examined to determine whether the proportions of each line had changed. The highly resistant (30Q) line appeared to possess a selective advantage in competition with the chloroquine- sensitive line from which it was derived. However, in Ix. competition with the chloroquine-sensitive line used in the cross the 30'Q line appeared to grow as well as the sensitive line; neither line was at a selective advantage. A highly resistant clone derived from the progeny of the cross was at a selective advantage over the parental (30CQ) highly resistant line obtained by selection.
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