Malaria disease severity and resilience: Plasmodium knowlesi infection of Macaca mulatta and Macaca fascicularis Öffentlichkeit
Peterson, Mariko (Spring 2020)
Abstract
Plasmodium knowlesi is an emergent zoonotic malaria parasite that has become the most common cause of malaria in Malaysia. Patients infected with P. knowlesi present across the clinical spectrum, and the accepted paradigm is that the natural monkey host in Southeast Asia, Macaca fascicularis (long-tailed macaques) does not exhibit severe disease, while the experimental host, Macaca mulatta (rhesus macaques, of Indian origin) experiences rapidly rising parasitemia and death if not treated. These striking differences provide a unique opportunity to examine determinants of disease severity, with direct application to understanding human P. knowlesi pathophysiology. Here we utilize M. fascicularis and M. mulatta and take a comparative approach to understand P. knowlesi disease severity and its resolution. A mixed cohort of these two species was infected with P. knowlesi Malayan strain sporozoites and studied longitudinally. Temperature was captured continuously via surgically implanted telemetry devices. As expected, M. fascicularis experienced much lower and controlled parasitemias compared to M. mulatta which required treatment. Recrudescent parasitemic peaks in M. fascicularis did not result in temperatures in the febrile range, suggesting that recrudescent parasitemias may not be clinically important. Yet, M. fascicularis did experience severe anemia, with hemoglobin nadirs as low as 4.8 g/dL and kidney injury. Chronicity and resolution of clinical conditions were achieved naturally in M. fascicularis, and only after subcurative treatment in M. mulatta. Twenty-two tissues were analyzed for the presence of parasites, their adhesion to the vascular endothelium, and pathology. Evidence for infected erythrocyte adhesion was observed in the gut tissues of M. mulatta, including by electron microscopy, while parasite adhesion was not clearly evident in M. fascicularis. Bone marrow, splenic and temperature responses occurred earlier in M. fascicularis infections, suggesting a role for immune response timing in disease resilience to this parasite. These findings contribute to better defining determinants of malarial disease in macaque hosts, understanding P. knowlesi pathophysiology, and improving diagnosis and treatment strategies in clinical settings.
Table of Contents
Table of Contents
Chapter 1: Introduction
1.1 Back to basics: biology of Plasmodium knowlesi ……………………………………………..1
1.2 A tale of two hosts: disease resilience and severity ……………………………………….......3
1.3 Making the jump: P. knowlesi in humans: history, ecology, and clinical importance …………5
1.4 A sticky situation: a role for sequestration in disease severity …………………………………9
1.5 The early bird and moderation: a role for host response in disease severity …………………11
1.6 Concluding Remarks ………………………………………………………………………...12
Chapter 2: Plasmodium vivax parasite load is associated with histopathology in Saimiri boliviensis with findings comparable to P. vivax pathogenesis in humans
2.1 Introduction ………………………………………………………………………………….14
2.2 Methods
Tissue acquisitions from Saimiri boliviensis infected with P. vivax ………………………..15
Histopathology, pathology scoring, and parasite quantification …………………………16
Immunohistochemistry …………………………………………………………………..17
Statistical Analysis ………………………………………………………………………17
2.3 Results
Parasite kinetics of P. vivax Brazil VII infection in S. boliviensis ………………………18
Plasmodium vivax Brazil VII infection of S. boliviensis causes lung, liver, and kidney tissue damage …………………………………………………………………………………..18
Figure 2.1 Lung tissue sections with representative micrographs showing
histopathology …………………………………………………………………………...20
Figure 2.2 Liver tissue sections of splenectomized monkeys with representative micrographs showing histopathology ……………………………………………………21
Figure 2.3 Kidney tissue sections of splenectomized monkeys with representative micrographs showing histopathology ……………………………………………………23
Tissue damage is associated with parasite prevalence in specific organs ……………….24
Figure 2.4 Histopathological and parasitological tissue analysis, in the organs of seven infected S. boliviensis ……………………………………………………………………25
2.4 Discussion …………………………………………………………………………………...28
Chapter 3: Disease resilience and malaria: host resistance and disease severity in Macaca mulatta and M. fascicularis to Plasmodium knowlesi infection
3.1 Introduction ………………………………………………………………………………….33
3.2 Methods
Experimental Design and Infections ……………………………………………………..34
Tissue Acquisition and Histopathology ………………………………………………….37
Telemetry Data Acquisition ……………………………………………………………...37
Calculation of Severity Metrics ………………………………………………………….38
Statistical Analysis ………………………………………………………………………39
3.3 Results
Parasitemia in P. knowlesi infection of a natural host and an experimental host: kinetics and burden ……………………………………………………………………………….39
Figure 3.1 Parasitemia in Rhesus and Kra monkeys ……………………………………..41
Kra monkeys develop a temperature response faster than rhesus monkeys …………….44
Figure 3.2 Temperature in P. knowlesi Infection ……………………………………….45
Figure 3.3 Temperature Response and Inflammation in P. knowlesi Infection ………….49
Kra monkeys develop and resolve severe anemia ………………………………………..51
Figure 3.4 Hemoglobin, Platelet Count, and Reticulocyte Production Index in P. knowlesi-Infected Macaques ……………………………………………………………………….52
Figure 3.5 Hemoglobin and Platelet Levels in P. knowlesi-Infected Macaques ………..54
Figure 3.6: Reticulocyte Production Index as it Relates to Parasitemia and
Hemoglobin ……………………………………………………………………………..57
Rhesus macaques and kra monkeys show minimal to moderate histopathological
changes …………………………………………………………………………………..58
Figure 3.7 Schematic Illustrating Tissue Collection in P. knowlesi-Infected Macaque Necropsies ……………………………………………………………………………….59
Figure 3.8 Splenic changes in macaques with P. knowlesi ………………………………61
Figure 3.9 The Bone Marrow Responds Earlier in Kra Monkeys ………………………64
Figure 3.10 Lung Involvement in P. knowlesi is Similar to Lung Involvement in Malaria Caused by Other Parasites ……………………………………………………………….66
Figure 3.11 Renal Involvement Occurs in Both Macaque Species ……………………...68
Figure 3.13 Hepatic Involvement in P. knowlesi Infection ……………………………..70
Chapter 4: Investigating Plasmodium knowlesi infected red blood cell cytoadhesion and sequestration in macaques
4.1 Introduction ………………………………………………………………………….81
4.2 Methods
Experimental Design and Infections ……………………………………………..83
Tissue analysis and parasite enumeration ………………………………………..84
Binding Studies ………………………………………………………………….85
RNAseq and Targeted Proteomics Analysis …………………………………….88
Statistical Analysis ………………………………………………………………88
4.3 Results
Parasites accumulation in tissues varies in host species and infection status ……88
Figure 4.1 Parasite tissue burdens in P. knowlesi infection ……………………..90
Figure 4.2 Parasite accumulation in the kra spleen in acute and chronic
infection ………………………………………………………………………….91
Figure 4.3 Parasite accumulation in the rhesus tissues in acute infection ………..92
Figure 4.4 Parasite accumulation in the kra bone marrow in acute and chronic infection ……………………………………………………………………….....95
Figure 4.5 Relative parasite tissue densities in kra and rhesus monkeys in acute and chronic infection …………………………………………………………………96
Degree of histopathology is weakly correlated with parasite tissue density …….97
Table 4.1: Fisher exact test for tissue score and parasite burden in rhesus acute infection ………………………………………………………………………….98
Table 4.2: Fisher exact test for tissue score and parasite burden in kra acute infection ………………………………………………………………………….98
Figure 4.6 Tissue score vs. parasite count ………………………………………99
Table 4.3 Linear Hierarchical Linear Regression Analysis …………………….101
SICA protein-expressing RBCs infected with mature parasites clearly sequester in rhesus monkeys ………………………………………………………………...103
Figure 4.7 Light microscopy: SICA protein expression and sequestration in the stomach ………………………………………………………………………...104
Figure 4.8 Light microscopy: SICA protein expression and sequestration in the duodenum ………………………………………………………………………105
Figure 4.9 Light microscopy: SICA protein expression and sequestration in the jejunum …………………………………………………………………………106
Figure 4.10 Light microscopy: SICA protein expression and sequestration in the colon ……………………………………………………………………………107
Table 4.4 Fisher exact test for sequestration and species ………………………108
Figure 4.11 Quantification of SICA[+] and SICA[-] parasite margination in the gastrointestinal tissues ………………………………………………………….110
Consideration of putative iRBC receptors, including Mucosal vascular Addressin Cell Adhesion Molecule 1 (MAdCAM1), which is expressed in the gastrointestinal tract………………………………………………………..……………………112
Figure 4.12 Electron microscopy: SICA protein expression and sequestration in the gut ………………………………………………………………………………113
Figure 4.13 SICAvar expression at necropsy SICA[+]-infected monkeys …….114
Figure 4.14 Soluble markers of endothelial activation …………………………116
Figure 4.15 ICAM1 Expression in the Stomach ……………………………….117
Figure 4.16 ICAM1 Expression in the Duodenum …………………………….118
Figure 4.17 ICAM1 Expression in the Colon …………………………………..119
Figure 4.18 MAdCAM1 Expression in the Stomach …………………………..120
P. knowlesi-infected RBCs do not bind CD36, ICAM, or
MAdCAM…..…………………………………………………………………..122
Figure 4.19 MAdCAM1 Expression in the Duodenum ………………………..123
Figure 4.20 MadCAM1 Expression in the Colon ………………………………124
Figure 4.21 The Effect of Anticoagulant and Binding Buffer on Binding of Rhesus RBCs to C32 Amelanotic Melanoma Cells …………………………………….125
Figure 4.22 The Effect of Trypsin on Binding of Rhesus RBCs to C32 Amelanotic Melanoma Cells ………………………………………………………………...126
Figure 4.23 The Effect of Hematocrit on Binding of Rhesus RBCs to C32 Amelanotic Melanoma Cells …………………………………………………...127
Figure 4.24 The Effect of BSA on Binding of Rhesus RBCs to C32 Amelanotic Melanoma Cells ……………………………………………….………….…….129
Figure 4.25 The Effect of FBS on Binding of Rhesus RBCs to C32 Amelanotic Melanoma Cells ………………………………………………….………….….130
Figure 4.26 The Effect of Naïve Monkey Serum on Binding of Rhesus RBCs to C32 Amelanotic Melanoma Cells ……………………………………………....131
Figure 4.27 Human RBC Binding to C32 Amelanotic Melanoma Cells …..…..132
Figure 4.28 Plasmodium-infected RBC Binding to Transfected CHO Cells …..133
Figure 4.29 Trypsinized Plasmodium-infected RBC Binding to Transfected CHO Cells ………………………………………….…………………………………134
Figure 4.30 Quantification of Untreated and Trypsinized Plasmodium-infected RBC Binding to Transfected CHO Cells ……………….………………………135
4.4 Discussion ………………………………………………………………………….136
Chapter 5: Conclusion ………………………………………………………………………...147
Chapter 6: Appendix
6.1 Appendix to Chapter 2: Plasmodium vivax parasite load is associated with histopathology in Saimiri boliviensis with findings comparable to P. vivax pathogenesis in humans …………………………………………………………………………………150
Figure 6.1 Parasitemia kinetics from seven S. boliviensis monkeys infected sequentially with P. vivax iRBCs …………………………………………………………………….150
Table 6.1: Review of experimental demographics and parasite kinetics ……………….152
Table 6.2: Summary of histopathological findings ……………………………………153
Table 6.3: Pairwise histology score comparison ………………………………………155
Table 6.4: Tukey HSD post-hoc pairwise comparison ……….………………………..159
Table 6.5: Fisher exact test …………………………………….………………………160
Table 6.6: Multiple linear regression analysis …………………………………………161
Table 6.7: Spearman’s Rank coefficient test …………………………………………..162
6.2 APPENDIX TO CHAPTER 3: Disease resilience and malaria: host resistance and disease severity in Macaca mulatta and M. fascicularis to Plasmodium knowlesi
Infection ………………………………………………………………………………..163
Table 6.8 Cohort Summary ……………………………………………………………163
Figure 6.2 Experimental Design and Parasitemia for E30 (Pilot acute P. knowlesi infection in rhesus macaques) …………………………………………………………………….168
Figure 6.3 Experimental Design and Parasitemia for E07 (Longitudinal P. knowlesi infection in kra monkeys) ……………….…………………………………………..….169
Figure 6.4 Experimental Design and Parasitemia for E06 (Acute P. knowlesi infection in rhesus macaques) ………………………………………………………………………170
Figure 6.5 Experimental Design and Parasitemia for E33 (Chloroquine treatment in acute P. knowlesi infection in macaques) …………………………………………………….171
Figure 6.6 Experimental Design and Parasitemia for E35 (Chronic P. knowlesi infection in macaques) …………………………………………………………………………...172
Figure 6.7 Area under the curve (AUC) as a measure of cumulative parasitemia in P. knowlesi infection of macaques ……………………………………………….………..173
Figure 6.8 Replication rate of P. knowlesi infection in rhesus macaques ……………..174
Figure 6.9 Replication rate of P. knowlesi infection in rhesus macaques ……….……..175
Figure 6.10 Telemetry and parasitemia for REd16 ……………………………………176
Figure 6.11 Telemetry and parasitemia for RKy15 …………………………...…..……177
Figure 6.12 Telemetry and parasitemia for 11C131 ………….………………..……….178
Figure 6.13 Telemetry and parasitemia for 11C166 …………………………………...179
Figure 6.14 Telemetry and parasitemia for 12C44 …………………………………….180
Figure 6.15 Telemetry and parasitemia for 12C136 ……………………………………181
Figure 6.16 Telemetry and parasitemia for 12C53 …………………………………….182
Figure 6.17 Telemetry and parasitemia for H12C8 ……………………………………183
Figure 6.18 Telemetry and parasitemia for H12C59 …………………………………..184
Figure 6.19 Telemetry and parasitemia for RCl15 …………………………………….185
Figure 6.20 Telemetry and parasitemia for RIh16 ……………………………………..186
Figure 6.21 Telemetry and parasitemia for RTe16 …………………………………….187
Figure 6.22 Telemetry and parasitemia for RUf16 …………………………………….188
Figure 6.23 Parasitemia and select hematological parameters for REd16 …………….189
Figure 6.24 Parasitemia and select hematological parameters for RKy15 …………….190
Figure 6.25 Parasitemia and select hematological parameters for 11C131 …………….191
Figure 6.26 Parasitemia and select hematological parameters for 11C166 ……..……..192
Figure 6.27 Parasitemia and select hematological parameters for 12C44 …..…………193
Figure 6.28 Parasitemia and select hematological parameters for 12C53 ……………..194
Figure 6.29 Parasitemia and select hematological parameters for 12C136 …………….195
Figure 6.30 Parasitemia and select hematological parameters for H12C8 …………….196
Figure 6.31 Parasitemia and select hematological parameters for H12C59 ……………197
Figure 6.32 Parasitemia and select hematological parameters for RCl15 ……………..198
Figure 6.33 Parasitemia and select hematological parameters for RIh16 ……………..199
Figure 6.34 Parasitemia and select hematological parameters for RTe16 ……………..200
Figure 6.35 Parasitemia and select hematological parameters for RUf16 …………..….201
Figure 6.36 Parasitemia and select hematological parameters for RFz15 ……………..202
Figure 6.37 Parasitemia and select hematological parameters for RNn9 …………..…..203
Figure 6.38 Parasitemia and select hematological parameters for 13C90 ……….……..204
Figure 6.39 Parasitemia and select hematological parameters for 14C3 ……….……...205
Figure 6.40 Parasitemia and select hematological parameters for 14C15 ……………..206
Figure 6.41 Parasitemia and select hematological parameters for H13C110 …….…….207
Figure 6.42 Parasitemia and select hematological parameters for 13_116 …….……….208
Figure 6.43 Parasitemia and select hematological parameters for 13_136 ……………..209
Figure 6.44 Parasitemia and select hematological parameters for RRz15 ………..…….210
Figure 6.45 Parasitemia and select hematological parameters for H13C101 …………..211
Figure 6.46 Parasitemia and select hematological parameters for H14C17 ……………212
Figure 6.47 Parasitemia and select hematological parameters for 13C33 …………..….213
Figure 6.48 Parasitemia and select hematological parameters for 13C74 ……………...214
Table 6.9 Summary of histopathology scores for P. knowlesi-infected macaques …….215
6.3 Appendix to Chapter 4: Chapter 4: Plasmodium knowlesi sequestration is concomitant with the expression of MAdCAM1 on the endothelium in macaques …..…218
Expanded methods for purified protein binding assay ………………………………….218
Figure 6.49 Plate set up for purified protein binding assay ………….………………….220
Figure 6.50 Humidified chamber, washing, and aspiration technique ……….…………221
Figure 6.51 Binding and staining ……….………………….…………………..………222
Expanded methods for cell binding assay ………………………………………….…..223
Figure 6.52 Plate set up and lawn seeding ……….……………………………..………225
Figure 6.53 Stained slide ……………………………………………………….………226
Figure 6.54 Technique for coverslip disc removal from well plate …………………….228
Table 6.10 Tissue parasite count means …….………………………………………….229
Table 6.11 Relative tissue parasite counts ……………………………..……………….231
Figure 6.55: Sequestration and parasite kinetics ………………………………………233
Figure 6.56 Fraction of vessels displaying margination ………………………………..234
Figure 6.57 Fraction of parasitized vessels …………………………………………….235
Figure 6.58 Fraction of vessels displaying margination normalized to parasitemia …..236
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