Background
Every year, 125 million women are exposed to the risk of malaria worldwide [
1]. In sub-Saharan Africa where malaria burden is high,
Plasmodium falciparum causes up to 10,000 cases of malaria-related deaths in pregnancy, mainly due to maternal anaemia, and approximately 200,000 infant deaths annually [
2]. In these women, malaria parasites accumulate and sequester in the placental intervillous space (IVS), a condition referred as placental malaria (PM) [
3]. PM leads to complications that threaten the lives of both mother and foetus, such as stillbirths, pre-term deliveries, low birth weights, reduction in gestation period, anaemia, and mortality [
4,
5]. This has socio-economic impact on the affected population [
6].
Over the years, substantial efforts have been made to prevent and control malaria by employing different strategies [
2,
6,
7]. These efforts have encountered major challenges, such as lack of effective vaccines, inadequate animal models and rampant drug resistance [
2]. Studies on PM immunopathology have been conducted in rodents. The shortcoming of these studies is that data obtained cannot be correlated to humans because of their difference in reproductive system [
8-
10]. To bridge this gap, animal models such as non-human primates whose reproductive system mimics the human situation are required in order to produce reliable data [
11-
13]. Baboons are good animal models for malaria in pregnancy because they have a similar host-pathogen interaction and a reproductive system that is physiologically similar to that of humans [
14-
18]. This study was designed to describe pathological features and clinical outcomes associated with PM in
Plasmodium knowlesi-infected baboons. Results from this study will contribute to the validation of the baboon-
P. knowlesi model of malaria in pregnancy.
Discussion
This study demonstrated, for the first time, the infiltration of parasitized RBC and inflammatory cells in the placentas of non-immune baboons. During P. knowlesi infection in pregnancy, symptomatic disease is manifested in combination with increased placental parasitaemia. Consequently, accumulation of parasites in the placental IVS combined with infiltration of inflammatory cells leads to placental damage.
In areas of stable malaria transmission, asymptomatic infections in pregnant women are common, with low clinical malaria presentation due to acquired immunity over time. In contrast, pregnant women in unstable malaria transmission areas, such as the Asia-Pacific region and South America present clinical disease because of low levels of acquired immunity [
23]. This study demonstrated higher levels (50%) of parasitaemia in nulligravids compared to pregnant infected baboons, although the difference was not significant. In addition, higher levels of placental parasitaemia were observed compared to peripheral parasitaemia in the same animal. The distribution of parasites in an infected pregnant woman varies with endemicity. The proportion of parasitized erythrocytes is often higher in the placenta than in the peripheral blood in endemic areas [
24,
25] because infected erythrocytes are preferentially retained in the placenta [
26,
27]. Haemozoin has also been frequently observed in placentas from malaria-infected women whether in the presence or absence of peripheral parasitaemia [
4,
28,
29]. These findings correlate well with human studies.
Placental falciparum malaria in humans increases neonatal mortality by lowering birth weight while fever is associated with premature birth [
30]. Increase in body temperature and reduction in RBC and Hb levels are also observed. According to human studies, it is documented that anaemia is associated with low birth weight, a major determinant of infant mortality associated with
P. falciparum [
30]. It is estimated that approximately 500,000 pregnant women develop severe anaemia due to
falciparum malaria [
31,
32] and up to 10,000 maternal anaemia-related deaths are as a result of malaria infection in sub-Saharan Africa [
33,
34]. Severe anaemia is common in children and pregnant women in regions of stable, high malaria transmission such as sub-Saharan Africa, while mild anaemia is common in semi-immune or non-immune populations [
35,
36]. Reduced level in Hb during malaria infection is an indicator of anaemia while high parasitaemia density has been implicated for reduced Hb levels [
37]. A similar observation was demonstrated in this study as displayed by the reduced Hb and RBC levels in
P. knowlesi-infected non-immune baboons.
Increased levels of WBC in peripheral circulation and inflammatory cell (monocytes, macrophages and neutrophils) infiltration in H&E-stained tissues obtained from
P. knowlesi-infected baboon placentas were observed in this study. Immunosuppression during pregnancy is important in maintaining the foetal allograft. Although some murine and human studies have demonstrated that suppression of cell-mediated immune responses plays a major role in the increased susceptibility to malaria during pregnancy [
24,
25,
38-
40], a study conducted in a
Plasmodium falciparum hyperendemic area in Tanzania revealed a marked increase in the levels of monocytes and macrophages and cytotoxic T cells in the IVS of placentas with active malaria infection [
41]. Findings in this baboon model indicate that placental malaria is not likely to be associated with cell-mediated immunosuppression. The role of immunosuppression in increasing risk to falciparum malarial infection as observed in pregnant women could not be clearly confirmed in this study.
Retroplacental transmission of falciparum malaria parasites from mother to her foetus is rare in humans particularly in CS deliveries. For instance, a study conducted in Burkina Faso, a region of stable malaria transmission, malaria rate in umbilical cord blood occurred in 1.4% of all newborns. This mechanism of congenital transmission was not clear, although it was attributed to several factors [
42,
43]. However, no such study has been conducted in areas of low or unstable malaria transmission although documented evidence indicates that malaria parasites affect the placenta selectively and are recognized in maternal erythrocytes. In addition, the placenta at full term presents an adequate barrier that prevents malaria parasites from crossing over to the foetal circulation [
41,
44]. This is similar to what was observed in this study. Although the sample size was small, the constant absence of parasites in placental foetal region and cord blood implies that congenital malaria in non-immune baboons is rare.
This study has also demonstrated accumulation of parasitized erythrocytes in the IVS of the baboon placenta, in addition to high levels of mature forms of the parasite in the placenta. This suggests possible cytoadherence of
P. knowlesi parasites in the baboon placenta. It is documented that only the mature falciparum malaria parasites show cytoadherence properties [
45]. These parasitized erythrocytes adhere to the endothelium via parasite-derived proteins expressed on their surface [
46]. In fact, syncytiotrophoblast cells of the human placenta expresses different and variable amounts of host cell receptors onto which the parasites can bind [
45,
46]. The principal molecule that mediates adhesion of infected erythrocytes is
P. falciparum erythrocyte membrane protein 1 (PfEMP-1), a large, highly variant parasite antigen protein, encoded by the
var multigene family [
47]. The adhesion phenotypes are not homologous and as a result different parasites can bind to various numbers and combinations of host molecules, such as chondroitin sulphate A (CSA), hyaluronic acid (HA) and Fc receptors [
48-
50]. In seeking to identify the ligand and receptor molecules associated with accumulation of
P. knowlesi infected erythrocytes in the baboon placenta, chondroitin sulphate proteoglycan (CSPG) 4 and HAPLN 1 were predicted as putative receptor molecules in the baboon with high similarity to human CSA and HA respectively. In addition,
P. knowlesi erythrocyte binding proteins (EBP-
alpha, EBP-
beta and EBP-
gamma) matched closely to the placental
P. falciparum ligand
Var2csa [
51]. Further work is required to demonstrate the precise ligand-receptor molecules responsible for accumulation of
P. knowlesi parasites in baboon placenta.
Histopathological findings demonstrated that PM in baboons is characterized by placental damage due to fibrinous necrosis of the villi, chorionic plate thrombosis, syncytiotrophoblast disruption, chorionic plate syncytiotrophoblast disruption, and thickening of trophoblastic basement membrane. The process was also accompanied with infiltration of inflammatory cells in the placental tissue was. These findings correlate well with human studies where placental falciparum malaria pathology is characterized by excess perivillous fibrinoid deposits, excessive syncytial knotting, trophoblastic membrane thickening, which have been associated with destruction/damage of placental tissues, and proliferation of cytotrophoblastic cells [
8,
10,
25,
46,
52]. It is hypothesized that placental damage, especially the thickening of the trophoblastic basement membrane, alters the maternal foetal exchange, leading to malaria-associated placental lesions and poor foetal outcomes [
47]. The same mechanism is likely to take place in
P. knowlesi associated PM in the baboon.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
FIO participated in study design, carrying out the experiments, data analysis and drafting the manuscript preparation. OWN and FM helped in sample collection, performing the experiment and drafting the manuscript. NWM and COAO supervised the study and participated in drafting the manuscript. IOF was the pathologist in the study. CLK was the pathologist consultant in malaria and pregnancy. JMM was the northern partner, who together with HSO developed the idea into a research project and obtained funding. HSO conceived the idea, obtained the funding, participated in study and coordination and reviewed the manuscript. All authors read and approved the final manuscript. This work was supported by the research capability strengthening WHO (Grant Number: A 50075) for malaria research in Africa under the Multilateral Initiative on Malaria /Special Programme for Research and Training in Tropical Diseases (WHO-MIM/TDR), awarded to HSO.