Background
Parasites belonging to the genus
Plasmodium are among the best studied parasites in the world, since they are responsible of causing malaria, the deadliest vector borne disease [
1]. Although malaria was targeted for elimination in 2030 in at least 35 countries, and the reduction of its incidence from 2005 until 2014 suggested this goal was achievable, in 2016 nine Latin American countries reported an increase in its incidence [
2]. In Colombia, the number of cases increased in 2016, but most importantly,
Plasmodium falciparum, the parasite responsible for cerebral malaria had an increase in its incidence, becoming more prevalent than
Plasmodium vivax, the predominant species in the country until 2013 [
2]. In 2018, the National System of Public Health Surveillance (SIVIGILA by its Spanish acronym) reported 61,339 cases of malaria in Colombia [
3],
P. vivax being the most prevalent (50%), followed by
P. falciparum (48%), and mixed infection
P. vivax and
P. falciparum (2%).
Changes in malaria eco-epidemiology can be related to shifts in sylvatic transmission cycles, since
Plasmodium parasites are also present in non-human primates (NHP) in tropical regions around the world [
4]. Until now, 26
Plasmodium species have been formally described in NHP, each found infecting from one to 29 species [
4].
In Latin America, NHP are potential reservoirs of
Plasmodium brasilianum/Plasmodium malariae [
5,
6], and some species have been found infected with
P. falciparum [
7] or with
Plasmodium simium [
5,
8], which is closely related to
P. vivax [
9,
10]. Although the risk of malaria zoonotic infection is of public health concern [
11], it still remains largely understudied.
Several studies were carried out between 1930 and 1985 in Brazil, Panama, Venezuela, Peru and Colombia, and blood samples were obtained from the genera
Alouatta, Aotus, Cacajao, Callicebus, Callithrix, Brachyteles, Chiropotes, Lagothrix, Pithecia, Saimiri, Saguinus, Cebus, Callicebus and
Ateles [
5,
8,
12‐
23]. They reported
P. simium infecting
Alouatta guariba and
Brachyteles arachnoides [
5,
8], and
P. brasilianum infecting
Callicebus brunneus, Chiropotes satanas, Saguinus midas, Phitecia monachus, Lagothrix cana, Cebus capucinus, Saimiri sciureus, Saimiri boliviensis, Ateles geoffroyi and
Alouatta palliata [
8,
16‐
18]. Most recent studies have been conducted in Brazil [
24‐
29] and few others in Costa Rica [
30] and Venezuela [
6]. In Colombia, recent reports of
Plasmodium infection on free-ranging primates are not available, and the last studies were conducted between 1952 and 1968. These early studies found evidence of
P. brasilianum infecting NHP, including
Ateles geoffroyi, Cebus albifrons, Cebus apella, Cebus capucinus, Lagothrix lagotricha and
Saimiri sciureus [
15,
17,
21].
The zoonotic risk of transmission between humans and NHP involves the presence of insect vectors feeding on both hosts; thus, mosquito feeding behaviour can influence
Plasmodium transmission between humans and NHP [
31]. In Africa, Makanga et al. documented that certain sylvatic mosquitoes infected with ape parasites also bite humans, being potential bridge vectors between humans and apes [
32].
In Colombia, the most important malaria vectors are
Anopheles (Nyssorhynchus) albimanus, Anopheles (Nys.) darlingi and
Anopheles (Nys.) nuneztovari [
33]. Studies on
Anopheles mosquitoes in the country have been mainly focused in urban transmission cycles. However, in a context of pervasive transformation of natural areas into agricultural fields and extensive cattle ranches, it is essential to identify malaria vectors in forested areas, as these species may transmit
Plasmodium from NHP to humans or vice versa [
31].
In a socio-ecological context, the increasing human population coupled with greater demand for agricultural land, has led to an incremental deforestation in tropical countries where malaria is endemic [
34]. It has been reported that deforestation and land use changes greatly influence malaria’s incidence [
35]. In Colombia, the Middle Magdalena River valley is a region where the natural forest has been reduced to less than 15% of the original coverage due to deforestation and land use change [
36]. This pervasive process might increase the contact between humans and NHP as well as affect parasite-host dynamics [
37].
In this context, this study aimed to identify the prevalence of Plasmodium in five fragmented forest patches in Colombia in order to infer a potential risk of malaria zoonotic transmission involving NHP. The risk can exist if infected NHP species and infected Anopheles species are present in the study sites. To achieve this goal, the main objectives were: (i) to determine the presence and infection rate of Plasmodium parasites in NHP and Anopheles, (ii) to establish if infection rates vary among infected Anopheles species, and (iii) to evaluate if sites with different degrees of habitat transformation exhibit variation in infection rates.
Discussion
In this study, three
Plasmodium species were found infecting NHP in the Magdalena River valley. As expected, the most prevalent parasite species was
P. malariae/P. brasilianum that naturally infects different NHP species in Central and South America [
6,
25,
30]. Interestingly,
P. brasilianum has been reported infecting humans living in close proximity with NHP in the Venezuelan Amazon [
6], which highlights the risk of parasite transmission from NHP to humans.
The finding of
P. vivax/P. simium in NHP is of great interest given that
P. vivax has been the most prevalent species historically recorded in the country. In 2017, after analysing 28 human blood samples from an outbreak in the Atlantic Forest coastal region of Brazil,
P. simium was detected infecting humans [
24]. Authors suggested that this species could be circulating in humans before, but was misdiagnosed as
P. vivax due to the absence of adequate diagnostic techniques to perform species identification. Additionally, Grigg and Snounou (2017) consider Brazilian monkeys as reservoirs for
P. vivax [
54]. The presence of sylvatic reservoirs is relevant as it can potentially threaten successful malaria elimination campaigns [
54]. In the Colombian context the presence of infected monkeys should be taken into account when elucidating the potential risk of human infection.
In our study, the unexpected presence of
P. falciparum infecting
Alouatta seniculus open very relevant questions and concerns. Although this species has been found in New World primates [
7,
55] it is not as prevalent as
P. brasilianum. The presence of
P. falciparum in NHP suggests parasite transmission from humans to NHP, which is relevant when evaluating human contribution to emerging infectious diseases in sylvatic NHP. Other vector borne diseases transmitted from humans to primates such as Yellow Fever in South America, have negatively impacted populations of
Alouatta guariba clamitans and
Alouatta caraya in Argentina and Brazil, reassessing their conservation status to Critically Endangered and Near Threatened respectively [
56]. Regarding the establishment of
P. falciparum in the wild, Araújo et al. considered that, due to the ability of the parasite to develop in NHP, mosquito infection from infected NHP is likely and so is the establishment of a sylvatic transmission cycle [
7].
Although infection with
Plasmodium species has been reported in the genera
Alouatta, Cebus, Aotus and
Ateles [
7,
8,
17], results obtained in this study constitute new records at the species level for
Cebus versicolor, Ateles hybridus and
Aotus griseimembra.
Fecal samples have been used in different studies mainly in Africa and Asia [
57,
58] for
Plasmodium detection as a simple, non-invasive and inexpensive alternative to blood samples. However, they degrade quickly [
58] and the presence of bacteria and polysaccharides from plant diet, which are potential inhibitors of PCR, makes it difficult to use these samples for diagnosis by PCR [
59]. To solve this inconveniences, there was used RNA
later for sample preservation and BSA in the PCR mix in order to stabilize the DNA [
60]. Since
Plasmodium detection from blood is more sensitive than from fecal samples [
57,
61], prevalence rates found in this study may be underestimated, given that blood samples could not be obtained for all sampled primates. Also, those differences in detection sensitivity according to the type of sample could partially explain the lack of coincidence in the obtained results for the tested paired fecal and blood samples. For future studies, in order to confirm
Plasmodium species circulating in zoonotic cycles, whole genome sequencing should be performed. In this way, it is possible to determine if
Plasmodium infections are caused by
P. simium or
P. vivax, and
P. malariae or
P. brasilianum.
Regarding mosquito collections, all the species found in this study were known records for the study sites:
An. nuneztovari, An. triannulatus, An. neomaculipalpus and
An. oswaldoi have been previously reported in Santander Department [
62,
63], and
An. punctimacula, An. neomaculipalpus and
An. triannulatus in Antioquia Department [
33], as well as
An. oswaldoi [
64].
Plasmodium vivax was found infecting three species,
An. neomaculipalpus, An. triannulatus and
An. nuneztovari, which is concordant with previous records [
65,
66].
Anopheles neomaculipalpus is known to be highly anthropophilic [
65] while
An. triannulatus has been collected resting on cattle and is known to colonize transformed environments and become very abundant [
67]. Lucitania, Rompederos and San Juan exhibit the presence of cattle and altered ecosystems mainly due to the progressive introduction of oil palm plantations. It has been reported that deforestation related to monoculture favours the presence of ponds which are frequent breeding sites of
An. nuneztovari [
68], a species recognized as primary malaria vector in Colombia [
69].
Infection with
P. malariae was found in
An. triannulatus and
An. oswaldoi as has also been previously recorded [
70,
71].
Anopheles triannulatus has been reported with zoophilic and anthropophilic activities [
33] and
An. oswaldoi has been incriminated as secondary vector in the country [
69].
Anopheles triannulatus and
An. oswaldoi were found in this study, supporting previous reports and confirming their presence in Santander and Antioquia Departments.
The most abundant species were
An. oswaldoi and
An. neomaculipalpus found infected with
P. vivax and
P. malariae, respectively; this suggests their potential role as vectors in the study sites. Rompederos and Lucitania were the localities with highest MIR.
Anopheles vectors and NHP exhibit different selection strategies; while the lifespan of NHP is long, probably supporting a long course of infection, mosquitoes have a short lifespan and marked fluctuation in population densities related to environmental variables [
72,
73]. Possibly the sampling time of this study coincided with low
Anopheles local abundances thus the number of collected individuals was below the detection threshold. Long-term studies including seasonal variation would allow a better understanding of hosts’ population dynamics and the parasite transmission system in the studied environment.
Regarding the diversity of Anopheles species it was higher in Rompederos and San Juan, compared to Lucitania. This could be explained partially by the fact that those fragments belong to flooded forests which could provide optimal mosquito breeding sites, while Lucitania is a terra firme fragment forest.
The analyses on the effect of habitat fragmentation and distance to nearest town over the prevalence of
Plasmodium in NHP didn’t show any significant association, while the fragmentation but not the distance to nearest town had an effect over the MIR. However, when evaluating malaria risk, it is important to consider that multiple factors and processes interact e.g. the environment (land cover use/change, landscape transformations), human populations (host susceptibility, movement patterns, forest-related activity), vector biology (vector activity and life cycle, mosquito species distribution) [
74]. This study only focused in some of those factors (e.g.:
Plasmodium infection rates), but the complexity of the malaria cycle is a fact that must be taken into account, and for further studies it is suggested to involve as many factors as possible.
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