Human malaria due to zoonotic transmission has been recorded in the Atlantic Forest, an extra-Amazonian area in Brazil, which are a challenge for malaria control. Naturally acquired humoral immune response against pre-erythrocytic and erythrocytic antigens of Neotropical primates (NP) was evaluated here to improve the knowledge about the exposure of those animals to the malaria transmission and support the identification of the potential reservoirs of the disease in the Atlantic Forest. Blood samples of 154 monkeys from three areas of the Atlantic Forest were used to identify IgG antibodies against peptides of the repeat region of the major pre-erythrocytic antigen, the circumsporozoite protein (CSP), of Plasmodium vivax (PvCSP), Plasmodium brasilianum/Plasmodium malariae (Pb/PmCSP), and Plasmodium falciparum (PfCSP) by ELISA. Antibodies against erythrocytic recombinant antigens of P. vivax, Apical membrane antigen 1 (PvAMA-1), Erythrocyte binding protein 2 (PvEBP-2) and domain II of Duffy binding protein (PvDBPII) were also evaluated. Parameters, such as age, sex, PCR positivity, and captivity, potentially associated with humoral immune response were analyzed. Eighty-five percent of NP had antibodies against at least one CSP peptide, and 76% against at least one P. vivax erythrocytic antigen. A high percentage of adults compared to non-adults were seropositive and showed increased antibody levels. Neotropical primates with PCR positive for P. simium had a significantly higher frequency of positivity rate for immune response against PvEBP-2, PvDBPII and also higher antibody levels against PvDBPII, compared to PCR negative NPs for this species. Monkeys with PCR positive for P. brasilianum/P. malariae showed higher frequency of seropositivity and antibody levels against Pb/PmCSP. Levels of antibodies against Pb/PmCSP, PvEBP-2 and PvDBPII were higher in free-living than in captive monkeys from the same area. All Platyrrhine families showed antibodies against CSP peptides, however not all showed IgG against erythrocytic antigens. These findings showed a high prevalence of naturally acquired antibodies against CSP repeats in all studied areas, suggesting an intense exposure to infected-mosquitoes bites of NP from all families. However, mainly monkeys of Atelidae family showed antibodies against P. vivax erythrocytic antigens, suggesting blood infection, which might serve as potential reservoirs of malaria in the Atlantic Forest.
Plasmodium falciparum and Plasmodium vivax have evolved with host switches between non-human primates (NHPs) and humans. Studies on the infection dynamics of Plasmodium species in NHPs will improve our understanding of the evolution of these parasites; however, such studies are hampered by the difficulty of handling animals in the field. The aim of this study was to detect genomic DNA of Plasmodium species from the faeces of New World monkeys. Faecal samples from 23 Alouatta clamitans from the Centre for Biological Research of Indaial (Santa Catarina, Brazil) were collected. Extracted DNA from faecal samples was used for molecular diagnosis of malaria by nested polymerase chain reaction. One natural infection with Plasmodium simium was identified by amplification of DNA extracted from the faeces of A. clamitans. Extracted DNA from a captive NHP was also used for parasite genotyping. The detection limit of the technique was evaluated in vitro using an artificial mixture of cultured P. falciparum in NHP faeces and determined to be 6.5 parasites/µL. Faecal samples of New World primates can be used to detect malaria infections in field surveys and also to monitor the genetic variability of parasites and dynamics of infection.
Plasmodium simium is a parasite from New World monkeys that is most closely related to the human malaria parasite Plasmodium vivax; it also naturally infects humans. The blood-stage infection of P. vivax depends on Duffy binding protein II (PvDBPII) and its cognate receptor on erythrocytes, the Duffy antigen receptor for chemokines (hDARC), but there is no information on the P. simium erythrocytic invasion pathway. The genes encoding P. simium DBP (PsDBPII) and simian DARC (sDARC) were sequenced from Southern brown howler monkeys (Alouatta guariba clamitans) naturally infected with P. simium because P. simium may also depend on the DBPII/DARC interaction. The sequences of DBP binding domains from P. vivax and P. simium were highly similar. However, the genetic variability of PsDBPII was lower than that of PvDBPII. Phylogenetic analyses demonstrated that these genes were strictly related and clustered in the same clade of the evolutionary tree. DARC from A. clamitans was also sequenced and contained three new non-synonymous substitutions. None of these substitutions were located in the N-terminal domain of DARC, which interacts directly with DBPII. The interaction between sDARC and PvDBPII was evaluated using a cytoadherence assay of COS7 cells expressing PvDBPII on their surfaces. Inhibitory binding assays in vitro demonstrated that antibodies from monkey sera blocked the interaction between COS-7 cells expressing PvDBPII and hDARC-positive erythrocytes. Taken together, phylogenetic analyses reinforced the hypothesis that the host switch from humans to monkeys may have occurred very recently in evolution, which sheds light on the evolutionary history of new world plasmodia. Further invasion studies would confirm whether P. simium depends on DBP/DARC to trigger internalization into red blood cells.
Acute chagasic encephalitis is a clinically severe central nervous system (CNS) manifestation. However, the knowledge of the nervous form of Chagas disease is incomplete. The role of the muscarinic acetylcholine receptor (mAChR) on mice behavior and brain lesions induced by Trypanosoma cruzi (Colombian strain) was herein investigated in mice treated with the mAChR agonist and antagonist (carbachol and atropine), respectively. Immunosuppressed or non-immunosuppressed mice were intracerebroventricularly (icv) or intraperitoneally (ip) infected. All groups were evaluated 15 d.p.i. (days post infection). Intraperitoneally infected animals had subpatent parasitemia. Patent parasitemia occurred only in icv infected mice. The blockade of mAChR increased the parasitemia, parasitism and lesions compared to its activation. Infected not treated (INT ip) mice did not present meningitis and encephalitis, regardless of immunosuppression. INT icv brains presented higher cellularity, discrete signs of cellular degeneration, frequent presence of parasites and focal meningitis. The immunosuppressed atropine + icv mice presented increased intracellular parasitism associated with degenerative parenchymal changes, while carbachol + icv mice presented discrete meningitis, preservation of the cortex and absence of relevant parasitism. Cholinergic receptor blockage increased impairment of coordination vs. receptor activation. Muscarinic cholinergic pathway seems to be involved in immune mediated cell invasion events while its blockade favored infection evolution, brain lesions, and behavioral alterations.
BackgroundHigh-copy genomic sequences could be used as PCR targets for the detection of Plasmodium infections, providing increased sensitivity over single- or low-copy genes. Mitochondrial genomes of malaria parasites are present in multiple copies in a single mitochondrion, and each parasite has many mitochondria. Here, we describe the development of seven species-specific qPCR assays for the diagnosis of Plasmodium vivax and Plasmodium falciparum, targeting coding and non-coding mitochondrial genomic regions.MethodsThe optimization of the qPCR protocols involved a gradient of annealing temperatures and concentrations of primers and probes, as well as the inclusion of PCR additives/enhancers [e.g., dimethyl sulfoxide (DMSO), glycerol, bovine serum albumin (BSA)] to improve the specificity of qPCR amplification.ResultsNon-specific amplification of other Plasmodium species and of human targets was observed in different levels for all assays. Regardless of the late Cq values for most non-specific amplifications, the application of a cutoff value did not completely exclude false-positive amplification, compromising the specificity and also the sensitivity of the assays.ConclusionsTherefore, although mitochondrial targets have higher sensitivity, they frequently lose specificity due to their high levels of sequence conservation. A screening to evaluate the cross-reaction between Plasmodium species and the non-specific amplification of human malaria-free samples must be performed for Plasmodium mitochondrial assays.
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