Malaria is an infectious disease caused by parasites of the genus Plasmodium, Class Sporozoa, and transmitted by mosquitoes belonging to the Anopheles genus (Bruce-Chwatt 1985). This disease is recognized as a major worldwide public health concern, with approximately 306,000 registered cases reported in Brazil in 2009 (Oliveira-Ferreira et al. 2010). More than 99% of human malaria cases in Brazil are concentrated in the endemic Amazon region. Outside of the Amazon, 92% of reported cases originate from endemic areas of Brazil and African countries (Brasil 2005a).

The identification of parasite species causing malaria has clear implications for human health, and, therefore, on the ability to treat and control malaria in affected areas (Deane 1992). Five Plasmodium species are responsible for human infections (P. vivax, P. falciparum, P. malariae, P. ovale, and P. knowlesi) (Greenwood et al. 2005, Cox-Singh et al. 2008) and are commonly targeted for malaria surveillance (Brasil 2005a). However, recent work has shown that Plasmodium species infecting primates, namely P. brasilianum and P. simium, are genetically similar to P. malariae and P. vivax of humans, respectively (Ayala et al. 1999; Tazi and Ayala 2011). This finding has led some investigators to conclude that primates could serve as reservoir hosts for Plasmodium infection (Fandeur et al. 2000, Hayakawa et al. 2009, Duarte et al. 2008). Infection surveillance in primates, including neotropical primates, has been suggested as being important for public health malaria screening and control programs (Duarte et al. 2008, Hayakawa et al. 2009).

Previous studies have shown that simian malaria is widespread in Brazil, with 14% infection prevalence among surveyed mostly belonging to the family Cebidae (Deane 1992). This is of particular concern for human health when anthropogenic induced change disrupts the ecosystem balance, generating opportunities for zoonotic disease transmission. With the Brazilian government’s recent initiative to reopen the BR-319 highway that connects the capital city of the state of Amazonas (Manaus) to the capital city of Rondônia (Porto Velho), there will be significant anthropogenic disturbance in areas that cross-protected lands. Other regions are also undergoing intense anthropogenic pressures through infrastructure development (e.g., the construction of a hydroelectric power station in Rondônia). Such anthropogenic actions have been implicated in increasing vector-borne diseases such as malaria or disseminating pathogens to new areas through the movement of wildlife (Hunter et al. 1982; Alemayehu et al. 1998; Bickel et al. 2003; Daszak et al. 2000). Therefore, Plasmodium infection surveillance among primates in these regions is important to better understand the potential epidemiologic implications.

The present study aimed to investigate the occurrence of Plasmodium spp. infection in free-ranging primates from Amazon regions under the influence of anthropogenic action.

From January 2010 to February 2011, venous blood was opportunistically collected from 19 primates in two locations of the Brazilian Amazon region (Fig. 1). The animals represented seven different genus and 52.6% (10/19—6♂; 4♀) were from the state of Amazonas (along the BR-319 road, 5°12′S, 61°50′W) and 47.4% (9/19—4♂; 5♀) from the state of Rondônia (9°47′S, 65°16′W; Madeira river, Abunã municipality) (Table 1).

Figure 1
figure 1

Capture sites and Plasmodium spp. survey localities. a Positive sites for P. brasilianum are shown with black dots. a Map of BR-319 (Amazonas State), showing the location of Plasmodium positive animals (P182 and P177). b Map of Rondônia State showing location of Plasmodium positive animals (P169 and P171). For all maps, negative animals (PCR and Smear) are shown with white dots.

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Table 1 Distribution of New World Primate Species Studied According to the Capture Location, Gender, Plasmodium spp. Results, 2010–2011, Brazil.
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Sampled animals from Amazonas (Fig. 1a) were collected as part of a taxonomy and biodiversity survey conducted by Wildlife Conservation Society (WCS-Brazil) to study the impact produced by the opening of the BR-319 highway. The animals from Rondônia (Fig. 1b) were collected by the Museum of Zoology, University of São Paulo, in order to study the resulting impact on biodiversity from construction of a hydroelectric power station on the Madeira River. All animals were sampled post-mortem and in good body condition.

Thin and thick blood smears from sampled animals as well as DNA extraction were performed to screen for Plasmodium. Blood smears were stained according to the Walker`s technique, (Brasil 2005b) and 200 microscopic fields were examined under light microscopy (1.000×). DNA was extracted from red blood cells with the GFXTM Genomic Blood DNA Purification Kit (Amersham Biosciences, GE Healthcare), following the manufacturer’s instructions. In order to detect Plasmodium, polymerase chain reaction (PCR) was carried out according to the previously described nested, genus-specific protocol (Santos et al. 2009). A second nested PCR using methods previously described (Perkins and Schall 2002) was performed to amplify a fragment of ~1.1 kb from the mitochondrial cytochrome b gene (cytb) to identify Plasmodium species in positive samples. Amplified products were purified from agarose gels and sequenced using a commercial kit (BigDye TM terminator mix, Applied Biosystems, Foster City CA, USA). Sequences obtained were aligned with sequences from GenBank using BLASTN (Basic Local Alignment Search Tool) available at http://www.ncbi.nlm.nih.gov/blast/Blast.cgi (Altschul et al. 1997).

All procedures were approved by the Ethical Principles in Animal Research, of the School of Veterinary Medicine and Animal Sciences—University of São Paulo (protocol number 1617/2009), and were in full compliance with federal permits issued by the Brazilian Ministry of the Environment (SISBIO # 24319-3 and 18861-3).

The estimated prevalence of Plasmodium spp. was 21.1% (4/19; 95% exact CI: 6.1–45.6%), with 22.2% (2/9; 95% exact CI: 2.8–60.0%) and 20% (2/10, 95% exact CI: 2.5–55.6%) of animals testing positive from Rondônia and Amazonas, respectively. All 4 animals were positive on both smears and PCR. In Rondônia, infection was detected in 01 Pithecia sp. (P171) and 01 Callicebus dubius (P169), while both animals were Callicebus caligatus (P177; P182) (Fig. 1) in Amazonas.

Plasmodium from all infected primates showed a 100% identity to P. brasilianum (Peruvian III, GenBank GQ355484) (Fig. 2). This isolate was originally obtained from a Saimiri sciureus peruviensis in Iquito, Peru, in 1987 (Collins et al., 1990).

Figure 2
figure 2

Photomicrographs of parasites visualized from thin blood smears obtained from neotropical primates: gametocytes (×1,000) of P. brasilianum in a Callicebus caligatus, Amazonas state and b Callicebus dubius, Rondônia state, 2010

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Our results suggest that there is a sylvatic cycle from P. brasilianum in these regions with anticipated anthropogenic impacts. The importance of this finding for human health arises from the genetic similarities between P. brasilianum of primates and P. malariae of humans. Some authors hypothesize that the origin of human Plasmodium is related to the species that infect non-human primates due to morphological and genetic similarities (Escalante et al. 1995; Ayala et al. 1999; Fandeur et al. 2000; Tazi and Ayala 2011). The high genetic identity found between P. malariae from humans and P. brasilianum from non-human primates suggests the occurrence of recent transfers between hosts (Tazi and Ayala 2011). In South America, sometimes these parasites are described as P. malariae/P. brasilianum, but they can also be described based on the parasite host (non-human primate = P. brasilianum; human = P. malariae) (Escalante et al. 1995; Fandeur et al. 2000). In recent work, our group compared the genetic diversity of Plasmodium from these positive primates with P. malariae from humans (Guimarães et al. 2012) and found a greater diversity among isolates of P. brasilianum compared to P. malariae indicating that P. malariae might be derived from P. brasilianum, as has been proposed by others (Tazi and Ayala 2011). Such similarities have led some authors to conclude that these are likely the same species of Plasmodium and primates may serve as an important reservoir host (Fandeur et al. 2000, Duarte et al. 2008).

Additional support for new world primates as zoonotic reservoirs has been previously discussed (De Arruda et al. 1989; Duarte et al. 2008).

The opportunistic nature of animal sampling limited the number of individuals that could be evaluated for this study. Although we found a higher prevalence (21%), our findings largely corroborated Deane (1992), which reports P. brasilianum infection in 10% of surveyed primates from the Amazon region. Previous studies have also identified which species of Plasmodium infect wild primates in the Brazilian Amazon; for example, P. brasilianum was reported infecting Cebus, Callicebus, Alouatta, Ateles, Lagothrix, and Cacajao (Coatney et al. 1971; Deane and Neto 1969). Despite the small sample size, we detected a substantial proportion of P. brasilianum infection in primates in this region. Additional studies with larger sample sizes could increase the precision of prevalence estimates.

The Brazilian Amazon is facing dramatic social, economic, and cultural changes. In this scenario, public and private economic activities may potentiate the occurrence of zoonosis. For example, after the construction of dams and highways epidemics of malaria can occur due to changes in environmental, ecological (dynamics of host-vector relationship), and social factors (Katsuragawa et al. 2008; Carvalho et al. 2009). Due to the nature of environmental disturbance around the two study sites, it is recommended that the potential for zoonotic disease transmission be considered by public health officials. Both human and primate populations should be closely monitored to mitigate risk of exposure and infection transmission and better understand the nature of the malaria risks post anthropogenic change. Along with anthropogenic changes arise wildlife rescue programs that strive to mitigate environmental impacts by releasing animals into adjacent areas or sending them to captivity in non-Amazonian states. The identification of P. brasilianum in primates from this region demonstrates that a substantial proportion of primates in the Amazon region are infected with simian malaria and could facilitate pathogen dissemination, as discussed by others (Daszak et al. 2000; Thoisy et al. 2001; Cunningham 1996; Woodford and Rossiter 1993). The authors recommend that all neotropical primates from the Amazon region be tested for Plasmodium spp. infection prior to any translocation or movement of animals in order to mitigate the risk of introducing malaria into non-endemic regions where competent invertebrate vectors are present.