2.1. Sera Sampling from Non-Human Primates

Serum samples were collected in two malaria endemic regions in Brazil, the Amazon and Atlantic Forest regions, and in a non-endemic region, the Cerrado region in Central Brazil. A total of 373 samples were collected from free-living animals and 122 from captive animals. Sera from free-living animals (Figure 1A) were obtained in different localities: (i) in the Amazon Region (n = 155), collected close to Porto Velho city in Rondônia state, from March 2009 to November 2012, during projects related to environmental management for the construction of hydroelectric power plants [7,8]; (ii) in the Atlantic Forest (n = 111), from October 1997 to July 2005, in forest fragments around the municipality of São Paulo [9] and the municipality of Indaial in Santa Catarina state, from June 2001 to February 2015; and (iii) in the Cerrado region (n = 107), from April 2000 to March 2001 and January to December 2009, in the canopy of woods in flooded areas of the lake at Porto Primavera dam during wildlife rescue operations [10]. Sera from captive animals (Figure 1B) were from the Atlantic Forest area (n = 103), 60 being from the São Paulo city Zoo, 31 from the Tietê Ecological Park and 12 from CETAS (Centro de Triagem de Animais Silvestres; Wildlife Rescue and Rehabilitation Center) in Lorena city (n = 08) and Unimonte in São Vicente city (n = 1), as well as from Bauru city Zoo (n = 1) and the Centre for Biological Research in Indaial city (n = 2). From the Amazon Region (n = 19), samples were collected from animals kept close to areas of human habitation, such as the Ecological Park of Porto Velho city, and animals rescued by IBAMA (Brazilian Institute of Environment and Renewable Natural Resources) which had been kept illegally as pets in rural or suburban areas.

Sera collection sites in the Amazon Region, Cerrado and the Atlantic Forest, Brazil, from free-living (A) and captive animals (B).

All procedures were approved by the Ethical Committee in Animal Research (CEUA) of the Institute of Tropical Medicine of São Paulo, University of Sao Paulo (number 2014/281A) and were in full compliance with federal permits issued by the Brazilian Ministry of the Environment (SISBIO numbers 14081, 17302, 18861, 24319, 44751, 47812, 50076).