New potential Plasmodium brasilianum hosts: tamarin and marmoset monkeys (family Callitrichidae)

NHPs from the Primate Centre of Rio de Janeiro (CPRJ, according to its initials in Portuguese) were studied in this survey. The CPRJ, part of the Brazilian Institute of Environment and renewable natural resources (Register Number 458460), is a unit for wild monkey conservation, which occupies an area of 259.54 hectares. It is located in the municipality of Guapimirim (between latitudes 22°27′ and 22°32′ S and longitudes 42°50′ and 42°56′ W), almost in the centre of RJ state, at the Serra dos Órgãos slopes, in the Atlantic forest (Fig. 1). The study had the authorization of the Fiocruz Research Ethical Committee. The Fiocruz Ethics Committee for the Use of Laboratory Animals agreed to the protocol for sample collection. The handling of monkeys was exclusively done by CPRJ technicians.

A total of 122 NHPs of the family Callitrichidae were sampled in June 2015 (n = 4), January 2016 (n = 115) and July 2016 (n = 4). The origin of all animals, sex, species and date of sample collection are shown in Additional file 1: Table S1. Of these animals, 10 belonged to the genus Callithrix (five Geoffroy marmosets—Callithrix geoffroyi, two common marmosets—Callithrix jacchus, and three hybrids); 23 of the genus Saguinus (nine golden-handed tamarins—Saguinus midas, eight pied bare-face tamarins—S. bicolor, 3 black-handed tamarins—Saguinus niger, one ochraceous bare-face tamarin—Saguinus martinsi ochraceus, one Martin’s bare-face tamarin—Saguinus martinsi martinsi and one hybrid); 85 of the genus Leontopithecus (eight golden lion tamarins—Leontopithecus rosalia, four black lion tamarins—Leontopithecus chrysopygus and 73 golden-headed lion tamarins—Leontopithecus chrysomelas); and five belonged to the genus Mico (four black-and-white tassel-ear marmosets—Mico humeralifer and one Maués marmoset—Mico mauesi) (Additional file 1: Table S1). The species were determined by the veterinaries from CPRJ based on Rylands et al. and Van Roosmalen et al. [26, 27]. Seventy-five NHPs (61%) were captive from conservation centers (CPRJ and Renabra nursery) or Zoos (Belo Horizonte Zoo, Mario Nardelli Zoo, Niteroi Zoo and São Carlos Zoo) and 47 (39%) were wild caught in different areas, including eight in the Brazilian Amazon (Additional file 1: Table S1). It is important to highlight that the wild animals from Serra da Tiririca Park in Niteroi (RJ state) were housed in the CPRJ Centre for a short period of time before transfer to be released in conservation areas of Bahia state or other states. No animal was splenectomized at the time of sample collection. Approximately 5 mL of blood were taken in vacuum tubes containing EDTA by femoral vein puncture. Packed red blood cells were separated by centrifugation, frozen and sent to the malaria laboratory at CPqRR, Fiocruz Minas.

Thick and thin blood smears stained with Giemsa’s solution were examined under 100× optical microscopy for morphological analysis by two expert microscopists using standard methods [37].

DNA extraction from blood samples was performed using the QIAamp DNA mini Kit (Qiagen, Venlo, The Netherlands) according to manufacturer’s recommendations. The DNA was eluted in 45 μL of Buffer AE and stored at −20 °C until it was used. The samples were subjected to nested PCR using primers for identification of human Plasmodium species (P. malariae/P. brasilianum, P. vivax/P. simium and Plasmodium falciparum), targeting the small subunit of 18S ribosomal RNA (18S SSU rRNA) gene [38]. Briefly, all PCR reactions were performed in 20 μL final volume containing 250 μM each oligonucleotide primer, 10 μL of master mix (Promega) (0.3 units of Taq Polymerase, 200 μM each deoxyribonucleotide triphosphates and 1.5 mM MgCl₂) and 1 μL DNA (5–10 ng). For all positive samples the DNA extraction was repeated and PCR performed using 2 μL whenever the use of 1 μL was negative. The PCR assays were performed using an automatic thermocycler (PTC-100TM v.7.0) (MJ Research Inc, USA) and the following cycling parameters were used: an initial denaturation at 95 °C for 5 min followed by 24 cycles of annealing at 58 °C for 2 min, extension at 72 °C for 2 min and denaturation at 94 °C for 1 min followed by a final annealing incubation at 58 °C for 2 min and extension at 72 °C for 5 min. The temperature was then reduced to 4 °C until the samples were removed from the cycler. The second round of PCR was performed with the same parameters of the first reaction, using 1 μL of the amplified DNA from the first reaction and 29 cycles of amplification. It is important to highlight that DNA extraction and master mix preparation were performed in “parasite DNA-free rooms” distinct from each other with different sets of pipettes and using plugged pipette tips to prevent cross-contamination. Every PCR reaction included positive controls for each pair of primers and a negative control with no DNA. The sources of genomic DNA samples that served as positive controls in the nested PCR assays were: i) DNA of P. brasilianum of MR4 (Malaria Research and Reference Reagent Resource Center—ATCC, USA); (ii) DNA of patient with high parasitaemia for P. vivax; (iii) P. falciparum DNA, strain 3D7 maintained in the malaria laboratory (CPqRR-Fiocruz Minas).

The amplified fragments were visualized using electrophoresis on 2% agarose gels in 1× TAE buffer (40 mM Tris–acetate, 1 mM EDTA) with 5 μg/mL ethidium bromide (Invitrogen) in a horizontal system (Bio-Rad) at 100 V for about 30 min. The gels were examined under a UV transilluminator and the image captured under digital system (UVP—Bio-Doc System it). Electrophoresis was performed in a room specifically assigned for analysis of amplified DNA, with appropriate sets of micropipettes and plugged pipette tips.

Two microlitre of PCR products were amplified using 2.0 μM of forward species-specific primer and 1 μL of Big Dye terminator kit for DNA sequencing. The following cycling parameters were used: 96 °C for 1 min, 35 cycles of 96 °C for 15 s, followed by the 58 °C for 15 s and 60 °C for 15 s. The fragments were precipitated using ammonium acetate, suspended in formamide HI-DI (Applied Biosystems) and electrophoretically separated in ABI 3730 DNA automatic sequencer.

The sequence data were analyzed using the Blast program and the sequences aligned using the Clustal W programs in Bioedit package v7.2.5 [39].