RESEARCH ARTICLE Outcomes of Trypanosoma cruzi and Trypanosoma evansi infections on health of Southern coati (Nasua nasua), crab-eating fox (Cerdocyon thous), and ocelot (Leopardus pardalis) in the Brazilian Pantanal Filipe Martins Santos1*, Gabriel Carvalho de Macedo1, Wanessa Teixeira Gomes Barreto2, Luiz Gustavo Rodrigues Oliveira-Santos2, Carolina Martins Garcia2, Guilherme de Miranda Mourão2,3, Grasiela Edith de Oliveira Porfı́rio1, Elizangela Domenis Marino1, Marcos Rogério André4, Lı́via Perles4, Carina Elisei de Oliveira1, Gisele Braziliano de Andrade1, Ana Maria Jansen1,5, Heitor Miraglia Herrera1,2 1 Programa de Pós-Graduação em Ciências Ambientais e Sustentabilidade Agropecuária, Universidade Católica Dom Bosco, Campo Grande, Mato Grosso do Sul, Brazil, 2 Programa de Pós-Graduação em Ecologia e Conservação, Universidade Federal de Mato Grosso do Sul, Campo Grande, Mato Grosso do Sul, Brazil, 3 Laboratório de Vida Selvagem, Centro de Pesquisa Agropecuária do Pantanal, Empresa Brasileira de Pesquisa Agropecuária, Corumbá, Mato Grosso do Sul, Brazil, 4 Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal, São Paulo, Brazil, 5 Laboratório de Biologia de Tripanosomatı́deos, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Rio de Janeiro, Brazil * filipemsantos@outlook.com Abstract The occurrence of Trypanosoma spp. in wild carnivore populations has been intensively investigated during the last decades. However, the impact of these parasites on the health of free-living infected animals has been largely neglected. The Pantanal biome is the world’s largest seasonal wetland, harboring a great diversity of species and habitats. This includes 174 species of mammals, of which 20 belong to the order Carnivora. The present study aimed to investigate the effect of Trypanosoma evansi and Trypanosoma cruzi infections and coinfections on the health of the most abundant carnivores in the Pantanal: coati (Nasua nasua), crab-eating fox (Cerdocyon thous), and ocelot (Leopardus pardalis). We captured 39 coatis, 48 crab-eating foxes, and 19 ocelots. Diagnostic tests showed T. cruzi infection in 7 crab-eating foxes and 5 coatis. Additionally, 7 crab-eating foxes, 10 coatis, and 12 ocelots were positive for T. evansi. We observed coinfections in 9 crab-eating foxes, 8 coatis, and 2 ocelots. This is the first report of T. evansi and T. cruzi infection on the health of free-living ocelots and crab-eating foxes. We showed that single T. evansi or T. cruzi infection, as well as coinfection, caused some degree of anemia in all animals, as well as an indirect negative effect on body condition in coatis and crab-eating foxes via anemia indica- tors and immune investment, respectively. Furthermore, the vigorous immune investment observed in sampled coatis, crab-eating foxes and ocelots infected by T. evansi, T. cruzi and coinfected can be highly harmful to their health. Overall, our results indicate that single PLOS ONE | https://doi.org/10.1371/journal.pone.0201357 August 15, 2018 1 / 15 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPENACCESS Citation: Martins Santos F, Carvalho de Macedo G, Teixeira Gomes Barreto W, Rodrigues Oliveira- Santos LG, Martins Garcia C, Miranda Mourão Gd, et al. (2018) Outcomes of Trypanosoma cruzi and Trypanosoma evansi infections on health of Southern coati (Nasua nasua), crab-eating fox (Cerdocyon thous), and ocelot (Leopardus pardalis) in the Brazilian Pantanal. PLoS ONE 13(8): e0201357. https://doi.org/10.1371/journal. pone.0201357 Editor: Érika Martins Braga, Universidade Federal de Minas Gerais, BRAZIL Received: May 28, 2018 Accepted: July 14, 2018 Published: August 15, 2018 Copyright: © 2018 Martins Santos et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The study was funded by Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT; grant PRONEX 006/2015) to GMM and https://doi.org/10.1371/journal.pone.0201357 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0201357&domain=pdf&date_stamp=2018-08-15 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0201357&domain=pdf&date_stamp=2018-08-15 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0201357&domain=pdf&date_stamp=2018-08-15 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0201357&domain=pdf&date_stamp=2018-08-15 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0201357&domain=pdf&date_stamp=2018-08-15 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0201357&domain=pdf&date_stamp=2018-08-15 https://doi.org/10.1371/journal.pone.0201357 https://doi.org/10.1371/journal.pone.0201357 http://creativecommons.org/licenses/by/4.0/ and combined infection with T. evansi and T. cruzi represent a severe risk to the health of wild carnivores in the Pantanal region. Introduction Although parasites are known to use resources from their hosts, thus affecting their energy bal- ance [1, 2], little is known about the effect of parasitism on the health of mammals that inhabit natural environments [3]. Loss of physical condition due to parasitism can negatively influence reproductive rates, movement, and survival of infected hosts [4–6]. The occurrence of Trypanosoma spp. in wild carnivores has sparked intensive investigation worldwide in the last decades [7–11]. Nevertheless, the impact of these parasites on the health of free-living infected animals has been largely neglected. Studies documenting the outcomes of Trypanosoma cruzi and T. evansi on the health of wild carnivores have shown that these par- asites can cause damage to the health of their hosts [12, 13]. In enzootic areas Trypanosoma species are maintained in a complex network transmission cycles including mammals and blood sucking vectors. While T. evansi transmission occur mechanically by hematophagus flies, such as tabanids and Stomoxys calcitrans, T. cruzi is cycli- cally transmitted through Triatominae faces [14,15]. Natural infection by T. evansi causes different degrees of anemia in several domestic and wild mammals. Infected animals display widespread subcutaneous edema, fever, lethargy, weight loss, abortion, nasal and ocular bleeding, and stiffness of the pelvic members [13,16,17]. Additionally, coatis and golden lion tamarins (Leontopithecus rosalia) infected with T. cruzi have been reported to present hematological disorders and cardiac diseases, respectively [18, 19, 20]. It has been suggested that coinfections play a central role in driving parasite dynamics [21, 22]. One such case is natural infection of the golden lion tamarin with T. cruzi: higher parasite- mia was observed in animals coinfected with nematode worms [23]. In fact, coinfections with multi-host parasites in free-living mammals may be highly dynamic and unpredictable because the ecological processes are stochastic. Cooccurrence of T. cruzi and T. evansi in wild carni- vores (e.g. Cerdocyon thous, Leopardus pardalis and Nasua nasua) has already been recorded [8, 13], but there is no knowledge about the impact of these parasites on the hosts’ health. Given the scarce knowledge about the health of free-living neotropical mammals infected by trypanosomatids, the present study aimed to investigate the effect of single and combined infection with T. evansi and T. cruzi on the hematological parameters of coatis (N. nasua), crab-eating foxes (C. thous), and ocelots (L. pardalis) in the Pantanal biome, the largest flood- plain of the world. Methodology Study area The study was carried out in a private ranch in the sub-region of Nhecolândia (19˚ 8’31.71"S 56˚47’40.97"O). The soil is sandy and vegetation is composed of deciduous and semi-decid- uous forests in “cordilheiras” (long strings of forest) and "capões" (forest patches surrounded by open native grasslands). The climate is marked by two distinct seasons: a warm rainy period (October to March) and a cold dry period (April to September). About 174 species of mam- mals was recorded in the Pantanal, of which 20 belongs to the order Carnivora [24]. Among Outcomes of Trypanosoma spp. infections on health of wild carnivores in the Pantanal PLOS ONE | https://doi.org/10.1371/journal.pone.0201357 August 15, 2018 2 / 15 FMS, and the Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Superior (CAPES) to the first author, FMS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. https://doi.org/10.1371/journal.pone.0201357 them, three species are considered abundant in the Pantanal: the southern coati, the crab-eat- ing fox, and the ocelot [25]. Sample collection Carnivores were sampled from November 2015 to October 2016. We used 50 Box-traps (90 × 45 × 50; EquiposFauna1) baited with bacon and tinned sardines to capture the target species. Once trapped, animals were sedated with an intramuscular injection of Zoletil 50 (containing tiletamine hydrochloride and zolazepan hydrochloride; Virbac) respecting the dosages currently recommended for each species, and marked with subcutaneous transpon- ders (AnimalTag1). Body condition (body size and mass) were recorded. Blood (~4 mL) was collected from the jugular vein, placed in tubes with and without ethylenediamine tetraacetic acid (EDTA), and stored in cool boxes until laboratory procedures. The animals were released at the capture site after recovery from anesthesia. Ethical approval All field procedures were conducted in accordance with a license granted by the Biodiversity Information and Authorization System of the Chico Mendes Institute for Biodiversity Conser- vation (license number 49662–5). The present study was approved by the Ethics Committee for Animal Use of Dom Bosco Catholic University, Campo Grande, MS (license number 19/ 2015). Health parameters The health of carnivores was inferred, mainly, by means of blood parameters. Packed cell vol- ume (PCV), red blood cell counts (RBC), and white blood cell counts (WBC) were measured up to 8 h after blood collection in Neubauer chambers, as described by Voigt [26]. Mean cor- puscular volume (MCV) was calculated based on the RBC and PCV values. The immunoglob- ulin concentration (IgG) was determined by titration with the indirect fluorescent antibody test (IFAT) [27,28] and by optical density using enzyme-linked immunosorbent assay (ELISA) [29]. Leukocyte (eosinophils, lymphocytes, monocytes, and neutrophils) counts were per- formed using blood smears fixed with methanol and stained with Giemsa [30]. We evaluated the health condition of sampled carnivores in terms of: (a) PCV, RBC, and MCV as anemia indicators; (b) monocyte and neutrophil counts as indicators of infection responses; and (c) lymphocyte counts and IgG concentration as indicators of immune invest- ment [30]. Diagnosis of T. evansi and T. cruzi infection Infections with T. evansi and T. cruzi were assessed by parasitological, molecular and serologi- cal tests. The parasitological test for T. evansi used the microhematocrit centrifuge technique (MHCT) according to Woo [31]. The absence of kinetoplast in buffy coat smears confirms T. evansi. For T. cruzi, the test was based on hemoculture by inoculating 300 μL of blood in Novy McNeal Nicole (NNN) medium with liver infusion tryptose (LIT), in duplicate. Hemoculture tubes were incubated at 27 ˚C for 30 days and monitored once a week. Molecular detection of Trypanosoma spp. infection was performed by nested polymerase chain reaction (nPCR). Genomic DNA was extracted from 200 μL of blood with EDTA using the QIAamp Blood DNA Mini Kit (Qiagen) according to the manufacturer’s instructions. Total DNA was diluted with 50 μL elution buffer and stored at -20 ˚C until molecular diagno- sis. We used as a target a variable region of the trypanosome 18S rRNA gene (600 bp), with Outcomes of Trypanosoma spp. infections on health of wild carnivores in the Pantanal PLOS ONE | https://doi.org/10.1371/journal.pone.0201357 August 15, 2018 3 / 15 https://doi.org/10.1371/journal.pone.0201357 external primers TRY927F and TRY927R, and internal primers SSU561F and SSU561R, according to Smith et al. [32]. TBR1 and TBR2 primers were applied to positive 18S rRNA samples to amplify a sequence of mini-chromosome satellite DNA for T. evansi, according to Masiga et al. [33]. Furthermore, D71 and D72 primers were used to amplify a conserved sequence of the large subunit of the ribosomal DNA gene (24Sα rDNA) in T. cruzi, according to Souto and Zingales [34]. Each reaction included sterile distilled water instead of DNA as negative control, and positive control samples from T. cruzi and T. evansi strains. PCR prod- ucts were visualized in 2% agarose gel after ethidium bromide staining under ultraviolet light. Serological tests were used to detect anti-T. evansi IgG antibodies in crab-eating foxes and ocelots by IFAT using a commercial fluorescein-conjugated antibody against dogs and cats IgGs, respectively. The cut-off value for IFAT was 1:40 [27]. There is presently no fluorescein- conjugated antibody against coatis’ IgGs. To detect anti-T. cruzi IgG antibodies, we used IFAT (fluorescein-conjugated antibody against dogs and cats IgGs) and ELISA (fluorescein-conju- gated antibody against raccoon’s IgGs), as described by Rocha et al. [28] and Alves et al. [29], respectively. The cut-off value for ELISA was defined as the mean optical absorbance of the negative controls +20%. We added two positive and two negative control sera to each reaction plate, as described by Alves et al. [29]. We considered an animal to be positive to Trypanosoma infection, when any of the four diagnostic tests used (hemoculture, MHCT, PCR or/and serological tests) was positive. Data analysis Descriptive statistic (mean ± standard deviation) was applied to obtain the mean health parameters of the specimens. The Shapiro-Wilk test served to establish whether the distribu- tion was normal. Finally, a Kruskal-Wallis test was applied to determine the differences between: no infection, T. evansi infection, T. cruzi infection, and coinfection. Post hoc Mann- Whitney tests were used to assess pair-wise results of the Kruskal-Wallis test. To determine the direct and indirect influences of infections and coinfections in relation to anemia, infection responses, immune investment and body condition, we carried out a path analysis. We assessed variation in body condition based on the standardized residuals from an ordinary linear regression between body mass (g) and head-body length (mm) of individuals, while accounting for age and sex effects (13). This should circumvent the effects of animal growth on the condition index. Therefore, the residuals were calculated for males and females separately. To perform dimensionality reduction of anemia, infection responses, and immune investment values, we used the principal coordinate analysis, a geometric technique that con- verts a matrix of distances between points in multivariate space into a projection that maxi- mizes the amount of variation along a series of orthogonal axes. We used an r value� 0.60 to interpret the results (positive or negative effect) of the path analysis. Path analysis describes two types of effects: direct and indirect. When the exogenous vari- able has an arrow directed towards the dependent variable, the effect is direct. When the effect is indirect, the arrow crosses one or more than one dependent variable until the final effect. The variables were considered to be statistically significant for p values� 0.05. All data were analyzed using R (version 3.4.2) [35]. Results We sampled 106 adult carnivores: 39 coatis (17 females and 22 males), 48 crab-eating foxes (22 females and 26 males), and 19 ocelots (eight females and 11 males). The different diagnostic tests showed T. evansi positivity in 12 ocelots (4 females and 8 males), 10 coatis (6 females and 4 males) and 7 crab-eating foxes (1 female and 6 males). Additionally, we found T. cruzi Outcomes of Trypanosoma spp. infections on health of wild carnivores in the Pantanal PLOS ONE | https://doi.org/10.1371/journal.pone.0201357 August 15, 2018 4 / 15 https://doi.org/10.1371/journal.pone.0201357 infection in 7 crab-eating foxes (4 female and 3 males), and 5 coatis (5 males). We observed coinfection in 9 crab-eating foxes (4 females and 5 males), 8 coatis (3 females and 5 males), and 5 ocelots (3 females and 2 males) (Table 1). Coatis Mean PCV values was significantly lower (χ2 = 11.94, df = 03, p< 0.05) in coatis infected with T. evansi (28.5±4.9) (U = 34.5, p< 0.05) and in coinfected animals (26.4±7.8) (U = 23, p< 0.05), when compared with non-infected coatis (35.8±5.2). Moreover, T. evansi infected and coinfected animals also presented lower means of RBC (T. evansi infection: 3.3×106 ±1.7 and coinfection: 3.3×106±1.2) and MCV (T. evansi infection: 94.2±19.7 and coinfection: 86.4 ±31.8) values, however without statistical significance (RBC: χ2 = 3.015, df = 03, p> 0.05; MCV: χ2 = 0.9674, df = 03, p> 0.05). Mean leukocyte values (χ2 = 11.07, df = 03, p< 0.05) were significantly higher in T. cruzi-infected (27,150±8,427) (U = 6.5, p< 0.05) and coinfected coatis (27,719±7,750) (U = 26, p< 0.05) (Table 2). Path analysis showed a negative direct effect of T. evansi infection (path coefficient = -0.30, p< 0.05) on anemia indicators, resulting in lower PCV (r = 0.84) and MCV (r = 0.65). Table 1. Number of positive coatis (Nasua nasua), crab-eating-foxes (Cerdocyon thous) and ocelots (Leopardus pardalis) for Trypanosoma cruzi and Trypanosoma evansi in the Pantanal. Samples were collected from November 2015 up to October 2016. Infection Diagnostic Test N. nasua (n = 39) C. thous (n = 48) L. pardalis (n = 19) T. cruzi HC 03 (8) - - PCR 06 (15) - - Serological tests 11 (28) 16 (33) 05 (26) T. evansi MHCT 10 (26) - - PCR 18 (46) 10 (21) 12 (63) Serological tests ND 7 (15) 15 (79) Coinfection HC/MHCT - - - PCR 03 (08) - - Serological tests - 03 (06) 04 (21) The data are expressed by number of captured animals/relative abundance (%). (−) negative results. ND: Not Done, HC: Hemoculture, PCR: Polymerase Chain Reaction, MHCT: Microhematocrit Centrifuge Technique https://doi.org/10.1371/journal.pone.0201357.t001 Table 2. Hematological mean values of coatis (Nasua nasua) infected with Trypanosoma evansi (TE), Trypanosoma cruzi (TC), and in coinfected (TE/TC) animals in the sub-region of Nhecolândia, Pantanal, between November 2015 and October 2016. Nasua nasua Non infected (n = 16) TE positive (n = 10) TC positive (n = 05) TE/TC positive (n = 08) RBC 4.2±1.6a 3.3±1.7a 3.9±1.1a 3.3±1.2a PCV 35.8±5.2a 28.5±4.9b 36.4±6.9a 26.4±7.8b MCV 97.7±44.7a 94.2±19.7a 98±34.3a 86.4±31.8a WBC 18,212±9,359a 15,595±6,297a 27,150±8,427b 27,719±7,750b Eosinophils 663±497a 444±310a 1,333±1,248b 1,681±1,013b Lymphocytes 3,785±4,739a 1,477±360a 2,474±2,122b 3,708±1,173c Monocytes 868±843a 997±486a 2,083±1,269b 2,260±1,068b Neutrophils 12,362±6,031a 12,414±6,035a 19,624±5,768b 19,816±6,086b Different letters denote statistical significance (p < 0.05). PCV: packed cell volume; RBC: red blood cell counts (×106); WBC: white blood cell counts; MCV: mean corpuscular volume. https://doi.org/10.1371/journal.pone.0201357.t002 Outcomes of Trypanosoma spp. infections on health of wild carnivores in the Pantanal PLOS ONE | https://doi.org/10.1371/journal.pone.0201357 August 15, 2018 5 / 15 https://doi.org/10.1371/journal.pone.0201357.t001 https://doi.org/10.1371/journal.pone.0201357.t002 https://doi.org/10.1371/journal.pone.0201357 Although we did not observe a direct effect on anemia indicators of coatis infected with T. cruzi, we found an increased negative direct effect on these values in coinfected animals (path coefficient = -0.47, p< 0.05). Additionally, our results showed that T. evansi infection had a negative influence on body condition via anemia indicators (path coefficient = 0.37, p< 0.05). Moreover, this effect was potentiated in coinfected animals (S1 Fig). We observed that T. cruzi positively affected infection responses (path coefficient = 0.44, p< 0.05). In contrast, T. evansi alone was unable to alter the infection response, but exhibited an increased effect (path coefficient = 0.52, p< 0.05) in coinfected animals, resulting in more monocytes (r = 0.60) and neutrophils (r = 0.89) (S2 Fig). We observed that T. cruzi (path coefficient = -0.43, p< 0.05) infection had a positive effect on immune investment, increasing further in coinfected animals (path coefficient of = -0.61, p< 0.05; path coefficient = -0.46, p< 0.05). This resulted in increased numbers of lympho- cytes (r = -0.60; r = -0.70) and anti-T. cruzi IgGs (r = -0.79; r = -0.60) (S3 Fig). Additionally, T. cruzi, T. evansi, and coinfection with both parasites had also indirect negative effects on body condition via immune investment (path coefficient = -0.34, p< 0.05) (S3 Fig). Crab-eating foxes No significant differences were observed for mean RBC (χ2 = 0.3187, df = 03, p> 0.05), PVC (χ2 = 2.552, df = 03, p> 0.05), and MCV (χ2 = 0.5056, df = 03, p> 0.05) values between infected and non-infected crab-eating foxes. Nevertheless, our data indicated a minor decrease of these values for T. evansi-infected animals. Furthermore, we observed a significant increase in WBC mean values (χ2 = 6.036, df = 03, p< 0.05) in T. cruzi-infected animals due to neutro- philia (10,823±4,745) (U = 40, p< 0.05) (Table 3). Path analysis revealed that infections with T. cruzi and T. evansi had no effect on anemia indicators of crab-eating foxes. However, we found a negative effect on the infection responses following T. cruzi infection (path coefficient = 0.27, p< 0.05) and coinfection (path coeffi- cient = 0.26, p< 0.05), resulting in fewer monocytes (r = -0.62) and neutrophils (r = -0.64) (S4 Fig). Moreover, we observed that T. cruzi (path coefficient = -0.72, p< 0.05) infection and coin- fection (path coefficient = -0.79, p< 0.05) had a positive influence on immune investment, as manifested by an increase in anti-T. cruzi IgGs (r = -0.97) (S5 Fig). Table 3. Hematological mean values for crab-eating foxes (Cerdocyon thous) infected with Trypanosoma evansi (TE), Trypanosoma cruzi (TC), and in coinfected (TE/TC) animals in the sub-region of Nhecolândia, Pantanal, between November 2015 and October 2016. Cerdocyon thous Non infected (n = 25) TE positive (n = 07) TC positive (n = 07) TE/TC positive (n = 09) RBC 3.1±1.3a 3±0.4a 3.2±1a 3±1a PCV 38.1±7.9a 37.7±3.7a 45.6±19.3a 40±3.6a MCV 143.4±66.1a 127.1±26.9a 154.4±73.2a 144.9±45.1a WBC 10,424±4,491a 12,428±6,897a 14,764±5,528b 12,905±3,563a Eosinophils 852±479a 1,299±1,303a 881±541a 1,048±1,046a Lymphocytes 2,006±1,318a 1,747±1,351a 2,138±1,118a 2,286±1,256a Monocytes 778±336a 686±450a 874±577a 780±517a Neutrophils 6,785±4,068a 8,638±5,429a 10,823±4,745b 8,677±3,547a Different letters denote statistical significance (p < 0.05). PCV: packed cell volume; RBC: red blood cell counts (×106); WBC: white blood cell counts; MCV: mean corpuscular volume. https://doi.org/10.1371/journal.pone.0201357.t003 Outcomes of Trypanosoma spp. infections on health of wild carnivores in the Pantanal PLOS ONE | https://doi.org/10.1371/journal.pone.0201357 August 15, 2018 6 / 15 https://doi.org/10.1371/journal.pone.0201357.t003 https://doi.org/10.1371/journal.pone.0201357 Finally, we observed that T. evansi infection (path coefficient = 0.31, p< 0.05) and coinfec- tion (path coefficient = 0.31, p< 0.05; path coefficient = 0.52, p< 0.05) influenced positively immune investment resulting in more lymphocytes (r = 0.70) and anti-T. evansi IgGs (r = 0.72 r = 0.60) (S6 Fig). Both, T. evansi infection and coinfection, had an indirect negative effect on body condition via immune investment on lymphocytes and anti-T. evansi IgGs (path coeffi- cient = -0.41, p< 0.05; path coefficient = -0.56, p< 0.05) (S6 Fig). Ocelots Although we found lower RBC and PCV mean values for ocelots parasitized by T. evansi and in those coinfected with both parasites than in non-infected animals, differences were not statistically significant (RBC: χ2 = 3.672, df = 03, p> 0.05; PVC: χ2 = 5.12, df = 03, p> 0.05; and MCV: χ2 = 0.2237, df = 03, p> 0.05) between non infected and T. evansi-infected ocelots (Table 4). Path analysis showed that T. evansi (path coefficient = 0.90, p< 0.05) and coinfection (path coefficient = 0.73, p< 0.05) resulted in lower PCV (r = -0.93) values (S7 Fig). Moreover, the decrease in body condition was influenced by fewer RBC (r = -0.91) and higher MCV (r = 0.88) values, irrespective of infection with T. evansi or coinfection (path coefficient = -0.41, p< 0.05) (S7 Fig). Infection with T. cruzi and T. evansi did not have any effect on the infection response in ocelots. Furthermore, we observed that coinfection (path coefficient = -1.03, p< 0.05) had a posi- tive effect on immune investment, marked by an increase in anti-T. cruzi IgGs (r = -0.97) (S8 Fig). A similar positive effect on immune investment was observed also with T. evansi infection (path coefficient = -0.99, p< 0.05; path coefficient = 0.94, p< 0.05) and coinfection (path coef- ficient = -0.77, p< 0.05; path coefficient = 0.96, p< 0.05) (S8 Fig). This resulted in more lym- phocytes (r = 0.75) and higher anti-T. evansi IgG (r = -0.87) values (S9 Fig). Additionally, T. evansi infection and coinfection had indirect positive effects on body condition via immune investment (path coefficient = 0.59, p< 0.05) (S9 Fig). Discussion Our results reveal that T. evansi infection in coatis, crab-eating foxes and ocelots causes some degree of anemia. Anemia has been recorded previously in coatis infected with T. evansi Table 4. Hematological mean values among ocelots (Leopardus pardalis) infected with Trypanosoma evansi (TE) and coinfected with T. evansi/Trypanosoma cruzi (TE/TC) in the sub-region of Nhecolândia, Pantanal, between November 2015 and October 2016. Leopardus pardalis Non infected (n = 02) TE positive (n = 12) TE/TC positive (n = 05) RBC 6.9±1.9a 4.5±1.8a 4.1±1.1a PCV 41.9±4.2a 32.1±4.7a 32.9±3.6a MCV 83±4.16a 81.5±34.3a 85.8±18.7a WBC 17,275±16,723a 14,650±4,479a 16,830±5,357a Eosinophils 54±77a 178±312a 331±161a Lymphocytes 1,545±1,107a 2,372±1,470a 2,493±907a Monocytes 600±386a 874±704a 1,229±796a Neutrophils 15,048±15,346a 11,027±3,513a 12,900±4,318a Different letters denote statistical significance (p < 0.05). PCV: packed cell volume; RBC: red blood cell counts; WBC: white blood cell counts; MCV: mean corpuscular volume. https://doi.org/10.1371/journal.pone.0201357.t004 Outcomes of Trypanosoma spp. infections on health of wild carnivores in the Pantanal PLOS ONE | https://doi.org/10.1371/journal.pone.0201357 August 15, 2018 7 / 15 https://doi.org/10.1371/journal.pone.0201357.t004 https://doi.org/10.1371/journal.pone.0201357 [13, 17–19], but the present study is the first report of T. evansi infection resulting in anemia also in free-living ocelots and crab-eating foxes. Anemia is characteristic of T. evansi infections [17, 36–38] and can represent a threat to the health of carnivores in the Pantanal wetland, as suggested by infection rates of 89% (17/19) in ocelots, 46% (18/39) in coatis, and 33% (16/48) in crab-eating foxes. Even though T. cruzi infection could not induce anemia in coatis, coinfection with T. evansi caused the degree of anemia to become more severe, a finding previously observed by Olifiers et al. 2015 [13]. The microcytic hypochromic anemia, characterized by the low MCV values in T. evansi-infected coatis and the even lower values in coinfected animals, may cor- relate to deficient hemoglobin synthesis due to iron deficiency [39–41], as observed in T. evansi infections [42, 43]. The low MCV values could also result from the influx of iron into the cell, which is necessary for the multiplication of intracellular amastigote forms of T. cruzi [44, 45]. Anemia was observed also in ocelots, as suggested by small differences in anemia indices in animals infected with T. evansi and coinfected with T. cruzi, as well as through direct effect of T. evansi infection and coinfection on PCV values tested by path analysis. Anemia has been recorded previously in domestic cats experimentally infected with T. evansi [46–48]. Moreover, lower RBC and higher MCV values indicated a megaloblastic anemia, which negatively influenced ocelots’ body condition, irrespective of Trypanosoma spp. infection. Although we have not investigated other pathogens or other causes, in natural environments animals are constantly and concomitantly exposed to a variety of parasites, particularly Ana- plasma spp., Mycoplasma spp., and piroplasmids, which cause lysis in parasited red blood cells and the consequent drop in RBC values. The same parasites have been described to infect oce- lots in the studied area [49–51]. Additionally, the observed increase in MCV values may have metabolic origin and be associated with deficiency of vitamin B12, which is found mainly in protein diets, or/and in hepatic dysfunction. Regarding crab-eating foxes, we observed a slight decrease in indicators of anemia only in T. evansi-infected animals. Importantly, domestic dogs that have been experimentally or natu- rally infected with T. evansi display evident signs of anemia and the course of infection is fatal if not treated [52, 53]. Therefore, free-living crab-eating foxes parasitized with T. evansi may become sick and prostrate, consequently they may die or are not collected. We observed discrete leucopenia due to fewer lymphocytes and eosinophils in coatis paras- ited with T. evansi. Immunosuppression in coatis infected with T. evansi has been described previously in natural and experimental studies [13, 18, 19, 54]. This phenomenon varies in nature due to different communities of parasites in their hosts, as well as the influence of marked seasonality of resources, which is characteristic of the Pantanal region [12, 27]. The leukocytosis observed here in T. cruzi-infected and coinfected coatis is typical of the acute phase of T. cruzi infection [55]. The increase in leucocytes during T. cruzi infection in wild mammals has been reported in Thrychomis pachyurus and coatis under experimental and natural conditions, respectively [12, 56]. We observed a notable infection response in coatis infected with T. cruzi and in coinfected animals. Monocytosis is a sign of immune response during the acute phase of T. cruzi infec- tion [57, 58]. Throughout the chronic phase of T. cruzi infection, neutrophils act together with monocytes and lymphocytes to repair the tissue damage caused by T. cruzi amostigote [59]. An increase in monocyte and neutrophil values is an important hallmark of infection by T. cruzi in naturally infected coatis, as already reported by Martı́nez-Hernández et al. 2016 [12]. We observed a decrease in lymphocytes in crab-eating foxes infected with T. evansi, con- firming the findings of Da Silva et al. 2011 [60] in the chronic phase of T. evansi infection in Outcomes of Trypanosoma spp. infections on health of wild carnivores in the Pantanal PLOS ONE | https://doi.org/10.1371/journal.pone.0201357 August 15, 2018 8 / 15 https://doi.org/10.1371/journal.pone.0201357 laboratory rodents. Indeed, domestic dogs naturally and experimentally infected with T. evansi displayed fewer WBCs and neutrophils [52, 53]. Additionally, the decrease in mono- cytes and neutrophils observed in crab-eating foxes infected with T. cruzi or in coinfected animals, was similar to that reported in dogs during the early stages of T. cruzi experimental infection [61]. Such immunosuppression, even if transient, can impair the health of the animal. An increase in immune investment in coatis, ocelots, and crab-eating foxes infected with T. cruzi, T. evansi, or in coinfected animals recorded in the present study may be associated with a potent stimulation of cellular and humoral immune response, characteristic of trypanosome infection [62–64]. The strong production of immunoglobulins results in an autoimmune hypersensitivity with consequent production of antigen-antibody molecules [65]. These immune complexes accumulate on the vascular wall, especially in the microcirculation, caus- ing damage to their thin layer of vascular endothelial cells, and resulting in widespread micro bleeding, a phenomenon known as disseminated intravascular coagulation (DIC). DIC has been associated with trypanosome infections in various host species [66–69] and has been observed in coatis infected with trypanosomes in the Pantanal region. Indeed, as observed here by path analysis, an increase in immune investment resulted in a worse body condition in ocelots and crab-eating foxes. DIC, together with the hypoferremic response discussed above, are the main causes of anemia observed in trypanosome-infected animals. Furthermore, oxida- tive stress due to oxidative damage in erythrocyte membranes are related to experimental and natural infection by T. evansi [70, 71]. Conclusions As T. cruzi is restricted to the New World, it had been interacting with its hosts over millions of years. On the contrary, T. evansi originates from the African continent, and has become a parasite of South American wild mammals only recently. In the Pantanal region, T. evansi was probably introduced together with horses and dogs in the late Eighteenth century when the first cattle farms were established. According to this scenario, while the course of T. cruzi infec- tion is known to be predominantly chronic probably due to ancient association with its hosts, T. evansi infection of endemic Neotropical fauna may cause great damage to the health of its hosts, particularly due to increased virulence and pathogenicity of present interactions. The anemia and immunosuppression evidenced by the present study, are associated with increasing habitat fragmentation and poaching [72], which poses a threat to wild coatis, ocelots and crab-eating foxes in the Pantanal wetland. Furthermore, due to epidemiological implica- tions and conservation importance, studies of T. cruzi and T. evansi infections in free-living mammals should be a priority for health surveillance organizations, research promotion agen- cies, and postgraduate programs. Supporting information S1 Fig. Path analysis on anemia indicators of coatis (Nasua nasua). Results of path analysis on anemia indicators of coatis (Nasua nasua) infected with Trypanosoma evansi and coin- fected with T. evansi/Trypanosoma cruzi in the sub-region of Nhecolândia, Pantanal, between November 2015 and October 2016. (TIF) S2 Fig. Path analysis on infection responses of coatis (Nasua nasua). Results of path analysis on infection responses of coatis (Nasua nasua) infected with Trypanosoma cruzi and coin- fected with Trypanosoma evansi/T. cruzi in the sub-region of Nhecolândia, Pantanal, between Outcomes of Trypanosoma spp. infections on health of wild carnivores in the Pantanal PLOS ONE | https://doi.org/10.1371/journal.pone.0201357 August 15, 2018 9 / 15 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0201357.s001 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0201357.s002 https://doi.org/10.1371/journal.pone.0201357 November 2015 and October 2016. (TIF) S3 Fig. Path analysis on immune investment of coatis (Nasua nasua). Results of path analy- sis on immune investment of coatis (Nasua nasua) infected with Trypanosoma cruzi and coin- fected with Trypanosoma evansi/T. cruzi in the sub-region of Nhecolândia, Pantanal, between November 2015 and October 2016. (TIF) S4 Fig. Path analysis on infection responses of crab-eating foxes (Cerdocyon thous). Results of path analysis on infection responses of crab-eating foxes (Cerdocyon thous) infected with Trypanosoma cruzi and coinfected with Trypanosoma evansi/T. cruzi in the sub-region of Nhe- colândia, Pantanal, between November 2015 and October 2016. (TIF) S5 Fig. Path analysis on immune investment against Trypanosoma cruzi of crab-eating foxes (Cerdocyon thous). Results of path analysis on immune investment against T. cruzi in crab-eating foxes (Cerdocyon thous) infected with Trypanosoma cruzi and coinfected with Try- panosoma evansi/T. cruzi in the sub-region of Nhecolândia, Pantanal, between November 2015 and October 2016. (TIF) S6 Fig. Path analysis on immune investment against Trypanosoma evansi of crab-eating foxes (Cerdocyon thous). Results of path analysis of immune investment against T. evansi in crab-eating foxes (Cerdocyon thous) infected with Trypanosoma cruzi and coinfected with Try- panosoma evansi/T. cruzi in the sub-region of Nhecolândia, Pantanal, between November 2015 and October 2016. (TIF) S7 Fig. Path analysis on anemia indicators in ocelots (Leopardus pardalis). Results of path analysis on anemia indicators in ocelots (Leopardus pardalis) infected with Trypanosoma evansi and coinfected with T. evansi/T. cruzi in the sub-region of Nhecolândia, Pantanal, between November 2015 and October 2016. (TIF) S8 Fig. Path analysis on immune investment against Trypanosoma cruzi of ocelots (Leopar- dus pardalis). Results of path analysis on immune investment against Trypanosoma cruzi in ocelots (Leopardus pardalis) infected with T. evansi and coinfected with Trypanosoma evansi/T. cruzi in the sub-region of Nhecolândia, Pantanal, between November 2015 and October 2016. (TIF) S9 Fig. Path analysis on immune investment against Trypanosoma evansi of ocelots (Leo- pardus pardalis). Results of path analysis on immune investment against Trypanosoma evansi in ocelots (Leopardus pardalis) infected with T. evansi and coinfected with T. evansi/Trypano- soma cruzi in the sub-region of Nhecolândia, Pantanal, between November 2015 and October 2016. (TIF) S1 File. Collection Data Set. Data of coatis (Nasua nasua), crab-eating-foxes (Cerdocyon thous) and ocelots (Leopardus pardalis) infected for Trypanosoma cruzi and Trypanosoma evansi in the Pantanal. Samples were collected from November 2015 up to October 2016. (XLSX) Outcomes of Trypanosoma spp. infections on health of wild carnivores in the Pantanal PLOS ONE | https://doi.org/10.1371/journal.pone.0201357 August 15, 2018 10 / 15 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0201357.s003 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0201357.s004 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0201357.s005 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0201357.s006 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0201357.s007 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0201357.s008 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0201357.s009 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0201357.s010 https://doi.org/10.1371/journal.pone.0201357 Acknowledgments First author thanks the Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Superior (CAPES) and Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT) Author Contributions Conceptualization: Filipe Martins Santos. Data curation: Filipe Martins Santos, Luiz Gustavo Rodrigues Oliveira-Santos, Guilherme de Miranda Mourão, Grasiela Edith de Oliveira Porfı́rio, Ana Maria Jansen. Formal analysis: Filipe Martins Santos, Luiz Gustavo Rodrigues Oliveira-Santos. Investigation: Filipe Martins Santos, Gabriel Carvalho de Macedo, Wanessa Teixeira Gomes Barreto, Carolina Martins Garcia, Guilherme de Miranda Mourão, Grasiela Edith de Oli- veira Porfı́rio, Elizangela Domenis Marino, Marcos Rogério André, Lı́via Perles, Carina Eli- sei de Oliveira, Gisele Braziliano de Andrade, Ana Maria Jansen, Heitor Miraglia Herrera. Methodology: Filipe Martins Santos, Gabriel Carvalho de Macedo, Wanessa Teixeira Gomes Barreto, Luiz Gustavo Rodrigues Oliveira-Santos, Carolina Martins Garcia, Guilherme de Miranda Mourão, Grasiela Edith de Oliveira Porfı́rio, Elizangela Domenis Marino, Marcos Rogério André, Lı́via Perles, Carina Elisei de Oliveira, Gisele Braziliano de Andrade, Ana Maria Jansen, Heitor Miraglia Herrera. 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