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OPEN Co‑infection by multiple

vector‑borne agents in wild
ring‑tailed coatis (Nasua nasua)
from Iguaçu National Park,
southern Brazil
L. Perles 1, M. F. Moraes 2, M. Xavier da Silva 3, R. F. C. Vieira 4,5,6, R. Z. Machado 1,
E. G. Lux Hoppe 2 & M. R. André 1*

The present study aimed to detect molecularly the presence of co-infections by vector-borne agents
(VBA) in ring-tailed coatis’ (Nasua nasua) blood samples from Iguaçu National Park (INP), southern
Brazil, and assess the phylogenetic positioning of the detected agents. DNA blood samples were
submitted to molecular screening and characterization for Anaplasmataceae agents, Piroplasmids,
Hepatozoon sp., hemotropic mycoplasmas, and Bartonella spp. In total, 42 (85.7%) coatis were
positive for hemotropic Mycoplasma sp., 12 (24.5%) for Bartonella machadoae, 7 (14.3%) for
Anaplasma sp. closely related to ‘Candidatus Anaplasma brasiliensis’, and 3 (6%) for Hepatozoon
procyonis. The most prevalent co-infections observed was from bacterial VBA: while 18.3% were
co-infected by hemotropic Mycoplasma sp. and Bartonella sp., 12.2% were co-infected by Anaplasma
sp. and hemotropic Mycoplasma sp. Only two animals (4%) presented co-infections by three VBA
(Bartonella sp., Anaplasma sp. and hemotropic Mycoplasma sp.). The coati is a wild carnivore found
in INP, mainly in areas visited by tourists. These animals are frequently seen searching for food in
garbage dumps or in tourists’ belongings. The present study expands the host specificity range of B.
machadoae, which has been isolated only from rodents until the present moment. Since the zoonotic
potential and transmission routes of the detected VBA are not yet known, surveillance in this area is
much needed.

Ring-tailed coatis (Nasua nasua Carnivora: Procyonidae) are widely distributed in South America. They are
known to be social animals, forming large familiar groups up to 30 individuals, composed mainly of females in
reproductive age and few mature ­males1,2. Coatis adapt very easily in urbanized areas, where they can interact
with domestic animals, such as dogs and ­cats2,3. In these environments they are used to explore and feed on
anthropogenic food sources (e.g. garbage)4,5. The Iguaçu National Park (INP) is one of the largest remaining
areas of the Atlantic Forest in Brazil, and is also an important tourist attraction, receiving annually 1.5 million
visitors from many regions of the world (https://​www.​icmbio.​gov.​br/​parna​iguacu/). At INP coatis are known to
interact constantly with tourists by locking for food, which eventually results in human i­ njuries6. Such mammals
can harbor several pathogens, such as rabies virus, Trypanosoma cruzi and Leishmania spp., zoonotic agents of
great importance in Public H ­ ealth7,8.

1
Vector‑Borne Bioagents Laboratory (VBBL), Department of Pathology, Reproduction and One Health, School
of Agricultural and Veterinarian Sciences, São Paulo State University (Unesp), Via de Acesso Prof. Paulo Donato
Castellane, s/n, Zona Rural, Jaboticabal, São Paulo  CEP: 14884‑900, Brazil. 2Laboratory of Parasitic Diseases
(LabEPar), Department of Pathology, Reproduction and One Health, School of Agricultural and Veterinarian
Sciences, São Paulo State University (Unesp), Jaboticabal, SP, Brazil. 3Iguaçu Carnivore Project, Iguaçu National
Park, BR‑469, Km 22.5, Foz do Iguaçu, Paraná 85851‑970, Brazil. 4Vector‑Borne Diseases Laboratory, Department
of Veterinary Medicine, Universidade Federal do Paraná – UFPR, Curitiba, Brazil. 5Department of Public Health
Sciences, University of North Carolina at Charlotte, Charlotte, USA. 6Center for Computational Intelligence
to Predict Health and Environmental Risks (CIPHER), University of North Carolina at Charlotte, Charlotte,
USA. *email: mr.andre@unesp.br

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Vector-borne agents (VBA) are pathogens (e.g. bacteria, virus, protozoa) transmitted by a plethora of vec-
tors, such as ticks, fleas, lice, mosquitoes, ­sandflies9,10. Some agents transmitted by vectors can cause important
economic losses and also important impacts in human h ­ ealth9,10. Epidemiological dynamics of VBAs are based
on complex interactions among vectors, pathogens and vertebrate hosts. Regarding VBAs in coatis from Brazil,
hemotropic Mycoplasma spp. has been detected in Mato Grosso do Sul and Paraná ­state11,12 and recently, ‘Can-
didatus Mycoplasma haematonasua’ has been described in coatis from “Parque Nacional do Iguaçu” (PARNA
Iguaçu)13. Regarding Apicomplexan protozoan, Hepatozoon procyonis has been widely detected in several Brazil-
ian ­regions14–18, while Piroplasmids agents were detected only in coatis sampled in the Pantanal of Mato Grosso
do Sul ­state19 and in Minas Gerais ­state17. Interestingly, Sousa et al.20 and Collere et al.13 detected Anaplasmataceae
agents in blood samples from coatis. While Ehrlichia 16S rRNA genotypes closely related to E. canis/E. chaffeensis,
and Anaplasma 16S rRNA genotypes closely related to A. bovis and A. phagocytophilum on coatis in the Pantanal
wetlands, state of Mato Grosso do ­Sul20, Collere et al.13 failed to sequence the detected agents. Recently, Perles
et al.21 described ‘Candidatus Ehrlichia dumleri’, a putative novel Anaplasmataceae agent, and Anaplasma sp.
closely related to ‘Candidatus Anaplasma brasiliensis’ in blood samples from coatis in peri-urbanized forested
areas in central-western Brazil.
Considering the ecological and zoonotic importance of the knowledge on VBA, the lack of information
regarding wild population’s diseases and the methodological complexity of field research with wild animals, the
present study aimed to detect molecularly the presence of VBA in coati’s blood samples and assess the phyloge-
netic positioning of the detected agents. Moreover, the INP is one of the most important Atlantic Forest Protected
area, that still harbor most of the terrestrial large mammals and it is also the second most visited National Park
in ­Brazil22. To the best of the authors’ knowledge, there is no report of Bartonella spp. in coatis from Brazil,
highlighting the importance of surveillance of vector-borne agents (VBA) in wild mammals.

Methods
Ethical aspects.  All methods were carried out in accordance with relevant guidelines and regulations and
were approved by the local Ethics Committee on Animal Use of São Paulo State University—Unesp, Agrarian
and Veterinarian Sciences School—FCAV (Protocol 07553/14) and by the Brazilian Authorization and Infor-
mation System—SISBIO (Protocol 38006-2); and “Sistema Nacional de Gestão de Patrimônio Genético e do
Conhecimento Tradicional Associado”—SISGEN (Protocol AAA5680); permit SISBIO (numbers: 48.141–5,
62.541–6, 38.006–2, 38.006–4, 38.006–6, and 38.006–8). All methods are reported at the present work are in
accordance with ARRIVE guidelines (https://​arriv​eguid​elines.​org).

Study area and animal trapping.  PARNA Iguaçu is located within the Atlantic Forest Biome in the west-
ern state of Paraná, Brazil, representing one of the last remnants of this important Biome (25°05′ S to 25°40′ S
and 54°30′ W to 54°40′ W)22 (Fig. 1). The park contains up to 185,000 hectares of semi-deciduous forest and its
fauna is highly diverse, with records of 102 species of mammals, 386 birds, 48 reptiles, 13 amphibians, 176 fishes,
839 invertebrates, and 28 species of parasite helminths, most of them endemic to the Atlantic r­ ainforest22.
Between August 2014 and December 2017, coatis (Nasua nasua) were captured with hand nets and Toma-
hawk traps baited with a mixture of fresh chicken meat, fruits (banana, pineapple or mango) and peanut butter,
during several campaigns, totaling 7200 h capture effort. Captures were performed at the Western region of the
INP. Following the captures, the animals were physically restrained, anesthetized with tiletamine-zolazepan

Figure 1.  Map showing Paraná State (light green) and Iguaçu National Park (dark green).

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association (5 mg/kg, IM), and identified with a small numbered ear tag to prevent recaptures. Blood samples
from 49 coatis (21 males; 28 females) were drawn from the jugular vein and then stored in EDTA-coated tubes
­ rocessing23.
in isothermal boxes with ice and then were transported to the laboratory for p

DNA extraction from blood samples and conventional PCR (PCR) for mammal glyceraldehyde
3‑phosphate dehydrogenase (gadph) endogenous gene.  DNA was extracted from 200 μL of blood
using Biopur Kit Mini Spin Plus, according to the manufacturer’s instructions. To evaluate the quality of the
extracted DNA and avoid false negative results, DNA samples were tested by a conventional PCR targeting the
mammal gapdh ­gene24. Ultra-pure sterile water (Life Technologies®, Carlsbad, CA, USA) was used as a negative
control in PCR assays.

Molecular assays for vector‑borne agents.  Coati DNA blood samples positive for gapdh gene were
submitted to PCR assays for Anaplasma, Ehrlichia, Neorickettsia, Bartonella, hemoplasmas, Hepatozoon, and
Piroplasmids. All agents, target gene, primer sequences, size of the amplicon (bp), thermal cycling conditions
and references are shown in Table 1 (and Figure SM1—Supplementary file).
Ehrlichia canis (Jaboticabal strain—maintained in DH82 cell culture), Anaplasma phagocytophilum (Webster
strain) and Neorickettsia risticii (kindly provided by Professor John Stephen Dumler (Uniformed Services Uni-
versity of the Health Sciences, Bethesda, MD, USA) DNA were used as positive controls for Anaplasmataceae
screening and characterization protocols. In the qPCR assays for Bartonella spp., serial dilutions were performed
to construct standard curves with different concentrations (2.0 × ­107 to 2.0 × ­100 copies) of a plasmid encoding
a fragment of the 83 bp nuoG gene of Bartonella henselae DNA (pIDTSMART; Integrated DNA Technolo-
gies). The number of plasmid copies was determined according to the formula (XG/μL DNA/ [Plasmid size
(BP) × 660]) × 6, 22 × ­1023 × plasmid copies/μL25. For hemotropic mycoplasmas, Mycoplasma parvum DNA from
a naturally infected domestic pig was u ­ sed26. DNA from Hepatozoon sp. detected in a naturally infected maned-
wolf (Chrysocyon brachyurus) was used as positive c­ ontrol27. For Piroplasms screening, Babesia vogeli DNA
(Jaboticabal strain) were used as a positive control. Ultra-pure sterile water (Life Technologies®, Carlsbad, CA,
USA) was used as negative control in all PCR assays.

Agarose gel electrophoresis, amplicon purification and Sanger sequencing.  The results of cPCR,
nPCR and semi-nPCR assays were visualized in 1% agarose gel stained by ethidium bromide solution and results
were visualized using a UV transilluminator (ChemiDoc MP Imaging System, Bio Rad®). Positive samples were
purified using ExoSAP-IT™ PCR Cleanup (ThermoFisher®) and sequenced. The sequencing was performed by
Sanger ­method28 using ABI PRISM 3730 DNA Analyzer (Applied Biosystems) at Human Genome and Stem Cell
Research Center, “Instituto de Biociências”, University of São Paulo (USP), Brazil.

Phylogenetic analyses.  Eletropherograms results were analyzed using Phred-Phrap software version
23, and the quality of each nucleotide sequence was checked for a score and considered of good quality when
Phred score was higher than ­2029. Consensus sequences obtained by the alignment of the forward and reverse
sequences were constructed using the same software. The BLASTn program was used to analyze consensus
sequences, aiming to browse and compare with sequences from the GenBank international d ­ atabase30. All
sequences obtained in the present study were deposited in NCBI platform GenBank (https://​www.​ncbi.​nlm.​nih.​
gov/​genba​nk/—Nucleotide) (Supplementary Information—Table SM1).
The obtained sequences were aligned with those retrieved from GenBank using MAFFT software, version
­731. For each studied agent and target gene, sequences were selected for phylogenetic inferences based on BLAST
results and other studies performed in Brazil and other countries. For phylogenetic analyses, initially, the best
model of evolution was selected by the program IQTREE (available at: http://i​ qtree.c​ ibiv.u
​ nivie.a​ c.a​ t/), under the
Akaike Information Criterion (AIC)32. Maximum likelihood (ML) analysis was performed with iqtree (available
at: http://​iqtree.​cibiv.​univie.​ac.​at/) and Bayesian analyses were performed using CIPRES g­ ateway33. The phylo-
genetic tree edition and rooting (outgroup) were performed using the Treegraph 2.0 beta ­software34.

Results
DNA extraction and quality evaluation.  Among the 49 ring-tailed coatis’ DNA blood samples, all were
positive in the cPCR based on the gapdh gene and were included in subsequent analysis.

PCR assays for Anaplasma spp.  Seven (7/49—14.3%) coatis were positive in the nPCR for Anaplasma
spp. based on the 16S rRNA gene. Six sequences (439-519  bp) were obtained and showed 99–100% identity
(ID), 89–97% query-coverage (QC) and 0.0 E-value (EV) with Anaplasma sp. sequences previously detected in
wild Nasua nasua from Mato Grosso do Sul ­State20,21 (GenBank accession numbers KY499184, KY499186-87,
KY499192-95); and 100% ID, 88% QC and 0.0 EV with Anaplasma sp. detected in a r­odent35. Four samples
amplified for ITS—23S–5S (339–415 bp) and the obtained sequences showed 94.7% ID, 100% QC and 2e-114
EV with ‘Candidatus Anaplasma brasiliensis’ (MT267343-44) previously detected in anteaters from São Paulo
­ razil36. Three samples amplified for gltA gene (569–599  bp) and the obtained sequences
state, southeastern B
showed 83% ID, 90% QC, and 1e-14 EV with Anaplasma sp. detected in Bos taurus in Japan (JX445933).
Maximum likelihood (ML) analyses based on the 16S rRNA gene (519 bp) and TIM2 + I + G4 evolutionary
model clustered the sequences detected in the present study in a large clade with other Anaplasma sp. sequences
previously detected in wild animals from Brazil (Fig. 2). ML analyses based on the gltA gene (alignment of 655 bp
and TPM3 + G4 as evolutionary model) (Fig. 3A) clustered the sequences detected in the present in a new clade,

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Fragment
Agents Aim Molecular assay Primers sequences size (bp) Thermal cycling Reference
5′-CAC​ATG​CAA​GTC​GAA​CGG​
Anaplasma spp. (16S rRNA gene) ATT​ATT​C-3′
External primers 932
5′-TTC​CGT​TAA​GAA​GGA​TCT​
gE3a AAT​CTC​C′-3′ 94 °C for 5 min 40 cycles: 94 °C for
gE10R Screening nPCR 30 s, 55 °C for 30 s and 72 °C for 25

Internal primers 5′-GGC​AGT​ATT​AAA​AGC​AGC​ 1 min 72 °C for 5 min

gE2 TCC​AGG​-3′
546
gE9f. 5′-AAC​GGA​TTA​TTC​TTT​ATA​
GCT​TGC​T-3′
Anaplasma spp. 5′-AGG​ATC​TGA​CTC​TAG​TAA​
94 °C for 2 min, 35 cycles; 94 °C
(ITS—23S–5S) CGAG-3′ 26
Characterization cPCR 300 for 30 s, 58 °C for 30 s, 72 °C for
ITS2F 5′-CTC​CCA​TGT​CTT​AAG​ACA​
1 min 72 °C for 5 min
ITS2R AAG-3′
Anaplasma spp.
5′-CCG​GGT​TTT​ATG​TCT​ACT​
(gltA gene)
GC-3′ 95 °C for min; 35 cycles: 95 °C for
External primers
5′-CGA​TGA​CCA​AAA​CCCAT-3′ 30 s, 55 °C for 30 s and 72 °C for
F4b Characterization nPCR 700 27
5′-TTY​ATG​TCY​ACT​GCT- 30 s and a final extension 72 °C
R1b
GCKTG-3′ for 10 min
EHR-C5136F
5′-GCNCCMCCATGMGCTGG-3′
HER-778R
Ehrlichia spp. 5′-GAT​GAT​GTC​TGA​AGA​TAT​ 95 °C for 2 min; 50 cycles: 95 °C
(dsb gene) GAA​ACA​AAT-3′ for 15 s, 58 °C for 30 s and 72 °C 28
Screening cPCR 409
dsb-330 5′-CTG​CTC​GTC​TAT​TTT​ACT​ For 30 s and a final extension 72 °C
dsb-728 TCT​TAA​AGT-3′ for 5 min
Neorickettsia ristici 5′-ATT​TGA​GAG​TTT​GAT​CCT​
(16S rRNA gene) GG-3′ 94 ºC for 5 min; 30 cycles: 94 ºC
ER3-F 5′-GTT​TTA​AAT​GCA​GTT​CTT​ for 1 min, 60 ºC for 2 min and 72
Screening nPCR 450 29
ER2-R GG-3′ ºC for 1 min, and a final extension
ER 3a-F 5′-CTA​GCG​GTA​GGC​TTAAC-3′ 72 ºC for 7 min
ER2a-R 5′-CAC​ACC​TAA​CTT​ACGGG-3′
5′-CAA​TCT​TCT​TTT​GCT​TCA​
Bartonella spp.
CC-3′
(nuoG gene) 95 °C for 3 min followed by 40
5′-TCA​GGG​CTT​TAT​GTG​AAT​
F-Bart Screening qPCR 83 cycles at 95 °C for 10 s and 52.8 °C 30
AC-3′
R-Bart for 30 s
TexasRed-5′-TTY GTC​ATT​TGA​
Probe
ACA​CG-3′ [BHQ2a-Q]-3′
94 °C for 2 min, followed by 54
Bartonella spp. 5′-GAC​TTC​TGT​TAT​CGC​TTT​
cycles of 94 °C for 15 s, 58 °C for
(pap-31 gene) GATTT-3′
Characterization cPCR 564 45 s, an extension at 72 °C for 45 s, 31
Pap31 1(s) 5′-CAC​CAC​CAG​CAA​MATA​AGG​
and a final extension at 72 °C for
Pap31 688 (as) CAT-3′
7 min
94 °C for 2 min, followed by 40
Bartonella spp. 5′-CAT​ATG​GTT​TTC​ATT​ACT​
cycles of 94 °C for 30 s, 53 °C for
(ftsZ gene) GCY​GGT​ATGG-3′ 32
Characterization cPCR 600 30 s, an extension at 72 °C for 15 s,
ftsZF 5′-TTC​TTC​GCG​AAT​ACG​ATT​
and a final extension at 72 °C for
ftsZR AGC​AGC​TTC-3′
5 min
94 °C for 5 min, followed by 35
Bartonella spp. 5′-GCT ATG TCT GCA TTC TAT
cycles of 94 °C for 45 s, 61 °C for
(gltA gene) CA-3′
Characterization cPCR 750 45 s, an extension at 72 °C for 45 s, 33
CS443F 5′-GAT CYT CAA TCA TTT CTT
and a final extension at 72 °C for
CS1210R TCA-3′
7 min
5′-AGA​GTT​TGA​TCC​TGG​CTC​
Hemotropic Mycoplasma spp. 95 °C for 5 min, followed by 35
AG-3′
(16S rRNA gene) cycles of 95 °C for 30 s, 57 °C for
5′-TAC​CTT​GTT​ACG​ACT​TAA​
HemoF1 Screening snPCR 1107 30 s, an extension at 72 °C for 34
CT-3
HemoR2 1 min, and a final extension at
5′-ATA​TTC​CTA​CGG​GAA​GCA​
HemoF2 72 °C for 10 min, for both rounds
GC-3′
5′ GCC​AGT​AGT CAT​ATG​CTT​
Hepatozoon spp. GTC​ 3′
95 °C for 3 min, followed by 40
(18S rRNA) 5′ GAC​TTC​TCC​TTC​GTC​TAA​
cycles of 95 °C for 1 min, 56 °C for
HAM-1 G 3′
Screening nPCR 1120 1 min, an extension at 72 °C for 35,36
HPF-2 5′ GCT​AAT​ACA​TGA​GCA​AAA​
1 min and 30 s, and a final exten-
4558 TCT​CAA​ 3′
sion at 72 °C for 7 min
2733 5′ CGG​AAT​TAA​CCA​GAC​AAA​
T 3′
5′-GGC​TCA​TTA​CAA​CAG​TTA​
Piroplasmids TAG-3′
(18S rRNA gene) 5′-CCC​AAA​GAC​TTT​GAT​TTC​ 94 °C for 3 min, followed by 45
BTF1 TCTC-3′ cycles of 94 °C for 30 seg, 58 °C for
Screening nPCR 800 37
BTR1 5′-CCG​TGC​TAA​TTG​TAG​GGC​ 20 seg, 72 °C for 30 seg, and a final
BTF2 TAA​TAC​-3′ extension at 72 °C for 7 min
BTR2 5′-GGA​CTA​CGA​CGG​TAT​CTG​
ATCG-3′

Table 1.  Molecular assays according to the involved agent, type of PCR, target gene, primer sequences, size
of the amplicons (bp), thermal cycling conditions and references, used in the present study for screening
and characterizing vector-borne agents DNA in coati’ blood sampled in Iguaçu National Park, Paraná state,
southern Brazil.

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Figure 2.  Phylogenetic trees inferred by the Bayesian method and based on 16S rRNA gene from Anaplasma
spp. sequences obtained from blood from ring-tailed coatis (Nasua nasua) from PARNA. Sequences
of Anaplasma sp. detected in the present study are highlighted in bold/underlined. Neorickettsia risticii was used
as outgroup.

Figure 3.  Phylogenetic trees inferred by the Bayesian method and based on Anaplasma spp. sequences obtained
from blood from ring-tailed coatis (Nasua nasua) from PARNA. Sequences of Anaplasma sp. detected in the
present study are highlighted in bold/underlined. (A) gltA gene; N. risticii, Wolbachia pipientis and Rickettsia
prowazekii were used as outgroup; (B) ITS—23S–5S; Ehrlichia muris and Ehrlichia chaffeensis were used as
outgroup.

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with the same origin of Anaplasma platys. ML analyses based on the ITS region (23S–5S) (alignment of 420 bp
and K3P + G4 as evolutionary model) (Fig. 3B) clustered the obtained sequences in a new clade with the same
origin of ‘Candidatus Anaplasma brasiliensis’.

PCR assay for Hepatozoon spp.  Three (3/49—6%) coatis were positive in the nPCR for Hepatozoon spp.
based on the 18S rRNA gene. The three obtained sequences (945, 985 and 1031 bp) showed 100% ID, 100% QC,
and 0.0 EV with Hepatozoon procyonis detected in coatis from Brazil (GenBank accession numbers MF685386-
MF685410 and MW862003-MW862006).
Phylogenetic analyses (ML method and GTR as evolutionary model) based on an alignment of 1017 bp of
the 18S rRNA gene clustered all three sequences detected in the present study in a high supported clade (100%
of bootstrap) with Hepatozoon procyonis sequences previously detected in ring-tailed coatis from other Brazilian
states (Figure SM2—Supplementary file).

PCR assay for hemotropic Mycoplasma spp.  Forty-two (42/49—85.7%) ring-tailed coatis samples
were positive in the snPCR for hemotropic Mycoplasma spp. based on the 16S rRNA gene. Eight samples were
randomly selected for sequencing (892–941 bp). All samples presented 99.8–100% ID, 100% QC, and 0.0 EV
with ‘Candidatus Mycoplasma haematonasua’ and hemotropic Mycoplasma sp. previously detected in coatis
from Mato Grosso do Sul state and Paraná state, Brazil. ML analyses (alignment of 941  bp and TN + I + G4)
clustered the sequences detected in the present study in a large clade with other sequences previously detected
in coatis from Brazil, with 100% of clade support (Figure SM3—Supplementary file).

PCR assays for Bartonella spp.  Twelve samples (12/49—24.5%) showed positivity in qPCR assays
for Bartonella spp. based on the nuoG gene. The number of copies of Bartonella-nuoG fragment/μL ranged from
1.38 × ­101 to 5.10 × ­101. The mean values of efficiency, correlation coefficient, and y-intercept of qPCR reactions
mean were 103.4%, 0.920, and 36.031, respectively. Five out of 12 qPCR-positive samples for  Bartonella  also
showed positivity in a PCR assay based on the pap-31 gene. The obtained sequences ranged from 382 to 532 bp
and showed 99.06–100% ID, 100% QC, 0.0 E-value and with Bartonella machadoae37. Seven out of 12 qPCR-
positive samples for Bartonella also showed positivity in a PCR assay based on the gltA gene, with sequences
ranging from 401 to 582 bp and showing 99.83–100% ID, 100% QC and 0.0 EC with Bartonella machadoae. Four
out of 12 qPCR-positive samples for Bartonella also showed positivity in a PCR assay based on the ftsZ gene,
with sequences ranging from 505 to 549 bp and showing 100% ID, 100% QC, and 0.0 EV and with Bartonella
machadoae. Bayesian phylogenetic analyses based on both the gltA gene (alignment of 578 bp and TIM2 + G as
evolutionary model) and the ftsZ gene (alignment of 538 bp and HKY + I + G4 as evolutionary model) clustered
all sequences detected in the present study in a clade with Bartonella machadoae (Fig. 4).

PCR assays for Ehrlichia spp., Neorickettsia risticii and Piroplasmids.  None of the 49 ring-tailed
coatis’ DNA blood samples were positive in the molecular assays for Ehrlichia spp., Neorickettsia risticii and
piroplasmids.

Co‑infection by vector‑borne agents.  Only four animals were negative for all the vector-borne agents
tested. None of the examined ring-tailed coatis were positive for all tested agents. The most co-infection
observed was from bacterial agents, in first place with hemotropic Mycoplasma sp. and Bartonella sp. (9 animals)
and in second place Anaplasma sp. with hemotropic Mycoplasma sp. (6 animals). Only two animals presented
co-infections by three agents (Bartonella sp., Anaplasma sp. and hemotropic Mycoplasma sp.) All information
about positive animals for the investigated agents regarding sampling date, sex and co-infections are shown in
Table SM2—Supplementary file). All co-infections among the investigated agents are presented in a Venn Dia-
gram (Figure SM4—Supplementary file).

Discussion
Herein, wild ring-tailed coatis were found to be infected with a potential novel genotype of Anaplasma sp.,
related to genotypes previously detected in ectoparasites (e.g. Amblyomma sculptum, Amblyomma ovale) and
wild animals (e.g. Sphiggurus villosus, Leopardus pardalis)20, and phylogenetically related to ‘Candidatus Ana-
plasma brasiliensis’36. Anaplasma spp. belong to Anaplasmataceae family and Order Rickettsiales and are obligate
intracellular α-proteobacteria that can cause important diseases in humans and a­ nimals38,39. Representatives of
Anaplasmataceae agents have been detected in several mammal species, and wild and domestic carnivores are
considered an important source of these agents, especially in anthropized areas, where a close contact among
domestic/wild animals and humans can ­occur39,40. In the USA, raccoons (Procyon lotor) play a role as reser-
voirs for Anaplasma phagocytophilum, a zoonotic ­agent39,41,42. When it comes to coatis, Sousa et al.21 detected
an Ehrlichia 16S rDNA genotype phylogenetically related to multiple Ehrlichia spp. previously detected in ticks
and wild rodents from Brazil. Additionally, two different Anaplasma 16S rRNA genotypes were detected in ring-
tailed coatis from the Brazilian Pantanal: while one was positioned in a clade related to A. phagocytophilum, the
other one clustered with A. bovis. Also, Ehrlichia/Anaplasma 16S rDNA was detected in ring-tailed coatis from
the same area (PARNA) where the present study was conducted, but the authors were not able to sequence the
positive ­sample13. In the present study, phylogenetic inferences based on the gltA gene positioned the Anaplasma
sp. Detected in coatis into a new clade, with the same origin of Anaplasma platys. In the ITS (23S–5S)-based
phylogeny, the Anaplasma genotypes detected showed to be closely related to ‘Candidatus Anaplasma brasiliensis’.

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Figure 4.  Phylogenetic trees based on the Maximum likelihood method from Bartonella spp. sequences
obtained from blood from ring-tailed coatis (Nasua nasua) from PARNA. Sequences of Bartonella sp. detected
in the present study are highlighted in bold/underlined (A) gltA gene; Brucella abortus and Ochrobactrum
anthropi were used as outgroup; (B) ftsZ gene; Brucella abortus and Ochrobactrum anthropi were used as
outgroup.

These findings suggest that a novel Anaplasma species, yet to be isolated and characterize, occurs in ring-tailed
coatis from Brazil.
In the present study, a lower occurrence for H. procyonis was found when compared to those previously
reported in ring-tailed coatis from other Brazilian regions. The genus Hepatozoon comprise apicomplexan
hemogregarinids with a heteroxene life cycle, that infects a wide range of vertebrates, including mammals, rep-
tiles, birds and ­amphibians43. Up to now, the only Hepatozoon species known to infect Procyonidae mammals is
H. procyonis, that was first described in 1961 infecting monocytes of raccoons (P. lotor) in the ­USA44. In Brazil,
occurrence rates of 42% (13/42), 25% (21/93), 32% (69/262), and 68% (112/165) were reported in ring-tailed
coatis from the Pantanal wetlands of Mato Grosso do Sul state, São Paulo state, Minas Gerais state, and peri-
urban forested areas (Cerrado biome) in Mato Grosso do Sul states, r­ espectively15–18. In the present study, only
3/49 (6%) samples tested positive for Hepatozoon sp. This lower prevalence might not be related to the applied
molecular technique, since a nested-PCR based on the 18S rRNA was used, which showed to be very sensitive
in the detection of H. procyonis with fluctuating p ­ arasitemia18. Considering that the vector for H. procyonis is
not known yet, the lower prevalence found herein might be due to the tick fauna presented (and consequently
vector competence) in the studied area when compared to the other studies. Amblyomma spp. larvae, and adults
of Haemaphysalis juxtakochi, Amblyomma brasiliense, Amblyomma coelebs and Amblyomma ovale have been
reported in coatis from P ­ ARNA45. When compared to other studies conducted in Mato Grosso do S­ ul14,15, São
­Paulo , and Minas ­Gerais17 states, where high occurrence of H. procyonis has been reported in the sampled
46,47

ring-tailed coatis, A. sculptum ticks were not found parasitizing the animals sampled h ­ erein45. The role of A.
sculptum and other tick species in the transmission of H. procyonis should be further evaluated in the future.
The most prevalent VBA detected in ring-tailed coatis from PARNA Iguaçu was Mycoplasma sp. closely related
to ‘Candidatus Mycoplasma haematonasua’, which was found in 85.7% of the studied animals. Hemoplasmas
(hemotropic mycoplasmas) are obligate epi-erythrocytic bacteria that infect a wide variety of mammalian hosts.
Infections with this agent can induce anemia and acute hemolysis and clinical signs, such as anorexia, lethargy,
dehydration, weight loss and, in some cases, sudden death in domestic ­animals48, while the infection appears to be
subclinical in ­wildlife49. In animals of the Procyonidae family, hemotropic Mycoplasma sp. 16S rRNA genotypes
phylogenetically related to “Candidatus Mycoplasma erythrodidelphis”, “Candidatus Mycoplasma haemozal-
ophi”, Candidatus Mycoplasma haemominutum” and “Candidatus Mycoplasma haemolamae” were detected in
59/95 (62.1%) raccoons in the U ­ SA50. In Brazil, Sousa et al.12 found 24/31 (77.4%) wild ring-tailed coatis in the
Brazilian Pantanal positive for hemotropic Mycoplasma sp. in a PCR based on the 16S rRNA gene. Interestingly,
authors detected two different genotypes: one closely related to hemotropic Mycoplasma sp. detected in raccoons
from the USA, and another one closely related to Mycoplasma haemofelis. More recently, Collere et al.13 detected
hemotropic Mycoplasma sp. in 44.5% (8/18) coatis in the same area (PARNA) where the present study was
conducted. Based on two molecular markers, namely 16S rRNA and 23S rRNA, a putative novel hemoplasma,

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‘Candidatus Mycoplasma haematonasua’, was proposed. It seems that hemoplasmas are the most prevalent VBA
detected in Procyonidae animals from the Americas. Although some hemotropic Mycoplasma species (e.g.
Mycoplasma suis, Mycoplasma haemocanis, Mycoplasma haemofelis)51 can cause moderate to severe disease in
their hosts, the pathogenicity of ‘Candidatus M. haematonasua’ is not yet known, requiring more studies, espe-
cially in animals infected with more than one VBA. The main route of transmission of hemoplasmas seems to
be through blood-sucking arthropods (e.g. fleas, ticks, etc.)48,52. However, few experimental studies have been
carried out in this sense, since such agents have not been yet cultivated in vitro48. Other routes of transmission
have also been pointed out, such as vertical, blood transfusion) 53,54 and through aggressive interactions among
­cats55 and wild rodents (Gerbillus andersoni)56. Based on multilayer network analysis, Alcantara et al.57 recently
demonstrated the lack of association among ectoparasites, non-hematophagous bats and hemoplasmas, suggest-
ing that ectoparasites may not play an important role in the transmission of hemotropic mycoplasmas among
bats. Considering these putative alternative transmission routes, Sousa et al.12 hypothesized that the gregarious
behavior of coatis associated to the fact that these mammals tend to form large g­ roups1 might contributed for a
higher incidence of hemotropic Mycoplasma spp. among this Procyonidae species.
The present study shows, for the first time, the molecular detection of Bartonella sp. in blood samples from
ring-tailed coatis from Brazil. The genus Bartonella (Rhizobiales: Bartonellaceae) comprises Gram-negative,
facultative intracellular alpha-proteobacteria, which have been identified in a wide variety of mammals, includ-
ing ­humans58–60. The transmission of bartonellae has been mainly associated with hematophagous arthropod
­vectors61. Up to now, Bartonella spp. has only been molecularly detected in raccoons from the USA. Hwang and
­Gottdenker62, when studying free-living raccoons from Georgia, USA, detected 16 positive samples (43%—16/37)
in a nested-PCR based on the 16S–23SrRNA intergenic spacer region (ITS). Thirteen positive samples were
sequenced and 12 samples were identified as B. henselae and one identified as B. koehlerae62. Bai et al.63, when
analyzing 186 spleen samples from raccoons during the enhanced rabies surveillance in Colorado, USA, obtained
14 samples positive in a PCR for Bartonella spp. based on the ITS (16S-23S) region. Sequencing results revealed
that 11 raccoons were infected with B. rochalimae and three with Bartonella vinsonii subsp. berkhoffii 63, recently
reclassified as Bartonella berkhoffii by Whole Genome Sequencing ­Analysis64.
When analyzing coatis from the Pantanal wetlands in Mato Grosso do Sul state, Brazil, Sousa et al.65 failed
to detect DNA from Bartonella sp. In blood samples from 31 sampled coatis. In the present study, 24.5% (12/49)
showed to be positive in a qPCR based on the nuoG gene. By sequencing three molecular markers (gltA, pap-31,
and ftsZ), B. machadoae was identified in ring-tailed coatis from PARNA. Bartonella machadoae has been recently
isolated and described from wild rodents from the Pantanal wetlands, Mato Grosso do Sul state, central-western
Brazil, and showed to be strictly related to species belonging to B. vinsonii ­complex37. The zoonotic potential is yet
unknown, and further studies should be carried out, since Bartonella species belonging to B. vinsonii complex are
associated with clinical manifestations in humans in different regions of the ­world37. Fleas have been suggested as
putative vector for Bartonellla among rodents in ­Brazil65,66. Indeed, Bartonella sp. has been molecularly detected
in Polygenis bohlsi bolshi fleas collected from rodents in the Pantanal wetlands biome, Mato Grosso do Sul state,
central-western ­Brazil65, as well as in Polygenis occidentalis, Polygenis platensis, and Craneopsylla minerva from
Pampa grasslands and Atlantic rainforest biomes, in the State of Rio Grande do Sul, southern B ­ razil66. Recently,
B. machadoae has been molecularly detected in Amblyomma sculptum nymphs collected from wild boars (Sus
scrofa), which showed to be negative for Bartonella spp. in a qPCR based on the nuoG gene, in southeastern Brazil.
According to the authors, A. sculptum nymphs may have acquired the Bartonella infection when fed as larvae
in a mammal (most probably rodents), and transestadially perpetuate the infection through the nymph stage.
It this is true, A. sculptum may play a role in the transmission of B. machadoae67. The present study expands the
host specificity range of B. machadoae, which has been isolated only from rodents until the present ­moment37.
A small percentage (8.1%) of animals were negative for the screened VBA in the present study. The most
prevalent co-infection observed was from bacterial VBA agents, in first place (18.3%) with hemotropic Myco-
plasma sp. and Bartonella sp., and Anaplasma sp. and hemotropic Mycoplasma sp. in second place (12.2%)
Only two animals (4%) presented co-infections by three VBA (Bartonella sp., Anaplasma sp. and hemotropic
Mycoplasma sp.). Although VBA infections seem to be associated with subclinical diseases in wild a­ nimals18,49,
VBA may play a role as potential opportunistic pathogens in immunocompromised animals, in stressful situ-
ations or in coinfections. For instance, Moraes et al.23 demonstrated that the infection by filarial nematodes in
ring-tailed coatis had no effects on the body condition and serum biochemistry parameters of the evaluated
coatis, albeit eosinophilia was observed. Also, Perles et al.18 showed that the H. procyonis infection seems not to
significantly influence clinical and hematological parameters. Co-infections may play an important role in these
infections. For instance, Alic et al.68 described a fatal case caused by coinfection by Leptospira spp. and H. canis in
a red fox cub (Vulpes vulpes) in Saravejo, Bosnia and Herzegovina. Also, Silveira et al.69 reported a fatal case of
co-infection by Rangelia vitalii, Hepatozoon sp., Leishmania sp., and Entamoeba and helminths (Ancylostoma
caninum, Ancylostoma braziliensis, Molineus sp., Trichuris sp. and Spirometra sp.) in the threatened maned-wolf
(Chrysocyon brachyurus) from Brazil.
Our results highlight the importance of assessing the health of wild animals in one of the largest remaining
areas of Atlantic forest in Brazil. Our observations also reveal the needs to deepen and expanding this type of
studies for other mammals and possible vectors inventories. The coati, besides common, play an important role in
food chain, being also the symbol of the company that provides tourism care and probably the only wild mammal
observed during the INP tourist visit. Since the zoonotic potential and transmission routes of the detected VBA
are not yet known, VBA surveillance in this area is much needed. Future studies are necessary to understand
the pathogenicity of the detected agents to coatis as well as their zoonotic potential. Also, new studies combin-
ing other methodologies with molecular detection, such as isolation, blood smears to investigate the target host
cells, and health analyses (e.g. hematological and biochemical analyses) would provide a better approach on the
surveillance and understanding of the pathogenicity of such agents.

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Conclusions
Ring-tailed coatis from Iguaçu National Park were found infected with different vector-borne agents. The most
prevalent VBA was Mycoplasma sp. closely related to ‘Candidatus Mycoplasma haematonasua’, followed by Bar-
tonella sp., Anaplasma sp., and Hepatozoon procyonis. A putative novel Anaplasma species, yet to be isolated
and characterized, occurs in ring-tailed coatis from Iguaçu National Park. This is the first molecular detection
of B. machadoae in coatis. Co-infections by Mycoplasma sp., Bartonella sp. and Anaplasma sp. are reported for
the first time in coatis.

Data availability
The datasets generated and analysed during the current study are available in the NCBI—GenBank—Nucleotide
platform (https://w
​ ww.n
​ cbi.n
​ lm.n ​ k/) and can be accessed through accession numbers: Anaplasma
​ ih.g​ ov/g​ enban
sp. (16SrRNA OM811667—OM811673), (ITS OP948063–OP948066), (gltA OM830713—OM830715); Hepato-
zoon sp. (18SrRNA OM812694—OM812696), Mycoplasma sp. (16SrRNA OP795503—OP795509), Bartonella
sp. (pap31 OP910252–OP910256), (ftsZ OP93382–OP933385), (gltA OP933386–OP933392).

Received: 14 November 2022; Accepted: 30 January 2023

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Author contributions
All authors contributed to the study conception and design. Material preparation, data collection and analysis
were performed by L.P., M.F.M., and M.X.d.S. The first draft of the manuscript was written by L.P. and M.R.A. and
all authors commented on previous versions of the manuscript. Funding acquisition was performed by M.R.A.
and E.G.L.H. All authors read and approved the final manuscript.

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Funding
This work was supported by FAPESP (Foundation for Research Support of the State of São Paulo) grants conceived
to M.R.A. (Process #2018/02753-0; #2020/12037-0) and E.G.L.H. (2016/15589-8), and CNPq (National Council
for Scientific and Technological Development; Productivity Grant to MRA [CNPq Process #303701/2021-8)]. LP
received scholarship from FAPESP (2019/15150-4); MFM received scholarship from FAPESP (2016/14886-9).

Competing interests 
The authors declare no competing interests.

Additional information
Supplementary Information The online version contains supplementary material available at https://​doi.​org/​
10.​1038/​s41598-​023-​29090-1.
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