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First molecular characterization of Sarcocystis neurona causingmeningoencephalitis in a domestic cat in Brazil

Márcia Elisa Hammerschmitt1 & Luan Cleber Henker1 & Juliana Lichtler2 & Fernanda Vieira Amorim da Costa2 &

Rodrigo Martins Soares3 & Horwald Alexander Bedoya Llano3& Saulo Petinatti Pavarini1

Received: 19 September 2019 /Accepted: 4 December 2019 /Published online: 4 January 2020# Springer-Verlag GmbH Germany, part of Springer Nature 2020

AbstractSarcocystis neurona is the main agent associated with equine protozoal myeloencephalitis (EPM). Apart from horses, S.neurona has been occasionally described causing neurologic disease in several other terrestrial animals as well as mortality inmarine mammals. Herein, we describe the clinical, pathological, and molecular findings of a fatal case of S. neurona-associatedmeningoencephalitis in a domestic cat. The causing agent was analyzed by multilocus genotyping, confirming the presence of S.neuronaDNA in the tissue samples of the affected animal. Significant molecular differences were found in relation to S. neuronaisolates detected in other regions of the Americas. In addition, the parasite was identical to Sarcocystis sp. identified in opossumsporocysts in Brazil at molecular level, which suggests that transmission of. S. neurona in Brazil might involve variants of theparasite different from those found elsewhere in the Americas. Studies including more samples of S. neurona would be requiredto test this hypothesis, as well as to assess the impact of this diversity.

Keywords Protozoalmeningoencephalitis . Feline . Genotyping . Polymerase chain reaction


Sarcocystis neurona, Sarcocystis speeri, and Sarcocystisfalcatula are very similar species, transmitted exclusively inthe Americas by opossums of the genus Didelphis spp.(Dubey et al. 2015). S. neurona and S. falcatula have alreadybeen described in Brazil. Birds of various species are suscep-tible to S. falcatula, while mammals, predominantly, are sus-ceptible to S. neurona/S. speeri (Dubey et al. 2015). It is worthmentioning that S. neurona and S. speeri are almost identicalspecies according to criteria based on molecular identification(Cesar et al. 2018; Acosta et al. 2018).

Sarcocystis neurona is the main agent associated withequine protozoal myeloencephalitis (EPM), an important neu-rologic disease of horses in the American continent (Dubeyet al. 2015). In addition, neurologic disease caused by S.neurona has been occasionally described in several other ter-restrial animals (Dubey et al., 2000), and this parasite hasarisen as a significant cause of mortality in marine mammals(Barbosa et al. 2015). Opossums, Didelphis virginiana inNorth America (Fenger et al. 1995) and Didelphis albiventrisin South America (Dubey et al., 2001a), are known definitive

Section Editor: Daniel K. Howe

Electronic supplementary material The online version of this article( contains supplementarymaterial, which is available to authorized users.

* Márcia Elisa [emailprotected]

1 Setor de Patologia Veterinária, Departamento de Patologia Clínica,Faculdade de Veterinária, Universidade Federal do Rio Grande doSul, Av. Bento Gonçalves, 9090, Bairro Agronomia, PortoAlegre, RS 91540-000, Brazil

2 Setor de Medicina Felina, Hospital de Clínicas Veterinárias,Faculdade de Veterinária, Universidade Federal do Rio Grande doSul, Av. Bento Gonçalves, 9090, Bairro Agronomia, PortoAlegre, RS 91540-000, Brazil

3 Departamento de Medicina Veterinária Preventiva e Saúde Animal,Faculdade deMedicina Veterinária e Zootecnia, Universidade de SãoPaulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, CidadeUniversitária, São Paulo, SP 05508-270, Brazil

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hosts of S. neurona, while intermediate hosts include domesticcats, sea otters, armadillos, raccoons, and skunks (Dubeyet al., 2001b). Central nervous system (CNS) disease causedby S. neurona has been rarely described in domestic cats(Dubey et al., 1994, 2003).

Therefore, the objective of this study is to describe theclinical, pathological, immunohistochemical, and molecularfindings of a fatal case of S. neurona-associated meningoen-cephalitis in a domestic cat in Southern Brazil.

Material and methods


A 1.7-year-old domestic cat, with free access to the street,presented a clinical history of anorexia and dyspnea, whichlasted 7 days. At clinical examination, hyperthermia and pleu-ral effusion were detected. Serological tests yielded positiveresults for feline leukemia virus (FeLV) and negative resultsfor feline immunodeficiency virus (FIV). Pleural effusion wassubmitted for cytological evaluation, and pyogranulomatousinflammation was observed. Thoracic effusion was subjectedfor coronavirus immunocytochemistry, yielding negative re-sults. Thoracic radiography was performed, and a radiopaquearea in the cranial mediastinal region was noted. Due to theclinical suspicion of mediastinal lymphoma, chemotherapeu-tic treatment was prescribed. Treatment with prednisolone(2 mg/kg, S.I.D., P.O.) was established, associated with vin-cristine administrations with a 7-day interval (0.75 mg/m2,I.V.). In addition, a single administration of cyclophospha-mide (250 mg/kg, P.O.), at the third week after treatment on-set, was prescribed. Twenty-one days after the first clinicalexamination, the cat returned to the hospital presenting neu-rological signs, such as compulsive walking, head pressing,and vocalization. The cat died and was referred for necropsy.At the necropsy, the animal showed severe dehydration, ema-ciation, as well as mild cerebellar herniation through the fora-men magnum. No mass consistent with lymphoma was evi-denced at the mediastinal region or any other organ. In addi-tion, no cavitary effusions or inflammatory lesions compatiblecom FIP were observed. At necropsy, multiple tissue sampleswere collected and fixed in a 10% neutral buffered formalinsolution. Samples were routinely processed for histopatholog-ical examination and stained with hematoxylin and eosin(HE). Fresh frozen samples of brain and cerebellum werecollected and posteriorly submitted for DNA extraction andmolecular analysis. At the histological examination, lesionswere restricted to the encephalon (spinal cord was not avail-able for evaluation). In the gray and white matter and some-times extending to the leptomeninges (Fig. 1a), there was amarked inflammatory infiltrate consisting of degenerate neu-trophils, macrophages, and lymphocytes (Fig. 1b). These

areas were often associatedwith parasitic structuresmeasuring10–15 μm in diameter (schizonts), which contained numerouselongated structures measuring around 1.5 × 4 μm (merozo-ites), morphologically consistent with S. neurona (Fig. 1c).Schizonts were observed freely in the midst of inflammationareas, as well as in the soma of neurons and in the cytoplasmof astrocytes. In addition, multifocal moderate to severeperivascular inflammatory infiltrate of lymphocytes, plasmacells, and macrophages was seen in the brain and in the cere-bellum. Furthermore, multifocal areas of gliosis associatedwith gemistocytic astrocytes, few gitter cells, and endothelialcell hypertrophy were seen. Lesions were marked in the tel-encephalon, diencephalon, cerebellum, and corpus striatum,and moderate in the mesencephalon and in the brainstem.


CNS sections were submitted for immunohistochemistry anti-S. neurona (polyclonal antibody non-commercial, 1:200), an-ti-Toxoplasma gondii (VRMD, Pullman, WA, USA, dilutionof 1:1000), and anti-Neospora caninum (VRMD, Pullman,WA, USA, dilution of 1:1000). Antigen retrieval was per-formed with proteinase K for 1 min for S. neurona and 0.1%trypsin for 10 min for N. caninum and T. gondii. Blocking ofnonspecific reactions was performed with 5% skim milk for15 min. The amplification signal was achieved by usingMACH 4 Universal HRP-Polymer (Biocare, Pacheco, CA,USA) for S. neurona and LSAB-HRP Universal kit(Dakocytomation, Carpinteria, CA, USA) for N. caninumand T. gondii. The reactions were visualized with 3-amino-9-ethylcarbazole chromogen (AEC; Sigma, St. Louis, Missouri,USA), and subsequently, all slides were counterstained withHarris’ hematoxylin. Positive control samples consisted ofknown cases of CNS disease caused by S. neurona,T. gondii, and N. caninum. In the negative control slides, pri-mary antibodies were replaced by PBS.

DNA extraction, PCR amplification, and sequencing

DNA was extracted from frozen samples of the brain of theinfected cat with DNeasy Blood & Tissue Kit (Qiagen,Hilden, Germany) as recommended by the manufacturer’s,except for elution of the final product in 50 μL of AE buffer.The DNA samples were tested for the presence of S. neuronaby means of multilocus genotyping as described below.

Sarcocystis spp. sequences of 18S small subunit rRNAregion, gene coding for cytochrome c oxidase subunit I, andinternal transcribed spacer 1, SAG2, SAG3, and SAG4 werenested PCR amplified. Nested PCR directed to 18S (nPCR-18S) was performed using primers 18S9L and 18S1H (Li et al.2002). DNA amplification of Sarcocystis spp. cytochrome coxidase subunit I (nPCR-COX1) was performed usingprimers designed by Gondim et al. (2019). Complete internal

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transcribed spacer 1 was nested PCR amplified using theprimer set described by Soares et al. (2011). Finally, geneticsequences of SAG2, SAG3, and SAG4 were nested PCR am-plified using the primers as designed in Monteiro et al. (2013),Valadas et al. (2016), and Gondim et al. (2019). Primers usedto amplify genetic sequences of Sarcocystis spp. using nestedPCR are depicted in Table 1.

DNA amplification was done in two rounds, in all cases.The first round of amplifications (external primers) was con-ducted by adding 4 μL of extracted DNA, 2.5 μL of 10x PCRBuffer (KCL 50 mM; Tris HCl 10 mM; pH 9.0), 1 μL ofMgCL2 (50 mM), 0.5 μL of mixed dNTPs (10 mM),0.35 μL of each primer (10 μM), 0.2 μL of Taq DNAPolymerase Platinum-Invitrogen (5 U/μL), and 16.1 μL ofultrapure autoclaved water to a volume of 25 μL per reaction.The PCR thermal cycling consisted of an initial incubation at94 °C for 3 min, followed by 40 cycles (94 °C for 30 s, 56 °Cfor 30 s, 72 °C for 50 s) and a final extension at 72 °C for5 min. For the second rounds of amplifications (internalprimers), 2 μL of template derived from the first reactionswas added to 2.5 μL of 10x PCR Buffer (KCL 50 mM; Tris

HCl 10 mM; pH 9.0), 1 μL of MgCL2 (50 mM), 0.5 μL ofmixed dNTPs (1.25 mM), 1 μL of each primer (10 μM),0.2 μL of Taq DNA Polymerase Platinum-Invitrogen (5 U/μI), and 16.8 μL of ultrapure autoclaved water to a volume of25 μL per reaction. The thermal cycling was the same used inthe first round.

The nested PCR products were analyzed under ultraviolettransilluminator after electrophoresis in 2% agarose gels andethidium bromide (0.5 μg/mL) staining for 60 min. Thetargeted bands were excised from the gel and purified usinga commercial purification kit (Illustra GFX PCR DNA andGel Band Purification, Amersham Biosciencies) accordingto the manufacturer’s instructions. The purified DNAwas bi-directionally sequenced using forward and reverse primerswith the Kit ABI PRISM Big Dye Terminator (AppliedBiosystem) following the manufacturer’s protocol.

Sequence edition and sequence analysis

The quality of nucleotide sequences, the contig assembly, andsequence edition were assessed using the program Phred-Phrad

Fig. 1 S. neurona-associated meningoencephalitis in a domestic cat. aCerebellum, marked diffuse mixed inflammatory infiltrate is observeddistending the leptomeninges as well as partially effacing the neuropileor the trilaminar aspect of the cerebellum. HE, bar 300 μm. bCerebellum,there is moderate inflammatory infiltrate of lymphocytes, plasmocytesand macrophages associated with parasitic schizonts (arrows). HE, bar

100 μm. c Cerebellum, multiple schizonts of S. neurona filled bynumerous elongated structures (merozoites) are seen in the neuropile.HE, bar 30 μm. d Cerebellum, marked multifocal anti-S. neuronaimmunostaing is noted in the neuropile. Numerous schizonts and mero-zoites are evidenced freely in the neuropile, as well as in the cytoplasm ofCNS cells. IHC, chromogen AEC, bar 45 μm

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in Codon Code Aligner, version 4.2.1. The final nucleotidesequences from each locus were analyzed using Blast searchtool ( ITS1 and SAGsequences were aligned with hom*ologous sequences availablein GenBank using the program Clustal W in BioEdit SequenceAlignment Editor v. (Hall 1999). ITS1 phylogenetic treewas inferred with maximum likelihood method, computingevolutionary distance using K2P +G method. The ITS1 phy-logeny was inferred with sequences of S. neurona, S. speeri, S.falcatula, and related organisms retrieved from GenBank afterBlast search tool using S. neurona from a cat as query. ITS1phylogeny was reconstructed using sequences with more than90% coverage containing at most one degenerate site by usingMEGA 7 (Kumar et al. 2016). The software PopART(Population Analysis with Reticulate Trees) (Leigh andBryant 2015) was used to infer evolutionary relationships andInteger NJ networks inference method among isolates of S.neurona and S. falcatula based on SAG loci. The genetic se-quences were deposited in GenBank under the following acces-sion numbers: SAG2 MN175964, SAG3 MN175965, SAG4MN175966, CO1 MN175967, ITS1 MN172273, and 18SMN169125.


Immunohistochemistry anti-S. neurona was strongly positive(Fig. 1d), and immunohistochemistry anti-T. gondii and anti-N. caninum were negative. Brain tissue samples were positiveby all nested PCRs and the results of the multilocus analysisrevealed the presence of S. neurona, which was called as S.neurona-07-2015-RS-BR. The complete nucleotide sequenceof the nPCR-18S amplicon of S. neurona-07-2015-RS-BR(783 nucleotides, primers excluded) disclosed 100% identityand 100% coverage to S. speeri detected in opossums fromArgentina (KT207459), and 99.87% identity and 100% cov-erage to S. falcatula detected in fatal Sarcocystis infection inthe Rainbow Lorikeets (Trichoglossus moluccanus) (isolateLorikeet #205850, MH626537) (Verma et al. 2018). A G-Asubstitution differs S. neurona-07-2015-RS-BR and S.falcatula isolate Lorikeet #205850 at nucleotide position 30(taking MH626537 as reference). Only two single nucleotidepolymorphisms differ S. falcatula isolate Lorikeet #205850from S. speeri among 1618 paired nucleotides (G-A and C-A substitutions at nucleotide positions 30 and 1563, respec-tively, taking MH626537 as reference). nPCR-COX1

Table 1 Primers for the detection of Sarcocystis spp. genetic sequences in brain tissue sample of an infected cat

Locus Primers Sequences PCR stepa Size (bp)b Reference

18S 18S9L GGATAACCTGGTAATTCTATG 1 + 2 825 Li et al. 2002

18S1H GGCAAATGCTTTCGCAGTAG 1 + 2 Li et al. 2002



COX1-275F22 TGTACCCACGAATTAATGCAGT 2 590 Gondim et al. 2019

COX1-844R21 GTGTGCCCATACTAGAGAACC 2 Gondim et al. 2019

ITS1 JS4 CGAAATGGGAAGTTTGAAC 1 Slapeta et al. 2002

CT2c CTGCAATTCACATTCGC 1 Soares et al. 2011

JS4b AGTCGTAACAAGGTTTCCGTAGG 2 ~ 1100 Soares et al. 2011

CT2b TTGCGCGAGCCAAGACATC 2 Soares et al. 2011


SAG2-R1 ACAACACTGTGAGAGATGCGA 1 Monteiro et al. 2013

SAG2-F2 GGTCAGAGCTTTGTGCTGAA 2 338 Monteiro et al. 2013

SAG2-459R21 CACATTGCAAGCASGACACCA 2 Gondim et al. 2019

SAG3 SAG3-F1 CTCGCAGTTGCCTGCCTTG 1 511 Monteiro et al. 2013


SAG3-589R21 TGGTCCTGTAGCAGTAACACA 1 + 2 Valadas et al. 2016

SAG4 SAG4-F2 CCGAGGTACAGTTCAAGGCG 1 Monteiro et al. 2013

SAG4-R1 CGACGACGATACCCAATGCC 1 Monteiro et al. 2013

SAG4-541F21 GGCAACGCCGCAGCMCTGCAA 2 282 Valadas et al. 2016

SAG4-803R20 CAATGCCGAMGCGGTACGAG 2 Gondim et al. 2019

a (1) Primers used only in the first round of amplification, (2) primers used only in the second round of amplification, and (1 + 2) primers used in the firstand second round of amplificationb Predicted size of the nested PCR product

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products of S. neurona-07-2015-RS-BR (547 nucleotides,primers excluded) differs from both S . falcatula(MH665257) and S. speeri (KT207461) at two nucleotidepositions (A-T and C-A substitutions at nucleotide positions347 and 497, respectively, taking KT207461 as reference).Curiously, S. falcatula MH665257 and S. speeri KT207461disclosed 100% identity and 100% coverage among 1002paired nucleotides. No gap was included in the alignmentsof 18S and COX1 from S. neurona-07-2015-RS-BR, S.falcatula isolate #205850, and S. speeri.

The almost complete nPCR-ITS1 amplicon was sequencedfrom S. neurona-07-2015-RS-BR (992 nucleotides). From theBlast search, the ITS1 sequence of S. neurona-07-2015-RS-BR is most similar to S. speeri detected in opossums inArgentina (KT207458), showing 97.89% identity, including10 gaps. From the sequences annotated as S. falcatula, themost similar to S. neurona-07-2015-RS-BR is S. falcatulaisolate Lorikeet #205850 (MH626538). These sequencesshare 97.08% nucleotide identity, including 16 gaps. ITS1phylogenetic tree including S. falcatula, S. neurona, S. speeri,and S. falcatula related sequences can be found in Fig. 2.Curiously, the topology of the ITS1 tree shows that S.neurona-07-2015-RS-BR is clearly divergent from S.neurona/S. speeri. The monophyly of S. neurona/S. speeri/S.neurona-07-2015-RS-BR clade is supported with low boot-strap value (< 70, not shown).

Evolutionary relationships among S. neurona and S.falcatula inferred with SAG loci revealed that S. neurona-07-2015-RS-BR clustered closer to S. neurona (Fig. 3). Inaddition to S. neurona, the networks in Fig. 3 included S.falcatula obtained from budgerigars that were experimentallyinfected with opossum’s sporocysts (Gondim et al. 2017,Cesar et al. 2018) and S. falcatula directly detected in inter-mediate hosts (Konradt et al. 2017, Acosta et al. 2018). At thethree SAG loci, S. neurona-07-2015-RS-BR was identical toSarcocystis sp. genotype II derived from opossum’s sporo-cysts in the state of Rio Grande do Sul, Brazil, described byMonteiro et al. (2013).


In the present study, the diagnosis of S. neurona-associatedmeningoencephalitis in a domestic cat was establishedthrough the association of the pathological, immunohisto-chemical, and molecular findings. Data regarding CNS dis-ease caused by S. neurona in domestic cats are scarce. InNorth America, a recent case report described encephalomy-elitis in an 8-year-old cat associated with S. neurona-like in-fection. The ITS1 sequence showed the most similarity with S.neurona and S. dasypi (Zitzer et al. 2017). The experimentalinfection of domestic cats by S. neurona, with subsequentformation of cysts in their muscular tissue, has been reported

(Dubey et al., 2001b), which suggests that cats may act as oneof the intermediate hosts of S. neurona, at least in experimen-tal conditions. Studies conducted in Michigan (Rossano et al.2002), Missouri (Turay et al. 2002), and Ohio (Stanek et al.2003) revealed antibodies to S. neurona in feral cats, indicat-ing possible exposure and natural infection by S. neurona indomestic felines with access to the outdoors in general, simi-larly to what was observed in the present case. However, insome of the mentioned studies, antibody cross-reactivityagainst other closely Sarcocystis species could not becompletely rule out (Rossano et al. 2002).

Rapid reproduction after oral infection of the intermediatehosts is likely responsible for the clinical manifestations(Wünschmann et al. 2009). Possibly, the use of corticoste-roids, FeLV infection, and chemotherapy had great impor-tance in the development of the clinical condition in the pres-ent case. Similarly, other immunosuppressive factors such aslesions, surgery, and stressful events have been associatedwith S. neurona infection in horses (Saville et al. 2001,Cooley et al. 2007). It has been suggested that the use ofcorticosteroids influences the proliferation of the protozoanin the CNS (Cooley et al. 2007), which may explain the ag-gravation of the clinical condition.

Microscopic lesions observed in the inspected organs wererestricted to the brain and cerebellum and were characterizedby meningoencephalitis associated with intralesional parasiticstructures compatible with S. neurona, confirmed by immu-nohistochemistry and PCR. Inflammatory lesions weremarked in the present case; however, unlike horses, in whichS. neurona is rarely observed (Dubey et al. 2015), numerousparasites were noted within areas of inflammation.Toxoplasma gondii and N. caninum, two coccidians candi-dates that could be also involved in neurological infections,were ruled out because nPCR-ITS1 are capable of detectingboth sarocystinae and toxoplasmatinae and genetic sequencesof the latter was not found. In addition, immunohistochemis-try anti-N. caninum and anti-T. gondii were negative.

To our knowledge, this is the first report on molecularcharacterization of S. neurona detected in a case of neurologicdisease affecting an animal in Brazil. Sarcocystis neurona andS. falcatula are closely related organisms exclusively transmit-ted within the American continents that use opossums of thegenus Didelphis as definitive hosts. Despite morphologicaland molecular similarities, S. neurona infects mammals pre-dominantly, while S. falcatula exclusively infects birds. Twoevolutionarily distant strains have been well recognizedamong isolates of S. falcatula in the Americas (Valadas et al.2016, Gondim et al. 2017, Cesar et al. 2018), and to ourknowledge, a similar situation has not yet been detected inS. neurona, which appeared to be formed by only one geneticlineage. However, from the results presented here, it is prob-able that this diversity also occurs within S. neurona. Thecharacterization of the ITS1 locus of the S. neurona-07-

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2015-RS-BR isolate revealed an ITS1 lineage, which is con-siderably distant from other organisms classified as S. neurona(including S. speeri). By ITS1-based analysis, S. speeri, foundin sporocysts of opossums in Argentina, is almost identical to

S. neurona isolates found in several other hosts in NorthAmerica and the clade that congregates all species of S.neurona and S. speeri with high statistical support excludesS. neurona-07-2015-RS-BR isolate.

Fig. 2 Evolutionary relationships among S. neurona, S. speeri, and S. falcatula inferred with ITS1. The tree is rooted with S. lindsayi. Bootstraps valuesless than 70 are not shown. S. neurona/S. speeri in blue, S. falcatula in red. The list of taxa presented in the tree is depicted in the supplementary table

Fig. 3 Phylogenetic networks onSAG2, SAG3, and SAG4genotypes from Sarcocystis spp.Sarcocystis neurona-07-2015-RS-BR is represented bygenotype #II in all networks. S.neurona in blue, S. falcatula inred. The size of the circles isproportional to the number ofsamples. Mutations are shown inhatch marks. The list of taxapresented in the networks isdepicted in the supplementarytable

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Although COX1-based analysis does not distinguish S.speeri and S. falcatula, both species differ from the S.neurona-07-2015-RS-BR isolate by two nucleotide substitu-tions. It is worth noting that two nucleotide substitutions (con-sidering the segment hom*ologous to nPCR-COX1 fragment)differentiate S. halieti (MH138308) from S. turdusi(KT588511) , S . lar i (MF596283) , and S. lutrae(MG273670), which are all recognized as distinct species (da-ta not shown).

Herein, the divergence of isolate S. neurona-07-2015-RS-BR from S. neurona was further investigated with the SAGloci and the isolate 07-2015-RS-BR reveals, although notidentical, very closely related to other samples of S. neurona,which allows concluding that the sarcocystid isolate found inthe cat investigated may in fact be classified as S. neurona.

From the analysis of SAG2, SAG3, and SAG4, S. neurona-07-2015-RS-BR was identical to Sarcocystis sp. identified inopossum sporocysts in the state of Rio Grande do Sul, Brazil(Monteiro et al. 2013), indicating that variants of S. neuronadifferent from those occurring elsewhere in the Americas arebeing transmitted in Brazil. Studies including more samples ofS. neuronawould be required to test this hypothesis, as well asto assess the impact of this diversity.

Acknowledgments We thank Dr. Marcele Bettim Bandinelliby performing immunohistochemistry. The authors thankConselho Nacional de Desenvolvimento Científico eTecnológico (CNPQ) and Coordenação de Aperfeiçoamentode Pessoal de Nível Superior (CAPES) for the financialsupport.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict ofinterest.


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