UNIVERSIDADE ESTADUAL PAULISTA - UNESP CÂMPUS DE JABOTICABAL MOLECULAR CHARACTERIZATION OF Sarcocystis spp. FROM OPOSSUMS (Didelphis spp.) AND SEROLOGICAL DETECTION OF ANTI-Sarcocystis spp. ANTIBODIES IN HORSES FROM SOUTHEASTERN AND MIDWESTERN BRAZIL Mariele De Santi Médica Veterinária 2023 UNIVERSIDADE ESTADUAL PAULISTA - UNESP CÂMPUS DE JABOTICABAL MOLECULAR CHARACTERIZATION OF Sarcocystis spp. FROM OPOSSUMS (Didelphis spp.) AND SEROLOGICAL DETECTION OF ANTI-Sarcocystis spp. ANTIBODIES IN HORSES FROM SOUTHEASTERN AND MIDWESTERN BRAZIL Discente: Mariele De Santi Orientadora: Profa. Dra. Rosangela Zacarias Machado Coorientadora: Profa. Dra. Karin Werther Tese de apresentada à Faculdade de Ciências Agrárias e Veterinárias – Unesp, Campus de Jaboticabal, como parte das exigências para a obtenção do título de Doutor em Medicina Veterinária. 2023 S235m Santi, Mariele De Molecular characterization of Sarcocystis spp. from opossums (Didelphis spp.) and serological detection of anti-Sarcocystis spp. antibodies in horses from Southeastern and Midwestern Brazil / Mariele De Santi. -- Jaboticabal, 2023 87 p. : il., tabs. Tese (doutorado) - Universidade Estadual Paulista (Unesp), Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal Orientadora: Rosangela Zacarias Machado Coorientadora: Karin Werther 1. Medicina veterinária. 2. Parasitologia veterinária. 3. Protozoologia veterinária. 4. Biologia molecular. 5. Sorologia veterinária. I. Título. Sistema de geração automática de fichas catalográficas da Unesp. Biblioteca da Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal. Dados fornecidos pelo autor(a). Essa ficha não pode ser modificada. IMPACTO ESPERADO DA PESQUISA De acordo com a INSTRUÇÃO AT/PROPG Nº 02, DE 22 DE DEZEMBRO DE 2022, abaixo estão descritos os impactos esperados a partir desta pesquisa. Mieloencefalite Protozoária Equina é uma doença grave que acomete os equinos. A doença é causada pelo protozoário Sarcocystis neurona, e é caracterizada pelo aparecimento de lesões em encéfalo e/ou medula espinhal. O quadro clínico cursa com sintomatologia neurológica, com sinais tais como dificuldade de ficar em pé, andar ou engolir podendo rapidamente progredir para a morte do animal. Trata-se, portanto, de uma enfermidade de elevada importância à qual se deve dar especial atenção. A presente pesquisa, realizada com base em biologia molecular e testes imunológicos, gerou dados importantes, que auxiliam no melhor entendimento da dinâmica da enfermidade. O conhecimento sobre a distribuição do protozoário nos seus hospedeiros definitivos (gambás) é importante pois pode auxiliar a identificar áreas com maior probabilidade de ocorrência da doença. O conhecimento das características genéticas do protozoário pode auxiliar no entendimento da patogênese da doença, ou seja, quais são os mecanismos utilizados pelo protozoário para causar a doença nos equinos. Com base nesse entendimento, metodologias mais efetivas e confiáveis para o diagnóstico precoce da doença podem ser desenvolvidas. Além disso, os dados gerados neste estudo podem auxiliar na criação de novas drogas para tratamento da enfermidade. A prevenção da doença também é um ponto importante, visto que que seu prognóstico é desfavorável. Sendo assim, os dados aqui gerados podem servir de base para o desenvolvimento de formas efetivas de prevenção, tais como as vacinas. IMPACT EXPECTED FROM THE STUDY In accordance with the INSTRUÇÃO AT/PROPG Nº 02, DE 22 DE DEZEMBRO DE 2022, here are the impacts expected from the study. Equine Protozoal Myeloencephalitis is a disease that affects horses. The disease is caused by the protozoa Sarcocystis neurona, and it is characterized by the occurrence of lesions in brain and/or in spinal cord. The clinical picture presents with neurological symptomatology, with signs such as difficulty on standing, walking, or swallowing, that could quickly progress to the death of the animal. It is, therefore, a very important disease, to which special attention should be given. The present study, based on molecular biology and immunological tests, lead to important knowledge that can help the better understanding of the dynamics of the disease. The information about the protozoa distribution in their definitive hosts (opossums) is important, as it could help to identify areas with higher probability of occurrence of the disease. Information about the protozoa’s genetic characteristics may help the better understanding of the pathogenesis of the disease, i.e., which are the mechanisms used by the protozoa to cause the disease in the horses. Based on that, more effective and reliable methodologies may be developed for the early diagnostic of the disease. Besides, the information obtained in the present study may help in the development of new drugs for the treatment of the disease. The prevention is also important, as the prognostic of the disease is not favorable. Therefore, the knowledge shared here may serve as base to the development of effective forms of prevention, such as the vaccines. DADOS CURRICULARES DO AUTOR MARIELE DE SANTI – Nascida em 13 de abril de 1988, em Concórdia, Santa Catarina. Filha de Ivanir Antonio De Santi e Geni Maria De Santi. Formada em Medicina Veterinária pelo Instituto Federal Catarinense – IFC Campus Concórdia em 2014. Em fevereiro de 2017 concluiu o Curso de Mestrado em Medicina Veterinária, na área de concentração - Patologia Animal, na Universidade Estadual Paulista “Júlio de Mesquita Filho” – Faculdade de Ciências Agrárias e Veterinárias – FCAV/UNESP, Jaboticabal, São Paulo, com bolsa concedida pelo Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq. Em março deste mesmo ano, ingressou no Programa de Aprimoramento Profissional do Hospital Veterinário “Governador Laudo Natel” da FCAV/UNESP, o qual foi finalizado em fevereiro de 2019. Em março de 2019, ingressou no Curso de doutorado em Medicina Veterinária, na área de concentração - Patologia Animal na mesma instituição sob a orientação da Profa. Dra. Rosangela Zacarias Machado e coorientação da Profa. Dra. Karin Werther. Durante o curso de doutorado usufruiu de bolsa da Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP. Entre dezembro de 2021 e junho de 2022, realizou Estágio de Doutorado Sanduíche na University of Kentucky, sob orientação do Prof. Dr. Daniel K. Howe, com bolsa concedida pela FAPESP. EPÍGRAFE Calma, Se você está no ponto de estourar mentalmente, silencie alguns instantes para pensar. Se o motivo é moléstia no próprio corpo, a intranquilidade traz o pior. Se a razão é enfermidade em pessoa querida, o seu desajuste é fator agravante. Se você sofreu prejuízos materiais, a reclamação é bomba atrasada, lançando caso novo. Se perdeu alguma afeição, a queixa tornará você uma pessoa menos simpática, junto de outros amigos. Se deixou alguma oportunidade valiosa para trás, a inquietação é desperdício de tempo. Se contrariedades aparecem, o ato de esbravejar afastará de você o concurso espontâneo. Se você praticou um erro, o desespero é porta aberta a faltas maiores. Se você não atingiu o que desejava, a impaciência fará mais larga a distância entre você e o objetivo a alcançar. Seja qual for a dificuldade, conserve a calma, trabalhando, porque, em todo problema, a serenidade é o teto da alma, pedindo o serviço por solução. Autor: André Luiz Psicografia de Francisco Cândido Xavier DEDICO... Aos meus pais, pelo esforço e apoio incondicionais. AGRADECIMENTOS A Deus, por seu infinito amor, bondade e justiça. À minha família, pelo suporte e pelos ensinamentos que moldaram meu caráter. Ao Professor Heitor Miraglia Herrera, pela confiança e por todo suporte oferecido durante os trabalhos de campo. Seu auxílio foi imprescindível para o desenvolvimento deste projeto. Ao Professor Rodrigo Martins Soares, pela prontidão e paciência. Ao Professor Marcos Rogério André, pelo incentivo e pelo exemplo de perseverança e competência. À professora Karin Werther por todo auxílio e ensinamentos. À professora Cláudia Momo pelo auxílio na realização dos trabalhos de campo. Ao Professor Daniel K. Howe, pela oportunidade, confiança e ensinamentos durante o estágio de Doutorado no exterior. Aos companheiros nos trabalhos de campo Filipe, Nayara, William, Wesley, Andreza, Oscar, João e Thiago, sempre dispostos a ajudar. Aos colegas do Laboratório de Imunoparasitologia. Aos amigos que fiz em Jaboticabal, com os quais compartilhei bons momentos. À FCAV/Unesp e ao Programa de Pós-graduação em Ciências Veterinárias pela oportunidade de crescimento acadêmico e científico proporcionado durante minha formação doutoral. À Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) pela bolsa de estudos (Processo 2019/08594-0) e pela bolsa de estágio de pesquisa no exterior (Processo 2021/06779-6), concedidos para a realização deste trabalho. À banca examinadora do exame geral de qualificação: Profa. Dra. Darci Moraes Barros-Battesti, Prof. Rodrigo Martins Soares e Profa. Rosangela Zacarias Machado e à banca examinadora da defesa, pelas valiosas críticas e contribuições. Aos funcionários da FCAV-Unesp/Jaboticabal, em especial, do Departamento de Patologia Veterinária, Seção de Pós-Graduação e Biblioteca, pela ajuda. A todos aqueles que um dia foram meus professores, e que contribuíram para minha formação. E por fim, a todos aqueles que de alguma forma participaram e me auxiliaram no desenvolvimento deste trabalho e crescimento pessoal durante o período do doutorado. O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001. AGRADECIMENTOS ESPECIAIS Minha imensa gratidão à Professora Dra. Rosangela Zacarias Machado. Orientadora no âmbito profissional e pessoal, me direcionando com competência, sabedoria e paciência em todos os momentos desta jornada. Sempre a terei como um exemplo a ser seguido. I CONTENTS Page CERTIFICADO DA COMISSÃO DE ÉTICA NO USO DE ANIMAIS............................III AUTORIZAÇÃO PARA ATIVIDADES COM FINALIDADE CIENTÍFICA (ICMBIO)………………………………………………………………….………………....VI RESUMO................................................................................................................. VIII ABSTRACT ................................................................................................................ X LIST OF ABBREVIATIONS ..................................................................................... XII CHAPTER 1 – General consideration ...................................................................... 1 1 INTRODUCTION ...................................................................................................... 1 2 LITERATURE REVIEW............................................................................................ 2 2.1 The genus Sarcocystis .................................................................................... 2 2.2 Sarcocystis spp. and opossums .................................................................... 6 2.2.1 Sarcocystis neurona .................................................................................. 8 2.2.2 Sarcocystis falcatula ............................................................................... 11 2.2.3 Other Sarcocystis that use opossums as definitive hosts .................. 12 2.2.4 Molecular identification of Sarcocystis spp. from opossums ............. 13 2.3 Serological studies on Sarcocystis neurona in South American horses . 15 3 GENERAL OBJECTIVES ...................................................................................... 17 4. REFERENCES ...................................................................................................... 17 CHAPTER 2 – Molecular diversity of Sarcocystis spp. in opossums (Didelphis spp.) from Southeastern and Midwestern Brazil ............................................... 29 Abstract .................................................................................................................... 29 Resumo .................................................................................................................... 29 Introduction ............................................................................................................. 30 Material and Methods .............................................................................................. 31 Sampling ............................................................................................................... 31 In vitro growth of Sarcocystis spp. .................................................................... 32 DNA extraction ..................................................................................................... 33 Molecular detection of Sarcocystis spp. based on ITS-1, cox1, SAG2, SAG3 and SAG4 gene fragments .................................................................................. 33 Cloning and sequencing ..................................................................................... 34 Sequence editing ................................................................................................. 34 Identification of genetic relationship of identified Sarcocystis ....................... 35 II Results ..................................................................................................................... 35 Discussion ............................................................................................................... 45 Conclusions ............................................................................................................. 47 Ethics statement ...................................................................................................... 48 References ............................................................................................................... 49 Supplementary material .......................................................................................... 53 CHAPTER 3 – Reactivity against Sarcocystis neurona and Sarcocystis falcatula-like in horse sera from Southeastern and Midwestern Brazil .............. 62 Abstract .................................................................................................................... 62 Resumo .................................................................................................................... 62 Introduction ............................................................................................................. 63 Material and methods ............................................................................................. 64 Samples ................................................................................................................ 64 Merozoites and Antigen Production ................................................................... 64 Indirect Fluorescent Antibody Test (IFAT) ......................................................... 65 Results and discussion .......................................................................................... 66 Ethics statement ...................................................................................................... 70 References ............................................................................................................... 71 Supplementary material .......................................................................................... 74 CHAPTER 4 – Final considerations ....................................................................... 87 III IV V VI VII VIII CARACTERIZAÇÃO MOLECULAR DE Sarcocystis spp. EM GAMBÁS (Didelphis spp.) E DETECÇÃO DE ANTICORPOS ANTI-Sarcocystis spp. EM EQUINOS DAS REGIÕES SUDESTE E CENTRO-OESTE DO BRASIL RESUMO - Gambás (Didelphis spp.) são hospedeiros definitivos de Sarcocystis neurona, Sarcocystis speeri, Sarcocystis lindsayi e Sarcocystis falcatula. No Brasil, diversos estudos vêm demonstrando a alta frequência de Sarcocystis falcatula-like em esporocistos recuperados dos intestinos de gambás, com grande diversidade nos genes codificadores de antígenos de superfície (SAGs). Neste estudo, a diversidade genética de Sarcocystis spp. oriundos de Didelphis albiventris e Didelphis aurita capturados nos municípios de Campo Grande e São Paulo, foi acessada por meio do sequenciamento de SAG2, SAG3 e SAG4, da primeira região espaçadora interna transcrita (ITS-1) e da subunidade I da citocromo c oxidase (cox1). Adicionalmente, reação de imunofluorescência indireta (RIFI) foi empregada para detecção de anticorpos (IgG) contra S. falcatula-like (Dal-CG23) e S. neurona (SN138) no soro de 342 equinos amostrados nos mesmos municípios. Identificação molecular foi realizada em 16 amostras de DNA obtidas de esporocistos recuperados do intestino de gambás ou merozoítos derivados de cultivo in vitro de Sarcocystis spp. Os fragmentos de ITS- 1, cox1, e SAG3 foram clonados, enquanto SAG2 e SAG4 foram sequenciados diretamente dos produtos de PCR. Vinte e sete alelos foram observados em ITS-1, todos filogeneticamente relacionados à S. falcatula-like previamente detectado em aves e/ou em gambás no Brasil. Sarcocystis sp. filogeneticamente relacionado à Sarcocystis rileyi foi evidenciado por cox1 em três gambás. Vinte e um novos alelos SAGs foram descritos, sendo quatro em SAG2 (n=4), 13 em SAG3 (n=13) e quatro em SAG4 (n=7). Nos Imunoensaios utilizando RIFI com ponto de corte de 1:25, anticorpos IgG anti-S. falcatula-like foram detectados em 177/342 amostras de soro equino (51.75%), e anticorpos IgG anti-S. neurona foram detectados em 239/342 amostras (69.88%). Anticorpos IgG contra ambos os isolados foram observados no soro de 132 equinos (38,59%). Ausência de reatividade foi verificada em 16.95% (58/342) animais. A elevada soroprevalência observada neste estudo pode estar relacionada ao baixo ponte de corte utilizado e à presença de gambás infectados com Sarcocystis falcatula-like e Sarcocystis spp. nas áreas onde os equinos foram IX amostrados. Devido à similaridade entre antígenos de superfície detectados nos imunoensaios, relatos de soropositividade contra S. neurona no Brasil podem resultar da exposição dos equinos à outras espécies de Sarcocystis. O papel de outras espécies de Sarcocystis como causa de doença neurológica nos equinos em Brasil permanece incerto. Palavras-chave: Didelphis spp., Sarcocystis spp., identificação molecular, Imunoensaios. X MOLECULAR CHARACTERIZATION OF Sarcocystis spp. FROM OPOSSUMS (Didelphis spp.) AND SEROLOGICAL DETECTION OF ANTI-Sarcocystis spp. ANTIBODIES IN HORSES FROM SOUTHEASTERN AND MIDWESTERN BRAZIL ABSTRACT – Opossums (Didelphis spp.) are definitive hosts of Sarcocystis neurona, Sarcocystis speeri, Sarcocystis lindsayi and Sarcocystis falcatula. In Brazil, several studies have demonstrated a high frequency of Sarcocystis falcatula-like in sporocysts derived from opossums, and high genetic diversity has been observed in surface antigen-encoding genes (SAGs). In this study, genetic diversity of Sarcocystis spp. derived from Didelphis albiventris and Didelphis aurita from the cities of Campo Grande and São Paulo, was accessed by sequencing SAG2, SAG3, SAG4, the first internal transcribed spacer (ITS-1) and cytochrome c oxidase subunit I (cox1). Additionally, indirect fluorescent antibody test (IFAT) was used to detect IgG antibodies against Sarcocystis falcatula-like (Dal-CG23) and S. neurona (SN138) in equine sera from 342 horses sampled in the same regions. Molecular identification was performed for 16 DNA samples obtained from sporocyst or culture-derived merozoites. The ITS- 1, cox1, and SAG3 fragments were cloned, whereas SAG2 and SAG4 were sequenced directly from PCR products. Twenty-seven allele variants were found for ITS-1, all phylogenetically related to S. falcatula-like previously described in birds and/or opossums in Brazil. Sarcocystis sp. phylogenetically related to Sarcocystis rileyi was evidenced by cox1 in three opossums. Twenty-one new SAG alelles were described, four in SAG2 (n=4), 13 in SAG3 (n=13) and four in SAG4 (n=7). On IFAT using 1:25 cutoff, IgG antibodies against S. falcatula-like were detected in 177/342 samples (51.75%), whereas IgG antibodies against S. neurona were detected in 239/342 samples (69.88%). IgG antibodies against both isolates were detected in 132 samples (38,59%). No reactivity was observed in 16.95% (58/342) horses. The high seroprevalence observed here may be related to the lower cutoff, and the presence of opossums infected with Sarcocystis falcatula-like and Sarcocystis spp. in the regions where the horses were sampled. Owing to the similarity among antigens targeted in immunoassays, reports on S. neurona-seropositive horses in Brazil might also derived from exposure of horses to other Sarcocystis species. The role of other Sarcocystis species in causing neurological diseases in horses in Brazil remains unclear. XI Keywords: Didelphis spp., Sarcocystis spp., molecular identification, immunoassays. XII LIST OF ABBREVIATIONS cox1: Cytochrome c oxidase 1 cPCR: Conventional Polymerase chain reaction DNA: Deoxyribonucleic acid ELISA: Enzyme-Linked Immunosorbent Assay EPM: Equine protozoal myeloencephalitis IFAT: Immunofluorescence antibody test IMDM: Iscove's Modified Dulbecco's Medium ITS-1: First internal transcribed spacer q.s.p: 'Quantidade Suficiente Para' SAGs: surface antigen-encoding genes SNC: Central Nervous System 1 CHAPTER 1 – General consideration 1 INTRODUCTION Tissue cysts-forming coccidia are a vast group of organisms within the Phylum Apicomplexa Levine, 1979. Apicomplexans from the family Sarcocystidae Poche, 1913, such as those in the genus Toxoplasma Nicolle and Manceaux, 1908, Neospora Dubey, Carpenter, Speer, Topper and Uggla, 1988 and Sarcocystis Lankester, 1882, have the ability of infecting a broad range of hosts. They multiply into tissue cysts inside muscle cells of intermediate hosts, and the ingestion of the cysts by definitive hosts propagates their life cycle (BLAZEJEWSKI et al., 2015). Sarcocystis is considered the most diverse genera withing the family Sarcocystidae, infecting birds, reptiles, amphibians, fish, and mammals, including humans (BLAZEJEWSKI et al., 2015). Currently, the genus Sarcocystis encompasses over 200 species, that vary considerably in their biology. Therefore, caution should be used when making generalized statements. Not all Sarcocystis spp. are pathogenic for the hosts. Notwithstanding, infections may have economic importance in farm animals, such as horses, cattle, sheep, goats, and pigs (DUBEY et al., 2016). In the last several years, substantial number of studies have been conducted on a global level in order to investigate the role of Sarcocystis species causing disease in vast number of animal species. Several genetic markers have been used to molecularly characterize Sarcocystis strains isolated from various host species, and considerable progress has been made regarding genetic and immunogenic aspects of these parasites. In the Americas, studies based on natural and experimental infections have contributed to the understanding of the biology of Sarcocystis spp., especially those using opossums (Didelphis Linnaeus, 1758) as definitive hosts. The genome annotation of North American S. neurona SO SN1 (BLAZEJEWSKI et al., 2015) and S. neurona SN3 provided important insights into the genetics of the parasite. Immunological techniques, such as Immunofluorescent antibody test (IFATs), Western blotting (WB) and various enzyme-linked immunosorbent assays (ELISAs) have been used in diagnosis and in seroepidemiological studies (DUBEY et al., 2016). 2 Despite the countless number of studies, and the undeniable advances achieved, several aspects of Sarcocystis species infecting opossums in the Americas remain poorly understood. The elucidation of the distribution, genetic diversity, and evolutionary relationships of the parasite is of great importance. Therefore, the present study sought: 1- to assess the genetic diversity of Sarcocystis spp. in Didelphis albiventris and Didelphis aurita sampled in the cities of Campo Grande (Midwestern) and São Paulo (Southeastern), Brazil; 2- to evaluate sera reactivity against S. falcatula-like (strain Dal23-CG) and a North American strain of S. neurona (strain SN138), among horses sampled in the above cited cities. 2 LITERATURE REVIEW Vast literature related to Sarcocystis spp. has been produced in the last several years. This review does not intend to exhaust the subject, simply representing an overview that aims to provide background, therefore contributing to the better understanding of the present work. 2.1 The genus Sarcocystis Sarcocystis spp. are two-host single-celled parasites of medical and veterinary importance. These protozoans can be found in muscle and central nervous system (CNS) of a broad range of poikilothermic and homeothermic animals (DUBEY et al., 2016). Sarcocystis was first reported by Miescher, in 1843, as “milky white threads” in skeletal muscle of a deer mouse in Switzerland. The genus was named as Sarcocystis by Lankester in 1882 (DUBEY et al., 2016). Sarcocystis species can be found in mammals, birds, reptiles, amphibians, and fishes (Figure 1) (PRAKAS; BUTKAUSKAS, 2012). In the last taxonomical review of the genus, 196 valid species were proposed (DUBEY et al., 2016). Since them, several new Sarcocystis spp. have been named and Sarcocystis-like parasites have been observed in muscles or nervous tissue of various animal species. At present, their number is estimated to be over 200. The majority of Sarcocystis species are described in intermediate host, and complete life cycle is ascertained to the majority of them. 3 Figure 1. Intermediate and definitive hosts of Sarcocystis species (PRAKAS; BUTKAUSKAS, 2012). Sarcocystis has an obligatory prey–predator two-host life cycle (Figure 2). Asexual stages develop in intermediate hosts, while sexual stages develop in definitive hosts. Definitive hosts become infected by ingesting mature cysts in muscle (sarcocysts) of intermediate hosts. The sarcocysts vary in size (from micro to macroscopic) and shape, according to the time of development, and the species of Sarcocystis. Also, more than one sarcocyst may be found within a host cell. Mature sarcocysts harbor banana-shaped zoites (bradyzoites), representing the infective form of the parasite. After sarcocyst ingestion by the definitive host, bradyzoites are released in the stomach and small intestines. Bradyzoites penetrate the enterocytes, converting into micro (male) and macro (female) gamonts. The fertilization originates an oocyst, which sporulate (sporocyst) in the lamina propria, is released into the intestinal lumen and excreted in feces. Each sporocyst contain four infective sporozoites (DUBEY et al., 2016). 4 Figure 2. Two-host life cycle of Sarcocystis spp. (Courtesy of Jamie K. Norris). Intermediate hosts become infected by ingesting sporocysts present in the environment. In the small intestines, sporozoites penetrate endothelial cells and initiate the first generation of schizogony. Sarcocystis schizonts divide by endopolygeny. The schizogonic cycle is asynchronous, therefore, schizonts of different maturity stages may be found in a single host cell. The number of generations of schizogony and the type of host cell invaded vary depending on the Sarcocystis species. Merozoites penetrate striated and cardiac muscle cells, and initiate cyst formation. Muscle cysts harbor asexual stages. After repeated cycles of endodyogeny, the sarcocyst is filled 5 with bradyzoites. Sarcocysts’ maturation time is considerable variate depending on the Sarcocystis species. Immature sarcocysts and schizonts are not infectious for the definitive host and tissue stages are not infectious between intermediate hosts. Merozoites may also achieve and penetrate cells from CNS, where they develop as schizonts (DUBEY et al., 2016). Intermediate and definitive hosts vary for each Sarcocystis species. Biological characteristics, such as sarcocysts morphology, and host specificity may be used to differentiate Sarcocystis species. Some Sarcocystis can infect numerous hosts, and some hosts may be infected by more than one Sarcocystis species. The parasite is generally more host-specific for intermediate hosts than for definitive hosts. This trait does not apply to Sarcocystis species shed by opossums (Didelphis spp.), as they have a wide range of intermediate hosts, while opossums are the only known definitive hosts (DUBEY et al., 2016). Not all species of Sarcocystis are pathogenic for intermediate hosts, and the severity of clinical sarcocystosis is dose dependent. Definitive hosts usually don’t present clinical disease (DUBEY et al., 2016). Sarcocystis infections have economic importance in farm animals. It can cause signs such as fever, anemia, muscle pain, and anorexia, reducing feed efficiency, weight gain, and milk yield. Abortion, neurologic impairment, and even death may also occur (DUBEY et al., 2016). Humans are definitive hosts for two Sarcocystis species, Sarcocystis hominis (Railliet and Lucet 1891) Dubey 1976 and Sarcocystis suihominis (Tadros and Laarman 1976) Heydorn 1977 (PENA; OGASSAWARA; SINHORINI, 2001) and serve as accidental intermediate or aberrant host for several other species that may cause muscular and intestinal disease (DUBEY et al., 2016). Several conditions account for the high prevalence of Sarcocystis spp. worldwide: a single host may harbor several species of Sarcocystis, and a wide number of species may serve as definitive hosts; oocysts and sporocysts are eliminated in the infective form, may be excreted in large number and over a long period; sporocysts remain viable in the environment for several months, being resistant to disinfectants (MCKENNA; CHARLESTON, 1992, 1994), freezing (FAYER; JOHNSON, 1975) and low humidity (SAVINI; ROBERTSON; DUNSMORE, 1996). 6 2.2 Sarcocystis spp. and opossums Opossums (Didelphis spp.) are marsupials from the order Didelphimorphia Gill, 1872. The species within the Didelphis genus are extremely adaptable to different habitats, inhabiting either forests or anthropized areas. They have lonely and nomad habits, traversing large areas, which enhances their role as pathogens spreaders. They are essentially terrestrial, however the presence of five digits in both hands and feet, including an opposable thumb, gives the opossums the ability to grab and escalate. Opossums are essentially crepuscular and nocturnal (CERQUEIRA, 1985; ROSSI; BIANCONI; PEDRO, 2006). Didelphis spp. inhabit exclusively the American continent. In the entire North America, a single species of opossum, named Didelphis virginiana, is observed, whereas in South America five species of opossum are described. South American opossums have been grossly divided into white-eared opossums (Didelphis albiventris Lund, 1840, Didelphis imperfecta Mondolfi and Pérez-Hernández, 1984 and Didelphis pernigra Allen, 1900) and black-eared opossums (Didelphis aurita Wied-Neuwied, 1826 and Didelphis marsupialis Linnaeus, 1758) (CERQUEIRA, 1985; LEMOS; CERQUEIRA, 2002). The most common and most widely distributed South American opossum is D. albiventris, especially in Argentina and Brazil (CERQUEIRA, 1985). Opossums act as definitive hosts for Sarcocystis neurona Dubey, Davis, Speer, Bowman, de Lahunta, Granstrom, Topper, Hamir, Cummings, and Suter 1991, Sarcocystis speeri Dubey and Lindsay 1999, Sarcocystis falcatula (Stiles 1893) Box, Meier, and Smith 1984, and Sarcocystis lindsayi Dubey, Rosenthal, and Speer 2001 (BOX; MEIER; SMITH, 1984; DUBEY et al., 1991a; DUBEY; ROSENTHAL; SPEER, 2001; DUBEY and LINDSAY, 1999). Sarcocystis neurona is the causative agent of equine protozoal myeloencephalitis (EPM), a neurological disorder affecting horses and some marine mammals (DUBEY et al., 1991a; WENDTE et al., 2010; ACOSTA et al., 2018). Sarcocystis speeri is biologically similar to S. neurona, being experimentally infective in immunodeficient mice (DUBEY; LINDSAY, 1999), but it was named a new species because of its morphological and antigenic differences. Sarcocystis speeri forms cysts in the tissues of infected mice, which do not occur with S. neurona (DUBEY; LINDSAY, 1999). Sarcocystis falcatula and S. lindsayi cause acute https://en.wikipedia.org/wiki/Peter_Wilhelm_Lund https://en.wikipedia.org/wiki/J._A._Allen https://en.wikipedia.org/wiki/10th_edition_of_Systema_Naturae 7 pulmonary sarcocystosis in birds (DUBEY; ROSENTHAL; SPEER, 2001; HILLIER et al., 1991; ACOSTA et al., 2018). Sarcocystis lindsayi is biologically similar to S. falcatula, however, it was named a new species predominantly because of the differences in ITS-1 locus of the rDNA (DUBEY; ROSENTHAL; SPEER, 2001). Extensive literature about the occurrence of Sarcocystis spp. in intestinal scrapings of D. virginiana in North America is currently available. Dubey (2000) reported 54.5% (24/44) of prevalence of Sarcocystis sporocysts in wild-caught opossums from diverse areas in the United States. In that work mixed Sarcocystis infections (S. falcatula, S. neurona and S. speeri) were present in 21 opossums. Subsequently, Dubey et al. (2001a) reported S. neurona sporocysts in intestinal scrapings of 24/72 (33%) D. virginiana from rural Mississippi. After bioassay in KO mice and immunostaining, the authors confirmed the presence of S. neurona in 19 opossums. Elsheikha, Murphy and Mansfield (2004a) reported S. neurona sporocysts in intestinal scrapings of 31/206 (15%) D. virginiana from Southern Michigan. Rejmanek et al. (2009) reported S. neurona sporocysts in intestinal scrapings of 53/288 (18.4%) D. virginiana from central California. In Brazil, several studies have reported the prevalence of Sarcocystis spp. sporocysts in intestinal scrapings of opossums. Casagrande et al. (2009) observed sporocysts from Sarcocystis spp. in intestinal scrapings of 6/66 (9.1%) D. aurita in São Paulo. Lins et al. (2011) observed sporocysts from Sarcocystis spp. in intestinal scrapings of 19/19 (100%) D. albiventris in Rio Grande do Sul. Valadas (2015) observed sporocysts from Sarcocystis spp. in intestinal scrapings of 33/50 (66%) Didelphis spp. in São Paulo and Rio Grande do Norte. Infected opossums may shed large numbers of Sarcocystis spp. sporocysts over a long period of time. Once infected, these animals may serve as a reservoir throughout their entire life span (2–3 years) (PORTER et al., 2001). Opossums fed muscles from brown-headed cowbird (Molothrus ater) infected with S. falcatula initiated to shed sporocysts 7-16 days after infection and continued to shed sporocysts until they were euthanized (46-200 days after infection) (PORTER et al., 2001). Dubey et al. (2001a) reported sporocysts in intestinal scrapings of opossums ranging from less than 100.000 to 187 million. In places with high population density, significant environmental loading rates of the parasite may occur. Sarcocystis sporocysts may be 8 found even in apparently healthy opossums (ELSHEIKHA; MURPHY; MANSFIELD, 2004b). This could evidence a nonharmful co-existence between parasite and opossums, indicating a long evolutionary history of host–parasite interaction (ELSHEIKHA; MURPHY; MANSFIELD, 2004b). The most frequently method applied to determine the prevalence of Sarcocystis in opossums is the microscopic observation of oocysts/sporocysts on feces. However, as sporocysts are trapped in the lamina propria, they are excreted irregularly and sometimes they are not detectable in feces although millions are present in the intestines (DUBEY et al., 2016). Casagrande et al. (2009) observed sporocysts in intestinal scrapings of 6/66 (9.1%) D. aurita, however, only in four of them sporocysts were observed in the feces. Structure of the oocyst and sporocysts has little or no taxonomic value because morphological characteristics are shared by several closely related species (DUBEY et al., 2016). Therefore, biologic and/or molecular methods are necessary to correct identification. Bioassay in immunodeficient mice and budgerigars can distinguish S. falcatula from S. neurona and S. speeri. Sarcocystis neurona and S. speeri are not infective to budgerigars, and S. falcatula is not infective to mice (DUBEY et al., 2016). Most of Sarcocystis derived from opossums in Brazil were identified as S. falcatula-like, due to their genetic characteristics and/or infectivity to birds (VALADAS et al., 2016; GONDIM et al., 2017, 2019; ACOSTA et al., 2018; CESAR et al., 2018; GALLO et al., 2018). It has been suggested that the diversity of Sarcocystis species in the intestine of opossums may enable sexual recombination, contributing to their allelic variability (MONTEIRO et al., 2013). 2.2.1 Sarcocystis neurona Sarcocystis neurona, first identified in the United States in 1991, is the main etiological agent of equine protozoal myeloencephalitis (EPM) (DUBEY et al., 1991a), a debilitating and potentially fatal neurologic syndrome, first described in equine. After some years from description in equids, an EPM-like fatal disease was also recognized in many other hosts, especially marine mammals (LINDSAY; THOMAS; DUBEY, 9 2000). This progressive disease is characterized by a wide range of neurological signs according to the parasite distribution in the CNS (DUBEY et al., 2015a). In Brazil, the disease was first observed several decades ago. One of the very first reports identified protozoal organisms in the spinal cord of a 10-year-old horse (LOMBARDO DE BARROS; BARROS; SANTOS, 1986). However, at that time, the causative agent was not elucidated. Few years later, mature schizonts and merozoites of a Sarcocystis sp. were observed in the CNS of two horses presenting neurological signs (MASRI; LOPEZ DE ALDA; DUBEY, 1992). Horses affected by EPM usually present progressive symmetrical ataxia that may be accompanied by focal muscular atrophy. Detailed information about clinical presentations and pathogenesis of EPM can be found on published reviews (DUBEY et al., 2001b; DUBEY et al. 2015a). Sarcocystis neurona presents a wide host range. White-nosed coati (Nasua narica molaris Merriam, 1902) skunks (Mephitis mephitis Schreber, 1776), raccoons (Procyon lotor Linnaeus, 1758), armadillos (Dasypus novencinctus Linnaeus, 1758), sea otter (Enhydra lutris Linnaeus, 1758), and cats (Felis catus Linnaeus, 1758), have already been described as intermediate hosts (DUBEY; HAMIR, 2000; CHEADLE et al., 2001a; CHEADLE 2001b; TANHAUSER et al., 2001; DUBEY et al., 2001c; DUBEY et al., 2001d; DUBEY et al., 2017; HAMMERSCHMITT et al., 2020). In addition, S. neurona-like sarcocysts have been reported in the muscles of horses (MULLANEY et al., 2005), dogs (VASHISHT et al., 2005; DUBEY et al., 2014), minks (Mustela vison Schreber, 1777) (RAMOS-VARA et al., 1997), and bobcats (Lynx rufus Schreber, 1777) (VERMA et al., 2015). Equines are considered aberrant hosts for S. neurona, with the parasite multiplying as schizonts in neural and inflammatory cells in the CNS, but failing to encyst (DUBEY et al., 2016). In North America, the parasite has arisen as a significant cause of mortality in marine mammals (WENDTE et al., 2010). A common described victim of the disease is the threatened southern sea otter (Enhydra lutris nereis Linnaeus, 1758). Between 1998 and 2001, S. neurona accounted for nearly 7% of all southern sea otter mortality (KREUDER et al., 2003). The North and the South American opossums, D. virginiana and D. albiventris, respectively, are the known definitive hosts for S. neurona (DUBEY et al., 2016). Multiple studies in the United States have reported varying levels of S. neurona 10 infection among opossum based on the detection of S. neurona sporocysts in their intestinal scrapings. A study performed by Dubey (2000) found 8/44 (18.1%) opossums infected with S. neurona sporocysts, compared to 19/72 (26%) opossums from rural Mississippi (DUBEY et al., 2001a), 31/206 (15%) opossums from Michigan (ELSHEIKHA; MURPHY; MANSFIELD, 2004b) and 17/288 (5.9%) opossums from central California (REJMANEK et al., 2009). Only on one occasion S. neurona was isolated from D. albiventris in Brazil (DUBEY et al., 2001e). South American opossums appears to present a lower incidence of S. neurona, comparing to its North American relatives. Sarcocystis neurona is highly prevalent in horses in the Americas. Antibodies to S. neurona were found in 1056 (53.6%) horses from Ohio (SAVILLE et al., 1997), 117(45.3%) horses from Pennsylvania (BENTZ; GRANSTROM; STAMPER, 1997), 334 (45%) horses from Oregon (BLYTHE et al., 1997), and in 5250 (78%) horses sampled across the United States (JAMES et al., 2017). In Brazil, seroepidemiological studies have been carried out in several states from North, Northeast, Center-West, Southeast and South regions, and seroprevalence as high as 84% were observed (RIBEIRO et al., 2016; BORGES et al., 2017; VALENÇA et al., 2019; KOCH et al., 2019; BORGES-SILVA et al., 2020). Seroprevalence may be associated to the age of the horse, the presence of opossums, and the serological test used (DUBEY et al., 2015a; BORGES et al., 2017). Immunofluorescent antibody test (IFAT), Western blotting (WB) and various enzyme-linked immunosorbent assays (ELISAs) have been widely used for EPM diagnosis and seroepidemiological studies. However, due to the presence of antibodies against other Sarcocystis spp. in equine sera, these tests may generate false-positive results. Studies using combinations of tests or immunoblotting as confirmatory after a positive IFAT or ELISA result, presented a lower number of “true positive” samples (BORGES et al., 2017; VALENÇA et al., 2019), denoting that, reports on S. neurona–seropositive horses in Brazil may also derived from exposure of horses to other Sarcocystis species, such as S. falcatula, S. lindsayi, S. speeri, S. fayeri and S. bertrami (BORGES-SILVA et al., 2020). 11 2.2.2 Sarcocystis falcatula Sarcocystis falcatula is one of the several species of Sarcocystis that affect birds. The parasite was first described by Stiles in muscles of the rose-breasted grosbeck (Pheucticus ludovicianus), and subsequently re-described by Box, Meier and Smith (1984). Similar to what occurs with other intermediate hosts, birds become infected with S. falcatula through ingestion of food or water contaminated with sporocysts. The extensive multiplication of the parasite in endothelial cells may cause physical damage and vasculitis (DUBEY et al., 2016). Respiratory alterations and death of heavily infected birds have been described in exotic species (VILLAR et al., 2008). Cases of encephalitis in birds have also been assumed to be caused by S. falcatula (JACOBSON et al., 1984; SIEGAL-WILLOTT et al., 2005; WÜNSCHMANN et al., 2009; KONRADT et al., 2017). Conversely, muscle cysts have been incidentally found in Brazilian native species, not associated with clinical disease (LLANO et al., 2022). Sarcocystis falcatula, just as its relative S. neurona, present an unusual wide host range. Numerous cases of acute sarcocystosis presumed to be caused by S. falcatula or related species (named S. falcatula-like) have been reported in captive passerine (DUBEY et al., 2001f), psittacine (SMITH et al., 1990; HILLIER et al., 1991; VILLAR et al., 2008; GODOY et al., 2009; ECCO et al., 2008), and pigeons (ECCO et al., 2008; SUEDMEYER et al., 2001) in the Americas. The parasite was also reported in a free-ranging great horned owl (Bubo virginianus) (WÜNSCHMANN et al., 2009), and in free-ranging eagles (Haliaeetus leucocephalus and Aquila chrysaetos) (DUBEY et al., 1991b; WÜNSCHMANN et al., 2010). The first description of S. falcatula outside the United States was in 1999, from the intestines of D. albiventris from Argentina (DUBEY et al., 1999). That isolate was later re-examined using enzyme restriction of a 1100-pb PCR product obtained from RAPD marker 33/54. The authors observed that fragment digestion with Dra I resulted in products similar to that produced by S. neurona digestion, and digestion with Hinf I resulted in products similar to that produced by S. falcatula digestion. The isolated was then named S. falcatula-like (DUBEY et al., 2000a). 12 Most Sarcocystis derived from opossums in Brazil were described as S. falcatula-like, as they were phylogenetically related to S. falcatula and infective for birds, but presented molecular differences compared with S. falcatula described in North America (GONDIM et al., 2017, 2019; ACOSTA et al., 2018; CESAR et al., 2018). Previous studies have shown that S. falcatula constitutes a heterogeneous population (MARSH et al., 1999; VALADAS et al., 2016; GONDIM et al., 2017, 2019; CESAR et al., 2018; LLANO et al., 2022). In vitro culture, single-cell cloning and molecular assays will be needed to resolve this speciation issue (DUBEY et al., 2016). 2.2.3 Other Sarcocystis that use opossums as definitive hosts Sarcocystis speeri and Sarcocystis lindsayi are the other species that use opossums as definitive hosts. Sarcocystis speeri was first described in 1999, and it was the third species recognized in the North American opossum (D. virginiana) (DUBEY; LINDSAY, 1999). Later D. albiventris and D. marsupialis were also described as definitive hosts for S. speeri (DUBEY et al., 2000b; 2000c). However, probably D. aurita was mistakenly identified as D. marsupialis, as the latter species does not occurs in the geographical area where it was described (Gondim et al., 2021). The natural intermediate hosts for S. speeri are not known. Sarcocystis speeri is infective for mammals and present high genetic similarity with S. neurona (DUBEY et al., 2015b). However, some characteristics differentiate the two parasites. Sarcocystis neurona does not produce sarcocysts in KO mice, whereas S. speeri does. Schizonts may be found in many organs, including liver, brain, and uterus (DUBEY; LINDSAY, 1999; DUBEY; SPEER; LINDSAY, 1999; DUBEY et al., 2015b). While few S. neurona sporocysts were lethal for KO mice (DUBEY et al., 2013), a thousand S. speeri sporocysts were necessary to be pathogenic for KO mice (DUBEY; LINDSAY, 1999). Also, S. speeri schizonts are antigenically and morphologically distinct from S. neurona schizonts (DUBEY; LINDSAY, 1999). Based on bioassays in KO mice and budgerigars, prevalence of S. speeri was 18.8% (44 opossums examined) (DUBEY, 2000). Sarcocystis lindsayi was described for the first time in D. albiventris from Brazil (DUBEY; ROSENTHAL; SPEER, 2001). Posteriorly, it was also described in D. aurita 13 (STABENOW et al., 2012). Sarcocystis lindsayi is biologically similar to S. falcatula, but it was proposed as a new species mostly due to differences in the first internal transcribed spacer 1 (ITS-1) locus of the rDNA. At ITS-1, S. lindsayi has 93.3% identity with S. falcatula, although at 28S rDNA these two species are almost identical (DUBEY; ROSENTHAL; SPEER, 2001). Budgerigars fed sporocysts of S. lindsayi from opossum feces died from acute sarcocystosis, similar to what occur in infection with S. falcatula. Natural intermediate hosts of Sarcocystis lindsayi are still unknown. 2.2.4 Molecular identification of Sarcocystis spp. from opossums Each Sarcocystis species has a particular spectrum of hosts and full knowledge of the host specificity is often difficult to obtain (GONDIM et al., 2021). Therefore, molecular data have become mandatory for identification of the species in the genus (GJERDE; VIKOREN; HAMNES, 2018). To compare and accurately describe closely related pathogens, the use of multiple genetic markers is essential. The annotation of the genomes of S. neurona SO SN1 (BLAZEJEWSKI et. al, 2015), and S. neurona SN3, considerably increased the repertoire of known genetic markers. The Sanger sequencing method directed to complete or partial gene segments, have been used to access the diversity of Sarcocystis spp. from opossums in the Americas (DUBEY et al., 2016; GONDIM et al., 2021). The gene encoding the small unit of ribosomal DNA (18S rDNA) is considered a universal marker for molecular identification of Sarcocystis spp., especially when first characterizing an isolate (MORRISON et al., 2004). This gene was already sequenced for several Sarcocystis species (DUBEY et al., 2016). Ribosomal RNA is ubiquitous and present highly conserved and highly variable domains. Conserved domains allow easy application of universal primers, whereas variable domains allow distinction among related biological species and lineages (MORRISON et al., 2004). However, closely related species, such as S. falcatula and S. neurona, and certain Sarcocystis species that use birds as intermediate hosts are almost identical at 18S rDNA (DAME et al., 1995; MARSH et al., 1999; OLIAS et al., 2010; PRAKAS et al., 2013). Therefore, other markers with better phylogenetic resolution, such as the first 14 internal transcribed spacer (ITS-1) and cytochrome c oxidase subunit 1 (cox1), have been associated with 18S rDNA (TANHAUSER et al., 1999; MARSH et al., 1999). The cox1 has shown good phylogenetic resolution for differentiation among Sarcocystis that use ruminants as intermediate hosts (GJERDE, 2013). However, it may not be variable enough to discriminate between Sarcocystis species that use birds or carnivorous mammals as intermediate hosts (GJERDE; VIKOREN; HAMNES, 2018, LLANO et al., 2022). The ITS-1 locus, located between 18S rDNA and 5.8S rDNA, enhances sensitivity of molecular assays, as it is present in several copies within the eukaryote genome. Besides, it present high evolutionary rates (HILLIS; DIXON, 1991; ALVAREZ; WENDEL, 2003). Sequencing of ITS-1 have allowed differentiation between S. falcatula and S. neurona, species that had been previously considered synonymous (MARSH et al., 1999). Isolates of a Sarcocystis sp. divergent from S. falcatula and S. neurona at ITS- 1 were detected in opossums in Brazil. Due to infectivity to budgerigars, the isolate was named Sarcocystis falcatula-like (DUBEY et al., 2001g). Over the years, this genotype has been molecularly detected in opossums (VALADAS et al., 2016; GONDIM et al., 2019), in budgerigars experimentally infected with opossums sporocysts (CESAR et al., 2018; GONDIM et al., 2017), and in naturally infected birds (ACOSTA et al., 2018, KONRADT, et al., 2017, LLANO et al., 2022) in the country. Surface antigen-encoding genes (SAGs) express immunodominant antigens present on the surface of merozoites in both extracellular and intracellular stages of development. These genes, first observed in S. neurona and therefore named SnSAG1-6, present differently depending on the strain. Some strains lack SAG1, expressing instead one or two alternative major surface antigens, SAG5 and SAG6. The SAGs have been assessed to evaluate diversity in Sarcocystis excreted by opossums (WENDTE et al., 2010; REJMANEK et al., 2010; GONDIM et al., 2017, 2019). The allelic combinations of SAG2, SAG3 and SAG4 observed among S. falcatula related isolates suggest that they may suffer sexual recombination processes with exchange of high divergent alleles (MONTEIRO et al., 2013). 15 2.3 Serological studies on Sarcocystis neurona in South American horses The first assay developed for detecting antibodies against S. neurona was a Western blot (WB) (GRANSTROM et al., 1993). S. neurona WB test was modified over the years to improve the diagnostic accuracy of EPM. However, WB is a fairly laborious technique that requires significant expertise to accurately interpretation of results. Therefore, other serologic assays, such as immunofluorescent antibody tests (IFATs), agglutination tests and enzyme-linked immunosorbent assays (ELISAs), more informative and with greater throughput, have been developed (DUBEY et al., 2015). IFATs and various ELISAs have been widely used for EPM diagnosis and seroepidemiological studies in South America. Most of these studies have been conducted in Brazil, where serum samples from horses tested using different methods have revealed variable results. Horse serum tested by IFAT in Brazil have shown a range of seropositivity varying from 2.8 to 84.1% (summarized by Gondim et al., 2021). It is important to stress that on IFAT the use of whole merozoites allow the detection of antibodies against surface antigens. Some of these antigens are suspect to be shared among different Sarcocystis spp., which could lead to the false assumption that a horse is seropositive for S. neurona due to seroconversion after infection with other Sarcocystis species. A recent study performed with 409 horses sampled in Bahia and Rio Grande do Sul states, used S. falcatula–like (Sarco-BA1) and S. neurona (SN138) as antigens for IFAT and WB. The authors observed that 43 (10.5%) and 70 (17.1%) samples tested by IFAT, were reactive to S. falcatula-like and S. neurona, respectively. A total of 25 samples (6.1%) were reactive for both parasites. The fair agreement observed between the two IFATs indicated that horses were exposed to more than one Sarcocystis species. Results from WB in the same work suggested that antigens in the range of 16 and 30 KDa are probably homologous in the two parasites (BORGES- SILVA et al., 2020). In a study performed with sera from 427 horses from the state of Alagoas, antibodies against S. neurona (strain SN37R) were observed in 2.8% (12/427) of the samples evaluated by IFAT (the authors used a more conservative cutoff of 1:80), and in 1.6% (7/427) of the samples evaluated by immunoblot. In the immunoblot, the 16 authors observed reactivity to antigens of 7-10KDa, 16 and 30 KDa. In that study confirmation of IFAT using immunoblot revealed a low number of “true positive” samples (VALENÇA et al., 2019). Cross-reactivity between S. neurona (strain SN138) and S. falcatula-like (strain Sarco-BA1) was also reported in experimentally infected Mongolian gerbils (Meriones unguiculatus) tested by WB (DE JESUS et al., 2019). The authors reported absence of cross-reactivity in samples tested using IFAT, while similar reactivity to proteins of 30 and 16 KDa was observed in both parasites. ELISAs using S. neurona merozoite surface antigens (SnSAGs), mainly expressed as recombinant proteins have been developed and applied in the EPM diagnosis and seroepidemiological studies (DUBEY et al., 2016). A study performed in Brazil evaluated sera from 961 horses from ten different states using an ELISA based on the SnSAG4. In that study the authors observed a positivity of 69.6% (669/991) (HOANE et al., 2006). To prevent from false-positive results derived from the use of a single antigen protein, improved SnSAG ELISAs were developed in North America. Chimeras combining two (YEARGAN; HOWE, 2011) and three (YEARGAN et al., 2015) of the major SnSAGs have been used in EPM diagnosis. In Brazil, seroepidemiological studies on S. neurona have been conducted using antigens and recombinant proteins based in North American strains (DUBEY et al., 2002; ONUMA et al., 2014; GENNARI et al., 2016; GOMES et al, 2019; VALENÇA et al., 2019), as no isolation of S. neurona has been obtained from affected horses in South America. Notably, despite the occurrence of a disease resembling EPM (MASRI et al., 1992) and the high seroprevalence of S. neurona observed in equids in Brazil, only on one occasion S. neurona was isolated from opossums in the country. Sporocysts of Sarcocystis spp. obtained from opossums (D. albiventris) were shipped to the United States and bioassayed in interferon gamma gene knock out (KO) mice. The mice developed neurologic sarcocystosis, and S. neurona was demonstrated in their brains by immunohistochemical staining (DUBEY et al., 2001e). No subsequent studies were published on that isolate. Several authors have discussed the hypothesis that horses in Brazil are being exposed and seroconverting to other Sarcocystis species shed by opossums (S. falcatula-like, S. neurona, S. lindsayi and S. speeri) as well as to S. bertrami (syn. S. fayeri) from which horses are intermediate hosts. However, the potential of other 17 Sarcocystis spp. present in south America in causing neurological disorders and EPM in horses remains uncertain. 3 GENERAL OBJECTIVES The current study aimed: 1- to assess the genetic diversity of Sarcocystis spp. in opossums (D. albiventris and D. aurita) sampled in the cities of Campo Grande (Midwestern) and São Paulo (Southeastern), Brazil; 2- to evaluate the horse sera reactivity against S. falcatula-like (strain Dal23-CG) and S. neurona (North American strain SN138), among horses sampled in the same cities. 4. REFERENCES ACOSTA, I. C. L.; SOARES, R. M.; MAYORGA, L. F. S. P.; ALVES, B. F.; SOARES, H. S.; GENNARI, S. M. Occurrence of tissue cyst forming coccidia in Magellanic penguins (Spheniscus magellanicus) rescued on the coast of Brazil. PLOS ONE, v. 14, n. 2, 2018. Available at: < https://doi.org/ 10.1371/journal.pone.0209007>. ALVAREZ, I.; WENDEL, J. F. Ribosomal ITS sequences and plant phylogenetic inference. 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Available at: 29 CHAPTER 2 – Molecular diversity of Sarcocystis spp. in opossums (Didelphis spp.) from Southeastern and Midwestern Brazil Abstract South American opossums (Didelphis spp.) are definitive hosts of Sarcocystis neurona, Sarcocystis speeri, Sarcocystis lindsayi and Sarcocystis falcatula. In Brazil, diverse studies have demonstrated a high frequency of Sarcocystis falcatula-like in sporocysts derived from opossums, and high genetic diversity has been observed in surface antigen-encoding genes (SAGs). In this study, genetic diversity of Sarcocystis spp. derived from Didelphis albiventris and Didelphis aurita from the cities of Campo Grande and São Paulo, was accessed by sequencing SAG2, SAG3, SAG4, the first internal transcribed spacer (ITS-1) and cytochrome c oxidase subunit I (cox1). Molecular identification was performed for 16 DNA samples obtained from sporocyst or culture-derived merozoites. The ITS-1, cox1, and SAG3 fragments were cloned, whereas SAG2 and SAG4 were sequenced directly from PCR products. Four alleles variants were found for SAG2, 13 for SAG3 and seven for SAG4, from which 04, 13 and 04, respectively, were novel. Twenty-seven allele variants were found for ITS-1, all phylogenetically related to S. falcatula-like previously described in Brazil. Sarcocystis sp. phylogenetically related to Sarcocystis rileyi was evidenced by cox1 in three opossums. Further studies are needed to clarify the role of Didelphis spp. as definitive hosts of Sarcocystis spp. other than those previous described. Keywords: Sarcocystis spp., opossums, molecular characterization, ITS-1, cox1, SAGs Resumo Gambás sul-americanos (Didelphis spp.) são hospedeiros definitivos de Sarcocystis neurona, Sarcocystis speeri, Sarcocystis lindsayi e Sarcocystis falcatula. No Brasil, diversos estudos têm demonstrado alta frequência de Sarcocystis falcatula- like em esporocistos derivados de gambás, com grande diversidade nos genes que codificam antígenos de superfície (SAGs). Neste estudo, a diversidade genética de Sarcocystis spp. oriundos de Didelphis albiventris e Didelphis aurita dos municípios de Campo Grande e São Paulo, foi acessada por meio do sequenciamento de SAG2, 30 SAG3 e SAG4, da primeira região espaçadora interna transcrita (ITS-1) e citocromo c oxidase subunidade I (cox1). Identificação molecular foi realizada em 16 amostras de DNA obtidas de esporocistos ou merozoítos derivados de cultivo. Os fragmentos de ITS-1, cox1, e SAG3 foram clonados, enquanto SAG2 e SAG4 foram sequenciados diretamente dos produtos de PCR. Quatro alelos foram observados em SAG2, 13 em SAG3 e sete em SAG4, sendo novos quatro, 13 e quatro, respectivamente. Em ITS- 1, 27 alelos foram observados, todos filogeneticamente relacionados à S. falcatula- like previamente detectados no Brasil. Sarcocystis sp. filogeneticamente relacionado à Sarcocystis rileyi foi evidenciado por cox1 em três gambás. Mais estudos são necessários para entender o papel de Didelphis spp. como hospedeiro definitivo de Sarcocystis spp. diferentes daqueles previamente descritos. Palavras-chave: Sarcocystis spp., gambás, caracterização molecular, ITS-1, cox1, SAGs Introduction Sarcocystis Lankester 1882, are obligate intracellular protozoa belonging to the phylum Apicomplexa Levine 1979. Species of the genus Sarcocystis have an obligatory prey-predator two-host life cycle. Opossums (Didelphis Linnaeus, 1758), which exclusively inhabit the American continents, act as definitive hosts for Sarcocystis neurona Dubey, Davis, Speer, Bowman, de Lahunta, Granstrom, Topper, Hamir, Cummings, and Suter 1991, Sarcocystis speeri Dubey and Lindsay 1999, Sarcocystis falcatula (Stiles 1893) Box, Meier, and Smith 1984, and Sarcocystis lindsayi Dubey, Rosenthal, and Speer 2001 (Box et al., 1984; Dubey et al., 1991, 2001a; Dubey & Lindsay, 1999). Sarcocystis neurona is the chief etiological agent of equine protozoal myeloencephalitis (EPM) (Dubey et al., 2001b). Sarcocystis falcatula has been associated with numerous cases of pulmonary sarcocystosis in free-living and captive birds (Smith et al., 1990; Hillyer et al., 1991; Clubb & Frenkel, 1992; Page et al., 1992; Dubey et al., 2001c; Suedmeyer et al., 2001; Villar et al., 2008; Wünschmann et al., 2009, 2010; Verma et al., 2018). Sarcocystis speeri and S. lindsayi are respectively, experimentally infective to mice and budgerigars (Melopsittacus undulatus), but their natural intermediate hosts are unknown (Dubey & Lindsay, 1999; Dubey et al., 2001a). 31 Several genetic markers have been used to molecularly characterize Sarcocystis spp. shed by opossums in the Americas. Genome annotation of the North American S. neurona SO SN1 (Blazejewski et al., 2015) and S. neurona SN3 provided important insights into the molecular biology of the parasite. In Brazil, various studies have demonstrated the presence of Sarcocystis spp. in sporocysts derived from opossums (Casagrande et al., 2009; Monteiro et al., 2013; Gallo et al., 2018; Valadas et al., 2016; Gondim et al., 2017, 2019; Cesar et al., 2018). The majority of the Sarcocystis identified in the country have been classified as Sarcocystis falcatula-like, due to genetic characteristics and/or experimental infectivity to budgerigars (Gondim et al., 2017, 2019; Acosta et al., 2018; Cesar et al., 2018). Extensive variability has been observed in surface antigen-encoding genes (SAGs) of Sarcocystis spp. derived from opossums in Brazil (Monteiro et al., 2013; Valadas et al., 2016; Gondim et al., 2017, 2019; Cesar et al., 2018). It has been suggested that the diversity of Sarcocystis species in the intestine of opossums could enable allele exchange through sexual recombination, contributing to their allelic variability (Monteiro et al., 2013). Considering the widespread occurrence of Sarcocystis spp. in opossums in Brazil and the wide genetic variation observed in previous studies, this study sought to assess the genetic diversity of Sarcocystis spp. in D. albiventris and D. aurita sampled in the cities of Campo Grande (midwestern) and São Paulo (southeastern), Brazil. The detection and molecular characterization of these agents in the opossums of these regions contribute to increasing the knowledge related to the genetic diversity of Sarcocystis spp. in Brazil. Material and Methods Sampling Between July 2019 and April 2021, five expeditions were performed for capturing free-ranging opossums. Four expeditions were performed in the city of Campo Grande, Mato Grosso do Sul state (Midwestern) and one expedition was performed in the city São Paulo, São Paulo state (Southeastern), Brazil. The animals were caught in six locations in the urban region of Campo Grande (1–20°41’37.51” S, 54°61’54.65” O; 2– 20°44’88.11” S, 54°57’95.99” O; 3–20°43’95.15” S, 54°57’43.24” 32 O; 4–20°49’96.79” S, 54°61’35.94” O; 5–20°47’17.08” S, 54°65’60.08” O; 6– 20°49’32.15” S, 54°58’09.15” O) and in an equestrian club in the city of São Paulo (23°38'31.3" S, 46°42'35.0" O), using Tomahawk and Sherman live traps baited with a mix of bananas, peanut butter, tinned sardines, and bacon. Together, these five expeditions resulted in the capture of 37 opossums: 26 Didelphis albiventris from Campo Grande and 11 Didelphis aurita from São Paulo (D. aurita was differentiated from the phenotypical similar species Didelphis marsupialis based on their distribution in the country). Trapped opossums were transported to the laboratory, where they were chemically restrained with a combination of cetamina and xilazina (30 mg/Kg and 5 mg/Kg, respectively, intramuscular), followed by euthanasia with T-61 (MSD) (0.3 mL/Kg, intravenously). Necropsy was performed. The small intestine was separated, longitudinally sectioned, and the internal surface was scraped and processed as previously described (Dubey et