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Protozoan parasites of the genus Sarcocystis are obligatory heteroxenous cyst-forming coccidia that infect a wide variety of animals and encompass approximately 200 described species. At least four Sarcocystis spp. (S. falcatula, S. neurona, S. lindsayi and S. speeri) use opossums (Didelphis spp.) as definitive hosts, and two of them, S. neurona and S. falcatula, are known to cause disease in horses and birds, respectively. Opossums are restricted to the Americas, but their distribution in the Americas is heterogeneous. Five Didelphis spp. are distributed in South America (D. aurita, D. albiventris, D. marsupialis, D. imperfecta and D. pernigra) whereas just one opossum species (D. virginiana) is found in North America. Studies conducted in the last decades show that Sarcocystis spp., derived from South American Didelphis spp., have biological and genetic differences in relation to Sarcocystis spp. shed by the North American opossum D. virginiana. The aim of this review was to address the peculiar scenario of Sarcocystis species shed by South American opossums, with a special focus on diagnosis, epidemiology, and animal infections, as well as the genetic characteristics of these parasites., Parasitos protozoários do gênero Sarcocystis são coccídios heteroxenos formadores de cistos, que infectam variadas espécies animais e compreendem cerca de 200 espécies descritas. Pelo menos quatro Sarcocystis spp. (S. falcatula, S. neurona, S. lindsayi e S. speeri) utilizam gambás (Didelphis spp.) como hospedeiros definitivos; e duas delas, S. neurona and S. falcatula são conhecidas por causarem doença em equinos e aves, respectivamente. Gambás didelfídeos são restritos ao continente americano, contudo são distribuídos de forma heterogênea nas Américas. Cinco Didelphis spp. são distribuídos na América do Sul (D. aurita, D. albiventris, D. marsupialis, D. imperfecta e D. pernigra), enquanto somente uma espécie (D. virginiana) é encontrada na América do Norte. Trabalhos conduzidos, nas últimas décadas, mostram que Sarcocystis spp. derivados de Didelphis spp. sulamericanos possuem diferenças biológicas e genéticas, quando comparados a Sarcocystis spp. excretados pelo gambá norte-americano D. virginiana. O objetivo desta revisão é discutir a situação peculiar das espécies de Sarcocystis na América do Sul com um foco especial em diagnóstico, epidemiologia e infecções animais, assim como nas características genéticas desses parasitos., Facultad de Ciencias Veterinarias

Sarcocystis neurona and Neospora spp. are related protozoa that can cause equine protozoal myeloencephalitis (EPM). The present study aimed to determine the frequency of antibodies to these parasites in 649 equids (351 horses, 267 donkeys, and 31 mules) from six departments in the North and Northwest of Colombia. For this purpose, the indirect fluorescent antibody test (IFAT) was used for detecting antibodies against S. neurona and Neospora spp. with a cut-off point of 1:20 and 1:50, respectively. A binomial logistic regression model was selected to predict variables associated with exposure. The frequency of anti-S. neurona antibodies was 14.24% (95% CI: 10.84-18.44) for horses, 2.99% (95% CI: 1.39-6.04) for donkeys, and 16.13% (95% CI: 6.09-34.47) for mules. The risk for S. neurona infection was significantly lower in donkeys (OR: 0.18 [0.08-0.38]; p0.001) than horses and mules, and higher in animals with a poor body condition (OR: 2.82 [1.45-6.05]; p0.05). Additionally, older animals (12y) had a higher risk of seropositivity (OR: 5.26 [1.88-19.1]; p0.05), as well as animals that inhabit climatic conditions associated with tropical very dry forest (OR: 1.85 [1.01-3.51]; p0.05). Córdoba and Antioquia departments presented the highest seropositivity to S. neurona with 13.01 and 8.3%, respectively. The frequency of anti-Neospora spp. antibodies was 1.42% (95% CI: 0.52-3.48) for horses, 1.12% (95% CI:0.29-3.52) for donkeys and 0% (95%, CI: 0-0) for mules. Atlántico was the state with the highest seropositivity to Neospora spp. (10%). No risks associated with Neospora spp. infection were found. These findings allow us to conclude that equids from these regions of Colombia are exposed to S. neurona, but antibodies to Neospora spp. are uncommon. Further studies are necessary to explore the presence of these two agents in other areas of the country. In addition, we need to prove the importance of the above-mentioned risk factors over the susceptibility of horses to these protozoal agents and the epidemiological impact of these underdiagnosed coccidia.

Simple Summary Sarcocystidae is a family of apicomplexan protozoa highly prevalent in vertebrates. The definitive hosts of sarcocystids eliminate oocysts or sporocysts that infect intermediate hosts. After infection, mature tissue cysts (sarcocysts) develop in intermediate hosts, mostly in muscle and neurological tissues. Sarcocysts are infectious for definitive hosts, which acquire them through carnivorous or scavenging habits. Intermediate hosts and definitive hosts are the natural hosts of sarcocystids in which infections are usually mildly or not symptomatic. In 2017, muscular and neurological tissues of 22 birds from Magdalena Islands, southern coast of Chile, were screened for the presence of DNA of sarcocystids. DNA of organisms of the genus Sarcocystis was identified in two Chilean skuas (Stercorarius chilensis). The genetic makeup of the parasite detected in skuas was unprecedented and probably represent a new species in the genus. It is well known that Sarcocystis may cause severe infections in aberrant hosts, which are susceptible animals that do not behave as natural hosts for the parasite and have low resistance to the infection, thus more studies are needed to characterize this parasitosis in skuas and other hosts to understand the epidemiology of the infection and its impact on the health of marine fauna. Abstract Evidence of sarcocystid infection was investigated in samples of 16 penguins (Spheniscus. magellanicus), four Dominican gulls (Larus dominicanus) and two Chilean skuas (Stercorarius chilensis) found in Madalenas Islands, Chile, in 2017. Samples of skeletal muscle, cardiac muscle and brain from all birds were screened by a pan-sarcocystid nested-PCR targeting a short fragment of the gene encoding the small ribosomal unit (nPCR-18Sa). The only two positive samples by nPCR-18Sa, both from skuas, were tested by a nested-PCR directed to the internal transcribed spacer 1 (nPCR-ITS1), also a pan-sarcocystidae nested-PCR, and to a nested-PCR directed to the B1 gene (nPCR-B1), for the exclusive detection of Toxoplasma gondii. The two nPCR-18Sa-positive samples were nPCR-ITS1-positive and nPCR-B1-negative. The nPCR-ITS1 nucleotide sequences from the two skuas, which were identical to each other, were revealed closely related to homologous sequences of Sarcocystis halieti, species found in seabirds of northern hemisphere. Larger fragments of genes encoding 18S and partial sequences of genes coding for cytochrome oxidase subunit 1 were also analyzed, corroborating ITS1 data. The haplotypes found in the skuas are unprecedent and closely related to species that use birds as the definitive host. Further studies need to be carried out to detect, identify and isolate this parasite to understand the epidemiology of the infection and its impact on the health of marine fauna.

Sarcocystis 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 in marine mammals. Herein, we describe the clinical, pathological, and molecular findings of a fatal case of S. neurona-associated meningoencephalitis in a domestic cat. The causing agent was analyzed by multilocus genotyping, confirming the presence of S. neurona DNA in the tissue samples of the affected animal. Significant molecular differences were found in relation to S. neurona isolates detected in other regions of the Americas. In addition, the parasite was identical to Sarcocystis sp. identified in opossum sporocysts in Brazil at molecular level, which suggests that transmission of. S. neurona in Brazil might involve variants of the parasite different from those found elsewhere in the Americas. Studies including more samples of S. neurona would be required to test this hypothesis, as well as to assess the impact of this diversity. Electronic supplementary material The online version of this article (10.1007/s00436-019-06570-w) contains supplementary material, which is available to authorized users.

Sarcocystis são parasitos coccidios intracelulares caracterizados por um ciclo de vida obrigatório em dois hospedeiros. Multiplicação assexuada do parasito ocorre em hospedeiros intermediários, geralmente onívoros, com formação de cistos na musculatura. A fase sexual, com formação de oocistos/esporocistos, ocorre no intestino delgado de hospedeiros definitivos, geralmente carnívoros. Mais de 30 espécies de Sarcocystis são reconhecidas em aves e algumas delas, como S. falcatula e S. calchasi, podem ser patogênicas para hospedeiros aberrantes. Em aves que atuam como hospedeiros intermediários, a infecção ocorre pela ingestão de alimentos ou água contaminados com esporocistos. Aves que atuam como hospedeiros definitivos se infectam pelo consumo de tecidos contendo cistos. Poucos estudos em sarcocistose aviária têm sido realizados na América do Sul. No presente estudo, investigamos a presença de coccidios formadores de cistos na musculatura de aves silvestres coletadas pela Divisão Técnica de Medicina Veterinária e Gestão da Fauna Selvagem (DEPAVE-3) no estado de São Paulo, Brasil. Amostras de músculo peitoral de 400 aves pertencentes a 103 espécies foram submetidas a extração e amplificação de DNA pela nPCR-ITS1 direcionada à sequência do primeiro espaço transcrito interno do DNA ribossômico para triagem das amostras. Estes primers permitem a detecção de uma ampla diversidade de organismos pertencentes a família Sarcocystidae, pois hibridizam em regiões conservadas dos genes codificadores de DNA ribossômico 18S e 5.8S. Em amostras positivas na nPCR-ITS1, outros fragmentos gênicos foram amplificados por PCR. Amostras relacionadas com S. falcatula foram caracterizadas com sequências gênicas codificadoras de antígenos de superfície (SAGs) e nas demais amostras, genes mais conservados como citocromo c oxidase subunidade I (COX1) e a fração do gene do DNA ribossômico (18S rDNA) foram amplificados. Os produtos amplificados foram sequenciados, em todos os casos. Com esta metodologia, 36 amostras positivas na nPCR-ITS1, foram relacionadas com S. falcatula, das quais 28 foram identificadas como S. falcatula (10 Piciformes, 8 Psittaciformes, 5 Columbiformes, 2 Accipitriformes, 1 Anseriformes, 1 Passeriformes, 1 Strigiformes), 7 como Sarcocystis sp. ex Cacicus haemorrhous (6 Passeriformes, 1 Piciformes), e uma como Sarcocystis sp. ex Guira guira (1 Cuculiforme). As duas últimas espécies são inéditas. Dez amostras de outros Sarcocystídeos também foram amplificadas na nPCR-ITS1, das quais uma foi identificada como S. halieti (1 Accipitriformes), seis como Toxoplasma gondii (3 Pelecaniformes, 2 Falconiformes, 1 Columbiformes), uma como Sarcocystis sp. ex Coragyps atratus (1 Cathartiformes) e uma como Atoxoplasma sp. ex Icterus jamacaii (1 Passeriformes). As duas últimas espécies são inéditas. Este estudo é a primeira pesquisa extensiva em aves silvestres no Brasil para espécies de Sarcocistídeos, relatando pela primeira vez infecção natural com S. falcatula em 14 espécies incluindo a Ordem Piciformes e mostrando a ampla diversidade de hospedeiros intermediários da América do Sul para Sarcocystis spp. Sarcocystis are intracellular coccidial parasites characterized by an obligatory life cycle in two hosts. Asexual multiplication of the parasite occurs in intermediate hosts, usually omnivores, with formation of cysts in the musculature. The sexual phase, with formation of oocysts / sporocysts, occurs in the small intestine of definitive hosts, usually carnivores. More than 30 species of Sarcocystis are recognized in birds and some of them, such as S. falcatula and S. calchasi, may be pathogenic for aberrant hosts. In birds that act as intermediate hosts, infection occurs by ingesting food or water contaminated with sporocysts. Birds that act as definitive hosts become infected by consuming tissue containing cysts. Few studies on avian sarcocystosis have been conducted in South America. Herein, we investigated the presence of cyst-forming coccidia in wild bird musculature collected by the Technical Division of Veterinary Medicine and Management of Wild Fauna (DEPAVE-3) in the state of São Paulo Brazil. Pectoral muscle samples from 400 birds belonging to 103 species were subjected to DNA extraction, and amplification by nPCR-ITS1 directed to the sequence of the first internal transcribed ribosomal DNA space for sample screening. These primers allow the detection of a wide diversity of organisms belonging to the Sarcocystidae family, as they hybridize in conserved regions of 18S and 5.8S ribosomal DNA coding genes. In nPCR-ITS1 positive samples, other gene fragments were amplified by PCR. S. falcatula-related samples were characterized with surface antigen coding gene sequences (SAGs), and in the other samples, more conserved genes such as cytochrome c oxidase subunit I (COX1) and the ribosomal DNA gene fraction (18S rDNA) were amplified. Amplified products were sequenced in all cases. With this methodology, 36 nPCR-ITS1 positive samples were related to S. falcatula, of which 28 were identified as S. falcatula (10 Piciformes, 8 Psittaciformes, 5 Columbiformes, 2 Accipitriformes, 1 Anseriformes, 1 Passeriformes, 1 Strigiformes), 7 as Sarcocystis sp. ex Cacicus haemorrhous (6 Passeriformes, 1 Piciformes), and one as Sarcocystis sp. ex Guira guira (1 Cuculiformes). The last two species are unpublished. Ten samples of other Sarcocystids were also amplified in nPCR-ITS1, one of which was identified as S. halieti (1 Accipitriformes), six as Toxoplasma gondii (3 Pelecaniformes, 2 Falconiformes, 1 Columbiformes), one as Sarcocystis sp. ex Coragyps atratus (1 Cathartiformes) and one as Atoxoplasma sp. ex Icterus jamacaii (1 Passeriformes). The last two species are unpublished. This study is the first extensive survey of wild birds in Brazil for Sarcocistydae species, reporting for the first-time natural S. falcatula infection in 14 species including the Order Piciformes and showing the wide diversity of South American intermediate hosts for Sarcocystis spp.

Sarcocistys -associated menigoencephalitis is virtually an unrecognized cause of neurological disease in chickens. An undescribed species of Sarcocystis cause fatal infection in two backyard chickens in the Midwest of Brazil. Infected chickens presented anorexia, weight loss, incoordination, ataxia and opisthotonos. Yellow necrotic foci in the gray and white matter of the telencephalon were the main gross lesion. Microscopically, necrotizing granulomatous and heterophilic meningoencephalitis with intralesional Sarcocystis -like schizonts and mezoites were observed in the central nervous system. Molecular analysis of frozen brain samples of the two chickens was identical and the protozoan was named Sarcocystis sp. Chicken-2016-DF-BR. Complete nested PCR- sequence of Sarcocystis sp. Chicken-2016-DF-BR was equally similar to Sarcocystis anasi (EU553477) and Sarcocystis albifronsi (EU502868). This is the first report of Sarcocistys -associated meningoencephalitis with molecular characterization in backyard chickens.

Neosporosis is caused by infection with the protozoa Neospora caninum. It manifests as various neurological symptoms and is considered as one of the main causes of abortion in cattle, and induces uncommon congenital infection in sheep. The standard diagnosis is based on indirect immunofluorescence (IFI); however, cross-reactivity with other protozoa proteins is common. Aiming a more specific diagnosis, recombinant antigens have been tested in several immunoassays; of these, NcSAG1 (surface antigen-1) and NcSRS2 (SAG1-related sequence 2) were the most promising. In this context, we developed an indirect ELISA with recombinant NcSRS2 (ELISA-rNcSRS2) and NcSAG1 (ELISA-rNcSAG1) proteins alone and in association (ELISA-rNcSRS2/rNcSAG1) for the diagnosis of cattle and ovine neosporosis. A total of 216 samples from cattle and 154 samples from sheep were used to evaluate the ELISAs. The sensitivity and specificity results of the ELISA-rNcSRS2 were 91.5 % and 96.4 % for cattle, and 89.6 % and 96.3 % for sheep, respectively. For the ELISA-rNcSAG1, the sensitivity and specificity were 84.9 % and 97.3 % for cattle, and 89.6 % and 92.6 % for sheep, respectively. The sensitivity and specificity of the ELISA-rNcSRS2/rNcSAG1 was 98.1 % and 99.1 % for cattle, 100 % and 97.2 % for sheep, respectively. These results indicated that indirect ELISA using the rNcSRS2 and rNcSAG1 proteins are a highly sensitive and specific method, especially when used in association, for detecting antibodies in cattle and ovine populations infected with N. caninum.

In a previous study in Brazil, six isolates of Sarcocystis spp. recovered from budgerigars fed sporocysts excreted by opossums of the genus Didelphis were characterized by means of sequencing fragments of gene coding cytochrome B (CYTB), internal transcribed spacer 1 (ITS1), and surface antigen genes (SAG2, SAG3 and SAG4). The isolates shared identical ITS1 and CYTB sequences, but differed at SAG2, SAG3 and SAG4: three allele variants of SAG2, 3 allele variants of SAG3 and 2 allele variants of SAG4 were encountered in three multilocus genotypes (MLGs) (MLG1, MLG2, and MLG3). At ITS1 and CYTB, all the isolates from budgerigars were identical to the Sarcocystis falcatula-like isolate 59-2016-RS-BR that was detected in a barefaced ibis (Phimosus infuscatus) causing necrotizing meningoencephalitis in Brazil. At ITS1 locus, all the above isolates were clearly distinct from Sarcocystis neurona, Sarcocystis falcatula, Sarcocystis lindsayi, and Sarcocystis speeri, the four known species of Sarcocystis that use opossums of the genus Didelphis as definitive hosts. Here, we replicated the experiment above to identify additional MLGs or other species of Sarcocystis. Fifteen budgerigars were experimentally infected with sporocysts of Sarcocystis spp. from 12 opossums of the genus Didelphis. All the birds died 9–19 days after infection and tissue samples containing merozoites and schizonts of Sarcocystis spp. were recovered. Fractions of sequences coding for 18S ribosomal RNA gene (18S), CYTB, ITS1, SAG2, SAG3 and SAG4 were PCR amplified and sequenced from the infected lungs. In addition, fractions of 18S, SAG2, SAG3 and SAG4 were sequenced from the isolate 59-2016-RS-BR and fractions of 18S were sequenced from the six isolates from budgerigars described above. From the results, all the isolates shared identical 18S, ITS1 and CYTB sequences. Among the 15 new isolates from budgerigars, three allele variants of SAG2, 3 allele variants of SAG3 and 2 allele variants of SAG4 were encountered in five MLGs, of which four were novel (MLG1, MLG4, MLG5, MLG6 and MLG7). Isolate 59-2016-RS-BR was assigned to an eighth MLG (MLG8). Molecular data pointed that Sarcocystis assigned to MLGs 1 to 8 are variants of the same species, but the SAG-based trees of the isolates conflicted, which supports genetic admixture among them. The sarcocystinae studied have high diversity of SAG alleles per locus and the correlation of such an abundant variety of SAG alleles to host specificity and pathogenicity needs to be assessed. Remains to be elucidated if the parasites studied here and S. falcatula are variants of the same species that have diverged to the point of possessing differences at ITS1 level, but that are still capable of exchanging genes.

Highlights • First risk factor-study on N. caninum infection in Antioquia department, Colombia. • Horizontal transmission is an important mode of infection in the studied region. • Abortion, age, origin and poor hygienic practices associated with manual milking are risk factors for N. caninum infection., Bovine neosporosis is a parasitic disease with worldwide distribution that causes important economic losses. Because of the limited information on the occurrence of Neospora caninum infection in Colombia, this study aimed to determine the seroprevalence and identify the risk factors associated with this infection in cattle in Antioquia, which is the largest milk-producing state in the country. We collected 1,038 blood samples from Holstein, Jersey and crossbred cows from 31 farms. An epidemiologic questionnaire was given to all the owners. A commercial ELISA kit was used as the diagnostic technique. The occurrence of anti-N. caninum antibodies was determined to be 28.3% (294/1038), and 100% of the screened farms were positive, indicating that all the properties had at least one positive animal. The seropositivity within each farm ranged from 5.5% to 50%. A multivariable logistic regression model identified the following as significant risk factors: history of abortion (OR = 5.33, p