Morphological and molecular identification of Sarcocystis falcatula from the emerald toucanet (Aulacorhynchus albivitta) in Colombia.

Sarcocystis spp. are cyst-forming coccidia characterized by a two-host predator-prey life cycle. Sarcocysts are formed in muscles or nervous system of the intermediate host, while sporocysts develop in the small intestine of the definitive host. The intermediate hosts of Sarcocystis falcatula are wild birds. Colombia is one of the countries with the greatest biodiversity of birds, however, there are few studies related to this parasite in wild birds. This study presents the morphological and molecular detection of Sarcocystis falcatula collected from the emerald toucanet (Aulacorhynchus albivitta), a wild bird species endemic to South America. Pectoral muscle samples were obtained, and microscopic and molecular detection was performed by light microscopy, transmission electron microscopy, and amplifying of the first internal transcribed spacer (ITS-1) and surface antigen-encoding genes (SAGs). Sarcocystis measured an average of 161  × 42 µm, with a cyst wall ∼0.4 µm thick. Ultrastructurally, the sarcocyst wall type 11b-like consisted of numerous villar protrusions of 850 nm wide on average. The ITS-1 sequence showed 97.0-99.7% identity to S. falcatula previously described from birds in the United States and Brazil, respectively. Concatenated phylogenetic analysis based on SAG2, SAG3 and SAG4 confirmed that the new isolate is grouped with other sequences of Sarcocystis from South America, but divergent from those isolates obtained in North America. The results of this study demonstrate for the first time the presence of S. falcatula in a wild bird from Colombia.

Acute, fatal Sarcocystis falcatula infection in rose-ringed parakeets (Psittacula krameri).

Sarcocystosis is an important avian disease that affects several intermediate host species. Birds not endemic from Americas, like Old World psittacine species, appear to be more susceptible to lethal infection than New World psittacine species. The aim of this study was to investigate the sudden death of rose-ringed parakeets (Psittacula krameri) in an exotic private parrot's aviary. Macroscopically, the most prevalent findings were severe lung congestion, slight superficial myocardial hemorrhagic lesions, enlarged liver and congestion of meningeal vessels. The initial diagnosis of sarcocystosis was made in all birds by microscopic observations of intravascular pulmonary schizonts, as well hepatitis, myocarditis, and nephritis. Immunohistochemistry for detection of Sarcocystis sp. antigen revealed an intense immunoreactivity in the lungs. Molecular identification of Sarcocystis falcatula were obtained by nested PCR and sequencing of amplified fragments of internal transcribed spacer 1 (ITS1) and three surface antigen-coding genes (SAG2, SAG3 and SAG4). SAG-based phylogenies showed a close relatedness of the isolate described here and S. falcatula previously detected in naturally infected native birds, which suggests that the isolates that affected ringnecks are a common isolate that circulates in Brazil.

Molecular screening for Sarcocystidae in muscles of wild birds from Brazil suggests a plethora of intermediate hosts for Sarcocystis falcatula.

The genus Sarcocystis and the species Toxoplasma gondii are the most prevalent sarcocystid organisms found in birds. Molecular phylogenies based on the first internal transcribed spacer of the ribosomal coding DNA (ITS1) have been widely used to identify them. Here, pectoral muscles from 400 wild birds from Brazil were screened by means of molecular methods using nested PCR, and Sanger sequencing yielded amplicons. A pan-sarcocystid ITS1-directed nested PCR revealed 28 birds infected by Sarcocystis falcatula (ten Piciformes, eight Psittaciformes, five Columbiformes, two Accipitriformes, one Anseriformes, one Passeriformes and one Strigiformes); one infected by Sarcocystis halieti (one Accipitriformes); nine infected by unknown or undescribed Sarcocystis (six Passeriformes, one Piciformes, one Cathartiformes and one Cuculiformes); and six harboring Toxoplasma gondii DNA (three Pelecaniformes, two Falconiformes and one Columbiformes). Samples harboring S. falcatula-related ITS1 sequences were further characterized by means of PCR and sequencing of genetic sequences of three surface antigen coding genes (SAGs). From this, 10 new allelic combinations of SAGs (SAG2, SAG3 and SAG4) were identified, in addition to 11 SAG allelic combinations already found in Brazil. Samples with S. falcatula-unrelated ITS1 sequences were further characterized by means of PCR and sequencing of cytochrome c oxidase subunit I coding sequences (CO1) and 18S ribosomal DNA gene (18S rDNA). This study was the first extensive survey of wild birds in Brazil for Sarcocystidae species. It provides the first molecular evidence of natural S. falcatula infection in 14 species, including in the order Piciformes, and shows the high genetic diversity of S. falcatula in intermediate hosts in South America. Evidence of occurrence of at least three non-described species of Sarcocystis was also presented in this study. This survey corroborated the ubiquity of T. gondii infection but revealed surprisingly low prevalence of this parasite (1.5%).

Seroepidemiology of Sarcocystis neurona and Neospora spp. in horses, donkeys, and mules from Colombia.

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]; p<0.001) than horses and mules, and higher in animals with a poor body condition (OR: 2.82 [1.45-6.05]; p<0.05). Additionally, older animals (>12y) had a higher risk of seropositivity (OR: 5.26 [1.88-19.1]; p<0.05), as well as animals that inhabit climatic conditions associated with tropical very dry forest (OR: 1.85 [1.01-3.51]; p<0.05). 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.

Molecular Characterization of New Haplotype of Genus Sarcocystis in Seabirds from Magdalena Island, Southern Chile.

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.

An undescribed species of Sarcocystis associated with necrotizing meningoencephalitis in naturally infected backyard chickens in the Midwest of Brazil.

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.

Sarcocystis falcatula-like derived from opossum in Northeastern Brazil: In vitro propagation in avian cells, molecular characterization and bioassay in birds.

Most reported isolates of Sarcocystis spp. derived from Brazilian opossums (Didelphis sp.) have genetic characteristics distinct from the known species of Sarcocystis, but behave similarly as Sarcocystis falcatula, as they are infective to budgerigars. In previous studies, these Brazilian isolates, classified as Sarcocystis falcatula-like, were originated from South and Southeast regions of Brazil. In the current work, we aimed to culture and to perform multilocus sequence analysis of Sarcocystis sp. derived from a Brazilian opossum (D. aurita/D. marsupialis) that inhabited the city of Salvador, Bahia, in the Northeast of Brazil. The parasite was isolated in Vero cells, referred here as Sarco-BA1, and propagated in avian cells (DF-1). Molecular analysis of Sarco-BA1 revealed that the nucleotide sequence of the internal transcribed spacer 1 (ITS1) of the rDNA was identical to all isolates (n = 19) of Sarcocystis spp. reported in two studies from South and Southeast regions of the country. Two budgerigars were inoculated with 10 and 1000 sporocysts of Sarco-BA1, respectively, and developed acute sarcocystosis, showing that the parasite behaves like S. falcatula. It was interesting to observe that Sarco-BA1 had almost identical ITS1 and SAG sequences to all 16 isolates of S. falcatula-like recently described in Magellanic penguins (Spheniscus magellanicus) rescued on the coast of Espírito Santo state, Brazil. Our results suggest that Sarco-BA1 and S. falcatula-like may represent a single species of Sarcocystis. Propagation of the parasite in a permanent avian cell line significantly improved the yield of merozoites in cell culture. To our knowledge, this is the first molecular study and in vitro isolation of S. falcatula-like derived from Northeastern Brazil. Studies are under way to determine the infectivity of Sarco-BA1 to other animal species, as well as to investigate serological cross-reactivity among Sarco-BA1, S. neurona and related species.