Multilocus characterization of Sarcocystis falcatula-related organisms isolated in Brazil supports genetic admixture of high diverse SAG alleles among the isolates.

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.

Diversity of Sarcocystis spp shed by opossums in Brazil inferred with phylogenetic analysis of DNA coding ITS1, cytochrome B, and surface antigens.

Although few species of Sarcocystis are known to use marsupials of the genus Didelphis as definitive host, an extensive diversity of alleles of surface antigen genes (sag2, sag3, and sag4) has been described in samples of didelphid opossums in Brazil. In this work, we studied 25 samples of Sarcocystis derived from gastrointestinal tract of opossums of the genus Didelphis by accessing the variability of sag2, sag3, sag4, gene encoding cytochrome b (cytB) and first internal transcribed spacer (ITS1). Reference samples of Sarcocystis neurona (SN138) and Sarcocystis falcatula (SF1) maintained in cell culture were also analyzed. We found four allele variants of cytB, seven allele variants of ITS1, 10 allele variants of sag2, 13 allele variants of sag3, and 6 allele variants of sag4. None of the sporocyst-derived sequences obtained from Brazilian opossums revealed 100% identity to SN138 at cytB gene, nor to SN138 or SF1 at ITS1 locus. In addition, none of the sag alleles were found identical to either SF1 or SN138 homologous sequences, and a high number of new sag allele types were found other than those previously described in Brazil. Out of ten sag2 alleles, four are novel, while eight out of 13 sag3 alleles are novel and one out of six sag4 alleles is novel. Further studies are needed to clarify if such a vast repertoire of allele variants of Sarcocystis is the consequence of re-assortments driven by sexual exchange, in order to form individuals with highly diverse characteristics, such as pathogenicity, host spectrum, among others or if it only represents allele variants of different species with different biological traits.

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%).

Toxoplasmosis in a free-ranging hairy dwarf porcupine (Sphiggurus spinosus) with a potential novel genotype.

Toxoplasmosis is a zoonotic disease caused by the ubiquitous coccidia Toxoplasma gondii. Rodents play an important role in maintaining its life cycle, as they are one of the main diet sources for felids (wild and domestic), the unique definitive hosts. However, reports of toxoplasmosis in porcupines (Order Rodentia) are uncommon, with gaps concerning its pathophysiology. South America is the continent with the greatest genetic diversity of rodents and T. gondii. A free-ranging hairy dwarf porcupine was admitted to a wildlife rescue centre with a history of trauma. During rehabilitation, the animal presented neurological symptoms (sporadic episodes of hind limbs paresis) and died 5 months later. The main findings during necropsy were brain congestion and severe incisor overgrowth associated with maxillary perforation. The histopathological exam showed moderate encephalitis, with variable-sized round cysts, positive for PAS stain and immunohistochemistry for T. gondii. Additionally, two cysts were observed in the medulla of the adrenal gland. Molecular techniques were performed to characterize the parasite load by qPCR (Cq = 30) and the genotype by PCR-RFLP with 11 markers, which revealed a potential new genotype. This case adds to the body of knowledge in comparative pathology of Neotropical Rodentia and reports a new potential genotype circulating in South America.

A retrospective pathology study of two Neotropical deer species (1995-2015), Brazil: Marsh deer (Blastocerus dichotomus) and brown brocket deer (Mazama gouazoubira).

This retrospective study describes the biological and epidemiological aspects, gross and microscopical findings, and most likely causes of death (CD) in two species of Neotropical deer in Brazil. The animals were collected between 1995 and 2015 and represented 75 marsh deer (MD) and 136 brown brocket deer (BBD). Summarized, pneumonia was diagnosed microscopically in 48 MD and 52 BBD; 76 deer suffered trauma, involving dog attack (14 BBD) and vehicle-collision (14 BBD). Pulmonary edema (50 MD; 55 BBD) and congestion (57 MD; 78 BBD) were the most common findings for both species. Additionally, we diagnosed ruminal and myocardial mycosis in MD and BBD, respectively; ovarian dysgerminoma and pancreatic trematodiasis in BBD; and lesions suggestive of malignant catarrhal fever and orbiviral hemorrhagic disease in both species. The main CD in MD was: respiratory (41/75), alimentary, nutritional, trauma and euthanasia (3/75 each). Correspondingly, in BBD were: trauma (34/131), respiratory (30/131) and euthanasia (9/131). Respiratory disease was often defined by pulmonary edema and pneumonia. We provide evidence that respiratory disease, mainly pneumonia, is a critical pathological process in these Neotropical deer species. Although no etiological agents were identified, there is evidence of bacterial and viral involvement. Our results show trauma, mainly anthropogenic, as a common ailment in BBD. We propose to prioritize respiratory disease in future research focused on South American deer health aspects. We believe anthropogenic trauma may be a primary threat for populations of BBD.

New host association and locality record for Pellonyssus gorgasi Yunker and Radovsky, 1966 (Mesostigmata: Macronyssidae) in the state of São Paulo, Brazil

The genus Pellonyssus Clark and Yunker, 1956 comprises 14 species worldwide that are associates of birds. One of these species, Pellonyssus gorgasi Yunker and Radovsky, 1966, parasitizes hummingbirds in the Americas. This study represents a new host association with Eupetomena macroura (Apodiformes: Trochilidae) and the first occurrence of this mite in the state of São Paulo.

Ticks (Acari: Ixodidae) on wild raptors in Brazil

This research reports ticks on wild raptors in Brazil. Between the years 1936 and 2019, 127 larvae, 230 nymphs and 34 adult ticks were collected on 92 raptors (among 27 different species) from 35 localities in distinct Brazilian biomes. Additionally, an extensive literature review on ticks on wild raptors has been carried out, demonstrating that from 1993 to 2016, 29 larvae, 81 nymphs, 29 adults and 186 indeterminate immature ticks (larvae or nymphs) were collected on 41 raptors (16 different species) in 17 distinct localities in the Brazilian territory. The following tick species were identified on wild raptors in the country: Amblyomma aureolatum (Pallas, 1772), Amblyomma auricularium (Conil, 1878), Amblyomma brasiliense Aragão, 1908, Amblyomma cajennense (Fabricius, 1787) sensu stricto, Amblyomma calcaratum Neumann, 1899, Amblyomma coelebs Neumann, 1899, Amblyomma dubitatum Neumann, 1899, Amblyomma longirostre (Koch, 1844), Amblyomma nodosum Neumann, 1899, Amblyomma ovale Koch, 1844, Amblyomma parkeri Fonseca & Aragão, 1952, Amblyomma sculptum Berlese, 1888, Haemaphysalis juxtakochi Cooley, 1946, Rhipicephalus microplus (Canestrini, 1888), and Rhipicephalus sanguineus (Latreille, 1806) sensu lato. This study is therefore a significant contribution to our knowledge of the ticks associated with Brazilian raptors.

New host association and locality record for Pellonyssus gorgasi Yunker and Radovsky, 1966 (Mesostigmata: Macronyssidae) in the state of São Paulo, Brazil

The genus Pellonyssus Clark and Yunker, 1956 comprises 14 species worldwide that are associates of birds. One of these species, Pellonyssus gorgasi Yunker and Radovsky, 1966, parasitizes hummingbirds in the Americas. This study represents a new host association with Eupetomena macroura (Apodiformes: Trochilidae) and the first occurrence of this mite in the state of São Paulo.

Borrelia burgdorferi antibodies in dogs from Cotia county, Säo Paulo State, Brazil

Dogs sera samples collected from Cotia County, Säo Paulo were tested using indirect immunoenzymatic test (ELISA) in order to study Lyme disease serology in dogs. ELISA method was standardized and G39/40 North American strain of Borrelia burgdorferi was used as antigen. Positive results were confirmed employing the Western blotting technique. Because of the possibility of cross-reactions, sera were also tested for different serological strains of Leptospira interrogans and L. biflexa using microscopic sera agglutination test. Twenty-three of 237 (9.7 percent) serum samples were positive in the ELISA; 20 of them (86.9 percent) were confirmed by the Western blotting, what suggests that Cotia may be a risk area for Lyme disease. Although 4 samples (1.7 percent) were positive for Lyme disease and leptospirosis, no correlation was found between the results (X² = 0.725; p = 0.394) what suggests absence of serological cross reactivity