Archives of Veterinary Science ISSN 1517-784X v.23, n.3, p.50-55, 2018 www.ser.ufpr.br/veterinary CONCOMITANT INFECTION OF HAEMOGREGARINA SP. AND STAPHYLOCOCCUS AUREUS IN FREE-LIVING YELLOW-SPOTTED RIVER TURTLE (PODOCNEMIS UNIFILIS): CASE REPORT (Infecção concomitante de Haemogregarina sp. e Staphylococcus aureus em tracajá (Podocmenis unifilis) de vida livre: relato de caso) Letícia Pereira Úngari1*, André Luiz Quagliatto Santos2, Guilherme Carrara Moreira Paiva2, Kelem Cristina Pereira Mota2, Lizandra Ferreira de Almeida Borges2, Márcia Cristina Cury2 1Universidade Estadual Paulista "Júlio de Mesquita Filho”, Botucatu, SP, Brazil. *Corresponding author: [email protected] 2Universidade Federal de Uberlândia, MG, Brazil ABSTRACT: A male adult yellow-spotted river turtle Podocnemis unifilis (Chordata: Testudines: Podocnemididae) was captured during field collections. Blood sample was collected for haemoparasite screening by conventional light microscopy. Morphometric and morphological analyzes of the parasite developmental stages were performed in the blood extensions, as well as the parasitemia intensity. Gametocytes of hemogregarines and innumerable bacterial cells were observed. To identify the bacteria present, a culture was performed and the presence of Staphylococcus aureus was observed. It was concluded that a co-infection of Staphylococcus aureus (Firmicutes: Bacillales: Staphylococcaceae) and Haemogregarina sp. (Apicomplexa: Adeleina: Haemogregarinidae) was observed in this free-living yellow-spotted river turtle Podocnemis unifilis. Keywords: co-infection; hemogregarines; occurrence; Podocnemis unifilis; Staphylococcus aureus. RESUMO: Durante coletas de campo, um macho adulto de tracajá Podocnemis unifilis (Chordata: Testudines: Podocnemididae) foi capturado. Amostra de sangue foi colhida para pesquisa de hemoparasitos pela microscopia óptica convencional. Nas extensões sanguíneas foram realizadas análises morfométricas e morfológicas das formas evolutivas encontradas, bem como o cálculo da parasitemia. Foram observados gametócitos de hemogregarinas e inúmeras células bacterianas. Para identificação das bactérias presentes foi realizada cultura, sendo observada a presença de Staphylococcus aureus. Conclui-se que havia co-infecção por Staphylococcus aureus (Firmicutes: Bacillales: Staphylococcaceae) e Haemogregarina sp. (Apicomplexa: Adeleina: Haemogregarinidae) no tracajá Podocnemis unifilis. Palavras-chave: co-infecção; hemogregarinas; ocorrência; Podocnemis unifilis; Staphylococcus aureus. Archives of Veterinary Science, v.23, n.3, p.50-55, 2018. 51 Úngari et al. (2018) INTRODUCTION Campbell, 1990; Orós et al., 2005). Nonetheless, there are no studies The yellow-spotted river turtle associating Staphylococcus aureus with Podocnemis unifilis Troschel 1848, is a parasitic diseases. semi-aquatic turtle widely distributed in Therefore, the aim of this study South America (Arraes and Tavares- was to identify the hemogregarines Dias, 2014). In the literature, there are (Apicomplexa: Adeleina) affecting few reports of pathogens affecting these aquatic turtles in their natural habitat, turtle species, being the hemogregarines unexpectedly, a concomitant infection by hemoparasites commonly reported Staphylococcus aureus and (Soares et al. 2014; Espinoza et al. Haemogregarina sp. was reported. 2017). The hemogregarine parasites MATERIALS AND METHODS (Apicomplexa: Adeleina) are This study was conducted under characterized as an intracellular the approval of the Ethics Committee for protozoan with heteroxenic lifecycle and Use of Animals of the University of frequently found in reptiles (Telford, Uberlândia (CEUA-UFU) (protocol 2009; Soares et al., 2014). The genus 032/16) and the Biodiversity Information Haemogregarina (Apicomplexa: and Authorization System (SISBIO) Adeleina: Haemogregarinidae) is (protocol 51398-1). common in aquatic turtles and has been In July 2016, a male adult yellow- documented in several countries spotted river turtle (Podocnemis unifilis) (Telford, 2009) However in Brazil, there was captured during field surveys in Red are only a few reports (Soares et al., River, Britânia district, Goiás, Brasil 2014; Picelli et al., 2015; Úngari et al., (coordinates 14°57'08,4" South latitude 2018). A recent study from Úngari et al. and 51°06'30,7" West longitude). The (2018) characterized through specimen was submitted to blood morphological, morphometric and collection with disposable and sterile 3 molecular analyses, a new species mL syringes and needles (22/19), by Haemogregarina podocnemis in puncturing the cervical paravertebral Podocnemis unifilis, being the first venous sinus (Zippel et al., 2001). At the Haemogregarina species described in a site, three blood smears were Brazilian turtle. performed, and the rest of the material Another pathogen observed in was stored at -4°C, in tubes containing reptiles, including turtles, although less EDTA as anticoagulant, for further common, is the bacteria Staphylococcus analyzes at the Laboratory of Serology aureus (Firmicutes: Bacilllales: and Molecular Biology of Parasites of Staphylococcaceae). It is present in a the Federal University of Uberlândia. wide variety of animal species, including The blood smears were fixed with mammals, birds, and reptiles, and may methanol, stained in 10% Giemsa (Eisen be commensal or pathogenic (Santoro et and Schall, 2000) and examined using al., 2006). S. aureus pathogenesis and, an optical microscope (Olympus Optical consequently, the host clinical condition Co) with 40x and 100x magnification. are associated with several factors, The hemoparasite parasitemia was immunity for example. estimated according to De Biasi et al. Studies in captive sea turtles have (1989) under 100x magnification. The demonstrated the association of S. morphometric analysis of the parasite aureus with bronchopneumonia, blood developmental stages was obstructive rhinitis, traumatic ulcer, and performed by measuring the length, ulcerative stomatitis (Glazebrook and Archives of Veterinary Science, v.23, n.3, p.50-55, 2018. 52 Concomitant infection of Haemogregarina sp. and Staphylococcus aureus in free-living yellow-spotted river turtle (Podocnemis unifilis): case report width, and area of the body and nucleus gametocytes were observed with nuclei of the parasites developmental stages. not evidenced, similar to a bean shape, For the bacteria analysis, an with body length, width, and area of the aliquot of the blood sample was sent to parasite 8.47 ± 1.58 µm, 4.2 ± 1.5 µm, the Laboratory of Research in and 15.1 ± 2.4 µm2, respectively. No Bacteriology of the Federal University of measures of the nuclei were performed, Uberlândia. To isolate the bacteria, the since their nuclei were not delimited. blood sample was cultured in 1 mL BHI The other type of gametocyte were (Brain Heart Infusion) broth and robust, causing deformations in incubated at 35 ± 2°C, for 24 hours. The erythrocytes and displacement of cell culture was then plated on TSA Agar nuclei, with nuclei of the parasite (Trypticase Soy Agar) and Minnitol salt delimited; the body length, width, and Agar, and the plates were incubated at area of the parasite were, respectively, 35 ± 2°C, for 24 hours. Colonies were 17.9 ± 1.3 µm, 5.4 ± 0.8 µm, and 39.1 ± subjected to Gram staining, catalase test 2.9 µm2 and the nuclei length, width, and (using 3% hydrogen peroxide), and area of the parasite were, respectively, coagulase exam (using 0.5 mL of rabbit 8.25 ± 1.30 µm, 3.59 ± 0.8 µm, and plasma). To test antimicrobial 13.97 ± 1.42 µm2 (Figure 1). susceptibility, a bacterial inoculum was prepared, and antimicrobial disks were placed on the agar surface and incubated at 35°C for 16 to 18 hours. After incubation, the plate was examined under light, and the inhibition halo diameters were compared to the “Clinical and Laboratory Standards Institute©” (CLSI) document to classify as resistant or sensitive (Santoro et al., 2006; Walther et al., 2008; Iverson et al., 2015). RESULTS AND DISCUSSION The occurrence of Figure 1 - Intraerithrocytic gametocytes of Haemogregarina sp. in turtles is Haemogregarina sp. (Apicomplexa: Adeleina: commonly reported in the literature. Haemogregarinidae) in Podocnemis unilifis. Jakes et al. (2001) reported a 100% Hemoparasites are indicated by arrows (Small arrows: immature gametocytes; big arrow: occurrence in the long-necked turtle mature gametocytes). Scale: 10 µm. (Chelodina longicollis) and the Brisbane short-necked turtle (Emydura signata), The same morphologies of the same way as Mihalca et al. (2002) in gametocytes were observed by Soares the lagoon turtle (Emys orbicularis). In et al. (2014) and Picelli et al. (2015) from Brazil, a high occurrence of 98% Brazilian P. unifilis and P. expansa (Soares et al., 2014) and 100% (Úngari turtles, respectively. The authors et al., 2018) were reported for this genus nominated the small gametocytes as of parasite in free-living P. unifilis. microgametocytes and the robust The morphological and gametocytes as macrogametocytes. The morphometric analyses showed morphometric values were similar too. gametocytes of different sizes and Therefore, from the morphological and morphologies. One type of small morphometric analyses of the Archives of Veterinary Science, v.23, n.3, p.50-55, 2018. 53 Úngari et al. (2018) hemoparasite blood developmental being a possible explanation for the stages, it was possible to identify turtle infection observed in this case, Haemogregarina sp. (Apicomplexa: since there are not many reports on this