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(Spheniscus Magellanicus) in South America Parasitology Research (2019) 118:599–606 https://doi.org/10.1007/s00436-018-6155-5 PROTOZOOLOGY - ORIGINAL PAPER Pathological and molecular characterization of avian malaria in captive Magellanic penguins (Spheniscus magellanicus)inSouthAmerica Paula Augusto Taunde1 & Matheus Viezzer Bianchi1 & Lívia Perles2 & Fernando Soares da Silva1 & Tainã Normanton Guim1 & Renan Alves Stadler3 & Marcos Rogério André2 & David Driemeier1 & Saulo Petinatti Pavarini1 Received: 7 May 2018 /Accepted: 12 November 2018 /Published online: 19 November 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018 Abstract Avian malaria is a mosquito-borne disease that affects multiple avian species and is caused by protozoans of the genus Plasmodium. An avian malaria infection caused by Plasmodium sp. in Magellanic penguins (Spheniscus magellanicus)with high mortality is described in a zoo in Southern Brazil. Clinically, three birds presented signs of inappetence, anorexia, pale mucosa, dyspnea, and opisthotonus, with death in a clinical course of 5–8 h. At the necropsy, all birds exhibited pale mucosa, marked splenomegaly and hepatomegaly, in addition to moderate leptomeningeal blood vessels ingurgitation in the brain. Microscopically, multiple exoerythrocytic meronts were observed in the cytoplasm of endothelial cells in the spleen, liver, heart, lungs, brain, kidneys, and pancreas. The spleen had a multifocal perivascular inflammatory infiltrate of lymphocytes, plasma cells, and macrophages, which also exhibited hemosiderosis and erythrophagocytosis. The liver had a multifocal periportal inflammatory infiltrate of lymphocytes, macrophages, and plasma cells, in addition to marked hemosiderosis in the hepatic sinusoids. Fragments of spleen, liver, brain, skeletal muscle, and lung were tested by the polymerase chain reaction technique for the detection of a fragment of the cytochrome B gene from haemosporidians, which resulted positive for Plasmodium spp. After sequencing, the samples were phylogenetically associated to Plasmodium sp. detected in Turdus albicollis (KU562808) in Brazil and matched to the lineage TURALB01 previously detected in T. albicollis. Avian malaria infections caused by Plasmodium sp. of lineage TURALB01 may occur in S. magellanicus with high mortality, and, thus, it is essential to detect and characterize the agent involved to obtain the differential diagnosis of the condition. Keywords Culicidae . Haemosporidian parasites . Morphologic analyses . Phylogenetic analyses . Plasmodium spp. Introduction Magellanic penguins (Spheniscus magellanicus) are native birds Southern Brazil from March to September. Annually, tens to from Argentina, Chile, and Falkland Islands, and migrate to hundreds of penguins, mainly juvenile, are admitted to rehabili- tation centers in the Brazilian coast (García-Borboroglu et al. 2006, 2010). During this period, these animals may be affected Section Editor: Larissa Howe by many diseases, such as aspergillosis, pododermatitis, and avi- an malaria (Cranfield et al. 1991). * Matheus Viezzer Bianchi Avian malaria is a mosquito-borne disease transmitted by [email protected] blood feeding insects from family Culicidae that affects mul- tiple bird species, and it is caused by protozoan of the genus 1 Departamento de Patologia Clínica Veterinária, Setor de Patologia Plasmodium (Valkiūnas 2005; Bueno et al. 2010;Sallaberry- Veterinária, Faculdade de Veterinária, Universidade Federal do Rio Pincheira et al. 2015). Despite that, these parasites are not Grande do Sul (UFRGS), Av. Bento Gonçalves 9090, Prédio 42505, associated with events of mass mortality in bird populations Porto Alegre, RS 91540-000, Brazil who co-evolve with species of Plasmodium (Grilo et al. 2 Departamento de Patologia Veterinária, Laboratório de 2016). Many species of penguins are, however, highly suscep- Imunoparasitologia, Faculdade de Ciências Agrárias e Veterinárias (FCAV), Universidade do Estado de São Paulo (UNESP), Via de tible to Plasmodium spp. both in the wild and in captivity Acesso Prof. Paulo Donato Castellani, Jaboticabal, SP 14884-900, (Vanstreels et al. 2016). Magellanic penguins of zoological Brazil gardens or undergoing rehabilitation, which are exposed for 3 GramadoZoo, Gramado, RS, Brazil the first time to the agent are mainly affected (Fix et al. 1988; 600 Parasitol Res (2019) 118:599–606 Cranfield et al. 1991; Bueno et al. 2010; Grilo et al. 2016), following the protocol previously described by Hellgreen with infections caused by Plasmodium relictum (Fix et al. et al. (2004). The first reaction was performed using the 1988), Plasmodium elongatum, Plasmodium tejerai (Silveira primers HaemNFI (5′-CATATATTAAGAGAAITATGGAG- et al. 2013), Plasmodium juxtanucleare (Grim et al. 2003), 3′) and HaemNR3 (5′-ATAGAAAGATAAGAAATACC Plasmodium cathemerium (Vanstreels et al. 2014), ATTC-3′) in a final volume of 25 μL, which contained Plasmodium nucleophilum,andPlasmodium unalis 50 ng of extracted DNA, 1.25 mM of each set of dNTP (Vanstreels et al. 2015), while avian malaria has not been (Invitrogen, Carlsbad, USA), 1.5 mM MgCl2, 10× concentrat- identified in free-living Magellanic penguins (Vanstreels ed buffer (Invitrogen®), 0.4 μM of each primer, and 0.5 U of et al. 2017). Taq DNA polymerase (Invitrogen, Carlsbad, USA). Cycling Clinically, avian malaria is characterized by an acute course conditions comprise an initial denaturation at 94 °C for 3 min, with anemia, lethargy, anorexia, fever, weight loss, and green- 20 cycles of 94 °C for 20s, 50 °C for 30s, 72 °C for 45 s, and a ish feces, followed by high mortality indexes (Valkiūnas final extension at 72 °C for 10 min. Two microliters of the 2005). Pathological findings are predominantly related to the amplification products from the first reaction were employed hemolytic disease and include hepatomegaly, splenomegaly, in the subsequent nested PCR reactions: 1 μLforthePCR hydropericardium, and pulmonary edema (Fix et al. 1988). product for the detection of Plasmodium spp. and Microscopically, splenic and hepatic hemosiderosis are char- Haemoproteus spp. (using the primers HaemF 5′-ATGG acteristic, with exoerythrocytic meronts inside endothelial TGCTTTCGATATATGCATG-3′ and HaemR2 5′-GCAT cells and macrophages (Valkiūnas 2005). The present study TATCTGGATGTGATAATGGT-3′), and 1 μL for the PCR aims to describe the clinical, pathological, and molecular as- product for the detection of Leucocytozoon spp. (using the pects of avian malaria infection in Magellanic penguins primers HaemFL 5′-ATGGTGTTTTAGATACTTACATT-3′ (S. magellanicus), which culminated with substantial mortal- and HaemR2L 5′-CATTATCTGGATGAGATAATGGIG ity at a zoo in the Southern Brazil. C-3′). These reactions were performed separately in a final volume of 25 μL, under the same cycling condition and re- agents concentration from the first reaction, except for 35 Materials and methods instead of 20 cycles. DNA samples obtained from an Orinoco goose (Neochen jubata) naturally infected with Three captive Magellanic penguins were kept in a zoo located Plasmodium sp.—MF043229 (Werther et al. 2017)and in the city of Gramado, Rio Grande do Sul state, Brazil (29° ultra-pure sterilized water were used as positive and negative 25′ 40″ S, 50° 51′ 18.8″ W), with an area of 20 ha that is controls, respectively. The results were visualized in 2% aga- surrounded by abundant secondary forests. Two adults (male rose gel stained by ethidium bromide solution. and female; penguins 1 and 2), and a juvenile male (penguin All the PCR products showing high intensity of the bands 3) were clinically examined and died a few hours after the of expected sizes were purified using the Silica Bead DNA onset of clinical signs. The birds were housed in this zoo for Gel Extraction Kit (Fermentas, São Paulo, Brazil). Purified at least 1 year and were previously admitted to a rehabilitation amplified DNA fragments from each positive sample were center located in the city of Florianópolis, Santa Catarina State sequenced in an automatic sequencer (ABI Prism 310 (27° 31′ 40″ S, 48° 25′ 44″ W). Although these birds were Genetic Analyzer; Applied Biosystems/Perkin Elmer, kept isolated in an indoor enclosure with controlled tempera- Waltham, USA) in both directions using Sanger’s method ture, they were fed at the outdoor enclosure where there was (Sanger et al. 1977). The electropherogram quality was ana- no mosquito control. Close to the penguin enclosure, there lyzed using the Phred Phrap software (Ewing et al. 1998), in was an enclosure with various species of Psittaciformes which only nucleotide sequences above 400 bp in size and (Amazona spp., Ara spp., and Anodorhyncus hyacinthinus). Phred quality ≥ 20 were used. Quality scores were assigned The penguins were submitted to necropsy, when multiple to each base call in automated sequencer traces. Additionally, fragments of tissues were collected, fixed in 10% neutral buff- consensus sequences were obtained through analysis on the ered formalin solution for 24–48 h, routinely processed for sense and antisense sequences using the Phred Phrap software histology, and stained with hematoxylin and eosin (HE). No (Ewing et al. 1998). The identity values among the nucleotide blood smears were available for evaluation in any of the cases. sequences were assessed by BLASTn tool (using default pa- Samples of spleen (penguins 1, 2, and 3), liver, brain, skel- rameters), available in MalAvi database (Bensch et al. 2009). etal muscle (penguins 2 and 3), and lungs (penguin 1) were The obtained sequences aligned
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