Parasitology International 63 (2014) 485–487

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Parasitology International

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Short communication Neotropical echinococcosis: Second report of vogeli natural infection in its main definitive host, the bush dog (Speothos venaticus)

Manoel do Carmo Pereira Soares a,⁎, Alex Junior Souza de Souza a,b, Andreza Pinheiro Malheiros a, Heloisa Marceliano Nunes a, Liliane Almeida Carneiro a,b, Max Moreira Alves a, Bernardo Farias da Conceição a, Michele Soares Gomes-Gouvêa c, Marinete Marins Póvoa d a Seção de Hepatologia, Instituto Evandro Chagas/SVS/MS, CEP 66093-020, Belém, PA, Brazil b Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, CEP 05508-270, São Paulo, SP, Brazil c Laboratório de Gastroenterologia e Hepatologia Tropical, Instituto de Medicina Tropical, Faculdade de Medicina, Universidade de São Paulo, CEP 05403-000, São Paulo, SP, Brazil d Seção de Parasitologia, Instituto Evandro Chagas/SVS/MS, CEP 67030-000, Ananindeua, PA, Brazil article info abstract

Article history: The bush dog (Speothos venaticus)actsasthenaturaldefinitive host in the life cycle of Echinococcus vogeli,the Received 30 July 2013 causative agent of polycystic hydatid disease, a zoonotic neglected disease in the South America. We report a Received in revised form 3 October 2013 case of natural infection by Echinococcus vogeli in a bush dog from the Brazilian Amazon, confirmed by the Accepted 11 October 2013 morphological and morphometric examination of adult parasites and their hooks obtained from the small Available online 19 October 2013 intestine of the canid. Additionally, mitochondrial DNA sequence analysis corroborated these findings. This is the second report of natural infection by E. vogeli in a bush dog. Keywords: Polycystic Echinococcosis © 2013 Elsevier Ireland Ltd. All rights reserved. Hydatidosis Echinococcus vogeli Speothos venaticus Zoonoses

Echinococcus vogeli is the main etiological agent of polycystic hydatid including capybara [4,5]. Despite a wide estimated geographical disease or echinococcosis, a disease caused by the development of the distribution, this species is considered rare in the wild making its larval stage of this tapeworm (metacestode) in man [1]. In the life study in the field difficult, and resulting in a poor understanding of its cycle of E. vogeli,thebushdog,Speothos venaticus (Lund, 1842), acts as real status distribution and habits [4–6]. A previous record of this canid's the definitive host, and the paca, Cuniculus paca (Linnaeus, 1766), is occurrence on the island of Marajó indicates a possible role for this the major intermediate host, whereas humans are considered an species in the natural cycle of E. vogeli in the region [6]. accidental intermediate host [2]. In intermediate hosts, E. vogeli Despite the bush dog's participation in the natural cycle of infection is characterized by the formation of cystic or tumorous-like neotropical echinococcosis caused by E. vogeli, its unusual status in the polycystic lesions in various organs such as the liver, spleen and lungs wild [1] and its protection by Brazilian environmental legislation have [1]. Due to its distribution – northern South America and parts of Central meant that, until now, only one instance of natural E. vogeli infection America – this disease is also called neotropical echinococcosis or in this canid species has been reported [2]. hydatidosis [1]. Brazil is the country with the highest incidence of In late 2007, an expedition from the Evandro Chagas Institute, neotropical echinococcosis in the world and the Marajó archipelago, Belém, Pará, Brazil, developed a fieldwork to investigate parasitic liver more recently, has been characterized as an enzootic and endemic diseases in wild rodents hosts of the Brazilian Amazon in the area of polycystic neotropical echinococcosis in the Brazilian Amazon municipality of Anajás, situated on Marajó island, Pará (00 ° 48′ 877″S [3]. 049 ° 42′ 06″ W). These activities were previously authorized by the The bush dog is a small wild canid, native to the rainforests that span Brazilian Institute of Environment and Renewable Natural Resources the extreme South of Panama, Colombia, Venezuela, Guyana, Brazil, (IBAMA) (Environmental License N° 006/2007). southern Bolivia, Paraguay and Northern Argentina, which has a diet Although the original aim of this work was to search for hydatid that includes fish, rodents such as paca and agouti, and larger mammals cysts in natural intermediate hosts of neotropical echinococcosis such as the paca and agouti, an adult specimen of Speothos venaticus, accidentally killed by a native hunter of the region during subsistence ⁎ Corresponding author at: Seção de Hepatologia, Instituto Evandro Chagas, Av. hunting, was delivered to the staff for study. Because of the potential Almirante Barroso, 492, CEP 66093-020, Belém, PA, Brazil. Tel.: +55 91 3214 2072; fax: +559132142139. importance of this mammal as the de finitive host of E. vogeli,the E-mail address: [email protected] (M. do Carmo Pereira Soares). canid was necropsied to collect stool, tissue and intestinal segment

1383-5769/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.parint.2013.10.004 486 M. do Carmo Pereira Soares et al. / Parasitology International 63 (2014) 485–487

Fig. 1. Segment of the small intestine of a bush dog with adult specimens of E. vogeli adherent to the mucosa (A); photomicrograph of a large hook (B); an egg of E. vogeli with hexacanth embryo within (C) and an adult E. vogeli specimen (D) obtained from a natural infection in a bush dog. samples, which were initially packaged for transport in liquid nitrogen specimen was deposited in the Emilio Goeldi Museum of Pará, located and subsequently stored at −70 °C until analyzed. After proper in Belém, Pará, Brazil (code: MPEG-41026). communication through a report to IBAMA, legal permission was In a suitable biosecurity laboratory environment, canid fecal samples obtained to continue the studies for scientific and sanitary purposes were submitted to coproparasitologic exams that indicated the using the previously obtained samples. Finally, the coat of this canid presence of eggs with chitinous double layer and hexacanth embryo.

Table 1 Morphometric measurements (range) of adult specimens of E. vogeli obtained from natural infection of S. venaticus on Marajo Island, Eastern Brazilian Amazon, 2007.

Parameters E. vogeli Marajó, Brazil E. vogeli Esmeraldas, Ecuadora (mm) (mm)

Total lenght of strobila 3.81 – 8.57 3.94 – 5.58 Length of gravid segment 2.1 – 5.47 2.94 – 4.2 Width of the gravid segment 0.53 – 0.86 0.285 – 0.33 Width of the penultimate segment 0.31 – 0.62 0.232 – 0.285 Rostellum (length × width) 0.13 – 0.18 × 0.18 – 0.23 0.103 – 0.134 × 0.154 – 0.188 Diameter of suckers 0.15 – 0.19 0.115 – 0.134 Genital pore position Unilateral or alternating/slightly posterior to the middle Unilateral or alternating/slightly posterior to the middle Number of segments 2 – 43 Scolex (lenght × width) 0.60 − 0.95 × 0.39 − 0.63 0.345 − 0.577 × 0.262 − 0.322 Ratio (anterior: posterior) 1:0.88 × 1;1.27 1:1.09 × 1:3

a Rausch & Bernstein (1972); mm, milimeters. M. do Carmo Pereira Soares et al. / Parasitology International 63 (2014) 485–487 487

described [8].Thepurified amplification products were sequenced with the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) with ABI 3500 DNA sequencer automatic analyzer. With the assistance of Bioedit 7.0.5.3 software, the consensus sequence obtained from the sample was aligned by CLUSTAL W and integrated with 9 other representative nucleotide sequences available in Genbank/NCBI of the genus Echinococcus and one representative of Taenia solium (GenBank Accession no. AB086256), which was used as an outgroup. The phylogenetic relationships between the sequences (396 bp) were developed using the MEGA program v.5 by the Neighbor-joining method with the Kimura 2-parameter substitution model. The reliability of the phylogenetic relationships was determined using bootstrap analysis (1000 replicates) and values above 70% were considered signifi cant for the construction of the tree groups (Fig. 2). Fig. 2. Phylogenetic tree inferred from the analysis of the partial region of the mitochondrial The nucleotide sequence obtained in this study (GenBank Accession COX-1 (396 bp) constructed by the neighbor-joining method, using the Kimura 2-parameter model. The sequences are identified by the GenBank number, followed by the identification no. JX315616) claded as clearly distinct from other Echinococcus species of the species. The isolate of E. vogeli in a bush dog is highlighted (♦). Numbers expressed in and had a close relationship with the sequence obtained from the nodes indicate bootstrap values obtained in 1000 replicates (expressed as a percentage). metacestode of the E. vogeli rodent host in Colombia [9].Thismolecular An isolated Taenia solium wasusedastheoutgroup. characterization was significant because it corroborated the parasitological examination and confirmed that cestodes of the species After opening a segment of the small intestine of the , with the E. vogeli had parasitized the bush dog. assistance of a magnifying glass, small whitish tapeworms were Additionally, the nucleotide sequence showed high phylogenetic observed adhered to the mucosa of the organ (Fig. 1,panelA). similarity with two other sequences of E. vogeli previously identified The taxonomic identification of these helminths was based on the in intermediate hosts (paca and human) from the same geographical microscopic observation of 11 uncolored specimens of tapeworm region of Marajó island [10], demonstrating the effective participation (mature and immature) and of 21 large rostellar hooks (Fig. 1,panel of the bush dog in the epidemiological cycle of neotropical B), mounted in histological laminas and submitted to morphological echinococcosis in the Brazilian Amazon. and morphometrical analysis for parasite species classification. These results represent the second record of natural infection by E. Parasitological assessment of adult worms indicated that these were vogeli in its main definitive host, the bush dog, and this is the first report cestodes with strobila composed by 2–4 segments, all longer than they of this type from the Brazilian Amazon or any location in Brazil. were wide. Eight parasites showed characteristics of fully developed strobilae (consisting of 3–4 segments) and 3 appeared to be immature Acknowledgments (only scolex and 2 segments). The location of a genital pore on the edges of the mature and gravid segment was posterior to the middle This study was performed with financial support from the Evandro in both, and the size ranged from unilateral to alternating in most Chagas Institute/Secretary of Health Surveillance/Ministry of Health. cases. The morphometric characteristics of the specimens described in We would like to thank to Igor Ricardo dos Santos Corrêa and Elisabete Table 1 gave support to the determination that they were cestodes of Maria de Figueiredo Brito without whose knowledge and assistance this the species E. vogeli. study would not have been successful. The analysis of the shape and size of the large hooks (n = 21) was important for taxonomic key identification because they demonstrated References morphological and morphometric characteristics compatible with E. vogeli, according to the parameters previously established [7]:the [1] D'Alessandro A, Rausch RL. New aspects of Neotropical Polycystic (Echinococcus total length ranged from 0.030 to 0.055 mm (0.044 mm average), the vogeli) and Unicystic (Echinococcus oligarthrus) Echinococcosis and Unicystic (Echinococcus oligarthrus) Echinococcosis. Clin Microbiol Rev 2008;21:380–401. handle length ranged from 0.010 to 0.025 mm (0.019 mm average) [2] Rausch RL, Bernstein JJ. Echinococcus vogeli sp.n. (: ) from the bush and the blade length ranged from 0.008 to 0.035 mm (0.025 mm dog, Speothus venaticus (Lund). Z Tropenmed Parasitol 1972;23:25–34. average). The measurement of 15 parasite eggs showed that their [3] Soares MCP, Moreira-Silva CA, Alves MM, Nunes HM, do Amaral IA, Móia LJMP, et al. Equinococose policística na Amazônia oriental brasileira: atualização da casuística. length ranged from 0.025 to 0.040 mm (0.0354 mm average), and Rev Soc Bras Med Trop 2004;37:75–83. their width from 0.025 to 0.035mm (mean 0.0304mm) (Fig. 1, Panel C). [4] Beisiegel BM, Zuercher GL. Speothos venaticus. Mamm Species 2005;783:1–6. Adult specimens of E. vogeli obtained in this study were, generally, of [5] DeMatteo KE, Loiselle BA. New data on the status and distribution of the bush dog (Speothos venaticus): evaluating its quality of protection and directing research a slightly larger size than had previously been described for this species efforts. Biol Conserv 2008;141:2494–505. [2] (Fig. 1, Panel D), although experimental studies in dogs have [6] Silva Jr JS, Soares MCP. An unexpected new record for the bush dog, Speothos demonstrated that E. vogeli strobila can reach lengths of up to 12 mm venaticus Lund, 1842, in the Brazilian Amazonia (Carnivora, canidae). Publ Avulsas – [1], which may explain the differences that exist between the Inst Pau Bras 1999;2:7 11. [7] Rausch RL, Rausch VR, D'Alessandro A. Discrimination of the larval stages of morphometric results of this study and the original description of the Echinococcus oligarthrus (Dieslng, 1863). Am J Trop Med Hyg 1978;27:1195–202. parasite. The number of adult specimens and rostellar hooks analyzed [8] Bowles J, Blair D, McManus DP. Genetic variants within the Genus Echinococcus fi – may also have affected this result because over 90 parasites specimens identi ed by mitochondrial DNA sequencing. Mol Biochem Parasitol 1992;54:165 74. [9] Nakao M, McManus DP, Schantz PM, Craig PS, Ito A. A molecular phylogeny of the were analyzed in the original description [2]. genus Echinococcus inferred from complete mitochondrial genomes. Parasitology DNA from a pool of adult worms was extracted using the QIAamp 2007;134:713–22. DNA mini kit (QIAGEN) and the molecular detection was made using [10] Santos GB, Soares MCP, Brito EMF, Rodrigues AL, Siqueira NG, Gomes-Gouvêa MS, fi et al. Mitochondrial and nuclear sequence polymorphisms reveal geographic a PCR-based ampli cation of a 446 bp fragment of mitochondrial structuring in Amazonian populations of Echinococcus vogeli (Cestoda: Taeniidae). cytochrome c oxidase subunit 1 gene (cox1) using primers previously Int J Parasitol 2012;42:1115–8.