Parasitology Identification of the avian tracheal trematode Typhlocoelum cucumerinum cambridge.org/par (Trematoda: Cyclocoelidae) in a host–parasite–environment system: diagnosis, Research Article life cycle and molecular phylogeny Cite this article: Assis JCA, López-Hernández D, Favoretto S, Medeiros LB, Melo AL, Martins 1 1 2 NRS, Pinto HA (2021). Identification of the Jordana C. A. Assis , Danimar López-Hernández , Samantha Favoretto , avian tracheal trematode Typhlocoelum 3 1 3 1 cucumerinum (Trematoda: Cyclocoelidae) in a Lilian B. Medeiros , Alan L. Melo , Nelson R. S. Martins and Hudson A. Pinto host–parasite–environment system: diagnosis, 1 life cycle and molecular phylogeny. Laboratório de Biologia de Trematoda, Department of Parasitology, Institute of Biological Sciences, Universidade Parasitology 148, 1383–1391. https://doi.org/ Federal de Minas Gerais, Belo Horizonte, Brazil; 2Department of Veterinary Medicine, Universidade Federal de 10.1017/S0031182021000986 Lavras, Lavras, Brazil and 3Laboratório de Doenças das Aves, Department of Preventive Veterinary Medicine, Veterinary School, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil Received: 27 April 2021 Revised: 1 June 2021 Accepted: 1 June 2021 Abstract First published online: 9 June 2021 Typhlocoelum cucumerinum is a tracheal parasite of birds widely distributed across the globe. Key words: Nevertheless, aspects of the biology of this cyclocoelid are still poorly understood. Herein, we Typhlocoelinae; life cycle; domestic waterfowl; report the finding of T. cucumerinum in definitive and intermediate hosts from an urban phylogeny waterbody of Brazil. The parasite was initially detected during the necropsy of domestic Muscovy ducks (Cairina moschata) found dead in the locality. Coproparasitological tests in Author for correspondence: Jordana C. A. Assis, live animals revealed that 12/47 (25.53%) Muscovy ducks and 2/8 (25%) mallards (Anas E-mail: [email protected] platyrhynchos platyrhynchos) were infected with T. cucumerinum. Moreover, rediae and metacercariae morphologically similar to T. cucumerinum were found in 3/248 (1.33%) Biomphalaria straminea collected in the same waterbody frequented by the birds. The conspe- cificity between the adult and the larval stages was confirmed molecularly (100% similarity in Cox-1). Moreover, the phylogenetic position of T. cucumerinum was determined for the first time based on partial fragments of the 28S, Cox-1 and Nad-1 genes. The species grouped with other members of the subfamily Typhlocoelinae with sequences available, but the data obtained do not support the distinctiveness of the genera Typhlocoelum and Tracheophilus. Further studies involving a broader range of species can result in taxonomic rearrangements in Typhlocoelinae. Introduction Typhlocoelum cucumerinum (Rudolphi, 1809) is a trematode of the upper respiratory system (mainly trachea) of waterfowls with worldwide distribution (Asia, Europe, Oceania and Americas) and currently included in the family Cyclocoelidae Stossich, 1902 (Gower, 1939; Barry, 1959; Schafranski et al., 1975; Soulsby, 1982; Tkach et al., 2016). Infection with these tracheal parasites can cause airway obstruction, resulting in dyspnoea, suffocation and even death (Schafranski et al., 1975; Soulsby, 1982; Taylor et al., 2017). Despite the potential veter- inary importance of typhlocoelosis in domestic anatids, aspects of the biology, epizoology and transmission dynamics of T. cucumerinum are still poorly understood. In fact, the countless reports of infection of birds with this cyclocoelid are based on post-mortem findings (Kinsella and Forrester, 1972; Schafranski et al., 1975; Hoyos et al., 2017). To the best of our knowledge, studies involving the in vivo diagnosis of this trematode are lacking, despite the potential importance of this approach to subside the treatment of infection. Moreover, no study reported the occurrence of T. cucumerinum in naturally infected snails so far. Studies on life cycles of species of the family Cyclocoelidae revealed that these trematodes have a single intermediate host, in which there is the development of rediae, followed by the production of cercariae (Cercariaeum type) and the formation of encysted metacercariae in the same mollusc (Taft, 1973, 1975, 1986; Schafranski et al., 1975; McLaughlin, 1976, 1986;Taft and Heard, 1978; Scott et al., 1982). Infection of birds occurs with the ingestion of infected molluscs (Schafranski et al., 1975; McLaughlin, 1976, 1986; Galaktionov and Dobrovolskij, 2003), a common food habit known to several species of waterfowls (Lavery, 1970; Middleton and van der Valk, 1987). Information on the molluscs that act as intermediate hosts of T. cucumerinum is scarce and restricted to the experimental life cycle studies carried out several decades ago (Schafranski et al., 1975; Scott et al., 1982). © The Author(s), 2021. Published by In another aspect, recent molecular studies have contributed to the advancement of knowl- Cambridge University Press edge on taxonomy and phylogeny of avian trematodes, including species of the family Cyclocoelidae (Sitko et al., 2017; Gomez-Puerta et al., 2018; López-Jiménez et al., 2018; Galosi et al., 2019;Liet al., 2020; Urabe et al., 2020). Despite this, molecular data are scarce for tracheal parasites belonging to the subfamily Typhlocoelinae, which are limited to sequences of isolates identified as Typhlocoelum sp. and Tracheophilus cymbium (Diesing, Downloaded from https://www.cambridge.org/core. University of Athens, on 01 Oct 2021 at 11:45:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0031182021000986 1384 Jordana C. A. Assis et al. 1850) from North America and Asia, respectively (Tkach et al., with the aid of a D-shaped hand net (50 × 30 cm, 1 mm mesh, 2016;Liet al., 2020). The acquisition of new molecular 20 cm depth bag) and sent to the laboratory, where they were information for members of the Typhlocoelinae, especially washed in running tap water, quantified and placed individually T. cucumerinum, the type species of the genus, is necessary to in 24-well polystyrene plates with approximately 2 mL of water. access taxonomic issues related to the validity of the genera cur- After a routine artificial photostimulation test to detect the emer- rently included in this subfamily. Moreover, such molecular gence of larval trematodes, the gastropods, recognized experimen- data can be useful to confirm the wide geographical distribution tal intermediate hosts of T. cucumerinum (Schafranski et al., 1975; previously reported to the species based on morphological data. Scott et al., 1982), were pressed between glass slides inside a Petri In the current study, a host–parasite–environment system dish and dissected under a stereomicroscope for the search of involving T. cucumerinum was identified in an urban lake from non-emergent larval stages. The taxonomic identification of the Brazil. Coproparasitological analysis was used for the first time molluscs was based on morphological traits according to different for the in vivo diagnosis of the infection in free-living domestic authors (Paraense, 1970, 1994; Paraense and Pointier, 2003). The waterfowl. Moreover, different parasite developmental stages, sampling of molluscs was made under the permission of the including larval forms in molluscs, were subjected to morpho- Brazilian Institute of Environment and Renewable Natural logical and molecular studies. This integrative taxonomic Resources (IBAMA, Biodiversity Authorization and Information approach resulted in insights and advances in taxonomy, System – SISBIO permit number 52870-1). phylogeny and biology of this avian tracheal trematode. Morphological study of developmental stages and taxonomic Materials and methods identification Study area, animals and adult parasites A live adult specimen recovered from C. moschata found dead in the locality was processed for morphological study. This trema- The study area comprises an urban lake located at the tode was placed in a Petri dish containing 0.9% saline to obtain ‘ Administrative Center of the State of Minas Gerais ( Cidade eggs. Then, the trematode was pressed between glass slides, killed ’ Administrativa Presidente Tancredo Neves ), Belo Horizonte with water at 70°C and fixed in 10% formalin. Subsequently, the city, Minas Gerais, Southeastern Brazil (19°47′06.20′′S and 43° ′ ′′ parasite was stained with alum acetocarmine, dehydrated in 57 11.41 W), where a population of about 150 domestic water- ascending ethanol series, diaphanized with beechwood creosote, fowl was maintained at the study time. During the necropsy of and mounted on a permanent slide with Canada balsam. The some specimens that died due to an outbreak of botulism between morphological study of the trematode was performed under a March and April 2016 (Silva et al., 2017), the presence of trema- light microscope. Photographs of the specimen in toto were todes in the trachea of Cairina moschata (L.) was verified for the taken using a Samsung ES 70® digital compact camera coupled first time (Fig. 1A). Based on this finding, a study was proposed to to a Leica EZ4® stereomicroscope. Details of internal structures – – characterize the host parasite environment system present in the were photographed with the aid of a Leica ICC50 HD® digital locality. camera attached to a microscope of the same brand. The captured images were analysed using the software Leica Application Suite® Coproparasitological