Cephenemyia stimulator (Diptera: Oestridae) myiasis in a roe deer (Capreolus capreolus) from Portugal

Veterinary Parasitology: Regional Studies and Reports 41 (2023) 100883 Available online 24 April 2023 2405-9390/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Case Report Cephenemyia stimulator (Diptera: Oestridae) myiasis in a roe deer (Capreolus capreolus) from Portugal N´ estor Martínez-Calabuig a , Madalena Vieira-Pinto b , c , d , Ceferino M. L´ opez a , * , Susana Remesar a , Rosario Panadero a a INVESAGA Group, Department of Animal Pathology, Faculty of Veterinary, University Santiago de Compostela, 27002 Lugo, Spain b Veterinary and Animal Research Centre (CECAV), Universidade de Tr´ as-os-Montes e Alto Douro (UTAD), Vila Real, Portugal c Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS), Portugal d Department of Veterinary Science, UTAD, Portugal ARTICLE INFO Keywords: Cephenemyia stimulator Bot fly Roe deer Portugal ABSTRACT Herein we present a nasopharyngeal myiasis case by Cephenemyia stimulator in a roe deer hunted in Tr´ as-osMontes (NE Portugal). Preliminary inspection showed one larva sorting through the nostrils and the examination of the nasopharyngeal cavity showed more than 15 larvae in the glottis and retropharyngeal recesses. Four larvae were collected and stored into 70% ethanol for morphological and molecular identification. Three of the larvae were identified as third instars and the other one as a prepupa of Cephenemyia stimulator, being the first confirmation of this species in roe deer from Portugal. C. stimulator is currently widely distributed in roe deer from central and northern Spain so, the transboundary natural dispersal of these cervids would explain the introduction of this myiasis in Portugal. Further studies are needed to monitor the spreading of this infection in the westernmost populations of European roe deer. 1. Introduction The roe deer (Capreolus capreolus) is one of the most abundant and widespread cervids in Europe. Its distribution ranges from Scandinavia to the Iberian Peninsula as its southwestern distribution limit (Apollonio et al., 2010). At the beginning of the 20th century, overhunting along with a substantial loss of forested areas caused by agricultural and pastoral activities, lead to the disappearance of this ungulate from almost all the Portuguese territory, with the exception of a few patches to the north of the Douro River. However, roe deer population size and density increased since the 70s, mainly due to a stricter hunting legislation and the reintroduction of individuals translocated from France and Spain for hunting purposes and also to increase prey availability for the endangered Iberian wolf (Torres et al., 2015). Natural colonization from nearby populations in north-western Spain was also recorded (Card˜ ao, 2009). Cephenemyiosis is an obligatory myiasis developing in the nasal and pharyngeal cavities of roe deer caused by Cephenemyia stimulator (Diptera: Oestridae) whose larvae are biontophagous, i.e., they feed on the living tissue of the host, producing inflammation of the nasal mucosa and an enlargement of the retropharyngeal recesses (Morrondo et al., 2021). In healthy animals, the presence of Cephenemyia spp. larvae rarely causes harm (McMahon, 1989). C. stimulator is widely spread in the range of distribution of roe deer in Europe. In Spain, located at the southern range margin of distribution of this ungulate, the first case of cephenemyiosis in roe deer was reported in 2001 in an animal imported from France in 1997 to a fenced hunting area in Castilla-La Mancha (central Spain) and the first autochthonous infestation was observed in a roe deer from northwestern Spain in 2005 (Pajares, 2016). Nowadays, this myiasis is widely distributed in north and central Spain with an average prevalence and intensity of 62.2% and 41.2 ±52.7 larvae per animal, respectively (Martínez-Calabuig et al., 2022). The aim of this study was to present the first case of a nasopharyngeal myiasis by C. stimulator in Portugal and to discuss the possible origin of the introduction of this myiasis into the country. 2. Case presentation In June 2021 a senior roe buck was hunted in the intersection * Corresponding author. E-mail address: [email protected] (C.M. L´ opez). Contents lists available at ScienceDirect Veterinary Parasitology: Regional Studies and Reports journal homepage: www.elsevier.com/locate/vprsr https://doi.org/10.1016/j.vprsr.2023.100883 Received 12 February 2023; Received in revised form 15 April 2023; Accepted 21 April 2023 Veterinary Parasitology: Regional Studies and Reports 41 (2023) 100883 2 between the districts of Bragança, Macedo de Cavaleiros and Vinhais (Tr´ as-os-Montes, NE Portugal). The hunter who killed the animal referred that it displayed a poor body condition and a nervous behavior, snorting and running for no apparent reason. Field preliminary examination of the animal showed one larva sorting through the nostrils and the examination of the nasopharyngeal cavity revealed more than 15 larvae in the glottis and retropharyngeal recesses. Most larvae were free on the mucosal surface and, only a few were firmly attached with their oral hooks. The larvae oriented their anterior end to the bottom of the pouches and the posterior spiracles towards the opening of the recesses. Apart from an enlargement of retropharyngeal recesses, no macroscopic lesions were observed around the larvae. Four larvae were collected and stored into 70% ethanol and submitted to the INVESAGA group for morphological and molecular identification. Furthermore, this hunter (personal communication) reported another two cases of nasopharyngeal myiasis in the area, but no larvae were recovered so the infestation with Cephenemyia could not be confirmed. Morphological identification for species and developmental stage was performed according to the keys of Zumpt (1965) and Quintela (2021), respectively. All the four recovered specimens were morphologically identified as Cephenemyia stimulator, according to the the distribution pattern of the cuticular spines (Fig. 1a), the reniform shape of the posterior spiracular plates (Fig. 1b), and the distance between the antennal lobes at the base of the antenna (Fig. 1c). Moreover, three of the larvae (24–26 mm long), showing yellowish to dark brown cuticle and dark pigmented peritremes, were classified as third instars and, the fourth larvae (22 mm long) with black cuticle was classified as a prepupa (Fig. 1a). To confirm larval morphological identification, DNA was extracted from three larvae using a commercial kit (High Pure PCR Template Preparation Kit, Roche Diagnostics GmbH®, Mannheim, Germany) following the manufacturer's instructions. Oestridae DNA was detected by two PCR assays targeting the COX1 partial gene (Otranto et al., 2000) and the D2 fragment of the 28S RNA (Stevens and Wall, 2001). Amplicons of the expected size were purified and sequenced on an ABI 3730xl sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were aligned and edited using ChromasPro (Technelysium, Brisbane, Australia) and consensus sequences were then scanned against the GenBank database using BLAST. Molecular identification confirmed the identification of C. stimulator. Unique sequences identified in this study were deposited in GenBank under accession numbers OQ791433OQ791434 for COI and OQ793045 for 28S. 3. Discussion Nasopharyngeal myiasis of roe deer is mainly the result of infection with larvae of bot flies of the genus Cephenemyia. All larval stages live on the surface of the host upper airways and, their bodies are covered with spines that, together with the oral hooks, serve to attach to the mucosa of the host. Affected animals manifest their discomfort through sneezing, coughing and sudden movements of the head trying to get rid of the larvae (Morrondo et al., 2021). Early larval stages of Cephenemyia spp. develops in the nasal mucosa of the host where they can enter in a phase of diapause in the winter months, whereas later stages invade the retropharyngeal recesses that distend from minute depressions of the pharyngeal wall into “pouches” or diverticula that can harbor up to 30 larvae (Cogley, 1987; Pajares, 2016). In this animal, all the larvae were finishing their development in the pharynx which coincides with the chronobiology described for this myiasis in Spain (Martínez-Calabuig et al., 2022) and other European countries (Kir´ aly and Egri, 2007). Despite the large size reached by the larvae, severe disease or death is unlikely because of the lack of involvement of surrounding tissues and Fig. 1. (a) Third instar larva (yellow) and a prepupa (black) of Cephenemyia stimulator found in a roe deer hunted in Portugal. Note the pattern of distribution of the cuticular spines in the third instar (b). Posterior end of a third instar larva of C. stimulator. Note the reniform shape of the posterior spiracular plates. (c) Anterior end of a third instar larva of C. stimulator showing the typical V-shape disposition of the antennal lobes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) N. Martínez-Calabuig et al. Veterinary Parasitology: Regional Studies and Reports 41 (2023) 100883 3 the ability of the nasopharynx to accommodate to greatly expanded retropharyngeal recesses (Cogley, 1987). In this study, all the four recovered specimens were identified as Cephenemyia stimulator, being the first citation of this species in Portugal. Through the study of the COX1 partial gene all the obtained sequences were identical to each other and to the deposited sequences MN212908.1-MN212913.1; MN212915.1-MN212919.1 and MN212921.1 to MN212924.1 of C. stimulator obtained in roe deer from Spain (De la Fuente et al., 2021). These sequences also had a percentage of identity between 99.67% to 99.83% with the sequences of C. stimulator AF497768.1; NC_059850; MN212914.1; MN212915.1; MN212920.1; MN212925.1-MN212928.1 and MN212930.1 obtained in roe deer from France and Spain (Otranto et al., 2003; Aleix-Mata et al., 2021; De la Fuente et al., 2021). In addition, a 97.72% identity was observed with the deposited sequence AF497769.1 of Cephenemyia trompe obtained in a reindeer from Norway (Otranto et al., 2003). The sequences obtained through the study of the 28S RNA partial gene were identical to the reference sequences MN203990.1; MN203995.1 and MN203999.1 and had a percentage of identity between 99.16% to 99.72% with the sequences MN204000.1; MN204003.1-MN204006.1 and MN2040010.1-MN204011.1 of C. stimulator obtained in roe deer from Spain (De la Fuente et al., 2021). The percentage of identity observed with other Cephenemyia spp. deposited sequences such as C. jellisoni (KP954359.1; Winkler et al., 2015) or C. auribarbis (MN204016.1; De la Fuente et al., 2021) was lower than 95%. Although roe deer specimens from France were reintroduced in central and south Portugal in the 1990s and early 2000s (Torres et al., 2015), C. stimulator infestations had never been reported in this country. The last documented reintroductions were performed in 2011 in Barrancos (South Portugal) and in 2013 in Arada mountain (Northwestern Portugal) with neighboring Spanish animals from C´ adiz and Le´ on, respectively. However, the place where the roe deer was hunted is only 30 km from the northwestern Spanish border where this myiasis is known to be widely distributed (Martínez-Calabuig et al., 2022). The natural expansion from Spanish roe deer populations to Portugal has been documented (Card˜ ao, 2009). Therefore, transboundary spreading of roe deer populations is the most probable hypothesis for explaining the introduction of this myiasis to Portugal. Further studies are needed to monitor the evolution of this infection in the westernmost populations of European roe deer. A desirable approach would be necessary to involve hunters and game keepers in scientific research activities, to which they can and should actively contribute with their intellectual effort, their knowledge, or their tools and resources. Declaration of Competing Interest All the authors declared no conflicts of interest with respect to the research, authorship, and/or publication of this article. Acknowledgements The authors are very grateful to Jorge Afonso (Carramo Safaris, Macedo de Cavaleiros, Portugal), the hunter who submitted the larvae for investigation. This work was supported by the projects UIDB7CVT700772/2020 and LA/P/0059/2020 funded by the Portuguese Foundation for Science and Technology (FCT) and by the Program for consolidating and structuring competitive research groups (ED4312019/04, Xunta de Galicia, Spain). References Aleix-Mata, G., L´ opez-Beceiro, A.M., Fidalgo, L.E., Per´ ez, J.M., Sanch´ ez, A., 2021. The complete mitochondrial genome of Cephenemyia stimulator (Diptera: Oestridae). Mitochondrial. DNA B Res. 6 (10), 2941–2942. Apollonio, M., Andersen, R., Putman, R.J., 2010. European Ungulates and their Management in the 21st Century. Cambridge University Press, New York. Card˜ ao, D., 2009. Distribuiç˜ ao e estatuto do veado e corço em portugal. Universidade de Aveiro, Departamento de Biologia. Cogley, T.P., 1987. Effects of Cephenemyia spp (Diptera: Oestridae) on the nasopharynx of black-tailed deer (Odocoileus hemionus columbianus). J. Wildl. Dis. 23 (4), 596–605. De la Fuente, A.M., Caparr´ os, N., Mora-Rodríguez, J.M., Molina, M., Aleix-Mata, G., Velarde, R., Fidalgo, L.E., L´ opez-Beceiro, A.M., Lorite, P., Boos, M., Faure, E., P´ erez, J.M., S´ anchez, A., 2021. Characterization of new molecular markers of three botflies parasitizing cervid hosts. J. Med. Entomol. 58 (3), 1463–1469. Kir´ aly, I., Egri, B., 2007. Epidemiological characteristics of Cephenemyia stimulator. (Clark, 1815) larval infestation in european roe deer (Capreolus capreolus) in Hungary. Acta Zool. Acad. Sci. Hung. 53 (3), 271–279. Martínez-Calabuig, N., Panadero, R., L´ opez, C.M., Díaz, P., Remesar, S., García Dios, D., Salda˜ na, A., L´ opez-Lorenzo, G., Díez-Ba˜ nos, P., Morrondo, P., 2022. Distribuci´ on y ciclo biol´ ogico de Cephenemyia stimulator (Diptera: Oestridae) en corzos del norte de Espa˜ na. In: XXII Congreso de la Sociedad Espa˜ nola de Parasitología. Madrid, 5–8 July 2022. McMahon, D.C., 1989. Bot fly larvae (Cephenemyia spp., Oestridae) in mule deer (Odocoileus hemionus) from Utah. J. Wildl. Dis. 25 (4), 636–638. Morrondo, P., Pajares, G., Arias, M.S., Martínez-Calabuig, N., Remesar, S., GarcíaDios, D., Díaz, P., L´ opez, C.M., Panadero, R., Díez-Ba˜ nos, P., 2021. An update on Cephenemyiosis in the European roe deer: emergent Myiasis in Spain. Animals 26;11 (12), 3382. Otranto, D., Tarsitano, E., Giangaspero, A., Puccini, V., 2000. Differentiation by polymerase chain reaction–restriction fragment length polymorphism of some Oestridae larvae causing myiasis. Vet. Parasitol. 90, 305–313. Otranto, D., Traversa, D., Guida, B., Tarsitano, E., Fiorente, P., Stevens, J.R., 2003. Molecular characterization of the mitochondrial cytochrome oxidase I gene of Oestridae species causing obligate myiasis. Med. Vet. Entomol. 17, 307–315. Pajares, G., 2016. Estudio sobre la infestaci´ on por larvas de Cephenemyia stimulator (Diptera: Oestridae) en corzos (Capreolus capreolus) del norte de Espa˜ na. PhD Dissertation,. Santiago de Compostela University, p. 143. Quintela, L., 2021. Estudio morfol´ ogico de las larvas nasofaríngeas (Diptera: Oestridae) encontradas en corzos de la provincia de Lugo. DMV Dissertation,. Santiago de Compostela University. Stevens, J., Wall, R., 2001. Genetic relationships between blowflies (Calliphoridae) of forensic importance. Forensic Sci. Int. 120 (1–2), 116–123. Torres, T.R., Miranda, J., Carvalho, J., Fonseca, C., 2015. Expansion and current status of roe deer (Capreolus capreolus) at the edge of its distribution in Portugal. Ann. Zool. Fenn. 52 (5–6), 339–352. Winkler, I.S., Blaschke, J.D., Davis, D.J., Stireman, J.O., O’Hara, J.E., Cerretti, P., Moulton, J.K., 2015. Explosive radiation or uninformative genes? Origin and early diversification of tachinid flies (Diptera: Tachinidae). Mol. Phylogenet. Evol. 88, 38–54. Zumpt, F., 1965. Myiasis in Man and Animals in the Old World. Butterworths, London. N. Martínez-Calabuig et al.