Pedicle myiasis by Lucilia caesar (Diptera, Calliphoridae): An emerging disease in roe deer from north‐western Spain

A total of 35 male roe deer (Capreolus capreolus) from Lugo province (north‐western Spain) were examined for pedicle myiasis between 2020 and 2022. All these animals had died by different causes and were examined or preserved just after their death to avoid post‐mortem infestations. After external and internal head inspection, five animals were diagnosed with severe myiasis at the basis of the antlers (14.29%; 95% confidence interval = 5.38–31.04). All the affected bucks presented a cutaneous wound (1.5–15 cm in diameter) around the pedicles with extensive tissue destruction, exposition of frontal and parietal bones, and massive infestation by dipteran larvae (2–12 mm long). Four of five roe deer showed whitish egg clusters adhered to the basis of the antlers. Moreover, in one animal, six larvae had penetrated the cranial cavity, and in two of them larvae were also found in the nasopharyngeal cavity. All the cases were recorded in summer (May–August). Morphological identification and subsequent molecular confirmation revealed that all animals were infested by different larval stages of Lucilia caesar (Diptera: Calliphoridae). This study represents the first report of pedicle myiasis in Spain. Since this disease is reported sporadically, the detection of five cases in a short period of time suggests an increase in the incidence of this myiasis.


INTRODUCTION
Blowflies (Diptera: Calliphoridae) include some of the most economically significant parasites of livestock worldwide, causing facultative myiasis (Lihou & Wall, 2019). Although most calliphorids are secondary flies that follow a pre-existent infestation, a small number of species such as Lucilia cuprina (Wiedemann, 1830), Lucilia sericata (Meigen, 1826) and Lucilia caesar (Linnaeus, 1758) can produce myiasis without previous existing injury (Zumpt, 1965). Blowflies are characterised by a rapid larval feeding stage, mainly in cutaneous infestations, and high pathogenicity (Stevens et al., 2006). The establishment of the first stage larvae (L1) on the dermis is facilitated by the excretion of digestive proteases that are even capable to digest bone sutures (Sandeman et al., 1987).
A wide range of wild and domestic vertebrates may suffer from blowfly myiasis, demonstrating low host specificity. Hosts became infected when gravid female flies, attracted by soiling of the moist wool or hair, wounds, or inflamed skin, lay eggs on a previously existing lesion (Broadmeadow et al., 1984;Sandeman et al., 1985). Almost any type of wound can become infested with calliphorid larvae, including those caused by management practices such as castration, dehorning, branding, shearing, and so forth. Moreover, it has been reported that gravid Wohlfahrtia magnifica (Diptera: Sarcophagidae) flies are strongly attracted to some chemical substances present in sexual fluids (Farkas et al., 1997;Ruiz-Martínez et al., 1987). It has been demonstrated that head-butting during dominance fights between males often results in wounds that can be easily infested by flies (Sotiraki et al., 2003). Consequently, in rams, the head is the second most frequently infested body region after the genitalia (Remesar et al., 2021;Sotiraki et al., 2005). Cervid antlers bleed easily during the velvet stage. Afterwards, a rise of androgen concentrations leads to the loss of the skin covering the antler, which is polished in preparation for the mating season. During both periods, the presence of blood and dead tissue attracts gravid flies that may deposit their eggs on the basis of the antlers. Therefore, newly hatched larvae can invade the tissues around the pedicles.
There is a lack of previous reports of pedicle myiasis in cervids from Spain. In north-western Spain, roe deer (Capreolus capreolus, Linnaeus, 1758) is the most abundant wild ruminant, and their population has significantly increased in size in the last two decades (Markina, 2017). In 2020, a first case of pedicle myiasis was observed in a roe deer from Lugo province (northwest Spain). From this first detection until 2022, roe deer were examined for investigating the incidence of this myiasis and for determining its causes.

Animals and study area
The study was carried out in Lugo province (north-western Spain, 43 0 0 44 00 N, 7 33 0 21 00 W), which is one of the Spanish geographical areas with the highest density of roe deer. This province is characterised by the presence of large areas of woodland, mainly composed of Quercus robur and Castanea sativa (Ortega, 2009) and the climate is temperate oceanic, with mild, rainy winters and warm and relatively sunny summers.
Between 2020 and 2022, a total of 35 roe deer bucks were submitted to the INVESAGA Laboratory at the School of Veterinary Medicine in Lugo as part of a project for studying the prevalence of nasopharyngeal myiasis by Cephenemyia stimulator (Diptera: Oestridae) in roe deer.
All the animals, except one whose head was provided directly by a hunter, were collected by the Natural Heritage Service in a moribund condition or recently dead. The cervids were immediately examined or preserved at À20 C to avoid post-mortem infestations. The age of the animals was determined according to their teeth features (Høye, 2006).

Roe deer head examination
Roe deer heads were thoroughly examined, particularly around the base of the antlers, to find skin wounds, ulcerations and ectoparasites, paying special attention to the presence of dipteran larvae. Moreover, the presence of dipteran eggs on the wounds or antlers was also recorded and a qualitative estimation of the number of eggs, from nil (À) to intense (+++), was established ( Table 1). The nasal and cranial cavities were subsequently opened and examined for assessing the presence of dipteran larvae. All the detected larvae were recovered, counted and stored in 70% ethanol for further identification.

Morphological and molecular identification
Larvae were identified under the stereomicroscope using morphological keys (Zumpt, 1965). Since accurate morphological identification can be difficult in some Calliphoridae species, molecular identification was carried out in a subset of larvae from each animal. DNA was extracted using a commercial kit (High Pure PCR Template Preparation Kit, Roche Diagnostics GmbH ® , Mannheim, Germany) following the manufacturer's instructions. DNA was subjected to two PCR protocols targeting a partial region of the genes encoding for the 28S RNA (Stevens & Wall, 2001) and the c oxidase subunit I (cox1) (Otranto et al., 2000).
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 scanned against the GenBank database using BLAST.

RESULTS
Five cases of pedicle myiasis were detected in 35 roe deer analysed between 2020 and 2022 (14.29%; 95% confidence interval = 5.38-T A B L E 1 Data and main findings in the roe deer bucks diagnosed with pedicle myiasis in Lugo province, NW Spain (2020-2022).  All the examined calliphorid larvae were molecularly identified as L. caesar, a facultative myiasis-causing agent. This species is widely distributed throughout Europe in both natural and synanthropic ecosystems (Szpila et al., 2013); their larvae mainly feed on animal carcasses, but also on living tissues as occurred in this study. Myiasis by L. caesar are mostly reported in captive wildlife (Gao et al., 2021), and the first case in free ranging wildlife was reported in a wild boar from Italy in 2021 (Pezzi et al., 2021). Agreeing with these observations, all the cases described in the present study were detected in summer months (May-August). In addition, Moneo and Bordas (2007) also proved that L. caesar is more abundant in shaded habitats of forest areas, which are very abundant in the studied area.

Lucilia caesar is a common necrophagic species in northern Spain
It should be noted that another emerging myiasis caused by Hypoderma actaeon, considered specific for red deer, has been reported in roe deer from Central Spain in recent years (Panadero et al., 2017). Changes in the pattern of distribution of red deer and roe deer could have favoured the spreading of this myiasis between different host species.
It must be also pointed out that difficulties in tracking wildlife hinder the detection of pedicle myiasis in these animals; thus, most cases go unnoticed, leading to an underestimation of the incidence of this myiasis in free ranging ungulates. According to Obanda et al. (2013), the vulnerability to predation of the infested animals may explain why data on the incidence or epidemiology of cutaneous myiasis in free ranging wildlife are limited. However, in the present study, the collection and inspection of heads as part of a survey to determine the incidence of nasopharyngeal myiasis may have allowed the detection of pedicle myiasis cases that otherwise would have remained undiagnosed.
Physical examination revealed that all the animals presented an open cutaneous wound around the pedicles with deep ulcerations that, in some cases, were not external. In most affected bucks, massive larval infestations with extensive destruction of deep tissues and exposition of the frontal and parietal bones were found. In one animal, larvae had penetrated in the cranial cavity through a cranial suture, causing severe neurological signs such as blindness, deafness and ataxia. Similar lesions were described in four cases of severe pedicle myiasis caused by L. illustris in roe deer from Sweden, including circumferential skin ulcerations at the base of the antlers, exposition of the frontal bone and partial lysis of cranial sutures (Nielsen et al., 2010).
It is worth noting that larvae of L. caesar were also present in the nasal cavity of two bucks co-infected with Cephenemyia stimulator. These findings might indicate that the presence of C. stimulator could have attracted the larvae of L. caesar towards the nasopharyngeal cavity.
Three animals showed emaciation and behavioural alterations that recommended their euthanasia. Since necropsies did not reveal other diseases, it is very probable that this myiasis was the cause of these alterations. Traumatic myiasis in domestic animals causes important health injuries that need veterinary attention. However, in wild or extensively reared animals, with minor or absence of veterinary care, spontaneous recovery is rare and complications with secondary infections may result in septicaemia or toxaemia (Azrolharith et al., 2022).
The severity of the cases described herein indicates that spontaneous recovery is unlikely, so further studies are needed to prevent and manage this myiasis in roe deer.