Presence of one ecto- and two endoparasite species of the black stork (Ciconia nigra) in Portugal

Background The black stork (Ciconia nigra Linnaeus, 1758) is a recognized endangered species in Europe and most of the specimens from the Western Palearctic region breed in the Iberian Peninsula. Available works regarding parasites in black storks are scarce. This work reports the presence one ecto- and two endoparasite species from a black stork in Portugal. Case presentation A black stork was found in southern Portugal after colliding against electric cables. The specimen did not survive its sustained injuries and a post-mortem exam was performed. During the procedure, several ecto- and endoparasite specimens were found. The collected parasites were lice (Neophilopterus tricolor), nematodes (Desportesius sagittatus) and trematodes (Cathaemasia hians). Conclusions Three different species of parasites are reported from a black stork in Portugal. Ecto- and endoparasites of C. nigra have not frequently been described in the literature, and this case report is a contribution to the field. Additional studies will be important to better understand the impact that parasites can have on C. nigra health and survival.

contact should be avoided in order to not disturb them in their natural habitat.
This report describes three different parasite species found in a black stork from Portugal.

Case presentation
An adult female black stork was found near Alqueva dam (38°11'51''N, 7°29'47''W), within the boundaries of the districts of Beja and Évora, southern Portugal, in March 2018, after colliding against electric cables. The specimen was received at the Wild Animal Rehabilitation Centre of Lisbon (LxCRAS) with several wounds, including an extensive hematoma in the pectoralis muscles, in the cranial zone of the keel and in the triceps muscle. After 3 days of treatment, the bird died due to the injuries and the corpse was sent to the Pathology Service of the Faculty of Veterinary Medicine of the University of Lisbon for post-mortem examination. During the procedure, trematodes that were present in the bird's mouth showed photophobic behaviour, moving back to the oesophagus when the beak was opened. Ectoparasites were collected in 70% ethanol and later placed on slides with lactophenol or Canada balsam [15] and observed under light microscopy. Collected endoparasites were placed in a Petri dish containing saline solution before microscopic examination.
Adult stages of three different parasites were found: lice on breast and belly feathers (n = 26), nematodes in the gizzard (n = 2) and trematodes in the oesophagus (n = 27). For parasite species' identification, several references were used [9,10,12,16]. Parasites measurements can be observed in Tables 1, 2 and 3.
After preparation of lice by the Canada balsam technique, they were identified as Neophilopterus tricolor (Burmeister, 1838) ( Fig. 1a and b), due to the presence of a fifth shorter marginal temporal seta when compared with the size of the other four setae. The performed measurements (Table 1) were in agreement with those registered for this species [10].
Following endoparasite observation, nematodes were recognized as Desportesius sagittatus (Rudolphi, 1809) ( Fig. 2a and b). Desportesius sagittatus can be identified by length and morphology of the male right spicule and the greatly reduced deirids [16]. Since only two female specimens were found, the identification was made by this species' type host (i.e. C. nigra), the presence of reduced deirids and the performed measurements (Table 2), which matched those previously observed for this species [16,17].
Trematodes were identified as Cathaemasia hians (Rudolphi, 1809) ( Fig. 3a and b). The observed specimens had flattened body, presence of scales on the ventral cuticle and the head crown was lacking (Cathaemasiidae family). The obtained measurements were in agreement with those observed for this species (Table 3). Eggs (n = 5) had a mean length of 98.3 µm and an average width of 52.8 µm. These findings, together with the host and their localization, allowed species identification [9,12,13,18,19].

Discussion and conclusions
In the present work, three different parasites of C. nigra were observed during necropsy of an adult specimen. The genus Neophilopterus (Ischnocera: Philopteridae) has already been described in black storks from Spain [10]. Ischnocera lice can affect host thermoregulation and induce feather breakage, reducing host fitness through the energetic consequences of that damage. Stress is also an indirect effect to the affected host [10]. Lice specimens found in this report's black stork had four marginal temporal setae of approximately the same length but the last one was several times shorter, thus being identified as Neophilopterus tricolor. This is the first reference of this species in a black stork in Portugal.
In what refers to nematodes, until the year 2015, only Austria, Czech Republic, Germany, Poland and Slovakia had information regarding helminth communities in storks [13]. The genus Desportesius (Nematoda: Acuarioidea) usually parasitizes birds of the order Ciconiiformes [17]. These nematodes occur under the gizzard lining [16], but nothing is known about their pathogenic effects on the hosts [14]. Desportesius sagittatus has also been reported in C. nigra [13]. However, this is the first reference of this species in a black stork caught in mainland Portugal.
Considering trematodes, Cathaemasia hians (Trematoda: Cathaemasiidae) is a well-known species which has members of family Ciconiidae as definitive hosts and are generally found in the oral cavity and sometimes in the oesophagus of these birds. However, reports of this species in Europe have been rare [12]. Black storks are more likely to be infected with this parasite than white storks (Ciconia ciconia), due to their different feeding behaviour [18]. In fact, white storks feed on arthropods and earthworms from permanent dry pastures [20,21], and black storks ingest amphibians and fish parasitized with metacercariae of Cathaemasia hians, which live in swamps and slowflowing waters [12,18]. Cathaemasia hians is generally considered as non-pathogenic to storks [12,13]. Nevertheless, this parasite can lead to irreversible alterations in the digestive tract of definitive hosts, lowering the birds' fitness. Massive infections by this parasite can cause serious health problems when combined with cachexia or lower immunity of the hosts [9,13]. This is also the first reference for this parasite in a black stork captured in Portugal.
Scientific studies regarding these birds are difficult to accomplish since they are hard to access and protected by European laws. Thus, living animals can only be found after sustaining serious injuries [9,12,18], like in the present study.
Since parasitological works in C. nigra are scarce, this work is considered as an important contribution to this study field, reporting three parasite species in a black stork caught in mainland Portugal.