Case report: First report of potentially zoonotic Gongylonema pulchrum in a free-living roe deer (Capreolus capreolus) in Slovenia

Adult female and male Gongylonema nematodes were found in the oesophagus of a free-living roe deer (Capreolus capreolus) in Slovenia during passive health surveillance of wildlife. The genus Gongylonema was determined by light microscopy based on the genus-specific cuticular bosses in the anterior part of the parasite. Molecular methods were used to confirm the species Gongylonema pulchrum, which has zoonotic potential. Although Gongylonema species are considered common and distributed worldwide, this is the first report of G. pulchrum in an animal on the territory of Slovenia and the first molecular report in a roe deer worldwide. The parasite is likely to be underdiagnosed, misdiagnosed or goes unnoticed as the animals show little or no clinical signs and minor pathological lesions. Slaughterhouse workers, hunters and veterinarians should be aware of this elusive parasite. Examination and evisceration of the upper digestive tract of animals should therefore be carried out more carefully.

The aim of this paper is to report the presence of Gongylonema sp. in the oesophagus of a free-living roe deer (C.capreolus) in Slovenia and its molecular identification as G. pulchrum using PCR and Sanger sequencing of the obtained PCR amplicons.

Case description
In March 2023, the necropsy of a juvenile female roe deer from a hunting ground near the town of Gornji Grad (Lower Styria, Slovenia) was performed at the Veterinary Faculty (Ljubljana, Slovenia) as part of a national passive surveillance programme.The death of the animal followed extensive tissue and organ damage caused by a predator.A detailed parasitological examination of the lungs (for lungworms) and surrounding tissues (heart, oesophagus) unexpectedly revealed serpentine-shape changes in the subserosa of the oesophagus (Figure 1, left).On extraction, the white thread-like worms were 5-12 cm long (Figure 1, right).Under the light microscope, cuticular bosses typical of the genus Gongylonema were observed in the anterior part of the parasites (Figure 2, left).A total of ten females and one male were collected.The male was 5 cm long and had asymmetrical caudal wings with two short, differently sized spicules (Figure 2, right), indicating a juvenile male (8).The females were about 12 cm long and had a pronounced uterus filled with embryonated oval eggs.
After morphological examination of the nematodes, molecular methods were used to determine the species; one female nematode was stored in sterile physiological saline solution at −20°C for subsequent molecular analysis.DNA was extracted from the mid-body section of the parasite using the iHelix kit (Institute of Metagenomic and Microbial Technologies, Slovenia; https://www.ihelix.eu/)according to the manufacturer's instructions.The extraction protocol included bead-beating (45 s at 6400 rpm) three times using a tissue homogenizer (MagNA Lyser Instrument; Roche, Switzerland), combined with enzymatic and heat-induced lysis between mechanical shearings.DNA was eluted to a final volume of 100 μL and stored at −20°C until further analysis.For species determination, PCR and Sanger sequencing were employed, targeting the overlapping segments of the ribosomal RNA (rRNA) genes (rDNA).Twelve universal eukaryotic primer pairs (Supplementary Table S1) were used for PCR amplification as previously described (1,9,31); each primer pair was used in a separate PCR reaction.In brief, 25-μl reaction mixtures contained 2.5 μL of DNA, 0.5 U of Platinum Taq DNA Polymerase (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA), 2.5 mM MgCl 2 and 1× PCR buffer supplied by the manufacturer, 1 μM of each primer, and 0.25 mM of each dNTP (Applied Biosystems by Thermo Fisher Scientific).Amplification was performed in the VeritiPro Thermal Cycler (Applied Biosystems by Thermo Fisher Scientific) according to the following protocol (applied for all PCR reactions/ primer pairs): initial denaturation at 94°C for 3 min, 35 cycles of denaturation at 94°C for 1 min, annealing at 63°C for 1 min, and extension at 72°C for 1 min, and final extension at 72°C for 10 min.The obtained PCR amplicons were analyzed with the QIAxcel capillary electrophoresis system (Qiagen, Germany) using the QIAxcel DNA High Resolution Kit, QX Alignment Marker 15-3,000 bp, QX Size Marker 100-2,500 bp, OM500 separation method and a sample injection time of 10 s according to the manufacturer's instructions.
Ribosomal PCR amplicons (n = 12) were sequenced in both directions (Eurofins Genomics Europe, Germany).The retrieved sequence fragments were imported into Geneious Prime v2022.1.1 (Biomatters, New Zealand) and mapped to a 6,091-bp reference G. pulchrum (GeneBank accession no.AB495389.2) to enable reconstruction of a nearly complete Gongylonema rDNA region containing also the internal transcribed spacers (ITS) 1 and 2; G. pulchrum was selected as suspected according to the origin of the isolate (16).A 6010-bp consensus 18S rDNA -ITS1-5.8SrDNA -ITS2 -28S rDNA sequence was obtained, which was aligned to three (of 22 available >6,000-bp long G. pulchrum sequences of rDNA in GenBank; accessed on 24 April 2024) selected G. pulchrum Serpentine-like structures in the subserosa of the oesophagus of a juvenile female roe deer (left); the scale bar represents 1 cm.From the subserosa, white thread-like worms were collected (right); the male (shorter nematode) was approximately 5 cm and the female (longer nematode) 12 cm long.To confirm the results of molecular identification, all >6,000-bp long rDNA sequences of the genus Gongylonema were retrieved from GenBank (accessed on 1 July 2024); a total of 31 rDNA sequences of G. pulchrum (n = 22 from Japan, China, Iran and Slovenia; the Gongylonema isolate from Slovenia was the only one of human origin), G. nepalensis (n = 4 from Nepal and Italy), G. neoplasticum (n = 2 from Japan) and G. aegypti (n = 3 from Egypt) were obtained.The sequences were complemented with the rDNA of G. pulchrum obtained in the present study and the phylogenetic tree was constructed in MEGA11 (32) (Figure 3); the maximum likelihood method and Tamura-Nei model were used with default parameters (33).The 6,705-bp long rDNA sequence of Stegophorus macronectes (HE793715.1),belonging to the same order (Rhabditida) and suborder (Spirurina) as Gongylonema spp., was used as an outgroup to root the tree.A clear clustering according to Gongylonema species was observed, but no sub-species clustering, indicating a high genetic similarity of G. pulchrum and the correct identification of the roe deer isolate as G. pulchrum.

Discussion
This is the first molecular report of G. pulchrum in a roe deer and the first report of a Gongylonema nematode found in an animal in Slovenia.The report also complements the recent human case of G. pulchrum reported from Slovenia, which was thought to be an autochthonous infestation (22).The molecular protocols and analyses are presented in detail to facilitate further use in diagnostic laboratories, as many warm-blooded animals are infested with Gongylonema nematodes, which are also potential zoonotic agents (1)(2)(3)(4)(5)(6).
Gongylonema infestation in the oesophagus was discovered at necropsy when a roe deer was found dead in the wild after attack by a predator and examined as part of a national passive health surveillance programme of wildlife in Slovenia.The roe deer population in Slovenia is estimated at around 110,000 animals, with a hunting quota of around 30,000-35,000 animals per year (20,34).Roe deer are considered the most widespread species of free-living wild ruminants and an important source of game meat in Slovenia (20).In twenty years of passive health surveillance of roe deer, a mortality rate of 26% was recorded for parasitic diseases.In addition to ectoparasites, endoparasites such as Haemonchus contortus, Chabertia ovina and lung parasites (Protostrongylidae, Dictyocaulus viviparus) were also detected during the post-mortem examination (35).Until now, not a single Gongylonema sp. has been found.The potential infestations in domestic animals and wildlife in Slovenia should be documented as there are no current prevalence reports.
Based on the morphology and the origin of the isolate, G. pulchrum was suspected (16).The species was confirmed by sequencing, as the reconstructed 18S rDNA -ITS1-5.8SrDNA -ITS2 -28S rDNA region was most similar to the rDNA region of G. pulchrum.Apart from the isolate obtained in the present study, there are no other Gongylonema isolates and corresponding sequences available from ungulates inhabiting Slovenia.Only one G. pulchrum rDNA was deposited from our country in 2019 (GenBank accession no.LR215834.1),but it was obtained as part of a report on G. pulchrum infestation in a human case (22).In addition, not many studies have generated Gongylonema rDNA sequences longer than 6,000 bp, and only two of these sequences (but not belonging to G. pulchrum) are from a neighboring country, namely the rDNA of G. nepalensis from Italy (GenBank accession nos.LC388743.1 and LC278392.1).The phylogenetic comparison of the rDNA sequences of G. pulchrum isolates obtained in Slovenia showed a high genetic similarity between the two sequences, but the same was true for all compared G. pulchrum sequences from four geographically distant countries.More sequences (partial rDNA) of Gongylonema spp.are available in GenBank, but most of them are shorter and therefore contain much less phylogenetic information (no additional discriminatory power).The high withinspecies similarity of Gongylonema rDNA was also previously described when it was reported that the nucleotide sequences of G. pulchrum rDNA were generally well conserved regardless of their host origin (9).We could achieve somewhat greater discriminatory power, if we sequenced the cytochrome c oxidase subunit I (COI) region of mitochondrial DNA (4,9); the COI sequences of G. pulchrum can be further subdivided into several haplotypes (9).
The first and only human case of G. pulchrum in Slovenia was self-diagnosed in 2015 and it was later molecularly identified and reported (22).The infestation was described as autochthonous and was most likely due to ingestion of food or water from natural sources thought to be contaminated with the nematode intermediate hosts, dung beetles and cockroaches (26); it was reported that the patient was drinking water from several local springs in the south-eastern part of Slovenia, where there are many grazing areas for livestock (22).Xiaodan et al. ( 25) reported that the parasite can be overlooked in a patient for more than ten years after infestation.It can also be misdiagnosed as candidiasis, burning mouth syndrome or even a delusional parasitic infestation, as patients report strange crawling sensations in the upper digestive tract (2,22,26,27,36,37).The parasite can also contribute to the development of squamous cell carcinoma (28, 29), which can have serious health implications.Therefore, more attention should be paid to Gongylonema species, especially G. pulchrum with a proven zoonotic potential.
Until the 1980s, Gongylonema sp. was frequently reported, with prevalence in domestic ruminants reaching, e.g., 49.7% (276/555) in Iran or up to 96.0% in some regions in Turkey (8,21).In free-living wild ruminants, a prevalence of 42.8% was reported in 1959 in roe deer from Romania (11,12) and recently a prevalence of 18.8% (25/133) in European fallow deer (D. dama) from Romania (12).In 2013, researchers from Japan reported varying prevalences (from no infestation to a 100% prevalence, depending on sampling location) in sika deer (C.nippon) (9).However, over the years, the prevalence in domestic ruminants in the same countries has decreased to, e.g., 4.6% (16/350; Iran) or 0% (0/848, Turkey) in sheep (8, 10) and 16.2% (96/680, Iran), 5.3% (34/638, Japan) or 0.5% (2/380, Turkey) in cattle (7)(8)(9).This decrease in prevalence has been attributed to the decline in grazing, the increased use of commercial feeds and the regular use of anthelmintics (8).In Slovenia, Gongylonema sp. is mentioned in veterinary parasitology textbooks as a common parasite in the oesophagus of ruminants (30).To our knowledge, there are no published data or reports indicating the prevalence of the parasite.The parasite may be under-reported or under-diagnosed as the clinical signs in animals are usually non-specific, mild and without obvious pathological changes at the site of infestation (7,10,25).In this study, the parasite would probably not have been discovered, if the animal had not been attacked by a predator and collected dead by the hunters.
The occurrence of Gongylonema sp. in roe deer prompts us to investigate the potential number of cases that may have been overlooked in domestic/captive and wild/free-living animals.This reminds us of the importance of passive health surveillance in wildlife.As regular monitoring activities are associated with high numbers of animals, passive health surveillance of wildlife is particularly important to detect diseases that might otherwise go unnoticed.Slaughterhouse staff, hunters and veterinarians should be educated Maximum likelihood phylogenetic tree of >6,000-bp long Gongylonema rDNA sequences.GenBank accession numbers are listed in addition to Gongylonema species and country of origin.In total, 32 nucleotide sequences of Gongylonema pulchrum (n = 22 and one from the present study), Gongylonema nepalensis (n = 4), Gongylonema neoplasticum (n = 2) and Gongylonema aegypti (n = 3) were included.The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.Stegophorus macronectes rDNA (GenBank accession no.HE793715.1)was used as an outgroup to root the tree.about this elusive parasite and be vigilant during evisceration or postmortem examinations.Further studies are essential to reassess the prevalence of Gongylonema species in domestic and wild ruminants in Europe and their zoonotic impact.

FIGURE 2
FIGURE 2 Cuticular bosses (CB) on the anterior part of the nematode, typical for genus Gongylonema sp.(left), and posterior part of the male Gongylonema pulchrum with caudal wings (CW) and two short spicules (S) of different sizes (right) at 100× magnification.The scale bar represents 100 μm.