First Detection of Cryptosporidium parvum in the Endemic Cyprus Mouflon (Ovis gmelini ophion)

Purpose Cryptosporidium is an intestinal zoonotic protozoan parasite that infects domesticated and wild animals. There are no reports on the prevalence and molecular characterisation of Cryptosporidium in the endemic Cyprus mouflon. The mouflon is strictly protected by national and international legislation. Its main distribution is Paphos State Forest and surrounding areas, where it may share the same water sources as free-ranging domestic goats. Therefore, the present study aimed to determine the prevalence of Cryptosporidium spp. and genotypes in mouflon and free-ranging goats within the mouflon range. Methods Faecal samples of 70 mouflons and 34 free-ranging goats were screened for Cryptosporidium by PCR amplification and sequencing. Results Only one sample (1/70) belonging to a mouflon was PCR positive for Cryptosporidium. Based on sequencing of the 18S rRNA locus, this species was identified as Cryptosporidium parvum (C. parvum). No positive sample was detected in the free-ranging goats (0/34). Conclusion This is the first report on the molecular identification of this Cryptosporidium species in a Cyprus mouflon. The results indicate that the prevalence of Cryptosporidium in Cyprus mouflon is low.

Identification of Cryptosporidium oocysts in animal stool samples can be performed using microscopic examination of the samples at 1000 × magnification after staining of the oocysts using the acid-fast Ziehl-Neelsen technique [25]; however, most Cryptosporidium species have morphologically identical oocysts; thus, they cannot be differentiated by microscopic methods.Instead, they can be differentiated by molecular biological techniques [26,27].Nested PCR amplification of the 18S rRNA gene using specific primers is a more sensitive method for the identification of Cryptosporidium in stools compared to microscopic examination [28][29][30].Nested PCR is more sensitive than the classic PCR and the PCR primers were designed to detect all Cryptosporidium species [20,26,28].
The Cyprus mouflon (Ovis gmelini ophion) is the largest wild terrestrial mammal on the island.It is endemic to Cyprus and classified as endangered and strictly protected [31].Its population is estimated at 2500-3000 animals, mainly located in the Paphos State Forest and surrounding areas.Low genetic variability [32] puts the population at significant risk of an illness outbreak transmitted from sympatric livestock such as free-ranging goats that frequent the same areas and share water sources [33].In a recent study, endoparasites were found in 97% of Cyprus mouflon faecal samples, including the lungworms Muellerius capillaris and Cystocaulus ocreatus strongylid eggs, Eimeria spp., Nematodirus spp., Trichuris spp., and Moniezia spp.[34].
The purpose of the present study was to determine the prevalence and molecular identification of Cryptosporidium species in the Cyprus mouflon and free-ranging domestic goats for the first time in Cyprus.

Study Area
Paphos State Forest is situated in the Northwest part of the Troodos Mountain range.It is a mainly coniferous forest of 600 km 2 and constitutes the most extensive and continuous forest ecosystem in Cyprus, ranging from 200 m up to the peak of Tripylos at 1352 m [35].The predominant vegetation is the Calabrian pine (Pinus brutia).Among other trees and shrubs, the area also includes the unique endemic Cyprus cedar (Cedrus brevifolia), as well as the endemic golden oak (Quercus alnifolia), along with the occasional strawberry tree (Arbutus andrachne), turpentine tree (Pistacia terebinthus) and sumac (Rhus coriaria) [36].Various flocks of livestock, mainly goats and sheep, utilise the forest and surrounding areas and mingle with the Cyprus mouflon population.

Sample Collection
Faecal samples from 70 Cyprus mouflon and 34 free-ranging goats were collected from the ground in Paphos State Forest (Cyprus) between November 2021 and August 2022.Sampling was carried out along mouflon trails, bedding areas, and near water points, including where livestock are regularly seen.Mouflon faecal material pellets differ from goat pellets, since the mouflon is a wild sheep versus free-ranging goats, and were identified in this study by experienced rangers.Each fresh faecal sample (4-10 g each) was placed in a sterile polystyrene tube (50-ml centrifuge tube) with records of the date, location, and identification number.The samples were transferred to the laboratory on the same day and were stored at 4 °C until DNA extraction within 3 weeks after collection.

DNA Extraction and PCR
The total genomic DNA of each faecal sample was extracted using a QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.Cryptosporidium species were determined by nested Polymerase Chain Reaction (PCR) amplification of an 18S rRNA locus fragment (600 bp) using previously described primers [20].The primers used for the first amplification were SHP1 (forward) 5ʹACC TAT CAG CTT TAG ACG GTA GGG TAT 3ʹ and SHP2 (reverse) 5ʹ TTC TCA TAA GGT GCT GAA GGA GTA AGG 3ʹ.The primers used for the second amplification were SHP3 (forward) 5ʹ ACA GGG AGG TAG TGA CAA GAA ATA ACA 3ʹ and SSU-R3 (reverse) 5ʹ AAG GAG TAA GGA ACA ACC TCC A 3ʹ.The cycling conditions used in both amplifications were 94 °C for 3 min, 35 cycles of 94 °C for 45 s, 56 °C for 45 s and 72 °C for 60 s, followed by a final extension of 72 °C for 7 min.

Sequencing and Phylogenetic Analysis
The DNA band of the positive PCR product was extracted from an agarose gel and purified using the Blirt ExtractMe DNA Kit (Blirt, Gdansk, Poland).The purified PCR product was sent for sequencing (using the forward primer of the nested PCR reaction) to Macrogen Ltd Europe, Amsterdam.For the determination of Cryptosporidium species, the sequence was subjected to BLAST (https:// blast.ncbi.nlm.nih.gov/ Blast) searches at NCBI GenBank.The sequence was deposited in the NCBI GenBank under the accession number: OQ287831.Using the Neighbour-Joining method in MEGA 11 software, a phylogenetic tree was constructed with Bootstrap 1000 replicates.Evolutionary distances were calculated using the Kimura two-parameter model (https:// www.megas oftwa re.net/) [37].

Results
Only one Cyprus mouflon sample (1/70 = 1.4%) was PCR positive for Cryptosporidium and was further identified by DNA sequencing and Blastn analysis as C. parvum.No positive sample was detected in the free-ranging goats (0/34).
The phylogenetic analysis using the Mega11 software indicated that the 18S rRNA sequence of the C. parvum isolated in this study (solid circle in Fig. 1) formed a welldefined cluster with the respective C. parvum reference sequences, regardless of whether these sequences were isolated from animals in Cyprus (solid squares in Fig. 1) or from other countries (Fig. 1).

Discussion
The present study is the first to identify Cryptosporidium species in the endemic Cyprus mouflon.Two other Cryptosporidium species, C. ubiquitum (previously Cryptosporidium cervine genotype) and C. muris have been found in the European mouflon (Ovis gmelini musimon) in the Czech Republic [38,39].The presence of C. parvum in the current study was low (1.4%), similar to C. muris (1/43 = 2.3%) found in faecal samples of wild European mouflon [39].
Due to the high genetic relationship between mouflon and sheep, C. parvum may cause severe symptoms in neonatal and young mouflon.In sheep, young infected animals are more likely to have clinical symptoms, while most of the infected adult animals are asymptomatic but still shed oocysts of the parasite [40].The clinical signs of cryptosporidiosis include watery diarrhoea, dehydration, significant weight loss, reduced growth rate and sometimes even death.Most symptoms usually last 1-2 weeks after infection [40].In a wild population of the Sardinian mouflon (Ovis gmelini musimon × Ovis sp.), reproductive females and young mouflon in poor body condition were found to be the main spreaders of gastrointestinal parasites [41].Therefore, the prevalence of C. parvum in relation to age and gender merits further study in the Cyprus mouflon and will provide vital information for its conservation management.
Data on the epidemiology of Cryptosporidium in Cyprus is limited.Hoque et al. [42] reported high occurrence of zoonotic subtypes of C. parvum in Cypriot dairy farms, almost 40% of the cattle at local farms were positive for Cryptosporidium.The species identified were C. bovis, C. ryanae and C. parvum.Similarly, Schou et al. [5] reported a high occurrence of Cryptosporidium in domestic sheep (9/32 = 28%) and goats (5/10 = 50%) at local farms, although the number of samples investigated was low.The species identified were C. xiaoi and C. ubiquitum in sheep The Cryptosporidium parvum sequences that were analysed from animals isolated in Cyprus (Schou et al. [5]) are marked with a solid square and C. parvum in goats.In comparison, none of the goats in the present study was infected.This may be a reflection of the different conditions faced by farmed livestock [5] compared to free-ranging animals.In a systematic review and meta-analysis of Cryptosporidium infections in domestic and wild ungulates, Hatam-Nahavandi et al. [43] found Cryptosporidium prevalence to be lower in wild compared to farmed populations of the same host species.Animal density and confinement to the same (contaminated) environment facilitate Cryptosporidium transmission in farmed animals.The outcomes of the present study support this assumption, with 1.4% C. parvum prevalence in the Cyprus mouflon and no positive samples in the free-ranging goats.Nevertheless, the investigated number of goat samples in the current study was low (n = 32) and research comparing farmed and free-ranging livestock at Paphos State Forest merits further attention.
Furthermore, infection between domestic and wild populations of closely related animal species is a common phenomenon [43].Therefore, Cryptosporidium may be transmitted from goats and sheep to mouflon, and vice versa.Importantly, the only Cryptosporidium species found in goats and the Cyprus mouflon is C. parvum.The transmission of cryptosporidiosis could occur through the faecaloral route and the contamination of shared water sources at Paphos State Forest and surrounding areas.More research is required to investigate this relationship.Sampling should focus primarily on areas where mouflon and domestic ungulates coexist, especially near artificial watering points constructed and maintained by the Game and Fauna Service in the driest parts of the mouflon range.Disease transmission risk within and between species has been identified as a factor to consider in such watering areas [44][45][46].Considering that C. parvum is the most dangerous Cryptosporidium species in animals and responsible for mortality in neonatal animals, actions should be taken to protect the Cyprus mouflon considering that it is an endemic and endangered species.

Conclusions
The results indicate a low prevalence of Cryptosporidium in the Cyprus mouflon and free-ranging goats.This is the first report of C. parvum in the endemic Cyprus mouflon.Further studies should identify the factors that influence parasite transmission in the Cyprus mouflon and the livestock it comes in contact with.

Fig. 1
Fig.1Phylogenetic analysis of Cryptosporidium species based on the 600 bp sequence of the 18S rRNA gene, using the Neighbour-Joining method with Bootstrap 1000 replicates.Evolutionary distances were calculated using the Kimura two-parameter model.The percentage of bootstrap samplings is indicated by the numbers above the branches.The phylogenetic tree was constructed using the MEGA 11.0 soft-