Molecular Detection and Distribution of <i>Giardia duodenalis</i> and <i>Cryptosporidium</i> spp. Infections in Wild and Domestic Animals in Portugal

Figueiredo, Ana M.; Köster, Pamela C.; Dashti, Alejandro; Torres, Rita T.; Fonseca, Carlos; Mysterud, Atle; Bailo, Begoña; Carvalho, João; Ferreira, Eduardo; Hipólito, Dário; Fernandes, Joana; Lino, Ana; Palmeira, Josman D.; Sarmento, Pedro; Neves, Nuno; Carrapato, Carlos; Calero-Bernal, Rafael; Carmena, David

doi:https://doi.org/10.1155/2023/5849842

Transboundary and Emerging Diseases

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Research Article | Open Access

Volume 2023 | Article ID 5849842 | https://doi.org/10.1155/2023/5849842

Molecular Detection and Distribution of Giardia duodenalis and Cryptosporidium spp. Infections in Wild and Domestic Animals in Portugal

Ana M. Figueiredo,^1,2Pamela C. Köster,³Alejandro Dashti,³Rita T. Torres,¹Carlos Fonseca,^1,4Atle Mysterud,²Begoña Bailo,³João Carvalho,¹Eduardo Ferreira,¹Dário Hipólito,^1,5Joana Fernandes,^1,6and Ana Lino¹ et al.

Academic Editor: Long-Xian Zhang

Received22 May 2023

Revised07 Sept 2023

Accepted30 Sept 2023

Published08 Nov 2023

Abstract

Enteric protozoan parasites Giardia duodenalis, Cryptosporidium spp., and, to a lesser extent, the ciliate Balantioides coli are responsible for severe human and animal intestinal disorders globally. However, limited information is available on the occurrence and epidemiology of these parasites in domestic, but especially wild species in Portugal. To fill this gap of knowledge, we have investigated G. duodenalis, Cryptosporidium spp., and B. coli occurrence, distribution, genetic diversity, and zoonotic potential by analyzing 756 fecal samples from several wild carnivores (n = 288), wild ungulates (n = 242), and domestic species (n = 226) collected across different areas of mainland Portugal. Overall, infection rates were 16.1% (122/756; 95% CI: 13.59–18.96) for G. duodenalis and 2.7% (20/756; CI: 1.62–4.06) for Cryptosporidium spp., while no ungulate sample analyzed yielded positive results for B. coli. Giardia duodenalis was found across a wide range of hosts and sampling areas, being most prevalent in the Iberian lynx (26.7%), the Iberian wolf (24.0%), and the domestic dog (23.9%). Cryptosporidium spp. was only identified in wild boar (8.4%), red fox (3.4%), Iberian lynx (3.3%), red deer (3.1%), and Iberian wolf (2.5%). Sequence analysis of G. duodenalis determined zoonotic assemblage A (subassemblage AI) in one roe deer sample, canine-specific assemblages C and D in Iberian wolf, red fox, and domestic dog, and ungulate-specific assemblage E in wild boar, sheep, cattle, and horse. Six Cryptosporidium species were identified: C. scrofarum in wild boar, C. canis in the Iberian wolf and red fox, C. ubiquitum in red deer and wild boar, C. felis in the Iberian lynx, and both C. ryanae and C. occultus in red deer. Giardia duodenalis and Cryptosporidium spp. coinfections were observed in 0.7% (5/756) of the samples. This is the first, most comprehensive, and largest molecular-based epidemiology study of its kind carried out in Portugal, covering a wide range of wild and domestic hosts and sampling areas. The detection of zoonotic Cryptosporidium spp. and G. duodenalis subassemblage AI demonstrates the role of wild and domestic host species in the transmission of these agents while representing a potential source of environmental contamination for other animals and humans.

1. Introduction

Giardia duodenalis (syn. G. lamblia, G. intestinalis) and Cryptosporidium spp. are two of the most common enteric protozoan parasites accountable for human and animal intestinal disorders worldwide [1, 2]. Near 200 million human symptomatic cases of giardiasis are reported annually [3]. Cryptosporidiosis is second only to rotavirus infection as a contributor to childhood diarrhea in poor-resource settings [4]. Acute to chronic diarrhea, abdominal pain, lack of appetite, malabsorption, and weight loss are the main clinical manifestations described for both protozoan infections [2, 5]. In children living in endemic areas, giardiasis and cryptosporidiosis are associated with growth retardation and cognitive impairment, extending their impact to life-threatening malnutrition and wasting [6, 7]. Notwithstanding, asymptomatic infections can also be frequent, depending on the parasite strain and the host’s immunological and health status [8, 9]. Cryptosporidium spp. and, to a lesser extent, G. duodenalis infections are linked to decreased growth rates and, in the case of Cryptosporidium, increased mortality in infected livestock species, especially neonatal individuals, triggering significant economic losses to the sector [2, 10]. Although its worldwide prevalence in humans usually does not exceed 1%, Balantioides coli (formerly known as Balantidium coli and Neobalantidium coli) is the only ciliate with public health importance, having domestic pigs as the primary animal reservoir, even though infections in this host are mostly asymptomatic [11]. Human infections by B. coli have a similar clinical picture to those previously described for giardiasis and cryptosporidiosis, with the aggravating factor of triggering colitis, an inflammatory bowel disease (IBD) [12].

While Cryptosporidium displays a complex life cycle comprising both sexual and asexual replication stages [2], G. duodenalis and B. coli life cycles involve two developmental stages, the replicative form (trophozoite) and the transmissive and infective form (cyst). Infection occurs through the fecal–oral route, which involves the ingestion of environmentally resistant cysts (G. duodenalis, B. coli) or oocysts (Cryptosporidium spp.) through the consumption of contaminated food or water or direct contact with an infected animal/human host [5, 12, 13].

Giardia duodenalis is currently regarded as a species complex consisting of eight assemblages (from A to H) with marked differences in host specificity and range [14]. Assemblages A and B are zoonotic, infecting humans, companion animals, livestock, and wildlife. Host-specific assemblages C and D are mainly reported in canids, E in artiodactyls, F in felids, G in rodents, and H in marine mammals [15, 16]. For the Cryptosporidium genus, at least 46 taxonomically valid species have been described [14, 17, 18]. Even though ca. 95% of human cases of cryptosporidiosis reported are due to C. hominis and C. parvum infections [19], over 20 Cryptosporidium species have been identified in humans, including host-adapted C. meleagridis, C. canis, and C. felis [14, 20]. As for B. coli, three genotypes (A, B, and C) have been described: genotypes A and B have a broad host range, whereas genotype C has only been identified in nonhuman primates [21].

Studies reporting G. duodenalis, Cryptosporidium spp., and, to a lesser extent, B. coli in wildlife have continuously contributed to improving our understanding of the epidemiology, host range, and zoonotic potential of these parasites [22]. In Europe, wild carnivores and ungulates have had an uprising in recent years, increasing the contact rate with domestic animals and humans due to hunting practices, overlapped distribution areas, and consequent synanthropic behaviors [23, 24]. The spatial overlap between wild and domestic species, particularly involving free-roaming livestock herds but also companion animals, is increasing the spillover of zoonotic strains/genotypes in the wildlife–domestic–human interface, perpetuating the transmission and spreading of these parasites. In Europe, G. duodenalis and Cryptosporidium spp. have been reported in wolf Canis lupus, red fox Vulpes vulpes, and stone marten Martes foina with prevalence rates ranging from 5% to 44% for G. duodenalis and 2% to 36% for Cryptosporidium spp., whereas for wild ungulates (red deer Cervus elaphus, roe deer Capreolus capreolus, and wild boar Sus scrofa), these values ranged from 1% to 41% for G. duodenalis and 1% to 18% for Cryptosporidium (Table 1). Zoonotic assemblages A and B and canid-specific C and D were the most frequent genetic variants identified within G. duodenalis, while C. parvum, C. canis, C. ubiquitum, and C. scrofarum were the predominant Cryptosporidium species circulating in such hosts. As for B. coli, the only reports were in wild boar and red deer, with the detection of genotypes A and B (Table 1). Giardia duodenalis reports in domestic dogs include prevalence rates ranging from 2% to 100% and 1% to 10% for Cryptosporidium spp., while for livestock species, these values range from 8% to 100% and 1% to 100%, respectively. Also, there were numerous reports across Europe of G. duodenalis assemblages A–E and zoonotic (e.g., C. parvum, C. canis) and host-adapted (e.g., C. ryanae) Cryptosporidium species (Table 2).

In Portugal, data on the occurrence and molecular diversity of these three enteric parasites in wild species are limited to the report of C. scrofarum in wild boar [51] and B. coli genotypes A and B in red deer and wild boar [51, 52] (Table 1). Regarding domestic animals, G. duodenalis assemblages B, C, and D have been reported in dogs Canis lupus familiaris [86, 87] and A, B, and E in cattle Bos taurus [101]. Cryptosporidium parvum was documented in horses Equus caballus [181], sheep Ovis aries [125], and cattle [101, 123–125], as well as C. meleagridis and C. andersoni [101] (Table 2). Considering the overall low number of studies evaluating the molecular diversity of these enteric parasites carried out in Portugal, especially in the wildlife counterpart, this study aims to determine the distribution, genetic diversity, and zoonotic potential of G. duodenalis, Cryptosporidium spp., and B. coli in wild and domestic animal species across different areas of mainland Portugal.

2. Materials and Methods

2.1. Study Area and Sampling Collection

This study was carried out in seven distinct areas of mainland Portugal (Figure 1), reflecting contrasting environmental and climate conditions and differences in their species’ community, covering European Union’s Natura 2000 Network sites (https://ec.europa.eu/environment/nature/natura2000/index_en.htm). Montesinho Natural Park (MNP), Central Portugal West (CPW), and Central Portugal East (CPE) are characterized by mountainous landscapes and a continental Mediterranean climate, even though CPW exhibits a strong Atlantic influence. The three areas comprise livestock herds raised under the traditional extensive grazing system and a large diversity of wild species (e.g., the Iberian wolf, Canis lupus signatus and roe deer). The Faia Brava Reserve (FBR) is a privately protected enclosed area encompassing semiwild herbivores (cattle and horses), co-occurring with other wildlife species. The Guadiana Valley (GV) is situated in the southern region of Portugal and features a continental Mediterranean climate and low-altitude mountains. This area is home to a variety of wild species, free-roaming livestock herds, and the recently reintroduced apex predator, the Iberian lynx (Lynx pardinus). Lousã Mountains (LM) and Malcata Nature Reserve (MNR) are both characterized by a Mediterranean climate, presenting a wide variety of wild ungulate and mesocarnivore species (e.g., red deer, the red fox), even though no apex predator and free-roaming livestock species overlap their territories.

2.2. Sampling Collection

Between 2017 and 2021, fresh fecal samples from a total of 12 mammal species (wild and domestic), which included wild carnivores (Iberian wolf, Iberian lynx, red fox, and stone marten), wild ungulates (red deer, roe deer, and wild boar), livestock species (cattle, horse, goat Capra hircus, and sheep), and domestic/feral dog, were collected in the prospected study areas (Figure 1). A total of 756 individual fecal specimens were sampled from wild carnivore (38.1%, 288/756), wild ungulate (32.0%, 242/756), and domestic (29.9%, 226/756, including livestock and domestic/feral dogs) species. Samples were opportunistically collected from (i) legally hunted animals, (ii) routine checkups/live-capture operations, (iii) free-roaming livestock herds, and (iv) transects or scats trails distributed across the different study areas. During field necropsies of hunted red deer and wild boar specimens and during routine checkups/live capture procedures of Iberian lynx individuals, fecal samples were directly collected from the animal’s rectum. Fecal samples from the Iberian lynx were obtained by compressing the intestinal tract of anesthetized individuals captured in the wild in the scope of ongoing projects. Concerning the remaining wild and domestic species, sampling collection was carried out whenever an animal was observed defecating or directly from the ground. For the latter case, samples were identified by experienced and field-trained personnel based on their morphology (e.g., content, size, shape) and deposition site. To reduce misleading identifications, Iberian wolf and domestic dog feces were genetically confirmed [191], together with a limited number of red fox (n = 49) and stone marten (n = 2) samples [192], as a regular procedure of ongoing monitoring projects of these species. Fecal samples were placed into 50 mL Corning-Falcon® containing 95% ethanol for preservation and transportation purposes and stored at −18°C for subsequent DNA extraction. The period between sample collection and DNA extraction varied from 3 to 24 months in retrospective samples from monitoring projects and a maximum of 5 months in prospective samples.

2.3. DNA Extraction and Purification

Genomic DNA was isolated from about 200 mg of feces using the QIAamp® Fast DNA Stool Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions at the Department of Biology & CESAM, University of Aveiro (Aveiro, Portugal) facilities. Extracted and purified DNA samples were eluted in 200 μl of polymerase chain reaction (PCR)-grade water or buffer ATE provided by the kit and sent to the Spanish National Centre for Microbiology (Majadahonda, Spain) for downstream molecular analyses.

2.4. Molecular Detection and Characterization of Giardia duodenalis, Cryptosporidium spp., and Balantioides coli

For the identification of G. duodenalis, a real-time PCR (qPCR) method was setup to amplify a 62-bp fragment of the small subunit of the rRNA (ssu RNA) gene of the parasite [193]. Samples that yielded cycle threshold (C_T) values <35 in qPCR were then analyzed through a nested PCR, used to amplify a 300-bp fragment of the ssu RNA gene [194] to assess G. duodenalis molecular diversity at the assemblage level. Samples that yielded qPCR C_T values <32 were additionally assessed using a sequence-based multilocus genotyping (MLST) scheme targeting the genes encoding for the glutamate dehydrogenase (gdh), β-giardin (bg), and triose phosphate isomerase (tpi) proteins to assess G. duodenalis molecular diversity at the subassemblage level. A 432-bp fragment of the gdh gene was amplified using a seminested PCR [195], while 511- and 530-bp fragments of the bg and tpi genes, respectively, were amplified through nested PCRs [55, 196].

Cryptosporidium spp. presence was investigated using a nested PCR protocol, amplifying a 587-bp fragment of the ssu RNA gene of the parasite [197]. Subtyping tools based on the amplification of partial sequences of the 60-kDa glycoprotein (gp60) gene were used to ascertain intraspecies genetic diversity in samples that tested positive for C. canis [198], C. felis [199], C. ryanae [200], and C. ubiquitum [201] by ssu-PCR.

B. coli occurrence in wild and domestic ungulates (as it does not naturally infect strict carnivores) was determined by a direct PCR assay targeting the ITS1-5.8s-rRNA-ITS2 region and the last 117 bp (3′ end) of the ssu-rRNA sequence of this ciliate, as previously described [21].

Detailed information on the PCR cycling conditions and oligonucleotides used for molecular identification and/or characterization of the abovementioned parasites can be found in Supplementary 1 and Supplementary 2, respectively. The previously described PCR protocols were conducted on a 2720 Thermal Cycler (Applied Biosystems). Reaction mixes included 2.5 units of MyTAQ^TM DNA polymerase (Bioline GmbH, Luckenwalde, Germany) and 5–10 μl 5× MyTAQ^TM Reaction Buffer containing 5 mM deoxynucleotide triphosphates and 15 mM MgCl₂. Negative and positive controls were included in all PCR runs. PCR amplicons obtained were examined on 1.5% D5 agarose gel stained with Pronasafe (Conda, Madrid, Spain) and sized using a 100-bp DNA ladder (Boehringer Mannheim GmbH, Mannheim, Germany).

2.5. Sequence Analysis

Positive PCR products with the expected size were directly sequenced in both directions with the corresponding internal primer pair (see Supplementary 2) in 10 μl reactions using BigDye^TM chemistries and an ABI 3730xl sequencer analyser (Applied Biosystems, Foster City, CA). Raw sequences were examined with the Chromas Lite version 2.1 software (http://chromaslite.software.informer.com/2.1) to generate consensus sequences. The BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to compare the newly generated sequences with reference sequences deposited at the National Center for Biotechnology Information (NCBI) GenBank database.

2.6. Statistical Analysis

Parasite prevalence was estimated using a binomial test in R software [202], establishing confidence limits with 95% confidence intervals (CI). A χ² test, using the chisq.test function, was used to compare parasite prevalence between hosts (wild carnivores, wild ungulates, and domestic animals) and study areas. A median-joining haplotype network [203] was constructed using PopART 1.7 (https://popart.maths.otago.ac.nz; Leigh and Bryant [204]) using the resulting G. duodenalis and Cryptosporidium spp. sequences from gdh, bg, and ssu. For the network construction, all positions containing gaps (indels) or ambiguities (R, Y, W, S) were disregarded, as the algorithm cannot handle those mutations. For the network’s representation, we considered the sampling area of each identified assemblage/genotype and its frequency.

3. Results

3.1. Prevalence of Giardia duodenalis and Cryptosporidium spp.

From a total of 756 analyzed fecal samples, 122 (16.1%, 95% CI: 13.59–18.96) were infected with G. duodenalis and 20 (2.7%, 95% CI: 1.62–4.06) with Cryptosporidium spp. (Table 3). None of the wild and domestic ungulate samples (n = 422) assessed for B. coli yielded positive results for this parasite.

Giardia duodenalis was detected across all the examined species, except for the domestic goat, and sampling areas, apart from the LM area. The occurrence of the protozoan varied across the sampled groups of animals (χ² (2, n = 756) = 9.779, ), with the highest prevalence of G. duodenalis found in the Iberian lynx (26.7%, 8/30), followed by the Iberian wolf (25.6%, 31/121) and the domestic dog (23.9%, 11/46). Regarding G. duodenalis geographic distribution, its occurrence varied among the seven sampled study areas (χ² (6, n = 756) = 19.81, ), and the GV (26.7%, 8/30), MNP (20.5%, 44/215), and CPW (19.3%, 48/249) were the areas where this protozoan was mainly detected (Table 3). Cryptosporidium spp. was only detected in wild boar (8.4%, 9/107), red fox (3.4%, 4/118), Iberian lynx (3.3%, 1/30), red deer (3.1%, 3/96), and Iberian wolf (2.5%, 3/121), demonstrating the significant differences on the occurrence of this parasite across host species (χ² (2, n = 756) = 10.661, ). Although this protozoan was not detected in FBR and MNR sampling areas, its occurrence varied among the remaining sampled locations (χ² (6, n = 756) = 14.971, ) (Table 3).

3.2. Molecular Diversity

Giardia duodenalis qPCR-positive samples generated C_T values that ranged from 22.6 to 39.8 (median: 33.9; SD: 3.5). Only samples with C_T values ≤35 (n = 63) were subsequently genotyped at the ssu-rRNA locus for assemblage identification. Overall, four G. duodenalis assemblages were identified in the investigated host species based on the information retrieved for one or more of the four genetic markers (ssu-rRNA, gdh, bg, and tpi) used for genotyping purposes. These include zoonotic assemblage A (5%, 1/20), canine-adapted assemblages C (20%, 4/20) and D (50%, 10/20), and ungulate-adapted assemblage E (25%, 5/20). Nucleotide sequence analysis at the ssu-rRNA locus allowed assemblage identification in 55% of the species (11/20), including assemblage A, subassemblage AI (n = 1), found in a roe deer sample, assemblage D (n = 5), found in samples belonging to Iberian wolf, red fox, and dog specimens, and ungulate-specific assemblage E (n = 5), found in wild boar and livestock species (Table 4, Figure 2). For assemblage confirmation and subassemblage identification, samples with C_T values ≤32 (n = 28) were reassessed at the gdh, bg, and tpi loci, allowing the identification of canine-specific assemblages C (n = 4) and D (n = 5) in Iberian wolf and red fox samples. Additionally, two Iberian wolves carried mixed infections involving assemblages C (detected at the gdh locus) and D (detected at the ssu-rRNA locus), and one red fox displayed a mixed infection by assemblages C (detected at the gdh and bg loci) and D (detected at the ssu-rRNA locus). MNP and CPW were the only sampling areas where it was possible to determine Giardia assemblages (A, C, D, and E), except for one horse sample from FBR, where assemblage E was possible to identify (Table 4, Figure 2).

Figure 2

Median-joining haplotype networks constructed in PopART. Giardia duodenalis assemblages are represented by circles, being the sizes proportional to the number of individuals where a given assemblage was sampled. Number of mutations among assemblages is represented by the number of slashes. Different colors represent the different wild and domestic hosts. Study areas where assemblages were detected—Montesinho Natural Park (MNP), Central Portugal West (CPW), and Faia Brava Reserve (FBR)—are also indicated.

Six Cryptosporidium species were identified: C. scrofarum (40.0%, 8/20), C. canis (35.0%, 7/20), C. ubiquitum (10.0%, 2/20), C. felis (5.0%, 1/20), C. ryanae (5.0%, 1/20), and C. occultus (5.0%, 1/20) (Table 4 Figure 3). Cryptosporidium scrofarum was exclusively found in wild boar, while C. canis was found in both the Iberian wolf (n = 3) and red fox (n = 4). Cryptosporidium ubiquitum was found in red deer (n = 1) and wild boar (n = 1). A single sample of red deer amplified C. ryanae and another C. occultus, while C. felis was identified on an Iberian lynx sample. Cryptosporidium canis was found across four sampling areas (MNP, CPW, CPE, and LM), while C. scrofarum was only detected in MNP and CPE. The two positive samples for C. ubiquitum were found in the same area (MNP) (Figure 3). Positive samples for C. canis, C. ubiquitum, C. ryanae, and C. felis could not be further genotyped at the gp60 gene.

Figure 3

Median-joining haplotype network constructed in PopART. Cryptosporidium spp. genotypes are represented by circles, being the sizes proportional to the number of individuals where a given Cryptosporidium genotype was sampled. Number of mutations among genotypes is represented by the number of slashes. Different colors represent the different wild and domestic hosts. Study areas where genotypes were detected—Montesinho Natural Park (MNP), Central Portugal West (CPW), Central Portugal East (CPE), Lousã Mountains (LM), and Guadiana Valley (GV)—are also indicated.

Coinfections with G. duodenalis and Cryptosporidium spp. were found in five specimens (0.7%, 5/756) of the analyzed samples, belonging to two Iberian wolf samples (G. duodenalis assemblage D + C. canis and G. duodenalis + C. canis), one red fox (G. duodenalis + C. canis), one Iberian lynx (G. duodenalis + C. felis), and one red deer (G. duodenalis + C. ubiquitum) sample. The full dataset of this study showing sampling, epidemiological, diagnostic, and molecular data can be found in Supplementary 3. The sequences obtained in this study were deposited in the GenBank database under accession numbers OQ818646–OQ818654 and OQ818103–OQ818108 (G. duodenalis), OQ818655–OQ818656 (C. canis), OQ818657 (C. felis), OQ818658 (C. ryanae), OQ818659–OQ818660 (C. scrofarum), OQ818661 (C. occultus), and OQ818662–OQ818663 (C. ubiquitum).

4. Discussion

As G. duodenalis and Cryptosporidium spp. have become major sources of enteric parasitic diseases worldwide, it is paramount to recognize the role played by domestic and wild animal reservoirs in the maintenance and spread of these protozoan pathogens of public and veterinary health relevance. This study is the first molecular-based survey ever carried out in Portugal to assess G. duodenalis and Cryptosporidium spp. occurrence, distribution, molecular diversity, and zoonotic potential in wild and domestic host species. For the first time, we were able (i) to genotype Cryptosporidium spp. in the Iberian wolf and the Iberian lynx, (ii) to detect G. duodenalis in the Iberian lynx, and (iii) to successfully genotype G. duodenalis in the Iberian wolf. In addition, we investigated the occurrence and host distribution of B. coli, a ciliate protozoan parasite whose epidemiology is poorly known in Portugal and was not detected in any of the species and areas screened here. This study comes as a follow-up to the one already developed for the microsporidia Enterocytozoon bieneusi, using the same range of host species and sampling areas [206].

4.1. Prevalence of Giardia duodenalis in Wild and Domestic Species

In our survey, G. duodenalis was the most prevalent enteric parasite found (16.1%), with the highest prevalence documented in the Iberian lynx (26.7%), followed by the Iberian wolf (25.6%) and the domestic dog (23.9%) (Table 3). In previous molecular studies carried out in Portugal, G. duodenalis was reported at prevalence rates of 16.9%–33.8% in dogs [86, 87] and 9.0% in cattle [101], the later found at a lower prevalence than we found in our study (14.9%) (Table 2). While infection rates documented in the three preceding studies were similar to those reported here, a higher figure was expected in those previous studies as canine and livestock samples were mainly from shelters and intensive commercial farms with high animal densities and reduced enclosures favoring the risk of infection and transmission. Discrepancies in prevalence rates among these surveys may be attributed to differences in the diagnostic performance of the screening method used, as light microscopy is usually a less sensitive technique than PCR for pathogen detection.

In neighboring Spain, G. duodenalis was reported in the Iberian wolf (16.7%), stone marten (12.5%) [30], red fox (9.6%) [34], and red deer (2.4%) [41], displaying lower prevalence values than those found in our study for the same evaluated hosts. The protozoan was described at similar prevalence rates (7.5%–8.9%) in roe deer [41, 43] (Table 1). As for domestic animal hosts, G. duodenalis has been reported across Europe, with prevalence ranging from 2.0% to 100% in dogs [75, 85] and 9.1% to 100% in cattle [55, 92] (see Table 2). Remarkably, G. duodenalis was apparently absent in domestic goats, the only host species analyzed in this study where this parasite was undetected. Caprine infections by G. duodenalis have been previously reported in a few European countries, namely Spain (19.8% [94]; 42.2% [102]) and Belgium (35.8%) [103]. Differences in environmental and anthropogenic pressures, the composition of wild species communities, and contact rates with livestock or companion animals in the sampled areas might explain the discrepant G. duodenalis results among studies.

4.2. Genetic Diversity of Giardia duodenalis Isolates

Nucleotide sequence analyses of G. duodenalis isolates at the ssu, gdh, and bg loci revealed the presence of zoonotic subassemblage AI in one roe deer (from MNP), canine-specific assemblages C and D in Iberian wolves, red foxes, and one dog (from CPW and MNP), and ungulate-specific assemblage E in wild boar, cattle, horse, and sheep (from different sampling locations). Assemblages B, C, and D were previously documented in dogs from Portugal [86, 87], while assemblages A, B, and E were reported in cattle [101]. In Spain, García-Presedo et al. [43] reported subassemblage AII in roe deer, which is considered the G. duodenalis genetic variant predominant in humans [14].

Giardia duodenalis assemblages C and D have been frequently reported in wolves (e.g., [25, 27]) and dogs (e.g., [61, 65]) populations across Europe (Table 1). In our study, assemblage D was found in wolves and a dog inhabiting the CPW area, displaying 100% identity with reference sequence AF199449 [205]. Interestingly, CPW sustains the most fragile and isolated subpopulation of Iberian wolves, which share their territory with feral dog packs and free-roaming shepherd dogs. As hybridization has already been confirmed between wolves and dogs in CPW [207], our finding indicates the possibility of a transmission route between the two hosts (Figure 2), which can occur either by direct contact or indirectly through environmental contamination of water or food resources with Giardia cysts. Additionally, a mixed infection of assemblages C + D was found in one red fox, suggesting that this host may also be involved in the sylvatic cycle of G. duodenalis (Figure 2). The report of assemblages C/D (in red fox) and E (in wild boar) is likely the first confirmation of these two species as hosts of these assemblages in Europe, representing another indicator of cross-species transmission. Additional evidence of overlapping sylvatic and domestic life cycles comes from the finding of assemblage E in a sheep from the same area (MNP) as the wild boar was reported, with both amplicons showing 100% identity with reference sequence AF199448 [205] (Table 4, Figure 2). Another interesting result was the identification of assemblage E in a horse from FBR since no other positive samples from cattle were typed in this geographical region. In this area, horses are subjected to a strict annual deworming scheme with ivermectin, while cattle have less rigorous protocols with occasional clorsulon–ivermectin administration. These antiparasitic drugs are essentially used to treat helminth (nematodes) and arthropod infections, while they proved ineffective against protozoan infections like giardiasis and cryptosporidiosis [208]. Therefore, the overall low prevalence of protozoan parasites found in FBR (8.3%) cannot be attributed to ongoing deworming protocols. Lower animal densities or environmental characteristics of the study area can be plausible explanations for reduced cyst contamination and transmission risk.

4.3. Prevalence of Cryptosporidium spp. in Wild and Domestic Species

Previous studies in Portugal have reported the presence of Cryptosporidium spp. in horses (21.4%) [181], sheep and cattle (17.6%–100%) [101, 123–125]. Nonetheless, information concerning this protozoan infection in wild reservoirs is restricted to a report in wild boar, where a prevalence of 1.4% (2/144) was detected [51]. This prevalence is lower than that reported here for the same host species (7.5%, 8/107) (Table 1). Across Europe, Cryptosporidium spp. has been described at highly variable prevalence (3.6%–35.7%) in wolves in eastern Slovakia [36] and Poland [35]. As for red fox, a similar prevalence to the one reported in this study was found in Poland (2.7%), the Czech Republic (1.7%), and Slovakia (2.1%) [36], while the highest reported prevalence was found in Ireland (20%) [38]. Even though we could not detect Cryptosporidium spp. in any of the analyzed stone martens, this protozoan was documented in this host species in Poland (29.4%) [39]. For wild ungulates, a similar prevalence to the one we reported for red deer was found in neighboring Spain (2.7%) [41]. Higher infection rates were described for wild boar in Austria (18.1%), Czech Republic (16.9%), Poland (8.5%) [49], and Spain (16.8%) [50] (Table 1). In domestic dogs, the highest prevalence was reported in Germany (10.0%) [89]. Furthermore, while Cryptosporidium spp. was not detected in any of the livestock samples analyzed, across Europe, literature reports are extensive, particularly for cattle, reporting highly variable prevalence rates (4.9%–100%) [119, 146] (Table 2). One of the reasons behind the lack of detection of this protozoan in our livestock analyzed samples may be related to the fact that we sampled adult individuals, and as previous studies have shown, Cryptosporidium infections are more frequent in younger animals, particularly neonatal calves [2, 94, 96, 209]. Furthermore, bovine Cryptosporidium infections are generally short-lived, with oocyst shedding lasting 1–2 weeks, decreasing the time frame where it would be possible to detect the parasite in the feces effectively [210].

4.4. Genetic Diversity of Cryptosporidium spp. Isolates

Six Cryptosporidium species were identified circulating in the wild, and domestic species investigated in the present survey (Table 4). Swine-adapted C. scrofarum (formerly known as pig genotype II) was the most prevalent species detected but was exclusively found in wild boars. Cryptosporidium scrofarum has been reported in wild boar across Europe [41, 44, 49], including Portugal [51]. Wild boars were also the reservoir where a higher prevalence of Cryptosporidium spp. (8.4%) was found, previously associated with their omnivorous diet and broader habitat selection requirements [209]. Canine-adapted C. canis was detected in the Iberian wolf, as previously described in wolves from Slovakia [36] and in red fox, as reported in Spain [30, 34] and Poland [39]. As for the identification of C. ryanae and C. occultus in red deer, these results agree with a report from Spain [41]. Interestingly, both studies found C. occultus in red deer, a species typically associated with rodents. This suggests the existence of a potential transmission route between these two hosts. Furthermore, the detection of C. ubiquitum in red deer and wild boar inhabiting the same area (MNP) suggests that both species are involved in the transmission of the parasite (Figure 3).

The first description of C. felis in the Iberian lynx has provided important insights into the potential pathogens that could threaten the successful reintroduction of this endangered species. However, the potential sources of infection remain unclear. Since domestic cats are the acknowledged reservoir of C. felis [14], one possibility is that Iberian lynxes acquire the parasite through spillover events between domestic and sylvatic transmission cycles [211]. Another possibility is that C. felis naturally circulates in the wild Iberian lynx population.

Apart from ungulate-adapted C. ryanae, all Cryptosporidium spp. identified in this study and G. duodenalis subassemblage AI have zoonotic potential [14]. This fact suggests that the wild and domestic host species can act as potential reservoirs of human cryptosporidiosis and giardiasis, in addition to a source of environmental contamination with infective (oo)cysts.

Even though none of our analyzed wild and domestic ungulate samples yielded positive results for B. coli, genotypes A and B were previously described in Portugal in red deer and wild boar [51, 52], as well as in wild boar populations from Spain [41, 44] and Poland [53] (Table 1). Contrary to the other free-ranging livestock species, pigs (B. coli primary host) are raised inside enclosures in the sampled areas, restricting their contact with wild boars, which can explain why we did not find this parasite in any of the analyzed samples.

The experiences made during our study may guide future research. Larger sample sizes from some species were due to stored-up collection or ongoing monitoring projects in the sampled areas (e.g., Iberian wolf, red fox, and wild boar), while smaller sample sizes can be attributed to the fact that we are working with endangered species (Iberian lynx) or species with limited distribution in the sampled areas (e.g., stone marten and domestic goat). Without targeted monitoring programs for the latter species, this will likely remain a limitation. The opportunistic sampling limited our ability to capture seasonal variability of pathogen occurrence or to compare potential effects of age, sex, and different sources of sampling. This is particularly important as cryptosporidiosis consistently occurs in younger animals, especially domestic animals, which we failed to sample. Nonetheless, it is unlikely this can be done for endangered species, and more in-depth studies could focus on the more common species. Last, our genotyping PCRs’ relatively low amplification was associated with the limited sensibility of the single-copy genes (gdh, bg, and tpi in G. duodenalis or gp60 in Cryptosporidium spp.) targeted in our PCR, associated with the small amount of parasite DNA in the analyzed samples, which hampered the attempts to assess the zoonotic potential and the public health significance in the analyzed samples.

5. Conclusions

Our findings contributed to bridging the knowledge gap regarding the epidemiology of protist species of public and veterinary health relevance in wild and domestic host species from Portugal. The identification of zoonotic Cryptosporidium spp. and G. duodenalis subassemblage AI highlights the role played by wild and domestic species in the maintenance of the sylvatic and domestic cycle of such organisms. These findings are a step forward to unraveling the epidemiological scenario in the Portuguese context while comparing it to other European studies (Tables 1 and 2), which is critical knowledge for understanding the possible infection risks that human populations may be facing in the sampled areas. Future studies should not only aim to cover additional ecological niches but also target host-dependent risk factors such as host age, as cryptosporidiosis consistently occurs in younger animals, especially in domestic species. Although not fully understood, the identification of G. duodenalis and Cryptosporidium infections in endangered species (e.g., Iberian wolf and Iberian lynx) may have important conservation implications, which should be addressed in future research. Therefore, it is essential to implement tailor-made conservation measures to attain the specific needs of these species, including the regular monitoring programs of these enteric protozoan parasites and other emerging infectious pathogens, with the ultimate goal of preserving biological diversity.

Data Availability

The authors confirm that the data supporting the findings of this study are available within the main body of the manuscript.

Disclosure

Funding agencies had no role in the design or conduct of the study, assessment of the data, or writing of the manuscript.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

A. M. Figueiredo, D. Hipólito, and J. Fernandes were supported by a PhD grant from Fundação para a Ciência e Tecnologia (SFRH/BD/144582/2019, SFRH/BD/144437/2019, PD/BD/150645/2020, respectively), cofinanced by the European Social Fund POPH-QREN program. A. Dashti is the recipient of a PFIS contract (FI20CIII/00002) funded by the Spanish Ministry of Science and Innovation and Universities. R. T. Torres and J. Carvalho were supported by a research contract (2021.00690.CEECIND and CEECIND/01428/2018, respectively) from the Fundação para a Ciência e a Tecnologia. Eduardo Ferreira is funded by national funds (OE) through FCT in the scope of the framework contract foreseen in article 23, Decree-Law 57/2016. This work was supported by Centre for Environmental and Marine Studies (CESAM) through FCT/MCTES (UIDP/50017/2020+UIDB/50017/2020+ LA/P/0094/2020), and national funds, Health Institute Carlos III (ISCIII) and Spanish Ministry of Economy and Competitiveness, under project PI19CIII/00029, EcoARUn (POCI-01-0145-FEDER-030310) and WildForests (POCI-01-0145-FEDER-028204) projects, funded by FEDER, through COMPETE2020-Programa Operacional Competitividade e Internacionalização (POCI), and by national funds (OE), through FCT/MCTES, project rWILD-COA: Ecological challenges and opportunities of trophic rewilding in Côa Valley—COA/BRB/0063/2019, funded by national funds (OE), through FCT/MCTES, LIFE WolFlux (LIFE17 NAT/PT/000554), Life+ Project Iberlince (LIFE10NAT/ES/570), and Life Nature and Biodiversity Lynxconnect (LIFE 19NAT/ES/001055), funded by the LIFE Programme of the European Union, the EU’s funding instrument for the environment and climate action. Additional funding was obtained by “Plano de Monitorização do Lobo Ibérico PMLDS-O–ACHLI.”

Supplementary Materials

Supplementary 1. PCR cycling conditions used for molecular identification and/or characterization of Giardia duodenalis, Cryptosporidium spp., and Balantioides coli in the present study.

Supplementary 2. Oligonucleotides used for molecular identification and/or characterization of Giardia duodenalis, Cryptosporidium spp., and Balantioides coli in the present study.

Supplementary 3. Dataset showing sampling, epidemiological, diagnostic, and molecular data of the present study.

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