Occurrence and Phylogenetic Analysis of Zoonotic Enteropathogenic Protist Parasites in Asymptomatic Domestic Ruminants from Portugal

Enteropathogenic parasites are of significant concern for public health due to their zoonotic potential and their impact on human and animal health. In this study, we investigated their occurrence and characterized these enteropathogens in asymptomatic domestic ruminants from Portugal. A total of 302 stool samples were collected from cattle (n = 166), sheep (n = 73), and goats (n = 63) in various regions of Portugal and tested for Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, Blastocystis sp., and Balantioides coli by PCR. The occurrence of Cryptosporidium spp. was found to be 12.7% (8/63, 95% confidence interval [CI]: 5.65–23.5) in goats; however, no sample was found to be positive for Cryptosporidium spp. in cattle and sheep. For E. bieneusi, 6.35% (4/63; 95%CI: 1.76–15.47) of goats were found to be positive; however, no cattle or sheep were found to be positive. Blastocystis sp. was found in sheep (9.59%; 7/73; 95% [CI]: 0.394–18.76) and goats (12.70%; 8/63; 95% [CI]: 5.65–23.50) but none was found in cattle. No positive results for G. duodenalis or B. coli were detected in this study. This study provides essential baseline information for understanding the silent shedding and epidemiology of these enteropathogens in Portugal, contributing to overall livestock health and related occupational safety. Raising awareness among consumers, veterinarians, and farm owners is crucial to minimize the risk of transmission and promote effective disease control strategies.


Introduction
Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, Blastocystis sp., and Balantioides coli are prevalent pathogens known to cause diarrhea not only in humans both also in wild and domestic animals globally, including cattle and small ruminants, such as sheep and goats [1][2][3].The fecal-oral transmission stands as the primary route of infection [4][5][6].As such, appropriate sanitation and hygiene practices are necessary in order to effectively mitigate the spread of these protozoa and reduce the associated public health risks [7].The protozoa can be transmitted through foodborne and waterborne routes, underscoring the importance of safe food handling and clean water sources to prevent outbreaks and infections [7].The zoonotic potential of these pathogens is an Pathogens 2023, 12, 1341 2 of 13 important public health concern as it contributes to human morbidity in both developed and developing countries [4] and domestic animals such as bovine, ovine, and caprine, which are known reservoirs for a wide array of fecal protozoa [1].
Recent studies report the existence of 1640.65 thousand bovines, 2237.97 thousand sheep, and 350.47 thousand goats in Portugal, ranking it as the twelfth bovine and seventh ovine and caprine producers in Europe [8][9][10].This substantial number underscores the country's importance in the European livestock industry and agriculture but also raises alerts on the potential impact of the silent dissemination of enteric pathogens from these animals.Despite the significance of this industry, there is currently a lack of information regarding the circulation of zoonotic enteropathogenic protists, particularly Cryptosporidium spp., G. duodenalis, E. bieneusi, Blastocystis sp., and B. coli in Portugal.Moreover, albeit many efforts have been made to ascertain the role of these protozoa, data on the asymptomatic shedding and the role of ruminants as silent shedders remain to be fully understood, thus a better understanding the impact of this spillover to animals and humans is needed.
Cryptosporidiosis is a major cause of moderate-to-severe diarrhea in humans and has been proven to be connected to infected calves and small ruminants [11,12], underscoring the vital role of ruminants as reservoirs for this pathogen [13].Among cattle, the prevalent Cryptosporidium species include C. parvum, C. bovis, C. ryanae, and C. andersoni [14], whereas in sheep and goats, the most prevalent Crypstosporidium species include C. parvum, C. ubiquitum, and C. xiaoi [12].Cryptosporidium spp.are intracellular protozoan that exist outside the host cell's cytoplasm and are recognized for inducing gastrointestinal disorders.Cryptosporidium follows a direct life cycle, enabling it to propagate within the gastrointestinal epithelial cells of diseased animals [15].Human infection can occur through the consumption of minute oocyst quantities, highlighting the pathogen's capacity for dissemination and interspecies transmission [16].Furthermore, a challenge posed by Cryptosporidium pertains to its impressive resistance to conventional water treatment methodologies, including chlorination [16].Giardia duodenalis is a zoonotic parasite found among calves, beef and dairy cattle, sheep, and goats [17][18][19].The trophozoite represents the vegetative stage during which noninvasive trophozoites replicate within the mucosa of the small intestine of the host [20].On the other hand, the cyst serves as the infective stage, with environmental resistance allowing cysts to be excreted in the host's stool and transmitted to a new host through ingestion [21,22].Giardia duodenalis exhibits eight flagella which contribute to its motility.Moreover, molecular methods have identified and categorized seven assemblages based on their genetic characteristics and host specificity [23].Enterocytozoon bieneusi is a microsporidium that can be found in various hosts, including cattle and humans, highlighting its zoonotic potential [24,25], Infection can arise from either direct or indirect contact with fecal matter [26].The progression of infection involves spores introducing the infective sporoplasm into host enterocytes via the discharged polar tube.This is succeeded by the formation of meronts, which mature into multinucleated types [27,28].These plasmodia then undergo sporogony, leading to the creation of sporoblasts.Eventually, fully developed spores emerge, subsequently exiting the infected cells and being expelled in the stool [27].Blastocystis sp. has emerged as the prevailing single-celled intestinal parasite in human populations, particularly in developing countries, where its prevalence often exceeds 50% [29].This widespread protozoan parasite is widely acknowledged as one of the most common intestinal pathogens, regularly encountered in both humans and various animal species [30] including cattle, sheep, goat, mammals, birds, reptiles, and insects [31,32].This unicellular, anaerobic protozoan parasite commonly resides in the large intestine, displaying a diverse life cycle that showcases its remarkable adaptability [33], and the infective cyst stage, known for its resilience, plays a pivotal role in facilitating the transmission of the parasite between hosts [34].Conversely, recent evidence suggests a potential association between the amoeboid form of Blastocystis and its ability to cause disease [34].Balantioides coli is the only ciliate parasite capable of infecting humans [4].Pigs have been identified as the main reservoir for this parasite [35].However, recent data suggest that other mammals, including cattle, sheep, and goats, can also serve as potential reservoirs for B. coli [4].The life cycle of this parasite is characterized by its simplicity, commencing with the formation of dormant cysts, and its ability to replicate potentially invasive, ciliated trophozoites [3].Upon excystation in the small intestine, ciliated, motile trophozoites are released, colonizing the lumen of the large intestine [3].Within the large intestine, the parasite undergoes multiplication by binary fission.Both cysts and trophozoites are excreted in stool, with only cysts retaining infectivity for a new host [34].
Despite the significance of this industry, there is currently a lack of information regarding the circulation of zoonotic enteropathogenic protists, particularly on asymptomatic animals which carry particular risk for silent shedding.Therefore, the primary objective of this study was to detect and characterize the circulation of Cryptosporidium spp., G. duodenalis, E. bieneusi, Blastocystis sp., and B. coli in domestic asymptomatic ruminants from Portugal.

Sample Collection
A total of 302 individual stool samples were collected.Bovine stool samples were collected in June/July 2015 from seven farms located in Setúbal, Évora, and Santarém districts, central/southern region of Portugal (Figure 1).Of the total bovine sampled (n = 166), 142 were from intensive and 24 were from extensive farming systems.
parasite commonly resides in the large intestine, displaying a diverse life cycle that showcases its remarkable adaptability [33], and the infective cyst stage, known for its resilience, plays a pivotal role in facilitating the transmission of the parasite between hosts [34].Conversely, recent evidence suggests a potential association between the amoeboid form of Blastocystis and its ability to cause disease [34].Balantioides coli is the only ciliate parasite capable of infecting humans [4].Pigs have been identified as the main reservoir for this parasite [35].However, recent data suggest that other mammals, including cattle, sheep, and goats, can also serve as potential reservoirs for B. coli [4].The life cycle of this parasite is characterized by its simplicity, commencing with the formation of dormant cysts, and its ability to replicate potentially invasive, ciliated trophozoites [3].Upon excystation in the small intestine, ciliated, motile trophozoites are released, colonizing the lumen of the large intestine [3].Within the large intestine, the parasite undergoes multiplication by binary fission.Both cysts and trophozoites are excreted in stool, with only cysts retaining infectivity for a new host [34].
Despite the significance of this industry, there is currently a lack of information regarding the circulation of zoonotic enteropathogenic protists, particularly on asymptomatic animals which carry particular risk for silent shedding.Therefore, the primary objective of this study was to detect and characterize the circulation of Cryptosporidium spp., G. duodenalis, E. bieneusi, Blastocystis sp., and B. coli in domestic asymptomatic ruminants from Portugal.

Sample Collection
A total of 302 individual stool samples were collected.Bovine stool samples were collected in June/July 2015 from seven farms located in Setúbal, Évora, and Santarém districts, central/southern region of Portugal (Figure 1).Of the total bovine sampled (n = 166), 142 were from intensive and 24 were from extensive farming systems.Stool samples from animals at farms were collected directly from the rectum of the animals or from freshly defecated stool.The collection of stool samples from the slaughterhouse focused on the posterior section of the large intestine.Stools were collected if they were found to have a well-structured texture and hence with no signs of gastrointestinal disease.None of the ruminants were sacrificed for the purpose of this study.All stool samples were promptly stored at 4 • C and transported to the laboratory within 12 h of collection.Upon arrival, the samples were subsequently preserved at −20 • C until the DNA extraction process, which was completed within 2 weeks after their initial collection.

DNA Extraction
Fecal suspensions (10%) were prepared in phosphate-buffered saline at pH 7.2 and subsequently centrifuged for 5 min at 8000× g.The extraction and purification of DNA was conducted simultaneously using the QIAamp Cador Pathogen Mini Kit (Qiagen, Hilden, Germany), following the manufacturer's instructions.Specifically, 200 µL of the clarified supernatants were processed using the QIAcube ® automated platform (Qiagen).The eluted DNA was then appropriately stored at −80 • C with RNase-free water.

Molecular Detection of Cryptosporidium spp.
To identify Cryptosporidium spp., a nested PCR was performed, amplifying the 587-bp fragment of the ssu rRNA gene with the primer sets CR-P1/CRP2 and CR-P3/CPB-DIAGR, as described in [36].

Molecular Detection of Giardia duodenalis
For the detection of G. duodenalis, a nested PCR strategy was employed.The initial primer pair RH11-derivates and Gia2150c was used to amplify a 497-bp product, followed by a secondary primer pair RH11-derivates and RH4-derivates, amplifying a 293-bp fragment.The method was used as described in [37].

Molecular Detection of Enterocytozoon bieneusi
The detection of E. bieneusi was attempted through a nested PCR, amplifying the 390-bp fragment using the primer sets EBITS3/EBITS4 and EBITS1/EBITS2-4, as described in [38].

Molecular Detection of Blastocystis sp.
The detection of Blastocystis sp. was conducted using the method developed by [39].This method employs a direct PCR approach that amplifies a 600-bp region of the SSU rRNA gene with the pan-Blastocystis barcode primer set RD5/BhRDr.

Molecular Detection of Balantioides coli
To attempt the detection of B. coli, a direct PCR was performed following the method outlined by [40].The method targets the complete ITS1-5.8s-rRNA-ITS2region and the last 117 bp (3' end) of the ssu rRNA gene, resulting in the amplification of a 400-bp product, utilizing the primer set B5D/RD5.

General Procedures
Oligonucleotides used for the molecular detection of the parasites described above are shown in Table 1.

Statistical Analysis
Information and first-data processing was conducted using Microsoft Office 365 Excel.Descriptive analysis was performed using IBM SPSS version 28.0.0.0 software for Windows (SPSS, Chicago, IL, USA)].For all of the analysis, the confidence interval (CI) was established at 95%.
All endpoint, nested, and semi-nested PCR reactions were performed on a T100 thermocycler (Bio-Rad).The reaction mixtures included Fast PCR Mastermix (GriSP ® ) and 2× Xpert Fast Hotstart Mastermix (GriSP ® ).After PCR amplification, the DNA fragments were separated and visualized through electrophoresis on 1.5% agarose gels stained with Xpert Green Safe DNA gel dye (GriSP ® ).Electrophoresis was carried out at 120 V for 25 min.To confirm the results, UV light was used for irradiation of the agarose gels.
In cattle, no evidence of parasitic presence was found; for goats, no evidence of G. duodenalis or B. coli was found; and for sheep, neither E. bieneusi, B. coli nor Cryptosporidium spp.were detected.
Overall, at least 26 of the 302 animals in this study were positive for at least one of the studied protozoa.One of the four goats positive for E. bieneusi was also positive for C. xiaoi.Phylogenetic trees inferred for Cryptosporidium spp., E. bieneusi, and Blastocystis sp.sequences confirmed the classifications via BLAST (Figures 2-4).
100% identity with sequences from goat and sheep from China, KM199758, MH059800, and OL376579), one sequence as C. parvum (98.37% identity with a sequence from a human in The Netherlands, MH796372), and one as C. ubiquitum (99.55% identity with a sequence from an alpaca from China, MN876847).
In cattle, no evidence of parasitic presence was found; for goats, no evidence of G. duodenalis or B. coli was found; and for sheep, neither E. bieneusi, B. coli nor Cryptosporidium spp.were detected.
Overall, at least 26 of the 302 animals in this study were positive for at least one of the studied protozoa.One of the four goats positive for E. bieneusi was also positive for C. xiaoi.Phylogenetic trees inferred for Cryptosporidium spp., E. bieneusi, and Blastocystis sp.sequences confirmed the classifications via BLAST (Figures 2-4).Life (iTOL).The samples in this study are in shown in bold and plus 22 strains of different Cryptosporidium species obtained from GenBank are shown without bold or shading and are identified with the accession number and its species and country of origin.
Comparing our findings with other studies on the same host, the present study found 12.7% of goats to be shedding Cryptosporidium spp., a number notably lower than that reported in other European countries, with 65.7% in Greece [43], 53.3% in France [44], and 19.1% in Spain [45].Alerts should be made to the fact that different detection methods (PCR, microscopic) were used in the different studies, yielding distinct sensitivities and specificities.Moreover, even when comparing studies also using molecular biology tools, distinct nucleic acid extraction methods and even the use of different oligonucleotide primers which target different genomic regions could have alone or combined produced results with different sensitivities.
Enterocytozoon bieneusi was only found in goats, occurring in 6.35% (4/63, 95% [CI]: 1.76-15.47) of these animals.Enterocytozoon bieneusi is one of the most frequently identified microsporidia in humans and domestic as well as wild animals worldwide [46], responsible for causing 90% of human microsporidiosis [47].The presence of E. bieneusi is of particular concern due to its zoonotic potential, posing a threat to both human and livestock health [27].
In this study, Blastocystis sp. was found in both sheep and goats, occurring in 9.59% and 12.70% of these animals, respectively.In comparison to the available data, the occurrence in sheep of 9.59% (7/73, 95% [CI]: 3.94-18.76)was lower than that reported in Greenland (14%), Iran (32.0%),Turkey (38.2%), and China (16.26%) [51][52][53][54].Interestingly, the prevalence of Blastocystis sp. in sheep remains relatively understudied in Europe.Also, interestingly, all Blastocystis sequences from sheep were classified as Blastocystis sp.In the present study, goats presented a higher occurrence of Blastocystis than sheep, 12.70% (8/63, 95% [CI]: 5.65-23.56),with all of cases being classified as Blastocystis sp.In this study, Blastocystis occurrence in goats was again lower when compared to Malaysia (30.9%) [55], Italy (44.4%) [56], and China (33.63%) [57].This could be due not only to differences in diagnostic performances but also to genetic and eco-epidemiological differences between sampled animals which could ultimately influence the circulation of this agent.Noteworthy, animals can serve as reservoirs for Blastocystis sp.due to its poor host specificity and zoonotic potential.This parasite is commonly detected in fecal samples from both animals and humans worldwide and is estimated to cause nearly one billion human infections, making it one of the most commonly found parasites in humans [31,52,58].Despite our findings, one cannot exclude the possibility of these numbers underrepresenting the true scenario of these enteropathogenic agents circulating in healthy domestic ruminants in Portugal.When analyzing stool samples, care should be taken since PCR inhibitors can be found, such as phenolic compounds, glycogen, fats, cellulose, constituents of bacterial cells, nontarget nucleic acids, and heavy metals, leading to false negatives [59].
In this study, the results suggest that within Portuguese farming systems, healthy ruminants pose limited zoonotic impact as reservoirs for enteropathogenic parasites.Unlike previous studies on the topic which have used microscopy to ascertain the circulation of fecal protozoa in ruminants, [1] we used molecular biology tools such as PCR and nested PCR, known for their superior sensitivity and specificity.Hence, despite the low occurrence found in the present study, the likelihood for false positives is low.
Overall, at least 26 of the 302 animals in this study were positive for at least one of the studied protozoa.Interestingly, the majority of the stools positive for protozoa were retrieved from the slaughterhouse processing facility, and this likely represents the highest risk of transmission from ruminants to humans.Factors such as disparities in animal management practices, variations in sample sizes utilized in research, local climatic and environmental conditions, potential contamination of feed and water sources, and the overall health and immune status of the animals may alone or in combination influence the data shown here.

Conclusions
The prevalence of gastrointestinal parasites in Portuguese ruminants (cattle, sheep, and goats) is relatively low, suggesting a limited role as enteropathogenic protist reservoirs.However, silent shedding of zoonotic parasites like C. parvum, C. ubiquitum, E. bieneusi, and Blastocystis sp. was found, thus requiring ongoing surveillance.These findings provide a baseline for understanding parasite epidemiology and improving livestock and food safety.Preventing pathogen transmission requires a comprehensive approach with biosecurity and raising awareness among consumers, veterinarians, and farm owners, which can mitigate the impact on human and animal health.The use of sensitive detection methods and regular surveillance will be essential for monitoring any changes in parasite prevalence over time and developing effective disease control strategies for Portuguese ruminants.

Figure 1 .
Figure 1.Geographical distribution of sampled animals from Portugal tested for Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, Blastocystis sp., and Balantioides coli.Ovine stool samples were collected in two periods, June/July 2015 and March 2023.The 2015 samples (n = 23) were from intensive farming systems.The 2023 samples (n = 50) were from intensive farming systems from Viseu and Guarda (n = 41) and from a slaughterhouse (n = 9) located in the central region of Portugal.Caprine stool samples (n = 63) were collected also in March 2023 and from the same slaughterhouse as sheep stools.Stool samples from animals at farms were collected directly from the rectum of the animals or from freshly defecated stool.The collection of stool samples from the slaughterhouse focused on the posterior section of the large intestine.Stools were collected if they were found to have a well-structured texture and hence with no signs of gastrointestinal disease.None of the ruminants were sacrificed for the purpose of this study.All stool samples

Figure 2 .
Figure 2. Phylogenetic analysis of Cryptosporidium found in ruminants.Tree was inferred using the MEGA X maximum likelihood method (Tamura 3-parameter model) and the Interactive Tree of

Figure 2 .
Figure 2. Phylogenetic analysis of Cryptosporidium found in ruminants.Tree was inferred using the MEGA X maximum likelihood method (Tamura 3-parameter model) and the Interactive Tree of Life (iTOL).The samples in this study are in shown in bold and plus 22 strains of different Cryptosporidium species obtained from GenBank are shown without bold and are identified with the accession number and its species and country of origin.

Figure 3 .
Figure 3. Phylogenetic analysis of E. bieneusi found in ruminants.Tree was inferred using the MEGA X maximum likelihood method (Hasegawa-Kishino-Yano) and the Interactive Tree of Life (iTOL).The samples in this study are in shown in bold and 25 strains of different Enterocytozoon species obtained from GenBank are shown without bold or shading and are identified with the accession number and its species and country of origin.

Figure 4 .
Figure 4. Phylogenetic analysis of Blastocystis sp.found in ruminants.Tree was inferred using the MEGA X maximum likelihood method (Tamura 3-parameter model) and the Interactive Tree of Life

Figure 3 .
Figure 3. Phylogenetic analysis of E. bieneusi found in ruminants.Tree was inferred using the MEGA X maximum likelihood method (Hasegawa-Kishino-Yano) and the Interactive Tree of Life (iTOL).The samples in this study are in shown in bold and 25 strains of different Enterocytozoon species obtained from GenBank are shown without bold and are identified with the accession number and its species and country of origin.

Figure 3 .
Figure 3. Phylogenetic analysis of E. bieneusi found in ruminants.Tree was inferred using the MEGA X maximum likelihood method (Hasegawa-Kishino-Yano) and the Interactive Tree of Life (iTOL).The samples in this study are in shown in bold and 25 strains of different Enterocytozoon species obtained from GenBank are shown without bold or shading and are identified with the accession number and its species and country of origin.

Figure 4 .
Figure 4. Phylogenetic analysis of Blastocystis sp.found in ruminants.Tree was inferred using the MEGA X maximum likelihood method (Tamura 3-parameter model) and the Interactive Tree of Life

Figure 4 .
Figure 4. Phylogenetic analysis of Blastocystis sp.found in ruminants.Tree was inferred using the MEGA X maximum likelihood method (Tamura 3-parameter model) and the Interactive Tree of Life (iTOL).The samples in this study are in shown in bold and 30 strains of different Blastocystis species obtained from GenBank are shown without bold and are identified with the accession number and its species and country of origin.

Table 1 .
Oligonucleotides used for the molecular detection of enteropathogenic protists in asymptomatic domestic ruminants from Portugal.
have been shown.It is worth noting that there are limited data available on the prevalence of B. coli in sheep, indicating the need for further research in this area.The prevalence of E. bieneusi in sheep has been reported in Sweden (45.0%),Brazil (19.2%), and Slovakia (4.4%) [78-80].