First report of Enterocytozoon bieneusi in Erinaceus europaeus and Hemiechinus auritus Hedgehogs from recovery centers of Portugal.

Enterocytozoon bieneusi microsporidia are emerging pathogens infecting a wide range of vertebrate and invertebrate hosts, known to have zoonotic features since they infect both wild and domestic animals, and humans. Despite their significance, there is very limited epidemiological data on microsporidia in hedgehogs, especially European hedgehogs (Erinaceus europaeus) and long-eared hedgehogs (Hemiechinus auritus), the former known as synantropic hedgehogs, and the latter suited as pets. As such, the present study aimed to assess the presence of E. bieneusi in hedgehogs from Portugal. For this purpose, fecal samples from 110 hedgehogs of three species - E. europaeus (n=106), H. auritus (n=1), and Atelerix albiventris (n=3) - were collected and tested for E. bieneusi by PCR targeting the ITS region and the flanking small and large subunit of the rRNA. We found an overall occurrence of 22.7% (25/110; 95% [CI]: 15.28-31.70), with 22.6% (24/106; 95% [CI]: 15.08-31.79) in E. europaeus, 100% (1/1) in H. auritus, and 0% in A. albiventris. Interestingly, three novel genotypes were identified, all belonging to the potentially zoonotic Group 1. Our findings highlight the importance of hedgehogs as potential reservoirs for E. bieneusi and emphasize the need for further research to understand their role in transmission dynamics and assess the associated risks to public and veterinary health.

Synanthropic hedgehogs were tested for Enterocytozoon bieneusi, the main cause of human microsporidiosis. Results showed 22.7% of hedgehogs were shedding E. bieneusi spores, with three new genotypes from the zoonotic Group 1. Hedgehogs may transmit to humans/animals, warranting more research.

Detection of Enterocytozoon bieneusi in Non-Human Primates in Portuguese Zoos.

Enterocytozoon bieneusi, an intracellular eukaryote closely related to fungi, is recognized as a significant pathogen affecting humans, particularly those with compromised immune systems. While its transmission routes are still not fully elucidated, fecal-oral transmission remains the primary one. With a wide host range, the zoonotic potential of E. bieneusi is a concern, albeit direct evidence of animal-to-human transmission remains scarce. Genotyping based on the internal transcribed spacer (ITS) region facilitates the delineation of genetic diversity, with potentially zoonotic genotypes predominantly associated with Groups 1 and 2. Despite the broad spectrum of susceptible animal hosts, research into microsporidian infection among zoo animals remains limited. This study aimed to evaluate the occurrence of E. bieneusi infection across diverse captive animals, focusing on zoo settings in Portugal. Fecal samples were collected from a variety of animals, and molecular detection of E. bieneusi was conducted using nested PCR targeting the ITS region. Of 127 fecal samples, 1.57% (95% CI: 0.19-5.57) tested positive for E. bieneusi, with non-human primates (NHP's) exhibiting an 18.18% (95% CI: 2.28-51.78) occurrence. Phylogenetic analysis revealed clustering within Group 2 genotypes, indicating potential zoonotic implications. This study highlights the need for further research to understand the epidemiology of E. bieneusi in zoo environments and its potential transmission pathways to humans.

Hepatitis E virus in wild and domestic rabbits from Portugal: a combined molecular and longitudinal serological study.

Hepatitis E virus (HEV), species Paslahepevirus balayani, poses a global public health threat, especially in developing countries, by causing acute enterically transmitted hepatitis. HEV infects various mammalian hosts and belongs to the genus Paslahepevirus in the family Hepeviridae. While swine are recognized as the main hosts of HEV, rabbits, which can also be affected by swine HEV-3 related strains, serve as the primary reservoir for the distinct emerging and zoonotic HEV-3ra subtype. In Portugal, where the European wild rabbit is abundant, their role in HEV epidemiology remains unclear. The primary aim of the present research was to evaluate the circulation and the potential for HEV infection within these species. This study employed a molecular and longitudinal serological approach to investigate HEV in Portuguese rabbits. Among the 205 wild rabbits tested, a seroprevalence of 2.44% (95% CI: 0.80-5.60) was found, with no significant associations with age, sex, localization, or sampling dates. Seropositive animals were found in the south and center regions of the country. HEV RNA was not detected in 120 fecal samples, suggesting a natural, low level, and widespread viral circulation. The study underscores the need for further research to comprehend HEV dynamics in these species, which is crucial for assessing potential transmission risks to humans.

Molecular evidence of sporadic Coxiella burnetii excretion in sheep milk, central Portugal.

Coxiella burnetii is the etiologic agent of Q fever, a worldwide zoonosis. Cattle, sheep and goats are considered the main reservoirs of the disease. Transmission to humans occurs mainly through the inhalation of infectious aerosols from milk, faeces, urine, and birth products from infected ruminants. In this study, a 2-year longitudinal approach was performed to ascertain the excretion of C. burnetii in bulk tank milk samples of sheep from a mountain plateau in central Portugal, with sampling conducted during the years 2015 and 2016. From a total of 156 bulk tank milk samples tested by qPCR, only one showed to be positive for C. burnetii (1.28% [95%CI: 0.03-6.94]), from 2015, the first year of collection. Bidirectional sequencing and phylogenetic analysis of IS1111 transposase partial region confirmed the presence of C. burnetii DNA. The presence of C. burnetii in raw milk samples highlights the necessity for additional research to determine if raw milk is a potential source for human infection. Animal health surveillance and prevention measures against this zoonotic disease should be considered.

Hunting for Answers: Assessing Brucella spp. Seroprevalence and Risks in Red Deer and Wild Boar in Central Portugal.

Between 2016 and 2023, a cross-sectional study was conducted in the central region of Portugal in order to better understand the epidemiology and public health risks resulting from the handling and consumption of game animals infected with Brucella spp. The seroprevalence and risk factors for Brucella spp. seropositivity were evaluated. Antibodies against Brucella spp. were determined using a commercial enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. Results showed that in the 650 serum samples collected from red deer (n = 298) and wild boars (n = 352) in Portugal, 21.7% (n = 141; 95% CI: 18.6-25.1%) tested positive. Wild boar had a significantly higher prevalence (35.5%; 95% CI: 30.5-40.8%) than red deer (5.4%, 95% CI: 3.1-8.6%; p ≤ 0.001). Risk factors for seropositivity were investigated using multivariable logistic regression models. The odds of being seropositive was 8.39 (95% CI: 4.75-14.84; p ≤ 0.001) times higher in wild boar than in red deer. Correlations between sex, age, body condition, and seropositivity could not be observed. The higher seroprevalence in wild boar suggests that this species may primarily contribute to the Brucella spp. ecology in central Portugal.

Molecular detection of Enterocytozoon bieneusi in bats from Portugal.

Enterocytozoon bieneusi is a microsporidia commonly found in the gastrointestinal tract of humans and a wide range of other animals, constituting a major cause of microsporidiosis in humans. Although E. bieneusi has been detected in humans, domestic, and wild animals in Portugal, and its presence in bats has been linked to zoonotic characteristics, its occurrence in bats within the country has not been reported. In this study, we investigated the presence of E. bieneusi in 380 bat fecal samples collected in mainland Portugal through a nested PCR assay targeting the internal transcribed spacer region and the flanking small and large subunits of the ribosomal RNA. Enterocytozoon bieneusi was detected in one bat sample (i.e., 0.26%; Pipistrellus pipistrellus). Additionally, another sample tested positive for Enterocytozoon sp. Phylogenetic analysis of the obtained ITS sequence of E. bieneusi revealed clustering within the potentially zoonotic Group 1. This study represents the first report of E. bieneusi in a bat from Europe. Findings presented here contribute to an enhanced understanding of E. bieneusi epidemiology.

Enterocytozoon bieneusi is the most frequent cause of microsporidiosis in humans. In this study, E. bieneusi, belonging to a potentially zoonotic Group, was detected in 0.26% bat samples from Portugal, highlighting bats' potential role in transmitting this microsporidia to humans and other animals.

Seroprevalence study of brucellosis in wild boar hunted for private consumption in northeast Portugal.

Brucellosis is an important infectious disease caused by bacteria of the genus Brucella. In the northeast region of Portugal, infection with Brucella melitensis is endemic in small ruminants, and there are also humans' cases. However, the epidemiological role of the wild boar in the dynamics of this disease in this region is unknown. In this study, a total of 332 blood samples were collected from wild boar hunted in thirty-six hunting areas during the 2022/2023 hunting season. All were taken by the hunters for private consumption, with no evisceration or examination in the field. Serum samples were tested by indirect ELISA (i-ELISA). It was observed that 88 wild boars were exposed to Brucella spp., pointing to a seroprevalence of 26.5% (95% CI: 21.8 - 31.3%). This high prevalence underlines the importance that wild boar may have in the dynamics of this disease in the region and its potential transmission to other animals, and to humans (for example, during the handling of carcasses). Increased awareness and knowledge of brucellosis in wild boar is essential for the implementation of effective practices and habits and, consequently, for the control and prevention of this important zoonosis.

Hepatitis E Virus in Individuals Undergoing Heparin Therapy: An Observational Serological and Molecular Study.

INTRODUCTION: Heparin is derived from swine and has been suggested as a possible source of HEV. To study the potential risk of HEV infection associated with heparin treatment, two groups of individuals were compared. Sera from heparinized (N=93) and non-heparinized individuals (N=111) were tested for markers of acute HEV infection and anti-HEV IgG seroprevalence. METHODS: An acute HEV case was defined by the presence of anti-HEV IgM and/or HEV RNA. From the 93 heparinized individuals, one was positive for IgM and IgG anti-HEV and two were positive for HEV RNA (for both ORF3 and ORF2), and there were a total of two (2.2%) cases of current or recent HEV infection. From the 111 non-heparinized individuals, three were positive for IgM anti-HEV, one was positive for both IgM and IgG anti-HEV, and none was positive for HEV RNA, and there were a total of three (2.7%) cases of current or recent HEV infection. The difference between HEV cases in the heparinized individuals and the non-heparinized individuals was not statistically significant (2.2% vs. 2.7%; p = 0.799). RESULTS: Concerning IgG anti-HEV, it was detected in 32 individuals from the heparinized group and in 18 from the non-heparinized control group. A statistically significant difference was observed in the presence of anti-HEV IgG in heparinized individuals and controls (p = 0.003). CONCLUSION: This study has not found any association between heparin treatment and acute HEV infection, but has shown the use of therapeutic heparin as a risk factor for IgG anti-HEV seropositivity.

Detection of hepatitis E virus genotype 3 in an Algerian mouse (Mus spretus) in Portugal.

Virus monitoring in small mammals is central to the design of epidemiological control strategies for rodent-borne zoonotic viruses. Synanthropic small mammals are versatile and may be potential carriers of several microbial agents. In the present work, a total of 330 fecal samples of small mammals were collected at two sites in the North of Portugal and screened for zoonotic hepatitis E virus (HEV, species Paslahepevirus balayani). Synanthropic small mammal samples (n = 40) were collected in a city park of Porto and belonged to the species Algerian mouse (Mus spretus) (n = 26) and to the greater white-toothed shrew (Crocidura russula) (n = 14). Furthermore, additional samples were collected in the Northeast region of Portugal and included Algerian mouse (n = 48), greater white-toothed shrew (n = 47), wood mouse (Apodemus sylvaticus) (n = 43), southwestern water vole (Arvicola sapidus) (n = 52), Cabrera's vole (Microtus cabrerae) (n = 49) and Lusitanian pine vole (Microtus lusitanicus) (n = 51). A nested RT-PCR targeting a part of open reading frame (ORF) 2 region of the HEV genome was used followed by sequencing and phylogenetic analysis. HEV RNA was detected in one fecal sample (0.3%; 95% confidence interval, CI: 0.01-1.68) from a synanthropic Algerian mouse that was genotyped as HEV-3, subgenotype 3e. This is the first study reporting the detection of HEV-3 in a synanthropic rodent, the Algerian mouse. The identified HEV isolate is probably the outcome of either a spill-over infection from domestic pigs or wild boars, or the result of passive viral transit through the intestinal tract. This finding reinforces the importance in the surveillance of novel potential hosts for HEV with a particular emphasis on synanthropic animals.

A Thorny Tale of Parasites: Screening for Enteric Protozoan Parasites in Hedgehogs from Portugal.

Enteric protozoan parasites, such as Blastocystis sp., Balantioides coli, Cryptosporidium spp., and Giardia duodenalis, may have implications for both animal and human health.Transmitted through the fecal-oral route, these parasites cause symptoms such as diarrhea, abdominal pain, and weight loss. This study investigated the presence of these enteric protozoan parasites and genetically characterized them in hedgehogs from Portugal. A total of 110 hedgehog stool samples were collected. Molecular detection methods showed an overall occurrence of protozoa in 1.82% (2/110 95% CI: 0.22-6.41) of hedgehogs, with Blastocystis being found in one hedgehog and Cryptosporidium being found in another. No evidence for the presence of B. coli or G. duodenalis was found. This study suggests that there is a need to stay aware of hedgehogs as potential hosts of enteric protozoa. Ongoing research and surveillance efforts are recommended to explore practical prevention and control strategies. The results contribute to the limited knowledge of these parasites in Portuguese hedgehog populations and underscore their potential relevance to both veterinary and public health.

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.

Prevalence and Risk Factors for Hepatitis E Virus in Wild Boar and Red Deer in Portugal.

Hepatitis E virus (HEV) is a zoonotic foodborne virus with an annual infection prevalence of 20 million human cases, which seriously affects public health and economic development in both developed and developing countries. To better understand the epidemiology of HEV in Central Portugal, a cross-sectional study was conducted from 2016 to 2023 with sera samples from wild ungulates. The seroprevalence and risk factors for HEV seropositivity were evaluated in the present study. Specifically, antibodies against HEV were determined by a commercial enzyme-linked immune-sorbent assay (ELISA). Our results show that in the 650 sera samples collected from 298 wild red deer and 352 wild boars in Portugal, 9.1% red deer and 1.7% wild boar were positive for antibodies to HEV. Regarding age, the seropositivity in juvenile wild ungulates was 1.3%, whereas it was 7.2% in adults. Logistic regression models investigated risk factors for seropositivity. The odds of being seropositive was 3.6 times higher in adults than in juveniles, and the risk was 4.2 times higher in red deer than in wild boar. Both wild ungulate species were exposed to HEV. The higher seroprevalence in red deer suggests that this species may make a major contribution to the ecology of HEV in Central Portugal. Further research is needed to understand how wildlife affects the epidemiology of HEV infections in Portugal.

Hepatitis E Virus in the Wild Boar Population: What Is the Real Zoonotic Risk in Portugal?

Hepatitis E virus (HEV) is an important zoonosis in wild boar. Reported zoonotic cases are mainly associated with the consumption of raw/undercooked meat and/or liver. This study aims to determine the occurrence of HEV in the Portuguese wild boar population. During the hunting season 2021/2022, 123-matched samples (liver, faeces, and blood) were collected from hunted wild boars throughout Portugal. An RT-PCR assay tested liver and faeces samples to detect HEV-RNA. From blood samples, an ELISA test was performed. Only one liver sample was positive for HEV (0,8%) and one other from faeces. A total of 34 sera were seropositive (26.7%). At the same time, in a survey of 106 hunters, 21 consumed/ate the liver of wild boars (19.8%). Only three recognised the possibility of consuming it undercooked. Contrary to previous studies in Portugal, the prevalence of HEV in liver and faeces is low, but the seropositivity is higher. But, when analyzing in detail, it could be observed that an HEV hotspot exists in the southeast of central Portugal and that it is a zoonotic risk for hunters of this region. The data of this study reinforce the importance of including HEV in surveillance programs for wildlife diseases to expand the potential zoonotic information.

Antibody-Based Assessment of Coxiella burnetii Circulation in Algerian Goat Herds.

Q fever is a zoonotic disease caused by Coxiella burnetii (C. burnetii), a pathogen with a high capability for infection. The disease primarily affects ruminants, leading to reproductive disorders, but can also be transmitted to humans through contact with infected animals or their products. In Algeria, Q fever is endemic, but little is known about the presence and circulation of C. burnetii in domestic goats. This study aimed to perform a multicentric serological analysis of C. burnetii antibodies in domestic goats from four provinces in the North East Region of Algeria. A total of 504 goat serum samples were collected from 77 herds, and serological analysis was performed using an indirect ELISA. The overall seroprevalence at the herd level was 35.06%, and 8.73% at the individual level. Herds with a history of abortions showed a high seropositivity rate of 88.9%. This research indicates the wide distribution of C. burnetii in goats in this region, suggesting the potential for zoonotic transmission to humans. Further studies and monitoring programs are essential to gain a comprehensive understanding of C. burnetii epidemiology in Algeria and to prevent or mitigate potential outbreaks. Awareness among practitioners and farmers is crucial to address this public health concern effectively.

Diversity of Rickettsia spp. in ticks from wild mammals of Morocco and Mauritania.

Ticks are known as vectors and reservoirs of rickettsiae and, wildlife vertebrate hosts as suitable dispersers of ticks contributing to the life cycle of rickettsial agents in nature. In the herein study, the presence of rickettsiae was investigated in ticks from wild mammals (Gerbillus and Jaculus, Vulpes rueppellii, Canis anthus, Felis lybica and Felis margarita) in Mauritania and Morocco. Morphological and molecular analysis of ticks allowed their identification as Rhipicephalus sanguineus sensu lato and Hyalomma impeltatum. A total of 126 partially engorged adult ticks, collected from 40 animals, were screened for the presence of rickettsial DNA by conventional PCR targeting the ompB gene, followed by ompA and gltA targets and bidirectional sequencing. As a result of the sequence analyses, that at least three different species of pathogenic spotted fever group rickettsiae were detected. Rickettsia parkeri-like was detected in a R. sanguineus s.l. (n=1) collected from an African wildcat from Morocco. Rickettsia aeschlimannii was detected in a H. impeltatum (n=1) collected from a gerbil rodent. Rickettsia massiliae was detected in R. sanguineus s.l. ticks (n=5) collected from two Ruppells' foxes. The herein study demonstrates that pathogenic Rickettsia species are circulating in Morocco and Mauritania wildlife.

Hepatitis E Virus in the Iberian Peninsula: A Systematic Review.

One of the most frequent causes of acute viral hepatitis is hepatitis E virus (HEV) causing 20 million infections worldwide each year and 44,000 deaths. Studies on HEV in the Iberian Peninsula have been increasing through time with HEV infection being identified in humans and animals. The aim of the present systematic review was to compile and evaluate all the published data on HEV from studies performed in humans, animals and environmental samples in the Iberian Peninsula. The electronic databases Mendeley, PubMed, Scopus, and Web of Science were thoroughly searched, and research published up until February 01, 2023 were included. Resulting in a total of 151 eligible papers by full reading and application of PRISMA exclusion/inclusion criteria. Overall, the present review shows that several HEV genotypes, namely HEV-1, 3, 4, and 6 as well as Rocahepevirus, are circulating in humans, animals, and in the environment in the Iberian Peninsula. HEV-3 was the most common genotype circulating in humans in Portugal and Spain, as expected for developed countries, with HEV-1 only being detected in travelers and emigrants from HEV endemic regions. Spain is the biggest pork producer in Europe and given the high circulation of HEV in pigs, with HEV-3 being primarily associated to zoonotic transmission through consumption of swine meat and meat products, in our opinion, the introduction of an HEV surveillance system in swine and inclusion of HEV in diagnostic routines for acute and chronic human hepatitis would be important. Additionally, we propose that establishing a monitoring mechanism for HEV is crucial in order to gain a comprehensive understanding of the prevalence of this illness and the various strains present in the Iberian Peninsula, as well as their potential impact on public health.

A Systematic Review of Hepatitis E Virus Detection in Camels.

Hepatitis E virus (HEV) represents a major cause of acute hepatitis and is considered an emerging public health problem around the world. In the Middle East's and Africa's arid regions, where camels frequently interact with human populations and camel-derived food products are a component of the food chain, camel-borne zoonotic HEV infection is a potential threat. To date, no review paper has been published on HEV in camels. As such, the purpose of the current work is to provide a scientific review of the identification of HEV genotypes seven and eight in camels worldwide to have a better understanding of the current status of this topic and to identify gaps in the current knowledge. Searches were carried out in the electronic databases PubMed, Mendeley, Web of Science, and Scopus, including studies published until 31 December 2022 (n = 435). Once the databases were checked for duplicate papers (n = 307), the exclusion criteria were applied to remove any research that was not relevant (n = 118). As a result, only 10 papers were found to be eligible for the study. Additionally, in eight of the ten studies, the rates of HEV infection were found to be between 0.6% and 2.2% in both stool and serum samples. Furthermore, four studies detected HEV genotype seven in dromedary camels, and two studies have shown HEV genotype eight in Bactrian camels. Interestingly, these genotypes were recently reported in camels from the Middle East and China, where one human infection with HEV genotype seven has been associated with the consumption of contaminated camel meat and milk. In conclusion, more research will be needed to determine the prevalence of HEV infection in camels around the world as well as the risk of foodborne transmission of contaminated camel products. As camels are utility animals in several countries, HEV in these animals may pose a potential risk to public health.

Morphologic and Genetic Analysis of Synhimantus (Synhimantus) laticeps from a Long-Eared Owl (Asio otus).

The long-eared owl (Asio otus) is a medium-sized owl species that is well-distributed in almost all of the territories in Portugal. Nematodes were found in the oral cavity of a long-eared owl (A. otus) admitted to CRASSA (Wildlife Rehabilitation Centre of Santo André). During a physical exam and stabilization of the bird, five nematodes were collected. The worms were examined and measured under light microscopy, and photos were taken. After a morphological analysis was conducted, all the nematodes (five females) were identified as Synhimantus (Synhimantus) laticeps. Two specimens were subjected to molecular analysis, which confirmed the result. This study provides a combined morphological and genetic approach to S. laticeps. To the authors' best knowledge, this is the first report including genetic sequencing of S. laticeps in a long-eared owl (A. otus) from Portugal.

A Systematic Review and Meta-Analysis on Hepatitis E Virus Detection in Farmed Ruminants.

Swine are widely recognized as the main reservoir of zoonotic HEV; however, a growing body of data on the HEV prevalence in farmed ruminants of different species also points to a potential route for HEV transmission through ruminants and ruminant products and by-products. Definite information on the zoonotic potential of ruminants is still absent or unclear, compelling the necessity for increasing knowledge on this. The aim of the current study was to analyze the state-of-the-art in this research topic and provide a summary of HEV detection and characterization in farmed ruminants. A total of 1567 papers were retrieved from four search databases that resulted in 35 eligible papers after application of exclusion/inclusion criteria. Studies on HEV in farmed ruminants were mainly based on the detection of HEV RNA and were reported in Africa (n = 1), America (n = 3), Asia (n = 18) and Europe (n = 13), and focused on a variety of ruminants species, namely cow, goat, sheep, deer, buffalo and yak. The overall pooled prevalence of HEV was 0.02% (0.01-0.03, 95% CI). The subgroup pooled prevalence of HEV RNA was 0.01% (0.00-0.02, 95% CI) in cow milk, stool, serum, liver, intestinal, bile, blood, spleen and rectal swab samples; 0.09% (0.02-0.18, 95% CI) in goat serum, bile, stool, milk, liver, rectal swab and blood samples; 0.01% (0.00-0.04, 95% CI) in sheep stool, serum, milk, blood and liver samples. Most of the HEV genotypes found in farmed ruminants belonged to the zoonotic HEV-3 (subtypes 3a, 3c) and HEV-4 (subtype 4d, 4h), with Rocahepevirus also found. The wide HEV circulation observed in different farmed ruminants raises concerns for the possibility of HEV transmission through products from infected ruminants and alerts for the potential zoonotic route for HEV in ruminant products, such as meat and dairy products. Also, contact exposure to infected farmed animals could be a risk factor. Further research should be conducted in order to understand the circulation of HEV in these animals and its zoonotic potential, as there is currently a lack of data on this topic.

Detection and Molecular Characterization of Giardia and Cryptosporidium spp. Circulating in Wild Small Mammals from Portugal.

Cryptosporidium spp. and Giardia spp. are important diarrhea-causing protozoan parasites worldwide that exhibit broad host ranges. Wild small mammals can harbor host-adapted and potentially zoonotic species of both parasites. The aim of this study was to investigate Cryptosporidium spp. and Giardia spp. in wild rodents and shrews in Portugal, focusing on the protist's occurrence and genetic diversity. Molecular screening by PCR at the small subunit (SSU) rRNA gene locus of 290 fecal samples from wood mice (Apodemus sylvaticus), southwestern water voles (Arvicola sapidus), Cabrera's voles (Microtus cabrerae), Lusitanian pine voles (Microtus lusitanicus), Algerian mice (Mus spretus) and greater white-toothed shrews (Crocidura russula) in Northeast Portugal revealed the low occurrence of Cryptosporidium spp. (1%) and high occurrence of Giardia spp. (32.8%). The analysis revealed that "species" was the only significant factor associated with the increasing probability of Giardia spp. infection, with the highest prevalence reported in southwestern water voles and Lusitanian pine voles. Cryptosporidium and Giardia species determination at the SSU rRNA gene locus revealed C. muris and G. microti as the only circulating species, respectively. Subtyping of the glutamate dehydrogenase (gdh) and beta-giardin (bg) genes provided evidence of the high genetic diversity within the G. microti clade. This study suggests that rodent-adapted G. microti occurs to a large extent in cricetid hosts and supports the limited role of wild rodents and shrews as natural sources of human infections in Northeast Portugal regarding the investigated parasites. Moreover, this is the first record of G. microti in southwestern water voles, Lusitanian pine voles, Algerian mice, wood mice and Cabrera's voles and C. muris in Cabrera's voles. Finally, this study improves the database of sequences relevant for the sequence typing of G. microti strains and provides new insights about the epidemiology of Giardia spp. and Cryptosporidium spp. in wild rodents and shrews, two parasite genera of high importance for public and animal health.