Epidemiologic Survey of Crimean-Congo Hemorrhagic Fever Virus in Suids, Spain.

We conducted a cross-sectional study in wild boar and extensively managed Iberian pig populations in a hotspot area of Crimean-Congo hemorrhagic fever virus (CCHFV) in Spain. We tested for antibodies against CCHFV by using 2 ELISAs in parallel. We assessed the presence of CCHFV RNA by means of reverse transcription quantitative PCR protocol, which detects all genotypes. A total of 113 (21.8%) of 518 suids sampled showed antibodies against CCHFV by ELISA. By species, 106 (39.7%) of 267 wild boars and 7 (2.8%) of 251 Iberian pigs analyzed were seropositive. Of the 231 Iberian pigs and 231 wild boars analyzed, none tested positive for CCHFV RNA. These findings indicate high CCHFV exposure in wild boar populations in endemic areas and confirm the susceptibility of extensively reared pigs to CCHFV, even though they may only play a limited role in the enzootic cycle.

Serosurvey of Blood Donors to Assess West Nile Virus Exposure, South-Central Spain.

We analyzed West Nile Virus (WNV) exposure from 1,222 blood donors during 2017-2018 from an area of south-central Spain. Results revealed WNV seroprevalence of 0.08% (95% CI 0.004%-0.4%) in this population. Our findings underscore the need for continued surveillance and research to manage WNV infection in this region.

Bluetongue Virus in the Iberian Lynx (Lynx pardinus), 2010-2022.

Clinical infection and death caused by bluetongue virus infection has been reported in the Eurasian lynx. Bluetongue virus surveillance in the Iberian lynx revealed widespread and repeated exposure to serotypes 1 and 4 in wild and captive populations of this species. This exposure is possibly from a spillover event from sympatric ruminants.

Detection of Rat Hepatitis E Virus in Pigs, Spain, 2023.

We identified rat hepatitis E virus (HEV) RNA in farmed pigs from Spain. Our results indicate that pigs might be susceptible to rat HEV and could serve as viral intermediaries between rodents and humans. Europe should evaluate the prevalence of rat HEV in farmed pigs to assess the risk to public health.

Acute Hepatitis in Children Due to Rat Hepatitis E Virus.

Two of 11 children with acute hepatitis of unknown origin were found to have rat hepatitis E virus infection. This infection should be considered in the differential diagnosis of children with acute hepatitis of unknown origin.

Survey of severe acute respiratory syndrome coronavirus 2 in captive and free-ranging wildlife from Spain.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), considered a zoonotic agent of wildlife origin, can infect various animal species, including wildlife in free-range and captive environments. Detecting susceptible species and potential reservoirs is crucial for preventing the transmission, spread, genetic evolution, and further emergence of viral variants that are major threats to global health. This study aimed to detect exposure or acute infection by SARS-CoV-2 in 420 animals from 40 different wildlife species, including terrestrial and aquatic mammals, from different regions of Spain during the 2020-2023 coronavirus disease 19 (COVID-19) pandemic. In total, 8/137 animals were positive for SARS-CoV-2 antibodies against the receptor binding domain and/or viral nucleoprotein according to independent ELISAs. However, only one ELISA-positive sample of a captive bottlenose dolphin (Tursiops truncatus) tested positive for SARS-CoV-2 neutralizing antibodies with a low titre (SNT50 38.15) according to a virus neutralization test. Cetaceans are expected to have a high risk of infection with SARS-CoV-2 according to early predictive studies due to the similarity of their angiotensin converting enzyme 2 cell receptor to that of humans. Moreover, of 283 animals analysed for SARS-CoV-2 RNA using RT-qPCR, none tested positive. Our results reinforce the importance of considering cetaceans at risk for SARS-CoV-2 infection and support taking preventive biosecurity measures when interacting with them, especially in the presence of individuals with suspected or confirmed COVID-19. Although most animals in this study tested negative for acute infection or viral exposure, ongoing surveillance of wildlife species and potentially susceptible animals is important to prevent future spillover events and detect potential novel reservoirs.

Detection and genotyping of zoonotic microsporidia in the endangered Iberian lynx (Lynx pardinus).

Microsporidia is a diverse group of obligate, intracellular, and spore-forming parasites that infect a wide range of animals. Among them, Enterocytozoon bieneusi and Encephalitozoon spp. are the most frequently reported species in humans. Limited information is available about the presence and molecular diversity of microsporidian species in the endangered Iberian lynx (Lynx pardinus). Presence of Enterocytozoon bieneusi and Encephalitozoon spp. was investigated by molecular methods in wild and captive Iberian lynxes from Spain. Overall, E. bieneusi was detected in 3.2% (8/251) of the animals examined. None of the samples tested were positive for Encephalitozoon spp. Four known (D, EbfelA, PigEBITS7, and Type IV) and a novel (named as LynxSpEb1) E. bieneusi genotypes were identified. All the genotypes found belonged to the zoonotic Group 1 of E. bieneusi. This study provides the first genotyping data of E. bieneusi in Iberian lynx in Spain. Our result indicate that the Iberian lynx does not seem to play a relevant role in the epidemiology of Encephalitozoon spp., and that this endangered felid is likely acting as spillover host rather than a true reservoir of E. bieneusi. Additional studies should be conducted to assess the impact of this parasite in the health status of the endangered Iberian lynx.

Hepatitis E Virus Infections in Free-Ranging and Captive Cetaceans, Spain, 2011-2022.

Epidemiologic surveillance of hepatitis E virus in over 300 free-ranging and captive cetaceans in waters off Spain revealed extensive exposure to this pathogen. We suggest the persistent and widespread presence of hepatitis E in the marine environment off the coast of Spain may be driven by terrestrial sources of contamination.

Development and Clinical Validation of a Pangenotypic PCR-Based Assay for the Detection and Quantification of Hepatitis E Virus (Orthohepevirus A Genus).

The objective of this study was to design a pangenotypic PCR-based assay for the detection and quantification of hepatitis E virus (HEV) RNA from across the entire spectrum of described genotypes belonging to the Orthohepevirus A genus. The optimal conditions and the performance of the assay were determined by testing the WHO standard strain (6219/10) and the WHO HEV panel (8578/13). Similarly, performance comparisons were made with two commercial assays (Real Star HEV RT-PCR 2.0 and ampliCube HEV 2.0 Quant) to detect HEV RNA at concentrations below 1,000 IU/ml with viral strains from the WHO and to test samples from 54 patients with acute hepatitis. The assay presented in this study was able to detect the entire spectrum of described genotypes belonging to the Orthohepevirus A genus, demonstrating better performance than both commercial kits. This procedure may represent a significant improvement in the molecular diagnosis of HEV infection.

Exposure to Coxiella burnetii in Wild Lagomorphs in Spanish Mediterranean Ecosystems.

Coxiella burnetii is an important zoonotic pathogen of worldwide distribution that can infect a wide range of wild and domestic species. The European wild rabbit (Oryctolagus cuniculus) can play a role as a reservoir for this bacterium in certain epidemiological scenarios, but, to date, a very limited numbers of large-scale serosurveys have been conducted for this species worldwide. Although exposure in hare species has also been described, C. burnetii in Iberian hare (Lepus granatensis) has never been assessed. Here, we aimed to determine the seroprevalence and risk factors associated with C. burnetii exposure in wild lagomorphs in the Mediterranean ecosystems of southern Spain. Between the 2018/2019 and 2021/2022 hunting seasons, blood samples from 638 wild lagomorphs, including 471 wild rabbits and 167 Iberian hares, were collected from 112 hunting grounds distributed across all eight provinces of Andalusia (southern Spain). The overall apparent individual seroprevalence was 8.9% (57/638; 95% CI: 6.8-11.4). Antibodies against C. burnetii were found in 11.3% (53/471; 95% CI: 8.4-14.1) of the wild rabbits and 2.4% (4/167; 95% CI: 0.1-4.7) of the Iberian hares. Seropositive animals were detected for 16 (14.3%; 95% CI: 7.8-20.8) of the 112 hunting grounds tested and in all the hunting seasons sampled. A generalized estimating equations model showed that the geographical area (western Andalusia) and presence of sheep were risk factors potentially associated with C. burnetii exposure in wild lagomorphs. A statistically significant spatial cluster (p < 0.001) was identified in the south-west of Andalusia. Our results provide evidence of moderate, endemic and heterogeneous circulation of C. burnetii in wild lagomorph populations in Spanish Mediterranean ecosystems. Risk-based strategies for integrative surveillance programs should be implemented in these species to reduce the risk of transmission of the bacterium to sympatric species, including humans.

Monitoring of Coxiella burnetii in the Iberian lynx (Lynx pardinus).

Coxiella burnetii is a multi-host bacterium of major public and animal health concern. This pathogen circulates among several wild species in the Iberian Peninsula, however, the role of the Iberian lynx (Lynx pardinus) in the epidemiology of this emerging pathogen is still unknown. The objective of this work was to assess the circulation of C. burnetii in Iberian lynx populations from the Iberian Peninsula and to study the molecular characterisation of this pathogen in lynxes and their feeding ticks. A total of 922 lynxes, including free-ranging and captive individuals, were sampled between 2010 and 2022 for the collection of sera (n = 543), spleen samples (n = 390) and ticks (n = 357 from 61 lynxes). The overall seroprevalence was 7.7 % (42/543; 95 %CI: 5.5-10.0 %), with age being significantly associated with the C. burnetii exposure in free-ranging lynxes. A longitudinal study was also carried out to assess the dynamics of the circulation of C. burnetii in this wild host, revealing that 7 of the 37 longitudinally surveyed individuals seroconverted during the study period. The PCR prevalence was 4.4 % (17/390, 95 %CI: 2.3-6.4 %) for spleen samples and 1.1 % (4/357; 95 % CI: 0.0-2.2) in ticks. This is the first study to evaluate the circulation of C. burnetii in the Iberian lynx and to confirm the infection in this felid. The results obtained show a moderate, wide, homogeneous, and endemic circulation of this bacterium in the Iberian lynx populations.

Prevalence of Paslahepevirus balayani in commercial swine food products from Spain.

Paslahepevirus balayani (formerly known as hepatitis E virus) is an emerging cause of foodborne disease in Europe, transmitted mainly by the consumption of raw or undercooked pork. Since little is known about the presence of the virus in several pork products that are eaten uncooked, our aim was to evaluate the prevalence of Paslahepevirus balayani in groups of commercial pork products intended for human consumption subjected to different processing techniques. A total of 1265 samples of pork products from Spain were divided into four groups and tested for the presence of Paslahepevirus balayani RNA: unprocessed pig and wild boar meat frozen at -20 °C (n = 389), dry-cured pork products (n = 391), dry-cured and salted pork products (n = 219), and boiled products (n = 266) (none of these products contained pork liver). Five samples were positive for Paslahepevirus balayani RNA (overall prevalence: 0.4%; 95% CI: 0.17% - 0.92%). All positive samples were from unprocessed meat stored at -20 °C, with a prevalence in this group of 1.3% (95% CI: 0.42-3.44); two samples came from pigs (1.1%; 95% CI: 0.13-3.81) and three from wild boar (1.5%; 95% CI: 0.31-4.28). None of the pork samples in the other groups was positive. In conclusion, Paslahepevirus balayani was found in unprocessed swine products form Spain, but not in processed products intended to be consumed undercooked, demonstrating that transmission of this zoonotic virus by eating these pork products should be more seriously considered.

Identification and characterization of a novel circovirus in Iberian lynx in Spain.

Circoviruses cause severe disease in pigs and birds. Canine circovirus has thus far only been associated with respiratory and gastrointestinal disorders and systemic disease in dogs. The Iberian lynx (Lynx pardinus) is one of the most endangered carnivores in Europe and the most endangered felid worldwide. Exploring the virome of these animals may be important in terms of virus discovery and assessing the interspecies-circulation of viruses from related carnivores. In this study, 162 spleen samples from Iberian lynx were screened for CRESS DNA viruses. Overall, 11 (6.8%) of 162 samples tested positive using a consensus PCR. Partial rep sequences were tightly related to each other (96.6-100%). Specific molecular protocols were designed on the partial rep sequences of the novel virus, Iberian lynx-associated circovirus-1 (ILCV-1). By screening a subset of 45 spleen samples, the infection rate of ILCV-1 in Iberian lynxes was 57.8% (26/45). ILCV-1 strains formed a separate cluster intermingled with bat, rodent, mongoose, and felid circoviruses. The genome of the novel virus displayed the highest nucleotide identity (64.3-65.3%) to mongoose circoviruses, thus representing a novel candidate circovirus species. The detection of these viruses in the spleen tissues could suggest systemic infection in the animal host. Overall, these findings suggest that this novel circovirus is common in the Iberian lynx. Further studies are warranted to assess the possible health implications of ILCV-1 in this endangered species.

Occurrence of Coxiella burnetii in wild lagomorphs and their ticks in Spanish Mediterranean ecosystems.

BACKGROUND: Coxiella burnetii, the causative agent of Q fever, is a zoonotic multi-host vector-borne pathogen of major public health importance. Although the European Food Safety Authority has recently made the monitoring of this bacterium in wildlife a priority, the role of wild lagomorphs in the transmission and maintenance of C. burnetii is poorly understood. AIMS: The aims of this study were to determine the prevalence and risk factors associated with C. burnetii circulation in European wild rabbits (Oryctolagus cuniculus) and Iberian hares (Lepus granatensis) and to assess the presence of this pathogen in ticks that feed on them in Mediterranean ecosystems in Spain, the country with the highest number of reported cases of Q fever in Europe. METHODS: A total of 574 spleen samples were collected from 453 wild rabbits and 121 Iberian hares, and 513 ticks (processed in 120 pools) between the 2017/2018 and 2021/2022 hunting seasons. RESULTS: C. burnetii DNA was detected in 103 (17.9%; 95% CI: 14.8-21.1) of the 574 wild lagomorphs tested. By species, prevalence was 16.3% (74/453; 95% CI: 12.9-19.7) in the European wild rabbit and 24.0% (29/121; 95% CI: 16.4-31.6) in the Iberian hare. At least one positive lagomorph was found on 47.9% of the 96 hunting estates sampled and in every hunting season since 2018/2019. Two risk factors associated with C. burnetii infection were as follows: outbreak of myxomatosis on the hunting estate in the month prior to sampling and high tick abundance observed by gamekeepers on the hunting estate. C. burnetii DNA was also found in 33 of the 120 (27.5%; 95% CI: 19.5-35.5) tick pools tested. The pathogen was detected in 66.7% (4/6), 29.2% (26/89) and 21.4% (3/14) of Haemaphysalis hispanica, Rhipicephalus pusillus and Hyalomma lusitanicum pools respectively. CONCLUSIONS: This study provides new epidemiological data on C. burnetii in European wild rabbits and is the first survey on this zoonotic pathogen performed in Iberian hares. Our results indicate widespread endemic circulation of C. burnetii and highlight the importance of both wild lagomorph species as natural reservoirs of this zoonotic bacterium in Mediterranean ecosystems in southern Spain, which may be of public and animal health concern. The high prevalence and wide diversity of positive tick species suggest the possible role of ticks in the epidemiological cycle of C. burnetii, with the potential risk of transmission to sympatric species, including humans.

Absence of Crimean-Congo hemorrhagic fever virus in wild lagomorphs and their ticks in Spanish Mediterranean ecosystems.

Crimean-Congo hemorrhagic fever virus (CCHFV) is an emerging tick-borne pathogen in different European countries. Since 2013, clinical and fatality cases associated with CCHFV infection have been reported in humans in Spain. During the last few years, endemic circulation of this virus has been detected in ticks and wild ungulates in this country, but the role of other sympatric wild species in the sylvatic cycle of this multi-host virus remains poorly understood. The aims of the present study were to assess exposure to CCHFV in wild lagomorphs in southern Spain, a CCHFV endemic area, and to determine the presence of the virus in ticks feeding on these species. Serum samples from 473 European wild rabbits (Oryctolagus cuniculus) and 162 Iberian hares (Lepus granatensis), and 120 ticks feeding on 85 of these wild lagomorphs were collected on 133 hunting grounds between 2018/2019 and 2021/2022 hunting seasons. The presence of antibodies against CCHFV was assessed in all serum samples using a commercial ELISA, whereas ticks were tested for the presence of CCHFV-RNA by a multiplex RT-qPCR that detects all known genotypes of this virus. None of the 635 (0.0 %; 95 %CI: 0.0-0.6) lagomorphs tested had anti-CCHFV antibodies and CCHFV-RNA was not found in any of the 120 (0.0 %; 95 %CI: 0.0-3.0) ticks analyzed. To the best of the authors knowledge, this is the first epidemiological study conducted on CCHFV in Iberian hare worldwide. Our findings indicate absence of exposure to CCHFV in European wild rabbit and Iberian hare populations, as well as in their ticks, which suggests that they do not seem to play a relevant role in the epidemiology of CCHFV in Mediterranean ecosystems of southern Spain.

Epidemiological survey and risk factors associated with Sarcoptes scabiei in wild lagomorphs in Spanish Mediterranean ecosystems.

Although mortality caused by Sarcoptes scabiei has been reported in European wild rabbit (Oryctolagus cuniculus) and Iberian hare (Lepus granatensis), there is a lack of detailed information regarding the exposure of wild lagomorph species to this parasite. Here, we aimed to determine the seroprevalence and potential risk factors associated with S. scabiei exposure in European wild rabbits and Iberian hares in Mediterranean ecosystems of southern Spain. Between 2018/2019 and 2021/2022 hunting seasons, serum samples from 464 wild rabbits and 132 Iberian hares were collected from 100 hunting grounds in Andalusia (southern Spain). Sera were tested using an in-house indirect ELISA to detect specific anti-S. scabiei antibodies based on the immunodominant protein Ssλ20ΔB3. The overall apparent individual seroprevalence was 15.9% (95/596; 95%CI: 13.0-18.9). Antibodies against S. scabiei were detected in 11.6% (54/464; 95%CI: 8.7-14.5) of the European wild rabbits and 31.1% (41/132; 95%CI: 23.2-39.0) of the Iberian hares. Species (Iberian hare), age (adults) and geographical area (western Andalusia) were identified as risk factors potentially associated with S. scabiei exposure using generalized estimating equation analysis. By applying spatial analysis, two significant cluster of high seropositivity were detected in western and central Andalusia, respectively. The seroprevalence values obtained provide evidence of endemic, widespread and heterogeneous exposure to S. scabiei among wild lagomorph populations in Spanish Mediterranean ecosystems. Our findings underscore the importance of implementing integrated surveillance programs for sarcoptic mange in wild lagomorphs as well as in other sympatric species.

Longitudinal survey of hepatitis E virus in extensively raised pigs in Spain.

Hepatitis E virus (HEV) is an emerging zoonotic virus of public health concern, of which pigs, wild boar and red deer are the main reservoirs. The European Food Safety Authority (EFSA) has recently prioritized the development of monitoring programs of HEV at different stages of the pig food chain, including outdoor pig farming. Pigs managed under these extensive production systems frequently share habitat and natural resources with wild boar and red deer during fattening stages and cross-species transmission of HEV among these species has previously been suggested. In this context, we aimed to (I) to evaluate the risk of HEV circulation within the production phases of extensively raised pigs and at the domestic-wildlife interface, and (II) to identify the genotypes circulating within these hosts. A total of 1452 pigs from seven different pig farms were longitudinally sampled during the breeding, rearing, and fattening production phases. In addition, 138 and 252 sympatric wild boar and red deer, respectively, were analysed. Anti-HEV antibodies were found in 1245 (85.7 %) out of the 1452 Iberian pigs sampled. The seroprevalence was 30.4 % in the breeding phase, 95.4 % in the rearing phase and 97.0 % in the fattening phase. Statistically significant differences (P < 0.05) were found among the three production phases. The seroprevalence was significantly higher (P < 0.001) in fattening pigs compared to those found in sympatric wild boar (31.9 %) and red deer (2.0 %). Three (1.0 %) out of the 293 serum pools analysed were positive for viral RNA. One of them was identified in pigs at the rearing phase (genotype 3 f) and two in wild boar (genotypes 3 f and 3 m). The high seroprevalence detected in extensively raised pigs, together with the detection of the zoonotic HEV-3 f and HEV-3 m subtypes in sympatric domestic and wild swine, highlights the risk of zoonotic transmission and the need to establish surveillance programs and control measures, particularly in breeding and rearing phase, in these epidemiological scenarios.

Rat hepatitis E virus (Rocahepevirus ratti) in people living with HIV.

Rat hepatitis E virus (ratHEV; species Rocahepevirus ratti) is considered a newly emerging cause of acute hepatitis of zoonotic origin. ratHEV infection of people living with HIV (PLWH) might portend a worse, as with hepatitis E virus (HEV; species Paslahepevirus balayani), and consequently this group may constitute a high-risk population. We aimed to evaluate the prevalence of ratHEV by measuring viral RNA and specific IgG antibodies in a large Spanish cohort of PLWH. Multicentre study conducted in Spain evaluating PLWHIV included in the Spanish AIDS Research Network (CoRIS). Patients were evaluated for ratHEV infection using PCR at baseline and anti-ratHEV IgG by dot blot analysis to evaluate exposure to ratHEV strains. Patients with detectable ratHEV RNA were followed-up to evaluate persistence of viremia and IgG seroconversion. Eight-hundred and forty-two individuals were tested. A total of 9 individuals showed specific IgG antibodies against ratHEV, supposing a prevalence of 1.1 (95% CI; 0.5%-2.1%). Of these, only one was reactive to HEV IgG antibodies by ELISA. One sample was positive for ratHEV RNA (prevalence of infection: 0.1%; 95% CI: 0.08%-0.7%). The case was a man who had sex with men exhibiting a slightly increased alanine transaminase level (49 IU/L) as only biochemical alteration. In the follow-up, the patients showed undetectable ratHEV RNA and seroconversion to specific ratHEV IgG antibodies. Our study shows that ratHEV is geographical broadly distributed in Spain, representing a potential zoonotic threat.

Serological and molecular survey of rat hepatitis E virus (Rocahepevirus ratti) in drug users.

ABSTRACTRat hepatitis E virus (ratHEV) is an emerging cause of acute hepatitis of zoonotic origin. Since seroprevalence studies are scarce, at-risk groups are almost unknown. Because blood-borne infections frequently occur in people with drug use, who are particularly vulnerable to infection due to lack of housing and homelessness, this population constitutes a priority in which ratHEV infection should be evaluated. Therefore, the aim of this study was to evaluate the ratHEV seroprevalence and RNA detection rate in drug users as a potential at-risk population. We designed a retrospective study involving individuals that attended drug rehabilitation centres. Exposure to ratHEV was assessed by specific antibody detection using ELISA and dot blot (DB) assay and the presence of active infection by ratHEV RNA detection using RT-qPCR. Three-hundred and forty-one individuals were included, the most of them being men (67.7%) with an average age of 45 years. A total of 17 individuals showed specific IgG antibodies against ratHEV (4.6%; 95% CI; 3.1%-7.9%). One case of active ratHEV infection was identified (0.3%; 95% CI: 0.1%-1.8%). This was a 57-year-old homeless woman with limited financial resources, who had active cocaine and heroin use via parenteral route. In conclusion, we identified a potential exposure to ratHEV among drug users. Targeted studies in drug users with proper control groups are necessary to evaluate high-risk populations and transmission routes more accurately.

Detection and Molecular Diversity of Cryptosporidium spp. and Giardia duodenalis in the Endangered Iberian Lynx (Lynx pardinus), Spain.

Cryptosporidium spp. and Giardia duodenalis are the main non-viral causes of diarrhoea in humans and domestic animals globally. Comparatively, much less information is currently available in free-ranging carnivore species in general and in the endangered Iberian lynx (Lynx pardinus) in particular. Cryptosporidium spp. and G. duodenalis were investigated with molecular (PCR and Sanger sequencing) methods in individual faecal DNA samples of free-ranging and captive Iberian lynxes from the main population nuclei in Spain. Overall, Cryptosporidium spp. and G. duodenalis were detected in 2.4% (6/251) and 27.9% (70/251) of the animals examined, respectively. Positive animals to at least one of them were detected in each of the analysed population nuclei. The analysis of partial ssu rRNA gene sequences revealed the presence of rodent-adapted C. alticolis (n = 1) and C. occultus (n = 1), leporid-adapted C. cuniculus (n = 2), and zoonotic C. parvum (n = 2) within Cryptosporidium, and zoonotic assemblages A (n = 5) and B (n = 3) within G. duodenalis. Subgenotyping analyses allowed for the identification of genotype VaA19 in C. cuniculus (gp60 locus) and sub-assemblages AI and BIII/BIV in G. duodenalis (gdh, bg, and tpi loci). This study represents the first molecular description of Cryptosporidium spp. and G. duodenalis in the Iberian lynx in Spain. The presence of rodent/leporid-adapted Cryptosporidium species in the surveyed animals suggests spurious infections associated to the Iberian lynx's diet. The Iberian lynx seems a suitable host for zoonotic genetic variants of Cryptosporidium (C. parvum) and G. duodenalis (assemblages A and B), although the potential risk of human transmission is regarded as limited due to light parasite burdens and suspected low excretion of infective (oo)cysts to the environment by infected animals. More research should be conducted to ascertain the true impact of these protozoan parasites in the health status of the endangered Iberian lynx.