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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. [ABSTRACT FROM AUTHOR]

Simple Summary: The Iberian lynx is an iconic feline species endemic to the Iberian Peninsula. Since the second half of the past century, its global population has decreased dramatically to the brink of extinction as a consequence of human-driven activities (habitat reduction and transformation, illegal hunting, road kills, density decrease in natural preys) and infectious diseases. Fortunately, the successful implementation of conservation programs has reversed this gloomy trend, allowing for an increase in the Iberian lynx population to over 1600 free-ranging animals in 2022. Regarding infectious diseases, very little is known on the epidemiology and health impact of the diarrhoea-causing intestinal protozoan parasites Cryptosporidium and Giardia in the Iberian lynx. To tackle these questions, we investigated the presence and molecular diversity of both pathogens in 256 collected faecal samples from 251 free-ranging and captive Iberian lynxes in Spain during the period 2017–2023. Our results demonstrate that Cryptosporidium (2.4%) and Giardia (27.9%) are present at different frequencies in the surveyed individuals. Our molecular analyses also indicate that a significant proportion of the Cryptosporidium infections detected are caused by strains that are typically found in the preys the Iberian lynx feed on. Interestingly, we also found that the Iberian lynx can harbour genetic variants of Cryptosporidium and Giardia with the potential to infect humans, although the likelihood of such events is judged low due to the light infections detected in the investigated animals. 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. [ABSTRACT FROM AUTHOR]

Hepatitis E virus (HEV) is an emerging zoonotic pathogen in Europe. In the Iberian Peninsula, wild boar (Sus scrofa) is considered the main wildlife reservoir of HEV. This wild ungulate shares habitat and resources with other potential HEV carriers in Iberian Mediterranean ecosystems, although information about the role of such sympatric species in the HEV epidemiological cycle is still very limited. The aims of the present large‐scale, long‐term study were: (1) to determine the seroprevalence and prevalence of HEV in both free‐living and captive populations of the Iberian lynx (Lynx pardinus), the most endangered felid in the world; (2) to determine potential risk factors associated with HEV exposure in this species and (3) to evaluate the dynamics of seropositivity in longitudinally sampled animals during the study period. Between 2010 and 2021, serum samples from 275 Iberian lynxes were collected in free‐ranging and captive populations across the Iberian Peninsula. Forty‐four of the 275 lynxes were also longitudinally sampled during the study period. A double‐antigen sandwich ELISA was used to test for the presence of antibodies against HEV. A subset of seropositive samples was analysed by Western blot (WB) assay to confirm exposure to HEV. In addition, serum, liver and/or faecal samples from 367 individuals were tested for orthohepevirus RNA by RT‐PCR. A total of 50 (18.2%; 95% CI: 14.1–23.2) of the 275 animals analysed had anti‐HEV antibodies by ELISA. Exposure to HEV was confirmed by WB in most of the ELISA‐positive Iberian lynxes analysed. Significantly higher seroprevalence was found in captive (33.6%) compared to free‐ranging (7.4%) individuals. Within captive population, the GEE model identified 'age' (senile, adult and subadult) as risk a factor potentially associated with HEV exposure in the Iberian lynx. Thirteen (29.5%) of 44 longitudinally surveyed individuals seroconverted against HEV during the study period. HEV RNA was detected in the faeces of one (1/364; 0.3%; 95% CI: 0.0–0.8) free‐ranging adult animal sampled in 2021. Phylogenetic analysis showed that the sequenced strain belongs to HEV‐3f subtype and shared a high nucleotide sequence identity (97–99.6%) with human HEV‐3f sequences from Spain and France. To the best of the authors' knowledge, this is the first survey study on HEV in the Iberian lynx and the first molecular report of HEV‐A infection in free‐ranging felines. Our results indicate high exposure to HEV‐3 in Iberian lynx populations, particularly those kept in captivity. The serological results suggest widespread but not homogeneous circulation of HEV in Iberian lynx populations. Further studies are required to assess the epidemiological role of this endangered species as a potential spillover host of HEV. [ABSTRACT FROM AUTHOR]