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.

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.

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.

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.

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.

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.

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.

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.

Monitoring the epidemic of West Nile virus in equids in Spain, 2020-2021.

The largest epidemic of West Nile virus (WNV) reported ever in Spain in both humans and equines occurred in 2020, affecting 77 humans and 139 equine herds. Here, we aimed to monitor the outbreaks detected in equid herds in Andalusia (southern Spain), the Spanish region where 89.9% of the outbreaks were reported, and to evaluate the virus circulation and risk factor associated with WNV exposure in the affected herds. The first WNV case was detected in mid-July 2020, the number of outbreaks peaked in mid-August and the last one was confirmed on 26th October 2020. WNV lineage 1 was detected in 12 clinically affected horses using real time RT-PCR. Molecular analysis evidenced high nucleotide identity with WNV sequences obtained from humans, birds and mosquitoes from Spain and Italy between 2020 and 2022. Between five and eight months after the WNV epidemic, a total of 724 equids (including 485 unvaccinated and 239 vaccinated animals) from 113 of the 125 affected herds in Andalusia were sampled. IgM and IgG antibodies against WNV were detected in 1.6% (8/485; 95%IC: 0.0-2.5) and 61.9% (300/485; 95%IC: 58.3-65.5) of the unvaccinated individuals, respectively. The seropositivity in vaccinated horses was 86.6% (207/239). The main risk factors associated with WNV exposure in unvaccinated equids were the breed (crossbreed), the location of animals in spring-summer (outside), and the presence of natural water ponds close to the surveyed herds. The high individual seroprevalence obtained in the affected herds indicates that WNV circulation was more widespread than the reported by passive surveillance during the WNV epidemic in 2020. The re-emergence of WNV in 2020 in southern Spain evidenced the needed to improve integrated surveillance systems, minimizing the impact of future cases in equids and humans in high-risk areas.

Butchering activity is the main risk factor for hepatitis E virus (Paslahepevirus balayani) infection in southwestern Nigeria: a prospective cohort study.

Introduction: Paslahepevirus balayani (Hepatitis E virus; HEV) is an emerging virus that poses as a public health threat. The virus is now reported to be the leading cause of acute viral hepatitis, with a unique impact on African settings. Our aim was to evaluate the prevalence and risk factors for HEV infection in three cohorts (animal handlers, villagers, and students). Methods: A prospective cross-sectional study was carried out on a total of 752 subjects from southwestern Nigeria. In all individuals, anti-HEV IgG and anti-HEV IgM antibodies were evaluated by using ELISA (confirming positive results via immunoblotting), and serum viral RNA was evaluated by using two RT-PCR assays. Results: The overall seroprevalence of HEV IgG and HEV IgM was 14.9% (95% CI: 12.5-17.6%) and 1.3% (95% CI: 0.7-2.5%), respectively. We observed the highest seroprevalence among animal contact individuals, with butchers being the population with the highest HEV IgG seroprevalence (31.1%). Similarly, HEV IgM was higher in the animal contact group (2.2%) than in the non-animal contact cohort (0%). Discussions: Viral RNA was not detected in any of the samples. Butchering was significantly associated with higher HEV prevalence. Although all efforts to prevent HEV in Africa have focused on the chlorination of water, our study suggests that most new infections could currently be linked to animal manipulation. Therefore, education and guidelines must be provided in southwest Nigeria to ensure that animal handling and processing methods are safe.

Lack of associations of microRNAs with severe NAFLD in people living with HIV: discovery case-control study.

Background & objective: Nonalcoholic fatty liver disease (NAFLD) is highly prevalent in people living with HIV (PLWH) and the expression of some microRNAs could be useful as biomarkers for the diagnosis of NAFLD. The aim of this study was to identify patterns of differential expression of microRNAs in PLWH and assess their diagnostic value for NALFD. Methods: A discovery case-control study with PLWH was carried out. The expression of miRNAs was determined using HTG EdgeSeq technology. Cases were defined as patients with severe NAFLD and controls as patients without NAFLD, characterized using the controlled attenuation parameter (CAP). Cases and controls were matched 1:1 for age, sex, BMI, CD4+ lymphocyte count, active HCV infection, and ART regimen. Results: Serum 2,083 simultaneous microRNA transcripts were analyzed using HTG technology and compared between cases and controls. Forty-five patients, 23 cases, and 22 controls were included in the study. In the analysis of the expression pattern of the 2,083 microRNAs, no differential expression patterns were found between both groups of patients included in the study. Conclusion: Analysis of the microRNA transcriptome profile of nonobese PLWH with severe NAFLD did not appear to differ from that of patients without NAFLD. Thus, microRNA might not serve as a proper biomarker for predicting severe NALFD in this population.

Epidemiological survey and risk factors associated with Paslahepevirus balayani in equines in Europe.

Paslahepevirus balayani (HEV) is an important emerging zoonotic virus in Europe. Although domestic pigs and wild boar are the main reservoirs of this pathogen, susceptibility to this virus has been confirmed in a growing number of animal species, including equines. However, their role in the epidemiology of this virus remains poorly understood. Our aim was to assess HEV circulation and identify potential risk factors associated with exposure in equid species in different European countries. A total of 596 equines, including 496 horses, 63 donkeys and 37 mules/hinnies bred in four European countries (Spain, Italy, United Kingdom and Ireland) were sampled. Thirty-three animals (5.5%; 95%CI: 3.7-7.4) had anti-HEV antibodies. Seropositivity was found in 4.6% of horses, 11.1% of donkeys and 8.1% of mules/hinnies tested. By country, 6.3%, 5.4%, 5.0% and 4.0% of the equines sampled in Spain, Italy, United Kingdom and Ireland, respectively, were seropositive, respectively. Statistical analysis showed that "species" and "drinking water from ponds and streams" were potential risk factors associated with HEV seropositivity in equines in Europe. HEV RNA was not detected in any (0.0%; 95%CI: 0.0-1.8) of the 202 equines tested. Our results provide evidence of a low, spatially homogeneous and widespread viral circulation that is not equal across species in equid populations in the European countries analyzed and indicate that these species appear to play a limited role in the epidemiology of this virus. Further studies are required to elucidate the differences in seroprevalence between donkeys, mules/hinnies and horses and to determine the risk of zoonotic transmission of this pathogen from equid species.

Iberian wild leporidae as hosts of zoonotic enteroparasites in Mediterranean ecosystems of Southern Spain.

Wild lagomorphs including rabbits and hares can act as natural carriers or reservoirs of bacterial and parasitic zoonotic diseases. However, little is known on the epidemiology and potential public health significance of intestinal eukaryotes in wild leporids. We examined faecal samples from European wild rabbits (Oryctolagus cuniculus, n = 438) and Iberian hares (Lepus granatensis, n = 111) collected in the Autonomous Region of Andalusia in southern Spain during 2012-2021. We searched for the presence of DNA from the main intestinal protist and microsporidial pathogens of veterinary and public health concerns using molecular methods (PCR followed by Sanger and next-generation sequencing). Giardia duodenalis was the most prevalent species found (27.8%, 153/550; 95% CI: 24.1-31.8), followed by Cryptosporidium spp. (1.3%, 7/550; 95% CI: 0.5-2.6), Blastocystis sp. (1.1%, 6/550; 95% CI: 0.4-2.4) and Encephalitozoon intestinalis (0.2%, 1/550; 95% CI: 0.0-10.1). All samples tested negative for Enterocytozoon bieneusi. Sequence analyses revealed the presence of sub-assemblage BIV (n = 1) within G. duodenalis, and Cryptosporidium cuniculus (n = 6) and Cryptosporidium andersoni (n = 1) within Cryptosporidium. The presence of ruminant-adapted C. andersoni is indicative of a potential cross-species transmission event, although a spurious infection (mechanical carriage) cannot be ruled out. Samples assigned to C. cuniculus belonged to the gp60 subtype families Va (n = 3) and Vb (n = 2). The six Blastocystis-positive samples were identified as ST2 (n = 3) and ST1 + ST2 (n = 3). Our molecular results suggest that wild rabbits and hares were primarily infected by leporid-adapted species of eukaryotic pathogens. However, the occasional findings of zoonotic G. duodenalis sub-assemblage BIV, Blastocystis sp. ST1 and ST2, and Encephalitozoon intestinalis could be of public health relevance.

Optimization of the molecular diagnosis of the acute hepatitis E virus infection.

To evaluate the diagnostic value of the combination of two broad-range PCR assays targeting two different and conserved regions of the viral genome for the diagnosis of acute Hepatitis E virus (HEV) infection. Patients with acute hepatitis were prospectively recruited. In all, HEV-IgM antibodies were tested together with evaluation of HEV viraemia by two PCR assays (ORF3 and ORF1). The number of individuals exhibiting negative IgM antibody results but carrying viral RNA was calculated by each PCR assay. Four-hundred and seventy individuals were included, of whom 145 (30.8%) were diagnosed as having acute HEV. Of them, 122 (84.1%) exhibited HEV-IgM antibodies, and 81 (55.8%) had detectable viral RNA for at least one PCR. Using the ORF3 molecular assay, 70 (48.3%) individuals were identified with HEV infection. When the ORF1 molecular assay was applied, 49 (33.8%) individuals were identified. The ORF3 assay detected viral RNA in 32 patients not detected by the ORF1 assay. In contrast, the ORF1 assay could amplify viral RNA in 11 patients who were not detected by the ORF3 assay. The parallel use of two broad-range PCR assays significantly increased the performance of the molecular diagnosis of HEV.

Temporal changes in the genotypes of Paslahepevirus balayani in southern Spain and their possible link with changes in pig trade imports.

Introduction: Paslahepevirus balayani (HEV) is an endemic zoonotic disease ranked as a major cause of acute hepatitis in Europe. Most infections occurring in Europe are due to the endemic several subtypes of genotype 3, through the consumption of raw or undercooked pork, observing a genotype geographical distribution pattern among countries Because of global changes in the pig and pork trading markets, subtype distribution might vary. We aimed to evaluate the temporal distribution of HEV genotypes in patients from southern Spain with acute hepatitis to determine whether these changes were related to the pig import trade during the study period between 2018 and 2022. Methods: Prospective longitudinal study including patients with acute hepatitis from southern Spain between 2018 and 2022. HEV RNA and antibodies was tested in all patients. In patients with detectable HEV RNA, genotype was obtained. To determine the number of imported pigs and their origins, we checked the official data from the Spanish statistics on international trade of Spanish Minister of Industry during by country of origin during the same study period. Results: A total of 659 patients with acute hepatitis were included in the study. Among them, 162 (24.5%) had at least one marker (IgM or RNA) of acute HEV infection. Among the 71 patients with detectable viral RNA, genotypes could be obtained for 58 (81.6%). The most prevalent HEV genotype was 3f (n = 48; 78.6%), showing a decreasing prevalence of over time, from 100% in 2018 to 70.6% in 2022. Since 2021, the emergence of other genotypes has been determined. A significant increase in the number of animals imported was observed since the beginning of the study. Denmark experienced a significant rise, from 0.03% in 2018 of total imports to 10.4% in 2022. Conclusions: HEV molecular diversity is changing in Spain, could be linked to changes in fattening pig import origin.

Prevalence of selected tick-borne pathogens in wild ungulates and ticks in southern Spain.

A survey study was carried out to assess the occurrence of selected tick-borne pathogens (TBP) in wild ungulates in Mediterranean ecosystems in southern Spain. Spleen samples were collected from 1,132 wild ungulates, including 578 red deer, 269 wild boar, 135 mouflon, 121 fallow deer and 29 roe deer, between 2009 and 2015. Eighty-nine ticks collected from TBP-positive animals were also analysed. Samples were tested by PCR and sequenced whenever possible. TBP DNA was detected in 127 of 863 wild ruminants (14.7%; 95% CI: 12.4-17.3) including the following: Anaplasma phagocytophilum (9.2%), Babesia divergens (2.9%), Theileria sp. OT3 (1.7%), Borrelia afzelii (0.7%) and Theileria capreoli (0.2%), but no positive samples were detected in wild boar (0/269). All the strains from mouflon were identified as Theileria sp. OT3, while B. divergens and T. capreoli were mainly found in red deer. Co-infection with A. phagocytophilum and B. divergens, and A. phagocytophilum and Theileria spp. was detected in red deer and mouflon, respectively. The risk factor analysis showed that the prevalences of A. phagocytophilum and piroplasms were species-related. Eighty-nine tick specimens collected from ungulates found to be infected with the selected TBP were identified as Hyalomma lusitanicum (95.5%) and Ixodes ricinus (4.5%). Thirty ticks were positive for Anaplasma/Ehrlichia spp. (33.7%), 25 for Babesia/Theileria (28.1%) and two for B. burgdorferi s.l. (2.3%). Eleven specimens showed co-infections with Anaplasma/Ehrlichia and Babesia/Theileria (10.1%) or Anaplasma/Ehrlichia and B. burgdorferi s.l. (2.3%). The estimated prevalences obtained in the present study suggest the possible contribution of wild ruminants to the maintenance of some selected TBP in Mediterranean ecosystems in southern Spain, while the role of wild boar in the epidemiology of these pathogens seems to be limited in this region.