Emergence of epizootic hemorrhagic disease in red deer (Cervus elaphus), Spain, 2022.

Epizootic hemorrhagic disease (EHD) virus serotype 8 (EHDV-8) emerged in Spain in autumn 2022. In this study, we aimed to (1) characterize the clinical and lesional presentation of EHDV infection in European red deer (Cervus elaphus), and (2) study the spatial spread of the virus in wild ruminants in Spain after its introduction, in 2022/2023. We confirmed EHDV infection in two clinically compatible sick red deer by PCR and detection of anti-EHDV specific antibodies. EHDV infection occurred in red deer with hyperacute to acute clinical signs and lesions associated to vascular changes leading to death of the animals. Partial sequences of variable segment 2 (VP2) and segment 5 (NS1) genes of the detected viruses had >99% nucleotide identity with EHDV-8 sequences from Tunisia and Italy. In a cross-sectional serological study of EHDV in 592 wild ruminants, mainly red deer (n=578), in southwestern Spain, we detected anti-EHDV antibodies in 37 of 592 samples (6.3%; 95% confidence interval: 4.3-8.2), all from red deer and from the localities where clinical cases of EHD were confirmed in red deer. We conclude that EHDV-8 infection causes severe EHD in European red deer. The serosurvey revealed a limited spread of EHDV-8 in Spanish wild ruminant populations in the first year of virus detection in Spain.

Crimean-Congo haemorrhagic fever virus in questing non-Hyalomma spp. ticks in Northwest Spain, 2021.

Crimean-Congo haemorrhagic fever (CCHF) unexpectedly emerged in humans in Northwest Spain in 2021, and two additional cases were reported in the region in 2022. The 2021 case was associated with a tick bite on the outskirts of the city where the patient lived. PCR analysis of 95 questing ticks collected in the outskirts of that city in 2021, none of the genus Hyalomma, revealed a prevalence of confirmed CCHF virus (CCHFV) infection of 10.5%. Our results in this emerging scenario suggest the need to consider that CCHFV may be effectively spreading to Northwest Spain and to urgently understand any possible role of non-Hyalomma spp. ticks in the eco-epidemiological dynamics of CCHFV.

Animal Exposure Model for Mapping Crimean-Congo Hemorrhagic Fever Virus Emergence Risk.

To estimate the determinants of spatial variation in Crimean-Congo hemorrhagic fever virus (CCHFV) transmission and to create a risk map as a preventive public health tool, we designed a survey of small domestic ruminants in Andalusia, Spain. To assess CCHFV exposure spatial distribution, we analyzed serum from 2,440 sheep and goats by using a double-antigen ELISA and modeled exposure probability with environmental predictors by using generalized linear mixed models. CCHFV antibodies detected in 84 samples confirmed low CCHFV prevalence in small domestic ruminants in the region. The best-fitted statistical model indicated that the most significant predictors of virus exposure risk were cattle/horse density and the normalized difference vegetation index. Model validation showed 99.7% specificity and 10.2% sensitivity for identifying CCHFV circulation areas. To map CCHFV exposure risk, we projected the model at a 1 × 1-km spatial resolution. Our study provides insight into CCHFV ecology that is useful for preventing virus transmission.

High Exposure to Livestock Pathogens in Southern Pudu (Pudu puda) from Chile.

A significant gap in exposure data for most livestock and zoonotic pathogens is common for several Latin America deer species. This study examined the seroprevalence against 13 pathogens in 164 wild and captive southern pudu from Chile between 2011 and 2023. Livestock and zoonotic pathogen antibodies were detected in 22 of 109 wild pudus (20.18%; 95% CI: 13.34-29.18) and 17 of 55 captive pudus (30.91%; 95% CI: 19.52-44.96), including five Leptospira interrogans serovars (15.38% and 10.71%), Toxoplasma gondii (8.57% and 37.50%), Chlamydia abortus (3.03% and 12.82%), Neospora caninum (0.00% and 9.52%), and Pestivirus (8.00% and 6.67%). Risk factors were detected for Leptospira spp., showing that fawn pudu have statistically significantly higher risk of positivity than adults. In the case of T. gondii, pudu living in "free-range" have a lower risk of being positive for this parasite. In under-human-care pudu, a Pestivirus outbreak is the most strongly suspected as the cause of abortions in a zoo in the past. This study presents the first evidence of Chlamydia abortus in wildlife in South America and exposure to T. gondii, L. interrogans, and N. caninum in wild ungulate species in Chile. High seroprevalence of livestock pathogens such as Pestivirus and Leptospira Hardjo in wild animals suggests a livestock transmission in Chilean template forest.

Testing the efficiency of capture methods for questing Hyalomma lusitanicum (Acari: Ixodidae), a vector of Crimean-Congo hemorrhagic fever virus.

Available methods to census exophilic tick populations have limitations in estimating true population size due to their inability to capture a high proportion of the actual tick population. We currently ignore the efficacy of these methods to capture questing Hyalomma spp. ticks, vectors of the Crimean-Congo hemorrhagic fever virus. To address the need of accurately estimating questing densities of Hyalomma spp., we designed a field experiment to test the efficacy of blanket dragging, blanket flagging, CO2-baited traps, and an ad hoc designed method, absolute surface counts, in capturing adult Hyalomma lusitanicum ticks from known numbers of preset fluorescent-marked ticks. The experiment was designed in 2 stages to estimate the point (1-day sampling) and cumulative (3-day serial sampling) efficacy of the methods under varying sampling effort and habitat. Tick survival, host interference, and weather effects on efficacy were controlled for in multiple regression models. There was high variability in method efficacy for capturing ticks, which was also modulated by effort, habitat, tick density, hosts, and soil temperature. The most effective method was absolute surface counts for both point estimates (39%) and cumulative efficacy (83%). CO2-baited traps reached a maximum efficacy of 37%, while blanket dragging and blanket flagging captured a maximum of the 8% of the marked ticks. Our results reveal the strengths and weaknesses of the different tick capture methods applied to adult H. lusitanicum and lay the groundwork for more accurate inferences about the true size of exophilic tick populations.

Mapping the risk of exposure to Crimean-Congo haemorrhagic fever virus in the Iberian Peninsula using Eurasian wild boar (Sus scrofa) as a model.

Crimean-Congo haemorrhagic fever (CCHF) virus (CCHFV) is a tick-borne zoonotic pathogen that can cause a lethal haemorrhagic disease in humans. Although the virus appears to be endemically established in the Iberian Peninsula, CCHF is an emerging disease in Spain. Clinical signs of CCHFV infection are mainly manifested in humans, but the virus replicates in several animal species. Understanding the determinants of CCHFV exposure risk from animal models is essential to predicting high-risk exposure hotspots for public health action. With this objective in mind, we designed a cross-sectional study of Eurasian wild boar (Sus scrofa) in Spain and Portugal. The study analysed 5,291 sera collected between 2006 and 2022 from 90 wild boar populations with a specific double-antigen ELISA to estimate CCHFV serum prevalence and identify the main determinants of exposure probability. To do so, we statistically modelled exposure risk with host- and environment-related predictors and spatially projected it at a 10 × 10 km square resolution at the scale of the Iberian Peninsula to map foci of infection risk. Fifty-seven (63.3 %) of the 90 populations had at least one seropositive animal, with seroprevalence ranging from 0.0 to 88.2 %. Anti-CCHFV antibodies were found in 1,026 of 5,291 wild boar (19.4 %; 95 % confidence interval: 18.3-20.5 %), with highest exposure rates in southwestern Iberia. The most relevant predictors of virus exposure risk were wild boar abundance, local rainfall regime, shrub cover, winter air temperature and soil temperature variation. The spatial projection of the best-fit model identified high-risk foci as occurring in most of western and southwestern Iberia and identified recently confirmed risk foci in eastern Spain. The results of the study demonstrate that serological surveys of CCHFV vector hosts are a powerful, robust and highly informative tool for public health authorities to take action to prevent human cases of CCHF in enzootic and emergency settings.

Insights into the spatiotemporal dynamics of West Nile virus transmission in emerging scenarios.

The incidence of West Nile fever (WNF) is highly variable in emerging areas, making it difficult to identify risk periods. Using clinical case records has important biases in understanding the transmission dynamics of West Nile virus (WNV) because asymptomatic infections are frequent. However, estimating virus exposure in sentinel species could help achieve this goal at varying spatiotemporal scales. To identify the determinants of inter-annual variation in WNV transmission rates, we designed a 15-year longitudinal seroepidemiological study (2005-2020) in five environmentally diverse areas of southwestern Spain. We modeled individual annual area-dependent exposure risk based on potential environmental and host predictors using generalized linear mixed models. Further, we analyzed the weight of predictors on exposure probability by variance partitioning of the model components. The analysis of 2418 wild ungulate sera (1168 red deer - Cervus elaphus - and 1250 Eurasian wild boar - Sus scrofa) with a highly sensitive commercial blocking ELISA identified an average seroprevalence of 24.9% (95% confidence interval (CI): 23.2-26.7%). Antibody prevalence was slightly higher in wild boar (27.5%; CI: 25.1-30.1%) than in deer (22.2%; CI: 19.8-24.7%). We observed a spatial trend in exposure, with higher frequency in the southernmost areas and a slight, although area-dependent, increasing temporal trend. Host-related predictors were important drivers of exposure risk. The environmental predictor with the highest weight was annual cumulative precipitation, while temperature variations were also relevant but with less weight. We observed a coincidence of spatiotemporal changes in exposure with the notification of WNF outbreaks in horses and humans. That indicates the usefulness of wild ungulates as sentinels for WNV transmission and as models to understand its spatiotemporal dynamics. These results will allow the development of more accurate predictive models of spatiotemporal variations in transmission risk that can inform health authorities to take appropriate action.

First Expert Elicitation of Knowledge on Possible Drivers of Observed Increasing Human Cases of Tick-Borne Encephalitis in Europe.

Tick-borne encephalitis (TBE) is a viral disease endemic in Eurasia. The virus is mainly transmitted to humans via ticks and occasionally via the consumption of unpasteurized milk products. The European Centre for Disease Prevention and Control reported an increase in TBE incidence over the past years in Europe as well as the emergence of the disease in new areas. To better understand this phenomenon, we investigated the drivers of TBE emergence and increase in incidence in humans through an expert knowledge elicitation. We listed 59 possible drivers grouped in eight domains and elicited forty European experts to: (i) allocate a score per driver, (ii) weight this score within each domain, and (iii) weight the different domains and attribute an uncertainty level per domain. An overall weighted score per driver was calculated, and drivers with comparable scores were grouped into three terminal nodes using a regression tree analysis. The drivers with the highest scores were: (i) changes in human behavior/activities; (ii) changes in eating habits or consumer demand; (iii) changes in the landscape; (iv) influence of humidity on the survival and transmission of the pathogen; (v) difficulty to control reservoir(s) and/or vector(s); (vi) influence of temperature on virus survival and transmission; (vii) number of wildlife compartments/groups acting as reservoirs or amplifying hosts; (viii) increase of autochthonous wild mammals; and (ix) number of tick species vectors and their distribution. Our results support researchers in prioritizing studies targeting the most relevant drivers of emergence and increasing TBE incidence.

The Impact of Host Abundance on the Epidemiology of Tick-Borne Infection.

Tick-borne diseases are an increasing global public health concern due to an expanding geographical range and increase in abundance of tick-borne infectious agents. A potential explanation for the rising impact of tick-borne diseases is an increase in tick abundance which may be linked to an increase in density of the hosts on which they feed. In this study, we develop a model framework to understand the link between host density, tick demography and tick-borne pathogen epidemiology. Our model links the development of specific tick stages to the specific hosts on which they feed. We show that host community composition and host density have an impact on tick population dynamics and that this has a consequent impact on host and tick epidemiological dynamics. A key result is that our model framework can exhibit variation in host infection prevalence for a fixed density of one host type due to changes in density of other host types that support different tick life stages. Our findings suggest that host community composition may play a crucial role in explaining the variation in prevalence of tick-borne infections in hosts observed in the field.

Risk Factors for Exposure of Wild Birds to West Nile Virus in A Gradient of Wildlife-Livestock Interaction.

West Nile virus (WNV) transmission rate is shaped by the interaction between virus reservoirs and vectors, which may be maximized in farm environments. Based on this hypothesis, we screened for WNV in wild birds in three scenarios with decreasing gradient of interaction with horses: (i) the farm (A1); (ii) the neighborhood (A2); and (iii) a wild area (A3). We captured wild birds and analyzed their sera for WNV antibodies by blocking ELISA and micro-virus neutralization test. Flavivirus infections were tested with generic and specific PCR protocols. We parameterized linear mixed models with predictors (bird abundance and diversity, vector abundance, vector host abundance, and weather quantities) to identify Flavivirus spp. and WNV exposure risk factors. We detected a low rate of Flavivirus infections by PCR (0.8%) and 6.9% of the birds were seropositive by ELISA. Exposure to Flavivirus spp. was higher in A1 (9%) than in A2 and A3 (5.6% and 5.8%, respectively). Bird diversity was the most relevant predictor of exposure risk and passerines dominated the on-farm bird community. Our results suggest that measures deterring the use of the farm by passerines should be implemented because the environmental favorability of continental Mediterranean environments for WNV is increasing and more outbreaks are expected.

The relevance of the wild reservoir in zoonotic multi-host pathogens: The links between Iberian wild mammals and Coxiella burnetii.

Q fever is a worldwide zoonosis caused by an obligate intracellular bacterium, Coxiella burnetii, with only anecdotal reports of human-to-human transmission. The cause of human Q fever infections is the circulation of C. burnetii in animal reservoirs. Infected livestock, particularly goats and sheep, may cause Q fever outbreaks in humans. However, wildlife is the origin of several human Q fever cases. Human impacts on habitats, biodiversity and climate are responsible for changes in the patterns of interaction between domestic animals, wildlife and humans, allowing wild animals to play an increasingly relevant role as Q fever reservoirs. In the Iberian Peninsula, human impacts on the environment combined with a high biodiversity, which could maintain high transmission rates of this multi-host pathogen, make wild reservoirs an important piece in Q fever epidemiology. In this study, we review the reporting of C. burnetii infections and exposure in Iberian wild mammals and analyse the link between the diversity of wild mammals and the frequency of C. burnetii notifications in wildlife. For it, the number of wild mammal species per UTM 10 × 10-km grid in mainland Spain and Portugal was estimated as a potential predictor of C. burnetii transmission. The results of non-linear regression analysis showed a quadratic relationship between the number of wild mammal species per grid and the presence of C. burnetii cases reported in the literature both by serology (R2  = 0.86) and polymerase chain reaction (R2  = 0.83). Increasing wild mammal diversity was linked to increasing C. burnetii transmission until an intermediate level when the relationship was inverted. Thus, at high levels of wild mammal diversity, the risk of C. burnetii transmission was lower. These observations show a role of wild mammal biodiversity in C. burnetii ecology that needs to be further explored to better prevent the negative impact of Q fever in livestock and human health in Iberia.

Patterns of adult tick parasitization of coexisting European (Erinaceus europaeus) and Algerian (Atelerix algirus) hedgehog populations in eastern Iberia.

The availability of data regarding population abundance and dynamics of ticks in wild vertebrate populations is crucial to understand the host-tick relationship and to assess the risk for domestic animals and humans from possible zoonotic diseases. In this study we analyse several host-intrinsic and environmental factors affecting the tick population of two sympatric hedgehog species (the European and the Algerian hedgehog) in two differently anthropized habitats in eastern Spain. We captured 215 hedgehogs and sampled 356 adult ticks of four different species (Rhipicephalus sanguineus, R. turanicus, R. bursa and R. pusillus). We analysed by General Linear Mixed Models how the hosts' species, sex, capture month and site and their interactions affected tick presence and tick load. Further, we carried out Spearman correlation tests to analyse how environmental temperature and precipitation affect tick presence and load on hedgehogs. We found that, in general, male hedgehogs were more infested than females. However, the effects of the different factors depended on the tick species, especially related to their endophilic or exophilic character. High values of general tick infestation can be explained by the coincidence of tick activity peaks and higher male host activity levels, especially within areas with higher habitat diversity and species richness. We also discuss how the potential immunosuppressive effect of testosterone could be affecting our results. Our results show that in highly anthropized environments hedgehogs potentially act as important mixing vessels for tick-borne zoonotic pathogens and that monitoring ticks in periurban wildlife is important to assess potential health risks for pets and humans.

Determinants of Crimean-Congo haemorrhagic fever virus exposure dynamics in Mediterranean environments.

Crimean-Congo haemorrhagic fever (CCHF) is an emerging tick-borne human disease in Spain. Understanding the spatiotemporal dynamics and exposure risk determinants of CCHF virus (CCHFV) in animal models is essential to predict the time and areas of highest transmission risk. With this goal, we designed a longitudinal survey of two wild ungulate species, the red deer (Cervus elaphus) and the Eurasian wild boar (Sus scrofa), in Doñana National Park, a protected Mediterranean biodiversity hotspot with high ungulate and CCHFV vector abundance, and which is also one of the main stopover sites for migratory birds between Africa and western Europe. Both ungulates are hosts to the principal CCHFV vector in Spain, Hyalomma lusitanicum. We sampled wild ungulates annually from 2005 to 2020 and analysed the frequency of exposure to CCHFV by a double-antigen ELISA. The annual exposure risk was modelled as a function of environmental traits in an approach to understanding exposure risk determinants that allow us to predict the most likely places and years for CCHFV transmission. The main findings show that H. lusitanicum abundance is a fundamental driver of the fine-scale spatial CCHFV transmission risk, while inter-annual risk variation is conditioned by virus/vector hosts, host community structure and weather variations. The most relevant conclusion of the study is that the emergence of CCHF in Spain might be associated with recent wild ungulate population changes promoting higher vector abundance. This work provides relevant insights into the transmission dynamics of CCHFV in enzootic scenarios that would allow deepening the understanding of the ecology of CCHFV and its major determinants.

Environmental factors driving fine-scale ixodid tick abundance patterns.

Tick abundance is an essential demographic parameter to infer tick-borne pathogen transmission risks. Spatiotemporal patterns of tick abundance are heterogeneous, so its determinants at small spatial scales need to be understood to reduce their negative effects on hosts. Current knowledge of these determinants is scarce, especially in Mediterranean environments, limiting the possibilities for designing efficient tick control strategies. With the goal of unravelling tick abundance determinants and informing new tick management strategies, we estimated tick burdens on 1965 wild ungulates in Doñana National Park, Spain, annually between 2010 and 2020. Under the hypothesis of a predominant host influence on tick abundance, we modelled the burdens of Rhipicephalus annulatus, Hyalomma lusitanicum, and Ixodes ricinus with relevant predictors grouped into four factors: i) environment; ii) host population; iii) host individual; and iv) land-use. Generalized linear mixed models with a zero-inflated negative binomial distribution were built. Additionally, we analysed the differential contribution to abundance of each factor by deviance partitioning. We finally estimated the similarity in the environmental space of tick species by analysing their niche overlap with the environmental principal component analysis method. Our work hypothesis was confirmed for R. annulatus and H. lusitanicum, but we found that tick abundance at a fine spatial scale is jointly driven by multiple drivers, including all four factors considered in this study. This result points out that understanding the demography of ticks is a complex multifactorial issue, even at small spatial scales. We found no niche differences between the three tick species at the study spatial scale, thus showing similar host and environmental dependencies. Overall results identify that host aggregation areas displaying environmentally favourable traits for ticks are relevant tick and vector-borne pathogen transmission hotspots. Our findings will facilitate the design of new strategies to reduce the negative effects of tick parasitism.

First serological evidence of Crimean-Congo haemorrhagic fever virus in transhumant bovines in Italy.

Crimean-Congo haemorrhagic fever (CCHF) is an emerging tick-borne disease caused by the arbovirus Crimean-Congo haemorrhagic fever virus (CCHFV; family Nairoviridae). Given the public health impact, CCHF is considered a priority disease for the European Union. This study describes the first detection of anti-CCHFV antibodies in transhumant bovines in Italy. Sera from 794 cattle collected across Basilicata region (Southern Italy) were screened using a commercial ELISA kit. The animal-level and herd-level seroprevalences detected were 1.89% [95%CI: 1.12-3.1] and 29.63% [95%CI: 15.68-48.65], respectively. Results of the χ2 test for trend show that the exposure to CCHFV was significantly associated with increasing age, with the odds 5 times higher in 11-22-year old cattle than 1-4-year old cattle. The detection of antibodies against CCHFV in indigenous cattle indicates that the infection occurred in the study area and may warrant further consideration. Additionally, no significant spatial clustering of CCHF infection was detected, supporting the hypothesis that the disease is widespread in the region. Further studies at larger scale are needed to identify the areas at higher risk of zoonotic infection. A One Health approach should be implemented to better understand the disease risk and dynamics in the country, which effectively address the related public health threat.

Wild micromammal host spectrum of zoonotic eukaryotic parasites in Spain. Occurrence and genetic characterisation.

Micromammals have historically been recognized as highly contentious species in terms of the maintenance and transmission of zoonotic pathogens to humans. Limited information is currently available on the epidemiology and potential public health significance of intestinal eukaryotes in wild micromammals. We examined 490 faecal samples, grouped into 155 pools, obtained from 11 micromammal species captured in 11 Spanish provinces for the presence of DNA from Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi and Blastocystis sp. The presence of Leishmania spp. was investigated in individual spleen samples. All micromammal species investigated harboured infections by at least one eukaryotic parasite, except Apodemus flavicollis, Myodes glareolus, Sorex coronatus and Sciurus vulgaris, but the sample size for these host species was very low. Cryptosporidium spp. was the most prevalent species found (3.7%, 95% confidence interval [CI]: 2.2-5.7), followed by G. duodenalis (2.8%, 95% CI: 1.6-4.6) and E. bieneusi (2.6%, 95% CI: 1.4-4.3). All pooled faecal samples tested negative for Blastocystis sp. Leishmania infantum was identified in 0.41% (95% CI: 0.05-1.46) of the 490 individual spleen samples analysed. Sequence analyses allowed the identification of Cryptosporidium andersoni (5.9%), C. ditrichi (11.7%), C. muris (5.9%), C. parvum (5.9%), C. tyzzeri (5.9%), rat genotypes CR97 (5.9%) and W19 (5.9%), vole genotypes V (11.7%) and VII (5.9%) and Cryptosproridium spp. (35.3%) within Cryptosporidium (n = 17). Known genotypes C (66.7%) and Peru11 (25.0%) and a novel genotype (named MouseSpEb1, 8.3%) were detected within E. bieneusi (n = 12). None of the G. duodenalis-positive samples could be genotyped at the assemblage level. Molecular data indicate that wild micromammals were primarily infected by rodent-adapted species/genotypes of eukaryotic pathogens and thereby have a limited role as a source of human infections. The presence of ruminant-adapted species C. andersoni along with finding C. parvum is indicative of an overlap between domestic/peri-domestic and sylvatic transmission cycles of these agents.

The Common Mosquito (Culex pipiens) Does Not Seem to Be a Competent Vector for Hepatitis E Virus Genotype 3.

An experimental infection approach was used to estimate the competence of the common mosquito, Culex pipiens, for hepatitis E virus replication and transmission, using an isolate of hepatitis E virus genotype 3 of human origin in varying infectious doses. The experimental approach was carried out in biosafety level 2 conditions on three batches of 120 Cx. pipiens females, each using an artificial feeding system containing the virus in aliquots of fresh avian blood. Mosquitoes from each batch were collected 1, 7, 14, and 21 days post-infection (dpi) and dissected. The proboscis was subjected to forced excretion of saliva to estimate potential virus transmission. HEV RNA presence in abdomen, thorax, and saliva samples was analyzed by PCR at the selected post-infection times. HEV RNA was detected in the abdomens of Cx. pipiens females collected 1 dpi in the two experimentally-infected batches, but not in the saliva or thorax. None of the samples collected 7-21 dpi were positive. Our results show that Cx. pipiens is not a competent vector for HEV, at least for zoonotic genotype 3.

Lack of Exposure to Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis in Chilean Cervids, and Evidence of a New Mycobacterium-Like Sequence.

Screening of serum and fecal samples from huemul (Hippocamelus bisulcus) and pudu (Pudu puda) from southern Chile for Mycobacterium bovis and Mycobacterium avium paratuberculosis (MAP) found all but four samples Mycobacterium-negative. The positive sequences showed only 92-93% similarity with MAP and were from remote Isla Riesco populations.