Beware with the backpack! New hosts and pathogens identified for Ixodes simplex ticks collected from bats in the Iberian Peninsula.

To improve the knowledge on the role of bats in the maintenance and transmission of tick-borne pathogens, a molecular approach was used to characterize Anaplasma spp., Rickettsia spp., Coxiella burnetii, Borrelia burgdorferi s.l., piroplasmids, Hepatozoon spp., flaviviruses and nairoviruses in ticks collected from Iberian bats. A total of 732 bats from 25 species were captured at 38 sampling sites distributed in seven provinces of Spain between 2018 and 2022. Seventy-nine Ixodes simplex ticks were collected from 31 bats (Eptesicus isabellinus, Hypsugo savii, Myotis capaccini, Myotis emarginatus, Myotis myotis, Miniopterus schreibersii, Pipistrellus pipistrellus and Rhinolophus ferrumequinum). Sixty of 79 I. simplex were positive for at least one pathogen tested and were collected from 23 bats captured in southeast Spain. We detected the presence of Rickettsia slovaca in 12 ticks collected from M. emarginatus, H. savii, M. schreibersii and E. isabellinus; Rickettsia aeschlimannii in 1 tick from M. schreibersii; Anaplasma ovis in 3 ticks from H. savii and M. schreibersii; C. burnetii in 2 ticks from H. savii; Occidentia massiliensis in 1 tick from H. savii; piroplasmids in 12 ticks from H. savii, M. schreibersii and E. isabellinus; and a novel nairovirus in 1 tick from M. schreibersii. Furthermore, blood samples obtained from 14 of the 31 tick-infested bats were negative in all PCR analyses. This study describes new host and pathogen associations for the bat-specialist I. simplex, highlights the risk of spread of these pathogens, and encourages further research to understand the role of Iberian bats in the epidemiology of tick-borne pathogens.

Effect of Myxoma Virus Species Jump on Iberian Hare Populations.

The myxoma virus species jump from European rabbits (Oryctolagus cuniculus) to Iberian hares (Lepus granatensis) has raised concerns. We assess the decline suffered by Iberian hare populations on the Iberian Peninsula and discuss the association between the effect of myxomatosis and the average abundance index, which we estimated by using hunting bags.

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

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

Phylogenomic analysis supports Mycobacterium tuberculosis transmission between humans and elephants.

Introduction: Tuberculosis is an infectious disease caused by a group of acid-fast bacilli known as Mycobacterium tuberculosis complex (MTC), which has a major impact on humans. Transmission of MTC across the human-animal interface has been demonstrated by several studies. However, the reverse zoonotic transmission from humans to animals (zooanthroponosis) has often been neglected. Methods: In this study, we used Nanopore MinION and Illumina MiSeq approaches to sequence the whole genome of M. tuberculosis strains isolated from two deceased Asian elephants (Elephas maximus) and one human in Chitwan, Nepal. The evolutionary relationships and drug resistance capacity of these strains were assessed using the whole genome data generated by the stand-alone tool Tb-Profiler. Phylogenomic trees were also constructed using a non-synonymous SNP alignment of 2,596 bp, including 94 whole genome sequences representative of the previously described M. tuberculosis lineages from elephants worldwide (lineages 1 and 4) and from humans in Nepal (lineages 1, 2 and 3). Results and Discussion: The new genomes achieved an average coverage of 99.6%, with an average depth of 55.67x. These M. tuberculosis strains belong to lineage 1 (elephant DG), lineage 2 (elephant PK) and lineage 4 (human), and none of them were found to have drug-resistant variants. The elephant-derived isolates were evolutionarily closely related to human-derived isolates previously described in Nepal, both in lineages 1 and 2, providing additional support for zooanthroponosis or bidirectional transmission between humans and elephants. The human-derived isolate clustered together with other published human isolates from Argentina, Russia and the United Kingdom in the lineage 4 clade. This complex multi-pathogen, multi-host system is challenging and highlights the need for a One Health approach to tuberculosis prevention and control at human-animal interface, particularly in regions where human tuberculosis is highly endemic.

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

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

A study of viral pathogens in bat species in the Iberian Peninsula: identification of new coronavirus genetic variants.

Bats have long been associated with multiple pathogens, including viruses affecting humans such as henipaviruses, filoviruses, bunyaviruses and coronaviruses. The alpha and beta coronaviruses genera can infect most mammalian species. Among them, betacoronavirus SARS-CoV, MERS-CoV and SARS-CoV-2, which have caused the three major pandemics in the last two decades, have been proposed to originate in bats. In this study, 194 oral swabs from 22 bats species sampled in 19 locations of the Iberian Peninsula were analysed and characterized by three different PCR tests (coronavirus generic real-time RT-PCR, multiplex conventional PCR, and SARS-CoV-2 specific real-time RT-PCR) to detect bat coronaviruses. Screening with coronavirus generic PCR showed 102 positives out of 194 oral swabs analysed. Then, metabarcoding with multiplex PCR amplified 15 positive samples. Most of the coronaviruses detected in this study belong to alphacoronavirus (α-CoV) genus, with multiple alphacoronaviruses identified by up to five different genetic variants coexisting in the same bat. One of the positive samples identified in a Miniopterus schreibersii bat positive for the generic coronavirus PCR and the specific SARS-CoV-2 PCR was classified as betacoronavirus (-CoV) through phylogenetic analysis. These results support the rapid evolution of coronaviruses to generate new genomic potentially pathogenic variants likely through co-infection and recombination.

Absence of anti-Besnoitia spp. specific antibodies in European wild lagomorphs from the Iberian Peninsula.

Besnoitia besnoiti is an apicomplexan parasite whose life cycle is not completely understood. It is assumed that this parasite might have an indirect life cycle with a carnivore as a definitive host able to shed oocysts after the ingestion of mature cysts in tissues of an infected intermediate host. Cattle and wild cervids on the Iberian Peninsula can act as intermediate hosts of B. besnoiti, and exposure to the parasite has been demonstrated in equids. In this study, we aimed to assess the presence of members of the genera Besnoitia in wild lagomorphs from the Iberian Peninsula and the potential role of these host species in the life cycle of B. besnoiti, as all the animals were sampled from 19 regions of the Iberian Peninsula where cases of bovine besnoitiosis have been previously detected. Serum samples (Oryctolagus cuniculus: n = 552; Lepus europaeus: n = 122) were first analysed by ELISA and subsequently confirmed by Western blot (WB). Specific antibodies against B. besnoiti were not found in any sampled animal by WB. In addition, lung samples from a subset of wild rabbits (n = 16) were tested by PCR and Besnoitia spp. DNA was not detected. These results suggest that Besnoitia spp. are unlikely to circulate in wild lagomorphs in the Iberian Peninsula. Thus, lagomorphs are not expected to play a key role in the biological cycle of B. besnoiti. Further studies are necessary to assess whether different micromammal species, such as rodents, can serve as natural reservoirs of Besnoitia spp. in other European regions.

Complementary roles of wild boar and red deer to animal tuberculosis maintenance in multi-host communities.

The contribution of wildlife species to pathogen maintenance in multi-host communities has seldom been quantified. To assess the relative contribution of the main wildlife hosts of animal tuberculosis (TB) to its maintenance, we estimated the basic reproduction number (R0) of Mycobacterium tuberculosis complex in wild boar and red deer at 29 sites in the Iberian Peninsula. Host abundance and true TB prevalence were estimated for each species at each site by sampling from distributions incorporating the uncertainty in the proportion of the population harvested each year, sensitivity, and specificity of the diagnostic methods, while excretion of mycobacteria was estimated using site-occupancy models. The distributions of these parameters were then used to estimate, at each site, the R0,wild boar (range 0.1 - 55.9, average 8.7, standard deviation 11.8), and the R0,red deer (0.1 - 18.9, 2.2, 3.9). Animal TB is maintained in epidemiological scenarios ranging from any single species acting as a maintenance host (the wild boar in 18 sites and the red deer in 5), to facultative multi-host disease (6 sites). The prevalence of TB in the red deer is likely an important driver of the epidemiology in multi-host communities. The wild boar was the main maintenance host of TB in most of the study sites and could have an epidemiological role linking the wildlife multi-host community and livestock.

Understanding Mycobacterium tuberculosis complex in elephants through a One Health approach: a systematic review.

BACKGROUND: Mycobacterium tuberculosis complex (MTC) that causes the chronic infectious disease- tuberculosis (TB), often presents with a complicated epidemiological pattern where the transmission chain may include humans, domestic animals and wildlife, including elephants. TB has been reported globally in both captive and wild elephants. The One Health approach might be the most effective way of understanding the shared MTC infection dynamics in captive and wild animals like Asian elephants. This systematic review accumulates evidence on occurrence, transmission pathways, and preventive measures of TB in elephants from a One Health perspective. RESULTS: The prevalence of TB reported in elephant populations ranges from 0 to 23.33% and high prevalence's are reported for elephants that are in close proximity to infected humans. The risk of elephant to human infection transmission increased significantly with exposure duration and contact with infected elephants. Some studies described the plausible TB transmission to captive elephants from other animals (wild and domestic), suggesting inter- and intra-species transmission. The results of this systematic review based on 27 relevant published works, suggest three overarching interrelated transmission pathways for M. tuberculosis infections in Asian elephants- i) humans and elephants, ii) other animals (wild or domestic) and elephants and iii) unclear sources of infection. CONCLUSIONS: The progress made with new TB diagnostic tools provides multiple methods to choose from. However, lack of harmonization of TB testing in elephants and their human contacts remains a challenge to prevent TB in those animals. Routine TB screening among elephants and caretakers by setting up an occupational health program for early diagnosis of infection through combined efforts of public health, veterinary medicine, and occupational health experts is suggested. This implies the need for a One Health approach to elephant TB control. This review reveals the need for more research on Mycobacterium tuberculosis complex transmission pathways at the human-animal interface.

Bagaza Virus in Wild Birds, Portugal, 2021.

Bagaza virus emerged in Spain in 2010 and was not reported in other countries in Europe until 2021, when the virus was detected by molecular methods in a corn bunting and several red-legged partridges in Portugal. Sequencing revealed high similarity between the 2021 strains from Portugal and the 2010 strains from Spain.

Meglumine antimoniate combination treatment for relapsing Kala-azar after treatment and secondary prophylaxis failure with liposomal amphotericin B in two HIV-coinfected patients.

Visceral leishmaniasis (VL) is a protozoan infection caused by Leishmania infantum and L. donovani with a higher incidence and severity in HIV-infected patients due to its synergistic effect on hampering the immune response, often leading to death after treatment failure. Literature regarding the management of relapsing VL in HIV-coinfected patients is lacking. Many experts recommend a combined therapy with liposomal amphotericin B and miltefosine. The use of pentavalent antimonials is often discouraged due to their toxicity. We report two cases of successful response to treatment with combined therapy with meglumine antimoniate followed by secondary prophylaxis with miltefosine and atovaquone on relapsing VL in two HIV-coinfected patients despite treatment and monthly prophylaxis with appropriate doses of liposomal amphotericin B.

A metaproteomics approach reveals changes in mandibular lymph node microbiota of wild boar naturally exposed to an increasing trend of Mycobacterium tuberculosis complex infection.

Constraints in the characterization of microbiota community that circulates in the host have limited the extent of co-infection studies in natural populations. In this study, we used a metaproteomics approach to characterize the mandibular lymph nodes microbiota of wild boar (Sus scrofa) naturally exposed to an increasing trend of Mycobacterium tuberculosis complex (MTC) infection. Our results showed a reduction in microbiota diversity and changes in the composition, structure and functionality of the microbiota community associated with an increase in tuberculosis prevalence, from 45% in 2002/06 to 83% in 2009/12. These temporal changes were accompanied by an increase in the relative abundance of Babesia, Theileria and Pestivirus genera and a decrease in the Ascogregarina and Chlorella. A positive association was also evidenced between the prevalence of tuberculosis and the presence of microbial proteins responsible for carbohydrate transport and metabolism. Our findings suggest MTC-host-microbiota interactions at the population level, which may occur in order to ensure sufficient metabolic resources for MTC survival, growth and transmission. We strongly recommend the use of metaproteomics when studying microbiota communities in wildlife populations, for which traditional diagnostic techniques are limited and in which new organisms with a pathogenic potential for domestic animals and humans may appear.

Red deer in Iberia: Molecular ecological studies in a southern refugium and inferences on European postglacial colonization history.

The red deer (Cervus elaphus) is a widespread wild ungulate in Europe that has suffered strong anthropogenic impacts over their distribution during the last centuries, but also at the present time, due its economic importance as a game species. Here we focus on the evolutionary history of the red deer in Iberia, one of the three main southern refugial areas for temperate species in Europe, and addressed the hypothesis of a cryptic refugia at higher latitudes during the Last Glacial Maximum (LGM). A total of 911 individuals were sampled, genotyped for 34 microsatellites specifically developed for red deer and sequenced for a fragment of 670 bp of the mitochondrial (mtDNA) D-loop. The results were combined with published mtDNA sequences, and integrated with species distribution models and historical European paleo-distribution data, in order to further examine the alternative glacial refugial models and the influence of cryptic refugia on European postglacial colonization history. Clear genetic differentiation between Iberian and European contemporary populations was observed at nuclear and mtDNA levels, despite the mtDNA haplotypes central to the phylogenetic network are present across western Europe (including Iberia) suggesting a panmictic population in the past. Species distribution models, fossil records and genetic data support a timing of divergence between Iberian and European populations that overlap with the LGM. A notable population structure was also found within the Iberian Peninsula, although several populations displayed high levels of admixture as a consequence of recent red deer translocations. Five D-loop sub-lineages were found in Iberia that belong to the Western European mtDNA lineage, while there were four main clusters based on analysis of nuclear markers. Regarding glacial refugial models, our findings provide detailed support for the hypothesis that red deer may have persisted in cryptic northern refugia in western Europe during the LGM, most likely in southern France, southern Ireland, or in a region between them (continental shelf), and these regions were the source of individuals during the European re-colonization. This evidence heightens the importance of conserving the high mitochondrial and nuclear diversity currently observed in Iberian populations.

Inbreeding shapes tuberculosis progression in female adult badgers (Meles meles).

In Focus: Beton, C. H., Delahay, R. J., Smith, F. A. P., Robertson, A., McDonald, R. A., Young, A. J., Burke T. A., & Hodgson, D. (2018). Inbreeding intensifies sex- and age-dependent disease in a wild mammal. Journal of Animal Ecology, 87, 1497-1499. https://doi.org/10.1111/1365-2656.12878 Increasing crossbreeding of relatives promotes inbreeding which, in turn, can cause a reduction in fitness and the emergence of a phenomenon known as inbreeding depression. Benton et al., used the badger (Meles meles)-Mycobacterium bovis model system to study the relationship between inbreeding, disease, ageing and sex. A link between multilocus homozygosity (general effect) and TB progression (measured as antibody response to infection) and between homozygosity at specific loci (local effect) and TB progression were established in this study, highlighting the importance of host genetic background in determining host disease outcomes, and thus on shaping disease dynamics in wild populations. Moreover, the authors demonstrated for the first time in a natural population that this positive association might be modulated by sex and age. In infected individuals, inbreeding only affects disease progression in adult females.

Genome-wide associations identify novel candidate loci associated with genetic susceptibility to tuberculosis in wild boar.

Tuberculosis (TB) affects a wide range of host species worldwide. Understanding host-pathogen co-evolution remains a global challenge owing to complex interactions among host genetic factors, pathogen traits and environmental conditions. We used an endemic wild boar population that had undergone a huge increase in Mycobacterium bovis infection prevalence, from 45% in 2002/06 to 83% in 2009/12, to understand the effects of host genetics on host TB outcomes and disease dynamics. Host genomic variation was characterized using a high-density single nucleotide polymorphism (SNP) array, while host TB phenotype was assessed using both gross pathology and mycobacterial culture. Two complementary genome-wide association (GWAS) analyses were conducted: (i) infected-uninfected; and (ii) 2002/06-2009/12. The SNPs with the highest allelic frequency differences between time-periods and TB outcomes were identified and validated in a large dataset. In addition, we quantified the expression levels of some of their closest genes. These analyses highlighted various SNPs (i.e. rs81465339, rs81394585, rs81423166) and some of the closest genes (i.e. LOC102164072, BDNF/NT-3, NTRK2, CDH8, IGSF21) as candidates for host genetic susceptibility. In addition to TB-driven selection, our findings outline the putative role of demographic events in shaping genomic variation in natural populations and how population crashes and drift may impact host genetic susceptibility to TB over time.

Tuberculosis-Associated Death among Adult Wild Boars, Spain, 2009-2014.

We investigated adult Eurasian wild boar (Sus scrofa) survival and death in 2 tuberculosis-endemic populations with different harvest pressure in Spain. Overall, tuberculosis accounted for 30% of total deaths. Increased survival in protected areas has direct implications for wild boar management and tuberculosis control.

Tuberculosis, genetic diversity and fitness in the red deer, Cervus elaphus.

Understanding how genetic diversity, infections and fitness interact in wild populations is a major challenge in ecology and management. These interactions were addressed through heterozygosity-fitness correlation analyses, by assessing the genetic diversity, tuberculosis (TB) and body size in adult red deer. Heterozygosity-fitness correlation models provided a better understanding of the link between genetic diversity and TB at individual and population levels. A single local effect was found for Ceh45 locus at individual level, enhancing the importance of its close functional genes in determining TB presence. At population level, the ability of the red deer to control TB progression correlated positively with population genetic diversity, indicating that inbred populations might represent more risk of deer TB severity. Statistical models also gained insights into the dynamics of multi-host interaction in natural environments. TB prevalence in neighbouring wild boar populations was positively associated with deer TB at both individual and population levels. Additionally, TB presence correlated positively with red deer body size, for which "general and local effect" hypotheses were found. Although body size might be correlated with age, an indirect genetic effect on TB presence could be implied. This study provides new insights towards understanding host-pathogen interactions in wild populations and their relation to fitness traits.

Comparative Proteomics Identifies Host Immune System Proteins Affected by Infection with Mycobacterium bovis.

Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly impact human and animal health worldwide. The mycobacterial life cycle is complex, and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Eurasian wild boar (Sus scrofa) are natural reservoir hosts for MTBC and a model for mycobacterial infection and tuberculosis (TB). In the wild boar TB model, mycobacterial infection affects the expression of innate and adaptive immune response genes in mandibular lymph nodes and oropharyngeal tonsils, and biomarkers have been proposed as correlates with resistance to natural infection. However, the mechanisms used by mycobacteria to manipulate host immune response are not fully characterized. Our hypothesis is that the immune system proteins under-represented in infected animals, when compared to uninfected controls, are used by mycobacteria to guarantee pathogen infection and transmission. To address this hypothesis, a comparative proteomics approach was used to compare host response between uninfected (TB-) and M. bovis-infected young (TB+) and adult animals with different infection status [TB lesions localized in the head (TB+) or affecting multiple organs (TB++)]. The results identified host immune system proteins that play an important role in host response to mycobacteria. Calcium binding protein A9, Heme peroxidase, Lactotransferrin, Cathelicidin and Peptidoglycan-recognition protein were under-represented in TB+ animals when compared to uninfected TB- controls, but protein levels were higher as infection progressed in TB++ animals when compared to TB- and/or TB+ adult wild boar. MHCI was the only protein over-represented in TB+ adult wild boar when compared to uninfected TB- controls. The results reported here suggest that M. bovis manipulates host immune response by reducing the production of immune system proteins. However, as infection progresses, wild boar immune response recovers to limit pathogen multiplication and promote survival, facilitating pathogen transmission.

Long-Term Dynamics of Coxiella burnetii in Farmed Red Deer (Cervus elaphus).

Several aspects of the dynamics of Coxiella burnetii that are relevant for the implementation of control strategies in ruminant herds with endemic Q fever are unknown. We designed a longitudinal study to monitor the dynamics of exposure to C. burnetii in a red deer herd with endemic infection in order to allow the design of Q fever-specific control approaches. Other relevant aspects of the dynamics of C. burnetii - the effect of herd immune status, age, season, and early infection on exposure, the average half-life of antibodies, the presence and duration of maternal humoral immunity, and the age of first exposure - were analyzed. The dynamics of C. burnetii in deer herds seems to be modulated by host herd and host individual factors and by particular host life-history traits. Red deer females become exposed to C. burnetii at the beginning of their second year since maternal antibodies protect them after birth and during the main pathogen shedding season - at the end of spring-early summer. Infection pressure varies between years, probably associated with herd immunity effects, determining inter-annual variation in the risk of exposure. These results suggest that any strategy applied to control C. burnetii in deer herds should be designed to induce immunity in their first year of life immediately after losing maternal antibodies. The short average life of C. burnetii antibodies suggests that any protection based on humoral immunity would require re-vaccination every 6 months.

Host and Environmental Factors Modulate the Exposure of Free-Ranging and Farmed Red Deer (Cervus elaphus) to Coxiella burnetii.

The control of multihost pathogens, such as Coxiella burnetii, should rely on accurate information about the roles played by the main hosts. We aimed to determine the involvement of the red deer (Cervus elaphus) in the ecology of C. burnetii. We predicted that red deer populations from broad geographic areas within a European context would be exposed to C. burnetii, and therefore, we hypothesized that a series of factors would modulate the exposure of red deer to C. burnetii. To test this hypothesis, we designed a retrospective survey of 47 Iberian red deer populations from which 1,751 serum samples and 489 spleen samples were collected. Sera were analyzed by enzyme-linked immunosorbent assays (ELISA) in order to estimate exposure to C. burnetii, and spleen samples were analyzed by PCR in order to estimate the prevalence of systemic infections. Thereafter, we gathered 23 variables-within environmental, host, and management factors-potentially modulating the risk of exposure of deer to C. burnetii, and we performed multivariate statistical analyses to identify the main risk factors. Twenty-three populations were seropositive (48.9%), and C. burnetii DNA in the spleen was detected in 50% of the populations analyzed. The statistical analyses reflect the complexity of C. burnetii ecology and suggest that although red deer may maintain the circulation of C. burnetii without third species, the most frequent scenario probably includes other wild and domestic host species. These findings, taken together with previous evidence of C. burnetii shedding by naturally infected red deer, point at this wild ungulate as a true reservoir for C. burnetii and an important node in the life cycle of C. burnetii, at least in the Iberian Peninsula.