Low sequence diversity of the prion protein gene (PRNP) in wild deer and goat species from Spain.

The first European cases of chronic wasting disease (CWD) in free-ranging reindeer and wild elk were confirmed in Norway in 2016 highlighting the urgent need to understand transmissible spongiform encephalopathies (TSEs) in the context of European deer species and the many individual populations throughout the European continent. The genetics of the prion protein gene (PRNP) are crucial in determining the relative susceptibility to TSEs. To establish PRNP gene sequence diversity for free-ranging ruminants in the Northeast of Spain, the open reading frame was sequenced in over 350 samples from five species: Iberian red deer (Cervus elaphus hispanicus), roe deer (Capreolus capreolus), fallow deer (Dama dama), Iberian wild goat (Capra pyrenaica hispanica) and Pyrenean chamois (Rupicapra p. pyrenaica). Three single nucleotide polymorphisms (SNPs) were found in red deer: a silent mutation at codon 136, and amino acid changes T98A and Q226E. Pyrenean chamois revealed a silent SNP at codon 38 and an allele with a single octapeptide-repeat deletion. No polymorphisms were found in roe deer, fallow deer and Iberian wild goat. This apparently low variability of the PRNP coding region sequences of four major species in Spain resembles previous findings for wild mammals, but implies that larger surveys will be necessary to find novel, low frequency PRNP gene alleles that may be utilized in CWD risk control.

Beyond tuberculosis: Diversity and implications of non-tuberculous mycobacteria at the wildlife-livestock interface.

Non-tuberculous mycobacteria (NTM) circulate between the environment, animals and humans entailing a double concern: their ability to interfere with tuberculosis diagnosis and their potential to cause infections in their hosts. However, published records on NTM infections in animals are still scarce. The aims of the present study were to describe the diversity of NTM circulating among wild and domestic species from Spain and to analyze their implications as potential pathogenic microorganisms or as sources of interferences in the diagnosis of bovine tuberculosis. Overall, 293 NTM isolates of 277 animals were obtained from tissue samples collected between 2012 and 2019, and analyzed through a multigene approach for mycobacteria identification. Thirty-one species were identified, being Mycobacterium avium subsp. avium (Maa) and M. avium subsp. hominissuis (Mah), but also M. bouchedurhonense, M. nonchromogenicum and M. lentiflavum, the most abundant ones. Maa and M. lentiflavum were isolated in several animals showing tuberculosis-like lesions. Maa, Mah and M. nonchromogenicum were recovered from many cattle that had reacted to the tuberculin skin test. Other NTM were also associated to these phenomena. These four mycobacterial species were geographically associated between wild boar and other hosts. The findings of the present study suggest that a high diversity of NTM circulates among wildlife and livestock. Wild boar and M. avium seem to play a relevant role in this epidemiological scenario.

Is serology a realistic approach for monitoring red deer tuberculosis in the field?

Tuberculosis (TB) is a zoonotic mycobacterial infection with great importance in human health, animal production, and wildlife conservation. Although an ambitious eradication programme in cattle has been implemented for decades, TB-free status has not yet been achieved in most of Spain, where animal TB persists in a multi-host system of domestic and wild hosts, including the red deer (Cervus elaphus). However, information on long time series and trends of TB prevalence in wildlife is scarce. The diagnosis of TB in wild red deer is often based on gross pathology and bacteriological culture confirmation, although recently serological assays have been developed to detect anti- Mycobacterium tuberculosis Complex (MTC) antibodies. Particularly, protein complex P22 has demonstrated to yield good specificity and sensitivity in the serological diagnosis of MTC for red deer, as well as cattle, goats, sheep, pigs, wild boar, and European badger. Thus, the objective of the present study was to compare the performance of the P22-ELISA with TB-compatible lesion detection, as well as to assess the potential application of each technique for determining spatiotemporal trends and risk factors of MTC infection in wild red deer from low and high TB prevalence areas of Spain over the last two decades. We tested 5095 sera from 13 wild populations by indirect ELISA using P22 as antigen. Mean seroprevalence (13.22%, CI95: 12.32-14.18) was compared with the prevalence of macroscopic TB-compatible lesions (6.94%, CI95: 6.18-7.79). The results evidenced a poor agreement between both techniques (K < 0.3), although generalized TB-lesions and anti-P22 antibodies showed a positive association (χ² = 9.054, P = 0.004). Consequently, TB-lesion based prevalence and seroprevalence cannot be considered as equivalent for TB surveillance in red deer. Regarding the spatiotemporal trend of TB in red deer in Spain, we observed a North-South gradient of TB occurrence [North: 1.23% (CI95: 0.77-1.97) of TB-lesions and 12.55% (CI95: 10.91-14.41) of P22-ELISA; Centre: 7.10% (CI95: 6.04-8.33) and 8.74% (CI95: 7.57-10.08); South: 21.04% (CI95:17.81-24.69) and 23.09% (CI95: 19.73-26.84), respectively]. Overall, there was a stability over time, with higher prevalence in adults belonging to densely populated sites. We conclude that the P22-ELISA alone is not sufficiently reliable for TB surveillance in red deer at large spatiotemporal scales. Instead, we recommend combining gross pathology and P22-ELISA.

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.

Serosurveillance of Schmallenberg virus in wild ruminants in Spain.

Schmallenberg disease (SBD) is an emerging vector-borne disease that affects domestic and wild ruminants. A long-term serosurvey was conducted to assess exposure to Schmallenberg virus (SBV) in all the wild ruminant species present in mainland Spain. Between 2010 and 2016, sera from 1,216 animals were tested for antibodies against SBV using a commercial blocking ELISA. The overall prevalence of antibodies was 27.1% (95%CI: 24.7-29.7). Statistically significant differences among species were observed, with significantly higher seropositivity found in fallow deer (Dama dama) (45.6%; 99/217), red deer (Cervus elaphus) (31.6%; 97/307) and mouflon (Ovis aries musimon) (28.0%; 33/118) compared to Barbary sheep (Ammotragus lervia) (22.2%; 8/36), Iberian wild goat (Capra pyrenaica) (19.9%; 49/246), roe deer (Capreolus capreolus) (17.5%; 34/194) and Southern chamois (Rupicapra pyrenaica) (10.2%; 10/98). Seropositive animals were detected in 81.4% (57/70; 95%CI: 70.8-88.8) of the sampled populations. SBV seroprevalence ranged from 18.8% (48/256) in bioregion (BR)2 (north-central, Mediterranean) to 32.3% (31/96) in BR1 (northeastern or Atlantic, Eurosiberian). Anti-SBV antibodies were not found before 2012, when the first outbreak of SBD was reported in Spain. In contrast, seropositivity was detected uninterruptedly during the period 2012-2016 and anti-SBV antibodies were found in yearling animals in each of these years. Our results provide evidence of widespread endemic circulation of SBV among wild ruminant populations in mainland Spain in recent years. Surveillance in these species could be a useful tool for monitoring SBV in Europe, particularly in areas where wild ruminants share habitats with livestock.

A Mother's Story, Mitogenome Relationships in the Genus Rupicapra.

Although the two species of chamois (Rupicapra rupicapra and R. pyrenaica) are currently classified as least-concern by the IUCN (International Union for Conservation of Nature), inconsistencies on the subspecies classification reported in literature make it challenging to assess the conservation status of the single subspecies. Previous studies relying on mitochondrial genes, sometimes in combination with nuclear or Y-chromosome markers, reported the presence of clusters corresponding to the geographic distribution but highlighting ambiguities in the genus phylogeny. Here we report novel de novo assembled sequences of the mitochondrial genome from nine individuals, including previously unpublished R. r. balcanica and R. r. tatrica subspecies, and use them to untangle the genus phylogeny. Our results based on the full mitogenome inferred phylogeny confirm the previously reported genus subdivision in three clades and its monophyletic positioning within the Caprinae. Phylogeny and taxonomy of Rupicapra species thus remain controversial prompting for the inclusion of archeological remains to solve the controversy.

Distribution of Pestivirus exposure in wild ruminants in Spain.

A large-scale study was carried out to determine the prevalence of antibodies against Pestivirus species in wild ruminants and describe their spatial variation in mainland Spain. Serum samples of 1,874 wild ruminants from different regions of this country were collected between the years 2000 and 2017. A total of 6.6% (123/1,874) animals showed antibodies against Pestivirus by both blocking ELISA (bELISA) and virus neutralization tests (VNT). The prevalence of antibodies against pestiviruses was different both among species and regions. Seroprevalence by species was 30.0% (75/250) in Southern chamois (Rupicapra pyrenaica), 7.0% (25/357) in fallow deer (Dama dama), 2.5% (10/401) in red deer (Cervus elaphus), 2.4% (8/330) in Iberian wild goat (Capra pyrenaica), 1.1% (4/369) in roe deer (Capreolus capreolus) and 0.8% (1/130) in mouflon (Ovis aries musimon), not detecting seropositivity (0/37) in Barbary sheep (Ammotragus lervia). The results confirm that exposure to pestiviruses was detected throughout mainland Spain, with significantly higher seroprevalence in Northern regions associated with the presence of Southern chamois. This indicates an endemic circulation of pestiviruses in Southern chamois and a limited circulation of these viruses in the remaining wild ruminant species during the last two decades, thus suggesting that non-chamois species are not true Pestivirus reservoirs in Spain. Nonetheless, the high spatial spread of these viruses points out that new epidemic outbreaks in naïve wild ruminant populations or transmission to livestock may occur, evidencing the usefulness of monitoring pestiviruses in wild ruminants, especially at the wildlife-livestock interface.

Tuberculosis outbreak caused by Mycobacterium caprae in a rabbit farm in Spain.

Animal tuberculosis remains a great source of socioeconomic and health concern worldwide. Its main causative agents, Mycobacterium bovis and Mycobacterium caprae, have been isolated from many different domestic and wild animals. Naturally, occurring tuberculosis is extremely rare in rabbits, and implication of M. caprae has never been reported earlier. This study describes a severe tuberculosis outbreak caused by M. caprae in a Spanish farm of rabbits raised for meat for human consumption. The disease was first identified in a cachectic dam, and then it was confirmed in ten does with similar clinical signs. Subsequently, a depopulation operation was ordered for public health, animal welfare and environmental reasons. To broaden knowledge of spontaneous tuberculosis in rabbits, a study focused on pathological, epidemiological and diagnostic aspects was carried out on 51 does and 16 kittens after receiving the necessary authorizations. These animals were subjected to a modified intradermal test. After being euthanized, rabbits were examined for the presence of visible tuberculosis-compatible lesions. Lung, kidney, caecal appendix and sacculus rotundus samples underwent microbiological and anatomopathological analysis. Infection was revealed by at least one of the methods used in 71% of dams and in 44% of kittens. The intradermal test was shown to be a good indicator of infection. Lung was the tissue for which more animals were positive but renal and intestinal tissues were also affected in many cases. Apparently, M. caprae spread mainly through the aerogenous route. Infection was pathologically characterized by the absence of evident fibrous capsules surrounding granulomas. A spoligotype (SB0415) frequently found in this area was considered responsible for the outbreak but the source could not be established. Regardless of the exceptional nature of animal tuberculosis in this host, rabbit industry might not escape from its effects and therefore, current biosafety and surveillance strategies should also consider this disease.

Phylogeography and epidemiology of Brucella suis biovar 2 in wildlife and domestic swine.

Swine brucellosis due to Brucella suis biovar 2 (bv2) is enzootic in wild boar and hare in continental Europe and may cause major economic losses to the pig industry, mainly in free-ranged pig farms. The high nucleotide identity found among the B. suis biovar 2 isolates has long hindered the full understanding of the epidemiology and the phylogeography of the disease. Here, we used multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) and whole-genome analysis to identify single-nucleotide polymorphisms (SNPs) in order to gain insights from the largest B. suis bv2 dataset analyzed so far composed of domestic pigs and wildlife isolates collected throughout Europe since the 1970s. We found four major clades with a specific phylogeographic pattern. The Iberian clade contains isolates exclusively from the Iberian Peninsula. The Central European clade includes most isolates from France, Northern Italy, Switzerland and an important proportion of those of Northern Spain. The Eastern European clade clustered isolates from Croatia and Hungary mainly but also from areas of France, Germany, Italy and Poland. Finally, a separated Sardinian clade grouped three isolates from this island. At fine scale, MLVA demonstrated an endemic status of the infection in Europe and it allowed tracking a large outbreak formed by different farms from Spain linked to the same infection source. The whole genome SNP analysis showed that the strains form genetically distinct clades, shared between wild boar and pigs, in agreement with the MLVA clades. Interestingly, all hare isolates clustered together within two groups composed exclusively of wildlife isolates. Our results support the hypothesis that maintenance and spread of B. suis bv2 in Europe is a dynamic process linked to the natural expansion of wild boar as the main wild reservoir of the infection, while spread over long distances is found largely dependent on anthropogenic activities.