Daniel Fernández de Luco

Spatial distribution and risk factors of Brucellosis in Iberian wild ungulates

BackgroundThe role of wildlife as a brucellosis reservoir for humans and domestic livestock remains to be properly established. The aim of this work was to determine the aetiology, apparent prevalence, spatial distribution and risk factors for brucellosis transmission in several Iberian wild ungulates.MethodsA multi-species indirect immunosorbent assay (iELISA) using Brucella S-LPS antigen was developed. In several regions having brucellosis in livestock, individual serum samples were taken between 1999 and 2009 from 2,579 wild bovids, 6,448 wild cervids and4,454 Eurasian wild boar (Sus scrofa), and tested to assess brucellosis apparent prevalence. Strains isolated from wild boar were characterized to identify the presence of markers shared with the strains isolated from domestic pigs.ResultsMean apparent prevalence below 0.5% was identified in chamois (Rupicapra pyrenaica), Iberian wild goat (Capra pyrenaica), and red deer (Cervus elaphus). Roe deer (Capreolus capreolus), fallow deer (Dama dama), mouflon (Ovis aries) and Barbary sheep (Ammotragus lervia) tested were seronegative. Only one red deer and one Iberian wild goat resulted positive in culture, isolating B. abortus biovar 1 and B. melitensis biovar 1, respectively. Apparent prevalence in wild boar ranged from 25% to 46% in the different regions studied, with the highest figures detected in South-Central Spain. The probability of wild boar being positive in the iELISA was also affected by age, age-by-sex interaction, sampling month, and the density of outdoor domestic pigs. A total of 104 bacterial isolates were obtained from wild boar, being all identified as B. suis biovar 2. DNA polymorphisms were similar to those found in domestic pigs.ConclusionsIn conclusion, brucellosis in wild boar is widespread in the Iberian Peninsula, thus representing an important threat for domestic pigs. By contrast, wild ruminants were not identified as a significant brucellosis reservoir for livestock.

Neospora caninum antibodies in wild carnivores from Spain

Serum samples from 251 wild carnivores from different regions of Spain were tested for antibodies to Neospora caninum by the commercial competitive screening enzyme linked immunosorbent assay (c-ELISA) and confirmed by Neospora agglutination test (NAT) and/or by indirect fluorescent antibody test (IFAT). Samples with antibodies detected by at least two serological tests were considered seropositive. Antibodies to N. caninum were found in 3.2% of 95 red foxes (Vulpes vulpes); in 21.4% of 28 wolves (Canis lupus); in 12.0% of 25 Iberian lynx (Lynx pardinus); in 16.7% of 6 European wildcats (Felis silvestris); in 6.4% of 31 Eurasian badgers (Meles meles); in 21.4% of 14 stone martens (Martes foina); in 66.7% of 3 pine martens (M. martes) and in 50% of 2 polecats (Mustela putorius). Antibodies to N. caninum in common genets (Genetta genetta) and Egyptian mongooses (Herpestes ichneumon) were only observed by c-ELISA but were not confirmed by IFAT and/or NAT. No antibodies were detected in 5 Eurasian otters (Lutra lutra) by any technique. Statistically significant differences were observed among species and among geographical areas. The highest seroprevalence of N. caninum infection was observed in the Cantabric Coastal region characterized by high humidity. To our knowledge, this is the first report of antibodies to N. caninum in free ranging wild carnivores, other than wild canids, in Europe. The existence of a possible sylvatic cycle could have important implications in both sylvatic and domestic cycles since they might influence the prevalence of infection in cattle farms in those areas.

Prevalence of antibodies against Toxoplasma gondii in roe deer from Spain.

Roe deer (Capreolus capreolus) is an important game animal in Spain. Sera from 278 roe deer from eight areas in mainland Spain were assayed for antibodies to Toxoplasma gondii by modified agglutination test (MAT). Titers of 1:25 or higher were found in 109 (39.2%) of 278 deer. No significant differences in antibody prevalence were found between sex or age categories. In contrast, significant differences in seroprevalence between locations were evident. Roe deer from the Northern coastal habitats (high humidity and roe deer density) had the highest prevalence, compared with low prevalence in Central Spain (arid areas and low roe deer density). There was a positive correlation between antibody prevalence and mean annual rainfall (r(s)=0.85, n=8, P<0.01). These findings have environmental and/or public health implications because venison can be an important meat source of T. gondii infections for humans and feral cats.

A large-scale survey of brown hare Lepus europaeus and Iberian hare L. granatensis populations at the limit of their ranges

Abstract The historical ranges of the European brown hare Lepus europaeus and the Iberian hare L. granatensis meet in Aragón in northeastern Spain. We studied the relative abundances and the population trends of the two species in 60 localities (13 for the brown hare, 38 for the Iberian hare, and nine from the transition zone where both species are present) by spotlighting in winter during 1992-2002. We carried out a total of 1,407 counts covering 41,511 km. Both the Iberian (132.2 ± 33.2 hares/100 km; range: 52-192) and the brown hare (106.7 ± 26.8; range: 53-136) were more abundant in their respective zones than both species combined in the transition zone (90.9 ± 50.5, range: 37-157). The highest Iberian hare abundances were recorded in the northern Iberian Mountains, an area with well-preserved cereal-dominated ecosystems and a less extreme climate than in other parts of the study area. The Iberian hare had significant inter-annual differences both locally and generally, which was mainly due to a peak in 1998, and this species showed a general positive trend during the study period, suggesting that Iberian hare numbers are increasing. Contrary to the marked declines reported from other European regions, the brown hare abundance indices obtained in the Spanish Pyrenees during our study period remained stable.

Dynamics of an infectious keratoconjunctivitis outbreak by Mycoplasma conjunctivae on Pyrenean Chamois Rupicapra p. pyrenaica.

Between 2006 and 2008, an outbreak of Infectious Keratoconjunctivitis (IKC) affected Pyrenean chamois Rupicapra p. pyrenaica, an endemic subspecies of mountain ungulate that lives in the Pyrenees. The study focused on 14 mountain massifs (180,000 ha) where the species’ population is stable. Cases of IKC were detected in ten of the massifs and, in five of them, mortality was substantial. The outbreak spread quickly from the first location detected, with two peaks in mortality that affected one (2007) and three (2008) massifs. In the latter, the peak was seasonal (spring to autumn) and, in the former, the outbreak persisted through winter. To identify the outbreak’s aetiology, we examined 105 Pyrenean chamois clinically affected with IKC. TaqMan rt-PCR identified Mycoplasma conjunctivae in 93 (88.5%) of the chamois. Another rt-PCR detected Chlamydophila spp. in 14 of chamois, and 12 of those had mixed infections with mycoplasmas. In the period 2000–2007, the chamois population increased slightly (λ 1.026) but decreased significantly during the IKC outbreak (λ 0.8, 2007–2008; λ 0.85, 2008–2009) before increasing significantly after the outbreak (λ 1.1, 2009–2010). Sex-biased mortality shifted the adult sex ratio toward males (from 0.6 to 0.7 males per female) and reduced productivity slightly. Hunting was practically banned in the massifs where chamois experienced significant mortality and allowed again after the outbreak ended. Long-term monitoring of wild populations provides a basis for understanding the impacts of disease outbreaks and improves management decisions, particularly when species are subject to extractive exploitation.

The role of wild ruminants as reservoirs of Besnoitia besnoiti infection in cattle.

Bovine besnoitiosis, a parasitic disease caused by Besnoitia besnoiti, has been reported mainly in beef cattle raised under extensive pastoral systems and is considered to be re-emerging in Western Europe. Horizontal transmission probably occurs either by means of blood sucking arthropods or as a consequence of direct contact between infected and non-infected cattle. However, the role that wild ruminants (e.g., red deer (Cervus elaphus) and roe deer (Capreolus capreolus)) may play in the parasite life cycle as putative reservoirs remains elusive. Thus, we investigated the presence of Besnoitia spp. infection in 2608 wild ruminants located in areas where bovine besnoitiosis is present and identified the Besnoitia species detected. First, a serosurvey was conducted in red deer (n=309), roe deer (n=417), Pyrenean chamois (Rupicapra p. pyrenaica, n=383) and Iberian wild goat (Capra pyrenaica hispanica, n=288) from two areas of Aragon, northeastern Spain, where bovine besnoitiosis is endemic. Second, red deer (n=820), roe deer (n=37), fallow deer (Dama dama, n=166), Iberian wild goat (n=86) and European mouflon (Ovis orientalis musimon, n=102) from southwestern Spain, where new outbreaks have recently been reported, were also sampled. The presence of Besnoitia spp.-specific antibodies was confirmed by western blot in one red deer and one roe deer from the Pyrenees, and Besnoitia spp. DNA was detected by ITS1-PCR in the seropositive red deer. Besnoitia genotyping based on 6 microsatellite (MS) analyses was carried out in red deer samples and compared with B. besnoiti genotypes from 7 in vitro isolates and 3 infected bovines, B. tarandi (1 isolate) and B. bennetti (from tissues of an infected donkey) for Besnoitia spp. assignation. Multilocus MS analysis of B. besnoiti, B. tarandi and B. bennetti showed specific genotypes for each species. A restricted genetic diversity with two genotypes by variation in a unique MS marker was revealed among the 7 B. besnoiti isolates. Incomplete Besnoitia spp. genotype of 3 MS markers from red deer samples entirely matched the B. besnoiti genotypes. Accordingly, this work gives clues for the presence of B. besnoiti infection in red deer from Western Europe. Further molecular genotyping is needed to confirm that red deer may act as an intermediate host of B. besnoiti, although the low prevalences that were found indicate that wild ruminant species do not pose a significant risk of transmitting the infection to cattle.

Effect of in vitro maedi-visna virus infection on adherence and phagocytosis of staphylococci by ovine cells.

This work was aimed at studying the effect of maedi-visna virus (MVV) infection in vitro on the ability of sheep cells to adhere to staphylococci (Staphylococcus aureus and Staphylococcus epidermidis), and phagocytose these bacteria. Adherence was studied in sheep choroid plexus cells (SCPC) using an ELISA test and phagocytosis was studied in pulmonary alveolar macrophages (PAM) by chemiluminescence. A 5- and 7-day of in vitro MVV infection resulted in syncytium formation and a significant increased adherence (P < 0.01) of SCPC to bacteria. SCPC endogenous fibronectin was significantly higher (P < 0.01) on days 5 and 7 than on day 0 of MVV infection. A significantly decreased phagocytosis (P < 0.05) was also observed on days 5 and 7 of MVV infection in PAM when compared to MVV-free controls. Comparatively, phagocytosis was highest for S. aureus non-slime producing strains, followed by S. epidermidis, and S. aureus slime producing strains, in that order. Finally, increased expression of both, class I and class II major histocompatibility antigens was also observed in MVV-infected PAM on days 5 and 7, whereas SCPC only demonstrated upregulation of MHC class I. These results, indicative of an alteration of some cell functions in MVV-infected cells, may help to understand interactions between MVV-infected cells and bacteria in simultaneous infections and may provide clues to the possible in vivo interactions of both pathogens.

The prevalence of proliferative kidney disease from the kidney and muscle of rainbow and brown trout in Aragón (Spain).

The prevalence and parasite density of PKX (the unknown myxosporean that causes proliferative kidney disease [PKD] of salmonids) were investigated in eight fishfarms in Aragón, Spain. Tissue sections stained with the biotynilated lectin GS-I revealed the presence of this protozoan in only one of the farms. In rainbow trout, the renal prevalence and parasite density peaked in July, but in brown trout the maximum renal prevalence and maximum renal parasite density were reached in May and in July, respectively. In rainbow trout, after the acute phase of the disease, the number of PKX decreases in the kidney but increases in the muscle. In this species of fish, the prevalence and parasite density are much higher in the muscles than in the kidney in October.

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.

Comments regarding Marco et al., 2015, 'The two sides of border disease in Pyrenean chamois (Rupicapra pyrenaica): silent persistence and population collapse'.

Dear Editor The recent paper of Marco et al. (2015) requires clarification regarding the origin of the Aragon data. Secondarily, but no less important to us, rectification is needed regarding certain aspects of the Rupicapra pyrenaica pyrenaica subspecies, its management and its distribution. Pestivirus, Pyrenean chamois Rupicapra pyrenaica pyrenaica and Aragon. Pestivirus has affected Pyrenean chamois Rupicapra p. pyrenaica in the eastern Pyrenees (Principality of Andorra, Ariège and Catalonia regions) since 2001–2002 (Alzieu et al., 2004; Arnal et al., 2004; Hurtado et al., 2004; Frölich et al., 2005). The disease agent is spreading to the western Pyrenees and was detected in Aragon in 2011, producing high mortalities (Arnal et al., 2013b). Chamois pestivirus in Aragon. We detected border disease in Benasque Game Reserve (Arnal et al., 2013b), in the frame of a long term monitoring of the Pyrenean chamois in the Aragon region (Arnal et al., 2013a). Although Marco et al., mentioned these unpublished data, they did not cite the source of information from the Spanish region of Aragon, Game Reserves 8 and 9, Benasque and Los Circos (Fig. 1 of Marco et al., 2015), which was from our abstract in the II International Rupicapra Symposium (Arnal et al., 2013b). Additional information about the Pyrenean chamois, R. p. pyrenaica. The subspecies affected is Pyrenean chamois R. p. pyrenaica, which is a commonly used name in English following the Caprinae Action Plan (Shackleton 1997). Following the same Action Plan, R. pyrenaica is considered the Southern chamois, with three subspecies: Pyrenean chamois R. p. pyrenaica, Cantabrian chamois Rupicapra pyrenaica parva and Apennine chamois Rupicapra pyrenaica ornata. Pyrenean chamois are not only found along the border between France and Spain. They also occupy areas far away from the border, including isolated massifs (Herrero et al., 2013). When considering the entire mountain range, Pyrenean chamois subspecies are managed mostly by local hunters, especially on the French side of the mountain chain, rather than mostly in game reserves and national parks. Such game reserves belong exclusively to Spain and Andorra (Pita et al., 2012; Solà and Riba, 2013). There are no game reserves as such in France, where most chamois populations are hunted and the majority of them are managed by local hunter societies. In Herrero et al. (2013), there is an update of the numbers and distribution of the subspecies in the whole Pyrenees. There are important ecological barriers along the Pyrenees, and some massifs are not connected with the main mountain chain, which limits animal dispersal rather than annual seasonal migrations. Figure 1 of Marco et al. (2015) showed only two Protected Areas: (1) Orlu Wildlife Reserve and (2) Aigües Tortes and Lake of Sant Maurici National Park. The former has the number 7, which was a mistake. The rest of the areas are game reserves, not protected areas. We hope these clarifications will help the readers to better understand authorship and certain aspects of Pyrenean chamois biology and management. Sincerely M. C. Arnal et al.