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CASE HISTORY: A 8.5x5.7x5 cm irregularly shaped mass was detected in the location of the left ovary of a 9-year-old Iberian red deer (Cervus elaphus hispanicus) hind shot during a hunting drive. PATHOLOGICAL FINDINGS: On the cut surface, the mass consisted of numerous fluid- or keratin-filled inter-communicating cysts, separated by connective tissue containing solid nodules of dark-coloured foci and hairy skin. The right ovary was of normal size, but contained a 1.5 cm mass of hairy skin and spongy structures. Histologically, the large mass comprised differentiated structures such as cartilage, fibrous connective tissue, smooth muscle, stratified squamous keratinised epithelium, cuboidal multi-layered and respiratory epithelium, clusters of pigment-laden cells, hair follicles, nervous tissue and glandular structures. The hind had a grossly and microscopically normal uterus that contained a 449.5 g female fetus. DIAGNOSIS: Bilateral mature ovarian teratomas. CLINICAL RELEVANCE: This case describes the first known description of an ovarian teratoma in a free-living red deer. The tumour had unique features, such as bilateral presence and concurrency with a normal pregnancy.

Serum samples from 693 hunted wild boar (Sus scrofa) were analysed by means of a blocking ELISA technique, and the mean (se) prevalence of antibodies to Aujeszky's disease virus was 44 (4) per cent. All the seropositive wild boar were from south central Spain, except for one from central Spain, close to the main positive area. In this area, where large game species are increasingly managed for hunting, the seroprevalence was affected by the type of management. More intensively managed populations had a higher prevalence than wild boar living in natural situations, and the seroprevalence increased with the age of the animals; the seroprevalence was higher in females in all age groups. The seroprevalence in males more than one year old peaked after the breeding season, whereas females of the same age had a higher and constant seroprevalence throughout the year.

Bovine tuberculosis (bTB) is an important re-emerging zoonotic disease, causing major economic losses and constraining international trade of animals and their products. Despite eradication programmes, some countries continue to encounter outbreaks, mainly due to wildlife acting as primary hosts or reservoirs. While the genetic component of tuberculosis in humans and cattle is well documented, the role of genetic factors as modulators of bTB resistance remains unclear for natural populations. To address this issue, we investigated the relative contribution of host genetic variability to susceptibility to bTB infection and disease progression in wild boars from southern Spain. We found that genetic heterozygosity is an important predictor of bTB, not only modulating resistance to infection but also influencing containment of disease progression in infected individuals. Our results provide further evidence that host genetic variability plays a central role in natural populations. Testing each marker separately reveals evidence of both general and single-locus associative effects on bTB and several loci reveal high homology to regions of the genome with known immune function. Our results may prove to be crucial for understanding outbreaks of bTB in wildlife that could potentially affect domestic livestock and humans. © 2005 Blackwell Publishing Ltd.

10 pages, 2 figures.-- PMID: 15610722 [PubMed].-- Article available Open Access at the publisher's site: http://www.vetres.org/articles/vetres/abs/2005/01/V4038/V4038.html.-- et al., The role of European wild ungulates in the epidemiology of tuberculosis (TB) is still under discussion. This study describes the geographical distribution and molecular typing of 77 Mycobacterium tuberculosis complex isolates belonging either to M. bovis or to M. caprae, cultivated from hunter harvested red deer (Cervus elaphus) and European wild boar (Sus scrofa) in 24 Spanish localities, and compares them with spoligotypes detected previously in humans, livestock or wild animals, as described in the literature. The distribution of the molecular type patterns suggests that the population of M. tuberculosis complex strains isolated from Spanish wild ungulates is spatially structured despite the lack of important geographical barriers and despite the increasingly frequent wildlife translocations. Red deer and the European wild boar can share the same molecular types in localities in which the M. tuberculosis complex was isolated from both species. Strains of bovine and caprine origin do circulate in the same local wildlife populations. Six out of 11 spoligotypes were similar to types described in human cases. The isolation of TB strains in fenced estates from wild animals that have not had contact with domestic livestock for at least the past two decades, strongly suggests that the M. tuberculosis complex is able to survive in these populations. Therefore, wildlife including cervids and the wild boar need to be considered in the epidemiology and control of tuberculosis., The study was supported by project AGL2001- 3947, Ministerio de Ciencia y Tecnología and FEDER. A wider study on wildlife TB is supported by INIA, MCYT RTA03-074-C2-2, and Grupo Santander. This is a contribution to the agreement between Yolanda Fierro and Universidad de Castilla–La Mancha. Joaquín Vicente had a grant from Junta de Comunidades de Castilla–La Mancha.

11 pages, 4 figures.-- PMID: 15099500 [PubMed].-- Article available Open Access at the publisher's site: http://www.vetres.org/articles/vetres/abs/2004/02/V4208/V4208.html, Porcine circovirus type 2 (PCV2) is considered as the causative agent of postweaning multisystemic wasting syndrome (PMWS) in domestic pigs, where the virus is ubiquitous as evidenced by serological surveys. We present the results of the first nationwide sero-survey on the presence of PCV2 antibodies in European wild boars, and report the first PMWS case in a wild boar from Spain. Sera from 656 hunter harvested wild boars from 45 different geographical sites and 22 additional imported animals were analysed by means of an immunoperoxidase monolayer assay (IPMA). We also examined the tissues from 55 healthy and one diseased wild boars for the presence of PCV2 nucleic acid and PMWS lesions by in situ hybridisation and histopathology, respectively. Additionally, abundance estimates of wild boars and field interviews were carried out on 30 sampling sites. The prevalence of medium to high PCV2 serological titres among the examined wild boars was 47.89 ± 1.9%. Seropositive wild boars appeared in all but one of the geographical regions analysed. Seroprevalence and titre of PCV2 antibodies were closely related to the management of the wild boar populations. Wild boars from intensively managed, farm-like populations had higher prevalence than wild boars living in more natural situations. The effect of wild boar abundance and management on PCV2 antibody prevalence was further evidenced by the high correlation existing between the relative abundance estimates of animals and the percentage of wild boars with medium to high levels of PCV2 antibodies. PCV2 nucleic acid was detected in the tissues of three wild boars. One of these was diagnosed as PMWS. The results, in addition to information on piglet mortalities, suggest a potential role of PMWS in piglet mortality in intensively managed wild boar populations., Part of this work has been a contribution derived from the agreements between Yolanda Fierro and UCLM, and between CSIC and Principado de Asturias. The study was supported by project AGL2001-3947, Ministerio de Ciencia y Tecnología and FEDER and partly funded by the Project QLRT-PL-199900307 from the Fifth Framework Programme 1998–2002 of the European Commission.

Large numbers of woodpigeons (Columba palumbus) winter each year in southwestern Spain and Portugal, where they feed on acorns. Although mortality in these wintering roosts is generally low, in winter and spring 2001 approximately 2,600 woodpigeons died from trichomoniasis. Diagnosis of the disease was based on postmortem examination, culture and identification of the parasite, Trichomonas gallinae, by PCR. Other viral, bacterial or fungal pathogens were ruled out as cause for the die-off. The gathering of the woodpigeons at game bird feeders set up for red-legged partridges may have contributed to the outbreak. Mortality ceased after treatment with dimetridazole via the game bird feeders. Supplementary feeding of game birds constitutes a risk factor for the appearance of disease outbreaks. Close monitoring of infectious diseases in these populations is important, but treatment of free-living birds should be avoided in favour of other measures, as it poses risks. © Springer-Verlag 2004., The authors would like to gratefully acknowledge the support of the Institut fu¨ r Geflu¨ gelkrankheiten, Justus- Liebig-University of Giessen, Germany. This is a contribution to FEDER-UE IF 1FD 1997-2299.

Organisms in the genus Anaplasma are obligate intracellular pathogens that multiply in both vertebrate and invertebrate hosts. The type species, Anaplasma marginale, causes bovine anaplasmosis and infects erythrocytes of the vertebrate host and undergoes a complex developmental cycle in ticks which serve as biological vectors. Infected cattle, wild ruminants and ticks can all serve as reservoirs of A. marginale. In this study, hunter killed Iberian red deer (Cervus elaphus hispanicus) from the region of Castilla-La Mancha in southwestern Spain were tested for Anaplasma infection. We found that 10% of the deer examined were seropositive for Anaplasma. Three A. marginale strains were subsequently obtained from salivary glands of Hyalomma marginatum that were removed from these deer, and the sequence of the major surface protein (msp)4 gene was determined for each strain and used for phylogenetic studies. Maximum parsimony analyses of msp4 sequences from H. marginatum ticks in comparison with New World cattle and bison isolates reported previously, suggested different origins for these Spanish A. marginale strains. The results of this study demonstrated that Iberian red deer are naturally infected with Anaplasma, and may therefore serve as a wildlife reservoir of the pathogen. Although the link between deer infection and the strains of A. marginale identified in ticks was not established, H. marginatum and Rhipicephalus bursa were identified as potential biological vectors for A. marginale in this region and may effect transmission of A. marginale between deer and cattle populations.

The helminth populations found in a group of wild boars collected in central Spain were compared to those in a group of animals imported from a French game farm that produces boars for restocking. Eleven helminth species, including ten nematodes and one acanthocephalan, were found. Gongylonema pulchrum and Macracanthorhynchus hirundinaceus were only detected in autochthonous wild boars, while Oesophagostomum dentatum, Ascaris suum, and Trichuris suis were detected in imported animals only. Autochthonous wild boars were more frequently and more intensely parasitised by Ascarops strongylina than the imported ones. No differences in prevalence nor intensity were found for the species Capillaria garfiai, Globocephalus urosubulatus, Metastrongylus sp., Physocephalus sexalatus and Simondsia paradoxa. To our knowledge, G. urosubulatus, G. pulchrum and S. paradoxa have not previously been described in wild boars in Spain. Our results highlight the risks of translocating wild animals, with regard to their helminth parasites. Until improved control measures are established, it would be wise to avoid long-distance translocations in order to prevent the potential introduction of foreign parasites.

The diagnosis and pathology of an avian pox outbreak in free-living red-legged partridges in Cadiz, Southern Spain, is described. Diagnosis of the disease was based on histopathology, ultrastructural examination of, and virus isolation from lesions of necropsied animals. Lesions were present mainly in juvenile partridges (41%), and were observed primarily on the dorsal part of the digits or on the hock joint. The lesions ranged from small wartlike nodules to large tumor-like lesions. The presence of acute lesions of any grade as opposed to absence of lesions or healed lesions adversely affected body condition of the partridges (P

Serologic testing of wild birds for avian influenza virus (AIV) surveillance poses problems due to species differences and nonspecific inhibitors that may be present in sera of wild birds. Recently available competitive enzyme-linked immunosorbent assay (cELISA) kits offer a new species-independent approach. In this study we compare two commercial competitive cELISAs, using a total of 184 serum and plasma samples from 23 species of wild birds belonging to 10 orders. Thirteen samples were from experimentally high pathogenicity AI and low pathogenicity AI infected red-legged partridges (Alectoris rufa), 77 samples were from a flock of sentinel hybrid ducks confirmed infected by AI by real-time PCR, and 94 samples were from wild birds admitted to a rehabilitation center. Both ELISAs detected AI antibodies in the experimentally infected partridges, whereas hemagglutination inhibition (HI) was negative. Concordance in results between the two ELISAs was 51.5%. When specific subtype-H5/H7 HI-positive samples were considered for comparison, ELISA 1 appeared to perform better on ducks, whereas ELISA 2 appeared to perform better in other wild bird species. Overall, 68.2% of H5/H7 positive samples tested positive by ELISA 1 and 36% by ELISA 2. Both ELISAs detected AIV-antibody-positive samples negative by specific HI against 9 of the 16 existing hemagglutinin (HA) subtypes. Presumably this reflects either higher sensitivity of cELISA when compared to HI, presence of antibodies against HA subtypes not tested, or unspecific reactions. Performance of ELISA 1 on ducks appears to be comparable to in-house cELISA previously used by other authors in wild birds, but requires a relatively large sample volume. Alternatively, although ELISA 2 required a smaller sample volume, it was less effective at identifying HI-positive samples. The results reflect the necessity of validation of cELISA tests for individual species or at least families, as required by the OIE., E. Pérez-Ramírez has an I3p fellowship from the Spanish Science Council (CSIC). P. Acevedo is currently funded by a Juan de la Cierva research contract awarded by the Ministerio de Ciencia e Innovación (MICINN) and is also supported by the project CGL2006-09567/BOS. This work was funded by project RTA2005-0111-00 and FAU2006-019-C03. We thank the JCCM for support, and the personnel of Wildlife Rehabilitation Centers of Castilla-La Mancha for their collaboration.

West Nile virus (WNV) is a globally distributed arthropod-borne flavivirus capable of infecting a wide variety of vertebrates, with birds as its natural reservoir. Although it had been considered a pathogen of little importance for birds, from the 1990's, and especially after its introduction in the North American continent in 1999, thousands of birds have succumbed to West Nile infection. This review summarizes the pathogenesis and pathology of WNV infection in birds highlighting differences in lesion and antigen distribution and severity among bird orders and families. Despite significant species differences in susceptibility to infection, WNV associated lesions and viral antigen are present in the majority of organs of infected birds. The non-progressive, acute or more prolonged course of the disease accounts for part of the differences in lesion and viral antigen distribution and lesion severity. Most likely a combination of host variables and environmental factors in addition to the intrinsic virulence and pathogenicity of the infecting WNV strain influence the pathogenesis of the infection., This review is part of the work supported by grant AG2008-02504GAN funded by the Spanish Ministry for Science and Innovation. V. Gamino (323/09) is a research fellow supported by the regional government of Castilla – La Mancha (JCCM).

Resumen del trabajo presentado al Joint Meeting of 30th Meeting of the European Society of Veterinary Pathology-Annual Meeting of the European College of Veterinary Pathology and 24th Annual Meeting of the Spanish Society of Veterinary Pathology, celebrado en Leon (Espana) del 5 al 8 de septiembre de 2012.-- et al.

European quail (Coturnix c. coturnix) may share with Japanese quail (Coturnix c. japonica) its potential as an intermediate host and reservoir of avian influenza viruses (AIV). To elucidate this question, European quail were experimentally challenged with two highly pathogenic AIV (HPAIV) (H7N1/HP and H5N1/HP) and one low pathogenic AIV (LPAIV) (H7N2/LP). Contact animals were also used to assess the viral transmission among birds. Severe neurological signs and mortality rates of 67% (H7N1/HP) and 92% (H5N1/HP) were observed. Although histopathological findings were present in both HPAIV-infected groups, H5N1/HP-quail displayed a broader viral antigen distribution and extent of microscopic lesions. Neither clinical nor pathological involvement was observed in LPAIV-infected quail. Consistent long-term viral shedding and effective transmission to naive quail was demonstrated for the three studied AIV. Drinking water arose as a possible transmission route and feathers as a potential origin of HPAIV dissemination. The present study demonstrates that European quail may play a major role in AI epidemiology, highlighting the need to further understand its putative role as an intermediate host for avian/mammalian reassortant viruses., This work was funded by The Network of Animal Disease Infectiology Research Facilities (NADIR), project 228394 (INFRA 2008), seventh framework programme, and by the coordinated project FAU2006-00019-C03-00 of the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) and the Ministerio de Educación, Cultura y Deporte of Spain.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Studies exploring the ecological interactions between avian influenza viruses (AIV), natural hosts and the environment are scarce. Most work has focused on viral survival and transmission under laboratory conditions and through mathematical modelling. However, more integrated studies performed under field conditions are required to validate these results. In this study, we combined information on bird community, environmental factors and viral epidemiology to assess the contribution of biotic and abiotic factors in the occurrence of low pathogenic AIV in Spanish wetlands. For that purpose, seven locations in five different wetlands were studied during two years (2007-2009), including seven sampling visits by location. In each survey, fresh faeces (n = 4578) of wild birds and water samples were collected for viral detection. Also, the vegetation structure, water physical properties of wetlands, climatic conditions and wild bird community composition were determined. An overall AIV prevalence of 1.7%±0.4 was detected in faecal samples with important fluctuations among seasons and locations. Twenty-six AIV were isolated from the 78 RRT-PCR positive samples and eight different haemagglutinines and five neuraminidases were identified, being the combination H3N8 the most frequent. Variation partitioning procedures identified the combination of space and time variables as the most important pure factor - independently to other factors - explaining the variation in AIV prevalence (36.8%), followed by meteorological factor (21.5%) and wild bird community composition/vegetation structure (21.1%). These results contribute to the understanding of AIV ecological drivers in Spanish ecosystems and provide useful guidelines for AIV risk assessment identifying potential hotspots of AIV activity., This work was financially supported by INIA (Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria), project FAU2006-00019-C03-00 and by Department of Environment, Territorial Planning, Agriculture and Fisheries of the Basque Government. P. Acevedo was supported by a Beatriu de Pinós fellowship funded by Comissionat per a Universitats i Recerca del Departament d'Innovació, Universitats i Empresa, of the Generalitat de Catalunya and the COFUND Programme-Marie Curie Actions under 7th Marc Programme of the European Community.

A collection of 259 staphylococci of 13 different species [212 coagulase-negative (CoNS) and 47 coagulase-positive (CoPS)] recovered from nasotracheal samples of 87 healthy nestling white storks was tested by the spot-on-lawn method for antimicrobial-activity (AA) against 14 indicator bacteria. Moreover, extracts of AP isolates were obtained [cell-free-supernatants (CFS) both crude and concentrated and butanol extracts] and tested against the 14 indicator bacteria. The microbiota modulation capacity of AP isolates was tested considering: (a) intra-sample AA, against all Gram-positive bacteria recovered in the same stork nasotracheal sample; (b) inter-sample AA against a selection of representative Gram-positive bacteria of the nasotracheal microbiota of all the storks (30 isolates of 29 different species and nine genera). In addition, enzymatic susceptibility test was carried out in selected AP isolates and bacteriocin encoding genes was studied by PCR/sequencing. In this respect, nine isolates (3.5%; seven CoNS and two CoPS) showed AA against at least one indicator bacteria and were considered antimicrobial-producing (AP) isolates. The AP isolates showed AA only for Gram-positive bacteria. Three of these AP isolates (S. hominis X3764, S. sciuri X4000, and S. chromogenes X4620) revealed AA on all extract conditions; other four AP isolates only showed activity in extracts after concentration; the remaining two AP isolates did not show AA in any of extract conditions. As for the microbiota modulation evaluation, three of the nine AP-isolates revealed intra-sample AA. It is to highlight the potent inter-sample AA of the X3764 isolate inhibiting 73% of the 29 representative Gram-positive species of the nasotracheal stork microbiota population. On the other hand, enzymatic analysis carried out in the two highest AP isolates (X3764 and X4000) verified the proteinaceous nature of the antimicrobial compound and PCR analysis revealed the presence of lantibiotic-like encoding genes in the nine AP isolates. In conclusion, these results show that nasotracheal staphylococci of healthy storks, and especially CoNS, produce antimicrobial substances that could be important in the modulations of their nasal microbiota.

AIM: To establish the optimal dose of the mitogen phytohaemagglutinin (PHA) and the optimal time for measuring increased skin-fold thickness in red deer following intradermal injection, as an indicator of cell-mediated immune response. METHODS: Three doses (10, 50 and 250 μg) of PHA were injected intradermally in the right side of the neck, and phosphate buffered saline (PBS) was injected at a fourth site as a control, in 20 captive Iberian red deer (Cervus elaphus hispanicus) hinds. Skin-fold thicknesses were measured at 0, 12, 24, 36, 48, 60, 72, 84 and 96 h following injection. RESULTS: The highest dose of PHA tested (250 μg) resulted in a clear and long-lasting cellular response; increases in skin-fold thickness between 48 and 84 h post-injection varied minimally and response correlated positively with liveweight. No correlations with liveweight and no clear increases in skin-fold thickness occurred at the lower doses of PHA or the PBS. CONCLUSIONS AND CLINICAL RELEVANCE: This technique could be applied with minimal training and without specialised equipment in deer, for immunological and ecological research.

AIM: To determine if there are sex- or age-related differences in the increase in skinfold thickness in response to the mitogen phytohaemagglutinin (PHA) in red deer. METHODS: One dose of 250 ug PHA was injected intradermally in the right side of the neck, and phosphate buffered saline (PBS) was injected at a second site as a control, in 110 (51 males and 59 females) captive Iberian red deer (Cervus elaphus hispanicus), ranging in age from 21 months to ≥5 years. Skinfold thicknesses were measured immediately before and 72 h following injection. RESULTS: There was a significant effect of gender on the average increase in skinfold thickness; males had greater increases (8.8 (SEM 0.57) mm) than females (4.23 (SEM 0.39) mm) after correcting for other confounding variables. No age-related differences were evident, but differences between sexes were more marked with increasing age. CONCLUSIONS AND CLINICAL RELEVANCE: Effects of gender, probably due to differences in energetic and reproductive constraints in red deer, should be taken into account when interpreting skinfold-test data, both in ecology and in the control of tuberculosis (Tb). Males tend to have a thicker skin than females, so skinfold increase relative to the thickness of the skin, rather than skinfold increase per se, should be used as a more appropriate measure of skinfold increase. This may also have clinical relevance in the interpretation of tuberculin skin testing.

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License.-- et al., [Background]: During the last decade, the spread of many flaviviruses (Genus Flavivirus) has been reported, representing an emerging threat for both animal and human health. To further study utility of wild ruminant samples in West Nile virus (WNV) surveillance, we assessed spatio-temporal trends and factors associated with WNV and cross-reacting flaviviruses exposure, particularly Usutu virus (USUV) and Meaban virus (MBV), in wild ruminants in Spain. Serum samples from 4693 wild ruminants, including 3073 free-living red deer (Cervus elaphus), 201 fallow deer (Dama dama), 125 mouflon (Ovis aries musimon), 32 roe deer (Capreolus capreolus) and 1262 farmed red deer collected in 2003-2014, were screened for WNV and antigenically-related flavivirus antibodies using a blocking ELISA (bELISA). Positive samples were tested for neutralizing antibodies against WNV, USUV and MBV by virus micro-neutralization tests. [Results]: Mean flavivirus seroprevalence according to bELISA was 3.4 ± 0.5 % in red deer, 1.0 ± 1.4 % in fallow deer, 2.4 ± 2.7 % in mouflon and 0 % in roe deer. A multivariate logistic regression model revealed as main risk factors for seropositivity in red deer; year (2011), the specific south-coastal bioregion (bioregion 5) and presence of wetlands. Red deer had neutralizing antibodies against WNV, USUV and MBV. [Conclusions]: The results indicate endemic circulation of WNV, USUV and MBV in Spanish red deer, even in areas without known flavivirus outbreaks. WNV antibodies detected in a free-living red deer yearling sampled in 2010, confirmed circulation this year. Co-circulation of WNV and USUV was detected in bioregions 3 and 5, and of WNV and MBV in bioregion 3. Sampling of hunted and farmed wild ruminants, specifically of red deer yearlings, could be a complementary way to national surveillance programs to monitor the activity of emerging flaviviruses., This study has been supported by projects PAC08-0296-7771 (JCCM), AG2008-02504GAN, FAU2008-00019-C03-01 and AGL2013-49159-C2-2-R. Gutierrez-Guzman AV, was a JCCM fellow (PAC08-0296-7771).

Since December 1997, 700 blood plasma samples from 31 different species of captive and free-living birds of prey from Spain were analyzed by hemagglutination inhibition (HI) test for the presence of antibodies to avian paramyxovirus (aPMV) 1,2, and 3. Out of 700 birds, 120 tested positive for aPMV-1, 10 birds had antibodies to aPMV-2, and 4 birds tested positive against aPMV-3. Prevalence of antibodies against aPMV-1 was significantly higher in captive than in free-living birds of prey and in Falconiformes than in Strigidae and Accipitridae. Infection or exposure in captive birds may be due to the use of avian-derived food in rehabilitation and captive-breeding centers. This may be of concern at the time of reintroduction of these birds into free-living populations.