Ovarian Sex Cord Stromal Tumor in a Free-Ranging Brown Bear (Ursus arctos).

Reports on neoplasms in bears are scarce, especially concerning ovarian tumors. A large primary ovarian neoplasm with multiple metastasis was found during the necropsy of a 14-year-old free-ranging Eurasian brown bear (Ursus arctos) from Northwestern Spain. Histopathology and immunohistochemistry allowed for the diagnosis of a sex cord stromal tumor. This is a complex group of neoplasms which differ in the predominant cell morphology and immunohistochemical features. The microscopic examination revealed two types of cells, one with eosinophilic cytoplasm, intermingled with larger vacuolated cells rich in lipids. The evaluation of the immunoreactivity to different markers, frequently used in the characterization of gonadal tumors (INHA, inhibin-alpha; PLAP, placental alkaline phosphatase; Ki-67; α-SMA, actin alpha-smooth muscle) and inflammation patterns (IBA1, ionized calcium-binding adapter molecule for macrophages; CD3 for T lymphocytes; CD20 for B lymphocytes), displayed significant INHA positive immunostaining of neoplastic cells, as well as inflammatory cell infiltration, mainly composed of macrophages and B lymphocytes. These findings were consistent with a malignant ovarian steroid cell tumor, not otherwise specified. The present study characterizes an unusual type of neoplasm, and also represents the first report of an ovarian sex cord stromal tumor in Ursidae.

Neuropathological lesions in intravenous BCG-stimulated K18-hACE2 mice challenged with SARS-CoV-2.

In the wake of the COVID-19 pandemic caused by SARS-CoV-2, questions emerged about the potential effects of Bacillus Calmette-Guérin (BCG) vaccine on the immune response to SARS-CoV-2 infection, including the neurodegenerative diseases it may contribute to. To explore this, an experimental study was carried out in BCG-stimulated and non-stimulated k18-hACE2 mice challenged with SARS-CoV-2. Viral loads in tissues determined by RT-qPCR, histopathology in brain and lungs, immunohistochemical study in brain (IHC) as well as mortality rates, clinical signs and plasma inflammatory and coagulation biomarkers were assessed. Our results showed BCG-SARS-CoV-2 challenged mice presented higher viral loads in the brain and an increased frequency of neuroinvasion, with the greatest differences observed between groups at 3-4 days post-infection (dpi). Histopathological examination showed a higher severity of brain lesions in BCG-SARS-CoV-2 challenged mice, mainly consisting of neuroinflammation, increased glial cell population and neuronal degeneration, from 5 dpi onwards. This group also presented higher interstitial pneumonia and vascular thrombosis in lungs (3-4 dpi), BCG-SARS-CoV-2 mice showed higher values for TNF-α and D-dimer values, while iNOS values were higher in SARS-CoV-2 mice at 3-4 dpi. Results presented in this study indicate that BCG stimulation could have intensified the inflammatory and neurodegenerative lesions promoting virus neuroinvasion and dissemination in this experimental model. Although k18-hACE2 mice show higher hACE2 expression and neurodissemination, this study suggests that, although the benefits of BCG on enhancing heterologous protection against pathogens and tumour cells have been broadly demonstrated, potential adverse outcomes due to the non-specific effects of BCG should be considered.

Non-invasive surveillance of shared pathogens in the Eurasian brown bear (Ursus arctos) human interface.

Multi-host communities are perfect scenarios for the emergence and spread of pathogens, threatening the recovery of endangered, isolated, or inbred populations, such as the brown bear (Ursus arctos) in northwestern Spain. The population recovery in recent years has forced bears to occupy highly anthropized areas, increasing their interaction with human and domestic animals, with potential consequences for global health. During 2022-2023 a survey of parasites, bacteria and viruses shared between wildlife, domestic animals and humans was performed in this population using non-invasive surveillance, i.e., bear fecal samples (n = 73) and sponge-based sampling of trees (n = 42; 14 rubbed trees and 28 control trees). Pathogen detection rates were defined as the percentage of qPCR or culture-positive samples. Generalized linear models were fitted to assess their relationship with environmental variables including dispersion of the human population, and percentage of agricultural and periurban habitats in a 6 km-buffer around each sample. Canine Adenovirus type 1 (45.2%), Giardia spp. (15.1%), Salmonella spp. (12.3%), and extended-spectrum-beta-lactamases (ESBL) Escherichia coli (1.4%) were identified in fecal samples. In contrast, only five sponges from three rubbed and two control trees resulted positive to E. coli (14.3%). The results suggest that several pathogens are common in the Cantabrian brown bear population and that anthropization of the territory modulates their prevalence and richness. The effective design of management programs for bear conservation will require a one-health approach, in which genetic analysis of non-invasive samples can be key tools for the sanitary surveillance at the wildlife-livestock-human interface.

Hypertrophic osteopathy in a common dolphin (Delphinus delphis) with concurrent pulmonary Halocercus delphini infestation.

Dolphins are marine mammals that often live in coastal habitats. Common causes of severe skeletal disorders among wild dolphins are congenital vertebral anomalities, collisions with sea vessels, trauma, hunting-related injury, infectious diseases, environmental pollution, and tumors. A free-ranging male, 3-year-old common dolphin (Delphinus delphis) was found dead in the coast of Asturias in northern Spain. Postmortem examination revealed lordosis in the caudal vertebral column, while X-ray imaging and computer tomography showed well-organized palisade-like periosteal proliferation, appearing as florid-like accretions, along the spinous apophysis of 26 lumbar-caudal vertebrae. The transverse apophysis was affected on only a few caudal vertebrae. The cortical layer remained intact. Histology of vertebra tissue showed periosteal proliferation of cancellous bone. The animal was diagnosed with hypertrophic osteopathy. The lungs showed diffuse parasitic granulomatous bronchointerstitial pneumonia caused by Halocercus delphini, consolidation of the pulmonary tissue, congestion, and alveolar edema. The animal was also afflicted by parasitic granulomatous gastritis caused by Anisakis simplex sensu lato and tattoo skin disease. The dolphin suffered from hypertrophic osteopathy associated with pulmonary Halocercus delphini infestation. This syndrome, known as hypertrophic pulmonary osteopathy, has been described in diverse terrestrial mammals, including domestic animals, wildlife and humans, but not in dolphins. This case reports the first description of hypertrophic osteopathy associated to a pulmonary disorder in dolphin, and it provides insights into factors that can induce column malformation in dolphins, suggesting the importance of taking thoracic lesions into account during differential diagnosis.

Occurrence and limited zoonotic potential of Cryptosporidium spp., Giardia duodenalis, and Balantioides coli infections in free-ranging and farmed wild ungulates in Spain.

Little information is currently available on the occurrence and molecular diversity of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Balantioides coli in wild ungulates and the role of these host species as potential sources of environmental contamination and consequent human infections. The presence of these three pathogens was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were retrospectively collected from free-ranging (n = 1058) and farmed (n = 324) wild ungulates from the five Spanish bioregions. Overall infection rates were 3.0% (42/1382; 95% CI: 2.1-3.9%) for Cryptosporidium spp., 5.4% (74/1382; 95% CI: 4.2-6.5%) for G. duodenalis, and 0.7% (9/1382; 95% CI: 0.3-1.2%) for B. coli. Cryptosporidium infection was detected in roe deer (7.5%), wild boar (7.0%) and red deer (1.5%), and G. duodenalis in southern chamois (12.9%), mouflon (10.0%), Iberian wild goat (9.0%), roe deer (7.5%), wild boar (5.6%), fallow deer (5.2%) and red deer (3.8%). Balantioides coli was only detected in wild boar (2.5%, 9/359). Sequence analyses revealed the presence of six distinct Cryptosporidium species: C. ryanae in red deer, roe deer, and wild boar; C. parvum in red deer and wild boar; C. ubiquitum in roe deer; C. scrofarum in wild boar; C. canis in roe deer; and C. suis in red deer. Zoonotic assemblages A and B were detected in wild boar and red deer, respectively. Ungulate-adapted assemblage E was identified in mouflon, red deer, and southern chamois. Attempts to genotype samples positive for B. coli failed. Sporadic infections by canine- or swine-adapted species may be indicative of potential cross-species transmission, although spurious infections cannot be ruled out. Molecular evidence gathered is consistent with parasite mild infections and limited environmental contamination with (oo)cysts. Free-ranging wild ungulate species would not presumably play a significant role as source of human infections by these pathogens. Wild ruminants do not seem to be susceptible hosts for B. coli.

Double immunofluorescence staining of whole-mount small intestinal mucosa samples as a tool for characterization of three-dimensional paratuberculosis granulomas.

Bovine paratuberculosis (PTB) is a chronic granulomatous enteritis, caused by Mycobacterium avium subsp. paratuberculosis (Map). The progression of PTB from subclinical to the clinical stage of the disease is determined locally at the level of the granuloma, a host defence hallmark against mycobacterial infection. Therefore, in-depth characterization of distinct cell populations controlling granuloma formation is critical to understanding PTB progression. Confocal laser scanning microscopy (CLSM) has been extensively used to visualize two or more proteins of interest concomitantly within a variety of cellular structures. As such, it is an invaluable tool for the correct identification and characterization of different cell populations. In this study, a novel approach, CLSM of whole-mount small intestinal mucosa samples, is used to characterize three-dimensional (3-D) paratuberculosis granulomas and epithelioid macrophages. Detailed optimized procedures to perform CLSM in whole mount small intestinal mucosa samples and also in formalin fixed paraffin embedded (FFPE) intestinal tissue sections of Holstein Friesian cows presenting different types of PTB-associated histological lesions are described.

Anencephaly and Severe Myelodysplasia in a Stillborn Brown Bear (Ursus arctos arctos).

Malformations in the development of the neural tube have been described to be associated with different aetiologies, such as genetic factors, toxic plants, chemical products, viral agents, or hyperthermia. A twenty-four-year-old female Eurasian brown bear (Ursus arctos arctos), permanently in captivity and kept under food and management control, gave birth to a stillborn cub at the end of gestation. Several malformations resulting from the anomalous development of the neural tube, not previously reported in bears, were observed, such as anencephaly, hypoplasia, micromyelia, severe myelodysplasia, syringomyelia, and spina bifida. Multiple canal defects (e.g., absence) were also observed in the spinal cord. In some regions, the intradural nerve roots surrounded the spinal cord in a diffuse and continuous way. The aetiology remains unidentified, although the advanced age of the mother and/or folic acid deficit might have been the possible causes of this disorder. Supplements of folate given to the mother before and during early pregnancy may have reduced the incidence of neural tube defects. That supplementation should be considered when the reproduction of bears is to occur in captivity, in order to prevent the loss of future generations of this endangered species.

Experimental Infection of Captive Red Foxes (Vulpes vulpes) with Mycobacterium bovis.

In Europe, animal tuberculosis (TB) due to Mycobacterium bovis involves multi-host communities that include cattle and wildlife species, such as wild boar (Sus scrofa), badgers (Meles meles) and red deer (Cervus elaphus). Red fox (Vulpes vulpes) infections have also been recently reported in some TB endemic regions in the Iberian Peninsula and France, with some of the infected animals shedding M. bovis in urine and feces. In order to understand the pathogenesis of M. bovis infection in foxes and the associated risk of transmission, 12 captive foxes (6 females and 6 males) were inoculated orally with 2 × 107 colony-forming units of a French field isolate of M. bovis. Clinical samples (urine, feces and oropharyngeal swabs) were collected every four weeks and tested for molecular diagnosis and bacteriology. Serological responses were measured by IDEXX M. bovis Ab Test and Multi Antigen Print Immunoassay (MAPIA). At a post-mortem examination performed 12 weeks post infection (wpi), tissues were tested for the presence of M. bovis and associated gross and microscopic TB-like lesions. M. bovis was detected by PCR in bladder swabs of 3 animals at 12 wpi. It was also detected pre-mortem at different time points of the experiment in the oropharyngeal mucus of three individuals and in the feces of nine foxes, with two of them confirmed by bacteriology. All 12 foxes had at least 4 PCR positive samples (out of the 23 tested), and all but 1 fox had at least 1 culture positive sample. The culture negative fox was PCR positive in both retropharyngeal and mesenteric lymph nodes, in line with the results of the other animals. Seroconversion was observed in all foxes except one during the experiment, and in nine at the final time point. No gross visible lesions were found in any animal at the post-mortem examination. The histology showed small granulomas within the lymph nodes, tonsils, liver and lungs from eight animals, with the presence of few acid-fast bacilli. These results confirmed that all orally-infected foxes developed mild TB lesions but they were able to shed mycobacteria in about 75% of cases, 1 month post-infection (9 out 12 foxes). These results show that it is possible to induce typical TB infection experimentally in captive foxes, with measurable M. bovis excretion; such an experimental system could be useful for future evaluations of diagnostics and vaccines in this species.

Wolf (Canis lupus) as canine adenovirus type 1 (CAdV-1) sentinel for the endangered cantabrian brown bear (Ursus arctos arctos).

Canine adenovirus type 1 (CAdV-1) causes infectious canine hepatitis (ICH) and has recently been described as a cause of death among endangered populations of European brown bear (Ursus arctos arctos) in the Cantabrian mountain range in Asturias, Spain. Sympatric wild and domestic carnivores can act as reservoirs of the virus and likely spread it into the environment and subsequently transmit it to brown bears. The present work investigates the prevalence and geo-temporal distribution of CAdV-1 among free-ranging wolves (Canis lupus) in Asturias from 2009 to 2018, during which three fatal cases of ICH were reported among brown bears in the region. A total of 149 wolves were analysed in this study, of which 21 (14%) were found to have CAdV-1 DNA based on real-time polymerase chain reaction (RT-PCR) of spleen samples. Prevalence of the virus was similar between males and females. All but one of the 20 CAdV-1-positive animals of estimable age were younger than 2 years, and only one of the 46 adult animals (>2 years) tested positive. Prevalence was highest in the western area of Asturias and during 2010 and 2011. Our results confirm that CAdV-1 is circulating in Asturian free-ranging wolves, supporting their possible role as virus reservoirs and sentinels in the region of this emerging disease in brown bears.

Seroepidemiology of Toxoplasma gondii in wild ruminants in Spain.

Toxoplasmosis is a parasitic zoonosis caused by Toxoplasma gondii which infects warm-blooded species worldwide. Humans can be infected through ingestion of tissue cysts from raw or undercooked meat, including game meat. A nationwide large-scale cross-sectional study was conducted to assess exposure to T. gondii in seven wild ruminant species in Spain. A total of 2,040 serum samples from 77 sampling sites randomly distributed in the five bioregions (BRs) covering mainland Spain were tested for antibodies against T. gondii using the modified agglutination test. The overall seroprevalence was 22.0% (449/2,040). Seroprevalence by species in decreasing order was as follows: 39.6% (141/356) in roe deer (Capreolus capreolus), 37.1% (138/372) in fallow deer (Dama dama), 16.6% (92/553) in red deer (Cervus elaphus), 14.0% (26/186) in Southern chamois (Rupicapra pyrenaica), 11.5% (24/209) in mouflon (Ovis aries musimon), 7.8% (27/346) in Iberian wild goat (Capra pyrenaica) and 5.6% (1/18) in Barbary sheep (Ammotragus lervia). Seropositivity was detected in 74.0% (57/77) of the sampling sites. Results indicate widespread but not homogeneous exposure to T. gondii in wild ruminant populations in Spain during the last two decades and highlight differences related to animal species and spatial distribution of these species in this country; this implies potential consequences of T. gondii for animal health, conservation and public health.

Bovine Intelectin 2 Expression as a Biomarker of Paratuberculosis Disease Progression.

Paratuberculosis (PTB), a chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP), is responsible for important economic losses in the dairy industry. Our previous RNA-sequencing (RNA-Seq) analysis showed that bovine intelectin 2 (ITLN2) precursor gene was overexpressed in ileocecal valve (ICV) samples of animals with focal (log2 fold-change = 10.6) and diffuse (log2 fold-change = 6.8) PTB-associated lesions compared to animals without lesions. This study analyzes the potential use of ITLN2, a protein that has been described as fundamental in the innate immune response to infections, as a biomarker of MAP infection. The presence of ITLN2 was investigated by quantitative immunohistochemical analysis of ICV samples of 20 Holstein Friesian cows showing focal (n = 5), multifocal (n = 5), diffuse (n = 5) and no histological lesions (n = 5). Significant differences were observed in the mean number of ITLN2 immunostained goblet and Paneth cells between the three histopathological types and the control. The number of immunolabelled cells was higher in the focal histopathological type (116.9 ± 113.9) followed by the multifocal (108.7 ± 140.5), diffuse (76.5 ± 97.8) and control types (41.0 ± 81.3). These results validate ITLN2 as a post-mortem biomarker of disease progression.

Mortality Causes in Free-Ranging Eurasian Brown Bears (Ursus arctos arctos) in Spain 1998-2018.

This work summarizes the mortality cases of twenty-five free-ranging Eurasian wild brown bears (Ursus arctos arctos) from the Cantabrian mountain range submitted for necropsy in Asturias and Castilla y León (northwestern Spain) from 1998 to 2018. Mortality cases were classified both caused by (i) "non-human intervention" or "human intervention" causes and based on (ii) "non-infectious" or "infectious" etiology. In four cases (16%) it was not possible to determine the cause of death due to the inadequate preservation of collected specimens or insufficient tissue availability. Based on "non-human intervention" or "human intervention" causes, fourteen of the 21 (66.7%) brown bears died as a consequence of "non-human intervention" due to traumatic lesions (fights, unknown traumas or infanticide), infectious canine hepatitis, neoplasia or mushroom poisoning. In contrast, seven (33.3%) brown bears died by "human intervention" due to illegal hunting (shooting or snare), handling (during transit in an attempt to reintroduce a bear back into the wild) or strychnine poisoning. Based on "non-infectious" or "infectious" etiology, twelve of the 21 (57.1%) brown bears died due to "non-infectious" causes, namely traumatic lesions such as shooting, snare, fighting or infanticide, handling, strychnine poisoning, mushroom poisoning or neoplasia. The remaining nine (42.9%) animals died due to "infectious" diseases which included gangrenous myositis, infectious canine hepatitis or septicemia. In six of those cases traumatic lesions caused by non-human or human activities were complicated with bacterial infection (clostridiosis and septicemia) which finally caused the death of those animals. Additionally, exertional myopathy was observed in the handled animal and in one bear found in a snare. In a free-ranging population of Eurasian brown bear from the Cantabrian mountain range, main causes of death are attributed to non-human related traumatic lesions and infectious diseases (primary developed such as infectious canine hepatitis or secondary developed such as clostridiosis or septicemia) which is in contrast to previously reported data for other bear populations. These data are valuable and may help in the conservation and management of this recovering population.

Detection of latent forms of Mycobacterium avium subsp. paratuberculosis infection using host biomarker-based ELISAs greatly improves paratuberculosis diagnostic sensitivity.

Bovine paratuberculosis (PTB) is a chronic granulomatous enteritis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), responsible for important economic losses in the dairy industry. Current diagnostic methods have low sensitivities for detection of latent forms of MAP infection, defined by focal granulomatous lesions and scarce humoral response or MAP presence. In contrast, patent infections correspond to multifocal and diffuse types of enteritis where there is increased antibody production, and substantial mycobacterial load. Our previous RNA-Seq analysis allowed the selection of five candidate biomarkers overexpressed in peripheral blood of MAP infected Holstein cows with focal (ABCA13 and MMP8) and diffuse (FAM84A, SPARC and DES) lesions vs. control animals with no detectable PTB-associated lesions in intestine and regional lymph nodes. The aim of the current study was to assess the PTB diagnostic potential of commercial ELISAs designed for the specific detection of these biomarkers. The ability of these ELISAs to identify animals with latent and/or patent forms of MAP infection was investigated using serum from naturally infected cattle (n = 88) and non-infected control animals (n = 67). ROC analysis revealed that the ABCA13-based ELISA showed the highest diagnostic accuracy for the detection of infected animals with focal lesions (AUC 0.837, sensitivity 79.25% and specificity 88.06%) and with any type of histological lesion (AUC 0.793, sensitivity 69.41% and specificity 86.57%) improving on the diagnostic performance of the popular IDEXX ELISA and other conventional diagnostic methods. SPARC and MMP8 showed the highest diagnostic accuracy for the detection of animals with multifocal (AUC 0.852) and diffuse lesions (AUC 0.831), respectively. In conclusion, our results suggest that quantification of ABCA13, SPARC and MMP8 by ELISA has the potential for implementation as a diagnostic tool to reliably identify MAP infection, greatly improving early detection of MAP latent infections when antibody responses and fecal shedding are undetectable using conventional diagnostic methods.

Local Lung Immune Response to Mycobacterium bovis Challenge after BCG and M. bovis Heat-Inactivated Vaccination in European Badger (Meles meles).

Tuberculosis (TB) vaccination could be used as a key part of integrated strategies for the disease's control if an effective and safe vaccine under field conditions is obtained. Recent studies in Spain have evaluated the protective efficacy of two oral vaccines against experimental challenge with live intra-bronchial Mycobacterium bovis in captive badgers: the live-attenuated M. bovis BCG vaccine (Danish strain) and a heat-inactivated M. bovis (HIMB) vaccine. With the objective of increasing the knowledge of the cellular development progress of infection and generating further tools to discriminate between mild and severe TB lesions between and within animals, the immunopathology of tuberculous lesions was studied to characterize the local immune response (cell type profile) within lung granulomas from control (non-vaccinated), BCG vaccinated and HIMB-vaccinated experimentally infected badgers with M. bovis. Four immunohistochemical protocols, for the specific detection of macrophages, T lymphocytes, B lymphocytes and plasma cells within TB granulomas in formalin fixed sections of the right middle lung lobe (lobe targeted for the M. bovis delivery), were performed. Immunolabelled sections were scanned and five randomly selected areas were analyzed with digital image analysis software. The results were expressed as the proportion of the positively immunolabelled area within the total area of the selected site. Data was analyzed using the statistical analysis software (SAS). In the three treatment groups, macrophages were the most abundant inflammatory cells within the granulomas, followed by B lymphocytes and plasma cells. T lymphocyes were absent in those granulomas. This would suggest a predominance of a non-specific innate response mediated by phagocytic cells over an adaptative humoral immune response. The proportion of macrophages and plasma cells was higher in BCG and HIMB-vaccinated badgers, respectively, suggesting the establishment of an adaptative humoral response in HIMB-vaccinated badgers. The lower bacterial load at the lung level, as well as the volume of lesions in lungs using magnetic resonance imaging in badgers with the HIMB vaccine in relation with local immune response presented, must be highlighted, since it would be an advantage in favor of its use under field conditions in terms of reducing TB transmission and environmental contamination.

Cholangiocarcinoma in a Free-Ranging Eurasian Brown Bear (Ursus arctos arctos) from Northern Spain.

A hepatic cholangiocarcinoma with metastases in the gallbladder, left elbow joint, adrenal glands, and lungs was observed in a female 21-yr-old free-ranging Eurasian brown bear (Ursus arctos arctos) found in the Principality of Asturias (northern Spain). Gross and histopathologic findings are described.

RNA-Seq analysis of ileocecal valve and peripheral blood from Holstein cattle infected with Mycobacterium avium subsp. paratuberculosis revealed dysregulation of the CXCL8/IL8 signaling pathway.

Paratuberculosis is chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Whole RNA-sequencing (RNA-Seq) is a promising source of novel biomarkers for early MAP infection and disease progression in cattle. Since the blood transcriptome is widely used as a source of biomarkers, we analyzed whether it recapitulates, at least in part, the transcriptome of the ileocecal valve (ICV), the primary site of MAP colonization. Total RNA was prepared from peripheral blood (PB) and ICV samples, and RNA-Seq was used to compare gene expression between animals with focal or diffuse histopathological lesions in gut tissues versus control animals with no detectable signs of infection. Our results demonstrated both shared, and PB and ICV-specific gene expression in response to a natural MAP infection. As expected, the number of differentially expressed (DE) genes was larger in the ICV than in the PB samples. Among the DE genes in the PB and ICV samples, there were some common genes irrespective of the type of lesion including the C-X-C motif chemokine ligand 8 (CXCL8/IL8), apolipoprotein L (APOLD1), and the interferon inducible protein 27 (IFI27). The biological processes (BP) enriched in the PB gene expression profiles from the cows with diffuse lesions included the killing of cells of other organism, defense response, immune response and the regulation of neutrophil chemotaxis. Two of these BP, the defense and immune response, were also enriched in the ICV from the cows with diffuse lesions. Metabolic analysis of the DE genes revealed that the N-glycan biosynthesis, bile secretion, one-carbon pool by folate and purine metabolism were significantly enriched in the ICV from the cows with focal lesions. In the ICV from cows with diffuse lesions; the valine, leucine and isoleucine degradation route, purine metabolism, vitamin digestion and absorption and the cholesterol routes were enriched. Some of the identified DE genes, BP and metabolic pathways will be studied further to develop novel diagnostic tools, vaccines and immunotherapeutics.

Canine adenovirus type 1 (CAdV-1) in free-ranging European brown bear (Ursus arctos arctos): A threat for Cantabrian population?

Canine adenovirus type 1 (CAdV-1) is responsible for infectious canine hepatitis. The disease has been described in captive American black bear (Ursus americanus) and European brown bear (Ursus arctos arctos), with just one recently reported case in a cub of a free-ranging brown bear (Ursus arctos horribilis) from Alaska. The aim of this work is to summarize findings related to presence and associated mortality of CAdV-1 in 21 free-ranging Cantabrian brown bears (Ursus arctos arctos) submitted to necropsy in Asturias and Castilla y León (northwestern Spain) from 1998 to 2018. On the basis of the anatomopathological findings and laboratory results three free-ranging brown bears died due to infectious canine hepatitis, which is to our knowledge the first description of death due to this disease in free-ranging bears in Europe. Gross lesions consisted of petechial haemorrhages and congestion in different internal organs, haemorrhagic fluid in internal cavities, friable and yellowish liver and thickening of gall bladder. Microscopic lesions were observed mainly in liver, kidney and brain and consisted of multifocal necrosis of cells with presence of basophilic intranuclear inclusions. Immunohistochemical (IHC) and real-time polymerase chain reaction (qPCR) techniques were used to assess the presence of CAdV-1 in paraffin-embedded liver samples. Viral antigens were detected by IHC labelling within hepatocytes and Küppfer cells in the three animals. The presence of viral DNA was confirmed by qPCR in one of them. In order to evaluate the circulation of CAdV-1 in brown bears, a retrospective study was performed using both IHC and qPCR techniques in 11 and 12 additional brown bears, respectively. An extra brown bear was found positive by IHC. This study shows that CAdV-1 surveillance of brown bears and sympatric carnivores should be considered as major concern for the monitoring the population evolution throughout time in this endangered species.

Immunohistochemical characterization of tuberculous lesions in sheep naturally infected with Mycobacterium bovis.

BACKGROUND: Sheep have been traditionally considered as less susceptible to Mycobacterium bovis (Mbovis) infection than other domestic ruminants such as cattle and goats. However, there is increasing evidence for the role of this species as a domestic Mbovis reservoir, mostly when sheep share grazing fields with infected cattle and goats. Nevertheless, there is a lack of information about the pathogenesis and the immune response of Mbovis infection in sheep. The goals of this study were to characterize the granuloma stages produced by the natural infection of Mbovis in sheep, to compare them with other species and to identify possible differences in the sheep immune response. Samples from bronchial lymph nodes from twelve Mbovis-naturally infected sheep were used. Four immunohistochemical protocols for the specific detection of T-lymphocytes, B-lymphocytes, plasma cells and macrophages were performed to study the local immune reaction within the granulomas. RESULTS: Differences were observed in the predominant cell type present in each type of granuloma, as well as differences and similarities with the development of tuberculous granulomas in other species. Very low numbers of T-lymphocytes were observed in all granuloma types indicating that specific cellular immune response mediated by T-cells might not be of much importance in sheep in the early stages of infection, when macrophages are the predominant cell type within lesions. Plasma cells and mainly B lymphocytes increased considerably as the granuloma developed being attracted to the lesions in a shift towards a Th2 response against the increasing amounts of mycobacteria. Therefore, we have proposed that the granulomas could be defined as initial, developed and terminal. CONCLUSIONS: Results showed that the study of the lymphoid tissue granulomata reinforces the view that the three different types of granuloma represent stages of lesion progression and suggest an explanation to the higher resistance of sheep based on a higher effective innate immune response to control tuberculosis infection.

Occurrence and molecular genotyping of Giardia duodenalis and Cryptosporidium spp. in wild mesocarnivores in Spain.

There is a surprisingly scarce amount of epidemiological and molecular data on the prevalence, frequency, and diversity of the intestinal protozoan parasites Giardia duodenalis and Cryptosporidium spp. in wildlife in general and mesocarnivore species in particular. Consequently, the extent of the cyst/oocyst environmental contamination attributable to these wild host species and their potential implications for public veterinary health remain largely unknown. In this molecular epidemiological survey a total of 193 individual faecal samples from badgers (Meles meles, n=70), ferrets (Mustela putorius furo, n=2), genets (Genetta genetta, n=6), Iberian lynxes (Lynx pardinus, n=6), beech martens (Martes foina, n=8), mongooses (Herpestes ichneumon, n=2), otters (Lutra lutra, n=2), polecats (Mustela putorius, n=2), red foxes (Vulpes vulpes, n=87), wildcats (Felis silvestris, n=2), and wolves (Canis lupus, n=6) were obtained from road-killed, hunted, and accidentally found carcasses, and from camera-trap surveys or animals entering rescue shelters, during the period December 2003-April 2016. Investigated specimens were collected in five Spanish autonomous regions including Andalusia (n=1), Asturias (n=69), Basque Country (n=49), Castile-La Mancha (n=38), and Extremadura (n=36). The presence of cysts/oocysts was confirmed by PCR-based methods targeting the small subunit (ssu) ribosomal RNA gene of these parasite species. Genotyping of the obtained isolates were attempted at appropriate markers including the glutamate dehydrogenase (G. duodenalis) and the 60-kDa glycoprotein (C. parvum and C. ubiquitum) loci. Overall, G. duodenalis was detected in 8% (7/87) of red foxes, a single beech marten, and a single wolf, respectively. Cryptosporidium was identified in 3% (2/70) of badgers, 8% (7/87) of red foxes, a single genet, and a single mongoose, respectively. None of the nine G. duodenalis isolates generated could be genotyped at the assemblage/sub-assemblage level. Out of the nine Cryptosporidium isolates successfully characterized, three were identified as C. canis (one in a mongoose and two in red foxes), and three as C. parvum (one in a badger and three in red foxes). The remaining three isolates were assigned to C. felis (in a red fox), C. hominis (in a badger), and C. ubiquitum (in a red fox), respectively. Two additional Cryptosporidium isolates infecting a badger and a genet, respectively, were untypable. The red fox was confirmed as a suitable host of potentially zoonotic Cryptosporidium species, mainly C. parvum and C. ubiquitum. The high mobility and wide home range of red foxes, together with their increasing presence in urban and peri-urban settings, may led to the overlapping of sylvatic and domestic cycles of the parasite, and consequently, to an increased risk of cryptosporidiosis in production animals and humans. The detection of C. hominis oocysts in a badger raises the question of whether this finding represents a true infection or a sporadic event of mechanical passage of C. hominis oocyst of anthroponotic origin.

Vaccination of rabbits with immunodominant antigens from Sarcoptes scabiei induced high levels of humoral responses and pro-inflammatory cytokines but confers limited protection.

BACKGROUND: Vaccination is an attractive ecological alternative to the use of acaricides for parasite control. However, effective anti-parasite vaccines against sarcoptic mange have not yet been developed. The purpose of this study was first to identify Sarcoptes scabiei immunodominant antigens and second to evaluate them as vaccine candidates in a rabbit/S. scabiei var. cuniculi model. METHODS: The S. scabiei Ssλ15 immunodominant antigen was selected by immunoscreening of a S. scabiei var. hominis cDNA. The full-length cDNA was sequenced and cloned into the pGEX vector and the recombinant protein expressed in BL21 (DE3) cells and purified. A vaccination trial was performed consisting of a test group (n = 8) immunised with recAgs (a mix of two recombinant antigens, Ssλ15 and the previously described Ssλ20∆B3) and a control group (n = 8) immunised with PBS. All analyses were performed with R Statistical Environment with α set at 0.050. RESULTS: The full-length open reading frame of the 1,821 nt cloned cDNA encodes a 64 kDa polypeptide, the sequence of which had 96 % identity with a hypothetical protein of S. scabiei. Ssλ15 was localised by immunostaining of skin sections in the tegument surrounding the mouthparts and the coxa in the legs of mites. Rabbit immunisation with recAgs induced high levels of specific IgG (P < 0.010) and increased levels of total IgEs. However, no significant clinical protection against S. scabiei challenge was detected. Unexpectedly, the group immunised with the recAgs mix had significantly higher lesion scores (P = 0.050) although lower mean mite densities than those observed in the control group. These results might indicate that the lesions in the recAgs group were due not only to the mites density but also to an exacerbated immunological response after challenge, which is in agreement with the specific high levels of pro-inflammatory cytokines (IL-1 and TNFα) detected after challenge in this group. CONCLUSIONS: The selected antigens delivered as recombinant proteins had no clinical protective efficacy against S. scabiei infestation although immunisation reduced mite density. However, these results pave the way for future studies on alternative production systems, adjuvants, delivery methods and combinations of antigens in order to manage stimulation of clinical protective immune responses.