Goats Naturally Infected with the Spanish Goat Encephalitis Virus (SGEV): Pathological Features and An Outbreak.

In autumn 2011, a disease outbreak caused by Spanish goat encephalitis virus (SGEV) was reported in a herd of goats from Asturias (north-western Spain), expanding the known geographic distribution of tick-borne encephalitis in Europe. The virus was classified as a new subtype (subspecies) within the Louping-ill virus species of the mammalian tick-borne flavivirus group. The aims of the present study were to describe the pathology in goats naturally infected with SGEV, as well as discuss the pathogenesis of the disease in that outbreak. A total of 22/85 (25.88%) goats (20 adults and 2 kids) died between October 2011 and June 2012, showing neurological clinical signs. Over three years, the mortality rate in the herd reached 100%. Neuropathological lesions caused by SGEV were severe and widespread throughout the central nervous system but were more severe and numerous in the proximal cervical spinal cord, medulla oblongata, pons and cerebellar cortex. They consisted of neuron necrosis, neuronophagia, mononuclear inflammatory cell perivascular cuffs (lymphocytes, plasma cells and macrophages) and gliosis. The distribution of viral antigens was restricted to the cytoplasm of neurons in several brain areas but not associated with inflammatory foci nor inflammatory cells. SGEV should be considered a significant pathogen of goats that results in severe neurological clinical disease and high mortality.

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.

Immunohistochemical Assessment of Immune Response in the Dermis of Sarcoptes scabiei-Infested Wild Carnivores (Wolf and Fox) and Ruminants (Chamois and Red Deer).

Sarcoptic mange is caused by the mite Sarcoptes scabiei and has been described in several species of domestic and wild mammals. Macroscopic lesions are predominantly hyperkeratotic (type I hypersensitivity) in fox, chamois and deer, but alopecic (type IV hypersensitivity) in wolf and some fox populations. To begin to understand the immune processes underlying these species differences in lesions, we examined skin biopsies from wolves (Canis lupus), foxes (Vulpes vulpes), chamois (Rupicapra rupicapra) and red deer (Cervus elaphus) naturally infested with S. scabiei. Twenty skin samples from five animals per species were used. Sections were immuno-stained with primary antibodies against Iba1 to detect macrophages, lambda chain to detect plasma cells, CD3 to detect T lymphocytes and CD20 to detect B lymphocytes. Skin lesions contained significantly more inflammatory cells in the fox than in the wolf and chamois. Macrophages were the most abundant inflammatory cells in the lesions of all the species studied, suggesting a predominantly innate, non-specific immune response. Lesions from the wolf contained higher proportions of macrophages than the other species, which may reflect a more effective response, leading to alopecic lesions. In red deer, macrophages were significantly more abundant than plasma cells, T lymphocytes and B lymphocytes, which were similarly abundant. The fox proportion of plasma cells was significantly higher than those of T and B lymphocytes. In chamois, T lymphocytes were more abundant than B lymphocytes and plasma cells, although the differences were significant only in the case of macrophages. These results suggest that all the species examined mount a predominantly innate immune response against S. scabiei infestation, while fox and chamois may also mount substantial humoral and cellular immune responses, respectively, with apparently scarce effectiveness that lead to hyperkeratotic lesions.

Phenotypic characterization of encephalitis in the brains of goats experimentally infected with Spanish Goat Encephalitis Virus.

Spanish goat encephalitis virus (SGEV) is a novel tick-borne flavivirus subtype, closely related to the flavivirus louping ill virus (LIV). SGEV caused a severe, acute and mortal neurological disease outbreak in northern Spain in a goat herd. In order to characterize the cell population in lesions and to determine the distribution of the inflammatory cells, central nervous system (CNS) samples of nine female Alpine goats challenged subcutaneously with SGEV over the right thorax behind the elbow were evaluated using immunohistochemistry (microglia-Iba1, T lymphocytes-CD3, B lymphocytes-CD20 and astrocytes-GFAP). The number of microglia (37.8 %) and T lymphocytes (21.5 %) was greater than the number of B lymphocytes (16.8 %). Goats were classified into clusters based on the severity of histological lesions in CNS (A-mild to moderate lesions and B-severe lesions). Microglia was significantly more abundant than T and B lymphocytes in cluster B (severe lesions). The total area occupied by glial foci revealed that medulla oblongata and spinal cord were the most affected tissues. Astrogliosis (GFAP+) was present in the majority of the CNS sections being near to the pial surface. The lesion predominance on the right side of the medulla oblongata, which could be associated to the site of challenge suggestive of neurotropic route was also statistically confirmed. Results suggest that the cellular immune response would be the most important response to the SGEV infection.

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.

Genetic Characterization and Phylogenetic Analysis of Small Ruminant Lentiviruses Detected in Spanish Assaf Sheep with Different Mammary Lesions.

Small Ruminant Lentiviruses (SRLVs) are widespread in many countries and cause economically relevant, slow, and persistent diseases in sheep and goats. Monitoring the genetic diversity of SRLVs is useful to improve the diagnostic tools used in the eradication programs. In this study, SRLVs detected in Spanish Assaf sheep with different grades of lymphoproliferative mastitis were sequenced. Genetic characterization showed that most samples belonged to type A and were closer to Spanish SRLV isolates previously classified as A2/A3. Four samples belonged to subtype B2 and showed higher homology with Italian B2 strains than with Spanish B2 isolates. Amino acid sequences of immuno-dominant epitopes in the gag region were very conserved while more alterations were found in the LTR sequences. No significant correlations were found between grades of mastitis and alterations in the sequences although samples with similar histological features were phylogenetically closer to each other. Broader genetic characterization surveys in samples with different grades of SRLV-lesions are required for evaluating potential correlations between SRLV sequences and the severity of diseases.

Mannose receptor may be involved in small ruminant lentivirus pathogenesis.

Thirty-one sheep naturally infected with small ruminant lentiviruses (SRLV) of known genotype (A or B), and clinically affected with neurological disease, pneumonia or arthritis were used to analyse mannose receptor (MR) expression (transcript levels) and proviral load in virus target tissues (lung, mammary gland, CNS and carpal joints). Control sheep were SRLV-seropositive asymptomatic (n = 3), seronegative (n = 3) or with chronic listeriosis, pseudotuberculosis or parasitic cysts (n = 1 in each case). MR expression and proviral load increased with the severity of lesions in most analyzed organs of the SRLV infected sheep and was detected in the affected tissue involved in the corresponding clinical disease (CNS, lung and carpal joint in neurological disease, pneumonia and arthritis animal groups, respectively). The increased MR expression appeared to be SRLV specific and may have a role in lentiviral pathogenesis.

Study of compartmentalization in the visna clinical form of small ruminant lentivirus infection in sheep.

BACKGROUND: A central nervous system (CNS) disease outbreak caused by small ruminant lentiviruses (SRLV) has triggered interest in Spain due to the rapid onset of clinical signs and relevant production losses. In a previous study on this outbreak, the role of LTR in tropism was unclear and env encoded sequences, likely involved in tropism, were not investigated. This study aimed to analyze heterogeneity of SRLV Env regions--TM amino terminal and SU V4, C4 and V5 segments--in order to assess virus compartmentalization in CNS. RESULTS: Eight Visna (neurologically) affected sheep of the outbreak were used. Of the 350 clones obtained after PCR amplification, 142 corresponded to CNS samples (spinal cord and choroid plexus) and the remaining to mammary gland, blood cells, bronchoalveolar lavage cells and/or lung. The diversity of the env sequences from CNS was 11.1-16.1% between animals and 0.35-11.6% within each animal, except in one animal presenting two sequence types (30% diversity) in the CNS (one grouping with those of the outbreak), indicative of CNS virus sequence heterogeneity. Outbreak sequences were of genotype A, clustering per animal and compartmentalizing in the animal tissues. No CNS specific signature patterns were found. CONCLUSIONS: Bayesian approach inferences suggested that proviruses from broncoalveolar lavage cells and peripheral blood mononuclear cells represented the common ancestors (infecting viruses) in the animal and that neuroinvasion in the outbreak involved microevolution after initial infection with an A-type strain. This study demonstrates virus compartmentalization in the CNS and other body tissues in sheep presenting the neurological form of SRLV infection.

Tuberculosis due to Mycobacterium bovis and Mycobacterium caprae in sheep.

Tuberculosis was diagnosed in three flocks of sheep in Galicia, Spain, in 2009 and 2010. Two flocks were infected with Mycobacterium bovis and one flock was infected with Mycobacterium caprae. Infection was confirmed by the comparative intradermal tuberculin test, bacteriology, molecular analysis and histopathology. Sheep have the potential to act as a reservoir for tuberculosis.

Nephrotoxicosis in Iberian piglets subsequent to exposure to melamine and derivatives in Spain between 2003 and 2006.

Between November 2003 and September 2006, 300 to 400 45-60-day-old Iberian piglets developed anorexia, polydipsia, and lethargy. Piglets were from 5 different farms in the western part of Spain. Morbidity was between 40% and 60%, and mortality ranged from 20% to 40% of the total population of postweaning piglets. In the 9 piglets in which postmortem examinations were conducted, kidneys were enlarged with yellow foci in the cortex and medulla. Microscopically, these foci were accumulations of crystals within the lumina of dilated distal tubules and collecting ducts, causing flattening of the renal tubular epithelial cells. The crystals displayed a multicolored birefringence under cross-polarized light. The multinucleated giant cells surrounding the crystals, interstitial fibrosis, and nonsuppurative infiltrates indicated a chronic inflammatory response. Toxicologic analysis of fixed kidney tissues from 4 piglets demonstrated the presence of melamine, ammeline, ammelide, and cyanuric acid. Ammelide concentrations were highest, ranging from 39,000 to 92,000 mg/kg, followed by ammeline (20,000-34,000 mg/kg), melamine (9,200-29,000 mg/kg), and cyanuric acid (2,200-9,100 mg/kg). The clinical, histologic, and toxicologic findings in affected piglets were similar to those reported in dogs and cats that died of melamine and melamine analogue-associated renal failure in 2004 and 2007. To the authors' knowledge, this is the first documented report of poisoning due to melamine and its analogues in pigs and demonstrates that contamination of pig feed occurred as early as 2003.

Closed-chest experimental porcine model of acute myocardial infarction-reperfusion.

INTRODUCTION: Progress in cardiovascular regenerative medicine research requires the availability of appropriate experimental animal models that are as close to humans as feasible. Our objective was to assess the validity of a porcine endovascular model of myocardial infarction and reperfusion. METHODS: Fifteen domestic pigs (Large White race) were anesthetized and pre-medicated with amiodarone. Endovascular fluoroscopy-guided coronary procedures were performed to occlude the mid-left anterior descending artery using a coronary angioplasty balloon. Occlusion was confirmed by angiography and electrocardiography. After 75 min the balloon catheter system was withdrawn and the presence of reperfusion flow was verified. The animals were sacrificed after 1 and 2 weeks of follow-up, the hearts were explanted, and the extent of myocardial infarction with respect to the left ventricle was quantified. RESULTS: Overall survival rate was 67%. Five animals died prematurely: 3 showing signs of heart failure, 1 had reperfusion failure (final TIMI flow grade 1) and 1 succumbed to acute stress. The most common adverse event was ventricular fibrillation (87% of the animals) and defibrillation was effective in all affected animals. The extent of myocardial infarct in the animals followed-up for 1 and 2 weeks was similar (20.4+/-4.3% vs. 20.9+/-2.8%, respectively; p=0.8) but was significantly greater in the animals that died prematurely (29.5+/-3.6%, p=0.02). CONCLUSIONS: The endovascular porcine model we have explored constitutes a feasible and reproducible alternative for the evaluation of human myocardial infarction and reperfusion.

Comparison of three immunological diagnostic tests for the detection of avian tuberculosis in naturally infected red deer (Cervus elaphus).

Cases of tuberculosis due to Mycobacterium avium subsp. avium in 52 adult red deer (Cervus elaphus) from a farm were studied using different diagnostic techniques. Immunological probes consisted of the comparative cervical tuberculin (CCT) skin test, the interferon-gamma (IFN-gamma) assay, and 2 enzyme-linked immunosorbent assays (ELISAs) employing either avian purified protein derivatives or protoplasmatic antigen (PPA-3) as antigens. Three of the animals were euthanized due to severe weakness, loss of weight, and emaciation. Macroscopically, the 3 animals showed tuberculous lesions located mainly in lymph nodes of the digestive system and small intestine but also in other organs and lymph nodes. Polymerase chain reaction was carried out on samples from the 3 deer using primers to detect IS901, IS900, and IS6110, specific for Mycobacterium avium subsp. avium, Mycobacterium avium subsp. paratuberculosis, and Mycobacterium tuberculosis complex, including Mycobacterium bovis, respectively. The last 2 agents cause pathologies very similar to avian tuberculosis in deer. The 3 deer were strongly positive by both ELISAs, slightly positive by the IFN-gamma test, and 1 of 2 was positive by the CCT test. As with domestic ruminants, ELISA could detect deer in an advanced stage of infection, with large numbers of mycobacteria.

Aleutian disease serology, protein electrophoresis, and pathology of the European mink (Mustela lutreola) from Navarra, Spain.

The European mink, Mustela lutreola, has suffered a dramatic decline in Europe during the 20th century and is one of the most endangered carnivores in the world. The subpopulation of European mink from Navarra, Spain, estimated to number approximately 420, represents approximately two thirds of the total number of mink in Spain. Aleutian Disease Virus (ADV) is a parvovirus with a high degree of variability that can infect a broad range of mustelid hosts. The pathogenesis of this virus in small carnivores is variable and can be influenced by both host factors (e.g., species, American mink genotype, and immune status) and viral strain. A cross-sectional study was conducted during the pre-reproductive period of February-March 2004 and 2005 and the postreproductive period of September-December 2004. Mink were intensively trapped along seven rivers that were representative of the European mink habitat in Navarra. Antibody counter immunoelectrophoresis against ADV was performed on 84 European mink blood samples. All the samples were negative. Protein electrophoresis was performed on 93 plasma samples. Nine of those samples (9.6%) had gamma globulin levels exceeding 20% of the total plasma protein. Complete necropsies were performed on 23 cadavers of European mink collected in the area between 2000 and 2005. Seventeen of the mink (74%) had traumatic and hemorrhagic lesions compatible with vehicular impact injuries. Although there were no histopathologic lesions associated with ADV, this study documents the first description of a naturally occurring canine distemper virus infection in a European mink. In addition, pulmonary adiaspiromycosis in three European mink from Spain was reported.