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.

New threats in the recovery of large carnivores inhabiting human-modified landscapes: the case of the Cantabrian brown bear (Ursus arctos).

Understanding mortality causes is important for the conservation of endangered species, especially in small and isolated populations inhabiting anthropized landscapes where both natural and human-caused mortality may hinder the conservation of these species. We investigated the mortality causes of 53 free-ranging brown bears (Ursus arctos) found dead between 1998 and 2023 in the Cantabrian Mountains (northwestern Spain), a highly human-modified region where bears are currently recovering after being critically threatened in the last century. We detected natural traumatic injuries in 52.63% and infectious diseases in 39.47% of the 38 bears for which the mortality causes were registered, with 21.05% of these cases presenting signs of both infectious diseases and traumas. More specifically, almost 30% of the bears died during or after intraspecific fights, including sexually selected infanticide (10.53%). In addition, primary infectious diseases such as infectious canine hepatitis, distemper, clostridiosis and colibacillosis caused the death of 15.79% of the bears. The number of direct human-caused deaths (i.e., shooting, poisoning, snare) decreased over the study period. This study also reveals three new mortality causes triggered by pathogens, two of which-Clostridium novyi and verotoxigenic Escherichia coli-not previously described in ursids, and the other one, canine distemper virus, never reported in brown bears as cause of death. New management strategies for the conservation of Cantabrian bears, which are urgently needed due to the rapid expansion of the population, should consider the mortality causes described in this study and must promote further research to elucidate how the high prevalence of infectious diseases may threaten the current recovery of the population.

MNAzymes and gold nanoparticles as isothermal signal amplification strategy for visual detection of miRNA.

MicroRNAs (miRNAs) represent a class of small noncoding RNAs that are considered a novel emerging class of disease biomarkers in a variety of afflictions. Sensitive detection of miRNA is typically achieved using hybridization-based methods coupled with genetic amplification techniques. Although their sensitivity has improved, amplification techniques often present erroneous results due to their complexity. In addition, the use of these techniques is usually linked to the application of protein enzymes, the activity of which is dependent on the temperature and pH of the medium. To address these drawbacks, an alternative genetic enzyme for the highly sensitive detection of miRNAs is proposed in this work. Multicomponent nucleic acid enzymes (MNAzymes), coupled with the use of DNA-functionalized gold nanoparticles (AuNPs), were used in this study to develop an isothermal signal amplification strategy for visual genetic detection. miR146a, a biomarker of bovine mastitis present in milk, was selected as a model analyte. The developed methodology is easily carried out in 80 min at 50 °C, generating a low visual limit of detection of 250 pM based on the observation of a color change. The methodology was successfully applied to the detection of miR146a in raw cow milk samples.

Mortality Causes in Captive Cantabrian capercaillie (Tetrao urogallus cantabricus) in Spain.

The Cantabrian capercaillie (Tetrao urogallus cantabricus) is one of the most severely threatened subspecies of capercaillie. Its current population range is restricted to a small area of the Cantabrian Mountains (northwestern Spain), with only around 200 individuals remaining. As part of the national strategy for the conservation of the subspecies, the Cantabrian capercaillie Captive Breeding Center of Sobrescobio opened in 2009. Here, we use the information provided by the necropsies performed in this facility on 29 individuals (11 males, 13 females and 5 undetermined; 16 chicks and 13 adults) in order to describe the main mortality causes of captive-bred Cantabrian capercaillies. After necropsy, tissue samples were taken for evaluation using standard methods in histology and microbiology. The majority of the captive animals (18/29, 62.07%) died due to infectious diseases, mainly due to Escherichia coli, Clostridium perfringens, or Aspergillus fumigatus infection. The remaining 11 animals died due to stress-related processes (i.e., rupture of the heart apex and cardiomyopathy or neurogenic shock) (8/29, 27.59%), duodenal obstruction and coelomitis (1/29, 3.45%), perforation of the proventriculus and heart with a briar branch (1/29, 3.45%) or euthanasia due to a valgus leg deformity that prevented proper animal welfare (1/29, 3.45%). Young animals (i.e., younger than 2 months) died mainly due to infectious diseases (14/16, 87.5%), while stress-related causes were responsible for most adult deaths (7/13, 53.85%). We additionally report that two free-ranging adult males died due to exertional myopathy. This study provides relevant information for reducing mortality in captive capercaillies and improving both living conditions in captivity and the adaptation of these animals to the wild.

Canine distemper virus in wildlife in south-western Europe.

Multi-host pathogens emerging and re-emerging at the wildlife-domestic animal interface affect wildlife management and conservation. This is the case of canine distemper virus (CDV), a paramyxovirus closely related to human measles virus and rinderpest virus of cattle. With an area of 10,603 km2 , Asturias region in Atlantic Spain is a hotspot of carnivore diversity, which includes the largest Eurasian brown bear (Ursus arctos arctos) population and one of the largest wolf (Canis lupus) populations in south-western Europe. In 2020-2021, we recorded mortality due to distemper in four carnivore species including three mustelids (Eurasian badger Meles meles, European marten Martes martes and European polecat Mustela putorius) and one canid (red fox, Vulpes vulpes). Clinical signs and pathology were similar across species and consistent with the emergence of a highly pathogenic viral strain, with CDV antigen mainly located in the central nervous system, lungs, spleen and lymph nodes. A molecular study in eight wild carnivore species, also including the Iberian wolf, Eurasian brown bear, American mink (Neovison vison) and stone marten (Martes foina), revealed 19.51% (16/82) of positivity. Phylogenetic analysis demonstrated that CDV belonged to the previously described European lineage. A retrospective serosurvey (2008-2020) showed a high seroprevalence of CDV antibodies (43.4%) in 684 analyzed badgers, indicating a long-term though not stable viral circulation in this multi-host community. The possible triggers of the 2020-2021 outbreak and the implications for carnivore management and conservation are discussed.

Visual detection of microRNA146a by using RNA-functionalized gold nanoparticles.

Gold nanoparticles of different sizes have been synthesized and surface-functionalized with selected RNA probes in order to develop a rapid, low-cost and sensitive method for detection of microRNA146a (miR146a). The strategy is based on the change of colour that can be observed visually after aggregation of the RNA modified-gold nanoparticles (AuNPs) in presence of miR146a. Experimental conditions have been carefully selected in order to obtain a good sensitivity that allows to perform visual detection of microRNA at the nM level, achieving a detection limit of 5 nM. Good repeatability and selectivity versus other sequences that only differ from miR146a in 3 bases was achieved. miR146a has been described as one of the main microRNA involved in the immune response of bovine mastitis, being expressed in tissue, blood and milk samples. The method was successfully applied to the detection of miR146a in raw cow milk samples. The present scheme constitutes a rapid and low-cost alternative to perform highly sensitive detection of microRNA without the need of instrumentation and amplification steps for the early detection of bovine mastitis in the agrofood industry. Graphical abstract Schematic representation of the assay based on aggregation of RNA-modified gold nanoparticles (blue) in presence of microRNA146a generating a dark blue spot onto a solid support, versus a pink spot observed in absence of miR146a due to dispersed gold nanoparticles (red).

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.

9-cis-retinoic acid during in vitro maturation improves development of the bovine oocyte and increases midkine but not IGF-I expression in cumulus-granulosa cells.

The isomer 9-cis of retinoic acid (9-cis-RA) exerts a beneficial effect on bovine in vitro development when added to in vitro maturation (IVM) culture. In the present work, 9-cis-RA 5 nM was found to be stimulatory as opposed to 500 nM (toxic). Cumulus-oocyte complexes (COCs) were treated with the found physiological dose 9-cis-RA 5 nM, and the next determinations performed: (1) relative expression of midkine (MK) and IGF-I, by reverse transcriptase-polymerase chain reaction (RT-PCR), in cumulus-granulosa cells detached from oocytes; (2) cytoplasmic granular migration, by labeling of oocytes with fluoroscein isothiocyanate lectins; and (3) in vitro survival of blastocysts after vitrification and warming. Gene expression of MK was enhanced by 9-cis-RA, but not by 1% ethanol (vehicle). However, we did not detect IGF-I expression, both in dependence on or in the absence of 9-cis-RA acting on cumulus-granulosa cells. The ability of vitrified blastocysts to survive in vitro was not improved by 9-cis-RA. Nevertheless, since only blastocysts obtained from oocytes matured with serum survived, more factors should be considered when evaluating cryopreservation survival. The complete granular migration observed in oocytes matured with 9-cis-RA anticipates the gain in developmental competence of the oocyte, being MK probably involved in this beneficial effect.

Modulating skeletal muscle mass by postnatal, muscle-specific inactivation of the myostatin gene.

By using a conditional gene targeting approach exploiting the cre-lox system, we show that postnatal inactivation of the myostatin gene in striated muscle is sufficient to cause a generalized muscular hypertrophy of the same magnitude as that observed for constitutive myostatin knockout mice. This formally demonstrates that striated muscle is the production site of functional myostatin and that this member of the TGFbeta family of growth and differentiation factors regulates muscle mass not only during early embryogenesis but throughout development. It indicates that myostatin antagonist could be used to treat muscle wasting and to promote muscle growth in man and animals.