The vomeronasal system of the wolf (Canis lupus signatus): The singularities of a wild canid.

Wolves, akin to their fellow canids, extensively employ chemical signals for various aspects of communication, including territory maintenance, reproductive synchronisation and social hierarchy signalling. Pheromone-mediated chemical communication operates unconsciously among individuals, serving as an innate sensory modality that regulates both their physiology and behaviour. Despite its crucial role in the life of the wolf, there is a lacuna in comprehensive research on the neuroanatomical and physiological underpinnings of chemical communication within this species. This study investigates the vomeronasal system (VNS) of the Iberian wolf, simultaneously probing potential alterations brought about by dog domestication. Our findings demonstrate the presence of a fully functional VNS, vital for pheromone-mediated communication, in the Iberian wolf. While macroscopic similarities between the VNS of the wolf and the domestic dog are discernible, notable microscopic differences emerge. These distinctions include the presence of neuronal clusters associated with the sensory epithelium of the vomeronasal organ (VNO) and a heightened degree of differentiation of the accessory olfactory bulb (AOB). Immunohistochemical analyses reveal the expression of the two primary families of vomeronasal receptors (V1R and V2R) within the VNO. However, only the V1R family is expressed in the AOB. These findings not only yield profound insights into the VNS of the wolf but also hint at how domestication might have altered neural configurations that underpin species-specific behaviours. This understanding holds implications for the development of innovative strategies, such as the application of semiochemicals for wolf population management, aligning with contemporary conservation goals.

First Immunohistochemical Demonstration of the Expression of a Type-2 Vomeronasal Receptor, V2R2, in Wild Canids.

The mammalian vomeronasal system enables the perception of chemical signals crucial for social communication via the receptor families V1R and V2R. These receptors are linked with the G-protein subunits, Gαi2 and Gαo, respectively. Exploring the evolutionary pathways of V1Rs and V2Rs across mammalian species remains a significant challenge, particularly when comparing genomic data with emerging immunohistochemical evidence. Recent studies have revealed the expression of Gαo in the vomeronasal neuroepithelium of wild canids, including wolves and foxes, contradicting predictions based on current genomic annotations. Our study provides detailed immunohistochemical evidence, mapping the expression of V2R receptors in the vomeronasal sensory epithelium, focusing particularly on wild canids, specifically wolves and foxes. An additional objective involves contrasting these findings with those from domestic species like dogs to highlight the evolutionary impacts of domestication on sensory systems. The employment of a specific antibody raised against the mouse V2R2, a member of the C-family of vomeronasal receptors, V2Rs, has confirmed the presence of V2R2-immunoreactivity (V2R2-ir) in the fox and wolf, but it has revealed the lack of expression in the dog. This may reflect the impact of domestication on the regression of the VNS in this species, in contrast to their wild counterparts, and it underscores the effects of artificial selection on sensory functions. Thus, these findings suggest a more refined chemical detection capability in wild species.

Biochemical Effects of Heavy Metals and Organochlorine Compounds Accumulated in Different Tissues of Yellow-Legged Gulls (Larus Michahellis).

In the present study, livers, kidneys and adipose tissue of Yellow-legged Gull (Larus michahellis) were collected. Samples were used to determine relationships between heavy metals/metalloids in liver and kidneys (Hg, Cd, Pb, Se and As) or persistent organic pollutants in adipose tissue (7 PCBs and 11 organochlorine pesticides) with biomarkers of oxidative stress (CAT, GPx, GR, GSH, GST, MDA) analysed in both internal organs. Three possible influencing variables have been studied: age, sex and sampling area. As a result, statistically significant differences (P < 0.05, P < 0.01) were only found according to the sampling area, with differences among the three studied areas found in both organs. Significant positive correlations (P < 0.01) were found in liver (Hg vs. GST; Se vs. MDA) and in kidney (As vs. GR; As vs. GPx; PCB52 vs. CAT; PCB138 vs. CAT). The scarcity in correlations suggests that the levels of pollutants found in animals were not high enough to trigger an effect at the oxidative level.

The vomeronasal organ of wild canids: the fox (Vulpes vulpes) as a model.

The vomeronasal system (VNS) has been extensively studied within specific animal families, such as Rodentia. However, the study of the VNS in other families, such as Canidae, has long been neglected. Among canids, the vomeronasal organ (VNO) has only been studied in detail in the dog, and no studies have examined the morphofunctional or immunohistochemical characteristics of the VNS in wild canids, which is surprising, given the well-known importance of chemical senses for the dog and fox and the likelihood that the VNS plays roles in the socio-reproductive physiology and behaviours of these species. In addition, characterising the fox VNS could contribute to a better understanding of the domestication process that occurred in the dog, as the fox would represent the first wild canid to be studied in depth. Therefore, the aim of this study was to analyze the morphological and immunohistochemical characteristics of the fox VNO. Tissue dissection and microdissection techniques were employed, followed by general and specific histological staining techniques, including with immunohistochemical and lectin-histochemical labelling strategies, using antibodies against olfactory marker protein (OMP), growth-associated protein 43 (GAP-43), calbindin (CB), calretinin (CR), α-tubulin, Gαo, and Gαi2 proteins, to highlight the specific features of the VNO in the fox. This study found significant differences in the VNS between the fox and the dog, particularly concerning the expression of Gαi2 and Gαo proteins, which were associated with the expression of the type 1 vomeronasal receptors (V1R) and type 2 vomeronasal receptors (V2R), respectively, in the vomeronasal epithelium. Both are immunopositive in foxes, as opposed to the dog, which only expresses Gαi2. This finding suggests that the fox possesses a well-developed VNO and supports the hypothesis that a profound transformation in the VNS is associated with domestication in the canid family. Furthermore, the unique features identified in the fox VNO confirm the necessity of studying the VNS system in different species to better comprehend specific phylogenetic aspects of the VNS.

Development and characterization of 15 novel polymorphic microsatellite loci for two important bot flies (Diptera, Oestridae) by next-generation sequencing.

Cephenemyia stimulator and Oestrus ovis are two important parasitic bot flies (Oestridae) species causing myiasis, with a potential negative impact on the welfare of the host. Using next-generation sequencing approach and bioinformatics tools, a large panel of possible microsatellites loci was obtained in both species. Primer pairs were designed for 15 selected microsatellite loci in C. stimulator and other 15 loci in O. ovis for PCR amplification. Loci amplification and analysis were performed in four populations of each species. The results demonstrated that all selected loci were polymorphic, with the number of alleles ranging from 2 to 6 per locus in C. stimulator and 3 to 13 per locus in O. ovis. This is the first time to describe these microsatellite loci for C. stimulator and O. ovis. These two sets of microsatellite markers could be further used for biogeographic and population genetics studies.

Dissecting the Effects of Cephenemyia stimulator on the Olfactory Turbinates and Nasopharynx of Roe Deers (Capreolus capreolus).

Nasopharyngeal myiasis in European roe deer (Capreolus capreolus) is a pathological condition caused by the larval stages of Cephenemyia stimulator, a fly from the Oestridae family. These larvae reside in the host's upper respiratory tract for months, inducing significant tissue damage and clinical symptoms. The lifecycle of Cephenemyia stimulator is complex, involving three larval stages before maturation into adult flies, with each stage contributing to the progressive pathology observed in the host. Despite their prevalence, the histopathological effects of these larvae in the nasal and nasopharyngeal cavities have been understudied. Our study fills this knowledge gap by providing a detailed histopathological analysis of the affected tissues, using various staining techniques to reveal the extent and nature of the damage caused by these parasitic larvae. This histopathological examination reveals significant alterations within the nasopharyngeal mucosa and nasal cavity, including erythematous changes, mucosal metaplasia, fibrosis, and tissue necrosis. Parasitic cysts and eosinophilic infiltration further characterize the impact of the infestation, compromising not only the mucosal integrity but also potentially the olfactory function of the affected animals. This research is crucial for understanding the impact of myiasis on both the health and olfactory capabilities of roe deer populations and could have significant implications for wildlife management and conservation.

Noninvasive heavy metal pollution assessment by means of Iberian wolf (Canis lupus signatus) hair from Galicia (NW Spain): a comparison with invasive samples.

The current study represents the first investigation of the suitability of wolf hair as indicator tissue for metal exposure and accumulation within NW Spanish ecosystems. Dead animals, which were not specifically killed for these purposes, were necropsied, and further toxicological analyses were performed in order to establish the heavy metal content (Pb, Cd, and Zn) in liver, kidney, and hair, by means of inductively coupled plasma optical emission spectrometry. Two different factors, gender and age, were considered in order to determine their influence on heavy metal accumulation. Mean liver, kidney and hair concentrations of both toxic element, Cd (0.528, 2.692, and 0.026 ppm) and Pb (4.108, 0.031, and 0.196 ppm) considered on a dry weight basis, were situated below the established as acute toxicity levels for mammals. The highest concentrations were quantified for Zn, with means ranging from a maximum in hair samples (150.9 ppm) to a minimum in kidney samples (25.81 ppm). When the variable of gender and age were considered, female and adult wolves had higher concentrations of Cd in all the considered organs, although differences were only statistically significant for kidney. Neither the sex nor the age had a significant effect on Pb and Zn concentrations. A significant positive correlation (p < 0.05) was identified between hair and liver Pb concentrations, as well as between hair and kidney Cd concentrations, thus suggesting the interest of the noninvasive sample for future ecotoxicological biomonitoring studies.

Metal and metalloid concentrations in wild mammals from SW Europe: European hedgehog (Erinaceus europaeus) and badger (Meles meles).

In recent years, there have been increasing ecological and global concerns associated to Potentially Toxic Elements (PTEs). Thus, the relevance of wild mammals as biomonitors has been globally recognised. In the present study, Cd, Pb, Hg, Zn and As concentrations were quantified in European hedgehog and badger inhabiting SW Europe, and cumulative trends in relation to age and sex were evaluated. Liver and kidney samples were collected, mineralised and PTE content was determined by ICP-MS. Zn was the most abundant element quantified in both organs (239 and 89.8 mg kg-1 for hedgehogs and 179 and 164 mg kg-1 dw for badgers). In hedgehogs, very high Hg concentration were quantified (4.35 and 15.5 mg kg-1 dw in liver and kidney), and Cd was the most abundant for badgers (4.70 and 7.61 mg kg-1 dw in liver and kidney). Positive correlations were observed for the concentrations of PTE in the organs of both species. Age-dependence increased only Cd concentration, with levels in adult kidneys being significantly higher. In this study, European hedgehog and badger were used as biomonitors for the determination of PTEs to provide current reference values in relatively non-polluted areas of SW Europe, and to enhance the use of these species for future ecotoxicological studies.

Seropathotypes, Phylogroups, Stx subtypes, and intimin types of wildlife-carried, shiga toxin-producing escherichia coli strains with the same characteristics as human-pathogenic isolates.

The objectives of this study were to investigate the presence of Shiga toxin-producing Escherichia coli (STEC) strains in wildlife that have spread in Europe, living near human settlements; to analyze their epidemiological role in maintenance and transmission to domestic livestock; and to assess the potential health risk of wildlife-carried strains. STEC strains were recovered from 53% of roe deer, 8.4% of wild boars, and 1.9% of foxes sampled in the northwest of Spain (Galicia). Of the 40 serotypes identified, 21 were classified as seropathotypes associated with human disease, accounting for 81.5% of the wildlife-carried STEC strains, including the enterohemorrhagic serotypes O157:H7-D-eae-γ1, O26:[H11]-B1-eae-ß1, O121:H19-B1-eae-ε1, and O145:[H28]-D-eae-γ1. None of the wildlife-carried strains belonged to the highly pathogenic serotype O104:H4-B1 from the recent Germany outbreak. Forty percent of wildlife-carried STEC strains shared serotypes, phylogroups, intimin types, and Stx profiles with isolates from human patients from the same geographic area. Furthermore, wildlife-carried strains belonging to serotypes O5:HNM-A, O26:[H11]-B1, O76:H19-B1, O145:[H28]-D, O146:H21-B1, and O157:H7-D showed pulsed-field gel electrophoresis (PFGE) profiles with >85% similarity to human-pathogenic STEC strains. We also found a high level of similarity among STEC strains of serotypes O5:HNM-A, O26:[H11]-B1, and O145:HNM-D of bovine (feces and beef) and wildlife origins. Interestingly, O146:H21-B1, the second most frequently detected serotype in this study, is commonly associated with human diarrhea and isolated from beef and vegetables sold in Galicia. Importantly, at least 3 STEC isolates from foxes (O5:HNM-A-eae-ß1, O98:[H21]-B1-eae-ζ1, and O146:[H21]-B1) showed characteristics similar to those of human STEC strains. In conclusion, roe deer, wild boar, and fox in Galicia are confirmed to be carriers of STEC strains potentially pathogenic for humans and seem to play an important role in the maintenance of STEC.

Comparative Neuroanatomical Study of the Main Olfactory Bulb in Domestic and Wild Canids: Dog, Wolf and Red Fox.

The sense of smell plays a fundamental role in mammalian survival. There is a considerable amount of information available on the vomeronasal system of both domestic and wild canids. However, much less information is available on the canid main olfactory system, particularly at the level of the main olfactory bulb. Comparative study of the neuroanatomy of wild and domestic canids provides an excellent model for understanding the effects of selection pressure associated with domestication. A comprehensive histological (hematoxylin-eosin, Nissl, Tolivia and Gallego's Trichrome stains), lectin (UEA, LEA) and immunohistochemical (Gαo, Gαi2, calretinin, calbindin, olfactory marker protein, glial fibrillary acidic protein, microtubule-associated protein 2) study of the olfactory bulbs of the dog, fox and wolf was performed. Our study found greater macroscopic development of the olfactory bulb in both the wolf and fox compared to the dog. At the microscopic level, all three species show a well-developed pattern of lamination and cellularity typical of a macrosmatic animal. However, greater development of cellularity in the periglomerular and mitral layers of wild canids is characteristic. Likewise, the immunohistochemical study shows comparable results between the three species, but with a noticeably higher expression of markers in wild canids. These results suggest that the reduction in encephalization experienced in dogs due to domestication also corresponds to a lower degree of morphological and neurochemical differentiation of the olfactory bulb.

The olfactory limbus of the red fox (Vulpes vulpes). New insights regarding a noncanonical olfactory bulb pathway.

Introduction: The olfactory system in most mammals is divided into several subsystems based on the anatomical locations of the neuroreceptor cells involved and the receptor families that are expressed. In addition to the main olfactory system and the vomeronasal system, a range of olfactory subsystems converge onto the transition zone located between the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB), which has been termed the olfactory limbus (OL). The OL contains specialized glomeruli that receive noncanonical sensory afferences and which interact with the MOB and AOB. Little is known regarding the olfactory subsystems of mammals other than laboratory rodents. Methods: We have focused on characterizing the OL in the red fox by performing general and specific histological stainings on serial sections, using both single and double immunohistochemical and lectin-histochemical labeling techniques. Results: As a result, we have been able to determine that the OL of the red fox (Vulpes vulpes) displays an uncommonly high degree of development and complexity. Discussion: This makes this species a novel mammalian model, the study of which could improve our understanding of the noncanonical pathways involved in the processing of chemosensory cues.

Heavy metals and metalloid levels in the tissues of yellow-legged gulls (Larus michahellis) from Spain: sex, age, and geographical location differences.

In the present study, mercury (Hg), cadmium (Cd), lead (Pb), selenium (Se), and arsenic (As) were measured in liver, kidney, and feathers of adult, juvenile, and chick seagulls (Larus michahellis) collected from the northwest of Spain. Age, sex, and the geographical location of samples were considered variables that can influence metal bioaccumulation, for which concentrations were determined by means of ICP-MS. The mean concentrations (dry weight) found in seagulls were 7.01 ± 0.37 mg Hg/kg, 22.82 ± 2.83 mg Cd/kg, 7.36 ± 1.36 mg Pb/kg, 18.64 ± 0.63 mg Se/kg, and 10.64 ± 0.59 mg As/kg. Regarding the different factors analyzed, Hg was the only metal showing sex-related differences, being significantly higher (p < 0.05) the concentrations found in feathers of males (1.26 ± 0.12 mg/kg) than those in females (0.99 ± 0.11 mg/kg). A highly significant (p < 0.01) increase in levels of some metals was found in liver related to the increase of age: Hg (adults (A) 3.33 ± 0.22 mg/kg vs chicks (C) 1.76 ± 0.28 mg/kg), Cd (A 4.74 ± 0.62 mg/kg vs C 1.79 ± 0.2), Pb (A 0.65 ± 0.12 mg/kg vs juveniles 0.4 ± 0.11 mg/kg), and Se (A 7.56 ± 0.43 mg/kg vs C 5.24 ± 0.53 mg/kg). Positive correlations between Cd-Hg and Se-Hg were found in liver (p < 0.001), kidney (p < 0.001), and feathers (p < 0.05 and p < 0.001, respectively). The associations found may reflect antagonistic interactions between Se and Cd on Hg toxicity. The results suggest that L. michahellis can reveal local contamination around the foraging and breeding sites and can be a very useful monitoring instrument for assessing heavy metal contamination and sentinel species of environmental health.

A High Prevalence of Cardiopulmonary Worms Detected in the Iberian Wolf (Canis lupus): A Threat for Wild and Domestic Canids.

Cardiopulmonary nematodes are highly pathogenic parasites affecting domestic and wild canids. As the result of conservation programs, the Iberian wolf (Canis lupus signatus) population has recently expanded, and its distribution range covers lands from where it had long disappeared. However, the exact epidemiological role of the wolf in the life cycle of zoonotic parasites causing diseases transmissible to pets and/or humans is largely unknown. This study sought to determine the diversity of cardiopulmonary nematode parasite species that affect wolves inhabiting northwestern areas of the Iberian Peninsula, and to estimate their prevalence and the relationship between these parasites and several epidemiological variables. For this purpose, we examined the cardiopulmonary systems of 57 wolves from Galicia (from the provinces A Coruña n = 15, Lugo n = 21, Ourense n =15 and Pontevedra n = 6) using techniques of dissection and cup sedimentation. Collected worms were then identified under a light microscope according to their morphological features. Three species of nematodes were detected: Angiostrongylus vasorum (the "French-heartworm"), Crenosoma vulpis and Eucoleus aerophilus, the latter being of zoonotic interest. The prevalence was 24.5% (14/57; 95% CI 13.3-35.6%) overall, 19.3% for A. vasorum (11/57; 95% CI 8.8-29.2%), 7% for C. vulpis (4/57; 95% CI 0.4-13.6%) and 3.5% for E. aerophilus (2/57; CI -1.1-9.1%). A significant relationship (p = 0.002) was found between age and the presence of C. vulpis, which was only found in juvenile animals. Furthermore, a higher prevalence of A. vasorum and/or C. vulpis was observed in wolves with a lower body condition score (40% and 20%, respectively), though the difference was not significant (p = 0.221 and p = 0.444, respectively). Our findings indicate a high "French-heartworm" and lungworm burden in the wolf population of northern Spain, and they identify a need for studies designed to elucidate the epidemiological role played by the Iberian wolf and to identify possible risks for veterinary and public health.

Can domestication shape Canidae brain morphology? The accessory olfactory bulb of the red fox as a case in point.

BACKGROUND: The accessory olfactory bulb (AOB) is the first integrative center of the vomeronasal system (VNS), and the general macroscopic, microscopic, and neurochemical organizational patterns of the AOB differ fundamentally among species. Therefore, the low degree of differentiation observed for the dog AOB is surprising. As the artificial selection pressure exerted on domestic dogs has been suggested to play a key role in the involution of the dog VNS, a wild canid, such as the fox, represents a useful model for studying the hypothetical effects of domestication on the AOB morphology. METHODS: A comprehensive histological, lectin-histochemical, and immunohistochemical study of the fox AOB was performed. Anti-Gαo and anti-Gαi2 antibodies were particularly useful, as they label the transduction cascade of the vomeronasal receptor types 1 (V1R) and 2 (V2R), respectively. Other employed antibodies included those against proteins such as microtubule-associated protein 2 (MAP-2), tubulin, glial fibrillary acidic protein, growth-associated protein 43 (GAP-43), olfactory marker protein (OMP), calbindin, and calretinin. RESULTS: The cytoarchitecture of the fox AOB showed a clear lamination, with neatly differentiated layers; a highly developed glomerular layer, rich in periglomerular cells; and large inner cell and granular layers. The immunolabeling of Gαi2, OMP, and GAP-43 delineated the outer layers, whereas Gαo and MAP-2 immunolabeling defined the inner layers. MAP-2 characterized the somas of AOB principal cells and their dendritic trees. Anti-calbindin and anti-calretinin antibodies discriminated neural subpopulations in both the mitral-plexiform layer and the granular cell layer, and the lectin Ulex europeus agglutinin I (UEA-I) showed selectivity for the AOB and the vomeronasal nerves. CONCLUSION: The fox AOB presents unique characteristics and a higher degree of morphological development compared with the dog AOB. The comparatively complex neural basis for semiochemical information processing in the fox compared with that observed in dogs suggests loss of AOB anatomical complexity during the evolutionary history of dogs and opens a new avenue of research for studying the effects of domestication on brain structures.

Use of native chicken breeds (Gallus gallus domesticus) for the development of suitable methods of Cantabrian capercaillie (Tetrao urogallus cantabricus) semen cryopreservation.

BACKGROUND: The Cantabrian capercaillie (Tetrao urogallus cantabricus) is critically endangered. This subspecies has the lowest genetic variability and it is in regression. It belongs to Phasianidae family; therefore, the domestic chicken (Gallus gallus domesticus) could be a good model for developing reproductive technologies for use in capercaillie populations with low availability of animals. OBJECTIVES: In this study, we analyzed the response of capercaillie sperm to the freezing-thawing process for contributing to the development of a semen cryobank of Cantabrian capercaillie. METHODS: We used domestic chicken as the animal model in order to obtain the freezing protocol before applying on capercaillie. In the first experiment, two different extenders (EK and LR84) and different concentrations [4% and 6% dimethyl-acetamide (DMA) v:v] of cryoprotectants were evaluated using in-straw freezing method in domestic chickens. A pilot study in capercaillie males, using the same conditions evaluated in chicken, was performed. RESULTS: In chicken, we found that the LR84-4% DMA media provided the best results for freezing semen. In capercaillie study, LR84 extender seemed to be the most appropriate diluent and 4% was the better dose of DMA cryoprotectant agent. Further, based on previous studies carried out in rooster samples, we also tested the glycerol (8% v/v) as a cryoprotectant for capercaillie semen cryopreservation. CONCLUSIONS: Our results suggest that sperm from both domestic and wild species had a similar response to freezing-thawing processes. Mediterranean chickens may be used as a suitable model for developing sperm freezing protocols that can be extrapolated to threatened capercaillie populations. In addition, LR84 media with glycerol was the most efficient extender to freeze capercaillie sperm native.

Sex- and age-dependent accumulation of heavy metals (Cd, Pb and Zn) in liver, kidney and muscle of roe deer (Capreolus capreolus) from NW Spain.

In the present study, bioaccumulation of lead, cadmium and zinc in liver, kidney and muscle of roe deer (Capreolus capreolus) from Galicia (NW Spain) was quantified. Metal analysis was performed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). In general the quantified concentrations were associated with background environmental levels, except for Pb, where some markedly elevated concentrations were obtained, but with no toxicological relevance. The effect of sex on Cd, Pb and Zn levels was considered, females showing a general trend to accumulate higher concentrations of these metals, with the exception of the hepatic content of Cd (which was significantly higher in males, P < 0.05) and Pb levels, which did not differ according to sex. The effect of age was analysed only in male animals and was clearly observed for Pb levels, with concentrations significantly higher in adult animals (liver: P < 0.01, kidney: P < 0.05 and muscle: P < 0.001) and for Cd levels in kidney samples (P < 0.001). This study provides the first data on heavy metal content in roe deer from the area of study and suggests that this species could be a good bioindicator of metal pollution, even though both factors, sex and age, could represent an important source of variation in the bioaccumulation of these metals in wild populations.

Holding power of three different pin designs in the femur and ulna of the common buzzard (Buteo buteo).

External skeletal fixation is generally considered the best stabilization technique for immobilizing avian long bone fractures, but one of its major complications is the failure of bone-fixation pin interface or the loss of holding power. Consequently, this study is aimed at elucidating which pin design offers more pull-out strength in certain bones of the common buzzard (Buteo buteo). To achieve this objective, three pin designs (a smooth design and two negative profile threaded designs, with different thread pitch) were placed in five positions along the femur and ulna of the common buzzard. The pin pull-out strength was measured with the purpose of comparing medullary and pneumatic bones, insertion sites, and pin designs. Threaded pins with negative profile showed greater holding power than smooth pins (P < 0.05). When comparing holding power between the ulna and femur, no differences were found for smooth pins, whereas threaded pins showed more pull-out strength in the ulna than in the femur (P < 0.05). There were no differences observed related to pin location along the same bone when considering the same pin type. These results suggest that negative profile threaded pins have more holding power than smooth pins and that pneumatic bones provide less pull-out strength to negative profile threaded pins than medullary bones.

The complete mitochondrial genome of Cephenemyia stimulator (Diptera: Oestridae).

Cephenemyia stimulator parasitizes roe deer (Capreolus capreolus) throughout its geographical distribution. The complete circular C. stimulator mitogenome was assembled, which is 16,407 bp in length, and encodes 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes. A phylogenetic tree was built with mitogenome sequences, including C. stimulator and 13 related Oestridae species, using Sarcophaga tuberosa as an outgroup.

Characterization of New Molecular Markers of Three Botflies Parasitizing Cervid Hosts.

Specific identification of oestrid larvae is usually problematic not only when using morphobiometric features, but also when applying molecular criteria, since very few molecular markers have been described for this group of flies. New molecular markers for oestrid are needed for more reliable species identification, diagnostic purposes, and epidemiological surveys; moreover, they can help in phylogenetic reconstruction. Here, we report the characterization of COI, 28S rDNA, ITS1, and ITS2 in Cephenemyia stimulator from roe deer and in Cephenemyia auribarbis and Pharyngomyia picta from red deer. The COI and 28S rDNA are very uniform in length, while the ITSs sequences are highly variable at both intraspecific and interspecific levels. The described ITSs sequences were longer than those described for other dipteran species by the presence of simple repeats and tandem repeat sequences. In C. auribarbis both ITS1 and ITS2 appeared as two variants, one short and the other long. In general, the analyzed markers present low intraspecific genetic variation and high interspecific variation. ITSs showed the greatest amount of intraspecific and interspecific variation. Phylogenetic analysis demonstrated that the characterized sequences differentiate the species and genera of Oestridae.

Genomic Characterization of Escherichia coli Isolates Belonging to a New Hybrid aEPEC/ExPEC Pathotype O153:H10-A-ST10 eae-beta1 Occurred in Meat, Poultry, Wildlife and Human Diarrheagenic Samples.

Different surveillance studies (2005-2015) in northwest Spain revealed the presence of eae-positive isolates of Escherichia coli O153:H10 in meat for human consumption, poultry farm, wildlife and human diarrheagenic samples. The aim of this study was to explore the genetic and genomic relatedness between human and animal/meat isolates, as well as the mechanism of its persistence. We also wanted to know whether it was a geographically restricted lineage, or whether it was also reported elsewhere. Conventional typing showed that 32 isolates were O153:H10-A-ST10 fimH54, fimAvMT78, traT and eae-beta1. Amongst these, 21 were CTX-M-32 or SHV-12 producers. The PFGE XbaI-macrorestriction comparison showed high similarity (>85%). The plasmidome analysis revealed a stable combination of IncF (F2:A-:B-), IncI1 (STunknown) and IncX1 plasmid types, together with non-conjugative Col-like plasmids. The core genome investigation based on the cgMLST scheme from EnteroBase proved close relatedness between isolates of human and animal origin. Our results demonstrate that a hybrid MDR aEPEC/ExPEC of the clonal group O153:H10-A-ST10 (CH11-54) is circulating in our region within different hosts, including wildlife. It seems implicated in human diarrhea via meat transmission, and in the spreading of ESBL genes (mainly of CTX-M-32 type). We found genomic evidence of a related hybrid aEPEC/ExPEC in at least one other country.