Mapping the risk of exposure to Crimean-Congo haemorrhagic fever virus in the Iberian Peninsula using Eurasian wild boar (Sus scrofa) as a model.

Crimean-Congo haemorrhagic fever (CCHF) virus (CCHFV) is a tick-borne zoonotic pathogen that can cause a lethal haemorrhagic disease in humans. Although the virus appears to be endemically established in the Iberian Peninsula, CCHF is an emerging disease in Spain. Clinical signs of CCHFV infection are mainly manifested in humans, but the virus replicates in several animal species. Understanding the determinants of CCHFV exposure risk from animal models is essential to predicting high-risk exposure hotspots for public health action. With this objective in mind, we designed a cross-sectional study of Eurasian wild boar (Sus scrofa) in Spain and Portugal. The study analysed 5,291 sera collected between 2006 and 2022 from 90 wild boar populations with a specific double-antigen ELISA to estimate CCHFV serum prevalence and identify the main determinants of exposure probability. To do so, we statistically modelled exposure risk with host- and environment-related predictors and spatially projected it at a 10 × 10 km square resolution at the scale of the Iberian Peninsula to map foci of infection risk. Fifty-seven (63.3 %) of the 90 populations had at least one seropositive animal, with seroprevalence ranging from 0.0 to 88.2 %. Anti-CCHFV antibodies were found in 1,026 of 5,291 wild boar (19.4 %; 95 % confidence interval: 18.3-20.5 %), with highest exposure rates in southwestern Iberia. The most relevant predictors of virus exposure risk were wild boar abundance, local rainfall regime, shrub cover, winter air temperature and soil temperature variation. The spatial projection of the best-fit model identified high-risk foci as occurring in most of western and southwestern Iberia and identified recently confirmed risk foci in eastern Spain. The results of the study demonstrate that serological surveys of CCHFV vector hosts are a powerful, robust and highly informative tool for public health authorities to take action to prevent human cases of CCHF in enzootic and emergency settings.

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.

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.

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.

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.

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.

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.

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.

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 Content in Great Shearwater (Ardenna Gravis): Accumulation, Distribution and Biomarkers of Effect in Different Tissues.

The purpose of this study was to explore the usefulness of Great Shearwater (Ardenna gravis) as a bioindicator for biomonitoring programs for metal pollution. Three different metals were analysed in liver, kidney, and feathers, including cadmium, lead, and zinc. Glutathione-S-transferase, malondialdehyde, reduced glutathione, and catalase were assessed as oxidative stress biomarkers. Sex-related trends in metal accumulation also were evaluated. In liver and kidney, the mean concentrations of Zn (146.1 ± 5.14 and 108 ± 2.70 mg/kg, respectively) and Pb (0.19 ± 0.01 and 0.13 ± 0.01 mg/kg, respectively) in A. gravis were generally comparable to values reported in other studies. However, animals presented slightly higher concentrations of Cd (9.67 ± 0.65 in liver and 17.41 ± 0.84 mg/kg in kidney) than those reported in the same species sampled in Southern Atlantic waters. The slightly higher levels of Cd found in this study compared with other studies are probably affected by the location in Northern Atlantic waters (with different diet intake). In feathers, levels of Zn (70.70 ± 1.76 mg/kg) were lower than in other Ardenna shearwaters, whereas higher levels were found for Cd (0.16 ± 0.01 mg/kg) and Pb (0.84 ± 0.06 mg/kg). The lack of differences found between males and females could be influenced by the migration status, because both sexes stay in similar physiological conditions, with no laying eggs. Levels found in the feathers of the present study were related to concentrations in internal tissues below those which cause adverse effects in birds. Thus, feathers would appear as a potential noninvasive tool for metals biomonitoring in seabirds, because it is possible to quantify them. Baseline data of oxidative stress levels have been reported, both in liver and kidney, presenting no correlations with the levels of metals in these tissues. The low internal metal levels and the lack of correlations between oxidative stress metrics suggest a low risk of the environmental concentrations for seabirds.

High Prevalence and Diversity of Cephalosporin-Resistant Enterobacteriaceae Including Extraintestinal Pathogenic E. coli CC648 Lineage in Rural and Urban Dogs in Northwest Spain.

The aim of this work was to assess the prevalence of extended spectrum-ß-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae in fecal samples recovered from rural and urban healthy dogs in Northwest Spain (Galicia) to identify potential high-risk clones and to molecularly characterize positive isolates regarding the genes coding for ESBL/pAmpC resistance and virulence. Thirty-five (19.6%) out of 179 dogs were positive for cephalosporin-resistant Enterobacteriaceae, including Escherichiacoli and Klebsiella pneumoniae (39 and three isolates, respectively). All the isolates were multidrug resistant, with high rates of resistance to different drugs, including ciprofloxacin (71.4%). A wide diversity of ESBL/pAmpC enzymes, as well as E. coli phylogroups (A, B1, C, D, E, F and clade I) were found. The eight isolates (20.5%) found to conform to the ExPEC status, belonged to clones O1:H45-clade I-ST770 (CH11-552), O18:H11-A-ST93-CC168 (CH11-neg), O23:H16-B1-ST453-CC86 (CH6-31), and O83:H42-F-ST1485-CC648 (CH231-58), with the latter also complying the uropathogenic (UPEC) status. The three K. pneumoniae recovered produced CTX-M-15 and belonged to the ST307, a clone previously reported in human clinical isolates. Our study highlights the potential role of both rural and urban dogs as a reservoir of high-risk Enterobacteriaceae clones, such as the CC648 of E. coli and antimicrobial resistance traits. Within a One-Health approach, their surveillance should be a priority in the fight against antimicrobial resistance.

Cardiopulmonary nematode infections in wild canids: Does the key lie on host-prey-parasite evolution?

Cardiopulmonary nematodes are among the most pathogenic parasites of domestic and wild canids. The aim of this study was to describe the species diversity, prevalence and infection intensity of these parasites in the Iberian wolf (Canis lupus signatus) and the red fox (Vulpes vulpes) in the northwest of the Iberian Peninsula. 257 foxes and 74 wolves were necropsied between 2008 and 2014. Four nematode species were identified: Angiostrongylus vasorum, Eucoleus aerophilus, Crenosoma vulpis and Filaroides hirthi. This last species was only found in wolves, being the first time that is cited worldwide in this wild canid. The overall parasite prevalence was significantly higher in foxes (70%) than in wolves (28%). Specifically, prevalences in foxes and wolves were, respectively, 43% and 22% for A. vasorum, 33% and 5% for E. aerophilus, and 30% and 9% for C. vulpis. The prevalence of F. hirthi was 16%. The A. vasorum intensity was significantly higher in foxes than in wolves. Differences between host species in the risk of infection would be associated to diverging feeding behavior, and possibly reflects a parasite-host adaptation related to host's hunting strategies and cardiorespiratory requirements. This study revealed an association between infection and environmental factors, and highlighted a wide variation in the spatial distribution of A. vasorum. Our results indicate that cardiopulmonary parasites are widespread in wild canids in northwest Spain, and further agrees with other studies indicating the expansion of A. vasorum in Europe and, therefore, the urgent need to investigate infection in dogs in sympatric areas.

Metal content in the liver, kidney, and feathers of Northern gannets, Morus bassanus, sampled on the Spanish coast.

The value of birds as bioindicators for monitoring the environmental inorganic elements has been globally recognized. In this context, due to its well-known ecology and population stability, the Northern gannet (Morus bassanus) could be particularly useful. Dead Northern gannets (n = 30) were collected and samples from the liver, kidney, and feathers were taken, dried, mineralized, and finally analyzed via ICP-MS. Metals and metalloids, namely As, Cd, Hg, Pb, and Zn, associated with environmental pollution and toxicity on living organisms, were evaluated. The mean highest concentrations of As, Hg, and Zn were found in the liver (0.916, 7.026, and 89.81 mg/kg dry weight, respectively). For Cd, the kidney showed the highest mean concentration (17.51 mg/kg dry weight), whereas for Pb, this value corresponded to the feathers (0.399 mg/kg dry weight). Significant differences were found between the age classes in terms of contaminant concentrations, with the adults exhibiting higher metal levels. This difference was significantly relevant for Pb and Hg, where the effect of age was observed for all the considered tissues. When considering the effect of gender, no significant differences were observed, in agreement with similar studies performed in other geographical regions. Finally, positive correlations between the concentrations of Hg and Pb in the feathers and in the liver (r = 0.688, p < 0.001 and r = 0.566, p < 0.001, respectively) were observed, as well as between the feather and kidney concentrations (r = 0.685, p < 0.001) indicating the possibility to use feathers, a non-invasive biomonitoring tissue, for better understanding Hg and Pb exposure in seabirds.

The role of healthy dog carriers of Babesia microti-like piroplasms.

BACKGROUND: While in Europe Babesia canis has been traditionally held responsible for canine piroplasmosis, Babesia microti-like piroplasm (Bml) infection is being ever more observed in dogs, with the first clinical cases reported in northwestern Spain. This study examines the epidemiological role of healthy dogs living in endemic areas of Bml infection in Spain. The data obtained were used to describe the clinical status and map the geographical distribution of Bml infection in healthy dogs in northwestern Spain. RESULTS: Blood samples and ticks were taken from 756 healthy dogs representatively across the whole Galicia region (northwestern Spain): stray (n = 211), hunting dogs (n = 333) and pets (n = 212). Blood samples were tested by microscopy parasite observation, nested PCR-RFLP and sequencing. Piroplasm infection prevalences in healthy dogs from northwestern Spain were 17.1% (129/756) by PCR and 3.4% (26/756) by microscopy observation. The species found by PCR were: 2.2% (17/756) for B. canis and 15.1% (114/756) for Bml. Co-infection with B. canis and Bml was noted in 2 dogs. The higher prevalences detected were Bml in hunting dogs (25.5%; 85/333) and B. canis in stray dogs (6.6%; 14/211). In fox-hunting dogs from any area and dogs from the A Coruña Province, significantly higher prevalences of Bml infection were detected (P < 0.001). Upon physical examination, tick infestation was observed: 130 ticks in 18 hunting and three pet dogs. These were subsequently identified as Rhipicephalus sanguineus (s.l.) (49.2%), Ixodes hexagonus (38.5%), Ixodes ricinus (6.9%) and Dermacentor reticulatus (5.4%). Among the more prevalent ticks infesting healthy carrier dogs were I. hexagonus, followed by D. reticulatus and I. ricinus. CONCLUSIONS: Babesia canis and Bml were the only piroplasm species found infecting healthy dogs in Galicia, the prevalence of Bml being higher than of B. canis. Factors correlated with a higher Bml infection risk were being a hunting dog and living in the A Coruña Province. Healthy dogs travelling to other countries could act as carriers and probably contribute to the spread of Bml infection in dogs and wild carnivores throughout Europe.

Babesia microti-like piroplasm (syn. Babesia vulpes) infection in red foxes (Vulpes vulpes) in NW Spain (Galicia) and its relationship with Ixodes hexagonus.

Piroplasmosis is caused by several species of protozoa such as the Babesia microti-like piroplasm (Bml), an emerging blood protozoan also known as Theileria annae or Babesia vulpes. Infection by Bml was first reported in dogs in Spain where it is endemic today. Recently, a high prevalence of Bml has been increasingly detected in red foxes (Vulpes vulpes) in European countries. The objective of this study was to determine infection levels of this parasite in foxes from Galicia, NW Spain, and ticks species infestation in these carnivores, where they are so far unknown. Samples of blood, spleen and ticks (if present) were taken from 237 hunted red foxes in the Galicia region. Blood smears were prepared for direct parasite observation, and spleen and tick samples were examined by nested PCR. Prevalences of Bml infection in Galician red foxes were estimated at 72% (171/237) by PCR and 38.23% (26/68) by direct observation. Among 837 ticks collected, the main tick identified was Ixodes hexagonus (present in 82.4% of the foxes) followed by Ixodes ricinus (12.3%), Dermacentor reticulatus (12.3%) and Rhipicephalus sanguineus sensu lato (3.5%). From 34 foxes testing positive for Bml, 616 ticks were collected: positive Bml PCR results were obtained in 55.6% (227/408) of ticks collected from 9 foxes, while the 208 ticks from the remaining 25 infected foxes returned negative PCR results. Given that canine piroplasmosis is endemic in this area, our observations point to the red fox as the main reservoir for Bml infection and the high proportion of I. hexagonus among ticks collected from red foxes suggests its likely role as vectors of B. microti-like piroplasm in this region. Further studies are needed for a better understanding of the link between the wild and domestic life cycles of this piroplasm.

Reference intervals for B-esterases in gull, Larus michahellis (Nauman, 1840) from Northwest Spain: influence of age, gender, and tissue.

Over the last years, cholinesterase (ChE) and carboxylesterase (CbE) activities have been increasingly used in environmental biomonitoring to detect the exposure to anticholinesterase insecticides such as organophosphorates (OPs) and carbamates (CBs). The aim of this study was to determine ChE and CbE enzymatic activities present in liver and muscle of yellow-legged gulls (Larus michahellis), a seabird species considered suitable to monitor environmental pollution. In order to provide reference data for further biomonitoring studies, the influence of different factors, such as gender, age, sampling mode, and tissue, was considered in the present study. Our data report a statistically significant difference in CbE enzymatic activity comparing liver and muscle samples (P < 0.05) along with an age-related CbE activity in liver samples (P < 0.05). Moreover, according to our results, capture method might influence CbE and ChE activity in both liver and muscle samples (P < 0.05). These findings underline the importance to assess basal levels of ChE and CbE activity considering, among other factors, gender-, age- and organ-related differences and confirm the suitability of Larus michahellis as a sentinel species especially within an urban environment.

A novel pathogenic mechanism for cerebellar lesions produced by Solanum bonariense in cattle.

Intoxication with Solanum bonariense in cattle causes cerebellar cortical degeneration with perikaryal vacuolation, axonal swelling, and death primarily of Purkinje cells, with accumulation of electron-dense residual storage bodies in membrane-bound vesicles. The pathogenesis of this disease is not fully understood. Previously, we proposed that inhibition of protein synthesis in Purkinje cells among other altered metabolic pathways could lead to cytoskeletal alterations, subsequently altering cell-specific axonal transport. In the present study, immunohistochemical and histochemical methods were used to identify neuronal cytoskeletal alterations and axonal loss, demyelination, and astrogliosis in the cerebellum of intoxicated bovines. Samples of cerebellum from 3 natural and 4 experimental cases and 2 control bovines were studied. Immunoreactivity against neurofilament (NF)-200KDa confirmed marked loss of Purkinje neurons, and phospho-NF protein, ß-tubulin, and affinity reaction against phalloidin revealed an altered perikaryal distribution of neuronal cytoskeletal proteins in the remaining Purkinje cells in intoxicated cattle. Reactive astrogliosis in every layer of the cerebellar cortex was also observed with anti-glial fibrillary acidic protein immunohistochemistry. In affected cattle, demyelination and axonal loss in the cerebellar white matter, as well as basket cell loss were demonstrated with Klüver-Barrera and Bielschowsky stains, respectively. Based on these results, we propose that neuronal cytoskeletal alterations with subsequent interference of the axonal transport in Purkinje cells may play a relevant role in the pathogenesis of this neurodegenerative disorder, and also that demyelination and axonal loss in the cerebellar white matter, as well as astrogliosis in the gray matter, likely occur secondarily to Purkinje cell degeneration and death.

Lead toxicity in captive and wild Mallards (Anas platyrhynchos) in Spain.

Forty captive Mallards (Anas platyrhynchos), of both sexes, were separated into five groups and dosed with lead shot via oral intubation; one group was used as a control. Lead dosage differed in terms of shot number and size, as well as administration time. One hundred and thirty-five wild mallards were trapped between 1998 and 2001 in the Boada and Nava lagoons near the Canal de Castilla, in the Spanish province of Palencia. Radiologic techniques (ventrodorsal and lateral views) were used to detect lead shot in the gizzard and to determine degradation in dosed birds over time. Heparinized blood samples were taken from wild and captive mallards and blood lead levels were determined using anodic stripping voltammetry with a dropping mercury electrode. Clinical signs, injuries, and body weight were recorded. In approximately 90% of the experimentally dosed mallards, administered shot stayed in their gizzard until it degraded; this took approximately 30 days. Peak lead levels in blood were observed between days 10 and 20, and 10 days following a repeat dosage; males were more sensitive than females to a repeat dosage. During the experimental phase, 34% of birds died, and those that survived had varying degrees of anorexia, lethargy, and a decreased response to external stimulus. Of 135 tested wild mallards, 41% had a blood lead concentration higher than 0.200 microg/g. Lead shot was found embedded in 3.6% of the wild birds and 1.2% had a lead shot pellet in their gizzard.