Oxidative Stress in Wild Boars Naturally and Experimentally Infected with Mycobacterium bovis

Reactive oxygen and nitrogen species (ROS-RNS) are important defence substances involved in the immune response against pathogens. An excessive increase in ROS-RNS, however, can damage the organism causing oxidative stress (OS). The organism is able to neutralise OS by the production of antioxidant enzymes (AE); hence, tissue damage is the result of an imbalance between oxidant and antioxidant status. Though some work has been carried out in humans, there is a lack of information about the oxidant/antioxidant status in the presence of tuberculosis (TB) in wild reservoirs. In the Mediterranean Basin, wild boar (Sus scrofa) is the main reservoir of TB. Wild boar showing severe TB have an increased risk to Mycobacterium spp. shedding, leading to pathogen spreading and persistence. If OS is greater in these individuals, oxidant/antioxidant balance in TB-affected boars could be used as a biomarker of disease severity. The present work had a two-fold objective: i) to study the effects of bovine TB on different OS biomarkers (namely superoxide dismutase (SOD), catalasa (CAT), glutathione peroxidase (GPX), glutathione reductase (GR) and thiobarbituric acid reactive substances (TBARS)) in wild boar experimentally challenged with Mycobacterium bovis, and ii) to explore the role of body weight, sex, population and season in explaining the observed variability of OS indicators in two populations of free-ranging wild boar where TB is common. For the first objective, a partial least squares regression (PLSR) approach was used whereas, recursive partitioning with regression tree models (RTM) were applied for the second. A negative relationship between antioxidant enzymes and bovine TB (the more severe lesions, the lower the concentration of antioxidant biomarkers) was observed in experimentally infected animals. The final PLSR model retained the GPX, SOD and GR biomarkers and showed that 17.6% of the observed variability of antioxidant capacity was significantly correlated with the PLSR X's component represented by both disease status and the age of boars. In the samples from free-ranging wild boar, however, the environmental factors were more relevant to the observed variability of the OS biomarkers than the TB itself. For each OS biomarker, each RTM was defined as a maximum by one node due to the population effect. Along the same lines, the ad hoc tree regression on boars from the population with a higher prevalence of severe TB confirmed that disease status was not the main factor explaining the observed variability in OS biomarkers. It was concluded that oxidative damage caused by TB is significant, but can only be detected in the absence of environmental variation in wild boar

Levels of organochlorine pesticides and polychlorinated biphenyls in the critically endangered Iberian lynx and other sympatric carnivores in Spain

Accumulation of organochlorine compounds is well studied in aquatic food chains whereas little information is available from terrestrial food chains. This study presents data of organochlorine levels in tissue and plasma samples of 15 critically endangered Iberian lynx (Lynx pardinus) and other 55 wild carnivores belonging to five species from three natural areas of Spain (Doñana National Park, Sierra Morena and Lozoya River) and explores their relationship with species diet. The Iberian lynx, with a diet based on the consumption of rabbit, had lower PCB levels (geometric means, plasma: <0.01 ng mL−1, liver: 0.4 ng g−1 wet weight, fat: 87 ng g−1 lipid weight) than other carnivores with more anthropic and opportunistic foraging behavior, such as the red fox (Vulpes vulpes; plasma: 1.11 ng mL−1, liver: 459 ng g−1, fat: 1984 ng g−1), or with diets including reptiles at higher proportion, such as the Egyptian mongoose (Herpestes ichneumon; plasma: 7.15 ng mL−1, liver: 216 ng g−1, fat: 540 ng g−1), or the common genet (Genetta genetta; liver: 466 ng g−1, fat: 3854 ng g−1). Chlorinated pesticides showed interspecific variations similar to PCBs. Organochlorine levels have declined since the 80s in carnivores from Doñana National Park, but PCB levels are still of concern in Eurasian otters (Lutra lutra; liver: 3873–5426 ng g−1) from the industrialized region of Madrid.This is a contribution to the “Programa de Actuaciones para la Conservación del Lince en Andalucía II”, Consejería de Medio Ambiente, Junta de Andalucía (CMA). J. Millán holds a “Ramón y Cajal” contract and M.E. Ortiz-Santaliestra holds a “Juan de la Cierva” contract, both awarded by the Ministerio de Ciencia e Innovación and the European Social Fund. J. Rodríguez-Estival is granted by the Junta the Comunidades de Castilla-La Mancha.Peer reviewe

Assessment of lead and pesticide exposure in the declining population of red breasted goose (Branta ruficollis) wintering in Eastern Europe

Presentado al 7th SETAC World Congress and SETAC North America 37th Annual Meeting, celebrados en Orlando (USA) del 6 al 10 de noviembre de 2016.Peer Reviewe

Mercury exposure and mechanism of response in large game using the Almadén mercury mining area (Spain) as a case study

Mercury (Hg) accumulation, transfer, defense mechanism and adverse effects were studied in red deer (Cervus elaphus) and wild boar (Sus scrofa) from the Almadén mining district (Spain), the largest (285,000. t of Hg) and the oldest (more than 2000 years) Hg mine/refining operation site in the world. Red deer (n=168) and wild boar (n=58) liver, kidney, bones (metacarpus), testis and muscle were analyzed for total Hg and selenium (Se) within a range of distances to the Almadén mining district. The highest Hg concentrations were found in kidney (0.092 and 0.103 οg/g d.w. for red deer and wild boar, respectively) followed by the levels in liver (0.013 and 0.023 οg/g d.w. for red deer and wild boar, respectively). A significant correlation (r=-0.609, p=0.007) was found between Hg concentrations and distance to the Almadén Hg mining district. However, both red deer and wild boar closest to the mining area still showed mercury concentrations well below the concentration associated with clinical signs of Hg poisoning. Highest Se concentrations were found in kidney (2.60 and 6.08 οg/g in red deer and wild boar, respectively) and testis (2.20 οg/g in red deer). For selenium, differences between red deer and wild boar were statistically significant (p0.001 for red deer and r=0.567, p=0.005 for wild boar). Liver GSSG concentrations were negatively correlated to total mercury and Hg:Se molar ratio in male deer, which could indicate a hormetic response to Hg exposure. Moreover, a positive association was found between the antioxidant element Se and antioxidant vitamins in red deer tissues.M.J. Patiño Ropero and J. Rodríguez-Estival acknowledge the Junta de Comunidades de Castilla-La Mancha for their Ph.D. fellowships.Peer Reviewe

Risk assessment of lead poisoning and pesticide exposure in the declining population of red-breasted goose (Branta ruficollis) wintering in Eastern Europe

The red-breasted goose Branta ruficollis is a globally threatened species (IUCN Vulnerable) and the only European goose species currently in decline. Working on the wintering grounds on the Black Sea Coast, we address two potential causes of decline of this species for the first time: lead poisoning, and contamination from pesticides. We quantified the densities of spent Pb shot in three wetlands used by the geese in north-east Bulgaria, and analysed the Pb concentration in the faeces of red-breasted geese and the more abundant greater white-fronted geese Anser albifrons, using Al concentration as an indicator of soil ingestion. Pb shot densities in sediments were low, and we found no evidence for Pb shot ingestion in red-breasted geese. On the other hand, we found that the geese were feeding on wheat whose seeds were treated with four fungicides: thiram, tebuconazole, difenoconazole and fludioxonil, and the two first were even detected in geese faecal samples. Using data on the daily food intake, we estimated the exposure levels of the geese to these fungicides, both by measuring the concentrations remaining on seeds and by estimating the amount used to coat the seeds at the time of sowing. We found that the exposure rates estimated during the sowing period for both geese species can exceed the recognized hazardous doses for thiram, and to a lesser extent for tebuconazole, which indicates that some pesticides may be playing a previously overlooked role in the decline of red-breasted geese.The field study was supported by the Life project LIFE/NAT/BG09/00230 “Safe Ground for Redbreasts”. Laboratory work was funded by the Spanish Ministry of Science and Innovation (grant CGL2010-17030) and FEDENCA (Real Federación Española de Caza) and Oficina Nacional de la Caza with the partnership of Fundación Biodiversidad.Peer Reviewe

Blood levels of heavy metals and arsenic in black-necked grebe Podiceps nigricollis during the moulting period in the Odiel Marshes, SW Spain

Resumen del trabajo presentado al Society of Environmental Toxicology and Chemistry (SETAC) Europe 27th Annual Meeting, celebrado en Bruselas (Bélgica) del 7 al 11 de mayo de 2017.Black-necked grebe (Podiceps nigricollis) has in the salt pans of Odiel Marshes (SW Spain) one of the most important moulting areas in Western Europe. However, Odiel Marshes are impacted by the mining and industrial activities located in the Odiel and Tinto River Basin and the city of Huelva, respectively. Grebes moulting in this wetland could be therefore exposed to heavy metals and metalloids during a critical period, such is moulting, when energy and protein requirements are elevated. Here we have studied the hypothesis that black-necked grebes would experience an increase in blood levels of heavy metals and metalloids during their stay at Odiel Marshes, between August and December. Alternatively, we also consider the hypothesis that grebes could reduce the concentration of some elements by excretion with the newly grown feathers. Blood levels of arsenic, lead, cadmium, mercury, copper and zinc were measured in blood of 180 black-necked grebes captured for a ringing program of the Doñana Biological Station. We studied the effect of sex, age (1styear, 2nd year and adults), moulting status (old plumage, active moult or new plumage), scaled mass index and date on the measured levels of metal(loids). Most of the studied elements showed significant temporal variations that could be associated with moulting status or with the time spent at the Odiel Marshes. Arsenic levels decreased during active moult (225 ng/ml) but increased afterwards to much higher blood concentrations during the stay at Odiel Marshes (488 ng/ml). Blood Pb levels were highest at the end of active moult (October: 18 ng/ml) and showed a slight increase with the age of birds. The analysis of recaptured birds in the same year also revealed an increase in blood Pb levels from October to November. One remarkable outlier with 404 ng/ml may correspond to the ingestion of a lead shot or a fishing weight. Mercury levels were lower in birds with new feathers (256 ng/ml) than in those with old feathers (303 ng/ml). Three birds showed extremely high levels of mercury (>10 ¿g/ml). Copper and zinc levels in blood were highest in birds with active moult. Our results show significant temporal variations of metal(loid) levels that can be associated with the requirements for feather formation (essential elements such as Cu and Zn) or with a detoxification due to the transfer of elements from blood to feathers (Hg).Peer Reviewe

Exposure of black-necked grebes (Podiceps nigricollis) to metal pollution during the moulting period in the Odiel Marshes, Southwest Spain

European populations of black-necked grebes (Podiceps nigricollis) congregate every year to moult at the salt ponds of the Odiel Marshes (SW Spain). However, the Odiel Marshes are part of one of the most metal-polluted coastal estuaries in the world, which may pose risks to wildlife. We assessed the exposure of grebes to metal pollution during the critical moulting period in the Odiel Marshes and its potential to cause adverse health effects. Levels of metals in red blood pellet (as a biomarker of exposure), plasma carotenoids, eye redness, and body condition (as biomarkers of effects) were studied. Metal content was also analyzed in the brine shrimp Artemia parthenogenetica, the most important food for grebes in this hypersaline ecosystem during the moulting period. Results showed that, in comparison to toxicity thresholds, grebes had relatively high blood levels of arsenic (As), mercury (Hg) and zinc (Zn). The high loads found in Artemia and the way blood levels vary during the moulting period indicate that shrimp consumption may be the main route of metal exposure for grebes. Plasma carotenoids and body condition showed a positive association with exposure to As, while the relationship of lutein-like carotenoids with Hg accumulation was negative at the beginning of the moulting period to become positive afterwards. Moreover, eye redness was negatively affected by As accumulation. Factors including food resource availability, seasonal fluctuations in physiological status, and interannual variations in the degree of environmental contamination should be considered in monitoring efforts when using moult migrant waterbirds as sentinel species.J. Rodríguez-Estival was supported by a Research and Innovation Strategy for Smart Specialisation (RIS3) postdoctoral contract (2017/11747) from the Junta de Comunidades de Castilla-La Mancha. The study was financially supported by Junta de Andalucía (Consejería de Innovación, Ciencia y Empresa, Project P07-CVI-02700).Peer reviewe