Transgenerational effects of triazole fungicides on gene expression and egg compounds in non-exposed offspring: A case study using Red-Legged Partridges (Alectoris rufa).

Triazole fungicides are widely used to treat cereal seeds before sowing. Granivorous birds like the Red-legged Partridge (Alectoris rufa) have high exposure risk because they ingest treated seeds that remain on the field surface. As triazole fungicides can act as endocrine disruptors, affecting sterol synthesis and reproduction in birds several months after exposure, we hypothesized that these effects could also impact subsequent generations of exposed birds. To test this hypothesis, we exposed adult partridges (F0) to seeds treated at commercial doses with four different formulations containing triazoles as active ingredients (flutriafol, prothioconazole, tebuconazole, and a mixture of the latter two), simulating field exposure during late autumn sowing. During the subsequent reproductive season, two to four months after exposure, we examined compound allocation of steroid hormones, cholesterol, vitamins, and carotenoids in eggs laid by exposed birds (F1), as well as the expression of genes encoding enzymes involved in sterol biosynthesis in one-day-old chicks of this F1. One year later, F1 animals were paired again to investigate the expression of the same genes in the F2 chicks. We found changes in the expression of some genes for all treatments and both generations. Additionally, we observed an increase in estrone levels in eggs from partridges treated with flutriafol compared to controls, a decrease in tocopherol levels in partridges exposed to the mixture of tebuconazole and prothioconazole, and an increase in retinol levels in partridges exposed to prothioconazole. Despite sample size limitations, this study provides novel insights into the mechanisms of action of the previously observed effects of triazole fungicide-treated seeds on avian reproduction with evidence that the effects can persist beyond the exposure windows, affecting unexposed offspring of partridges fed with treated seeds. The results highlight the importance of considering long-term chronic effects when assessing pesticide risks to wild birds.

Diet and Spatial Ecology Influence Red-Legged Partridge Exposure to Pesticides Used as Seed Treatment.

Seed treatment with pesticides is an extended agricultural practice with a high risk to granivorous birds that consume those seeds. To characterize that risk, it is necessary to understand the ecological factors that determine the exposure chances of birds to treated seeds. We investigated how pesticide uptake by red-legged partridges was related to cultivated plant ingestion and to the use of recently sown fields. We analyzed pesticide residues in 144 fecal samples from 32 flocks and determined the plant diet composition using DNA metabarcoding. Habitat use was studied through the monitoring of 15 GPS-tagged partridges. We confirmed, through the analysis of seeds, that >80% of cereal fields from the area had seeds treated with triazole fungicides. Tebuconazole was detected in 16.6% of partridges' feces. During the sowing season, cultivated plants accounted for half of the plant diet, but no association was found between cultivated plant consumption and pesticide intake. GPS tracking revealed that tebuconazole was detected in feces when partridges had recently used sown fields, whereas nonexposed partridges showed no overlap with recently sown areas. Our results highlight the need to incorporate field ecology into the characterization of pesticide exposure to improve the efficacy of environmental risk assessment.

A non-invasive method to monitor farmland bird exposure to triazole fungicides.

The treatment of seeds with pesticides is an extended practice in current agriculture. There is a high risk of exposure in granivorous birds, such as the red-legged partridge (Alectoris rufa), that can consume those seeds remaining on the surface during sowing. Fungicide exposure could in turn affect bird reproductive capacity. To better understand to what extent triazole fungicides are a threat to granivorous birds, we need an easy and reliable method to quantify field exposure. In this study, we tested a novel non-invasive method to detect the presence of triazole fungicide residues in farmland bird faeces. We experimentally exposed captive red-legged partridges to validate the method, and then applied it in a real scenario to assess exposure of wild partridges. We exposed adult partridges to seeds treated with two formulations containing triazole fungicides as active ingredients: Vincit®Minima (flutriafol 2.5%) and Raxil®Plus (prothioconazole 25% and tebuconazole 15%). We collected two types of faeces (caecal and rectal samples) immediately after exposure and 7 days later and quantified the concentrations of the three triazoles and their common metabolite (1,2,4-triazole). The three active ingredients and 1,2,4-triazole were only detected in faeces collected immediately after exposure. Triazole fungicide detection rates in rectal stool were 28.6%, 73.3% and 80% for flutriafol, prothioconazole and tebuconazole, respectively. In caecal samples, detection rates were 40%, 93.3% and 33.3%, respectively. 1,2,4-triazole was detected in 53% of rectal samples. For an applied use of the method in the field, we collected 43 faecal samples from wild red-legged partridges during autumn cereal seed sowing and found detectable levels of tebuconazole in 18.6% of the analysed wild partridges. The results of the experiment were then used to estimate actual exposure levels from this prevalence value found in wild birds. Our study shows that faecal analysis can be a useful tool to assess farmland bird exposure to triazole fungicides, when samples are fresh and the method has been validated for the detection of target molecules.

Bird exposure to fungicides through the consumption of treated seeds: A study of wild red-legged partridges in central Spain.

Sown seeds are a key component of many farmland birds' diets due to natural food shortages in autumn and winter. Because these seeds are often treated with pesticides, their ingestion by birds can result in toxic effects. For risk assessment, data on treated seed toxicity should be combined with information about exposure risk for wild birds and the factors that modulate it. We characterized the exposure of red-legged partridges to pesticide-treated seeds through the analysis of digestive contents of birds shot by hunters (n = 194) in an agricultural region in central Spain. We measured the contribution of sown seeds to the partridges' diet and how it related to pesticide exposure. Moreover, we evaluated the influence of landscape composition on the intake of sown seeds and pesticides by partridges. During peak sowing time, seeds constituted half (50.7%) of the fresh biomass ingested by partridges, which consumed mostly winter cereal seeds (42.3% of biomass). Residues of seven fungicides and one insecticide (active ingredients) were detected in 33.0% of birds. The presence of pesticides in digestive contents was linked to the ingestion of cereal sown seeds. Moreover, dietary exposure of birds to pesticides was modulated by landscape characteristics, being lower in areas with heterogeneous landscapes, greater habitat mosaic and more natural vegetation. The estimated dietary intake of pesticides resulting from our field observations, in combination with experimental data on pesticide toxicity, raise concerns about the risks that pesticide-treated cereal seeds pose to granivorous bird populations. Our results highlight the importance of farming landscape composition and diversification, which should be considered as a priority in the agricultural policy to mitigate pesticide risks to farmland birds through the consumption of treated seeds.

Detection of Stephanurus dentatus in wild boar urine using different parasitological techniques.

Stephanurus dentatus is a nematode that parasitizes the urinary tract of domestic and wild Suidae, especially in tropical areas. However, there is a lack of information about stephanurosis in wild boar (Sus scrofa), thus making it necessary to develop sensitive techniques with which to diagnose this pathogen in order to carry out further research. In Spain, the high prevalence of this nematode has been evidenced in Doñana National Park (DNP). The objective of the present work is twofold. The first is to compare the efficacy of three parasitological techniques to detect S. dentatus eggs in the urine of infected wild boar: (i) gravity sedimentation, (ii) sedimentation by centrifugation, and (iii) flotation techniques, while the second is to determine whether the quantification of eggs can serve as an indicative value of the host's parasite intensity. In order to accomplish these purposes, 27 wild boars from DNP were necropsied, and the urinary system of each animal was examined in order to determine parasite intensity. While all the aforementioned techniques can be used to detect eggs in urine, the most effective in terms of egg quantification are sedimentation by gravity and by centrifugation, as they allow a greater number of S. dentatus eggs to be detected. However, none of the results obtained with these techniques significantly correlated with the number of adult nematodes parasitizing the host, signifying that counts in urine can provide guidance on only the parasite intensity of wild boar.

Multi-level analysis of exposure to triazole fungicides through treated seed ingestion in the red-legged partridge.

Triazole fungicides are the most widely used products to treat cereal seeds. Granivorous birds, such as red-legged partridges (Alectoris rufa), which consume seeds left on the surface of fields after sowing, have a high risk of exposure. As triazole fungicides can affect sterol synthesis, we tested the hypothesis that treated seed consumption could alter the synthesis of sex hormones and reduce the reproductive capacity of partridges. We exposed adult partridges to seeds treated with four different formulations containing triazoles as active ingredients (flutriafol, prothioconazole, tebuconazole, and a mixture of the latter two) simulating a field exposure during the late autumn sowing season. All treatments produced biochemical changes and an overexpression of genes encoding for enzymes involved in the biosynthesis of sterols and steroid hormones, such as PMVK, ABCA1, MVD, PSCK9, DHCR7 and HSD17B7. Plasma levels of oestradiol were reduced in partridges exposed to tebuconazole. We also monitored reproduction 3 months after exposure (laying date, egg fertilization and hatching rates). We observed a 14-day delay in the laying onset of partridges that had been exposed to flutriafol as compared to controls. These results show that the consumption of seeds treated with triazole fungicides has the potential to affect granivorous bird reproduction. We recommend the evaluation of lagged reproductive effects as part of the protocols of environmental risk assessment of pesticides in wild birds in light of the effects resulting from the exposure to triazole-treated seeds.

Liposarcoma retroperitoneal gigante con afectación renal: reporte de un caso y revisión de la literatura / A giant retroperitoneal liposarcoma with renal involvement: A case report and literature review

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Long-term risk of cardiovascular events after detecting silent coronary atheromatosis using computed tomography.

BACKGROUND: Coronary calcium score (CCS) and coronary computed tomography angiography (CTA) assessments using multidetector computed tomography are invaluable for atheromatosis screening. We studied their usefulness in cardiovascular risk assessments, and compared evaluations using the Systematic COronary Risk Evaluation (SCORE) algorithm with those from CTA and CSS assessments in terms of their ability to predict cardiovascular events in Mediterranean patients. PATIENTS AND METHODS: Two hundred and sixty-six asymptomatic patients whose mean age was 55.4 years, 89.5% of whom were men, were evaluated using CTA and CCS and followed for more than 10 years. The CTA and CCS risk predictions were compared with those determined using the SCORE algorithm designed for low-risk populations. RESULTS: Coronary lesions were present in 140 (53.4%) patients. Of the lesions, 17% were noncalcified, 17% were mixed, and 66% were calcified; in addition, 24.2% of the patients who had lesions had cardiovascular events during follow-up (P<0.00001), but just 2.9% of the patients without lesions. Detection of atheromatosis using computed tomography was associated with an increased risk of cardiovascular disease events at more than 10 years [odds ratio (OR): 6.828; 95% confidence interval (CI): 2.001-23.305; P=0.002]. This OR was higher than that obtained for intermediate-risk individuals (OR: 4.818; 95% CI: 1.360-17.075; P=0.015) and lower than that determined for high-risk individuals (OR: 9.395; 95% CI: 2.489-35.460; P=0.001) using the SCORE algorithm, and higher that that determined for CCS assessments (OR: 3.916; 95% CI: 1.572-9.751; P=0.03). More cardiovascular events were associated with higher amounts of calcium. CONCLUSION: The detection of atheromatosis using the CCS and CTA was associated with an increased risk of cardiovascular events at more than 10 years. CTA and CCS assessments had a higher OR than that associated with assessments of patients at intermediate risk using the SCORE algorithm.

The use of acute phase proteins for monitoring animal health and welfare in the pig production chain: the validation of an immunochromatographic method for the detection of elevated levels of pig-MAP.

The serum concentration of acute phase proteins (APPs) increases in the presence of disease or stress, which makes APPs notable parameters for the global assessment of animal health and welfare. A rapid, immunochromatographic test (ICT) for the detection of elevated levels of pig Major Acute-phase Protein (pig-MAP), one of the main APPs in pigs, was evaluated in more than 1400 pig serum samples obtained from commercial farms. The ICT showed a good performance with a relative sensitivity (Sn) and specificity (Sp) of 94 and 97%, respectively, for a threshold of 1.5mg/mL (comparison with ELISA). Differences in the pig-MAP levels and the number of positive samples with the ICT were observed within the season of sampling, farms, and age groups at one farm, according to the presence of disease or lesions. The ICT was also evaluated in blood samples obtained at slaughter in association with the carcase inspection. The results from this study indicate that the ICT may be used for the evaluation of groups of pigs, after analysing one sub-sample of these pigs, and might be a useful tool in routine health and welfare monitoring programmes aimed to improve the quality of pig production.

Chronic ß-adrenergic stimulation increases ErbB receptors and cell proliferation in mouse kidney.

Although the sympathetic nervous system is involved in injury caused to the kidney by several stressors such as hypertension or ischemia/reperfusion, little is known about the effect of chronic adrenergic stimulation in the kidneys. Upon injury, however, the kidney possesses a high capacity for tubular cell regeneration and functional recovery. The ErbB1 receptor and its ligands play an essential role in this process. We studied the effects of chronic isoproterenol (ISO) administration (ß-adrenoceptor agonist) in the mouse kidney. ISO induced a moderate and reversible loss of kidney weight and protein content that was not associated with renal dysfunction. We observed an increase in tubular cell proliferation (bromodeoxyuridine labeling) in ISO-treated mice in both the outer and inner cortex. ErbB1 (epidermal growth factor receptor) along with ErbB2 and ErbB3 (neuregulin receptor) were transiently overexpressed in ISO-treated mice, with an increase in protein but not mRNA content. All receptors were localized in the same nephron segments and cell types. Immunoprecipitation studies after epidermal growth factor or neuregulin-1ß stimulation showed dynamic interaction of all four ErbB receptors. Therefore, we conclude that ErbB receptors may cooperate in the response to chronic ß-adrenergic stimulation.

Heparin-binding EGF-like growth factor in human serum. Association with high blood cholesterol and heart hypertrophy.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) belongs to the EGF family of growth factors which are ligands of the ErbB receptors. Studies in animals suggest the role of HB-EGF in several pathogenic processes such as atherosclerosis and heart hypertrophy. Here, we set up an assay to measure HB-EGF in human serum. Our ELISA determined serum HB-EGF in the range of 0.03-3 nM. It did not cross-react with EGF or with transforming growth factor-alpha. The mean serum HB-EGF was 0.26 nM (confidence interval: 0.13-0.39) in women and 0.28 nM (confidence interval: 0.09-0.47) in men. In a cohort of 121 healthy volunteers, we identified nine individuals with high serum HB-EGF (above 0.47 nM). These individuals had higher left ventricle mass (determined by Colour Doppler echocardiography) and greater total and low density lipoprotein cholesterol than control. On the basis of our results, we propose that increased serum HB-EGF is associated with heart hypertrophy and elevated blood cholesterol.