Urine proteome in animals of veterinary interest: species comparison and new biomarkers of nephropathy

Urine is considered an ideal source of biomarkers, however in veterinary medicine a complete study on the urine proteome is still lacking. The present work aimed to apply proteomic techniques to the separation of the urine proteome in dogs, cats, horses, cows and some non-conventional species. High resolution electrophoresis (HRE) was also validated for the quantification of albuminuria in dogs and cats. In healthy cats, applying SDS-PAGE and 2DE coupled to mass spectrometry (MS), was produced a reference map of the urine proteome. Moreover, 13 differentially represented urine proteins were linked with CKD, suggesting uromodulin, cauxin, CFAD, Apo-H, RBP and CYSM as candidate biomarkers to be investigated further. In dogs, applying SDS-PAGE coupled to MS, was highlighted a specific pattern in healthy animals showing important differences in patients affected by leishmaniasis. In particular, uromodulin could be a putative biomarker of tubular damage while arginine esterase and low MW proteins needs to be investigated further. In cows, applying SDS-PAGE, were highlighted different patterns between heifers and cows showing some interesting changes during pregnancy. In particular, putative alpha-fetoprotein and b-PAP needs to be further investigated. In horses, applying SDS-PAGE, was produced a reference profile characterized by 13±4 protein bands and the most represented one was the putative uromodulin. Proteinuric horses showed the decrease of the putative uromodulin band and the appearance of 2 to 4 protein bands at higher MW and a greater variability in the range of MW between 49 and 17 kDa. In felids and giraffes was quantified proteinuria reporting the first data for UTP and UPC. Moreover, by means of SDS-PAGE, were highlighted species-specific electrophoretic patterns in big felids and giraffes

Supplementation of Boswellia serrata and Salix alba Extracts during the Early Laying Phase: Effects on Serum and Albumen Proteins, Trace Elements, and Yolk Cholesterol

Abstract: Extracts from Boswellia serrata (Bs) and Salix alba (Sa) are used as supplement in poultry feed. The aims of this research were to study possible effects of a dietary supplementation with Bs and Sa on serum and albumen proteins, zinc and iron, and yolk cholesterol content in Leg-horn hens during the critical phase of the onset of laying. A total of 120 pullets, 17 weeks of age, were assigned to 2 groups (Control (C) and Treated (T), n = 60 each). The T group received a diet supplemented with 0.3% of dry extracts of Bs (5%) and Sa (5%) for 12 weeks. The study lasted 19 weeks. Serum proteins were fractionated using agarose gel electrophoresis (AGE) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Trace elements were determined in serum using atomic absorption spectrometry and yolk cholesterol was determined using a colorimetric test. No significant differences were observed between control and supplemented hens for ana-lyzed biochemical indices. Moreover, the supplementation with phytoextracts did not negative-ly affect the physiological variations of serum proteins therefore it can be safely used as a treatment to prevent inflammatory states at onset and during the early laying phase

Essential (Mg, fe, zn and cu) and non-essential (cd and pb) elements in predatory insects (vespa crabro and vespa velutina): A molecular perspective

The recent introduction of the Asian yellow-legged hornet, Vespa velutina, into Europe has raised concern regarding the threat to honeybees and the competition with the European hornet, Vespa crabro. The aim of this study was to investigated essential (Mg, Fe, Zn, Cu) and non-essential (Cd and Pb) elements in these two species. Element concentrations were determined in the whole body and separately in the head, thorax and abdomen using atomic absorption spectrometry (AAS). The changes in essential element concentration and speciation during metamorphosis were also studied using size exclusion chromatography followed by AAS and proteomic analysis. In both species, the essential elements were more concentrated in the abdomen due to the presence of fat bodies. Magnesium, Fe and Zn concentrations were significantly higher in V. crabro than in V. velutina and could have been related to the higher aerobic energy demand of the former species required to sustain foraging flight. Low concentrations of Cd and Pb were indicative of low environmental exposure. The concentration and speciation of essential elements, particularly Fe, varied among the developmental stages, indicating a modification of ligand preferences during metamorphosis. Overall, the results in the present study provide a better understanding of the hornet metal metabolism and a foundation for additional studies

A machine learning approach to study demographic alterations in honeybee colonies using SDS\u2013PAGE fingerprinting

Honeybees, as social insects, live in highly organised colonies where tasks reflect the age of individuals. As is widely known, in this context, emergent properties arise from interactions between them. The accelerated maturation of nurses into foragers, stimulated by many negative fac-tors, may disrupt this complex equilibrium. This complexity needs a paradigm shift: from the study of a single stressor to the study of the effects exerted by multiple stressors on colony homeostasis. The aim of this research is, therefore, to study colony population disturbances by discriminating overaged nurses from proper aged nurses and precocious foragers from proper aged foragers using SDS-PAGE patterns of haemolymph proteins and a machine-learning algorithm. The KNN (K Near-est Neighbours) model fitted on the forager dataset showed remarkably good performances (accu-racy 0.93, sensitivity 0.88, specificity 1.00) in discriminating precocious foragers from proper aged ones. The main strength of this innovative approach lies in the possibility of it being deployed as a preventive tool. Depopulation is an elusive syndrome in bee pathology and early detection with the method described could shed more light on the phenomenon. In addition, it enables countermeas-ures to revert this vicious circle

Trace elements in home-processed food obtained from unconventional animals

Wild animals have been used as food since ancient times and, currently, the consumption of unconventional animals is increasing worldwide. The process of cooking meat using traditional recipes includes a variety of ingredients, which can influence the total metal intake from the diet. In this study, the concentrations of eight essential (Fe, Zn, Cu, Mn, Se, Ni, Mo, and Co) and six non-essential (Pb, Cd, Hg, Al, As, and Cr) trace elements were determined in home-processed food obtained from snails and from three common species of game animals (woodcock, pheasant, and hare), seasoned with anchovies, mushrooms, and different vegetables using inductively coupled plasma mass spectrometry (ICP-MS). In general, Fe was the most abundant trace element, ranging from 18 \ub1 8 \u3bcg/g in pheasant to 99 \ub1 76 \u3bcg/g in snail, and Co was the least abundant, ranging from 0.007 \ub1 0.003 \u3bcg/g in hare to 0.093 \ub1 0.048 \u3bcg/g in snail. Regarding the non-essential trace elements, Pb concentrations showed wide variations, reaching a concentration of 17.30 \u3bcg/g in hare, while Cd concentrations were higher in snail, ranging from 0.18 to 0.46 \u3bcg/g. These alternative food sources can offer an important contribution to the human nutritional requirements of essential trace elements, in particular of Fe. The high concentrations of Pb and Cd present in some samples should be considered as potentially dangerous for the consumers

Urinary reference values and first insight into the urinary proteome of captive giraffes

Urinalysis is widely recognized to be a useful tool in routine health investigations, since it can diagnose numerous pathologies. Considering the paucity of knowledge concerning giraffes, urine from 44 giraffes (Giraffa camelopardalis) (18 males and 26 females, from 3 months of age to 21 years of age) underwent routine urinalysis, 1D-electrophoresis, and protein identification using mass spectrometry, with the aim of identifying the urinary reference values and the urine proteome. The urine specific gravity (USG), urine total proteins (uTP), urine creatinine (uCr), and urine protein:creatinine ratio (UPC) reference values, reported as the median, and lower limit (LL) and upper limit (UL), were 1.030 (1006\u20131.049), 17.58 (4.54\u201335.31) mg/dL, 154.62 (39.59\u2013357.95) mg/dL, and 0.11 (0.07\u20130.16), respectively. Mass spectrometry, together with electrophoresis, revealed a pattern of common urinary proteins; albumin, lysozyme C, and ubiquitin were the most represented proteins in the giraffe urine. It has been hypothesized that these proteins could act as a defense against microbes. Moreover, in giraffes, urinalysis could be a valid tool for gauging renal function and physiological status changes

SDS-PAGE-Based Quantitative Assay of Hemolymph Proteins in Honeybees: Progress and Prospects for Field Application

In human and veterinary medicine, serum proteins are considered to be useful biomarkers for assessing the health and nutritional status of the organism. Honeybee hemolymph has a unique proteome that could represent a source of valuable biomarkers. Therefore, the aims of this study were to separate and identify the most abundant proteins in the hemolymph of worker honeybees to suggest a panel of these proteins that could represent useful biomarkers for assessing the nutritional and health status of the colonies and, finally, to analyze them in different periods of the year. Four apiaries were selected in the province of Bologna, and the bees were analyzed in April, May, July, and November. Thirty specimens from three hives of each apiary were sampled and their hemolymph was collected. The most represented bands obtained after 1D sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were cut from the gel, and the proteins were identified using an LC-ESI-Q-MS/MS System. A total of twelve proteins were unmistakably identified; the two most abundant proteins were apolipophorin and vitellogenin, which are known biomarkers of bee trophic and health status. The two other proteins identified were transferrin and hexamerin 70a, the first being involved in iron homeostasis and the second being a storage protein. Most of these proteins showed an increase from April to November, mirroring the physiological changes of honeybees during the productive season. The current study suggests a panel of biomarkers from honeybee hemolymph worth testing under different physiological and pathological field conditions

Essential (Mg, Fe, Zn and Cu) and non-essential (Cd and Pb) elements in predatory insects (Vespa crabro and Vespa velutina): a molecular perspective

The recent introduction of the Asian hornet, Vespa velutina, into Europe has raised concern regarding the threat to honeybees and the competition with the European hornet, Vespa crabro. The aim of this study was to investigated essential (Mg, Fe, Zn, Cu) and non-essential (Cd and Pb) elements in these two species. Element concentrations were determined in the whole body and separately in the head, thorax and abdomen using atomic absorption spectrometry (AAS). The changes in essential element concentration and speciation during metamorphosis were also studied using gel filtration chromatography followed by AAS and proteomic analysis. In both species, the essential elements were more concentrated in the abdomen due to the presence of fat bodies. Magnesium, Fe and Zn concentrations were significantly higher in V. crabro than in V. velutina and could have been related to a higher aerobic energy demand of the former species required to sustain foraging flight at higher speeds and to cover longer distances. Low concentrations of Cd and Pb were indicative of low environmental exposure. The concentration and speciation of essential elements, particularly Fe, varied among the developmental stages, indicating a modification of ligand preferences during metamorphosis. Overall, the results in the present study provide a better understanding of the hornet metal metabolism and a foundation for additional studies

Effects of exercise on urinary biochemical parameters and proteins in a group of well-trained military working dogs

Exercise-induced proteinuria has been widely investigated in humans, also in relation to intensity and duration of activity. Instead, there are only limited publications regarding urinary biochemical parameters and urinary proteins before and after physical activity in dogs. This paper aimed to investigate the effects of exercise on urinary biochemistry and proteins in military dogs. Twenty-four dogs were enrolled in this study. All the dogs were clinically sound, and they were examined before and after activity. Pulse rates (PR) and respiratory rate (RR) were monitored. Urine was sampled before and after a training session of search activity. Standard urinalysis was carried out, urine total proteins and creatinine were measured and the urinary protein:creatinine ratio was calculated; finally, the urinary proteins were separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Clinical examination before and after activity did not reveal any pathological finding. After activity, the PR was slightly increased, while the RR was notably increased (p < 0.05). Total proteins, albumin, and their ratio with creatinine were significantly higher after exercise when considering all the dogs included or only the females while, when considering only the males no significant difference was detected. The clinical relevance of this study was related to the possibility of using urine as a non-invasive sample for monitoring health status after training activity and exercise in dogs. An increase in microalbuminuria after search activity, measured using SDS-PAGE could be considered an early biomarker of renal function during training sessions

Trace Elements (Pb, Zn, Cu) in Blood of Mute Swan (Cygnus olor) from the Isonzo River Nature Reserve (Italy)

Lead concentrations in blood of 45 specimens of mute swan from the molting area of the Isonzo River Mouth Nature Reserve (Italy) were determined in two consecutive years (2006-2007), some birds were neck ringed to identify their homing behavior. The second sampling included whole body X-ray radiography and Cu and Zn plasma analyses to investigate the health impact of putative Pb exposure. X-ray images of all investigated specimens did not show any radiopacity due to the ingestion of metal bodies. Lead levels (0.08-0.44 g/ml) were in the range of those reported for swans living in unpolluted or slightly polluted environments and excluded acute intoxication, as confirmed by clinical investigation. Zinc concentrations ranged between 2.93 and 7.59 g/ml and were one order of magnitude higher than Cu concentrations (0.21-0.42 g/ml). The negative correlation between Pb and Zn concentrations could be indicative of adverse health effects caused by chronic lead exposure. To our knowledge this is the first study reporting Pb, Zn and Cu blood levels, X-ray radiographies and data on the origin of swan populations