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The quantification of circulating inflammatory biomarkers is relevant in the assessment of canine SIRS. The aim of this prospective study was to investigate the role of selected cytokines (IL6, IL8, IL10, IL15, MCP-1, GM-CSF; Millipore) during SIRS in dogs and their potential to differentiate sepsis from SIRS of non-infectious origin. Furthermore, a potential correlation between cytokines and other clinical and clinicopathological variables was investigated. Twenty dogs admitted to a Veterinary Teaching Hospital with a diagnosis of SIRS related to 3 different diseases (parvovirus, n=8; septic peritonitis, n=7; acute pancreatitis; n=5) were included. Ten blood donor dogs were included as healthy controls. Blood was collected upon admission by venipuncture, immediately processed at +4°C, and at least two aliquots of EDTA-plasma were stored at -80°C upon analysis. Additional laboratory analysis included measurement of C-reactive protein (CRP). Illness severity was calculated by the acute patient physiologic and laboratory evaluation fast score (APPLE). Cytokines were assayed by a multiparametric xMAP Luminex. CRP was assayed on serum using an immunoturbidimetric method on an automated chemistry analyzer. Results were analyzed by non-parametric statistic analysis. SIRS dogs have significantly higher serum CRP compared to controls (p

Heavy metals are toxic elements naturally present in the environment which can be bioconcentrated by plants and animals and incorporated into food cycles. Thus the use of wildlife species as monitors is a useful tool to assess environmental pollution. Because of its ethological characteristics, wild boar is highly probable to be exposed to pollution, mainly by heavy metals. These pollutants, when present at high enough concentrations, can induce histological lesion in various tissues. The purpose of the present work was to investigate the levels of five metals (cadmium, copper, iron, lead and zinc) in wild boar, Sus scrofa, originating from an Apennine area of Emilia Romagna, Northern Italy, and to evaluate any possible correlation with histological lesions eventually observed. Samples of several organs (liver, heart, kidney, diaphragm, abductor muscle, masseter, eye, testis, brain, lung and spleen) were collected from hunted animals during winter 2001. Levels of metals were determined on freeze dried tissues employing microwave wet digestion. Toxicological analysis were performed by atomic absorption spectrophotometry and data expressed on a dry weight basis. Histopatological analyses were performed on fixed samples of the same tissue. Highest mean values were found in kidney for cadmium and copper (7 ± 1 ppm and 30 ± 2.6 ppm respectively) and in liver for copper (21.16 ± 3.28 ppm), lead (2139 ± 680.6 ppb), iron (415.92 ± 24.41 ppm) and zinc (94.76 ± 6.11 ppm). In all other tissues, mean concentrations of each metal were at low levels, and were comparable one another. A statistical difference was found for iron (p00.05), copper (p00.05) and cadmium (p00.01) in kidney, in testis (p00.01) for zinc and in liver for copper (p00.05) and cadmium (p00.01) as function of age cohorts. When sex was considered, a statistical difference was found for iron in eye, for copper in heart and for cadmium in heart and kidney. Histopatological analysis allowed to find lesions of different gravity, which anyhow didn?t appear so severe to compromise the welfare of the animals. In particular the lungs show episode of chronic interstitial pneumonia; in liver it was constantly observed vacuolar degeneration, mainly perilobular, with frequent focal dissociation of the hepatic laminas. The kidneys show high glomerular cellularity and occasionally growth of the mesangial matrix and, rarely, chronic interstitial nephritis. In all the studied organs congestion was constant. It was not possible to find any correlation between the lesions found and heavy metals? levels. However it is possible to suppose that poor intoxication?s facts occurred, anyhow not specific. Starting from obtained data it is possible to define that the exposure to heavy metals in animals from the studied area could be considered very low so that mean concentrations defined for the tissues can be taken as indicative of a ?background? contamination.

We have focused our study on the concentrations of non essential trace metals Pb, Cd and Cr and essential heavy metals Zn and Cu, in the liver and kidney of raptor species from different areas of the Calabria region in Italy, with the aim of obtaining information concerning the quality and the contamination level of their habitat. No significant differences related to sampling areas and gender were found, while significant differences were found in metal concentrations among species. Mean Cd concentrations in the kidneys were generally 1.5-10 fold higher than hepatic ones, while Pb was mostly concentrated in the liver; a different pattern in tissue Cr concentration was observed between species. Referring to essential metals, Cu levels were higher in the liver, whereas higher Zn concentrations were constantly present in the kidneys. The ratio between kidney and liver metal concentrations (K:Ls) was also evaluated. The measured concentrations in toxic elements can be considered as indicative of chronic exposure to low amounts of pollutants. The essential metal concentrations were in the range of physiological values and did not show the presence of any deficiency or excess of metal. The present study can be considered as a starting point in the study of a possible correlation between levels of pollutants and the appearance of any adverse effects in raptor species in the Calabria area.

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.

The matrix metalloproteinases (MMPs) are a family of enzymes involved in extracellular matrix remodeling. MMPs are secreted in a latent form and activated by local and infiltrating cells. MMP-2 and -9 are the most studied in reproduction and have been detected in bovine, ovine, equine and human placenta. There is only one study on MMPs in the equine amniotic fluid (AF) reporting a decrease in the activity of MMP-2 in case of premature delivery. The aim of this study was focused on MMP-2 and -9 activity in AF collected at parturition from mares with normal or high-risk pregnancy. High-risk pregnancy was defined as a history of premature udder development/lactation, increase of combined thickness of the uterus and placenta, vulvar discharge and/or mare's systemic illness. The diagnosis of placental insufficiency was confirmed retrospectively after macroscopic and histopatologic examination of the placenta. AF was collected by needle puncture of the amnion within 5 min after its appearance through the vulva. The activity of MMP-2 and -9 was analyzed by in-gel zymography allowing the evaluation of both latent and active forms. Twenty mares with normal pregnancy (group 1) and 8 mares with high-risk pregnancy (group 2) were included. All mares in group 2 had a high-risk pregnancy with a diagnosis of placental insufficiency associated with placental villous hypoplasia, placentitis or placental edema. The bands relative to latent and active forms of MMP-2 were clearly visible in both groups and the activity of latent (P = 0.010) and active (P = 0.004) forms was lower in the AF samples of group 2. The band of the latent form of MMP-9 was visible in 17/20 samples of group 1, while it was completely absent in all samples of group 2. In contrast, the band of the active form was clearly visible and with a greater activity in AF samples of group 2 (P = 0.002). Placental dysfunction seems to induce a lower MMP-2 activity and a higher MMP-9 activity through the release of pro-inflammatory cytokines. Because fetal pulmonary secretions are a likely source of gelatinases in AF during late gestation, the increased MMP-9 activity could be related to fetal distress. These data provide a starting point to better understand the role of MMPs in equine pregnancy, although it should be confirmed in a larger and more homogeneous population of mares with high-risk pregnancy.

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.

Urinalysis could be helpful to investigate the health status of giraffes held in captivity using noninvasive methods to avoid animal handling or anesthesia. We collected 52 voided urine samples from 20 giraffes of different ages, sexes, and subspecies from the ground. To evaluate potential interference by soil contaminants, a pilot study was performed using 20 urine samples obtained from 10 cows. All bovine and 29 giraffe samples were subjected to routine urinalysis including urine specific gravity (USG). All samples were analyzed for urine total protein (uTP), urine creatinine (uCrea) concentration, and urine protein-to-urine creatinine ratio (UPC). Urinary proteins were separated by SDS-PAGE electrophoresis. No significant differences were determined between free-catch and urine sampled from the ground in cows. Giraffe urine was pale-yellow, with alkaline pH (>8.0) and a mean USG of 1.035 ± 0.013. The uTP, uCrea, and UPC expressed as median (range) were 0.20 (0.08–0.47) g/L, 2.36 (0.62–5.2) g/L, and 0.08 (0.05–0.15), respectively. SDS-PAGE allowed the separation of protein bands with different molecular masses, including putative uromodulin at 90 kD, putative albumin at 64 kD, and putative immunoglobulin heavy and light chains at 49 kD and 25 kD, respectively. Urine collection from the ground appears to be a reliable technique for urinalysis and urine electrophoresis in giraffes.

Simple Summary Honeybees are vital pollinators for the human food chain. Colony depopulation is a serious threat to Apis mellifera populations and unfortunately it is also one of the most elusive and difficult to study. This research deals with the problem at its foundation: population imbalances. The proposed method allows to discriminate, with remarkably good performances, precocious foragers from proper aged ones using SDS-PAGE patterns of haemolymph proteins. Implications and future perspectives are discussed. Abstract 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 factors, 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 Nearest Neighbours) model fitted on the forager dataset showed remarkably good performances (accuracy 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 countermeasures to revert this vicious circle.

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 &plusmn, 8 &micro, g/g in pheasant to 99 &plusmn, 76 &micro, g/g in snail, and Co was the least abundant, ranging from 0.007 &plusmn, 0.003 &micro, g/g in hare to 0.093 &plusmn, 0.048 &micro, g/g in snail. Regarding the non-essential trace elements, Pb concentrations showed wide variations, reaching a concentration of 17.30 &micro, g/g in hare, while Cd concentrations were higher in snail, ranging from 0.18 to 0.46 &micro, g/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.

Chronic kidney disease (CKD) is a progressive and irreversible disease. Although urine is an ideal biological sample for proteomics and metabolomics studies, sensitive and specific biomarkers are currently lacking in dogs. This study characterised dog urine proteome and metabolome aiming to identify and possibly quantify putative biomarkers of CKD in dogs. Twenty-two healthy dogs and 28 dogs with spontaneous CKD were selected and urine samples were collected. Urinary proteome was separated by SDS-PAGE and analysed by mass spectrometry, while urinary metabolome was analysed in protein-depleted samples by 1D 1H NMR spectra. The most abundant proteins in urine samples from healthy dogs were uromodulin, albumin and, in entire male dogs, arginine esterase. In urine samples from CKD dogs, the concentrations of uromodulin and albumin were significantly lower and higher, respectively, than in healthy dogs. In addition, these samples were characterised by a more complex protein pattern indicating mixed glomerular (protein bands ≥65 kDa) and tubular (protein bands

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&ndash, 1.049), 17.58 (4.54&ndash, 35.31) mg/dL, 154.62 (39.59&ndash, 357.95) mg/dL, and 0.11 (0.07&ndash, 0.16), 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.

Urinalysis offers important clinical information regarding not only the kidney function but also the general health status of an organism.The aims of this research were to obtain preliminary data on urine chemistry and electrophoretic protein profile from cows and heifers,to compare electrophoretic profiles of not pregnantwith pregnant animals and to evaluate their changes as the pregnancy progresses. Eight heifers and six cows were included in the study and 97 urine samples were collected. Complete urinalysis was performed and urinary proteins were separated by electrophoresis. Considering the pregnancy as a source of variability, significant differences were reported between pregnant and not pregnantheifers for the urine specific gravity (P=0.005), urine total proteins (P=0.009) and urine total proteins to urine creatinine ratio (UPC) (P=0.008). The majority of urine samples analysed in this study showed common protein bands. A mean of protein bands of 17±3 was detected in heifers, while a mean of 13±3 protein bands was recorded in cows. The putative proteins were uromodulin, transferrin, albumin, heavy and light chains of immunoglobulins. The comparison between pregnant and not pregnantanimals showed qualitative differences, with the absence of threebands in not pregnantcows including the putative alpha-fetoprotein. In conclusion, urinalysis is an economical and a non-invasive diagnostic protocol, which should be routinely used for the clinical evaluation of large animals. The data reported in the present study could be considered suggestive of healthy animals and they confirmed those previously reported in the literature.

Magnesium (Mg) is the fourth most-prevalent cation in the body and it is found predominantly in bone but also in muscle and neuronal tissue; plasma and red blood cells contain less than 1% of the total body Mg. Disorders of Mg regulation are rarely reported in critically ill foals. The aims of this study were: 1- to evaluate total plasma Mg concentration in healthy foals during the first 72 hours of life; 2- to evaluate total plasma Mg concentration during the first 72 hours of hospitalization in sick foals affected by perinatal asphyxia syndrome, prematurity/dismaturity, or sepsis; 3- to compare total plasma Mg concentration between healthy and sick foals; 4- to evaluate the association of Mg concentration at admission with survival and primary clinical diagnosis. Mg concentration measured in healthy foals (n=20) did not show any time-dependent changes. In sick foals (n=97), Mg concentration evaluated at T0 was statistically higher than Mg concentrations measured at subsequent times. Foals affected by PAS (n=20) had a Mg concentration significantly higher (p

Aquatic organisms may suffer from exposure to high Cu concentrations, since this metal is widely used in feed supplementation, in pesticide formulation and as antifouling. Chronic exposure to Cu, even at sub-lethal doses, may strongly affect fish physiology. To date, several biomarkers have been used to detect Cu exposure in fish producing contrasting results. Therefore, we used a proteomic approach to clarify how Cu exposure may affect the serum proteome of gilthead sea bream (Sparus aurata), since serum could be considered a good source of early-biomarkers of Cu toxicosis. For this purpose we exposed juvenile gilthead sea bream to waterborne Cu (0.5 mg/L). Our results indicate that fish tightly regulate circulating Cu levels, which are not affected by metal exposure. This homeostatic control is mainly achieved by the liver, able to excrete high amounts of the metal via bile. Cu exposure caused differential expression of several serum proteins, 10 of which were identified by Mascot and BLAST search. All these proteins, with the exception of growth hormone receptor and γ-glutamyl-carboxylase, can be related to: 1) Cu-induced hepatotoxicity (cytochrome oxidase subunit I, alanine aminotransferase, glutathione S-transferase); 2) potential immunosuppression due to interference of Cu with the inflammation/immunity network (α-1 antitrypsin, angiotensinogen, complement component C3, recombination-activating protein-1 and warm temperature acclimation-related 65 kDa protein).

The aims of this work were: (1) identification of the metallothionein (MT) gene coding sequence in order to prepare an MT probe in Scapharca inaequivalvis and (2) quantification of Cd, Zn, Cu, MT and MTmRNA expression in tissues of molluscs from three areas along the Northern Adriatic coast of Italy. By RT-PCR we cloned the MTcDNA of S. inaequivalvis using the RNA extracted from hepatopancreas of specimens exposed to Cd. The 61 amino acids sequence of MT was deduced and was 70% identical to S. brughtonii MT. Cd concentration in molluscs from the wild was significantly higher in gills from specimens sampled near Ravenna. Zn concentration in the same tissue was significantly higher in Ravenna with respect to Porto Garibaldi while no difference with respect to Cesenatico was detected. Cu levels showed significant differences among sites in gills and mantle whereas values in the hepatopancreas were similar in all sites. The low MT levels were indicative of a low metal exposure; few differences were found in MTmRNA concentrations, which resulted significantly higher in hepatopancreas of molluscs from Porto Garibaldi.

This paper aimed to evaluate cat seminal plasma protein profile (with SDS-page) and determine differences in seminal plasma composition from ejaculates obtained using urethral catheterization after pharmacological induction (UrCaPI) and electroejaculation (EE). In addition, this study evaluates whether the recovery method affected seminal plasma protein and zinc concentrations. A single ejaculation was collected from 17 mixed-breed cats by EE (5/21) or UrCaPI (12/21), while 4/21 cats underwent four sperm collections once every four days using EE and UrCaPI techniques alternately. The semen parameters evaluated were: volume, percentage of motility and progressive motility, morphology, and sperm concentration. After centrifugation, the seminal plasma obtained was stored at -80 °C and later used to measure protein and zinc concentrations, and to determine protein profile by SDS-polyacrylamide gel electrophoresis (PAGE). The results obtained indicate that cat seminal plasma protein profile is characterized by many protein bands (>30) with a molecular weight ranging from 3.5 to 200 kDa, and that the recovery method influences the seminal plasma protein profile: EE is related to the absence of two proteins (P55 and P14), and alters three protein bands (P200, P80, P28). The collection technique also affected zinc concentration (mg/dL) and protein concentration (g/dL) which were significantly higher (P < 0.01) in samples collected by UrCaPI; on the contrary the total Zn and protein amount/ejaculate were not significantly different in samples collected by both technique (P < 0.05).

Trace elements normally occur in living tissues in low concentrations, even though elements like zinc and copper are essential for growth, health and fertility. Metallothioneins (MTs) play an important role in controlling tissue concentrations of Zn, Cu and Cd and tissue functions. In this paper are reported studies on trace elements and MTs in different animal species. The liver was riche in essential and non-essential metals, due to the physiological role played in the processes of detoxification and storage. Among the investigated metals, Cd reached high concentrations in the kidney of wild birds mainly bound to MT. Preliminary studies that we performed in the mammary gland tumors of dog indicated higher expression of the MT2A isoform. Much remains to be learnt about the role of MTs in organisms and the integrated approach proposed by systems biology could help in this stimulating challenge

With the introduction of molecular biology techniques in medicine, new biochemical tools are available for determining a diagnosis. Transcriptional profiling and proteomics represent a major subject in translational clinical research. Metallothionein is a small molecular weight protein, having a strong affinity for heavy metals. However, despite to the increasing experimental data, the true function of this elusive protein remains to be identified. Due to the induction by a variety of stimuli, metallothioneins are considered valid biomarkers in environmental and medical fields. In this respect we present data related to the isolation and quantification of metallothionein in tissues of reptiles and mammals naturally exposed to heavy metals. In green turtles from the Caribbean Sea, copper-metallothionein in the liver was 10 times higher than the concentration found in the liver of loggerhead turtles from the Mediterranean Sea. As regards to mammals, the badger kidney metallothionein was rich in zinc, copper and cadmium, while in fox from the same area, only traces of the protein were found. These results are indicative of a different environmental metal exposure or a different mechanism in the control of metallothionein transcription. Moreover, in our laboratory, preliminary studies were conducted to evaluate the expression of metallothionein in mammary tumors of bitches. Western blotting patterns failed to identify the monomeric form of the protein; immunoreactive bands were present in correspondence of higher molecular weight molecules, which could be related to polymeric forms of metallothionein caused by disulphide bridges.

Metallothioneins (MTs), firstly isolated in the equine kidney, are ubiquitous low molecular weight proteins and polypeptides of extremely high metal and cysteine content which give rise to metal-thiolate clusters. Any protein or polypeptide resembling mammalian MTs can be classified as MT. MTs constitute a protein superfamily of 15 families which comprise a large number of sequences inferred from both aminoacid and polynucleotide sequences obtained from all animal phyla examined so far and also from certain fungi, plants and cyanobacteria. In mammals, the MT gene family consists of four subfamilies designated MT-1 through MT-4. The study of MTs needs the competences in different fields, which range from analytical chemistry and structural spectroscopy to molecular biology and medicine. Till now does not exist a simple quantification method whereas the sophisticated 2D NMR spectroscopy shows that notwithstanding MTs have different aminoacid sequences they have similar spatial structure with metal-thiolate clusters containing 3 or 4 bivalent metal ions. Increasing body of evidence shows that mammalian MT-1 / MT-2 isoforms are involved in zinc homeostasis, protection against heavy metal toxicity and oxidative stress. MT-3 is expressed mainly in neurons but also in glia while MT-4 is mostly present in differentiating stratified squamous epithelial cells. Due to the induction by a variety of different stimuli the MT determination is considered a valid biomarker in environmental and medical field. However the true biological role of these proteins is still enigmatic.