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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.

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.

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.

The urine from 35 giraffes was studied by untargeted 1H-NMR, with the purpose of obtaining, for the first time, a fingerprint of its metabolome. The metabolome, as downstream of the transcriptome and proteome, has been considered as the most representative approach to monitor the relationships between animal physiological features and environment. Thirty-nine molecules were unambiguously quantified, able to give information about diet, proteins digestion, energy generation, and gut-microbial co-metabolism. The samples collected allowed study of the effects of age and sex on the giraffe urinary metabolome. In addition, preliminary information about how sampling procedure and pregnancy could affect a giraffe’s urinary metabolome was obtained. Such work could trigger the setting up of methods to non-invasively study the health status of giraffes, which is utterly needed, considering that anesthetic-related complications make their immobilization a very risky practice.

Objective To assess the agreement between point-of-care and laboratory analysers in measuring biochemical and blood gas analytes in venous samples from tortoises and to define preliminary reference intervals for venous blood gas analysis in Hermann's tortoises (Testudo hermanni). Materials and methods Jugular venous blood samples from 47 Hermann's tortoises underwent paired analysis with a portable gas analyser (i-STAT 1, Abaxis), a portable chemical analyser (VetScan VS2, Abaxis), and with the respective reference analysers. Agarose gel electrophoresis was used to determine albumin concentrations on 12 specimens. Agreement was evaluated with Bland-Altman plots and regression analysis using the Passing-Bablok method. Results Point-of-care analysers had variable agreement with the reference analysers, presenting constant or proportional bias depending on the analyte. Relevant analytes in reptiles, such as ionised and total calcium, had acceptable agreement. The method for determining albumin concentration currently available in both point-of-care and laboratory analysers significantly overestimated albumin concentrations as compared to protein electrophoresis. Clinical significance While the use of POC analysers is extremely advantageous in small animal primary care facilities, agreement between point-of-care and laboratory analysers varies depending on the analyte. For certain analytes, interchangeability of results is limited and specific reference intervals for point-of-care analysers are required. Veterinarians should be aware of the size and the direction of the bias of each analyte.

This study was conducted to determine heavy metals concentrations in tissues of 94 birds belonging to different species from coastal areas of Naples and Salerno (Southern Italy) in order to provide baseline data concerning trace element levels in wild birds living in or close to an area characterized by high anthropogenic impact. Additional aim of this study was to verify if diet influenced metal accumulation, so birds were classified as birds of prey, fish eating birds and insectivorous birds. Kidney was the primary organ for Cd accumulation in all groups considered, Pb was accumulated preferentially in bone, whereas Hg showed high values in liver of fish eating birds. Zn showed the highest mean concentrations, while Cu levels were one order of magnitude less. The variance analysis with respect to feeding habits disclosed only a lower accumulation of Hg in insectivores with respect to the other groups. For all metals, the concentrations measured in tissues should be considered indicative of chronic exposure to low, “background” environmental levels and/or to the presence of low bioavailable metals in the environment. In addition, metal levels were not of toxicological concern, being always well below the toxic thresholds defined for each metal.

Trace metals such as Zn, Cu, and Fe are essential for life; differently, no biochemical function is known for Cd. Changes in dietary metal concentrations can cause deficiency or toxicity. Studies on trace elements in cat are lacking. This paper aimed to analyze Zn, Cu, Fe and Cd concentrations in liver and kidney of pathological domestic cat and to isolate metallothionein (MT) in these tissues. It was not possible to explore a possible correlation between metal concentrations and pathologies because the incidence for each of them was too low. Fe was the most abundant metal; in particular, the liver accumulates average Fe concentrations one order of magnitude higher than Zn and Cu, ranging from 66.75 and 1,444.23 microg/g. Significantly, higher levels of Fe were found in the liver of elder animals. Zn concentrations varied between 26.31 and 84.78 microg/g in the liver whereas in the kidney, ranged between 7.69 and 71.15 microg/g. Cu concentrations were between 2.37 and 112.91 microg/g in liver and between 2.12 and 9.85 microg/g in kidney. Cd was the least abundant metal with the exception of the kidney of the oldest cats where it reached a maximum of 13.71 microg/g. Gel-filtration metal distribution profiles from cytosolic extracts revealed the presence of Cd, Cu, Zn thioneins either in the liver or in the kidney. Because tissue samples were taken from pathological cats from different breed and age, care must be taken to use these data as a baseline profile of trace elements in healthy animals. Our results are indicative that for some specimens the feed levels of Fe and Cu could be higher than the optimal dietary intake and in few cats, there was also an exposure to Cd that was counteracted by MT biosynthesis.

One of the main biotic threat to honey bees all over the world is the ectoparasitic mite Varroa destructor (Acari: Mesostigmata). Without proper treatment, colonies are doomed and collapse within two years. Many treatments are used by beekeepers, with well-established acaricidal efficacy; however very little is known on the impact of the above-mentioned treatments on the wellbeing of the colony. Biochemical markers represent an interesting tool to assess the animal and human welfare. Particularly promising for our purposes seems to be the evaluation of vitellogenin in the hemolymph of a pool of bees. The potential of this protein can be foreseen considering his pivotal role in the homeostasis of social insect colonies. In fact, the function of vitellogenin in these species is not limited to egg yolk constitution: trophic, antioxidant and hormonal function are also described. The aim of this research was to test the impact of two different Varroa control techniques by analyzing the vitellogenin content in the hemolymph. Brood interruption by queen caging (five colonies) and brood removal (five colonies) followed by trickling of Api-Bioxal (Oxalic acid based acaricide) were performed. The hemolymph of 30 bees per colony was sampled and pooled. Four samplings were considered: pre manipulation, after manipulation, autumn (wintering phase) and winter. All the samples underwent SDS-PAGE and quantification of vitellogenin, qualitative analysis of other relevant proteins was also performed. Vitellogenin was successfully isolated and identified by mass spectrometry. Our data are confirmatory of the abundance of this protein in the hemolymph of worker bees and interesting differences were found depending on the season. Major differences were also found between the experimental groups, suggesting the expected different impact of the techniques and stressing the need of an objective tool to better evaluate and understand the techniques for Varroa control.

Metallothioneins (MTs) are low molecular weight proteins characterized by a high cysteine content and give rise to metal-thiolate clusters. Most MTs have two metal clusters containing three and four bivalent metal ions, respectively. The MT gene family in mammals consists of four subfamilies designated MT-1 through MT-4. MT-3 is expressed predominantly in brain and MT-4 in differentiating stratified squamous epithelial cells. Many reports have addressed MT structure and function, but despite the increasing experimental data several topics remain to be clarified, and the true function of this elusive protein has yet to be disclosed. Owing to their induction by a variety of stimuli, MTs are considered valid biomarkers in medicine and environmental studies. Here, we will discuss only a few topics taken from the latest literature. Special emphasis will be placed on MT antioxidant functions, the related oxidation of cysteines, which can give rise to intra/intermolecular bridges, and the relations between MTs and diseases which could be originated by metal dysregulation.

Chronic kidney disease (CKD) is a major cause of mortality in cats, but sensitive and specific biomarkers for early prediction and monitoring of CKD are currently lacking. The present study aimed to apply proteomic techniques to map the urine proteome of the healthy cat and compare it with the proteome of cats with CKD. Urine samples were collected by cystocentesis from 23 healthy young cats and 17 cats with CKD. One-dimensional sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) was conducted on 4-12% gels. Two-dimensional electrophoresis (2DE) was applied to pooled urine samples from healthy cats (n = 4) and cats with CKD (n = 4), respectively. Sixteen protein bands and 36 spots were cut, trypsin-digested and identified by mass spectrometry. 1D-SDS-PAGE yielded an overall view of the protein profile and the separation of 32 ± 6 protein bands in the urine of healthy cats, while CKD cats showed significantly fewer bands (P

Data on proteinuria are lacking in giraffes, therefore the aim of our research was to study urine proteome with sensitive analytical methods to obtain important information regarding not only the kidney function but also the general health status. Twenty-nine samples were collected during 2013 and 2014, through non-invasive methods (from the ground by a syringe) from 4 specimens of Giraffa camelopardalis. Urinary total proteins (UTP), creatinine and the urine protein to creatinine ratio (UP/C) were measured by automated methods (Olympus AU400) routinely used in small animal practice. Urinary proteins were separated by sodium-dodecyl-sulphate polyacrilamide gel electrophoresis (SDS-PAGE) and gels were stained with silver nitrate. Mean UTP±SD was 12.1±0.6 mg/dL ranging from 2.4 to 30 mg/dL, while mean UP/C values were 0.06±0.01. SDS-PAGE yielded an overall view of the protein profile allowing the separation of 7±2 protein bands. Electropherograms showed few protein bands as compared to dog and cat and big felids. The most represented band had an apparent molecular weight (MW) of 67-69 kDa and could be identified as putative albumin. Between 65 and 14 kDa only few low abundance bands were present, while more intense bands had a MW lower than 14 kDa. Our data could be considered as representative of animals with no clinical signs of disease resulted in a useful diagnostic tool that could help clinicians in quantitative and qualitative evaluation of proteinuria also in giraffes.

Data on proteinuria in giraffes are lacking, therefore the aim of our research was to study urine proteome with sensitive analytical methods to obtain important information regarding not only the kidney function but also the general health status. Fifty urine samples were collected during 2013 and 2014, through non-invasive methods from 21 specimens of Giraffa camelopardalis. Urinary total proteins (UTP), creatinine and urine protein to creatinine ratio (UP/C) were measured by automated methods routinely used in small animal practice. Urinary proteins were separated by sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and gels were stained with silver nitrate. Mean UTP±SD was 16.1±8.8 mg/dL (range 2.4-30 mg/dL), while mean UP/C was 0.08±0.03 (range 0.04-0.2); these values can be considered representative of healthy animals. SDS-PAGE yielded an overall view of the urine protein profile allowing the separation of 6±2 protein bands. Electropherograms showed few protein bands as compared to dogs, cats (Ferlizza et al., 2012) and big felids (Di Cerbo et al., 2013). The most represented band had an apparent molecular weight (MW) of 67-69 kDa and could be identified as putative albumin. Between 65 and 14 kDa only few low abundance protein bands were present, while more intense bands had a MW lower than 14 kDa. This is the first study reporting UTP and UPC ranges in giraffes. 1-D-SDS-PAGE yielded an overall view of the protein profile and resulted in a useful diagnostic tool that could help clinicians in qualitative evaluation of proteinuria. References Di Cerbo P., Ferrari G., Isani G., Ferlizza E., Friedrich K.G., 2013. Urine proteome in big cats (Panthera leo, Panthera tigris, Panthera pardus). In: proceedings of the International Conference on Diseases of Zoo and Wild Animals, Vienna, A. Ferlizza E., Armuzzi E., Buono A., Carpenè E., Andreani G., Dondi F., Isani G., 2012. Urine proteome from healthy dogs, cats, horses and cows. In: proceedings of the European Society of Veterinary Clinical Pathology Conference, Ljubljana, SLO.

Background Hermann's tortoise, Testudo hermanni, is currently on the International Union for Conservation of Nature (IUCN) red list of endangered species. Reptile medicine relies also on laboratory analyses to evaluate health status, but reference ranges for hematology and biochemistry variables and protein electrophoresis in plasma of healthy tortoises are not available. Objective The purposes of this study were to establish reference ranges for select hematologic and biochemical variables in clinically healthy Hermann's tortoises, and evaluate the impact of sex and season. Methods Blood samples were collected from 34 healthy tortoises at the end of September and beginning of July. Blood smears, HCT, concentrations of HGB and select plasma biochemical analytes, select enzyme activities, and plasma protein fractions were evaluated. Reference ranges were determined and checked for influence of sex and sampling time point. Results Typical reptilian RBC and WBC were observed in blood smears. HCT and concentrations of HGB, uric acid and urea, and ALT and AST activities were significantly higher in males than in females. Concentrations of glucose, uric acid, and phosphate, and AST activity were significantly higher at the beginning of July, whereas concentrations of urea and Cl were higher at the end of September prior to hibernation. The electrophoretic protein fractions included albumin, and α, β, and γ globulins. Conclusions The reference ranges defined in the present study are useful for clinical tortoise medicine and conservation. Sex and seasonal sampling were identified as factors significantly affecting hematology and blood chemistry analytes; they should be taken into consideration when assessing tortoise health status.

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

A comparison of IL-1β and IL-8 mRNA and their respective proteins in equine bronchoalveolar lavage fluid during recurrent airway obstruction, Online J Vet Res., 15 (3): 215-228, 2011. The role of different cytokines in triggering airway inflammation during recurrent airway obstruction (RAO) has become the focus of recent investigations. The majority of published papers are principally concerned with cytokine mRNA while there are fewer papers which deal with their respective proteins alone or their correlation with mRNA in the same trial. In the present study IL-1β and IL-8 mRNAs and their respective proteins were quantified by Real Time RT-PCR and western blot and related to bronchoalveolar lavage fluid (BALF) cytology in 8 horses during an RAO attack induced by stabling on straw. The results were compared to those obtained from 4 susceptible horses kept in a pasture. Significant changes in cytokine profile and BALF pattern were observed in the challenge group during the study (12 days) while no modifications were detected in the control group. An increase in IL-1β mRNA and protein preceded the increase of the same parameters as IL-8, but the related mRNA and respective protein levels did not strictly follow the same pattern. The rise in IL-1β protein, but not mRNA, continued over the entire period and was correlated with BALF neutrophilia. On the contrary, the increase in both IL-8 mRNA and protein were transient, and the peak of the protein (day 8) followed the peak of BALF neutrophilia. The increase of IL-1β, paralleled by neutrophil and epithelial cell accumulation in the BALF, is indicative of the active production of the cytokine by these cells. This reinforces the pivotal role of cells other than the macrophages in driving airway inflammation during RAO

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).

L’aumento generalizzato delle attività umane ha generato una continuità spaziale, che si è tradotta in contiguità di ambienti, fra animali selvatici, animali domestici ed insediamenti umani. Purtroppo questa stretta convivenza (promiscuità?) è in grado di favorire malattie contagiose nelle tre comunità di selvatici, domestici ed umani. Inoltre la riduzione dell’habitat silvestre, la sua frammentazione ed il disturbo antropico che si assommano all’eccessivo sfruttamento di molte specie rappresentano un trend in ascesa che pone crescenti problemi di biologia della conservazione. Questa erosione di habitat ha inoltre importanza come causa di patologie non infettive ma può anche favorire la colonizzazione di agenti trasmissibili. La consapevolezza della necessità di tutelare l’ambiente è una priorità epocale e la salute degli animali selvatici è uno dei punti chiave per la conservazione delle specie selvatiche. L’interesse per la salute degli animali selvatici si interfaccia con barriere legislative fra stati nella movimentazione di animali e prodotti di origine animale quando particolari malattie sono presenti nella fauna selvatica di una determinata area geografica. Le più recenti tendenze nella gestione di importanti malattie degli animali domestici non sottovalutano l’importanza degli animali selvatici come componenti della biocenosi (patocenosi) in cui dovrà realizzarsi l’eradicazione. In accordo con queste regole internazionali di polizia veterinaria, i veterinari dovranno occuparsi di malattie dei selvatici con lo scopo di migliorare la salute dell’ambiente e di evitare la diffusione di malattie dagli animali domestici ai selvatici (e vice versa) dimostrando alla Comunità Europea l’efficacia delle misure di controllo applicate. Il coinvolgimento dei veterinari nella protezione dell’ambiente, in relazione alle loro competenze, dovrebbe essere migliorato anche alla luce degli importanti risultati scientifici ottenuti da alcuni gruppi di ricerca negli ultimi decenni. Il ruolo dei veterinari nella conservazione e nella salute degli animali selvatici non è una nuova moda; in particolare l’ affermazione che le malattie degli animali selvatici non rappresentano ancora uno sbocco professionale ma potrebbero e dovrebbero divenirlo è tuttora valida. A tuttoggi, in un contesto di salute degli ecosistemi la medicina dei selvatici non può essere considerata appannaggio di un ristretto gruppo di ricercatori o l’interesse “hobbystico” di alcuni colleghi considerando che le malattie trasmissibili e quelle non infettive sono uno degli aspetti più rilevanti per assicurare il successo in termini di conservazione ambientale.

Gli enzimi superossido dismutasi (SOD) e catalasi (CAT) sono importanti in quanto svolgono, insieme ad altre molecole ad attività catalitica, attività antiossidante nelle cellule e nei tessuti di numerosi organismi. Il grande interesse intorno a queste molecole catalitiche va cercato principalmente in due fattori: 1)sono utilizzati come biomarkers per quantificare il grado di stress ossidativo cellulare; 2) sono coinvolti in malattie degenerative del sistema nervoso centrale. Il presente lavoro è volto ad individuare metodiche per l’evidenziazione, in tessuti di orata (Sparus aurata), degli enzimi Cu/Zn SOD e CAT. SOD: è stata osservata positività diffusa nel citoplasma ed in alcuni casi nel nucleo di cellule di fegato, rene, enterociti, pirenofori dei plessi enterici, miocardiociti del tessuto ventricolare e macrofagi sub-endocardici nel bulbo cardiaco, epitelio branchiale e pirenofori del sistema nervoso centrale. CAT: il segnale era citoplasmatico sottoforma di granulosità di dimensioni e quantità differenti in funzione dei diversi tessuti: granuli citoplasmatici ben apprezzabili e diffusi nel fegato e nei tubuli renali; positività granulare più fine negli enterociti, nell’epitelio branchiale, neuroni e cellule gliali. Le differenti caratteristiche nell’espressione del segnale fra i due enzimi indagati nel presente lavoro confermerebbero la specificità dei risultati. La Cu/Zn SOD è prevalentemente citoplasmatica mentre la CAT è confinata nei perossisomi e/o microperossisomi in funzione delle cellule e tessuti interessati.

GLUTs are a family of proteins that facilitate the transport of glucose and other hexoses through the plasma membrane of the cells. GLUTs are present in mammalian spermatozoon's membrane in different isoforms and they supply metabolic substrates for all the cell's activities such as motility, homoeostasis and fertilization. As studies about donkey spermatozoa and their metabolism are lacking, this study was aimed at detecting GLUTs 1, 2, 3 and 5 presence by western blotting technique and at determining their localization on the plasma membrane by indirect immunofluorescence. Each protein showed a typical localization on the sperm cells' plasma membrane, differencing the one to the other on the basis of the hexose they transport. We also highlighted some differences between GLUTs distribution and molecular weight in donkey spermatozoa and its nearest relative, the horse.

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

Cadmium (Cd), a heavy metal with limited biological function, is widely distributed in the aquatic environment as a result of natural and anthropogenic activities. The effect of 4 and 11 days exposure of gilthead sea bream Sparus aurata to sub-lethal concentrations of Cd was evaluated as levels of Cd content and Cd-metallothionein (MT) presence in different organs. The possible genotoxic effect was also evaluated in erythrocytes by using the "comet assay", a promising tool for estimating DNA damage at the single-cell level. The results obtained show that in the controls, Cd content was significantly higher in gills compared to in liver, but the treatment of fish with 0.1mg/l Cd induced a stronger accumulation of metal in liver depending on the length of the exposure period. Cd traces were found in plasma, muscle and kidney. Cd forms complexes in the cytosol with MT only in the liver but Cd-MT content significantly increased after 11 days of exposure to the metal, while after 4 days of treatment the protein level was similar to the control. The "comet assay" performed on S. aurata eryhtrocytes isolated from fish treated for 4 and 11 days with 0.1mg/l Cd, showed that there was no DNA damage at both exposure periods.

The first aim of our study was to determine the concentrations of selected trace elements (Zn, Cu, Fe, Mn, Cd and Pb) in tissues of green turtles from Tortuguero National Park on the North Caribbean coast of Costa Rica and of loggerheads from the Mediterranean Sea. Zn, Cu, Fe, Mn and Cd were present at detectable concentrations in all samples and showed clear organotropism, whereas Pb was not always over the detection limit and did not show any particular tissue distribution. The two species presented significant differences: Cu and Cd in liver and kidney of Chelonia mydas were significantly higher with respect to the concentrations found in Caretta caretta. The second and major goal of our study was to evaluate hepatic and renal metallothionein (MT) as a biomarker of environmental metal exposure. The present paper is the first to describe and quantify MT in kidney and liver of loggerhead turtles and in kidney of green turtles. MT concentrations were higher in green than in loggerhead turtles. In addition, positive correlations were found between Cu and Cd concentrations and Cu-MT and Cd-MT in liver and kidney in both species, suggesting a pivotal role of MT in metal storage and detoxification. The quantification of metals and MT in liver and kidney may be a valid biomarker of metal exposure in the aquatic environment to assess the health of marine sea turtles as long as accurate analytical methods are adopted.

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.

We have measured the concentrations f heavy metals in soils, earthworms and tissues of woodcocks in Quaderna Valley, Northern Italy. The soil concentration of metals analysed in this research is consistent with data reported by other authors for uncontaminated or slightly contaminated soils. In earthworms, metals were mostly accumulated in the encapsulating chloragogenous tissue; the positive correlation between Cu concentration in the soil and in earthworms is noteworthy. Heavy metals distribution in the tissues of woodcock showed that Cd accumulation in the kidney was linked to the diet. Cu and Fe were preferentially concentrated in the liver and Zn in the testis. Kidney Cd and Zn concentrations were higher in adults than in juvenile. In addition, a main kidney metallothionein isoform, containing Cd and Zn, was isolated. In the kidney, Cd levels were linearly correlated with the concentration of metallothionein. Of the investigated metals, Cd raises the greatest concern, due to the increasing soil contamination by human activities.

(AChE) forms were detected and recovered from foot or gill tissues of the benthonic bivalve mollusc Scapharca inaequivalvis. A study was performed to investigate changes in catalytic and hydrodynamic features of these enzymes as well as in their expression levels after a 4 or a15 day exposure to a sublethal 79 concentration (0.1 ppm) of the pesticide chlorpyrifos (CPF). Both considered organs hold, in either CPF exposed or untreated animals, two non-amphiphilic AChE forms, G2 and G4, which copurified on a procainamide-containing affinity gel, and were separated by density gradient centrifugation. A third AChE form, an amphiphilic membrane-anchored G2, was also purified on the same affinity matrix from both organs. All enzymatic forms are true AChEs and are poorly inhibited by CPF. They show differently sized increases in the maximum velocity (Vmax) and in the Michaelis constant (Km) values after CPF exposure. Consequently, catalytic efficiency of AChEs, as defined by the ratio Vmax / Km, rises in the gills and drops in the foot. This would produce an over-expression of AChE specific mRNAs. The effect was longer lasting in the foot. The combined results suggest that over-expression of enzymes in the presence of organophosphate (OP) may be a consequence of OP-resistance itself. Again, the resistance of the organism to CPF seem to depend mainly on the resulting increase in AChE content.

Copper is an essential micronutrient, but in excess it is highly toxic due to reaction with intracellular thiol groups and the initiation of oxidative damage. Recent studies in mammals have shown that cellular uptake and efflux are mediated by high affinity carriers, whilst cellular toxicity is minimised both by a series of specific chaperone proteins which carry and deliver the metal to apometalloenzymes and by sequestration of excesses by metallothionein. In aquatic organisms anthropogenic copper discharges (eg. from viniculture) are a serious problem in some areas and in this preliminary study we have investigated copper homeostasis in a copper-exposed fish, the Sea Bream, Sparus aurata. Further studies on two fish species which show an apparent defect in hepatic copper efflux have also been initiated. cDNAs for a high affinity copper uptake transporter (saCTR1), a copper chaperone (saATOX1), an efflux pump (saATP7) and the heavy metal detoxification protein metallothionein (MT) were cloned, the first three for the first time from a lower vertebrate. Quantitative assays for transcript expression were developed and used to study effects of exposure to both waterborne copper and different dietary copper levels. The deduced amino acid sequences of the four investigated protein showed very high structural conservation between fish and man. Responses to waterborne copper exposure were most marked. Expression of saCTR1mRNA was reduced by copper exposure in gill and liver, there was an initial induction of hepatic saATP7mRNA expression, and hepatic MTmRNA levels were induced, indicating regulation of intracellular copper. Moreover induction of glutathione reductase indicated that it elicited an oxidative stress response. Dietary exposure reduced CTR1 mRNA expression in intestine but at low dose levels there was an initial elevation in both CTR1 and ATP7 mRNA levels in liver. These results indicate tissue differences in response and one explanation which is being investigated is that this may be attributable to differences in copper speciation and availability. AKNOWLEDGEMENTS: Work supported by EU LSF and grant from the Ministero Politiche Agricole, VI piano