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The present study investigated the in vitro effects on caprine monocytes of two ω-3 PUFAs, namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on lipid droplet formation, an emerging process of fundamental importance in innate immunity regulation. The mRNA abundance of PAT protein family (PLIN1, PLIN2 and PLIN3), involved in the formation and trafficking of the droplets, was also assessed. The effects of EPA and DHA on monocyte apoptosis were studied as well. The number of lipid droplets per cell was found to be dependent on both type and concentration of fatty acid. ω-3 PUFAs upregulated PLIN3 and PLIN2 gene expression, as well as apoptosis rate. The present findings suggest that PUFA might modify innate immune functions of goat monocytes by interfering with the formation of lipid droplets and by upregulating proteins belonging to PAT protein family.

The serum amyloid A (SAA) and haptoglobin (Hp) are the most prominent acute phase proteins (APPs) in cow. Liver mainly produces APPs, but extra hepatic expression has also been demonstrated in some tissues. The major aim of the present study was to assess the constitutive SAA and Hp mRNA expression by quantitative PCR (qPCR) in a wide panel of 33 bovine tissues, including gastrointestinal tract, respiratory system, urogenital system, mammary gland, hematopoietic system, central nervous system, eye, thyroid and heart. Normalization of gene expression in different samples requires reference genes, which are stably expressed. Therefore, seven reference genes were investigated (ACTB, GAPDH, HMBS, SDHA, YWHAZ, SF3A1, EEF1A2) and three genes, namely SF3A1, HMBS and ACTB, were selected after assessing their stability with geNorm™ and NormFinder(©) softwares. The qPCR analysis confirmed liver as the principal source of SAA and Hp, but also identified both APPs' mRNA in almost all tissues. The highest expression rate of SAA was found in thyroid, followed by pancreas and submandibulary gland. Hp mRNA expression was detected at high concentration in pancreas and submandibulary gland. The present data indicated a widespread expression of SAA and Hp also in non pathological conditions, thus envisaging a possible role as immunomodulatory and protective molecules. To understand where SAA and Hp come from is the prerequisite to their utilization as Acute Phase Reaction biomarkers.

The acute phase proteins (APP) are a group of serum proteins that change their concentration in animals following external or internal challenges, such as infection, inflammation or stress. The concentrations of four APPs, including serum amyloid A (SAA), haptoglobin (Hp), alpha(1)-acid glycoprotein (AGP) and ceruloplasmin (Cp) were determined in serum collected from healthy Alpine ibexes (Capra ibex) and ibexes with Sarcoptes scabiei mange. Primary structures of all four APPs were determined by cDNA sequencing. The concentrations of all four APPs were higher in serum of animals with clinical signs of sarcoptic mange when compared to healthy animals. Two of the APPs, including SAA and AGP, acted as major APPs, since their serum concentrations were increased more than 10-folds when compared to healthy animals (P0.001). The other two APPs, including Hp and Cp, acted as minor acute phase proteins, as their concentrations were increased from two to five folds (P0.001). These findings provide a remarkable potential as diagnostic markers for the early detection of sarcoptic mange in free ranging animals.

An in vitro study was carried out to examine the influence of two fish-oil-derived long chain omega-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on goat polymorphonuclear leukocytes (PMN). Twelve Saanen healthy goats were used as blood donors. Neutrophils were isolated from blood and incubated with increasing concentration of EPA and DHA (25, 50, 100, 200muM). Control samples were incubated in the absence of omega-3 PUFAs. Phagocytosis was evaluated by fluorescein-labeled Escherichia coli incorporation, while extracellular Reactive Oxygen Species (ROS) production was determined by cytochrome c reduction assay, which was selected among the others due to its specificity for extracellular superoxide anion release. Phagocytic activity was significantly increased by EPA (P

Background: The tawny owl (Strix aluco) is a protected species in Italy. Orphaned, injured, and ill owls often are sheltered and treated in rehabilitation centers, where hematologic and biochemical analyses would be helpful to evaluate and monitor the status of their health. Objectives: The major aim of this work was to assess hematologic and biochemical constituents together with protein electrophoretic fractions in healthy tawny owls. In addition, we compared laboratory methods for determining hemoglobin (Hgb), total protein, and albumin concentrations. Methods: Heparinized blood samples were collected from 10 clinically healthy adult captive tawny owls between March 2001 and November 2003 for CBC, routine biochemical analysis, and protein electrophoresis. Alternate methods for Hgb (estimation as HCT÷3 vs spectrophotometry), total protein (biuret vs refractometry), and albumin (bromcresol green vs electrophoresis) concentrations were compared in 34 samples from 16 unhealthy adult owls and 8 nestlings. Results: Results were reported as mean, median, and range (minimum–maximum). Significant differences and poor concordance were observed between methods for Hgb, total protein, and albumin. Conclusions: Hematologic and plasma biochemical values in captive tawny owls may be useful in evaluating and monitoring the health of this species in captivity.

The chemotactic activity of zymosan-activated serum (ZAS) and of two concentrations of recombinant human IL-8 (IL-8(25), 25 ng/ml; IL-8(50), 50 ng/ml) for ovine polymorphonuclear granulocytes (PMNs) was tested in a modified Boyden chamber. Thick cellulose acetate filters and the leading front method were used to quantify the movements of the cells. Both ZAS and IL-8(25) exerted a chemotactic effect on ovine PMNs (P < 0.01): IL-8(50) induced a more homogeneous response (P < 0.001). To verify the characteristics of the responsiveness to the chemokines after short-term (st) or long-term (lt) repeated samplings, chemotaxis was investigated 1 (T1st), 2 (T2st), 24 (T3st) and 48 h (T4st) after the basal sampling (T0st) and 15 days (T1lt) after the basal sampling (T0lt). No differences in chemotaxis were found in long-term repeated samplings. In contrast an increase in the responsiveness to IL-8(25) and to IL-8(50) (P < 0.05) was detected at T2st in comparison with T0st. Furthermore, the significance of the distance run by activated PMNs compared with the controls, increased from T0st to T2st, as a sign of a more homogeneous response to the chemokines. In the absence of evident changes in circulating leucocyte numbers and in serum cortisol concentrations, these findings could be interpreted as a consequence of a different expression of chemoattractant receptors on the membrane of PMNs collected at different times.

The effects of the ketone bodies beta-OH-butyrate and acetoacetate (2.4 or 4.8 mmol/l), administered singly or simultaneously in vitro, on adherence and superoxide anion (SO) production in ovine neutrophils were investigated by simultaneous assay in 96-well microplates. Because the acetoacetate used was a lithium salt, the effect of 2.4 and 4.8 mmol/l lithium chloride was also tested. Neutrophils from eight non-lactating, non-pregnant ewes were used. SO release from neutrophils was found to be very low in basal conditions and was apparently not stimulated by contact with plastic. Administration of 10(-7) mol/l phorbol myristate acetate (PMA) caused a rapid increase and release of SO production, but smaller than that induced by co-stimulation with plastic and 10(-7) mol/l PMA. LiCl (2.4 and 4.8 mmol/l) significantly increased PMA-stimulated release, but inhibited plastic and PMA co-stimulated SO release. Administration of 2.4 mmol/l ketone bodies inhibited plastic and PMA-costimulated SO release, but the effect of acetoacetate could be due to the lithium component. Administration of 4.8 mmol/l ketone bodies had no effect. Adherence was significantly increased by contact with plastic, and moreover by 10(-7) mol/l PMA. The effect was similar when PMA was acting alone or with plastic. Neither basal nor stimulated adherence were affected by 2.4 or 4.8 mmol/l ketone bodies. LiCl at a concentration of 4.8 mmol/l increased PMA and plastic co-stimulated adherence. The results suggest that, in sheep, only the ketone body beta-OH butyrate at concentrations seen in mild ketosis, could decrease bactericidal activity, while adherence is not affected. In addition to other factors that could impair the efficiency of the immune system in ketotic ruminants, the reduced bactericidal activity may contribute to the higher occurrence of infectious disease in these animals.

The in vitro effects of the ketone bodies beta-OH-butyrate (2.4 or 4.8 mmol/l) and acetoacetate (2.4 or 4.8 mmol/l) on the uptake of latex particles (1.09 microns) and chemotaxis were investigated in ovine neutrophils. Because the acetoacetate used was a lithium salt, the effect of 2.4 or 4.8 mmol/l lithium chloride was also tested. Neutrophils from eight non-lactating, non-pregnant ewes were studied. The uptake of latex particles, as measured by a spectrophotometric method, showed wide individual variation. The phagocytotic activity was unaffected by 2.4 mmol/l ketone bodies and LiCl, but it was significantly inhibited by 4.8 mmol/l beta-OH-butyrate and activated by 4.8 mmol/l LiCl. The latter result could be masking an inhibitory effect of acetoacetate. Chemotactic movements of neutrophils, as evaluated in a modified Boyden chamber using homologous zymosan-activated serum (ZAS) as a chemoattractant, were slightly but significantly reduced by a 2.4 mmolar concentration of the ketone bodies, administered singly or simultaneously, and by LiCl. We conclude therefore that the inhibitory effect of lithium-acetoacetate could be due to its lithium component. The 4.8 mmol/l dose of acetoacetate and beta-OH butyrate significantly decreased chemotaxis only when both compounds were added simultaneously. No effect of 4.8 mmol/l LiCl was observed. These results suggest that ketone bodies, in particular beta-OH butyrate, could directly influence particle uptake and chemotaxis in neutrophils. Although other factors could decrease the efficiency of the immune system in ketotic ruminants, the effects of the ketone bodies on neutrophils functions may explain the high frequency of infectious disease during 'ketotic syndrome'. The immunomodulatory effect of lithium needs to be evaluated further and it should be considered when testing lithium compounds.

Summary The effects on some hematochemical stress indices of previously administered metyrapone (750 mg given orally six times every 4 hours) were evaluated in calves submitted to simulated transport for 30 min. The plasma Cortisol increases were significantly lower than those observed in untreated calves of the same age, sex and breed. Plasma adrenaline and NEFA increased similarly in both groups of calves during simulated transport and were unaffected by metyrapone administration. The results indicate that in cattle, as in other animals, metyrapone inhibits Cortisol biosynthesis. Under our experimental conditions, the rise of NEFA, a well known stress index, seems mainly to be related to adrenaline release.

BACKGROUND Increasing interest in wildlife care leads to the need for new tools to evaluate animal health. Laboratory investigations require reference intervals against which to compare the results obtained. For common buzzards, only a few studies have been performed to establish hematologic and biochemical reference intervals. OBJECTIVES The aim of this work was to develop reference values for routine hematologic and biochemical constituents and protein electrophoretic fractions and evaluate possible seasonal differences in values for healthy common buzzards. METHODS Heparinized blood samples were collected from 23 captive, clinically healthy common buzzards between February 2001 and June 2003. A CBC, routine biochemical analysis, and protein electrophoresis were performed. Data distribution was assessed and results from birds sampled in spring, summer, and winter were compared. Results from alternative methods for hemoglobin (Hgb; estimated as HCT / 3 vs spectrophotometry), total protein (biuret vs refractometry), and albumin (bromcresol green vs electrophoresis) concentrations also were compared. RESULTS Reference intervals were calculated as 10-90th percentiles. In spring and summer, total WBC and heterophil counts, and urea, total protein, prealbumin, and beta- and gamma-globulins concentrations were significantly different from winter values. Results obtained by alternative methods for Hgb, total protein, and albumin concentrations were significantly different from those obtained by standard methods, although estimated and spectrophotometric Hgb values were significantly correlated. CONCLUSIONS The reference values obtained in this study for hematologic and plasma biochemical constituents and their seasonal variation in healthy, captive common buzzards will be useful in the clinical evaluation of these birds in rehabilitation settings.