Paola Sartorelli

Adrenal response in the calf to repeated simulated transport

The adrenal responses in calves submitted to simulated transport on three occasions for 30 min were evaluated. Plasma adrenaline, cortisol and NEFA increased significantly during simulated transport but became less marked in successive trials. Haematological stress-related parameters (Hb, PCV) increased to the same extent on repeated exposure to simulated transport. Plasma noradrenaline, glucose and cholesterol values were unchanged throughout the study.

In vitro modulatory effect of ω-3 polyunsaturated fatty acid (EPA and DHA) on phagocytosis and ROS production of goat neutrophils

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<0.05) and DHA (P<0.01). Treating PMN with EPA does not affect extracellular ROS production which is, on the contrary, down-regulated by DHA. This effect was increased in experimental conditions which mimic pro-inflammatory challenges (stimulation with PMA). This study demonstrates that EPA and DHA may have beneficial effect on neutrophil function by increasing their phagocytosis activity and, in the meanwhile, decreasing the tissue damages due to extracellular release of ROS.

Acute phase protein response in Alpine ibex with sarcoptic mange

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 (P<0.001). The other two APPs, including Hp and Cp, acted as minor acute phase proteins, as their concentrations were increased from two to five folds (P<0.001). These findings provide a remarkable potential as diagnostic markers for the early detection of sarcoptic mange in free ranging animals.

Alpha1-acid glycoprotein is contained in bovine neutrophil granules and released after activation

The present study was designed to investigate the capability of bovine neutrophil granulocytes to produce the minor acute phase protein alpha(1)-acid glycoprotein (AGP, Orososmucoid). Bovine neutrophils contain a high MW (50-60kDa) AGP isoform (PMN-AGP), as determined by Western blotting and confirmed by fluorescence microscopy. The presence of AGP in bovine neutrophils has been confirmed by fluorescence immunocytometry. In addition, bovine neutrophils contain also a 42-45kDa isoform, which has the same MW as plasma-, liver-delivered, AGP. cDNA sequence of plasma- and PMN-AGP revealed that (i) the two proteins are products of the same gene; (ii) the differences in molecular weight are due do different post-translational modifications. This result was confirmed by deglycosylation of the two glycoforms. Exocytosis studies showed that isolated neutrophils exposed to several challengers, including Zymosan activated serum (ZAS) and phorbol 12-myristate 13-acetate (PMA), which mimic the inflammatory activation, released PMN-AGP as early as 15min. AGPs mRNA is physiologically expressed by mature resting neutrophils. Real-time PCR on LPS, ZAS and PMA challenged cells revealed that the level of expression apparently does not increase after inflammatory activation. Collectively, the findings reported in this paper proved that PMN-AGP: (i) is a hyperglycosylated glycoform of plasma AGP, (ii) is stored in granules, and (iii) is released by neutrophils in response to activation. Due to its anti-inflammatory activity, PMN-AGP may work as a fine tuning of the neutrophils functions in the inflammatory focus, i.e. it can reduce the damages caused by an excess of inflammatory response.

Hematologic, biochemical, and protein electrophoretic values in captive tawny owls (Strix aluco)

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.

Distribution of acute phase proteins in the bovine forestomachs and abomasum

Acute phase proteins (APPs) are produced mainly by the liver and their concentration is increased during the systemic inflammatory response. Expression of haptoglobin (Hp), serum amyloid A (SAA), lipopolysaccharide-binding protein (LBP) and α-1 acid glycoprotein (AGP) was determined in the mucosa of the normal bovine forestomachs and abomasum by qualitative and quantitative reverse transcriptase-PCR for mRNA and by Western blot analysis and immunohistochemistry for proteins. Although expression of SAA mRNA was evident in the forestomachs and abomasum, SAA protein was identified only in the abomasum. Expression of Hp protein was high in the forestomachs and abomasum, even though expression of Hp mRNA was negligible. The main site of expression of LBP mRNA was the omasum, whereas the highest protein expression was evident in the abomasum. AGP was expressed at low levels in the bovine forestomachs. Western blot analysis revealed a heterogeneous electrophoretic pattern for AGP, LBP and Hp, indicating that different stomach compartments produce isoforms that are different to those expressed by the liver. Expression of APPs by the bovine forestomachs and abomasum may contribute to regulation of the innate immune response against pathogens.

Lipopolysaccharide-binding protein: Local expression in bovine extrahepatic tissues

Lipopolysaccharide-binding protein (LBP) is an acute phase protein involved in host response to Gram-negative and Gram-positive pathogens. It is synthesized by hepatocytes and released as 60-65kDa glycoprotein in plasma. Little is known about the distribution of LBP in non-pathological bovine tissues. The aim of the present study was to investigate the extra hepatic expression of LBP in different bovine tissues by qualitative and quantitative real time (RT) PCR. The presence of the protein was also confirmed by immunohistochemistry using an anti-human LBP antibody preliminarily validated by cross-reactivity in bovine tissues. While a wide panel of organs and tissues was investigated, the attention was focused on the digestive tract and mammary gland. Moderate amount of mRNA was detected in most of the tissues involved in this study. Extra hepatic LBP mRNA expression was particularly high in parotid and submandibular salivary glands. Remarkably, LBP mRNA was found in rumen, reticulum and omasum. High expression was also found in the mammary gland. Intensity of protein staining paralleled mRNA expression in most tissues, with the exception of lung, ovary and thyroid gland. The presence of LBP throughout epithelial mucosal tissues is indicative of an important role of LBP in mucosal immunity at sites of bacterial exposure. These results suggest that ruminant forestomachs may mount a local acute phase reaction.

Differential expression and secretion of alpha1-acid glycoprotein in bovine milk.

alpha1-Acid glycoprotein (AGP) is a lipocalin that is produced mainly by the liver and secreted into plasma in response to infections and injuries. In this study, we evaluated AGP isoforms that can be detected in bovine milk. We found that milk-AGP content is made up of at least two isoform groups, a low MW group (44 kDa) that is produced in the mammary gland (MG-AGP), and a higher MW group (55-70 kDa), that is produced by somatic cells (SC-AGP). Identical SC-AGP isoforms can be found both in milk and blood PMN cells. Analysis of the mammary tissue cDNA showed that the sequence of the MG-AGP isoform is identical to that of plasma AGP. Each group contains several proteins with different MWs and different isoelectric points, as shown by 2D-electrophoresis. The glycosylation patterns of these isoforms were analysed by means of specific lectin binding, to evaluate the degree of sialylation, fucosylation and branching. The MG-AGP glycan pattern was identical to plasma AGP produced by the liver. Several differences were detected, however, between plasma and SC-AGP isoforms, the most evident being the strong degree of fucosylation and the elevated number of di-antennary glycans in SC-AGP. Immunohistochemistry showed that AGP is found in all tissues that make up the mammary gland, but that it is most likely produced for the main part by the alveoli.

Bovine alpha-1 acid glycoprotein can reduce the chemotaxis of bovine monocytes and modulate CD18 expression

The acute phase protein alpha(1)-acid glycoprotein (AGP--Orosomucoid) is a lipocalin with immunomodulatory functions. The present study provides evidence that the plasma glycoforms of AGP inhibit the migration of bovine monocytes in response to classical chemoattractants. The inhibition is specific, since neutrophils are apparently not affected. To investigate the molecular basis of this finding, the expression of the molecules mostly involved in chemotaxis, including CD18, CD11b and CD47 was studied. It was found that the incubation of activated monocytes with acute phase concentration of AGP (0.9 mg/mL) induces a down-regulation of CD18, and has no apparent influence on CD11b and CD47. RT-PCR expression studies on CD18, CD11b and CD47 mRNA revealed that AGP treatment does not modify the expression rate of these genes. Since AGP treatment is related to a down-regulation of CD18 on the surface of the monocytes, the authors suggest that one of its possible functions consists in specifically reducing the firm adhesion phase of bovine monocytes to the endothelium.

Effects of EPA and DHA on lipid droplet accumulation and mRNA abundance of PAT proteins in caprine monocytes.

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.