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Haemorrhagic enteritis (HE) is a viral disease affecting intestinal integrity and barrier function in turkey (Meleagris gallopavo) and resulting in a significant economic loss. Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra (SWATH-MS) was applied to identify crucial proteins involved in HE infection. A total of 938 proteins were identified and used to generate a reference library for SWATH-MS analysis. In total, 523 proteins were reliably quantified, and 64 proteins were found to be differentially expressed, including 49 up-regulated and 15 down-regulated proteins between healthy and HE-affected intestinal mucosa. Functional analysis suggested that these proteins were involved in the following categories of cellular pathways and metabolisms: 1) energy pathways; 2) intestine lipid and amino acid metabolism; 3) oxidative stress; 4) intestinal immune response. Major findings of this study demonstrated that natural HE infection is related to the changes in abundance of several proteins involved in cell-intrinsic immune defense against viral invasion, systemic inflammation, modulation of excessive inflammation, B and T cell development and function and antigen presentation. mRNA quantitative expression demonstrated that most of the proteins involved in innate immunity that were found to be differentially abundant were produced by intestinal mucosa, suggesting its direct involvement in immune defences against HE infection.

Acute phase proteins (APP) are plasma proteins that can modify their expression in response to inflammation caused by tissue injury, infections, immunological disorders or stress. Although APP are produced mainly in liver, extrahepatic production has also been described. As a prerequisite to get insight the expression of APP in chicken during diseases, this study investigated the presence of five APP, including alpha1-acid glycoprotein (AGP), Serum Amyloid A (SAA), PIT54, C-Reactive protein (CRP) and Ovotransferrin (OVT) in twenty tissues collected from healthy chicken (Gallus gallus) by quantitative Real Time PCR and immunohistochemistry. As expected, APP gene abundance was higher in liver compared with other tissues. The mRNA coding for CRP, OVT and SAA was detected in all analyzed tissues with a higher expression in gastrointestinal tract, respiratory and lymphatic samples. SAA expression was particularly high in cecal tonsil, lung, spleen and Meckel's diverticulum, whereas OVT in lung, bursa of Fabricius and pancreas. AGP and PIT54 mRNA expression were detected in all tissues but at negligible levels. Immunohistochemical expression of AGP and OVT was variably detected in different organs, being identified in endothelium of every tissue. Positive cells were present in the epithelium of the mucosal layer of gastrointestinal tract and kidney. Lung and central nervous system stained for both proteins. No positive staining was detected in lymphoid tissues and muscle. These results suggest that most tissues can express different amount of APP even in healthy conditions and are therefore capable to mount a local acute phase reaction.

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 TIR8 receptor (also called SIGIRR) is an orphan member of the TIR superfamily. Its function is still elusive, but it is believed to trigger a negative pathway of regulation of the Toll-like/IL-1 receptor system, crucial for modulating inflammation in gastrointestinal (GI) tract and in other tissues (lung and kidney). The expression pattern of TIR8 in bovine tissues is unknown. Given the importance of GI diseases in cattle, the aim of this investigation was to study the distribution of TIR8 in a wide panel of non-pathologic tissues and organs. TIR8 expression was assessed by Northern blot analysis and further confirmed and comparatively quantified by qualitative and quantitative (Real-Time) PCR. The possible presence of tissue-specific isoforms was determined by Western blot immunodetection, using an anti-human TIR8 polyclonal antibody previously validated in bovine tissues. Similarly to humans and mice, bovine TIR8 was found in the GI tract and kidney. Expression of TIR8 mRNA was also detected in lymph nodes, thymus and thyroid gland. Interestingly, several isoforms of bovine TIR8 were detected in the same organs, suggesting the occurrence of different post-translational processings.

The present study was carried out in order to investigate the systemic and local expression of the acute-phase protein alpha(1)-acid glycoprotein (AGP, Orosomucoid) during Caprine Arthritis-Encephalitis Virus (CAEV) natural infection. The aminoacid sequence of goat AGP (gAGP), which was unknown, was determined by cDNA sequencing of the gene. AGP serum concentration was analyzed from 40 healthy and 36 CAEV-induced arthritis-affected goats. The mean concentration of AGP in healthy goats was of 219.8 microg/ml (+/-178.6 s.d.) and did not statistically differ from that of arthritis-affected goats (157 microg/ml, +/-137.8 s.d.). In a second set of experiments, AGP was purified to homogeneity from the serum of healthy and unhealthy goats, and the glycan pattern modifications were analyzed by means of specific binding with lectins. In particular, branching, fucosylation and alpha(1-6)- and alpha(1-3)-linked sialylation were analyzed. Goat AGP is not fucosylated in neither physiological nor pathological status. On the contrary, both major (branching) and minor (sialylation) microheterogeneities increase during arthritis. Finally, the possible local synovial origin of gAGP was determined by means of in vitro expression studies (real-time PCR) which used goat synovial membrane (GSM) as cellular model. It was found that gAGP's mRNA can be constitutively produced by GSM cells, but real-time PCR experiments revealed that the expression of AGP was not influenced by in vitro infection with CAEV.

The present study was designed to investigate the capability of bovine neutrophil granulocytes to produce the minor acute phase protein α 1 -acid glycoprotein (AGP, Orososmucoid). Bovine neutrophils contain a high MW (50–60 kDa) 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 42–45 kDa 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 15 min. AGP's 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.