Hemagglutinin protein (HA) was considered to be the primary target for monoclonal antibody production. This protein not only plays an important role in viral infections, but can also be used to differentiate H5N1 virus from other influenza A viruses. Hence, for diagnostic and therapeutic applications, it is important to develop anti-HA monoclonal antibody (MAb) with high sensitivity, specificity, stability, and productivity. Nine unique Fab MAbs were generated from chimeric chicken/human Fab phage display library constructed from cDNA derived from chickens immunized with recombinant hemagglutinin protein constructed from H5N1 avian influenza virus (A/Vietnam/1203/04). The obtained Fab MAbs showed several characteristics for further optimization and development—three clones were highly specific to only H5N1 virus. This finding can be applied to the development of H5N1 diagnostic testing. Another clone showed neutralization activity that inhibited H5N1 influenza virus infection in Madin–Darby canine kidney (MDCK) cells. In addition, one clone showed strong reactivity with several of the influenza A virus subtypes tested. The conversion of this clone to whole IgG is a promising study for a cross-neutralization activity test.

Thirteen thirty-week-old layers (Lohman Silver) that showed lameness were necropsied. Sera were collected and bacterial isolation was performed. Gross examination revealed amyloid deposits in the joints. The lesion was limitedly present only in knee joints. Enterococcus faecalis was the predominant microorganism isolated. Tissues from joints, liver, spleen, and kidney were positive with anti-chicken AA antisera using EnvisionTM + Peroxidase. SAA levels were generally increased in amyloidotic sera using direct ELISA but relatively low compared to acute phase sera. Moreover, Western Blot analysis revealed a low signal of SAA. From these findings, it was concluded that the birds suffered from amyloid arthropathy.

The highly pathogenic avian influenza (HPAI) is a viral disease of poultry and is a zoonosis. The eighth segment of the viral genomic RNA encodes a nonstructural protein, NS1. NS1 is a viral regulatory protein responsible for virus virulence. This study, produced the 6xHis-tag fusion protein. The full length NS1 cDNA of A/Chicken/TH/KU14/04 (H5N1) was cloned into an expression vector, pQE80L. The recombinant NS1 was expressed in the E. coli strain DH5_ . The results showed that the polyhistidine-tagged NS1 fusion protein was successfully expressed in bacterial cells, but the fusion protein accumulated in the inclusion bodies. The fusion protein was isolated as either a native or denatured form and further purified by precipitation or immobilization on a nickel ion affinity column. Purification using affinity chromatography under denaturing conditions yielded greater amounts of purer NS1 protein. However, sufficient purity and concentration of the native NS1 protein was obtained after purification using a precipitation method. Both native and denatured, purified NS1 protein reacted specifically with the anti-NS1 chicken serum in a western blot assay. The results indicated that the NS1 protein may be useful for further applications.

In domestic brown layer fowl, reactive amyloidosis of internal organs, such as liver and spleen, and of the joints is a common disorder. In a variety of amyloid types including the AA-amyloid of the chicken, in addition to amyloid fibrils, proteoglycans and glycosaminoglycans (GAGs) are found on immunohistochemistry or after extraction. The aim of the present report is to study amyloid fibrils for the ultrastructural location of GAGs by cuprolinic blue staining and immunogold labeling. Rabbit antichicken AA antiserum was used for the immunogold labeling on conventionally embedded and cryoembedded liver tissue and revealed similar results. Therefore conventional blocks could be used for further analysis. Cuprolinic blue staining was performed on blocks of joint tissue in which clearly discernable rod-shaped glycoproteins were encountered in between collagen fibrils. Moreover, it appeared to stain larger deposits which might represent amyloid. Postlabeling with the immunogold method of the cuprolinic blue-stained tissue proved that cuprolinic blue positive fibrils represented AA-amyloid fibrils. Therefore, it was concluded that the GAGs which appeared to colocalize with the fibrillar microanatomy of amyloid, represent a structural part of the amyloid fibrils and that the avian amyloid fibrils may be considered as a pathological proteoglycan.

The future of acute phase proteins (APPs) in science is discussed in this paper. Many functions and associated pathological processes of APPs are unknown. Extrahepatic formation in local tissues needs attention. Local serum amyloid A (SAA) formation may be involved in deposition of AA-amyloid induced by conformational change of SAA resulting in amyloid formation, having tremendous food safety implications. Amyloidogenesis is enhanced in mouse fed beta pleated sheet-rich proteins. The local amyloid in joints of chicken and mammary corpora amylacea is discussed. Differences in glycosylation of glycoproteins among the APPs, as has been shown for α1-acid glycoprotein, have to be considered. More knowledge on the reactivity patterns may lead to implication of APPs in the diagnostics and staging of a disease. Calculation of an index from values of several acute phase variables increases the power of APPs in monitoring unhealthy individuals in animal populations. Vaccinations, just as infections in eliciting acute phase response seem to limit the profitability of vaccines because acute phase reactions are contraproductive in view of muscle anabolism. Interest is focused on amino acid patterns and vitamins in view of dietary nutrition effect on sick and convalescing animals. When inexpensive methodology such as liquid phase methods (nephelometry, turbidimetry) or protein array technology for rapid APPs measurement is available, APPs have a future in routine diagnostics. Specific groups of patients may be screened or populations monitored by using APPs.

In brown chicken with chronic inflammatory processes of the joints amyloid arthropathy easily develops. The amyloid has been shown to be of the AA type which is derived from serum amyloid A (SAA). The aim of the present study was to investigate whether fibroblast-like synoviocytes (FLS) originating from brown chicken and other chicken breeds express SAA mRNA and produce SAA protein. FLS were isolated from the knee joint synovium of healthy brown chickens, white chickens, and broilers. The absence of macrophages in FLS cultures was confirmed by assessment of the phagocytic capability and by immunohistochemistry. Additionally, cultured cells were identified by electron microscopy and immunohistochemical staining. Expression of SAA mRNA in normal and lipopolysaccharide (LPS)-stimulated cells was assessed by in situ hybridization, Northern blot analysis, reverse transcriptase-polymerase chain reaction (RT-PCR), Southern blot analysis and real-time quantitative PCR. SAA protein production was analyzed by Western blotting and ELISA. SAA mRNA was detected in unstimulated FLS isolated from the three different chicken breeds and more abundantly in those stimulated with LPS. However, SAA protein production was only detected in culture medium and cell lysate of LPS-stimulated FLS. Furthermore, FLS produced SAA in a concentration-dependent manner after stimulation with different amounts of LPS. The data suggest that during infection and inflammation chicken FLS may act as a source of articular SAA. This process may enhance development of amyloid from SAA in the joint.

Amyloidosis is a group of diseases characterized by the extracellular deposition of protein that contains non-branching, straight fibrils on electron microscopy (amyloid fibrils) that have a high content of beta-pleated sheet conformation. Various biochemically distinct proteins can undergo transformation into amyloid fibrils. The precursor protein of amyloid protein A (AA) is the acute phase protein serum amyloid A (SAA). The concentration of SAA in plasma increases up to 1000-fold within 24 to 48 h after trauma, inflammation or infection. Individuals with chronically increased SAA levels may develop AA amyloidosis. SAA has been divided into two groups according to the encoding genes and the source of protein production. These two groups are acute phase SAA (A-SAA) and constitutive SAA (C-SAA). Although the liver is the primary site of the synthesis of A-SAA and C-SAA, extrahepatic production of both SAAs has been observed in animal models and cell culture experiments of several mammalian species and chicken. The functions of A-SAA are thought to involve lipid metabolism, lipid transport, chemotaxis and regulation of the inflammatory process. There is growing evidence that extrahepatic A-SAA formation may play a crucial role in amyloidogenesis and enhances amyloid formation at the site of SAA production.

ABSTRACTTo investigate whether superantigen (SAG) from endogenous mouse mammary tumor virus functions as an immunogenic or a tumorigenic factor in tumor development, the BALB/c myeloma cell line FO was transfected with the SAG gene from the 3′Mtv-50long terminal repeat (LTR) open reading frame (ORF), the product of which was specific for Vβ6. All five transfectants expressingMtv-50LTR ORF mRNA showed stimulatory activity for Vβ6 T-cell hybridomas in vitro; this activity was inhibited by the addition of anti-Mtv-7monoclonal antibody (MAb) or anti-major histocompatibility complex class III-AdandI-EdMAb. All transfectants with the SAG gene grew more rapidly than did mock transfectants in BALB/c mice after subcutaneous inoculation, whereas all clones, including mock transfectants, grew equally well in athymic nude mice. A significant fraction of Vβ6 T cells selectively expressed activation markers, including CD44high, CD62Llow, and CD69high, and produced large amounts of interleukin 5 (IL-5) and IL-6 in BALB/c mice inoculated with transfectants. These results suggested that the expression of viral SAG enhances the tumorigenicity of a myeloma cell line through the stimulation of SAG-reactive T cells.

We previously reported that a mouse mammary tumor virus (MMTV(II-TES14)), encoding a superantigen specific for TCR V beta 14, can infect lymph node (LN) cells of mice in an I-E-independent manner, Here we examined the kinetics of cell types infected with exogenous MMTV in the draining LN after s.c. injection of II-TES milk containing MMTV(II-TES14), The infectivity was assessed in LN cells sorted into each cell subset by a semiquantitative analysis of MMTV provirus using PCR with a primer specific for MMTV(II-TES14). Only B cells in the LN were infected by the MMTV on day 6 after injection, but CD8(+) T cells and, to a lesser extent, CD4(+) T cells were also found to be detectably infected on day 14 after the injection of II-TES milk, Among the T cells we examined, V beta 8 T cells were most preferentially infected with MMTV, but no V beta 14 T cells specific for MMTV(II-TES14) superantigen were infected on day 14 after infection, The transfer of V beta 8 T cells sorted from mice injected with II-TES milk 14 days previously resulted in the deletion of CD4(+)V beta 14 T cells and in the MMTV infection of normal B6 mice, No MMTV infection of T cells occurred in IgM knockout mice, which lack a mature B cell compartment, These results suggest that MMTV surviving in B cells is transferred to V beta 8 T cells, which may play an important role in MMTV longevity.