Elemental imbalance elicited by arsenic and copper exposures leads to oxidative stress and immunotoxicity in chicken gizzard, activating the protective effects of heat shock proteins.

Arsenic (As) and copper (Cu) are ubiquitous pollutants that pose a threat to the environment. Our aim is to study the underlying mechanisms by which As and Cu act on the chicken gizzard. In order to detect ionic disorders in chicken gizzard under chronic treatment with As3+ and/or Cu2+ and whether they can induce oxidative damage as well as immune disorders, 30 mg/kg arsenic trioxide (As2O3) and/or 300 mg/kg copper sulfate (CuSO4) were added to the chicken's basal diet. After 12 weeks of exposure, trace elements were found to have significant interference, accompanied by damage to the antioxidant system. In addition, As3+ and/or Cu2+ activated the nuclear factor kappa B (NF-κB), inducing severe inflammation. At the same time, damaged structural integrity which might be caused by inflammation was discovered after hematoxylin and eosin (H&E) staining. Moreover, symbolic Th1/Th2 (Th, helper T cell) drift was also observed in treatment groups, meaning that immune function is left to be affected, and the increment in heat shock proteins may be a self-protective mechanism of gizzard. Interestingly, we found that the damage to the gizzard of chicken was aggravated in a time-dependent manner, and the combined exposure was more pathogenic than the single exposure, of which the mechanism needs further exploration. Together, this work helps move us toward a better understanding of the molecular mechanisms that mediate the interactions between Cu excess and As3+ exposures and possible health consequences in susceptible species.

Infection with an apathogenic fowl adenovirus serotype-1 strain (CELO) prevents adenoviral gizzard erosion in broilers.

Gizzard erosion in broilers due to an infection with virulent fowl adenovirus serotype 1 (FAdV-1) is an emerging disease. Although experimental studies were performed, a possible prevention strategy was not reported so far. The present study was set up to determine (i) a possible influence of birds' age at time of inoculation on the pathogenicity of a European FAdV-1 field strain (PA7127), (ii) the virulence of a apathogenic FAdV-1 strain (CELO), and (iii) its capability to protect SPF broilers from adenoviral gizzard erosion caused by the field virus. Oral infection of birds with PA7127 at 1-, 10- and 21-days of life, resulted in reduced weight gain compared to non-infected birds, with significance for birds infected at day-old. Independent of the birds' age at time of inoculation, clinical signs appearing approximately one week after challenge coincided with gizzard lesions. Birds infected exclusively with CELO at the first day of life did not show any clinical signs or pathological changes in the gizzard, confirming the apathogenicity of this European FAdV-1. A similar result was obtained for birds orally infected at the first day of life with CELO and challenged three weeks later with the pathogenic PA7127 strain. Therefore, complete protection of adenoviral gizzard erosion in broilers by vaccination of day-old birds could be demonstrated for the first time, although virus excretion was detected post challenge. Establishment of an amplification refractory mutation system quantitative PCR (ARMS-qPCR) facilitated the identification of the FAdV-1 strain and presence of challenges virus was confirmed in one sample.

Pathogenicity by parenteral injection of fowl adenovirus isolated from gizzard erosion and resistance to reinfection in adenoviral gizzard erosion in chickens.

The pathogenicity of a serotype-1 fowl adenovirus (FAV-99ZH), which causes adenoviral gizzard erosion by oral inoculation in chickens, was investigated in specific pathogen-free white leghorn chickens. In trial 1, 14 chickens were inoculated intravenously with the virus at 21 days of age and euthanatized for necropsy within 1-14 days of inoculation. Gizzard erosion was grossly observed from day 7 postinoculation (PI), and histologically, FAV-99ZH antigen-positive, basophilic intranuclear inclusion bodies were seen in the gizzard lesions from day 7 to 11 PI. Necrotizing pancreatitis, and cholecystitis and cholangitis associated with the inclusions were observed from day 3 to 14 PI (pancreatitis) and from day 5 to 9 PI (cholecystitis and cholangitis), respectively. The inclusions were also observed in the epithelial cells of the cecal tonsils from day 3 to 5 PI. The virus was recovered from samples of the lesions. It was revealed that FAV-99ZH causes not only gizzard erosion but also pancreatitis, cholecystitis, and cholangitis by intravenous inoculation in chickens. In trial 2, 10 chickens were inoculated orally with the virus twice, at 13 and 36 days of age, and euthanatized for necropsy within 4-17 days after reinfection. Macroscopically, focal gizzard lesions were observed; however, neither necrosis nor inclusions were observed by microscopy. Moreover, FAV was not recovered from the gizzard or rectum of any of the chickens at necropsy. This suggests that the gizzard lesions occurred as a result of the primary infection, and that the chickens were able to resist reinfection.

Characterization of monoclonal antibodies to chicken gizzard talin.

Eleven monoclonal antibodies against chicken gizzard talin, a major focal adhesion protein, have been produced. In order to determine the degree of homology between talin molecules from different sources, these antibodies were used to immunolocalize talin in five different vertebrate species. Three representative talin antibodies are described in this report.

Inhibition of energy-dependent Ca2+ accumulation in fibroblasts by smooth muscle antibodies.

An antibody prepared against smooth muscle myosin interferes with active Ca2+ accumulation of fibroblasts. This provides further evidence for the existence of myosin at the cell surface.

Striated myofibrils in anti-myosin stained, isolated chicken gizzard smooth muscle cells.

Highly purified chicken gizzard myosin was used to induce antibody production in rabbits. The IgG fraction was separated from the antisera and coupled to fluorescein isothiocyanate (FITC). Specific antibody (AGM) was isolated from the IgG fraction by affinity purification. Comparisons of the specificity of IgG and AGM for chicken smooth muscle myosin revealed a much greater specificity by AGM. Staining with IgG led to an apparent cross-reactivity with guinea pig smooth muscles which was not seen with AGM staining. Therefore, staining of cells for localization of myosin was performed with AGM. Isolated cells were obtained from chicken gizzards either by collagenase digestion or by agitation of glycerinated pieces. Stained cells and cell fragments revealed the presence of myofibrils as structural units with diameters of about 1.0 micrometer. Stained myofibrils occasionally displayed regular banding patterns with a repeating period of about 1.5 +/- 0.2 micrometer. The presence of banded myofibrils in non-cultured cells shows that the organization of the contractile material is similar to that previously reported for cultured cells by Gröschel-Stewart.

Selective binding of antibody against gizzard 10-nm filaments to different cell types in myogenic cultures.

Antibody against the intermediate-sized filaments from gizzard smooth muscle was used to determine the presence or absence of reacting 10-nm filaments in different cell types. The antibody against gizzard 10-nm filaments reacted with filaments in cultured smooth muscle cells, skeletal myotubes and postmitotic skeletal myoblasts. It did not bind to the 10-nm filaments present in replicating presumptive myoblasts and fibroblasts, or the 10-nm filaments in spinal ganglion cells.

Anti-actomyosin. Influence on adhesive behaviour of eukaryotic cells and of Cuvierian tubules.

The effect of antisera against chicken gizzard smooth-muscle actomyosin and against pectoralis striated-muscle actomyosin on adhesive behaviour of eukaryotic cells (from sea urchin embryos and from a silicious sponge) and of Cuvierian tubules has been studied. The results with sea urchin cells, which require divalent cations for aggregation, showed that antiserum to chicken gizzard smooth-muscle actomyosin inhibited reaggreagation of trypsin-treated cells better than mechanically dissociated cells, while anti-chicken pectoralis striated-muscle had no effect. Primary reaggreagation of trypsin-dissociated sponge cells, in the presence of calcium and magnesium, is also inhibitable by anti-gizzard smooth-muscle but not by anti-pectoralis straited muscle. Anti-gizzard smooth-muscle had no effect on secondary reaggregation of sponge cells mediated by a soluble aggregation factor. Anti-gizzard smooth-muscle inhibited Cuvierian tubule adhesion.

Antibody to myosin: the specific visualization of myosin-containing filaments in nonmuscle cells.

Myosin in human, rat, mouse, and chicken fibroblasts was localized by indirect immunofluorescence microscopy using antibodies prepared in rabbits against highly purified chicken gizzard myosin. Filaments containing myosin span the interior of the cells and are often parallel to each other. The majority of the fibers are concentrated toward the adhesive side of the cell. Most of the myosin-containing filaments show "interruptions" or "striations." From a comparison of these fibers in fluorescence and phase microscopy and from previous results on actin-containing fibers, we conclude that at least some of the cytoplasmic myosin can be found in the actin-containing fibers, which themselves have been shown to be very similar or identical to the microfilament bundles. The occurrence of both myosin and actin in the microfilament bundles provides a basis for the motility and contractility of the cell.