LPS-induced NLRP3 gene expression in chicken.

Background: NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) is a cytosolic sensor that detects many microbial pathogen-associated molecular patterns and damage-associated molecular patterns. It works as a pro-inflammatory cytokine that is encoded in the NLRP3 gene. This protein is profoundly expressed in macrophage and other innate immune system cells, and participates in the assembly of NLRP3 inflammasome. NLRP3 inflammasome activates caspase-1, which in turn renders the inactive precursors of the pro-inflammatory cytokines, the interleukin-1ß (IL-1ß) and the IL-18, into active forms. This cytokine may trigger many inflammatory responses and cell signaling pathways as well. Little is known about this cytokine in chickens, especially its role in vaccines or induced immune responses. Aim: In this article, we sought to determine the presence of this gene mRNA in selected organs of male and female commercial, brown leghorn egg-type chickens. In addition, we sought to determine this gene's potential expression in these organs upon stimulation. Methods: Using real-time polymerase chain reaction, first we tested the presence of the NLRP3 gene in the chickens. Second, the levels and the time course of NLRP3 gene expression have been tested after stimulation with bacterial lipopolysaccharide (LPS) for 12, 24, and 48 hours postinoculation (pi). One-hundred twenty, day-old males and females egg-type brown leghorn chickens were used for the study. Results: Our results showed that the gene mRNA is actually present in chickens solely. Also, there were no significant differences in the density of the expression and the distribution of the expression of the NLRP3 between male and female chickens and among different organs. Upon stimulation with LPS administration, however, there were marked elevations in the gene expression rates in small intestine, large intestine, gizzard, liver, lung, spleen, and Peyer's patches 12 hours pi. This elevation continued to elevate 24 hours pi. However, the significance of the expression was only recorded in the small intestine, large intestine, and with less significance, in Peyer's patches and spleen. This elevation in expression subsided and almost returned to normal within these organs 48 hours pi. Conclusion: The results suggest that there were no significant differences in the NLRP3 gene expression between male and female. Upon stimulation, the course of the gene expression showed a time-dependent response. First, the dominance in the NLRP3 gene mRNA expression was in the small intestine and gizzard. Similarly, but less profoundly, the large intestine, Peyer's patches, and spleen expressed NLRP3 mRNA 12 and 24 hours pi with LPS. Secondary immune organs, lungs, small intestine, and large intestine expressed the NLRP3 mRNA significantly 24 hours pi. All the levels had diminished and almost returned to normal 48 hours pi.

Single-stranded DNA aptamer-based rolling circle amplification as anti-chicken Salmonella bacteriostatic.

Background and Aim: Salmonella is a major foodborne pathogen in the poultry industry, wherein the control measures may include sanitation and antibacterial and vaccines. However, there have been severe global restrictions on using anti-Salmonella antibacterial agents in livestock. This situation, along with rapidly increasing drug-resistant bacterial species, has led to the exploration of unconventional methods to control Salmonella infection in poultry. In recent years, selection techniques of promising DNA aptamers have begun to permeate several medical branches, resulting in the development of numerous anti-Salmonella DNA aptamers, most of which are used as sensing molecules for diagnostic purposes. These DNA aptamers have been demonstrated to interfere with bacterial growth, multiplication, and viability. Aptamers formed in rolling circle amplification products (RCA-p) could improve the potential action of aptamer interference with bacteria. This study aimed to test the use of single-stranded DNA aptamers in the form of RCA-p as a bacteriostatic to Salmonella in vitro. Materials and Methods: Salmonella Typhimurium and Salmonella Enteritidis isolates were subjected to the action of anti-ST and anti-SE DNA aptamers in the form of RCA-p. Each isolate was grown on MacConkey and Luria-Bertani agar media separately in different concentrations in the presence or absence of the cognate RCA-p. Results: The anti-Salmonella species DNA aptamer-based RCA-p were capable of reducing bacterial growth to significant levels in vitro. Conclusion: We describe a potential solution for the rapidly developing drug resistance of several bacterial species. Our findings suggested that the use of non-toxic, non-immunogenic, and low-cost DNA aptamers targeting Salmonella in the form of RCA-p could inhibit the bacterial growth rate. Unlike polymerase chain reaction, RCA yields tandem repeats of single-stranded DNA at isothermal conditions, which would increase the probability of receptor-ligand clustering and increase affinity. Furthermore, as our RCA template was bivalent with two DNA aptamer sequences, we could target multiple sites or antigens on a bacterial cell.

Development of lymphocyte subpopulations in local breed chickens.

BACKGROUND AND AIM: Local breeds of chicken are known to have relatively higher disease resistance to many endemic diseases and diseases that are highly virulent in commercial chickens. This study aimed to address the lymphocyte subpopulations in three constitutive immune system organs (thymus, bursa of Fabricius, and spleen) in 30, 8-week-old, male local breed chickens. MATERIALS AND METHODS: The T (CD3+) and B lymphocytes (Bu-1+) were identified through one-color, direct immunofluorescent staining of the thymus, bursa, and spleen lymphocytes. Likewise, two-color, direct immunofluorescent staining was performed to identify the CD4- and/or CD8-defined T lymphocytes. The proportions of T and B lymphocytes and CD4- and/or CD8 defined chicken lymphocyte subsets in lymphoid suspensions prepared from the thymus, bursa, and spleen were determined by flow cytometry. RESULTS: CD3+ cells, particularly those positive for CD4+CD8-, were dominant in the thymus, whereas cells expressing the Bu-1 marker were predominant in the bursa of Fabricius. The proportion of T and B cells was almost equal in the spleen, with more cells expressing the CD4-CD8+ marker in the red pulp. CONCLUSION: These findings indicate that local breeds of chicken could serve as a reliable model for studying the immune system of commercial light chicken breeds, due to the similarity in the presence and the distribution of the immune cells.

Studies on immunity and immunopathogenesis of parrot bornaviral disease in cockatiels.

We have demonstrated that vaccination of cockatiels (Nymphicus hollandicus) with killed parrot bornavirus (PaBV) plus recombinant PaBV-4 nucleoprotein (N) in alum was protective against disease in birds challenged with a virulent bornavirus isolate (PaBV-2). Unvaccinated birds, as well as birds vaccinated after challenge, developed gross and histologic lesions typical of proventricular dilatation disease (PDD). There was no evidence that vaccination either before or after challenge made the infection more severe. Birds vaccinated prior to challenge largely remained free of disease, despite the persistence of the virus in many organs. Similar results were obtained when recombinant N, in alum, was used for vaccination. In some rodent models, Borna disease is immune mediated thus we did an additional study whereby cyclosporine A was administered to unvaccinated birds starting 1day prior to challenge. This treatment also conferred complete protection from disease, but not infection.