In recent years, emerging strains of pathogenic arthrogenic avian reovirus (ARV) have become a challenge to the chicken industry across USA and Canada causing significant economic impact. In this study, we characterized emerging variant ARV strains and examined their genetic and antigenic relationship with reference strains. We isolated 37 emerging variant ARV strains from tendons of broiler chickens with clinical cases of arthritis/tenosynovitis at commercial farms in Saskatchewan, Canada. Viral characterization using immunocytochemistry, gold-immunolabeling and electron microscopy revealed distinct features characteristic of ARV. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses of the viral Sigma C gene revealed genetic heterogeneity between the field isolates. On phylogenetic analyses, the Sigma C amino acid sequences of the isolates were clustered into four distinct genotypic groups. These ARV field strains were genetically diverse and quite distant from the vaccine and vaccine related field strains. Antibodies produced against a commercial Reo 2177^®vaccine did not neutralize these variants. Moreover, structure based analysis of the Sigma C protein revealed significant antigenic variability between the cluster groups and the vaccine strains. To the best of our knowledge, this is the first report on the genetic, phenotypic and antigenic characterization of emerging ARVs in Canada.

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) induce innate immunity against bacterial infections. Despite recent advances, how CpG-ODN alone protects against bacterial infections remained elusive. Here, we report for the first time, to our knowledge, that CpG-ODN orchestrates anti-microbial protective immunity by inducing a rapid enrichment of various immune compartments in chickens. In this study, eighteen-day-old embryonated eggs were injected with either 50 µg of CpG-ODN or saline (~n = 90 per group). In the first experiment, four days after CpG-ODN treatment, chicks were challenged subcutaneously with a virulent strain ofEscherichia coli(E.coli) and mortality was monitored for 8 days. We found significant protection, and reduced clinical scores in CpG-ODN treated chicks. To gain insights into mechanisms of protection induced by CpG-ODN, first we investigated cytokine expression kinetics elicited by CpG-ODN. The spleen and lung were collected from embryos or chicks (n = 3–4 per group) at 10 time points post-CpG-ODN inoculation. Multiplex gene analysis (interleukin (IL)-1, IL-4, IL-6, IL-10, IL-18, interferon (IFN)-γ, IFN-α, and lipopolysaccharide induced tumor necrosis factor (LITAF), revealed a significantly higher expression of pro-inflammatory cytokines following CpG-ODN treatment compared to the saline controls. In our study, LITAF stands out in the cytokine profiles of spleen and lungs, underscoring its role in CpG-ODN-induced protection. The third experiment was designed to examine the effects of CpG-ODN on immune cell populations in spleen, lungs, and thymus. Flow cytometry analysis was conducted at 24, 48 and 72 hrs (thymus only collected at 72 hr) after CpG-ODN administration to examine the changes in CD4⁺and CD8⁺T-cell subsets, monocyte/macrophage cell populations and their expression of maturation markers (CD40 and CD86). Flow cytometry data indicated a significant enrichment of macrophages, CD4⁺and CD8⁺T-cell subsets in both spleen and lungs of CpG-ODN treated embryos and chicks. Macrophages in spleen and lungs showed an upregulation of CD40 but not CD86, whereas thymocytes revealed significantly high CD4 and CD8 expression. Overall, the present study has demonstrated that CpG-ODN provides protection in neonatal chicks againstE.coliinfection not only by eliciting cytokine responses and stimulating immune cells but also through enriching immunological niches in spleen and lungs.