Roles of intestinal intraepithelial lymphocytes in specific-pathogen-free chickens infected with lentogenic and velogenic newcastle disease virus strains

There are increasing reports of Newcastle disease virus (NDV) shedding in vaccinated poultry flocks. Two intraepithelial lymphocytes (IEL), the Natural Killer (NK) cells and T cells, play a critical role in the control of virus shedding. The main objective of this study is to investigate the role of these cells in specific-pathogen-free (SPF) chickens following inoculation with NDV lentogenic strain LaSota, and/or velogenic strains, genotypes VII and VIII. The SPF chickens were divided into groups of inoculated with lentogenic strain (LaSota); genotype VII (GVII); genotype VIII (GVIII); lentogenic and challenged with genotype VII (LSGVII); lentogenic and challenged with genotype VIII (LSGVIII); and the group of uninfected control. Immunophenotyping of NK and T cells subtypes was conducted using a flow cytometry. Furthermore, CD3-/CD25+/CD45+IEL NK cell was purified, and the expression profile of immune and apoptosis-related genes was quantified using Reverse Transcriptase Quantitative Polymerase Chain Reaction (RTqPCR). Virus shedding was then measured using RT-qPCR. Data were analysed using Two-way Analysis of Variance (ANOVA). The percentage of CD3+ cells showed a decreasing pattern in GVII and GVIII challenged groups compared with LSGVII and LSGVIII. Similarly, a steady decrease of CD3+/CD4+ cells in GVII and GVIII challenged groups was seen as the infection progressed. LSGVII and LSGVIII challenged groups, however, showed a statistically significant increase of these cells. A similar finding was obtained with CD3+/CD8+ cells. Among all the experimental groups, the highest virus shedding occurred at 60 hrs postchallenge with GVII. There was a strong negative correlation between an increase in GVII shedding and a decrease in CD25+IEL NK cells. Following inoculation of LGVII, there was a statistically significant decrease in virus shedding in all the time points; however, there was no significant correlation between GVII shedding and CD25+IEL NK cells. There was no statistically significant difference in GVIII shedding between the time points of the GVIII challenge group and its corresponding LSGVIII group. There was a strong positive correlation between CD25+IEL NK cells and GVIII shedding in a LSGVIII challenged group. The expression profiles of CD69, FasL and granzyme A, NK-lysin, and IFN-γ were generally upregulated in LSGVII and LSGVIII challenged groups. In contrast, B-NK, was downregulated. In NDV GVII and GVIII challenged groups, however, B-NK was upregulated, whereas the remaining receptors were generally downregulated except for CHIR- AB1. Taking together, the findings of this study showed that CD25+IEL NK cell showed a strong negative correlation with GVII shedding, but no correlation with GVIII shedding. Furthermore, there was a moderate negative correlation between CD25+IEL NK cells and GVII shedding in LSGVII challenge group and a strong positive correlation between CD25+IEL NK and GVIII shedding in LSGVIII challenged chickens. The CD3- /CD25+/CD45+IEL NK cells in the LSGVII and LSGVIII showed enhanced NK cell activity through upregulation of the activating receptors, peptides and interferon-γ. In contrast, the function of CD3-/CD25+/CD45+IEL NK was downgraded through upregulation of inhibitory receptors following inoculation of GVII and GVIII NDV. In addition, enriched CD3-/CD25+/CD45+IEL NK cells may use both receptor and granules-mediated apoptosis pathways in killing virus-infected cells.

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