Antimicrobial resistance (AMR) is a global health challenge and antimicrobial use (AMU) in the livestock sector has been considered as one of the contributing factors towards the development of AMR in bacteria. This study summarizes the results of a point prevalence survey conducted to monitor farm-level AMU in commercial broiler chicken farms in Punjab and Khyber Pakhtunkhwa (KPK) provinces of Pakistan. A cross-sectional study was conducted to quantify AMU and to check seasonal variations of AMU in 12 commercial broiler chicken farms (six from each province) during the summer and winter seasons of the year 2020–2021. AMU was recorded using three AMU metrics: kg, mg per population correction unit (mg/PCU), and mg/kg of final flock weight. A total of 22 antimicrobial drugs (348.59 kg) were used for therapeutic or prophylactic purposes in surveyed broiler chicken farms. The total combined AMU for all the broiler chicken farms was 462.57 mg/PCU. The use of most of the antimicrobials increased during winter flocks compared to summer. The top three antimicrobial drugs used during the summer were neomycin (111.39 mg/PCU), doxycycline (91.91 mg/PCU), and tilmicosin (77.22 mg/PCU), whereas doxycycline (196.81 mg/PCU), neomycin (136.74 mg/PCU), and amoxicillin (115.04 mg/PCU) during the winter. Overall, 60% of the antibiotics used in broiler chicken were critically important antimicrobial classes (CIA) for human medicine as characterized by the World Health Organization. Our findings showed high AMU in broiler chicken production and a call for urgent actions to regulate CIA use in food animals in Pakistan. This baseline survey is critical for the design and implementation of a subsequent national level AMU surveys that can include additional farming types, animals’ species, and geographical locations over a longer period of time.

The Cytosine–phosphate–guanosine (CpG) motif, which is specifically recognized intracellularly by dendritic cells (DCs), plays a crucial role in regulating the innate immune response. MicroRNAs (miRNAs) can strongly influence the antigen-presenting ability of DCs. In this study, we examine the action of miRNAs on CpG-stimulated and control DCs, as well as their effect on cyclic guanosine monophosphate-adenosine monophosphate (GMP–AMP) synthase (cGAS) and the stimulator of interferon genes (STING) signal pathway. Firstly, we selected miRNAs (miR-29a and miR-378b) based on expression in CpG-stimulated mouse bone marrow-derived dendritic cells (BMDCs). Secondly, we investigated the functions of miR-29a and miR-378b on CpG-stimulated and unstimulated BMDCs. The results showed that miR-29a and miR-378b increased expression of both the immunoregulatory DC surface markers (CD86 and CD40) and the immunosuppressive molecule CD273 by DCs. Thirdly, cytokine detection revealed that both miR-29a and miR-378b enhanced interferon-β (IFN-β) expression while suppressing tumor necrosis factor-α (TNF-α) production. Finally, our results suggest that miR-378b can bind TANK-binding kinase binding protein 1 (TBKBP1) to activate the cGAS/STING signaling pathway. By contrast, miR-29a targeted interferon regulatory factor 7 (IRF7) and promoted the expression of STING. Together, our results provide insight into the molecular mechanism of miRNA induction by CpG to regulate DC function.

Background: The PD-1/PD-L1 pathway is an inhibitory signaling pathway that maintains the balance between the immune response and immunotolerance, and its overactivation in cancer and viral infections inhibits T cell function. The target cells of various viruses, microvascular endothelial cells (MECs) have been shown to be key regulatory points in immune regulation and virion diffusion in vivo during infection with multiple influenza virus subtypes. Furthermore, avian influenza virus (AIV) infection can induce immunosuppression by causing imbalances in immune responses and immune organ damage. Thus, the aim of this study was to investigate whether the H9N2 virus inhibited the immune function of T cells that migrated across MECs by upregulating PD-L1 expression on MECs. Methods: The susceptibility of rat pulmonary microvascular endothelial cells (RPMECs) to the H9N2 virus was evaluated by a plaque-forming assay and immunofluorescence staining. Then, we quantified the mRNA and protein levels of PD-L1 in RPMECs induced by H9N2 virus infection using quantitative real-time PCR and flow cytometry. The interaction between the activated T cells and RPMECs infected with the H9N2 virus was revealed using a coculture system. The effect of endothelial-derived PD-L1 on T cell function was investigated by using ELISA and flow cytometry with or without a PD-L1-specific antibody. Results: Surface staining and the plaque-forming assay showed that the H9N2 virus infected and replicated in RPMECs. Both the PD-L1 mRNA level and PD-L1 protein level were upregulated in RPMECs infected with the H9N2 virus. H9N2 virus-induced PD-L1 expression significantly reduced the secretions of IL-2, IFN-γ and granzyme B and perforin expression in T cells. The above data were significantly increased after treatment with an anti-PD-L1 antibody, confirming the above mentioned findings. In addition, the induction of PD-L1 expression decreased the proliferative capacity of the cocultured T cells but did not affect the apoptosis rate of T cells. Conclusions: Taken together, the results suggest that the H9N2 virus is able to inhibit the T cell immune response by upregulating PD-L1 expression in pulmonary microvascular endothelial cells.

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