Establishment of a novel probe-based RT-qPCR approach for detection and quantification of tight junctions reveals age-related changes in the gut barriers of broiler chickens

Abstract

Tight junctions (TJs) play a dominant role in gut barrier formation, therefore, resolving the structures of TJs in any animal species is crucial but of major importance in fast growing broilers. They are regulated in molecular composition, ultrastructure and function by intracellular proteins and the cytoskeleton. TJ proteins are classified according to their function into barrier-forming, scaffolding and pore-forming types with deductible consequences for permeability. In spite of their importance for gut health and its integrity limited studies have investigated the TJs in chickens, including the comprehensive evaluation of TJs molecular composition and function in the chicken gut. In the actual study sequence-specific probes to target different TJ genes (claudin 1, 3, 5, 7, 10, 19, zonula occludens 1 (ZO1), occludin (OCLN) and tricellulin (MD2)) were designed and probe-based RT-qPCRs were newly developed. Claudin (CLDN) 1, 5, ZO1 and CLDN 3, 7, MD2 were engulfed in multiplex RT-qPCRs, minimizing the number of separate reactions and enabling robust testing of many samples. All RT-qPCRs were standardized for chicken jejunum and caecum samples, which enabled specific detection and quantification of the gene expression. Furthermore, the newly established protocols were used to investigate the age developmental changes in the TJs of broiler chickens from 1–35 days of age in the same organ samples. Results revealed a significant increase in mRNA expression between 14 and 21days of age of all tested TJs in jejunum. However, in caecum, mRNA expression of some TJs decreased after 1 day of age whereas some TJs mRNA remained constant till 35 days of age. Taken together, determining the segment-specific changes in the expression of TJ- proteins by RT-qPCR provides a deeper understanding of the molecular mechanisms underpinning pathophysiological changes in the gut of broiler chickens with various etiologies.

Enzymatically treated yeast bolstered growth performance of broiler chicks from young broiler breeders linked to improved indices of intestinal function, integrity, and immunity

“…Tumor necrosis factor ( TNFα ) expression was quadratically reduced by yeast perhaps linked to yeast components activation of phagocytes macrophages and monocytes ( Silva et al, 2009 ; Eom et al, 2021 ). Yeast increased occludin expression an indication of role in gut permeability ( Shen and Turner, 2006 ; von Buchholz et al, 2021 ). Physiological oxidative stress in broiler chickens is indicated by superoxide dismutase (SOD1).…”

Supplemental magnolol or honokiol attenuates adverse effects in broilers infected with Salmonella pullorum by modulating mucosal gene expression and the gut microbiota

“…Consistently, magnolol and honokiol effectively alleviated the damage to intestinal villi caused by S. pullorum . The genes claudin-1 ( CLDN1 ) and claudin-5 ( CLDN5 ), key component of tight junction protein genes, play important roles in gut barrier formation [ 25 ]. Reduced gene expression of tight junction proteins is usually associated with pathogenic challenges and pathological conditions [ 26 ].…”

Influence of heat stress on intestinal integrity and the caecal microbiota during Enterococcus cecorum infection in broilers

“…RNA was isolated from the intestinal tissue using the Analytik Jena RNA Mini Kit 2.0 (Analytik Jena AG) in accordance with the manufacturer's instructions, and RNA was stored at −80 °C until further use. Primers and probes were designed exactly as described by von Buchholz et al [42], and RT-qPCR was performed with some minor modifications. The Luna ® Universal Probe One-Step RT-qPCR Kit (New England Biolabs GmbH, Frankfurt, Germany) and QuantStudio 3 Real-Time-PCR-System (Thermo Fisher Scientific Inc., Wilmington, NC, USA) were used for RT-qPCR, and the setup was adapted to the given conditions.…”