RNA-seq reveals downregulated osteochondral genes potentially related to tibia bacterial chondronecrosis with osteomyelitis in broilers

Abstract

Background Bacterial chondronecrosis with osteomyelitis (BCO) develops in the growth plate (GP) of the proximal femur and tibia and is initiated by damage to the less mineralized chondrocytes followed by colonization of opportunistic bacteria. This condition affects approximately 1% of all birds housed, being considered one of the major causes of lameness in fast growing broilers. Although several studies have been previously performed aiming to understand its pathogenesis, the molecular mechanisms involved with BCO remains to be elucidated. Therefore, this study aimed to generate a profile of global differential gene expression involved with BCO in the tibia of commercial broilers, through RNA sequencing analysis to identity genes and molecular pathways involved with BCO in chickens. Results Our data showed 192 differentially expressed (DE) genes: 63 upregulated and 129 downregulated in the GP of the tibia proximal epiphysis of BCO-affected broilers. Using all DE genes, six Biological Processes (BP) were associated with bone development (connective tissue development, cartilage development, skeletal system development, organ morphogenesis, system development and skeletal system morphogenesis). The analyses of the upregulated genes did not indicate any significant BP (FDR < 0.05). However, with the downregulated genes, the same BP were identified when using all DE genes in the analysis, with a total of 26 coding genes explaining BCO in the tibia: ACAN, ALDH1A2, CDH7, CHAD, CHADL, COL11A1, COMP, CSGALNACT1, CYR61, FRZB, GAL3ST1, HAPLN1, IHH, KIF26B, LECT1, LPPR1, PDE6B, RBP4A, SERINC5, SFRP1, SOX8, SOX9, TENM2, THBS1, UCHL1 and WFIKKN2. In addition, seven transcription factors were also associated to BCO: NFATC2, MAFB, HIF1A-ARNT, EWSR1-FLI1, NFIC, TCF3 and NF-KAPPAB. Conclusions Our data show that osteochondral downregulated genes are potential molecular causes of BCO in broilers, and the bacterial process seems to be, in fact, a secondary condition. Sixteen genes responsible for bone and cartilage formation were downregulated in BCO-affected broilers being strong candidate genes to trigger this disorder.

Downregulation of growth plate genes involved with the onset of femoral head separation in young broilers

“…When looking at the 12 exclusively DE genes between normal and FHS-affected broilers at 21 days of age ( SHISA3, FNDC1, ANGPTL7, LEPR, OXTR, COL16A1, BOC, E NSGALG00000049529, RASD2, GDF10 ENSGALG00000045154, and THSD7B) , they were mainly related to WNT and FGF signaling ( Amanatullah et al, 2014 ), bone metabolism ( Nilsson et al, 2007 ; Di Benedetto et al, 2014 ; Lui et al, 2018 ), angiogenesis ( Shang et al, 2015 ; Huang et al, 2021 ), chondrogenesis ( Sekiya et al, 2002 ; Paradise et al, 2018 ) and Hedgehog signaling pathway ( Kavran et al, 2010 ; Xavier et al, 2016 ). In this way, it is possible to highlight that DE genes between normal and affected broilers at 21 days of age were mainly related to endochondral ossification, while when GP and AC at 35 and 42 days of age were evaluated ( Peixoto et al, 2019 ; Oliveira et al, 2020 ; Hul et al, 2021 ; Goldoni et al, 2022 ), there were several DE genes related to other biological processes, such as inflammation, defense response, chemotaxis, within others. Therefore, the expression profiles found at 21 days of age are in agreement with other studies that found that one of the main issues regarding FHS/FHN is related to the femur maturation and mineralization ( Prisby et al, 2014 ; Wilson et al, 2019 ), possibly due to fast-growth and lack of increasing bone volume in the same rate.…”

“…FHS occurrence is also affected by broilers age, increasing at older ages ( Prisby et al, 2014 ). Our research group has been evaluating the FHS/FHN gene expression in several ages in femur ( Paludo et al, 2017 ; Hul et al, 2021 ; Goldoni et al, 2022 ; Santos et al, 2022 ) and in tibia ( Oliveira et al, 2020 ). In this way, trying to understand the genes more related with FHS onset, we have compared the common DE genes and also those exclusive to 21, 35, and 42 days of age in GP and AC.…”

“…Additionally, in order to verify the FHS gene expression profile among different ages, Ensembl IDs of DE genes identified in this study were compared with results from transcriptome studies of Peixoto et al (2019) , Oliveira et al (2020) , Hul et al (2021) and Goldoni et al (2022) . These studies evaluated early stages of FHS at 35 days in growth plate and articular cartilage ( Peixoto et al, 2019 ; Hul et al, 2021 ; Goldoni et al, 2022 ) and tibia head separation at 42 days of age ( Oliveira et al, 2020 ).…”

“…RNA sequences obtained in this study, as well as sequences from other GP and cartilage from normal and FHS-affected samples from our previous studies ( Peixoto et al, 2019 ; Oliveira et al, 2020 ; Hul et al, 2021 ; Goldoni et al, 2022 ) were used to verify the presence of variants involved with FHS. To this, sequences from femoral GP and AC of 35-day-old and tibia GP from 42-day-old broilers were downloaded from SRA database bioprojects # PRJNA352962, PRJNA350521, and PRJNA352716, respectively, totaling 24 samples (9 normal and 15 FHS-affected).…”

“…From transcriptomes of the femur articular cartilage (AC) and growth plate (GP) of 35-day-old broilers ( Peixoto et al, 2019 ; Hul et al, 2021 ; Goldoni et al, 2022 ), several candidate genes were prospected. Oliveira et al (2020) also pointed out that osteochondral downregulated genes are potentially triggering BCO in tibia of 42-day-old broilers. Most of the studies with FHN/FHS in chickens are focused on understanding the ossification processes and, generally, in animals older than 35 days of age ( Li et al, 2015 ; Paludo et al, 2017 ; Peixoto et al, 2019 ; Hul et al, 2021 ; Ramser et al, 2021 , 2022 ), since the highest incidences of this condition occur at these ages ( McNamee and Smyth, 2000 ; Paxton et al, 2014 ; Packialakshmi et al, 2015 ).…”