Differential expression of miRNAs associated with pectoral myopathies in young broilers: insights from a comparative transcriptome analysis.

INTRODUCTION: White Striping (WS) and Wooden Breast (WB) pectoral myopathies are relevant disorders for contemporary broiler production worldwide. Several studies aimed to elucidate the genetic components associated with the occurrence of these myopathies. However, epigenetic factors that trigger or differentiate these two conditions are still unclear. The aim of this study was to identify miRNAs differentially expressed (DE) between normal and WS and WB-affected broilers, and to verify the possible role of these miRNAs in metabolic pathways related to the manifestation of these pectoral myopathies in 28-day-old broilers. RESULTS: Five miRNAs were DE in the WS vs control (gga-miR-375, gga-miR-200b-3p, gga-miR-429-3p, gga-miR-1769-5p, gga-miR-200a-3p), 82 between WB vs control and 62 between WB vs WS. Several known miRNAs were associated with WB, such as gga-miR-155, gga-miR-146b, gga-miR-222, gga-miR-146-5p, gga-miR- 29, gga-miR-21-5p, gga-miR-133a-3p and gga-miR-133b. Most of them had not previously been associated with the development of this myopathy in broilers. We also have predicted 17 new miRNAs expressed in the broilers pectoral muscle. DE miRNA target gene ontology analysis enriched 6 common pathways for WS and WB compared to control: autophagy, insulin signaling, FoxO signaling, endocytosis, and metabolic pathways. The WS vs control contrast had two unique pathways, ERBB signaling and the mTOR signaling, while WB vs control had 14 unique pathways, with ubiquitin-mediated proteolysis and endoplasmic reticulum protein processing being the most significant. CONCLUSIONS: We found miRNAs DE between normal broilers and those affected with breast myopathies at 28 days of age. Our results also provide novel evidence of the miRNAs role on the regulation of WS and in the differentiation of both WS and WB myopathies. Overall, our study provides insights into miRNA-mediated and pathways involved in the occurrence of WS and WB helping to better understand these chicken growth disorders in an early age. These findings can help developing new approaches to reduce these complex issues in poultry production possibly by adjustments in nutrition and management conditions. Moreover, the miRNAs and target genes associated with the initial stages of WS and WB development could be potential biomarkers to be used in selection to reduce the occurrence of these myopathies in broiler production.

Upregulated genes in articular cartilage may help to counteract femoral head separation in broilers with 21 days of age.

Femoral head necrosis (FHN) is one of the most common conditions in fast growing broilers, being characterized by separation of articular cartilage from epiphysis and classified as femoral head separation (FHS) or FHS with laceration (FHSL) depending on severity. Although molecular mechanisms involved with this disorder have been observed, its etiology is still unclear. Therefore, the expression of 15 candidate genes, chosen based on previous transcriptomic studies, was evaluated in the articular cartilage (AC) of normal and FHS-affected broilers at 21 days of age. Samples were collected based on the absence or presence of FHS for physical-chemical and qPCR analysis. The AvBD2, RHAG, COL28A1, ADA and ANGPTL7 were upregulated in FHS-affected broilers compared to the healthy group. These genes are involved in immune response, defense against pathogens, inflammation, cellular migration and adhesion, indicating different molecular mechanisms to control FHS progression at early age. Our results can contribute to improve the knowledge on FHN etiology in chickens and other species, such as horse and pigs that are severely affected by bone disorders.

Comprehensive Analyses of Bone and Cartilage Transcriptomes Evince Ion Transport, Inflammation and Cartilage Development-Related Genes Involved in Chickens' Femoral Head Separation.

Femoral head separation (FHS) is usually a subclinical condition characterized by the detachment of articular cartilage from the bone. In this study, a comprehensive analysis identifying shared and exclusive expression profiles, biological processes (BP) and variants related to FHS in the femoral articular cartilage and growth plate in chickens was performed through RNA sequencing analysis. Thirty-six differentially expressed (DE) genes were shared between femoral articular cartilage (AC) and growth plate (GP) tissues. Out of those, 23 genes were enriched in BP related to ion transport, translation factors and immune response. Seventy genes were DE exclusively in the AC and 288 in the GP. Among the BP of AC, the response against bacteria can be highlighted, and for the GP tissue, the processes related to chondrocyte differentiation and cartilage development stand out. When the chicken DE genes were compared to other datasets, eight genes (SLC4A1, RHAG, ANK1, MKNK2, SPTB, ADA, C7 and EPB420) were shared between chickens and humans. Furthermore, 89 variants, including missense in the SPATS2L, PRKAB1 and TRIM25 genes, were identified between groups. Therefore, those genes should be more explored to validate them as candidates to FHS/FHN in chickens and humans.

Differentially expressed genes in the femur cartilage transcriptome clarify the understanding of femoral head separation in chickens.

Locomotor problems are among one of the main concerns in the current poultry industry, causing major economic losses and affecting animal welfare. The most common bone anomalies in the femur are dyschondroplasia, femoral head separation (FHS), and bacterial chondronecrosis with osteomyelitis (BCO), also known as femoral head necrosis (FHN). The present study aimed to identify differentially expressed (DE) genes in the articular cartilage (AC) of normal and FHS-affected broilers by RNA-Seq analysis. In the transcriptome analysis, 12,169 genes were expressed in the femur AC. Of those, 107 genes were DE (FDR < 0.05) between normal and affected chickens, of which 9 were downregulated and 98 were upregulated in the affected broilers. In the gene-set enrichment analysis using the DE genes, 79 biological processes (BP) were identified and were grouped into 12 superclusters. The main BP found were involved in the response to biotic stimulus, gas transport, cellular activation, carbohydrate-derived catabolism, multi-organism regulation, immune system, muscle contraction, multi-organism process, cytolysis, leukocytes and cell adhesion. In this study, the first transcriptome analysis of the broilers femur articular cartilage was performed, and a set of candidate genes (AvBD1, AvBD2, ANK1, EPX, ADA, RHAG) that could trigger changes in the broiler´s femoral growth plate was identified. Moreover, these results could be helpful to better understand FHN in chickens and possibly in humans.

Differential Expression of Myogenic and Calcium Signaling-Related Genes in Broilers Affected With White Striping.

White Striping (WS) has been one of the main issues in poultry production in the last years since it affects meat quality. Studies have been conducted to understand WS and other myopathies in chickens, and some biological pathways have been associated to the prevalence of these conditions, such as extracellular calcium level, oxidative stress, localized hypoxia, possible fiber-type switching, and cellular repairing. Therefore, to understand the genetic mechanisms involved in WS, 15 functional candidate genes were chosen to be analyzed by quantitative PCR (qPCR) in breast muscle of normal and WS-affected chickens. To this, the pectoral major muscle (PMM) of 16 normal and 16 WS-affected broilers were collected at 42 days of age and submitted to qRT-PCR analysis. Out of the 15 genes studied, six were differentially expressed between groups. The CA2, CSRP3, and PLIN1 were upregulated, while CALM2, DNASE1L3, and MYLK2 genes were downregulated in the WS-affected when compared to the normal broilers. These findings highlight that the disruption on muscle and calcium signaling pathways can possibly be triggering WS in chickens. Improving our understanding on the genetic basis involved with this myopathy might contribute for reducing WS in poultry production.

Stable reference genes for expression studies in breast muscle of normal and white striping-affected chickens.

The normalization with proper reference genes is a crucial step to obtain accurate mRNA expression levels in quantitative PCR (qPCR) studies. Therefore, in this study, 10 reference candidate genes were evaluated to determine their stability in normal pectoralis major muscle of broilers and those counterparts affected with White Striping (WS) myopathy at 42 days age. Four different tools were used for ranking the most stable genes: GeNorm, NormFinder, BestKeeper and Comparative Ct (ΔCt), and a general ranking was performed using the RankAggreg tool to select the best reference genes among all tools. From the 10 genes evaluated in the breast muscle of broilers, 8 were amplified. Most of the algorithms/tools indicated the same two genes, RPL30 and RPL5, as the most stable in the broilers breast muscle. In addition, there was agreement among the tools for the least stable genes: MRPS27, GAPDH and RPLP1 in the broilers breast muscle. Therefore, it is interesting to note that even with different tools for evaluating gene expression, there was consensus on the most and least stable genes. These results indicate that the Ribosomal protein L30 (RPL30) and Ribosomal protein L5 (RPL5) can be recommended for accurate normalization in qPCR studies with chicken pectoralis major muscle affected with White Striping and other myopathies.

Structure analysis of capsid protein of Porcine circovirus type 2 from pigs with systemic disease

Abstract Economic losses with high mortality rate associated with Porcine circovirus type 2 (PCV2) is reported worldwide. PCV2 commercial vaccine was introduced in 2006 in U.S. and in 2008 in Brazil. Although PCV2 vaccines have been widely used, cases of PCV2 systemic disease have been reported in the last years. Eleven nursery or fattening pigs suffering from PCV2 systemic disease were selected from eight PCV2-vaccinated farms with historical records of PCV2 systemic disease in Southern Brazil. PCV2 genomes were amplified and sequenced from lymph node samples of selected pigs. The comparison among the ORF2 amino acid sequences of PCV2 isolates revealed three amino acid substitutions in the positions F57I, N178S and A190T, respectively. Using molecular modeling, a structural model for the capsid protein of PCV2 was built. Afterwards, the mutated residues positions were identified in the model. The structural analysis of the mutated residues showed that the external residue 190 is close to an important predicted region for antibodies recognition. Therefore, changes in the viral protein conformation might lead to an inefficient antibody binding and this could be a relevant mechanism underlying the recent vaccine failures observed in swine farms in Brazil.

Structure analysis of capsid protein of Porcine circovirus type 2 from pigs with systemic disease.

Economic losses with high mortality rate associated with Porcine circovirus type 2 (PCV2) is reported worldwide. PCV2 commercial vaccine was introduced in 2006 in U.S. and in 2008 in Brazil. Although PCV2 vaccines have been widely used, cases of PCV2 systemic disease have been reported in the last years. Eleven nursery or fattening pigs suffering from PCV2 systemic disease were selected from eight PCV2-vaccinated farms with historical records of PCV2 systemic disease in Southern Brazil. PCV2 genomes were amplified and sequenced from lymph node samples of selected pigs. The comparison among the ORF2 amino acid sequences of PCV2 isolates revealed three amino acid substitutions in the positions F57I, N178S and A190T, respectively. Using molecular modeling, a structural model for the capsid protein of PCV2 was built. Afterwards, the mutated residues positions were identified in the model. The structural analysis of the mutated residues showed that the external residue 190 is close to an important predicted region for antibodies recognition. Therefore, changes in the viral protein conformation might lead to an inefficient antibody binding and this could be a relevant mechanism underlying the recent vaccine failures observed in swine farms in Brazil.

Detection of porcine Circovirus type 2 (PCV2) variants PCV2-1 and PCV2-2 in Brazilian pig population.

In the present study whole genome of six Brazilian isolates of PCV2 were sequenced, analyzed and compared with 35 other sequences (24 from other countries and 17 from Brazil). The phylogenetic analysis showed that mostly Brazilian variants of PCV2 were grouped as PCV2-1. Two isolates among the six analyzed here could not be grouped with any other PCV2-2 analyzed in this study. One of these isolates was from an aborted fetus with myocarditis and the other from a PMWS affected pig. The results pointed here showed that both groups of PCV2 are present in Brazilian pig population without any clear geographical correlation.