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.

Clinical study of skin changes in low and high risk pregnant women

Abstract: BACKGROUND: During pregnancy there is immunological, metabolic, endocrine and vascular changes responsible for physiological and pathological skin changes. OBJECTIVES: determine the prevalence of specific physiological changes and pregnancy, comparing the period of gestation of their appearances and compare type of prenatal care as the skin changes. METHODS: A cross-sectional study with 905 pregnant women. RESULTS: The prevalence of physiological skin changes was 88.95% and the most common was pigment. The prevalence of specific dermatoses was 8.72% and atopic eruption was the most common. CONCLUSION: Physiological changes were seen more in the 3rd quarter, as well as the specific dermatoses. No statistical difference in prenatal low risk compared to high risk was observed, whereas the cutaneous physiological changes and specific pregnancy dermatoses.

Clinical study of skin changes in low and high risk pregnant women.

BACKGROUND: During pregnancy there is immunological, metabolic, endocrine and vascular changes responsible for physiological and pathological skin changes. OBJECTIVES: determine the prevalence of specific physiological changes and pregnancy, comparing the period of gestation of their appearances and compare type of prenatal care as the skin changes. METHODS: A cross-sectional study with 905 pregnant women. RESULTS: The prevalence of physiological skin changes was 88.95% and the most common was pigment. The prevalence of specific dermatoses was 8.72% and atopic eruption was the most common. CONCLUSION: Physiological changes were seen more in the 3rd quarter, as well as the specific dermatoses. No statistical difference in prenatal low risk compared to high risk was observed, whereas the cutaneous physiological changes and specific pregnancy dermatoses.

Unraveling the associations of osteoprotegerin gene with production traits in a paternal broiler line.

Improvements on growth and carcass traits in the poultry industry have been achieved by intense selection for heavier chickens at early ages. This faster growth has caused serious problems due to insufficient skeletal structure development needed to support the musculature of modern broilers. The osteoprotegerin gene (OPG), located on GGA2, is an important regulator of bone metabolism and reabsorption, being suggestive as a possible functional candidate gene associated with bone integrity in chickens. This study reports associations of a single nucleotide polymorphism (SNP) in the OPG gene with production traits in a parental broiler line. Different phenotypic groups were evaluated: performance, carcass and skeletal traits. SNPs were identified within the OPG gene and the most informative SNP g.9144C > G was chosen for association analyses. Chickens (n = 1230) were genotyped using PCR-RFLP. The association was carried out with QxPaK v4.0 software using a mixed model including sex, hatch and SNP as fixed effects, and the infinitesimal and residual as random effects. The OPG SNP was associated with important traits as body weight at 21 days, weights of tibia and drumstick skin, leg muscle yield, and tibia breaking strength (P < 0.05). Associations were explained by the additive effect of the SNP and the additive effect within sex. This SNP could be considered a potential marker to improve bone resistance in chickens; however, caution should be taken because of its negative effect in other important traits evaluated in this study. Furthermore, these findings suggest a possible involvement of the OPG gene in fat deposition in poultry.

Alterações dermatológicas na gravidez: revisão da literatura / Skin changes in pregnancy: literature review

Durante o período gestacional ocorrem profundas alterações imunológicas, metabólicas, endócrinas e vasculares, que são responsáveis por alterações cutâneas, fisiológicas e patológicas. O objetivo deste artigo foi verificar as alterações dermatológicas que ocorrem durante a gravidez. Para isso, foi realizada uma revisão da literatura, de artigos publicados entre 1982 e 2013, indexados no PubMed e SciELO. Em relação a pele, os achados indicam que as alterações gestacionais são divididas em: alterações fisiológicas gestacionais, dermatoses específicas e dermatoses alteradas na gestação. Este apanhado engloba as características clínicas e o prognóstico das alterações fisiológicas da pele durante a gravidez, as dermatoses influenciadas pela gravidez e as dermatoses específicas da gravidez.(AU)

During pregnancy, deep immunological, metabolic, endocrine and vascular changes responsible for physiological and pathological skin changes occur. The objective of this paper was to check the skin changes that occur during pregnancy. For this, a literature review of articles published between 1982 and 2013, indexed in PubMed and SciELO was performed. Regarding the skin, the findings indicate that gestational changes are divided into gestational physiologic changes, specific skin diseases and skin conditions changed during pregnancy. This overview covers the clinical features and prognosis of physiological skin changes during pregnancy, dermatoses affected by pregnancy specific dermatoses of pregnancy.(AU)

Langerhans cells histiocytosis with vulvar involvement and responding to thalidomide therapy--case report.

Langerhans cell histiocytosis is a member of a group of rare histiocytic syndromes and is characterized for the proliferation of histiocytes called Langerhans'cells. Its manifestations vary from a solitary injury to systemic involvement, and vulvar lesions are uncommon. We describe a refractory case of cutaneous limited disease in a 57-year-old woman. She presented with a 6-year history of an erythematous papular eruption of the scalp, face, vulva, trunk and axillae. The diagnosis is difficult and in this case it was confirmed through immunohistochemical study and clinical improvement was achieved with thalidomide.

Comparative dermatology: elephantiasis nostra in verrucous form comparable to coral.

Study of a rare case of Elephantiasis Nostra in verrucous form on the dorsum of the foot of an 80 year-old male with a history of recurrent erysipelas infection. The vegetant, confluent lesions on the foot resemble Trumpet Coral (Caulastrea curvata).

Dermatologia comparativa: similaridade entre elefantíase nostra verrucosa e coral / Comparative dermatology: elephantiasis nostra in verrucous form comparable to coral

Demonstra-se quadro raro de Elefantíase Nostra, na sua forma verrucosa, no dorso de pé de homem de 80 anos por episódios prévios de erisipela de repetição. As lesões confluentes vegetantes e difusas em dorso de pé são comparáveis aos corais Trumpet Coral (Caulastrea curvata).

Study of a rare case of Elephantiasis Nostra in verrucous form on the dorsum of the foot of an 80year-old male with a history of recurrent erysipelas infection. The vegetant, confluent lesions on the foot resemble Trumpet Coral (Caulastrea curvata).

Histiocitose de células de Langerhans com acometimento vulvar e com resposta terapêutica à talidomida: relato de caso / Langerhans cells histiocytosis with vulvar involvement and responding to thalidomide therapy: case report

A histiocitose de células de Langerhans é representante de um raro grupo de síndromes histiocitárias, sendo caracterizada pela proliferação das células de Langerhans. Suas manifestações variam de lesão solitária a envolvimento multissistêmico, sendo o acometimento vulvar incomum. Segue-se o relato de caso refratário da doença limitada à pele, em mulher de 57 anos. A paciente apresentava história de pápulas eritematosas ulceradas em couro cabeludo, face, vulva, tronco e axila há seis anos. O diagnóstico da doença é difícil, sendo confirmado neste caso através de estudo imuno-histoquímico e se obteve resposta terapêutica e eficaz, com a administração de talidomida.

Langerhans cell histiocytosis is a member of a group of rare histiocytic syndromes and is characterized for the proliferation of histiocytes called Langerhans'cells. Its manifestations vary from a solitary injury to systemic involvement, and vulvar lesions are uncommon. We describe a refractory case of cutaneous limited disease in a 57-year-old woman. She presented with a 6-year history of an erythematous papular eruption of the scalp, face, vulva, trunk and axillae. The diagnosis is difficult and in this case it was confirmed through immunohistochemical study and clinical improvement was achieved with thalidomide.

Quantitative proteomics by 2-DE, 16O/18O labelling and linear ion trap mass spectrometry analysis of lymph nodes from piglets inoculated by porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) has been identified as the essential causal agent of postweaning multisystemic wasting syndrome. However, little is known regarding the mechanism(s) underlying the pathogenesis of PCV2-induced disease and the interaction of the virus with the host immune system. Here, we present a proteomics study on inguinal lymph nodes of piglets inoculated with PCV2, in order to better understand the pathogenesis of postweaning multisystemic wasting syndrome and the pathways might be affected after infection. We used two proteomics strategies, 2-DE and 1-DE followed by (16)O/(18)O peptide labelling and peptide identification and quantification by MS. More than 100 proteins were found to be differentially regulated and the results obtained by the two strategies were fairly concordant but also complementary, the (18)O labelling approach being a more robust alternative. Analysis of these proteins by systems biology tools revealed the implication of acute phase response and NrF2-mediated oxidative stress, suggesting a putative role for these pathways in the pig immune response. Besides, CD81 was found to be up-regulated, suggesting a possible role in the internalization of the virus. The use of proteomics technologies together with biology analysis systems opens up the way to gain more exhaustive and systematic knowledge of virus-pathogen interactions.

Time course differential gene expression in response to porcine circovirus type 2 subclinical infection.

This study was aimed at characterizing the potential differences in gene expression in piglets inoculated with Porcine circovirus type 2 (PCV2), the essential causative agent of postweaning multisystemic wasting syndrome. Seven-day-old caesarean-derived, colostrum-deprived piglets were distributed into two groups: control (n = 8) and pigs inoculated with 10(5.2) TCID(50) of the Burgos PCV2 isolate (n = 16). One control and three inoculated pigs were necropsied on days 1, 2, 5, and 8 post-infection (p.i.). The remaining pigs (four of each group) were sequentially bled on days 0, 7, 14, 21, and 29 p.i. (necropsy). Total RNA from the mediastinal lymph node (MLN) and lysed whole blood (LWB) samples were hybridized to Affymetrix Porcine GeneChip. Forty-three probes were differentially expressed (DE) in MLN samples (FDR < 0.1, fold change > 2) and were distributed into three clusters: globally down-regulated genes, and up-regulated genes at early (first week p.i.) and late (day 29 p.i.) stages of infection. In LWB samples,maximal differences were observed at day 7 p.i., with 54 probes DE between control and inoculated pigs. Main Gene Ontology biological processes assigned to upregulated genes were related to the immune response. Six common genes were found in both types of samples, all of which belonged to the interferon signaling antiviral effector pathway. Down-regulated genes were mainly related to cell adhesion and migration in MLN, and cellular organization and biogenesis in LWB. Microarray results were validated by quantitative real-time PCR. This study provides, for the first time, the characterization of the early and late molecular events taking place in response to a subclinical PCV2 infection.

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.