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.

A polyvalent virosomal influenza vaccine induces broad cellular and humoral immunity in pigs.

BACKGROUND: Influenza A virus (IAV) is endemic in pigs globally and co-circulation of genetically and antigenically diverse virus lineages of subtypes H1N1, H1N2 and H3N2 is a challenge for the development of effective vaccines. Virosomes are virus-like particles that mimic virus infection and have proven to be a successful vaccine platform against several animal and human viruses. METHODS: This study evaluated the immunogenicity of a virosome-based influenza vaccine containing the surface glycoproteins of H1N1 pandemic, H1N2 and H3N2 in pigs. RESULTS: A robust humoral and cellular immune response was induced against the three IAV subtypes in pigs after two vaccine doses. The influenza virosome vaccine elicited hemagglutinin-specific antibodies and virus-neutralizing activity. Furthermore, it induced a significant maturation of macrophages, and proliferation of B lymphocytes, effector and central memory CD4+ and CD8+ T cells, and CD8+ T lymphocytes producing interferon-γ. Also, the vaccine demonstrated potential to confer long-lasting immunity until the market age of pigs and proved to be safe and non-cytotoxic to pigs. CONCLUSIONS: This virosome platform allows flexibility to adjust the vaccine content to reflect the diversity of circulating IAVs in swine in Brazil. The vaccination of pigs may reduce the impact of the disease on swine production and the risk of swine-to-human transmission.

Toltrazuril-Loaded Polymeric Nanocapsules as a Promising Approach for the Preventive Control of Coccidiosis in Poultry.

Coccidiosis is a disease caused by intracellular protozoan parasites of the genus Eimeria that affect the intestinal tract of poultry. However, strain resistance and drug residue in the carcass have drawn the attention of the productive sector. The nanotechnology can improve the biological effect of drugs, reducing of administered doses and toxic effects. Due to this, toltrazuril-load polymeric nanoparticles based on Eudragit® S100 (NCt) or poly-ε-caprolactone (LNCt) were developed to prevent coccidiosis in broilers. Nanoformulations were produced and showed homogeneous particle diameter distribution in the nanometer range (z-average and D (4.3) < 200 nm), negative zeta potential (<-8.93 mV), drug content ~100%, and encapsulation efficiency >90%. Cell viability assays using avian fibroblasts showed that LNCt presented no relevant toxicity up to 72 h. LNCt was then prophylactically administrated to chicken followed by challenge with Eimeria oocysts. The evaluation of the small intestine and cecum showed that the treatment with LNCt (3.5 mg/kg/day) in drinking water reduced the lesion scores and oocysts excretion, similar to the reference medicine containing toltrazuril (Baycox®, 7 mg/kg/day). The current study shows the potential protective use of nanoencapsulating anticoccidial drugs as a promising approach for the control of coccidiosis in poultry.

Role of p21 and oxidative stress on renal tubular resistance after acute ischaemic injury.

BACKGROUND: Subsequent ischaemic episodes may induce renal resistance. P21 is a cell cycle inhibitor that may be induced by oxygen-free radicals and may have a protective effect in ischaemic acute kidney injury (AKI). This study aimed at evaluating the role of oxidative stress and p21 on tubular resistance in a model of acquired resistance after renal ischaemia and in isolated renal tubules. METHODS: Wistar rats were divided into: Group 1--sham; Group 2--sham operated and after 2 days submitted to 45-min ischaemia; and Group 3--45-min ischaemia followed after 2 days by a second 45-min ischaemia. Plasma urea was evaluated on Days 0, 2 and 4. Serum creatinine, creatinine clearance and oxidants (thiobarbituric acid-reactive substances) were determined 48 h after the second procedure (Day 4). Histology, immunohistochemistry for lymphocytes (CD3), macrophages (ED1), proliferation (PCNA) and apoptosis (TUNEL) were also evaluated. Rat proximal tubules (PTs) were isolated by collagenase digestion and Percoll gradient from control rats and rats previously subjected to 35 min of ischaemia. PTs were submitted to 15-min hypoxia followed by 45-min reoxygenation. Cell injury was assessed by lactate dehydrogenase release and hydroperoxide production (xylenol orange). RESULTS: Ischaemia induced AKI in Group 2 and 3 rats. Subsequent ischaemia did not aggravate renal injury, demonstrating renal resistance (Group 3). Renal function recovery was similar in Group 2 and 3. Plasma and urine oxidants were similar among in Group 2 and 3. Histology disclosed acute tubular necrosis in Group 2 and 3. Lymphocyte infiltrates were similar among all groups whereas macrophages infiltrate was greater in Group 3. Cell proliferation was greater in Group 2 compared with Group 3. Apoptosis was similar in groups 2 and 3. The p21 expression was increased only in Group 3 whereas it was similar in groups 1 and 2. PTs from the ischaemia group were sensitive to hypoxia but resistant to reoxygenation injury which was followed by lower hydroperoxide production compared to control PT. CONCLUSION: Renal resistance induced by ischaemia was associated with cell mechanism mediators involving oxidative stress and increased p21 expression.

A haploinsuficiência de Pkd1 aumenta a lesão renal e induz formação de microcistos após isquemia/reperfusão em camundongos / Pkd1 haploinsufficiency increases renal damage and induces microcyst formation following ischemia/reperfusion in mice

A maior parte dos casos de doença renal policística autossômica dominante (DRPAD) é causada por mutações no gene PKD1 (Polycystic Kidney Disease 1). O insulto por isquemia/reperfusão (IR) constitui-se em uma causa freqüente de lesão renal aguda, incluindo a população de pacientes com DRPAD, mas a relação entre policistina-1 e IR é essencialmente desconhecida. Uma vez que a policistina-1 modula proliferação, diferenciação celular e apoptose em sistemas de cultura de células, sua menor atividade biológica na DRPAD poderia favorecer um maior grau de lesão renal. Utilizamos uma linhagem endogâmica de camundongos 129Sv com uma mutação nula em Pkd1 para testar esta hipótese. Camundongos Pkd1+/- não apresentam cistos renais até 12 semanas de vida, constituindo-se em um modelo puro de haploinsuficiência para este gene. Um insulto IR bilateral de 32 min foi induzido em camundongos machos de 10-12 semanas de idade, heterozigotos e selvagens, por meio do clampeamento reversível de ambos os pedículos renais. Os animais foram analisados 48 h, 7 dias (d) e 14 d após o insulto. Camundongos Pkd1+/- apresentaram FENa, FEK e SCr mais elevadas que animais Pkd1+/+ 48 h após IR. O dano cortical residual foi mais severo em heterozigotos que em selvagens em todos os tempos avaliados. A marcação para PCNA também foi mais alta em camundongos Pkd1+/- que Pkd1+/+ 48 h e 7 d pós-IR, enquanto a taxa de apoptose e a infiltração inflamatória intersticial foram maiores em heterozigotos que em selvagens nos seguimentos de 48 h, 7 d e 14 d pós-IR. A expressão renal de p21 foi menor nos camundongos Pkd1+/- que Pkd1+/+ no tempo de 48 h pós-insulto, tanto no nível transcricional como traducional. Análises adicionais realizadas 6 semanas após o insulto IR revelaram dilatação tubular e formação de microcistos nos camundongos haploinsuficientes para Pkd1, assim como fibrose renal aumentada nesses animais, comparados aos camundongos selvagens...

The majority of autosomal dominant polycystic kidney disease (ADPKD) cases are caused by mutations in the PKD1 gene. Ischemia/reperfusion is a frequent cause of acute kidney injury, including the ADPKD patient population, but the relationship between polycystin-1 and ischemia/reperfusion is essentially unknown. Since polycystin-1 modulates cell proliferation, cell differentiation and apoptosis in cell culture systems, its lower biological activity in ADPKD might amplify the degree of renal injury. Using an inbred 129Sv mouse line with a Pkd1-null mutation, 32-min renal ischemia/reperfusion was induced in 10-12 week-old male non-cystic mice, heterozygotes and wild types. The animals were analyzed at 48h, 7 days (d) and 14d after the insult. Pkd1+/- mice showed higher FENa, FEK and SCr than Pkd1+/+ animals at 48h of follow-up. The residual cortical damage was more severe in heterozygotes than wild types at all evaluated time points. The PCNA staining was also higher in Pkd1+/- than Pkd1+/+ mice at 48h and 7d, while cell apoptotic rates and the interstitial inflammatory infiltration were higher in heterozygotes than wild types at 48h, 7d and 14d postischemia/ reperfusion. The expression of p21 was lower in Pkd1+/- than Pkd1+/+ kidneys at 48h, both at the transcriptional and translational levels. Additional analyses performed 6 weeks after the insult showed tubular dilatation and microcyst formation in the haploinsufficient mice, and increased renal fibrosis in these animals compared to wild types. Thirty-fivemin ischemia/reperfusion, at last, was accompanied by a substantially higher early mortality of Pkd1+/- animals. These findings suggest that ischemia/reperfusion induces a more severe injury in kidneys of Pkd1- haploinsufficient mice, a process that is apparently dependent on a relative deficiency of p21 activity, as well as tubular dilatation and microcyst formation. Altogether, our results suggest that mouse Pkd1-null heterozygosity...