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-"-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) 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

Avaliação da resposta de bovinos Nelore e cruzados, Senepol x Nelore e Angus X Nelore, infestados com carrapato Rhipicephalus (Boophilus) microplus

Infestations caused by ectoparasites are among the main problems that affect stock raising in tropical countries, and in Brazil, the Rhipicephalus (Boophilus) tick is responsible for substantial losses in the animal production. The aim of this study was to evaluate the response of three genetic groups of cattle artificially infested with Rhipicephalus (Boophilus) microplus. For this, skin samples from Nellore (NX) and crossbreeds, Senepol x Nellore (SN) and Angus x Nellore (TA) heifers were collected before and 24 hours after the last artificial infestation with Rhipicephalus (Boophilus) microplus larvae for gene expression analysis and histology. We collected hair and performed behavioral analysis in order to relate them to the cattle parasite load. There were significant differences (P <0.01) for the tick count within three studied groups. TA animals (0.92 ± 0.07) had higher mean and the NX group (0.28 ± 0.07), the lowest mean. In the differential leukocyte count, TA animals had higher numbers of monocytes that NX (P <0.05), not differing from SN. No significant differences were evaluated for other leukocytes. However, the NX genetic group had higher mean for mast cells than SN and TA (P <0.01). NX had more CD4 cells that TA that did not differ from SN. Negative correlation was observed between the number of ticks and CD4 T cells. In the hair and haircoat analysis, TA showed the higher values of haircoat mean (CP) and mass density (MD) of hair than SN and NX (p <0.05), which did not differ from each other. For the number of hairs per sample and number of hair/cm2, NX had larger mean (p <0.05) than SN and TA, which did not differ. Results of Pearson correlations indicated that CP and DM have a positive correlation (p <0.05) with tick count. Regarding grooming behavior it has been observed that the total of self-grooming, as well as, allogrooming events influenced the tick count evaluated, and that there is a negative correlation with the tick number, helping to reduce this parasite, especially in Nellore. On 1, 4, 6 and 7 days after infestation and at the times 7:00, 9:00 and 12:00 h animals performed more grooming events when compared to other periods. According to the large scale gene expression results, there was no difference within genetic groups. The comparison between the groups before and after 24 hours infestation showed that 1,502 genes were differentially expressed. Of these, 851 were activated and 651 had reduced expression after challenge. The microarray experiment was validated by quantitative real time PCR. 12 Among the genes and pathways activated in response to tick, the chemokine genes, MAPK signaling pathways and Jak-Stat, cytokine receptors, complement and focal adhesion molecules as well as calcium channel genes stood out. Such genes and pathways can be identified as candidates to play the role of host protection at this time of bovine parasitic cycle. In this way, it was observed that mechanisms as grooming and increase of mast cells should confer resistance to tick. Furthermore, we found metabolic pathways that were not previously described in the literature may be novel targets for the development of strategies prevent tick infestation, as vaccines and drugs.Financiadora de Estudos e ProjetosEntre os principais problemas da pecuária brasileira está o parasitismo por carrapatos da espécie Rhipicephalus (Boophilus) microplus. Assim, o objetivo deste trabalho foi avaliar a resposta de bovinos de três grupos genéticos infestados com R. microplus. Para isso, amostras de pele de fêmeas Nelore (NX) e cruzadas, Senepol x Nelore (SN) e Angus x Nelore (TA), foram coletadas, antes e após a infestação artificial com larvas de R. microplus para análises de expressão gênica e de histologia. Foram também coletados pelos e realizada análise comportamental visando relacioná-los com a carga parasitária dos animais. Foi verificada diferença significativa (P<0,01) para a contagem de carrapatos entre os três grupos avaliados, sendo que os animais TA (0,92±0,07) apresentaram maiores médias e o grupo NX (0,28±0,07), a menor média. Na contagem diferencial de leucócitos, animais TA tiveram maiores (P<0,05) quantidades de monócitos que NX, não diferindo de SN. Não houve diferença significativa para os outros leucócitos avaliados. Entretanto, o grupo genético NX teve maiores médias (P<0,01) de mastócitos que SN e TA. NX apresentou mais células TCD4 que TA, não diferindo de SN. Foi observada correlação negativa entre o número de carrapatos e de células TCD4. Nas análises de pelo e pelame, o grupo genético TA apresentou maiores valores das características comprimento médio de pelo (CP) e densidade de massa (DM) dos pelos que SN e NX (p < 0,05), que não diferiram entre si. Já para as características de número de pelos por amostra e número de pelos/cm2, NX apresentou maiores médias (p < 0,05) que SN e TA, que não diferiram entre si. Os resultados da correlação de Pearson indicaram que CP e DM têm correlação positiva (P<0,05) com a contagem de carrapatos. Na análise de comportamento foi possível observar que o total dos eventos de auto-limpeza (grooming) avaliados influenciou a contagem de carrapatos e que há correlação negativa com o número de carrapatos, auxiliando a redução desse parasita, especialmente na raça Nelore e que nos dias 1, 4, 6 e 7 após a infestação e nos horários das 7:00, 9:00 e 12:00 h os animais realizaram mais eventos de grooming, quando comparados aos outros períodos. De acordo com os resultados de expressão gênica em larga escala das amostras de pele, não foi possível observar influência do grupo genético na expressão dos genes. A comparação entre os grupos antes e depois da infestação mostrou que 10 1502 genes foram diferencialmente expressos. Desses, 851 foram ativados e 651 tiveram sua expressão reduzida após o desafio. Os resultados encontrados no experimento de microarranjos foram validados por PCR quantitativo em tempo real. Entre os genes e vias metabólicas ativados na resposta ao carrapato destacaram-se as quimiocinas, os genes de sinalização das vias MAPK e Jak-Stat, os receptores de citocinas, do complemento e de moléculas de adesão focal. Tais vias e genes podem ser apontados como candidatos a desempenhar papel de proteção do hospedeiro bovino nesse período do ciclo parasitário. Dessa maneira, foi possível observar que mecanismos como, comportamento de auto-limpeza e aumento no número de mastócitos devem conferir resistência ao carrapato. Além disso, foram encontradas vias metabólicas ainda não descritas na literatura que podem ser novos alvos para o desenvolvimento de estratégias de combate ao carrapato, como vacinas e fármacos

Reference genes for proximal femoral epiphysiolysis expression studies in broilers cartilage.

The use of reference genes is required for relative quantification in gene expression analysis and the stability of these genes can be variable depending on the experimental design. Therefore, it is indispensable to test the reliability of endogenous genes previously to their use. This study evaluated nine candidate reference genes to select the most stable genes to be used as reference in gene expression studies with the femoral cartilage of normal and epiphysiolysis-affected broilers. The femur articular cartilage of 29 male broilers with 35 days of age was collected, frozen and further submitted to RNA extraction and quantitative PCR (qPCR) analysis. The candidate reference genes evaluated were GAPDH, HMBS, HPRT1, MRPS27, MRPS30, RPL30, RPL4, RPL5, and RPLP1. For the gene stability evaluation, three software were used: GeNorm, BestKeeper and NormFinder, and a global ranking was generated using the function RankAggreg. In this study, the RPLP1 and RPL5 were the most reliable endogenous genes being recommended for expression studies with femur cartilage in broilers with epiphysiolysis and possible other femur anomalies

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

Association of apolipoprotein B and adiponectin receptor 1 genes with carcass, bone integrity and performance traits in a paternal broiler line

Apolipoprotein B (APOB) and Adiponectin Receptor 1 (ADIPOR1) are related to the regulation of feed intake, fat metabolism and protein deposition and are candidate genes for genomic studies in birds. In this study, associations of two single nucleotide polymorphisms (SNPs) g.102A>T (APOB) and g.729C>T (ADIPOR1) with carcass, bone integrity and performance traits in broilers were investigated. Genotyping was performed on a paternal line of 1,454 broilers. The SNP detection was carried out by PCR-RFLP technique using the restriction enzymes HhaI for the SNP g.729C>T and MslI for the SNP g.102A>T. The association analyses of the two SNPs with 85 traits were performed using the restricted maximum likelihood (REML) and Generalized Quasi-Likelihood Score (GQLS) methods. For REML the model included the random additive genetic effect of animal and fixed effects of sex, hatch and SNP genotypes. In the GQLS method, a logistic regression was used to associate the genotypes with phenotypes adjusted for fixed effects of sex and hatch. The SNP g.729C>T in the ADIPOR1 gene was associated with thickness of the femur and breast skin yield. Thus, the ADIPOR1 gene seems implicated in the metabolism and/or fat deposition and bone integrity in broilers.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Genes and SNPs Involved with Scrotal and Umbilical Hernia in Pigs

Hernia is one of the most common defects in pigs. The most prevalent are the scrotal (SH), inguinal (IH) and umbilical (UH) hernias. We compared the inguinal ring transcriptome of normal and SH-affected pigs with the umbilical ring transcriptome of normal and UH-affected pigs to discover genes and pathways involved with the development of both types of hernia. A total of 13,307 transcripts was expressed in the inguinal and 13,302 in the umbilical ring tissues with 94.91% of them present in both tissues. From those, 35 genes were differentially expressed in both groups, participating in 108 biological processes. A total of 67 polymorphisms was identified in the inguinal ring and 76 in the umbilical ring tissue, of which 11 and 14 were novel, respectively. A single nucleotide polymorphism (SNP) with deleterious function was identified in the integrin α M (ITGAM) gene. The microtubule associated protein 1 light chain 3 γ (MAP1LC3C), vitrin (VIT), aggrecan (ACAN), alkaline ceramidase 2 (ACER2), potassium calcium-activated channel subfamily M α 1 (KCNMA1) and synaptopodin 2 (SYNPO2) genes are highlighted as candidates to trigger both types of hernia. We generated the first comparative study of the pig umbilical and inguinal ring transcriptomes, contributing to the understanding of the genetic mechanism involved with these two types of hernia in pigs and probably in other mammals

Marker, number of animals (N), genotypic and allelic frequencies for the SNPs studied in the broiler line.

<p>Allelic and genotypic frequencies, percentages are between parentheses.</p

P-values for associations of SNPs in the <i>APOB</i> and <i>ADIPOR1</i> genes with broiler traits, using a Generalized Quasi-Likelihood Score method (traits are defined in Tables 1 and 2).

<p>**Significance at 5% experiment-wise level by Bonferroni correction.</p><p>*A trend of significance at 15% experiment-wise level by Bonferroni correction.</p><p>P-values for associations of SNPs in the <i>APOB</i> and <i>ADIPOR1</i> genes with broiler traits, using a Generalized Quasi-Likelihood Score method (traits are defined in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136824#pone.0136824.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136824#pone.0136824.t002" target="_blank">2</a>).</p

Bone integrity and bird performance traits recorded in the broiler line andcorresponding abbreviations used.

<p>Weight in grams; thickness, distance and length in centimeters; force in kgf/mm².</p><p>Bone integrity and bird performance traits recorded in the broiler line andcorresponding abbreviations used.</p