Toll-Like Receptor 6 differential expression in two pig genetic groups vaccinated against Mycoplasma hyopneumoniae

<p>Abstract</p> <p>Background</p> <p><it>Mycoplasma hyopneumoniae</it> is the etiologic agent of enzootic pneumonia, which causes important economic losses to swine industry. The Toll-like receptors (TLRs) are pattern-recognition receptors which detect microbial presence and initiate the innate as well as the adaptative immune defense. Toll-like receptor 6 is a type I transmembrane protein that recognizes bacterial components. The aim of this study was to compare mRNA expression pattern of TLR6 gene in two genetically distinct groups of pigs vaccinated against <it>Mycoplasma hyopneumoniae.</it></p> <p>Methods</p> <p>For each genetic group, peripheral blood was collected just before and 10 days after vaccination from 10 Naturalized Brazilian Piau breed and 10 Commercial White Line serum-negative female piglets. RNA was extracted from peripheral blood mononuclear cells (PBMCs), reverse transcripted and the qRT-PCR performed using SYBR green fluorescence system, using GAPDH gene as endogenous control. Analyses were performed by UNIVARIATE (Shapiro-Wilk test) and MIXED procedures of SAS software (version 9.0).</p> <p>Results</p> <p>It was observed significant interaction between breed and vaccination, being the TLR6 mRNA expression higher in the Commercial White line than in the Piau breed after vaccination. Furthermore, there was differential expression before and after vaccination in the Commercial White line.</p> <p>Conclusions</p> <p>Analysis of in TLR6 gene expression showed difference between the two distinct genetic groups, however, other TLRs gene expression must be evaluated for a better understanding of innate resistance in the pig concerning <it>Mycoplasma hyopneumoniae</it> infection.</p

Serum Epidemiological Analysis and Risk Factors Associated with Leptospira spp. in Cattle in the State of Piauí

Background: Leptospirosis is a cosmopolitan zoonosis caused by pathogenic spirochetes of the genus Leptospira spp. and it is considered one of the main causes of reproductive problems in cattle. Therefore, the aim of this study was to determine the occurrence of anti-Leptospira antibodies and identify the prevalent serovars and risk factors associated with infection in cattle herds, in the microregion of Floriano, Piaui State, Brazil.Materials, Methods & Results: A total of 414 bovine sera samples were collected (390 females aged over 24 months and 24 bulls) from 22 properties (farms) in the municipalities that compose the study area. The samples were analyzed using the Microscopic Agglutination Test (MAT) to detect anti-Leptospira antibodies from 23 pathogenic serovars. An epidemiological questionnaire was applied in each farm to evaluate the risk factors, using a univariate analysis of the variables of interest, by Pearson’s Chi-square test (χ2) or Fisher’s exact test, when it was necessary. Then, each independent variable was crossed with the dependent variable and those that presented statistical significance 1: 100) in the 22 evaluated farms; all of them had at least one positive animal, resulting in a prevalence of 34.54%, with 32,8% females (136) and 1,7% males (07), and 8,93% (37) of co-agglutination. Nineteen of the 23 tested serovars were identified; among them, Icterohaemorrhagiae (42.48%), Hardjo (31.2%), Pomona (4.3%), and Castellonis (4.3%) stood out. Absence of quarantine (OR = 16.172, P = 0.024), vaccination (OR = 0.090, P = 0.037) and isolation of diseased animals (OR = 0.006, P = 0.030) were identified, by the multivariate logistic regression analysis, as risk factors for any serovar of Leptospira spp.Discussion: The results of the present study showed that leptospirosis is present in all studied municipalities, in which the prevalence may be related to the variety of factors that influence the occurrence of the disease, such as climatic and environmental conditions, transit of animal, practices of management adopted in the herd, and the lack of information about the disease. The occurrence of co-agglutination can be explained by the possibility of multiple and concomitant contaminations with several serovars. Icterohaemorrhagiae was the most prevalent serovar, which has significant importance to public health since it is considered the main serovar of leptospirosis in humans, associated with the presence of rodents. On the other hand, the serovar Hardjo is related to the chronic leptospirosis in cattle, demonstrated through reproductive problems. The serovars Castellonis and Pomona were also observed in bovine herds, suggesting a possible transmission of the microorganism between animal species, probably due to exposure to the same sources of infection. In fact, the large number of serovarieties of Leptospira spp. indicates the presence of one or more maintenance hosts, which are natural reservoirs of this infection. The risk factors confirmed in the logistic regression analysis probably occur due to failures in sanitary management. It is concluded that Leptospira spp. is disseminated in cattle in the studied region, with the presence of several serovars, which reinforces the need for intensifying the prevention and control measures, such as vaccination

Expression profile of genes associated with mastitis in dairy cattle

In order to characterize the expression of genes associated with immune response mechanisms to mastitis, we quantified the relative expression of the IL-2, IL-4, IL-6, IL-8, IL-10, IFN-γ and TNF- α genes in milk cells of healthy cows and cows with clinical mastitis. Total RNA was extracted from milk cells of six Black and White Holstein (BW) cows and six Gyr cows, including three animals with and three without mastitis per breed. Gene expression was analyzed by real-time PCR. IL-10 gene expression was higher in the group of BW and Gyr cows with mastitis compared to animals free of infection from both breeds (p < 0.05). It was also higher in BW Holstein animals with clinical mastitis (p < 0.001), but it was not significant when Gyr cows with and without mastitis were compared (0.05 < p < 0.10). Among healthy cows, BW Holstein animals tended to present a higher expression of all genes studied, with a significant difference for the IL-2 and IFN- γ genes (p < 0.001). For animals with mastitis no significant difference in gene expression was observed between the two breeds. These findings suggest that animals with mastitis develop a preferentially cell-mediated immune response. Further studies including larger samples are necessary to better characterize the gene expression profile in cows with mastitis

Imunomodulação de suínos de linhagem comercial e da raça naturalizada brasileira Piau em resposta à vacinação contra Mycoplasma hyopneumoniae e Pasteurella multocida tipo D

Doenças respiratórias continuam a ser uma grande causa de perdas econômicas na produção de suínos. Tem havido relativamente pouco progresso no controle de doenças respiratórias, apesar do desenvolvimento contínuo de novas vacinas e antibióticos. Objetivou-se no presente estudo, comparar a resposta imune inata e adaptativa de suínos da raça naturalizada brasileira Piau e de uma linhagem comercial frente a vacinação contra Micoplasma hyopneumoniae e Pasteurella multocida tipo D. Para cada grupo genético, o sangue periférico das células mononucleares (PBMC) foi coletado antes e 10 dias depois de cada dose de vacinação de 12 leitões fêmeas soro-negativas para os patógenos em estudo para análise de RTqPCR. Às dez semanas de idade, os baços dos animais foram coletados para ensaio de proliferação celular e, amostras de lavado broncoalveolar (BALF) foram coletadas para mensurar a produção de óxido nítrico e RT-qPCR. RNA foi extraído de células de PBMC e BALF, foi feita transcrição reversa e o RT-qPCR foi feito usando o sistema de detecção de fluorescência SYBR Green, usando como controle endógeno os genes GAPDH e HPRT1 para PBMC e BALF, respectivamente. Para o experimento com Micoplasma hyopneumoniae como patógeno, a linhagem comercial teve maior expressão de mRNA para TLR2, TLR4, TLR6 e TLR10 e as citocinas IL6 e TNF&#945; depois da vacinação que antes da vacinação, enquanto os animais da raça Piau mostraram maior expressão para TLR6, TLR10, TNF&#945; e TGF&#946;. Em relação às amostras de BALF, os animais vacinados da raça Piau tiveram maior diferença de expressão para os genes TLR4, TLR10, TNF&#945; e TGF&#946; que os animais não vacinados da mesma raça. Por outro lado, os animais comerciais vacinados mostraram diferença significativa para TNF&#945;. Quando se estudou a resposta específica para Pasteurella multocida, os animais comerciais tiveram expressão significativa para os genes TLR2, TLR4, TLR6, TLR10, IL2, IL6, IL8, IL10, IL12, IL13 e TNF&#945; entre os tempos estudados, após duas doses da vacina; enquanto os animais Piau tiveram somente diferença de expressão para a citocina antiinflamatória TGF&#946;. Para expressão gênica em células de BALF dos animais comerciais vacinados, houve maiores níveis de mRNA de IL6 e TNF&#945; que os não vacinados da mesma raça. Por outro lado, os animais vacinados Piau tiveram um aumento significativo para TNF&#945; e TGF&#946; quando comparados aos não vacinados Piau. Portanto, foram observadas diferenças entre os animais Piau e os comerciais frente à vacinação contra Micoplasma hyopneumoniae e Pasteurella multocida tipo D, tais como, expressão de mRNA de receptores TLR2, TLR4, TLR6 e TLR10 e citocinas IL2, IL6, IL8, IL10, IL12, IL13, TNF&#945; e TGF&#946;. Esses resultados são sugestivos de diferenças genéticas entre as raças que podem influenciar na susceptibilidade e na resistência às doenças.Conselho Nacional de Desenvolvimento Científico e TecnológicoRespiratory diseases continue to be the greatest cause of economic losses to swine production. There has been relatively little progress in the control of respiratory diseases, despite the continuous development of new vaccines and antibiotics. The objective of this study was to compare innate and adaptive immune response in Piau Brazilian Naturalized breed and commercial line in response to vaccination against Mycoplasma hyopneumoniae and Pasteurella multocida type D. For each genetic group, peripheral blood mononuclear cells (PBMC) were collected before and 10 days after vaccination against each pathogen from 12 serum-negative female piglets for RT-qPCR analysis. At 10 weeks of age, bronchoalveolar lavage fluid BALF samples were collected to measure nitric oxide production and RT-qPCR. Spleens samples were collected for lymphocytes proliferation studies. RNA was extracted from PBMCs and BALF cells, reverse transcripted and the RT-qPCR was performed using SYBR green fluorescence system, using GAPDH and HPRT1 genes as endogenous control for PBMC and BALF cells, respectively. For the Mycoplasma hyopneumoniae experiment, commercial line had higher expression of TLR2, TLR4, TLR6, TLR10, IL6 and TNF&#945; mRNA after vaccination in comparison to before vaccination, while Piau animals showed higher expression only for TLR6, TLR10, TNF&#945; e TGF&#946;. In relation to BALF samples, vaccinated Piau pigs showed higher differential expression for TLR4, TLR10, TNF&#945; e TGF&#946; than unvaccinated Piau. In contrast, vaccinated commercial piglets showed significant difference for TNF&#945;. When specific response in PBMC was studied in Pasteurella multocida analysis, commercial animals had stronger mRNA expression for TLR2, TLR4, TLR6, TLR10, IL2, IL6, IL8, IL10, IL12, IL13 and TNF&#945;, comparing the time points studied after two doses of vaccine; on the other hand, Piau breed had only differential expression for TGF&#946; anti-inflammatory cytokine. For mRNA expression in BALF, vaccinated commercial group had higher levels of IL6 and TNF&#945; than unvaccinated animals. In contrast, vaccinated Piau animals had increased TNF&#945; and TGF&#946; mRNA in comparison to the unvaccinated Piau piglets. In conclusion, it was observed breed differences between Piau Brazilian Naturalized and commercial pigs in response to vaccination against Mycoplasma hyopneumoniae and Pasteurella multocida type D, such as expression of mRNA TLR2, TLR4, TLR6 and TLR10 and IL2, IL6, IL8, IL10, IL12, IL13, TNF&#945; and TGF&#946;. These results are suggestive of genetic differences that may influence in disease susceptibility/ resistance

Imunomodulação de suínos de linhagem comercial e da raça naturalizada brasileira Piau em resposta à vacinação contra Mycoplasma hyopneumoniae e Pasteurella multocida tipo D

Doenças respiratórias continuam a ser uma grande causa de perdas econômicas na produção de suínos. Tem havido relativamente pouco progresso no controle de doenças respiratórias, apesar do desenvolvimento contínuo de novas vacinas e antibióticos. Objetivou-se no presente estudo, comparar a resposta imune inata e adaptativa de suínos da raça naturalizada brasileira Piau e de uma linhagem comercial frente a vacinação contra Micoplasma hyopneumoniae e Pasteurella multocida tipo D. Para cada grupo genético, o sangue periférico das células mononucleares (PBMC) foi coletado antes e 10 dias depois de cada dose de vacinação de 12 leitões fêmeas soro-negativas para os patógenos em estudo para análise de RT- qPCR. Às dez semanas de idade, os baços dos animais foram coletados para ensaio de proliferação celular e, amostras de lavado broncoalveolar (BALF) foram coletadas para mensurar a produção de óxido nítrico e RT-qPCR. RNA foi extraído de células de PBMC e BALF, foi feita transcrição reversa e o RT-qPCR foi feito usando o sistema de detecção de fluorescência SYBR Green, usando como controle endógeno os genes GAPDH e HPRT1 para PBMC e BALF, respectivamente. Para o experimento com Micoplasma hyopneumoniae como patógeno, a linhagem comercial teve maior expressão de mRNA para TLR2, TLR4, TLR6 e TLR10 e as citocinas IL6 e TNF&#945; depois da vacinação que antes da vacinação, enquanto os animais da raça Piau mostraram maior expressão para TLR6, TLR10, TNF&#945; e TGF&#946;. Em relação às amostras de BALF, os animais vacinados da raça Piau tiveram maior diferença de expressão para os genes TLR4, TLR10, TNF&#945; e TGF&#946; que os animais não vacinados da mesma raça. Por outro lado, os animais comerciais vacinados mostraram diferença significativa para TNF&#945;. Quando se estudou a resposta específica para Pasteurella multocida, os animais comerciais tiveram expressão significativa para os genes TLR2, TLR4, TLR6, TLR10, IL2, IL6, IL8, IL10, IL12, IL13 e TNF&#945; entre os tempos estudados, após duas doses da vacina; enquanto os animais Piau tiveram somente diferença de expressão para a citocina antiinflamatória TGF&#946;. Para expressão gênica em células de BALF dos animais comerciais vacinados, houve maiores níveis de mRNA de IL6 e TNF&#945; que os não vacinados da mesma raça. Por outro lado, os animais vacinados Piau tiveram um aumento significativo para TNF&#945; e TGF&#946; quando comparados aos não vacinados Piau. Portanto, foram observadas diferenças entre os animais Piau e os comerciais frente à vacinação contra Micoplasma hyopneumoniae e Pasteurella multocida tipo D, tais como, expressão de mRNA de receptores TLR2, TLR4, TLR6 e TLR10 e citocinas IL2, IL6, IL8, IL10, IL12, IL13, TNF&#945; e TGF&#946;. Esses resultados são sugestivos de diferenças genéticas entre as raças que podem influenciar na susceptibilidade e na resistência às doenças.Conselho Nacional de Desenvolvimento Científico e TecnológicoRespiratory diseases continue to be the greatest cause of economic losses to swine production. There has been relatively little progress in the control of respiratory diseases, despite the continuous development of new vaccines and antibiotics. The objective of this study was to compare innate and adaptive immune response in Piau Brazilian Naturalized breed and commercial line in response to vaccination against Mycoplasma hyopneumoniae and Pasteurella multocida type D. For each genetic group, peripheral blood mononuclear cells (PBMC) were collected before and 10 days after vaccination against each pathogen from 12 serum-negative female piglets for RT-qPCR analysis. At 10 weeks of age, bronchoalveolar lavage fluid BALF samples were collected to measure nitric oxide production and RT-qPCR. Spleens samples were collected for lymphocytes proliferation studies. RNA was extracted from PBMCs and BALF cells, reverse transcripted and the RT-qPCR was performed using SYBR green fluorescence system, using GAPDH and HPRT1 genes as endogenous control for PBMC and BALF cells, respectively. For the Mycoplasma hyopneumoniae experiment, commercial line had higher expression of TLR2, TLR4, TLR6, TLR10, IL6 and TNF&#945; mRNA after vaccination in comparison to before vaccination, while Piau animals showed higher expression only for TLR6, TLR10, TNF&#945; e TGF&#946;. In relation to BALF samples, vaccinated Piau pigs showed higher differential expression for TLR4, TLR10, TNF&#945; e TGF&#946; than unvaccinated Piau. In contrast, vaccinated commercial piglets showed significant difference for TNF&#945;. When specific response in PBMC was studied in Pasteurella multocida analysis, commercial animals had stronger mRNA expression for TLR2, TLR4, TLR6, TLR10, IL2, IL6, IL8, IL10, IL12, IL13 and TNF&#945;, comparing the time points studied after two doses of vaccine; on the other hand, Piau breed had only differential expression for TGF&#946; anti-inflammatory cytokine. For mRNA expression in BALF, vaccinated commercial group had higher levels of IL6 and TNF&#945; than unvaccinated animals. In contrast, vaccinated Piau animals had increased TNF&#945; and TGF&#946; mRNA in comparison to the unvaccinated Piau piglets. In conclusion, it was observed breed differences between Piau Brazilian Naturalized and commercial pigs in response to vaccination against Mycoplasma hyopneumoniae and Pasteurella multocida type D, such as expression of mRNA TLR2, TLR4, TLR6 and TLR10 and IL2, IL6, IL8, IL10, IL12, IL13, TNF&#945; and TGF&#946;. These results are suggestive of genetic differences that may influence in disease susceptibility/ resistance

Detection of Quantitative Trait Loci (QTL) on Pig Chromosome 5, 7 and 8

Marcadores moleculares podem ser usados para identificar regiões cromossômicas que contêm locos de características quantitativas (QTL) que controlam fenótipos de importância econômica em animais de produção. Para estudá-los em suínos, foi gerada uma população de um cruzamento entre dois varrões da raça naturalizada Piau e dezoito fêmeas da linha comercial (Landrace x Large White x Pietrain). Uma progênie de 614 animais na F2 foi produzida de 50 acasalamentos da F1. Os dados fenotípicos de desempenho, carcaça, órgãos internos, vísceras, corte de carcaça e qualidade de carne foram coletados nos animais F2. Os animais parentais, F1 e F2 foram genotipados para 17 marcadores tipo microssatélites cobrindo os cromossomos 5, 7 e 8. Os locos foram considerados adequados para estudos de características quantitativas, quando foram analisados os valores de heterozigosidade observada (Ho), heterozigosidade esperada (He) e conteúdo de informação polimórfica (PIC). No cromossomo 5, os valores das médias de Ho, He e PIC encontrados foram 0,73; 0,66 e 0,61; no cromossomo 7, os valores encontrados foram 0,81, 071 e 0,67 e no cromossomo 8 os valores foram 0,65; 0,65 e 0,61. Com o resultado da genotipagem foi construído o mapa de ligação específico dos marcadores para a população desenvolvida. As análises de associação foram feitas utilizando mapeamento por intervalo por regressão para detecção de QTL. Foram detectados 17 QTL para características de carcaça e corte de carcaça nos três cromossomos, enquanto para característica de desempenho apenas um QTL para conversão alimentar foi encontrado no cromossomo oito. Não foram encontrados nas literaturas consultadas QTL para Peso de Carré e Espessura de Bacon no cromossomo sete. As informações dos QTL significativos encontrados servem para estudos futuros como o mapeamento fino e identificação de genes que ajudam no melhor entendimento da fisiologia e características de produção de suínos.Molecular markers can be used to identify chromosomal regions harboring quantitative trait locos (QTL) that control traits of economic importance in farm animals. To study these locos in the pig, a resource population has been generated from a cross between two Naturalized Brazilian Piau grand sires and eighteen Commercial grand dams (Landrace x Large White x Piétrain). A total of 614 F2 progeny from 50 matings of F1 parents were produced. Phenotypic data on performance, carcass, internal organs, viscera, carcass cuts and meat quality traits were collected on the F2 animals. Parentals, F1 e F2 animals were genotyped for 17 microsatellite markers covering the chromosomes 5, 7 e 8. The loci were considered appropriate for quantitative analysis, when it was analysed their values for observed heterogosity (Ho), expected heterozigosity (He) and polimorfic information content (PIC). In the chromosome 5, the average values of the Ho, He; and PIC found were 0,73; 0,66 and 0,61; in the chromosome 7, the values found were 0,81, 071 and 0,67 and in the chromosome 8 had the average values for Ho, He and PIC of 0,65; 0,65 and 0,61 respectively. After the genotype scoring, it was constructed the linkage map for these markers in the population. Association analyses were performed using interval mapping by regression for QTL detection. Seventeen QTL were mapped for carcass and cuts carcass traits on the three chromosomes, while just one QTL for feed intake were found for performance trait in the eight chromosome. One QTL for Total (bone-in) loin weight (Kg) and for bacon depth were mapped and not yet described in the literature on the seven chromosome. The generated information of significant QTL will be useful for future studies dealing fine mapping with and identification of genes that could provide a better understanding of physiology and production traits of pigs.Conselho Nacional de Desenvolvimento Científico e Tecnológic

Comparison of gene expression of Toll-like receptors and cytokines between Piau and commercial line (Landrace × Large White crossbred) pigs vaccinated against Pasteurella multocida type D

We aimed to compare Toll-like receptors (TLR) and cytokines expression in local Piau breed and a Commercial line (Landrace × Large White crossbred) pigs in response to vaccination against Pasteurella multocida type D. Seronegative gilts for Pasteurella multocida type D and Mycoplasma hyopneumoniae were used, from which peripheral blood mononuclear cells (PBMC) were collected in four time points (T0, T1, T2 and T3; before and after each vaccination dose). For bronchoalveolar lavage fluid cells (BALF), we set groups of vaccinated and unvaccinated animals for both genetic groups. Gene expression was evaluated on PBMC and BALF. In PBMC, when we analyzed time points within breeds, significant differences in expression for TLRs and cytokines, except TGFβ, were observed for Commercial animals. For the Piau pigs, only TGFβ showed differential expression. Comparing the expression among genetic groups, the Commercial pigs showed higher expression for TLRs after first vaccination dose, while for IL2, IL6, IL12 and IL13, higher expression was also observed in T3 and IL8 and IL10, in T1 and T3. Still comparing the breeds, the crossbred animals showed higher expression for TNFα in T1 and T2, while for TGFβ only in T2. For gene expression in BALF, vaccinated Commercial pigs showed higher expression of TLR6, TLR10, IL6, IL8, IL10, TNFα and TGFβ genes than vaccinated Piau pigs. The Commercial line pigs showed higher sensitivity to vaccination, while in local Piau breed lower responsiveness, which may partly explain genetic variability in immune response and will let us better understand the tolerance/susceptibility for pasteurellosis