Evaluation of reference genes for real-time PCR studies of Brazilian Somalis sheep infected by gastrointestinal nematodes

Precise normalization with reference genes is necessary, in order to obtain reliable relative expression data in response to gastrointestinal nematode infection. By using sheep from temperate regions as models, three reference genes, viz., ribosomal protein LO (RPLO), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and succinate dehydrogenase complex subunit A (SDHA), were investigated in the abomasum, abomasal lymph nodes and small intestine of Brazilian Somalis sheep, either resistant or susceptible to gastrointestinal nematodes infections. Real time PCR was carried out by using SYBR Green I dye, and gene stability was tested by geNorm. RPLO was an ideal reference gene, since its expression was constant across treatments, presented lower variation, and was ranked as the most stable in abomasum and lymph node tissues. On the other hand, SDHA was the most stable in the small intestine followed by RPLO and GAPDH. These findings demonstrate the importance of correctly choosing reference genes prior to relative quantification. In addition, we determined that reference genes used in sheep from temperate regions, when properly tested, can be applied in animals from tropical regions such as the Brazilian Somalis sheep

Descovery and study of genes involved in immune response against gastrointestinal nematodes in cattle

Este trabalho teve por objetivo identificar e estudar a expressão de genes envolvidos na resposta imune a endoparasitas gastrintestinais em bovinos. Contagem periódica de ovos por grama de fezes (OPG) e análises de coproculturas foram realizadas para identificar animais resistentes e susceptíveis a endoparasitas gastrintestinais. A contagem de OPG permitiu identificar estes animais, sendo que o grupo susceptível apresentou uma contagem 20 vezes superior ao grupo resistente (P<0,001). Análises de coproculturas permitiram concluir que a infestação em bovinos foi mista, com a predominância dos gêneros Cooperia e Haemonchus e menor incidência de Oesophagostomum. Para a identificação dos genes, foi utilizada a metodologia EST (Expressed Sequence Tag) e foram construídas duas bibliotecas de clones de cDNA obtidos a partir da mucosa do abomaso, intestino delgado e nódulos linfáticos abomasais de bovinos resistentes (ER1) e susceptíveis (ES1) a nematódeos gastrintestinais. Foram geradas 2496 ESTs de cada biblioteca. Destas, 1664 e 1898 foram válidas para as bibliotecas ER1 e ES1, respectivamente. Dentre as 2323 sequências únicas obtidas foram identificados vários produtos gênicos relacionados à resposta imune, tais como moléculas de classe I e II do MHC, imunoglobulinas, interleucinas, lisozima e pepsinogênio. Para a quantificação da expressão de RNA mensageiro, foi utilizada a metodologia de transcrição reversa e reação em cadeia da polimerase em tempo real, onde foram analisados 10 genes relacionados à polarização da resposta imune (as interleucinas IL- 2, IL-4, IL-8, IL-12p35 e IL-13, as citocinas TNF-&#945;, IFN-&#947;, MCP-1&#945; e MCP-2 e a glicoproteína mucina 1-MUC1). As análises de expressão gênica realizadas na mucosa do abomaso revelaram aumento nos níveis de RNAm para IL-4 (P<0,018), IL-13 (P<0,002) e diminuição de TNF-? (P<0,0001) nos animais resistentes; nos nódulos linfáticos abomasais houve aumento de IL-4 (P<0,019) nos animais resistentes e de IFN-&#947; (P<0,007) nos animais susceptíveis; no intestino delgado houve aumento de IL-4 (P<0,01) e IL-13 (P<0,045) nos animais resistentes, ao contrário de IL-2 (P<0,047), IL-12p35 (P<0,029), IFN-&#947; (P<0,004) e MCP-1 (P<0,03), cujos níveis de RNAm foram maiores nos animais susceptíveis. No abomaso dos animais resistentes houve menor expressão de pepsinogênio (P<0,025) e maior de lisozima (P<0,042). Em conclusão, a estratégia utilizada permitiu a identificação de vários genes envolvidos na resposta imune e a descoberta inédita de que Bos indicus resistentes a parasitas gastrintestinais apresentam uma resposta TH2 polarizada, em contraste aos animais susceptíveis, que apresentam uma resposta TH1.The aim of this work was identify genes related to immune response to gastrointestinal nematodes in cattle. The periodic counts of eggs per gram (EPG) of feces and coproculture analysis were done to identify the resistant and susceptible animals. The EPG counts allowed us to identify these animals. It was twenty-fold higher in susceptible group (P<0.001). The coproculture analysis allowed us to conclude that the infestation is predominantly characterized byCooperia spp. and Haemonchus spp. and a low incidency of Oesophagostomum. To identify the genes, it was used the EST (Expressed Sequence Tags) methodology and constructed two cDNA libraries from abomasum, abomasum lymph nodules and small intestine from resistant (ER1) and susceptible (ES1) cattle. It was generated 2496 ESTs from each library. From these, 1664 and 1898 ESTs were valids to ER1 and ES1 libraries, respectively. Among the 2323 unique sequences were identifyed several genes related to immune response, such as MHC class I and II molecules, immunoglobulins, interleukins, lysozyme and pepsinogen. To study the gene expression, it was used the reverse transcription and real-time polymerase chain reaction methodology to quantify the messager RNA expression of 10 genes related to polarization of immune response (the interleukins IL-2, IL-4, IL-8, IL-12p35 e IL-13, the cytokines TNF-&#946;, IFN- &#947;, MCP-1&#946; and MCP-2 and the glycoprotein mucin 1-MUC1). The gene expression analysis in the abomasum reveled that IL-4 (P<0,018) and IL-13 (P<0,002) were up-regulated and TNF-&#946; (P<0,0001) was down-regulated in resistant group; in the abomasum lymph nodules IL-4 (P<0,019) and IFN-&#947; (P<0,007) were both up-regulated in resistant and susceptible group, respectively; in the small intestine IL-4 (P<0,01) and IL-13 (P<0,045) were up-regulated in resistant group and IL-2 (P<0,047), IL-12p35 (P<0,029), IFN-&#947; (P<0,004) and MCP-1 (P<0,03) were down-regulated in susceptible one. In the abomasum from resistant group, pepsinogen was down-regulated (P<0,025) and lysozyme was up-regulated (P<0,042). In conclusion, the strategy used allowed us to identify several genes involved in immune response and the inedit discovery that Bos indicus resistant to gastrointestinal nematodes present a TH2-type response, in contrast to susceptible animals that present a TH1-type response

Formação inicial de professores de ciências numa perspetiva humanista

O ensino de ciências com enfoque CTS deve contribuir para preparar todos os alunos para, perante os desafios e necessidades contemporâneos, participarem fundamentadamente em processos de tomada de decisões tecnocientíficas de interesse social. Esta perspetiva implica a necessidade de os professores de ciências terem formação adequada na área específica do curso, em natureza das ciências e das tecnologias e sobre problemas sociais associados a desigualdades com que convivem e afetam os seus alunos. No Brasil, a maior parte da população pobre e com baixa escolaridade é afrodescendente e constatam-se preconceitos raciais em vários contextos, inclusive educativos. Os preconceitos afastam os jovens das escolas impedindo-os de aceder a conhecimentos científicos que os capacitem para participarem em decisões sobre questões CTS. Neste artigo abordam-se alguns problemas que configuram a presente situação de emergência planetária, discutem-se dificuldades de formação de professores, relaciona-se racismo com outras desigualdades e exclusão escolar e apresenta-se um estudo de caso integrado em formação inicial de professores de ciências, que contemplou questões étnico-raciais, num Curso de Licenciatura em Ciências Biológicas a Distância, de uma Universidade brasileira, em que participaram 54 estudantes. A reflexão de futuros professores de ciências sobre um problema social muito comum em escolas brasileiras pode contribuir para a tomada de consciência da necessidade de se superar atitudes e comportamentos racistas e de se utilizar abordagens de ensino de ciências que visem estimular todos os alunos a aprender e, assim, reduzir a evasão escolar e melhorar a alfabetização científica da população

Quantification of bovine cytokine gene expression using real-time RT-PCR methodology

T cells produce cytokines that affect host response to infection. This paper reports real-time RT-PCR conditions and validation steps for accurate quantification of Bos indicus cytokines, interleukin (IL)-2, IL-4, IL-8, IL12p-35, IL-13, tumoral necrosis factor (TNF)-alpha, interferon (IFN)-gamma, monocyte chemoattractant proteins (MCP)-1 and MCP-2, and the glycoprotein mucin (MUC)-1 in two groups of Nelore cattle, one resistant and the other susceptible to gastrointestinal nematode infections. RPL-19 was shown to be an ideal internal control gene, since its expression was constant across treatments and presented lower variation when compared to the GAPDH gene. The optimized conditions established in the present study can be used to determine the immune response of cattle under different experimental conditions, such as viral, bacterial and parasite infections.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Analysis of the stability of the reference genes GAPDH, SDHA and RPL-19 in sheep from a semi-arid region infected by gastrointestinal nematodes

Abstract Analyzing the stability of reference genes already described as universal is an important methodology to lead gene expression analysis because different studies have shown that the expression of universal reference genes may vary between experimental treatments. In this sense, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Succinate dehydrogenase complex subunit A (SDHA) and Ribosomal Protein L-19 (RPL-19) reference genes (already described in other studies with sheep from different regions, breeds and infectious agents or in organisms evolutionarily close to sheep) were investigated in the abomasum, small and large intestines of resistant and susceptible crossbred sheep groups to gastrointestinal nematode infections in the Semi-arid region in Northeast of Brazil. The animals were naturally infected to determine the resistance or susceptibility status by counting eggs per gram (EPG) of feces from the gastrointestinal tract after 33 weeks of observations of infection evolution. Relative gene expression was performed by RT-qPCR methodology using Sybr green and relative gene expression stability was tested by different software programs such as REST, BestKeeper, geNorm and Normfinder. Our results showed the susceptible animals had increase in egg counts per gram of feces than resistant animals (p < 0.001), and both groups showed a mixed infection by nematodes of the genus Haemonchus, Trichostrongylus, Oesophagostomum and Trichuris. Furthermore, we show the importance of analyzing different genes in different software programs and the importance to choose ideal reference genes. In this sense, GAPDH was the most stable gene in the abomasum, whereas SDHA was the most stable in the small and large intestines. In addition, we discuss about variables which can interfere in relative expression such as breed, species, climate and tissue. However, utilizing other reference genes already described in other studies with the same and different variables should be performed

The effect of Brachiaria brizantha cultivars on host-parasite-environment interactions in sheep naturally infected with gastrointestinal nematodes.

The objective was to evaluate the effect of different cultivars of Brachiaria brizantha on the dynamics and concentration of the larval stages of gastrointestinal nematodes in the soil and forage strata, as well as their effects on the performance of naturally infected lambs. Overall, 48 90-day-old lambs with an initial weight of 19.04 ± 0.96 kg were observed. Moreover, a randomised block factorial design with four cultivars of Brachiaria brizantha (Marandu, Xaraés, Piatã and Paiaguás grasses) under intermittent stocking (with a pre-grazing canopy height of 40 cm and post-grazing canopy height of 20 cm) for two grazing cycles was used. The following variables were analysed: faecal egg counting, faecal culture, mean corpuscular volume, FAMACHA© score, weight and body condition score, the recovery of larvae from pasture and soil samples, nutritional value and the production and structural components of forage. Lambs grazing Marandu grass demonstrated the highest level of nematode infection (P < 0.05). However, the nutritional value did not differ between cultivars. Marandu grass had the highest pasture density (P < 0.05), while Paiaguás grass had the highest percentage of dead material (P < 0.05). The various genera of gastrointestinal nematodes found in the faecal cultures, regardless of the cultivars, include Haemonchus (92.01%), Trichostrongylus (4.55%), Strongyloides (3.06%) and Oesophagostomum (0.37%). Lambs grazing Xaraés grass had the lowest body weight (P < 0.05). Furthermore, larvae concentrations were highest in Marandu and Paiaguás pastures; infective Haemonchus and Trichostrongylus larvae were recovered from pasture and soil samples. The different cultivars of Brachiaria brizantha produce diverse and relevant microclimatic conditions to contaminate soil, pastures and animals. Animal performance was not compromised despite the Marandu and Paiaguás cultivars having the highest levels of contamination and infection. Based on parasitological aspects, the Brachiaria brizantha cultivars Xaraés and Piatã are recommended for grass-based sheep production systems over the other cultivars since they contribute to the reduction of larval contamination and infection