Molecular epidemiology of Streptococcus agalactiae isolated from mastitis in Brazilian dairy herds

Abstract Streptococcus agalactiae is one of the most common pathogens leading to mastitis in dairy herds worldwide; consequently, the pathogen causes major economic losses for affected farmers. In this study, multilocus sequence typing (MLST), genotypic capsular typing by multiplex polymerase chain reaction (PCR), and virulence gene detection were performed to address the molecular epidemiology of 59 bovine (mastitis) S. agalactiae isolates from 36 dairy farms located in the largest milk-producing mesoregions in Brazil (Minas Gerais, São Paulo, Paraná, and Pernambuco). We screened for the virulence genes bac, bca, bibA, cfb, hylB, fbsA, fbsB, PI-1, PI-2a, and PI-2b, which are associated with adhesion, invasion, tissue damage, and/or immune evasion. Furthermore, five capsular types were identified (Ia, Ib, II, III, and IV), and a few isolates were classified as non-typeable (NT). MLST revealed the following eight sequence types (STs): ST-61, ST-67, ST-103, ST-146, ST-226, ST-314, and ST-570, which were clustered in five clonal complexes (CC64, CC67, CC103, CC17, and CC314), and one singleton, ST-91. Among the virulence genes screened in this study, PI-2b, fbsB, cfb, and hylB appear to be the most important during mastitis development in cattle. Collectively, these results establish the molecular epidemiology of S. agalactiae isolated from cows in Brazilian herds. We believe that the data presented here provide a foundation for future research aimed at developing and implementing new preventative and treatment options for mastitis caused by S. agalactiae.

Seleção, caracterização e clonagem dos genes fljB e groEL agonistas dos receptores de reconhecimento de padrão do sistema imune inato das aves / Selection, characterization and cloning of the fljB and groEL genes agonists of the innate immune system pattern recognition receptors of birds

The recombinant production of innate immune system pattern recognition receptor agonists has provided a new tool for the production of immunostimulants for animals. The molecular pattern associated with the pathogen (PAMP), flagellin, coded by the fljB gene from Salmonella Typhimirium, and the molecular pattern associated to the damage (DAMP), HSP60, coded by the groEL gene from S. Typhimurium and S. Enteritidis, are recognized by pattern recognition receptors (PRRs) of the innate immune system of birds. In the present study, we performed the cloning of genetic fragments of the genes fljB, from S. Typhimurium, and groEL from S. Typhimurium and S. Enteritidis inserted in expression vector pET100/D-TOPO and transformed in E. coli TO10 cells. The clones were evaluated by colony PCR, plasmidial DNA PCR and genome sequencing in order to confirm the presence of these genes. In the colony PCR, we identified the presence of genes groEL (S. Enteritidis), groEL (S. Typhimurium) and fljB (S. Typhimurium) in 80%, 60% and 80% of the transformed colonies, respectively. The cloning system adopted allowed the production of HSP60 genetic fragment clones and flagellin of Salmonella strains, allowing the posterior use of these clones in gene expression trials, with the future potential of being used as non-specific immunostimulants for birds.

A produção recombinante de agonistas dos receptores do reconhecimento de padrão do sistema imune inato tem fornecido uma nova ferramenta para a produção de imunoestimulantes para animais. O padrão molecular associado ao patógeno (PAMP), flagelina, codificado pelo gene fljB de Salmonella Typhimurium e o padrão molecular associado ao dano (DAMP) HSP60, codificado pelo gene groEL da S. Typhimurium e S. Enteritidis, são reconhecidos por receptores de reconhecimento de padrões (RRPs) do sistema imune inato das aves. No presente estudo, foi feita a clonagem de fragmentos genéticos dos genes fljB de S. Typhimurium e groEL de S. Typhimurium e S. Enteritidis inseridos no vetor de expressão pET100/D-TOPO e transformados em células de E. coli TOP10. Os clones foram avaliados pela PCR de colônia, PCR de DNA plasmidial e sequenciamento genômico para a confirmação da presença desses genes. Na PCR de colônia, foram identificadas em 80%, 60% e 80% das colônias transformadas, a presença dos genes groEL (S. Enteritidis), groEL (S. Typhimurium) e fljB (S. Typhimurium) respectivamente. O sistema de clonagem adotado possibilitou a produção de clones dos fragmentos genéticos da HSP60 e flagelina das cepas de Salmonella, permitindo a utilização posterior desses clones em ensaios de expressão gênica, com potencial futuro de serem utilizados como imunoestimulante inespecífico das aves.

Divergência genética entre cultivares de gérbera utilizando marcadores RAPD / Genetic divergence among cultivars of gerbera using RAPD markers

No processo de produção comercial de mudas de gérbera, a cor da flor é uma das principais características morfológicas de interesse agronômico, sendo uma característica importante em programas de melhoramento genético. A utilização de marcadores moleculares pode servir para direcionar cruzamentos, confirmar novos híbridos ou genótipos mutantes e identificar novos genótipos para fins comerciais. Nesse contexto, o objetivo deste trabalho foi analisar a divergência genética entre seis cultivares de Gerbera jamesonii ('Jaguar Yellow', 'Jaguar Cream', 'Jaguar Lemon', 'Jaguar Salmon Pastel', 'Jaguar Red', 'Jaguar Deep Rose'). A análise de divergência genética entre as cultivares de gérbera foi realizada utilizando-se 21 primers, os quais amplificaram 37 fragmentos polimórficos de DNA, que foram usados para estimar o coeficiente de Jaccard, o qual apresentou uma média de 0,38, variando de 0,28 a 0,56. A estrutura genética entre as cultivares foi estimada pelo UPGMA, revelando dois grupos distintos, a 38 por cento de similaridade genética. A maior similaridade genética encontrada (56 por cento) foi entre as cultivares 'Jaguar Yellow' e 'Jaguar Lemon'. Os resultados demonstram que a técnica RAPD oferece uma maneira rápida, relativamente barata e útil para a caracterização da divergência genética entre as diferentes cultivares de Gerbera jamesonii com relação à cor da flor.

During the commercial production of gerbera seedlings, flower color is one of the main morphological aspects that have an agronomic interest and becoming an important feature in genetic breeding programs. The use of molecular markers may serve to direct crossings, new hybrids and mutants, besides confirm and identify new genotypes for commercial purposes. In that context, this work aimed to analyze the genetic divergence among six cultivars of Gerbera jamesonii ('Jaguar Yellow', 'Jaguar Cream', 'Jaguar Lemon', 'Jaguar Salmon Pastel', 'Jaguar Red', 'Jaguar Deep Rose'). The genetic divergence among cultivars of gerbera was carried out with 21 primers, which amplified 37 DNA polymorphic fragments, used to estimate the Jaccard index and presented an average of 0,38, ranging from 0,28 to 0,56. The genetic structure among cultivars was estimated by UPGMA and revealed two distinct groups, at 38 percent genetic similarity. The largest genetic similarity found (56 percent) was between cultivars 'Jaguar Yellow' and 'Jaguar Lemon'. The results showed that the RAPD is a fast, relatively inexpensive and useful technique for genetic divergence characterization between different cultivars of Gerbera jamesonii.

Identificação de variantes somaclonais em bananeiras 'Prata Anã', utilizando técnicas moleculares e citogenéticas / Identification of somaclonal variants in 'Prata Anã' banana using molecular and cytogenetic techniques

Variação somaclonal é uma variação fenotípica de origem genética, ou seja, uma variação cromossômica que se torna herdável nas gerações seguintes, ou epigenética, que é uma variação transitória devido ao estresse fisiológico que o material sofre, quando submetido ao cultivo in vitro. Um problema específico envolvendo a variação somaclonal em bananeiras 'Prata Anã' foi observado em Andradas, Minas Gerais, em plantas oriundas de micropropagação. A maior dificuldade na separação dos indivíduos normais e variantes é que os caracteres morfológicos, que são inerentes a este tipo de variação, só se tornam evidentes quando a planta está adulta, o que impossibilita a eliminação dos indivíduos variantes ainda em viveiro. Com o objetivo de identificar, ainda em viveiro aqueles indivíduos variantes somaclonais, técnicas moleculares (RAPD e SSR) e citogenéticas (contagem cromossômica e citometria de fluxo) foram utilizadas. Cento e três primers RAPD, 11 combinações de dois primers RAPD, e 33 pares de primers SSR foram utilizados na tentativa de se encontrar marcadores polimórficos capazes de distinguir os indivíduos normais dos variantes, além de distinguir bananeiras 'Prata Anã' de 'Prata'. O primer OPW-08 gerou um fragmento polimórfico que distinguiu uma planta variante de todas as demais, provando que a variação não ocorre de maneira uniforme no genoma dos indivíduos variantes e que não há um retorno à cultivar Prata. As análises com marcadores SSR e a contagem cromossômica não possibilitaram a distinção dos indivíduos variantes, nem a separação das cultivares Prata e Prata Anã. As análises de citometria de fluxo evidenciaram a grande instabilidade cromossômica das bananeiras, porém elas não foram eficientes na identificação de variantes somaclonais.

Somaclonal variation is a phenotypical variation of genetic origin, that is, a chromosomal variation that becomes inheritable in the generations to follow, or of epigenetic origin, in this case being a transitory variation due to the physiological stress suffered when the material is submitted to in vitro cultivation. A specific problem involving somaclonal variation in 'Prata Anã' banana was observed in Andradas, Minas Gerais, in plants originated from tissue culture. The main difficulty in the distinction between the normal and variant plants is the fact that the morphological characters that allow the separation of these two types are only visible and distinguishable when the plants are in their adult phase, which makes it impossible to eliminate the variant seedlings at the nursery stage. For the early distinction of the variants, molecular (RAPD - Random Amplified Polymorphic DNA and SSR - Simple Sequence Repeat) and cytogenetic (chromosome counting and flow cytometry) techniques were used. In the attempt to find polymorphic markers that distinguished the normal plants from the variants as well as the Prata cultivar from the Prata Anã cultivar, 103 RAPD primers, 11 combinations of two RAPD primers, and 33 pairs of SSR primers were used. Primer OPW-08 generated a polymorphic fragment that distinguished a variant from all of the other plants, proving that the variation does not occur uniformly in the genome of all variants, and that there is no return to Prata cultivar. Analyses with SSR markers and chromosome counting were not efficient in separating normal plants from variants or 'Prata' from 'Prata Anã'. Flow cytometry analyses showed an evident instability in the banana genome in terms of number of chromosomes, however, they were not efficient in identifying the somaclonal variants.

Organogênese em capítulos florais e avaliação de características anatômicas da folha de Gerbera jamesonii Adlam / Capitulum organogenesis and anatomical characterization of Gerbera jamesonii Adlan leaves

Objetivou-se determinar um protocolo de micropropagação por organogênese indireta em capítulos florais de gérbera (Gerbera jamesonii Adlam) e comparar as características anatômicas de folhas de gérbera obtidas in vitro com as mantidas em condições in vivo. Capítulos florais de gérbera foram utilizados como fonte inicial de explantes para a indução de calos e regeneração. As brotações obtidas foram enraizadas in vitro e, após 30 dias, as plântulas foram aclimatizadas. Posteriormente, foram realizados estudos anatômicos de folhas provenientes do cultivo in vivo e in vitro. Obtiveram-se em média 3,2 brotações e 6,6 folhas a partir da indução de calos em capítulos florais de gérbera. Foi observada a formação de raízes na ausência e na presença de ANA, obtendo-se 100 por cento de enraizamento. A suplementação do meio de cultura com doses crescentes de ANA promoveram um aumento linear no número de raízes e no comprimento médio de raízes. As plântulas provenientes do cultivo in vitro apresentaram taxa de 100 por cento de sobrevivência na aclimatização. As estruturas foliares desenvolvidas in vivo apresentaram as epidermes adaxial e abaxial, parênquimas paliçádico e esponjoso mais espessos que no cultivo in vitro. O sistema vascular em folhas produzidas in vivo é mais desenvolvido que in vitro.

The objective was to determine a micropropagation protocol for indirect organogenesis and to compare the anatomical characteristics of leaves of gerbera (Gerbera jamesonii Adlam) obtained in vitro with the leaves maintained in vivo conditions. Capitulum explants of gerbera were taken as an initial source of explants to induce callus and regeneration. The obtained shoots were rootted in vitro and after 30 days seedlings were acclimatized.Thus, anatomical studies of leaves originating from of the in vivo and in vitro cultivation were taken. On average it was obtained 3,2 shoots and 6,6 leaves from the induction of callus in capitulum explants of gerbera. The formation of roots was observed in the presence and absence of NAA, obtaining 100 percent of rooting. The supplementation of NAA to the medium promoted a linear increase in the number of roots and in the mean length of roots. Seedlings from the in vitro cultivation showed rate of 100 percent of survival in the acclimatization. The foliar structures developed in vivo showed adaxial epidermis, palisade parenchyma, spongy parenchyma and abaxial epidermis thicker than in the in vitro cultivation. The vascular system in leaves produced in vivo is more developed than in vitro.

Analysis of the SHP2 enhancer for the use of tissue specific activation tagging in Arabidopsis thaliana

Activation tagging is a powerful tool to identify new mutants and to obtain information about possible biological functions of the overexpressed genes. The quadruple cauliflower mosaic virus (CaMV) 35S enhancer fragment is a strong enhancer, which is most commonly used for this purpose. However, the constitutive nature of this enhancer may generate lethal mutations or aberrations in different plant organs by the same overexpressed gene. A tissue-specific activation tagging approach may overcome these drawbacks and may also lead more efficiently to the desired phenotype. For this reason the SHATTERPROOF2 (SHP2) promoter fragment was analysed for enhancer activity. The SHP2 gene is involved in dehiscence zone development and expressed during silique development. The aim of the experiments described here was to identify a dehiscence zone specific enhancer that could be used for tissue-specific activation tagging. The chosen SHP2 enhancer fragment was found to be expressed predominantly in the dehiscence zone and showed enhancer activity as well as ectopic expression activity. This activity was not influenced by its orientation towards the promoter and it was still functional at the largest tested distance of 2.0 kb. Based on these results, the SHP2 enhancer fragment can potentially be used in a tissue-specific activation tagging approach to identify new Arabidopsis mutants with an altered dehiscence zone formation.