Sensitivity evaluation of a single-step PCR assay using Ehrlichia canis p28 gene as a target and its application in diagnosis of canine ehrlichiosis / Avaliação da sensibilidade da PCR em uma etapa com base no gene p28 de Ehrlichia canis e sua aplicação no diagnóstico da erliquiose canina

The aim of this study was to optimize a PCR assay that amplifies an 843 pb fragment from the p28 gene of Ehrlichia canis and compare it with two other PCR methods used to amplify portions of the 16S rRNA and dsb genes of Ehrlichia. Blood samples were collected from dogs suspected of having a positive diagnosis for canine ehrlichiosis. Amplification of the p28 gene by PCR produced an 843-bp fragment and this assay could detect DNA from one gene copy among 1 billion cells. All positive samples detected by the p28-based PCR were also positive by the 16S rRNA nested-PCR and also by the dsb-based PCR. Among the p28-based PCR negative samples, 55.3 percent were co-negatives, but 27.6 percent were positive in 16S rRNA and dsb based PCR assays. The p28-based PCR seems to be a useful test for the molecular detection of E. canis, however improvements in this PCR sensitivity are desired, so that it can become an important alternative in the diagnosis of canine ehrlichiosis.

O objetivo deste estudo foi aperfeiçoar um ensaio de PCR que amplificasse um fragmento de 843 pares de bases do gene p28 da Ehrlichia canis e compará-lo com outros dois métodos de PCR utilizados para amplificar partes do gene 16S rRNA e dsb do gênero Ehrlichia. Amostras sanguíneas foram colhidas de cães com diagnóstico clínico de erliquiose. A amplificação do gene p28 pela PCR produziu um fragmento de 843pb e esse ensaio permitiu a detecção do DNA de um parasita dentre 1 bilhão de células. Todas as amostras positivas detectadas pela PCR baseada no gene p28 foram também positivas pela nested PCR para detecção do gene 16S rRNA e também pela PCR dsb. Dentre as amostras negativas para a PCR p28, 55,3 por cento foram co-negativas, mas 27,6 por cento foram positivas pela PCR baseada nos genes 16S rRNA e dsb. A PCR p28 parece ser um teste útil para detecção molecular de E. canis, entretanto otimizações na sensibilidade nesta PCR são necessárias, para que esta técnica se torne uma importante alternativa no diagnóstico da erliquiose canina.

Structure and genetic relationships between Brazilian naturalized and exotic purebred goat domestic goat (Capra hircus) breeds based on microsatellites

The genetic relationships and structure of fourteen goat (Capra hircus) populations were estimated based on genotyping data from 14 goat populations (n = 410 goats) at 13 microsatellite loci. We used analysis of molecular variance (AMOVA), principal component analysis (PCA) and F statistics (F IS, F IT and F ST) to evaluate the genetic diversity (Ho, He and ad) of the goats. Genetic distances between the 14 goat populations were calculated from allelic frequency data for the 13 microsatellite markers. Moderate differentiation was observed for the populations of the undefined breeds (including the Anglo-Nubian-M breed), the naturalized Brazilian breeds (Moxotó, Canindé), the exotic purebred breeds (Alpine, Saanen, Toggenbourg and Anglo-Nubian) and the naturalized Brazilian Graúna group. Our AMOVA showed that a major portion (88.51 percent) of the total genetic variation resulted from differences between individual goats within populations, while between-populations variation accounted for the remaining 11.49 percent of genetic variation. We used a Reynolds genetic distance matrix and PCA to produce a phenogram based on the 14 goat populations and found three clusters, or groups, consisting of the goats belonging to the undefined breed, the naturalized breeds and the exotic purebred breeds. The closer proximity of the Canindé breed from the Brazilian state of Paraíba to the Graúna breed from the same state than to the genetically conserved Canindé breed from the Brazilian state of Ceará, as well as the heterozygosity values and significant deviations from Hardy-Weinberg equilibrium suggests that there was a high number of homozygotes in the populations studied, and indicates the importance of the State for the conservation of the local breeds. Cataloguing the genetic profile of Brazilian goat populations provides essential information for conservation and genetic improvements programs.

Hepatic mRNA expression and plasma levels of insulin-like growth factor-I (IGF-I) in broiler chickens selected for different growth rates

The hepatic expression and plasma concentrations of IGF-I were investigated in three broiler chicken strains selected for different growth rates (HP-Hubbard-Pettersen, a fast growing strain; NN-Naked-neck, a strain with an intermediate growth rate and a heterozygous genotype, and C-Caipira, a slow growing crossbred strain). The chickens were studied at 1, 21 and 42 days of age and had free access to food throughout the study. Hepatic IGF-I mRNA expression was assessed by dot blot analysis using a randomly labeled chicken IGF-I cDNA as the probe and plasma IGF-I concentrations were assayed by radioimmunoassay. The hepatic levels of IGF-I mRNA increased from 1 to 21 days of age in all strains, with NN chickens showing a higher (p < 0.05) IGF-I expression than the other strains. Plasma IGF-I concentrations increased (p < 0.05) with broiler chicken age, but there were no significant differences among the strains. These results indicate that despite differences in the growth rates among the strains, the changes in the expression of IGF-I mRNA in liver and in the plasma levels of IGF-I were independent of broiler chicken strain, but varied with chicken age.

Carbohydrate metabolism of Xylella fastidiosa: Detection of glycolytic and pentose phosphate pathway enzymes and cloning and expression of the enolase gene

The objective of this work was to assess the functionality of the glycolytic pathways in the bacterium Xylella fastidiosa. To this effect, the enzymes phosphoglucose isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase of the glycolytic pathway, and glucose 6-phosphate dehydrogenase of the Entner-Doudoroff pathway were studied, followed by cloning and expression studies of the enolase gene and determination of its activity. These studies showed that X. fastidiosa does not use the glycolytic pathway to metabolize carbohydrates, which explains the increased duplication time of this phytopatogen. Recombinant enolase was expressed as inclusion bodies and solubilized with urea (most efficient extractor), Triton X-100, and TCA. Enolase extracted from X. fastidiosa and from chicken muscle and liver is irreversibly inactivated by urea. The purification of enolase was partial and resulted in a low yield. No enzymatic activity was detected for either recombinant and native enolases, aldolase, and glyceraldehyde-3-phosphate dehydrogenase, suggesting that X. fastidiosa uses the Entner-Doudoroff pathway to produce pyruvate. Evidence is presented supporting the idea that the regulation of genes and the presence of isoforms with regulation patterns might make it difficult to understand the metabolism of carbohydrates in X. fastidiosa

Polymorphism analysis of the hsp70 stress gene in Broiler chickens (Gallus gallus) of different breeds

The promoter region and the beginning of the coding region of the hsp70 stress gene were analysed in broiler chickens of a commercial breed (Hubbard-Pettersen), a breed selected for weight gain (PP1) and a non-selected breed (naked-neck Label Rouge). The naked neck gene (Naked neck, Na), which reduces feathering in birds and is thus related to heat resistance, was present in both PP1 and Label Rouge breeds. Genomic DNA was restricted with PstI and Southern blotting analysis of the samples revealed the absence of polymorphic sites for that enzyme in the promoter region and beginning of the coding region of the hsp70 gene of studied birds. PCR-SSCP analysis of these regions, however, indicated the presence of polymorphisms in the beginning of the coding region and the sequencing of the PCR products confirmed and identified two polymorphic sites in this region: a transition A ® G in position +258 and a transversion C ® G in position +276. Both mutations were considered to be silent, since they did not modify the aminoacid sequence of the protein Hsp70. The promoter region of the hsp70 gene was identical in all studied birds, indicating that the regulation pattern of this gene must be the same in all birds at the promoter region. Three different alleles (hsp70-1, hsp70-2 and hsp70-3) were identified for the hsp70 gene from the observed mutations. The allele hsp70-3 was detected in only two breeds, Hubbard-Pettersen and PP1, but at a low frequency (0,016 and 0,006, respectively)

Sexagem de embriöes bovinos fecundados in vitro pela técnica de PCR multiplex / Sexing of in vitro fertilized bovine embryos by multiplex PCR

Neste trabalho, a técnica de PCR ("polymerase chain reaction") foi utilizada para a sexagem de 92 embriöes bovinos fertilizados in vitro. Os embriöes originaram-se de fertilizaçäo in vitro de oócitos aspirados de ovários de fêmeas bovinas, provenientes de abatedouros comerciais. Os oócitos foram maturados, fertilizados e cultivados até o estádio de blastocisto. Os embriöes foram lavados em soluçäo de PBS, transferidos para tubos de polipropileno contendo água ultrapura, e imediatamente congelados a -196§C. Os embriöes foram descongelados sobre isopor contendo gelo picado e tratados com proteinase K. Para a reaçäo de PCR, utilizaram-se alíquotas de 34 µl de cada tudo, onde foram acrescidos dois pares de primers, seqüência BC1.2 e seqüência satélite 1.715, desoxinucleotídeos, MgCl2, tampao PCR 10X, TaqDNA polimerase e água, em um volume final de 50 µl. As amostras foram amplificadas e a eletroforese realizada em gel de poliacrilamida a 8 por cento. Os géis foram corados com soluçäo de brometo de etídio e analisados em transiluminador de luz ultravioleta. Um índice de 93,47 por cento de amplificaçäo foi atingido, com 41 embriöes (47,67 por cento) machos e 45 (52,32 por cento) embriöes fêmeas. O uso de gel de poliacrilamida a 8 por cento foi eficaz na separaçäo de fragmentos de DNA muito próximos

Variaçäo das características físico-químicas e microbiológicas das salmouras empregadas na salga de queijo tipo mussarela durante o período de sua utilizaçäo / Variation of physical, chemical and microbiological characteristics of brines applied in the salting of mozzarella cheese during the period of utilization

Foram analisadas 40 amostras de salmouras empregadas na salga por submersäo de queijos tipo mussarela, em uma indústria de laticínios do Estado de Säo Paulo, Brasil, com o objetivo de se conhecer a variaçäo das características físico-químicas e microbiológicas durante o período de sua utilizaçäo. Os valores médios do pH, concentraçäo de cloreto de sódio e de proteínas solúveis, desde o preparo de salmoura até o 21§ dia de utilizaçäo, variaram de 7,21 a 5,76, 27,1 a 24,5 e de zero a 0,126 mg/ml, respectivamente. Por outro lado, os valores médios das contagens de microrganismos mesófilos e do número mais provável de coliformes totais e de origem fecal variaram de 5,8 x 10 UFC/ml a 6,9 x 10*4 UFC/ml, zero a 1,6 x 10*5/100ml e zero a 1,1 x 10*5/10ml, respectivamente. Além disso, os valores médios das contagens dos bolores e leveduras e de Staphylococcus coagulase positiva, variaram de 0,4 x 10 UFC/ml a 2,0 x 10*3 UFC/ml e de zero a 1,3 x 10 UFC/ml, respectivamente. Os resultados encontrados sugerem a existência de condiçöes higiênicas inadequadas durante o preparo e utilizaçäo das salmouras, de modo a representar uma importante fonte de contaminaçäo para os queijos. Em decorrência deste fato, existe a possibilidade do comprometimento da qualidade dos queijos, de modo a representar risco potencial à populaçäo consumidora