Rui Fernando Felix Lopes

Polymerase chain reaction (PCR) for the detection of Salmonella in artificially inoculated chicken meat

The aim of this study was to develop a polymerase chain reaction (PCR) protocol for the detection of Salmonella in artificially contaminated chicken meat. Tests were performed with different dilutions of Salmonella Typhimurium or Salmonella Enteritidis cells (10(-7), 10(-8) or 10(-9) CFU/mL) inoculated in chicken meat samples, in order to establish the limits of detection, incubation times (0, 6, 8 and 24 hours of pre-enrichment in PBW 1%) and three DNA extraction protocols (phenol-chloroform, thermal treatment and thermal treatment and Sephaglass). The assay was able to detect until 10(-9) CFU/mL of initial dilution of Salmonella cells inoculated in chicken meat, which allows detection of Salmonella within 48 hours, including 24 hours of pre-enrichment and using the phenol-chloroform DNA extraction protocol. As the results are obtained in a shorter time period than that of microbiological culture, this procedure will be useful in the methodology for detection of Salmonella in chicken.

Efeito do genótipo halotano e de diferentes sistemas de produção na qualidade da carne suína

The effect of halothane genotypes (heterozygous and dominant homozygous) and intensive rearing systems (indoor, wood shavings bedding and outdoor) on pork quality were determinated. Ninety six castrated male pigs were used for the trial. Identification of the halothane genotype was determined in blood samples using the DNA-test, based on the polymerase chain reaction (PCR) amplification of the critical region of the ryanodine receptor and subsequent restricion of the amplifield fragment by the restriction fragment lengh polymorfism (RFLP) technique. The pH at 45 minutes and 24 hours after slaughter was measured on the Longissimus dorsi and Semimembranosus muscles. At the laboratory, the Longissimus dorsi muscle was used for the evaluation of the color, marbling, drip loss, defrosting loss and cooking loss and shear force. The muscle of heterozygous pigs for the halothane gene had lower pH and higher drip loss and the incidence of the PSE condition in this genotype was three times higher. The rearing system did not affect the initial and ultimate pH or water holding capacity. Indoor reared pigs had greater frequency of PSE carcass. The interaction between halothane genotypes and rearing systems had no significant effect on any trait studied.

Influência do gene halotano sobre a qualidade da carne suína

O objetivo deste trabalho foi avaliar o efeito do gene halotano sobre as caracteristicas de qualidade da carne suina. Foram utilizadas 151 carcacas de suinos hibridos comerciais, sendo 93 carcacas com genotipo halotano normal (HalNN), 51 heterozigotas (HalNn) e 7 recessivas (Halnn). As medidas efetuadas foram peso da carcaca, refletância atraves da fibra optica GP4-Hennessy, temperatura muscular aos 45 minutos e pH aos 45 minutos e 24 horas apos o abate no musculo Longissimus dorsi, cor e identificacao do genotipo halotano em amostras de gordura atraves de PCR-RFLP. Houve diferenca significativa entre suinos HalNn e HalNN e entre Halnn e HalNN quanto ao pH inicial e a cor. Em relacao a temperatura muscular e pH, final nao houve diferenca significativa entre os genotipos. A frequencia de carcacas PSE foi mais elevada nos suinos Halnn e HalNn do que nos suinos HalNN (85,71; 58,82 e 36,56%, pelo pH inicial; 71,43; 47,06 e 17,20%, pela cor, respectivamente). A qualidade da carne de suinos Halnn e HalNn foi inferior a de suinos HalNN, em termos de pH e cor.

Selection of Rattus norvegicus oocytes for in vitro maturation by brilliant cresyl blue staining

The objective of this work was to evaluate the rate of meiosis resumption and nuclear maturation of rat (Rattus norvegicus) oocytes selected for in vitro maturation (IVM) after staining of cumulus-oocyte complexes (COCs) with blue cresyl brilliant (BCB) using different protocols: exposure for 30, 60 or 90 min at 26 μM BCB (Experiment 1), and exposure for 60 min at 13, 20 or 26 μM BCB (Experiment 2). In Experiment 1, the selection of oocytes exposed to BCB for 60 min was found to be the most suitable, as meiosis resumption rates in the BCB(+) group (n = 35/61; 57.37%) were the closest to the observed in the control (not exposed) group (n = 70/90; 77.77%) and statistically higher than the values observed for the BCB(-) group (n = 3/41; 7.32%). Additionally, the more effective evaluation of diagnostic tests (sensitivity and negative predictive value 100%) was observed in COCs exposed for 60 min. In Experiment 2, the 13 μM BCB(+) group presented rates of meiosis resumption (n = 57/72; 72.22%) similar to the control group (n = 87/105; 82.86%) and higher than other concentration groups. However, this results of the analysis between BCB(-) oocytes was also higher in the 13 μM BCB group (n = 28/91; 30.78%) when compared with BCB(-) COCs exposed to 20 μM (n = 3/62; 4.84%) or 26 μM (n = 3/61; 4.92%) BCB. The nuclear maturation rate in the 13 μM BCB group was similar between BCB(+) or BCB(-) oocytes. The 20 μM BCB group had a lower rate of nuclear maturation of BCB(-) oocytes than other groups. Thus, our best results in the selection of Rattus norvegicus oocytes by staining with BCB were obtained using the concentration of 13 μM and 20 μM, and an incubation period of 60 min.

Análise da contaminação por Salmonella em ovos do tipo colonial através da reação em cadeia da polimerase

The identification of salmonella infection in commercial poultry has been one of the strong points of prophylaxis and consequent reduction of salmonellosis outbreaks in humans associated to consumption of eggs, considering that the analysis of the eggs can be one more point of detection of infection, which for many times appear without clinical signs. The Polymerase Chain Reaction (PCR) seems to be a useful strategy for Salmonella detection, because various authors have used the PCR to verify the presence of bacteria in meat, feces, tissues, blood, milk and eggs, with different methods of manipulation of samples. We have analyzed 360 eggs from ten farms, producers of free range-eggs, in the district of Camobi, in Santa Maria n RS n Brasil. The eggs were grouped in pools of six, totaling sixty samples. The bacteriological exam was done in compliance with the method preconized by the technical rules and the method for extraction of DNA was by phenol-chloroform. The PCR was performed for the amplification of a 284 bp DNA fragment. The analysis of the results do not show significant difference between the PCR and the bacteriological exam. All positive samples in the

Utilização de biópsias da terceira pálpebra e mucosa retal em ovinos para diagnóstico de scrapie em uma propriedade da região sul do Brasil

Scrapie, a form of transmissible spongiform encephalopathy (TSEs) is a fatal neurodegenerative disorder that affects sheep and goats. The disease is characterized by an accumulation of the abnormal prionic protein (PrPSc) in the encephalic and lymphoreticular tissues. This paper describes the use of anti-prionic protein immunohistochemical (IHC) procedure as a method of pre-clinical diagnosis of scrapie.The test was carried out in biopsied lymphoreticular tissues from third eyelid and rectal mucosa. Anti-prion protein monoclonal antibodies F89/160.1.5 and F99/97.6.1 were used. Scrapie diagnosis in lymphoreticular tissues through IHC was achieved when the samples had a minimum of three lymphoid follicles in well delimited germinal centre. Positive immunostaining was identified in 19 out of 318 samples of the third eyelid. Material sampled at post-mortem examination in 18 of these scrapie-positive sheep, which were previously verified by biopsy, and in 21 of its relatives, was confirmed with IHC tests. Positive immunostaining from rectal mucosa tissue was not observed. Third eyelid and tonsil were the organs with the larger amount of positive immunostaining (18/18 and 8/18 respectively) at post-mortem examination. None positive result was obtained along the 21 animals related to the positive ones, and none of the positive cases showed IHC labeling in the brain. The use of lymphoid tissues for scrapie diagnosis by IHC through biopsies showed to be a viable and efficient method for pre-clinical diagnostic.

MÉTODOS DE EXTRAÇÃO DE DNA PARA A DETECÇÃO DE Salmonella EM OVOS DE GALINHAS, COM E SEM CASCA, ATRAVÉS DA REAÇÃO EM CADEIA PELA POLIMERASE

The Salmonella sp detection in feed samples is time consuming, it has five stages and requires 120 hours for final results. The use of polimerase chain reaction technique can reduce this time considerably, however it can be affected by substances from the sample. This study had the objective of comparing two methods of DNA extraction, by heating process and by phenol-chloroform in samples of 100 chicken eggs experimentally infected with a sample of Salmonella enterica sorovar typhimurium in stationary phase. After the two extraction methods a PCR was done using a pair of oligonucleotides that amplifies a fragment of 284pb in the InvA gene de Salmonella sp. Comparing the extraction methods it was noted a difference of 12% favorably to the phenol-chloroform method when the extraction was done from eggs with shell. The same method using just the egg internal part resulted in a difference of 26% with high significance (P<0.003), showing that the method used was determinant to improve the technique efficiency. However, comparing the positive percentage independent of the DNA extraction method a significant difference (P<0.047) was noted for outshell eggs. This fact suggests that for Salmonella egg analysis, only the egg internal part should be used because the shell can determine interference in technique.

A CONTRIBUIÇÃO DO GENE HALOTANO SOBRE AS CARACTERÍSTICAS DE QUALIDADE DA CARNE SUÍNA

). Asmedidas efetuadas foram: espessura de toucinho e de musculo,percentagem de carne, peso da carcaca, pH aos 45 minutos e 24horas apos o abate, no musculo Longissimus dorsi, cor, perda deliquido por gotejamento e identificacao do genotipo halotano emamostras de gordura atraves de PCR-RFLP. Suinos Hal

Scrapie diagnosis in a goat and four Santa Inês sheep from the same herd in Brazil

Scrapie is a fatal and progressive transmissible spongiform encephalopathy (TSE) of natural occurrence in sheep and goats. The suspicion of scrapie may be based on clinical signs; however, the detection of pathological features of the prionic protein (PrP) in target tissues is necessary to diagnose the disease. The presence of an abnormal protein form (PrPSc) in lymphoreticular and nervous tissues is an important characteristic in diagnosis. This paper reports a case of scrapie in a flock of 55 Suffolk crossbred sheep, 19 Santa Ines sheep and 21 goats in the Mato Grosso state, midwestern Brazil. The animals were euthanized after the confirmation of a scrapie case with clinical signs in a Suffolk sheep in the same farm. Samples of brainstem at the level of the obex and lymphoid issues like palatine tonsils, mesenteric lymph nodes, third eyelid fixed in formalin 10% were processed for histological examination. Histological examination with hematoxylin and eosin did not show any microscopic changes in samples. Immunohistochemistry (IHC) examination to detect anti-prion PrPSc was performed in lymphoid tissues. Scrapie diagnosis was confirmed based on IHC positive results for PrPSc in lymphoid tissues of a crossbreed goat and four Santa Ines sheep, without any clinical scrapie signs. IHC showed positive staining in at least three lymphoid germinal centers in goat mesenteric lymph node, palatine tonsil, and third eyelid samples. The mesenteric lymph node, and tonsil samples of all sheep showed positive immunostaining, and only one sheep showed positive staining in lymphoid follicles in the third eyelid. Scrapie diagnosis using IHC in fixed samples of lymphoreticular tissue is technically feasible to detect the disease in both goats and sheep, as a form of pre-clinical diagnosis. The results indicate that the herd was infected by a sheep coming from another herd where scrapie had been diagnosed before.