Reação em cadeia pela polimerase (PCR) para detecção de Salmonella em carne de frango artificialmente contaminada

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.O objetivo deste trabalho foi adequar um protocolo de reação em cadeia pela polimerase (PCR) para detecção de Salmonella em carne de frango artificialmente contaminada. Foram realizados ensaios com amostras de carne de frango inoculadas com diluições de Salmonella Typhimurium ou Salmonella Enteritidis (10-7, 10-8 ou 10-9 UFC/mL), procurando-se determinar o limite de detecção da técnica, intervalos de incubação das amostras (0, 6, 8 ou 24 horas de pré-enriquecimento em água peptonada 1%) e três protocolos de extração de DNA (fenol-clorofórmio, tratamento térmico ou tratamento térmico e Sephaglass). Foi possível amplificar DNA de Salmonella nas amostras de carne de frango inoculadas inicialmente com diluições de até 10-9 UFC/mL, utilizando-se protocolo de extração de DNA por fenol-clorofórmio e após um período de 24 horas de pré-enriquecimento, totalizando 48 horas de análise. Como os resultados são obtidos mais rapidamente que a cultura, este procedimento será útil na metodologia para detecção de Salmonella em carne de frango

Comportamento de células do sistema imune frente ao desafio com Salmonella Enteritidis em aves tratadas e não tratadas com ácidos orgânicos

A Salmonelose é uma importante zoonose, considerada a principal causa de infecções bacterianas, sendo associada ao consumo de produtos avícolas. Como alternativa de controle, ácidos orgânicos têm sido amplamente usados. No entanto, pouco se conhece sobre o estado imunológico de aves de produção, e uma avaliação deste status é necessária para proteger frente a enfermidades e para garantir à aplicação segura de agentes terapêuticos ou imunização profilática. Este trabalho teve como objetivo verificar o comportamento do sistema imunológico das aves previamente infectadas com Salmonella Enteritidis (SE) tratadas com um composto de ácidos orgânicos em diferentes concentrações administrado via água e ração comparando com as aves infectadas e não tratadas. Foram inoculados 120 frangos de corte com 1mL de SE, via oral, na concentração de 1,0 x 108 UFC/mL, no 1º e 2º dia de idade, divididos em seis tratamentos com duas repetições, utilizando 200, 400, 500 e 1000ppm do ácido orgânico. Aos 35 dias de vida das aves, foram coletados, de todos os grupos, alíquotas de sangue de 3mL em tubo contendo EDTA para a avaliação das células imunes através de citometria de fluxo. Foram analisadas as porcentagens circulantes de células CD4+, CD8β+, MHC I+, MHC II+, TCRVβ1+, TCRVβ2+ e CD28+. Para análise microbiológica foram coletadas tonsilas cecais destas aves. Observou-se com esse estudo que os ácidos orgânicos nas dosagens 1000ppm na água e 500ppm na ração durante, dois e sete dias respectivamente antes do abate, foram eficazes na redução da infecção por SE em frangos de corte, comprovadas pelo método microbiológico e demonstradas através do comportamento das células do sistema imune. No presente estudo as aves infectadas apresentaram uma proporção menor de células T auxiliares circulantes quando comparadas às aves infectadas, mas tratadas com o AO ou com o grupo não infectado. A mesma tendência pode ser observada para as células CD28+, TCRVβ1+ e MHC IIbright+, e, com menor resolução, para CD8β+

Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): Study protocol for a randomized controlled trial

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH(2)O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure <= 30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method.Hospital do Coracao (HCor) as part of the Program 'Hospitais de Excelencia a Servico do SUS (PROADI-SUS)'Brazilian Ministry of Healt

Detecção de fatores de virulência de Escherichia coli e análise de Salmonella spp. em psitacídeos

A flora entérica dos psitacídeos é composta principalmente por bactérias Gram positivas. Bactérias Gram negativas, como Escherichia coli e Salmonella spp., apresentam elevado potencial patogênico, sendo consideradas indicativo de problemas de manejo, que poderão culminar em manifestação de doenças em decorrência de fatores estressantes, dietas deficientes e superlotação, combinados com alta carga bacteriana no ambiente. O objetivo deste trabalho foi avaliar a presença de Salmonella spp., Escherichia coli e os fatores de virulência dos genes iss e iutA dos isolados de E. coli. Analisou-se um total de 44 amostras provenientes de psitacídeos criados em cativeiro, sendo estas 15 fragmentos de órgãos de aves submetidas a exame de necropsia e também 29 amostras de swabs de cloaca e inglúvio de papagaios-charão (Amazona pretrei) criados em cativeiro. Nenhuma amostra foi positiva para Salmonella spp. Nas amostras de E. coli detectou-se ambos os fatores de virulência pesquisados

Detecção de fatores de virulência de Escherichia coli e análise de Salmonella spp. em psitacídeos Detection of virulence factors in Escherichia coli and analysis of Salmonella spp. in psittacines

A flora entérica dos psitacídeos é composta principalmente por bactérias Gram positivas. Bactérias Gram negativas, como Escherichia coli e Salmonella spp., apresentam elevado potencial patogênico, sendo consideradas indicativo de problemas de manejo, que poderão culminar em manifestação de doenças em decorrência de fatores estressantes, dietas deficientes e superlotação, combinados com alta carga bacteriana no ambiente. O objetivo deste trabalho foi avaliar a presença de Salmonella spp., Escherichia coli e os fatores de virulência dos genes iss e iutA dos isolados de E. coli. Analisou-se um total de 44 amostras provenientes de psitacídeos criados em cativeiro, sendo estas 15 fragmentos de órgãos de aves submetidas a exame de necropsia e também 29 amostras de swabs de cloaca e inglúvio de papagaios-charão (Amazona pretrei) criados em cativeiro. Nenhuma amostra foi positiva para Salmonella spp. Nas amostras de E. coli detectou-se ambos os fatores de virulência pesquisados.The enteric flora of psittacines is mainly composed of Gram positive bacteria. Gram negative bacteria, like Escherichia coli and Salmonella spp., have a high pathogenic potential and can be considerate as an indicative of management problems that may culminate in disease manifestation due to stress factors, poor diets and overcrowding, in combination with a high bacterial load on the environment. The objective of this study was evaluated the presence of Salmonella spp., Escherichia coli and the virulence genes iss and iutA from E. coli isolates. Forty-four samples were analyzed from psittacines living in captivity, which fifteen samples were from organs fragments of necropsied birds, and twenty-nine were from cloacal and crop swabs of red-spectacled parrots (Amazona pretrei) keeping in captivity. No samples were positive for Salmonella spp. In the samples in which E. coli was detected, both virulence factors (genes iss and iutA) were present

Efficacy of phosphine gas against the darkling beetle (Alphitobius diaperinus)

Background: The darkling beetle, Alphitobius diaperinus is the most commonly beetle found in poultry sheds and causes economic and sanitary impact in the poultry industry. The life cycle of the mealworm can vary from one to three months depending on environmental conditions, and adults can survive for up to one year. The insect lives in the poultry litter where it eats feed and organic waste. The temperature in the poultry house and the accumulation of feed and organic matter promote ideal conditions for beetle infestation. The consumption of beetles affects feed conversion in poultry, especially in the first days of life and it is often cited as a vector of viral, bacterial and parasitic pathogens. The control of its populations is generally achieved by insecticide application on the walls and floor, but resistant populations of beetles are often reported. Phosphine gas is used as a fumigant to control insects in stored grain. In this study the efficacy of phosphine gas against this beetle was evaluated. Materials, Methods & Results: Two experiments were conducted: one in vitro trial, and a trial simulating field conditions. The in vitro trial aimed to evaluate the exposure time required (ETR) to obtain 100% insect mortality, in the presence and absence of wood shavings. Adults and larvae were tested separately. In treatment T1, 100 adult beetles were placed in a petri dish without poultry litter; treatment T2, had 100 adult beetles per plate and filled with sterilized poultry litter. Treatments T3 and T4 had 100 A. diaperinus larvae per plate, in absence and presence of poultry litter, respectively. Three repetitions were performed for each treatment. Insect mortality in plates was monitored at 5 min intervals. The absence of beetle movements after shaking the plate was considered an indicator of insect mortality. The field evaluation was carried out in a poultry house with litter infested with A. diaperinus. The evaluation was made in areas of 1 m2 where 2 gm-3 of aluminum phosphide was applied, according to manufactures recommendation. The area of product application was covered with a plastic cover to seal the litter. Under in vitro conditions the ETR for total mortality of insects in plates with presence of litter was 15 min for larvae and 20 min for adults. In plates without poultry litter the ETR for A. diaperinus mortality was 25 and 35 min, for larvae and adults, respectively. It was observed that the presence of poultry litter in the plates reduced the volume and so increased the phosphine gas concentration, causing more rapid inset death. The mortality of adults and larvae insects occurred approximately 30 s after the exposure to the product. All the insects exposed to the phosphine gas died, resistant insects were not observed. In the field test the ETR to obtain 100% of mortality was 90 min for larvae and adults. Discussion: The phosphine gas showed good potential to be used in strategic control of A. diaperinus. The product was effective in the killing adults and larvae beetles on in vitro and in field trials. However, more studies are needed to establish the issues related to safe use of PH3 in poultry facilities