The objective of the current study was to evaluate various RNA extraction and polymerase chain reaction (PCR) protocols for the detection of Porcine reproductive and respiratory syndrome virus ...(PRRSV) in porcine oral fluids. Extraction protocols were selected based on ease of use and compatibility with high-throughput, automated systems. The results showed marked differences among extraction protocols, PCR protocols, and combinations thereof in detecting PRRSV in the oral fluid matrix. An important finding was that PCR reactions were partially inhibited by unknown factors in the oral fluid matrix and that inhibition was reduced by use of a higher concentration of PCR enzymes. The results suggest that further optimization of PCR assays for porcine oral fluids is needed and that laboratories should not assume that methods optimized for detection of virus in serum will perform equally with porcine oral fluids.
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NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
While real-time–polymerase chain reaction (RT PCR) has been used as a rapid test for detection of Salmonella Enteritidis in recent years, little research has been done to assess the feasibility of ...pooling poultry environmental samples with a Salmonella Enteritidis–specific RT PCR assay. Therefore the objective of this study was to compare RT PCR Salmonella Enteritidis detection in individual and pooled (in groups of two, three, and four) poultry environmental drag swab samples to traditional cultural methods. The drag swabs were collected from poultry facilities previously confirmed positive for Salmonella Enteritidis and were cultured according to National Poultry Improvement Plan guidelines. Initial, Salmonella Enteritidis–specific RT PCR assay threshold cycle cutoff values of ≤36, ≤30, and ≤28 were evaluated in comparison to culture. The average limit of detection of the RT PCR assay was 2.4 × 103 colony-forming units (CFUs)/ml, which corresponded to an average threshold cycle value of 36.6. Before enrichment, samples inoculated with concentrations from 102 to 105 CFUs/ml were detected by RT PCR, while after enrichment, samples inoculated from 100 to 105 CFUs/ml were detected by RT PCR. Threshold cycle cutoff values were used in the subsequent field trial from which Salmonella Enteritidis was cultured in 7 of 208 environmental samples (3.4%). Individual samples were 99.0%, 100%, and 100% in agreement with the RT PCR at threshold cycle (Ct) cutoff values of ≤36, ≤30, and ≤28 respectively. The agreement for pooled samples also followed the same trend with highest agreement at Ct ≤ 28 (pool of 2 = 100.0%, pool of 3 = 100.0%, pool of 4 = 100.0%), midrange agreement at Ct ≤ 30 (pool of 2 = 99.0%, pool of 3 = 100.0%, pool of 4 = 100.0%), and lowest agreement at Ct ≤ 36 (pool of 2 = 98.1%, pool of 3 = 97.1%, pool of 4 = 98.1%). In conclusion, regardless of the level of pooling after tetrathionate enrichment, sensitivity was very good, and results would be comparable to what would have been found with individual culture or individual RT PCR at Ct ≤ 36. Detección de Salmonella Enteritidis en muestras ambientales avícolas agrupadas, utilizando un ensayo de reacción en cadena de la polimerasa en tiempo real específico de serotipo. No obstante que en los años recientes el método de reacción en cadena de la polimerasa en tiempo real (RT-PCR) ha sido utilizado como una prueba rápida para la detección de Salmonella Enteritidis, se ha realizado poca investigación para evaluar la viabilidad de agrupar las muestras ambientales avícolas para realizar un ensayo específico de RT-PCR para Salmonella Enteritidis. Por lo tanto, el objetivo de este estudio fue comparar el método de RT- PCR para la detección de Salmonella Enteritidis con muestras de hisopos de arrastre de instalaciones avícolas individuales y agrupadas (en grupos de dos, tres o cuatro hisopos) con los métodos de cultivo tradicionales. Los hisopos se obtuvieron de instalaciones avícolas que fueron previamente confirmadas positivas para Salmonella Enteritidis y se cultivaron de acuerdo con las especificaciones del Plan Nacional para el Mejoramiento Avícola. Se evaluaron los valores iniciales de corte de los ciclos umbrales de ≤36, ≤30, y 28 ≤ del método específico de RT-PCR para S. Enteritidis en comparación con el cultivo. El límite promedio de detección del ensayo de RT- PCR fue de 2.4 × 103 unidades formadoras de colonias (UFC)/ml, lo cual corresponde a un valor promedio de ciclo umbral de 36.6. Las muestras inoculadas con concentraciones de 102 a 105 UFC/ml fueron detectadas por RT-PCR antes del enriquecimiento, mientras que las muestras inoculadas con concentraciones de 100 a 105 UFC/ml fueron detectadas por la RT PCR después del enriquecimiento. Los valores de corte de los ciclos umbrales se utilizaron en una prueba de campo posterior donde se cultivó S. Enteritidis en 7 de 208 muestras ambientales (3.4%). Las muestras individuales mostraron una concordancia de 99.0%, 100%, y 100% con los valores de corte de los ciclo umbrales (Ct) de ≤36, ≤30, y ≤28, respectivamente. Las muestras agrupadas mostraron la misma tendencia con un mayor nivel de concordancia con los valores de Ct ≤ 28 (grupo de 2 = 100.0%, grupo de 3 = 100.0%, grupo de 4 = 100.0%), se observó una concordancia intermedia con un Ct ≤ 30 (grupo de 2 = 99.0%, grupo de 3 = 100.0%, grupo de 4 = 100.0%) y la concordancia más baja con un Ct ≤36 (grupo de 2 = 98.1%, grupo de 3 = 97.1%, grupo de 4 = 98.1%). En conclusión, independientemente del grado de agrupación después de enriquecimiento con caldo tetrationato, la sensibilidad fue muy buena, y los resultados fueron comparables a lo que se habrían encontrado con un cultivo individual o con un procesamiento individual por RT-PCR con un Ct ≤ 36.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Objective: To evaluate the stability of porcine reproductive and respiratory syndrome virus (PRRSV) and anti-PRRSV antibodies in oral fluid as a function of time and temperature. Materials and ...methods: A 4-L pool of swine oral fluid was collected from 16-week-old finisher pigs. To ensure uniform, quantifiable levels of virus and antibody over time, 4 mL of PRRSV isolate ISU-P containing 1 × 1012 RNA copies per mL and 10 mL of concentrated hyperimmune anti-PRRSV antibodies were added to the pool. The pool was then divided into three equal portions: no treatment, chlorhexidine digluconate at 0.01% by volume, and isothiazolinone at 3 ppm. Each treatment was tested in triplicate at each of five temperatures (-20°C, 4°C, 10°C, 20°C, and 30°C). Samples were removed at specific intervals (0, 12, 24, 48, 72, 144, 216, and 288 hours), stored at -80°C, and then assayed for PRRSV RNA; IgM, IgA, and IgG PRRSV-specific antibody; and culturable bacteria per mL. Results: The stabilities of anti-PRRSV antibody and detectable PRRSV were temperature-dependent, with antimicrobial treatment providing no improvements in stability at lower temperatures. Both virus and antibody were stable at ≤ 10°C over 12 days of storage. Implication: Conventional serum storage protocols (freezing or refrigeration at 4°C) preserves PRRSV and anti-PRRSV antibody in oral-fluid diagnostic samples without the use of preservatives.
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