Erin Strait

Influenza A Pandemic (H1N1) 2009 Virus Infection in Domestic Cat

Influenza A pandemic (H1N1) 2009 virus continues to rapidly spread worldwide. In 2009, pandemic (H1N1) 2009 infection in a domestic cat from Iowa was diagnosed by a novel PCR assay that distinguishes between Eurasian and North American pandemic (H1N1) 2009 virus matrix genes. Human-to-cat transmission is presumed.

Comparative virulence of clinical Brachyspira spp. isolates in inoculated pigs

Classical swine dysentery is associated with the presence of the strongly beta-hemolytic Brachyspira hyodysenteriae. However, multiple Brachyspira spp. can colonize the porcine colon. Since 2008, several Brachyspira spp. not identified as B. hyodysenteriae by genotypic and/or phenotypic methods have been isolated from the feces of pigs with clinical disease typical of swine dysentery. In the current study, 8 clinical isolates, including 5 strongly beta-hemolytic and 3 weakly beta-hemolytic Brachyspira strains, and a reference strain of B. hyodysenteriae (B204) were inoculated into pigs (n = 6 per isolate) to compare pathogenic potential following oral inoculation. Results revealed that strongly beta-hemolytic isolates induced significantly greater typhlocolitis than those that are weakly beta-hemolytic, regardless of the genetic identification of the isolate, and that strongly beta-hemolytic isolates identified as “Brachyspira sp. SASK30446” and Brachyspira intermedia by polymerase chain reaction (PCR) produced lesions similar to those caused by B. hyodysenteriae. The results suggest that phenotypic culture characteristics of Brachyspira spp. may be a more sensitive indicator of potential to induce dysentery-like disease in pigs than molecular identification alone based on currently available PCR assays. Additionally, culture of mucosal scrapings obtained at necropsy was more sensitive than direct PCR on the same samples for detection of Brachyspira spp.

Comparison of RNA Extraction and Real-Time Reverse Transcription Polymerase Chain Reaction Methods for the Detection of Porcine Reproductive and Respiratory Syndrome Virus in Porcine Oral Fluid Specimens

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.

Identification of Mycoplasma suis antigens and development of a multiplex microbead immunoassay

The aims of the current study were to identify Mycoplasma suis antigens and develop a multiplex microbead immunoassay (MIA). A M. suis–expression library was screened for immunogens using sera from infected pigs. Based on bioinformatics, putative antigens were identified within positive inserts; gene fragments were expressed and purified as polyhistidine fusion proteins, and immunoreactivity was confirmed by Western blot. Selected antigens were used to develop a MIA. Sera from noninfected and infected pigs were used to set the median fluorescent intensity (MFI) cutoffs and as positive controls, respectively. Assay specificity was tested using sera from pigs seropositive for other pathogens (2 different pigs seropositive for each pathogen). Samples from 51 field pigs and 2 pigs during the course of acute (pig 1) and chronic (pig 2) infections were tested using MIA, indirect hemagglutination assay (IHA), and quantitative polymerase chain reaction (qPCR). Sixteen reactive plaques (52 genes) were detected. A heat-shock protein (GrpE), a nicotinamide adenine dinucleotide–dependent glyceraldehyde 3-phosphate dehydrogenase (GAPN), and 4 proteins from paralogous gene families (PGFs) were identified as antigens by Western blot. While GrpE, GAPN, and 1 PGF protein were strong antigens, the others were not suitable as MIA targets. A MIA using GrpE, GAPN, and the strongly reactive PGF protein was developed. Cross-reactivity with sera from pigs infected with Mycoplasma hyopneumoniae, Porcine circovirus-2, Porcine parvovirus, Porcine reproductive and respiratory syndrome virus, and Porcine respiratory coronavirus with this MIA was not observed. Pig 2 was consistently positive by MIA and qPCR, whereas pig 1, initially negative, seroconverted before becoming qPCR positive. Only 2 samples (from pig 1) were IHA positive. Five (9.8%) field samples were qPCR positive and 40 (78.43%) were positive for all 3 MIA antigens; however, all were IHA negative. In summary, the MIA is specific and more sensitive than qPCR and IHA, providing simultaneous evaluation of antibody response to M. suis antigens.

Detection of Salmonella Enteritidis in Pooled Poultry Environmental Samples Using a Serotype-Specific Real-Time-Polymerase Chain Reaction Assay

SUMMARY. 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. RESUMEN. 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.

Development of a real-time polymerase chain reaction assay for detection of Actinobacillus suis in porcine lung

In the current study, the development and validation of a real-time polymerase chain reaction (PCR) assay using a TaqMan-labeled probe for the detection of Actinobacillus suis from porcine lung samples is described. This real-time PCR amplified a 110-bp region of the 23S ribosomal RNA gene from A. suis but not from other bacteria. First, the assay was validated with 183 bacterial strains representing different species of bacteria. Subsequently, 85 porcine lung specimens that were declared A. suis–positive and –negative by bacterial culture and identification were tested to assess whether it can be performed directly on tissue specimens. The bacterial culture results and real-time PCR results agreed across all the samples tested assigning 100% positive and negative predictive values to the PCR. Further, the detection limit of the assay was 380 colony-forming units (CFU) per ml or approximately 1 CFU per reaction. In conclusion, the TaqMan real-time PCR assay described herein is a highly specific, sensitive, and reproducible test, which can be used to detect A. suis DNA in porcine lung specimens, thus providing a timely diagnosis.

Two clinical isolates of Mycoplasma hyosynoviae showeddiffering pattern of lameness and pathogen detection inexperimentally challenged pigs

Mycoplasma (M.) hyosynoviae is known to colonize and cause disease in growing-finishing pigs. In this study, two clinical isolates of M. hyosynoviae were compared by inoculating cesarean-derived colostrum-deprived and specific-pathogen-free growing pigs. After intranasal or intravenous inoculation, the proportion and distribution pattern of clinical cases was compared in addition to the severity of lameness. Tonsils were found to be the primary site of colonization, while bacteremia was rarely detected prior to the observation of clinical signs. Regardless of the clinical isolate, route of inoculation, or volume of inocula, histopathological alterations and tissue invasion were detected in multiple joints, indicating an apparent lack of specific joint tropism. Acute disease was primarily observed 7 to 10 days post-inoculation. The variability in the severity of synovial microscopic lesions and pathogen detection in joint cavities suggests that the duration of joint infection may influence the diagnostic accuracy. In summary, these findings demonstrate that diagnosis of M. hyosynoviae-associated arthritis can be influenced by the clinical isolate, and provides a study platform to investigate the colonization and virulence potential of field isolates. This approach can be particularly relevant to auxiliate in surveillance and testing of therapeutic and/or vaccine candidates.