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In the last decade livestock-associated methicillin-resistant S. aureus (LA-MRSA) has become a public health concern in many parts of the world. Sequence type 398 (ST398) has been the most commonly reported type of LA-MRSA. While many studies have focused on long-term exposure experienced by swine workers, this study focuses on short-term exposures experienced by veterinary students conducting diagnostic investigations. The objectives were to assess the rate of MRSA acquisition and longevity of carriage in students exposed to pork farms and characterize the recovered MRSA isolates. Student nasal swabs were collected immediately before and after farm visits. Pig nasal swabs and environmental sponge samples were also collected. MRSA isolates were identified biochemically and molecularly including spa typing and antimicrobial susceptibility testing. Thirty (30) veterinary students were enrolled and 40 pork farms were visited. MRSA was detected in 30% of the pork farms and in 22% of the students following an exposure to a MRSA-positive pork farm. All students found to be MRSA-positive initially following farm visit were negative for MRSA within 24 hours post visit. Most common spa types recovered were t002 (79%), t034 (16%) and t548 (4%). Spa types found in pork farms closely matched those recovered from students with few exceptions. Resistance levels to antimicrobials varied, but resistance was most commonly seen for spectinomycin, tetracyclines and neomycin. Non-ST398 MRSA isolates were more likely to be resistant to florfenicol and neomycin as well as more likely to be multidrug resistant compared to ST398 MRSA isolates. These findings indicate that MRSA can be recovered from persons visiting contaminated farms. However, the duration of carriage was very brief and most likely represents contamination of nasal passages rather than biological colonization. The most common spa types found in this study were associated with ST5 and expands the range of livestock-associated MRSA types.

Managing the disposal of infectious animal carcasses from routine and catastrophic disease outbreaks is a global concern. Recent research suggests that burial in lined and aerated trenches provides the rapid pathogen containment provided by burial, while reducing air and water pollution potential and the length of time that land is taken out of agricultural production. Survival of pathogens in the digestate remains a concern, however. A potential answer is a 'dual'-barrier approach in which ammonia is used as a secondary barrier treatment to reduce the risk of pathogen contamination when trench liners ultimately leak. Results of this study showed that the minimum inhibitory concentration (MIC) of NH3 is 0.1 M (~1,468 NH3-N mg/L), and 0.5 M NH3 (~7,340 NH3-N mg/L) for ST4232 & MRSA43300, respectively at 24 h and pH = 9±0.1 and inactivation was increased by increasing NH3 concentration and/or treatment time. Results for digestate treated with NH3 were consistent with the MICs, and both pathogens were completely inactivated within 24 h.

Livestock associated methicillin-resistant Staphylococcus aureus (LA-MRSA) draws concern from the public health community because in some countries these organisms may represent the largest reservoir of MRSA outside hospital settings. Recent studies indicate LA-MRSA strains from swine are more genetically diverse than the first reported sequence type ST398. In the US, a diverse population of LA-MRSA is found including organisms of the ST398, ST9, and ST5 lineages. Occurrence of ST5 MRSA in swine is of particular concern since ST5 is among the most prevalent lineages causing clinical infections in humans. The prominence of ST5 in clinical disease is believed to result from acquisition of bacteriophages containing virulence or host-adapted genes including the immune-evasion cluster (IEC) genes carried by β-hemolysin converting bacteriophages, whose absence in LA-MRSA ST398 is thought to contribute to reduced rates of human infection and transmission associated with this lineage. The goal of this study was to investigate the prevalence of IEC genes associated with β-hemolysin converting bacteriophages in MRSA ST5 isolates obtained from agricultural sources, including swine, swine facilities, and humans with short- or long-term swine exposure. To gain a broader perspective, the prevalence of these genes in LA-MRSA ST5 strains was compared to the prevalence in clinical MRSA ST5 strains from humans with no known exposure to swine. IEC genes were not present in any of the tested MRSA ST5 strains from agricultural sources and the β-hemolysin gene was intact in these strains, indicating the bacteriophage's absence. In contrast, the prevalence of the β-hemolysin converting bacteriophage in MRSA ST5 strains from humans with no exposure to swine was 90.4%. The absence of β-hemolysin converting bacteriophage in LA-MRSA ST5 isolates is consistent with previous reports evaluating ST398 strains and provides genetic evidence indicating LA-MRSA ST5 isolates may harbor a reduced capacity to cause severe disease in immunocompetent humans.

Cystic fibrosis (CF) is a recessive disease that affects multiple organs. It is caused by mutations in CFTR. Animal modeling of this disease has been challenging, with species- and strain-specific differences in organ biology and CFTR function influencing the emergence of disease pathology. Here, we report the phenotype of a CFTR-knockout ferret model of CF. Neonatal CFTR-knockout ferrets demonstrated many of the characteristics of human CF disease, including defective airway chloride transport and submucosal gland fluid secretion; variably penetrant meconium ileus (MI); pancreatic, liver, and vas deferens disease; and a predisposition to lung infection in the early postnatal period. Severe malabsorption by the gastrointestinal (GI) tract was the primary cause of death in CFTR-knockout kits that escaped MI. Elevated liver function tests in CFTR-knockout kits were corrected by oral administration of ursodeoxycholic acid, and the addition of an oral proton-pump inhibitor improved weight gain and survival. To overcome the limitations imposed by the severe intestinal phenotype, we cloned 4 gut-corrected transgenic CFTR-knockout kits that expressed ferret CFTR specifically in the intestine. One clone passed feces normally and demonstrated no detectable ferret CFTR expression in the lung or liver. The animals described in this study are likely to be useful tools for dissecting CF disease pathogenesis and developing treatments.

Managing the disposal of infectious animal carcasses from routine and catastrophic disease outbreaks is a global concern. Recent research suggests that burial in lined and aerated trenches provides the rapid pathogen containment provided by burial, while reducing air and water pollution potential and the length of time that land is taken out of agricultural production. Survival of pathogens in the digestate remains a concern, however. A potential answer is a 'dual'-barrier approach in which ammonia is used as a secondary barrier treatment to reduce the risk of pathogen contamination when trench liners ultimately leak. Results of this study showed that the minimum inhibitory concentration (MIC) of NH.sub.3 is 0.1 M (~1,468 NH.sub.3 -N mg/L), and 0.5 M NH.sub.3 (~7,340 NH.sub.3 -N mg/L) for ST4232 & MRSA43300, respectively at 24 h and pH = 9±0.1 and inactivation was increased by increasing NH.sub.3 concentration and/or treatment time. Results for digestate treated with NH.sub.3 were consistent with the MICs, and both pathogens were completely inactivated within 24 h.

Livestock associated methicillin-resistant Staphylococcus aureus (LA-MRSA) draws concern from the public health community because in some countries these organisms may represent the largest reservoir of MRSA outside hospital settings. Recent studies indicate LA-MRSA strains from swine are more genetically diverse than the first reported sequence type ST398. In the US, a diverse population of LA-MRSA is found including organisms of the ST398, ST9, and ST5 lineages. Occurrence of ST5 MRSA in swine is of particular concern since ST5 is among the most prevalent lineages causing clinical infections in humans. The prominence of ST5 in clinical disease is believed to result from acquisition of bacteriophages containing virulence or host-adapted genes including the immune-evasion cluster (IEC) genes carried by [beta]-hemolysin converting bacteriophages, whose absence in LA-MRSA ST398 is thought to contribute to reduced rates of human infection and transmission associated with this lineage. The goal of this study was to investigate the prevalence of IEC genes associated with [beta]-hemolysin converting bacteriophages in MRSA ST5 isolates obtained from agricultural sources, including swine, swine facilities, and humans with short- or long-term swine exposure. To gain a broader perspective, the prevalence of these genes in LA-MRSA ST5 strains was compared to the prevalence in clinical MRSA ST5 strains from humans with no known exposure to swine. IEC genes were not present in any of the tested MRSA ST5 strains from agricultural sources and the [beta]-hemolysin gene was intact in these strains, indicating the bacteriophage's absence. In contrast, the prevalence of the [beta]-hemolysin converting bacteriophage in MRSA ST5 strains from humans with no exposure to swine was 90.4%. The absence of [beta]-hemolysin converting bacteriophage in LA-MRSA ST5 isolates is consistent with previous reports evaluating ST398 strains and provides genetic evidence indicating LA-MRSA ST5 isolates may harbor a reduced capacity to cause severe disease in immunocompetent humans.

Nicholson TL, Shore SM, Smith TC, Frana TS (2013) Livestock-Associated Methicillin-Resistant Staphylococcus aureus (LA-MRSA) Isolates of Swine Origin Form Robust Biofilms. PLoS ONE 8(8): e73376. doi:10.1371/journal.pone.0073376 Citation: Nicholson TL, Shore SM, Smith TC, Frana TS (2013) Correction: Livestock-Associated Methicillin-Resistant Staphylococcus aureus (LA-MRSA) Isolates of Swine Origin Form Robust Biofilms.

Methicillin-resistant Staphylococcus aureus (MRSA) colonization of livestock animals is common and prevalence rates for pigs have been reported to be as high as 49%. Mechanisms contributing to the persistent carriage and high prevalence rates of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) strains in swine herds and production facilities have not been investigated. One explanation for the high prevalence of MRSA in swine herds is the ability of these organisms to exist as biofilms. In this report, the ability of swine LA-MRSA strains, including ST398, ST9, and ST5, to form biofilms was quantified and compared to several swine and human isolates. The contribution of known biofilm matrix components, polysaccharides, proteins and extracellular DNA (eDNA), was tested in all strains as well. All MRSA swine isolates formed robust biofilms similar to human clinical isolates. The addition of Dispersin B had no inhibitory effect on swine MRSA isolates when added at the initiation of biofilm growth or after pre-established mature biofilms formed. In contrast, the addition of proteinase K inhibited biofilm formation in all strains when added at the initiation of biofilm growth and was able to disperse pre-established mature biofilms. Of the LA-MRSA strains tested, we found ST398 strains to be the most sensitive to both inhibition of biofilm formation and dispersal of pre-formed biofilms by DNaseI. Collectively, these findings provide a critical first step in designing strategies to control or eliminate MRSA in swine herds.

Background Bibersteinia trehalosi causes respiratory disease in ruminants particularly in wild and domestic sheep. Recently, there has been an increased number of B. trehalosi isolates obtained from diagnostic samples from bovine respiratory disease cases. This study evaluated the role of B. trehalosi in bovine respiratory disease using an intra-tracheal inoculation model in calves. Thirty six cross bred 2–3 month old dairy calves were inoculated intra-tracheally with either leukotoxin negative B. trehalosi , leukotoxin positive B. trehalosi isolate, Mannheimia haemolytica , a combination of leukotoxin negative B. trehalosi and M. haemolytica or negative control. Calves were euthanized and necropsy performed on day 10 of study. Results B. trehalosi inoculated calves did not have increased lung involvement compared to control calves. Additionally, B. trehalosi was only cultured once from the lungs of inoculated calves at necropsy. Conclusions Based on these findings B. trehalosi may not be a primary pathogen of respiratory disease in cattle. Culture of B. trehalosi from diagnostic submissions should not be immediately identified as a primary cause of respiratory disease.

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.