Browsing by Subject "quantitative PCR"

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    Lawsonia intracellularis disease control and epidemiology.
    (2009-09) Wattanaphansak, Suphot
    Proliferative enteropathy (PE) is an important enteric disease in grower and finisher pigs caused by an obligate intracellular bacterium, Lawsonia intracellularis. The overall aims of this thesis were to obtain more information about the in vitro activity of antimicrobials and disinfectants against L. intracellularis and to develop a new quantitative PCR assay. To determine the minimum inhibitory concentration (MIC) of antimicrobials against L. intracellularis, ten isolates obtained from North America and Europe were tested against 6 antimicrobials using a modified tissue culture assay. In vitro results found that carbadox, tiamulin, and valnemulin were the most active antimicrobials, chlortetracycline and tylosin were intermediately active, lincomycin was the least active against L. intracellularis, and the antimicrobial sensitivity patterns differed across isolates. We next evaluated the effectiveness of the modified tissue culture and direct count method with special fluorescence to determine in vitro disinfectant activity against L. intracellularis. The outcomes of both methods predicted similar in vitro bactericidal activities against L. intracellularis with a high degree of correspondence. This suggests that either assay would be appropriate in determining the bactericidal activity of disinfectants against L. intracellularis. Based on our in vitro results, we predict that a powder disinfectant (Stalosan® F), DC&R®, Roccal®-D, Synergize®, and Virkon®-S would perform well under field conditions, while Certi-Dine®, Nolvasan®-S, and Tek-Trol® would be less active against L. intracellularis. Finally, a new quantitative PCR assay was developed using a SYBR green system. The assay showed negative results when DNA from 16 other species of enteric bacteria as well as 20 negative control samples of pig feces was tested. Quantitative estimates from the assay were roughly 2-fold lower than the expected values across all dilutions of pure culture and spiked feces. Validation results indicated that this new qPCR is sensitive, specific, reliable, accurate, and precise for the detection and quantification of L. intracellularis in different types of samples. In conclusion, the results of these investigations update and expand upon existing information about the in vitro antimicrobial and disinfectant activities against L. intracellularis. Further, the development of a new, accurate, and precise qPCR assay will facilitate future epidemiology studies.