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Whitney Briggs Master's Thesis.pdf (1.09 MB)

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Evaluation and optimization of quantitative analysis methods for Clostridium perfringens detection in broiler intestinal samples to use with necrotic enteritis challenge models

Briggs, Whitney
http://rave.ohiolink.edu/etdc/view?acc_num=osu1587491319889086

Abstract Details

2020, Master of Science, Ohio State University, Animal Sciences.
Necrotic enteritis (NE) is a multifactorial disease caused by Clostridium perfringens. A way to examine treatments on NE is through methods like body weight gain or lesion scores. Quantification of C. perfringens could be additional measurement. In experiment 3.1, four published extraction protocols, and a new protocol were compared to determine which would be ideal for extracting bacterial DNA. After extraction, DNA concentrations were measured with quality assessed, and a qPCR standard curve was established for each protocol. For Protocols 1 to 5, mean DNA yields and A260/280/A260/230 ratios were 459.02μg/g and 2.18/1.26, 989.66μg/g and 2.07/2.06, 110.71μg/g and 1.58/0.89, 437.03μg/g and 2.07/1.93 and 1,794.90μg/g and 1.91/1.42, respectively. Amplification efficiency percentages and standard curve regression coefficient values for Protocols 1 to 5 were 84.69%/1.00, 79.76%/0.99, 100.30%/1.00, 80.93%/0.99 and 95.76%/0.99, respectively. There was extensive variation in DNA quantity, quality, and PCR efficiency, highlighting challenges of DNA extraction from intestinal samples. Protocol 2 was identified as ideal based on DNA quantity and quality, and low standard deviations between qPCR samples. Experiments 4.1 and 4.2 compared qPCR and direct plating of fecal, ileal and cecal samples for C. perfringens quantification. In experiment 4.1, two groups were compared; an un-inoculated control and an inoculated group, while in experiment 4.2, the two groups compared were an un-inoculated control, fed a corn-based diet and an inoculated rye-based diet group. For quantifying C. perfringens, qPCR and two recently developed selective agar types were compared, with results analyzed by ANOVA factorials and Tukey’s Honestly Significant Difference test. All methods were further evaluated using relative standard deviation of CFU/g percentage (RSD%) values. In experiment 4.1, quantification method was a significant variable (p<0.05), with significant differences between qPCR and C. perfringens ChromoSelect plates for both fecal and ileal samples. For experiment 4.2 quantification method was a significant variable (p<.0001), with significant differences between quantification results from qPCR and both agar types. In almost all cases, RSD% were lower for qPCR than CHROMagarTM C. perfringens or C. perfringens ChromoSelect agar, suggesting that qPCR was more precise. High RSD% for the agars may have been influenced by samples having C. perfringens quantities below the plate’s limit of detection. Based on comparisons between qPCR and CHROMagarTM C. perfringens, it was concluded that CHROMagarTM C. perfringens could be used in place of qPCR for C. perfringens quantification. From these studies, a DNA extraction protocol has been identified, and two new selective C. perfringens agars compared against qPCR. Results highlight new avenues for quantifying C. perfringens in future NE research trials.
Lisa Bielke (Advisor)
Thaddeus Ezeji (Committee Member)
Zhongtang Yu (Committee Member)
98 p.
Animal Diseases; Microbiology; Molecular Biology
Quantitative methods; qPCR; direct agar plating; Clostridium perfringens; necrotic enteritis

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Citations

  • Briggs, W. (2020). Evaluation and optimization of quantitative analysis methods for Clostridium perfringens detection in broiler intestinal samples to use with necrotic enteritis challenge models [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1587491319889086

    APA Style (7th edition)

  • Briggs, Whitney. Evaluation and optimization of quantitative analysis methods for Clostridium perfringens detection in broiler intestinal samples to use with necrotic enteritis challenge models. 2020. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1587491319889086.

    MLA Style (8th edition)

  • Briggs, Whitney. "Evaluation and optimization of quantitative analysis methods for Clostridium perfringens detection in broiler intestinal samples to use with necrotic enteritis challenge models." Master's thesis, Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1587491319889086

    Chicago Manual of Style (17th edition)

osu1587491319889086
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