Browsing by Subject "Resistance genes"

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    Antibiotic resistance in the lower intestinal microbiota of dairy cattle: longitudinal analysis of phenotypic and genotypic resistance.
    (2012-02) Boyer, Timothy Charles
    This research focused on methods of measuring antibiotic resistance and analysis of antibiotic resistance data in dairy cattle that were sampled repeatedly over time. Specific objectives included: characterization and longitudinal analysis of phenotypic antibiotic resistance of commensal Escherichia coli, development of a statistical model for the analysis of low quantity resistance genes measured by quantitative real-time polymerase chain reaction (qPCR), measurement of antibiotic resistance genes in the lower intestinal bacterial communities of dairy cattle that received a short-term therapeutic dose of antibiotic and untreated cattle, and measurement and longitudinal analysis of the quantities of six antibiotic resistance genes in the lower intestinal bacterial communities of dairy cattle. Enteric E. coli collected from dairy cattle over 1.5 years were tested for phenotypic resistance to 17 antimicrobials. A total of 93 phenotypic patterns were observed among 3,402 isolates tested, with a majority (67%) susceptible to all 17 antimicrobials. The most prevalent resistances were to tetracycline, sulfamethoxazole, and streptomycin. Latent class and latent transition analyses were carried out to group the animals into classes according to their resistance phenotypes and to estimate the probabilities of transitioning into and out of classes over time. Probabilities of transitioning to a pan-susceptible class were high, as were the probabilities of remaining in the pan-susceptible class. Probabilities of transitioning form a pan-susceptible class to a resistant class were very low. Measurement of antibiotic resistance genes by qPCR presents challenges for genes that are present in very low quantities. A statistical model was developed to analyze qPCR data made up of a significant proportion of observations that fall below the limit of quantification of a qPCR assay. Computer simulations showed that the statistical model produced less biased estimates of regression parameters than common methods of handling low quantity qPCR data. qPCR was applied to a cohort of dairy cattle that received a five day course of ceftiofur and matched untreated cattle. Quantities of a gene (blaCMY-2) that confers resistance to ceftiofur were measured and analyzed using the statistical model developed for low quantity genes. Treated animals had significantly higher quantities of blaCMY-2 during treatment than untreated animals. By the first day post-treatment, gene quantities had returned to pre-treatment levels. The quantities of six different antibiotic resistance genes were measured by qPCR in the fecal community bacterial DNA of a cattle population that was sampled repeatedly over 2.5 years. Significantly increasing trends over time were observed for three of the six genes conferring resistance to tetracyclines, macrolides, and cephalosporins. Comparison of phenotypic resistance and qPCR data showed that qPCR performed on community DNA is a more sensitive method of detection that phenotypic testing of cultured isolates.
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    Characterizing the host response and genetic control in 'Honeycrisp' to apple scab (Venturia inaequalis)
    (2014-01) Clark, Matthew Daniel
    Two novel apple scab resistance loci have been identified in the apple cultivar Honeycrisp, an emerging cultivar in North America that is utilized in apple breeding programs worldwide. Greenhouse inoculation experiments with the apple scab fungal pathogen, Venturia inaequalis, identified a resistance defense response in `Honeycrisp' and its ancestors `Keepsake', `Frostbite', and `Northern Spy' seven days after inoculation. The defense response ranged from necrotic and chlorotic lesions to stellate necrosis and class 3b lesions (with sporulation). A hallmark of the resistance defense response is autofluorescence at the infection site in cleared leaf tissue. Several `Honeycrisp' progeny populations were screened with monoconidial isolates of V. inaequalis and segregated 3:1 for resistance, suggesting two resistance genes inherited from `Honeycrisp'. A consensus `Honeycrisp' linkage map with 1091 SNP markers was constructed for use in mapping. Two resistance loci were mapped using linkage mapping and quantitative trait loci mapping approaches. Marker haplotypes were constructed to trace the inheritance of resistance loci. Rvi19 mapped on linkage group 1 at ~50 cM. In the Rvi19 haplotype, the 138 bp allele for the Ch-Vf1 marker cosegregates with resistance, and is identical by state (IBS) with the Rvi17 resistance in `Antonovka'. Rvi19 is transmitted from `Frostbite' to `Keepsake' to `Honeycrisp', and into the resistant progeny of `Honeycrisp'. The other locus, Rvi20, mapped onto linkage group 15, and is IBS with a marker haplotype found in the susceptible cultivar Golden Delicious. Rvi20 is transmitted to `Honeycrisp' from an unknown parent. Molecular marker haplotypes were used to identify advanced selections in the University of Minnesota breeding program with pyramided scab resistance. Candidate genes were identified at each haplotype that can serve as starting points for identifying the functional genes conferring resistance. A collection of V. inaequalis isolates was assembled and curated from six locations in Minnesota for screening scab resistance. These 80+ isolates were examined for genetic diversity and population structure and provide a snapshot of the diversity of the pathogen present at this time.