Canturri, Albert2024-02-092024-02-092023-12https://hdl.handle.net/11299/260688University of Minnesota Ph.D. dissertation. December 2023. Major: Veterinary Medicine. Advisor: Maria Pieters. 1 computer file (PDF); 1 xi, 159 pages.Mycoplasma hyopneumoniae (M. hyopneumoniae) is one of the most significant pathogens in swine, causing a respiratory disease that affects animal welfare and decreases animal production. Disease diagnostics are the cornerstone for the implementation of measures aiming at the detection and control of pathogens, like M. hyopneumoniae. However, some aspects of disease diagnostics, such as PCR result interpretation or bacterial viability determination, are challenging and subject to improvement. The general objective of this thesis was to contribute to the advancement of M. hyopneumoniae diagnostic capabilities, to be applied in disease control. Unforeseen laboratory testing results, such as the detection of M. hyopneumoniae by PCR in processing fluids (PF), can occur and require further analysis. The detection dynamics and the possible origin of the genetic material present in PF were evaluated in different farm conditions. Detection of the bacterium was a consistent finding in positive farms, and the origin of the genetic material could be traced down to cross-contamination, highlighting the importance that the persistence of nucleic acids in the environment can have for proper PCR result interpretation. Although the extensive resistance to degradation of DNA in the environment has been established in certain fields of knowledge, there was limited information on the detection dynamics of DNA from non-viable M. hyopneumoniae in various conditions. Thus, the detection of inactivated M. hyopneumoniae was explored both in vitro, using cultured bacteria in the closed environment of the culture tubes, and in vivo, inoculating pigs with non-viable cells. Combined results indicated that, while DNA detection of non-viable bacterial cells persisted in vitro, this did not occur in vivo, as DNA was not detectable by PCR in the organism of the pigs, not even soon after inoculation. Based on the obtained results, the development of a culture-independent method to assess the viability of M. hyopneumoniae in clinical samples was considered essential. A viability, RNA-based PCR assay, targeting M. hyopneumoniae messenger RNA (mRNA) was developed and compared to DNA-based PCR in regard to their ability to differentiate viable from inert bacteria. The developed RNA-based PCR assay detected only viable or very recently inactivated M. hyopneumoniae, while the DNA-based PCR consistently detected cells irrespective of their viability status. Changes in in vitro growth activity over time were only observable via RNA-based PCR. Furthermore, the applicability of the viability PCR assay to evaluate M. hyopneumoniae viability post-antibiotic treatment in infected pigs was investigated. Results showed differences between the detection of bacterial DNA and mRNA, suggesting that DNA-based PCR assays may overrepresent the population of viable bacteria. Thus, the use of diagnostic methods that allow the relative quantification of viable cells may be more suitable to determine the efficacy of antibiotic treatments than DNA-based methods. However, the clinical or epidemiological implications of such differences, especially in the chronic phase of infection, are unknown, and further research is warranted. Overall, the new information gathered in this thesis indicate that improved diagnostics are critical for the control of the disease caused by M. hyopneumoniae. The data presented here will impact the work of swine practitioners and producers, and will contribute to a more sustainable pork production.enThesis or Dissertation