Development of a point-of-care diagnostic tool for the early detection of bovine respiratory disease associated agents in feedlot cattle

The purpose of this thesis is to determine the optimal in-field methodology and to develop an in-field diagnostic technique to bridge a technology gap in the current diagnosis of bovine respiratory disease (BRD). The PCR technology developed enabled the survey of cattle at a location or pen level to better understand the role of agent presence and load on the clinical presentation of BRD presentation and explored the role of known and novel agents in coinfection and active BRD in Australian feedlot cattle.
A meta-analysis of published PCR approaches for the detection and / or quantification of BRD associated agents was undertaken using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. This analysis was carried out as a result of uncovering discrepancies in the reporting of PCR based tests for BRD agents in literature. The application of the Minimum Information for Publication of Quantitative Real-Time PCR Experiment (MIQE) guidelines was examined to determine the reliability and reproducibility of report assays. The analysis revealed that the in silico specificity of some reported primers / probes was rarely stringently tested, as well as there being major inconsistencies in reporting. It was found that the MIQE guidelines were not widely utilised in the analysed studies. Results from this meta-analysis demonstrated that the sensitivity and specificity or published PCR-based tests for BRD are highly variable and the lack of application of the MIQE guideline could be significantly impeding the reader’s ability to interpret results whilst raising questions around the reliability and reproducibility of published assays.
To determine the optimal methodology process for in-field testing of BRD associated agents from nasal swabs, a direct comparison of two extraction techniques, and three detection methodologies: microbiological culture, BioSprint® or phosphate-buffered saline boil (PBSB) extractions for Mic™ PCR analysis, was undertaken. It was found that Mic™ PCR was more sensitive at detecting BRD associated agents than microbiological culture. While the simpler, field deployable PBSB extraction technique facilitated DNA recovery for PCR analysis better than the BioSprint® method. Therefore, the PCR methodology, including the Mic™ PCR thermocycler in conjunction with PBSB extraction form the basis of the experiments described in the subsequent chapters of this thesis.
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In order to determine the relationship between prevalence and load of BRD associated DNA microbe in cattle from two Australian feedlot locations, efficiency-corrected (EC) PCR quantification was used on nasal swabs extracted using the PSBS extraction methodology.. The findings from this study indicate there may be a relationship between agent load and combination of agents present and BRD in feedlot cattle. These findings and the use of the screening approach could be highly informative for better management of BRD treatment and / or prevention in Australian feedlot systems and worldwide. This approach presented a potentially useful tool that could bridge the technology gap for current BRD diagnostics and enabled in-field detection, quantification and analysis.
PCR methodologies developed earlier in this thesis were then optimised and multiplexed to facilitate the testing of a large number of individual animals. While assays for RNA viral BRD associated agents were also developed to enable testing of animals for the full panel of common BRD associated agents. These were then used to determine the prevalence and load of BRD associated microbial agents in the upper airways at feedlot induction (Day 0) and after 14 days on feed and whether this was related to lung health at the time of slaughter. It was found that Mycoplasma bovis, Trueperella pyogenes and Bovine alphaherpesvirus 1 (BoAHV-1) were all implicated in disease outcomes, with coinfection with numerous BRD agents potentially playing a role. Bovine respiratory syncytial virus (BRSV) was the most commonly detected agent in lung tissues, even though it was rarely detected in the upper respiratory tract. Findings from this study suggest that the prevalence and load of bacterial and viral microorganisms need to be considered together when investigating disease risk for BRD in cattle.
The final experimental chapter of this thesis presents a preliminary study in which PCR tests were developed for the detection of novel BRD agents, Ureaplasma diversum, Bibersteinia trehalosi and the extended-spectrum β-lactamase (ESBL) gene blaCTX-M1. This study was undertaken as these agents appear to be increasingly detected in cattle diseases globally. The samples collected in Chapter 5 were used for this study; briefly animals sampled at feedlot induction, on Day 0, and Day 14 as well as an independent cohort of hospital animals. It was found that the prevalence of these targets was higher in hospital pen animals though their detection was not dependent on treatment reason (BRD or non BRD).U. diversum was more prevalent in the hospital pen steers compared to
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induction animals, regardless of treatment reason, and was most commonly identified in combination with M. bovis. This chapter provides proof of concept that these agents are able to be successfully detected in bovine nasal swabs from an Australian feedlot and confirms that a wider study is warranted to confirm the role that agents not commonly associated with BRD, may play in cattle respiratory diseases.
Together, these findings demonstrate the methodologies developed in this thesis are useful tools for determining the prevalence and load of agents in the bovine upper respiratory tract. The extraction, PCR and analysis methodologies developed in this thesis can be adopted in-field, for the investigation, analysis and identification of BRD associated viral and bacterial agents, thus dramatically reducing turnaround times and providing more timely information for management and treatment practices in feedlots.