Development of molecular-based diagnostics

Molecular methods can detect very small quantities of target nucleic acid in complex samples and have been used in veterinary diagnostics for decades (Lauerman, Reference Lauerman1998). However, recent advancements in technologies and chemistries have enabled robust and cost-effective assays that allow for simultaneous quantitative detection of multiple targets in a single test reaction. With these advances have come efficient nucleic acid extraction chemistries that can be utilized on high-throughput extraction platforms that co-purify RNA and DNA (Berensmeier, Reference Berensmeier2006). PCR-based approaches were quickly adapted to and utilized to detect viral pathogens associated with BRD. PCR methods were superior in turnaround time and interpretation compared to classical approaches such as cell culture and antibody-based detection, and even singleplexed conventional PCR testing has significant advantages over classic methods (Vilcek et al., Reference Vilcek, Elvander, Ballagi-Pordány and Belák1994; Schmitt et al., Reference Schmitt, Lopez, Ridpath, Galeota-Wheeler and Osorio1994; Masri et al., Reference Masri, Olson, Nguyen, Prins and Deregt1996). Highly multiplexed real-time PCR assays (rtPCR) that include reverse transcription to detect RNA and DNA pathogens are now widely used across US diagnostic labs for virus detection (Horwood and Mahony, Reference Horwood and Mahony2011; Fulton et al., Reference Fulton, D'offay, Landis, Miles, Smith, Saliki, Ridpath, Confer, Neill, Eberle, Clement, Chase, Burge and Payton2016).

However, widespread implementation of these methods for the detection of bacterial pathogens of BRD has lagged. There are several challenges to the development and implementation of these assays. These include culture-based approaches that labs have existing capacity for, the need for isolated pathogens for downstream testing (typing, susceptibility, etc.), and relative cost. In contrast to culture, PCR-based testing is also inherently narrow in scope, in that the test is limited to the sequence of the assay targets. However, newer technologies using 16S rRNA amplification and sequencing may hold promise for a more comprehensive and broad-based molecular diagnostics tool (Johnston et al., Reference Johnston, Earley, Cormican, Murray, Kenny, Waters, Mcgee, Kelly and Mccabe2017; Timsit et al., Reference Timsit, Workentine, Van Der Meer and Alexander2018). Additionally, only recently have high-quality complete whole-genome sequences from diverse sources become available, greatly improving the ability to identify and select robust targets for assay design (Clawson et al., Reference Clawson, Murray, Sweeney, Apley, Dedonder, Capik, Larson, Lubbers, White, Kalbfleisch, Schuller, Dickey, Harhay, Heaton, Chitko-Mckown, Brichta-Harhay, Bono and Smith2016; Harhay et al., Reference Harhay, Harhay, Bono, Smith, Capik, Dedonder, Apley, Lubbers, White and Larson2017).

Another challenge is that diagnostically, interpretation of results from molecular testing may be misleading. Many pathogens are opportunistic in nature, and are present in both normal and diseased animals; therefore, direct detection of pathogens through culture and antibody-based approaches was preferred by some as more interpretable (Fulton and Confer, Reference Fulton and Confer2012). However, the combination of being readily able to assess the relative abundance of bacteria using real-time platforms, combined with rapid cycling rotary-based real-time platforms and robust enzyme mixes, has enhanced the utility of molecular methods for the detection of bacterial pathogens of BRD (Reynisson et al., Reference Reynisson, Josefsen, Krause and Hoorfar2006; Loy et al., Reference Loy, Leger, Workman, Clawson, Bulut and Wang2018a).

We developed a real-time-based assay for the most frequently detected bacterial BRD pathogens. Newly available BRD pathogen genome sequences were used to establish robust targets, and the assay was developed and validated on multiple instrument platforms (Clawson et al., Reference Clawson, Murray, Sweeney, Apley, Dedonder, Capik, Larson, Lubbers, White, Kalbfleisch, Schuller, Dickey, Harhay, Heaton, Chitko-Mckown, Brichta-Harhay, Bono and Smith2016; Loy et al., Reference Loy, Leger, Workman, Clawson, Bulut and Wang2018a). As the advantages over culture-based approaches were not immediately apparent, an extensive comparative analysis was done to evaluate PCR-based detection compared to culture on samples that are routinely submitted to diagnostic labs. Both antemortem (nasal and nasopharyngeal swabs) and postmortem diagnostic samples (lungs) were included. Limits of detection for the assay are quite low (1.2–12 CFU ml⁻¹) and had a near-perfect agreement with culture for lung tissues with high overall levels of specificity and sensitivity. One large advantage over culture is the number of co-detections found. Co-detections were extremely under-represented when relying on culture alone, with only 25 found in the data set, with 125 co-detections using PCR-based approaches, indicating a fivefold increase in the detection of these types of infections (Loy et al., Reference Loy, Leger, Workman, Clawson, Bulut and Wang2018a). Agreement between culture and rtPCR was found to be highest in lungs and lowest in nasal swabs, likely due to the limitations of culture on samples more likely to contain environmental bacteria, such as nasal swabs. The instrumentation used did not adversely affect overall method sensitivity and specificity; however, the rotary-based instrument had significantly lower Cq thresholds, indicating more efficient PCR reactions. The overall conclusions of this work indicate the multiplexed rtPCR panels are rapid, sensitive, and diagnostically useful in multiple relevant sample types and have several advantages over classical methods.

Development of proteomic-based diagnostics

Another emerging technology, matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) has revolutionized clinical microbiology labs and veterinary diagnostics (Seng et al., Reference Seng, Drancourt, Gouriet, La Scola, Fournier, Rolain and Raoult2009; Clark et al., Reference Clark, Kaleta, Arora and Wolk2013). These instruments enable the accurate identification of a single colony of bacterial growth in minutes. Many platforms are also flexible allowing users to add locally relevant strains and species to their databases. Mass spectrum data can be examined for biomarkers that may enable differentiation of phenotypes or genotypes (Mani et al., Reference Mani, Thachil and Ramachandran2017; Pérez-Sancho et al., Reference Pérez-Sancho, Vela, Horcajo, Ugarte-Ruiz, Domínguez, Fernández-Garayzábal and De La Fuente2018). Recently, we have demonstrated the utility of MALDI-TOF MS, using a bioinformatics approach for biomarker discovery, to distinguish amongst two major genotypes of M. haemolytica (Loy and Clawson, Reference Loy and Clawson2017). This method enables near-real-time typing of these isolates by mining data collected during the MS identification process. This method has been validated on whole-cell bacteria ‘direct smears’ so no extraction or other processing is required and isolates can be rapidly screened and selected for downstream testing.

Applications using molecular-based diagnostics

One application of rtPCR BRD panels has been epidemiological investigations to examine the contributions of emerging pathogens to BRD. Workman et al. have utilized rtPCR to estimate BRD pathogen shedding throughout the beef production cycle to examine the role respiratory coronaviruses play in increasing the risk of BRD (Workman et al., Reference Workman, Kuehn, Mcdaneld, Clawson, Chitko-Mckown and Loy2017; Workman et al., Reference Workman, Kuehn, Mcdaneld, Clawson and Loy2019). One challenge to interpreting results from these panels is unlike viruses, where any detection of virus shedding may be clinically significant, the detection of bacteria that normally resides in the nasopharynx may not be clinically relevant. Further work to determine Cq cutoffs or levels of relative abundance that may be clinically significant is important to enable these tests so they be more readily interpreted from antemortem samples. In one study, neither the Cq level nor the numbers of animals classified as cases or controls which had a detected pathogen were significantly different at and following feedlot entry for bacterial pathogens. However, nasal shedding of bovine coronavirus (BCV) both in Cq values and the number of animals shedding was higher in those that were classified as cases. A follow-up study using these same diagnostic tools following BRD in pre-weaned beef calves was able to determine in one longitudinal study, H. somni, in addition to BCV, was potentially contributing to clinical cases, as H. somni was detected during BRD outbreaks and not at other time points (Workman et al., Reference Workman, Kuehn, Mcdaneld, Clawson and Loy2019). These observational studies demonstrate that molecular detection tools may be useful to examine risk factors and contributions of pathogens during BRD outbreaks.

Molecular workflows also provide for any number of culture-independent nucleic acid tests in addition to pathogen detection. One approach is to evaluate the extracted clinical samples for the presence of antimicrobial resistance genes to develop a rapid and culture-independent resistance detection method. Recent work has shown that the detection of macrolide and tetracycline-resistant genes in BRD clinical samples have a high agreement with the isolation of M. haemolytica with increased MIC values to these antimicrobials (Loy et al., Reference Loy, Payne, Deal, Dutta, Bulut, Clawson and Wang2018b). Such an approach could provide clinicians with information about the presence of potential resistant pathogens in samples more rapidly than culture and susceptibility testing.

Applications using proteomic-based diagnostics

Another challenge with the interpretation of detection results from antemortem samples is the presence of mixed intra-species populations that may not be representative of the causative organisms deeper in lung tissues. Capik et al. have found using pulsed-field gel electrophoresis that M. haemolytica populations in the nasopharynx do not always match those found in the lungs of cattle with BRD (Capik et al., Reference Capik, White, Lubbers, Apley, Mosier, Larson and Murray2015). One potential diagnostic approach to assist microbiologists in finding those populations most likely associated with disease from NP samples is to use MALDI-TOF MS profiles to screen isolates for downstream testing. Genotype 2 M. haemolytica is more likely to be associated with lung invasion and contain AMR genes and ICE elements, which can carry antimicrobial resistance genes. Therefore, preferential selection of these genotypes using MALDI-TOF prior to MIC testing and other downstream assays may be useful.