Evaluation of Nanopore Sequencing as a Diagnostic Tool for the Rapid Identification of Mycoplasma bovis from Individual and Pooled Respiratory Tract Samples

Abstract

Rapid identification of Mycoplasma bovis infections in cattle is a key factor to guide antimicrobial therapy and biosecurity measures. Recently, nanopore sequencing became an affordable diagnostic tool for both clinically relevant viruses and bacteria, but the diagnostic accuracy for M. bovis identification is undocumented. Therefore, in this study nanopore sequencing was compared to rapid identification of M. bovis with MALDI-TOF MS (RIMM), and triplex real-time PCR in a Bayesian latent class model (BLCM) for M. bovis in bronchoalveolar lavage fluid (BALf) obtained from calves. In practice, pooling of samples is often used to save money, but the influence on diagnostic accuracy has not been described for M. bovis . Therefore, a convenience sample of 17 pooled samples containing 5 individual BALf samples per farm was analyzed as well. The results of the pooled samples were compared to the individual samples, to determine sensitivity (Se) and specificity (Sp). The BLCM showed a good Se (77.3%; 95% Credible Interval: 57.8%-92.8%) and high Sp (97.4%; 91.5%-99.7%) for nanopore sequencing compared to RIMM (Se: 93.0%; 76.8%-99.5%, Sp: 91.3; 82.5%-97.0%) and real-time PCR (Se: 94.6%; 89.7%-97.7%, Sp: 86.0%; 76.1-93.6%). Se and Sp of pooled analysis for M. bovis were 85.7% (95% confidence interval: 59.8-111.6%) and 90.0% (71.4-108.6%%) for nanopore sequencing and 100% (100%-100%) and 88.9% (68.4-109.4%) for RIMM, respectively. In conclusion, nanopore sequencing is a rapid, reliable tool for the identification of M. bovis . To reduce costs and increase the chance of M. bovis identification, pooling of 5 samples for nanopore sequencing and RIMM is possible.

Evaluating the potential of third generation metagenomic sequencing for the detection of BRD pathogens and genetic determinants of antimicrobial resistance in chronically ill feedlot cattle

“…A recent investigation by Bokma et al . using a Bayesian latent class model described a lower sensitivity for identification of M. bovis with nanopore sequencing (77.3% [95% credible interval, 57.8 to 92.8%]) as compared to the described method of rapid identification of M. bovis by MALDI-TOF MS [RIMM] (93.0% [76.8 to 99.5%]) [ 70 ]. The investigation by Bokma et al .…”

Bovine respiratory microbiota of feedlot cattle and its association with disease

“…However, recent studies using NGS techniques have shown the complex microbial composition and found the common bacterial pathogens for BRD in the lungs of both healthy and sick cattle [ 1 , 16 , 23 , 43 , 52 ]. With the improvement of techniques, nanopore sequencing and multiple omics [i.e., metagenomics (the study of a collection of genomes and genes from the members of a microbiota by shotgun sequencing), metaproteomics, metatranscriptomics and metabolomics] are becoming more effective to investigate the composition and functions of the respiratory microbiome [ 59 , 60 ]. In cattle, the bacterial community and BRD-associated pathogens were found when using metagenomics to measure all the genes of all the microbes in an environment [ 20 , 25 , 61 – 63 ], but still no reports using other omics were found.…”

Pathogen-oriented approaches for neonatal calf diarrhea

“…However, to the authors' knowledge, there are no peer-reviewed Bayesian latent class evaluations of PCR tests for NCD pathogens available. Previous work on PCR for bovine respiratory disease pathogens has shown that these tests do not have a perfect sensitivity and specificity (Bokma et al, 2021).…”

“…The viral metagenome can be identified in a sample, as well as certain bacteria. The technique has already shown its added value for diagnostics in diarrheic pigs and bovine respiratory disease (Theuns et al, 2018;Bokma et al, 2021). Other techniques like immunofluorescence assay or the carbolfuchsine smear method for microscopic examination for C. parvum are either not feasible on farm level or no longer widely offered by veterinary laboratories, and hence outside the scope of this review.…”