Advancing animal tuberculosis surveillance using culture-independent long-read whole-genome sequencing.

Animal tuberculosis is a significant infectious disease affecting both livestock and wildlife populations worldwide. Effective disease surveillance and characterization of Mycobacterium bovis (M. bovis) strains are essential for understanding transmission dynamics and implementing control measures. Currently, sequencing of genomic information has relied on culture-based methods, which are time-consuming, resource-demanding, and concerning in terms of biosafety. This study explores the use of culture-independent long-read whole-genome sequencing (WGS) for a better understanding of M. bovis epidemiology in African buffaloes (Syncerus caffer). By comparing two sequencing approaches, we evaluated the efficacy of Illumina WGS performed on culture extracts and culture-independent Oxford Nanopore adaptive sampling (NAS). Our objective was to assess the potential of NAS to detect genomic variants without sample culture. In addition, culture-independent amplicon sequencing, targeting mycobacterial-specific housekeeping and full-length 16S rRNA genes, was applied to investigate the presence of microorganisms, including nontuberculous mycobacteria. The sequencing quality obtained from DNA extracted directly from tissues using NAS is comparable to the sequencing quality of reads generated from culture-derived DNA using both NAS and Illumina technologies. We present a new approach that provides complete and accurate genome sequence reconstruction, culture independently, and using an economically affordable technique.

Leptospira in livestock in Madagascar: uncultured strains, mixed infections and small mammal-livestock transmission highlight challenges in controlling and diagnosing leptospirosis in the developing world.

In developing countries, estimates of the prevalence and diversity of Leptospira infections in livestock, an important but neglected zoonotic pathogen and cause of livestock productivity loss, are lacking. In Madagascar, abattoir sampling of cattle and pigs demonstrated a prevalence of infection of 20% in cattle and 5% in pigs by real-time PCR. In cattle, amplification and sequencing of the Leptospira-specific lfb1 gene revealed novel genotypes, mixed infections of two or more Leptospira species and evidence for potential transmission between small mammals and cattle. Sequencing of the secY gene demonstrated genetic similarities between Leptospira detected in Madagascar and, as yet, uncultured Leptospira strains identified in Tanzania, Reunion and Brazil. Detection of Leptospira DNA in the same animal was more likely in urine samples or pooled samples from four kidney lobes relative to samples collected from a single kidney lobe, suggesting an effect of sampling method on detection. In pigs, no molecular typing of positive samples was possible. Further research into the epidemiology of livestock leptospirosis in developing countries is needed to inform efforts to reduce human infections and to improve livestock productivity.