Simple and cost-effective SNP genotyping method for discriminating subpopulations of the fish pathogen, Nocardia seriolae

Nocardia seriolae has caused significant fish losses in Asia and the Americas in recent decades, including in Vietnam, which has witnessed devastating economic and social impacts due to this bacterial pathogen. Surveillance strategies are urgently needed to mitigate N. seriolae dissemination in Vietnamese aquaculture and mariculture industries. Whole-genome sequencing (WGS) offers the highest level of resolution to discriminate closely related strains and to determine their putative origin and transmission routes. However, WGS is impractical for epidemiological investigations and pathogen surveillance due to its time-consuming and costly nature, putting this technology out-of-reach for many industry end-users. To overcome this issue, we targeted two previously characterised, phylogenetically informative single-nucleotide polymorphisms (SNPs) in N. seriolae that accurately distinguish: i) Vietnamese from non-Vietnamese strains, and ii) the two Vietnamese subclades. Using the mismatch amplification mutation assay (MAMA) format, we developed assays that genotype strains based on differences in amplicon melting temperature (melt-MAMA) and size (agarose-MAMA). Our MAMA assays accurately genotyped strains both from culture and fish tissues at low cost, using either real-time (~AUD$1/per sample) or conventional (~AUD$0.50/per sample) PCR instrumentation. Our novel assays provide a rapid, reproducible, and cost-effective tool for routine genotyping of this pathogen, allowing faster identification and treatment of nocardiosis-effected permit fish within Vietnamese aquaculture/mariculture facilities, an essential step in mitigating N. seriolae-associated losses.

Guidance for populating GenomeTrakr metadata templates (BioSample and SRA) v2

PURPOSE: Guidance on how to populate NCBI's metadata packages, maximizing interoperability for foodborne pathogen surveillance. SCOPE: This protocol provides detailed instructions for populating the following two templates: 1. BioSample metadata: guidelines to populate the GenomeTrakr-extended pathogen package. 2. SRA metadata: NCBI's generic sequence metadata template for SRA submissions. Versions: v6: Added the One Health Enteric package presented at IAFP 2021 meeting. v7: Updated the picklists in the GenomeTrakr-extended pathogen package, "GT-pathogen package-OHE v0.2.2.xlsx" and added an incremental update file for the DRAFT One Health Enteric Package that includes extensive edits compared to v6.

A panel of real-time PCR assays for the detection of Bourbon virus, Heartland virus, West Nile virus, and Trypanosoma cruzi in major disease–transmitting vectors

Vector-borne pathogens, such as Bourbon virus (BRBV), Heartland virus (HRTV), West Nile virus (WNV), and Trypanosoma cruzi (TCZ) are a great threat to public health and animal health. We developed a panel of TaqMan real-time PCR assays for pathogen surveillance. PCR targets were selected based on nucleic acid sequences deposited in GenBank. Primers and probes were either designed de novo or selected from publications. The coverages and specificities of the primers and probes were extensively evaluated by performing BLAST searches. Synthetic DNA or RNA fragments (gBlocks) were used as PCR templates in initial assay development and PCR positive controls in subsequent assay validation. For operational efficiency, the same thermocycling profile was used in BRBV, HRTV, and WNV reverse-transcription quantitative PCR (RT-qPCR) assays, and a similar thermocycling profile without the initial reverse-transcription step was used in TCZ qPCR. The assays were optimized by titrating primer and probe concentrations. The analytical sensitivities were 100, 100, 10, and 10 copies of gBlock per reaction for BRBV (Cq = 36.0 ± 0.7), HRTV (Cq = 36.6 ± 0.9), WNV (Cq = 35.5 ± 0.4), and TCZ (Cq = 38.8 ± 0.3), respectively. PCR sensitivities for vector genomic DNA or RNA spiked with gBlock reached 100, 100, 10, and 10 copies per reaction for BRBV, HRTV, WNV, and TCZ, respectively. PCR specificity evaluated against a panel of non-target pathogens showed no significant cross-reactivity. Our BRBV, HRTV, WNV, and TCZ PCR panel could support epidemiologic studies and pathogen surveillance.

Leveraging natural history biorepositories as a global, decentralized, pathogen surveillance network

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic reveals a major gap in global biosecurity infrastructure: a lack of publicly available biological samples representative across space, time, and taxonomic diversity. The shortfall, in this case for vertebrates, prevents accurate and rapid identification and monitoring of emerging pathogens and their reservoir host(s) and precludes extended investigation of ecological, evolutionary, and environmental associations that lead to human infection or spillover. Natural history museum biorepositories form the backbone of a critically needed, decentralized, global network for zoonotic pathogen surveillance, yet this infrastructure remains marginally developed, underutilized, underfunded, and disconnected from public health initiatives. Proactive detection and mitigation for emerging infectious diseases (EIDs) requires expanded biodiversity infrastructure and training (particularly in biodiverse and lower income countries) and new communication pipelines that connect biorepositories and biomedical communities. To this end, we highlight a novel adaptation of Project ECHO’s virtual community of practice model: Museums and Emerging Pathogens in the Americas (MEPA). MEPA is a virtual network aimed at fostering communication, coordination, and collaborative problem-solving among pathogen researchers, public health officials, and biorepositories in the Americas. MEPA now acts as a model of effective international, interdisciplinary collaboration that can and should be replicated in other biodiversity hotspots. We encourage deposition of wildlife specimens and associated data with public biorepositories, regardless of original collection purpose, and urge biorepositories to embrace new specimen sources, types, and uses to maximize strategic growth and utility for EID research. Taxonomically, geographically, and temporally deep biorepository archives serve as the foundation of a proactive and increasingly predictive approach to zoonotic spillover, risk assessment, and threat mitigation.

Global genomic pathogen surveillance to inform vaccine strategies: a decade-long expedition in pneumococcal genomics

Vaccines are powerful agents in infectious disease prevention but often designed to protect against some strains that are most likely to spread and cause diseases. Most vaccines do not succeed in eradicating the pathogen and thus allow the potential emergence of vaccine evading strains. As with most evolutionary processes, being able to capture all variations across the entire genome gives us the best chance of monitoring and understanding the processes of vaccine evasion. Genomics is being widely adopted as the optimum approach for pathogen surveillance with the potential for early and precise identification of high-risk strains. Given sufficient longitudinal data, genomics also has the potential to forecast the emergence of such strains enabling immediate or pre-emptive intervention. In this review, we consider the strengths and challenges for pathogen genomic surveillance using the experience of the Global Pneumococcal Sequencing (GPS) project as an early example. We highlight the multifaceted nature of genome data and recent advances in genome-based tools to extract useful information relevant to inform vaccine strategies and treatment options. We conclude with future perspectives for genomic pathogen surveillance.