Development of a Rapid and Inexpensive Assay for Farm-Based Detection of Four Pathogenic Vibrio Strains Linked to Shellfish Hatchery Failures

Progress report for LNE22-457R

Project Type: Research Only
Funds awarded in 2022: $199,985.00
Projected End Date: 08/31/2024
Grant Recipient: Mook Sea Farm
Region: Northeast
State: Maine
Project Leader:
Dr. Meredith White
Mook Sea Farm
Co-Leaders:
Steve Zimmerman
Mook Sea farm, Inc
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Project Information

Summary:

We propose to develop and commercialize loop-mediated isothermal amplification (LAMP) assays for farm-based detection of six virulence marker genes from four Vibrio species associated with shellfish hatchery failures, along with an easy-to-understand Hatchery Instructions Guide for the assays. This proposal has been developed in response to an increased number of shellfish hatchery failures, which ultimately impact hundreds of oysters farms in the Northeast who depend on hatcheries for seed. Vibriosis, infection of larval or juvenile oysters by pathogenic Vibrio spp. is frequently a cause of these failures, reducing dependability of seed production. From a survey of hatchery managers, 86.7% indicated they would adopt the use of this assay into their hatchery operations to help diagnose production problems or verify that no pathogens are present in the algae they are growing as food for the larvae and seed. LAMP assays will be developed at Bigelow Laboratory for Ocean Sciences in Year 1 of the project, resulting in rapid, inexpensive molecular assays that can be implemented directly by hatchery operators, instead of sending samples out for expensive, long-turnaround PCR assays. The quick return of presence/absence of pathogens will let hatchery operators quickly develop management plans and mitigation strategies to remove the pathogens. The developed assays will be tested in the Mook Sea Farm (MSF) Hatchery in Maine, to verify that the protocols are appropriate for hatchery use. The MSF team will develop clear protocols and supplies lists in the Hatchery Instructions Guide, which will be shared with the Project Advisory Committee and a Hatchery Feedback Group to ensure the guide is user-friendly for those without molecular biology backgrounds. MSF and Bigelow Laboratory will seek commercialization opportunities for the custom LAMP assays, including but not limited to ‘kit production’, to ensure that high-quality LAMP assays are readily available to end users in a convenient, reliable, and contamination-free ‘easy to use’ format. Lessons learned through the development of these hatchery-focused LAMP assays will simplify the steps needed to develop LAMP assays for additional pathogens that are of concern to shellfish hatcheries and grow-out operations.

Project Objective:

We propose to develop and commercialize loop-mediated isothermal amplification (LAMP) assays for farm-based detection of six virulence marker genes from four Vibrio species associated with shellfish hatchery failures, along with an easy-to-understand Hatchery Instructions Guide. The LAMP method allows for rapid, inexpensive, colorimetric detection of pathogens with minimal sample preparation, giving shellfish hatcheries straightforward diagnostic tools to manage hatchery production. These tools can be used to detect infections and determine management strategies, as well as to check shellfish seed viability prior to shipping to farms from the hatcheries, giving confidence to those purchasing the seed for their farms.

Introduction:

The oyster aquaculture industry depends on hatchery-produced seed. Northeast oyster farms annually purchase their seed from one of 40 Northeast hatcheries, which raise larvae and seed in land-based facilities, typically with 8-12 employees per hatchery. When a hatchery has production failure, farms lose access to seed that would have been their market product in 2-3 years, potentially putting thousands of jobs at risk. The seed supply for shellfish farming is increasingly challenged by hatchery failures (Walker 2017), with myriad potential causes (Jones 2006). Due to difficulty gathering production crash information from commercial hatcheries, recent research compiled production crash case studies from research hatcheries along the East Coast (Gray et al. 2022). Causes of crashes at these research hatcheries included vibriosis, infection of larval and juvenile shellfish by one of several pathogenic Vibrio species that cause mortality (Gray et al. 2022). When there is concern about pathogens, hatcheries currently must send samples to commercial laboratories to identify pathogenic Vibrios through PCR-based tests, waiting 2-4 weeks for results, at which point, the opportunity to control the pathogen has passed. These tests cost upwards of $200/sample, depending on how many genes are targeted, which can be cost-prohibitive for a small business losing product and sales.

This project will develop rapid, inexpensive loop-mediated isothermal amplication (LAMP) assays that can be performed onsite with basic equipment, allowing hatcheries to diagnose the presence of pathogenic Vibrio spp. within an hour, and immediately take proactive measures to control the pathogen spread. This project will develop LAMP assays to detect six virulence marker genes of four pathogenic Vibrio species known to cause production crashes at shellfish hatcheries (Gharaibeh et al. 2009, Ushijima et al. 2020, Xu et al. 2017, Yang et al. 2021). A recent survey to Northeast hatcheries elicited anonymous feedback such as ‘This work will certainly help shellfish hatcheries’ and ‘It will be a terrific asset for our industry.’ This technology represents a novel tool for shellfish hatchery pathogen management, and will likely be adopted across hatcheries to create more dependable production. That dependability will increase production efficiency, sustainability, and profitability for hatcheries adopting the approach, and a dependable seed source will increase efficiency and profitability for hundreds of oyster farms purchasing seed from these hatcheries.

Cooperators

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  • Dr. Meredith White (Researcher)
  • Dr. Peter Countway (Researcher)
  • Dr. Robin Sleith (Researcher)

Research

Hypothesis:

Pathogenicity varies between strains of identical species of Vibrio bacteria. Pathogenic strains of Vibrio are known to cause significant losses in oyster aquaculture. Timely and accurate detection of pathogenic strains is therefore necessary to detect and isolate infected seed and feed. This proposal seeks to address a need in the aquaculture community by developing a sensitive and rapid test for pathogenic Vibrio. We hypothesize that loop-mediated isothermal amplification (LAMP) methods will be ideal for detecting pathogenic Vibrio in aquaculture settings across three substrates: water, algae, and oyster seed.

Materials and methods:

In August 2022, our Project Advisory Committee (PAC) met to discuss the start of the project. The PAC consists of two hatchery operators, two farmer outreach specialists, and one technical advisor. We heard from the hatchery operators and one of the outreach specialist that in addition to the assays we are developing to detect hatchery pathogens, farmers would greatly benefit from LAMP assays to detect Aliiroseovarius Crassostrea (Juvenile Oyster Disease; JOD) as well as pathogenic strains ST36 and ST631 of Vibrio parahaemolyticus, which causes human health issues, as opposed to the oyster health issues caused by the Vibrio species targeted for our hatchery assays. As time and funding allows, we will aim to incorporate this into our work as well.

In September 2022, we used LampDesigner software to design LAMP assays for Aliiroseovarius (a target for which we already had samples, qPCR standards, and results to compare with LAMP) and Vibrio alginolyticus collagenase (clg) target. We ordered the primers and NEB WarmStart LAMP Master Mix and began testing on existing samples and a culture of Vibrio alginolyticus (B51) from the National Center for Marine Algae (NCMA) at Bigelow. Our LAMP reactions for Aliiroseovarius initially worked for the qPCR standard, but failed for the environmental samples. We returned to our alignment and noticed that the 3 prime end of the alignment, where LAMPDesigner had picked primers, was variable and could differ between the environmental samples and our standard. We then selected another set of primers in the more conserved 5 prime end of the region and tested these against our standard and environmental samples, with great success!. For Vibrio alginolyticus we tested against the B51 culture and saw very strong amplification after just 10 minutes. We are now cloning the clg gene from the culture to be able to test the sensitivity of the assay.

Research results and discussion:

We have demonstrated the ability to design LAMP assays that are specific to the species of interest. These assays gave rapid results in as little as 10 minutes, demonstrating their potential for the aquaculture community. For Aliiroseovarius we used a dilution series of qPCR standards to determine that we are able to detect as few as 2,552 copies of the target, or 850 cells per reaction after 30 minutes, with further sensitivity testing ongoing. The next steps are to continue to calculate the sensitivity and confirm the specificity of our assays as well as begin development of the other assays listed in our proposal. These early successes have energized our team and we look forward to tackling the next steps in the project.

 

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Figure 1 Geneious alignment of Vibrio alginolyticus clg gene, with LAMP primers annotated
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Figure 2 Fluoresence accumulation of LAMP reaction over 60 minutes for Vibrio alginolyticus clg target
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Figure 3 Dilution series of JOD standard demonstrates sensitivity of LAMP assay.

Participation Summary
4 Farmers participating in research

Education & Outreach Activities and Participation Summary

Educational activities:

1 Consultations
1 Webinars / talks / presentations

Participation Summary:

4 Farmers participated
2 Number of agricultural educator or service providers reached through education and outreach activities
Outreach description:

Project Advisory Committee Meeting

Project Outcomes

1 Grant received that built upon this project
$20,000.00 Dollar amount of grant received that built upon this project
Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.