July 17, 2025
July 17, 2025
Arun Venugopalan1,2, Adithya Bindhu Sreekumar1,2, and Rebecca J Gast3.
1Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, Stoneville, MS 38776; 2Gulf Coast Aquatic Health Lab, Global Center for Aquatic Health and Food Security, Gautier, MS 39553; 3 Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543
Juvenile oyster disease (JOD), caused by the bacterium Aliiroseovarius crassostreae, can lead to mortality rates of up to 100%, causing devastating losses to the oyster industry. Current PCR methods are time-consuming and cannot distinguish between newly reported strains isolated from biofilms—which may not be pathogenic to oysters—and the clinical isolates from diseased oysters. To address these limitations, a new qPCR protocol was developed and validated according to MIQE guidelines for sensitivity, specificity, and reproducibility. Further, a complete, gap-free genome of the bacterium was assembled using long-read nanopore technology. The complete genome and new qPCR will enable rapid detection of pathogenic oyster isolates of this bacterium. This work will support timely interventions to mitigate economic losses and enhance aquaculture sustainability.
1Amber E. Johnston, 2Thomas F. Rounsville Jr., 1Deborah A. Bouchard, 3Mark P. Polinski
1University of Maine Cooperative Extension, Aquaculture Research Institute, Aquatic Animal Health Laboratory, 17 Godfrey Drive, Orono, ME 04473; 2University of Maine Cooperative Extension, Pest Management Unit, 17 Godfrey Drive, Orono, ME 04473; 3United States Department of Agriculture – Agricultural Research Service, National Cold Water Marine Aquaculture Center, 25 Salmon Farm Rd, Franklin, ME 04634
Infectious Salmon Anemia Virus (ISAV; Family Orthomyxoviridae), causative agent of Infectious Salmon Anemia (ISA), is one of the most intensively managed and regulated finfish pathogens due to its capacity to cause severe epizootic events. Like other segmented single-stranded RNA viruses such as influenza, ISAV demonstrates high genetic diversity between isolates. This diversity is captured within two main genotypes – North American and European – and two virulence phenotypes based on a deletion in the hyper-variable region of the genome – the virulent HPR-delete (HPRΔ) or the avirulent HPR-replete (HPR0). Despite nearly three decades of ISAV research and detection in North America, little progress has been made in defining the complete genetic diversity of catalogued isolates or tracking potential deletion events that lead to disease outbreaks, largely due to both resource constraints and the lack of a broadly-applicable, multi-genotype-capturing whole genome sequencing (WGS) method. Further, not much is known about ISAV co-infections, where different genotypes may be present in the same host, which is an important consideration given the known process of reassortment in other Orthomyxoviruses. Herein we aimed to develop a WGS method using Nanopore sequencing technologies that will enable us to 1) sequence whole genome directly from ISAV-infected cells and tissue, 2) capture all current genotypes and phenotypes, and 3) identify potential co-infections in ISAV infected Atlantic salmon. Here we discuss and compare different primer design strategies for detection and pre-sequencing enrichment of viral RNA to encompass the high genetic diversity of ISAV, while limiting the common impediment of excess host genetic material to virus-specific WGS efforts. Further understanding of the diversity of ISAV isolates in the Northeastern Atlantic will allow researchers to address critical questions about ISAV epidemiology in the region, and our ongoing development of a broadly-applicable WGS method is the first step.
Daniel DeLap1,2, Sarah M. Turner1,3, Deborah Bouchard1,3
1University of Maine Aquaculture Research Institute, 17 Godfrey Drive, Orono, Maine, 04473; 2 University of Maine School of Marine Science, 360 Aubert Hall University of Maine, Orono, Maine, 04469; 3University of Maine Cooperative Extension, 17 Godfrey Drive, Orono, Maine, 04473
Diseases are one of the limiting factors contributing to sustainable aquaculture production. Fortunately, vaccines have been developed to combat numerous pathogens that plague aquaculture species and farmers. Aquaculture vaccinations are administered through injection, oral/feed, or dip/immersion. Immersion vaccines offer a proactive approach to increasing immunity against pathogens without the cost and labor-intensive practices of injectable vaccination. However, immersion vaccines are known to provide a short duration of protection and low adaptive immune response, often requiring booster doses. Innovative delivery systems have shown that nanoparticles can improve immersion vaccine efficacy through specific mechanisms. For instance, chitin or chitosan has been effective in immersion vaccines against Flavobacterium in salmonid species. Furthermore, mannosylated chitosan(MCA), created by attaching mannose to chitosan, has been developed and effective in antigen delivery for various animal species. To explore the potential for enhancing the efficacy of dip vaccines, the addition of mannosylated chitosan (MCA) was evaluated as a means to improve immune response and protection against pathogens. Aeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, a disease in wild and cultured salmonid species. In this research, juvenile Atlantic salmon parr were vaccinated using an immersion vaccine delivery method that included MCA and bacterin formulations and a nasal vaccine treatment group containing MCA and bacterin. All nine vaccinated treatment groups were housed in a common garden design, with each treatment placed into one of six cohabitation tanks (10 fish per treatment). After 600-degree days, a cohabitation challenge was conducted using Atlantic salmon parr. In this challenge, the vaccinated fish were exposed to naive salmon that had been injected with A. salmonicida subsp. salmonicida. Mortality rates were recorded, and a survival curve was generated. Additionally, tissue samples were collected to assess IgM and IgT antibody responses. Mortalities of injected cohabiting fish began on day four post-injection, with mortalities across all treatment groups occurring from days seven to eleven. The study concluded when 60% of the PBS negative control groups succumbed to the disease on day eleven. Survival curves and specific antibody responses (IgM and IgT) of all treatment groups showed no statistically significant differences. While this study did not demonstrate improved efficacy of the MCA formulation, this outcome is likely due to the highly robust nature of the bacterial challenge used, which may have overwhelmed potential differences among treatments. Therefore, these findings do not necessarily negate the potential utility of MCA as a novel adjuvant. Rather, additional studies under varying challenge conditions and using alternative delivery protocols are necessary to assess the efficacy of MCA in aquaculture vaccine strategies more accurately.