Performing Department
Medicine & Epidemology
Non Technical Summary
Infectious diseases pose a major threat to aquaculture production, causing millions of dollars in annual losses. In addition to direct mortality, disease leads to reduced growth, increased labor costs and additional treatment expenditures. Veronaea botryosa is the causative agent of a systemic fungal disease known as "fluid belly" in cultured sturgeon, one of the most important emergent fungal diseases in US aquaculture. Streptococcus iniae infections are also regarded as emerging bacterial diseases in cultured sturgeon. Although these pathogens can cause significant losses alone, they are frequently found in co-infections with Acipenserid Herpesvirus 2 (AciHV2). It is well established that herpesviruses have multiple pathways for regulating host cellular immune competency, resulting in immune system evasion and in some cases even oncogenesis. A better understanding of the host immune response during infection is essential for the development of prophylactic, therapeutic and biosecurity regimens. Therefore, the purpose of this study is to provide insight how emerging bacterial and fungal pathogens cause severe disease in the presence of AciHV2.
Animal Health Component
100%
Research Effort Categories
Basic
60%
Applied
30%
Developmental
10%
Goals / Objectives
Objective 1) Gain insights to the pathogenesis of AciHV2 and S. iniae or V. botryosa co-infections in White sturgeon.Objective 2) Compare whole genome sequences (WGS) of AciHV2­ isolated from cultured white sturgeon in California and identify potential virokines involved in white sturgeon immunosuppression. Additionally, WGS analysis will be used to identify potential immunogenic antigens shared between AciHV2.
Project Methods
Objective 1. To investigate the role of AciHV2 during co-infections with S. iniae and V. botryosa, in vivo controlled co-infection challenges in white sturgeon fingerlings will be utilized following the protocols of Ma et al. (24). Briefly, white sturgeon fingerlings will be divided into six treatments, with five-replicate 100L flow?through tanks (20 fish/tank) per treatment. Fish in AciHV2, AciHV2/S. iniae, and AciHV2/V. botryosa groups will be injected (intacoelomically) at day 0 with an AciHV2 (2019 isolate) suspension containing an infectious dose of ~1,000 plaque?forming units (PFU)/fish. Fish in the S. iniae, V. botryosa, AciHV2/S. iniae, and AciHV2/V. botryosa groups will be intramuscularly injected lateral to the dorsal fin at day 30 with S. iniae or V. botryosa suspensions containing an infectious dose of 10e7 CFU/fish. Mock?infected controls will be injected with 100μl of sterile cell culture media or phosphate buffer saline at day 0 and 30, respectively. Mortality will be recorded daily for 60days and cumulative percent mortality (CPM) of all replicate tanks determined. Two fish per tank (ten fish per treatment) will be euthanized at days 15, 30, 45 and 60 post-initial injection. Spleens will be collected, and analysis of genes involved in innate and cellular immunity will be investigated through quantitative reverse-transcription PCR (qRTPCR) according to methods recently developed in our laboratory. For statistical analyses, the proposed sample size (n= 5 tanks per treatment, 20 fish per tank) is appropriate assuming an effect size (f) in ANOVA of 0.7, Type I error of 0.05 and a power of 80%. Differences in CPM will be determined using a one-way ANOVA (α = 0.05) after confirming residuals are normally distributed and variances are equal for both data sets. If differences are significant (p < 0.05), Tukey's post-hoc test will be used to identify differences between individual groups. Statistical analysis will be performed in GraphPad® Prism v5.03 (GraphPad, San Diego, CA, USA). Additionally, survival curves will be generated and compared via the log-rank test. Nonparametric statistical significance of relative gene expression will be calculated with REST software by a pairwise fixed reallocation randomization test. This calculation will be based on the probability of observing ratios equal to or greater than no effect in randomly assigned control and challenged sample groups. The significance of differences in relative gene expression levels between treatments will be calculated by Student's t-test, assuming equal variation. Results for both objectives will be considered significant at p < 0.05.Objective 2. Next generation sequencing will be employed to genetically characterize AciHV2 isolates recovered from fish in CA, predict novel immunogenic antigens shared between AciHV2, and investigate the presence of AciHV2 virokines. Objective 2 will be achieved via high-throughput short-read (Illumina HiSeq 2x150 bp) and long-read (Oxford Nanopore Technologies; ONT) sequencing and bioinformatic analysis of six isolates (two isolated prior to 2000, two isolated from 2000-2010 and two isolated between 2010-2020) that have been recovered by our laboratory from disease outbreaks in California from 1980-2020. At present, limited work has been conducted on the protein and antigenic diversity of AciHV2 and only one genome has been partially sequenced (25). Still, proteomes will be acquired either from NCBI or through WGS. Retrieved sequences compiled in a local database will be subjected to an "all vs. all" protein BLAST to obtain proteins common to all AciHV2. The generated WGS will then be used to predict vaccine targets using the Vaxign system for reverse vaccinology (21).