GENERATION OF A LIVE-ATTENUATED VACCINE TO PISCINE STREPTOCOCCOSIS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 1018829
• Project No.: CALV-AH-394
• Project Start Date: Feb 26, 2019
• Project End Date: Jun 30, 2020

Project Director
Soto Martinez, ES, .

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
Medicine & Epidemology

Non Technical Summary
If funded, we will define an S. iniae LAV capable of inducing protective immunity to tilapia against piscine streptococcosis. This will be used to develop and validate mucosal vaccination protocols, and, potentially lead to the first commercial LAV against S. iniae in fish.

Animal Health Component

100%

Research Effort Categories

Basic

30%

Applied

40%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	0810	1090	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
0810 - Finfish;

Field Of Science
1090 - Immunology;

Keywords

aquaculture

vaccines

Goals / Objectives
The main objectives are to: 1. Generate LAV for each one of the S. iniae genogroups affecting cultured fish in the US (n=3); 2. Investigate the efficacy of the LAV against piscine streptococcosis using the Nile tilapia model, and 3. Inform and disseminate results obtained in this project to the aquaculture industry.

Project Methods
Objective 1. We will initiate steps to generate rifampicin resistant strains of one S. iniae isolate per genogroup (n=3) following the methods outlined by Wise et al.26 Briefly, strains will be grown on agar and a single colony will be transferred to plates containing 0.25 µg/mL rifampicin. The MIC of the selected wild type strains is <0.5mg/ml. Individual colonies (max of 3) that grow on this first plate will be designated as individual passage lines and kept separate. Each passage line will be serially transferred to agar plates containing increasingly higher concentrations of rifampicin, culminating in a final concentration of 250 µg/mL rifampicin. Virulence of passaged strains (n=30 per genogroup; n=90 total) will be compared to its WT parental strain measuring cytotoxicity (assessed by measuring the release of cytosolic lactate dehydrogenase into the supernatant of challenged cells) of tilapia cell line.21 The SAS® (version 9.4) GLM procedure will be used to conduct an analysis of variance in a factorial arrangement of treatments for data obtained in cytotoxicity assays. When overall significance is found, post hoc comparisons will be conducted with pairwise t tests of least-squares means. All tests will be considered significant at p ≤ 0.05. Cross-reactivity of attenuated vaccine candidates (n=3 for each genogroup) will also be evaluated (total of 9). For each genogroup (n=3), 30 adult Nile tilapia (n=10 per strain) will be immunized by injection.20 Thirty days post-immunization, blood will be drawn from each fish and serum will be purified.19 Anti-S. iniae IgM produced by immunized tilapia will be quantified by ELISA11 and/or agglutination.19,20 Antigen preparations will be prepared from 3 strains per genogroup (total 9) in efforts to evaluate cross-reactive potential of each vaccine strain and establish whether serum antibody activity in tilapia vaccinated with one genogroup is similar, enhanced or diminished when presented against antigens derived from other genogroups. A group of 10 adult tilapia will be sham immunized and serum collected and analyzed similarly to serve as controls. Objective 2. Attenuated strains selected from objective 1 (n=3 per genogroup), will be used to immunize tilapia fingerlings (n=100 fish per treatment; 20 fish per tank) by immersion.20 Control groups will be exposed to sterile media. Protection conferred by the different vaccine candidates will be evaluated by challenging fish with homologous wild type S. iniae following published protocols at 30 days post-immunization.20 Thirty days post-challenge, relative percent survival will be calculated for each treatment. For statistical analyses, a sample size (n= 5 tanks per treatment, 20 fish per tank) is necessary, assuming an effect size (f) in ANOVA of 0.7, Type I error of 0.05 and a power of 80%. The SAS statistical package (version 9.3) GLM procedure will be used to analyze the data as an analysis of variance in a factorial arrangement of treatments in a fixed effects linear model. The main effects that will be included are the live attenuated vaccines used and all interactions. When overall significance is found in either a main effect or interaction term, post hoc comparisons will be conducted with pairwise t tests of least-squares means. All tests will be considered significant at p < 0.05. Finally, LAV isolates resulting in significant protection to vaccinated tilapia will be subjected to whole-genome sequence and compared to alignment generated from parental WT strains to identify single nucleotide polymorphisms resulting in attenuation.

Progress 02/26/19 to 06/30/20

Outputs
Target Audience:Aquaculturists, pharmaceutical companies, regulatory agencies, biologists, microbiologists, veterinarians Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Taylor Heckman (graduate student at the Microbiology Graduate Group at UC Davis and Khalid Shahin (post-doctoral researcher at UC Davis) participated in this project. Taylor served as the leading trainee in this project and helped in the design and execution of the study. Additionally, she analyzed data and wrote manuscrip (currently under review). Moreover, she presented this work in 4 different national meetings to make sure results were disseminated as proposed. How have the results been disseminated to communities of interest?The results of this project were presented in 4 different national meetings with wide range of attendance. Additionally, results are also summarized in a peer-reviewed manuscript submitted for publication. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Streptococcus iniae is a re-emerging bacterial pathogen in freshwater and marine aquaculture worldwide. There are no commercial vaccines available for S. iniae in the United States, and autogenous vaccines are restricted to inactivated whole-cell preparations with limited protection against heterogenous strains. Live-attenuated vaccines (LAV) represent an advantageous alternative to these bacterins, as they induce robust cellular and humoral immunity, and may provide longer lasting protection through less stressful routes of administration. We investigated whether accumulation of mutations in S. iniae by serial passage in the presence of rifampin can generate immunogenic LAV conferring protection against challenge with heterologous wild-type (WT) S. iniae strains in Nile tilapia (Oreochromis niloticus). Three lineages of rifampin-resistant S. iniae strains were generated from three genetically distinct parent strains (n=9) by multiple passages in increments of Rifamycin SV sodium salt. Growth in liquid media, extent of capsulation, antimicrobial susceptibility, survival in Nile tilapia whole blood, and cytotoxicity in an O. mossambicus endothelial cell line were compared between the passaged and parental strains. Nile tilapia challenges were used to assess strain virulence, generation of anti-S. iniae IgM, and the protection conferred by LAV candidates against virulent S. iniae. Rifampin-resistant strains demonstrated changes in growth rate and cytotoxicity in endothelial cells, as well as significant reductions in whole blood survival (p < 0.05). Selected strains showed attenuated virulence in the Nile tilapia challenge model, and anti-S. iniae IgM generated against these strains demonstrated considerable cross-reactivity. Immunization by intracoelomic injection induced protection against a virulent WT strain of S. iniae, with relative percent survival ranging from 4.22 to 95.05%.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Heckman TI, Shahin K, Henderson EE, Griffin MJ, Soto E. Development and efficacy of Streptococcus iniae live-attenuated vaccines in Nile tilapia, Oreochromis niloticus. Submitted