Performing Department
Pathology, Microbiology & Immunology
Non Technical Summary
Lactococcus garviae is one of the most important emergent pathogens of farmed fish in the USA. Outbreaks of lactococcosis have occurred in at least three states where freshwater fish are cultured for human consumption, causing significant economic losses to conservation programs as well as private aquaculture operations. A lack of efficacious vaccines, mounting antimicrobial resistance, and limited treatment options have led to mortalities approaching 90% in some US epizootics. Direct economic losses are compounded by the costs of delayed production and growth, chemotherapeutants, and added labor. Furthermore, when antibiotic therapy is attempted, treatment failure is common. Lack of fish host specificity poses an additional risk, should the pathogen escape into natural habitats. These factors illustrate that development of effective prophylactic measures are urgently needed. A substantial body of peer-reviewed literature provides strong evidence for the effectiveness of vaccination, particularly under intensive culture practices, and its contribution to economic sustainability in the aquaculture industry. To address this need, we propose to: 1) Investigate protection conferred to rainbow trout by a formalin-killed vaccine augmented by injectable and immersion adjuvants upon L. garviae challenge, 2) Investigate the immune response of rainbow trout after immunization with a formalin-killed vaccine augmented by injectable and immersion adjuvants upon L. garviae challenge, and 3) Disseminate the results obtained to the aquaculture industry. The overarching goal of this study is to provide scientifically validated protocols to state, federal, and private aquaculture facilities in the US for control of lactococcosis.
Animal Health Component
100%
Research Effort Categories
Basic
20%
Applied
50%
Developmental
30%
Goals / Objectives
1) Investigate protection conferred to rainbow trout by a formalin-killed vaccine, using injectable and immersion adjuvants, followed by challenge with virulent L. garviae, and 2) Investigate the immune response of trout to vaccination with a formalin-killed vaccine against L. garvieae. 3) Disseminate the results obtained to the aquaculture industry.
Project Methods
Vaccine preparation: In aquaculture, formaldehyde is commonly used to produce whole-cell killed vaccines (12). Protocols by Fukushima et al (6), the standard method to generate formalin-killed autogenous vaccines, will be followed to inactivate L. garvieae recovered from a CA trout aquaculture facility in 2020. Bacteria grown at 30°C in broth media for 24 h to a final concentration of 109 CFU/mL will be suspended in sterile phosphate-buffered saline (PBS) and inactivated by the addition of 1% (v/v) formalin while incubated at 4°C for 24 h. Killed bacteria will then be resuspended in sterile PBS at a final concentration of 109 CFU/mL (bacterin) and sterility confirmed by plating the bacterin on agar media and incubating at 30°C for 72 h.Intracoelomic injection immunization: As a proof of concept and to evaluate the systemic immune response of trout, fish will initially be immunized with a CA recovered L. garviae isolate by intracoelomic injection (IC). Briefly, rainbow trout fingerlings (n=20 fish per tank, n=4 tanks per treatment) kept at 18 C will be immunized with 107 CFU/fish formalin killed L. garviae suspended in the mineral oil based adjuvant Montanide ISA 763 A (Seppic, France) via IC injection. Fish will be monitored daily and fed a commercial pelleted diet at 1% of their body weight per day. Control groups will be injected with adjuvant alone or PBS to investigate protection conferred by adjuvant immunostimulation.Immersion immunization: Protocols by Soltani et al. (11) will be followed for immersion vaccination (Imm.) of fingerlings using Montanide IMS 1312 VG adjuvant to enhance immunogenic responses. Rainbow trout. fingerlings (n=20 fish per tank, n=4 tanks per treatment) kept at 18 C will be vaccinated with 107 CFU/ml suspended in MontanideTM IMS 1312 VG at a final concentration of 10% in the bath for 1h. Control groups will be exposed to adjuvant alone or PBS to investigate protection conferred by adjuvant immunostimulation. .Treatment A: IC bacterin+adjuvant; exposed to L. garviaeTreatment B: IC bacterin+adjuvant; non-exposed to L. garviaeTreatment C: IC PBS; exposed to L. garviae (Positive Control/PBS)Treatment D: IC PBS; non-exposed to L. garviae (Negative Control/PBS)Treatment E: IC Adjuvant; exposed to L. garviae (Positive Control/Adjuvant)Treatment F: IC Adjuvant; non-exposed to L. garviae (Negative Control/Adjuvant)Treatment G: Imm. bacterin+adjuvant; exposed to L. garviaeTreatment H Imm. bacterin+adjuvant; non-exposed to L. garviaeTreatment I: Imm. PBS; exposed to L. garviae (Positive Control/PBS)Treatment J: Imm. PBS; non-exposed to L. garviae (Negative Control/PBS)Treatment K: Imm. Adjuvant; exposed to L. garviae (Positive Control/Adjuvant)Treatment L: Imm. Adjuvant; non-exposed to L. garviae (Negative Control/Adjuvant)Challenge: Protection conferred by the different treatments will be evaluated by IC challenge with 105 CFU/fish L. garviae following protocols developed in our laboratory at 750 degree-days (temperature in Celsius x days post-immunization) post-immunization. Thirty days post-challenge, cumulative percent mortality (CPM) will be calculated for each treatment as shown below. The relative percent survival (RPS) will be calculated for the vaccinated groups using the following formula (1): (1 - [CPM in vaccinated group/CPM in unvaccinated group]) x 100Additionally, 6 random survivors per treatment will be collected and formalin-fixed for histopathological analysis. Posterior kidney will be collected to evaluate for persistence of L. garviae infection by plating on agar media. Moreover, since we hypothesize the primary mechanism of protection against L. garviae is antibody based, serum and mucus IgM antibodies against L. garviae will be quantified from 10 different surviving fish per treatment following protocols outlined by Soto et al. (13).Statistical analysis Differences in CPM and antibody titer (log10 transformed) 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 the differences are significant (P < 0.05), a Tukey's post-hoc test will be carried out to determine which groups are different. Statistical analysis will be performed in GraphPad® Prism v5.03 (GraphPad, San Diego, CA, USA). Additionally, survival curves will be generated and compared via log-rank test.