Elsevier

Fish & Shellfish Immunology

Volume 105, October 2020, Pages 350-358
Fish & Shellfish Immunology

Full length article
Efficacy of two adjuvants administrated with a novel hydrogen peroxide-inactivated vaccine against Streptococcus agalactiae in Nile tilapia fingerlings

https://doi.org/10.1016/j.fsi.2020.07.051Get rights and content

Highlights

  • H2O2 inactivated vaccines for S. agalactiae can be used as an alternative to formaldehyde-inactivated vaccines.

  • Inactivated vaccine by H2O2 provided moderate protection against S. agalactiae in Nile tilapia.

  • H2O2 inactivated vaccine against S. agalactiae when used with adjuvants showed high protection in Nile tilapia.

Abstract

Streptococcus agalactiae is considered the main bacterial pathogen in cultured Nile tilapia. Formaldehyde-inactivated vaccines are the most accepted method for prevention and control of the disease. However, alternative inactivation methods for S. agalactiae vaccines have not been fully explored. Recently, we developed a hydrogen peroxide-inactivated vaccine against S. agalactiae with moderate efficacy, with the possibility to improve vaccine efficacy by adding adjuvants. The current study compared the efficacy of aluminum hydroxide and Freund's incomplete adjuvant (FIA) incorporated into a novel hydrogen peroxide-inactivated intraperitoneal vaccine against S. agalactiae for Nile tilapia fingerlings. The relative percentage survival (RPS) for aluminum hydroxide-adjuvanted vaccine (59.3%), and FIA-adjuvanted vaccine (77.8%) were higher than the vaccine without adjuvant (40.7%). In addition, fish immunized with aluminum hydroxide-adjuvanted vaccine had significantly higher levels of specific antibodies than control fish at 4 weeks post vaccination (wpv). Blood lymphocytes counts showed a decrease in vaccinated groups when compared to control fish, suggesting white cells migration to the tissues where antigen presentation is ongoing. Fish that received FIA-adjuvanted vaccine exhibited persistence of adjuvant deposits on intraperitoneal surfaces for at least 4 wpv that may be related to its superior performance compared to aluminum hydroxide adjuvanted vaccine, which did not evidence any type of deposit at any sampling times. The results observed in this study demonstrate that hydrogen peroxide-inactivated vaccine administered with either aluminum hydroxide or FIA induce optimal levels of protection, with a superior performance for FIA vaccine, which could be a good alternative to conventional formaldehyde-inactivated vaccines against S. agalactiae, due to its shorter manufacture time, and less toxicity.

Introduction

Streptococcus agalactiae, a group B Streptococcus, is a Gram-positive bacterium [1], that can infect many freshwater, estuarine and marine fish species, particularly Nile tilapia Oreochromis niloticus [1,2]. Streptococcosis is characterized by septicemia and severe meningoencephalitis [1], and causes huge economic losses in tilapia culture [3], due to the high rates of infection and mortality that can reach up to 50–90% [1]. S. agalactiae has been responsible for several outbreaks of disease worldwide [4]. In Brazil, S. agalactiae infection occurs continuously, and is widely distributed geographically in the states of Paraná, São Paulo, Minas Gerais, Santa Catarina, Bahia, Ceará, Mato Grosso, Pernambuco, Goiás and Espírito Santo [5].

Sustainable aquaculture is not possible without disease prevention. Therefore, vaccination has become a significant prevention tool [6]. Vaccination is a widely accepted and effective method to control S. agalactiae infection and prevent mass mortality in tilapia [7]. Live attenuated vaccines have a great risk for reversion to virulence, thus spreading pathogenic bacteria into the environment [8]. For this reason, the use of inactivated or purified antigens is preferable.

Alternative methods for pathogen inactivation using hydrogen peroxide (H2O2) have been standardized in recent years for mice vaccines against Staphylococcus aureus and Pseudomonas auruginosa [9], as well as lymphocytic choriomeningitis virus [10], and rabies virus [11] with great success. Additionally, H2O2-inactivated vaccines have been tested in rhesus monkeys and mice against West Nile virus [12,13], reporting high efficacy and improvement of the adaptive immune response. H2O2 inactivation presents lower toxicity and preserves epitopes better than formaldehyde [9]. Recently, our research group compared the performance of a novel H2O2-inactivated vaccine against S. agalactiae in Nile tilapia showing equivalent efficacy to formaldehyde-inactivated vaccine and pH-manipulated vaccine [14]. During the vaccination trial, the vaccine proved to induce moderate efficacy against challenge using a homologous strain, despite no adjuvant was added to its formulation, suggesting that its efficacy can still be widely improved.

Inactivated vaccines are less immunogenic compared to live vaccines [15]. As a result, adjuvants have been indispensable for improvement of vaccine efficacy, by increasing the strength and length of protection [16]. Adjuvants increase the adaptive immune response [[17], [18], [19]] and can be classified as signal 1 and signal 2 facilitators [20]. Oily emulsions, also known as signal 1 facilitators, are the most commonly used [15]. Such adjuvants include the Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA) and Montanide [19], and have reported to potentiate vaccine efficacy when used in fish vaccines [21]. FIA is a water-oil (W/O) emulsion, that has proved to boost vaccine efficacy by stimulating higher antibody responses both humoral and mucosal when used in inactivated vaccines against S. agalactiae incorporated into the diet of red tilapia, Oreochromis sp. [22], and in polyvalent inactivated vaccines against Edwardsiella tarda and Vibrio anguillarum for Japanese flounder, Paralichthys olivaceus, evidencing high relative percent survival (RPS), of 70.9% and 67.4%, respectively [23]. Additionally, subunit vaccines against S. agalactiae, adjuvanted with FIA or Montanide, reported high RPS of 76.7% and 74.4%, respectively [24].

On the other hand, signal 2 facilitators activate cytokines and regulate costimulatory molecules during the antigen recognition phase, thus increasing the immune response during the vaccination period [19]. These facilitators include aluminum compounds such as aluminum hydroxide and aluminum phosphate [19]. So far, there are no studies relating to aluminum adjuvants to potentiate vaccines for tilapias. Previous studies involving vaccines against E. tarda in Japanese flounder with aluminum hydroxide or aluminum phosphate adjuvants revealed RPS of 53% and 69%, respectively and higher antibody titers for both vaccines when compared to the vaccine without adjuvant [25].

Therefore, the aim of the present study was to refine the efforts developed in our previous work [14] with a novel H2O2-inactivated vaccine against S. agalactiae for Nile tilapia, by enhancing its efficacy using adjuvants, and compare the efficacy of two adjuvants (FIA and aluminum hydroxide).

Section snippets

Bacterial strain

Streptococcus agalactiae strain (serotype Ib) used in this study was isolated from naturally infected Nile tilapia with clinical signs of streptococcosis in the northern region of Paraná State, Brazil [26]. The strain (SA43) was previously used to develop a H2O2-inactivated vaccine against S. agalactiae for Nile tilapia [14]. Bacteria were preserved in BHI broth (Kasvi, Brazil) containing 15% glycerol (v/v) in ultra-freezer at −80 °C until further use.

Fish and experimental conditions

Nile tilapia fingerlings used during the

Results

No fish died during the vaccination period (four weeks). Naïve and non-vaccinated, non-challenged groups did not evidence any clinical sign during the challenge period. Regarding the challenged groups, mortality started at the 6th, 5th and 2nd day after challenge in groups non-vaccinated, aluminum hydroxide and FIA, respectively, and at 5th day in groups H2O2-vaccine and H2O2-vaccine + aluminum hydroxide. Finally, in the H2O2-vaccine + FIA group mortality started at the 4th day after bacterial

Discussion

The present study tested the efficacy of an experimental H2O2-inactivated vaccine against S. agalactiae with two different adjuvants (FIA and aluminum hydroxide). Most fish vaccination studies use formaldehyde for bacterial inactivation [[38], [39], [40]]. Despite formaldehyde being a strong inactivating agent, it can have negative effects on physical and chemical characteristics of superficial antigens and on the immune response [10] by modifying the integrity of epitopes, affecting antigenic

Conclusions

The results observed in this study are promising on the use of H2O2 as an alternative methodology to conventional formaldehyde bacterial inactivation. In addition, this type of vaccine proved to be highly effective when administered with two of the most common adjuvants used in aquaculture. Future studies should focus on improving the vaccine efficacy.

CRediT authorship contribution statement

Fernando Carlos Ramos-Espinoza: Conceptualization, Methodology, Investigation. Victor Alexander Cueva-Quiroz: Methodology, Investigation, Formal analysis. Jefferson Yunis-Aguinaga: Visualization, Writing - review & editing. Norquis Caled Alvarez-Rubio: Investigation, Formal analysis. Nicoli Paganoti de Mello: Investigation, Formal analysis. Julieta Rodini Engrácia de Moraes: Supervision, Project administration, Funding acquisition.

Acknowledgements

This work was supported by the São Paulo Research Foundation (Fapesp) - Grant 2018/06137-1 and 2019/02339-1; and the National Council for Scientific and Technological Development (CNPq) - Scholarship 141835/2018-4. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

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