High efficacy and economical procedure of oral vaccination against Lactococcus garvieae/Streptococcus iniae in rainbow trout (Oncorhynchus mykiss)

Highlights

• Chitosan-alginate coated oral vaccines increased the survival rate in rainbow trout challenged with Lactococcus garvieae(72.18 ± 9.8%) and Streptococcus iniae(83 ± 9.1%).

• Chitosan-alginate coated oral vaccines enhanced specific immunity by increasing antibody titer.

• Chitosan-alginate coated oral vaccines induced the expression of immune-related genes (IL-6 and IgM).

• These oral vaccines induced bactericidal activity and complement system 20 days after vaccination.

Abstract

The present study was aimed to examine the efficacy of chitosan-alginate coated vaccines against pathogenicity of Lactococcus garvieae and Streptococcus iniae in rainbow trout. Fish were divided into four groups including: Group A: fish immunized by chitosan-alginate coated vaccine, Group B: fish immunized by non-coated vaccine, Group C: fish feed by chitosan-alginate coated pellets without vaccine and Group D: fish feed by basic diet (non-coated and without vaccine). In groups A and B, the vaccination was carried out for 14 days and after that supplemented with fundamental diet (control diet). Comparable to groups A and B, fish of group C were also fed 14 days with test diets and after that fed control food. On day 0, 20, 40 and 60 of the experiment, serum samples were given. Fish have been challenged with live L. garvieae and S. iniae after 60 days. The levels of bactericidal activity and complement activity among innate immunity components extended on day 20 of the research and after that decreased in group A and B (P < 0.05) all through the examination. The relative expression of IL-6 and IgM in groups A and B extended on examination day 20. The expression of these genes illustrated no advancements in different groups in during the examination (P > 0.05). In group A, the serum antibody titer against L. garvieae and S. iniae broadly raised on day 40 and 60 of examination, whereas in group B, the immune response titer against S. iniae and L. garvieae illustrated a significant elevation on day 60 of the trial (P < 0.05). After challenge with live bacteria, survival rate of 83 ± 9.1%(challenged with S. iniae) and 72.18 ± 9.8% (challenged with L. garvieae) were gotten independently in group A, which were higher than survival of other exploratory groups (P < 0.05). In conclusion, the results of the present examination appear that the orally vaccination of rainbow trout with chitosan-alginate covered vaccine stimulates immunity system and also efficiently protects rainbow trout against Lactococcus garvieae and Streptococcus iniae.

Introduction

Intensive culture of fish has always increased the risk of various diseases in aquaculture [1].Today, a wide variety of diseases have been reported in fish, causing significant losses in fish production. Over the last decade, the gram-positive cocci, Streptococcus iniae and Lactococcus garvieae have been recognized as responsible for many outbreaks in fish farms, especially salmonid farms [2,3]. Streptococcosis induced by Streptococcus iniae was reported to be responsible for huge outbreaks in aquaculture throughout the world [4]. Furthermore, 60–70% of mortality in salmonid farms have been attributed to Lactococcosis caused by Lactococcus garvieae [3]. From a pathogenicity point of view, the emergence of these diseases usually occur in warm seasons (>15 °C) with signs including exophthalmia, accumulation of ascetic fluid in peritoneal cavity, erratic swimming, necrosis in the liver and spleen and a wide spectrum of surface and internal hemorrhages [3,[5], [6], [7]]. Today, a wide variety of vaccines are used to immunize domestic animals against bacterial diseases [8,9]. Vaccines have also been used in fish, however their efficacy has been different depending on fish species, pathogen agent and method of administration [[10], [11], [12], [13], [14], [15], [16], [17]]. Fish vaccines usually are applied by immersion, injection and oral administration. Although the injection and immersion methods have high efficiency, the excessive manipulation of fish and the high labour costs limit the use of these methods, particularly when fish are to be vaccinated on a large scale [10,13,18,19]. Oral administration allows the vaccination of fish in large scale, thus is considered as a less time consuming and cost effective method [10,11,20,21]. However, this method has shown less efficacy compared to injection or immersion methods of vaccination. Today, many studies have focused on coating of oral vaccines to enhance the efficacy of vaccine delivery [[22], [23], [24], [25]]. In this regard, polymers have been successfully used to protect vaccines against the acidic condition of stomach [26]. Chitosan-alginate capsules are of efficient materials for coating of oral vaccines in aquaculture. Chitosan is a biopolymer of glucosamine and N-acetyl glucosamine residues, obtained from the wastes of seafoods such as shrimp, crab shell and also cell wall of fungi [27,28]. As natural polymer, nanoparticles of chitosan have showed appropriate efficacy in coating of oral fish vaccines [[29], [30], [31]]. In addition to chitosan, alginate microparticles were also used for coating of an oral vaccine. Alginates are polysaccharides extracted from brown seaweeds and have consisted of chains of ß-D-mannuronic (M) and ar-L-guluronic (G) acids [24,25,27]. In the present study, a chitosan-alginate-coated oral vaccine (by the procedure of our research) was used for the first time against S. iniae and L. garvieae in rainbow trout. Furthermore, the efficiency of vaccination was evaluated by measuring serum immunological components, the expression of immune related genes and also by challenging fish with live bacteria and following estimation of fish mortality.