Oral vaccination of Nile tilapia ( Oreochromis niloticus ) against francisellosis elevates specific antibody titres in serum and mucus

Although Nile tilapia (Oreochromis niloticus) is a well-established aquaculture species globally, there are a limited number of commercial vaccines available or are used for this species. The majority of diseases affecting farmed tilapia are bacterial, with antibiotics frequently used to treat fish. The current study was performed to optimise the use of mucosal vaccines for tilapia by adapting an existing bacterin vaccine against Francisella noatunensis subsp. orientalis (Fno) as a proof of concept. This vaccine has previously provided excellent protection by injection, however, the preference for tilapia farmers would be to vaccinate fish by immersion or orally, due to the lower cost and ease of application. These vaccination routes, however, are often less efficacious probably due to the lack of adjuvants in immersion and oral vaccines. The aims of this study, therefore, were to optimise the formulation and dose of the Fno vaccine with mucosal adjuvants for oral and immersion delivery. Tilapia fry (av. 6 g) were given three concentrations (high, medium, low; i.e. 1×109, 1×108 and 1×107 CFU mL-1) of antigen combined with the oral adjuvant by oral gavage, to optimise the dose needed to induce an immune response to Fno, and the immune response obtained compared with fish vaccinated by immersion (with and without an immersion adjuvant). Fry were boosted by the same route at 420 degree days (DD), and samples (serum, mucus ) taken at 840 DD for specific antibody responses measured by ELISA and western blotting. Specific IgM titres were significantly elevated in serum and mucus of fish given the high dose adjuvanted vaccine by gavage. In addition, by western blotting with serum, a significant immunogenic reaction was evident between 20 and 37 kDa in the fish given the high dose oral vaccine by gavage. As protection against Fno provided by the injection vaccine was correlated with specific antibody responses these findings suggest the oral vaccine also has potential to provide protection. Further studies are needed to optimise delivery of the vaccine via feed. A significant proportion of the diseases affecting farmed Nile tilapia, Oreochromics niloticus, are bacterial, with antibiotics frequently used to treat fish. Of the few vaccines available commercially for tilapia, most tend to be administered by injection. Due to the lower cost and ease of their application, mucosal vaccines (immersion/oral) are an ideal route of vaccine delivery for this species. There is, however, a need to develop and optimise mucosal adjuvants to enhance the immunogenicity and length of protection elicited by mucosal vaccines [1–3]. This study was performed to give preliminary data on a novel oral adjuvant (Essai GR01, Seppic Courbevoie, France) by adapting a protective injectable vaccine [4] against a common bacterial pathogen of tilapia, Francisella noatunensis subsp. orientalis (Fno). Nile tilapia fry were maintained in a recirculation system within the research aquarium facility of Benchmark R&D Ltd., in Thailand. An isolate of Fno obtained from a francisellosis outbreak in the UK was used to formulate the vaccine as previously described [4]. Tilapia (5.97 ± 0.19 g) were given an Fno antigen/Seppic oral adjuvant (30% antigen:70% adjuvant) by oral gavage. Three concentrations of Fno antigen were tested in the vaccine (high, medium, low, i.e., 1 × 109, 1 × 108 and 1 × 107 CFU mL 1, respectively) and the antibody response compared with fish vaccinated by immersion (1 × 108 CFU mL 1 inactivated Fno with and without an immersion adjuvant; IMS 1312 VG PR, Seppic). Groups of tilapia (n = 30/dose) were sedated using benzocaine (100 mg mL 1, 10%, Sigma-Aldrich Company Ltd., UK) and a 0.05 mL dose of oral vaccine applied into the stomach of the fish using a 20 gauge implantation tubing attached to a 2 mL syringe. The immersion adjuvant (IMS 1312 VG PR) was mixed with the formalin-inactivated vaccine concentrated to 2 × 109 CFU mL-1 (1:1) before diluting with tank water to give a final adjuvant concentration of 5%. Non-adjuvanted vaccine was prepared in same way, using sterile PBS in place of adjuvant. For * Corresponding author. Institute of Aquaculture, University of Stirling, Stirling, FK9 4LA, UK. E-mail address: rowena.hoare1@stir.ac.uk (R. Hoare). 1 these authors contributed equally to this work.

A significant proportion of the diseases affecting farmed Nile tilapia, Oreochromics niloticus, are bacterial, with antibiotics frequently used to treat fish. Of the few vaccines available commercially for tilapia, most tend to be administered by injection. Due to the lower cost and ease of their application, mucosal vaccines (immersion/oral) are an ideal route of vaccine delivery for this species. There is, however, a need to develop and optimise mucosal adjuvants to enhance the immunogenicity and length of protection elicited by mucosal vaccines [1][2][3]. This study was performed to give preliminary data on a novel oral adjuvant (Essai GR01, Seppic Courbevoie, France) by adapting a protective injectable vaccine [4] against a common bacterial pathogen of tilapia, Francisella noatunensis subsp. orientalis (Fno).
Nile tilapia fry were maintained in a recirculation system within the research aquarium facility of Benchmark R&D Ltd., in Thailand. An isolate of Fno obtained from a francisellosis outbreak in the UK was used to formulate the vaccine as previously described [4]. Tilapia (5.97 ± 0.19 g) were given an Fno antigen/Seppic oral adjuvant (30% antigen:70% adjuvant) by oral gavage. Three concentrations of Fno antigen were tested in the vaccine (high, medium, low, i.e., 1 × 10 9 , 1 × 10 8 and 1 × 10 7 CFU mL − 1 , respectively) and the antibody response compared with fish vaccinated by immersion (1 × 10 8 CFU mL − 1 inactivated Fno with and without an immersion adjuvant; IMS 1312 VG PR, Seppic). Groups of tilapia (n = 30/dose) were sedated using benzocaine (100 mg mL − 1 , 10%, Sigma-Aldrich Company Ltd., UK) and a 0.05 mL dose of oral vaccine applied into the stomach of the fish using a 20 gauge implantation tubing attached to a 2 mL syringe. The immersion adjuvant (IMS 1312 VG PR) was mixed with the formalin-inactivated vaccine concentrated to 2 × 10 9 CFU mL-1 (1:1) before diluting with tank water to give a final adjuvant concentration of 5%. Non-adjuvanted vaccine was prepared in same way, using sterile PBS in place of adjuvant. For immersion vaccination, two groups of fry (n = 30) were removed from the holding tank with a net and placed in the container of vaccine-±adjuvant for 30 s at 28 • C ±1. Fry were maintained at 28 • C ±1 throughout the vaccination period. Tissue samples were taken at day 1 and 2 post-vaccination (pv) for antigen uptake by immunohistochemistry. Fry were booster vaccinated by the respective methods (oral or immersion) as described above at 420 degree DD. Fish were sampled (n = 6) for skin mucus and blood at 0 and 30-days post-vaccination (dpv) (840 DD). An indirect ELISA was used to measure the specific antibody response in serum and mucus as previously described [4]; mucus samples were applied to the ELISA plate at a 1/5 dilution. Western blotting was also performed as described in Ref. [4]. Tilapia given the adjuvanted oral vaccine by gavage containing the highest dose of antigen (1 × 10 9 , CFU mL − 1 ), had significantly higher levels of anti-Fno IgM in both serum and mucus than pre-vaccinated control fish at 0 dpv, fish vaccinated orally with the medium or low dose of antigen, and the immersion vaccinated fish (Table 1).
Tissue samples (0, 1, 2 dpv; n = 3) were analysed by immunohistochemistry (IHC) for uptake of the vaccine antigen (Fno) according to Ref. [5]. The presence of vaccine antigen was observed in immersion vaccinated fish at 1 dpv in gills, skin, gut and spleen and in orally vaccinated fish it was observed at 2 days post-oral vaccination in the gut and spleen, with the antigen in the spleen only seen in fish given the high dose of oral vaccine (data not shown).
By western blotting with serum, a significant immunogenic reaction was evident between 20 and 37 kDa in the fish given the high dose oral vaccine by gavage (Fig. 1, lane 3). This region was also strongly stained with serum from fish given intraperitoneal (i.p.) vaccination with an oil adjuvanted vaccine against Fno (obtained from the study of [4]) and is associated with the pathogenicity island of Fno [6] (Fig. 1, Lane 4). Previously, protection observed with the injectable Fno vaccine (relative percentage survival of 82%) was linked to specific serum antibodies [4]. Further studies are needed to optimise delivery of the oral vaccine by feeding and to the determine efficacy of the adjuvanted oral vaccine presented here.

Declaration of competing interest
None.