Evaluation of increasing bioavailability of oral vaccines against pasteurellosis in reared sea bass (Dicentrarchus labrax L.).

Pasteurellosis or photobacteriosis, sustained by Photobacterium damselae subsp. piscicida (Phdp), affects many natural populations and cultures of marine fish in many parts of the world and results in very high losses in the cultured sea bass (Dicentrarchus labrax L.) in the Mediterranean sea.Vaccination of farmed fish is recognised as the best procedure to prevent diseases and is preferred to antibiotic therapy due to drug-resistance and environmental impact. Immersion and intraperitoneal injection are the two most common methods used for vaccination in fish. However, these methods require handling of fish and, thereby, stress generation. The oral vaccination represents the ideal and most convenient route of delivery. Recently, the research has been focused on techniques devoted to including vaccines in nanoparticles/ microparticles of different size to increase the protection in the intestinal tract and the bioavailability of the antigen. The aim of this research was to evaluate the oral absorption of a vaccine anti-Phdp from two forms of microcapsules by immunohistochemical and electron microscopy investigations. The oral vaccinations were conducted on 48h-fasted sea bass at 24.6°C by 5 days in-feed vaccination, 5 days vaccination-free and 5 days re-vaccination. Samples of hindgut, spleen and kidney, collected during the first and the second vaccination period, were fixed in 10% neutral buffered formalin and embedded in paraffin wax. The sections (4 μm) placed on poly-lysine coated slides were subjected to immunohistochemical procedures after deparaffination in xylene. Antigen retrieval with microwave and immunohistochemistry (IHC) using the primary antibody (Aquatic Diagnostic Ltd.-Stirling), revealed colonies of bacteria in the intestinal tract of all experimental groups receiving the second administration of vaccines, while the primary antibody Microtek did not appear to react with bacteria present in the samples from our experimental groups. As positive control, spleen tissue sections of infected fish from another provenience were used. No staining were observed in negative control. Trials were repeated five times to validate the results. For ultrastructural analysis samples were fixed with 4% p-formaldehyde and 0.5% glutaraldehyde and embedded in LR White resin polymerized with UV light. Immunocytochemistry was performed with the post–embedding technique using the primary antibody (Stirling) undiluted and the antigen, revealed by gold grains, was present along the vacuole borders and over the amorphous material confirming the results obtained by immunohistochemical investigations. The presence of Phdp antigen in the intestinal epithelium bring us to consider that microencapsulation represent a promising result for the oral vaccination in that the first contact with pathogens mostly occur through mucosal surfaces.