Antigens of Aeromonas salmonicida subsp. salmonicida specifically induced in vivo in Oncorhynchus mykiss

Furunculosis is a major fish disease, associated with high mortality rates, and its effect is particularly significant in farmed salmonids (Hiney & Olivier 1999). Efficacious vaccines, generally injected alongside an oil adjuvant, are available. However, certain side effects have been reported (Midtlyng, Reitan & Speilberg 1996; Anderson et al. 1997) and protection can lapse in stressed fish and in non-salmonid species (Gudmundsd ottir & Bj€ornsd ottir 2007). In vivo-induced antigen technology (IVIAT) uses antibodies raised by individuals exposed to the pathogen of interest. These antibodies are then adsorbed against an in vitro culture of both the pathogen and, to avoid cross-reaction, the organism in which the genomic library will be expressed (Handfield et al. 2000). This removes antibodies binding antigens expressed under culture conditions. The remaining antibodies, recognizing antigens specifically expressed during growth within the host, are used to probe a genomic library expressing random segments from the genome of the pathogen of interest. Reactive clones are sequenced and identified based on homology, identifying genes overexpressed in vivo (Rollins et al. 2005). Antigens associated with the infectious phenotype are likely to act as virulence factors and constitute interesting targets for vaccine development. Consequently, we have previously applied IVIAT to Aeromonas salmonicida subsp. salmonicida (clinical isolate A14390) infecting rainbow trout weighing an average of 92 grams (Menanteau-Ledouble et al. 2014a; Menanteauledouble et al. 2014b) in a study approved by the university institutional ethics committee and the national authority according to §26 of the Austrian Law for Animal Experiments (Tierversuchsgesetz 2012–TVG 2012-91 under the No. GZ 68.205/140-II/3b/2012). Sera were harvested at multiple time points to sample a wide array of the immune response and screening identified four antigens: UDP-3-O-acyl-N-acetylglucosamine deacetylase (involved in cell wall synthesis), RNA polymerase sigma factor RpoD (a regulator of gene expression) as well as TonB (that provides energy for transport across the cell membrane) and a hypothetical protein (Menanteau-ledouble et al. 2014b). In this study, we report on further screening of this library using the same pool of adsorbed sera. A significant difference between both studies is that more time points were included in the RTqPCR to confirm that the genes discovered were overexpressed throughout the course of the infection: This time, six time points were included: 1, 6, 12 and 48 h as well as a 1 and 2 weeks postinfection. Mean fold changes in gene expression were calculated between in vitro cultures and the Correspondence M El-Matbouli, Clinical Division of Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterinarplatz 1, Vienna 1210, Austria (e-mail: [email protected])