Fish have numerous pathogens to contend with in the course of their life. Some of these dangers are reduced through their innate immunity, although others are more virulent and require the assistance of adapted immunity to fight them.

This adapted or acquired immunity can be trained to obtain better results, although the product that induces this immunisation is just as important as the application protocol.

Vaccination is the safest and most effective way to achieve this specific acquired immunity against certain pathogens. In fish, the vaccination technique that achieves a stronger and longer-lasting immunity is vaccination by injection, and intraperitoneal injection is the most common route.

By means of intraperitoneal injection, a vaccine for a specific pathogen can be introduced into the fish to generate protection against the pathogen. This protection is called active immunity, and it consists of training the immune system to recognise future infections by the same pathogen and to fight them with a faster and stronger immune reaction.

For this active immunisation process to be successful, several parameters must be considered:

• The moment of vaccination: when choosing when to vaccinate, it should be taken into account that vaccination is followed by a period of immunisation. During this period (Onset of Immunity), the fish's immune system is learning to generate a response against the pathogen targeted by the vaccine, and it is important that this response is optimal to ensure proper immunisation. It is measured in degree-days, so temperature can be decisive in developing the expected protection against the pathogen (Soto et al. 2014).
• The anaesthesia: a human anaesthetist has a great responsibility; this is no different in fish anaesthesia, as this process is extremely important for the vaccine to be injected correctly and for the animal's recovery and subsequent immunisation.
• The needle: needle size (diameter and length) is important to minimise damage when penetrating the skin and depositing the vaccine in the appropriate place within the peritoneum.
• The injection site: this must be within the tolerance area, otherwise the vaccine may be injected into an organ, muscle wall or cartilage.
• The size of the fish: larger fish are known to have a faster and longer-lasting immunological response (Fender and Amend, 1978; Amend and Eshenour, 1980). The smaller the fish, the smaller the injection area and the more difficult vaccination becomes, leading to more mistakes, such as injecting the vaccine into vital organs, the muscle or even outside the animal. This is compounded by the fact that working with smaller fish makes the anaesthesia, the time out of water, and the grip and pressure applied when injecting even more delicate, requiring much greater care.

These points indicate that reducing the recommended vaccination size may yield a weaker immune response or lead to side effects in the fish, such as developmental problems (internal organ necrosis) or the death of the animal. A good injection technique means always keeping these indications in mind when vaccinating.

Tolerance area for the injection site in sea bass, trout, cobia, grouper, etc.

A poor vaccine injection technique may cause low immunisation, organ necrosis, adhesions, melanisation or even the death of the fish.

One factor that makes the vaccination technique vitally important is the vaccine composition, and more specifically the excipient. A vaccine can have an aqueous or an oily excipient.

The aqueous excipient allows for less precision in the injection technique because it does not usually generate side effects, although the immune response achieved in the fish is weak and does not last long. There are two oily excipients that are mostly used in the formulation of fish vaccines, one is mineral and the other is non-mineral. Liquid paraffin is the most widely used mineral oil excipient in fish vaccines, and Montanide™ is the most common non-mineral oil excipient.

The mineral oil excipient generates a powerful immune response, but it is a very aggressive adjuvant that usually generates adhesions and melanisation (Midtlyng et al., 1996), so the technique, dose and size of the fish are very important for vaccination to be successful. It has been proven that with the same dose of mineral oil vaccine, fish vaccinated at a smaller size develop more adverse effects (Berg et al., 2006), whereas fish vaccinated at a larger size will develop fewer adverse effects and will therefore have greater growth (Berg et al., 2007).

A non-mineral oil adjuvant is used to achieve a powerful and lasting but less aggressive immune response (Aucouturier et al., 2001). This adjuvant is less aggressive than a mineral oil one because it is easily metabolised.

The test conducted by Tziouvas and Varvarigos (2021) shows that vaccines with mineral oil (non-metabolizable) adjuvants generate greater side effects such as adhesions, granulomas and pigmentations than vaccines with non-mineral oil (metabolizable) adjuvants.

According to this same study, side effects are more common in smaller fish. Similarly, they verified that with non-mineral oil vaccines, side effects are usually resolved at 3,000-3,500 dd, whereas in the case of mineral oil vaccines it is 9,500 dd.

References

Aucouturier, J., Dupuis, L., Ganne, V., Adjuvants designed for veterinary and human vaccines, Vaccine 19 (2001) 2666–2672.

Amend, D.F. and Eshenour, R.W.; 1980. Development and use of commercial fish vaccines. Salmonid. Vol. III, No. 6: S-12.

Aucouturier, J., Dupuis, L., Ganne, V. Adjuvants designed for veterinary and human vaccines, Vaccine 19 (2001) 2666–2672.

Berg, A., Rødseth, O.M., Tangerås, A., Hansen, T.J., 2006. Time of vaccination influences development of adherences, growth and spinal deformities in Atlantic salmon (Salmo salar L). Dis. Aquat. Org. 69, 239–248.

Berg, A., Rødseth, O.M., Hansen, T., 2007. Fish size at vaccination influence the development of side-effects in Atlantic salmon (Salmo salar L.). Aquaculture 265, 9–15.

Fender, D. C. and Amend, D. I.; 1978. Hyperosmotic infiltration: factors influencing uptake of bovine serum albumin by rainbow trout. J. Fish. Res. Board Can. 35: 871.874.

Midtlyng, P.J., Reitan, L.J., Speilberg, L., 1996. Experimental studies on the efficacy and side-effects of intraperitoneal vaccination of Atlantic salmon (Salmo salar L.) against furunculosis. Fish Shellfish Immun. 6, 335–350.

Soto, E., Brown, N., Gardenfors, Z., Yount, S., Revan, F., Francis, S., Kearney, M., Camus, A., (2014). Effect of size and temperature at vaccination on immunization and protection conferred by a live attenuated Francisella noatunensis immersion vaccine in red hybrid tilapia. Fish & shellfish immunology. 41. 593-599.

Tziouvas, H., and P. Varvarigos. 2021. “Intensity Scale of Side Effects in European Sea Bass (Dicentrarchus Labrax) Post Intraperitoneal Injection with Commercial Oil-Adjuvanted Vaccines.” Bulletin of the European Association of Fish Pathologists 41 (3): 103–10.