Abstract
Neobenedenia melleni is a highly infectious monogenean ectoparasite that impacts aquaculture and public aquaria, with a reported host record of over 100 marine teleost species.1 In Hawaii, N. melleni was originally identified on tilapia (Oreochromis mossambicus) held in sea cages,2 and also affected the open-ocean culture of amberjack (S. rivoliana). While the development of immunity after exposure to live N. melleni is well established, the underlying mechanisms remain unclear.3-6 Initially, we observed low parasite numbers in tilapia with high levels of anti-parasite mucus antibodies. A series of experiments were conducted to study the development of antibodies against N. melleni in tilapia.7,8
A vaccination experiment was conducted to confirm the development of specific antibody assessed via ELISA, modified from published techniques.4,5 Intraperitoneal (IP) injection of tilapia with whole sonicated parasites with adjuvant at days 0, 14 and 28 resulted in the production at day 35 of both mucus antibodies and plasma antibodies when compared to the pre-vaccination controls. These data imply that multiple parasite exposure induces both systemic and mucosal antibodies.
A multiple exposure experiment was conducted by exposing tilapia to parasites in a single exposure protocol (‘1x’) or a double exposure protocol (‘2x’), followed by a parasite challenge and then sample collection at 3, 7, 10 and 13 days post-challenge (DPC). Parasites dislodged in freshwater immersion were enumerated and mucus and plasma antibodies were assessed. The 1x group displayed significantly lower levels of parasites than the control group at all time points. The 2x group displayed significantly lower levels of parasites than the control group at 3 DPC and 13 DPC. However, for all groups, antibodies remained at baseline levels.
A continuous parasite exposure experiment was conducted to replicate the pilot study where mucus antibody and protection was observed. Tilapia were exposed to N. melleni for 120 days and assessed for parasites and antibodies at several time points. Fish displayed high parasite loads at 45 days post-exposure (DPE) (146.45±99.44 parasites/fish) and subsequent decreases at 102 DPE (12.19±13.92 parasites/fish, p<0.05) and 120 DPE (2.19±1.97 parasites/fish, p<0.05). Mucus antibodies at 45 DPE (OD 0.0382±0.4) increased at 102 DPE (OD 0.8578±0.36, p<0.05) and were sustained at 120 DPE (OD 0.2600±0.26, p<0.05). Plasma antibody levels were increased at 102 DPE (OD 0.6681±0.20) and 120 DPE (0.8890±0.19). Here, antibody and immunity were associated during continuous exposure.
These results suggest that local antibody production may be a key mechanism in the development of immunity against N. melleni as seen in other fish parasites9,10, but also that other arms of the mucosal immune response may be involved8. Overall, immunity against N. melleni may be possible in certain species where the host-parasite relationship or parasite-immune response is in balance. These species may serve as indicator species to monitor and also be a source of outbreaks in closed systems such as public aquaria.
Acknowledgements
The authors wish to thank Mr. Neil Sims, formerly of Kona Blue Water Farms and current founder of Ocean Era, for his contributions to this study, including providing the initial parasite sources and allowing his team to share techniques for enumeration of parasites. The authors also thank Dr. Gordon Grau and his laboratory at the Hawaii Institute of Marine Biology, for providing the tilapia stocks.
*Presenting author
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