Surviving fish developed specific IgM against MDRA. hydrophila
All surviving fish in three groups challenged with MDR A. hydrophila had significantly higher levels of specific antibody (IgM) compared to the three unchallenged groups (p < 0.05) as measured by indirect ELISA, Kruskal - Wallis test: H (5) = 35.218,p = 0.000 (Figure 7). The serum from fish in the Ah + no phage, Ah + phage 0.1, and Ah + phage 1.0 groups had OD readings of 0.18 ± 0.09, 0.22 ± 0.17, and 0.22 ± 0.12, respectively. The IgM level was slightly higher in 2 phage treated groups but not statistically significant difference. In contrast, the low level of OD450 readings were recorded in the remaining groups (0.06 ± 0.003 to 0.08 ± 0.03) (Figure 7).
DISCUSSION
The Myoviridae phages specific to A. hydrophila are highly diverse in nature (Chandrarathna et al., 2020; Cheng et al., 2021; Jun et al., 2013). The lytic pAh6.2TG isolated in this study had genome characteristics most closely related to phage PVN02 (99.33% nt. identity) in the GenBank database, previously isolated from Vietnam (Tu et al., 2020). The origins of two phages from the closed geographical area of Mekong basin, although from different rivers, may explain the high genomic similarity of pAh6.2TG and PVN02. Compared to previously reported A. hydrophila -specific phages, pAh6.2TG (51,780 bp) had similar genome size with the phage PVN02 (51,668 bp) from Vietnam (Tu et al., 2020), and pAh6-C (53,744 bp) from Korea (Jun et al., 2015), but is larger than phage AhyVDH1 (39,175 bp) from China (Cheng et al., 2021), and smaller than phage LAh10 (260,310 bp) from Australia. The latter is the largest known phage infecting A. hydrophila (Kabwe et al., 2020). Genome analysis indicated that pAh6.2TG does not contain potential virulent genes or antimicrobial resistant genes, suggesting it is highly relevant as a biocontrol agent in aquaculture systems without concern of antimicrobial-resistant gene transmission.
Climate change has affected aquaculture environments by perturbing chemical and physical properties of water, particularly in the increase of water temperature and salinization (Maulu et al., 2021; Seggel & De Young, 2016). The stability of pAh6.2TG under a wide range of temperatures (4 – 40 °C) and salinity (0 – 40 ppt) might be important characteristics for its wider application in diverse aquaculture environments. Relatively high stability of pAh6.2TG in fish-rearing water suggests that immersion route is practical. However, low viability of pAh6.2TG at pH 3 – 5 suggests that oral administration might not be applicable due to the low pH in gastrointestinal tract of aquatic animals, e.g. pH in Nile tilapia stomach range from 1.4 – 2.0 (Moriarty, 1973).
One of the major limitations of phage application is its narrow host range and geographical specificity (Culot et al., 2019; Pérez-Sánchez et al., 2018; Ross et al., 2016). Although the newly isolated phage pAh6.2TG could lyse multiple isolates of MDR A. hydrophila from Vietnam, however, it does not lyse the isolates from Thailand and other bacterial species from the same or different genera. Therefore, to expand wider application of phage in aquaculture, a cocktail of multiple phage strains from different geographical locations might be the better approach to tackle not only AMR A. hydrophila but also other important bacterial pathogens in freshwater fishes. In addition, the specific infection of pAh6.2TG to A. hydrophila and not probiotic bacteria suggest the potential combination of phage therapy and probiotics to combat MDR A. hydrophila infection in aquaculture.
Carps, tilapias, and catfishes are crucial inland freshwater fish that play a vital role for food system transformation to tackle micronutrient deficiencies in LMICs (FAO, 2020; Hicks et al., 2019). A. hydrophila infection is one of the most important bacterial diseases responsible for the loss of millions of dollars in the global freshwater aquaculture industry (Hossain et al., 2014; Peterman & Posadas, 2019; Pridgeon & Klesius, 2012). Increasing prevalence of pathogenic MDRA. hydrophila in aquaculture poses the high risk for serious uncontrollable disease outbreaks and public health concern due to spread of AMR. Non-antibiotic approach using lytic phages, therefore, was explored to control disease caused by MDR A. hydrophila in aquaculture systems. In this study, we provided in vivo evidences for the efficacy of phage application in rearing water which is effective at suppressing bacterial concentration in water as well as reducing the bacterial load in fish liver. The presence of phages in the fish liver also suggests that immersion administration could deliver considerably large number of phages into the fish tissue. These factors may contribute to improvement of survivability (RPS = 50 – 75%) of tilapia. Importantly, not only was there higher survival in phage treated groups, but all surviving fish also developed specific IgM against A. hydrophila . This suggests that phages possibly weakened the bacteria which allowed the fish immune system to respond more effectively and saved the fish from death. Similarly, there were several studies using phages as therapeutic agent to control A. hydrophila infection. Le et al. (2018) used phage cocktails (ɸ2 and ɸ5) with MOI = 0.01, 1.0, and 100 to control A. hydrophila infection in striped catfish (Pangasianodon hypophthalmus ) by injection administration and obtained RPS of 16.33%, 44.9%, and 100%, respectively. Immersion treatment of 1 x 108 PFU/mL phage Akh-2 improved survivability of Nile tilapia with RPS of 41.1% (Akmal et al., 2020). Cao et al. (2020) applied phage MJG by injection, immersion, and oral administration to control a pathogenic A. hydrophila in rainbow trout and the fish gained RPS of 100%, 66.7%, and 50%, respectively. Dang et al. (2021) showed protective efficacy of phage PVN02-sprayed feed against A. hydrophila 4.4T in striped catfish with RPS from 75.6 – 87.8%.
The findings in this study suggest a potential approach using phage as prophylactic agent that was effective in protecting Nile tilapia from a MDR A. hydrophila . This approach provided comparable RPS to other promising alternatives to antibiotics, such as probiotic-based or plant-based products (Dawood et al., 2020; Kuebutornye et al., 2020; Naliato et al., 2021; Neamat-Allah et al., 2021). Apart from tilapia, pAh6.2TG has great potential to be applied in catfish aquaculture industry due to the lytic activity of pAh6.2TG against multiple MDRA. hydrophila strains isolated from diseased striped catfish.
In summary, this study reported a newly isolated lytic phage pAh6.2TG that infects several isolates of MDR A. hydrophila . The phage was classified as a member of Myoviridae based on a combination of morphology and genomic characterization. In vitro tests showed that pAh6.2TG was relatively stable at different environmental conditions. Using this phage as prophylactic agent was successful at reducing mortality in Nile tilapia. Phage pAh6.2TG application in rearing water not only suppressed MDR A. hydrophila loads in the rearing water and colonization of the bacteria in fish liver, but also improved fish survivability. These findings supported that pAh6.2TG could be used in rearing water for biocontrol of MDR A. hydrophila infection towards sustainable aquaculture.