A phytobiotic-based additive to support immunocompetence and reduce the impact of co-infections

Ruth Garcia Gomez, Maria Mercè Isern-Subich, Martha Mamora, Waldo G. Nuez Ortin, Adisseo

Shrimp immunocompetence and co-infections

The immune system, in general terms, is founded in three major “control barriers”, namely the “physical barriers” or first line of defense, the innate immunity based on the recognition and destruction of pathogens without specific “memory” against them, and the “adaptative immunity”, in which the host fights against microorganisms through pathogen-specific actions. This adaptative barrier is not present in shrimp; therefore, immunocompetence is greatly supported by the activation of hemocytes as a major part of the innate response.

When a pathogen gets in touch with the host shrimp, it is recognized by receptors located in the membranes of hemocytes and cell tissues. After this first recognition, a series of complementary responses are unchained, including phagocytosis, apoptosis, nodulation, encapsulation, melanization, clotting, and production of antimicrobial peptides (AMPs). Two types of AMPs exclusive to shrimp are penaidins and crustins.

• Penaidins have a strong antimicrobial action against gram + bacteria and fungi, and opsonic activity against gram – bacteria, releasing a signal to activate phagocytosis by hemocytes.
• Crustins present antimicrobial activity against both gram + and -, and opsonic activity to help in the phagocytosis of bacteria by hemocytes.

AMPs play a crucial role in supporting shrimp survival under infection. Therefore, by promoting the effective production of key AMPs, we can stimulate a faster and better-coordinated response, thus increasing survival and productivity.

Co-infections caused by bacteria and virus (e.g. Vibrio spp. + White-Spot Disease Virus (WSDV)), or by bacteria and parasite (e.g. Vibrio spp. + Enterocytozoon hepatopenaei (EHP) such as White Feces Syndrome (WFS)) are common in shrimp farming. It has been demonstrated that shrimp previously exposed to WSDV is more easily infected by Vibrio parahaemolyticus (VP) (i.e. causative agent of Acute Hepatopancreatic Necrosis Disease (AHPND)) and shows a suppressed capacity for recovery (de la Peña et al., 2017). In addition, WSDV infection can be accelerated and exacerbated by the secondary AHPND infection (Han et al., 2019). Therefore, not only WSDV shrimp can easily develop AHPND, but shrimp infected with the two pathogens at the same time will face a reduced recovery rate and higher mortality. AMPs including crustins and penaidins have been identified as an important humoral defense mechanism in co-infections such as the WFS (Tassanakajon et al., 2017).

Co-infections can only be minimized from a preventive approach in shrimp aquaculture – there is no other option for success: single bullets will no longer be effective in multifactorial disease scenarios. The first approach to control multiple infections is based on improved farming and biosecurity practices. On-farm biosecurity should consider high PL health status, as well as control of active and passive vectors. A second approach is to reinforce prevention strategies via functional nutrition devoted to supporting immunocompetence and gut health/integrity and reducing the impact of systemic infection.

This article presents the efficacy of a phytobiotic- based functional additive (Sanacore® GM) towards enhancing shrimp immunocompetence through the promotion of antimicrobial peptides (AMP) as a mechanism to reduce the severity of co-infections and therefore enhance survival and productivity. Laboratory and field trial data are presented to demonstrate the efficacy of the additive under two different co-infection scenarios.

Sanacore® GM stimulates AMP production and improves shrimp survival under co-infections

A laboratory trial evaluated the effect of Sanacore® GM to boost penaidin and crustin production in shrimp and improve survival under a co-infection challenge based on WSDV and VP.

Shrimp specimens below one gram were stocked in tanks and fed with a preventive dose of the additive for 28 days. At the end of this period, shrimp hepatopancreas of nine shrimp per group was sampled for gene expression analysis of penaidins and crustins (Soonthornchai et al., 2010; Rahimnejad et al., 2018). Subsequently, a total of 21 shrimp per treatment were selected in triplicate groups and subjected to WSDV infection, followed by VP infection two days after (Han et al., 2019). Infection by WSDV aimed to weaken shrimp, while the subsequent infection by VP triggered mortality. Mortality was monitored for 24h after infection.

Figure 1. Penaidin and crustin mRNA relative gene expression at day 28 pre-challenge. Data were analyzed by Independent t-test (significance at p<0.05, n = 9)

Results showed a statistically significant increase of three times and seven times in the expression of penaidin and crustin (Fig. 1), respectively, in shrimp supplemented with Sanacore® GM. Penaidins and crustins have proven antimicrobial effects against VP (Mai et al., 2021). Interestingly, increased AMP expression was correlated with a numerically 40% improvement in the survival rate at 6-12 hours after the co-infection challenge (Fig. 2).

Figure 2. Percentage of survival in control and Sanacore® GM groups 24h post-challenge. Each time point was statistically evaluated by independent t-test (significance at p<0.05; n = 3)

Table 1. Comparison between supplementation strategies of Sanacore® GM on survival, feed conversion ratio (FCR) and pond production.

Different letters within the same row represent statistically significant differences (significance at p<0.05, n = 10). Historical data represented by the average of four crops were not included in the statistical analysis.

These results confirm that additive supplementation supports immunocompetence and enhances protection against co-infections.

Sanacore® GM improves shrimp survival and productivity in ponds

A field test evaluated the efficacy of Sanacore® GM to reduce the impact of co-infections in a shrimp farm located in Bratasena Lampung (Indonesia). During the previous four crops, ponds tested positive for EHP and showed water Vibrio load over 10² CFU/ mL along with typical signs of WFS. Historical data of the previous four crops was compared with the current crop under two application strategies of the additive in feed:
1) a strategy based on preventive supplementation at a low dosage from the start of the cycle combined with corrective supplementation at a high dose during the high-risk period (around DOC 25), and 2) only corrective at high dose during the high-risk period. A total of 10 ponds were selected for each application strategy. Historical farm data were used only for comparison purposes and were not included in the statistical analysis. Independent t-test was used to assess the difference in efficacy between the two application strategies.

Survival was improved by 58% and 169% by the corrective and the combined preventive and corrective strategies, respectively, as compared with historical data (Table 1). The preventive use of Sanacore® GM resulted in an additional and numerical improvement of 48% in relation to only the corrective application. Feed conversion ratio (FCR) was also improved by supplementation; corrective application showed an improvement of 11% in relation to previous crops, while the combined preventive and corrective strategy resulted in an additional and significant 21% improvement (Table 1).

Overall, the corrective strategy improved the pond biomass by 10% when compared to previous crops, while the combined preventive and corrective strategy achieved a 64% improvement. Remarkably, the use of a preventive dosage resulted in an additional and significant improvement of biomass by 50% as compared with just corrective supplementation (Table 1). These results are in line with previous field evaluations showing that corrective application of the additive reduces the signs of infection, but only preventive supplementation has proved to maximize post-outbreak growth rates. Sanacore® GM increased feed cost per ha when compared to the farm’s historical data, but this investment was well paid off by the biomass gain and economic returns. The return on investment (ROI) calculation indicated that for each US dollar invested in the additive application, USD18 and USD40 were additionally gained for the corrective and combined preventive and corrective strategies, respectively.

The present article demonstrates the efficacy of the phytobiotic-based additive Sanacore® GM to support shrimp survival and productivity under co-infection challenges. An important mechanism of the additive to promote immunocompetence and reduce the impact of infections is via increased production of AMPs. Preventive application of the additive seems to be a more successful strategy to modify the course of the infection and maximize post-outbreak growth rates.

References available on request

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