by Maxime Hugonin and Stéphane Frouel, MiXscience, France

 

Ubiquitous antimicrobial activity of a new feed additive against several pathogens in aquaculture farming systems

 

As a potential protein source of tomorrow, for a population always growing, the aquaculture industry is facing several challenges. To reach the demand, the yield production must be maximised. In this way, farmers always increase their stocking densities, going from intensive culture to super-intensive ones, leading to new pathogens appearances and propagations with a multiplication of disease outbreaks.

The first people impacted by these issues are the farmers. This pathogenic pressure significantly impacts the economics of farming. The main solution to this issue remains the use of antibiotics, thanks to their easy use in curative treatment and their visible and rapid effects. Unfortunately, the use and abuse of chemicals raises public health concerns, because of antibiotic resistance, and adverse effects on the environment. Then, this remedy participates to the bad image associated to aquaculture production and produces shifts in the public opinion.

Active researches are ongoing in their hard work to explore alternatives. This article reports on the use of a natural phytogenic, based on specifically selected plant extracts, to control a broad spectrum of pathogens in aquaculture systems. The story of the product started from a laboratory, associated with RID trials, and ended in-field at larger and commercial scale. Thus, the antimicrobial effects of this phytogenic have been investigated both in vitroand in vivo, which provides a robust and pragmatic feedback on its benefits.

 

Mechanisms of action

The large spectrum of the antimicrobial activity of this feed additive is based on particular mechanisms of action with common targets among pathogens: the proteins. The anti-microbial properties of this phytogenic are provided by Sulfur Organic Compounds (SOCs) from Alliaceae extracts. The Alliaceae family includes 13 genera and 600 species. Main representatives are onion, garlic, leek, shallots and chives.

Some research studies raise the possibility that, in biological systems, SOCs can penetrate very rapidly into different compartments of the cells where they exert their biological effects. Depending on the pathogen, there are several ways for SOCs to penetrate cells (See figure 1)

Due to their low molecular weight, SOCs can easily diffuse by different processes into the internal volume of vesicles, in the cytoplasm of bacteria, (Gram – or Gram +), or into viruses. That is the case in Gram – where the peptidoglycan layer is small.

SOCs give the phytogenic antibacterial properties, due to different interactions with cell compounds. Once in the cell, SOCs combine with certain proteins to alter fixation and to dislocate thiol functions, contained in disulfide bridges involved in the structure of proteins and enzymes. Without their 3D conformation, the denatured proteins are not functionable anymore, (see figure 2).

Among the altered functions, gene expression, energetic metabolism and protein synthesis are some associated functions, whose alteration leads to a global malfunctioning of the cell, to its final apoptosis and then the death of the pathogen, (see figure 3).

For bacteria, SOCs appear to target multiple pathways including the modulation of enzyme activities (e.g. glutathione S-transferase, involved in several vital pathways), the inhibition of DNA enzymes (gyrase, polymerase), the affection of the intrinsic pathway for apoptotic cell death and cell cycle machinery. SOCs can also block the synthesis of polyamines, as well as disrupt cellular microtubules (that form the cytoskeleton and the mitotic spindle in cells), requested for cell division.

The antiproliferative and antimicrobial effects of SOC compounds appears to be related to the induction of cell apoptosis, resulting from the alteration of pathogenic cells.

For viruses, SOC"s will alter the protein of their capsid. Without the genome protection provided by this protein envelop, the viruses will die on the same model as microbial cell apoptosis.

 

Product potential: A three scales" evaluation

In vitro, the product"s efficacy has been evaluated through minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) tests.

Using microdilution methods, the growth inhibition effect of the phytogenic against a wide range of pathogens, from seawater and freshwater aquaculture systems, has been compared to MIC of natural extracts known to have high antimicrobial potential such as carvacrol, from oregano and thyme oils, citral, isolated from citrus oil and eugenol, from clove oil.

Moreover, in order to evaluate the real potential of this product as an antibiotic alternative, MIC and MBC were compared to common antibiotics used in aquaculture (oxytetracycline, erythromycin and enrofloxacin).

In vitro results indicated that this feed additive showed a wide bactericidal action, since it exhibited high efficiency against both gram-positive and gram-negative bacteria. In addition, it showed the strongest antimicrobial activity, compared to in-kind products. The experimental phytogenic presented the lowest MIC from 16 to 125 ppm versus 32 to 250 ppm for carvacrol, 64 to 1,000 ppm for citral and 64 to 2000 ppm for eugenol (see Table 1).

It also demonstrated a minimal inhibitory and bactericidal concentration in the same order of magnitude (less than one log unit of difference) than tested antibiotics (Table 2).

Based on these promising studies, the product was then applied in challenge trials.

The in vivo laboratory trials have been conducted on different species: fresh water fish (Seabass), warm water fish (Tilapia) and marine shrimp (White Leg Shrimp) which have been challenged for different pathogens in a preventive use protocol of the product.

To start with, animals have been acclimated to experimental condition (from between one to four weeks), before being fed continuously with the experimental feed, containing the phytogenic, at a concentration of 1-2Kg/ton of feed (See figure 4).

After a period of three or four weeks, according to the species, the fish and the shrimps have been challenged with the selected pathogen and fed with the product for at least two more weeks post-challenge. Survival was then observed (see figure 5).

The results presented in figure 5 clearly show a significative survival improvement (ANOVA p<0.05), regardless of farmed species and associated pathogens (bacteria or virus). The use of the phytogenic increased the shrimp survival rate of 54 percent against Vibrio Parahaemolyticus, and 52 percent against the white spot syndrome, the two main diseases encountered by the industry.

In fish, even if the results are a little bit less impressive (due to existing Immune System for fish then lower mortality rate for control), they are still significative, and the mortality reduction can also represent a reliable economic gain with a survival increase of 18 percent for seabass against Pasteurella and 19 to 12 percent for tilapia against Streptococcus and Francissella, respectively.

During research trials, the significant results confirmed the antimicrobial effect of the phytogenic observed at laboratory scale. To definitely validate these benefits, a last but no least step has been undertaken: under farming real conditions trials.

 

Commercial field scale

The effect of the phytogenic was tested under commercial farming conditions for two species in Vietnam, (shrimp and tilapia), and in Turkey for seabream and seabass. For these last fishes, five trials have been carried out to evaluate the effects of the phytogenic on disease control, randomly met under natural conditions, and compare them to antibiotics" ones. Fish have faced either Vibriosis, Flexibacteriosis or parasitic infections (see table 3). Interestingly, the use of the product at 5Kg/tons of feed, during 20 days after the first symptoms appearance, lead to a complete control of the disease (at least as efficient as antibiotics) and a total recovery with a comeback to initial metabolic state of the animals.

For tilapia in cages, the phytogenic was applied temporarily at a disease control dosage of 4kg/ton of feed during 14 days after the emergence of a streptococcal infection. It was applied at the same amount, for a duration of 35 days, in shrimp farmed in outdoor ponds after the appearance of Vibriosis. Antimicrobial effect of the phytogenic was confirmed, under farming conditions, where it significantly supported resistance of tilapia and shrimp (ANOVA p <0.05) when challenged with Streptococcus spp. and Vibrio spp. respectively (see figures 6 and 7).

We concluded that this new feed additive provides efficient control against a variety of pathogens and could be considered as a holistic and natural approach of reducing the use of antibiotics in aquaculture systems. Trial data also showed the efficacy of the functional additive to counterbalance disease outbreaks and to maintain a reliable growth performance and farm profit.

Moreover, this new phytogenic can be applied in a wide range of conditions, either continuously as a prophylactic agent, or during certain critical periods as a curative agent. The optimal duration of application is at least 14 days before any known critical period, or after the first appearance of disease symptoms.

Recently, the product efficiency was extended to new species against new pathogen: Rickettsia (Salmonid RickettsialSepticaemia) in Chile. Great scale use of the product has shown benefits, in terms of survival rate, and then economics return of investment.

New trials have also been conducted against the devastating and emergent Tilapia lake Virus (TiLV) and have shown positive results to be confirmed under field conditions. In Vietnam, out of 219 shrimp ponds of 219 ponds using the phytogenic, 75 percent didn"t show any mortality and only 15 percent have shown mortality due to EMS, 4 percent due to WSSD and only 2 percent white feces syndrome.

 

The success story is ongoing, we need to keep the momentum going!

Figure 1: (Below) Nature of potential ways of penetration of SOCs into cell

Figure 2: (Top right) Denaturation of microbial proteins by fixation of SOCs on disulfide bridges

Figure 3: (Bottom right) Functional metabolic alterations by SOCs contained in the phytogenic

Figure 4: Preventive product application protocol for In vivo laboratory trials for three tested aquaculture species

Figure 5: Overall effect of the phytogenic on the final survival (control vs preventive dosage application)

Figure 6: Curative effect of phytogenic on tilapia after streptococcal challenge

Figure 7: Curative effect of phytogenic on shrimp after Vibriosis challenge

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