In response farmers have dedicated more attention to pond management techniques to reduce the Vibrio presence in their ponds. Introduction of nursery systems, disinfection, use of probiotics, polyculture with tilapia and, more alarmingly, the return to wide use of antibiotics have presented no relief to the EMS problem. This could be related to the fact that these efforts are, in many cases, generic solutions to eliminate all bacteria present in the pond and not ones designed to specifically target the precise Vibrio strain and its ability to survive or become virulent. In this article we focus on three main strategies for combating Vibrio: probiotics for pond, in-feed tools such as phytogenics and acids, and quorum quenching compounds.
The bacterial culprit
Vibrio spp. are Gram-negative bacteria indigenous to water. They are difficult to eradicate because they adapt well to different environmental conditions and can adopt a dormant state when facing adverse conditions. The pathogenicity of the EMS/AHPND agent varies greatly. There are many strains of V. parahaemolyticus, some virulent for EMS/AHPND, others not. Even among those that can cause EMS/AHPND there is a wide range of virulence levels, with some mild strains that can cause mortalities when they reach concentration levels of 106 to 107 colony-forming units per milliliter (CFU/ml), while other more virulent strains can cause mortalities at lower levels 104 to 105 CFU/ml.
A bacteria’s capacity to cause disease, or virulence, is a complex process affected by many variables, including host, Vibrio strain, developmental stages, physiological conditions, environmental stress, and infection method.
Probiotics for pond
The use of probiotic bacteria to improve pond environment and control Vibrio populations has been one of the most common strategies used by farmers to fight EMS outbreaks. Yet, not all probiotic bacteria are effective.
According to recent analysis from BIOMIN Research Center, certain probiotic species seems to be better than others in inhibiting the growth of the pathogenic V. parahaemolyticus. As Figure 1 shows, probiotic strains such as Lactobacillus reuteri, Pediococcus acidilactici, Enterococcus faecium and B. subtilis (proprietary BIOMIN probiotics) were shown to inhibit V. parahaemolyticus. This shows that not every menace can be targeted with Bacillus bacteria. In fact, results demonstrate that pathogen inhibition is most effective with the three lactic acid bacteria, L. reuteri, P. acidilactici, and E. faecium. Within the Bacillus family, pathogen inhibition is both species-specific as well as strain-specific. Among different strains of the same species, B. subtilis, one out of six strains was able to inhibit the growth of virulent V. parahaemolyticus.
Figure 1. Varying effectiveness of probiotic bacteria against pathogenic Vibrio parahaemolyticus after 8 hours.
This variation highlights the importance of selecting effective probiotics in combating pathogens or pathogenic bacteria.
Animal gut health is crucial to animal performance. While pond contamination by pathogenic Vibrio is cause for alarm, ultimately its effects are going to be exerted in the animal’s digestive system, for shrimp in the hepatopancreas. As such, strategies to reduce the effects of the V. parahaemolyticus in the shrimp digestive system can help protect the animal.
Certain essential oil mixtures and organic acid mixtures have been shown effective for their inhibitory potential towards V. parahaemolyticus. These compounds can be added to the feed to have an effect in the digestive system of the animal. The acid mixture in Figure 2 inhibited V. parahaemolyticus growth by 80% to 95% at a concentration of 5000 ppm. The minimal effective dose is between 1000 and 5000 ppm. Essential oils mixtures have also been demonstrated to possess an inhibitory potential, such as the one in Figure 3 which inhibits V. parahaemolyticus growth by 80% to 85%. The minimal effective dose is between 100 and 500 ppm.
Figure 2. In vitro inhibition potential of an organic acid mixture against virulent V. parahaemolyticus.
Figure 3: Growth inhibition of virulent V. paraemolyticus after exposure to an essential oil mixture.
Vibrio bacteria possess the ability to communicate with each other by excreting small chemical molecules that allow them to sense the density of surrounding Vibrios—bacterial chatter known as quorum sensing. Once the bacteria reach a critical mass they then switch on their virulence factors, allowing them to cause disease. Preventing Vibrios from reaching critical mass can therefore be a useful way to prevent EMS.
Quorum quenching compounds
Whereas high dosages of phytogenic compounds restrain bacteria growth, shown in Figures 2 and 3, lower dosages can restrain their virulence. A wide range of phytogenic substances have been shown to inhibit quorum sensing function of bacteria—a silencing effect known as quorum quenching. Compounds found in several types of marine algae, spices, herbs and essential oils have all been found to possess quorum quenching capabilities. Figure 4 shows how one such phytogenic substance reduces bacterial chatter in vitro without inhibiting growth as measured by optical density. (Higher optical density indicates higher growth: lower optical density; lower growth). Luminescence activity, or glowing, strongly correlates with quorum sensing and virulence. Suppression of luminescence at uninhibited growth therefore indicates quorum quenching activity of the phytogenic preparation. Th is quorum quenching can be seen in the lower luminescence of the low dosage phytogenic groups versus the control group.
Figure 4. A phytogenic substance’s quorum quenching in V. parahaemolyticus means less virulence of the pathogen.
Early mortality syndrome/acute hepatopancreatic necrosis (EMS/AHPND) caused by Vibrio parahaemolyticus presents a recent and serious risk to shrimp production in many countries. Afflicting younger shrimp, it has the potential to wipe out entire pond populations. While tools to counter EMS are available, to date no single solution has proven to be 100% effective. A number of tools offer hope. Some probiotic strains significantly inhibit growth of virulent V. parahaemolyticus. Certain essential oil mixtures and organic acid mixtures have been shown effective. Cutting the bacteria’s phone lines using quorum quenching compounds can also help in restraining virulence. A more holistic approach to fight the menace both in the pond and within the animal’s digestive system is needed. Effective solutions must integrate many variables and test their effectiveness both in controlled laboratory conditions and also under the challenging field conditions.