What is the Safe Level of Total Vibrio Count?

There is no validated information on safe level of vibrio count. Different studies have shown varying counts of vibrio in various habitats. Bacterial population dynamics, including those of vibrio are complex. Ten different samples from the same pond will give different counts. One paper suggests that vibrio count of 1.0±0.1×10⁵ CFU/mL exceeds the safe limit so anything below this should be safe. This is not a peer reviewed paper and I have not been able to find a validated source so far. 

Supono et al (2019) AACL Bioflux, 2019, Volume 12, Issue 2. http://www.bioflux.com.ro/docs/2019.417-425.pdf

Yellow vs. Green Colonies

Vibrio harveyi and V. parahemolyticus produce green colonies on TCBS agar, whereas V. alginolyticus produces yellow colonies. All three are opportunistic pathogens of shrimp. The concept of yellow and green colony proportion as an indicator of pathogen load is a fallacy for many reasons. 

1. If the shrimp is stressed (environmental, physical, or viral diseases) any marine bacterium can proliferate to become an opportunistic pathogen. Vibrios are endemic to marine waters and are predominant in comparison with other bacterial communities, so the presence of vibrio (both yellow and green) is an obvious fact. 

2. Studies have shown some vibrios can shift between yellow and green, due to genetic or various biophysiochemical factors. 

3. Studies have shown that only 2% of bacteria from natural habitats are cultivable in laboratory agar plates i.e. 98% of bacteria cannot be cultured with the media we use. When we isolate bacteria for total heterotrophic count or vibrio count, yes we will get vibrios and other bacteria which grow on TCBS and general purpose media, but can we really tell whether the disease was caused by these 2% cultivable bacteria or 98% unculturable bacteria? 

In short, whichever vibrio is predominant when the shrimp is stressed or affected by WSV, will dominate to form the secondary and opportunistic pathogen. The vibrio can be either yellow or green, it doesn’t really matter.

Some Additional Notes on Vibrio 

There are totally 147 vibrio species and 4 sub species. Out of the 147 species, around 15 of them have been associated as opportunistic pathogen of shrimps and other marine crustaceans. The major pathogens frequently isolated from diseased shrimp are, Vibrio harveyi, Vibrio parahemolyticus and Vibrio alginolyticus. Other vibrio species have also been isolated to a lesser extent from diseased shrimp.

Pathogenesis of Vibrios 

As in the current scenario let us accept the fact that vibrio is the main pathogen, then how does it cause disease? Vibrios proliferate well under high salinity (~ 30ppt) and high temperature (30-37 ⁰C). Under these conditions vibrios proliferate to a certain population level and through quorum sensing collectively produce several virulence factors, such as toxins, enzymes and the most important; triggering of a bacterial pathogenic mechanism specific to only Gram negative bacteria. This mechanism is operated through the Type III Secretion System (Henke & Bassler, 2004). V. harveyi, V. parahemolyticus and V. alginolyticus and other pathogenic vibrios have this specific virulence mechanism. V. alginolyticus in addition has a Type VI secretion system through which they share and exchange toxins with other marine bacteria (Salomon et al 2015). High temperature is the main trigger for the virulence of vibrios associated with already stressed shrimp. 

References

Henke, J. M., & Bassler, B. L. (2004). Quorum sensing regulates type III secretion in Vibrio harveyi and Vibrio parahaemolyticus. Journal of bacteriology, 186(12), 3794–3805. doi:10.1128/JB.186.12.3794-3805.2004 

Salomon, D., Klimko, J. A., Trudgian, D. C., Kinch, L. N., Grishin, N. V., Mirzaei, H., & Orth, K. (2015). Type VI Secretion System Toxins Horizontally Shared between Marine Bacteria. PLoS pathogens, 11(8), e1005128. doi:10.1371/journal.ppat.1005128

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Dr. Sathish Prasad

Senior Scientist – Aquatic Animal Health

Growel Innovation Center,

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Email: sathish.prasad@growelfeeds.com