Typical pond side culture
Perhaps the single largest biosecurity gap, right after not ensuring that broodstock are clean, in dealing with AHPNS is the continued pond side growout of bacteria without appropriate quality control and oversight This poses a serious biosecurity risk: AHPNS causing bacteria thrive under these conditions. Please note that I am not saying that the products being activated are problematic; this I have no way of knowing one way or another. We sell products that are used this way as well. What I am saying is that without appropriate oversight there is a strong possibility of contamination of these tanks with vibrios and quite possibly the vibrios that cause AHPNS. This makes this practice a serious biosecurity risk. There are products that avoid this, such as the tableted blend of Bacillus species, PRO4000X which is in wide use in Ecuador and other countries.
Vibrios are ubiquitous in aquatic environments. They serve an important role in degrading chitin. Most are benign, although they can infect weakened animals. Only a few are associated with disease as obligate pathogens, i.e., they infect and affect healthy animals. The vibrios that is of greatest concern globally are those that cause AHPNS. This is a toxicosis in that the presence of the PIRa and PIRb toxins are responsible for this syndrome. This has stimulated a great deal of interest among a wide range of researchers. The genes that encode for the production of these toxins are located on a plasmid (pVA1), a small circular piece of DNA that is extrachromosomal. Plasmids can carry many different genes including, among many others, those for antibiotic resistance, outer membrane proteins and a variety of the toxins. These particular plasmids and parts of them are readily transferred between different strains and species. We are only just beginning to learn how widespread it is. Originally found in a strain of Vibrio parahaemolyticus (VP) in China, since then it has been found in various strains in culture collections that predate the Chinese reports. It has also been found in a number of other vibrios and other species of bacteria. It is not unreasonable to expect that this trend will continue as long as cultural practices encourage this.
Among the many findings are that there are variable quantities of plasmids within different strains. It is important to understand that the process by which these genes move between bacteria is not highly efficient and pieces of the genes, genes that encode for only one of the toxins and many variations thereof will occur. Some strains will have many copies of the plasmid and not produce much toxin while others may produce high levels of toxins. Some may produce both toxins while others may only produce one or the other. These toxins are responsible for the pathology although recent data suggests that some of the other properties of the bacteria are likely contributing as well. Studies with cell free purified toxins have shown that by themselves, i.e., in the absence of the bacteria, these toxins can cause the damage observed in the target organ.
Bacteria are ubiquitous in and on shrimp and the environment that they live in. For many, vibrios are thought of as being undesirable bacteria. This has typically focused on those that cannot degrade the sugar sucrose. On the selective media TCBS this property is responsible for the colonies appearing green when they cannot utilize it and yellow when they can. This is factually incorrect in that there is no correlation between the ability to produce disease and the ability to degrade sucrose. Selective elimination of any given bacterial species in the absence of failing to address what are usually the true reasons for many problems in the shrimp, including but not limited to the presence of stressors and the carry-over of pathogens from broodstock into PLs is more than likely not wise. Microbiomes are complex and selectively eliminating elements of them may lead to other problems. The widespread use of disinfectants appears to enable the strains that cause AHPNS by eliminating competitive bacteria.
Preparing pond side culture
Most vibrios grow very quickly. Their generation times (time to doubling) under the right conditions can be 12 minutes or less. When bacillus spore products are cultured on pond side it can take hours for the spores to germinate with some species and strains being quicker than others although many take 12 or more hours. This is temperature and salinity dependent. The strains of VP responsible for AHPNS have some properties that allow them to readily outgrow and compete against many other bacteria.
The PIRa and PIRb toxins can be present in the absence of the bacteria. Bacteria can be protected by biofilms and sequestered in areas that further protect them. The use of disinfectants disrupts the microbiome and may actually create a selective advantage for the faster growing bacteria to dominate. There is increasing evidence that even low levels of the toxins may damage the HPs. This in turn increases the chances of animal health challenges affecting growth rates, feed conversions and of course profitability.
Screening for the presence of the specific bacteria that is producing the toxins is problematic. One cannot be certain what species are producing the toxins. There are practical limits to how much material can be sampled cost effectively. Using the most sensitive techniques available one can still have false negatives. For various reasons the limitations that exist in sampling complex aquatic ecosystems ensure that there will always be some false negatives. It is not accurate to state that when samples are negative that this always means that the environment being tested is free of the toxin.
Pond side grow out systems allow rapidly growing strains/species such as the vibrios to proliferate at the expense of all others. This is why quality control of any activation or grow out scheme that entails growing bacteria up on pond side is essential. For many years farmers have decided that the best way to add beneficial bacteria to their ponds is to grow the bacteria up on pond side. They have been persuaded that adding high levels of the bacteria that they think they are culturing is how to gain the greatest benefit from their usage. In typical production systems, which are open, many factors impact what is going on. Adding large numbers of bacterial species to ponds is not as straightforward as it seems. Moreover, it is not necessarily better. These are complex ecosystems that one is adding the bacteria to. The added bacteria are limited by the availability of nutrients and competition for them by the existing microbiome. There are bacterial viruses and wide variety of antimicrobial substances which in concert all act to limit the ability of these exogenous bacteria to grow. Thus, the need to add them repeatedly. There is no data that supports the idea that more is necessarily better in your average pond.
Pond side culture; without professional oversight and proper SOPs to ensure the culture is free from contaminates there is no way to truly know what you are adding to the pond.
This however is but one aspect of the process. The vibrios that contain the specific plasmid and genes for the production of PirA and PirB thrive when there is little competition. Blending molasses with seawater and culturing products that contain spores and many other non-spore forming bacteria (currently being offered to farmers as beneficial bacteria) without making sure that the Pir toxins are not present when this ferment is added to ponds is the norm. Many farmers are unwittingly adding AHPNS causing bacteria to their ponds via this route. While data showing this is not readily available, my personal experience with growing bacteria up outdoors suggests that vibrios are common contaminants as well as the experience of others who have bothered to look. While there may be some benefit from the other bacteria present, it is not worth the risk to the farmer, his family and to the industry as a whole.
This lack of control is a guarantee that AHPNS will continue to cause serious challenges to shrimp farmers everywhere. While it is possible that some tolerance and perhaps eventually resistance may develop to these toxins, the damage that is occurring today and certain to continue into the near future is huge and being made worse. This approach to the addition of bacteria to the ponds belies the overall lack of understanding on the part of most farmers and elements of the supply chain about the risks. Furthermore, strains of VP are also pathogens of human beings. It is grossly irresponsible to be taking an approach that as a result of its massive scale, ensures that there are selective pressures in place that can produce even more virulent strains, endangering the health of the farmers and those around them. Growing bacteria is best left to professionals with the tools to ensure that cultures are not being routinely contaminated with bacteria that could harm the crop and potentially the farmers as well.
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