Strategies to Control Acute Hepatopancreatic Necrosis Syndrome (AHPNS/EMS) and the Highly Lethal Vibrio Disease (HLVD/GPD/TPD) (Part 1)

By Ph.D. Stephen Newman

Some countries deny that the strain of Vibrio parahaemolyticus (VP) that causes acute hepatopancreatic necrosis syndrome or disease (AHPNS or AHPND) is present in their stocks, further increasing the chances of spreading it. More than likely this perspective will persist with highly lethal vibrio disease (HLVD/GPD/TPD).

Much as with EHP and WSSV and other obligate pathogens that impact farmed shrimp, early mortality syndrome (EMS), acute hepatopancreatic necrosis syndrome or disease (AHPNS or AHPND) continues to harm shrimp farmers in almost every country that farms shrimp. Recently, novel strains of Vibrio parahaemolyticus (VP) have been associated with another serious disease.

The strains that cause HLVD (highly lethal vibrio disease) also known as translucent post-larvae disease (TPD) or glass post-larval disease (GPD) do not contain the toxins responsible for AHPNS. Some estimates are that AHPNS alone has caused over USD 40 billion in losses to date. It has shaped the global industry into what it is today and is impacting how it will evolve in years to come.

This new VP strain is wreaking havoc and is causing significant losses in many countries. Recent reports suggest that a newly discovered virus may also be associated with some cases of GPD. These issues reiterate the need to keep pathogens out of production systems whenever possible.

Some countries deny that the strain of VP that causes AHPNS is present in their stocks, further increasing the chances of spreading it. More than likely this perspective will persist with GPD. For AHPNS, the conditions under which the toxins are produced, the relative roles of the two toxins, PIRa and PIRb, and what the options are in terms of eliminating the impact have been the subject of intensive study.

For GPD, studies are also being conducted to better understand how the cluster of toxins causes the disease. These vibrios should be considered to be super-pathogens. While eradication starts with the brood stock, this by itself is not usually sufficient to keep them entirely out of one’s production system. The data suggests that AHPNS can be considered an environmental disease.

Cultural practices damage the microbiome allowing this unusual bacterial strain to proliferate. More than likely, this will be similar with GPD.

High levels of mortality have been reported due to GPD. These strains of VP carry Tc toxins. These are well characterized and much as with PIRa and PIRb toxins appear to have originated in non-vibrios. These strains are lethal and can rapidly disseminate through out aquatic ecosystems and dominate.

It is probable that GPD will become firmly entrenched along with AHPNS intensifying the negative impact of these vibrios. This makes it imperative to control the presence of these vibrios to the greatest extent possible.

Background

Vibrio species are critical for the normal functioning of marine ecosystems. They play an important role in the recycling of chitin, a component of arthropod exoskeletons. More than 140 species of vibrios have been identified to date with many more likely to be characterized. As is typical of all bacteria, each species has many different and distinctive strains within it.

These strains are often characterized by the production of compounds that are unique to the specific strain. These may be toxins or the ability to grow in the presence of what would be growth limiting conditions for other strains.

The species Vibrio parahaemolyticus is widespread throughout all aquatic ecosystems. Some strains are human pathogens. These can cause acute food poisoning (usually from eating contaminated shellfish) while others are responsible for rapid fulminating septicemias that can result in death in less than 24 hours after an initial infection, often from a minor puncture wound. Some are unique fish and shrimp pathogens.

The presence of VP, in of itself, in production systems is not necessarily a problem as most strains are opportunistic and not obligate pathogens. Obligate pathogens produce disease in healthy animals while opportunistic pathogens produce disease in weakened animals.

Published observations state that EMS/AHPNS first appeared in China in 2009. This strain spread and was subsequently reported in Vietnam in 2010, Malaysia in 2011, Thailand in 2012, Mexico in 2013, the Philippines in 2015, and South America in 2016. It is likely that almost every country that farms shrimp has this obligate pathogen present. Geographical isolation may help keep it out.

When the disease first appeared, it killed primarily post larval shrimp (PLs) in the hatchery, and eventually shrimp post within the first three to four weeks post stocking. Thus, the name “early mortality syndrome” (EMS). As the pathogen spread mortality was not confined solely to this early stage of production.

The pathology seen is consistent between geographic areas and likely contributes to secondary infections due to opportunistic pathogens. Affected animals do not eat and the hepatopancreas shows extensive damage to the tubules.

The hepatopancreas is pale because of this. While this damage is indicative of the disease process (the technical term is pathognomonic), there are other things that can damage the tubules. Accurate diagnosis requires isolation of the specific pathogen or the use of real time PCR.

Vibrios, much as with most bacteria, readily exchange genetic material within their own genus as well as with other genera. Dogmatic approaches towards marine microbiology state that TCBS green colonies (of which most strains of VP are an example) should be kept out of hatcheries or farms.

The reality is somewhat different. Focusing solely on TCBS green colonies is at best naive and at worst a formula for failure. Many TCBS yellow vibrios are obligate pathogens including highly virulent strains of Vibrio alginolyticus. The strains of VP responsible for GPD have been reported as being weakly yellow unlike the strains that cause AHPNS.

The goal should be to focus on producing clean, healthy, stress free, genetically improved animals which have been shown to be refractory to infection. Minimizing the presence of vibrios overall regardless of whether they are yellow or green on TCBS is not always practical and it can result in the presence of other opportunistic pathogens.

Bacteria produce disease in many ways. The strain of VP that causes AHPNS produces two pore forming toxin proteins, PIRa and PIRb, that are very similar amino acid wise to a toxin that kills insects produced by Photorhabdus luminescens strains. They are also very similar in terms of structure and activity to the Bacillus thuringiensis crystal insecticidal (Cry) toxin.

These genes are present on a conjugative plasmid, a small circular piece of DNA that is easily transferred between other vibrios and potentially other genera. This plasmid has been found not just in this specific strain of V. parahaemolyticus but also in strains of V. owensii, V. harveyi, V. campbellii, V. punensis and Micrococcus and Shewenella. It has also been introduced into Escherichia coli in the lab.

Since bacteria exchange genetic material very freely with other bacteria, there are many different strains of VP that carry this plasmid. Some produce only one of the toxins. Others produce very small amounts. Some produce large amounts. Some produce a lot of one and a little of the other. Some have one copy of the plasmid and others have many.

These toxins are excreted by the bacteria and damage the shrimp eventually to the point where the shrimp are unable to gain adequate nutrition and in this weakened state, they are easily invaded by the strain producing the toxin and other opportunistic pathogens.

The toxins are water borne. Although the VP strains associated with AHPNS form biofilms and there are reports of biofilm formation in the immediate production environment, this does not appear to be an absolute requirement for the shrimp to be affected as lab studies have shown that the formation of biofilms may not always be a critical element for toxin production.

There is substantial evidence that another trait of this strain, an antibacterial type VI secretion system (T6SS) allows it to dominate in environments over other vibrios and other bacteria. This is a potent tool by which the vibrio wreaks havoc on the microbiome allowing it to dominate quickly.

This trait is critical for the ability of these bacterial strains to spread, and it appears necessary for the ability to produce disease. It also appears to be present only in the plasmid carrying strains and not in other wild type VP strains. The VP strains that cause GPD also have this trait. These properties make them super-pathogens.

GPD was first reported in China in 2019. It has spread to many shrimp farming countries although the extent to which it is impacting these is not well understood.

GPD is much more lethal than AHPNS. When PLs are infected in the hatchery more than 90% die within 24 to 48 hours after the pathogen is observed to be impacting animals. Infected animals have a pale to colorless hepatopancreas and digestive tracts that appear very similar to shrimp with AHPNS.

Unlike the PIRa and PIRb toxins the Tc toxin complex is not excreted and requires the bacteria to die for the toxin to be present in the environment. Originally characterized in Photorhabdus luminescens, Tc toxins have an important role in the control of certain plant pathogens.

More than likely, they made their way into VP via horizontal gene transfer much as the PIRa and PIRb toxins are thought to have found their way into VP strains as well. As mentioned, these strains also express T6SS.

The best way to control pathogens is to exclude them. This is the basis of proactive animal health manage ment strategies. The concept is to reduce the environmental load by reducing the numbers of potential carriers and thus spreaders. This would seem to be simple but with the specific strain of VP that causes AHPNS, it is only part of the solution.

By keeping it out of broodstock and thus PLs the occurrence of an early acute phase of the disease is less likely. Since GPD can cause also cause acute mortality it is also imperative to keep it out of the brood stock as well. Chronic mortality can occur from exposure to low levels of PIRa and PIRb toxins. This is probably similar in GPD.

In theory one could stimulate an immune response against these strains that might be useful, although the nature of the toxins (at least the PIRa and PIRb toxins) are such that they can produce pathology even when the pathogen is not present. Shrimp cannot be immunized in the classic sense of the word. There is no specificity to their immune response.

They can be stimulated in a largely non-specific manner which will increase their ability to tolerate higher pathogen loads and there is evidence that this might be the case with many of the vibrios. There is data that supports that this could afford some protection, enough to require higher levels of exposure before shrimp are impacted.

Overall animal fitness is critical for ensuring that shrimp will grow as fast as they are genetically capable of doing so and that they can resist infection. The best production environments ensure that the animal has all that it needs to protect itself from disease. Eradication of pathogens is rarely easily accomplished (when it is even possible) so a logical place to start is to gradually reduce the environmental load.

Cultural practices need to ensure that every reasonable step is being taken to lower the levels of obligate pathogens. In the case of water borne toxins this is challenging. Since one can have strains that produce very high levels of the toxin and there are all too often design flaws in production facilities that preclude readily being able to eliminate all biofilms,
there are going to be instances where there is evidence of damage from the toxins, but the VP strain responsible cannot be found using PCR or culture.

Although the Tc toxins produced by VP strains that cause GPD are not excreted like the PIRa and PIRb toxins are it is likely that they could also cause pathology in the absence of the bacteria being present.

These vibrios must be eliminated from the broodstock. If broodstock are held in non-biosecure environments in an area where the disease is endemic, there is a good probability that they will be carrying the bacteria. Elimination can only be accomplished via individual testing of animals to ensure that broodstock are free of pathogens and held in properly constructed maturation facilities with no movement of animals from outside and the high levels of biosecurity required to maintain the pathogen free status.

Historically this is not the norm and if this is ignored the diseases will not be eradicated. Hatcheries often have several possible entry points. Even if the broodstock are free of the pathogen, there are other ways for vibrios to enter the hatchery. Vibrios can be windborne. They are in the air in environments near the ocean or other bodies of water that might be carrying them, such as discharge canals or ponds.

Hatcheries that do not have positive air flow systems, i.e., air pressure in the hatchery that ensures that airborne bacteria cannot enter, are at risk. Careless biosecurity measures can move it with employees or equipment that is not properly disinfected.

The toxins can be present even when standard techniques such as PCR, RT PCR and culture fail to find the bacteria. Low level exposure to PIRa and PIRb toxins is not invariably fatal although it will damage the HP and increase the likelihood of problems with secondary pathogens.

Animals will grow poorly and succumb to infections that they would not other wise. With GPD although it stands to reason that it could be similar.

Enrichment is a standard technique employed in microbiology. When one is looking for certain types of bacteria that might be present at very low levels, samples are added to a generic broth and allowed to grow up. These are then tested for the organism of interest via standard techniques.

Since the plasmid that encodes for the toxin genes in AHPNS can be present in many copies, a relatively small number of cells could be responsible for pathology.

If routine animal health screening shows that there is damage to the HPs that is characteristic of the presence of these toxins and testing fails to reveal the presence of VP, enrich ment may be needed to confirm its presence. Of course, it is also possible that another species of bacteria is producing the toxin.

It is not possible to consistently eliminate all vibrios or to selectively eliminate some and not others although there are some who will try and convince you otherwise. They are ubiquitous and while there have been no observations yet of these Tc toxins being transferred to non-vibrios in the wild, we know that this is possible.

The goal should be to keep the loads as low as is practical and avoid those activities that might allow the strains to proliferate. The following edition will cover the environmental component related to AHPND and GDP, along with recommendations for controlling these diseases.

*Stephen G. Newman has a bachelor’s degree from the University of Maryland in Conservation and Resource Management (ecology) and a Ph.D. from the University of Miami, in Marine Microbiology. He has over 40 years of experience working within a range of topics and approaches on aquaculture such as water quality, animal health, biosecurity with special focus on shrimp and salmonids.
He founded Aquaintech in 1996 and continues to be CEO of this company to the present day.
It is heavily focused on providing consulting services around the world on microbial technologies and biosecurity issues.
sgnewm@aqua-in-tech.com
www.aqua-in-tech.com
www.bioremediationaquaculture.com
www.sustainablegreenaquaculture.com