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AHPNS

What is the biggest disease issue facing shrimp farming today? AHPNS?

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By: Ph.D. Stephen G. Newman*

The global shrimp farming industry faces many challenges. It is widely agreed that disease is the most serious of these. It is imperative that farmers learn how to do everything possible to reduce stress and ensure the highest possible levels of biosecurity.

The global shrimp farming industry faces many challenges. It is widely agreed that disease is the most serious of these. Many diseases occur with regularity and the impact on overall productivity is large. New pathogens appear with dismal regularity. Unfortunately, the very nature of shrimp farming ensures that this will continue to be the case.

There are literally hundreds if not more uncharacterized viruses, bacteria, and fungi that are present in production environments. Given the right conditions any number of these could become obligate pathogens. This is why it is imperative that farmers learn how to do everything possible to reduce stress and ensure the highest possible levels of biosecurity.

“Those farmers without the resources to do this properly pose a risk to everybody else. Vibrio parahaemolyticus (VP) is water borne pathogen that causes diarrhea in humans when it is consumed in raw seafood including shrimp.”

It can also cause a rapidly developing fulminating septicemia from minor wounds causing death in 24 hours even in healthy individuals.

Its primary virulence factors for humans are hemolysis (proteins that lyse red blood cells). It is ubiquitous in marine environments and increasing in prevalence in a warming planet. There are over 150 species of vibrios with new species being classified regularly. Of these there are many strains (different phenotypes with enough genetic overlap for them to be considered the same species).

Most are not pathogenic although a number can be found in dead and dying shrimp where some presume that they are the cause of the issue. Vibrios are critical in the recycling of chitin which explains their affinity for animals such as shrimp whose chitinous exoskeleton is shed constantly as shrimp grow.

Strains of VP can be found in dead and dying shrimp, often as secondary pathogens. That is, they may not be the primary cause for mortality, but they infect weak animals, and the vibrios contribute to the mortality. However, in the last decade strains have appeared that have been the primary (i.e., they are obligate pathogens) cause of massive mortalities in farmed shrimp.

“Plasmids are circular extrachromosomal pieces of DNA that can encode for many different traits. Among these are virulence factors and antibiotic resistance.”

One example is found in salmon pathogenic strains of Vibrio anguillarum (some strains have also been implicated in primary disease in shrimp) where the presence of a plasmid that encodes for genes that produce outer membranes that allow the host to sequester iron, an obligate growth factor, is responsible for virulence. If the plasmid is cured the strains are not virulent.

The strains of VP associated with acute mortality in shrimp have been found to harbor plasmids (copy’s range in number from a few to many) that encode for an unusual pair of toxins that disrupt membrane integrity in the hepatopancreatic (HP) tubules, affecting the ability of the animals to gain nutrition and to grow.

It also makes them much more susceptible to secondary infections. Animals that are exposed to high levels of the toxins typically appear sluggish; have reduced appetites and empty guts with the HP appearing pale or white.

The disease was first reported in China in 2009 and spread rapidly throughout SE Asia. The first appearance of what is now known as Acute Hepatopancreatic Necrosis Disease (AHPND) or syndrome (AHPNS) manifested itself as what was coined early mortality syndrome (EMS).

Massive numbers of animals died within the first month or so of stocking. It is more than likely present today in almost every country that farms shrimp. It spread into the Americas in 2013 and moved into South America and the US.

 shrimp farming

Initially the focus was on strains of VP as being the sole source of the disease. Subsequently it has been determined that this plasmid has been found in several other species of vibrios and even in a few non-vibrios.

These toxins are known as PirA and PirB. They are structurally similar to toxins produced by several bacteria which impact insects, including Photorhabdus and Xenorhabdus sp among others, although it is not clear where they originated and how they ended up in marine vibrios.

“Historically, this plasmid was found in a strain of Vibrio campbellii in a culture collection that preceded the first reported incidence of the disease.”

Likely vibrios containing the toxins have been around for a while (there are also reports of similar pathologies seen decades ago) and something changed that allowed them it to proliferate in the manner that they have.

There are at least three possible reasons aside from changes in the strains themselves.

a. The misguided culture of bacteria (probiotics) on pond sides without quality control. Vibrios are ubiquitous. Shrimp farming areas will have vibrios in the sediments, the water and even the air. The toxin containing strains grow rapidly and a mixture of pond water with added nutrients such as molasses contains all that they need to thrive.

Thrive they do, easily out growing any bacteria that are added by the farmer. Bacillus spores take time to germinate during which the vibrios proliferate.

b. The excessive use of chlorine. Using chlorine to “kill” off the microbiome leaves a depleted microbiome that is readily colonized by rapidly growing vibrios including the strains that cause AHPNS. These vibrios have traits besides rapid growth that allow them to prevent other bacteria from growing.

c. A failure to appreciate the nature of the disease process. This disease is a toxicities. The toxins and not the presence of the specific bacteria cause the tissue damage. The bacteria don’t have to be present for the toxin to be present although they are the source of the toxins being secreted into the environment.

Opportunistic bacteria take advantage of weakened stressed animals and cause secondary infections that kill the animals.

Studies have determined there are many variants of the strains that carry the plasmids. This is not uncommon. It is the nature of plasmids. Some strains carry one copy of the genes encoding the toxins while others may carry many. Some produce no toxins, and some produce variable levels of either or both of them.

“Since vibrios can be VBNC (viable but non cultivable) not finding the bacteria does not mean that it is not present. When the toxins are present, they, without the bacteria being present, can damage the HP.”

There is evidence that suggests that many shrimp can be affected, weakened, and show signs of secondary disease processes after even a low-level toxin exposure. In many hatcheries, there is the distinct possibility that the toxins are present at low levels, high enough to cause pathology, but not high enough to cause mortality.

 shrimp farming

This is a result of efforts to control the vibrio without understanding that the toxins are how the shrimp are affected.

A culprit VP strain (or other vibrio that carries the plasmid) cannot always be found using traditional approaches such as PCR, RT PCR, antibody-based tests, isolation on media, etc. because it is sequestered in some part of the ecosystem that is not being sampled or it is present at low levels.

“Biofilms are important for many microbes to protect them and serve as the site of quorum sensing. VP and many other vibrios (and non-vibrios) do form biofilms.”

There is no absolute relationship between the number of bacteria present and the toxin load. A strain could be present that produces huge quantities of the toxins but is not present at very high numbers. It is prudent when testing for the presence of the bacteria that samples are enriched.

This entails growing samples up in broth for 12 to 18 hours and then seeing if VP is present. Also avoid the use of chlorine and the addition of home brewed batches of probiotics. PRO4000x, the first tableted bacterial product for shrimp farming, should be used instead.

There are currently no commercial tools available to determine if the toxin is present, although the technology does exist, and test strips have been developed and evaluated. Getting rid of this vibrio is not easy and the aim should be to minimize it and provide a production environment that is harder for it to thrive in. Avoid the use of outdoors cultures, whether algae, artemia or probiotic bacteria.

“Use ecofriendly probiotics such as PRO4000x and bacteriophages that are specific for VP (although they likely cannot differentiate harmless strains from those that carry the plasmid with active genes). PCR, while extremely sensitive, does have detection limits.”

Enrichment should be used for validation that these protocols have been effective in eliminating the culprit vibrios. Keeping it out of the hatchery and nursery tanks/ponds is essential to avoid low levels of damage to the HP, which can lead to increased sensitivity to a variety of pathogens.

Limiting the impact of stress is the biggest challenge facing shrimp farmers. Minimizing stress on the animals is critical for ensuring that susceptibility to disease is not increased, whether it be from obligate pathogens such as viruses, examples of which are WSSV and IMNV (there are many others) and fungi, the foremost example of which is Enterocytozoan hepatopenaei, the etiologic agent of EHP or any number of other pathogens.

Until farmers understand that stress is not an acceptable component of a sustainable production model and ensure that every step is taken to minimize both the presence and impact of stress, disease will continue to be a challenge.

“AHPNS appears to have moderated in the sense that we are not seeing the large mortality in early stocking that was characteristic of the disease when it first became widespread.”

Overall toxin loads may be less, but they are not gone. Animals will develop tolerance and eventually even resistance to these toxins. Even with this, pushing productivity beyond the carrying capacity of the production environment will continue to result in animal health challenges.

 Ph.D. Stephen G. Newman

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

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