How to Manage the New Microsporidian Disease

The March/April 2015 issue of The Global Aquaculture Advocate (“The Global Magazine for Farmed Seafood”) contains an excellent article by Dr. Stephen Newman on how to manage the new microsporidian diseasecaused by Enterocytozoon hepatopenaei (EHP), a parasite widely found in Asia and other areas that severely retards the growth of farmed shrimp.  EHP infects the tubules in the shrimp’s hepatopancreas and damages the organ’s ability to gain nutrition from feed.  Total elimination of EHP may not be possible.  The best approach is to lessen the amount of EHP coming into ponds and then to control its levels in the ponds.  It is widely understood that EHP limits growth, but does not cause mortality.

EHP is now endemic throughout China, Malaysia, Thailand, Indonesia and Vietnam, and likely present in India and possibly Mexico.  It can likely be found anywhere that has imported live feeds from China and animals from areas where EHP is endemic.  EHP is very difficult to eradicate.  More than likely, we will only be able to control its levels.  The carriers of EHP have not been identified.

Diagnosis

The pathogen can be detected using gene-based tools such as polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) testing of feces from broodstock.  These methods can also be used with postlarvae.  Light microscopy can be used, as well, although it can be very difficult to visualize the very small spores.  Effective, screening of broodstock requires examination of individual animals, a costly practice.  In some areas, there may be no animals totally free of the pathogen.

Treatment

Other microsporidian infections are typically treated with drugs, but those drugs are unlikely to be effective against EHP because they target specific tissues.  Dealing with EHP requires a three-pronged strategy: hatchery biosecurity, pond preparation and pond management.

Hatchery Biosecurity

No Live Feeds: Pond-reared broodstock, as well as those fed infected live feeds, can be infected and spread EHP through their feces.  The use of live animals—including polychaetes, clams, warmwater squid and locally produced Artemia—in broodstock maturation facilities poses a significant biosecurity risk and should be discouraged.  Krill does not pose a risk.  If live feeds are used, they should be frozen, pasteurized or even irradiated.

Maturation facilities and hatcheries should be dried out completely, washed and then disinfected with a caustic solution of sodium hydroxide.  It has been suggested that all equipment, pipes and tanks should be soaked in a 2.5% sodium hydroxide solution for at least three hours.  After this, the remaining caustic solution should be washed away, and all the treated materials allowed to dry for an extended period.  Then before using them, rinse with acidified chlorine at 200 parts per million and pH less than 4.5.  Microsporidian spores are extremely resistant to most treatments, and complete elimination will prove challenging.  The goal is to substantially lower the load.

Clean Eggs, Nauplii: Proven strategies for washing and rinsing nauplii with the appropriate mix of freshwater and chemicals (iodine and formaldehyde, among others) that can weaken the passive attachment of spores to eggs and nauplii—thus lessening transmission—must become routine.  This is an effective tool against EHP, as well as for lowering the loads of the bacteria that cause early mortality syndrome that pass from broodstock to postlarvae.

Pond Preparation

High organic loads are typically associated with high spore counts.  There is likely some intermediate carrier, and until we are sure what it is, use strategies to properly treat sediments before stocking.

As spores typically are resistant to a wide variety of environmental conditions, with different species displaying differential susceptibility, the general suggestions are to physically remove accumulated organic matter and treat pond bottoms with a very caustic material to increase the pH to 12, which will kill many of the spores.  Killing all of them may not be possible.

It has been recommended that earthen ponds be disinfected with very heavy use of calcium oxide, or quick lime, applied at a level of 6,000 kilograms per hectare or greater.  Pond bottoms must be completely dry.  Plow the quick lime into the dried sediments to a depth of 10 to 12 centimeters, and then moisten the sediments to activate the lime.  If the application is done properly, the pH of the soils will rise to 12 or more within days and then gradually return to normal as the quick lime becomes calcium carbonate.

Pond Management

After the soils have recovered, use suitable commercial products [probiotics] from the early stages of culture to prevent the accumulation of large amounts of organic matter.  These can be used alone or in combination with water exchange.  The goal is to lessen the amount of accumulated organic matter and thus reduce the potential reservoir for spores that will be ingested and continue to infect shrimp.  Consistent use at levels that lessen the amount of organic matter is important.

Editors Note: Dr. Tim Flegel kindly provided the following additional comments on detecting EHP.

Microscopy can be used to detect EHP with hepatopancreas tissue smears or tissue sections, but scanning using at least a 40x objective is needed, followed by confirmation by inspection using a l00x objective.  Since this is very tedious and depends on seeing mature or nearly mature spores, the process can take a lot of time and may fail if no spores are seen.  This often happens with Penaeus vannamei.

Thus, PCR detection is preferred, using DNA extracted from hepatopancreas tissue as the best option.  With nested PCR, DNA from feces can also be used, but may give negative results if the tested shrimp have light infections.