EHP has become the major threat and some measures to contain the spread are proposed
At Infofish Shrimp 2019, held from 12-14 November, there was a panel on “Updates on shrimp disease threats”. Three presenters discussed recent knowledge: Dr Loc Tran, ShrimpVet Lab, Minh Phu AquaMekong, Vietnam; Dr Kallaya Sritunyalucksana-Dangtip, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Thailand and Dr Jumroensri Thawonsuwan, from the Songkhla Aquatic Animal Health Research Center (SAAHRC), Department of Fisheries, Thailand (DOF). Jumroensri gave results from the surveillance conducted by DOF Thailand as well as some nationwide measures to reduce pathogens in shrimp farming.
Both Loc Tran and Jumroensri discussed threats from several diseases but in this report, the focus is on Enterocytozoon hepatopenaei (EHP) which Kallaya said, “Overall, infections have reached epidemic proportions in Asian shrimp aquaculture. Specifically, in Thailand, there is a high prevalence of EHP at 60.1% of ponds affected.” Jumroensri showed that in Thailand, DOF focuses on detecting disease pathogens in samples. In the surveillance in 2019, the presence in post larvae and growout was: EHP >VpAHPND>WSSV>IHHNV>YHV. The presence of EHP have increased to 26.5% in 2019, from 25.1% in 2017 and 17.5% in 2018 whereas detections of Vibrio parahaemolyticus (VpAHPND) went down to 2.1% in 2019 from 3.6% in 2018 and 6.4% in 2017. Furthermore, EHP detections were highest in Penaeus vannamei juveniles at almost double that in post larvae.
Loc Tran who is Founder- Director of ShrimpVet reviewed lessons learnt from the emergence of three disease threats affecting productivity and margins for Asia’s shrimp industry. These are the early mortality syndrome/acute hepatopancreatic necrosis disease (EMS/AHPND), EHP and white feces disease (WFD). Besides these, there are of course challenges from white spot syndrome virus (WSSV), shrimp haemocyte iridescent virus (SHIV) as well as the antibiotic residues, as farms resort to quick remedies. Based on samples tested at ShrimpVet Laboratory, Loc Tran showed an increasing trend in PCR positive results for EHP. In 2019, EHP was positive in 60.3% of grow-out shrimp samples versus 43% in 2018. In contrast, it was only 33% for the AHPND pathogen in 2019. He said, “There is an accumulation of this pathogen in farms and incoming water and living with EHP is becoming a norm.”
Kallaya who heads the Aquatic Animal Health Research Team (AQHT) at BIOTEC gave updates from research done at BIOTEC and in collaboration with experts on microsporidia at CEFAS and University of Exeter, UK. EHP is a microsporidia related to fungi and infected shrimp grows slowly. However, growth retardation is not clearly evident before two months of culture. The research team seeks to answer several questions listed below. Answers will help them design possible control strategies.
Transmission route of EHP and its mechanism of infection
For the first time, “we have showed that EHP is transmitted horizontally when we added infected shrimp to a tank of naive shrimp. At the laboratory, infection occurred within 14 days. This is through faeces of the infected shrimp which contained the spores. It has taken us a long time to purify the spore for a better understanding of the infection mechanism. The EHP spore germination is an important process for EHP infection in shrimp. Recently, we can purify the EHP spores and develop the assay to induce spore germination via polar tube extrusion. The assay allows us to test chemicals and disinfectants that can be used to inhibit the germination process in the hatchery and grow-out pond (Aldama-Cano et al., 2018),” said Kallaya. Figure 1 shows the germinated EHP spores (active spores) and the non-germinated spores (inactive spores).
What causes shrimp to grow slowly
With the purification of the spore, Kallaya said that the team then went on to sequence the genome of the microsporidian (Wiredu Boakye, et al., 2017). “The genome showed missing genes encoding for the enzymes involved in the glycolytic pathway in EHP. As glycolysis is the fundamental pathway of ATP generation in eukaryotes, when the genes of the pathway are lost, energy for EHP growth is obtained directly from the shrimp resulting in shrimp slow growth.”
New PCR method
Arising from the work on the EHP genome, BIOTEC and Centex Shrimp has developed a method (SWP-PCR) to detect EHP. It is recommended to use for detection of EHP in non-shrimp samples (faeces, feed and environmental samples) for potential EHP carriers (Jaroenlak et al., 2016). The new SWP-PCR method did not produce false positive results from closely related microsporidia whereas the existing PCR detection methods (SSU-PCR) can give false positive test results due to cross reactivity of the SSU-PCR primers with DNA from closely related microsporidia that infect other aquatic organisms.
Environment reservoir of EHP
“Despite that SWP-PCR methods has been established and that we have a better understanding of the mechanism of EHP infection, the prevalence of EHP is still high in our last survey in Thailand. We hypothesize that there might be the environmental reservoir(s) of EHP in the pond that has been overlooked. Our on-going task is to use plankton nets with different mesh sizes to filter the water collected from the EHP heavily infected ponds. By using metagenomics approach, we hope to find the reservoir(s) of EHP in the pond, if there are any, “added Kallaya.
WFD-EHP: A perfect combo
Loc Tran gave this description when the shrimp is first infected by EHP. WFD is bacterial linked and the result of sloughing of microvilli and cells of the hepatopancreas. Vibrio has been isolated from WFD infected animals. In some laboratory trials, he showed that a pre-infection with EHP may increase the severity of WFD in a Vibrio challenge. Similar phenomenon is observed in the field leading to two types of WFD, a treatable one when there is vibriosis only and an untreatable vibriosis + EHP.
Some practical solutions specifically for EHP
From the current molecular understanding, Kallaya gave some practical suggestions on the control the EHP including.
- Check the post larvae with PCR-SWP before purchase.
- Laboratory trials showed a complete inhibition of spore germination occurred when the spores were frozen at -20°C for at least 2 hours. The chemicals that yielded 100% inhibition were 15 ppm KMnO4 (potassium permanganate) for 15 minutes, 40 ppm of 65% active chlorine for 15 minutes or 10 ppm of 65% active chlorine for 24 hours and 20% ethanol for 15 minutes (Aldama-Cano et al, 2018). There was no loss of fatty acids profile of frozen polychaetes after 3 months.
- Spore germination occurs at high pH (≥9). Pre-treatment of grow-out ponds by increasing pH before stocking might help to induce spore that are contaminated in the pond from the previous crop to germinate. Germination without the host will cause the spore to be inactivated and finally die.
According to Loc Tran, it is important to control EHP at the hatchery level. He recommends PCR checks of all inputs (brood stock, brood stock feed) and importantly to freeze live polychaetes. “This is a very effective control, but the downside is reduction in fecundity which brings up nauplii production costs by 30%. The total cost implication is small; at the hatchery, nauplii production cost is only 10% of total production cost and for the farmer, the post larvae cost only account for 5% of total farming costs. Therefore, the hatchery owner and farmer need to collaborate and share costs to prevent EHP in the farming system.”
Nationwide control on pathogens
Over in Thailand, Jumroensri who is Director, SAAHRC said that the DOF has set up several measures and legislations to reduce disease impacts in hatchery and nursery stages. There is the mandatory standard for pathogen-free P. vannamei nauplii production system where compliant farms are accredited. Today there are 50 farms accredited. The “cleaning up project” for marine shrimp hatchery and nursery using real time PCR is for eight diseases (WSSV, IHHNV, YHV, TSV, IMNV, VpAHPND, EHP, SHIV) and in this 245 farms are in the white list where DOF does the “Lot by Lot project” where these eight pathogens are tested in post larvae before stocking in grow out ponds.
In terms of control strategies DOF works with BIOTEC- once information is obtained, some protocols are developed for the transfer to farmers. One example is on pond treatments, using fertilizers and increasing alkalinity to induce spore shooting. The DOF has a Bacillus probiotics and herb extract (galangal extract) to reduce infections.