Source: UNIVERSITY OF ARIZONA submitted to NRP
ACUTE HP NECROSIS DISEASE (AHPND) AND ENTEROCYTOZOON HEPATOPENAEI: GAME CHANGERS IN PENAEID SHRIMP FARMING ON A GLOBAL SCALE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
ANIMAL HEALTH
Reporting Frequency
Annual
Accession No.
1006512
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2015
Project End Date
Sep 30, 2020
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF ARIZONA
888 N EUCLID AVE
TUCSON,AZ 85719-4824
Performing Department
Animal & Comparative Biomedical Sciences
Non Technical Summary
For AHPND: The laboratory in the School of Animal and Comparative Biomedical Sciences (SACBS) at the University of Arizona will confirm AHPND (Acute hepatopancreatic necrosis disease) cases in Mexico, Guatamela, Nicragua and Honduras. This confirmation will include the PirA and PirB that toxins are present in reportable episodes of post-larvae (PLs) occurrences in the Americas. The way that AHPND works is that PirA and PirB toxins are produced in the stomachs of PLs which are the first to assume residence in a newly completed shrimp pond immediately after it has been stocked. Because PLs prefer bacterial detritus to normal shrimp feed, there is little or no consumption of any shrimp feed. The Vibrio parahaemolyticus strain that causes AHPND can thencoat the stomach lining and gastric mill to produce extremely virulent toxins, which we now know is mostly PirB. The PirB toxin then causes a massive loss of desmosomes and tight junctions of hepatopancreatic epithelial cells into the lumens of the hepatopancreas tubules. This stage is the most remarkable phase of AHPND, because of the massive sloughing of HP tubule epithelial cells into the lumens of the HP tubules. This is followed by a massive secondary presumed vibriosis infection which will terminate any surviving PLs. For EHP: Likewise, my laboratory at the University of Arizona is uniquely situated to confirm cases of a microsporidian parasite that infects the shrimp's hepatopancreas (EHP = Enterocytoozon hepatopenaei).
Animal Health Component
80%
Research Effort Categories
Basic
10%
Applied
80%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113721104020%
3113721110020%
3113721110120%
3113721110320%
3113721116020%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3721 - Marine shrimp;

Field Of Science
1040 - Molecular biology; 1101 - Virology; 1103 - Other microbiology; 1160 - Pathology; 1100 - Bacteriology;
Goals / Objectives
1). Confirm the nature and pathology of AHPND (acute hepatopancreatic necrosis disease) to Penaeus vannamei using pirA and pirB to determine how the toxin works on the shrimp's hepatopancreas.2). Determine if sloughing of HP tubule epithelial cells always leads to the secondary massive Vibrio spp. infections that identify the massive hemocytic response and bacterial involvement of AHPND.3). Confirm the pathology of EHP (Enterocytozoon hepatopenaei) to other penaeids including P. monodon, P. stylirostris and P. vannamei. (This work will be done at our West Campus Aquaculture facility).4). Determine if the reduction of B-cells vesicles in the hepatopancreas is a precursor to infection by EHP. (This work will be done at our West Campus Aquaculture facility).5). Confirm if EHP (and when) EHP has entered the Americas.
Project Methods
1). Much of the work on AHPND will be limited to determination of the nature of the toxin forming units called pirA and pirB in the plasmid pVPA3-1. It contains 69,168 bp, along with the insecticidal related genes called pirA and pirB.2). Determine how the sloughing of AHPND tubule cells contributes to the failure of the hepatopancreas in affected shrimp. Is it because of the loss of desmosomes or tight junctions, or is the massive sloughing that is seen with AHPND that accompanies some other function of desmosomes and tight junctions? 3). Confirm the pathology of EHP to other penaeids including P. monodon, P. stylirostris and P. vannamei. If this is the situation (and we believe it to be) then the former species (P. monodon) should be much more resistant to EHP than are P. stylirostris and P. vannamei.4). Determine if the reduction in B-cell vesicles in the HP is a precursor to infection by EHP. This may be accomplished using a standard microscope fitted with a camera that detects variations in B-cell counts.5). My lab has the primers listed with Table 1 (above) which are capable of detecting both AHPND and EHP with high sensitivity.

Progress 10/01/15 to 09/30/20

Outputs
Target Audience:The target audience for this project will be shrimp industry in the US and elsewhere in the world, seafood industry personnel, aquatic animal health professionals, VMOs, researchers in academia, industry, government and non-governmental agencies worldwide. Two infectious diseases of shrimp that are the foci of this project,Acute Hepatopancreatic Necrosis Disease (AHPND) and Hepatopancreatic Microsporidiosis (HPM) have had profound negative impacts on shrimp industry since their emergence around 2009-2010. AHPND is caused by a bacterium, Vibrio parahaemolyticus carryingbinary toxin producing genes and HPM is caused by a fungus,Enterocytozoon hepatopenaei (EHP). Considering the fact that there is no therapy against either of these two diseases, it is critical that the professionals engaged in shrimp aquaculture and aquatic animal health and stakeholders are aware of the risk that these two diseases pose to shrimp aquaculture in the US and the rest of the world and all stakeholders take steps to prevent the introduction of the disease in shrimp aquaculture operations including captive breeding programs, hatchery and grow-out operations. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Trainings on bioassay, histopathology and molecular diagnostics on AHPND and EHP were provided to undergraduate and graduate students and postdoctoral fellows working in Aquaculture Pathology Laboratory in The University of Arizona. In addition, trainings were provided through "Shrimp Pathology Short Course" to aquaculture professionals from industry, academia, government and non-governmental agencies from many countries in the Americas, Europe, Asia, Middle-East and Australia. The trainings were conducted on July 17-22, 2017 (19participants), June 18-23, 2018 (15 participants), and June 10-15, 2019 (25 participants). How have the results been disseminated to communities of interest?The findings from this project were disseminated through publications in peer-reviewed journals including Aquaculture, Aquaculture Reports, Diseases of Aquatic Organisms, Journal of Invertebrate Pathology, Journal of Microbiological Methods, Microorganisms, and Molecular and Cellular Probes. Oral presentations were given in World Aquaculture Society (WAS) Meetings: Aquaculture America, Las Vegas-2016; Aquaculture America, San Antonio-2017; Aquaculture America, San Antonio-2019; WAS Meeting in Cape Town, South Africa-2017; WAS Asia Pacific Meeting, AQUA INDIA, Chennai- 2019. Presentations were given in other professional meetings including in 10th International Symposium on Aquatic Animal Health & Food Safety, Ensenada, Mexico, September-2018; International Society of Aquatic Animal Health, Charlottetown, PEI, Canada, September-2018; and in Guatemala Aquaculture Symposium, Antigua, Guatemala, June 2018. Findings from this project were also presented during invited lectures in the School of Aquatic & Fishery Sciences, University of Washington, Seattle, May 2019; and Department of Biomedical Sciences, Oregon State University, Corvallis, May 2019. Finally, the research findings were also disseminated through publications in an industry magazine, Global Aquaculture Advocate. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The virulence factor of Vibrio parahaemolyticus causing AHPND is a binary toxin, PirA/PirB. The genes that encode PirA/PirB are located in alarge plasmid, ~70 kb residing within the bacterial cells. The plasmid DNA sequence revealed that the toxin encoding genes are flanked by transposes making this region of the bacterial genome mobile. It was determined that AHPND-causing V. parahaemolyticus isolates from Mexico, but not the isolates from Asia, carry aTn3-like transposon. Based on this genetic difference a polymerase chain reaction (PCR)-based method was developed to delineate V. parahaemolyticus isolates from these two geographic regions. The etiologic bacteria upon enteringshrimp gut produces PirA/PirB binary toxin. The binary toxin eventually gets into hepatopancreas through gastric sieve and exerts cellular changes.The main histological features of AHPND in the early to middle stage of the disease are progressive degeneration of HP tubule from medial to distal end of the tubule. Prominent necrosis & sloughing of the tubule epithelial cells is observed with no detectable causative pathogen. At the initial stage, the disease is characterized by medial to distal dysfunction of HP tubule cells such as B (blister like), F (fibrillar), and R (resorptive) cells, prominent karyomegaly, and lack of mitotic activity in E cells (embryonic). In the terminal stage, massive secondary infections occur. This is characterized by a massive accumulation of hemocytes and the formation of melanized granulomas followed by infection in tubule lumen with opportunistic bacteria.The sloughing of epithelial cells in hepatopancreatic tubule is followed by massive secondaryinfections by other opportunistic bacteria including non-AHPND causing Vibrio spp. This leads toan increase of Septic Hepatopancreatic Necrosis (SHPN) in shrimp.Both polyclonal and monoclonal antibodies were developed against PirA and PirB toxin. Real-time PCR using TaqMan and SYBR Green chemistries were developed to detect the AHPND. In addition, enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies against PirA/ PirB toxin were developed for detection of AHPND in acute and chronic phase of infection. Like AHPND, EHP poses threat to shrimp aquaculture globally. EHP infects not only Pacific white shrimp (Penaeus vannamei)but also other penaeid species such as P. mondon and P. stylirostris. At farm level, chronic mortalities are observed due to EHP infectionwhich occurnot due to direct EHP infection alone but due to EHP infection followed by the secondary Vibrio infection. Using histopathology, in situ hybridization and PCR-diagnosis targeting the 18S rRNA gene, it was shown that EHP has been introduced in the Americas (in Venezuela). We also compared the nucleotide sequence similarities in 18S rRNA gene (1095- bp), in β-tubulin (583-bp) and sporewall protein (431-bp) genes between the Venezuela EHP and SE Asia isolates; and the results showed 99%, 93% and 91%identities, respectively. In order to prevent further spread of EHP, it is critical to screen broodstock and post larvae, shrimp feed and feed ingredients for EHP. Conventional PCRand real-time PCR-based diagnostic tools were developed to detect the pathogen.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: 1. Schofield, Paul J., Noble, Brenda L., Aranguren Caro, L. Fernando, Mai, Hung N., Padilla, Tanner J., Millabas, J., and Dhar, Arun K. 2020. Impact of Acute Hepatopancreatic Necrosis Disease (AHPND) on the freshwater prawn (Macrobrachium rosenbergii) and Pacific white shrimp (Penaeus vannamei) at various salinities. Aquaculture Research 52: 1480-1489.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: 2. Aranguren Caro, L. Fernando, Mai, Hung N., Kanrar, Siddhartha, Cruz-Flores, Roberto, and Dhar, Arun K. 2020. Isolation of a novel mutant of Vibrio parahaemolyticus pirABVP (+) that carries binary toxin genes but does not cause acute hepatopancreatic necrosis disease (AHPND) in Pacific white shrimp (Penaeus vannamei). Microorganisms 8: 1549.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: 3. Mai, Hung N., Cruz-Flores, Roberto, and Dhar, Arun K. 2020. Development of an indirect Enzyme Linked Immunoassay (iELISA) using monoclonal antibodies against Photorhabdus insect related toxins, PirA and PirB released from Vibrio spp. Journal of Microbiological Methods 176: 106002.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: 4. Aranguren Caro, L. Fernando, Mai, Hung N., Noble, Brenda, and Dhar, Arun K. 2020. Acute hepatopancreatic necrosis disease (VPAHPND), a chronic disease in Penaeus vannamei population raised in Latin America. Journal of Invertebrate Pathology, 174, 107424.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: 5. Mai, Hung, N., Cruz-Flores, R., Aranguren Caro, L. Fernando, Noble White, B., and Dhar, Arun K. 2020. A comparative study of Enterocytozoon hepatopenaei (EHP) challenge methods in Penaeus vannamei. Journal of Invertebrate Pathology 171: 107336.


Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The stakeholders of the project include(1) shrimp breeding companies in the US that areproducing genetically superior and disease free broodstock, (2) shrimp industry in the US, (2) USDA-APHISand US Department of Commerce, (3) researchers working on crustacean diseases especially those working on to developing therapies against acute hepatopancreatic necrosis disease andEnterocytozoon hepatopenaei (EHP). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The results were presented in an international confernece held in Ilo Ilo, Philippines and in a meeting in Sultan Qaboos University in Oman. Arun K. Dhar. 2019. "Emerging Diseases & Disease Management in Shrimp Aquaculture", Department of Marine Sciences & Fisheries, Sultan Qaboos University, August 21, 2019, Muscat, Oman. Arun K. Dhar. 2019. "Acute hepatopancreatic necrosis disease and Enterocytozoon hepatopenaei - Threats to Sustainable Shrimp Aquaculture", In a Workshop on the "Promotion of Sustainable Aquaculture, Aquatic Animal Health and Resource Enhancement in Southeast Asia", SEAFDEC, 25-27 June 2019, Iloilo City, Philippines. What do you plan to do during the next reporting period to accomplish the goals?1. Plan to develop an enzyme-linked immunosorbant assay (ELISA) for the detection of toxin in AHPND-affected shrimp. 2. Compare the efficacy of ELISA to PCR-based diagnostic method to assess the utility of ELISA as method to detect AHPND since level of toxin produced by the etiologic bacteria, Vibrio parahaemolyticus appears to be critical in ausing clinical manifestation that presence or absence of the binary toxin gene. 3. Assessing EHP suseptibility of crustaceans other than P. vannamei shrimp.

Impacts
What was accomplished under these goals? A non-invasive sampling method was employed to collect hepatopancreas tissue samples from P. vanname shrimp andpathology of EHP was confirmed in the biopsy samples by H&E histology and real-time PCR.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Arun K. Dhar. 2019. Acute hepatopancreatic necrosis disease and Enterocytozoon hepatopenaei - Threats to Sustainable Shrimp Aquaculture, In a Workshop on the Promotion of Sustainable Aquaculture, Aquatic Animal Health and Resource Enhancement in Southeast Asia, SEAFDEC, 25-27 June 2019, Iloilo City, Philippines.
  • Type: Other Status: Other Year Published: 2019 Citation: Arun K. Dhar. 2019. Emerging Diseases & Disease Management in Shrimp Aquaculture, Department of Marine Sciences & Fisheries, Sultan Qaboos University, August 21, 2019, Muscat, Oman.


Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The results of the project will be of interest to (1) the US shrimp companies producing genetically superior and disease free broodstock, and shrimp farmers, (2) USDA-APHIS, and US Department of Commerce, (3) researchers working on diseases of shrimp and other crustaceans, in particular those working on acute hepatopancreatic necrosis disease and Enterocytozoon hepatopenaei (EHP) diseases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The findings were published in peer reviewed journals and presented in national and international conferences. What do you plan to do during the next reporting period to accomplish the goals?1. Develop a protocol for the detection of pirA and pirB toxin in shrimp affected by AHPND. 2. Determine the effect of AHPND is crayfish 3.Compare pathology of EHP (Enterocytozoon hepatopenaei) in penaeid shrimp(e.g. P. vannamei) and fresh water prawn/ crayfish.

Impacts
What was accomplished under these goals? 1. A multplex real-time PCR-based assay using SYBR Green dye was developed for the detection and quantification of pirA and pirB genes in Vibrio parahaemolyticus bacteria and in shrimp infected with V. parahaemolyticus causing AHPND. 2. Histopathology of Penaeus vannamei geneticallyresistant or susceptible to AHPND was studies.A shrimp genetic line (APE1) of Penaeus vannamei displayed significantly higher survival compared to susceptible lines in a VPAHPND challenge test. AVPAHPND chronic phase was described for the first time. Our findings indicated that it is possible to develop genetically resistant lines of shrimp against AHPND. 2. Currently developing monoclonal antibodies to detect pirA and pirB toxins in bacteria and in shrimp infected with AHPND. The antibodies will be used to determine the level of toxin present in moribund animals. This information will be useful in determining the level of toxin needed to cause mortality in shrimp.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Cruz-Flores, R., Mai, Hung N., and Dhar, Arun K. 2019. Multiplex SYBR Green Real-Time PCR for the detection of Photorhabdus Insect-Related (Pir) Toxin Genes PirA and PirB. Molecular & Cellular Probes, 43: 20-28
  • Type: Other Status: Published Year Published: 2019 Citation: Invited Lecture: Arun K. Dhar. 2019.Emerging Diseases in Shrimp Aquaculture, School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA, May 30, 2019.
  • Type: Other Status: Published Year Published: 2019 Citation: Invited lecture: Arun K. Dhar. 2019.Emerging Diseases in Shrimp Aquaculture, Department of Biomedical Sciences, College of Veterinary medicine, Oregon State University, Corvallis, OR, May 14, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Arun K. Dhar, Michelle Garfias, Jasmine Millabas, Joshua Lin, Gregory Lyons, Roberto Cruz-Flores, Hung Mai, Siddhartha Kanrar & L. Fernando Aranguren Caro. 2019. Evolving Trends in Shrimp Disease Diagnostics, Aquaculture America, New Orleans, LA, March 08-11, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Invited Lecture: Arun K. Dhar. 2018. The current status of knowledge on viral disease and its impact for commerce", 10th International Symposium on Aquatic Animal Health and Food Safety, September 18, 2018, Ensenada, Mexico.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Arun K Dhar, Luis Fernando Aranguren Caro, Siddhartha Kanrar, Brenda Noble White, Jasmine Millabas, Hung N. Mai, Roberto C. Flores, and Paul Schofield. 2018. Current status of acute hepatopancreatic necrosis disease in shrimp: biology, diagnostics and disease management, International Society of Aquatic Animal Health, Charlottetown, PEI, Canada, Sept 03-06, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Arun K. Dhar. 2018. AHPND and EHP and Their Control in Shrimp Farming Guatemala Aquaculture Symposium, Antigua, Guatemala, June 06-08, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Arun K. Dhar. 2018. Acute Hepatopancreatic Necrosis Disease (AHPND) and Enterocytozoon hepatopenaei (EHP): Two emerging threats to shrimp aquaculture, AQUAINDIA-2018, Chennai, India, Feb 02-03, 2018


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The results of the project will be of interest to (1) the US shrimp producers (breeding companies and grow out farms); (2) scientists in the areas of crustacean diseases, in particular Vibrio bacteria and microsporidia; (3) regulatory agencies, such as US-APHIS, US Department of Commerce, as our results described the crustacean diseases status in the US and in other countries, this provides information for shrimp diseases to be monitored and screened for import/export purposes. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?An Associate Research Professor, a Post-Doctorate Associate and a Research Specialist are the researchers on the project. Two undergraduate student workers are also working on the project to aid in setting up tanks, stocking and maintaining shrimp, tissue sampling, and DNA and RNA extractions. How have the results been disseminated to communities of interest?The results were published as peer-reviewed articles and in an aquaculture popular press, Global Aquaculture Alliance, also presented in (3) World Aquaculture Society annual meetings in Las Vegas, Nevada, (2) shrimp pathology short-course organized by University of Arizona-Aquaculture Pathology Laboratory (2017 & 2018). What do you plan to do during the next reporting period to accomplish the goals?Objective 1: To determine how the pirA&B work on the shrimp's hepatopancreas. The polyclonal anti-PirA and anti-PirB antibodies will be used in ELISA and Western blot analyses in (a) identifying genes that might be involved in AHPND pathogenesis (b) investigating their binding sites (receptors) of AHPND binary toxin in hepatopancreas cells. Objective 2: to determine if sloughing of HP tubule epithelial cells always leads to the secondary massive Vibrio spp. infections. We will focus on the relationship between AHPND (its infection causes the sloughing of hepatopancreas tubule epitheilial cells) and septic hepatopancreatic necrosis (SHPN, which is a result of secondary Vibrio infections). We will review the cases of AHPND submitted to our Lab, and perform a case control study to determine the association between SHPN and the AHPND histology. The cases those shrimp display histological signs of SHPN and the controls will be those shrimp from the same ponds without SHPN. The presence of AHPND will be as a risk factor for assessment. The association between SHPN and AHPND will be analyzed by calculating the Odds Ratio (OR) and 95% Confidence intervals (CI). Objective 3: To continue study of EHP. We will develop a qPCR assay for this pathogen. This assay will be used to evaluate the control and treatments for this pathogen. In addition, we will develop a non-lethal tissue sampling method for the detection of the pathogen that will eliminate the need to sacrificing broodstock. Objective 4 (year 2-5): Determine if the reduction of B-cells vesicles in the hepatopancreas is a precursor to infection by EHP. This objective will be started in 2019. We plan to set up treatment tanks in which shrimp will not be fed for 1-week and monitor the reduction of B-cells in hepatopancreas by histology. The control shrimp will be fed twice a day, so that they will have normal quantities of B-cells. Then these shrimp will be fed with EHP-tissue and sampled every 3 days (or 7-day) for 1 month to determine if the reduction of B-cells will increase the severity of EHP infection. We acquired and have been maintaining a population of EHP-infected shrimp from Thailand, their hepatopancreas (contain EHP) will be used as EHP feeding tissues. Objective 5 (year 2-5): Determine the prevalence of EHP in the Americas. For this objective, we will monitor the presence of EHP in the samples submitted to our laboratory by PCR and histological examination. Our laboratory is a US- APHIS approved diagnostic lab and receive samples including shrimp (cultured and wild caught), Artemia biomass and cysts, etc.

Impacts
What was accomplished under these goals? Objective 1: To determine how the pirA&B work on the shrimp's hepatopancreas. We have found that not only V. parahaemolyticus is able to cause AHPND, but also some other bacteria such as V. campbelli carrying the plasmid that contain the binary toxin PirA and PirB can cause AHPND. In addition, it was found that the Vibrio spp. carrying the plasmid that cause AHPND is part of a composite transposon named as Tn6264 that is flanking upstream and downstream by a transposase. Due to this transposition activity, we have discovered some strain of V. parahaemolyticus positive for AHPND by PCR that are not able to cause AHPND. After further sequencing we discovered that some of these mutants present a PirA deletion/ their mutants contain both, PirA and PirB but, due to an 1000 bp insertion sequence are unable to produce the AHPND in susceptible shrimp. Objective 2: to determine if sloughing of HP tubule epithelial cells always leads to the secondary massive Vibrio spp. infections. We have seen an increase of SHPN (Septic Hepatopancreatic Necrosis) in shrimp farming from areas where EHP is endemic. Based on the results we found in the previous year, we have been able to confirm that EHP is a primary pathogen that later on, when sloughing occurs due to EHP replication, some other Vibrio spp are able to colonize these cells and cause SHPN, which is a result of secondary Vibrio infections. At farm level some chronic mortalities occur due not directly by the EHP infection but also by the secondary Vibrio infection. Objective 3: To determine the pathogen of EHP in P. vannamei, P. mondon, and P. stylirostris. All 3 major species of farmed penaeid shrimp, P. vannamei, P. stylirostris and P. monodon, can be infected by EHP based on histology results. Our laboratory confirmed the EHP infection through in situ hybridization (ISH) using a cloned 18S rRNA gene fragment. This probe was labeled with digoxigenin and hybridized to EHP-infected P. vannamei collected in Vietnam during 2014-2015. The probe reacted intensely to the basophilic inclusions within the cytoplasm. Our P. stylirostris samples collected from Brunei in 2006 also showed the presence of microsporidium spores within the hepatopancreas, where the tubule epithelial cells had a positive reaction by ISH. These EHP detection methods were used in the study of white feces syndrome of P. vannamei, EHP was detected in all the samples analyzed from white feces syndrome-affected ponds, but not in those sampled from healthy shrimp ponds, suggesting that EHP is associated with white feces syndrome. This EHP 18S rRNA gene clone was patented by University of Arizona and licensed to a Taiwanese Biotechnology company for manufacturing an EHP PCR detection kit. Objective 4: Determine if the reduction of B-cells vesicles in the hepatopancreas is a precursor to infection by EHP. No progress was made in year 2. Objective 5: Confirm if EHP (and when) EHP has entered the Americas. We described the first case of EHP-infected Penaeus vannamei cultured in Latin America shrimp farming. Its histopathology is very similar to that of SE Asia EHP, with infected shrimp showing basophilic inclusions in hepatopancreas. Upon in situ hybridization of an EHP 18S rRNA gene fragment labeled with digoxigenin, the probe reacted intensely to the basophilic inclusions within the cytoplasm of the infected cells. We also compared the nucleotide sequence similarities in 18S rRNA gene (1095- bp), in β-tubulin (583-bp) and spore wall protein (431-bp) genes between the Venezuela EHP and SE Asia isolates; and the results showed 99%, 93% and 91% identities, respectively. In the previous report, we detected the presence of EHP in frozen Artemia biomass from a US provider. In recent samples of Artemia biomass, we found also positive results by PCR. After amplified and sequenced the 18S rRNA the 1.1 kb fragment we found a different microsporidia specie that has been previously reported in Artemia salina with an identity 99% suggesting the need of improving the EHP detection method by PCR using a different gene.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Aranguren LF, Han JE, Tang KFJ (2017) Enterocytozoon hepatopenaei (EHP) is a risk factor for acute hepatopancreatic necrosis disease (AHPND) and septic hepatopancreatic necrosis (SHPN) in the Paci?c white shrimp Penaeus vannamei. Aquaculture. 471. 37-42.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Han JE, Tang KFJ, Pantoja CP, Piamsomboom P. (2017) Evaluation of a reliable non-invasive molecular test for the diagnosis of the causative agent of acute hepatopancreatic necrosis disease (AHPND) of shrimp. Aquaculture Report 5: 58-61.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Isolation of novel mutant strains of Vibrio parahaemolyticus from pacific white shrimp Penaeus vannamei in Latin america that contains Pir a and b genes but does not cause acute hepatopancreatic necrosis disease (AHPND). World Aquaculture Society meeting. South Africa, June 2017
  • Type: Other Status: Published Year Published: 2017 Citation: Studies on The Hepatopancreatic Microsporidian EHP: A risk factor for AHPND and SHPN diseases in Pacific white shrimp Global Aquaculture Advocate, March 2017
  • Type: Other Status: Published Year Published: 2017 Citation: Four AHPND strains identified on Latin American shrimp farms. Global Aquaculture Advocate, February 2017
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Han JE, Tang KFJ, Piamsomboom P, Aranguren LF (2017). Characterization and pathogenicity of acute hepatopancreatic necrosis disease natural mutants, pirABvp (-) V. parahaemolyticus, and pirABvp (+) V. campbellii strains. Aquaculture 470: 84-90.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Tang. KF, L, Aranguren LF, Piamsomboon P, Han JE, Maskaykina I, Schmidt M 2017. Detection of the microsporidian Enterocytozoon hepatopenaei (EHP) and Taura syndrome virus in Penaeus vannamei cultured in Venezuela. Aquaculture. 480, 17-21
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Histopathology and in situ detection of the microsporidian Enterocytozoon hepatopenaei (EHP) in Penaeus vannamei cultured in a Latin America country. World Aquaculture Society meeting. San Antonio Texas, February 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Biosecurity and The Relevance of The Proficiency Test (Pt Test) World Aquaculture Society meeting. San Antonio Texas, February 2017


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The results of the project will be of interest to (1) the US shrimp producers (breeding companies and grow out farms); (2) scientists in the areas of crustacean diseases, in particularVibrio bacteria and microsporidia; (3) regulatory agencies, such as US-APHIS, US Department of Commerce, as our results described the crustacean diseases status in the US and in other countries, this provides information for shrimp diseases to be monitored and screened for import/export purposes. Changes/Problems:The Principal Investigator. Dr. Donald Lightner, retired in October, 2015. The project was managed by Dr. Kathy Tang-Nelson during this reporting period. What opportunities for training and professional development has the project provided?An Associate Research Professor, a Post-Doctorate Associate and a Research Specialist are the researchers on the project. Two undergraduate student workers are also working on the project to aid in setting up tanks, stocking and maintaining shrimp, tissue sampling, and DNA and RNA extractions. How have the results been disseminated to communities of interest?The results were published as peer-reviewed articles andin an aquaculture popular press, Global Aquaculture Alliance, also presented in (1) World Aquaculture Society annual meetings in Las Vegas, Nevada, (2) shrimp pathology short-course organized by University of Arizona-Aquaculture Pathology Laboratory. What do you plan to do during the next reporting period to accomplish the goals?Objective 1:To determine how the pirA&B work on the shrimp's hepatopancreas.The polyclonal anti-PirA and anti-PirB antibodies will be used in ElISA and Western blot analyses in (a) evaluating the effects of potential therapeutic agents for APHND, including probiotics, application of bacteriophages, organic acids, QS inbibitors, etc. (b) investigating their binding sites (receptors) in hepatopancreas cells. Objective 2: to determine if sloughing of HP tubule epithelial cells always leads to the secondary massive Vibrio spp. infections. For year 2, we will focus on the relationship between AHPND (its infection causes the sloughing of hepatopancreas tubule epitheilial cells) and septic hepatopancreatic necrosis (SHPN, which is a result of secondary Vibrio infections). We will review the cases of AHPND submitted to our Lab, and perform a case control study to determine the association between SHPN and the AHPND histology. The cases those shrimp display histological signs of SHPN and the controls will be those shrimp from the same ponds without SHPN. The presence of AHPND will be as a risk factor for assessment. The association between SHPN and AHPND will be analyzed by calculating the Odds Ratio (OR) and 95% Confidence intervals (CI). Objective 3: To continue study of EHP. In year 2, we will develop a qPCR assay for this pathogen. This assay will be used to evaluate the control and treatments for this pathogen. The qPCR will be based on EHP's beta tubulin and spore wall protein genes. In addition, we will develop EHP genotyping methods to differentiate among isolates. Objective 4 (year 2-5): Determine if the reduction of B-cells vesicles in the hepatopancreas is a precursor to infection by EHP. This objective will be started in 2016. We plan to set up treatment tanks in which shrimp will not be fed for 1-week and monitor the reduction of B-cells in hepatopancreas by histology. The control shrimp will be fed twice a day, so that they will have normal quantities of B-cells. Then these shrimp will be fed with EHP-tissue and sampled every 3 days (or 7-day) for 1 month to determine if the reduction of B-cells will increase the severity of EHP infection. We acquired and have been maintaining a population of EHP-infected shrimp from Thailand, their hepatopancreas (contain EHP) will be used as EHP feeding tissues. Objective 5 (year 2-5):Confirm if EHP (and when) EHP has entered the Americas. For this objective, we will monitor the presence of EHP in the samples submitted to our laboratory by PCR and histological examination. Our laboratory is a US-APHIS approved diagnostic lab and receive samples includingshrimp (cultured and wild caught), Artemia biomass and cysts, etc.

Impacts
What was accomplished under these goals? Objective 1: To determine how the pirA&B work on the shrimp's hepatopancreas. The pirA&B genes are located in a large plasmid residing within the bacterial cells. During 2015, we analyzed the plasmid sequence from the whole genome sequences of AHPND-V. parahaemolyticus isolates and identified 2 regions that exhibit a clear geographical variation: a 4243-bp Tn3-like transposon and a 9-bp small sequence repeat (SSR). The Tn3-like transposon was only found in the isolates from Mexico and 2 Central American countries, but not in SE Asian isolates. We developed PCR methods to characterize AHPND-V. parahaemolyticus isolates as either Mexican-type or SE Asian-type. In addition, we generated polyclonal antibodies against pirA and PirB, both antibodies were validated by ELISA against AHPND-toxin secreted into the bacterial broth. These antibodies will be used to evaluate the effects of potential therapeutic treatments for AHPND. Objective 2: to determine if sloughing of HP tubule epithelial cells always leads to the secondary massive Vibrio spp. infections. For year 1, we focused on the relationship between EHP (a microsporidia parasite,its infection causes the sloughing of hepatopancreas tubule epitheilial cells) and septic hepatopancreatic necrosis (SHPN, which is a result of secondary Vibrio infections). From histology samples, we performed a case control study to determine the association between SHPN and the EHP. The cases were shrimp displaying histological signs of SHPN and the controls were shrimp from the same ponds without SHPN. The presence of EHP as a risk factor was assessed. A strong association was found between the presence of SHPN-infected shrimp and the presence of EHP-infected shrimp (P<0.05) (OR: 4.2), our data shows that cases of SHPN are more likely to have been affected by EHP than the controls, suggesting severe infections by EHP can increase the susceptibility for other bacterial infections. Objective 3: To determine the pathogen of EHP in P. vannamei, P. mondon, and P. stylirostris. All 3 major species of farmed penaeid shrimp, P. vannamei, P. stylirostris and P. monodon, can be infected by EHP based on histology results. Our laboratory confirmed the EHP infection through in situ hybridization (ISH) using a cloned 18S rRNA gene fragment. This probe was labeled with digoxigenin and hybridized to EHP-infected P. vannamei collected in Vietnam during 2014-2015. The probe reacted intensely to the basophilic inclusions within the cytoplasm. Our P. stylirostris samples collected from Brunei in 2006 also showed the presence of microsporidium spores within the hepatopancreas, where the tubule epithelial cells had a positive reaction by ISH. These EHP detection methods were used in the study of white feces syndrome of P. vannamei,EHP was detected in all the samples analyzed from white feces syndrome-affected ponds, but not in those sampled from healthy shrimp ponds, suggesting thatEHP is associated with white feces syndrome.This EHP 18S rRNA gene clone was patented by University of Arizona and licensed to a Taiwanese Biotechnology company for manufacturing an EHP PCR detection kit. Objective 4: Determine if the reduction of B-cells vesicles in the hepatopancreas is a precursor to infection by EHP. No progress was made in year 1. Objective 5:Confirm if EHP (and when) EHP has entered the Americas. So far, we have not detected EHP, by PCR and histology, in the US yet. But unexpectedly, we detected EHP in samples of frozen Artemia biomass sent from a US provider. From EHP-positive Artemia biomass, its 18S rRNA gene was amplified and sequenced, and the 1.1 kb fragment was 99.9% identical (different in 2 nucleotides) to that of EHP from Vietnam, suggesting the EHP present in Artemia biomass may have originated from SE Asia. Up to date, there are no reports on EHP from Latin America.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Han JE, Tang KFJ, Lightner DV (2015) Genotying of the virulence plasmid harbored in the Vibrio parahaemolyticus isolates causing acute hepatopancreastic necrosis disease (AHPND)in shrimp. Dis Aquat Org. 115: 245-251.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Han JE. Tang KFJ, Pantoja CR, White BL, Lightner DV (2015) qPCR assay for detecting and quantifying a virulence plasmid in acute hepatopancreatic necrosis disease (AHPND)due to pathogenic Vibrio parahaemolyticus. Aquaculture 442: 12-15.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Han JE, Tang KFJ, Pantoja CR, Mohney L, Lightner DV (2015) Plasmid mediated tetracycline resistance of Vibrio parahaemolyticus associated with acute hepatopancreatic necrosis disease (AHPND). Aquaculture Reports 2: 17-21.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Tang KFJ, Pantoja CR, Redman RM, Han JE, Tran LH, Lightner DV (2015) Development of in situ hybridization and PCR assays for the detection of Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp. J Invertebrate Pathology 130: 37-41.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Jun JW, Han JE, Tang KFJ, Lightner DV, Kim J, Seo SW, Cheng C, Giri SS, Yun S, Kim, HJ, Kim SG (2016) Abundant potential of bacteriophage pVp-1 application: agent combating Vibrio parahaemolyticus related to acute hepatopancreatic necrosis disease (AHPND) in shrimp. Aquaculture 457: 100-103
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Tang KFJ, Han JE, Aranguren LF, White-Noble B, Schmidt MM, Piamsomboon P, Risdiana E, Hanggono B (2016) Dense populations of the microsporidian Enterocytozoon hepatopenaei (EHP) in feces of Penaeus vannamei exhibiting white feces syndrome and pathways of their transmission to healthy shrimp. J Invertebrate Pathology 140: 1-7.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Han JE, Tang KFJ, Lightner DV (2016) RECENT STUDIES ON ACUTE HEPATOPANCREATIC NECROSIS DISEASE (AHPND, ALSO KNOWN AS EARLY MORTALITY SYNDROME, EMS). AQUACULTURE 2016, Las Vegas, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Han JE, Tang KFJ, Lightner DV (2016) RECENT STUDIES ON SHRIMP MICROSPORIDIAN INFECTION: ENTEROCYTOZOON HEPATOPENAEI (EHP) AND COTTON SHRIMP DISEASE. AQUACULTURE 2016, Las Vegas, USA.
  • Type: Other Status: Published Year Published: 2016 Citation: Han JE, Tang KFJ, Lightner DV (2016) In situ hybridization and PCR effective at detecting microsporidian parasite. Global Aquaculture Alliance April issue
  • Type: Other Status: Published Year Published: 2016 Citation: Han JE, Tang KFJ, Lightner DV (2016) PCR typing methods based on sequence variation of the virulence plasmid for acute hepatopancreatic necrosis disease (AHPND) in shrimp. Global Aquaculture Alliance May issue.