Emerging Diseases of Concerns in Shrimp Aquaculture: Biology, Genomics and Management Practices

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1 Emerging Diseases of Concerns in Shrimp Aquaculture: Biology, Genomics and Management Practices Arun K. Dhar, PhD Aquaculture Pathology Laboratory The University of Arizona Tucson, Arizona, USA

2 Agenda Overview of the Aquaculture Pathology Laboratory, The University of Arizona, Tucson, Arizona, USA Chronology on the emergence of diseases in shrimp OIE-listed diseases in shrimp and their economic impact Emerging diseases in shrimp aquaculture AHPND-Etiology, genomics and pathogen detection EHP-Etiology, genomics and pathogen detection Managing diseases in shrimp aquaculture Evolution of disease management practices in shrimp aquaculture and future perspectives

3 Aquaculture Pathology Laboratory The World Organization for Animal Health Reference Laboratory (Office International des Epizooties, OIE, Paris, France) USDA-APHIS Approved & ISO Accredited Laboratory

4 Aquaculture Pathology Laboratory MISSIONS Shrimp Disease Research- Building 90 Disease diagnostic services to shrimp industry. Educational & Training Services: Shrimp Pathology Short Course. Conducting Inter-laboratory Calibration- Ring Test Basic research in shrimp virology, microbiology & genomics. Shrimp Research Facility- WCAC Disease challenge study, testing therapeutics, feed & feed additives.

5 Aquaculture Pathology Laboratory Disease Diagnostic Services Development of shrimp diseases detection tools Bioassay Histopathology In situ Hybridization Molecular detection PCR Real-time PCR M C ntc M M ntc +C AHPND WSSV 620 bp 325 bp 848 bp 620 bp 325 bp NHP PCR WSSV PCR ntc +C TSV H&E AHPND H&E IHHNV In situ EHP In situ EHP qpcr

6 Aquaculture Pathology Laboratory Educational & Training Services Shrimp Pathology Short Course 2017 Conducted 27 Courses & trained ~1400 researchers worldwide on shrimp disease diagnostics.

7 Aquaculture Pathology Laboratory Shrimp Research Facility : West Campus Agricultural Center Developed challenge models for shrimp viral and bacterial pathogens. Served shrimp industry with disease diagnostics, quarantine facility, screening therapeutics against viral and bacterial diseases, and testing feed and feed additives.

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10 Chronology of Shrimp Pathogen Emergence 1970s 1980s 1990s BP: Baculovirus penaei MBV: Monodon baculovirus IHHNV: Infectious hypodermal & haematopoietic necrosis virus HPV: Hepatopancreatic parvovirus WSSV: White spot syndrome virus YHV: yellow head virus NHP: Necrotizing hepatopancreatitis 1990s 2000s 2010s TSV: Taura MrNV: syndrome virusmacrobrachium rosenbergii nodavirus IMNV: Infectious myonecrosis virus MoV: Mourilyan virus PvNV: Penaeus vannamei nodavirus LSNV: Laem-Singh Virus CMNV: Covert Mortality Nodavirus Vibriosis: Vibrio harveyi, V. damsela, V. alginolyticus, V. vulnificus, V. penaeicida, V. alginoyticus EHP: Enterocytozoon hepatopenaei AHPND: V. parahaemolyticus 10

11 Loss (%) Proportional Losses Attributed to Different Shrimp Pathogens Virus Bacteria Fungi Parasites Pathogen Type Unknown East West Since 2009, AHPND has resulted ~$1.0 billion losses in Asia. Global production loss estimates: Viruses ~65% Bacteria ~20% Viruses are the most important disease agents by almost 3:1 over bacterial diseases. 11

12 OIE Listed Crustacean Pathogens-2017 ( Viral Diseases Infection with yellow head virus Infectious hypodermal and haematopoietic necrosis Infectious myonecrosis Taura syndrome White spot disease White tail disease Bacterial Diseases Acute hepatopancreatic necrosis disease (2016) Necrotising hepatopancreatitis OIE-listed Shrimp Diseases Six viral diseases Two bacterial diseases One fungal disease Fungal Diseases Crayfish plague (Aphanomyces astaci)

13 AHPND Geographic Distribution 2009= China 2010= Vietnam 2011= Malaysia 2012= Thailand 2015= Philippines 2017= Myanmar 2017= Bangladesh 2013= Mexico 2017= USA 13

14 Unusual mortalities in China, 2009 Evolutionary Progress in Identifying the Etiologic Agent of EMS/ AHPND EMS Lightner, 2011 AHPNS NACA, 2012 AHPND Tran et al., 2013 Plasmid encoding binary toxins, pir A & pir B, from V. parahaemolyticus Han et al., 2015 Toxin plasmid can be passed to other bacteria Lee et al., 2015 Toxin from plasmid in V. owensii, V campbellii.. Lieu et al., 2015; Dong et al., 2017

15 Virulence Factor In AHPND: Toxin Genes (pira & pirb) in the Plasmid Vibrio spp. AHPND Vibrio parahaemolyticus. pir A pirab pir B Others.. V. owensii Vibrio campbellii. Modified from Huan et al., 2014

16 Histopathology of AHPND Acute phase >HP tubule cells (R, B, F & later E-cells) show acute loss of function. >Bacteria (of any kind) are not easily demonstrated by in situ hybridization with a 16S rrna universal probe. >Progressive degeneration of hepatopancreas from medial to distal with dysfunction of all HP cells, prominent necrosis & sloughing of these tubule epithelial cells. AHPND-infected: Acute Phase Healthy

17 Histopathology of AHPND Terminal phase: >Shows marked inter- & intra-tubular hemocytic inflammation >Development of massive secondary bacterial infections that occur in association with necrotic & sloughed HP tubule cells. Healthy AHPND-infected: Terminal Phase

18 Causative agents of AHPND V. parahaemolyticus, V. owensii, V. campbellii, Vibrio spp. Halophilic bacteria Common bacteria in marine /brackish water environments Facultative anaerobic Temp: o C Salinity:10-40 ppt High replication rate (higher than E. coli) Vp does not use Sucrose as source of carbohydrate (*). Green color in TCBS

19 AHPND detection by PCR V. parahaemolyticus Transposase 1 ORF23 pir A ORF24 pir B 3462 nt Transposase pirb-392-bp pira amplicon (284-bp) pirb amplicon (392-bp) 69 kb Plasmid pira-284-bp

20 Pathogenic vs. Non pathogenic strains of AHPND Pathogenic strain Non-Pathogenic strain

21 Evolution of AHPND detection by PCR pirb-392-bp pira-284-bp R14: Both pir A and pir B positive but non- pathogenic Confirm by histopathology Bacterial challenge test Determine AHPND by alternative method?

22 Comparison of the toxin gene loci of pathogenic and non-pathogenic Vibrio parahaemolyticus isolates Genetic Features A3-1 R13 R14 XN81 Size (kb) pir A pir B + Truncated (nt ) + + Deletion/Insertion - Deletion Insertion Insertion Mortality (%)/ Histopathology 100/ Yes 0/ No 0/ No 47/ No A3-1 pira pirb R-14 pira pirb R kb del. pirb XN87 pira pirb

23 Whole Genome Sequences of V. parahaemolyticus Isolates R13 and R-14 and Comparison with Reference Strain A3 Instruments: Pacific Biosciences RSII System, Library Size: 20 kb, Sequence Coverage: X Strain Size (MB) GC Content # Coding Sequence # RNA (trna, 5S RNA) Plasmids (kb) Vp-A3 I R , 55, 19 R , 55, 19

24 Comparative Genomics in V. parahaemolyticus V. parahaemolyticus RMID (Ref. Strain: Human Clinical ) Gene Annotation & Category Distribution Vp R-13 Vp R-14 Cofactors, Vitamins, Prosthetic Groups, Pigments (331) Cell Wall and Capsule (163) Virulence, Disease and Defense (106) Potassium metabolism (45) Photosynthesis (0) Miscellaneous (43) Phages, Prophages, Transposable elements, Plasmids (4) Membrane Transport (262) Iron acquisition and metabolism (65) RNA Metabolism (242) Nucleosides and Nucleotides (107) Protein Metabolism (307) Cell Division and Cell Cycle (55) Motility and Chemotaxis (169) Regulation and Cell signaling (125) Secondary Metabolism (4) DNA Metabolism (145) Fatty Acids, Lipids, and Isoprenoids (147) Nitrogen Metabolism (51) Dormancy and Sporulation (5) Respiration (140) Stress Response (188) Metabolism of Aromatic Compounds (12) Amino Acids and Derivatives (547) Sulfur Metabolism (34) Phosphorus Metabolism (56)

25 Why Study Comparative Genomics of Vibrio parahaemolyticus? To compare genome structure of pathogenic vs. nonpathogenic strains Factors responsible for virulence or avirulence To predict the role of DNA ( e.g. Transposon/Mobile Elements) and bacterial protein in pathogenicity How bacterial chromosomally encoded protein(s) interact with extra-chromosomally (plasmid) encoded protein(s) To study the role of bacterial immune factors (e.g. T6SS) in AHPND pathogenicity.

26 Enterocytozoon hepatopenaei (EHP) First described in Penaeus monodon from Thailand. Severely retarded growth of cultured shrimp & Size Variability. It infects only the tubules of the hepatopancreas, which damages the ability of this critical organ to gain nutrition from feed. Causes chronic mortality in severe cases. EHP Infection increases susceptibility to AHPND and secondary vibriosis (Aranguren et al., 2016). 26

27 EHP GEOGRAPHIC DISTRIBUTION EHP GEOGRAPHIC DISTRIBUTION NHP GEOGRAPHIC DISTRIBUTION (YEAR 2003) 5 27

28 Histopathology of EHP Infection in P. vannamei shrimp- Giemsa Staining EHP Spores

29 H& E Histology of Hepatopancreas in P. vannamei shrimp with EHP Infection EHP Spores EHP Spores Epithelial cells about to slough off in the lumen Nucleus

30 Transmission Electron Microscopy of EHP Photo Credit: Kanokporn Chayaburakul

31 EHP Genomics: A Novel Pathogen EHP genome has been sequenced recently (Boakye et al., 2017). EHP Genome size 3.26 Mb, G+C% Content 25.45%, Contains 2540 genes and ~71% of the genome has protein coding capacity. Genome sequence revealed that EHP parasite does not have all the genes involved in glycolysis pathways (mechanism for producing energy in a cell); thus depend on it host for energy (i.e. ATP). EHP Genome contains genes for ATP/ADP translocases that are involved in transport of energy from host cell. It has been proposed that EHP enters host cells by phagocytosis; if translocases are expressed upon phagocytosis, there will readily available energy for the spores to geminate.

32 PCR Detection of EHP in Penaeus vannamei Hepatopancreas Tissue, Feces and Tank Water

33 Management of Emerging Diseases in Shrimp Aquaculture Good management practices including routine sanitation and biosecurity are key tools for management. Use of SPF broodstocks & PLs [and SPR-lines, if available] Pond management: Avoid high concentration of organic matter /sediment in the pond Water exchange to reduce organic matter, lined pond Low stocking density, Biofloc Chemotherapeutics, probiotics, prebiotics, organic acids, immunostimulants & many other products are in common use. 33

34 Host-Pathogen Evolution in Shrimp Diseases Resistance Disease Host Evolutionary Dance of Host & Pathogen Pathogen Susceptibility No Disease Managing a disease with (in endemic areas) or without a pathogen (in disease free area) should be the goal.

35 Evolution in Disease Management in Shrimp Aquaculture Technological advancement and its impact on shrimp disease control Identifying etiology of shrimp diseases a& developing detection methods Bioassay, Pathogen Culture, Histopathology Immunoassay PCR, qpcr Feed & Feed Ingredients High quality feed Feed additives, Probiotics, prebiotics, Antibiotics Functional feed (e.g. AHPND Control feed) Genetic Improvements Development of SPF lines of P. vannamei Efforts in developing SPR lines of P. vannamei Breeding program in P. monodon & M. rosenbergii Genomics, Nutrigenomics & Therapeutics Genomic applications to develop SPR lines Application of nutrigenomics and functional feed Development of therapeutics (alternatives to antibiotics) Microbial management in the pond Past & Present Efforts in Disease Management Future Efforts

36 Thank You