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1 "Incremento del riesgo de Necrosis Aguda del Hepatopáncreas (AHPND) y Necrosis Séptica del Hepátopacreas (SHPN) en Penaeus vannamei infectados con Enterocytozoon hepatopenaei (EHP) Luis Fernando Aranguren Caro lfarangu@ .arizona.edu Aquaculture Pathology Laboratory OIE Reference Laboratory of Crustacean (Shrimp) Diseases University of Arizona September 28 of 2017

2 AHPND EHP SHPN INTERACTION Agenda

3 World Shrimp Aquaculture EMS/AHPND, EHP, Vibriosis Sources: FAO (2016); GOAL (2014) & GOAL (2016)

4 Shrimp production In the SE Asian countries AHPND No AHPND Sources: FAO (2013); GOAL (2016)

5 Shrimp production In the American countries AHPND No AHPND Sources: FAO (2013); GOAL (2016)

6 Acute Hepatopancreatic Necrosis Disease (AHPND)

7

8 Spread of EMS/AHPND in East and SE Asia ?

9 Spread of EMS/AHPND in the Americas www. Guardian Belize; shrimp news Jun et al Aquaculture Restrepo et al 2016 Genomic data

10 Evolution of Acute hepatopancreatic necrosis disease AHPND EMS AHPNS AHPND Toxin from V. parahaemolyticus Toxin from plasmid in VP (pir A pir B) Toxin from plasmid in V. parahaemolyticus, V. harveyi, V campbellii

11 AHPND An acute phase Direct effect of the toxin HP tubule cells (R, B, F & later E-cells) show acute loss of function. Bacteria not present Acute progressive degeneration of hepatopancreas (HP) from medial to distal with dysfunction of all HP cells, prominent necrosis & sloughing of these tubule epithelial cells.

12 12 Case P. monodon. Vietnam; Severe sloughing & moderate inflammation

13

14 AHPND Terminal phase shows marked inter- & intra-tubular hemocytic inflammation Massive secondary bacterial infections that occur in association with necrotic & sloughed HP tubule cells. 14

15 15

16 Case P. vannamei. USA; Terminal phase of AHPND/ EMS. Most HP tubules are destroyed. Massive bacterial infection by a probable Vibrio spp. 4x.

17 Shrimp microsporidia Enterocytozoon hepatopenaei (EHP) 17

18 Enterocytozoon hepatopenaei (EHP) A microsporidian parasite that has been widely found in Asia and other parts of the world. Recently it was found a similar strain in South America Impacting aquaculture production by severely retarding the growth of cultured shrimp It only infects the tubules of the hepatopancreas in shrimp and anterior midgut It cause chronic mortality in severe cases EHP do not require intermedia host 18

19 Spread of EHP in East and SE Asia

20 Spread of EHP in the Americas 2016

21

22 H&E histology and in situ hybridization of EHP in the infected P. vannamei from an Indonesia shrimp farm. (A) H&E staining of hepatopancreas tissue. (B) In situ hybridization of the consecutive section.

23 SEPTIC HEPATOPANCREATIC NECROSIS (SHPN) Vibriosis in hepatopancreas Reduced feeding. Extreme lethargy; accumulate on pond edges & surface. In severe case cause chronic mortality. Muscle opacity & pigmentation changes. HP atrophied, whitish, often with black spots, streaks.

24 SHPN Vibriosis of HP

25 SHPN Vibriosis of HP

26 Interaction of EHP, AHPND & SHPN These disease are caused by enteric pathogens: Target organ: Hepatopancreas More than one pathogen could be present at the same time in a given population AHPND: EHP: SHPN: Toxin/Vibrio Microsporidio Vibrio spp.

27 Interaction of EHP and AHPND Several SE Asian countries affected by AHPND have been affected in the before by EHP Country EHP (First) AHPND/EMS Thailand Yes Yes Vietnam Yes Yes China Yes Yes Malaysia Yes Yes Indonesia india Philippines Brunei Yes Yes Yes Yes Chayaburakul et al., 2004, Tourtip et al 2009, Ha et al 2010

28 Is EHP a risk factor for AHPND? Pre-infected Experimental group Treatment Dosage (CFU/mL of water) NO AHPND Low dose 2.4 x 10 5 EHP AHPND Low dose 2.4 x 10 5 NO Positive control High dose 2.4 x 10 6 EHP Tank Control EHP Negative control 0 NO Tank Control SPF Negative control 0 Aranguren et al., 2017

29 Survival (%) Negative control Low doses AHPND 80% Low doses EHP-AHPND 56% Positive control First trial % vs 40% Low doses AHPND Low doses EHP-AHPND Second trial Aranguren et al., 2017

30 Histopathology AHPND vs. EHP-AHPND groups Time post infection (h) Group Dosis 0 AHPND Nº shrimp tested AHPND AHPND AHPND 1.5x10 9 UFC/ml AHPND 4 1 (G1) AHPND Lesions 12 AHPND 4 1 (G1) 2/18 (11.1%) 0 EHP/AHPND EHP/AHPND 3 1 (G1) 6 EHP/AHPND 1.5x10 9 UFC/ml 3 2(G1) 9 EHP/AHPND 3 2(G1) 12 EHP/AHPND 3 2(G2) 8/14 (57.1%)

31 0 h.p.i 12 h.p.i 6 h.p.i AHPND A B C EHP AHPND D E F EHP AHPND ISH G H I

32 HP Sloughing EHP & AHPND Both diseases cause HP sloughing The sloughing of epithelial cells from basal membrane (BM) is typical in both cases however the mechanism is different EHP: Infected cells: Depletion of cell nutrients death and detachment from (BM) AHPND: PirAB toxin affect cytoskeletal proteins involved in cell attachment Combination of two mechanisms increase damage to the HP Tissue failure

33 Is EHP a risk factor for SHPN? Shrimp samples from an EHP endemic area In severe cases: EHP affected shrimp ended up with SHPN (Vibriosis in HP) 33

34 Case control analysis: Cases of Septic hepatopancreatic necrosis (SHPN) and association with EHP in juvenile P. vannamei shrimp farming in SE Asia on EHP SHPN (Vibriosis in HP) Control Total Yes No ponds with history of EHP (3 countries) SHPN Aranguren et al., 2017 EHP There is a strong association between SHPN and EHP (P<0.05)

35 Shrimp displaying SHPN presented EHP

36 PERSPECTIVES EHP is a risk factor for SHPN ( HP Vibriosis) EHP is a risk factor for AHPND It is possible that any enteric pathogen is a risk factor for AHPND NHP?

37 Thank you for your attention!

Dr. Celia R. Lavilla-Pitogo

Dr. Celia R. Lavilla-Pitogo Current situation of emerging shrimp diseases in Asia and some recommendations to prevent their arrival and spread Hilton Hotel, Guayaquil, Ecuador September 25-28, 2017 Dr. Celia R. Lavilla-Pitogo celia.pitogo@fulbrightmail.org

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