"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@email.arizona.edu

Aquaculture Pathology Laboratory

OIE Reference Laboratory of Crustacean (Shrimp)

Diseases

University of Arizona

September 28 of 2017

Agenda

• AHPND

• EHP

• SHPN

• INTERACTION

EMS/AHPND, EHP, Vibriosis

Sources: FAO (2016); GOAL (2014) & GOAL (2016)

World Shrimp Aquaculture 1995-2018

Shrimp production In the SE Asian countries

AHPND No AHPND

Sources: FAO (2013); GOAL (2016)

AHPND

No AHPND

Shrimp production In the American countries

Sources: FAO (2013); GOAL (2016)

Acute Hepatopancreatic Necrosis Disease (AHPND)

2009

2010

20112011

20122015

Spread of EMS/AHPND in East and SE Asia

2017

2016?

Spread of EMS/AHPND in the Americas

www. Guardian Belize; shrimp news

Jun et al 2016. Aquaculture 457. 100-103

Restrepo et al 2016 Genomic data 9 143-144

20172013

2015

2015-2017

Evolution of Acute hepatopancreaticnecrosis 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

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.

Case 11-214. P. monodon. Vietnam; Severe sloughing & moderate inflammation12

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.

15

Case 17-327. P. vannamei. USA; Terminal phase of AHPND/ EMS. Most HP tubules are destroyed. Massive bacterial infection by a probable Vibrio spp. 4x.

Shrimp microsporidia

Enterocytozoon hepatopenaei (EHP)

17

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

➢ In the pond affected, more than

18

Spread of EHP in East and SE Asia

Spread of EHP in the Americas

2016

H&E histology and in situ hybridization of EHP in the infected P. vannameifrom an Indonesia shrimp farm. (A) H&E staining of hepatopancreas tissue.

(B) In situ hybridization of the consecutive section.

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.

SHPN – Vibriosis of HP

SHPN – Vibriosis of HP

Interaction of EHP, AHPND & SHPN

➢ These disease are caused byenteric pathogens:

➢ Target organ: Hepatopancreas

➢ More than one pathogen couldbe present at the same time ina given population

➢ AHPND: Toxin/Vibrio

➢ EHP: Microsporidio

➢ SHPN: Vibrio spp.

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 Yes

india Yes

Philippines Yes

Brunei Yes

Chayaburakul et al., 2004,

Tourtip et al 2009,

Ha et al 2010

Is EHP a risk factor for AHPND?

Pre-infectedExperimental

groupTreatment

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 ControlEHP

Negative control0

NO Tank ControlSPF

Negative control0

Aranguren et al., 2017

0

10

20

30

40

50

60

70

80

90

100

Low doses EHP-AHPND

Surv

ival

(%

) 100%

vs

40%

Aranguren et al., 2017

Low doses AHPND

Negative control

Low doses AHPND

80%

Low doses EHP-AHPND

56%

Positive control

First trial

Second trial

Histopathology AHPND vs. EHP-AHPND groups

Time post infection

(h) Group DosisNº shrimp

tested AHPNDAHPND Lesions

0 AHPND

1.5x109 UFC/ml

2 0

3 AHPND 4 0

6 AHPND 4 0

9 AHPND 4 1 (G1)

12 AHPND 4 1 (G1) 2/18 (11.1%)

0 EHP/AHPND

1.5x109 UFC/ml

2 0

3 EHP/AHPND 3 1 (G1)

6 EHP/AHPND 3 2(G1)

9 EHP/AHPND 3 2(G1)

12 EHP/AHPND 3 2(G2) 8/14 (57.1%)

A B C

D E F

G H I

0 h.p.i 12 h.p.i6 h.p.i

AHPND

EHP

AHPND

ISH

EHP

AHPND

HP Sloughing EHP & AHPND

• Both diseases cause HP sloughing• The sloughing of epithelial cells

from basal membrane (BM) istypical in both cases however themechanism is different

• EHP: Infected cells: Depletion ofcell nutrients death anddetachment from (BM)

• AHPND: PirAB toxin affectcytoskeletal proteins involved incell attachment

• Combination of two mechanismsincrease damage to the HP

• Tissue failure

Shrimp samples from an EHP endemic area

33

In severe cases: EHP

affected shrimp ended up

with SHPN (Vibriosis in

HP)

Is EHP a risk factor for SHPN?

Case control analysis:Cases of Septic hepatopancreatic necrosis (SHPN) and

association with EHP in juvenile P. vannamei shrimp farmingin SE Asia on 2015–2016.

SHPN (Vibriosis in HP)

Control Total

EHPYes 17 46 63

No 3 34 37

20 80 100

19 ponds with history of EHP (3 countries)

Aranguren et al., 2017

SHPN EHP

There is a strong

association

between SHPN

and EHP

(P<0.05)

Shrimp

displaying

SHPN presented

EHP

➢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?

PERSPECTIVES

Thank you for your attention!