Acute hepatopancreatic necrosis disease (AHPND) outbreaks in Penaeus vannamei and P. monodon cultured in the Philippines

Abstract

Acute hepatopancreatic necrosis disease (AHPND) has recently emerged as a serious disease of cultured shrimp. It has also been described as early mortality syndrome (EMS) due to mass mortalities occurring within 20 to 30 d after stocking of ponds with postlarvae. Here, Penaeus vannamei and Penaeus monodon from shrimp farms in the Philippines were examined for the toxin-producing strain of Vibrio parahaemolyticus due to AHPND-like symptoms occurring in marketable size shrimp. In the P. vannamei, histology revealed typical AHPND pathology, such as sloughing of undifferentiated cells in the hepatopancreatic tubule epithelium. Analysis using the IQ2000 AHPND/EMS Toxin 1 PCR test generated 218 bp and 432 bp amplicons confirmative of the toxin-producing strain of V. parahaemolyticus among shrimp sampled from 8 of 9 ponds. In the P. monodon, histology revealed massive sloughing of undifferentiated cells of the hepatopancreatic tubule epithelium in the absence of basophilic bacterial cells. PCR testing generated the 2 amplicons confirmatory for AHPND among shrimp sampled from 5 of 7 ponds. This study confirms the presence of AHPND in P. vannamei and P. monodon farmed in the Philippines and suggests that the disease can also impact late-stage juvenile shrimp.

Full text

DISEASES OF AQUATIC ORGANISMS
Dis Aquat Org
Vol. 116: 251–254, 2015
doi: 10.3354/dao02919 Published October 27
A disease described initially as early mortality syn-
drome (EMS), and subsequently as acute hepa to -
pancreatic necrosis disease (AHPND) based on its
characteristic histopathology, has recently been the
cause of mass mortalities and significant production
losses of shrimp farmed in China since 2009, Vietnam
since 2010, Malaysia since 2011 and Thailand since
2012 (Bondad-Reantaso et al. 2012, Lightner et al.
2012, Joshi et al. 2014). Shrimp affected by AHPND
characteristically display a pale to white discoloured
hepatopancreas as well as atrophy of > 50% of the
organ mass. Due to melanin deposits forming as a
result of hemocyte activity, black spots in the hepa -
topancreas become visible in the terminal phase of
disease (Tran et al. 2013). Disease signs associated
with mortality typically appear within 20 to 30 d after
stocking ponds with postlarvae (Lightner et al. 2012).
The causative agent for AHPND has been identi-
fied as a toxin-producing strain of Vibrio parahaemo -
lyticus (Tran et al. 2013). V. parahaemolyticus strains
© Inter-Research 2015 · www.int-res.com*Corresponding author: leobertd@seafdec.org.ph
NOTE
Acute hepatopancreatic necrosis disease (AHPND)
outbreaks in Penaeus vannamei and P. monodon
cultured in the Philippines
Leobert D. de la Peña1,*, Nikko Alvin R. Cabillon1, Demy D. Catedral1,
Edgar C. Amar1, Roselyn C. Usero2, Wilberto D. Monotilla3, Adelaida T. Calpe4,
Dalisay DG. Fernandez4, Cynthia P. Saloma3
1Aquaculture Department, Southeast Asian Fisheries Development Center (SEAFDEC/AQD), Tigbauan, Iloilo 5021,
Philippines
2Negros Prawn Producers Cooperative (NPPC), Bacolod City, Negros Occidental 6100, Philippines
3Philippine Genome Center (PGC), University of the Philippines Diliman, Quezon City 1101, Philippines
4Philippine Council for Agriculture, Aquatic and Natural Resources Research and Development (PCAARRD),
Paseo de Valmayor, Timugan Los Baños, Laguna 4030, Philippines
ABSTRACT: Acute hepatopancreatic necrosis disease (AHPND) has recently emerged as a seri-
ous disease of cultured shrimp. It has also been described as early mortality syndrome (EMS) due
to mass mortalities occurring within 20 to 30 d after stocking of ponds with postlarvae. Here,
Penaeus vannamei and Penaeus monodon from shrimp farms in the Philippines were examined
for the toxin-producing strain of Vibrio parahaemolyticus due to AHPND-like symptoms occurring
in marketable size shrimp. In the P. vannamei, histology revealed typical AHPND pathology, such
as sloughing of undifferentiated cells in the hepatopancreatic tubule epithelium. Analysis using
the IQ2000 AHPND/EMS Toxin 1 PCR test generated 218 bp and 432 bp amplicons confirmative
of the toxin-producing strain of V. parahaemolyticus among shrimp sampled from 8 of 9 ponds. In
the P. monodon, histology revealed massive sloughing of undifferentiated cells of the hepatopan-
creatic tubule epithelium in the absence of basophilic bacterial cells. PCR testing generated the 2
amplicons confirmatory for AHPND among shrimp sampled from 5 of 7 ponds. This study confirms
the presence of AHPND in P. vannamei and P. monodon farmed in the Philippines and suggests
that the disease can also impact late-stage juvenile shrimp.
KEY WORDS: AHPND · P. vannamei · P. monodon · PCR · Histopathology · Philippines
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Dis Aquat Org 116: 251–254, 2015
are found commonly in diseased P. monodon (Ruang-
pan & Kitao 1991) and, together with other Vibrio
species, have been associated with red disease syn-
drome (Alapide-Tendencia & Dureza 1997). To gether
with V. alginolyticus and Pseudomonas sp., V. para-
haemolyticus strains have also been associated with
bacterial septicaemia (Anderson et al. 1988). More-
over, a strain of V. parahaemolyticus has been associ-
ated with red-leg disease characterized by an expan-
sion of red chromatophores in the swimmerets and
walking legs of P. monodon (Chen 1992). V. parahae -
mo lyticus has also been isolated in low numbers from
haemolymph sampled from healthy P. vannamei
(Gomez-Gil et al. 1998).
EMS/AHPND was first reported in China in 2009.
However, it was ignored initially until disease out-
breaks became more serious early in 2011, resulting
in ~80% production losses in the provinces of Hai -
nan, Guangdong, Fujian and Guangxi (Leaño & Mo -
han 2012). Later in 2011, AHPND outbreaks occurred
in P. vannamei farmed in Malaysia that resulted in
significant production losses valued at 100 million
USD (FAO 2013), and unprecedented losses of
farmed P. monodon valued at 1.5 trillion Vietnamese
Dong occurred in the province of Soc Trang in the
Mekong Delta in Vietnam (Lyon et al. 2013). The
onset of AHPND in Thailand in 2012 to 2013 de -
creased shrimp production ~54% through a combi-
nation of disease and a reluctance of farmers to stock
ponds due to the fear of disease. Up to 2013, direct
and indirect losses to the Asian shrimp culture sector
due to AHPND have been estimated to be in the
order of 1 billion USD (FAO 2013). Moreover, since
2013, several countries have suspended or banned
imports of live shrimp and shrimp products from
countries affected by AHPND. In the Philippines, the
Bureau of Fisheries and Aquatic Resources (BFAR)
has also banned im ports of other crustaceans that
might act as carriers of AHPND.
Between 2013 and 2014, production volumes of
P. vannamei and P. monodon in the Philippines de -
creased due to disease outbreaks primarily in Lu zon
and the Visayas areas (Bureau of Agricultural Statis-
tics 2014) suspected to have been caused by AHPND.
The present study was undertaken to identify
whether AHPND contributed to this lost production.
P. monodon were sampled from a farm in late 2014
when production of P. vannamei and P. monodon
notably decreased, and P. vannamei were sampled in
early 2015 from farms in Bohol experiencing disease
problems (Bureau of Agricultural Statistics 2014). At
the P. monodon farm, mortality rates of up to 40%
occurred after 56 to 94 d of culture (DOC) among
ponds stocked at a density of 23 shrimp m−2. At the
P. vannamei farm, mortality rates of up to 60%
occurred after 56 to 94 DOC among ponds stocked at
50 shrimp m−2. The numbers of dead shrimp recov-
ered at the pond bottom increased daily, and clinical
signs of AHPND, including weakness as well as pale
to white discoloration and atrophy of the hepatopan-
creas, coincided with the mortalities. After sampling,
the affected ponds were emergency harvested.
Groups of 10 moribund P. vannamei were sampled
from each of 9 ponds, and groups of 5 moribund
P. monodon were sampled from each of 7 ponds
(Table 1). Tissue samples for PCR were either frozen
at −80°C or fixed in 95% ethanol and stored at 4°C,
and samples for histology were fixed in Davidson’s
fixative and transported to the Southeast Asian Fish-
eries Development Center Aquaculture Department
(SEAFDEC/AQD), Tigbauan, Iloilo, Philippines, for
processing.
Among the moribund P. vannamei and P. monodon
examined (Figs. 1 & 2), histology on 5 to 7 µm thick
sections of hepatopancreas tissue stained with haema -
toxylin and eosin (H&E) revealed manifestations of
typical AHPND pathology (Tran et al. 2013). These
manifestations included severe tissue necrosis asso-
ciated with sloughing of undifferentiated cells of he -
pato pancreatic tubules and the absence of baso philic
bacterial cells. Among the P. vanna mei, histopathol-
ogy was evident in 4 of 6 shrimp examined from
Pond 16 and 5 of 6 shrimp examined from Pond 27.
252
Pond DOC ABW (g)
Penaeus vannamei
17110
7719
12 94 12
13 94 13
16 58 7
19 56 7
20 60 6
21 63 7
27 67 8
Penaeus monodon
33 66 15
35 60 14
36 60 14
37 40 6
38 46 6
40 52 8
41 40 7
Table 1. Pond details of the P. vannamei and P. monodon
from a farm in Bohol, Philippines, sampled in early 2015 and
late 2014, respectively. DOC: days of culture; ABW: average
body weight
Author copy

de la Peña et al.: AHPND in Philippine cultured shrimp
Among the P. monodon, histo pa thology was evident
in all 5 shrimp examined from Ponds 33 and 38.
Using DNA extraction and PCR methods recom-
mended for the IQ2000 AHPND/EMS Toxin 1 Detec-
tion and Prevention System (GeneReach Bio tech nol -
ogy, Taiwan), the presence of V. para haemo lyticus
(432 bp amplicon) together with the toxin- producing
plasmid (218 bp amplicon) associated with AHPND
was evident among P. vannamei sampled from 8 of 9
ponds and P. monodon sampled from 5 of 7 ponds
(Fig. 3).
Based on the observed histopathology and PCR
data, the presence of AHPND was confirmed for the
first time to be affecting P. vannamei and P. monodon
being cultured in the Philippines. However, unlike
classical AHPND in which disease commonly arises
within 20 to 30 d of ponds being stocked with postlar-
vae (Lightner et al. 2012), disease symptoms and
mortality were identified here to be present signifi-
cantly later in the culture cycle, between 46 and 96 d
after pond stocking. While other factors might have
been involved in the mortalities, our data suggest
that older shrimp life stages remain susceptible to the
disease.
Acknowledgements. We are very grateful to DOST-PCAARRD
and SEAFDEC/AQD for funding this work and to the
Shrimp Pathogenomics team, especially Dr. N. R. L. Rojas
and Dr. E. P. Enriquez. We thank the shrimp farms that pro-
vided the samples and the staff of Fish Health Section Diag-
nostic Services and Microtechnique Lab for the technical
support. We also thank T. W. Flegel for confirming our histo-
logical analysis and for assistance in editing the manuscript.
253
Fig. 1. H&E stained histological sections of the hepatopancreas of P. vannamei (Pond 27, 67 DOC) sampled from a farm in
Bohol. (A) Normal shrimp hepa topancreatic histology; (B) AHPND pathology characterized by sloughing of undifferentiated
epithelial cells of the hepatopancreatic tubule epithelia. Scale bars = 100 µm
Fig. 2. H&E stained histological sections of the hepatopancreas of moribund P. monodon sampled from a farm in Bohol. (A)
Massive necrotic sloughing of tubule epithelial cells in the hepatopancreas (Pond 38, 46 DOC); (B) typical AHPND at the acute
stage of disease showing sloughing of tubule epithelial cells in the absence of basophilic bacterial cells (Pond 33, 66 DOC).
Scale bars = 50 µm
Author copy

Dis Aquat Org 116: 251–254, 2015
254
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Editorial responsibility: Jeff Cowley,
Brisbane, Queensland, Australia
Submitted: June 4, 2015; Accepted: August 25, 2015
Proofs received from author(s): October 9, 2015
Fig. 3. Agarose gel electrophoresis of DNA products amplified using the IQ2000 AHPND PCR test. (A) P. vannamei samples,
DNA marker (Lane 1), AHPND positive control (Lane 2), Ponds 1, 7, 12, 13, 16, 19, 20, 21 and 27 (Lanes 3 to 11, respectively)
and negative control (Lane 12). (B) P. monodon samples, DNA marker (Lane 1), AHPND positive control (Lane 2), Ponds 33,
35, 36, 37, 38, 40 and 41 (Lanes 3 to 9, respectively) and negative control (Lane 10). The AHPND DNA positive control con-
sisted of a plasmid containing the AHPND toxin 1 gene and an AHPND plasmid pAP1 marker that yielded PCR amplicons of
218 bp and 432 bp, respectively. Non-virulent isolates of V. parahaemolyticus that carry the AHPND plasmid pAP1 containing
no toxin gene generate the 432 bp amplicon only. Negative samples generate an internal 712 bp control amplicon only to con-
firm the integrity of the shrimp DNA extract
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