Shotgun Sequencing of Bacteria from AHPNS
A New Shrimp Disease Threat for Thailand
SUMMARY
The most threatening new problem in Asian shrimp aquaculture at this time is serious mortality from
unknown causes that began in China in 2009, then Vietnam since 2010 and more recently in 2011 to the
eastern coast of Malaysia and the eastern coast of the gulf of Thailand. So far no potential causative
pathogen has been found and possible etiologies include toxins (biotic or abiotic), bacteria and viruses.
A case definition for the syndrome affecting shrimp in the outbreak ponds has been described by D.V.
Lightner from the University of Arizona (unpublished) and this is consistent with the specimens that have
been received and analyzed by Centex Shrimp in Thailand (T.W. Flegel, unpublished). Dr. Lightner has
referred to this syndrome as acute hepatopancreatic necrosis syndrome (AHPNS) that can be described
as acute progressive degeneration of the hepatopancreas (HP) with the following histological features:
lack of mitotic activity in generative E cells of the HP; central dysfuncton of hepatopancreatic B, F and R
cells; prominent karyomegaly and massive sloughing of central HP tubule epithelial cells; terminal
stages including massive untertubular hemocytic aggregation followed by secondary bacterial infections.
To test for the possible involvement of bacteria in causing AHPNS, we carried out shotgun sequencing
of bacterial ssrDNA fragments amplified from HP of shrimp from AHPNS ponds. From analysis of
approximately 100,000 fragments from 6 disease ponds and one control pond, a total of 8327 unique
sequences (taxonomic units or TU) of approximately 400 bp were obtained. Eliminating singleton TU left
1205 TU with reads from 2 or more ponds. A comparison of the read frequency for these yielded 5 TU
with the highest difference between test ponds and the control pond. These sequences will be used to
design specific probes for in situ hybridization assays with HP tissues from AHPNS shrimp.
AHPNS histopathology
Sloughing of HP cells
Lack of E-cell mitosis
Lack of B, F & R cells
INTRODUCTION
Since the serious mortality from unknown causes that began in China in 2009, there have been rumors
that infectious myonecrosis (IMN) caused by the virus IMNV may have been the cause. IMNV was first
described from Indonesia (Senapin et al. 2007. Aquaculture 266:32-38) as a probable example for
disease translocation with aquaculture stocks from Brazil. However, continual testing at Centex Shrimp
has revealed that these have been false rumors (Senapin et al. 2011. BMC J Negative Results in
Biomedicine. 10:10). Most of these rumors were probably caused by muscle cramp syndrome that can
commonly cause whitened muscles in stressed Penaeus (Litopenaeus) vannamei. Additonal rumors
may have resulted from light, false-positive RT-PCR results using the IQ2000 test kit with DNA extracts
from shrimp pleopods rather than excised, infected muscle tissue or hemolymph (i.e., internal material).
Since samples from the outbreaks in Vetnam and China have tested negative for IMNV by RT-PCR,
histopathology and immunohistoghemistry, IMNV can be ruled out as the cause of the problems in
Vietnam and China.
In addition, other pathogens newly discovered in P. vannamei from Thailand and Vietnam [including
Macrobrachium rosenbergii nodavirus (MrNV) and the microsporidian Enterocytozoon hepatopenaei]
have also proven not to be the cause of the massive disease outbreaks based on low prevalence in
tested specimens from the outbreak ponds as revealed by histological analysis, PCR testing and
immunohistochemistry. In addition, tests for nectotizing hepatopancreatic necrosis (NHP) have proven
negative by PCR testing, histological analysis and transmission electron microscopy (TEM).
karyomegaly
Enlarged HP nuclei
Hemocytic infiltration
2o bacterial infection
Semi-thin HP tissue sections
Thai samples of P. vannamei collected from Chantaburi and Rayong provinces in late 2011 and early
2012 showed histological signs of AHPNS and HP tissues were embedded in epoxy resin to prepare for
transmission electron microscopy. In prelmnary semi-thin sections stained with toluidine blue, abnormal
blebbing of the HP tubule margins was seen. Since it occurred in many tubules in the absence of visible
bacteria, supportng Dr. Lightner’s earlier proposal for a possible toxic origin. This phenomenon was
also seen in similar HP tissue sections of AHPNS shrimp specimens from Vietnam and Malaysia,
indicating that the phenomenon could serve as an additional characteristic of the AHPNS case
definition.
We are cooperating with Dr. Niti Chuchird and Dr. Chalor Limsuwan at Kasetsart Universty to investigate
the possibility of novel bacteria that might be present in shrimp from AHPNS ponds using “shotgun”
samples originating from normal ponds and AHPNS ponds. Since this is a new technological approach,
we have already begun with the simplest method of screening for bacterial pathogens. Briefly, the
process involves use of universal bacterial primers to amplify a 500 bp small subunit ribosomal RNA
(ssu rRNA) gene fragment from all bacteria present in shrimp HP tissue samples. These are then
sequenced in mass followed by computer analysis to collate the sequences. The objective is to be able
to compare the sequences from normal and disease ponds to search for any unusual bacteria
characteristic of the disease ponds only.
RESULTS
Sequencing and bioinformatics
Using sets of shrimp pooled from each of 7 ponds (3 diseased and one control pond from Vietnam, and
5 diseased ponds from Thailand), DNA was extracted and used as the template for PCR amplification of
an rRNA gene fragment of an expected size of approximately 500 bp. The amplified bands from each
pond were cut from the agarose gel, eluted from the gel and subjected to DNA barcode addition for each
pond sample. Next, the pooled samples were subjected to Roche 454 sequencing. A total number of 41
million bases were read with an average read of 450 bp yielding approximately 100,000 sequences. At
97% identity for sequences of approximately 400-500 bp with primers at both ends, a total of 8327
unique sequences (taxonomic units or TU) were obtained. Of these, 7123 TU were singletons (i.e.,
arising from only one pond as single or as multiple reads).
Eliminating the singleton TU left 1205 TU with reads from 2 or more ponds. A comparison of the read
frequency for each of the 1205 TU for each of the ponds yielded the following 5 TU with the highest
difference between test ponds and the control pond. The genus names are numbered and shown in
yellow with example species names for Blast hits given in brackets. The latter may or may not be the
actual species in the target samples.
CONCLUSIONS
The bacterial sequences obtained from AHPNS shrimp by shotgun sequencing were not related to
sequences normally reported from diseased shrimp, particularly with respect to Vbrio species. Of the
genera showing significant homology, Delftia was the most prevalent in our samples, but aside from
one report for a new species (Delftia litopenaei) described from normal shrimp in Taiwan (Chen et al.
2011. IJSEM, in press doi:10.1099/ijs.0.037507-0), no species of Delftia has previously been reported
from shrimp. Similarly, none of the other 4 genera from the homology search have previously been
reported from shrimp and all 5 are most frequently reported from water or wastewater samples. Their
possible role in AHPNS will be assessed by preparation of labeled probes for in situ hybridization tests
using AHPNS shrimp samples.
Acknowledgements
Order Burkholderiales:
Family Burkholderiaceae
Genus Ralstonia (1)
Family Comamonadaceae (all found were former Pseudomonas species)
Genus Delftia (acidovorans & tsuruhatensis) (2)
Genus Pelomonas (aquatica) (3)
This work was supported by Mahidol Universty, Charoen Pokphand Co. Ltd., the Higher Education
Research Promotion and National University Development, Office of the Thailand Higher Education
Commission, the Surathani Shrimp Farmer’s Club, the Thai Frozen Foods Association and the National
Research Council of Thailand.