http://www.fao.org/documents/card/en/c/28b6bd62-5433-4fad-b5a1-8ac61eb671b1/ FAO Second International Technical Seminar/Workshop on Acute hepatopancreatic necrosis disease (AHPND) There is a way forward! FAO Technical Cooperation Programme: TCP/INT/3501 and TCP/INT/3502.

1. Update June 2016
on EMS/AHPND
and EHP research
in Thailand
Tim Flegel
tim.flegel@gmail.com
Centex Shrimpa member of NSTDA

2. Dividing Penaeus into 6 genera is invalid
 I have consistently opposed sub-division of
Penaeus into six genera
– Flegel, T.W., 2007. The right to refuse revision in the genus Penaeus.
Aquaculture. 264, 2-8.
– Flegel, T.W., 2008. Confirmation of the right to refuse revision in the
genus Penaeus. Aquaculture. 280, 1-4.
 It is scientifically unsupportable and must be
abandoned

3. Acute hepatopancreatic necrosis
disease (AHPND)

4. AHPND is only part of EMS, not = EMS
AHPND by histology
or PCR
No AHPND but
bacterial
lesions
Early mortality
but No HP
lesions
Collapsed HP
epithelium
Normal HP
03, 06 07, 11, 12, 16,
17, 19, 20, 32, 40, 47,
50, 52, 54, 74, 84, 91,
93, 110, 112, 116, 119,
122, 126, 131, 136,
144, 150, 156, 160,
161, 162, 166, 167,
168, 169, 172, 181,
191, 199, 200
04, 05, 21, 24,
44, 45, 51, 58,
69, 75, 79, 86,
87, 88, 96, 99,
101, 117, 130,
133, 139, 143,
170, 174, 186,
192, 196, 197,
198
39, 57, 70, 98,
100, 111, 194
02, 15, 25, 26, 29,
30, 37, 38, 46, 48,
49, 53, 64, 65, 76,
80, 81, 82, 83, 90,
95, 97, 104, 109,
113, 114, 115, 123,
125, 127, 128, 134,
135, 158, 159, 163,
165, 171, 173, 175,
178, 179, 182, 184,
185, 187, 188, 189,
193
01, 08, 09, 10, 13, 14, 18,
22, 23, 27, 28, 31, 33, 34,
35, 36, 41, 42, 43, 55, 56,
59, 60, 61, 62, 63, 66, 67,
68, 71, 72, 73, 77, 85, 89,
92, 94, 102, 103, 105, 106,
107, 108, 118, 120, 121,
124, 129, 132, 137, 138,
140, 141, 142, 145, 148,
149, 152, 153, 154, 155,
157, 164, 176, 177, 180,
183, 190, 195
42/196
(21.4%)
29/196
(14.8%)
7/196
(3.6%)
49/196
(25.0%)
69/196
(35.2%)
Mortality <35 days (study definition of EMS = 29/196 = 14.8%)
Infected with the microsproidian E. hepatopenaei (EHP)(PCR)(119/196 = 61%)
Infected with white spot syndrome virus (WSSV)(PCR) # = 8/196 = 4%
Covert mortality nodavirus (CMNV) (PCR) = 64/148 = 43%
Aggregated, transformed microvilli (ATM) (Histology) 153/196 = 78%
Altogether 130/196 ponds not normal = 66.3%
Specimens 146, 147, 151 un-readable (poor fixation) and 78 no histology

5. AHPND spread since June 2015
 OIE report of two outbreaks in northern Australia
with Pirvp toxin genes in the chromosome
 Rumors of unreported outbreaks in India and
Central America other than Mexico
 Report of an isolate (tentatively V. harveyi) with
pAP1 plasmid containing Pirvp toxin genes (Kondo
et al. 2015. Genome Announc. 3, e00978-00915)
 Shows that transfer to other Vibrio species is
possible and perhaps other genera as well
 pAP1 found in many VP serotypes in Thailand
(Chonsin et al. 2015. FEMS microbiology letters, fnv222; Kongrueng et al.
2015. J Fish Dis. 38, 957-966)
 A Malaysian colleague apparently has isolates
of 4 other Vibrio species that carry pAP1

6. Medial sloughing of HP cells
Normal
hepatopancreas
AHPNS
hepatopancreas
(pathognomic lesion)
The key diagnostic feature needed for confirmation

7. Variation in VP virulence on a single farm
(Joshi et al. 2014. Aquaculture 428–429: 297-302)
Including 2HP with 50% mortality but no AHPND histopathology

8. VP Isolate 2HP
 50% mortality but not
AHPND pathology;
instead collapsed
epithelia
 Later found to give a
positive PCR reaction
with AP2 but negative
with AP4
 This indicated
presence of pAP1
plasmid but absence
of the Pirvp toxin genes
(Joshi et al. 2014. Aquaculture 428–429: 297-302)

9. Collapsed epithelia with VPAHPND 5HP
Also seen with diluted AHPND isolate 5HP in immersion
bioassays (106, 105, 104 103/ml)
(Joshi et al. 2014. Aquaculture 428–429: 297-302)

10. Collapsed epithelia with dilute Pir toxins
 Both Pir toxins are needed to cause AHPND
 Diluted, expressed Pir toxins also cause collapsed HP
tubule epithelia (Sirikharin et al. 2015. PLoS ONE. 10, e0126987)
5 µg each
collapsed epithelia
10 µg each
AHPND sloughing

11. AHPND potentiating factors
 Our expressed proteins caused AHPND at 10 µg
per g shrimp each (total 20 µg)
 But, (NH4)2SO4 precipitate from culture broth
required only 1 µg/g shrimp (20x less)
 Thus, other broth components must potentiate
ToxA and ToxB virulence
 Two additional proteins in the (NH4)2SO4
precipitate are being examined
 They occur only in our 3 VPAHPND isolates and
not in a non-AHPND VP isolate
 It is important to identify such potentiators and
determine if they play any role in AHPND

12. pAP1 Plasmid other toxin genes?
(Han et al. 2015. Dis Aquat Org. 113, 33-40)
Chromosomal
toxin genes
too?

13. A virulent AP plasmid (pAP) mutant
Dr. Saengchan Senapin
at Centex Shrimp
working with Dr. Ha T.
Dong from Vietnam
A Vietnam VP isolate
XN87 gave unusual PCR
amplicons with the AP4
detection method
Unlike the other typical
VPAHPND isolates
It gave the B toxin gene
amplicon but not the A
toxin gene amplicon

14. XN87 PirvpA/B toxin region amplified
XN89 gave the
expected 2020
amplicon
XN87 gave an
amplicon more than
1000 bp larger
Sequencing revealed
that it contained a
transposable element
inserted into the
coding sequence of
the A toxin
Primers upstream of A and downstream of B coding

15. Diagram of the XN87 mutant genes
The sequence explained the unusual PCR amplicons
from the Pir A/B region of pAP and suggested that B toxin
alone might still be produced by XN87

16. No Pir mRNA from XN87
VPAHPND isolate 5HP produces a bicystronic mRNA for
the Pir A/B toxins while XN87 does not. This suggests that
the toxins are translated as a single protein and then
cleaved after synthesis

17. No Pirvp A or B toxins in XN87 cultures
Pirvp A and B toxins are absent in the concentrated
ammonium sulfate precipitate from broth of XN87
by Coomasie-stained gel and by western blotting

18. XN87 bioassays
50% mortality with XN87 but no AHPND pathology, only
some regions of collapsed HP epithelia in moribund
shrimp. A good candidate to study other toxins
The pathology and mortality resembles that of Thai 2HP, so its
pAP1may be mutated similar to that of Vietnamese mutant XN87

19. Conclusions for AHPND
 A range of bacterial isolates of variable virulence
carry the pAP1 plasmid (“pAP1 on the move”!)
 Some produce Pirvp toxins but vary in virulence that
needs to be explained
 Some produce no Pirvp toxins nor cause AHPND
pathology but still cause significant mortality
 Do we need to change the case definition of
AHPND to include these isolates?
 Is virulence based on plasmid genes only?
 pAP1 is now present in many serotypes of VP in
Thailand (Chonsin et al. 2015. FEMS microbiology letters, fnv222;
Kongrueng et al. 2015. J Fish Dis. 38, 957-966)
 Other speakers have covered AHPND control
 Dr. Kallaya will discuss VPAHPND partner bacteria

20. Hepatopancreatic
microsporidiosis (HPM) caused by
Enterocytozoon hepatopenaei
(EHP)

21. Emerging microsporidian in HP
 First seen in 2001 in the HP of P. monodon
(Chayaburakul et al. 2004. Dis Aquat Org 60:89-96)
 The disease is called hepatopancreatic
microsporidiosis (HPM)
 Considered to be of no impact at the time
 The causative agent was named
Enterocytozoon hepatopenaei (EHP)(Tourtip et al.
2009. J. Invertebr. Pathol. 102, 21-29)
 Now reported from China, Vietnam, Thailand,
India; anecdotal, Indonesia, Malaysia, Australia
 Probably endemic in Australasia but not yet
reported from some countries

22. Surprised by high Thai prevalence 2014
AHPND by histology
or PCR
No AHPND but
bacterial
lesions
Early mortality
but No HP
lesions
Collapsed HP
epithelium
Normal HP
03, 06 07, 11, 12, 16,
17, 19, 20, 32, 40, 47,
50, 52, 54, 74, 84, 91,
93, 110, 112, 116, 119,
122, 126, 131, 136,
144, 150, 156, 160,
161, 162, 166, 167,
168, 169, 172, 181,
191, 199, 200
04, 05, 21, 24,
44, 45, 51, 58,
69, 75, 79, 86,
87, 88, 96, 99,
101, 117, 130,
133, 139, 143,
170, 174, 186,
192, 196, 197,
198
39, 57, 70, 98,
100, 111, 194
02, 15, 25, 26, 29,
30, 37, 38, 46, 48,
49, 53, 64, 65, 76,
80, 81, 82, 83, 90,
95, 97, 104, 109,
113, 114, 115, 123,
125, 127, 128, 134,
135, 158, 159, 163,
165, 171, 173, 175,
178, 179, 182, 184,
185, 187, 188, 189,
193
01, 08, 09, 10, 13, 14, 18,
22, 23, 27, 28, 31, 33, 34,
35, 36, 41, 42, 43, 55, 56,
59, 60, 61, 62, 63, 66, 67,
68, 71, 72, 73, 77, 85, 89,
92, 94, 102, 103, 105, 106,
107, 108, 118, 120, 121,
124, 129, 132, 137, 138,
140, 141, 142, 145, 148,
149, 152, 153, 154, 155,
157, 164, 176, 177, 180,
183, 190, 195
42/196
(21.4%)
29/196
(14.8%)
7/196
(3.6%)
49/196
(25.0%)
69/196
(35.2%)
Mortality <35 days (study definition of EMS = 29/196 = 14.8%)
Infected with the microsproidian E. hepatopenaei (EHP)(PCR)(119/196 = 61%)
Infected with white spot syndrome virus (WSSV)(PCR) # = 8/196 = 4%
Covert mortality nodavirus (CMNV) (PCR) = 64/148 = 43%
Aggregated, transformed microvilli (ATM) (Histology) 153/196 = 78%
Altogether 130/196 ponds not normal = 66.3%
Specimens 146, 147, 151 un-readable (poor fixation) and 78 no histology

23. Significant correlations by 2 test
AHPND Bacterial
lesions
EMS but no
HP lesions
Collapsed HP
epithelia
Normal
HP
Observed 59 62 14 75.6 47.8
Expected 50 50 50 50 50
χ2
1.62 2.88 25.92 13.12 0.097
p value 0.20 0.90 3.6 e-7
0.0003 0.76
The frequency of HPM in the EMS group with normal HP
histology is Significantly lower than expected.
The frequency of HPM in the collapsed epithelium group is
significantly higher than expected.
Note that this means collapsed epithelia (seen also with
AHPND) have low diagnostic value for either AHPND or HPM

24. Review of what we know about EHP
 SPF stocks of P. monodon and P. vannamei can be
infected in hatcheries and produce infected PL
 Live feeds like polychaetes and mollusks probably
transmit EHP to broodstock (killed at -20 Celcius for 48 h)
 Uninfected PL can be infected after stocking
ponds, especially in former HPM ponds
 We have effective PCR tools and pond
management protocols to reduce its impact
 No effective therapeutic methods are currently
known
 Recommendations for control in hatcheries and
ponds at the NACA web site www.enaca.com

25. Laboratory infection models
 EHP can be transmitted by feeding infected
HP tissue to naïve shrimp
 It can also be transmitted by co-habitation of
naïve shrimp separated from infected shrimp
 The co-habitation model may be best to test
preventative treatments
 So far, transmission by oral administration of
purified spores from HP tissue has failed

26. What is the impact of EHP
 May severely retard shrimp growth
 Degree of retardation is proportional to
severity of infection as measured by qPCR (Liu
et al. 2016. Prog Fish Sci. In press. In Chinese, Abstract in English)
 Infection up to 103 copies/µg total DNA by
PCR seems to have no negative effect
 Above this level, retardation is directly
proportional to copy number
 Copy numbers above 107 per µg DNA may
give slow cumulative mortality
 Infections may predispose shrimp to bacterial
pathogens (but may protect against AHPND?)

27. Diagnosis of HPM
 No clear gross signs for diagnosis but may be
suspected with severe growth retardation
 Diagnostic confirmation requires histological
analysis or molecular methods
 Histological diagnosis depends on finding the
presence of EHP spores in HP tissue
 This is difficult by light microscopy because:
– Spores are very small and a 100x lens is required
– Sometimes spores are present in low numbers, even
with heavy infections

28. Diagnosis by wet mount
 Place a drop of a 1-2% Phloxine-B stain in
distilled water on a microscope slide
 Remove tissue from the middle of the HP of a
suspected shrimp specimen
 Put it in the staining solution, macerate it with
a needle and then remove any large pieces
 Cover with a coverglass and inspect using a
100x objective lens
 Slightly egg-shaped spores of about 1.7 x 0.9µ
indicate the presence of EHP

29. EHP spores in a wet mount
Egg-shaped to elliptical spores ~ 1.7 x 0.9 µ (microns)
indicate presence of EHP (larger than in dehydrated sections)
Stained with 2% phloxine B

30. Diagnosis of HPM by H&E staining
 Use the 100x objective lens with H&E stained
tissue sections or faster HP smears
 A major problem is that spores are sometimes
produced in low quantities, even in heavily
infected specimens
 Spores are about 1.1 x 0.7 µm from shrinkage
during tissue processing (smaller than in wet mounts)

31. H&E stained section
Histology of HPM
shrimp by H&E

32. Also chromotrope stain
 First used for EHP by Tourtip et al. (2009. J.
Invertebr. Pathol. 102, 21-29)
 Obtained from Weber et al. (1992. N Engl J Med.
326, 161-166)
 A more rapid “hot” method was developed
later (Moura et al. 1997. Arch Pathol Lab Med. 121, 888-
893)
 These methods reveal spores better but still do
not detect infected cells without spores
 Can lead to an underestimation of HPM
severity due to infected cells without spores
 These sometimes greatly outnumber cells with
spores

33. Chromotrope staining
Weber et al., 1992. N Engl
J Med. 326, 161-166
Normal method Hot method
Moura et al. 1997. Arch Pathol
Lab Med. 121, 888-893

34. Spores poorly indicate HPM severity

35. PCR is better than microscopy
 These examples show that PCR is the best way
to detect EHP infections
 qPCR is needed in growout ponds to know
whether EHP exceeds ~1000 copies per µg DNA
 A local qPCR technology is available for about
$3000 (http://www.mobilis.co.th/products/realamp.html)

36. Real-time
LAMP
These figures from LSNV as a model.
Narong Arunrut, Rungkarn Suebsing
Boonsirm Withyachumnarnkul
Wansika Kiatpathomchai. 2014.
PLoS ONE 9:9 e108047
Local Thai technology, very
low cost, real-time estimation
of DNA target quantity

37. PCR detection of EHP by ssu rRNA
 Detection methods targeting ssu rRNA:
– 1-step PCR (Tourtip et al. 2009. J. Invertebr. Pathol. 102, 21-29
– Nested PCR (Tangprasittipap et al. 2013. BMC Veterinary
Research. 9, 139)
– LAMP method (Suebsing et al. 2013. J Appl Microbiol. 114
1254-1263.
– 1-step PCR (Tang et al. 2015. J Invertebr Pathol. 130, 37–41)
– Real-time PCR (Liu et al. 2016. Progress in Fishery Sciences. In
press. In Chinese with abstract in English)
 Some of these methods were adapted for in
situ detection (Tourtip et al., Tangprasittipap et al., Tang et al.)
 These methods are OK for checking shrimp
 They are not suitable for environmental
samples because of possible cross reactions

38. Possible ssu rRNA cross reactions
 Some newly discovered microsporidians have
ssu rRNA sequences very similar to EHP
 They infect crabs and fish and other potential
carriers of EHP
 Thus, a unique gene of EHP was needed to
screen suspected carriers specifically for EHP
 Ornchuma Itsathitphaisarn and student Pattana
Charorenlak are working on this problem
 They are cooperating with CEFAS and Exeter
University to sequence the EHP genome
 As a result, they have found a unique spore
wall protein gene specific for EHP

39. Spore purification
EH
P

40. PCR detection of spore-wall protein gene
 Pattana used this gene to develop a specific,
nested PCR method for EHP (SPW-PCR)
 It is more specific and more sensitive than the
ssu rRNA PCR (SSU-PCR) method
 Thus, SPW-PCR is now recommended as the
best method for detecting EHP
 Suitable for environmental samples and solves
difficulty of detecting spores by microscopy
 Suitable for non-destructive detection of EHP
in shrimp feces (broodstock, PL, juveniles)

41. What is still unknown about EHP
 Are there life stages in other host species?
– These may be identified by PCR screening, microscopy and in
situ hybridization
 What are the modes of transmission in shrimp?
– These may be determined by co-habitation, feeding and
bath exposure to purified spores
– Do spore types exist for internal reinfection and external
transmission? (Vávra, J., Lukeš, J., 2013. Adv Parasitol. 82, 253-319)
 How can the spores be inactivated?
– This may be determined using viability assays and/or an
infection model
 Is therapeutic treatment possible?
– This may be determined using naturally infected shrimp or
using an infection model

42. Conclusions for EHP
 It does not cause WFS but may sometimes be
present in WFS shrimp
 Broodstock and PL should be monitored by PCR
to ensure freedom from EHP
 Broodstock should not be fed with live, non-SPF
polychaetes, mollusks, etc.
 Reservoir species need to be identified and
eliminated from the culture system
 It would be good if a therapeutic method could
be found

43. The King’s project at Kungkabaen