Presentation 04 HUE diagnostic studies

FAO/MARD Joint Final Technical and National
Consultations on Acute Hepatopancreatic
Necrosis Syndrome (AHPNS) of Cultured
Shrimp” (TCP/VIE/3304)

Diagnostic studies
(histopathology & PCR)
Dang Thi Hoang Oanh, Pham Phong Vu, Dao Thi Thanh Hue,
Don Lightner, Tim Flegel, Le Van Khoa, Melba B. Reantaso

Prestige Hotel, Hanoi, Viet Nam, 25 June 2013


Contents
1. A case definition for AHPNS
2. Locations and date of collecting samples
3. Collection of samples
4. Testing of samples
5. Results of histopathology & PCR


1. A case definition for AHPNS
Prof. Don Lightner proposed the following case definitions at the
animal level for AHPNS, which was generally agreed on by the
consultation:
•Idiotpathic - no specific disease causing agent (infectious or toxic)
has been identified.
•Pathology: Acute progressive degeneration of hepatopancreas from
medial to distal with dysfunction of B, F, R and E cells, prominent
karyomegaly and necrosis and sloughing of these tubule epithelial
cells. The terminal stage shows marked inter- and intra-tubular

hemocytic inflammation and development of secondary bacterial
infections that occur in association with necrotic and sloughed
hepatopancreas tubule cells.



1. A case definition for AHPNS (Cont.)
At the pond level, the following clinical signs could be used for
presumptive diagnosis which can be further confirmed by
histopathology at the animal level by using the above case definition.
•Often pale to white within HP connective tissue capsule.
•Significant atrophy of HP.
•Often soft shells and partially full to empty guts.
•Black spots or streaks within the HP sometimes visible.
•HP does not squash easily between thumb & finger.
•Onset of clinical signs and mortality starting as early as 10 days post
stocking
•Moribund shrimp sink to bottom.

Please visit NACA website www.enaca.org


2.Locations and date of collecting samples


Province

Locations

Soc Trang 1. Tran De Dist

Lieu Tu Commune

-

Lich Hoi Thuong Commune RAHO6

-

Ca Mau

Soc Trang Sub Animal Health

-

2. Vinh Chau Dist
Bac Lieu

Person
CTU
RAHO7

Vinh Tan Commune

1. Hoa Binh Dist

Bac Lieu Sub Aquaculture

-

Vinh Hau Commune

CTU

-

Vinh Thinh Commune

RAHO6

-

Vinh Hau A Commune

RAHO7

1. Dam Doi Dist

Ca Mau Sub Animal Health

-

Quach Pham Bac Commune CTU

-

Ta An Khuong Commune

RAHO6

-

Tan Duyet Commune

RAHO7


Round 1
11--18 Sept. 2012
11

Round 2

Round 3

20--27 Sept. 2012 29 Sept. - 6 Oct. 2012
20


3. Collection of samples
1. 60 samples (each sample has more than 20 shrimp) were
preserved in Davidson’ fixative
2. 60 samples (pooled gills and pleopods) were preserved in
95% ethanol
3. 175 bacterial isolates (~ Vibrio) were recovered from
hepatopancreatic of shrimp specimens (- 80 °C at CTU)
4. 10 fungal isolates were recovered from hepatopancreatic
of shrimp specimens (4°°C at CTU).
5. 60 frozen samples are kept at -80°°C at CTU.

4. Testing of samples
Sampling pond (~ 100100-300 shrimp/pond)

Hepatopancreatic

Bacterial isolation
(5 shrimp)

Davidson’s fixative
(~30
(~30 shrimp)

Fungal isolation
(5 shrimp)

Fresh smear
(10 shrimp)

EtOH (10 shrimp/ Liquid nitrogen,
gill & pleopod)
pleopod)
then -80
80°°C

Extract RNA/DNA

PCR/Seq

Indentification
ISH

Infectivity studies

Aetiology

Transmission/
Infectivity studies


4. Testing of samples (Cont.)
1. Histologycal analysis: RAHO6, UAZ and CENTEX
2. PCR analysis: RAHO7
3. Bacterial and fungal identification: CTU
4. Bacterial metagenomics: CENTEX
5. Infectivity: UAZ


Gross signs

P.monodon

Source: RAHO6/DAH


Gross signs

Astrophy of HP, pale to white

Emty gut

Source: RAHO6/DAH

Gross signs

P.vanamei
Abnormal HP

Normal HP


Necrostic HP from inside to outside


Astrophied Epithelium tubule of HP
with no R,B,F cells (BL2-A4)


Necrosis and sloughing of the tubule epithelial cells


Accompanying the development of a secondary bacterial infection destroy HP
structure with a lot hemocyte encapsulation of necrotic HP tubule and the lesion areas


MBV &HPV

MBV
HPV

WSSV


Gregarine ( ) and Gregarine-like ( )


5. Results of samples
5.1. Results of histopathology
Laboratory

UAZ

CENTEX

RAHO6

60 ponds were
ST BL CM SUM ST BL CM SUM ST BL CM SUM
examined
EMS/AHPNS
WSSV
MBV
HPV
Gregarine

3 16 10
7 4 1
0 18 1
0 2 0
- -

Gregarine like

-

-

-

29
12
19
2
-

6 17 7
7 1 1
0 8 2
0 1 0
- - -

30
9
10
2
-

4
10
1

0
1

18 9
7 5
7 0
1 0
6 1

31
22
8
1
8

12 8

33

6

2

10

15

2

BL: Bac Lieu

CM: Ca Mau
ST: Soc Trang


5. Results of samples (Cont.)
5.2. Results of PCR by RAHO7
Test
WSSV
YHV
IMNV

Results
Positive
23
0
0

Negative
37
60
60



Acknowledgements
FAO
MARD
DAH
Bac Lieu Sub Aquaculture
Ca Mau Sub Animal Health

Soc Trang Sub Animal Health

Thank you for your attention