Information about Infectious hematopoietic necrosis virus in finfish (IHNV)

1. INFECTIOUS HAEMATOPOIETIC NECROSIS IN
FINFISH
PRESENTED BY:
AVIJIT PRAMANIK
AAH-MA-09-02
B.F.SC, W.B.U.A.F.S.
M.F.SC, CIFE, MUMBAI

2. • IHN is a viral disease affecting most species of salmonid fish reared
in fresh water or sea water.
• First recognized in 1950s in sockeye salmon and chinook salmon.
• The virus was introduced to Japan in 1968 by eggs from Alaska.
• IHN virus spread across North America in the 1970s in rainbow trout.
• Apparently originating from fry or egg shipments from
a single source.
INTRODUCTION

3. • IHNV is commonly found in the Pacific Coast of Canada and the
United States, and has also been found in Europe and Japan.
• KOREA
• PAKISTAN
• EUROPE
• BELGIUM
• GERMANY
• ITALY
• FRANCE
• NETHERLANDS
• POLAND
• SLOVENIA Officially reported from China, Iran, Japan and the Republic of
Korea.

4. • The causative agent of IHN is a rhabdovirus of the genus
novirhabdovirus, known as IHN virus (IHNV).
• Bullet-shaped RNA virus.
• IHNV can be isolated from spawning fish,pyloric caeca,
intestine and ovarian/seminal fluid.
Visible at the cell surface or intracellular
spaces after budding through cellular
membranes

5. Sockeye salmon (O. nerka),
Pink salmon (O. rhodurus)
Chinook salmon (O. tshawytscha),
Atlantic salmon (Salmo salar)
Rainbow trout (O.
mykiss)
Chum salmon (Oncorhynchus keta)
Coho salmon (O. kisutch)
Masou salmon (O. masou)

6. ØNot all salmonid species are susceptible to the virus.
ØWith fry and small fingerlings becoming infected very readily,
ØBecoming more resistant as they mature.
ØOlder fish are typically more resistant to clinical disease.

7. üDepending on temperature,microflora, IHNV survives in
freshwater from 3 days to several months(Wedemeyer et
al.,1978).
üInfection is influenced by environmental condition,including
temperature and salinity.Epizootics occur at 8-14℃ and have
not been seen17℃.(Bootland and Leong,1999)
üIHNV survives longer in salt water than
in fresh water.
SURVIVAL OUTSIDE THE HOST

8. Virus entry is occur through:
§ The gills
§ At bases of fins
§ While kidney,spleen,and other
internal organs are the infectious
sites.
ü Fish body length: 4 cm to 6 cm, kidney should be taken
ü Fish is less than 4 cm long, these should be minced with
sterile scissors or a scalpel.

9. Mostly Horizontally:
Infected juveniles will shed IHN virus particles in the
faeces, sexual fluids and external mucus
Vertical suspected:
egg-associated transmission have been recorded.
Those fish that survive an outbreak of IHNV:
Some fish become covert carriers of the virus,
providing a reservoir of infection.
Ø Insects, annelids, and crustaceans may act as viral vectors
MORBIDITY & MORTALITY : 90-95% in fry. Not significant in
market-size fish

10. ØIHNV is very sensitive to degradation.
ØSampling tissues with high enzymatic activities or large
numbers of contaminating bacteria such as the intestine or skin
should be avoided when possible.
ØMuscle tissue is also less useful as it typically contains a lower
virus load.
SAMPLES/TISSUES THAT ARE
NOT SUITABLE
Gastrointestinal tract of Atlantic
Salmon

11. The disease is typically characterised by gross signs that include
a. lethargy
b. abnormal activity
c. darkening of the skin
d. pale gills
e. distended abdomen
f. exophthalmia
FIELD DIAGNOSTIC METHODS
Clinical signs:

12. a. Fish appear anaemic
b. Lack food in the gut
c. The liver, kidney and spleen are pale
d. Ascitic fluid is present
e. Petechiae are observed in the organs of the body cavity.
INTERNAL OBSERVATION:

13. CLINICAL SYMPTOMS OF RAINBOW TROUT INFECTED WITH IHNV

14. The liver, spleen, kidney was swollen with a milky white liquid.

15. vHemorrhage and exophthalmia (pop-eye)
vRainbow trout fry - darkening and
exophthalmia (Popeye) as shown by
the fish.
Signs of IHNV disease in rainbow trout (Oncorhynchus mykiss)

16. Chinook salmon fry with IHNV
ü Darkening from the tail region,
ü swollen stomach,
ü haemorrhaging at base of fins.

17. Examples of clinical signs observed in
IHNV-infected rainbow trout :-
Absence of muscular petechiae in trout
Catarrh of anterior intestine but normal spleen in
trout
Splenomegaly in trout

18. In sockeye salmon, 5% or more of surviving fish may have spinal
deformities.
A trailing faecal cast is observed in some species.

19. CLINICAL PATHOLOGY
qCauses changes in cellular and chemical blood constituents, primarily because of
renal damage.
qMost diagnostic change - presence of remnants of necrotic cells(“ necrobiotic
bodies ” ), probably erythrocytes, in kidney smears.

20. 7-8 months old sockeye salmon naturally
infected with IHNV
Kidney imprint, necrobiotic body (arrow).

21. Observed under an electron microscope

22. HISTOLOGICAL ANALYSIS OF RAINBOW TROUT
EXPERIMENTALLY INFECTED WITH IHNV
o Arrows showed that
• The liver cells haemorrhaged
and necrosed
• The cells nucleus is
prominant,the chromatin is
condensed
• The liver cytoplasm exhibited
extensive vacuole formation.

23. Kidney tissue hemorrhaged and
necrosed.

24. vRT-PCR is established for detection and identification of IHNV.
vA set of primers is prepared for amplification of a 510 bp
nucleotide which encodes a region of IHNV nucleoprotein (N)
gene.
vThe first primer (5＇ -TCATTGCAGAGACGGTCCAT- 3＇) and
the second primer (5＇- TGGTTGAACAGTCCCACCAT- 3＇)
vThe second primer was used to generate first-strand cDNA in a
reverse transcription reaction mixture.
PCR FOR IHNV

25. SOUTHERN HYBRIDIZATION
§ The PCR product was confirmed as the IHNV specific nucleotide by
southern hybridization with an oligonucleotide probe synthesized
from the N gene.
§ DNA fragments in agarose gels were transferred overnight to
nitrocellulose membrane.
§ (5＇CTTGTTTTGGC AGTATGTGGCCATCTTGTC-3＇)-
Oligonucleotide probe was synthesized and labeled at the 3' end
using the DIG oligonucleotide

26. Serological Identification
§ An alkaline phosphatase immunocytochemical (APIC) assay was
adapted for direct detection of (IHNV) in infected tissue culture cells.
§ The APIC assay provided a means of confirming the diagnosis of
IHNV after the tissue culture plates had been fixed with formalin
and stained with crystal violet.
§ The assay used a broadly reactive monoclonal antibody
(lNDW14D) to the IHNV nucleoprotein to detect viral antigen in cells
infected by virus isolates representing the five known IHNV types.

27. LAMP
§ LAMP primers were designed according to the published sequence
of IHHNV structural gene (GenBank accession no. AF218266)
using the Primer Explorer version 3
§ Targeted positions 1957–2185 of the reference sequence.
§ Details of the final primers are shown in Fig. 1

28. • As the IHNV can be transmitted through water,
contact with contaminated waste material in areas
where IHN is endemic, it can be controlled by
ü good hygiene
ü the use of virus-free water supplies
vVACCINATION – A DNA vaccine has been
registered in Canada to be used in salmon industry
(Salmo salar) .
vEgg associated transmission reduced by surface
disinfection of eggs with Iodophor solution.
PREVENTION AND CONTROL

29. REFERENCES
• FISH DISEASES AND DISORDER by Woo P.T.K
• FISH PATHOLOGY fourth edition edited by RONALD J. ROBERTS.
• Detection and Identification of Infectious Hematopoietic Necrosis Virus (IHNV) by Reverse
Transcription (RT)-polymerase Chain Reaction (PCR),Toko Yoshinaka, Mamoru Yoshimizu,
Tomoo Sawabe, and Yoshio Ezura ,Laboratory of Microbiology, Faculty of Fisheries,
Hokkaido University, Hakodate, Hokkaido 041, Japan
• Bhatnagar Anita and Pooja Devi.,2013, Water Quality Guidelines for the Management of
pond fish culture. International journal of Environmental sciences, Vol 3(6).
• B. P. Gupta, M. Muralidhar, K.K.Krishnani, M.Jayanthi,p.Nilarekha and R. Saraswathy,
august 2004. Soil and water quality management in brackish water aquaculture, CIBA
special publication no. 13.
• Rouse denis.,2002,water quality management in pond fish culture. Aburn universi.