EHN IN FISHES

1. EHN (Epizootic hematopoietic
necrosis)
BY
R.PRATHISHA

2. Importance
• Epizootic hematopoietic necrosis (EHN) is a
systemic iridoviral disease of fish.
• redfin perch and rainbow trout.
• This disease is highly fatal in redfin perch;
• Rainbow trout are less likely to be infected, and
the cumulative mortality rate is usually low.
• Closely related viruses cause serious outbreaks of
epizootic hematopoietic necrosis in catfish and
sheatfish.

3. Etiology
• Epizootic hematopoietic
necrosis is a systemic
disease characterized by
necrosis of the liver,
spleen and hematopoietic
tissues within the kidney
• genus Ranavirus and
family Iridoviridae.
• Iridoviruses are large
cytoplasmic DNA viruses
with an icosahedral
morphology

4. To date, the causative viruses include
• EHNV in redfin perch and rainbow trout,
• European sheatfish iridovirus (ESV)
• European catfish iridovirus (ECV) in catfish and
sheatfish.
Genetic studies suggest that ECV and ESV are
isolates of the same virus; EHNV is a different
virus.

5. •Iridoviridae family is divided into four genera
including Iridovirus, Chloriridovirus,
Ranavirus, and Lymphocystisvirus.
Rock bream infected with iridovirus.
A; Darkness of the body color , B; Congested eye

6. C; Congested internal organs, D; Enlarged spleen.

7. Electron micrograph showing virions (120-130 nm in diameter) in
the cytoplasm of infected cells from the spleen of diseased fish. Bar
is 100 nm.

9. Mass mortality of redfin perch (Perca fluviatilis); note the small size of
individuals affected and the swollen abdomen of the fish at the centre of the
photograph

10. Species Affected
• In nature, EHNV has
been reported only
in redfin perch
(Perca fluviatilis)
• rainbow trout
(Oncorhynchus
mykiss)

11. • Species that have been infected experimentally include
Macquarie perch , mosquito fish, silver perch ,
mountain galaxias , Murray cod, and Atlantic salmon
Other species may also be susceptible.
• ESV has been reported from sheatfish/ wels catfish,
• while ECV can infect European catfish, channel catfish,
goldfish and short finned eels
• Systemic necrotizing iridovirus syndromes have also
been reported in other species of fish including turbot
in Denmark.

13. Geographic Distribution
• EHNV is endemic only in Australia. Outbreaks
of EHN have occasionally been reported in
other countries including Kuwait, Pakistan and
Peru.
• ECV/ ESV is endemic in Europe

14. Disease signs at the farm, tank or
pond level are:
• Typically many (hundreds or thousands) small,
dead fish less than 10 cm long found on the
downwind bank of the water body
Mass mortality of redfin
perch (Perca fluviatilis);
note the small size of
individuals affected and
the swollen abdomen of
the fish at the centre of
the photograph

15. • large numbers of fish-eating birds (e.g.
seagulls) at the water surface
seagulls at the
water surface

16. • loss of appetite
• juveniles (<25 mm long) often swimming in a
disorientated fashion at the surface
• occasional adults affected when the disease
first arrives in an area

17. Gross pathological signs are:
• swollen abdomen
• darkened skin colour
• petechial (pinpoint) haemorrhages at the base
of the fins
• haemorrhaging of the gills
• dead fish
• enlargement of the kidney, liver and spleen

18. • focal white to yellow liver lesions
Experimentally
infected Redfin Perch
showing multiple
white spots in the
liver

19. Microscopic pathological signs are:
• coagulative or liquefactive necrosis of the
liver, kidney and spleen
• necrotic lesions in the heart, pancreas,
gastrointestinal tract and gills
• small numbers of basophilic intracytoplasmic
inclusion bodies surrounding necrotic areas of
the liver and kidney.

20. Transmission
• Fish can be infected by bath inoculation, and
spread through the water is likely.
• Oral transmission may occur;
• naturally infected fish have gastrointestinal
lesions that are not reported after
intraperitoneal inoculation.
• Infection through the gills or skin has also
been proposed.

21. • The EHN group of viruses can be transmitted on
fomites, and birds may act as mechanical vectors.
• EHNV can survive in the avian digestive tract for a
few hours, and might be transmitted in
regurgitated food.
• It could also be carried on the feathers, feet and
bill.
• EHNV is highly resistant to drying

22. LONGEVITY
• This virus can remain infective for more than
97 days in the water and for at least 113 days
in dried fish tissues
• It can also survive for more than 300 days in
cell cultures at 4°C
• and for two years in fish tissues stored at –
20°C

23. Incubation Period
• For experimentally infected rainbow trout is
3-10 days in water temperatures of 19-21°C ,
and 14-32 days in water temperatures of 8-
10°C
• For experimentally infected redfin perch, the
incubation period is 10-11 days at 19-21°C,
and 10-28 days at 12-18°C

24. Clinical Signs
The clinical signs are nonspecific
• In perch, sudden death is the most common
sign.
• Darkening of the body surface, ataxia,
lethargy and erythema around the nostrils and
brain region have also been seen.
• Hemorrhages may occur in the gills and at the
base of the fins.

25. • Symptoms reported in experimentally
infected rainbow trout include darkening of
the body surface, lethargy, inappetence,
abdominal distension and loss of equilibrium.

26. Post Mortem Lesions
redfin perch
• In redfin perch, there may be swelling of the
kidney, spleen and/ or liver, hemorrhages at the
base of the fins and focal hemorrhages in the
gills.
• The spleen is often swollen, but it is occasionally
pale and shrunken.
• Petechiae may be found on the viscera. Multiple
white to yellow areas of focal necrosis are
sometimes found in the liver

27. rainbow trout
• In rainbow trout, the lesions may include
abdominal distension with serosanguineous
ascitic fluid, and swelling of the spleen or
kidney.
• Petechial hemorrhages have been seen on the
viscera in a few fish.
• Focal necrosis in the liver is rare. The gross
lesions may be minimal in this species.

28. TEMPERATURE
• EHN epizootics in redfin perch are most
common in the spring and summer. In this
species, disease has not been reported at
water temperatures below 12°C (54°F).
• In rainbow trout, outbreaks have been
reported in water temperatures between 11°C
and 17°C

29. MORBIDITY AND MORTALITY
• Redfin perch are highly susceptible to epizootic
hematopoietic necrosis. The morbidity rate is
very high in this species, and most infected fish
die;
• Rainbow trout are relatively resistant to EHN
• it is most common in young fingerlings up to 125
mm fork-length.
• Generally, OIE recommended samples from
asymptomatic animals include the kidney, liver,
spleen, and heart;

30. • ESV/ ECV can cause high morbidity and
mortality rates in susceptible species.
• ESV outbreaks have been associated with
mortality rates up to 100% in sheatfish

31. Diagnosis
Clinical
Epizootic hematopoietic necrosis should be
suspected in redfin perch when an epidemic is
characterized
• by sudden high mortality
• histological evidence of necrosis in the renal
hematopoietic tissue, spleen and liver.
During outbreaks in rainbow trout, far fewer fish
are usually affected
• there may be evidence of poor husbandry

32. Laboratory tests
Diagnosis:
• Epizootic hematopoietic necrosis can be
diagnosed by isolating EHNV or ECV/ ESV in
cell cultures; many fish cell lines including
CHSE-214 (Chinook salmon embryo), FHM
(fathead minnow), EPC (epithelioma
papulosum cyprini), and BF-2 (bluegill fry)
cells can be used

33. Confirmation:
• The identity of the virus can be confirmed by
immunostaining, enzyme-linked
immunosorbent assay (ELISA),
immunoelectron microscopy, polymerase
chain reaction (PCR) or other methods.

34. • Cross-reactions occur between EHNV, ECV/ ESV
and other fish and amphibian ranaviruses when
antibody-dependent techniques are used.
• PCR combined with either restriction
endonuclease analysis (REA) or sequence analysis
can differentiate these viruses.
• Real-time PCR assays to distinguish closely
related ranaviruses have recently been published.

35. Rainbow trout, anterior kidney:
intracytoplasmic inclusion bodies (arrows).

36. Rainbow trout, liver: tiny foci of acute necrosis;
abundant, often multiple, intracytoplasmic
inclusion bodies (arrows).

37. Redfin perch, liver: focal necrosis; abundant
intracytoplasmic inclusions in adjacent
hepatocytes
(arrows).

38. Transmission electron micrograph of a fathead
minnow cell (FHM cell line) infected with
epizootic hematopoietic necrosis virus (EHNV)
Paracrystalline arrays are indicated by the
arrow. An enveloped virus that has budded
from the host cell plasma membrane can also
be seen. Nu = nucleus. Scale bar represents
200 nm.

39. (D) Hematoxylin and eosin (H&E)-
stained section of an adult
zebrafish kidney corresponding to
mock-infected
(a) or ESV-infected (b) animals at
48 h p.i. Arrows indicate nuclear
chromatin margination,
necrosis, and vacuolation in
segments of the renal tubular
epithelium. Scale bars are shown
in panels C and D.

40. (A) Spleen: numerous
virus particles among
spleen cells and a
macrophage which has
phagocytosed the viral
particles (arrow). (Bar = 1
lm.)
Electron micrographs of spleen, kidney and
gut of sheatfish 4±8 days after waterborn
infection with the sheatfish iridovirus.

41. (B) Spleen: viral
particles receiving an
envelope by budding
through the plasma
membrane of a spleen
cell
(a) and a released
enveloped virion (b).
(Bar = 100 nm.)

42. (C) Spleen: viral
particles near to
swollen
mitochondria of an
endothelial cell
within the spleen.
(Bar = 200 nm.)

43. (D) Gut: viral
particles (arrow)
in a necrotic area.
(Bar = 1 lm.)

44. (E) Kidney: viral
particles (arrows)
in renal
interstitium
showing different
stages of virus assembly
(Bar = 1 lm.)

45. (F) Kidney: budding of
viruses
(a), viruses in a lytic
area
(b) and entry of
viruses
(c) in kidney cells.
(Bar = 1 lm.)

46. • Outbreaks in rainbow trout are usually related
to poor husbandry. In this species, good
management (low stocking rates; adequate
water quality) can reduce the risk of disease.
• In redfin perch, high morbidity and mortality
rates can be expected in an outbreak,
regardless of husbandry conditions. Vaccines
are not available for any species.

47. • EHNV is highly resistant to drying
and disinfection.
• In dried surface films, this virus
can be destroyed by 70% ethanol
for two hours but it is resistant to
sodium hypochlorite.
• In liquid suspension, EHNV can
be destroyed by sodium
hypochlorite, heating to 60°C
(140°F) for 15 minutes, or pH of
4.0 or 12.0.
• Farm equipment should be
scrubbed to remove dried films,
then disinfected with sodium
hypochlorite.
• Lime may be effective in earthen
ponds.

48. THANKYOU
THANKOU FOR
YOUR ATTENTION