INFECTIOUS HAEMATOPOIETICNECROSIS

Infectious haematopoietic necrosis (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

Caused by the rhabdovirus

the principal clinical and economic consequences of IHN

occur on farms rearing rainbow trout where acute

outbreaks can result in very high mortality.

However, both Pacific and Atlantic salmon can be

severely affected.

Aetiological agent Agent strains The fish rhabdovirus, IHNV, has a bullet-shaped virion containing a non-segmented, negative-sense, single-stranded RNA genome

single-stranded RNA genome of approximately 11,000

nucleotides that encodes six proteins in the following order

a nucleoprotein (N),

a phosphoprotein (P),

a matrix protein (M),

a glycoprotein (G),

a non-virion protein (NV),

a polymerase (L).

The causative agent of IHN is a Rhabdovirus , genus

Novirhabdovirus 3 main genotypes described ( Kurath

et al., 2003)

GROUPS ORIGIN

U Isolates from Alaska, British Columbia

Washington,Oregon, California and Japan

obtained from :

Sockeye salmon ( O. nerka )Chinook salmon ( O. tshawytscha)

M: isolates from Idaho, Washington, France and Italy obtained from rainbow trout (O. mikiss )

L: isolates from California, Oregon and Japan obtained

from Giappone, obtained from

Survival outside the host IHNV is heat, acid and ether labile.

The virus will survive in fresh water for at least 1 month at

cooler temperatures, especially if organic material is present.

Stability of the agent (effective inactivation methods)

IHNV is readily inactivated by common disinfectants and

drying

Life cycle Reservoirs of IHNV are

clinically infected fish and

covert carriers among

cultured, feral or wild fish.

Virus is shed via urine,

sexual fluids and from

external mucus, whereas

kidney, spleen and other

internal organs are the sites

in which virus is most

abundant during the course

of overt infection

SUSCEPTIBLE HOSTS

rainbow or steelhead trout (O. mykiss),

Chum salmon (Oncorhynchus keta),

coho salmon (O. kisutch),

Masou salmon (O. masou),

sockeye salmon (O. nerka),

pink salmon (O. rhodurus)

Chinook salmon (O.tshawytscha),

Atlantic salmon (Salmo salar

Susceptible stages of the hostIHN occurs among several species of salmonids with fry being

the most highly susceptible stage.

Older fish are typically moreresistant to clinical disease, but among individuals, thereis a high degree of variationin susceptibility to IHNV.

Survivors of IHN demonstrate a strong protective immunity with the synthesis of circulating antibodies to the virus

Target organs and infected tissueVirus entry is thought to occur through

the gills and

at bases of fins

while kidney, spleen and other internal organs are the

sites in which virus is most abundant during the course of

overt infection.

Salmon affected by IHN virus, ventral congestion and pale gill

Disease pattern Infection with IHNV often leads to mortality due to the

impairment of osmotic balance, and occurs within a

clinical context of oedema and haemorrhage.

Virus multiplication in endothelial cells of blood

capillaries, haematopoietic tissues, and cells of the

kidney underlies the clinical signs.

Transmission mechanismsThe transmission of IHNV between fish is primarily

horizontal and high levels of virus are shed from infected

juvenile fish, however, cases of vertical or egg-associated

transmission have been recorded.

Although egg-associated

transmission is

significantly reduced by

the now common practice

of surface disinfection of

eggs with an iodophor

solution, it is the only

mechanism accounting for

the appearance of IHN in

new geographical

locations among alevins

• CANADA

• USA

• DOMINICAN REP.

• JAPAN

• KOREA

• PAKISTAN

• EUROPE

• BELGIUM

• CZECH REPUBLIC

• GERMANY

• ITALY

• FRANCE

• NETHERLANDS

• POLAND

• SLOVENIA

GEOGRAPHICAL DISTRIBUTION

officially reported from China, Iran, Japan and the Republic of Korea.

Mortality and morbidity

Depending on the

species of fish,

rearing conditions,

temperature, and, to some extent,

the virus strain, outbreaks of IHN

may range from explosive to chronic.

Losses in acute outbreaks will exceed several per cent of the

population per day and cumulative mortality may reach 90–

95% or more .

In chronic cases, losses are protracted and fish in various

stages of disease can be observed in the pond.

IHN mortality in market size Atlantic salmon

TRASMISSION AND PATHOGENESISVIRUS ENTRY : Gills , skin, oral

VIRUS SHEDDING : Feces , urine , sessual fluids, mucus

TRANSMISSION : Mostly orizontally

Vertical suspected

Confirmed by vectors ( invertebrates)

TEMPERATURE : Most of the outbreaks at 8-15°C

REPLICATION : Viremia AT 5-10 days

TARGET ORGANS : haematopoietic tissues ( kidney, spleen ) ,

brain and gastro- intestinal.

MORBIDITY & MORTALITY : 90-95% in fry . Not significant in

market-size fish

BTSF

Best organs or tissues The optimal tissue material to be examined is spleen,

anterior kidney, and either heart or encephalon.

In some cases, ovarian fluid and milt must be examined.

Infectious hematopoietic necrosis ( IHN ) in chinook salmon

Collecting-sperm

If a sample consists of whole fish with a body length between

4 cm and 6 cm, the viscera including kidney should be

collected.

If a sample consisted of whole fish less than 4 cm long,

these should be minced with sterile scissors or a scalpel,

after removal of the body behind the gut opening.

If a sample consisted of whole fish more than 6 cm

long, tissue specimens should be collected as

described above. The tissue specimens should be

minced with sterile scissors or a scalpel,

homogenised and suspended in transport medium.

Samples/tissues that are not suitable

IHNV is very sensitive to degradation, therefore

sampling tissues with high enzymatic activities or

large numbers of contaminating bacteria such as the

intestine or skin should be avoided when possible.

Muscle tissue is also less useful as it typically contains

a lower virus load.

Field diagnostic methods

Clinical signs The disease is typically characterised by gross signs

that include

lethargy interspersed with bouts of frenzied,

abnormal activity,

darkening of the skin,

pale gills,

ascites,

distended abdomen,

exophthalmia, and

petechial haemorrhages internally and externally.

photo of affected fry with ascites

Black body Color with exophthalmos(a), Abdominal enlargement (b), Pale and weak gill filaments. There were bleeding in cephalosome(c), Actinost (d), abdominal(e)And the muscular tissue of the dorsal fin (f). The liver(g),

Clinical symptoms Of rainbow trout infected with IHNV

Spleen and kidney were pale, the stomach was swollen with a milky white liquid, And the bowel released a yellowish liquid. There were no clinical symptoms in rainbow trout of control group(−).

Salmon affected by IHN virus exhibiting peritoneal

and caecal fat haemorrhage

Rainbow trout fry -darkening and exopthalmia (popeye) as shown by the fish in the lower portion of the photo.

Signs of IHNV disease in rainbow trout (Oncorhynchus mykiss)

include hemorrhage and exophthalmia (pop-eye)(photograph at left),

skin darkening relative tolighter colored healthy fish(photograph at right)

Chinook salmon fry with

infectious haematopoietic

necrosis.

Note characteristic darkening

from the tail region, swollen

stomach and haemorrhaging at

base of fins

Rainbow trout fry with (left)

and without (right)

infectious haematopoietic

necrosis.

Note the darker colour of

the infected fish

Examples of clinical signs observed in IHNV-infected

rainbow trout.

Behavioural changes

During outbreaks, fish are

typically lethargic with bouts of frenzied,

abnormal activity,

such as spiral swimming and flashing.

A trailing faecal cast is observed in some species.

Spinal deformities are present among some of the surviving fish.

Rainbow_trout_fingerling

Clinical methods Gross pathology

Affected fish exhibit

darkening of the skin,

pale gills,

ascites,

distended abdomen,

exophthalmia, and

petechial haemorrhages internally and externally.

Internally, fish appear anaemic and lack food in the gut.

The liver, kidney and spleen are pale.

Ascitic fluid is present and petechiae are observed in the

organs of the body cavity.

Larger, more robust individuals die first.

Fry are lethargic (swim feebly and avoid current by moving to

the edge of the raceway) with sporadic hyperactivity.

A long, thick, off - white fecal pseudocast trailing from the

rectum is diagnostic.

Other clinical signs include darkening, abdominal distension,

exophthalmos, and hemorrhage at the base of the

fins.

Gills are pale, and internally, there is visceral pallor, caused

by anemia.

There is no food in the gastrointestinal tract, which is

distended with an off - white, translucent, mucoid, fluid.

There may be petechiation of the visceral fat, mesenteries,

peritoneum, swim bladder, meninges, and pericardium.

In sockeye salmon, 5% or more

of surviving fish may have

spinal deformities.

Clinical signs are less severe in

older fish and may be absent or

simply appear as lateral

compression because of

anorexia .

Microscopic pathology Histopathological findings reveal degenerative necrosis

in haematopoietic tissues, kidney, spleen, liver,

pancreas, and digestive tract.

Necrosis of eosinophilic granular cells in the

intestinal wall is pathognomonic of IHNV infection

CLINICAL PATHOLOGYIHN causes profound changes in cellular and chemical blood

constituents, primarily because of renal damage.

The most diagnostic change is the presence of remnants

of necrotic cells (“ necrobiotic bodies ” ), probably

erythrocytes, in kidney smears.

These cells are less frequent in peripheral blood.

Fish are anemic and leukopenic, and there is evidence of

osmotic imbalance (hypoosmolality).

7-8 months old sockeye salmon naturally infected with IHN.

Fig 1-Kidney imprint. Note necrobiotic body (arrow). Fig. 2. Kidney imprint. Note necrobiotic bodies (arrows).

HISTOPATHOLOGYIn affected fry, major changes are necrosis of

the kidney,

hematopoetic tissue,

pancreas,

gastrointestinal tract, and

interrenal tissue (adrenal cortex).

.

Splenic and renal

hematopoetic

tissues are usually

affected first and

most severely

Fig. 3. Kidney section, showing area of focal degeneration

and necrosis (arrow).

Fig. 4. Spleen section, showing area of focal necrosis

involving few cells (arrows)

Interrenal tissue may eventually

be involved, as well as glomeruli

and tubules.

Pancreatic necrosis is

common.

Pleiomorphic

intracytoplasmic and

intranuclear inclusions are

present in the pancreaticacinar and islet cells.

Hepatic necrosis has been reported in some cases.

Necrosis of the eosinophilic granule cells of the intestinal

submucosa (Fig) is highly diagnostic but is only evident in fish at least 3 – 4 months old

In older fingerlings, lesions are similar (splenic and renal

hematopoetic necrosis, moderate sloughing of intestinal

mucosa, degeneration of pancreas) but more subtle.

One distinguishing feature may be the presence of gill lesions

(branchial hyperplasia and fusion)

Peripheral blood smear.

Note large cell in the center,

probably a monocyte, showing

foamy cytoplasm.

10-13 month old sockeye salmon experimentally infected with IHN

10-13 month old sockeye salmon experimentally infected with IHN.

Fig. 7. Spleen section. Note some degeneration and necrosis and the lack of lymphoid cells.Fig. 8. Intestine just posterior to the caeca. Sloughing of the epithelial layer of the intestine is clearly evident.

Histopathology should be supported by at least confirmation

with immunological or molecular probes, when possible.

Virus - infected cells can be immunologically identified in

histological sections or tissue smears from target organs or

blood

Kidney section.

Note the hyaline droplets in

the epithelium of tubules

(arrows).

(A) Arrows showed that the

liver cells hemorrhaged

and necrosed, the cell

nucleus was prominent, the

chromatin was condensed,

and the liver cytoplasm

exhibited extensive vacuole

formation.

Histological analysis of rainbow trout experimentally

infected with IHNV

(C) Kidney tissue hemorrhaged and necrosed extensively

with characteristics similar to those of the liver.

Renal tubular necrosis led to red blood cells and cell debris

appearing in the lumen.

The interspace was blocked between the necrotic epithelial

cells of the glomerulus and the renal corpusculum

(E) The myocardium showed

bleeding, and there were

erythrocytes between muscle

fibers

(G) Some back muscle fibers were broken, and the fiber

streak dis-appeared. (H) The muscle fibers showed

bleeding, and red blood cell clusters were visible. (I)

normal muscles

Histopathology and immunohistochemistry of Atlantic

salmon infected with the high virulence IHNV

(a) shows kidney necrosis H&E, and panels

show

immunohistochemical

staining (red) of viral

nucleocapsid antigen in

necrotic lesions of exocrine

pancreas cells,

immunohistochemic

al staining (red) of

viral nucleocapsid

antigen in necrotic

lesions of kidney

hematopoietic tissue

immunohistochemical staining (red) of viral nucleocapsid

antigen in necrotic lesions of(d) skin – subepidermally below

the basement membrane, (e) granular layer of pyloric cecae

IHNV is a relatively weak immunogen.

However, there is little antigenic variation among various

isolates, making serological identification of the virus

relatively simple.

SUBCLINICAL CARRIERS OF IHNV

Adult carriers are asymptomatic.

In female carriers the most sensitive tissues for virus

isolation are ovarian fluid, gills, pyloric ceca, and

kidney.

Postspawning examination of a carrier’s ovarian fluid

is best, since no virus may be detectable for as little as 2

weeks before spawning.

Diagnosis

Wet mounts

Wet mounts have limited

diagnostic value.

Tissue imprints and smears

Necrobiotic bodies and foamy

macrophages, indicative of a

clinical manifestation of IHN,

can be best observed using

tissue imprints obtained from

the kidney and spleen rather

than smears.

Electron microscopy/cytopathology

Electron microscopy of virus-infected cells reveals bullet-

shaped virions of approximately 150–190 nm in length and

65–75 nm in width.

The virions are visible at the

cell surface or within vacuoles

or intracellular spaces after

budding through cellular

membranes.

The virion possesses an outer

envelope containing host

lipids and the viral

glycoprotein spikes that react

with immunogold staining to decorate the virion surface.

Electron micrograph of IHN virus (the rod-like particles)

Identification of IHN viruses under transmission

electron microscopy (TEM).After inoculation with viruses, the EPC cells were craped off.

The virions were cut into slices and observed under TEM.

The virions of rIHNV-EGFP were showed in

A (scale bar 2000 nm), B(scale bar 500 nm)

C(scale bar 1000 nm) and D(scale bar 1000 nm).

The virions of IHNV were showed in E(scale bar 500 nm), F(scale bar 100 nm),G(scale bar 300 nm) andH(scale bar 100 nm).

IHNV immunofluorescence pattern in EPC cells.

DIAGNOSISThe suspicion of IHN may be confirmed by virus

isolation and identification of the causativeagent

Select at least 10 symptomatic specimen and test virologically according to the following methods :

virus isolation on epc/bf-2 or fhm/rtg-2followed by identification virus isolation and serological identification by ifat or elisa or pcr

PREVENTION AND CONTROL

In addition to the disinfection of eggs , the control of IHN may be obtained by

ERADICATION METHODS

– harvest and eliminate all

the fish population

– dry all the basins simultaneously

(6 weeks)

– disinfect

– restoke with free fish

VACCINATION

– a dna vaccine has been registerd in canada to be used in salmon industry ( salmo salar) .

BTSF