First isolation and identification of the
Infectious Pancreatic Necrosis (IPN) virus from rainbow trout
Onchorhynchus mykiss
fingerlings farmed in
|
Authored by Dr. PANOS VARVARIGOS |
CONTENTS:
|| Abstract
|| Introduction || Fish samples || Gross clinical signs || Internal lesions & microscopy ||
|| Bacterial cultures || Virological examination || Conclusion || References ||
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Copyright (c) Dr. Panos Varvarigos.
During February 2000, two trout farms, situated
in NW Greece, reported unusually high losses of hatchery reared rainbow trout
fry and fingerlings. Live fish and chilled samples of diseased whole fish were
delivered to our veterinary laboratory in Athens for diagnostic investigation.
Infections with the fungus Ichthyophonus hoferi as well as the Gram negative bacterium Aeromonas hydrophila were
diagnosed in some of the samples. However, the reported clinical signs,
mortality levels, age and size of the fish and the necropsy findings suggested
that IPNV might also be present. The presence of the virus was detected
initially by means of a rapid ELISA test kit and later confirmed by virus
isolation and identification from tissue and whole fish samples by an
independent virology laboratory. In neutralisation tests the virus was found to
relate more closely to the A2 (formerly Sp) serotype of aquatic birnaviruses suggesting that the original source of the
virus is elsewhere in Europe. This is the first published report of IPN virus
affecting farmed fish in Greece.
Trout farms in Greece are mainly situated in
the north of the country where the natural water supplies are of a good quality
and favourable to aquaculture. Trout for the table are commonly raised in
concrete walled raceways with a sand, gravel or concrete bottom. Water is
sourced directly without any treatment either from adjacent springs,
maintaining a constant temperature of around 15°C, or pumped from rivers with
temperatures that vary seasonally from 14°C in the winter to 18°C in the
summer. The water is returned to the river systems untreated and poor farming
practices and hygiene measures allow for easy entry and establishment of trout
pathogens. Most farms keep their own broodstock and
operate small hatcheries in order to cover their own needs for fry. When the
quantity or quality of home produced fry is insufficient to meet production
needs, eyed ova are imported, mainly from the US or Denmark.
Perennial disease problems that are responsible
for considerable losses on most trout farms in Greece, comprise primarily
Enteric Red Mouth (ERM) disease caused by the bacteria Yersinia
ruckeri (Savvidis,
1991; Varvarigos, 1999) and Ichthyophonosis
caused by the fungus Ichthyophonus hoferi (Varvarigos, 2000). A
secondary pathogen of concern is cold water disease attributed to Flexibacter psychrophilus
(Schlotfeldt and Alderman, 1995; Varvarigos,
personal observation).
During February 2000 two trout farms in NW
Greece reported mortality rates of 30%-55% among fry and fingerling rainbow
trout in their hatcheries. Mortalities were seen to rise with the increase in
fish handling and crowding during transfer between raceways and vaccination
against ERM. A total of three disease outbreaks at the two farms were reported
to the AquaHealthä laboratory in Athens and samples were submitted for diagnostic
investigation. Although bacterial and fungal pathogens were observed in some of
the fish, the gross clinical signs and mortality levels suggested an underlying
viral aetiology.
Moribund diseased fish, ranging in size from 1g
- 8g average wet weight, were collected from the farms and transported on ice
to the AquaHealthä laboratory. Other fish were delivered live to the laboratory and
observed in an aquarium for a few hours before undergoing post mortem. The
internal organs were examined for the presence of gross lesions and then swab
samples from the liver, spleen and kidney were taken for bacteriological
examination and kidney tissue was excised for virological
examination. Microscopic examinations were also made of fresh smears and
squashes from liver, kidney, spleen and gill tissues from the fish.
Diseased fish were notably darker in colour and
appeared weak and lethargic. Many fish were seen maintaining an almost still
position in the water near the sides or bottom of the raceways and others
showed uncoordinated spiral swimming. Exophthalmia and distension of the
abdomen were evident but no haemorrhagic lesions, ulcers, or fin rot were
observed. Closer inspection revealed that some fry showed darker coloration of
the rear half or third of the body and small, but evident, swellings on the
head (Fig. 1 and Fig. 2). These findings were more evident on the live
specimens during observation in the aquarium and together with the distinct
localised abdominal distension, raised suspicions about the possibility of IPNV
infection (Post, 1987; Schlotfeldt and Alderman,
1995).
Fig. 1. Trout fry of about 1.5g
suffering IPNV infection, February 2000, showing darker colouration of the rear
third of the body (top fish)
and slight swellings on the head (bottom
fish).
Fig. 2. Trout fry from 2g to 7g
suffering IPNV infection, February 2000, showing abdominal distension. Some
fish with slight swellings
on the head. The latter were more
visible while the fish were kept live in an aquarium for observation.
Internal lesions and microscopy 
Internally, the alimentary tract was empty of
food or faeces but moderately distended and filled with greyish mucus. The liver
was dark and inflamed and the kidney and spleen were pale and swollen.
Microscopic examination of tissue imprints and wet mounts from fry originating
from one of the two farms revealed a widespread infection by the fungus Ichthyophonus hoferi.
Large numbers of spherical quiescent cysts of varying sizes, with the
characteristic thick double wall as well as maturing fungal cysts demonstrating
distinct nuclei and active cysts showing plasmodial
germination were observed on all slides (Fig. 3).
Fig. 3. Fresh kidney tissue squash
(unstained) from trout fry suffering Ichthyophonosis
as well as an IPNV infection, February 2000,
showing the characteristic double walled
cysts of the fungus Ichthyophonus hoferi as well as active germinating cysts (x100).
Swab
samples from the liver, spleen and kidney of fish, received live at the AquaHealthä laboratory, were plated onto Tryptone Soya Agar
(TSA). The cultures were incubated at 25 oC for 36 hours. Colonies
appearing were examined by Gram stain and biochemical reactions performed on
API-20E test strips. The more prominent isolates were tested for resistance to
selected antibiotics.
Examination of bacterial cultures
showed growth of round, 1-2mm, pale colonies of Gram negative bacteria from one
third of the fry sampled. These bacteria gave an API-20E profile of 3047165 or:
+ + - | - - -
| - - + | + + + |
+ - - | - + + | + - +
According to Austin and Austin (1999) this
identifies Aeromonas hydrophila.
The bacteria were found resistant in vitro to ampicillin,
amoxycillin and potentiated
sulfonamides, slightly sensitive to furazolidone, oxolinic acid and oxytetracycline and adequately sensitive to flumequine.
Rapid detection of virus: Kidney tissue was sampled from a total of 30
trout fry from the three disease outbreaks and tested with a rapid IPNV test
kit (Diagxotics Inc., 27 Cannon Rd., Wilton, CT
06897, USA). The kit is based on a modified antigen capture enzyme linked immunosorbent assay (ELISA) and tissue extracts were tested
according to the manufacturer's instructions.
Virus isolation and identification: Pools of whole kidney tissue, from 6-8 fry,
were transported, unfrozen, in viral transport medium (Glasgow modification of
minimal essential medium (GMEM) supplemented with 10% new-born calf serum, 2 mM L-glutamine and 1% antibiotic + antimycotic
solution (all Sigma)) to the CEFAS laboratory (Weymouth, UK). In addition,
samples of 30 whole fry packed in dry ice were also submitted for virological examination which followed procedures
recommended for the isolation and identification of IPNV in the OIE Diagnostic
Manual for Aquatic Animal Diseases (OIE 1997). Pools of viscera from each of
five whole fry and the kidney tissue pools were homogenised with a pestle,
mortar and sterile sand, re-suspended in transport medium and clarified by
centrifugation at 2000 rcf for 20 minutes.
Supernatants were then diluted to 1:100 and 1:1000 (w/v) with culture medium
(GMEM supplemented with 10 % foetal bovine serum, 500 I.U./ml penicillin, 500 m
g/ml of streptomycin and 2 mM L-glutamine) and
inoculated onto monolayers of the established fish
cells lines, BF-2 (Bluegill fry) and CHSE-214 (Chinook Salmon Embryo) in
12-well multidishes (Falcon). Inoculated monolayers were incubated at 15°C and examined daily for a cytopathic effect (cpe).
Virus neutralisation: Aquatic birnavirus
strains, West Buxton, Sp (Spjarup), Ab (Abild), He (Hecht) and TV-2
were used to represent the A1, A2, A3, A4 and A5 reference serotypes,
respectively. Rabbit antisera to purified
preparations of these reference viruses had been produced previously as
detailed in Hill and Way (1995). Neutralisation tests were carried out using
96- well cultures of BF-2 cells. Two-fold serial dilutions of each antiserum
from 1/1000 - 1/2,560,000 were made in maintenance medium (MM - GMEM
supplemented with 2% foetal bovine serum) and mixed with an equal volume of
virus diluted in MM to approximately 200 TCID50 /ml. The mixtures
were then incubated at 22°C and, after 1h, 100m l aliquots of each mixture were
transferred to 4 wells of the 96 well culture plates. Control virus wells were
also included containing equal volumes of the virus dilution mixed with MM. The
neutralisation end point was taken as the last dilution where no cpe was observed after 5 days incubation at 15°C.
Kidney tissue sampled from diseased fry and
fingerlings gave strong positive signals when tested with the rapid IPNV ELISA
test kit. This identification of IPNV infection was then confirmed by isolation
of the virus in cell culture. At the CEFAS laboratory, extracts from the
sampled tissue and fry showed a cytopathic effect in
CHSE-214 cells after 48h and in BF-2 cells at 72h post inoculation. Preliminary
neutralisation tests using polyclonal rabbit antiserum raised against the West
Buxton (A1 serotype) and Sp (A2 serotype) reference aquatic birnaviruses
indicated that the Greek virus was more closely related to the A2 serotype than
it was to the A1 serotype. The A2 serotype is the most prevalent of the
European serotypes while A1 is the major North American serotype (Hill and Way,
1995).
More extensive neutralisation tests were then
carried out which confirmed that the Greek IPNV isolate falls into the A2
serotype of the aquatic birnaviruses (Table 1).
Table 1.
Cross-neutralisation
titres of aquatic birnavirus reference serotypes
and the IPN virus from Greece.
|
|
|
|
Antiserum |
|
|
|
Virus |
A1 |
A2 |
A3 |
A4 |
A5 |
|
A1 |
1280000* |
20000 |
<1000 |
1000 |
<1000 |
|
A2 |
4000 |
640000 |
2000 |
20000 |
2000 |
|
A3 |
8000 |
2000 |
1280000 |
1000 |
20000 |
|
A4 |
1000 |
4000 |
1000 |
1280000 |
20000 |
|
A5 |
4000 |
1000 |
40000 |
<1000 |
640000 |
|
Greek IPNV |
20000 |
320000 |
2000 |
4000 |
2000 |
* - Neutralisation end point titre taken as the last antiserum dilution
where no cpe was observed at the end of the
incubation period.
This report represents the first confirmed
isolation of IPN virus from farmed rainbow trout in Greece. The only other
record of the presence of IPN disease in Greece was a personal communication
from C.Carlson cited by Wolf (1988). However, this
finding has not been confirmed in a published report. The IPNV infected fry
populations almost certainly originated from imported, eyed ova. On one farm,
adjacent raceways holding fry that were progeny of the farm's own brood-stock
were seen to suffer much lower rates of morbidity and mortality. The Greek IPNV
isolate was found to belong to the A2 serotype of aquatic birnaviruses and the presence of this serotype, in combination with
the records of introductions at the farm sites, strongly suggest that the
origin of the imported ova is Denmark.
The two trout farms, where IPN has been
verified, are situated at different locations. One is sourcing water directly
from a spring, the other from an unconnected river system. It is possible that
IPN disease has already spread, with fish transfers, to other farms on other
river systems in Greece. In order to implement control measures and prevent
re-infections of IPN it is important to assess the magnitude of the disease
spread. There will be a need to screen wild fish as well as the farmed
populations in order to achieve this.
Unless disease control measures are implemented
to prevent re-infections, IPN has to be considered in the future together with
ERM and Ichthyophonosis as a potential major cause of
economic loss. If the disease is allowed to become widespread then Greek trout
producers will have to introduce increasing numbers of fry each year in order
to achieve their planned production.
Austin, B. and Austin, D.
A. (1999) Bacterial fish pathogens: Diseases of farmed and wild fish. Third
(Revised) edition. Springer, London, p 176-180.
Hill, B. J. and Way, K.
(1995) Serological classification of IPN virus and other aquatic birnaviruses. Annual Review of Fish Diseases. 5: 55-77.
Post, G. (1987) Textbook of
Fish Health. Revised edition. T.F.H. Publications Limited, Berkshire, England,
p 94-100
Savvidis, G. K. (1991) Yersinia
ruckeri in trout. First isolation in Greece.
Bulletin of the Hellenic Veterinary Medical Society. 42(3): 169-173 (In
Greek).
Schlotfeldt, H. J. and Alderman, D. J. (1995)
What should I do? A practical guide for the fresh water fish farmer. ISBN
0-9526242-0-6. The European Association of Fish Pathologists, Weymouth, 60pp.
Varvarigos, P. (1999) Enteric red mouth. A
detrimental disease for the Greek trout farming industry. Fishing News. 211:
51-54 (In Greek)
Varvarigos, P. (2000) Ichthyophonosis.
A killer of farmed fish in fresh and marine waters. Fishing News. 231: 65-72 (In
Greek)
Varvarigos, P., and Way, K. (2002) First
Isolation and identification of the Infectious Pancreatic Necrosis (IPN) virus
from rainbow trout Onchorhynchus mykiss fingerlings in Greece. Bull. Eur.
Ass. Fish Pathol., 22(3) 2002, 195-200
Wolf, K. (1988) Fish
viruses and fish viral diseases. Cornell University Press, Ithaca and London.
VETCARE Ô
VETERINARY SERVICES TO AQUACULTURE AND
DISTRIBUTION OF FISH HEALTH PRODUCTS
Copyright (c) Dr. Panos Varvarigos.