First isolation and identification of the
Infectious Pancreatic Necrosis (IPN) virus from rainbow trout
Onchorhynchus mykiss
fingerlings farmed in
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Authored by Dr. PANOS VARVARIGOS Freelance
Veterinarian – Fish Pathologist |
CONTENTS:
|| Abstract
|| Introduction || Fish samples || Gross clinical signs || Internal lesions & microscopy ||
|| Bacterial cultures || Virological examination || Conclusion || References ||
Copyright © Dr. Panos Varvarigos.
AquaHealthTM
Laboratory.
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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.
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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.
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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.
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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.
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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).
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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.
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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.
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Antiserum |
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|
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.
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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.
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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.
Author:
Dr. Panos Varvarigos
Freelance
Veterinarian – Fish Pathologist, Athens, Greece.
AquaHealthTM Laboratory.
Reproduction
of this website (or parts of it) is illegal and strictly forbidden.
No rights can be derived from this website.
Disclaimer:
Every effort has been made to ensure that the
information is accurate until the date of last editing. It is based upon the
accumulated personal experience of applied veterinary work. The author cannot
take responsibility for incorrect interpretation or any resulting consequences.
The contents may be used as an educational guide and are definitely not meant
to become a stand-alone diagnostic tool or operations manual.
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