Virus Research 63 (1999) 131134

Short communication
Dual challenges of infectious pancreatic necrosis virus and
Vibrio carchariae in the grouper, Epinephelus sp.
K.-K. Lee
a,
*, T.-I. Yang
a
, P.-C. Liu
a
, J.-L. Wu
b
, Y.-L. Hsu
b
a
Department of Aquaculture, National Taiwan Ocean Uni6ersity, 2, Pei -Ning Road, Keelung, Taiwan, ROC
b
Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan, ROC
Abstract
The grouper industry in Taiwan faces serious threats from various disease problems. The present study investigated
dual challenges with infectious pancreatic necrosis virus (IPNV) and Vibrio carchariae in the grouper (Epinephelus
sp.). The sh were infected with IPNV for 2 weeks prior to a secondary infection with the bacteria, or vice versa, by
either immersion (10
3
10
4
TCID
50
IPNV per ml, 10
6
10
7
colony forming units (CFU) Vibrio per ml) or by
intraperitoneal injection (10
3
10
4
TCID
50
IPNV per g sh or 10
7
CFU Vibrio/g sh) challenges. Mass mortalities
occurred in sh infected with IPNV for 2 weeks prior to the infection with the bacteria, or vice versa, in either
immersion or intraperitoneal injection challenges. The bacterium could only survive in seawater or brackish water
similar to that of cultured groupers. 1999 Elsevier Science B.V. All rights reserved.
Keywords: Dual challenge; Grouper; IPNV; Pathogenicity; Vibrio carchariae
www.elsevier.com/locate/virusres
Vibrio species are natural habitants of seawater
and brackish water and widely distributed
throughout the world. However, some species
have exhibited clinical signicance for aquatic an-
imals and are recognized as potential pathogens.
Vibriosis is one of the most serious diseases in
cultured sh and shellsh (Ezura et al., 1980;
Brisinello et al., 1985; Egidius, 1987; Lightner,
1988; Rasheed, 1989; Kusuda and Salati, 1993;
Lee, 1995; Liao et al., 1996). Various Vibrio spe-
cies have been demonstrated to be the causative
agents of disease in cultured marine sh in Japan
and Taiwan (Kusuda and Salati, 1993; Liao et al.,
1996). Recently, we isolated and characterised
Vibrio carchariae as a causative agent of gastroen-
teritis in groupers (Yii et al., 1997), but which
caused mass mortality of the sh only after chal-
lenge dosage.
Infectious pancreatic necrosis virus (IPNV) is a
member of a group of aquatic birnaviruses which
have been isolated from a wide range of aquatic
animals and which are recognised as important
sh pathogens (Mcknight and Roberts, 1976;
Chen et al., 1984; Sorimachi and Hara, 1985;
Wolf, 1988; Kimura and Yoshimizu, 1991; Hill
* Corresponding author. Tel.: +886-2-24622192; fax: +
886-2-24633150.
0168-1702/99/$ - see front matter 1999 Elsevier Science B.V. All rights reserved.
PII: S0168- 1702( 99) 00066- 0
K.-K. Lee et al. / Virus Research 63 (1999) 131134 132
and Way, 1995; Johansen and Sommer, 1997).
The virus is shed by carriers with their sex prod-
ucts and in their faeces (Mcknight and Roberts,
1976; Wolf, 1988). The virus has been frequently
diagnosed in sh in Japan and Taiwan (Chen et
al., 1984; Sorimachi and Hara, 1985; Kimura and
Yoshimizu, 1991), especially in marine sh such
as yellowtail, Japanese ounder and sea bream.
As the production and scale of marine sh culture
in Taiwan has expanded this decade, the control
of diseases caused by viral and bacterial patho-
gens has become increasingly important. The
present study investigates the effect of dual chal-
lenges of both IPNV and vibrio in the grouper.
Infectious pancreatic necrosis virus strain T42G
was a gift from Dr Y.-L. Hsu, Institute of Zool-
ogy, Academia Sinica, Taipei, Taiwan. Chinook
salmon embryo (CHSE-214) cell lines were grown
in minimum essential medium (MEM) in Earles
salt (Flow Laboratories), supplemented with
foetal bovine serum to 10% (MEM-10), penicillin
(100 IU/ml), streptomycin (100 mg/ml), gentam-
icin (25 mg/ml) and fungizone (0.25 mg/ml). The
strain T42G was propagated in CHSE-214 cells
for 7 days at 1820C (Hsu et al., 1993). Vibrio
carchariae strain EmI82KL was previously iso-
lated from the transparent yellow uid of the
swollen intestine of diseased grouper, Epinephelus
coioides (Yii et al., 1997).
Groupers (Epinephelus sp.) were held in tanks
(2500 l) supplied with air-lifted 3% salinity seawa-
ter at 2528C. Fish weighing 0.120.32 g and
1.52.0 g were challenged by immersion for 2 h in
a suspension of IPNV (10
3
10
4
TCID
50
/ml) or V.
carchariae (10
6
10
7
colony forming units (CFU)/
ml) and held for 2 weeks prior to the second
immersion with the other pathogen (Table 1).
Groupers weighing 46 g and 1520 g were chal-
lenged by intraperitoneal (i.p.) injection with
IPNV (10
3
10
4
TCID
50
/g sh) or vibrio (10
7
CFU/g sh) and held for 2 weeks prior to the
second intraperitoneal injection with vibrio or
IPNV.
Suspensions of Vibrio carchariae strain
EmI82KL were held at 25C in seawater (34 ppt),
brackish water (15 and 25 ppt) or freshwater,
without supplementing nutrients. At intervals, the
concentration of viable bacteria was determined
in duplicate by plate count on tryptic soy agar
(supplemented with 2% NaCl) for a duration of
14 weeks.
Among the small groupers (0.120.32 g), all
sh survived initial challenge with IPNV (10
3
TCID
50
/ml) for 2 weeks, but then died within 2
weeks following a second challenge using 10
7
CFU/ml V. carchariae (Table 1). Of the larger
groupers (1.52.0 g) used in the immersion chal-
lenge 92% survived initial challenge with IPNV
(10
4
TCID
50
/ml) for 2 weeks. Of these, 64% sur-
vived for 2 weeks following the second challenge
using V. carchariae (10
7
CFU/ml) (Table 1). The
results reveal that size of the grouper was in-
versely related to its degree of susceptibility to V.
carchariae. This was further conrmed by chal-
Table 1
Challenges of IPNV strain T42G and V. carchariae strain EmI82KL in grouper fry by immersion
Surviving sh after Number (size) of groupers Agent for second Agent for rst Surviving sh after
2 weeks challenge used challenge 2 weeks
50 IPNV (10
3
TCID
50
/ml) 0 Vibrio (10
7
CFU/ml) 50 (0.120.32 g)
IPNV (10
4
TCID
50
/ml) 11 12 (1.52.0 g) Vibrio (10
7
CFU/ml) 7
Vibrio (10
7
CFU/ml) 50 (0.120.32 g) 0
0 IPNV (10
3
TCID
50
/ml) 12 20 (0.120.32 g) Vibrio (10
6
CFU/g sh)
Vibrio (10
7
CFU/ml) 12 (1.52.0 g) 12 IPNV (10
4
TCID
50
/ml) 0
K.-K. Lee et al. / Virus Research 63 (1999) 131134 133
Table 2
Inoculation of IPNV strain T42G and V. carchariae strain EmI82KL in grouper fry by intraperitoneal injection
Agent for rst Surviving sh after Surviving sh after Number (size) of Agent for second
groupers used 2 weeks challenge challenge 2 weeks
10 Vibrio (10
7
CFU/g sh) 10 (46 g) 0 IPNV (10
3
TCID
50
/g sh)
10 Vibrio (10
7
CFU/g sh IPNV (10
4
TCID
50
/g sh) 0 10 (46 g)
10 10 (1520 g) IPNV (10
4
TCID
50
/g sh) Vibrio (10
7
CFU/g sh 0
10 IPNV (10
3
TCID
50
/g sh) Vibrio (10
7
CFU/g sh) 0 10 (1520 g)
immersion or intraperitoneal injection cause mass
mortality in the groupers. In the immersion study,
a higher dosage of V. carchariae EmI82KL exhib-
ited similar pathogenicity in the groupers as that
described previously (Yii et al., 1997). Recently,
Johansen and Sommer (1997) demonstrated that
challenge with Vibrio salmonicida (intraperitoneal
injection) 3 weeks after IPNV infection (bath
challenge) gave a cumulative mortality of 39.7%
in the group of Atlantic salmon post-smolts in-
fected with both IPNV and the bacterium while
signicantly fewer sh (15%) died in a control
group infected with only the bacterium. It seems
that challenge with both IPNV and V. salmonicida
were necessary to cause higher cumulative mortal-
ity in these post-smolts. Similarly, in our present
study, we obtained mass mortality in both immer-
sion and intraperitoneal injection challenges only
in dual infections if the pathogens were delivered
lenging 50 smaller groupers with a dose of 10
7
CFU/ml of V. carchariae which resulted in 100%
mortality (Table 1).
Of the small groupers (0.120.32 g) 60% sur-
vived for 2 weeks after initial challenge with V.
carchariae (10
6
CFU/ml). All the sh died within
2 weeks after a second challenge using IPNV at a
dose of 10
3
TCID
50
/ml (Table 1). In a further
study using larger groupers, all the sh (1.52.0 g)
survived for 2 weeks after challenge with V. car-
chariae (10
7
CFU/ml), while all sh died within 2
weeks following a second challenge using IPNV at
a dose of 10
4
TCID
50
/ml (Table 1).
These results reveal that mass mortalities occur
in groups of sh challenged by immersion with
both IPNV and V. carchariae in either order, but
not by a single immersion challenge with either
agent when administered at moderate doses.
All the smaller groupers (46 g) used in the
injection challenge survived for 2 weeks after in-
oculation with IPNV (10
3
or 10
4
TCID
50
/g sh),
but then died within 2 weeks following the second
inoculation with V. carchariae at 10
7
CFU/g sh
(Table 2). In a further experiment where larger
(1520 g) sh were injected in the opposite order,
all the groupers survived for 2 weeks after initial
inoculation with V. carchariae (10
7
CFU/g sh),
while all the sh died within 2 weeks after the
subsequent injection with IPNV at 10
3
or 10
4
TCID
50
/g sh (Table 2). Thus no mortality oc-
curred in the sh challenged by intraperitoneal
injection with either IPNV or V. carchariae alone
and held for 2 weeks. However, if a second infec-
tion with the other agent was given, 100% of all
sh died in the subsequent 2-week period.
The above results showed that dual infections
with IPNV and V. carchariae initiated by either
Fig. 1. Survival of Vibrio carchariae strain EmI82KL in seawa-
ter (34 ppt) and brackish water (25 and 15 ppt) at 25C
without supplementing nutrients. Each data point represents
the mean of two similar determinations.
K.-K. Lee et al. / Virus Research 63 (1999) 131134 134
at moderate doses. Our results conrmed the syn-
ergistic effects of these sh pathogens and showed
that a second challenge with either IPNV or V.
carchariae caused mass mortality.
Vibrio carchariae strain EmI82KL did not sur-
vive in freshwater, while it remained viable in
seawater or brackish water without supplementing
nutrients for at least 14 weeks (Fig. 1). An appar-
ent increase of viable counts, with highest counts
in 15 ppt brackish water, was determined at week
1, indicating that numbers of the bacteria in-
creased as a response to nutrient deprivation espe-
cially in lower salinity water in the early phase.
This is in agreement with ndings by Morita
(1982) who suggested that a response to starva-
tion by marine bacteria might be rapid division.
The present results suggest that this Vibrio spe-
cies might survive in marine or brackish systems
much longer because of the presence of high
amounts of organic matter in systems where
marine sh are reared. While IPNV is generally
recognised as a primary pathogen and frequently
diagnosed in cultured marine sh, our results
indicate the control of secondary invaders such as
Vibrio species may be crucial to successful sh
health management.
Acknowledgements
The work was supported by a grant NSC-86-
2311-B-019-002-B23 from the National Science
Council, ROC. We thank W.-M. Su and C.-C. Tu
for their technical assistance.
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