Isolation of Vagococcus salmoninarum in Rainbow Trout,

Oncorhynchus mykiss (Walbaum), Broodstocks:

Characterization of the Pathogen
I. Ruiz-Zarzuela
1
*, I. de Blas
1
, O. Girone s
1
, C. Ghittino
2
and J.L. Mu zquiz
1
1
Fish Pathology Laboratory, Veterinary Faculty, University of Zaragoza, c/Miguel Servet
177, 50013 Zaragoza, Spain;
2
Fish Pathology Laboratory, IZS ^ State Veterinary
Institute, Perugia, Italy
*Correspondence: E-mail: imaruiz@posta.unizar.es
Ruiz-Zarzuela, I., de Blas, I., Girone s, O., Ghittino, C. and Mu zquiz, J.L., 2005. Isolation of Vagococcus
salmoninarum in rainbow trout, Oncorhynchus mykiss (Walbaum), broodstocks: characterization of the
pathogen. Veterinary Research Communications, 29(7), 553^562
ABSTRACT
Coldwater `streptococcosis', caused by Vagococcus salmoninarum, is an emerging disease of rainbow
trout in the European Union, causing mortality rates up to 50% in broodstock during the spawning
period, with water temperature of 10^128C. A study to determine the presence and role of this
bacterium was undertaken using classical bacteriological techniques conrmed with polymerase chain
reaction. This is the rst report of isolation of V. salmoninarum in relation to outbreaks of mortality in a
rainbow trout farm devoted exclusively to broodstock rearing in Spain. A total of 10 isolates of V.
salmoninarum were characterized by their morphological, cultural, physiological, biochemical and
enzymatic traits. Some dierences were observed in parameters such as growth on MacConkey agar,
H
2
S production, acid production from starch, and some other minor variations. Isolates were sensitive
to erythromycin and oxytetracycline tested in vitro, but treatments conducted in the eld were
ineective. An attempt at vaccination did not provide encouraging results.
Keywords: antibiotics, diagnosis, rainbow trout, Vagococcus salmoninarum
Abbreviations: cfu, colony-forming unit; PCR, polymerase chain reaction; SxT, sulfamethoxazole^
trimethoprim; TSI, triple-sugar^iron medium
INTRODUCTION
The increasing level of international trade in aquatic cultured species is a signicant
cause of the recent increase in emerging diseases. One of the most important
pathological conditions aecting freshwater and marine sh is `streptococcosis', a
systemic process causing high mortality rates, particularly in large sh, and responsible
for heavy economic losses in aquaculture (Mu zquiz et al., 1999).
Aetiologically, `streptococcosis' has been dened as a complex of diseases caused by
several agents belonging to dierent genera and species of Gram-positive cocci. From a
clinical point of view, two groups of infections may be distinguished: warmwater
infections, caused by those cocci that are pathogenic for both freshwater and marine
sh at water temperatures above 158C, and coldwater infections, caused by those
Veterinary Research Communications, 29 (2005) 553^562
#2005 Springer. Printed in the Netherlands
553
coccus species that are pathogenic exclusively for salmonid sh at water temperatures
below 128C (Ghittino and Mu zquiz, 1998; Ghittino and Pedroni, 2001). Warmwater
Gram-positive coccal infections are more widespread and severe, and include
lactococcosis caused by Lactococcus garvieae (formerly Enterococcus seriolicida)
(Kusuda et al., 1991; Eldar et al., 1996) and three Streptococcus infections: Strepto-
coccus dicile (formerly Streptococcus agalactiae) (Eldar et al., 1994; Vandamme et al.,
1997), Streptococcus iniae (formerly Streptococcus shiloi) (Eldar et al., 1995) and
Streptococcus parauberis (Dome nech et al., 1996; Austin and Austin, 1999). Coldwater
Gram-positive coccal infections are less widespread, and include vagococcosis caused
by Vagococcus salmoninarum (Wallbanks et al., 1990), carnobacteriosis due to
Carnobacterium piscicola (formerly Lactobacillus piscicola) (Collins et al., 1987) and
Lactococcus piscium infection (Williams et al., 1990). A wide range of cultured sh
species may be aected by `streptococcosis', such as yellowtail, Seriola quinqueradiata
(Temminck and Schlegel), rainbow trout, Oncorhynchus mykiss (Walbaum), tilapia,
Oreochromis sp., turbot, Scophthalmus maximus (L.), European sea bass, Dicentrarchus
labrax (L.) and gilthead sea bream, Sparus aurata (L.) (Kusuda et al. 1991; Ghittino
and Prearo, 1992; Eldar et al., 1994; Schmidtke and Carson, 1994; Toranzo et al.,
1994).
Vagococcosis is now considered an emerging disease for the European trout industry
(Daly, 1999), aecting either subadult or adult rainbow trout (4150^200 g), with
mortality rates of 20^50%. Outbreaks occur with a water temperature range of 10^
128C and in broodstock are generally associated with post-spawning stress (Michel et
al., 1997; Ghittino et al., 1999). In addition to France and Italy, V. salmoninarum has
frequently been isolated during recent years in Spain. Isolates were resistant to most of
the registered antibiotics for aquacultural use in the European Union (CEC, 1990,
1996), vaccination therefore represents an alternative strategy that can be used to
reduce the impact of the disease.
The aim of this work was the characterization of the V. salmoninarum isolated from a
Spanish farm during a period of three years as part of an investigation of an outbreak.
MATERIALS AND METHODS
This work was undertaken during the period from 1999 to 2002 in a farm devoted
exclusively to broodstock rearing for the production of rainbow trout eggs. The farm is
located in north-eastern Spain and takes part in a health-improving programme,
undergoing regular health monitoring.
During this period, amoxicillin, oxytetracycline, sulfamethoxazole^trimethoprim
(SxT) and erythromycin were used to treat disease outbreaks by oral administration.
The medicated feed was commercially prepared by Trouw, S.A. (Spain). The dose rates
of the respective antibiotics were 90 mg, 80 mg, 30 mg and 100 mg of active ingredient
per kilogram of sh body weight (bw) per day. All feed was hand-fed, twice per day, at
1% bw per day. Administration of medicated feed started 1 day after disease outbreaks
and continued for 8^10 consecutive days. Water was supplied from a spring with a
forced oxygenation system.
554
Sample collection
When an abnormal increase of mortality was observed in the farm, at least 14 shes
were sampled in order to detect V. salmoninarum in a population of 1500 animals with
a prevalence of more than 20% in a random sample and 95% of level of condence,
calculated using Win Episcope 2.0 (Thruseld et al., 2001). However, sampling was not
completely random, since moribund sh were preferentially collected to improve the
probability of detection of bacterium. Moreover, samples were also collected within the
periodic health-monitoring programme, with at least 14 shes in each one. Thus 15
samplings were performed during the study period (the total number of collected shes
was 241): six samplings were part of the sanitary programme and the rest corresponded
to outbreak investigations.
Necropsy of all collected shes was made in situ; samples for microbiological
analysis were collected and also organs from symptomatic shes, which were xed in
10% formalin for histopathology.
Microbiological examination
Samples of kidney, brain and eye, from each sh collected, were inoculated onto blood
agar supplemented with 5% debrinated sheep's blood (bioMe rieux, France) and bile^
aesculin^azide agar (Difco, USA) and incubated in air for 48^72 h at 208C.
Isolated colonies were identied by physiological, biochemical and enzymatic
characterization. Gram staining and cell morphology, motility, oxidase and catalase
activity, acid production under aerobic and anaerobic conditions with O/F basal
medium supplemented with 0.5% of glucose (Difco), H
2
S production on triple-sugar^
iron medium (TSI) (Difco), growth at dierent temperatures (10, 20, 37 and 428C),
growth on trypticase^soy agar containing 6.5% NaCl (Difco), growth on brain^heart
infusion agar at pH 9.6 (Difco) and haemolysin production were assayed. Additional
tests were performed using API 20NE, API 20STREP and API 50CHL systems
(bioMe rieux).
Conrmation by PCR
An experimental PCR assay was developed to conrm the biochemical diagnostics.
The method used to lyse Gram-positive cocci and to extract the DNA is described
elsewhere (Eldar et al., 1994). V. salmoninarum species-specic PCR assay was
performed on all isolates (the target sequence is the highly specic 16S rRNA gene),
using the following diagnostic primers produced by the Hebrew University of
Jerusalem, Israel: pSal-1 (5'-GTTTTAGCCGCATGGCTGAGATAT-3') and pSal-2
(5'-AGGTGGGAACAGTTACTCTCCCA-3'). The process included denaturation at
948C for 3 min and amplication performed with 35 cycles of 948C for 60 s, 558C for
60 s and 728C for 60 s. The reaction was terminated by an extension for 10 min at 728C.
PCR reactions were then analysed using 2% agarose gel electrophoresis stained with
ethidium bromide A segment of 300 bp indicates a positive reaction (data not shown).
555
Antimicrobial susceptibility
Strains were tested for sensitivity to 10 antibiotics on Mueller^Hinton agar (Difco) at
208C for 48^72 h and disc-diusion assay to amoxicillin (30 mg), ampicillin (10 mg),
erythromycin (15 mg), lincomycin (2 mg), neomycin (30 mg), oxytetracycline (30 mg),
penicillin G (10 IU), streptomycin (10 IU), SxT (23.75/1.25 mg) and vancomycin (30
mg). The zones of inhibition were measured and susceptibility was determined.
Histopathology
Eyes, heart, liver and the upper part of the head from 42 symptomatic shes were
processed using standard techniques of histopathology (Hibiya, 1994).
RESULTS
Clinical and histopathological ndings
Vagococcus salmoninarum was isolated from 10 samplings out of 15. Isolation occurred
during four disease outbreaks and six samplings along with the periodic health
monitoring programme (Table I). Mortality attributed to this pathogen was between
11% and 36% during the disease outbreaks, but no increase beyond the expected
mortality rate was observed in the other cases. No other pathogen was detected during
the study. All outbreaks were recorded in post-spawning sh, with a water temperature
range of 10.5^11.58C.
TABLE I
Main variables related to V. salmoninarum isolation
Water
Mortality Sexual maturity temperature
Collection date Aim of sample rate (%) status (8C)
May^1999 Outbreak 11 Post-spawning 9.1
July^1999 Outbreak 34 Post-spawning 10.5
Sept^1999 Outbreak 27 Post-spawning 10.8
Jan^2000 Health monitoring 0 Pre-spawning 6
Jan^2000 Health monitoring 0 Yearling 6.2
April^2000 Health monitoring 0 Pre-spawning 7.5
April^2000 Health monitoring 0 Post-spawning 7.5
July^2000 Outbreak 36 Post-spawning 11.5
March^2002 Health monitoring 0 Pre-spawning 7.3
March^2002 Health monitoring 0 Post-spawning 7.5
556
External signs observed in populations of diseased trout were lethargy, anorexia,
melanosis, marked monolateral or bilateral exophthalmos leading to frequent disrup-
tion of the eyeball, keratitis, haemorrhaging in the periocular area and pale gills. Some
sh showed petechial haemorrhages at the base of ns, the mouth and around the anus.
Macroscopically, internal signs such as pale liver with small petechial haemorrhages,
pericarditis, spleen enlargement and/or congestion of meninges were frequently
observed.
Histologically, a severe panophthalmitis with clusters of bacteria in the vitreous
humour (34 out of 42), and meningitis (25 out of 42) were frequently observed in
symptomatic shes. Inammation and necrosis of epicardium, endocardium and
myocardium, characterized by the presence of diuse accumulation of leukocytes and
lymphocytes in the spaces among the muscle bres (7 out of 42), as well as hyperaemia
and oedema of the liver with degeneration of the hepatocites (5 out of 42), were also
observed.
Phenotypic and genomic characterization
Isolated strains showed the phenotypic traits of V. salmoninarum, which are compara-
tively summarized in Table II.
Species-specic PCR conrmed the identication of all isolates as V. salmoninarum.
The amplied 300 bp products indicate a positive reaction (Figure 1).
Antimicrobial susceptibility
All isolates (n = 10) were sensitive to erythromycin and oxytetracycline, and were
resistant to lincomycin, penicillin, streptomycin and SxT. Eight isolates were resistant
to ampicillin and amoxycillin, and two showed an intermediate antibiotic sensitivity.
Results obtained with vancomycin were also variable (Table III).
DISCUSSION
This is the rst isolation of V. salmoninarum in Spain, related to a disease outbreak in
broodstock rainbow trout that occurred during May 1999. Clinical and anatomo-
histopathological ndings were very similar to those previously described by Michel
and colleagues (1997) in France and Ghittino and colleagues (1999) in Italy. However,
mortality rates were lower than those indicated by these authors.
According to the epidemiological data collected during the disease outbreaks and
along with the periodic sh health monitoring, we can suggest that water temperature
between 10 and 118C and the spawning period are the most important risk factors for
high mortality in adult rainbow trout females.
With respect to cultural, morphological and biochemical characterization of the
Spanish isolates of V. salmoninarum, the most important dierences from the ones
557
TABLE II
Variable biochemical and physiological reactions of V. salmoninarum isolates from rainbow
trout
a
Published results

V. salmoninarum
b
V. salmoninarum
c
Present study
Colony size NA 0.5^1 mm 51 mm
Gram + + +
Shape sr/cb cb cb
Motility ^ ^ ^
Haemolysis a a (weak) a
Oxidase ^ ^ ^
Catalase ^ ^ d (0/2/8)
Oxidative/fermentative F F F
Urease NA ^ ^
Indole NA ^ ^
NO
3
reduction ^ ^ ^
Aesculin hydrolysis + + +
H
2
S production on TSI + + d (2/0/8)
Growth:
at 108C + NA +
at 208C NA NA +
at 378C NA + ^
at 428C ^ ^ -
at pH 9.6 + + +
at 6.5% NaCl ^ ^ ^
on MacConkey agar + NA d (0/2/8)
Vogues^Proskauer NA + +
Hippurate hydrolysis ^ + d (0/2/8)
Pyrrolidonyl arylamidase + + d (8/2/0)
a-Galactosidase NA ^ d (3/0/7)
b-Glucuronidase NA ^ ^
b-Galactosidase NA ^ ^
Alkaline phosphatase NA + d (0/2/8)
Leucine arylamidase NA + +
Arginine dihydrolase ^ ^ ^
Acid production from:
Ribose NA d +
Mannitol ^ ^ d (2/0/8)
Sorbitol ^ d d (2/0/8)
Lactose ^ ^ d (0/3/7)
Trehalose + + +
Inulin ^ ^ ^
Ranose ^ NA ^
Starch ^ ^ d (8/0/2)
Glycogen NA ^ ^
558
TABLE II (continued)
Published results

V. salmoninarum
b
V. salmoninarum
c
Present study
Glycerol ^ ^ ^
Erythritol NA ^ ^
d-Arabinose NA ^ ^
l-Arabinose ^ ^ d (2/0/8)
d-Xylose ^ [+] d (0/1/9)
l-Xylose NA ^
Adonitol ^ NA ^
b-Methylxylidose NA NA ^
Galactose ^ ^ ^
d-Glucose NA + +
d-Fructose NA + +
d-Mannose NA + +
l-Sorbose NA d d (0/3/7)
Rhamnose NA ^ d (0/3/7)
Dulcitol ^ ^ ^
Inositol NA ^ d (0/2/8)
a-Methyl-d-mannoside NA ^ ^
a-Methyl-d-glucoside NA ^ ^
N-Acetylglucosamine NA + +
Amygdalin NA + +
Arbutin NA + +
Salicin NA + +
Cellobiose NA d +
Maltose d d +
Melibiose ^ ^ ^
Saccharose NA NA d (2/4/4)
Melezitose NA ^ d (0/2/8)
Xylitol NA ^ ^
b-gentiobiose NA d +
d-Turanose NA ^ ^
d-Lyxose NA ^ ^
d-Tagatose NA + +
d-Fucose NA ^ ^
l-Fucose NA d ^
d-Arabitol NA ^ ^
l-Arabitol NA ^ ^
Gluconate NA ^ ^
2-Ceto-gluconate NA [+] ^
5-Ceto-gluconate NA ^ ^
a
+, positive; ^, negative; [+], delayed or weak; d, variable character (positive/weak/negative indicated in
parentheses); cb, coccobacilli; sr, short rods; NA, not available
b
Data for V. salmoninarum from Schmidtke and Carson (1994)
c
Data for V. salmoninarum from Michel and colleagues (1997)
559
Figure 1. PCR analyses of V. salmoninarum isolates. Agarose gel electrophoretic analyses of
PCR products of 300 pb were amplied. Lane M, length marker; lanes 1^10, samples
TABLE III
Comparison of results for sensitivity to antibiotic discs of the bacteria isolated (n = 10) from
the rainbow trout farm
a
Antibiotic Zone size (mm) Sensitivity
Amoxicillin (30 mg) 0^17 R(8), I(2)
Ampicillin (10 mg) 0^15 R(8), I(2)
Erythromycin (15 mg) 22^24 S
Lincomycin (2 mg) 8 R
Neomycin (30 mg) 16^18 I
Oxytetracycline (30 mg) 22-24 S
Penicillin G (10 IU) 0 R
Streptomycin (10 IU) 0 R
SxT (23.75/1.25 mg) 0 R
Vancomycin (30 mg) 15^21 I(7), S(3)
a
R, resistant; I, intermedium; S, sensitive; number of strains with feature is given in parentheses
560
described by Schmidtke and Carson (1994) and Michel and colleagues (1997) are the
following: growth on MacConkey agar and H
2
S production (our culture medium was
TSI instead of lead acetate medium) were mostly negative, while acid production from
starch was mostly positive. Dierences were also detected on tests for catalase,
hippurate hydrolysis, pyrrolidonyl arylamidase, a-galactosidase, alkaline phophatase
and metabolism of d-xylose. Other minor variations were observed for mannitol, l-
arabinose and l-sorbose.
It is interesting to note that sensitivity to erythromycin and oxytetracycline was
constant in V. salmoninarum strains isolated during the study period, but treatments
with these antibiotics (at dose and time indicated) were eective only for short periods
(5^7 days), and a continuous delivery was necessary to reduce mortality, with
consequent increase of antibiotic resistance risk (especially observed with amoxicillin
and SxT).
Vaccination was seen as an alternative method for controlling the disease, but during
an additional experiment no signicant results were obtained with 0.2 ml intraper-
itoneal injection of V. salmoninarum bacterin containing 1.8610
8
cfu (unpublished
data). Also, an empirical relationship between the correlation of survival curves and
water temperature can be observed, allowing the conclusion that a constant water
temperature higher than 10.58C during daytime hours could be a trigger factor for
increase of mortality due toV. salmoninarum. An incubation period of 10 days from the
occurrence of this factor until the mortality peak is suggested.
ACKNOWLEDGEMENTS
The authors express their thanks to S. Stockermans and A. Mun oz, who helped with
the English, and for the technical assistance of J. Oro s and R. Claver in microbiologi-
cal diagnostics.
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