Diversity and pathogenecity of Vibrio species in cultured bivalve molluscs: Vibrio spp., bivalve molluscs, pathogens

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Diversity and pathogenecity of Vibrio species in cultured bivalve molluscs.

Journal: Environmental Microbiology and Environmental Microbiology

Reports

Manuscript ID: EMI-2009-0874.R1

Manuscript Type: EMIR - Minireview

Journal: Environmental Microbiology Reports

Date Submitted by the Author:

12-Dec-2009

Complete List of Authors: Hidalgo, Roxana; Universitat Rovira i Virgili, Ciencias Mèdiques Bàsiques Balboa, Sabela; Universidad de Santiago de Compostela, Microbiologia y Parasitologia Romalde, Jesús; Universidad de Santiago de Compostela, Microbiologia y Parasitologia Figueras, Maria; Rovira i Virgili, Microbiology

Keywords: bacteria, microbial communities, pathogen ecology

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Diversity and pathogenecity of Vibrio species in cultured bivalve molluscs. 4

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Roxana Beaz-Hidalgo1, Sabela Balboa 2, Jesús L. Romalde2 & Maria José Figueras1* 7

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1 Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina i Ciències de la Salut. 9

IISPV. Universitat Rovira i Virgili, Reus, Spain. 10

2 Departamento de Microbiología y Parasitología. CIBUS. Facultad de Biología. Universidad de 11

Santiago de Compostela. Santiago de Compostela, Spain. 12

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Running title: Vibrio spp., bivalve molluscs, pathogens. 16

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Revised version Ms. EMI-2009-0874. December, 2009 19

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*Corresponding author. Mailing address: Unitat de Biologia y Microbiologia. Facultat de 25

Medicina i Ciencia de la Salut. Universitat Rovira i Virgili. Sant Llorenc 21, 43201 Reus, 26

Barcelona, Spain. Phone: +34-97-759321. E-mail: [email protected] 27

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Summary 30

Shellfish production is seriously affected by bacterial pathogens that cause high losses in 31

hatcheries and in the aquaculture sector. A number of Vibrio species are considered important 32

pathogens and have provoked severe mortality outbreaks. The pathologies caused by vibrios in 33

bivalves have been described since the 1960s; however, over recent years, successive episodes 34

of high mortality have been recorded due to these microorganisms. The present work provides 35

an updated overview of the different studies performed in relation with the diversity of Vibrio 36

spp. associated to bivalves. Special attention is given to the main Vibrio diseases and implicated 37

species affecting the different life stages of cultured bivalves. 38

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Introduction 40

Coastal and estuarine environments are growing areas of bivalve molluscs with hatcheries for 41

seed production of commercialised species, this being an important industry in many countries. 42

Due to their filter-feeding habit, bivalves normally accumulate a rich bacterial microbiota, 43

composed of various species belonging to different genera like Vibrio, Pseudomonas, 44

Acinetobacter, Photobacterium, Moraxella, Aeromonas, Micrococcus and Bacillus (Murchelano 45

and Brown, 1970; Kueh and Chan, 1985; Prieur et al., 1990). One of the main problems in the 46

culture of bivalve molluscs is the repeated episodes of mortality due to bacterial infections that 47

reduce the production and cause high economical losses. Some members of the genus Vibrio 48

have been described as the main causal agents of diseases affecting all life stages of bivalve 49

molluscs: larval, juveniles and adults (Liu et al., 2000; 2001; Allam et al., 2002; Waechter et 50

al., 2002; Lee et al., 2003; Paillard et al., 2004 and references therein; Anguiano-Beltrán et al., 51

2004; Estes et al., 2004; Gay et al., 2004 a, b; Gómez-León et al., 2005; Prado et al., 2005; 52

Labreuche et al., 2006 a, b; Garnier et al., 2007; 2008; Gómez-León et al., 2008). The diversity 53

of Vibrio species associated with bivalves in different geographical areas has also been the 54

subject of various studies (Montilla et al., 1994; Hariharan et al., 1995; Arias et al., 1999; 55

Pujalte et al., 1999; Maugeri et al., 2000; Caballo and Stabili 2002; Castro et al., 2002; 56

Guisande et al., 2004; Lafisca et al., 2008; Beaz-Hidalgo et al., 2008). It is well known that 57

environmental parameters, such as variations in the water temperature and salinity, can 58

influence the diversity of Vibrio spp. in the environment as well as the physiological state of the 59

bivalve and its susceptibility to bacterial infections (Arias et al., 1999; Pujalte et al., 1999; 60

Maugeri et al., 2000; Paillard et al., 2004; Garnier et al., 2007). 61

Paillard et al. (2004) provided an excellent and complete overview of the bacterial diseases in 62

marine bivalves, including an updated reference of pathogenic Vibrio spp. They recognized that 63

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the emergence of vibrios as etiological agents in cultured bivalves is likely to increase over the 64

coming years due to ocean warming. The present study provides an updated overview of studies 65

performed on the diversity of Vibrio species associated with bivalve molluscs. Special emphasis 66

is made on the species that produce disease in the different bivalve mollusc’s life stages (larvae 67

vs. juveniles and adults) describing the characteristic signs and diseases caused by Vibrio spp. in 68

different hosts. 69

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Diversity of Vibrio species associated with bivalves 71

Studies on the diversity of Vibrio species found that the predominating species associated with 72

bivalves from different geographical locations (Spain, Canada, Italy or Brazil) were either V. 73

splendidus, V. alginolyticus, V. harveyi or any combination of these species. Regarding this, 74

Pujalte et al. (1999) and Arias et al. (1999) found V. splendidus and V. harveyi as the prevailing 75

species in wild bivalves. Montilla et al. (1995) agreed in the dominance of V. splendidus, but 76

also recovered a high proportion of V. tubiashii strains, as did Castro et al. (2002) who also 77

recognized V. harveyi as an abundant species. However, other studies have found V. 78

alginolyticus as the dominant species (Hariharan et al., 1995; Caballo and Stabili, 2002; Lafisca 79

et al., 2008). Other species such as V. fluvialis, V. vulnificus and V. mimicus have also been 80

associated with mussels (Mytilus galloprovincialis) (Maugeri et al., 2000; Caballo and Stabili, 81

2002). In those mentioned studies, the prevalence of Vibrio species was established using 82

phenotypic identification methods which are known to underestimate the real diversity present 83

in bivalves. For instance, the high variability among the phenotypic characteristics within the V. 84

splendidus-like and the V. harveyi-like groups makes it virtually impossible to discriminate the 85

many species masked under these groups (Thompson et al., 2005; Le Roux and Austin, 2006; 86

Pascual et al., 2009). 87

Some of the above mentioned studies described the influence of environmental parameters (i.e. 88

salinity and water temperature) on the diversity of Vibrio species (Kaspar and Tamplin, 1993; 89

Motes et al., 1998; Arias et al., 1999; Pujalte et al., 1999). In wild bivalves (Ostrea edulis, M. 90

edulis, Chaemelea gallina, Donax trincili and D. semistriatus) from the Mediterranean Sea, V. 91

harveyi has been found to be dominant during the warmer months and V. splendidus during 92

winter and spring (Arias et al., 1999; Pujalte et al., 1999). Densities of V. vulnificus in shellfish 93

growing areas of the US Northern Gulf Coast were high and were not changing at temperatures 94

above 26°C and/or at salinity below 25 ppt, but were drastically reduced below this temperature 95

and/or above this salinity (Motes et al., 1998). Previous experiments in sterile microcosms 96

(Kaspar and Tamplin, 1993) demonstrated that salinity greater than 25 ppt reduced the survival 97

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of V. vulnificus in seawater. The presence of V. vulnificus in brackish water and mussels was 98

documented by Maugeri et al. (2000), but a low incidence of V. vulnificus was found in bivalves 99

from the western Mediterranean coast where the salinity values fluctuated between 30.7 and 100

38.3 ppt (Arias et al., 1999). These facts suggest that bivalves (oysters, clams, mussels, etc) 101

harvested from areas with high water temperature and low salinity may harbour high densities 102

of V. vulnificus, thereby increasing the risk of transmission to humans. The latter species 103

together with V. parahaemolyticus and V. cholerae are considered the most important human 104

pathogens and have produced important outbreaks associated with the consumption of 105

contaminated shellfish, mainly oysters, and therefore have been the subject of many studies (see 106

reviews by Morris, 2003; Cheng Su and Liu, 2007; Jones and Oliver, 2009). The public health 107

importance of these species will not be evaluated in this study. 108

The introduction of molecular techniques such as the amplified fragment length polymorphism 109

(AFLP) and multilocus sequence analysis (MLSA) have allowed a more precise identification of 110

Vibrio species which were previously masked under other taxa (Thompson et al., 2001; 2005; 111

Beaz-Hidalgo et al., 2008; Pascual et al., 2009). In this sense, phenotypically identified V. 112

harveyi strains were re-classified as V. campbellii by AFLP, DNA-DNA hybridization and 113

MLSA (Gómez-Gill et al., 2004; Thompson et al., 2007). Furthermore, molecular studies have 114

demonstrated the genetic diversity and the polyphyletic nature of V. splendidus (Thompson et 115

al., 2001; Le Roux et al., 2002; Thompson et al., 2005) and have enabled many new species to 116

be described, such as V. kanaloae, V. pomeroyi, V. chagasii and V. gallaecicus (Thompson et 117

al., 2003; Beaz-Hidalgo et al., 2009 c). In a previous study (Beaz-Hidalgo et al., 2008) we 118

analysed the diversity of Vibrio spp. in clams (Ruditapes decussatus and R. philippinarum) 119

cultured in Galicia (north-western coast of Spain) with phenotypic and genotypic methods 120

(AFLP). The latter method enabled us to identify 39% (57/145) of the isolates to the species 121

level. The predominant species that accounted for 66.6% of the total identified strains were V. 122

cyclitrophicus (17/57), V. splendidus (16/57) and V. alginolyticus (5/57), and we found only a 123

29.8% concordance between phenotypic and AFLP results (Beaz-Hidalgo et al., 2008). There 124

were major discrepancies for the V. splendidus-like strains, from which many were genetically 125

identified as V. crassostreae, V. cyclitrophicus, V. chagasii or could not be identified to species 126

level. The latter two species together with V. diabolicus and V. ichthyoenteri were associated for 127

the first time with cultured bivalves (Beaz-Hidalgo et al., 2008). Further studies using the 16S 128

rRNA gene and a MLSA with 4 genes (rpoD, recA, pyrH and atpA) enabled us to recognize 129

three new species within the family Vibrionaceae: Aliivibrio finisterrensis, Vibrio breoganii and 130

the already mentioned Vibrio gallaecicus (Beaz-Hidalgo et al., 2009 a, b, c). Pascual et al. 131

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(2009) investigated the usefulness of an MLSA approach with 6 protein genes (recA, pyrH, 132

rpoD, gyrB, rctB and toxR) to discriminate six species, V. harveyi, V. campbellii, V. 133

rotiferianus, V. parahaemolyticus, V. alginolyticus and V. natriegens that are tightly related 134

because they have DNA-DNA reassociation values very close to 70%. They recognized the 135

genes toxR and rpoD as the most reliable for species identification. Furthermore they proposed a 136

threshold value (> 90.3%) for species definition on the basis of the similarities of the 137

concatenated sequences of the 3 most resolving genes (toxR, rpoD and rctB). 138

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Pathogenic Vibrio species in bivalve larvae 140

Larval development of bivalves includes a planktonic embryonic stage that evolves by 141

metamorphosis into a free-living veliger larva, first developing a velum to swim and during the 142

late stage developing a “foot” to become a pediveliger larva (settling stage). In a hatchery, 143

although all larval stages are vulnerable, during the temporary fixation of the larvae on the 144

bottom of the tank they are exposed to a high concentration of potential pathogenic bacteria 145

associated with the tank surface, moribund larvae or organic detritus (Sutton and Garrick, 1993). 146

Guillard (1959) was the first to report evidence of the involvement of a Vibrio sp. in the 147

disruption of the velum and internal tissues of the clam larvae Mercenaria mercenaria, which 148

produced a mortality of 70% of the population. In the study performed by Tubiash et al. (1965), 149

they observed “swarms” of bacteria appearing on the margins of the larvae (Fig. 1A), which 150

became progressively denser and after 8 hours, mortality occurred as a result of granular 151

necrosis. This study was the first one to describe the term “bacillary necrosis” affecting 152

numerous bivalves: Crassostrea virginica, O. edulis, M. mercenaria, Argopecten irradians and 153

Teredo navalis. Typical signs of “bacillary necrosis” included the extension of the velum, 154

motility reduction or erratic movements in circles that appeared after 4-5 hours of exposure to 155

Vibrio spp. The species V. alginolyticus, V. tubiashii and V. anguillarum were recognised as the 156

main causal agents of the “bacillary necrosis” in later studies (Tubiash et al., 1970; Tubiash and 157

Otto, 1986). Elston and Leibovitz (1980) described three patterns of bivalve larvae disease 158

produced by Vibrio spp. termed Pathogenesis I, II and III. Pathogenesis I affects all larval 159

stages, the larvae become sedentary showing signs of colonization of the mantle and invasion of 160

the visceral cavity. Pathogenesis II affects the early stage of the veliger larvae, producing velum 161

disruption and extension and abnormal swimming. The larvae remain active, showing visceral 162

atrophy before invasion into the organs of the digestive tract occurs. Pathogenesis III affects the 163

late veliger larval stage (pediveliger larva) and as in Pathogenesis I the larvae become 164

sedentary. However, in Pathogenesis III there is a progressive and extensive visceral atrophy 165

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and lesions in the organs of the digestive tract. Another characteristic sign of larval vibriosis in 166

hatcheries is the appearance of the phenomenon called “spotting” (Fig. 1B), defined as an 167

accumulation of moribund larvae agglutinated at the bottom of the tanks (Di Salvo et al., 1978). 168

All the studies mentioned so far have set up the typical signs produced by vibriosis in bivalve 169

larvae, which are still being used for the recognition and description of other pathogenic Vibrio 170

spp. Table 1 provides a summary of the most recent studies on bivalve larvae vibriosis, updating 171

the information presented in an earlier review (Paillard et al., 2004). As commented originally, 172

V. anguillarum, V. alginolyticus, V. tubiashii (Tubiash et al., 1970, Tubiash and Otto 1986) 173

together with V. splendidus (Sugumar et al., 1998) have been recognized as the main pathogenic 174

species associated to larval vibriosis. However, other species have been described in the last two 175

decades (Nicolas et al., 1996; Lambert et al., 1998; Prado et al., 2005). This is the case of V. 176

pectenicida (Lambert et al., 1998), originally described as a Vibrio sp. strain able to produce a 177

high mortality of the scallop larvae Pecten maximus after 48 hours of exposure due to the 178

release of bacteria toxins which interrupt the digestive transit and degrade the tissues of the 179

larvae (Nicolas et al., 1996). It is worth mentioning that V. pectenicida does not grow on the 180

thiosulfate-citrate-bile salts sucrose (TCBS) selective medium classically used for vibrios. A 181

number of studies have been conducted with this species to reproduce experimentally the 182

infection and to evaluate the pathogenesis and the interactions with haemocytes (cited and 183

discussed in the review by Paillard et al., 2004). 184

Prado et al. (2005) investigated 3 Vibrio strains isolated from oyster larvae (O. edulis) in three 185

different hatchery outbreaks in Galicia that had been identified with the 16S rRNA gene 186

sequences. One strain was identified as V. neptunius, and the other two classified as Vibrio sp., 187

because despite showing similarities to V. orientalis and V. vulnificus they could not be 188

unequivocally assigned to these species. Further studies showed that one of the latter Vibrio sp. 189

strains belonged to a new species, still unpublished, named V. ostreicida (S. Prado and J. L. 190

Romalde, personal communication). The experimental challenges on oyster larvae performed 191

with the three mentioned isolates caused a very high mortality (98-100%) in less than 48h. 192

Microscopical examination showed abnormal swimming behaviour, closed valves with internal 193

movement and velum abnormalities (Prado et al., 2005). Interestingly, this is the first study that 194

demonstrated the pathogenicity of V. neptunius in oyster larvae. The disease was compatible 195

with a “bacillary necrosis” and was associated with the Pathogenesis II described by Elston and 196

Leibovitz (1980). Other signs observed by Prado et al. (2005) included the previously described 197

“spotting” phenomenon (Di Salvo et al., 1978, Nottage and Birkbeck 1987) and “swarms” of 198

bacteria around the larvae (Tubiash et al., 1965). 199

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The species V. tubiashii reported as one of the causative agents of the “bacillary necrosis” 200

(Tubiash et al., 1970; Tubiash and Otto 1980) has recently been described as a re-emergent 201

pathogen in North America causing a decline of approximately 59% in larvae oyster production 202

(Elston et al., 2008). In addition, Hasegawa et al. (2008) demonstrated that one of the critical 203

factors in the pathogenicity of V. tubiashii in oysters (C. gigas) is the presence of a 204

metalloprotease (VtpA). These authors found that the gene encoding this protease is widespread 205

among Vibrio species and that the protein is regulated by quorum sensing (i.e. the gene is 206

activated at high cell population densities). The purified VtpA protein was found to be more 207

toxic for 6-day-old larvae than for 16-day-old larvae (Hasegawa et al., 2009). 208

It has been suggested that agents (pathogenic or opportunistic) responsible for the development 209

of disease in bivalve larvae seem to act synergistically and infection occurs as a result of 210

stressed larvae (Di Salvo et al., 1978; Paillard et al., 1994; Prado et al., 2005). 211

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4. Pathogenic Vibrio species in juvenile and adult bivalves. 213

Tubiash et al. (1965) were also the first to perform research studies on pathogenic vibrios in 214

juvenile and adult bivalves. Their studies associated moribund adult bivalves (M. mercenaria, 215

C. virginica, M. edulis and Mya arenaria) with the species V. tubiashii and V. alginolyticus. 216

Elston and Leibovitz (1980) described further cases of vibriosis in oysters, detecting anomalies 217

in the shell and alterations in the function of the ligament and digestive processes. The classical 218

Vibrio infections are “summer mortality” in juvenile oysters and “brown ring disease” in adult 219

clams. Table 2 lists recent studies of Vibrio spp. that have caused pathologies in juvenile and 220

adult bivalves, completing the information provided by Paillard et al. (2004). 221

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“Summer mortality” of juvenile oysters 223

“Summer mortality” affects juvenile populations of the Pacific oyster (C. gigas) during the 224

warmer months when the water temperature is ≥18ºC and reproduction takes place (Berthelin et 225

al., 2000). This phenomenon has been associated to stress situations, low dissolved oxygen or 226

presence of toxic substances in the sediment (Cheney et al., 2000). Lipp et al. (1976) were the 227

first to observe that the oyster haemolymph had high levels of vibrios that were causing death. 228

Later studies in France identified V. splendidus as the causal agent of “summer mortality” 229

(Lacoste et al., 2001; Waechter et al., 2002; Gay et al., 2004 a, b). Recently, Garnier et al. 230

(2007) analysed the bacterial populations in the haemolymph of moribund oysters by 231

phenotypic and molecular methods (RFLP of gyrB gene and partial sequences of the 16S rRNA 232

gene) and found that the prevalent species were, among others, V. aestuarianus (56%) and V. 233

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splendidus (25%). The pathogenicity of V. aestuarianus for the oyster was demonstrated by 234

Labreuche et al. (2006 b), that described the immunosuppressive activity of the extracellular 235

products of this species on the haemocyte functions. The susceptibility of bivalve microbiota to 236

the bactericidal activity of the haemolymph is one of the factors that regulate their persistence in 237

bivalve tissues (Canesi et al., 2002, Pruzzo et al., 2005). Bacterial surface ligands, soluble 238

haemolymph components and the ability of vibrios to activate distinct stress signalling pathways 239

involved in the haemocyte response are considered the factors that determine their presence 240

within a bivalve host (Pruzzo et al., 2005). 241

Garnier et al. (2008) characterized several strains of V. aestuarianus recovered from oyster (C. 242

gigas) and described them as V. aestuarianus subsps. francensis. They further demonstrated 243

(challenging studies on oysters) that 45% (5/11) of the strains of the new subspecies were highly 244

virulent producing a mortality above 50%, while 27% (3/11) of the strains caused moderate 245

mortality (15-50% mortality) and 27% were innocuous (< 15% mortality), considering virulence 246

to be a strain-specific character. More recently, Allain et al. (2009) suggested the possible role 247

of V. harveyi as aetiological agent of “summer mortality”, since it was detected in most samples 248

of affected oysters during the 2008 warm season, and was able to produce mortality in 249

experimental challenges. 250

The most accepted theory today is that “summer mortality” of the oyster (C. gigas) is not 251

attributed to only one bacterial pathogen but to a complex interaction between the physiological 252

and/or genetic state of the host, environmental factors and the presence of various opportunistic 253

infectious agents (Paillard et al., 2004; Pruzzo et al., 2005; Labreuche et al., 2006 b), among 254

which Vibrio spp. may play an important role. 255

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Brown Ring Disease 257

Brown Ring Disease (BRD) caused by V. tapetis has been widely studied and the pathogenic 258

aetiology in adult clams (R. phillipinarum and R. decussatus) has been reviewed previously 259

(Paillard et al., 1994; Paillard, 2004). Bivalve’s susceptibility to V. tapetis infections is species 260

specific, causing greater physiological disturbances and mortality in R. philippinarum than in 261

other species of clam (R. decussatus and M. mercenaria) or in the oyster C. virginica (Allam et 262

al., 2001; 2006). This microorganism alters the clam’s haemocytes decreasing their viability and 263

phagocytic activity in experimental studies (Allam and Ford, 2006). The disease is characterized 264

by the alteration of the calcification process of the inner surface of the valves and the 265

appearance of a characteristic brown deposit consisting of conchiolin between the edge of the 266

shell and the pallial line (Paillard and Maes, 1990; Borrego et al., 1996). This brown deposit 267

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resembles that which appears in the Juvenile Oyster Disease (or Roseovarius Oyster Disease) 268

produced by the bacterial species Roseovarius crassostreae, although in this case the conchiolin 269

is deposited over the entire mantle and the attachment of the adductor muscle (Paillard et al., 270

2004; Boardman et al., 2008). 271

BRD is considered one of the main limiting factors when culturing Manila clams (R. 272

philippinarum) on many European coasts: France, Italy, Spain, Portugal, England, Ireland and 273

has been recently introduced in Norway (Paillard and Maes, 1990; Figueras et al., 1996; Allam 274

et al., 2000; Drummond et al., 2007; Paillard et al., 2009). The pathogen has also been recently 275

detected in Manila clams cultured in Korea (Park et al., 2006). It has been demonstrated that 276

clam sensitivity to V. tapetis varies according to the geographical origin of the clams. 277

Populations of French Manila clams have been described as more susceptible to BRD than 278

populations in the United States (Allam et al., 2001) as are Manila clams grown in Galicia 279

compared to those of Ireland (Drummond et al., 2007). Environmental factors (i.e. temperature 280

and salinity) play a role in the development of BRD, which tends to be more frequent in the 281

spring and winter as the optimum growth temperature for V. tapetis is 15ºC (Paillard et al., 282

1994; 2004). Reid et al. (2003) demonstrated when challenging BRD infections on Manila 283

clams, that the disease was more severe when performed at 20 ppt salinity than at 40 ppt. The 284

diagnostic of BRD is currently based on the examination of the characteristic brown ring on the 285

inner edge of the shell. For an early diagnosis in the absence of the brown ring, specific PCR 286

detection protocols targeting the 16S rRNA gene of V. tapetis have been designed (Paillard et 287

al., 2006; Park et al, 2006; Romalde et al., 2007). Biochemical, serological and genetic 288

intraspecific variability within V. tapetis had been described as well as three main subgroups 289

that correlate with the host type (Rodríguez et al., 2006). This heterogeneity has been further 290

demonstrated with a MLSA and proteomic analysis by 2D-PAGE (Balboa et al., 2007, 2008). 291

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Control and preventive measures 293

Control of diseases is a difficult task in the culture of bivalve molluscs, both at the hatcheries 294

and at adult stages, the maintenance of optimum water quality and culture density being 295

important for the prevention of mortality outbreaks in hatcheries (Prado et al., 2009). Once the 296

bivalves begin to die, chemotherapy is the only possible approach, but it has been shown to be 297

inefficient and to have disadvantages, like the development of antibiotic resistances 298

(Karunasagar et al., 1994; Kemper, 2008). On the other hand, vaccines, an alternative useful in 299

fish cultures, are not viable in bivalves (Bachère, 2003). Furthermore none of these methods can 300

be used when bivalve are in the growing beds in the natural environment. 301

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One promising approach is the use of probiotics defined in this context as ‘those live microbial 302

adjuncts which have a beneficial effect on the host’ (Riquelme et al., 2000; Verschuere et al., 303

2000; Prado et al., 2009). The use of live bacteria with activity against pathogens is especially 304

interesting in bivalve hatcheries, because the larvae are released and exposed to environmental 305

microbiota in early ontogenic stages. The filter feeding behaviour of these animals increases the 306

influence of the surrounding water. In the last years a number of strains have been proposed as 307

good candidates to be used as probiotics in aquaculture systems. They are usually marine 308

bacteria with a wide spectrum of antibacterial activity, also effective against the main 309

pathogenic Vibrio spp., being able to develop their inhibitory activity in seawater. Their 310

biotechnological potential is based on the ability to control the proliferation of opportunistic 311

pathogens in a hatchery and farm environments. 312

Other research area of increasing interest is the selective breeding of molluscs to obtain genetic 313

families resistant to diseases (Tanguy et al., 2008). The main advantage of breeding selection is 314

that it offers long-term increased resistance, from hatchery through all live stages up to the 315

commercial size. This approach has been developed against parasitic diseases, like QX disease 316

(specific to Sydney Rock oysters caused by the protozoan parasite Marteilia sydneyi) and 317

bonamiasis, where hemocytes of flat oysters are affected (Bezemer et al., 2006; da Silva et al., 318

2008; Simonian et al., 2009). However, in the last years some attemps have been made to get 319

families of bivalves, mainly Pacific oyster (C. virginica), resistant to bacterial pathogens 320

(Gómez-León et al., 2008) with promising results. Thus, some oyster families have been 321

selected with increased resistance, not only to protistan, but also to bacterial pathogens 322

including Roseovarius crassostreae and several Vibrio species. 323

Other promising research line which has been scarcely studied is the genomic characterization 324

through the analysis of EST (Expressed Sequence Tags) collections, which are short sub-325

sequences of a transcribed cDNA sequences that represent the genes expressed in an organism 326

under a given condition. Using this strategy host defence genes have been identified in 327

numerous bivalves including Argopecten irradians, Chlamys farreri, C. gigas, C. virginica, M. 328

edulis or R. decussatus, which may help to understand the host-parasitic relationship and the 329

molecular mechanisms by which these marine bivalves respond to pathogens (Song et al., 2006, 330

Gestal et al., 2007, Guegen et al., 2007, Wang et al., 2009; Fleury et al., 2009 and references 331

therein). In this sense, the expression of several genes implicated in the resistance and 332

susceptibility of C. gigas to the “summer mortality” have been recognized and compiled in a 333

publicly-available database (Fleury et al., 2009 and references therein). Further investigation in 334

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this area will help to elucidate of the physiological and genetic basis for the survival of selected 335

progeny. 336

337

Conclusions and future prospects 338

The present study overviews the diversity of Vibrio species associated with bivalve molluscs. 339

Ongoing studies on the disease and pathogenicity of bivalves primarily relies on the use of 340

phenotypic and molecular methods for an exact species identification following challenging in 341

vivo and in vitro tests. However, studies that establish the molecular mechanisms involved in the 342

development of bivalve disease, such as the detection of virulent genes, are scarce and therefore 343

needed in order to better understand the host-pathogen interactions. Major advances are being 344

made in the study of toxicity of bacterial extracellular products to bivalves as well as in the 345

study of the cellular mechanisms in relation to bacteria–hemocyte interactions. These new 346

insights will explain how a bacterial pathogen is able to avoid phagocytic activity and multiply 347

to high levels inside the bivalve, leading to death. A complete description of how the 348

environment influences these bacteria-host interactions and health status of the bivalve may lead 349

towards a better understanding of the infection routes of the pathogen and mode of action in the 350

natural conditions. 351

It remains to be investigated to what extent some of the recently discovered species are 352

commensal, opportunistic or pathogenic organisms. Knowledge of the infection mechanisms 353

used by classical and emerging Vibrio spp. to develop disease in bivalve molluscs will help to 354

establish adequate preventive measures to control the transmission of these pathogens in 355

hatcheries and in coastal growing areas. Furhermore, research on the selection of genetic 356

resistant bivalve populations and on the potential use of probiotic bacteria may be greatly useful 357

for the improvement of cultured bivalve production. 358

359

360

361

362

363

364

365

366

367

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necrosis, a disease of larval and juvenile bivalve molluscs. J Bacteriol 103: 272-273. 634

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Tubiash, H.S., and Otto, S.V. (1986). Bacterial problems in oysters. A review. In Pathology in 635

Marine Aquaculture. Vivarès C.P., Bonami J.R., and Jasper E. (eds) Bredene (Belgium): 636

European Aquaculture Society, Spec. Publ. 9, pp. 233-242. 637

Verschuere L., Rombaut G., Sorgeloos P., and Verstraete W. (2000) Probiotic bacteria as 638

biological control agents in aquaculture. Microbiol Mol Biol Rev 64: 655-671. 639

Waechter M., Le Roux F., Nicolas J.L., Marissal E., and Berthe F. (2002). Characterization of 640

pathogenic bacteria of the cupped oyster Crassostrea gigas. C R Biol 325: 231. 641

Wang, L., Song, L., Zhao, J., Qiu, L., Zhang, H., Wei, X. et al. (2009). Expressed sequence tags 642

from the zhikong scallop (Chlamys farreri): Discovery and annotation of host defense genes. 643

Fish & Shellfish Immunol 26: 744-750. 644

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Figure 1. Signs of disease in experimentally infected larvae: (A) Swarms of bacteria on

the margins of the larvae, the so called “bacterial swarming” (arrow) surrounding the

larvae (Bar 200 µm). (B) Moribund larvae agglutinating at the bottom of the tanks, the

so called the “spotting” phenomenon (Bar 200 µm).

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Table 1. Experimental infections of bivalve larvae with pathogenic Vibrio spp. *Abbreviations: C = Crassostrea, M = Mytilus, O = Ostrea, P=

Pecten, R = Ruditapes. NS= Not specified.

Pathogenic species Vibrio identification

method

Origin of the tested

strain/s Host*

Experimental

observations Reference

V. alginolyticus NS Culture collection M. galloprovincialis Sublethal, abnormal larvae Anguiano-Beltrán et al., 2004

Vibrio sp. Phenotypic Mortality outbreak C. gigas

O. edulis Mortality Estes et al., 2004

V. splendidus biovar II

V. alginolyticus

Phenotypic,

16S rDNA Mortality outbreaks R. decussatus

Mortality, reduced

haemocyte survival Gómez-León et al., 2005

V. neptunius

Vibrio sp.

Phenotypic,

16S rDNA Mortality outbreaks O. edulis Mortality Prado et al., 2005

V. pectenicida

V. splendidus-like PCR identification Mortality outbreaks P. maximus Mortality Sandlund et al., 2006

Vibrio sp. NS Pacific oyster C. virginica Mortality Gómez-León et al., 2008

V. tubiashii NS Pacific oyster C. gigas

Destruction of tissues,

toxicity due to an

extracellular

metalloprotease

Hasegawa et al., 2008

V. tubiashii

V. splendidus

V. cholerae

NS NS C. gigas

Toxicity due to an

extracellular

metalloprotease

Hasegawa et al., 2009

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Table 2. Experimental infections of juvenile and adult bivalves with pathogenic Vibrio spp. *Abbreviations: C = Crassostrea, R = Ruditapes.

BRD= Brown Ring Disease, ECP= Extracellular products, NS= Not specified.

Pathogenic species

Vibrio

identification

method

Origin of the

tested strain/s

Host* Life

stage

Experimental observations Reference

V. tapetis

NS

Symptomatic

clams

R. philippinarum

R. decussatus

Adults

Mortality, BRD, reduced

haemocyte phagocitosis

Mortality

Decreasing viability and

reduced haemocyte

phagocitosis

Host cell damage

Allam et al., 2001

Allam et al., 2002

Allam and Ford 2006

Allam et al., 2006

V. splendidus Phenotypic and

genotypic (NS)

Moribund

oysters

C. gigas Juveniles Mortality Lacoste et al., 2001

V. splendidus biovar II

(V. chagasii)

Phenotypic,

16S rDNA

Mortality

outbreak

C. gigas Juveniles Mortality Waechter et al., 2002

V. splendidus-like gyrB Moribund/

healthy oysters

C. gigas

R. philippinarum

Adults

Seed

Weakness of adductor

muscle

Gay et al., 2004b

V. aestuarianus Phenotypic, 16S

rDNA and gyrB

gyrB-RFLP,

16S rDNA

Mortality

outbreak

Diseased/

healthy oysters

C. gigas

Adults

Reduced haemocyte

adhesive capacities and

phagocitosis.

Mortality depending on the

strain

Labreuche et al., 2006 a, b

Garnier et al., 2007

V. aestuarianus subsp.

francensis

gyrB, toxR,

DNA-DNA

Mortality

outbreak

C. gigas Adults Mortality depending on the

strain

Garnier et al., 2008

Vibrio sp. DNA-DNA

NS

Moribund/

healthy oysters

Pacific oyster

C. gigas

C. virginica

Juveniles

Juveniles

Mortality depending on the

strain

Mortality, ECP toxic to

haemocytes

Gay et al., 2004 a

Gómez-León et al., 2008

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Table 1. Experimental infections of bivalve larvae, juvenile and adults with pathogenic Vibrio spp. *Abbreviations: C = Crassostrea, M =

Mytilus, O = Ostrea, P= Pecten, R = Ruditapes, BRD= Brown Ring Disease, ECP= Extracellular products, NS= Not specified.

Pathogenic species Vibrio identification

method

Origin of the tested

strain/s Host*

Life stage Experimental observations Reference

V. alginolyticus NS Culture collection M. galloprovincialis Larvae Sublethal, abnormal larvae Anguiano-Beltrán et al., 2004

Vibrio sp. Phenotypic Mortality outbreak C. gigas

O. edulis

Larvae Mortality Estes et al., 2004

V. splendidus biovar II

V. alginolyticus

Phenotypic,

16S rDNA Mortality outbreaks R. decussatus

Larvae Mortality, reduced haemocyte

survival Gómez-León et al., 2005

V. neptunius

Vibrio sp.

Phenotypic,

16S rDNA Mortality outbreaks O. edulis

Larvae Mortality Prado et al., 2005

V. pectenicida

V. splendidus-like PCR identification Mortality outbreaks P. maximus

Larvae Mortality Sandlund et al., 2006

Vibrio sp. NS Pacific oyster C. virginica Larvae Mortality Gómez-León et al., 2008

V. tubiashii NS Pacific oyster C. gigas Larvae Destruction of tissues, toxicity due

to an extracellular metalloprotease Hasegawa et al., 2008

V. tapetis

NS

Symptomatic clams R. philippinarum

R. decussatus

Adults

Mortality, BRD, reduced

haemocyte phagocitosis

Mortality

Decreasing viability and reduced

haemocyte phagocitosis

Host cell damage

Allam et al., 2001

Allam et al., 2002

Allam and Ford 2006

Allam et al., 2006

V. splendidus Phenotypic and

genotypic (NS)

Moribund oysters C. gigas Juveniles Mortality Lacoste et al., 2001

V. splendidus biovar II

(V. chagasii)

Phenotypic,

16S rDNA

Mortality outbreak C. gigas Juveniles Mortality Waechter et al., 2002

V. splendidus-like gyrB Moribund/ healthy

oysters

C. gigas

R. philippinarum

Adults

Seed

Weakness of adductor muscle Gay et al., 2004b

V. aestuarianus Phenotypic, 16S

rDNA and gyrB

Mortality outbreak

C. gigas

Adults

Reduced haemocyte adhesive

capacities and phagocitosis.

Labreuche et al., 2006 a, b

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Pathogenic Vibrio species mainly cause mortality of bivalve larvae but they are able to cause different disease symptoms in juvenile and adults

where mortality seems to be strain specific in many cases.

gyrB-RFLP,

16S rDNA

Diseased/ healthy

oysters

Mortality depending on the strain

Garnier et al., 2007

V. aestuarianus subsp.

francensis

gyrB, toxR,

DNA-DNA

Mortality outbreak C. gigas Adults Mortality depending on the strain Garnier et al., 2008

Vibrio sp. DNA-DNA

NS

Moribund/ healthy

oysters

Pacific oyster

C. gigas

C. virginica

Juveniles

Juveniles

Mortality depending on the strain

Mortality, ECP toxic to haemocytes

Gay et al., 2004 a

Gómez-León et al., 2008

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