Chapter 66

Vibriosis in larval scallops

Roger Sie-Maen Chong1, 2
1
Registered Veterinary Specialist in Fish Health and Production, Royal College of Veterinary Surgeons, London, United Kingdom; 2Registered
Veterinary Specialist in Veterinary Aquatic Animal Health, Queensland Veterinary Surgeons Board, Brisbane, QLD, Australia

66.1 Overview
Scallop species affected by vibriosis (bacillary necrosis) in the larval stage include Argopecten irradians, A. purpuratus,
Patinopecten yessoensis, Pecten maximus, and Euvola (¼Pecten) ziczac (Bower, 2009). While this chapter is focused on
scallops, vibriosis also occurs in other bivalve larvae, including clams and oysters (Bower, 2009).

66.2 Etiological agents

Various Vibrio spp. are involved in infection of scallops, including V. pectenicida, V. splendidus, V. alginolyticus,
V. lentus, V. anguillarum-related larval pathogen, and Vibrio sp. (Bower, 2009). Other bacteria may coinfect with the
vibrios, and these belong to Pseudomonas and Moraxella genera (Bower, 2009). V. splendidus produces 80% mortality in
larval A. purpuratus (Rojas et al., 2015).

66.3 Clinical signs

The disease is rapid and severe, causing 100% mortality in under 18 h of infection in a heavily infected culture of larval
scallops (Bower, 2009). Mortalities of P. maximus due to bacterial strains like V. splendidus are preceded by velar necrosis
and detachment of velar cells and settlement of sick larvae (Nicolas et al., 1996).

66.4 Histopathology
The bacteria cause a systemic infection of the soft tissues which become necrotized, followed by secondary invasion with
ciliated protozoa (Bower, 2009). Rod-shaped bacteria which are slightly curved (typical of vibrios) occur within the tissues
(Bower, 2009).

66.5 Disease risk factors

Adult scallops are more resistant to vibrios and do not have significant mortalities when experimentally challenged with
vibrios from larval scallops (Bower, 2009). The virulence of bacterial isolates can be assayed by inoculating 24 h sus-
pensions of the isolate into 15-mL cell culture containers with 30e45 early stage scallop larvae (Bower, 2009). For
V. splendidus, the bacterial concentration that is moderately pathogenic to A. purpuratus is 104 CFU/mL which causes
about 27%e35% mortality or highly pathogenic at 106 CFU/mL resulting in 79%e88% mortality (Rojas et al., 2015).
Different strains of V. splendidus result in different mortality rates at the same exposure concentration of bacteria, with the
VPA16 and VPA18 strains being more pathogenic than the VPAP23 strain (Rojas et al., 2015). Lower density of larvae
(1 larva/mL compared to 2.5e5 larvae/mL) of P. maximus results in lower mortalities (50% vs. 100%) when infected with
V. splendidus-like vibrios (Nicolas et al., 1996).

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66.6 Clinical pathology

Wet mount light microscopy of diseased larvae shows masses of invading bacteria throughout the visceral soft tissues of
the animals (Bower, 2009). Larval vibriosis is likely a combination of invasion by pathogenic vibrios and by the pro-
duction of exotoxins (Nicolas et al., 1996). Exotoxin is triggered by threshold bacterial concentrations in the gut which are
“lyzed by digestive enzymes of the host to release ciliostatic toxin, proteases, or endotoxins” (Nicolas et al., 1996). The
toxins inhibit movement of ingesta in the digestive tract and facilitate degradation of host tissues (Nicolas et al., 1996),
leading to rapid mortality.
V. splendidus also causes bacillary necrosis where swarms of bacteria occur on the margins of larval A. purpuratus, and
there is detachment of velum ciliated cells followed by digestive gland necrosis after 24 h of bacterial bath exposure
(Fig. 66.1AeD) (Rojas et al., 2015). There is in addition, pathology of extension of the velum and loss of cilia from velar
cells observed which is not reported for bacillary necrosis in oysters or other scallops (Rojas et al., 2015). Using 5-DTAF
fluorescence microscopy, invasion by V. splendidus occurred at 30 mine1-hour postexposure and invaded the digestive
gland and internal tissues within 24 h (Fig. 66.2AeD), which is similar for V. tubiashii (Rojas et al., 2015). ECPs of
V. splendidus, heat-stable ciliostatic toxins or proteinases are involved in causing larval necrosis and high levels
of mortality after 24 h of exposure (Rojas et al., 2015). The pathogenic strains of V. spendidus produced a low level of
siderophores and may contribute to pathogenicity (Rojas et al., 2015).

66.7 Pathogen isolation and identification

Vibrios can be cultured on thiosulfate citrate bile salts sucrose and Zobell bacterial culture agar using tissues of diseased
scallop larvae (Bower, 2009). V. spendidus is similar to V. tubiashii for the above biochemical phenotyping but is different

FIGURE 66.1 V. splendidus challenge of Argopecten purpuratus larvae and developing pathology. “Main symptoms of pathogenic activity of
V. splendidus strains on experimentally infected A. purpuratus larvae after 24 h exposure. (A) Swarms of bacteria on the margins of the larvae (bacterial
swarming), (B) velum disruption, (C) extension of the velum, and (D) detachment of the cilia cells of the velum and necrosis of digestive tissue stained
with trypan blue (dark gray in print version). Scale bars: 50 mm”. (For interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.) Reproduced with permission from Rojas, R., Miranda, C.D., Opazo, R., Romero, J., 2015. Characterization and
pathogenicity of Vibrio splendidus strains associated with massive mortalities of commercial hatchery-reared larvae of scallop Argopecten purpuratus
(Lamarck, 1819). J. Invertebr. Pathol. 124, 61e69, Fig. 2.
 Vibriosis in larval scallops Chapter | 66 503

FIGURE 66.2 V. splendidus challenge of Argopecten purpuratus larvae and epifluorescence microscopy. “Scallop larvae inoculated with the VPAP16
strain not stained (A), and stained with 5-DTAF after 1 h (B), 24 h (C) and 30 h (D) of exposure. Scale bars: 50 mm.” Reproduced with permission from
Rojas, R., Miranda, C.D., Opazo, R., Romero, J., 2015. Characterization and pathogenicity of Vibrio splendidus strains associated with massive
mortalities of commercial hatchery-reared larvae of scallop Argopecten purpuratus (Lamarck, 1819). J. Invertebr. Pathol. 124, 61e69, Fig. 4.

by being positive for b-galactosidase, and produces acid from mannitol, D-mannose, inositol, sorbitol, melibiose, rham-
nose, and arabinose, while V. tubiashii does not (Rojas et al., 2015).

66.8 Molecular diagnostics

Polymerase chain reaction of isolated bacteria targeting the 16S ribosomal DNA, small subunit ribosomal DNA, “rpoA,
recA, pyrH, and gyrase B subunit (gyrB)” genes are used to speciate the vibrios (Bower, 2009). V. spendidus can be
identified using the same primer pair as for V. tubiashii and the primers for rpoA gene: rpoAF: ATGCAGGGTTCTGT-
DACAG and rpoAR: GHGGCCARTTTTCHARRCGC, followed by sequencing of the amplicon (Rojas et al., 2015).

66.9 Disease control

Larval vibriosis in scallops requires management of the culture system to ensure that low numbers of bacteria and hygiene
are maintained. This involves identifying sources of pathogenic vibrios in the broodstocks, algal cultures, seawater,
hatchery system biofilm on surfaces, taking steps to disinfect and dry out the hatchery periodically (Bower, 2009), so as to
avoid the continual transfer of bacteria onto new batches of larvae. The application of beneficial bacteria in the form of
probiotics, e.g., Alteromonas haloplanktis, which is isolated from the gonad of A. purpuratus and has inhibitory effects
against V. alginolyticus and V. anguillarum, may have a role in prevention of outbreaks, although there are significant
variables to consider, which may be site- and species-specific (Bower, 2009). No single probiotic will protect against all
strains of vibrios in every scallop hatchery. The use of antibiotics is generally not effective in an outbreak of larval vibriosis
and may encourage the development or selection of more virulent vibrios. Furthermore, more antibiotic-resistant bacteria
arise, and antibiotics can reduce the population of beneficial bacteria (Bower, 2009). On the other hand, continuous use of
antibiotic treatment based on chloramphenicol at 8 ppm for 15 years was necessary to support culture of P. maximus in
France because without antibiotics, bacterial disease involving strains similar to V. splendidus occurred some 12e19 days
504 SECTION | XI Bacterial diseases

into culture (Nicolas et al., 1996). Ultraviolet treatment of incoming seawater and antibiotic treatment of scallop eggs could
protect larval cultures (Nicolas et al., 1996). For V. spendidus, maintaining a bacterial concentration less than 103 CFU/mL
may be important to minimizing the mortality levels if infection of scallop larvae occurs (Rojas et al., 2015).

References
Bower, S.M., 2009. Synopsis of Infectious Diseases and Parasites of Commercially Exploited Shellfish: Vibrio spp. (Larval Vibriosis) of Scallops.
Available from: http://www.dfo-mpo.gc.ca/science/aah-saa/diseases-maladies/vibriosc-eng.html. (Accessed 26 June 2018).
Nicolas, J.L., Corre, S., Gauthier, G., Robert, R., Ansquer, D., 1996. Bacterial problems with scallop Pecten maximus larval culture. Dis. Aquat. Org. 27,
67e76.
Rojas, R., Miranda, C.D., Opazo, R., Romero, J., 2015. Characterization and pathogenicity of Vibrio splendidus strains associated with massive mortalities
of commercial hatchery-reared larvae of scallop Argopecten purpuratus (Lamarck, 1819). J. Invertebr. Pathol. 124, 61e69.