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Se-Kwon Kim, and Peer B. Jacobson, Academic Editor

Abstract

A variety of bivalve mollusks (phylum Mollusca, class Bivalvia) constitute a prominent commodity in fisheries and aquacultures, but are also crucial in order to preserve our ecosystem's complexity and function. Bivalve mollusks, such as clams, mussels, oysters and scallops, are relevant bred species, and their global farming maintains a high incremental annual growth rate, representing a considerable proportion of the overall fishery activities. Bivalve mollusks are filter feeders; therefore by filtering a great quantity of water, they may bioaccumulate in their tissues a high number of microorganisms that can be considered infectious for humans and higher vertebrates. Moreover, since some pathogens are also able to infect bivalve mollusks, they are a threat for the entire mollusk farming industry. In consideration of the leading role in aquaculture and the growing financial importance of bivalve farming, much interest has been recently devoted to investigate the pathogenesis of infectious diseases of these mollusks in order to be prepared for public health emergencies and to avoid dreadful income losses. Several bacterial and viral pathogens will be described herein. Despite the minor complexity of the organization of the immune system of bivalves, compared to mammalian immune systems, a precise description of the different mechanisms that induce its activation and functioning is still missing. In the present review, a substantial consideration will be devoted in outlining the immune responses of bivalves and their repertoire of immune cells. Finally, we will focus on the description of antimicrobial peptides that have been identified and characterized in bivalve mollusks. Their structural and antimicrobial features are also of great interest for the biotechnology sector as antimicrobial templates to combat the increasing antibiotic-resistance of different pathogenic bacteria that plague the human population all over the world.

Keywords: marine bivalve mollusks, antimicrobial peptides, bivalve immune system

1. Introduction

Marine bivalve mollusks may be affected by numerous infectious diseases. In this review, we will consider the most important diseases caused by viruses, bacteria and protistans, which are responsible for mortality outbreaks and have a substantial commercial impact. To a lesser extent, other diseases are also caused by fungi ( Aspergillus, Penicillium and Fusarium) [ 1], Porifera ( Cliona spp.) [ 2], and helminth parasites, such as trematodes, cestodes and nematodes [ 3].

Currently, the main infectious diseases of marine bivalve mollusks, such as herpes virus infection and bonamiasis, have taken on a worldwide distribution due to trade globalization. Since the transmission among bivalve mollusks is direct and horizontal, high-density production systems and environmental changes might have contributed to increasing the spread of diseases [ 4]; therefore, to avoid the current risk of further spreading of illnesses throughout the world, the World Organisation for Animal Health (OIE) Aquatic Code has set out standards and recommendations to improve the safety of international trade in aquatic animals, including marine bivalve mollusks. Nowadays, the aim is to prevent the pathogen's introduction into an importing country, so as to avoid the onset of disease outbreak rather than to eradicate the pathogen, which would be more difficult and expensive because of the high-density production systems used in commercial hatcheries and nurseries and the continuous stock movements around the world. Due to the absence of effective and specific chemotherapy and anti-viral treatments or vaccines available to prevent illnesses [ 5], the surveillance and control plan of these diseases based on their prevention have a key role. For this reason, it is important to implement high levels of on-farm and live-holding facility biosecurity and to restrict stock movements. Avoiding stressors, such as exposition to intense temperatures, a high or low level of salinity, handling, substantial co-infection with other parasites, as well as decreasing density, should help to reduce the impact of diseases [ 4]. The present review will describe bacterial and viral pathogens that affect bivalve mollusks and will illustrate the immune responses generated in bivalves and the repertoire of immune cells and their activation upon infection. An aspect of importance in the immune defense mechanisms operated by bivalve mollusks is the expression of antimicrobial peptides; therefore, the second part of the review will be dedicated to providing an outline of the antimicrobial peptides that have been identified and characterized in bivalve mollusks.

2. Infectious Diseases of Marine Bivalve Mollusks

Taking into account all of these considerations, the development of reliable and useful diagnostic tools is of paramount importance for the prevention and control of diseases. Considering that the lack of bivalve molluscan cell lines has greatly limited the possibility for viral isolation and the study of the experimental transmission of these pathogenic microorganisms, currently, the detection of the causative agent of microbial disease is mainly based on direct diagnostic methods. Moreover, classic serological methods are not suitable for diagnostic purposes since mollusks do not produce antibodies; in fact, histology is considered as the standard screening diagnostic method because it supplies a wider amount of information, but macroscopic examination usually gives no pathognomonic signs or indicative information. It is difficult to diagnose an infection based exclusively on morphological differences between species that have similar morphological characteristics, such as for example Bonamia ostreae and Bonamia exitiosa, when observed under the microscope [ 6]. To overcome these problems, nowadays, more specific and sensitive molecular diagnostic techniques are used in addition to electron microscopy for specific identification of the pathogen. The OIE Manual of Diagnostic Tests for Aquatic Animals describes specific protocols designed to detect a certain pathogen agent, to be employed to confirm histological examination results and provides a species-specific diagnosis. Methods used in targeted surveillance programs should not be time consuming; in fact, in most cases, PCR and subsequently the sequencing of 16S or other candidate loci are recommended for the identification of the isolate. For a presumptive diagnosis of a disease, the standard method is histopathology, but for a confirmatory diagnosis, the standard methods are polymerase chain reaction (PCR) and in situ hybridization (ISH); however, sequencing and transmission electron microscope (TEM) are recommended.

Viral infectious diseases: the most important viruses associated with disease outbreaks and the major cause of mortality in bivalves are currently members of the families of Herpesviridae and Iridoviridae; nevertheless there are also other viruses that can infect bivalves belonging to the families of Picornaviridae, Papovaviridae, Birnaviridae, Retroviridae and Reoviridae [ 7]. Viral pathogens are often highly infectious and easily transmissible. High-density production systems and environmental changes might have contributed to increasing the spread of the disease [ 4]. Molecular tools such as PCR, ISH and immunochemistry are used to detect viral pathogens in mollusks [ 8, 9, 10].

Herpes-like viruses: The first description of herpes-like viral infection was reported by Farley et al. (1972) [ 11] in Crassostrea virginica from the east coast of the USA. Afterwards, disease outbreaks, associated with high mortality rates, particularly in larvae and spawn during the summer period, have been reported from Pacific oysters ( Crassostrea gigas) in France, where higher mortalities were observed in 2008 and increased in 2009 and 2010 [ 12]. Oyster herpesvirus type 1 variant μvar (OsHV-1 μvar) was also found associated with Pacific oyster mass mortalities in Ireland, Italy, The Netherlands, Spain, the U.K. and in Australia, New Zealand and Korea, but is known to be detected elsewhere in the absence of oyster mortalities (e.g., Japan) [ 13]. Recently, OsHV-1 was detected in C. gigas in Japan and South Korea associated with mass mortality rate [ 14, 15] and in Sweden and Norway [ 16]. In the Thau Lagoon (France), OsHV-1 and, secondarily Vibrio splendidus are responsible for mass mortality of C. gigas [ 17]. During the summer of 2012 and 2013, OsHV-1 caused high mortality of Scapharca broughtonii in China [ 10]. TEM analysis showed that larvae exhibited generalized infections, whereas focal infections usually occurred in juveniles. Adult stages were less sensitive than younger stages. Infected larvae showed a reduction in feeding and swimming activities, and mortality can reach 100% in a few days. The effects of the disease on the hosts manifested in velar and mantle lesions, and the attitude of larvae to swim weakly in circles. Histologically, fibroblastic-like cells exhibited abnormal cytoplasmic basophilia and enlarged nuclei with marginated chromatin; other cell types including hemocytes and myocytes showed extensive chromatin condensation [ 18].

Irido-like viruses: Maladie des branchies or gill disease is a disease caused by gill necrosis virus (GNV), responsible for recurrent mass mortalities in adult Portuguese oysters ( Crassostrea angulata), from 1966 until the early 1970s along French coasts. To a lesser extent, gill disease also affected the Pacific oyster, C. gigas, imported in France, but with a negligible mortality. Because of its natural resistance to infection, C. gigas is currently the main species bred in Europe. Another Irido-like virus, hemocyte infection virus (HIV), caused mass mortality of Portuguese adult oysters ( C. angulata) in France between 1970 and 1973. This virus is similar to GNV, and viral particles can be observed in the cytoplasm of atypical infected hemocytes in connective tissues. A third type of Irido-like virus, the oyster velar virus (OVV), caused a high mortality rate of Pacific larval oysters on the west coast of North America (Washington State, USA) from 1976-1984 [ 19]. Currently, Irido-like virus infections are uncommon in Europe.

Bacterial infectious diseases: Due to the marine bivalve filter-feeding habit, they concentrate a rich and diverse bacterial commensal microbiota, composed of various species belonging to different genera like Vibrio, Pseudomonas, Acinetobacter, Photobacterium, Moraxella, Aeromonas, Micrococcus and Bacillus [ 20]; some of them may be pathogenic in larval rearing systems and not in the wild; in fact, pathogenicity depends on the host species, their life stage, amount of bacteria and on environmental factors. The rate of larval mortality can reach 100%, especially if larvae are reared in static systems at high temperature and density. Diagnosis of bacterial diseases is based on macroscopic inspection of shell valves in combination with PCR, which is the most sensitive and rapid method [ 21].

Gram-negative bacteria: Most bacterial diseases of bivalves are caused by a large range of Vibrio species ( Vibrio alginolyticus, V. splendidus, Vibrio anguillarum, Vibrio tubiashi, Vibrio tapetis, Vibrio aestuarianuns, Vibrio neptunius and other Vibrio spp.), Pseudomonas and Aeromonas [ 22, 23, 24, 25]. These bacteria are responsible for bacillary necrosis in a wide range of species of bivalve larvae [ 26]. Vibrio spp. produce exotoxins (ciliostatic factors and hemolysins), which cause deciliation, loss of velar epithelial and abnormal swimming behavior [ 27]. Necrosis has been well described with histological, immunofluorescent and ultrastructure techniques [ 28]. V. tapetis is the activating agent of an epizootic infection described in adult clams called brown ring disease (BRD); the most sensitive species is Ruditapes philippinarum [ 21, 29]. The disease has been detected in France, Spain, Portugal, Italy, the United Kingdom, Ireland, Norway [ 30] and, less frequently, in the Mediterranean and Adriatic seas [ 31]. In 2006, V. tapetis was reported from Manila clams on the west coast of Korea [ 32]. Transmission passes through direct contact between infected clams [ 33]. V. tapetis adheres to and colonizes the surface of the periostracal lamina at the mantle edge of the shell, causing anomalous deposition of periostracum, an abnormal calcification process and the accumulation of brown organic material, which is a striking sign of the disease. From the extrapallial space, the bacteria can penetrate the mantle epithelium and the soft tissues, where they reproduces themselves and cause severe damage and subsequent death [ 34]. Juvenile oyster disease (JOD) is a similar syndrome to BRD that appeared in 1988 in juvenile C. virginica [ 35] caused by Roseovarius crassostreae; nowadays, the disease is known as Roseovarius oyster disease [ 36]. Major mortal outbreaks have been reported in cultured oysters from New York to Maine (USA). Symptoms, such as reduced growth rates, fragile shell development, cupping on the left valve, anomalous conchiolin deposit around the periphery of the mantle on the inner valves [ 37], occur when water temperature exceeds about 21-25 °C, in high salinity and high-density culture conditions. Mortality reaches up to 90% in animals <25 mm; instead, larger juveniles forms show lower rates of mortality [ 38].

Gram-positive bacteria: Few Gram-positive bacteria cause diseases in bivalves; the main pathogenic agent is represented by Nocardia crassostreae, the etiological agent of Pacific oyster nocardiosis (PON) infecting C. gigas and Ostrea edulis cultivated near infected C. gigas along the west coast of North America from the Strait of Georgia, British Columbia to California and Japan (Matsushima Bay) [ 39]. Carella et al. (2013) [ 40] have notified nocardiosis in Mediterranean bivalves. Mortality rate reaches up to 35%; infected animals show yellow-green pustules in the mantle, gills, adductor and cardiac muscle associated with intense hemocyte infiltration around the colonies of Nocardia [ 41].

Protozoan infectious diseases: The most important protozoan pathogens belong to the genera Bonamia, Perkinsus, Haplosporidium and Marteilia; they can mainly infect oyster and clam species, causing enormous damage to commercial productions. In particular, in this review, we will discuss in detail the diseases caused by Perkinsus marinus, Perkinsus olseni, Marteilia refringens, B. ostreae and B. exitiosa that are currently under surveillance and require mandatory notification by the World Organization for Animal Health. Prevalence and intensity of infections tend to increase during the warm season, depending on temperature and high salinity rates [ 42]. For correct identification of the pathogen, histological examination is needed, as well as ISH or PCR. However, for unknown susceptible species and unknown geographical range, confirmation by sequencing and description by TEM are recommended. Bonamiosis represents 63% of protozoan diseases in Europe [ 43] and is caused by a group of protists in the genus Bonamia. B. ostreae has spread in Europe (France, Ireland, Netherlands, Portugal, Spain and the U.K.), but also on the west coast of Canada and both coasts of the USA [ 44]. B. ostreae was found in O. edulis imported to China [ 45]; recently, this species was detected in New Zealand infecting the flat oyster Ostrea chilensis [ 46]. B. exitiosa was found in O. chilensis in South Island, New Zealand [ 47], and in Ostrea angasi in southeastern Australia [ 48]. Since 2003, the parasite has been observed in both the Atlantic and Pacific coasts of the USA [ 49], including California [ 50]; B. exitiosa was also detected in O. edulis from the Galician coast (Spain) and the Manfredonia Gulf, Italy (Adriatic Sea), including concurrent infections with B. ostreae [ 51] and in Ostrea stentina in Tunisia [ 52]. B. exitiosa was found, as well, on the Spanish Mediterranean coast [ 53], in southwestern England [ 54] and in southern Portugal [ 55]. Others species, Bonamia perspora and Bonamia roughleyi, have been found on the east coast of the USA [ 56] and in southeastern Australia [ 57]. With the exception of B. perspora, all of the other species can be normally observed within hemocytes of the host [ 58]; B. perspora occurs within connective tissues [ 56]. The pathogen infects the hemocytes, multiplies within blood cells and spreads to all tissues. In highly infected adult oysters, we can find a yellow discoloration of the tissue, extensive lesions on the gill and mantle, breakdown of connective tissue and significant mortality (>90%). Larvae can be infected and contribute to the spread of the parasite. A lack of resistance to infection and, therefore, high densities of oysters in closely-spaced beds favor the development of epizootics.

In the late 1960s, two protozoans, both in the genus Marteilia, Marteilia refringens and Marteilia sydneyi, were identified as the causative agents of disease and heavy mortalities in the flat oyster: O. edulis, in France, and in Saccostrea glomerata, in Australia, respectively [ 59]; afterward, the parasite become widespread: M. refringens, infecting O. edulis, has, to date, mainly been found in Europe (Albania, Croatia, France, Greece, Italy, Morocco, Portugal, Spain, Sweden, Tunisia and the United Kingdom); Mytilus galloprovincialis, in the Gulf of Thermaikos, northern Greece [ 60], on the north coast of the Adriatic Sea [ 61] and along the Campanian coast (Tyrrhenian Sea, South of Italy) [ 62]. Marteilia was also previously found in M. galloprovincialis bred in Puglia [ 63]. M. sydneyi, infecting Saccostrea glomerata and possibly other Saccostrea spp. [ 64], has been reported in New South Wales, Queensland and Western Australia [ 65]. Marteiliosis is also known as Aber disease ( M. refringens, two types: M and O, as defined by Peruzzi et al. [ 66], and Queensland Unknown (QX) disease ( M. sydneyi). Both infect the digestive system and sporulate in epithelial cells of the digestive gland causing paleness of the digestive glands, emaciation of the oyster, dissipation of its reserves of energy, tissue necrosis, cessation of growth and mortality up to 90% in summer. Juveniles and older life stages are susceptible to infection, but prevalence and infection intensity are generally higher in individuals of two years old or more [ 67]. By TEM analysis, M. sydneyi can be differentiated from M. refringens by a paucity of striated inclusions within the plasmodia, the formation of eight to sixteen sporangial primordia in each plasmodium (instead of eight for M. refringens), the occurrence of two or three spores in each sporangium (rather than four in M. refringens) and the presence of a thick coat of concentric biological membranes surrounding mature M. sydneyi spores.

Perkinsosis: Perkinsosis is caused by Perkinsus marinus, responsible for dermo disease in C. virginica and, to a lesser extent, in C. gigas, Crassostrea rhizophorae and Crassostrea corteziensis [ 68]. The pathogen, uncommon in Europe, was first described in the Gulf of Mexico [ 69]. It was found along the southeast coast of the USA from Maine to Florida [ 70], along the Pacific coast of Mexico [ 71], in the Gulf of California (northwest Mexico) [ 72] and in Brazil [ 73]. Perkinsus olseni causes perkinsosis in many clam species with distribution in Australia, Korea, China, Japan and Europe [ 32]. Both parasites were also found associated with ten oyster species collected from both Panamanian coasts, including the Panama Canal and Bocas del Toro [ 74]. Transmission is direct from oyster to oyster; viable cells are released in host feces or on the death of the host [ 32] and are acquired through host feeding mechanisms. Every life stage is susceptible to disease [ 75]. The pathogen infects and proliferates in the digestive epithelium, connective tissue of all organs and hemocytes causing hemocytosis and tissue lysis with a consequent severe emaciation; mortality arrives up to 80% based on environmental factors [ 76]. In order to simplify all microbiological diseases, we generated tables with details of the infections (, and ).

Table 1

Disease
(Pathogenic Agent)

Host Species

Effects on Host

Geographical Distribution

References

Herpes virus infection
(oyster herpes virus)

Mainly hatchery-reared larvae of Crassostrea gigas and Ostrea spp.

Velar and mantle lesions; deterioration; swim in circles

Europe (France, Ireland, Italy, The Netherlands, Spain); U.K.; Australia; New Zealand, Mexico, USA, Japan, South Korea, China

Gill necrosis virus (GNV)

Crassostrea angulata and C. gigas

Destruction of gill filaments

France, Portugal, Spain, U.K.

Hemocyte infection virus (HIV)

Virus infected hemocytes

France, Spain

Oyster velar virus disease (OVV)

C. gigas larvae

Larval movement affected through loss of infected epithelial cells from velum

Washington State, USA

Table 2

Disease
(Pathogenic Agent)

Host Species

Effects on Host

Geographical Distribution

References

Larval and juvenile vibriosis
( Vibrio anguillarum, V. tubiashi,
V. alginolyticus, V. splendidus,
V. aestuarianus, V. neptunius)

Wide range of hatchery-reared species

Tissue necrosis (due to production of exotoxin by the bacteria), up to 100% larval mortality

In all marine waters where bivalve hatchery culture is practiced

Brown ring disease
( Vibrio tapetis)
Ruditapes philippinarum
Brown deposit on shell; degeneration of digestive gland followed by metabolic disorder and death

Entire European Atlantic coast to North Africa, including coasts of France, Portugal, Spain, Italy, U.K., Ireland and Norway, west coast of Korea

Roseovarius oyster disease
( Roseovarius crassostreae)

Crassostrea virginica juveniles <25 mm shell length

Reduced growth rates, fragile shell development, cupping on the left valve, mantle lesions, up to 90% mortalities

USA

Pacific oyster nocardiosis
( Nocardia crassostreae)

Crassostrea gigas, Ostrea edulis cultivated near infected C. gigas

Yellow-green pustules in the mantle, gills, adductor and cardiac muscle, up to 35% mortalities

West coast of North America from the Strait of Georgia, British Columbia to California, and Japan (Matsushima Bay), Mediterranean Sea

Table 3

Disease
(Pathogenic Agent)

Host Species

Effects on Host

Geographical Distribution

References

Bonamiasis
( Bonamia ostreae, B. exitiosa, B. perspora, B. roughleyi)

Wide range of oyster species

Yellow discoloration of tissue, extensive lesions on gill and mantle, breakdown of connective tissue, significant mortality (up to 90%)

Europe, U.K., west coast Canada, east and west coasts of USA, New Zealand and SE Australia

Digestive gland (or Aber) disease
( Marteilia refringens)

Ostrea edulis and Mytilus galloprovincialis

Pale digestive gland, severe emaciation, tissue necrosis, cessation of growth, mortalities up to 90% in summer

In O. edulis: Europe (Albania, Croatia, France, Greece, Italy, Morocco, Portugal, Spain, Sweden, Tunisia, U.K.); in M. galloprovincialis: northern Greece, in Italy, along the Adriatic Sea and the Campanian coast (Tyrrhenian Sea)

QX disease
( Marteilia sydneyi)

Saccostrea glomerata and Saccostrea spp.

Necrosis of digestive gland, loss of condition, gonad absorption, mortalities up to 90% in summer

New South Wales, Queensland and Western Australia.

Dermo disease
( Perkinsus marinus)
Crassostrea virginica
Severe emaciation, loss of condition, high mortality rate depending on temperature and salinity

Gulf of Mexico, southeast coast of USA, Pacific coast of Mexico, Gulf of California, Brazil

In conclusion, we must as well remember that for a disease to occur, the synergy among three factors is required. This synergy is commonly named the epidemiological triangle, which is composed by a host, a pathogenic agent and the environment. In fact, some of the infectious agents may be pathogenic or nonpathogenic based on the host species, its life stage (larval, juvenile or adult form) and the immune system, on which the environmental factor plays a key role. We can, therefore, assert that, in general, the exposition to extreme temperatures, a too high or too low level of salinity, human handling, an increasing density in rearing systems and co-infection with other parasites may reduce the immune defenses of the host, as well as increase the pathogenic agent rate of growth and, hence, its pathogenicity, making the host more susceptible to illness.

Based on the knowledge we have today on how a disease can spread and acknowledging the key role of the environment, it is clear that an improvement of the surveillance on the environment of rearing systems is essential.

3. Defense Mechanisms in Marine Bivalve Mollusks

During the last few decades, the immunology of marine bivalve mollusks (MBM) has been investigated with great interest, leading to the development of different branches for basic and applied research. The hemocyte phagocytosis constitutes the major immune response in MBM, and the study of this highly conserved process has contributed to better understanding not only the pathogenetic mechanisms of infectious diseases in MBM [ 81], but also the role of the filter-feeding organisms as passive carriers of pathogens to humans, considering the involvement of the hemocytes and hemolymph factors on the microbial clearance from mollusk's tissues [ 82].

The study of the hemocyte properties as biomarkers for monitoring the biological effects of anthropogenic stressors in polluted sites [ 83], as well as measuring the economic impact of environmental stressors on shellfish productions [ 84] represents another interesting immunological field of investigation for MBM. Moreover, the basic investigation of the hemocyte/hemolymph system represents a simplified phylogenetic model for understanding the ancestral interactions and integrations that occur between immunity and neuroendocrine response [ 85] and, more generally, between defense mechanisms and host homeostasis.

In the present section, we have mainly described the most important phases that characterize the hemocyte phagocytosis in MBM, giving, in parallel, importance to the humoral factors that participate in the recognition and opsonization of foreign particles, thus focusing on the common features of the innate immunity that are shared by invertebrate hemocytes and phagocytic cells of higher vertebrates.

Hemocyte-mediated immunity: The cell-mediated immunity represents the main internal defense response of marine bivalve mollusks. The hemocyte phagocytosis constitutes the key activity, leading to the recognition, engulfment and demolition of biotic and abiotic foreign particles [ 86]. The innate immune properties of the hemocytes rely on their ancestral role in food digestion and nutrient transport [ 87]. Indeed, the interplay between phagocytosis and nutrition in invertebrates has been ascribed to the primary function of the hemocytes phagosome as the digestive organelle, where microorganisms are degraded as nutrients source, then evolving into a more specialized compartment to kill pathogens () [ 88].

Marine bivalve mollusks possess an open circulatory system, and the hemocytes are found either in hemolymph or in tissues, respectively as circulating or infiltrating cells [ 89]. From an ontogenetic point of view, some authors suggested that hemocytes are derived from connective tissue cells [ 90]; however, different models have been proposed about the types of progenitor cell lines [ 91]. The nomenclature of mollusks hemocytes still represents a subject of debate, and the efforts at developing a uniform classification have resulted in the recognition of two main types of cells, such as granulocytes and hyalinocytes, based on morphological appearance and granularity under microscopic examinations. However, flow cytometry [ 92], electron microscopy [ 93, 94] and monoclonal-antibodies based assays [ 95] have suggested the presence of various hemocyte sub-types. Such diversity may reveal a broad array of activities, and in particular, it is widely accepted that granulocytes play the most active role in the phagocytosis response [ 96]. Nevertheless, all of the hemocyte populations contribute to the overall immune response, operating in a differential fashion on the basis of the different stimulations [ 97]. The relative concentration of the various circulating hemocyte types can be exposed to reversible and selective modifications through physiological and molecular mechanisms comparable to the margination/demargination processes, which take place in humans and other mammals [ 98]. Therefore, the quantitative diversity of the cellular hemolymph configuration represents an important factor for the modulation of the immune response [ 99].

The interest for the study of the hemocyte immunity mainly derives from the role of marine bivalve mollusks as sentinel organisms in environmental monitoring [ 100]. Indeed, many hemocyte parameters have been investigated as biomarkers in field or laboratory studies [ 101, 102, 103], and detailed data are available in the literature about the strong influence of natural and anthropic stressors on the hemocyte activity. In particular, the disruption of their morpho-functional properties has been described after exposure to low [ 104] and high [ 105] temperatures, pH acidification [ 106], mechanical stress [ 107], salinity changes [ 108], exposure to air [ 109], harmful algal bloom [ 110], organic and inorganic contaminants [ 111]. Nevertheless, some endogenous factors seem to have also an important influence on the modulation of the hemocyte activity, such as age [ 112], gender [ 113] and reproductive stage [ 114].

The hemocyte phagocytosis, mechanisms and kinetics: Bivalve hemocytes resemble the vertebrate monocyte/macrophage lineage, both in structure and function [ 115]. The chemotactic ability of hemocytes to migrate toward foreign particles and to incorporate them inside phagosomes is closely dependent on morphological activation through the projections of membrane ruffles or pseudopodia [ 116]. The cytoskeleton re-arrangement following proper stimulation represents the pivotal mechanism that allows the hemocytes to acquire a morphological spreading, from roundish to irregular shape [ 117]. Previous studies demonstrated that bacterial products, such as lipopolysaccharides and formylated tripeptide (N-FMLP), were able to elicit chemotactic and/or chemokinetic reactions in hemocytes [ 118], and the type of cell movement appeared as dependent on the nature of chemoattractant, thus hypothesizing a receptor-dependent mechanism [ 119]. Indeed, a differential migration activity was detected in hemocytes on the basis of the bacteria types that were encountered [ 120]. Both chemotaxis and chemokinesis augment the probability of physical association between hemocytes and foreign particles, but to date, the knowledge about pattern recognition receptors (PRRs) is still limited in bivalve hemocytes. PRRs recognize the conserved highly repeated microbial structures, termed pathogen-associated molecular patterns (PAMPs), and the Toll-like receptors (TRLs) have a prominent role within PRRs group, being traced to the most ancestral multicellular invertebrates [ 121]. TLRs belong to type I membrane receptors and contain an extracellular leucine-rich repeat (LRR) domain mediating the recognition of PAMPs [ 122]. Unlike mammal TLRs, invertebrate TLRs could not directly recognize PAMPs, but they seem to require the cytokine-like molecule Spatzle as an assistant [ 123]. Indeed, some authors suggested a hybrid function in pattern recognition for the primitive mollusk TLR, being characterized by broader ligands affinity and involving the assistance of some serum components [ 124].

In the Pacific oyster C. gigas, a putative TLR was cloned and named CgToll-1, showing upregulation in the hemolymph after challenge with V. anguillarum [ 125]. Similarly, in the scallop Chlamys farreri, a Toll homologue was detected and named CfToll-1, revealing transcripts modulation in the hemocytes after exposure to LPS [ 126]. In the mussel Mytilus edulis, the transcriptome analysis indicated a wide repertoire of innate recognition receptors, including transcripts for 27 TLR, particularly expressed in hemocytes [ 127]. Following binding of the ligand to the extracellular segment of TLR, signal transduction takes place by the intracellular toll-interleukin domain (TIR) containing adaptor molecules [ 128]. Each TLR recognizes distinct microbial components and activates different signaling pathways using selected adaptor molecules, then leading to the engagement of the signaling cascade of protein kinases that ultimately activate transcription factors and the expression of genes involved in the immune response [ 129, 130]. In contrast to the large amount of data on TLR signaling systems from higher vertebrates, relatively little is known in bivalve mollusks. The existence of genes/transcripts mediating the Toll signaling pathway in hemocytes was reported in M. galloprovincialis, showing upregulation after bacterial challenge, particularly by Gram-negative, whereas a marginal response was detected following stimulation with purified PAMPs (LPS, β-glucans) [ 131]. Intermediate transcripts of the Toll signaling pathway were also detected in scallop [ 132], clams [ 133] and oysters [ 134]. Moreover, the intensity and duration of intermediate components activation, such as kinase-mediated cascade, appeared as dependent on the type of extracellular stimuli [ 135]. This kind of evidence contributes to support the existence of a differential hemocyte response depending on the bacteria types that are used for challenge [ 136]. Although most of the studies focused on the presence of Toll pathways in marine bivalve mollusks, other types of receptors have been investigated. Recently, the mRNA transcripts of a new putative phagocytic receptor (C gNimc), belonging to the Nimrod superfamily, were identified in hemocytes of C. gigas, revealing upregulation after bacterial challenge, whereas the recombinant protein showed higher binding affinity toward LPS rather than peptidoglycan [ 137]. In the scallop Argopecten irradians, a peptidoglycan recognition protein (PGRP) was cloned, sharing high identity with PGRPs of higher organisms and showing upregulation in the hemocytes exposed to peptidoglycan, but not to LPS [ 138]. Two short PGRPs were also detected in the bivalve Solen grandis, and they were particularly induced by peptidoglycan and β-1,3-glucan [ 139]. Following an engulfment of foreign particles within hemocyte phagosome, the activation of lysosomes granules leads to the formation of phago-lysosomes vacuoles where intracellular digestion takes place [ 140]. The lysosomal enzymes strongly participate in the degradation of ingested, material and the hydrolytic activity of β-glucuronidase, phosphatases, esterases and sulfatases has been detected in mussels [ 141], clams [ 142], cockles [ 143] and oysters [ 58]. Oxidative enzymes, such as peroxidase and phenoloxidase, are also involved in degradative mechanisms, but their presence is not a common feature in any marine bivalve mollusks [ 144, 145]. The respiratory burst represents another heavy microbicidal mechanism, and the generation of the highly oxidant reactive oxygen species (ROS) inside phago-lysosomal vacuoles of stimulated hemocytes was suggested as an NADPH-oxidase-dependent mechanism [ 146, 147, 148]. The ROS synthesis has been widely reported in mussels [ 149], oysters [ 150], scallops and clams [ 151], although some authors indicated the lack of the NADPH-oxidase activity in the family Veneridae. The detection of ROS has been mostly investigated in terms of defense mechanism; however, the role of these molecules has been also considered in cellular and tissue homeostasis [ 152]. Indeed, previous reports indicated the ability of mussel hemocytes to generate ROS in the absence of phagocytic stimulation [ 153], and more recent evidence has suggested that mitochondria represent the main source of ROS in the unstimulated hemocytes, rather than the activity of lysosomal NADPH-oxidase [ 154].

Humoral defense factors: The hemocyte degranulation and the extracellular release of lysosomal enzymes represent the first humoral defense mechanism that was investigated in marine bivalve mollusks [ 155], a strategy commonly described as a response to pathogens [ 156]. However, marine bivalve mollusks possess more selective extracellular tools to contrast invaders, including recognition and effector proteins, such as lectins, complement-like molecules, lipopolysaccharide- (LBP) and β-1,3-glucan-binding proteins (β-GBP), fibrinogen-related proteins (FREPs) and antimicrobial peptides (AMPs) [ 156, 157, 158]. Lectins represent carbohydrate-recognition proteins, and their agglutinating and opsonizing activities have been previously described in marine bivalve mollusks, revealing heterogeneous binding specificity towards microbial surface sugars [ 159, 160]. In particular, C-type lectins can recognize and bind terminal sugars on glycoproteins and glycolipids in a calcium-dependent manner. Recent studies in different marine bivalve mollusks have demonstrated both their gene upregulation following bacterial challenge and the binding activity of the recombinant proteins towards purified PAMPs [ 161, 162, 163]. Galectins, formerly known as S-type lectins, represent another conserved and ubiquitous family of carbohydrates-binding proteins, particularly characterized by their affinity for β-galactosides [ 164, 165, 166, 167]. In the clam R. philippinarum [ 168] and scallop A. irradians [ 169], galectins have been cloned and characterized, showing gene upregulation and agglutination activity following bacterial challenge. Galectins seem to possess also an opsonizing role by promoting the hemocyte phagocytosis through cross-linking between extracellular glycocalyx and hemocyte surface, as observed for the oyster galectin CvGal1 [ 170]. Homologues of the vertebrate complement cascade have been investigated in marine bivalve mollusks for their immune role against pathogens. C1q represents the first sub-component of the classical complement pathway, and to date, C1q domain-containing proteins have been characterized at molecular level in oysters [ 171], mussels [ 172], scallops [ 173] and clams [ 174], revealing high molecular diversification of this family [ 175]. From a functional point of view, the recombinant proteins showed binding activity towards whole bacterial cells, as well as isolated PAMPs [ 176]. In addition, some authors have identified in oyster a complement component C3-like gene, particularly expressed in the hemocytes [ 177]. Although AMPs represent the most examined group of antimicrobial proteins, further discussed in the present review, other bactericidal compounds have been identified in marine bivalve molluscan integrative components of their humoral defense system. Member homologues of the bactericidal/permeability-increasing protein (BPI) family were isolated, showing binding activity toward LPS and bactericidal properties against Gram-negative bacteria [ 178, 179, 180]. Members of the lysozyme families (N-acetylmuramide glycanhydrolase) have been characterized in mucosal tissues and secretions of several bivalve species, as described in mussels [ 181], clams [ 182], scallops [ 183] and oysters [ 184], displaying a broad spectrum of antimicrobial activity and playing a dual role both in nutrition and immunity [ 185]. Moreover, the presence of plasma proteases' activity was previously described in marine bivalve mollusks as a microbicidal mechanism [ 186, 187], and such evidence has accounted for the isolation and characterization of genes encoding proteases, such as cathepsins [ 188].

In conclusion, the great part of the studies on bivalve immunity has been directed to investigating the morpho-functional properties of circulating hemocytes and the humoral defense factors, providing limited information about the spatial and temporal heterogeneity of the immune response. In the future, a better understanding of microbe-bivalve interactions at mucosal interfaces is required, considering the interplay between mutualistic, commensal and pathogenic microbes at the initial encounter/colonization sites [ 189].

4. AMPs and Their Mechanism of Action

Marine ecosystems constitute more than 70% of the Earth's surface, are associated with astonishing species diversity and, therefore, represent an enormous resource of pharmacologically-active molecules. Marine living beings can be reconsidered as a potentially unlimited reservoir of bioactive molecules, either derived by complex metabolic reactions or gene-encoded peptides [ 190, 191]. For each milliliter of seawater, approximately 10 ⁶ bacteria and 10 ⁹ viruses are generally present; therefore, seawater is to be considered an abundant source of pathogens. Most marine organisms reside in intimate coexistence with pathogenic microbes, and their survival in such a hostile surrounding is directly dependent on the development of a vigorous and successful immune system. In fact, living marine organisms are continuously exposed to microbial hazards, and to maintain their safeguard in such a harsh environment, they need a strong defensive mechanism to control all microbial pathogens that are inglobated with nutrients [ 192, 193]. In fact, microbes are accumulated in bivalves, and microbial densities in their tissues are generally greater than in seawater. If the filtered microbes are pathogenic, their concentration in bivalve tissues can be deleterious. In order to defend themselves against such detrimental pathogens, bivalves depend on cellular defense mechanisms, as earlier described, and humoral defense factors, among which AMPs play an important role.

As a matter of fact, bivalves having evolved in the constant proximity of microorganisms must rely on their innate immune system effector molecules to contrast microbial pathogens. An ancient mechanism of innate immunity is represented by the production of anti-microbial substances, primarily peptides or polypeptides, which are produced by different types of cells and secretions and are either constitutively synthesized or induced at the time of infection [ 191]. In this regard, marine bivalves represent a valuable and scarcely delved source for novel antimicrobials [ 194].

Since the innate immunity system is supposed to represent the primary line of host defense against invading pathogens, it is of paramount importance to maintain host-microbe homeostasis and AMPs as ancient evolutionary molecules universally distributed in most of the multicellular organisms, which perform a broad-spectrum antimicrobial activity and, often, also present an immunomodulatory capacity. Therefore, AMPs play a crucial role in host defense against a wide range of microorganisms including Gram-positive and Gram-negative bacteria, viruses, fungi and parasites. Since their first discovery, with the isolation of a peptide named cecropin [ 195, 196, 197], from the insect Hyalophora cecropia, almost 2000 sequences encoding putative AMPs have been described and included in "The Antimicrobial Peptide Database" ( http://aps.unmc.edu/AP/main.php).

AMPs are relatively small peptides (<60 amino acids) and may play polyvalent roles, which expand beyond their ability to serve as antibiotics [ 198]. Several of these peptides have been proven to possess anticancer activity, to be able to stimulate the immune system by promoting cytokine release, promote chemotaxis, antigen presentation, angiogenesis, inflammatory responses and adaptive immune induction [ 199, 200, 201]. During the last few decades, they have been purified from plants, invertebrates and vertebrates and are consequently considered to be part of the immune process probably in all Metazoa, representing innate immunity actors conserved along evolution in all biological kingdoms [ 202, 203, 204]. Despite their ample variation in biophysical characteristics, such as mass, composition and primary structure, several functional correlates have been identified [ 205]. In fact, the embracing features of most AMPs include rapid killing mechanisms, broad spectra of action, a clear net cationic charge and a strong propensity to give rise to amphipathic surfaces able to promote peptide: membrane interactions [ 206]. Although the primary structures of AMPs are diverse, based on genomic and protein sequences analysis coupled with structural and functional studies, AMPs have been sorted into several groups, including: (i) linear peptides able to adopt an α-helical conformation in a membrane-mimetic environment, (ii) peptides stabilized by one or several pairs of cysteine residues able to form disulfide bridges that have structures predominantly composed of β-sheets, (iii) peptides with a high content of specific amino acids, such as prolines, arginines, tryptophans, histidines and glycines, but with no uniform secondary structures, and (iv) peptides derived by partial hydrolysis of bulky precursor proteins with unknown or limited antimicrobial activities before the enzymatic degradation. AMPs' antimicrobial activity derives from membrane disruption and osmotic lysis of bacteria as opposed to the usual mechanism of action of most antibiotics where specific sites during bacterial growth and replication are targeted. Moreover, some AMPs proved to be also efficient in inhibiting viral infections. The putative mechanism for exerting an antiviral activity seems to be: (i) blocking early steps of viral entry by surface carbohydrate interaction, (ii) blocking viral attachment or penetration into the host cells by interactions with specific cellular receptors, (iii) interaction and inactivation of viral envelope glycoproteins, (iv) modulation of host cell antiviral responses, (v) blocking intracellular expression of viral genes and/or production of viral proteins. However, no unequivocal correlation between AMP structures and microbial inhibition or killing mechanisms has so far become obvious; in fact, striking differences from peptide to peptide and specificity for particular AMP-microbe combinations are generally observed. The forthright antibiotic action of AMPs is considered to hinge on their cationic and amphiphilic nature, which empowers these molecules with the ability of interact with negatively-charged bacterial surfaces and membranes, therefore leading to membrane disruption or alteration [ 207]. In fact, AMPs essentially take advantage of the broad differences found in the organization of bacterial against eukaryotic membranes in order to promote damage of the membrane. Several differences, such as the absence of cholesterol, the abundance of anionic lipids and an electric field with a strong inward direction, are decisive for the correlation of specificity in favor of AMPs action against bacterial pathogens and a lower toxicity toward host cells [ 208, 209]. Moreover, it is unlikely that bacteria can spoil these features and develop resistance since it would necessitate a profound modification of the bacterial membranes and their functions. Even though the exact AMP mechanisms of action remain a subject of discussion, the majority of them share similar biophysical characteristics that allow peptides to interact with microbes. Importantly, AMPs' antibacterial power is directly attributable to some of these features, such as a net positive charge, enabling AMP-bacterial membrane interactions via electrostatic forces and the propensity to form amphipathic structures in hydrophobic environments allowing penetration into the bacterial phospholipid bilayer. Therefore, regardless of any variations in size and structure, AMPs are often portrayed with an intrinsic cationic and hydrophobic nature that is the key for their first interaction with target bacterial cells. In agreement with literature data, AMPs' mode of action seems to proceed similarly to a pore-forming action or to a detergent effect. Several models have been put forth to explain such mechanisms, namely the barrel-stave model, the toroidal model and the carpet-like model () [ 210]. The three models were all elaborated following the assumption that AMPs have the tendency of being attracted by the bacterial membrane in virtue of electrostatic bonding forming between the peptide cationic feature and the low electric charge conferred to outer bacterial membranes by their surface components, such as phosphate groups within the lipopolysaccharide (LPS) of Gram-negative bacteria or lipoteichoic acids abundant on the exterior of Gram-positive bacteria [ 211].

These models can be described in brief as follows: in the barrel-stave model, peptides (more often α-helical peptides with marked hydrophobic-hydrophilic domains), once attached to the phospholipids, aggregate and enter inside the membrane bilayer with the hydrophilic peptide domains forming the inside of the pore, and the hydrophobic peptide parts lined up facing the lipidic region of membrane phospholipids [ 212]. As a result, transmembrane pores, made up of a bundle of amphipathic helices, are created in a perpendicular topology within the membrane. A toroidal model with lipids intercalating between helices can be envisioned when peptides remain linked with lipid head groups, also when peptides are located upright within the highly-curved lipid bilayer. In this case, diffusion of lipids between the outer and inner membrane layers is granted by the continuous surface formed by both membrane leaflets [ 203, 213]. A carpet-like model revealed instrumental for describing AMPs mechanism devoid of the straight insertion within the hydrophobic core of the membrane, but with peptides accumulation in an oriented array on the membrane surface forming a real carpet-like structure. When a threshold concentration is attained, the formation of transitory pores with disruption of lipid assembly and a detergent-like cell lysis ensues. This also leads to the micellization of the bilayer. Hence, all models considered, membrane perturbation is driven by cationic and hydrophobic residues shaping the interactions between peptides and phospholipids [ 214]. Nevertheless, an oversimplification of the hypothetical pore models is generally applied to the description of the mechanisms of action of AMPs [ 215]. More likely, a disordered or chaotic pore can be envisaged where peptides may shift their conformation, mutating the charges and relative interactions with lipids and, therefore, allowing the flickering of pores [ 216].

In conclusion, the interfacial activity is a leading determinant of the permeabilizing activity of several peptides [ 217], and it also provides a useful means to differentiate between peptides with antibacterial power that have a detrimental effect on membrane bilayers and cell-penetrating peptides, which seem to move past the bilayer without producing serious damages [ 218, 219]. A further possibility for the antibacterial mechanisms is the action AMPs can exert on microbial intracellular targets where peptides can block cell-wall and/or nucleic acid synthesis, protein production and enzymatic activities [ 203].

5. Marine Bivalve Antimicrobial Peptides

Marine AMPs have been discussed thoroughly in other reviews [ 220, 221, 222, 223]. However, in the present review, we describe AMPs derived from marine mollusks and their application in fighting infectious diseases. One unanswered enigma remains: the understanding of how mollusk survive in the absence of an acquired immune system since they are in close contact with a magnitude of putative pathogens such as viruses, bacteria, fungi and parasites, as a consequence of their filtering activities. Fortunately, mollusks encode for several antimicrobial molecules, and several AMPs have been isolated from marine mollusks, such as mussels (major species analyzed: M. galloprovincialis, M. edulis), clams (major species analyzed: Venerupis philippinarum), scallops (major species analyzed: A. irradians, Argopecten purpuratus and Chlamys nobilis) and oysters (major species analyzed: C. virginica and C. gigas).

Initial identification of AMPs in bivalves dates almost 20 years ago with the pioneering studies that led to the characterization of the first AMPs in mussels [ 224, 225, 226, 227]. To date, several AMPs have been described from mussels and other bivalves. The majority of them rank along to the group of cysteine-containing peptides, which include a huge variety of defensins and defensin-like peptide and larger proteins. The discovery of the first molluscan AMP was performed by Hubert et al., 1996, from the M. galloprovincialis. Subsequently, several AMPs have been identified and extensively studied in two main mussels species, M. galloprovincialis and M. edulis. These molecules with antimicrobial properties have been classified into four groups following a primary structure classification parameter: defensins, mytilins, myticins and mytimycin. Overall, mussels' AMPs are characterized by possessing strong hydrophobic and cationic properties and a signature amphipathic structure (α-helix, β-hairpin-like β-sheet, β-sheet or α-helix/β-sheet mixed structures), all considered fundamental for the antimicrobial activity displayed. The principal defensin from mussels is a 39-long peptide present in two isoforms MGD1 (PDB 1FJN) and MGD2 sharing significant sequence homology. Furthermore, the three-dimensional structure ( shows the 3D structure of several mollusk-derived AMPs) has been solved using NMR analysis [ 228] and has shown the presence of a side helical part (spanning from Asn7 to Ser16) and two anti-parallel β-strands (spanning fromArg20 to Cys25 and from Cys33-Arg37), which constitute the common cysteine-stabilized motif.

A similar structural icon has been recently described in the attempt to unify all known classes of Cys-stabilized antimicrobial peptides. Yount and Yeaman identified this common structural signature and named the "γ-core motif" [ 229, 230]. Conservation of the γ-core motif across all living organisms suggests it may represent an antimicrobial peptide archetype; in fact, several structural topologies resembling a γ-core motif can be described in a wide range of organisms, from unicellular organisms to humans. The γ-core present in many AMPs is not the only structural determinant to confer an antimicrobial activity, but in many instances, it can be used as a scaffold, to which further antimicrobial determinants (e.g., α-helices or β-sheets) can be attached in a modular fashion to yield various configurations. Higher organisms showed the most diversified range of γ-core polypeptides. This is, though, expected considering the necessity to provide protection to diverse tissues and to cooperate with other useful immune-system components. For example, several studies have been recently conducted in the analysis of such determinant in human β-defensins (HBDs), and the γ-core alone has been proven sufficient for retaining substantial antimicrobial activity [ 231, 232, 233, 234]. Nevertheless, there is a significant difference with most of the know AMPs bearing the γ-core signature; that is, the fact that mussels defensins are characterized from being stabilized by four disulfide bonds (Cys4-Cys25, Cys10-Cys33, Cys14-Cys35 and Cys21-Cys38 in MGD-1), instead of the three disulfide bonds generally described in most other molecules, including arthropod defensins. MGD1 [ 224], a member of the arthropod defensin family from edible Mediterranean mussels ( M. galloprovincialis), and MGD2 [ 227] share the same size and sequence showing 80% identity with amino acids. Both contain an ORF encoding 81 amino acids including a 21-residue N-terminal sequence with a highly hydrophobic core representing a signal domain, followed by a 39-amino acid sequence corresponding to the active defensin and a 21-residue C-terminal extension. MGDs are principally active against Gram-positive bacteria, but MGD2 showed increased activity also against Gram-negative bacteria. Structural features of MGD1, cardinal for the supply of antimicrobial activity, were analyzed by Romestand et al., 2003 [ 235], by producing a set of synthetic peptides analogous to the described secondary structures of the molecule. The nonapeptide from residue 25 to residue 33 (CGGWHRLRC) displayed a consistent bacteriostatic activity, especially when cyclized by a disulfide bridge between Cys25 and Cys33.

The second group of mussels-derived AMPs is represented by the mytilins family, comprising five isoforms (A, B, C, D and G1). Isoforms A and B were found in M. edulis plasma [ 236], while isoforms B, C, D and G1 were isolated from M. galloprovincialis hemocytes [ 237]. Mytilin B (PDB 2EEM) is produced from a precursor molecule, which contains an initial region (22-amino acid residues) signal peptide region, a mature peptide 34 amino acids long, followed by a C-terminal domain of 48 residues rich in acidic amino acids [ 236]. The assorted mytilin isoforms have been shown to possess distinctive antimicrobial activities. In fact, mytilins A, B, C, and D showed a considerable activity against both Gram-positive and Gram-negative bacteria while mytilin G1 was revealed to be functioning only against Gram-positive bacteria. Mytilins B and D have also shown potency against the filamentous fungus Fusarium oxysporum. Moreover, experiments for describing the kinetics of bactericidal effects showed that, at high concentrations, several hours of incubation were needed for mytilins D and G1 to kill all bacteria in contrast to the few minutes necessary in the presence of mytilin B. A potent antiviral activity was also observed for mytilin B. Therefore, the different mytilin isoforms are endowed with complementary properties, which altogether contribute to the defense mechanisms, increasing the antimicrobial potential of mussels living in the context with a high diversity of pathogens.

A cysteine-rich peptide has also been isolated from mussels ( M. galloprovincialis) and named myticin [ 226]. The mature molecule is 40 residues long and shows four intra-molecular disulfide bridges. Three different isoforms of myticin have been described (A, B and C) with the isoform C being the most abundantly expressed transcript in adult mollusks. All isoforms are highly active against Gram-positive bacteria and sometimes against Gram-negative bacteria, but myticin C is also a potent antiviral compound [ 238, 239]. Constitutively-expressed myticin C-peptides in naive mussels render oysters resistant to ostreid herpesvirus 1 (OsHV-1) infections when oyster hemocytes are incubated with mussel hemolymph. Moreover, myticin C molecules retain antiviral activity in vitro against human herpes simplex viruses 1 (HSV-1) and 2 (HSV-2), showing a high potential for biotechnological applications [ 240].

A strictly antifungal peptide named mytimycin (MytM) containing 12 cysteines with a molecular weight of 6.2 KDa was derived [ 225] from the plasma of M. edulis. A novel cysteine-rich peptide with noteworthy antibacterial activity was recently isolated from Mytilus coruscus and was named myticusin-1 [ 241]. This is a 104-amino acid long polypeptide including 10 cysteine residues. Antimicrobial studies showed that myticusin-1 presented a more pronounced anti-microbial activity against Gram-positive bacteria compared to Gram-negative bacteria and fungus. From the same mussel ( M. coruscus), a novel antimicrobial peptide with 55 amino acid residues was also identified [ 242]. This new antimicrobial peptide is endowed by predominant activity against fungi and Gram-positive bacteria and is characterized by possessing a chitin-biding domain and by six Cys residues forming three intra-molecular disulfide bridges. The recent advent of genome sequencing technologies has also allowed the identification of two previously uncharacterized mussel AMP families, big defensins and macins. A recent analysis [ 243] brought to light the existence of eight novel big defensins (MgBDs) and five novel macins (mytimacins) in the transcriptome of the Mediterranean mussel M. galloprovincialis, therefore further extending the vast antimicrobial peptides range present in this marine bivalve organism.

The Manila clam, V. philippinarum, is a meaningful marine bivalve for commercial purposes, and an amino acid sequence has been identified (named VpBD) that shares common features with other AMPs, such as an α-helical structure, a net positive charge and a high hydrophobic residue ratio. The display and spacing of cysteine residues and their flanking amino acid residues indicated that VpBD represents a member of the big defensin family. The structure of big defensins, generally, comprises a highly hydrophobic region located at the N-terminal, one C-terminal cysteine-rich and positively-charged region, as well as six cysteine residues arranged to form 1-5, 2-4, 3-6 disulfide bonds in the mature peptide, in a similar pattern to mammalian β-defensins. The microbicidal activities of VpBD (expressed in Escherichia coli) in vitro have been investigated and demonstrated a strong antibacterial activity towards various bacterial species, namely Gram-negative ( Pseudomonas putida) and Gram-positive ( Staphylococcus aureus) [ 244]. A further big defensin has been isolated from the ark shell, Scapharca broughtonii [ 245] and clam myticin isoforms 1, 2 and 3, and clam mytilin, (similar to myticins and mytilins from mussels) have been identified and characterized in Ruditapes decussatus [ 246].

Extracts from acidified gills of the American oyster C. virginica [ 247] delivered the first defensin molecules to be purified by oysters. The peptide (named Cv-Def) was 38 amino acids long with six cysteines, and the molecular mass was 4.2 KDa. The antimicrobial spectrum covered by Cv-Def included both Gram-positive bacteria and Gram-negative bacteria [ 247]. Successively, three more defensins were found and characterized from the mantle, denoted as Cg-Def, or hemocytes, designated as Cg-defh1 (PDB 2B68) and Cg-defh2, of the Pacific oyster C. gigas [ 248, 249], which shared the cystine-stabilized alpha-beta motif (CS-αβ) [ 250]. Other AMPs are produced in C. gigas, such as Cg-Prp, which belong to the family of proline-rich peptides and has been identified from hemocytes [ 251, 252]. A 5.5-kDa antimicrobial peptide 55 amino acids long, and named cgMolluscidin, has been recently purified from the acidified gill extract of C. gigas. This sequence has no homology with any known AMPs and showed a strong antimicrobial effect against both Gram-positive bacteria ( Bacillus subtilis, Micrococcus luteus and S. aureus) and Gram-negative bacteria ( E. coli, Salmonella enterica and Vibrio parahaemolyticus). Finally, the last group of AMPs, described in the present review, has been identified in scallops, mainly from A. irradians (AiBD) and A. purpuratus (Ap). The scallop AiBD consisted of 531 nucleotides and produced a peptide of 122 amino acids. Recombinant AiBD was able to block the growth of both Gram-positive and Gram-negative bacteria and also presented a strong fungicidal power [ 253]. AP was isolated from A. purpuratus hemocytes, consists of 47 residues and shares partial homology with reported effective AMPs. A modified version of 30 residues designed to increase hydrophobicity and cationicity was used in antimicrobial experiments and showed an excellent activity against Saprolegnia sp., a parasitic pathogen fungus that attacks the culture of fish in different stages of their life, from the egg stage to grown-up animal [ 254].

In order to summarize all those peptides in an intelligible overview, we generated a table with their detailed characteristics ().

Table 4

6. Applications of AMPs in Medicine and in Preventing Diseases of Aquatic Animals

In the present review, we first outlined the infectious diseases of marine bivalve and their defense mechanisms including the classification and mode of action of AMPs. In the remaining and concluding part of the review, we will focus on the potential utilization of AMPs as substitutes for antibiotics in aquaculture and in the medical field.

The economic development of several countries relies on the aquaculture of mollusks since a considerable amount of shellfish (mainly mollusks) for human consumption is produced by aquaculture. Clam, scallop, oyster and mussel farming represents a noticeable share of the aquaculture market worldwide, accounting for more than 50% of the shellfish present on the global market. The increasing demand for seafood, including bivalves, will surely extend in the forthcoming years, and higher capability productions will need to be accomplished to meet this demand. The major obstacle to the thriving of the aquaculture industry is posed by the emergence and spread of many infectious diseases, which are exacerbated by the densely-populated culture conditions in limited space. Future research needs to include the development of novel methods to control diseases in hatcheries in order to minimize the occurrences of mass mortalities caused by either obligate or opportunistic pathogens. Research efforts are mandatory to explore inexpensive and effective treatments when diseases occur in a hatchery situation to avoid production losses. Moreover, safer compounds are highly desired considering the environmental risks and that bivalve production is mainly devoted to human consumption. The possibilities to contain the spread of diseases in mollusks is not facilitated by the fact that there is limited information to fully understand the physiology of marine bivalves, in particular concerning their immune defense system. A detailed description of the immune defense mechanisms of mollusks has been provided in the first part of the review with the intent to foster the characterization of immune effectors to provide new understanding into healthiness and disease management in mollusk aquaculture. Since the introduction of antibiotics for the treatment of infectious diseases, they have been widely used in medicine and animal breeding, as well as in aquaculture with successful regimens. However, due to the overuse and misuse of antibiotics, the resistance of bacteria to antibiotics has dramatically increased, posing new challenges to human health and to the sustainability of the aquaculture industry. Moreover, the worldwide threat of a rapid increase in pathogenic multidrug-resistant (MDR) bacteria is paralleled by the environmental risks with antibiotic compounds being distributed throughout the environment. The quest for novel compounds with antibacterial activity that could overcome resistance emergence events is, therefore, an urgent societal challenge. Nature-derived AMPs are regarded as convenient templates for the development of substitutes to traditional antibiotic [ 255], and bivalve AMPs have been shown to be structurally different from their analogue peptides derived from the terrestrial habitat and usually present novel and unexploited structures [ 223, 256]. AMPs' antimicrobial activity relies on the early electrostatic interactions with the negatively-charged surface of the bacteria, therefore, free ions produced by the high salt concentrations in the surrounding medium, typical of some illnesses, could efficiently decrease interaction and antimicrobial activity. In general, marine AMPs have evolved to easily adapt to the high salt concentration of seawater, and probably, this has been obtained by the substitution of lysines with arginines. Therefore, marine bivalves are a hopeful reservoir of novel bioactive molecules for the development of alternative antimicrobials. In fact, AMPs can be regarded as encouraging candidates for selecting new and more environmentally-friendly antimicrobials. Notwithstanding the abundance of scientific knowledge on their activities in vitro, major challenges need to be outflanked to allow for their clinical application. The major obstacles to be considered are: (1) rapid degradation by proteases; (2) uncertainty of the antimicrobial activities under physiological salt, pH and serum conditions; (3) poor oral availability; (4) laborious routes of administration; (5) burdensome transportation across cell membranes; (6) non-selective receptor binding; and (7) costs associated with their production [ 223]. Excessive amounts of common antibiotics are used in aquaculture in some countries for both therapeutic and prophylactic purposes [ 257, 258, 259]. Moreover, veterinary antimicrobials also include compounds used clinically in human medicine [ 260]. Current knowledge regarding the genetic aspects of antimicrobial resistance in aquatic bacteria is highly suggestive of the possibility that antibiotics used in fish farming are likely to select antimicrobial-resistant bacteria in aquacultural environments and of their subsequent diffusion to terrestrial counterpart [ 261, 262, 263]. Therefore, in order to safeguard public health, it is of paramount importance to adopt novel methodologies used in aquaculture. The use of AMPs is one of the main strategies currently under deep investigation. It is, though, imperative to devote future efforts to design improved antimicrobial molecules with a broad spectrum of activity against a wide array of pathogenic microorganisms through the modification of native AMPs in order to achieve more selective and efficient drugs that could substitute conventional antibiotics both in aquaculture and in clinical human medicine.

One of the most awaited goals is the use of rational design to produce AMPs with improved characteristics, such as: (1) stronger antibacterial activity, (2) lower cytotoxicity and (3) ease of production on an industrial scale for obtaining a marketable drug. Native AMPs can be used as templates for the design of new antibacterial agents through peptidomimetics, where only structural key elements of the native peptide are conserved to provide a scaffold able to preserve the AMP characteristic of easy interaction with the biological target and produce an enhanced antimicrobial biological response [ 264]. Domains responsible for activity are, therefore, analyzed in detail and modified to obtain functionally-improved AMPs. Several authors have described the modification of primary AMP sequences to enhance their effectiveness and stability in order to obtain promising lead compounds for the development of therapeutic agents [ 265]. Attempt to increase AMP activities have generally involved the methodical change of amino acid residues or alternative chemical alterations, which permit the achievement of improved activities, such as: chemical modification of terminal ends of peptides [ 266], development of analogues containing unnatural amino acids [ 267], β-peptides, shortening of the native sequence, modifications of their amphipathic character, cyclization [ 234], hybrid peptide-peptidomimetic structures, lipidation, etc. [ 268]. One of the reasons for the high interest in AMPs derived from marine peptides is their ability to sustain physiological salt concentration and protease activity. In fact, a foremost role for the improvement of AMPs therapeutic impact is represented by the suitable harmonization of their hydrophobicity, amphipathicity and positive charge [ 269, 270]. Peptides with sufficient positive charge can be modified to decrease interactions with mammalian cells, while favoring the preferential binding to bacterial cell membranes by reducing the overall hydrophobicity of the molecule [ 271, 272]. Exploiting the variety of post-translational modifications displayed by marine AMPs could be instrumental in the design of AMPs with enhanced stability and efficacy, for therapeutic utilization in human medicine [ 273]. For example, the high salinity (up to 600 mM) of the marine habitat may have forged marine AMPs to be naturally endowed with a sharpened salt resistance, allowing them to conserve strong antibacterial effectiveness in relatively high-salt environments, such as in saliva, gastrointestinal fluid, serum or other body fluids [ 231, 232]. Understanding the chemical propriety backing this salt independent activity could subside in the construction of novel AMPs that could be better endowed for facing pathogens notwithstanding the wide range of salt concentrations that could be encountered. For example, human beta defensin-1 (hBD-1) being unable to inhibit P. aeruginosa due to a 120 mM concentration of NaCl in the lungs of cystic fibrosis patients [ 274] has been engineered by constructing a chimera with β-defensin-3 (hBD-3), which shows antibacterial activity also at high salt concentrations [ 231, 232]. The C-terminal domain of hBD-3 presents an abundance of arginine residues considered to be involved in the activity at high ionic conditions. Therefore, marine AMPs, dictating many of the universal rules featuring the ability of an AMP to inhibit microorganisms under physiological salt concentrations (120-150 mM), are of paramount importance for indicating novel strategies for the improvement of native AMP sequences [ 275].

7. Conclusions

Bivalve farming has recently reached a large portion of the fish market worldwide; therefore, from both a human health perspective and for reducing economic losses, huge efforts are devoted to the improvement of the microbiological quality of the product. We are awaiting a comprehensive understanding and knowledge of all of the infectious diseases that could affect bivalve mollusks, and insights on their defense mechanisms are only recently being deeply investigated. Several studies on bivalve immunity have expanded our understanding of the morpho-functional properties of circulating hemocytes and the humoral defense factors, but most of the patterns for microbe recognition and downhill immune pathways activation are still to be explored. At the same time, in the last decade, a large amount of data on mollusk-derived AMPs has been gathered, to allow the beginning of the initial studies to attempt to modify these lead compounds in search of a wider applicability of their antimicrobial properties in both aquaculture and human medicine.

Acknowledgments

We thank Albert Bonaminio (M. Sci.) for linguistic editing. This work was supported by: Regional funds "Pepmar".

Author Contributions

All Authors contributed equally.

Conflicts of Interest

The authors declare no conflict of interest.

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Microbial Diseases of Bivalve Mollusks: Infections, Immunology and Antimicrobial Defense

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Se-Kwon Kim, and Peer B. Jacobson, Academic Editor A variety of bivalve mollusks (phylum Mollusca, class Bivalvia) constitute a prominent commodity in fisheries and aquacultures, but are also crucial in order to preserve our ecosystem's complexity and function.

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Article 1.2

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Summary: Seafood is part of a healthful diet, but seafood consumption is not risk-free. Seafood is responsible for an important proportion of food-borne illnesses and outbreaks in the United States. Seafood-associated infections are caused by a variety of bacteria, viruses, and parasites; this diverse group of pathogens results in a wide variety of clinical syndromes, each with its own epidemiology. Some seafood commodities are inherently more risky than others, owing to many factors, including the nature of the environment from which they come, their mode of feeding, the season during which they are harvested, and how they are prepared and served. Prevention of seafood-associated infections requires an understanding not only of the etiologic agents and seafood commodities associated with illness but also of the mechanisms of contamination that are amenable to control. Defining these problem areas, which relies on surveillance of seafood-associated infections through outbreak and case reporting, can lead to targeted research and help to guide control efforts. Coordinated efforts are necessary to further reduce the risk of seafood-associated illnesses. Continued surveillance will be important to assess the effectiveness of current and future prevention strategies.

Bacteria

Vibrio species.

Vibrio organisms are Gram-negative, halophilic bacteria that are widespread and naturally present in marine and estuarine environments. Environmental factors influence their growth, and their numbers are highest when the water is warm. The genus Vibrio includes 30 species, of which at least 14 are recognized as pathogenic in humans. Vibrio infections are acquired through ingestion of contaminated seafood or through exposure of an open wound to seawater. Vibrio parahaemolyticus and Vibrio vulnificus are the species most commonly associated with reported infection. V. parahaemolyticus has been associated with sporadic infections and outbreaks of gastroenteritis, while V. vulnificus infections occur almost exclusively as sporadic cases in the United States. Clinical features most often associated with V. parahaemolyticus infection include watery diarrhea, abdominal cramps, nausea, and vomiting; wound infections and septicemia occur less commonly ( 18, 39, 45). V. vulnificus is particularly virulent, especially among patients with liver disease and iron storage disorders, who are at increased risk of invasive disease ( 4, 33, 39). V. vulnificus infections can lead to sepsis and severe wound infections ( 33, 39, 50, 60). Severe infections, such as bloodstream and wound infections, require prompt antimicrobial therapy. The case fatality rate is about 50% for bloodstream infections and 25% for wound infections ( 21, 33, 35, 60). Laboratory diagnosis is made by isolation of the organism from clinical specimens, including blood, stool, and wound samples. Because they can be overlooked on standard agar plates, Vibrio organisms in stool or wound samples are best identified on selective media, such as thiosulfate-citrate-bile salts-sucrose (TCBS) agar ( 1, 34).

Cases of Vibrio infections have a marked seasonal distribution; most occur during summer and early fall, corresponding to the period of warmer temperatures. Almost all cases of food-borne Vibrio infection are associated with a recent history of seafood consumption, primarily raw oyster consumption. Like other organisms found in water, vibrios can be concentrated in the tissues of filter-feeding bivalve mollusks. Measures to prevent food-borne Vibrio infections include consumer education regarding the dangers of eating raw or undercooked shellfish, particularly among persons with medical conditions, such as liver disease, that predispose them to severe illness. Thoroughly cooking shellfish and preventing raw seafood from cross-contaminating other foods are effective measures for consumers to reduce risk ( 25). Regulatory control measures have included monitoring of harvest waters and microbiological sampling of oysters. However, it is important that the presence of vibrios is not associated with fecal contamination. Therefore, monitoring waters for fecal coliform bacteria is not effective as an indicator of the presence of Vibrio in harvest environments. Finally, postharvest processing methods, such as high-pressure treatment, irradiation, quick-freezing, and pasteurization, are available to make oysters safer. Vibrio infections are reportable to state health departments, and traceback of oysters associated with human Vibrio infection is strongly encouraged ( 16).

Salmonella.

Salmonellae are Gram-negative bacilli. Approximately 2,500 Salmonella serotypes have been identified, causing a variety of clinical syndromes ranging from asymptomatic carriage to invasive disease ( 6). Salmonella most commonly causes acute gastroenteritis, with symptoms including diarrhea, abdominal cramps, and fever. Other clinical manifestations can include enteric fever, urinary tract infections, bacteremia, and severe focal infections. Isolation of Salmonella organisms from cultures of stool, blood, or other clinical samples is diagnostic; isolates are referred to public health laboratories for serotype characterization. Salmonella is a leading cause of food-borne illness, causing approximately 1.4 million illnesses annually in the United States ( 44). Incidence is highest among infants and the elderly, and infections are more likely to occur during summer and early fall ( 52). Each Salmonella serotype has its individual biology and ecological niche. Many natural reservoirs for the different Salmonella serotypes have been identified. Humans are the only known reservoir for Salmonella serotype Typhi; many animal hosts (birds, reptiles, and mammals) serve as reservoirs for nontyphoidal serotypes. With improvements in sanitation over the past several decades, Salmonella serotype Typhi went from being the leading cause of Salmonella infection to becoming relatively uncommon. However, nontyphoidal Salmonella infections have emerged as a public health problem since the 1950s ( 52, 63, 64).

Routes of Salmonella transmission include food-borne and waterborne routes, person-to-person contact, and contact with animals, particularly reptiles. Most cases of nontyphoidal salmonellosis are caused by ingestion of contaminated food, and outbreaks have been associated with a wide range of food vehicles in the United States. Seafood-associated outbreaks have been caused by fish, shrimp, oysters, and clams. Studies to determine the prevalence of Salmonella spp. in oysters from domestic bays and testing of sampled oysters from domestic seafood samples by the U.S. Food and Drug Administration (FDA) have demonstrated the presence of salmonellae in a variety of fish and shellfish, including seafood intended for consumption without further preparation upon distribution, requiring minimal cooking, and shellfish eaten raw ( 5, 30). Fish and shellfish can acquire Salmonella from polluted waters. Historically, sewage contamination of shellfish harvest beds led to large shellfish-associated outbreaks of Salmonella serotype Typhi infections. Control measures aimed at detecting contamination of harvest waters, such as monitoring fecal coliform counts in these waters, have been effective at reducing the risk of Salmonella contamination of seafood occurring before harvest. Additionally, seafood can become contaminated with Salmonella during storage and processing ( 7, 29). Salmonella infection can be prevented by adequate cooking, proper storage and processing after harvest, and avoidance of cross-contamination during seafood handling ( 24, 25).

Shigella species.

Shigella species are Gram-negative bacilli. Four species have been identified, and clinical presentations vary by species. Clinical manifestations of Shigella infection range from watery, loose stools to more severe symptoms, including fever, abdominal pain, tenesmus, and bloody diarrhea. Complications are rare and include seizures in young children, toxic megacolon, bacteremia, Reiter's syndrome, and hemolytic-uremic syndrome. Diagnosis is made by isolation of Shigella from feces or rectal swabs. Cases occur worldwide, in endemic and epidemic forms. Most cases occur among children aged <10 years.

Humans are the primary reservoir of Shigella. Transmission occurs through direct or indirect contact with feces of infected persons. Shigella infection is highly communicable, because ingestion of as few as 10 viable organisms is sufficient for infection to occur. The low infectious dose contributes to the potential for large outbreaks. Outbreaks have been associated with person-to-person transmission in crowded or unhygienic environments and with ingestion of contaminated food and water. Foods can become contaminated during handling or preparation by an infected food handler. With seafood, contamination can occur if seafood is harvested from sewage-contaminated water, as occurred in an outbreak caused by consumption of raw oysters harvested from waters where sewage was dumped overboard from the oyster harvest boat ( 57). Shigella organisms can survive outside the host, but they are killed readily by cooking. Control strategies to prevent shigellosis associated with seafood have included monitoring of harvest water for fecal coliforms, prohibition of harvesting from sewage-contaminated areas, enforced control of dumping sewage overboard, and guidelines for seafood handling in restaurants.

Clostridium botulinum.

Clostridium botulinum is a spore-forming, anaerobic, Gram-positive bacillus that is widespread in the environment. The bacterium produces a potent neurotoxin under anaerobic, low-acid conditions. Seven types of botulism toxin have been identified; toxin types A, B, and E cause most human illnesses. Food-borne botulism is caused by the ingestion of food contaminated with preformed toxin produced by spores of C. botulinum. Botulism is characterized by an acute, symmetric, descending flaccid paralysis. Early signs and symptoms of botulism often include cranial nerve palsies, with diplopia, ptosis, slurred speech, and difficulty swallowing progressing to descending weakness and paralysis. Symptoms can progress to cause paralysis of the respiratory muscles, requiring ventilatory support. Cases of botulism are rare but serious; an estimated 60 food-borne cases occur each year in the United States ( 44). Most cases are sporadic, but food-borne botulism outbreaks are reported each year.

Food-borne botulism cases are most often associated with home-canned foods. Other food vehicles identified in outbreak investigations have included fermented or salted seafood, potatoes baked in aluminum foil, garlic in oil, onions held under butter, and homemade salsa ( 61). Most seafood-associated cases are caused by toxin type E, which is associated most commonly with eating traditional Alaska Native foods, such as fermented salmon heads, salmon eggs, and blubber and skin from marine animals (muktuk) ( 14). C. botulinum type E spores are commonly found in fish and aquatic animals, and implicated seafood has been fermented under anaerobic conditions that favor the germination of C. botulinum ( 43, 66). Measures to prevent seafood-borne botulism have included educational efforts to promote proper methods to ferment foods and to boil fermented foods before consumption, especially if they were stored in tightly sealed plastic or glass containers ( 10).

Other toxin-forming bacteria.

Staphylococcus aureus, Clostridium perfringens, and Bacillus cereus can form enterotoxins that cause acute gastrointestinal illness. The illness typically seen with S. aureus and B. cereus intoxication is the onset of nausea, vomiting, and mild diarrhea within 1 to 6 h after ingestion of contaminated food. Incubation periods are very short because preformed toxin is present in the ingested food. Intoxication with C. perfringens has a slightly longer incubation period, because toxin is not preformed but is produced in the gastrointestinal tract. Symptoms include watery diarrhea and abdominal cramps starting 8 to 16 h after ingestion of contaminated food. Duration of symptoms is usually less than 24 h; therefore, many cases likely go undiagnosed. Diagnosis is confirmed by isolation of the organism in samples of stool, vomitus, or food. Additionally, S. aureus enterotoxin can be detected in food samples, which can be useful in situations in which the organism has been killed in food processing or preparation and therefore cannot be cultured.

Only certain strains of S. aureus produce a toxin that causes gastrointestinal illness. The main reservoir is humans, who carry the bacterium in their nasal passages, skin, or wounds. S. aureus contamination of food, including seafood, is usually due to contamination by a food worker during food preparation ( 7, 65). B. cereus and C. perfringens are found in the soil and are ubiquitous. Only a few reports of illness due to the presence of these organisms in seafood have been published, but the mechanisms in these cases appear to be similar to those for other foods ( 7). To prevent outbreaks of toxin-mediated illnesses, it is important to keep foods refrigerated and to ensure proper cooling of hot foods to refrigerator temperatures. Outbreaks are usually associated with foods left at inappropriate temperatures for prolonged periods, allowing multiplication of the organism and enterotoxin production ( 7, 51).

Viruses

Norovirus.

Norovirus is a small structured RNA virus that is among the caliciviruses recognized as human pathogens. Norovirus infection causes a self-limited illness characterized by diarrhea, vomiting, nausea, abdominal cramps, and sometimes headache, myalgias, and low-grade fever. The virus may be identified by electron microscopy or reverse transcriptase PCR of stool samples and by enzyme immunoassay for detection of viral antigen in serum samples. Humans are the only known reservoir, and norovirus is transmitted by person-to-person spread through the fecal-oral route or through contaminated food or water. The infectious dose is low, and attack rates are high. Noroviruses can persist in the environment and can infect large numbers of people of all ages. Most outbreaks are reported from child care centers, cruise ships, long-term care facilities, and other closed populations. Norovirus is a leading cause of food-borne illness and outbreaks ( 44, 68), although the number of reported outbreaks is likely grossly underestimated because of a lack of routine availability or use of diagnostic testing. The adoption of reverse transcriptase PCR by the CDC and state public health laboratories in the 1990s has greatly improved and increased the identification of norovirus as the etiologic agent of food-borne outbreaks ( 68).

Seafood harvested from sewage-contaminated waters has caused outbreaks of norovirus gastroenteritis. Large outbreaks have been associated with consumption of raw or inadequately cooked shellfish, such as a series of oyster- and clam-associated outbreaks in which northeastern coastal waters were implicated ( 46). Shellfish, particularly bivalve mollusks that are filter feeders, can accumulate large numbers of viruses. Contamination of harvest areas with human sewage, which has occurred following overboard sewage dumping by oyster harvest boats and from sewage runoff into harvest areas following heavy rains, has been an important source of outbreaks. Additionally, insufficient cooking, such as steaming clams only until they open rather than to higher temperatures that kill noroviruses, has contributed to illness and outbreaks ( 23, 46). Control measures have focused on monitoring harvest waters for fecal coliforms and encouraging adequate cooking of seafood; however, continued outbreaks of illness have brought renewed attention to the risks associated with oyster consumption ( 36, 41, 46).

Hepatitis A virus.

Hepatitis A virus is an RNA virus that is classified as a picornavirus. Although clinical manifestations vary in severity, hepatitis A virus infection is the most serious viral infection associated with seafood consumption. Most commonly, it is an acute, self-limited illness associated with fever, malaise, jaundice, anorexia, and nausea; symptoms may last from several weeks to several months. However, deaths from fulminant hepatitis can occur. Severity increases with age, and in rare cases, a prolonged, relapsing hepatitis can occur. In infants and young children, infection is often asymptomatic. Diagnosis is made using serologic tests that measure immunoglobulin M antibody against hepatitis A virus. The illness's long incubation period (2 to 8 weeks) can make the identification of the source of infection difficult, and hepatitis A virus infection associated with seafood is likely underreported. Sporadic and epidemic cases occur worldwide. In the United States, since the implementation of routine hepatitis A vaccination of children, rates of reported illnesses, especially among children, have declined considerably ( 67).

Humans are the only reservoir of hepatitis A virus, and transmission is generally by the fecal-oral route. Common-source food-borne outbreaks occur, and outbreaks of hepatitis A virus infections associated with oysters and clams have been described in the United States since the 1960s ( 22, 40, 55). The infectious dose is thought to be low, and the virus is taken up by the mollusk during feeding. Hepatitis A virus is heat resistant and able to withstand steaming, and appropriate cooking of seafood lessens the chance of ingesting live hepatitis A virus. But because shellfish is commonly prepared in ways that are insufficient to inactivate the virus, many prevention strategies are aimed at controlling contamination prior to food preparation. Contamination most commonly occurs when shellfish growing areas are contaminated with human sewage ( 22, 55). The largest reported outbreak occurred in China in 1988; almost 300,000 persons were infected after consuming clams harvested from a sewage-contaminated area ( 56). Fewer outbreaks of hepatitis A virus infection associated with seafood consumption have been reported in the United States in the past 2 decades, which may be attributable, at least in part, to implementation of increased control measures to prevent fecal contamination of harvest beds during the 1980s. This decrease suggests that control strategies may have been effective in reducing the risk of infection via seafood. Recently, the availability of molecular sequencing methods to enhance epidemiologic data has been critical in linking dispersed cases in multiple states to contaminated food products ( 3). Increases in public health laboratory capacities for molecular sequencing will improve our ability to identify outbreaks of hepatitis A virus infections.

Parasites

Helminths.

Helminths are large, worm-like parasites. Helminthic infections associated with the consumption of raw seafood include infections caused by nematodes, trematodes, and cestodes, notably eustrongyloides, species of Diphyllobothrium ( 58), and members of the family Anisakidae ( 59, 69). Clinical manifestations associated with helminthic infections range from no symptoms to mild, chronic gastrointestinal symptoms, allergic reactions, and, rarely, intestinal perforation and invasive disease. Diagnosis of most parasitic infections is confirmed by detection of ova or segments of worms in stool samples. Many worms are present naturally in marine and fresh waters, and most marine animals are infected. Despite the high prevalence of helminths in seafood, however, outbreaks associated with seafood are rarely reported. This observation is likely due to several factors: first, helminths are not able to multiply in food; second, long incubation periods make identification of the source of infection difficult; and third, infections are likely underdiagnosed and underreported, due in part to a lack of routine diagnostic tests ( 51, 59). Nonetheless, the potential for helminthic infection exists with the consumption of raw seafood. Helminths are killed by adequate freezing and by cooking. Control strategies for industry to reduce the risk of helminthic infections also include visually inspecting fish, by means such as candling, for parasites that are large enough to be detected visually.

Protozoa.

Protozoa are an uncommon cause of food-borne illnesses in the United States, and common-source outbreaks are almost always waterborne ( 44, 70). Protozoa causing recognized seafood-associated illnesses have included Giardia spp. ( 54). Giardia is one of the most commonly identified intestinal parasites in the United States. Clinical manifestations may include abdominal pain, flatulence, anorexia, and a protracted course of watery, malodorous diarrhea; asymptomatic infection is common. Laboratory diagnosis is most often made by identification of trophozoites or cysts in stool specimens or by detection of stool antigens by enzyme immunoassay. Humans and other animals are the reservoir of infection. Food-borne outbreaks of giardiasis have been reported in which transmission occurred through contamination of food by infected food handlers ( 54). Protozoa are sensitive to heat and to prolonged freezing ( 8). Strategies to prevent food-borne transmission include adequate cooking and preventing contamination of food during preparation.

Sources of Data and Methods

We examined two data sources to understand the epidemiology of seafood-associated illnesses in the United States: the Food-Borne Disease Outbreak Surveillance System and the Cholera and Other Vibrio Illness Surveillance System (COVIS). Determining the food that caused an individual illness is difficult, except during investigations of outbreaks or of certain infections which are almost always associated with a specific type of food. Investigations of outbreaks of food-borne illnesses are used to determine the food vehicles for pathogens that cause illness and to improve our understanding of the epidemiology of these outbreaks. State and local health departments identify and investigate food-borne outbreaks and, in turn, report these outbreaks to the CDC. Since the early 1970s, the CDC has collected reports from all states and territories on recognized food-borne disease outbreaks, defined as 2 or more cases of a similar illness resulting from ingestion of a common food ( 53). Information reported includes the number of persons affected, the etiologic agent identified, characteristics of illnesses, the month of illness onset, the implicated food item, possible sources of contamination, and other contributing factors. These surveillance data have been maintained in a central, electronic database since 1973. In 1998, the reporting procedures were modified to provide feedback to reporting states, and the CDC began verifying outbreak reports with states on a yearly basis.

For this analysis, we included outbreaks with an implicated seafood vehicle and a single confirmed infectious etiologic agent. Seafood vehicles were categorized into the following three commodity groups: (i) mollusks (e.g., oysters, clams, and scallops); (ii) crustaceans (e.g., shrimp, crab, and lobster); and (iii) finfish (e.g., salmon, tuna, and finfish eggs) and other aquatic vertebrates (e.g., whale and seal), or "fish" for brevity. Outbreaks caused by multiple etiologic agents and outbreaks for which implicated foods could not be classified into the single overarching food commodity class "seafood" were not included in this analysis, unless a single seafood was identified as the contaminated ingredient responsible for the outbreak.

Additionally, we examined surveillance case reports of Vibrio illnesses reported through COVIS, which is a collaborative effort of state and county health departments, the FDA, and the CDC. For the investigation of cases of vibriosis, a standard report form is used to collect detailed information on the Vibrio species causing illness; demographic information, medical history, and travel and food history of the patient; and shellfish traceback information, when available. COVIS data are used to help define Vibrio-related syndromes, describe the epidemiology of vibriosis, and identify vehicles of infection. Surveillance for vibriosis began in 1988 in Gulf Coast states and subsequently expanded nationally. In January 2007, vibriosis became a nationally notifiable disease ( 16).

Summary of Seafood-Associated Outbreaks and Infections

General epidemiologic characteristics.

During 1973 to 2006, 188 outbreaks of seafood-associated infections, causing 4,020 illnesses, 161 hospitalizations, and 11 deaths, were reported to the Food-Borne Disease Outbreak Surveillance System. Most of these seafood-associated outbreaks (143 [76.1%]) were due to a bacterial agent; 40 (21.3%) outbreaks had a viral etiology, and 5 (2.6%) had a parasitic cause (Table ). The number of confirmed outbreaks reported increased greatly during 1998, when enhanced surveillance of food-borne outbreaks began (Fig. ). Otherwise, the number of outbreaks fluctuated from year to year during the study period, with no significant increasing or decreasing trends seen. Outbreaks ranged in size from 2 to 400 affected persons, with an overall median outbreak size of 5 persons affected. The median size of outbreaks was stable through the 1970s and 1980s. Starting in the early 1990s, the median outbreak size varied substantially, with large peaks seen during 1993, 1995, and 2004.

TABLE 1.

No. (%) of outbreaks

No. (%) of illnesses

No. (%) of hospitalizations

No. (%) of outbreaks

No. (%) of illnesses

No. (%) of hospitalizations

No. (%) of outbreaks

No. (%) of illnesses

No. (%) of hospitalizations

No. (%) of outbreaks

No. (%) of illnesses

No. (%) of hospitalizations

Seafood-associated outbreaks occurred throughout the year, with a peak during late summer (Fig. ). However, seasonality differed by etiology. Bacterial pathogens, especially vibrios, caused more outbreaks during warmer months, whereas outbreaks of seafood-associated norovirus infections occurred more frequently during colder months. Seafood-associated outbreaks from all causes were reported most commonly from the following coastal states (Fig. ): Alaska ( 39), New York ( 19), California ( 17), Florida ( 16), Oregon ( 11), and Washington ( 16). All other states reported fewer than 10 outbreaks during the study period. Etiologic agents and implicated seafood vehicles varied by state. Almost all outbreaks reported from Alaska, which reported 39 (20.7%) outbreaks, were due to botulism. States in the Pacific Northwest reported a disproportionate number of Vibrio and norovirus outbreaks, largely associated with molluscan shellfish ( 11, 12). Notably, in recent years, outbreaks of infections due to some pathogens usually associated with seafood harvested from warm waters were reported from more northerly areas of the country that had not previously reported outbreaks. For example, the first outbreak of Vibrio parahaemolyticus infections in Alaska was reported in 2004, during a period when waters in the area experienced historically high temperatures ( 42). Nine multistate seafood-associated outbreaks were reported to the CDC, and almost all of these outbreaks occurred after the mid-1990s. Each of these multistate outbreaks was associated with oysters that were widely distributed.

Seafood-associated outbreaks of infection varied by seafood commodity. Mollusks were implicated in 85 (45.2%) outbreaks, followed by fish in 73 (38.8%) outbreaks and crustaceans in 30 (16.0%) outbreaks (Table ). All of the mollusk-associated outbreaks were associated with bivalve mollusks, including oysters (implicated in 72 outbreaks) and clams (11 outbreaks.) In fish-associated outbreaks, the most commonly implicated fish was salmon (15 outbreaks); "salmon" included fermented fish parts and fermented salmon eggs, which accounted for 10 of these outbreaks. Other commonly reported fish vehicles were tuna (10 outbreaks), seal meat (9 outbreaks), whale (7 outbreaks), and whitefish (not further specified) (4 outbreaks). Among crustacean-associated outbreaks, the most commonly implicated items were crab (15 outbreaks) and shrimp (15 outbreaks). Comparing seafood categories within different periods, the number and proportion of mollusk-associated outbreaks increased greatly during the study period, from 10% of outbreaks during 1973 to 1979 to 56.6% of outbreaks during 1998 to 2006 (Fig. ). The proportion of outbreaks due to fish decreased during the study period, from 90.0% of outbreaks to 24.2% of outbreaks during the last part of the study period. The proportion due to crustaceans varied, ranging from no outbreaks during the first decade to 19.2% of outbreaks.

Outbreaks and infections caused by bacteria.

Bacteria were reported as the etiologic agent in 143 (76.1%) outbreaks, causing 2,646 illnesses, 136 hospitalizations, and 11 deaths. The median size of seafood-associated outbreaks caused by bacteria was 4 cases, with a range of 2 to 400 cases. Almost all outbreaks associated with fish (89.0%) and crustaceans (93.3%) were caused by a bacterial agent; among outbreaks associated with mollusks, 50 outbreaks (58.8%) were caused by a bacterium.

Vibrios were the most commonly reported cause of seafood-associated outbreaks. Specifically, Vibrio parahaemolyticus caused more outbreaks and illnesses than any other pathogen (45 outbreaks [23.9% of all infectious disease outbreaks], 1,393 illnesses, and no deaths) during the study period. Other Vibrio species reported included toxigenic V. cholerae (3 outbreaks, 10 illnesses, and no deaths), non-O1, non-O139 V. cholerae (4 outbreaks, 12 illnesses, and no deaths), and V. vulnificus (1 outbreak, 2 illnesses, and 1 death). Among outbreaks of Vibrio illness, 15 (27.8%) were associated with crustaceans, and the remaining 39 (72.2%) were associated with mollusks, particularly oysters. Outbreaks of Vibrio illnesses were sharply seasonal, with 44 (81.5%) occurring during the warmer months of May to September. The number of reported outbreaks of Vibrio infections increased over the study period, with the greatest number reported during the last decade, 1997 to 2006. The largest reported seafood-associated outbreak was an outbreak in 1998 of V. parahaemolyticus infections associated with oyster consumption, causing 416 illnesses among persons in 13 states ( 19). During 1997 to 2006, there was substantial year-to-year variability in the number of outbreaks of Vibrio infections, with large multistate outbreaks associated with oysters occurring during 1998 and 2006 ( 12, 19).

During the past decade, the increases in the number and size of Vibrio outbreaks mirrored the trends in reports of sporadic cases of infections nationally, as reported to COVIS (Fig. ). During 1997 to 2006, 4,755 cases of Vibrio illness were reported to COVIS; of these, 3,406 (71.6%) were classified as food-borne infections. The most commonly reported Vibrio species causing food-borne infections were V. parahaemolyticus (1,931 [56.7%] food-borne Vibrio infections) and V. vulnificus (459 [13.5%] infections). V. parahaemolyticus infections were most often food borne (89.0% [1,931/2,170 infections]). Among patients with food-borne V. parahaemolyticus infections who reported food exposures, almost all (94.5%) patients reported eating seafood during the week before illness onset, most commonly oysters (72.5% [1,143/1,577 patients]). Larger numbers of sporadic cases of Vibrio infection were reported in years during which multistate outbreaks were also reported. During these years, warmer sea temperatures were documented ( 12, 18, 42). Similarly, the distribution of sporadic cases was sharply seasonal, with most cases (72.8%) occurring during summer months, corresponding to warmer water temperatures. Of the 944 V. vulnificus cases, 459 (47.0%) were classified as food-borne infections; among patients with food-borne V. vulnificus infection who reported food exposures, 91.1% reported seafood consumption during the week before illness, most commonly oysters (80.5% [289/361 patients]). Nearly all patients (92.2%) with food-borne V. vulnificus infection were reported to have a preexisting medical condition, most commonly liver disease. The case fatality rate for food-borne V. vulnificus infections was 48.3%.

During the study period, the second most commonly reported bacterial pathogen causing seafood-associated outbreaks was Clostridium botulinum, which caused 43 outbreaks, 152 illnesses, 61 hospitalizations, and 9 deaths. Most (86.0% [37/43 reports]) reports came from Alaska. Most of these outbreaks occurred among Alaska Natives and were associated with the consumption of traditional Alaska Native seafood dishes, which are often fermented under anaerobic conditions where botulinum spores may germinate ( 66). Botulinum toxin type E was most frequently identified in these seafood-associated outbreaks of botulism. Seafood associated with the outbreaks included fish, particularly salmon eggs and fish heads, seal, and whale meat ( 43). No botulism outbreaks were associated with consumption of mollusks or crustaceans.

Other bacterial etiologies of seafood-associated outbreaks included Salmonella, which caused 18 outbreaks, 374 illnesses, and 28 hospitalizations. Fish were implicated in 10 outbreaks; specifically, gefilte fish, a traditional dish made from ground deboned fish, was implicated in 4 outbreaks, with whitefish implicated in 2 outbreaks, bass in 1 outbreak, and unspecified fish in the rest ( 3). Mollusks and crustaceans were each implicated in 4 outbreaks. Most outbreaks of Salmonella infection occurred during warmer months. Shigella was the etiologic agent in 12 outbreaks, causing 402 illnesses and 20 hospitalizations. Implicated seafood included fish in 6 outbreaks, raw oysters in 4 outbreaks, scallops in 1 outbreak, and crab in 1 outbreak.

Outbreaks caused by viruses.

During the study period, 40 (21.3%) seafood-associated outbreaks were caused by viruses. Seafood associated with these outbreaks included mollusks (34 outbreaks [85.0% of outbreaks caused by viruses]), fish (5 outbreaks [12.5%]), and crustaceans (1 outbreak [2.5%]). The median size of viral outbreaks was 10 cases (range, 2 to 380 cases). Overall, norovirus was the third most commonly reported pathogen associated with seafood and the most common viral agent, causing 31 (77.5%) outbreaks of viral illness. Mollusk-associated, especially raw oyster-associated, outbreaks of norovirus were common (27 outbreaks) and made up almost one-third (31.8% [27/85 outbreaks]) of all molluscan outbreaks. Unlike outbreaks of bacterial illness, outbreaks of norovirus illness were more common during cooler months, with more than three-fourths (77.4%) of outbreaks occurring during October to March. Confirmed outbreaks of norovirus infection were reported more commonly during the past decade; 26 of 31 (83.8%) outbreaks were reported during 1998 to 2006. However, the etiology of outbreaks changed over the course of the study period. During the early 1980s, large outbreaks of viral gastroenteritis caused by molluscan shellfish ( 46) were prominent. These were followed by a decrease in reported outbreaks of viral illness that lasted until 2000, when reports again increased, likely due to better diagnostic testing, an improved ability to confirm etiology, and improved public health surveillance for outbreaks.

Hepatitis A virus was the cause of 9 seafood-associated outbreaks. Most outbreaks of hepatitis A virus infection (7 outbreaks [77.7%]) were associated with mollusks. Tuna and crab were each implicated in one outbreak. Most outbreaks occurred during the 1980s ( 22, 46), none were reported during the 1990s, and only 2 were reported after 2000 ( 3).

Outbreaks caused by parasites.

Seafood-associated outbreaks caused by parasites were rare, with 5 outbreaks reported during the study period. Outbreaks ranged in size from 3 to 29 persons affected; the median size of outbreaks was 14 cases. Reported outbreaks included 2 outbreaks of giardiasis, one caused by salmon ( 54) and one by oysters, an outbreak of Paragonimus infections associated with the consumption of live crabs ( 15), an outbreak of Diphyllobothrium infection associated with salmon consumption ( 9, 58), and an outbreak of anisakiasis associated with consumption of an unspecified fish.

Martha Iwamoto is a medical epidemiologist with the Division of Healthcare Quality Promotion, National Center for Zoonotic and Emerging Infections, Centers for Disease Control and Prevention (CDC). Previously, she was a medical epidemiologist with the Enteric Diseases Epidemiology Branch and worked with surveillance of food-borne bacterial infections. She is board certified in pediatrics and preventive medicine. Her undergraduate education was at Davidson College, Davidson, North Carolina; she received her medical degree at Medical College of Georgia in Augusta, Georgia; and she received a Master of Public Health degree from the Rollins School of Public Health, Atlanta, Georgia. She completed her clinical training in pediatrics at the University of Virginia, Charlottesville, Virginia. She trained in CDC's Epidemic Intelligence Service and Preventive Medicine Residency Program.

Tracy Ayers is an analytical epidemiologist in the Division of Food-Borne, Bacterial and Mycotic Diseases at CDC. She served as a surveillance coordinator for the Hawaii State Department of Health for 4 years, where she had the opportunity to work on several large-scale surveillance systems and analytical projects. She began her employment with the CDC in 2005 and has been involved in several cross-cutting activities within the division, with a focus on food-borne attribution.

Barbara E. Mahon received her M.D. and clinical training in pediatrics from the University of California, San Francisco, and her M.P.H. from the University of California, Berkeley. She trained in CDC's Epidemic Intelligence Service (EIS). Dr. Mahon leads the FoodNet and Outbreak Surveillance Team in the Enteric Diseases Epidemiology and Surveillance Branch at the Centers for Disease Control and Prevention (CDC). The team is responsible for the FoodNet active food-borne disease surveillance system and for national surveillance for outbreaks of food-borne disease. It conducts epidemiologic studies of the burden, trends, and food source attribution of food-borne diseases caused by Salmonella, Listeria, and Escherichia coli O157, among other pathogens. Dr. Mahon has a broad background in infectious disease epidemiology, as she has worked on food-borne diseases, vaccine-preventable diseases, and sexually transmitted diseases in academic and industry positions as well as in government. She holds an adjunct faculty appointment at the Boston University School of Public Health.

David L. Swerdlow is Senior Advisor for Epidemiology and Emergency Response, National Center for Immunizations and Respiratory Diseases, Centers for Disease Control and Prevention. He has served in the pandemic influenza H1N1 response since April 2009, as co-team leader of the Regional and Epidemiologic Field Investigations Team and co-team leader of the Epidemiology and Laboratory Task Force. Previously, he was team leader of the National Surveillance Team, Enteric Diseases Epidemiology Branch. The team conducted national surveillance for food-borne bacterial infections, including seafood-associated diseases. He is board certified in internal medicine and infectious diseases. His undergraduate education was at the University of California, San Diego, and he graduated from Harvard Medical School in 1986. His internal medicine residency was at the University of Washington, Seattle, and he completed his infectious diseases fellowship at the Massachusetts General Hospital in Boston. He was in the Epidemic Intelligence Service at CDC from 1989 to 1991.

1. Abbott, S. L., J. M. Janda, J. A. Johnson, and J. J. Farmer. 2007. Vibrio and related organisms, 9th ed. ASM Press, Washington, DC.

2. Ayers, T., M. Iwamoto, D. L. Swerdlow, and I. Williams. 2008. Epidemiology of seafood-associated outbreaks in the United States, 1973-2006, abstr. P3-70. Abstr. Int. Assoc. Food Prot. Annu. Meeting, Columbus, OH.

7. Bryan, F. L. 1980. Epidemiology of foodborne diseases transmitted by fish, shellfish and marine crustaceans in the United States, 1970-1978. J. Food. Prot. 43 : 859-876.

9. CDC. 1981. Diphyllobothriasis associated with salmon. MMWR Morb. Mortal. Wkly. Rep. 30 : 331-338.

24. FDA. 2001. Fish and fisheries products hazards and controls guide, 3rd ed. FDA, Rockville, MD.

27. FDA. 2005. Quantitative risk assessment on the public health impact of pathogenic Vibrio parahaemolyticus in raw oysters. FDA, College Park, MD.

34. [ Janda, J. M., C. Powers, R. G. Bryant, and S. L. Abbott. 1988. Current perspectives on the epidemiology and pathogenesis of clinically significant Vibrio spp. Clin. Microbiol. Rev. 1 :245-267. PMC free article]

49. NOAA. 2005. Fisheries of the United States, 2005. National Marine Fisheries Service, Silver Spring, MD.

51. Olsen, S. J., J. N. Aucott, and D. L. Swerdlow. 2002. Food poisoning, p. 199-214. In M. Blaser, P. Smith, J. Ravdin, H. Greenberg, and R. Guerrant (ed.), Infections of the gastrointestinal tract, 2nd ed. Lippincott Williams and Wilkins, Philadelphia, PA.

63. Tauxe, R. V. 1991. Salmonella: a postmodern pathogen. J. Food Prot. 54 : 563-568.

64. Tauxe, R. V., and A. T. Pavia. 1998. Salmonellosis: nontyphoidal, p. 613-630. In A. S. Evans and P. S. Brachman (ed.), Bacterial infection of humans: epidemiology and control, 3rd ed. Plenum Publishing Corporation, New York, NY.

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Epidemiology of Seafood-Associated Infections in the United States

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Summary: Seafood is part of a healthful diet, but seafood consumption is not risk-free. Seafood is responsible for an important proportion of food-borne illnesses and outbreaks in the United States. Seafood-associated infections are caused by a variety of bacteria, viruses, and parasites; this diverse group of pathogens results in a wide variety of clinical syndromes, each with its own epidemiology.

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DFO - Vibrio spp. (Larval and Juvenile Vibriosis) of Clams

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Vibrio anguillarum, Vibrio alginolyticus, Vibrio tubiashii, Vibrio splendidus (specifically V. splendidus biovar II), and sp. Sutton and Garrick (1993) reported bacteria with phenotypic characteristics 80% or greater similar to Vibrio orientalis, Vibrio campbelli, Vibrio metschnikovii, Vibrio harveyi and Photobacterium () as pathogenic to larval Tridacna gigas from a research hatchery in Australia.

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This Research Topic brings together 24 articles that highlight the most recent research findings concerning the biology of the genus Vibrio and covers pathogenicity and host interaction, genome plasticity and evolution, and the dynamics of factors influencing the ecology of vibrios.

Vibrio comprises one of the most diverse marine bacterial genera (Gomez-Gil et al., 2014), and its diversity is emphasized in two of the articles opening this set of Research Topic papers. Sawabe et al. ( 2013) present a molecular phylogeny of 86 Vibrio species based on genome sequencing that provides insight into Vibrio biodiversity and evolutionary history. In a study of more than 300 Vibrio genome sequences, Lukjancenko and Ussery ( 2014) conclude that the Vibrio pan-genome comprises 17,000 gene families, differentially present and/or expressed in any given species.

A remarkable feature of all Vibrio species is an highly plastic genome, a feature examined in five papers. The two chromosomes are shaped by horizontal gene transfer involving, among others, antibiotic resistance, virulence, and niche adaptation (Rowe-Magnus et al., 2001; Kirkup et al., 2010). V. vulnificus biotype 3 is a notable example. Efimov et al. ( 2013) suggest biotype 3 evolved from biotype 1 by acquisition of unique genes from other bacterial species, such as Shewanella, sharing the same ecological niche. Carraro et al. ( 2014) employ molecular and functional characterization of pVCR94, to identify the role of IncA/C plasmids in antibiotic resistance in a Rwandan V. cholerae isolate. A retrospective analysis of epidemic V. cholerae in Angola is reported by Valia et al. ( 2013), showing unexpected genomic variability among variants, highlighting the role of genomic islands, phages, and integrative conjugative elements in the genetic diversity of V. cholerae in a single epidemic. Rivas et al. ( 2013) describe acquisition by Photobacterium damselae subsp. damselae of virulence plasmid pPHDD1 that encodes pore-forming toxins and hemolysins which play a key role in virulence for both fish and humans. A review by Rapa and Labbate ( 2013) describes the role of integrons in Vibrio species for which gene cassettes comprise approximately 1-3% of the entire genome and are very likely involved in bacterial adaptation and evolution.

Nine of the manuscripts analyze Vibrio pathogenicity, disease development, specificity, and adaptation in both human and animal hosts. Tan et al. ( 2014) deciphered the biosynthetic network of the siderophore vulnibactin, essential in iron uptake from host proteins, the importance of which in V. vulnificus pathogenicity has been clinically demonstrated. Inhibition/resistance mechanisms developed by V. salmonicida, the causative agent of hemorrhagic septicemia in Atlantic salmon, is described by Bjelland et al. ( 2013), who show that it overcomes the salmon innate immune system to a point where the infection overwhelms the host. The role in bacterial virulence of diverse extracellular proteolytic enzymes secreted by human pathogenic Vibrio species is the focus of a review by Miyoshi ( 2013). The engagement of type VI secretion systems by V. cholerae is suggested as a means of intra- and inter-species predation and nutrient acquisition, inducing rapid multiplication in the human host (Pukatzki and Provenzano, 2013). The bioluminescent marine bacterium V. campbellii is used by Wang et al. ( 2013) to analyze the pyomelanin-pigmented phenotype, known to provide Vibrio species with greater UV and oxidative stress resistance and enhanced intestine colonization. The relationship between pathogenicity and motility in Vibrio species and the contribution of flagella to adhesion and biofilm formation are discussed by Zhu et al. ( 2013). The largely unexplored V. fluvialis mechanisms of pathogenesis, survival and fitness are reviewed by Ramamurthy et al. ( 2014). Twenty new Vibrio species associated with molluscans are described and their pathogenic potential for molluscs elucidated by Romalde et al. ( 2014). The exquisite bacteria-host interaction between V. fisheri and its squid host, Euprymna scolopes, is described in detail, as are the molecular pathways of biofilm formation, motility, and chemotaxis (Norsworthy and Visick, 2013).

The capacity of Vibrio species to persist in the aquatic environment, their ecology and association with abiotic and biotic factors, as well as environmental surveillance for public health (Lipp et al., 2002; Grimes et al., 2009; Johnson, 2013) comprise a section in the Research Topic that opens with a review by Lutz et al. ( 2013) elucidating complex mechanisms enabling V. cholerae to withstand starvation, temperature fluctuation, salinity variation, and predation. Haley et al. ( 2014) report water temperature increase can be correlated with rise of a diverse population of V. parahaemolyticus, some of which carry pandemic markers, in water and plankton along the Georgian coast of the Black Sea. V. parahaemolyticus and V. vulnificus populations associated with oyster, sediment, and surface water related to a hurricane event in the Chesapeake Bay are concluded to be influenced by wave energy and sediment resuspension (Shaw et al., 2014). Canesi et al. ( 2013) show the serum of Mytilus galloprovincialis promotes phagocytosis and killing by hemocytes of both V. cholerae O1 and non-O1/non-O139 in edible bivalves. Chakraborty et al. ( 2013) evaluate a sensitive and specific dipstick test to detect toxigenic V. cholerae in water, validating a simple, inexpensive method for use in areas at risk of cholera.

Three articles addressing Vibrio environmental diversity and dynamics complete this Research Topic. Mansergh and Zehr ( 2014) suggest that the natural shift of Vibrio populations in Monterey Bay is affected by larger oceanographic conditions (flow velocities and wind patterns), rather than individual environmental factors. Meta-analysis of environmental variables and Vibrio association with plants, algae, zooplankton, and animals are reviewed by Takemura et al. ( 2014). As a final point concerning environmental distribution, Constantin De Magny et al. ( 2014) propose temporal shifts, zooplankton community variability, and occurrence of V. cholerae in the aquatic environment are related to cholera dynamics. These factors, analyzed by metagenomics, permit greater understanding of community structure, function, and competition.

In summary, the collection of manuscripts provided in this Research Topic offers a comprehensive exploration of Vibrio biology, from the single gene to the bacterial community, elucidating Vibrio molecular pathways and evolutionary history. This special issue shows the significant progress achieved in understanding the complex biology of the genus Vibrio and should both stimulate discussion and offer a challenge to researchers in microbial ecology and evolution.

References

Bjelland A. M., Fauske A. K., Nguyen A., Orlien I. E., Østgaard I., Sørum H. (2013). Expression of [Vibrio salmonicida virulence genes and immune response parameters in experimentally challenged Atlantic salmon (Salmo salar L.). Front. Microbiol. 4:401 10.3389/fmicb.2013.00401PMC free article]

Canesi L., Pezzati E., Stauder M., Grande C., Bavestrello M., Papetti A. F. (2013). [Vibrio cholerae interactions with Mytilus galloprovincialis hemocytes mediated by serum components. Front. Microbiol. 4:371 10.3389/fmicb.2013.00371PMC free article]

Gomez-Gil B., Thompson C. C., Matsumura Y., Sawabe T., Iida T., Christen R., et al. (2014). Family Vibrionaceae (Chapter 225), in The Prokaryotes, 4th Edn. eds Rosenberg E., DeLong E., Thonpson F. L., Lory S., Stackebrandt E., editors. (New York, NY: Springer; ), 88

Grimes D. J., Johnson C. N., Dillon K. S., Flowers A. R., Noriea N. F., 3rd., Berutti T. (2009). What genomic sequence information has revealed about Vibrio ecology in the ocean-a review. Microb. Ecol. 58, 447-460 10.1007/s00248-009-9578-9

Johnson C. N. (2013). Fitness factors in Vibrios: a mini-review. Microb. Ecol. 65, 826-851 10.1007/s00248-012-0168-x

Sawabe T., Ogura Y., Matsumura Y., Gao F., Amin A. R., Mino S., et al. (2013). Updating the [Vibrio clades defined by multilocus sequence phylogeny: proposal of eight new clades, and the description of Vibrio tritonius sp. nov. Front. Microbiol. 4:414 10.3389/fmicb.2013.00414PMC free article]

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Vibrio ecology, pathogenesis, and evolution

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This Research Topic brings together 24 articles that highlight the most recent research findings concerning the biology of the genus Vibrio and covers pathogenicity and host interaction, genome plasticity and evolution, and the dynamics of factors influencing the ecology of vibrios.

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Bonamia exitiosa (Haplosporidia) observed infecting the European flat oyster Ostrea edulis cultured on the Spanish Mediterranean coast - ScienceDirect

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Bonamia exitiosa and Bonamia ostreae are parasites that reproduce within the haemocytes of several oyster species. In Europe, the host species is the flat oyster Ostrea edulis. The parasite B. ostreae has been responsible for mortalities since the late 1970s throughout the European Atlantic coast. B.

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Article 4.1

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In Vitro Propagation of the Protozoan Perkinsus Marinus, A Pathogen of the Eastern Oyster, Crassostrea Virginica - MSU Scholars

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ABSTRACT. Perkinsus marinus, a pathogen of eastern oysters (Crassostrea virginica), has been successfully propagated in vitro. Cultures of the parasite were initiated from heart fragments of an infected oyster. the cultured protozoan (designated Parkinsus‐1) was similar in morphology at both the light and transmission electron microscopy levels to histozoic stages of P.

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Website 5.1

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A deadly virus threatens to decimate oyster populations around the world

smithsonian.com

Oysters, a delicacy eaten on most coastlines of the world, are a multi-billion-dollar industry. They also are intriguing to study from a health perspective. Oysters feed by filtering tiny plankton from the surrounding water, processing up to 50 gallons per oyster daily. In doing so, they improve water quality and make their ecosystems healthier. But the water that they grow can be filled with disease-causing microorganisms that can affect both oysters and humans.

Today a deadly herpes virus, Ostreid herpesvirus 1 (OsHV-1), is threatening Pacific oysters ( Crassostrea gigas), the world's most popular and valuable oyster species. It is almost certain to spread more widely in our globally connected world.

I know what you're thinking: "Oysters get herpes??" Yes, and they can also can get sick from other types of pathogens and stresses. But you won't contract this virus from eating an oyster, whether you enjoy them on the half-shell or cooked. OsHV-1 can infect other bivalve species, like some animal herpes viruses that can cross species barriers, but it is genetically distinct from other animal herpes viruses and does not infect humans.

With support from the NOAA Sea Grant aquaculture program, I'm working with a diverse team that includes researchers, regulators and outreach specialists in the United States and abroad to better prepare the U.S. oyster industry for the spread of this virus.

**********

Pacific oysters are native to Asia and are the most popular and valued oyster for aquaculture globally. Humans transferred them from their native range to multiple grow-out areas globally, including France, the United States and Australia. They are the primary species grown on the U.S. West Coast, whereas both wild and cultured Eastern oysters grow on the East and Gulf coasts. In contrast to Eastern oysters, Pacific oysters were relatively resistant to infectious diseases until OsHV-1 emerged in the early 1990s.

Herpes is often fatal to Pacific oysters. That's especially true for OsHV-1 microvariants - mutant variants of OsHV-1 which are more virulent than the original reference strain. These viruses are spreading globally, causing mass mortalities of Pacific oysters.

An OsHV-1 microvariant was first detected in France in 2008, where it killed 80 to 100 percent of affected oyster beds. Since then, similar variants have caused mass mortalities of oysters in many European countries. A 2010 outbreak in England killed over eight million oysters.

OsHV-1 microvariants also infect Pacific oysters in New Zealand and Australia. Their spread in Australia, in particular to Tasmania, has crippled the Australian Pacific oyster aquaculture industry.

**********

U.S. oyster growers are strongly concerned about the spread of OsHV-1 microvariants globally. I was part of the team that first detected OsHV-1 in Tomales Bay, California. To date the virus has been detected only in oysters in Tomales Bay and an adjacent bay, and no microvariants have been found yet in U.S. waters. The California OsHV-1 causes mortalities of young Pacific oysters, but is thought to be less virulent than OsHV-1 microvariants.

Given the spread of the OsHV-1 microvariants elsewhere around the world, it may only be a matter of time until they reach U.S. coastal bays or other nonimpacted oyster growing areas. We spent the summer of 2017 conducting experiments in Tomales Bay to determine whether any cultured U.S. oysters species are resistant to OsHV-1, and soon will also conduct laboratory challenges with OsHV-1 microvariants.

Once OsHV-1 is established within a bay, mass oyster deaths typically occur each year during the summer when water temperatures are warm. The situation is analogous to a human who is infected with herpes and periodically get cold sores. Normally the virus is latent (present at a low level) and does not cause cold sores. But after a stressful situation, the virus replicates and cold sores emerge.

Not all oysters die of herpes, and if OsHV-1 behaves like other herpes viruses, it probably remains present latently within infected oysters' tissues and is reactivated after a stressful event. For oysters, most of the evidence for virus reactivation points to warm summer water conditions.

**********

We can't vaccinate oysters, and even if antibiotics were effective against viruses, they are not permitted for treating oysters in the United States. Though oysters have an innate immune system that destroys foreign invaders, it lacks an adaptive response, including cells that "remember," recognize and destroy specific pathogens, as human B or T lymphocytes do. Most vaccines rely on this "immune memory" to be effective. Recent research indicates that oysters' innate immune systems can be stimulated by a virus mimic, but we do not know whether this effect is long-lasting.

The most effective strategy to date has been developing disease-resistant oyster lines, which can limit both mortalities and oysters' susceptibility to infection. But this approach involves exposing healthy oysters to the virus - and moving oysters infected with OsHV-1 to naive (disease-free) areas could spread the virus. This means that we can use this approach only in places where OsHV-1 already exists.

Toward that end, breeding programs in locations including France, New Zealand and Australia are working to develop OsHV-1-resistant Pacific oysters. A complementary approach is to expose oysters and determine genes involved in OsHV-1 resistance. I am currently working with two strains of OsHV-1 - the California virus and a microvariant in France - to determine OsHV-1 resistance genes, including a collaboration with the Ifremer station in La Tremblade, France.

The most effective way to limit damage in new locations from OsHV-1 is to limit its spread. However, we also want to be ready in case OsHV-1 microvariants spread to the United States. Beyond their cash value and the benefits that oysters provide by filtering water, oyster reefs provide food and habitat for many commercial fish species. Oysters can't move themselves out of harm's way, nor can we move all susceptible oysters, so we need to protect them where they grow.
Colleen Burge, Assistant Professor, Institute of Marine and Environmental Technology, University of Maryland, Baltimore County

This article was originally published on The Conversation.

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# data[].attributes.attachment_link.title
Oysters Can Get Herpes, And It's Killing Them

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smithsonian.com Oysters, a delicacy eaten on most coastlines of the world, are a multi-billion-dollar industry. They also are intriguing to study from a health perspective. Oysters feed by filtering tiny plankton from the surrounding water, processing up to 50 gallons per oyster daily. In doing so, they improve water quality and make their ecosystems healthier.

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Website 5.2

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Bivalve molluscs can bioaccumulate, in their tissues, well known viruses that infect humans and higher animals. However, since 1969 there have been reports, based largely on ultrastructural studies,...

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Article 5.1

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Article 4.2

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Perkinsus marinus in pleasure oyster Crassostrea corteziensis from Nayarit, Pacific coast of México

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Culture of the pleasure oyster Crassostrea corteziensis is emerging as an alternative to the Pacific oyster (Crassostrea gigas) for oyster producers, ...

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Detection of Bonamia exitiosa (Haplosporidia) in European flat oysters Ostrea edulis cultivated in mainland Britain. - PubMed - NCBI