Journal of Invertebrate Pathology 106 (2011) 18–26

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Journal of Invertebrate Pathology

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Minireview Bacterial diseases of : A review ⇑ W. Wang

Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Diseases, College of Life Sciences, Nanjing Normal University, Nanjing 210046, China article info abstract

Keywords: Bacterial diseases of crabs are manifested as bacteremias caused by organisms such as Vibrio, Aeromo- Bacteria nas, and a Rhodobacteriales-like organism or tissue and organ tropic organisms such as chitinoclastic Disease bacteria, Rickettsia intracellular organisms, Chlamydia-like organism, and Spiroplasma. This paper pro- vides general information about bacterial diseases of both marine and freshwater crabs. Some bacteria pathogens such as Vibrio cholerae and Vibrio vulnificus occur commonly in blue crab haemolymph and should be paid much attention to because they may represent potential health hazards to human beings because they can cause serious diseases when the crab is consumed as raw sea food. With the development of aquaculture, new diseases associated with novel pathogens such as spiroplasmas and Rhodobacteriales-like organisms have appeared in commercially exploited crab species in recent years. Many potential approaches to control bacterial diseases of crab will be helpful and practicable in aquaculture. Ó 2010 Published by Elsevier Inc.

Contents

1. Introduction ...... 18 2. Vibrio and bacteremia ...... 19 3. Chitinoclastic bacteria (other than Vibrio) associated with shell disease ...... 20 4. Aeromonas ...... 21 5. Rickettsia intracellular organisms or Rickettsia-like organisms (RLOs) ...... 21 6. Chlamydia-like organism ...... 22 7. Spiroplasma ...... 22 8. Rhodobacteriales-like organism ...... 23 9. Potential approaches to control bacterial diseases of crab ...... 23 References ...... 24

1. Introduction by other chitinolytic or chitinoclastic bacteria which were first described in 1967 (Rosen, 1967). Shell disease continues to re- Bacterial diseases of crabs are not common when compared main a topic of significant research (Comely and Ansell, 1989; to virus and protozoa diseases. A review of the literature indi- Vogan et al., 1999, 2001, 2002) as several decapod species of cates that the majority of studies focus upon bacterial diseases commercial importance are affected. With the development of marine crabs that produce bacteremias or affect the exoskel- and intensification of aquaculture, new bacterial diseases have eton. The genus Vibrio is frequently cited, especially Vibrio para- recently appeared in commercially exploited crab species. These hemolyticus, Vibrio cholerae and Vibrio vulnificus, which novel pathogens have inflicted heavy losses on the aquaculture commonly occur in blue crab, but are also associated with hu- industry. For example, Spiroplasma sp. in the Chinese mitten man diseases (Krantz et al., 1969; Tubiash and Krantz, 1970; crab, Eriocheir sinensis, is a recent discovery and is the first spi- Sizemore et al., 1975; Davis and Sizemore, 1982; Huq et al., roplasma isolated from a crustacean (Wang et al., 2003b, 2004, 1986). Shell disease is caused by other Vibrio spp. as well as 2010). In this paper, bacterial diseases of crabs, including the most recent new findings, are discussed. In order to present the information clearly, an outline of bacterial diseases is ⇑ Fax: +86 858915265. E-mail address: [email protected] presented in Table. 1.

0022-2011/$ - see front matter Ó 2010 Published by Elsevier Inc. doi:10.1016/j.jip.2010.09.018 W. Wang / Journal of Invertebrate Pathology 106 (2011) 18–26 19

Table 1 Bacteria from different species of crabs, the tissues in which they reside and key references selected by the authors.

Parasite Global location Host Tissue Key reference Bacteria Clostridium botulinum type F York River in Virginia Blue crab sapidus Haemolymph Williams-Walls (1968) Vibrio parahemolyticus Chesapeake Bay Krantz et al. (1969) Vibrio parahemolyticus Chesapeake Bay Colwell et al., (1975) Sizemore et al. (1975), Sizemore and Davis (1985), Welsh and Sizemore (1985) Galveston Bay, Texas Davis and Sizemore (1982) Vibrio cholerae Potomac River, Washington Gut Huq et al. (1986) Porto Novo Coast. Indian Venkateswaran et al. (1981) Hepatopancreas Johnson (1983) Gills Babinchak et al. (1982) Vibrio spp. Hoseshoe crab, Limulus polyoemus Haemolymph Bang (1956) Puerto Rico Callinectes bocourti, Rivera et al. (1999) Connecticut USA Rock crab, Newman and Fen (1982) Rock crab, Cancer irroratus; Shore Spindler-Barth et al. (1976) crabs Carcinus maenas L Chitinoclastic bacteria New York Bight Blue crab Shell Rosen (1967, 1970), Overstreet and Cook (1972), Cook and Lofton (1973), Sandifer and Eldridge (1974), Young and Pearce (1975) Iversen and Beardsley (1976), Noga et al. (1994) Rowan Bay, Alaska , Cancer magister Morado et al., 1988 Shore crab, C. pagurus Gordon (1966) Bakke (1973) Brittany, France Leglise (1976) Langland Bay, Swansea, UK. Ayre and Edwards, (1982), Vogan et al. (1999), (2001, 2002) Powell and Rowley (2005) New York Bight Rock crab, C. irroratus Shell Young and Pearce (1975), Sawyer (1982), Sawyer (1991) Mid-Atlantic Bight , C. borealis Haefner (1977) South Florida, USA Stone crab, Menippe mercenaria Iversen and Beardsley (1976) New York Bight Red crab, Geryon quinquedens Young (1991), Bullis et al. (1988) South Atlantic Bight Golden crab, C. fenneri Wenner et al. (1987) Eastern Canada Snow crab opilio Bower et al. (1994) Southern Gulf of St.Lawrence, Benhalima et al. (1998) Canada Tanner crab, Chionoecetes tanneri Baross et al., 1978 Aeromonas trota Brittany France Edible crab Haemolymph Leglise and Raguenes (1975) Hangzhou, China Eriocheir Haemolymph, Xu and Xu (2002) sinensis hepatopancreas, muscle Rickettsia-like organism Mediterranean coast of France Carcinus mediterraneus Connective tissue of Bonami and Pappalardo (1980) hepatopancreas, gut, gills and gonads Alaska, USA Blue Paralithodes Hepatopancreatic Johnson (1984) platypus epithelium Southeast Alaska, USA Golden king crabs lithodes Haemolymph, connective Meyers and Shorts (1990) aequispina tissue of hepatopancreas, gut, gills and gonads Jiangsu, China Cheses mitten crab Eriocheir Wang et al. (2001); Zhang et al. sinensis (2002) Chlamydia-like organism Willapa Bay and northern Puget Dungeness crab Cancer magister Connective tissue and Sparks et al. (1985) Sound, Washington, USA connective tissue cells Laboratory-reared Rock crab Cancer irroratus jonah Haemocytes and Leibovitz (1988) crab Cancer borealis haematopoietic tissue Spiroplasma Jiangsu, China Cheses mitten crab Eriocheir Haemolymph and all Wang et al. (2003), Wang et al. (2004) sinensis connective tissue of hepatopancreas, gut, gills and gonads Rhodobacteriales-like Swansea, Wales, UK European shore crab, Carcinus Connective tissue and the Eddy et al. (2007) organism maenas blood vessels

2. Vibrio and bacteremia 1982; Sizemore and Davis, 1985; Welsh and Sizemore, 1985), Cal- linectes bocourti,(Rivera et al., 1999) and rock crab, Cancer irroratus Members of the genus Vibrio are ubiquitous throughout the (Newman and Fen, 1982). Under conditions of Vibrio-caused bac- world and are found associated with many marine and freshwater teremias, a marked reduction in hemocyte numbers and intravas- . However, the pathological and physiological effects of cular clotting is observed; the apparent result of an endotoxin in Vibrio infections are best documented in marine crabs where Vibrio the cell wall of the bacterium (Sritunyalucksana and Söderhäll, infections typically cause or produce bacteremias and shell disease. 2000). Vibrio-caused bacteremias have been reported from the blue crab, The blue crab, C. sapidus, soft-shell industry is a major fishery Callinectes sapidus (Krantz et al., 1969; Tubiash and Krantz, 1970; throughout the eastern United States (NOAA Fisheries Office of Colwell et al., 1975; Sizemore et al., 1975; Davis and Sizemore, Sustainable Fisheries, 2010), which leads the top of the species 20 W. Wang / Journal of Invertebrate Pathology 106 (2011) 18–26 rankings by its value. V. parahemolyticus was isolated from lethar- growth and physiological activity of heterotrophs. Under experi- gic and moribund blue crabs, C. sapidus, in commercial tanks dur- mental feeding conditions, Huq et al. (1986) observed the attach- ing ‘‘shedding” of soft crabs in Chesapeake Bay (Krantz et al., ment of V. cholerae only to the mucosal surface of the hindgut of 1969; Tubiash and Krantz, 1970). The disease produced a mortality the blue crab, C. sapidus. This study demonstrated the potential rate in excess of 50% in short-term shedding facilities. Affected importance of crustaceans in the epidemiology and transmission crabs were weak and their haemolymph contained many bacteria of cholera in the aquatic environment. when examined by phase microscopy. Using biochemical and sero- logical techniques, and computer-based taxonomic analysis, the 3. Chitinoclastic bacteria (other than Vibrio) associated with causative agent was identified as V. parahemolyticus, which has shell disease previously been associated with outbreaks of food poisoning in hu- mans. The bacterium is naturally found in the marine environment, Shell disease is common and frequently reported in various spe- occasionally invades marine and has been the etiological cies of commercially exploited crabs: the blue crab, C. sapidus in agent of ‘‘shirasu” food poisoning in Japan (Fujino et al., 1953). Fur- New York Bight (Rosen 1967, 1970; Overstreet and Cook 1972; thermore, other common Vibrio human pathogens, such as V. chol- Cook and Lofton, 1973; Sandifer and Eldridge, 1974; Young and erae and V. vulnificus, have been reported to regularly occur in blue Pearce, 1975; Iversen and Beardsley, 1976; Noga et al., 1994); crab haemolymph (Sizemore et al., 1975; Davis and Sizemore, Dungeness crab, Cancer magister (Morado et al., 1988); edible crab, 1982). Two proteolytic strains of Clostridium botulinum type F, Cancer pagurus (Gordon, 1966; Bakke, 1973; Leglise, 1976; Ayre which were involved in an outbreak of human botulism, have been and Edwards, 1982; Vogan et al., 1999, 2001, 2002; Powell and isolated from the blue crab, C. sapidus, from the York River in Vir- Rowley, 2005); rock crab, C. irroratus (Young and Pearce, 1975; ginia (Williams-Walls, 1968). These disease outbreaks in crabs, Sawyer, 1982, 1991); Jonah crab, Cancer borealis (Haefner, 1977; regardless of degree of infection, should be closely monitored be- stone crab, Menippe mercenaria (Iversen and Beardsley, 1976); cause they may represent potential threats to human health. red crab, Geryon quinquedens (Young, 1991; Bullis et al., 1988); Members of the genus Vibrio has also been found in healthy blue golden crab, Geryon fenneri (Wenner et al., 1987) and snow crab crabs. Colwell et al. (1975) determined that the haemolymph of Chionoecetes opilio in eastern Canada (Bower et al., 1994; Benhali- healthy blue crabs was not sterile, but instead contained large and ma et al., 1998). In general, shell disease of crabs occurs at such a variable numbers of viable, aerobic, heterotrophic bacteria. Com- high incidence that this syndrome has been the most studied dis- puter-based taxonomic analysis revealed that the predominant ease of commercially exploited crabs. genus was Vibrio, but members of the genera Bacillus, Acinetobacer A complex population of chitinolytic or chitinoclastic bacteria are and Flavobacterium were also present. However, Johnson (1976a) generally associated with shell disease because they possess the en- has argued that ‘‘naturally” infected crab acquired bacterial infec- zyme chitinase that is capable of degrading carapace chitin. This deg- tions during the stress of capture and transport, not before. Dr. John- radation generally results in the typical erosion and pigmentation of son questioned whether any could tolerate the presence of a lesions in the exoskeleton of crabs which are often viewed as box known pathogen such as V. paraheamolyticus within its body fluids burnts, black-spots on the exoskeleton (Fig. 1). In addition to the and tissues. Indeed, most of the collected crabs were heavily external gross signs, this bacterial complex can penetrate the cuticle stressed, having been purchased from commercial sources. The ef- and establish infections in the blood and cause host tissue damage fect of commercial capture and handling stresses on the prevalence (Comely and Ansell, 1989; Vogan et al., 1999, 2001, 2002). Histolog- and levels of infection in blue crab were studied by Sizemore and ical studies on shell disease of the edible crab, C. pagurus, showed Davis (1985) and Welsh and Sizemore (1985). They found Vibrio that the gills, hepatopancreas and heart of diseased crabs were sig- spp. to be the predominant bacterium present in heavily infected nificantly affected (Vogan et al., 2001). In the gills, epithelial inflam- crabs and were primarily responsible for progressive infections in mation and necrosis and cuticular erosion in affected crabs leads to commercially stressed crabs. As a result, physiological stressors the formation of hemocyte plugs termed nodules while inflamma- including capture, handling and transport may reduce the effective- tion and other irreversible changes were common in the hepatopan- ness of host defensive reactions and may lead to increased numbers creas and heart. The disease resulted in unsuccessful molting of bacteria within the haemolymph. As an example, the haemo- (Smolowitz et al., 1992) or septicaemic infections by opportunistic lymph of crabs with missing appendages had significantly higher pathogenic bacteria, which entered through the lesion sites (Baross counts than uninjured crabs (Tubiash et al., 1975). et al., 1978; Vogan et al., 2001). The Vibrio species in the haemolymph of the rock crab, C. irrora- Initially thought to be restricted to the exterior surfaces of the tus, from Connecticut USA, was demonstrated to be pathogenic over exoskeleton, recent studies show that shell disease is not a disease a wide range of temperatures (Newman and Feng, 1982). Another caused by a single pathogen and solely restricted to the exoskele- blue crab, C. bocourti, from a eutrophic system in Puerto Rico, was ton. Chitinolytic bacteria are members of several genera including studied in an effort to quantify total bacterial densities and identify Vibrio, Aeromonas, Pseudomonas, Alteromonas, Flavobacterium, Spi- bacterial species in the haemolymph (Rivera et al., 1999). The re- rillum, Moraxella, Pasteurella and Photobacterium (Getchell, 1989) sults showed that C. bocourti could tolerate very high densities of As a result, a number of bacteria are involved in production of bacteria in their hemopymph (average = 8.89 Â 1010 cells mlÀ1). the lesions which may invade the body tissues of crab. The nature However, hemocoelic bacterial infections in green, Carcinus maenas, of the bacterial complex is not entirely understood, but may result and fiddler, Uca pugilator, crabs greatly increased the length of the in varying degrees of bacterial colonization, shell erosion and tis- intermolt stage by three times when compared to uninfected crabs sue invasion. Host responses differ depending on the nature of (Spindler-Barth, 1976). But acute hemocoelic bacterial infections the penetrating bacteria and this may ultimately lead to death of can often kill the majority of infected animals in 1–6 days (Johnson, the animal (Vogan et al., 2002). Unexpectedly, the severity of shell 1976a; Spindler-Barth, 1976). disease in C. pagurus did not cause dramatic changes to the major- In addition to an active bacteremia, Vibrio bacteria can be found ity of immune parameters tested, including total hemocyte counts in other tissues, such as gills (Babinchak et al., 1982), gut (Huq (Vogan and Rowley, 2002). Vogan et al. (2002) found that extracel- et al., 1986; Venkateswaran et al., 1981), hepatopancreas (Johnson, lular products (ECP) produced by chitinolytic bacteria from the edi- 1983) and exoskeleton (discussion to follow in following section). ble crab (C. pagurus) with shell disease could cause rapid death of The research of Babinchak et al. (1982) indicates that the gills of the crab upon injection. Costa-Ramos and Rowley (2004) examined blue crab, C. sapidus, provide a protective ecological niche for the the nature of the active lethal factor(s) in ECP. They found that W. Wang / Journal of Invertebrate Pathology 106 (2011) 18–26 21

4. Aeromonas

There are a few reports about Aeromonas infections causing dis- eases in crabs. Leglise and Raguenes (1975) isolated a species of Aer- omonas from the haemolymph of moribund edible crabs, C. pagurus, with mortality rates of 50–70%, in commercial ponds in Brittany, France. Experimental inoculations of crabs with the isolated bacte- rium caused death within 24 h. Wounded crabs immersed in water containing the bacterium died in about 8 days, while non-wounded crabs in the same water remained healthy (Leglise and Raguenes, 1975). Aeromonas trota was isolated from the haemolymph, hepato- pancreas and muscle of diseased Chinese mitten crabs, E. sinensis, from aquaculture ponds in Zhuantang, Hangzhou, in China (Xu and Xu, 2002). The bacterium was Gram-negative, motile by polar flagella and facultatively anaerobic. Glucose was catabolized with the production of acid and gas. Oxidase and catalase actively were positive but metabolism of esculin, sucrose and salicin were nega- tive. The bacterium was sensitive to ampicillin and carbenicillin. Experimental infection reached the 100% mortality rate. The iso- lates showed the same morphological, physiological and biochem- ical characteristics with A. trota.

5. Rickettsia intracellular organisms or Rickettsia-like organisms (RLOs)

Rickettsia intracellular organisms or rickettsia-like organisms (RLOs) are small, pleomorphic, rod-shaped coccoid prokaryotes containing ribosomes, fibrils and nuclear material, most of which are obligate intracellular Gram-positive organisms (Sparks, 1985). The microorganisms differ from bacteria because they lack a true bacterial wall. Shortly after the turn of the 21st century, rickettsia have been identified as causing chronic and acute diseases in in- sects, birds, man and other animals (Wen, 1999), but their pres- ence in crustaceans is a recent development. In 1970, the first rickettsial disease of a crustacean was reported from a terrestrial isopod from France (Vago et al., 1970). There are some reports of Fig. 1. (a) The ventral surfaces of C. pagurus displaying the characteristic black-spot lesions of shell disease. (b) Low power scanning electron micrograph of a ventral pathogenic rickettsia-like organisms (RLOs) causing serious dis- carapace lesion. Boxed regions are enlarged in (c) and (d). Note the columnar eases in crustaceans (Frederici et al., 1974; Brock et al., 1986; Ket- pattern of degradation in the central regions of the lesion (c) which contrasts with terer et al., 1992; Owens et al., 1992; Bower et al., 1994; Bower, the lamellar cleavage planes at lesion peripheries (d). Bars: 5 cm, (a); 100 lm (b); 1996; Nunan et al., 2003). Mass mortality of has been asso- 10 lm (c, d). (From Vogan et al. (2002). Microbiology. 148, 743–754.) ciated with RLOs (Krol et al., 1991). However, diseases caused by RLOs are rare in crabs and have only been reported from Carcinus lipopolysaccharide (LPS), either alone or in combination with other mediterraneus,(Bonami and Pappalardo, 1980), Paralithodes platy- heat-stable factors, was the main virulence factor of Pseudoaltero- pus (Johnson, 1984) and Lithodes aequispina (Meyers and Shorts, monas atlantica for the crabs. 1990) and E. sinensis, a freshwater crab (Wang et al., 2001, 2002; Degraded environmental conditions are generally considered to Wang and Gu, 2002; Zhang et al., 2002). be associated with shell disease in crabs because diseased crabs, C. RLOs infect the shore crab, C. mediterraneus, and have caused irroratus, were often found in waste disposal areas with sewage fatal disease in the Sète region on the Mediterranean coast of sludge and dredge spoils in the New York Bight (Young and Pearce, France (Bonami and Pappalardo, 1980). The organism produced 1975). But Powell and Rowley (2005) detailed an unchanged prev- Feulgen-positive microcolonies (10–20 lm in diameter) within alence of shell disease in the edible crab, C. pagurus, 4 years after intracytoplasmic vacuoles and was widely dispersed in the cyto- the decommissioning of a sewage out fall at Langland Bay, UK. plasm of connective tissue cells of the hepatopancreas, gut, gills and concluded that sewage pollution is probably not a major con- and gonads. Individual RLOs were approximately 2 Â 0.7 lmin tributory factor to this disease (Powell and Rowley, 2005). Some size and multiplied within host cell cytoplasmic vacuoles. Replica- researchers have investigated physiological changes that may oc- tion of the microbe eventually caused lysis of infected host cells cur in crabs with shell disease. Noga et al. (1994) reported low ser- afterwhich the organisms were released into extracellular spaces. um antibacterial activity coincided with increased prevalence of Experimental infection caused death in 15 days. shell disease in blue crabs, C. sapidus, from the Albemarle–Pamlico Johnson (1984) found RLOs in the hepatopancreatic epithelium of Estuary, North Carolina, USA. This antibacterial activity may be an an immature female blue king crab, P. platypus, from around Kodiak important mechanism protecting crabs against shell disease. How- Island, Alaska, USA. The infection was massive and apparently ever, as previously noted, Vogan and Rowley (2002) reported few caused necrosis and encapsulation of some hepatopancreatic changes in haemocyte counts and humoral defences in C. pagurus tubules, arrested ovarian development, and abnormal syn- affected by shell disease; their results indicate that no dramatic chrony of the molt cycle. The rickettsia-like organisms mea- changes in prophenoloxidase and antibacterial activity occurred. sured 0.3 Â 0.6–1.0 lm. Lightly Feulgen-positive microcolonies 22 W. Wang / Journal of Invertebrate Pathology 106 (2011) 18–26

(10–40 lm diameters) were observed within the cytoplasm of known genus of the family Spiroplasmataceae that is currently hepatopancreas epithelium. placed in the order Entomoplasmatales, class Mollicutes, phylum Infections in blue king crabs P. platypus (Johnson, 1984; Meyers Firmicutes, field Bacteria or empire Eubacteria (Euzéby, 2005). and Shorts, 1990), golden king crabs L. aequispina (Meyers and Members of the genus Spiroplasma are unique microbes and are Shorts, 1990) and shore crabs C. mediterraneus (Bonami and Pappa- characterized by the absence of a cell wall, a helical shape that is lardo, 1980) were thought to be fatal. Prevalences from aquacul- evident during its exponential growth phase and tiny size (one of ture systems have rarely been reported. In blue crabs, C. sapidus, the smallest prokaryotes in the world). These bacteria can pass the prevalence of RLO was 2.3% in a Maryland shedding facility, through membrane filters with pores 220 nm in diameter and but heavy infections were not fatal (Messick and Kennedy, 1990) can be cultivated in vitro in artificial medium. and were associated with little pathology (Messick, 1998). How- Spiroplasmas were first found in 1973 in the United States of ever, the RLO infections in the Chinese mitten crab, E. sinensis, America and commonly infect plants and insects (Williamson and are significant due to the high prevalence (30%-90%) in pond sys- Whitcomb, 1975). Historically, spiroplasmas have been associated tems, and the rapid and high mortality (above 70%) associated with with insects, ticks and plants (Tully et al., 1977, 1983; Whitcomb the tremor disease (TD)osses (Yang and Cai, 1998) of cultured crabs and Williamson, 1979; Whitcomb, 1980; Daniels, 1979; Moulder in southeast China (Wang et al., 2001, 2002; Zhang et al., 2002). et al., 2002; Gasparich, 2002, 2010). However, a spiroplasma was However, the RLOs pathogen in E. sinensis was later confirmed to isolated from the Chinese mitten crab E. sinensis associated with be a spiroplasma rather than rickettsia when 16S rRNA gene anal- highly pathognomonic signs, of tremor disease (TD) in 2003 (Wang ysis was applied (Wang et al., 2004. see Section 7). et al., 2003a, 2004). This was the first Spiroplasma isolated from crustaceans and has begun to change our understanding of the host range of spiroplasmas (Christensen et al., 2005; Regassa and 6. Chlamydia-like organism Gasparich, 2006). E. sinensis is a commercially important decapod that is widely cultured in China and supports an aquaculture indus- Chlamydia-like organism (CLOs) differ from rickettsia-like try that is valued in excess of US$2 billion every year. But from 1994, organisms (RLOs) by their special characteristics in multiplication, uncontrolled epizootics have occurred throughout the region; prev- including small, rigid-walled ‘‘elementary bodies”, the infectious alences range from 30 to 90%, and resulting mortalities may exceed forms, that change into larger, thin-walled ‘‘initial bodies” dividing 70% and approach 100% (Wei, 1999). Diseased crabs showed weak- by fission and resulting in the formation of daughter cells which ness and anorexia in the early stage of infection. As the disease eventually reorganize and condense (intermediate bodies) to be- progresses, uncontrolled shaking of the pereiopods is observed come elementary bodies (Sparks, 1985; Bower, 1996). High mortal- which led its description as ‘‘tremor disease” by culturists (Fig. 2). ities of Dungeness crabs, C. magister, which occurred each winter and The agent was originally thought to be a rickettsia-like organism spring in Willapa Bay and northern Puget Sound, Washington, USA, (RLO) based on morphological and pathological studies (Wang were found to be associated with a Chlamydia-like organism (Sparks et al., 2002; Zhang et al., 2002). et al., 1985). The disease appeared to be associated with cold water The causative agent is distributed systemically in E. sinensis temperatures. Affected crabs displayed lethargy. The disease was which likely explains the gross signs and behaviour of the disease. well documented histologically and ultrastructurally. In the histo- Infection in periopod skeletal musculature and connective tissue logical survey, diseased crabs exhibited systemic infections with a and in the heart and gastrointestinal tract likely results in weak- small Gram-negative or basophilic microorganism. Cell necrosis ness (lethargy) and anorexia. The presence of the pathogen in the and moderate-to-dense accumulation of hemocytes accompanied thoracic ganglion and myoneural junctions likely affect nerve the chlamydial infection in some tissues. The agent demonstrated transmission leading to the characteristic paroxymal tremors of strong affinity for connective tissue and connective tissue cells while the pereiopods (Wang et al., 2002). only infrequently infecting epithelial cells. TEM observation re- 16S rRNA gene sequence analysis of the agent from diseased vealed different developmental stages of CLOs within infected host crabs led to the molecular identification of Spiroplasma. In order cells. Microcolonies were membrane-bound and compressed and to confirm the taxonomic position of the agent, Wang et al. used displaced cytoplasmic organelles. (Sparks et al., 1985). bacterial universal primers for the 16S rRNA gene to amplify Other chlamydial infections have been reported in the rock crab non-host DNA from blood cells, nerves, muscles and connective tis- (C. irroratus) and Jonah crab (C. borealis). Louis Leibovitz devoted 6 sues from affected crabs. In addition, DNA sequencing was also years (1983–1988) to studying the highly fatal transmissible disease performed on isolates inoculated and isolated from the yolk sac of laboratory-maintained populations of C. irroratus and C. borealis of embryonated chicken eggs (Wang et al., 2003b). PCR amplicons (Leibovitz, 1988). Primary infections were associated with the hae- were compared to those in GenBank using the programme BLASTN. molymph, and crab hemocytes were filled with fine basophilic chla- The results showed that tremor disease agent (TDA) was not a rick- mydia, which were easily examined by light microscopic ettsia, but rather a spiroplasma belonging to the genus Spiroplasma observation of fresh and fixed-stained blood smears. The Chla- and closely related to S. mirum having 98% similarity with the lat- mydia-like organism altered the morphology of infected haemo- ter. In addition to molecular identity, characteristics such as the cytes. Ultrastructural examination of infected tissue revealed the absence of a cell wall, helical morphology during exponential presence of typical chlamydial life history stages; reticulate stages growth (Fig. 3), motility, in vitro culture and the ability to pass (560–475 nm), intermediate (422 nm) and condensing stages, ultrafilters have been confirmed (Wang et al., 2004). The Chinese (354 nm) and elementary bodies (214 nm). The crab’s hematopoi- mitten crab pathogen has been formally named as Spiroplasma eri- etic tissues were destroyed as the disease progressed (Leibovitz, ocheiris sp. nov. (Wang et al., 2010). 1988). Since the initial discovery, other spiroplasma-caused diseases have been identified in crayfish, ,(Wang et al., 7. Spiroplasma 2005) and in shrimp, Penacus vannamei (Nunan et al., 2004) and Rimicaris exoculata (Zbinden and Cambon-Bonavita, 2003), and in Spiroplasma is a new pathogen in crustaceans that causes seri- the sediment where susceptible hosts are encountered (Ding ous disease in commercially exploited crustaceans in both fresh- et al. 2007). In Columbia, in cultured P. vannamei, Spiroplasma pe- water (Wang et al., 2003a,b, 2004, 2005, 2010) and marine naei is responsible for significant mortality (up to 90%) (Nunan (Nunan et al., 2004, 2005) environments. Spiroplasma is the only et al., 2004, 2005). These studies indicate that spiroplasmas have W. Wang / Journal of Invertebrate Pathology 106 (2011) 18–26 23

significant reductions in free blood cell numbers and total serum protein. Such animals also displayed raised levels of glucose and ammonium in blood. Although the study has revealed much about this novel disease of the European shore crab, C. maenas, many questions still remain unanswered, including how the disease is transferred in the wild and whether other economically important species of crustaceans, such as the edible crab, C. pagurus, are susceptible to infection. The fact that the causative agent cannot be maintained in the absence of susceptible cells, coupled with the lack of available crustacean cell lines, will unfortunately hamper further studies (Eddy et al., 2007).

9. Potential approaches to control bacterial diseases of crab

Generally, diseases are the consequence of interactions of the disease agent (bacteria), the crab and the environment. Crabs are continuously affected by environmental fluctuations and manage- Fig. 2. Chinese mitten crab (Eriocheir sinensis) with tremor disease. ment practices such as handling, crowding, transporting, fluctuat- ing temperatures and poor water quality. All of these factors can a wide distribution in the aquatic environment and may be serious impose considerable stress on the homeostatic mechanisms of crab threats to aquatic crustaceans, especially cultured decapods. rendering them susceptible to a wide variety of pathogens. Because spiroplasmas are very small and have the ability to pass Many reported aspects of the crab immune reaction have been through 0.22-lm filters, just like viruses, and also have polymor- published (Smith and Ratcliffe, 1978, 1980a,b,1981; McDonald phic features during development, diseases associated with these et al., 1979; White and Ratcliffe, 1982; Smith et al., 1984; Bell bacteria are difficult for diagnoses. However, a DNA-based simple, and Smith, 1993; Schnapp et al., 1996; Hauton et al., 1997; Iwana- efficient, rapid, sensitive and low-cost method based on a human ga and Kawabata, 1998; Iwanaga et al., 1998; Relf et al., 1999; Na- medicine protocol has been developed for the detection of Spiropl- gai et al., 2001; Hammond and Smith, 2002; Iwanaga, 2002; asma from aquatic animals and environment samples. The protocol Gokudan et al., 1999; Holman et al., 2004; Burnett et al., 2006; yields accurate results within 4 h (Ding et al., 2007). In addition, an Towle and Burnett, 2007; Brockton et al., 2007; Mu et al., ELISA method for field diagnose of spiroplasma in aquatic animals 2009a,b; Meng et al., 2010). As for other invertebrates, crabs have was recently developed (Wang et al., 2009). no specific, adaptive and long-term ‘memory-based’ immunity. Crab defenses are dependent upon innate immunity that responds to common antigens on the cell surfaces of potential pathogens 8. Rhodobacteriales-like organism (Iwanaga and Lee, 2005). The crustacean host defence system has been well characterized and summarized by Smith (Smith et al., The European shore crab, C. maenas, which is valued as bait for 2003). Circulating hemocytes play an extremely important role in sport angling and as food in continental Europe, was recently re- the defence reactions by phagocytosis, hemocyte clumping, the ported to have an emerging disease christened ‘‘milky” disease be- production of reactive oxygen metabolites and the release of cause of the unusual milky appearance of the haemolymph (Eddy microbicidal proteins (Smith and Chisholm, 1992, 2001; Smith et al., 2007). The disease appears to be more prevalent during sum- et al., 2003). In addition, gills serve another important role in the mer months when water temperatures are higher and a Gram-neg- immune response (Johnson, 1976a,b; White et al., 1985; Martin ative bacterium was identified as the putative causative agent. The et al., 2000; Burnett et al., 2006). Johnson (1976) provided histolog- bacterium could not be cultured on a range of agar-based growth ical evidence that nodule formation in response to stress-induced media. Phylogenetic analysis of the 16S rRNA gene from the bacte- bacteremia in blue crabs might have adverse effects on gill func- rium indicated that it is a previously undescribed species of a-pro- tion, including distention of gill lamellae and disruption of haemo- teobacteria, with little phylogenetic similarity to members of the lymph flow. In C. maenas injected with bacteria, Smith and Ratcliffe order Rickettsiales. Eddy et al. used full-length of 16S rRNA gene (1980a) observed the formation of compact hemocyte clumps or and in situ hybridization probes to identify the agent. It was similar diffuse hemocyte networks that appeared to occlude the lumen to several uncultured marine bacteria (DQ009292, Brown et al., of the lamellar sinus, leading several authors, including White 2005; DQ071145, Lau and Armhurst, 2006). Diseased crabs showed et al. (1985) and Martin et al. (2000), to suggest that hemocyte aggregates nodules could interfere with respiration and ion regula- tion. Burnett found that Vibrio campbellii interfered with the respi- ratory function of the gills of the Atlantic blue crab C. sapidus.He concluded that a healthy blue crab can eliminate most invading bacteria, unless the respiratory function of the gills is impaired (Burnett et al., 2006). Immune stimulation can be achieved by dif- ferent kinds of microorganisms or compounds. Live bacteria, killed bacteria (bacterins or bacterial antigen), glucans, peptidoglycans and lipopolysaccharides (LPS) are thought to act as ‘immunostim- ulants’ because of their known effects on the crustacean immune system (Teunissen et al., 1998; Alabi et al., 2000; Vici et al., 2000; Sung et al., 1994, 1996; Song et al., 1997; Chang et al., Fig. 3. A spiroplasma isolated from tremor disease in the Chinese mitten crab 2000; Takahashi et al., 2000). 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Gene characterisation, isoforms and recombinant expression of carcinin, an antibacterial protein from the shore Another potential approach for the control crab pathogenic bac- crab, Carcinus maenas. Mol. Immunol. 44, 943–949. teria is to make use of a bacterial predator Bdellovibrio. Bdellovib- Brown, M.V., Schwalbach, M.S., Hewson, I., Fuhrman, J.A., 2005. Coupling 16S-ITS rios can attack other Gram-negative cells, penetrate their rDNA clone libraries and automated ribosomal intergenic spacer analysis to periplasm, multiply in the periplasmic space and finally burst the show marine microbial diversity: development and application to a time series. Environ. Microbiol. 7, 1466–1479. cell envelope to start the cycle anew (Jurkevitch, 2000). This lytic Bullis, R., Leibovitz, L., Swanson, L., Young, R., 1988. Bacteriologic investigation of action can rapidly reduce bacterial populations therefore this shell disease in deep sea red crab, Geryon quinquedens. Biol. 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