Gram-negative bacteria are predominant bacteria in marine environment and usually constitute the majority in the normal microflora of farmed and wild penaeid shrimp (Lightner, 1993; Bright Sing et al., 1998). Bacteria of the genus Vibrio are ubiquitous in the marine and estuarine aquatic ecosystem in which shrimp occur naturally and are farmed (Dempsey and Kitting, 1987; Ruangpan and Kitao,1991).
For nearly as long as penaeid shrimp have been cultured , reports of infections and disease caused by Vibrio spp. have been by far the most numerous of the reported bacterial agents of penaeid shrimp (Sindermann, 1990a; Lightner, 1993). Vibrio diseases have been reported as a limiting factor in penaeid aquaculture (Delves-Broughton and Poupard, 1976). Mortalities up to 100% because of vibriosis have been reported (Lightner, 1993). A 70-95% reduction in the expected harvest because of these vibrio infections has also been reported (Anderson et al., 1988). Most of the reported species of bacterial pathogens of penaeid shrimp (especially the vibrios), have also been reported to be part of their normal microflora (Lightner, 1993). Gomez-Gil et al. (1998) analysed healthy juvenile Penaeus vannamei for Vibrio spp. and found that hepatopancreas contained several Vibrio species, including V. alginolyticus, V.damsela and other Vibrio spp. They further suggested that normal penaeid shrimp might have a diverse population of bacteria as also reported by Bright Singh et al. (1998). This differs from the situation in diseased shrimps where one or two species of bacteria predominate (de la - Pena, 1993; Gomez -Gil et al., 1998).
Since, relatively large numbers of Vibrio spp. are normally present in the gut microflora of shrimp, several scientists have found species of Vibrio as frequent and apparently opportunistic pathogens of penaeid shrimp. These opportunistic shrimp pathogens take advantage of such conditions as toxicoses, nutrient deficiencies or viral infections (Lightner, 1993). It is generally accepted that isolates of Vibrio spp. from diseased shrimp may not always produce experimental infection to complete Koch’s postulate, except when challenged with massive doses (Lightner, 1993). However, some of the recently occurring disease syndromes of penaeid shrimp have been caused by Vibrio spp. which behave more like true pathogens than opportunistic invaders (Anderson et al., 1988; de la Pena et al., 1993; Abraham et al., 1996).
Most bacterial isolates from diseased penaeid shrimp have been Vibrio species, usually V. alginolyticus, V. parahaemolyticus , V. harveyi , V.splendidus, V.vulnificus and V.damsela (Lightner , 1988; Baticados et al., 1990; Lavilla - Pitogo et al., 1990; Lightner, 1993). Other gram negative rods including Flavobacterium spp., Pseudomonas spp. and Aeromonas spp., as well as the gram positive bacteria, Mycobacterium marinum and an occasional Micrococcus sp., were also reported to be involved in bacterial disease syndromes in penaeid shrimp .
Vibriosis, a principal bacterial disease in shrimp, causes shell disease, localized infections and bacterial septicemia. (Sindermann,1990a; Nash, 1992; Lightner, 1993; Otta et al., 1998). Vibrios cause disease and mortality among cultured penaeids in all stages.
Luminescent vibrios are autochthonous flora of coastal waters and they act as opportunistic pathogens and often are responsible for large scale mortalities in hatcheries (Lavilla -Pitogo et al., 1990; Karunasagar et al., 1994). Luminescent vibriosis have been reported in hatcheries of Indonesia (Sunaryanto and Mariam, 1986); Thailand (quoted by Lightner, 1993); the Philippines (Lavilla -Pitogo, 1990); Taiwan (quoted by Lightner, 1993) and India (Karunasagar et al., 1994).
Vibrio harveyi and V. splendidus were reported as responsible for luminescent vibriosis in Penaeus monodon larvae in the Philippines (Lavilla-Pitogo et al., 1990). Luminescent vibrios were first noted in Ecuador during 1988 (Lightner et al., 1993). Larvae infected by these bacteria displayed massive colonisation of the appendages and foregut, followed by infection of the midgut, hepatopancreas and a terminal septicemia. Serious epizootics in Penaeus japonicus by Vibrio spp. were reported by Takahashi et al. (1985) from postlarvae and juveniles . Diseased postlarvae displayed cloudiness of the hepatopancreas, while juveniles displayed cloudiness of muscle in the sixth abdominal segment and brown spots in the gills and lymphoid organ.
Lightner (1988) stated that most isolates reported from bacterial septicemias in penaeids have been Vibrio species. Anderson and Shariff (1987) reported that bacteria of the same genus, most commonly V.alginolyticus was isolated from penaeids affected with bacterial septicemias.
Anderson et al. (1988) reported vibriosis in market sized P. monodon from brackishwater farms of Malaysia. Specimens exhibited body reddening, extended gill covers and slight melanisation of uropods, pleopods and pereopods. “Syndroma Gaviota” (Seagull syndrome - ‘SGS’) a massive vibriosis epizootic syndrome affected nursery and grow-out ponds in shrimp farms of Guayaquil , Ecuador (Aiken, 1990). The dominant organisms isolated from SGS vibriosis affected shrimp include V.parahaemolyticus, V.alginolyticus, V.vulnificus, V. fluvialis and V. damsela. Nash (1992) investigated moribund shrimps in Thailand and found the dominant presence of the V.parahaemolyticus, V.anguillarum, V.vulnificus, V.damsela and V. alginolyticus. Diseased samples of P. monodon collected from intensive shrimp farms showed abundance of V.parahaemolyticus, V.vulnificus, V. alginolyticus and other Vibrio spp. (Ruangpan and Kitao, 1991).
Alfaro et al. (1993) reported V.alginolyticus as responsible for infection of reproductive system in Penaeus setiferus. The blackening of reproductive system and the subsequent infertility were observed in the shrimp. Vibrio spp. especially V.alginolyticus were predominantly isolated from the necrotic lesions on the appendages of larvae and adult shrimp and adult lobster affected by shell disease or black spot disease. (Hameed, 1994). Manley (1994) reported luminescent V. harveyi from P. indicus affected by bacterial shell disease.
Since 1993, significant mortality outbreaks of Penaeus stylirostris usually during winter, have been reported from grow out ponds in New Caledonia, France. Costa et al. (1998) carried out studies to describe the ‘Syndrome 93’ and found that bacteria of genus Vibrio as major culprit. Lavilla –Pitogo et al. (1998) observed severe mortalities of pond-cultured juvenile shrimp, Penaeus monodon, associated with dominance of luminescent vibrios in the rearing environment. Lavilla –Pitogo and de la Pena (1998) reported the luminescent virbosis in the hatchery system in the Philippines.
Pathogenicity tests are conducted to know the virulence of the pathogen. Lavilla-Pitogo et al. (1990) conducted pathogenicity tests using V. harveyi isolate obtained from a postlarva and found that exposure to V.harveyi induced significant mortalities in the larval and postlarval stages of P. monodon within 48h. They also found that relatively small quantity of inoculum can elicit the disease, in contrast to Lightner’s (1988) observation based on Koch’s postulation indicating the massive inoculum needed for expression of a disease condition. Vera et al. (1992) tested virulence of three species of Vibrio bacteria in juveniles of P. japonicus and found V. alginolyticus to be the most virulent species followed by V. parahaemolyticus and V. anguillarum in that order. High dose of V. parahaemolyticus , V. alginolyticus and V. anguillarum at concentrations of 108 cfu/shrimp induced mortalities of 100%, 80% and 40% respectively within 4 hrs. Nash (1992) conducted pathogenicity studies in P.monodon juveniles using V. parahaemolyticus and found that with 107 colony-forming units per shrimp (cfu/Shrimp) death occurred in 1 hour, with 106cfu/shrimp death occurred in 6-9 hours and with 105cfu/shrimp death occurred in 12-13 hours. Below 105 cfu/shrimp shrimp were weak for 2 days but usually recovered within 3-5 days after injection. He noted that 105 cfu/shrimp was threshold level for the development of disease problems. Alfaro et al. (1993) carried out experiments to study the disease associated with blackening of the male reproductive system in captive Penaeus setiferus. When the authors challenged the shrimp using V.alginolyticus (isolated from diseased shrimp) by injecting 5-fold diluted culture into the gonopore, they observed 83% mortality in less than 24 hours with no blackening of reproductive tissue in P. setiferus. The pathogenicity test conducted by de la Pena et al. (1993) by injecting a specific Vibrio sp. isolated from vibriosis affected kuruma shrimp (Panaeus japonicus) the experimental animals were killed at a median lethal dose (LD50) between 1 x 102 and 1 x 103 cfu/shrimp.
Virulence studies using Vibrio harveyi isolates from larval tank and sea water were carried out in P. monodon larvae by Karunasagar et al. (1994) and they found that V. harveyi isolates from the larval tanks showed high virulence (LD50 of 2.6 x 103) whereas isolates from sea water had LD50 of 1.5 x 105. Jiravanichpaisal et al. (1994) performed infectivity trials with black tiger shrimps (Penaeus monodon) to confirm pathogenicity of two isolates of V.harveyi (Chitinase positive and negative). They found that both types of isolates were pathogenic to shrimp, intramuscular injection of 105 - 106 cfu/shrimp resulted in 53-100% mortality, and the Chitinase – negative isolate was found to be virulent than the Chitinase – positive isolate. Manley (1994) carried out pathogenicity test of six isolates including V. alginolyticus-H1, V.parahaemolyticus and V.harveyi and found V. alginolyticus-H1 to be the most virulent one, causing 60% mortality in 24 h even at 1.90 x 103 cfu / shrimp in juveniles (mean weight of 2.9 g) of P. indicus.
Pathogenicity experiment was carried out by Hameed (1994) on the mysis larvae of P. indicus using V. alginolyticus. Immersion method was followed to infect the larvae. At concentrations of 2.8 x 106 and 2.8 x 108 cells/ml exposure for 48 horus, V. alginolyticus caused 20 and 36.6% mortality in the larvae respectively. Based on the pathogenicity test conducted through immersion, Prayitno and Latchford (1995) observed several strains of Vibrio harveyi as pathogenic to P. monodon larvae at 103 cfu/ml concentration. Further, they noted that the virulence of the bacteria was related to the age of the larvae, such that 25.28% of zoea, 47.08% of mysis and 51.5% of postlarvae survived after 48 h exposure to the pathogen. Uma (1995) observed that V.alginolyticus – HPY2 caused 85% mortality at 106 cfu/ml in P. indicus juveniles (mean weight 150 – 200 mg) in 7 days when challenged through immersion technique.
Abraham and Shanmugam (1996) challenged Penaeus indicus juveniles of 3 g average weight by intramuscular injection with Vibrio alginolyticus strain and found the death of all challenged shrimp within 8 h at a higher dose of 2x107 and 2 x 108 cfu/shrimp. The mean lethal dose (LD50) was < 2 x 103 cfu/shrimp. Pradeep Kumar (1996) conducted pathogenicity test of bacterial strain V.alginolyticus-PH1 on P. indicus juveniles (mean weight 1.25 – 1.5g) and found the LD50 value of this strain at 1.425 x 105 cfu/shrimp. Pereira (1996) reported the LD50 values of a V. harveyi strain on P. monodon postlarvae (mean weight 80 mg) at 6.1 x 104 cfu / ml. Alapide–Tendencia and Dureza (1997) observed that injection with V. parahaemolyticus 107 cfu/shrimp induced significantly higher mortalities (100%, 10 h post-injection) in P. monodon juveniles than that with V.harveyi (20%).
Pathogenicity tests were carried out by Ravi et al. (1998) to determine different levels of virulence in V. harveyi isolated from the sea water and reisolated strain from the haemolymph of experimentally infected shrimp by challenging against juveniles of P. monodon and P. indicus. In the case of P.monodon, they found that sea water strain caused 100% mortality (at the end of 48h) at 105 cfu/shrimp whereas the strain reisolated from haemolymph caused 100% mortality at 103 cfu/shrimp, indicating that the isolates from the shrimp source were highly pathogenic to shrimp. In the case of P. indicus the authors observed similar mortality pattern, however at the end of 84 h.
Costa et al. (1998) studied about the virulence of Vibrio spp. strains isolated from ‘Syndrome 93’ affected P. stylirostris by intramuscular injection. The author observed very high virulence (LD100 = 10 cfu/g of shrimp). Roque et al. (1998) observed that juveniles of marine shrimp Penaeus vannamei when exposed to V.parahaemolyticus through immersion method at a dose of 105 cfu/ml resulted in 0 to 13% mortality within 4 days. Goarant et al. (1998) carried out experimental infection with V. penaeicida (a strain isolated from ‘Syndrome 93’ diseased juvenile shrimp) at different concentrations ranging from 7 x 105 to 7 x 106 cfu / ml and found out increased sensitivity to V. penaeicida after the animal reached PL9 stage. Robertson et al. (1998) found out that a culture of Vibrio harveyi isolated from diseased (Bolitas negricans affected) Penaeus vannamei was pathogenic to penaeid shrimp larvae when exposed to a bath treatment containing 105 cells / ml for 2 h. According to the authors, longer period of exposure of the pathogen to the shrimp led to multiplication of the pathogen and extremely rapid mortalities.
IMMUNE SYSTEM OF CRUSTACEANS
The crustaceans have ways and means to defend themselves against pathogens. Although the hard cuticle forms a structural and chemical barrier to parasites, there is still a need for an efficient internal immune defence (also denoted as defense by some authors) network to deal with opportunistic or pathogenic microorganisms that can gain entry into the body cavity through wounds or during the moulting. Besides they exhibit the capacity of rapid wound healing which prevents loss of haemolymph. Following the tradition established for vertebrates, the host defences of invertebrates are often categorized as either humoral or cellular, i.e. mediated by factors in the plasma or brought about by activities of intact blood cells (Sindermann, 1990b; Soderhall and Cerenius, 1992; Smith and Chisholm, 1992; Azad et al., 1995; Bachere et al., 1995a)
CELLULAR DEFENCE MECHANISM
Crustaceans are not different from other animals in that the host defence is largely based on activities of the blood cells or haemocytes . Three main types of circulating haemocytes have been identified and isolated by isopycnic centrifugation on percoll gradient (Soderhall and Cerenius, 1992). In the crab (Carcinus maenas) and crayfish (Pacifastacus leniusculus), the hyaline cells are characterized by the absence of granules, spreading capability and phagocytosis. Whereas the semi-granular cell contains small granules and it lyses very rapidly when manipulated in vitro or reacting with microbial polysaccharides and the third cell type is the granular haemocyte containing large granules and whose main function, in crayfish, is the storage of the prophenoloxidase system (Soderhall and Cerenius, 1992; Bachere et al., 1995b).
Haemocyte types, particularly the hyaline cells, are not analogous between different crustacean species, both morphologically and functionally. In the shrimp Penaeus japonicus the hyaline cells are relatively less numerous than in other decapods. In vitro studies revealed that the hyaline cells are very labile and non-phagocytic; morphologically corresponding to undifferentiated haemocytes (Bachere et al., 1995a). In a study on penaeid shrimp Sicyonia ingentis haemopoiesis and based on typical ultrastructural features, two types of stem cells have been distinguished, giving rise to a line of granular cells and a line of hyaline cells, characterised by cytoplasmic deposits (Hose et al., 1992). Moullac et al. (1997) studied about the haemocytes of P.stylirostris and classified the haemocytes in three groups designated as hyaline cells (HC), characterized by their small size and absence of granules in cytoplasm and semi-granular cells (SGC) and large granular cells (LGC) easily recognisable by the intracytoplasmic granular content. The authors attributed the variation in total haemocyte count (THC) during the moult cycle to the variation in HC representing about 80% of THC. They also confirmed the origin of prophenoloxidase (PO) activity to LGC.
Kobayashi and Soderhall (1990) carried out experiments to compare the behaviour of isolated haemocytes from parasite-infected and non-infected crayfish using an in vitro assay system. By using a lectin as a legand the authors demonstrated that haemocytes of parasite-infected crayfish bound the lectin to the haemocyte surface in a manner similar to vertebrate lymphocytes (i.e. capping). They noted that animals infected by the parasite (Psorospermium haeckeli) exhibited a higher degree of capping, indicative of haemocyte activation, than non-infected animals.
Hose et al. (1992) carried out experiments to study the patterns of haemocyte production and release throughout the moult cycle in the penaeid shrimp Sicyonia ingentis and observed the higher production of granulocytes and hyaline cells at premoult stage. They reasoned the abundance of granulocytes as well as hyaline cells to repair the exoskeleton and fight infection as advantageous during the critical moulting period due to the soft exoskeleton and most susceptible nature of the animal. Lee et al. (1995) conducted experiments to know about the interaction between crustaceans and pathogens and their products. They conducted a novel haemocytolytic assay using haemolymph of tiger shrimp (Penaeus monodon) and found the assay to be useful for pathogenesis studies.
Phagocytosis is the most common of the cellular defence reactions, and together with humoral components, constitutes the first line of defence once a parasite or an intruder has overcome the physicochemical barrier of the cuticle (Soderhall and Cerenius, 1992). Phagocytosis is an unicellular defence reaction that occurs when the foreign object is smaller than the haemocyte, whereas, encapsulation and nodule formation are multicellular defences reaction that occur when the foreign material is larger than the haemocyte (Sindermann, 1990b).
Phagocytosis by fixed and mobile cells in the gills, pericardial sinus and sinuses at the base of appendages seems to be a principal cellular defence reaction in many crustaceans. Further efficacy of phagocytes in destroying invading microorganisms varies, depending on the species of microorganisms as well as host physiology and environmental factors. Phagocytic activity is enhanced by haemolymph factors, which in addition to immobilizing and agglutinating the invading organisms, also sensitize them to phagocytosis (Sindermann, 1990b).
The phagocytic cell number in crustacean haemocytes vary from 2 to 28% (Soderhall and Cerenius , 1992). Krol et al. (1989) studied the haemocytic response to an acid fast bacterial infection in cultured P. vannamei and found evidence of both cellular defence processes, phagocytosis and nodule formation. They found that clumps of bacteria were surrounded by multicelluar layers of haemocytes, and some individual bacteria were phagocytized by a single haemocyte. According to them, only small and large-granular haemocytes participated in phagocytosis and nodule formation in P. vannamei.
Itami et al. (1994) observed that oral administration of a b-1,3- glucan (Schizophyllan, SPG) resulted in high phagocytic activities in the haemocytes of shrimps ( Penaeus japonicus). It was also noted that the blood homogenate of the SPG fed shrimp was able to activate the phagocytosis of the granulocytes isolated from a normal shrimp. They further reported that phagocytic activity of the haemocytes increased after vaccination against vibrosis by spray and immersion methods.
Vargas-Albores (1995) studied the phagocytic activity of brown shrimp (Penaeus californiensis) by conducting assay using glutaraldehyde-treated mouse erythrocytes and found the increased phagocytic activity by addition of purified LPS- binding agglutinin.
When the body cavity is invaded by large number of microorganisms, in excess of those that can be removed by phagocytosis, nodule or cell clumping occurs in several invertebrates, including the crustaceans. The end result of nodule formation is that the microorganisms become entrapped in several layers of haemocytes, and normally the nodule becomes heavily melanized because of the host’s phenoloxidase activity (Soderhall and Cerenius, 1992).
Krol et al. (1989) studied the histopathology and ultrastructure of the haemocytic response of P. vannamei to an acid-fast bacterial infection and described the nodule thus formed as composed of flattened haemocytes in concentric layers surrounding a core of rod-shaped, acid-fast bacteria and degenerating cells. They also reported nodules randomly distributed throughout the affected tissues of shrimp.
When a parasite is too large to be engulfed by phagocytosis, several haemocytes collaboratively seal off the foreign particle from circulation (Soderhall and Cerenius, 1992). The onset of encapsulation may be rapid, and the cellular aggregates may be resolved only very slowly (Sindermann, 1990b). Semi-granular haemocytes are the first to react to a foreign particle or organism by encapsulation and the 76kDa protein acts as cell adhesion factor and encapsulation promoting factor in the encapsulation process. (Soderhall and Cerenius, 1992).
Sindermann (1990b) reported that the involvement of haemocytes in coagulation or clot formation as a complex one, indicating the formation of either cellular or extracellular clots. He observed that the intravascular cellular clots adhered to the walls of blood vessels and spaces, producing stasis. According to him, the intravascular clots once formed, would persist for some time. Whereas the extra-cellular clots, resulting from release of the constituents of haemocytes, could inhibit microbial motion and thus rendering microorganisms more vulnerable to phagocytosis.
Cyctotoxicity is the mechanism by which some cells of the haemocytes specialize to kill the target cells or organism by their toxic properties. In crustaceans few studies have been devoted to this process (Soderhall and Cerenius, 1992).
PHAGOCYTOSIS AND OXIDATIVE DEFENCE MECHANISM
Phagocytosis is generally recognised as a central and important way to eliminate microorganisms or foreign particles. But little is known about the post-phagocytic events and haemocytic killing mechanisms, or about oxidative metabolism in particular. In vertebrates, stimulated phagocytes are able to generate reactive oxygen intermediates (ROIs ) with powerful microbicidal activity. The phenomenon is known as the respiratory burst (Bachere et al., 1995a). The killing substances include highly reactive oxygen species such as superoxide anions (O2-), hydrogen peroxide (H2O2), hydroxide ions (OH- ), singlet oxygen (O21), myeloperoxidase (MPO)- catalyzed hypochlorite and digestive enzymes within cytoplasmic granules (Song and Hsieh, 1994). In crustacea, only limited investigations are available concerned with oxidative metabolism in shore crab Carcinus maenas (Bell and Smith, 1993) and in the tiger shrimp P.monodon (Song and Hsieh, 1994). Bell and Smith (1993) demonstrated the generation of O2- by separated hyaline cells but not the semi-granular or granular cells of the shore crab. Song and Hsieh (1994) analysed the reactive oxygen species and MPO generated by the tiger shrimp (Penaeus monodon) haemocytes and found that b-glucan had the strongest stimulative effect on haemocytes in terms of generating O2-- and H2O2, but phorbol myristate acetate (PMA) had the strongest stimulative effect on OCl-- production and MPO activity, thus underscoring the fact that different immunostimulants affect different stages of the phagocytic process. Bachere et al. (1995a) used the technique of chemiluminescence (CL) for the demonstration of the respiratory burst in the haemocytes of P.japonicus upon stimulation by either PMA or zymosan and found the response to be dose dependent for the two elicitors.
THE PROPHENOLOXIDASE SYSTEM (PRO PO-SYSTEM)
Phenoloxidase is the most important component of the pro Po-system. Phenoloxidase is responsible for the blackening or melanization commonly observed in crustaceans infected with parasites (Sindermann, 1990b). Biochemistry of prophenoloxidase (proPo) system activation and regulation is well known in crayfish (Soderhall and Cerenius, 1992). According to them, proPo is activated by a prophenoloxidase -activating enzyme (PPA), which is serine protease previously activated in turn by microbial cell walls. They also observed that two protease inhibitors, a2-macroglobulin and a trypsin inhibitor, could block PPA. A protein, 76kDa cell adhesive factor, released by the haemocytes, amplifies the generation of the proPo system by inducing degranulation of semigranular and granular cells and by stimulating phagocytes by hyaline cells (Soderhall and Cerenius, 1992).
Prophenoloxidase in its activated state functions also as redox, oxidizing phenols to quinones sub-sequently resulting in melanin formation (Bachere et al., 1995a). They reasoned that the quinones are thought to be antimicrobial, possibly because of their strong oxidizing properties. Krol et al. (1989) observed the melanized cores and intercellular material in nodules and confirmed the diffused nature of melanin in Penaeus vannamei.
Ashida and Soderhall (1984) studied the proPo activating system in crayfish. They obtained a preparation containing proPo and a stable proPo activating system from crayfish haemocytes and they found that proPo in the preparation can be activated by elicitors such as b-1,3-glucan, heat, SDS or trypsin. They further observed that not all the elicitors activated proPo through the same mechanism and that the Ca2+ is required for the activation by all the elicitors.
Cerenius et al. (1991) found that preparations of the crayfish pathogen Psorospermium haeckeli were found to activate the proPo system in haemocyte lysates of two crayfish species Pacifastacus leniusculus and Astacus astacus. Vargas-Albores (1995) isolated a plasma protein (b-glucan binding protein) from brown shrimp (Penaeus californiensis) which enhanced the activation of proPo by b-1,3-glucan. Vargas –Albores et al. (1996) reported that these recognition proteins are capable of activating cellular functions only after reaction with the microbial substances (LPS or glucans) and paradoxically the signal to activate the defence mechanism is given by the pathogen through their surface components like LPS or glucans. The author further observed that these recognition proteins and their mechanisms could help to design strategies for immunoprophylaxis by using microbial products to stimulate the shrimp immune system.
Moullac et al. (1997) studied the haematological and PO activity changes in the shrimp (P. stylirostris) in relation with the moult cycle protection against vibriosis and found that the PO activity in haemocytes was significantly higher in intermoult than in premoult period. Further according to them the variations in the proPo system were correlated with sensitivity of shrimp to infection by the Vibrio AM23.
HUMORAL DEFENCE MECHANISM
Humoral immunity is interpreted to include those defence strategies which serve to reduce, neutralise or overcome invasion by non-self material without the direct intervention of intact haemocytes (Smith and Chisholm, 1992). According to the authors, the humoral defence strategies of invertebrates included a multiplicity of serum or plasma factors for acting against microorganisms, foreign cells or biotic materials. These factors include naturally occurring or inducible bioactive molecules which agglutinate, precipitate or inactivate non-self particles as well as those that have bactericidal, lytic or bacteriostatic properties. Various fungitoxic, antiviral or cytotoxic agents are also considered as humoral factors (Smith and Chisholm, 1992).
Bachere et al. (1995a) reported that apart from haemocyanin (the respiratory protein which is quantitatively the most important of the circulating proteins in crustaceans), other haemolymph compounds with potential immune functions have been identified in P. japonicus.
Factors that bind to and cause the aggregation or agglutination of foreign particles are denoted as agglutinins (Smith and Chisholm, 1992). For most crustaceans, agglutinins appear to occur naturally in the plasma or serum although enhanced titres following prior exposure of the host to the test material, as reported for Penaeus mondon by Adams (1991) and for Penaeus californiensis by Vargas-Albores (1995). As far as agglutinins against microorganisms are concerned, these factors must assist in the sequestration of invasive organisms from the haemolymph, thereby facilitating their interaction with the circulating blood cells and preventing the spread of potentially infective agents around the body (Smith and Chisholm, 1992).
The function of the haemagglutinins is less obvious. Typically, they are inhibited by polysaccharides sugar and this affinity for polysaccharides has encouraged several workers to consider haemagglutinins also as lectins (Smith and Chisholm, 1992). (Lectins are defined by the Nomenclature committee of the International Union of Biochemists as carbohydrate binding proteins of non-immune origin that agglutinate cells or precipitate polysaccharides or glycoconjugates. Lectins are of non-immune origin whereas haemagglutinins are of immune origin). Bachere et al. (1995a) isolated a molecule with agglutinating activity, from the haemolymph of P. japonicus which was found to be closely related to monodin, a lectin of a 420 kDa size purified from the shrimp P.monodon.
A wide variety of substances that kill foreign cells or microorganisms have been described in invertebrates. They are ubiquitous throughout the invertebrates and may act against bacteria, fungi, viruses, protozoan or other foreign cells (Smith and Chisholm, 1992). In general, these factors are non-specific and comprise of a heterogenous collection of molecules. Adams (1991) reported killing factors from the haemolymph of penaeid shrimp acting against bacteria (Vibrio sp.). The killing activity was induced by pretreatment of the host (penaeid shrimp) with live or heat -killed bacteria (Vibrio sp.). However, the response seemed to possess limited specificity and the time taken to reach maximum killing effect varied between 36 h and 48 h. Biochemically, little is known about the killing factors in haemolymph of crustaceans.
Since arthropods have open circulatory system, wounds must be sealed rapidly to prevent-blood loss and also to hinder pathogenic micro- organisms from entering through the wound and causing infections (Soderhall and Cerenius, 1992). Wound caused clotting could generate signals that mediate cell to cell communication in bringing together the cell -derived immune factors to the point of action for effecting aggregation, killing, neutralising the invasive microorganisms (Smith and Chisholm, 1992). A plasma clotting protein was isolated from P. japonicus by Bachere et al. (1995a). The above mentioned protein has a molecular weight of about 360 kDa . Besides plasma clotting, cellular coagulation was also observed in shrimp.
The clotting system represents an important component in host defence. Administration of LPS or b-1-3-glucan to crustaceans result in the formation of clot which entraps invading microorganism (Soderhall and Cerenius, 1992). This process is also linked to the triggering of the prophenoloxidase activating system.
Vaccines are preparations of antigens derived from pathogenic organisms, rendered non-pathogenic by various means, which will stimulate the immune system in such a way as to increase the resistance to disease from subsequent infection by a pathogen (Ellis, 1988a).
The first attempt to vaccinate fish against disease were published by Duff (1942) who attempted to orally vaccinate cut-throat trout (Salmo clarkii) against furunculosis with a diet containing chloroform-killed Aeromonas salmonicida cells. The first commercial product licence for a vaccine for fish was granted in the USA during 1976 for use against Yersinia ruckeri for preventing enteric red moulth (ERM) disease. Since then product licences have been granted for Vibrio vaccines in many parts of the world and the above two vaccines assumed greater commercial value (Ellis, 1988a).
Various techniques in administration of vaccine to fish have been developed and these include injection, immersion, hyperosmotic immersion, bath, spray, and oral administration. Johnson and Amend (1983a) and Ellis (1988a) have evaluated the efficiency of several delivery methods. Johnson and Amend (1983b) administered the bacterin of Yersinia ruckeri to rainbow trout (Salmo gairdneri) through various delivery methods and reported intraperitoneal injection method as the most efficient delivery method. For Salmo gairdneri of 2.2 g size, the effective delivery system next to intraperitoneal injection was reported to be immersion, followed by shower and spray delivery. Moreover, according to them, injection method was found to be most costly due to the work force involved in assessment of the weight of individual fish and the dose of vaccine administered. On the other hand immersion method was found to be advantageous since small dose of vaccine is needed to vaccinate larger number of small fish group. In the case of shower or spray method of administration of vaccine, it is difficult to ascertain the total surface area of fish and exposure time for known quantity / volume of bacterin.
According to Ellis (1988a), oral vaccination was reported to be the only suitable method for extensive pond rearing of fish where catching of fish for injection or immersion vaccination could not be done practically. However, according to him the most important limitation of oral vaccines was reported to be their poor potency. He further reported that immersion vaccination was proved very efficacious for immunizing fish against vibriosis and enteric redmouth disease, but found to be not so successful for other diseases (Ellis, 1988a).
The selection of the most efficient vaccine delivery method should be based on the size of fish at the time of disease outbreak, ability to gather the farmed fish for vaccination, time and workforce involved (Johnson and Amend, 1983a).
Many authors have reported about fish vaccines and their efficacy. Fletcher and White (1973) reported that antibody to heat killed Vibrio antigen was detectable in higher titres in the intestinal extract from orally immunized plaice (Pleuronectes platessa), than in its serum. Further, parenteral immunization with V.anguillarum, bacterin along with Freund’s complete adjuvant resulted in high and persistent antibody titres in the serum, while only small quantity of antibody were found in the intestinal and cutaneous secretions and no circulating antibodies were detectable one year after injection. Paterson et al. (1981) observed that both under-yearling and post-yearling of Atlantic salmon (Salmo salar) parr produced high agglutinating antibody titres in response to a single intraperitoneal injection of killed Renibacterium salmoninarum cells emulsified in Freund’s complete adjuvant (FCA).
Agius et al. (1983) noted that intraperitoneal vaccination of V. anguillarum extract vaccine resulted in virtually 100% protection within two weeks whereas oral vaccination gave a maximum protection of 50% to 70% after eight weeks against vibriosis. Johnson and Amend (1983b) found that a single anal vaccination with bacterins of V. anguillarum and Y. ruckeri bacterin resulted in the best protection and immunity in salmonids when challenged by V. anguillarum. Amend et al. (1983) evaluated several factors which affected the potency of the vaccine (Y. ruckeri bacterin) in Salmo gairdneri. McCarthy et al. (1983) found that the efficacy of Aeromonas salmonicida bacterin (vaccine) was high when the vaccine was administered through injection method, while the same through immersion resulted in only satisfactory level of protection.
Holm and Jorgensen(1987) vaccinated Atlantic salmon, Salmo salar, against ‘Hitra - disease’ or coldwater vibriosis by direct immersion in a formalized bacterin prepared from a Vibrio sp. associated with 'Hitra disease' and found that the vaccine gave excellent protection. The efficacy of adjuvanted vaccine (administered intraperitoneally) was evaluated in rainbow trout Salmo gairdneri by Adams et al. (1988) and they noted that simple mineral oil-based adjuvants resulted in protection against furunculosis, vibriosis and enteric red mouth disease. Ellis (1988b) described different optimizing factors for fish vaccination which affected the development of effective immunity.
According to Smith (1988) Vibrio vaccines were denoted as the most successful and most promising vaccine among the vaccines available for the major diseases of fish. Ellis (1988c) described about vaccination against enteric redmouth and the advantages and drawbacks of the vaccine. Different types of furunculosis vaccines and their administration were described by Hastings (1988). Stevenson (1988) reviewed about vaccination against Aeromonas hydrophila and its development. Munro and Bruno (1988) described the prospects of developing a bacterial kidney disease vaccine and its limitations. Salati (1988) reported about the limitation of present vaccine available against Edwardsiella tarda and prospects of development of a commercial vaccine against it. Plump (1988) reported about the efficacy of E. ictaluri vaccine Houghton et al. (1988) dealt with about vaccination against protozoan and helminth parasites of fish. Horne and Ellis (1988) reported about the strategies of fish vaccination.
Lillehaug (1989a) carried out a survey on different procedures available for vaccinating salmonids against vibriosis in Norwegian fish-farming and found that out of intraperitoneal injection, direct immersion and prolonged bathing injection was preferred for rainbow trout and direct immersion was the method of choice for Atlantic salmon. Lillehaug (1989b) prepared two oral vaccines namely the protected vaccine (against digestive degradation) and unprotected vaccine. The latter was found to be promising. According to the author the vaccine protected against digestive degradation did not release the lipopolysaccharides and hence exhibited poor performance.
Effects of oral vaccination against vibriosis in turbot, Scophthalmus maximus and sea bass, Dicentrarchus labrax were studied by Dec et al. (1990) using Vibrio anguillarum bacterin. They observed the superiority of intraperitoneal administration over the oral route. Moore et al. (1990) attempted to control Flexibacter columnaris epizootics in pond-reared channel catfish Ictalurus punctatus by vaccination through immersion, using formalin inactivated F. columnaris bacterins. They also observed the reduction in F. columnaris epizootics in the vaccinated group. Lillehaug (1991) documented protective immunity in Atlantic salmon extended even up to 1.5 to 2 years after vaccination against cold water vibriosis and further noted gradual decline in immunity with time. Santos et al. (1991) observed the potency of a whole cell bacterin and a toxoid enriched whole cell vaccine administered intraperitoneally into the rainbow trout, Oncorhynchus mykiss and found that both vaccines conferred good protection, with the toxoid enriched vaccine yielding better result. Immunological response of Indian major carps to Aeromonas hydrophila vaccine was observed by Karunasagar et al. (1991) and they reported that very high titres of antibodies were induced in Catla catla followed by Cirrhinus mrigala and Labeo rohita. They further found that immunized fish showed good protection against homologous challenge and moderate protection against heterologous challenge was observed in C. mrigala and L. rohita.
According to Lillehaug et al. (1992) vaccination of Atlantic salmon by injection with adjuvanted vaccines against furunculosis reduced the mortality during disease outbreaks by approximately two-third compared to unvaccinated controls. Schroder et al. (1992) observed that an immersion vaccine based on formalin inactivated V. salmonicida elicited 90-100% protection against coldwater vibriosis in cod (Gadus morhua ). Ling et al. (1993) reported that fish vaccinated with Ichthyophthirius multifiliis and Tetrahymena pyriformis by immersion or intraperitoneal injection showed effective protection not only against I. multifiliis but also against various types of parasitic protozoans commonly found in that region. Sakai et al. (1993) tested the immune responses elicited in the rainbow trout, Oncorhynchus mykiss by using five bacterins such as formalin–killed, heat-killed, pH-lysed and UV-killed cells of Renibacterium salmoninarum and formalin-killed mixture of R. salmoninarum with Streptococcus sp.
They found that pH-lysed bacterin showed best performance. A comparative study of the efficacy of two vaccine formulations namely a whole - cell bacterin and a toxoid enriched whole-cell vaccine, against Pasteurella piscicida was conducted by immersion method in the gilt head seabream Magarinos et al. (1994). They observed that the antibody response exhibited by the fish immunized with both vaccines was very low and protection was also moderate.
The rainbow trout (Oncorhynchus mykiss) were vaccinated with a whole cell, formalin-killed Vibrio anguillarum bacterin by injection, immersion and oral routes by Palm et al. (1994). According to them, injection method of stimulated the high level of antibody production. On the other hand, immersion provided intermediate level and oral delivery resulted in the lowest level of circulating specific antibody.
Gutierrez and Miyazaki (1994) observed the responses of Japanese eels to oral challenge with Edwardsiella tarda after vaccination with formalin- killed cells (FKC at a dose of 10 mg/100 g body weight) or lipopolysaccharides (LPS at a dose of 1mg /100g body weight) of the same bacterium and found that the LPS vaccination provided considerably more protection than the FKC vaccination. Sakai et al. (1995) examined the efficacies of combined vaccine consisting of killed Vibrio anguillarum and Streptococcus sp. in rainbow trout (Oncorhynchus mykiss) and observed increased resistance against challenge with virulent bacteria.
Quentel and Ogier de Baulny (1995) carried out intraperitoneal vaccination in the juvenile turbot, Scophthalmus maximus, against vibriosis using formalin -killed Vibrio anguillarum bacterin and found that vaccination protected juvenile turbot during a challenge with same species performed one month after a single immunization. He et al. (1997) developed a recombinant vaccine against I. multifiliis infection for goldfish. They observed that the average survival rate of the immunized fish was 95% when compared to 55% for the control fish when challenged with infectious tomites of I. multifiliis. Leung et al. (1997) reported that attenuated growth-deficient mutants as promising candidates for the development of live vaccines against Aeromonas hydrophila infection in fish. Eggset et al. (1999) carried out vaccination in Atlantic salmon (Salmo salar) before and during smoltification using oil-adjuvanted vaccine, protective against Aeromonas salmonicida and Vibrio salmonicida and found that vaccination close to the start of smoltification yielded high immunological protection.
Immunization of culture shrimps against several bacterial pathogens were tested for their efficacy by different workers. Cultured kuruma shrimps (Penaeus japonicus ) were experimentally vaccinated against vibriosis with formalin-killed Vibrio sp. NU-1 by injection, immersion, and spray techniques by Itami et al. (1989). They found that all delivery methods reduced mortality of the shrimp when they were challenged by Vibrio sp. NU-1 injection. Itami et al. (1991) conducted an experiment to know the survival of larval giant tiger shrimp, Penaeus monodon after addition of killed Vibrio cells to a microencapsulated diet and it was found that percentage of survival was higher in the groups fed with killed bacterial cells. Adams (1991) observed that greater than 99% of Vibrio alginolyticus were cleared from the haemolymph of P. monodon within 4h of exposure to the heat-killed bacteria. Itami et al. (1992a) carried out experiments to study the efficacy of several vaccines prepared by different methods and observed the response of the vaccinated shrimp. An effective vaccine against vibriosis (Vibrogen-S), was developed by Aqua Health(Asia) Ltd., a joint venture company formed between Charoen Pokphand Feedmill Co. Ltd., Thailand and Aqua Health Ltd., Canada. The vaccine, Vibrogen-S, was proved to be effective under laboratory and field conditions against virulent Vibrio parahaemolyticus. The vaccine Vibrogen-S was administered by immersion, injection and by oral techniques, to black tiger shrimp, P. monodon and all the three delivery methods exhibited effective protection against virulent V.parahaemolyticus when compared to unvaccinated control.
According to Latchford et al. (1995), the shrimp administered with formalin-killed bacterins and vaccines consisting of live attenuated strains of pathogenic bacteria (produced by UV light mutagenesis) showed significantly higher survival rate than the non-vaccinated group of shrimp and a significantly enhanced growth rate. Preliminary studies of the immunization of shrimp (Penaeus monodon ) against vibrio infections were carried out by Bechteler and Holler (1995). Karunasagar et al. (1996) conducted a trial on vaccination of shrimp larvae and results indicated that vaccination significantly improved survival of larvae in hatchery systems. In areas where outbreak of white spot disease occurred, farmers who administered regular booster doses of the above vaccine noted delayed onset of disease and significant reduction in mortality rates. Bechteler and Holler (1996) developed a vaccine consisting of formalin-killed bacterin of three Vibrio strains Vibrio parahaemolyticus, V. alginolyticus and V.vulnificus and tested it in field and laboratory in the shrimp. They observed that vaccinated group showed better survival than that of the untreated control group.
Pereira (1996) tested killed vaccine against vibriosis in culture shrimp. According to them the vaccine was effective against virulent V. harveyi when challenged 30 days after vaccination. Sung and Song (1996) observed the tissue location of Vibrio antigen in tiger shrimp after challenging by immersion technique. Teunissen et al. (1998) observed the influence of vaccination with polyvalent vaccine prototypes on vibriosis resistance in the giant black tiger shrimp, P. monodon. The authors observed that the vaccinated P.monodon post-larvae showed significant increase in resistance against vibriosis when challenged with a virulent V.alginolyticus strain on 10th, 20th and 30th day after vaccination.