Shrimp Disease – a review
(From V.N. Biju Thesis)
2.1. VIBRIO INFECTION IN CULTURE SHRIMP
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.
2.2. PATHOGENICITY TESTS
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.
2.3.
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)
2.3.1. 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.
2.3.1.1. PHAGOCYTOSIS
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.
2.3.1.2. NODULE FORMATION
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.
2.3.1.3. ENCAPSULATION
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).
2.3.1.4. COAGULATION
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.
2.3.1.5. CYTOTOXICITY
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).
2.3.1.6. 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.
2.3.1.7. 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.
2.3.2. 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.
2.3.2.1. AGGLUTININS
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.
2.3.2.2. KILLING FACTORS
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.
2.3.2.3. CLOTTING FACTOR
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.
2.4. FISH VACCINES
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.
2.5.
SHRIMP VACCINE
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.