Review of Literature - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/10439/9/09_chapter 2.pdf · the exoskeleton of diseased animals, but these can also be caused by environmental

Review of Literature__________

Review of Literature

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Disease is a condition of a living animal that impairs performance of vital

function and can be induced by environmental and nutritional factors or infection

by pathogenic microbes (Gallo, 1991). In the recent years, the shrimp culture

was suffering with prevalence of several endemic diseases (Common in a

particular area or region) particularly with recently introduced L. vannamei and

several emerging diseases in the native popular species, P. monodon. These

emerging diseases could be an endemic and occurring only in this particular

region. Baldock (2002) defined several trans-boundary animal diseases as

epidemic diseases that are highly contagious or transmissible with potential for

very rapid spread irrespective of national boarders and causing serious socio-

economic and possibly public health consequences . Some of the most serious

problems currently faced by the aquaculture sector are caused by those

pathogens and diseases that are spread through movement of hatchery

produced stocks, new species introduced for aquaculture and via ornamental fish

trade (wyban et al., 2010). The spread of pathogens with trans-boundary

movements of live aquatic animals has been clearly associated with the disease

outbreaks and significant losses of aquaculture production and revenue. The

Asian aquatic food production has already been seriously affected by the

outbreak of several diseases. In view of this, it is necessary to take appropriate

practical measure which can minimizes the risk of introduction and spread of

pathogens is of urgent need for sustainable growth of this sector (Humphrey et

al., 1996).


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In the recent days more than 20 viruses have been described in shrimp

culture practices of this area. The white spot syndrome virus (WSSV) has had

the greatest impact on shrimp culture and at present causing the major disease

problem (Rosenberry, 2001). Other important viruses are infectious hypodermal

and haematopoietic necrosis (IHHN) virus, hepatopancreatic parvovirus (HPV),

baculoviral midgut gland necrosis (BMN) virus, baculovirus penaei (BP), yellow

head virus (YHV), monodon baculovirus (MBV), lymphoid organ vacuolization

virus (LOVV) and Taura syndrome virus (TSV) (Lightner, 1996).

Viral diseases are often accompanied by bacterial infestations (Lightner

and Redman, 1998). Only a small number of bacterial species have been

diagnosed as infectious agents in penaeid shrimp. Vibrio spp. Is being the

major bacterial pathogens and can cause severe mortalities, particularly in

hatcheries. Vibriosis is often considered to be a secondary (opportunistic)

infection, which usually occurs when shrimp are weak (Johnson, 1989; Lightner

et al., 1992). Primary pathogens can cause mortality even when other

environmental factors are adequate, whereas opportunistic pathogens normally

present in the natural environment of the host and only can cause mortality when

other physiological or environmental factors are very poor. In practice, the

differences in effects are marginal between primary pathogens and opportunistic

pathogens. As the semi-intensive shrimp culture practice is relatively a new and

emerging one, basic knowledge about the interaction between the pathogens of

cultured shrimp and the reaction of the hosts is still poor (Flegel, 1997), which


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complicates the development of intervention strategies. Therefore, during the

last decade, infectious diseases constitute a main barrier for the development of

shrimp aquaculture, both in terms of product quality and regular supply, thereby

threatening the continuity of the development (Flegel, 1997).

Disease outbreak could be a consequence of complex interaction between

the host, pathogen and environment. As the environment of aquatic animals is

abound with infectious microbes, the transmission of disease in this environment

is relatively easy, especially under dense cultured conditions. Losses due to

disease outbreak, whether by slow continuous attrition or by sudden catastrophic

epizootics, are now being the familiar problems that are to be confront with the

aquaculture sector (Lightner 1996).

3.1 Disease control

Correct diagnosis, including the life cycle and ecology of the pathogen, is

the very critical step in any disease control program. Epidemiological surveys of

viruses are not been performed totally, might be due to the lack of proper

diagnostic methods. However, there was a rapid progress in technological

development, particularly for quick recognition of pathogens in shrimp culture and

diagnostic probes for screening of captured brood stock and their post larvae are

now available for many of the shrimp pathogens (Lightner, 1996). Chemotherapy,

preferably combined with preventive measures, is widely applied now in control

of many infectious diseases in aquaculture. However, this type of chemical


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control should be considered as a last resort because of the growing concern for

food quality, accumulation of such substances in the environment may cause

increase in spread of antibiotic or drug resistant pathogenic strains. In shrimp

culture practices, it is a regular phenomenon that a ‘new’ and often ‘difficult’

pathogens are frequently emerging to replace the existing tackled pathogens i.e.

pathogens of yesterday. Therefore, preventive measures are always necessary

for control of various diseases. Prevention include management of

environmental parameters, use of immunostimulants and manipulation of culture

conditions, gaining importance in aquaculture (soderhall et al., 1992).

Shrimp pathogens are been classified into two categories: (i) those for

which shrimp are the natural host in which they have existed in for, perhaps,

millions of years; and (ii) those that have been introduced to shrimp as a result of

a recent cross-species transmission (walker et al.,2009). Among them, the

major viral pathogens of marine shrimp, WSSV, TSV and IMNV have almost

certainly emerged through cross species transmission. Each of these viral

pathogens appears to have spread from a single focal origin in Asia or South

America and had not been detected in cultured or wild shrimp, prior to their

disease emergence. It was observed that each of these pathogens appears to

represent a single evolving genetic lineage that reflects its focal origin .

However, neither the original sources of infection nor the mechanism of first

exposure have been determined for the prevalence of these important

pathogens. White spot syndrome virus has a very broad susceptible host range


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in decapod crustaceans and is now detected commonly in wild shrimp and crabs

in many locations where shrimp are farmed (wang et al., 2009). However,

occurrence of WSSV in crustaceans outside the shrimp-farming regions is rare

and the virus has also not been detected in in farmed shrimp of Australia (East et

al., 2004). The apparent absence of widespread WSSV infection in wild decapod

crustaceans prior to the emergence of WSD in 1992, despite their susceptibility,

suggests that the original source was a non-decapod invertebrate from which the

virus has ‘crossed’ and adapted to shrimp and other crustaceans. Alternatively,

WSSV may have been introduced to shrimp from a decapod host that is relatively

isolated from other susceptible species and in which disease does not occur

(East et al., 2004). There is an evidence of rapid evolution of WSSV since its

emergence in shrimp and some strains contain large deletions of the DNA

sequence, indicating that these regions of the genome are non-essential for

efficient replication and transmission in shrimp (Marks et al., 2005). The high

rate of evolution and the severity of disease are characteristics often displayed

by infectious agents that have undergone a temporary loss of ecological balance

as a result of cross-species transmission (Steinhauer and Holland 1987).

3.2 Diseases and problems in L. vannamei

o White Spot Syndrome Virus (WSSV)

White spot syndrome first emerged in Fujian Province of China in 1992

(Zhan et al., 1998). Soon after it was reported in Taiwan and Japan and has


24

become panzootic throughout shrimp farming regions of Asia and the Americas

(Walker et al., 2009). It is the most devastating disease of farmed shrimp with

social and economic impacts over 15 years on a scale that is seldom seen, even

for the most important diseases of terrestrial animals. White spot syndrome virus

(WSSV) is a large, enveloped, ovaloid DNA virus with a flagellum-like tail and

helical nucleocapsid that has been classified as the only member of the new

family Nimaviridae, genus Whispovirus (Valk et al., 2005). The 300 kbp viral

genome contains at least 181 ORF, most of which encode polypeptides with no

detectable homology to other known proteins (Van Hulten et al., 2001). Although

first emerged in farmed Kuruma shrimp (Penaeus japonicus), WSSV has a very

broad host range amongst decapod crustaceans (marine and freshwater shrimp,

crabs, lobsters, crayfish, etc.), all of which appear to be susceptible to its

infection (Leu et al., 2009). However, the susceptibility to disease varies from

species to species, some crustacean species have been reported to develop

very high viral loads without any clinical symptoms and almost all the cultured

crustaceans are reported to be highly susceptible to white spot disease, with

mass mortalities (80–100%) in ponds within a period of 3–10 days (Cho et al.,

1995, Lightner et al., 1998). Persistent, low level infections in shrimp and other

crustaceans occur very commonly, sometimes at levels that are not detectable

even by nested PCR. It was reported that the amplification of viral loads and

onset of disease can be induced by environmental or physiological stress (Peng

et al., 1998; Liu et al., 2006) or at ambient temperatures below 300C (Vidal et al.,

2001).


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The susceptibility of all decapods and absence of evidence of replication

in other organisms suggests the virus is of crustacean origin but it remains a

mystery why a virus with such broad host range and ease of transmission was

not long established globally in crustacean populations prior to the advent of

aquaculture (Walker et al., 2009). The origin of the virus may lie in China (Chou

et al., 1995; Tapay et al., 1996), but WSSV was first described in cultured P.

monodon and P. penicillatus in Taiwan in 1993. And WSSV is known to cause

disease in P. japonicus and to experimentally infect larval L. stylirostris (Tapay et

al., 1996). The most obvious sign of WSD is the appearance of white spots in

the exoskeleton of diseased animals, but these can also be caused by

environmental conditions and bacterial infections (Wang et al., 2000).

Pathological changes associated with WSD include development of characteristic

basophilic nuclei and necrosis of subcuticular epithelium and other tissues of

ectodermal and mesodermal origin (Lo et al., 1997). WSD of farmed penaeid

shrimp is characterized by high and rapid mortality but infection of prawns in the

wild is usually sub-clinical and appears to be exacerbated by stress (Lo et al.,

1996).

o Infectious hypodermal and haematopoietic necrosis (IHHNV)

Infectious hypodermal and haematopoietic necrosis was first detected in

Hawaii in 1981, causing mass mortalities in blue shrimp (Penaeus stylirostris)

cultured in super-intensive raceways (Lightner et al., 1983). Infectious


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hypodermal and haematopoietic necrosis virus (IHHNV) is a small, naked,

ssDNA virus that has been classified with several insect viruses in the family

Parvoviridae, Genus Brevidensovirus (Christian et al., 2005). Infectious

Hypodermal and Hematopoietic Necrosis (IHHN) is caused by a non-enveloped

icosahedral virus, Infectious Hypodermal and Hematopoietic Necrosis Virus

(IHHNV), average 22 nm in diameter, with a density of 1.40 g/ml in CsCl,

containing linear ssDNA with an estimated size of 4.1 kb, and a capsid that has

four polypeptides with molecular weights of 74, 47, 39 and 37.5 kD. Because of

these characteristics, IHHNV has been classified as a member of the family

Parvoviridae. Following its initial detection in Hawaii, IHHNV was found to be

widely distributed in both P. stylirostris and P. vannamei shrimp throughout the

farming regions of the Americas and in the wild shrimp population of the Gulf of

California where some reports suggest that it may have contributed to the

collapse of the capture fishery (Morales Covarrubias et al., 1999; Pantoja et al.,

1999). Although it does not cause mortalities in P. vannamei, IHHNV has been

shown to reduce growth up to 30% and cause deformities of the rostrum and

anterior appendages in a condition called ‘‘runt deformity syndrome’’ (Kalagayan

et al.,1991). In Asia, IHHNV is endemic and occurs commonly in P. monodon

shrimp which appears to be the natural host and in which it does not cause

disease and has no impact on growth or fecundity (Chayaburakul, 2005;

Withyachumnarnkul, 2006). Four genotypes of IHHNV have been identified, of

which two have been identified to be integrated into host genomic DNA, and the


27

experimental transmission studies suggest they may not be infectious for P.

monodon or P. vannamei shrimp (Tang et al., 2003a; Tang et al., 2006).

The other two genotypes can be transmitted horizontally by injection,

ingestion or exposure to infected water or vertically from infected females

(Lightner, 1983). Genetic evidence suggests that P. monodon imported from the

Philippines were the source of the epizootic in the Americas, indicating that

disease emergence has been the consequence of an expanded host range

providing opportunities for pathogenicity and a vastly expanded geographic

distribution (Tang et al., 2002). Interestingly, sequence information from various

geographical isolates of IHHNV (a DNA virus) suggests that there are at least

three strains of this virus, although it is not clear if these strains differ in virulence

and host range (Tang and Lightner, 2002).

IHHNV infected shrimp diagnosis histological methods are currently used

for the confirmation of the clinical signs that result from IHHN syndrome.

However, these methods are laborious, time-consuming and not suitable for

detecting low levels of pathogens that occur in acute viral infection. More robust

and sensitive techniques, based on DNA technology, such as polymerase chain

reaction (PCR) and in situ hybridization, have emerged as routine methods for

molecular diagnosis of IHHNV, principally when the aim is to detect chronic

infection or to obtain pathogen free shrimp broods tock (OIE, 2009). It was

previously reported that IHHNV related sequences were detected in the genome

of P. monodon from Africa (Madagascar) and Australia (Tanzania). These IHHNV


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sequences were found to be associated with genomic integrated non infective

type A and B forms of IHHNV (Tang and Lightner, 2006).

IHHNV infection cause acute epizootics and mass mortality (> 90%) in

Penaeus stylirostris. Although vertically infected larvae and early post larvae do

not become diseased, juveniles more than 35 days old appear susceptible

showing gross signs followed by mass mortalities. In horizontally infected

juveniles, the incubation period and severity of the disease appears size and/or

age dependent, with young juveniles always being the most severely affected but

in adult seldom show signs of the disease or mortalities. Litopenaeus vannamei

is a chronic disease, “runt deformity syndrome” (RDS) is caused by IHHNV

infection of P. vannamei. Juveniles with RDS exhibit wide ranges of sizes and

comparatively smaller than the average (“runted”) shrimp. Size variations

typically exceed 30% from the mean size and may reach 90%. Uninfected

populations of juvenile P. vannamei usually show size variations of < 30% of the

mean and comparable RDS signs also observed in cultured P. stylirostris

Anatomical imperfections such as twisted, stunted antennae, truncated or

skewed rostrums missing rostral teeth, convoluted intestinal tracts, asymmetrical

tail segments, swollen cephalothorax and rough cuticle are often symptomatic of

the IHHN virus. These abnormalities can also be caused by genetically originated

malfunctions in the shrimp’s molting processes. An acceptable level of incidence

of physical deformities would be 5% (Henry Clifford et al., 2002).


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o Vibriosis

In shrimp’s bacteria species can cause an array of diseases or problems

ranging from mass mortalities to growth retardation and sporadic mortalities. But

bacterial spp. requires an additional focus and authentication in experimentally

demonstrating the possible involvement during pathogenecity and disease

commencement. The high density of animals in hatchery tanks and ponds is

conducive to spread the bacterial pathogens, and the aquatic environment, with

regular applications of protein rich feed, is ideal for the extensive growth of

bacteria (Moriarty, 1999). Extensive use of antibiotics such as prophylactics in

large quantities, even when pathogens are not evident has lead to an increase in

vibrios, and presumably other bacteria, having multiple antibiotic resistance and

to an increase in more virulent pathogens. Many of the pathogens appear to

have mutated to more virulent forms than were present a decade ago, and thus

even when the shrimps are not stressed by poor water quality they succumb to

attack (Moriarty, 1999). Bacterial diseases, mainly owed to 11 species of Vibrio

have been reported in Asian penaeid shrimp culture systems by Lavilla Pitogo et

al., (1990) and Saulnier et al.,(2000). Hence there is a need to frequent

involvement of Vibrio species as stressors in dissimilar shrimp diseases provoke

special attention in the design of experimental protocols to judge their role in the

diseases (Sung et al., 1999; Saulnier et al., 2000).

Mass transience in cultured tiger shrimps by Vibrio illnesses are

extensively reported subsequent to stress, such as viral infection in specific

(Song et al., 1993; Chou et al., 1995). The Vibrio spp. is one of the most


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important causative driving forces of shrimp diseases such as shell disease,

localized infection and bacterial septicemia (Lightner and Lewis, 1975; Liu and

Chen, 1988), which account for about 80% of the bacteria isolated from sample

of diseased tiger shrimp were reported (Liu and Chen 1988; Song et al., 1993).

In this milieu, bacterial diseases owed largely to Vibrio species are

habitually associated with stumpy survival rates in hatchery or grow out farms.

Infected hepatopancreatic epithelial cells are hypertrophied and contain large

basophilic masses of bacteria in the cytoplasm. Infected cells may appear

cubical, contain little stored lipid and have reduced or no secretory vacuoles

(Lightner, 1996). Infected cells become necrotic, cease to function and evoke a

host inflammatory response, which results in the formation of multiple

glaucomatous lesions in affected hepatopancreas (Lightner, 1993). Larval

mortalities coupled with the existence of V. harveyi have been portrayed in

P. monodon and P. vannamei in Indonesia (Sunaryanto and Mariam, 1986) with

a sickness signs array of localized cuticular lesions, oral and enteric infections

reported in India (Karunasagar et al., 1994), Philippines (Baticados et al., 1990;

Lavilla-Pitogo et al., 1990), Australia (Pizzutto and Hirst, 1995), Taiwan (Song

and Lee, 1993) and Ecuador (Robertson et al., 1998). Early macrobrachium

larval stages are also susceptible to vibriosis caused by Vibrio harveyi. The

unique clinical sign of Vibrio harveyi is the luminescence of infected larvae, which

can be observed at night. Infected larvae also show fouling, opacity, swim slowly,

aggregation and they ultimately die. Mortalities may reach 100%. Sae-oui et al.,

(1987) tested antibiotic sensitivity of V. harveyi found in P. merguiensis and


31

reported that the bacterium was sensitive to chloramphenicol and novobiocin but

resistant to streptromycin. They also found that the bacteria were completely

killed by treating with Ca (HOCl) 2 at 20-30 ppm or formalin at 50 ppm.

In normal conditions the haemolymph and hepatopancreas do not contain

bacteria. The crustacean immune system is formed by strong physical barriers

(thick cuticle) further to cellular and humoral defenses mediated by the

haemolymph (Jiravanichpaisal et al., 2006). When the physical barrier is broken

by an infectious agent, the Immune system is responsible for reducing the

circulating microorganisms (Van De Braak et al., 2002). These microorganisms

attach to other organs such as the hepatopancreas (Sung et al., 1999) and

lymphoid organ (Van De Braak et al., 2002). The reduction in the total haemocyte

count in the vibrio infected shrimp is possibly related to haemocytes migration to

the infected area as assumed by Lorenzon et al., (2002). In addition, the

haemocytes can agglutinate in several layers to capture microorganisms

(Alvarenga et al., 1990) to be removed from the circulation of the gills (Martin et

al., 2000).

2.3 Black and brown gill

Black gill syndrome is also known as black gill disease or burned gills

disease or black spot disease and also called as branchiostegite melanization. It

is a manifestation of a number of disease syndromes including ascorbic acid

deficiency. Penaeid shrimp reared in semi-intensive, intensive, or super-intensive

culture systems, or in systems of poor water quality, often develop some form of


32

gill or surface fouling disease. All, or nearly all, of the organisms involved in gill

and surface fouling disease syndromes are free-living organisms, and are not

true pathogens. Because they use shrimp as a substrate for attachment, these

organisms are called epicommensals or epibionts. For the most part, these

organisms do no direct damage to the shrimp, but instead cause problems

indirectly by attaching to the gill or cuticular surfaces. They may kill shrimp by

either preventing proper water flow over the gills, by interfering with gas

exchange across the gill surfaces, or by interfering with molting, feeding, or

locomotion. Various epicommensal organisms may also produce exotoxins that

may cause different levels of tissue damage. Shrimp from ponds with black flocs

always develop black gills. It has been determined that black flocs have much

higher iron levels than do brown flocs (Rod McNeil, Personal Communication).

Black gills are the result of colonization in the gill tissue of shrimp by the same

bacteria that make up the black flocs. The bacteria deposit iron on the shrimps’

gill lamellae fouling organisms, protozoa and bacteria settle on gill surfaces and

cause inflammation of tissues, which then turn black. The fouling organisms may

become numerous and problematic when shrimp are weak and environmental

conditions not good. Gill fouling causes slow growth and lowered survival of

shrimp (Ilse Silva-Krott et al., 1997).

Some of the fusarium species cause black gill disease in kuruma prawn

Penaeus japonicus (Khoa et al., 2005). Black gill disease due to fusarium

infection also occurred in blacktiger shrimp Penaeus monodon cultured in

Vietnam (Khoa et al., 2004).


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Generally there are multifocal black or brown spots in, or general

discoloration of, the gills, due to melanization at sites of tissue necrosis. Gill

melanization may be visible through the side of the carapace. There is massive

hemocyte accumulation or inflammation, tissue necrosis, and melanin deposition

in affected areas of gills. Secondary infections of bacteria, fungi, and protozoa

may occur.

The presence (or absence) of epicommensals such as filamentous

bacteria, protozoans, or algae on the gills of the shrimp is regarded as a general

indicator of shrimp health. Biofouled or damaged gill lamella can affect the

respiratory and osmoregulatory capacity of the shrimp, and can precipitate

asphyxia in severely affected animals, or generate sub-lethal effects at dissolved

oxygen concentrations that would normally not be deleterious to healthy shrimp.

The most conspicuous symptom of compromised gill function is a change in the

coloration of the gill tissue. Thus, in the sampled shrimp the percentage of shrimp

with darkened or discolored gills should be recorded. Black or brown colored gills

can be caused by 1) biofouling by epicommensals, 2) melanized bacterial

lesions, 3) a melanization reaction to toxins in the water, 4) iron precipitation, or

5) silt or detritus. Discoloration due to detritus or silt accumulation in the gill

lamella can be easily diagnosed by forcefully shaking the shrimp in clean water,

which will generally clean the gills of any unattached debris. A confirmed

diagnosis of the other causes of darkened gills can only be confirmed via

microscopic examination of the gill lamella. If it is confirmed that the discolored


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gills are colonized by fouling organisms such as ciliate protozoans or filamentous

bacteria, this is generally symptomatic of excessive organic matter in the water,

or slow growth (i.e. reduced molting frequency). An acceptable level of incidence

of shrimp with black or brown gills in a random sample from a pond would be 5%

(Henry Clifford et al., 2002).

Very few reaseach reports were observed red-brown mineral deposits in

the gill chamber of Rimicaris exoculata, including mouthparts and

branchiostegites (Gloter et al., 2004, Zbinden et al., 2004).These deposits have

been identified as hydrous iron oxide in the form of ferrihydrite (Gloter et al.,

2004). Because of their close association with the bacterial cell walls, these

minerals have been hypothesised to result from bacterial metabolism,

suggesting, therefore, the presence of chemoautotrophic iron-oxidisers among

the bacterial community (Zbinden et al., 2004). Macroscopically, these iron

oxides deposited in the gill chamber generate different shrimp colours (Zbinden

et al., 2004). For instance, shrimps from the Rainbow vent site exhibit rusty or

red hues, while those from the TAG vent site appear mostly dark or black and

rusty brown (Gebruk et al., 1993, Zbinden et al., 2004). Changes in iron oxide

organization, structure, and abundance are probably responsible for these colour

changes. Severely affected shrimp often die during molting, and thus, will be

found with a "clean" but soft cuticle. Such shrimp may have been feeding and

behaving normally until attempting to molt. Hypoxia due to low morning dissolved

oxygen levels certainly exacerbates the problem.


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Tea brown colour of the gill in Penaeus monodon is caused by Vibrio

harveyi and bacteriophase associated with it (Ruangpan et al., 1998), shrimp

exhibit tea brown pigment at their gills. The only obvious histopathological

changes evident are devastation of the hepatopancreas which contains both

bacteria and phage. It is possible that the tea brawn pigment in the gills is a

coincident symptom, and not the main cause of shrimp death.

Black gill disease is due to the presence of chemical contaminants, heavy

siltation and elevated ammonia or nitrite levels in rearing water. It is also due to

high organic load as a result of residual feed debris and fish fecal matter on the

pond bottom (i.e., black soil). Signs the gills of affected shrimps show reddish or

brownish-to-black discoloration, and atrophy on the tips of the gill filaments. In

advanced cases, most of the gill filaments become totally black and the dorsal

side of the body may be covered with a fog-like substance. Onset of physical

deformities coincides with, or is followed by, loss of appetite and mortalities.

2.4 Body cramp

Cramped tail occasionally, a percentage of shrimp collected with a cast

net will exhibit cramped tail syndrome (CTS). Although the complete etiology of

CTS has not been definitively ascertained, it is thought to be a stress reaction in

susceptible shrimp, caused by one or more of the following conditions: 1) high

temperature stress, 2) vibriosis, 3) mineral imbalances and 4) toxins in the water.

Normal, healthy shrimp should not cramp even when handled. Even though it is

believed that CTS occurs only after handling, it is symptomatic of reduced


36

tolerance to stress, and indicates a fundamental deterioration in the overall

health of the shrimp. An acceptable level of CTS would be 5%, or less (Henry

Clifford et al., 2002)

Body cramp has an uncertain etiology occurred due to imbalance of

nutritional factors such as an in the Ca: Mg ratio and vitamin B deficiency have

been implicated as etiologic agents. Body cramp and muscle necrosis often

occur together, with the striated muscles as the target organ. Body cramp

describes a functional lesion in which the muscle contracts, but is unable to relax

hence, the abdomen or tail remains flexed for an extended period. Partially

cramped shrimp swim with a humped abdomen and fully cramped individuals lie

on their sides at the tank/pond bottom. The condition may result in death or

canabalism of cramped shrimps by unaffected ones. Injured muscle fibers may

turn brown as an outcome of the wound repair process.

Informal observations point out that shrimps exposed to temperatures

above 30oC and those that are abruptly raised from the water with the feeding

trays instantaneously show the condition. Gentle massaging brings back the

original state. This occurs during handling and harvesting on hot days. The body

of cramped shrimp curves and becomes rigid. Mortality is high. The real cause of

this condition is unknown, but mortality is reduced if shrimp are handled during

cool weather (Liao et al., 1977).


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2.5 Blisters and Tail rot

Blister is a thin vesicle, especially on the skin, containing watery matter

and it containing cyanotic gelatinous fluid may develop on the carapace or

abdominal segment. The blister may extend to the underside of the ventrolateral

section of the carapace creating a bulge on the underside. Edema is

characterized by an inflammation of tissue in appendages or extremities of the

shrimp, and frequently Vibrio is the culprit. For example, in the tail it may feel like

a small blister at the extreme end of the uropod, or a swelling of the opercular

membrane that covers the gill chamber. An acceptable incidence of edema in

sampled shrimp would be 5%. (Henry Clifford et al., 2002).

2.5 Low dissolved oxygen

Oxygen is required by shrimp for respiration, the physiological process in

which cells oxidise carbohydrates and release energy needed to metabolise

nutrients from feed. If oxygen s in short supply, the ability of shrimp to metbolise

feed wll be limited, causing growth rates and feed conversion to suffer. Best

growth and feed conversion ratios are obtained when DO levels are maintained

at or above 80% of the saturation level. As a general rule no stress will be placed

upon aquatic organisms including shrimp if DO levels are maintained above

5 ppm. prolonged periods of low oxygen concentration (less than 1.5 ppm) are

lethal, although shrimp can survive for short periods of time with as little as


38

1ppm. If the levels of 3ppm or lower is found preventive measures should be

taken to correct the problem (Peter Van Wyk et al., 2005).

Low dissolved-oxygen concentration is recognized as a major cause of

stress, poor appetite, slow growth, disease susceptibilityand mortality in

aquaculture animals. It is generally accepted that the minimum daily dissolved-

oxygen concentration in pond culture systems is of greatest concern. Dissolved-

oxygen levels can be high during most of a 24-hour period, but the response of

culture species appears to be affected primarily by the lowest dissolved oxygen

concentration during the night. Never the less, accepted minimum dissolved

oxygen criteria for warm water aquaculture species have not been developed

(Boyd, 2010).

The white necrotic areas redden, Similar to the color of cooked shrimp. In

IMNV infection, the major clinical sign is the appearance of whitish, opaque

lesions in the skeletal muscle, and infected shrimp eventually become lethargic.

Infection can cause 40 to 60% mortality in affected ponds, (lightner et al.,

unpublished) Current methods for diagnosis of IMNV infection rely on clinical

signs and histological examination. These methods are, however, not adequate

for diagnosis, since a number of factors (e.g. hypoxia, crowding, or sudden

changes in temperature or salinity) can cause similar signs (Lightner, 1988).


39

2.6 Gas bubble disease

Gas-bubble disease is caused by super saturation of atmospheric gases,

usually nitrogen, but occasionally oxygen (Lightner et al.,., 1974). Nitrogen is

most often responsible, partly because air is 80% nitrogen. Because nitrogen

super saturation above a threshold of only 104% can result in the disease.

Nitrogen is not metabolized (like oxygen is) so its presence in the shrimp's

gills for example causes circulation blockage and hypoxia (Suplee et al., 1976).

Mortality rates may be high up to 100% if corrective measures (vigorous aeration

to release supersaturated gasses), plumbing repairs, etc. are not taken when

condition is first recognized. The disease often occurs in tanks receiving water

with supersaturated gas content. The gas can come from pumps with faulty

plumbing that impinge air through leaks on the intake side of the pump, from

seawater wells where pump cavitation is occurring, from pressurized heated

water tanks, from heated effluents from power plants, etc. Oxygen has

occasionally been found to cause the disease. In systems where oxygen is

injected under pressure to achieve supersaturated levels. When oxygen

produced from photosynthesis by abundant algae is not released by aeration,

wind action, or biological uptake metabolism.

Dissolved oxygen at levels of 250% above saturation was documented to

cause gas-bubble disease. Damage to shrimp from oxygen gas-bubble disease

is mostly physical trauma, and mortality rates may be low in affected shrimp


40

(relative to expected losses in nitrogen gas-bubble disease) if corrective actions

are taken when the disease is first observed.

2.7 White fecal matter

White feces disease exibit clinical sign of long strings of white feces

floating on the water surface. Additional clinical signs of some diseased shrimp

found along the pond edges included loose shells with protozoan epibiont

cuticular body fouling, as well as darker or paler discoloration. Two species of

bacteria, Vibrio alginolyticus and V. parahaemolyticus were isolated from

moribund shrimp. Histopathological examination revealed hemocyte

encapsulation, nodule formation and melanization in the hepatopancreas. White

feces disease outbreaks in this study were determined to have been associated

with deteriorated pond bottom conditions. This was assessed by measuring the

soil redox potential at the feeding area and in the middle of the ponds at 30, 60,

90 and 120 days post-stocking. Redox potential values at the feeding areas of

diseased pond were statistically significantly lower than those in the normal

ponds from day 90 until the shrimp were harvested. Redox potential values from

the diseased ponds indicated that more severe anaerobic conditions in the

feeding area after 60 days led to the increased susceptibility of some shrimp to

bacterial infection and white feces production (Niti Chuchird et al., 2009).

2.8 Protozoan infestation

Epicommensal fouling disease is a condition caused by a variety of agents

and affects all life stages of L. vannamei. It occurs when respiratory; feeding or


41

locomotory functions are impaired by excessive colonization of the cuticle

surface by bacteria, protozoans, diatoms or blue green algae. The infestation

usually involves a mixed population of organisms with one dominant species. In

juveniles and sub adult shrimps the gills are commonly affected, which can inhibit

respiration. Shrimp appears outwardly normal but die rapidly during or

immediately following exercise, handling or exposure to low oxygen conditions.

The symptoms of epicommensal fouling disease include reduced growth, and

reduced feed consumption, gill discoloration, abnormal swimming behavior, and

intolerance to excise of low dissolved oxygen.

The agents for this condition are commonly found in the culture

environment, but they will increase in numbers and cause disease problems

when environmental conditions are not suitable. High densities and high

concentrations of nutrients have been associated with the occurrence of this

condition (Kevan et al., 2002).

2.9 Different pigmentations

Color changes in the appendages and chromatophores of the shrimp

usually are symptomatic of viral or bacterial diseases. Shrimp infected with TSV

or WSSV often display reddish uropods and expanded chromatophores. A pale

pinkish coloration of the muscle is also an early symptom of WSSV. Reddish

pleopods or pereiopods are often associated with TSV or vibriosis. Considering

the seriousness of the diseases that are typically associated with abnormal


42

coloration of appendages and chromatophores, there is no acceptable level of

incidence for these symptoms.

Occasionally, the pleopods and pereiopods of the shrimp will take on a

dark brown, black or rusty coloration. This is usually caused by adherence of

detritus or other substances to the setae of the appendages, and is associated

with poor pond bottom conditions. An acceptable prevalence for this condition

would be 15%, as long as the harvest of the pond is not imminent, in which case

a much lower incidence should be tolerated so as to not substantially diminish

the market value of the harvested product. (Henry Clifford et al., 2002).

2.10 Melanized lesions on the body

Melanized cuticular lesions (MCL) can be caused by: 1) Vibrio sp., 2) TSV,

and 3) physical injury. Although MCL’s caused by Vibrio sp. may be symptomatic

of poor water quality, they are generally not considered to be life threatening to

the shrimp unless the necrotic lesions have penetrated through the exoskeleton

into tissue. Animals in the transitional or chronic phases of TSV will often display

classic TSV-related MCL’s. Regardless of the etiology, MCL’s will affect the

commercial value at harvest of the afflicted shrimp (unless they are marketed in

the peeled form). An acceptable level of incidence of shrimp with melanized

cuticular lesions in a random sample would be 5-10% during the production cycle

and less than 2% prior to harvest (Henry Clifford et al., 2002). Fusarium solani

has been reported as causing black spots on the carapace of M. rosenbergii

cultured in Florida, USA (Burns et al., 1979). In that case F. solani did not affect


43

healthy prawns, but only those which had sustained previous cuticular damage

(Burns et al., 1979).

2.11 Growth rate in Litopenaeus vannamei

In one paddlewheel pond, shrimp grew from 2.3 to 20.9 g between weeks

7 and 16. This is an average growth rate of 2.1 g / week, which exceeds any

previously published growth rates for P. vannamei in earthen ponds (reviewed in

Wyban et al.,1987). In nature, P. vannamei growth rates can approach 1.5

g/week at densities of 2-3 animals/m2 (Menz and Blake, 1980). In South

Carolina P. vannamei Production trials, with continuous paddle wheel use and

stocking densities of 45 postlarvae /m2, mean shrimp size at 14 weeks was about

12 g (Sandifer et al., 1987). Shrimp growth is a discontinuous process regulated

by the moult cycle, which is made up of short moult periods of rapid growth and

of longer intermoult periods when no growth occurs. The duration of the moult

cycle depends on species and size, and it influences themorphology, physiology

and behaviour of these animals (Bureau et al., 2000). Growth depends on sex,

stage and environmental factors such as food quantity and quality, water

temperature and salinity (Dall et al., 1990). Due to the economic importance of

penaeid shrimp worldwide, particularly in aquaculture, a great effort to

understand the growth biology of Penaeus spp. has been made in recent years.

This includes studies on the influence of environmental factors such as

temperature (Wyban et al., 1995; Miao and Tu, 1996; Ye et al., 2003; López-

Martínez et al., 2003), salinity (Lemos et al., 2001) and lunar cycles (Griffith and


44

Wigglesworth, 1993) on shrimp growth. Moulting process results in discontinuous

size increases (Chang, 1992).

Litopenaeus vannamei like all crustaceans is poikilothermic. This means

that they are not able to regulate their body temperature .the shrimps body

temperature will normally be in equilibrium with water temperature. This has

profound consequences for the physiology of the shrimp because the rates of

biochemical processes are temperature dependent. According to van huff’s law,

100 c temperature increases will roughly double the rate of most biochemical

reactions. This means that the temperature of water directly affects the

metabolism of the shrimp.The rate of feed consumption, oxygen consumption;

ammonia excretion and growth are directly related to the metabolic rate of the

shrimp (Peter Van Wyk et al., 2002).

2.12 Diseases and problems in Penaeus monodon

2.12.1 Swollen Hind Gut (SHG)

The SHG was first reported by Lavilla Pitogo et al., (2002) in P. monodon

post larvae. SHG mainly affected the hindgut and to some extent, the posterior

midgut. Postlarvae infected with SHG shows enlargement and distension of the

hindgut folds and its junction with the midgut. In some cases, swollen midgut was

also noticed. SHG causes gradual mortality in affected post larvae, but larvae

shows no abnormal swimming behavior. SHG has direct impacts on the hatchery

and may also affect grow out systems (Lavilla-Pitogo et al., 2002). It has been

suspected that natural food and artificial feed quality, husbandry practices, water


45

quality and presence of toxic substances from chemical prophylactics are

responsible for SHG, but no specific cause has been pinpointed so far (Lavilla-

Pitogo et al., 2002). Direct microscopic examination of PL is a must for the

detection of SHG. SHG can be controlled by good water quality and feed

management. Use of newly hatched batches of brine shrimp should be

maximized to avoid left over on sanitary procedures for Nauplii production and

enrichment should be employed (Dhont et al., 1993).

2.12.2 Calcification

In intensive aquaculture, high-density algal blooms can lead to high water

Ph. The relationship of photosynthesis and respiration to pond pH has been well

documented (Tucker and Boyd, 1985). In some waters, evening pH readings

have been observed which were well into the lethal range for many species.

Water that has been low buffering capacity has an increased probability of

causing fish mortality due to high pH (Boyd, 1982; Durborow, 1986). Boyd (1982)

noted that high pH (i.e. sufficiently high to inhibit growth or induce mortality) is

most frequently found in water, which has its alkalinity anions associated with

sodium or potassium rather than calcium. In water quality testing, this is reflected

by a calcium hardness that is low in comparison to the alkalinity.

Suggested methods of pH control in ponds have included injection

ofcarbon dioxide, addition of acid-forming fertilizers, addition of strong acids, and

reduction of the algal bloom by use of flushing or herbicides (Boyd, 1982). Most

of these alternatives are too expensive, too dangerous, and/or too temporary to


46

be practical. The most commonly accepted alternative is the addition of various

salts in order to develop a good buffering system. Boyd (1979) tested a series of

chemical additives for efficacy in controlling pH. Among those tested were lime,

ammonia fertilizer, aluminum sulfate and calcium sulfate (gypsum). Only calcium

sulfate appeared to provide effective control. It was felt that, by increasing the

hardness-to-alkalinity ratio, pH fluctuations would be moderated and the

maximum daily pH would be reduced. Boyd also stated that 'further research on

reducing pH in ponds is badly needed (Boyd, 1982). In order to address this

need, a series of tests were conducted during the summer of 1987. The purpose

of the tests was to further explore the efficacy of various chemical treatments for

the control of high pond pH.

2.12.3 Calcium and magnesium imbalance

Penaeid shrimp inhabit many aquatic habitats; from marine through

estuarine to fully freshwater environments, and their inherent ability to tolerate a

broad range of salinity variations is key to their survival. Many shrimp producers

are now taking advantage of this distinctive characteristic to rear some species in

oligohaline or low-salinity waters with less than 1 ppt salinity. Hardness is a

measure of the amount of calcium and magnesium in water, in oligohaline

waters, unlike seawater, calcium and magnesium can be limiting. This may result

in animals with soft exoskeletons or stunted growth as a result of molting

difficulties. Concentrations of calcium, magnesium, and potassium are critical

because these ions have specific physiological functions. The other ions –

sodium, chloride, and sulfate contribute to salinity and osmotic pressure. Thus


47

their proportions are probably less important than those of calcium, magnesium,

and potassium Magnesium sulfate often is put in fertilizers as a source of the

secondary nutrients magnesium and sulfate. The most common fertilizer-grade

material is magnesium sulfate heptahydrate, or Epsom salt, which contains about

10% magnesium and 0.39% sulfate. When applied to water at 1 g/m3, the

increase in magnesium concentration will be 0.1 mg/l, and sulfate concentration

will increase by 0.39 mg/l concentration of elements in sea water which is the

natural habitat of marine shrimps (Table 24) shows that the magnesium

concentration is almost three times to the calcium concentration. The ionic

composition of inland well water can vary from suitable to toxic to cultured

animals. Reliable data on concentrations of major cations (calcium, magnesium,

potassium, and sodium) and major anions (bicarbonate, sulfate, and chloride) is

therefore important in the management of waters for inland shrimp farming

(Claude Boyd, 2000).

Shrimp are able to absorb calcium directly from the water, and shrimp

living in seawater do not need calcium supplements in the diet (Davis, 1991).

However, diets for shrimp cultured in near-freshwater systems should contain up

to 2.5% calcium. Higher levels of calcium should be avoided because in high

concentrations calcium appears to interfere with the bioavailability of phosphorus

(Davis, 1990). Phosphorus is required for exoskeleton formation and is an

essential component of phospholipids, nucleic acids, ATP, and many metabolic

intermediates and coenzymes. Davis (1990) demonstrated that the phosphorus


48

requirement for Litopenaeus vannamei was dependent upon the calcium content

of the diet, and that in the absence of calcium, 0.34% phosphorus was sufficient

for normal growth and development. Shrimp diets often contain up to 1% dietary

phosphorus. Unlike calcium, phosphorus is not absorbed in significant quantities

from the water and must be supplied in the feed (Davis, 1991). Calcium and

phosphorus are often added to the diet in the form of dicalcium phosphate.

2.12.4 Loose shell syndrome /white gut and white faecal matter

Nutritional deficiency, pesticide contamination and poor pond water and

soil condition, Exposure of normal hard-shelled shrimps to very low levels of

chemical pesticides such as Aquatin or Gusathion A or to higher levels of

rotenone (10-50 ppm) and saponin (100 ppm) for 4 days resulted in significant

soft-shelling of the stock. Pond surveys also indicated that the occurrence of soft-

shelling could be predicted with 98% accuracy under condition of high soil pH,

low water phosphate, and low organic matter content in the soil. Of the pond that

were surveyed and had softshelling of shrimps, 70% had high soil pH (>6), low

water phosphate (<1 ppm), low organic matter content (< 7%). Insufficient or

infrequent water exchange was highly correlated with soft-shelling. Inadequate

feeding practices like improper storage of feed, use of rancid or low-quality feeds,

and lack of supplementaryfeeding in pond with relative higher stocking densities

were also highly correlated with significantly high incidence of softshelling.


49

2.12.5 Control of gregarines

The apicomplexan Nematopsis rosenbergii has been recorded from

M. rosenbergii in Asia (Shanavas et al.,. 1989). At least three genera of

gregarines (Protozoa, Apicomplexa) infect the penaeid shrimp. These are:

Nematopsis spp., Cephalolobus spp., Paraophioidina spp. Gregarines (Protozoa,

Apicomplexa) are common parasites of many invertebrate groups, especially

arthropods, annelids, and mollusks. In these groups gregarines may occur as

inter- or intracellular parasites, and although individual host cells may be

destroyed by the intracellular stages, most species are not considered to be

highly pathogenic.

In shrimp, ingestion of an infected intermediate host that contains spores

of the gregarine, results in infection. In the shrimp the ingested spores germinate

to become sporozoites which attach to the chitinous lining of the walls and

terminal lappets of the gastric filter, or invade or attach to a midgut or anterior

midgut caecum epithelial cell with their specialized epimerite (holdfast) process.

Once attached, they develop into the trophozoite stage, which consists of a

primite consisting of the epimerite holdfast and a protomerite with a distinctive

centrally located nucleus. Up to three trophonts in syzygy may occur. Trophonts

release from their attachment in the stomach or midgut, become the sporadin

stage of the parasite, and pass to the hindgut where they lodge in the folds of

that organ. In the hindgut each individual cell of the sporadin develops into a

gametocyst, with some cells forming microgametes and others forming

macrogametes. When the gametocysts rupture, gametes intermingle and fuse


50

forming zygotes, which are released into the external environment. Zygospores

are ingested by a bivalve mollusk or an annelid worm like Polydora cirrhosa, a

very common polychaete worm that lives in burrows in shrimp pond bottoms.

The gut of the mollusk or polychaete becomes infected by the gregarine,

and sporogony occurs in the infected epithelial cells. Sporocysts are either

released in the pseudofeces of the mollusca or ingested by a shrimp, or the

infected polychaete is consumed by a shrimp. Sporozoites are released in the

shrimp gastrointestinal tract and these infect the posterior stomach or midgut by

attaching to the cuticle of the posterior stomach or by attaching to or penetrating

the cell membrane of the host cell. Sporozoites develop into trophozoites in the

midgut (Nematopsis spp. and Paraophioidina spp.) or posterior stomach

(Cephalolobus spp.).

Unless heavily infected, gregarine-infected shrimp are very difficult to

discern from uninfected shrimp. However, shrimp with very heavy infections (by

Nematopsis sp.; perhaps with >100 trophozoites per cm of midgut) may show a

yellowish coloration of the midgut, severe lesions of the midgut, and physical

blockage of the lumen by masses of the parasite. Damage to the midgut mucosa

may provide a route of entry for a potentially lethal bacteremia by opportunistic

Vibrio spp, large numbers of this protozoan could interfere with particle filtration

through the hepatopancreatic duct. Infection rate in pond-grown prawns was

reported to reach 94 %.


51

2.13 Immunostimulant properties of Tinospora cordifolia

Plant Tinospora cordifolia is locally called as Gudichi (Sanskrit) means

one who protects the body. It is used as an antioxidant supplement and also

supplement for improving memory, detoxify liver and blood purifier. Gudichi is a

famous plant of traditional use and also a powerful rasayana mentioned in Indian

Ayurveda. It is considered as a bitter tonic and powerful immuno modulator.

Guduchi is very much useful to enhance memory. Guduchi acts as a diuretic and

found to be effective against renal obstruction like calculi and other urinary

disorders. Guduchi acts as a memory booster, develops inteligence, and

promotes mental clarity. It is described as one of the Medhya Rasayana (mental

rejuvenative) in the Charak Samhita (The oldest and most potent book of

Ayurvedic Medicine). Guduchi is regarded as a liver protector. Guduchi is

considered helpful in eye disorders as a tissue builder and promotes mental

clarity. The stem of guduchi is used in general debility, dyspepsia and urinary

diseases. Guduchi is anti-pyretic and act as a tonic after fever, also has action

against alternative fever like Malaria.

2.13.1 Immuno stimulation in shrimp

An immuno-stimulant is a chemical, drug, stressor or action that enhances

the defence mechanisms or immune response (Anderson, 1992), thus rendering

the animal more resistant to diseases. In cases where disease outbreaks are

cyclic and can be predicted, immuno-stimulants may be used in anticipation of

events to elevate the nonspecific defence mechanism, and thus prevent losses


52

from diseases. A vaccine is a compound that induces a specific immune

response against one pathogen. Non-specific immuno-stimulants may be

administered together with a vaccine to activate non-specific defence

mechanisms as well as to enhance a specific immune response (Anderson,

1992). In vertebrates, the principle of vaccination is primarily based on two key

elements of adaptive immunity, namely specificity and memory, which are

mediated by the lymphocytes. The formed memory cells allow the immune

system to mount a much stronger and faster response upon a second encounter

with the antigen. Therefore, this secondary response is more effective than the

primary response.

Unfortunately, invertebrates do not produce lymphocytes and/or specific

antibodies and, accordingly, do not possess an adaptive immune system like

vertebrates do. The invertebrate defence system is often described as based

only on innate immunity, which excludes the possibility of vaccination. However,

defence stimulation in invertebrates is often called ‘vaccination’ too, but this

‘vaccination’ is not equal to vertebrate vaccination, therefore, this term will be

used between quotation marks when used for shrimp. Several reports have been

published about experiments to enhance the invertebrate defence mechanisms

with great potential (Schapiro et al., 1974; Itami and Takahashi, 1991; Sung et

al., 1991; Teunissen et al.,1998; Alabi, 1999; Vici et al., 2000). As a stimulant,

most studies used killed (Vibrio) cells, yeast glucans or derived elements or a

combination of those two components, which are also widely used for fish (Sakai,

1999). Promising tests have also been realised on a small commercial scale


53

(Böhnel et al., 1999). Enhancement of the defence system in the practice of

shrimp culture is most feasible by oral administration.

However, for efficient and effective research on defence stimulation,

practically applicable parameters are needed. These should be based on

scientific data, and they are of major importance to qualify and quantify

stimulation of the defence system. Only haemolymph clotting time and changes

in total haemocyte count are used by shrimp disease diagnosticians

2.13.2 The prophenoloxidase activating system

It has been recognised that defence reactions in many invertebrates are

often accompanied by melanization. In arthropods, melanin synthesis is involved

in the process of sclerotization and wound healing of the cuticle as well as in

defence reactions nodule formation andror encapsulations.against invading

microorganisms entering the hemocoel (Ratcliffe et al., 1985). The enzyme

involved in melanin formation is phenoloxidase (PO), monophenol, L-DOPA and

oxygen oxidoreductase has been detected in the hemolymph blood. PO is a

bifunctional copper containing enzyme, which catalyses both the o-hydroxylation

of monophenols and the oxidation of phenols to quinines (Sugumaran,

1996).Thus, this enzyme is able to convert tyrosine to DOPA, as well as DOPA to

DOPA-quinone, followed by several intermediate steps that lead to the synthesis

of melanin, a brown pigment. PO is the terminal enzyme of the so-called proPO

system, a non-self recognition system present in arthropods and other inverte-

brates (So¨derh¨all et al., 1996). During the formation of melanin, toxic


54

metabolites are formed which have fungistatic activity (Nappi and Vass, 1993 in

the penaeid shrimp; enzymes of the proPO system are localized in the

semigranular and granular cells (Perazzolo and Barracco, 1997).

The clotting mechanism entraps foreign material and prevents loss of

haemolymph. The transglutaminase (TGase)-dependent clotting reaction of

crustaceans is best described in the freshwater crayfish Pacifastacus leniusculus

(Hall et al., 1999). The clotting reaction is induced when TGase is released from

the haemocytes or tissues. The Ca2+ dependent TGase catalyses polymerisation

of the clotting protein, found in the plasma and to form a gel (Yeh et al., 1998).

Haemocytes constitute mainly the first line of internal defence against

invaders (Bachère et al., 1995) and are crucial in the immune reactions of

crustaceans. The cells are capable of phagocytosis, encapsulation, nodule

formation and mediation of cytotoxicity (Chisholm and Smith, 1995). As infectious

diseases and the composition of the ambient water reflect back on the

haemolymph of crustaceans (Ferraris et al., 1986), individual haemograms

related to physiological, environmental or stress parameters might be a

parameter for sensitivity to pathogens and other stress factors. Haemolymph

composition and function is not well understood in P. monodon. However,

studying haemocytes and haemograms in terms of haemocyte types and

numbers, individual and strain variability and microbial activities is a possibility to

monitor shrimp health and should be developed further. Phagocytosis is

believed to be one of the major cellular defence mechanisms in crustaceans. The

semigranular haemocytes are the primary cells involved in the phagocytosis of


55

foreign particles in shrimp (Bodhipaksha and Weeks Perkins, 1994). Granular

haemocytes are also capable of phagocytosis of foreign material but with less

frequency than the smaller ones (Hose and Martin, 1989). Granular cells have

been proven to play a significant role in the shrimp defence system because of

their antibacterial activity (Chisholm and Smith, 1995). The smallest and least

numerous haemocytes are the hyaline cells. They are also considered as

phagocytes (Söderhäll and Cerenius, 1992).

Three main types of circulating haemocytes are usually identified in

crustaceans, i.e. the hyaline (H), the semigranular (SG) and the granular (G)

haemocytes (Söderhäll and Cerenius, 1992). This classification is mainly based

on the number of cytoplasmic granules in the haemocytes and different staining

techniques and, to a much lesser extent, on density, functions and enzyme

distribution. Clearance from the circulation is induced by humoral factors, causing

bacterial aggregation in the circulating haemolymph, which enhances the

clearance rate.

2.13.3 Treatments for iron and manganese deposits

Manganese exists in the aquatic environment in two main forms: Mn (II)

and Mn (IV). Movement between these two forms occurs via oxidation and

reduction reactions that may be abiotic or microbially mediated. The

environmental chemistry of manganese is largely governed by pH and redox

conditions; Mn (II) dominates at lower pH and redox potential, with an increasing

proportion of colloidal manganese oxyhydroxides above pH 5.5 in non-dystrophic


56

waters. Primary chemical factors controlling sedimentary manganese cycling are

the oxygen content of the overlying water, the penetration of oxygen into

thesediments, and benthic organic carbon supply Microbial oxidation of Mn (II) to

Mn (IV) by spores of the marine Bacillus sp.was observed by Bargar et al.,

(2000), whereas Stein et al., (2001) found three freshwater bacterial isolates

capable of manganese oxidation. In the marine environment, manganese can be

taken up and accumulated by organisms during hypoxic releases of dissolved

manganese from manganese-rich sediments. In the field, the high frequency of

shell disease in crabs in a metal-contaminated estuary was ascribed to

manganese toxicity, and the deposition of manganese dioxide on the gills of

lobsters gave rise to a brown or black discoloration of the gills and black

corroded areas on the carapace following hypoxic conditions in the south-east

Kattegat, Sweden (Howe et al., 2005.)

2.14 False positives in MBV- PCR

Penaeus monodon-type baculovirus or Monodon baculovirus (MBV), also

called spherical baculovirus, was first observed in 1977 in adult P. monodon that

were laboratory reared in Mexico from postlarvae imported from Taiwan (Lightner

and Redman, 1981). Monodon baculovirus can cause high mortalities in larvae

(protozoea and mysis) and early postlarval stages (Natividad and Lightner,

1992a). There are varied reports of the effect of MBV on the survival of juvenile

and adult stages. Although MBV was implicated in the collapse of the

P. monodon culture industry in Taiwan in 1987 ⁄ 1988 (Lin, 1989) and as the

cause of mortalities in pond-reared juveniles in Indonesia and Malaysia in the


57

mid-1980s (Nash et al., 1988), concomitant bacterial, parasitic or viral infections

are now thought to be the likely cause of disease (Lightner et al., 1987).

Persistent infections in juvenile and adult stages occur commonly and appear to

be tolerated without apparent signs of disease (Natividad and Lightner, 1992b).

Environmental or other stress has been identified as a significant factor in

disease associated with MBV. Co-infection with other viruses, such as HPV,

IHHNV and WSSV, has also been reported to occur commonly (Natividad et al.,

2006) and multiple infections may result in retarded growth (Flegel et al., 2004).

Monodon baculovirus is a large, bacilliform dsDNA virus that is classified

as the tentative species Penaeus monodon nuclear polyhedrosis virus

(PemoNPV) in the genus Nucleopolyhedrovirus of the family Baculoviridae. In the

late stages of infection, virions in the nucleus are observed within spherical

inclusion bodies comprising a paracrystalline network of small polyhedrin

subunits, each 20 nm in diameter (Mari et al., 1993). The spherical shape of

these inclusion bodies is the origin of the alternative common name of MBV (i.e.

spherical baculovirus), although by scanning electorn microscopy they appear

polyhedral (Flegel, 2006).

Monodon baculovirus can be transmitted by co-habitation, feeding or

exposure to homogenates of infected tissue. All life stages, except eggs and

nauplii, are susceptible to MBV infection. Infection occurs in the epithelial cells of

the hepatopancreatic tubules and anterior midgut from which virus particles and

inclusion bodies are released to enter the intestinal tract via the hepatopancreatic

lumen. As for other baculo viruses, faecal oral transmission is the most likely


58

route of infection, with polyhedron providing environmental protection of virus

released in inclusion bodies. Monodon baculovirus can be transmitted from

broodstock to progeny, but there is no evidence of transovarial transmission and

the evidence indicates that infection occurs by faecal contamination of eggs

during spawning. Good husbandry practices, including washing of fertilized eggs

or Nauplii with filtered seawater, formalin and iodophores, can be effective in

breaking the transmission cycle (Chen et al., 1992).

Documents

Review of Literature - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/10439/9/09_chapter 2.pdf · the exoskeleton of diseased animals, but these can also be caused by environmental