HISTOPATHOLOGICAL SURVEY OF CULTURED SHRIMPS IN MODIFIED
EXTENSIVE SYSTEMS OF COCHIN, KERALA
D IS S E R T A T IO N S U B M IT T E D
IN P A R T IA L F U L F IL M E N T O F T H E R E Q U IR E M E N TS FOR T H E D E G R E E OF
M ASTER OF FISH ERIES SCIENCE (MARICULTURE)
O F TH E
C E N T R A L IN S T IT U T E O F FISH ERIES E D U C A TIO N (D E E M E D U N IVER SITY)
M U M B A M O O 061
by
LIYA AMBJPIUAJ (Mari. 50)
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CENTRAL MARINE FISH ER IES RESEARCH INSTITUTE
INDIAN COUNCIL OF AGRICULTURAL RESEARCH C O C H IN -682 014
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JUNE 2001
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Dated: 30 June 2001
CERTIFICATE
C ertified tha t th e thesis entitled
H IS T O P A T H O L O G IC A L S U R V E Y OF C U LT U R E D S H R IM P S IN M O D IF IE D E X T E N S IV E S Y S T E M S OF COCHIN, K E R A L A ” is a record o f in d e p e n d e n t bonafide research w ork carried out by Ms. Liya Am bipillai during the period o f study from S e ptem be r 1999 to August 2001 under our supervision and guidance fo r th e degree o f M aster of Fisheries S c ie n c e (M ariculture) and that the thesis has not previously form ed th e ba sis fo r the aw ard o f any degree, diplom a, associateship, fellow sh ip or any other sim ilar title.
(K. C. George) Sr. Scientist PNP Division CMFRI
M ajo r Advisor/C hairm an
Advisory comm ittee
(K. S. Sobhana) Scientist PNP Division
CMFRI
(N. K. Sanil) Scientist PNP Division CMFRI
DECLARATION
I hereby declare that the dissertation entitled
" H IS T O P A T H O L O G IC A L S U R V E Y O F C U L T U R E D S H R IM P S IN M O D IF IE D E X T E N S IV E S Y S T E M S O F C O C H IN , K E R A L A ” is an authentic record of the work done by me and that no part thereof has been presented for the award o f any degree, diploma, associateship, fellowship or any other similar title.
30*'' June 2001 Cochin
Liya Am bipiliai
M.F.Sc, Student C entral Marine Fisheries Research InstituteACKNOWLEDGEMENT
1 e xp re ss my deep sense of gratitude and indebtedness to my supervising guide, Dr. (Mrs.) K. S. Sobhana, Scientist, CMFRI, Cochin fo r the v a lu a b le guidance, unstinted support, constant encouragem ent and fo r the va luable suggestions through out the period of my study as well as in the preparation of th e m anuscript. I am greatly indebted to the members of the advisory com m ittee, Dr, K. C. George, Sr. Scientist and Shri. N. K. Sanil, Scientist, CMFRI fo r th eir guidance, supervision and encouragem ent through out the work.
I am grateful to th e Director, CMFRI, Cochin for providing all the facilities to carry o u t th e study.
I wish to record m y thanks to Dr. R. Paul Raj, O fficer in charge, PGPM for the tim ely help and encouragem ent throughout the M .F.Sc course.
I take this opportunity to express my sincere thanks to Dr. C. V. Mohan, Associate Professor, College o f fisheries, Mangalore fo r the critical and valuable suggestions.
I also express my gratitude to Dr. K. Sunil Kumar Mohamed and Dr. P.
R adhakrishnan N air fo r the tim ely help.
1 express m y sincere gratitude and thanks to Mr. John, TTC, fo r the help rendered during collection of sam ples,
I am thankful to Ms. Nisha and Ms. Radhika fo r the help and cooperation rendered to m e during the course of m y work.
The help rendered b y Mr Anilkumar, Mr Surendran and Mr Nandakumar.
technical assistants throughout th e period of study is greatfully acknowledged, I express m y sincere thanks to all farm ers for the kind cooperation, interest and help extended to m e at the farm sites
! take this o p p o rtu n ity to express my thanks to all my classm ates, seniors and juniors fo r the help, cooperation, suggestions and support.
The award of a Ju n io r Research Fellowship by the Indian council of Agricultural research. N ew Delhi throughout the M.F.Sc. program m e is greatfully acknow ledged.
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ABSTRACT
A disease survey w as conducted using histopathologicai tools in a cross section o f shrim p farm ing area in Cochin during March to June, 2001.
S hrim p sam ples collected from 26 farm s comprised of Penaeus indicus, P e n ae u s m onodon. and M etapenaeus dobsoni. The m ajor pathological condition recorded w as the presence o f chronic inflammatory (esions characteristic of system ic bacterial infection and w as obsen/ed in 42% of the farms sun/eyed. In 19% of the fa rm s hepatopancreatic pathology, typical of oral/enteric vibriosis was recorded. M ortality o f P .m onodon due to mixed infection of IVIBV and vibriosis w as recorded in one farm . Fouling of shrimps by th e ciliate protozoan, Zootham nium could be detected only in 8% of the cases. Abnorm al conditions such as sp o ntan e o us m uscle necrosis, dark coloured gills and brown discolouration of the shell were recorded in 36% of the farm s. Absence o f white s p o t viral inclusions in any of the target tissues of the sam ples examined was one of the m ost im portant observations during th e study. No apparent pathological conditions w ere recorded in 27% of the fa rm s surveyed.
CONTENTS
Page no
IN T R O D U C T IO N 1
R E V IE W OF LITER A TU R E 4
M A TER IA LS AN D M ETH O D S 19
R ESU LTS 21
DISCUSSION 34
S U M M A R Y 45
R E F E R E N C E S 46
Page No.
T a b ie 1 ; S um m ary of the w ater quality parameters 25 recorded in the farm s surveyed during
March - June, 2001.
Table 2 : S um m ary o f the major disease/ histopathological 26 fe a tu re s identified in the shrim p samples collected
during the sun/ey.
Page No.
Plate 1 : S pontane ous m uscle necrosis in P.indicus 29 Plate 2 : H istological section showing haem ocytic nodules 29
in the gills.
P late 3 ; H aem ocytic nodules in the connective tissue. 29 P late 4 to 7 : H istological s e c tio i, show ing pathology of 30
hepa topancreas due to bacterial infection.
P la te s : H istological section of hepatopancreas from 30 norm al shrimp.
Plate 9 T u b u la r epithelial cells in hepatopancreas from 31 n o rm a l shrimp,
P late 10 ; EosinophiJic, intranuclear occlusion bodies 31 characteristic of M B V in the hepatopancreas
P l a t e n : H epatopancreas pathology showing necrosis 31 and vacuolation
Plate 12 : M idgut section from norm al shrimp. 31 P late 13 ; C uticular epiderm is o f norm al shrim p w ith underlying 32
connective tissue.
P late 14 ; Section of gill from norm al shrimp. 32 Plate 15 : H istological section of lym phoid organ from 32
norm al shrimp.
Plate 16 ; H aem atopoietic tissue from normal shrimp. 32 Plate 17 : H aem atopoietic tissue around the lateral arterial 33
vessel, from norm al shrimp,
Plate 18 ; H istlological section o f ventral nerve cord from 33 norm a] shrimp,
Plate 19 ; Z o o tham nium infestation in gill of P.indicus. 33 Plate 20 ; Z o o th a m niu m infestation on cuticular surface 33
o f P.indicus.
INTRODUCTION
1. INTRODUCTION
T he farm ing of penaeid shrimp is practised in several countries around th e world. But th e industry is concentrated m ainly in the S outheast A sian and the Latin A m erican countries. The production is dominated by the tw o species, th e black tiger shrim p {Penaeus monodon) in Indo-pacific region and th e pacific white leg shrim p {Litopenaeus vannamei) in the W estern hem isphere. In th e ye a r 1998, global production of farm ed marine shrim p was 7,48,460 mt, w ith th e Asian continent contributing about 71% of the w orld’s shrim p production through aquaculture (Aquaculture magazine, 1999).
India has got vast potential for developm ent of commercial shrimp farm ing, with a coastal brackishw ater area of 1.2 million ha. Traditional shrimp trapping syste m s have been in practice fo r several years in the pokkali prawn filtration fields o f Kerala, bheries of W e s t Bengal, gasani lands of Karnataka and khar lands of Goa. Autostocking aided by tidal action w as m ostly being practised with m inim al m anagerial inputs. Later, improved practices like pond preparation, screening the w ate r source and stocking with desired species were introduced.
Subsequently, m ore intensive culture practices evolved in response to evolution of scientific fa rm in g technologies, ea sy availability of hatchery raised seed in adequate quantities and also due to th e high profitability and increasing demand fo r shrim ps in th e export market.
T h e re w as a rapid expansion of the industry between 1988 and 1994. R ealising th e enorm ous potential fo r investment opportunities and taking advantage of th e liberalisation policies of the government, several national and Internationa! corporate houses, private com panies and individual entrepreneurs ventured into th e field. About 0.18 m illion ha. has been brought under shrimp culture until 1995 - ’96 (Karunasagar and Karunasagar, 1999).
W ith the expansion of the industry, aquaculture waste m a n a g e m en t becam e a very serious issue, Majority of the farms were forced to use intake w a te r source fo r waste discharge. Raw effluents rich in organic matter and w aste fe e d w ere released directly into the water source w ithout any
treatm en t or se ttlem ent. This has led to disease problem s connected with bad w ater quality, w here iittle attention only was being given to pond drying, disinfection and w aste rem oval between crops (Mohan, 1996). The unorganised and unregulated expansion w ith iack of scientific consideration resulted in the o u tb rea k o f d ise a se s and the industry collapsed between 1994 and ’96. During 19 9 4 -’95 a lo n e th e loss was estim ated at 10,000 - 12,000 tons valued at Rs.
250 - 350 billion (K arunasagar and Karunasagar, 1999).
W o rld over, the growth of shrim p culture industry was accom panied w ith increased incide nce of various typ es of diseases. Viral diseases in particular have severely im pacted m any leading shrim p farm ing nations o f the world causing sign ifica nt production and econom ic losses. Am ong the shrim p viruses recorded, th e w h ite spot virus is the m ost serious pathogen. Since 1993, this disease has b e en causing significant production losses in cultured shrim ps all over S outheast and South Asia. T his epizootic probably began in China in 1993 and subsequently spread from there to Japan, Taiw an and the rest of the A sia as fa r as India (Flegel and Alday-Sanz, 1998). In India this epidem ic first erupted in A ndhra Pradesh, in O ctober 1994, and caused m ass m ortalities of Peneaus m o nodon and P e n ea u s indicus. In N ovem ber - D ecem ber 1994, heavy shrimp m ortality reaching up to 100% w as noticed in shrim p culture system s of Nellore and E ast G o d a vari districts (M ohan and Shankar, 1995). The disease then spread along th e entire East coast and also in th e W e s t coast (Krishna et al., 1997),
D iseases are usually related to the type and m anagem ent of the culture system s, w hich influence the health and environm ent of the shrim ps being cultured. D isease outbreaks occur, when a susceptible host com es in contact with a p a thogenic organism in a stressful environm ent. Many diseases in cultured sh rim p result from a com bination of environm ental and infectious com ponents. T h e principal agents causing diseases are viruses such as W hite spot virus (W S V ), Y ellow head virus (YHV), M onodon baculovirus (MBV), H ep a to p a ncre a tic parvo-like virus (HPV) etc.. bacteria like vibrios and filam e n to u s bacteria, fungi, rickettsia and parasites like m icrosporidians and gregarines (L/ghtner, 1996).
M any a tim es, outbreak of the disease m ay be the sole factor limiting the success of th e culture. Often these outbreaks can be avoided by proper m a na g e m en t m easures. Rapid detection of a disease condition before reaching th e epizootic level will help to contain it by appropriate m anagem ent strategies. A nalysing the disease history of an area will also be helpful in planning proper m anagem ent m easures. In order to form ulate useful control strategies, it is essential to identify the diseases prevalent in an area. Knowledge o f current disease problem s is also essential if the changing pattern of disease associated with w idespread intensification is to be monitored and controlled (Tumbull e ta l., 1994).
C rop losses due to disease outbreaks have been reported at several fa rm s in Cochin area during the past fe w years. However detailed stu dies regarding diseases affecting farm ed shrim p along the brackish water fa rm s in this a rea are limited, Histopathology is an ideal tool for routine health m onitoring and fo r routine diagnosis, where the changes at the cellular and tissue JeveJ due to the pathogen is interpreted to arrive at diagnosis. The present histopathological survey on cultured shrim ps in Cochin area was undertaken with th e follow ing objectives ;
1) T o identify th e occurrence of disease conditions and pathogens involved, in a cross section of shrim p farm ing area at a given period during peak culture season and also to study
2} the tissue level pathological changes caused and host responses in different d ise a se conditions
3) the relationship between case history, clinical signs and pathological conditions
4) the occurrence of disease problem s in relation to environment.
REVIEW OF
LITERATURE
2. REVIEW OF LITERATURE
In less than 30 years, the penaeid shrim p culture industries o f the world developed from their experim ental beginnings into major industries providing im m e n se job opportunities, billions of U.S dollars in revenue and augm entation o f th e w orld’s food supply with a high value crop. Concomitant with the growth o f shrim p culture industry has been the recognition of the ever increasing im portan ce of disease, especially those caused by infectious agents.
C onventional m ethods fo r th e disease diagnosis include traditional m ethods of m o rphological pathology (light microscopy, histopathology, electron m icroscopy), tra d itio n a l m icrobiology and the application of serological methods (Lightner and Redm an, 1998). The industry n o w has modern diagnostic genom ic p robes with non radioactive labels fo r viral pathogens like IHHNV (Infectious hypoderm al and hem atopoietic necrosis virus), HPV (H epatopancreatic parvo-lfke virus), T S V (Taura syndrom e virus), W S V (White spot virus), M B V (Monodon baculovirus) and BP (Baculovirus penaei), for bacterial pathogens like NHP (Necrotising hepatopancreatitis) and certain Vibrio spp. and also fo r m icrosporidia (Lightner and Redman, 1998; Karunasagar and
Karunasagar, 1999).
D isease investigations in shrim ps have gone much ahead of other crustaceans, obviously due to th eir econom ic value and high demand world over.
D iseases in sh rim p s have been review ed a num ber o f tim es by various authors (O verstreet, 1973; 1983; Lightner, 1977; 1983; Couch, 1978; 1983; Johnson, 1978; Johnson, 1983; Otta ef a/., 1998). The most significant pathogens th a t are co m m only a ssociate d with cultured shrim ps belong to viruses, bacteria, fungi and parasites. A p a rt from these, non-infectious diseases like those caused by epicom m ensals, environm ental stress factors, nutritional deficiencies and toxic factors are afso reported to cause m ortalities in shrim p ponds.
2 .1 .0 Viral d is ea se s
A m o n g th e diseases of intensively cultured shrimp, viruses are the m ost im porta nt pathogens, characterised by high infection rates and ability to
cause high m ortalities. V iruses spread from one host to another through horizontal and vertical transm ission. Infection and disease developm ent In the host d epends on viral load in the environnnent (infectious dose). More often anim als surviving infection becom e assymptomatic carriers. Viruses being obligatory pathogens, cell culture is essential fo r their study and propagation, But at present shrim p virus infection is m ostly detected by histopathology, electron m icroscopy and PCR,
Viral aetiology is being attributed to nearly 20 diseases in crustaceans (Lightner, 1983) and fourteen o f these viruses have been reported fo rp e n a e id sh rim p s {O tta et al., 1998), Based on the type of nucleic acid, shrimp viruses can be b ro a d ly grouped as R NA and DNA viruses.
2.1.1 W h ite s p o t syn d ro m e virus
M ass mortalities due to white spot virus had occurred in the cultured kurum a shrim p. P enaeus japonicus, in Japan and Penaeus monodon in Taiwan during 1992 (fnouye e t a i, 1994; Nakano e ta l., 1994; Chen, 1995) and the virus caused a w ide spread destruction of shrim p culture in Asia (Fiegel.
1996). Disease w as characterised by th e presence o f w hite spots on th e inside surface of the carapace and w as suggested to be caused by a ds-DNA virus (Chou e ta l., 1995). Later it w as nam ed as W S D V (Chen, 1995). Similar disease, variously called as SEMBV, system ic ectodermal and m esoderm al baculovirus w a s reported in Thailand (W ongteerasupaya e t al., 1995) and in China as HHNBV, hypoderm al and haem atopoietic necrosis baculovirus (Cai etal., 1995), P A V (Penaeid acute virem ia) reported to cause m ass m ortality in farm raised kurum a shrim p s in Japan {N akano e t al., 1994; Takahashi e t al., 1994; Inouye et al., 1996) w as also a sim ilar disease. The causative agent being designated as RV-PJ, rod shaped nuclear virus of P enaeus japonicus (Inouye etal., 1994) and later as rod shaped D N A virus (PRDV) (Inouye etal., 1996). The clinical signs of w hite spot disea se included m any w hite spots on the inside surface of the carapace, in severe cases of infection, spots were also seen on the appendages.
A n abnorm al red colouration or discolouration was usually seen in the diseased shrim p. W S V affected shrim ps w ere found lethargic and inappetant. The white spots w ere representing a protrusion on th e inside surface o f the carapace and
its com position w as sim ilar to that of cuticle (Wang et a i, 1997). H istopathology dem onstrated d e ge n e ra te d cells characterised by basophilic inclusion bodies in hypertrophied nuclei in various tissues including cuticular epidermis, gills, connective tissue, haem ocytes, haem opoietic tissue, lym phoid organ, cuticular epithelium o f fo re g u t and hindgut, heart, antennal gland, nervous tissue, striated m uscle (M om oyam a e t a i, 1997; W ang e t al., 1997), The disease shows a wide h o st range (W ongteerasupaya et al., 1995; Otta et al., 1998), The environm ental stressors such as am m onia may enhance the severity o f white spot disease virus infection in cultured shrim p (W ang e ta l., 1997),
2.1.2 B a c u lo v iru s penaei (BP)
B aculovirus penaei, w as the first virus pathogen to be reported from penaeids. It w as reported from the pink shrim p {Penaeus duorarum) by C ouch (1974), Infection was by horizontal transm ission to all life stages.
Epizootics w ere chronic to acute with high cum ulative m ortality but presence of virus did not alw ays result in disease. Feeding and grow th rates reduced; gill and surface fouling increased with the occurrence of the disease. The target organs o f BP virus w ere th e epithelial cells o f hepatopancreas and anterior m idgut giand of larval and a d u lt stages of several species of penaeid shrimps and caused m ass m ortalities in larval stages (Couch, 1974; Lightner, 1983), Single or m ultiple e osin o ph ilic intranuclear polyhedral inclusion bodies .were seen in the affected cells (Couch, 1978; Lightner, 1983), It w as reported from species like P.
duorarum, P. aztecus, P. setiferus, P.vannamei, P. stylirostris, P. marginatus, P.pencillatus, P .schm itti and P. subtilis over a wide geographic range (Couch, 1974; Brock e t al., 1986a; O verstreet e t al., 1988; Lightner et al., 1989;
S ergioluiz e ta l.. 1990; Leblanc eta!., 1991),
2 .1 .3 M o n o d o n b aculovirus (M BV)
M onodon baculovirus w as named so because of its first detection from P e n ae u s nnonodon by Lightner and Redman (1981) in Taiwan. From 1987, serious m ortalities up to 35-70% of the cultured populations of P enaeus monodon, occurred in Taiwan due to this virus (Rosenberry, 1988). The ap pa re nt ta rg e t o rgans and tissues fo r M B V were the hepatopancreatic tubule
and duct epithelium of postlarvae, juveniles and adults and the anterior midgut epithelium o f very young postlarvae (Lightner et al., 1983a). MBV occlusion bodies appeared as prom inent eosinophilic, single to multiple round bodies within the hypertrophied nuclei o f hepatopancreatic tubule or m idgut epithelial cells. A ffected shrim ps showed lethargy, anorexia, dark colouration and heavy surface fouling. A cu te M BV caused loss o f hepatopancreatic tubule and midgut epithelium and consequently, dysfunction of these organs, often follow ed by se co nd a ry bacterial infections (B ow er e t al., 1994). Environmental stressors such as crowding and biological stressors, including the effect of other p athog ens have been dem onstrated to enhance th e severity o f M BV infection in P enaeus m o nodon (Chen e ta l., 1989; Fegan etal., 1991).
2.1 .4 In fe c tio u s hypoderm al and haem atopoietic necrosis virus (IH H N V )
Infectious hypoderm al and haem atopoetlc necrosis virus, a parvovirus, causative agent fo r a n othe r viral disease in shrimps was first reported from P. stylirostris (Lightner e t al., 1983b). Affected shrim ps showed reduced food consum ption, cannibalism and increased mortality. Shrimp repeatedly rose slow ly to surface, rolled over and sank to the bottom. Mottled colouration w as a diagnostic feature. Disease w as particularly severe in high d ensity culture. T he histopathoiogy o f acute and subacute IHHNV was dom inated by conspicuous eosinophilic intranuclear inclusion bodies of the cow dry type A variety in ectoderm ally and m esoderm aliy derived tissues that w ere undergoing necrosis (Lightner e t al., 1983b), The inclusion bodies were com m on in a cu te infections, later decreasing in num b er and followed by necrosis and inflam m ation of target tissue. Affected cells also had highly vacuolated cytoplasm w ith cytoplasm ic bodies th a t range from eosinophilic to basophilic (Bower e t al., 1994). Distinctive histopathological lesion patterns were observed betw een sp e cie s (Bell and Lightner, 1984) and within the sam e species the disease prevalence as num ber of inclusion bodies decreased with increase in size (Bell a n d Lightner, 1987). S im ilar pattern in disease prevalence w as found in the case o f BP in P. vannam ei (Leblanc and Overstreet, 1990) and M BV in P e n ae u s m o n o d o n (Fegan e t a/., 1991), RDS (Runt deform ity syndrom e),
characterised by reduced growth and deformities of the rostrum, anterior appendages or other parts of shrim ps, a frequent disease problem of cultured Penaeus va n na m e i w as found to be caused by IHHN virus (Kalagayan e t a!., 1991).
2.1 .5 H e p a to p a n c re a tic parvo-like virus (HPV)
A second parvovirus, found to affect shrim ps is the hepatopancreatic parvo-like virus. It w as reported from P. merguiensis and P.
sem isulcatus by Lightner and Redm an (1985). It was found to affect other species like P. chinensis, P. esculentus, P. monodon, P. indicus, P. penicillatus a n d P. vannam ei. A ffected shrim ps showed poor growth rate, anorexia, reduced preening activity, increased surface fouling and occasional opacity of tail m usculature. S igns w ere accom panied by morta(ities during juvenile stages after apparently n orm al developm ent through larval and post larval stages.
H istopathology show ed necrosis and atrophy of the hepatopancreas, typically of m id-juvenile stages. Single prom inent basophilic spherical intranuclear inclusion bodies were present in the affected epithelial cells of hepatopancreatic tubules and epigastric caecal epithelial cells with lateral displacem ent and compression of the host cell nucleolus and chrom atin margination o f the nucleus. Early in their developm ent, H P V inclusions w ere seen as small eosinophilic bodies centrally located within th e nucleus and closely associated with the nucleolus (Bower et a!., 1994).
2 .1 .6 B acu loviral m idgut gland necrosis virus (BMNV)
B aculoviral m idgut gland necrosis virus (BMNV) disease was first reported by Sano e t al. (1981). T he disease was also reported as m idgut gland cloudy disease, w hite turbid fiver disease and white turbidity disease. Disease w as characterised by sudden onset of white turbid midgut gland in larvae and postlarve with associated high mortality. Larvae float inactively on the surface and exhibit a w hite m idgut line through the abdomen (Bower etal., 1994). The virus belong to baculoviridae do not form occlusion bodies. Midgut and intestine w ere the ta rg e t organs. Diagnosed by greatly hypertrophied nucleus within hepatopancreatic epitheJial cells th a t were undergoing necrosis (Sano et a/.,
1981). Latently infected spawners and cultured animals were suggested to be the vertical and horizontal sources o f infection respectively (Momoyama, 1988).
BMNV generally infected lan/ae and early post larval stages causing high mortalities.
2 .1 .7 R e o -lik e v iru s (REO)
A non-enveioped, paraspherical virus, affecting P. japonicus was reported by T sing and Bonam i (1987). it belong to Reoviridae group and w as a do ub le stranded R NA virus. T he external signs of this viral disease involved behavioral change (the shrim ps did not hide them selves in the sand) and color change (the te lso n, uropods and hepatopancreas becam e reddish). Affected shrim ps sho w e d p o o r feeding, growth, anorexia, lethargy, reduced preening and increased su rfa ce fouling. G ut and nen/e syndrome (GNS), an idiopathic condition found in chronically ill populations of P. japonicus cultured in Hawaii w as suspected to be caused by this virus. Occasionally eosinophilic cytoplasm ic inclusions w ere seen in hepatopancreatocytes (Bower eta l.. 1994).
2.1.8 R h ab d o vlru s of penaeid s h rim p (RPS)
R habdovirus of penaeid shrim p w as the first reported rhabdovirus to be isolated from cultured penaeid shrim p populations (Lu e ta l., 1991), RPS w as found to infe ct P.stylirosths with th e viral infection being localised in the lymphoid organ (Oka organ) and resulting in cellular alteration and necrosis (Nadala e ta l., 1992).
2 .1 .9 Y e llo w liead v iru s (YHV)
T h e ye llow head disease of the black tiger shrimp, in Thailand since 1990, w as reported to be caused by a baculo-like virus (Boonyaratpalin et a i, 1993). A ffected shrim p had pale body and light yellow colouration o f the hepatopancreas and gills. The virus particles obsen/ed in necrotic tissues of the gills and lym phoid organ were bacilliform in shape (Boonyaratpalin e ta l., 1993).
Viral particles w ere found In the cytoplasm of the infected cells and were contained within th e cytoplasm ic vacuoles (Lu e t a l, 1994). Gross observations showed an increased feeding initially, follow ed by a reduction in later stages of
th e disease (Bower e t a i, 1994). The disease w as found to be an acute and lethal one. M assive system ic necrosis were seen in numerous tissues, apparently of ectoderm al and m esoderm al in origin, of the affected shrim ps and nearly spherical basophilic cytoplasm ic inclusions were present in those cells (C hantanachookin eta!., 1993; Bower eta L , 1994).
2 .1 .1 0 L ym p h o id O rgan vacuolisation virus ( L O W )
Lym phoid Organ vacuolisation virus also known as the Lymphoid organ parvo-like virus, LOPV, is a togavirus, LOPV showed a similar histopathology as those reported from th e cultured penaeid shrimps in Australia and Taiwan by a parvo-like virus (Bonami e t ai., 1992). In histology the cytoplasm o f th e lym phoid organ cells were with highly vacuolated and intracytoplasm ic inclusion bodies, th a t ranged in characteristic from eosinophilic to basophilic (Lightner e ta L . 1992),
2.1.11 T au ra S yndrom e v iru s (TSV)
First noticed in Penaeid shrim p farm s located near the mouth of Taura River in Ecuador (Jimenez, 1992). Taura syndrom e virus (TSV) had been th e causative agent of econom ically disastrous epizootics in P. vannamei, causing m ass m ortalities of 40-95% in affected post larval and juvenile populations (Lightner e t a!.. 1995). T S V caused three distinguishable phases in infected shrim ps, the peracute and acute phase, characterised by an overall pale reddish coloration and a recovery phase characterised by multifocal, melanised cuticular lesions (Lightner, 1996).
2.2 .0 B acterial d is ea se s
M ortalities due to bacterial diseases w ere observed in all life stages, (an/ae, juveniles and adults o f penaeids (LavlKa-Pitogo, 1995). Most of th e bacterial disease outbreaks w ere caused by species of Vibrio, Pseudomonas and A erom onas (Lightner, 1977).
Bacterial diseases had been limiting factors in penaeid culture system s, th e ir effects becom ing directly proportional to the growth of the industry
in term s of severity and impact. Although eight genera had been reported to be associated with th e se problems, only two groups occur quite commonly, the filam entous bacteria and vibrios, with the later being m ore important (Lightner, 1988). Though m any \/(ibr/o species had been reported in penaeids, V. vulnificus, V. parahaem oliticus, and V. harveyi, were the m ost im portant species. Bacterial infections in shrim p to o k two form s: Localised pits in the cuticle (shell disease) or localised infections w ithin the body and generalised septicem ia (Lightner, 1983).
Dfagnosis is mostly done by conventional bacteriology and histopathology. More rapid detection methods like indirect fluorescent antibody technique and other enzyme immunoassays are being developed to improve monftoring and surveillance.
2.2.1 V ib rio s is
V ibriosis, the disease condition associated with the genus vibrio, w as found to cause m ass m ortality in shrimp (Linsuwan, 1988; Karunasagar et a!., 1994; Leano e ta !., 1994). Pathological changes associated with vibriosis w ere extensive necrosis by severe bacterial invasion and multiple form ation of me/anised no'dules in the lym phoid organ (Egusa e t a/.,1988). Significant necrosis and inflam m ation occured especially in the oka/lymphoid organ, frequ ently but usually less severe on gills, heart, hepatopancreas and some tim e s other tissu e s (Nash e t al.. 1990; Jiravanichpaisal and Miyazaki, 1994). A num ber of Vibrio species were found associated with vibriosis and the major ones reported w e re V. parahaemoiyticus, V. harveyi, V/. vulnificus, I/, damseia,
V. alginolyticus, V. anguillarum a n d [/. Campbell/ (Lightner, 1977; 1983; Nash et a/,, 1990; K a runasagar et al., 1994; Lavilla-Pitogo, 1995). But prior to the outbreak of vibriosis, a decrease in the diversity of th e vibrio com m unity was obsen/ed (Laviila-Pitogo et al., 1998; Sung e t al., 1999). Vibrio was found to affect all developm ental stages from larvae in hatchery tanks to juveniles and brood stock in grow out. H owever bacterial strains responsible for vibriosis in successive stages w ere usually considered to be different and the virulence also changed both at th e species and at the developm ental stage levels (Gorant et al., 1998).
V ibriosis of larvae and post larvae
Larval vibriosis w as caused by luminescent form s like V. harveyi, V.vulnificus and V. parahaem olyticus and also by related nonlum inescent forms like alginolyticus {Karunasagar e ta l., 1994; Leano et a i, 1998), In luminous vibriosis, hepato pancreas was the target organ w here severe inflammatory responses w ere seen in the intertubular sinuses (LavKla-Pitogo et a!., 1998), Diseased postlarvae displayed cloudiness of the hepatopancreas or midgut gland (Takahashi e t al., 1984). V irulence of the strains and susceptibility of hosts w ere im portant factors leading to lan/al mortalities by luminous bacteria in hatcheries. E pibiontic infestation of luminous bacteria, V. harveyi was also observed in hatchery reared larvae of P. indicus (Abraham et a i, 1997).
Bacterial flora associated with larvae w as not very stable and is influenced by th e bacterial flo ra of the adm inistered food and by the environm ent (V andenberghe e f a/,,1998).
V ib rio s is of Juvenile and ad u lt shrim p
(n juveniles the Infection displayed cloudiness of muscle in the sixth abdom inal se g m e n t and brown spots in the gills and lymphoid organ (Takahashi e t a!., 1985). O ften affected shrim ps were small and stunted, swimming lethargically at th e w ater surface or lying motionless at the pond edges. Such severely affected shrim ps lost their escape reflex, had a darkened cuticular co lo u r and h e a vy fouling by epibionts. Early signs included body reddening, extended gill covers and slight m elanised erosions of the uropods, pleopods, periopods (Anderson e t al., 1988). In localised cuticular vibriosis, the infections w ere typically w ell circumscribed by haemocytes forming nodules which later becam e m elanised. System ic vibriosis showed extensive necrosis and bacterial invasion of the lymphoid organ, with multiple m elanised or nonmelanised haem ocytic nodules. These nodules were m ost com m only com posed o f a bacterial colony in the center surrounded by a m elanised zone, and multiple layers of haem ocytes attem pting to encapsulate the bacterial colony. W hile such nodu(es were fo u n d in other tissues such as the heart, gills, hepatopancreas, gonads, and m uscle, necrosis equivalent to that observed in the lymphoid organ w ere not observed (Egusa e t at., 1988). Enteric vibriosis or septic
hepatopancreatitis included the characteristic features like haem ocytic infiltration, m elanisation and basophilic masses of bacterial colony and tissue debris in th e hepatopancreatlc tubule lumen (Lightner et al.,1992).
2.2.2 F ila m e n to u s bacterial disease
Filam entous bacterial disease, caused by Leucothrix infestation w as often associated with poor w ater quality (Lightner, 1977). Filamentous grow th w ere observed on setae of uropods and pleopods on gill filam ents and on tip o f epipodites (Lightner, 1977), T h e bacteria m ay cause m ortality due to hypoxia and im pairm ent of molting process (Karunasagar and Karunasagar, 1996),
2 .2 .3 N e c ro tis in g hep ato pancreatitis (NHP)
First described in penaeid shrim ps cultured in Texas (Krol eta!., 1991), later from Peru in 1993 (Lightner and Redman, 1994), It was caused by a small, highly pleom orphic, intracellular. Gram -negative bacterium. Gross signs included reduced feed intake, elevated food conversion ratios, reduced growth, poor length-w eight ratios, soft shells, black or darkened gills, expansion of chrom atophores of the pleopods and uropods, em pty guts, fouling and lethargy.
M oderately affected individuals showed a m oderate atrophy of the h e patopancreas and a white colouration when dissected. Severely affected individuals show ed a m arkedly atrophied hepatopancreas and contained more flu id than tis s u e (Jim enez et a!., 1997).
2.3.0 R ickettsial Diseases
Rickettsia or rickettsial like organisms (RLO) had been reported to cause diseases in cultured shrim p (Brock eta!., 1986b; Anderson eta!., 1987).
B ut these agents were not isolated, cultured or characterised from shrimps.
Shrim ps lightly infected were assym ptom atic carriers while heavily infected ones becam e lethargic, w ent off feed and show pale colouration and atrophy of hepatopancreas.
R ickettsial infections w ere diagnosed by the demonstration of RLO in the cytopfasm o f infected cefis (Anderson e t al., 1987), Rickettsial m icrocolonies w ere intracytoplasm ic, m em brane bound and basophilic. Infected shrim ps displayed gross signs of disease that included lethargy, inappetence, p oor escape responses and w hite coloured hepatopancreas. In P.marginatus, P.m erguiensis, and P.stylirostris, the infection occurred in the hepatopancreas epithelium . In P. monodon, the infection was wide spread in m esodermally and ectoderm ally derived tissues. It form ed large m em brane bound basophiiic cytoplasm ic m asses in hypertrophied hepatopancreatocytes. Systemic Rickettsia like organisam s localised within th e fixed phagocytes, connective tissue cells and lymphoid organ. Rickettsial infection evoked marked haem ocytic response.
2.4.0 Fungal d is e a s e s
Fungal pathogens invaded shrimps th a t have been injured or exposed to stressful conditions(Lightner and Fontaine, 1973). It affected the larval stages, juve n ile s and adults. Prim ary mycosis also was reported from shrimps, Laginidium and Sirolpidium w ere reported to be responsible fo r major epizootics o f th e larvae o f m ost species o f penaeid shrimps through out the world (Lightner 1977; Chin and Ching, 1985), Diagnosis w as by wet m ount or histological dem onstration of hyphae with in the body and appendages and sporangia with discharge tubes and m otile zoospores. Diagnosis can afso be done by isolation and culture of the fungus from infected shrimp. The Fusarium caused black gill disease was a serious problem in adult penaeid shrimps (Ishikawa, 1968; Egusa and Ueda, 1972), The causative agent was identified as Fusarium s o la n i (Hatai e t al., 1978). The inflammatory responses of Penaeus jap o n icu s a g ain st Fusarium solani w ere haemocytic infiltration and collagen like fibre deposition. These lesions w ere m ore pronounced in the exoskeleton than in th e gill lam ellae (Bian and Egusa, 1981). The hyphae and tissue destruction due to black gill disease were obsen/ed in the gills and also in the maxillipeds, periopods, thoracic body wall, thoracic central nerve and occasionally in the ventral thoracic artery (Momoyama, 1987). Fusarium moniHformae was another species, isolated from the gills o f Penaeus japonicus, that showed similar sym ptom s to th a t o f Fusarium so/an/(R hoobunjonde etal., 1991),
Lag'midium callinects infections in laA/al tiger shrimp w as th e first described lan/al m ycosis in shrim ps in lndia(Ramasamy e t al.. 1996), Mycelium invaded and em beded itself in tissues or alternatively replaced the muscle tissues.
2 .5 .0 M icro s p o rid ia sis
In shrim p, m icrosporidiasis causes a condition known as milk or cotton shrim p. C om m on m icrosporidians which cause this disease condition w ere A gm asom a spp., A m esom a spp. and Pleistophora spp.. On the infected shrim ps the developing trophozoites replaced the striated muscle causing it to becom e opaque and white. T he gonands enlarged and became opaque and white, M icrosporidians while replacing the host tissue did not invoke any host Inflam m atory response . D epending on the type o f microsporidian. the site of infection w a s th ro u g h o u t the m usculature or in particular organs and tissues.
M icrosporidians w ere present in th e affected shrim p in the form o f spores (Overstreet, 1973; Johnson, 1978), It w as found to affect a number of species like P enaeus duorarum, P enaeus setiferus, Penaeus aztecus, Penaeus breziliansis, P andalas jordani, P andalus borealis, Crangon alaskensis and P enaeus m o nodon (Lightner, 1977; Olson and Lannan,, 1984; Parsons and Khan, 1986; A n derson e ta l., 1989).
2 .6 .0 E p ic o m m e n s als
Surface and/or gill fouling was very common in shrimp reared in high density culture system s or in system s with poor water quality (Lightner, 1988). T hese fo u lin g organism s w ere called epicom m ensals because they use shrim p as a su b stra te fo r attachm ent. These organism s caused problems indirectly b y attaching to gills o r cuticular surfaces. They kill the shrim ps by interfering with w ater flo w over gill surface, moulting, feeding and locomotion.
Diagnosis of epicom m ensals were usually done by examining whole animal wet m ounts o r w et m ounts prepared from biopsied gill or appendages under m icroscope. Serious losses in cultured penaeids occured when the gills of the host becam e fouled by heavy infestations of epicommensal ciliates such as Z o o th a m niu m sp.. Epistylis sp., Vorticella sp. An increase in the intensity of
infestation w ith Z o o tham nium sp. was found with an increase in stocking density (Overstreet, 1973), D ifferent species of epicommensals prefered particular body parts of shrim p as sites of attachm ent (Johnson, 1978),
2.7 .0 N o n -in fe c tio u s Disease
T h e rapid w orldwide developm ent of the shrim p culture industry has been accom panied by the occurrence of numerous significant diseases of non- infectious aetiologies. S om e non-infectious diseases had sim ple aetiology while o thers had com plex aetiologies in which the affected animals became susceptible to infection by opportunistic pathogens after initial insult by other predisposing fa ctors th a t may be environm ental or intrinsic. Several nutritional disease syn d ro m e s had also been reported such as ascorbic acid deficiency syndrom e ca lled black death (Lightner, 1993); cramped m uscle syndrome (CMS) or cram ped tail presum ed to be due to mineral im balance / physiological or nutritional fa cto rs th a t were enhanced by physical or environm ental stressors
(Lightner, 1988),
2.7.1 C h ro n ic soft shell syn d ro m e
F requently observed in cultured penaeid shrim ps (Baticados e t a i, 1986; B aticados and Tendencia, 1991), Affected shrimps displayed thin and persistently soft, rough and w rinkled shells, Soft shelled shrim ps were lethargic, w eak, susceptible to w ounding and cannibalism, showed poor growth rate and e ventually died (Baticados e t at., 1990). It was suggested that soft shell syndrom e is a m etabolic disease involving calcium and phosphorous m etabolism , b u t with m ultiple etiologies (Baticados and Tendencia, 1991), Inadequate fe e d and feeding practices such as im proper storage of feeds, rancid o r low quality fe e d s and exposure to certain pesticides had been linked to soft shell syndrom e in penaeids (Baticados e ta !., 1986; Baticados and Tendencia, 1991). S tudies had indicated that occurrence of soft shelling was more under conditions o f high p^, Jow phosphate in the water and low organic matter in the soil (Baticados e t a i , 1990),
2.7.2 S p o n ta n e o u s necrosis (Idiopathic m uscle necrosis)
M uscle necrosis was the nam e given to a condition in all species o f penae'id and ca ride an shrim p th a t w as characterised by whitish opaque areas in the striated m usculature, especially of the distal abdominal segm ents (Lightner, 1993), T h e condition typica lly followed periods of severe stress from over-crow ding, low dissolved oxygen levels, sudden temperature or salinity changes and rough handling (Lakshm i et al., 1978). It was reversible in its initial stages, but lethal if large areas w ere affected (Lightner, 1993).
2 .8 .0 D isease in In d ian sh rim p farm ing
Inform ation regarding diseases affecting comm ercially important penaeid shrim p of India is limited, Rao and Soni (1988) had reviewed the diseases and para site s of penaeid praw ns of India. There were also some published reports on incidence of disease problems (Soni, 1986; Felix and Devaraj, 1993; A b ra ha m and S hanm ugam , 1994; Karunasagar et al., 1994;
Sahul ham eed and Rao, 1994; S hankar e ta l., 1994; CIBA, 1995; R am asam y et a/.,1995; R uby e t al., 1998; Shankar and Mohan, 1998; Sudha e ta l., 1998), M ortalities due to lum inous vibriosis and filam entous bacterial infections and larval m ycosis w ere reported in hatcheries (Karunasagar et al., 1994; Abraham e ta l., 1997 and Felix, 2000),
Panchayuthapani (1997) had published a report on survey of sh rim p dise a se s in India, It covered both P. monodon and P. Indicus and was based on o bservations of a large num ber of shrimp farm s practising both extensive and sem i-intensive culture along both coasts o f India, for a period of around tw o m onths. He had recorded m ass mortalities due to viral diseases like MBV, IHHNV, Y H V and W SV. Bacterial diseases due to pathogenic vibrios and filam ento us bacteria, fungal infection (Laginidium) and epicom m ensal infestation,
abnorm al conditions such as black gill and tali rot had also been reported.
Jasm in and Manissery (2000) had reported prevalence o f white spot virus disease in shrim p farm s around Cochin area. Deepa (1997) and Nisha (1997) had studied the occurrence o f epicom m ensal ciliates in relation to water quality param eters in penaeid shrimps, Metapenaeus dobsoni and P. indicus in culture ponds. The ciiiate protozoans such as Zoothamnium, Eplstylis, Vorticella
and the suctorian, Acineta were recorded in both the species, Zoothamnium was recorded as th e m o s t dom inant species, They couid observe correlation between the incidence of epicom m ensal ciliates and the water quality parameters such as total suspended so lid s and dissolved oxygen. Ciliate infestations w ere m ore at higher levels o f to ta l suspended solids and low levels o f dissolved oxygen.
MATERIALS AND
METHODS
3. MATERIALS AND METHODS
3.1. S h rim p s a m p le collection
T h e survey was conducted over a period of four months from March to June, 2001, S am ples w ere collected from 26 farm s mainly comprising o f the m odified extensive system s in and around Cochin. The period was coinciding with th e peak culture season of the prawn filtration fields when the biom ass of th e culture system is believed to be the highest. The samples collected co m prise d o f P e n ae u s indicus, P enaeus monodon and M etapenaeus dobsoni.
S h rim p sam ples w ere collected random ly by operating cast net.
Both healthy looking shrim ps and th o se appeared to be suffering from disease (those o b se rve d to be w eak o r w ith abnorm al gill or shell coloration) were collected. A m inim um of five shrim ps w as collected from each farm. Following gross e xam ination, the sam ples w ere fixed using neutral buffered form alin or D avidsons fixa tive (Bell and Lightner, 1988), either at the farm site or carried live and fixed in th e laboratory. T he specim ens were fixed as whole and larger specim ens w e re injected w ith the fixative in the cephalothorax and abdomen.
A t th e tim e of sam ple collection, information regarding water source, pond preparation details, seed source, stocking rate, feed composition, fe edin g schedu le, m anagem ent practices followed, history of disease occurrence and current sta te o f the culture system w ere gathered either from the farm ers or by direct observation,
3.2. W a te r q u a lity analysis
Hydrological parameters like salinity and temperature were measured at the farm site using a portable refractometer and an ordinary mercury therm om eter respectively. W ater samples were collected from each farm to analyse the water quality parameters like pH, dissolved oxygen, total alkalinity, ammonia, nitrite, nitrate, chemical oxygen demand and particulate organic matter.
pH w a s m easure d using a digital pH m e le r after standardising ttie instrument with a b u ffe r solution of known pH. O ther w ater quality param eters were determ ined fo llo w in g standard procedures (Strickland and Parsons, 1972).
3.3. H istological te c h n iq u e s
T h e s h rim p s fixed in D avidson ’s fixative were stored in 50%
ethanol after 48 hrs o f fixation. T he tissu e s fixed in neutral buffered form alin were given o ve rn ig h t w ash in g in ta p w ater.
S h rim p s w e re cut in to fo u r sections fo r fu rther processing. The routine m ethod o f tis s u e cutting w as follo w ed, which ensured representation of all major tissu e s and organ system s in th e sections. Longitudinal sections of cephalothorax, c ro ss section o f a b d o m e n and sla n t section of the last fwo abdominal s e g m e n ts w ere used. T h e tissu e s were then properly labelled and processed fo llo w in g sta n d ard p ro ce d u re s (Bell and Lightner, 1988), Tissues were passed th ro u g h a sce nd ing g ra d e s o f alcohol fo r dehydration and then cleared in tw o c h a n g e s o f xylene. T issu e s w ere then transferred to molten paraffin w ax fo r infiltration. T h re e changes w ere given in molten wax. The processed tis s u e sa m p fe s were e m b e d d e d in m olten paraffin wax and blocks were prepared. S e c tio n s w ere cut at 5 — 6 jam thickness in a rotary microtome.
The de pa ra ffin ise d s e c tio n s w ere stain ed with Haem atoxylin and Eosin (Bell &
Lightener, 1988).
P h o to m ic ro g ra p h s w ere ta ke n using a Carl Zeiss Axiostar 1061 - 130 trinocular re s e a rc h m icro sco p e w ith digital cam era attachment.
3.4. B a cte rio lo g y
S h rim p s a m p le s sh o w in g cfinical signs o f disease were subjected to bacteriological e xa m in a tio n of th e ha em o lym ph and hepatopancreas (Elston,
1989), The body s u rfa c e w as sw a b b e d w ith alcohol, Haem olym ph w as then withdrawn using a s te rile syringe and stre a ke d on to thiosulphate citrate bile salt sucrose agar (TC BS), T h e ca ra p a ce w a s then opened asceptically and a loopful of inoculum from th e h e pa to p a n c re a s w as streaked on to TCBS a gar and incubated, fo r p re su m p tive ide n tifica tion o f vibrios.
RESULTS
4. RESULTS
4.1. Sample d e ta ils
S a m p le s w e re collected from 26 farm s having populations reported to be suffering fro m d ise a se , with a previous history of disease outbreak or having reportedly h e a fth y p o p u latio n s. P. indicus a ccounted fo r 70% o f the samples, whife P. m o n o d o n fo rm e d 24% and M. d o b s o n i 6% of the total. The samples varied in size fro m 8 -10 cm, 12 - 16 cm and 3 - 5 cm fo r P.indicus, P.
monodon and M. d o b s o n i respectively.
4.2. W ater q u a lity
T h e d a ta p e rta in in g to th e w a te r quality param eters recorded from the farm s s u rv e y e d a re prese n te d in T a b le l. A ir and w ater tem perature fluctuated betw een 30 - 34®C and 31 — 36°C respectively. Salinity values ranged between 1 5 - 2 7 %<>. T h e va lues o f pH recorded w ere m ostly in the range 7,0 • 8,5, However, pH v a lu e s b e lo w 7,0 w ere also recorded in tw o farms. Dissolved oxygen varied b e tw ee n 1.5 -7.5 ml/I, but m ajority w ere in the range of 2 - 5 ml/I, Alkalinity w as in th e ra n g e of 20 - 30 ppm except fo r th e fa rm s having pH below 7.0, where th e a lk a in ity v a lu e s fell b e lo w 12 ppm . A m m o n ia values show ed w ide variations and ra n g e d fro m 0 - 4 .6 jjg a to m N as am m onia/l. However, in three farms am m onia v a lu e s reached 13.37, 15.93 and 26.25 fag atom N as ammonia/l, N itrate levels ranged betw een 0.13 -1.66 Mg atom N as nitrate/i.
Nitrite was u n d e te c ta b le in m o s t o f th e fa rm s except fo r three farm s w here levels of 0,02, 0.06 and 0 ,0 8 |ag atom N as nitrite/1 w ere recorded. Particulate organic matter and the c h e m ic a l o xyg e n d e m a n d values ranged from 1 2 - 6 2 mg/l and 40
•162 mg/l respectively.
4.3. M a n a g e m e n t p ra c tic e s
M ost o f th e fa rm s su rveye d are follow ing crop rotation with paddy and only seven fa rm s fa rm no.5, 6, 9, 10,11, 12 and 16 are perennial fields.
Modified extensive s yste m o f fa rm ing practices is follow ed in all the farm s. All farms are e ith er c o n n e c te d d ire c tly o r th ro u gh tidal creeks to the V em ban ad
backwaters. T he fa rm s surveyed are m ostly tide fed and in ten farms pum ps are also used w hen tid a l a m p litu d e is not sufficient. Pond preparation m easures followed in the fa rm s a re alm ost sim ilar. T he drainable ponds are dried fo r one week before a p plica tio n o f lime. In farm s, which ca n n o t be drained completely, mahuva oil cake o r C ro to n tiglium is applied to eradicate predatory and weed fishes. Pond fertilization is m ostly done w ith cow dung and three farm s viz. farm no. 1, 13 and 16 also use chem ical fertilize rs like su p er phosphate and urea. On development of s u ffic ie n t plankton bloom , stocking is done with post-larvae (PL 18 - 25). H atchery ra ise d seed Is used in all the fa rm s except in farm no. 17 where only seed c o lle c le d from wild is used. S tocking density used in the farm s ranged from 2 0 00 -1 0 ,0 0 0 /a cre . T he larvae are initially fe d with Higashi starter feed {H igashim aru F e e d s Pvt. Ltd., C ochin) and replaced in the later stages, either with farm m a d e fe e d s or H igashi g ro w e r along with o r without clam meat.
In farm s no.1 and 13 an tibiotics is given in feed as a prophylactic measure.
Feeding w as e ith e r by d ispensing in fe e d in g trays (only in farm no.1 and 15.) or by casting by hand. W a te r is exch an g e d once d a ily in all the farms e xce pt in farm no. 1 and 16 w h e re it is done tw ice a day. T h e tim ing of water exchange varied depending on th e location of the fa rm s and tidal am plitude In the area.
4.4. Case h is to ry a n d c lin ic al s ig n s
M o st o f th e sa m p le s collected w ere appearing normal on gross examination (94% ) and only 6% w ere show ing clinical signs of disease or abnormal co n d itio n s. N ineteen p ercent of th e fa rm s w ere reported to have history of disease o u tb re a k during th e previous crop. Sam ples collected from farms no. 4, 8 and 15 w e re from th e sto ck surviving disease outbreak. Shrim p mortality during th e p e rio d of study w as reported from fa rm s no, 16 and 26, In farm no, 16, m o rta lity w a s reported in P. m o n o d o n ju ve n ile s ranging in size from 3 - 5 cm, after one m o n th o f stocking. A ffe cte d sh rim p s w ere surfacing at the pond edges during d a ytim e . No o th e r e xternal clinical signs were observed except for the b reaking o f th e tip o f righ t antenna. In farm no. 26 P . indicus ranging in size from 4 - 6 cm w ere a ffected . A ffe cte d shrim ps were com ing to pond margins and had re dd ish co lo u ra tion o f cephalothoraxes. Another case of stunted growth w a s o b se rve d in farm no. 14, Brown discolouration of gills was
noticed in the sam ples fro m farm no. 3 and darkened gills in samples from farms no. 9, 10,12 and 13. S am ples from about 36% farm s had several whitish, opaque areas in th e m usole resem bling instantaneous m uscle necrosis (P la te l), There were no a p p a re n t h isto pa th o log icai features in sam ples examined from 27% of the fa rm s surveyed.
4.5. H isto p a th o lo g y
T he d e ta ils o f o b serva tio ns from the histopathologicai studies are summarised in T a b le 2.
C hron ic in fla m m a to ry lesions in the form of melanised or non- melanised h a e m o cytic nodu ies w ere identified in th e gills, connective tissue, muscle and h a e m o c o e l sp a ces in sa m p le s from 42% o f the farms surveyed (Plates 2 & 3). P a th o lo g y of h e pa to p a ncre a s showing severe necrosis, loss of structure, a tro p h y o f tu b u le epithelia l cells, vaouolation and rounding and sloughing of cells into th e lum en w ere recorded in 19% cases (Plates 4 to 7). No marked h a em o cytic infiltration w as noticed in any o f these cases. Normal hepatopancreas (m id g u t gland) in sh rim p s contain num erous tubules with star shaped lum en lined w ith various typ es o f epithelial cells (Plates 8 and 9),
P re s e n c e of eosinophilc, spherical, intranuclear occlusion bodies characteristic of M B V w as recorded in th e hepatopancreatic tubule epithelial cells (Plate 10) of s a m p le s from farm n o .1 6, w here m ortality was recorded during the period of study. T h e sam ples also show ed hepatopancreatic pathology of severe necrosis, ro u n d in g and slou g h in g o f cells into the lumen, vacuolation and loss of structure (P late 11). M idgut epithelium of sam ples from all other cases appeared n orm a l (P late 12), O th er th a n this, there w as no histopathologicai evidence of viral in clu sio n s in any of th e sam ples exam ined. The target tissues of W SV, such as c u tic u la r epiderm is, connective tissue, gills, lymphoid organ, haem atopoietic tissue, ventral ne rve cord etc. were fre e from inclusion bodies characteristic fo r W S V (Plates 13 to 18).
Infestation by the e p ico m m e n sa l ciliate protozoan, Zootham nium in the gills and c u tic u la r s u rfa c e w as o bserve d in histological sections of sam ples collected from 8% o f th e fa rm s s u rve ye d (Plates 19 and 20).
4.6. B acteriology
B a cte riolo gica l investigations were carried out only for sampies from farms facing m o rta lity during th e survey period, i.e., in farm no. 16 and 26.
Haemolymph and h e pa to p a n c re a s from both cases w ere positive fo r pathogenic vibrios.
