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STUDY ON NEW SHRIMP DISEASE “Internal mortality syndrome” OF EXOTIC SPECIES litopenaeus vannamei
Anil Kumar Moola
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Shrimp farming has a long and colorful history whose development can be divided into three distinct periods; start up era (1982-87), hatchery era (1988-96) and breeding era (1997-present). The breeding era
1997 to the present, saw a resumption of growth, which jumped to more than 20% year for more than 10
years. This rapid industry growth was primarily driven
by the domestication, breeding and world wise spread- ing of L.vannamei from the west Indonesia. World shrimp production using L.vannamei expanded from only 10% total production in 1998-75% of total world production in 2006. [1]
In India commercial shrimp farming started gaining roots only during the mid eighties. It was relatively late start in India by this time shrimp farming had reached peak in most of the neighboring Asian coun- tries .the boom period of commercial scale shrimp cul- ture in India started in 1900 and the best came in
1995-96 with the outbreak of viral disease (sept 2002
aquaculture authority).
According to aquaculture, national portal of India, Andhrapradesh ranked in coastal aquaculture, fresh water aquaculture. Andhrapradesh contributes nearly
40% of the total marine exports of the country.
In Andhrapradesh region Nellore the species L.vannamei and p.monodon (black tiger) has been cul- tured .In present study we have targeted on the pre- dominant problems of high mortality and white feaces in both species. But majorly high mortality rate ob- served in vannamei and white feces Morley seen in black tiger shrimp .so we have diagnosed for the virus diseases like (YHV, TSV, WSBV and GAV) and bacte- rial diseases like VIBRIOSIS .We have targeted on the major organs for white feces (Shown in figure 1 and
2).
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For the study of presumably diseased shrimp, select those that shrimp are moribund, discolored, dis-
playing abnormal behavior, or otherwise abnormal, except in the case of intentional random sampling for
estimation of diseased prevalence. Have ready an ade- quate supply of fixative. A general rule is that a mini- mum of 10 volumes of fixative should be used for one volume of tissue sample (i.e. a 10 g sample of shrimp would require 100 ml of fixative).
Davidson’s AFA (Alcohol, Formalin, Acetic acid) fix- ative composition contains Strong formalin (37%)
=500 ml, Alcohol =750 ml, Glacial acetic acid =250 ml and distilled water = 750 ml (Do not substitute oth-
er acids, such as HCL, for acetic acid. Histological sections prepared from HCL-Davidson’s solution are not suitable for routine haematoxylin and eosin stain- ing.)
The presence of spherical inclusion bodies, staining
from eosinophilic to basophilic, in the cytoplasm of target cells (cuticular epithelium, gills, hind and fore gut, connective tissue, muscle etc.. The pathogenic lesions are associated with pyknotic, karyorrhectic and hypertrophied nuclei of tissue of ectodermal and en- dodermal in origin. Abescence of haemocyte infiltra- tion in characteristic of the acute phase, compared to the recovery chronic phase. [2] ,[ 3],][ 4 ] In case of YHV lymphoid organ tubules contain necrotic cells
with feulgen positive inclusions and occluded lumens.
The staining method involves application of hem alum, which is a complex formed from alumini- um ions and oxidised haematoxylin, these colours nu-
clei of cells are blue. The nuclear staining is followed by counter staining with an aqueous or alcoholic solu-
tion of eosin, which colours eosinophilic other struc- tures with various shades of red, pink, and orange.
The staining of nuclei by hem alum does not require the presence of DNA and is probably due to binding of
the dye metal complex to arginine rich basic nucleo- proteins such as histones. Feulgen –positive intra nu-
clear inclusion bodies in hypertrophied nuclei of hepatopancreatic tubule epithelial cells.
Consequent lateral displacement and compression of
the host cell nucleolus, which becomes hypertrophied inclusion bodies. Is a key morphological feature char- acteristic of an HPV infected nucleus chromatin mari- nating is also a prominent characteristic of an HPV infected nuclei.
Early in their development, HPV inclusion is small
eosinophilic bodies centrally located within the nucle- us and closely associated with the nucleolus. Later in virogenesis the inclusion body becomes intensely ba- sophilic, affected cells are most common in the distal portion of tubules in F or E type epithelial cells.
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Antibiotic sensitivity is a term used to describe the susceptibility of bacteria to antibiotics. Antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful in treating a bacterial infection (Table: 1). Testing for antibiotic sensitivity is often done by the Kirby-braver method. Small wafers containing antibiotics are placed on a plate upon which bacteria are growing. If the bac- teria are sensitive to the antibiotics a clear ring or zone ofinhibition, in seen around the wafer indicating poor growth.
Collect fresh shrimp sample (50 ul), mix immediately with 500 ul of TrizolT reagent and extract RNA ac- cording to the protocol. Resuspend RNA (2ul) in 20 ul of PCR buffer (10 mM Tris/HCL, Ph 8.3,50 mM Kcl) containing 2.5 U of M-MLV (moloney murine leu- kaemia virus) reverse transcriptase, 1.0 u of ribonucle- ase inhibitor, 0.75 um of antisense primer, 1mM each of dATP, dTTP, dCTP, and dGTP , and 5 mM OF
Mgcl2 and incubate at 420C for 15 minutes to synthe- sise cDNA .Incubate the mixture at 100oC for 5
minutes to inactivate the reverse transcriptase and al- low the mixture to cool to 5oc. Add the PCR mixture containing 2.5 U of Taq DNA polymerase (Perkin Elmer Cetus) 2mM Mgcl2 and 0.75 um of sense pri-
mer to give a final volume of 100 ul. Overlay the tubes with 100 ul of mineral oil and carry out PCR amplifi- cation for 40 cycles at 940C for 30 seconds, 580C for
30 seconds, and finishing at 720C for 30 seconds. And finishing at 720C for 30 seconds. Include a negative
control containing a diethyl pyro-carbonate (DEPC) - treated distilled water instead of RNA extract, and a positive control containing YHV RNA. The sequences of the PCR primers are
10F: 5’-CCG-CTA-ATT-TCA-AAA-ACT-ACG-3’
144R: 5’-AAG-GTG-TTA-TGT-CGA-GGA-AGT-3’
The RT-PCR method of Wongteerasupaya et al. Will not detect GAV due to sequence difference from YHV at the PCR primer sites. The nested RT-PCR proce- dure that allows detection of both YHV and GAV in the first amplification and discrimination of the geno- types in the nested amplification.
The sequence of the nested PCR primers is
GY1 5’-GAC-ATC-ACT-CCA-GAC-AAC-ATC-TG-3’ GY2 5’-CAT-CTG-TCC-AGA-AGG-CGT-CTA-TGA-3’ GY4 5’-GTG-AAG-TCC-ATG-TGT-GTG-AGA-CG-3’ GY5 5’-GAG-CTG-GAA-TTC-AGT-GAG-AGA-ACA-3’ Y3 5’-ACG-CTC-TGT-GAC-AAG-CAT-GAA-GTT-3’ G6 5’-GTA-GTA-GAG-ACG-AGT-GAC-ACC-TAT-3’
Detection of viruses in carrier shrimp or other
crustaceans cannot be realizably accomplished by his- tological methods, and more sophisticated techniques are required. The protocol mentioned here is adapted from Wongteerasupaya et.al[5]. An alternative assay for YHV is reported by Tang & Lightener [6]. A nest- ed PCR method, which is more sensitive and will de- tect and discriminate both YHV & GAV.
The sequence of above RT-PCR primers generic for GAV and YHV (or) specific for GAV or YHV are as follows.
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In your beautiful pond has turned into a green eyesore, or if you want to prevent your pond from turning green, you have to increase plankton and stop weedfrom growing with a little pond care. Fertilising your pond is an absolute aquatic weed.
Fertilising your pond increases plankton, the plankton will grow and cover the bottom of your pond, which prevent sunlight from penetrating through to the soil bottom of the pond and stops weed growth.In the early spring, feeds input to aquatic ponds usually are low, and there is not enough nitrogen and phosphorous concentration for the (purpose of promoting plant growth) in ponds water to promote growth of micro- scopic plant known as ‘phytoplankton’.
As water temperature increases, feed input in- creases due to increased fish activity. Phytoplankton becomes more abundant as a result of increased food input and warm temperature an abundance of phyto- plankton is called phytoplankton bloom.Fertilisation encourages excessive phytoplankton and a greater ox- ygen demand. Unnecessary fertilisation is detrimental to water. Quality in ponds may lead to a higher con- centration of nitrogen and phosphorous effluents [7].
In good quality and toxic ponds three types of blue
green algae were identified that is micocystis, nostoc,
and phormediom.
Antibiotic sensitivity is a term used to describe the susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful- ly in treating a bacterial infection. Testing for antibi- otic sensitivity is often done by the Kirby-bauer meth- od. Small wafers containing antibiotic are placed on a plate upon which bacteria are growing. If the bacteria are sensitive to the antibiotics a clear ring or zone of inhibition, in seen around the wafer indicating poor growth. Agar and broth dilution methods for minimum inhibition concentration. From our results for poly- merase chain reaction and nested PCR for all viruses and bacterial diseases are showing negative result for YHV.
In our challenge test, culture pond mass mor- tality rates were gradually increasing after affecting the infection and mortalities mostly occurs at the re- gions of the aerator. According to couch, 1974. Light- ener et.al, 1983[9],[10]., baculovirus associate with the diseases infected the Hepatopancreas. And all bac- terial and viral diseases targeted the major organs like Hepatopancreas, gills and lymphoid organ. Therefore the Hepatopancreas is thought to be the target organ for these viruses.
In present study sign and symptoms are not matching with any of these diseases reported so far for cultured shrimp species, the symptoms like no mori- bund and white to yellowish faecal matter it was named as “Internal mortality syndrome”. And micro- biological, molecular and histological study de-
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picts/reveals there is no virus involved. Still there may be an approach to study on prevalence of new shrimp diseases of exotic species.
One of the typical external signs of infected shrimp was yellow colour observed inside surface of the cara- pace (fig. 1). There was a dark lesions observed under the light microscope. And cephalothorax region is yel- lowish colour in some infected shrimp of the L.vannamei. Light yellow decolourisation of the ceph- alothorax region and bleached appearance was not ob- served in naturally affected shrimp [8].
From our light microscopic observations for two ef- fected culture shrimps i.e. L.vannamei and p.monodon by the process of histopathology diagnosis, In case of vannamei species hepatopancreas was affected (Fig 3 and 4).Our study depicts for bacterial and viral diseas- es in rapid gill staining process a very clear nucleus was observed under light microscope (Fig 5 and 6). However there are pyknotic and karyorrhectic nuclei was seen
However in water quality analysis huge number of yellow colonies developed by streak plate method with in 8hrs in the infected pond water. After 8-12 hrs fermented bacteria converted into non fermented bac- teria. In case of plankton analysis three types of dia- toms were found. Still there may be an important ap- proach to study on prevalence of new shrimp disease of exotic species.
The author acknowledged Dr.A.Ravikumar, Alpha-biologicals, Nellore to provide laboratory and equipment to carry out total research work and I wish to thank Shri.Gopakumar to provide accommodation in Nellore during my project/Research work. It is with immense gratitude that I acknowledge the support and help of my professor K.R.S.Sambasivarao, Depart- ment of Biotechnology, Acharya Nagarjuna Universi- ty. I wish to thank Ch.Eswar Rao, my colleague to help in my part of work and last but not least I am dedicating this (my first) paper to my parents.
[1]. Jim.wyban PhD. High health aquaculture, Global aq- uaculture advocate.
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[3]. Lightner, D.V. 1996b. Epizootiology, distribution and the impact on international trade of two penaeid shrimp viruses in the Americas. Revue Scientifique et Technique Office International des Epizooties 15: 579-
601.
[4]. Hassan, R.B., Fajamanickam, L.D., 1990. Preliminary investigation on the total sediment oxygen demand in brackishwater fish/shrimp ponds in Malaysia. Fish. Bull., Dept. Fish. Malaysia 64 (6 pp.).
[5]. Wongteerasupaya C, Vickers JE, Sriurairatana S, Nash GLand 6 others (1995) A non-occluded, systemic bac- ulovirus that occurs in cells of ectodermal and meso- dermal origin and causes high mortality in the black tiger prawn Penaeus monodon. Dis Aquat Org 21:69–
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[6]. Tang, K.F.J. and Lightner, D.V. (1999). A yellow-head virus gene probe: application to in situ hybridization and determination of its nucleotide sequence. Diseas- es of Aquatic Organisms (in press).
[7]. Boyd, C.E. (1981). “Fertilisation of warmwater fish ponds”, Journal of Soil and Water Conservation, vol
36, pp 142-145.
[8]. ChantanachookinC, Boonyaratpalin S, Kasornchandra J, Direkbusarakom, S, Ekpanithanpong U, Supama- taya K, Sriurairatana S, Flegel TW (1993) Histology and ultrastructure reveal a new granulosis-like virus in Penaeus monodon affected by yellow-head dlsease. Dis aquat Org 17:145-157.
[9]. Couch, J.A. (1974a). Free and occluded virus similar to Baculovirus in hepatopancreas of pink shrimp. Na- ture 247, 229-231.
[10]. Lightner, D.V., Redman, R.M. and Bell, T.A. (1983b). Observations on the geographic distribution,
pathogenesis and morphology of the baculovirus
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from Penaeus monodon Fabricius. Aquaculture 32,
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