International Journal of Pharma and Bio Sciences DIAGNOSIS OF EMERGING DISEASES IN COMMERCIALLY IMPORTANT SHRIMP ABSTRACT

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1 Research Article Biotechonology International Journal of Pharma and Bio Sciences ISSN DIAGNOSIS OF EMERGING DISEASES IN COMMERCIALLY IMPORTANT SHRIMP R.CAROLINE JEBA*AND MARIA SHIRLEY Department of Bio-technology, Dr.M.G.R.Educational And Research Institute University, Chennai, India. ABSTRACT As emerging diseases are known to cause a serious setback in shrimp aquaculture due to production and economic losses, it is essential to screen for such diseases. Hence, this study was formulated with the objective to understand the prevalence of emerging diseases affecting commercially important cultured shrimp White spot syndrome virus (WSSV), Infectious hypodermal and hematopoietic necrosis (IHHNV), Acute Hepatopancreatic Necrosis Diseases (AHPND)/ Early Mortality Syndrome(EMS), Enterocytozoon hepatopenaei (EHP), Hepatopancreatic parvovirus (HPV), Yellow head disease (YHV), Monodon baculovirus (MBV). KEYWORDS: Emerging diseases, PCR, Wet mount, Microscopy, Histopathology. R.CAROLINE JEBA Department of Bio-technology, Dr.M.G.R.Educational And Research Institute University, Chennai, India. *Corresponding author B - 937

2 INTRODUCTION Aquaculture is one of the fastest growing food production sectors in the world (Subasinghe et al., 1998). Shrimp aquaculture has emerged as a valuable foreign exchange earner for developing countries and as an alternative to meet the protein requirement of the increasing world population. Aquaculture shrimps have been the primary contributors to the seafood industry s growth, contributing 47 per cent of the total exports. Shrimp exports were $3.2 billion in , which was 64 per cent of total exports, of which aquaculture shrimp alone was worth $2.3 billion. Shrimp exports from capture fisheries contributed only 27 per cent of total exports with a value of $900 million 1. Shrimp is the largest single seafood commodity by value, accounting for 17% of all internationally traded fishery products. Approximately 75% of production is from aquaculture which is now almost entirely dominated by two species the black shrimp (Penaeus monodon) and the white Pacific shrimp (Penaeus vannamei) that represent the most important invertebrate food animals 2. India s Marine Products Exports Development Authority (MPEDA, 2013) reports that 22,715 hectares of ponds are dedicated to vannamei farming, but seafood exporters, some of whom are themselves involved in shrimp farming, estimate that there may at least be 50,000 hectares of vannamei ponds 3. The major causes of shrimp diseases include microbial pathogens, inadequate control of the culture environment leading to stress and susceptibility to disease. Diseases are caused by various factors of which pathogenic and non pathogenic factors forms the major cause. Pathogenic diseases include bacterial, viral and parasitic infection. Non pathogenic includes water quality and other factors during the maintenance of the shrimp farm 4. According to Snieszsko (1997), disease is an end result of an interaction of the shrimp, its environment and the pathogens itself Viral diseases are major threat to shrimp farming operation worldwide 5.Of all the known shrimp viruses, White Spot Syndrome Virus (WSSV), Yellow Head Virus (YHV) and Taura Syndrome Viruses (TSV) are the most important viral pathogens affecting the shrimp culture industry. Hence it is important for screening virus and other emerging diseases to avoid the production and economic loss 6. Of all the known shrimp viruses, White Spot Syndrome Virus (WSSV), Yellow Head Virus (YHV) and Taura Syndrome Viruses (TSV) are the most important viral pathogens affecting the shrimp culture industry. Hence it is important for screening virus and other emerging diseases to avoid the production and economic loss 7. MATERIALS AND METHODS MATERIALS (i) Samples and codes Shrimp samples were collected from various shrimp farms in and around Ponneri, Thiruvallur district, Tamilnadu. Samples were brought either in live condition or fixed live at the site in aerated bags to lab facilities at Shrimp Disease Diagnostic Laboratory of Tamilnadu Fisheries University. B - 938

3 Table 1 Details of the shrimp samples used in the study S.NO Sample Code Date Sample Source Organs used for DNA extraction 1. FRF FREC Farm, Chennai Gill, Whole head 2. FRF FREC Farm, Chennai HP 3. FRF FREC Farm,Chennai Gill, HP 4. MA Manoj-Thevampattu, Ponneri Gill 5. MA Manoj-Thevampattu, Ponneri Gill 6. MA Manoj-Thevampattu, Ponneri Gill 7. SK Kanish, Ponneri Gill 8. SE Shrimp aqua farm,sirkazhi Gill 9. CD Cheenu aqua farm, Ponneri Gill 10. SD Sudhakar aqua farm, Ponneri Gill 11. M Manikandan farm,ponneri Gill, HP 12. AD Sirkazhi farm Gill, HP 13. FRF FREC Farm,Chennai Gill, HP 14. FRF Shrimp farm,chennai Gill, Muscle 15. S Pulicat,Chennai Gill, HP (ii) Chemicals and molecular biologicals used in the study 1. Ethyl alcohol Fixing of samples was done either with ethyl alcohol or Davidson s fixative. DNA extraction kit was used with the samples fixed in 70% alcohol (70% ethyl alcohol, 30ml distilled water). 2. Davidson s fixative Davidson s fixative solution used for fixing the samples for histopathology can be prepared by mixing the following ingredients (total, 900 ml) in an appropriate size container and mixing well. a. 300 ml distilled water b. 200 ml formalin (37% formaldehyde) c. 100 ml acetic acid d. 300 ml 95% alcohol 2. Malachite green preparation Malachite green was used to stain the wet mounts of hepatopancreas tissues in screening for MBV, HPV, EH in Shrimp. Malachite Green: 0.1g Distilled Water: 1000ml Wet mount of squashed hepatopancreatic tissue were prepared in clean glass slides and stained with 0.05% malachite green (Lightner, 1996). The slides were observed under the microscopic for the histological changes specific to MBV, HPV or EH. 3. DNA Extraction chemicals Commercially available DNA extraction kit was used to perform the DNA extraction of required parts from the samples collected for the study (QIAGEN DNA extraction kit, USA). 4. PCR chemicals PCR Primers Primers used for the diagnosis of the diseases of Shrimp are listed in table 3. The primers included self designed and published primers. B - 939

4 Table 2 PCR Primers for emerging diseases used for screening the samples Virus WSSV EMS IHHNV HPV MBV EH YHV Primers WSS330F: 5 -TGG-TCC-CGT-CCT-CAT-CTC-AG-3 WSS330R: 5 -GCT-GCC-TTG-CCG-GAA-ATT-A-3 Product Size 330 AP3F:5 -ATGAGTAACAATATAAAACATGAAAC-3 AP3R:5 -GTGGTAATAGATTGTACAGAA IHHNV703F:5 -TTG-GGG-ATG-CAG-CAA-TAT-CT-3 IHHNV703R:5 -GTC-CAT-CCA-CTG-ATC-GGA-CT-3 441F:5 CTA-CTC-CAA-TGG-AAA-CTT-CTGAGC-3 441R:5 -GTG-GCG-TTG-GAA-GGC-ACT-TC-3 261F: 5 -AAT-CCT-AGG-CGA-TCT-TAC-CA-3 261R: 5 -CGT-TCG-TTG-ATG-AAC-ATC-TC-3 MF1: 5 -CCG GAG AGG GAG CCT GAGA-3 MR1: 5 -GAC GGG CGG TGT GTA CAA A-3 10F: 5 -CCG-CTA-ATT-TCA-AAA-ACT-ACG-3 144R: 5 -AAG-GTG-TTA-TGT-CGA-GGA-AGT-3 Reference SRFDLA Kallaya Sritunyalucksana et al., 703 SRFDLA 441 SRFDLA 261 SRFDLA 951 Flegel et al., SRFDLA PCR Reaction Mix (PCR master mix) Commercial PCR master mix with optimized concentration if bases, buffer and Mgcl2 was used in the study (GeNei master mix, Bangalore, India). DNA marker Commercially available 100bp molecular marker was also separated parallely to compare the PCR amplified product (medoxbio molecular marker, Chennai, India). Agarose Molecular biology grade Agarose gel was prepared in TBE buffer with 4 µl ethidium bromide for gel electrophoresis. Gel documentation The amplified product in gel were observed under UV and documented using a gel documentation unit (BioRad). METHODS (i) Collection and fixing of sample Shrimp samples were collected from different places like Minjur, Ponneri, Sirkazhi, Nagpattinam and from the farm facilities available in Tamilnadu Fisheries University Madhavaram campus. Shrimp sample were brought to the laboratory in live condition in aerated polythene bags or they were fixed in ethyl alcohol immediately after collection. The shrimp samples were selected based on the wet mount/pcr results, coded and stored in deep freezer (-70 C) to be used for the experiments. (ii) Maintenance of the sample Shrimp samples were maintained with good aeration; adequate feeding and maintenance of water quality parameters at ideal levels in the wet lab of state referral laboratory, Tamilnadu Fisheries university (TNFU), Madhavaram. (iii) Screening of shrimp samples for diseases The samples were screened for the presence of White spot syndrome (WSSV), Monodon baculovirus (MBV), Hepatopancreatic Parvovirus (HPV), Yellow Head Virus (YHV), Early mortality syndrome (EMS), Enterocytozoan hepatopenaei (EH). The following methodologies were followed for screening. B - 940

5 Table 3 Methodologies followed for screening of emerging diseases Disease White spot syndrome virus Infectious hypodermal and haematopoietic necrosis Acute Hepato pancreatic Necrosis Disease Enterocytozoon hepatopenaei Hepatopancreatic parvovirus Yellow head virus Monodon baculovirus Methodology followed Wet mount Histopathology PCR Microbiology a) Wet mount diagnosis of HPV, EH Wet mount of squashed hepatopancreatic tissue from the collected sample were prepared in clean glass slides and stained with 0.05% malachite green and observed under the microscope for disease specific changes. b) Histopathology diagnosis of samples for IHHNV, WSSV Samples are fixed with Davidson s fixative for carrying out the histopathological study for IHHNV, WSSV. The fixing of samples were done with Davidson s fixative processing of samples, sectioning and staining were carried out following standard methods utilizing the services of the Department of veterinary pathology, Madras Veterinary college, Chennai-7. The diseases were identified by histopathology and they were documented for the result analysis. c) PCR diagnosis for emerging diseases WSSV, IHHNV, EH, YHV, EMS, MBV, HPV Shrimp samples were screened by PCR using the primers. Primers were selected for the diagnosis of the diseases of shrimp listed in the study (table 4). The primers included were self designed and published primers. PCR standard protocols were followed in case of self designed primers and protocols based on the published papers in case of published primers. Specific primers were designed for the diagnosis of WSSV, IHHNV, YHV, MBV and HPV with the sequence information available in the Gen Bank, NCBI. DNA Extraction Organ such as Gill, hepato pancrease and whole head were exercised from the Shrimp sample were used for the extraction as the target organ of the pathogens of interest vary. The extracted DNA was coded and stored in a deep freezer (- 70 C) for the further study. Specific extracted DNA part was selected for each disease and the target organ varies for every disease. Preparation of PCR mixture Reagents Quantity/concentration required/reaction PCR master mix 22 µl(1x) Forward primer 1 µl(30 picomoles/reaction) Reverse primer 1 µl(30 picomoles/reaction) DNA of target organ 1 µl(50mg) Total 25 µl B - 941

6 Agarose gel electrophoresis The amplified products from first step were separated by agarose gel electrophoresis. 2% agarose gel was prepared in 1X tris-borate EDTA buffer (TBE buffer) added with 4 µl ethidium bromide around 250ml was poured on the agarose gel electrophoresis apparatus.4 µl of PCR amplified product were loaded into performed wells of agarose gel.2 µl of 100bp molecular weight DNA ladder was also added in the gel comparing the size of the PCR products. Gel electrophoresis was carried out at 100 volts for 30min. After the run,the gel was viewed under UV gel documentation system. The results were documented, transferred and stored in the computer. RESULTS 1. Collection, fixing and maintenance of samples P.vannamei samples collected and maintained in the study is shown in Figure 1. Collection and maintenance of shrimp Figure 1 Collection and maintenance of shrimp samples used in the study with aeration in the wet lab during the study Shrimp samples were collected from various shrimp farms in and around Ponneri, Thiruvallur district, Tamilnadu. Samples were brought either in live condition or fixed live at the site in aerated bags to lab facilities at Shrimp Disease Diagnostic Laboratory of Tamilnadu Fisheries University. Fixing of samples is done either with 70% ethyl alcohol or Davidson s Fixative. Samples fixed with ethyl alcohol are used in DNA extraction and they are preserved for further study. Samples fixed with Davidson s Fixative are used in histopathological study. B - 942

7 2. Isolation of Hepatopancreas Isolation of Hepatopancreas and gill Int J Pharm Bio Sci 2015 July; 6(3): (B) Figure 2 Hepatopancreas and gill of shrimp used for the extraction of DNA in molecular diagnosis by PCR 3. Wet mount squash preparation Monodon baculovirus Screening of the samples for MBV by wet mount squash method showed that none of the sample screened in this study showed characteristic occlusion bodies as shown in the Figure 3. Staining with malachite green revealed that the squash preparation contains occlusion bodies in the hepatopancreas of infected shrimp. Wet mount microscopic observation of hepatopancreas Figure 3 Hepatopancreas wet mount squash preparation showing MBV specific occlusion bodies The samples were showing HPV inclusion bodies when observed in the wet mount squash preparation in (100x magnification) of hepatopancreatic shrimp is shown in Figure 3.Hence the results of the squash preparation showed the presence of hepatopancreatic parvovirus in the cultured shrimp collected. B - 943

8 Wet mount microscopic observation of hepatopancreas Figure 4 HPV inclusion bodies observed in samples (100x magnification) compared with the results of previous study. Enterocytozoon Hepatopenaei The samples screened for E. hepatopenaei showed inclusion bodies observed in the hepatopancreas of shrimp in the wet mount preparation(100x magnification).the observed inclusion bodies were compared with the published literatures (Flegel et al.,) Wet mount microscopic observation of hepatopancreas Figure 5 E. hepatopenaei inclusion bodies observed in the hepatopancreas of shrimp (100x magnification) compared the results of previous study 4. Screening for diseases a) WSSV Shrimp samples showed white musculature which is being compared with the healthy shrimp is shown in (Figure 6 and 7) this shows that the white musculature is due to the shrimp affected by white spot syndrome. Shrimp samples were showing size variation which is due to the slow growth in the infected shrimps and mortality is shown in (Figure 8). B - 944

9 Shrimp showing mortality Figure 6 White musculature in WSSV infected P.vannamei compared to healthy shrimp which has a clear strature Figure 7 Shrimp showing mortality during the maintenance due to WSSV Infection. Shrimps showing size variation Figure 8 Size variation observed in the infected P.vannamei (slow growth) 5. Agarose gel electrophoresis WSSV PCR screening showed that the PCR amplified products of WSSV DNA. Presence of band formation relevant to the specific primers used 330 and 615 showed the presence of white spot syndrome in the cultured shrimps (Figure 9 and 10). B - 945

10 Agarose Gel Figure 9 Agarose gel shows the presence of Ethidium bromide dye (shown with an arrow) for WSSV 330 which is compared with the standard DNA marker run at the same time on the same gel cast (shown with an arrow M WSSV Figure 10 Agarose gel shows the presence of Ethidium bromide dye (shown with an arrow) for WSSV 615 which is compared with the standard DNA marker run at the same time on the same gel cast (shown with an arrow). WSSV M M WSSV IHHNV PCR screening for IHHNV showed amplification of IHHNV (703 bp) specific for IHHNV. B - 946

11 Figure 11 Agarose gel shows the presence of Ethidium bromide dye (shown with an arrow) for IHHNV 703 which is compared with the standard DNA marker run at the same time on the same gel cast (shown with an arrow). MBV, HPV IHHNV Although HPV inclusions were observed in a sample analysed by wet mount analysis, none of the samples screened for HPV/ MBV showed PCR amplification (Figure 12) which could be due to low level of infection or low detection limit of the PCR assay followed. M Figure 12 Agarose gel shows the absence of Ethidium bromide dye for MBV and HPV which is compared with the standard DNA marker run at the same time on the same gel cast (shown with an arrow). AHPND EMS was screened for the presence of vibrio bacteria on the TCBS plate is shown (Figure 13). B - 947

12 Figure 13 Formation of yellow and green colonies of vibrio species on TCBS agar plate due to the presence of parasite in P.vannamei showing AHPND infection. None of the samples screened for AHPND by PCR technique showed the PCR amplification of AHPND specific DNA hence showing the absence of the disease. Agarose Gel Figure 14 Agarose gel shows the absence of Ethidium bromide dye for AHPND which is compared with the standard DNA marker run at the same time on the same gel cast (shown with an arrow). 6. Histopathological studies Eosin - haematoxylin-counterstaining (blue) revealed distinct populations of stained cells with IHHNV showing absence of hemocyte aggregation and viral inclusions in the gills of shrimp. These staining of cells were completely absent for other formulation. The Blue staining (Haematoxylin) for the nuclei and the pink (Eosin) for the cytoplasm were clearly visualized for all the formulation under 100 X magnification. The eosin haematoxylin staining showed WSSV showing condensed B - 948

13 chromatin in the gills of shrimp blue large sized granular shaped structures which are a clear evident for the presence of Evans blue dye. Histology slides Figure 15 IHHNV showing absence of hemocyte aggregation and viral inclusions in the gills of shrimp (100x magnification) compared with the previous study. Figure 16 WSSV showing condensed chromatin in the gills of shrimp (100x magnification) compared with the previous study. DISCUSSION The results from the wet mount squash preparation showed that the hepatopancreas of none of the sample screened in this study showed characteristic occlusion bodies. Staining with malachite green revealed that the squash preparation contains occlusion bodies in the hepatopancreas of infected shrimp. The shrimp samples were showing HPV inclusion bodies when observed in the wet mount squash preparation in (100x magnification) of hepatopancreatic shrimp. Hence the results of the squash preparation showed the presence of hepatopancreatic parvovirus in the cultured shrimp collected.shrimp samples showed white musculature which is being compared with the healthy shrimp is shown in (Figure 6 and 7) this shows that the white musculature is due to the shrimp affected by white spot syndrome. Shrimp samples were showing size variation which is due to the slow growth in the infected shrimps and mortality is shown in (Figure 8). PCR screening showed that the PCR amplified products of WSSV DNA. Presence of band B - 949

14 formation relevant to the specific primers used 330 and 615 showed the presence of white spot syndrome in the cultured shrimps (Figure 9 and 10). PCR screening for IHHNV showed amplification of IHHNV (703 bp) specific for IHHNV. Although HPV inclusions were observed in a sample analysed by wet mount analysis, none of the samples screened for HPV/ MBV showed PCR amplification (Figure 12) which could be due to low level of infection or low detection limit of the PCR assay followed. EMS was screened for the presence of vibrio bacteria on the TCBS plate is shown (Figure 13). Eosin - haematoxylin-counterstaining (blue) revealed distinct populations of stained cells with IHHNV showing absence of hemocyte aggregation and viral inclusions in the gills of shrimp. These staining of cells was completely absent for other formulation. The Blue staining (Haematoxylin) for the nuclei and the pink (Eosin) for the cytoplasm were clearly visualized for all the formulation under 100 X magnification.the eosin haematoxylin staining showed WSSV showing condensed chromatin in the gills of shrimp blue large sized granular shaped structures which are a clear evident for the presence of Evans blue dye (Figure 15 and 16). CONCLUSION Thus, the shrimp aquaculture is facing a serious threat due to the emerging diseases which causes huge economic losses. Though the brooders which are transported stating viral free brooders may also contain a certain silent pathogen which emerges out once they attain maturity. Diseases in shrimp are mainly caused due to the stress which is caused during the maintenance of the shrimp culture. Hence it is very much important to screen the diseases which are more prevalently found and also the uncommon viral emerging diseases which bring about the major mortality range which causes a drastic loss both economically and commercially. REFERENCES 1. Lightner D.V, Biosecurity in shrimp farming: Pathogen exclusion through the use of SPF stock and routine surveillance. Shrimp aquaculture, 13 (12): , (2005). 2. Phromjai J, Sukhumsirichart W, Pantoja C, Lightner D.V. & Flegel T.W, Different reactions obtained using the same DNA detection reagents for Thai and Korean hepatopancreatic parvovirus of penaeid shrimp. Diseases of Aquatic Organisms, 46, , (2001). 3. Bondad-Reantaso M.G, Mcgladdery S.E, East.I & Subasinghe R.P, Asia Diagnostic Guide to Aquatic Animal Diseases. FAO Fisheries Technical Paper 402,(2001). 4. Bonnichon V, Bonami J.R.& Lightner D.V, Viral interference between infectious hypodermal and hematopoietic necrosis virus (IHHNV) and white spot syndrome virus in Litopenaeus vannamei. Dis. Aquat. Org., 72, , (2006). 5. Bray W.A., Lawrence A.L. & Leung-Trujillo J.R, The effect of salinity on growth and survival of Penaeus vannamei, with observations on the interaction of IHHN virus and salinity. Aquaculture, 122, , (1998). 6. Brock J.A. & Lightner D.V, Diseases of Crustaceans. Diseases Caused by Microorganisms. In: Diseases of Marine Animals, Vol. III, Kinne O., ed. Biologische Anstalt Helgoland, Hamburg, Germany, , (2006). 7. Brock J.A., Lightner D.V. & Bell T.A, A review of four virus (BP, MBV, BMN, and IHHNV) diseases of penaeid shrimp with particular reference to clinical significance, diagnosis and control in shrimp aquaculture, C.M. 1983/Gen:10/1 18, (2008). 8. Paynter, Bondad-Reantaso, Tourtip mari, Poultes, International journal of pharma and Bio sciences, Soc. 36, (2011). B - 950