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ThesisItem Open Access Development of Molecular based Diagnostics for Simultaneous Detection of Viruses of Crustaceans(Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar, 2011-12-21) Mohan, S.A.; Venugopal, M.N.; Pradeep, B.; Karunasagar, I.; Shamsunder, B. A.; Maragal, M.M.; Krishna Bhatt, C.H.Among infectious diseases of cultured shrimp, diseases caused by certain viruses are highly significant due to the loss they cause to farmers. The present study was carried out to understand the incidence and prevalence of DNA and RNA viruses in cultured as well as wild caught shrimps from both west and east coast of India. Of the one hundred samples of Penaeus. monodon from both west and east coast analyzed for the presence of DNA viruses White spot syndrome virus (WSSV), Hepatopancreatic parvovirus (HPV), Monodon baculovirus (MBV) and Infectious hypodermal and hematopoietic necrosis virus (IHHNV), 39 were found to have multiple viral infections. Analysis of twelve samples of cultured P. vannamei from east coast showed dual/triple viral infection in 7 samples. Multiple viral infection in P. vannamei is being reported for the first time from India. Among 65 samples of P. monodon postlarvae analyzed for the presence of DNA viruses from both the coast, 22 samples were positive with multiple infections. Of fifty two samples of P. monodon analyzed for the presence of RNA viruses Taura syndrome virus (TSV), Yellow head virus (YHV), Leam singh virus (LSNV) and Infectious myonecrosis virus (IMNV) from both the coasts, only 3 samples from west coast were positive for LSNV. For the first time multiple viral infection in wild caught shrimp (M. brevicornis and P. canaliculatus) is being reported. Eleven different combinations of multiplex PCR were developed and detection protocol standardized for DNA viruses which could detect two, three or four viruses at a time. The developed multiplex PCR was as sensitive as single step PCR and could detect upto pictogram level of viral DNA. Uniplex real-time PCR for detection and quantification of DNA viral particles developed could detect as low as 10 in case of WSSV, 105 for HPV, 104 for MBV and 104 copies/μl for IHHNV, and a standard curve was created for all the four viruses. Duplex real-time PCR was developed for the shrimp virus combination of HPV-MBV, HPVIHHNV and MBV-IHHNV.ThesisItem Open Access Emerging Diseases of Cultured Crustaceans in India(Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar, 2007-08-15) Prakasha, B.K.; Indrani Karunasagar; Karunasagar, I.; Venugopal, M.N.; Udupa, K.S.; Maragal, M.M.Shrimp aquaculture is one of the most rapidly growing food producing sectors in the world. Fast expansion together with the intensification of farming practices in the last two decades has created several problems which are threatening the sustainability of shrimp industry. Of these, disease outbreaks in cultured animals are of major concern which results in severe economic losses. In addition to existing disease problems, several new diseases of unknown etiology remain to be studied to determine the causative agent and route of infection. Diseases spread across continents as a result of trafficking of farmed species leading to sudden emergence of a disease in a geographical region where such a disease problem was previously unknown. In India, though many diseases of unknown etiology have been frequently observed in hatcheries and grow out systems, no studies have been conducted to determine the possible association of viral/bacterial pathogens. A new disease called the white tail disease has been observed in hatcheries and nursery ponds in India, threatening the success of the culture of Macrobrachium rosenbergii. Even though the causative agents [Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV)] have been identified, there are no reports on the healthy carriers or natural reservoirs for these viruses. With this background information, the present study was conducted to understand the prevalence and causative agents of some new diseases affecting the culture of Penaeus monodon and Macrobrachium rosengbergii. Majority of shrimp (Penaeus monodon) samples (healthy and moribund shrimps) analyzed in this study were collected during the disease outbreak. Of the 135 shrimp samples analyzed, 49% (67) were positive for White spot syndrome virus (WSSV) (37 in single step PCR and 30 in nested PCR). Monodon baculovirus (MBV) was detected in 29% (40) of the samples of which 21 were found positive in single step PCR and 19 were positive in nested PCR. Hepatopancreatic parvovirus (HPV) was detected in 34% (47) of the samples (23 were found positive in first step PCR and 24 in nested PCR). Presence of multiple viral infections was observed in many samples. Twenty-one samples (15%) showed the presence of triple viral infections (WSSV, MBV and HPV). Dual viral infection with WSSV and HPV were seen in 17 (12.5%) samples. Dual viral infection with WSSV and MBV were found in 6 and with MBV and HPV in 2 samples. Loose shell syndrome (LSS) was one of the emerging diseases that were noticed during this study. The LSS affected shrimps show hard or leathery shell but not soft; and shrunken tail meat with gap between the shell and the muscle tissue (causing the “loose shell”). The moribund shrimp exhibited bacterial, fungal and algal parasite infections on the exterior. In many of the farms, the water colour was dark-greenish and in some farms LSS was also noticed in normal bluish-grey colour ponds. The histopathological studies revealed extensive degenerative changes in hepatopancreas including sloughing of the hepatopancreatic tubules from the basement membrane. Necrotized areas showed deep basophilia, karyorhexis and pyknosis. Deeply basophilic, inclusion-like bodies were observed in the cells of tubular epithelium as well as in the inter-tubular space. Secondary bacterial infections were also observed but not consistent. The bacteriological analysis also showed the presence of Vibrio fluvialis, V. cincinnatiensis, V. fischeri, V. harveyii and V. natriegens. Six samples (three from LSS and three from slow growth syndrome) were positive for an RNA virus called Laem-Singh virus (LSNV) by RT-PCR. None of the samples showed positive for necrotizing hepatopancreatitis (NHP) bacteria by PCR. Still the causative agent is not yet clear and more studies have to be conducted. Another new disease, the whitegut syndrome or swollen hindgut syndrome of adults was also studied. The analysis of the affected shrimp showed multiple viral infections with WSSV, MBV and HPV but is not consistent. The histopathological studies revealed mild vacuolar degenerations in the tegmental gland of the hind gut but the chitin lining of the hindgut remained unaffected. Hepatopancreas showed degenerative changes. The bacteriological analysis showed the presence of V. fluvialis, V. proteolyticus, V. carchariae, V. logei, V. metschnikovii and Bacillus sp. Another important emerging disease in focus of this study was the swollen hindgut (SHG) in postlarvae. Post larvae with SHG showed enlargement and distention of the hindgut folds and its junction with the midgut, although in some cases swelling also occurred in the midgut of the sixth abdominal segment in contrast to healthy PL. The abnormality caused cessation of the rhythmic movements of the hindgut–midgut junction resulting to failure of affected post-larvae to excrete fecal pellets. Hindgut appeared normal histologically but slight degenerations were seen in midgut epithelium anterior to the midgut-hindgut junction. Nevertheless, the significant pathology observed was in hepatopancreas, where severely necrotized tubules were evident with occasional deeply basophilic inclusion-like bodies. Only dual viral infections (WSSV/HPV or WSSV/MBV) were noticed in the samples but not consistent. Bacteriological analysis of post larval samples showed the presence of V. alginolyticus. Berried Freshwater prawns (M. rosenbergii) from Netravathi estuary, juveniles from a farm in Hasson (Karnataka) and Nellore (Andra Pradesh) were analyzed for M. rosenbergii nodavirus (MrNV) and extra small virus (XSV) by RT-PCR and were found to be negative. Artemia nauplii and Artemia flakes investigated in this study did not carry MrNV. Prevalence of viruses in post larval samples from West Coast of India was analyzed by PCR. The year-wise analysis revealed an increasing trend in the level of WSSV infection over the years but a decreasing trend in the level of individual infections with MBV and HPV. Analysis of data on multiple virus infections revealed fluctuations in the dual and triple viral infections during the study period.ThesisItem Open Access Expression of White Spot Syndrome Viral Coat Proteins and Study of their Immunogenic Potential(Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar, 2007-06-14) Girisha, S.K; Indrani Karunasagar; Karunasagar, I.; Venugopal, M.N.; Shamsunder, B. A.; Maragal, M.M.; Krishna Bhatt, C.H.White spot syndrome virus following its first appearance in Taiwan in the 1990s has spread to all the shrimp farming countries including India. Due to its ability to spread quickly and cause 100% mortality in 3-10 days causing large scale economic losses, this virus has threatened the very sustainability of shrimp aquaculture. Therefore, this virus has attracted vigorous research interest in the last one decade. Despite the application of numerous strategies, the problem of WSSV infection still remains unabated. Unlike in higher animals, where many microbial diseases have been controlled by vaccination, till recently vaccination has not been a well accepted strategy in invertebrates owing to the assumed lack of adaptive immune system. However, the recent discovery of “quasi immune response” in invertebrates including shrimps has opened up new frontiers towards development of vaccine-based protection strategies against WSSV infection in shrimps. Though the whole and inactivated viral particles are known to enhance disease resistance, the practical approach has been to identify viral components interacting with host immune components and stimulate adaptive immune response. Envelope proteins of virus are thus considered potential vaccine candidates, since these first come in contact with the host immune system. The present study was initiated with the aim of identifying useful vaccine candidate proteins of WSSV that would serve to protect P. monodon against infection by this virus. To achieve this, the genes coding for 4 envelope proteins were cloned and expressed in E. coli. The efficacy of these recombinant proteins as vaccines to protect P. monodon against WSSV infection was investigated.ThesisItem Open Access Molecular Characterization of Salmonella Isolated from Seafood(Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar, 2006-07-12) Shabarinatha, S.; Karunasagar, I.; Indrani Karunasagar; Venugopal, M.N.; Udupa, K.S.; Maragal, M.M.The prevalence of Salmonella in seafood samples collected from the southwest coast of India was studied by conventional culture and by a DNA based molecular technique, polymerase chain reaction (PCR). While conventional culture techniques detected Salmonella in only 20 out of the 100 samples analyzed, direct enrichment lysate PCR detected 52 as positive for Salmonella. A set of three different PCR primers viz., hns, invA and invE were used. It was observed that hns primer detected Salmonella in a significantly higher number of samples. Fourteen out of nineteen isolates belonged to serovar S. enterica Weltevreden. S. Weltevreden isolates were genotyped yielding 4 different patterns both by RAPD and ERICPCR but when combined, the overall results discriminated the isolates of S. Weltevreden into 6 different types. This suggests that genetically diverse Salmonella Weltevreden are prevalent in seafood. The intracellular survival of Salmonella enterica serovar Virchow, S. Bareilly, S. Weltevreden, S. Newport, S. Typhi and S. Paratyphi were studied using the murine macrophage cell line J774.A1. In case of S. Bareilly, S. Weltevreden and S. Newport survival up to 72 hours was observed and was related to macrophage viability. In case of S. Virchow, the bacterial counts became undetectable in 24 hours, though macrophages remained viable up to 72 hours. The human adapted serovars S. Typhi and S. Paratyphi was found to survive much longer even though macrophage viability came to nil by 48 hours. In the case of nontyphoidal serovars of S. enterica the loss of viability in macrophages was accompanied by bacterial cell death. The seafood isolates had a very low frequency of resistance. The phenotypic expression of resistance in antibiogram was always accompanied by the presence of the corresponding gene encoding for the particular resistance determinant. The observation of class I integrons for the very first time in S. Paratyphi C and S. Weltevreden indicates that the aquatic medium of these seafood isolates may promote dissemination of antibiotic resistance in future.ThesisItem Open Access Human Enteric Viruses Associated with Molluscan Shellfish in Coastal Waters(Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar, 2007-01-12) Umesha, K.R.; Venugopal, M.N.; Indrani Karunasagar; Karunasagar, I.; Maragal, M.M.Rapid population growth and urbanization occurring along the coast have led to the increased discharge of sewage into coastal surface waters in many parts of the world. Although this discharge is mitigated by wastewater treatment plants in industrialized countries, the effluents from these plants and uncontrolled spillage still release substantial amounts of pathogens. Bivalve mollusks (including muscles, clams and oyster) growing in such contaminated waters accumulate pathogenic bacteria and viruses, and can present a significant health risk when consumed raw or lightly cooked. The present study was carried out to know the occurrence of enteric viruses in bivalve mollusks and cultured shrimp along west coast of India. Samples comprising of oysters, clams and water were collected biweekly from two sites (Site 1: Sasthan, Site 2: Mulki). Shrimp (P. monodon) samples were collected from culture ponds along the west coast of India and were analyzed for the presence of human pathogenic enteric viruses, fecal coliforms and MS-2 phage. Molecular techniques like RT-PCR and PCR were used for the detection of viruses while soft agar overlay method for the detection of MS-2 phage and MPN methods for fecal coliforms were employed. Enteroviruses were detected in 38% of oyster and 49% of clam samples from site 1 and 36% of oyster and 43% of clam samples from site 2. Enteroviruses were also detected in 15% of shrimp samples. However, none of these samples were found positive for polioviruses. This result indicates that the shellfish growing waters are contaminated with non-polio enteroviruses. Adenoviruses were detected in 18% of oysters and 27% of clam samples from site 1 and 16% of oyster and 27% of clam samples from site 2 by nested PCR. The sequence analysis of nested PCR products revealed that the strain isolated from shellfish share 96% sequence identify with adenovirus 41 and 91% with adenovirus 40. It has been reported that adenoviruses are more often detected than enteroviruses in coastal waters and domestic sewage. However, our results showed that the enteroviruses are distributed more frequently in bivalve molluscan shellfish than adenovirus along southwest coast of India. The presence of both Adenoviruses and Enteric viruses was detected in 26 samples, 45 samples were positive only for enteroviruses and 11 samples were positive only for adenoviruses. This result indicates that adenoviruses might not serve as an indicator of viral pollution. None of the samples analyzed were found positive for noroviruses, rotaviruses and HAV. Physiological functions, accumulation and elimination of microbes in bivalves are affected by temperature and salinity. In the present study, the frequency of occurrence of enteroviruses and adenoviruses was high between May to December. It was interesting to note that the salinity of shellfish harvesting waters was low (Avg. 11.7 ‰ in site 1 and 9.25 ‰ in site 2) and river inflow was high during the same period. However, the variation in temperature was minimal during this period (Average 29C). The total and fecal coliform counts in the shellfish growing areas failed to meet the standards set by NSSP of US and fecal coliforms showed negative associated with both MS-2 phage and enteric viruses. Hence fecal coliforms alone may not serve as an indicator of human enteric viral pollution. The detection of MS-2 phage as indicator of viral pollution in shellfish and their growing water was carried out in two sites, and we found association between the occurrence of MS-2 phage and enteroviruses in oyster samples and no association in clam samples. MS-2 phage showed no association with the occurrence of adenoviruses in both oyster and clam samples from both the sampling sites.ThesisItem Open Access A Study on Impact of Dam on Bacterial Profile of Coastal Waters(Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar, 2005-09-12) Sunil, R.; Karunasagar, I.; Indrani Karunasagar; Venugopal, M.N.; Udupa, K.S.; Maragal, M.M.The effect of dams built on rivers and their influence on the microbial Biodiversity Was studied by comparing the microbial profile of two rivers of Karnataka. one with dam (River Sharavathi) and another without (River Ncthravathi), Surface. Bottom and sediment samples "were drawn from the riverine, estuarine and coastal region. It was found bacterial counts were higher in sediment samples compared la water samples from both the rivers at the sites. Presumptive and fecal Streptococcal counts were high in riverine and estuarine waters but low in seawater. Fecal streptococcal counts were high in estuarine and seawater. The ratio of fecal of coliform to fecal streptococci (FC/FS) indicated that there was a fecal contamination in these aquatic habitats by human and animals. Bacterial counts were found to be higher in surface water samples from in Nethravathi when compared to samples from river Sharavathi ANOVA (statistical analysis) showed significance of difference between rivers, stations. Seasons and depths with respect to various bacterial groups found in these aquatic habitats. Prevalence of Salmonella in samples from river Ncthravathi and Sharavathi was quite low (0.5). All the four isolates, which were biochemically confirmed, were also positive by PCR targeting hns gene coding for DNA biding protein. The variations in different bacterial groups in river Sharavathi cannot be attributed solely to the presence of dam across this river but other physico-chemical, biological, geological and climatic factors that might have influenced bacterial diversity and abundance in these aquatic habitats. It is concluded that there was no significant effect of dam on microbial diversity as seen from monthly sampling of the two rivers during the two year study period (May 2001 to April 2003).ThesisItem Open Access Immune Gene Expression in Tiger Shrimp(Penaeus Monodon) to Vibrio Harveyi Infection(Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar, 2015-01-15) Dechamma, M. M.; Indrani Karunasagar; Otta, S.K.; Venugopal, M.N.; Girish, S.K.; Maragal, M.M.; Krishna Bhatt, C.H.Culture of penaeid shrimp is an important economic activity in most of the Asian and Latin American countries. Crustacean aquaculture is showing an increasing trend but disease problems are a major constraint. Vibriosis is the most significant bacterial infectious disease in the aquaculture sector caused by members of Vibrio harveyi clade. Crustaceans resist the pathogen by the innate immune response as they lack the classical adaptive immune system though a primitive form of memory is being unravelled. Thus, in view of developing strategies to disease management, the present study throws light on the identification and expression of immune gene in black tiger shrimp P. monodon to V. harveyi infection to harness their potential. The shrimps were immune stimulated with β-glucan to see if the immune molecules were enhanced. Following immune-stimulation they were infected with V. harveyi. Selected novel immune genes were tested for the tissue distribution and expression profiling using semi- quantitative reverse transcriptase PCR. Akirin and thrombospondin genes were found to be constitutively expressed in all tissue samples such as gill, hepatopancreas, lymphoid organ, hemolymph, eye stalk, stomach, heart, muscle of healthy P. monodon. Akirin, HSP 60, HSP 70, HSP 90, thrombospondin and toll-like receptors were checked for their expression level in gill, hepatopancreas, lymphoid tissue and hemolymph in both post V. harveyi infected and also in immunemodulated cum infected shrimps. The present study gives an idea of the relative expression levels of immune related gene in normal, infected and immune-stimulated animals.ThesisItem Open Access Development of Recombinant Protein and RNAI to Protect Shrimp from White Spot Syndrome Virus (WSSV) Infection(Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar, 2014-05-15) Akhila, D.S.; Indrani Karunasagar; Karunasagar, I.; Pradeep, B.; Venu Gopal, M.N.; Girish, S.K.; Krishna Bhatt, C.H.White spot syndrome virus (WSSV) is a highly virulent shrimp pathogen, causing huge economic loss to the aquaculture industry. The possibility of protecting Litopenaeus vannamei against white spot syndrome virus (WSSV) infection via vaccination and RNA interference technology by intramuscular and oral administration was explored. Shrimp orally vaccinated with rVP39 and rVP28 showed a cumulative mortality of 50 and 60% respectively following challenge. rVP39 had a better protective effect against WSSV infection compared to rVP28. Vaccination by intramuscular injection with rVP39 and rVP28 resulted in survival rate of 70 and 60% respectively. Various genes of WSSV envelop protein (VP28 and VP39), structural protein (RR1) and the shrimp protein PmRab7 were targeted for the production of dsRNA. The results of both oral and injection mode of delivery of dsRNA showed higher percentage and longer period of survival when using RR1 in comparison with the other targeted genes of dsRNA. The present study demonstrates the protective efficacy of vaccination and RNAi strategy which mainly depends on the role and significance of protein encoded by the target gene in the viral life cycle.ThesisItem Open Access Efficacy of Recombinant Protein and Double Stranded RNA (DSRNA) for the Protection of Shrimp against Hepatopancreatic Parvovirus (HPV)(Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar, 2014-05-15) Safeena, M.P.; Indrani Karunasagar; Karunasagar, I.; Venugopal, M.N.; Shamsunder, B. A.; Maragal, M.M.; Krishna Bhatt, C.H.Hepatopancreatic parvovirus (HPV) is a relatively common pathogen of wild and farmed penaeid shrimp, especially in the Indo Pacific region. HPV infects penaeid shrimp species and widely distributed in many parts of the world. Heavy infection can result in poor growth and reduced production for shrimp farmers. Since there was no information on complete genome of Indian strain of HPV, we have sequenced whole genome of this virus to characterize further. The complete nucleic acid sequence of HPV from India was obtained by cloning and sequencing different DNA fragments of the virus. Analysis of the whole genome, consisting of 6310 bp revealed the presence of three open reading frames (ORFs), comprising 1281 bp, 1734 bp and 2460 bp, respectively. Up to date, there is no efficient therapeutic or preventive method has been found to control the HPV infection in shrimp. In this regard, recombinant proteins and viral specific dsRNA were developed and used to reduce the HPV replication in shrimp. For protein vaccination trials the recombinant protein against NS2 and Structural protein components were used and the shrimps injected with recombinant proteins showed significant reduction in viral load compared with control group. Furthermore, we have developed HPV gene specific dsRNA and evaluated the potential of virus specific dsRNA to inhibit the HPV replication in shrimp. In addition, Monoclonal antibodies (MAbs) were raised against highly antigenic N terminal part of recombinant structural protein of HPV for the specific detection of HPV infection. The MAbs produced in this study can be used for developing a field friendly, rapid, immunobased kit for the diagnosis of HPV infection.
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