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1.
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2.
The present study evaluated the role of recombinant RNA‐dependent RNA polymerase (RdRp) protein of Macrobrachium rosenbergii nodavirus (MrNV) in modulating the immune response and in reducing MrNV load in infected prawn. In the first experiment, prawns (25–30 g) were injected with recombinant RdRp protein (RP) at a concentration of 0, 1.0 and 10 μg, and immune parameters and expression of some immune‐related genes were measured up to 14 days post injection (p.i.). In the second experiment, early juveniles were injected with a similar dose of RdRp and animals were challenged by immersion with MrNV. The infection status was detected in muscles by nested RT‐PCR up to 21 days post challenge. Prawn injected with higher concentration of RP showed significantly higher total haemocyte count at different period post injection. Significant up‐regulation of immune‐related genes was observed within 24 h in prawn treated with lower dose of RP and after 7 days p.i. at higher level of RP injection compared with adult control. Most of the tested samples (63%) were found to be RT‐PCR positive for MrNV at 48 h of post‐immersion challenge. After 14 days, MrNV was detected only in control prawn, while both RP‐injected groups were MrNV negative. This study elucidated the potential viral load reduction role played by RdRP in MrNV‐infected prawn.  相似文献   

3.
The giant freshwater prawn, Macrobrachium rosenbergii, is an economically important and extensively cultured crustacean worldwide. The viral pathogens, Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) are responsible for causing severe mortalities in the hatchery and nursery phases. This study investigates the protection of postlarvae of freshwater against white tail disease (WTD) using plant extract derived from Cyanodon dactylon and the modulation of the prawn non‐specific immunity. To determine the immunomodulatory effect of C. dactylon extract, the prawn was injected with plant extract and various immunological parameters were estimated. The immunological parameters such as proPO, SOD, THC and clotting time were found to be significantly higher in the plant extract‐injected prawn when compared with control groups. The results of real time PCR analysis revealed up regulation on the expression proPO, SOD and lysozyme genes in MrNV and XSV challenged prawn postlarvae treated with C. dactylon extract. Infectivity experiment showed high relative per cent survival in MrNV and XSV‐challenged prawn postlarvae treated with C. dactylon extract. These results strongly indicate that the administration of C. dactylon plant extract enhances immunity of the prawn. Based on the results, this study recommends that the immersion of postlarvae in C. dactylon plant extract is a potential prophylactic agent against WTD.  相似文献   

4.
RNA‐dependent RNA polymerase (RdRp), B2 and capsid genes of Macrobrachium rosenbergii nodavirus (MrNV) of Indian isolate were polymerase chain reaction amplified, cloned and sequenced. Expression of the MrNV fusion recombinant proteins of RdRp (44.5 kDa), B2 (32.2 kDa) and capsid (58.4 kDa) was confirmed by Western blot analysis using anti‐His mouse monoclonal antibodies. Polyclonal antibodies specific to purified recombinant MrNV capsid protein showed specificity against the capsid protein by Western blot. The protein sequence analysis of the partial RdRp gene of MrNV revealed the signature sequence along with the conserved core residues of the catalytic domain and indicated the presence of active sites, metal ion‐binding site and nucleic acid‐binding site residues. The Indian isolate of MrNV showed high RdRp and capsid gene sequence homology with the other MrNV geographical isolates. However, the Belize isolate was found to be the most distinct among the different geographical prawn nodavirus isolates due to the host specificity. Secondary structure prediction analysis of the MrNV capsid predicted it to be a DNA‐binding protein consisting of α helix (22.91%), extended strand (24.80%), β turn (5.39%) and random coil (46.90%) regions.  相似文献   

5.
White tail disease (WTD) is found to cause immense economic losses in hatcheries, with mortalities often reaching 100% within 4 or 5 days. The pathogenic agents have been identified as Macrobrachium rosenbergii nodavirus (MrNV) associated with extra small virus (XSV), which are 27 and 15 nm in diameter respectively. The effects of some chemical disinfectants hydrogen ions (pH), heat and ultraviolet (UV) irradiation on the inactivation of MrNV and XSV were investigated. The viral inoculum exposed to UV irradiation for a period of 5 min and more was totally inactivated and failed to cause mortality in postlarvae of prawn. The viruses were totally inactivated by this high pH (8.5, 9 and 10). The viral suspension treated with sodium hypochloride, formalin, Benzalkonium chloride and Benzethonium chloride at the concentration of 200 ppm caused 100% mortality in postlarvae of prawn. Iodine was found to be effective to inactivate MrNV and XSV at the concentration of 100 ppm or more, whereas the viral suspension treated with iodine at the concentration of 50 ppm or less caused mortality in postlarvae. The infected postlarvae in treated and positive control groups showed positive by RT‐PCR for these viruses.  相似文献   

6.
An attempt was made to determine the replication efficiency of hepatopancreatic parvo‐like virus (HPV) of shrimp in different organs of freshwater rice‐field crab Paratelphusa hydrodomous (Herbst) using bioassay, PCR, RT‐PCR, ELISA, Western blot and q‐PCR analyses. Another attempt was made to use this crab as an alternative to penaeid shrimp for the large‐scale production of HPV. This crab was found to be highly susceptible to HPV by intramuscular injection. The systemic HPV infection was confirmed by PCR and Western blot analyses in freshwater crab. The expression of capsid protein gene in different organs of infected crab was revealed by RT‐PCR analysis. Indirect ELISA was used to quantify the capsid protein in different organs of the crab. The copy number of HPV in different organs of the infected crab was quantified by q‐PCR. The results revealed a steady decrease in CT values in different organs of the infected crab during the course of infection. The viral inoculum that was prepared from different organs of the infected crab caused significant mortality in post‐larvae of tiger prawn, Penaeus monodon (Fabricius). The results revealed that this rice‐field crab could be used as an alternative host for HPV replication and also for large‐scale production of HPV.  相似文献   

7.
The freshwater prawn, Macrobrachium rosenbergii naturally lives in the freshwater, though it migrates to the brackish water environment during spawning that claimed to be resistant on a broad range of saline fluxes. However, little is known about the osmoregulatory patterns and the effect of an enzyme glutamine synthetase (GS) in M. rosenbergii under stress. Here, we described the identification and functional characterization of GS from M. rosenbergii (Mr‐GS) at molecular and protein levels. The identified Mr‐GS was comprised of 361 amino acids that phylogenetically shared the highest identity with other crustaceans and predicted to contain Gln‐synt_C and Gln‐synt_N domains at the respective terminal regions. Tissue distribution analysis in M. rosenbergii revealed that the Mr‐GS was highly expressed in muscle, and commonly existed in other examined tissues in the following order gills > heart > stomach > brain > haemolymph. Whereas, the mRNA of Mr‐GS was significantly up‐regulated in the muscle and gill tissues following challenges with either hyper (0 → 13‰), or hypo (13 → 0‰) osmotic stress at 3, 6 and 12 hr. Furthermore, the level of Glutamine concentration was positively correlated with the GS mRNA and protein expression patterns in hyper‐osmotic stress, whereas in hypo‐osmotic stress a slight decrease in the gills and maintained a level in the muscle tissues at 3, 6 and 12 hr post‐treatments. Our findings suggest that Mr‐GS potentially exhibited the osmoregulation responses in the gill and muscle tissues of M. rosenbergii throughout the time of osmotic stress, which will benefit for future study on osmoregulation.  相似文献   

8.
Iridoviruses infect a wide variety of wild and cultured fish. Those iridoviruses belonging to the genus Ranavirus, in the Iridoviridae family, cause systemic disease in infected animals with a high morbidity and mortality. This paper reports the cloning, sequencing, and expression of the rock bream iridovirus (RBIV) major capsid protein (MCP) in an Escherichia coli expression system for subsequent immunological studies. The completeness of the expressed protein was confirmed by peptide mass fingerprinting (PMF) analysis using MALDI-TOF MS. The recombinant MCP (rMCP)-specific mouse polyclonal antibody reacted with the viral 52 kDa protein, indicating that this rMCP induces an immunological response. Fish antibodies induced against iridovirus infection were also detected using ELISA when rMCP was used as an antigen. As a result, it was found that many cultured rock bream (92.5%) were naturally infected with iridovirus and that the rMCP might be useful for serological tests.  相似文献   

9.
Haemocyanin is a multi‐subunit protein complex found in the haemolymph and is involved in the immune system of crustaceans. In this study, a haemocyanin gene of Macrobrachium rosenbergii, designated MrHc, was successfully isolated. The MrHc gene contained an open reading frame (ORF) of 1,992 nucleotides, encoding a protein of 663 amino acid residues with a molecular mass of 76.5 kDa. The deduced amino acid sequence contained distinct structural motifs of the haemocyanin superfamily, including an all‐alpha domain, a copper‐containing domain and an immunoglobulin‐like domain. Based on the phylogenetic analysis, the MrHC protein demonstrated a close relationship with the haemocyanins of Palaemon carinicauda and Macrobrachium nipponense. The MrHc gene was expressed in various shrimp tissues, including the hepatopancreas, gill, haemocytes, stomach and muscle. After Macrobrachium rosenbergii nodavirus (MrNV) challenge tests, the MrHc gene was up‐regulated 237‐fold at day 2. A recombinant protein of the MrHc immunoglobulin‐like domain exhibited antibacterial activity against Vibrio vulnificus, V. parahaemolyticus, Aeromonas caviae, A. veronii, A. hydrophila and Bacillus cereus. This study suggested that MrHc may play important roles in the shrimp innate immune response to MrNV infection and bacterial infection.  相似文献   

10.
An attempt was made to determine the replication efficiency of white spot syndrome virus (WSSV) of shrimp in different organs of freshwater rice‐field crab, Paratelphusa hydrodomous (Herbst), using bioassay, PCR, RT‐PCR, ELISA, Western blot and real‐time PCR analyses, and also to use this crab instead of penaeid shrimp for the large‐scale production of WSSV. This crab was found to be highly susceptible to WSSV by intramuscular injection. PCR and Western blot analyses confirmed the systemic WSSV infection in freshwater crab. The RT‐PCR analysis revealed the expression of VP28 gene in different organs of infected crab. The indirect ELISA was used to quantify the VP28 protein in different organs of crab. It was found that there was a high concentration of VP28 protein in gill tissue, muscle, haemolymph and heart tissue. The copy number of WSSV in different organs of infected crab was quantified by real‐time PCR, and the results revealed a steady increase in copy number in different organs of infected crab during the course of infection. The viral inoculum prepared from different organs of infected crab caused significant mortality in tiger prawn, Penaeus monodon (Fabricius). The results revealed that this crab can be used as an alternate host for WSSV replication and production.  相似文献   

11.
Giant river prawn (Macrobrachium rosenbergii) farming plays an important role in the economy of Bangladesh. Presently, it is cultured in around 50 000 ha area with total annual production of 23 240 t. Traditional extensive prawn farming has been expanding over the last three decades through the introduction and adoption of improved culture systems, such as culture of prawn‐carps, prawn‐shrimp‐fish and prawn‐fish‐rice as concurrent and rotational systems. Efforts for the development of improved techniques on broodstock management, seed production and rearing and grow‐out of prawn have been made over the last decade. The outcomes are low‐cost feed for broodstock, production of post‐larvae in net cages (hapa), all‐male prawn culture, periphyton based prawn‐tilapia culture, C/N based prawn culture, organic prawn farming, prawn‐mola culture and prawn‐carp‐mola polyculture. Despite the development of culture technologies, a number of challenges for sustainable development of prawn farming need to be overcome to realize the potentials of this promising sector. Good aquaculture practises at all levels and application of measures for quality control and food safety would ensure sustainable development of prawn farming in Bangladesh.  相似文献   

12.
The protective efficacy of a DNA construct containing extra small virus antisense (XSVAS) gene of nodavirus encapsulated with chitosan nanoparticles (NPs) was investigated in giant freshwater prawn Macrobrachium rosenbergii (De Man, 1879). The delivery was carried out using oral and immersion methods. A plasmid concentration of 100 ng μL?1 when conjugated with chitosan NPs was found to be more effective in increasing the survivability of the infected prawn. The particle mean size, zeta potential and loading efficiency percentage were 297 nm, 27 mV and 85%, respectively. The ability of the chitosan to form a complex with the plasmid was studied by agarose gel electrophoresis. The NPs were characterized by atomic force microscopy (AFM). Persistence study showed the presence of the DNA construct up to 30th day post‐treatment. The oral treatment was found to be better than the immersion treatment for delivery of the chitosan‐conjugated DNA construct. This is probably the first report on the delivery of nanoconjugated DNA construct in M. rosenbergii, against nodavirus.  相似文献   

13.
The VP 28 gene encoding a structural envelope protein of the white spot syndrome virus (WSSV) was cloned into a pET32a(+) expression vector for the production of the recombinant VP28 protein. A purified recombinant protein of 39.9 kDa size was used for polyclonal antibody production in rabbit. Specific immunoreactivity of the rabbit anti rVP28 antiserum to the viral antigen was confirmed by a Western blot. The specificity of this polyclonal anti‐rVP28 antiserum to detect the presence of the virus in WSSV‐infected Penaeus monodon was verified using a immunodot blot assay. Immunodot blot showed a positive reaction in infected shrimp tissues with prominent colour development using 3,3′,5,5′‐tetramethylbenzidine (TMB) as a chromogenic substrate when compared with 3–3′ diaminobenzidine tetrahydrochloride (DAB). Highest signal intensities of the immunodots were observed in infected shrimp pleopod extracts and haemolymph. On comparison with polymerase chain reaction (PCR), immunodot blot could detect 76% of PCR‐positive WSSV‐infected shrimp samples. Immunodot blot was found to be equivalent to first‐step PCR sensitivity to detect WSSV particles estimated to contain 1.0 × 105 viral DNA copies.  相似文献   

14.
Myostatin (MSTN) is an interesting negative growth‐regulating gene that has been well characterized in vertebrates but scantly described in invertebrates. The current study focuses on the downregulation of the MrMSTN gene and subsequently records any histological changes for giant freshwater prawn, Macrobrachium rosenbergii (Mr). In addition, the study also deals with the MrMSTN gene's influence on other growth‐related genes, which include myosin heavy chain, dystrophin‐dystroglycoprotein complex, tropomyosin, farnesoic acid o‐methyl transferase, arginine kinase, cyclophilin, and acyl CoA desaturase. The preliminary histological analysis following MrMSTN silencing favors muscle regeneration, which supports its functional role as a negative growth regulator and its significant effect on the expression of other growth‐related genes. Overall, our results show that the MrMSTN gene could therefore be a potential target for gene manipulation aimed at enhancing the growth and muscle development of M. rosenbergii, which could be beneficial in increasing the total mass production in the postlarva phase at the hatchery level.  相似文献   

15.
Fish iridovirus causes systemic disease with high morbidity and mortality in various species of wild and farm‐raised fish, resulting in severe economic losses. Recently, frequent outbreaks of iridovirus infection have occurred among cultured fish in many Asian countries, emphasizing the need for a protective vaccine programme or the development of a suitable therapy. In this study, we expressed a recombinant major capsid protein (rMCP) of rock bream iridovirus (RBIV) from yeast using codon optimization. The rMCP in yeast was added to feed in an attempt to induce intestinal mucosal immunity for protection against and/or to reduce the severity of fish iridovirus infection. We found that fish immunized orally with rMCP underwent a successful induction of antibodies (P < 0.05) and were protected (P = 0.0001) against viral challenge. Based upon these results, oral administration of immunogenic protein as an antigen can be considered a useful method for implementation of vaccine programmes against iridovirus as well as other marine viral diseases.  相似文献   

16.
The simple immunoprecipitation method was used to isolate tilapia immunoglobulin (Ig) for immunization in order to produce monoclonal antibodies (MAbs) specific to tilapia Ig. First, the tilapia antiserum against bovine serum albumin (BSA) was prepared by peritoneal injection of BSA into tilapia, and the tilapia anti‐BSA antiserum was used to precipitate BSA to form the Ig/BSA immune complex. The Ig/BSA immune complex was then injected into Swiss mice for hybridoma production. After fusion, three hybridoma clones producing MAbs specific to the tilapia antibody were selected by dot blot and Western blot. All MAbs (101A, 59G, and 11A) were bound specifically to the heavy chain of immunoglobulin M (IgM). The MAbs 101A and 59G demonstrated twofold higher affinity than MAb 11A and the commercialized antibody. However, MAbs 11A could also bind to the heavy chain of IgM in Asian seabass, Lates calcarifer, as well. These MAbs can be used to monitor the immune responses of individual fish by indirect ELISA upon exposure to various antigens.  相似文献   

17.
The different products of Eichhornia crassipes leaves including dried E. crassipes powder (DEP), hot‐water treated E. crassipe (HTE), hot‐water extract of E. crassipe (ECE) and dreg of hot‐water extract of E. crassipe extract (dECE) were produced and incorporated into the diet of the prawn, Macrobrachium rosenbergii, as an immunostimulant. Results showed that prawn fed the HTE‐, ECE‐ and dECE‐containing diets for 4 months had increased total haemocyte count, different haemocyte count, phenoloxidase activity, respiratory bursts, superoxide dismutase activity, glutathione peroxidase activity especially in HTE and ECE treatments. The phagocytic activity and clearance efficiency against Lactococcus garvieae of prawn fed the HTE‐ and ECE‐containing diets were significantly higher than those of prawn fed the control diet at 2–4 months. The relative percentage survival of prawn fed the DEP‐, HTE‐, ECE‐ and dECE‐containing diets for 4 months following 144 h challenging with L. garvieae were 19.0%, 38.1%, 38.1% and 33.3%. It was concluded that E. crassipes leaves containing an active component which was easily extracted by hot water can enhance innate immunity and resistance against pathogen of M. rosenbergii by dietary long‐term administration, and the administration of HTE in the diet was the best strategy due to the availability and convenience.  相似文献   

18.
The purpose of this study was to evaluate the resistance to vibriosis, growth, survival and tolerance to stress of the selected prawn, second generation, compared to a non‐selected control. The first generation of selected giant freshwater prawn, which has 10.4% higher of resistance, was used to attain disease resistant freshwater prawn (Macrobrachium rosenbergii) generation through challenge test‐based selection. Resistance test was conducted by infecting the prawn (mean body weight of 10.29 ± 1.40 g) with pathogenic Vibrio harveyi (5 × 105 cfu prawn?1). The growth and survival of the prawn were evaluated by rearing the two populations of prawn in both nursery and grow‐out phases. Stress tolerance test was done by evaluating the viability of postlarvae exposed to environmental stressors, i.e. temperature, salinity, NH3 and formaldehide. Post‐challenge survival of the selected prawn (55.0 ± 5.0%) was about 46% higher than that of the control (37.5 ± 7.5%). The survival of the selected prawn in nursery culture (77.16 ± 0.841%) was significantly higher (p < .05) than that of the control (51.31 ± 2.938%), while the survival in grow‐out culture was similar (p > .05). The growth of selected prawn (4.99 ± 0.03% day?1) was significantly higher than that of the control (4.81 ± 0.05% day?1). There was no difference between treatments on the tolerance level against the tested environmental stressor. Overall data suggested that the selected prawn showed better performance in growth and resistance against vibriosis.  相似文献   

19.
β‐glucan binding protein (βGBP), a pattern recognition protein was purified from the haemolymph of freshwater prawn Macrobrachium rosenbergii by heparin affinity chromatography that showed a single band in native gradient PAGE. The β‐glucan binding property of the purified protein was confirmed in a phenoloxidase (PO) assay, where addition of βGBP along with β‐glucan increased the specific PO activity compared with that of β‐glucan alone. The molecular weight of the βGBP was found to be ~316 kDa on gel filtration chromatography. In SDS‐PAGE, βGBP molecule was reduced to one polypeptide chain of molecular weight ~113 kDa. Thus the βGBP in M. rosenbergii is possibly a homotrimeric molecule. The purified sample run on unreduced condition in SDS‐PAGE also revealed a similar size band (~113 kDa) and hence, the polypeptide chains of βGBP are held by non‐covalent interactions. The purified βGBP samples run in native PAGE was stained positively with alcian blue for carbohydrates and Sudan black for lipids indicating the βGBP to be a glycolipoprotein. With rabbit polyclonal anti‐βGBP serum developed, an indirect ELISA was standardized and the normal βGBP concentration in adult M. rosenbergii serum was quantified to be ~2 mg mL?1. Furthermore, the applicability of the developed ELISA is discussed.  相似文献   

20.
Freshwater prawn production in India that includes farming and wild capture of the giant freshwater prawn, Macrobrachium rosenbergii and the monsoon river prawn, M. malcolmsonii has increased steadily since 1999 reaching a peak output of 42 780 t in 2005, but then declined to 6568 t in 2009–2010. Stunted growth and diseases in ponds because of poor seed quality and the broodstock which had been inbred over several generations; pond water quality issues; and increased cost of production on account of feed, labour and the mandatory certification requirements are suggested to be some of the factors leading to the production declines. While majority of the output occurs in Andhra Pradesh, single crop paddy–prawn production systems in the low‐lying fields of Kerala have helped gradual transformation to a sustainable, organic mode of farming of both rice and prawns, suitable for other states of India. Although the trends by June 2011 indicate that the sector is set to a revival, future prospects of freshwater prawn farming in India will also depend on the expansion of whiteleg shrimp Litopenaeus vannamei that was introduced recently in India and provided a more profitable opportunity for farming.  相似文献   

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