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Expression analysis of heat shock protein genes during Aeromonas hydrophila infection in rohu, Labeo rohita, with special reference to molecular characterization of Grp78.

ABSTRACT: Heat shock protein (Hsp) genes are stress-related genes that activate the host immune system during infection. Hsp genes expression in fish, studied during bacterial infections, is mostly confined to Hsp70 and Hsp90. The present study is an expression analysis of seven Hsp genes: Apg2, Hsp90, Hsp70, glucose-regulated protein 78 (Grp78), heat shock cognate 70 (Hsc70), Grp75, and Hsp30 during Aeromonas hydrophila infection in rohu, Labeo rohita. Forty-eight rohu juveniles were challenged with 3?×?10(7) cfu bacteria per 20 g of body weight intraperitoneally. The expression of these genes was studied in infected liver, anterior kidney, and spleen tissues of rohu at different time periods: 0, 1, 3, 6, 12, 24, 48, 72 h, 7, and 15 days post-infection by qPCR. The Hsp gene modulation was greater in liver as compared to spleen and kidney tissues. Induced expression of Apg2, Hsp90, Grp78, Grp75, and Hsc70 was noticed during peak periods (3 to 24 h post-challenge) of bacterial infectivity. Hsp70 was found to be down-regulated during the process of infection. In contrast to the other six genes, Hsp30 showed a variable expression pattern in all three tissues. Grp78 was found to be the most highly (1,587-fold) expressed gene in liver at 12 h post-challenge. Further, molecular characterization of Grp78 revealed it to be a highly conserved Hsp gene, essential not only during infection but also during early developmental stages of rohu, and its expression was noticed in all organs studied except in gill tissues, which indicated its potential immune regulatory role during infection process.

SUBMITTER: Das S

PROVIDER: S-EPMC4255248 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Expression analysis of heat shock protein genes during Aeromonas hydrophila infection in rohu, Labeo rohita, with special reference to molecular characterization of Grp78.

Das Sweta S Mohapatra Amruta A Sahoo P K PK

Cell stress & chaperones 20140719 1

Heat shock protein (Hsp) genes are stress-related genes that activate the host immune system during infection. Hsp genes expression in fish, studied during bacterial infections, is mostly confined to Hsp70 and Hsp90. The present study is an expression analysis of seven Hsp genes: Apg2, Hsp90, Hsp70, glucose-regulated protein 78 (Grp78), heat shock cognate 70 (Hsc70), Grp75, and Hsp30 during Aeromonas hydrophila infection in rohu, Labeo rohita. Forty-eight rohu juveniles were challenged with 3 × ...[more]

PMID: 25037476

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Project description:Aeromonas hydrophila is a Gram-negative bacterium that causes high mortality in different fish species and at different growth stages. Although vaccination has significantly contributed to the decline of disease outbreaks in aquaculture, the use of oral vaccines has lagged behind the injectable vaccines due to lack of proven efficacy, that being from primary immunization or by use of boost protocols. In this study, the outer membrane protein W (OmpW) of A. hydrophila was cloned, purified, and encapsulated in poly d,l-lactide-co-glycolic acid (PLGA) nanoparticles (NPs) for oral vaccination of rohu (Labeo rohita Hamilton). The physical properties of PLGA NPs encapsulating the recombinant OmpW (rOmpW) was characterized as having a diameter of 370-375 nm, encapsulation efficiency of 53% and -19.3 mV zeta potential. In vitro release of rOmpW was estimated at 34% within 48 h of incubation in phosphate-buffered saline. To evaluate the efficacy of the NP-rOmpW oral vaccine, two antigen doses were orally administered in rohu with a high antigen (HiAg) dose that had twice the amount of antigens compared to the low antigen (LoAg) dose. Antibody levels obtained after vaccination showed an antigen dose dependency in which fish from the HiAg group had higher antibody levels than those from the LoAg group. The antibody levels corresponded with post challenge survival proportions (PCSPs) and relative percent survival (RPS) in which the HiAg group had a higher PCSP and RPS than the LoAg group. Likewise, the ability to inhibit A. hydrophila growth on trypticase soy agar (TSA) by sera obtained from the HiAg group was higher than that from the LoAg group. Overall, data presented here shows that OmpW orally administered using PLGA NPs is protective against A. hydrophila infection with the level of protective immunity induced by oral vaccination being antigen dose-dependent. Future studies should seek to optimize the antigen dose and duration of oral immunization in rohu in order to induce the highest protection in vaccinated fish.

Project description:BACKGROUND: Production of carp dominates world aquaculture. More than 1.1 million tonnes of rohu carp, Labeo rohita (Hamilton), were produced in 2010. Aeromonas hydrophila is a bacterial pathogen causing aeromoniasis in rohu, and is a major problem for carp production worldwide. There is a need to better understand the genetic mechanisms affecting resistance to this disease, and to develop tools that can be used with selective breeding to improve resistance. Here we use a 6 K SNP array to genotype 21 full-sibling families of L. rohita that were experimentally challenged intra-peritoneally with a virulent strain of A. hydrophila to scan the genome for quantitative trait loci associated with disease resistance. RESULTS: In all, 3193 SNPs were found to be informative and were used to create a linkage map and to scan for QTL affecting resistance to A. hydrophila. The linkage map consisted of 25 linkage groups, corresponding to the number of haploid chromosomes in L. rohita. Male and female linkage maps were similar in terms of order, coverage (1384 and 1393 cM, respectively) and average interval distances (1.32 and 1.35 cM, respectively). Forty-one percent of the SNPs were annotated with gene identity using BLAST (cut off E-score of 0.001). Twenty-one SNPs mapping to ten linkage groups showed significant associations with the traits hours of survival and dead or alive (P <0.05 after Bonferroni correction). Of the SNPs showing significant or suggestive associations with the traits, several were homologous to genes of known immune function or were in close linkage to such genes. Genes of interest included heat shock proteins (70, 60, 105 and "small heat shock proteins"), mucin (5b precursor and 2), lectin (receptor and CD22), tributyltin-binding protein, major histocompatibility loci (I and II), complement protein component c7-1, perforin 1, ubiquitin (ligase, factor e4b isoform 2 and conjugation enzyme e2 c), proteasome subunit, T-cell antigen receptor and lymphocyte specific protein tyrosine kinase. CONCLUSIONS: A panel of markers has been identified that will be validated for use with both genomic and marker-assisted selection to improve resistance of L. rohita to A. hydrophila.

Project description:The current study aimed at assessing the immunostimulatory properties of silver nanoparticles (AgNPs) on Labeo rohita, and understanding how it affects the growth, cellular ultrastructure, the expression level of immune genes, and infection risk from Aeromonas hydrophila. Fish (avg wt: 30.1±3.26 g) were fed diets with four separate AgNP inclusion levels (0 µgKg-1 [basic diet, T1], 10 µgKg-1 [T2], 15 µgKg-1 [T3], and 20 µgKg-1 [T4]) for 56 days. After the feeding trial, growth, histological, immunological parameters, and protective immune response against A. hydrophila were assessed. The fish in the treatment groups including T1(control), the T3 growth indices, such as specific growth rate (7.56±0.26) and percent weight gain (231.05±3.21), was statistically higher (P < 0.05). In the immunological and oxidative parameters, levels of SOD and catalase decreased in correlation with a rise in the inclusion doses of AgNP in the liver, and a reduction in catalase values was recorded in the gill. With the addition of AgNP, the NBT value was decreased in the gill, and T3 had a considerably larger (P<0.05) value in the liver (0.493±0.02). The kidney of the L. rohita fed AgNP (0 and20 µgKg-1 AgNP) showed expansion through Bowman's gaps, severing of glomeruli with haemorrhage, as well as atrophic spots between its gaps. The liver showed fibrosis, karyolysis, and the removal of the hepatocytes wall. The gill, liver, kidney, and muscle of fish-fed diets supplemented with AgNP, showed that interleukin-8 (IL-8), and cyclooxygenase-2 (COX-2), were up-regulated. Expression was considerably higher in T3 compared with the control. However, the control group that wasn't given AgNP supplemented diet had increased levels of TGF-beta. Additionally, fish on the T3 diet showed much greater post-challenge survival rates (90%). These findings strongly suggest that dietary inclusion of AgNP (at 10 and 15 µgKg-1 feed) enhances growth, health, and protective immune response against A. hydrophila.

Dataset Information

Expression analysis of heat shock protein genes during Aeromonas hydrophila infection in rohu, Labeo rohita, with special reference to molecular characterization of Grp78.

Publications

Expression analysis of heat shock protein genes during Aeromonas hydrophila infection in rohu, Labeo rohita, with special reference to molecular characterization of Grp78.

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