Influence of selected Indian immunostimulant herbs against white spot syndrome virus (WSSV) infection in black tiger shrimp, Penaeus monodon with reference to haematological, biochemical and immunological changes

Immunostimulants are the substances, which enhance the non-specific defence mechanism and provide resistance against the invading pathogenic micro-organism. In order to increase the immunity of shrimps against the WSSV, the methanolic extracts of five different herbal medicinal plants like Cyanodon dactylon, Aegle marmelos, Tinospora cordifolia, Picrorhiza kurooa and Eclipta alba were selected and mixed thoroughly in equal proportion. The mixed extract was supplemented with various concentrations viz. 100 (A), 200 (B), 400 (C), and 800 (D) mg kg(-1) through artificial diets individually. The prepared diets (A-D) were fed individually to WSSV free healthy shrimp Penaeus monodon with an average weight of 8.0 +/- 0.5 g for 25 days. Control diet (E), devoid of herbal extract was also fed to shrimps simultaneously. After 25 days of feeding experiment, the shrimps were challenged with WSSV, which were isolated and propagated from the infected crustaceans. The shrimps succumbed to death within 7 days when fed on no herbal immunostimulant diet (E). Among the different concentrations of herbal immunostimulant supplemented diets, the shrimps fed on diet D (800 mg kg(-1)) significantly (P < 0.0001) had more survival (74%) and reduction in the viral load. Also the better performance of haematological, biochemical and immunological parameters was found in the immunostimulant incorporated diets fed shrimps. The present work revealed that the application of herbal immunostimulants will be effective against shrimp viral pathogenesis and they can be recommended for shrimp culture. (c) 2006 Published by Elsevier Ltd.

Differential expression of three Paralichthys olivaceus Hsp40 genes in responses to virus infection and heat shock

Heat shock proteins (Hsps) are a family of highly conserved cellular proteins present in all organisms, mediating a range of essential housekeeping and cytoprotective functions as well-known molecular chaperons and recently as regulators of the immune response. By subtractive suppression hybridization, three Hsp40 homologues have been identified in the flounder (Paralichthys olivaceus) embryonic cells (FEC) after treatment with UV-inactivated turbot (Scophthalmus maximus L.) rhabdovirus (SMRV), termed PoHsp40A4, PoHsp40B6 and PoHsp40B11, whose encoded proteins all possess the conserved DnaJ domain, a signature motif of the Hsp40 family. Based on different protein structure and phylogenetic analysis, they can be categorized into two subfamilies, PoHsp40A4 for Type I Hsp40, PoHsp40B6 and PoHsp40B11 for Type 11 Hsp40. Further expression analysis revealed two very different types of kinetics in response either to heat shock or to virus infection, with a marked induction for PoHsp4OA4 and a weak one for both PoHsp40B6 and PoHsp40B11. A very distinct tissue distribution of mRNA was also revealed among the three genes, even between PoHsp40B6 and PoHsp40B11. This is the first report on the transcriptional induction of Hsp40 in virally stimulated fish cells, and the differential expressions might reflect their different roles in unstressed and stressed cells. (c) 2005 Elsevier Ltd. All rights reserved.

Optimization by orthogonal array design and humoral immunity of the bivalent vaccine against Aeromonas hydrophila and Vibrio fluvialis infection in crucian carp (Carassius auratus L.)

Aeromonas hydrophila and Vibrio fluvialis are the causative agents of a serious haemorrhagic septicaemia that affects a wide range of freshwater fish in China. In order to develop a bivalent anti-A. hydrophila and anti-V. fluvialis formalin-killed vaccine to prevent this disease, an orthogonal array design (OAD) method was used to optimize the production conditions, using three factors, each having three levels. The effects of these factors and levels on the relative per cent survival for crucian carp were quantitatively evaluated by analysis of variance. The final optimized formulation was established. The data showed that inactivation temperature had a significant effect on the potency of vaccine, but formalin concentration did not. The bivalent vaccine could elicit a strong humoral response in crucian carp (Carassius auratus L.) against both A. hydrophila and V. fluvialis simultaneously, which peaked at 3 or 5 weeks respectively. Antibody titres remained high until week 12, the end of the experiment, after a single intraperitoneal injection. The verification experiment confirmed that an optimized preparation could provide protection for fish at least against A. hydrophila infection, and did perform better than the non-optimized vaccine judged by the antibody levels and protection rate, suggesting that OAD is of value in the development of improved vaccine formulations.

Identification of immune genes in grass carp Ctenopharyngodon idella in response to infection of the parasitic copepod Sinergasilus major

The parasitic copepod Sinergasilus major is an important pathogen of grass carp Ctenopharyngodon idella. To understand the immune response of grass carp to the copepod infection, suppression subtractive hybridization method was employed to characterize genes up-regulation during the copepod infection in liver and gills of the fish. One hundred and twenty-two dot blot positive clones from infected subtracted library were sequenced. Searching available databases by using these nucleotide sequences revealed that 23 genes are immune-related, including known acute-phase reactants, and four novel genes encoding proteins such as source of immunodominant MHC-associated peptides (SIMP), TNF receptor-associated factor 2 binding protein (T2BP), poliovirus receptor-related protein 1 precursor, glycoprotein A repetitions predominant (GARP). The differential expression of seven immune genes, i.e. GARP, alpha-2-macroglobulin, MHC class I, C3, SIMP, T2BP, transferrin, as a result of infection was further confirmed by RT-PCR, with the up-regulation of alpha-2-macroglobulin, MHC class I, C3, SIMP and T2BP in the liver of infected fish, and down-regulation of SIMP in the gills of infected fish. The present study provides foundation for understanding grass carp immune response and candidate genes for further analysis.

Enhanced resistance to Aeromonas hydrophila infection and enhanced phagocytic activities in human lactoferrin-transgenic grass carp (Ctenopharyngodon idellus)

Human lactoferrin (hLF) is an iron-binding protein with antimicrobial and immunomodulatory activities. hLF cDNA was transferred into grass carp via electroporated sperm. The production of transgenic fish was as high as 55% tinder the best parameters. 2(11) pulses and 20-min incubation. The expression of the transgene was demonstrated by the detection of hLF mRNA by RT-PCR. We also investigated the response of G(0) transgenic grass carp to Aeromonas hydrophila infection. Serum lysozyme activities (P>0.05) and phagocytic activities of kidney cells (P<0.05) were measured in transgenic individuals. The transgenic fish not only cleared A. hydrophila significantly faster than the control carp (P<0.05), but also showed enhanced phagocytic activities. The result shows that hLF has immunomodulatory activities in hLF-transgenic grass carp. The transgenic grass carp exhibited enhanced immunity to A. hydrophila infection. These results reveal that the mechanisms of disease resistance are different between hLF-transgenic plants and hLF-transgenic grass carp. (C) 2004 Elsevier B.V. All rights reserved.

Rickettsia-like organism infection in a freshwater cultured fish Ophiocephalus argus C. in China

From 2001 to 2002, a new and emergent infectious disease of Ophiocephalus argus occurred in a fishery in Hubei Province, China, with an incidence of 60% similar to 70% and a mortality as high as 100 %. The diseased fish showed an enlarged abdomen, the millet-like nodules in internal organs, and the swollen kidney which was composed of 5 similar to 10 sarcoma-like bodies in cream or gray-white colour or ulcerated into beandregs-like substance. Light microscopic observation revealed the basophilic or acidphilic inclusions in cytoplasm of the cells and the granulomas, a diffusive chronic inflammation in internal organs. Further analysis under an electron microscope indicated that the intracytoplasmic inclusions were rickettsia-like organisms (RLOs) that are either spherical or coccoid, with variable size, ranging from 0.5 similar to 1.5 mum in diameter, and enclosed within membrane-bound cytoplasmic vacuoles. RLO had a central nucleoid region with some fine filamentous structures and an electron-dense granule. Its cytoplasm contained abundant ribosomal bodies. Occasionally, RLO appeared to be divided by binary fission. RLOs were also observed in the homogenized tissue of infected fish. The results suggested that the death of cultured O. argus was caused by RLO infection.

Infection and propagation of lymphocystis virus isolated from the cultured flounder Paralichthys olivaceus in grass carp cell lines

The causative agent of lymphocystis disease that frequently occurs in cultured flounder Paralichthys olivaceus in China is lymphocystis virus (LV). In this study, 13 fish cell lines were tested for their susceptibility to LV. Of these, 2 cell lines derived from the freshwater grass carp Ctenopharyngodon idellus proved susceptible to the LV, and 1 cell line, GCO (grass carp ovary), was therefore used to replicate and propagate the virus. An obvious cytopathic effect (CPE) was first observed in cell monolayers at 1 d post-inoculation, and at 3 d this had extended to about 75% of the cell monolayer. However, no further CPE extension was observed after 4 d. Cytopathic characteristics induced by the LV were detected by Giemsa staining and fluorescence microscopic observation with Hoechst 33258 staining. The propagated virus particles were also observed by electron microscopy. Ultrastructure analysis revealed several distinct cellular changes, such as chromatin compaction and margination, vesicle formation, cell-surface convolution, nuclear fragmentation and the occurrence of characteristic 'blebs' and cell fusion. This study provides a detailed report of LV infection and propagation in a freshwater fish cell line, and presents direct electron microscopy evidence for propagation of the virus in infected cells. A possible process by which the CPEs are controlled is suggested.

Molecular characterization and IFN signal pathway analysis of Carassius auratus CaSTAT1 identified from the cultured cells in response to virus infection

Type I interferon (IFN) exerts its pleiotropic effects mainly through the JAK-STAT signaling pathway, which is presently best described in mammals. By subtractive suppression hybridization, two fish signaling factors, JAK1 and STAT1, had been identified in the IFN-induced crucian carp Carassius auratus L. blastulae embryonic (CAB) cells after treatment with UV-inactivated grass carp hemorrhagic virus (GCHV). Further, the full-length cDNA of STAT1, termed CaSTAT1, was obtained. It contains 2926 bp and encodes a protein of 718 aa. CaSTAT1 is most similar to rat STAT1 with 59% identity overall and displays all highly conserved domains that the STAT family possesses. Like human STAT1beta, it lacks the C-terminus acting as transcriptional activation domain in mammals. By contrast, only a single transcript was detected in virus-induced CAB cells. Expression analysis showed that CaSTAT1 could be activated by stimulation of CAB cells with poly I:C, active GCHV, UV-inactivated GCHV or CAB IFN, and displayed diverse expression patterns similar to that of mammalian STATI. Additionally, the expression of an antiviral gene CaMx1 was also induced under the same conditions, and expression difference between CaSTAT1 and CaMx1 was revealed by induction of CAB IFN. These results provide molecular evidence supporting the notion that the fish IFN signaling transduction pathway is similar to that in mammals. Fish IFN exerts its multiple functions, at least antiviral action, through a JAK-STAT pathway. (C) 2004 Elsevier Ltd. All rights reserved.

Three tetraspanins from Chinese shrimp, Fenneropenaeus chinensis, may play important roles in WSSV infection

Three members of the tetraspanin/TM4SF superfamily were cloned from Chinese shrimp, Fenneropenaeus chinensis. The deduced amino acid sequences of the three proteins have typical motifs of the tetraspanin/TM4SF superfamily. Phylogenetic analysis of the proteins, together with the known tetraspanins of invertebrates and vertebrates, revealed that they belong to different tetraspanin subfamilies: CD9, CD63 and tetraspanin-3. The three cloned genes of CD9, CD63 and tetraspanin-3 showed apparently different tissue distributions. The CD9 gene (FcCD9) was specifically expressed in the hepatopancreas. While for the CD63 gene (FcCD63), the highest expression was detected in nerves, epidermis and heart, with low expression in haemocytes, ovary, gill, hepatopancreas and stomach and no expression in intestine, muscle and lymphoid organ. Compared with FcCD9 and FcCD63, the tetraspanin-3 gene (FcTetraspanin-3) was more broadly expressed and its highest expression was detected in the intestine. Its expression in nerves was lower than in the intestine, but was higher than in other tissues. Expression in haemocytes, ovary and muscle was much lower than in other tissues. The expression profiles of FcCD9, FcCD63 and FcTetraspanin-3 in different tissues, including haemocytes, lymphoid organ and hepatopancreas, were compared by real-time PCR when shrimp were challenged by live white spot syndrome virus (WSSV) and heat-inactivated WSSV. All three tetraspanins were markedly up-regulated in the live WSSV-challenged shrimp tissues. The data suggested that the three cloned members of TM4SF superfamily in Chinese shrimp may play a key role in the route of WSSV infection.