Experimental infection of Enterocytozoon hepatopanaei parasite (EHP) of penaeid shrimp in Indian marine crabs.

Enterocytozoon hepatopanaei parasite (EHP) is identified as an emerging pathogenic microsporidium parasite in shrimp culture industry. Though the etiology, disease pattern and sustainability of shrimp are well known, significantly less research has been carried out about the disease transmission and symptoms of infected aquatic animals. The present study aims is to determine the disease carrier status of five different species of Indian marine crabs (Scylla olivacea, Scylla serrata, Portunus pelagicus, Ocypode quadrata and Portunus sanquinolentus) using EHP. At the first instance, oral infection and intramuscular injection were performed to determine the susceptibility of the parasite at 50 days post-infection and it was observed that there was no mortality. The experimental infected crabs were confirmed by polymerase chain reaction, bioassay and histopathology. The crabs were EHP-PCR positive at 5th day post-infection (d.p.i) in gills, heart, hepatopancreas, haemolymph and muscle tissue. However, after 5th d.p.i EHP was PCR negative in all the tissue samples. There were no mortalities and histological changes in the negative group and experimental group. Therefore, marine crabs are found to be not suitable hosts for replicating EHP spores but crabs fecal matters are PCR positive till 5th d.p.i. Therefore, marine crabs are having the possibilities of acceptance as a vector for Enterocytozoon hepatopanaei in shrimp. Shrimp farmers need to take necessary action to control this deadly infection in shrimp ponds.

The Oxidative Injury of Extracellular Hemoglobin Is Associated With Reactive Oxygen Species Generation of Grass Carp (Ctenopharyngodon idella).

Intravascular hemolysis is a fundamental feature of hemorrhagic venereal infection or tissue and releases the endogenous damage-associated molecular pattern hemoglobin (Hb) into the plasma or tissues, which results in systemic inflammation, vasomotor dysfunction, thrombophilia, and proliferative vasculopathy. However, how the cytotoxic Hb affects the tissues of grass carp remains unclear. Here, we established a hemolysis model in grass carp by injecting phenylhydrazine (PHZ). The data revealed that the PHZ-induced hemolysis increased the content of Hb and activated the antioxidant system in plasma. The histopathology analysis data showed that the PHZ-induced hemolysis increased the accumulation of Hb and iron both in the head and middle kidney. The results of quantitative real-time PCR (qRT-PCR) detection suggested that the hemolysis upregulated the expressions of iron metabolism-related genes. In addition, the immunofluorescence and immunohistochemistry data revealed that the hemolysis caused an obvious deposition of collagen fiber, malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE) accumulation and increased the content of oxidative-related enzymes such as ß-galactosidase (ß-GAL), lipid peroxide (LPO), and MDA in both the head and middle kidney. Furthermore, the PHZ-induced hemolysis significantly increased the production of reactive oxygen species (ROS), which resulted in apoptosis and modulated the expressions of cytokine-related genes. Taken together, excess of Hb released from hemolysis caused tissue oxidative damage, which may be associated with ROS and inflammation generation.

Induction of apoptosis in SSN-1cells by Snakehead Fish Vesiculovirus (SHVV) via Matrix protein dependent intrinsic pathway.

An increasing important area in immunology is the process cell death mechanism, enabling the immune system triggered thru extrinsic or intrinsic signals to effectively remove unwanted or virus infected cells called apoptosis. A recently isolated infectious Snakehead fish vesiculovirus (SHVV), comprising negative strand RNA and encoded viral matrix (M) proteins, is responsible for causing cytopathic effects in infected fish cells. However, the mechanism by which viral M protein mediates apoptosis has not been elucidated. Therefore, in the present experiments, it was investigated the regulatory potential of apoptosis signals during SHVV infection. By employing the model of SHVV infection in SSN-1 cells, the accelerated apoptosis pathway involves an intrinsic pathway requiring the activation of caspase-9 but not caspase-3 or -8. In the groups of infection (SHVV) or treatment (hydrogen peroxide) were induced apoptotic morphological changes and indicated the activation of the main caspases, i.e.; executioner caspase-3, initiators caspase-8 and caspase-9 using colorimetric assays. Turning to the role of viral M protein when it was overexpressed in SSN-1 cells, it was indicated that the viral M gene alone has the ability to induce apoptosis. To elucidate the mechanism of apoptosis in SSN-1 cells, the activation inhibitors of main caspases were used showing that inhibiting of caspase-3 or caspase-8 activation did not seize induction of apoptosis in virus-infected SSN-1 cells. However, the inhibiting of caspase-9 activation reduced significantly the apoptosis initiation process and sharply the expression of viral M gene, suggesting that SHVV plays a major role in the early induction of apoptosis by caspase-9. Interestingly, there were also differences in the mitochondrial membrane potential after the apoptotic induction of caspases, which confirm that caspase-9 is primarily responsible for the cleavage of caspases during apoptosis. Taken together, these findings can therefore be assumed that viral M protein induces apoptosis via the intrinsic apoptotic pathway in SHVV infecting SSN-1 cells.

Immediate Effect of Cervico-thoracic Mobilization on Deep Neck Flexors Strength in Individuals with Forward Head Posture: A Randomized Controlled Trial.

INTRODUCTION: Forward head posture is the most frequently observed postural deviations and is said to be associated with shortening of posterior cervical extensors and weakening of the anterior deep cervical flexors. Manual therapy has the potential to achieve reflexogenic changes in muscle and enhance the motor activity and strength. PURPOSE OF THE STUDY: To evaluate the immediate effect of grade IV cervicothoracic Maitland mobilization on deep neck flexors strength in individuals with forward head posture. STUDY DESIGN: A Single-blinded randomized placebo-controlled trial. METHOD: Sixty individuals were randomly divided into two groups. Placebo-controlled (PBO) group (n = 30) received the grade I and experimental (EXP) group (n = 30) received grade IV posteroanterior central and unilateral Maitland mobilization from the upper cervical to the upper thoracic spine. Outcome measure: Clinical Cranio-cervical flexion test (CCFT) was used to measure the motor activity and the strength of deep neck flexors. RESULTS: The strength of deep neck flexors effectively increased (p = <0.0001) after advocating grade IV mobilization. CONCLUSION: This study concluded that grade IV central and unilateral posteroanterior Maitland mobilization demonstrated significant increase in the deep neck flexors strength in individuals with forward head posture.

Effects of oligochitosan on the growth, immune responses and gut microbes of tilapia (Oreochromis niloticus).

The immunomodulatory effects of oligochitosan have been demonstrated in several fish. However, the underlying mechanisms are not well characterized. The profound interplay between gut microbes and aquaculture has received much scientific attention but understanding the alternations of microbes populating in gut of tilapia (Oreochromis niloticus) fed with oligochitosan remains enigmatic. In this study, the effects of oligochitosan on the growth, immune responses and gut microbes of tilapia were investigated. The feeding trial was conducted in triplicates with the control diet supplemented with oligochitosan at different concentrations (0, 100, 200, 400 or 800 mg/kg). Following a six-week feeding trial, body weights of the fish supplemented with 200 mg/kg and 400 mg/kg oligochitosan were significantly higher than that of the control group. To address the immune responses stimulated by oligochitosan, by the quantitative real time PCR (qRT-PCR), the mRNA expression levels of CSF, IL-1ß, IgM, TLR2 and TLR3 genes from head kidney were all significantly up-regulated in the 400 mg/kg group compared to the control. To characterize the gut microbes, bacterial samples were collected from the foregut, midgut, and hindgut, respectively and were subjected to high-throughput sequencing of 16S rDNA. The results showed that significantly lower abundance of Fusobacterium was detected in the hindgut of 400 mg/kg group compared to the control. Additionally, beta-diversity revealed that both gut habitat and oligochitosan had effects on the gut bacterial assembly. To further elucidate the mechanism underlying the effects of oligochitosan on bacterial assembly, the results showed that difference dosages of dietary oligochitosan could alter the specific metabolic pathways and functions of the discriminatory bacterial taxa, resulting in the different bacterial assemblies. To test the antibacterial ability of tilapia fed with oligochitosan, when the tilapias were challenged with Aeromonas hydrophila, the mortality of groups fed with dietary oligochitosan was significantly lower than that of the control. Taken together, appropriate dietary oligochitosan could improve growth, immune responses and alter the bacterial flora in the intestine of tilapia, so as to play a role in fighting against the bacterial infection.

The RAG2 gene of yellow catfish (Tachysurus fulvidraco) and its immune response against Edwardsiella ictaluri infection.

Recombination-activating gene 2 (rag 2) allies with recombination-activating gene 1 (rag 1) and regulates the V(D)J recombination of immunoglobulin (Ig) and T-cell receptor (TCR) genes. Being a key player in the adaptive immune response of vertebrates, functional characterization of rag 2 from yellow catfish is beneficial for understanding the biological response towards the pathogens. In this report, we have cloned and characterized the rag 2 gene of yellow catfish, and a particular pattern of expression was analysed in the major tissues of yellow catfish. The results showed that the open reading frame (ORF) of yellow catfish rag 2 was 1596 bp in length, which encodes a peptide of 531 amino acids. The multiple sequence alignment and phylogenetic analysis of rag 2 of yellow catfish with other species showed the conserved regions and the classical taxonomic evolution among the different vertebrate species. The qRT-PCR and Western blot results revealed that rag 2 transcripts and proteins were present in various tissues of yellow catfish with relatively high expression in the tissues of the thymus, head-kidney, and spleen. The systematic distribution analysis of the rag 2 expression by immunohistochemistry (IHC) using the rabbit polyclonal antibody, exposed relatively high expression in head kidney, spleen and thymus tissues after infected with Edwardsiella ictaluri. Moreover, the temporal expression of rag 2 and pro-inflammatory cytokines (IL-1ß and TNF-α) were significantly upregulated at different time points in the specific lymphoid tissues of yellow catfish following E. ictaluri infection. Our findings suggest that rag 2 potentially exhibited the immunological response in primary lymphoid tissues of yellow catfish against bacterial infection. This study will provide an essential source about rag 2 gene and its relationship with the inflammatory cytokines during infection.

Transcriptomic profiles of striped snakehead cells (SSN-1) infected with snakehead vesiculovirus (SHVV) identifying IFI35 as a positive factor for SHVV replication.

Snakehead vesiculovirus (SHVV) has caused great economic loss in snakehead fish culture in China. However, there is no effective strategy to prevent the epidemic of the virus. Understanding the host factors in response to virus infection is the basis for the prevention of viral disease. In this study, the transcriptomic profiles of SHVV-infected and mock-infected SSN-1 cells (derived from striped snakehead, Channa striatus) at 3 and 24 h (h) post of infection (poi) were obtained using high-throughput sequencing technique. A total of 93,372 unigenes were obtained. The differently expressed genes (DEGs) of SSN-1 cells upon SHVV infection were thereby identified, including 3668 and 3536 DEGs at 3 and 24 h poi, respectively. These DEGs were involved in many pathways of viral pathogenesis, including retinoic acid-inducible gene I (RIG-I) like receptors pathway, Toll-like receptor signaling pathway, NF-kappa B signaling pathway, PI3K-Akt signaling pathway and MAPK signaling pathway. Therefore, several immune-related DEGs were randomly selected and confirmed by quantitative real-time PCR (qRT-PCR). In addition, the effects of the interferon inducible protein 35 (IFI35) on SHVV replication were further investigated. Over-expression or inhibition of IFI35 significantly promoted or reduced SHVV replication at the level of viral gene expression, which indicated that IFI35 might be a positive factor for SHVV replication in SSN-1 cells. Our findings presented some valuable information, which will benefit for future study on SHVV-host interactions.

MicroRNAs profiles of Chinese Perch Brain (CPB) cells infected with Siniperca chuatsi rhabdovirus (SCRV).

MicroRNAs are non-coding RNAs, which widely participate in biological processes. In recent years, Siniperca chuatsi rhabdovirus (SCRV) has caused mass mortality in Chinese perch (Siniperca chuatsi). To identify specific miRNAs involved in SCRV infection, deep sequencing of microRNA on Chinese perch brain cell line (CPB) with or without SCRV infection were performed at 6 and 12 h post of infection (hpi). Totally 382 miRNAs were identified, including 217 known miRNA aligned with zebrafish miRNAs and 165 novel miRNAs by MiRDeep2 program. Of which 15 and 35 differentially-expressed miRNAs were determined respectively to 6 and 12 hpi. Nine miRNAs were selected randomly from the differentially-expressed miRNAs and validated by quantitative real-time PCR (qRT-PCR). These results were consistent with the microRNA sequencing results. Besides, target genes of 98 differentially-expressed miRNAs were predicted. Three of miRNAs (miR-122, miR-214, miR-135a) were selected, and its effects were analyzed in CPC cells transfected with appropriate miRNA mimics/inhibitors to evaluate its regulation effects by qRT-PCR and western blot. The results demonstrated that miR-214 inhibited the replication of SCRV, while miR-122 promoted the replication of SCRV and there was no correlation between the miR-135a and SCRV replication. These results will pave a new way for the development of effective strategies against the SCRV infection.

Glutamine starvation inhibits snakehead vesiculovirus replication via inducing autophagy associated with the disturbance of endogenous glutathione pool.

Autophagy is a degradation cellular process which also plays an important role in virus infection. Glutamine is an essential substrate for the synthesis of glutathione which is the most abundant thiol-containing compound within the cells and plays a key role in the antioxidant defense and intracellular signaling. There is an endogenous cellular glutathione pool which consists of two forms of glutathione, i.e. the reduced form (GSH) and the oxidized form (GSSG). GSH serves as an intracellular antioxidant to maintain cellular redox homeostasis by scavenging free radicals and other reactive oxygen species (ROS) which can lead to autophagy. Under physiological conditions, the concentration of GSSG is only about 1% of total glutathione, while stress condition can result in a transient increase of GSSG. In our previous report, we showed that the replication of snakehead fish vesiculovirus (SHVV) was significant inhibited in SSN-1 cells cultured in the glutamine-starvation medium, however the underlying mechanism remains enigmatic. Here, we revealed that the addition of L-Buthionine-sulfoximine (BSO), a specific inhibitor of the GSH synthesis, could decrease the γ-glutamate-cysteine ligase (GCL) activity and GSH levels, resulting in autophagy and significantly inhibition of the replication of SHVV in SSN-1 cells cultured in the complete medium. On the other hand, the replication of SHVV was rescued and the autophagy was inhibited in the SSN-1 cells cultured in the glutamine-starvation medium supplemented with additional GSH. Furthermore, the inhibition of the synthesis of GSH had not significantly affected the generation of reactive oxygen species (ROS). However, it significantly decreased level of GSH and enhanced the level of GSSG, resulting in the decrease of the value of GSH/GSSG, indicating that it promoted the cellular oxidative stress. Overall, the present study demonstrated that glutamine starvation impaired the replication of SHVV in SSN-1 cells via inducing autophagy associated with the disturbance of the endogenous glutathione pool.

Features of chicken egg yolk immunoglobulin (IgY) against the infection of red-spotted grouper nervous necrosis virus.

Red-spotted grouper nervous necrosis virus (RGNNV) is one of the most important viruses which mainly infects the larva of marine and freshwater fish with high mortality and affects the fishery industry worldwide. Currently, there are no effective vaccines available for the fish larva infected with NNV. Immunoglobulin yolk (IgY) origin of oviparous animals is passed from the blood serum and concentrated in the egg yolk. With the advantages of high yield, cost-effectiveness, and high stability, IgY can be widely used in passive immunization, especially in young animals in which adaptive immunity is not fully developed. In this study, we have cloned and expressed the recombinant capsid protein of RGNNV in Escherichia coli and used as an immunogen for generating specific anti-RGNNV IgY antibody in laying hens. Water-soluble fractions (WSF) of the specific IgY were isolated from egg yolk and purified by two-step precipitation with saturated ammonium sulfate salting. By Enzyme linked immunosorbent assay (ELISA), the titer of the IgY reached a peak at the 6th week post of immunization and had a strong stability at a wide range of temperature, pH, and pepsin enzyme digestion. The purified IgY was competent to neutralize and completely inhibited the RGNNV replication in the grouper fin cell line (GF-1), indicating that it was highly specific and effectively recognized RGNNV. The results will pave a new way for the prevention of RGNNV infection.

Distribution of mannose receptor in blunt snout bream (Megalobrama amblycephala) during the embryonic development and its immune response to the challenge of Aeromonas hydrophila.

The mannose receptor (MR) is a type I transmembrane protein. Its ectodomain has eight C-type lectin-like domains, which are able to recognize and mediate the phagocytosis of a wide range of pathogens. Comprehensive studies have revealed that mammalian MR is widely distributed in the mononuclear phagocyte system (MPS, previously known as the reticuloendothelial system) and play a key role both in the physiological clearance and cell activation. Hitherto, neither the MR distribution, nor the function of clearance and cell activation has been investigated in fish. In the previous study, we have reported the full-length cDNA of blunt snout bream MR, analyzed its structure and relative mRNA expression during embryogenesis and in the liver, head kidney, spleen and intestine of fish after stimulation with killed Aeromonas hydrophila. In the present study, we developed a rabbit polyclonal antibody against MR and undertook a systematic survey of the expression of MR at the protein level by immunohistochemistry. To get more information about MR function, the mRNA expression of MR, pro-inflammatory factor TNF-α and anti-inflammatory factor ARG2 genes was measured by qRT-PCR in the liver, head kidney, and spleen after A. hydrophila challenge. We first observed MR expression in the yolk sac at the fertilized egg stage and possibly MR was expressed by early macrophages. We also showed the MR distribution in head kidney, body kidney, spleen, liver, intestine, muscle, brain, heart, and gills. Following A. hydrophila challenge the MR immunoreactive cells became more widespread in head kidney and spleen, which are the major reticuloendothelial systems of fish. The quantitative studies at mRNA levels showed that there exists a high correlation between MR expression and immune cytokine expressions after bacteria challenge.

Protective effects of chicken egg yolk immunoglobulins (IgY) against experimental Aeromonas hydrophila infection in blunt snout bream (Megalobrama amblycephala).

The emergence of multi antibiotic resistance by the pathogens and toxic impacts on host metabolism has opened new perspectives to rational novel vaccine techniques. Outbreaks of Aeromonas hydrophila in aquaculture caused high mortality throughout the world and resulted in the extensive economic loss in the aquaculture industry. In this study, we report the efficacy of anti-A. hydrophila IgY antibodies by passive vaccination and its prophylactic or therapeutic effects against A. hydrophila in blunt snout bream. Inactivated A. hydrophila immunized hens produced effective IgY antibodies that were stable at temperatures less than 60 °C or the pH value was >4. The specific IgY can be bound directly to A. hydrophila that efficiently agglutinated and inhibited the bacterial growth in a dose-dependent manner. The specific IgY had significantly enhanced the phagocytosis activity of macrophages and resulted in rapid bacterial clearance. Anti-A. hydrophila IgY antibodies significantly increased macrophage mediated respiratory burst, including nitric oxide and superoxide anion production and subsequently killed the pathogen. Histopathological studies of intestine and spleen from vaccinated blunt-snout bream challenged with A. hydrophila showed the structural integrity of the organs was maintained intact from the bacterial injury. In addition, the prophylactic and therapeutic immunization, protected the blunt snout bream and the survival is approximately about 60% and 50%, respectively. These data suggest that specific IgY has the potential for protecting blunt snout bream against A. hydrophila infection and show promise for the future development of harmless vaccines.

Transcriptome analysis of Pacific white shrimp (Litopenaeus vannamei) challenged by Vibrio parahaemolyticus reveals unique immune-related genes.

Pacific white shrimp (Litopenaeus vannamei) is an important cultural species worldwide. However, Vibrio spp. infections have caused a great economic loss in Pacific white shrimp culture industry. The immune responses of Pacific white shrimp to the Vibrio spp. is not fully characterized. In this study, the transcriptomic profiles of L. vannamei hemocytes were explored by injecting with or without Vibrio parahaemolyticus. Totally, 42,632 high-quality unigenes were obtained from RNAseq data. Comparative genome analysis showed 2258 differentially expressed genes (DEGs) following the Vibrio challenge, including 1017 up-regulated and 1241 down-regulated genes. Eight DEGs were randomly selected for further validation by quantitative real-time RT-PCR (qRT-PCR) and the results showed that are consistent with the RNA-seq data. Due to the lack of predictable adaptive immunity, shrimps rely on an innate immune system to defend themselves against invading microbes by recognizing and clearing them through humoral and cellular immune responses. Here we focused our studies on the humoral immunity, five genes (SR, MNK, CTL3, GILT, and ALFP) were selected from the transcriptomic data, which were significantly up-regulated by V. parahaemolyticus infection. These genes were widely expressed in six different tissues and were up-regulated by both Gram negative bacteria (V. parahaemolyticus) and Gram positive bacteria (Staphylococcus aureus). To further extend our studies, we knock-down those five genes by dsRNA in L. vannamei and analyzed the functions of specific genes against V. parahaemolyticus and S. aureus by bacterial clearance analysis. We found that the ability of L. vannamei was significantly reduced in bacterial clearance when treated with those specific dsRNA. These results indicate that those five genes play essential roles in antibacterial immunity and have its specific functions against different types of pathogens. The obtained data will shed a new light on the immunity of L. vannamei and pave a new way for fighting against the bacterial infection in Pacific white shrimp.

Antibacterial activity of hemocyanin from red swamp crayfish (Procambarus clarkii).

Hemocyanins (HMC): the copper-containing respiratory proteins present in invertebrate hemolymph, which plays many essential roles in the immune system. Currently, little is known about the HMC domains of Procambarus clarkii (P. clarkii) and their function in antimicrobial immune response. In this present study, we comparatively studied the expression pattern of native PcHMC with the three recombinant proteins of variable domains of crayfish hemocyanin (PcHMC-N, N-terminal domain of hemocyanin; PcHMC-T, tyrosinase domain of hemocyanin; PcHMC-C, C-terminal domain of hemocyanin). The results showed that three purified recombinant proteins had a strong binding to various bacteria and lipopolysaccharides that further highly agglutinated. The HMCs recombinant proteins showed strong antibacterial activity against V. parahaemolyticus and S. aureus by bacterial growth inhibition, phenoloxidase (PO) and phagocytosis assays. Specifically, rPcHMC1-T and rPcHMC1-C inhibited both the bacteria efficiently, rPcHMC1-T was highly upregulated the PO activity than the other recombinant proteins. Whereas, recombinant proteins pretreated crayfish hemocytes participated in phagocytosis activity, rPcHMC1-N and rPcHMC1-C proteins had a profound effect than the rPcHMC1-T on S. aureus and V. parahaemolyticus phagocytosis. The crayfish hemocyanin domains clearly exhibited antibacterial and phagocytic activities against both the bacteria, suggesting that its variable domains of hemocyanin have the different function on specific pathogen during the assault of pathogens.

Detection and neutralization of cobra venom using rabbit antiserum in experimental envenomated mice.

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect the venom of Indian cobra (Naja naja naja) in various tissues (brain, heart, lungs, liver, spleen, blood, kidneys, and tissue at the site of injection) of mice after cobra venom injected at different time intervals (0, 2, 4, 6, 8, and 12 h intervals up to 24 h). Whole venom antiserum or individual venom protein antiserum (14, 29, 65, 72, and 99 kDa) could recognize N. n. naja venom by Western blotting and ELISA, and antibody titer was also assayed by ELISA. Antiserum raised against cobra venom in rabbit significantly neutralized the toxicity of venom-injected mice at different time intervals after treatment. The assay could detect N. n. naja venom levels up to 2.5 ng/ml of tissue homogenate, and the venom was detected up to 24 h after venom injection. Venom was detected in brain, heart, lungs, liver, spleen, kidneys, tissue at the bite area, and blood. As observed in mice, tissue at the site of bite area showed the highest concentration of venom and the brain showed the least. Moderate amounts of venoms were found in liver, spleen, kidneys, heart, and lungs. Development of a simple, rapid, and species-specific diagnostic kit based on this ELISA technique useful to clinicians is discussed.

Antivenom activity of triterpenoid (C34H68O2) from Leucas aspera Linn. against Naja naja naja venom induced toxicity: antioxidant and histological study in mice.

The isolated and identified triterpenoid, 1-hydroxytetratriacontane-4-one (C34H68O2), obtained from the methanolic leaf extract of Leucas aspera Linn. was explored for the first time for antisnake venom activity. The plant (L. aspera Linn.) extract significantly antagonized the spectacled cobra (Naja naja naja) venom induced lethal activity in a mouse model. It was compared with commercial antiserum obtained from King Institute of Preventive Medicine (Chennai, Tamil Nadu, India). N. naja naja venom induced a significant decrease in antioxidant superoxide dismutase, glutathione (GSH) peroxidase, catalase, reduced GSH and glutathione-S-transferase activities and increased lipid peroxidase (LPO) activity in different organs such as heart, liver, kidney and lungs. The histological changes following the antivenom treatment were also evaluated in all these organs. There were significant alterations in the histology. Triterpenoid from methanol extract of L. aspera Linn. at a dose level of 75 mg per mouse significantly attenuated (neutralized) the venom-induced antioxidant status and also the LPO activity in different organs.

Development and characterization of cell lines derived from rohu, Labeo rohita (Hamilton), and catla, Catla catla (Hamilton).

Two new cell lines, designated RE and CB, were derived from the eye of rohu, Labeo rohita, and the brain of catla, Catla catla, respectively. The cell lines were maintained in Leibovitz's L-15 supplemented with 20% foetal bovine serum. The RE cell line was sub-cultured for more than 70 passages and the CB cell line for more than 35 passages. The RE cells are rounded and consist predominantly of epithelial cells. The CB cell line consists of predominantly fibroblastic-like cells. Both cell lines are able to grow at temperatures between 25 and 32 degrees C with an optimum of 28 degrees C. The growth rate of the cells increased as the foetal bovine serum concentration increased from 2% to 20% at 28 degrees C, with optimum growth at concentrations of 15% or 20% foetal bovine serum. The cells were successfully cryopreserved and revived at different passage levels. The cell lines were not susceptible to four marine fish viruses. Extracellular products from Aeromonas sp. were toxic to the cell lines. When the cells were transfected with plasmid eukaryotic green fluorescent protein (pEGFP [Clontech, Carlsbad, CA, USA]) vector DNA, a significant fluorescent signal was observed suggesting that these cell lines could be a useful tool for transgenic and genetic manipulation studies. Polymerase chain reaction amplification of mitochondrial 12S rRNA from rohu and catla confirmed that the cell lines originated from these fish species. The cell lines were further characterized by immunocytochemistry using confocal laser scanning microscopy.

Natural aquatic insect carriers of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV).

Five different species of aquatic insects were collected from nursery ponds containing the freshwater prawn Macrobrachium rosenbergii infected with Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV). The insects were screened as potential natural carriers of MrNV and XSV. RT-PCR (reverse transcription polymerase chain reaction) analysis gave positive results for MrNV and XSV in Belostoma sp., Aesohna sp., Cybister sp. and Notonecta sp., and negative results for Nepa sp. An Aedes albopictus mosquito cell line (C6/36) was used for infectivity assays, with viral inoculum prepared from the aquatic insects, since C6/36 cells have recently been shown to be susceptible to infection with MrNV and XSV. The C6/36 cells were harvested 4 d post-challenge for examination by electron microscopy. This revealed aggregation of viral particles throughout the cytoplasm for cells challenged with inocula from all the insect species except Nepa sp. Our results indicate that several aquatic insect species may present a risk for MrNV and XSV transmission to M. rosenbergii.