A.S. Sahul Hameed

Potential use of chitosan nanoparticles for oral delivery of DNA vaccine in Asian sea bass (Lates calcarifer) to protect from Vibrio (Listonella) anguillarum.

In recent years, attention has been focused on the possibility of utilizing DNA vaccines in fish aquaculture. A successful regime for intramuscular injection of naked DNA into fish has been developed and novel methods to deliver this DNA to fish are under investigation. The potential of chitosan as a polycationic gene carrier for oral administration has been explored since 1990s. The present study examines the potential efficacy of DNA vaccine against Vibrio anguillarum through oral route using chitosan nanoparticles encapsulation. The porin gene of V. anguillarum was used to construct DNA vaccine using pcDNA 3.1, a eukaryotic expression vector and the construct was named as pVAOMP38. The chitosan nanoparticles were used to deliver the constructed plasmid. In vitro and in vivo expression of porin gene was observed in sea bass kidney cell line (SISK) and in fish, respectively by fluorescent microscopy. The cytotoxicity of chitosan encapsulated DNA vaccine construct was analyzed by MTT assay and it was found that the cytotoxicity of pVAOMP38/chitosan was quite low. Distribution of gene in different tissues was studied in fish fed with the DNA (pVAOMP38) encapsulated in chitosan by using immunohistochemistry. The results indicate that DNA vaccine can be easily delivered into fish by feeding with chitosan nanoparticles. After oral vaccination Asian sea bass were challenged with Vibrio anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 46% was recorded. The results indicate that Sea bass (Lates calcarifer) orally vaccinated with chitosan-DNA (pVAOMP38) complex showed moderate protection against experimental V. anguillarum infection.

Tolerance of Macrobrachium rosenbergii to white spot syndrome virus.

The susceptibility of Macrobrachium idella, M. lamerrae and M. rosenbergii to white spot syndrome virus (WSSV) was tested by immersion challenge, oral route and intramuscular injection. Their susceptibility to WSSV was compared with that of Penaeus indicus and P. monodon. The WSSV caused 43.3% and 53.3% mortality in M. lamerrae and M. idella, respectively, by immersion method and 53.3% and 66.7% mortality in M. lamerrae and M. idella, respectively, by oral route. This virus caused 100% mortality in M. idella, M. lamerrae, P. indicus and P. monodon when the animals were injected WSSV intramuscularly. Moribund animals were screened for the presence of WSSV by western blot or histopathology. The results indicated the susceptibility of marine shrimp and freshwater prawn to this virus except M. rosenbergii. This virus failed to produce mortality with any of the methods of infection applied in M. rosenbergii. The exact mechanism of tolerance of M. rosenbergii to WSSV is not known at present and the possibilities for this tolerance are discussed.

Oral delivery of DNA construct using chitosan nanoparticles to protect the shrimp from white spot syndrome virus (WSSV)

The protective efficacy of oral delivery of a DNA construct containing the VP28 gene of WSSV encapsulated in chitosan nanoparticles was investigated in black tiger shrimp (Penaeus monodon). The results showed that significant survival was obtained in WSSV-challenged shrimp at 7, 15 and 30 days post-treatment (relative survival, 85%, 65% and 50%, respectively) whereas 100% mortality was observed in the control shrimp fed with feed containing chitosan/pcDNA 3.1 or chitosan/PBS complex. The ability of the chitosan to form a complex with the pVP28 and to stabilize it from endonuclease degradation was studied by agarose gel electrophoresis. Cytotoxicity of chitosan-encapsulated pVP28 was also evaluated by the MTT assay, which showed 90% viability of SISK cells incubated with the pVP28/chitosan complexes. Transcription analysis of the chitosan-encapsulated pVP28 gene in different tissues of DNA-treated shrimp and SISK cell line was confirmed by an RT-PCR reaction. The present study also measured the changes in the level of important immunological parameters such as prophenoloxidase, superoxide dismutase and superoxide anion in hemolymph of chitosan-encapsulated VP28 DNA-treated and controls shrimp. The study also correlated the changes in the level of immunological parameters with the survival percentage and protective efficacy of oral route of DNA construct against WSSV in shrimp.

White spot syndrome virus (WSSV) in two species of freshwater crabs (Paratelphusa hydrodomous and P. pulvinata)

The susceptibility of two species of freshwater crabs, Paratelphusa hydrodomous and P. pulvinata to white spot syndrome virus (WSSV) was tested by oral route and intramuscular injection. The results revealed that these freshwater crabs were as highly susceptible as marine shrimp. WSSV caused 100% mortality in both P. pulvinata and P. hydrodomous, at 60 and 84 h, respectively, after intramuscular injection. It also caused 100% mortality when given orally. The presence of WSSV in the moribund crabs was confirmed by PCR and histological analysis. It was found in gill, heart tissue, eyestalk, abdominal muscle and hemolymph.

Antibiotic resistance in bacteria isolated from Artemia nauplii and efficacy of formaldehyde to control bacterial load.

Abstract A study was carried out to determine the antibiotic resistance in bacteria isolated from Artemia nauplii and the efficiency of formaldehyde to control the bacteria associated with Artemia nauplii. The total aerobic heterotrophic bacteria of Artemia nauplii was determined on seawater nutrient agar and TCBS agar, and ranged from 3.8×10 3 to 8.1×10 3 and 9.4×10 2 to 4.3×10 3 colony forming units (CFU) per nauplius on seawater nutrient agar and TCBS agar plates, respectively. Among these bacteria, 336 isolates were tested for their resistance to five antibiotics. The minimum inhibiting concentrations of chloramphenicol, erythromycin, nitrofurazone, oxytetracycline, tetracycline, formaldehyde and sodium hypochlorite for 336 isolates were recorded. The LC50 values of oxytetracycline, formaldehyde and sodium hypochlorite for Artemia nauplii were determined as 540.5, 293.1 and 5.6 mg/l, respectively, after 24 h of exposure. The efficacy of formaldehyde was compared with that of antibiotics and formaldehyde was found to be very effective in controlling the bacteria of Artemia nauplii.

Immunological responses of Penaeus monodon to DNA vaccine and its efficacy to protect shrimp against white spot syndrome virus (WSSV).

White spot disease is an important viral disease caused by white spot syndrome virus (WSSV) and is responsible for huge economic losses in the shrimp culture industry worldwide. The VP28 gene encoding the most dominant envelope protein of WSSV was used to construct a DNA vaccine. The VP28 gene was cloned in the eukaryotic expression vector pcDNA3.1 and the construct was named as pVP28. The protective efficiency of pVP28 against WSSV was evaluated in Penaeus monodon by intramuscular challenge. In vitro expression of VP28 gene was confirmed in sea bass kidney cell line (SISK) by fluorescence microscopy before administering to shrimp. The distribution of injected pVP28 in different tissues of shrimp was studied and the results revealed the presence of pVP28 in gill, head soft tissue, abdominal muscle, hemolymph, pleopods, hepatopancreas and gut. RT-PCR and fluorescence microscopy analyses showed the expression of pVP28 in all these tissues examined. The results of vaccination trials showed a significantly higher survival rate in shrimp vaccinated with pVP28 (56.6-90%) when compared to control groups (100% mortality). The immunological parameters analyzed in the vaccinated and control groups revealed that the vaccinated shrimp showed significantly high level of prophenoloxidase and superoxide dismutase (SOD) when compared to the control groups. The high levels of prophenoloxidase and superoxide dismutase (SOD) might be responsible for developing resistance against WSSV in DNA vaccinated shrimp.

Antibiotic resistance in bacteria isolated from hatchery-reared larvae and post-larvae of Macrobrachium rosenbergii

A study was carried out to determine the bacterial load of larvae and post-larvae of Macrobrachium rosenbergii and their resistance to various antibiotics used commonly in aquaculture. Total aerobic heterotrophic bacteria associated with larvae and post-larvae of M. rosenbergii were determined on nutrient agar and thiosulphate citrate bile salt agar (TCBS), and the results showed that the bacterial numbers differed in different larval stages. A total of 523 bacterial isolates isolated from animal and tank water was examined for their resistance to six antibiotics. Incidence of bacterial resistance to oxytetracycline was highest followed by furazolidone and erythromycin among the antibiotics tested. Incidence of resistance to chloramphenicol was the least observed among the individual antibiotics. Vibrio species were found to be the dominant taxon in eggs, larvae and post-larvae of M. rosenbergii. The minimum inhibitory concentrations of six antimicrobials against the bacterial isolates were determined. Formalin was found to be very effective in controlling the bacteria isolated from larvae and post-larvae of freshwater prawn.

Biochemical, physiological and hematological changes in white spot syndrome virus-infected shrimp, Penaeus indicus

The biochemical and hematological changes provoked by white spot syndrome virus (WSSV) in hemolymph, hepatopancreas and muscle of Penaeus indicus were examined. Total carbohydrate, glucose, total protein, amino acids, fatty acids and hemocyanin were measured in healthy and WSSV-infected shrimp. There was a significant increase in glucose and total carbohydrate levels in the hemolymph of WSSV-infected shrimp in comparison to values for healthy shrimp and reductions in muscle and hepatopancreas of infected shrimp. The average total protein and free amino acids were significantly different between two groups in hemolymph, muscle and hepatopancreas. The fatty acid level increased significantly in the hepatopancreas of WSSV-infected shrimp, but was reduced in the hemolymph and muscle of WSSV-infected shrimp in comparison to healthy shrimp. Significant reductions in total hemocyte counts (THC) and hemocyanin contents were observed in WSSV-infected shrimp; hemolymph from WSSV-infected shrimp failed to clot. Some physiological responses such as oxygen consumption and ammonia excretion were examined in both healthy and WSSV-infected shrimp and the results showed reductions in oxygen consumption and ammonia excretion in WSSV-infected shrimp.

RETRACTED: Chitosan tripolyphosphate (CS/TPP) nanoparticles: Preparation, characterization and application for gene delivery in shrimp

The present study examines the use of CS/TPP nanoparticles for gene delivery in different tissues of shrimp through oral route. The viral gene of WSSV was used to construct DNA vaccines using pcDNA 3.1, a eukaryotic expression vector and the constructs were named as pVP28. The CS/TPP nanoparticles were synthesized by ionic gelation process and these particles were characterized. The structure and morphology of the nanoparticles were studied by field emission scanning electron microscopy (FE-SEM) and FTIR (Fourier Transform Infrared Spectra). The cytotoxicity of CS/TPP nanoparticles was evaluated by MTT assay using fish cell line. The expression of gene was confirmed by Immuno-dot blot, ELISA and RT-PCR analyses. The results indicate that DNA can be easily delivered into shrimp by feeding with CS/TPP nanoparticles.

Screening the organs for early detection of white spot syndrome virus in Penaeus indicus by histopathology and PCR techniques

PCR and histopathological observations were carried out at different time intervals to detect white spot syndrome virus (WSSV) in shrimp samples obtained from time-course experiments. Histopathological observations revealed the presence of intranuclear inclusion bodies in gill tissue, eyestalk, appendages and connective tissue at 36 h post-infection (p.i.) and in heart and stomach at 48 h p.i. The PCR analysis showed that hemolymph was positive for WSSV at 6 h p.i. and all other organs at 12 h p.i. For both techniques, the use of eyestalk as material for WSSV detection was suitable and allows for sample collection without sacrificing the shrimp. Eyestalk samples can be used for nonlethal screening of Penaeus indicus to detect WSSV in positive samples as early as 12 h p.i. by PCR, or 36 h p.i. by histology.