Application of Novel Gill Cell Line from Lates calcarifer for Recognizing Metals Using Probes.

Lates calcarifer (Bloch) is a potential candidate fish species for culture in marine and brackishwater. A continuous gill cell line was derived from L. calcarifer by the explant culture method and has been passaged for 132 times, in Leibovitz's L-15 medium containing 10% fetal bovine serum (FBS) at 28 °C. The cells showed a rate of recovery between 90 and 95% after being successfully cryopreserved at various passage levels and formed monolayer in 2-3 days without any morphological changes. Immunophenotypic analysis of the SBG cell line revealed that they are of epithelial origin. Polymerase chain reaction assay using mitochondrial 12S rRNA primer specific to L. calcarifer was used to confirm the authenticity of the established gill cell line origin from seabass. The transfection efficiency was evaluated in Seabass Gill (SBG) cell line using pEGFP-N1 and Lipofectamine™ 3000. Transfection efficiency was found to be between 13 and 16%. The cytotoxicity of three different metal detecting probes was evaluated by MTT and Alamar blue assays to determine safe concentration. The result revealed that SBG cell line can be applied for recognition of metals using probes. The current study established, for the first time, a gill-derived cell line (SBG) from Lates calcarifer and its application for the detection of intracellular indium, mercury, and lutetium ions by specific fluorescent probes.

First report on the occurrence of cyprinid herpesvirus 3 in koi carp (Cyprinus carpio koi) in India.

This study reports the occurrence of cyprinid herpesvirus 3 (CyHV-3) in koi carp (Cyprinus carpio koi) for the first time in India. The koi carp, with clinical signs of ulcer with haemorrhage on body surface, necrosis of fin and discolouration of gill associated with huge mortality, were observed in aquarium shops, rearing tanks and grow-out ponds located in Chennai, India. The PCR assay carried out on infected fish samples using different primer sets specific to CyHV-3 confirmed its presence in the infected fish. Sequence analysis of partial thymidine kinase gene revealed 100% similarity with the sequence of CyHV-3 available in GenBank. Cell lines of koi carp and catla were found to be susceptible to CyHV-3 and its replication was confirmed by viral-specific cytopathic effect, PCR and bioassay. The CyHV-3 infection was reproduced by intramuscular injection of inoculum prepared from CyHV-3-infected fish to satisfy Koch's postulates. Tissue tropism of CyHV-3 in infected fish by PCR assay revealed the presence of CyHV-3 in all vital organs with prominent band in gill and gut tissue.

First report on the occurrence of white spot syndrome virus, infectious myonecrosis virus and Enterocytozoon hepatopenaei in Penaeus vannamei reared in freshwater systems.

Samples of white leg shrimp, Penaeus vannamei, were collected on a monthly basis from freshwater ponds with the salinity of 0 ppt located at Tiruvannamalai and Villupuram districts in Tamil Nadu, India for screening of viral and fungal pathogens. Totally, 130 shrimp samples were collected from 67 freshwater ponds and screened for white spot syndrome virus (WSSV), infectious myonecrosis virus (IMNV), infectious hypodermal and haematopoietic necrosis virus (IHHNV) and Enterocytozoon hepatopenaei (EHP) by PCR and RT-PCR using pathogen-specific primers. Among the samples screened, one sample was found to be positive to WSSV, two samples showed positive to IMNV and two samples positive for EHP. No sample showed positive to IHHNV. The WSSV detected in the sample was found to be a new strain of WSSV and highly virulent. The inoculum prepared from freshwater reared WSSV or IMNV-infected shrimp caused 100% mortality in experimental infection studies. The PCR and RT-PCR results revealed the presence of WSSV and IMNV in different organs of experimentally infected shrimp, respectively. No clinical signs were observed in experimentally EHP-injected shrimp, although the PCR results revealed the presence of EHP in experimentally infected shrimp.

In vitro propagation of tilapia lake virus in cell lines developed from Oreochromis mossambicus.

Tilapia lake virus (TiLV)-suspected samples of tilapia were collected from grow-out ponds located with clinical signs and mortality ranged from 5% to 50%. The reverse transcription-polymerase chain reaction (RT-PCR) assay revealed the presence of TiLV in the disease outbreak ponds. Cell lines were developed from heart, gill and eye of Mozambique tilapia and characterized. Morphologically, these cell lines are composed of epithelioid cells. The optimum growth of these cells was observed at 28°C and 20% concentration of FBS. After cryopreservation, 70%-90% of cells were found to be viable. The cells of all three cell lines were found to be positive to fibronectin and pancytokeratin. PCR amplification of 16S rRNA and COI of O. mossambicus confirmed the origin of these cell lines from O. mossambicus. Heart and gill cell lines were found to be highly susceptible to TiLV and found to be useful for its isolation from infected fish samples. The experimental infection was carried out in O. niloticus and O. mossambicus using the TiLV propagated in susceptible cell lines. The RT-PCR results revealed the presence of TiLV in brain, gill, liver, kidney, spleen, eye, muscle, intestine and heart of experimentally infected O. niloticus and O. mossambicus.

Field-Usable Lateral Flow Immunoassay for the Rapid Detection of White Spot Syndrome Virus (WSSV).

BACKGROUND: White spot disease (WSD), a major threat to sustainable aquaculture worldwide, is caused by White spot syndrome virus (WSSV). The diagnosis of WSD relies heavily on molecular detection of the virus by one-step PCR. These procedures are neither field-usable nor rapid enough considering the speed at which the virus spreads. Thus, development of a rapid, reliable and field-usable diagnostic method for the detection of WSSV infection is imperative to prevent huge economic losses. METHODS/PRINCIPAL FINDINGS: Here, we report on the development of a lateral flow immunoassay (LFIA) employing gold nanoparticles conjugated to a polyclonal antibody against VP28 (envelope protein of WSSV). The LFIA detected WSSV in ~20 min and showed no cross-reactivity with other shrimp viruses, viz. Monodon Baculovirus (MBV), Hepatopancreatic parvovirus (HPV) and Infectious Hypodermal and Hematopoietic Necrosis virus (IHHNV). The limit of detection (LOD) of the assay, as determined by real-time PCR, was 103 copies of WSSV. In a time course infectivity experiment, ~104 WSSV particles were injected in Litopenaeus vannamei. The LFIA could rapidly (~ 20 min) detect the virus in different tissues after 3 h (hemolymph), 6 h (gill tissue) and 12 h (head soft tissue, eye stalk, and pleopod) of infection. Based on these findings, a validation study was performed using 75 field samples collected from different geographical locations in India. The LFIA results obtained were compared with the conventional "gold standard test", viz. one-step PCR. The analysis of results in 2x2 matrix indicated very high sensitivity (100%) and specificity (96.77%) of LFIA. Similarly, Cohen's kappa coefficient of 0.983 suggested "very good agreement" between the developed LFIA and the conventional one-step PCR. CONCLUSION: The LFIA developed for the rapid detection of WSSV has an excellent potential for use in the field and could prove to be a boon to the aquaculture industry.

Development and use of retinal pigmented epithelial cell line from zebrafish (Danio rerio) for evaluating the toxicity of ultraviolet-B.

Danio rerio retinal pigmented epithelial (DrRPE) cell line, derived from the RPE tissue, was established and characterized. The cells were able to grow at a wide range of temperatures from 25°C to 32°C in Leibovitz's L-15 medium. The DrRPE cell line consists of epithelial cells with a diameter of 15-19 µm. The cell line was characterized by mitochondrial 12S rRNA gene, immunocytochemical analysis, and karyotyping. DrRPE cells treated with 10 µM of all-trans-retinol for 24 h readily formed lipid droplets. DrRPE cells were irradiated with narrowband ultraviolet-B (UV-B) radiation at different time periods of 0, 10, 20, and 40 min. The cells were subsequently examined for changes in morphology, cell viability, phagocytotic activity, mitochondrial distribution, nuclei morphology, generation of reactive oxygen species, and expression of apoptotic-related genes p53 and Cas3 by quantitative polymerase chain reaction. The results demonstrate that UV-B radiation can cause a considerable decrease in DrRPE cell viability as well as in phagocytotic activity. In addition, the results demonstrate that UV-B radiation can induce the degradation of mitochondria and DNA in cultured DrRPE cells.