Susceptibility of betanodavirus in a newly established vertebra-derived cell line from Mosquitofish (Gambusia affinis)

In the present study, a new cell line from the vertebra of mosquitofish Gambusia affinis was successfully established and characterized. The cell line is named as bone Gambusia affinis (BGA) and subcultured for more than 55 passages in Leibovitz's/L15 medium supplemented with 15% FBS at 28°C. The cell line has a modal chromosome number of 48. Molecular characterization of the partial sequence of the coi gene confirmed the origin of the BGA cell line from mosquitofish. These cells exhibited epithelial morphology confirmed by the cytokeratin marker. The BGA cells showed mineralization of their extracellular matrix when stained with alizarin red and von Kossa stain. BGA cells were found to be susceptible to RGNNV and SJNNV strains of betanodavirus (NNV) showing cytopathic effect with multiple vacuolations in the cells. The RT-PCR confirmed the betanodavirus infections in BGA cells. The SEM micrograph showed the morphological changes observed in the cell during virus infection. The in vivo challenge experiment also showed the viral replicating efficiency in the Gambusia affinis with increasing viral titre. Thus, our present results show that the BGA cell line is a useful tool for isolating betanodavirus and could be used to investigate bone cell differentiation and extracellular matrix mineralization.     

Anti-viral activity of methyl 1-chloro-7-methyl-2-propyl-1h-benzo[d] imidazole-5-carboxylate against white spot syndrome virus in freshwater crab (Paratelphusa hydrodromous)

Shrimp farming industries are subjected to severe economic loss due to a disease called white spot syndrome, a viral disease caused by white spot syndrome virus (WSSV) in penaeid shrimp. Numerous active compounds in the market possess anti-viral activity against the white spot syndrome virus, yet the issue remains unsolved. The present study was carried out to determine the anti-viral activity of methyl 1-chloro-7-methyl-2-propyl-1h-benzo[d] imidazole-5-carboxylate (C₁₃H₁₅N₂O₂Cl) against WSSV. The anti-viral activity of the synthetic compound was determined in freshwater crabs. Crabs were divided into three different experimental groups: healthy control groups (N.C.) received NTE buffer, positive control group (P.C.) crabs received WSSV, and treatment group crabs received WSSV along with synthetic weight compound. Experimental groups were observed for 30 days post-infection. Three different organs (gills, muscles, and head soft tissue (HST)) were dissected from all three groups and analyzed using molecular-based techniques, including polymerase chain reaction (PCR), Western blot, and histopathology. Clinical signs of WSSV were observed in the positive and N.C. groups; however, the treatment group showed a 100% survival rate. Confirmation was done using PCR, Western blot, and histopathology. These results demonstrated that the given synthetic compound has significant anti-viral activity against WSSV.

     

Stable Isotope (δ13C and δ15N) Signatures and Their Relationship among Soil Enzyme Activities in Indian Semi-arid Agricultural Soils

ABSTRACT

A field evaluation of the stable isotopes (δ¹³C and δ¹⁵N) and their relationship among physicochemical and enzyme activities was conducted in Indian semi-arid agricultural soils. Composite soil samples were collected based on organic management (ORG), inorganic management (IM), integrated crop management (ICM) and precision farming (PF) experimental plots from the fall of September 2017 to October 2018. δ¹⁵N was significantly higher (13.85 %) in ORG soils compared with ICM (13.28 %), IM (12.84 %) and PF (12.75 %). In contrast δ¹³C was higher (?13.25%) in PF soils than IM (?13.6 %), ICM (?15.07 %) and ORG (?15.23 %). Soils from ORG had significantly higher levels of total N, total C, total S, organic carbon, available N, extractable P, Soil organic carbon stock, exchangeable K and enzyme activities compared to IM, ICM and PF. Urease, β – glucosidase, acid phosphatase, alkaline phosphatase, invertase, cellulase and dehydrogenase activities significantly increased the δ¹⁵N and reduced δ¹³C in agricultural soils. Our results suggested that organic management had improved the δ¹⁵N, plant available nutrients and soil enzyme activities. Stable δ¹³C and δ¹⁵N isotopes are good indicators of monitor the soil health, carbon, and nitrogen biogeochemical cycles in Indian semi-arid agricultural soils.     

A Simple PCR Procedure to Detect White Spot Syndrome Virus (WSSV) of Shrimp, <Emphasis Type="Italic">Penaeus monodon</Emphasis> (Fabricious)

A non-stop, single tube and semi-nested polymerase chain reaction (PCR) technique with simple procedure was developed for simultaneous detection and grading the level of white spot syndrome infection in penaeid shrimp, Penaeus monodon. In this PCR procedure, three sense primers and one antisense primer with uniform annealing temperature of 55 °C were used. These primers amplify three PCR products (500, 300 and 200 base pairs [bp]) depending upon the severity of infection. Quantities of WSSV-DNA at 10 pg and greater gave three PCR products of 500, 300, 200 bp. A moderate concentration of WSSV-DNA, around 100 fg, gave two products of 300 and 200 bp and a low concentration of 1 fg or more gave only one PCR product of 200 bp. This PCR technique was assessed for early detection of WSSV in shrimp. In time-course infectivity experiments conducted on shrimp with WSSV, one PCR product (200 bp) was seen in hemolymph, tail tissue and gill at 3 h post infection (p.i.); two PCR products (300 and 200 bp) were seen in tail tissue, hemolymph, heart tissue and gill at 18 h p.i. At 30 h p.i., three PCR products (500, 300, 200 bp) were seen in all the organs tested. The samples collected from control animals showed negative for WSSV.     

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 °C with an optimum of 28 °C. The growth rate of the cells increased as the foetal bovine serum concentration increased from 2% to 20% at 28 °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.     

Production and economic effects of in-pond grading of channel catfish

Periodic grading and harvesting of fish has been shown to increase production of some species by removing larger fish throughout the growing season, reducing overall biomass, and increasing growth of smaller fish. Two two-year studies measured production and economic effects of the UAPB grader as compared to traditional live car grading and assessed effects of frequent harvesting and grading. Multi-batch production systems were used in both studies. In Study 1, fingerlings grew significantly better in ponds graded with the UAPB grader (0.97 g d⁻¹) as compared to the traditional live car (0.80 g d⁻¹) and resulted in 14% fewer under-sized (<0.57 kg) fish sent to the processor. In Study 2, increased frequency of harvest up to four harvests a year resulted in significantly fewer over-sized fish (>1.51 kg), more frequent receipt of revenue, and improved cash flow. Economic analysis showed that: (1) it was economically advantageous for catfish farmers to switch to the UAPB grader under a variety of farm size and processor dockage scenarios; and (2) increasing the frequency of harvest up to four harvests/year resulted in fewer over-sized fish, less dockage at the processing plant, improved cash flow, and more favorable economic outcomes.