Padmaja Jayaprasad Pradeep

Detection of natural infection of infectious spleen and kidney necrosis virus in farmed tilapia by hydroxynapthol blue-loop-mediated isothermal amplification assay.

Infectious spleen and kidney necrosis virus (ISKNV) has recently been recognized as a causative agent of serious systemic disease in tilapia. Our objective was to establish a new colorimetric loop‐mediated isothermal amplification (LAMP) assay with pre‐addition of hydroxynapthol blue (blue‐LAMP) to investigate ISKNV transmission in tilapia.

Duplex PCR assay and in situ hybridization for detection of Francisella spp. and Francisella noatunensis subsp. orientalis in red tilapia.

Conventional isolation and identification based on phenotypic characteristics is challenging with the highly fastidious, intracellular bacterium Francisella noatunensis subsp. orientalis (Fno). Here, we developed a duplex PCR method for simultaneous detection of the Francisella genus and Fno in one PCR reaction and an in situ hybridization method for paraffin section based diagnosis of Fno. The PCR results showed genus- and species-specific bands (1140 and 203 bp) from Fno but only one genus-specific band (1140 bp) from F. noatunensis subsp. noatunensis. Sensitivity of the duplex PCR assay revealed a detection limit of 20 to 200 fg genomic DNA (~10 to 100 genome equivalents) depending on DNA template extraction methods. The newly developed duplex PCR assay could be used to detect Fno from clinically sick fish exhibiting signs of visceral granulomas and would also be able to detect Fno infection in naturally diseased fish without symptoms of francisellosis, indicating potential application for diagnosis of field samples. The in situ hybridization assay using Fno species-specific probe revealed positive signals in multiple organs including the spleen, liver, kidney, gills and intestine of infected fish.

Shewanella putrefaciens in cultured tilapia detected by a new calcein-loop-mediated isothermal amplification (Ca-LAMP) method

Shewanella putrefaciens is being increasingly isolated from a wide variety of sources and is pathogenic to many marine and freshwater fish. For better control of this pathogen, there is a need for the development of simple and inexpensive but highly specific, sensitive, and rapid detection methods suitable for application in field laboratories. Our colorogenic loop-mediated isothermal amplification (LAMP) assay combined with calcein (Ca-LAMP) for unaided visual confirmation of LAMP amplicons is a simple method for fish pathogen detection in cultured tilapia. Here, we describe the detection of S. putrefaciens using the same platform. As before, the method gave positive results (orange to green color change) in 45 min at 63°C with sensitivity 100 times higher than that of a conventional PCR assay, with no cross-amplification of other known fish bacterial pathogens tested. Using the assay with 389 samples of gonads, fertilized eggs, and fry of farmed Nile and red tilapia Oreochromis spp., 35% of samples were positive for S. putrefaciens. The highest prevalence was found in samples of gonads (55%) and fertilized eggs (55%) from adult breeding stocks, indicating that S. putrefaciens could be passed on easily to fry used for stocking production ponds. Tissue tropism assays revealed that the spleen showed the highest colonization by S. putrefaciens in naturally infected tilapia and that it would be the most suitable organ for screening and monitoring fish stocks for presence of the bacteria.

Growth Performance of Triploid Red Tilapia Reared Under Laboratory Conditions

Triploidy could reduce breeding activity in tilapia without the use of hormones. In this study, the effect of triploidy on survival, growth, and gender of a line of red hybrid tilapia (Oreochromis mossambicus X Oreochromis niloticus) was assessed relative to the performance of diploid siblings. Triploidy was induced by preventing second polar body extrusion by applying either heat or cold shock. Growth was similar for both ploidies during the first 90 days of culture. However, at the age of 120 days, the average body weight of triploids produced by heat shock (215.5 ± 3.61 g) was significantly higher than that of cold shock (192.7 ± 2.6 g) and the diploid control (191.9 ± 1.74 g). Survival among triploids was inferior to diploids. Percentage of males in the triploid population was 82.9% in the heat-shocked treatment group, 54.8% in the cold-shock treatment, and 50% in the diploid control. Maximum attainable weight of red tilapia was calculated by applying the Ford-Walford growth plot: 650 g (heat-shocked triploids), 490 g (cold-shocked triploids), and 440 g in the diploid control.