Development of a TaqMan quantitative reverse transcription PCR assay to detect tilapia lake virus.

Tilapia lake virus disease (TiLVD) is an emerging viral disease associated with high morbidity and mortality in cultured tilapia worldwide. In this study, we have developed and validated a TaqMan quantitative reverse transcription PCR (RT-qPCR) assay for TiLV, targeting a conserved region within segment 10 of the genome. The RT-qPCR assay was efficient (mean ± SD: 96.71 ± 3.20%), sensitive with a limit of detection of 10 RNA viral copies per reaction, and detected TiLV strains from different geographic regions including North America, South America, Africa, and Asia. The intra- and inter-assay variability ranged over 0.18-1.41% and 0.21-2.21%, respectively. The TaqMan RT-qPCR assay did not cross-react with other RNA viruses of fish, including an orthomyxovirus, a betanodavirus, a picornavirus, and a rhabdovirus. Analysis of 91 proven-positive and 185 proven-negative samples yielded a diagnostic sensitivity of 96.7% and a diagnostic specificity of 100%. The TaqMan RT-qPCR assay also detected TiLV RNA in infected Nile tilapia liver tissue extracts following an experimental challenge study, and it successfully detected TiLV RNA in SSN-1 (E-11 clone) cell cultures displaying cytopathic effects following their inoculation with TiLV-infected tissue homogenates. Thus, the validated TaqMan RT-qPCR assay should be useful for both research and diagnostic purposes. Additionally, the TiLV qPCR assay returns the clinically relevant viral load of a sample which can assist health professionals in determining the role of TiLV during disease investigations. This RT-qPCR assay could be integrated into surveillance programs aimed at mitigating the effects of TiLVD on global tilapia production.

Comparative Morphology and Morphometry of Blood Cells in Zebrafish (Danio rerio), Common Carp (Cyprinus carpio carpio), and Tilapia (Oreochromis niloticus).

Several diagnostic tools are available for veterinarians and fish health professionals to evaluate fish health and their abnormalities. However, reference data regarding the character and size of fish blood cells are limited. The purpose of this study was to analyze the morphology and morphometry of normal blood cells in zebrafish (Danio rerio), common carp (Cyprinus carpio carpio), and tilapia (Oreochromis niloticus). To get a representative sample, we took blood from ten 6-mo old healthy fish from each species. Fish were purchased from an ornamental fish market and the local government fish breeding center in Yogyakarta, Indonesia. A total of 100 erythrocytes and a maximum of 30 leukocytes (neutrophils, eosinophils, basophils, lymphocytes and monocytes) were randomly sampled and analyzed qualitatively and quantitatively, including morphometric analysis of both the long axis (LA) and short axis (SA) of these cells. All data obtained were analyzed using ANOVA statistical tests to compare the blood cells in each species with SPSS software. The findings revealed distinct differences in both morphology and morphometry of the blood cells among the species. Basic knowledge obtained from this research will aid in the development of biomarkers and other ancillary diagnostic tools for further hematology research, conservation, and clinical diagnosis in these 3 fish species.