Development and evaluation of the polymerase chain reaction (PCR) method for diagnosis of spring viremia of carp virus (SVCV)

Aquaculture has been expanded rapidly to become a major commercial and food-producing sector worldwide in recent decade. In parallel, viral diseases rapidly spread among farms causing enormous economic losses. The accurate detection of pathogens at early stages of infection is a key point for disease control in aquaculture. Spring Viraemia of Carp Virus (SVCV) is a very severe pathogen of carp fishes in different parts of the world and is categorized as a reportable listed disease in the annual published list of World Organization for animal Health (OIE). The objective of this study was to develop and evaluate RT- PCR test for detecting SVC virus and also the sensitivity and specificity of this test. A semi nested RT- PCR was designed using combination of three primers: two external (SVCF , SVCR) and one internal (SVCS) primers which based on conserved region of G gen. The specificity of designed primers (only external ones) by examination on Viral Hemorrhagic Septicemia Virus (VHSV) and Infectious Hematopoietic Necrosis Virus (IHNV) was confirmed. For optimizing of the PCR test, primer concentration, primer annealing temperature, cycle number and Mgcl2 concentration were surveyed. Also for validity test, prevention of false negative and Assurance of its accuracy, a competitive internal control (mimic) designed and its suitable concentration was defined. Evaluation of the sensitivity of designed test were conducted first by comparing the different commercially available RNA isolation guidelines, two guidelines: isotiocyanate phenol–chloroform based protocols (RNX–Plus Iran, Iq2000 kit Taiwan ) and two column based protocols (Cinna pure RNA Iran , high pure viral RNA kit, Roche Germany ). The results indicated that the column based protocols (Roche method and Cinna pure), yield 36.77 ng/μl and 16/47 ng/μl RNA concentration respectively, which were significantly higher than other protocols(P<0.05). Then for evaluation of extracted RNA sensitivity, Serial dilution of SVCV strain 56.70 grown in EPC (1.9×105 TCID50/ml) was examined To compare sensitivity. Extracted RNA from serial dilution with stone's primers and commercial IQ-2000 kit were examined simultaneously. The result indicated that designed semi- nested RT- PCR was able to recognize SVC virus to 10-4 dilution and stone's primer recognize to 10-3 dilution whereas Iq-2000 commercial kit did not recognized in any dilution. In high virus titer in designed test two DNA band (462 bp and 266 bp) produced, and by decreasing virus titer 462 bp was omitted. In low virus titer or lack of virus, just DNA band (mimic) 729 bp can propagate. After designing and optimizing PCR test, a total of 400 suspected cultured Cyprinus carpio with high mortality from 4 aquaculture zone of Khuzestan province were collected and tested for SVCV during 2012- 2013 using developed PCR method and IQ- 2000. The results indicated that SVC virus was not observed in samples using both methods.

A video method for quantifying size distribution, density, and three-dimensional spatial structure of reef fish spawning aggregations

There is a clear need to develop fisheries independent methods to quantify individual sizes, density, and three dimensional characteristics of reef fish spawning aggregations for use in population assessments and to provide critical baseline data on reproductive life history of exploited populations. We designed, constructed, calibrated, and applied an underwater stereo-video system to estimate individual sizes and three dimensional (3D) positions of Nassau grouper (Epinephelus striatus) at a spawning aggregation site located on a reef promontory on the western edge of Little Cayman Island, Cayman Islands, BWI, on 23 January 2003. The system consists of two free-running camcorders mounted on a meter-long bar and supported by a SCUBA diver. Paired video “stills” were captured, and nose and tail of individual fish observed in the field of view of both cameras were digitized using image analysis software. Conversion of these two dimensional screen coordinates to 3D coordinates was achieved through a matrix inversion algorithm and calibration data. Our estimate of mean total length (58.5 cm, n = 29) was in close agreement with estimated lengths from a hydroacoustic survey and from direct measures of fish size using visual census techniques. We discovered a possible bias in length measures using the video method, most likely arising from some fish orientations that were not perpendicular with respect to the optical axis of the camera system. We observed 40 individuals occupying a volume of 33.3 m3, resulting in a concentration of 1.2 individuals m–3 with a mean (SD) nearest neighbor distance of 70.0 (29.7) cm. We promote the use of roving diver stereo-videography as a method to assess the size distribution, density, and 3D spatial structure of fish spawning aggregations.

A Bayesian method for identification of stock mixtures from molecular marker data

Molecular markers have been demonstrated to be useful for the estimation of stock mixture proportions where the origin of individuals is determined from baseline samples. Bayesian statistical methods are widely recognized as providing a preferable strategy for such analyses. In general, Bayesian estimation is based on standard latent class models using data augmentation through Markov chain Monte Carlo techniques. In this study, we introduce a novel approach based on recent developments in the estimation of genetic population structure. Our strategy combines analytical integration with stochastic optimization to identify stock mixtures. An important enhancement over previous methods is the possibility of appropriately handling data where only partial baseline sample information is available. We address the potential use of nonmolecular, auxiliary biological information in our Bayesian model.