A simple non-enzymatic method for the preparation of white spot syndrome virus (WSSV) DNA from the haemolymph of Marsupenaeus japonicus using FTA matrix cards.

White spot syndrome virus (WSSV) is an important shrimp pathogen responsible for large economic losses for the shrimp culture industry worldwide. The nucleic acids of the virus must be adequately preserved and transported from the field to the laboratory before molecular diagnostic analysis is performed. Here, we developed a new method to isolate WSSV-DNA using Flinders Technology Associates filter paper (FTA matrix card; Whatman) without centrifugation or hazardous steps involved. FTA technology is a new method allowing the simple collection, shipment and archiving of nucleic acids from haemolymph samples providing DNA protection against nucleases, oxidation, UV damage, microbial and fungal attack. DNA samples prepared from 10-fold dilutions of moribund shrimp haemolymph using FTA matrix cards were analysed using semi-quantitative and quantitative polymerase chain reaction (PCR) and were compared with two commercially available DNA isolation methods, the blood GenomicPrep Mini Spin Kit (GE Healthcare) and the DNAzol (Invitrogen). Sequence analysis was performed for the DNA samples prepared using the various isolation procedures and no differences in the sequence among these methods were identified. Results based on the initial copy number of DNA prepared from the GenomicPrep Mini Spin Kit are a little more sensitive than the DNA prepared from FTA matrix cards, whereas the DNAzol method is not suitable for blood samples. Our data shows the efficiency of retention capacity of WSSV-DNA samples from impregnated FTA matrix cards. Matrix cards were easy to store and ship for long periods of time. They provide ease of handling and are a reliable alternative for sample collection and for molecular detection and characterization of WSSV isolates.

Real-time quantitative loop-mediated isothermal amplification as a simple method for detecting white spot syndrome virus.

AIMS: White spot syndrome virus (WSSV) continues to be the most pathogenic virus among the crustacean aquaculture causing mass mortality. In the present study, we established a one-step, single tube, real-time accelerated loop-mediated isothermal amplification (real-time LAMP) for quantitative detection of WSSV. MATERIALS AND METHODS: A set of six specially designed primers that recognize eight distinct sequences of the target. The whole process can be completed in 1 h under isothermal conditions at 63 degrees C. Detection and quantification can be achieved by real-time monitoring in an inexpensive turbidimeter based on threshold time required for turbidity in the LAMP reaction. A standard curve was constructed by plotting viral titre against the threshold time (T(t)) using plasmid standards with high correlation coefficient (R(2) = 0.988). CONCLUSIONS: Sensitivity analysis using 10-fold dilutions (equivalent to 35 ng microl(-1) to 35 ag microl(-1)) of plasmid standards revealed this method is capable of detecting upto 100 copies of template DNA. Cross-reactivity analysis with DNA/cDNA of IHHNV, TSV, YHV-infected and healthy shrimp showed this method is highly specific for quantitative detection of WSSV. SIGNIFICANCE AND IMPACT OF THE STUDY: WSSV real-time LAMP assay appears to be precise, accurate and a valuable tool for the detection and quantification of WSSV in large field samples and epidemiological studies.

Light and electron microscope observations on presporogonic and sporogonic stages of Sphaerospora epinepheli (Myxosporea) in grouper (Epinephelus malabaricus).

Presporogonic (blood) stages of Sphaerospora epinepheli Supamattaya, Fischer-Scherl, Hoffmann, Boonyaratpalin, 1990 were observed in the circulating blood, sinus of kidney, glomerurar capillaries and liver arteries of grouper Epinephelus malabaricus. The earliest detectable stage was a primary cell with one secondary cell. After cell divisions, nine to 16 secondary cells were found in one primary cell. Ultrastructural examination revealed electron-dense bodies (118-145 nm) in the cytoplasm of primary cells. Sporogonic stages and spores were located in Bowman's space and in kidney tubule lumens. Electron micrographs revealed a similar pattern of spore development as described from other Sphaerospora spp. Kidneys infected with S. epinepheli showed highly vacuolated tubular epithelial cells and severely affected renal corpuscles.

Sphaerospora epinepheli n. sp. (Myxosporea: Sphaerosporidae) observed in grouper (Epinephelus malabaricus).

Sphaerospora epinepheli n. sp. is described from grouper, Epinephelus malabaricus, in cage-cultured and wild fish collected from both coastal lines of southern Thailand. Subspherical to spherical spores and mono- or disporous pseudoplasmodia were observed in the lumen of kidney tubules. Pseudoplasmodia were round to elongate, size range 15.6-22.9 microns (length) x 8.4-21.6 microns (width). Spores were 7.8-10.0 microns (length) x 12.3-14.5 microns (thickness), and 7.0-9.5 microns (width) with two spherical polar capsules of equal size measuring 2.9-4.4 microns in diameter and containing polar filaments with six or seven windings. Two uninucleate sporoplasms showed iodine vacuoles. Blood stages, similar to C-blood protozoans observed from freshwater fish in Europe, were found from peripheral blood smears of grouper. Ultrastructural studies of blood stages showed a similar structure to unidentified mobile protozoans from the blood of carp. Electron dense bodies were observed in the cytoplasm of the primary cell blood stages. Infected proximal-tubular epithelial cells showed highly vacuolated cytoplasm and pycnotic nuclei.