Comparison of three molecular methods for the detection of Pilchard herpesvirus in archived paraffin-embedded tissue and frozen tissue

Abstract

Two epizootics affecting pilchards Sardinops sagax neopilchardus have been observed over their entire geographical range off the Australian coastline. The first occurred in 1995, involving high mortality (at least 10%) that devastated the pilchard population. The second occurred in 1998 and involved even higher mortality (70%). Both epizootics moved rapidly against the prevailing Leeuwin and East Australian currents from a defined point of origin. A herpesvirus, pilchard herpesvirus (PHV), was determined to be the cause of the epizootics, but the source of the virus remains unknown. In this research, in situ hybridization (ISH), polymerase chain reaction (PCR), and realtime PCR were compared for the detection of PHV in archived paraffin-embedded tissue and frozen tissue collected before, during, and after the 1995 epizootic. Results show that the conventional PCR failed to detect PHV in archived paraffin-embedded tissue, and that real-time PCR was the most sensitive of the 3 techniques and the best method for the detection of PHV. KEY WORDS: PHV · Epizootic · Real-time PCR · PCR · ISHResale or republication not permitted without written consent of the publisher

Comparative Analysis of a Conserved Gene Block from the Genome of the Members of the Genus<i> Ictalurivirus</i>

“…The topology was also identical with that of the Bayesian or distance matrix trees (not shown). Although from most alloherpesviruses, another part of the terminase gene has been amplified by PCR, choosing this region allowed us to include the HV described from the Australasian pilchard [25] into a phylogenetic analysis for the very first time. From the putative PHV, this is the only nt sequence available [12] .…”

Herpesviruses of Fish, Amphibians and Invertebrates

“…Involvement of an infectious agent was suggested, and PCR analysis revealed the putative involvement of a herpesvirus (Tham & Moon, 1996), which was soon confirmed by EM . Diagnostic tools for detection of the pilchard herpesvirus have been developed (Crockford et al, 2005;Crockford et al, 2008), revealing that the virus is now endemic in Australian pilchard populations . Although the pilchard herpesvirus has not yet been isolated in cell culture, hampering further studies, limited phylogenetic analysis showed its relation to other fish and frog herpesviruses (Doszpoly et al, 2011a).…”

Testing for the occurrence of pilchard herpesvirus (PHV) in South African sardine Sardinops sagax

“…Real-time PCR analysis was conducted using a Bio-Rad CFX96TM real-time PCR detection system on a C1000TM thermal cycler. Real-time PCR primers and cycling conditions were as described by Crockford et al (2008). Data collection and real-time analysis occurred at the annealing step of each cycle and melt-curve data collection and analysis occurred at each increment in the latter 40 cycles.…”

“…Samples that failed to record a Cq value or failed to produce a specific melt peak were considered to be negative. For each PCR, a standard curve was constructed by spiking known quantities of plasmid DNA, purified from a PHV clone obtained from the Department of Fisheries in Western Australia (see Crockford et al 2008), into the homogenised gill tissues prepared from a PHV-negative sardine. DNA added for sensitivity testing was spiked into tissues in order to achieve final concentrations that ranged from 13 ng μl-1 to 1.3 × 10-11 ng μl-1 in the reaction tubes.…”

“…The most recent mass-mortality events occurred off Australia and New Zealand in 1995and then again in 1998/1999, where an estimated 70% of the spawning biomass of sardine off Australia was lost (Crockford et al 2005Whittington et al 2008). In both instances, the causative agent was identified as a herpesvirus, the pilchard herpesvirus (PHV), which is postulated to have been introduced into Australian waters through the importation of PHV-infected sardine to feed sea-caged southern bluefin tuna Thunnus maccoyii in South Australia (Gaughan 2002;Crockford et al 2008). Pilchard herpesvirus is now considered to be endemic in Australian sardine .…”