A polymerase chain reaction (PCR) protocol has been developed for the early detection of white-spot syndrome virus (WSSV), one of the most widespread and devastating infectious agents that have hit the shrimp aquaculture industry.
Dr. Lourdes Tapay of BIOTECH_UPLB, Dr. Cesar B. Nadala, Jr. And Dr Philip C. Loh of the University of Hawaii, with support from the Department of Agriculture’s Bureau of Fisheries and Aquatic Resources (BFAR) and the Oplan Sagip Sugpo Task Force, were the developers of the method. The details of the protocol were included in an article entitled “A polymerase chain reaction protocol for the detection of various geographic isolates of the white spot virus” by the Journal of Virological Methods 82 (1999) 39-43, which won for Dr. Tapay an International Publication Award from the University of the Philippine System.
This development comes in an opportune time. Shrimp aquaculture used to be a lucrative industry until disease outbreaks wreaked havoc worldwide, especially in Asian countries, including the Philippines. Among the infectious agents reported to affect the shrimp, viruses remain the most potent threat simply because viral infections remain untreatable. It is thus important that these viruses, especially those which cause massive disease outbreaks, such as the yellow head virus (YHV) and the WSSV are detected early.
Conventional diagnostic methods, which are widely used are not as specific and sensitive. They fail to detect viral infections during the early stage of infection. Thus, a continuos effort is being made to develop sensitive and specific diagnostic protocols through biotechnology for the early detection of the virus, especially among broodstock and postlarval (fry) populations, including asymptomatic animals of harvestable “age” so that the shrimp farmer can harvest the infected crop prior to total infestation. The PCR protocol was developed based on a unique cloned DNA fragment of the viral genome determined by Dr. Nadala and Dr. Loh. Several set of primers were designed to amplify the target sequence but Primers C (forward) and D (reverse) were chosen since with these were able to amplify the target sequence even a a stringent annealing temperature of 65 degrees Celsius. The protocol involves several rounds of amplification , rendering it sensitive enough to detect low levels of the virus (10 to 100 picograms of WSSV DNA) by producing 211 bp product which can be analysed by agarose gel electrophoresis. Furthermore, the WSSV-specific primers could detect various geographic isolates f the virus, e.g. US , japan China, Indonesia and India. The protocol was used here in the Philippines to determine the prevalence of WSSV in shrimp farms and hatcheries.
In a nationwide survey conducted from January to May 1999, 72% were found positive for WSSV after 1-step and 39% after 2-step , non-nested PCR. Of the post larvae (PL) and the Juvenile /adult shrimp samples tested, 50 and 79% were positive for WSSV, respectively. Twelve percent (12%) of the PCR-positive samples also tested positive by Western blot assay, an antibody-biased assay developed for WSSV using a hyperimmune polyclonal anti-wssg IgG. This is the first report of the occurrence of WSSV in the Philippines. Results of this survey are presented in a paper entitled “white spot syndrome virus (WSSV) in cultured Penaeus monodon in the Philippines” which has been accepted for publication by the Disease of Aquatic Organisms.
In an effort to contain the spread of the infectious agent in farms and hatcheries, teh Bureau of Fisheries and Aquatic Resources has modified the current fry quality assessment to include screening for WSSV int the routine testing for Fry. Eight (8) satellite laboratories strategically located throughout the country have been identified to conduct PCR testing for WSSV: Luzon: Fish Health Section, BFAR central office; Panay Island:Seafdec AQD; Negros Island: Negros Prawn Production and Marketing Cooperative, inc.; Cebu regional Fisheries Office VII; Bohol : Bohol Aquaculture Research Foundation, inc.; Mindanao: RFO XIII (Agusan, Surigao and Misamis ), RFO IX (Zambanga) RFO XII (Pangil Bay Area).
BIOTECH at UPLB will serve as teh national reference laboratory to validate , intercalibrate and monitor the satellite laboratories and at the same time conduct training of personnel in the satellite laboratories
Source: BAR Today July – September 2000, Dr. Lourdes Mahilum-Tapay
For more information contact Dr Tapay at the Philippine National Collection of Microorganisms, National Institute of Molecular Biology and Biotechnology (BIOTECH). UP Los Banos College Laguna.