Optimized multiplex PCR assays for simultaneous detection of viruses infecting hatchery-reared shrimp, Penaeus monodon in the Philippines

Introduction There are more than 20 viruses that affect penaeid shrimps, and these infect both cultured and wild stock in various geographical locations worldwide (Hernandez-Rodriguez et al 2001). The white spot syndrome virus (WSSV) is one of the widespread and devastating viruses that has affected the shrimp culture industry. However, there are other viruses, which are considered less virulent but can cause significant reduction in the profitability of the culture operations. Examples of these are the Penaeus monodon-type baculovirus (MBV), which is implicated in the stunted growth of the shrimp (Flegel et al 1999) and the infectious hypodermal and hematopoietic necrosis virus (IHHNV), which can lead to growth reduction and severe cuticular deformities in the affected shrimp especially Litopenaeus vannamei and Penaeus monodon, known as the runt deformity syndrome (Kalagayan et al 1991). These deformities and decrease in growth can reduce the market value of the shrimp by 10-50%, depending on the severity of the disease (Lightner & Redman 1998). These viruses are not only prevalent in shrimp farms but also infect shrimp postlarvae in hatcheries. Dual infections of MBV and WSSV have been observed in wild black tiger shrimp from several sampling sites in the Philippines using PCR (de la Pena et al 2005). Infections of WSSV and MBV were also seen in P. monodon hatcheries in the Philippines (Natividad et al 2006). In similar cases, mixed infections of WSSV and MBV were also reported in other Asian countries like Vietnam (Hao et al 1999) and Thailand (Flegel et al 2004). In India, Manivannan et al (2002) reported that P. monodon post-larvae were simultaneously infected by three different viruses including MBV, hepatopancreatic parvovirus (HPV) and WSSV as detected by histology and non-nested PCR. In an effort to minimize the spread of viruses in shrimp farms and hatcheries, the Philippine government through the Bureau of Fisheries and Aquatic Resources (BFAR) has modified routine fry quality assessment to include screening for WSSV. Definitive diagnosis and certification of the infection status of P. monodon broodstock and fry by WSSV, the use of polymerase chain reaction (PCR) assay is recommended (Natividad et al 2006). However, the inclusion of other shrimp viruses including MBV and IHHNV during routine PCR analysis is also done because these viruses can result in reduction in growth of infected shrimp and thus decrease productivity. The simultaneous detection of these shrimp viruses in shrimp postlarvae can be done in a single PCR analysis known as multiplex PCR. There have been several multiplex PCR assays that have been developed to detect commercially important viruses of shrimp. Tsai et al (2002) developed multiplex reverse transcription-polymerase chain reaction (mRT-PCR) for simultaneous detection of WSSV and Taura syndrome virus (TSV) in pacific white shrimp P. vannamei. Xie et al (2007) standardized a multiplex RT-PCR assay to detect IHHNV, TSV and WSSV in penaeid shrimp. Recently, Khawsak et al (2008) used multiplex RT-PCR to detect simultaneously six shrimp viruses including, yellow-head virus (YHV), WSSV, TSV, IHHNV, HPV and MBV. Abstract. Multiple viral infections occur in most shrimp hatcheries and early detection of the pathogens is necessary to ensure efficient management of the hatchery facilities. The presence of the white spot syndrome virus, Penaeus monodon-type baculovirus and the infectious hypodermal and hematopoietic necrosis virus in shrimp, Penaeus monodon postlarvae was confirmed simultaneously using multiplex PCR assays based on primers from previously published studies. Different primer combinations and the optimum annealing temperatures were standardized that could be applicable for routine diagnoses of these viral diseases in shrimp. The optimized multiplex PCR assays could be used for regular monitoring and surveillance of these viruses in shrimp hatcheries as well as tracing the movement of these viruses in different shrimp-farming regions in the country during transport of shrimp postlarvae for stocking as well as broodstock for hatchery purposes.