Department of Fishery Microbiology, University of Agricultural Sciences,
College of Fisheries, Mangalore - 575 002, India
Rapid detection of pathogens would be very essential for effective health management in aquaculture. While conventional microbiological isolation methods are used in case of bacterial pathogens, histopathology is widely used to detect viral infections. However, these methods are time consuming and lack sensitivities to detect latent pathogens. Polymerase chain reaction (PCR) on the other hand is highly sensitive and rapid and can be used to detect latent pathogens.
In our laboratory, we have been studying the application of PCR for rapid detection of shrimp pathogens such as whitespot syndrome virus (WSSV) and Vibrio spp. WSSV is a serious pathogen that has caused extensive mortalities in shrimp culture systems in India (Karunasagar et al., 1997, 1998; Karunasagar and Karunasagar, 1999). We have studied the application of PCR primers reported by Lo et al. (1996) for detection of WSSV. Our results indicate that one step PCR is able to detect infection in case of clinically symptomatic animals. Two step PCR was necessary to detect WSSV in clinically asymptomatic shrimps and in other carrier animals (Otta et al., 1999). Using two step PCR, a large number of apparently healthy P.monodon post larval stages were screened for the presence of WSSV. Only 5% apparently healthy PL gave positive reaction in one step PCR whereas 48% showed positive reaction in two step PCR (Otta et al., 1999). These results suggest that two step PCR is very essential for detection of WSSV in asymptomatic animals and carriers.
Using PCR, WSSV could be detected in carrier animals such as crabs, Acetes spp and even in water samples. An evaluation of the relation between PCR positivity and infectivity was studied in the case of Penaeus monodon showing clinical signs of white spot syndrome. Viral extracts from freshly harvested shrimp (22-25g) were highly infective causing clinical signs and mortality in healthy shrimp within 48 h. Viral extracts prepared from clinically symptomatic animals, which were stored under frozen conditions (-20°C) for two months, showed positive reaction in PCR. However, the viral extracts from the frozen specimens failed to induce clinical signs or mortality in healthy animals. This suggests that WSSV might lose infectivity during frozen storage in shrimp tissue. Therefore, PCR positivity in frozen shrimp should be interpreted with caution with respect to its potential to spread the virus.
In our laboratory, PCR is also being used to detect Vibrio parahaemolyticus in shrimp. Comparision of culture and PCR methods show that PCR would be very useful in detecting this organism and the technique has potential to detect even atypical strains showing variation in biochemical reactions (Karunasagar et al., 1997). Thus PCR would be a very useful tool for rapid and sensitive detection of pathogens in shrimp. The technique has applications in diagnostic laboratories, for monitoring health, to identify environmental reservoirs of infections, to detect the presence of pathogens in animals in quarantine etc.
References
Lo, C.F., Leu, J.H., Ho, C.H., Chen, C.H., Peng, S.E., Chen, Y.T., Chou, C.M., Yeh, P.Y., Huang, C.J., Chou, H.Y., Wang, C.H. and Kou, G.H. (1996). Detection of white spot syndrome baculovirus (WSSBV) in penaeid shrimp using polymerase chain reaction. Diseases of Aquatic Organisms 25, 133-141.
Karunasagar, I., Otta, S.K. and Karunasagar, I. (1997). Histopathological and bacteriological study of white spot syndrome of Penaeus monodon along west coast of India. Aquaculture 153, 9-13.
Karunasagar, I., Nayak, B.B. and Karunasagar, I. (1997). Rapid detection of Vibrio parahaemolyticus from fish by polymerase chain reaction. In Diseases in Asian Aquaculture III. T.W. Flegel,. et al (Ed). Pp. 119-122. Asian Fisheries Society, Manila..
Karunasagar, I., Otta, S.K. and Karunasagar, I. (1998). Disease problems affecting cultured penaeid shrimp in India. Fish Pathology 33, 413-419.
Karunasagar, I. and Karunasagar, I. (1999). Diagnosis, treatment and prevention of microbial diseases of fish and shellfish. Current Science 76, 387-399.
Otta, S.K., Shubha, G., Biju, J., Karunasagar, I. and Karunasagar, I. (1999). Polymerase chain reaction based detection of whitespot syndrome virus in cultured and wild crustaceans in India. Diseases of Aquatic Organisms (in press).
