DNA-BASED DIAGNOSTIC AND DETECTION METHODS FOR PENAEID SHRIMP VIRAL DISEASES - DONALD V. LIGHTNER

Department of Veterinary Science and Microbiology

University of Arizona, Tucson, AZ 85721 USA

The most important diseases, in terms of economic impact, of cultured penaeid shrimp in Asia, the Indo-Pacific, and the Americas have infectious etiologies. Among the infectious diseases of cultured shrimp, certain virus-caused diseases stand out as the most significant. Since the first report of a penaeid shrimp virus disease by Couch in 1974, at least 20 more viruses have been described from the penaeids (Table 1). The earliest diagnostic methods developed for these pathogens included the traditional methods of morphological pathology (direct light microscopy, histopathology, and electron microscopy), as well as enhancement and bioassay methods. While tissue culture is considered to be a standard tool in medical and veterinary diagnostic labs, it has never been developed as a useable, routine diagnostic tool for shrimp pathogens. Likewise, there are few antibody-based diagnostic tests available for the penaeid virus diseases (Lightner and Redman, 1998). The need for rapid and sensitive diagnostic methods has led to the application of modern biotechnology to penaeid shrimp diseases. DNA-based detection methods for the most important viral pathogens (IHHNV, HPV, SMV, TSV, YHV, GAV/LOV, WSSV, MBV, and BP) have been reported in the literature and some DNA-based diagnostic methods are commercially available. PCR or RT-PCR methods are available for several of these viruses and some are in routine use by certain sectors of the industry. For others, specific DNA probes tagged with non-radioactive labels provide highly specific detection methods for application in dot blot formats with hemolymph or tissue extracts, and with routine histological sections using in situ hybridization (Lightner, 1996; OIE, 1997; Lightner and Redman, 1998).

The OIE Fish Disease Commission at its September 1998 meeting voted to recommend to the OIE that three of the penaeid shrimp viruses diseases from the "listed" category be upgraded to the "notifiable" category. If the recommendations of the Fish Disease Commission are approved by the OIE's General Assembly in May 1999, the notifiable and listed viral pathogens of crustacea will be those shown in Table 2. All of these viral diseases affect cultured penaeid shrimp. Before a disease may be included on the OIE lists of notifiable and listed diseases, several criteria must be met: 1) the etiological agent must be known, 2) reliable diagnostic(s) methods must be available, and 3) the disease must be a significant disease of local, regional, or international importance.

The accompanying Tables in this report list the known penaeid shrimp viruses and summarize the available traditional and DNA-based diagnostic and detection methods available for the recently proposed OIE notifiable and listed pathogens of penaeid shrimp (OIE, 1997; OIE, unpublished report). The diagnostic and detection methods for these viruses are listed in Table 3. There is a growing need to standardize and validate the DNA-based diagnostic methods and the laboratories that use them. Standardization of DNA-based diagnostic methods is almost inherent in the nature of the tests. That is, a specific DNA probe, or a specific set of primers, that is used to demonstrate the presence of absence of a unique DNA or RNA sequence does not vary from batch to batch. Hence, with proper controls, these DNA-based methods are readily standardized (Reddington and Lightner, 1994). However, despite the growing dependence of the shrimp culture industry on DNA-based diagnostic methods, none of the tests that are available from commercial sources nor from the literature have been validated using controlled field tests. Likewise, there are no formal accreditation or certification programs yet in place to assure that test results from technicians and laboratories running the tests are indeed accurate and properly controlled (Lightner and Redman, 1998). The implementation of a formal program by appropriate international agencies or professional societies is needed to validate new diagnostic methods and to periodically review the accreditation and certification of diagnosticians and diagnostic laboratories. The establishment of regional reference laboratories for DNA-based diagnostic methods of penaeid shrimp/prawn pathogens would fit well into such a program with the goal of making these methods uniform, reliable, and readily applicable to disease control and management strategies for viral diseases of cultured penaeids.

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Table 1a. DNA Viruses of Penaeid Shrimp (as of February 1999; modified from Lightner, 1996; Lightner and Redman, 1998)

Table 1b. RNA Viruses of Penaeid Shrimp (as of February 1999; modified from Lightner, 1996; Lightner and Redman, 1998).

Table 2. Proposed list of OIE notifiable and listed penaeid shrimp diseases and their current, presently known distribution in wild and cultured stocks (modified from Lightner, 1996; Lightner and Redman, 1998

Table 3. Diagnostic and pathogen detection methods for the OIE notifiable and listed viral diseases of penaeid shrimp (modified from Lightner, 1996; Lightner and Redman, 1998

* Definitions for each virus:

- = no known or published application of technique.

+ = application of technique known or published, but not commonly practiced or readily available.

++ = application of technique considered by authors of present paper to provide sufficient diagnostic accuracy or pathogen detection sensitivity for most applications.

+++ = technique provides a high degree of sensitivity in pathogen detection.

Methods:

BF = bright field LM of tissue impression smears, wet-mounts, stained whole mounts;

LM = light microscopy; PH = phase microscopy, DF = dark-field microscopy,

EM = electron microscopy of sections or of purified or semi-purified virus;

ELISA = enzyme-linked immunosorbent assay; PAbs = polyclonal antibodies;

MAbs = monoclonal antibodies; DBH = dot blot hybridization, ISH = in situ hybridization