Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER I.3.

GENERAL INFORMATION

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1.	Diseases of Crustaceans Listed by the OIE
	Crustaceans are adversely affected by a number of diseases. This is especially evident in penaeid shrimp from aquaculture. All of the crustacean diseases that have significant social or economic notoriety are infectious diseases. The crustacean diseases and their aetiological agents that are included in the Aquatic Animal Health Code (the Aquatic Code) have a restricted geographical range, have no therapeutic remedies or treatments, are potentially excludable, and are of significant social and economic importance. There are currently eight diseases of crustaceans listed by the OIE. Seven of these eight crustacean diseases are listed because of the size and importance of the penaeid shrimp aquaculture industry. Therefore, the principles and methods discussed in this chapter will, of necessity, emphasise the penaeid shrimp.
	The OIE listed crustacean diseases, the nature of their respective aetiological agents, and their principal hosts are:
	.	Diseases of crustaceans listed by the OIE:
		Taura syndrome (viral/penaeid shrimp)
		White spot disease (viral/penaeid shrimp and other decapod crustaceans)
		Yellowhead disease (viral/penaeid shrimp)
		Tetrahedral baculovirosis (Baculovirus penaei) (viral/penaeid shrimp)
		Spherical baculovirosis (Penaeus monodon-type baculovirus) (viral/penaeid shrimp)
		Infectious hypodermal and haematopoietic necrosis (viral/penaeid shrimp)
		Crayfish plague (Aphanomyces astaci) (fungal/freshwater crayfish)
		Spawner-isolated mortality virus disease (viral/penaeid shrimp)
2.	Diagnostic Methods
	The methods available for diagnosis of the above-listed diseases include the traditional methods of morphological pathology (direct light microscopy, histopathology, and electron microscopy), bioassay methods with susceptible indicator hosts, and molecular methods (gene probes and polymerase chain reaction [PCR]). While tissue culture is considered to be a standard tool in medical, veterinary, and fish diagnostic laboratories, it has yet to be developed as a usable, routine diagnostic tool for crustacean pathogens. Clinical chemistry has not become a routinely used diagnostic tool by crustacean pathologists.
	2.1.	Diagnostic methods for diseases of crustaceans
		As of the time of writing this section of the Aquatic Manual, the available diagnostic methods that may be selected for diagnosis of the OIE listed crustacean diseases or detection of their aetiological agents are based on:
		.	Gross and clinical signs
		.	Direct bright-field, phase-contrast or dark-field microscopy with whole stained or unstained tissue wet-mounts, tissue squashes, and impression smears; and wet-mounts of faecal strands
		.	Histology of fixed specimens
		.	Bioassays of suspect or asymptomatic carriers using a highly susceptible host (life stage or species) as the indicator for the presence of the pathogen
		.	Transmission or scanning electron microscopy
		.	Antibody-based tests for pathogen detection using immune sera polyclonal antibodies (PAbs) or monoclonal antibodies (MAbs)
		.	Molecular methods
			DNA probes in dot-blot hybridisation assays directly with fresh tissue samples or with extracted DNA
			DNA probes or RNA probes for in situ hybridisation assays with histological sections of fixed tissues
			PCR and reverse-transcription (RT)-PCR for direct assay with fresh tissue samples or with extracted DNA or RNA.
		The detailed procedures for each of the available methods (screening, presumptive, and confirmatory) for diagnosis of each of the OIE listed crustacean diseases are outlined in the individual disease chapters of this Aquatic Manual.
		There is a paucity of antibody-based diagnostic tests available for the pathogens that cause crustacean diseases. As crustaceans do not produce antibodies, antibody-based diagnostic tests are limited in their application to pathogen detection. While a number of antibody-based diagnostic methods have been developed and are described in the literature, these were developed with mouse or rabbit antibodies generated to viruses purified from infected hosts. Because crustacean viruses cannot be routinely produced in tissue culture, purified virus from infected hosts must be used to produce antibody. This has severely limited the development and availability of this diagnostic tool. The recent application of MAb technologies to this problem has begun to provide a few antibody-based tests. MAbs are available for three of the OIE listed crustacean diseases (for Taura syndrome virus [TSV], infectious hypodermal and haematopoietic necrosis virus [IHHNV], and white spot syndrome virus [WSSV]). Antibody-based diagnostic kits/reagents for TSV and WSSV infections are currently available from commercial sources.
		Molecular methods have been developed and some methods are in widespread use for the detection of many of the viral, bacterial, and protozoan pathogens of the penaeid shrimp. DNA-based detection methods are readily available from the literature and some are available in kit form from commercial sources for the OIE listed pathogens TSV, WSSV, and yellowhead disease virus (YHV/GAV), IHHNV, Penaeus monodon-type baculovirus (MBV), Baculovirus penaei (BP), and spawner-isolated mortality virus (SMV). PCR or RT-PCR methods are available for several of these viruses and some are in routine use by certain sectors of the crustacean aquaculture industry. For other OIE listed viral pathogens, specific DNA probes tagged with nonradioactive labels are either reported in the literature or available commercially for application in dot-blot formats with haemolymph or tissue extracts, or for use with routine histological sections using in-situ hybridisation.
		Despite the growing dependence of the shrimp aquaculture industry on DNA-based diagnostic methods, few of the tests that is available from commercial sources or reported in the literature has been validated using controlled field trials. Likewise, there are few formal accreditation or certification programmes yet in place to assure that test results from technicians and laboratories are indeed accurate and the tests properly controlled. There is a growing need to standardise and validate the DNA-based diagnostic methods and the laboratories that use them. Standardisation 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 or 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 standardised. The implementation of a formal programme 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 programme with the goal of making these methods uniform, reliable, and readily applicable to disease control and management strategies for viral diseases of cultured penaeids.
3.	Sampling
	There are at least three purposes for which crustacean stocks may be sampled with regard to the OIE listed crustacean pathogens. These are: 1) surveillance; 2) stock or facility 'certification'; and 3) disease diagnosis. The number and type of samples to be taken for analysis varies greatly according to which of these purposes applies.
	A general approach to surveillance and sampling is given in Chapter 1.1.4. of this Aquatic Manual. The sampling should be designed in order to enable detection, at a 95% confidence level, of infected animals. The following section gives information relevant to sampling crustaceans. Until disease-specific details are included in the individual disease chapters in this Aquatic Manual, Table 1 can be used to calculate sample size.
	3.1.	Diagnosis in disease situations
		In clinical disease episodes, carefully selected quality specimens with representative lesions should be obtained from live or moribund crustaceans. Every effort should be made to sample those specimens for diagnosis that are representative of the disease(s) that is (are) affecting the crustacean stock of interest, and that are moribund or clinically diseased. Collection of dead specimens should be avoided. When cultured or wild crustacean stocks are presenting clinical signs of an active disease that are consistent with, or suggestive of, any one of the OIE listed crustacean diseases, care should be taken to ensure that the samples collected are preserved appropriately for the anticipated diagnostic tests (see sample preservation section for recommended methods).
		The recommended minimum numbers of specimens to collect for diagnostic testing are 100 for the larval stages of most crustaceans; 50 for the postlarval stages; and 10 for juveniles and adults. Sample numbers may be greater if clinically diseased specimens are readily apparent and collected. Nonetheless, these recommended 'minimum' sample numbers are provided as guidelines, and it must be emphasised that carefully selected, quality specimens are far more valuable (and cost-effective) diagnostic specimens than dozens or hundreds of specimens taken at random to 'fill out' the sample.
	3.2.	Diagnosis in asymptomatic crustaceans
		When samples are to be taken for surveillance, for testing of asymptomatic carriers of previous disease epizootics, for 'certification' of specific pathogen free (SPF) status, or for freedom of particular disease within a country, zone, or facility the sample size to be taken should be determined using a statistical table. The minimum sample size for each lot tested should provide a 95% level of confidence that infected specimens, if present, will be in the sample, assuming a defined minimum prevalence of infection equal or greater than 2%, 5% or 10%. For surveillance and certification purposes for OIE listed diseases, the samples taken for diagnostic tests at any given aquaculture site or from wild stocks, should include the appropriate number of specimens from each lot to be tested according to Table 1. For the OIE listed diseases it is highly recommended that the scheduling of sampling be planned (i.e. by farm schedule, season, etc.) so that the particular life-stage(s) are sampled at a time when the pathogen of concern is most likely to be detected. This is especially important when the available diagnostic methods are dependent on simple microscopy or histological methods and do not include molecular methods. For the baculoviruses BP and MBV larval and early postlarval are the most appropriate samples; for TSV, IHHNV, WSSV and YHV/GAV, juveniles and subadults provide the best samples; and for crayfish plague, juveniles and adults are suitable samples.
		Samples taken for molecular or antibody-based tests for OIE-listed crustacean diseases may be combined as pooled samples of no more than five specimens per pooled sample.
	3.3.	Testing for verification or maintenance of freedom from specific diseases
		Once a crustacean production facility has been recognised to be free of all or certain diseases listed in the Aquatic Code after 2 years of surveillance with laboratory tests and in the absence of any suspect clinical signs, twice-yearly inspections should continue. However, collection of specimens for testing may be reduced to 30 crustaceans (shrimp), including especially broodstock. Moribund shrimp observed during inspection visits must, however, be collected for further laboratory examination

Table 1. Sample size based on assumed pathogen prevalence in lot

Lot size	At 2% prevalence, size of sample	At 5% prevalence, size of sample	At 10% prevalence, size of sample
50	50	35	20
100	75	45	23
250	110	50	25
500	130	55	26
1000	140	55	27
1500	140	55	27
2000	145	60	27
4000	145	60	27
10,000	145	60	27
100,000 or more	150	60	30