Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER I.1.

GENERAL INFORMATION

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GENERAL BASIS FOR FISH HEALTH SURVEILLANCE/CONTROL PROGRAMMES

1.	Target Pathogens and Diseases
	Target pathogens and fish diseases are included in the Aquatic Animal Health Code (the Aquatic Code) according to the following basic considerations: they resist or respond poorly to therapy, have a restricted geographical range, are of high socio-economic importance, and occur in species involved in international trade. The list of fish diseases considered for notification and certification is currently restricted to the following diseases:
	Epizootic haematopoietic necrosis (EHN)
	Infectious haematopoietic necrosis (IHN)
	Oncorhynchus masou virus disease (OMVD)
	Spring viraemia of carp (SVC)
	Viral haemorrhagic septicaemia (VHS)
	Channel catfish virus disease
	Viral encephalopathy and retinopathy
	Infectious pancreatic necrosis
	Infectious salmon anaemia
	Epizootic ulcerative syndrome
	Bacterial kidney disease (Renibacterium salmoninarum)
	Enteric septicaemia of catfish (Edwardsiella ictaluri)
	Piscirickettsiosis (Piscirickettsia salmonis)
	Gyrodactylosis (Gyrodactylus salaris)
	Red sea bream iridoviral disease
	White sturgeon iridoviral disease
2.	Overall Approach for Animal Health Control in Fish Culture
	A comprehensive approach for animal health control in fish culture requires:
	.	Assessment of the health status of animals using methods based on the provision in Chapter 1.1.4.
	.	The constraint of restocking open waters and farming facilities only with aquatic animals having a health status higher than or equal to that of animals already living in the considered areas.
	.	Eradication of disease when possible, by slaughtering infected stocks, disinfecting facilities and restocking with fish from approved disease-free sources.
	.	Notification by every Member Country of its particular requirements, besides those provided by the Aquatic Code, for importation of aquatic animals and aquatic animal products.
	If the above procedures are followed, it becomes possible to give adequate assurance of the health status of aquaculture products for specified diseases, according to their country, zone or site of origin.
	The issue of a health certificate by the appropriate official, based on a health status report and examinations of aquatic animals, provides assurance that the aquaculture products in a defined consignment originate from a whole country, a zone or a farm/harvesting site free of one or more of the specified diseases listed in the Aquatic Code and possibly of other specified diseases (see model of international certificate in the Aquatic Code).
	The assessment of the health status of fish stocks is based on inspection of fish production sites and further laboratory examination of samples originating from fish specimens taken among the stock of a defined fish population. This endeavour requires the fish sample to be collected according to defined sampling size charts and the samples to be processed according to accepted methods.
	Several techniques are applicable for aquatic animal pathogens. For screening and diagnostic purposes, the Aquatic Manual has established two types of examination procedures that will be acceptable for such work; 1) Screening methods, and 2) Diagnostic methods. The accepted methods are listed under each disease chapter.

B. SAMPLING PROCEDURES

1.	Collection of Fish Specimens
	1.1.	Diagnosis in a disease situation
		A minimum number of ten moribund fish or ten fish exhibiting clinical signs of the diseases in question must be collected: fish should be alive when collected, and should be sent to the laboratory alive or killed and packed separately in sealed aseptic refrigerated containers or on ice. The freezing of collected fish must be strictly avoided. However, it is highly preferable and recommended to collect organ samples from the fish immediately after they have been selected at the fish production site and to store and process the samples as described in Sections 2 and 3. An identification label that includes information on the place and time of sampling must be attached to the sample.
	1.2.	Fish appear to be clinically normal
		Fish collection must encompass a statistically significant number of specimens, but it is obvious that failure to detect certain pathogens from the sample does not guarantee the absence of these agents in the specimen examined or in the stock. This is particularly true of free-ranging or feral stocks from which it is difficult to collect a representative and random sample. However, the risk of a pathogen escaping the surveillance system is reduced in fish farms whose fish stocks have been inspected and checked for pathogens for several years (at least 2), insofar as they are not exposed to possible recontamination by feral fish.
		When a given fish production site harbours a broodstock, it is essential for one of the sample collections made each year to be focused on the sexual products (sperm and ovarian fluid) released by broodfish at the time of spawning (see below). If an adult broodstock includes fish of different ages, the older fish should be selected for sampling:
		.	Samples must comprise all susceptible species on the site (see relevant chapters of this Aquatic Manual for the list of species susceptible to each disease), with each lot of a species being represented in the sample group. A lot is defined as a group of the same fish species that shares a common water supply and that originates from the same broodfish or spawning population.
			The geographical origin of samples should be defined by the name of the sampling site associated with either its geographical co-ordinates or its location along a river course or body of water.
		.	If any moribund fish are present in the fish population to be sampled, they should be selected first for sample collection and the remainder of the sample should comprise randomly selected live fish from all rearing units that represent the lot being examined.
		.	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 finfish. Until disease-specific details are included in the individual disease chapters in this Aquatic Manual, Table 1 can be used to calculate sample size.
		.	As in the case of clinically infected fish, organ and fluid samples must be taken and processed as soon as possible after fish specimen collection. Sample freezing must be avoided.

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

After Ossiander & Wedemeyer, 1973.
 

	1.3.	Sampling specifications according to the objectives of a given fish surveillance programme
		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 finfish. Until disease-specific details are included in the individual disease chapters in this Aquatic Manual, Table 1 can be used to calculate sample size.
		a)	Achievement of the health status of a fish stock/population at a given inspection site
			.	A fish culture unit must be inspected twice a year for 2 years at the appropriate life stage of the fish and at times of the year when temperature and season offer the best opportunity for observing clinical signs and isolating pathogens. On each occasion, fish of the susceptible species listed in the Aquatic Code for the disease in question must be collected in order to detect a prevalence of infection equal to or higher than 2% at 95% confidence level. Most often, 150 fish will thus be collected on each occasion. If broodfish are present during one of the two inspections, up to 150 ovarian fluid samples will be taken from broodfish in the given fish culture unit.
			.	If fish health surveillance is focused on wild fish populations at a given site of inspection or on rearing ponds without holding facilities in which different fish crops may be pooled, 150 fish specimens must be collected once a year for 2 years. Insofar as it is possible, specimens of the oldest fish and/or ovarian fluid must be collected as a priority.
			.	During this 2-year period, the fish production unit may only receive fish from a unit whose health status has already been approved and is equal to or higher than the health status sought for the facility being inspected.
		b)	Maintenance of the health status
			.	Once a fish production unit, including pond fish production units equipped with holding facilities, has been recognised to be free from 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 must continue. However, collection of fish specimens may be reduced to 30 fish, including broodfish when available. Moribund fish observed during inspection visits must, however, be collected for further laboratory examination.
			.	Maintenance of health status of wild fish populations relevant to diseases listed in the Aquatic Code at a given site of inspection, can only be ascertained by annual collection of 150 individuals including as many broodfish as possible.
			.	The fish production unit may only receive fish having a health status higher than or equal to that of those already present.
			.	If, during viral testing of samples, a cytopathic effect (CPE) appears in cell cultures inoculated with dilutions of the samples being tested, virus identification procedures have to be undertaken immediately (see the relevant chapters). Provisions must be taken to suspend the approved health status of the production unit and/or the zone (if it was approved previously) from which the virus-positive sample originated. The suspension of approved status will be maintained until it is demonstrated that the virus in question is not the one referred to in the granting of disease-free status.

The above sampling specifications for the achievement and maintenance of the health status of fish at given fish production sites imply that all provisions given in Section A.2. (Overall approach for animal health control in fish culture) are in force.
 

2.	Sample Material to be used in Viral and Bacteriological Tests
	Sample material depends both on the size of animals and the objective of testing, i.e. diagnosis of overt disease or detection of fish that are asymptomatic pathogen carriers.
	2.1.	Specifications according to fish size
		Alevin and sac fry: sample the entire fish but remove the yolk sac if present.
		Fish 4-6 cm: take the entire viscera including the kidney. A piece of encephalon can be obtained after severing the head at the level of the rear edge of the operculum and pressing it laterally.
		Fish over 6 cm: take the kidney, spleen and encephalon.
		Broodfish: take the ovarian fluid and/or tissues.
	2.2.	Specifications according to clinical status
		In the case of clinical infection, besides whole alevin or entire viscera, organs to be sampled are anterior kidney, spleen and encephalon for virus screening, and kidney and spleen for bacterial screening. Samples from ten diseased fish will thus be taken and combined to form pools of a maximum of five fish each. The amount of material should be approximately 1.5 g/pool of material from five fish.
		For detecting asymptomatic carriers, samples may be combined as pools of no more than five fish/pool, for a total weight of about 1.5 g. Pools of ovarian fluid from five broodfish should not exceed a total volume of 5 ml, i.e. 1 ml/broodfish. These ovarian fluid samples are to be taken individually from every sampled female and not collected following the pooling of ova.
		Once aseptically removed from fish, the organs and/or ovarian fluid sampled are each split into two parts if both bacteriological and virological examinations are to be done.
3.	General Processing of Organs/Fluid Samples for Virological Examination
	3.1.	Transportation and antibiotic treatment of samples
		Pools of organs or of ovarian fluids are placed in sterile vials and stored at 4°C until virus extraction is performed at the laboratory. Virus extraction should optimally be carried out within 24 hours after fish sampling, but is still acceptable for up to 48 hours.
		Organ samples may also be transported to the laboratory by placing them in vials containing cell culture medium or Hanks' balanced salt solution (HBSS) with added antibiotics to suppress the growth of bacterial contaminants (one volume of organ in at least five volumes of transportation fluid). Suitable antibiotic concentrations are: gentamycin (1000 µg/ml) or penicillin (800 International Units [IU]/ml) and streptomycin (800 µg/ml). The antifungal compounds Mycostatin® or Fungizone® may also be incorporated into the transport medium at a final concentration of 400 IU/ml. Serum or albumen (5-10%) may be added to stabilise the virus if the transport time will exceed 12 hours.
	3.2.	Virus extraction
		.	This procedure is conducted below 15°C and preferably at between 0 and 4°C.
		.	Decant antibiotic-supplemented medium from organ sample.
		.	Homogenise organ pools in transport medium at a final dilution of 1/10 using a mortar and pestle or electric homogeniser until a paste is obtained.
		.	If organ samples have not been treated with antibiotics prior to homogenisation, organ homogenates are to be resuspended in antibiotic-supplemented medium and incubated in this medium for 2-4 hours at 15°C or overnight at 4°C. Likewise, ovarian fluid samples may be treated with antibiotics to control microbial contamination. In neither case can homogenates or ovarian fluid samples be diluted more than twofold.
		.	Clarify the diluted homogenates by centrifugation at 2000 g for 15 minutes and collect the supernatants.
		.	Ovarian fluid samples should be centrifuged in the same way as organ homogenates, and their supernatants used directly in subsequent steps.
	3.3.	Treatment to neutralise enzootic viruses
		In some countries, fish are often asymptomatic carriers of enzootic viruses, such as infectious pancreatic necrosis virus (IPNV), which induce a CPE in susceptible cell cultures and thus complicate isolation and identification of target pathogens. In such situations, the infectivity of the enzootic viruses must be neutralised before testing for the viruses listed in the Aquatic Code. However, when it is important to determine whether one of the enzootic viruses is present, samples must be tested with and without the presence of neutralising antibodies (NAbs).
		To neutralise birnaviruses, mix equal volumes (200 µl) of a solution of NAbs against the indigenous birnavirus serotypes with the supernatant to be tested. Allow the mixture to react for 1 hour at 15°C or overnight at 4°C prior to inoculation on to susceptible cell monolayers. The titre of the NAb solution used (it may be a multivalent serum) should be at least 2000 in a 50% plaque reduction test versus the viral serotypes present in the given geographical area.
		When samples are from a country, region, fish population or production unit considered to be free from enzootic viral infections, this treatment of the organ homogenate should be omitted.
		This approach can also be used to neutralise other viruses enzootic to the area being tested.
4.	General Processing of Samples Intended for Bacteriological Examination
	As in viral infections, internal organs may be used as a source of isolation whenever systemic infection is suspected. However, active proliferation of saprophytic microorganisms is such a disadvantage that live fish are preferred for bacteriological examination. The fact that no antibiotic substances may be added to the transport medium in which the samples are collected reinforces this preference.

C. MATERIALS AND BIOLOGICAL PRODUCTS REQUIRED FOR THE ISOLATION AND IDENTIFICATION OF FISH PATHOGENS

1.	Fish Viruses
	1.1.	Fish cell lines
		The following five fish cell lines will be required to test for the fish pathogens mentioned in the Aquatic Code:
		Bluegill fry (BF-2)
		Channel catfish ovary (CCO)
		Chinook salmon embryo (CHSE-214)
		Epithelioma papulosum cyprini (EPC)
		Rainbow trout gonad (RTG-2)
		Technical information on the use of these cells for the isolation of the fish pathogens listed in the Aquatic Code is given in Table 2.
	1.2.	Culture media
		Traditional Eagle's minimal essential medium (MEM) with Earle's salt supplemented with 10% fetal calf serum (FCS), antibiotics and 2 mM l-glutamine is the most widely used medium for fish cell culture.
		Stoker's medium, however, which is a modified form of the above medium comprising a double-strength concentration of certain amino acids and vitamins, is particularly recommended to enhance cell growth, using the same supplementations as above +10% tryptose phosphate.
		These media are buffered with either sodium bicarbonate, 0.16 M tris-hydroxymethyl aminomethane (Tris) HCl, or, preferably, 0.02 M N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (HEPES). The use of sodium bicarbonate alone is restricted to those cell cultures made in tightly closed cell culture vessels.

Table 2. Technical information on the most suitable fish cell lines for detection of the
viral agents listed in the Aquatic Code

Cell line nomenclature   Properties - Culture characteristics   BF-2   CCO   CHSE-214   RTG-2   EPC   Cell morphology   Fibroblastic   Fibroblastic/ epithelioid   Epithelioid   Fibroblastic   Epithelioid   Temperature range (°C)   15-28   15-35   4-25   4-25   10-33   Optimum growth temperature (°C)   20   30   20   20   30   Inoculum (number of cells x 104/cm2) to achieve loose confluency within 24 hours   20   35   50   40   30   Saturation density (number of cells x 104/cm2)   150   300   300   200   300

 

		For cell growth, the fetal bovine serum content of the medium is usually 10%, whereas for virus isolation or virus production it may be reduced to 2%. Similarly, the pH of the culture medium for cell growth is 7.3-7.4 and is adjusted to 7.6 for virus production or virus assay.
		The composition of the most frequently used antibiotic mixture is penicillin (100 IU/ml) and dihydrostreptomycin (100 µg/ml). Mycostatin (50 IU/ml) may be used if fungal contamination is expected. Other antibiotics or antibiotic concentrations may be used as convenient for the operator depending on the antibiotic sensitivity of the bacterial strains encountered.
	1.3.	Virus positive controls and antigen preparation
		a)	Virus nomenclature
			.	Epizootic haematopoietic necrosis virus (EHNV)
			.	European catfish virus (ECV)
			.	European sheatfish virus (ESV)
			.	Infectious haematopoietic necrosis virus (IHNV)
			.	Oncorhynchus masou virus (OMV) (snonym Salmonid herpesvirus type 2)
			.	Spring viraemia of carp virus (SVCV)
			.	Viral haemorrhagic septicaemia virus (VHSV) (snonym Egtved virus)
		b)	Virus production
			For the production of most of these viruses, the susceptible cell cultures (see relevant sections in this Aquatic Manual) must be inoculated with fairly low multiplicities of infection (m.o.i.), i.e. 10-2 to 10-3 plaque-forming units (PFU) per cell. However, the m.o.i. for OMV must be increased up to 0.1-1 PFU/cell. Furthermore, the best yields for OMV are obtained using inocula containing cell debris from a monolayer previously infected with the virus.
		c)	Virus preservation and storage
			.	Dilute virus-containing cell culture fluids in order to obtain virus titres averaging 1-2 x 106 PFU/ml.
			.	Dispense the resulting viral suspensions into sterile vials at volumes of 0.3-0.5 ml each.
			.	Freeze and store each series of standard virus stocks at -80°C or liquid nitrogen, and check the titre of each virus stock at regular intervals if it has not been used during that time period. This is especially important for OMV, which is relatively labile, even when stored at -80°C.
			Lyophilisation: long-term storage (decades) of the seeds of standard virus strains is achievable by lyophilisation. For this purpose, viral suspensions in cell culture medium supplemented with 10% FCS are mixed (v/v) with a 50% sodium glutamate solution in distilled water before processing. Seal or plug under vacuum and store at 4°C, in the dark.
2.	Fish Bacteria
	2.1.	Culture media
		Few species of fish pathogenic bacteria require special media for cultivation, and most of the commonly used isolation peptones (trypsin-soya, brain-heart, etc.) can conveniently be employed. However, the low optimal temperatures of some species result in slow growth, and small colonies are frequently obtained during isolation. In these cases, it is usual to add enrichment factors, such as serum or blood at 5-10%, in order to improve cultivation. Conversely Renibacterium salmoninarum is a very fastidious organism and requires special media enriched with cysteine (No. A11B).
		Bacteriology of fish is generally conducted at temperatures between 20 and 26°C. It is sometimes necessary or useful to have access to several incubators. Renibacterium salmoninarum, Flavobacterium psychrophilus and others need 15°C for optimal growth and many of the bacteria isolated from warm water fish may be incubated at 30 or 37°C to accelerate the diagnostic steps.
	2.2.	Storage of cultures
		Bacterial strains can be stored for a short term on ordinary media, placing the slants or broths at 4°C. For most strains, the use of commercial media or agar slants with mineral oil overlay will extend viability to 1-2 years under the same conditions without further special requirements.
		Freezing is probably the best way to preserve bacterial suspensions of high titre. However, it does not always prevent some phenotype characteristics from changing. When stability of characteristics such as virulence is important, it may be better to use lyophilisation, although the number of viable bacteria may be decreased dramatically.
		Different kinds of supports have been proposed to improve the efficacy of freezing and lyophilisation, namely glycerol (5-15%) in the former case, and skim milk, lactose, and dextran (5-10%) in the latter. There is no general rule, and convenient conditions have to be determined in prior trials for all species. The addition to fresh cultures of one volume of support containing Bactopeptone 11% + Dextran 4% has provided excellent results for lyophilisation of certain fish bacteria, but other formulae would be worth testing in many cases.
3.	Serology
	3.1.	Production of rabbit antisera and polyclonal antibodies to fish viruses
		There are various ways in which antibodies against fish viruses can be raised in rabbits. Titre and specificity are influenced, however, by the inoculation programme used. The following immunisation protocols may be used to produce antisera for use in the virus isolation and/or identification procedures described later.
		a)	Antisera to infectious pancreatic necrosis virus
			Intravenous injection with 50-100 µg of purified virus on day 0, followed by an identical booster on day 21, and bleeding 5-7 days later. Rabbits may be reused if not bled completely.
		b)	Antisera to other viruses
			The immunisation protocols alternate an intramuscular or intra-dermal injection with further intravenous boosters:
			Day 0: primary injection, 500-1000 µg of virus is mixed (v/v) with adjuvant (Freund's incomplete or other; use of Freund's complete adjuvants may be restricted on animal welfare grounds - alternative synthetic adjuvants include trehalose dimycolate and monophosphate lipid A) giving a total volume of 1.2 ml. This antigen is delivered to the rabbit as multipoint intradermal injections (20 points on each side) after the animal has been shaved.
			Day 21: collect about 20 ml of blood and check for reactivity (neutralisation, fluorescence); boost intravenously with the same amount of virus as in the primary injection.
			Prior to the intravenous booster injection, the rabbit must be treated with promothazine (12 mg intramuscularly) to prevent possible anaphylactic response.
			Day 28: sample the blood, check the serum reactivity and bleed or boost according to the results.
			For rhabdoviruses, this immunisation procedure is well suited to production of antisera to be used in immunofluorescence and enzyme-linked immunosorbent assay. However, a more efficient method for production of neutralising antisera is regular intravenous injection without adjuvant (0.2 ml) every 3-4 days (twice a week). As many as 15 injections may be necessary; 1 week after the last injection, a serum sample should be collected and tested.
	3.2.	Antisera to fish bacteria
		It is still difficult to obtain antimicrobial sera in large amounts from commercial sources, and it will often be necessary to prepare such antisera. The general methods are the same as for viruses. Bacterial antigens are often used as crude preparations killed by heating or by formalin (0.35% formalin). Rabbit injection at increasing doses can be either intramuscular, each week, with adjuvant the first time, or intravenous at 3-4-day intervals without adjuvant. A booster injection is often required after 15 days.
		A special multipoint intradermal schedule has proven very efficient for anti-Renibacterium sera production, and is also valuable for other bacteria of low antigenicity in rabbits.
		The antigen is heat killed (60°C, 45 minutes) and adjusted to 2 mg/ml. The flanks of the animals are thoroughly shaved, and multipoint intradermal injections are performed using total amounts of 1 mg bacteria/animal, according to the following schedule:
		Day 1	1 mg + complete Freund's adjuvant (CFA) (v/v)
		Day 21	1 mg + incomplete Freund's adjuvant (IFA) (v/v)
		Day 42	1 mg + IFA
		Day 63	1 mg antigen
		Day 68	Collecting of blood samples (about 30 ml)
		Withdrawal injections and bleeding for serum collection may be repeated at 1-month intervals.
	3.3.	Processing and storage of immune sera
		After blood clotting, collect and centrifuge the serum at 20°C and heat it for 30 minutes at 56°C. Filter the resulting heat-inactivated serum through a membrane filter (450 nm pore size) and temporarily store it at 4°C for the time necessary for the screening of its reactivity and specificity and for checking that these properties are not affected by preservation conditions (e.g. freezing or lyophilisation). Sterile rabbit sera can be kept for at least 2 months at 4°C without any change in their properties. Dispense (usually as small volumes) and freeze at -20°C or lyophilise.
		Immunoglobulins (Ig) may be extracted from antisera using conventional methods suitable for Ig purification. Selective attachment to protein A constitutes a reliable and effective method. The concentration of Ig solutions is adjusted to the values required for further conjugate preparation or storage.
		Preservation of Ig: Mix a solution of Ig of concentration 2 mg/litre with sterile pure glycerol (v/v) and keep at -20°C. Solutions of Ig with a higher concentration may also be prepared in glycerol.
	3.4.	Mouse monoclonal antibodies to fish viruses and bacteria
		Monoclonal antibodies (MAbs) to most of the fish viruses have been raised over the past years. Some of them, singly or as two or three associated MAbs, have given rise to biological reagents suitable for the identification of virus groups (IPN, VHS, IHN). Other MAbs, taken individually or as components of Ab panels, allow accurate typing of VHSV and IHNV. These MAbs can be obtained from the Reference Laboratories listed at the end of this Aquatic Manual.
		The production of MAbs to bacteria has also been described. It has resulted in the development of commercial diagnostic kits for Renibacterium salmoninarum, but in most cases remains limited to specialised laboratories.
		In theory, mouse monoclonal IgGs can be processed and stored as for polyclonal IgGs. However, the reactivity of certain MAbs may be impaired by processes such as enzymatic- or radio-labelling or lyophilisation. It is thus necessary to test various MAbs for the conditions under which they will be used.
	3.5.	Use of molecular techniques for confirmatory testing and diagnosis
		Molecular techniques including DNA probes and the polymerase chain reaction (PCR) have been developed for the identification of many pathogens of aquatic animals. However, as is the case with several other diagnostic techniques, an advantage in sensitivity is frequently offset by problems in interpretation or susceptibility to technical problems. Whereas methods based on direct culture or serology are relatively robust, PCR can be quite dependent on the conditions under which it is run and can be highly subject to laboratory contamination by previous PCR products, yielding false-positive results. Thus, while several DNA probe and PCR protocols are included in this version of this Aquatic Manual as diagnostic or confirmatory methods, where possible, well-established techniques (e.g. virus isolation) are specified as standard screening methods. Whenever these newer molecular techniques are used, they should be performed with caution and with special attention to the inclusion of adequate positive and negative controls.

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