Manual of Diagnostic Tests for Aquatic Animals 2003

PART 2
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SECTION 2.1.
  

CHAPTER I.1.
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Summary
? - Index

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:
 

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Assessment of the health status of animals using methods based on the provision in Chapter 1.1.4.
 

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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.
 

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Eradication of disease when possible, by slaughtering infected stocks, disinfecting facilities and restocking with fish from approved disease-free sources.
 

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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.
 

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:
 

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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.
 

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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.
 

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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.
 

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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.
 

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
 

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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.
 

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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.
 

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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
 

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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.
 

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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.
 

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The fish production unit may only receive fish having a health status higher than or equal to that of those already present.
 

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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.
 

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 as described in Chapters 2.1.1.-2.1.5.
 

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
 

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This procedure is conducted below 15°C and preferably at between 0 and 4°C.
 

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Decant antibiotic-supplemented medium from organ sample.
 

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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.
 

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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.
 

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Clarify the diluted homogenates by centrifugation at 2000 g for 15 minutes and collect the supernatants.
 

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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.
 

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.