| PART II | DECLARATION |
[to be completed by the exporter or his agent]
1. Species of fish
2. Country of origin or export
3. Country of destination
4. Name and address of exporter
5. Type 1 and address of establishment of origin
6. Name and address of importer
7. Type 1 and address of establishment of destination
8. Total number of eggs 2/fish 2 in the present consignment
9. Size of fish in the present consignment
I hereby DECLARE that:
[for consignments of EGGS only]
1. This consignment of eggs has been disinfected immediately prior to despatch in accordance with the procedures specified in Annex III of the International Convention for the Control of Communicable Fish Diseases 19 ..
[for ALL consignments]
2. All tanks or containers other than disposable tanks or containers used in this consignment have been disinfected immediately prior to use in accordance with the procedures specified in Annex III (in the case of live fish) of the International Convention for the Control of Communicable Fish Diseases 19 ..
3. No disposable tanks or containers used in this consignment have been previously used.
I hereby DECLARE that the above information is true and accurate.
Signature of exporter or agent
Date
1 for example: Governmental or institutional or private hatchery or fish culture establishment
INTERNATIONAL CONVENTION FOR THE CONTROL OF COMMUNICABLE FISH DISEASES
INTERNATIONAL FISH HEALTH CERTIFICATE FOR EYED EGGS OR LIVE FISH OF THE FAMILY SALMONIDAE AND EYED EGGS OR LIVE FISH OF THE FAMILY CYPRINIDAE
[To be completed by the Certifying Officer]
PART I I hereby CERTIFY that:
11. The establishment of origin of this consignment has been inspected in conformity with the provisions of Annex III of the International Convention for the Control of Communicable Fish Diseases 19.. and has been classified as a Minimal Disease establishment. As a result of such inspections the following diseases have been found absent:
12. The fish or eggs comprising the present consignment have been taken from waters inspected in conformity with section 4.4 (iii) of Annex III and exhibit no clinical signs of the diseases indicated:
| for SALMONIDAE | 1. | VHS |  | for CYPRINIDAE | 4. | SV | |
| 2. | IHN | | |||||
| 3. | IPN | |
3. The establishment of origin of this consignment meets the minimum certification requirements notified by the country of destination.
Name and stamp of Certifying Authority
Signature and title of Certifying Officer
Name of Certifying Officer (in CAPITALS)
Place of issue Date of issue
FOR INFORMATION PURPOSES ONLY:
The following diseases are absent:
| (a) | Myxosomiasis | |
| (b) | Furunculosis | |
| (c) | Erythrodermatitis | |
THIS CERTIFICATE IS VALID ONLY IF ISSUED NOT MORE THAN .... DAYS BEFORE THE DATE OF COMMENCEMENT OF SHIPMENT, AND IF BOTH PARTS HAVE BEEN DULY COMPLETED AND SIGNED
PART II
DECLARATION
[to be completed by the exporter or his agent]
1. Species of fish
2. Country of origin or export
3. Country of destination
4. Name and address of exporter
5. Type 1 and address of establishment of origin
6. Name and address of importer
7. Type 1 and address of establishment of destination
8. Name of source of wild fish or eggs
9. Name of major river into which this source drains
10. Total number of eggs 2/fish 2 in the present consignment
11. Size of fish in the present consignment
I hereby DECLARE that:
[for consignments of EGGS only]
1. This consignment of eggs has been disinfected immediately prior to despatch in accordance with the procedures specified in Annex III of the International Convention for the Control of Communicable Fish Diseases 19 ..
[for ALL consignments]
2. All tanks or containers other than disposable tanks or containers used in this consignment have been disinfected immediately prior to use in accordance with the procedures specified in Annex III (in the case of live fish) of the International Convention for the Control of Communicable Fish Diseases 19 ..
3. No disposable tanks or containers used in this consignment have been previously used.
Signature of exporter or agent Date
I hereby DECLARE that the above information is true and accurate.Signature of exporter or agent Date
1 for example: Governmental or institutional or private hatchery or fish culture establishment
Certification is provided after classification of the farm and following clinical examination and/or laboratory tests.
Inspectors should consider the geographical situation and health state of the farm as well as the hygiene measures and husbandry techniques practised, and base an estimate of the condition of the farm on those factors as well as on general clinical grounds. These precautions are particularly important in certificates for CPF (coded pathogen free), CDF (coded disease free) and SDF (specified disease free) farms.
Appropriate laboratory tests must be carried out as stipulated in Section 14 of this Annex, and sampling should be carried out as specified in Section 10 of this Annex.
2.1 CPF establishments (see Tables 1 and 2)
These must be free of all pathogens listed in Table 1 of this Annex, and must be located in such a geographical and ecological situation that contamination from any source would be highly improbable. The farm should be fed from a spring, bore-hole, well or other similar uncontaminated source which must be strictly under the control of the exporter or his agent and must not be subject to contamination by re-stocking or by virus carriers.
Re-stocking must be from other CPF establishments only.
2.2 CDF establishments (see Tables 1 and 2)
These must be free of all diseases listed in Table 1 of this Annex, but the geographical and ecological situation is not so strictly controlled as in CPF establishments (2.1 above). CDF establishments may be supplied by water from streams or rivers. Upstream there must be no fish culture establishment other than CPF or CDF establishments, and re-stocking of the establishment and any other establishment upstream must be controlled.
2.3 SDF establishments
Certification requirements for these establishments are less rigorous than for CDF and CPF establishments. SDF establishment may be infected with one or more of the coded diseases. Upstream there must be no establishment of lower category and re-stocking of the establishment and any other establishment upstream must be controlled.
2.4 MD (minimum disease) establishments (see Tables 1 and 2)
These establishments may be supplied from open waters and there must be no overt signs of disease on the establishment.
3.1 Certificate 1
This Certificate can be issued only for CPF establishments and the Certifying Officer must satisfy himself that the criteria for this category of establishment are satisfied.
A. For Salmonidae or salmonid eggs (see also Section 4 of this Annex). The establishment of origin:
must have been clinically inspected at least twice in each of the first two consecutive years and on each occasion appropriate samples taken as in Section 7 of this Annex and appropriate laboratory tests carried out as specified in Sections 9 and 10 of this Annex and on the basis of clinical examination and corresponding laboratory tests has been found to be free of the coded pathogens in Table 1 of this Annex.
must thereafter be inspected clinically and subjected to the appropriate laboratory tests as specified in 3.1 A(i) above twice per year at the appropriate time.
on any suspicion of disease, appropriate laboratory tests as specified in Sections 9 and 10 of this Annex must be carried out.
B. For Cyprinidae (see also Section 5 of this Annex). The establishment:
must have been inspected clinically at least once in every year, when the water temperature is between 13°C and 18°C. Samples as specified in Section 7 of this Annex must be taken and subjected to laboratory tests as specified in Sections 9 and 10 of this Annex.
must be inspected clinically at least once a year at the appropriate time thereafter, and appropriate samples taken as in (i) above if there is any suspicion of disease.
3.2 Certificate 2
This Certificate may be issued for CDF farms FREE of all coded diseases in Table 1 of this Annex.
The criteria as laid down for CPF farms must be satisfied except that CDF farms may be situated on rivers or other natural waters in which there has been no evidence of any of the diseases caused by the coded pathogens.
Sampling, clinical examination and laboratory tests must be carried out as stipulated for CPF farms in 3.1 A and, in the case of cyprinids 3.1 B, respectively, of this Annex.
If another farm is situated upstream of the one under investigation, diseases present on that farm must be determined and the certification category of that farm must be applied to the farm under investigation.
3.3 Certificate 3
This Certificate may be issued for salmonid fish and eggs from SDF establishments with some of the coded diseases present (see Table 1). It is not valid for Cyprinidae.
The criteria laid down must be satisfied, but it should be noted that one or more of the listed diseases may be present on such farms. It is important that the boxes should be marked when the diseases are ABSENT. The presence of a disease is not noted: only its absence.
If another SDF farm is situated upstream of the one under investigation, diseases of that farm must be determined. If any coded disease (see Table 1) is present on the upstream farm or farms, it cannot be noted as absent in the farm under investigation.
Certificates can be issued only on completion of the first two years of inspection and may continue to be issued only if inspection has taken place at least twice in each subsequent year and if there is no suspicion of coded diseases on clinical or other grounds. Laboratory tests must be made at each inspection and at any other time when the occurrence of any of the coded diseases is suspected.
3.4 Certificate 4
This Certificate may be issued for MD farms or for wild fish or eggs from open waters.
MD farms differ from others in their ecological situation and in their susceptibility to infection. There must be no clinical signs of disease on the farm. The certificate may be completed as a result of clinical inspection only, though if there is suspicion of disease appropriate laboratory tests must be carried out as specified in Sections 9 and 10 of this Annex and the Certificate marked accordingly.
When wild fish or their eggs have been taken from open waters and are to be inspected for issue of Certificate 4, it is essential that samples (in accordance with Section 7 of this Annex) of wild fish of the same species, from the same waters, must have been subjected to clinical examination and laboratory tests as specified in Sections 9 and 10 of this Annex for the previous two years, on at least four occasions, and found to be free of those coded diseases so indicated on the Certificate.
4.1 For CPF establishments
The initial Certificate can be issued only if, after two years continuous inspection as in 3.1 A (i), there has been no evidence of the presence of coded pathogens.
Subsequent certificates may be issued each year after inspection as in 3.1 A (ii) has shown no evidence of coded pathogens.
If clinical examination arouses suspicion of any of the coded diseases, certification must immediately be suspended, and if this suspicion is confirmed by laboratory tests the Certificate must immediately be cancelled and the farm may not export until the situation has been satisfactorily remedied.
For re-certification, the farm must be disinfected (see recommendations in this Annex Section 16) and then re-stocked with fish from another certificated CPF establishment. In this case, for the first year after re-stocking, two inspections must be made as in 3.1 A (i) and if these inspections and tests have all been negative a new Certificate may then be issued. In the following years, inspections must be carried out as in 3.1 A (ii).
If presence of a disease suspected on clinical ground is not confirmed by laboratory tests, a second series of laboratory tests must be made as soon as possible: the Certificate cannot be re-issued until after the results of a second laboratory test have proved negative for this disease.
4.2 CDF establishments
In CDF farms all the procedures as specified for CPF farms must be followed. It should be noted that if an infection is detected on an CDF farm, it may be due to presence of the disease in the river or in some other uncontrollable source, in which case discussion with the farmer is necessary to assess whether disinfection is economically practicable. If it is not considered worthwhile, the Certificate must be downgraded to an appropriate level.
4.3 SDF establishments
Inspection procedures are as for CDF establishments.
4.4 Re-assessment of classification
If for any of the above categories of establishment, circumstances occur that cause an alteration to the water supply to the establishment, or if in any other way its ecological or geographical situation becomes altered (e.g. by mining or forestry) then the situation must be re-assessed by the Certifying Officer.
Every mortality must be checked, particular attention being given in springtime, at water temperatures between 10° and 20°C. If the presence of SV virus is suspected, virological examination is obligatory.
If mortalities are not apparent or if they do not appear to be caused by SV virus, the appropriate number of fish as specified in Section 7 of this Annex must be taken in springtime, preferably at water temperatures between 13° and 18°C and subjected to clinical examination and laboratory tests as specified in Sections 9 and 10 of this Annex. One sampling per year for a minimum period of two consecutive years must be carried out before certification may be made.
For issue of Certificate 1, the inspector must have evidence that fish were reared in an environment uncontaminated by the virus of pring viraemia.
If criteria for re-issue of Certificate 1 cannot be met, the number of fish sampled must be increased as specified in this Annex (Section 7). After clinical and laboratory tests as laid down in Sections 9 and 10 of this Annex Certificates 2 or 4 may be issued.
Where clinical signs of disease are observed, certification must immediately be suspended: if clinical suspicion is confirmed by the appropriate laboratory tests the Certificate must immediately be cancelled and the farm may not export until the situation has been satisfactorily remedied.
If the presence of SV is confirmed, CPF establishments may be disinfected according to the instructions in this Annex (Section 16) but in other categories of establishments, if disinfection is not possible, Certificate 4 only may be re-issued.
If the presence of SV is confirmed and disinfection procedures as specified in this Annex (Section 16) are carried out, certification may be made after negative tests carried out in two successive spring seasons.
Inspections are carried out initially for a period of two years for all certificates, and thereafter at bi-annual intervals or preferably more often (or as shall from time to time be determined by International Agreement).
It should be noted that it is the farm (that is, as formulated on the certificates, the “Establishment of Origin”) whose health status is established, and not individual batches of fish. The validity of the certifying procedure depends, therefore, not only on clinical diagnosis and laboratory tests where necessary but on the geographical and ecological situation of the farm as well as on its re-stocking policy.
As has previously been pointed out, farms should re-stock only from fish certificated at the same level, or at a higher level, as the farm in question. If any farm accepts stocks or eggs from a farm of a lower certification category it can be certified only at that lower category in future, until the inspections and tests necessary to re-establish it at its previous category have been carried out for a minimum period of two years. Some aspects of the health status of farms are provided in Table 3 of this Annex.
Table 1
CODED PATHOGENS AND DISEASES
| The coded pathogenic organisms are: | ||
| 1. | IPN virus | |
| 2. | IHN virus | |
| 3. | VHS virus | |
| 4. | SV virus (for cyprinid fish) | |
| The coded diseases are: | ||
| 1. | IPN | |
| 2. | IHN | |
| 3. | VHS | |
| 4. | SV (for cyprinid fish) | |
Table 2
CATEGORIES OF ESTABLISHMENT
| Category | Description | Certificate |
| CPF | Establishment free of coded pathogenic organisms | 1 |
| CDF | Establishment free of coded diseases | 2 |
| SDF | Establishment free of those coded diseases as indicated on the certificate | 3 |
| MD | Establishments free of coded diseases on clinical inspection and laboratory tests on suspicion, and showing no evidence of those coded diseases as indicated on the certificate | 4 |
Table 3
HEALTH STATUS OF FARMS
| Category of farm | Occurrence of | Infected Environment | Purpose of Production | ||
| Clinical Disease | Pathogen | Antibody | |||
| CPF | - | - | - | - | International traffic, breeding, egg production |
| CDF | - | - | - | ± | -ditto- |
| SDF | - | - | ± | ± | Breeding, stocking of open waters, consumption or processing |
| MD | - | ± | ± | ± or + | Immediate consumption and infected areas |
7.1 When selecting fish for sampling, severely diseased, moribund and weak fish must be taken; apparently healthy fish will be used only to complete the sample if necessary.
7.2 CPF and CDF salmonid establishments
The minimum sample size for each water supply on the farm will be as follows: during the first and the second year of inspection samples of 40 fish will be taken twice yearly; during the consecutive years sample size will be reduced to 20 fish while the sampling frequency will be the same. For the first inspection in the year, fish of 4–7 cm (… g) will be taken while for the second inspection (3–6 months later) fish of the same year of 10–15 cm (… g) are used. If both sizes are available at the time of inspection, the sample taken should comprise fish from both size groups.
7.3 CPF and CDF carp farms
In springtime, at water temperatures between 10 and 20°C, weak carp in each pond can be attracted to the water inlet by a slow inflow of water. All the fish caught should be examined clinically. For virological examination, a total of 80 fish representing all ponds must be taken. The procedure is repeated at each annual inspection.
7.4 SDF trout farms
Farms of this category may be certified free from one or more of the specified diseases.
Regardless of the disease from which freedom is certified, clinical and virological control through two years must be carried out as stipulated for CPF and CDF trout farms.
During the following years the type of examination required is different for the various diseases:
IPN: As in CPF and CDF trout farms after the initial two-year period.
VHS: Clinical examination at least twice a year. Virological examination whenever symptoms resembling those of the disease are observed.
IHN: As for VHS.
7.5 MD farms
These farms are in the lowest category of certification.
They are certified on clinical grounds free from one or more of the following: VHS, IHN, SV.
Clinical inspection is carried out twice per year every year at approximately six-month intervals. Sampling for virological examination is necessary only on suspicion of a specified disease. Certification can be commenced after two inspections.
Although histopathological examinations may provide extremely useful diagnostic information, they should not be used as determinative procedures, but rather as presumptive indications.
The exception is myxosomiasis, in which trophozoites can be detected only by histological methods. The histopathological changes associated with this disease are related to the stage of infestation with the parasite.
During the first seventy days, there is a slight erosion of the head cartilage without evidence of tissue proliferation. Only the trophozoites of Myxosoma cerebralis are present at this stage.
At three to four months post-infection, there is a lesion-like cavity containing cellular debris in the head cartilage, due to erosion of the tissues but without inflammation or tissue proliferation. Myxosoma cerebralis spores are present at this stage.
At eight to twelve months post-infection, there is proliferation of epithelioid granulation tissue around the mass of Myxosoma cerebralis spores. This epithelioid tissue replaces the normal tissue, and gives rise to a “cyst” resembling a granuloma: the process affects the normal osteogenesis.
9.1 Transport to the diagnostic laboratory
Ideally, live moribund fish should be delivered by aerated or oxygenated tanks, or in plastic bags with oxygen. If transport of live material is not possible specimens should be carried on ice. They should not be frozen or stored in glycerol.
All fish should be carried whole, but only in exceptional circumstances should fish be eviscerated and samples of kidney, spleen, liver and other organs should be pooled in batches not exceeding five in number and appropriately packaged.
In the case of brood stock, ovarian or seminal fluid is collected at spawning time into appropriate sterile tubes or other suitable vessels.
Faeces : 0.5–1.0 ml fresh faeces are collected into 5.0 ml of physiological saline.
Neither faeces nor reproductive fluids may be frozen: they should be stored for not longer than 24 hours on ice or at 4°C.
When testing for myxosomiasis, fish less than four months old should not be used, as spore formation is unlikely to have occurred. Heads should be removed from older fish and sent with the other samples.
For furunculosis, swabs of lesions should be taken into TSA broth or streaked directly onto TSA agar and sent with the other samples. On arrival at the laboratory kidney smears should be Gram-stained.
9.2 Laboratory procedures
On arrival of samples at the laboratory, material for the diagnosis of myxosomiasis and furunculosis should be removed. The remaining pooled specimens should be homogenized with an equal volume of a buffered (pH 7.6–7.8) isotonic saline (Earle's or Hanks' solution). Ovarian fluid remains undiluted or may be diluted 1:2 when turbid. Homogenized tissue or ovarian fluid samples are then centrifuged at 1 000–2 000 g for 10–15 minutes. Bacteria and fungi are removed from the supernatant fluid either by filtration or by antibiotic treatment.
If desired, the material may be centrifuged in a narrow tube at 5 000 g for..minutes, and the upper surface of the supernatant fluid removed, the lower levels and the pellet being discarded.
If necessary the pH should not be adjusted to 7.6–7.8 and the filtrate or antibiotic treated or centrifuged fluid should be inoculated onto duplicate flasks or tubes of appropriate cells.
In routine tests for all viruses except SV, RTG2 cells should be used, incubation being at 15°C. RTG2 cells, however, are not particularly susceptible to IHN virus, and if the presence of this disease is suspected or if isolation of IHN virus is specifically desired, FHM cells must be used. For isolation of SV virus, FHM cells are essential: the virus grows only poorly in RTG2 cells.
In all cases FHM cells must be incubated at 20°C.
It should be noted that some cell lines of RTG2 and FHM are not necessarily susceptible to the strain of virus inoculated. Positive controls should, therefore, always be used.
After inoculation onto the appropriate cells, an absorption period of one hour should be allowed at room temperature and then the normal volume of maintenance medium added.
It is important that only young, actively-growing cell cultures seeded 1–4 days previously and at 75–95 percent confluence should be used for virus tests.
Incubation should be continued for ten days, examining regularly for evidence of CPE.
Inoculated cell cultures showing no evidence of CPE following ten days incubation are sub-cultured once (blind passages) and the sub-culture incubated as described above for a further ten-day period and examined regularly for CPE.
Absence of CPE in both inoculated cell cultures and following blind passage shall be interpreted as a negative finding for certification purposes.
If strong CPE develops within the first 24 hours of incubation it usually indicates toxicity of the inoculated fish extract for the cells. In such cases the cells should be harvested by shaking the tube or flask vigorously and the harvest ultra-sonicated for ten seconds, diluted 1:5 in maintenance medium and re-inoculated onto fresh cell cultures.
If bacterial or fungal contamination of the cell culture occurs, the cell culture should be harvested, ultra-sonicated for 20–30 seconds, centrifuged at 1 000 g for 15 minutes and the supernatant filtered through a 0.5 μ membrane and re-inoculated onto fresh cultures.
10.1 It is important that the identity of any virus isolated in tissue culture should be confirmed unequivocally in the shortest possible time. The methods described below are those most usually used at present.
Whichever serological test is carried out, it will require the use of standardized antisera1 of known specificity against the different fish viruses. In view of the serological diversity of the IPN group of viruses the serological confirmation of an IPN virus isolation should be based on a positive reaction with a polyvalent antiserum against the major serotypes of the virus. Tubes or microtitre plates may conveniently be used.
Cell cultures showing viral CPE should be harvested, ultra-sonicated for 10 seconds and diluted 1:1 000 with maintenance medium. An aliquot of the diluted harvest should be mixed with an equal volume of specific antiserum at an appropriate dilution (depending on its specific titre, but usually 10-2 or 10-3), and a second aliquot mixed with an equal volume of maintenance medium. Incubate for one hour at 20°C. Inoculate at normal maintenance medium volume onto fresh cell cultures and incubate at the appropriate temperature. Cultures should be examined after one, two and three days for appearance of CPE.
If CPE appears in the cultures receiving harvest mixed with antiserum this indicates either that the identity of the virus is not that suspected or that the concentration of virus at 10-3 dilution was too high to be neutralized by the specific antiserum at 10-2. This should not occur with IPN since high titre antiserum is easy to prepare, but may be a problem in neutralization tests for the three Rhabdoviruses (VHS, IHN and SV), against which it is difficult to prepare high titre antisera. In these cases, the plaque reduction method is recommended. Immunofluorescent techniques may be used for virus identification.
10.2 Plaque reduction test - This test may, if preferred, be used for all viruses to be tested.
The culture fluid from the cell cultures showing typical CPE is diluted to contain approximately 103 PFU/ml. The dilution is mixed with an equal volume appropriate antiserum, the dilution of which will depend on the titre of the antiserum used, but the neutralizing titre should not be less than 100. A further amount of the diluted culture fluid is mixed with an equal volume of maintenance medium. Equal amounts of antiserum and negative control respectively are each mixed with a reference virus strain to provide positive controls.
The mixtures prepared as described above are maintained at room temperature for about one hour. Aliquots of each mixture are added to duplicate cell cultures of known sensitivity in Petri dishes, at the appropriate volume. Following half-hour adsorption, cell cultures are overlayed with a medium containing 0.5 percent agarose. Two uninoculated cell cultures in dishes are used as negative controls.
The Petri dishes containing the cell cultures are incubated for 48–72 hours at 15°C or 20°C as appropriate and the cell layers stained with neutral red (0.2 mgm/ml) and the number of plaques enumerated. A reduction of 40 percent or more in the number of plaques in the Petri dishes inoculated with the virus-antiserum mixture is considered as evidence of the presence of the relevant virus in the material examined.
Recommended procedures for detection of Myxosoma cerebralis spores are given in the following references:
11.1 Digestion techniques
11.2 Plankton centrifugation method
O'Grodnick, J.J. (1975), J.Wildl.Dis., 11, pp. 54–57
12.1 Routine bacteriological identification
Presumptive diagnosis of furunculosis on the basis of clinical signs and histopathological findings must be confirmed in the laboratory by isolation of Aeromonas salmonicida, the causative agent of the disease. The following procedures are acceptable for detection of the bacterium.
Material from lesions and tissues of diseased fish is examined as wet mounts and Gramstained smears. Material from lesions and tissues is also streaked onto plates of furunculosis agar or TSA on which A. salmonicida typically produces a brownish-coloured water-soluble pigment following 48 hours incubation at 20–25°C. The presence of non-motile, asporogenous, Gram-negative rods in the material taken from lesions and tissues of diseased fish, and the isolation of colonies producing a brown coloured diffusible pigment on furunculosis agar or TSA is strong presumptive evidence for the existence of A. salmonicida in the samples which have been examined. It is important to note, however, that achromogenic strains of A. salmonicida are known to occur.
Identification of all colonies suspected of being A. salmonicida should be made by the following procedures:
Cytochrome oxidase test on 24–48 hour cultures (A. salmonicida is cytochrome oxidase positive).
12.2 Serological identification
Slide agglutination test: a 24-hour-old colony is emulsified in physiological saline. Two drops of the resulting suspension are placed separately on a slide. Both drops must now be examined with a hand lens for an auto-agglutination. If there is no auto-agglutination a drop of A. salmonicida antiserum is added to one of the drops of suspension, and a drop of normal saline is added to the second drop of suspension. Occurrence of agglutination in the drop of suspension to which the antiserum has been added, together with no agglutination in the saline-suspension control, provides evidence that the bacterium isolated is A. salmonicida. If there is auto-agglutination the test is not valid.
A. salmonicida antiserum is not available commercially but may be prepared in the laboratory. Depending on its titre, it may need dilution. Care should be taken that the antiserum is added to the drop - not the drop to the antiserum.
Two recommended tests for the detection of A. salmonicida are given in the following papers:
McCarthy (1975), J.Gen.Microbiol., 88, pp. 384–386
The first is a passive haemagglutination test and the second is a test using antibody-coated latex.
NOTES:
The diagnosis of furunculosis in asymptomatic carrier fish, on a basis of attempts to isolate A. salmonicida, is an unreliable procedure, particularly where only small numbers of the bacterium are present. Caution should therefore be exercised in the certification of apparently healthy salmonids as being free from furunculosis.
In all cases, identification of Aeromonas salmonicida should be confirmed using classical biochemical tests.
In conformity with the requirements for certification contained in Annex II of the International Convention for the Control of Communicable Fish Diseases 19.. each batch of eyed eggs of cultivated fish of the family Salmonidae must be disinfected immediately prior to shipment. The disinfectant used shall be an approved organic iodine1 compound of proven efficacy in the destruction of viruses (IPN, IHN, VHS) and certain bacterial fish pathogens (e.g. aeromonads, vibrios, myxobacteria) which may be carried on the surface of such eyed eggs.
The approved organic iodine compound is freshly diluted in water to give a final effective concentration of 75–100 ppm available iodine (as free I2)2. The eyed eggs are exposed to this freshly prepared disinfectant solution for a period of five minutes. To ensure maximum virucidal activity, the pH of the water with which the disinfectant is diluted is adjusted to approximately neutral (pH 7.0). Organic matter in the water should be absent, or reduced to a minimum, during the time in which the eggs are immersed in the disinfectant solution.
1 or an equivalent substance
2 or to 50 ppm for 15 minutes
When disinfecting eggs a sufficient volume of disinfectant solution must be used. In preparing the solution, it is important that the quantity of the final dilution of the approved compound, in relation to the volume of eggs should be 50 l (15 gallons) of solution per 100 000 eggs (or 10 volumes of disinfectant to one volume of eggs).
NOTES:
An approved organic iodine compound is marketed by a number of commercial firms but the “approved iodine compound” is polyvinylpyrrolidoneiodine (PVP-I) containing 1 percent available iodine.
Freshly prepared aqueous solutions of the approved iodine compound are amber in colour. When the amber colour fades, due to inactivation or elimination of the available iodine, the solution is considered to have lost its effectiveness as a disinfectant and must not be used again.
Tanks and other containers used for the transportation of live fish must be thoroughly cleaned and disinfected prior to each shipment, by an effective method of disinfection. The following procedure is acceptable:
Thorough mechanical cleaning with a rinsing machine under pressure, using a 0.2 percent sodium hydroxide solution;
OR
by thorough manual scrubbing with a brush, using a 0.2 percent sodium hydroxide solution;
OR
by other methods of equivalent efficiency.
Following cleaning, the tank or other container is thoroughly disinfected by spraying with formalin (commercial grade) diluted 1:50 with water.
Containers used in the transportation of fish eggs must be of a disposable type, and these must always be destroyed by incineration immediately following their use. When disposable containers are used it is essential that they should be new, and previously unused.
The establishment is drained or pumped dry and all mud and other detritus cleared.
Ponds, basins and other holding facilities are covered liberally with quick lime, using at least ½ kg/m2.
Basins, sluices, screens and all other hatchery equipment, as well as the interior of the hatchery and food preparation rooms or other rooms used for hatchery equipment are carefully cleaned with a 2% solution of NaOH (20 g NaOH/10 litres water) by means of a brush or preferably using a pressure-spraying machine used at a minimum temperature of 60°C.
All equipment and machines used in 1), 2) and 3) are dried and sprayed with a 2% solution of formalin (200 ml commercial grade formalin per 10 litres water) or with an iodophore solution containing 50–100 ppm free iodine.
In cases where holding facilities cannot be properly drained they must be pumped dry on at least two occasions during the whole cleaning period and on each occasion on which they are pumped dry, an amount of quick lime as in 2) above is distributed.
Equipment for transportation of fish must be cleaned each time before use according to the instructions given in Annex III (Section 14).
When vehicles are used for the transportation of fish, and water has to be changed during the trip, the water from the containers must be discharged only into approved localities which can be subsequently disinfected.
Fish diseases included for certification purposes
Family Salmonidae
Viral haemorrhagic septicaemia (VHS)
Family Cyprinidae
Spring viraemia (SV)
Fish diseases and disease conditions included for information purposes only
Family Salmonidae
Erythrodermatitis
(end of amended technical annexes)
