2002/106/EC: Commission DecisionShow full title

F1 F2 F3 F4 F5 CHAPTER VI U.K. Principles and use of virological tests and evaluation of their results

A. Detection of virus antigen U.K.

1. Fluorescent antibody test (FAT) U.K.

The principle of the test is the detection of viral antigen on thin cryosections of organ material from pigs suspected of being infected with classical swine fever virus. The intracellular antigen is detected by using a FITC conjugated antibody. A positive result should be confirmed by repeating the staining with a specific monoclonal antibody.

Suitable organs are tonsils, kidney, spleen, different lymph nodes and ileum. A smear of bone marrow cells might also be used in case of feral pigs, if these organs are not available or are autolysed.

The test can be performed within one day. As organ samples can only be obtained from dead animals its use for screening purposes is limited. Confidence in the test result may be limited by doubtful staining, particularly where considerable experience in performing the test has not been acquired or if the organs tested are autolysed.

2. ELISA for antigen detection U.K.

Viral antigen is detected by using various ELISA techniques. The sensitivity of the antigen ELISA should be high enough to score a positive result from animals showing clinical signs of classical swine fever.

The use of ELISA for antigen detection is recommended on samples from animals with clinical signs or pathological lesions of disease. It is not suitable for the investigation of individual animals. Suitable samples are leukocytes, serum, non-coagulated blood as well as suspensions of the organs referred to in subparagraph 1 taken from pigs suspected of being infected with classical swine fever virus(1).

The ELISA can be carried out within one day and can be performed by automatic equipment. The most important advantage is that large numbers of samples can be processed in a short period of time. It is recommended that ELISA antigen which give satisfactory results on reference material are used. However, at present all commercial ELISA are less sensitive than the virus isolation on cell culture and their sensitivity is significantly better on blood samples from piglets than from adult pigs.

B. Virus isolation U.K.

1. Virus isolation is based on the incubation of sample material on susceptible cell cultures of porcine origin. If classical swine fever virus occurs in the sample, it will replicate in the cells to an amount that can be detected, by immunostaining of the infected cells with conjugated antibodies. Classical swine fever specific antibodies are required for differential diagnosis with respect to other pestiviruses. U.K.

2. The preferred samples for isolation of classical swine fever virus are leukocytes, plasma or whole blood obtained from non-coagulated blood samples or the organs referred to in A.1. U.K.

3. Virus isolation is best suited for the investigation of samples from small numbers of animals rather than mass surveillance. The virus isolation procedure is labour intensive and requires at least three days before results are available. Two further cell culture passages may be necessary in order that a small amount of virus in the sample is detected. This may lead to an investigation time of up to 10 days before a final result is obtained. Autolysed samples can be cytotoxic to the cell culture and consequently limit its use. U.K.

4. It is recommended to perform virus isolation also in case of previous confirmation of classical swine fever by other methods. It must be used as reference test for the confirmation of positive results of prior antigen ELISA, PCR or FAT, indirect peroxidase-staining methods respectively. U.K.

Classical swine fever virus isolates obtained in this way are useful for virus characterisation including genetic typing and molecular epidemiology.

5. All classical swine fever virus isolates from all primary outbreaks, primary cases in feral pigs or cases in slaughterhouse or means of transport must be genetic typed by [F1 the United Kingdom National Reference Laboratory or any other laboratory authorised by the Secretary of State, with the consent of each other authority who in relation to any constituent part of the United Kingdom is the appropriate Minister, in accordance with Section E]. U.K.

In any case, these virus isolates must be sent to the Community Reference Laboratory for virus collection without delay.

C. Detection of virus genome U.K.

1. The polymerase chain reaction (PCR) is applied to detect virus genome in blood, tissues or organ samples. Small fragments of viral RNA are transcribed into DNA fragments which are amplified by PCR to detectable quantities. Since this test detects only a genome sequence of the virus, the PCR may be positive, even when there is no infectious virus present (e.g. in autolysed tissues or samples from convalescent pigs). U.K.

2. PCR can be used on small numbers of samples which have been carefully selected from suspect animals or on material from aborted fetuses. In carcasses from wild boar it might be the method of choice, if the material is autolysed and virus isolation is not possible any more due to cytotoxicity. U.K.

3. Suitable sample material for diagnostic PCR are the organs described for virus isolation or unclotted blood. U.K.

4. PCR can be performed within 48 hours. It requires appropriate laboratory equipment, separated facilities and skilled staff. An advantage is that infectious virus particles need not be replicated in the laboratory. The method is highly sensitive, but contamination may easily occur, which leads to false positive results. Therefore stringent quality control procedures are essential. Some methods are pestivirus rather than classical swine fever specific, requiring further confirmatory tests, such as sequencing of the PCR product. U.K.

D. Evaluation of the results of virological tests U.K.

1. Virological tests are essential for the confirmation of classical swine fever. U.K.

Virus isolation must be considered as the reference virological test and must be used as confirmatory test when necessary. Its use is particularly recommended in case positive FAT, ELISA or PCR results are not associated with the detection of clinical signs or lesions of disease and in any other doubtful case.

However, a primary outbreak of classical swine fever can be confirmed if clinical signs or lesions of disease have been detected in the pigs in question and at least two antigen or genome detection tests have given a positive result.

A secondary outbreak of classical swine fever can be confirmed if, in addition to the epidemiological link to a confirmed outbreak or case, clinical signs or lesions of disease have been detected in the pigs in question and an antigen or genome detection tests has given a positive result.

A primary case of classical swine fever in feral pigs can be confirmed after virus isolation or if at least two antigen or genome detection tests have given a positive result. Further cases of classical swine fever in feral pigs for which an epidemiologial link with previously confirmed cases have been found can be confirmed if an antigen or genome detection test has given a positive result.

2. A positive result for classical swine fever to a genome or antigen detection test requires that the test in question has been performed using classical swine fever virus-specific antibodies or primers. If the test used was not specific for classical swine fever virus but only pestivirus specific, it must be repeated using classical swine fever specific reagents. U.K.

E. Genetic typing of classical swine fever virus isolates U.K.

1. Genetic typing of classical swine fever virus isolates is achieved by determining the nucleotide sequence of portions of the virus genome, namely specific parts of the 5'noncoding region and/or of the E2 glycoprotein gene. The similarity of these sequences with those already obtained from previous virus isolates can indicate whether or not outbreaks of disease are caused by new or already recognised strains. This can support or refute hypotheses on transmission routes that have been provided by epidemiological tracing. U.K.

Genetic typing of classical swine fever virus isolates is of major importance to determine the source of disease. However, a close relationship between viruses obtained from different outbreaks is not an absolute proof for a direct epidemiological link.

2. F2 ... U.K.

The data on typing and sequencing of classical swine fever virus isolates available to the laboratories authorised [F3 by the Secretary of State with the consent of the other appropriate Ministers,] to diagnose classical swine fever must be forwarded to the [F4 National] Reference Laboratory in order that this information is entered into the database kept by this laboratory.

F5 ...

Textual Amendments

F1 Words in Annex Ch.6 Section B para. 5 substituted (31.12.2020) by The Exotic Disease (Amendment etc.) (EU Exit) Regulations 2018 (S.I. 2018/1410), regs. 1, 7(7)(a); 2020 c. 1, Sch. 5 para. 1(1)

F2 Words in Annex Ch. 6 Section E para. 2 omitted (31.12.2020) by virtue of The Exotic Disease (Amendment etc.) (EU Exit) Regulations 2018 (S.I. 2018/1410), regs. 1, 7(7)(b)(i); 2020 c. 1, Sch. 5 para. 1(1)

F3 Words in Annex Ch. 6 Section E para. 2 inserted (31.12.2020) by The Exotic Disease (Amendment etc.) (EU Exit) Regulations 2018 (S.I. 2018/1410), regs. 1, 7(7)(b)(ii)(aa); 2020 c. 1, Sch. 5 para. 1(1)

F4 Word in Annex Ch. 6 Section E para. 2 substituted (31.12.2020) by The Exotic Disease (Amendment etc.) (EU Exit) Regulations 2018 (S.I. 2018/1410), regs. 1, 7(7)(b)(ii)(bb); 2020 c. 1, Sch. 5 para. 1(1)

F5 Words in Annex Ch. 6 Section E para. 2 omitted (31.12.2020) by virtue of The Exotic Disease (Amendment etc.) (EU Exit) Regulations 2018 (S.I. 2018/1410), regs. 1, 7(7)(b)(iii); 2020 c. 1, Sch. 5 para. 1(1)

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Several Classical swine fever ELISA antigen are commercially available, which are validated with different types of samples.