xmlns:atom="http://www.w3.org/2005/Atom" xmlns:atom="http://www.w3.org/2005/Atom"

ANNEXU.K.CLASSICAL SWINE FEVER DIAGNOSTIC MANUAL

F1F2F3F4F5F6CHAPTER VIIU.K.Principles and use of serological tests and evaluation of their results

A.Basic principles and diagnostic valueU.K.

1.In classical swine fever virus infected pigs, antibodies are usually detectable in serum samples from two to three weeks after infection. In pigs that have recovered from the disease, protective neutralising antibodies can be detected for several years or even for their lifetime. Antibodies are also sporadically detectable in the terminal stage of lethally diseased animals. In some pigs with chronic form of classical swine fever, antibodies might be detectable for a few days at the end of the first month post-infection.U.K.

Pigs infected in utero may be immunotolerant against the homologue classical swine fever virus and produce no specific antibodies. However, antibodies of maternal origin can be detected during the first days of life. The half-life of maternal antibodies in non-viraemic healthy piglets is about two weeks. If found in piglets older than three months, classical swine fever antibodies are very unlikely to be of maternal origin.

2.The detection of antibodies against classical swine fever virus in serum or plasma samples is carried out to assist the diagnosis of classical swine fever in suspected holdings, for establishing the age of infection in case of a confirmed outbreak and for monitoring and surveillance purposes. However, serological tests are of limited value for the detection of classical swine fever in the case of a recent infection in a holding.U.K.

A few seropositive pigs with a low neutralisation titre can be indicative of a recent infection (two to four weeks). Many pigs with high neutralisation titre could indicate that virus entered the holding more than one month before. The location of seropositive pigs in the holding can provide valuable information on how classical swine fever virus entered the holding.

However, an accurate evaluation of the results of the serological tests must be carried out taking into account the whole clinical, virological and epidemiological findings, in the framework of the enquiry to be carried out in case of suspicion or confirmation of classical swine fever, in accordance with Article 8 of Directive 2001/89/EC.

B.Recommended serological testsU.K.

1.The virus neutralisation test (VNT) and the ELISA are the tests of choice for the serological diagnosis of classical swine fever.U.K.

The quality and efficiency of the serological diagnosis performed by the national [F1laboratory] must be regularly checked in the framework of the inter-laboratory comparison test F2....

2.The VNT is based on the determination of the virus neutralising activity of the antibodies of the serum sample, expressed as neutralising 50 % end point.U.K.

A constant amount of classical swine fever virus is incubated at 37 °C with diluted serum. For screening purposes, the sera are initially diluted 1/10. When a full titration is necessary two-fold dilutions of serum starting at 1/2 or 1/5 can be prepared. Each dilution is mixed with an equal volume of a virus suspension containing 100 infectious doses (TCID 50).

After incubation the mixture is inoculated onto cell cultures which are incubated for three to five days. After this incubation period the cultures are fixed and any viral replication in the infected cells is detected by an immune labelling system. Either the neutralisation peroxidase-linked antibody (NPLA) or the neutralisation-immunofluorescence (NIF) assays may be used.

The results of the VNT are expressed as the reciprocal of the initial serum dilution at which half the inoculated cell cultures (50 % end point) fail to show viral replication (no specific labelling). A point between two dilution levels is estimated. The final dilution system is based on the actual dilution of serum during the neutralisation reaction, i.e. after addition of virus, but before adding the cell suspension.

3.The VNT is the most sensitive and reliable test to detect antibodies against classical swine fever virus. Therefore, it is recommended for the serological examination of a single animal as well as on a herd basis. However, cross-neutralising antibodies specific for ruminant pestiviruses infections of pigs may be detected by this test.U.K.

The VNT for the detection of antibodies against BVD virus and BD virus follow the same principals mentioned above and are conducted for the differential diagnosis of classical swine fever.

4.The pestiviruses strains to be used in the neutralisation tests shall be in accordance with the recommendation of the [F3National] Reference Laboratory.U.K.
5.Several ELISA techniques using specific monoclonal antibodies have been developed, which are based on two formats: competitive or blocking ELISA and non-competitive ELISA.U.K.

The competitive or blocking ELISA is usually based on monoclonal antibodies. If the serum sample contains antibodies to classical virus, the binding of a selected peroxidase-conjugated monoclonal antibody to virus antigen will be inhibited resulting in a reduced signal.

In non-competitive ELISA the binding of serum antibodies to antigen is measured directly using peroxidase-conjugated anti-pig antibodies.

6.Quality control on sensitivity and specificity of each batch of an ELISA must be regularly performed by the national [F4laboratory], making use of [F5a panel] of reference sera F6.... This panel shall include:U.K.

The ELISA to be used for the serological diagnosis of classical swine fever must recognise all reference sera from the convalescent pigs. All results obtained with the reference sera must be repeatable. It is further recommended that they detect all positive sera from the early phase and to show a minimum of cross-reactions with the sera from pigs infected with ruminant pestiviruses.

The results obtained with the reference sera from pigs in the early phase of infection give an indication of the sensitivity of the ELISA.

7.The sensitivity of the ELISA is regarded as lower than that of the VNT, and it is recommended to use it as a screening test on a herd basis. However, the ELISA require less-specialised facilities and can be performed much more rapidly, thanks to automated systems, than the VNT.U.K.

The ELISA must ensure identification of all classical swine fever infections at the convalescence stage and need to be as free as possible from interference by cross-reacting antibodies to ruminant pestiviruses.

C.Interpretation of serological results and differential diagnosis with infections due to ruminant pestiviruses (BVDV and BDV)U.K.

1.Without prejudice to the provisions of Article 4(3)(a) or Article 7(2) of Directive 2001/89/EC, in case of detection of a classical swine fever virus neutralisation titre equal to or higher than 10 ND50 in serum samples collected from one or more pigs or a positive ELISA result in serum samples from a group of pigs, the measures referred to in Article 4(2) of Directive 2001/89/EC shall immediately apply or shall continue to apply in the holding in question.U.K.

The samples already collected from this holding must be re-tested by VNT by comparative end point titration of the neutralising antibodies against classical swine fever virus and ruminant pestiviruses.

2.If the comparative tests show antibodies to ruminant pestiviruses and no or evidently lower (less than three-fold) antibody titres to classical swine fever virus, the suspicion for classical swine fever shall be ruled out, unless other reasons exist which warrant the continued application of the measures referred to in Article 4(2) of Directive 2001/89/EC in the holding in question.U.K.
3.If the comparative tests show a virus neutralisation titre in more than one pig equal to or higher than 10 ND50 and this titre is equal or higher than the titres to other pestiviruses, the competent authority shall ensure that classical swine fever shall be confirmed, provided that epidemiological evidence of disease has been found in the holding in question.U.K.
4.Without prejudice to the provisions of Article 4(3) of Directive 2001/89/EC, if epidemiological evidence of disease has not been found or if the results of the previous tests are inconclusive, the competent authority shall ensure that in the holding in question:U.K.
5.However, if the further checks and tests referred to in subparagraph 4 do not allow classical swine fever to be ruled out, further blood sampling for serological testing shall be carried out in the holding after at least two weeks have elapsed from the previous checks.U.K.

In the framework of this further sampling, the pigs already sampled and tested shall be re-sampled for a comparative serological testing with the previously collected samples to detect sero-conversion for classical swine fever virus or for ruminant pestiviruses, if any.

If these further checks and tests do not allow classical swine fever to be confirmed, the measures referred to in Article 4 of Directive 2001/89/EC may be lifted.

Textual Amendments