求 非洲猪瘟间接免疫荧光试验操作规程

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CHAPTER 2.1.12.

AFRICAN SWINE FEVER


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SUMMARY

African swine fever (ASF) is an infectious disease of domestic and wild pigs that affects animals of all breeds and ages, and which is caused by a virus that produces a range of syndromes. Acute disease is characterised by high fever, haemorrhages in the reticuloendothelial system, and a high mortality rate. Infectious virus can survive for several months in fresh and salted dried-meat products.

ASF virus is the only member of the Asfarviridae family.

Laboratory diagnostic procedures for ASF fall into two groups: the first contains the tests for virus isolation and the detection of virus antigens and genomic DNA, while the second contains the tests for antibody detection. The selection of the tests to be carried out depends on the disease situation in the area or country.

In countries free from ASF but suspecting its presence, the laboratory diagnosis must be directed towards isolation of the virus by simultaneously carrying out the inoculation of pig leukocyte or bone marrow cultures, the detection of antigen in smears or cryostat sections of tissues by fluorescent antibody test (FAT) and, where possible, the detection of genomic DNA by the polymerase chain reaction (PCR). The detection of antibodies in tissue fluids by the indirect fluorescent antibody test should also be carried out at the same time.

Identification of the agent: Tissues submitted from suspected cases of disease in the field should be examined for specific antigen by the FAT on smears or cryostat sections, and for the presence of virus by inoculation of primary pig leukocyte cultures, which are examined daily for haemadsorption and cytopathic effects. The cells from negative cultures are examined for antigen by FAT and subinoculation into fresh leukocyte cultures.

The PCR can be used to detect virus genome in tissues and is especially useful if these are unsuitable for virus isolation and antigen detection.

In doubtful cases, the material is passaged and the procedures described above are repeated.

Serological tests: Where the disease is endemic, or where a primary outbreak is caused by a strain of low virulence, the investigation of new outbreaks should include the detection, using the enzyme-linked immunosorbent assay, of specific antibodies in serum or extracts of the tissues submitted.

Requirements for vaccines and diagnostic biologicals: At present, there is no vaccine for ASF.


A. INTRODUCTION

African swine fever virus (ASFV) was originally classified as a member of the family Iridoviridae, but the structure of the genome and the replication strategy of the virus have been shown to have many features in common with members of the Poxviridae (14). The proposal that ASFV be placed in a family separate from the Iridoviridae was accepted at the Sixth Meeting of the International Committee on the Taxonomy of Viruses (ICTV) in Sendai (Japan) in 1984. This virus is currently classified as the only member of a family called Asfarviridae.

ASF viruses produce a range of syndromes varying from peracute to chronic disease, and apparently healthy virus carriers. The more virulent strains produce peracute or acute haemorrhagic disease characterised by high fever, loss of appetite, haemorrhages in the skin and internal organs, and death in 2-10 days. Mortality rates may be as high as 100%. Less virulent strains produce mild clinical signs - slight fever, reduced appetite and depression - which can be readily confused with many other conditions in pigs and may not lead to suspicion of ASF. In some countries, avirulent, nonhaemadsorbing strains produce mainly subclinical nonhaemorrhagic infection and seroconversion, but some animals may develop discrete lesions in the lungs or on the skin in areas over bony protrusions and other areas subject to trauma.

ASF cannot be differentiated from classical swine fever (hog cholera) by either clinical or post-mortem examination, and both diseases should be considered in the differential diagnosis of any acute febrile haemorrhagic syndrome of pigs. Bacterial septicaemias may also be confused with ASF and classical swine fever. Laboratory tests are essential to distinguish between these diseases.

In countries free from ASF but suspecting its presence, the laboratory diagnosis must be directed towards isolation of the virus by simultaneously carrying out the inoculation of pig leukocyte or bone marrow cultures, and detection of antigen in smears or cryostat sections of tissues by the fluorescent antibody test (FAT). However, the detection of antibodies in serum or tissue fluids by the enzyme-linked immunosorbent assay (ELISA), immunoblotting or indirect fluorescent antibody (IFA) test should also be carried out at the same time in order to avoid a delay in detecting infection by an unexpected virus of low virulence. Serology can be an invaluable tool for helping to confirm an outbreak as antibody can often be detected in animals that die of acute disease.

An additional technique now available is the polymerase chain reaction (PCR), which can be used to detect the virus genome in blood or tissues and is particularly useful if samples submitted are unsuitable for virus isolation and antigen detection because they have undergone putrefaction.
B. DIAGNOSTIC TECHNIQUES

1.    Identification of the agent


      Where ASF is suspected, the following samples should be sent to the laboratory: blood in anticoagulant (heparin or ethylene diamine tetra-acetic acid [EDTA]), spleen, tonsil, kidney, lymph nodes. These should be kept as cold as possible, without freezing, during transit. After the samples arrive at the laboratory, they should be stored at -70°C if processing is going to be delayed. As maintaining a cold chain is not always possible, samples can be submitted in glycerosaline; this may slightly decrease the likelihood of virus identification, but it may facilitate the submission of samples to the laboratory so that an outbreak can be confirmed.


      .    Sample preparation for haemadsorption and pig inoculation


            i)    Prepare suspensions of tissues by grinding small pieces with a pestle and mortar containing sterile sand, then add 5-10 ml of a buffered salt solution or tissue culture medium containing antibiotics.


            ii)    Clarify the suspensions by centrifugation at 1000 g for 5 minutes.


            Use the supernatant for haemadsorption (Section B.1.a. below) and pig inoculation (Section B.1.d. below), although pig inoculation is not recommended.


      a)    Haemadsorption test


            The haemadsorption (HAD) test (6) is definitive for ASF and depends on the fact that pig erythrocytes will adhere to the surface of pig monocyte or macrophage cells infected with ASFV, and that most virus isolates produce this phenomenon of haemadsorption. A very small number of 'nonhaemadsorbing' viruses have been isolated, most of which are avirulent, but some do produce typical acute ASF. The test is carried out by inoculating blood or tissue suspensions from suspect pigs into primary leukocyte cultures (Procedure 1 below) or by preparing leukocyte cultures from the blood of pigs inoculated at the laboratory or from the blood of suspect pigs collected in the field (Procedure 2 below). Up to 300 cultures can be prepared from each 100 ml of defibrinated or heparinised blood collected. It is essential to carry out all procedures in such a way as to prevent contamination of the cultures.


            .    Procedure 1: Haemadsorption test in primary leukocyte cultures


            i)    Collect the required volume of fresh pig blood in heparin (100 International Units [IU]/ml blood).


            ii)    Centrifuge at 700 g for 30 minutes, remove the buffy coat cells and wash in medium.


            iii)    Resuspend the cells at a concentration of 107 cells/ml in tissue culture medium containing 10-30% pig serum and antibiotics. In order to prevent nonspecific haemadsorption, the medium should contain serum or plasma from the same pig from which the leukocytes were obtained. If a large volume of samples is to be tested, the homologues serum can be replaced by serum that has been identified by pre-screening as capable of preventing the nonspecific auto-rosette formation.


            iv)    Dispense the cell suspension in aliquots of 1.5 ml in 160 x 16 mm tubes and incubate in a sloping position (5-10° from the horizontal) at 37°C.


                  Note: For routine diagnosis, only 2-4-day-old cultures are sufficiently sensitive.


            v)    Inoculate three tubes of cells by adding 0.2 ml of prepared samples of tissue per tube. It is advisable to inoculate ten-fold and hundred-fold dilutions into cultures, and this is especially important when the field material submitted is in poor condition.


            vi)    Inoculate positive control cultures with haemadsorbing virus. Uninoculated negative controls are essential to monitor the possibility of nonspecific haemadsorption.


            vii)    After 3 days, add 0.2 ml of a fresh preparation of 1% pig erythrocytes in buffered saline to each tube.


            viii)    Examine the cultures daily for 7-10 days under a microscope for cytopathic effect (CPE) and haemadsorption.


            ix)    Reading the results: Haemadsorption consists of the attachment of large numbers of pig erythrocytes to the surface of infected cells. A CPE consisting of a reduction in the number of adherent cells in the absence of haemadsorption may be due to the cytotoxicity of the inoculum, Aujeszky's disease virus or nonhaemadsorbing ASFV, which can be detected by the FAT on the cell sediment or by use of PCR (see below). If no change is observed, or if the results of the immunofluorescence and PCR tests are negative, subinoculate the supernatant into fresh leukocyte cultures.


      .    Procedure 2: Haemadsorption 'autorosette' test with peripheral blood leukocytes from infected pigs


            This procedure is quicker than the preparation and inoculation of primary pig leukocyte cultures (described in Procedure 1 above) and will give more rapid results in positive cases. It can be performed in laboratories that are not equipped for routine virological examinations; the minimum requirements are slides and cover-slips, a microscope and sterile medium, tubes or bottles and pipettes. Blood from suspect pigs in the field, or those inoculated in the laboratory, is collected in heparin and leukocyte cultures are prepared for direct examination for haemadsorption. However, the results of the test are difficult to evaluate and it is now being replaced by the PCR.


            i)    Collect 20 ml of whole blood in a syringe containing 2000 IU heparin in 2 ml of saline, mix and transfer to a glass tube or narrow bottle.


            ii)    Place the tube/bottle vertically in an incubator or water bath at 37°C, and allow the cells to settle. Sedimentation is improved by the addition of 2 ml of a plasma volume expander, such as 'Dextravan 150' which is a solution of Dextran 150 in 0.9% NaCl for injection (Fisons, United Kingdom).


            iii)    Incubate the cultures for 6-8 hours at 37°C, and then examine the cultures at 2-3-hour intervals by transferring small aliquots of the white-cell-rich supernatant, together with some erythrocytes, on to a glass slide and identify haemadsorbing cells under a microscope.


      b)    Antigen detection by fluorescent antibody test


            The FAT (2) can be used as an additional method to detect antigen in tissues of suspect pigs in the field or those inoculated at the laboratory. By itself, it is not enough for ASF diagnosis. It can also be used to detect ASFV antigen in leukocyte cultures in which no HAD is observed and can thus identify nonhaemadsorbing strains of virus. It also distinguishes between the CPE produced by ASFV and that produced by other viruses, such as Aujeszky's disease virus or a cytotoxic inoculum.

Test procedure


            i)    Prepare cryostat sections or impression smears of test tissues, or spreads of cell sediment from inoculated leukocyte cultures on slides, air dry and fix with acetone for 10 minutes at room temperature.


            ii)    Stain with fluorescein isothiocyanate (FITC)-conjugated anti-ASFV immunoglobulin at the recommended or pretitrated dilution for 1 hour at 37°C in a humid chamber.


            iii)    Fix and stain positive and negative control preparations similarly.


            iv)    Wash in phosphate buffered saline (PBS), mount stained tissues in PBS/glycerol, and examine under an ultraviolet light microscope with suitable barrier and exciter filters.


            v)    Reading the results: Tissues are positive if specific granular cytoplasmic fluorescence is observed in paracortical tissue of lymphoid organs or in fixed macrophages in other organs.


      c)    Detection of virus genome by the polymerase chain reaction


            PCR techniques have been developed, using primers from a highly conserved region of the genome, to detect and identify a wide range of isolates belonging to all the known virus genotypes, including both nonhaemadsorbing viruses and isolates of low virulence. The PCR techniques are particularly useful for identifying virus DNA in pig tissues that are unsuitable for virus isolation or antigen detection because they have undergone putrefaction, or when there is good reason to believe that virus may have been inactivated before samples are received in the laboratory. Two PCR procedures are described and consist of a sample preparation procedure followed by the test procedure.


            .    PCR method


            This procedure and procedure 2 serve as a general guideline and a starting point for the PCR protocol. Optimal reaction conditions (incubation times and temperatures, models and suppliers of equipment, concentrations of assay reagents such as the primers and dNTPs) may vary so the described conditions should be evaluated first. Details of PCR validation and procedures to help ensure test validity are given in Chapter I.1.4 of this Terrestrial Manual. Alterations can then easily be made to any aspect of the protocol to achieve better performance.


            .    Sample preparation


            i)    Prepare suspensions of tissue by grinding up small pieces of tissue with a pestle and mortar containing sterile sand, and make a 1/10 dilution by adding 5-10 ml of PBS containing 1% ox serum and antibiotics.


            ii)    Centrifuge at 500 g for 5 minutes.


            iii)    Extraction for control samples: 1/10 tissue homogenates (same tissue as the samples to be analysed): (a) a negative control: use 500 µl of a homogenate of ASFV-negative tissue; (b) a positive control: use 500 µl of a homogenate of ASFV-positive tissue.


            iv)    Transfer 500 µl to a screw-capped Eppendorf tube and boil for 10 minutes.


            v)    Centrifuge at 13,000 g in a microfuge for 5 minutes.


            The tissue supernatant is used in the PCR test.


            The sample preparation procedure given above is simple and inexpensive, but may produce false-negative results due to the presence of PCR inhibitors. An alternative extraction procedure using the NucleoSpin Virus Kit (Macherey Nagel) is described below. This kit includes the reagents, RAV1, RAV3, and NucleoSpin filter columns.


            Working procedure for fluid samples: plasma, serum, cell culture medium.


            (Note that for organ and tissue samples, first prepare a 1/10 homogenate of the material in PBS, then centrifuge to clarify at 12,000 g for 5 minutes. Use the supernatant fluid.)


            Extraction for control samples: 1/10 tissue homogenates (same tissue as samples to be analysed): (a) a negative control: use 150 µl of a homogenate of ASFV-negative tissue; (b) a positive control: use 150 µl of a homogenate of ASVF-positive tissue.


            i)    Add 600 µl of RAV1 (carrier RNA included) to 150 µl of sample. Pipette up and down several times and vortex well. Incubate for 5-10 minutes at room temperature.


            ii)    Optional: If the resulting solution is turbid, centrifuge the mixture for 1 minute to clarify. Transfer the supernatant to a new tube.


            iii)    Add 600 µl ethanol to the clear solution and mix by vortexing.


            iv)    Load 700 µl of the sample on to a NucleoSpin column, placed in a 2 ml centrifuge tube.


            v)    Centrifuge for 60 seconds at 6000 g at room temperature. Discard the flowthrough.


            vi)    Load the remaining sample (about 600 µl) on to the same NucleoSpin column and centrifuge as above. Discard the flowthrough.


            vii)    Add 500 µl of buffer RAV3 to the NucleoSpin column.


            viii)    Centrifuge for 30 seconds at 6000-8000 g. Discard the flowthrough and repeat this washing step.


            ix)    Discard the flowthrough, then place the NucleoSpin column in a fresh 2 ml tube and centrifuge for 5 minutes at maximum speed to completely remove buffer RAV3.


            x)    Elution of nucleic acids: Place the NucleoSpin column in a sterile 1.5 ml centrifuge tube, add 50 µl of elution buffer (5 mM Tris/HCl, pH 8.5, preheated to 70°C), and incubate for 2 minutes.


            xi)    Centrifuge for 60 seconds at 8000 g.


            xii)    Keep the 50 µl of eluted DNA at -20°C until use.


            .    Stock solutions


            i)    Taq DNA polymerase and PCR amplification buffer (10x) are commercially available.


            ii)    Stock 1.25 mM dNTP: Prepare 50 mM stock solutions of each of the following nucleotides: dATP, dCTP, dGTP and dTTP. Add 10 µl of each of these stock solutions to 360 µl sterile distilled water.


            iii)    Primers at a concentration of 20 pmol/µl: Primer 1 sequence 5'-ATGGA-TACCG-AGGGA-ATAGC-3' (positive strand); Primer 2 sequence 5'-CTTAC-CGATG-AAAAT-GATAC-3' (negative strand).


            iv)    Loading buffer: 0.25% Orange G in an aqueous solution of 30% glycerol.


            v)    TAE buffer (50x) for agarose gel: Tris base (242 g); glacial acetic acid (57.1 ml); 0.5 M EDTA, pH 8.0 (100 ml); distilled water (to 1 litre).


            vi)    Marker DNA: 100 base-pair ladder is



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