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SUMMARY
Enzootic bovine leukosis (EBL) is a disease of adult cattle caused by the retrovirus, bovine leukaemia virus (BLV). Cattle may be infected at any age, including the embryonic stage. Most infections are subclinical, but a proportion of cattle (approx. 30%) over 3 years old develop persistent lymphocytosis, and a smaller proportion develop lymphosarcomas (tumours) in various internal organs. Natural infection has also been recorded in buffaloes, sheep and capybaras. Clinical signs, if present, depend on the organs affected. Cattle with lymphosarcomas almost invariably die either suddenly, or weeks or months after the onset of clinical signs.
Identification of the agent: Virus can be isolated by cultivation of peripheral lymphocytes, followed by demonstration of the virus by electron microscopy or by BLV antigen detection tests. Proviral DNA can be detected in peripheral blood or tumours by the polymerase chain reaction.
Serological tests: The methods most widely used are agar gel immunodiffusion (AGID) on serum and the enzyme-linked immunosorbent assay (ELISA) on serum or milk. These tests have formed the basis for successful eradication policies in many countries. Other tests, such as radio-immunoassay, can also be used. A number of AGID and ELISA kits are available commercially.
Requirements for vaccines and diagnostic biologicals: No vaccine against BLV is available.
A. INTRODUCTION
There may be several causes of lymposarcomas in cattle, but the only definitely known cause is the retrovirus, bovine leukaemia virus (BLV), which causes enzootic bovine leukosis (EBL). The term sporadic bovine leukosis (SBL) is usually reserved for calf, cutaneous and thymic types of lymphoma, which are defined by the age of the animal affected and the distribution of the tumours. The cause or causes of SBL are not known. There may also be lymphosarcomatous conditions that do not fall into either the SBL or EBL categories, i.e. adult multicentric lymphoma with sporadic occurrence of unknown aetiology. Only lymphomas caused by BLV infection should be termed leukosis or enzootic bovine leukosis.
Although animals can become infected with BLV at any age, tumours (lymphosarcomas) are seen typically in animals over 3 years of age. Infections are usually subclinical; only 30-70% of infected cattle develop persistent lymphocytosis, and 0.1-10% develop tumours. Signs will depend on the site of the tumours and may include digestive disturbances, inappetance, weight loss, weakness or general debility and sometimes neurological manifestations. Superficial lymph nodes may be obviously enlarged and may be palpable under the skin and by rectal examination. At necropsy, lymph nodes and a wide range of tissues are found to be infiltrated by neoplastic cells. Organs most frequently involved are the abomasum, right auricle of the heart, spleen, intestine, liver, kidney, omasum, lung, and uterus. The susceptibility of cattle to persistent lymphocytosis, and perhaps also to tumour development, is genetically determined. There is conflicting evidence of the role of the virus as a cause of immunological deficiency or increased cull rate. In one study it was demonstrated that BLV-infected herds have lower milk production (2.5-3% on a herd level), an increased cull rate, and are more susceptible to other diseases with infectious aetiology, e.g. mastitis, diarrhoea and pneumonia, but the effect on fertility is only minor (9).
B. DIAGNOSTIC TECHNIQUES
Virus can be detected by in-vitro cultivation of peripheral blood lymphocytes. The virus is present in blood lymphocytes and in tumour cells as provirus integrated into the DNA of the cell. Virus is also found in the cellular fraction of various body fluids (nasal and bronchial fluids, saliva, milk). Natural transmission depends on the transfer of infected cells, for example during parturition. Artificial transmission occurs, especially by blood-contaminated needles, surgical equipment, gloves used for rectal examinations, etc. Lateral transmission in the absence of these contributory factors is usually slow. In regions where blood-sucking insects occur in large numbers, especially tabanids, these may transmit the virus mechanically.
Although several species can be infected by inoculation of the virus, natural infection occurs only in cattle (Bos taurus and Bos indicus), water buffaloes, and capybaras. Sheep are very susceptible to experimental inoculation and develop tumours more often and at a younger age than cattle. Persistent antibody can also be detected after experimental infection in deer, rabbits, rats, guinea-pigs, cats, dogs, sheep, rhesus monkeys, chimpanzees, antelope, pigs, goats and buffaloes.
BLV was probably present in Europe during the 19th century, from where it spread to the American continent in the first half of the 20th century. It may then have spread back into Europe and on into other countries for the first time, by the importation of cattle from North America (11). A number of countries are recognised as officially free from BLV infection.
Several studies have been carried out in an attempt to determine whether BLV causes disease in humans, especially through the consumption of milk from infected cows. There is, however, no conclusive evidence of transmission, and it is now generally thought that BLV is not a hazard to humans.
| 1. | Identification of the agent
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| | BLV is an exogenous retrovirus related structurally and functionally to human T-lymphotropic viruses 1 and 2 (HTLV-1 and HTLV-2). The major target cells of BLV are B lymphocytes. The virus particle consists principally of single-stranded RNA, nucleoprotein p12, capsid (core) protein p24, transmembrane glycoprotein gp30, envelope glycoprotein gp51, and several enzymes, including reverse transcriptase. Proviral DNA, which is generated by reverse transcription of most of the virus genome, integrates at random into the nuclear DNA of the host cell where it remains, but does not give rise to free virus in vivo. When infected cells are cultured in vitro, usually by co-cultivation of lymphocytes and an indicator cell line, infectious virus is produced, most readily through stimulation with mitogens (16).
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| | a) | Virus isolation
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| | | Mononuclear cells from 1.5 ml of blood in ethylene diamine tetra-acetic acid (EDTA) are separated out on a ficoll/sodium metrizoate density gradient, then cultured with 2 x 106 fetal bovine lung (FBL) cells, and grown for 3-4 days in 40 ml of minimal essential medium (MEM) containing 20% fetal calf serum. Virus causes syncytia to develop in the cell sheet. Short-term cultures can be prepared by culturing mononuclear cells in the absence of FBL cells in 24-well plastic trays for 3 days (13). The p24 and gp51 antigens can be detected in the supernatant of the cultures by radio-immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunoblot or agar gel immunodiffusion (AGID), and the presence of the BLV particles and of the BLV-provirus can be demonstrated by electron microscopy and by PCR, respectively.
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| | b) | Polymerase chain reaction
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| | | The use of the polymerase chain reaction (PCR) to detect BLV provirus has been described by various workers (3-5, 19). Primers constructed to match the gag, pol and env regions of the genome have all been used with variable success. Double (nested) PCR followed by gel electrophoresis and staining is the most rapid and sensitive method (4, 12). The method described is based on primer sequences from the env gene, coding for gp51. This gene is highly conserved, and the gene and the antigen are generally present in all infected animals throughout the course of infection. The technique is restricted to those laboratories that have the facilities for molecular virology, and the usual precautions and control procedures must be in place to ensure validity of the test results (see Chapters I.1.4. and I.1.8.). Several other PCR protocols for the detection of BLV provirus sequences have been published (3, 4, 19).
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| | | The nested PCR is applicable to the detection of BLV infection in individual animals in the following circumstances:
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| | | . | Young calves with colostral antibodies,
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| | | . | Tumour cases, for differentiation between sporadic and infectious lymphoma,
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| | | . | Tumour tissue from suspected cases collected at slaughterhouses,
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| | | . | New infections, before development of antibodies to BLV,
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| | | . | Cases of weak positive or uncertain results in ELISA,
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| | | . | The systematic screening of cattle in progeny-testing stations (before introduction into artificial-insemination centres),
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| | | . | Cattle used for production of vaccines, ensuring that they are BLV free.
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| | | PCR is not suitable for use as a herd test, but may be used as an adjunct to serology for confirmatory testing.
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| | | . | Sensitivity and reliability of the method
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| | | i) | Analytical sensitivity
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| | | | Although the nested PCR assay has a theoretical sensitivity of one target molecule, in practice the analytical sensitivity is somewhat lower, around five to ten target molecules of proviral DNA per sample.
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| | | ii) | False-positive samples
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| | | | The high sensitivity of the nested PCR method may cause problems of false-positive samples due to contamination between samples. To minimise this, several special procedures are adopted during the analysis, such as the use of laminar air-flow hoods, separate rooms for different steps of the analysis, new gloves or the use of special tube openers for each individual assay, negative controls (water blanks), etc. These precautions against contamination have been described (5).
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| | | iii) | False-negative samples
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| | | | It should be noted that only a small proportion of the peripheral lymphocytes can be infected, thus limiting the sensitivity of the assay. The presence of inhibitory substances in some samples may cause false-negative results. To detect this, at least one positive control is used on every test run. In addition, internal controls (mimics) are added to each sample. The mimic is a modified target molecule that is amplified with the same primers as the real target, but that generates a longer PCR product, which can be visualised by agarose gel electrophoresis. The mimic is added at a low concentration, and this, together with its larger PCR-product size, favour an amplification of the real target (2). However, it is possible for the mimic to compete with the true target. It may therefore be necessary to analyse each sample with or without the mimic.
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| | | . | Sample preparation
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| | | Peripheral blood lymphocytes (PBL) are separated from EDTA blood samples by using the Ficoll-Paque separation method (Pharmacia & Upjohn, Uppsala, Sweden). Alternatively buffy coat may be used, or even whole blood, e.g. where samples have been frozen.
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| | | Tumours or other tissues should be homogenised to a 10% suspension.
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| | | . | DNA extraction
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| | | Purification of total DNA is a prerequisite for achieving optimal sensitivity. Various purification methods are commercially available, e.g. NucleoSpin (Macherey-Nagel) or Chelating resin treatment (BioRad).
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| | | The following method is based on studies by Singer-Sam et al. (17) and Walsh et al. (20). Special precautions should be taken during all steps to minimise the risk of contamination (5).
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| | | i) | Approximately 100 µl chelating resin (Sigma C-7901 or Chelex from Bio-Rad) is added for each sample in a 1.5 ml eppendorf tube.
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| | | ii) | 100 µl of the samples and 10 µl of the mimic are added to the tubes with chelating resin. The samples are vortexed.
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| | | iii) | The eppendorf tubes are closed and incubated at 56-60°C for 20 minutes.
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| | | iv) | The tubes are vortexed for 10 seconds.
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| | | v) | The tubes are incubated at 98°C for 8-10 minutes.
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| | | vi) | The tubes are vortexed for 10 seconds and immediately put on ice.
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| | | vii) | Optional: all samples are equilibrated to a standard amount of DNA (500 ng/reaction) applying, for example, the Beta Globin-method (19).
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| | | viii) | The tubes are centrifuged at 15,000 g for 2 minutes.
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| | | ix) | 5 µl is used in the PCR assay.
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| | | . | Nested PCR procedure
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| | | i) | Primer design and sequences
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| | | | Several PCR protocols for the detection of BLV provirus sequences have been published (3, 4, 19). As an example, a PCR assay based on the one developed by Ballagi-Pordany et al. (3) is described in detail. The BLV region used as target is the gp51 (env) gene. The sequence used for designing the primers is available from GenBank, accession No. K02120. The sequences of the primers are:
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Oligo
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Sequence
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Position in K02120
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OBLV1A
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(5'-CTT-TGT-GTG-CCA-AGT-CTC-CCA-GAT-ACA-3')
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5029
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OBLV6A
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(5'-CCA-ACA-TAT-AGC-ACA-GTC-TGG-GAA-GGC-3')
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5442
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OBLV3
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(5'-CTG-TAA-ATG-GCT-ATC-CTA-AGA-TCT-ACT-GGC-3')
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5065
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OBLV5
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(5'-GAC-AGA-GGG-AAC-CCA-GTC-ACT-GTT-CAA-CTG-3')
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5376
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| | | | PCRI-product size: 440 bp; PCRII-product size: 341 bp; Mimic-product size: 761 bp.
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| | | ii) | Reaction mixtures
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| | | | Reaction mixtures are blended (except sample and mimic) before adding to the separate reaction tubes. One negative control (double distilled H2O) per five samples, and one positive control should be added. Total volumes of mixtures are calculated by multiplying the indicated volumes by the total number of samples, including controls, plus one. Taq polymerase is used in a premade 1/10 dilution.
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| | | | DNA samples and mimic (available from Dr S. Belák, Department of Virology, National Veterinary Institute, Box 585, Biomedical Centre, S-751 23, Uppsala, Sweden) (2) should be added in separate rooms in the laboratory: laboratory room 1 for DNA preparations and mimics, and laboratory room 2 for PCRII-products, to minimise contamination.
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| | | | a) | Reagents added in clean laboratory room
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| | | | This mixture may be prepared in advance and stored at 4°C for up to 1 month.
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Reagents per reaction (conc.)
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PCRI reaction
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PCRII reaction
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Double-distilled H2O (standardised)
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21 µl
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21 µl
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10 x PCR buffer (Perkin Elmer)
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5 µl
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5 µl
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dNTP (10 mM)
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4 x 1 µl
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4 x 1 µl
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Bovine serum albumin (1 mg/ml)
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5 µl
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5 µl
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Primers (10 pmol/µl):
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OBLV1A
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1.5 µl
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-
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OBLV6A
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1.5 µl
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-
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OBLV3
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-
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1.5 µl
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OBLV5
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-
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1.5 µl
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In total:
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38 µl
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38 µl
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| | | | The following should be added just before starting the PCR
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Reagents per reaction (conc.)
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PCRI reaction
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PCRII reaction
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MgCl2 (25 mM)
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5 µl
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5 µl
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Taq polymerase (1 unit/reaction)
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2 µl
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2 µl
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Mineral oil
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2 drops
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2 drops
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In total:
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45 µl
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45 µl
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| | | | b) | Reagents added in laboratory room 1 (DNA) or 2 (PCRII)
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Reagents per reaction (conc.)
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PCRI reaction
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PCRII reaction
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DNA sample* (or water*)
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5 µl
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-
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PCRI product
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-
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5 µl
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In total:
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50 µl
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50 µl
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| | | iii) | PCR thermoprofiles
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| | | | PCRI-thermoprofile
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5 x
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94°C/45 seconds, 60°C/60 seconds, 72°C/90 seconds
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30 x
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94°C/45 seconds, 55°C/60 seconds, 72°C/90 seconds
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1 x
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72°C/420 seconds >/= 20°C
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5 x
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94°C/45 seconds, 58°C/60 seconds, 72°C/90 seconds
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30 x
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94°C/45 seconds, 53°C/60 seconds, 72°C/90 seconds
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1 x
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72°C/420 seconds >/=20°C
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| | | iv) | Laboratory procedure
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| | | | Mix PCRI-reagents as described in step ii. Use separate gloves or tube openers for each individual tube when adding the DNA samples. Put the samples on ice. Heat the thermoblock to 80°C. Put samples in the thermoblock and start the PCRI-programme (step iii).
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| | | | Mix PCRII-reagents as described in step ii. Use separate gloves or tube openers for each individual tube when adding the PCRI -product. Put the samples on ice. Heat the thermoblock to 80°C. Put samples in the thermoblock and start the PCRII-programme (step iii).
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| | | . | Agarose gel electrophoresis
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| | | | Take the PCRII-products to the electrophoresis laboratory. Load approximately 10-15 µl of the samples and 23 µl loading buffer on a 2% agarose gel containing ethidiumbromide at 0.01%. Using 0.5 x Tris/borate/EDTA (TBE) buffer, electrophoresis is performed with 90 mA for 2 hours. To control the size of the amplification products, a 100 bp ladder is recommended. Analysis of PCR products is done by UV transilluumiunation.
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| | | . | Interpretation of the results
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| | | i) | Positive samples
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| | | | Positive samples should have PCR products of the expected size (341 bp), similar to the positive control.
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| | | ii) | Negative samples
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| | | | Negative samples should have no PCR products of the expected size (341 bp), but mimic product (144 bp) should be present.
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| | | iii) | Unclear results
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| | | | The assay must be repeated if the positive controls (mimic or external positive control) are negative, or if the negative water controls are positive.
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| | | . | Confirmatory testing
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| | | For confirmatory identification, the PCR products can be sequenced, hybridised to a probe, or analysed by restriction fragment length polymorphism (RFLP) analysis (10).
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| | 2. | Serological tests
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| | | Infection with the virus in cattle is lifelong and gives rise to a persistent antibody response. Antibodies can first be detected 3-16 weeks after infection. Maternally derived antibodies may take up to 6 or 7 months to disappear. There is no way of distinguishing passively transferred antibodies from those resulting from active infection. Active infection, however, can be confirmed by the detection of BLV provirus by the PCR. Passive antibody tends to protect calves against infection. During the periparturient period, cows may have serum antibody that is undetectable by AGID because of an antibody shift from the dam's circulation to her colostrum. Therefore, when using the AGID test, a negative test result on serum taken at this time (2-6 weeks pre- and 1-2 weeks post-partum) is not conclusive and the test should be repeated. However, the AGID can be performed at this stage with first-phase colostrum.
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| | | The antibodies most readily detected are those directed towards the gp51 and p24 of the virus. Most AGID tests and ELISAs in routine use detect antibodies to the glycoprotein gp51, as these appear earlier. Methods of performing these tests have been published (6, 8).
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| | | Weak positive and negative OIE Standard Sera for use in ELISA are available in freeze-dried, irradiated form from the OIE Reference Laboratory in the UK (please consult the OIE Web site at: http://www.oie.int/eng/OIE/organisation/en_LR.htm). These sera may be used to establish ELISA sensitivity. It should be noted that, although the weak positive serum has been calibrated to be equivalent to E4 diluted 1/10; it is not suitable for use in the AGID test. Because supplies are limited, it is suggested that ELISA working standards, calibrated against the Standard Sera, be derived locally.
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a) |
Enzyme-linked immunosorbent assay (a prescribed test for international trade)
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Either an indirect or blocking ELISA may be used. Assays based on both of these are available commercially; different kits may be required for serum or milk samples. Some ELISAs are sufficiently sensitive to be used with pooled samples. ELISAs are carried out in solid-phase microplates. BLV antigen is used to coat the plates either directly or by the use of a capture polyclonal or monoclonal antibody (MAb). The antigen is prepared in a similar manner to the antigen used in the AGID test and is used at a predetermined dilution (e.g. 1/10) in phosphate buffered saline (PBS). In kit form, the plates are sometimes purchased precoated. Some preservatives may be added to milk samples to prevent souring. Preserved samples will not usually deteriorate significantly if stored for up to 6 weeks at 4°C.
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Indirect enzyme-linked immunosorbent assay - Milk ELISA
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The following method is suitable for antibody detection in pooled milk samples.
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Controls
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Strong positive, weak positive, negative milk and diluent controls should be included in each assay. A strong positive control should be prepared by diluting the OIE positive Standard Serum (E4 1/10) 1/25 in negative milk. A weak positive control should be prepared by diluting, in negative milk, the OIE positive Standard Serum (E4 1/10) 25 times the number of individual milk samples in the pool under test. The milk used for diluting the Standard Serum controls should be unpasteurised, cream free and preserved.
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Example test procedure
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i) |
Milk samples must be stored, undisturbed in a refrigerator until a definite cream layer has formed (24-48 hours), or alternatively, centrifuged at 2000 rpm for 10 minutes, the cream layer should be removed prior to testing.
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ii) |
A BLV antigen and a control negative antigen are precoated in alternate columns in the plate. 100 µl of test sample is added to 100 µl wash buffer in the plate to make a 1/2 dilution, adding to two control antigen wells and two BLV antigen wells.
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iii) |
The plate is sealed and mixed on a shaker.
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iv) |
The plate is incubated between 14 and 18 hours at 2-8°C.
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v) |
300 µl per well of wash diluent is added and discarded, and then 200 µl per well wash diluent is added, shaken for 10 seconds and discarded. Finally, 300 µl of wash diluent is added and soaked for 3 minutes and discarded.
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vi) |
200 µl per well of anti-bovine IgG-horseradish peroxidase affinity-purified conjugate diluted in wash diluent is added and the plate is incubated for 90 minutes at room temperature.
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vii) |
The plate is washed by adding 300 µl of wash diluent per well; this is then discarded and a further 300 µl of wash diluent is added. This is left to soak for 3 minutes and discarded. Steps vi and vii are repeated.
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viii) |
200 µl of ABTS (2,2'-azino-bis-[3-ethylbenzothiazoline-6-sulphonic acid]) substrate (prewarmed to 25°C) is added and the plate is incubated for 20 minutes at room temperature in the dark. The reaction may be stopped by adding 50 µl of stopping solution.
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Reading and interpreting the results
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The plate reader is blanked on air and the absorbance is read at 405 nm. All microplate wells must be read within 2 hours of addition of stopper. The absorbance readings of the wells containing negative antigen are subtracted from the readings of wells containing the positive antigen. The two net absorbance values for each test sample should be averaged. The same applies for the replicate weak positive controls. Replicates should be within 0.1 absorbance units of each other.
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For the test to be considered valid, the averaged net absorbance of the weak positive (WP) controls should be 0.2-0.6 absorbance units. The net absorbance of the strong positive control should be >1.0 absorbance units. The net absorbance of the negative and diluent controls should be less than the lower limit of the inconclusive range.
Assuming that the above criteria are met:
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i) |
Test samples are positive if their net absorbance value is greater than or equal to that of the WP control.
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ii) |
Test samples are inconclusive if their net absorbance value is 75% or less of the net absorbance value of the WP control.
i.e. if the WP control net absorbance = 0.40
then the lower limit of the inconclusive range = 0.40 x 0.750 = 0.30
the inconclusive range in this example would be 0.30-0.39
and samples of greater than or equal to 0.40 are considered positive.
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| | | iii) | Test samples are negative if their net absorbance value is less than the lower limit of the 'inconclusive' range (<0.30 in the example).
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| | . | Blocking enzyme-linked immunosorbent assay - Serum ELISA
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| | | The following method is suitable for antibody detection in single or pooled serum samples.
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| | | . | Test procedure
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| | | i) | Coating the plate
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| | | | All wells are coated with BLV antibody, prediluted in coating buffer (100 µl/well), the plate is sealed and incubated for 18 hours at 4°C. A wash cycle (standard wash) is performed, which is three washes filling wells to the top, with a 3-minute soak in between each wash, and then the plate is blotted. BLV antigen is added, prediluted in wash buffer (100 µl/well), the plate is sealed and incubated for 2 hours at 37°C. A standard wash cycle is performed.
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| | | ii) | Preparation and addition of samples and controls
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| | | | The positive and negative control sera are prediluted (1/2) in wash buffer and the solution is added to four wells per control (100 µl/well). For testing pooled samples, 80 sera may be bulked then diluted (1/2) using wash buffer and the solution is added to two wells (100 µl/well) per sample. Single samples should be diluted 1/100 using wash buffer and the solution added to two wells (100 µl/well) per sample. After plating out the samples, the plate is sealed and incubated for 18 hours at 4°C. A brief wash is performed by filling the wells and immediately emptying them.
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| | | iii) Preparation and addition of conjugates and substrate
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| | | | Prediluted biotinylated antibody is added (100 µl/well) to all wells - predilute using wash buffer + 10% fetal calf serum - the plate is sealed and incubated on a rocking table for 1 hour at 37°C. A standard wash is performed as described earlier. The peroxidase-conjugated avidin is prediluted in wash buffer and the solution is added to all wells (100 µl/well). The plate is sealed and incubated on a rocking table for 30 minutes at 37°C. A standard wash is performed. 100 µl orthophenylamine diamine substrate is added to all wells, the plate is covered and left in the dark for 9 minutes. The reaction is stopped with 100 µl of 0.5 M sulphuric acid per well.
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| | | . | Reading and interpretation of results
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| | | The plate reader is blanked on air and the absorbance is read at 490 nm. For dual wave-length readers a reference filter between 620 nm and 650 nm is used. Results are read within 60 minutes after the addition of stop solution.
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| | | The absorbance of the negative control should be about 1.1 +/- 0.4; if the absorbance is below 0.7, the colour development time in step iii above (preparation and addition of conjugates and substrate) should be increased. Conversely, the time should be shortened if the absorbance is above 1.5. The absorbance of the positive control should be less than the absorbance of the negative control x 0.25.
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| | | A sample is positive when the absorbance of each of the two test wells is identical with or less than the mean absorbance of the four negative wells x 0.5.
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| | | A sample is negative when the absorbance of each of the two test wells is identical with or higher than the mean absorbance of the four negative control wells x 0.65.
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| | | For samples giving values between the absorbance of the negative control x 0.5 and x 0.65 it is recommended to retest the animal, using a sample taken 1 month later.
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| | . | Sensitivity of the enzyme-linked immunosorbent assay
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| | | The sensitivity of pooled milk ELISAs can be evaluated using the OIE weak positive and negative Standard Sera. Assays should give a positive result on E4 diluted in negative milk 250 times more than the number of individual milks in the pool (EU Directive 88/406). For example, for pools of 60 milks, E4 should be diluted 1/250 x 60 = 1/15000. Using the weak positive OIE Standard Serum currently available (E4 1/10) in the above example, the dilution used should therefore be 1/25 x 60 = 1/1500. For individual milk samples the weak positive OIE Standard Serum diluted 1/25 (E4 1/250) in negative milk must be positive.
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| | | Where pooled serum samples are tested, the weak positive OIE Standard Serum must test positive at a dilution equal to the number of individual animals in the pool. For example, for a pool of 50 individual samples, the weak positive OIE Standard Serum diluted 1/50 in negative serum should give a positive result. In assays where serum samples are tested individually, undiluted weak positive OIE Standard Serum must be positive.
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| | b) | Agar gel immunodiffusion (a prescribed test for international trade)
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| | | The AGID test is a specific, but not very sensitive, test for detecting antibody in serum samples from individual animals. It is, however, unsuitable for milk samples (except first colostrums) because of lack of specificity and sensitivity. The AGID is simple and easy to perform and has proven to be highly useful and efficient as a basis for eradication schemes.
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| | | Reference sera are included with commercial AGID test kits, but there are no OIE Standard Sera currently available for this test. Further advice may be obtained from the OIE Reference Laboratories ( please consult the OIE Web site at: http://www.oie.int/eng/OIE/organisation/en_LR.htm).
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| | | i) | Agar gel: A 0.8-1.2% solution of agar or agarose is prepared in 0.2 M Tris buffer, pH 7.2, with 8.5% NaCl. One method of preparing the agar is to dissolve 24.23 g of Tris methylamine in 1 litre of distilled water and adjust to pH 7.2 with 2.5 M HCl. Sodium chloride (85 g) is dissolved in 250 ml Tris/HCl and made up to 1 litre. Agarose (8 g) is added and the mixture is heated in a pressure cooker or autoclave at 4.55 kg/sq. cm for 10 minutes. The mixture is dispensed in 15 ml aliquots, which can be stored at 4°C for up to approximately 6 weeks.
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| | | ii) | Antigen: The antigen must contain specific glycoprotein gp51 of BLV. Antigen is prepared in a suitable cell culture system, such as permanently infected fetal lamb kidney (FLK) cell monolayers. The cells used to produce the BLV antigen should be free from noncytopathic bovine viral diarrhoea virus and of bovine retroviruses, bovine immunodeficiency-like virus (lentivirus), and bovine syncytial virus (spumavirus). After 3-4 days' culture at 37°C, the growth medium is replaced with maintenance medium. The cells are harvested after 7 days using standard trypsin/versene solution. The cell suspension is centrifuged at 500 g for 10 minutes. Cells are resuspended in growth medium; 30% of the cells is returned to the culture vessel and the remainder is discarded. All culture supernatants are collected. The supernatants are concentrated 50-100-fold by available methods. This can be done by concentration in Visking tubing immersed in polyethylene glycol, or by precipitation with ammonium sulphate followed by ultrafiltration, or by precipitation in polyethylene glycol followed by desalting and size separation on a polyacrylamide bead column. The antigen contains gp51 predominantly, but may also contain p24.
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| | | | The antigen may be standardised for glycoprotein gp51 by titration against E4 as follows: a twofold dilution of the antigen preparation is made. The highest dilution that, when tested against undiluted standard serum E4, gives a precipitation line equidistant between the antigen and the serum will contain one unit. Two units of antigen are used in the test.
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| | | iii) | Known positive control serum: The positive control serum comes from a naturally or experimentally infected animal (cattle or sheep). The precipitation line formed should be a sharp distinct line midway between the antigen and the control serum wells. A dilution of the control positive serum that gives a weak positive result should be included in the test as an indicator of the test's sensitivity.
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| | | iv) | Known negative control serum: Serum from uninfected animals (cattle, sheep) is used.
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| | | v) | Test sera: Sera from any species of animal are suitable.
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| | | . | Test procedure
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| | | i) | The agar is melted by heating in a water bath and poured into Petri dishes (15 ml per Petri dish of diameter 8.5 cm). The poured plates are allowed to cool at 4°C for about 1 hour before holes are cut in the agar. A punch is used that cuts a hexagonal arrangement of six wells round a central well. Various dimensions of wells can be used; one satisfactory pattern has been produced using wells of 6.5 mm in diameter with 3 mm between wells. For best results, agar plates are used the same day that they are poured and cut.
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| | | ii) | Antigen is placed in the central wells of the hexagonally arranged patterns. Test sera are placed alternately with positive control serum in the outer wells. There should be one control pattern per plate with positive control serum, weak positive control serum and negative control serum in the place of test sera.
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| | | iii) | The test plates are kept at room temperature (20-27°C) in a closed humid chamber, and read at 24, 48 and 72 hours.
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| | | iv) | Interpretation of the results: A test serum is positive if it forms a specific precipitation line with the antigen and forms a line of identity with the control serum. A test serum is negative if it does not form a specific line with the antigen and if it does not bend the line of the control serum. Nonspecific lines may occur; these do not merge with or deflect the lines formed by the positive control. A test serum is a weak positive if it bends the line of the control serum towards the antigen well without forming a visible precipitation line with the antigen; the reaction is inconclusive if it cannot be read either as negative or positive. A test is invalid if the controls do not give the expected results. Sera giving inconclusive or weak positive results can be concentrated and retested.
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C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS
In young calves born to a BLV-infected mother, maternal antibodies to BLV gp51 are important in protection against BLV infection (12). However, experiments using inactivated BLV, fixed infected FLK cells, and purified gp51 have indicated that these give only short-term protection. It was also found that vaccination of cattle with live cells from a cell line BL3, established from an animal with sporadic bovine leukosis, resulted in short-term protection. The nature of the antigen conferring protection was possibly a tumour-associated transplantation antigen (18). Ovine cells synthesising only the env gene products gp51 and gp30 and the main structural protein p24, induced a serological response in cattle (1); cattle were protected after repeated vaccination with these cells.
Vaccination in sheep with a recombinant vaccinia virus expressing BLV gp51 induced protection (14). However, protection was achieved without production of detectable levels of neutralising antibodies. Also, Portetelle et al. found that vaccinia virus recombinants protected sheep even though anti-gp51 antibody waned (15). It is therefore thought that a cell-mediated immune response may play a role in protective immunity against BLV infection. Expression of gp51 has been obtained by recombinant vaccinia virus and yeast containing the coding sequence of BLV env gene. These have resulted in protection of sheep (7). Despite these advances in knowledge, there is as yet no vaccine available commercially for the control of EBL.
REFERENCES
| 1. | Altaner C., Barr J., Altanerova V. & Janik V. (1991). Protective vaccination against bovine leukaemia virus infection by means of cell-derived vaccine. Vaccine, 9, 889-895.
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| 2. | Ballagi-Pordany A. & Belak S. (1996). The use of mimics as internal standards to avoid false negatives in diagnostic PCR. Mol. Cell. Probes, 10, 159-164.
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| 3. | Ballagi-Pordany A., Klintevall K., Merza M., Klingeborn B. & Belak S. (1992). Direct detection of bovine leukaemia virus infection: practical applicability of a double polymerase chain reaction. J. Vet. Med. [B], 39, 69-77.
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| 4. | Beier D., Blankenstein P. & Fechner H. (1998). Chances and limitations for the use of the polymerase chain reaction in the diagnosis of bovine leukemia virus (BLV) infection in cattle. Dtsch Tierartzl. Wochenschr., 105, 408-412.
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| 5. | Belak S. & Ballagi-Pordany A. (1993). Experiences on the application of the polymerase chain reaction in a diagnostic laboratory. Mol. Cell. Probes, 7, 241-248.
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| 6. | Commission of the European Communities (1991). Council Directive of Amending Directive 64/432/EEC as Regards the Diagnosis of Bovine Brucellosis and Enzootic Bovine Leukosis. Off. J. European Communities Council, 14 May 1991.
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| 7. | Daniel R.C.W., Gatei M.H., Good M.F., Boyle D.B. & Lavin M.F. (1993). Recombinant viral vaccines for enzootic bovine leucosis. Immunol. Cell Biol., 71, 399-404.
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| 8. | Dimmock C.K., Rodwell B.J. & Chung Y.S. (1987). Enzootic bovine leucosis. Pathology, Virology and Serology. Australian standard diagnostic techniques for animal disease. No. 49. Australian Agricultural Council.
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| 9. | Emanuelsson U., Scherling K. & Pettersson H. (1992). Relationships between herd bovine leukemia virus infection status and reproduction, disease incidence, and productivity in Swedish dairy herds. Prev. Vet. Med., 12, 121-131.
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| 10. | Fechner H., Blankenstein P., Looman A.C., Elwert J., Geue L., Albrecht C., Kurg A., Beier D., Marquardt O. & Ebner D. (1997). Provirus variants of the bovine leukemia virus and their relation to the serological status of naturally infected cattle. Virology, 237, 261-269.
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| 11. | Johnson R. & Kaneene J.B. (1992). Bovine leukaemia virus and enzootic bovine leukosis. Vet. Bull., 62, 287-312.
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| 12. | Klintevall K. (1995). Bovine Leukaemia Virus: Course of Infection and Means of Detection. Thesis, SLU Info/repro, Uppsala, Sweden, 1-38.
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| 13. | Miller L.D., Miller J.M., Van Der Maaten M.J. & Schmerr M.J.F. (1985). Blood from bovine leukaemia virus-infected cattle: antigen production correlated with infectivity. Am. J. Vet. Res., 46, 808-810.
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| 14. | Ohishi K., Suzuki H., Yamamoto T., Maruyama T., Miki K., Ikawa Y., Numakunai S., Okada K., Ohshima K. & Sugimoto M. (1991). Protective immunity against bovine leukaemia virus (BLV) induced in carrier sheep by inoculating with a vaccinia virus - BLV env recombinant: Association with cell mediated immunity. J. Gen. Virol., 72, 1887-1892.
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| 15. | Portetelle D., Limbach K., Burny A., Mammerickx M., Desmettre P., Riviere M., Zavada J. & Paoletti E. (1991). Recombinant vaccinia virus expression of the bovine leukaemia virus envelope gene and protection of immunized sheep against infection. Vaccine, 9, 194.
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| 16. | Ressang A.A., Mastenbroek N., Quak J., Van Griensven L.J.L.D., Calaft J., Hilgers J., Hageman C., Souissi T. & Swen S. (1974). Studies on bovine leukaemia 1. Establishment of type C virus producing cell lines. Zentralbl. Veterinarmed. [B]., 21, 602-617.
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| 17. | Singer-Sam J., et al. (1989). Use of Chelex to improve the PCR signal from a small number of cells. Amplifications: A forum for PCR users, 3, 11.
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| 18. | Theilen G.H., Miller J.M., Higgins J., Ruppaner R.N. & Garrett W. (1982). Vaccination against bovine leukaemia virus infection. Curr. Top. Vet. Med. Anim. Sci., 15, 547-559.
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| 19. | Venables C., Martin T.C. & Hughes S. (1997). Detection of bovine leukosis virus proviral DNA in whole blood and tissue by nested polymerase chain reaction. Fourth International Congress of Veterinary Virology, Edinburgh, UK, Abstracts, 202.
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| 20. | Walsh P.S., Metzger D.A. & Higuchi R. (1991). Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques, 10, 506-513.
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NB: There are OIE Reference Laboratories for Enzootic bovine leukosis (please consult the OIE Web site at: http://www.oie.int/eng/OIE/organisation/en_LR.htm).
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