A PCR assay for the detection of small ruminant lentiviral gag DNA (provirus) in the white blood cells of sheep and goats was developed and compared with a ...
Veterinary Research Communications, 22 (1998) 355^362 # 1998 Kluwer Academic Publishers. Printed in the Netherlands
PCR DETECTION OF LENTIVIRAL GAG SEGMENT DNA IN THE WHITE BLOOD CELLS OF SHEEP AND GOATS L.H.A. WAGTER1*, A. JANSEN1, N.M.C. BLEUMINK-PLUYM2, J.A. LENSTRA2 AND D.J. HOUWERS2 1 Animal Health Service, PO Box 361, 9200 AJ Drachten; 2University of Utrecht, Faculty of Veterinary Medicine, Veterinary Microbiological Diagnostic Centre, PO Box 80165, 3508 TD Utrecht, The Netherlands *Correspondence ABSTRACT Wagter, L.H.A., Jansen, A., Bleumink-Pluym, N.M.C., Lenstra, J.A. and Houwers, D.J., 1998. PCR detection of lentiviral GAG segment DNA in the white blood cells of sheep and goats. Veterinary Research Communications, 22(5), 355^362 A PCR assay for the detection of small ruminant lentiviral gag DNA (provirus) in the white blood cells of sheep and goats was developed and compared with a serological test (AGIDT). A sample of the DNA prepared from the white blood cells in 3 ml of blood from 208 sheep and goats from 18 di¡erent £ocks was subjected to PCR assay. One of 85 animals from £ocks accredited under the Dutch national MVV/CAEV control programme was positive by PCR while none was positive by AGIDT. In infected £ocks, the AGIDT appeared slightly more sensitive, but preliminary results show that the sensitivity of the PCR assay may be further improved by increasing the number of monocytes tested. The PCR assay, however, was clearly more sensitive in detecting animals in the early stages of infection. With the use of a set of mixed primers and probes, the assay was able to detect the variety of CAEV and MVV strains occurring in the ¢eld. Keywords: caprine arthritis-encephalitis, diagnosis, goat, leukocyte, maedi-visna, polymerase chain reaction, sheep, virus Abbreviations: AGIDT, agar gel immunodi¡usion test; CAEV, caprine arthritis-encephalitis virus; CTB-ELISA, complex trapping blocking^enzyme-linked immunosorbent assay; GAG, general antigen gene; MVV, maedi-visna virus; PBS, phosphate-bu¡ered saline; PCR, polymerase chain reaction; SRLV, small ruminant lentivirus; WBC, white blood cell
INTRODUCTION Detection of infection with the small ruminant lentiviruses (SRLV) maedi-visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) in sheep and goats, respectively, is usually based on the demonstration of antibodies directed against the structural viral proteins (Dawson et al., 1982). Detection of the virus by either virus isolation or otherwise is both laborious and insensitive. Induction of antibodies is slow; in some cases seroconversion only occurs several months after infection. This frustrates serology-based control programmes. Over the years, recurrent detection of seropositive animals has been observed in a low number of £ocks, which is generally attributed to the presence of seronegative carriers (Houwers 355
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et al., 1987). These may be detected by a PCR assay, but the published assays (Haase et al., 1990; Zanoni et al., 1991; Brodie et al., 1992; Johnson et al., 1992; Reddy et al., 1993; Barlough et al., 1994; Leroux et al., 1997; Russo et al., 1997) are not suitable for routine application. Here we describe the development of a practical PCR assay targeted at the lentiviral gag-segment DNA (provirus of SRLV) in white blood cells and its comparison to serology using blood samples from accredited sheep and goat £ocks, samples from sheep collected after exposure to natural infection, and samples from infected sheep and goat £ocks. MATERIALS AND METHODS Blood samples Both serum samples and EDTA blood were collected from 150 sheep and 58 goats. The ovine samples were taken from 12 £ocks, six of which (72 sheep) were accredited under the Dutch national MVV-CAEV accrediation programme. Of the 6 £ocks of goats, two (13 goats) were accredited. The £ocks used came from di¡erent parts of the Netherlands so as to test the ability of the PCR assay to detect genetic variants of the virus strains. A set of 32 bu¡y coats (5^8 ml of blood) were available from sheep which had been exposed to natural infection for 4 months (Pekelder et al., 1994). Bu¡y coats had been stored in liquid nitrogen for 5 years. Serological tests Sera were harvested from clotted blood samples by centrifugation (10 min; 200g) and were stored at ^208C. Antibodies to MVV/CAEV were tested using the agar gel immunodi¡usion test (AGIDT; Central Veterinary Laboratory, Weybridge, UK) following the instructions of the manufacturer. Plasma from blood samples yielding the stored bu¡y coats from the naturally exposed sheep was tested using the CTBELISA (Houwers and Schaake, 1987). DNA preparation from whole blood WBCs were collected by mixing 3 ml fresh EDTA blood with 6 ml sterile 0.83% NH4Cl. The mixture was left for 3 min at room temperature and centrifuged (10 min; 2000g; 48C). The pellet was resuspended in 5 ml sterile 0.83% NH4Cl and centrifuged again. The pellet was resuspended in 1 ml ultrapure water containing 1 mg proteinase K and digested at 558C for 2 h. The enzyme was inactivated at 948C for 15 min and the suspension was stored at ^708C. Before use, the suspension was clari¢ed (5 s; 10 000g) and a volume of 10 ml of the supernatant was used for PCR analysis.
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DNA preparation from collected monocytes For additional experiments, monocytes were speci¢cally collected by using their ability to adhere to the surface of tissue culture £asks. Equal volumes (5 ml) of fresh EDTA blood and sterile PBS were mixed and pipetted into two wells of a 6-well tissue culture plate (Costar). After 2 h of incubation at 378C, the wells were thoroughly washed with PBS and the remaining monocytes were incubated overnight with 1 ml proteinase K (1 mg/ml) at 608C. The contents of both wells were resuspended, collected and brie£y centrifuged (5 s; 10 000g). The DNA of 0.5 ml of the supernatant was extracted once with an equal volume of a mixture of phenol^chloroform^isoamylalcohol (49.5:49.5:1.0 v/v) and once with 0.5 ml water saturated with ether. The DNA was isopropanol-precipitated, washed with 0.5 ml of 70% ethanol (^208C), dried, and dissolved in 50 ml of ultrapure sterile water. Ten ml was subjected to PCR. PCR Primers and probes Several primers and probes were derived from the gag sequences of three ovine lentivirus strains of Dutch, French and Icelandic origin, respectively, and one Swiss caprine strain (EMBL database; Heidelberg; X54379, OLVCG, REV:XX and CAEVCG, respectively). A mix of six primers amplifying the same 300-base-pair product, and a mix of four probes, were composed to cover the ¢eld strains in our samples (Table I). TABLE I Mix of primers and probes for detection by PCR of small ruminant lentivirus proviral gag DNA in white blood cells from sheep and goats Nucleotide number Primers Forward Dutch Forward CAEV Reverse French Reverse Icelandic Reverse Dutch Reverse CAEV
512^532 512-532 789^811 789^811 789^811 789^813
5'-ATG-CAG-CAT-GGA-CTT-GTG-TCC-3' 5'-ATG-CAG-CAT-GGC-CTC-GTG-TCT-3' 5'-CA-AGG-CTG-TTA-TTA-CCC-ACT-GCA-3' 5'-TA-ACG-CTG-TTA-TTA-CCC-ACT-GCA-3' 5'-TA-AGG-CTG-TTA-TTA-CCC-ATT-GCA-3' 5'-C-TTA-ATG-CAT-TTA-TTA-CCC-ATT-GCA-3'
Probes (Mix of the four strains)
749^771
5'-GCA-TCG-CAA-GCT-AAT-ATG-GAT-CA-3' 5'-GCA-TGT-CAA-GCC-AAT-ATG-GAT-CA-3' 5'-GCA-TCA-CAG-GCT-AAT-ATG-GAT-CA-3' 5'-GCA-GCA-CAA-GCT-AAC-ATG-GAT-CA-3'
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Assay An ampliwax gem (Perkin Elmer) was placed in a reaction vial containing 50 ml of a mix with 10 ml bu¡er (106), 10 ml dNTPs (10 mmol/L) and 200 pmol of primer mix. This premix was incubated for 10 min at 808C in a Gene Amp 9600 thermocycler (Perkin Elmer). After cooling, 40 ml ultrapure water containing 0.5 units of DNApolymerase (Amplitaq, Perkin Elmer) and 10 ml sample were added. After 30 s at 948C, 35 cycles of denaturation for 15 s at 948C, annealing for 15 s at 638C and extension for 60 s at 728C were performed. A ¢nal extension was 5 min at 728C. Ampli¢cation products were hybridized with probes labelled with digoxigenin using a DIG oligonucleotide 3'-end labelling kit (Boehringer Mannheim). PCR product (2 ml) was spotted on a nylon membrane (Boehringer Mannheim), dried, denatured (0.5 mol/L NaOH; 1.5 mol/L NaCl), neutralized (1 mol/L NH4Ac), equilibrated (SSC, 26, Mercke 11371) and cross-linked by exposure to UV radiation (1200 mJ/cm2). After preincubation with hybridization bu¡er (56 SSC-bu¡er, 1% blocking reagent, 0.1% sarkosyl and 0.02% SDS), the membrane was sealed in a plastic bag containing 100 pmol of labelled probe mix in 10 ml hybridization bu¡er and hybridized overnight at 508C. The membrane was washed once with 26 SSC, 0.1% SDS, once with 0.16 SSC, 0.1% SDS and once with 0.1 mol/L maleic acid bu¡er (pH 7.5) + 0.15 mol/L NaCl + 0.3% Tween-20. The bound digoxigenin was detected using the DIG luminescent detection kit for nucleic acids according to the manufacturer's instructions (Boehringer Mannheim). An X-ray ¢lm (Fuji 036270) was exposed to the sealed membrane for 1 h at 208C and developed in photo developer (B&W Polycon 180128). A pEX-gag construct and ultrapure water were incorporated in each assay as positive and negative controls, respectively. RESULTS The speci¢city of the primers and probes was determined by subjecting bovine viral diarrhoea virus, bovine herpesvirus 1, parain£uenza 3 virus, bovine respiratory syncitial virus, bovine immunode¢ciency virus, equine herpesvirus 1, porcine herpesvirus, porcine parvovirus, and DNA from 15 bacterial species to the PCR assay including hybridization. No ampli¢cation was observed. Table II shows the results of samples from 6 sheep and 2 goat £ocks accredited under the o¤cial control programme. Both tests show good £ock speci¢city. Whereas all the animals were negative on serology, as expected, PCR revealed one positive sheep in a small £ock of 9 animals. The results of both PCR and CTB-ELISA from sheep that had recently been exposed to maedi-visna virus infection are shown in Table III. Of the 9 PCR-positive but seronegative sheep (one had died) that were tested 4 months later, 6 appeared to have seroconverted. PCR and AGIDT results of blood samples from 4 £ocks of goats and 6 of sheep, which were infected with SRVL, are given in Table IV. The chi-square and kappa values show a fair to very good test agreement between the PCR and AGIDT. The overall test
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TABLE II Number of positive reactions from accredited £ocks by PCR and AGIDT
Test
Sheep (n = 72)
Goats (n = 13)
0 1
0 0
AGIDT PCR
TABLE III A comparison of the results using samples collected from sheep which had been exposed to natural horizontal MVV infection during the preceding 4 months
PCR +ve PCR ^ve Total
CTB-ELISA +ve
CTB-ELISA ^ve
Totals
2 1 3
10 19 29
12 20 32
TABLE IV Numbers of positive reactions from infected £ocks by PCR and by AGIDT. Kappa values were calculated by performing McNemar's test for correlated proportions Test
AGID PCR Chi-square Kappa
Sheep (n = 78) 40 38 0.222 0.539
Goats (n = 45) 20 15 3.571 0.677
kappa value for goats as well as sheep indicated fair to very good agreement by the two tests. Of 123 animals, 60 were positive by AGIDT and 53 by PCR. PCR was positive in samples from 8 seronegative animals (1 goat and 7 sheep). Samples from 16 seropositive animals (6 goats and 10 sheep) were negative by PCR. To assess whether the sensitivity of the PCR assay was improved by a more elaborate method of sample preparation, i.e. isolation of DNA from the monocyte fraction instead of the whole WBC fraction, we performed an initial study using monocytes
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isolated from the blood samples of 16 sheep and 14 goats from two infected £ocks. Ten samples from animals with weakly positive serological results were negative by PCR on WBCs, but 5 of these animals were positive when the selectively collected monocyte fractions were tested. The remaining 20 samples were from animals not suspected of infection; their monocyte fractions were negative in PCR. To assess the e¡ect of the size of the WBC fraction, i.e. the volume of the initial blood sample, we comparatively tested di¡erent blood volumes sequentially collected from an infected sheep on six occasions over a period of 3 months. The WBC fractions were prepared as above using NH4Cl volumes appropriate to the blood volume. Because the ¢nal amount of DNA was resuspended in the same end volume (1 ml), the 5 ml and 10 ml blood samples resulted in higher concentrations of WBC DNA (and thus also of monocyte DNA). The six 3 ml samples, i.e. the standard volume, were all negative, while ¢ve of the six 5 ml samples were positive. With 10 ml samples, the sheep was positive on all six sampling occasions. DISCUSSION Initially we evaluated single primer sets by testing a set of samples originating from several infected £ocks. It appeared that the results were £ock dependent, which suggested that the primers were too speci¢c to recognize di¡erent lentivirus strains, the genetic window being too narrow. We therefore combined several primer sets in one primer mix. Our ¢nal primer mix combined a high overall sensitivity with no ampli¢cation of the other viruses or bacteria. The positive results from the di¡erent sheep and goat £ocks tested indicate that our assay covers at least the major part of the array of SRLV present in The Netherlands. This also suggests that our assay could be used in other parts of the world. The results with the samples from the accredited £ocks, which are considered to be free from lentivirus infection, demonstrate the diagnostic speci¢city of the PCR assay (Table II). It is not clear why one sheep in a small accredited £ock was positive to PCR. Retesting of this sheep was impossible because it was immediately removed by the owner. Serological follow-up testing of the £ock over a period of 18 months showed no evidence of infection, but this does not entirely exclude infection in this single animal. Although the negative test-controls have always been negative, contamination of the sample during the preparation phase can never by totally excluded. PCR detected 12 infected sheep in the early stages of infection, whereas serology only detected 3 (Table III). That 6 out of 8 of these PCR-positives were seropositive 4 months later indicates that they were truly infected. This underlines the impression that the serological response may be very slow; 3 out of 9 sheep remained seronegative despite being infected for a period of at least 4 months. In the infected £ocks, 8 seronegatives were positive by PCR. One of these animals, a ewe, was isolated and monitored; she turned seropositive 18 months later. This also shows that seroconversion may be very slow. Of the 60 seropositive animals, PCR detected only 53. Preliminary experiments clearly indicate that the sensitivity of the PCR assay is limited by the sample size and
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may be improved by extracting the DNA from isolated monocytes. As the monocytes most probably harbour the SRLV proviral DNA, it is likely that a higher concentration of monocytes in the starting material improves the sensitivity of the PCR. We observed, however, that the yield of monocytes from 5 ml of blood varied considerably from sample to sample. In addition, isolating monocytes is a laborious procedure. Increasing the starting blood volume may be an alternative, because testing 10 ml volumes of blood appeared to considerably improve sensitivity without loss of practicality. Using either procedure, it appeared that the sample size is the primary limiting factor in the sensitivity of our PCR assay and that practical solutions are feasible. The current assay (3 ml blood) proved reproducible and robust in our hands. Retesting of the samples gave identical results. This will probably also hold for 10 ml samples. We conclude that the PCR assay we present is speci¢c for multiple SRLV strains owing to the use of a mix of primers and probes and that it has superior diagnostic sensitivity during the early stages of infection. The assay proved valuable in detecting SRLV-infected animals that were missed by the serological test. Hence the test is of considerable value for MVV and CAEV accrediation programmes. ACKNOWLEDGEMENTS This work was supported by a grant from the I.S.P. (local support from the Dutch Government). REFERENCES Barlough, J., East, N., Rowe, J.D., van Hoosear, K., DeRock, E., Bigornia, L. and Rimstad, E., 1994. Double-nested polymerase chain reaction for detection of caprine arthritis-encephalitis virus proviral DNA in blood, milk, and tissues of infected goats. Journal of Virological Methods, 50, 101^114. Brodie, S.J., Pearson, L.D., Snowder, G.D. and DeMartini, J.C., 1992. Host^virus interaction as de¢ned by ampli¢cation of viral DNA and serology in lentivirus-infected sheep. Archives of Virology, 130, 413^428 Dawson, M., Biront, P. and Houwers, D.J., 1982. Comparison of serological tests used in three state veterinary laboratories to identify maedi-visna virus infection. Veterinary Record, 111, 432^434 Haase, A.T., Retzel, E.F. and Staskus, K.A., 1990. Ampli¢cation and detection of lentiviral DNA inside cells. Proceedings of the National Academy of Sciences of the USA, 87, 4971^4975 Houwers, D.J. and Schaake, J., Jr, 1987. An improved ELISA for the detection of antibodies to ovine and caprine lentiviruses, employing monoclonal antibodies in a one-step assay. Journal of Immunological Methods, 98, 151^154 Houwers, D.J., Ko«nig, C.D.W., Bakker, J., de Boer, M.J., Pekelder, J.J., Sol, J., Vellema, P. and de Vries, G., 1987. Maedi-visna control in sheep. III. Results and evaluation of a voluntary control program in the Netherlands over a period of four years. Veterinary Quarterly, 9, 29S^36S Johnson, L.K., Meyer, A.L. and Zink, M.C., 1992. Detection of ovine lentivirus in seronegative sheep by in situ hybridization, PCR, and cocultivation with susceptible cells. Clinical Immunology and Immunopathology, 65, 254^260 Leroux, C., Lerondelle, C., Chastang, J. and Mornex, J.F., 1997. RT-PCR detection of lentiviruses in milk or mammary secretions of sheep or goats from infected £ocks. Veterinary Research, 28, 115^121 Pekelder, J.J., Veenink, G.J., Akkermans, J.P.W.M., van Eldik, P., Elving, L. and Houwers, D.J., 1994. Ovine lentivirus induced indurative lymphocytic mastitis and its e¡ect on the growth of lambs. Veterinary Record, 134, 348^350
362 Reddy, P.G., Sapp, W.J. and Heneine, W., 1993. Detection of caprine arthritis-encephalitis virus by polymerase chain reaction. Journal of Clinical Microbiology, 31, 3042^3043 Russo, P., Vitu, C., Bourgogne, A., Vignoni, M., Abadie, G., David, V. and Pepin, M., 1997. Caprine arthritis-encephalitis virus: detection of proviral DNA in lactoserum cells. Veterinary Record, 140, 483^ 484 Zanoni, R.G., Nauta, I.M., Pauli, U. and Peterhans, E., 1991. Expression of E. coli and sequencing of the coding region for the capsid protein of Dutch maedi-visna strain ZZV 1050: application of the recombinant protein in enzyme-linked immunosorbent assay for the detection of caprine and ovine lentiviruses. Journal of Clinical Microbiology, 29, 1290^1294 (Accepted: 4 March 1998)
