Colorimetric diagnosis of prolonged bluetongue viremia in sheep, using an enzyme-linked oligonucleotide sorbent assay of amplified viral nucleic acids

Jonathan B. Katz From the Diagnostic Virology Laboratory, National Veterinary Services Laboratories, Veterinary Services, Animal and Plant Health Inspection Service, USDA, Ames, IA 50010 (Katz, Gustafson, Alstad, Moser) and Dupont Biotechnology Systems Division, Research and Development, 331 Treble Cove Rd, Billerica, MA 01862 (Adler).

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Gary A. Gustafson From the Diagnostic Virology Laboratory, National Veterinary Services Laboratories, Veterinary Services, Animal and Plant Health Inspection Service, USDA, Ames, IA 50010 (Katz, Gustafson, Alstad, Moser) and Dupont Biotechnology Systems Division, Research and Development, 331 Treble Cove Rd, Billerica, MA 01862 (Adler).

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A. David Alstad From the Diagnostic Virology Laboratory, National Veterinary Services Laboratories, Veterinary Services, Animal and Plant Health Inspection Service, USDA, Ames, IA 50010 (Katz, Gustafson, Alstad, Moser) and Dupont Biotechnology Systems Division, Research and Development, 331 Treble Cove Rd, Billerica, MA 01862 (Adler).

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Karl A. Adler From the Diagnostic Virology Laboratory, National Veterinary Services Laboratories, Veterinary Services, Animal and Plant Health Inspection Service, USDA, Ames, IA 50010 (Katz, Gustafson, Alstad, Moser) and Dupont Biotechnology Systems Division, Research and Development, 331 Treble Cove Rd, Billerica, MA 01862 (Adler).

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Kathryn M. Moser From the Diagnostic Virology Laboratory, National Veterinary Services Laboratories, Veterinary Services, Animal and Plant Health Inspection Service, USDA, Ames, IA 50010 (Katz, Gustafson, Alstad, Moser) and Dupont Biotechnology Systems Division, Research and Development, 331 Treble Cove Rd, Billerica, MA 01862 (Adler).

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Summary

Each of 5 US-origin serotypes of bluetongue virus (btv) was inoculated into a separate pair of sheep. The duration of each animal's ensuing viremia was monitored, using a btv serogroup-specific nested polymerase chain reaction (pcr) method and an embryonating chicken egg (ece) inoculation procedure. Mean duration of viremia was 100 and 38 days for the pcr and ece methods, respectively. This difference was significant (P < 0.001) and documents a more prolonged viremia in virus-exposed sheep than has been reported. A dual internal oligonucleotide solution hybridization procedure was developed for the rapid (2 hours) colorimetric detection and identification of btv-specific pcr products. This enzyme-linked oligonucleotide sorbent assay (elosa) relied on annealing of separate biotinylated and fluores-ceinated probes to the amplified btv nucleic acid; these complexes were captured on streptavidin-coated microtitration wells and were detected, using a horseradish peroxidase-labeled antifluorescein antibody conjugate. End-point dilution analyses of pcr products indicated that the elosa was more sensitive than gel electrophoretic or comparable colorimetric slot-blot hybridization techniques. The btv pcr-elosa system represents a more sensitive and expeditious means of diagnosing btv-induced viremia than does the ece procedure currently used. The combination of elosa with pcr should facilitate practical application of nucleic acid technology to diagnostic veterinary medicine.

Summary

Each of 5 US-origin serotypes of bluetongue virus (btv) was inoculated into a separate pair of sheep. The duration of each animal's ensuing viremia was monitored, using a btv serogroup-specific nested polymerase chain reaction (pcr) method and an embryonating chicken egg (ece) inoculation procedure. Mean duration of viremia was 100 and 38 days for the pcr and ece methods, respectively. This difference was significant (P < 0.001) and documents a more prolonged viremia in virus-exposed sheep than has been reported. A dual internal oligonucleotide solution hybridization procedure was developed for the rapid (2 hours) colorimetric detection and identification of btv-specific pcr products. This enzyme-linked oligonucleotide sorbent assay (elosa) relied on annealing of separate biotinylated and fluores-ceinated probes to the amplified btv nucleic acid; these complexes were captured on streptavidin-coated microtitration wells and were detected, using a horseradish peroxidase-labeled antifluorescein antibody conjugate. End-point dilution analyses of pcr products indicated that the elosa was more sensitive than gel electrophoretic or comparable colorimetric slot-blot hybridization techniques. The btv pcr-elosa system represents a more sensitive and expeditious means of diagnosing btv-induced viremia than does the ece procedure currently used. The combination of elosa with pcr should facilitate practical application of nucleic acid technology to diagnostic veterinary medicine.

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