title: Effects of pre-culture holding time and temperature on interferon-gamma responses in whole blood cultures from Mycobacterium bovis-infected cattle.
abstract: The Bovigam assay is approved for use within the United States as a complementary tuberculosis test. Prior to whole blood culture and the ensuing ELISA to detect interferon-(IFN)-gamma, samples are subjected to various holding time/temperature combinations due, in part, to practical constraints associated with shipment of samples to approved laboratories. To evaluate these effects, 5-month-old Holstein calves (n = 7) received 10(3) cfu Mycobacterium bovis by aerosol. Heparinized blood was collected 2 months after challenge and held at 4 or 22 degrees C for 0, 8 or 24 h prior to culture with mycobacterial antigens or pokeweed mitogen (PWM). Responses of samples held for 8 or 24 h were comparable and lower than responses of cultures prepared immediately after collection, regardless of holding temperature. Differences in responses of samples held at 4 degrees C versus 22 degrees C were also minimal. A subset of samples was held for 2 h at 37 degrees C at the beginning of the holding period. This subset of samples had diminished responses to all stimulants and increased holding times (i.e., 24 h versus 8 h) negatively impacted the response. Pre-processing conditions, particularly delays in set-up and initial high sample temperatures, reduces IFN-gamma responses of cells from infected cattle increasing the risk of false negatives in this assay of regulatory importance.
authors.affiliation: Bacterial Diseases of Livestock, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA 50010, USA. rwaters@nadc.ars.usda.gov
authors.forename: W R
authors.initials: WR
authors.lastname: Waters
authors.forename: B J
authors.initials: BJ
authors.lastname: Nonnecke
authors.forename: S C
authors.initials: SC
authors.lastname: Olsen
authors.forename: M V
authors.initials: MV
authors.lastname: Palmer
mesh_terms: D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D007371:Interferon-gamma / Q000032:analysis* / Q000096:biosynthesis; D008297:Male; D009163:Mycobacterium bovis / Q000276:immunology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000592:standards / Q000662:veterinary*; D013696:Temperature; D013997:Time Factors; D014374:Tuberculin Test / Q000379:methods / Q000662:veterinary; D014380:Tuberculosis, Bovine / Q000097:blood / Q000175:diagnosis*
chemical_list: D007371:Interferon-gamma