title: Detection of murine typhus infected fleas with an enzyme-linked immunosorbent assay.
abstract: An enzyme-linked immunosorbent assay (ELISA) for the detection of Rickettsia typhi antigen in homogenates of pooled or individual laboratory infected fleas is described. The assay uses a double sandwich technique, employing a pool of monoclonal antibodies to capture the antigen and a hyperimmune rabbit serum for antigen detection. Using pools of R. typhi infected Xenopsylla cheopis, Ctenocephalides felis, and Leptopsylla segnis, the sensitivity of the ELISA was compared with direct fluorescent antibody examination of individual fleas for rickettsiae and with rickettsial titers determined by plaque enumeration on primary chicken embryo fibroblasts (PFU). Pooled samples with less than 4 PFU of viable rickettsiae gave ELISA results which were not significantly above background. Both ELISA OD and ELISA titer (last dilution giving an OD that was 2 SD above the control) of a 1:10 dilution of homogenate (4 fleas/ml) were linearly related to rickettsial titer up to 10(6.8) PFU/sample. Multiple freeze-thaws of pools of infected fleas led to a rapid loss of ELISA sensitivity. ELISA assays on single fleas demonstrated large individual variability in rickettsial content. This was independent of the number of days postinfectious feeding or the mean number of PFU/flea (10(1.7-6.9) found for pooled fleas in the same cohort. The sensitivity and ease of performance of ELISA should make it usable under field conditions.
authors.affiliation: Rickettsial Diseases Division, Naval Medical Research Institute, Bethesda, Maryland.
authors.forename: M E
authors.initials: ME
authors.lastname: Dobson
authors.forename: A F
authors.initials: AF
authors.lastname: Azad
authors.forename: G A
authors.initials: GA
authors.lastname: Dasch
authors.forename: L
authors.initials: L
authors.lastname: Webb
authors.forename: J G
authors.initials: JG
authors.lastname: Olson
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000942:Antigens, Bacterial / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D007303:Insect Vectors / Q000382:microbiology*; D011237:Predictive Value of Tests; D012286:Rickettsia typhi / Q000276:immunology / Q000302:isolation & purification*; D005423:Siphonaptera / Q000382:microbiology*
chemical_list: D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial