Volume 7, Number 3—June 2001
Transmission of an Arenavirus in White-Throated Woodrats (Neotoma albigula), Southeastern Colorado, 1995-1999
aTwelve-well IFA spot slides were coated with test antigens consisting of a mixture of Vero E6 cells infected with WWAV prototype strain AV 9310135 and Vero E6 cells infected with Amapari virus prototype strain BeAn 70563; uninfected Vero E6 cells were controls. Blood samples were diluted 1:100 and tested for positivity at that dilution, providing somewhat less sensitivity but greater specificity than using a lower starting dilution. Each sample was scored as a negative (0 or 1+) or positive (2+, 3+, or 4+) serologic reaction. Antibody-positive samples were titrated in twofold dilutions to determine endpoints.
bAll samples were tested for immunoglobulin (Ig) G antibody by ELISA. The infected cells were sonicated and detergent (Triton X-100; Sigma Chemical Co., St. Louis, MO) treated; control antigens were prepared from uninfected Vero E6 cells. The working dilution of the test antigen was determined by box-titration against a homologous hyperimmune mouse ascitic fluid prepared against WWAV. Test and control antigens were diluted in 0.01 M phosphate-buffered saline, pH 7.4, and coated onto flat-bottom wells in 96-well polyvinyl chloride assay plates (Becton Dickinson Labware, Oxnard, CA). For screening purposes, 1:100 dilutions of bloods were tested; positives were titrated in serial twofold dilutions. Bound antibody was detected by using a mixture of goat anti-Rattus rattus IgG peroxidase conjugate and goat anti-Peromyscus leucopus IgG peroxidase conjugate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and ABTS (Microwell Peroxidase Substrate System, Kirkegaard & Perry) was used as the enzyme indicator. Optical densities (OD) at 410 nm (reference = 490 nm) were measured with a Dynatech MR 5000 microplate reader (Dynatech Industries, Inc., McLean, VA). A blood sample was considered positive if the OD with the test antigen was at least 0.300 and at least twice that of the same sample with the control antigen, providing the OD with the control antigen was <0.150.