Western immunoblot analysis of the antigens of Haemobartonella felis with sera from experimentally infected cats

Abstract

Cats were experimentally infected with a Florida isolate of Haemobartonella felis in order to collect organisms and evaluate the immune response to H. felis. Cryopreserved organisms were thawed and injected intravenously into nonsplenectomized and splenectomized cats. Splenectomized animals were given 10 mg of methylprednisolone per ml at the time of inoculation. Blood films were evaluated daily for 1 week prior to infection and for up to 60 days postinfection (p. i.). Blood for H. felis purification was repeatedly collected from splenectomized animals at periods of peak parasitemias. Organisms were purified from infected blood by differential centrifugation, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes for immunoblot analysis. Serum was collected from nonsplenectomized animals prior to and for up to 60 days p.i. and was used on immunoblots to identify antigens. The combination of splenectomy and corticosteroid treatment resulted in marked, cyclic parasitemias without concurrent severe anemia, providing an opportunity to harvest organisms in a manner that was not lethal to the animals. Several antigens (150, 52, 47, 45, and 14 kDa) were identified. An antigen with a molecular mass of approximately 14 kDa appeared to be one of the most immunodominant and was consistently recognized by immune sera collected at various times during the course of infection. These data suggest that one or more of these antigens might be useful for the serologic diagnosis of H. felis infections in cats.

FIG. 1

PCV and percent parasitemia for each of two splenectomized cats (A and B) from 0 to 60 days postinoculation with an FL isolate of H. felis. Asterisks indicate periods when the organisms were harvested from the peripheral blood.

FIG. 2

PCV and percent parasitemia for each of two spleen-intact cats (A and B) from 0 to 60 days postinoculation with an FL isolate of H. felis. Asterisks indicate periods when doxycycline administration was necessary to prevent mortality.

FIG. 3

Electron micrograph of H. felis bodies purified from infected whole blood by differential centrifugation. Bar, 1 μm.

FIG. 4

Immunoblot of an FL isolate of H. felis and noninfected, feline erythrocytes (RBC) with preinfection serum (Pre) or immune serum (Post) collected from an experimentally infected animal 21 days postinoculation. The fractions (1/300 to 1/3,000) indicate the dilutions of serum used. Molecular size standards (in kilodaltons) are given on the left.

FIG. 5

Immunoblot of an FL isolate of H. felis and noninfected, feline erythrocytes (RBC) with preinfection serum (Pre) or immune serum collected from an experimentally infected animal at 14 days (Post 14), 21 days (Post 30), 45 days (Post 45), and 60 days (Post 60) postinoculation. The fraction (1/1,000) indicates the dilution of serum used. Molecular size standards (in kilodaltons) are given on the left.