Circulating Antibodies to Hepatozoon Canis Demonstrated by Immunofluorescence

Abstract

Hepatozoon canis is a protozoan parasite of dogs that has a worldwide distribution. Dogs become infected by ingesting the infected tick vector, Rhipicephalus sanguineus. Natural clinical infections range in severity from asymptomatic to fatal. 6,14 The life cycle in the canine host includes the development of macroand microschizonts in various tissues, with gamonts appearing in the peripheral blood. Diagnosis is made by detecting the appropriate stages in blood smears, tissue impressions, or sections from biopsy materials. Humoral antibody maybe stimulated by H. canis infection, but a serologic test has not been developed because of the unavailability of a satisfactory antigen. This paper describes the detection of antibodies in infected dogs by the indirect fluorescent antibody (IFA) test. Results of a limited survey indicate that this technique may be of diagnostic value in canine hepatozoonosis. Blood from a dog heavily infected with H. canis was drawn into 0.4% sodium citrate, The titrated blood was centrifuged at 600 x g for 10 minutes, and the plasma and buffy coat were removed. The erythrocyte pellet was discarded. The plasma and buffy coat were recentrifuged, and the buffy coat was removed and washed 3 times with phosphate-buffered saline (PBS), (pH 7.2) by successive 10-minute centrifugations at 700 x g. The plasma was saved for use as a possible positive serum control in the IFA test. The final pellet was resuspended in a mixture of equal volumes of PBS and 3% bovine serum albumin (BSA), and thin blood smears were prepared on glass slides. After drying at room temperature, the slides were immersed for 10 minutes in acetone, or 80% acetone/PBS containing 1% formalin, or methanol. The slides were wrapped individually in filter paper and stored at -80 C. Some of the slides were wrapped and frozen before fixation. Twenty-nine sera were tested: 12 were from healthy, tickfree, laboratory colony-bred dogs in which H. canis was not detected in Giemsa-stained blood smears; 10 were from dogs showing gametocytes in blood smears; and 7 were from dogs negative for gametocytes at the time of blood sampling but with a confirmed history of H. canis infection up to 4 months previously. Antigen slides were removed from the freezer and warmed to 37 C in a container with silica gel for about 30 minutes. Dry slides that were not fixed before freezing were immersed in 1 of the 3 fixatives for 10 minutes. Circles were marked on the smears with cosmetic nail polish to prevent intermixing of serum dilutions. Two-fold dilutions of serum in PBS, from 1:16 to 1:1,024, were applied to the smears, and