An immunoglobulin binding protein (antigen 5) of the stable fly (Diptera: Muscidae) salivary gland stimulates bovine immune responses

Abstract

The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), is an economically important pest of livestock. Previous studies demonstrated lymphocyte suppression by crude salivary gland extract (SGE) of the stable fly. A dominant 27-kDa protein identified in the SGE was reported to stimulate immunodominant antibody responses in exposed cattle. The purpose of this study was to determine whether this protein, now identified as ahomolog of insect proteins named antigen 5 (Ag5), was responsible for the lymphocyte suppression and whether naive calves can mount an immune response to it. Calves raised in the winter were immunized with recombinant Ag5 (rAg5) expressed in Drosophila S2 cells or with "natural" Ag5 protein isolated by preparative gel electrophoresis of SGE. Control calves were immunized with adjuvant alone. Rising antibody concentrations to rAg5 were detected in two of three calves immunized with rAg5 and one of three calves immunized with natural Ag5. Recall lymphocyte responses to rAg5 were detected at 21 and 28 d postimmunization in calves immunized with rAg5 but not in calves immunized with the natural Ag5 or those exposed to adjuvant alone. Mitogen-stimulated bovine lymphocyte responses were not suppressed by rAg5. Further investigation using immunoblotting revealed that rAg5 binds to the Fc and F (ab')2 portions of bovine IgG, but not to an Fab fragment. These findings suggest that Ag5 of the stable fly salivary gland is not immunosuppressive but that it has immunoglobulin binding properties and can invoke specific antibody and memory lymphocyte responses in immunized calves.

Fig. 1

SDS-PAGE analysis of rAg5 stained with Coomassie blue (A) and SGE stained with silver stain (B) depicting the dominant 27 KD protein (native Ag5) of the extract.

Fig. 2

Lymphocyte proliferation assay determined by incorporation of tritiated thymidine defined as counts per minute of radioactivity. Lymphocytes from four steers were treated with five separate conditions: no stimulation (no mitogen), stimulated with Con A alone, Con A plus 3.0 μg/mL of rAg5, control protein (CP) or crude SGE. Error bars represent one standard error of the mean of three replicate experiments.

Fig. 3

Detection of rising antibody concentration against rAg5 in immunized calves following 21, 28, and 42 days post immunization. Serum samples were diluted 1:1000 and reacted with rAg5 (1 μg/mL) on the solid phase of ELISA plates. Each point represents the average value of the OD₄₅₀ obtained from duplicate ELISA results.

Fig. 4

Lymphocyte proliferation assay determined by incorporation of tritiated thymidine defined as counts per minute of radioactivity. Lymphocytes collected from eight calves at 21 and 28 DPI were not stimulated (medium alone) or stimulated with rAg5 (3.0 μg/mL). Three calves (Nos. 22, 23, 73) were immunized with rAg5, and control calf 18 received adjuvant with control protein (A), whereas three calves (Nos. 19, 20, 21) were immunized with natural Ag5 isolated from a preparative acrylamide gel, and control calf 19 received adjuvant plus preparative gel lacking protein. Error bars represent one standard error of the mean (n = 3).

Fig. 5

Western blot immunoassay demonstrates immunoglobulin-binding properties of the natural Ag5 from SGE (A) and rAg5 (B). Lanes 1 and 2 were incubated with serum from two different steers that had been exposed to stable fly bites; lanes 3 and 4 were incubated with serum from two newborn calves collected prior to ingestion of colostrum. Lanes 1−5 were incubated with HRP-sheep-anti-bovine IgG (H + L). Lanes 6−8 were incubated with HRP-bovine IgG Fab, HRP-bovine IgG Fc, or HRP-bovine IgG F(ab’)₂, respectively. Molecular weight markers (Rainbow markers; Amersham, Piscataway, NJ) included 35, 30, and 20 kDa.