Testing independent and interactive effects of corticosterone and synergized resmethrin on the immune response to West Nile virus in chickens

Abstract

Public health agencies utilize aerial insecticides to interrupt an active West Nile virus (WNV) transmission cycle, which may expose WNV-infected birds to these agents. Although resmethrin has been considered benign to birds, no studies have evaluated whether the environmentally employed form of resmethrin with PBO synergist (synergized resmethrin (SR)) can suppress avian immunity to WNV infection and enhance a bird's host competence. Recognizing that wild birds confront toxicological stressors in the context of various physiological states, we exposed four groups (n=9-11) of 9-week-old chickens (Gallus domesticus) to drinking water with either SR (three alternate days at 50 microg/l resmethrin+150 microg/l piperonyl butoxide), CORT (10 days at 20mg/l to induce subacute stress), the combination of SR and CORT, or 0.10% ethanol vehicle coincident with WNV infection. Compared to controls, SR treatment did not magnify but extended viremia by 1 day, and depressed IgG; CORT treatment elevated (mean, 4.26 log(10)PFU/ml) and extended viremia by 2 days, enhanced IgM and IgG, and increased oral virus. The combination of SR and CORT increased the number of chickens that shed oral virus compared to those treated with CORT alone. None of the chickens developed a readily infectious viremia to mosquitoes (none >or=5 log(10)PFU/ml), but viremia in a CORT-exposed chicken was up to 4.95 log(10)PFU/ml. Given that SR is utilized during WNV outbreaks, continued work toward a complete risk assessment of the potential immunotoxic effects of SR is warranted. This would include parameterization of SR exposures with immunological consequences in wild birds using both replicating (in the laboratory) and non-replicating (in the field) antigens. As a start, this study indicates that SR can alter some immunological parameters, but with limited consequences to primary WNV infection outcome, and that elevated CORT mildly enhances SRs immunotoxicity in chickens.

Figure 1. Viremia and oral shedding profiles

(a) Mean ± SEM viremia for all subjects. Means presented as on the Y-axis in log₁₀ PFU/ml when > minimum detection limit (MDL); or, as 0.00 log10 PFU/ml when < MDL (MDL for Vero cell plaque forming assay = 1.70 log₁₀ PFU/ml). Viremia statistics summarize total viremia response curves. P < 0.0001. (b) Percent of chickens within a treatment group with a viremia greater than the MDL for the Vero cell plaque-forming assay (i.e., % positive for WN viremia within a treatment group). P < 0.05 between different letter superscripts for 3 DPI (c) Percent of chickens within a treatment group that were shedding oral virus on a given DPI. (d) Mean ± SEM fraction of days a chicken shed virus while it was alive from 1–5 DPI. P < 0.05 between different letter superscripts for panel c and d.

Figure 2. WNV_EIgM and WNV_EIgG profiles

(a) IgM and (b) IgG were considered WNV_E-antibody positive by ELISA when the OD of sample wells were > 2.0 times the OD of negative control wells. The mean ± SEM log₂ pseudotiters of (c) IgM and (d) IgG were calculated by inserting a sample's OD into a dilution curve of WNV_E-antibody positive chicken sera. P < 0.0001 between different letter superscripts.

Figure 3. Effect of antigen on antibody production

(a) In a pilot experiment, CORT-exposed chickens were inoculated (i.p.) with 10% sheep red blood cells (SRBC) in sterile PBS and tested for antibody production on day 6 post inoculation (by hemagglutination inhibition assay) and this was compared to CORT-exposed chickens' production of anti-WNV_E IgM (by ELISA) on day 7 post inoculation (* indicates statistical significance at P < 0.0005). (b) Correlation between viremia and anti-WNV_E IgM production (F=69.6, R²=0.67, P < 0.0001). (WNV_EIgG correlation not shown, F=21.3, R²=0.38, P < 0.0001.)

Figure 4

Correlation between FGM and maximum WN viremia (F=83.8, R²=0.69, P < 0.0001).