Assessing antibody stability in filter paper-preserved blood samples for wildlife disease surveillance in tropical forests

Dried blood spots (DBS) on filter paper are widely used for serological diagnosis because they require minimal blood volumes and enable storage and transport without refrigeration, making them particularly useful in remote and resource-limited settings. However, DBS-based serology may be compromised by methodological variability and the lack of standardized preservation protocols. We evaluated the effects of preservation factors and quality indicators on DBS serological performance for Toxoplasma gondii, a pathogen with a broad host range and limited seasonal variability. We analyzed 787 dB samples from 17 mammal species collected by subsistence hunters in an Indigenous community in the northern Peruvian Amazon between 2008 and 2020. Recorded variables included filter paper type, time to cold-chain initiation, time to analysis, blood saturation, fungal contamination, and colorimetric values of reconstituted blood. Linear models with Type II ANOVA and AIC-based model selection were used to identify factors associated with seropositivity. Filter paper type was the primary determinant of serological outcomes, while severe fungal contamination was associated with reduced seropositivity. Colorimetric values were significantly related to serological results and may serve as a simple indicator of sample quality. These findings highlight the importance of standardized preservation protocols to minimize false negatives under tropical conditions. DBS-based serological studies should prioritize Protein Saver cards, limit fungal growth, and incorporate basic quality assessments to improve the reliability of wildlife disease surveillance within a One Health framework.