Validation of an ELISA test based on Cephenemyia stimulator first instar antigen and their application for monitoring the seroprevalence of this myiasis in roe deer (Capreolus capreolus) in the Iberian Peninsula

Nasopharyngeal myiasis caused by Cephenemyia stimulator is prevalent among roe deer in northwestern Spain and has recently been reported in other regions of the Iberian Peninsula. Postmortem detection of larvae in the nasopharyngeal cavity, particularly first instars (L1), can be challenging and time-consuming. Therefore, a serological test to detect anti-C. stimulator antibodies could be a more practical option for monitoring the prevalence and distribution of this myiasis. Between 2022 and 2024, a total of 314 roe deer from different Spanish and Portuguese regions were analysed for nasal bot fly infestation. Diagnosis was based on the detection of antibodies with an in-house indirect ELISA using a crude somatic antigen from C. stimulator L1 (CsL1) and the results were compared to necropsy as the gold standard. The overall seroprevalence by indirect ELISA was 57%, showing a sensitivity of 82.7% and specificity of 80.7%. A good agreement was observed between ELISA and the necropsy (kappa index = 0.69); in addition, absorbance values correlated positively with the total number of larvae found at necropsy. Seroprevalence increased significantly with the age of the animals, and varied according to the region, being highest in northern areas of Spain. Given the high and rising occurrence of this myiasis in the Iberian Peninsula, monitoring is crucial to assess its impact on roe deer health and to develop effective control strategies. In this sense, our results highlight the usefulness of CsL1 ELISA as a valuable tool for assessing the prevalence and expansion of C. stimulator.