Avian influenza surveillance in wild birds using different diagnostic techniques in Costa Rica

Vigilância da influenza aviária em aves silvestres utilizando diferentes técnicas diagnósticas na Costa Rica

Avian influenza virus (AIV) surveillance is essential for monitoring outbreaks in wild bird populations. This study evaluated the performance of a rapid immunochromatographic test (RIT) compared to real-time PCR (qRT-PCR) and the agar gel immunodiffusion (AGID) test for AIV detection. RIT demonstrated greater sensitivity than AGID, detecting antigens at dilutions up to 1:512, whereas AGID only reached 1:16. At 1:512 dilution, qRT-PCR confirmed RIT results with a Ct value of 34.5, equivalent to 22 viral DNA copies/µL. The qRT-PCR detection limit for the AIV matrix gene was 2.57 DNA copies/µL. A total of 61 wild birds were reported to the National Animal Health Service in Costa Rica in 2023. Of these, 47 (77.0 %) exhibited clinical signs of avian influenza, while 14 (23.0 %) were dead. Thirteen taxonomic orders and 22 bird species were identified. with 32.2 % of the samples belonging to Pelecaniformes. Using qRT-PCR, 17 birds (27.9 %) tested positive for influenza A H5N1 virus. 76.5 % were Pelecanus occidentalis, 11.8 % Falco peregrinus, 5.9 % redfooted boobies (Sula sula), and 5.9 % great frigatebirds (Fregata minor). Fourteen (7 positive and 7 negative) were tested by RIT and qRT-PCR, showing 100 % concordance between the two methods. Brain tissue showed the highest viral load (918,847 copies/µL), while cloacal swabs had the lowest (1,450 copies/µL). Tracheal samples were most frequently submitted (94.1 %) and had a 93.8 % positive rate. Geospatial analysis revealed most positive cases were from the Pacific Coast. Organspecific testing highlighted the risk of false negatives, emphasizing the need for optimized tissue sampling. Additionally, 200 cloacal swabs from birds in four rescue centers in Costa Rica’s Central Valley tested negative by RIT. These findings support the complementary use of RIT with qRT-PCR for effective field and laboratory AIV surveillance. Monitoring and improved diagnostic strategies are critical for early detection and control of outbreaks. in Costa Rica.

Keywords:
Zoonosis; avian; H5N1; wild birds.