Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods

Background: Dirofilaria immitis is a cosmopolitan zoonotic, vector-borne parasite of carnivorous animals causing dirofilariasis in human beings. Common commercial serodiagnostic tests for canine dirofilariasis usually lead to different results in their sensitivity and specificity. The present study reports development of recombinant DgK (rDgK) antigen of D. immitis for accurate immunodiagnosis of D. Immitis-infected dogs using indirect ELISA test. Methods: The rDgK coding sequence was successfully sequenced, codon optimized and cloned into pET-24a(+) expression vector and then expressed in Escherichia coli. The recombinant DgK was affinity purified using Ni²+-charged HiTrap chelating column, followed by testing in Western blotting and enzyme-linked immunosorbent assays (ELISA) with dog sera from a dirofilariasis endemic area. The performance of rDgK ELISA was evaluated using 60 sera collected from suspected dogs, while molecular technique was used as a reference test. Results: Sera from positive control D. immitis infection produced a strong IgG antibody response to rDgK both in ELISA and Western blotting tests. The sensitivity and specificity related to diagnostic potential of rDgK for ELISA were 92.5% and 87.5%, respectively. The results of rDgK ELISA showed a high agreement (0.764) with molecular identification. Conclusions: The findings revealed that the developed new rDgK antigen is sensitive and specific for immunodiagnosis of canine dirofilariasis using ELISA test.