Dirofilaria repens is a parasitic nematode causing vector-borne disease (dirofilariasis), considered an emerging problem in veterinary and human medicine. Although main hosts are carnivores, particularly dogs, D. repens shows high zoonotic potential. The disease spreads uncontrollably, affecting new areas. Since there is no vaccine against dirofilariasis, the only way to limit disease transmission is an early diagnosis. Currently, diagnosis depends on the detection of microfilariae in the host bloodstream using modified Knott's test or multiplex PCR. However, the efficacy of tests relying on microfilariae detection is limited by microfilariae periodic occurrence. Therefore, a new reliable diagnostic test is required. Our study aimed to select new diagnostic markers for dirofilariasis with potential application in diagnostics. We focused on single epitopes to ensure high specificity of diagnosis and avoid cross-reactivity with the other parasite infections common in dogs. Using phage display technology and 12-mer peptides library, we selected epitopes highly reactive with IgG from sera of infected dogs. Additionally, our study presents the possibility of detecting D. repens specific cell-free DNA in dogs with no microfilaria but high IgG and IgM antibody levels against parasite somatic antigen.

Dirofilariarepens is a parasitic nematode causing a vector-borne zoonotic infection (dirofilariosis), considered an emerging problem in human and veterinary medicine. Currently, diagnosis is based on the detection of the adult parasite and microfilariae in the host tissues. However, the efficacy of tests relying on microfilariae detection is limited by microfilariae periodic occurrence. Therefore, a new reliable and affordable serological diagnostic method is needed. Better characteristic of the parasite biology and its interaction with host immune system should help to achieve this goal. This study analyzes adult and microfilariae proteomes, and the use of one-dimensional electrophoresis (1-DE) and two-dimensional electrophoresis (2-DE) proteomics, immunoproteomics, and LC-MS/MS mass spectrometry allowed us to identify 316 potentially immunogenic proteins (75 belong to adult stage, 183 to microfilariae, and 58 are common for both). Classified by their ontology, the proteins showed important similarities and differences between both parasite stages. The most frequently identified proteins are structural, metabolic, and heat shock proteins. Additionally, real-time PCR analysis of some immunogenic targets revealed significant differences between microfilariae and adult life stages. We indicated molecules involved in parasite-host interactions and discussed their importance in parasite biology, which may help to reveal potential diagnostic antigens or select drug and vaccine targets.