Application of Dirofilaria immitis immunoreactive proteins in serodiagnosis

Abstract

Dirofilariasis is a zoonotic global vector-borne disease caused by Dirofilaria immitis.The present study focuses on the somatic and excretory/secretory (E/S) proteins released from adult D. immitis. We aimed to fractionate and identify adult D. immitis immunoreactive proteins. Somatic and E/S extracts were immunoblotted to identify the immunoreactive proteins. In the current study, we used matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF/MS) to characterize the immunogenic proteins. Additionally, we used fast protein liquid chromatography (FPLC) to fractionate and evaluate the immunogenicity of the D. immitis secretome. The most immunoreactive proteins were between 10 and 48 kDa. Six proteins including polyprotein antigen, P22u, pepsin inhibitor Dit33, neutrophil chemotactic factor (DiNCF) precursor, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and heat-shock protein 70 (HSP70) were found in both somatic and E/S extracts. Eluting the FPLC column with NaCl resolved two peaks in which the immunoreactivities of the purified proteins were conserved. Characterization of these proteins could provide a novel perspective for understanding the pathogenesis and diagnosing of this disease. K E Y W O R D S Dirofilaria immitis, dirofilariasis, FPLC, mass spectrometry, serodiagnosis How to cite this article: Khanmohammadi M, Falak R, Meamar AR, et al. Application of Dirofilaria immitis immunoreactive proteins in serodiagnosis. Parasite Immunol.

Immunoproteomic Analysis of Dirofilaria repens Microfilariae and Adult Parasite Stages

“…To our knowledge, this study represents the first simultaneous identification of immunogenic antigens of D. repens adult worms and microfilariae in serum collected from naturally infected dogs. Molecules that we identified in D. repens tissue extracts have been previously considered a potential diagnostic target, vaccine candidates, and drug targets for treating, e.g., schistosomiasis [33][34][35], cestodiasis [36][37][38][39][40][41][42], and parasitic nematodes [43][44][45][46][47][48][49][50][51][52]. Our analysis also confirmed the existence of molecules for which antigenic properties have never been considered in Dirofilaria genus.…”

“…Cytoskeleton proteins (actin, tubulin, myosin, and paramyosin) were previously found in D. immitis [51,53,[65][66][67][68]. In the present study, cytoskeleton proteins were identified as potential antigens in both developmental stages (actin).…”

“…Parasitic enzymes, apart from being engaged in metabolic processes, are considered to exhibit moonlighting activity, including immunomodulatory functions [42,49,51,[71][72][73]. For example, GAPDH found in both D. repens developmental stages was identified in other helminths as a potential immunomodulator and an interesting drug target.…”

Recent Advances and Potential Future Applications of MALDI-TOF Mass Spectrometry for Identification of Helminths

“…Apart from using MALDI-TOF MS as a new tool for the identification of adult worms or larvae, other studies focused on the characterization and identification of specific nematode proteins. Khanmohammadi and colleagues [ 30 ] studied the characterization of immunogenic proteins of D. immitis using MALDI-TOF MS. Herein, authors collected adult worms from six infected dogs and cultivated them in cell culture flasks containing roswell park memorial institute (RPMI)-1640 medium and other substances. Afterwards, the medium was submitted to successive steps of centrifugation, filtration, homogenization, and sonication in order to retrieve the in vivo secretome (somatic and excretory/secretory (E/S) protein extracts).…”

Canine Filariasis Outbreak in Southwestern Colombia: A Molecular and Epidemiological Study

“…In the initial diagnosis stage, blood smears were stained with Wright and a thick drop stained with Giemsa or Knott´s test for a microscopic examination that allowed us to recruit positive cases. All cases detected by microscopy were also analyzed with a serological test that specifically detects the secreted 14KDa antigen of D. immitis [50] (Canine Heartworm Ag 2.0 Bionote, Inc, Korea).…”