Immunoproteomic Analysis of Dirofilaria repens Microfilariae and Adult Parasite Stages

Abstract

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.

Selection of new diagnostic markers for Dirofilaria repens infections with the use of phage display technology

“…Adult and microfilariae lysates were prepared as we previously described 59 . Adult worms were washed with PBS to remove debris and homogenized manually in lysis buffer (8 M Urea, 40 mM Tris, 4% CHAPS).…”

Prospects of Using High-Throughput Proteomics to Underpin the Discovery of Animal Host–Nematode Interactions

“…Recently, significant progress has been made in the identification of somatic biomarkers or therapeutic targets in nematodes, employing proteomics and immunoproteomic approaches. Nevertheless, most investigations have focused on studying one or two developmental stages [ 54 , 73 , 74 , 75 , 76 , 77 , 78 ], and global investigations of developmental proteomes have only recently been tackled using high-throughput methods. Since the first systematic investigation of the somatic proteome of free-living and parasite stages of the human filarioid nematode, Brugia malayi , approximately a decade ago [ 23 ], there have been only two studies of other filarioids— Onchocerca volvulus and Onchocerca ochengi [ 79 , 80 ], one of T. spiralis (trichina) [ 81 ] and another of Haemonchus contortus [ 22 ].…”

Selection of New Diagnostic Markers for Dirofilaria Repens Infections With the Use of Phage Display Technology

“…Adult and micro laria lysates were prepared as we previously described 62 . Adult worms were washed with PBS to remove debris and homogenized manually in lysis buffer (8 M Urea, 40 mM Tris, 4% CHAPS).…”