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

Abstract

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.

Subcutaneous Dirofilariosis of the Eyelid Brought to Poland from the Endemic Territory of Ukraine

“…Experimental infections of mosquitoes carried out in Ukraine, relevant to Poland, revealed the development of D. repens to the L3 stage within 13–14 days at 18–28 °C after ingestion an infectious blood meal [ 19 ]. In recent years, dirofilariasis has spread to Central and Eastern European countries, such as Ukraine, Poland, and Slovakia as well as Northern Europe and the Baltic States: Lithuania and Estonia, as shown by the latest data [ 20 , 21 ].…”

Characterization of Dr20/22: Molecular Insights and Antibody Response in Naturally Infected Dogs withDirofilaria repens

“…All blood samples from dogs were collected and stored following the procedures detailed in our previous study [25]. For the immunohistochemistry staining, the histological slides were deparaffinized in xylene, rehydrated through a gradient of ethanol (from absolute alcohol to 70 %), and then subjected to antigen retrieval by incubation for 5 min at 82 °C in 10 mM citric acid (pH 6).…”

The long-distance relationship between Dirofilaria and the UK: case report and literature review

“…While for D. immitis , one can rely on rapid and straightforward in-clinic test kits together with several serological laboratory tools, no similar tests are available for D. repens ( 12 ). Encouragingly, epitopes of D. repens have recently been identified by Pękacz and colleagues ( 22 ), using phage display technology in combination with a 12-mer peptide library. These antigenic peptides have been shown to be strongly recognised by IgG from sera of infected dogs ( 22 ) and these may form the basis of a new generation of serological tests for Dirofilaria spp.…”

“…Encouragingly, epitopes of D. repens have recently been identified by Pękacz and colleagues ( 22 ), using phage display technology in combination with a 12-mer peptide library. These antigenic peptides have been shown to be strongly recognised by IgG from sera of infected dogs ( 22 ) and these may form the basis of a new generation of serological tests for Dirofilaria spp.…”

“…According to the American Heartworm Society (AHS) and the European Society of Dirofilariosis and Angiostrongylosis (ESDA), the diagnosis of D. immitis should be demonstrated by the presence of circulating mfs and/or adult antigens ( 24 , 33 ). However, each diagnostic method has different sensitivity and specificity, and each of them has its own advantages and disadvantages which may lead to false positive or negative results ( 22 – 25 , 31 – 33 ). Indeed, studies on the incidence and/or prevalence of Dirofilaria spp.…”