Characterization and Proteomic Analysis of Plasma EVs Recovered from Healthy and Diseased Dogs with Canine Leishmaniosis

Abstract

Dogs are highly valued companions and work animals that are susceptible to many life-threatening conditions such as canine leishmaniosis (CanL). Plasma-derived extracellular vesicles (EVs), exploited extensively in biomarker discovery, constitute a mostly untapped resource in veterinary sciences. Thus, the definition of proteins associated with plasma EVs recovered from healthy and diseased dogs with a relevant pathogen would be important for biomarker development. For this, we recovered, using size-exclusion chromatography (SEC), EVs from 19 healthy and 20 CanL dogs’ plasma and performed proteomic analysis by LC-MS/MS to define their core proteomic composition and search for CanL-associated alterations. EVs-specific markers were identified in all preparations and also non-EVs proteins. Some EVs markers such as CD82 were specific to the healthy animals, while others, such as the Integrin beta 3 were identified in most samples. The EVs-enriched preparations allowed the identification of 529 canine proteins that were identified in both groups, while 465 and 154 were only identified in healthy or CanL samples, respectively. A GO enrichment analysis revealed few CanL-specific terms. Leishmania spp. protein identifications were also found, although with only one unique peptide. Ultimately, CanL-associated proteins of interest were identified and a core proteome was revealed that will be available for intra- and inter-species comparisons.

Emerging role of extracellular vesicles in veterinary practice: novel opportunities and potential challenges

“…In veterinary medicine, sporadic studies have been conducted to identify biomarkers for parasite disease. A study used proteomic analysis and miRNA sequencing to identify the difference between diseased dogs with Canine leishmaniosis and healthy dogs, stating that several proteins, such as myo-inositol and carboxylesterase 5 A, might be proteins of interest ( 58 , 85 ). However, most studies only provide description results instead of testified biomarkers.…”

Host-Derived Extracellular Vesicles in Blood and Tissue Human Protozoan Infections

“…Increased concentrations of miR-21-5p and miR-146a-5p were reported in the serum of the same dogs; unfortunately, only a small selection of miRNAs was assessed, and their abundance within circulating EVs was not investigated. The protein cargo of circulating EVs during canine leishmaniasis was instead investigated by Esteves et al, who compared healthy and L. infantum -infected dogs (CanL) [ 58 ]. Among the 1148 identified proteins, 46.1% were common to both groups, while 154 proteins (13.41%) were present only in the CanL group.…”

“…It could thus be hypothesised that EVs released in the circulation during leishmaniasis participate in CD8 + T cell activation. Twelve L. infantum proteins were also identified within plasma-circulating EVs, and putative DNA polymerase epsilon subunit b (LINJ_35_1780) was the most frequently detected (six dogs), even though protein identification was achieved with only one unique peptide [ 58 ]. A broader identification of pathogen-derived proteins was likely hampered by the presence of highly abundant plasma proteins.…”