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Experimental pathogenesis of Anisakis pegreffii in Sprague-Dawley rats inferred by RNA-Seq: Clues to elucidate infection in humans

Anisakiasis is an emerging fish-borne parasitic disease in humans caused by accidental ingestion of live third stage Anisakis sp. larvae. The aim of this study was to explore early infection mechanisms of Anisakis pegreffii larvae and the response of its accidental host, Sprague-Dawley rat, representing a mammalian model mimicking human infection. High-throughput next generation RNA sequencing was used to evaluate both in vivo experimental rat infection and in vitro, assessing transcriptoms of peripheral blood leukocytes (PBLs) stimulated with parasite extract. Seven rats were sampled at 6, 10, 18, 24 and 32h post-infection after being orally intubated with 10 A. pegreffii larvae (N=5) or 1.5 ml of NaCl (N=2). Total RNA was extracted from stomach and muscle tissues penetrated with Anisakis larvae, as well as the surrounding healthy tissue of infected rats (internal control) and non-infected external controls using TriReagent (Ambion, USA). RNA was also extracted from parasites found in different stages of infection ; non-migrating larvae in stomach and intestine, larvae in the process of stomach/intestine/muscle penetration and larvae freshly isolated from fish, as a control. In vitro effect of A. pegreffii protein extract was tested on rat PBLs, 1 and 12h post-stimulation, with non-treated PBLs as a control. Sequencing using Illumina NextSeq 500 of 16 pooled rat, 16 pooled A. pegreffii and 7 pooled PBLs samples resulted in approx. 30 million paired-end reads per sample, 75 bases long and of good average quality. Reads were filtered and mapped to Rattus norvegicus and A. simplex genomes. The host response is strongly directed towards inflammatory reaction, congruent with observed gross and histopathological findings, showing pathways of stimulated neutrophil chemotaxis, leukocyte migration, peptide secretion, as well as intense tissue remodelling marked by keratinocyte proliferation and apoptosis. De novo assembly of A. pegreffii transcriptome has shown a myriad of secretary and excretory products, previously noted as potent allergens. Gaining an improved understanding of development of anisakiasis in accidental host offers a possible pathway for discovering new methods of treatment and control of this disease.

experimental infection, RNA sequencing, anisakid, nematode, NGS

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