Development of a Novel Method for Identification of Alaria alata Mesocercariae by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Carolyn Kästner,Ralph Schönfelder,Anne Mayer-Scholl,Annette Johne,Zanda Ozoliņa,Peter Bahn,Gunita Deksne,Martin Heinrich Richter,Laura Alksne

doi:10.3390/microorganisms9081664

Abstract

Alaria (A.) alata mesocercariae (AM) have increasingly appeared as incidental findings during the mandatory inspection of wild boars for Trichinella in many European countries. An Alaria spp.-specific PCR is available for the identification of AM; however, it is time- and cost-intensive. Therefore, we propose a rapid and cost-efficient MALDI-TOF assay for the identification of AM in wild boar meat that can be applied in routine diagnostics. In this study, a fast and methodologically simple protocol for the protein extraction of AM from different host species in different countries was established, and an AM-specific reference spectra database was created as part of the ongoing development of an existing Trichinella spp. database. A formic acid protein extraction was performed after pooling 10 AM from the same host individual. In total, 61 main spectra profiles (MSPs) from different host individuals were stored in an AM-specific MSP library. The cluster analysis of these 61 MSPs indicated a possible variation within the A. alata species with a tentative association with the geographical origin of the host, but not the host species. This MALDI-TOF assay allows for a fast verification of the AM isolates, which is the next step in the development of a universal database for the identification of several parasites isolated from meat.

Highlights

To ensure that meat from livestock or game is safe for human consumption, European legislation lays down rules for mandatory Trichinella testing such as CommissionImplementing Regulation (EU) No 2015/1375 [1] and subsequent amendments such as Commission Implementing Regulation (EU) No 2020/1478 [2]
The 61 AM positive muscle samples came from naturally AM infected wild boars (n = 54), European water frogs (n = 4), common frogs (n = 2) and a Eurasian lynx (n = 1). 41 wild boars originated from Germany (Brandenburg (25/41), Saxony (15/41) and Schleswig-Holstein
(1/41)), one wild boar came from Poland (Brodnica). 19 samples originated from different regions in Latvia and were isolated from wild boars (12/19), European water frogs (4/19), common frogs (2/19) and a Eurasian lynx (1/19) (Table 1)

Summary

Introduction

To ensure that meat from livestock or game is safe for human consumption, European legislation lays down rules for mandatory Trichinella testing such as CommissionImplementing Regulation (EU) No 2015/1375 [1] and subsequent amendments such as Commission Implementing Regulation (EU) No 2020/1478 [2]. To ensure that meat from livestock or game is safe for human consumption, European legislation lays down rules for mandatory Trichinella testing such as Commission. During the inspection of game meat, wide varieties of parasites, which do not belong to the Trichinella genus, are frequently detected [3]. One such parasite is Alaria (A.) alata, whose mesocercariae (AM) have been found with increased frequency in wild boars in Europe during the past few years [4,5,6,7,8,9,10,11,12,13]. Odening [14] demonstrated that primates can function as paratenic hosts for A

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