Abstract
Human trichinellosis occurs through consumption of raw or inadequately processed meat or meat products containing larvae of the parasitic nematodes of the genus Trichinella. Currently, nine species and three genotypes are recognized, of which T. spiralis, T. britovi and T. pseudospiralis have the highest public health relevance. To date, the differentiation of the larvae to the species and genotype level is based primarily on molecular methods, which can be relatively time consuming and labor intensive. Due to its rapidness and ease of use a matrix assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF MS) reference spectra database using Trichinella strains of all known species and genotypes was created. A formicacid/acetonitrile protein extraction was carried out after pooling 10 larvae of each Trichinella species and genotype. Each sample was spotted 9 times using α-cyano 4-hydoxy cinnamic acid matrix and a MicroFlex LT mass spectrometer was used to acquire 3 spectra (m/z 2000 to 20000 Da) from each spot resulting in 27 spectra/species or genotype. Following the spectra quality assessment, Biotyper software was used to create a main spectra library (MSP) representing nine species and three genotypes of Trichinella. The evaluation of the spectra generated by MALDI-TOF MS revealed a classification which was comparable to the results obtained by molecular methods. Also, each Trichinella species utilized in this study was distinct and distinguishable with a high confidence level. Further, different conservation methods such as freezing and conservation in alcohol and the host species origin of the isolated larvae did not have a significant influence on the generated spectra. Therefore, the described MALDI-TOF MS can successfully be implemented for both genus and species level identification and represents a major step forward in the use of this technique in foodborne parasitology.
Highlights
Nematodes of the genus Trichinella are one of the ten foodborne parasites with the greatest global impact [1]
The four Trichinella species (T. spiralis, T. britovi, T. pseudospiralis and T. nativa), which play the most important role in world-wide human infection clearly displayed different peaks, at least in terms of peak patterns and their intensities which indicates that the spectra are suitable for the creation of distinct reference spectra for each species (Fig 1)
As shown in a heat map of all 33 tested Trichinella strains (Fig 2), all tested isolates of T. spiralis, T. pseudospiralis, T. nelsoni, T. papuae and T. zimbabwensis could be distinguished with high confidence
Summary
Nematodes of the genus Trichinella are one of the ten foodborne parasites with the greatest global impact [1]. Human trichinellosis has been documented in 55 countries with an average yearly incidence of approximately ten-thousand cases [2]. Infections occur through consumption of raw or inadequately processed meat or meat products containing the parasitic larvae [3]. Trichinellosis is mainly controlled through the examination of each animal carcass which may be infested with Trichinella and intended for human consumption [5]. Several countries have monitoring programs for assessing the prevalence of infection in wildlife and to identify the circulating etiological agents. According to the International Commission on Trichinellosis all positive samples should be forwarded to a qualified reference laboratory for the determination of the Trichinella species involved [6]
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