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Trichinella spiralis - New method for sample preparation and objective detection of specific antigens using a chemiluminescence immunoassay.

ABSTRACT: The parasitic roundworm Trichinella spiralis is most commonly transmitted to humans through consumption of raw or undercooked meat of infected pigs or game. To prevent human infection, slaughterhouses perform meat safety surveillance using the gold standard "Magnetic Stirrer Method". We introduce a fast and objective method using automated detection of specific Trichinella spiralis antigens by a newly developed immunoassay based on chemiluminescence (ChLIA). Panel A comprised muscle tissue samples from non-infected pigs (n = 37). Panel B comprised muscle tissue samples from non-infected pigs spiked with different amounts of Trichinella larvae without collagen capsules (n = 56). Panel C contained muscle tissue samples from experimentally infected pigs including Trichinella larvae encapsulated in collagen (n = 32). Each sample was shredded with PBS buffer in a knife mill, destroying Trichinella larvae. Following centrifugation, the supernatant (muscle tissue extract containing released excretory and secretory Trichinella spiralis antigens) was used for Trichinella-specific antigen detection by the new Trichinella ChLIA. The overall accuracy of the Trichinella ChLIA was 97.6 %. The specificity of the Trichinella ChLIA was 100 % (panel A). The sensitivity in samples from experimentally infected pigs was 100 % representing a detection limit of 0.01 larvae per gram. Cross-reactivity with parasites other than Trichinella spp. was not observed. This new meat inspection method for the detection of Trichinella spiralis antigens presents high specificity and high sensitivity, especially in truly infected samples. In contrast to the gold standard, this new approach to meat safety surveillance does not require longsome digestion or microscopy by trained personnel.

SUBMITTER: Braasch J

PROVIDER: S-EPMC7711141 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Trichinella spiralis - New method for sample preparation and objective detection of specific antigens using a chemiluminescence immunoassay.

Braasch Jana J Ostermann Stefanie S Mackiewicz Monika M Bardot Catherine C Pagneux Caroline C Borchardt-Lohölter Viola V Lattwein Erik E

Veterinary parasitology: X 20201008

The parasitic roundworm <i>Trichinella spiralis</i> is most commonly transmitted to humans through consumption of raw or undercooked meat of infected pigs or game. To prevent human infection, slaughterhouses perform meat safety surveillance using the gold standard "Magnetic Stirrer Method". We introduce a fast and objective method using automated detection of specific <i>Trichinella spiralis</i> antigens by a newly developed immunoassay based on chemiluminescence (ChLIA). Panel A comprised muscl ...[more]

PMID: 33305254

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Project description:Publication of the genome from the clade I organism, Trichinella spiralis, has provided us an avenue to address more holistic problems in parasitology; namely the processes of adaptation and the evolution of parasitism. Parasitism among nematodes has evolved in multiple, independent events. Deciphering processes that drive species diversity and adaptation are keys to understanding parasitism and advancing control strategies. Studies have been put forth on morphological and physiological aspects of parasitism and adaptation in nematodes; however, data is now coming available to investigate adaptation, host switching and parasitism at the genomic level. Herein we compare proteomic data from the clade I parasite, Trichinella spiralis with data from Brugia malayi (clade III), Meloidogyne hapla and Meloidogyne incognita (clade IV), and free-living nematodes belonging to the genera Caenorhabditis and Pristionchus (clade V). We explore changes in protein family birth/death and expansion/reduction over the course of metazoan evolution using Homo sapiens, Drosophila melanogaster and Saccharomyces cerevisiae as outgroups for the phylum Nematoda. We further examine relationships between these changes and the ability and/or result of nematodes adapting to their environments. Data are consistent with gene loss occurring in conjunction with nematode specialization resulting from parasitic worms acclimating to well-defined, environmental niches. We observed evidence for independent, lateral gene transfer events involving conserved genes that may have played a role in the evolution of nematode parasitism. In general, parasitic nematodes gained proteins through duplication and lateral gene transfer, and lost proteins through random mutation and deletions. Data suggest independent acquisition rather than ancestral inheritance among the Nematoda followed by selective gene loss over evolutionary time. Data also show that parasitism and adaptation affected a broad range of proteins, especially those involved in sensory perception, metabolism, and transcription/translation. New protein gains with functions related to regulating transcription and translation, and protein family expansions with functions related to morphology and body development have occurred in association with parasitism. Further gains occurred as a result of lateral gene transfer and in particular, with the cyanase protein family In contrast, reductions and/or losses have occurred in protein families with functions related to metabolic process and signal transduction. Taking advantage of the independent occurrences of parasitism in nematodes, which enabled us to distinguish changes associated with parasitism from species specific niche adaptation, our study provides valuable insights into nematode parasitism at a proteome level using T. spiralis as a benchmark for early adaptation to or acquisition of parasitism.

Project description:BackgroundTrichinellosis is a meat-borne zoonotic disease caused by parasites of the genus Trichinella. To date, 12 taxa have been described. The identification of Trichinella species is crucial in order to identify the possible source of infection, the geographical origin of the parasite and to assess risk of infection for domestic pigs and humans. Specific identification of the etiological agent is not always feasible using direct methods since the source of infection can be untraceable. The aim of this study was to develop a diagnostic tool to infer the causative Trichinella species using western blot patterns of sera derived from infected animal and human hosts.MethodsSera from mice experimentally infected with Trichinella spiralis, Trichinella britovi, Trichinella pseudospiralis and Trichinella papuae were tested by western blot using homologous and heterologous crude worm extracts (CWE) and a highly sensitive detection system based on chemiluminescence. In addition, sera from pigs experimentally infected with T. spiralis, T. britovi and T. pseudospiralis and from patients with confirmed T. spiralis, T. britovi and T. pseudospiralis infections, were also included.ResultsSera from mice infected with one Trichinella species reacted with CWE proteins from all four investigated species. Likewise, sera derived from pigs and humans infected with one Trichinella species reacted with CWE proteins from all the three investigated species. Using T. spiralis CWE, sera from T. pseudospiralis-infected hosts yielded a characteristic pattern of reactivity using Wb, which differed to that produced by T. spiralis/T. britovi- or T. papuae-infected host sera.ConclusionsThe present study suggests that western blot using T. spiralis CWE may be a useful tool to distinguish Trichinella infections caused by T. pseudospiralis from those caused by T. spiralis or T. britovi. This method may support epidemiological investigations, particularly when the source of infection is not traceable.

Project description:BackgroundTrichinellosis is a zoonotic disease in humans caused by Trichinella spp. The present study was undertaken to discover excretory-secretory (E-S) proteins from T. spiralis and T. britovi muscle larvae (ML) that hold promise for species-specific diagnostics. To that end, the purified E-S proteins were analyzed by fluorescent two-dimensional difference gel electrophoresis (2-D DIGE) coupled with protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). To search for immunoreactive proteins that are specifically recognized by host antibodies the E-S proteins were subjected to two-dimensional (2-DE) immunoblotting with antisera derived from pigs experimentally infected with T. spiralis or T. britovi.ResultsAccording to 2-D DIGE analysis, a total of twenty-two proteins including potentially immunogenic proteins and proteins produced only by one of the two Trichinella species were subjected to LC-MS/MS for protein identification. From these proteins seventeen could be identified, of which many were identified in multiple spots, suggesting that they have undergone post-translational modification, possibly involving glycosylation and/or proteolysis. These proteins included 5'-nucleotidase, serine-type protease/proteinase, and p43 glycoprotein (gp43) as well as 49 kDa E-S protein (p49). Our findings also suggest that some of the commonly identified proteins were post-translationally modified to different extents, which in certain cases seemed to result in species-specific modification. Both commonly and specifically recognized immunoreactive proteins were identified by 2-DE immunoblotting; shared antigens were identified as gp43 and different protease variants, whereas those specific to T. britovi included multiple isoforms of the 5'-nucleotidase.ConclusionsBoth 2-D DIGE and 2-DE immunoblotting approaches indicate that T. spiralis and T. britovi produce somewhat distinctive antigen profiles, which contain E-S antigens with potential as species-specific diagnostic markers for Trichinella. Our results also demonstrate the value of 2-D DIGE as a versatile tool to compare secretomes of different Trichinella species for pinpointing factors contributing to the interaction with the host.

Dataset Information

Trichinella spiralis - New method for sample preparation and objective detection of specific antigens using a chemiluminescence immunoassay.

Publications

Trichinella spiralis - New method for sample preparation and objective detection of specific antigens using a chemiluminescence immunoassay.

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