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Intraspecific genetic variation in Trichinella spiralis and Trichinella britovi populations circulating in different geographical regions of Poland.

ABSTRACT: Trichinella spiralis and Trichinella britovi are species of nematodes which are responsible for the majority of Trichinella infections in the world and the most prevalent in Poland. The most abundant species - T. spiralis, is considered to be more genetically homogeneous in Europe than T. britovi. The aim of the present study was to determine the genetic variability in T. spiralis and T. britovi populations based on nuclear 5S rDNA intergenic spacer region (5S rDNA) and cytochrome c oxidase 1 (COX1) gene sequences. For the study, 55 isolates of T. spiralis and 50 isolates of T. britovi isolated from wild boars, pigs, brown rat and a red fox were analyzed. Based on the analysis of both genes, the genetic variability within populations of T. spiralis and T. britovi differed. In T. spiralis, two single nucleotide polymorphisms (SNPs) were observed in the 612 bp 5S rDNA gene fragment, and one SNP was detected in the 700 bp COX1 gene fragment. In T. britovi, 17 single nucleotide variations (SNVs) were detected in the 5S rDNA gene fragment (among them 16 SNPs), while COX1 sequence analysis revealed the occurrence of 20 SNVs between the sequences tested (among them 19 SNPs). For the majority of T. spiralis isolates the investigated larvae presented uniform haplotypes. In contrast, most of the isolates of T. britovi consisted of larvae of different haplotypes. Geographical analysis showed that each region exhibited different haplotype composition and richness. Warmi?sko-Mazurskie and Zachodniopomorskie regions were the richest in haplotypes (15 and 16 haplotypes, respectively). We used heatmaps showing a characteristic pattern for each region graphically. This may allow to differentiate regions based on the occurrence of particular haplotypes. Furthermore, a PCA analysis on the SNP level yielded biplots that show that certain haplotypes/genotypes are associated with (clusters of) regions.

SUBMITTER: Bilska-Zajac E

PROVIDER: S-EPMC6698805 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Intraspecific genetic variation in Trichinella spiralis and Trichinella britovi populations circulating in different geographical regions of Poland.

Bilska-Zając Ewa E Franssen Frits F Różycki Mirosław M Swart Arno A Karamon Jacek J Sroka Jacek J Zdybel Jolanta J Ziętek-Barszcz Anna A Cencek Tomasz T

International journal for parasitology. Parasites and wildlife 20190731

Trichinella spiralis and Trichinella britovi are species of nematodes which are responsible for the majority of Trichinella infections in the world and the most prevalent in Poland. The most abundant species - T. spiralis, is considered to be more genetically homogeneous in Europe than T. britovi. The aim of the present study was to determine the genetic variability in T. spiralis and T. britovi populations based on nuclear 5S rDNA intergenic spac ...[more]

PMID: 31440446

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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:There is little or even no data in the global literature on the distribution of different species of Trichinella in the individual parts of the diaphragms and tongues in infected pigs. This is of particular importance from the food safety point of view and for the conduct of routine testing of pig carcasses for Trichinella as well as epidemiological surveys. Therefore, the aim of the present study was to evaluate the distribution of Trichinella spiralis (T. spiralis), Trichinella britovi (T. britovi), and Trichinella pseudospiralis (T. pseudospiralis) ML in various parts of the diaphragm (the pillars, costal, and sternal part) and the distribution of encapsulated species of Trichinella (T. spiralis and T. britovi) in various parts of the tongues (the tip, body, and root) of experimentally infected pigs. The diaphragm pillars were the most heavily parasitized part of the diaphragm both in groups of pigs infected with particular species of Trichinella and in groups of pigs presenting different levels of infection; however, statistical differences were observed only in the group of pigs with moderate (21-35 larvae per gram-lpg) or moderately high (35-55 lpg) intensity of Trichinella spp. infection in the entire diaphragm. In all groups of pigs, regardless of the infecting Trichinella species or infection level, larvae showed a homogeneous distribution on both sides of the diaphragm and excluding those of T. pseudospiralis, also in all three parts of the tongue. Histological examination showed features of a differential inflammatory response around larvae of the different Trichinella species. This study confirmed that for mandatory examination of pig carcasses using a pooled-sample digestion assay in which each pig is intended to be represented by a 1 gram sample taken from the diaphragm pillars, if that tissue is not available, the mass of the sample taken from the remaining diaphragm parts (costal or sternal) should be at least double that from the pillars. Histological findings confirmed that the inflammatory pattern of pig muscles varies depending on the Trichinella species triggering the infection and is less intense in the case of infections with T. pseudospiralis than in infections with encapsulated species of Trichinella (T. spiralis and T. britovi).

Project description:The present study compares the immunogenic patterns of muscle larvae excretory-secretory proteins (ML E-S) from T. spiralis and T. britovi recognized by Trichinella-infected human sera. Samples were analyzed using two-dimensional electrophoresis (2-DE) coupled with 2D-immunoblot and liquid chromatography-tandem mass spectrometry LC-MS/MS analysis, two ELISA procedures and a confirmatory 1D-immunoblot test. Sera were obtained from nine patients with a history of ingestion of raw or undercooked meat who presented typical clinical manifestations of trichinellosis and from eleven healthy people. Specific anti-Trichinella IgG antibodies were detected in all samples tested with the Home-ELISA kits, but in only four samples for the commercially-available kit. The 1D-immunoblot results indicated that all nine serum samples were positive for T. spiralis ML E-S antigens, expressed as the presence of specific bands. In contrast, eight of the serum samples with T. britovi E-S ML antigens were positive, with one serum sample taken from a patient at 33dpi (days post infection) being negative. To identify immunoreactive proteins that are specifically recognized by host antibodies, both species of ML E-S proteins were subjected to 2D-immunoblotting with human serum taken at 49 dpi. The sera recognized 22 protein spots for T. spiralis and 18 for T. britovi in 2D-immunoblot analysis. Their molecular weights (MW) ranged from 50 to 60 kDa. LC-MS/MS analysis identified both common and specifically-recognized immunoreactive proteins: transmembrane serine protease 9, serine protease, antigen targeted by protective antibodies and Actin-1 partial were shared for both Trichinella species; hypothetical protein T01_7775 and P49 antigen, partial those specific to T. spiralis; deoxyribonuclease-2-alpha and hypothetical protein T03_17187/T12_7360 were specific to T. britovi. Our results demonstrate the value of 2-DE and 2D-immunblot as versatile tools for pinpointing factors contributing to the parasite-host relationship by comparing the secretomes of different Trichinella species.

Project description:BACKGROUND:Domesticated and wild swine play an important role as reservoir hosts of Trichinella spp. and a source of infection for humans. Little is known about the survival of Trichinella larvae in muscles and the duration of anti-Trichinella antibodies in pigs with long-lasting infections. METHODS:Sixty pigs were divided into three groups of 20 animals and infected with 10,000 larvae of Trichinella spiralis, Trichinella britovi or Trichinella pseudospiralis. Four pigs from each group were sacrificed at 2, 6, 12, 18 and 24 months post-infection (p.i.) and the number of larvae per gram (LPG) of muscles was calculated. Serum samples were tested by ELISA and western blot using excretory/secretory (ES) and crude antigens. RESULTS:Trichinella spiralis showed the highest infectivity and immunogenicity in pigs and larvae survived in pig muscles for up to 2 years p.i. In these pigs, the IgG level significantly increased at 30 days p.i. and reached a peak at about 60 days p.i., remaining stable until the end of the experiment. In T. britovi-infected pigs, LPG was about 70 times lower than for T. spiralis at 2 months p.i. and only very few infecting larvae were detected at 6 months p.i., whereas no larvae were detected at 12, 18 and 24 months p.i. At 6 months p.i., degenerated/calcified larvae and cysts were detected in the muscles by trichinoscopy and histology. The IgG pattern showed by T. britovi-infected pigs was similar to that of T. spiralis-infected pigs, although seroconversion occurred some days later. The larval burden of T. pseudospiralis was slightly greater than for T. britovi at 2 months p.i., but no larvae were detected at 6 and 12 months p.i. In T. pseudospiralis-infected pigs, seroconversion occurred slowly, as in T. britovi-infected pigs. The IgG level showed a significant drop at 6 months p.i. and declining to the cut-off value at 12 months p.i. CONCLUSIONS:The longer survival of T. spiralis in pigs in comparison with the other two species highlights its exceptional dissemination potential. These results provide an explanation of the controversial data collected by parasitological and serological tools in the course of epidemiological investigations.

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.

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Intraspecific genetic variation in Trichinella spiralis and Trichinella britovi populations circulating in different geographical regions of Poland.

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Intraspecific genetic variation in <i>Trichinella spiralis</i> and <i>Trichinella britovi</i> populations circulating in different geographical regions of Poland.

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