Spiralis Isolates Research Articles

Hematological, biochemical, and serological parameters of experimentally infected rabbits with Trichinella nativa and Trichinella spiralis for early identification of trichinellosis.

Trichinellosis remains a dangerous disease for humans and animals, which can lead to a lethal outcome. The study of specific body reactions in response to invasion by different types of Trichinella can help in the early diagnosis of the disease. This study aimed to investigate the hematological, biochemical, and serological characteristics of rabbits experimentally infected with trichinellosis, as well as the possibility of using changes in these parameters at various disease stages for early hematological, biochemical, and serological diagnosis of trichinellosis. Three groups of rabbits were orally infected with Trichinella nativa and Trichinella spiralis derived from encysted T. spirtalis larvae in pork muscle samples. The first and second groups were infected with T. nativa and T. spiralis, respectively, while the third group served as control by receiving a physiological solution. An ADVIA 2120i automatic hematology analyzer with a blood smear staining module was used to determine the hematological parameters of rabbits. Antigens were used in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in the sera of infected rabbits that were supernatants containing excretory-secretory antigens (ES-Ag) and somatic antigen (S-Ag). The detection of biochemical responses to the invasion of T. nativa and T. spiralis isolates was detected and hematological parameters were featured in two cases. Trichinella nativa increased the number of erythrocytes, neutrophils, eosinophils, monocytes, basophils, and thrombocytes on day 7 in rabbits. Creatine kinase (CK) is regarded as the most important indicator for the early detection of parasite invasion. Blood biochemistry showed no active response to T. spiralis infection. However, counts of erythrocytes, neutrophils, lymphocytes, and CK rose significantly. In both color indicators, the number of thrombocytes decreased. Enzyme-linked immunosorbent assay with ES-Ag and S-Ag of these isolates demonstrated the ability to detect antibodies as early as 7 days after infection, with a significant increase in the marker up to 70 days. On the 7th day after infection, blood tests of infected animals revealed CK-N-acetyl-cysteine (18.2%) and neutrophils (43%) when infected with T. nativa and neutrophils (26.7%) and lymphocytes (20%) when infected with T. spiralis. These indicators may serve as specific parameters for the early detection of Trichinella spp. invasion.

Genetic evidence substantiates transmission of Trichinella spiralis from one swine farm to another

BackgroundTrichinella spiralis ranks seventh in the risk posed by foodborne parasites. It causes most human cases of trichinellosis and is the most frequent cause of Trichinella outbreaks on pig farms and in wild boar, worldwide. Veterinary inspectors seek the source of outbreaks in hopes of limiting the spread. Established molecular tools are inadequate for distinguishing among potential T. spiralis infection sources because genetic variability in these zoonotic pathogens is limited in Europe. Microsatellite markers proved successful in tracing an outbreak of T. britovi, a related parasite harboring much more genetic variation. Here, we successfully employed microsatellite markers to determine the genetic structure of T. spiralis isolates from two pig outbreaks, discovering notable uniformity among parasites within each farm and discovering an epidemiological link between these two outbreaks.MethodsThe individual larvae from five isolates of T. spiralis from two pig farms and from ten wild boars were genotyped using nine microsatellite markers to examine their genetic structure.ResultsNotably uniform parasite populations constituted each farm outbreak, and the parasites from the first and second outbreaks resembled each other to a notable degree, indicating an epidemiological link between them. Wild boar harbored more genetically variable larval cohorts, distinguishing them from parasites isolated from domestic pigs.ConclusionsMicrosatellite markers succeeded in distinguishing isolates of the highly homogeneous T. spiralis, aiding efforts to track transmission. Each outbreak was composed of a homogenous group of parasites, suggesting a point source of contamination.Graphical abstract

Divergence at mitochondrial and ribosomal loci indicates the split between Asian and European populations of Trichinella spiralis occurred prior to swine domestication.

Available evidence suggests that Trichinella spiralis first originated in Asia and subsequently spread to the rest of the world. Notably limited genetic diversity in European T. spiralis isolates indicates that the parasite went through a dramatic genetic bottleneck at some point in its history. Did this genetic bottleneck result from the transport of a limited number of T. spiralis infected pigs from Asian centers of domestication, or was the parasite resident in Europe far earlier than the domestication of pigs there? In order to explore this hypothesis, we generated complete mitochondrial genomes and ribosomal DNAs from seventeen European T. spiralis isolates, six North American isolates and seven Asian isolates using next generation sequencing. A total of 13,858 base pairs of mitochondrial DNA and 7431 nucleotides of the nuclear ribosomal DNA sequence from each isolate were aligned and subjected to phylogenetic analysis using T. nelsoni as an outgroup. We confirmed that North American and European isolates were tightly clustered within a single "western clade" and all Chinese T. spiralis isolates were placed within a well-supported sister clade. These results indicate that European T. spiralis did not directly descend from extant Chinese parasite populations. Furthermore, the amount of nucleotide divergence between the two clades suggests that they diverged before pigs were domesticated. Over evolutionary time periods, Chinese and European T. spiralis were likely maintained as separate populations. The data presented here indicates the genetic bottleneck observed in European T. spiralis did not result from a small number of founders introduced with Chinese pigs in the recent past, but derives from an earlier bottleneck in host populations associated with the end of the last glacial maximum.

Genotyping and Phylogenetic Position of Trichinella spiralis Isolates from Different Geographical Locations in China.

In China, the nematode Trichinella spiralis is the main aetiological agent of human trichinellosis. We performed multi-locus microsatellite typing of T. spiralis isolates to improve the current knowledge of the evolution and population diversity. First, seven polymorphic microsatellite loci were used to infer the genetic diversity of T. spiralis collected in 10 endemic regions. Then, a Bayesian model-based STRUCTURE analysis, a clustering based on the neighbor-joining method, and a principal coordinate analysis (PCA) were performed to identify the genetic structure. Finally, the phylogenetic position of Chinese isolates was explored based on six mitochondrial and nuclear genetic markers (cox1, cytb, 5S ISR, ESV, ITS1, and 18S rDNA) using the maximum likelihood and Bayesian methods. In addition, the divergence time was estimated with multiple genes using an uncorrelated log-normal relaxed molecular-clock model. A total of 16 alleles were detected in 2,310 individuals (1,650 muscle larvae and 660 adult worms) using seven loci. The STRUCTURE analysis indicated that the T. spiralis isolates could be organized and derived from the admixture of two ancestral clusters, which was also substantiated through the clustering analysis based on the allelic data. PCA separated most samples from Tiandong, Guangxi (GX-td), and Linzhi, Tibet (Tibet-lz), from the remaining isolates. However, both maximum likelihood and Bayesian inference supported the close relationship between Xiangfan, Hubei (HB-xf), and GX-td. The molecular dating analysis suggested that the Chinese isolates started to diverge during the Late Pleistocene (0.69 Mya). Generally, T. spiralis was observed to harbor low genetic variation, and further investigation with deeper sampling is needed to elucidate the population structure.

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

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. Warmińsko-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.

Molecular identification and characterization of Trichinella spiralis from a leopard in India

TPresent study describes species level identification of Trichinella spiralis of leopard origin from India using multiplex PCR and molecular characterization of the parasite based on sequencing of multiple genetic markers viz. 5S ribosomal DNA intergenic spacer region (5S ISR), partial mitochondrial large ribosomal subunit (Mt-lsr) and partial mitochondrial cytochrome c oxidase I (COI) genes. A single amplicons of 173 bp, indicative of T. spiralis was obtained in multiplex PCR. Further, specific PCR amplifications viz. 750 bp (5S ISR), 445 bp (Mt-lsr) and 850 bp (COI) were obtained for selected genetic markers. Homology search analysis of 5S-ISR, Mt-lsr gene and COI gene showed highest 99.6% identity with sequences originating from China (KT894074, T. spiralis), 98.6% similarity with T. spiralis China isolates (GU339127, GU339147) and 99.8% sequence homology with T. spiralis sequences originating from Belarus (MH119334), respectively. In the phylogenetic analysis, sequences of each selected genetic marker clustered together with published T. spiralis isolates only, which further confirmed species level identification of detected larvae as T. spiralis, although very few differences were noted with reference to relative positions. This is the first study from India, which provide molecular evidence on circulation of T. spiralis in wild animals.

Ribo HRM — Detection of inter- and intra-species polymorphisms within ribosomal DNA by high resolution melting analysis supported by application of artificial allelic standards

Ribo HRM, a single-tube PCR and high resolution melting (HRM) assay for detection of polymorphisms in the large subunit ribosomal DNA expansion segment V, was developed on a Trichinella model. Four Trichinella species: T. spiralis (isolates ISS3 and ISS160), T. nativa (isolates ISS10 and ISS70), T. britovi (isolates ISS2 and ISS392) and T. pseudospiralis (isolates ISS13 and ISS1348) were genotyped. Cloned allelic variants of the expansion segment V were used as standards to prepare reference HRM curves characteristic for single sequences and mixtures of several cloned sequences imitating allelic composition detected in Trichinella isolates.Using the primer pair Tsr1 and Trich1bi, it was possible to amplify a fragment of the ESV and detect PCR products obtained from the genomic DNA of pools of larvae belonging to the four investigated species: T. pseudospiralis, T. spiralis, T. britovi and T. nativa, in a single tube Real-Time PCR reaction. Differences in the shape of the HRM curves of Trichinella isolates suggested the presence of differences between examined isolates of T. nativa, T. britovi and T. pseudospiralis species. No differences were observed between T. spiralis isolates. The presence of polymorphisms within the amplified ESV sequence fragment of T. nativa T. britovi and T. pseudospiralis was confirmed by sequencing of the cloned PCR products. Novel sequences were discovered and deposited in GenBank (GenBank IDs: JN971020–JN971027JN971020JN971021JN971022JN971023JN971024JN971025JN971026JN971027, JN120902.1, JN120903.1, JN120904.1, JN120906.1, JN120905.1).Screening the ESV region of Trichinella for polymorphism is possible using the genotyping assay Ribo HRM at the current state of its development. The Ribo HRM assay could be useful in phylogenetic studies of the Trichinella genus.

Prevalence of Trichinella spp. in North Spain wild fauna and new variety of Trichinella britovi identification

Trichinella spiralis and Trichinella britovi live in apparent sympatry among wild fauna of the Iberian Peninsula. In the present study 105 Trichinella isolates from wild mammals were typed by inter-sequence simple repeat PCR (ISSR-PCR). All isolates identified as T. spiralis were indistinguishable from the ISS48 reference strain. Among those belonging to T. britovi, four variations were clearly distinguishable; two of them, ISS11 C-76 and ISS86 MON, had been previously detected while the ISS2 reference strain and Trichinella Rioja 3, (MVUL/SP/02/R3) had not been reported before. The newly distinguished genotype of T. britovi was analyzed by ISSR-PCR, multiplex-PCR, UARR sequencing, and single larva cross-breeding with the other T. britovi genotypes including Trichinella T8 (ISS49). Among all of them, the ISS11 and ISS2 isolates were found to be the most frequent. The uniformity found within T. spiralis isolates is consistent with its recent introduction in Iberian Peninsula, whereas the presence of four variations within T. britovi suggests that this species is an endemic species. Orographical diversity of the West-End of Eurasian Region could act to preserve population diversity observed within T. britovi.

Identification of Spanish Trichinella isolates by ISSR-PCR: Intra-specific variability of Trichinella britovi

In the present work, we investigated genetic variability of the Spanish Trichinella isolates by ISSR-PCR (inter-simple sequence repeat polymerase chain reaction), a technique that is being successfully used to study diversity among related populations. We recovered a total of 43 isolates from different host and geographic localization and identified them by molecular techniques (RAPD and multiplex-PCR) and by Western blot with monoclonal antibodies US5 and US9. Nineteen (44.2%) out of 43 were identified as Trichinella spiralis and 24 (55.8%) as Trichinella britovi. When these samples were analysed by the ISSR technique, all the T. spiralis isolates presented a pattern similar to the T. spiralis ISS116. By contrast, the ISSR-PCR analysis of the isolates identified as T. britovi, showed two different banding profiles compatible with the European T. britovi isolate pattern (ISS2), and the autochthonous Spanish T. britovi isolate (ISS11). Three of these 43 isolates were involved in human outbreaks; the three were identified as T. britovi and showed a pattern similar to the European isolate ISS2. As conclusion, we highlight that an intra-species variability within the Spanish T. britovi isolates analysed was observed, with a predominant group similar to T. britovi ISS2, while T. spiralis group isolates were more homogeneous. No correlations were found between the different ISSR-PCR T. britovi types and the host/geographical origin of the isolates.

Genetic diversity among isolates of Trichinella spiralis from the Province of Buenos Aires, Argentina

AbstractRandom Amplified Polymorphic DNAs, (RAPDs) are used to study the occurrence of Trichinella britovi and T5 among domestic animals in the Province of Buenos Aires, Argentina and to assess the genetic diversity among isolates of T. spiralisfrom this area in a number of infected hosts. All the local isolates proved to be T. spiralis. Six of the eight primers used indicate that the Buenos Aires isolates are distinct from each other as they produce a considerable number of polymorphic bands. Our overall estimates are relatively higher than other intraspecific distances previously estimated within species of this genus and among T. spiralis isolates. Such high degrees of variability observed among local isolates and between isolates from Buenos Aires and Spain should be taken into account when defining isolates within this species, and considering differences in the epidemiology of T. spiralis.

Genetic diversity among isolates of Trichinella spiralis from the Province of Buenos Aires, Argentina

Random Amplified Polymorphic DNAs, (RAPDs) are used to study the occurrence of Trichinella britovi and T5 among domestic animals in the Province of Buenos Aires, Argentina and to assess the genetic diversity among isolates of T. spiralisfrom this area in a number of infected hosts. All the local isolates proved to be T. spiralis. Six of the eight primers used indicate that the Buenos Aires isolates are distinct from each other as they produce a considerable number of polymorphic bands. Our overall estimates are relatively higher than other intraspecific distances previously estimated within species of this genus and among T. spiralis isolates. Such high degrees of variability observed among local isolates and between isolates from Buenos Aires and Spain should be taken into account when defining isolates within this species, and considering differences in the epidemiology of T. spiralis.

Seroepidemiology of trichinellosis in Florida swine

Surveys for swine trichinellosis, conducted in Florida using a commercial enzyme-linked immunosorbent assay (ELISA) kit with a modified protocol for screening and Western blot as a definitive test, revealed significantly different seroprevalences of 0.3% (four of 1294 samples) and 2.8% (five of 179 samples) in domestic breeding swine and feral swine, respectively. Seropositive swine were identified in four of 114 (3.5%) domestic swine herds and in three of six feral swine regions. The location of two domestic herds with seropositive swine within approximately 2 miles of one another, and adjacent to a region where a seropositive feral hog was identified, suggests that a focus of trichinellosis exists in this area. The occurrence of singleton reactors in domestic herds may be explained by the absence of the common modes of transmission and suggests that swine were infected incidentally by feeding on the infected carcasses of small wild mammals or rodents. Although the presence of Trichinella spiralis spiralis isolates cannot be excluded, it is more likely that a sylvatic Trichinella isolate occurs in Florida swine. This conclusion is supported by the low seroprevalence in domestic swine, the failure to detect larvae in domestic or feral swine and the high prevalence in Florida panthers.

Infectivity, antigenicity and host responses to isolates of the genusTrichinella

Comparisons were made of the infectivity and antigenicity of 4 Trichinella spiralis isolates (S, D, Y, W), of quite different geographical origins, and T. pseudospiralis (P) in rapid- and slow-responder inbred mice. Infectivity was measured by the Index of Reproductive Capacity (ICR) expressed as the ratio between the number of muscle larvae recovered on day 30 post-infection (p.i.) and the numbers of larvae given at infection. Antigen recognition was measured by the degree of proliferation of mesenteric lymph node cells (MLNC) to in vitro stimulation with crude muscle larvae antigen (CMLA) and by the total antibody responses to CMLA at day 25 p.i. as measured by ELISA. Regarding infectivity the isolates fell into two groups, high infectivity (S, D and Y) and low infectivity (W and P). Analysis of CMLA, detergent-stripped (CTAB) and 125I-labelled surface larval proteins was made by SDS-PAGE under reducing conditions. Differences in antigen profiles were seen in all antigen preparations, being most noticeable in CTAB and 125I-labelled proteins from W and P isolates. Antigen recognition by polyclonal infection-derived antisera and by monoclonal antibodies raised against the T. spiralis London strain (L) was studied in the W (Arctic) and S (Spanish) isolates. Polyclonal antisera recognized different antigens in the S and W isolates, as did the monoclonal antibody, although recognition was more restricted. Neither antibody recognized a 64 kDa band in the W isolate which was clearly visible in the others tested.

Trichinella spiralis Infections of Inbred Mice: Immunologically Specific Responses Induced by Different Trichinella Isolates

The immune response of inbred mice was studied following infection with Trichinella spiralis var. pseudospiralis (TP) or with isolates of T. spiralis derived from a pig or from an arctic fox. Animals given a primary infection with 1 isolate of Trichinella and challenged 21 days later with the same or different isolates responded more quickly by expelling worms from the homologous challenge. In addition, although mesenteric lymph node cells from mice infected with each isolate of Trichinella would proliferate in vitro when cultured with antigen derived from each of the others, the strongest proliferation response always occurred when cells were cultured in the presence of antigen prepared from the specific isolate used to infect the mouse from which the cells were derived. In addition, it was possible to prepare monoclonal antibodies that recognized an antigen expressed by TP which was not shared by T. spiralis isolates and vice versa. Collectively, these data support the conclusion that the differences observed in the kinetics of immune responsiveness to different Trichinella isolates are referable, at least in part, to differences among the isolates in the expression of functionally relevant antigens.

Trichinella spiralis: Genetic evidence for synanthropic subspecies in sylvatic hosts

Isolates of the nematode genus Trichinella from sylvatic hosts differ in their potential to reproduce in domestic swine. The structure of the genomic DNA from 13 sylvatic isolates from North America and 5 pig isolates, 4 from North America and 1 from Asia, was examined and correlated with the infectivity of the isolate for domestic pigs. DNA restriction fragment length differences, identified by ethidium bromide staining and by hybridization with 32P-labeled ribosomal RNA, served as molecular markers to classify each isolate. All 5 pig isolates and 8 of 13 sylvatic isolates had a high infectivity and reproductive capacity in pigs. All isolates that were highly infectious for pigs regardless of host origin had similar DNA characteristics and were classified operationally as T. spiralis spiralis (pig) and those of the second group as T. spiralis ssp. A DNA clone of repetitive DNA from T. s. spiralis, pBP2, was selected from a library of genomic DNA in plasmid pUC8. When used as a probe, pBP2 hybridized only to the DNA of T. s. spiralis isolates, thus making it a useful diagnostic reagent to predict whether new isolates are highly infectious for pigs (i.e., T. s. spiralis). These results show that T. s. spiralis occurs in wild mammals and this should be considered a serious obstacle to efforts to eradicate trichinellosis from domestic swine.

Prevalence and Intensity of Trichinella spiralis Infection in Illinois Wildlife

Recent evidence that Trichinella spiralis isolates from wildlife may act as reservoirs for domestic swine trichinosis (Murrell et al., 1985, In Trichinellosis, C. W. Kim (ed.), State University of New York Press, Albany, pp. 301-305) complicates the epidemiology of the disease. The infectivity of T. spiralis isolates from wildlife for domestic swine has not been uniform, but several isolates have been shown to be highly infective (Dick, 1983, Journal of Wildlife Diseases 19: 333336; Murrell et al., 1985, loc. cit.). The purpose of this study was to determine the prevalence and intensity of T. spiralis from 3 common Illinois fur-bearers, the raccoon (Procyon lotor), coyote (Canis latrans), and red fox (Vulpesfulva), collected during the annual hunting/trapping