Non-encapsulated Species Research Articles

First Identification of Trichinella pseudospiralis in a Golden Jackal (Canis aureus) in Romania.

Trichinella spp. are etiological zoonotic agents that spread throughout the world and affect mammals, birds, and reptiles. Within this genus, Trichinella pseudospiralis is the only recognized non-encapsulated species known to infect mammals and birds. This species has been reported in the majority of European countries, and the real epidemiological scenario of this species remains to be defined because its detection in mammals is much lower than that of the capsulated species. The aim of this study was to examine the presence of Trichinella larvae isolated from the muscles of a jackal from the hunting fund of 36 Murfatlar, Constanta County, Romania. The muscle samples were examined by artificial digestion, and the larvae were identified at the species level by multiplex PCR. The presence of larvae belonging to T. pseudospiralis, a species more frequently reported in carnivorous birds, was observed. This study describes the first identification of T. pseudospiralis in a jackal. The results suggest that there is an urgent need to investigate which species of mammals and/or birds act as reservoirs for this zoonotic nematode in Romania.

Identification of Trichinella taxa by ITS-1 amplicon next-generation sequencing with an improved resolution for detecting underrepresented genotypes in mixed natural infections

BackgroundAmplicon-based next-generation sequencing (NGS) has rapidly gained popularity as a powerful method for delineating taxa in complex communities, including helminths. Here, we applied this approach to identify species and genotypes of zoonotic nematodes of the Trichinella genus. A known limitation of the current multiplex PCR (mPCR) assay recommended by the International Commission on Trichinellosis is that it does not differentiate Trichinella nativa from T. chanchalensis.MethodsThe new assay entails deep sequencing of an amplified variable fragment of the ribosomal cistron's (rDNA) internal transcribed spacer 1 using the Illumina platform. The assay was evaluated using first-stage larvae (L1) of select laboratory strains of various Trichinella taxa mixed in known proportions and then validated using archived L1 from 109 wildlife hosts. The species/genotypes of these L1 isolates from wildlife were previously determined using mPCR.ResultsNGS data analysis for Trichinella laboratory strains selected as representative of North American fauna revealed a sequence representation bias. Trichinella pseudospiralis, a non-encapsulated species, was the most underrepresented when mixed with T. spiralis, T. murrelli, T. nativa and Trichinella T6 in equal quantities. However, five L1 of T. pseudospiralis were readily revealed by NGS in a mix with 2000 L1 of T. nativa (1:400 ratio). From naturally infected wildlife, all Trichinella taxa revealed by mPCR were also identified by NGS in 103 of 107 (96.3%) samples amplified on both assays. NGS identified additional taxa in 11 (10.3%) samples, whereas additional taxa were revealed by mPCR in only four (3.7%) samples. Most isolates comprised single or mixed infections of T. nativa and Trichinella T6. On NGS, T. chanchalensis (T13) was detected in combination with Trichinella T6 in a wolverine (Gulo gulo) and in combination with T. nativa and Trichinella T6 in a marten (Martes americana) from the Northwest Territories, Canada.ConclusionsThis new NGS assay demonstrates strong potential as a single assay for identifying all recognised Trichinella taxa as well as improved sensitivity for detecting under-represented and novel genotypes in mixed infections. In addition, we report a new host record for T. chanchalensis in American marten.Graphical

Phylogenomic Analysis of the Phylum Nematoda: Conflicts and Congruences With Morphology, 18S rRNA, and Mitogenomes

Phylogenetic relationships within many lineages of the phylum Nematoda remain unresolved, despite numerous morphology-based and molecular analyses. We performed several phylogenomic analyses using 286 published genomes and transcriptomes and 19 new transcriptomes by focusing on Trichinellida, Spirurina, Rhabditina, and Tylenchina separately, and by analyzing a selection of species from the whole phylum Nematoda. The phylogeny of Trichinellida supported the division ofTrichinellainto encapsulated and non-encapsulated species and placed them as sister toTrichuris. The Spirurina subtree supported the clades formed by species from Ascaridomorpha and Spiruromorpha respectively, but did not support Dracunculoidea. The analysis of Tylenchina supported a clade that included all sampled species from Tylenchomorpha and placed it as sister to clades that included sampled species from Cephalobomorpha and Panagrolaimomorpha, supporting the hypothesis that postulates the single origin of the stomatostylet. The Rhabditina subtree placed a clade composed of all sampled species from Diplogastridae as sister to a lineage consisting of paraphyletic Rhabditidae, a single representative of Heterorhabditidae and a clade composed of sampled species belonging to Strongylida. It also strongly supported all suborders within Strongylida. In the phylum-wide analysis, a clade composed of all sampled species belonging to Enoplia were consistently placed as sister to Dorylaimia + Chromadoria. The topology of the Nematoda backbone was consistent with previous studies, including polyphyletic placement of sampled representatives of Monhysterida and Araeolaimida.

Reprint of: Gamma radiation effect on Trichinella pseudospiralis and Trichinella spiralis infected wild boar meat

Trichinellosis is a foodborne parasitic disease caused by Trichinella spp. Different methods, such as cooking, freezing and irradiation, have been suggested to inactivate the parasite in meat infected with Trichinella spp. The International Commission on Trichinellosis (ICT) recommends an irradiation dose of 0.3 kGy to inactivate Trichinella spp. larvae, but its effectiveness in all Trichinella species has not yet been demonstrated. Therefore, the objective of the present study was to determine the effectiveness of gamma radiation in treating wild boar meat to inactivate Trichinella pseudospiralis and Trichinella spiralis. Two wild boars (Sus scrofa) were each inoculated per os (PO) with 20,000 muscle larvae (ML) of Trichinella. One wild boar was inoculated with T. pseudospiralis and the other one with T. spiralis. Both wild boars were euthanized 20 weeks post infection (pi). A 250░g sample from each selected muscle type (side ribs, shoulder and upper forelegs) was obtained in duplicate from each animal. One group of samples was not irradiated (Gc; n░=░6) while the other group of samples was irradiated (Gt; n░=░6) at the Atomic Centre of the Argentinean National Commission of Atomic Energy (CNEA) with a minimum and maximum dose of 0.32–0.41 kGy. Twenty gram of each muscle from Gt and Gc were taken at 24░h, 7 days, 14 days and 21 days post-irradiation, and all samples were individually processed by means of artificial digestion. The ML were then inoculated into mice to evaluate the intestinal phase and the muscular phase. All recovered larvae from Gt and Gc samples showed integrity of the cuticle and active motility. Adult worms and ML were recovered from all mice inoculated with ML from Gc. However, no adult worms or ML were obtained in mice inoculated with ML from Gt.These results show the efficacy of irradiation to inhibit the development of T. pseudospiralis and T. spiralis in the host, which could possibly be extended to other non-encapsulated species, but further studies are needed to demonstrate this hypothesis.

First report of Trichinella pseudospiralis in a wolf (Canis lupus italicus).

Within the genus Trichinella, Trichinella pseudospiralis is the only recognized non-encapsulated species known to infect mammals and birds. In October 2020, larvae recovered from muscle tissues of a wolf (Canis lupus italicus) originating from Molise Region, Central Italy, were molecularly confirmed as those of Trichinella britovi and T. pseudospiralis. This is the first detection of T. pseudospiralis from a wolf. In Italy, this zoonotic nematode was detected in a red fox (Vulpes vulpes), three birds (Strix aluco, Athene noctua, Milvus milvus) and five wild boars (Sus scrofa), and was also identified as the etiological agent of a human outbreak of trichinellosis in 2015. Since T. pseudospiralis is rarely reported from carnivore mammals in comparison to the encapsulated species frequently detected in these hosts, this finding opens the question of the role of carnivores as reservoirs for this parasite.

Trichinella pseudospiralis in a red kite (Milvus milvus) from Italy.

Trichinella pseudospiralis is a non-encapsulated species infecting both mammals and birds. In Italy, this species has been reported so far only in central regions (two nocturnal birds of prey, one red fox, and one wild boar) and in northeast regions (four wild boars). In November 2020, Trichinella sp. larvae were isolated by enzymatic digestion from muscle tissues of a red kite (Milvus milvus) specimen belonging to a population residing in the Basilicata region (Southern Italy). The parasite was identified as T. pseudospiralis by multiplex PCR, and the sequencing of the expansion segment V (ESV) region of the nuclear large subunit ribosomal DNA showed, in the microsatellite region, the polymorphism characteristic of the Palearctic population. This represents the first record of T. pseudospiralis in a red kite and the first report of this parasite in Southern Italy. The isolation of the parasite in a resident bird confirms that T. pseudospiralis is present, even if at low prevalence, in the Italian avifauna.

Gamma radiation effect on Trichinella pseudospiralis and Trichinella spiralis infected wild boar meat

Trichinellosis is a foodborne parasitic disease caused by Trichinella spp. Different methods, such as cooking, freezing and irradiation, have been suggested to inactivate the parasite in meat infected with Trichinella spp. The International Commission on Trichinellosis (ICT) recommends an irradiation dose of 0.3 kGy to inactivate Trichinella spp. larvae, but its effectiveness in all Trichinella species has not yet been demonstrated. Therefore, the objective of the present study was to determine the effectiveness of gamma radiation in treating wild boar meat to inactivate Trichinella pseudospiralis and Trichinella spiralis. Two wild boars (Sus scrofa) were each inoculated per os (PO) with 20,000 muscle larvae (ML) of Trichinella. One wild boar was inoculated with T. pseudospiralis and the other one with T. spiralis. Both wild boars were euthanized 20 weeks post infection (pi). A 250 g sample from each selected muscle type (side ribs, shoulder and upper forelegs) was obtained in duplicate from each animal. One group of samples was not irradiated (Gc; n = 6) while the other group of samples was irradiated (Gt; n = 6) at the Atomic Centre of the Argentinean National Commission of Atomic Energy (CNEA) with a minimum and maximum dose of 0.32 – 0.41 kGy. Twenty gram of each muscle from Gt and Gc were taken at 24 h, 7 days, 14 days and 21 days post-irradiation, and all samples were individually processed by means of artificial digestion. The ML were then inoculated into mice to evaluate the intestinal phase and the muscular phase. All recovered larvae from Gt and Gc samples showed integrity of the cuticle and active motility. Adult worms and ML were recovered from all mice inoculated with ML from Gc. However, no adult worms or ML were obtained in mice inoculated with ML from Gt.These results show the efficacy of irradiation to inhibit the development of T. pseudospiralis and T. spiralis in the host, which could possibly be extended to other non-encapsulated species, but further studies are needed to demonstrate this hypothesis.

Trichinella pseudospiralis in a wolverine (Gulo gulo) from the Canadian North.

Species of Trichinella are a globally distributed assemblage of nematodes, often with distinct host ranges, which include people, domestic, and wild animals. Trichinella spp. are important in northern Canada, where dietary habits of people and methods of meat preparation (drying, smoking, fermenting as well as raw) increase the risk posed by these foodborne zoonotic parasites. Outbreaks in the arctic and subarctic regions of Canada and the United States are generally attributed to T. nativa (T2) or the T6 genotype, when genetic characterization is performed. We report the discovery of Trichinella pseudospiralis (T4), a non-encapsulated species, in a wolverine (Gulo gulo) from the Northwest Territories of Canada. This parasite has been previously reported elsewhere from both mammals and carnivorous birds, but our findings represent new host and geographic records for T. pseudospiralis. Multiplex PCR and sequencing of fragments of Cytochrome Oxidase Subunit I (COI) and D3 rDNA confirmed the identification. Phylogenetically, Canadian isolates linked with each other and others derived from Palearctic or Neotropical regions, but not elsewhere in the Nearctic (continental USA). We suggest that migratory birds might have played a role in the dispersal of this pathogen 1000's of km to northwestern Canada. Wolverines are not typically consumed by humans, and thus should not pose a direct food safety risk for trichinellosis. However, the current finding suggests that they may serve as an indicator of a broader distribution for T. pseudospiralis. Along with infection risk already recognized for T. nativa and Trichinella T6, our observations emphasize the need for further studies using molecular diagnostics and alternative methods to clarify if this is a solitary case, or if T. pseudospiralis and other freeze susceptible species of Trichinella (such as T. spiralis) circulate more broadly in wildlife in Canada, and elsewhere.

Immuno-proteomic analysis of Trichinella spiralis, T. pseudospiralis, and T. papuae extracts recognized by human T. spiralis-infected sera.

The present study explored potentially immunogenic proteins of the encapsulated (Trichinella spiralis) and non-encapsulated (T. pseudospiralis, T. papuae) species within the genus Trichinella. The somatic muscle larval extracts of each species were subjected to immunoblotting analysis using human T. spiralis-infected serum samples. Fifteen reactive bands of all three species were selected for further protein identification by liquid chromatography-tandem mass spectrometry, and their possible functions were ascertained using the gene ontology. Our findings showed immunogenic protein patterns with molecular mass in the range of 33-67kDa. Proteomic and bioinformatic analysis revealed a wide variety of functions of 17 identified proteins, which are associated with catalytic, binding, and structural activities. Most proteins were involved in cellular and metabolic processes that contribute in the invasion of host tissues and the larval molting processes. The parasite proteins were identified as actin-5C, serine protease, deoxyribonuclease-2, and intermediate filament protein ifa-1. This information may lead to alternative tools for selection of potential diagnostic protein markers or aid in the design of vaccine candidates for prevention and control of Trichinella infection.

First finding of Trichinella pseudospiralis in the Neotropical region

Prior to this study, only encapsulated species of Trichinella had been found in South America, i.e., T. spiralis and T. patagoniensis. Here we report the molecular identification of a non-encapsulated isolate of Trichinella from a domestic pig in Argentina. The multiplex PCR technique and the analysis of mitochondrial and nuclear DNA sequences revealed that it belongs to T. pseudospiralis, which parasitises birds and mammals from Australian, Nearctic, and Palaearctic regions. Interestingly, the isolate is closely related to the Palaearctic population. This is the first report of a non-encapsulated species of Trichinella from the Neotropical region.

First reports of Trichinella pseudospiralis in wild boars ( Sus scrofa) of Italy

Trichinella pseudospiralis is a non-encapsulated species infecting both mammals and birds. In Italy, this parasite was reported only in two night-birds of prey of Central Italy. In January 2010, Trichinella larvae were detected in three wild boars ( Sus scrofa) of two regions of Northern Italy by enzymatic digestion. The parasites were identified as T. pseudospiralis by multiplex-PCR. The first infected wild boar was hunted in the Emilia Romagna region and the other two infected wild boars were bred outdoors in a small family farm of the Friuli Venezia Giulia region. These new epidemiological data reinforce the role of the wild boar as the main reservoir of T. pseudospiralis in Europe.

Infection of non-encapsulated species of Trichinella ameliorates experimental autoimmune encephalomyelitis involving suppression of Th17 and Th1 response

Epidemiological and experimental studies have indicated that helminth infections can ameliorate autoimmune diseases. The present study investigated the amelioration effect of the Trichinella pseudospiralis infection on experimental autoimmune encephalomyelitis (EAE), a T-cell-mediated autoimmune disease of central nervous system (CNS), and expression kinetics of Th17 and Th1 cytokine which play a crucial role in the pathogenesis of EAE. The results indicated that the infection of helminth T. pseudospiralis obviously ameliorated clinical severity and greatly delayed the onset of EAE induced by myelin oligodendrocyte glycoprotein (MOG) immunization. Infection caused much lesser inflammatory infiltration and demyilination in the CNS of infected EAE mice than uninfected EAE mice. The reduced infiltration was also suggested by the expressions of the inflammation cytokines, IL-17, IL-6, IL-1β, IFN-γ, and TNF-α, which were high in the spinal cords of the uninfected EAE mice, but was nearly normal or low in the infected EAE mice. The increased production of MOG-induced IL-17 and IFN-γ and the expression of IL-6, IL-1β, TGF-β in splenocytes after restimulation with MOG was inhibited in the infected EAE mice. On the other hand, the greatly induced Th2 response was observed in the splenocytes of the infected EAE mice. The present study showed that T. pseudospiralis infection can suppresses EAE by reducing the inflammatory infiltration in CNS, likely associated with the suppression of Th17 and Th1 responses by the infection.

Trichinella: Differential expression of angiogenic factors in macrophages stimulated with antigens from encapsulated and non-encapsulated species

The newborn larval stage of Trichinella spiralis enters the host striated skeletal muscle cell resulting in the formation of the nurse cell. Vascular Endothelial Growth Factor (VEGF) was detected in cells in the area immediately surrounding the nurse cells. However, no data are available on the antigens involved, the role of other angiogenic factors or the relationship of angiogenesis with Nitric Oxide (NO) production. Using macrophage cell culture we study the effect of different Trichinella L1 antigens from one encapsulated ( T. spiralis) and one non-encapsulated ( Trichinella pseudospiralis) on the expression of VEGF and basic Fibroblast Growth Factor (FGF2). Also, we investigate the relationship between the production of NO and angiogenic mediators. The results show that encapsulated and non-encapsulated Trichinella species are different in their capacity to stimulate the expression of VEGF and FGF2 from host macrophages. Finally, there is no relationship between angiogenic factors and NO production by T. spiralis antigen.

Evaluation of inflammatory responses against muscle larvae of different Trichinella species by an image analysis system

The aim of this study was to evaluate the inflammatory response in the muscle tissue against Trichinella larvae of encapsulated (T. spiralis, T. britovi) and non-encapsulated (T. pseudospiralis) species. The inflammatory response was estimated in histological sections of muscle tissues from Trichinella-infected CD1 mice by a newly developed method. Nuclei were stained with one fluorescent probe, which binds nucleic acids with high affinity, and fluorescence was analysed by a software program. Evaluation of the relative fluorescence units was performed in both peri-capsular (close to the nurse cell–parasite complex) and extra-capsular (where the parasite was not visible) areas. The increase in the number of nuclei in the muscle tissues of Trichinella-infected mice was considered an inflammation marker, since uninfected muscles show low nucleus density. In order to evaluate differences in the nitrosylation pattern between encapsulated (T. spiralis, T. britovi) and non-encapsulated (T. pseudospiralis, Trichinella papuae, Trichinella zimbabwensis) species, L1 larvae were tested by immunoblotting with an anti-nitrotyrosine polyclonal antibody. Inflammation induced by T. spiralis larvae in muscle tissues is statistically higher than that elicited by the other species, both in peri- and extra-capsular areas. Nitrosylation occurs at a higher level in encapsulated than in non-encapsulated species. The method developed in this work allows demonstration of differences in the host inflammatory response against encapsulated and non-encapsulated Trichinella species.

Survival of Trichinella papuae muscle larvae in a pig carcass maintained under simulated natural conditions in Papua New Guinea

In Papua New Guinea, Trichinella papuae, a non-encapsulated species, is circulating among wild and domestic pigs and saltwater crocodiles. Since an important phase of the life cycle of nematodes of the genus Trichinella is the time of survival of infective larvae in decaying muscle tissues of the hosts, the carcass of a pig, experimentally infected with larvae of T. papuae, was exposed to the environmental conditions of Papua New Guinea to establish how long these larvae would survive and remain infective to a new host. Larvae retained their infectivity in the pig carcass up to 9 days after slaughtering, during which time the temperature within the carcass reached 35.0 degrees C on 2 days; the average relative humidity was 79.0%. A low number of larvae survived up to day 14 after the pig was killed, when the carcass temperature reached 38.0 degrees C, but they lost their infectivity to laboratory mice. This result suggests that the larvae of T. papuae can survive in a tropical environment for a time, favouring their transmission to a new host in spite of the lack of a collagen capsule.

Genetic relationships among Trichinella pseudospiralis isolates from Australian, Nearctic, and Palearctic regions

The study of the genetic polymorphism of pathogens is important for phylogenetic and biogeographic studies and, in the case of foodborne pathogens, to trace the origin of food infection. Since its discovery in 1972, the nonencapsulated species Trichinella pseudospiralis has been detected in mammals and birds, and human infection has occurred, in some cases resulting in death. We studied DNA polymorphism among ten T. pseudospiralis isolates from the Palearctic, Nearctic, and Australian regions, screening the sequences of nine genes [18sRNA, a random amplified polymorphism DNA derived sequence, mitochondrial cytochrome oxidase subunit I (COI), cytochrome P450, cynate lyase, epithelial fusion failure-1, and three unknown genes of Tp3, Tp8, and Tp26]. A high identity of sequence for the nine gene loci was obtained among the seven isolates from the Palearctic region and between the two isolates from the Nearctic region. Genetic identity analysis indicated the distinct polymorphism among the three geographical origins. To easily identify T. pseudospiralis genotypes, a polymerase chain reaction-restriction fragment length polymorphism analysis of COI gene was performed, and the results confirmed the DNA polymorphism within T. pseudospiralis, corresponding to the three regions of origin. We have named the three genotypes as "T. pseudospiralis Palearctic genotype" (code T4P), "Nearctic genotype" (code T4N), and "Australian genotype" (code T4A). To further investigate polymorphism among the nonencapsulated Trichinella species, the sequences of four gene loci (COI, P450, cynate lyase, and SB147D) of T. pseudospiralis, T. papuae, and T. zimbabwensis were analyzed, and the results showed high polymorphism among the three species, strongly supporting their classification as separate species.

Human Trichinellosis From Laos

There are biblical references to an illness that could have been trichinellosis and which was associated with consumption of pork, but the nematode Trichinella spiralis was first described in 1835. The genus Trichenella contains at least seven species: T spiralis , Trichenella nativa , Trichenella britovi , Trichenella pseudospiralis , Trichenella murrelli , Trichenella nelsoni , and Trichenella papuae. 1 The geographical distribution of the different species is related to ecology, climate, and host behavior.2 The host range of Trichinella is wider than previously believed. New species are being continuously recognized. Three classes of vertebrates are known to act as hosts (ie, mammals, birds, and reptiles). Mammals represent the most important hosts, and all Trichinella species are able to develop in this class. The nonencapsulated species T pseudospiralis , detected in both mammals and birds, show a cosmopolitan distribution. They tend to have a more prolonged clinical course. Three cases of trichinellosis, after a saltwater turtle dinner, were reported from Songkhla, Thailand, 14 years ago. The species of the turtle and of the Trichinella were not identified. Two additional nonencapsulated species, T papuae , detected in wild pigs and saltwater crocodiles of Papua, New Guinea, and Trichenella zimbabwensis , found in farmed Nile crocodiles and in sylvatic monitor lizards of Zimbabwe, have been reported.3,4 The life cycle of Trichinella is completed when first‐stage larvae mature in striated muscle. Such larvae are immunogenic, as evidenced by sustained antibody production against larval glycans. In contrast to a vigorous systemic immune response, local inflammation is limited. Nonencapsulating species of Trichinella such as T pseudospiralis induce little to no local inflammatory responses in reptilian, avian, and mammalian hosts. They also appear to remain viable longer. Encapsulated species induce limited local inflammation in a variety of mammalian hosts, which contributes to isolation and …

Trichinella: Differing effects of antigens from encapsulated and non-encapsulated species on in vitro nitric oxide production

Trichinellosis is a cosmopolitan zoonotic disease affecting a wide variety of animals, including man. Non-encapsulated and encapsulated species diverge with respect to their developmental strategies. Little is known at the molecular level about parasite-derived mediators responsible for host muscle cell transformation occurring during trichinellosis. In this context, host–parasite relationships in Trichinella-infected animals could be related to different host-immune and cell mediators, e.g. nitric oxide (NO). Here, we investigate the stimulatory/inhibitory role of L1 antigens from four encapsulated ( T. spiralis, T. britovi, T. nelsoni and T. nativa) and one non-encapsulated ( T. pseudospiralis) Trichinella species on NO production from rat macrophages in vitro. Our results demonstrate that encapsulated and non-encapsulated Trichinella species differ in their capacity to stimulate the secretion of NO from host macrophages. Biological significance of these differences should be further assessed in the available experimental models.

MOLECULAR CHARACTERIZATION OF TRICHINELLA GENOTYPES BY INTER-SIMPLE SEQUENCE REPEAT POLYMERASE CHAIN REACTION (ISSR-PCR)

A bulk analysis of inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) provides a quick, reliable, and highly informative system for DNA banding patterns that permit species identification. The present study evaluates the applicability of this system to Trichinella species identification. After a single amplification carried out on a single larva with the primer 816([CA]nRY) under high stringency conditions, which provide high reproducibility, we were able to identify by consistent banding patterns 5 sibling species: Trichinella spiralis (ISS48), 2 Trichinella britovi isolates (ISS11 and ISS86), Trichinella murrelli (ISS35), Trichinella nativa (ISS71), Trichinella nelsoni (ISS29); 3 additional Trichinella genotypes: T8 (ISS149), T9 (ISS408 and ISS409), and T6 (ISS34); and the nonencapsulated species Trichinella pseudospiralis (ISS13). Moreover, 33 new Trichinella isolates from 2 zoogeographical regions were unequivocally identified. All Trichinella isolates have shown an identical pattern with those produced by the reference strain. According to these data, we have demonstrated that ISSR-PCR is a robust technique that emerges as a useful new application for the molecular identification of Trichinella isolates in epidemiological studies.

Trichinella spiralis and Trichinella pseudospiralis mixed infection in a wild boar ( Sus scrofa) of Germany

A wild boar ( Sus scrofa) from the island Usedom in Mecklenburg-Western Pomerania (north-east Germany) was detected as Trichinella-positive during routine meat inspection. Encapsulated and non-encapsulated larvae were detected in the muscle tissue by trichinoscopy. In the diaphragm, 922 larvae per g were detected by artificial digestion. Muscle larvae displayed two different sizes of about 700 and 1100 μm. By a multiplex PCR analysis, larvae with a large size were identified as Trichinella spiralis, whereas those of a smaller size were identified as Trichinella pseudospiralis. This is the first finding of a mixed infection of T. spiralis and T. pseudospiralis in a naturally infected animal and it supports the tendency of more frequent detection of the non-encapsulated species T. pseudospiralis in Europe.