Outbreak of Human Trichinellosis - Arizona, Minnesota, and South Dakota, 2022.

Trichinellosis is a parasitic zoonotic disease transmitted through the consumption of meat from animals infected with Trichinella spp. nematodes. In North America, human trichinellosis is rare and is most commonly acquired through consumption of wild game meat. In July 2022, a hospitalized patient with suspected trichinellosis was reported to the Minnesota Department of Health. One week before symptom onset, the patient and eight other persons shared a meal that included bear meat that had been frozen for 45 days before being grilled and served rare with vegetables that had been cooked with the meat. Investigation identified six trichinellosis cases, including two in persons who consumed only the vegetables. Motile Trichinella larvae were found in remaining bear meat that had been frozen for >15 weeks. Molecular testing identified larvae from the bear meat as Trichinella nativa, a freeze-resistant species. Persons who consume meat from wild game animals should be aware that that adequate cooking is the only reliable way to kill Trichinella parasites and that infected meat can cross-contaminate other foods.

Quantitative label-free proteomic analysis of excretory-secretory proteins in different developmental stages of Trichinella spiralis.

Trichinella spiralis (T. spiralis) is a zoonotic parasitic nematode with a unique life cycle, as all developmental stages are contained within a single host. Excretory-secretory (ES) proteins are the main targets of the interactions between T. spiralis and the host at different stages of development and are essential for parasite survival. However, the ES protein profiles of T. spiralis at different developmental stages have not been characterized. The proteomes of ES proteins from different developmental stages, namely, muscle larvae (ML), intestinal infective larvae (IIL), preadult (PA) 6 h, PA 30 h, adult (Ad) 3 days post-infection (dpi) and Ad 6 dpi, were characterized via label-free mass spectrometry analysis in combination with bioinformatics. A total of 1217 proteins were identified from 9341 unique peptides in all developmental stages, 590 of which were quantified and differentially expressed. GO classification and KEGG pathway analysis revealed that these proteins were important for the growth of the larvae and involved in energy metabolism. Moreover, the heat shock cognate 71 kDa protein was the centre of protein interactions at different developmental stages. The results of this study provide comprehensive proteomic data on ES proteins and reveal that these ES proteins were differentially expressed at different developmental stages. Differential proteins are associated with parasite survival and the host immune response and may be potential early diagnostic antigen or antiparasitic vaccine candidates.

Diversity of Trichinella species in carnivores from Bosnia and Herzegovina.

BACKGROUND: In Bosnia and Herzegovina, domestic and wild carnivores represent a significant driver for the transmission and ecology of zoonotic pathogens, especially those of parasitic aetiology. Nevertheless, there is no systematic research of Trichinella species in animals that have been conducted in Bosnia and Herzegovina, even though trichinellosis is considered the most important parasitic zoonosis. The available results of the few studies carried out in Bosnia and Herzegovina are mainly related to the confirmation of parasitic larvae in the musculature of domestic pigs and wild boars or data related to trichinellosis in humans. The objective of our study was to present the findings of a comprehensive investigation into the species composition of Trichinella among 11 carnivorous species within the territory of Bosnia and Herzegovina, as follows: red fox (Vulpes vulpes), grey wolf (Canis lupus), brown bear (Ursus arctos), wildcat (Felis silvestris), pine marten (Martes martes), European badger (Meles meles), weasel (Mustela nivalis), European polecat (Mustela putorius), Eurasian lynx (Lynx lynx), but also dog (Canis lupus familiaris) and cat (Felis catus). RESULTS: In the period 2013-2023, carnivore musculature samples (n = 629), each consisting of 10 g of muscle tissue, were taken post-mortem and individually examined using the artificial digestion method. In the positive samples (n = 128), molecular genotyping and identification of parasitic larvae of Trichinella spp. were performed using a PCR-based technique up to the species/genotype level. Positive samples were used for basic PCR detection of the genus Trichinella (rrnS rt-PCR technique) and genotyping (rrnl-EVS rt-PCR technique). The Trichinella infection was documented for the first time in Bosnia and Herzegovina among red foxes, grey wolves, brown bears, dogs, badgers and Eurasian lynx, with a frequency rate of 20.3%. Additionally, the presence of T. britovi infection was newly confirmed in Bosnia and Herzegovina, marking the initial documented cases. Furthermore, both T. britovi and T. pseudospiralis infections were observed in the wildcat population, whereas T. britovi and T. spiralis infections were detected in pine martens. Consistent with previous research, our findings align particularly regarding carnivores, with data from other countries such as Germany, Finland, Romania, Poland and Spain, where T. britovi exhibits a wider distribution (62.5-100%) compared to T. spiralis (0.0-37.5%). T. britovi is more common among sylvatic carnivores (89.0%), while T. spiralis prevails in wild boars (62.0%), domestic swine (82.0%) and rodents (75.0%). CONCLUSION: The results of our study represent the first molecular identification of species of the genus Trichinella in Bosnia and Herzegovina. Additionally, our findings underscore the necessity for targeted epidemiological studies to thoroughly assess trichinellosis prevalence across diverse animal populations. Considering the relatively high frequency of trichinellosis infection in investigated animal species and its public health implications, there is an evident need for establishing an effective trichinellosis surveillance system in Bosnia and Herzegovina.

Enhanced Genomic and Transcriptomic Resources for Trichinella pseudospiralis and T. spiralis to Underpin the Discovery of Molecular Differences between Stages and Species.

Nematodes of the genus Trichinella are important pathogens of humans and animals. This study aimed to enhance the genomic and transcriptomic resources for T. pseudospiralis (non-encapsulated phenotype) and T. spiralis (encapsulated phenotype) and to explore transcriptional profiles. First, we improved the assemblies of the genomes of T. pseudospiralis (code ISS13) and T. spiralis (code ISS534), achieving genome sizes of 56.6 Mb (320 scaffolds, and an N50 of 1.02 Mb) and 63.5 Mb (568 scaffolds, and an N50 value of 0.44 Mb), respectively. Then, for each species, we produced RNA sequence data for three key developmental stages (first-stage muscle larvae [L1s], adults, and newborn larvae [NBLs]; three replicates for each stage), analysed differential transcription between stages, and explored enriched pathways and processes between species. Stage-specific upregulation was linked to cellular processes, metabolism, and host-parasite interactions, and pathway enrichment analysis showed distinctive biological processes and cellular localisations between species. Indeed, the secreted molecules calmodulin, calreticulin, and calsyntenin-with possible roles in modulating host immune responses and facilitating parasite survival-were unique to T. pseudospiralis and not detected in T. spiralis. These insights into the molecular mechanisms of Trichinella-host interactions might offer possible avenues for developing new interventions against trichinellosis.

Trichinella infection in Serbia from 2011 to 2020: a success story in the field of One Health.

In Serbia, modern pork production systems with implemented control measures, including the detection of Trichinella larvae in meat (ISO18743), have eliminated farmed pork from pigs slaughtered at abattoirs as a source of trichinellosis. Epidemiological data from 2011 to 2020 indicate that the number of human cases and the number of infected domestic pigs has decreased significantly. Over the years, pork was the most frequent source of human infection. Cases generally occurred in small family outbreaks, and the infection was linked to consumption of raw or undercooked pork from backyard pigs. In most of the outbreaks, T. spiralis was the aetiological agent of infection, but in 2016, a large outbreak was caused by consumption of uninspected wild boar meat containing T. britovi larvae. To achieve safe pork, it is important that consumers of pork from animals raised in backyard smallholdings and of wild game meat are properly educated about the risks associated with consumption of untested meat. Laboratories conducting Trichinella testing should have a functional quality assurance system to ensure competency of analysts and that accurate and repeatable results are achieved. Regular participation in proficiency testing is needed.

Establishment of a method for detecting Trichinella spiralis in ovine muscle tissues using real-time fluorescence quantitative PCR.

Trichinellosis is caused by Trichinella spiralis, a meat-borne zoonotic disease transmitted to humans through the consumption of infected undercooked or raw meat. Surveillance using safe and precise diagnostic tools to diagnose T. spiralis in sheep is needed to assess the incidence and probability of transmission from sheep to humans. In this study, we developed a real-time PCR assay to detect T. spiralis DNA in ovine muscle samples that can be used as an alternative surveillance tool to ensure food safety using newly designed primers. The assay is specific for the Scfld4 gene of Trichinella (T1) and enables the detection of larvae in ovine muscle tissue samples with high sensitivity and specificity. Trichuris ovis, Oesophagostomum dentatum, Haemonchus contortus, and Bunostomum trigonocephalum showed no nonspecific amplification. The assay could detect Trichinella DNA concentrations as low as 0.0026 ng/µL, equivalent to 0.0064 larvae, indicating a high sensitivity for T. spiralis detection. We used this real-time PCR to detect 73 ovine muscle samples from an ovine abattoir, and five samples tested positive via real-time PCR but negative via microscopy. This assay may provide a more specific and sensitive method for rapidly detecting Trichinella larvae in ovine muscle tissues.

Immunomodulatory in vitro effects of Trichinella cystatin-like protein on mouse splenocytes.

Trichinella parasites have developed specific mechanisms allowing successful completion of their life cycle. These mechanisms are in a great part involved in immunomodulation and studying them may provide a valuable insight into the functioning of the immune system. Trichinella products may be also used as potential therapeutic agents to treat immune diseases. This study investigates the immunomodulatory potential of recombinant multi cystatin-like protein (CLP) derived from T. britovi to determine whether CLP has anti-inflammatory properties in vitro. CLP is a highly antigenic glycoprotein present in Trichinella excetory-secretory (ES) products. AlphaFold structure prediction confirms that it consists of three type-two cystatin-like domains. Mouse splenocytes were stimulated in vitro with lipopolysaccharide (LPS) and co-stimulated with recombinant CLP. The culture supernatants were collected and tested for secreted cytokine levels using ELISA. CLP was found to reduce LPS-induced secretion of inflammatory cytokines TNFα and IL-6. On the contrary, in some experimental groups, co-stimulation with CLP resulted in increased secretion of the regulatory cytokine IL-10. The obtained results indicate that CLP has anti-inflammatory properties and future research on its function is advisable, specifically in the context of the therapy of inflammatory disorders.

Epidemiology and risk factors of trichinellosis in Bosnia and Herzegovina from 1961 to 2021.

This study is a retrospective analysis of social, economic, political, and cultural events on the annual incidence of trichinellosis in Bosnia and Herzegovina (BiH). It highlights the importance of specific regulations, which have been incorporated into developing preventive protection against this human disease for the last 61 years since it has been monitored by law. In the period, 1961-2021, 3828 people became infected. The first outbreak of trichinellosis was documented in 1965, and the incidence peaked during the war and post-war period at the end of the twentieth century. At that time, outbreaks with hundreds of cases and five deaths were recorded. In the first decade of the twenty-first century, the incidence decreased, and at the end of the second decade, two consecutive years without trichinellosis cases were finally registered. The analysis shows that a significantly higher incidence rate was recorded between the two political entities of BiH, the Republic of Srpska (4.97% 000) versus the Federation of BiH (0.9% 000) and that the occurrence of trichinellosis in humans in BiH is related to the presence of Trichinella spp. in animals, of a degree of biosecurity on pig farms, population's awareness of safe food, the impact of tradition and religion on eating habits, doctors' awareness of the symptomatology, and the Trichinella spp. larva detection method in muscle samples. The spread risk of trichinellosis requires meat inspection for Trichinella spp. larvae to be mandatory in BiH.

Expression of Trichinella spiralis serpin Tsp_01570 in Pichia pastoris: a first insight of its biomodulatory activity.

Serpins represent one of the most diverse families of serine protease inhibitors. Despite their complexity, they are virtually found in all organisms and play an important role in homeostasis processes such as blood coagulation, inflammation, fibrinolysis, immune responses, chromatin condensation, tumor suppression, and apoptosis. There has recently been particular interest in studying serpin functions in infection and inflammation, especially since more serpins from parasites have been identified and characterized. Among helminths, Trichinella spiralis is one of the few parasites with an extremely strong ability to induce host immune suppression. Previous studies show that serpins are present in Trichinella and are expressed differentially at different parasite stages. More interesting, there is evidence of a recombinant serpin from Trichinella pseudospiralis that alters macrophage polarization in vitro. This finding could be relevant to comprehend the modulation process of the immune response. We expressed Tsp_01570, a putative serpin gene from Trichinella spiralis, in the eukaryotic system Pichia pastoris SMD1168H and evaluated its presence at different parasite stages, finding the serine protease inhibitor in the crude extract of adult worms. The effect of recombinant serpin on THP-1 cells was tested by quantification of IL-12p40, TNF-α, IL-4, and IL-10 cytokines released by ELISA. We also evaluated the expression of the M1 markers, CCR7 and CD86, and the M2 markers, CD163 and CD206, by immunofluorescence staining. This study represents the first insight in elucidating the importance of serpin Tsp_01570 as a potential molecular modulator.

Metabolomics (Non-Targeted) of Induced Type 2 Diabetic Sprague Dawley Rats Comorbid with a Tissue-Dwelling Nematode Parasite.

Type 2 diabetes is a non-communicable metabolic syndrome that is characterized by the dysfunction of pancreatic ß-cells and insulin resistance. Both animal and human studies have been conducted, demonstrating that helminth infections are associated with a decreased prevalence of type 2 diabetes mellitus (T2DM). However, there is a paucity of information on the impact that helminths have on the metabolome of the host and how the infection ameliorates T2DM or its progression. Therefore, this study aimed at using a non-targeted metabolomics approach to systematically identify differentiating metabolites from serum samples of T2DM-induced Sprague Dawley (SD) rats infected with a tissue-dwelling nematode, Trichinella zimbabwensis, and determine the metabolic pathways impacted during comorbidity. Forty-five male SD rats with a body weight between 160 g and 180 g were used, and these were randomly selected into control (non-diabetic and not infected with T. zimbabwensis) (n = 15) and T2DM rats infected with T. zimbabwensis (TzDM) (n = 30). The results showed metabolic separation between the two groups, where d-mannitol, d-fructose, and glucose were upregulated in the TzDM group, when compared to the control group. L-tyrosine, glycine, diglycerol, L-lysine, and L-hydroxyproline were downregulated in the TzDM group when compared to the control group. Metabolic pathways which were highly impacted in the TzDM group include biotin metabolism, carnitine synthesis, and lactose degradation. We conclude from our study that infecting T2DM rats with a tissue-dwelling nematode, T. zimbabwensis, causes a shift in the metabolome, causing changes in different metabolic pathways. Additionally, the infection showed the potential to regulate or improve diabetes complications by causing a decrease in the amino acid concentration that results in metabolic syndrome.

Laboratory Features of Trichinellosis and Eosinophilia Threshold for Testing, Nunavik, Quebec, Canada, 2009-2019.

Prolonged eosinophilia is characteristic of trichinellosis. To determine the optimal eosinophil threshold for reflex Trichinella testing, we examined all 43 cases in Nunavik, Quebec, Canada, during 2009-2019. Using receiver operating characteristic analysis, we determined that eosinophil counts >0.8 × 109 cells/L should prompt consideration of trichinellosis and testing to rapidly identify potential outbreaks.

Trichinella britovi recombinant proteins produced in Pichia pastoris expression system for specific IgG antibody detection in the sera of mice and pigs infected with Trichinella spp.

Trichinellosis, a disease caused by infection with Trichinella spp, poses an economic problem in the animal sector and a recurrent health problem for humans. Discovering the new diagnostic tests may be achieved by identification and production of species- and stage-specific recombinant proteins of Trichinella genus which are recognized by the host antibodies after infection. In this study the T. britovi proteins identified earlier in excretory-secretory (ES) products: CTRL, ES21 and HSP20, were cloned and produced using a eukaryotic Pichia pastoris system. Their immunodiagnostic properties were verified by measuring the abundance of specific IgG antibodies in sera from mice and pigs experimentally infected with T. britovi or T. spiralis. The rTbCTRL and the rTbES21 proteins were more effectively produced and stable than rTbHSP20. The most sensitive protein for serodiagnostic purposes occurred to be CTRL; anti-rTbCTRL IgG level increased at 41 days post infection (dpi) in pigs infected with T. britovi and 45 dpi for those infected with T. spiralis. The rTbES21 protein was the most specific for the T. britovi species, as no antibody titers were observed in pigs infected with T. spiralis. Following the multiple-antigen strategy, the combination of rTbCTRL + rTbES21 was applied in ELISA, but no significant difference in IgG level was detected in the tested conditions.

Trichinella Infection Ameliorated Vincristine-Induced Neuroinflammation in Mice.

Vincristine (VCR) is a chemotherapeutic agent widely used in treatment of malignancies. However, VCR has a limitation in use since it commonly causes a painful neuropathy (VCR-induced peripheral neuropathy, VIPN). Inflammatory cytokines secreted by immune cells such as macrophages can exacerbate allodynia and hyperalgesia, because inhibiting the inflammatory response is a treatment target for VIPN. In this study, we investigated whether Trichinella spiralis, a widely studied helminth for its immunomodulatory abilities, can alleviate VCR-induced allodynia. Von Frey test showed that T. spiralis infection improved mechanical allodynia at 10 days after VCR injection. We further observed whether the difference was due to mitigated axon degeneration, but no significant difference between the groups in axonal degeneration in sciatic nerves and intra-epidermal nerve fibers was found. Conversely, we observed that number of infiltrated macrophages was decreased in the sciatic nerves of the T. spiralis infected mice. Moreover, treatment of T. spiralis excretory-secretory products caused peritoneal macrophages to secrete decreased level of IL-1ß. This study suggests that T. spiralis can relieve VCR-induced mechanical allodynia by suppressing neuroinflammation and that application of controllable degree of helminth may prove beneficial for VIPN treatment.

Long lasting immunity in trichinellosis - insight from a small study group.

Trichinellosis in humans is most often caused by the parasite Trichinella spiralis. The clinical course of the disease is diverse and the symptoms can vary from mild to severe. Symptoms usually disappear within a few months, but encysted larvae in the muscles can cause myalgia and weakness that last for years. However, the existence of chronic trichinellosis as a disease is still debatable. This study presents the results obtained at the National Reference Laboratory for Trichinellosis - INEP, Serbia. The study was conducted to assess the immunoserological, biochemical and symptomatic disease parameters of twelve patients who acquired trichinellosis 13 and 18 years ago, respectively. They were involved in two T. spiralis outbreaks in Serbia, at the village of Kumane and the city of Belgrade (nine and three patients, respectively). Results indicated the presence of specific anti-Trichinella antibodies in 83% of the total number of patients. However, while the humoral immune response to Trichinella lasted for more than a decade reaching almost two decades after the acute infection phase (7/9 and 3/3 respectively, in two outbreaks), persistence of chronic muscular pain, as the most prolonged symptom of trichinellosis, could be found in the majority of patients from the Kumane outbreak (7/9). As a consequence, these patients suffered from limitations in daily living activities for the same period of time. The results presented in this paper are our contribution to the view that trichinellosis as a chronic disease with symptoms exists and may be related to the severity of the disease in the acute phase.

Two trichinellosis outbreaks in Serbia - challenging diagnosis due to a potential co-infection with Toxocara spp.

In Serbia, in most cases, small family outbreaks of trichinellosis occur due to the consumption of untested infected meat from domestic pigs that are raised and slaughtered in the backyards of small individual farms. The aim of this study is to present data regarding 24 patients involved in two outbreaks that occurred in two neighbouring districts in Serbia during a closely related period of time in August 2014. The source of infection in the first outbreak was undercooked pork, while raw pork sausages caused the second outbreak. Meat samples and sausages were Trichinella spp. positive by artificial digestion. With the aim to obtain a second opinion and validate the serological findings discovered at the Public Health Institute Nis, all samples were sent to the National Reference Laboratory for Trichinellosis, INEP. Serodiagnosis showed that 21 persons were positive (87.5%) and three (12.5%) were negative for anti-Trichinella antibodies, while 15 patients fulfilled the trichinellosis case definition. Western blot analysis (using an epitope unique for the muscle larvae stage of the Trichinella genus) confirmed the diagnosis of trichinellosis in five patients. Six patients also had specific antibodies against Toxocara canis (T. canis). Due to the fact that in endemic foci in Serbia there is the presence of Trichinella spiralis and T. canis and that these two infections could be asymptomatic, we consider that trichinellosis cases were irrefutably proven. The dilemma about the existence of co-infection with T. canis remained open due to the lack of clinical findings.

Efficacy of an aerosol-resistant pepsin powder used in artificial digestion for the detection of Trichinella larvae in meat.

Trichinellosis is an important worldwide foodborne zoonosis. The gold standard test to detect Trichinella spp. larvae in muscle samples of animals intended for human consumption is the artificial digestion method. Handling and dispensing of conventional pepsin powder present significant safety risks for analysts. The use of pepsin powder that is resistant to aerosolization should alleviate these safety concerns. The aim of this study was to compare the efficacy of an aerosol-resistant pepsin powder to conventional pepsin powder in the artificial digestion method. Proficiency samples of pork diaphragm containing specific numbers of viable Trichinella spiralis larvae were tested in two laboratories. The results revealed that aerosol-resistant pepsin was simple, effective and convenient to use, and showed good solubility and larval recovery that met the requirements of the European Union regulation EU 2015/1375. Overall, the efficacy of the aerosol-resistant pepsin was comparable to the conventional pepsin and safer for analysts.

Inactivation of Trichinella muscle larvae at different time-temperature heating profiles simulating home-cooking.

BACKGROUND: Trichinellosis is caused by consumption of raw or undercooked meat containing infective Trichinella muscle larvae (ML). Only few studies on heat-inactivation of Trichinella ML are available in literature and more validated data concerning heat inactivation is needed to improve the risk estimation. OBJECTIVE AND METHODS: The aim of the present study was to evaluate the two in vitro methods "staining" and "morphological examination" as proxies for Trichinella ML heat inactivation in comparison with the mouse bioassay method to get more insight in the relationship between heat, heating time and inactivation of Trichinella ML. The second aim was to evaluate whether these methods could replace the bioassay in the light of ongoing animal use reduction in lifescience research. Tubes containing quantified live Trichinella ML were exposed to heat profiles ranging from 40 to 80 °C. Subsequently, inactivation was evaluated using both methylene blue staining and morphological examination, which was validated by bioassay. Results were used to model Trichinella inactivation. RESULTS: Trichinella muscle larvae exposed to 60 °C or higher for 12-12.5 min were not infective to mice. We found that morphological examination was more consistent with the bioassay than methylene blue staining. Modelled inactivation fitted experimental data consistently. Moreover, this study shows that larval Trichinella morphology may be used in situations where bioassays are not possible or prohibited. CONCLUSIONS: The relationship between heat and inactivation of larvae obtained from this study could be used in Trichinella QMRA models to improve quantification of the risk of Trichinella infection.

Prevalence of meat-transmitted Taenia and Trichinella parasites in the Far East countries.

Taenia and Trichinella parasites are globally distributed foodborne zoonotic pathogens transmitted from animal to humans via consumption of raw or undercooked meats. This short review is intended to provide the parasites community a snapshot of the literature on the current and recent prevalence of taeniasis and trichinellosis in humans and animals in the Far East countries. Prevalence rates in these countries are highly diverse due to differences in development, culture, ethnic and religious background, animal forming practices, and eating habits. Taenia and Trichinella remain as important meat-transmitted pathogens in the Far East. A One Health approach is needed to eliminate or continuously reduce the foodborne zoonotic taeniasis and trichinellosis in the Far East.

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.

Molecular Mechanism of Thymidylate Synthase Inhibition by N4-Hydroxy-dCMP in View of Spectrophotometric and Crystallographic Studies.

Novel evidence is presented allowing further clarification of the mechanism of the slow-binding thymidylate synthase (TS) inhibition by N4-hydroxy-dCMP (N4-OH-dCMP). Spectrophotometric monitoring documented time- and temperature-, and N4-OH-dCMP-dependent TS-catalyzed dihydrofolate production, accompanying the mouse enzyme incubation with N4-OH-dCMP and N5,10-methylenetetrahydrofolate, known to inactivate the enzyme by the covalent binding of the inhibitor, suggesting the demonstrated reaction to be uncoupled from the pyrimidine C(5) methylation. The latter was in accord with the hypothesis based on the previously presented structure of mouse TS (cf. PDB ID: 4EZ8), and with conclusions based on the present structure of the parasitic nematode Trichinella spiralis, both co-crystallized with N4-OH-dCMP and N5,10-methylenetetrahdrofolate. The crystal structure of the mouse TS-N4-OH-dCMP complex soaked with N5,10-methylenetetrahydrofolate revealed the reaction to run via a unique imidazolidine ring opening, leaving the one-carbon group bound to the N(10) atom, thus too distant from the pyrimidine C(5) atom to enable the electrophilic attack and methylene group transfer.