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Genome assemblies provide a powerful basis of comparative multi-omics analyses that offer insight into parasite pathogenicity, host-parasite interactions, and invasion biology. As a unique intracellular nematode, Trichinella consists of two clades, encapsulated and non-encapsulated. Genomic correlation of the distinct differences between the two clades is still unclear. Here, we report an annotated draft reference genome of non-encapsulated Trichinella, T. pseudospiralis, and perform comparative multi-omics analyses with encapsulated T. spiralis. Genome and methylome analyses indicate that, during Trichinella evolution, the two clades of Trichinella exhibit differential expansion and methylation of parasitism-related multi-copy gene families, especially for the DNase II members of the phospholipase D superfamily and Glutathione S-transferases. Further, methylome and transcriptome analyses revealed divergent key excretory/secretory (E/S) genes between the two clades. Among these key E/S genes, TP12446 is significantly more expressed across three life stages in T. pseudospiralis. Overexpression of TP12446 in the mouse C2C12 skeletal muscle cell line could induce inhibition of myotube formation and differentiation, further indicating its key role in parasitism of T. pseudospiralis. This multi-omics study provides a foundation for further elucidation of the mechanism of nurse cell formation and immunoevasion, as well as the identification of pharmacological and diagnostic targets of trichinellosis.Xiaolei Liu, Yayan Feng, et al. report an annotated draft reference genome of the non-encapsulated Trichinella pseudospiralis, and perform comparative multi-omics analyses with encapsulated T. spiralis. Overexpression of TP12446, a key excretory/secretory gene, in a mouse C2C12 skeletal muscle cell line could induce inhibition of myotube formation and differentiation. These results provide insight into the mechanism of nurse cell formation and immunoevasion, as well as the identification of potential pharmacological and diagnostic targets for trichinellosis.

Trypanosoma cruzi causes Chagas disease, the most important parasitic infection in Latin America. Major pathologies include severe damage to the heart and digestive tract, although symptoms do not usually appear until decades after infection. Research has been hampered by the complex nature of the disease and technical difficulties in locating the extremely low number of parasites. Here, using highly sensitive imaging technology, we reveal the sites of parasite persistence during chronic-stage infections of experimental mice at single-cell resolution. We show that parasites are frequently located in smooth muscle cells in the circular muscle layer of the colon and that skeletal muscle cells and the skin can also be important reservoirs. This information provides a framework for investigating how the parasite is able to survive as a lifelong infection, despite a vigorous immune response. It also informs drug development strategies by identifying tissue sites that must be accessed to achieve a curative outcome. Infections with Trypanosoma cruzi are usually lifelong despite generating a strong adaptive immune response. Identifying the sites of parasite persistence is therefore crucial to understanding how T. cruzi avoids immune-mediated destruction. However, this is a major technical challenge, because the parasite burden during chronic infections is extremely low. Here, we describe an integrated approach involving comprehensive tissue processing, ex vivo imaging, and confocal microscopy, which allowed us to visualize infected host cells in murine tissue with exquisite sensitivity. Using bioluminescence-guided tissue sampling, with a detection level of <20 parasites, we showed that in the colon, smooth muscle myocytes in the circular muscle layer are the most common infected host cell type. Typically, during chronic infections, the entire colon of a mouse contains only a few hundred parasites, often concentrated in a small number of cells each containing >200 parasites, which we term mega-nests. In contrast, during the acute stage, when the total parasite burden is considerably higher and many cells are infected, nests containing >50 parasites are rarely found. In C3H/HeN mice, but not BALB/c mice, we identified skeletal muscle as a major site of persistence during the chronic stage, with most parasites being found in large mega-nests within the muscle fibers. Finally, we report that parasites are also frequently found in the skin during chronic murine infections, often in multiple infection foci. In addition to being a site of parasite persistence, this anatomical reservoir could play an important role in insect-mediated transmission and have implications for drug development. IMPORTANCE Trypanosoma cruzi causes Chagas disease, the most important parasitic infection in Latin America. Major pathologies include severe damage to the heart and digestive tract, although symptoms do not usually appear until decades after infection. Research has been hampered by the complex nature of the disease and technical difficulties in locating the extremely low number of parasites. Here, using highly sensitive imaging technology, we reveal the sites of parasite persistence during chronic-stage infections of experimental mice at single-cell resolution. We show that parasites are frequently located in smooth muscle cells in the circular muscle layer of the colon and that skeletal muscle cells and the skin can also be important reservoirs. This information provides a framework for investigating how the parasite is able to survive as a lifelong infection, despite a vigorous immune response. It also informs drug development strategies by identifying tissue sites that must be accessed to achieve a curative outcome.

Toxoplasma gondii belongs to a large and diverse group of obligate intracellular parasitic protozoa. Primary culture of mice skeletal muscle cells (SkMC) was employed as a model for experimental toxoplasmosis studies. The myogenesis of SkMC was reproduced in vitro and the ability of T. gondii tachyzoite forms to infect myoblasts and myotubes and its influence on SkMC myogenesis were analyzed. In this study we show that, after 24 h of interaction, myoblasts (61%) were more infected with T. gondii than myotubes (38%) and inhibition of myogenesis was about 75%. The role of adhesion molecules such as cadherin in this event was investigated. First, we demonstrate that cadherin localization was restricted to the contact areas between myocytes/myocytes and myocytes/myotubes during the myogenesis process. Immunofluorescence and immunoblotting analysis of parasite-host cell interaction showed a 54% reduction in cadherin expression at 24 h of infection. Concomitantly, a reduction in M-cadherin mRNA levels was observed after 3 and 24 h of T. gondii-host cell interaction. These data suggest that T. gondii is able to down regulate M-cadherin expression, leading to molecular modifications in the host cell surface that interfere with membrane fusion and consequently affect the myogenesis process.

Philometroides Yamaguti, 1935 is a genus of dracunculoid nematodes of the family Philometridae Baylis & Daubney, 1926 comprising tissue-infecting species worldwide. In the present study, a new species of Philometroides is described from the Tyrrhenian Sea (central-western Mediterranean Sea) using integrative approaches such as light and scanning microscopy, histopathology and 18S rRNA sequencing. Philometroides giginosantoroi n. sp. found in the skeletal muscles of the Mediterranean moray Muraena helena can be distinguished from its congeners by a combination of morphological traits and in particular by having the oral aperture with 3 large sclerotized triangular oesophageal teeth. The molecular analyses and phylogenetic reconstructions support its status as a new taxon and placed it within a clade of tissue-infecting species, although also confirmed mismatches in the generic assignment of several species. Philometroides giginosantoroi n. sp. is the second species of the genus found in the Mediterranean Sea and in general in the European marine waters and the third species of the family known to infect the family Muraenidae.

The spotted seatrout, Cynoscion nebulosus , is a popular game fish in the southeastern USA. It is estimated that nearly 90% of the adult population in South Carolina estuaries are infected in their skeletal muscle by the myxosporean, Kudoa inornata . However, little is known about this parasite’s biology, including the distribution and densities of myxospores within tissues of infected fish, which we expect affect the physiology of their hosts. In order to correlate densities with physiological parameters in future studies, we quantified the myxospores density in muscle and characterized the variation among individual fish. Naïve juvenile seatrout was experimentally infected via presumed K. inornata actinospores exposure to raw seawater. A plug of muscle was extracted from two bilaterally symmetrical regions in the epaxial fillet from fresh and frozen carcasses. Variation in density data was calculated both within and among individuals. Within individuals, density counts were compared between left- and right-side biopsies. There was no significant difference between fresh and frozen plugs, and variation among individuals accounted for the greatest proportion of variation at 68.8%, while variation within individuals was substantial at 25.6%. Simulation and correlation tests confirmed that bilaterally symmetrical replicates varied significantly within individuals. When sampled from areas surrounding the initial biopsies, myxospore density estimates were more similar than between sides. Our findings have important implications for sampling design, particularly for studies investigating physiological parameters at the cellular or molecular level in association with parasite infection.

Background Early diagnosis of neosporosis in dogs is challenging. Objectives To evaluate the feasibility of a compound multimodal testing approach for diagnosing in dogs neuromuscular and combined forms of neosporosis. Animals A total of 16 dogs diagnosed with solely neuromuscular neosporosis or with a combination of neuromuscular and central nervous system neosporosis. Methods Retrospective review of clinical signs, laboratory findings, treatment, and outcome with focus on the diagnostic utility of different tests. Development of a chromogenic in situ hybridization (ISH) assay for the identification of Neospora caninum in paraffin‐embedded muscle samples. Results 13/16 dogs had only neuromuscular signs of neosporosis, 3/16 had disease signs with concomitant central nervous system (CNS) involvement. Serology was performed in 15/16, with 10/15 showing titers >1 : 160 at admission. PCR on muscle samples detected N. caninum DNA in 11/16. Immunohistochemistry (IHC) detected N. caninum in 9/16 and ISH in 9/16. Histopathology revealed inflammatory myopathy in 10/16, necrotizing myopathy in 5/16, borderline changes in 1/16 and tachyzoites in 9/16. In 4 cases, N. caninum infection was confirmed with all 5 diagnostic methods, 3 cases with 4, 2 with 3, 6 with 2, and 1 animal with 1. Conclusions and Clinical Importance Diagnosis of N. caninum infection should rely on a multimodal diagnostic approach and negativity of 1 single test should not allow for exclusion. Serology in combination with direct parasite identification via histopathology, DNA via PCR, or both modalities, appears a reliable diagnostic approach.

is a worldwide intestinal nematode that can parasitize the striated muscles of its hosts at the larval stage. This study aims to evaluate potential of vitamin C for treating trichinellosis-related pathological problems in the infected muscles of mice. Thirty CD1 male Albino mice were divided into three groups (10 mice per group). Negative and positive control groups (0.9% NaCl) and the infected vitamin C group (10 mg/kg body weight). Two weeks post-infection, each group was intraperitoneally injected daily for two weeks with Vitamin C or saline. The performance of the muscles was assessed both before and after the treatment. After dissection, constant parts of striated muscles were removed for further assays. The scoring of the histological changes of infected muscles was carried out. In addition to muscle malondialdehyde levels, superoxide dismutase and catalase activities were measured for the oxidative and antioxidant states. Creatine kinase and aspartate aminotransferase were also measured in tissues to reflect the degree of muscular damage. Vitamin C enhances the weakness of the muscular performance resulting from the infection. Vitamin C was able to repair some of the histological lesions that resulted from the infection. Trichinellosis caused severe changes in the biochemical markers in positive control animals. Muscle damage biomarkers and, besides, oxidative and antioxidant conditions were greatly ameliorated in infected vitamin C animals. Summing up, vitamin C can be used as a complementary drug due to its efficiency in improving pathogenesis following a trichinellosis infection. The supplement also must be tested in the intestinal stage of infection after showing promising results in the muscular stage.

Cachexia is a progressive muscle wasting disease that contributes to death in a wide range of chronic diseases. Currently, the cachexia field lacks animal models that recapitulate the long-term kinetics of clinical disease, which would provide insight into the pathophysiology of chronic cachexia and a tool to test therapeutics for disease reversal. Toxoplasma gondii ( T. gondii ) is a protozoan parasite that uses conserved mechanisms to infect rodents and human hosts. Infection is lifelong and has been associated with chronic weight loss and muscle atrophy in mice. We have recently shown that T. gondii -induced muscle atrophy meets the clinical definition of cachexia. Here, the longevity of the T. gondii -induced chronic cachexia model revealed that cachectic mice develop perivascular fibrosis in major metabolic organs, including the adipose tissue, skeletal muscle, and liver by 9 weeks post-infection. Development of cachexia, as well as liver and skeletal muscle fibrosis, is dependent on intact signaling through the type I IL-1R receptor. IL-1α is sufficient to activate cultured fibroblasts and primary hepatic stellate cells (myofibroblast precursors in the liver) in vitro, and IL-1α is elevated in the sera and liver of cachectic, suggesting a mechanism by which chronic IL-1R signaling could be leading to cachexia-associated fibrosis.

Background For several decades, the European wildcat ( Felis silvestris ) has gradually been returning to the forests of Germany, mainly in the central and southwestern regions. To increase the knowledge about this threatened species, the endoparasite status of dead found specimens from Germany was surveyed. Methods A total of 118 wildcats were examined for endoparasites in peritoneal organs and skeletal muscles. Owing to decomposition or incomplete carcasses, 104 gastrointestinal tracts (stomachs and intestines), 101 livers with gallbladders, 99 urinary bladders, as well as kidneys of 95 and skeletal muscles of 112 specimens were available for examination. All detected parasites were identified morphologically to genus or species level, followed by molecular examinations of one to ten specimens of each parasite species. Results Overall endoparasite prevalence in peritoneal organs was 99.0% (103/104). Among the 99.0% (103/104) infected gastrointestinal tracts, the most frequent species were Toxocara cati (95.2% [99/104]), Hydatigera kamiyai (84.6% [88/104]), Mesocestoides litteratus (69.2% [72/104]), Strongyloides spp. (58.7% [61/104]), Cylicospirura petrowi (37.5% [39/104]), Ancylostoma tubaeforme (31.7% [33/104]), Capillaria putorii (24.0% [25/104]), and Echinococcus multilocularis (18.3% [19/104]). In 77.8% (77/99) of the urinary bladders, Capillaria plica and/or Capillaria feliscati were detected. Moreover, the liver fluke Metorchis bilis occurred in 2.0% (2/101) of the livers, and roundworm larvae (presumably Toxocara spp.) were detected in 33.0% (37/112) of the muscle samples. Conclusions These results show a broad spectrum of endoparasite species infecting European wildcats in Germany. It might be assumed that some of the endoparasites could pose a risk to domestic cats ( Felis catus ) and humans through spillover events, or may be transmitted from domestic cats to the free-ranging population, posing a potential risk to wildcats. Graphical Abstract

Background Sarcocystosis is a parasitic disease found worldwide, resulting from various Sarcocystis species. The current research was carried out in three significant economic areas in Egypt: Greater Cairo, the Nile Delta, and Upper Egypt. It aimed to investigate the occurrence of Sarcocystis spp. in locally bred water buffaloes Bubalus bubalis . Methods To achieve this objective, 317 buffalos were slaughtered in different slaughterhouses in various regions of Egypt. Samples of heart, skeletal muscle, esophagus, and tongue were assessed using macroscopic and microscopic tests. Examination methods included direct optical observation of tissues as well as digestion and examination of the sediment obtained from the tissues. Additionally, ultrastructural features were analyzed using scanning and transmission electron microscopy. Molecular characterization was conducted through PCR, followed by nucleotide sequencing and phylogenetic analysis. Results A total of 317 slaughtered buffaloes were examined for Sarcocystis during the period from September 2021 to October 2023. The prevalence of infection was recorded with 229 out of 317 (72.2%) infected with Sarcocystis spp. The results also showed that the prevalence of Sarcocystis species in females was higher than males. Based on the age of carcasses, adults (> 2 years) had a higher infection rate compared to young ones (< 2 years). Regarding seasonal variation, the highest prevalence of infection was recorded during the summer followed by spring, and then autumn, while winter had the lowest prevalence of infection. Additionally, the skeletal muscle was the most susceptible organ to sarcocystosis (87.3%) followed by the esophageal muscle (8.3%), the tongue (4.4%), and no infection in the heart muscle. The use of scanning and transmission electron microscopy allowed the identification of S. fusiformis and S. cruzi in buffaloes in Egypt. Furthermore, the Sarcocystis 18 S rRNA genes from skeletal tissue samples were cloned and sequenced under accession numbers OQ507387, OQ507388, and OQ507389 for S. fusiforms , and one OQ507391 for S. cruzi. Conclusion The findings revealed a notably high prevalence of Sarcocystis infection (72.2%) in buffaloes from Egypt, with skeletal muscle identified as the organ most susceptible to the parasite. Two Sarcocystis species were detected: S. fusiformis and S. cruzi.