The plerocercoid larvae of Spirometra mansoni are etiological agents of human and animal sparganosis. Annexins are proteins with important roles in parasites. However, our knowledge of annexins in S. mansoni is still inadequate. In this study, 18 new members of the Annexin (ANX) family were characterized in S. mansoni. The clustering analysis demonstrated that all the SmANXs were divided into two main classes, consistent with the patterns of conserved motif organization. The 18 SmANXs were detected at all developmental stages (plerocercoid, adult, and egg) and displayed ubiquitous but highly variable expression patterns in all tissues/organs studied. The representative member rSmANX18 was successfully cloned and expressed. The protein was immunolocalized in the tegument and parenchyma of the plerocercoid and in the tegument, parenchyma, uterus and egg shell of adult worms. The recombinant protein can bind phospholipids with high affinity in a Ca2+-dependent manner, shows high anticoagulant activity and combines with FITC to recognize apoptotic cells. Annexin gene polymorphism and conservative core motif permutation were found in both cestodes and trematodes. SmANXs also revealed high genetic diversity among Platyhelminthes of medical interest. Our findings lay a foundation for further studies on the biological functions of ANXs in S. mansoni as well as other taxa in which ANXs occur.
UNASSIGNED: La famille des gènes des annexines chez Spirometra mansoni (Cestoda : Diphyllobothriidae) et son schéma phylogénétique parmi les Plathelminthes d’intérêt médical.
UNASSIGNED: Les larves plérocercoïdes de Spirometra mansoni sont des agents étiologiques de la sparganose humaine et animale. Les annexines sont des protéines jouant un rôle important chez les parasites. Cependant, nos connaissances sur les annexines chez S. mansoni sont encore insuffisantes. Dans cette étude, 18 nouveaux membres de la famille des annexines (ANX) ont été caractérisés chez S. mansoni. L’analyse de regroupement a démontré que tous les SmANX étaient divisées en deux classes principales, ce qui correspond aux modèles d’organisation des motifs conservés. Les 18 SmANX ont été détectées à tous les stades de développement (plérocercoïde, adulte et œuf) et présentaient des modèles d’expression omniprésents mais très variables dans tous les tissus/organes étudiés. Le membre représentatif rSmANX18 a été cloné et exprimé avec succès. La protéine a été immunolocalisée dans le tégument et le parenchyme du plérocercoïde ainsi que dans le tégument, le parenchyme, l’utérus et la coquille d’œuf des vers adultes. La protéine recombinante peut se lier aux phospholipides avec une affinité élevée de manière dépendante du Ca2+, présente une activité anticoagulante élevée et se combine avec le FITC pour reconnaître les cellules apoptotiques. Un polymorphisme du gène de l’annexine et une permutation conservatrice du motif central ont été trouvés chez les cestodes et les trématodes. Les SmANX ont également révélé une grande diversité génétique parmi les Plathelminthes d’intérêt médical. Nos résultats jettent les bases pour des études plus approfondies sur les fonctions biologiques des ANX chez S. mansoni ainsi que dans d’autres taxons dans lesquels les ANX sont présents.

Two adult male Puerto Rican crested anoles (Anolis cristatellus cristatellus) housed in a research facility were presented with debilitation and were euthanized. On autopsy, anole 1 had a large cystic white structure in the left pelvic limb, which protruded through the ruptured epidermis, and a large, poorly demarcated swelling in the right caudal abdomen. Anole 2 had masses in the mid-dorsum, caudal dorsum, left pelvic limb, and tail. These masses contained variably sized cestode larvae, which ruptured into the coelomic cavity. Evaluation of the larvae revealed a thickened and wrinkled anterior end, with a cleft-like invagination, consistent with either a plerocercoid sparganum or a tetrathyridium. Histologically, several cestode larvae were contained in the body wall of both anoles. These were up to 650 μm in diameter, with a thin tegument and a spongy parenchyma. The spongy parenchyma contained numerous, up to 30 μm diameter, sharply demarcated, basophilic-to-black structures (calcareous corpuscles). There was pneumonia and hepatitis in anole 2, suggestive of potential secondary infection subsequent to immunosuppression. Molecular amplification of the cytochrome C oxidase subunit 1 revealed 100% homology for the COX1 gene of the diphyllobothriid tapeworm Spirometra erinaceieuropaei, also known as Spirometra mansoni.

BACKGROUND: Spirometra mansoni can parasitize animals and humans through food and water, causing parasitic zoonosis. Knowledge of the developmental process of S. mansoni is crucial for effective treatment; thus, it is important to characterize differential and specific proteins and pathways associated with parasite development.
METHODS: In this study, we performed a comparative proteomic analysis of the plerocercoid and adult stages using a tandem mass tag-based quantitative proteomic approach. Additionally, integrated transcriptomic and proteomic analyses were conducted to obtain the full protein expression profiles of different life cycle stages of the tapeworm.
RESULTS: Approximately 1166 differentially expressed proteins (DEPs) were identified in adults versus plerocercoids, of which 641 DEPs were upregulated and 525 were downregulated. Gene Ontology (GO), Clusters of Orthologous groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that most DEPs related to genetic information processing and metabolism of energy in adults seem to be more activated. In the plerocercoid stage, compared to metabolism, genetic information processing appears more dynamic. Protein-protein interaction (PPI) revealed six key proteins (phosphomannomutase, glutathione transferase, malate dehydrogenase, cytoplasmic, 40S ribosomal protein S15, ribosomal protein L15 and 60S acidic ribosomal protein P2) that may play active roles in the growth and development of S. mansoni. Finally, the combination of transcriptomic and proteomic data suggested that three pathways (ubiquitin-mediated proteolysis, phagosome and spliceosome) and five proteins closely related to these pathways might have a significant influence in S. mansoni.
CONCLUSIONS: These findings contribute to increasing the knowledge on the protein expression profiles of S. mansoni and provide new insights into functional studies on the molecular mechanisms of the neglected medical tapeworm.

Morphological characteristics and DNA sequencing were used to identify plerocercoids of a Schistocephalus sp. infecting slimy sculpin (Cottus cognatus) from northern New Brunswick and plerocercoids of Ligula intestinalis infecting blacknose dace (Rhinichthys atratulus) in Fundy National Park (FNP, New Brunswick). To our knowledge, no previous publications documented either cestode from New Brunswick, Canada. Blacknose dace represent a new host record for L. intestinalis. Identifications were made based on the presence or absence of segmentation and sequencing partial nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1; mitochondrial DNA) and/or partial cytochrome c oxidase subunit 1 (COI; mitochondrial DNA). Plerocercoids from blacknose dace in FNP were identified as Ligula intestinalis based on >99% nucleotide identity with COI for this species in the NCBI GenBank database. Plerocercoids in slimy sculpin from northern New Brunswick were identified as a Schistocephalus sp. based on high nucleotide identity with congenerics in the NCBI GenBank database. The absence of GenBank entries with sufficient high percent identity to our specimens, and potential species hybrids in this genus, prevents species-level identification of Schistocephalus sp. plerocercoids currently. The absence of previous documentation of these cestodes might reflect recent environmental change promoting the transmission of these parasites that can modulate host fish behavior, induce sterility of host fishes, and contribute to epizootics.

We report a case of Spirometra infection in a Samar cobra (Naja samarensis) imported from the Philippines, belonging to a zoological collection in the southern United States. Under a poor post-surgical prognosis, the snake was euthanized, and at necropsy plerocercoids of a Diphyllobotriidea were found in its subcutaneous tissues and musculature. Molecular and phylogenetic analyses of the complete cytochrome oxidase c subunit I (cox1) gene of the mitochondrial DNA confirmed that the isolate belonged to the genus Spirometra and was closely related to Spirometra mansoni isolates from Asian countries (bootstrap support = 99.4%). Considering the origin and clinical history and handling of the animal, the snake probably arrived infected in America. We suggest the inclusion of diagnostic imaging in the investigation of sparganosis in research and disease surveillance protocols applied in the pre- and post-quarantine period to asymptomatic animals imported from endemic areas.

BACKGROUND: In China, the plerocercoid of the cestode Spirometra mansoni is the main causative agent of human and animal sparganosis. However, the population genetic structure of this parasite remains unclear. In this study, we genotyped S. mansoni isolates with the aim to improve current knowledge on the evolution and population diversity of this cestode.
METHODS: We first screened 34 perfect simple sequence repeats (SSRs) using all available omic data and then constructed target sequencing technology (Target SSR-seq) based on the Illumina NovaSeq platform. Next, a series of STRUCTURE. clustering, principal component, analysis of molecular variance and TreeMix analyses were performed on 362 worm samples isolated from 12 different hosts in 16 geographical populations of China to identify the genetic structure.
RESULTS: A total of 170 alleles were detected. The whole population could be organized and was found to be derived from the admixture of two ancestral clusters. TreeMix analysis hinted that possible gene flow occurred from Guizhou (GZ) to Sichuan (SC), SC to Jaingxi (JX), SC to Hubei (HB), GZ to Yunnan (YN) and GZ to Jiangsu (JS). Both neighbor-joining clustering and principal coordinate analysis showed that isolates from intermediate hosts tend to cluster together, while parasites from definitive hosts revealed greater genetic differences. Generally, a S. mansoni population was observed to harbor high genetic diversity, moderate genetic differentiation and a little genetic exchange among geographical populations.
CONCLUSIONS: A Target SSR-seq genotyping method was successfully developed, and an in-depth view of genetic diversity and genetic relationship will have important implications for the prevention and control of sparganosis.

The protein composition of the cestode Schistocephalus solidus was measured in an experiment simulating the trophic transmission of the parasite from a cold-blooded to a warm-blooded host. The first hour of host colonisation was studied in a model experiment, in which sticklebacks Gasterosteus aculeatus infected with S. solidus were heated at 40°C for 1 h. As a result, a decrease in the content of one tegument protein was detected in the plerocercoids of S. solidus. Sexual maturation of the parasites was initiated in an experiment where S. solidus larvae were taken from fish and cultured in vitro at 40°C for 48 h. Temperature-independent changes in the parasite proteome were investigated by incubating plerocercoids at 22°C for 48 h in culture medium. Analysis of the proteome allowed us to distinguish the temperature-induced genes of S. solidus, as well as to specify the molecular markers of the plerocercoid and adult worms. The main conclusion of the study is that the key enzymes of long-term metabolic changes (glycogen consumption, protein production, etc.) in parasites during colonisation of a warm-blooded host are induced by temperature.

Oarfish (Regalecus russelii Cuvier) are mesopelagic fish with little known about their life history. Oarfish live in deep water, making it difficult for researchers to collect specimens; thus, records of their parasitic helminths are limited. Two plerocercoids were found for the first time in an oarfish stranded on the coast of Akita Prefecture, Japan. These plerocercoids were identified as Clistobothrium sp. RR-1 using morphological and molecular analyses. It was revealed that oarfish represent one of the intermediate hosts of the genus Clistobothrium, and large sharks are the definitive hosts for these parasites.

We report three cases of sparganosis due to plerocercoids of the tapeworm Spirometra sp. in captive meerkats (Suricata suricatta) from a zoo exhibit in the southeastern United States. Two meerkats were euthanized, one due to an uncontrollable seizure and the other due to trauma, and at necropsy cysts containing cestode larvae were observed. A third meerkat had a subcutaneous nodule surgically removed, which contained similar larvae. The third animal died years later, and had numerous cestode larvae in the pleural and peritoneal cavities. The larvae were morphologically identified as plerocercoids of diphyllobothriidean cestodes. On necropsy, multiple nodules, ranging in size from 2.5 to 3.0 cm, were observed in the subcutaneous tissue and muscles. Multifocally, separating skeletal muscle fibers were longitudinal and transversal sections of cestode larva. Histologically, parasitic cysts contained large numbers of neutrophils and macrophages, admixed with proteinaceous material. Molecular and phylogenetic analyses confirmed that specimens from one of the meerkats belonged to the genus Spirometra and was closely related to Spirometra plerocercoids isolated from a snake from the United States and wild felids from South America. Meerkats likely became infected by ingesting infected second intermediate hosts, such as amphibians and reptiles that may have entered the exhibit. Management practices that minimize access of meerkats and other susceptible hosts to intermediate hosts should be implemented.

A platyhelminth, Spirometra erinaceieuropaei, belonging to the class Cestoda, causes human sparganosis, and infection with its larva results in subtle inflammation in the body of its host. We previously reported the purification of a glycoprotein, plerocercoid-immunosuppressive factor (P-ISF) from the excretory/secretory products of S. erinaceieuropaei plerocercoids that may be involved in immuno-modification. We determined the sequence of P-ISF from the N-terminal and the internal 10 amino acids of P-ISF using degenerate PCR and 5\'- and 3\'-RACE methods. The putative gene encoding P-ISF was 1443 bp long and the gene contained 10 exons and 9 introns in a genomic DNA of size 5205 bp. P-ISF consists of 480 amino acids including the N-terminal signal peptide sequence, and has two unknown domains,-cestoda cysteine-rich domains (CCDs) and a fibronectin type III domain between the two CCDs. All cysteine residues were conserved in the two CCDs, which shared 62% amino acid identities. Homologous analysis revealed that the CCDs were homologous with an unknown protein of Diphyllobothrium latum. To produce specific antibodies, we expressed recombinant P-ISF (rP-ISF) using wheat germ protein synthetic system. P-ISF was localized in the sub-cutaneous tissues and the parenchymal tissues of plerocercoids. Transcription of P-ISF was detected only in plerocercoid stage, but not in adult stage. Western blotting also showed a band in plerocercoide stage but not in adult. The rP-ISF did not suppress nitrite production in RAW 264.7 cells stimulated with LPS, and this might be due to lack of carbohydrate chains in the recombinant protein.