Integrated transcriptomic and proteomic analyses of plerocercoid and adult Spirometra mansoni reveal potential important pathways in the development of the medical tapeworm.

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.

First report of Spirometra (Eucestoda; Diphyllobothriidae) naturally occurring in a fish host.

Spirometra Faust, Campbell et Kellogg, 1929 is a genus of cestodes belonging to the family Diphyllobothriidae. To date, amphibians, reptiles, and mammals are known second intermediate hosts of these parasites; humans can also be infected (the zoonotic disease is known as sparganosis or spirometrosis). Although the number of phylogenetic studies on Spirometra spp. has increased worldwide in recent years, there are few in South America. Specifically in Uruguay, molecular studies have shown that tapeworms of S. decipiens (Diesing, 1850) complexes 1 and 2 are present in this country. In this study, we characterised the larvae of Spirometra present in the annual fish Austrolebias charrua Costa et Cheffe. Phylogenetic analysis of the cytochrome c oxidase subunit I (COI) sequences of these larvae showed that they belong to S. decipiens complex 1. This is the first report of teleost fishes serving as a second intermediate host for tapeworms of the genus Spirometra in nature.

Extracellular vesicles derived from Spirometra erinaceieuropaei plerocercoids inhibit activation of murine macrophage RAW264.7 cells.

Parasitic helminths modify host immune reactions to promote long-term parasitism. We previously purified a glycoprotein, plerocercoid-immunosuppressive factor (P-ISF), from the excretory/secretory products of Spirometra erinaceieuropaei plerocercoids and reported its cDNA and genomic DNA sequences. In this study, we isolated extracellular vesicles (EVs) from the excretory/secretory products of S. erinaceieuropaei plerocercoids and found that they suppressed the production of nitric oxide and the gene expression of tumor necrosis factor-α, interleukin-1ß, and interleukin-6 in lipopolysaccharide-stimulated macrophages. EVs are membrane-bound vesicles 50-250 nm in diameter and are localized in the whole bodies of plerocercoids. EVs from plerocercoids encapsulate a variety of unidentified proteins and microRNAs (miRNAs), which are non-coding RNAs that play essential roles in post-transcriptional gene regulation. The miRNAs of the EVs were analyzed, and 334,137 sequencing reads were mapped to the genomes of other organisms. A total of 26 different miRNA families were identified, including miR-71, miR-10-5p, miR-223, and let-7-5p, which have been reported to have immunosuppressive effects. We confirmed that P-ISF was present in the supernatant but not in the EVs by western blotting with an anti-P-ISF antibody. These results suggest that S. erinaceieuropaei plerocercoids suppress host immunity by releasing P-ISF and EVs.

Spirometra mansoni sparganosis identified by metagenomic next-generation sequencing: a case report.

A 30-year-old male patient had a cyst on the left hip and progressive enlargement for more than 2 months. Combined blood tests, magnetic resonance imaging, and pathology findings, cysticercosis infection was suspected. However, the treatment for cysticercosis was ineffective. We conducted a metagenomic next-generation sequencing (mNGS) analysis on the formalin-fixed, paraffin-embedded specimen of the patient's surgically excised tissue, and the results suggested Spirometra mansoni, mNGS was further confirmed by polymerase chain reaction and phylogenetic analysis of cytochrome c oxidase subunit 1 (cox1) gene. Based on these results, we found that mNGS provided a better method of diagnosing parasitic infections.

Genetic structure of Spirometra mansoni (Cestoda: Diphyllobothriidae) populations in China revealed by a Target SSR-seq method.

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.

Presence of Spirometra mansoni, Causative Agent of Sparganosis, in South America.

We report molecular identification of an adult Spirometra mansoni tapeworm retrieved from a crab-eating fox (Cerdocyon thous) in Colombia, confirming presence of this parasite in South America. This tapeworm is the causative agent of human sparganosis, commonly reported from Southeast Asia, and represents the second congeneric species with known zoonotic potential in the Americas.

The global prevalence of Spirometra parasites in snakes, frogs, dogs, and cats: A systematic review and meta-analysis.

BACKGROUND: Spirometra infection is aneglected food- and waterborne disease with worldwide distribution. OBJECTIVES: The present study aims to estimate the global prevalence of Spirometra species in snakes, frogs, dogs and cats. METHODS: Multiple databases (PubMed, Scopus, ProQuest, Web of Science and Google Scholar) were searched for relevant literatures published up to March 2022. RESULTS: Among 131 data sets (including 113 articles) that met the inclusion, 15 investigations reported Spirometra infection in snakes, 23 in frogs, 41 in dogs and 52 in cats. The pooled prevalence (95% confidence interval) in intermediate hosts and definitive hosts was found to be 0.313% and 0.089%, respectively. Based on continent, the infection was most prevalent in Asia for studies on snakes (0.696%) and frogs (0.181%), while Africa (0.224%) and Oceania (0.203%) were the regions with the highest pooled prevalence rates of the infection in dogs and cats, respectively. Among different diagnostic methods, the highest pooled prevalence was related to morphological method for studies on snakes, frog and cats with rate of 0.665%, 0.189% and 0.104%, respectively. Regarding studies on dogs, the highest pooled prevalence was observed for molecular technique (0.101%). CONCLUSIONS: The results presented here revealed the importance of establishing a prevention and control measure focused on protection of aquaculture systems from being contaminated with faeces of dogs and cats, and raising awareness of parasitic zoonotic diseases to decrease the transmission risk.

Surgical treatment of a patient with live intracranial sparganosis for 17 years.

BACKGROUND: The incidence of sparganosis, especially intracranial live sparganosis is very low in China. Due to the lack of typical clinical manifestations, it is difficult to make a clear preoperative diagnosis of the disease, which often leads to delays the disease and serious consequences. CASE PRESENTATION: A 23-year-old man presented with a 17-year history of intermittent seizures and right extremity numbness and weakness. Magnetic resonance imaging (MRI) showed patchy, nodular and line-like enhancement. Enzyme-linked immunosorbent assay (ELISA) detected positive antibodies to Spirometra mansoni in peripheral blood and cerebrospinal fluid (CSF). In addition, during the operation, an ivory-colored live sparganosis was removed under the precise positioning of neuronavigation, and the patient was diagnosed with cerebral sparganosis. The patient began praziquantel and sodium valproate treatment after the operation, and was followed up for 3 months. There was no recurrence of epilepsy, and the weakness and numbness of the right limb improved. CONCLUSION: Nonspecific clinical manifestations often make the diagnosis of cerebral sparganosis difficult, and a comprehensive diagnosis should be made based on epidemiological history, clinical manifestations, ELISA results and imaging findings. Surgery is the preferred method for the treatment of cerebral sparganosis, and more satisfactory results can be achieved under the precise positioning of neuronavigation.

Transcriptome profiling of plerocercoid and adult developmental stages of the neglected medical tapeworm Spirometra erinaceieuropaei.

The plerocercoid larvae of the tapeworm Spirometra erinaceieuropaei can parasitize humans and animals and cause serious parasitic zoonosis. However, our knowledge of the developmental process of S. erinaceieuropaei is still inadequate. To better characterize differential and specific genes and pathways associated with parasite development, a comparative transcriptomic analysis of the plerocercoid stage and the adult stage was performed using RNA-seq and de novo analysis. Approximately 13,659 differentially expressed genes (DEGs) were identified in plerocercoids versus adults, of which 6455 DEGs were upregulated and 7204 were downregulated. DEGs involved in parasite immunoevasion were more active in plerocercoid larvae than in adults, while DEGs associated with metabolic activity were upregulated in adults. Gene Ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) analyses revealed that most DEGs involved in protein phosphorylation/dephosphorylation and the Wnt signalling pathway were much more active in plerocercoid larvae. The molecular functions of upregulated unigenes in adults were mainly enriched for metabolic activities. qPCR validated that the expression levels of 10 selected DEGs were consistent with those in RNA-seq, confirming the accuracy of the RNA-seq results. Our results contributed to increasing the knowledge on the S. erinaceieuropaei gene repertoire and expression profile and also provide valuable resources for functional studies on the molecular mechanisms of S. erinaceieuropaei.

Genetic interrelationships of Spirometra erinaceieuropaei (Cestoda: Diphyllobothriidea), the causative agent of sparganosis in Europe.

The geographic distribution of Spirometra erinaceieuropaei (Cestoda: Diphyllobothriidea), the causative agent of food/water-borne sparganosis, is restricted to Europe, where infected canids, felids, mustelids, suids, and reptiles have been documented from Poland, Ukraine, Belarus, Russia, Serbia, Estonia, Latvia, and Finland. The main objective of the current study was to map the molecular divergence of S. erinaceieuropaei from Finland using the complete sequences of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1 mtDNA). Seven cox1 haplotypes were determined in 15 tapeworms from Eurasian lynx (Lynx lynx) from three localities in southern Finland. In addition, the first inter-population study of S. erinaceieuropaei based on currently obtained data on cox1 from Finland and previously published data from Finland, Latvia, Ukraine, and Poland, was performed. The haplotype network showed a star-like pattern without specific subdivision of lineages according to the locality. Samples from Finland, Latvia, and Poland shared several haplotypes and formed the common Baltic lineage. The haplotype of S. erinaceieuropaei from Ukraine was unique and placed on a separate mutational pathway, suggesting a different lineage of the parasite.

TITLE: Interrelations génétiques de Spirometra erinaceieuropaei (Cestoda, Diphyllobothriidea), l'agent causal de la sparganose en Europe. ABSTRACT: La distribution géographique de Spirometra erinaceieuropaei (Cestoda : Diphyllobothriidea), l'agent causal de la sparganose d'origine alimentaire/hydrique, est limitée à l'Europe, où des canidés, félidés, mustélidés, suidés et reptiles infectés ont été documentés en Pologne, Ukraine, Biélorussie, Russie, Serbie, Estonie, Lettonie et Finlande. L'objectif principal de la présente étude était de cartographier la divergence moléculaire de S. erinaceieuropaei de Finlande à l'aide des séquences complètes du gène mitochondrial de la sous-unité 1 de la cytochrome c oxydase (ADNmt cox1). Sept haplotypes cox1 ont été déterminés chez quinze cestodes du Lynx d'Eurasie (Lynx lynx) de trois localités du sud de la Finlande. En outre, la première étude inter-populationnelle de S. erinaceieuropaei basée sur les données actuellement obtenues sur cox1 de Finlande et sur des données précédemment publiées de Finlande, Lettonie, Ukraine et Pologne, a été réalisée. Le réseau d'haplotypes a montré un motif en étoile sans subdivision spécifique des lignées selon la localité. Des échantillons de Finlande, Lettonie et Pologne partagent plusieurs haplotypes et forment la lignée commune de la Baltique. L'haplotype de S. erinaceieuropaei d'Ukraine est unique et placé sur une voie de mutation distincte suggérant une lignée différente du parasite.

Morphological observation and molecular phylogeny of Spirometra decipiens complex 1 (Cestoda: Diphyllobothriidae) found in cat from Chile.

The systematics of tapeworms in the genus Spirometra has been progressing with the accumulation of molecular genetics data, but the taxonomic status of many nominal species remains under debate. We report morphological and molecular-phylogenetic data for a Spirometra species collected from a domestic cat (Felis silvestris catus) in Chiloé Island, Chile. The Spirometra species was shown to be genetically conspecific with Spirometra decipiens complex 1 found in a Pampas fox (Lycalopex gymnocercus) from Argentina, and was closely related to a Hoary fox (Lycalopex vetulus) and rattlesnake (Crotalus durissus) from Brazil. Therefore, the presence of S. decipiens complex 1 was molecularly confirmed for the first time in Chile. The findings of the present study add useful information for the systematics of poorly known Spirometra species in South America.

A Case of Ocular Sparganosis in China: Episode of Migration from Muscle Cone to Subconjunctiva.

To improve our understanding of the migration of sparganum in humans, we report a case of ocular sparganosis having the migratory episode from the muscle cone to the subconjunctiva. A 34-year-old woman was admitted to the Hospital of Anhui Medical University (Hefei, China), in December 2019. She presented with conjunctival hemorrhage and recurrent pain in the left eye. A foreign body was found in the muscle cone of the eye. Two months later, a ribbon-like white material was found under the conjunctiva on slit-lamp examination. A long and slender, actively moving parasite was extracted by surgery. The extracted worm was approximately 8 cm long and 2 mm wide. The worm was whitish, wrinkled, ribbon shaped, and had a slightly enlarged scolex. The worm sample was morphologically identified as a plerocercoid larva (sparganum) of the Spirometra tapeworm. Her conjunctival blood suffusion and eye pain ceased within 1 week after operation. She has been in good health without any symptoms during the 2-year follow-up. A case of ocular sparganosis, in which larval worm migrated from the muscle cone to the subconjunctiva is reported from China.

Genome of the fatal tapeworm Sparganum proliferum uncovers mechanisms for cryptic life cycle and aberrant larval proliferation.

The cryptic parasite Sparganum proliferum proliferates in humans and invades tissues and organs. Only scattered cases have been reported, but S. proliferum infection is always fatal. However, S. proliferum's phylogeny and life cycle remain enigmatic. To investigate the phylogenetic relationships between S. proliferum and other cestode species, and to examine the mechanisms underlying pathogenicity, we sequenced the entire genomes of S. proliferum and a closely related non-life-threatening tapeworm Spirometra erinaceieuropaei. Additionally, we performed larvae transcriptome analyses of S. proliferum plerocercoid to identify genes involved in asexual reproduction in the host. The genome sequences confirmed that the S. proliferum has experienced a clearly distinct evolutionary history from S. erinaceieuropaei. Moreover, we found that nonordinal extracellular matrix coordination allows asexual reproduction in the host, and loss of sexual maturity in S. proliferum are responsible for its fatal pathogenicity to humans. Our high-quality reference genome sequences should be valuable for future studies of pseudophyllidean tapeworm biology and parasitism.

A case of human breast sparganosis diagnosed as Spirometra Type I by molecular analysis in Japan.

A 92-year-old Japanese woman presented with a mass in the left breast, and sparganosis was suspected by biopsy of the mass. The mass disappeared once, but it reappeared at the same site one year later. For a definitive diagnosis, the mass was surgically removed, and a sparganum-like worm was detected. The causative agent was confirmed as Spirometra Type I (most probably Spirometra mansoni) by mitochondrial DNA analysis. The serological examination also proved the case as sparganosis. Considering the presence of two Spirometra species (Type I and II) in Asia, particularly Japan, molecular analysis of the causative agents is highly recommended to understand the epidemiology, infection sources, and pathogenicity in humans in both species, if the parasite specimens are available.

Mitochondrial Genome of Spirometra theileri Compared with Other Spirometra Species.

This study was carried out to provide information on the taxonomic classification and analysis of mitochondrial genomes of Spirometra theileri. One strobila of S. theileri was collected from the intestine of an African leopard (Panthera pardus) in the Maswa Game Reserve, Tanzania. The complete mtDNA sequence of S. theileri was 13,685 bp encoding 36 genes including 12 protein genes, 22 tRNAs and 2 rRNAs with absence of atp8. Divergences of 12 protein-coding genes were as follow: 14.9% between S. theileri and S. erinaceieuropaei, 14.7% between S. theileri and S. decipiens, and 14.5% between S. theileri with S. ranarum. Divergences of 12 proteins of S. theileri and S. erinaceieuropaei ranged from 2.3% in cox1 to 15.7% in nad5, while S. theileri varied from S. decipiens and S. ranarum by 1.3% in cox1 to 15.7% in nad3. Phylogenetic relationship of S. theileri with eucestodes inferred using the maximum likelihood and Bayesian inferences exhibited identical tree topologies. A clade composed of S. decipiens and S. ranarum formed a sister species to S. erinaceieuropaei, and S. theileri formed a sister species to all species in this clade. Within the diphyllobothridean clade, Dibothriocephalus, Diphyllobothrium and Spirometra formed a monophyletic group, and sister genera were well supported.

First Clinical Cases of Spirometrosis in Two Cats in Korea.

This study reports the first two clinical cases of spirometrosis caused by Spirometra sp. in cats in Korea. In these two cases, the cats vomited, and long proglottids of tapeworm were recovered. The sick cats presented with anorexia and lethargy. However, they unexpectedly showed no diarrhea, which is the main symptom of spirometrosis. Based on a fecal floatation test as well as morphological and molecular analyses, the parasite was diagnosed as Spirometra sp. The 2 cases were treated with praziquantel. This study suggests regular monitoring of health and deworming in companion animals, even when animals are well cared for, with regular preventive medication. Additionally, spirometrosis should be considered in the differential diagnosis in cases of gastrointestinal symptoms in Spirometra endemic areas.

Identification of an enolase gene and its physiological role in Spirometra mansoni.

Enolase is a crucial enzyme involved in the glycolytic pathway and gluconeogenesis in parasites. It also has been reported to function as a plasminogen receptor and may be involved in tissue invasion. In this study, the biochemical properties of the enolase of Spirometra mansoni (Smenolase) were investigated. The Smenolase gene was found to cluster closely with the enolase genes of Clonorchis sinensis and Echinococcus granulosus, and some functional motifs were identified as conserved. Smenolase was confirmed to be a component of the secretory/excretory products (ESPs) and a circulating antigen of spargana. Recombinant Smenolase expressed in vitro was able to bind to human plasminogen. Smenolase was detected in the eggs, testicles, and vitellaria of adult worms and the tegument of spargana. The transcription level of Smenolase was highest at the gravid proglottid stage. When spargana were cultured with glucose of different concentration in vitro, it was observed that the expression levels of Smenolase in the low-glucose groups were consistent with that of Smenolase in vivo. These results indicate that Smenolase is a critical enzyme involved in supplying energy to support the development and reproduction of the parasite, and it may also play a role in sparganum invasion.

Helminth infracommunity in a maned wolf, Chrysocyon brachyurus, from the humid Chaco, Argentina.

Parasitism is an important factor in conservation worldwide, especially for endangered species, as it can affect host populations by reducing growth rates, fecundity and affecting nutritional status. The maned wolf Chrysocyon brachyurus, a near-threatened species, is distributed across different habitats from the south Amazonian forest in Brazil to northern Argentina, Paraguay and eastern Bolivia. Most studies on gastrointestinal parasites in wild maned wolves are based on coprological findings, and there is little information about adult parasites and parasite species richness. Therefore, this study aims to expand the knowledge about the helminth infracommunity of a maned wolf, describe adult parasites, and explore parasitic coinfections. We performed a necropsy of an adult individual found road killed in Chaco province, Argentina. We collected adult worms from subcutaneous tissues, from the right ventricle, kidneys, and intestine. The parasites were morphologically identified as Dirofilaria immitis, Dioctophyma renale and Spirometra sp. respectively. To our knowledge, this is the first record for adults of Dirofilaria immitis in maned wolves. Filarioids were located within the heart as well as in subcutaneous tissues with mature females containing uterine microfilariae; this study also adds C. brachyurus as a new host for Spirometra sp. in Argentina. Considering that the three helminths found herein are zoonotic and that the maned wolf is an endangered canid, it is essential provide baseline information to determine the risk factors involved in the transmission and extend the studies to sympatric wild and domestic canids in the area.