Cestodiasis in 2 Puerto Rican crested anoles.

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.

Molecular identification of sparganum of Spirometra mansoni isolated from the abdominal cavity of a domestic cat in Vietnam.

Cats normally play a role of the definitive host in which the plerocercoid (sparganum), the second larval form of Spirometra spp., develops into an adult in the intestines. However, some cases of cats with visceral or subcutaneous sparganosis were sporadically reported worldwide. We herein documented the discovery of a sparganum in abdominal cavity of a domestic cat during a surgery of dystocia. The larva was molecularly identified as Spirometra mansoni, belonging to Type I, that was recently misidentified to be S. erinaceieuropaei in several Asian countries. This is the first report for sparganum of S. mansoni in the cat. The future study is necessary to provide further insights into the species of Spirometra causing sparganosis and spirometrosis in humans and other animals.

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.

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.

Ocular Sparganosis: The First Report of Spirometra ranarum in Thailand.

A 22-year-old Thai man from the Northeast region presented with acute eye swelling, itching, and discharge on his left eye. He was suspected of having gnathostomiasis and treated with albendazole and prednisolone for 3 weeks. Nine months later, he was treated with high-dose oral prednisolone for the preliminary and differential diagnoses with thyroid-associated orbitopathy and lymphoma. He had been administered prednisolone intermittently over a few years. Then he developed a painless movable mass at the left upper eyelid and recurrent pseudotumor oculi was suspected. The surgical removal of the mass was performed. A white pseudosegmented worm revealed a definite diagnosis of ocular sparganosis by a plerocercoid larva. Molecular diagnosis of the causative species was made based on the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. Proper technique of extraction and amplification of short fragments DNA from formalin-fixed paraffin-embedded tissue successfully identified parasite species. The result from the sequencing of the PCR-amplified cox1 fragments in this study showed 99.0% sequence homology to Spirometra ranarum. This is the first report of S. ranarum in Thailand.

Molecular cloning and characterization of plerocercoid-immunosuppressive factor from Spirometra erinaceieuropaei.

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.

Subcutaneous Sparganosis on Abdomen Mimicking Multiple Lipomas.

Human sparganosis is a food-borne zoonosis mainly caused by the plerocercoid belonging to the genus Spirometra. The most common clinical sign of sparganosis is a subcutaneous mass in the trunk including abdominal or chest wall. The mass may be mistaken for a malignant tumor, thereby causing difficulty in terms of diagnosis and treatment. A 66-year-old woman visited our clinic for the removal of a lipoma-like mass. It was movable, hard, and painless. We identified 2 white mass, measuring 0.2×4 cm and 0.2×1 cm. Pathologic findings indicated the white mass was a sparganum. She recalled having eaten a raw frog approximately 60 years before. A 35-year-old who lived North Korea was also presented to our clinic with an asymptomatic nodule on her abdomen. Intraoperatively, we found sparganum approximately 24 cm size. Subcutaneous masses are associated with clinical signs of inflammation or they may mimic a soft tissue neoplasm. While the incidence rate of sparganosis has decreased with economic development and advancements in sanitation, surgeons still encounter patients with sparganosis in the clinical setting. Therefore, a careful history is required in order to diagnose sparganosis.

Identification of sparganosis based on next-generation sequencing.

The incidence of sparganosis, a parasitic disease caused by plerocercoid larvae of the genus Spirometra, has gradually risen worldwide (especially in remote areas) in recent years. Pulmonary and pleural sparganosis, as well as other sites of infestation, including the subcutaneous tissues, the abdominal viscera, brain and eyes, has been reported. In clinical practice, due to the atypical signs and symptoms as well as limited laboratory approaches for the specific detection of sparganum, sparganosis is often misdiagnosed. In the present study, an 11-year-old girl visited the Department of Infectious Diseases in Shanghai Children's Medical Center for recurrent shoulder and chest pain and shortness of breath. Imaging tests demonstrated bilateral pleural and pericardial effusion, enlarged lymph nodes in front of the tracheal carina, and infection of the left lower lobe. Sparganum were not observed in the dissected soft tissue at the root of the right thigh with naked-eye and light microscopy examination. Histologic examination revealed granulomatous inflammation and tunnel-like necrosis with eosinophilic, neutrophilic and lymphocytic infiltration. Although the patient's serum was positive for sparganum antibodies, the diagnosis of sparganosis was not confirmed for more than three months. Ultimately, genomic DNA of Spirometra erinaceieuropaei was detected in the mass at the root of the right thigh using next-generation sequencing (NGS), confirming the diagnosis of sparganosis. The patient was treated with praziquantel (150 mg/kg/day) without recurrence after an eight-month follow-up. We present, for the first time, a study of human sparganosis diagnosed using NGS, which provided a clinically actionable diagnosis of a specific infectious disease from an uncommon pathogen.

Using the small subunit of nuclear ribosomal DNA to reveal the phylogenetic position of the plerocercoid larvae of Spirometra tapeworms.

Although medically important, the systematics of Spirometra and the taxonomic position of S. erinaceieuropaei remain unclear. In this study, the 18S rDNA gene of S. erinaceieuropaei sparganum from naturally infected frogs caught in 14 geographical locations of China was sequenced. In addition, all available 18S sequences of the family Diphyllobothriidae in the Genbank database were included to reconstruct the phylogeny of diphyllobothriid tapeworms. The secondary structure model of the 18S rDNA was also predicated to further explore the sequence variation. Phylogenetic analyses were performed using maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) methods. The intraspecific divergences of 18S rDNA in Chinese sparganum isolates ranged from 0.0 to 0.4%. Regions of V2, V4 and V7 were the most variable regions in the secondary structure of 18S rDNA. With the exception of genera Duthiersia and Probothriocephalus, other genera (i.e., Adenocephalus, Diphyllobothrium, Diplogonoporus, Duthiersia, Schistocephalus and Spirometra) selected in the Diphyllobothriidae shared similar topologies of V2, V4 and V7 structures. The topology of generated phylogenetic trees revealed close relationships among Adenocephalus, Digramma, Diphyllobothrium, Diplogonoporus, Ligula, Sparganum and Spirometra. The exact phylogenetic position of Spirometra species should be further analyzed with more sampling and more useful molecular markers.

The genome of the sparganosis tapeworm Spirometra erinaceieuropaei isolated from the biopsy of a migrating brain lesion.

BACKGROUND: Sparganosis is an infection with a larval Diphyllobothriidea tapeworm. From a rare cerebral case presented at a clinic in the UK, DNA was recovered from a biopsy sample and used to determine the causative species as Spirometra erinaceieuropaei through sequencing of the cox1 gene. From the same DNA, we have produced a draft genome, the first of its kind for this species, and used it to perform a comparative genomics analysis and to investigate known and potential tapeworm drug targets in this tapeworm. RESULTS: The 1.26 Gb draft genome of S. erinaceieuropaei is currently the largest reported for any flatworm. Through investigation of ß-tubulin genes, we predict that S. erinaceieuropaei larvae are insensitive to the tapeworm drug albendazole. We find that many putative tapeworm drug targets are also present in S. erinaceieuropaei, allowing possible cross application of new drugs. In comparison to other sequenced tapeworm species we observe expansion of protease classes, and of Kuntiz-type protease inhibitors. Expanded gene families in this tapeworm also include those that are involved in processes that add post-translational diversity to the protein landscape, intracellular transport, transcriptional regulation and detoxification. CONCLUSIONS: The S. erinaceieuropaei genome begins to give us insight into an order of tapeworms previously uncharacterized at the genome-wide level. From a single clinical case we have begun to sketch a picture of the characteristics of these organisms. Finally, our work represents a significant technological achievement as we present a draft genome sequence of a rare tapeworm, and from a small amount of starting material.

Diagnostic efficacy of a recombinant cysteine protease of Spirometra erinacei larvae for serodiagnosis of sparganosis.

The mature domain of a cysteine protease of Spirometra erinacei plerocercoid larva (i.e., sparganum) was expressed in Escherichia coli, and its value as an antigen for the serodiagnosis of sparganosis was investigated. The recombinant protein (rSepCp-1) has the molecular weight of 23.4 kDa, and strongly reacted with the sparganum positive human or mice sera but not with negative sera by immunoblotting. ELISA with rSepCp-1 protein or sparganum crude antigen (SeC) was evaluated for the serodiagnosis of sparganosis using patient's sera. The sensitivity and specificity of ELISA using rSepCp-1 protein were 95.0% (19/20) and 99.1% (111/112), respectively. In contrast, the sensitivity and specificity of ELISA with SeC were 100% (20/20) and 96.4% (108/112), respectively. Moreover, in experimentally infected mice, the sensitivity and specificity of both ELISA assays were 100% for the detection of anti-sparganum IgG. It is suggested that the rSepCp-1 protein-based ELISA could provide a highly sensitive and specific assay for the diagnosis of sparganosis.

Bioinformatic analysis for structure and function of TCTP from Spirometra mansoni.

OBJECTIVE: To predict structure and function of translationally controlled tumor protein (TCTP) from Spirometra mansoni by bioinformatics technology, and to provide a theoretical basis for further study. METHODS: Open reading frame (ORF) of EST sequence from Spirometra mansoni was obtained by ORF finder and was translated into amino acid residue by DNAclub. The structure domain was analyzed by Blast. By the method of online analysis tools: Protparam, InterProScan, protscale, SignalP-3.0, PSORT II, BepiPred, TMHMM, VectorNTI Suite 9 packages and Phyre2, the structure and function of the protein were predicted and analyzed. RESULTS: The results showed that the EST sequence was Sm TCTP with 173 amino acid residues, theoretical molecular weight was 19 872.0 Da. The protein has the closest evolutionary status with Clonorchis sinensis, Schistosoma mansoni, and Schistosoma japonicum. Then it had no signal peptide site and transmembrane domain. Secondary structure of TCTP contained two α -helices and eight ß -strands. CONCLUSIONS: Sm TCTP was a variety of biological functions of protein that may be used as a vaccine candidate molecule and drug target.

Spirometra erinacei / S. erinaceieuropaei in a feral cat in Manawatu with chronic intermittent diarrhoea.

CASE HISTORY: A feral cat captured in the Manawatu region of New Zealand was treated for worms and fleas, and kept confined in a metabolic cage. It showed good appetite and weight gain but had intermittent watery, yellow diarrhoea. CLINICAL FINDINGS: Clinical examination under sedation was unremarkable and routine blood tests showed no significant abnormalities. The cat was negative for feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV). Different canned cat foods did not alter the course of the diarrhoea, and the cat was euthanised 6 months after capture. At necropsy, two sections of adult Spirometra tapeworms were found in the jejunum and typical Spirometra eggs were found in colonic contents. Molecular identification of the parasite was undertaken, using the cytochrome-c oxidase subunit-1 gene (cox1) sequence. DIAGNOSIS: Chronic intermittent diarrhoea associated with Spirometra erinacei / S. erinaceieuropaei infection. CLINICAL RELEVANCE: Spirometra has not been reported in New Zealand before but has been associated with gastrointestinal disease in cats in other parts of the world. It requires species targeted treatment to be eliminated effectively, and is zoonotic. Diagnosis could be difficult for clinicians who are not familiar with the parasite and its life cycle.

Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress interleukin-1beta gene expression in murine macrophages.

The present study shows that ES products from plerocercoids of Spirometra erinaceieuropaei suppressed interleukin-1beta mRNA expression in lipopolysaccharide-stimulated RAW 264.7 macrophages in the absence or presence of a cyclic AMP analogue, dibutyryl cyclic AMP. Investigation using the inhibitors of mitogen-activated protein kinase (MAPK) pathways revealed that extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase pathways are crucial for full induction of interleukin-1beta mRNA expression. ES products additionally suppressed interleukin-1beta mRNA expression in the cells treated with p38 mitogen-activated protein kinase inhibitor (SB203580) or extracellular signal-regulated protein kinase 1/2 inhibitor (PD98059). Western blot analysis showed that dibutyryl cyclic AMP enhanced lipopolysaccharide-induced phosphorylation of extracellular signal-regulated protein kinase 1/2, p38 mitogen-activated protein kinase and cyclic AMP responsive element binding protein (CREB) and, in turn, we demonstrated that ES products reduced the lipopolysaccharide and dibutyryl cyclic AMP-induced phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase, but not cyclic AMP responsive element binding protein. These data demonstrate that ES products from the plerocercoids of S. erinaceieuropaei may evade induction of interleukin-1beta mRNA by inhibiting extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase pathways in lipopolysaccharide and/or dibutyryl cyclic AMP-stimulated macrophages.

[Molecular cloning of a cDNA encoding an antigenic polypeptide containing repeat units of Spirometra erinaceieuropaei plerocercoid].

AIM: To study the gene structure encoding some antigenic polypeptides of plerocercoid of Spirometra erinaceieuropaei (SEP). METHODS: A cDNA library constructed from SEP was immunoscreened using mouse anti-SEP polyclonal antibody. The gene structure was analyzed by computer after the insert of positive clone was subcloned and the nucleotide sequences of the insert were determined. The length of mRNA hybridized with cDNA was detected by Northern blotting. RESULTS: A cDNA clone of 1,084 bp encoding an antigenic polypeptide was isolated. The clone contained one open reading frame composed of 828 bp encoding 276 amino acids. The open reading frame contained tandem repeating unit of 123 bp which appeared 5 times in this clone. The 41 amino acids deduced from 123 bp repeating unit consisted of 53.7% of hydrophobic amino acid residues. In Northern blot assay of poly (A)+RNA, a strong band of about 1.1 kb and a weak band of about 1.7 kb were found in the plerocercoids but no band was found in the adult worms. CONCLUSION: The repeating element derived from the plerocercoids may be associated with the migration of the plerocercoids in the host tissue. The polypeptides with repeating element may act as evasive antigens of the parasite to escape from the destruction of host immune reactions.

Molecular cloning and expression of the gene encoding a cysteine proteinase of Spirometra erinacei.

A cDNA library constructed from plerocercoid of Spirometra erinacei (SEP) was immunoscreened using rabbit anti-plerocercoid proteinase polyclonal antibody. A 1.0-kb cDNA clone encoding a cysteine proteinase composed of 336 amino acids was isolated. The amino acid sequence predicted from the cDNA showed significant homology with human and mouse cathepsin L. N-terminal amino acid sequence of the native cysteine proteinase extracted from SEP was the same as that of mature proteinase predicted from the cloned gene. The gene encoding the proteinase was characterized by Southern and Northern blot analysis using the cDNA as a probe. The proteinase with a molecular mass of 34 kDa was demonstrated in in vitro translation products using anti-proteinase polyclonal antibody. A fusion protein derived from the cDNA synthesized by Escherichia coli (TB1) using the expression vector, pMAL-c2 was identified as an immunodominant antigen by epitope-selection method and had no cross-reactivity with other parasite-infected sera. A genomic DNA library derived from SEP was screened by the colony hybridization technique using the cDNA probe. A gene with 4.5 kb encoding the proteinase was obtained, which comprised three exons and two introns.