The first records of Spirometra erinaceieuropaei (Cestoda: Diphyllobothriidae), a causative agent of human sparganosis, in Latvian wildlife.

Diphyllobothriid tapeworms of the genus Spirometra are causative agents of sparganosis, food-borne zoonotic parasitic disease. They have been recorded in broad spectrum of hosts, including humans, in all continents except Antarctica. Spirometra tapeworms have been intensively studied in several Asian countries; however, they have been rather neglected in Europe. The aim of this study was to provide a pilot screening of Spirometra spp. in Latvia, where data on sparganosis are not available. Tapeworms morphologically identified as diphyllobothriid species were isolated from grey wolves Canis lupus and Eurasian lynxes Lynx lynx from Latvia during the hunting periods 2013-2019. The parasites were subjected to molecular genotyping using sequences of the partial large (LSU rDNA; 615 bp) and small (SSU rDNA; 720 bp) subunits of the nuclear ribosomal RNA gene and complete (1566 bp) cytochrome c oxidase subunit I gene of the mitochondrial DNA (cox1 mtDNA). Analyses of both ribosomal subunits of 13 tapeworms revealed no intraspecific variation within the respective rDNA subunits. On the other hand, sequence analysis of mitochondrial cox1 revealed intraspecific polymorphism displayed by 12 cox1 haplotypes. Comparison of the current data with sequences of the corresponding DNA regions deposited in the GenBank revealed 99.3-99.5% (LSU rDNA), 99.2% (SSU rDNA) and 99.6-100% (cox1 mtDNA) identity of studied tapeworms with Spirometra erinaceieuropaei, which provided the first confirmation of this diphyllobothriid tapeworm in Latvia. Since S. erinaceieuropaei is probably prevalent in Latvian wildlife and may also occur in other potential host species, further studies are needed in order to acquire complex data on its geographic distribution and transmission in the natural environment of Latvia, as well as on the spectrum of its intermediate, paratenic, and definitive hosts.

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.

Morphological and Molecular Identification of Spirometra Tapeworms (Cestoda: Diphyllobothriidae) from Carnivorous Mammals in the Serengeti and Selous Ecosystems of Tanzania.

Spirometra tapeworms (Cestoda: Diphyllobothriidae) collected from carnivorous mammals in Tanzania were identified by the DNA sequence analysis of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and internal transcribed spacer 1 (ITS1), and by morphological characteristics. A total of 15 adult worms were collected from stool samples and carcasses of Panthera leo, Panthera pardus, and Crocuta crocuta in the Serengeti and Selous ecosystems of Tanzania. Three Spirometra species: S. theileri, S. ranarum and S. erinaceieuropaei were identified based on morphological features. Partial cox1 sequences (400 bp) of 10 specimens were revealed. Eight specimens showed 99.5% similarity with Spirometra theileri (MK955901), 1 specimen showed 99.5% similarity with the Korean S. erinaceieuropaei and 1 specimen had 99.5% similarity with Myanmar S. ranarum. Sequence homology estimates for the ITS1 region of S. theileri were 89.8% with S. erinaceieuropaei, 82.5% with S. decipiens, and 78.3% with S. ranarum; and 94.4% homology was observed between S. decipiens and S. ranarum. Phylogenetic analyses were performed with 4 species of Spirometra and 2 species of Dibothriocephalus (=Diphyllobothrium). By both ML and BI methods, cox1 and ITS1 gave well supported, congruent trees topology of S. erinaceieuropaei and S. theileri with S. decipiens and S. ranarum forming a clade. The Dibothriocephalus species were sisters of each other and collectively forming successive outgroups. Our findings confirmed that 3 Spirometra species (S. theileri, S. ranarum, and S. erinaceieuropaei) are distributed in the Serengeti and Selous ecosystems of Tanzania.

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.

A Retrieved Sparganum of Spirometra erinaceieuropaei from a Korean Man during Mechanical Thrombectomy.

Human sparganosis is a zoonotic disease caused by infection and migration of the plerocercoid of Spirometra spp. Although sparganosis were reported from most parts of the body, the sparganum parasitizing inside cerebral artery is remarkably uncommon. We report a case of cerebral intravascular sparganosis in an elderly patient with acute ischemic stroke who was diagnosed by retrieving sparganum during mechanical thrombectomy. Finally, the parasites were identified as Spirometra erinaceieuropaei using multiplex PCR and cox1 gene sequencing.

Prevalence and molecular characterization of Spirometra erinaceieuropaei spargana in snakes in Hunan Province, China.

Sparganosis is an important foodborne parasitic zoonosis; however, few reports on the prevalence of snake-infecting plerocercoids from Hunan province in China are available. Therefore, we investigated the prevalence of spargana infection in wild snakes from this region in 2018, and identified an astonishing prevalence rate of 91.7% (344/375). Spargana parasites were found in 99.1% of Zaocys dhumnades, 94.1% of Elaphe carinata and 86.7% of Elaphe taeniura. Parasites exhibited various distributions: 50% were located in muscular tissue, 32.1% in subcutaneous tissue and 17.9% in the coelomic cavity. To identify the specific status of spargana collected from wild snakes, partial mitochondrial cytochrome c oxidase subunit 1 (cox1) gene sequences were amplified, sequenced and analysed. Sequence variations for cox1 among all the examined plerocercoids ranged between 0.0 and 2.9%, with 21 variable sites identified (4.71%, 21/446). Phylogenetic analyses identified that all plerocercoids isolated from Hunan province were Spirometra erinaceieuropaei. This is the first report of S. erinaceieuropaei infection in snakes in Hunan province. The risks and harms of sparganosis should be publicized, and illegal wildlife trade should be controlled.

Cerebral sparganosis presenting with atypical postcontrast magnetic resonance imaging findings: a case report and literature review.

BACKGROUND: Sparganosis, a rare and severe parasitic infection caused by the larvae of Spirometra species or simply sparganum, generally involves subcutaneous tissue or muscle. But occasionally, sparganum can also invade the human brain, resulting in cerebral sparganosis. CASE PRESENTATION: A 33-year-old woman presented with a 10-day history of headache. Postcontrast magnetic resonance imaging (MRI) revealed an irregular lesion with enhancement and the tunnel-shaped focus extending to the contralateral hemiphere. Cerebrospinal fluid (CSF) analysis disclosed pleocytosis (166 cells/µL) and an elevated protein concentration (0.742 g/L). Enzyme-linked immunosorbent assay (ELISA) revealed positive sparganum-specific antibody in both blood and CSF. Finally, the diagnosis of cerebral sparganosis was comfirmed. She received praziquantel treatment and got a favorable outcome during six-month follow-up. CONCLUSIONS: Irregular enhancement and the tunnel sign that extends to the contralateral hemisphere on postconstrast MRI are unusual presentations of cerebral sparganosis. ELISA for sparganum-specific antibody can help confirm the diagnosis. Although surgery is the preferred treatment for cerebral sparganosis, praziquantel might also achieve satisfying outcomes.

Mitochondrial DNA Sequence Variability of Spirometra Species in Asian Countries.

Mitochondrial DNA sequence variability of Spirometra erinaceieuropaei in GenBank was observed by reinvestigation of mitochondrial cox1 and cytb sequences. The DNA sequences were analyzed in this study, comprising complete DNA sequences of cox1 (n=239) and cytb (n=213) genes. The 10 complete mitochondrial DNA sequences of Spirometra species were compared with those of Korea, China and Japan. The sequences were analyzed for nucleotide composition, conserved sites, variable sites, singleton sites and parsimony-informative sites. Phylogenetic analyses was done using neighbor joining, maximum parsimony, Bayesian inference and maximum-likelihood on cox1 and cytb sequences of Spirometra species. These polymorphic sites identified 148 (cox1) and 83 (cytb) haplotypes within 239 and 213 isolates from 3 Asian countries. Phylogenetic tree topologies were presented high-level confidence values for the 2 major branches of 2 Spirometra species containing S. erinaceieuropaei and S. decipiens, and S. decipiens sub-clades including all sequences registered as S. erinaceieuropaei in cox1 and cytb genes. These results indicated that mitochondrial haplotypes of S. erinaceieuropaei and S. decipiens were found in the 3 Asian countries.

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.

Genetic Identification of Spirometra erinaceieuropaei Spargana in Liaoning and Hubei Provinces, PR China.

Spargana were collected from human and frogs in Liaoning and Hubei Provinces, China. PCR amplification and direct sequencing of A cox1 fragment was PCR-amplified from genomic DNA extracted from 7 specimens (5 from humans and 2 from frogs). The cox1 fragment (390 bp) showed 97-100% similarity to the reference sequence of S. erinaceieuropaei and 88-89% to the reference sequence of S. decipiens. There were 1-12 bases different between these worms, but no obvious genetic variation (0-3.3%) to the references. There was little difference of cox1 gene between sparganum samples of humans and frogs (1-3%). This study is the first report on S. erinaceieuropaei spargana from humans in Liaoning and Hubei Provinces.

Identity of Spirometra theileri from a Leopard (Panthera pardus) and Spotted Hyena (Crocuta crocuta) in Tanzania.

In the present study, a Spirometra species of Tanzania origin obtained from an African leopard (Panthera pardus) and spotted hyena (Crocuta crocuta) was identified based on molecular analysis of cytochrome c oxidase I (cox1) and NADH dehydrogenase subunit I (nad1) as well as by morphological observations of an adult tapeworm. One strobila and several segments of a Spirometra species were obtained from the intestine of an African male leopard (Panthera pardus) and spotted hyena (Crocuta Crocuta) in the Maswa Game Reserve of Tanzania. The morphological characteristics of S. theileri observed comprised 3 uterine loops on one side and 4 on the other side of the mid-line, a uterine pore situated posterior to the vagina and alternating irregularly either to the right or left of the latter, and vesicular seminis that were much smaller than other Spirometra species. Sequence differences in the cox1 and nad1 genes between S. theileri (Tanzania origin) and S. erinaceieuropaei were 10.1% (cox1) and 12.0% (nad1), while those of S. decipiens and S. ranarum were 9.6%, 9.8% (cox1) and 13.0%, 12.6% (nad1), respectively. The morphological features of the Tanzania-origin Spirometra specimens coincided with those of S. theileri, and the molecular data was also consistent with that of S. theileri, thereby demonstrating the distribution of S. theileri in Tanzania. This places the leopard (Panthera pardus) and spotted hyena (Crocuta Crocuta) as new definitive hosts of this spirometrid tapeworm.

The first case of genetically confirmed sparganosis (Spirometra erinaceieuropaei) in European reptiles.

Sparganosis is a zoonosis caused by the spargana (larvae) of Spirometra sp. (Diphyllobothriidae). Reptiles are particularly important vectors for the transmission of this parasite in Asia; however, their role in sparganosis spread in European wildlife is unrecognized. We investigated the infection of reptiles with Spirometra sp. in NE Poland, where several mammalian hosts have been identified recently and in the past. Of the 59 dead reptiles, plerocercoids were found in two grass snakes (Natrix natrix) from the Bialowieza Primeval Forest (BPF). The Spirometra erinaceieuropaei species was genetically confirmed using the evolutionary conserved nuclear 18S rRNA gene, and then compared to GenBank deposits. The sequences were identical to previously investigated Spirometra sp. found in Eurasian badger and wild boar from BPF. Our finding is the first genetically confirmed record of Spirometra sp. in reptiles in Europe. Since reptiles are often a component of mammalian diet, they can be a source of Spirometra tapeworm infection in European wildlife; however, further studies are needed to investigate the prevalence of infection in reptiles and other non-mammalian hosts.

Molecular and Morphologic Identification of Spirometra ranarum Found in the Stool of African Lion, Panthera leo in the Serengeti Plain of Tanzania.

The present study was performed with morphological and molecular analysis (cox1 and nad1 mitochondrial genes) to identify the proglottids of spirometrid tapeworm found in the stool of an African lion, Panthera leo, in the Serengeti plain of Tanzania. A strand of tapeworm strobila, about 75 cm in length, was obtained in the stool of a male African lion in the Serengeti National Park (34˚ 50' E, 02˚ 30' S), Tanzania, in February 2012. The morphological features of the adult worm examined exhibited 3 uterine coils with a bow tie appearance and adopted a diagonal direction in the second turn. The posterior uterine coils are larger than terminal uterine ball and the feature of uteri are swirling rather than spirally coiling. The sequence difference between the Spirometra species (Tanzania origin) and S. erinaceieuropaei (GenBank no. KJ599680) was 9.4% while those of S. decipiens (GenBank no. KJ599679) differed by 2.1% in the cox1 and nad1 genes. Phylogenetic tree topologies generated using the 2 analytic methods were identical and presented high level of confidence values for the 3 major branches of the 3 Spirometra species in the cox1 gene. The morphological and molecular findings obtained in this study were nearly coincided with those of S. ranarum. Therefore, we can know for the first time that the African lion, Panthera leo, is to the definitive host of this tapeworm.

Genetic and Morphologic Identification of Spirometra ranarum in Myanmar.

In the present study, we identified a Spirometra species of Myanmar origin (plerocercoid) by molecular analysis using mitochondrial cox1 and nad1 genes, as well as by morphological observations of an adult tapeworm. Spargana specimens were collected from a paddy-field in Taik Kyi Township Tarkwa Village, Yangon, Myanmar in December 2017. A total of 5 spargana were obtained from 20 frogs Hoplobatrachus rugulosus; syn: Rana rugulosa (Wiegmann, 1834) or R. tigrina (Steindachner, 1867). The plerocercoids were used for experimental infection of a dog. After 4 weeks of infection, an adult tapeworm was recovered from the intestine of the dog. Morphologically, the distinct features of Spirometra sp. (Myanmar origin) relative to S. erinaceieuropaei and S. decipiens include a uterine morphology comprising posterior uterine coils that larger than the terminal uterine ball and coiling of the uteri diagonally (swirling) rather than spirally. The cox1 sequences (1,566 bp) of the Myanmar-origin Spirometra species showed 97.9% similarity to a reference sequence of S. decipiens (GenBank no. KJ599679) and 90.5% similarity to a reference sequence of S. erinaceieuropaei (GenBank no. KJ599680). Phylogenetic tree topologies were identical and presented high confidence level of values for the 3 major branches of the 3 Spirometra species in cox1 and nad1 genes. These results indicated that Myanmar-origin Spirometra species coincided with those of S. ranarum and may be considered as a valid species.

Molecular Genetic Findings of Spirometra decipiens and S. ranarum in Korea.

The taxonomy of Spirometra species has been controversial despite the medical and veterinary importance. Currently, only a few Spirometra species are considered valid species in the genus Spirometra. In the present study, the distribution of Spirometra species obtained from animals in Korea were identified by molecular analysis of the mitochondrial cytochrome c oxidase I (cox1) gene. A total of 28 Spirometra species specimens were analyzed. These were all collected between 1973 and 2008 in the Republic of Korea. Mitochondrial cox1 sequences were examined for a total of 28 specimens comprising 14 S. decipiens and 14 S. ranarum. The difference in partial cox1 sequences (316 bp) between S. erinaceieuropaei (KJ599680) and S. ranarum (this study) was 9.3%, while that between S. decipiens (KJ599679) and S. ranarum (this study) was 2.2%. Genetic analyses identified 2 Spirometra species in animals such as cat, leopard cat, dog, duck and snake in Korea as S. decipiens and S. ranarum. S. decipiens and S. ranarum were present in Gyeongnam Province (P), Jeonnam P, Gangwon P, Chungbuk P, and Seoul. S. decipiens was found in tadpoles, snakes, ducks, cats, leopard cats and dogs, while S. ranarum was found in cats and dogs. The ratio of S. decipiens:S. ranarum calculated from the molecular data was 14:14 (or 1:1). These results indicate that S. decipiens and S. ranarum are sympatrically distributed in Korea.

Spirometra decipiens (Cestoda: Diphyllobothriidae) Collected in A Heavily Infected Stray Cat from the Republic of Korea.

Morphological and molecular characteristics of spirometrid tapeworms, Spirometra decipiens, were studied, which were recovered from a heavily infected stray cat road-killed in Eumseong-gun, Chungcheongbuk-do (Province), the Republic of Korea (=Korea). A total of 134 scolices and many broken immature and mature proglottids of Spirometra tapeworms were collected from the small intestine of the cat. Morphological observations were based on 116 specimens. The scolex was 22.8-32.6 mm (27.4 mm in average) in length and small spoon-shape with 2 distinct bothria. The uterus was coiled 3-4 times, the end of the uterus was ball-shaped, and the vaginal aperture shaped as a crescent moon was closer to the cirrus aperture than to the uterine aperture. PCR amplification and direct sequencing of the cox1 target fragment (377 bp in length and corresponding to positions 769-1,146 bp of the cox1 gene) were performed using total genomic DNA extracted from 134 specimens. The cox1 sequences (377 bp) of the specimens showed 99.0% similarity to the reference sequence of S. decipiens and 89.3% similarity to the reference sequence of S. erinaceieuropaei. In the present study, we report a stray cat heavily infected with S. decipiens identified by mitochondrial cox1 sequence analysis and morphological examinations of the adult worms.

Molecular Identification of Spirometra erinaceieuropaei Tapeworm in Cases of Human Sparganosis, Hong Kong.

Human sparganosis is a foodborne zoonosis endemic in Asia. We report a series of 9 histologically confirmed human sparganosis cases in Hong Kong, China. All parasites were retrospectively identified as Spirometra erinaceieuropaei. Skin and soft tissue swelling was the most common symptom, followed by central nervous system lesions.

Molecular Detection of Spirometra decipiens in the United States.

The genus Spirometra belongs to the family Diphyllobothriidae and order Pseudophyllidea, and includes intestinal parasites of cats and dogs. In this study, a plerocercoid labeled as Spirometra mansonoides from the USA was examined for species identification and phylogenetic analysis using 2 complete mitochondrial genes, cytochrome c oxidase I (cox1) and NADH dehydrogenase subunit 3 (nad3). The cox1 sequences (1,566 bp) of the plerocercoid specimen (USA) showed 99.2% similarity to the reference sequences of the plerocercoid of Korean Spirometra decipiens (GenBank no. KJ599679), and 99.1% similarity in regard to nad3 (346 bp). Phylogenetic tree topologies generated using 4 analytical methods were identical and showed high confidence levels with bootstrap values of 1.00, 100%, 100%, and 100% for Bayesian inference (BI), maximum-likelihood (ML), neighbor-joining (NJ), and maximum parsimony (MP) methods, respectively. Representatives of Diphyllobothrium and Spirometra species formed a monophyletic group, and the sister-genera status between these species was well supported. Trapezoic proglottids in the posterior 1/5 region of an adult worm obtained from an experimentally infected cat were morphologically examined. The outer uterine loop of the uterus coiling characteristically consisted of 2 complete turns. The results clearly indicated that the examined Spirometra specimen from the USA matched to S. decipiens very well, and indicated possible presence of the life cycle of this species in this region.

Genetic Identification of Spirometra decipiens Plerocercoids in Terrestrial Snakes from Korea and China.

Human sparganosis is a zoonotic disease caused by infection with larval forms (procercoid/plerocercoid) of Spirometra spp. The purpose of this study was to identify Spirometra spp. of infected snakes using a multiplex PCR assay and phylogenetic analysis of mitochondrial DNA sequence data from the spargana of terrestrial snakes obtained from Korea and China. A total of 283 snakes were obtained that included 4 species of Colubridae comprising Rhabdophis tigrinus tigrinus (n=150), Dinodon rufozonatum rufozonatum (n=64), Elaphe davidi (n=2), and Elaphe schrenkii (n=7), and 1 species of Viperidae, Agkistrodon saxatilis (n=60). The snakes were collected from the provinces of Chungbuk, Chungnam, and Gyeongbuk in Korea (n=161), and from China (n=122). The overall infection rate with spargana was 83% (235/283). The highest was recorded for D. rufozonatum rufozonatum (100%), followed by A. saxatilis (85%) and R. tigrinus tigrinus (80%), with a negative result for E. davidi (0%) and E. schrenkii (0%). The sequence identities between the spargana from snakes (n=50) and Spirometra erinaceieuropaei (KJ599680) or S. decipiens (KJ599679) control specimens were 90.8% and 99.2%, respectively. Pairwise genetic distances between spargana (n=50) and S. decipiens ranged from 0.0080 to 0.0107, while those between spargana and S. erinaceieuropaei ranged from 0.1070 to 0.1096. In this study, all of the 904 spargana analyzed were identified as S. decipiens either by a multiplex PCR assay (n=854) or mitochondrial cox1 sequence analysis (n=50).