Diphyllobothrium sprakeri n. sp. (Cestoda: Diphyllobothriidae): a hidden broad tapeworm from sea lions off North and South America.

BACKGROUND: The systematic of several marine diphyllobothriid tapeworms of pinnipeds has been revised in recent years. However, 20 species of Diphyllobothrium from phocids and otariids are still recognized as incertae sedis. We describe a new species of Diphyllobothrium from the intestine of California sea lions Zalophus californianus (Lesson) (type-host) and South American sea lions Otaria flavescens (Shaw). METHODS: Zalophus californianus from the Pacific coast of the USA and O. flavescens from Peru and Argentina were screened for parasites. Partial fragments of the large ribosomal subunit gene (lsrDNA) and the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene were amplified for 22 isolates. Properly fixed material from California sea lions was examined using light and scanning electron microscopy. RESULTS: A total of four lsrDNA and 21 cox1 sequences were generated and aligned with published sequences of other diphyllobothriid taxa. Based on cox1 sequences, four diphyllobothriid tapeworms from O. flavescens in Peru were found to be conspecific with Adenocephalus pacificus Nybelin, 1931. The other newly generated sequences fall into a well-supported clade with sequences of a putative new species previously identified as Diphyllobothrium sp. 1. from Z. californianus and O. flavescens. A new species, Diphyllobothrium sprakeri n. sp., is proposed for tapeworms of this clade. CONCLUSIONS: Diphyllobothrium sprakeri n. sp. is the first diphyllobothriid species described from Z. californianus from the Pacific coast of North America, but O. flavescens from Argentina, Chile and Peru was confirmed as an additional host. The present study molecularly confirmed the first coinfection of two diphyllobothriid species in sea lions from the Southern Hemisphere.

Molecular and morphological identification of Adenocephalus pacificus (Cestoda) isolated from South American sea lion Otaria byronia stranded on the northern Peruvian coasts.

The most frequent etiologic agent of diphyllobothriosis in South America and the only one confirmed by molecular data in human cases in Peru is Adenocephalus pacificus (syn. Diphyllobothrium pacificum). This cestode is transmitted by ingestion of the plerocercoids found in marine fish, causing a parasitic zoonosis. The objective of the present study was to identify two cestodes isolated from two specimens of the South American sea lion (Otaria byronia) stranded on the beaches of Huacho and Barranca cities, located on the northern Peruvian coasts, in the department of Lima. Tapeworms were confirmed by morphological characteristics due to the presence of transverse papilla-like tegumental protuberances in proglottids and small sized eggs, as well as by sequencing of the partial cytochrome c oxidase subunit 1 (mtDNA-COI) gene that are congruent with additional available A. pacificus sequences. Even though sea lions in Peru are distributed along the coast and in areas of difficult access, generally located in protected natural areas, the fortuitous finding represented an opportunity to confirm the presence of A. pacificus in South American sea lions. This report of tapeworm A. pacificus could allow future monitoring of the occurrence and geographical distribution of this causative agent in epidemiological studies, since it is one of the main species of zoonotic importance in Peru.

Complex insight on microanatomy of larval "human broad tapeworm" Dibothriocephalus latus (Cestoda: Diphyllobothriidea).

BACKGROUND: In Europe, the tapeworm Dibothriocephalus latus (syn. Diphyllobothrium latum) is a well-known etiological agent of human diphyllobothriosis, which spreads by the consumption of raw fish flesh infected by plerocercoids (tapeworm's larval stage). However, the process of parasite establishment in both intermediate and definitive hosts is poorly understood. This study was targeted mainly on the scolex (anterior part) of the plerocercoid of this species, which facilitates penetration of the parasite in intermediate paratenic fish hosts, and subsequently its attachment to the intestine of the definitive host. METHODS: Plerocercoids were isolated from the musculature of European perch (Perca fluviatilis) caught in Italian alpine lakes. Parasites were examined using confocal microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Immunofluorescence tagging was held on whole mount larvae. RESULTS: The organisation of the central and peripheral nervous system was captured in D. latus plerocercoids, including the ultrastructure of the nerve cells possessing large dense neurosecretory granules. Two types of nerve fibres run from the body surface toward the nerve plexus located in the parenchyma on each side of bothria. One type of these fibres was found to be serotoninergic and possessed large subtegumental nerve cell bodies. A well-developed gland apparatus, found throughout the plerocercoid parenchyma, produced heterogeneous granules with lucent core packed in a dense layer. Three different types of microtriches occurred on the scolex and body surface of plerocercoids of D. latus: (i) uncinate spinitriches; (ii) coniform spinitriches; and (iii) capilliform filitriches. Non-ciliated sensory receptors were observed between the distal cytoplasm of the tegument and the underlying musculature. CONCLUSIONS: Confocal laser scanning microscopy and electron microscopy (SEM and TEM) showed the detailed microanatomy of the nervous system in the scolex of plerocercoids, and also several differences in the larval stages compared with adult D. latus. These features, i.e. well-developed glandular system and massive hook-shaped uncinate spinitriches, are thus probably required for plerocercoids inhabiting fish hosts and also for their post-infection attachment in the human intestine.

Molecular Identification of Diphyllobothrium latum from a Pediatric Case in Taiwan.

Human diphyllobothriasis is a parasitic disease caused by ingestion of larvae (plerocercoids) in raw or undercooked fish and commonly found in temperate areas. Rare cases were reported in tropical or subtropical areas especially in children. The first documented case of pediatric diphyllobothriasis in Taiwan had been reported 11 years ago. Here, we report another 8-year-old girl case who presented with a live noodle-like worm hanging down from her anus, with no other detectable symptoms. We pulled the worm out and found the strobila being 260 cm in length. Examination of gravid proglottids showed that they were wider than their lengths, containing an ovoid cirrus sac in the anterior side and the rosette-shaped uterus. Eggs extracted from the uterus were ovoid and operculated. Diphyllobothrium latum was confirmed by molecular analysis of the mitochondrial DNA cytochrome c oxidase subunit 1 (cox1) gene. The girl was treated with a single oral dose of praziquantel, and no eggs or proglottids were observed from her stool in the subsequent 3 months. The reemergence of human diphyllobothriasis in non-endemic countries is probably due to prevalent habit of eating imported raw fish from endemic areas. This pediatric case raised our concern that human diphyllobothriasis is likely underestimated because of unremarkable symptoms.

Four Human Cases of Diphyllobothrium nihonkaiense (Eucestoda: Diphyllobothriidae) in China with a Brief Review of Chinese Cases.

We described 4 human infection cases of zoonotic fish-tapeworm, Diphyllobothrium nihonkaiense, identified with morphological and molecular characters and briefly reviewed Chinese cases in consideration of it as an emerging parasitic disease in China. The scolex and mature and gravid proglottids of some cases were seen, a rosette-shaped uterus was observed in the middle of the mature and gravid proglottids, and the diphyllobothriid eggs were yellowish-brown in color and displayed a small knob or abopercular protuberance on the opposite end of a lid-like opening. The average size of the eggs was recorded as 62-67×42-45 µm. The parasitic materials gathered from 4 human cases were morphologically identified as belonging to the genera Diphyllobothrium and Adenocephalus. The phylogenetic analysis based on the nucleotide sequences of cytochrome c oxidase subunit 1 gene of the etiologic agents confirmed that the 4 cases were D. nihonkaiense infection. The finding of 4 additional D. nihonkaiense cases suggests that D. nihonkaiense might be a major causative species of human diphyllobothriasis in China. A combined morphological and molecular analysis is the main method to confirm D. nihonkaiense infection.

Eggs as a Suitable Tool for Species Diagnosis of Causative Agents of Human Diphyllobothriosis (Cestoda).

BACKGROUND: Tapeworms of the order Diphyllobothriidea are parasites of tetrapods and several species may infect man and cause neglected human disease called diphyllobothriosis. Identification of human-infecting diphyllobothriid cestodes is difficult because of their morphological uniformity, which concerns also their eggs in stool samples. METHODS: In the present study, we analysed by far the largest dataset of more than 2,000 eggs of 8 species of diphyllobothriid cestodes that may infect humans, including the most frequent human parasites Diphyllobothrium latum, D. nihonkaiense and Adenocephalus pacificus (syn. Diphyllobothrium pacificum). Size (length, width and length/width ratio) and the surface of the egg shell from naturally and experimentally infected hosts were studied using light and scanning electron microscopy. RESULTS: A high degree of intraspecific and host-related size variability has been detected, but combination of morphometrical and ultrastructural data made it possible to distinguish all of the studied species, including otherwise quite similar eggs of the 3 most common species infecting man, i.e. D. latum, D. nihonkaiense and D. dendriticum. The surface of all marine species is covered by numerous deep pits with species-specific density, whereas the surface of freshwater species is smooth or with isolated shallow hollows or wrinkles.

High morphological plasticity and global geographical distribution of the Pacific broad tapeworm Adenocephalus pacificus (syn. Diphyllobothrium pacificum): molecular and morphological survey.

The most important causative agent of human diphyllobothriosis in South America, Diphyllobothrium pacificum, is transferred to the original genus Adenocephalus Nybelin, 1931; revised and redescribed on the basis of the evaluation of an extensive material collected mainly from northern fur seal, Callorhinus ursinus, from St. Paul Island, Alaska. Detailed analysis of morphological and morphometrical data shows a high variability in most of the characteristics traditionally used in diagnosis of diphyllobothriid tapeworms. Phylogenetic analyses based on newly characterised sequences of mitochondrial cytochrome c oxidase subunit 1 and nuclear large subunit ribosomal RNA genes consistently reveal Adenocephalus pacificus as a sister lineage to the clade formed of the remaining Diphyllobothrium species and other genera (Digramma, Diplogonoporus, Ligula). Despite the generally similar morphology, A. pacificus can be differentiated from the closely related taxa in the presence of transverse papilla-like tegumental protuberances distributed anteriorly, separated by narrow semicircular grooves on the ventral surface of proglottids between their anterior margin and the anterior edge of the male gonopore, and relatively small eggs. A. pacificus displays a relatively low host specificity (found in 9 of 16 otariids, and in accidental hosts such as man, dog and jackal, the latter representing a new host) and a uniquely wide geographical distribution on both hemispheres. In addition, suitability of morphological criteria used in diagnostics of diphyllobothriid cestodes is discussed.

Validity of the bear tapeworm Diphyllobothrium ursi (Cestoda: Diphyllobothriidae) based on morphological and molecular markers.

The bear tapeworm Diphyllobothrium ursi is described based upon the morphology of adult tapeworms recovered from the brown bear (Ursus arctos middendorffi) and larval plerocercoids found in sockeye salmon (Oncorhynchus nerka) from Kodiak Island in Alaska in 1952. However, in 1987 D. ursi was synonymized with Diphyllobothrium dendriticum, and the taxonomic relationship between both species has not subsequently been revised. In this study mitochondrial cytochrome c oxidase subunit 1 gene (cox1) sequences of holotype and paratype D. ursi specimens that had been preserved in a formalin-acetic acid-alcohol solution since the time the species was initially described approximately 60 yr ago were analyzed. Molecular and phylogenetic analysis of the cox1 sequences revealed that D. ursi is more closely related to D. dendriticum than it is to Diphyllobothrium nihonkaiense and Diphyllobothrium latum. In addition to molecular evidence, differences in the life cycle and ecology of the larval plerocercoids between D. ursi and D. dendriticum also suggest that D. ursi is a distinct species, separate from D. dendriticum and D. nihonkaiense, and also possibly from D. latum .

Four human cases of Diphyllobothrium latum infection.

Diphyllobothrium latum infections in 4 young Korean men detected from 2008 to 2012 are presented. Three were diagnosed based on spontaneously discharged strobila of the adult worm in their feces, and 1 case was diagnosed by finding the worm at colonoscopy examination in a local clinic. The morphologic characteristics of the gravid proglottid and eggs were consistent with D. latum. All patients were treated with praziquantel 15 mg/kg, and follow-up stool examinations were done at 2 months after the medication. The main clinical complaints were intermittent gastrointestinal troubles such as indigestion, abdominal distension, and spontaneous discharge of tapeworm's segments in their feces. The most probable source of infection was the flesh of salmon or trout according to a patient's past history. These are the 45th to 48th recorded cases diagnosed by the adult worm in the Republic of Korea since 1971.

First molecular identification of Diphyllobothrium dendriticum plerocercoids from feral rainbow trout (Oncorhynchus mykiss) in Chile.

Between April and June 2009, 1,075 feral rainbow trout from 10 different lakes involved with aquaculture activities in Los Lagos Region, Chile, were inspected for Diphyllobothrium species. All viscera and muscles of the fish were examined using stereomicroscopy; pyloric cecae and stomachs infected with plerocercoids were checked by histology and scanning electron microscopy. Plerocercoids of Diphyllobothrium dendriticum were confirmed by PCR and sequencing of COI and 18S rRNA + ITS1 + 5.8S rRNA + ITS2 genes for the first time in Chile. Overall prevalence of plerocercoids of D. dendriticum was 9.2% (99/1,075) in Los Lagos Region and 17.4% (99/570) for Chiloe Island. Plerocercoids were not detected in the continental lakes of the Los Lagos Region (Chapo, Rupanco, and Llanquihue). Tarahuín Lake exhibited a prevalence of 50.9% (81/159), Cucao Lake 5.1% (4/79), Natri Lake 4.7% (5/107), Huillinco Lake 3.6% (5/138), and San Antonio Lake 66.7% (4/6). Abundance was 1.1 plerocercoid larvae per fish (1,169 larvae/1,075 fish). All the plerocercoids were found encysted in the viscera of the fish. Plerocercoids were 10.9 ± 3 (7-16) mm long by 0.4 ± 0.2 (0.2-0.6) mm wide. The scolex was enlarged, with 2 bothria and a frontal pit. The body was covered with short capilliform filitriches, 4-6 mm long. The Chilean COI and 18SrRNA + ITS1 + 5.8SrRNA + ITS2 gene sequences indicated 96.34-96.52% and 99% similarity with D. dendriticum sequences, respectively. Diphyllobothrium dendriticum is reported for the first time in freshwater ecosystems as far as 43 ° S on Chiloe Island. These findings and previous reports of plerocercoids of Diphyllobothrium spp. in farmed rainbow trout at Tarahuín Lake support the putative life cycle of this parasite in lakes of southern Chile where there are aquaculture activities.

Four Human Cases of Diphyllobothrium latum Infection

Diphyllobothrium latum infections in 4 young Korean men detected from 2008 to 2012 are presented. Three were diagnosed based on spontaneously discharged strobila of the adult worm in their feces, and 1 case was diagnosed by finding the worm at colonoscopy examination in a local clinic. The morphologic characteristics of the gravid proglottid and eggs were consistent with D. latum. All patients were treated with praziquantel 15 mg/kg, and follow-up stool examinations were done at 2 months after the medication. The main clinical complaints were intermittent gastrointestinal troubles such as indigestion, abdominal distension, and spontaneous discharge of tapeworm's segments in their feces. The most probable source of infection was the flesh of salmon or trout according to a patient's past history. These are the 45th to 48th recorded cases diagnosed by the adult worm in the Republic of Korea since 1971.

Long fish tapeworm in the intestine: an in situ observation by capsule endoscopy.

We present the case of a patient who was referred to our hospital after she reported having passed a long, whitish object per rectum. Accordingly, capsule endoscopy was performed using the PillCam(®) SB video capsule. A tapeworm of the species Diphyllobothrium nihonkaiense was detected; it appeared to be freely floating and unfolded in the jejunum and sometimes tangled or irregularly folded in the ileum. The stretching of the strobila by strong peristalsis in the ileum may have resulted in the separation of the caudal portion of the strobila, which descended into the colon and was eventually passed per rectum.

Molecular identification of the Diphyllobothrium species causing diphyllobothriasis in Chilean patients.

Diphyllobothriasis caused by the infection of adult Diphyllobothrium tapeworms sporadically occurs in Chile. The occurrence of the disease is closely linked to the consumption of raw or undercooked freshwater and marine fishes. Diagnosis of diphyllobothriasis has been based on laboratory examinations of the morphological characteristics of proglottids and eggs passed in the feces. Although determination of the parasite to the species level is possible through histologic examination of proglottid specimens, the parasites of patients who only discharge eggs cannot be diagnosed to the species level. Determining the species responsible for the infection of humans and other animals in affected areas is an important component of understanding the epidemiologic and enzootic characteristics of any infectious disease. We therefore compared the classification results obtained using a molecular approach with those obtained from morphological and histopathological examination of proglottids or eggs from five Chilean individuals with diphyllobothriasis. DNA analysis confirmed that the causative Diphyllobothrium species in Chile were first identified as Diphyllobothrium latum and Diphyllobothrium pacificum at least. Furthermore, mitochondrial cytochrome c oxidase subunit 1 gene analysis also supported the hypothesis that D. latum from Chile originated from Europe.

Identity of Diphyllobothrium spp. (Cestoda: Diphyllobothriidae) from sea lions and people along the Pacific coast of South America.

Host specificity evidently is not expressed by various species of Diphyllobothrium that occur typically in marine mammals, and people become infected occasionally when dietary customs favor ingestion of plerocercoids. This report mainly concerns 2 species, Diphyllobothrium pacificum and Diphyllobothrium arctocephalinum, for which sea lions (Otariidae) are final hosts. The taxonomic status of those cestodes has not been clearly discernible because of misinterpretation of relationships; complex synonymies have resulted from misidentification(s). Stiles and Hassall in 1899 obtained, but did not describe, cestodes from the northern fur seal (Otariidae; Pribilof Islands). That taxon was subsequently studied by several investigators, with diverse conclusions. The valid designation is D. pacificum (Nybelin, 1931). In 1937, Johnston and Drummond described separately 2 conspecific cestodes from sea lions near Australia, designated D. arctocephalinum and Diphyllobothrium arctocephali. Both names have been listed incorrectly as synonyms of D. pacificum.

Anomalous segmentation of Diphyllobothrium nihonkaiense.

An anomalous tapeworm with abnormal segmentation was obtained from a 6-year-old boy in Japan. The tapeworm consisted of proglottids with slanted anterior and posterior margins of proglottids and 4-6 sets of reproductive organs arranged between the margins. The morphology of the tapeworm did not correspond to any of the described cestodes. However, molecular identification based on nuclear and mitochondrial genes clearly showed the tapeworm was Diphyllobothrium nihonkaiense.

Morphologic and genetic identification of Diphyllobothrium nihonkaiense in Korea.

Diphyllobothrium nihonkaiense was first described by Yamane in 1986 but the taxonomical features have been obscure due to lack of critical morphologic criteria in its larval and adult stages. In Korea, this tapeworm had long been known as Diphyllobothrium latum. In this study, we observed 62 specimens collected from Korean residents and analyzed them by morphological features and nucleotide sequences of mitochondrial cox1 gene as well as the ITS1 region. Adult tapeworms were examined after carmine or trichrome stain. Longitudinal sections of the gravid proglottids showed an obtuse angle of about 150 degree between the cirrus sac and seminal vesicle. This angle is known as a major differential point compared with that of D. latum. Nucleotide sequence differences between D. latum and the specimens from Koreans represented 17.3% in mitochondrial DNA cox1 gene. Sequence divergence of ITS1 among 4 Korean isolates was 0.3% and similarity was 99.7% with D. nihonkaiense and D. klebanovskii. All of the Korean specimens analyzed in this study were identified as being D. nihonkaiense (n = 62). We propose its Korean name as "Dong-hae-gin-chon-chung" which means 'long tapeworm of the East Sea' for this newly analyzed diphyllobothriid tapeworm in Korea.

Morphologic and Genetic Identification of Diphyllobothrium nihonkaiense in Korea

Diphyllobothrium nihonkaiense was first described by Yamane in 1986 but the taxonomical features have been obscure due to lack of critical morphologic criteria in its larval and adult stages. In Korea, this tapeworm had long been known as Diphyllobothrium latum. In this study, we observed 62 specimens collected from Korean residents and analyzed them by morphological features and nucleotide sequences of mitochondrial cox1 gene as well as the ITS1 region. Adult tapeworms were examined after carmine or trichrome stain. Longitudinal sections of the gravid proglottids showed an obtuse angle of about 150 degree between the cirrus sac and seminal vesicle. This angle is known as a major differential point compared with that of D. latum. Nucleotide sequence differences between D. latum and the specimens from Koreans represented 17.3% in mitochondrial DNA cox1 gene. Sequence divergence of ITS1 among 4 Korean isolates was 0.3% and similarity was 99.7% with D. nihonkaiense and D. klebanovskii. All of the Korean specimens analyzed in this study were identified as being D. nihonkaiense (n = 62). We propose its Korean name as "Dong-hae-gin-chon-chung" which means 'long tapeworm of the East Sea' for this newly analyzed diphyllobothriid tapeworm in Korea.

A case of human diphyllobothriasis in northern Taiwan after eating raw fish fillets.

The consumption of raw fish fillets is increasing in Taiwan. A male Taiwanese aged 30 years presented after passing a flat, white noodle-like worm. Strobila examination showed that most proglottids were wider than they were long, with the genital pore located at the posterior edge of the cirrus. Histological and coprological findings confirmed the diagnosis of Diphyllobothrium latum; ova were ellipsoidal with operculate characteristics, and had a small knob in the anti-operculum side. Hematological data, including vitamin B12 levels, were normal, except for a low folate level. The patient was treated with a single dose of praziquantel 600 mg and 196 cm of proglottids were expelled during the 3 days following treatment. Further follow-up was declined. Consumption of raw and undercooked fish (especially salmon) poses a risk of D. latum infection.