Molecular identification and prevalence of plerocercoid larvae (Cestoda: Diphyllobothriidae) in some commercial fish species from Peru.

Diphyllobothriosis, a fish-borne zoonosis in South America, is mainly caused by the Pacific broad tapeworm Adenocephalus pacificus Nybelin, 1931, a parasite of considerable concern in fishery resources due to its impact on public health. A new diphyllobothrid, Diphyllobothrium sprakeri Hernández-Orts et al. Parasites Vectors 14:219, 2021, was recently described from sea lions from the Pacific Coast, but marine fish acting as intermediate hosts are unknown. The objective of this study was to confirm the presence of plerocercoid larvae of Diphyllobothriidae Lühe, 1910 (Cestoda: Diphyllobothriidea) in nine fish species of commercial importance in Peru. Of a total of 6999 fish (5861 Engraulis ringens, 853 Sciaena deliciosa, 6 Sciaena callaensis, 171 Scomber japonicus, 40 Trachurus murphyi, 40 Ariopsis seemanni, 18 Merluccius peruanus, 5 Sarda chiliensis, and 5 Coryphaena hippurus), 183 were infected with plerocercoid larvae, representing a total prevalence of 2.61% and a mean intensity of 3.2. Based on mtDNA cox1 sequences of 43 plerocercoids, a phylogenetic analysis revealed that 41 belong to A. pacificus and two to D. sprakeri. These findings are first molecular data for D. sprakeri larvae, and the infections of E. ringens and T. murphyi by plerocercoid larvae represent the first records of intermediate/paratenic hosts for this species. Hence, the findings of the current study enhance our understanding of the presence of diphyllobothriid species in commercial fish from the Southeastern Pacific Ocean and their potential impact on seafood safety for local human populations.

Human diphyllobothriasis in Argentina: assessing the epidemiological significance from historical records and reports of new cases.

Between 1898 and 1940, eight human cases of diphyllobothriasis were reported in Argentina, always in recently arrived European immigrants. In 1982, the first autochthonous case was detected, and since then, 33 other autochthonous cases have been reported, totaling 42 cases of human diphyllobothriasis in Argentina before the present study. Our aim is to update the information on diphyllobothriasis in Argentina by identifying specimens from new cases using morphometrical and/or molecular methods. We also aim to assess the epidemiological relevance of this food-borne disease in the country. Anamnestic data were obtained from patients or professionals, along with 26 worms identified using morphometrical (21 samples) and molecular techniques (5 samples). All the patients acquired the infection by consuming freshwater salmonids caught in Andean lakes in Northern Patagonia. Morphometrics and DNA markers of worms were compatible with Dibothriocephalus latus. In total, 68 human cases have been detected in Argentina, 60 of which were autochthonous. The human population living North-western Patagonia, whose lakes are inhabited by salmonids, is increasing. Similarly, the number of other definitive hosts for Dibothriocephalus dendriticus (gulls) and for D. latus (dogs) is also increasing. In addition, salmonid fishing and the habit of consuming home-prepared raw fish dishes are becoming widespread. Therefore, it is to be expected that diphyllobothriasis in Argentina will increase further.

Diphyllobothrium stemmacephalum (Cestoda: Diphyllobothriidae) found from a harbor porpoise in northern Japan, with comments on a geographical gap with human infection cases in southern Japan.

Even though the cetacean tapeworm Diphyllobothrium stemmacephalum occurs in both cold and warm waters, human infections and final host occurrences have been confined to temperate areas in and near Japan. We recently obtained a strobila of this cestode that was excreted from a harbor porpoise accidentally caught offshore of Hokkaido of northern Japan. Genetic analysis of 28S rDNA and cox1 genes confirmed that the cestode was D. stemmacephalum. Our finding sets the northernmost record of D. stemmacephalum in the western Pacific, suggesting that the risk of human infections by this parasite in northern Japan deserves further attention.

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.

Endohelminths of European Perch (Perca fluviatilis) from Selected Localities in Poland with an Emphasis on Search of the Broad Fish Tapeworm Dibothriocephalus latus.

INTRODUCTION: A piscivorous fish European perch (Perca fluviatilis) is present in all types of lakes and brackish waters in Poland. Previous ichthyoparasitological surveys revealed broad spectrum of endohelminths in perch from different aquatic environments. Among them, detection of Diphyllobothrium sp. and Dibothriocephalus latus (syn. Diphyllobothrium latum) in the Pomeranian Bay is of particular interest, since D. latus is one of the causative agents of diphyllobothriasis, fish-borne parasitic zoonosis. Besides, D. latus eggs were previously detected in coprological samples of otter, wolf and lynx from the Bialowieza Primeval Forest. AIM: To conduct parasitological examinations of European perch from the Pomeranian Bay in order to detect the spectrum of its endohelminths and to provide a pilot study on helminths of perch from different water bodies in the Bialowieza Primeval Forest. Due to zoonotic character of D. latus, we have focused our attention to this tapeworm. RESULTS: The larvae of tapeworm Triaenophorus nodulosus and thorny-headed worm Acanthocephalus lucii were detected in perch from the Pomeranian Bay. In perch from different localities in the Bialowieza Primeval Forest, T. nodulosus, A. lucii and tapeworm Proteocephalus percae were detected. D. latus plerocercoids were found neither in musculature nor in peritoneal cavity and other internal organs of any of the fish examined from both studied localities in Poland. CONCLUSION: Future screening implementing morphological and molecular markers is needed in order to understand the current distribution of D. latus in Europe.

Archaeoparasitological Analysis of Samples from the Cultural Layer of Nadym Gorodok dated Back to the 14th-Late 18th Centuries.

An archaeoparasitological analysis of the soil samples from Nadym Gorodok site of Western Siberia has been carried out in this study. The archaeological site was dated as the 13 to 18th century, being characterized as permafrost region ensuring good preservation of ancient parasite eggs. Parasite eggs as Opisthorchis felineus, Alaria alata, and Diphyllobothrium sp. were found in the archaeological soil samples, which made clear about the detailed aspects of Nadym Gorodok people's life. We found the Diphyllobothrium sp. eggs throughout the 14 to 18th century specimens, allowing us to presume that raw or undercooked fish might have been commonly used for the foods of Nadym Gorodok inhabitants and their dogs for at least the past 400 years. Our study on Nadym Gorodok specimens also demonstrate that there might have been migratory interactions and strong economic ties between the people and society in Western Siberia, based on archaeoparasitological results of Opisthorchis felineus in Western Siberia.

Development of microsatellite loci in zoonotic tapeworm Dibothriocephalus latus (Linnaeus, 1758), Lühe, 1899 (syn. Diphyllobothrium latum) using microsatellite library screening.

The broad fish tapeworm Dibothriocephalus latus is a causative agent of human food-borne disease called diphyllobothriosis. Medical importance, scattered geographical distribution and unknown origin of D. latus in Europe and North America make this species to be an interesting model for population genetics. Microsatellite markers were originally designed by library screening using NGS approach and validated as tools for future studies on population genetics of D. latus. Out of 122 candidates selected after NGS analysis, 110 yielded PCR products of the expected size, and in 78 of them, a declared repetitive motif was confirmed by Sanger sequencing. After the fragment analysis, six loci were proved to be polymorphic and tested for observed (Ho) and expected (He) heterozygosity, and deviations from Hardy-Weinberg equilibrium (HWE). They promise future application in studies on genetic interrelationships, origin and migratory routes of this medically important emerging tapeworm.

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.

First confirmed human case of Diphyllobothrium stemmacephalum infection and molecular verification of the synonymy of Diphyllobothrium yonagoense with D. stemmacephalum (Cestoda: Diphyllobothriidea).

Several tapeworm species in the genus Diphyllobothrium Cobbold, 1858 have uncertain taxonomic positions, leading to taxonomic confusion as well as misdiagnosis of infections. Taxonomic revision based on DNA sequence analysis is considered necessary to resolve the taxonomy of several cases, including that between Diphyllobothrium stemmacephalum, the type species of the genus, and Diphyllobothrium yonagoense. Diphyllobothrium yonagoense was synonymized with D. stemmacephalum based on morphological observations by Andersen (1987), however no molecular studies have been undertaken to verify the validity of this synonymization. In the present study, the first human case confirmed molecularly as D. stemmacephalum infection is reported, and the validity of the synonymization of D. yonagoense with D. stemmacephalum was assessed based on molecular phylogenetics. Diphyllobothrium stemmacephalum and D. yonagoense grouped into the same clades with high bootstrap confidence values for both cox1 and nad3. Genetic distances between the two taxa were very small (0.000-0.012 and 0.000-0.017 for cox1 and nad3, respectively) and were considered to fall within the range of intraspecific variation. Using these molecular analyses, this study verified molecularly that D. yonagoense is a junior synonym of D. stemmacephalum. Further, the closer phylogenetic relationship between D. stemmacephalum and Diplogonoporus species rather than other diphyllobothriids, including Diphyllobothrium nihonkaiense and Diphyllobothrium latum, was corroborated. The genus name for D. nihonkaiense and D. latum is also discussed.

Invalidation of Diphyllobothrium hottai (Cestoda: Diphyllobothriidae) based on morphological and molecular phylogenetic analyses.

Diphyllobothrium hottai Yazaki, Fukumoto & Abe, 1988 was described based on the morphology of adult worms recovered from golden hamsters that had been experimentally infected with plerocercoids obtained from Japanese surf smelts (Hypomesus pretiosus japonicus) and olive rainbow smelts (Osmerus eperlanus mordax). Although D. hottai was considered to be distinct from Diphyllobothrium ditremum (Creplin, 1825), their taxonomic relationship requires further clarification. In our study, D. hottai and D. ditremum obtained from hamsters experimentally infected with plerocercoids isolated from Japanese surf smelts were compared using morphological and molecular methods. The criterion usually used to differentiate between D. hottai and D. ditremum is the difference in the angle between the long axis of the cirrus sac and that of the seminal vesicle. However, we found variation of the angle within the same individual and, one specimen showed both of the different angles that were supposedly unique to each of the species. Furthermore, phylogenetic analysis of the complete sequences of the mitochondrial cytochrome c oxidase subunit 1 and cytochrome b genes revealed that both species were genetically indistinguishable. Therefore, D. hottai is considered to be a junior synonym of D. ditremum.

Characterisation of the relationship between Spirometra erinaceieuropaei and Diphyllobothrium species using complete cytb and cox1 genes.

Spirometra erinaceieuropaei is an important diphyllobothriid cestode whose larval plerocercoid (sparganum) can parasitise humans, causing sparganosis. In China, sparganosis is an important foodborne parasitic zoonosis and poses a serious threat to human health. However, our knowledge of the taxonomic classification of S. erinaceieuropaei and its relationship with other diphyllobothriid cestodes, such as Diphyllobothrium, remains fragmentary. In this study, we determined the complete sequences of cytochrome b (cytb) and cytochrome c oxidase subunit I (cox1) genes from 61 isolates of S. erinaceieuropaei collected from 11 geographical locations in eastern China and compared them to the sequences of Diphyllobothrium species from the GenBank database. Then, we evaluated the genetic variation of S. erinaceieuropaei and the phylogenetic relationship between S. erinaceieuropaei and Diphyllobothrium. Both negative Tajima's D and Fu's FS values of neutrality tests and unimodal curve of mismatch distribution analyses supported demographic population expansion. Bayesian skyline plot (BSP) analyses indicated that a population expansion occurred in the Pleistocene. Phylogenetic analysis based on cytb, cox1, and cytb+cox1 under all three methods tested (neighbour-joining, maximum parsimony, and maximum likelihood) suggested that the S. erinaceieuropaei isolates from eastern China and the genus Diphyllobothrium are separate monophyletic groups. Furthermore, the uncorrelated log-normal relaxed molecular-clock model suggested that divergence between Spirometra and Diphyllobothrium began in the late Miocene.

Four additional cases of Diphyllobothrium nihonkaiense infection confirmed by analysis of COX1 gene in Korea.

Most of the diphyllobothriid tapeworms isolated from human samples in the Republic of Korea (= Korea) have been identified as Diphyllobothrium nihonkaiense by genetic analysis. This paper reports confirmation of D. nihonkaiense infections in 4 additional human samples obtained between 1995 and 2014, which were analyzed at the Department of Parasitology, Hallym University College of Medicine, Korea. Analysis of the mitochondrial cytochrome c oxidase 1 (cox1) gene revealed a 98.5-99.5% similarity with a reference D. nihonkaiense sequence in GenBank. The present report adds 4 cases of D. nihonkaiense infections to the literature, indicating that the dominant diphyllobothriid tapeworm species in Korea is D. nihonkaiense but not D. latum.

Diphyllobothrium nihonkaiense infections in a family.

Diphyllobothrium latum and Diphyllobothrium nihonkaiense are morphologically similar to each other, and only genetic method can differentiate clearly between the 2 species. A strobila of diphyllobothriid tapeworm discharged from a 7-year-old boy was analyzed to identify the species by mitochondrial cytochrome c oxidase subunit 1 (cox1) gene sequencing. He and his family (total 4 persons) ate slices of 3 kinds of raw fish 16 days before visiting our outpatient clinic. All family members complained of abdominal pain and watery diarrhea. They all expelled tapeworm strobilae in their stools. They were treated with a single oral dose of praziquantel and then complained of no more symptoms. The cox1 gene sequencing of the strobila from the boy revealed 99.9% (687/688 bp) similarity with D. nihonkaiense and only 93.2% (641/688 bp) similarity with D. latum. Thus, we assigned this tapeworm as D. nihonkaiense. This is the first report of D. nihonkaiense infection in a family in Korea, and this report includes the 8th pediatric case in Korea. The current report is meaningful because D. nihonkaiense infection within a family is rare.

Molecular Identification of Diphyllobothrium nihonkaiense from 3 Human Cases in Heilongjiang Province with a Brief Literature Review in China.

Human diphyllobothriasis is a widespread fish-borne zoonosis caused by the infection with broad tapeworms belonging to the genus Diphyllobothrium. In mainland China, so far 20 human cases of Diphyllobothrium infections have been reported, and the etiologic species were identified as D. latum and D. nihonkaiense based on morphological characteristics or molecular analysis. In the present study, proglottids of diphyllobothriid tapeworms from 3 human cases that occurred in Heilongjiang Province, China were identified as D. nihonkaiense by sequencing mitochondrial cytochrome c oxidase subunit I (cox1) and NADH dehydrogenase subunit 5 (nad5) genes. Two different cox1 gene sequences were obtained. One sequence showed 100% homology with those from humans in Japan. The remaining cox1 gene sequence and 2 different nad5 gene sequences obtained were not described previously, and might reflect endemic genetic characterizations. D. nihonkaiense might also be a major causative species of human diphyllobothriasis in China. Meanwhile, the finding of the first pediatric case of D. nihonkaiense infection in China suggests that infants infected with D. nihonkaiense should not be ignored.

Three cases of Diphyllobothrium nihonkaiense infection in Korea.

Until 2012, a total of 48 cases of diphyllobothriasis had been reported in Korea, all of which were morphologically identified as Diphyllobothrium latum. However, some of these specimens were analyzed by nucleotide sequencing of the mitochondrial cox1 gene, which showed that all were D. nihonkaiense, not D. latum. After that, 3 further cases of diphyllobothriasis were confirmed as D. nihonkaiense. In the present study, 3 new cases of D. nihonkaiense were detected from 2011 through 2013. The hosts were infected through consumption of salmonid fishes, such as the trout or salmon, and 2 of them experienced severe diarrhea prior to proglottid passage. All of the tapeworms were confirmed to be D. nihonkaiense by genetic identification. This proved again that most diphyllobothriasis in Korea have been caused by D. nihonkaiense.

Extracorporeal worm extraction of Diphyllobothrium nihonkaiense with amidotrizoic acid in a child.

Infection cases of diphyllobothriid tapeworms are not much in the below teen-age group. We report a case of Diphyllobothrium nihonkaiense infection in a 13-year-old boy. He presented with severe fatigue, occasional abdominal pain at night time. He also had several episodes of tapeworm segment discharge in his stools. By his past history, he had frequently eaten raw fish including salmon and trout with his families. Numerous eggs of diphyllobothriid tapeworm were detected in the fecal examination. We introduced amidotrizoic acid as a cathartic agent through nasogastroduodenal tube and let nearly whole length (4.75 m) of D. nihonkaiense be excreted through his anus. After a single dose of praziquantel, the child's stool showed no further eggs, and his symptoms disappeared. The evacuated worm was identified as D. nihonkaiense by mitochondrial cox1 gene analysis. Here we report a successful extracorporeal worm extraction from an infection case of D. nihonkaiense by the injection of amidotrizoic acid.

Molecular detection of Diphyllobothrium nihonkaiense in humans, China.

The cause of diphyllobothriosis in 5 persons in Harbin and Shanghai, China, during 2008-2011, initially attributed to the tapeworm Diphyllobothrium latum, was confirmed as D. nihonkaiense by using molecular analysis of expelled proglottids. The use of morphologic characteristics alone to identify this organism was inadequate and led to misidentification of the species.

Human infection by a "fish tapeworm", Diphyllobothrium latum, in a non-endemic country.

We document a case of locally acquired "broad- or fish-tapeworm" infection caused by Diphyllobothrium latum in a 27-year-old Spanish man, confirmed by molecular analysis (COI gene). The patient had naturally expelled a worm of 110 cm in length, but the physical examination did not yield any remarkable findings, and the patient did not suffer from any particular symptoms. Laboratory test results were normal except for a remarkable increase in the red blood cell count, and an evident decrease in the mean corpuscular volume and mean corpuscular hemoglobin. Vitamin B12 and folic acid values were in the normal range without signs of anemia. It was suggested that these anomalies in erythrocyte formation might not be related to the parasite, and analysis of the patient's anamnestic data revealed that the infection could only have been caused by the ingestion of imported fish, although no light could be shed on the specific source of infection. From a public health viewpoint, this human case of fish-borne zoonosis is exemplary, suggesting that not only is control of fish and fish product quality essential, but also increased awareness of the general population with regard to changes in culinary habits.