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.

Genetic characterization of Dibothriocephalus latus and Dibothriocephalus dendriticus (Cestoda: Diphyllobothriidae) from Chile based on haplotype analysis using mitochondrial DNA markers.

Dibothriocephalus latus and Dibothriocephalus dendriticus are found throughout the temperate and sub-arctic zones of the northern hemisphere, but they are also found in the southern core countries of South America, Chile and Argentina. Genetic characteristics of D. latus and D. dendriticus from South America have yet to be fully defined. The present study aimed to understand the genetic characteristics of D. latus and D. dendriticus from Chile by haplotype network analysis of mitochondrial cytochrome c oxidase subunit I gene (cox1) and cytochrome b gene (cob), as well as their origins. Dibothriocephalus latus and D. dendriticus plerocercoid larvae were obtained from feral and/or wild salmonids captured in Lake Llanquihue in Región de Los Lagos, and Lake Panguipulli in Región de Los Ríos, located south of central Chile. Haplotype analysis of D. latus revealed that H1 in cox1 and H2 in cob are the key haplotypes common to D. latus across the world, including Chile, and both genes exhibited limited genetic diversity in D. latus. It was assumed that D. latus was brought into South America by European and Russian immigrants in the 19th century as previously reported. In contrast, both the cox1 and cob of D. dendriticus display considerable genetic diversity, with no common haplotypes between D. dendriticus populations from Chile and the northern hemisphere. More intriguingly, two cob haplotypes (H24, H25) detected in Chilean D. dendriticus were closely linked to haplotypes (H30, H31) detected in North American D. dendriticus, strongly implying that D. dendriticus in Chile was brought by piscivorous migrating birds from North America. It has also been estimated that the D. dendriticus from Chile genetically diverged from the D. dendriticus from the northern hemisphere approximately 1.11 million years ago, long before humans migrated to the southern parts of South America.

Plerocercoids of Adenocephalus pacificus in Argentine hakes: Broad distribution, low zoonotic risk.

Adenocephalus pacificus is a tapeworm parasitic of marine mammals and the main agent of human diphylobothriosis caused by consumption of raw or undercooked marine fishes, being considered as a reemerging disease. Despite having a broad distribution in marine mammals in both hemispheres, plerocercoid larvae in fish have only been reported in the Pacific Ocean, in Peruvian waters, from where most human cases are known. In Argentine waters larval stages of Diphyllobothriidae have been recorded in Merluccius hubbsi, a main fish resource mostly exported frozen, headed and gutted (H&G) or as fillets; therefore, the possible presence of A. pacificus in edible products, and the extent of the risk of parasitism for humans becomes of health and commercial relevance. With the aim of detecting and identifying potentially zoonotic diphyllobothriids and quantifying infection levels in viscera and fillets of hakes, 43 entire fish, 471 H&G, and 942 fillets obtained from research cruises in 2019 and 2021 from the southern Argentine Sea (44-53°S; 63-68°W) were examined by transillumination and under stereoscopic microscopy. Plerocercoids were recovered at low prevalence and mean abundance in entire fish (13.95 % and 0.35) and H&G fish (2.76 % and 0.03) but no larval worms were found adhered to musculature or peritoneum, furthermore, no larvae were found in the fillets. Larvae were genetically identified, based on sequences of the large subunit ribosomal RNA nuclear gene (lsrDNA) and the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene, as members of A. pacificus, representing the first report of this species in a fish host outside South American Pacific coasts. No spatial nor bathymetric patterns in parasite burdens were observed across sampling sites, but prevalence increased with fish size. The recorded low parasite burdens, the absence of infective stages in fillets and the fact that most products are commercialized deeply frozen, diminish the risk of parasitism for consumers to a minimum. However, the identification of this zoonotic agent and the assessment of its distribution in fish products are a first indispensable step for the design of efficient and suitable measures, such as freezing or cooking meet, to ensure the prevention of human infections.

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.

Diphyllobothriidea in the north area of the Andean Patagonia: Epidemiology in urban dogs, morphometrical and molecular identification, with comments on wild carnivores.

Diphyllobothriidea are the principal agents of Diphyllobothriosis, a widespread food-borne cestodosis. Accurate identification of the species in samples is therefore crucial for diagnosis and epidemiology in wild and domestic animals, and also humans. We aim to identify at specific level the causative agent, and provide an observational, descriptive, and transversal study of the epidemiology of this zoonosis in urban dogs. Also data on wild carnivores from Northwestern Patagonia are presented. Dog feces were collected in thirteen neighborhoods of varying socioeconomic status, and stools were analyzed by two concentration methods. Adult worms were collected and identified by molecular methods. The population of free-roaming dogs in each neighborhood was estimated, and surveys were conducted at all veterinary clinics registered in the Veterinary Medical College of Bariloche city. A total of 36 wild carnivores road killed or found dead in three National Parks were analyzed. Molecular and morphometric analyses of proglottids and eggs from dogs indicate they are infected with D. latum. Twenty out of 118 dog feces were positive for Diphyllobothrium, from 9 out of 13 neighborhoods, with infection values between 10% and 66%. Percentage of infection was correlated positively with the number of free roaming dogs per block, and with Unsatisfied Basic Needs (UBN %), but not with distance to nearest water body. Infection by D. latum in dogs is widely distributed throughout the city. Not all local veterinarians know the occurrence of Diphyllobothriosis in the dogs of the city, and it is evident that this zoonosis is underdiagnosed in relation to the percentage of infection found in this study. None of the analyzed wild carnivores were positive for Diphyllobothrium.

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.

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.

Is the human-infecting Diphyllobothrium pacificum a valid species or just a South American population of the holarctic fish broad tapeworm, D. latum?

Using ITS2 gene sequences, the validity of the tapeworm Diphyllobothrium pacificum (Nybelin, 1931), infecting humans on the Pacific coast of South America and in Japan, was assessed. ITS2 sequences of this cestode differed markedly (sequence similarity 79.0-80.2%) from those of the most common human-infecting cestode, the broad fish tapeworm Diphyllobothrium latum (L.), as well as other four species of Diphyllobothrium, including potential human parasites (D. cordatum, D. dendriticum, and D. lanceolatum) and two species of Spirometra (sequence similarity 77.5-81.9%). Interspecific sequence similarity between all but one (D. pacificum) species was 86.1-99.6%, whereas individual isolates of D. dendriticum and D. ditremum exhibited intraspecific sequence similarity of 97.0-98.0% and 98.2-99.9%, respectively. Phylogenetic trees constructed from ITS2 sequences show a markedly distant position of D. pacificum from other species analyzed and also indicate the possible paraphyly of Spirometra.