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.

PALEOEPIDEMIOLOGY OF DIPHYLLOBOTHRIOSIS: CULTURAL AND ENVIRONMENTAL FACTORS AFFECTING ADENOCEPHALUS INTENSITY AND PREVALENCE.

Peruvian and Chilean mummies and coprolites provide a source of population-based parasitological information. This is especially true of the fish tapeworm, Adenocephalus pacificus. Our analysis of Chinchorro and Chiribaya mummies and diversified coprolite samples from Chile and Peru show variation in infection. There is a statistically significant difference in prevalence between Chinchorro hunter-gatherer and Chiribaya mixed-subsistence contexts. Furthermore, the most pronounced differences occur between populations within these groups. Chinchorro differences in cemeteries at the same location can be related to El Niño-Southern Oscillation variations. Pronounced prevalence variations between 3 Chiribaya villages within 7 km of each other relate to fish distribution and preparation variation. As with other recent archaeoparasitology studies, eggs-per-gram data exhibit overdispersion.

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.

Mitochondrial and microsatellite data show close genetic relationships between Dibothriocephalus latus from South America (Argentina) and Europe (the Alpine lakes region).

The most frequent causative agent of diphyllobothriosis, a fish-borne parasitic zoonosis, is the broad fish tapeworm Dibothriocephalus latus distributed mainly throughout the Holarctic region. The larval stages of the tapeworm were also detected in native and introduced freshwater fish in several lakes in South America, particularly in the north-western Patagonia in Argentina. The main objective of the present study was to determine the genetic structure of D. latus from rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), and brook trout (Salvelinus fontinalis) from Gutiérrez Lake and Alicura Reservoir in Argentina using the sequences of three mitochondrial genes (cox1, cob, and nad3) and six polymorphic microsatellite loci. The results were compared with the corresponding molecular data of D. latus from Europe (Switzerland and Italy; the Alpine lakes region) and Asia (Siberia, Russia). Only one concatenated haplotype identical with the con-Ha1 specific for D. latus from the Alpine lakes region was detected in all individuals from Argentina. Three different alleles were detected in five out of six analysed microsatellite loci, indicating the presence of three sets of chromosomes. The same structure of microsatellite loci was recently observed in D. latus from Switzerland and Italy, in which triploidy was previously confirmed by cytogenetic study. The data on the mitochondrial genes, the allelic structure of microsatellite loci, and the principal coordinate analysis showed close genetic relationships between D. latus from Argentina and the Alpine lakes region, thus supporting the hypothesis of the European origin of the broad fish tapeworm from South America.

A regional study of the zoonotic broad tapeworm Dibothriocephalus spp. in Northwestern Patagonia (Argentina): origin of fishes and coastal cities as factors affecting infection in fishes.

Diphyllobothriosis was first recorded in humans in Argentina in 1892 and in introduced salmonids in 1952. The aim of this work is to assess factors influencing the values of prevalence and abundance of plerocercoids in fishes that could increase the risk of transmission of Dibothriocephalus spp. in Andean Patagonian lakes. We analysed two key issues potentially related to the occurrence of tapeworms in fish: the presence of cities on coastlines (as potential sources of eggs to nearby lakes) and the difference between native and exotic fishes in susceptibility to infection. We investigated the probability of finding parasites in fish, the variation in parasite abundance in different environments and the relationship between host length and occurrence of plerocercoids. A total of 3226 fishes (belonging to six autochthonous and four introduced species) were analysed between 2010 and 2019 in eight environments. Plerocercoids were counted, and a subset was determined molecularly to species level. Two species, Dibothriocephalus latus and Dibothriocephalus dendriticus, were identified from both salmonids and native fishes, this being the first molecular confirmation of these tapeworm species parasitizing native South American fishes. Salmonids had higher levels of infection than native fishes, and these levels were higher in aquatic environments with a city on their coastline. Transmission to humans seems to occur mainly through Oncorhynchus mykiss, which showed the highest infection values and is the species most captured by fishers. Based on previous data and the present results, eggs shed by humans, dogs and gulls in cities could be the principal factors in maintaining the life cycle of this parasite in surrounding aquatic environments.

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.

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.

Pacific Broad Tapeworm Adenocephalus pacificus as a Causative Agent of Globally Reemerging Diphyllobothriosis.

The Pacific broad tapeworm Adenocephalus pacificus (syn. Diphyllobothrium pacificum) is the causative agent of the third most common fish-borne cestodosis among humans. Although most of the nearly 1,000 cases among humans have been reported in South America (Peru, Chile, and Ecuador), cases recently imported to Europe demonstrate the potential for spread of this tapeworm throughout the world as a result of global trade of fresh or chilled marine fish and travel or migration of humans. We provide a comprehensive survey of human cases of infection with this zoonotic parasite, summarize the history of this re-emerging disease, and identify marine fish species that may serve as a source of human infection when eaten raw or undercooked.

Diphyllobothrium sp. en Canis familiares de la región del subtrópico argentino (Puerto Iguazú, Misiones) / Diphyllobothrium sp. in Canis familiaris from the subtropical area of Argentina (Puerto Iguazú, Misiones)

En este trabajo se informa el hallazgo de huevos de Diphyllobothrium sp. en ejemplares de Canis familiaris (perro doméstico) de Puerto Iguazú, una ciudad subtropical de la provincia de Misiones, Argentina. Durante 2013, en el marco de un relevamiento de la fauna parasitológica de los perros de Puerto Iguazú, se detectaron dos casos positivos en la búsqueda de huevos de Diphyllobothrium sp. La materia fecal de los perros fue recolectada en frascos con formol al 10% y procesada mediante las técnicas de sedimentación de Telemann y de flotación de Sheather. Dado que Misiones no forma parte de la zona endémica de difilobotriasis y considerando, además, su ubicación fronteriza, este hallazgo reviste gran importancia para la salud pública. Se señala la necesidad de actualizar el estado de conocimiento sobre el ciclo de vida de estos parásitos identificando el rango de hospederos intermediarios y definitivos, su potencial zoonótico y la situación epidemiológica en áreas no endémicas

This paper reports the first finding of Diphyllobothrium sp. eggs in Canis familiaris (domestic dog) from Puerto Iguazú, a subtropical city of Misiones province, Argentina. In 2013, two positive cases of Diphyllobothrium sp. eggs were detected during an annual parasitological survey of dogs. Dog feces were collected in vials containing 10% formalin and processed using Telemann's sedimentation and Sheather's flotation techniques. The two cases were detected in rural areas of the municipality. Since Misiones is not a part of the endemic area of diphyllobothriasis and given the fact that it is located in the three-border area of Argentina, Brazil and Paraguay, we consider this finding of great importance to public health. We stress the need for updating the current knowledge about the life cycle of these parasites considering the range of intermediate and definitive hosts, their zoonotic potential, and the epidemiological situation in non-endemic areas

Diphyllobothrium sp. in Canis familiaris from the subtropical area of Argentina (Puerto Iguazú, Misiones).

This paper reports the first finding of Diphyllobothrium sp. eggs in Canis familiaris (domestic dog) from Puerto Iguazú, a subtropical city of Misiones province, Argentina. In 2013, two positive cases of Diphyllobothrium sp. eggs were detected during an annual parasitological survey of dogs. Dog feces were collected in vials containing 10% formalin and processed using Telemann's sedimentation and Sheather's flotation techniques. The two cases were detected in rural areas of the municipality. Since Misiones is not a part of the endemic area of diphyllobothriasis and given the fact that it is located in the three-border area of Argentina, Brazil and Paraguay, we consider this finding of great importance to public health. We stress the need for updating the current knowledge about the life cycle of these parasites considering the range of intermediate and definitive hosts, their zoonotic potential, and the epidemiological situation in non-endemic areas.

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.

Difilobotriosis humana: Un caso en área no endémica de la Argentina / Human diphyllobothriosis: A case in a non-endemic area of Argentina

La difilobotriosis es una parasitosis intestinal causada por la infección de cestodos del genero Diphyllobothrium. En la Argentina, la Patagonia Andina es considerada una zona endémica para esta parasitosis. La infección por Diphyllobothrium latum no ha sido previamente notificada en la provincia de Mendoza; en este trabajo comunicamos un caso de esta parasitosis que fue confirmada por el análisis de las características morfológicas de los huevos eliminados con la materia fecal de un paciente infectado. Se destaca la necesidad de información y capacitación de los profesionales de la salud en el diagnóstico y tratamiento de parasitosis no endémicas.

Diphyllobothriosis is an intestinal parasitosis caused by cestodes infection of the genus Diphyllobothrium. In Argentina, the Andean Patagonia is considered an endemic area for this parasitosis. Diphyllobothrium latum infection has not been previously reported in the province of Mendoza, Argentina. We are now reporting then the first case. Diphyllobothriosis was confirmed by examination of morphologic characteristics of the eggs eliminated in the patients' feces. These results suggest the requirement of a more specific training of health workers in the diagnosis and treatment of non endemic parasitosis. We want to emphasize the need of health workers' education on diagnosis and treatment of endemic and non-endemic parasitosis.

Difilobotriosis humana: Un caso en área no endémica de la Argentina / Human diphyllobothriosis: A case in a non-endemic area of Argentina

La difilobotriosis es una parasitosis intestinal causada por la infección de cestodos del genero Diphyllobothrium. En la Argentina, la Patagonia Andina es considerada una zona endémica para esta parasitosis. La infección por Diphyllobothrium latum no ha sido previamente notificada en la provincia de Mendoza; en este trabajo comunicamos un caso de esta parasitosis que fue confirmada por el análisis de las características morfológicas de los huevos eliminados con la materia fecal de un paciente infectado. Se destaca la necesidad de información y capacitación de los profesionales de la salud en el diagnóstico y tratamiento de parasitosis no endémicas.(AU)

Diphyllobothriosis is an intestinal parasitosis caused by cestodes infection of the genus Diphyllobothrium. In Argentina, the Andean Patagonia is considered an endemic area for this parasitosis. Diphyllobothrium latum infection has not been previously reported in the province of Mendoza, Argentina. We are now reporting then the first case. Diphyllobothriosis was confirmed by examination of morphologic characteristics of the eggs eliminated in the patients feces. These results suggest the requirement of a more specific training of health workers in the diagnosis and treatment of non endemic parasitosis. We want to emphasize the need of health workers education on diagnosis and treatment of endemic and non-endemic parasitosis.(AU)