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.

A possible circulation of a dominant Dibothriocephalus nihonkaiensis haplotype in Japan revealed by molecular analysis of clinical tapeworm samples.

Human diphyllobothriasis, caused by Dibothriocephalus nihonkaiensis, is prevalent globally, especially in regions where raw fish is consumed. Recent molecular diagnostic techniques have made species identification of tapeworm parasites and the determination of genetic variations among parasite populations possible. However, only a few studies done over a decade ago, have reported on the genetic variation among D. nihonkaiensis in Japan. The present study employed PCR-based mitochondrial DNA analysis to specifically detect D. nihonkaiensis from archived clinical samples, and to determine any genetic variation that may exist among the Japanese broad tapeworms from patients of Kanagawa Prefecture, Japan. Target genes were amplified from DNA extracted from the ethanol- or formaldehyde-fixed samples by PCR. Further sequencing and comparative phylogenetic analyses based on mitochondrial COI and ND1 sequences were also performed. In our results, all PCR-amplified and sequenced samples were identified as D. nihonkaiensis. Analysis of COI sequences revealed two haplotype lineages. However, clustering of almost all COI (and ND1) sample sequences into one of the two haplotype clades, together with reference sequences from different countries worldwide, revealed a common haplotype among D. nihonkaiensis samples in our study. Our results suggest a possible presence of a dominant D. nihonkaiensis haplotype, with a global distribution circulating in Japan. Results from this study have the potential to improve the management of clinical cases and establish robust control measures to reduce the burden of human diphyllobothriasis in Japan.

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.

An examination of nervous system revealed unexpected immunoreactivity of both secretory apparatus and excretory canals in plerocercoids of two broad tapeworms (Cestoda: Diphyllobothriidea).

Dibothriocephalus ditremus and Dibothriocephalus latus are diphyllobothriidean tapeworms autochthonous to Europe. Their larval stages (plerocercoids) may seriously alter health of their intermediate fish hosts (D. ditremus) or cause intestinal diphyllobothriosis of the final human host (D. latus). Despite numerous data on the internal structure of broad tapeworms, many aspects of the morphology and physiology related to host­parasite co-existence remain unclear for these 2 species. The main objective of this work was to elucidate functional morphology of the frontal part (scolex) of plerocercoids, which is crucial for their establishment in fish tissues and for an early attachment in final hosts. The whole-mount specimens were labelled with different antibodies and examined by confocal microscope to capture their complex 3-dimensional microanatomy. Both species exhibited similar general pattern of immunofluorescent signal, although some differences were observed. In the nervous system, FMRF amide-like immunoreactivity (IR) occurred in the bi-lobed brain, 2 main nerve cords and surrounding nerve plexuses. Differences between the species were found in the structure of the brain commissures and the size of the sensilla. Synapsin IR examined in D. ditremus occurred mainly around FMRF amide-like IR brain lobes and main cords. The unexpected finding was an occurrence of FMRF amide-like IR in terminal reservoirs of secretory gland ducts and excretory canals, which has not been observed previously in any tapeworm species. This may indicate that secretory/excretory products, which play a key role in host­parasite relationships, are likely to contain FMRF amide-related peptide/s.

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.

Comparative transcriptomic analysis of the larval and adult stages of Dibothriocephalus dendriticus (Cestoda: Diphyllobothriidea).

Tapeworms of the genus Dibothriocephalus are widely distributed throughout the world, some of which are agents of human diphyllobothriasis, one of the most important fish-borne zoonoses caused by a cestode parasite. Genomic and transcriptomic data can be used to develop future diagnostic tools and epidemiological studies. The present work focuses on a comparative analysis of the transcriptomes of adult and plerocercoid D. dendriticus and the identification of their differentially expressed genes (DEGs). Transcriptome assembly and analysis yielded and annotated 35,129 unigenes, noting that 16,568 (47%) unigenes were not annotated in known databases, which may indicate a unique set of expressed transcripts for D. dendriticus. A total of 8022 differentially expressed transcripts were identified, including 3225 upregulated and 4797 downregulated differentially expressed transcripts from the plerocercoid and adult animals. The analysis of DEGs has shown that among the most differentially expressed genes, there are important genes characteristic of each stage. Thus, several genes are characteristic of D. dendriticus plerocercoids, including fatty acid-binding protein and ferritin. Among the most highly expressed DEGs of the adult stage of D. dendriticus is the Kunitz-type serine protease inhibitor, in two putative isoforms. The analyses of GO and KEGG metabolic pathways revealed that a large number of the DEGs of D. dendriticus are associated with the biosynthesis of various substances such as arginine and folate, as well as with various metabolic pathways such as galactose metabolism, selenocompound metabolism, and phosphonate and phosphinate metabolism. This will contribute to further research aimed at identifying targets for new generation drugs and the development of specific vaccines.

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.

Fish tapeworms (Cestoda) in the molecular era: achievements, gaps and prospects.

The tapeworms of fishes (Chondrichthyes and Actinopterygii) account one-third (1670 from around 5000) of the total tapeworm (Platyhelminthes: Cestoda) species diversity. In total 1186 species from 9 orders occur as adults in elasmobranchs (sharks, rays and chimaeras), and 484 species from 8 orders mature in ray-finned fishes (referred to here as teleosts). Teleost tapeworms are dominated by freshwater species (78%), but only 3% of elasmobranch tapeworms are known from freshwater rays of South America and Asia (Borneo). In the last 2 decades, vast progress has been made in understanding species diversity, host associations and interrelationships among fish tapeworms. In total, 172 new species have been described since 2017 (149 from elasmobranchs and 23 from teleosts; invalidly described taxa are not included, especially those from the Oriental region). Molecular data, however, largely limited to a few molecular markers (mainly 28S rDNA, but also 18S and cox1), are available for about 40% of fish tapeworm species. They allowed us to significantly improve our understanding of their interrelationships, including proposals of a new, more natural classification at the higher-taxonomy level (orders and families) as well as at the lower-taxonomy level (genera). In this review, we summarize the main advances and provide perspectives for future research.

Unique genetic structure of the human tapeworm Dibothriocephalus latus from the Alpine lakes region - a successful adaptation?

Dibothriocephalus latus is the most frequent causative agent of fish-borne zoonosis (diphyllobothriosis) in Europe, where it is currently circulating mainly in the Alpine lakes region (ALR) and Russia. Three mitochondrial genes (cox1, cob and nad3) and 6 microsatellite loci were analysed to determine how is the recently detected triploidy/parthenogenesis in tapeworms from ALR displayed at the DNA level. A geographically distant population from the Krasnoyarsk Reservoir in Russia (RU-KR) was analysed as a comparative population. One or 2 alleles of each microsatellite locus was detected in plerocercoids from RU-KR, corresponding to the microsatellite pattern of a diploid organism. In contrast, 1­3 alleles were observed in tapeworms from ALR, in accordance with their triploidy. The high diversity of mitochondrial haplotypes in D. latus from RU-KR implied an original and relatively stable population, but the identical structure of mitochondrial genes of tapeworms from ALR was probably a consequence of a bottleneck typical of introduced populations. These results indicated that the diploid/sexually reproducing population from RU-KR was ancestral, located within the centre of the distribution of the species, and the triploid/parthenogenetically reproducing subalpine population was at the margin of the distribution. The current study revealed the allelic structure of the microsatellite loci in the triploid tapeworm for the first time.

Intestinal parasites in the Neolithic population who built Stonehenge (Durrington Walls, 2500 BCE).

Durrington Walls was a large Neolithic settlement in Britain dating around 2500 BCE, located very close to Stonehenge and likely to be the campsite where its builders lived during its main stage of construction. Nineteen coprolites recovered from a midden and associated pits at Durrington Walls were analysed for intestinal parasite eggs using digital light microscopy. Five (26%) contained helminth eggs, 1 with those of fish tapeworm (likely Dibothriocephalus dendriticus) and 4 with those of capillariid nematodes. Analyses of bile acid and sterol from these 5 coprolites show 1 to be of likely human origin and the other 4 to likely derive from dogs. The presence of fish tapeworm reveals that the Neolithic people who gathered to feast at Durrington Walls were at risk of infection from eating raw or undercooked freshwater fish. When the eggs of capillariids are found in the feces of humans or dogs it normally indicates that the internal organs (liver, lung or intestines) of animals with capillariasis have been eaten, and eggs passed through the gut without causing disease. Their presence in multiple coprolites provides new evidence that internal organs of animals were consumed. These novel findings improve our understanding of both parasitic infection and dietary habits associated with this key Neolithic ceremonial site.

Reconstructing the history of helminth prevalence in the UK.

Intestinal helminth parasites (worms) have afflicted humans throughout history and their eggs are readily detected in archaeological deposits including at locations where intestinal parasites are no longer considered endemic (e.g. the UK). Parasites provide valuable archaeological insights into historical health, sanitation, hygiene, dietary and culinary practices, as well as other factors. Differences in the prevalence of helminths over time may help us understand factors that affected the rate of infection of these parasites in past populations. While communal deposits often contain relatively high numbers of parasite eggs, these cannot be used to calculate prevalence rates, which are a key epidemiological measure of infection. The prevalence of intestinal helminths was investigated through time in England, based on analysis of 464 human burials from 17 sites, dating from the Prehistoric to Industrial periods. Eggs from two faecal-oral transmitted nematodes (Ascaris sp. and Trichuris sp.) and the food-derived cestodes (Taenia spp. and Diphyllobothrium latum syn Dibothriocephalus latus) were identified, although only Ascaris was detected at a high frequency. The changing prevalence of nematode infections can be attributed to changes in effective sanitation or other factors that affect these faecal-oral transmitted parasites and the presence of cestode infections reflect dietary and culinary preferences. These results indicate that the impact of helminth infections on past populations varied over time, and that some locations witnessed a dramatic reduction in parasite prevalence during the industrial era (18th-19th century), whereas other locations continued to experience high prevalence levels. The factors underlying these reductions and the variation in prevalence provide a key historical context for modern anthelmintic programs.

Population genetic structure of diphyllobothriid tapeworms (Cestoda: Diphyllobothriidea) parasitising fish in the Baikal Rift Zone.

Tapeworms of the genus Dibothriocephalus are widely distributed throughout the world, and some are agents of human diphyllobothriasis, one of the most important fish-borne zoonoses caused by a cestode parasite. Until now, the population genetic structure of diphyllobothriid tapeworms in the Baikal Rift Zone (BRZ) has remained unexplored. The major aim of this study was to analyse the population genetic structure of D. dendriticus and D. ditremus parasitising fish in the BRZ based on internal transcribed spacer 1 (ITS1) and mitochondrial gene cytochrome oxidase subunit I (cox1) sequences. We found that both species had complex population genetic structures. Each species formed 2 clades (D. dendriticus: Clade 1 & 2; D. ditremus Clade A & B) that differed in genetic diversity. D. dendriticus haplotypes in Clade 1 formed a star-like sub-network with a main haplotype, whereas the haplotypes in Clade 2 formed a diffuse network. We assumed that the complex population genetic structure of D. dendriticus was a consequence of populations evolving under different palaeoecological conditions during the Last Glacial Maximum. In contrast to D. dendriticus, both clades in the D. ditremus samples formed a diffuse network. Our findings revealed hypothetical pathways in the formation of the population genetic structure of diphyllobothriids in the BRZ. On one hand, isolation by distance played an important role; on the other hand, lake recolonisation from refugia and a genetic bottleneck after the end of the Last Glacial Maximum had a possible influence.

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.

Ups and downs of infections with the broad fish tapeworm Dibothriocephalus latus in Europe from 1900 to 2020: Part I.

The broad fish tapeworm, Dibothriocephalus latus (Diphyllobothriidea), is the most frequent causative agent of diphyllobothriosis, a fish-borne zoonosis, in Europe. Diphyllobothriosis is characterized by the transmission of D. latus larvae to humans via the consumption of raw, marinated, smoked or inadequately cooked fish products. The most important European foci of diphyllobothriosis have been Fennoscandia, the Baltic region, the Alpine lakes region, the Danube River region, and several endemic regions in Russia. This review provides basic data on the biology, life cycle, host specificity, methods of identification of D. latus, and a detailed summary of its occurrence in intermediate and definitive hosts in Fennoscandia and the Baltic, Alpine, and Danube regions during the last 120 years (1900-2020). Deeper insight into the unique pattern of distribution of D. latus in endemic regions is provided. The numbers of records are associated with several milestones of particular time periods. The first milestone (historical), which influenced studies on D. latus in Europe, was the period during and after World War II (1941-1950). The second milestone (epidemiological) was the decade 1981-1990, when previous massive health campaigns led to a marked decline of diphyllobothriosis in Europe and less published data on D. latus. Based on recent data, the broad fish tapeworm is either absent or present at very low prevalences in Fennoscandia and the Baltic and Danube regions, but the Alpine lakes region represents a continuous ongoing circulation of the parasite in the natural environment and humans.

Relationship between the prevalence of Dibothriocephalus latus (Cestoda: Diphyllobothriidea) and the load of Escherichia coli: New findings in a neglected fish-borne parasitic zoonosis.

The sub-Alpine lakes of Switzerland, Italy and France have long been reported as an endemic area of diphyllobothriosis, a parasitic zoonosis caused by Dibothriocephalus latus. With this study, we explored the hypothesis for a relationship between the prevalence of D. latus in Perca fluviatilis and the Escherichia coli load in lake water. To do this, we identified eleven sampling sites in three areas (north, centre and south) of Lake Iseo (north Italy) to determine E. coli load and the prevalence of D. latus in P. fluviatilis. Prevalence and 95% confidence interval (CI) of D. latus infestation ranged from 0% (95% CI: 0.71-0.0) in Sarnico (southern area) to 20% (95% CI: 33.0-11.2) in Pisogne (northern area). There were significant differences in prevalence between the sites (χ2  = 31.12; p-value = .0006) and in E. coli load (Kruskal-Wallis test; p-value = .0005). There was decreasing gradient of E. coli load and prevalence of D. latus infestation from north to south. A significant positive correlation (r = .881; p-value = .003) was found between E. coli load and prevalence of D. latus. Also, linear regression showed a significant relationship between E. coli load and prevalence of infestation (R2  = .775). Our findings offer an explanation for the link between E. coli load in water and D. latus prevalence. The potential factors in this link are the efficiency of the local wastewater treatment plant, the bathymetric profile of the lake and the life cycle of D. latus, which is mainly affected by light and water temperature.

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.