Dicrocoeliidae Family: Major Species Causing Veterinary Diseases.

This chapter analyses the taxonomic position of the Dicrocoeliidae family and several of its genera and species. The chapter reviews the biology of major veterinary disease-causing species, including Dicrocoelium dendriticum, Dicrocoelium hospes, Dicrocoelium chinensis, Eurytrema pancreaticum and Platynosomum fastosum. All these species have indirect life cycles with two intermediate hosts: molluscs as the first host and ants, grasshoppers and lizards as the second host. Dicrocoelium dendriticum is a widespread liver fluke found in ruminants across Europe, Asia, North Africa and North America. It can also infect humans. Dicrocoelium hospes is widely distributed in the savanna areas of Africa south of the Sahara, while D. chinensis is mainly found in ruminants in East Asia and some European countries (probably imported from Asia). Eurytrema pancreaticum is a common parasite that lives in the bile ducts, gall bladder, pancreatic ducts and intestines of ruminants. It is found in Europe, Madagascar, Asia and South America. Adults of P. fastosum live in the liver, gall bladder and pancreas of birds and mammals in Europe, Africa, Asia, North, Central and South America. Information on the epidemiology, pathology, clinical aspects, diagnosis, treatment, control, prevention and economic impact of Dicrocoeliosis caused by D. dendriticum, Eurytrematodosis and Platynosomiosis have been included.

Interrelationships of Anenterotrema (Digenea: Dicrocoeliidae) from Neotropical bats (Mammalia: Chiroptera) with description of a new species from Molossus molossus in Brazil.

Anenterotrema is a small genus of dicrocoeliids (Digenea: Dicrocoeliidae) containing 6 species found in Neotropical bats. Members of this genus are characterized by the lack of digestive system organs and, unlike the majority of dicrocoeliids, parasitize the intestine of their definitive hosts. In this study, we have morphologically examined newly collected specimens belonging to four species of Anenterotrema from Brazil, Ecuador, and Panama. Based on the data in original descriptions and our analysis of quality new specimens, we resurrected Anenterotrema freitasi, previously synonymized with A. eduardocaballeroi. We also described a new species of Anenterotrema from Molossus molossus in the Brazilian Amazon. The new species differs from congeners in several morphological features, most prominently in the size and structure of its highly muscular, protuberant ventral sucker. It is also characterized by the lack of the semi-circular thickening of the tegument around the oral sucker typical for some members of the genus. We used partial DNA sequences of the large ribosomal subunit gene (28S) and mitochondrial cytochrome c oxidase subunit 1 gene (cox1) to test the monophyly of Anenterotrema, and study the interrelationships and determine the inter- and intraspecific variation of three Anenterotrema spp. collected from different bat species in Brazil, Ecuador and Panama.

First molecular assessment of two digenean parasites of the lancehead snake Bothrops moojeni Hoge, 1966 (Serpentes, Viperidae) in Brazil.

Two digenean species, Infidum infidum Faria, 1910 (Dicrocoeliidae) and Travtrema stenocotyle Cohn, 1902 (Plagiorchiidae), were collected in the large pit viper Bothrops moojeni Hoge, 1966 from Reserva Particular do Patrimônio Natural Cisalpina, municipality of Brasilândia, Mato Grosso do Sul State, Brazil. In this study, we provide the first molecular characterisation using the 28S rDNA and phylogenetic position data of these two common digeneans from B. moojeni. The molecular framework revealed topologies with strongly supported clades using maximum likelihood and Bayesian inference methods, positioned I. infidum among Plagiorchiidae and not among Dicrocoeliidae as expected and T. stenocotyle (Plagiorchiidae) surprisingly grouped as a sister group to Allassogonoporidae, Microphallidae, Pleurogenidae, and Prosthogonimidae, not related to plagiorchids. Our molecular phylogenetic data showed that these species may not correspond to their assigned families and encourage future studies on the systematic of these understudied groups.

Dicrocoeliidae Family: Major Species Causing Veterinary Diseases.

This chapter analyses the taxonomic position of Dicrocoeliidae family and several of its genus and species. The biology of the major species causing veterinary diseases such Dicrocoelium dendriticum, Dicrocoelium hospes, Dicrocoelium chinensis, Eurytrema pancreaticum and Platynosomum fastosum, has been reviewed. All these species have an indirect life cycle, involving two intermediate hosts (molluscs as first and ants, grasshoppers and lizards as second). Dicrocoelium dendriticum is a very widespread hepatic trematode in the ruminants of many countries in Europe, Asia, North Africa and North America, even affecting humans. Dicrocoelium hospes is widely distributed in the savanna areas of Africa south of the Sahara, whilst D. chinensis has mainly been found in ruminants in East Asia and some European countries (probably imported from Asia). Eurytrema pancreaticum is a common parasite whose adults live in ruminant bile ducts, gall bladder, pancreatic ducts and intestines in Europe, Madagascar, Asia and South America. Adult P. fastosum live in the liver, gall bladder and pancreas of birds and mammals in Europe, Africa, Asia, North, Central and South America. Information about the epidemiology, pathology, clinical aspect, diagnosis, treatment, control, prevention and economic impact mainly of Dicrocoeliosis produced by D. dendriticum, as well as of Eurytrematodosis and Platynosomiosis, has been included.

Phylogenetic relationships within Dicrocoeliidae (Platyhelminthes: Digenea) from birds from the Czech Republic using partial 28S rDNA sequences.

Partial (D1-D3) 28S rRNA gene sequences from 16 isolates of digenean parasites of the family Dicrocoeliidae recovered from 16 bird species from the Czech Republic were used for phylogenetic reconstruction. Comparison with sequences available from GenBank suggests that the genus Brachylecithum is paraphyletic, requiring further validation and possible systematic revision. Although partial 28S rDNA is relatively conserved, analyses suggest that the following taxa are synonymous: Lutztrema attenuatum = L. monenteron = L. microstomum, Brachylecithum lobatum = B. glareoli. Zonorchis petiolatus is reassigned back to the genus Lyperosomum with L. collurionis as a junior synonym. The study revealed how complicated the systematics of the family Dicrocoeliidae is currently. The morphology of the group is variable, and the current distinguishing characters at species and even generic level are not sufficiently distinctive; it is difficult to identify the specimens correctly and identification of GenBank isolates is not reliable. Extensive sampling of isolates for both molecular and morphological studies is necessary to resolve the relationships within the family.

Digital Image Analysis to Estimate the Minimum Number of Eurytrema Coelomaticum Eggs in the Uterus of Adult Specimens.

This study was carried out to estimate the minimal number of eggs present in adult E. coelomaticum uterus. Samples were collected during post-mortem inspection and were submitted to light microscopy (bright field). The length, width, the total area of the parasite, uterus, and eggs were measured. The ImageJ software was used to calculate the area of the different parameters analyzed in this study. It was possible to observe that the uterus corresponds on average to 51.9 % of the total area of the parasite (ranging from 45 to 64 %). The number of eggs present in the uterus of parasites ranged from 5,946 to 15,813. To estimate the number of eggs three scenarios were considered, where the first taken into account the number of whole eggs observed in the image. In the second way to estimate the number of eggs, all the structures were considered (whole eggs and fractions that could be delimited) and compared with manual counting. Finally, in the last scenario, was considered an occupancy rate of 100 % of the uterine area per eggs, since there are overlapping eggs and these cannot be correctly delimited and accounted for. This study describes an important tool for quantifying eggs in a nondestructive manner and aggregate information until then is not explained by other works.

First Report of Athesmia Foxi Goldberger and Crane, 1911 (Digenea, Dicrocoeliidae) from Chrysocyon Brachyurus (Illiger, 1815) (Carnivora, Canidae) and Pathological Findings.

Chrysocyon brachyurus, the largest South American canid, is a native species of the Brazilian cerrado. The present study is aimed to report the occurrence of the trematode, Athesmia foxi, in the liver of a new host, C. brachyurus, and to describe its morphology and pathology. One C. brachyurus individual was necropsied and examined for the presence of parasites. Worms were collected from the bile ducts and based on morphological and morphometrical characteristics, such as a relatively large, slender, aspinose, elongated shape with vitellarium present on the upper left side of the body were identified as A. foxi. On the host, hepatic lesions limited to the bile ducts and periportal regions, were characterized as chronic-active cholangitis, biliary hyperplasia, and fibrosis . This is the first report of A. foxi parasitizing C. brachyurus, demonstrating that this parasite has no host specificity and can be widely distributed. A. foxi lesions noted in C. brachyurus are similar to those noted in various other mammalian hosts.

Molecular characteristics of representatives of the genus Brachylecithum Shtrom, 1940 (Digenea, Dicrocoeliidae) with comments on life cycle and host specificity.

The genus Brachylecithum was for the first time subject to molecular taxonomic phylogenetic analysis in order to ascertain relationships among its component taxa. We used two markers-the nuclear ribosomal 28S ribosomal DNA (rDNA) gene and the mitochondrial cox1 gene, for six species of the genus; 11 sequences of partial 28S rDNA and partial cox1 were obtained from adult B. capilliformis, B. glareoli, B. kakea, B. laniicola, B. lobatum, and B. strigis, and from larval stages obtained from snails of the genus Cepaea. We propose to synonymize B. strigis with B. lobatum, while the genetic differences in the 28S rDNA gene and mitochondrial cox1 gene confirm the species status of B. capilliformis and indicate a distinct group within Brachylecithum, including B. kakea and B. laniicola. Cercarial and metacercarial isolates from the snails showed 100 % similarity to B. lobatum; thus, it is the first record of Cepaea snails as intermediate hosts of this species and the first report on life cycle abbreviation within the Dicrocoeliidae.

New molecular data help clarify the taxonomy of central European avian dicrocoeliidae Looss, 1899 (Trematoda: Plagiorchiida).

Avian Dicrocoeliidae are difficult to identify, even in their adult stages. Molecular analyses have been conducted for only a few species, complicating the accurate identification of juvenile stages. The taxonomy of the family is unresolved, and the status of many dicrocoeliid species is uncertain. Sequences of nuclear and mitochondrial DNA loci of Central European avian Dicrocoeliidae were generated and analyzed. These included representatives of the genera Lyperosomum, Platynosomum, Stromitrema, Brachylecithum, Brachydistomum, and Lutztrema. All the sequences were obtained from morphologically identified adult specimens of dicrocoeliids isolated from avian hosts. Molecular support was obtained to validate Lyperosomum turdia, confirm the rejection of Lyperosomum dujardini and Lyperosomum alagesi, and resurrect Lyperosomum longicauda and Lyperosomum collurionis. The identity of European Platynosomum illiciens from avian hosts with American vouchers of the same species from avian and mammalian hosts was confirmed. Brachylecithum fringillae is not considered valid; the individuals that matched its diagnosis were subadult Brachydistomum ventricosum. Descriptions and comparative data for five new species are provided. These are Lyperosomum hirundinis sp. n., Lyperosomum tenori sp. n., Lyperosomum atricapillae sp. n., Stromitrema acrocephali sp. n., and Lutztrema atricapillae sp. n.. Based on the molecular data, suggestions are provided regarding the validity of dicrocoeliid species that parasitize Central European birds. Further research should address the polyphyletic status of Brachylecithum.

Phenoloxidase is involved in the immune reaction of Helix lucorum to parasitic infestation by dicrocoeliid trematode.

INTRODUCTION: Phenoloxidases are known to play a role in the immune defences of arthropods and molluscs. In the invertebrates, phenoloxidases mediate three major physiologically important processes: sclerotization, wound healing, and defence reactions. Helix lucorum serve as the first intermediate host for the larval stages of dicrocoeliid trematodes which infects animals as well as human beings. OBJECTIVE: The aim of the study is to investigate the effect of larval forms of dicrocoeliid trematodes to phenoloxidase acitivity in H. lucorum, Linneaus, 1758, in Bitlis, Turkey. The effect of the snail's shell colour to phenoloxidase activity was also investigated. MATERIAL AND METHODS: Land snails (n=200) were collected by hand from their natural habitats during the period May - June 2019 in Bitlis, Turkey. Evaluation of the process was performed by measuring immune reaction of the snails against larval forms of dicrocoeliid trematodes. Phenoloxidase activity assay was carried out using a spectrophotometer device based on 3,4-Dihydroxy-L-phenylalanine (L-dopa) hydrolysis. RESULTS: The natural infection rate of the land snails with the developmental stages of dicrocoeliid trematodes was 20%. Phenoloxidase activity was found to be significantly higher (*p<0.05) in larval forms of dicrocoeliid trematodes infected snails when compared with non-infected snails. No effect of shell colours to phenoloxidase activity was observed. CONCLUSIONS: To the best of the authors' knowledge, this study is the first to report that the phenoloxidase system is involved in the immune reaction of Helix lucorum to parasitic infestation by larval forms of dicrocoeliid trematodes.

Molecular phylogenetics and mitogenomics of three avian dicrocoeliids (Digenea: Dicrocoeliidae) and comparison with mammalian dicrocoeliids.

BACKGROUND: The Dicrocoeliidae are digenetic trematodes mostly parasitic in the bile ducts and gall bladder of various avian and mammalian hosts. Until recently their systematics was based on morphological data only. Due to the high morphological uniformity across multiple dicrocoeliid taxa and insufficient knowledge of relative systematic value of traditionally used morphological characters, their taxonomy has always been unstable. Therefore, DNA sequence data provide a critical independent source of characters for phylogenetic inference and improvement of the system. METHODS: We examined the phylogenetic affinities of three avian dicrocoeliids representing the genera Brachylecithum, Brachydistomum and Lyperosomum, using partial sequences of the nuclear large ribosomal subunit (28S) RNA gene. We also sequenced the complete or nearly complete mitogenomes of these three isolates and conducted a comparative mitogenomic analysis with the previously available mitogenomes from three mammalian dicrocoeliids (from 2 different genera) and examined the phylogenetic position of the family Dicrocoeliidae within the order Plagiorchiida based on concatenated nucleotide sequences of all mitochondrial genes (except trnG and trnE). RESULTS: Combined nucleotide diversity, Kimura-2-parameter distance, non-synonymous/synonymous substitutions ratio and average sequence identity analyses consistently demonstrated that cox1, cytb, nad1 and two rRNAs were the most conserved and atp6, nad5, nad3 and nad2 were the most variable genes across dicrocoeliid mitogenomes. Phylogenetic analyses based on mtDNA sequences did not support the close relatedness of the Paragonimidae and Dicrocoeliidae and suggested non-monophyly of the Gorgoderoidea as currently recognized. CONCLUSIONS: Our results show that fast-evolving mitochondrial genes atp6, nad5 and nad3 would be better markers than slow-evolving genes cox1 and nad1 for species discrimination and population level studies in the Dicrocoeliidae. Furthermore, the Dicrocoeliidae being outside of the clade containing other xiphidiatan trematodes suggests a need for the re-evaluation of the taxonomic content of the Xiphidiata.

New dicrocoeliid digeneans from mammals in Ecuador including a highly genetically divergent new genus from an ancient marsupial lineage.

The Dicrocoeliidae is a highly diverse and broadly distributed family of digeneans typically parasitic in the gall bladder and liver of their tetrapod hosts. So far, no study has reported dicrocoeliids, or any digeneans, from the ancient marsupial family Caenolestidae. Herein, we describe a new genus of dicrocoeliids (Otongatrema n. gen.) from Tate's shrew opossum Caenolestes fuliginosus and a new species of Metadelphis (Metadelphis cesartapiai n. sp.) from a phyllostomid bat Anoura peruana collected in Ecuador. Otongatrema can be readily distinguished from the morphologically closest dicrocoeliid genera Concinnum, Conspicuum and Canaania based on the position of the genital pore, distribution/position of the uterus and extent of vitellarium. Metadelphis cesartapiai can be easily differentiated from other Metadelphis spp. based on a combination of morphological characters including body shape and size, distribution of vitellarium, shape of the gonads as well as size of suckers and cirrus sac. In addition, we used newly generated partial sequences of the nuclear 28S rRNA gene and mitochondrial cox1 genes to examine phylogenetic affinities of the new taxa within the Dicrocoeliidae. Both the 28S and cox1 phylogenies confidently positioned Otongatrema as a sister/basal group to all other dicrocoeliids sequenced so far. The phylogenetic position of Otongatrema may be explained by a close co-evolutionary relationship with Caenolestidae, one of the most basal and most ancient groups of marsupials. In addition, our 28S phylogeny provides evidence that the complete or partial loss of intestinal structures has likely occurred independently at least 3 times in the evolutionary history of the Dicrocoeliidae.

Molecular phylogeny provides new insights on the taxonomy and composition of Lyperosomum Looss, 1899 (Digenea, Dicrocoeliidae) and related genera.

Lyperosomum Looss, 1899 is one of the largest genera of the Dicrocoeliidae and is one of the best examples of the systematic complexity and taxonomic instability within this family. We present the molecular analyses based on novel sequences of nuclear and mitochondrial genes obtained from 56 isolates of adult flukes and larval stages of dicrocoeliids belonging to Lyperosomum, Skrjabinus, Zonorchis as well as previously available sequence data. According to obtained results we propose to return Zonorchis clathratus and Z. petiolatus into Lyperosomum, and to recognize L. alagesi as a synonym of L. petiolatum. Our study shows that L. petiolatum commonly occurs in Europe in corvids as well as in several species of migratory songbirds, e.g. Sylvia atricapilla. At the same time, the Turdidae appear to host a distinct species of Lyperosomum. The phylogenetic analysis has clearly demonstrated the paraphylepic nature of Lyperosomum and indicated the need of its thorough revision preferably using specimens from type hosts and type territories of nominal species. In addition, inclusion of numerous not yet sequenced dicrocoeliid genera into future phylogenetic studies is necessary to clarify the interrelationships of taxa within the family and stabilize its system.

Description and Phylogenetic Relationships of Pojmanskatrema balcanica n. gen., n. sp. (Digenea: Dicrocoeliidae) from the Eurasian Water Shrew Neomys fodiens (Mammalia: Soricidae) in Bulgaria.

BACKGROUND: We describe a new dicrocoeliid collected from the Eurasian water shrew Neomys fodiens in Bulgaria based on the combination of morphological characteristics and molecular data. RESULTS: Pojmanskatrema balcanica n. sp., n. gen. is distinguished from all other species of family Dicrocoeliidae Looss, 1900 in having a postovarian vitellarium situated in the posterior half of the body, postacetabular, nearly tandem, lobed testes, and relatively short body. CONCLUSIONS: Molecular phylogenetic analysis based on 28S rDNA sequences of the new species and previously sequenced dicrocoeliid taxa has demonstrated close relationship of Pojmanskatrema n. gen. with the genus Eurytrema. Considering the non-monophyletic nature of previously recognized dicrocoeliid sub-families we support the recently proposed dissolution of the sub-families within this family.

Convoluted history and confusing morphology: Molecular phylogenetic analysis of dicrocoeliids reveals true systematic position of the Anenterotrematidae Yamaguti, 1958 (Platyhelminthes, Digenea).

The Dicrocoeliidae is a highly diverse family of digeneans parasitic in amniotic tetrapods. Detailed molecular phylogenetic analysis of dicrocoeliids is lacking and only a few dicrocoeliids from mammals have been included in previous studies. Sequence data were previously absent for the Anenterotrematidae that shares several morphological characteristics with dicrocoeliids. We examined phylogenetic affinities of several newly sequenced (nuclear 28S rDNA) taxa of dicrocoeliids and anenterotrematids collected from small mammals in Ecuador, Panama, Peru, USA and Vietnam. Our analyses demonstrated that the two anenterotrematid genera (Anenterotrema, Apharyngotrema) belong to the Dicrocoeliidae, placing the Anenterotrematidae into synonymy with the Dicrocoeliidae. Molecular data combined with morphological examination of type and new specimens provided evidence that Parametadelphis and Apharyngotrema are junior synonyms of Metadelphis, with all Metadelphis species lacking a digestive system. Phylogenetic analysis demonstrates that reduction of the alimentary tract in Lutztrema and its loss in Anenterotrema and Metadelphis represent at least two independent evolutionary events. Genera Brachylecithum, Brachydistomum, and Lyperosomum proved to be non-monophyletic, each likely representing more than a single genus. Furthermore, phylogenetic analysis did not support monophyly of the two largest subfamilies of the Dicrocoeliidae (Dicrocoeliinae and Leipertrematinae) with the other two subfamilies not included in this study. Therefore, we propose to abandon the current subfamily division of the Dicrocoeliidae. Analysis of host associations indicates multiple host-switching events throughout evolution of dicrocoeliids. Lastly, analysis of dicrocoeliid geographic distribution revealed that nearly all major clades included taxa from more than a single zoogeographic realm with the exception of the clade Anenterotrema + Metadelphis, found only in the Neotropics.