Characterization of the Complete Mitochondrial Genome and Phylogenetic Analyses of Eurytrema coelomaticum (Trematoda: Dicrocoeliidae).

Eurytrema coelomaticum, a pancreatic fluke, is recognized as a causative agent of substantial economic losses in ruminants. This infection, commonly referred to as eurytrematosis, is a significant concern due to its detrimental impact on livestock production. However, there is a paucity of knowledge regarding the mitochondrial genome of E. coelomaticum. In this study, we performed the initial sequencing of the complete mitochondrial genome of E. coelomaticum. Our findings unveiled that the mitochondrial genome of E. coelomaticum spans a length of 15,831 bp and consists of 12 protein-coding genes, 22 tRNA genes, two rRNA genes, and two noncoding regions. The A+T content constituted 62.49% of the genome. Moreover, all 12 protein-coding genes of E. coelomaticum exhibit the same arrangement as those of E. pancreaticum and other published species belonging to the family Dicrocoeliidae. The presence of a short string of additional amino acids (approximately 20~23 aa) at the N-terminal of the cox1 protein in both E. coelomaticum and E. pancreaticum mitochondrial genomes has contributed to the elongation of the cox1 gene in genus Eurytrema, surpassing that of all previously sequenced Dicrocoeliidae. The phylogenetic analysis displayed a close relationship between E. coelomaticum and E. pancreaticum, along with a genus-level association between Eurytrema and Lyperosomum. These findings underscore the importance of mitochondrial genomic data for comparative studies of Dicrocoeliidae and even Digenea, offering valuable DNA markers for future investigations in the systematic, epidemiological, and population genetic studies of this parasite and other digenean trematodes.

Eurytrematosis in cattle in southern Espírito Santo State, Brazil - case report.

This work reports an outbreak of eurytrematosis in cattle in the municipality of Ibitirama, southern Espírito Santo State, Brazil. Six cattle were necropsied from August to December 2019, with finding of Eurytrema coelomaticum in the pancreas. A survey of epidemiological data was carried out on the farms along with coproparasitological examination of cattle from the same herd. Parasites were found in all necropsied animals, with different degrees of parasitism, ranging from mild to massive infection (6 - 2000 specimens). Macroscopic analyses of the pancreas revealed changes in 83.33% (5/6) of the cases, and by microscopy, pancreatic fibrosis ranging from Grade I to Grade III was observed. Inspection of the grazing areas confirmed the presence of two intermediate hosts, a terrestrial snail of the Bradybaena genus, with larval forms of the trematode in histological findings, and a grasshopper of the Conocephalus genus. Although none of the cattle showed clinical signs in the coproparasitological examination, 73.80% (31/42) tested positive for E. coelomaticum eggs. This is the first record of an outbreak of eurytrematosis in cattle in Espírito Santo State, indicating the importance of carrying out diagnosis based on epidemiology and necroscopic and parasitological examinations in animals in the region so that appropriate control measures can be adopted.

Este trabalho objetivou relatar um caso de euritrematose em bovinos no município de Ibitirama, Sul do Estado do Espírito Santo, Brasil. Foram necropsiados seis bovinos de agosto a dezembro de 2019, que apresentaram Eurytrema coelomaticum no pâncreas. Foi realizado levantamento de dados epidemiológicos nas propriedades e exames coproparasitológico em bovinos do mesmo plantel. Em todos os animais necropsiados foram encontrados parasitos, com diferentes graus de parasitismo, variando de infeção branda a maciça (6 - 2000 exemplares). Análises macroscópicas dos pâncreas revelaram alterações em 83,33% (5/6) dos casos e, na microscopia, observou-se fibrose pancreática variando de Grau I a Grau III. A inspeção das áreas de pastejo constatou a presença dos dois hospedeiros intermediários, moluscos terrestres do gênero Bradybaena com formas larvares do trematoda em achados histológicos e gafanhotos do gênero Conocephalus. Nenhum dos bovinos apresentou sinais clínicos, no entanto, no exame coproparasitológico, 73,80% (31/42) testaram positivo para ovos de E. coelomaticum. Este é o primeiro registro de surto de euritrematose em bovinos no estado do Espírito Santo, mostrando a importância da realização do diagnostico a partir da epidemiologia e de exames necroscópicos e parasitológicos em animais da região para que sejam adotadas medidas adequadas de controle.

Morphological and molecular characterization of Eurytrema spp. Looss, 1907 detected in domestic water buffaloes and cattle in northern Vietnam.

Eurytrema spp. are pancreatic flukes belonging to the Dicrocoeliidae family. They are the cause of neglected diseases in Vietnam and are responsible for economic losses in ruminant production, particularly in water buffaloes and cattle. Eurytrema spp. have been widely reported in several Asian countries. Recently, morphological and molecular analyses to discriminate Eurytrema spp. have been conducted in Brazil, China, Bangladesh, Nepal, and Indonesia; however, similar analyses have not been performed in Vietnam. In the present study, we identified Eurytrema flukes collected from water buffaloes and cattle in northern Vietnam based on their morphology. Morphometric analyses were conducted on 15 samples each of Eurytrema cladorchis and Eurytrema coelomaticum. Representative samples from both species were selected for molecular analyses, and the nucleotide sequences of the 18S ribosomal RNA (18S rRNA) gene and internal transcribed spacer 2 (ITS2) were determined. Phylogenetic analyses based on 18S rRNA sequences revealed that E. cladorchis from Vietnam belongs to the same clade as that from Bangladesh. Similarly, E. coelomaticum isolates from Vietnam and China belonged to the same clade. Both clades were isolated from E. pancreaticum. This is the first study to describe the coexistence of E. cladorchis and E. coelomaticum in Vietnam and the first report of the ITS2 nucleotide sequence for E. coelomaticum, which can be used for molecular species discrimination.

Eurytrema coelomaticum infection: correlation between parasite burden and impairment of pancreatic exocrine enzyme secretion / Eurytrema coelomaticum: correlação entre carga parasitária e insuficiência pancreática exócrina

Eurytrema coelomaticum is a trematode reported in the pancreatic ducts of ruminants. It is conjectured that may cause disorders in the pancreas, as well as digestive and metabolic processes dependent on them. This study, determined if there is an impairment of exocrine pancreatic function, and correlated it with parasite burden. Pancreas, blood, and fecal samples were collected from 119 bovines at a abattoir. Stool samples were subjected to the gelatin and x-ray film digestion tests (to detect the presence of trypsin in feces). Using blood samples, the following biochemical tests were performed: amylase, lipase, glucose, fructosamine, cholesterol, triglycerides, total protein, albumin, and globulins. Analyses were correlated with pancreatic parasite burden. Cattle with a high parasitic load presented higher incidence of negative tests in both gelatin digestion and x-ray film digestion tests (P < 0.001) when compared to non-parasitized animals and those with a low parasitic load. Changes in those tests only occurred if the parasitemia was moderate or severe. The activity of the amylase and lipase enzymes was significantly higher in animals with low parasitemia (P < 0.05), compared to non-parasitized animals and with a high parasitic burden. In this study, in cases of high parasitemia, negative results were observed in both gelatin and x-ray film in the feces digestion tests. However, the low infection of E. coelomaticum, higher levels of serum amylase and lipase that also indicated loss of pancreatic exocrine functions were reported.

Eurytrema coelomaticum, um trematódeo de ductos pancreáticos de ruminantes. Conjectura-se que possa ocasionar transtornos nas funções pancreáticas, mais especificamente nos processos digestivos e metabólicos dependentes destas. Neste estudo, o objetivo foi determinar se há comprometimento da função pancreática exócrina, correlacionado-a a carga parasitária. Foram utilizados pâncreas e respectivas amostras de sangue e fezes de 119 bovinos. As amostras de fezes foram submetidas aos testes de digestão da gelatina em tubo e digestão de filme radiográfico, ambos para detecção de tripsina nas fezes. Foram realizados os seguintes exames bioquímicos em amostras de sangue: amilase, lipase, glicemia, frutosamina, colesterol, triglicerídeos, proteínas totais, albumina e globulinas. Após isto, as análises bioquímicas foram correlacionadas com a quantidade numérica de parasitas encontrados no pâncreas (post-mortem). Houve maior quantidade de testes negativos (digestão do filme radiográfico e prova de digestão da gelatina) nos animais com alta carga parasitária (P < 0.001), quando comparados aos animais não parasitados e com baixa carga parasitária. Portanto, os exames supracitados se alteram somente se a quantidade de parasitas for moderada ou severa. As atividades das enzimas amilase e lipase foram significativamente maiores nos animais que apresentavam baixa parasitemia (P < 0.05), em comparação com os animais com alta carga parasitária e não parasitados. Conclui-se que em quadros de alta parasitemia há alteração significativa nos testes de digestão nas fezes, e que em quadros de baixa parasitemia há alterações significativas nos valores de amilase e lipase séricas, ambos comprovando alterações pancreáticas importantes, de acordo com o quadro de parasitemia.

Eurytrema coelomaticum: updated morphology of adult worms using advanced microscopy experiments.

Eurytrema coelomaticum is a digenean flatworm of ruminants that is the causative agent of eurytrematosis, a disease of veterinary health concern. Although modern techniques of morphological analysis have provided new insights about the morphology and anatomy of parasitic helminths, most studies on E. coelomaticum adults are based on conventional light microscopy. In the present study, a combined approach using brightfield, fluorescence, confocal and scanning electron microscopies (SEMs), together with the cryofracture technique, have updated morphological data on E. coelomaticum recovered from cattle in Rio de Janeiro State, Brazil. Light microscopy confirmed the presence of several structures present in the current description, such as suckers, pharynx, oesophagus, intestinal bifurcation and the cirrus-sac. Fluorescence stereomicroscopy revealed for the first time the cubic crystal protein inclusions in the forebody, which were further detailed by confocal and SEMs. Confocal microscopy provided detailed information of the muscular architecture associated with the attachment structures (suckers), digestive system (pharynx and oesophagus), egg-forming complex (ovary, Mehlis' gland and Laurer's canal) and male reproductive system, which are similar to those found in other digenean flukes. SEM images of cryofractured parasites showed mucus and developing eggs within uterine loops. It was demonstrated that the combination of advanced tools generated complementary information, confirming the importance of experimental morphology in parasitology. Therefore, the knowledge of the adult structural organization of E. coelomaticum was improved and this work has contributed to propose new morphological criteria to evaluate the effects of antiparasitic drugs on flukes of medical and veterinary importance.

Comments on eurytrematosis in Brazil and the possibility of human infection.

The manuscript "Eurytrematosis: An emerging and neglected disease in South Brazil" discusses some aspects of Eurytrema sp. fluke as an animal pathogen and based in some aspects of the parasitism in cattle and the life cycle of Eurytrema sp. Authors suggest the possibility of human infection, once there is no research on this subject in Brazil. In human cases reported, the mechanism of infection was not disclosed, so it keeps the discussion opened. Although we focused on animal eurytrematosis, we speculated the possibility of human infection by Eurytrema sp. in Brazil, but after all, the only way to determine it, would be a study searching for people infected through coprological or serological tests.

Changes in the lipid profile of Bradybaena similaris (Férussac, 1821) (Gastropoda, Xanthonychidae) during the development of Eurytrema coelomaticum (Giard and Billet, 1892) (Digenea, Dicrocoeliidae).

The effect of experimental exposure of Bradybaena similaris by Eurytrema coelomaticum on the cholesterol and triglycerides total levels circulating in the hemolymph and the neutral lipids in the digestive gland-gonad (DGG) complex of the host snail were studied. In this study, snails were dissected after one, two, and three months of infection to collect the hemolymph and DGG to measure the cholesterol and triacylglycerol levels in the hemolymph and neutral lipid fractions in the tissues. The results for the hemolymph showed that the infection by this trematode resulted in a significant decrease in the concentrations of triacylglycerol during all periods analyzed, as well as, increase in the levels of cholesterol observed after the second and third months of study. This decrease suggests the possible use of triacylglycerol by both parasite and host as alternative substrate in maintaining their energy metabolisms. In parallel, the increase in the contents of cholesterol results from a severe cellular disorganization in snail, especially DGG, as a consequence of the intense plastic processes showed by parasite. Additionally, changes in the neutral lipid profile in the DGG of the infected snails were observed, indicating the importance of these molecules for successful infection.

Observação in vitro da ação dos fungos nematòfagos Duddingtonia flagrans, Monacrosporium thaumasium e Pochonia chlamydosporia sobre ovos de Eurytrema coelomaticum / In vitro evaluation of the action of the nematophagous fungi Duddingtonia flagrans, Monacrosporium thaumasium and Pochonia chlamydosporia on Eurytrema coelomaticum eggs

Objetivou-se a observação in vitro da ação dos fungos nematófagos Duddingtonia flagrans (AC001), Monacrosporium thaumasium (NF 34) e Pochonia chlamydosporia (VC1 e VC4) sobre ovos de Eurytrema coelomaticum. Os ovos foram vertidos em superfície de ágar-água 2 por cento contendo os isolados fúngicos e em AA 2 por cento sem fungo como controle. Ao completarem sete, 10 e 14 dias, aproximadamente os ovos foram removidos e classificados de acordo com os seguintes parâmetros: efeito tipo 1, efeito lítico sem prejuízo morfológico a casca do ovo; tipo 2, efeito lítico com alteração morfológica da casca e embrião e tipo 3, efeito lítico com alteração morfológica do embrião e da casca, além de penetração de hifas e colonização interna do ovo. Os isolados AC001 e NF34 não demonstraram percentuais para o efeito do tipo 3, contudo o isolado VC1 apresentou resultados percentuais para o efeitodo tipo 3 que determinam a atividade ovicida de um fungo: 27,2 por cento aos sete dias, 23,1 por cento aos 10dias e 25,0 por cento aos 14 dias. Da mesma forma que isolado VC4 apresentou: 15,0 por cento aos sete dias, 25,4 por cento aos 10 dias e 21,8 por cento aos 14 dias respectivamente. Pochonia chlamydosporia é um fungo promissor que pode ser usado no controle biológico de E. coelomaticum.

The present study assessed in vitro action of nematophagous fungi species Duddingtonia flagrans (AC 001), Monacrosporium thaumasium (NF 34) and Pochonia chlamydosporia (VC1 and VC4) on eggs of Eurytrema coelomaticum. Eggs were placed on Petri dishes with fungus isolate grown in water- agar 2 percent and in the control (no fungus). After seven, 10 and 14 days, the eggs were removed and classified according to the following parameters: type 1, lytic effect without morphological damage to eggshell; type 2, lytic effect with morphological alteration of embryo and eggshell; and type 3, lytic effect with morphological alteration of embryo and eggshell, besides hyphal penetration and internal egg colonization. The isolate AC001 andNF34 had not demonstrated percentages to type 3 effects, however isolated VC1 presented results percentages for the type 3 effect that it determines the ovicida activity of one fungus: 27.2 percent to the seven days, 23.1 percent to the 10 days and 25.0 percent to the 14 days. The isolated VC4 presented: 15.0 percent to the seven days, 25.4 percent to the 10 days and, 21.8 percentto the 14 days. P. chlamydosporia is a promising fungus can be used in the biological control of E. coelomaticum.