Morphological and molecular data confirm the occurrence of Paramphistomum leydeni (Trematoda: Paramphistomidae) in ruminants from Southern Brazil.

Species belonging to the family Paramphistomidae Fischoeder, 1901, commonly known as "rumen flukes", are a group of parasites frequently related to Brazilian livestock production. They inhabit the digestive tract of ruminants and have recognized pathogenicity during the early stages of infection, which can be responsible for economic losses. These trematodes are often associated with Southern Brazil, a region heavily focused on animal farming, which also makes it ideal for the life cycle of paramphistomes. Despite their aforementioned importance, studies regarding their distribution, molecular taxonomy and biology are still scarce in the country. In the present study, rumen flukes collected from cattle (n = 22) and sheep (n = 3) from 9 batches of ruminants from the cities of Jaguarão, Pelotas and Rio Grande, state of Rio Grande do Sul, Brazil, between May and July 2022, were subjected to morphological and molecular study. The microscopic analysis of histological and manual cuts revealed diagnostical traits compatible with Paramphistomum leydeni Näsmark, 1937, including the presence of tegumental papillae, pharynx of the liorchis type and acetabulum of the leydeni type. Molecular data corroborated the morphological identification, with ITS-2 and cox-1 sequences here obtained presenting 100% and 96.8-99.8% similarity, respectively, to P. leydeni samples previously characterized in different countries from Asia, Europe, and South America. Intensity of infection ranged from 5 to 458 and 1 to3 specimens of P. leydeni in sampled cattle and sheep, respectively. The present study contributes to a better understanding of the taxonomy of the flukes involved in cattle and sheep paramphistomosis in Brazil, suggesting that P. leydeni could be the main paramphistome species found in ruminants in the studied region.

Idiopyrgus souleyetianus (Gastropoda: Tomichiidae) as an intermediate host of the liver fluke Amphimerus sp. (Trematoda: Opisthorchiidae) in Brazil.

Despite the importance of fish-borne trematodes of the family Opisthorchiidae as causative agents of human liver fluke disease, studies on these parasites outside Asia are relativally scarce. In South America, human focus of amphimerosis is known in Ecuador since the mid-20th century, and Amphimerus spp. have also been reported in wild and domestic mammals. Nevertheless, the knowledge on the snails that act as the first intermediate host of these potentially zoonotic parasites are scarce. Herein, a new cercaria of the pleurolophocercous morphotype found in the freshwater snail Idiopyrgus souleyetianus from Brazil was subjected to morphological and molecular studies. Multigene phylogenetic analyses based on 28S, 5.8S-ITS-2 and Cox-1 sequences enabled the identification of Amphimerus sp., a species distinct from that reported in humans from Ecuador. This cercariae was morphologically compared with other opisthorchiid cercariae known. The possible occurrence of human amphimerosis in Brazil is discussed.

A worm in the toilet bowl! What does it tell us about the challenges in the identification of supposed human parasites in practical parasitology?

Non-parasitic vermiform organisms can circumstantially be associated with humans and their identification can be challenging for medical professionals. The present report describes the finding of a worm in the toilet bowl by a patient from Brazil, who thought he had expelled it in his feces. The gross analyses in a clinical laboratory reveal the worm was different from other macroscopic organisms routinely identified, and the laboratory staff requested assistance in an academic laboratory specialized in helminthology. After preliminary analysis in a stereomicroscope, the supposed human worm was identified as an oligochaete annelid (earthworm). The patient was contacted to investigate a possible case of pseudoparasitism. However, we were informed that the organism had been collected in a toilet bowl from a rural environment where the untreated water comes from a cistern indicating our finding was circumstantial. The methodology revisited herein allowed a quick microscopic analysis of easy-to-view morphological structures, which are useful to separate oligochaete annelids from helminths and can prevent misdiagnosis in similar situations. We discuss the overly restricted view on human parasites by health professionals in collecting clinical history and laboratory analysis, providing some epistemological insights on the necessary interdisciplinarity between parasitology and other basic knowledge with health practice.

A new cryptic species of Echinostoma (Trematoda: Echinostomatidae) closely related to Echinostoma paraensei found in Brazil.

Echinostoma paraensei, described in Brazil at the end of the 1960s and used as a biological model for a range of studies, belongs to the 'revolutum' complex of Echinostoma comprising species with 37 collar spines. However, molecular data are available only for a few isolates maintained under laboratory conditions, with molecular prospecting based on specimens originating from naturally infected hosts virtually lacking. The present study describes Echinostoma maldonadoi Valadão, Alves & Pinto n. sp., a species cryptically related to E. paraensei found in Brazil. Larval stages (cercariae, metacercariae and rediae) of the new species were found in the physid snail Stenophysa marmorata in the State of Minas Gerais, Brazil, the same geographical area where E. paraensei was originally described. Adult parasites obtained experimentally in Meriones unguiculatus were used for morphological (optical microscopy) and molecular [28S, internal transcribed spacer (ITS), nad1 and cox1] characterization. The morphology of larval and adult parasites (most notable the small-sized dorsal spines in the head collar), associated with low (0­0.1%) molecular divergence for 28S gene or ITS region, and only moderate divergence for the mitochondrial cox1 gene (3.83%), might suggest that the newly collected specimens should be assigned to E. paraensei. However, higher genetic divergence (6.16­6.39%) was found in the mitochondrial nad1, revealing that it is a genetically distinct, cryptic lineage. In the most informative phylogenetic reconstruction, based on nad1, E. maldonadoi n. sp. exhibited a strongly supported sister relationship with E. paraensei, which may indicate a very recent speciation event giving rise to these 2 species.

Biomphalaria straminea as an Intermediate Host of a Renal Trematode Species of the Genus Tanaisia (Trematoda: Eucotylidae) in Brazil.

PURPOSE: Eucotylid trematodes are parasites of the urinary system of birds with a cosmopolitan distribution. Despite the importance of these flukes, fundamental aspects of their biology, such as intermediate hosts and larval morphology, are poorly known. Herein, the potential involvement of aquatic mollusks in the transmission of a species of Tanaisia is reported for the first time. METHODS: During the search of non-emergent larval stages of trematodes in mollusks collected from an urban waterbody from Brazil in February of 2021, 1 out of 18 specimens (5.5%) of Biomphalaria straminea was found harboring sporocysts, cercariae and encysted metacercariae morphologically compatible with those described for eucotylid species. Sequences generated for 28S, ITS-2, and cox1 molecular markers were compared with sequences available in GenBank and subjected to phylogenetic analyses. RESULTS: Molecular analyses revealed parasite affiliation with members of the genus Tanaisia, given it groped in a strongly supported clade with species of this genus included in the 28S phylogenetic tree. The larvae tentatively identified as Tanaisia sp. can be conspecific with an unpublished isolate of Tanaisia valida found in birds in South Brazil (100% similarity in 28S and ITS-2). CONCLUSION: Biomphalaria straminea is reported as a natural host of a species of Tanaisia for the first time. This finding highlights the possibility, so far unknown, of transmission of species of the family Eucotylidae in aquatic environments.

Survey on Limnic Gastropods: Relationships between Human Health and Conservation.

The present work aimed to study ecological aspects related to the distribution pattern of medically important and native freshwater mollusks, found in a rural municipality in the state of Minas Gerais, Brazil. Malacological captures were carried out in aquatic environments (lentic and lotic) from 46 locations between October 2018 and September 2019. The collected specimens were subjected to taxonomic identification and evaluation for infection with trematode larvae. Qualitative data were used to analyze the similarity and the odds ratios between the environmental variables. In total, 1125 specimens were sampled, belonging to the following species: Biomphalaria glabrata, B. tenagophila, B. straminea, B. kuhniana, B. cousini, Biomphalaria sp., and Drepanotrema cimex (Planorbidae), Stenophysa marmorata (Physidae), Omalonyx sp. (Succineidae), Pseudosuccinea columella (Lymnaeidae), and Pomacea sp. (Ampullaridae). Echinostome, strigeocercaria, and xiphidiocercaria types of larval trematodes were detected in S. marmorata and D. cimex. Of note was the similarity in the distribution of S. marmorata, a supposedly endangered species, with that of the medically important Biomphalaria species, with the two sharing environments. This complex scenario led us to reflect on and discuss the need for the control of important intermediate hosts, as well as the conservation of endangered species. This relevant issue has not yet been discussed in detail, in Brazil or in other countries that recommend snail control.

Size does not matter: molecular phylogeny reveals one of the largest trematodes from vertebrates, the enigmatic Ithyoclinostomum dimorphum, as a species of Clinostomum (Trematoda: Clinostomidae).

Despite the recent advances raised in the molecular era to the taxonomic knowledge of species of the family Clinostomidae, especially those belonging to the specious genus Clinostomum, some groups of these vertebrate parasites remain poorly studied. This is the case of species of the enigmatic genus Ithyoclinostomum Witenberg, 1926, until recently monotypic and restricted to South America, but with its occurrence expanded to North America after the description of I. yamagutii Rosser et al., 2020. Nevertheless, molecular data for the type species of the genus, Ithyoclinostomum dimorphum (Diesing, 1850), is lacking so far. In the present study, large clinostomid metacercariae morphologically indistinguishable from I. dimorphum were obtained from two erythrinid fishes from the Rio Doce River, Southeast Brazil. Samples of the parasites were subjected to a multigene (28S rDNA, ITS and cox1) molecular characterization followed by phylogenetic reconstructions. Phylogenies based on single-gene and concatenated datasets revealed unequivocally that I. dimorphum falls in a well-supported clade together with species of the genus Clinostomum. Moreover, the molecular divergences observed in relation to Clinostomum spp. [ranges of 2.4-6%, 2.4-3.8% and 14.7-19.3% for the ITS, 28S and cox1 genes, respectively] are compatible with a congeneric status with these species. Therefore, the genus Ithyoclinostomum is here synonymized with Clinostomum Leidy, 1856 and C. dimorphum (Diesing, 1850) Braun, 1899 re-established. In the phylogenetic analysis, the recently described 'Ithyoclinostomum' yamagutii, presented as an isolated, independent lineage, showing significant molecular divergences to C. dimorphum (12.6%, 7.6%, 18,6% for the ITS, 28S and cox1 genes, respectively). However, given the complex scenario raised in the morphology-based taxonomy of Clinostomidae, we took a conservative approach by not proposing a new genus to 'I.' yamagutii until molecular data of other clinostomid genus from birds, Clinostomatopsis, become available. Data here presented reveals that body size is not a useful criterion for higher-level classification in Clinostomidae. Finally, we highlighted the importance of the availability of molecular data for the type species of trematode genera proposed from South America to support a trans- or intercontinental distribution.

Phylogenomics and Diversification of the Schistosomatidae Based on Targeted Sequence Capture of Ultra-Conserved Elements.

Schistosomatidae Stiles and Hassall 1898 is a medically significant family of digenetic trematodes (Trematoda: Digenea), members of which infect mammals or birds as definitive hosts and aquatic or amphibious gastropods as intermediate hosts. Currently, there are 17 named genera, for many of which evolutionary interrelationships remain unresolved. The lack of a resolved phylogeny has encumbered our understanding of schistosomatid evolution, specifically patterns of host-use and the role of host-switching in diversification. Here, we used targeted sequence capture of ultra-conserved elements (UCEs) from representatives of 13 of the 17 named genera and 11 undescribed lineages that are presumed to represent either novel genera or species to generate a phylogenomic dataset for the estimation of schistosomatid interrelationships. This study represents the largest phylogenetic effort within the Schistosomatidae in both the number of loci and breadth of taxon sampling. We present a near-comprehensive family-level phylogeny providing resolution to several clades of long-standing uncertainty within Schistosomatidae, including resolution for the placement of the North American mammalian schistosomes, implying a second separate capture of mammalian hosts. Additionally, we present evidence for the placement of Macrobilharzia at the base of the Schistosoma + Bivitellobilharzia radiation. Patterns of definitive and intermediate host use and a strong role for intermediate host-switching are discussed relative to schistosomatid diversification.

A putative new genus of avian schistosome transmitted by Biomphalaria straminea (Gastropoda: Planorbidae) in Brazil, with a discussion on the potential involvement in human cercarial dermatitis.

Human cercarial dermatitis (HCD) caused by avian schistosomes is an emerging health issue in different parts of the world. Nevertheless, parasite diversity, life cycle, and involvement in HCD remain poorly known or neglected in South America. Herein, we reported data obtained during a long-term malacological survey carried out in Pampulha Reservoir, an urban eutrophic waterbody from Brazil between 2009 and 2012. An ocellate brevifurcate cercaria emerged from 55 of 16,235 (0.34%) specimens of Biomphalaria straminea. Samples of the cercariae were subjected to morphological, experimental, and molecular study (analysis of partial sequences of nuclear 28S and mitochondrial cox1 genes). The molecular analysis revealed that the larva corresponds to an avian schistosome; however, it does not correspond to any named genus. A close related isolate was previously reported in Biomphalaria sudanica from Kenya (molecular divergences of 0.54% and 9.62% for 28S and cox1, respectively). The morphology of this cercaria was compared with other avian schistosome larvae from Biomphalaria spp. Attempts to infect experimentally ducks (Cairina moschata) and mice revealed cutaneous manifestations after exposure to cercariae, but adult parasites were not obtained in these hosts. Phylogenetic analysis suggests this parasite is a putative new genus and species of avian schistosome. The potential involvement of the larvae herein described in cases of HCD in Brazil cannot be ruled out. Surprisingly, HCD was not reported in the country so far, which can be related to difficulties in its diagnosis in areas of overlap with human schistosomes.

Acute infection with Platynosomum illiciens (Trematoda: Dicrocoeliidae) as a clinically relevant and potentially fatal disease in Falco sparverius (Aves: Falconidae) in Brazil.

Platynosomum illiciens is a dicrocoeliid trematode from the biliary tract of warm-blooded vertebrates (felines, primates, marsupials, and birds) reported in different parts of the world. Although the veterinary relevance of platynosomosis in mammals, especially in domestic felines, has been increasingly evidenced in the scientific literature, studies involving avian disease caused by P. illiciens are comparatively scarce. In the present study, a female specimen of the American kestrel, Falco sparverius L., found dead, in November 2019, in Brazil, was necropsied. Parietal biliary effusion in the celomatic cavity was observed, suggesting biliary transudation and gallbladder stasis, which possibily preceded the distension and rupture of gallbladder noted during necropsy. In the microscopic analysis of the bile content, small trematodes were found and characterized as immature stages of Platynosomum after the morphological study. Partial sequences of the cox-1 gene enabled the identification of P. illiciens, with 100% similarity with previously sequenced sympatric isolates from mammals. The finding of immature specimens in a ruptured gallbladder strongly suggests a role for the parasite in biliary flow dysfunction, indicating acute platynosomosis as a clinically relevant and potentially fatal condition that has not yet been discussed.

A new species of Echinostoma (Trematoda: Echinostomatidae) from the 'revolutum' group found in Brazil: refuting the occurrence of Echinostoma miyagawai (=E. robustum) in the Americas.

Although Echinostoma robustum (currently a synonym of E. miyagawai) was reported in the Americas based on molecular data, morphological support on adult parasites is still required. Herein, a new species of Echinostoma is described based on worms found in a chicken from Brazil. Molecular phylogenetic analyses based on 28S (1063 bp), ITS (947 bp) and Nad-1 (442 bp) datasets reveal the inclusion of the new species within Echinostoma 'revolutum' species complex. Moreover, it was verified the conspecificity between cercariae previously identified as E. robustum in Brazil [identical ITS and only 0.3% of divergence (1 nucleotide) in Nad-1]. Species discovery analyses show that these two isolates form an independent lineage (species) among Echinostoma spp. Compared to E. miyagawai, the new species presents relatively high divergence in Nad-1 (7.88­9.09%). Morphologically, the specimens are distinguished from all nominal species from the 'revolutum' species complex by the more posterior position of the testes (length of post-testicular field as a proportion of body length about 20%). They further differ from E. miyagawai and South American Echinostoma spp. by the higher proportion of forebody to the body length. Therefore, combined molecular and morphological evidence supports the proposal of the species named here as Echinostoma pseudorobustum sp. nov.

The life cycle of Philophthalmus aylacostoma n. sp. (Trematoda: Philophthalmidae), a new eye fluke species transmitted by Aylacostoma spp. (Gastropoda: Thiaridae) in Brazil.

Philophthalmus is a cosmopolitan genus of digeneans that includes ocular parasites of birds and mammals. Despite broad distribution and veterinary importance of these digeneans, there are still gaps in knowledge about their diversity and biology, especially in South America. Herein, we conducted morphological, life cycle, and molecular studies of megalurous cercariae found in aquatic gastropod molluscs Aylacostoma chloroticum and A. tuberculatum collected in the São Francisco River, Brazil. Adult parasites reared experimentally in the eyes of chicks are described here as Philophthalmus aylacostoma n. sp. The new species differs from its congeners known in the Americas by a combination of traits, including the sucker width ratio, the oral sucker to pharynx width ratio, egg size, and the type of vitellarium in adult forms. The new species is morphologically closest to Philophthalmus megalurus, from which it differs by the smaller body and larger eggs, as well as by the measurements of cercariae and the family of snails that act as the intermediate host. Molecular phylogenetic analysis based on 28S rDNA and comparison of cox1 sequences confirm that P. aylacostoma n. sp. is distinct from four previously sequenced named species of the genus. Moreover, cox1 sequences revealed conspecificity of our specimens with an isolate of Philophthalmus sp. previously reported, also in thiarid snails, in Paraná River, Brazil. The interspecific divergence in cox1 between the new species and other species with sequences available for comparison varied between 12 and 15%.

NEW RECORDS OF NEMATODES IN THE YELLOW-BILLED CUCKOO COCCYZUS AMERICANUS (CUCULIFORMES: CUCULIDAE) BASED ON A MIGRANT SPECIMEN FOUND IN BRAZIL.

The yellow-billed cuckoo, Coccyzus americanus, is a threatened passerine bird native to North America that migrates to overwinter in South America. Although migratory birds have drawn broad attention, given their assumed role in the long-distance dispersal of parasites, studies on the helminth fauna of this cuculid are scarce. In the present study, nematodes found in the gastrointestinal tract of a specimen of C. americanus found in Belo Horizonte, State of Minas Gerais, southeastern Brazil, were characterized morphologically. Five species of gastrointestinal nematodes belonging to 4 families were identified: Synhimantus (Dispharynx) nasuta (Rudolphi, 1819) and Synhimantus (Dispharynx) resticulaCanavan, 1929 (Acuariidae), Microtetrameres sp. (Tetrameridae), and Cyrnea piayaeSandground, 1929 (Habronematidae) and Subulura halli Barreto, 1918 (Subuluridae). Except for Microtetrameres sp., all other nematodes are reported in this host for the first time. Although it is difficult to accurately determine the geographical origin of infections, it is important to note that Sy. (D.) resticula and Su. halli are reported for the first time in Brazil. Additionally, the absence of the former species infecting hosts where the widespread and generalist Sy. (D.) nasuta was found may indicate that Sy. (D.) resticula have either been overlooked in previous studies or that this nematode is rare indeed. Finally, the geographical distribution of Su. halli is greatly expanded and now includes the Americas. Aspects related to the dispersion of these parasites, both in migrating and native hosts, are briefly discussed.

Identification of the avian tracheal trematode Typhlocoelum cucumerinum (Trematoda: Cyclocoelidae) in a host-parasite-environment system: diagnosis, life cycle and molecular phylogeny.

Typhlocoelum cucumerinum is a tracheal parasite of birds widely distributed across the globe. Nevertheless, aspects of the biology of this cyclocoelid are still poorly understood. Herein, we report the finding of T. cucumerinum in definitive and intermediate hosts from an urban waterbody of Brazil. The parasite was initially detected during the necropsy of domestic Muscovy ducks (Cairina moschata) found dead in the locality. Coproparasitological tests in live animals revealed that 12/47 (25.53%) Muscovy ducks and 2/8 (25%) mallards (Anas platyrhynchos platyrhynchos) were infected with T. cucumerinum. Moreover, rediae and metacercariae morphologically similar to T. cucumerinum were found in 3/248 (1.33%) Biomphalaria straminea collected in the same waterbody frequented by the birds. The conspecificity between the adult and the larval stages was confirmed molecularly (100% similarity in Cox-1). Moreover, the phylogenetic position of T. cucumerinum was determined for the first time based on partial fragments of the 28S, Cox-1 and Nad-1 genes. The species grouped with other members of the subfamily Typhlocoelinae with sequences available, but the data obtained do not support the distinctiveness of the genera Typhlocoelum and Tracheophilus. Further studies involving a broader range of species can result in taxonomic rearrangements in Typhlocoelinae.

Exploring Neotropical anuran parasites: a morphological, life cycle and phylogenetic study of Catadiscus marinholutzi (Trematoda: Diplodiscidae).

Amphistome species belonging to the genus Catadiscus are poorly studied intestinal trematodes found primarily in Neotropical anurans. Herein, developmental stages of an amphistome species found during herpetological and malacological surveys in a temporary marsh pond from Brazil were subjected to morphological (light and scanning electron microscopy) and molecular analyses. Adult parasites recovered from anurans were identified as Catadiscus marinholutzi. Amphistome cercariae found in the planorbid snails Drepanotrema depressissimum and Drepanotrema lucidum from the same waterbody were used for experimental and molecular studies. Immature parasites, morphologically compatible with members of Catadiscus, were experimentally obtained in laboratory-reared tadpoles. Sequencing of a partial region of 28S rDNA gene of both adult and cercariae revealed 100% similarity between these developmental stages, confirming their conspecificity. Phylogenetic analyses were attempted for the first time to reveal the position of a species of Catadiscus in the superfamily Paramphistomoidea. Catadiscus marinholutzi falls in a virtual polytomy together with other paramphistomoids, which leaves its phylogenetic relationships within the group unclear. Moreover, the high genetic divergence to Diplodiscus spp. (10.06­10.84%) cast doubts on the placement of Catadiscus within Diplodiscidae. Hence the species composition of the Diplodiscidae should be re-evaluated in further studies using a broader spectrum of related taxa.

Pathology of Free-Ranging and Captive Brazilian Anteaters.

There is little information on the anatomical pathology of Brazilian anteaters. Considering the relevance of knowledge of diseases of these species for their conservation, the aim of this study was to describe pathological changes in 99 captive and free-ranging anteaters from the Brazilian states of São Paulo, Minas Gerais, Bahia, Mato Grosso do Sul and Amazonas. Forty-two animals were killed on roads and 10 died from burns injuries. Other significant conditions included the metabolic diseases of iron storage disease, tissue mineralization and taurine deficiency, protozoan and metazoan infections, candidiasis, sporotrichosis, clostridiosis and proliferative disorders including squamous cell carcinoma.

Haplorchis pumilio (Trematoda: Heterophyidae) as a new fish-borne zoonotic agent transmitted by Melanoides tuberculata (Mollusca: Thiaridae) in Brazil: A morphological and molecular study.

The fishborne zoonotic trematode Haplorchis pumilio (Looss, 1896) is here reported for the first time in Brazil based on morphological, experimental and molecular studies. Pleurolophocercous cercariae emerged from the invasive snail Melanoides tuberculata collected in the municipality of Ceará-Mirim, state of Rio Grande do Norte, Northeast region of Brazil, in November 2018, were used for experimental infection of guppies, Poecilia reticulata. Metacercariae were extracted from the caudal musculature of these fishes and were used to infect mice. Adult parasites recovered in the small intestine of the mice were morphologically identified as H. pumilio. Molecular sequences were obtained for the Brazilian and Peruvian isolates of H. pumilio, and were compared with data available in GenBank. Analyses of fragments of the nuclear genes 28S (1219 bp) and ITS-2 (290 bp) revealed 98.48-100% similarity between the South American and Asian isolates of H. pumilio. Moreover, new sequences of the mitochondrial gene cox-1 obtained for the Brazilian (797 bp) and Peruvian (646 pb) isolates were 100% similar to a Mexican isolate of this species and 97.54% similar to an isolate from Thailand. This finding reveals the potential for occurrence of human haplorchiasis in Brazil, especially because of the increasing popularity of raw fish dishes in the country.