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.

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.

A phylogenetic study of the cecal amphistome Zygocotyle lunata (Trematoda: Zygocotylidae), with notes on the molecular systematics of Paramphistomoidea.

Zygocotyle lunata inhabits the caecum of birds and mammals from the American continent. This amphistome parasite is easily maintained in the laboratory and serves as a model organism in life-cycle studies, but it has seldom been studied using molecular data. Neither the position of Z. lunata in the superfamily Paramphistomoidea nor the monophyly of the Zygocotylidae has been evaluated with molecular phylogenetic methods. In the present study, adult specimens of Z. lunata obtained experimentally in mice from Brazil were submitted to molecular studies. Partial sequences of nuclear (1261 bp of 28S and 418 bp of 5.8S-ITS-2) and mitochondrial (1410 bp of cytochrome c oxidase 1, cox1) markers were compared with published data. In the most well-resolved phylogeny, based on 28S sequences, Z. lunata clustered in a well-supported clade with Wardius zibethicus, the only other species currently included in the Zygocotylidae, thus confirming the validity of this family. Divergence of 28S sequences between these species was 2.2%, which falls in the range of intergeneric variation (0.9-5.6%) observed in the other two monophyletic groups in the 28S tree, i.e., representatives of Gastrodicidae and Neotropical cladorchiids (Cladorchiidae). Analysis of ITS-2 and two parts of the cox1 gene placed Z. lunata within poorly resolved clades or large polytomies composed of several paramphistomoid families, without clarifying higher-level phylogenetic relationships. The cox1 of a Brazilian isolate of Z. lunata is 99.6% similar to a Canadian isolate, confirming the pan-American distribution of the species. Finally, our phylogenetic reconstructions of Paramphistomoidea revealed a complex scenario in the taxonomic composition of some amphistome families, which highlights a need for further integrative studies that will likely result in rearrangements of traditional morphology-based classifications.

A morphological, molecular and life cycle study of the capybara parasite Hippocrepis hippocrepis (Trematoda: Notocotylidae).

Hippocrepis hippocrepis is a notocotylid that has been widely reported in capybaras; however, the molluscs that act as intermediate hosts of this parasite remain unknown. Furthermore, there are currently no molecular data available for H. hippocrepis regarding its phylogenetic relationship with other members of the family Notocotylidae. In the present study, we collected monostome cercariae and adult parasites from the planorbid Biomphalaria straminea and in the large intestine of capybaras, respectively, from Belo Horizonte, Minas Gerais, Brazil. We subjected them to morphological and molecular (amplification and sequencing of partial regions of 28S and cox-1 genes) studies. Adult parasites collected from the capybaras were identified as H. hippocrepis and the sequences obtained for both molecular markers showed 100% similarity with monostome cercariae found in B. straminea. The sequences obtained for H. hippocrepis were compared with data available in public databases; analysis revealed this species differs from other notocotylids with available sequences (1.5-3.8% with respect to 28S and 11.4%-13.8% with respect to cox-1). On the phylogenetic analyses, H. hippocrepis appeared to be a distinct lineage in relation to other notocotylids. Some ecological aspects related to the infection of capybaras with H. hippocrepis are briefly discussed.

Zygocotyle lunata as a model for in vivo screening of anthelmintic activity against paramphistomes: Evaluation of efficacy of praziquantel, albendazole and closantel in experimentally infected mice.

Paramphistomes are important parasites in veterinary medicine. There are few anthelmintic drugs available against them. The development of new drugs is urgently needed and this process can be accelerated through the development of rodent models for in vivo testing. Among the few paramphistomes that develop in rodents is the caecal fluke Zygocotyle lunata, a species with which several biological studies have been performed over several decades. Nevertheless, its use as a model for evaluation of anthelmintic drugs had not yet been evaluated. In the present study, we evaluated the efficacy of praziquantel (PZQ 300 mg/kg 5x), albendazole (ABZ 200 mg/kg 5x) and closantel (CLO 50 mg/kg single dose, 50 mg/kg 3x and 25 mg/kg 3x) for treatment of mice experimentally infected with Z. lunata. The animals were infected with 20 metacercariae of the parasite and were treated 30 days post-infection. Untreated groups were maintained as controls. Seven days after the treatments, the animals were euthanized for recovery and counting of parasites. We found that PZQ and ABZ, at the dosages and therapeutic schedule employed here, did not cause significant alterations in worm burden [worm counts 16.0 ±â€¯2.8 (13-19), 17.6 ±â€¯2.1 (14-19) and 16.2 ±â€¯1.9 (13-18) (p = 0.51) in PZQ, ALB and control, respectively]. CLO 50 mg/kg in a single dose caused significant reduction in the number of parasites [treated: 1.8 ±â€¯0.9 (1-3); control: 15.6 ±â€¯2.5 (12-19)], although it did not result in complete elimination of the parasites in any animal. Despite the fact that three doses of CLO 50 mg/kg or CLO 25 mg/kg caused complete elimination of the parasites in most surviving animals, there was significant host mortality. In general, results here obtained are concordant with those of studies performed on ruminant paramphistomes. Given that Z. lunata can be maintained in laboratory rodents, it is a suitable model for screening anthelmintic drugs against paramphistomes.

Experimental avian philophthalmosis: Evaluation of diagnosis and treatment of chickens infected with Philophthalmus gralli (Trematoda: Philophthalmidae).

The trematodes of the genus Philophthalmus are eye flukes that cause damage to ocular structures of animals and humans. Despite the increasing number of cases reported in birds, studies related to the diagnosis of subclinical philophthalmosis are lacking, and there are no effective therapeutic regimens available. In the present study, we evaluated the diagnosis and treatment of philophthalmosis in specific pathogen-free chickens (Gallus gallus domesticus) experimentally infected with Philophthalmus gralli. Four chickens were inoculated with metacercariae of P. gralli (20 per eye) obtained from cercariae emerged from naturally infected Melanoides tuberculata. From 90 days post-infection, the chickens were subjected to direct ophthalmic examination (DOE) and conjunctival sac lavage (CSL). The latter technique consisted of lavage of each eye with 200 µL sterile saline solution and subsequent microscopical examination of the collected fluid for the presence of eggs of P. gralli. The anthelminthic treatment protocols included praziquantel (PZQ) at 10, 50, or 100 mg/kg (single dose given intramuscularly), or fenbendazole (FBZ) at 50 mg/kg (three doses at 24 h-intervals given per os). The treatment protocols were performed at 14 day-intervals between each dosage of PZQ. Chickens developed a minimum of one to more than five adult P. gralli per eye, except for one chicken that had a single eye with one parasite. No ocular clinical signs or changes in behavior were noted in any chickens. DOE and CSL were considered techniques with similar sensitivity for the diagnosis of avian philophthalmosis. The data suggested that PZQ and FBZ, at the dosages and schedules employed, are not effective for the complete elimination of P. gralli. CSL is proposed as a complementary technique for the diagnosis and monitoring of philophthalmosis post-treatment, especially in subclinical cases. The evaluation of new protocols, routes of administration, and anthelmintic drugs are needed for successful pharmacological treatment of philophthalmosis.