Using DNA barcoding to link cystacanths and adults of the acanthocephalan Corynosoma australe of the Southeastern Pacific Ocean (off Peru coast).

The objective of this study is to use DNA barcoding to link cystacanths and adults belonging to the acanthocephalans Corynosoma australe found in the Southeastern Pacific Ocean off the coast central from Peru. We sampled three species of commercial fish (Paralichthys adspersus (Steindachner), Paralabrax humeralis (Valenciennes), and Cheilodactylus variegatus (Valenciennes)) and two South American sea lions, Otaria byronia, stranded on the beaches of the city of Huacho and Barranca, Lima province. A total of 509 acanthocephalan larvae were found in the body cavity of 95 fish (prevalence 54.28%, total mean intensity 8.64). A total of 127 adult worms were found in the large intestine from two South American sea lions (P= 100%, MI= 63.5). A total of 203 larvae from P. humeralis were isolates (P=65.71%; MI= 8.83; MA=5.8), 235 (P=54.29%; MI= 12.37; MA= 6.71) from C. variegatus, and 71 (P=42.86%; MI= 4.73; MA= 2.03) from P. adspersus. All adult and larval specimens were morphologically identified as C. australe. They were generated cytochrome c oxidase subunit 1 (cox1) gene sequences of specimens and were compared with available data from GenBank. Molecular phylogenetic analysis supported our morphological identification, where the Peruvian isolates formed a clade with other isolates of C. australe from other countries of the American continent. Of the sequences obtained, two haplotypes were detected and were not identical with previous reports. Based on both DNA barcoding and morphological analyses, our finding represents the first molecular data of C. australe from Peru and the report of Cheilodactylus variegatus as a new paratenic host on the central coast, extending the knowledge and distribution range of this acanthocephalan in Southeastern Pacific Ocean.

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%.

Caracterización del patrón proteico y perfil antigénico de larvas L 3 de anisákidos de importancia médica aisladas de peces comerciales de la costa peruana / Characterization of the protein pattern and antigen profile of L 3 larvae of medical importance in anisakids isolated from commercial fish of the peruvian coast

RESUMEN Introducción. La anisakidosis es una zoonosis causada por la ingestión accidental de larvas (L3) de anisákidos. Objetivo. Caracterizar el patrón proteico y perfil antigénico de la L3 de Anisakis simplex s.l. (tipo I), A. physeteris s.l. (tipo II) y Contracaecum osculatum s.l. aisladas de peces comerciales. Métodos. Se realizó una corrida electroforética en SDS-PAGE de los antígenos somáticos. Se inmunizó conejos experimentalmente y se evaluó por EITB. Resultados. El patrón proteico de Anisakis tipo I mostró 12 bandas, 18 Anisakis tipo II y C. osculatum 13, con las bandas 10 y 35 kDa específicas para Anisakis tipo II, 28 y 77 para C. osculatum no presentes en Anisakis tipo I. Conclusión. Se determinó bandas inmunogénicas específicas para Anisakis tipo I con las proteínas de peso molecular 11, 14, 25 y 40 kDa, para el tipo II de 9, 10, 12, 35 y 41 kDa, y C. osculatum 13, 15, 17, 30 y 47 kDa.

ABSTRACT Introduction. Anisakidosis is a zoonosis caused by accidental ingestion of anisakid larvae (L3). Objective. To characterize the protein pattern and antigenic profile of the L3 of Anisakis simplex s.l. (type I), A. physeteris s.l. (type II) and Contracaecum osculatum s.l. commercial3 fish isolated. Methods. An SDS-PAGE electrophoretic run of the somatic antigens was performed. Rabbits were immunized experimentally and evaluated by EITB. Results. The protein pattern of Anisakis type I showed 12 bands, 18 Anisakis type II and C. osculatum 13, with bands 10 and 35 kDa specific for Anisakis type II, 28 and 77 for C. osculatum, not present in Anisakis type I. Conclusion. Specific immunogenic bands were determined for Anisakis type I with the molecular weight proteins 11, 14, 25 and 40 kDa, for type II of 9, 10, 12, 35 and 41 kDa and C. osculatum 13, 15, 17, 30 and 47 kDa.

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.

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.

DNA sequences confirm low specificity to definitive host and wide distribution of the cat pathogen Platynosomum illiciens (= P. fastosum) (Trematoda: Dicrocoeliidae).

Although feline platynosomosis has been commonly reported in several parts of the world, the taxonomy and epidemiological chain related to cat liver flukes remain controversial. In this study, nuclear ribosomal ITS, 28S, and mitochondrial cox1 sequences obtained for Platynosomum illiciens from cat, marmoset, lizard, and snail found naturally infected in Brazil reveal no significant molecular differences between these isolates. Moreover, sequence data confirm that Brazilian P. illiciens from different hosts is conspecific with parasites obtained from cats in Vietnam, supporting wide distribution of the species. The lack of pronounced specificity of P. illiciens to definitive hosts is confirmed here for the first time using molecular approach. The results are discussed in context of the epizootology of platynosomosis.

The life cycle of a zoonotic parasite reassessed: Experimental infection of Melanoides tuberculata (Mollusca: Thiaridae) with Centrocestus formosanus (Trematoda: Heterophyidae).

Centrocestus formosanus is a foodborne intestinal trematode that is native to Asia and has been introduced into the Americas and Europe. Although there are several studies of C. formosanus in definitive vertebrate hosts (birds and mammals, including humans), and in intermediate vertebrate hosts (fish and amphibians), there is little published information regarding interaction with its transmitting mollusc. In this study we studied the miracidial development of C. formosanus using a mouse as a source of eggs. Adult parasites were maintained in water in order to develop miracidia in intrauterine eggs. Miracidia appeared at 12 days of incubation, with no hatching observed for up to 40 days. Subsequently, we placed dead C. formosanus containing eggs with miracidia individually in contact with 48 specimens of Melanoides tuberculata, and observed the absence of the parasites after 1h of exposure, suggesting that they were ingested by the snails. Of the 33 experimentally-infected snails that were alive after 84-89 days post-infection (DPI), seven (21%) shed cercariae. We detected young C. formosanus rediae in 21/33 (64%) M. tuberculata at 90 DPI. To our knowledge, this report is the first to show that, in the life cycle of C. formosanus, infection of molluscs occurs passively by ingestion of eggs, followed by a long intramolluscan phase. We compare these data with those described for other Heterophyidae, and discuss on the phylogenetic background of the pattern of miracidial development verified in these parasites.

Fishborne Zoonotic Trematodes Transmitted by Melanoides tuberculata Snails, Peru.

We investigated the transmission of the fishborne trematodes Centrocestus formosanus and Haplorchis pumilio by Melanoides tuberculata snails in Peru. We report on results of experimental, morphological, and molecular approaches and discuss the potential risk for future human cases, given the existence of food habits in the country involving the ingestion of raw fish.

Putative new genera and species of avian schistosomes potentially involved in human cercarial dermatitis in the Americas, Europe and Africa.

New larval avian schistosomes found in planorbid snails from Brazil and USA were used for morphological and molecular studies. Eggs with a distinctive long polar filament were found in ducks infected experimentally with Brazilian cercariae. Similar eggs were reported previously in wild or experimentally infected anatids from Brazil, South Africa, and the Czech Republic. Molecular phylogenetic analyses showed that the North American and European schistosomes are sister taxa, which are both sister to the Brazilian species. However, these clades do not group with any named genus. Molecular data plus egg morphology suggest that these are new putative genera and species of avian schistosomes that can cause human cercarial dermatitis in the Americas, Africa and Europe.