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.

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.

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.

Evaluation of Culex quinquefasciatus wings asymmetry after exposure of larvae to sublethal concentration of ivermectin.

The surviving insects submitted to chemical control have morphological alterations that impact on their mechanisms of resistance and their final development. Those changes are detected and measured using physical features related to symmetry, specifically named fluctuating asymmetry. This is detected when deviations from the perfect bilateral symmetry for specific morphological characteristic is influenced by genetics or environmental stress. Thus, in this paper we analyze the wing in adult of Culex quinquefasciatus (Diptera - Culicidae) after larvae exposure to ivermectin LC50. Three hundred larvae of C. quinquefasciatus were exposed to ivermectin in 1.5 µg/L (LC50) concentration during 30 min, and three hundred larvae were exposed to distilled pure water as control group. For fluctuating asymmetry, adult males and adult females were selected from each group (n = 83) from the untreated group and (n = 79) from treated group. Wings from adults of each group were mounted in glass microscope slides and coverslip in Canada's balsam and analyzed with a stereomicroscope with a video camera attached. The treatment effect on M3 + 4 was marginally significant with higher asymmetry values in the control group. The data obtained here suggest the importance of future experiments to elucidate the mechanisms associated with FA. Moreover, according to the results obtained, it may be suggested that FA is present in females in ornaments, or secondary sexual characters, as an indicator of phenotypic quality of the partners.

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.

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.

Platynosomum illiciens (Trematoda: Dicrocoeliidae) in Captive Black-Tufted Marmoset Callithrix penicillata (Primates: Cebidae) from Brazil: A Morphometric Analyses with Taxonomic Comments on Species of Platynosomum from Nonhuman Primates.

The trematodes belonging to the genus Platynosomum are biliary parasites of birds and mammals (domestic and wildlife) in tropical and subtropical areas of the globe. Despite several reports on platynosomosis in captive nonhuman primates, mainly in South America, the taxonomy of species of Platynosomum that infect these hosts remains confused, and it is not clear whether the species found in cats is the same that infects nonhuman primates. Because a detailed morphological study of Platynosomum from nonhuman primates is lacking, in this study we analyzed specimens of Platynosomum recovered from the biliary system of Callithrix penicillata kept in captivity in an animal facility. The helminths were submitted to morphological and morphometric analyses in a light microscope and measurements of 16 morphological traits were taken. A kernel density estimation (KDE) was used to estimate density distributions of the measurements obtained as well as the occurrence of overlap with the ranges of the measurements known to 2 other species of Platynosomum previously described from South American marmosets, Platynosomum amazonensis and Platynosomum marmoseti. A principal component analysis (PCA) was also performed in order to evaluate the position of each of the 3 species in the multivariate gradient of morphometric measurements. The occurrence of a growth gradient was also evaluated by analysis of correlation between the measurements. Besides a great morphological variability, all specimens obtained from marmosets in this study were identified as Platynosomum illiciens (Braun, 1901). In addition, the published ranges of the measurements of P. amazonensis and P. marmoseti were completely contained within the ranges found in this study as revealed by KDE. The PCA did not show the formation of groups, and the 3 species were distributed along a growth continuum, also corroborated by correlation analysis. Therefore, P. amazonensis and P. marmoseti are here synonymized with P. illiciens. The involvement of wildlife hosts to the epidemiology of feline platynosomosis and implications for its control are briefly discussed.

Exploring possibilities for an alternative approach in experimental schistosomiasis mansoni: the peritoneal cavity of mice.

The schistosome oviposition and granuloma constitution in the peritoneal cavity of AKR/J mice were evaluated. Groups of mice intraperitoneally infected with cercariae of Schistosoma mansoni were weekly euthanized during the acute (56 to 84 days post-infection (DPI)) and chronic (147 to 175 DPI) phase of infection. Schistosome developmental stages obtained via peritoneal lavage and perfusion of the portal system were inspected, counted and fixed, and peritoneal granulomata were then processed for histology. The morphological characterization and quantitative analysis of peritoneal schistosome eggs and granulomata were for the first time performed, such as the demonstration of the viability of miracidia obtained there from. Eutopic and ectopic mature schistosomes and normal pattern of worm oviposition were observed in all periods studied. However, the size of schistosome eggs from peritoneal cavity was smaller than observed for eggs laid by female worms from the portal system. The numbers of S. mansoni eggs and/or granulomata recovered from the peritoneal cavity was higher in chronic than acute infection, while the mean diameter of peritoneal chronic granulomata was smaller than for peritoneal acute granulomata. The constitution and evolution of these cellular reactions at histology were similar to that of hepatic granuloma, and peritoneal granulomata were subject to the host immunomodulation. In addition to the standardization of this experimental approach, which allows the obtaining of free schistosomal granulomata from peritoneal cavity of AKR/J mice, the potential use of these granulomata in ex vivo and in vivo studies is discussed.

Biomphalaria straminea (Mollusca: Planorbidae) as an intermediate host of Drepanocephalus spp. (Trematoda: Echinostomatidae) in Brazil: a morphological and molecular study.

Species of trematodes belonging to the genus Drepanocephalus are intestinal parasites of piscivorous birds, primarily cormorants (Phalachrocorax spp.), and are widely reported in the Americas. During a 4-year malacological study conducted on an urban lake in Brazil, 27-collar-spined echinostome cercariae were found in 1665/15,459 (10.7 %) specimens of Biomphalaria straminea collected. The cercariae were identified as Drepanocephalus spp. by sequencing the 18S (SSU) rDNA, ITS1/5.8S rDNA/ITS2 (ITS), 28S (LSU) rDNA region, cytochrome oxidase subunit 1 (CO1), and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1) markers. In experimental life cycle studies, metacercariae developed in laboratory-reared guppies (Poecilia reticulata); however, attempts to infect birds and rodents were unsuccessful. Two closely related morphotypes of cercariae were characterized. One species, identified by molecular markers as a genetic variant of Drepanocephalus auritus (99.9 % similarity at SSU, ITS, LSU; 97.2 % at CO1; 95.8 % at ND1), differs slightly from an archived North American isolate of this species also sequenced as part of this study. A second species, putatively identified as Drepanocephalus sp., has smaller cercariae and demonstrates significant differences from D. auritus at the CO1 (11.0 %) and ND1 (13.6 %) markers. Aspects related to the morphological taxonomic identification of 27-collar-spined echinostome metacercariae are briefly discussed. This is the first report of the involvement of molluscs of the genus Biomphalaria in the transmission of Drepanocephalus and the first report of D. auritus in South America.

Experimental platynosomosis: Characterization of parasite development in the mouse model.

Despite the veterinary importance of species of Platynosomum, biliary trematode parasites of birds and mammals with worldwide distribution and a growing role in feline practice, the basic parasitological aspects of platynosomosis is still not completely understood due to the scarcity of studies in experimental models. In the present study, metacercariae of Platynosomum illiciens obtained from naturally infected tropical house geckos (Hemidactylus mabouia) in an urban area of Brazil were force-fed to mice of the AKR/J strain (100 metacercariae/animal). Groups of mice were euthanized at 60, 120, 160 and 240 days post-infection (DPI), and the biliary tree of the animals (intrahepatic biliary ducts, common hepatic and bile ducts, cystic duct and gallbladder) were examined for the presence of adult parasites. Recovered flukes were counted, classified by their site of origin (i.e., intrahepatic or extrahepatic biliary ducts) and morphologically analyzed under light microscope. The number of adult parasites obtained at 60, 120, 160 and 240 DPI was 22 ± 6 (16-32), 41 ± 14 (18-48), 27 ± 11 (18-40) and 20 ± 6 (13-30), respectively, and no significant differences in total worm burden at the different experimental times were observed. However, 41%, 51%, 75% and 95% of the parasites were found in the common hepatic and bile ducts at 60, 120, 160 and 240 DPI, respectively, suggesting the occurrence of parasitic migration to the extrahepatic biliary tree during infection; however, no parasites were observed in the gallbladder or cystic duct. Regarding the morphometric analysis, progressive growth of P. illiciens during the experimental time was observed, and the parasites collected from the extrahepatic bile ducts were larger than those obtained from the intrahepatic ducts at the same time of infection. Parasites obtained from the extrahepatic biliary tree of the mice at 160 DPI had similar measurements to those of parasites obtained at 240 DPI, and those measurements were equivalent to those reported for parasites from natural hosts (cats, birds and nonhuman primates). The results obtained provide new insights into the biology of P. illiciens, and the kinetics of the parasite development of this species is presented here for the first time. The potential use of mice as an experimental model for P. illiciens is presented and the implications of the results obtained in that model for feline platynosomosis are briefly discussed.

The Apple Snail Pomacea maculata (Caenogastropoda: Ampullariidae) as the intermediate host of Stomylotrema gratiosus (Trematoda: Stomylotrematidae) in Brazil: the first report of a mollusc host of a Stomylotrematid Trematode.

Trematodes belonging to the family Stomylotrematidae are intestinal parasites of birds. Despite the worldwide distribution and diversity of host species, the first intermediate host remains unknown. For a survey of parasites of Pomacea maculata , snails were collected from the municipality of São Vicente Férrer, state of Maranhão, northeastern Brazil. In the present study, the xiphidiocercariae shed from these snails were used in the experimental infection of the water bug Belostoma plebejum. The insect mortality was observed 30 days post-infection, and the metacercariae recovered in the body cavity of B. plebejum were identified as Stomylotrema gratiosus. This is the first report of an ampullariid snail as intermediate host of stomylotrematid trematodes.

Metacercarial infection of wild Nile tilapia (Oreochromis niloticus) from Brazil.

Fingerlings of Oreochromis niloticus collected in an artificial urban lake from Belo Horizonte, Minas Gerais, Brazil, were evaluated for natural infection with trematodes. Morphological taxonomic identification of four fluke species was performed in O. niloticus examined, and the total prevalence of metacercariae was 60.7% (37/61). Centrocestus formosanus, a heterophyid found in the gills, was the species with the highest prevalence and mean intensity of infection (31.1% and 3.42 (1-42), resp.), followed by the diplostomid Austrodiplostomum compactum (29.5% and 1.27 (1-2)) recovered from the eyes. Metacercariae of Drepanocephalus sp. and Ribeiroia sp., both found in the oral cavity of the fish, were verified at low prevalences (8.2% and 1.6%, resp.) and intensities of infection (only one metacercaria of each of these species per fish). These species of trematodes are reported for the first time in O. niloticus from South America. The potential of occurrence of these parasites in tilapia farming and the control strategies are briefly discussed.

Physa marmorata (Mollusca: Physidae) as a natural intermediate host of Trichobilharzia (Trematoda: Schistosomatidae), a potential causative agent of avian cercarial dermatitis in Brazil.

Species of Trichobilharzia are the main etiological agents of cercarial dermatitis in humans, which is considered a re-emerging disease. Despite the diversity and global distribution of species of this genus, studies of Trichobilharzia are scarce in South America. The goal of our investigations is better understand the diversity, distribution and life cycle of avian schistosomes and their likely role in causing dermatitis in Brazil. As part of this effort, cercariae found in naturally infected Physa marmorata were identified by morphological and molecular (mitochondrial cox1, nuclear ITS1 and 28S gene regions) methods as Trichobilharzia sp. These cercariae are similar morphologically to T. jequitibaensis described previously from Brazil and similar genetically to the North American physid transmitted species T. querquedulae and T. physellae. This is the first report of a potential agent of cercarial dermatitis from naturally infected snails from Brazil and first molecular characterization of a South American species of Trichobilharzia. A discussion follows concerning the potential role of this species has in outbreaks of dermatitis in Brazil.