Morphological and Molecular Characterization of Larval and Adult Stages of Eustrongylides excisus (Nematoda: Dioctophymatoidea) with Histopathological Observations.

The genus Eustrongylides includes nematodes known as the etiological agent of the "big red worm disease." The aim of this work was to identify Eustrongylides spp. larvae from fish and adults from great cormorant (Phalacrocorax carbo) sampled at Lake Trasimeno, Italy, by morphological and molecular analysis. Histopathological description of the lesions in birds was also provided. We described adults of Eustrongylides excisus for the first time in Italy, and we also linked larval stages 3 and 4 to adults. The use of molecular tools combined with the traditional taxonomy will help the identification of the species, including species inquirendae. Moreover, molecular analysis can also help to investigate the role of intermediate and paratenic hosts, to deepen the knowledge about geographical distribution of the different Eustrongylides spp. and to define the zoonotic potential of E. excisus, which has not yet been identified as causal agent of human cases.

The role of freshwater fish in the life cycle of Dioctophyme renale in Southern Brazil.

Brazil stands out by the diverse records of Dioctopphyme renale in different hosts; however, there is little information about the life cycle of the nematode in the region. This study aims to report on third-stage larvae infections in fish in southern Brazil. In this context, 324 fish of different species belonging to Characiformes, Cyprinodontiformes and Siluriformes were collected in an urban area of Rio Grande do Sul State, where domestic and wild hosts were reported with the nematode. Of the total fish examined, 25(7.7%) were found to be parasitized by third-stage larvae of D. renale which were found encysted in the stomach serous membrane and in the celoma cavity of Hoplosternum littorale (Siluriformes) with a prevalence of 53.2% (25/47) and mean intensity of infection of 4.4 larvae/host (1 to 13 larvae). The occurrence of larvae in H. littorale indicates the presence of parasitosis in the region; however, the contribution of this fish species as a source of infection for dogs in urban areas must be considered with caution given the difficulties these dogs may face in the capture and predation of the fish to the point of effectively maintaining the urban cycle of D. renale. In addition, the low level of larvae registered in the total sample of fish examined indicates that these hosts are unlikely to play an important role in the transmission of D. renale to domestic animals in the region of the study.

Third-stage larvae of the enoplid nematode Dioctophyme renale (Goeze, 1782) in the freshwater turtle Trachemys dorbigni from southern Brazil.

The giant kidney worm Dioctophyme renale is normally found in wild carnivores and domestic dogs, with aquatic oligochaetes acting as intermediate hosts. In the present study a prevalence of 50% of third-stage larvae of D. renale was recorded in 60 specimens of the freshwater turtle Trachemys dorbigni from southern Brazil. Larvae were encysted in muscles, the coelomic cavity and mesentery, the serous lining of the stomach and on the surfaces of the lung, heart, liver, pancreas, spleen and intestines. There are no previous records of reptiles being part of the life cycle of D. renale, although fish and amphibians normally act as paratenic hosts. This is the first report of third-stage D. renale larvae in the freshwater turtle, T. dorbigni.

Development of Eustrongylides ignotus (Nematoda: Dioctophmida) in domestic ducks (Anas platyrhynchos domestica (L.)).

Investigating the development of Eustrongylides ignotus in its definitive host would enable us to trace the complete life cycle of this nematode. Fourth-stage larvae isolated from naturally infected swamp eels (Monopterus albus) were used to infect domestic ducks (Anas platyrhynchos domestica [L.]). We observed that male and female worms exhibited different developmental patterns in host ducks. In males, the fourth molt occurred at day 1-2 post-infection (PI), after which they attained maturity on day 4 PI and died between day 7 and 9 PI. However, females underwent the fourth molt at day 2-4 PI, produced eggs from day 9 to 17 PI, and then degenerated and died. When compared to fourth-stage female larvae, adult females demonstrated a considerable increase in total body size with a 151% increase in average body width and a 17% increase in average body length. However, the increase in size of the male larvae was not as significant as that in females. The average body width in adult males exhibited only a 45% increase over that in the larval stage.

Severe granulomatous lesions in several organs from Eustrongylides larvae in a free-ranging dice snake, Natrix tessellata.

During an extensive study regarding the epidemiology of larval Eustrongylides infestation in a free-ranging endangered population of dice snakes (Natrix tessellata) from Histria, Romania, an adult female was euthanized to evaluate pathologic changes. Parasites appeared as nodules at various locations: in subcutaneous connective tissues, on the serosae of the intestines and liver. Histologic sections revealed nematode larvae surrounded by a capsule, forming a parasitic granuloma with 3 layers: macrophage layer, lymphocyte layer, and fibrous capsule. Differences between newly formed and mature granulomas consisted mainly in the eosinophilic infiltration. Other types of parasitic granulomas of reptiles are discussed in comparison with our findings.

Geographic and host range of the nematode Soboliphyme baturini across Beringia.

The nematode Soboliphyme baturini Petrov, 1930, was found to represent a single species with a relatively broad geographic range across Beringia and northwestern North America on the basis of the assessment of molecular sequence data for adult and juvenile parasites. Refuted are hypotheses suggesting that several cryptic species could be partitioned either among an array of mustelid definitive hosts or across the vast region that links North America and Eurasia. Host specificity for this species is examined on the basis of a comprehensive list for definitive hosts, derived from new field surveys and existing literature for S. baturini. Only 5 mustelids (Gulo gulo, Martes americana, M. caurina, M. zibellina, and Neovison vison) appear to have significant roles in the life history, persistence, and transmission of this nematode. Soboliphyme baturini readily switches among M. americana, M. caurina, Mustela erminea, or N. vison at any particular locality throughout its geographic range in North America, although Martes spp. could represent the source for nematodes in a broader array of mustelids. Molecular analyses (243 base pairs of mitochondrial gene nicotinamide dehydrogenase [ND4]) suggest that hypotheses for host specificity across an array of mustelid definitive hosts are not supported. The life cycle of S. baturini is explored through a review of diet literature for 2 marten species, M. americana and M. caurina, and other mustelids across the Holarctic. Shrews (Soricomorpha: Soricidae) comprise >8% of prey for these species of Martes, suggesting their putative role as paratenic hosts. Juvenile nematodes found in the diaphragms of soricids are genetically identical to adult S. baturini found in the stomachs of mustelids at the same locations in both Asia and North America, corroborating a role in transmission for species of Sorex.

Epizootiology of Eustrongylides ignotus in Florida: transmission and development of larvae in intermediate hosts.

Under laboratory conditions, 2 modes of transmission of Eustrongylides ignotus (Nematoda: Dioctophymatoidea) to fish were identified. Eastern mosquitofish (Gambusia holbrooki) became infected after ingestion of either eggs of E. ignotus containing first-stage larvae or aquatic oligochaetes (Limnodrilus hoffmeisteri) containing third-stage larvae of E. ignotus. After removal from the uterus of gravid E. ignotus females and incubation for 17-28 days, depending on temperature, it was found that parasite eggs contained first-stage larvae that were infective to fish and oligochaetes. Larvae developed to the third stage in oligochaetes and were infective to fish 35-77 days postinfection (PI) and when fed to fish, developed to the fourth stage between 127 and 184 days PI. Eggs containing first-stage larvae fed directly to fish developed to the fourth stage between 84 and 105 days PI. The amount of time for development from the undifferentiated egg to the fourth-stage larva was 78-156 days shorter when fish ingested eggs containing first-stage larvae than when fish ingested oligochaetes containing third-stage larvae. Three species of large piscivorous fish, including black crappie (Pomoxis nigromaculatus), largemouth bass (Micropterus salmoides), and warmouth (Lepomis gulosus), were fed mosquitofish containing fourth-stage larvae. At necropsy, live E. ignotus larvae were recovered from all 3 species. Several fish had multiple infections after ingesting > 1 larva, indicating that bioaccumulation of the parasite in the food chain may occur.

Altered predation susceptibility of mosquitofish infected with Eustrongylides ignotus.

Eustrongylides ignotus is a parasitic nematode whose definitive hosts are often piscivorous wading birds (Ciconiiformes). Several species of small fishes are intermediate hosts, while larger predatory fish may be paratenic (transport) hosts. We examined predation susceptibility of infected mosquitofish (Gambusia holbrooki) to three species of predatory fishes, including juvenile largemouth bass (Micropterus salminoides), warmouth (Lepomis gulosus), and bluegill (Lepomis macrochirus). A 250 L aquarium with removable plexiglass divider and remote observation windows was constructed. Aquatic macrophytes were placed in the tank to provide refuge for the fishes. Predatory fish were allowed to acclimate to one half of the tank, while one infected and one uninfected mosquitofish were placed in the other. The divider was removed and an observer recorded the number of capture attempts and time required for capture. Predators were observed for behavioral alterations for 4 days after ingestion of infected mosquitofish, then examined at necropsy. Infected prey were selected preferentially in 31 of 38 (82%) trials. The number of capture attempts was 2.7+/-0.2 (x +/- SE) for infected fish and 3.9+/-0.4 for uninfected fish. Mean time of capture was 12.4+/-1.6 min for infected fish and 21.7+/-2.9 for uninfected fish. Because of these differences, infected mosquitofish were more susceptible to predation (P < 0.01) than uninfected fish. Aberrant behavior including lethargy, convulsions, and buoyancy abnormalities was observed in eight (67%) predatory fish. At necropsy, larvae of E. ignotus were found in the coelomic cavity, viscera, and swim bladders of predators. Parasite-induced behavior modification of intermediate hosts may predispose them to predation by wading birds and thereby facilitate the transmission of this nematode in natural populations.