Integrative species delimitation and community structure of nematodes in three species of Australian flathead fishes (Scorpaeniformes: Platycephalidae).

This study aimed to determine the integrative characterisation of nematodes from three species of edible flathead fishes (Scorpaeniformes: Platycephalidae) in New South Wales, Australia, and describe nematode communities within three species of flatheads. Tiger (Platycephalus richardsoni (Castelnau); n = 20) and sand flatheads (Platycephalus bassensis (Cuvier); n = 20), sourced from the Nelson Bay area, and dusky flathead (Platycephalus fuscus (Cuvier); n = 20) from the Manning River, Taree, were examined for the presence of nematodes. The nematodes were initially classified morphologically as 12 different morphotypes belonging to the families Anisakidae (Anisakis types I, II, and III, Contracaecum type II, Terranova types I and II), Raphidascarididae (Hysterothylacium types IV, VI, VIII, and H. zhoushanense larva), and Gnathostomatidae (Echinocephalus sp. larva), Capillariidae (Capillaria sp.), followed by genetic identification through sequencing of the internal transcribed spacer (ITS-1, 5.8S, ITS-2) regions. Phylogenetic analyses revealed the evolutionary relationship between the identified larval specimens in the present study with available GenBank larval and adult nematodes. Sand flathead was 90% infected with nematodes followed by tiger flathead at 85% and dusky flathead at 15%. Nematodes infecting estuarine dusky and oceanic sand and tiger flatheads contrasted markedly. The analysis of similarities (ANOSIM) showed significant differences (p < 0.001) in the composition of taxa within nematode communities between the three species of flatheads (global R = 0.208) with the highest difference being between sand and dusky flatheads (R = 0.308, p < 0.001). The findings of the present study provide a foundation for future investigations of the community composition, life cycles, and distribution of nematode populations in edible fish in Australia and explore and clarify their significance to public health.

New insights into the coexistence of Contracaecum rudolphii A and Contracaecum rudolphii B (Nematoda: Anisakidae) in Phalacrocorax carbo sinensis from Sardinia: genetic variability and phylogenetic analysis.

Contracaecum sp. nematodes are important parasites of fish eating birds that can cause animal health problems. In the present study, specimens of Contracaecum rudolphii sensu lato, from the great cormorant Phalacrocorax carbo sinensis from Sardinia, were characterized based on morphological and molecular data. The morphological analysis allowed to identify all the fourth stage larvae (n = 1918) as Contracaecum sp., and adults, male (n = 5845) and female (n = 8312), as C. rudolphii sensu lato. Population genetics and phylogenetic relationships were inferred based on mitochondrial and nuclear markers. Multiple sequence alignment of the ribosomal internal transcribed spacer showed the coexistence of C. rudolphii A (n = 157), C. rudolphii B (n = 22) and a rare heterozygote of these species. Moreover, mitochondrial markers, namely NADH dehydrogenase subunits I (nad1), cytochrome c oxidase subunit (cox1 and cox2) and small subunit of rRNA (rrnS), showed that the studied C. rudolphii A populations had undergone bottleneck, or founder effect event, subsequent to a rapid population growth and expansion. The observed heterozygote is with a mitochondrial pattern of C. rudolphii B. Although, both Contracaecum species showed high genetic diversity, no genetic structure between localities was detected. Phylogenetic reconstructions supported the paraphyly of the avian Contracaecum species including C. ogmorhini (parasite of otariids).

Morphological and molecular identification of Hysterothylacium larvae (Nematoda: Raphidascarididae) in marine fish from Tunisian Mediterranean coasts.

The taxonomy of Hysterothylacium genus in Mediterranean waters remains incomplete and unresolved. The aim of the current study was to investigate the morphological and molecular identification of selected species of Hysterothylacium larvae in marine fish from the Tunisian Mediterranean coasts. A total of 192 marine fish samples were examined. In total, thirty-seven third-stage larvae of Hysterothylacium were morphologically identified as Hysterothylacium type V. In the present study, representatives of this type from the Mediterranean Sea were genetically characterized for the first time by sequencing the rDNA ITS (ITS1-5.8S-ITS2) regions and mtDNA cox2 gene. This study represents the first report of Hysterothylacium type V from the Mediterranean Sea. We also report Mullus barbatus, M. surmuletus, and Pagellus erythrinus as new hosts for this larval type. Based upon molecular and phylogenetic analyses considering the rDNA ITS regions, the Hysterothylacium type V described here was classified as a new genotype, named Genotype B. The valid genetic data of the described Hysterothylacium type V in the present study can be used to establish the phylogenetic relationships among Hysterothylacium species from the Mediterranean Sea and worldwide for future research.

Differential survival of 3rd stage larvae of Contracaecum rudolphii type B infecting common bream (Abramis brama) and common carp (Cyprinus carpio).

The main fish host reaction to an infection with third stage anisakid nematode larvae is a response in which host immune cells (macrophages, granulocytes, lymphocytes) in affected internal organs initially are attracted to the parasite whereafter fibroblasts may enclose the parasite forming granuloma. Generally, the reaction is non-lethal to the parasite which may survive for years in the fish host retaining infectivity to the final host. This may also apply for the anisakid nematode Contracaecum rudolphii (having the adult stage in cormorants, using copepods as first intermediate/paratenic host and zooplankton feeding fish as paratenic hosts). The present study has shown that most Contracaecum rudolphii larvae survive in bream (Abramis brama) (from Lake Balaton, Hungary) whereas the majority of the nematode larvae die in Cyprinus carpio (from Lake Hévíz, directly connected to Lake Balaton). Both cyprinid host species interacted with the nematode larvae through establishing a marked cellular encapsulation around them but with different effects. The differential survival in common carp and bream may theoretically be explained by ecological factors, such as the environmental temperature which either directly or indirectly affect the development of nematode larvae, and/or intrinsic host factors, such as differential immune responses and host genetics.

A new species of Raphidascaris (Nematoda: Raphidascarididae) infecting the fish Gymnogeophagus balzanii (Cichlidae) from the Pantanal wetlands, Brazil and a taxonomic update of the subgenera of Raphidascaris based on molecular phylogeny and morphology.

Raphidascaris (Sprentascaris) andersoni n. sp. (Nematoda: Raphidascarididae) collected in the intestine of the humphead cichlid Gymnogeophagus balzanii (Perugia) from the Pantanal wetlands, State of Mato Grosso do Sul (Brazil) is described and genetically characterized. The new species differs from its congeners mainly by having a conspicuous papilla-like formation slightly anterior to the cloacal aperture. Furthermore, males of R. (S.) lanfrediae and R. (S.) mahnerti have caudal alae, and R. (S.) hypostomi and R. (S.) pimelodi lack lateral alae, whereas in the new species caudal alae are absent and lateral alae present. The remaining congeners, namely, R. (S.) marano and R. (S.) saltaensis differ from Raphidascaris (Sprentascaris) andersoni n. sp. mainly because males have three pairs of postcloacal papillae (vs five pairs). In the phylogenetic reconstructions, using three nuclear genetic markers (18S, ITS1-5.8S-ITS2 and 28S rDNA) and one mitochondrial (cox1 mtDNA), the new species was separated from other representatives of Raphidascarididae, and the absence of monophyly in Hysterothylacium and Raphidascaroides was confirmed. Moreover, the subgenera Sprentascaris and Ichthyascaris appeared to be monophyletic. Therefore, even though Raphidascaris (Raphidascaris) was apparently not monophyletic, the subgenera of Raphidascaris should be re-erected as valid genera. The updated diagnoses of Ichthyascaris, Raphidascaris and Sprentascaris are given. The present study represents the first parasitological survey in G. balzanii.

Early development and life cycle of Contracaecum multipapillatum s.l. from a brown pelican Pelecanus occidentalis in the Gulf of California, Mexico.

The initial developmental stages of Contracaecum multipapillatum (von Drasche, 1882) Lucker, 1941 sensu lato were studied using eggs obtained from the uteri of female nematodes (genetically identified) found in a brown pelican Pelecanus occidentalis from Bahía de La Paz (Gulf of California, Mexico). Optical microscopy revealed a smooth or slightly rough surface to the eggs. Egg dimensions were approximately 53 × 43 µm, although after the larvae had developed inside, egg size increased to 66 × 55 µm. Hatching and survival of the larvae were greater at 15°C than at 24°C, and increased salinity resulted in a slight increase in hatching but seemed to reduce survival at 24°C, but not at 15°C. The recently hatched larvae measured 261 × 16 µm within their sheath. When placed in culture medium, the larvae grew within their sheath, and a small percentage (~2%) exsheathed completely (314 × 19 µm). The larvae continued to grow and develop once they had exsheathed, attaining mean dimensions of 333 × 22 µm. Although they did not moult during culture, optical microscopy revealed a morphology typical of third-stage larvae. Finally, the genetic identity between the larval parasites collected from mullet Mugil curema and adult female parasites collected from the brown pelican suggests a life cycle of C. multipapillatum in which the mullet are involved as intermediate/paratenic hosts and the brown pelicans as final hosts in the geographical area of Bahía de La Paz.

Anguilla anguilla intestinal immune response to natural infection with Contracaecum rudolphii A larvae.

The European eel, Anguilla anguilla, is a major warm-water fish species cultured in North and South Europe. Seventy-one A. anguilla collected between 2010 and 2015 from the Comacchio lagoons were examined. Fish were infected and damaged by larvae (L3) of the nematode Contracaecum rudolphii A, which were encapsulated within the thickness of the intestinal wall and within the external visceral peritoneum (serosa). Conspicuous granulomas, visible at sites of infection, were arranged in a trilayer, formed by a series of concentric whorls. The cells involved in the immune response and their distribution in the granuloma layers were assessed by immunohistochemical, immunofluorescence, and ultrastructural techniques. The outer part of the granuloma contained macrophages, macrophage aggregates, and mast cells (MCs) scattered among fibroblasts. This layer was vascularized, with degranulation of MCs occurring in close proximity to the capillaries. The middle layer was rich in MCs and fibroblasts. The inner layer, closest to the parasite larva, consisted mainly of dark epithelioid cells, some of which were necrotic. Non-necrotic epithelioid cells formed desmosomes between themselves or with fibroblasts. Within the granulomas, numerous cells of different types were positive to proliferative cell nuclear antigen antibody, indicating a high degree of cellular proliferation around the larvae.

Analysis of the effect of soil saprophytic fungi on the eggs of Baylisascaris procyonis.

Baylisascaris procyonis is a soil-transmitted helminth mainly found in raccoons (Procyon lotor) which can also affect other domestic and sylvatic animals, as well as humans, when the eggs released in the feces of parasitized raccoons are accidentally ingested. Three assays have been conducted to assess the effect of three saprophytic fungi, Mucor circinelloides, Paecilomyces lilacinus, and Verticillium sp., on the eggs of B. procyonis. Firstly, their ovicidal effect was in vitro ascertained by placing 1 mL with 2 × 10(6) spores of each fungus in Petri plates with water-agar (2 %) and simultaneously adding 200 eggs of Baylisascaris/plate. Two in vivo probes were carried out, by spraying the fungal spores (3 mL containing about 2 × 10(6) spores/mL) on the feces of raccoons and coatis (Nasua narica) passing eggs of B. procyonis in a zoological park; the other assay consisted of evaluating the activity of the fungi after adding sand to fecal samples from raccoons. An ovicidal type 3 activity characterized by morphological damage of the eggshell with hyphal penetration, internal egg colonization, and embryo alteration was observed for all the tested fungi. In the plate assays, viability of Baylisascaris eggs reduced significantly by 53-69 % with Mucor, 45-62 % with Paecilomyces, and 52-67 % with Verticillium. A similar ovicidal effect was detected in the feces with sand. These results demonstrate the usefulness of spraying spores of M. circinelloides, Pa. lilacinus, or Verticillium sp. on the feces of animals infected by Baylisascaris to decrease the numbers of viable eggs and, thus, the risk of infection.

Reducing Baylisascaris procyonis roundworm larvae in raccoon latrines.

Baylisascaris procyonis roundworms, a parasite of raccoons, can infect humans, sometimes fatally. Parasite eggs can remain viable in raccoon latrines for years. To develop a management technique for parasite eggs, we tested anthelmintic baiting. The prevalence of eggs decreased at latrines, and larval infections decreased among intermediate hosts, indicating that baiting is effective.

Hysterothylacium larvae (Nematoda, Anisakidae) in the freshwater mussel Diplodon suavidicus (Lea, 1856) (Mollusca, Unioniformes, Hyriidae) in Aripuanã River, Amazon, Brazil.

Larvae of Hysterothylacium use various invertebrates as intermediate hosts. Definite hosts include fish, birds, reptiles or marine mammals. This study describes the occurrence of Hysterothylacium (Nematoda, Anisakidae) larvae parasitizing the pericardic cavity of Diplodon suavidicus (Unioniformes, Hyriidae) specimens collected in the Amazon basin, Brazil. This is the first record of this nematode parasitizing freshwater bivalves in South America. The high prevalence, medium intensity and medium abundance suggest that D. suavidicus acts as intermediate host for Hysterothylacium species in that environment.

Direct competitive enzyme-linked immunosorbent assay (ELISA) for rapid screening of anisakid larvae in seafood.

BACKGROUND: Anisakid larvae are one of the most important pathogenic parasites in marine products; however, simple and rapid analytical techniques for them are still very limited. In this research, based on specific rabbit polyclonal antibodies which were raised against crude extracts of Anisakis larvae, purified by protein A affinity chromatography and labeled with horseradish peroxidase, a direct competitive enzyme-linked immunosorbent assay (ELISA) was developed and validated for detection of anisakid larvae in seafood. RESULTS: The established method exhibited a broad selectivity to Anisakis larvae and Pseudoterranova larvae, and the lowest detection limit to them was estimated to be about 5 parasites kg(-1) in food matrix. Using Pseudopleuronectes yokohamae, Scomberomorus niphonius and Ommastrephes bartrami as samples and within spiking concentrations from 20 to 100 larvae kg(-1), the determination recovery for Anisakis larvae and Pseudoterranova larvae ranged from 77.8% to 107.0%, with relative standard deviations all less than 20%. CONCLUSION: The results allowed us to suggest the established direct competitive ELISA as an effective analytical tool for fast screening of anisakid larvae in sea foods.

Cold tolerance in sealworm ( Pseudoterranova decipiens) due to heat-shock adaptations.

Third-stage larvae of Pseudoterranova decipiens commonly infect whitefish such as cod, and the parasite can be transferred to humans through lightly prepared (sushi) meals. Because little is known about the nematode's cold tolerance capacity, we examined the nematode's ability to supercool, and whether or not cold acclimation could induce physiological changes that might increase its ability to tolerate freezing conditions. Even if third-stage Pseudoterranova decipiens larvae have some supercooling ability, they show no potential for freezing avoidance because they are not able to withstand inoculative freezing. Still, they have the ability to survive freezing at high subzero temperatures, something which suggests that these nematodes have a moderate freeze tolerance. We also show that acclimation to high temperatures triggers trehalose accumulation to an even greater extent than cold acclimation. Trehalose is a potential cryoprotectant which has been shown to play a vital role in the freeze tolerance of nematodes. We suggest that the trehalose accumulation observed for the cold acclimation is a general response to thermal stress, and that the nematode's moderate freeze tolerance may be acquired through adaptation to heat rather than coldness.

Lagochilascaris minor: experimental infection of C57BL/6 and BALB/c isogenic mice reveals the presence of adult worms.

The nematode Lagochilascaris minor is the causative agent of lagochilascariosis, a human disease that affects the neck region causing exudative abscesses with eggs, larvae, and adult parasites. Mice are currently considered intermediate hosts for the parasite. To determine the pattern of infection and the possibility of mice as definitive hosts for L. minor, experimental lagochilascariosis was studied in two distinct isogenic mouse strains: BALB/c and C57BL/6. Our results indicate that BALB/c mice are more resistant to L. minor infection than C57BL/6, having less intense lesions in the lungs, a lower number of nodules with encysted larvae and fewer adult worms, and displaying a higher serum level of IFN gamma. Both mouse strains had low levels of serum IL-10. We also observed adult parasites in both mouse strains, raising the possibility that mice are definitive hosts of L. minor. This is the first description of adult parasite development of L. minor in mice.

Hysterothylacium fabri (Nematoda: Raphidascarididae) in Mullus surmuletus (Perciformes: Mullidae) and Uranoscopus scaber (Perciformes: Uranoscopidae) from the Mediterranean.

Raphidascarididae are among the most abundant and widespread parasitic nematodes in the marine environment. The life-cycle of most raphidascaridid species is poorly known and information about their distribution and host range is lacking in many geographical areas, as is the taxonomy of several species. A study of larval and adult stages of Hysterothylacium fabri (Rudolphi, 1819) Deardorff and Overstreet, 1980 (Nematoda: Raphidascarididae) infecting the striped goatfish Mullus surmuletus Linnaeus, 1758 (Mullidae) and the Mediterranean stargazer Uranoscopus scaber Linnaeus, 1759 (Uranoscopidae) from the Ionian Sea (central Mediterranean) has been carried out by combining light and scanning electron microscopy observations and molecular analyses through polymerase chain reaction-restriction fragment length polymorphism and sequencing of the ITS rDNA gene. Results indicate that U. scaber and M. surmuletus represent suitable definitive and intermediate/paratenic hosts of H. fabri, respectively, in the Mediterranean and highlight the importance of combining genetic and morphological data to study the taxonomy and epidemiology of parasites widely distributed in different fish species and aquatic ecosystems.

Similar yet different: co-analysis of the genetic diversity and structure of an invasive nematode parasite and its invasive mammalian host.

Animal parasitic nematodes can cause serious diseases and their emergence in new areas can be an issue of major concern for biodiversity conservation and human health. Their ability to adapt to new environments and hosts is likely to be affected by their degree of genetic diversity, with gene flow between distinct populations counteracting genetic drift and increasing effective population size. The raccoon roundworm (Baylisascaris procyonis), a gastrointestinal parasite of the raccoon (Procyon lotor), has increased its global geographic range after being translocated with its host. The raccoon has been introduced multiple times to Germany, but not all its populations are infected with the parasite. While fewer introduced individuals may have led to reduced diversity in the parasite, admixture between different founder populations may have counteracted genetic drift and bottlenecks. Here, we analyse the population genetic structure of the roundworm and its raccoon host at the intersection of distinct raccoon populations infected with B. procyonis. We found evidence for two parasite clusters resulting from independent introductions. Both clusters exhibited an extremely low genetic diversity, suggesting small founding populations subjected to inbreeding and genetic drift with no, or very limited, genetic influx from population admixture. Comparison of the population genetic structures of both host and parasite suggested that the parasite spread to an uninfected raccoon founder population. On the other hand, an almost perfect match between cluster boundaries also suggested that the population genetic structure of B. procyonis has remained stable since its introduction, mirroring that of its raccoon host.

Human antibody recognition of Anisakidae and Trichinella spp. in Greenland.

High levels of total IgE are observed among children in Greenland. To evaluate the extent to which Anisakidae and Trichinella spp. contribute to the high total IgE level, an ELISA and a western blot were developed for the detection of IgG antibodies to Anisakidae, based on excretory/secretory antigens from Anisakidae larvae. Western blots with Anisakidae and Trichinella antigens discriminated between Anisakidae and Trichinella infections, enabling cross-reactivity between the two parasite infections to be eliminated. Serum samples from 1012 children in Greenland were analysed for specific antibodies to Anisakidae and Trichinella. Eleven children were IgG-positive for Trichinella and nine were IgG-positive for Anisakidae, indicating a relatively low prevalence of both infections among children in Greenland. Faecal samples from 320 children were also examined for other intestinal parasites. Enterobius vermicularis was found in one sample and Blastocystis hominis in 32 samples, but no other intestinal parasites were identified. In total, 304 children had elevated total IgE levels. There was a significant association between Trichinella seropositivity and high levels of total IgE, but not between Anisakidae seropositivity and total IgE. The data indicate that parasitic infections alone do not explain the high level of total IgE observed among children in Greenland.

Cases of reduced cyathostomin egg-reappearance period and failure of Parascaris equorum egg count reduction following ivermectin treatment as well as survey on pyrantel efficacy on German horse farms.

In 2003 and 2004, on a total of 63 different German horse farms, a survey using the faecal egg count reduction (FECR) test was performed to investigate the efficacy of ivermectin (IVM, Ivomec) and pyrantel (PYR, Banminth) treatment against gastro-intestinal nematodes in a total of 767 horses. IVM treatment resulted in 100% reduction of the cyathostomin egg production 14 and 21 days post-treatment (d.p.t.) on 37 farms. On the remaining five farms, the mean faecal egg count reduction ranged between 97.7 and 99.9%. The mean cyathostomin FECR following PYR treatment ranged between 92.2 and 100% on the 25 farms tested. Therefore, based on the 90% FECR threshold suggested for detection of anthelmintic resistance in horses, neither IVM nor PYR anthelmintic resistance was detected. However, if the thresholds recommended for the detection of resistance in small ruminants were applied, on one and four farms signs of reduced IVM and PYR efficacy, respectively, were observed. In 2005, to further investigate these findings, the cyathostomin egg-reappearance period (ERP) following IVM treatment was examined on six selected farms, two of which were found to show less than 99.8% FECR in the previous survey. On these two latter farms, the ERP was less than 5 weeks, while on the other four it was at least 8 weeks. Earlier investigations described IVM cyathostomin ERP of at least 9 weeks. The efficacy of IVM to reduce Parascaris equorum egg excretion was also studied. On one farm in 2 consecutive years, IVM treatment did not lead to a significant reduction in P. equorum faecal egg counts in one and five young horses, respectively.

Molecular detection of sibling species in anisakid nematodes.

The number of sibling species of anisakid nematodes detected over the last two decades has been increased, fuelled by the use of genetic/molecular methodologies. In the present review, we summarize the biological species discovered within most of the nominal species belonging to the genera Anisakis, Contracaecum and Pseudoterranova by the use of allozyme (20-24 loci studied) and recently confirmed by us using mitochondrial cox-2 gene sequence analysis (mtDNA cox-2). Ecological evidence relating to the distributional range of the genetically detected sibling species and their host preferences, which represent data sets that can be utilized for species delimitation and definition, are summarized.

Lagochilascaris minor third-stage larvae secrete metalloproteases with specificity for fibrinogen and native collagen.

Dissemination of parasitic infections depends on migration through tissues and evasion from both hemostatic processes and immune responses from hosts. Metalloproteases play major roles in these mechanisms of pathogen-host interactions and, thus, are considered drug targets. In this study, we characterized metalloprotease activities in excretory/secretory (ES) products from third stage larvae (L3) of the ascarid Lagochilascaris minor, the causative agent of lagochilascariosis, which demonstrates an impressive migrating capacity across host tissues, including bone. Gel enzymography showed that ES products of L3 display two major gelatinolytic activities. Optimal proteolytic activity was found to occur at neutral/alkaline pH and was associated with two L. minor-secreted metalloproteases of 59 (SM59(Lm)) and 114kDa (SM114(Lm)). We next showed that ES products of L3 were able to hydrolyze fibrinogen and collagen I at neutral pH, but not BSA, in an extensive manner. Furthermore, we demonstrated that ES products of L3 mediate hydrolysis of the triple helical structure of collagen I fibers in mouse mesentery. These results suggest that ES proteases of L3 might facilitate both L. minor migration through host tissues by hydrolyzing collagens of the extracellular matrix and evasion from host hemostatic mechanisms by degrading fibrinogen.

Effects of low and high temperatures on viability of Parascaris equorum eggs suspended in water.

Microcentrifuge tubes containing 5000 eggs of Parascaris equorum suspended in water were frozen at -5, -10, -15, -20, and -80 degrees C for 1-168 h and then thawed at a room temperature. Other samples of P. equorum eggs suspended in water were inserted into wells in the heated metal block of a thermal DNA cycler. Block temperatures were set at 5 degrees C incremental temperatures from 40 to 100 degrees C. At each temperature setting microcentrifuge tubes containing P. equorum eggs were removed 1 and 5 min later. Both, frozen and heated egg suspensions as well as untreated control suspensions were then incubated to test of viability based on the development of infective larvae inside viable eggs. We found out that eggs of P. equorum in water can retain viability and infectivity after freezing and that eggs survive longer at higher freezing temperatures. Our results also indicated that when water containing P. equorum eggs reached temperatures of 60 degrees C or higher within 1 min, the viability of eggs was lost.