Identification of antiparasitic drug targets using a multi-omics workflow in the acanthocephalan model.

BACKGROUND: With the expansion of animal production, parasitic helminths are gaining increasing economic importance. However, application of several established deworming agents can harm treated hosts and environment due to their low specificity. Furthermore, the number of parasite strains showing resistance is growing, while hardly any new anthelminthics are being developed. Here, we present a bioinformatics workflow designed to reduce the time and cost in the development of new strategies against parasites. The workflow includes quantitative transcriptomics and proteomics, 3D structure modeling, binding site prediction, and virtual ligand screening. Its use is demonstrated for Acanthocephala (thorny-headed worms) which are an emerging pest in fish aquaculture. We included three acanthocephalans (Pomphorhynchus laevis, Neoechinorhynchus agilis, Neoechinorhynchus buttnerae) from four fish species (common barbel, European eel, thinlip mullet, tambaqui). RESULTS: The workflow led to eleven highly specific candidate targets in acanthocephalans. The candidate targets showed constant and elevated transcript abundances across definitive and accidental hosts, suggestive of constitutive expression and functional importance. Hence, the impairment of the corresponding proteins should enable specific and effective killing of acanthocephalans. Candidate targets were also highly abundant in the acanthocephalan body wall, through which these gutless parasites take up nutrients. Thus, the candidate targets are likely to be accessible to compounds that are orally administered to fish. Virtual ligand screening led to ten compounds, of which five appeared to be especially promising according to ADMET, GHS, and RO5 criteria: tadalafil, pranazepide, piketoprofen, heliomycin, and the nematicide derquantel. CONCLUSIONS: The combination of genomics, transcriptomics, and proteomics led to a broadly applicable procedure for the cost- and time-saving identification of candidate target proteins in parasites. The ligands predicted to bind can now be further evaluated for their suitability in the control of acanthocephalans. The workflow has been deposited at the Galaxy workflow server under the URL tinyurl.com/yx72rda7 .

The impact of Anguillicoloides crassus (Nematoda) on European eel swimbladder: histopathology and relationship between neuroendocrine and immune cells.

The swimbladder functions as a hydrostatic organ in most bony fishes, including the European eel, Anguilla anguilla. Infection by the nematode Anguillicoloides crassus impairs swimbladder function, significantly compromising the success of the eel spawning migration. Swimbladders from 32 yellow eels taken from Lake Trasimeno (Central Italy) were analysed by histopathology- and electron microscopy-based techniques. Sixteen eels (50%) harboured A. crassus in their swimbladders and intensity of infection ranged from 2 to 17 adult nematodes per organ (6.9 ± 1.6, mean ± s.e.). Gross observations of heavily infected swimbladders showed opacity and histological analysis found a papillose aspect to the mucosa and hyperplasia of the lamina propria, muscularis mucosae and submucosa. Inflammation, haemorrhages, dilation of blood vessels and epithelial erosion were common in infected swimbladders. In the epithelium of parasitized swimbladders, many empty spaces and lack of apical junctional complexes were frequent among the gas gland cells. In heavily infected swimbladders, we observed hyperplasia, cellular swelling and abundant vacuolization in the apical portion of the gas gland cells. Numerous mast cells and several macrophage aggregates were noticed in the mucosal layer of infected swimbladders. We found more nervous and endocrine elements immunoreactive to a panel of six rabbit polyclonal antibodies in infected swimbladders compared to uninfected.

Intestinal Histopathology due to an Acanthocephalan in Two Corvid Species from Northern Italy.

Carnivorous birds maintain parasites in the sylvatic cycle and have a role in their diffusion. The histopathology and ultrastructure of the intestine of 29 Hooded Crows (Corvus corone cornix) and 51 Eurasian Magpies (Pica pica), from the Province of Ferrara (Northern Italy), naturally infected with Sphaerirostris picae (Acanthocephala), were investigated. In both bird species, the prevalence of infection was around 10%, and the intensity of the infection in the Hooded Crows ranged from two to 12 acanthocephalans per host, whereas in the Eurasian Magpies it ranged from one to nine worms per bird. Previous records on the histopathology of acanthocephalans in birds do not provide information on the type of cells involved in the host's reaction. We aimed to gain information on the effects of acanthocephalans on the structural integrity of the birds' intestine and to describe the type of immune cells in the hosts against the parasite. Our results showed that S. picae disrupted the intestinal wall at the site of attachment by means of its neck and proboscis, and three main types of bird intestinal reactions were noticed. The most severe response of the hosts was against the proboscis because of the action of its hooks with recruitment of macrophages, giant cells, eosinophils, and heterophils. Sphaerirostris picae perforated the birds' entire intestinal wall, reaching the peritoneal visceral serosa, but it did not provoke a diffuse peritonitis.

Differential mucins secretion by intestinal mucous cells of Chelon ramada in response to an enteric helminth Neoechinorhynchus agilis (Acanthocephala).

Intestinal mucous cells produce and secrete mucins which hydrate, lubricate and protect the intestinal epithelium from mechanical injuries due to the transition of digesta or action of pathogens. Intestinal mucous cells are considered elements of the innate immune system as they secrete lectins, toxins, immunoglobulins, and anti-microbial peptides. Acid mucins can surround and eliminate many pathogenic microorganisms. We performed a quantitative analysis of the density and mucus composition of different intestinal mucous cell types from mullet (Chelon ramada) that were infected solely with Neoechinorhynchus agilis. Most N. agilis were encountered in the middle region of the intestine. Mucous cell types were identified with Alcian Blue (pH2.5) and Periodic acid-Schiff (PAS) histochemistry, and by staining with a panel of seven lectins. Mucus enriched for high viscosity acid mucins was accumulated near points of worm attachment. Parasites were surrounded by an adherent mucus layer or blanket. Ultrastructural examination showed intestinal mucous cells typically possessed an elongated, basally positioned nucleus and numerous electron dense and lucent vesicles in the cytoplasm. The results show both an increase in mucus production and changes in mucin composition in infected mullet in comparison with uninfected conspecifics.

Temporal and spatial changes in the composition and structure of helminth component communities in European eels Anguilla anguilla in an Adriatic coastal lagoon and some freshwaters in Italy.

The composition and diversity of the helminth component communities in eels Anguilla anguilla were determined in three separate localities in Italy: an Adriatic coastal lagoon, Comacchio and two freshwater localities, the River Po and the Lake Piediluco. Data from Comacchio lagoon were analysed over 15 years to determine whether community composition and diversity changed significantly overtime. The community was species rich (nine species, all marine except Proteocephalus macrocephalus) and was dominated by a suite of digeneans: Deropristis inflata, Helicometra fasciata, Lecithochirium musculus and Bucephalus anguillae. The community showed little change in composition over the period, but the relative abundance and dominance of the species did alter. By contrast, the component communities in the freshwater localities were species poor and the dominant species were freshwater acanthocephalans, Pomphorhyncus laevis in River Po and Acanthocephalus rhinensis in Lake Piediluco. The helminth community of Lake Piediluco with five species was richer than that of the River Po with only three species, but was poorer than that of Comacchio lagoons. Similarity indices between samples from Comacchio were high; between the lagoon and the freshwater localities and between the two freshwater localities, similarity indices were very low. Helminth component community structure in coastal lagoons was comparable across Europe. The helminth community in the River Po was similar to those in the River Tiber and other European rivers whilst that in Lake Piediluco was similar to that in other European lakes. Levels of the pathogenic Anguillicoloides crassus in swim bladders were consistently lower in prevalence and abundance in the coastal lagoons than in freshwater localities. This suggests that this parasite may have little impact on migrating eels if they are indeed primarily of marine origin and so it may be of little importance in the recent decline of eel populations throughout Europe.

Acidophilic granulocytes in the gills of gilthead seabream Sparus aurata: evidence for their responses to a natural infection by a copepod ectoparasite.

Immunohistochemical and ultrastructural studies were conducted on the gills of gilthead seabream, Sparus aurata L., naturally infected with the copepod ectoparasite Ergasilus lizae (Krøyer, 1863) in order to assess pathology and the host immune cell response. Gills of 56 gilthead seabream were screened for ectoparasites; 36 specimens (64.3%) harbored E. lizae. Intensity of infection was 32.7 ± 8.7 (mean ± SE). Pathological alterations to the gills of the host were more pronounced in close proximity to the copepod site of attachment. The parasite attached to the gills by means of its modified second antennae, occluded the arteries, provoked epithelial hyperplasia and hemorrhages and most often caused lamellar disruption. Numerous granular cells were encountered near the site of E. lizae attachment. In both infected and uninfected gills, the granular cells lay within the filaments and frequently occurred within the connective tissue inside and outside the blood vessels of the filaments. The type of granular cell was identified by immunohistochemical staining by using the monoclonal antibody G7 (mAb G7), which specifically recognizes acidophilic granulocytes (AGs) of S. aurata and with an anti-histamine antibody (as a marker for mast cells, MCs) on sections from 13 uninfected gills and 21 parasitized gills. The use of mAb G7 revealed that, in gills harboring copepods, the number of G7-positive cells (i.e., AGs; 32.9 ± 3.9, mean number of cells per 45,000 µm2 ± SE) was significantly higher than the density of the same cells in uninfected gills (15.3 ± 3.8; ANOVA, P < 0.05). Few histamine-positive granular cells (i.e., MCs) were found in the uninfected and parasitized gills. Here, we show, for the first time in S. aurata infected gills, that AGs rather than MCs are recruited and involved in the response to E. lizae infection in seabream.

Proliferative cell nuclear antigen (PCNA) expression in the intestine of Salmo trutta trutta naturally infected with an acanthocephalan.

BACKGROUND: Changes in the production of proliferating cell nuclear antigen (PCNA), a 36 kd protein involved in protein synthesis, within intestinal epithelia can provide an early indication of deviations to normal functioning. Inhibition or stimulation of cell proliferation and PCNA can be determined through immunohistochemical staining of intestinal tissue. Changes in the expression of PCNA act as an early warning system of changes to the gut and this application has not been applied to the fields of aquatic parasitology and fish health. The current study set out to determine whether a population of wild brown trout, Salmo trutta trutta (L.) harbouring an infection of the acanthocephalan Dentitruncus truttae Sinzar, 1955 collected from Lake Piediluco in Central Italy also effected changes in the expression of PCNA. METHODS: A total of 29 brown trout were investigated, 19 of which (i.e. 65.5%) were found to harbour acanthocephalans (5-320 worms fish-1). Histological sections of both uninfected and infected intestinal material were immunostained for PCNA. RESULTS: The expression of PCNA was observed in the epithelial cells in the intestinal crypts and within the mast cells and fibroblasts in the submucosa layer which is consistent with its role in cell proliferation and DNA synthesis. The number of PCNA-positive cells in both the intestinal epithelium and the submucosa layer in regions close to the point of parasite attachment were significantly higher than the number observed in uninfected individuals and in infected individuals in zones at least 0.7 cm from the point of parasite attachment (ANOVA, p < 0.05). CONCLUSIONS: An infection of the acanthocephalan D. truttae within the intestinal tract of S. t. trutta effected a significant increase in the number of PCNA positive cells (mast cells and fibroblasts) at the site of parasite attachment when compared to the number of positive cells found in uninfected conspecifics and in tissue zones away from the point of parasite attachment.

Ultrastructural effects of cisplatin on the inner ear and lateral line system of zebrafish (Danio rerio) larvae.

Zebrafish, Danio rerio, has been a prominent model vertebrate for the study of chemical toxicity and human disease. Zebrafish hair cells (HCs) show significant structural, functional and molecular similarities to the mammalian inner ear HCs. We examined the effects of cisplatin, an anti-cancer drug, on HCs of the inner ear and on HCs and support cells (SCs) of neuromasts in zebrafish using transmission and scanning electron microscopy. Forty-five zebrafish larvae, 12 days post-fertilization, were assessed: 15 unexposed controls, 15 exposed to 10 µM cisplatin solution, and 15 exposed to 50 µM cisplatin solution. Hair cells in the cristae and maculae of the inner ear and of neuromasts were extremely sensitive to cisplatin. The drug was associated with vacuolization and the presence of myelinoid bodies in HC cytoplasm and with a condensation of the nuclear chromatin. The predominant pattern of injury was widespread degeneration of mitochondria, which appeared swollen and less electron-dense with disorganized or reduced cristae. Severity of damage seemed to be concentration-dependent, and the inner ear suffered more damage than the lateral line. Alterations similar to those in HCs were also observed in SCs of the neuromasts. Scanning electron microscopy showed loss of kinocilia in neuromasts of fish exposed to the higher concentration of cisplatin.

Histological damage and inflammatory response elicited by Monobothrium wageneri (Cestoda) in the intestine of Tinca tinca (Cyprinidae).

BACKGROUND: Among the European cyprinids, tench, Tinca tinca (L.), and the pathological effects their cestodes may effect, have received very little or no attention. Most literature relating to Monobothrium wageneri Nybelin, 1922, a common intestinal cestode of tench, for example, has focused on aspects of its morphology rather than on aspects of the host-parasite interaction. RESULTS: Immunopathological and ultrastructural studies were conducted on the intestines of 28 tench, collected from Lake Piediluco, of which 16 specimens harboured tight clusters of numerous M. wageneri attached to the intestinal wall. The infection was associated with the degeneration of the mucosal layer and the formation of raised inflammatory swelling surrounding the worms. At the site of infection, the number of granulocytes in the intestine of T. tinca was significantly higher than the number determined 1 cm away from the site of infection or the number found in uninfected fish. Using transmission electron microscopy, mast cells and neutrophils were frequently observed in close proximity to, and inside, the intestinal capillaries; often these cells were in contact with the cestode tegument. At the host-parasite interface, no secretion from the parasite's tegument was observed. Intense degranulation of the mast cells was seen within the submucosa and lamina muscularis, most noticeably at sites close to the tegument of the scolex. In some instances, rodlet cells were encountered in the submucosa. In histological sections, hyperplasia of the mucous cells, notably those giving an alcian blue positive reaction, were evident in the intestinal tissues close to the swelling surrounding the worms. Enhanced mucus secretion was recorded in the intestines of infected tench. CONCLUSIONS: The pathological changes and the inflammatory cellular response induced by the caryophyllidean monozoic tapeworm M. wageneri within the intestinal tract of an Italian population of wild tench is reported for the first time.

Crustacean-acanthocephalan interaction and host cell-mediated immunity: parasite encapsulation and melanization.

Host-parasite interactions of Pomphorhynchus laevis (Müller, 1776) in naturally infected amphipod, Echinogammarus stammeri (Karaman), from the Brenta River (northern Italy) are described. A fully developed acanthocephalan larva occupies a large portion of an amphipod's haemocoelic space; thus, the parasite frequently induces displacement of host digestive tract and other internal organs. However, no apparent damage to the host's internal structures was observed. Within the haemocoel of E. stammeri, each larva of P. laevis is surrounded with a membranous layer, formed by microvilli, which maintains intimate contact with the amphipod's internal organs and haemocytes. Three types of circulatory haemocytes were identified based upon their distinct appearance: hyaline cell, semi-granular cell and granular cell. Echinogammarus stammeri haemocytes surrounded acanthocephalan larvae and in some instances a partially and/or totally melanized P. laevis larva was noticed. Interestingly, no melanized larvae were found in E. stammeri parasitized with other acanthocephalans namely Echinorhynchus truttae (Schrank, 1788), Polymorphus minutus (Goeze, 1782) and Acanthocephalus clavula (Dujardin, 1845).

Histopathological and ultrastructural observations of metacercarial infections of Diplostomum phoxini (Digenea) in the brain of minnows Phoxinus phoxinus.

The spatial distribution and histopathological changes induced by metacercariae of the digenean trematode Diplostomum phoxini (Faust, 1918) in the brains of European minnows Phoxinus phoxinus (L.) from the River Endrick, Scotland, were studied by light and electron microscopy. Post-mortem examination of a sample of 34 minnows revealed that 50% (n = 17) of the population was infected with 13.7 +/- 2.6 (mean +/- SE; range 1 to 38) metacercariae per infected host. Serial histological sections of the infected minnow brains revealed that the metacercariae were unevenly distributed throughout the brain, with aggregations occurring in the cerebellum, the medulla oblongata and the optic lobes. In fish with highest intensities of infection, over 40% of the cerebellar area and about 30% of the medulla oblongata area were occupied by larvae. Metacercariae disrupt the integrity of brain tissue, with individuals being found in small pockets surrounded by cellular debris. Metacercariae were rarely encountered on the surface of the brain. Electron microscopic examination of infection sites revealed that the granular layer surrounding metacercariae was necrotic, exhibited nuclear degradation and was marked by vacuolation of the cytoplasm. Rodlet cells, the only inflammatory cell types recorded in this study, were found only in parasitized brains and in close proximity to the teguments of metacercariae. It is hypothesised that secretions released from the teguments of metacercariae are a counter response to protect the metacercariae from the fish brain's cellular defence mechanisms.

Rodlet cells and the sensory systems in zebrafish (Danio rerio).

This preliminary work was designed to study, using routine procedures for light and transmission electron microscopy, the presence of rodlet cells (RCs) in or near the sensory systems of 12 adult specimens (4.0 +/- 1.2 cm, LT +/- SD) of zebrafish, Danio rerio Hamilton, 1822. Rodlet cells, characterized by a distinctive cell cortex (range, 0.4-1.5 microm in thickness) and conspicuous inclusions named "rodlets," have a round to ovoid nucleus with irregular outline. Mature RCs are 11.5 +/- 1.2 microm (mean +/- SD) long and 7.8 +/- 1.1 microm (mean +/- SD) wide. These cells are more numerous near neuromasts enclosed by an epithelial roof and/or ossified canal wall. In contrast, very few RCs were noticed near superficial neuromasts. Based on the presence of RCs around the two cranial neuromasts of each fish, a variable number from 1 to 15 rodlet cells was found (10.4 +/- 3.6, mean +/- SD). The RCs were located 1.5 microm (minimal) to 73.3 microm (maximal) from the neuromast (27.9 +/- 17.2, mean +/- SD). Moreover, RCs were found in olfactory epithelium and in proximity to some taste buds. Interestingly, RCs were absent in the inner ear, eye, and brain.

Histopathology, ultrastructure and immunohistochemistry of Coregonus lavaretus hearts naturally infected with Ichthyocotylurus erraticus (Trematoda).

Histopathological, ultrastructural and immunohistochemical investigations were conducted on 26 specimens of powan Coregonus lavaretus (L.) from Loch Lomond (Scotland). The hearts of all 26 powan (15 females and 11 males) investigated harboured metacercariae of the digenean trematode Ichthyocotylurus erraticus (Rudolphi, 1809). The vast majority of metacercariae were located either singly or as an aggregation of white cysts on the surface of the bulbus arteriosus. The intensity of infection ranged from 2 to 200 larvae heart(-1), although the number of metacercariae found on male powan did not exceed 13. Histochemically, the parasite cyst wall gave a strong positive reaction with periodic acid schiff (PAS) and a faint positive signal with Azan-Mallory stain. All the metacercariae cysts were embedded in a granulomatous proliferation of heart epicardium tissue, forming a reactive fibroconnective capsule around the parasite. The capsule enclosing the parasite (produced by the host's reaction to the parasite) measured 13.57 to 90.20 microm (37.43 +/- 3.56) in thickness. Within the capsule wall, eosinophilic granular cells (EGCs), granulocytes, melanocytes and, in some instances, partially degenerated or vacuolated epithelioid cells were observed in close proximity to the cyst wall. Pigment-bearing macrophages were scattered throughout the granulomatous host-tissue reaction and as macrophage aggregates (MAs) within the capsules surrounding parasites. Immunohistochemical tests were applied to infected heart sections using 12 different antisera. Nerve fibres immunoreactive to bombesin, substance P (SP), and atrial natriuretic peptide (ANP) antisera were observed in close proximity to the parasite larvae. The presence of a serotonin-like substance was also observed within host immune-cells surrounding trematode cysts. Large cells of the epicardium were found to be immunoreactive to met-enkephalin and vasoactive intestinal peptide (VIP) antisera but not immunoreactive to anti-protein gene-product 9.5 (PGP9.5) sera.

Immunohistochemistry, ultrastructure and pathology of gills of Abramis brama from Lake Mondsee, Austria, infected with Ergasilus sieboldi (Copepoda).

Immunohistochemical, ultrastructural and pathological studies were carried out on the gills of bream Abramis brama (L.) from Lake Mondsee, Austria, that were naturally infected with Ergasilus sieboldi Nordmann, 1832. Of a total of 14 specimens of bream examined, the gills of 7 (50%) were parasitized with this copepod and the intensity of infection ranged from 1 to 23 crustaceans per host. Histopathological investigations of infected gill showed extensive tissue damage due to attachment and feeding of the crustacean. Parasites attached close to the base of filaments near the gill arch. Pressure exerted by the ectoparasite attached to the lateral margin of the gill filaments induced atrophy of the secondary lamellae. Tissue reactions included hyperplasia and mucous cell proliferation of the respiratory epithelium. Mucous cells displayed an intense immunohistochemical reactivity with the anti-nitric oxide synthase antibody. In parasitized primary and secondary lamellae, a high number of eosinophilic granular cells and rodlet cells were noticed. Rodlet cells represent an inflammatory cell type closely linked to other piscine inflammatory cells. Presence of a high number of inflammatory cells at the site of E. sieboldi attachment is related to intense host cellular reaction.

Lernanthropus kroyeri infections in farmed sea bass Dicentrarchus labrax: pathological features.

Twenty sea bass Dicentrarchus labrax L. from a fish farm (floating cage) in Greece were examined for the presence of parasites. The gills of 7 (35%) fish were infected with adult female specimens of the parasitic copepod Lernanthropus kroyeri van Beneden, 1851, and the intensity of infection ranged from 1 to 24 parasites per host. The most infected portion of the gills appeared to be the primary lamellae. Erosion, desquamation and necrosis of the secondary lamellae were noticed near the site of copepod attachment; furthermore, the terminal claw of the second antennae lacerated tissue and vessels of infected gill. Parasitism by L. kroyeri affected the host's condition factor (mean +/- SE in uninfected vs parasitized; 1.88 +/- 0.04 vs 1.66 +/- 0.12; p < 0.05).

Effect of Pomphorhynchus laevis (Acanthocephala) on putative neuromodulators in the intestine of naturally infected Salmo trutta.

Immunohistochemical and pathological studies were carried out on the digestive tract of parasitized and uninfected specimens of Salmo trutta (L.). A total of 124 brown trout were collected on several occasions from 3 tributaries of the Brenta River, northern Italy. Twenty-eight individuals of S. trutta (22.6%) were parasitized with Pomphorhynchus laevis (Miller, 1776). The occurrence of P. laevis in the trout gut significantly increased the number of endocrine cells immunoreactive to calcitonin gene-related peptide (CGRP), beta-endorphin, met-enkephalin, neuropeptide Y (NPY) and Substance P (SP) antisera. Moreover, bombesin-, cholecistokinin-8- (CCK-8), leu-enkephalin- and serotonin- (5-HT)-like immunoreactive cells were less numerous in the intestine of the parasitized brown trout. A strong positive immunoreactivity was observed in nerve fibres and neurones of the myenteric plexus of the parasitized fish; the antisera involved in this positive reactivity were bombesin, met-enkephalin, SP and vasoactive intestinal peptide (VIP). More neurones immunoreactive to anti-CGRP and anti-5-HT sera were noted in the myenteric plexus and in the inner layer of the tunica muscularis of the infected fish. Most of the above-mentioned neuromodulators are known to control gut motility, digestive/absorptive processes, as well as the immune response. The changes induced by parasites in the neuroendocrine system of the brown trout are discussed.