A role for the enteric nervous system in the response to helminth infections

The enteric nervous system (ENS) in the gut contains a particularly high concentration of nerve cells, and effectively functions as an independent 'minibrain'. Interactions between nerve, endocrine, immune and other cell types allow the sophisticated regulation of normal gut physiology. They can also bring about a co-ordinated response to parasitic infection, possibly leading to expulsion of the parasite. In this review, Derek McKay and Ian Fairweather will consider, in brief, data pertaining to changes in the ENS following intestinal helminth infections and speculate on the role that these alterations may have in the expulsion of the parasite burden and the putative ability of the parasite to modulate these events.

Nematode neuropeptides: Localization, isolation and functions

Historically, peptidergic substances (in the form of neurosecretions) were linked to moulting in nematodes. More recently, there has been a renewal of interest in nematode neurobiology, initially triggered by studies demonstrating the localization of peptide immunoreactivities to the nervous system. Here, David Brownlee, Ian Fairweather, Lindy Holden-Dye and Robert Walker will review progress on the isolation of nematode neuropeptides and efforts to unravel their physiological actions and inactivation mechanisms. Future avenues for research are suggested and the potential exploitation of peptidergic pathways in future therapeutic strategies highlighted.

Immunocytochemical localization of glutamate-like immunoreactivity within the nervous system of the cestode Mesocestoides corti and the trematode Fasciola hepatica

The localization and distribution of glutamate-like immunoreactivity (IR) in the nervous system of both the cestode Mesocestoides corti and the trematode Fasciola hepatica has been determined by an indirect immunofluorescent technique, in conjunction with confocal scanning laser microscopy (CSLM). Immunostaining was widespread in the central (CNS) and peripheral (PNS) nervous systems of both species examined. In the CNS, IR was evident in nerve cells and fibres in the cerebral ganglia, the cerebral commissure and the dorsal, ventral and longitudinal nerve cords. In the peripheral nervous system (PNS) of M. corti, IR was apparent in nerve plexuses associated with the subtegmental musculature and the musculature associated with the anteriorly positioned suckers. In F. hepatica, IR was evident in the innervation of both the oral and the ventral suckers, In the reproductive system of F. hepatica, glutamate-IR was observed around the ootype/Mehlis' gland complex.

Cellular and subcellular localization of SALMFamide (S1)-like immunoreactivity within the central nervous system of the nematode Ascaris suum (Nematoda, Ascaroidea)

The localization and distribution of SALMFamide (S1)-like immunoreactivity (IR), was determined at both the cellular and subcellular level in the central nervous system (CNS) of the nematode roundworm Ascaris suum. The techniques of indirect immunofluorescence in conjunction with confocal scanning laser microscopy and post-embedding, IgG-conjugated colloidal gold immunostaining were used, respectively. Immunostaining was widespread in the CNS of adult A. suum, with immunoreactivity (IR) being localized in nerve cells and fibres in the ganglia associated with the anterior nerve ring and in the main nerve cords and their commissures. At the subcellular level, gold labeling of peptide was localized exclusively over dense-cored vesicles within nerve cell bodies, nerve axons and nerve terminals of the neuropile of the anterior nerve ring, main ganglia and nerve cords in the CNS. Double-labeling demonstrated an apparent co-localization of S1- and FMRFamide-IR-together IR-together with S1- and pancreatic polypeptide (PP)-IR in the same dense-cored vesicles. Antigen preabsorption experiments indicated little cross-reactivity, if any, between the three antisera; indeed, neither FMRFamide nor PP antigens abolished S1 immunostaining.

THE ACTION OF SEROTONIN AND THE NEMATODE NEUROPEPTIDE KSAYMRFAMIDE ON THE PHARYNGEAL MUSCLE OF THE PARASITIC NEMATODE, ASCARIS-SUUM

The pharyngeal component of the enteric nervous system of the parasitic nematode, Ascaris suum exhibits immunoreactivity for serotonin (5-hydroxytryptamine or 5-HT) and for FMRFamide-like peptides. This paper describes the application of an in vitro pharmacological approach to investigate the functional role of 5-HT and FMRFamide-like peptides. The pharyngeal pumping behaviour of Ascaris suum was monitored using a modified pressure transducer system which measures pharyngeal pressure changes and therefore pumping. The pharynx did not contract spontaneously; however, 5-HT (10-1000 mu M) stimulated pumping at a frequency of 0 . 5 Hz. FMRFamide had no apparent effect on pharyngeal pumping. The native nematode FMRFamide-related peptide (FaRP), KSAYMRFamide inhibited the pumping elicited by 5-HT. The duration of inhibition was dose-dependent (0 . 1-1000 nM) with a threshold of 0 . 1 nM. In 4 preparations, the inhibition of the pharyngeal muscle was preceded by an initial excitation and increase in the amplitude of pharyngeal pressure changes. The pharynx is involved in various nematode processes, including feeding, regulation of hydrostatic pressure and excretion. The role of 5-HT and KSAYMRFamide in the pharyngeal function of nematodes is discussed.

IONIC COMPOSITION OF THE LIVER FLUKE FASCIOLA-HEPATICA FROM DIFFERENT MAMMALIAN HOSTS AND COMPARISON WITH HOST BILE

A qualitative analysis of the cationic profile of bovine and ovine biles and of bovine, ovine and rat liver flukes has been carried out by DC are emission spectrography. A quantitative assessment of the concentrations of Na+, K+, Ca2+ and Mg2+ ions in bovine, ovine and rat flukes has been determined by atomic absorption spectrophotometry. The levels of these ions in bovine and ovine bile samples have also been assessed and compared with those of Hedon-Heig saline. The ionic composition of the two biles is similar and the concentration of each ion is greater than that in Hedon-Heig saline. Despite the similarity in biles, ion levels in bovine flukes are generally higher than those in ovine flukes. Ion levels in rat flukes are different again but show closer similarity to those in bovine, not ovine, flukes. The results are discussed in relation to the proposed operation of the osmoregulatory system in the fluke.

FASCIOLA-HEPATICA - ULTRASTRUCTURAL-CHANGES TO THE TEGUMENT OF JUVENILE FLUKES FOLLOWING INCUBATION IN-VITRO WITH THE DEACETYLATED (AMINE) METABOLITE OF DIAMPHENETHIDE

Ultrastructural changes to the tegument of 5-week-old, 3-week-old and freshly-excysted Fasciola hepatica following in vitro incubation with the deacetylated (amine) metabolite of diamphenethide (DAMD, 10 mu gml(-1)) were examined by transmission electron microscopy, A similar sequence of tegumental changes occurred in all three age groups of fluke, although, with increasing fluke age, the time before onset increased and the damage became more extensive. The 5-week-old flukes showed an initial stress response after 3 h, typified by blebbing of the apical plasma membrane, formation of microvilli and an accumulation and accelerated release of secretory bodies at the tegumental apex, as well as swelling of the basal infolds, The swelling increased in extent with progressively longer periods of incubation in DAMD, leading to extreme edema and sloughing of the tegument after 9 h. The 3-week-old flukes showed a stress response and swelling of the basal infolds after only 1.5 h, although sloughing of the tegument did not occur until after 9 h. In the freshly-excysted metacercaria, a stress response and some sloughing of the tegument were evident after only 0.5 h. At all stages of development, the ventral tegument was more severely affected than the dorsal, Changes also occurred to the tegumental cells which were indicative of a disruption in the synthesis and release of tegumental secretory bodies: the amount of GER became reduced, the cisternae became swollen and their ribosomal covering decreased, the Golgi complexes disappeared from the cells and the numbers of secretory bodies in the cells also decreased, The heterochromatin content of the nuclei increased and eventually the tegumental cells began to break down, Again, the changes became apparent more rapidly at the earlier stages of development. The ultrastructural changes to the tegument are linked to a possible mode of action for diamphenethide as an inhibitor of protein synthesis. In turn, the results may help to explain the drug's high efficacy against juvenile stages of F. hepatica.

IMMUNOCYTOCHEMICAL DEMONSTRATION OF A SALMFAMIDE-LIKE NEUROPEPTIDE IN THE NERVOUS-SYSTEM OF ADULT AND LARVAL STAGES OF THE HUMAN BLOOD FLUKE, SCHISTOSOMA-MANSONI

The localization and distribution of SALMFamide immunoreactivity (IR), SI(GFNSALMFamide), in the nervous system of both the adult and larval stages of the trematode Schistosoma mansoni has been determined by an indirect immunofluorescent technique in conjunction with confocal scanning laser microscopy (CSLM). Immunostaining was widespread in the nervous system of adult male and female S. mansoni. In the central nervous system (CNS), IR was evident in nerve cells and fibres in the anterior ganglia, cerebral commissure and dorsal and ventral nerve cords. In the peripheral nervous system (PNS), IR was apparent in nerve plexuses associated with the subtegmental musculature, oral and ventral suckers, the lining of the gynaecophoric canal, and in fine nerve fibres innervating the dorsal tubercles of the male worm. In the reproductive system of male and female worms, S1-IR was only observed around the ootype/Mehlis' gland complex in the female. Immunostaining was also evident in the nervous system of both miracidium and cercarial larval stages. A post-embedding, IgG-conjugated colloidal gold immunostaining technique was employed to examine the subcellular distribution of SALMFamide-IR in the CNS of S. mansoni. Gold labelling of peptide was localized over dense-cored vesicles within nerve cell bodies and fibres constituting the neuropile of the anterior ganglia, cerebral commissure and nerve cords of the CNS. Antigen pre-absorption studies indicated that the results obtained do suggest S1-like immunostaining and not cross-reactivity with other peptides, in particular FMRFamide.

SEROTONINERGIC AND PEPTIDERGIC NERVE ELEMENTS IN THE PROTOSCOLEX OF ECHINOCOCCUS-GRANULOSUS (CESTODA, CYCLOPHYLLIDEA)

The localisation and distribution of 5-hydroxytryptamine (5-HT, or serotonin) and neuropeptides in the nervous system of the protoscolex of the hydatid organism Echinococcus granulosus were determined by an indirect immunofluorescence technique. Nerve-cell bodies immunoreactive for 5-HT occurred in the lateral ganglia and in association with the lateral longitudinal nerve cords. 5-HT immunostaining was also evident in the central nerve ring, in the rostellar nerves and in the nerve plexus innervating the suckers. Of the antisera used to screen the protoscolex for neuropeptide immunoreactivity (IR), immunostaining was obtained with those raised against pancreatic polypeptide (PP), peptide YY (PYY), substance P (SP), peptide histidine isoleucine (PI-II) and vasoactive intestinal peptide (VIP). The most extensive pattern of IR occurred with antisera to PP and PYY. Immunoreactive nerve elements were evident in the lateral ganglia, central nerve ring, rostellar nerves, rostellar ganglia, sucker plexus and longitudinal nerve cords. The distribution of SP-, PHI- and VIP-IRs was more restricted: SP-IR occurred in the lateral ganglia and sucker nerves, whilst PHI- and VIP-immunoreactive nerve elements were associated with the lateral longitudinal nerve cords. Protoscoleces cultured in vitro for 29 days were also examined and neuroanatomical changes noted. A greater development of the longitudinal nerve cords and their cross-connectives in the body of the worm was evident, and a group of nerve cells were seen to develop at the posterior end of the main lateral nerve cords.

IMMUNOCYTOCHEMICAL LOCALIZATION OF SEROTONIN (5-HT) IN THE NERVOUS-SYSTEM OF THE HYDATID ORGANISM, ECHINOCOCCUS-GRANULOSUS (CESTODA, CYCLOPHYLLIDEA)

The localization and distribution of the serotoninergic components of the nervous system in the hydatid organism, Echinococcus granulosus, were determined by immunocytochemical techniques in conjunction with confocal scanning laser microscopy (CSLM). The distribution of serotonin immunoreactivity (IR) paralleled that previously described for cholinesterase activity, although it was more widespread. Nerve cell bodies and nerve fibres immunoreactive for 5-HT were present throughout the central nervous system (CNS), occurring in the paired lateral, posterior lateral and rostellar ganglia, their connecting commissures and nerve rings in the scolex and in the ten longitudinal nerve cords that run posteriorly throughout the body of the worm. A large population of nerve cell bodies was associated with the lateral nerve cords. In the peripheral nervous system (PNS), immunoreactive nerve fibres occurred in well-developed nerve plexuses innervating the somatic musculature and the musculature of the rostellum and suckers. The genital atrium and associated reproductive ducts were richly innervated with serotoninergic nerve cell bodies and nerve fibres.

THE EFFECT OF THE SULFOXIDE METABOLITE OF TRICLABENDAZOLE (FASINEX) ON THE TEGUMENT OF MATURE AND IMMATURE STAGES OF THE LIVER FLUKE, FASCIOLA-HEPATICA

The effects of the novel benzimidazole, triclabendazole (TCBZ) ('Fasinex', Ciba-Geigy), in its active sulphoxide metabolite form (TCBZ-SX), on the tegumental ultrastructure of Fasciola hepatica were determined in vitro by transmission electron microscopy (TEM), using both intact flukes and tissue-slice material. At a concentration of 15 mu g/ml, the tegument of the whole adult fluke showed ultrastructural changes only after prolonged time-periods, with vacuolation at the base of the syncytium and accumulation of T2 secretory bodies in the tegumental cells. At a concentration of 50 mu g/ml, with both whole flukes and tissue-slices, the tegument appeared extremely abnormal with accumulation of secretory bodies towards the base of the syncytium. With longer incubation times, the tegument was completely sloughed away and the tegumental cells became synthetically inactive. The tegument of the 3-week-old juvenile became progressively convoluted at the apex, while in the basal regions there was severe vacuolation. In the tegumental cells, there were accumulations of T1 secretory bodies. These results confirm TCBZ as a potent fasciolicide, being very effective in disrupting the fluke tegument. They may go some way to explain the mode of action of this important fasciolicide.

ULTRASTRUCTURAL-LOCALIZATION OF PANCREATIC POLYPEPTIDE AND FMRFAMIDE IMMUNOREACTIVITIES WITHIN THE CENTRAL-NERVOUS-SYSTEM OF THE NEMATODE, ASCARIS-SUUM (NEMATODA, ASCAROIDEA)

A post-embedding immunogold technique has been used to examine the subcellular distribution of immunoreactivities to vertebrate pancreatic polypeptide (PP) and to the invertebrate peptide, FMRFamide within the central nervous system (CNS) of the nematode, Ascaris suum. Gold labelling of peptide was localized exclusively over dense-cored vesicles within nerve cell bodies, nerve axons and nerve terminals of the main ganglia and nerve cords in the CNS. Double-labelling of peptides demonstrated an apparent co-localization of PP and FMRFamide immunoreactivities in the same dense-cored vesicles, although populations of dense-cored vesicles that labelled solely for FMRFamide were also evident. Antigen preabsorption studies indicated little or no cross-reactivity between the two antisera.

ULTRASTRUCTURAL-LOCALIZATION OF FMRFAMIDE-IMMUNOREACTIVITY AND PANCREATIC POLYPEPTIDE-IMMUNOREACTIVITY WITHIN THE CENTRAL-NERVOUS-SYSTEM OF THE LIVER FLUKE, FASCIOLA-HEPATICA (TREMATODA, DIGENEA)

A post-embedding immunogold technique was used to examine the subcellular distribution of immunoreactivities to the invertebrate peptide, FMRFamide, and to vertebrate pancreatic polypeptide (PP) within the central nervous system of the trematode, Fasciola hepatica. Gold labeling of peptide was localised exclusively over both dense-cored and ellipsoidal electron-dense vesicles (with a homogeneous matrix) present within nerve cell bodies, small and 'giant' nerve processes of the neuropile in the cerebral ganglia and transverse commissure, as well as in the main longitudinal nerve cords. Double labeling demonstrated an apparent co-localisation of FMRFamide and PP immunoreactivities in the same dense-cored vesicles, although populations of ellipsoidal electron-dense vesicles that labeled solely for FMRFamide were also evident. Antigen pre-absorption studies indicated little, if any, cross-reactivity of the two antisera.

IMMUNOCYTOCHEMICAL DEMONSTRATION OF PEPTIDERGIC AND SEROTONINERGIC COMPONENTS IN THE ENTERIC NERVOUS-SYSTEM OF THE ROUNDWORM, ASCARIS-SUUM (NEMATODA, ASCAROIDEA)

The localization and distribution of neuropeptides and an indoleamine (serotonin or 5-hydroxytryptamine) in the enteric nervous system (ENS) of the pig roundworm, Ascaris suum, have been determined by the application of an indirect immunofluorescence technique in conjunction with confocal scanning laser microscopy. Whole-mount preparations of pharyngeal, intestinal and rectal regions were screened with antisera to 23 vertebrate peptides, 2 invertebrate peptides and serotonin(= 5-HT). Positive immunoreactivity (IR) was obtained with antisera to pancreatic polypeptide (PP), peptide YY (PYY), FMRFamide, gastrin and serotonin. The only IR observed in the ENS was that evident in the nerve supply to the pharynx and rectal region; no IR was associated with any region of the intestine. The most extensive patterns of IR occurred with antisera to PW, FMRFamide and serotonin. In the pharyngeal component of the ENS, IR was evident in the lateral and dorsal longitudinal pharyngeal nerves, pharyngeal commissures, nerve plexus, and associated nerve cells and fibres. In contrast, the distribution of IR to the PP and gastrin antisera was more restricted and displayed a lower intensity of immunostaining. The other component of the ENS, the rectal enteric system, only yielded immunostaining to FMRFamide. The possible role of neuropeptides and serotonin in the nutritional biology of nematodes is discussed.

FASCIOLA-HEPATICA - TEGUMENTAL SURFACE CHANGES IN ADULT AND JUVENILE FLUKES FOLLOWING TREATMENT IN-VITRO WITH THE SULFOXIDE METABOLITE OF TRICLABENDAZOLE (FASINEX)

The effects of the novel benzimidazole, triclabendazole (Fasinex, Ciba-Geigy), in its active sulphoxide metabolite form (TCBZ-SX), on the tegumental surface of Fasciola hepatica has been examined in vitro. The tegument of adult flukes incubated in TCBZ-SX (50 mug/ml) appeared swollen and blebbed after only 6 h. In addition, progressive spine loss at the oral cone was evident following 12 h treatment. After 24 h, the tegumental syncytium and spines had completely sloughed away, leaving an exposed basal lamina and empty spine sockets. Juvenile flukes (3 weeks old) also demonstrated tegumental alterations after treatment with TCBZ-SX (20 mug/ml). The syncytium became extremely roughened and corrugated on both dorsal and ventral surfaces after only 3 h. Following 6- and 9-h incubations, there were many deep furrows, which were especially pronounced on the ventral surface, and by 18 h, the juvenile tegument was severely disrupted, especially on the ventral surface. In all cases, the effects were more marked than in the previous incubation periods. The results confirm the potent activity of triclabendazole against F. hepatica and suggest that the tegument of adult and juvenile flukes may be a target organ for this important fasciolicide.