Enteric neuromodulators and mucus discharge in a fish infected with the intestinal helminth Pomphorhynchus laevis.

BACKGROUND: In vertebrates, the presence of enteric worms can induce structural changes to the alimentary canal impacting on the neuroendocrine system, altering the proper functioning of the gastrointestinal tract and affecting the occurrence and relative density of endocrine cells (ECs). This account represents the first immunohistochemistry and ultrastructure-based study which documents the intimate relationship between the intestinal mucous cells and ECs in a fish-helminth system, investigating the potential effects of enteric neuromodulators on gut mucus secretion/discharge. METHODS: A modified dual immunohisto- and histochemical staining technique was applied on intestinal sections from both infected and uninfected fish. Sections were incubated in antisera to a range of neuromodulators (i.e. leu-enkephalin, met-enkephalin, galanin and serotonin) and the glycoconjugate histochemistry of the mucous cells was determined using a subsequent alcian blue - periodic acid Schiff staining step. Dual fluorescent staining on sections prepared for confocal laser scanning microscopy and transmission electron microscopy were also used to document the relationship between ECs and mucous cells. RESULTS: From a total of 26 specimens of Squalius cephalus sampled from the River Paglia, 16 (i.e. 62 %) specimens were found to harbour an infection of the acanthocephalan Pomphorhynchus laevis (average intensity of infection 9.2 ± 0.8 parasites host(-1), mean ± standard error). When acanthocephalans were present, the numbers of mucous cells (most notably those containing acidic or mixed glycoconjugates) and ECs secreting leu-enkephalin, met-enkephalin, galanin, serotonin were significantly higher than those seen on sections from uninfected fish. The relationship between met-enkephalin-like or serotonin-like ECs and lectin DBA positive mucous cells was demonstrated through a dual fluorescent staining. The presence of tight connections and desmosomes between mucous and ECs in transmission electron micrographs provides further evidence of this intimate relationship. CONCLUSIONS: The presence of P. laevis induces an increase in the number of enteric ECs that are immunoreactive to leu- and met-enkephalin, galanin, and serotonin anti-sera. The mucous cells hyperplasia and enhanced mucus secretion in the helminth-infected intestines could be elicited by the increase in the number of ECs which release these regulatory substances.

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.

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.

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.