Infection of endemic chub Squalius tenellus with the intestinal tapeworm Caryophyllaeus brachycollis (Cestoda): histopathology and ultrastructural surveys.

The endemic chub Squalius tenellus (Heckel, 1843) was introduced more than 100 years ago to Lake Blidinje (Bosnia-Herzegovina). Only 1 species of enteric helminth was found in a sample of 35 chubs, the tapeworm Caryophyllaeus brachycollis (Janiszewska, 1953). The paper includes histopathological investigation with identification of innate immune cells involved in host reaction and molecular data allowed correct designation of the cestode species. Of 35 specimens of chub examined, 21 (60%) harboured individuals of C. brachycollis and a total of 1619 tapeworms were counted, the intensity of infection ranged from 1 to 390 worms per fish (46.2 ± 15.3, mean ± s.e.). Histopathological and ultrastructural investigations showed strict contact between the worm's body and the epithelia and increase in the number of mucous cells, rodlet cells among the epithelial cells. Within the tunica propria-submucosa, beneath the site of scolex attachment, numerous neutrophils and mast cells were noticed. This is the first study of the occurrence of C. brachycollis in chub from Lake Blidinje and on the response of the innate immune cells of S. tenellus to this tapeworm. Interestingly, in 3 very heavily infected chubs, perforation of the intestinal wall was documented; this is uncommon among cestodes which use fish as a definitive host.

Teleost innate immunity, an intricate game between immune cells and parasites of fish organs: who wins, who loses.

Fish, comprising over 27,000 species, represent the oldest vertebrate group and possess both innate and adaptive immune systems. The susceptibility of most wild fish to parasitic infections and related diseases is well-established. Among all vertebrates, the digestive tract creates a remarkably favorable and nutrient-rich environment, which, in turn, renders it susceptible to microparasites and macroparasites. Consequently, metazoan parasites emerge as important disease agents, impacting both wild and farmed fish and resulting in substantial economic losses. Given their status as pathogenic organisms, these parasites warrant considerable attention. Helminths, a general term encompassing worms, constitute one of the most important groups of metazoan parasites in fish. This group includes various species of platyhelminthes (digeneans, cestodes), nematodes, and acanthocephalans. In addition, myxozoans, microscopic metazoan endoparasites, are found in water-dwelling invertebrates and vertebrate hosts. It is worth noting that several innate immune cells within the fish alimentary canal and certain visceral organs (e.g., liver, spleen, and gonads) play active roles in the immune response against parasites. These immune cells include macrophages, neutrophils, rodlet cells, and mast cells also known as eosinophilic granular cells. At the site of intestinal infection, helminths often impact mucous cells number and alter mucus composition. This paper presents an overview of the state of the art on the occurrence and characteristics of innate immune cells in the digestive tract and other visceral organs in different fish-parasite systems. The data, coming especially from studies employed immunohistochemical, histopathological, and ultrastructural analyses, provide evidence supporting the involvement of teleost innate immune cells in modulating inflammatory responses to metazoan and protozoan parasitic infections.

Platelet-rich plasma and ovarian quiescence: a bovine in vitro model for regeneration of the ovary.

CONTEXT: Ovarian quiescence can be due to hormonal deficiency usually caused by apoptosis of granulosa cells responsible for oestrogen synthesis. AIM: This study evaluated the regenerative effect of platelet rich plasma (PRP) on bovine in vitro models to understand its effect on granulosa cells. METHODS: Quiescent and healthy ovarian sections were cultured in the presence/absence of PRP for 72h and, at different times (0, 24, 48 and 72h), hematoxylin-eosin and immunohistochemical detection of Ki-67 were performed. Additionally, granulosa cells collected from healthy bovine ovaries were stressed with 100ng/mL of lipopolysaccharide (LPS) in presence/absence of PRP and evaluated at 0, 4, 8 and 24h for apoptosis by acridine orange and propidium iodide staining. Enzyme-linked immunosorbent assay tests were performed to evaluate oestrogen (E2) and anti-Müllerian hormone (AMH) concentrations on cultures of ovarian slices and granulosa cells. KEY RESULTS: In slides of quiescent ovaries treated with PRP, a marked and widespread positivity to Ki-67 was expressed by 40-60% of the follicular wall cells at 48h of culture. Levels of E2 and AMH were significantly higher compared to untreated quiescent samples reaching the levels of healthy control samples. PRP counteracted the LPS effect and apoptosis (at 24h, there were 93.44±3.51% live cells with LPS+PRP compared to 37±1.32% with LPS) and significantly increased concentrations of E2 and AMH. CONCLUSIONS: PRP can stimulate granulosa cell proliferation and counteract inflammatory processes in vitro . IMPLICATIONS: This treatment could improve the reproductive ability of quiescent females.

Amniotic Mesenchymal-Derived Extracellular Vesicles and Their Role in the Prevention of Persistent Post-Breeding Induced Endometritis.

Persistent post-breeding induced endometritis (PPBIE) is considered a major cause of subfertility in mares. It consists of persistent or delayed uterine inflammation in susceptible mares. There are many options for the treatment of PPBIE, but in this study, a novel approach aimed at preventing the onset of PPBIE was investigated. Stallion semen was supplemented with extracellular vesicles derived from amniotic mesenchymal stromal cells (AMSC-EVs) at the time of insemination to prevent or limit the development of PPBIE. Before use in mares, a dose-response curve was produced to evaluate the effect of AMSC-EVs on spermatozoa, and an optimal concentration of 400 × 106 EVs with 10 × 106 spermatozoa/mL was identified. At this concentration, sperm mobility parameters were not negatively affected. Sixteen susceptible mares were enrolled and inseminated with semen (n = 8; control group) or with semen supplemented with EVs (n = 8; EV group). The supplementation of AMSC-EVs to semen resulted in a reduction in polymorphonuclear neutrophil (PMN) infiltration as well as intrauterine fluid accumulation (IUF; p < 0.05). There was a significant reduction in intrauterine cytokine levels (p < 0.05) for TNF-α and IL-6 and an increase in anti-inflammatory IL-10 in mares in the EV group, suggesting successful modulation of the post-insemination inflammatory response. This procedure may be useful for mares susceptible to PPBIE.

Extracellular vesicles from seminal plasma to improve fertilizing capacity of bulls.

Seminal plasma contains extracellular vesicles (EVs) that vehicle RNA, proteins, and other molecules able to influence the biological function of sperm. The aim of this study was to improve the fertilizing capacity of male gametes of low-fertility bulls using EVs isolated by ultracentrifugation from the seminal plasma of a bull of proven fertility. After dose-response curve, 10×106 sperm of low-fertility bulls were co-incubated for an hour with 400×106 EVs/ml. In addition, it has been verified that the incorporation of EVs, which takes place in the sperm midpiece, is maintained for 5 hours and even after cryopreservation. Subsequently, the spermatozoa of low-fertility bulls, with EVs incorporated, were used for the in vitro production of embryos. The rate of blastocyst at seventh day yield in vitro, with the use of sperm with EVs incorporated, increased by about twice the yield obtained with the same sperm in the absence of EVs: bulls having an average embryonic yield of 6.41±1.48%, 10.32±4.34% and 10.92±0.95% improved their yield to 21.21±1.99%, 22.17±6.09% and 19.99±5.78%, respectively (P<0.05). These encouraging results suggest that it might be possible to keep breeding bulls with poor fertility. Further studies will be needed to evaluate the in vivo fertility of sperm treated with EVs and understand how the content of EVs is involve in the sperm-vesicle interaction and in the improved sperm performance.

Endometrial and oviduct extra-cellular vescicles for in vitro equine sperm hyperactivation and oocyte fertilization.

Unlike humans and many other mammalian species, conventional in vitro fertilization (IVF) in equine species is not successful. To mimic in vitro equine spermatozoon-oviduct interaction as close as possible to that which occurs in vivo, extracellular vesicles (EVs) secreted by the female genital tract were used. Three female genital tracts were collected at slaughterhouse from mares in late estrus. Ipsilateral proximal and apical horn endometrial explants were digested with collagenase and trypsin and cells obtained were cultured on insert system to allow their polarization. Ipsilateral oviducts were squeezed out to obtain spheroids. To produce EVs, proximal and apical horn endometrial cells and oviductal spheroids were cultured for three days in serum free medium. To trace interaction between spermatozoa and EVs by fluorescence microscopy, EVs were differently labeled. Pooled samples of ejaculated spermatozoa from three stallions were incubated in capacitating medium (CM) for 6 h and to induce hyperactivation for other 6 h in CM supplemented with different kind of EVs alone or in combination. A control was performed in absence of EVs. Sperm were assessed for motility by CASA system, EV incorporation by confocal microscopy and acrosomal reaction (AR) by staining with FITC-PNA/PI. In vitro fertilization was performed, and presumed zygotes were subjected to chromatin configuration. The results show that incorporation of EVs of the proximal horn does not take place, while apical horn EVs are incorporated in the head of the spermatozoon in 4 h. The EVs of oviductal spheroids are incorporated in the middle tract in 1 h. The rate of AR with EVs of the apical horn and oviductal spheroids were respectively 50.25% and 57.14%. When these EVs were added in combination, the rate of AR was 71.42%. In the control, the rate of AR was of 15%. After in vitro fertilization, 44% of oocytes showed male and female pronuclei, whereas no fertilization is obtained in the control. In conclusion, EVs from apical horn and oviduct could be involved in cell trafficking during equine semen hyperactivation, and their possible use in vitro could facilitate the development of equine reproductive biotechnologies.

Parasites and the neuroendocrine control of fish intestinal function: an ancient struggle between pathogens and host.

Most individual fish in wild and farmed populations can be infected with parasites. Fish intestines can harbour protozoans, myxozoans and helminths, which include several species of digeneans, cestodes, nematodes and acanthocephalans. Enteric parasites often induce inflammation of the intestine; the pathogen provokes changes in the host physiology, which will be genetically selected for if they benefit the parasite. The host response to intestinal parasites involves neural, endocrine and immune systems and interaction among these systems is coordinated by hormones, chemokines, cytokines and neurotransmitters including peptides. Intestinal fish parasites have effects on the components of the enteric nervous and endocrine systems; mechanical/chemical changes impair the activity of these systems, including gut motility and digestion. Investigations on the role of the neuroendocrine system in response to fish intestinal parasites are very few. This paper provides immunohistochemical and ultrastructural data on effects of parasites on the enteric nervous system and the enteric endocrine system in several fish­parasite systems. Emphasis is on the occurrence of 21 molecules including cholecystokinin-8, neuropeptide Y, enkephalins, galanin, vasoactive intestinal peptide and serotonin in infected tissues.

Rodlet cells, fish immune cells and a sentinel of parasitic harm in teleost organs.

Rodlet cells (RCs) are the enigmatic and distinctive pear-shaped cells had found in many tissues of marine and freshwater teleosts. They have a distinctive fibrous capsule or the cell cortex that envelopes conspicuous inclusions called rodlets, basally situated nucleus, and poorly developed mitochondria. The contraction of the cell cortex results in the expulsion of the cell contents through an apical opening. One hundred and thirty years since rodlet cells were first reported, many questions remain about their origin and a function. This review will present new evidence regarding the relationship between RCs and metazoan parasites, and a protozoan infecting organs of different fish species, and update the state of knowledge about the origin, structure and the function of these intriguing fish cells.

Survival of metazoan parasites in fish: Putting into context the protective immune responses of teleost fish.

Defence mechanisms of fish can be divided into specific and non-specific that act in concert and are often interdependent. Most fish in both wild and cultured populations are vulnerable to metazoan parasites. Endoparasitic helminths include several species of digeneans, cestodes, nematodes, and acanthocephalans. Although they may occur in large numbers, helminth infections rarely result in fish mortality. Conversely, some ectoparasites cause mass mortality in farmed fish. Given the importance of fish innate immunity, this review addresses non-specific defence mechanisms of fish against metazoan parasites, with emphasis on granulocyte responses involving mast cells, neutrophils, macrophages, rodlet cells, and mucous cells. Metazoan parasites are important disease agents that affect wild and farmed fish and can induce high economic loss and, as pathogen organisms, deserve considerable attention. The paper will provide our light and transmission electron microscopy data on metazoan parasites-fish innate immune and neuroendocrine systems. Insights about the structure and functions of the cell types listed above and a brief account of the effects and harms of each metazoan taxon to specific fish apparati/organs will be presented.

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.

Mucosal Hallmarks in the Alimentary Canal of Northern Pike Esox lucius (Linnaeus).

On the basis of trophic behavior, fish are classified as herbivores, carnivores, omnivores, or detritivores. Epithelial mucous cells secrete mucin types specific to diet and digestive function. Mucus secretion is regulated mainly by molecular modulators produced by epithelial endocrine cells in response to luminal or tissue stimuli. These modulators are involved in control of food intake and digestive functions. Immunohistochemical and immunofluorescence studies were conducted on 10 adult northern pike (Esox lucius Linnaeus, 1758) from Lake Piediluco (Central Italy) to quantify distribution of sub-types of mucous and endocrine cells in alimentary mucosal epithelium. Neutral mucins predominated in the esophagus, and mixed and acidic mucins predominated in stomach and intestine. The gastric epithelium contained endocrine cells secreting somatostatin, tyrosine hydroxylase, and substance P. Mucous cells secreting neutral mucins increased in number from proximal to distal intestine, with endocrine cells containing substance P in the proximal intestine and those containing Leu-enkephalin throughout the intestine. Lectin histochemistry of gut sections revealed an abundance of N-acetyl-glucosamine and N-acetyl-galactosamine as carbohydrate residues on the mucin chain. The quantity and content of endocrine and mucous cells in the alimentary canal of E. lucius showed a direct relationship with its diet.

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.

Description of epithelial granular cell in catshark spiral intestine: Immunohistochemistry and ultrastructure.

We evaluated the histology of the spiral intestine of the blackmouth catshark (Galeus melastomus), a small shark distributed in the eastern Atlantic and Mediterranean Sea basin. Entire digestive tracts of 10 G. melastomus were studied using histochemical, immunohistochemical, and ultrastructural methods. Our studies identified a unique, large granular cell type in the intestinal epithelium. Transmission electron microscopy showed that the epithelial granular cell type made intimate contact, by means of junctional complexes, with adjacent epithelial and mucous cells. Several histochemical staining methods showed that the cytoplasmic granules were strongly eosinophilic. Immunostaining of intestinal sections revealed immunoreactivity of the granular cell to tumor necrosis factor-α (TNF-α) antibody. However, no reactivity to inducible-nitric oxide synthase (i-NOS), interleukin-6 (IL-6), interleukin IL-1ß, lysozyme, serotonin 5-HT antibodies was detected.

Pike intestinal reaction to Acanthocephalus lucii (Acanthocephala): immunohistochemical and ultrastructural surveys.

BACKGROUND: The Northern pike, Esox lucius, is a large, long-lived, top-predator fish species and occupies a broad range of aquatic environments. This species is on its way to becoming an important model organism and has the potential to contribute new knowledge and a better understanding of ecology and evolutionary biology. Very few studies have been done on the intestinal pathology of pike infected with helminths. The present study details the first Italian record of adult Acanthocephalus lucii reported in the intestine of E. lucius. RESULTS: A total of 22 pike from Lake Piediluco (Central Italy) were examined, of which 16 (72.7%) were infected with A. lucii. The most affected areas of gastrointestinal tract were the medium and distal intestine. The intensity of infection ranged from 1 to 18 parasites per host. Acanthocephalus lucii penetrated mucosal and submucosal layers which had a high number of mast cells (MCs) with an intense degranulation. The cellular elements involved in the immune response within the intestine of pike were assessed by ultrastructural techniques and immunohistochemistry using antibodies against met-enkephalin, immunoglobulin E (IgE)-like receptor (FCεRIγ), histamine, interleukin-6, interleukin-1ß, substance P, lysozyme, serotonin, inducible-nitric oxide synthase (i-NOS), tumor necrosis factor-α (TNF-α) and the antimicrobial peptide piscidin 3 (P3). In intestines of the pike, several MCs were immunopositive to 9 out of the 11 aforementioned antibodies and infected fish had a higher number of positive MCs when compared to uninfected fish. CONCLUSIONS: Pike intestinal tissue response to A. lucii was documented. Numerous MCs were seen throughout the mucosa and submucosal layers. In infected and uninfected intestines of pike, MCs were the dominant immune cell type encountered; they are the most common granulocyte type involved in several fish-helminth systems. Immunopositivity of MCs to 9 out of 11 antibodies is of great interest and these cells could play an important key role in the host response to an enteric helminth. This is the first report of A. lucii in an Italian population of E. lucius and the first account on positivity of MCs to piscidin 3 and histamine in a non-perciform fish.

Peculiarity of Porcine Amniotic Membrane and Its Derived Cells: A Contribution to the Study of Cell Therapy from a Large Animal Model.

The aim of this work was to provide, for the first time, a protocol for isolation and characterization of stem cells from porcine amniotic membrane in view of their potential uses in regenerative medicine. From three samples of allanto-amnion recovered at delivery, the amniotic membrane was stripped from overlying allantois and digested with trypsin and collagenase to isolate epithelial (amniotic epithelial cells [AECs]) and mesenchymal cells, respectively. Proliferation, differentiation, and characterization studies by molecular biology and flow cytometry were performed. Histological examination revealed very few mesenchymal cells in the stromal layer, and a cellular yield of AECs of 10 × 10(6)/gram of digested tissue was achieved. AECs readily attached to plastic culture dishes displaying typical cuboidal morphology and, although their proliferative capacity decreased to the fifth passage, AECs showed a mean doubling time of 24.77 ± 6 h and a mean frequency of one fibroblast colony-forming unit (CFU-F) for every 116.75 plated cells. AECs expressed mesenchymal stem cell (MSC) mRNA markers (CD29, CD166, CD90, CD73, CD117) and pluripotent markers (Nanog and Oct 4), whereas they were negative for CD34 and MHCII. Mesodermic, ectodermic, and endodermic differentiation was confirmed by staining and expression of specific markers. We conclude that porcine amniotic membrane can provide an attractive source of stem cells that may be a useful tool for biomedical research.

Occurrence of immune cells in the intestinal wall of Squalius cephalus infected with Pomphorhynchus laevis.

A sub-population of 34 specimens of chub, Squalius cephalus, was sampled from the River Brenta (Northern Italy) and examined for ecto- and endo-parasites. Pomphorhynchus laevis (Acanthocephala) was the only enteric helminth encountered. Immunofluorescence and ultrastructural studies were conducted on the intestines of chub. Near the site of parasite's attachment, mucous cells, mast cells (MCs), neutrophils and rodlet cells (RCs) were found to co-occur within the intestinal epithelium. The numbers of mucous cells, MCs and neutrophils were significantly higher in infected fish (Mann-Whitney U test, p < 0.05). Dual immunofluorescence staining with the lectin Dolichos Biflorus Agglutinin (DBA) and the macrophage-specific MAC387 monoclonal antibody, with parallel transmission electron microscopy, revealed that epithelial MCs often made intimate contact with the mucous cells. Degranulation of a large number of MCs around the site of the acanthocephalan's attachment and in proximity to mucous cells was also documented. MCs and neutrophils were abundant in the submucosa. Immune cells of the intestinal epithelium have been described at the ultrastructural level and their possible functions and interactions are discussed.

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.

Responses of Squalius cephalus intestinal mucous cells to Pomphorhynchus laevis.

Intestinal mucous cell numbers and their glycoconjugate composition were investigated by histochemical methods in uninfected chub, Squalius cephalus, and in conspecifics naturally parasitised with the acanthocephalan Pomphorhynchus laevis. A sub-population of 42 chub from the River Tiber (Perugia, Italy) were sampled and screened for ecto and endoparasites. No parasites were found in gills and in other visceral organs of chub and P. laevis appeared to be the only enteric worm encountered. In all infected chub (twenty-eight out of 42) this acanthocephalan was encountered mainly in the mid-gut. In situ, an excessive yellowish mucus or catarrh was observed around each acanthocephalan. Hyperplasia and hypertrophy of the mucous cells were only evident near the site of P. laevis attachment where the total number of mucous cells and the number of those containing acidic, particularly non-sulphated mucins, or mixed glycoconjugates were significantly higher. In intestinal regions of infected fish far away from the point of parasite attachment, there were no statistical differences in the density of mucous cells in comparison to uninfected fish. Interestingly, in parasitised chub, the length of intestinal folds was significantly larger close to the sites at which P. laevis attach when compared to the length of the intestinal folds located further away from the acanthocephalans and/or in uninfected intestines. The effect of P. laevis on intestinal mucous cells of S. cephalus was compared to other parasite-host systems and the role of enhanced mucus production in parasitized intestines was discussed.

Follicular fluid leptin concentrations and expression of leptin and leptin receptor in the equine ovary and in vitro-matured oocyte with reference to pubertal development and breeds.

There is no published information about follicular-fluid leptin concentrations or the presence of leptin and leptin receptor in the equine ovary or oocyte. Three groups of mares - adult draft mares, draft fillies and adult Standardbred mares - were included in the study. Leptin and leptin receptor were detected in all immature oocytes by immunofluorescence with higher intensity in oocytes from draft mares compared with draft fillies and Standardbred mares. After in vitro maturation a higher proportion of oocytes reached metaphase II in draft mares than in draft fillies and Standardbred mares, and in all groups both leptin and leptin receptor became localised in the oocyte cortex but with higher immunopositivity in draft mares compared with draft fillies and Standardbred mares. These intensities were confirmed by the expression profiles of leptin and leptin receptor mRNA. Moreover, leptin was detected in ovarian blood vessels in all three types of animal and within the corpora lutea in adult mares. Serum and follicular-fluid concentrations of leptin were similar in draft and Standardbred mares but higher in draft mares than in draft fillies. This study supports the hypothesis that expression of leptin and leptin receptor mRNA and the rate of maturation can be related either to adiposity or to puberty.

Identification of C-kit-positive interstitial cells in the dog lower urinary tract and relationship with smooth muscle and nerves. Hypotheses for a likely pacemaker role.

The aim of this work was to give an evidence of the likely presence of interstitial cells in the canine lower urinary tract and to study their possible interactions with the musculature and the intramural innervation. Cryosections of normal canine bladder and urethra were immunofluorescently labelled with c-kit, a transmembrane, tyrosine kinase growth factor receptor, known to be expressed on the interstitial cells of Cajal (ICCs) of the gut. The relationship with antiactin positive smooth muscle cells and PGP9.5-positive intramural innervation was also investigated by confocal microscopy. Anti-c-kit labelling demonstrated a network of elongated and branched c-kit positive cells, which were located in interstitial spaces, oriented in parallel to the smooth muscle bundles that form the bladder muscular layer, irrespective of dog sex. Cells with a similar localization were also PAS- and NADPH-diaphorase-positive. A contact between c-kit immunofluorescent cells and intramural innervation was demonstrated, too. The roles of interstitial cells might include regulation of smooth muscle activity of the bladder detrusor, integrating neuronal signals during urine storage and voiding.