Immunohistochemical detection of neuromodulators in the intestine of Salmo trutta L. naturally infected with Cyathocephalus truncatus Pallas (Cestoda)

Pathological and immunohistochemical investigations were carried out on the middle intestine of uninfected and parasitized brown trout, Salmo trutta L., from the Ceresone Channel in North Italy. Eighty-six brown trout were sampled by electrofishing, and 32 (37.2%) were infected with Cyathocephalus truncatus Pallas, 1781 (Cestoda). The intensity of infection ranged from 1 to 82 parasites per host and the most infected segments were the anterior (near the pyloric caeca region) and the central part of the middle intestine. Immunohistochemical tests were applied on sections of intestinal tissue of healthy and infected fish, and the presence of substance P (SP), calcitonin gene related peptide (CGRP), met-enkephalin, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY) and serotonin (5-HT) was documented. Endocrine epithelial cells of the tunica mucosa were positive to SP-, CGRP-, met-enkephalin-, and NPY-like peptides and 5-HT antisera; moreover, a higher number of these cells were recorded in the intestine of infected trout in comparison to uninfected fish. In addition, in parasitized S. trutta , SP-like and 5-HT immunoreactivities were found in likely immuno-related cells of the tunica propria-submucosa. Nerve cell bodies and terminals in the myenteric plexus were immunoreactive to almost all the tested peptides and 5-HT antisera. These data provide evidence for the role of the neuroendocrine system of S. trutta in the modulation of inflammatory responses to C. truncatus . Results are discussed with respect to a peptidergic involvement and host immune response to an intestinal tapeworm.     

An immunohistochemical study on endocrine cells in the neuroendocrine system of the digestive tract of milkfish Chanos chanos (Forsskal, 1775)

In this study, several kinds of endocrine cells which produce neuropeptides and hormones have been identified and located in the digestive tract of milkfish (Chanos chanos) by the immunohistochemical technique. The oesophagus, stomach (cardiacus, fundic and pyloric regions), pyloric caeca, intestine (anterior, middle and posterior regions) and rectum were divided from the digestive tract of milkfish. The distribution and frequency of the endocrine cells produced these neuropeptides and hormones including gastrin, cholecystokinin, leu‐enkephalin, neuropeptide Y and somatostatin were described in these segments. Endocrine cells containing all neuropeptides and hormones immunoreactivities were present in the anterior intestine region, but no reaction was found in oesophagus. Only a few L‐ENK‐ and SST‐immunoreactive cells were detected in the pyloric caeca. Large numbers of SST‐immunoreactive cells were observed in the stomach with no immunoreactivities to other neuropeptides and hormones. Two different types of endocrine cells were founded in the intestine and pyloric caeca. These results indicate that the anterior intestine region may be the main region of food digestion and absorption, and the neuropeptides and hormones regulate the food intake process by different ways in milkfish.     

Influence of enteric helminths on the distribution of intestinal endocrine cells belonging to the diffuse endocrine system in brown trout,Salmo trutta L

The presence of intestinal helminths in the alimentary canal of brown trout, Salmo trutta L., can alter the number of cells that synthesize modulatory peptides. A total of 167 brown trout were collected from tributaries of the River Brenta (northern Italy), of which 119 (71.3%) specimens were infected with enteric helminths, 28 with the acanthocephalan Pomphorhynchus laevis Müller, 1776 with intensity of infection ranging from 1 to 162 (18.57 +/- 30.79) worms per host and 67 fish with the cestode Cyathocephalus truncatus Pallas, 1781. Intensity of infection with C. truncatus ranged from 1 to 85 (6.87 +/- 12.59) per fish. In 24 fish there were concurrent infections of both species of helminths. The caecal and middle regions of the intestine were the most heavily parasitized. Immunohistochemical tests showed a decrease in endocrine cells (ECs) of the diffuse endocrine system (DES) positive to gastrin, cholecystokinin-8, bombesin and secretin antisera in the intestine of the infected trout. The number of ECs immunoreactive to anti-glucagon serum did not show differences in the digestive tract of uninfected brown trout and in conspecifics parasitized with P. laevis. The density of cells containing glucagon-like material was low in the fish parasitized with C. truncatus. The results suggest that endoparasitic helminths induce alterations in the DES of infected S. trutta.     

Expression of insulin-like growth factor-I in the gastrointestinal tract of Atlantic salmon (Salmo salar L.)

The expression of insulin-like growth factor (IGF-I) in the gut of Atlantic salmon (Salmo salar L.) smolt was investigated at the cellular level using in situ hybridization. A hybridization protocol was adapted for use on formalin fixed, paraffin embedded tissues and was based on a digoxigenin-labelled oligoprobe that recognised a conserved region of IGF-I mRNA. The specificity of the anti-sense IGF-I probe and the performance of the hybridization protocol were evaluated using a sense IGF-I probe, an Ep stein-Barr virus probe and an insulin probe cocktail on serial sections of fish gut, mouse thyroid and human lymph node tissue. In the gut of Atlantic salmon smolt, IGF-I was found to be expressed in single epithelial cells or small clusters of epithelial cells in the pyloric ampulla and in the pyloric caeca. Expression was not detected in the lamina propria of the gut or in epithelial cells of the stomach, midgut or hindgut. The restriction of IGF-I expression to the pyloric ampulla and pyloric caeca is consistent with the role of these gut segments in osmoregulation and the scattered epithelial expression supports the putative autocrine/paracrine mechanism of action of IGF-I in osmoregulation.     

Genetic variation of body size, condition and pyloric caeca number in juvenile brown trout, Salmo trutta L.

Parental and individual variance components of body length, weight, condition (estimated as the second principal component of the length–weight relationship) and pyloric caeca number were investigated in 6‐month‐old brown trout (Salmo trutta L.) by the mean of two sib analyses, which provided consistent results. The average heritabilities (±SE) were 0.12 (±0.08) for length, 0.16 (±0.08) for weight, 0.47 (±0.14) for condition and 0.38 (±0.12) for pyloric caeca number. Maternal effects were also observed, although short of significance, in length, weight and caeca number. Correlations between caeca number and body size averaged +0.10 among individuals within lots, but genetic correlations were negative, i.e. about ?0.9 for length and ?0.7 for weight. There was no significant correlation between caeca number and condition. These results lead to question the role that pyloric caeca may play in growth, as well as their usefulness in fish breeding.     

Histopathological changes induced by the digenean intestinal parasite Masenia nkomatiensis Dumbo,Dos Santos, & Avenant‐Oldewage, 2019 of the catfish Clarias gariepinus (Burchell) from Incomati Basin,Mozambique

The intestines of 154 Clarias gariepinus were examined of which 29 were naturally infected with Masenia nkomatiensis, and of these, seven (intensity ranging from 8 to 231) were examined for pathology. Destruction of the epithelium covering the villi, detachment of epithelial cells and parts of villi were observed. Excessive mucus secretion occurred in the vicinity of the worm and catarrh was observed, indicative of an inflammatory response. The number of mucous and mast cells was higher at the attachment site than at an area 5,000 µm away and in uninfected individuals, suggesting that the parasite triggered a localized innate immune response. The number of neutrophils, basophils and lymphocytes in infected tissue was not significantly different from uninfected tissue confirming that no acquired immune response was produced against the maseniid. The caecae in the anterior part of the parasites' intestine consisted of convoluted epithelium forming invaginations or “crypts.” Contraction of the thick layer of circular muscle fibres of the caeca facilitates the movement of digested material. Observation of digested host cells and cell debris within the caecae provides further evidence that M. nkomatiensis is consuming host cells.     

Scanning and transmission electron microscopical observations on Hexamita salmonis (Moore, 1922) related to mortalities in rainbow trout fry Salmo gairdneri Richardson

Abstract. Examination of the digestive tract of rainbow trout fry infected with Hexamita salmonis revealed no pathological changes in the epithelial cells of the pyloric caeca or upper intestine even where the parasites were closely apposed to the brush border of these cells. No invasion of the epithelium by the parasites was observed, although examples of shrinkage necrosis (apoptotic bodies) of epithelial cells were often found. The ultrastructure of the parasite resembled that of the closely related Giardia lamblia . Despite the lack of pathological changes to the intestinal epithelium, treatment of the fish to remove the parasites greatly reduced mortalities.     

Selected pathological, immunohistochemical and ultrastructural changes associated with an infection by Diphyllobothrium dendriticum (Nitzsch, 1824) (Cestoda) plerocercoids in Coregonus lavaretus (L.) (Coregonidae)

The pathological changes induced by an infection of Diphyllobothrium dendriticum (Nitzsch, 1824) plerocercoids in powan, Coregonus lavaretus (L.), from Loch Lomond, Scotland, were assessed using immunohistochemical and ultrastructural techniques. In a sample of 26 powan, the occurrence of encysted plerocercoids of D. dendriticum on the outer surface of the stomach was 38.5% (n = 10) with the number of cysts ranging from 4 to 15 and measuring 4.2 +/- 1.0 mm x 3.4 +/- 0.9 mm (mean +/- SD). Histological examination of intestinal samples also revealed plerocercoids (2-21) encapsulated within a proliferation of mesenteric fibrous tissues of the gastric wall and, occasionally, by the gut lamina propria-submucosa and lamina muscularis. In section, cysts were tri-layered and were formed from a series of concentric whorls of fibroblast and collagen fibre-based connective elements. The extent of necrosis within each muscle layer and the serosa of the stomach differed, notably within the latter that was marked by a chronic inflammatory reaction and fibrosis. Within the cyst and around it, a large number of degranulating mast cell/eosinophilic granule cells were seen, in addition to melano-macrophage centres. Immunohistochemical staining of sections of infected stomach revealed a high density of elements, in close proximity to plerocercoids, staining positive for serotonin, bombesin, substance P and galanin. Uninfected material did not present the same levels of activity. Sections through both infected and uninfected tissue were also tested for elements containing vasoactive intestinal peptide, met-enkephalin, calcitonin gene-related peptide, neuropeptide Y and nitric oxide synthase, but these were absent.     

Composition of the microbial communities in the gastrointestinal tract of perch (Perca fluviatilis L. 1758) and cestodes parasitizing the perch digestive tract

Using the approach of sequencing the V3-V4 region of the 16S rRNA gene, we have analysed the bacterial diversity associated with the distinct compartments of the gastrointestinal tract of perch (Perca fluviatilis) and cestodes (Proteocephalus sp.) parasitizing their digestive tract. The dominant microbiota associated with cestodes (Proteocephalus sp.) was represented by bacteria from the genera Serratia, Pseudomonas and Mycoplasma. By comparing the associated microbiota of perch and cestodes, a clear difference in bacterial composition and diversity was revealed between the community from the stomach content and other parts of the gastrointestinal tract of fish. Microbiota associated with cestodes was not significantly different in comparison with microbiota of different subcompartments of perch (mucosa and content of intestine and pyloric caeca) (ADONIS, p > .05) excluding microbiota of stomach content (ADONIS, p ≤ .05). PICRUSt-based functional assessments of the microbial communities of perch and cestodes indicated that they mainly linked in terms of metabolism and environmental information processing and could play an important role in the nutrition and health of host.     

Infection of the epaulette shark, Hemiscyllium ocellatum (Bonnaterre), by the nematode parasite Proleptus australis Bayliss (Spirurida: Physalopteridae)

Seventy-two epaulette sharks, Hemiscyllium ocellatum (Bonnaterre), were infected with the nematode parasite Proleptus australis Bayliss, 1933. The parasite population was overdispersed. Infection intensity ranged from 3 to 1002 worms per fish stomach, and there was a positive correlation between shark length and number of parasites present. The majority of worms were attached to the stomach wall, and scanning electron microscopy and histological examination showed that worms penetrated the stomach lining. Worms were observed within the lamina propria of the stomach and occasionally penetrated the muscularis mucosa. Little to no inflammatory or cellular immune reaction to the presence of the parasites was observed, except in one case where a worm was being degraded by a host tissue response. There was a large amount of connective tissue proliferation as a result of nematode attachment, but no obvious effects on the overall health of the sharks were seen. Three sharks were also found to be infected by the cestode Callitetrarhynchus sp.     

Morphometric and histochemical assessment of the branchial tissue response of rainbow trout, Oncorhynchus mykiss (Walbaum), associated with chloramine-T treatment

Twelve tanks of juvenile rainbow trout, Oncorhynchus mykiss (Walbaum), were treated twice weekly for an 11- week period with a one-hour static bath of chloramine-T (10 mg l^–1). The morphometric indices of gill damage and the histochemical characteristics of gill mucous cells of fish from treated and paired untreated tanks were compared. The use pattern of chloramine-T in this study evoked a slight increase in mean lamellar width, but it did not induce a greater degree of lamellar oedema, lamellar fusion, tissue infiltration, epithelial hyperplasia, chloride cell metaplasia or thrombosis of pillar channels in treated fish. Treatment caused a trend towards an increased number of mucous cells on lamellae, associated with a significant shift from neutral mucin to acid mucin production based on histochemical characteristics.     

Effect of buffers on the gill structure of common carp, Cyprinus carpio L., and rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. The effects of phosphate and tris buffers on the gill structure of common carp, Cyprinus carpio L, and rainbow trout, Oncorhynchus mykiss (Walbaum), were investigated. Common carp maintained in phosphate-buffered (a combination of sodium di-hydrogen phosphate and di-sodium hydrogen phosphate at a final concentration of 0.01 m ) standard dilution water for a period of 8 days developed severe pathological changes in the gill tissues, while carp maintained in tris-buffered (0.01 m ) dilution water and trout subjected to either of these buffers showed no such changes. Histological changes in the gills of carp exposed to phosphate buffered dilution water included an extensive mucous cell proliferation on the lamellar and filarnental epithelium, and an increased infiltration of eosinophilic granular cells into the gill tissues. A moderate epithelial hyperplasia and an increase in the chloride cell densities on the gill epithelia were also noted. The implications of these findings on the widely accepted practice of the use of phosphate salts and various other buffers in pH control of experimental water are highlighted.     

不同规格瘤背石磺胃盲囊组织学初步研究

通过组织学观察与组织化学染色,研究了野生瘤背石磺不同体重(2～25 g)与胃盲囊的组织结构和PAS阳性细胞及肥大细胞的分布关系,为进一步探讨瘤背石磺消化生理和食性提供理论依据。结果发现:瘤背石磺胃盲囊组织主要由浆膜层、粘膜下层、固有膜和粘膜上皮层构成。瘤背石磺胃盲囊绒毛主要有两种类型,分别为非纺锤型绒毛和纺锤型绒毛,其中非纺锤型绒毛比例有随瘤背石磺体重增高而降低的趋势。胃盲囊横截面积、绒毛长度和粘膜上皮高度均有随瘤背石磺体重增高而增高的趋势,其中尤以低体重组(<8 g)和高体重组(>18 g)差异明显(P<0.05)。经PAS染色和肥大细胞染色后,发现仅在高体重组(>18 g)瘤背石磺胃盲囊绒毛粘膜上皮有大量的PAS染色阳性细胞和肥大细胞分布。     

Host-parasite interface of Caryophyllaeus laticeps (Eucestoda: Caryophyllidae) in three species of fish

Abstract. The host-parasite interface of Caryophyllaeus laticeps has been investigated in naturally infected bream, Abramis brama (L.), chub, Leuciscus cephalus (L.), and roach, Rutilus rutilus (L.), by light and electron microscopy. The fish were caught in the Ruhr-lake near Bochum. Bream showed the highest rate of infection and also harboured the largest worms compared to the two other host species. In all three cyprinids, the worms' scolices caused local compression of the host's intestinal epithelium. At the attachment site of scolices of medium- to large-sized worms, the intestinal epithelial cells were vacuolized and their brush border ruptured. These pathological effects were most pronounced in bream, which also showed conspicuous granulomas in the tunica propria adjacent to the scolices. A heavy infiltration of eosinophilic granulocytes was noted in chub and roach at the worms' attachment site. In bream, only the smallest worms provoked a slight eosinophilic reaction. It is concluded that the worms within the three host species were of different ages and that the chronic stage of infection, associated with the formation of granulomas, was only reached in bream, the major host.     

Light and electron microscopic observations of sporogenesis in the myxosporida, Ceratomyxa shasta (Noble, 1950)

Abstract. The stages of development leading to sporogenesis of Ceratomyxa shasta (Noble) were investigated by light and electron microscopy. Salmonid fishes were infected by exposing them to water containing the infectious stage and intestinal material was fixed at weekly intervals. Signs of intestinal infection were barely detectable by 7 days following exposure where trophozoites and later developmental forms were present, but by 14 days a large number of pansporoblasts could be detected in varying stages of development. By 21 days the majority of caeca were completely occluded and infection had spread throughout the connective tissues attached to the caeca.
The early developing trophozoites contained two or more nucleated cells within a mother cell. There was some evidence of multiplication of nuclei by fission. The sporoblasts usually contained twelve nucleated cells that gave rise to two groups of six cells (sporonts) and resulted in the formation of two spores in each mother cell. Each spore was formed by two sets of bilaterally arranged cells consisting of the main germinative cell or sporoplasm, the anteriorly placed capsule cells and the outer envelope or spore valve cell that surrounded the others and formed the spore covering.
As the spore matured the two germinative cells interacted with each other by pseudopodial extensions and appeared to fuse to form a diploid cell. The position of the cells laterally and slightly posteriorly to the central suture line formed a bilaterally curved spore. Mature spores when examined with the electron microscope were condensed, dark staining and relatively featureless, with a lateral measurement of 15 μm and an anterior-posterior measurement of 7μm.     

鲻鱼胃肠道内分泌细胞免疫组织化学的定位

用7种哺乳类胃肠激素抗体对鲻鱼消化道内分泌细胞进行免疫组织化学定位。结果表明5-HT、SST、VIP、GAS和P物质免疫活性内分泌细胞均存在于鲻鱼胃肠粘膜中，而胰高血糖素和胰岛则显免疫阴性反应。文中还描述了鲻鱼胃肠道免疫活性内分泌细胞的形态学特点及在胃肠各部的分布密度，并对其可能的分泌方式和功能进行了讨论。     

Recruitment and effects of Discocotyle sagittata (Monogenea) infection on farmed trout

Farmed rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta were monitored over 3 years for infection with the blood-feeding gill fluke Discocotyle sagittata. Parasite transmission is seasonal: new infections take place during summer/autumn, and transmission is generally negligible during winter/spring. There are 2 sources of infection for naïve fish-of-the-year: limited invasion when fish are in the raceways by riverborne larvae originating external to the farm; and internally, within the farm, when 0+ fish are transferred to ponds previously occupied by older cohorts of infected fish. Thereafter, infection levels continue to increase in rainbow trout primarily through transmission within the farm. Prevalence rose to 100% in 1+ fish by the end of their second summer. In O. mykiss, mean abundance reached 194 worms/host for 1+ fish (up to 489 worms/host) and 160 worms/host for 2+ fish. By contrast, in S. trutta, parasite prevalence never exceeded 85% and, after the first year's invasions, infection levels decreased over time: in 1+ and 2+ brown trout, parasite mean abundance was < 4 (maximum 15) worms/host. We present evidence of the detrimental effects of D. sagittata on the host: high burdens are associated with pale gills, decreased body condition and host mortality. Parasite burdens become overdispersed during the warmer part of the year, as prevalence and mean abundance increase. However, the degree of parasite overdispersion decreases over winter; we cannot distinguish whether decreased aggregation is due to parasite losses from infected fish (including immune-mediated parasite mortality) or parasite-induced host mortality.     

Time after seawater transfer influences immune cell abundance and responses to SAV3 infection in Atlantic salmon

Pancreas disease (PD) caused by salmonid alphavirus (SAV) severely affects salmonid aquaculture during the seawater phase. To characterize immune cells in target tissues for SAV infection, heart, pancreas and pyloric caeca were analysed from two groups of fish adapted to seawater for 2 and 9 weeks. The sections were scored for the relative abundance of cells expressing MHC class II, IgM, CD3, CD8 or neutrophil/granulocyte markers using immuno‐histochemical techniques. In general, necrosis of tissue was more severe in fish infected at 2 weeks post‐seawater transfer (wpt) compared with those infected at 9 wpt. At 9 wpt, there were higher numbers of MHC II⁺ cells in heart, pancreas and pyloric caeca, IgM⁺ cells in heart and pancreas, and CD3⁺ cells in pancreas compared to those infected at 2 wpt. The majority of the immune cells infiltrating PD‐affected tissues were MHC II⁺ and CD3⁺ cells suggesting that antigen‐presenting cells and T lymphocytes are the main types of immune cells responding to SAV infection. All the investigated cell types were also observed in pyloric caeca of infected fish, suggesting that this tissue may play a role in the immune response to SAV.     

Replication of the infectious salmon anaemia virus (ISAV) in rainbow trout, Oncorhynchus mykiss (Walbaum)

Infectious salmon anaemia (ISA) is a viral disease of farmed Atlantic salmon, Salmo salar L., in Norway. However, in laboratory experiments, the virus has also been found to propagate in trout, Salmo trutta L., where it is a persistent infection. It is crucial for the management of ISA in Norway, and for the prevention of spread to fish in other countries, that possible carriers of the virus are found. Another possible salmonid reservoir species is the rainbow trout, Oncorhynchus mykiss (Walbaum). After experimental infection, the ISA virus was found to be present in O. mykiss as late 28 days after infection, with a peak around day 20. The infection resulted in a significant drop in the haematocrit and haemorrhages on the liver in some specimens. The ISA virus was seen budding from endothelial cells lining blood vessels in the heart ventricle of challenged rainbow trout. It is concluded that the ISA virus is able to propagate in O. mykiss and that this species may function as a reservoir when cultured in areas where the virus is found. The virus does not seem to cause any significant mortalities of infected rainbow trout.     

虹鳟肠炎红嘴病病理模型的构建

为探讨鲁氏耶尔森菌侵染虹鳟的致病机制,本实验建立了鲁氏耶尔森菌感染虹鳟引起的肠炎红嘴病的病理模型,制定相应的临床症状及组织病理学评分系统,并对该模型进行研究。将43尾平均体质量约为12 g的健康虹鳟随机分成5组:3个实验组(n=30)、对照组(n=10)和哨兵组(n=3)。3个实验组分别采用2.0×106、2.0×107和2.0×108 CFU/m L的鲁氏耶尔森菌感染浓度,通过腹腔注射方式进行人工感染试验。对感染鲁氏耶尔森菌的虹鳟肠、肝脏、脾脏和肾脏组织进行镜检及临床症状、剖检病变判断,结合细菌学检测,按制定的评分系统评价各组肠炎红嘴病造模效果,确定最佳造模方案。结果显示,各攻毒组虹鳟感染后72 h均出现不同程度死亡,临床症状表现为红嘴、肛门红肿、鳍(胸鳍、腹鳍、臀鳍)等出现不同程度充血,下颌部、腹部出现出血点等。组织病理学可见肝脏、脾脏、肾脏及肠组织均有炎性细胞浸润现象出现,肝细胞、肠上皮细胞和肾小管上皮细胞等实质细胞变性、坏死,脾脏部位淋巴细胞减少、红细胞死亡堆积。综合各组得分发现,2.0×107 CFU/m L组的鲁氏耶尔森菌感染虹鳟造模效果最佳,患病虹鳟的临床症状显著且组内差异较小,病程迁延较长,便于研究。研究表明,对体质量约12 g的虹鳟幼鱼腹腔注射0.1 m L浓度为2.0×107 CFU/m L的鲁氏耶尔森菌可成功构建肠炎红嘴病病理模型。