Changes in the neuromodulators of the diffuse endocrine system of the alimentary canal of farmed rainbow trout, Oncorhynchus mykiss (Walbaum), naturally infected with Eubothrium crassum (Cestoda)

A histopathological and immunohistochemical study on the intestines of 45 specimens of farmed rainbow trout, Oncorhynchus mykiss (Walbaum), from Loch Awe, Scotland, revealed a number of cellular deviations in individuals naturally infected with the pseudophyllidean cestode Eubothrium crassum (Bloch, 1779). Twenty-five individuals (55.5%) were infected with an average worm burden of 18.84 +/- 4.06 (mean +/- SE) cestodes per host (range, 2-80 worms; total 471 worms). The cestodes, measuring an average 8.23 +/- 1.10 cm (mean +/- SE; range, 5.3-13.0 cm) in length, were found attached by their scolices to the mucosal lining of the distal portion of the pyloric caeca. Within the caeca, the strobila evoked a mild catarrhal enteritis, namely an enhanced mucus production with epithelial cellular desquamation, a leucocytic infiltration of the lamina propria-submucosa and vacuolization of the intestinal epithelial cells. Eosinophilic granular cells of the stratum granulosum exhibited granular depletion, while within the catarrh, the presence of a high number of rodlet cells was noticed. Immunohistochemically, the occurrence of E. crassum caused a significant reduction in the number of bombesin-, gastrin-releasing peptide and glucagon-like immunoreactive endocrine cells, but an increase in the relative densities of endocrine cells containing cholecystokinin-8- and gastrin-like substances. There were, however, no significant differences in the number of endocrine cells that were immunoreactive to secretin, neuropeptide Y and peptide histidine-isoleucine antisera in the digestive tracts of either the infected or non-infected O. mykiss.     

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.     

Powan, Coregonus lavaretus (L.), ova predation by newly introduced ruffe, Gymnocephalus cernuus (L.), in Loch Lomond, Scotland

Abstract. Loch Lomond, one of the few British strongholds of the powan, Coregonus lavaretus (L.), has recently been colonized by the ruffe, Gymnocephalus cernuus (L.). The ruffe are now widespread, abundant and one of the commonest fish in Loch Lomond. Analysis of the gut contents of these fish caught on powan spawning grounds in Loch Lomond showed that they, as well as native brown trout, Salmo trutta L., and powan themselves, prey upon powan ova. Ruffe maintained a high winter feeding rate compared with powan and trout and fed on a broad range of benthic prey, of which powan ova formed the greatest biomass (84% of total diet) and dominated the diet numerically (57% of prey items). The observed incidence of powan ova consumption by ruffe was significantly greater than that of both brown trout and powan (P <0·001).     

Melanotic visceral fibrosis associated with larval infections of Posthodiplostomum minimum and Proteocephalus sp. in bluegill, Lepomis macrochirus Rafinesque, in central Iowa, U.S.A.

Abstract. Severe melanotic liver and visceral fibroses in bluegill associated with infections of plerocercoids of Proteocephalus sp. and metacercariae of the digenean Posthodiplostomum minimum , were studied by light and transmission electron microscopy. Fibrosis was most evident in bluegill carrying heavy worm burdens. Tubular and multilocular melanotic and non-melanotic cysts were formed of concentric layers of non-cellular eosinophilic connective tissue surrounding central spaces containing basophilic material. Moribund and deteriorating plerocercoids and metacercariae were surrounded by, and sometimes invested with, fibrous tissue. Living plerocercoids in the liver were surrounded by necrotic and compressed hepatocyte zones. Fibro-blasts, epithelioid cells and eosinophilic granulocytes were common in compressed cell zones. Intrahepatic fibrotic cysts were surrounded by granulomatous zones. Parenchyma in fibrotic livers showed increased numbers of macrophages, melano-macrophage centres and engorged blood vessels with thickened walls. Histochemical tests for lipofuscin and haemosiderin were strongly positive in melanotic liver tissue. Ultrastructural changes in hepatocytes adjacent to fibrotic cysts and epithelioid zones included intranuclear lipid and glycogen inclusions, chromatin alterations and mitochrondrial degeneration. Bile canaliculi, sinusoids and perisinusoidal spaces showed reduced micro villarsurfacesand a decrease in luminar diameter. Degenerating hepatocytes contained phagolysosomes and myelin bodies.     

Cutaneous myxidiosis in European eel,Anguilla anguilla (Linnaeus, 1758): histopathology,histochemistry and laminin immunohistochemistry

Histopathology, histochemistry and immunohistochemistry of the integument of European eel, Anguilla anguilla (Linnaeus, 1758), infected by Myxidium sp. are reported. Skin samples from affected and unaffected eels were dissected, formalin fixed, paraffin embedded, sectioned and stained with H&E, Periodic acid–Schiff's staining method, Alcian Blue 8 GX pH 2.5/Periodic acid–Schiff's and McCallum‐Goodpasture's Gram stain. Moreover, immunohistochemistry was performed using a primary polyclonal laminin antibody. Histologically, cysts (diameter 2–3 mm) were observed mainly under the scale pockets, encircled by a thin collagen layer, lined by elongated, flattened fibroblasts and containing bipolar, PAS‐ and Gram‐positive spores with opposite polar capsules. The epidermis stretched by the underlying cyst appeared dysplastic, thinned with a significant reduction in mucous cells number. Only inconsistent and aspecific inflammatory reaction was noted around the cysts at the dermis/epidermis interface. Intense laminin‐like protein immunolabel was documented in the plasmodial ectoplasm and related to host anergia. This was the first report of laminin immunolabel in a member of the Myxozoa. Epidermal dysplasia represents likely an aspecific response against the underlying tensile force exerted by the developing parasite cyst, while fibroblast and collagen encapsulation denote a parasite‐driven host response protecting, rather than harming, the encircled parasite.     

Oral transmission as a route of infection for viral haemorrhagic septicaemia virus in rainbow trout, Oncorhynchus mykiss (Walbaum)

Surveys among wild marine fish have revealed occurrence of viral haemorrhagic septicaemia virus (VHSV) infections in a high number of diverse fish species. In marine aquaculture of rainbow trout, preying on invading wild fish might thus be a risk factor for introduction and adaptation of VHSV and subsequent disease outbreaks. Our objective was to determine whether an oral transmission route for VHSV in rainbow trout exists. Juvenile trout were infected through oral, waterborne and cohabitation transmission routes, using a recombinant virus strain harbouring Renilla luciferase as reporter gene. Viral replication in stomach and kidney tissue was detected through bioluminescence activity of luciferase and qRT-PCR. Replication was detected in both tissues, irrespective of transmission route. Replication patterns, however, differed among transmission routes. In trout infected through oral transmission, replication was detected in the stomach prior to kidney tissue. In trout infected through waterborne or cohabitation transmission, replication was detected in kidney prior to stomach or in both tissues simultaneously. We demonstrate the existence of an oral transmission route for VHSV in rainbow trout. This implies that preying on invading infected wild fish is a risk factor for introduction of VHSV into marine cultures of rainbow trout.     

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.     

Study of the distribution of active caspase‐3‐positive cells in turbot,Scophthalmus maximus (L.), enteromyxosis

Enteromyxosis caused by Enteromyxum scophthalmi is one of the parasitizations with a higher economic impact on turbot, Scophthalmus maximus (L.), aquaculture. This myxosporean produces severe catarrhal enteritis with abundant inflammatory infiltrates in the lamina propria‐submucosa (LP), epithelial detachment and leucocyte depletion of the lymphohaematopoietic organs. Some advances made on the pathogenesis pointed to a role of apoptosis in the enteromyxosis. Therefore, the main aim of this work was to employ the TUNEL assay and the anti‐(active caspase‐3) immunohistochemical assay to detect apoptotic cells in both healthy and E. scophthalmi‐infected turbot in order to establish the presence and distribution of apoptotic cells during development of the disease. More apoptotic cells located within the gastrointestinal epithelium were observed in the initial stages of the infection in E. scophthalmi‐infected turbot compared with non‐infected turbot. As the infection progressed, a higher degree of apoptosis occurred in the epithelium of folds heavily parasitized. In the severely infected turbot, apoptosis was also found among the leucocytes of the intestinal inflammatory infiltrates. Moreover, the number of active caspase‐3‐positive cells in the lymphohaematopoietic organs tended to increase with disease severity. In view of the results, increased apoptosis in the epithelium may favour the scaling that occurs during enteromyxosis and cell death of leucocytes in the intestinal LP, contributing to leucocyte depletion in severe cases.     

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.     

中国养殖斑石鲷(Oplegnathus puncatus)上皮囊肿病的发现及显微观察

2015年,山东省某养殖场工厂化养殖的斑石鲷(Oplegnathus puncatus)幼鱼(全长为15 cm左右)因病陆续死亡,15d内累积死亡率达40％以上.现场调查发现,发病池水温为21℃,盐度为30.患病鱼群散开、不聚集.病鱼身体侧偏,活力差,常贴底或者贴壁,严重者随着水流漂流.病鱼呼吸困难,口部持续张开,鳃盖开合频繁,对投喂的食物无反应.但病鱼反应灵敏,难以捕捉.临床检查和剖检可见病鱼鳃表面覆盖着大量黏液,鳃丝有损伤,肠道无食物.取病鱼鳃丝制成水浸片,在光学显微镜下观察,鳃丝上可见到许多直径约为30-70 μm的囊肿物,外观圆形或卵圆形,呈浅黄棕色.在苏木精-伊红染色的石蜡切片中,病鱼次级鳃丝末端粘连,许多上皮细胞膨大呈囊肿状.囊肿嗜碱性,内部均质化.在扫描电子显微镜下观察,病鱼鳃丝呈棍棒化,鳃小片被大量黏液覆盖,表面光滑的囊肿细胞镶嵌其间.通过上述疾病现场调查、病鱼的临床检查、鳃组织的光学显微镜和扫描电子显微镜病理观察,可以初步确定该病为斑石鲷上皮囊肿病.这是上皮囊肿病在中国养殖斑石鲷中首次被发现和记载.     

The production and characterization of monoclonal antibodies against Photobacterium damselae ssp. piscicida and initial observations using immunohistochemistry

Five monoclonal antibodies (MAbs: F2B1, 1E4, 13B10, 4D4 and F3G12) were produced against lysed Photobacterium damselae ssp. piscicida ( Ph. d . ssp. piscicida ). The MAbs recognized three antigens of differing molecular weight on the Western blot of Ph. d . ssp. piscicida . They also cross-reacted with five different species of Vibrio . An enzyme linked immunosorbent assay (ELISA) with MAbs, F3G12 and 4D4 demonstrated differences between Ph. d . ssp. piscicida and three Ph. d . ssp. damselae type strains, indicating differences in the surface antigenicity between these two groups of bacteria. Antigen retrieval in conjunction with immunohistochemistry (IHC) using MAb 13B10, revealed colonies of bacteria in the kidney, spleen and liver of sea bass, Dicentrarchus labrax , infected with pasteurellosis. A number of positive colonies were observed around the mucosal layers of the intestinal tissue, especially within the lamina propria. In addition, a number of bacterial colonies were associated with red blood cells and blood vessels of the organs examined.     

Infection of the yellow head baculo-like virus (YBV) in two species of penaeid shrimp, Penaeus stylirostris (Stimpson) and Penaeus vannamei (Boone)

Abstract. Yellow head baculo-like virus infection and disease were demonstrated experimentally in the two main species of penaeid shrimp cultured in Hawaii and the Western hemisphere. Viral infection was induced by intramuscular inoculation of a 10% suspension of cephalothorax tissue filtrate prepared from two tiger shrimp, Penaeus monodon Fabricius, infected with yellow head disease, into sub-adult (3–10g) P. stylirostris (Stimpson) and P. vannamei (Boone). Signs of disease appeared as early as 2 days post infection (p.i.), and in most cases mortality reached 100% within 5–7 days p.i. Histopathological examination of the infected animals revealed extensive cellular necrosis in ectodermal and some mesenchymal tissues. Electron microscopical examination of thin sections of the gill and hepatopancreas from the infected shrimp revealed non-occluded rod-shaped baculo-like virus particles measuring 130–197 & 45–58 nm which were primarily localized within the cytoplasm of infected cells. The virus particles were contained within cytoplasmic vacuoles, and occurred singly or in small groups of two or more particles.     

Histopathology, immunohistochemistry and ultrastructure of the intestine of Leuciscus cephalus (L.) naturally infected with Pomphorhynchus laevis (Acanthocephala)

The histopathology, immunohistochemistry and ultrastructure of the alimentary canal of chub, Leuciscus cephalus (L.), from the River Brenta, naturally infected with the acanthocephalan Pomphorhynchus laevis Müller, 1776, was studied and described. Of 62 chub examined, 54 (87%) were infected with P. laevis ; the intensity of infection ranged from five to 130 parasites per host, and a density of 8 P. laevis per cm² was common. Examination of histological material of infected chub revealed that both male and female acanthocephalans deeply penetrated all layers of the gut wall by means of their slender neck, bulb and proboscis. As a result, a capsule was formed around the bulb and proboscis on the external surface of the host intestine. In parasitized chub, four main types of reaction against the body of the acanthocephalan were recognized. Pomphorhynchus laevis caused local damage to the intestinal wall, eliciting catarrhal-erosive enteritis in the lumen and a fibroblastic-collagenous and fibro-epithelioid encapsulation in its thickness with tissue zonation according to the depth of parasite penetration. Furthermore, eosinophilic granular cells (EGC) within the inflammatory tissue were identified by immunohistochemical methods and transmission electron microscopy.     

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.     

Gastrointestinal histopathology of paddlefish, Polyodon spathula (Walbaum), infected with larval Hysterothylacium dollfusi Schmidt, Leiby & Kritsky, 1974

Abstract. Gastrointestinal histopathology of the paddlefish, Polyodon spathula (Walbaum), infected with larval nematodes, Hysterothylacium dollfusi Schmidt, Leiby & Kritsky 1974, was studied. Larval H. dollfusi invaded the gastric mucosa and caused ulcers with accompanying infiltration of eosinophilic granulocytes. The deeper penetration into the stomach and intestine caused the formation of capsular granulation tissues around the worms. Larval worms were also histologicahy studied. The intestinal epithelium of larvae contained glycogen, haemosiderin and ceroid, indicating their blood-feeding capability.     

The pathology of Haplorchis pumilio (Looss, 1896) infections in cultured tilapias

Abstract. Haplorchis pumilio (Looss, 1896) is a common digenean parasite of many species of fish of economic importance and has been found to infect cultured Sarotherodon spp. Fry of S. spilurus (Günther), S. mossambicus (Peters) and S. galilaea (L.) were experimentally infected with cercariae of H. pumilio from natural infections of Melanoides tuberculata (Müller) from an East African fish farm. Large numbers of cercariae were lethal to the fry within a few hours. Histopathological examination showed that the most significant pathological effect was focal haemorrhages in skeletal muscles in heavily infected fish. Migration of cercariae was through connective tissue and the final location of the metacercariae was connective tissue associated with skeletal structures. Clusters of cysts occurred at the bases of fins and in the loose connective tissue of the head, but the reaction to encysted metacercariae was insignificant. The overall effect on growth performance and thus economic importance of H. pumilio infection was assessed. Growth studies showed no significant differences between specific growth rate, food conversion ratios and condition of infected and uninfected fish.     

Ontogenetic diet shifts of age-0 smallmouth bass (Micropterus dolomieu Lacepède) in the New River, West Virginia, USA

Larval and juvenile smallmouth bass (Micropterus dolomieu Lacepède; 8.5–85.0 mm total length (TL)) were collected from 2 sites in the New River, West Virginia, in the late spring and early summer to document diet shifts during early ontogeny. The first foods of smallmouth bass (TL = 9 mm) were primarily Chironomidae and microcrustaceans (primarily Copepoda). Other aquatic insects, primarily Ephemeroptera, became more abundant in the stomachs as TL increased. A shift in the proportions and types of prey consumed began at approximately 15 mm TL and was primarily attributed to fin development and increased mouth size. Foraging success, measured by number of prey per stomach, stomach fullness, and average prey volume was lower for first-feeding smallmouth bass than for later life stages. Mouth width appeared to restrict the size of prey consumed by larval smallmouth bass, but the maximum prey width never approached the mouth width for fish longer than 35 mm TL.     

Epitheliocystis in silver perch, Bidyanus bidyanus (Mitchell)

Cultured juvenile silver perch, Bidyanus bidyanus (Mitchell), were affected by epitheliocystis, with a prevalence of up to 75%. The condition affected 2–96% of gill filaments. Granular basophilic cysts ranged from 10 to 87 μm and had a round-to-oval shape. The infected cells were positioned centrally within a lamella, often close to its tip, and their identity was difficult to determine, but the involvement of pillar cells could not be precluded. The size of pathogen was within the range previously reported. The pathogen had two morphological forms, which were positioned differently within the cyst. The form located more in the centre was rod-shaped with an electron-dense core and electron-lucent vesicles on the sides of the core. Its mean length was 498.14 nm (SD = 47.68 nm), and its mean width was 145.14 nm (SD = 9.53 nm). The form present in a more peripheral position had an irregular shape, often did not show the electron-dense core and did not have the electron-lucent vesicles. Its mean length was 704 nm (SD = 170.29 nm) and its mean width was 152.4 nm (SD = 16.40 nm). Both forms were enclosed by a double trilaminar membrane. This is the first confirmed report of epitheliocystis in Australian freshwater fish.     

Coccidiosis due to Goussia lomi (Protista: Apicomplexa) in aquarium-reared Murray cod, Maccullochella peeli (Mitchell), (Percichthyidae)

Abstract. Intestinal parasitism by the coccidian Goussia lomi was associated with morbidity and histological lesions in aquarium-reared Murray cod fry, Maccullochella peeli (Mitchell), at a hatchery near Narrandera, New South Wales, Australia. Coccidial infection was not found in pond-reared Murray cod of the same age, nor in trout cod, M. macquariensis (Cuvier & Valenciennes), reared under similar conditions at the same hatchery. Overcrowding may have predisposed Murray cod in aquaria to heavy infection with G. lomi. Clinical coccidiosis was expressed in a small proportion of infected fish. Large numbers of coccidial developmental forms were detected histologically in the intestinal mucosa of affected fish in association with a necrotic enteropathy. Using a method of intestinal trypsin digestion and flotation, 2000 coccidial oocysts were counted in one 0-05g fish.