Control of Salmincola californiensis (Copepoda: Lernaeapodidae) in rainbow trout, Oncorhynchus mykiss (Walbaum): a clinical and histopathological study

A stock of rainbow trout, Oncorhynchus mykiss, held at an experimental facility, was found to be heavily infested with the lernmaeapodid copepod Salmincola californiensis. The efficacy and effects of treatment were compared with ivermectin or manual removal of parasites as a means of control of S. californiensis. One group of fish was orally intubated with 0.2 mg ivermectin active ingredient kg-1 fish. A second treatment was administered after a further 14 days. In a second group of fish, parasites were manually removed from the gills using forceps. These fish were sampled for up to 21 days post-first removal of parasites. In the ivermectin-treated fish adult parasites became inactive and changed colour within 18 h of the initial treatment. Copepods began to disappear by day 3 post-treatment and by day 31 almost all embedded female parasites had disappeared. Gills were clinically normal apart from cavitation deformity resulting from parasite attachment. Post-ivermectin treatment, there was an increase in the number of eosinophilic granular cells surrounding the bulla of attached S. californiensis, but from day 31 post-treatment these were replaced by macrophages and epithelioid cells to form a necrotic focus. In manually picked fish there was extensive haemorrhage in the interlamellar spaces as a result of parasite removal. At sites of parasite removal tissue necrosis was minimal and healing was rapid. At the end of the sampling period the structure of the gill was improved. The use of oral dosage with ivermectin is an effective treatment for S. californiensis and could be particularly beneficial for use with endangered salmon broodstocks infested with the parasite.     

Ichthyophthiriasis in carp, Cyprinus carpio L.: fate of parasites in immunized fish

Abstract. The host-parasite relationship between O-group carp and the ciliatc Ichthyophthirius multifiliis Fouquet was investigated, with the specific aim of characterizing the fate of parasites encountering immunized fish. Carp were immunized by repeated controlled infections; immunized fish and control fish naive to I. multifiliis were then infected in the caudal fin epidermis by a single controlled exposure to theronts, which were applied in a droplet suspension to the tail surface. The number of parasites present within the caudal fin was monitored over a subsequent 5-day period by means of in situ parasite mapping. Results indicated that, contrary to previous reports, theronts penetrated the skin of immunized fish in numbers comparable to those of fish receiving a primary infection. However, the majority of parasites which penetrated immune skin did not complete normal development; 79% of the parasites which had initially penetrated the immune skin were not relocated within 2h of exposure, and since no parasite material was detected at penetration sites, it was concluded that these parasites had prematurely exited the skin rather than been killed in situ. Subsequently, these sites became populated by leucocytes, predominantly macrophages, and the infiltrations continued for up to 5 days after the initial exposure. In contrast, at sites where mature trophonts had exited the skin of fish following a primary infection, more diffuse leucocytic infiltrations were recorded, and these were predominated by neutrophils. Differences in the response to parasite exit from immunized and previously unexposed control fish skin are discussed, with particular reference to the mode of protection and the fate of parasites encountering immune fish.     

The process of infection, migration, growth and development of Sanguinicola inermis Plehn, 1905 (Digenea: Sanguinicolidae) in carp, Cyprinus carpio L.

Abstract. Sanguinicoliasis is a serious disease of cultured carp causing high mortality in early fry. Experimental infections were used to study the pattern of penetration, and subsequent behaviour and development of the parasite. Maximum penetration of cercariae was achieved within 30 min, followed by a period of rapid development and growth marked by a doubling in length within the first 12 h. Subsequent growth was slower, during which major organ development took place, with gonadal maturity being completed between 60 and 90 days p.i. Penetration occurred mainly through the fins, particularly the caudal fin, and large numbers of worms remained in the skin where they continued to develop to maturity. A small proportion of the worms were found in the heart and gill vessels soon after infection. However, migration from the skin began at 60 days p.i. and greater numbers (57%) of the worms were found in heart and gill vessels at 90 days, coinciding with onset of egg production. Decline in worm numbers in the gill/heart region began after 120 days. Gill/heart worms differed in a number of features from skin worms, but egg production occurred simultaneously in both sites.     

Bromodeoxyuridine DNA labelling reveals host and parasite proliferation in a fish‐myxozoan model

Enteromyxum leei is a myxozoan parasite responsible for enteritis in gilthead sea bream (Sparus aurata). The parasite proliferates in the paracellular space of the intestinal epithelium and induces an inflammatory reaction. To assess intestinal cell turnover and parasite proliferation, fish were infected with the parasite by anal intubation; after 17 and 64 days, the cell proliferative marker bromodeoxyuridine (BrdU) was administered; and after 24 hr, tissue samples were taken for immunohistochemical detection. Parasite exposure induced increased epithelial and immune cell proliferation in all intestinal segments at all time points, even before parasite establishment. This increased turnover was triggered early after intubation and mainly at a local level, as shown by an increased proliferating cell nuclear antigen (pcna) gene expression only at the posterior intestine after 17 days (not found in lymphohaematopoietic organs). Incorporation of BrdU in parasite secondary and tertiary daughter cells indicated that parasite endogeny is not by schizogonial division, which uses de novo synthesis pathway of pyrimidines. Altogether, BrdU immunolabelling and pcna gene expression showed the rapid proliferative response of the fish intestines upon a myxozoan infection and how this response is effectively triggered even before the parasite reaches or establishes in the site.     

一种虹彩病毒感染大菱鲆的病理学研究

对我国虹彩病毒感染的大菱鲆Scophthalmus maximus进行的组织病理和超微病理学研究发现,该病典型的病理学特点是在病鱼的脾脏、肾脏、肠、肝脏、鳃、心脏和皮肤等器官组织内出现嗜碱性的肿大细胞。病毒感染导致患病大菱鲆多个器官组织发生了不同程度的病理变化,其中以脾脏组织的病理变化最为显著,表现为造血组织的严重坏死。此外,肾脏造血组织发生坏死、肠固有膜和黏膜下层出血和水肿、肝细胞水样变性、心肌局灶性坏死以及皮肤真皮层出血并伴有水肿和炎性渗出也是该病常见的组织病理学变化。超微病理研究表明,肿大细胞内有虹彩病毒粒子存在。病毒分布于受感染细胞的胞质、组织间隙以及血管腔内。受感染细胞出现线粒体和内质网等细胞器肿胀、崩解等细胞病理变化。研究认为,病毒感染造成皮下组织血管损伤出血,是虹彩病毒感染的大菱鲆发生＂红体病＂的原因所在。虹彩病毒感染所致的机体严重贫血是患病大菱鲆死亡的主要原因,而主要器官组织的病变使得病鱼器官功能衰竭则可加速鱼的死亡。     

Observations on the intestinal pathology caused by three caryophyllid tapeworms of the white sucker Catostomus commersoni Laécpéde

Abstract. The structure of the parasite-host interface at the site of attachment was studied for Glaridacris catostomi, G. laruei and Hunterella nodulosa , caryophyllid tapeworms of the white sucker Catostomus commersoni . Light microscope observation of fish infected with H. nodulosa showed a loss of intestinal epithelium and lamina propria, and a chronic inflammatory response characterized by infiltration of lymphocytes and extensive hyperplasia of the submucosal tissue; intestinal pathology associated with G. catostomi and G. laruei was minimal. Electron microscope examination of the parasite-host interface revealed that in no case does parasite tissue come into direct contact with host tissue. H. nodulosa is separated from host tissue by an amorphous layer of electron lucent material (the 'eosinophilic matrix' seen with the light microscope) which appears to act as an adhesive; this material is a secretory product of the scolex glands. In G. catostomi and G. laruei a layer of material separating parasite from host tissue was also found, but it is not clear whether this layer is adhesive or protective.     

Characterization of a host response to the myxosporean parasite, Ceratomyxa shasta (Noble), by histology, scanning electron microscopy and immunological techniques

Abstract. The tissue response of Salmo gairdneri Richardson, against the myxosporean parasite. Ceratomyxa shasta (Noble), was investigated using histological techniques, scanning electron microscopy and immunological methods. The progress of infection in C. shasta -susceptible and resistant steelhead and rainbow trout was examined by standard histological techniques and by indirect fluorescent antibody methods using monoclonal antibodies directed against C. shasta antigens. Trophozoite stages were first observed in the posterior intestine and there was indication that resistance was due to the inability of the parasite to penetrate this tissue rather than to an inflammatory response. Examination of a severely infected intestine by scanning electron microscopy showed extensive destruction of the mucosal folds of the posterior intestine. Western blotting and indirect fluorescent antibody techniques were used to investigate the immunological component of the host response. No antibodies specific for C. shasta were detected by either method.     

Lymphocystis disease virus persists in the epidermal tissues of olive flounder, Paralichthys olivaceus (Temminch & Schlegel), at low temperatures

Olive flounder artificially infected with lymphocystis disease virus (LCDV) were reared at 10, 20 and 30 °C for 60 days, to compare LCD-incidence. In the fish reared at 20 °C, lymphocystis cells appeared on the skin and fins at 35 days post-challenge, and the cumulative LCD-incidence was 80% at 60 days. High levels of LCDV, with a mean polymerase chain reaction (PCR) titre of 10⁶ PCR-U mg⁻¹ tissue, were detected in the fins and skin of LCD-affected fish at 20 °C, but were not detected in the spleen, kidney, brain and intestinal tissues of these fish. No LCD clinical signs were observed in the fish reared at 10 °C and 30 °C; however, a low level of LCDV (10³ PCR-U mg⁻¹ tissue) was detected in the fins and skin of these fish. By increasing the rearing temperature from 10 to 20 °C, lymphocystis clusters appeared on the skin and fins of the fish with no previous LCD clinical signs within 33 days after the temperature change. It was shown that permissive cells for LCDV infection exist in the epidermis of olive flounder. At low temperatures, small amounts of LCDV were able to persist over a period extended for a further 45 days in the fish epidermis, even though the fish showed no LCD clinical signs. The optimum growth temperature of LCDV is near 20 °C.     

Outbreaks of ulcerative disease associated with ranavirus infection in barcoo grunter,Scortum barcoo (McCulloch & Waite)

In 2013, an outbreak of ulcerative disease associated with ranavirus infection occurred in barcoo grunter, Scortum barcoo (McCulloch & Waite), farms in Thailand. Affected fish exhibited extensive haemorrhage and ulceration on skin and muscle. Microscopically, the widespread haemorrhagic ulceration and necrosis were noted in gill, spleen and kidney with the presence of intracytoplasmic eosinophilic inclusion bodies. When healthy barcoo grunter were experimentally challenged via intraperitoneal and oral modes with homogenized tissue of naturally infected fish, gross and microscopic lesions occurred with a cumulative mortality of 70–90%. Both naturally and experimentally infected fish yielded positive results to the ranavirus‐specific PCR. The full‐length nucleotide sequences of major capsid protein gene of ranaviral isolates were similar to largemouth bass virus (LMBV) and identical to largemouth bass ulcerative syndrome virus (LBUSV), previously reported in farmed largemouth bass (Micropterus salmoides L.), which also produced lethal ulcerative skin lesions. To the best of our knowledge, this is the first report of a LMBV‐like infection associated with skin lesions in barcoo grunter, adding to the known examples of ranavirus infection associated with skin ulceration in fish.     

Wound‐healing potential of curcumin in the carp,Labeo rohita

In this investigation, the effect of dietary administration of curcumin on the healing of skin wound in fish, Labeo rohita, has been reported. Fish were divided into three groups: control group (fish without skin wound), sham group (fish with skin wound without curcumin treatment) and curcumin‐treated group (fish with skin wound and subjected to dietary administration of 1% curcumin). Experiments were conducted for 30 days to assess the healing of skin wounds at different time intervals using scanning electron microscopy, histology, and mucopolysaccharide and enzyme histochemistry. In the curcumin‐treated group, healing of skin wounds was found to be enhanced than in the sham group as indicated by early restoration of morphology of the surface layer of epithelial cells; the density of the mucous goblet cells; the density of club cells in epidermal layer; and early granular tissue formation, collagen deposition and tissue remodelling in dermal layer. Furthermore, peroxidase and catalase enzyme activity showed increased endogenous defence system in the curcumin‐treated group compared with the sham group. It could be concluded that dietary administration of curcumin is beneficial in rapid healing of skin wounds in fish. Early healing of wounds could be considered to prevent the invasion of pathogens and to maintain the integrity of the surrounding tissue.     

The histopathology of the eye of big head carp, Aristichthys noblis (Richardson), infested with Lernaea piscinae Harding, 1950

Abstract. The histopathological changes in the eye due to the penetration and growth of the parasite, Lernaea piscinae , in the host tissue of big head carp is described. Distinct pathological changes were hyperplasia of the corneal epithelium, severe inflammatory response mainly around the anchor of the parasite, in the anterior chamber, the corneal stroma and the vitreous body and detachment of the vitreous and retinal layers. Rupture of the lens capsule and fibrous tissue response around the process of the anchor structure of the parasite were also observed. The development of an immune response to the parasite within the body of the host is thought to have led to the infection of the cornea, an avascular site.     

The influence of Gyrodactylus salaris Malmberg 1957 (Monogenea) on the epidermis of Atlantic salmon,Salmo salar L., and brook trout,Salvelinus fontinalis (Mitchill): experimental studies

The effect of Gyrodactylus salaris on the epidermal structure of hatchery-reared brook trout parr and Norwegian Atlantic salmon parr was evaluated. Both species were initially susceptible to this parasite, but whereas populations on salmon increased until the host died, brook trout responded to, and eventually eliminated, their infections. Salmon skin samples taken 14 days p.i. showed a reduced mucous cell concentration (less than 1500 cells mm^–2 on the head compared with 2000 cells mm^–2 in controls; the same trend was also seen in other sites) and the epidermis was thinner (48 μm compared with 60 μm on pectoral fins; similar trend seen in other sites) than in uninfected controls kept under identical conditions. Brook trout skin samples were taken 50 days p.i., when the hosts had responded to, and almost eliminated, their infections. No change was then observed in mucous cell density, while the number of epidermal cell layers and the epidermal thickness of brook trout had increased slightly following infection. These results are related to the nature of the host response, and the thinning of the epidermis and loss of mucous cells may in some way be related to the inability of Norwegian salmon parr to respond to the parasite.     

Effects of infection with the ectoparasite Argulus japonicus (Thiele) and administration of cortisol on cellular proliferation and apoptosis in the epidermis of common carp,Cyprinus carpio L., skin

The host‐parasite interaction between juvenile carp, Cyprinus carpio, and the ectoparasitic branchiuran, Argulus japonicus, together with the role of cortisol in this interaction, was examined at the level of the host skin epidermis. Epidermal mucous cell numbers, and proliferation and apoptosis of the epithelial cells were studied over 32 days. Apoptotic cell numbers in the uppermost epidermis were reduced at 26 days post‐infection with A. japonicus, while the other parameters were unaffected. Administration of cortisol‐containing food resulted in reduced apoptosis in the cells in the upper skin epidermis at 24 h and at 28 days post‐feeding. Cortisol feeding combined with A. japonicus infection reduced numbers of apoptotic cells in the upper epidermis more than either individual treatment. Further, combining the treatments also significantly increased apoptosis in the lower epidermis in cells morphologically identified as leucocytes apparently migrating macrophages and lymphocytes. Using immunohistochemistry, we demonstrated cortisol receptor presence and cellular localization in the teleost epidermis. Receptors only occurred in pavement cells in the upper epidermis and in leucocytes in the lower parts of the epidermis. The ectoparasites, or administered cortisol, induced effects which may be functionally adaptive in the upper pavement cells, while combining the two treatments also induced changes indicative of immunosuppression.     

Studies on the morphology of Uronema marinum Dujardin (Ciliatea: Uronematidae) with a description of the histopathology of the infection in marine fishes

Abstract. A normally free-living hymenostomatid ciliate was found to be the cause of heavy infections of gills, viscera and body muscle in Atlantic and Pacific marine fishes kept in the New York Aquarium. Of special interest is the fact that the parasites, usually with food vacuoles filled with blood cells and cellular debris, caused extensive damage to the body muscle; the other infected organs showed no characteristic tissue reactions. The immediate cause of death is attributed to the exceptional load of the parasite on the gills, thus interfering with normal respiratory function.
The ciliate, isolated from a recent outbreak, is described in detail, and shows many characteristics of Uronema marinum Dujardin, a species that has been reported as free-living in salt water; the form reported here differs from the type and other members included in this species, by the presence of less than three rows of bipolar meridians. This facultative parasite is described in detail and compared with other genera.     

The relationship between flesh quality and numbers of Kudoa thyrsites plasmodia and spores in farmed Atlantic salmon, Salmo salar L

Atlantic salmon, Salmo salar L., were exposed to Kudoa thyrsites (Myxozoa, Myxosporea)-containing sea water for 15 months, and then harvested and assessed for parasite burden and fillet quality. At harvest, parasites were enumerated in muscle samples from a variety of somatic and opercular sites, and mean counts were determined for each fish. After 6 days storage at 4 degrees C, fillet quality was determined by visual assessment and by analysis of muscle firmness using a texture analyzer. Fillet quality could best be predicted by determining mean parasite numbers and spore counts in all eight tissue samples (somatic and opercular) or in four fillet samples, as the counts from opercular samples alone showed greater variability and thus decreased reliability. The variability in both plasmodia and spore numbers between tissue samples taken from an individual fish indicated that the parasites were not uniformly distributed in the somatic musculature. Therefore, to best predict the probable level of fillet degradation caused by K. thyrsites infections, multiple samples must be taken from each fish. If this is performed, a mean plasmodia count of 0.3 mm(-2) or a mean spore count of 4.0 x 10(5) g(-1) of tissue are the levels where the probability of severe myoliquefaction becomes a significant risk.     

鱼类抗刺激隐核虫感染的黏膜免疫研究进展

刺激隐核虫是一种原生纤毛类寄生虫,可以感染几乎所有海水硬骨鱼类并导致死亡,给海水鱼类养殖业造成巨大经济损失。由于刺激隐核虫体表寄生的特性,使其成为研究鱼类黏膜免疫机制的良好病原模型,可为高效疫苗的研发提供理论依据。本文综述了鱼类抗刺激隐核虫感染的黏膜免疫研究进展,以期为开展海水鱼类抗刺激隐核虫感染的免疫防控措施研究提供理论支撑。已有研究表明,受刺激隐核虫感染后,斜带石斑鱼皮肤黏液或其培养上清液能使幼虫发生阻动,由皮肤中的抗体分泌细胞产生的特异性IgM抗体在抗寄生虫感染中发挥重要作用;同时在刺激隐核虫感染时,多种免疫细胞如NCC细胞、中性粒细胞等在寄生虫周围聚集,趋化因子以及趋化因子受体表达量在寄生虫感染部位上调,暗示其调节的免疫细胞也参与抗虫免疫;此外,研究发现黄斑蓝子鱼对刺激隐核虫具有天然抗性,其血清和皮肤黏液对刺激隐核虫幼虫和滋养体均具有杀灭作用,已从其血清中分离到一种天然抗虫蛋白—L-氨基酸氧化酶,为刺激隐核虫病的防控提供新的途径;在理论研究的基础上,通过免疫实验证实疫苗防控刺激隐核虫病是可行的。     

大菱鲆仔鱼皮肤发育及超微结构

The histological development and ultrastructure of larval turbot skin were studied by both light and transmission electron microscopes.The early larvae showed a complete bisymmetry of skin.The skin of 1-25days old larvae developed slowly and was composed of the thin epidermis of 1-2layers epidermal cells and undeveloped dermis before the metamorphosis.But the skin developed significantly when the.metamorphosis began.Till the completion of metamorphosis(about 60days old),the skin contained 3-4layers epidermal cells and very developed collagenous strata.Wisth the process of metamorphosis,the ocular side skin and blind side skin became different in ultrastructure.Ultrastructural observation showed the epidermis of turbot contained three types of epidermal cells;filament-containing cells,mucus cells and chloride cells.The ocular side epidermis with looser structure consisted one type of filament-containing cells,but the blind side epidermis was structured densely and two types of filament-containing cells were observed in it.Melanophores,iridophores,fibroblasts and other cell types and tisssues were found in the spongiosum of dermis.The distribution of melanophores depended on the larval developing stage and the pigmentation of skin.The differentiation of skin seems to be an adaptation for the change of life style of turbot larvae from pelagic living to benthonic living after the metamorphosis.     

Evidence of enhanced bacterial invasion during Diplostomum spathaceum infection in European grayling, Thymallus thymallus (L.)

Farmed grayling, Thymallus thymallus (L.), are susceptible to atypical Aeromonas salmonicida (aAS) infections. Interactions between bacteria and parasites were studied using grayling subjected to concomitant exposure to aAS bacteria and the digenean parasite Diplostomum spathaceum. Atypical AS was detected from fish by a combination of bacterial cultivation and polymerase chain reaction techniques. A detection level of 17 aAS cells per 100 mg intestine tissue sample was obtained. Concomitant bacterial exposure did not enhance the severity of grayling eye rupture and nuclear extrusion induced by D. spathaceum, but D. spathaceum invasion into grayling increased the proportion of fish carrying aAS in their heart tissue. However, the number of aAS cells detected in heart tissue was low. Atypical AS did not cause acute disease or mortality during 15 days post-exposure. There was a higher prevalence of aAS in grayling heart samples than in intestinal samples, indicating that the intestine is not favoured by aAS. We suggest that heart tissue would be a good organ from which to isolate aAS when tracing latent carrier fish. We conclude that penetrating diplostomids can enhance bacterial infections in fish and that diplostomids can cause serious eye ruptures in grayling.     

Histopathology of respiratory organs of certain air-breathing fishes of India

Air-breathing fishes have evolved bimodal respiratory mechanisms for exploitation of water (through gills and highly vascularized skin) as well as atmospheric air (through aerial respiratory organs, ABO). Mucous cells in these respiratory organs of variously stressed fishes exhibit periodic fluctuations in their density and staining properties. The main types of damage in the gills include congestion of blood capillaries (BLCs), periodic lifting and sloughing of respiratory epithelia of the secondary lamellae causing haemorrhage, extensive fusion of secondary lamellae and hyperplasia of the respiratory epithelia due to uncontrolled regeneration leading to asphyxiation, altered excretion, and death of the fish. Haemolysis has also been observed following lead exposure. The damage in the ABO of Heteropneustes fossilis includes sloughing of the epithelial cells, leading to haemorrhage causing decreased red blood corpuscles density and degeneration of the secondary gill lamellae with reduced respiratory area. Subsequent hyperplasia of the respiratory epithelia and fusion of gill lamellae increase the respiratory barrier distance. The BLCs often bulge out and protrude into the lumen, bringing blood nearer to air. The ladder-like pillar cell (PLC)-BLC components of the gill lamellae frequently collapse. Damage to the ABO of Channa striata is less severe. Often haemorrhaging due to bursting of extensively stretched BLCs causes aerial respiratory failure. Chloride cells of the ABOs also show hyperplasia. While the highly mucogenic epidermis of C. striata shows less damage, the epidermis of Clarias batrachus and H. fossilis shows severe wear and tear, sloughing, and haemorrhage. Side-by-side regeneration continues, causing altered histomorphology of the epidermis. The different gland cells also show periodic fluctuations in their density and staining. The dermis also shows severe damage with loosening of their connective tissue fibres. These fibres give stronger reactions for sulfated mucin that not only retain additional water molecules for continuance of skin breathing, but also bind the toxic ambient pollutants.     

Pathogenesis and autoradiographic studies of the epidermis of salmonids infested with Ichtyobodo necator (Henneguy, 1883)

Abstract. The sequential pathology of Ichtyobodo necator infestations of the skin of 0+ and 1+ salmon and rainbow trout was studied. Areas of greatest shelter from water currents were found to be most commonly infested and no parasites were found attached to the epidermis on the head of the fish. The parasite caused hyperplasia of the malphigian cells and exhaustion of the goblet cells below infestations, followed by spongiosis of the underlying epidermis. The epidermal plaque then sloughed off leaving a single layer of cells attached to the basement membrane. Cell kinetic studies showed that I. necator caused the cells immediately below infestations to divide, a markedly different pattern from that of normal teleost epidermal cell proliferation. The possibility that the parasite secretes some form of digestive enzyme is postulated. In areas where sloughing had occurred, the remaining malphigian cells were seen to be in the process of division.