ArticleLiterature Review

Amoebic gill disease and host response in Atlantic salmon (Salmo salar L.): A review

Authors:
  • FishVet Group Ireland
  • VAI Consulting
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Abstract

Gill health is one of the main health challenges for Atlantic salmon (Salmo salar L.) mariculture worldwide, and amoebic gill disease (AGD), caused by the marine ectoprotozoan Neoparamoeba perurans, is currently one of the most significant diseases in terms of prevalence and economic impact. This review describes the host response of Atlantic salmon to the disease, focusing on the pathological changes, immune response, and mechanisms underlying the prominent epithelial proliferation and mucus hypersecretion occurring in affected fish. Health management strategies and risk factors are also discussed.

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... It is also one of the most relevant gill diseases in this species [57]. The disease and particularly the fish response, as it plays a major role in the pathogenesis and effects of the disease, has recently been extensively reviewed [58]. Although the main impact of the disease is in Atlantic salmon, other salmonids such as coho salmon (Oncorhynchus kisutch), chinook salmon (Oncorhynchus tshawytscha), rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) can be affected. ...
... In these species, the development of AGD is probably due to their close contact with potentially affected Atlantic salmon in cages, due to the frequent use of these species as cleaner fish as a system for the biological control of sealice (Lepeophtheirus salmonis) in Atlantic salmon farms. All these affected species are also widely reviewed in Oldham et al. [67] and Marcos and Rodgers [58]. ...
... There is a relevant interest in the study of the development of the immune response in AGD mainly to find an efficient protective vaccine, but also to understand the full host response of the salmon immune system against the parasite [58], as the proliferative response triggered by the parasite is itself, one of the problems associated with the disease. Some experimental vaccines were tested in the past and nowadays there are several projects with different vaccine development strategies. ...
Article
Full-text available
Parasitic and amphizoic amoebae are ubiquitous and can affect a huge variety of hosts, from invertebrates to humans, and fish are not an exception. Most of the relationships between amoebae and fish are based on four different types: ectocommensals, ectoparasites, endocommensals and endoparasites, although the lines between them are not always clear. As ectocommensals, they are located specially on the gills and particularly the amphizoic Neoparamoeba perurans is the most relevant species, being a real pathogenic parasite in farmed salmon. It causes amoebic gill disease, which causes a progressive hyperplasia of epithelial cells in the gill filaments and lamellae. Nodular gill disease is its analogue in freshwater fish but the causative agent is still not clear, although several amoebae have been identified associated to the lesions. Other species have been described in different fish species, affecting not only gills but also other organs, even internal ones. In some cases, species of the genera Naegleria or Acanthamoeba, which also contain pathogenic species affecting humans, are usually described affecting freshwater fish species. As endocommensals, Entamoebae species have been described in the digestive tract of freshwater and marine fish species, but Endolimax nana can reach other organs and cause systemic infections in farmed Solea senegalensis. Other systemic infections caused by amoebae are usually described in wild fish, although in most cases these are isolated cases without clinical signs or significance.
... Amoebic gill disease (AGD), caused by the amphizoic protozoan amoeba Neoparamoeba perurans, and sea lice (mainly Lepeophtheirus salmonis and Caligus elongates in Northern Europe) infestations occur rapidly, cannot be predicted reliably, and cannot be completely palliated with current anti-parasite treatments. 15,16 The infection of fish by both parasites results in weakening of the immune system, poor welfare and can lead to death. Parasite control is made harder when several farms occupy the same region, such as the same fjordic enclosure, particularly if farms from different companies fail to coordinate their anti-parasite treatments. ...
... 30 For example, gill health will likely be affected positively by offshore environments due to increased oxygen and water exchange but negatively by higher salinity. 16 The following sections provide a state-of-the-art review of knowledge to assess the above claims. Knowledge gaps, where further research is needed, are also identified. ...
... [177][178][179][180] These results likely reflect differences in the stage of the disease, with more severe cases resulting in reduced gas transfer, which can be especially harmful in poorly oxygenated water. 16 As a result of this reduction in maximum oxygen uptake due to AGD, the aerobic scope of Atlantic salmon is severely affected (from 406 mg O 2 per kg per h in healthy fish to 203 mg O 2 per kg per h). 175 In turn, high-intensity swimming performance in strong currents is negatively affected (from 3 body lengths per s in healthy fish to 2.5 body lengths per s) and cortisol levels increase during exercise. ...
Article
Full-text available
Offshore aquaculture has gained momentum in recent years, and the production of an increasing number of marine fish species is being relocated offshore. Initially, predictions of the advantages that offshore aquaculture would present over nearshore farming were made without enough science-based evidence. Now, with more scientific knowledge, this review revisits past predictions and expectations of offshore aquaculture. We analysed and explained the oceanographic features that define offshore and nearshore sites. Using Atlantic salmon (Salmo salar) as a case study, we focussed on sea lice, amoebic gill disease, and the risk of harmful algal blooms, as well as the direct effects of the oceanography on the health and physiology of fish. The operational and licencing challenges and advantages of offshore aquaculture are also considered. The lack of space in increasingly saturated sheltered areas will push new farms out to offshore locations and, if appropriate steps are followed, offshore aquaculture can be successful. Firstly, the physical capabilities of the farmed fish species and infrastructure must be fully understood. Secondly, the oceanography of potential sites must be carefully studied to confirm that they are compatible with the species-specific capabilities. And, thirdly, an economic plan considering the operational costs and licencing limitations of the site must be developed. This review will serve as a guide and a compilation of information for researchers and stakeholders.
... The aetiological agent of the condition is Neoparamoeba perurans [13], a marine free-living amphizoic amoeba, which upon attachment to the gill surface elicits clinical responses including grossly visible white mucoid patches upon the gills, respiratory distress, hypernatremia, inappetence and mortality if untreated [14,15]. Multifocal gill lesions are dominated by hyperplasia of the respiratory mucosa with variable inflammatory infiltration [16][17][18][19][20][21][22]. While the host response to AGD is well characterized, the impact (and interactions) that AGD may have with the commensal bacterial community is largely unknown to date. ...
Article
Full-text available
Amoebic gill disease is a parasitic condition that commonly affects marine farmed Atlantic salmon. The causative agent, Neoparamoeba perurans, induces a marked proliferation of the gill mucosa and focal superficial necrosis upon branchial lesions. The effect that amoebic branchialitis has upon gill associated commensal bacteria is unknown. A 16S rRNA sequencing approach was employed to profile changes in bacterial community composition, within amoebic gill disease (AGD)-affected and non-affected gill tissue. The bacterial diversity of biopsies with and without diseased tissue was significantly lower in the AGD-affected fish compared to uninfected fish. Furthermore, within the AGD-affected tissue, lesions appeared to contain a significantly higher abundance of the Flavobacterium, Tenacibaculum dicentrarchi compared to adjunct unaffected tissues. Quantitative PCR specific to both N. perurans and T. dicentrarchi was used to further examine the co-abundance of these known fish pathogens. A moderate positive correlation between these pathogens was observed. Taken together, the present study sheds new light on the complex interaction between the host, parasite and bacterial communities during AGD progression. The role that T. dicentrarchi may play in this complex relationship requires further investigation.
... Teleost fish have only three Ig isotypes: IgM, IgT/Z, and IgD (39), there is no switch of Igs in the secondary response, and the production of specific antibodies is temperature dependent (40,41) (see section 3 for more details about the different functions of Igs). In the last decade our knowledge of the immune system of fish has notably increased (42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53), and in parallel, that of the immune responses against fish parasitic flagellates (54, 55), microsporidians (56), ciliates (57)(58)(59)(60), helminths (61)(62)(63)(64), amoeba (65,66) and crustaceans (67)(68)(69). However, there are methodological difficulties that limit our understanding of fish immune responses in comparison to other vertebrates. ...
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Myxozoans are microscopic, metazoan, obligate parasites, belonging to the phylum Cnidaria. In contrast to the free-living lifestyle of most members of this taxon, myxozoans have complex life cycles alternating between vertebrate and invertebrate hosts. Vertebrate hosts are primarily fish, although they are also reported from amphibians, reptiles, trematodes, mollusks, birds and mammals. Invertebrate hosts include annelids and bryozoans. Most myxozoans are not overtly pathogenic to fish hosts, but some are responsible for severe economic losses in fisheries and aquaculture. In both scenarios, the interaction between the parasite and the host immune system is key to explain such different outcomes of this relationship. Innate immune responses contribute to the resistance of certain fish strains and species, and the absence or low levels of some innate and regulatory factors explain the high pathogenicity of some infections. In many cases, immune evasion explains the absence of a host response and allows the parasite to proliferate covertly during the first stages of the infection. In some infections, the lack of an appropriate regulatory response results in an excessive inflammatory response, causing immunopathological consequences that are worse than inflicted by the parasite itself. This review will update the available information about the immune responses against Myxozoa, with special focus on T and B lymphocyte and immunoglobulin responses, how these immune effectors are modulated by different biotic and abiotic factors, and on the mechanisms of immune evasion targeting specific immune effectors. The current and future design of control strategies for myxozoan diseases is based on understanding this myxozoan-fish interaction, and immune-based strategies such as improvement of innate and specific factors through diets and additives, host genetic selection, passive immunization and vaccination, are starting to be considered.
... The function of glucans have been extensively studied in aquatic animals and the findings indicate that β -glucans improved growth and significantly reduced mortality rate in certain species of aquatic animals [162,163]. β-glucans have been mentioned for their valuable role in several fish species such as red sea bream (Pagrus major) [164], Atlantic Salmon (Salmo salar L.) is utilized to improve growth, survival, immune system, disease resistance against aquatic pathogens and reduced mortality rate in aquatic animals [165]. ...
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Bioactive immunostimulants could be derived from different sources like plants, animals, microbes, algae, yeast, etc. Bioactive immunostimulants are the most significant role to enhance aquatic production, as well as the cost of this method, which is effective, non-toxic, and environment-friendly. These immunostimulants are supportive to increase the immune system, growth, antioxidant, anti-inflammatory, and disease resistance of aquatic ani- mals’ health and also improve aquatic animal feed. Diseases are mainly targeted to the immune system of aquatic organisms in such a way that different processes of bioactive immunostimulants progress are considered imperative techniques for the development of aquaculture production. Communicable infections are the main problem for aquaculture, while the mortality and morbidity connected with some outbreaks significantly limit the productivity of some sectors. Aquaculture is considered the mainly developing food production sector globally. Protein insists is an important issue in human nutrition. Aquaculture has been an exercise for thousands of years, and it has now surpassed capture fisheries as the most vital source of seafood in the world. Limited study reports are available to focal point on bioactive immunostimulants in aquaculture applications. This review report provides information on the nutritional administration of bioactive immunostimulants, their types, functions, and beneficial impacts on aquatic animals’ health as well as for the feed quality development in the aquaculture industry. The scope of this review combined to afford various kinds of natural derived bioactive molecules utilization and their beneficial effects in aquaculture applications.
... Despite the growing number of gene expression and proteomic studies being carried out on AGD-affected salmon (reviewed in Marcos-Lopez & Rodger 2020) 4 , there are still knowledge gaps remaining regarding host immune response in early stage disease. The progression of AGD on the gill can be monitored visually and scored based on the absence or presence of mucoid patches and their distribution on the gills 6 . ...
Article
Full-text available
Amoebic Gill Disease (AGD), caused by the protozoan extracellular parasite Paramoeba perurans ( P. perurans ) is a disease affecting Atlantic salmon ( Salmo salar ). This study investigated the gill transcriptomic profile of pre-clinical AGD using RNA-sequencing (RNA-seq) technology. RNA-seq libraries generated at 0, 4, 7, 14 and 16 days post infection (dpi) identified 19,251 differentially expressed genes (DEGs) of which 56.2% were up-regulated. DEGs mapped to 224 Gene Ontology (GO) terms including 140 biological processes (BP), 45 cellular components (CC), and 39 molecular functions (MF). A total of 27 reference pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and 15 Reactome gene sets were identified. The RNA-seq data was validated using real-time, quantitative PCR (qPCR). A host immune response though the activation of complement and the acute phase genes was evident at 7 dpi, with a concurrent immune suppression involving cytokine signalling, notably in interleukins, interferon regulatory factors and tumour necrosis factor-alpha ( tnf- α) genes. Down-regulated gene expression with involvement in receptor signalling pathways (NOD-like, Toll-like and RIG-1) were also identified. The results of this study support the theory that P. perurans can evade immune surveillance during the initial stages of gill colonisation through interference of signal transduction pathways.
... Diet composition and osmotic stress were the factors that alter the gut microbiota composition (24,64,65). On the other hand, studies of gill bacteria were extremely limited and were mainly focused on the pathological infection issues (66,67). Currently, a study of gills of reef fish has suggested that the gill microbiome composition differed significantly from that of the gut for both adults and juveniles across 15 teleost fish families (68). ...
Article
Full-text available
Aquatic fishes face osmotic stress continuously, and the gill is the first tissue that senses and responds to the external osmotic challenges. However, the understandings of how the gill microbiota could respond to osmotic stress and their potential host-bacterium relationships are limited. The objectives of the current study are to identify the hypotonic responsive genes in the gill cells and profile the gill microbiota communities after fresh water transfer experiment via transcriptome sequencing and 16S rRNA gene sequencing. Transcriptome sequencing identified 1,034 differentially expressed genes (DEGs), such as aquaporin and sodium potassium chloride cotransporter, after the fresh water transfer. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis further highlighted the steroid biosynthesis and glycosaminoglycan biosynthesis pathways in the gill. Moreover, the 16S rRNA gene sequencing identified Vibrio as the dominant bacterium in the seawater, which changed to Pseudomonas and Cetobacterium after the fresh water transfer. The alpha diversity analysis suggested that the gill bacterial diversity was lower in the fresh water transferred group. The KEGG and MetaCyc analysis further predicted the alteration of the glycosaminoglycan and chitin metabolisms in the gill bacteria. Collectively, the common glycosaminoglycan and chitin pathways in both the gill cells and gill microbiota suggest the host-bacterium interaction in gill facilitates the fresh water acclimation. IMPORTANCE This is the first study using the transcriptome and 16S rRNA gene sequencing to report the hypotonic responsive genes in gill cells and the compositions of gill microbiota in marine medaka. The overlapped glycosaminoglycan- and chitin-related pathways suggest host-bacterium interaction in fish gill during osmotic stress.
... Following the infection of ionocytes, in more advanced stages of the infection we observed hyperplasia of the lamellar epithelium and proliferation of epithelial cells. Proliferation of undifferentiated epithelial cells in this area is commonly reported in fish exposed to heavy metals [41][42][43][44], drugs [45] and organic compounds [46,47], as well as during different kinds of gill infections [48,49], where it has been correlated with the downregulation of p53 tumour suppression protein and the overexpression of proliferating cell nuclear antigen PCNA [50]. This reaction was suggested to be a fish immune strategy aiming to increase the width of the thin tissue layer that separates the shallow respiratory capillaries from the harmful agent, preventing its permeation into the blood stream [44]. ...
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Epitheliocystis is a fish gill disease caused by a broad range of intracellular bacteria infecting freshwater and marine fish worldwide. Here we report the occurrence and progression of epitheliocystis in greater amberjack reared in Crete (Greece). The disease appears to be caused mainly by a novel Betaproteobacteria belonging to the Candidatus Ichthyocystis genus with a second agent genetically similar to Ca. Parilichlamydia carangidicola coinfecting the gills in some cases. After a first detection of the disease in 2017, we investigated epitheliocystis in the following year’s cohort of greater amberjack juveniles (cohort 2018) transferred from inland tanks to the same cage farm in the open sea where the first outbreak was detected. This cohort was monitored for over a year together with stocks of gilthead seabream and meagre co-farmed in the same area. Our observations showed that epitheliocystis could be detected in greater amberjack gills as early as a month following the transfer to sea cages, with ionocytes at the base of the gill lamellae being initially infected. Cyst formation appears to trigger a proliferative response, leading to the fusion of lamellae, impairment of gill functions and subsequently to mortality. Lesions are characterized by infiltration of immune cells, indicating activation of the innate immune response. At later stages of the outbreak, cysts were no longer found in ionocytes but were observed in mucocytes at the trailing edge of the filament. Whole cysts appeared finally to be expelled from infected mucocytes directly into the water, which might constitute a novel means of dispersion of the infectious agents. Molecular screening indicates that meagre is not affected by this disease and confirms the presence of previously described epitheliocystis agents, Ca. Ichthyocystis sparus, Ca. Ichthyocystis hellenicum and Ca. Similichlamydia spp., in gilthead seabream. Prevalence data show that the bacteria persist in both gilthead seabream and greater amberjack cohorts after first infection.
... Following the infection of ionocytes, in more advanced stages of the infection we observed hyperplasia of the lamellar epithelium and proliferation of epithelial cells. Proliferation of undifferentiated epithelial cells in this area is commonly reported in fish exposed to heavy metals [41][42][43][44], drugs [45] and organic compounds [46,47], as well as during different kinds of gill infections [48,49], where it has been correlated with the downregulation of p53 tumour suppression protein and the overexpression of proliferating cell nuclear antigen PCNA [50]. This reaction was suggested to be a fish immune strategy aiming to increase the width of the thin tissue layer that separates the shallow respiratory capillaries from the harmful agent, preventing its permeation into the blood stream [44]. ...
Article
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Treatment development for parasitic infestation is often limited to disease resolution as an endpoint response, and physiological and immunological consequences are not thoroughly considered. Here we report the impact of exposing Atlantic salmon affected with amoebic gill disease (AGD) to peracetic acid (PAA), an oxidative chemotherapeutic. AGD-affected fish were treated with PAA either by exposing them to 5 ppm for 30 mins or 10 ppm for 15 mins. Unexposed fish from both infected and uninfected groups were also included. Samples for molecular, biochemical and histological evaluations were collected at 24 h, 2 weeks and 4 weeks post treatment. Behavioural changes were observed during PAA exposure and post-treatment mortality was higher in the infected and PAA treated groups, especially in 10ppm-15mins. Plasma indicators showed that liver health was affected by AGD, though PAA treatment did not exacerbate the infection-related changes. Transcriptome profiling in the gills showed significant changes triggered by AGD and PAA treatments, and the effects of PAA were more notable 24 h after treatment. Genes related to immune pathways of B- and T- cells and protein synthesis and metabolism were downregulated, where the magnitude was more remarkable in 10ppm-15mins group. Even though treatment did not fully resolve the pathologies associated with AGD, 5ppm-30mins group showed lower parasite load at 4 weeks post-treatment. Mucous cell parameters (i.e., size and density) increased within 24 h post-treatment and were significantly higher at termination, especially in AGD-affected fish, with some treatment effects influenced by the dose of PAA. Infection and treatments resulted in oxidative stress – in the early phase in the gill mucosa, while systemic reactive oxygen species (ROS) dysregulation was evident at the later stage. Infected fish responded to elevated circulating ROS by increasing antioxidant production. Exposing the fish to a crowding stress revealed interference in the post-stress responses. Lower cortisol response was displayed by AGD-affected groups. Collectively, the study established that PAA, within the evaluated treatment protocols, could not provide a convincing treatment resolution and thus require further optimisation. Nonetheless, PAA treatment altered the mucosal immune and stress responses of AGD-affected Atlantic salmon, shedding light on the host-parasite-treatment interactions.
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Marine farmed Atlantic salmon (Salmo salar) are susceptible to recurrent amoebic gill disease (AGD) caused by the ectoparasite Neoparamoeba perurans over the growout production cycle. The parasite elicits a highly localized response within the gill epithelium resulting in multifocal mucoid patches at the site of parasite attachment. This host-parasite response drives a complex immune reaction which remains poorly understood. To generate a model for host-parasite interaction during pathogenesis of AGD in Atlantic salmon the local (gill) and systemic transcriptomic response in the host, and the parasite during AGD pathogenesis was explored. A dual RNA-seq approach together with differential gene expression and system-wide statistical analyses of gene and transcription factor networks was employed. A multi-tissue transcriptomic data set was generated from the gill (including both lesioned and non-lesioned tissue), head kidney and spleen tissues naïve and AGD-affected Atlantic salmon sourced from an in vivo AGD challenge trial. Differential gene expression of the salmon host indicates local and systemic upregulation of defense and immune responses. Two transcription factors, znfOZF-like and znf70-like, and their associated gene networks significantly altered with disease state. The majority of genes in these networks are candidates for mediators of the immune response, cellular proliferation and invasion. These include Aurora kinase B-like, rho guanine nucleotide exchange factor 25-like and protein NDNF-like inhibited. Analysis of the N. perurans transcriptome during AGD pathology compared to in vitro cultured N. perurans trophozoites, as a proxy for wild type trophozoites, identified multiple gene candidates for virulence and indicates a potential master regulatory gene system analogous to the two component PhoP/Q system. Candidate genes identified are associated with invasion of host tissue, evasion of host defense mechanisms and formation of the mucoid lesion. We generated a novel model for host-parasite interaction during AGD pathogenesis through integration of host and parasite functional profiles. Collectively, this dual transcriptomic study provides novel molecular insights into the pathology of AGD and provides alternative theories for future research in a step towards improved management of AGD.
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Bioactive immunostimulants could be derived from different sources like plants, animals, microbes, algae, yeast, etc. Bioactive immunostimulants are the most significant role to enhance aquatic production, as well as the cost of this method, which is effective, non-toxic, and environment-friendly. These immunostimulants are supportive to increase the immune system, growth, antioxidant, anti-inflammatory, and disease resistance of aquatic animals' health and also improve aquatic animal feed. Diseases are mainly targeted to the immune system of aquatic organisms in such a way that different processes of bioactive immunostimulants progress are considered imperative techniques for the development of aquaculture production. Communicable infections are the main problem for aquaculture, while the mortality and morbidity connected with some outbreaks significantly limit the productivity of some sectors. Aquaculture is considered the mainly developing food production sector globally. Protein insists is an important issue in human nutrition. Aquaculture has been an exercise for thousands of years, and it has now surpassed capture fisheries as the most vital source of seafood in the world. Limited study reports are available to focal point on bioactive immunostimulants in aquaculture applications. This review report provides information on the nutritional administration of bioactive immunostimulants, their types, functions, and beneficial impacts on aquatic animals’ health as well as for the feed quality development in the aquaculture industry. The scope of this review combined to afford various kinds of natural derived bioactive molecules utilization and their beneficial effects in aquaculture applications.
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Amoebic gill disease (AGD) is a significant issue in Atlantic salmon mariculture. Research on the development of treatments or vaccines uses experimental challenges where salmon is exposed to amoebae concentrations ranging from 500 to 5,000/L. However, the water concentrations of N. perurans on affected salmon farms are much lower. The lowest concentration of N. perurans previously reported to cause AGD was 10/L. Here, we report that concentrations as low as 0.1/L of N. perurans can cause AGD. We propose that concentrations of N. perurans that reflect those measured on salmon farms should be used for future experimental challenges.
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Offshore aquaculture has gained momentum in recent years and the production of more marine farmed fish species is moving offshore. Initially, predictions of the advantages that offshore aquaculture would present over near shore farming were made without enough science-based evidence. Now, with more scientific knowledge, this review revisits past predictions and expectations of offshore aquaculture. The oceanographic features that define offshore and near shore sites are analysed and explained. Using Atlantic salmon (Salmo salar) as a case study we focused on sea lice, amoebic gill disease, and harmful algal blooms risk, as well as the direct effects of the oceanography on the health and physiology of fish. The operational and licencing challenges and advantages of offshore aquaculture are also explained in depth. Overall, moving aquaculture offshore is a risky and capital-intensive enterprise that still seems unadvisable. Regardless, the lack of space in sheltered areas will push new farms out to offshore locations. But if appropriate steps are followed, offshore aquaculture can be successful. First, the physical capabilities of the farmed fish species and infrastructure must be fully understood. Secondly, the oceanography of potential sites must be carefully studied to confirm that they are compatible with the fish species-specific physiological capabilities. And third, an economic plan considering the operational costs and licencing limitations of the site must be elaborated. This review will serve as a guide and compilation of this information for researchers and stakeholders.
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Amoebic gill disease (AGD) is one of the main health issues impacting farmed Atlantic salmon. Neoparamoeba perurans causes AGD; however, a diversity of other amoeba species colonizes the gills and there is little understanding of whether they are commensal or potentially involved in different stages of gill disease development. Here, we conduct in vivo challenges of naïve Atlantic salmon with cultured Nolandella sp. and Pseudoparamoeba sp. to investigate their pathogenicity to Atlantic salmon gills. Additionally, we assessed whether the presence of Nolandella sp. and Pseudoparamoeba sp. influences the onset and/or severity of N. perurans‐induced AGD. All three strains attached and multiplied on the gills according to qPCR analysis. Furthermore, minor gross gill lesions and histological changes were observed post‐exposure. While N. perurans was found associated with classical AGD lesions, Nolandella sp. and Pseudoparamoeba sp. were not found associated with lesion sites and these lesions did not meet the expected composite of histopathological changes for AGD. Moreover, the presence of these non‐N. perurans species did not significantly increase the severity of AGD. This trial provides evidence that cultured Nolandella sp. and Pseudoparamoeba sp. do not induce AGD and do not influence the severity of AGD during the early stages of development.
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The present study investigated the involvement of key molecular regulators of oxidative stress in amoebic gill disease (AGD), a parasitic infestation in Atlantic salmon. In addition, the study evaluated how these molecular biomarkers responded when AGD-affected fish were exposed to a candidate chemotherapeutic peracetic acid (PAA). Atlantic salmon were experimentally infected with the parasite Neoparameoba perurans, the causative agent of AGD, by bath exposure and after 2 weeks, the fish were treated with three commercial PAA products (i.e., Perfectoxid, AquaDes and ADDIAqua) at a dose of 5 ppm. Two exposure durations were evaluated – 30 min and 60 min. Sampling was performed 24 h and 2 weeks after PAA treatment (equivalent to 2- and 4-weeks post infection). At each sampling point, the following parameters were evaluated: gross gill pathology, gill parasitic load, plasma reactive oxygen species (ROS) and total antioxidant capacity (TAC), histology and gene expression profiling of genes with key involvement in oxidative stress in the gills and olfactory organ. AGD did not result in systemic oxidative stress as ROS and TAC levels remained unchanged. There were no clear patterns of AGD-mediated regulation of the oxidative stress biomarkers in both the gills and olfactory organ; significant changes in the expression were mostly related to time rather than infection status. However, the expression profiles of the oxidative stress biomarkers in AGD-affected salmon, following treatment with PAA, revealed that gills and olfactory organ responded differently – upregulation was prominent in the gills while downregulation was more frequent in the olfactory organs. The expression of catalase, glutathione S-transferase and thioredoxin reductase 2 was significantly affected by the treatments, both in the gills and olfactory organ, and these alterations were influenced by the duration of exposure and PAA product type. Parasitic load in the gills did significantly increase after treatment regardless of the product and exposure duration. However, PAA treated groups for 30 min showed lower macroscopic gill scores than the infected-untreated fish. Histology disclosed the classic pathological findings such as multifocal hyperplasia and increased number of mucous cells in AGD-affected fish. Microscopic scoring of gill injuries showed that AGD-infected-PAA-treated fish had lower scores, however, an overall trend could not be established. The morphology and structural integrity of the olfactory organ were not significantly altered by parasitism or PAA treatment. Collectively, the results indicate that AGD did not affect the systemic and mucosal oxidative status of Atlantic salmon. However, such a striking profile was changed when AGD-affected fish were exposed to oxidative chemotherapeutics. Moreover, the gills and olfactory organ demonstrated distinct patterns of gene expression of oxidative stress biomarkers in AGD-infected-PAA-treated fish. Lastly, PAA treatment did not fully resolve the infection, but appeared not to worsen the mucosal health either.
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The utilization of feed resources in Norwegian salmon farming in 2010 and 2012 has been reported previously. The present study is an update for 2016, along with data on whole body composition of slaughter sized salmon. In 2016, in total 1,252,573 tonnes of salmon were produced. Fillet production was estimated to 814,172 tonnes. Given ‘as is’, 1,627,478 tonnes of feed ingredients were used (1,520,358 tonnes on dry matter basis). Marine ingredients constituted 405,921 tonnes (25%), 1,156,135 tonnes (71%) were of plant origin and 65,422 tonnes (4%) were other ingredients. The estimated retention of energy, protein, lipid, DHA + EPA and phosphorus was 41.3%, 36.6%, 49.4%, 37.3% and 18.5%, respectively, in whole salmon. In fillet, the corresponding retention values were 23.0%, 26.1%, 24.6%, 21.8% and 9.5%, respectively. Whole body of slaughter sized salmon (mean body weight 5276 g) contained 12.71 MJ/kg energy, 16.9% crude protein, 21.5% total lipids (0.44% EPA, 0.72% DHA) and 1.8% ash (0.31% phosphorus). The salmon production and use of feed ingredients in 2016 were of similar volumes as in 2012, but the use of marine protein sources was further reduced and replaced by plant ingredients. Keywords: Feed ingredients, Ingredient origin, Annual salmon production, Nutrient retention, Whole body analysis
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Amoebic gill disease (AGD) is emerging as one of the most significant health challenges affecting farmed Atlantic salmon in the marine environment. It is caused by the amphizoic amoeba Neoparamoeba perurans, with infestation of gills causing severe hyperplastic lesions, compromising overall gill integrity and function. This study used histology, transmission electron microscopy (TEM), immunohistochemistry and transcript expression to relate AGD‐associated pathological changes to changes in the morphology and distribution of chloride cells (CCs) in the gills of Atlantic salmon (Salmo salar L.) showing the progression of an AGD infection. A marked reduction in numbers of immunolabelled CCs was detected, and a changing pattern in distribution and morphology was closely linked with the level of basal epithelial hyperplasia in the gill. In addition, acute degenerative ultrastructural changes to CCs at the lesion site were observed with TEM. These findings were supported by the early‐onset downregulation of Na+/K+‐ATPase transcript expression. This study provides supportive evidence that histological AGD lesion assessment was a good qualitative tool for AGD scoring and corresponded well with qPCR genomic Paramoeba perurans quantification. Ultrastructural changes induced in salmon CCs as a result of AGD are reported here for the first time.
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ABSTRACT: Amoebic gill disease (AGD) is the primary health concern for Atlantic salmon farmed in Tasmania, Australia. Neoparamoeba perurans is the aetiological agent of AGD, however a diversity of other amoebae colonise the gills, and their role in AGD is unknown. Previous studies which document these accompanying amoebae on AGD-affected farmed Atlantic salmon relied on culture-based techniques which do not accurately determine the prevalence and abundance of these species nor whether they correlate with AGD pathology. Drawing on our previous culture-based study, here we develop and apply 5 new Taqman quantitative PCR assays to profile the prevalence of multiple amoeba species on the gills of AGD-affected Atlantic salmon held at 2 Tasmanian farm sites over a 1 yr period. The prevalence and abundance of N. perurans was also assessed using a previously established qPCR method. N. perurans was the dominant species, and its abundance positively correlated with the progression of gross gill pathology. Only a small number of sporadic detections of Pseudoparamoeba and Vannellida species were observed. Nolandella spp. was the notable exception, as it was the most prevalent amoeba (92%) at 1 site at 1 sample time, during which no N. perurans were detected on gills but low levels of gross gill pathology were observed. N. perurans is the predominant species and primary pathogen of AGD, however there were instances when they were not detected on diseased gills and Nolandella spp. were highly prevalent. The significance of Nolandella spp. in relation to AGD is not yet understood.
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Estimates of genetic parameters of susceptibility of Atlantic salmon to amoebic gill disease (AGD) were obtained from a bath challenge test with two repeated infections (1612 and 1582 fish, the offspring of 50 sires and 100 dams) and from a field test of their sibs (1156 fish) that were naturally infected and scored once for AGD. A third sibling group were reared in an AGD-free environment and their body weights recorded at harvest. In both challenge tests, susceptibility to AGD were measured using an adapted Taylor gill-score (0–5) where gill-score 3 was divided into three sub-classes 3A, 3B and 3C. In the field test, one gill arch of each animal was swabbed to quantify the amount of Paramoeba perurans by RT-qPCR, and a random sample of 126 of the fish were also analysed by RT-qPCR for Paranucleospora theridion and Branchiomonas cysticola. In the field test, body weights of the fish were recorded at time of gill-scoring and seven months later. In both tests, the distributions of gill-score was quite narrow (large proportion with gill-score 2 and 3A, and none with gill-score 4 and 5). In the field test, average body weight of fish with gill-score 1, 2, 3A and 3B was 17.6, 9.4, 17.9 and 22.2% lower, respectively than fish with gill-score 0. The genetic correlation between gill-score in the bath and the field test was close to zero. Therefore, the present bath challenge test for susceptibility to AGD cannot replace a field test in a selective breeding program. In the AGD-affected environment, the genetic correlation of gill-score with CT was −0.81 ± 0.16 and with body weight − 0.88 ± 0.09. These high genetic correlations indicate that CT and growth may be used as indirect trait measures of susceptibility to AGD. The high genetic correlation between body weights in the AGD-affected and the AGD-free environment (0.86 ± 0.05) indicate a true favourable genetic correlation between susceptibility to AGD and growth in Atlantic salmon. Consequently, selection for increased growth rate will result in a favourable genetic correlated response in susceptibility to AGD. The magnitude of these correlations need to be verified, in particular as the negative effect of decreasing CT-values of P. theridion on body weight was found to be larger than that of P. perurans and that growth of the fish in the AGD-free environment may be affected by other gill pathogens with negative effect of growth.
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Gill diseases are a growing health challenge in salmon farming worldwide, but many gaps remain in our knowledge. Gill diseases are generally complex and multifactorial disorders, often with presumable spatial and temporal distribution patterns, but are highly difficult to effectively prevent and control. The term complex gill disease (CGD) includes a wide range of clinical disease presentations on the gills of farmed salmon; usually, CGD presents from the end of summer to early winter. The pathogens involved include Neoparamoeba perurans, Tenacibaculum maritimum, Candidatus Piscichlamydia salmonis, Candidatus Branchiomonas cysticola, Desmozoon lepeophtherii (syn. Paranucleospora theridion) and viruses, such as the Atlantic salmon paramyxovirus (ASPV) and salmon gill poxvirus (SGPV). Amoebic gill disease (AGD) is perhaps the most significant disease in terms of gill health and economic impact. AGD results in high mortality, reduced production performance and impaired fish welfare. This review summarizes and analyses CGD research, outbreaks and treatment, with a focus on AGD, as well as on knowledge gaps and avenues for future research.
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Amoebic gill disease (AGD) is one of the most significant infectious diseases affecting marine farmed Atlantic salmon and is caused by the marine amoeba Neoparamoeba perurans . Atlantic halibut are also farmed in Norway, Canada, Chile and Scotland; however, the total production is approximately 0.1 per cent that of salmon. Halibut reared in a net pen sea site in Scotland presented with low-level mortalities and pale, raised patches on the gills. Fresh microscopy of the gills demonstrated amoeboid organisms and histology of the gills confirmed typical AGD pathology. Gill samples tested by PCR confirmed the presence of N perurans . Freshwater baths of three hours duration are the preferred treatment for AGD in salmon; however, hydrogen peroxide has also been used and as halibut do not tolerate freshwater low-level hydrogen peroxide baths (1000 mg/l) were administered as a 15-minute bath. Net changes were also undertaken, and mortalities subsequently reduced.
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Amoebic gill disease (AGD) is one of the most important parasitic diseases of farmed Atlantic salmon. It is a source of major economic loss to the industry and poses significant threats to animal welfare. Previous studies have shown that resistance against this disease has a moderate, heritable genetic component, although the genes and the genetic pathways that contribute to this process have yet to be elucidated. In this study, to identify the genetic mechanisms of AGD resistance, we first investigated the molecular signatures of AGD infection in Atlantic salmon through a challenge model, where we compared the transcriptome profiles of the naïve and infected animals. We then conducted a genome-wide association analysis with 1,333 challenged tested fish to map the AGD resistance genomic regions, supported by the results from the transcriptomic data. Further, we investigated the potential of incorporating gene expression analysis results in genomic prediction to improve prediction accuracy. Our data suggest thousands of genes have modified their expression following infection, with a significant increase in the transcription of genes with functional properties in cell adhesion and a sharp decline in the abundance of various components of the immune system genes. From the genome-wide association analysis, QTL regions on chromosomes ssa04, ssa09, and ssa13 were detected to be linked with AGD resistance. In particular, we found that QTL region on ssa04 harbors members of the cadherin gene family. These genes play a critical role in target recognition and cell adhesion. The QTL region on ssa09 also is associated with another member of the cadherin gene family, protocadherin Fat 4. The associated genetic markers on ssa13 span a large genomic region that includes interleukin-18-binding protein, a gene with function essential in inhibiting the proinflammatory effect of cytokine IL18. Incorporating gene expression information through a weighted genomic relationship matrix approach decreased genomic prediction accuracy and increased bias of prediction. Together, these findings help to improve our breeding programs and animal welfare against AGD and advance our knowledge of the genetic basis of host-pathogen interactions.
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An in vitro model to study the host response to Neoparamoeba perurans, the causative agent of amoebic gill disease (AGD), was evaluated. The rainbow trout gill derived cell line, RTgill-W1, was seeded onto permeable cell culture supports and maintained asymmetrically with apical seawater. Cells were inoculated with either a passage attenuated or a recent wild clone of N. perurans. Amoebae, loaded with phagocytosed fluorescent beads, were observed associated with host cells within 20 min post inoculation (pi). By 6 h small foci of cytopathic effect appeared and at 72 h cytolysis was observed, with total disruption of the cell monolayer at 96 h pi. Due to cell monolayer disruption, the platform could not support proliferation of amoebae, which showed a 3-log reduction in parasite 18S rRNA mRNA after 72 h (10⁶ copies at 1 h to 10³ at 72 h pi). SEM observations showed amoebae-like cells with either short pseudopodia and a malleiform shape, or, long pseudopodia embedded within the gill cells and erosion of the cell monolayer. To study the host immune response, inoculated gill cells were harvested from triplicate inserts at 0, 1, 3, 6, 24 and 48 h pi, and expression of 12 genes involved in the Atlantic salmon response to AGD was compared between infected and uninfected cells and between amoebic clones. Both clones induced similar host inmate immune responses, with the up-regulation of proinflammatory cytokine IL1β complement C3 and cell receptor MHC-1. The Th2 pathway was up-regulated, with increased gene expression of the transcription factor GATA3, and Th2 cytokines IL10, IL6 and IL4/13A. PCNA and AG-2 were also up-regulated. The wild clone induced significantly higher up-regulation of IL1β MHC-1, PCNA, lysozyme and IL10 than the attenuated clone for at least some exposure times, but AG-2 gene expression was higher in cells inoculated with the attenuated one. A principal component analysis showed that AG-2 and IL10 were key genes in the in vitro host response to N. perurans. This in vitro model has proved to be a promising tool to study host responses to amoebae and may therefore reduce the requirement for in vivo studies when evaluating alternative therapeutants to AGD control.
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Amoebic gill disease (AGD) is one of the main diseases affecting Atlantic salmon (Salmo salar L.) mariculture. Hallmarks of AGD are hyperplasia of the lamellar epithelium and increased production of gill mucus. This study investigated the expression of genes involved in mucus secretion, cell cycle regulation, immunity and oxidative stress in gills using a targeted 21-gene PCR array. Gill samples were obtained from experimental and natural Neoparamoeba perurans infections, and sampling points included progressive infection stages and post-freshwater treatment. Up-regulation of genes related to mucin secretion and cell proliferation, and down-regulation of pro-inflammatory and pro-apoptotic genes were associated with AGD severity, while partial restoration of the gill homeostasis was detected post-treatment. Mucins and Th2 cytokines accoun ted for most of the variability observed between groups highlighting their key role in AGD. Two mucins (muc5, muc18) showed differential regulation upon disease. Substantial up-regulation of the secreted muc5 was detected in clinical AGD, and the membrane bound muc18 showed an opposite pattern. Th2 cytokines, il4/13a and il4/13b2, were significantly up-regulated from 2 days post-infection onwards, and changes were lesion-specific. Despite the differences between experimental and natural infections, both yielded comparable results that underline the importance of the studied genes in the respiratory organs of fish, and during AGD progression.
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While research into the growth, survival, nutrition and, more recently, disease susceptibility of triploid Atlantic salmon has expanded, there remains an overall lack of studies assessing the response of triploids to chemical treatments. It is essential that the response of triploids to disease treatments be characterised to validate their suitability for commercial production. This study aimed to investigate and compare the stress and immune responses of triploid and diploid Atlantic salmon following an experimental treatment with hydrogen peroxide (H2O2). A dose response test was first undertaken to determine a suitable test dose for both diploid and triploid Atlantic salmon. Following this, diploids and triploids were exposed to H2O2 (1800 ppm) for 20 min, as per commercial practices, after which blood glucose and lactate, and plasma cortisol and lysozyme were measured, along with the expression of oxidative stress and immune-related genes. In the first 6 h post-exposure to H2O2, comparable mortalities occurred in both diploid and triploid Atlantic salmon. Cortisol, glucose and lactate were not significantly influenced by ploidy suggesting that, physiologically, triploid Atlantic salmon are able to cope with the stress associated with H2O2 exposure as well as their diploid counterparts. Exposure to H2O2 significantly elevated the expression of cat and sod2 in diploid livers and gr, il1β and crp/sap1b in diploid gills, while it significantly decreased the expression of saa5 and crp/sap1a in diploid gills. In triploids, the expression levels of cat, hsp70, sod1, saa5, crp/sap1a and crp/sap1b in liver was significantly higher in fish exposed to H2O2 compared to control fish. The expression of gr, sod1 and il1β in triploid gills was also elevated in response to H2O2 exposure. This study represents the first experimental evidence of the effects of H2O2 exposure on triploid Atlantic salmon and continues to support their application into commercial production.
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This study examines the potential implications of biofouling management on the development of an infectious disease in Norwegian farmed salmon. The hydroid Ectopleura larynx frequently colonises cage nets at high densities (thousands of colonies per m²) and is released into the water during regular in-situ net cleaning. Contact with the hydroids’ nematocysts has the potential to cause irritation and pathological damage to salmon gills. Amoebic gill disease (AGD), caused by the amoeba Paramoeba perurans, is an increasingly international health challenge in Atlantic salmon farming. AGD often occurs concomitantly with other agents of gill disease. This study used laboratory challenge trials to: (1) characterise the gill pathology resulting from the exposure of salmon to hydroids, and (2) investigate if such exposure can predispose the fish to secondary infections–using P. perurans as an example. Salmon in tanks were exposed either to freshly ‘shredded’ hydroids resembling waste material from net cleaning, or to authentic concentrations of free-living P. perurans, or first to ‘shredded’ hydroids and then to P. perurans. Gill health (AGD gill scores, non-specific gill scores, lamellar thrombi, epithelial hyperplasia) was monitored over 5 weeks and compared to an untreated control group. Nematocysts of E. larynx contained in cleaning waste remained active following high-pressure cleaning, resulting in higher non-specific gill scores in salmon up to 1 day after exposure to hydroids. Higher average numbers of gill lamellar thrombi occurred in fish up to 7 days after exposure to hydroids. However, gill lesions caused by hydroids did not affect the infection rates of P. perurans or the disease progression of AGD. This study discusses the negative impacts hydroids and current net cleaning practices can have on gill health and welfare of farmed salmon, highlights existing knowledge gaps and reiterates the need for alternative approaches to net cleaning.
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Amoebic gill disease (AGD) is one of the largest threats to salmon aquaculture, causing serious economic and animal welfare burden. Treatments can be expensive and environmentally damaging, hence the need for alternative strategies. Breeding for disease resistance can contribute to prevention and control of AGD, providing long-term cumulative benefits in selected stocks. The use of genomic selection can expedite selection for disease resistance due to improved accuracy compared to pedigree-based approaches. The aim of this work was to quantify and characterise genetic variation in AGD resistance in salmon, the genetic architecture of the trait, and the potential of genomic selection to contribute to disease control. An AGD challenge was performed in ~1,500 Atlantic salmon, using gill damage and amoebic load as indicator traits for host resistance. Both traits are heritable (h2 ~ 0.25-0.30) and show high positive correlation, indicating they may be good measurements of host resistance to AGD. While the genetic architecture of resistance appeared to be largely polygenic in nature, two regions on chromosome 18 showed suggestive association with both AGD resistance traits. Using a cross-validation approach, genomic prediction accuracy was up to 18 % higher than that obtained using pedigree, and a reduction in marker density to ~2,000 SNPs was sufficient to obtain accuracies similar to those obtained using the whole dataset. This study indicates that resistance to AGD is a suitable trait for genomic selection, and the addition of this trait to Atlantic salmon breeding programs can lead to more resistant stocks.
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The parasitic amoeba Paramoeba perurans is an aetiological agent of amoebic gill disease (AGD), a serious problem in seawater salmonid aquaculture globally. Other finfish species are also infected and infection events may be associated with periods of unusual high temperatures. Currently little is known about the impact of AGD on wild fish, but in a time with global warming and increasing aquaculture production this potential threat could be on the rise. A better understanding of the pathophysiology of infected fish is therefore warranted. In this study, groups of Atlantic salmon with and without AGD were tested in a large swim tunnel respirometer in seawater at 13°C to assess oxygen uptake, swimming capacity and blood parameters. Standard metabolic rates were similar between groups, but the maximum rate of oxygen uptake was drastically reduced in infected fish, which resulted in a smaller aerobic scope (AS) of 203 mg O 2 kg −1 h −1 compared to 406 mg O 2 kg −1 h −1 in healthy fish. The critical swimming speed was 2.5 body lengths s −1 in infected fish and 3.0 body lengths s −1 in healthy ones. Furthermore, AGD fish had lower haematocrit and [haemoglobin], but similar condition factor compared to healthy fish. Prior to swim trials infected fish had higher plasma osmolality, elevated plasma [Na + ], [Cl-] and [cortisol], indicating reduced capacity to maintain ionic homoeostasis as well as chronic stress during routine conditions. These results demonstrate that AGD compromises gill function both in terms of gas exchange and ion regulation, and consequently the capacity for aerobic activity is reduced. Reduced AS due to the P. perurans infections is likely to interfere with appetite, growth and overall survival, even more so in the context of a warmer and more hypoxic future.
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This study aims to demonstrate the presence of oxidative stress in farmed Atlantic salmon (Salmo salar L.) infected with Neoparamoeba perurans and undergoing clinical amoebic gill disease (AGD). Hydrophilic antioxidant activity (HAA), activity of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR),and gene expression of SOD and CAT, were determined in gills. Sequential samples were obtained from a natural infection at gill score (GS) 0, GS 2, and post freshwater treatment and separate samples comprising gill areas without and with lesions were taken. HAA analysis revealed a significant depletion of the antioxidant capacity in infected gills, and a recovery to previous antioxidant levels after freshwater treatment. SOD activity did not differ between lesion and non-lesion areas, CAT activity was diminished in lesion areas of both infected and treated fish, whereas GR activity at GS 2 was increased in lesion areas respect to non-lesions. Furthermore, gene expression of SOD and CAT were up-regulated in gill lesion areas of GS 2 fish. These results indicate the involvement of oxidative stress in AGD pathogenesis, and show that enzymatic activity and gene expression are focally regulated in the gills.
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Few studies have focussed on the health and immunity of triploid Atlantic salmon and therefore much is still unknown about their response to commercially significant pathogens. This is important if triploid stocks are to be considered for full-scale commercial production. This study aimed to investigate and compare the response of triploid and diploid Atlantic salmon to an experimental challenge with Neoparamoeba perurans , causative agent of amoebic gill disease (AGD). This disease is economically significant for the aquaculture industry. The results indicated that ploidy had no significant effect on gross gill score or gill filaments affected, while infection and time had significant effects. Ploidy, infection and time did not affect complement or anti-protease activities. Ploidy had a significant effect on lysozyme activity at 21 days post-infection (while infection and time did not), although activity was within the ranges previously recorded for salmonids. Stock did not significantly affect any of the parameters measured. Based on the study results, it can be suggested that ploidy does not affect the manifestation or severity of AGD pathology or the serum innate immune response. Additionally, the serum immune response of diploid and triploid Atlantic salmon may not be significantly affected by amoebic gill disease.
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Amoebic gill disease (AGD), caused by the protozoan parasite Neoparamoeba perurans, is one of the most significant infectious diseases for Atlantic salmon (Salmo salar L.) mariculture. The present study investigated the humoral immune response (both local in gill mucus and systemic in serum) of farmed Atlantic salmon naturally infected with N. perurans in commercial sea pens, at two different stages of the disease and after freshwater treatment. Parameters analysed included activity of immune related enzymes (i.e. lysozyme, peroxidase, protease, anti-protease, esterase, alkaline phosphatase), IgM levels, and the terminal carbohydrate profile in the gill mucus. Overall, greater variations between groups were noted in the immune parameters determined in gill mucus than the equivalent in the serum. In gill mucus, IgM levels and peroxidase, lysozyme, esterase and protease activities were decreased in fish showing longer exposure time to the infection and higher disease severity, then showed a sequential increase after treatment. Results obtained highlight the capacity of gills to elicit a local response to the infection, indicate an impaired immune response at the later stages of the disease, and show partial reestablishment of the host immune status after freshwater treatment. In addition to providing data on the humoral response to AGD, this study increases knowledge on gill mucosal humoral immunity, since some of the parameters were analysed for the first time in gill mucus.
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The gene encoding IgH δ has been found in all species of teleosts studied to date. However, catfish (Ictalurus punctatus) is the only species of fish in which a secretory form of IgD has been characterized, and it occurs through the use of a dedicated δ-secretory exon, which is absent from all other species examined. Our studies have revealed that rainbow trout (Oncorhynchus mykiss) use a novel strategy for the generation of secreted IgD. The trout secretory δ transcript is produced via a run-on event in which the splice donor site at the end of the last constant domain exon (D7) is ignored and transcription continues until a stop codon is reached 33 nt downstream of the splice site, resulting in the production of an in-frame, 11-aa secretory tail at the end of the D7 domain. In silico analysis of several published IgD genes suggested that this unique splicing mechanism may also be used in other species of fish, reptiles, and amphibians. Alternative splicing of the secretory δ transcript resulted in two δ-H chains, which incorporated Cμ1 and variable domains. Secreted IgD was found in two heavily glycosylated isoforms, which are assembled as monomeric polypeptides associated with L chains. Secretory δ mRNA and IgD(+) plasma cells were detected in all immune tissues at a lower frequency than secretory IgM. Our data demonstrate that secretory IgD is more prevalent and widespread across taxa than previously thought, and thus illustrate the potential that IgD may have a conserved role in immunity.
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Elucidation of the role of infectious agents putatively involved in gill disease is commonly hampered by the lack of culture systems for these organisms. In this study, a farmed population of Atlantic salmon pre-smolts, displaying proliferative gill disease with associated Candidatus Branchiomonas cysticola, Ca. Piscichlamydia salmonis and Atlantic salmon gill pox virus (SGPV) infections, was identified. A subpopulation of the diseased fish was used as a source of waterborne infection towards a population of naïve Atlantic salmon pre-smolts. Ca. B. cysticola infection became established in exposed naïve fish at high prevalence within the first month of exposure and the bacterial load increased over the study period. Ca. P. salmonis and SGPV infections were identified only at low prevalence in exposed fish during the trial. Although clinically healthy, at termination of the trial the exposed, naïve fish displayed histologically visible pathological changes typified by epithelial hyperplasia and subepithelial inflammation with associated bacterial inclusions, confirmed by fluorescent in situ hybridization to contain Ca. B. cysticola. The results strongly suggest that Ca. B. cysticola infections transmit directly from fish to fish and that the bacterium is directly associated with the pathological changes observed in the exposed, previously naïve fish.
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Amoebic gill disease (AGD) caused by Neoparamoeba perurans, has emerged in Europe as a significant problem for the Atlantic salmon farming industry. Gross gill score is the most widely used and practical method for determining AGD severity on farms and informing management decisions on disease mitigation strategies. As molecular diagnosis of AGD remains a high priority for much of the international salmon farming industry, there is a need to evaluate the suitability of currently available molecular assays in conjunction with the most appropriate non-destructive sampling methodology. The aims of this study were to assess a non-destructive sampling methodology (gill swabs) and to compare a range of currently available real-time polymerase chain-reaction (PCR) assays for the detection of N. perurans. Furthermore a comparison of the non-destructive molecular diagnostics with traditional screening methods of gill scoring and histopathology was also undertaken. The study found that all molecular protocols assessed performed well in cases of clinical AGD with high gill scores. A TaqMan based assay (protocol 1) was the optimal assay based on a range of parameters including % positive samples from a field trial performed on fish with gill scores ranging from 0 to 5. A higher proportion of gill swab samples tested positive by all protocols than gill filament biopsies and there was a strong correlation between gill swabs tested by protocol 1 and gross gill score and histology scores. Screening for N. perurans using protocol 1 in conjunction with non-destructive gill swab samples was shown to give the best results.
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Intracellular parasites can alter the cellular machinery of host cells to create a safe haven for their survival. In this regard, microsporidia are obligate intracellular fungal parasites with extremely reduced genomes and hence, they are strongly dependent on their host for energy and resources. To date, there are few studies into host cell manipulation by microsporidia, most of which have focused on morphological aspects. The microsporidia Nosema apis and Nosema ceranae are worldwide parasites of honey bees, infecting their ventricular epithelial cells. In this work, quantitative gene expression and histology were studied to investigate how these two parasites manipulate their host’s cells at the molecular level. Both these microsporidia provoke infection-induced regulation of genes involved in apoptosis and the cell cycle. The up-regulation of buffy (which encodes a pro-survival protein) and BIRC5 (belonging to the Inhibitor Apoptosis protein family) was observed after infection, shedding light on the pathways that these pathogens use to inhibit host cell apoptosis. Curiously, different routes related to cell cycle were modified after infection by each microsporidia. In the case of N. apis, cyclin B1, dacapo and E2F2 were up-regulated, whereas only cyclin E was up-regulated by N. ceranae, in both cases promoting the G1/S phase transition. This is the first report describing molecular pathways related to parasite-host interactions that are probably intended to ensure the parasite’s survival within the cell.
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Gill diseases cause considerable losses in Norwegian salmon farming. In 2015, we characterized salmon gill poxvirus (SGPV) and associated gill disease. Using newly developed diagnostic tools, we show here that SGPV infection is more widely distributed than previously assumed. We present seven cases of complex gill disease in Atlantic salmon farmed in seawater and freshwater from different parts of Norway. Apoptosis, the hallmark of acute SGPV infection, was not easily observed in these cases, and qPCR analysis was critical for identification of the presence of SGPV. Several other agents including Costia-like parasites, gill amoebas, Saprolegnia spp. and bacteria were observed. The studied populations experienced significant mortalities, which increased to extreme levels when severe SGPV infections coincided with smoltification. SGPV infection appears to affect the smoltification process directly by affecting the gills and chloride cells in particular. SGPV may be considered a primary pathogen as it was often found prior to identification of complex gill disease. It is hypothesized that SGPV-induced gill damage may impair innate immunity and allow invasion of secondary invaders. The distinct possibility that SGPV has been widely overlooked as a primary pathogen calls for extended use of SGPV qPCR in Atlantic salmon gill health management.
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The B cell receptor and secreted antibody are at the nexus of humoral adaptive immunity. In this review, we summarize what is known of the immunoglobulin genes of jawed cartilaginous and bony fishes. We focus on what has been learned from genomic or cDNA sequence data, but where appropriate draw upon protein, immunization, affinity and structural studies. Work from major aquatic model organisms and less studied comparative species are both included to define what is the rule for an immunoglobulin isotype or taxonomic group and what exemplifies an exception.
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What is known about the biological activity of fish cytokines is reviewed. Most of the functional studies performed to date have been in teleost fish, and have focused on the induced effects of cytokine recombinant proteins, or have used loss- and gain-of-function experiments in zebrafish. Such studies begin to tell us about the role of these molecules in the regulation of fish immune responses and whether they are similar or divergent to the well-characterised functions of mammalian cytokines. This knowledge will aid our ability to determine and modulate the pathways leading to protective immunity, to improve fish health in aquaculture.
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Gas-exchange structures are critical for acquiring oxygen, but they also represent portals for pathogen entry. Local mucosal immunoglobulin responses against pathogens in specialized respiratory organs have only been described in tetrapods. Since fish gills are considered a mucosal surface, we hypothesized that a dedicated mucosal immunoglobulin response would be generated within its mucosa on microbial exposure. Supporting this hypothesis, here we demonstrate that following pathogen exposure, IgT + B cells proliferate and generate pathogen-specific IgT within the gills of fish, thus providing the first example of locally induced immunoglobulin in the mucosa of a cold-blooded species. Moreover, we demonstrate that gill microbiota is predominantly coated with IgT, thus providing previously unappreciated evidence that the microbiota present at a respiratory surface of a vertebrate is recognized by a mucosal immunoglobulin. Our findings indicate that respiratory surfaces and mucosal immunoglobulins are part of an ancient association that predates the emergence of tetrapods.
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Amoebic gill disease (AGD) caused by the ectoparasite Paramoeba perurans affects several cultured marine fish species worldwide. In this study, the morphology and ultrastructure of P. perurans in vitro and in vivo was investigated using scanning and transmission electron microscopy (SEM and TEM, respectively). Amoebae cultures contained several different morphologies ranging from a distinct rounded cell structure and polymorphic cells with pseudopodia of different lengths and shapes. SEM studies of the gills of AGD-affected Atlantic salmon, Salmo salar L., revealed the presence of enlarged swellings in affected gill filaments and fusion of adjacent lamellae. Spherical amoebae appeared to embed within the epithelium, and subsequently leave hemispherical indentations with visible fenestrations in the basolateral surface following their departure. These fenestrated structures corresponded to the presence of pseudopodia which could be seen by TEM to penetrate into the epithelium. The membrane-membrane interface contained an amorphous and slightly fibrous matrix. This suggests the existence of cellular glycocalyces and a role for extracellular products in mediating pathological changes in amoebic gill disease.
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Amoebic gill disease (AGD)is an increasing problem in Northern Europe and selective breeding has been shown as a good strategy to manage this disease in Tasmania. To explore the possibilities to perform selective breeding for AGD-resistance in Norwegian Atlantic salmon, two controlled challenge tests and one field (on-farm)test were performed with fish from two different breeding companies. For the field test, full-sibs of the same families from one of the controlled challenge tests were exposed to AGD naturally in a net-cage in the sea in Ireland. In all three experiments, two rounds of AGD infection were run, and all fish were assessed when the average gill score had progressed to an advanced threshold. Following assessment, all fish were bathed in freshwater. Heritability for resistance against both first and second AGD infection in the challenge tests and the field test ranged from 0.09 to 0.20. Estimated genetic correlation between gill score in the challenge test and field test resistance ranged from 0.02 to 0.34 and was not significantly different from 0. The genetic correlation between gill score in the first and second infection of the field test was 0.69 and ranged from 0.09 to 0.18 in the two challenge tests. These results indicate that AGD resistance in Norwegian Atlantic salmon can be improved by selective breeding. However, further work to optimize the controlled challenge test to better predict field test resistance is required.
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In this study we show that four arginase isoforms (arg1a, arg1b, arg2a, arg2b) exist in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). We have characterised these molecules in terms of a) sequence analysis, b) constitutive expression in different tissues, and modulated expression following c) stimulation of head kidney macrophages in vitro, or d) vaccination/infection with Yersinia ruckeri and e) parasite infection (AGD caused by Paramoeba perurans and PKD caused by Tetracapsuloides bryosalmonae). Synteny analysis suggested that these arginase genes are paralogues likely from the Ss4R duplication event, and amino acid identity/similarity analyses showed that the proteins are relatively well conserved across species. In rainbow trout constitutive expression of one or both paralogues was seen in most tissues but different constitutive expression patterns were observed for the different isoforms. Stimulation of rainbow trout head kidney macrophages with PAMPs and cytokines also revealed isoform specific responses and kinetics, with arg1a being particularly highly modulated by the PAMPs and pro-inflammatory cytokines. In contrast the type II arginase paralogues were induced by rIl-4/13, albeit to a lesser degree. Vaccination and infection with Y. ruckeri also revealed isoform specific responses, with variation in tissue expression level and kinetics. Lastly, the impact of parasite infection was studied, where down regulation of arg1a and arg1b was seen in two different models (AGD in salmon and PKD in trout) and of arg2a in AGD. The differential responses seen are discussed in the context of markers of type II responses in fish and paralogue subfunctionalization.
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Amoebic gill disease (AGD), caused by the marine ectoparasite Neoparamoeba perurans, is one of the most significant infectious diseases for Atlantic salmon (Salmo salar) aquaculture. Upon colonisation of the host gills, the parasite induces a marked hyperplasia and fusion of the lamellar epithelium, which can severely compromise the host if left untreated. In order to investigate the host response during the disease at a protein level, sequential gill samples were collected and analysed using two-dimensional electrophoresis (2DE), with peptides of interest subjected to LC-MS/MS. Samples were obtained from an experimental challenge using naïve Atlantic salmon smolts and cultured N. perurans. Sampling points were set at 1, 2, 3, 7, 14 and 21 days post-infection, and included both sub-clinical and clinical stages of the disease. A total of 23 proteins differentially expressed between non-infected and infected individuals were successfully identified by LC-MS/MS. Findings included upregulation of prohibitin, cyclophilin A, apolipoprotein A1, ictacalcin, RhoGDP dissociation inhibitor α, components of the heat shock proteins 70 family and histones H3a and H4, and downregulation of peroxiredoxin-5 and cofilin. Among the most significant protein functions identified were cell cycle regulation, cytoskeletal regulation, oxidative metabolism and immunity. This is the first sequential proteomic analysis of gills during N. perurans infection. It is believed that the use of non-target screening techniques can contribute to the knowledge of gill responses to injury and pathogenic insults, including AGD.
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Gill disease (GD) in farmed Atlantic salmon may be due to the impact of several agents that may interact. We followed the infections of four gill disease-associated agents during the first year of production in six farms; examining the correlation between their densities in the gills. These farms were located in an area with high risk of developing gill disease, and three of the farms were diagnosed with GD in the autumn. In the present study we show temporal changes in pathogens in salmon from all 6 farms, revealing recurring infection patterns by Desmozoon lepeophtherii and Candidatus Branchiomonas cysticola (C. B. cysticola), and large variation without an apparent pattern in densities of Ichthyobodo salmonis. Infections with the microsporidian D. lepeophtherii show a clear seasonal pattern, and high densities are associated with GD. Ca. B. cysticola appears not to be directly connected to the disease, albeit densities of this bacterium may coincide with high D. lepeophtherii densities. Amoebae (Paramoeba spp.) were only sporadically detected with low densities. Salmon with low condition had elevated densities of D. lepeophtherii and Ichthyobodo spp., while no such pattern was seen for the other pathogens. Challenge studies are needed to substantiate the role of D. lepeophtherii in GD, but realistic challenge model (i.e. bath) has so far not been developed. Based on the present molecular screening, we suggest that D. lepeophtherii infections both may be responsible for GD, and cause runting in farmed salmon populations. Statement of relevance This is an epizootiological study of Atlantic salmon and gill diseases. There is much known about pathogen in connection with GD outbreak, less is known about the occurrence of potential pathogen in healthy farmed salmon or how such infections develop over time. High densities of the microsporidian parasite Paranucleospora theridion in the gills coincide with gill disease, making P. theridion a possible primary source to this condition. Densities of Paranucleospora theridion, Candidatus Branchiomonas cysticola and Ichthyobodo salmonis may be correlated in farmed salmon populations with gill disease. P.theridion and I. salmonis densities are elevated in Atlantic salmon with low condition (‘runts’).
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Amoebic gill disease (AGD) is caused by Neoparamoeba perurans and represents a significant threat to Atlantic salmon marine farming in several countries worldwide. Sequential natural reinfection with N. perurans after treatment occurs after the first AGD outbreak during the grow-out phase of Atlantic salmon culture. Little is known about the immune response of Atlantic salmon following reinfection with N. perurans. In a previous single exposure study, using gill biopsies with various severity of AGD-lesion, we reported that the immune signalling in AGD-affected gills was highly dependent on the ratio of normal to hyperplastic gill tissues. Here, following experimental reinfection we investigated the transcriptional immune response in the gills of AGD-affected Atlantic salmon. Furthermore, we reported the inflammatory and immune response following a single exposure to N. perurans during late infection and compared it to the reinfected fish. Fish groups were selected based on the gross gill scores carried out during the trial. Two gill biopsies were collected from each AGD-affected individual, one with no lesion and one partly including AGD-lesion. Furthermore, gill biopsies were collected from uninfected controls. Pro-inflammatory and immune-related genes were characterised at the mRNA level by using a quantitative RT-PCR. Targeted immune genes included IL-1β, TCR-α chain, CD8, CD4, MHC-IIα, MHC-I, IgM and IgT. Histopathology and image analysis were used to assess the severity and to verify the reliability of the gross gill score as AGD severity assessment method to select fish groups for gene expression studies. Overall the expression at the mRNA level of the immune and pro-inflammatory genes analysed showed little change in AGD-affected gills of experimentally reinfected fish and of fish exposed once to N. perurans.
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Daily variation in the absorption, metabolism and excretion of toxic substances will ultimately determine the actual concentration to which the cells and tissues are exposed. In aquaculture, Atlantic salmon (Salmo salar) can be frequently exposed to hydrogen peroxide (H2O2) to treat topical skin and gill infections, particularly in relation to parasitic infections (e.g. sea lice Lepeophtheirus salmonis and amoebic gill disease caused by Neoparamoeba perurans). It is well accepted that the time of administration influences pharmacodynamics and pharmacokinetics of drugs which in turn affects their efficacy and toxicity. Consequently, a better understanding of drug side effects as a function of time of day exposure would help to improve treatment efficacy and fish welfare. To this end, salmon were exposed to H2O2 (1500 mg/L) for 20 min at six different times of the day during a 24-h cycle and we investigated the time-dependent effects of exposure on physiological stress (glucose, lactate and cortisol) and antioxidant enzyme expression (gpx1, cat, Mn-sod and hsp70) in liver and gills. In addition, at each sampling point, 8 control fish were also sampled. Our results revealed that the time of administration of H2O2 caused significant differences in the induction of both physiological and oxidative stress responses. Glucose and lactate were higher in the treated fish during daytime whereas cortisol levels appeared to be systematically increased (>1000 ng/mL) after H2O2 treatment irrespective of exposure time, although differences with control levels were higher during the day. In liver, gene expression of antioxidant enzymes displayed daily rhythmicity in both treated and control groups and showed higher mRNA expression levels in salmon treated with H2O2 at ZT6 (6 h after lights onset). In gills, rhythmic expression was only found for gpx1 in the control fish and for hsp70 and Mn-sod in the treated groups. However, in the treated salmon, higher gene expression levels of all the investigated enzymes were also observed at ZT6-10. Clock gene expression showed rhythmicity only in the liver in accordance with the daily rhythm of enzyme expression observed in this tissue. Altogether, this study provides first evidence of chronotoxicity in Atlantic salmon treated with H2O2 and suggests increased sublethal toxic effect during the first half of the day. These results have direct relevance to the salmon and broader aquaculture industry by optimising the timing of treatment administration, opening the door to chronotherapy to treat fish diseases.
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Presence of Paramoeba perurans, causative agent of amoebic gill disease was determined by qPCR in wild fish from Scottish coastal waters (n = 2. 348). Overall, the apparent prevalence was low. A single fish, a horse mackerel Trachurus trachurus, tested positive. This is the first report of detection of P. perurans in horse mackerel.
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As part of the mucosal immune system, the intestine plays a paramount role in the immune response in all vertebrates. The mucosal immunity includes inductive and effector tissues, and effector cells and molecules, all part of both innate and adaptive immunity and their connections. The digestive tract is the target organ of many parasites. In ectotherms, intestinal parasites include mainly flagellates, apicomplexans, myxozoans and helminths. The gut mucosal epithelium acts as a protective barrier against pathogens. Intestinal mucins and terminal carbohydrate residues are involved in the first contact pathogen-host, in some occasions through interaction with parasite lectins, as illustrated by mammalian and reptile Entamoeba spp. Mucins and their glycosylation patterns can change in reponse to intestinal parasites, including several piscine myxozoans and helminths. In addition, different pathogen recognizing receptors (PRRs) are sensed by the pathogen-associated molecular patterns (PAMPs) of invading pathogens. Although several PRRs have been thus far identified in ectotherms, little or no information is available on their presence in the intestine or on their involvement in parasite recognition. However, in mammals, several PRRs, mainly Toll-like receptors (TLRs) and nucleotide-binding oligomerization domains (NODs) play a significant role at the intestinal level by sensing pathogens and controlling inflammation and apoptosis, thus contributing to the maintenance of homeostasis. Several molecules and factors related to acute phase response (APR), such as AMPs, lysozyme, lectins, complement, cytokines and antiproteases can be activated at the gut level, in connection with innate and adaptive immune factors. Cellular components are also crucial in the intestinal immune response, and they include intestinal epithelial leucocytes (IECs), dendritic cells, macrophages, granulocytes and intraepithelial lymphocytes (IELs), all of them interacting at both the mucosal and systemic levels, to orchestrate an integrated immune response. The cross-talk between the different cellular types, involving also TLRs, AMPs, cytokines and other factors is well known in endotherms. The cellular reactions to several mammalian intestinal parasites are also well characterized. However, information is much scarcer for ectotherms. Although several types of immune cells (EGC/mast cells, other ganulocytes, rodlet cells (RCs), T and B cells, neuroendocrine cells) have been identified (mainly in teleosts), these cells are not well characterized and the knowledge of the different T cells subsets is in its infancy. In addition, though inflammatory reactions to several intestinal studied parasites have been reported, the cellular responses have been characterized for few species, mainly Enteromyxum spp. Available information points to the relevant role of RCs and EGCs/mast cells, but the cellular types have not been fully characterized, and the involvement of other granulocytes remains to be elucidated. In addition, the T and B cell responses and the role of secretory immunoglobulins are poorly known.
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Background: Benign prostatic hyperplasia (BPH) is characterized by increased tissue mass in the transition zone of the prostate, which leads to obstruction of urine outflow and significant morbidity in the majority of older men. Plasma markers of oxidative stress are increased in men with BPH but it is unclear whether oxidative stress and/or oxidative DNA damage are causal in the pathogenesis of BPH. Methods: Levels of 8-OH deoxyguanosine (8-OH dG), a marker of oxidative stress, were measured in prostate tissues from normal transition zone and BPH by ELISA. 8-OH dG was also detected in tissues by immunohistochemistry and staining quantitated by image analysis. Nox4 promotes the formation of reactive oxygen species. We therefore created and characterized transgenic mice with prostate specific expression of Nox4 under the control of the prostate specific ARR2PB promoter. Results: Human BPH tissues contained significantly higher levels of 8-OH dG than control transition zone tissues and the levels of 8-OH dG were correlated with prostate weight. Cells with 8-OH dG staining were predominantly in the epithelium and were present in a patchy distribution. The total fraction of epithelial staining with 8-OH dG was significantly increased in BPH tissues by image analysis. The ARR2PB-Nox4 mice had increased oxidative DNA damage in the prostate, increased prostate weight, increased epithelial proliferation, and histological changes including epithelial proliferation, stromal thickening, and fibrosis when compared to wild type controls. Conclusions: Oxidative stress and oxidative DNA damage are important in the pathogenesis of BPH. Prostate. © 2015 Wiley Periodicals, Inc.