Novel DNA-based in situ hybridization method to detect Desmozoon lepeophtherii in Atlantic salmon tissues.

The microsporidian Desmozoon lepeophtherii Freeman and Sommerville, 2009 is considered significant in the pathogenesis of gill disease in Atlantic salmon (Salmo salar Linnaeus, 1758). Due to the difficulty in detecting D. lepeophtherii in tissue sections, infections are normally diagnosed by molecular methods, routine haematoxylin and eosin (H&E) stained gill tissue sections and the use of other histochemical stains and labels to confirm the presence of spores. An in situ hybridization (ISH) protocol specific for D. lepeophtherii was developed using DIG-labelled oligonucleotide probes. Diseased Atlantic salmon gills were analysed by ISH, calcofluor white (CW) and H&E. All methods showed high levels of specificity (100%) in their ability to detect D. lepeophtherii, but the sensitivity was higher with ISH (92%), compared with CW (64%) and the presence of microvesicles on H&E stained sections (52%). High levels of D. lepeophtherii spores were significantly associated (p < .05) with the development of D. lepeophtherii-associated pathology in the gills, with Ct values below 19 and over 100 microsporidia/10 mm2 of gill tissue (from the ISH counts) seemingly necessary for the development of microvesicles. The ISH method has the advantage over other histological techniques in that it allows all life stages of the microsporidian to be detected in infected salmon gill tissue sections.

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

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.

Multilocus Sequence Typing (MLST) and Random Polymorphic DNA (RAPD) Comparisons of Geographic Isolates of Neoparamoeba perurans, the Causative Agent of Amoebic Gill Disease.

Neoparamoba perurans, is the aetiological agent of amoebic gill disease (AGD), a disease that affects farmed Atlantic salmon worldwide. Multilocus sequence typing (MLST) and Random Amplified Polymorphic DNA (RAPD) are PCR-based typing methods that allow for the highly reproducible genetic analysis of population structure within microbial species. To the best of our knowledge, this study represents the first use of these typing methods applied to N. perurans with the objective of distinguishing geographical isolates. These analyses were applied to a total of 16 isolates from Australia, Canada, Ireland, Scotland, Norway, and the USA. All the samples from Australia came from farm sites on the island state of Tasmania. Genetic polymorphism among isolates was more evident from the RAPD analysis compared to the MLST that used conserved housekeeping genes. Both techniques consistently identified that isolates of N. perurans from Tasmania, Australia were more similar to each other than to the isolates from other countries. While genetic differences were identified between geographical isolates, a BURST analysis provided no evidence of a founder genotype. This suggests that emerging outbreaks of AGD are not due to rapid translocation of this important salmonid pathogen from the same area.

Detection and characterisation of haplosporidian parasites of the blue mussel Mytilus edulis, including description of the novel parasite Minchinia mytili n. sp.

The edible mussel Mytilus edulis is a major aquaculture commodity in Europe, with 168000 t produced in 2015. A number of abundant, well characterised parasites of the species are known, though none are considered to cause significant mortality. Haplosporida (Rhizaria, Endomyxa) is an order of protistan parasites of aquatic invertebrates, the best studied of which are the oyster pathogens Haplosporidium nelsoni and Bonamia ostreae. While these species are well characterised within their hosts, the diversity, life-cycle and modes of transmission of haplosporidians are very poorly understood. Haplosporidian parasites have previously been reported from Mytilus spp., however the majority of these remain uncharacterised, and no molecular data exist for any species. In this study, we identified 2 novel haplosporidian parasites of M. edulis present in the UK. The first of these, observed by light microscopy and in situ hybridisation infecting the gills, mantle, gonadal tubules and digestive connective tissues of mussels in the Tamar estuary, England, we describe as Minchinia mytili on the basis of 18S sequence data. The second, observed infecting a single archive specimen collected in Loch Spelve, Mull, Scotland, infects the foot muscle, gills and connective tissue of the digestive gland. Sequence data places this parasite in an uncharacterised clade of sequences amplified from tropical bivalve guts and water samples, sister to H. nelsoni. Screening of water and sediment samples collected at the sample site in the Tamar estuary revealed the presence of both sequence types in the water column, suggesting host-free or planktonic life stages.

Gene expression analysis of Atlantic salmon gills reveals mucin 5 and interleukin 4/13 as key molecules during amoebic gill disease.

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.

Local and systemic humoral immune response in farmed Atlantic salmon (Salmo salar L.) under a natural amoebic gill disease outbreak.

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.

Characterization of a novel alloherpesvirus from Atlantic cod (Gadus morhua).

Alloherpesviruses affect freshwater and marine fish species. The aim of the current study was to characterize a novel alloherpesvirus in Atlantic cod (Gadus morhua). Samples were processed for histopathology, transmission electron microscopy (TEM), virus isolation, molecular characterization, and in situ hybridization (ISH). Histopathology revealed that the infection was restricted to the gills and that it induced cytomegaly in infected cells. By TEM, numerous viral particles with morphology compatible with a herpesvirus were observed inside the cytomegalic cells. To characterize this new agent, polymerase chain reaction amplified regions of the ATPase subunit of the terminase, and DNA polymerase genes were sequenced. Phylogenetic analysis revealed strongest similarity with alloherpesviruses belonging to the genus Ictalurivirus and Salmonivirus. The ISH showed specific labeling of nuclear inclusions in the cytomegalic cells. While virus isolation was unsuccessful, the results obtained through different diagnostic tests in the present study confirm the discovery of a new alloherpesvirus affecting Atlantic cod. The authors propose the formal species designation Gadid herpesvirus 1 (GaHV-1) to be considered for approval by the International Committee on the Taxonomy of Viruses.

In vitro assessment of the chemotherapeutic action of a specific hydrogen peroxide, peracetic, acetic, and peroctanoic acid-based formulation against the free-living stages of Ichthyophthirius multifiliis (Ciliophora).

Traditionally, malachite green administrated as in-bath treatment was the most effective and common strategy used in freshwater aquaculture systems to control infections of the ciliate protozoan parasite Ichthyophthirius multifiliis Fouquet, 1876. After the ban of malachite green in the USA and Europe to be used in fish for human consumption, there has been extensive research destined to find efficacious replacements. Recently, peracetic acid-based compounds have demonstrated a strong cytotoxic effect in vitro and in vivo against I. multifiliis. In the present study, we tested the efficacy of a hydrogen peroxide, peracetic, acetic and peroctanoic acid-based formulation (HPPAPA) to eliminate the free-living stages of I. multifiliis (tomonts, cysts and theronts). The results obtained showed that the administration of low doses (8, 12 or 15 mg/l) of a specific HPPAPA-based product during a short window of exposure (60 min) kills nearly all free-living stages of I. multifiliis (theronts, tomonts and cysts) within the window of treatment (∼100% mortality for all the stages; one-way ANOVA, P ≤ 0.001). Of note, even the lowest concentration of HPPAPA tested (8 mg/l) was able to disrupt normal cyst development and therefore theront release. The demonstrated in vitro efficacy of the peracetic acid-based product tested on the present study suggests its great potential to control I. multifiliis infections in commercial aquacultural systems.