Biolayer interferometry is insufficiently sensitive for direct measurement of IgM quantity and avidity in Atlantic salmon (Salmo salar) serum.

Quantification of specific antibodies underpins the assessment of adaptive immunity in response to vaccination or infection and is performed by enzyme-linked immunosorbent assay (ELISA). A biolayer interferometry (BLI) assay was recently developed that simultaneously quantifies IgM antibodies and their avidity in giant grouper (Epinephelus lanceolatus) sera and proved to be a robust, repeatable and more high-throughput alternative to ELISA [1]. Here we attempted to optimise a similar single-step BLI assay using an Octet HTX instrument to quantify IgM specific to the hapten 2,4-dinitrophenol (DNP) in serum from Atlantic salmon (Salmo salar) primed and boosted with DNP conjugated to keyhole limpet hemocyanin.

Immersion challenge of naïve Atlantic salmon with cultured Nolandella sp. and Pseudoparamoeba sp. did not increase the severity of Neoparamoeba perurans-induced amoebic gill disease (AGD).

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.

Seawater transmission and infection dynamics of pilchard orthomyxovirus (POMV) in Atlantic salmon (Salmo salar).

The Tasmanian salmon industry had remained relatively free of major viral diseases until the emergence of pilchard orthomyxovirus (POMV). Originally isolated from wild pilchards, POMV is of concern to the industry as it can cause high mortality in farmed salmon (Salmo salar). Field observations suggest the virus can spread from pen to pen and between farms, but evidence of passive transmission in sea water was unclear. Our aim was to establish whether direct contact between infected and naïve fish was required for transmission, and to examine viral infection dynamics. Atlantic salmon post-smolts were challenged with POMV by either direct exposure via cohabitation or indirect exposure via virus-contaminated sea water. POMV was transmissible in sea water and direct contact between fish was not required for infection. Head kidney and heart presented the highest viral loads in early stages of infection. POMV survivors presented low viral loads in most tissues, but these remained relatively high in gills. A consistent feature was the infiltration of viral-infected melanomacrophages in different tissues, suggesting an important role of these in the immune response to POMV. Understanding POMV transmission and host-pathogen interactions is key for the development of improved surveillance tools, transmission models and ultimately for disease prevention.

Defining the aetiology of amoebic diseases of aquatic animals: trends, hurdles and best practices.

Disease caused by parasitic amoebae impacts a range of aquatic organisms including finfish, crustaceans, echinoderms and molluscs. Despite the significant economic impact caused in both aquaculture and fisheries, the aetiology of most aquatic amoebic diseases is uncertain, which then affects diagnosis, treatment and prevention. The main factors hampering research effort in this area are the confusion around amoeba taxonomy and the difficulty proving that a particular species causes specific lesions. These issues stem from morphological and genetic similarities between cryptic species and technical challenges such as establishing and maintaining pure amoeba cultures, scarcity of Amoebozoa sequence data, and the inability to trigger pathogenesis under experimental conditions. This review provides a critical analysis of how amoebae are commonly identified and defined as aetiological agents of disease in aquatic animals and highlights gaps in the available knowledge regarding determining pathogenic Amoebozoa.

A High Throughput Viability Screening Method for the Marine Ectoparasite Neoparamoeba perurans.

The marine protozoan parasite Neoparamoeba perurans has been established as the causative agent for amoebic gill disease (AGD) in Atlantic salmon (Salmo salar). Freshwater bathing is the only routinely used treatment for AGD in Australia while hydrogen peroxide (H2O2) is used in countries with cooler water temperatures. The identification of new treatments that do not rely on either freshwater or H2O2 bathing is highly sought. However, in vitro based methods for high throughput screening of antiparasitic compounds have not been established for this parasite. To this end the present study evaluated two in vitro bioassays based on metabolic energy production and cellular membrane integrity to distinguish between amoebistatic (crenated or pseudocyst forms with recovery possible) and amoebicidal (death) activity. Amoebae were subject to either freshwater, H2O2 or chloramine-T for 4h treatment and assessed 24h after recovery. Visualization by microscopy and bioassay assessment 24h post-treatment confirmed that H2O2 and freshwater are 95% amoebicidal albeit due to different mechanisms of action. These data are consistent with other studies where amoebae have been observed to recover following exposure to these compounds and provide evidence for the inclusion of a recovery component to differentiate between the mechanism of action of amoebicidal and amoebistatic treatments. Together these bioassays are a critical tool for high throughput screening of novel and more effective treatments against AGD.

A diversity of amoebae colonise the gills of farmed Atlantic salmon (Salmo salar) with amoebic gill disease (AGD).

Neoparamoeba perurans is the aetiological agent of amoebic gill disease (AGD) in salmonids, however multiple other amoeba species colonise the gills and their role in AGD is unknown. Taxonomic assessments of these accompanying amoebae on AGD-affected salmon have previously been based on gross morphology alone. The aim of the present study was to document the diversity of amoebae colonising the gills of AGD-affected farmed Atlantic salmon using a combination of morphological and sequence-based taxonomic methods. Amoebae were characterised morphologically via light microscopy and transmission electron microscopy, and by phylogenetic analyses based on the 18S rRNA gene and cytochrome oxidase subunit I (COI) gene. In addition to N. perurans, 11 other amoebozoans were isolated from the gills, and were classified within the genera Neoparamoeba, Paramoeba, Vexillifera, Pseudoparamoeba, Vannella and Nolandella. In some cases, such as Paramoeba eilhardi, this is the first time this species has been isolated from the gills of teleost fish. Furthermore, sequencing of both the 18S rRNA and COI gene revealed significant genetic variation within genera. We highlight that there is a far greater diversity of amoebae colonising AGD-affected gills than previously established.