Rn Morrison

Neoparamoeba perurans is a cosmopolitan aetiological agent of amoebic gill disease

Previously we described a new member of the Neoparamoeba genus, N. perurans, and showed that it is an agent of amoebic gill disease (AGD) of Atlantic salmon Salmo salar cultured in southeast Tasmania, Australia. Given the broad distribution of cases of AGD, we were interested in extending our studies to epizootics in farmed fish from other sites around the world. Oligonucleotide probes that hybridise with the 18S rRNA of N. perurans, N. branchiphila or N. pemaquidensis were used to examine archival samples of AGD in Tasmania as well as samples obtained from 4 host fish species cultured across 6 countries. In archival samples, N. perurans was the only detectable amoeba, confirming that it has been the predominant aetiological agent of AGD in Tasmania since epizootics were first reported. N. perurans was also the exclusive agent of AGD in 4 host species across 6 countries. Together, these data show that N. perurans is a cosmopolitan agent of AGD and, therefore, of significance to the global mariculture industry.

Development of a diagnostic PCR to detect Neoparamoeba perurans , agent of amoebic gill disease

The recent description of Neoparamoeba perurans as an aetiological agent of amoebic gill disease (AGD) advanced our understanding of the condition and has forced a re-evaluation of methods used for the diagnosis of AGD. Currently, there are no tools available that are both specific for N. perurans and suitable for a routine diagnostic procedure. Therefore, in this study we describe an assay to detect N. perurans. The assay, which utilizes PCR to amplify the N. perurans 18S rRNA gene, was shown to be specific and highly sensitive. Neoparamoeba perurans was detected in both gill samples and primary isolates of non-cultured gill-derived amoebae obtained during necropsy or biopsy from AGD-affected Atlantic salmon, Salmo salar L. The PCR-based assay provides a simple, flexible tool that will be a useful addition to the diagnostic repertoire for AGD. It may also be used for the genotypic screening of trophozoites during culture and could facilitate further epidemiological and ecological studies of AGD.

The histopathological effects of a levamisole-adjuvanted Vibrio anguillarum vaccine on Atlantic salmon (Salmo salar L.)

Abstract Atlantic salmon (Salmo salar L.) were administered a levamisole-adjuvanted or non-adjuvanted Vibrio anguillarum vaccine by either bath or intraperitoneal (IP) injection. The potential side effects of vaccination were investigated at a histological level, with the skin, gills, anterior kidney and spleen all examined. At a gross level, no pathological changes were evident in any organs. Observations of histological structures found that the skin, anterior kidney and spleen were not affected by vaccination. However pathological changes were observed in the gills of fish treated with the adjuvanted vaccine by both routes of administration. Pathology of the gills was multifocal and included hyperplasia of mucous cells, lamellar oedema, proliferation of chloride cells, inflammation and necrosis. Hyperplasia and hypertrophy of epithelial cells resulted in lamellar fusion. The abundance of lamellar associated mucous cells significantly increased in the gills of fish from groups administered the levamisole-adjuvanted vaccine (IP and bath) (P 0.05).

Description of an Atlantic salmon (Salmo salar L.) type II interleukin-1 receptor cDNA and analysis of interleukin-1 receptor expression in amoebic gill disease-affected fish.

Previously, we showed that IL-1β transcription is induced in the gills of amoebic gill disease (AGD)-affected fish in an AGD lesion-restricted fashion. However, in this environment, there is very little evidence of inflammation on histopathological or transcriptional levels and we hypothesised that aberrant signalling may occur. As a first step in investigating this issue, we cloned and sequenced the Atlantic salmon IL-1 receptor type II (IL-1RII) mRNA, and then examined the expression of both the IL-1RI (IL-1 receptor-like protein) and II during Neoparamoeba perurans infection. In gill lesions from AGD-affected fish, a step-wise temporal increase in the relative expression of IL-1β coincided with a significant reduction in IL-1RI, whereas the IL-1RII mRNA remained unchanged. Down-regulation of IL-1RI could explain the paucity of inflammation in affected tissue, although simultaneous up-regulation of IL-1β-inducible transcripts indicated that this is not due to a complete blockage of the IL-1RI pathway. Rather, it appears that IL-1RI transcription is reduced and this rate limits the effects of chronic IL-1β over-expression.

Snapper (Pagrus auratus) leucocyte proliferation is synergistically enhanced by simultaneous stimulation with LPS and PHA

Important channels of communication between mammalian leucocytes have long been recognised. Here, data are reported that suggest similar integrations may occur between snapper leucocytes upon mitogen stimulation. Cell surface immunoglobulin (IgM) expression was used in conjunction with intracellular fluorescence staining and flow cytometry to differentiate proliferating peripheral blood leucocyte subsets (PBLs). Independent activation using phytohaemagglutinin (PHA) or lipopolysaccharide (LPS) drove both mIg(-)and mIg(+)cells into cycle. It is not known if the proliferation of mIg(+)cells was mediated by a mutually exclusive effect of the mitogen on each cell population, cognate cellular interaction or a soluble growth factor. Simultaneous activation of PBLs with PHA and LPS consistently induced significantly more cells to proliferate than the sum of proliferating cells stimulated solely with PHA or LPS. Together, the results suggest that different leucocyte subsets have the capability to influence their respective responses to mitogenic stimulation. Therefore, like in the mammalian immune system, communication may occur between snapper leucocyte subsets.

A screen of mammalian antibodies on snapper (Pagrus auratus, Sparidae) peripheral blood leukocytes reveals cross reactivity of an anti-human CD3 antibody with a population of mIg− cells

Detailed immunological studies of the teleosts have been hampered by a lack of antibodies against cell-specific markers. Furthermore, where antibodies have been raised, in many instances they have been found to be species-specific. In comparison, many monoclonal and polyclonal antibodies exist with specificities for mammalian proteins and glycoproteins that effectively differentiate leukocyte sub-populations. In this study, we have tested a panel of 54 commercial antibodies against human and murine cell surface receptors for their ability to bind leukocytes isolated from the peripheral blood of snapper (Pagrus auratus). From this panel, one antibody, A452, which is specific for the intracytoplasmic tail of the epsilon (epsilon) chain of the T cell receptor-associated CD3 complex (CD3epsilon) bound to a subpopulation of peripheral blood leukocytes. Mutually exclusive counterstaining was observed when this antibody was used in conjunction with a monoclonal anti-snapper immunoglobulin antibody. This suggests that A452 may be binding to putative snapper T cells.

Affinity purification and partial characterisation of systemic immunoglobulin of the snapper (Pagrus auratus)

Immunoglobulins (Ig) from tank housed wild caught snapper (Pagrus auratus, Bloch and Schneider) were single step purified from serum using staphylococcal protein A (SpA) affinity chromatography. Purified proteins were analysed using SDS-PAGE under reducing, non-reducing denaturing and PAGE under native conditions. Under native conditions, a single population of Ig was identified and using gel filtration chromatography was found to have an approximate molecular weight of 766 kDa. Further, using SDS-PAGE under non-denaturing reducing conditions the single population of Ig was found to be heterogeneous in subunit linkages. Ig subjected to fully reducing conditions dissociated into heavy (H) and light (L) chain polypeptides. Two H and two L chain variants based differences in electrophoretic mobility were detected by SDS-PAGE, however evidence of an isotypic disparity was not proven. The L chains were shown to be approximately 30.2 and 29.0 kDa in molecular weight while the H chains were 71.8 and 67.7 kDa, suggesting that the native molecule was likely to be tetrameric in structure. Polyclonal antisera against snapper Ig were produced and screened by indirect ELISA, Western blot and flow cytometry. Specificity of the antisera was demonstrated by probing against purified Ig, whole snapper serum and heterologous serum in Western blots. Antisera reacted predominantly with the H chains of purified Ig however antisera reacted with both H and L chain variants in reduced serum. A lack of cross-reactivity with five of six heterologous sera tested, demonstrated a high degree of specificity of both antiserum. In flow cytometry, both antiserum bound to the putative B cell population, reacting with 40.8% (rabbit 1) and 29.5% (rabbit 2) of the gated lymphocytes in the peripheral blood.

Support for the coevolution of Neoparamoeba and their endosymbionts, Perkinsela amoebae-like organisms.

Some of the species from the genus Neoparamoeba, for example N. perurans have been shown to be pathogenic to aquatic animals and thus have economic significance. They all contain endosymbiont, Perkinsela amoebae like organisms (PLOs). In this study we investigated phylogenetic ambiguities within the Neoparamoeba taxonomy and phylogenetic congruence between PLOs and their host Neoparamoeba to confirm the existence of a single ancient infection/colonisation that led to cospeciation between all PLOs and their host Neoparamoeba. DNA was extracted and rRNA genes from host amoeba and endosymbiont were amplified using PCR. Uncertainties in the Neoparamoeba phylogeny were initially resolved by a secondary phylogenetic marker, the internal transcribed spacer 2 (ITS2). The secondary structure of ITS2 was reconstructed for Neoparamoeba. The ITS2 was phylogenetically informative, separating N. pemaquidensis and N. aestuarina into distinct monophyletic clades and designating N. perurans as the most phylogenetically divergent Neoparamoeba species. The new phylogenetic data were used to verify the tree topologies used in cophylogenetic analyses that revealed strict phylogenetic congruence between endosymbiotic PLOs with their host Neoparamoeba. Strict congruence in the phylogeny of all PLOs and their host Neoparamoeba was demonstrated implying that PLOs are transmitted vertically from parent to daughter cell.

Isolation of RAG-1 and IgM transcripts from the striped trumpeter (Latris lineata), and their expression as markers for development of the adaptive immune response

A partial sequence of the recombination activating gene-1 (RAG-1) and several full length sequences of the immunoglobulin M (IgM) heavy chain mRNA were obtained from the striped trumpeter (Latris lineata). The RAG-1 fragment consisted of 205 aa and fell within the core region of the open reading frame. The complete IgM heavy chain sequences translated into peptides ranging between 581 and 591 aa. Both genes showed good homology to other vertebrate sequences. The expression of the two genes was assessed throughout the early developmental stages of striped trumpeter larvae (5-100 dph) and used as markers to follow the ontogeny of the adaptive immune response. Using RT-PCR, RAG-1 mRNA expression was detectable at 5 dph and remained so until 80 dph, before becoming undetectable at 100 dph. IgM expression was also detectable at 5 dph, and remained so throughout. These patterns of expression may suggest that the striped trumpeter possess mature B cells with surface IgM at 100 dph. However, complete immunological competence is likely not reached until some time later. The early detection of IgM mRNA at 5 dph led to the investigation of its presence in oocytes. Both RAG-1 and IgM mRNA transcripts were detected in unfertilized oocytes, suggesting that they are maternally transferred. The biological significance of such a phenomenon remains to be investigated.

Immunohistochemical detection of anterior gradient‐2 in the gills of amoebic gill disease‐affected Atlantic salmon, Salmo salar L.

Sea-farmed Atlantic salmon, Salmo salar L. , rainbow trout, Oncorhynchus mykiss , Chinook salmon, O. tshawytscha, coho salmon, O. kisutch and turbot, Psetta maxima (L.) are all susceptible to a condition known as amoebic gill disease