Generation of Paramoeba perurans clonal cultures using flow cytometry and confirmation of virulence.

Amoebic gill disease (AGD) in farmed Atlantic salmon is caused by the amoeba Paramoeba perurans. The recent establishment of in vitro culture techniques for P. perurans has provided a valuable tool for studying the parasite in detail. In this study, flow cytometry was used to generate clonal cultures from single-sorted amoeba, and these were used to successfully establish AGD in experimental Atlantic salmon. The clonal cultures displayed differences in virulence, based on gill scores. The P. perurans load on gills, determined by qPCR analysis, showed a positive relationship with gill score, and with clonal virulence, indicating that the ability of amoebae to proliferate and/or remain attached on gills may play a role in virulence. Gill scores based on gross signs and histopathological analysis were in agreement. No association between level of gill score and specific gill arch was observed. It was found that for fish with lower gill scores based on histopathological examination, gross examination and qPCR analysis of gills from the same fish were less successful in detecting lesions and amoebae, respectively.

Experimental transmission of segmented filamentous bacteria (SFB) in rainbow trout Oncorhynchus mykiss.

Rainbow trout gastroenteritis (RTGE) has been the cause of acute mortality in farmed rainbow trout in Europe since 1992. Epidemiological analysis has indicated a strong association with high production levels and suggested an infectious aetiology. The condition is characterised by the presence of large numbers of segmented filamentous bacteria (SFB) in the intestine, but the role of these in the disease has not been confirmed, in part because the organisms cannot be cultured. Therefore, other approaches need to be developed to investigate the role of SFB in RTGE. Faecal material from clinically affected RTGE trout, either untreated or heat-inactivated, was administered to fish from a susceptible stock, to determine whether the SFB could be transferred artificially and survive in or colonise the new host. Using histology and nested PCR, SFB were detected in the pyloric caeca of fish 23 to 30 d after challenge with untreated faeces. Histological changes in the intestine and the presence of an unidentified Gram-negative coccus were also significantly associated with exposure to untreated faeces. Upregulation of IFN-γ, IL-17A/F and IL-22 gene expression in proximal intestine suggested a low-level immune response to the challenge.

Red mark syndrome in rainbow trout Oncorhynchus mykiss: investigation of immune responses in lesions using histology, immunohistochemistry and analysis of immune gene expression.

Red mark syndrome (RMS) is an economically significant disease which affects farmed rainbow trout in the United Kingdom, in the US and in mainland Europe. From the pattern of incidence, it appears to be transmissable, although no causative agent has yet been identified. RMS presents as a severe lymphocytic infiltration centred on the dermis and an alternative, host-focused approach was taken to understand the disease through investigating immune responses occurring in the lesion. Lesion and non-lesion skin at different stages of lesion development were examined using histochemistry and immunohistochemistry on paraffin sections. Expression of immune-related genes was compared between lesion and non-lesion skin. Investigation of early stage lesions suggested that the initial immune response is targeted at the region of the scale pocket, with lymphocyte infiltration and anti-tumour necrosis factor (TNF)-α staining of the stratum spongiosum, and increased numbers of major histocompatibility complex (MHC) II-positive cells immediately adjacent to the scale pocket. Gene expression analysis suggested a counterbalancing T helper (Th)1 and T regulatory (Treg) - type response is occurring in the lesion, with repression of Th2 and Th17-type responses.

An improved in situ hybridization method for the detection of fish pathogens.

A fluorescent in situ hybridization (FISH) method was developed for detection of infectious pancreatic necrosis virus (IPNV) in paraffin-embedded tissues of Atlantic salmon, Salmo salar L. Several methods of probe labelling and detection were evaluated and found unsuitable for FISH because of tissue autofluorescence. Likewise, the use of avidin to detect biotin-labelled probe was obviated by the presence of endogenous biotin. An existing approach, using digoxigenin (DIG)-labelled probes and detection by anti-DIG antibody-labelled with alkaline phosphatase, was modified to use a fluorescent substrate, 2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate/4-chloro-2-methylbenzene diazonium hemi-zinc chloride salt (HNPP/Fast Red TR). This improved method allowed sensitive detection of IPNV target, without interference from autofluorescence or endogenous alkaline phosphatase. Furthermore, the reporter produces a discrete, non-fading signal, which is particularly suitable for analysis by confocal microscopy.

Confirmation of Piscirickettsia salmonis as a pathogen in European sea bass Dicentrarchus labrax and phylogenetic comparison with salmonid strains.

European sea bass Dicentrarchus labrax from the Mediterranean were diagnosed with a severe encephalitis. Rickettsia-like organisms (RLOs) were associated with brain lesions in routine paraffin sections. These were found to share common antigens with the Piscirickettsia salmonis type-strain, LF-89, by indirect fluorescent antibody test (IFAT) and by immunohistochemistry (IHC). In addition, we compared the DNA sequences of the 16S rDNA and 16S-23S internal transcribed spacer region (ITS) with those published for P. salmonis strains and found that the sea bass piscirickettsia-like organism (SBPLO) was another strain of P. salmonis, closely related to the salmonid pathogens. Furthermore, we showed that the SBPLO possessed at least 2 ITS regions, 1 of which contained tRNA genes.