Multi-agent in situ hybridization confirms Ca. Branchiomonas cysticola as a major contributor in complex gill disease in Atlantic salmon.

Gill diseases may cause high mortalities in farmed Atlantic salmon. In seawater reared fish co-infections involving the epitheliocystis associated bacterium Ca. Branchiomonas cysticola, the microsporidian Desmozoon lepeophtherii, the causative agent of amoebic gill disease Paramoeba perurans and salmon gill poxvirus are common and histopathological lesions may be complex. Here, we report detection of these agents utilising multiplex real-time PCR and link the presence of agents to histopathologically visible gill lesions by in situ hybridisation (ISH) utilising RNAscope®. We show that Ca. Branchiomonas cysticola infections may remain undetected if diagnostic investigations are restricted to histopathology alone. Further, positive in situ labelling of Ca. Branchiomonas cysticola was observed within epitheliocysts, but also in small foci within areas of inflammation and necrosis in which histologically detectable epitheliocysts were not visible. In situ labelling of D. lepeophtherii corresponded well with tissue distribution patterns previously associated with this microsporidian. Salmon gill poxvirus was associated with apoptotic gill epithelial cells, while Ca. Piscichlamydia salmonis could not be associated with pathological changes. The multiplex real-time PCRs utilised were rapid and sensitive diagnostic tools and the results corresponded well with ISH. This study shows that the agents involved in complex gill disease can be linked to lesions using ISH and suggests that Ca. B. cysticola plays a crucial role in the development of gill disease in the farming of salmon in Norway.

First record of experimentally induced salmon gill poxvirus disease (SGPVD) in Atlantic salmon (Salmo salar L.).

Salmon gill poxvirus (SGPV) infection is a common denominator in many cases of complex gill disease in the Norwegian salmon farming industry and may, as a single agent infection, result in salmon poxvirus disease (SGPVD). Experiences from the field suggest that stress may be a decisive factor for the induction of SGPVD. Here we investigated the effect of stress hormone treatment on SGPV kinetics and disease development. In our experiment, Atlantic salmon were divided into four groups. Two groups of fish received an intraperitoneal injection of hydrocortisone dissolved in a fatty vehicle, whereas fish in the other two groups received a sham injection of the vehicle. After 24 h, one group with hydrocortisone injection and one with sham injection were exposed to dead SGPV-infected fish. Plasma cortisol level, virus kinetics, virus localization, and pathological gill were monitored for 4 weeks post-exposure. Hydrocortisone injected fish displayed higher plasma cortisol and SGPV loads than non-hydrocortisone treated fish. Signs of SGPVD and ensuing mortality appeared only in fish exposed to the virus and injected with hydrocortisone around 2 weeks post-exposure. No clinical signs of disease or mortality were recorded in the other groups. Further, gill histopathology in diseased fish correlated well with SGPV load, with the infection apparently confined to gill epithelial cells. The current findings suggest elevated plasma cortisol being a prerequisite for the development of SGPVD and recommend minimization of stressful farming activities, particularly if SGPV infection has been previously identified.

A case study of Desmozoon lepeophtherii infection in farmed Atlantic salmon associated with gill disease, peritonitis, intestinal infection, stunted growth, and increased mortality.

BACKGROUND: In September 2008, a disease outbreak characterized by acute, severe gill pathology and peritonitis, involving the gastrointestinal tract, was observed in an Atlantic salmon (Salmo salar L.) farm in north-western Norway. During subsequent sampling in November 2008 and January 2009, chronic proliferative gill inflammation and peritonitis was observed. Cumulative mortalities of 5.6-12.8% and severe growth retardation were observed. Routine diagnostic analysis revealed no diseases known to salmon at the time, but microsporidian infection of tissues was observed. METHODS: To characterize the disease outbreak, a combination of histopathology, in situ hybridization (ISH), chitin, calcofluor-white (CFW) staining, and real-time PCR were used to describe the disease progression with visualization of the D. lepeophtherii stages in situ. RESULTS: The presence of the microsporidian Desmozoon lepeophtherii was confirmed with real-time PCR, DNA sequencing and ISH, and the parasite was detected in association with acute lesions in the gills and peritoneum. ISH using a probe specific to small subunit 16S rRNA gene provided an effective tool for demonstrating the distribution of D. lepeophtherii in the tissue. Infection in the peritoneum seemed localized in and around pre-existing vaccine granulomas, and in the gastrointestinal walls. In the heart, kidney and spleen, the infection was most often associated with mononuclear leucocytes and macrophages, including melanomacrophages. Desmozoon lepeophtherii exospores were found in the nuclei of the gastrointestinal epithelium for the first time, suggesting a role of the gastrointestinal tract in the spread of spores to the environment. CONCLUSIONS: This study describes the progression of D. lepeophtherii disease outbreak in an Atlantic salmon farm without any other known diseases present. Using different methods to examine the disease outbreak, new insight into the pathology of D. lepeophtherii was obtained. The parasite was localized in situ in association with severe tissue damage and inflammation in the gills, peritoneal cavity and in the gastrointestinal (GI) tract that links the parasite directly to the observed pathology.

A sequential study of incomplete Freund's adjuvant-induced peritonitis in Atlantic cod.

Development of diagnostic and prophylactic methodologies is dependent on knowledge of the host's defence system and reaction to different vaccine adjuvants. Here we present a sequential morphological study of peritonitis and inflammatory cell processing of incomplete Freund's adjuvant (IFA) in intraperitoneally injected Atlantic cod. The peritoneal tissue responses were characterised using necropsy, histology and electron microscopy. An extensive inflammatory response as characterised by leukocyte morphology and contents of enzymes, presence of apoptotic cells and IFN-γ-expressing cells was observed. Three days post injection, IFA droplets were surrounded by different types of inflammatory cells and two different patterns could be discerned. The first was characterised by flattened and concentrically arranged interdigitating cells connected by desmosomes and with macrophage-like cells (MLCs) predominant in the periphery. The second type possessed four stratified layers with an inner layer containing many apoptotic MLCs; a second layer containing flattened and shrunken cells and outer layers comprising moderately flattened cells and an outermost layer of mononuclear cells expressing IFN-γ. Oil was detected both inside and outside MLCs. The two types of processes, of which the second was clearly stratified, were similar to those observed in other teleosts, indicating a variety of reaction modes or alternatively sequential process development. The numerous dead MLCs contributed to inflammation.

Exophiala angulospora causes systemic inflammation in atlantic cod Gadus morhua.

Species of Exophiala are opportunistic fungal pathogens that may infect a broad range of warm- and cold-blooded animals, including salmonids and Atlantic cod. In the present study, we observed abnormal swimming behaviour and skin pigmentation and increased mortality in cod kept in an indoor tank. Necropsy revealed foci of different sizes with a greyish to brownish colour in internal organs of diseased fish. The foci consisted of ramifying darkly pigmented fungal hyphae surrounded by distinct layers of inflammatory cells, including macrophage-like cells. In the inner layer with many hyphae, the macrophage-like cells were dead. We observed no apparent restriction of fungal growth by the inflammatory response. A darkly pigmented fungus was repeatedly isolated in pure culture from foci of diseased fish and identified as Exophiala angulospora using morphological and molecular characters. This species has not been previously reported to cause disease in cod, but has been reported as an opportunistic pathogen of both marine and freshwater fish. Based on the morphology and sequence analysis presented here, we conclude that E. angulospora caused the observed chronic multifocal inflammation in internal organs of cod, leading to severe disease and mortality.

Francisella noatunensis in Atlantic cod (Gadus morhua L.); waterborne transmission and immune responses.

This is the first report that confirms waterborne transmission of francisellosis in Atlantic cod. To investigate the transmission of disease, particle reduced water was transferred from a tank with intraperitoneally infected cod to a tank with healthy cod. Waterborne transmission of Francisella noatunensis was confirmed in the effluent group using immunohistochemistry and real-time quantitative PCR (RT-qPCR). The bacteria were located inside the accumulated macrophage-like cells. Specific and high antibody responses against live and inactivated bacteria were observed. Oil adjuvant had no effect on the antibody responses against inactivated F. noatunensis compared to saline formulation. The antigen epitope was a 20-25 kDa component of F. noatunensis suggested to be lipopolysaccharide detected by Western blot, Sypro Ruby and Silver staining. Systemic immune reactions were investigated by measuring the expression of IFN-γ, IL-1ß and IL-10 genes with RT-qPCR. After i.p. injection of live bacteria, a significant up-regulation of IFN-γ and IL-1ß expression was observed from 15 to 60 days post infection in spleen and head kidney. In intestine, IFN-γ was significantly up-regulated after 30 days whereas rectum showed no significant differences in expression. Elevated expression of IL-10 was observed in all the organs tested but was only significantly up-regulated at 60 days post infection in intestine from i.p. infected fish. For the cohabitant group, IL-1ß and IFN-γ was up-regulated in spleen whereas intestine and rectum showed a down-regulation after 60 days. IL-10 was up-regulated in intestine of cohabitant fish from day 30 to day 60. These results indicate that F. noatunensis infection provokes both specific antibody responses and long term inflammatory responses in cod. The present study provides new knowledge about infection routes and shows that both humoral and cellular defence mechanisms are triggered by F. noatunensis in cod.