Agnar Kvellestad

A Novel Betaproteobacterial Agent of Gill Epitheliocystis in Seawater Farmed Atlantic Salmon (Salmo salar)

Epitheliocystis, a disease characterised by cytoplasmic bacterial inclusions (cysts) in the gill and less commonly skin epithelial cells, has been reported in many marine and freshwater fish species and may be associated with mortality. Previously, molecular and ultrastructural analyses have exclusively associated members of the Chlamydiae with such inclusions. Here we investigated a population of farmed Atlantic salmon from the west coast of Norway displaying gill epitheliocystis. Although ‘Candidatus Piscichlamydia salmonis’, previously reported to be present in such cysts, was detected by PCR in most of the gill samples analysed, this bacterium was found to be a rare member of the gill microbiota, and not associated with the observed cysts as demonstrated by fluorescence in situ hybridization assays. The application of a broad range 16 S rRNA targeted PCR assay instead identified a novel betaproteobacterium as an abundant member of the gill microbiota. Fluorescence in situ hybridization demonstrated that this bacterium, tentatively classified as ‘Candidatus Branchiomonas cysticola’, was the cyst-forming agent in these samples. While histology and ultrastructure of ‘Ca. B. cysticola’ cysts revealed forms similar to the reticulate and intermediate bodies described in earlier reports from salmon in seawater, no elementary bodies typical of the chlamydial developmental cycle were observed. In conclusion, this study identified a novel agent of epitheliocystis in sea-farmed Atlantic salmon and demonstrated that these cysts can be caused by bacteria phylogenetically distinct from the Chlamydiae.

From Chronic Feed-Induced Intestinal Inflammation to Adenocarcinoma with Metastases in Salmonid Fish

Neoplasms in fish normally show poor abilities for metastasis, and there are no reports on intestinal cancer with metastasis to other organs. In aquaculture production, carnivorous salmonids in Northern Europe receive commercial feeds with plant ingredients. Such contents have been shown to cause chronic intestinal inflammation. Inflammation provokes carcinogenesis in the human gut, and here, we report a similar pathologic progression in salmonids. Nine commercially farmed groups of Atlantic salmon and rainbow trout (n = 39,160) and one experimental positive group (n = 789) fed the same commercial feed and two negative control groups (n = 3009) were investigated for the occurrence of intestinal tumors and metastases. Exposure period, gender, and sexual maturation were registered. Autopsy revealed an overall intestinal tumor occurrence of 10.62%, of which liver metastasis varied from 0% to 11.35% between the groups. Intestinal cancer prevalence increased from 0.50% to 14.81% during 4 months of feeding in the experimental group. A significant gender effect was registered in the commercially farmed groups but not in the experimental group. Histologic examination showed adenocarcinomas evolving through progressive epithelial dysplasia associated with severe chronic inflammation. One intestinal tumor was registered in one individual in the negative control groups. This is the first report on feed-induced intestinal carcinogenesis and metastasizing adenocarcinomas in fish fed an approved commercial diet. The pathogenesis was associated with a certain commercial diet provoking the inflammation-dysplasia-carcinoma sequence. The histologic progression was analogous to that of human colorectal cancer associated with inflammatory bowel disease.

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 hosts defence system and reaction to different vaccine adjuvants. Here we present a sequential morphological study of peritonitis and inflammatory cell processing of incomplete Freunds 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.

Nodavirus provokes subclinical encephalitis and retinochoroiditis in adult farmed Atlantic cod, Gadus morhua L.

Viral nervous necrosis (VNN) caused by beta-nodavirus affects many species of farmed marine fish, in particular juveniles. Apparently healthy, normally feeding, adult farmed Atlantic cod, Gadus morhua, were sampled in a farm 14 months after an outbreak of VNN with clinical signs. Following necropsy, brain and eye tissues were examined by histology, immunohistochemistry and polymerase chain reaction (PCR). Nodavirus-provoked cell death and inflammation was detected in eye and brain, particularly in the retina and cerebellum and differed from that previously described in Atlantic cod during clinical stages of VNN. Virus was detected both by PCR and immunohistochemistry. This is, to the best of our knowledge, the first description of pathological changes associated with chronic subclinical nodavirus infection in Atlantic cod. Our observations suggest that severe infection and pathological changes may go undetected if investigations are restricted to clinical examination and macroscopic evaluation at necropsy.

Piscine orthoreovirus (PRV) in red and melanised foci in white muscle of Atlantic salmon (Salmo salar).

Melanised focal changes (black spots) are common findings in the white skeletal muscle of seawater-farmed Atlantic salmon (Salmo salar). Fillets with melanised focal changes are considered as lower quality and cause large economic losses. It has been suggested that red focal changes (red spots) precede the melanised focal changes. In the present work, we examined different populations of captive and wild salmon for the occurrence of both types of changes, which were investigated for the presence of different viruses by immunohistochemistry and RT-qPCR. The occurrence of red or melanised foci varied significantly between the populations, from none in wild fish control group, low prevalence of small foci in fish kept in in-house tanks, to high prevalence of large foci in farm-raised salmon. Large amounts of Piscine orthoreovirus (PRV) antigen were detected in all foci. No other viruses were detected. Red focal changes contained significantly higher levels of PRV RNA than apparently non-affected areas in white muscle of the same individuals. Some changes displayed a transient form between a red and melanised pathotype, indicating a progression from an acute to a chronic manifestation. We conclude that PRV is associated with the focal pathological changes in the white muscle of farmed Atlantic salmon and is a premise for the development of focal melanised changes.

Molecular detection and genotyping of Aphanomyces astaci directly from preserved crayfish samples uncovers the Norwegian crayfish plague disease history.

Aphanomyces astaci causes crayfish plague in European freshwater crayfish, but most historical epizootics lack agent isolation and identification. Although declared as crayfish plague outbreaks by the Norwegian Competent Authorities, only presumptive diagnoses without agent isolation exist from Norwegian epizootics until 2005. Molecular methods now allow both A. astaci detection and genotype determination from preserved samples. We therefore aimed to (1) investigate molecularly if A. astaci was involved in a selection of mass-mortality events in Norwegian noble crayfish populations from 1971 to 2004, and (2) determine the eventually involved A. astaci genotype groups both from these historical and also more recent mass-mortality events. DNA was extracted directly from presumptively infected crayfish tissues, and screened by A. astaci specific qPCR. A representative selection of positive samples was confirmed by ITS-sequencing. Finally, genotype determination was performed with microsatellite markers that distinguish all known A. astaci genotype groups. The molecular examination detected A. astaci in crayfish materials from all examined mass-mortality events. The first event in 1971-1974 was caused by the A. astaci genotype group A, presumably the first genotype group that entered Europe more than 150 years ago. All later outbreaks were caused by the A. astaci genotype group B which was introduced to Europe by importation of signal crayfish in the 1960s. The results suggest that molecular methods can verify the involvement of A. astaci in the vast majority of observed crayfish mass mortalities in Europe whenever preserved materials exist. Moreover, microsatellite genotyping can reveal at least parts of the underlying epidemiology.

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.

Presence and interaction of inflammatory cells in the spleen of Atlantic cod, Gadus morhua L., infected with Francisella noatunensis

Serious infectious diseases, accompanied by macrophage-dominated chronic inflammation, are common in farmed Atlantic cod. To increase knowledge relating to morphological aspects of such inflammatory responses, cod were challenged with Francisella noatunensis, an important bacterial pathogen of this fish species. Tissue and cell dynamics in the spleen were examined sequentially over 60 days. Small clusters of mainly macrophage-like cells (MLCs) staining for non-specific esterase and acid phosphatase developed with time. These foci were transiently infiltrated by pleomorphic proliferating cells of unknown nature and by granulocyte-like cells (GCLCs) staining for peroxidase and lysozyme. The latter cell type, which appeared to be resident in the red pulp of control fish, migrated into the inflammatory foci of infected fish. Cells expressing genes encoding IFN-γ and IL-8 increased in number during the study period. Bacteria were detected only in the MLCs and their number increased despite the extensive inflammation. Our results demonstrate an intimate spatial relationship in inflammatory foci between at least three cell types. The presence of GCLCs, together with MLCs, suggests pyogranulomatous inflammation as a more appropriate descriptive term than granulomatous inflammation.

Fish mucosal immunity: gill

The gills of fish are covered with a thin mucous membrane, which has a large external surface area that is in continuous contact with ambient water irrigating the organ during respiration. Large amounts of soluble and particulate substances, including various pathogens, may penetrate this mucosal barrier to cause local and/or systemic infections followed by mucosal and systemic immune responses. While substances that do not pose a threat to the fish may be tolerated by the gill immune system, pathogens have been found to induce innate and adaptive immune responses when entering the fish through the gills. Adaptive responses provide the basis for immune memory and consequently vaccination. Recently, investigations on the molecular level have added to our understanding of how immune responses of the gills are triggered. Such studies have heightened our understanding of host–pathogen interactions and are enhancing the development of immersion vaccines. This chapter describes teleost gill development, physiology, cellular composition, and immune responses to selected pathogens known to affect the gills.

Morphological and functional development of the interbranchial lymphoid tissue (ILT) in Atlantic salmon (Salmo salar L)

The interbranchial lymphoid tissue (ILT) of Atlantic salmon originates from an embryological location that in higher vertebrates gives rise to both primary and secondary lymphoid tissues. Still much is unknown about the morphological and functional development of the ILT. In the present work a standardized method of organ volume determination was established to study its development in relation to its containing gill and the thymus. Based on morphological findings and gene transcription data, the ILT shows no signs of primary lymphoid function. In contrast to the thymus, an ILT-complex first became discernible after the yolk-sac period. After its appearance, the ILT-complex constitutes 3-7% of the total volume of the gill (excluding the gill arch) with the newly described distal ILT constituting a major part, and in adult fish it is approximately 13 times larger than the thymus. Confined regions of T-cell proliferation are present within the ILT. Communication with systemic circulation through the distal ILT is also highly plausible thus offering both internal and external recruitment of immune cells in the growing ILT.