Passive Immunization of Farmed Fish.

The fast growth and potential of global aquaculture has necessitated the adoption of sustainable and welfare-oriented therapeutics and prophylactic strategies. Knowledge gathered from studies about maternal passive immunity in fish and fish-to-fish passive immunization experiments supports the concept of using therapeutic Abs (of piscine and other vertebrate origin) in aquaculture. Traditional Ab formats (IgG, IgM) are expensive and laborious to produce; however, the introduction of new rAb fragments and single-domain Abs have reinvigorated the concept of passive immunization. This review will focus primarily on farmed salmonids (salmon and trout) within a comparative context and will give an overview of the basic principles and scientific premises for the passive immunization strategy, including existing and emerging Ab therapeutics.

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.

Transcriptional characterization of the T cell population within the salmonid interbranchial lymphoid tissue.

Previously, our group has shown that the interbranchial lymphoid tissue (ILT) is a distinct structure largely consisting of T cells embedded in a meshwork of epithelial cells, with no direct resemblance to previously described lymphoid tissues. In this study, we aim to focus on the T cell population and the possibility of the ILT being a thymus analog. By characterizing structural responsiveness to Ag challenge, the presence of recombination activating genes, and different T cell-related transcripts, we attempt to further approach the immunological function of the ILT in salmonid gills. In addition to eight healthy individuals, a group of eight infectious salmon anemia virus-challenged fish were included to observe T cell responses related to infection. The results showed reduced size of ILT in the infected group, no expression of RAG-1 and -2, and a high degree of T cell diversity within the ILT. Taking into account that the ILT can be regarded as a strategically located T cell reservoir and possibly an evolutionary forerunner of mammalian MALTs right at the border to the external environment, the alteration in transcription observed may likely represent a shift in the T cell population to optimize local gill defense mechanisms.

Substitution of dietary fish oil with plant oils is associated with shortened mid intestinal folds in Atlantic salmon (Salmo salar).

BACKGROUND: Fish meal and fish oil are increasingly replaced by ingredients from terrestrial sources in the feeds for farmed salmonids due to expanding production and reduced availability of marine feed raw material. Fish oil that is rich in n-3 polyunsaturated fatty acids is considered beneficial to human health in general and to prevent intestinal inflammation and carcinogenesis in particular. In contrast, n-6 fatty acids that are present in many vegetable oils have been associated with increased risk of colitis and colon cancer in rodents and humans, as well as lowered transcription levels of certain stress and antioxidant-related genes in Atlantic salmon.The aim of the present study was to investigate the intestinal health in Atlantic salmon fed with different vegetable oils as partial substitutes of fish oil in the diet. A feed trial lasting for 28 weeks included one reference diet containing fish oil as the sole lipid source and three diets where 80% of the fish oil was replaced by a plant oil blend with either olive oil, rapeseed oil or soybean oil as the main lipid source. These plant oils have intermediate or low n-3/n-6-ratios compared to fish oil having a high n-3/n-6-ratio. The protein and carbohydrate fractions were identical in all the feeds. RESULTS: Morphometric measurements showed significantly shorter folds in the mid intestine in all groups fed vegetable oils compared to the group fed fish oil. In the distal intestine, the complex folds were significantly shorter in the fish fed soybean oil compared to the fish fed rapeseed oil. Histological and immunohistochemical examination did not show clear difference in the degree of inflammation or proliferation of epithelial cells related to dietary groups, which was further confirmed by real-time RT-PCR which revealed only moderate alterations in the mRNA transcript levels of selected immune-related genes. CONCLUSIONS: Shortened intestinal folds might be associated with reduced intestinal surface and impaired nutrient absorption and growth, but our results suggest that partial substitution of dietary fish oil with vegetable oils does not have any major negative impact on the intestinal health of Atlantic salmon.

Pathological pigmentation in cardiac tissues of Atlantic salmon (Salmo salar L.) with cardiomyopathy syndrome.

It is widely accepted that melanin formation may play an immunologic role in invertebrates and ectothermic vertebrates. In farmed Atlantic salmon, cardiomyopathy syndrome (CMS) is a common viral disease associated with severe cardiac inflammation that may be accompanied by heavy melanisation of the heart. By the use of histology, laser capture microdissection and transcription analysis of tyrosinase genes, we here show that this melanisation is linked to de novo melanogenesis by melanomacrophages, suggesting an active part in the inflammatory reaction. No general systemic activation of the extracutaneous pigmentary system in response to viral infections with affinity to the heart was observed.

Immune parameters correlating with reduced susceptibility to pancreas disease in experimentally challenged Atlantic salmon (Salmo salar).

Two strains of Atlantic salmon (Salmon salar) with different susceptibility to infectious salmon anaemia (ISA) were challenged with salmon pancreas disease virus (SPDV), the etiological agent of salmon pancreas disease (PD), by cohabitation. Serum and tissues were sampled at 0, 1, 3, 6 and 8 weeks post-challenge. Experimental challenge with SAV did not cause mortality, but virus loads and assessment of histopathology indicated that the fish more resistant to ISAV (ISAHi) also was more resistant to PD. Eight weeks post-challenge, the ISAHi strain had higher titres of SAV-neutralising antibodies than the less resistant strain (ISALo). Transcript levels of four adaptive and six innate immune parameters were analysed by real-time RT-PCR in heart, head kidney (HK) and gills of both strains. Secretory IgM (sIgM) and CD8 levels differed most between the two salmon strains. The ISAHi strain had significantly higher levels of sIgM in HK at all samplings, and significantly higher CD8 levels in gills at most samplings. In heart, both sIgM and CD8 levels increased significantly during the challenge, but the increase appeared earlier for the ISALo strain. By hierarchical clustering analysis of mRNA levels, a clear segregation was observed between the two strains prior to the virus challenge. As the viral infection developed, the clustering divide between fish strains disappeared, first for innate and later for adaptive parameters. At eight weeks post-challenge, the divide had however reformed for adaptive parameters. Possible pair-wise correlation between transcript levels of immune parameters was evaluated by a non-parametric statistical test. For innate parameters, the extent of correlation peaked at 3 wpc in all tissues; this came rapidly for ISALo and more gradual for ISAHi. The ISAHi strain tended to show higher correlation for innate parameters in heart and gill than ISALo at early sampling times. For adaptive immune parameters, little correlation was observed in general, except for ISAHi in heart at 6 wpc. Overall, the observed differences in immune parameters may provide important clues to the causes underlying the observed difference in susceptibility to PD.

Healthy goats naturally devoid of prion protein.

Prion diseases such as scrapie in small ruminants, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in man, are fatal neurodegenerative disorders. These diseases result from the accumulation of misfolded conformers of the host-encoded prion protein (PrP) in the central nervous system. To date naturally-occurring PrP free animals have not been reported. Here we describe healthy non-transgenic animals, Norwegian Dairy Goats, lacking prion protein due to a nonsense mutation early in the gene. These animals are predicted to be resistant to prion disease and will be valuable for research and for production of prion-free products.

Pigment-producing granulomatous myopathy in Atlantic salmon: a novel inflammatory response.

Melanin comprises a complex group of pigmented polymers whose primary function is ascribed to dermal solar protection, but may also have an interesting role in innate immunity. In ectothermic vertebrates, melanogenesis is reported in leukocyte populations, but it is not known if this occurs in connection with inflammatory reactions. Melanin accumulations in ectopic locations, in particular muscle, represent a serious quality problem in salmon production. Here, we investigated such changes for the expression of dopachrome tautomerase and tyrosinase as well as some important immune genes and pathogens. Furthermore, the nature of the pathological changes was addressed by morphological methods. Gene transcripts encoding key enzymes in melanogenesis, suggesting a de novo melanin synthesis in pigmented muscle, were found. MHC class II transcripts were up-regulated and there was no indication of bacterial or viral infection. The histological examination revealed granulomatous inflammation with distribution of MHC class II positive cells and T cells, analogous to the pattern found in mammals. Importantly, in contrast to mammals pigmented cells were contributing in the inflammation. We demonstrate that melanin production occurs in granulomatous inflammation in salmon, revealing a close and hitherto unreported link between the pigmentary and immune systems.

Transcription of reference genes used for quantitative RT-PCR in Atlantic salmon is affected by viral infection.

Relative quantification using RT-qPCR is a widely used method for transcription profiling. Transcript levels of target genes in fish after experimental infection is often reported without documentation of stably transcribed reference genes. We present results demonstrating that transcription of typically used reference genes in Atlantic salmon is not stable during experimental infection with salmon pancreas disease virus (SPDV). Transcript levels 0 to 6 weeks after challenge revealed statistically significant changes between time-points that corresponded with a peak in viral load 3 weeks after challenge. The results emphasize the need for thorough method validation prior to transcriptional studies during viral infections.

Whole genome sequencing reveals possible host species adaptation of Streptococcus dysgalactiae.

Streptococcus dysgalactiae (SD) is an emerging pathogen in human and veterinary medicine, and is associated with several host species, disease phenotypes and virulence mechanisms. SD has traditionally been divided into the subspecies dysgalactiae (SDSD) and subsp. equisimilis (SDSE), but recent molecular studies have indicated that the phylogenetic relationships are more complex. Moreover, the genetic basis for the niche versatility of SD has not been extensively investigated. To expand the knowledge about virulence factors, phylogenetic relationships and host-adaptation strategies of SD, we analyzed 78 SDSD genomes from cows and sheep, and 78 SDSE genomes from other host species. Sixty SDSD and 40 SDSE genomes were newly sequenced in this study. Phylogenetic analysis supported SDSD as a distinct taxonomic entity, presenting a mean value of the average nucleotide identity of 99%. Bovine and ovine associated SDSD isolates clustered separately on pangenome analysis, but no single gene or genetic region was uniquely associated with host species. In contrast, SDSE isolates were more heterogenous and could be delineated in accordance with host. Although phylogenetic clustering suggestive of cross species transmission was observed, we predominantly detected a host restricted distribution of the SD-lineages. Furthermore, lineage specific virulence factors were detected, several of them located in proximity to hotspots for integration of mobile genetic elements. Our study indicates that SD has evolved to adapt to several different host species and infers a potential role of horizontal genetic transfer in niche specialization.

Molecular detection and genotype characterization of Streptococcus dysgalactiae from sheep flocks with outbreaks of infectious arthritis.

Outbreaks of infectious arthritis in young lambs are a growing concern for the Norwegian sheep industry. In other countries, Streptococcus dysgalactiae subspecies dysgalactiae (SDSD) is a frequent cause of such outbreaks. The objectives of this study were to investigate the causes of outbreaks of infections arthritis in Norwegian sheep flocks, and describe the sources, colonization patterns and genetic diversity of SDSD in affected and healthy sheep flocks. Almost 2000 samples from joints, animal body sites and the indoor environment were analysed by qPCR and culturing for SDSD, which was detected in 27 of 30 flocks. The proportion of positive samples was greater in outbreak flocks compared to healthy flocks. Altogether, SDSD was detected in 48 % of the samples from lambs, 27 % of the samples from ewes and 48 % of environmental samples. A relatively high proportion (67 %) of ear tag wounds were SDSD positive. These wounds may provide a port of entry for SDSD. Whole genome sequencing revealed a clonal distribution of SDSD-isolates, and identified four different multi locus sequence types (STs), among which two STs, ST454 and ST531, dominated. These STs were found in geographically distant flocks. ST454 was almost exclusively found in outbreak flocks. The current study points to skin, wounds and mucous membranes of animals as the main reservoir of SDSD in sheep flocks. However, a significantly higher proportion of SDSD-positive environmental samples in outbreak flocks compared to healthy flocks suggests that also indirect transmission may play a role.

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.

Identification of differentially expressed genes associated with osteochondrosis in standardbred horses using RNA arbitrarily primed PCR.

The aim of this study was to investigate genes for differential expression in cartilage of foals predisposed to osteochondrosis (OC). Tissue was sampled from the cranial part of the distal intermediate ridge of the tibia in the tarso-crural joint. Foals were considered predisposed to OC when parents had OC at the distal intermediate ridge of the tibia. RNA was isolated and subjected to arbitrarily primed PCR (RAP-PCR) followed by fingerprinting to screen for differentially expressed genes. By verification of results from the RAP-PCR fingerprint screening using real-time RT-PCR, we identified two genes not previously correlated with OC as differentially expressed. The two genes, which were identical to TLK2 and an equine EST, are good targets for future research on OC.

Identification of differentially expressed genes in ileal Peyer's patch of scrapie-infected sheep using RNA arbitrarily primed PCR.

BACKGROUND: In scrapie and prion diseases, the knowledge concerning genes involved in host response during the early infection period in the lymphoid tissues, still remains limited. In the present study, we have examined differential gene expression in ileal Peyer's patches and in laser microdissected follicles of sheep infected with scrapie. METHODS: Ileal Peyer's patches and laser microdissected follicles were of scrapie and control lambs with susceptible genotypes for classical scrapie. Potential regulated genes were found using RNA arbitrarily primed polymerase chain reaction (RAP-PCR) and fingerprinting. The differentially expressed genes were confirmed using real-time RT-PCR. RESULTS: The expression of three genes (MAPRE3, LOC729073 and DNAJC3), were found to be significantly altered in scrapie infected lambs (P < 0.05). CONCLUSION: The three genes have not previously been associated with prion diseases and are interesting as they may reflect biological processes involved in the molecular pathogenesis of prion diseases.

The interbranchial lymphoid tissue likely contributes to immune tolerance and defense in the gills of Atlantic salmon.

Central and peripheral immune tolerance is together with defense mechanisms a hallmark of all lymphoid tissues. In fish, such tolerance is especially important in the gills, where the intimate contact between gill tissue and the aqueous environment would otherwise lead to continual immune stimulation by innocuous antigens. In this paper, we focus on the expression of genes associated with immune regulation by the interbranchial lymphoid tissue (ILT) in an attempt to understand its role in maintaining immune homeostasis. Both healthy and virus-challenged fish were investigated, and transcript levels were examined from laser-dissected ILT, gills, head kidney and intestine. Lack of Aire expression in the ILT excluded its involvement in central tolerance and any possibility of its being an analogue to the thymus. On the other hand, the ILT appears to participate in peripheral immune tolerance due to its relatively high expression of forkhead box protein 3 (Foxp3) and other genes associated with regulatory T cells (Tregs) and immune suppression.

Cloning and expression analysis of an ovine PAP-like protein cDNA, a gene differentially expressed in scrapie.

In a previous study, the mRNA level of a pancreatitis-associated protein (PAP)-like protein was found to be elevated in the ileal Peyer's patch of lambs during the early phase of scrapie infection. Here, we report the isolation of the ovine PAP-like protein cDNA which encodes a putative 178 amino acid protein with a signal peptide and a C-lectin binding domain. Comparisons of REG/PAP proteins between various species showed that the deduced amino acid sequences were conserved. The overall amino acid identity between the ovine PAP-like protein and bovine, human and rat REG/PAP proteins varied from 23% to 85%. In Northern blot analysis the expression of the ovine PAP-like protein mRNA was restricted to the ileal and jejunal Peyer's patches. The cellular expression of the PAP-like protein mRNA in the ovine intestine was further characterized by in situ hybridization. PAP-like protein mRNA was detected in cells of the epithelial lining in most crypts and in some intestinal villi in the ileum and jejunum while in the colon and rectum, the PAP-like protein mRNA expression was only detected in the deep portion of a few crypts. The data provided will offer the possibility to search for a link between this PAP-like protein and early events in the development of scrapie.

The interbranchial lymphoid tissue of Atlantic Salmon (Salmo salar L) extends as a diffuse mucosal lymphoid tissue throughout the trailing edge of the gill filament.

The teleost gill forms an extensive, semipermeable barrier that must tolerate intimate contact with the surrounding environment and be able to protect the body from external pathogens. The recent discovery of the interbranchial lymphoid tissue (ILT) has initiated an anatomical and functional investigation of the lymphoid tissue of the salmonid gill. In this article, sectioning of gill arches in all three primary planes revealed an elongation of the ILT outward along the trailing edge of the primary filament to the very distal end, a finding not previously described. This newly found lymphoid tissue was investigated using a range of morphological and transcriptional tools. Avoiding potential salinity-related effects, the study focused on two fresh-water life stages-smoltifying juveniles and mature adults. Aggregates of T-cells continuous with the ILT were found within the thick epithelial lining of the trailing edge of the filament in considerably larger numbers than seen in the epithelium of the leading edge and of the interlamellar area. Only a few of these cells were identified as CD8α(+) -cells, and there was a significantly (P < 0.05) higher relative expression of CD4- than of CD8- related genes in all gill segments investigated. Numerous major histocompatibility complex class II(+) -cells were distributed uniformly throughout the filament epithelial tissue. Few Ig(+) -cells were detected. Overall, the morphological features and comparable immune gene expression of the previously described ILT and the filament trailing edge lymphoid tissue suggest a close functional and anatomical relationship. We propose that the anatomical definition of the ILT must be broadened to include both the previously described ILT (to be renamed proximal ILT) and the trailing edge lymphoid tissue (to be named distal ILT). This extended anatomical localisation identifies the ILT as a widely distributed mucosal lymphoid tissue in the gill of Atlantic salmon.

Uptake of yeast cells in the Atlantic salmon (Salmo salar L.) intestine.

The intestinal mucosa is an important port of entry for many pathogens. Information of antigen uptake mechanisms is essential to understand and to possibly prevent infections. In teleosts, several studies have aimed at investigating particulate uptake in the gastrointestinal system that seems to vary dependent on fish species and antigen. In the present study, particulate uptake in the Atlantic salmon intestine by anal intubation of yeast cells has been investigated. In the anal intubated fish, yeast were found in the epithelium close to nuclei of macrophage-like cells and inside large mononuclear cells in the intestinal lumen, indicating uptake and possible transport of large antigen particles over the epithelium by macrophage-like cells.

Transcriptional response of immune genes in gills and the interbranchial lymphoid tissue of Atlantic salmon challenged with infectious salmon anaemia virus.

Previously, it has been assumed that fish lack organized mucosa-associated lymphoid structures. Recently, an interbranchial lymphoid tissue (ILT) was described in salmonid gills at a site with substantial exposure to antigen. In this study, immune responses were examined in gills, mid-kidney and the laser-dissected ILT of Atlantic salmon (Salmo salar L.) infected with infectious salmon anaemia virus (ISAV). A strong innate response was observed in gills and mid-kidney and even in the laser-dissected ILT, despite the fact that no virus could be traced in this tissue. A small delayed increase in IgT transcripts, exclusively in the ILT, could indicate that this tissue has a role as a secondary lymphoid organ with clonal expansion of IgT expressing B-cells. Compared to the other examined tissues, gills displayed the earliest replication of the virus, further supporting this tissue as the main entry route for infection with ISAV.