Specific Gene Expression Responses to Parasite Genotypes Reveal Redundancy of Innate Immunity in Vertebrates

Vertebrate innate immunity is the first line of defense against an invading pathogen and has long been assumed to be largely unspecific with respect to parasite/pathogen species. However, recent phenotypic evidence suggests that immunogenetic variation, i.e. allelic variability in genes associated with the immune system, results in host-parasite genotype-by-genotype interactions and thus specific innate immune responses. Immunogenetic variation is common in all vertebrate taxa and this reflects an effective immunological function in complex environments. However, the underlying variability in host gene expression patterns as response of innate immunity to within-species genetic diversity of macroparasites in vertebrates is unknown. We hypothesized that intra-specific variation among parasite genotypes must be reflected in host gene expression patterns. Here we used high-throughput RNA-sequencing to examine the effect of parasite genotypes on gene expression patterns of a vertebrate host, the three-spined stickleback (Gasterosteus aculeatus). By infecting naïve fish with distinct trematode genotypes of the species Diplostomum pseudospathaceum we show that gene activity of innate immunity in three-spined sticklebacks depended on the identity of an infecting macroparasite genotype. In addition to a suite of genes indicative for a general response against the trematode we also find parasite-strain specific gene expression, in particular in the complement system genes, despite similar infection rates of single clone treatments. The observed discrepancy between infection rates and gene expression indicates the presence of alternative pathways which execute similar functions. This suggests that the innate immune system can induce redundant responses specific to parasite genotypes

Use of functional feeding strategies to protect Atlantic salmon from virally-induced inflammatory diseases- mechanistic insights revealed by transcriptomic analysis

Over the past few years one of the major concerns in the Atlantic salmon (Salmo salar) farming industry has been the increasing incidence and severity of inflammatory viral diseases. Heart and skeletal muscle inflammation (HSMI) and cardiomyopathy syndrome (CMS) are currently two of the most prevalent viral diseases in commercial Atlantic salmon farms in Norway. Mortality levels in both diseases are generally low but morbidity can be very high with the associated chronic inflammatory response lasting for several months. The consequent reduced growth performance is causing considerable financial impact as HSMI has become increasingly widespread in recent years. The impact of CMS is further exacerbated as it generally affects large fish close to harvest. HSMI lesions occur in the atrium and ventricle in the heart including inflammation and necrosis in epi- endo- and myocardium along with myositis of red skeletal muscle. CMS lesions are commonly observed in the spongy myocardium in the atrium and ventricle of the heart with severe mononuclear inflammation and necrosis. Furthermore, circulatory disturbances associated with reduced cardiac function cause multifocal liver steatosis and necrosis in both diseases. Currently there are no vaccines or any other effective treatments for these diseases and so alternative therapies that could potentially modulate the intensity of the inflammatory response could be crucial to improve the clinical manifestation of the diseases. Therefore, the overall aim of the present study was to evaluate the concept of “clinical nutrition” to improve the clinical symptoms of both viral diseases, HSMI and CMS, through the use of functional feeds formulated with reduced lipid content and increased proportions of anti-inflammatory fatty acids to moderate the apparently uncontrolled inflammatory response in the heart tissue associated with both diseases and also alleviate the secondary hepatic lesions. The experimental work consisted of three major dietary trials in Atlantic salmon in seawater. Two large trials investigated the effects of functional feeds in Atlantic salmon challenged with Atlantic salmon piscine reovirus (ASRV) and piscine myocarditis virus (PMCV), the causal agents of HSMI and CMS, respectively. In both trials, heart transcriptome, heart and liver histopathology and tissue lipid and fatty acid compositions and metabolism were determined post-infection in fish fed with the functional feeds in comparison with fish fed with a standard commercial feed formulation considered as a reference diet. All the functional feeds were formulated to have reduced digestible energy through lower dietary lipid and higher protein contents, and increased levels and proportions of anti-inflammatory long-chain polyunsaturated fatty acids (LC-PUFA), particularly eicosapentaenoic acid (EPA) compared with the reference diets. Histopathology, fatty acid composition and gene expression of heart were assessed over a long time-period of 16 weeks and 14 weeks post-challenge with ASRV and PMCV, respectively. Viral load in heart tissue, hepatic histopathology and fatty acid composition of liver and head kidney along with expression of the genes involved in the eicosanoid and LC-PUFA and eicosanoid biosynthesis pathways were also determined in the HSMI trial. The third trial was a nutritional trial evaluating the effects of dietary digestible energy content on lipid and fatty acid metabolism in salmon fed diets containing graded amounts of lipid. Fatty acid composition of liver and heart were assessed over 12 weeks, along with the hepatic expression of genes of lipid and fatty acid metabolism. The results of this research are presented in four chapters (Chapters 2-5) as four paper manuscripts. The manuscripts/Papers are either published (Chapter 2), in review (Chapter 3 and 4) or drafted for submission (Chapter 5) in appropriate peer-reviewed international journals. Chapter 2 and 3 correspond to the HSMI trial, Chapter 4 to the nutritional trial, and Chapter 5 to the CMS trial. Chapter 2 showed that viral load and histopathology scores were lower in fish fed the functional feeds, especially diet FF1, which displayed better performance. Diet strongly influenced the expression of genes related with the immune and inflammatory responses, with delayed expression in fish fed the functional feeds. Up-regulation of pro-inflammatory genes was correlated with the higher viral load observed at early-mid stages of the disease in fish fed the reference diet (ST). Expression of genes related with the immune response at 16-weeks post challenge reflected the differences in immunomodulation between the functional feeds, with fish fed diet FF1 showing lower expression. Therefore, severity of the heart lesions was correlated with the intensity of the immune response and could be associated with tissue anti-inflammatory LC-PUFA levels. Chapter 3 was focused on liver histopathology, fatty acid composition and LC-PUFA biosynthesis, along with phospholipid fatty acid composition and eicosanoid production in head kidney and heart tissue at early and late stages of ASRV infection. Liver was severely affected by the virus at the beginning of the infection in fish fed the reference ST diet, but the level of lesions were similar in all dietary groups at the end of the trial. Hepatic expression of fatty acyl desaturases was significantly depressed in fish fed the ST diet compare with fish fed the functional feeds despite the lower levels of dietary LC-PUFA in that feed. Thus endogenous production and bioavailability of anti-inflammatory LC-PUFA was potentially enhanced in fish fed the functional feeds. Changes in tissue lipid content, mobilization of fatty acids involved in inflammatory responses and changes in expression of transcription factors and genes involved in eicosanoid biosynthesis were more prominent in head kidney, confirming the important role of this organ in dietary immunomodulation after viral infection. To a lesser extent similar changes were observed in heart tissue, suggesting in situ production of eicosanoids could also be important. The unexpected effects of diet on expression of genes of LC-PUFA biosynthesis were specifically investigated in the trial described in Chapter 4. One aim of this study was to clarify whether dietary lipid content or viral infection was the cause of altered expression of desaturase genes between the different diets. Hepatic expression of other genes of lipid and fatty acid metabolism were also determined to evaluate metabolic changes associated with dietary lipid/energy level. In general, reduction of dietary energy and lipid contents while maintaining similar proportions of dietary fatty acids, led to a general up-regulation of genes involved in lipid biosynthetic pathways. Thus salmon fed lower energy diet showed increased liver expression of fatty acyl desaturases in comparison with fish fed higher energy levels. Heart transcriptomic data in Chapter 5 showed a similar delay in the inflammatory response in fish fed the functional feeds after PCMV infection as observed in the HSMI study. Modulation of inflammatory responses, similar to that previously described after ASRV infection, was also observed in fish fed the functional feeds. However, the differences in the expression of immune related genes and the level of heart lesions were not as prominent at mid-late stages of the disease as in fish fed FF1 in the HSMI trial. The present study demonstrated the beneficial effects of a clinical nutrition approach via functional feeds in two viral inflammatory diseases, HSMI and CMS, currently affecting farmed Atlantic salmon. Dietary immunomodulation increased the availability of anti-inflammatory LC-PUFA and significantly influenced the expression of the genes related with the immune/inflammatory response reducing the level and severity of cardiac and liver lesions and therefore improving the performance of fish suffering the diseases

Infectious hematopoietic necrosis virus (IHNV) persistence in Sockeye Salmon: influence on brain transcriptome and subsequent response to the viral mimic poly(I:C)

BACKGROUND: Sockeye Salmon are an iconic species widely distributed throughout the North Pacific. A devastating pathogen of Sockeye Salmon is infectious hematopoietic necrosis virus (IHNV, genus Novirhabdovirus, family Rhabdoviridae). It has been postulated that IHNV is maintained in salmon populations by persisting over the life of its host and/or by residing in natural reservoirs other than its susceptible hosts. Herein we demonstrate the presence of IHNV in the brain of Sockeye Salmon that survived an experimentally-induced outbreak, suggesting the presence of viral persistence in this susceptible species. To understand the viral persistent state in Sockeye Salmon we profiled the transcriptome to evaluate the host response in asymptomatic carriers and to determine what effects (if any) IHNV exposure may have on subsequent virus challenges. RESULTS: A laboratory disease model to simulate a natural IHNV outbreak in Sockeye Salmon resulted in over a third of the population incurring acute IHN disease and mortality during the first four months after initial exposure. Nine months post IHNV exposure, despite the absence of disease and mortality, a small percentage (<4 %) of the surviving population contained IHNV in brain. Transcriptome analysis in brain of asymptomatic virus carriers and survivors without virus exhibited distinct transcriptional profiles in comparison to naïve fish. Characteristic for carriers was the up-regulation of genes involved in antibody production and antigen presentation. In both carriers and survivors a down-regulation of genes related to cholesterol biosynthesis, resembling an antiviral mechanism observed in higher vertebrates was revealed along with differences in nervous system development. Moreover, following challenge with poly(I:C), survivors and carriers displayed an elevated antiviral immune response in comparison to naïve fish. CONCLUSIONS: IHN virus persistence was identified in Sockeye Salmon where it elicited a unique brain transcriptome profile suggesting an ongoing adaptive immune response. IHNV carriers remained uncompromised in mounting efficient innate antiviral responses when exposed to a viral mimic. The capacity of IHNV to reside in asymptomatic hosts supports a virus carrier hypothesis and if proven infectious, could have significant epidemiological consequences towards maintaining and spreading IHNV among susceptible host populations. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1759-y) contains supplementary material, which is available to authorized users