Immune defence White Spot Syndrome Virus infected shrimp, Penaeus monodon

White spot syndrome virus (WSSV) is the most important viral pathogen of cultured penaeid shrimp worldwide. Since the initial discovery of the virus inTaiwanin 1992, it has spread to shrimp farming regions in Southeast Asia, theAmericas, Europe and theMiddle Eastcausing major economic losses. The virus has a wide host range among crustaceans and induces distinctive clinical signs (white spots)on the inner surface of the exoskeletonof penaeid shrimps.Limited data is available about the immune response genes of P. monodon upon a WSSV infection. This thesis describes the results of our study into the generation of tools, like the generation of a dedicated microarray enabling the analysis of induction and regulation of (innate) immune defence genes in the host that are activated upon infection. Moreover, a putative vaccination strategy to protect shrimp against lethal WSSV infections has been developed previously. We have also analysed the induction of protective vaccination for induction and regulation of gene expression using this microarray.The first focus had been on the haemocyte response of the shrimp upon an immersion infection (chapter 2). Immunocytochemistry and electron microscopy has been used to study the infection route of WSSV in gills and gut up to 3 days after immersion infection. Using a mouse haemocyte specific monoclonal antibody (WSH8) and a rabbit VP28 polyclonal antibody, double immunoreactivity could be observed. Differential haemocyte characteristics in the gills and the midgut of P. monodon were determined.An invasion of haemocytes in the gills was observed in Penaeus monodon upon WSSV-infection, possibly caused by the adherence of haemocytes to the haemolymph vessels. Although many infected cells were found in the gills, haemocytes were not WSSV-infected in this organ. Gills appear to be an important site of haemocyte invasion after immersion infection. In the midgut, uptake of WSSV in the epithelium could be detected, however, infected nuclei of epithelial cells were not observed. In contrast to the gills, the gut connective tissue shows a clear increase in degranulation of haemocytes, resulting in the appearance of WSH8-immunoreactive thread-like material at later time points during the infection. The significance for the different reaction of haemocytes in both organs studied remains to be investigated further. The observation that haemocytes are not the main target for WSSV suggests that free virions circulating in the haemolymph lead to systemic infection in vivo.We conclude that the most likely natural infection route for WSSV is through the gills rather than through themidgut,and that the shrimp have an evolutionary deficit in killing the virus or virus-infected cells effectively.A combination of suppression subtractive hydridisation (SSH) and cDNA microarray analysis was used to enrich for those genes that are differentially expressed upon a WSSV infection (chapter 3). The construction of SSH libraries and subsequent selection of differentially expressed genes is described in detail. The selected clones were used to generate a dedicated WSSV infection-related cDNA microarray comprising 750 differentially expressed genes.The approach to combine suppressive subtraction hybridisation with microarray analysis has resulted in a read-out system for the detection of shrimp genes involved in the defence reaction upon a WSSV-infection. This approach has good potential for identifying genes involved in shrimp defences in the future. Further studies on these gene transcripts involved in the defence mechanism have to be initiated.The focus of chapter 4 is to determine the expression profile of the genes selected in chapter 3. By using the generated microarray it was possible to follow a few hundred clones during the first day of infection. In addition, the immune response of the shrimp upon "vaccination" was studied with the microarray. The results obtained in this investigation provide insight into the previously unknown complexities of host-WSSV molecular interactions. The discovery of differential expression of genes in WSSV infected shrimp allowed the visualisation of several pathways and potential mechanisms that may play a role in WSSV pathogenesis. Identification of regulated genes in WSSV infected shrimp enabled the development of a model depicting several ways in which host cell responds to infection. Gene expression changes also provided clues about the possible mechanisms involved in the development of pathological changes that are characteristic of the disease. Most importantly, the data obtained in this study identifies several genes whose mRNA is regulated on virus infection suggesting an array of hypotheses which could be tested to reveal their role in WSSV molecular pathogenesis. This study also provides insight in "vaccine"-host interactions. Microarray studies coupled with in vivo experiments obtain relevant data about the functionality of "vaccines" in shrimp and invertebrates in general. The combination of host immune response genes and "vaccination" can reveal the route of WSSV infection and may unravel the immune system of the giant tiger shrimp. Taken together, the present investigation demonstrates the application of a powerful approach of combining the high throughput technologies of SSH and microarray to study differential expression of genes in response to virus infection. SSH could be used for initial isolation of differentially expressed transcripts, a large-scale confirmation of which can be accomplished very efficiently by microarray analysis. The detailed methods described herein could be potentially applied to any biological system.With information available of Drosophila it is possible to look more thoroughly into immune related genes. Toll receptors are known to play a substantial role in detecting pathogens, both in invertebrates as well as in vertebrates, where they are called Toll-like receptors (TLR). Therefore, in chapter 5, a new Toll receptor was identified and described and expression studies upon WSSV infection were performed. The absence of regulation in different organs upon a viral challenge suggests that PmToll is not directly involved in the defense against WSSV. However, the Toll pathway can be regulated at a higher level (PGRPs and GNBPs; extra cellular) and regulation of PmToll might not be necessary. Currently we are investigating the effect of bacterial challenges on the regulation of PmToll.Finally, the results presented, are summarised and discussed in chapter 6. We provide an evolutionary framework for the virus-host response and describe the relevance of differentially expressed and regulated innate immune response genes. We integrate the characterisation of a shrimp-specific Toll receptor with results from the microarray analysis and provideaintegrative immune defence of P. monodon to exposure with WSSV. Moreover, we describe the immunological background known so far with respect to the vaccination strategy for WSSV infection

Immune functions in crustaceans: lessons from flies

In recent years insects, notably Drosophila, have emerged as a popular model for studying immune responses to bacterial and fungal pathogens. Due to the availability of the complete genome sequence, genome-wide scans of immune responses have been performed using microarray analyses. These analyses have revealed the presence of two major pathways: Toll and Imd. Each pathway consists of four steps: (i) recognition through pattern recognition receptors; (ii) modulation by serine proteases; (iii) signal transduction leading to translocation of transcription factors and (iv) humoral and cellular responses. We have compared the information from insects with those currently available in crustaceans, and have identified commonalities and differences. Remarkably, in both insects and crustaceans, little is known about their anti-viral responses. Future research will have to focus on these anti-viral immune responses ultimately to control viral diseases, which are at present a major threat to culturing penaeid shrimp

Maternal Transfer of Natural (Auto-) Antibodies in Chickens

The presence and relative levels (titers) of IgM and IgG natural antibodies (NAb) binding keyhole limpet hemocyanin (KLH), and natural (auto-) antibodies (N(A)Ab) binding salmon double-stranded DNA (dsDNA), (oxidated-) phosphatidyl (phosphoryl) choline-conjugated bovine serum albumin (PC-BSA), PC-conjugated ovalbumin (PC-OVA), and OVA, respectively, were studied in adult hen plasma, egg yolk, egg albumen, plasma of their hatchlings, and in 8-day-old chick plasma. Birds and eggs were from 2 lines divergently selected for high or low NAb levels binding KLH. This study aimed to determine 1) correlated phenotypic responses of selection for NAb to KLH, 2) transfer of maternal NAb and N(A)Ab via egg compartments, 3) levels of likely maternal NAb and N(A)Ab in hatchlings and 8-day-old chicks, and 4) whether a composite trait: IgM anti-PC-BSA/IgG anti-dsDNA ratio in the compartments could be used as a parameter for health or immune status.NAb and N(A)Ab to all tested antigens were found in adult hens, but low or no levels were found for IgM in yolk and IgG in albumen. Depending on the antigen, NAb and N(A)Ab were found in hatchlings and day 8 birds. Divergent selection and breeding based on NAb binding KLH affected antibody titers of almost all antigens in almost all compartments, in a similar way. Maternal transfer of NAb and N(A)Ab from the adult hen to offspring was via specific routes for specific antigens and isotypes, especially for IgG as suggested by cluster analyses and significant correlations. There was little indication of production of new NAb and N(A)Ab to the studied antigens in either the egg compartments or the hatchlings. A composite trait of IgM PC-BSA/IgG dsDNA ratio was as yet not indicative for immune status, as no significant differences were found between the lines for all compartments.In conclusion, hens provide neonatal chickens with natural (self-) binding IgG antibodies that have been proposed to perform homeostatic functions during the period in which neonates do not produce these antibodies themselves

Haemocyte reactions in WSSV immersion infected Penaeus monodon

White spot syndrome virus (WSSV) has been a major cause of shrimp mortality in aquaculture worldwide in the past decades. In this study, WSSV infection (by immersion) and behaviour recruitment of haemocytes is investigated in gills and midgut, using an antiserum against the viral protein VP28 and a monoclonal antibody recognising haemocytes (WSH8) in a double immunohistochemical staining and in addition transmission electron microscopy was applied. More WSH 8+ haemocytes were detected at 48 and 72 h post-infection in the gills of infected shrimp compared to uninfected animals. Haemocytes in the gills and midgut were not associated with VP28-immunoreactivity. In the gills many other cells showed virus replication in their nuclei, while infected nuclei in the gut cells were rare. Nevertheless, the epithelial cells in the midgut showed a clear uptake of VP28 and accumulation in supranuclear vacuoles (SNV) at 8 h post-infection. However, epithelial nuclei were never VP28-immunoreactive and electron microscopy study suggests degradation of viral-like particles in the SNV. In contrast to the gills, the midgut connective tissue shows a clear increase in degranulation of haemocytes, resulting in the appearance of WSH8-immunoreactive thread-like material at 48 and 72 h post-infection. These results indicate recruitment of haemocytes upon immersion infection in the gills and degranulation of haemocytes in less infected organs, like the midgut

Transgenerational Effects of Maternal Immune Activation on Specific Antibody Responses in Layer Chickens

Activation of the maternal immune system may affect innate and adaptive immune responses in the next generation and may therefore have implications for vaccine efficacy and dietary immune modulation by feed additives. However, transgenerational effects on immune responses in chickens have been investigated to a limited extend. The present study investigated effects of intratracheal (i.t) specific and aspecific immune activation of laying hens on specific antibody production in the next generation. In two experiments laying hens received intratracheally an immune stimulus with human serum albumin (HuSA) or lipopolysaccharide (LPS). In experiment 1, hatchlings of the immune activated hens were at 4 weeks i.t. immunized with HuSA or HuSA+LPS. Maternal immune activation with LPS increased HuSA specific IgY and IgM responses in offspring. These results suggest a transgenerational effect of the maternal immune system on the specific antibody response in the next generation. In experiment 2 hatchlings received either β-glucan-enriched feed or control feed and were i.t. immunized with HuSA. Maternal immune activation with LPS decreased IgY anti-HuSA responses after HuSA immunization within hatchlings that received β-glucan enriched feed. The results of Experiment 2 suggest a transgenerational link between the innate immune system of mother and specific antibody responses in offspring. Despite variabilities in the outcomes of the two experiments, the observations of both suggest a link between the maternal innate immune system and the immune system of the offspring. Furthermore, our results may imply that maternal activation of the innate immune system can influence immune modulating dietary interventions and vaccine strategies in the next generation

Detection of candidate regions affecting bovine IgM natural antibodies in milk

Two genomic regions were found to be associated with IgM antibody titers in milk binding lipoteichoic acid (LTA); one on chromosome 17 and the other on chromosome 21. Phenotypes were measured by ELISA and genotypes consisted of imputed 777k SNP. Single SNP analyses were run using an animal model to retrieve significant SNP. In BTA21, a gene for the heavy chains of immunoglobulins, IGHV is proposed as a candidate gene as was true for VPREB3 in BTA17 related to B-cell maturation. These findings provide a further step in better understanding the genetic background of natural antibodies (NAbs), that may be relevant for relevant for the estimation of dairy cattle health. Keywords: dairy cattle, natural antibodies, genome-wide associatio

Functional analysis of carp interferon-gamma: Evolutionary conservation of classical phagocyte activation

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Molecular cloning and expression of a Toll receptor in the giant tiger shrimp, Penaeus monodon

Invertebrates rely completely for their protection against pathogens on the innate immune system. This non-self-recognition is activated by microbial cell wall components with unique conserved molecular patterns. Pathogen-associated molecular patterns (PAMPs) are recognised by pattern recognition receptors (PRRs). Toll and its mammalian homologs Toll-like receptors are cell-surface receptors acting as PRRs and involved in the signalling pathway implicated in their immune response. Here we describe a novel partial Toll receptor gene cloned from a gill library of the giant tiger shrimp, Penaeus monodon, using primers based on the highly conserved Toll/IL-1R (TIR) domain. The deduced amino acid sequence of the P. monodon Toll (PmToll) shows 59% similarity to a Toll-related protein of Apis mellifera. Analysis of the LRRs of shrimp Toll contained no obvious PAMP-binding insertions. Phylogenetic analysis with the insect Toll family shows clustering with Toll1 and Toll5 gene products, and it is less related to Toll3 and Toll4. Furthermore, RT-qPCR shows that PmToll is constitutively expressed in gut, gill and hepatopancreas. Challenge with white spot syndrome virus (WSSV) shows equal levels of expression in these organs. A role in the defence mechanism is discussed. In conclusion, shrimp possess at least one Toll receptor that might be involved in immune defence

Effects of early life dextran sulfate sodium administration on pathology and immune response in broilers and layers

Intestinal pathology early in life may affect immune development and therefore immune responses later in life. Dextran sulfate sodium (DSS) induces colitis in rodents and is a widely used model for inflammatory bowel diseases. The present study investigated DSS as a model for early life intestinal pathology and its consequences on intestinal pathology, ileal cytokine, and immunoglobulin mRNA expression levels as well as the antibody response towards an immunological challenge later in life in chickens. Broiler and layer chicks received 2.5% DSS in drinking water during d 11 through d 18 post hatch or plain drinking water as a control. As an immunological challenge all birds received a combination of Escherichia coli lipopolysaccharide (LPS) and human serum albumin (HuSA) intramuscularly (i.m.) at d 35, and antibody titers against LPS, HuSA, and keyhole limpet hemocyanin (KLH) were determined to investigate effects of intestinal inflammation early in life on humoral immunity later in life. DSS treated birds showed a decrease in BW from which broilers quickly recovered, but which persisted for several weeks in layers. Histological examination of intestinal samples showed symptoms similar to those in rodents, including shortening and loss of villi and crypts as well as damage of the epithelial cell layer of different parts of the intestine. Effects of DSS on intestinal morphology were less severe in broilers that also showed a lower mortality in response to DSS than layers. No effect of DSS on ileal cytokine expression levels could be observed, but ileal immunoglobulin expression levels were decreased in DSS treated broilers that also showed lower antibody titers against LPS in response to the challenge. In conclusion, DSS may serve as a model for intestinal pathology early in life, although more research on the appropriate dose is necessary and is likely to differ between breeds. Results from the present study could indicate that broilers are less susceptible to DSS compared with layers or have a better capacity to recover from intestinal pathology.</p