Comparison of mouse models of microbial experience reveals differences in microbial diversity and response to vaccination.

Specific pathogen-free (SPF) laboratory mice dominate preclinical studies for immunology and vaccinology. Unfortunately, SPF mice often fail to accurately model human responses to vaccination and other immunological perturbations. Several groups have taken different approaches to introduce additional microbial experience to SPF mice to better model human immune experience. How these different models compare is unknown. Here, we directly compare three models: housing SPF mice in a microbe-rich barn-like environment (feralizing), adding wild-caught mice to the barn-like environment (fer-cohoused), or cohousing SPF mice with pet store mice in a barrier facility (pet-cohoused); the two latter representing different murine sources of microbial transmission. Pet-cohousing mice resulted in the greatest microbial exposure. Feralizing alone did not result in the transmission of any pathogens tested, while fer-cohousing resulted in the transmission of several picornaviruses. Murine astrovirus 2, the most common pathogen from pet store mice, was absent from the other two model systems. Previously, we had shown that pet-cohousing reduced the antibody response to vaccination compared with SPF mice. This was not recapitulated in either the feralized or fer-cohoused mice. These data indicate that not all dirty mouse models are equivalent in either microbial experience or immune responses to vaccination. These disparities suggest that more cross model comparisons are needed but also represent opportunities to uncover microbe combination-specific phenotypes and develop more refined experimental models. Given the breadth of microbes encountered by humans across the globe, multiple model systems may be needed to accurately recapitulate heterogenous human immune responses.IMPORTANCEAnimal models are an essential tool for evaluating clinical interventions. Unfortunately, they can often fail to accurately predict outcomes when translated into humans. This failure is due in part to a lack of natural infections experienced by most laboratory animals. To improve the mouse model, we and others have exposed laboratory mice to microbes they would experience in the wild. Although these models have been growing in popularity, these different models have not been specifically compared. Here, we directly compare how three different models of microbial experience impact the immune response to influenza vaccination. We find that these models are not the same and that the degree of microbial exposure affects the magnitude of the response to vaccination. These results provide an opportunity for the field to continue comparing and contrasting these systems to determine which models best recapitulate different aspects of the human condition.

Antigen surface display in two novel whole genome sequenced food grade strains, Lactiplantibacillus pentosus KW1 and KW2.

BACKGROUND: Utilization of commensal bacteria for delivery of medicinal proteins, such as vaccine antigens, is an emerging strategy. Here, we describe two novel food-grade strains of lactic acid bacteria, Lactiplantibacillus pentosus KW1 and KW2, as well as newly developed tools for using this relatively unexplored but promising bacterial species for production and surface-display of heterologous proteins. RESULTS: Whole genome sequencing was performed to investigate genomic features of both strains and to identify native proteins enabling surface display of heterologous proteins. Basic characterization of the strains revealed the optimum growth temperatures for both strains to be 35-37 °C, with peak heterologous protein production at 33 °C (KW1) and 37 °C (KW2). Negative staining revealed that only KW1 produces closely bound exopolysaccharides. Production of heterologous proteins with the inducible pSIP-expression system enabled high expression in both strains. Exposure to KW1 and KW2 skewed macrophages toward the antigen presenting state, indicating potential adjuvant properties. To develop these strains as delivery vehicles, expression of the mycobacterial H56 antigen was fused to four different strain-specific surface-anchoring sequences. CONCLUSION: All experiments that enabled comparison of heterologous protein production revealed KW1 to be the better recombinant protein production host. Use of the pSIP expression system enabled successful construction of L. pentosus strains for production and surface display of an antigen, underpinning the potential of these strains as novel delivery vehicles.

Microbial experience through housing in a farmyard-type environment alters intestinal barrier properties in mouse colons.

To close the gap between ultra-hygienic research mouse models and the much more environmentally exposed conditions of humans, we have established a system where laboratory mice are raised under a full set of environmental factors present in a naturalistic, farmyard-type habitat-a process we have called feralization. In previous studies we have shown that feralized (Fer) mice were protected against colorectal cancer when compared to conventionally reared laboratory mice (Lab). However, the protective mechanisms remain to be elucidated. Disruption of the protective intestinal barrier is an acknowledged player in colorectal carcinogenesis, and in the current study we assessed colonic mucosal barrier properties in healthy, feralized C57BL/6JRj male mice. While we found no effect of feralization on mucus layer properties, higher expression of genes encoding the mucus components Fcgbp and Clca1 still suggested mucus enforcement due to feralization. Genes encoding other proteins known to be involved in bacterial defense (Itln1, Ang1, Retnlb) and inflammatory mechanisms (Zbp1, Gsdmc2) were also higher expressed in feralized mice, further suggesting that the Fer mice have an altered intestinal mucosal barrier. These findings demonstrate that microbial experience conferred by housing in a farmyard-type environment alters the intestinal barrier properties in mice possibly leading to a more robust protection against disease. Future studies to unravel regulatory roles of feralization on intestinal barrier should aim to conduct proteomic analyses and in vivo performance of the feralized mice intestinal barrier.

Comparison of the Immunogenic Properties of Lactiplantibacillus plantarum Carrying the Mycobacterial Ag85B-ESAT-6 Antigen at Various Cellular Localizations.

The bacille Calmette-Guèrin (BCG) vaccine has been used for a century; nonetheless, tuberculosis (TB) remains one of the deadliest diseases in the world. Thus, new approaches to developing a new, more efficient vaccine are desirable. Mucosal vaccines are of particular interest, considering that Mycobacterium tuberculosis first enters the body through the mucosal membranes. We have previously demonstrated the immunogenicity of a recombinant Lactiplantibacillus plantarum delivery vector with TB hybrid antigen Ag85B-ESAT-6 anchored to the cell membrane. The goal of the present study was to analyze the impact of antigen localization in the immune response. Thus, we assessed two novel vaccine candidates, with the TB antigen either non-covalently anchored to the cell wall (LysMAgE6) or located intracellularly (CytAgE6). In addition, we compared two expression systems, using an inducible (LipoAgE6) or a constitutive promoter (cLipoAgE6) for expression of covalently anchored antigen to the cell membrane. Following administration to mice, antigen-specific CD4+ T-cell proliferation and IFN-γ and IL-17A secretion were analyzed for lung cell and splenocyte populations. Generally, the immune response in lung cells was stronger compared to splenocytes. The analyses showed that the type of expression system did not significantly affect the immunogenicity, while various antigen localizations resulted in markedly different responses. The immune response was considerably stronger for the surface-displaying candidate strains compared to the candidate with an intracellular antigen. These findings emphasize the significance of antigen exposure and further support the potential of L. plantarum as a mucosal vaccine delivery vehicle in the fight against TB.

Evaluation of Immune Status in Two Cohorts of Atlantic Salmon Raised in Different Aquaculture Systems (Case Study).

Assessment of immune competence of farmed Atlantic salmon is especially important during smoltification and the first several months in the sea. Recently developed tools were applied to salmon raised in a traditional flow-through facility (FT, cohort 1) and in a recirculation aquaculture system (RAS, cohort 2). Fish were sampled at four time-points: parr, smolt, and at three weeks and three months after seawater transfer (SWT); expression of 85 selected immune and stress genes, IgM transcripts (Ig-seq), and circulating antibodies were analyzed. A steady increase in gene expression was seen over time in gill and spleen in both cohorts, and especially in antiviral and inflammatory genes in the gill. Differences between the cohorts were greatest in the dorsal fin but later leveled off. Comparison with a gill reference dataset found a deviation in only three of 85 fish, suggesting a good immune status in both cohorts. Levels of both specific and nonspecific antibodies were higher in cohort 2 in smolts and in growers three weeks after SWT; however, levels evened out after three months in the sea. Ig-seq indicated association between antibody production, expansion of the largest clonotypes, and massive migration of B cells from spleen to gill in smolts. The results suggested greater agitation and higher reactivity of the immune system in RAS-produced salmon, but the difference between the cohorts leveled off over time.

Identification of novel biomarkers of inflammation in Atlantic salmon (Salmo salar L.) by a plasma proteomic approach.

Monitoring fish welfare has become a central issue for the fast-growing aquaculture industry, and finding proper biomarkers of stress, inflammation and infection is necessary for surveillance and documentation of fish health. In this study, a proteomic approach using mass spectrometry was applied to identify indicators of the acute response in Atlantic salmon blood plasma by comparing Aeromonas salmonicida subsp. salmonicida infected fish and non-infected controls. The antimicrobial proteins cathelicidin (CATH), L-plastin (Plastin-2, LCP1) and soluble toll-like receptor 5 (sTLR5) were uniquely or mainly identified in the plasma of infected fish. In addition, five immune-related proteins showed significantly increased expression in plasma of infected fish: haptoglobin, high affinity immunoglobulin Fc gamma receptor I (FcγR1, CD64), leucine-rich alpha 2 glycoprotein (LRG1), complement C4 (C4) and phospholipase A2 inhibitor 31 kDa subunit-like protein. However, various fibrinogen components, CD209 and CD44 antigen-like molecules decreased in infected fish. Selected biomarkers were further verified by Western blot analysis of plasma and real time PCR of spleen and liver, including CATH1, CATH2 and L-plastin. A significant increase of L-plastin occurred as early as 24 h after infection, and a CATH2 increase was observed from 72 h in plasma of infected fish. Real time PCR of selected genes confirmed increased transcription of CATH1 and CATH2. In addition, serum amyloid A mRNA significantly increased in liver and spleen after bacterial infection. However, transcription of L-plastin was not consistently induced in liver and spleen. The results of the present study reveal novel and promising biomarkers of the acute phase response and inflammation in Atlantic salmon.

Naturalizing laboratory mice by housing in a farmyard-type habitat confers protection against colorectal carcinogenesis.

Living in a farm environment in proximity to animals is associated with reduced risk of developing allergies and asthma, and has been suggested to protect against other diseases, such as inflammatory bowel disease and cancer. Despite epidemiological evidence, experimental disease models that recapitulate such environments are needed to understand the underlying mechanisms. In this study, we show that feralizing conventional inbred mice by continuous exposure to a livestock farmyard-type environment conferred protection toward colorectal carcinogenesis. Two independent experimental approaches for colorectal cancer induction were used; spontaneous (Apc Min/+ mice on an A/J background) or chemical (AOM/DSS). In contrast to conventionally reared laboratory mice, the feralized mouse gut microbiota structure remained stable and resistant to mutagen- and colitis-induced neoplasia. Moreover, the feralized mice exhibited signs of a more mature immunophenotype, indicated by increased expression of NK and T-cell maturation markers, and a more potent IFN-γ response to stimuli. In our study, hygienically born and raised mice subsequently feralized post-weaning were protected to a similar level as life-long exposed mice, although the greatest effect was seen upon neonatal exposure. Collectively, we show protective implications of a farmyard-type environment on colorectal cancer development and demonstrate the utility of a novel animal modeling approach that recapitulates realistic disease responses in a naturalized mammal.

Mast Cells Are Identified in the Lung Parenchyma of Wild Mice, Which Can Be Recapitulated in Naturalized Laboratory Mice.

Background: It is well documented that laboratory mice bred and maintained in ultra-hygienic specific pathogen-free (SPF) barriers display reduced richness and complexity of microbiota compared with wild mice. The laboratory mice profoundly lack lung parenchymal mast cells. Hence, we aimed to investigate the lung distribution of mast cells in free-living wild mice. Methods: Wild house mice were trapped in South-Eastern Norway and Hemtabad, West Bengal, India. C57BL/6 laboratory mice were bred in a purposefully built, closed environment with bedding material obtained from the natural environment in order to normalize the gut microbiota of these laboratory mice to that of the wild mice, and the offspring were collected for study at eight weeks of age. Results: Mast cells were easily identified at a substantial density in the lung parenchymal tissues of wild mice from both Norway and India, which stands in clear contrast to the rare distribution of lung parenchymal mast cells in the conventional laboratory SPF mice. Consistently, wild mice also expressed higher pulmonary levels of stem cell factor, a critical growth factor for mast cell survival. Higher levels of histamine were recorded in the lung tissues of the wild mice. Interestingly, "naturalized" C57BL/6 laboratory mice which spent their entire life in a semi-natural environment developed lung parenchymal mast cells at an appreciable density. Conclusion: Our observations support that environmental factors, possibly through modulation of microbiota, may impact the tissue distribution of mast cells in mouse lung parenchyma.

Effect of two constant light regimens on antibody profiles and immune gene expression in Atlantic salmon following vaccination and experimental challenge with salmonid alphavirus.

Before seawater transfer, farmed Atlantic salmon are subjected to treatments that may affect the immune system and susceptibility to pathogens. E.g., exposure to constant light (CL) stimulates smoltification, which prepares salmon to life in sea water, but endocrine changes in this period are associated with suppression of immune genes. Salmon are vaccinated towards end of the freshwater period to safeguard that adequate vaccine efficacy is achieved by the time the fish is transferred to sea. In the present study, we investigated how the responses to vaccination and viral infection varied depending on the time of CL onset relative to vaccination. The salmon were either exposed to CL two weeks prior to vaccination (2-PRI) or exposed to CL at the time of vaccination (0-PRI). A cohabitant challenge with salmonid alphavirus, the causative agent of pancreatic disease, was performed 9 weeks post vaccination. The immunological effects of the different light manipulation were examined at 0- and 6-weeks post vaccination, and 6 weeks post challenge. Antibody levels in serum were measured using a serological bead-based multiplex panel as well as ELISA, and 92 immune genes in heart and spleen were measured using an integrated fluidic circuit-based qPCR array for multiple gene expression. The 2-PRI group showed a moderate transcript down-regulation of genes in the heart at the time of vaccination, which were restored 6 weeks after vaccination (WPV). Conversely, at 6WPV a down-regulation was seen for the 0-PRI fish. Moreover, the 2-PRI group had significantly higher levels of antibodies binding to three of the vaccine components at 6WPV, compared to 0-PRI. In response to SAV challenge, transcription of immune genes between 2-PRI and 0-PRI was markedly dissimilar in the heart and spleen of control fish, but no difference was found between vaccinated salmon from the two CL regimens. Thus, by using labor-saving high throughput detection methods, we demonstrated that light regimens affected antibody production and transcription of immune genes in non-vaccinated and virus challenged salmon, but the differences between the light treatment groups appeared eliminated by vaccination.

Induction of colorectal carcinogenesis in the C57BL/6J and A/J mouse strains with a reduced DSS dose in the AOM/DSS model.

BACKGROUND: Colorectal cancer (CRC) is one of the most frequently diagnosed cancers worldwide and thus mouse models of CRC are of significant value to study the pathogenesis. The Azoxymethane/Dextran sulfate sodium (AOM/DSS) model is a widely used, robust initiation-promotion model for chemical induction of colitis-associated CRC in rodents. However, the dosage of chemicals, treatment regimens and outcome measures vary greatly among studies employing this model. Thus, the aim of this study was to examine an AOM/DSS model involving a reduced (1%) dose of DSS for induction of carcinogenesis in A/J and C57BL/6J (B6) mice. RESULTS: We show that colonic preneoplastic lesions can be reliably detected in A/J and B6 mice by use of a AOM/DSS model involving a single injection of 10 mg/kg AOM followed by three 7-day cycles of a low-dose (1%) DSS administration. Supporting existing evidence of A/J mice exhibiting higher susceptibility to AOM than B6 mice, our AOM/DSS-treated A/J mice developed the highest number of large colonic lesions. Clinical symptoms in both strains subjected to the AOM/DSS treatment did not persist in-between treatment cycles, demonstrating that the animals tolerated the treatment well. CONCLUSIONS: Our findings suggest that a reduced dose of DSS in the AOM/DSS model can be considered in future studies of early phase colorectal carcinogenesis in the A/J and B6 mouse strains using preneoplastic lesions as an outcome measure, and that such regimen may reduce the risk of early trial terminations to accommodate human endpoints. Overall, our data emphasize the importance of devoting attention towards choice of protocol, outcome measures and mouse strain in studies of CRC in mice according to the study purpose.

IgM+ and IgT+ B Cell Traffic to the Heart during SAV Infection in Atlantic Salmon.

B cells of teleost fish differentiate in the head kidney, and spleen, and either remain in the lymphatic organs or move to the blood and peripheral tissues. There is limited knowledge about piscine B cell traffic to sites of vaccination and infection and their functional roles at these sites. In this work, we examined the traffic of B cells in Atlantic salmon challenged with salmonid alphavirus (SAV). In situ hybridization (RNAScope) showed increased numbers of immunoglobin (Ig)M+ and IgT+ B cells in the heart in response to SAV challenge, with IgM+ B cells being most abundant. An increase in IgT+ B cells was also evident, indicating a role of IgT+ B cells in nonmucosal tissues and systemic viral infections. After infection, B cells were mainly found in the stratum spongiosum of the cardiac ventricle, colocalizing with virus-infected myocardial-like cells. From sequencing the variable region of IgM in the main target organ (heart) and comparing it with a major lymphatic organ (the spleen), co-occurrence in antibody repertoires indicated a transfer of B cells from the spleen to the heart, as well as earlier recruitment of B cells to the heart in vaccinated fish compared to those that were unvaccinated. Transcriptome analyses performed at 21 days post-challenge suggested higher expression of multiple mediators of inflammation and lymphocyte-specific genes in unvaccinated compared to vaccinated fish, in parallel with a massive suppression of genes involved in heart contraction, metabolism, and development of tissue. The adaptive responses to SAV in vaccinated salmon appeared to alleviate the disease. Altogether, these results suggest that migration of B cells from lymphatic organs to sites of infection is an important part of the adaptive immune response of Atlantic salmon to SAV.

Detection of specific Atlantic salmon antibodies against salmonid alphavirus using a bead-based immunoassay.

Salmonid alphavirus (SAV) is the etiological cause of pancreas disease (PD) in Atlantic salmon (Salmo salar). Several vaccines against SAV are in use, but PD still cause significant mortality and concern in European aquaculture, raising the need for optimal tools to monitor SAV immunity. To monitor and control the distribution of PD in Norway, all salmonid farms are regularly screened for SAV by RT-qPCR. While the direct detection of SAV is helpful in the early stages of infection, serological methods could bring additional information on acquired SAV immunity in the later stages. Traditionally, SAV antibodies are monitored in neutralization assays, but they are time-consuming and cumbersome, thus alternative assays are warranted. Enzyme-linked immunosorbent assays (ELISAs) have not yet been successfully used for anti-SAV antibody detection in aquaculture. We aimed to develop a bead-based immunoassay for SAV-specific antibodies. By using detergent-treated SAV particles as antigens, we detected SAV-specific antibodies in plasma collected from both a SAV challenge trial and a field outbreak of PD. Increased levels of SAV-specific antibodies were seen after most fish had become negative for viral RNA. The bead-based assay is time saving compared to virus neutralization assays, and suitable for non-lethal testing due to low sample size requirements. We conclude that the bead-based immunoassay for SAV antibody detection is a promising diagnostic tool to complement SAV screening in aquaculture.

Is clinical effect of autologous conditioned serum in spontaneously occurring equine articular lameness related to ACS cytokine profile?

BACKGROUND: Biologic' therapies, such as autologous conditioned serum (ACS), are gaining popularity in treating orthopaedic conditions in equine veterinary medicine. Evidence is scarce regarding ACS constituents, and large inter-individual differences in cytokine and growth factor content have been demonstrated. The objective of the current study was to investigate the potential association between cytokine and growth factor content of ACS and clinical effect in harness racehorses with spontaneously occurring low-grade articular lameness. Horses received 3 intra-articular injections of ACS administered at approximately 2-week intervals. Lameness evaluation consisting of a trot-up with subsequent flexions tests was performed at inclusion and approximately 2 weeks after the last treatment (re-evaluation); horses were classified as responders when there was no detectable lameness on trot-up and a minimum of 50% reduction in flexion test scores at re-evaluation. Association between clinical outcome (responders vs. non-responders) and age, lameness grades at inclusion (both initial trot-up and after flexion tests), treatment interval, follow-up time and the ACS content of IL-1Ra, IGF-1 and TGF-ß was determined by regression modelling. RESULTS: Outcome analysis was available for 19 of 20 included horses; 11 responded to treatment whereas 8 did not. There was considerable inter-individual variability in cytokine/growth factor content of ACS, and in the majority of the horses, the level of IL-10, IL-1ß and TNF-α was below the detection limit. In the final multivariate logistic regression model, ACS content of IGF-1 and IL-1Ra was significantly associated with clinical response (P = 0.01 and P = 0.03, respectively). No association with clinical response was found for the other tested variables. CONCLUSIONS: The therapeutic benefit of ACS may be related to higher levels of IL-1Ra and IGF-1. Our study corroborates previous findings of considerable inter-individual variability of cytokine- and growth factor content in ACS.

A Model System for Feralizing Laboratory Mice in Large Farmyard-Like Pens.

Laboratory mice are typically housed under extremely clean laboratory conditions, far removed from the natural lifestyle of a free-living mouse. There is a risk that this isolation from real-life conditions may lead to poor translatability and misinterpretation of results. We and others have shown that feral mice as well as laboratory mice exposed to naturalistic environments harbor a more diverse gut microbiota and display an activated immunological phenotype compared to hygienic laboratory mice. We here describe a naturalistic indoors housing system for mice, representing a farmyard-type habitat typical for house mice. Large open pens were installed with soil and domestic animal feces, creating a highly diverse microbial environment and providing space and complexity allowing for natural behavior. Laboratory C57BL/6 mice were co-housed in this system together with wild-caught feral mice, included as a source of murine microbionts. We found that mice feralized in this manner displayed a gut microbiota structure similar to their feral cohabitants, such as higher relative content of Firmicutes and enrichment of Proteobacteria. Furthermore, the immunophenotype of feralized mice approached that of feral mice, with elevated levels of memory T-cells and late-stage NK cells compared to laboratory-housed control mice, indicating antigenic experience and immune training. The dietary elements presented in the mouse pens could only moderately explain changes in microbial colonization, and none of the immunological changes. In conclusion, this system enables various types of studies using genetically controlled mice on the background of adaptation to a high diversity microbial environment and a lifestyle natural for the species.

Consequences of Haemorrhagic Smolt Syndrome (HSS) for the Immune Status of Atlantic salmon (Salmo salar L.) (Case Study).

Haemorrhagic smolt syndrome (HSS) is a disorder of unknown aetiology causing losses in the fresh water phase of Atlantic salmon farming. Normally, the mortality is limited and symptoms disappear upon seawater exposure. In this case study, classical HSS pathology with internal organ haemorrhages and nephrocalcinosis was diagnosed, and the losses were substantial. Microarray analyses of head kidney revealed association between HSS and enhanced expression of stress genes and proteins reducing bioavailability of iron, heme, and retinol. In parallel, suppression of multiple metabolic pathways was observed. Up-regulation of genes encoding acute phase proteins, complement, and lectins indicated mild inflammation but without characteristic features of viral or bacterial infections. Microarray analyses highlighted several members of tumor necrosis factor receptor superfamily that may control development of B-cell immunity. Examination of IgM at the mRNA and protein levels showed the impact of HSS on vaccine responses. In fish without HSS symptoms (non-HSS), titres of vaccine specific antibodies to A-layer of Aeromonas salmonicida subsp. salmonicida and Moritella viscosa and antibodies binding to DNP-keyhole limpet hemocyanin (DNP-KLH), which are presumably polyreactive, were respectively four- and 14-fold higher than in HSS-diseased fish. Parallel sequencing of variable regions of immunoglobulin Mrevealed a larger size of most abundant clonotypes shared by multiple individuals in the non-HSS group. The results of the current case study indicated that, in addition to direct damage, HSS suppresses humoral immune responses including the production of specific and polyreactive antibodies.

Detection of Salmonid IgM Specific to the Piscine Orthoreovirus Outer Capsid Spike Protein Sigma 1 Using Lipid-Modified Antigens in a Bead-Based Antibody Detection Assay.

Bead-based multiplex immunoassays are promising tools for determination of the specific humoral immune response. In this study, we developed a multiplexed bead-based immunoassay for the detection of Atlantic salmon (Salmo salar) antibodies against Piscine orthoreovirus (PRV). Three different genotypes of PRV (PRV-1, PRV-2, and PRV-3) cause disease in farmed salmonids. The PRV outer capsid spike protein σ1 is predicted to be a host receptor binding protein and a target for neutralizing and protective antibodies. While recombinant σ1 performed poorly as an antigen to detect specific antibodies, N-terminal lipid modification of recombinant PRV-1 σ1 enabled sensitive detection of specific IgM in the bead-based assay. The specificity of anti-PRV-1 σ1 antibodies was confirmed by western blotting and pre-adsorption of plasma. Binding of non-specific IgM to beads coated with control antigens also increased after PRV infection, indicating a release of polyreactive antibodies. This non-specific binding was reduced by heat treatment of plasma. The same immunoassay also detected anti-PRV-3 σ1 antibodies from infected rainbow trout. In summary, a refined bead based immunoassay created by N-terminal lipid-modification of the PRV-1 σ1 antigen allowed sensitive detection of anti-PRV-1 and anti-PRV-3 antibodies from salmonids.

Transcription Profiling of Monocyte-Derived Macrophages Infected In Vitro With Two Strains of Streptococcus agalactiae Reveals Candidate Pathways Affecting Subclinical Mastitis in Cattle.

Macrophages are key cells of innate immune response and serve as the first line of defense against bacteria. Transcription profiling of bacteria-infected macrophages could provide important insights on the pathogenicity and host defense mechanisms during infection. We have examined transcription profiles of bovine monocyte-derived macrophages (bMDMs) isolated from the blood of 12 animals and infected in vitro with two strains of Streptococcus agalactiae. Illumina sequencing of RNA from 36 bMDMs cultures exposed in vitro to either one of two sequence types of S. agalactiae (ST103 or ST12) for 6 h and unchallenged controls was performed. Analyses of over 1,656 million high-quality paired-end sequence reads revealed 5,936 and 6,443 differentially expressed genes (p < 0.05) in bMDMs infected with ST103 and ST12, respectively, versus unchallenged controls. Moreover, 588 genes differentially expressed between bMDMs infected with ST103 versus ST12 were identified. Ingenuity pathway analysis of the differentially up-regulated genes in the bMDMs infected with ST103 revealed significant enrichment for granulocyte adhesion and diapedesis, while significant enrichment for the phagosome formation pathway was found among down-regulated genes. Moreover, Ingenuity pathway analysis of the differentially up-regulated genes in the bMDMs infected with ST12 showed significant enrichment for type 1/type 2 T helper cell activation, while the complement activation pathway was overrepresented in the down-regulated genes. Our study identified pathogen-induced regulation of key genes and pathways involved in the immune response of macrophages against infection but also likely involved in bacterial evasion of the host immune system. These results may contribute to better understanding of the mechanisms underlying subclinical infection such as bovine streptococcal mastitis.

Evidence of functional Cd94 polymorphism in a free-living house mouse population.

The CD94 receptor, expressed on natural killer (NK) and CD8+ T cells, is known as a relatively non-polymorphic receptor with orthologues in humans, other primates, cattle, and rodents. In the house mouse (Mus musculus), a single allele is highly conserved among laboratory strains, and reports of allelic variation in lab- or wild-living mice are lacking, except for deficiency in one lab strain (DBA/2J). The non-classical MHC-I molecule Qa-1b is the ligand for mouse CD94/NKG2A, presenting alternative non-americ fragment of leader peptides (Qa-1 determinant modifier (Qdm)) from classical MHC-I molecules. Here, we report a novel allele identified in free-living house mice captured in Norway, living among individuals carrying the canonical Cd94 allele. The novel Cd94LocA allele encodes 12 amino acid substitutions in the extracellular lectin-like domain. Flow cytometric analysis of primary NK cells and transfected cells indicates that the substitutions prevent binding of CD94 mAb and Qa-1b/Qdm tetramers. Our data further indicate correlation of Cd94 polymorphism with the two major subspecies of house mice in Europe. Together, these findings suggest that the Cd94LocA/NKG2A heterodimeric receptor is widely expressed among M. musculus subspecies musculus, with ligand-binding properties different from mice of subspecies domesticus, such as the C57BL/6 strain.

B7H6 is a functional ligand for NKp30 in rat and cattle and determines NKp30 reactivity toward human cancer cell lines.

NK cells kill cancer cells and infected cells upon activation by cell surface receptors. Human NKp30 is an activating receptor expressed by all mature NK cells. The B7 family member B7H6 has been identified as one ligand for NKp30. Several alternative ligands have also been reported, and the field remains unsettled. To this end, we have identified full-length functional B7H6 orthologs in rat and cattle, demonstrated by phylogenetic analysis and transfection experiments. In cell-cell contact-dependent assays, chimeric NKp30 reporter cells responded strongly to B7H6 in rat and cattle. Likewise, rat NKp30 expressing target cells induced strong activation of B7H6 reporter cells. Together, these observations demonstrate that B7H6 is conserved as a functional ligand for NKp30 in mammalian species separated by more than 100 million years of evolution. B7H6 and NKp30 are pseudogenes in laboratory mice. The rat thus represents an attractive experimental animal model to study the NKp30-B7H6 interaction in vivo. B7H6 was widely expressed among human cancer cell lines, and the expression level correlated strongly with the activation of human NKp30 reporter cells. Furthermore, siRNA knockdown of B7H6 abolished NKp30 reporter responses, suggesting that B7H6 is the major functionally relevant expressed ligand for NKp30 on these cancer cell lines.

A time-course study of gene expression and antibody repertoire at early time post vaccination of Atlantic salmon.

The majority of studies of vaccine responses in Atlantic salmon have focused on several weeks after vaccination, and employed a limited number of marker genes. In this study, novel techniques were used to examine a broad panel of expressed genes and antibody repertoire of Atlantic salmon following vaccination. Salmon parr were vaccinated with a multivalent oil-based vaccine, and blood plasma and head kidney were sampled at several time-points between 0-35 days post vaccination. Saline-injected fish were used as control at all time-points. Microarray analyses showed increased expression of immune genes from the first day to the end of study in the head kidney of vaccinated fish. Genes up-regulated in the late phase included several leukocyte markers and components of the oxidative burst complex. A suite of genes that can take part in B cells differentiation were up-regulated from day 14, at which time secretory IgM transcripts also peaked. This coincided with marked increased plasma titres of non-vaccine specific antibodies binding to a hapten-carrier antigen DNP-KLH, while antibodies to bacterial components of the vaccine, Moritella viscosa and Aeromonas salmonicida, first showed significantly elevated antibody levels at day 21, and at a markedly lower magnitude than the non-vaccine specific titres. Sequencing of the variable region of IgM heavy chain (CDR3) revealed higher cumulative frequencies of unique clonotypes in vaccinated salmon starting from day 14 when specific antibodies were first detected. Reduced sequence variance of CDR3 suggested expansion of recently emerged clonotypes. Overall, the results presented here follow a broad panel of gene expression, immunoglobulin sequencing and plasma antibody titres in the first few weeks after vaccination of Atlantic salmon, pointing to a potentially important contribution of non-vaccine specific antibody responses early in the vaccine response.