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Defects in gut barrier function are implicated in gastrointestinal (GI) disorders like inflammatory bowel disease (IBD), as well as in systemic inflammation. With the increasing incidence of IBD worldwide, more attention should be paid to dietary interventions and therapeutics with the potential to boost the natural defense mechanisms of gut epithelial cells. The current study aimed to investigate the protective effects of Limosilactobacillus reuteri ATCC PTA 4659 in a colitis mouse model and delineate the mechanisms behind it. Wild-type mice were allocated to the control group; or given 3% dextran sulfate sodium (DSS) in drinking water for 7 days to induce colitis; or administered L. reuteri for 7 days as pretreatment; or for 14 days starting 7 days before subjecting to the DSS. Peroral treatment with L. reuteri improved colitis severity clinically and morphologically and reduced the colonic levels of Tumor necrosis factor-α (TNF-α) (Tnf), Interleukin 1-β (Il1β), and nterferon-γ (Ifng), the crucial pro-inflammatory cytokines in colitis onset. It also prevented the CD11b+Ly6G+ neutrophil recruitment and the skewed immune responses in mesenteric lymph nodes (MLNs) of CD11b+CD11c+ dendritic cell (DC) expansion and Foxp3+CD4+ T-cell reduction. Using 16S rRNA gene amplicon sequencing and RT-qPCR, we demonstrated a colitis-driven bacterial translocation to MLNs and gut microbiota dysbiosis that were in part counterbalanced by L. reuteri treatment. Moreover, the expression of barrier-preserving tight junction (TJ) proteins and cytoprotective heat shock protein (HSP) 70 and HSP25 was reduced by colitis but boosted by L. reuteri treatment. A shift in expression pattern was also observed with HSP70 in response to the pretreatment and with HSP25 in response to L. reuteri-DSS. In addition, the changes of HSPs were found to be correlated to bacterial load and epithelial cell proliferation. In conclusion, our results demonstrate that the human-derived L. reuteri strain 4659 confers protection in experimental colitis in young mice, while intestinal HSPs may mediate the probiotic effects by providing a supportive protein–protein network for the epithelium in health and colitis.

Immunoassays are widely used for detection and quantification of analytes in biological samples, but are vulnerable to analytical errors caused by interfering sample substances. Of particular interest are endogenous anti-animal antibodies that may bind to the immunoassay antibodies and cause erroneous test results. This phenomenon is a hazard to patient safety in both human and veterinary medicine. Here, we demonstrate that anti-mouse antibodies in dogs bind selectively to different regions of the murine IgG molecule, cross-react with IgG from different species, and consist of all major antibody classes present in canine serum (IgA, IgG and IgM). The antibody characteristics varied among individuals and their prevalence differed between two dog breeds. The selective binding to different IgG regions suggests that the antibodies might not originate from immunization through exposure to mice or other species. These findings show that canine anti-mouse antibodies are highly heterogeneous in nature and therefore require a combination of strategies to be counteracted.

Colostrum with sufficient IgG content is essential for the newborn calf, as it requires this passive immunity to survive during its rearing. Failure of passive transfer (FPT) occurs when a calf does not absorb enough antibodies (

Significance Animal NLRs form wheel-like structures called inflammasomes upon perception of pathogen-associated molecules. The induced proximity of the signaling domains at the center of the wheel is hypothesized to recruit caspases for the first step of immune signal transduction. We expressed a plant-animal NLR fusion to demonstrate that induced proximity of TIR signaling domains from plant NLRs is sufficient to activate plant immune signaling. This demonstrates that a signaling-competent inflammasome can be formed from known, minimal components. The intrinsic NADase activity of plant TIRs is necessary for immune signaling, but fusions to a bacterial or a mammalian TIR domain with NADase activity, which also lead to accumulation of NAD+ hydrolysis products (e.g. cyclic ADP-ribose), were unable to activate immune signaling., Plant and animal intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors detect pathogen-derived molecules and activate defense. Plant NLRs can be divided into several classes based upon their N-terminal signaling domains, including TIR (Toll-like, Interleukin-1 receptor, Resistance protein)- and CC (coiled-coil)-NLRs. Upon ligand detection, mammalian NAIP and NLRC4 NLRs oligomerize, forming an inflammasome that induces proximity of its N-terminal signaling domains. Recently, a plant CC-NLR was revealed to form an inflammasome-like hetero-oligomer. To further investigate plant NLR signaling mechanisms, we fused the N-terminal TIR domain of several plant NLRs to the N terminus of NLRC4. Inflammasome-dependent induced proximity of the TIR domain in planta initiated defense signaling. Thus, induced proximity of a plant TIR domain imposed by oligomerization of a mammalian inflammasome is sufficient to activate authentic plant defense. Ligand detection and inflammasome formation is maintained when the known components of the NLRC4 inflammasome is transferred across kingdoms, indicating that NLRC4 complex can robustly function without any additional mammalian proteins. Additionally, we found NADase activity of a plant TIR domain is necessary for plant defense activation, but NADase activity of a mammalian or a bacterial TIR is not sufficient to activate defense in plants.

Rift Valley fever (RVF) is a zoonotic disease affecting humans and animals. It is caused by RVF virus transmitted primarily by Aedes mosquitoes. The data presented in this article propose environmental layers suitable for mapping RVF vector habitat zones and livestock migratory routes. Using species distribution modelling, we used RVF vector occurrence data sampled along livestock migratory routes to identify suitable vector habitats within the study region which is located in the central and the north-eastern part of Kenya. Eleven herds monitored with GPS collars were used to estimate cattle utilization distribution patterns. We used kernel density estimator to produce utilization contours where the 0.5 percentile represents core grazing areas and the 0.99 percentile represents the entire home range. The home ranges were overlaid on the vector suitability map to identify risks zones for possible RVF exposure. Assimilating high spatial and temporal livestock movement and vector distribution datasets generates new knowledge in understanding RVF epidemiology and generates spatially explicit risk maps. The results can be used to guide vector control and vaccination strategies for better disease control. Keywords: Home range estimation, Vector distribution, Rift Valley fever

Lactobacillus reuteri, a ubiquitous inhabitant of the mammalian gastrointestinal (GI) tract has known health-promoting effects and various strains are commercially available as probiotics. Several probiosis mechanisms have been suggested in L. reuteri’s mode of action, but the mediators and factors involved are not well understood. This thesis examined the function of probiotics, particularly L. reuteri, in the GI tract by equipping L. reuteri ATCC PTA 6475 and R2LC with reporter genes (luminescence and fluorescence) and a mutant of strain 6475 was generated by inactivation of chaperon dnaK. Different in vitro and in vivo applications of fluorescent and luminescent strains were evaluated, and it was demonstrated that flow cytometry can be a powerful method for determination of plasmid persistence. Biophotonic imaging (BPI) enabled low doses (~1x10^5) of luminescent bacteria to be monitored in the GI tract and revealed retention of large numbers of bacteria in the stomach up to 3 hours post-gavage. The effect of four strains of L. reuteri (6475, R2LC, DSM 17938, 1563F) was examined in an epithelial infection model using IPEC-J2 cells induced by enterotoxigenic E. coli. By analysing transepithelial electrical resistance (TEER) and leakage of FITC-dextran, it was shown that L. reuteri pre-treatment prevented damage by ETEC to epithelial monolayer integrity. The strains also reduced expression of pro-inflammatory cytokines (TNF-alfa and IL-6) and maintained expression of adherens junction(E-cadherin) and upregulated tight junction (ZO-1) proteins. To further explore the L. reuteri mode of action, five mutants were evaluated in DSS-induced acute colitis and IPEC-J2 models. It was found that dnaK-, pduC-, cmbA-, amidase- and srtA- may not play major roles in the mechanisms by which 6475 maintains mucosal integrity and counteracts inflammation. However, mutants 6475 pduC- and 6475 cmbA- had a tendency to weaken the protective effect of 6475 in the colitis model. Studies on the effects of L. reuteri strains on mast cell activation and inflammatory response revealed no inhibition of degranulation mediated by IgE-antigen activation, but down-regulated expression of the pro-inflammatory cytokines IL-6 and IL-13, irrespective of degranulation. Thus pre-treatment with L. reuteri strains can protect against intestinal barrier dysfunction and mucosal inflammation, partly through altering junctional complex proteins, mediators of immunity and mast cells.

Alkylresorcinols (AR) are amphiphilic phenolic lipids that are extensively metabolized in the liver and form metabolites that can be detected in plasma and urine. Two AR metabolites, 3,5-dihydroxybenzoic acid (DHBA) and 3-(3,5-dihydroxyphenyl)-propanoic acid (DHPPA), have been evaluated as urinary biomarkers of whole grain wheat and rye intake. AR metabolite concentrations are currently measured by chromatographic techniques but for samples from large epidemiological studies, immunoassay techniques are preferred due to rapid and cheap analysis. The aim of this thesis was to develop high-throughput analytical methods for the analysis of alkylresorcinol metabolites and to evaluate recently discovered putative metabolites 3,5-dihydroxycinnamic acid (DHCA), 5-(3,5-dihydroxyphenyl) pentanoic acid (DHPPTA), 3,5-dihydroxycinnamic acid amide (DHCA-amide) and 2-(3,5-dihydroxybenzamido) acetic acid (DHBA-glycine) as biomarkers of whole grain wheat and rye intake. Polyclonal antibodies against DHBA and DHPPA were produced and characterized in terms of cross-reactivity, assay sensitivity, precision and accuracy. A developed ELISA method was used for analysis of urine samples and results were compared with GC-MS. A lack of agreement was found. Several putative AR metabolites were identified by LC-MS/MS, including DHPPTA, DHCA, DHBA-glycine and DHCA-amide. These compounds showed high cross reactivity in ELISA and likely explains the lack of agreement between the two methods. The medium-term reproducibility and relative validity of DHCA, DHPPTA and DHBA-glycine were found to be moderate to excellent in 24h urine collections. Moreover, the reproducibility was good for these metabolites also in spot-urine samples. Age and sex were found to be important determinants besides whole grain intake. The results suggest that determination of AR metabolites in 24h urine and single or duplicate spot urine samples appear to be suitable medium to long-term biomarkers of whole grain wheat and rye intake. These findings need to be confirmed in other populations before using these biomarkers in epidemiological studies.

Sepsis is difficult to diagnose and remains a common mortality cause worldwide in both humans and animals. The uterine infection pyometra causes sepsis in more than half of affected dogs and therefore allows the natural physiological development of sepsis to be studied. To find a sepsis-specific biochemical marker that could be combined with conventional clinical criteria for a more robust and quick diagnosis of sepsis, we measured systemic concentrations of high-mobility group box 1 (HMGB1) in 23 healthy control dogs and in 27 dogs with pyometra, 74% of which had sepsis. We also measured concentrations of the major acute phase protein C-reactive protein (CRP) and an indicator for endotoxaemia, prostaglandin F-2 alpha metabolite (PGM) to assess the relative contribution of HMGB1 to the detection of systemic inflammation and endotoxaemia. We found that HMGB1 concentrations, in line with concentrations of CRP and PGM, were significantly increased in dogs with pyometra, and that concentrations of CRP, but not HMGB1, were significantly higher in dogs with sepsis compared to dogs without sepsis. Although serum HMGB1 did not differ between dogs with or without sepsis and was not correlated with either CRP or PGM concentrations, HMGB1 was correlated with the total white blood cell counts, suggesting an independent regulation and involvement in inflammation. (C) 2013 Elsevier B.V. All rights reserved.

A total of 961 harvested and 241 unharvested moose (Alces alces) carcasses and parts from throughout Sweden were examined for Elaphostrongylus alces from 1985 to 1989. When available, the central nervous system and skeletal muscles were searched for adult nematodes, and lungs and feces were examined for first-stage larvae. The parasite was distributed throughout Sweden with highest prevalence (56%) in the central region and lowest in the south (13%). Prevalence was highest in calves and old moose (>9 years) and lowest in middle-aged animals (5–9 years), with no statistical difference between sexes, although prevalence trended higher in young males. Body condition and abundance of Elaphostrongylus alces were negatively correlated, and condition was poorer in unharvested than harvested moose. A short (39–73 days) prepatent period was documented, and calves as young as 1.5 months were infected. These results indicate the importance of continued surveillance of Elaphostrongylus alces, particularly because a warming climate will likely increase abundance of intermediate mollusk hosts and possibly cause increased infection of moose.

Animal by-products and manure contain valuable plant nutrients that could be recycled onto arable land, as fertiliser. If these materials contain pathogenic microorganisms, such as viruses, transmission to domestic animals, wildlife and the food chain could occur. Virus contamination of food may further occur during all production phases, from slaughter to packaging and distribution. To reduce virus hazards, control measures such as physical and chemical treatments could be applied. As many important food-borne viruses are non-culturable, model viruses are often used to evaluate the effect of virus inactivation methods. As models for swine hepatitis E virus (HEV) in food treatments, feline calicivirus, murine norovirus and bacteriophages were evaluated. MS2 and ø6 were used as models for highly pathogenic avian influenza virus (HPAIV) in ammonia inactivation and composting of animal by-products, respectively. In laboratory scale, controlling the factors considered to be the most important for virus inactivation, reduction of relevant and model viruses was assessed as a function of these factors. Recommendations regarding continuously measurable process conditions that should be kept over a certain time to reach sufficient viral reductions could be given, both for normal conditions and in an out-break situation. Bacteriophages could further be used as potential indicators for verification or validation in pilot or full scale processes. Regimes to assure a 3 log10 reduction for Category 3 materials (2011/142/EC) for ammonia and heat treatment were determined. Further protocols based on pH and temperature to be kept during a certain time for management of HPAIV in outbreak situations were provided based on statistical evaluations of the laboratory results. In high pressure treatment of pork products, pressure and time were defined as critical control points for feline calicivirus and murine norovirus, used as models for HEV. MS2 and ø6 were successfully used for verification of ammonia treatment and composting, respectively, in larger scale. In food treatments, MS2 was the most conservative indicator of noro and calicivirus inactivation in high pressure and intense light pulse treatments, and øX174 in lactic acid treatments, with potential as models for these types of viruses for verification in production scale.

Sepsis is a syndrome with high morbidity, mortality and astronomical health care costs and it is challenging to diagnose both in humans and animals due to the lack of suitable diagnostic biomarkers. Although several types of proteins have been suggested as diagnostic biomarkers of sepsis, none of them were shown to be reliable for routine use in the clinical practice. Dogs with uterine bacterial infection called pyometra often develop sepsis and have been suggested as a natural model of sepsis. To investigate whether there is a pattern of biomarkers that can be useful to diagnose bacterial sepsis on early stages in addition to existing clinical criteria, we measured both local gene expression and serum levels of cytokines in dogs with pyometra and compared these levels with known inflammatory markers and blood clotting parameters. Serum concentrations of keratinocyte-derived chemokine (KC)-like protein and the global clot strength were significantly increased both in dogs with pyometra compared to healthy dogs and in dogs with sepsis compared to dogs without sepsis in pyometra. Moreover, the expression levels of the chemokines interleukin (IL)-8 and C-X-C motif ligand 5 (CXCL5) mRNA were significantly higher in uteri from dogs with pyometra compared to healthy dogs and in cultured stromal endometrial cells derived from uteri of healthy dogs and cocultured with LPS or pathogenic Escherichia coli compared to unstimulated cells. Although serum concentrations of IL-8, high-mobility group box 1 (HMGB1), prostaglandin F2α, IL-2, IL-15, IL-18, interferon (IFN)-γ and monocyte-macrophage colony stimulating factor (MG-CSF) were not different between dogs with or without sepsis in the presence of pyometra, some of these cytokines correlated significantly with clinical parameters such as total white blood cell count (correlated with HMGB1) and KC-like (correlated with IL-8). Measurements of serum IL-10, CXCL10, tumor necrosis factor (TNF)-α, IL-6 and IL-4 will require a more sensitive method in dogs with pyometra. Our findings suggest that KC-like, CXCL5 and IL-8 may be useful as early diagnostic biomarkers of sepsis in dogs with pyometra. Further investigation of these chemokines in sepsis may help to improve routines in sepsis diagnosis in dogs and possibly also humans.

Mast cells are implicated in immunity towards bacterial infection, but the molecular mechanisms by which mast cells contribute to the host response are only partially understood. Previous studies have examined how mast cells react to purified bacterial cell wall components, such as peptidoglycan and lipopolysaccharide. To investigate how mast cells react to live bacteria we co-cultured mast cells and the gram-positive bacteria Streptococcus equi (S. equi) and Staphylococcus aureus (S. aureus). Gene array analysis showed that mast cells upregulate a number of genes in response to live bacteria. Many of these corresponded to pro-inflammatory cytokines, but also numerous additional genes, including transcription factors, signaling molecules and proteases were upregulated. The release of cytokines was confirmed on the protein level by antibody-based cytokine/chemokine arrays and/or ELISA. Granzyme D, a protease mainly expressed in cytotoxic T cells, was one of the genes that were upregulated by S. equi. We showed that granzyme D is expressed by murine mast cells and that its level of expression correlated positively with the extent of mast cell maturation. Granzyme D expression was also induced by stem cell factor, IgE receptor cross-linking and calcium ionophore stimulation. Previous studies investigating the role of mast cells in bacterial infection in vivo have used mice that are mast cell deficient due to mutations in Kit-signaling. However, these mutations also influence other cell types than mast cells. Thus, to study the role of mast cells during in vivo infection with S. aureus we used the newly developed Mcpt5-Cre+ x R-DTA mice whose expression of diphteria toxin under the Mcpt5 promoter selectively depletes mast cells. S. aureus was injected intraperitoneally into Mcpt5-Cre+ x R-DTA mice using littermate mast cell-sufficient mice as controls. We did not observe any difference between mast cell-deficient and control mice in regard to weight loss, bacterial clearance, inflammation or cytokine production. We conclude that, despite mast cells being activated by S. aureus in vitro, mast cells do not influence the in vivo manifestations of S. aureus intraperitoneal infection. However, to make more general conclusions about the role of mast cells in bacterial infections, more studies in the new mast cell-deficient mice are needed using other bacterial strains and other routes of administration.

Life-history theory predicts that the trade-off between parasite defense and other costly traits such as reproduction may be most evident when resources are scarce. The strength of selection that parasites inflict on their host may therefore vary across environmental conditions. Collared flycatchers (Ficedula albicollis) breeding on the Swedish island Öland experience a seasonal decline in their preferred food resource, which opens the possibility to test the strength of life-history trade-offs across environmental conditions. We used nested-PCR and quantitative-PCR protocols to investigate the association of Haemosporidia infection with reproductive performance of collared flycatcher females in relation to a seasonal change in the external environment. We show that despite no difference in mean onset of breeding, infected females produced relatively more of their fledglings late in the season. This pattern was also upheld when considering only the most common malaria lineage (hPHSIB1), however there was no apparent link between the reproductive output and the intensity of infection. Infected females produced heavier-than-average fledglings with higher-than-expected recruitment success late in the season. This reversal of the typical seasonal trend in reproductive output compensated them for lower fledging and recruitment rates compared to uninfected birds earlier in the season. Thus, despite different seasonal patterns of reproductive performance the overall number of recruits was the same for infected versus uninfected birds. A possible explanation for our results is that infected females breed in a different microhabitat where food availability is higher late in the season but also is the risk of infection. Thus, our results suggest that another trade-off than the one we aimed to test is more important for explaining variation in reproductive performance in this natural population: female flycatchers appear to face a trade-off between the risk of infection and reproductive success late in the season.

Bluetongue virus (BTV) causes the primarily vector-borne bluetongue disease of ruminants, which poses a permanent threat to Europe since new serotypes and strains are frequently introduced. Vaccination of cattle is essential to control BTV outbreaks. Commercial attenuated and inactivated vaccines are efficacious in reducing BTV spread and disease, but do not fulfil all safety, adaptability, or production requirements. Additionally, no current vaccines allow the differentiation of infected from vaccinated animals (DIVA). DIVA vaccines enable surveillance of BTV epidemiology and vaccine efficacy, and facilitate a quick return for countries to a BTV-free status. This thesis presents the development and evaluation of a novel subunit DIVA vaccine against BTV serotype 8 (BTV-8) in cattle. Five His-tagged recombinant BTV proteins (VP2, VP5 of BTV-8; NS1, NS2, NS3 of BTV-2) were produced in baculovirus or E. coli expression systems. Purification protocols were optimized for all but VP5. Based on the feasibility of protein production and the capability of the remaining four proteins to induce humoral or cellular immune responses in mice, VP2, NS1, and NS2 were selected to formulate an experimental vaccine combined to an ISCOM-matrix adjuvant (SubV). Next, cattle were immunized twice at a three-week interval with SubV, a commercial inactivated vaccine, or a placebo. SubV induced humoral immune responses, including virus-neutralizing antibodies, against all three proteins, as well as a cellular immune response directed against NS1. These responses were of similar type and comparable magnitude between both vaccines, suggesting that SubV might provide protection that is at least as effective as the commercial vaccine. Finally, the protective efficacy of SubV was evaluated and complete virological and clinical protection against virulent BTV-8 challenge was observed following vaccination in calves. This was likely due to the induction of virus-neutralizing antibodies directed against VP2 of BTV-8 and cross-serotype T cell responses directed against NS1 and NS2 of BTV-2. Furthermore, SubV was shown to be DIVA-compliant based on the detection of antibodies directed against VP7, by using commercially-available diagnostic assays. This novel BTV subunit vaccine is a promising candidate and should be further developed.

Mast cells (MCs) are key effector cells in allergic reactions, through the release of a wide variety of granule-stored and de novo synthesized inflammatory mediators. The MC secretory granules contain exceedingly high levels of serglycin proteoglycan and the heparin-binding proteases chymase, tryptase and carboxypeptidase A. In this thesis the contribution of mouse mast cell protease (mMCP)-4, which is thought to be the functional homolog to the human chymase, was studied in the context of allergic airway inflammation. Using two models of allergic airway inflammation, wild-type (WT) and mMCP-4 deficient (mMCP-4-/-) mice were treated with ovalbumin (OVA) or with house dust mite (HDM) extract. We found that the OVA challenged mMCP-4-/- mice displayed increased airway hyperreactivity and lung eosinophilia and in the HDM model they displayed increased serum IgE levels. Moreover, the level of IL-33, a pro-inflammatory cytokine, was enhanced in the lung tissue in mMCP-4-/- mice compared to WT mice after HDM-treatment. The active proteases stored in MC granules have the ability to cleave a number of components upon degranulation. We could demonstrate that proteolytic degradation of IL-13 by MCs is mediated by a serine protease, dependent on serglycin proteoglycan for its storage. Permeabilization of lysosomal membranes often leads to apoptosis and the released proteases take part in this process, activating pro-apoptotic compounds. We have found that serglycin-/- MCs are more resistant to apoptosis induced by secretory granule damage. We showed that serglycin-/- MCs exhibited reduced caspase-3 activity and protease activity in the cytosol compared to WT cells. Taken together, the studies in this thesis suggest that MCs chymase plays a protective role in the development of allergic airway inflammation and this could possibly be explained by chymases ability to degrade the pro-inflammatory cytokine, IL-33. In addition, we also suggest that serglycin proteoglycan and serglycin-dependent MC proteases participate in IL-13 degradation as well as in MC apoptosis induced by secretory granule damage.

Pattern recognition receptors play an important role in insect immune defense. We cloned the β-1,3-glucan recognition protein, lectin-5 and C-type lectin 1 genes of Antheraea pernyi and examined the expression profiles of immune-stimulated pupae. After infection with Bacillus subtilis, Escherichia coli, Antheraea pernyi nuclear polyhedrosis virus (ApNPV) and Saccharomyces cerevisiae, respectively, the pupae showed different gene expression levels in the different tissues examined (midgut, fatbody, epidermis, testis, and hemocytes). ApβGRP and Aplectin-5 was induced by all the microorganisms, and mainly in epidermis and hemocytes, but not in testis; Aplectin-5 was also expressed in fatbody. Ap C-type lectin 1 was, on the contrary, highly expressed in testis and also in fatbody, but not in hemocytes. Unlike ApβGRP and Aplectin-5, Ap C-type lectin 1 was not induced by Gram-positive bacteria. The results suggest that the cloned lectins may have different functions in different tissues of A. pernyi.