Agaricus subrufescens fermented rye affects the development of intestinal microbiota, local intestinal and innate immunity in suckling-to-nursery pigs.

BACKGROUND: Agaricus subrufescens is considered as one of the most important culinary-medicinal mushrooms around the world. It has been widely suggested to be used for the development of functional food ingredients to promote human health ascribed to the various properties (e.g., anti-inflammatory, antioxidant, and immunomodulatory activities). In this context, the interest in A. subrufescens based feed ingredients as alternatives for antibiotics has also been fuelled during an era of reduced/banned antibiotics use. This study aimed to investigate the effects of a fermented feed additive -rye overgrown with mycelium (ROM) of A. subrufescens-on pig intestinal microbiota, mucosal gene expression and local and systemic immunity during early life. Piglets received ROM or a tap water placebo (Ctrl) perorally every other day from day 2 after birth until 2 weeks post-weaning. Eight animals per treatment were euthanized and dissected on days 27, 44 and 70. RESULTS: The results showed ROM piglets had a lower inter-individual variation of faecal microbiota composition before weaning and a lower relative abundance of proteobacterial genera in jejunum (Undibacterium and Solobacterium) and caecum (Intestinibacter and Succinivibrionaceae_UCG_001) on day 70, as compared to Ctrl piglets. ROM supplementation also influenced gut mucosal gene expression in both ileum and caecum on day 44. In ileum, ROM pigs showed increased expression of TJP1/ZO1 but decreased expression of CLDN3, CLDN5 and MUC2 than Ctrl pigs. Genes involved in TLR signalling (e.g., TICAM2, IRAK4 and LY96) were more expressed but MYD88 and TOLLIP were less expressed in ROM pigs than Ctrl animals. NOS2 and HIF1A involved in redox signalling were either decreased or increased in ROM pigs, respectively. In caecum, differentially expressed genes between two groups were mainly shown as increased expression (e.g., MUC2, PDGFRB, TOLLIP, TNFAIP3 and MYD88) in ROM pigs. Moreover, ROM animals showed higher NK cell activation in blood and enhanced IL-10 production in ex vivo stimulated MLN cells before weaning. CONCLUSIONS: Collectively, these results suggest that ROM supplementation in early life modulates gut microbiota and (local) immune system development. Consequently, ROM supplementation may contribute to improving health of pigs during the weaning transition period and reducing antibiotics use.

Optimization of Capture ELISAs for Chicken Cytokines Using Commercially Available Antibodies.

Cytokines like interferon (IFN)-γ, interleukin (IL)-2, IL-6, IL-10, and IL-12p40 are important biomarkers for characterizing the nature and strength of immune responses. It is important to be able to quantify the cytokines at the protein level in biological samples. Quantification of chicken cytokines is generally performed on the level of messenger RNA (mRNA) by quantitative polymerase chain reaction (qPCR) because very few capture ELISAs for the quantification of chicken cytokine proteins are commercially available. Here, we describe the optimization and validation of capture ELISAs for chicken IL-2, IL-6, IL-10, IL-12p40, and IFN-γ using commercially available antibodies and reagents. First, we determined the optimal concentrations of the antibodies. We then verified the ELISAs' performance and established that the lower limit of detection (LLOD) for all cytokines was below 32 pg/mL. The ELISAs show the same binding characteristics for recombinant and native cytokines (parallelism was <15.2% CV). Values for inter-assay variation were consistently low and mostly <20% CV. Overall, the optimized capture ELISAs are sensitive (<32 pg/mL) and reliable tools to quantify chicken cytokines. These ELISAs can easily and inexpensively be utilized in any immunological lab and may therefore have wide applicability in immunological research for poultry.

Highly Specific Protein Identification by Immunoprecipitation-Mass Spectrometry Using Antifouling Microbeads.

A common method to study protein complexes is immunoprecipitation (IP), followed by mass spectrometry (thus labeled: IP-MS). IP-MS has been shown to be a powerful tool to identify protein-protein interactions. It is, however, often challenging to discriminate true protein interactors from contaminating ones. Here, we describe the preparation of antifouling azide-functionalized polymer-coated beads that can be equipped with an antibody of choice via click chemistry. We show the preparation of generic immunoprecipitation beads that target the green fluorescent protein (GFP) and show how they can be used in IP-MS experiments targeting two different GFP-fusion proteins. Our antifouling beads were able to efficiently identify relevant protein-protein interactions but with a strong reduction in unwanted nonspecific protein binding compared to commercial anti-GFP beads.

Effects of E scherichia coli Nissle 1917 on the Porcine Gut Microbiota, Intestinal Epithelium and Immune System in Early Life.

Early in life and particularly around weaning, piglets are susceptible to infections because of abrupt social, environmental, and dietary changes. Dietary interventions with probiotic bacteria have gained popularity because of the increased awareness of the direct link between diet and health. In this study, piglets received the probiotic strain Escherichia coli Nissle 1917 (EcN) or a control treatment perorally from day 2 after birth until 2 weeks post-weaning. To investigate spatio-temporal effects of EcN on the gut microbiota composition, intestinal epithelial gene expression and immune system, feces, digesta, blood, scraping material and mesenteric lymph node tissue were collected at different time points. In addition, oral vaccinations against Salmonella enterica serovar Typhimurium were administered on days 21 and 45 of the study to assess the immunocompetence. EcN-treated pigs showed a reduced diversity of taxa within the phylum Proteobacteria and a lower relative abundance of taxa within the genus Treponema during the pre-weaning period. Moreover, EcN induced T cell proliferation and Natural Killer cell activation in blood and enhanced IL-10 production in ex vivo stimulated mesenteric lymph node cells, the latter pointing toward a more regulatory or anti-inflammatory state of the local gut-associated immune system. These outcomes were primarily observed pre-weaning. No significant differences were observed between the treatment groups with regards to body weight, epithelial gene expression, and immune response upon vaccination. Differences observed during the post-weaning period between the treatment groups were modest. Overall, this study demonstrates that the pre-weaning period offers a 'window of opportunity' to modulate the porcine gut microbiota and immune system through dietary interventions such as EcN supplementation.

Location and expression kinetics of Tc24 in different life stages of Trypanosoma cruzi.

Tc24-C4, a modified recombinant flagellar calcium-binding protein of Trypanosoma cruzi, is under development as a therapeutic subunit vaccine candidate to prevent or delay progression of chronic Chagasic cardiomyopathy. When combined with Toll-like receptor agonists, Tc24-C4 immunization reduces parasitemia, parasites in cardiac tissue, and cardiac fibrosis and inflammation in animal models. To support further research on the vaccine candidate and its mechanism of action, murine monoclonal antibodies (mAbs) against Tc24-C4 were generated. Here, we report new findings made with mAb Tc24-C4/884 that detects Tc24-WT and Tc24-C4, as well as native Tc24 in T. cruzi on ELISA, western blots, and different imaging techniques. Surprisingly, detection of Tc24 by Tc24-C/884 in fixed T. cruzi trypomastigotes required permeabilization of the parasite, revealing that Tc24 is not exposed on the surface of T. cruzi, making a direct role of antibodies in the induced protection after Tc24-C4 immunization less likely. We further observed that after immunostaining T. cruzi-infected cells with mAb Tc24-C4/884, the expression of Tc24 decreases significantly when T. cruzi trypomastigotes enter host cells and transform into amastigotes. However, Tc24 is then upregulated in association with parasite flagellar growth linked to re-transformation into the trypomastigote form, prior to host cellular escape. These observations are discussed in the context of potential mechanisms of vaccine immunity.

Component-resolved microarray analysis of IgE sensitization profiles to Culicoides recombinant allergens in horses with insect bite hypersensitivity.

BACKGROUND: Allergy to bites of blood-sucking insects, including biting midges, can affect both human and veterinary patients. Horses are often suffering from an IgE-mediated allergic dermatitis caused by bites of midges (Culicoides spp). With the aim to improve allergen immunotherapy (AIT), numerous Culicoides allergens have been produced as recombinant (r-) proteins. This study aimed to test a comprehensive panel of differently expressed Culicoides r-allergens on a cohort of IBH-affected and control horses using an allergen microarray. METHODS: IgE levels to 27 Culicoides r-allergens, including 8 previously unpublished allergens, of which 11 were expressed in more than one expression system, were determined in sera from 347 horses. ROC analyses were carried out, cut-offs selected using a specificity of 95% and seropositivity rates compared between horses affected with insect bite hypersensitivity (IBH) and control horses. The combination of r-allergens giving the best performing test was determined using logistic regression analysis. RESULTS: Seropositivity was significantly higher in IBH horses compared with controls for 25 r-allergens. Nine Culicoides r-allergens were major allergens for IBH with seven of them binding IgE in sera from > 70% of the IBH-affected horses. Combination of these top seven r-allergens could diagnose > 90% of IBH-affected horses with a specificity of > 95%. Correlation between differently expressed r-allergens was usually high (mean = 0.69, range: 0.28-0.91). CONCLUSION: This microarray will be a powerful tool for the development of component-resolved, patient-tailored AIT for IBH and could be useful for the study of allergy to biting midges in humans and other species.

Impact of Yeast-Derived ß-Glucans on the Porcine Gut Microbiota and Immune System in Early Life.

Piglets are susceptible to infections in early life and around weaning due to rapid environmental and dietary changes. A compelling target to improve pig health in early life is diet, as it constitutes a pivotal determinant of gut microbial colonization and maturation of the host's immune system. In the present study, we investigated how supplementation of yeast-derived ß-glucans affects the gut microbiota and immune function pre- and post-weaning, and how these complex systems develop over time. From day two after birth until two weeks after weaning, piglets received yeast-derived ß-glucans or a control treatment orally and were subsequently vaccinated against Salmonella Typhimurium. Faeces, digesta, blood, and tissue samples were collected to study gut microbiota composition and immune function. Overall, yeast-derived ß-glucans did not affect the vaccination response, and only modest effects on faecal microbiota composition and immune parameters were observed, primarily before weaning. This study demonstrates that the pre-weaning period offers a 'window of opportunity' to alter the gut microbiota and immune system through diet. However, the observed changes were modest, and any long-lasting effects of yeast-derived ß-glucans remain to be elucidated.

Differential immunomodulation of porcine bone marrow derived dendritic cells by E. coli Nissle 1917 and ß-glucans.

In early life and around weaning, pigs are at risk of developing infectious diseases which compromise animal welfare and have major economic consequences for the pig industry. A promising strategy to enhance resistance against infectious diseases is immunomodulation by feed additives. To assess the immune stimulating potential of feed additives in vitro, bone marrow-derived dendritic cells were used. These cells play a central role in the innate and adaptive immune system and are the first cells encountered by antigens that pass the epithelial barrier. Two different feed additives were tested on dendritic cells cultured from fresh and cryopreserved bone marrow cells; a widely used commercial feed additive based on yeast-derived ß-glucans and the gram-negative probiotic strain E. coli Nissle 1917. E. coli Nissle 1917, but not ß-glucans, induced a dose-dependent upregulation of the cell maturation marker CD80/86, whereas both feed additives induced a dose-dependent production of pro- and anti-inflammatory cytokines, including TNFα, IL-1ß, IL-6 and IL-10. Furthermore, E. coli Nissle 1917 consistently induced higher levels of cytokine production than ß-glucans. These immunomodulatory responses could be assessed by fresh as well as cryopreserved in vitro cultured porcine bone marrow-derived dendritic cells. Taken together, these results demonstrate that both ß-glucans and E. coli Nissle 1917 are able to enhance dendritic cell maturation, but in a differential manner. A more mature dendritic cell phenotype could contribute to a more efficient response to infections. Moreover, both fresh and cryopreserved bone marrow-derived dendritic cells can be used as in vitro pre-screening tools which enable an evidence based prediction of the potential immune stimulating effects of different feed additives.

Genomic Regions Associated with IgE Levels against Culicoides spp. Antigens in Three Horse Breeds.

Insect bite hypersensitivity (IBH), which is a cutaneous allergic reaction to antigens from Culicoides spp., is the most prevalent skin disorder in horses. Misdiagnosis is possible, as IBH is usually diagnosed based on clinical signs. Our study is the first to employ IgE levels against several recombinant Culicoides spp. allergens as an objective, independent, and quantitative phenotype to improve the power to detect genetic variants that underlie IBH. Genotypes of 200 Shetland ponies, 127 Icelandic horses, and 223 Belgian Warmblood horses were analyzed while using a mixed model approach. No single-nucleotide polymorphism (SNP) passed the Bonferroni corrected significance threshold, but several regions were identified within and across breeds, which confirmed previously identified regions of interest and, in addition, identifying new regions of interest. Allergen-specific IgE levels are a continuous and objective phenotype that allow for more powerful analyses when compared to a case-control set-up, as more significant associations were obtained. However, the use of a higher density array seems necessary to fully employ the use of IgE levels as a phenotype. While these results still require validation in a large independent dataset, the use of allergen-specific IgE levels showed value as an objective and continuous phenotype that can deepen our understanding of the biology underlying IBH.

Leveraging the Medicines for Malaria Venture malaria and pathogen boxes to discover chemical inhibitors of East Coast fever.

Chemotherapy of East Coast fever, a lymphoproliferative cancer-like disease of cattle causing significant economic losses in Africa, is largely dependent on the use of buparvaquone, a drug that was developed in the late 1980's. The disease is caused by the tick-borne protozoan pathogen Theileria parva. Buparvaquone can be used prophylactically and it is also active against tropical theileriosis, caused by the related parasite Theileria annulata. Recently, drug resistance was reported in T. annulata, and could occur in T. parva. Using a 3H-thymidine incorporation assay we screened 796 open source compounds from the Medicines for Malaria Venture (MMV) to discover novel chemicals with potential inhibitory activity to T. parva. We identified nine malaria box compounds and eight pathogen box compounds that inhibited the proliferation of F100TpM, a T. parva infected lymphocyte cell line. However, only two compounds, MMV008212 and MMV688372 represent promising leads with IC50 values of 0.78 and 0.61 µM, respectively, and CC50 values > 5 µM. The remaining compounds exhibited a high degree of toxicity (CC50 values < 1.09 µM) on the proliferation of bovine peripheral blood mononuclear cells stimulated with concanavalin A. We also tested the anti-cancer drug, dasatinib, used in the chemotherapy of some leukemias. Dasatinib was as active and safe as buparvaquone in vitro, with an IC50 of 5 and 4.2 nM, respectively, and CC50 > 10 µM. Our preliminary data suggest that it may be possible to repurpose compounds from the cancer field as well as MMV as novel anti-T. parva molecules.

Systematic Comparison of Zwitterionic and Non-Zwitterionic Antifouling Polymer Brushes on a Bead-Based Platform.

Nonspecific adsorption of biomolecules to solid surfaces, a process called biofouling, is a major concern in many biomedical applications. Great effort has been made in the development of antifouling polymer coatings that are capable of repelling the nonspecific adsorption of proteins, cells, and micro-organisms. In this respect, we herein contribute to understanding the factors that determine which polymer brush results in the best antifouling coating. To this end, we compared five different monomers: two sulfobetaines, a carboxybetaine, a phosphocholine, and a hydroxyl acrylamide. The antifouling coatings were analyzed using our previously described bead-based method with flow cytometry as the read-out system. This method allows for the quick and automated analysis of thousands of beads per second, enabling fast analysis and good statistics. We report the first direct comparison made between a sulfobetaine with opposite charges separated by two and three methylene groups and a carboxybetaine bearing two separating methylene groups. It was concluded that both the distance between opposite charges and the nature of the anionic groups have a distinct effect on the antifouling performance. Phosphocholines and simple hydroxyl acrylamides are not often compared with the betaines. However, here we found that they perform equally well or even better, yielding the following overall antifouling ranking: HPMAA ≥ PCMA-2 ≈ CBMAA-2 > SBMAA-2 > SBMAA-3 ≫ nonmodified beads (HPMAA being the best).

Antibodies to in silico selected GPI-anchored Theileria parva proteins neutralize sporozoite infection in vitro.

East Coast fever (ECF) caused by Theileria parva kills cattle in East, Central and Southern Africa leading to significant economic losses. Vaccination is used as a control strategy against ECF and is presently dependent on deliberate infection with live sporozoites and simultaneous treatment with a long-acting oxytetracycline. Although effective, this method has serious limitations; the immunity is parasite strain specific and immunized cattle can become life-long asymptomatic carriers of the parasite, posing risk for the spread of the disease. In efforts to develop a subunit vaccine, the role of antibodies in the neutralization of T. parva sporozoites infection of host cells has been investigated and a circumsporozoite protein, p67, is able to induce such neutralizing antibodies. However, the p67 protein only protects a proportion of immunized cattle against T. parva challenge and such protection might be improved by inclusion of additional parasite antigens that neutralize sporozoite infection. In an attempt to identify such antigens, we searched the re-annotated T. parva genome for genes predicted to contain GPI anchor signals, since they are likely to be located on the cell surface, and expressed fragments of six of the selected genes in E. coli. The recombinant proteins were used to raise antisera in mice. Antisera to two proteins, TpMuguga_01g00876 and TpMuguga_01g00939, neutralized sporozoite infectivity to a high degree, while antisera to two additional proteins, TpMuguga_01g00095 and TpMuguga_04g00437, exhibited moderate neutralizing capacity. We conclude that these four antigens are potential vaccine candidates, which should be evaluated further in cattle.

Characterization of the Theileria parva sporozoite proteome.

East Coast fever is a lymphoproliferative disease caused by the tick-borne protozoan parasite Theileria parva. The sporozoite stage of this parasite, harboured and released from the salivary glands of the tick Rhipicephalus appendiculatus during feeding, invades and establishes infection in bovine lymphocytes. Blocking this initial stage of invasion presents a promising vaccine strategy for control of East Coast fever and can in part be achieved by targeting the major sporozoite surface protein p67. To support research on the biology of T. parva and the identification of additional candidate vaccine antigens, we report on the sporozoite proteome as defined by LC-MS/MS analysis. In total, 4780 proteins were identified in an enriched preparation of sporozoites. Of these, 2007 were identified as T. parva proteins, representing close to 50% of the total predicted parasite proteome. The remaining 2773 proteins were derived from the tick vector. The identified sporozoite proteins include a set of known T. parva antigens targeted by antibodies and cytotoxic T cells from cattle that are immune to East Coast fever. We also identified proteins predicted to be orthologs of Plasmodium falciparum sporozoite surface molecules and invasion organelle proteins, and proteins that may contribute to the phenomenon of bovine lymphocyte transformation. Overall, these data establish a protein expression profile of T. parva sporozoites as an important starting point for further study of a parasitic species which has considerable agricultural impact.

Highly Specific Binding on Antifouling Zwitterionic Polymer-Coated Microbeads as Measured by Flow Cytometry.

Micron- and nano-sized particles are extensively used in various biomedical applications. However, their performance is often drastically hampered by the nonspecific adsorption of biomolecules, a process called biofouling, which can cause false-positive and false-negative outcomes in diagnostic tests. Although antifouling coatings have been extensively studied on flat surfaces, their use on micro- and nanoparticles remains largely unexplored, despite the widespread experimental (specifically, clinical) uncertainties that arise because of biofouling. Here, we describe the preparation of magnetic micron-sized beads coated with zwitterionic sulfobetaine polymer brushes that display strong antifouling characteristics. These coated beads can then be equipped with recognition elements of choice, to enable the specific binding of target molecules. First, we present a proof of principle with biotin-functionalized beads that are able to specifically bind fluorescently labeled streptavidin from a complex mixture of serum proteins. Moreover, we show the versatility of the method by demonstrating that it is also possible to functionalize the beads with mannose moieties to specifically bind the carbohydrate-binding protein concanavalin A. Flow cytometry was used to show that thus-modified beads only bind specifically targeted proteins, with minimal/near-zero nonspecific protein adsorption from other proteins that are present. These antifouling zwitterionic polymer-coated beads, therefore, provide a significant advancement for the many bead-based diagnostic and other biosensing applications that require stringent antifouling conditions.

Cyprinid Herpesvirus 3 Il10 Inhibits Inflammatory Activities of Carp Macrophages and Promotes Proliferation of Igm+ B Cells and Memory T Cells in a Manner Similar to Carp Il10.

Cyprinid herpesvirus 3 (CyHV-3) is the causative agent of a lethal disease of carp and encodes for an Il10 homolog (ORF134). Our previous studies with a recombinant ORF134-deleted strain and the derived revertant strain suggested that cyprinid herpesvirus 3 Il10 (CyHV-3 Il10 [cyhv3Il10]) is not essential for viral replication in vitro, or virulence in vivo. In apparent contrast, cyhv3Il10 is one of the most abundant proteins of the CyHV-3 secretome and is structurally very similar to carp Il10 and also human IL10. To date, studies addressing the biological activity of cyhv3Il10 on cells of its natural host have not been performed. To address the apparent contradiction between the presence of a structurally conserved Il10 homolog in the genome of CyHV-3 and the lack of a clear phenotype in vivo using recombinant cyhv3Il10-deleted viruses, we used an in vitro approach to investigate in detail whether cyhv3Il10 exerts any biological activity on carp cells. In this study, we provide direct evidence that cyhv3Il10 is biologically active and, similarly to carp Il10, signals via a conserved Stat3 pathway modulating immune cells of its natural host, carp. In vitro, cyhv3Il10 deactivates phagocytes with a prominent effect on macrophages, while also promoting proliferation of Igm(+) B cells and memory T cells. Collectively, this study demonstrates a clear biological activity of cyhv3Il10 on cells of its natural host and indicates that cyhv3Il10 is a true viral ortholog of carp Il10. Furthermore, to our knowledge, this is the first report on biological activities of a nonmammalian viral Il10 homolog.

Recombinant Culicoides obsoletus complex allergens stimulate antigen-specific T cells of insect bite hypersensitive Shetland ponies in vitro.

BACKGROUND: Ponies may suffer from Insect bite hypersensitivity (IBH), an allergic IgE-mediated pruritic skin disorder, induced by allergens from biting midges of the Culicoides spp. HYPOTHESIS/OBJECTIVES: To determine whether recombinant Culicoides obsoletus allergens are able to activate T cells of ponies exposed to C. obsoletus and whether these allergen-specific responses differ between IBH-affected and healthy ponies. ANIMALS: Ten IBH-affected Shetland ponies and 10 age-matched healthy controls taken from the same stables, to ensure similar exposure to midges. METHOD: Peripheral blood mononuclear cells (PBMC) were cultured with two different pools of recombinant C. obsoletus complex allergens to expand the allergen-specific T cells. These PBMC cultures were subsequently co-cultured with mature dendritic cells (DCs) loaded with the same antigens. Induction of Th1, Th2 and regulatory T (Treg) cells in these DC/PBMC co-cultures was assessed by analysis of IFN-γ, IL-4, IL-10 and FoxP3 expression levels using quantitative RT-PCR and phenotyping by flow cytometry. RESULTS: Recombinant C. obsoletus allergens increased IFN-γ mRNA expression levels, percentages of IFN-γ expressing (Th1) cells and CD25(high) FoxP3(+) IL-10(+) Tregs compared to unstimulated DC/PBMC co-cultures. Stimulation of IL-4 expressing Th2 cells by the recombinant allergens was far less pronounced. The DC/PBMC co-cultures did not reveal significant differences between healthy and IBH-affected ponies for any of the analysed parameters, except for higher IL-4 mRNA levels in IBH affected ponies after stimulation with one of the two allergen pools. CONCLUSION AND CLINICAL IMPORTANCE: The recombinant C. obsoletus complex allergens can stimulate antigen-specific Th1 and IL10 producing Treg cells and are therefore promising candidates for the immunotherapy of IBH.

Factors associated with Culicoides Obsoletus complex spp.-specific IgE reactivity in Icelandic horses and Shetland ponies.

Insect bite hypersensitivity (IBH) is a common allergic skin disease in horses, caused by biting insects of the Culicoides spp. In The Netherlands, Culicoides spp. of the Obsoletus complex are the most important midges involved in IBH. The aim of the present study was to identify and quantify associations between several endogenous (host) and exogenous (environmental) factors and immunoglobulin E (IgE) reactivity against Obsoletus complex-derived whole body extract or seven recombinant allergens, measured by ELISA. Data from 143 Icelandic horses and 177 Shetland ponies were analysed using multivariable models. In addition, the relationship between IgE reactivity and severity of clinical signs in IBH-affected horses was examined. Positive correlations were found between Obsoletus complex-specific IgE and severity of clinical signs. Disease status (IBH affected or control), breed and the interaction between IBH status and breed were significantly associated with IgE reactivity against several Obsoletus complex allergens. Significantly greater IgE reactivity was seen in IBH-affected horses compared to controls. The differences in IgE values between cases and controls were most pronounced in Icelandic horses. Shetland pony controls had significantly greater IgE reactivity compared to Icelandic horse controls, while differences in IgE values comparing Shetland pony cases and Icelandic horse cases were not significant. Severity of clinical signs and IgE reactivity in IBH-affected horses against several Obsoletus complex allergens appeared to be related. Consideration of the factors associated with Obsoletus complex-specific IgE in horses might further improve interpretation and accuracy of IgE ELISA test results within these breeds, although further research is required.

Expression of the iron hormone hepcidin distinguishes different types of anemia in African children.

Childhood anemia is a major global health problem resulting from multiple causes. Iron supplementation addresses iron deficiency anemia but is undesirable for other types of anemia and may exacerbate infections. The peptide hormone hepcidin governs iron absorption; hepcidin transcription is mediated by iron, inflammation, and erythropoietic signals. However, the behavior of hepcidin in populations where anemia is prevalent is not well established. We show that hepcidin measurements in 1313 African children from The Gambia and Tanzania (samples taken in 2001 and 2008, respectively) could be used to identify iron deficiency anemia. A retrospective secondary analysis of published data from 25 Gambian children with either postmalarial or nonmalarial anemia demonstrated that hepcidin measurements identified individuals who incorporated >20% oral iron into their erythrocytes. Modeling showed that this sensitivity of hepcidin expression at the population level could potentially enable simple groupings of individuals with anemia into iron-responsive and non-iron-responsive subtypes and hence could guide iron supplementation for those who would most benefit.

Evaluation of a diagnostic ELISA for insect bite hypersensitivity in horses using recombinant Obsoletus complex allergens.

Culicoides spp. of the Obsoletus complex belong to the most important species of midge, involved in causing insect bite hypersensitivity (IBH) in horses in The Netherlands. The aim of the current study was to evaluate seven different Obsoletus complex-derived recombinant allergens (Cul o 1-Cul o 7) and to compare these with Obsoletus complex whole body extract (WBE) in an IgE ELISA, using sera of 194 clinically-confirmed cases of IBH and 175 unaffected horses. The highest test accuracy was obtained with WBE, followed by Cul o 2, 3 and 5. Two ELISAs with a combination of recombinant allergens, Combi-1 (Cul o 3, 5 and 7) and Combi-2 (Cul o 1, 2, 5 and 7) were additionally performed and both resulted in high test accuracies close to that obtained with WBE. Combi-1 resulted in the best sensitivity and specificity, both 89%. Both Combi-1 and Combi-2 performed less well with samples collected in winter, but over 70% of the IBH-affected horses could still be identified. In conclusion, a combination of three Obsoletus complex recombinant allergens (Cul o 3, 5 and 7) could potentially replace Obsoletus complex WBE in an IgE ELISA for diagnosis of IBH in horses.

Cloning and expression of candidate allergens from Culicoides obsoletus for diagnosis of insect bite hypersensitivity in horses.

Insect bite hypersensitivity (IBH) is an IgE-mediated (Type I) hypersensitivity reaction induced by allergens from biting midges of the Culicoides spp. The aim of the present study was to identify, clone and express recombinant allergens from C. obsoletus, the main species found feeding on horses in the Netherlands, by sequence homology searches on the C. obsoletus specific RNA database, with previously described allergens from C. nubeculosus and C. sonorensis. BLAST searches with these described allergens resulted in similarity hits with 7 genes coding for C. obsoletus allergens. These allergens were expressed as hexahistidine tagged recombinant proteins in E. coli. Allergens were termed Cul o 1-Cul o 7. A maltase (Cul o 1) plus Cul s 1 (maltase of C. sonorensis) were additionally expressed in insect cells using the baculovirus expression system to compare homologous allergens from different species produced with different expression systems in diagnostic in vitro and in vivo tests. We demonstrate that IBH affected horses in the Netherlands show higher IgE levels to Cul o 1 than to Cul s 1, as determined by an IgE ELISA. Furthermore, we show that Cul o 1 produced in E. coli is at least as suitable for in vitro diagnosis of IBH affected horses as Cul o 1 produced in the baculovirus/insect cell expression system. The resulting proteins were evaluated for their ability to discriminate IBH affected and healthy horses by ELISA and intradermal testing. The frequency of positive test results by ELISA within IBH affected horses ranged from 38% to 67% for the different allergens. When results of IgE-binding to Cul o 1-Cul o 7 were combined the test had a sensitivity of 92% and specificity of 85%. The capability of the allergens to induce Type I hypersensitivity reaction in IBH affected horses was demonstrated by an intradermal test. The results show that E. coli expressed recombinant allergens from C. obsoletus are valuable tools to determine the allergen specific sensitisation profile (component resolved diagnosis) in horses with IBH in countries were C. obsoletus is the most abundant species and may facilitate in the development of future immunotherapy.