Cytokines and chemokines skin gene expression in correlation with immune cells in blood and severity in equine insect bite hypersensitivity.

Background: Insect bite hypersensitivity (IBH) is the most frequent skin allergy of horses and is highly debilitating, especially in the chronic phase. IBH is caused by IgE-mediated hypersensitivity reactions to culicoides midge bites and an imbalanced immune response that reduces the welfare of affected horses. Objective: In the present study, we investigated the pathological mechanisms of IBH, aiming to understand the immune cell modulation in acute allergic skin lesions of IBH horses with the goal of finding possible biomarkers for a diagnostic approach to monitor treatment success. Methods: By qPCR, we quantified the gene expression of cytokines, chemokines, and immune receptors in skin punch biopsies of IBH with different severity levels and healthy horses simultaneously in tandem with the analysis of immune cell counts in the blood. Results: Our data show an increase in blood eosinophils, monocytes, and basophils with a concomitant, significant increase in associated cytokine, chemokine, and immune cell receptor mRNA expression levels in the lesional skin of IBH horses. Moreover, IL-5Ra, CCR5, IFN-γ, and IL-31Ra were strongly associated with IBH severity, while IL-31 and IL-33 were rather associated with a milder form of IBH. In addition, our data show a strong correlation of basophil cell count in blood with IL-31Ra, IL-5, IL-5Ra, IFN-γ, HRH2, HRH4, CCR3, CCR5, IL-12b, IL-10, IL-1ß, and CCL26 mRNA expression in skin punch biopsies of IBH horses. Conclusion: In summary, several cytokines and chemokines have been found to be associated with disease severity, hence contributing to IBH pathology. These molecules can be used as potential biomarkers to monitor the onset and progression of the disease or even to evaluate and monitor the efficacy of new therapeutic treatments for IBH skin allergy. To our knowledge, this is the first study that investigated immune cells together with a large set of genes related to their biological function, including correlation to disease severity, in a large cohort of healthy and IBH horses.

First clinical expression of equine insect bite hypersensitivity is associated with co-sensitization to multiple Culicoides allergens.

BACKGROUND: Insect bite hypersensitivity (IBH) is an IgE-mediated allergic dermatitis in horses incited by salivary allergens from Culicoides spp. IBH does not occur in Iceland, as the causative agents are absent, however a high prevalence is seen in horses exported to Culicoides-rich environments. AIMS: To study the natural course of sensitization to Culicoides allergens and identify the primary sensitizing allergen(s) in horses exported from Iceland utilizing a comprehensive panel of Culicoides recombinant (r-) allergens. METHOD: IgE microarray profiling to 27 Culicoides r-allergens was conducted on 110 serological samples from horses imported to Switzerland from Iceland that subsequently developed IBH or remained healthy. Furthermore, a longitudinal study of 31 IBH horses determined IgE profiles the summer preceding first clinical signs of IBH (TIBH-1), the summer of first clinical signs (TIBH) and the following summer (TIBH+1). In a group of Icelandic horses residing in Sweden, effects of origin (born in Iceland or Sweden) and duration of IBH (<4 years, 4-7 years, >7 years) on Culicoides-specific IgE was evaluated. Sero-positivity rates and IgE levels were compared. RESULTS: At TIBH, horses were sensitized to a median of 11 r-allergens (range = 0-21), of which nine were major allergens. This was significantly higher than TIBH-1 (3, 0-16), as well as the healthy (1, 0-14) group. There was no significant increase between TIBH and TIBH+1(12, 0-23). IBH-affected horses exported from Iceland had a significantly higher degree of sensitization than those born in Europe, while duration of IBH did not significantly affect degree of sensitization. CONCLUSION: Significant sensitization is only detected in serum the year of first clinical signs of IBH. Horses become sensitized simultaneously to multiple Culicoides r-allergens, indicating that IgE-reactivity is due to co-sensitization rather than cross-reactivity between Culicoides allergens. Nine major first sensitizing r-allergens have been identified, which could be used for preventive allergen immunotherapy.

Comparison of recombinant Culicoides allergens produced in different expression systems for IgE serology of insect bite hypersensitivity in horses of different origins.

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses, an IgE-mediated reaction to Culicoides midges. Causative Culicoides spp. are not indigenous in Iceland resulting in high prevalence of IBH in horses born in Iceland and exported as compared to Icelandic horses born in a Culicoides rich environment. The aims were (i) to compare IgE levels in sera of IBH-affected horses born in Iceland (n = 47) with horses of the Icelandic breed (n = 23) and of other breeds (n = 27) born in Culicoides infested area; (ii) to investigate if barley could be a useful production system of allergens for IBH immunoassays. IgE binding in sera was tested by ELISA on two recombinant Culicoides allergens, rCul n 3 and rCul n 4, each produced in E. coli, insect cells and barley. Significantly more IgE was detected against all allergens in sera from IBH-affected compared to healthy horses. Icelandic-born Icelandic horses stand out with higher IgE levels against the allergens and higher area under the curve (AUC) on rCul n 4 as compared to the European-born horses. The barley and E.coli produced allergens had very similar performance in distinguishing between IBH-affected and healthy horses.

Peripheral blood basophils are the main source for early interleukin-4 secretion upon in vitro stimulation with Culicoides allergen in allergic horses.

Interleukin-4 (IL-4) is a key cytokine secreted by type 2 T helper (Th2) cells that orchestrates immune responses during allergic reactions. Human and mouse studies additionally suggest that basophils have a unique role in the regulation of allergic diseases by providing initial IL-4 to drive T cell development towards the Th2 phenotype. Equine Culicoides hypersensitivity (CH) is a seasonal immunoglobulin E (IgE)-mediated allergic dermatitis in horses in response to salivary allergens from Culicoides (Cul) midges. Here, we analyzed IL-4 production in peripheral blood mononuclear cells (PBMC) of CH affected (n = 8) and healthy horses (n = 8) living together in an environment with natural Cul exposure. During Cul exposure when allergic horses had clinical allergy, IL-4 secretion from PBMC after stimulation with Cul extract was similar between healthy and CH affected horses. In contrast, allergic horses had higher IL-4 secretion from PBMC than healthy horses during months without allergen exposure. In addition, allergic horses had increased percentages of IL-4+ cells after Cul stimulation compared to healthy horses, while both groups had similar percentages of IL-4+ cells following IgE crosslinking. The IL-4+ cells were subsequently characterized using different cell surface markers as basophils, while very few allergen-specific CD4+ cells were detected in PBMC after Cul extract stimulation. Similarly, IgE crosslinking by anti-IgE triggered basophils to produce IL-4 in all horses. PMA/ionomycin consistently induced high percentages of IL-4+ Th2 cells in both groups confirming that T cells of all horses studied were capable of IL-4 production. In conclusion, peripheral blood basophils produced high amounts of IL-4 in allergic horses after stimulation with Cul allergens, and allergic horses also maintained higher basophil percentages throughout the year than healthy horses. These new findings suggest that peripheral blood basophils may play a yet underestimated role in innate IL-4 production upon allergen activation in horses with CH. Basophil-derived IL-4 might be a crucial early signal for immune induction, modulating of immune responses towards Th2 immunity and IgE production.

Viral infection and allergy - What equine immune responses can tell us about disease severity and protection.

Horses have many naturally occurring diseases that mimic similar conditions in humans. The ability to conduct environmentally controlled experiments and induced disease studies in a genetically diverse host makes the horse a valuable intermediate model between mouse studies and human clinical trials. This review highlights important similarities in the immune landscape between horses and humans using current research on two equine diseases as examples. First, equine herpesvirus type 1 (EHV-1) infection initiates a series of innate inflammatory signals at its mucosal entry site in the upper respiratory tract. These inflammatory markers are highly synchronized and predictable between individuals during viral respiratory infection and ultimately lead to adaptive immune induction and protection. The timing of early inflammatory signals, followed by specific adaptive immune markers correlating with immunity and protection, allow accurate outbreak tracking and also provide a foundation for understanding the importance of local mucosal immunity during other viral respiratory infections. Second, rare peripheral blood immune cells that promote allergic inflammation can be analyzed during Culicoides hypersensitivity, a naturally occurring type I IgE-mediated allergic disease of horses. Rare immune cells, such as IgE-binding monocytes or basophils, can be studied repeatedly in the horse model to unravel their larger mechanistic role in inflammation during allergic and other inflammatory diseases. We conclude with a survey of all other common equine inflammatory conditions. Together, this review serves as a reference and rationale for the horse as a non-rodent model for immunological research.

IgE-Binding Monocytes Have an Enhanced Ability to Produce IL-8 (CXCL8) in Animals with Naturally Occurring Allergy.

IL-8 is a potent chemokine that recruits neutrophils and basophils to promote inflammation in many species. IL-8 is produced by many cell types, including monocytes. In this study, we report a novel role for IgE-binding monocytes, a rare peripheral immune cell type, to promote allergic inflammation through IL-8 production in a horse model of natural IgE-mediated allergy. We developed a mAb with confirmed specificity for both recombinant and native equine IL-8 for flow cytometric analysis. Equine IL-8 was produced by CD14+/MHC class II+/CD16- monocytes, including a subpopulation of IgE-binding monocytes, following stimulation with LPS. In addition, IgE cross-linking induced IL-8 production by both peripheral blood basophils and IgE-binding monocytes. IL-8 production was compared between healthy horses and those with a naturally occurring IgE-mediated skin allergy, Culicoides hypersensitivity. Allergic horses had significantly higher percentages of IL-8+ IgE-binding monocytes after IgE cross-linking. In contrast, frequencies of IL-8+ basophils after IgE cross-linking were similar in all horses, regardless of allergic disease, highlighting IgE-binding monocytes as a novel source of IL-8 during allergy. We concluded that IgE-binding monocytes from allergic individuals have an increased capacity for IL-8 production and likely contribute to the recruitment of innate immune cells during IgE-mediated allergy and promotion of inflammation during repeated allergen contact.

Cul o 2 specific IgG3/5 antibodies predicted Culicoides hypersensitivity in a group imported Icelandic horses.

BACKGROUND: Culicoides hypersensitivity (CH) is induced in horses by salivary allergens of Culicoides midges. In Iceland, the causal Culicoides species for CH are not present. Previous epidemiological data indicated that Icelandic horses are more susceptible to CH when they are exported from Iceland and first exposed to Culicoides at adult age. Horses born in countries where Culicoides is endemic, develop the disease less frequently. Here, we established a longitudinal allergy model to identify predictive and diagnostic serological biomarkers of CH. RESULTS: Sixteen adult Icelandic horses from Iceland were imported to the Northeastern United States (US) during the winter and were kept in the same environment with natural Culicoides exposure for the next two years. None of the horses showed clinical allergy during the first summer of Culicoides exposure. In the second summer, 9/16 horses (56%) developed CH. Allergen specific IgE and IgG isotype responses in serum samples were analysed using nine potential Culicoides allergens in a fluorescent bead-based multiplex assay. During the first summer of Culicoides exposure, while all horses were still clinically healthy, Cul o 2 specific IgG3/5 antibodies were higher in horses that developed the allergic disease in the second summer compared to those that did not become allergic (p = 0.043). The difference in Cul o 2 specific IgG3/5 antibodies between the two groups continued to be detectable through fall (p = 0.035) and winter of the first year. During the second summer, clinical signs first appeared and Cul o 3 specific IgG3/5 isotypes were elevated in allergic horses (p = 0.041). Cul o 2 specific IgG5 (p = 0.035), and Cul o 3 specific IgG3/5 (p = 0.043) were increased in late fall of year two when clinical signs started to improve again. CONCLUSIONS: Our results identified IgG5 and IgG3/5 antibodies against Cul o 2 and Cul o 3, respectively, as markers for CH during and shortly after the allergy season in the Northeastern US. In addition, Cul o 2 specific IgG3/5 antibodies may be valuable as a predictive biomarker of CH in horses that have been exposed to Culicoides but did not yet develop clinical signs.

Phenotype and function of IgE-binding monocytes in equine Culicoides hypersensitivity.

Human IgE-binding monocytes are identified as allergic disease mediators, but it is unknown whether IgE-binding monocytes promote or prevent an allergic response. We identified IgE-binding monocytes in equine peripheral blood as IgE+/MHCIIhigh/CD14low cells that bind IgE through an FcεRI αÉ£ variant. IgE-binding monocytes were analyzed monthly in Culicoides hypersensitive horses and nonallergic horses living together with natural exposure to Culicoides midges. The phenotype and frequency of IgE-binding monocytes remained consistent in all horses regardless of Culicoides exposure. All horses upregulated IgE-binding monocyte CD16 expression following initial Culicoides exposure. Serum total IgE concentration and monocyte surface IgE densities were positively correlated in all horses. We also demonstrated that IgE-binding monocytes produce IL-10, but not IL-4, IL-17A, or IFN-γ, following IgE crosslinking. In conclusion, we have characterized horse IgE-binding monocytes for the first time and further studies of these cells may provide important connections between regulation and cellular mechanisms of IgE-mediated diseases.

Therapeutic DNA vaccine attenuates itching and allergic inflammation in mice with established biting midge allergy.

Forcipomyia taiwana is a tiny hematophagous midge that attacks en masse. It is responsible for the most prevalent biting insect allergy in Taiwan. For t 2 is its major allergen. The intense itchy reactions can prevent allergic individuals from performing their regular daily outdoor activities. This study aimed to investigate whether the For t 2 DNA vaccine was effective in treating mice with established biting midge allergy. Mice were sensitized with recombinant For t 2 proteins or whole midge extracts. Two to four consecutive shots of various concentrations of For t 2 DNA vaccine, with or without CpG adjuvants, were then administered to midge-sensitized mice. Mice that received two shots of 50-100 µg For t 2 DNA vaccine showed a significant reduction in allergen-induced bouts of scratching, For t 2-specific IgE, specific IgG1/IgG2a ratio in sera, skin eosinophil infiltration, and IL-31 production, as well as IL-4 and IL-13 production by splenocytes. Two doses of For t 2 DNA vaccine one week apart was sufficient to treat mice with established biting midge allergy. The treatment resulted in clinical, immunological, and histopathological improvements. We recommend that this low-cost, convenient treatment strategy be developed for use in humans who are allergic to biting midges.

Effects of hypersensitivity disorders and environmental factors on the equine intestinal microbiota.

Background: Recent evidence suggests that an altered intestinal microbiota, specifically a reduction of bacterial diversity or a shift in microbial composition, is associated with the development of hypersensitivity disorders in humans, but this is unknown for horses.Objectives: In this study we hypothesized that horses affected by either Culicoides hypersensitivity or severe equine asthma or both show a decreased diversity of their intestinal microbiota. We also investigated environmental effects.Methods: Rectal swab samples of a total of 140 horses were collected and the owners completed a detailed questionnaire about their horse. For each allergic horse, a healthy peer from the same stable was equally sampled as an environmentally matched control. Microbiota in the swabs was determined by assessing the V4 region of the bacterial 16S rRNA gene. Structures of bacterial communities were investigated by means of alpha and beta diversity indices.Results: Group wise comparisons between healthy and allergic horses showed no significant differences regarding alpha (p = 0.9) and beta diversity (p = 0.5). However, the microbial structure was associated with environmental factors such as the type of stable (p = 0.001), access to pasture (p = 0.001) or the type of feeding (p = 0.003). There was also a strong location effect meaning that the microbiota was more similar within the same as compared between farms within this study.Conclusion: Our observations suggest that hypersensitivity disorders in adult horses are not associated with an alteration of the intestinal microbiota, but environmental and/or location factors strongly influence these bacteria.

Transfection of Culicoides sonorensis biting midge cell lines with Wolbachia pipientis.

BACKGROUND: Biting midges of the genus Culicoides vector multiple veterinary pathogens and are difficult to control. Endosymbionts particularly Wolbachia pipientis may offer an alternative to control populations of Culicoides and/or impact disease transmission in the form of population suppression or replacement strategies. METHODS: Culicoides sonorensis cell lines were transfected with a Wolbachia infection using a modified shell vial technique. Infections were confirmed using PCR and cell localization using fluorescent in situ hybridization (FISH). The stability of Wolbachia infections and density was determined by qPCR. qPCR was also used to examine immune genes in the IMD, Toll and JACK/STAT pathways to determine if Wolbachia were associated with an immune response in infected cells. RESULTS: Here we have transfected two Culicoides sonorensis cell lines (W3 and W8) with a Wolbachia infection (walbB) from donor Aedes albopictus Aa23 cells. PCR and FISH showed the presence of Wolbachia infections in both C. sonorensis cell lines. Infection densities were higher in the W8 cell lines when compared to W3. In stably infected cells, genes in the immune Toll, IMD and JAK/STAT pathways were upregulated, along with Attacin and an Attacin-like anti-microbial peptides. CONCLUSIONS: The successful introduction of Wolbachia infections in C. sonorensis cell lines and the upregulation of immune genes, suggest the utility of using Wolbachia for a population replacement and/or population suppression approach to limit the transmission of C. sonorensis vectored diseases. Results support the further investigation of Wolbachia induced pathogen inhibitory effects in Wolbachia-infected C. sonorensis cell lines and the introduction of Wolbachia into C. sonorensis adults via embryonic microinjection to examine for reproductive phenotypes and host fitness effects of a novel Wolbachia infection.

Comparison of three clinical scoring systems for Culicoides hypersensitivity in a herd of Icelandic horses.

BACKGROUND: Culicoides hypersensitivity (CH), an intensely pruritic and seasonal allergic dermatitis, is a common allergic disease affecting horses worldwide. Currently, there is no validated clinical scoring system for the quantification of clinical signs associated with CH. OBJECTIVES: To (i) determine the best cut-off point of three scoring systems, (ii) test the accuracy of each system when compared to the clinical diagnosis of an experienced veterinarian and (iii) assess agreement between systems. ANIMALS: Icelandic horses (n = 20); eight with CH and 12 unaffected, from a research herd receiving no treatments for allergic dermatitis. METHODS AND MATERIALS: Lesion scores were recorded biweekly from April until September with three clinical scoring systems (A, B and C) by a single observer initially blinded to CH status. Separate logistic regression analyses for each time point were used to determine appropriate cut-offs for CH classification. Spearman's rho and Cohen's kappa were calculated to analyze correlation of scores and agreement of CH categorization between systems, respectively. RESULTS: The best allergic cut-off scores for system A, B and C were determined to be three, eight and 12, respectively. For each system median areas under the curve (>0.85) were excellent and discriminatory ability for correctly classifying CH status was strong. Excellent correlation between scores for each system (Spearman's rho > 0.96) and excellent intersystem agreement for CH categorization (kappa ≥ 0.73) were found across scoring time points. CONCLUSION AND CLINICAL IMPORTANCE: Results support the use of these scoring systems as templates for the future standardization of a CH clinical scoring system.

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.

Allergen-specific immunoglobulin E in sera of horses affected with insect bite hypersensitivity, severe equine asthma or both conditions.

BACKGROUND: Genetic, epidemiologic, and clinical evidence suggests that, in horses, there are manifestations of hypersensitivity that can occur together. OBJECTIVES: To investigate whether concurrent insect bite hypersensitivity (IBH) and severe equine asthma (EA) is associated with higher allergen-specific and total serum immunoglobulin E (IgE) concentrations than only EA or IBH. ANIMALS: Healthy control horses (C, n = 40), horses with IBH (IBH, n = 24), severe EA (EA, n = 18), and both conditions (IBH/EA, n = 23) were included. METHODS: In our retrospective comparative study, sera from horses with signs of severe EA, IBH, and control animals were used. IgE specific for 15 recombinant (r) allergens as well as total serum IgE concentrations were measured by enzyme-linked immunosorbent assay. RESULTS: Group IBH (median sum r-Culicoides IgE: optical density at 405 nm [OD405 ] = 3.54 [0.48-15.07]) and group IBH/EA (OD405 = 4.55 [0.46-17.15]) had significantly (P < .001) higher IgE against Culicoides r-allergens than groups C (OD405 = 0.44 [0.21-2.05]) and EA (OD405 = 0.6 [0.2-2.9]). There were no significant (P > .05) differences between group IBH and group IBH/EA. No significant differences among the groups were found for the other r-allergens or total serum IgE concentration. Compared to controls, horses with severe IBH had significantly increased IgE concentration to 5 Culicoides r-allergens (P < .05), whereas horses with moderate IBH had significantly increased IgE concentration to only 3 Culicoides r-allergens (P < .05). CONCLUSIONS AND CLINICAL IMPORTANCE: Susceptibility of IBH-affected horses to develop EA is likely not associated with IgE-mediated immune reactions but with other immunopathological mechanisms.

The genome of the biting midge Culicoides sonorensis and gene expression analyses of vector competence for bluetongue virus.

BACKGROUND: The new genomic technologies have provided novel insights into the genetics of interactions between vectors, viruses and hosts, which are leading to advances in the control of arboviruses of medical importance. However, the development of tools and resources available for vectors of non-zoonotic arboviruses remains neglected. Biting midges of the genus Culicoides transmit some of the most important arboviruses of wildlife and livestock worldwide, with a global impact on economic productivity, health and welfare. The absence of a suitable reference genome has hindered genomic analyses to date in this important genus of vectors. In the present study, the genome of Culicoides sonorensis, a vector of bluetongue virus (BTV) in the USA, has been sequenced to provide the first reference genome for these vectors. In this study, we also report the use of the reference genome to perform initial transcriptomic analyses of vector competence for BTV. RESULTS: Our analyses reveal that the genome is 189 Mb, assembled in 7974 scaffolds. Its annotation using the transcriptomic data generated in this study and in a previous study has identified 15,612 genes. Gene expression analyses of C. sonorensis females infected with BTV performed in this study revealed 165 genes that were differentially expressed between vector competent and refractory females. Two candidate genes, glutathione S-transferase (gst) and the antiviral helicase ski2, previously recognized as involved in vector competence for BTV in C. sonorensis (gst) and repressing dsRNA virus propagation (ski2), were confirmed in this study. CONCLUSIONS: The reference genome of C. sonorensis has enabled preliminary analyses of the gene expression profiles of vector competent and refractory individuals. The genome and transcriptomes generated in this study provide suitable tools for future research on arbovirus transmission. These provide a valuable resource for these vector lineage, which diverged from other major Dipteran vector families over 200 million years ago. The genome will be a valuable source of comparative data for other important Dipteran vector families including mosquitoes (Culicidae) and sandflies (Psychodidae), and together with the transcriptomic data can yield potential targets for transgenic modification in vector control and functional studies.

Physiological and immunological responses to Culicoides sonorensis blood-feeding: a murine model.

BACKGROUND: Hematophagous Culicoides spp. biting midges are of great agricultural importance as livestock, equine, and wildlife pests and as vectors of the orbiviruses bluetongue, epizootic hemorrhagic disease and African horse sickness. To obtain a blood meal, midges deposit saliva containing allergens, proteases, and anti-hemostatic factors, into the dermis to facilitate feeding. Infected midges deposit virus along with the myriad of salivary proteins during feeding. The extreme efficiency with which midges are able to transmit orbiviruses is not clearly understood, as much is still unknown about the physiological trauma of the bite and immune responses to saliva deposited during feeding. Of particular interest are the first few hours and days after the bite; a critical time period for any midge-transmitted virus to quickly establish a localized infection and disseminate, while avoiding the hosts' immune responses. RESULTS: A mouse-midge feeding model using colonized Culicoides sonorensis midges was used to characterize innate mammalian immune responses to blood-feeding. Histological analysis of skin, and cellular and cytokine profiles of draining lymph nodes show Culicoides midge feeding elicited a potent pro-inflammatory Th-mediated cellular response with significant mast cell activation, subcutaneous hematomas, hypodermal edema and dermal capillary vasodilation, and rapid infiltration of leukocytes to the bite sites. Mast cell degranulation, triggered by bite trauma and specifically by midge saliva, was key to physiological and immunological responses and the ability of midges to feed to repletion. CONCLUSIONS: Midge feeding causes physiological and immunological responses that would be highly favorable for rapid infection and systemic dissemination orbiviruses if delivered during blood-feeding. Recruitment of leukocytic cells to bitten skin brings susceptible cell populations in proximity of deposited virus within hours of feeding. Infected cells would drain to lymph nodes, which become hyperplastic in response to saliva, and result in robust viral replication in expanding cell populations and dissemination via the lymph system. Additionally, saliva-induced vasodilation and direct breaches in dermal capillaries by biting mouthparts exposes susceptible vascular endothelial cells, thereby providing immediate sites of virus replication and a dissemination route via the circulatory system. This research provides insights into the efficiency of Culicoides midges as orbivirus vectors.

Barley produced Culicoides allergens are suitable for monitoring the immune response of horses immunized with E. coli expressed allergens.

Insect bite hypersensitivity is an allergic dermatitis of horses caused by bites of Culicoides midges. Sufficient amount of pure, endotoxin-free allergens is a prerequisite for development and monitoring of preventive and therapeutic allergen immunotherapy. Aims of the study were to compare the Culicoides nubeculosus (Cul n) allergens Cul n 3 and Cul n 4, produced in transgenic barley grains with the corresponding E. coli or insect cells expressed proteins for measuring antibody and cytokine responses. Allergen-specific IgG responses were measured by ELISA in sera from twelve horses not exposed to Culicoides, before and after vaccination with E. coli-rCul n 3 and 4. Before vaccination no IgG binding to the barley and insect cell produced proteins was detected and a similar increase in specific IgG was observed after vaccination. While IgG levels to the E.coli expressed proteins were higher in the post-vaccination sera, some background binding was observed pre-vaccination. In vitro re-stimulation of PBMC was performed for measurements of cytokines. E. coli expressed proteins resulted in high background in PBMC from non-vaccinated controls. The barley and insect cell expressed proteins induced similar amount of IFN-γ and IL-4 in PBMC from vaccinated horses. Barley produced allergens are promising tools for use in immunoassays.

Treating insect-bite hypersensitivity in horses with active vaccination against IL-5.

BACKGROUND: Insect-bite hypersensitivity is the most common allergic dermatitis in horses. Excoriated skin lesions are typical symptoms of this seasonal and refractory chronic disease. On a cellular level, the skin lesions are characterized by massive eosinophil infiltration caused by an underlying allergic response. OBJECTIVE: To target these cells and treat disease, we developed a therapeutic vaccine against equine IL-5 (eIL-5), the master regulator of eosinophils. METHODS: The vaccine consisted of eIL-5 covalently linked to a virus-like particle derived from cucumber mosaic virus containing the tetanus toxoid universal T-cell epitope tt830-843 (CMVTT). Thirty-four Icelandic horses were recruited and immunized with 400 µg of eIL-5-CMVTT formulated in PBS without adjuvant (19 horses) or PBS alone (15 horses). RESULTS: The vaccine was well tolerated and did not reveal any safety concerns but was able to induce anti-eIL-5 autoantibody titers in 17 of 19 horses. This resulted in a statistically significant reduction in clinical lesion scores when compared with previous season levels, as well as levels in placebo-treated horses. Protection required a minimal threshold of anti-eIL-5 antibodies. Clinical improvement by disease scoring showed that 47% and 21% of vaccinated horses reached 50% and 75% improvement, respectively. In the placebo group no horse reached 75% improvement, and only 13% reached 50% improvement. CONCLUSION: Our therapeutic vaccine inducing autoantibodies against self IL-5 brings biologics to horses, is the first successful immunotherapeutic approach targeting a chronic disease in horses, and might facilitate development of a similar vaccine against IL-5 in human subjects.

Longitudinal analysis of allergen-specific IgE and IgG subclasses as potential predictors of insect bite hypersensitivity following first exposure to Culicoides in Icelandic horses.

BACKGROUND: Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of Culicoides spp. IBH does not occur in Iceland because of the absence of Culicoides, but the prevalence is high in horses imported from Iceland to environments where Culicoides are present. HYPOTHESIS/OBJECTIVE: Test, in a longitudinal study before and after Culicoides exposure, whether a primary sensitizing Culicoides allergen can be identified and if an increase of allergen-specific immunoglobulin (Ig)E or IgG subclasses precedes clinical signs of IBH. ANIMALS: Thirty two horses imported from Iceland to Europe; 16 developed IBH and 16 remained healthy. METHODS: Determination of IgE and IgG subclasses against recombinant (r)-Culicoides allergens and Culicoides extract in sera taken before first exposure to Culicoides and yearly over a period of 3-4 years. RESULTS: Before Culicoides exposure, there were no significant differences in Culicoides-specific serum IgE levels between horse that developed IBH or remained healthy. Culicoides exposure induced an individual IgE response pattern (to a median of 4.5 r-allergens) in the IBH but not in the healthy end-point group. The increase in serum IgE levels to Culicoides r-allergens was concurrent with the initial onset of clinical signs of IBH. IBH-affected horses displayed significantly higher allergen-specific IgG1 and IgG5 levels than healthy controls. Recombinant Culicoides obsoletus 1 (Cul o1) and Cul o3-specific IgG5 was significantly higher in the IBH compared to the healthy end-point group, before clinical signs of IBH. CONCLUSION/CLINICAL RELEVANCE: Allergen-specific serum IgE cannot be used as predictor for IBH, whereas allergen-specific IgG5 levels may have a predictive value.

Culicoides-virus interactions: infection barriers and possible factors underlying vector competence.

In the United States, Culicoides midges vector arboviruses of economic importance such as Bluetongue Virus and Epizootic Hemorrhagic Disease Virus. A limited number of studies have demonstrated the complexities of midge-virus interactions, including dynamic changes in virus titer and prevalence over the infection time course. These dynamics are, in part, dictated by mesenteron infection and escape barriers. This review summarizes the overarching trends in viral titer and prevalence throughout the course of infection. Essential barriers to infection and dissemination in the midge are highlighted, along with heritable and extrinsic factors that likely contribute to these barriers. Next generation molecular tools and techniques, now available for Culicoides midges, give researchers the opportunity to test how these factors contribute to vector competence.