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During 2021-22 High Pathogenicity Avian Influenza (HPAI) killed thousands of wild birds across Europe and North America, suggesting a change in infection dynamics and a shift to new hosts, including seabirds. Northern Gannets (Morus bassanus) appeared especially severely impacted, but limited understanding of how the virus spread across the metapopulation, or the demographic consequences of mass mortality limit our understanding of its severity. Accordingly, we collate information on HPAIV outbreaks across most North Atlantic gannet colonies and for the largest colony (Bass Rock, UK), provide impacts on population size, breeding success, adult survival, and preliminary results on serology. Unusually high numbers of dead gannets were first noted in Iceland during April 2022. Outbreaks in May occurred in many Scottish colonies, followed by colonies in Canada, Germany and Norway. By the end of June, outbreaks had occurred in five Canadian colonies and in the Channel Islands. Outbreaks in 12 UK and Ireland colonies appeared to follow a clockwise pattern with the last infected colonies recorded in late August/September. Unusually high mortality was recorded at 40 colonies (75% of global total colonies). Dead birds testing positive for HPAIV H5N1 were associated with 58% of these colonies. At Bass Rock, the number of occupied sites decreased by at least 71%, breeding success declined by ∼66% compared to the long-term UK mean and adult survival between 2021 and 2022 was 42% lower than the preceding 10-year average. Serological investigation detected antibodies specific to H5 in apparently healthy birds indicating that some gannets recover from HPAIV infection. Further, most of these recovered birds had black irises, suggestive of a phenotypic indicator of previous infection. Untangling the impacts of HPAIV infection from other key pressures faced by seabirds is key to establishing effective conservation strategies for threatened seabird populations, HPAIV being a novel and pandemic threat.

Publisher's version (útgefin grein), 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., The importation and maintenance of the horses in this study were funded by research support from the Harry M. Zweig Memorial Fund for Equine Research at Cornell University. The equine isotype reagent described in this article were developed and characterized with funding from Agriculture and Food Research Initiative Competitive Grants no. #2005–01812 (The US Veterinary Immune Reagent Network). The serological allergen multiplex analysis of the samples was supported by research funds provided by Boehringer Ingelheim.

Peptidylarginine deiminases (PADs) are phylogenetically conserved calcium-dependent enzymes which post-translationally convert arginine into citrulline in target proteins in an irreversible manner, leading to functional and structural changes in target proteins. Protein deimination can cause the generation of neo-epitopes, affect gene regulation and also allow for protein moonlighting and therefore facilitate multifaceted functions of the same protein. PADs are furthermore a key regulator of cellular release of extracellular vesicle (EVs), which are found in most body fluids and participate in cellular communication via transfer of cargo proteins and genetic material. In this study, post-translationally deiminated proteins and EVs were assessed in sera of two seal species, grey seal and harbour seal. We report a poly-dispersed population of serum-EVs, which were positive for phylogenetically conserved EV-specific markers and characterised by transmission electron microscopy. A number of deiminated proteins critical for immune and metabolic functions were identified in the seal sera and varied somewhat between the two species under study, while some targets were in common. EV profiles of the seal sera further revealed that key microRNAs for inflammation, immunity and hypoxia also vary between the two species. Protein deimination and EVs profiles may be useful biomarkers for assessing health status of sea mammals, which face environmental challenges, including opportunistic infection, pollution and shifting habitat due to global warming.

Purpose of Review Treatment of equine insect bite hypersensitivity (IBH) needs to be improved. Allergen-specific immunotherapy (ASIT), the only curative treatment of allergy, currently has only a limited efficacy for treatment of IBH. This review highlights the latest findings in prophylactic and therapeutic strategies. Recent Findings Prophylactic vaccination against IBH using recombinant Culicoides allergen has been developed in unexposed Icelandic horses and is ready to be tested. Therapeutic virus-like particle (VLP)–based vaccines targeting equine interleukin- (IL-) 5 or IL-31 improved clinical signs of IBH by induction of anti-cytokine antibodies thus reducing eosinophil counts or allergic pruritus, respectively. Summary First studies for development of ASIT using pure r-Culicoides allergens have yielded promising results and need now to be tested in clinical studies for both prevention and treatment of IBH. Therapeutic vaccines inducing neutralizing antibodies against IL-5 or IL-31 will be valuable future treatments for reduction of clinical signs of IBH.

The Icelandic horse has been maintained as a closed population in its eponymous homeland for many generations, with no recorded introductions of new horses of any breed since the year 1000 CE. Here we determined the diversity of major histocompatibility complex (MHC) haplotypes in 156 Icelandic horses from two groups, based on a panel of 12 polymorphic intra-MHC microsatellites tested in families of various composition. We identified a total of 79 MHC haplotypes in these two groups, including one documented intra-MHC recombination event from a total of 147 observed meioses. None of these MHC haplotypes have been previously described in any other horse breed. Only one MHC homozygote was found in the entire population studied. These results indicate a very high level of MHC heterozygosity and haplotype diversity in the Icelandic horse. The environment in Iceland is remarkable for its lack of common agents of equine infectious disease, including equine herpesvirus type 1, influenza virus, and streptococcus equi. The driving forces for maintenance of MHC heterozygosity in Icelandic horses must thus be sought outside of these major horse pathogens. Based on our results, we propose that intra-MHC recombination may play a major role in the generation of novel haplotypes.

Equine coital exanthema (ECE) caused by equid alphaherpesvirus 3 (EHV-3) is a contagious venereal disease. It is characterized by the formation of papules, vesicles, pustules and ulcers on the external genitals of both mares and stallions. The Icelandic horse is the only breed in Iceland and has lived isolated in the country for over 1000 years. Three types of equine herpesviruses (EHV) have been found in Iceland, EHV-4, EHV-2 and EHV-5, while EHV-1 has never been detected. Symptoms resembling ECE have previous been observed in horses in Iceland, arousing suspicion of EHV-3 infection, but this has never been confirmed using virological methods. Samples were collected from a mare with papules on the vulva and inoculated in primary equine kidney cells. Cytopathic effects developed as rounded cells and syncytial formation. Polymerase chain reaction and sequencing of the partial glycoprotein G and DNA polymerase genes identified the isolated virus as EHV-3. On the basis of the findings, EHV-3 infection was verified for the first time in the native Icelandic horse population.

Horses in Iceland have been isolated for more than 1,000 yr but still harbor a similar range of gastrointestinal parasites as do horses across the world. The long isolation of the horses and their parasites presumably means that no resistance genes have been introduced into the Parascaris spp. population. It is therefore of particular interest to investigate the efficacy of ivermectin on Parascaris spp. infecting Icelandic foals. Potential treatment failure of ivermectin in Iceland will add substantial new information on how resistance can arise independently. This study aimed to determine the efficacy of subcutaneous injection of ivermectin for the treatment of Parascaris spp. infection in foals and to identify the Parascaris species present in the west and north of Iceland. A fecal egg count reduction (FECR) test (FECRT) was performed on 50 foals from 8 farms, including an untreated control group of 6 foals, from September to November 2019. The foals were between 3 and 5 mo of age at the start of the study and had not previously been treated with anthelmintic drugs. Each foal was treated subcutaneously with off-label use of Ivomec (R) injection 10 mg/ml or Noromectin (R) 1% at a dose of 0.2 mg/kg. The FECR for each farm was calculated in 2 ways, by the eggCounts package in R and by the Presidente formula (FECRT). Both calculation methods resulted in efficacy levels between 0% and 80.78%, indicating ivermectin resistance on all farms. We also confirmed, by karyotyping, that the species of equine ascarid present in the west and north of Iceland is Parascaris univalens. This study provides evidence for treatment failure of ivermectin against P. univalens infection in foals. Since Icelandic horses have been isolated on the island for more than 1,000 yr, this implies that resistance alleles have developed independently in the Icelandic Parascaris population. The actual clinical impact of ivermectin resistance is unknown but another drug of choice should be considered to treat Parascaris infection in foals in Iceland.

Peptidylarginine deiminases (PADs) are a family of phylogenetically conserved calcium-dependent enzymes which cause post-translational protein deimination. This can result in neoepitope generation, affect gene regulation and allow for protein moonlighting via functional and structural changes in target proteins. Extracellular vesicles (EVs) carry cargo proteins and genetic material and are released from cells as part of cellular communication. EVs are found in most body fluids where they can be useful biomarkers for assessment of health status. Here, serum-derived EVs were profiled, and post-translationally deiminated proteins and EV-related microRNAs are described in 5 ceataceans: minke whale, fin whale, humpback whale, Cuvier's beaked whale and orca. EV-serum profiles were assessed by transmission electron microscopy and nanoparticle tracking analysis. EV profiles varied between the 5 species and were identified to contain deiminated proteins and selected key inflammatory and metabolic microRNAs. A range of proteins, critical for immune responses and metabolism were identified to be deiminated in cetacean sera, with some shared KEGG pathways of deiminated proteins relating to immunity and physiology, while some KEGG pathways were species-specific. This is the first study to characterise and profile EVs and to report deiminated proteins and putative effects of protein-protein interaction networks via such post-translationald deimination in cetaceans, revealing key immune and metabolic factors to undergo this post-translational modification. Deiminated proteins and EVs profiles may possibly be developed as new biomarkers for assessing health status of sea mammals.

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.

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.

Publisher's version (útgefin grein), Two types of gammaherpesviruses (γEHV) are known to infect horses, EHV-2 and EHV-5. Foals become infected early in life, probably via the upper respiratory tract, despite maternal antibodies. In this study, we analyzed samples from a herd of mares and their foals. The foals were followed from birth to 22 months of age and the dams during the first 6 months postpartum. Blood and nasal swab samples were taken regularly for evaluation of antibody responses, virus isolation and viral load by qPCR. EHV-2 was isolated on day 5, and EHV-5 on day 12, earlier than previously reported. γEHV specific antibodies were not detectable in serum of foals before colostrum intake but peaked a few days after colostrum. Overall, EHV-2 viral load peaked in nasal swab at three to four months of age, paralleled with decline in maternal antibodies, but EHV-5 viral load did not peak until month 12. Maternal antibodies had a notable effect on the viral load and induction of endogenous antibody production. Foals were grouped in two groups depending on the mare’s γEHV specific total IgG levels in serum at birth, group-high and group-low. Group-high had higher levels of maternal γEHV specific total IgG and IgG4/7 for the first 3 months, but when the endogenous production had superseded maternal antibodies, group-low was higher. The maternal antibodies had an effect on the γEHV viral load. Group-low peaked in EHV-2 viral load one month earlier than group-high. These effects were more evident for EHV-5, as there were seven months between the viral load peaks for the groups. The study provides information on how maternal antibody transfer affects γEHV shedding and antibody production in offspring. It also extends our knowledge on the occurrence of EHV-2 and EHV-5 infection in foals during the first two years of life., This work was supported by: The Icelandic Research Fund (VS, 130446), The Developmental Fund for Icelandic Horse Breeding (VS, 10190951), The Icelandic Horse Conservation Fund (VS, 10190942).

SummaryReasons for performing study Rhodococcus equi (Rhodococcus hoagii/Prescottella equi) is a common cause of foal pneumonia, but its diagnosis remains a challenge for equine veterinarians. While the VapA-specific (virulence-associated protein A) immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) has low sensitivity and specificity for detecting pneumonic foals, little is known about VapA-specific IgG subclasses. Objectives To evaluate the performance of VapA-specific ELISA for IgG and its subclasses IgGa, IgGb and IgG(T) in the early diagnosis of pneumonia caused by R. equi. Study design Assay validation followed by assessment of diagnostic performance using archived samples from animals of known status. Methods Serum samples from exposed (n = 125) and nonexposed adult horses (n = 10) and from experimentally challenged and naturally infected foals were used for ELISA validation. Post mortem and tissue culture records of the last 24 years from the Institute for Experimental Pathology at the University of Iceland in Keldur, Iceland laboratory were evaluated to confirm the absence of R. equi cases in Iceland. The diagnostic performance of VapA-specific IgG and its subclasses was evaluated using banked serum samples from pneumonic (n = 21) and healthy foals (n = 80). To evaluate each IgG assay, a cut-off value was selected based on receiver operating characteristic curve analysis and used to calculate sensitivity and specificity. The intra- and interassay coefficients of variation were calculated for each ELISA. Results Using sera from Iceland, where R. equi infection has not been reported, the VapA-specific IgG ELISA differentiated exposed from nonexposed horses. When used to identify infected foals, VapA-specific IgG, IgGa and IgGb had no diagnostic value. In contrast, IgG(T) had high sensitivity and specificity. Conclusions Horses from Iceland are not exposed to VapA+ R. equi and can serve as negative controls. VapA-specific IgG subclasses, with the exception of IgG(T), are poor predictors of disease. Further investigation on the use of IgG(T) as a diagnostic tool in field conditions is needed.

Neonatal foals respond poorly to conventional vaccines. These vaccines typically target T-helper (Th) cell dependent B-cell activation. However, Th2-cell immunity is impaired in foals during the first three months of life. In contrast, neonatal basophils are potent interleukin-4 (IL-4) producers. The purpose of this study was to develop a novel vaccine triggering the natural capacity of neonatal basophils to secrete IL-4 and to evaluate if vaccination resulted in B-cell activation and antibody production against EHV-1 glycoprotein C (gC). Neonatal vaccination was performed by oral biotinylated IgE (IgE-bio) treatment at birth followed by intramuscular injection of a single dose of streptavidin-conjugated gC/IL-4 fusion protein (Sav-gC/IL-4) for crosslinking of receptor-bound IgE-bio (group 1). Neonates in group 2 received the intramuscular Sav-gC/IL-4 vaccine only. Group 3 remained non-vaccinated at birth. After vaccination, gC antibody production was not detectable. The ability of the vaccine to induce protection was evaluated by an EHV-1 challenge infection after weaning at 7 months of age. Groups 1 and 2 responded to EHV-1 infection with an earlier onset and overall significantly increased anti-gC serum antibody responses compared to control group 3. In addition, group 1 weanlings had a decreased initial fever peak after infection indicating partial protection from EHV-1 infection. This suggested that the neonatal vaccination induced a memory B-cell response at birth that was recalled at weanling age after EHV-1 challenge. In conclusion, early stimulation of neonatal immunity via the innate arm of the immune system can induce partial protection and increased antibody responses against EHV-1., Funding for this project was provided by the Harry M. Zweig Memorial Fund for Equine Research at Cornell University ‘A Novel Strategy to Boost Antibody Production to EHV-1 in Neonates’ (http://vet.cornell.edu/research/Zweig/). Monoclonal antibody development for horse cell surface markers and cytokines was supported by USDA grant #2005-01812 ‘The US Veterinary Immune Reagent Network’ and #2015-67015-23072 ‘Equine Immune Reagents: Development of monoclonal antibodies to improve the analysis of immunity in horses’ (https://nifa.usda.gov/).

The native horse population of Iceland has remained free of major infectious diseases. Between May and July 2010 an epidemic of respiratory disease swept through the population. Initial microbiological investigations ruled out known equine viral agents as the cause of the infections, but identified the opportunistic pathogenStreptococcus zooepidemicusas being frequently isolated from diseased animals. This diverse bacterial species has a broad host range and is usually regarded as a commensal of horses. By genome sequencingS. zooepidemicusrecovered from horses during the epidemic we show that although multiple clones ofS. zooepidemicuswere present in the population, one particular clone, ST209, was responsible for the epidemic. Concurrent with the epidemic, ST209 caused zoonotic infections, highlighting the pathogenic potential of this clone. Phylogenetic analysis suggests that the original ST209 strain entered Iceland in late 2008 or early 2009. Epidemiological investigation revealed that the incursion of this strain into a training yard that utilized a submerged treadmill between the 5thand 19thof February 2010 was a critical trigger for the ensuing epidemic of disease, provided a nidus for the infection of multiple horses, and subsequent distribution of these animals to multiple sites in Iceland.

SummaryBackground Insect bite hypersensitivity is an IgE-mediated dermatitis of horses initiated by bites of midges of the genus Culicoides. Culicoides spp. are not indigenous to Iceland and the prevalence of insect bite hypersensitivity is much higher in horses born in Iceland and exported as compared to Icelandic horses born in a Culicoides rich environment. Immunotherapy is therefore needed. Objectives The aim of the study was to express an allergen from Culicoides in barley grain and investigate whether an immune response could be obtained in healthy Icelandic horses by oral treatment with transgenic barley expressing the allergen. Study design In vivo experiment. Methods The allergen was expressed in barley grain with the Orfeus™ technique. A device was developed to orally treat horses with barley flour. Four Icelandic horses were treated with transgenic barley and three with control barley, in total 500 g in seven feedings. Serum and saliva samples were collected for measuring specific antibodies. Results The allergen Cul n 2, a hyaluronidase originating from the salivary gland of C. nubeculosus, was expressed in barley. Horses treated with the transgenic barley mounted a Cul n 2 specific IgG1 and IgG4/7 response in serum and saliva. The serum response was significantly different between the transgenic and control barley treated horses for both subclasses and the saliva response for IgG1. The induced serum antibodies bound to the corresponding allergen from Culicoides obsoletus, rCul o 2 and were able to partially block binding of Cul n 2 as well as Cul o 2 specific IgE from insect bite hypersensitivity affected horses. Main limitations Small number of horses. Conclusion This study shows that specific antibody response can be induced in horses not exposed to Culicoides, using oral treatment with transgenic barley expressing an allergen. Further studies will determine whether this approach is a useful alternative for prevention and treatment of equine insect bite hypersensitivity. This article is protected by copyright. All rights reserved.

Equine herpesvirus type 1 (EHV-1) continues to cause severe outbreaks of abortions or myeloencephalopathy in horses despite widely used vaccination. The aim of this work was to determine the effects of frequent vaccination with an inactivated EHV vaccine on immune development in horses. Fifteen EHV-1 naive mares were vaccinated a total of 5 times over a period of 8 months with intervals of 20, 60, 90 and 60 days between vaccine administrations. Total antibody and antibody isotype responses were evaluated with a new sensitive EHV-1 Multiplex assay to glycoprotein C (gC) and gD for up to 14 months after initial vaccination. Antibodies peaked after the first two vaccine doses and then declined despite a third administration of the vaccine. The fourth vaccine dose was given at 6 months and the gC and gD antibody titers increased again. Mixed responses with increasing gC but decreasing gD antibody values were observed after the fifth vaccination at 8 months. IgG4/7 isotype responses mimicked the total Ig antibody production to vaccination most closely. Vaccination also induced short-lasting IgG1 antibodies to gC, but not to gD. EHV-1-specific cellular immunity induced by vaccination developed slower than antibodies, was dominated by IFN-γ producing T-helper 1 (Th1) cells, and was significantly increased compared to pre-vaccination values after administration of 3 vaccine doses. Decreased IFN-γ production and reduced Th1-cell induction were also observed after the second and fourth vaccination. Overall, repeated EHV vaccine administration did not always result in increasing immunity. The adverse effects on antibody and cellular immunity that were observed here when the EHV vaccine was given in short intervals might in part explain why EHV-1 outbreaks are observed worldwide despite widely used vaccination. The findings warrant further evaluation of immune responses to EHV vaccines to optimize vaccination protocols for different vaccines and horse groups at risk.

Due to the slow growth of equine gammaherpesviruses, isolation of these viruses requires cells that can be propagated long term and show clear cytopathy following infection. Equine cell lines with extended lifespan were established from primary cells originating from equine fetal kidney and lung by transfecting the cells with the retroviral vector LXSN116E6E7 containing the human papilloma virus oncogenes 16 E6 and E7. The transfected equine kidney cell line and equine lung cell line can be propagated for more than 40 passages, whereas the corresponding primary cells only for 10–12 passages. The primary cells and the derived cell lines can be infected with equine gammaherpesvirus 2 (EHV-2) with similar efficiency. However EHV-5 can be grown to a substantially higher titer in the kidney cell line than their primary counterpart, with cytopathic effect visible three days earlier than in the primary cells. Due to rapid cell growth the lung cell line is difficult to use for virus production. The kidney cell line was four times more susceptible to transfection as compared to the primary kidney cells. On the other hand no difference was between the lung cell line and the primary lung cells in transfection efficiency. The cell lines can be a valuable tool for investigating gammaherpesviruses, and possibly other viruses infecting 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.

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of midges (Culicoides spp.). IgE-mediated reactions are often involved in the pathogenesis of this disease. IBH does not occur in Iceland due to the absence of Culicoides, but it occurs with a high frequency in Icelandic horses exported to mainland Europe, where Culicoides are present. We hypothesize that immunization with the Culicoides allergens before export could reduce the incidence of IBH in exported Icelandic horses. The aim of the present study was therefore to compare intradermal and intralymphatic vaccination using four purified recombinant allergens, in combination with a Th1 focusing adjuvant. Twelve horses were vaccinated three times with 10μg of each of the four recombinant Culicoides nubeculosus allergens. Six horses were injected intralymphatically, three with and three without IC31(®), and six were injected intradermally, in the presence or absence of IC31(®). Antibody responses were measured by immunoblots and ELISA, potential sensitization in a sulfidoleukotriene release test and an intradermal test, cytokine and FoxP3 expression with real time PCR following in vitro stimulation of PBMC. Immunization with the r-allergens induced a significant increase in levels of r-allergen-specific IgG1, IgG1/3, IgG4/7, IgG5 and IgG(T). Application of the r-allergens in IC31(®) adjuvant resulted in a significantly higher IgG1, IgG1/3, IgG4/7 allergen-specific response. Intralymphatic injection was slightly more efficient than intradermal injection, but the difference did not reach significance. Testing of the blocking activity of the sera from the horses immunized intralymphatically with IC31(®) showed that the generated IgG antibodies were able to partly block binding of serum IgE from an IBH-affected horse to these r-allergens. Furthermore, IgG antibodies bound to protein bands on blots of C. nubeculosus salivary gland extract. No allergen-specific IgE was induced and there was no indication of induction of IgE-mediated reactions, as horses neither responded to Culicoides extract stimulation in a sulfidoleukotriene release test, nor developed a relevant immediate hypersensitivity reaction to the recombinant allergens in skin test. IL-4 expression was significantly higher in horses vaccinated intralymphatically without IC31(®), as compared to horses intradermally vaccinated with IC31(®). Both routes gave higher IL-10 expression with IC31(®). Both intralymphatic and intradermal vaccination of horses with recombinant allergens in IC31(®) adjuvant induced an immune response without adverse effects and without IgE production. The horses were not sensitized and produced IgG that could inhibit allergen-specific IgE binding. We therefore conclude that both the injection routes and the IC31(®) adjuvant are strong candidates for further development of immunoprophylaxis and therapy in horses.

Curiously, of the 16 culture positive occasions, 12 of these horses remained seronegative to antigen C. In summary, despite all weanlings being exposed to and/or infected by S. equi in this outbreak, minimal to onlymild disease ensued, possibly related to by their failure of a robust immunologic response to portions of the SeM protein (antigen C). Sequencing studies of the S. equi strains involved are ongoing to determine whether genetic deletion events (3) could help explain the reduced clinical severity.

Íslenski hesturinn er eini hestastofninn á Íslandi. Hestar voru fluttir hingað við landnám og hafa verið hreinræktaðir síðan. Vegna landfræðilegrar einangrunar er stofninn berskjaldaður fyrir mörgum sjúkdómsvöldum sem herja á hesta í öðrum löndum. Fimm gerðir herpesveira eru þekktar hjá hestum (equine herpesvirus, EHV), þrjár alfa () EHV 1, 3 og 4 og tvær gamma () EHV 2 og 5. Meirihluti fullorðinna hesta á Íslandi hafa mótefni gegn EHV-4. Sjúkdómseinkenni sem líkjast EHV-3 sýkingu hafa komið fram í hrossum og báðar gammaherpesveirurnar eru algengar í stofninum. Ísland er samkvæmt okkar bestu vitund, eina landið þar sem EHV-1 veiran finnst ekki. Við staðfestum, með einangrun, EHV-3 veiruna í fyrsta skiptið hjá íslenska hestastofninum. Út frá eðli herpesveira má álykta að allar fjórar landlægu herpesveirurnar hafi komið til landsins við landnám. Við rannsóknir á herpesveirum er mikilvægt að notast við frumur úr sama hýsli og veiran. Með því að innleiða æxlisgen í prímer nýrna- og lungnafrumur bjuggum við til hestafrumulínur. Hægt er að rækta frumulínurnar 40 sinnum, ólíkt upprunalegu frumunum sem einungis er hægt að umrækta 10-12 sinnum. EHV-5 veiran hafði mun hærri títer í nýrnafrumulínunni og innleiðslugeta hennar var um fjórum sinnum betri, borið saman við prímer nýrnafrumurnar. Þessar frumulínur nýtast við rannsóknir á hestaveirum. Sýkiferill EHV-2 og EHV-5 og ónæmissvar gegn EHV var skoðað með því að fylgja eftir folöldum frá köstun fram að 22 mánaða aldri. Mæðrum þeirra var fylgt eftir í 6 mánuði. Sértækt EHV mótefnasvar var mælt, veirumagn var greint með qPCR og veirueinangrun var reynd. EHV-2 var einangruð frá 5 daga gömlu folaldi og EHV-5 á degi 12, fyrr en áður hefur fundist. Sértækt EHV mótefnasvar var ekki mælanlegt hjá folöldunum áður en þau komust á spena, en hækkaði nokkrum dögum síðar. Mótefnin frá móður lækkuðu síðan fram að 3-4 mánaða aldri og samhliða jókst EHV-2 magn. EHV-5 náði hins vegar ekki hámarki fyrr en folöldin voru eins árs. Folöldin voru flokkuð í tvo hópa, eftir sértæku EHV heildar IgG mótefnasvari móður í sermi við fæðingu, hátt-svar og lágt-svar. Það var greinileg fylgni milli magns mótefna hjá móður og veirumagns og mótefnaframleiðslu folaldanna. Þetta sást betur hjá EHV-5 þar sem 7 mánuðir voru á milli hópanna tveggja með tilliti til þess hvenær veiran náði hámarki. Fyrir EHV-2 var aðeins mánuður á milli. Með qPCR fékkst mögnun á erfðaefni EHV frá hluta af folaldasýnunum sem tekin voru áður en þau komust á spena. Þetta gæti bent til þess að einhver hluti erfðaefnis veiranna sé innlimaður í litninga hestsins. Þessar heildarniðurstöður gefa innsýn í hvernig flutningur á mótefnum frá móður hefur áhrif á EHV sýkingu og mótefnaframleiðslu afkvæmis. Einnig auka niðurstöðurnar þekkingu okkar á sýkiferli EHV-2 og EHV-5 fyrstu tvö æviárin. Hönnuð var tjáningarkasetta sem tjáir EGFP prótein samhliða tjáningu EHV-2 glýkópróteins B. Framleidd var endurröðuð EHV-2, rEHV2-gB-egfp, sem gat sýkt bæði hestafrumur og kanínufrumur (RK13). Þessi flúrljómandi veira gæti reynst gagnleg í in vitro rannsóknum á EHV-2. Endurraðaðar baculoveirur með tjáningarkassettu sem hefur stýril sem virkar í spendýrum hafa nýst í tilraunabólusetningum, bæði in vitro og in vivo. Útbúnar voru sex ólíkar baculoveiruferjur og prófaðar in vitro. Þrjár voru síðan prófaðar áfram með bólusetningartilraun í folöldum. Bólusett var í eitla, undir húð og í vöðva. Sértækt mótefnasvar gegn markpróteininu var ekki mælanlegt hjá neinu folaldi. Niðurstöður verkefnisins veita gleggri innsýn í herpesveirusýkingar í íslenska hrossastofninum og jafnframt voru hönnuð tól sem auðvelda áframhaldandi rannsóknir á sviðinu., The native Icelandic horse is the only horse breed in Iceland. The horses were brought to the country in the 9th and 10th centuries and have been purebred ever since. Due to this geographic isolation the horses are immunologically naïve to various agents known to infect horses in other countries. Five types of herpesviruses are known in horses, three EHVs; 1, 3 and 4 and two EHVs; 2 and 5. In Iceland, the majority of adult horses are antibody positive against EHV-4, clinical symptoms resembling an infection with EHV-3 have been detected and EHV-2 and EHV-5 are endemic and ubiquitous. To our knowledge, Iceland is the only country free of EHV-1. In this study we isolated and confirmed EHV-3 for the first time in our native population. In light of the nature of herpesviruses it is likely the four EHVs endemic in Iceland arrived with the founders of the breed. Cell lines originating from equines are important when working with EHVs. We established equine cell lines, both kidney and lung, with extended life span by transfecting primary cells with a retroviral vector containing oncogenes. The cell lines can be passaged approximately 40 times, as opposed to the primary cells 10-12 times. EHV-5 grew to a substantially higher titer and the transfection efficiency was four times higher in the kidney cell line than in the primary cells. These cells could have further advantages for equine virus research. The course of EHV-2 and EHV-5 infections and the immune response against EHV was studied by following foals from birth to 22 months of age and their dams during the first 6 months postpartum. The EHV specific antibody responses were evaluated, the viral load was measured with qPCR and virus isolation attempted. Both EHV-2 and EHV-5 were isolated earlier than previously reported on day 5 and 12, respectively. EHV specific antibodies were not detected before colostrum intake but peaked a few days after birth. The maternal antibodies declined when the foals were 3–4 months of age, followed by a peak in the EHV-2 viral load, whereas the EHV-5 viral load peaked when the foals were one year old. Depending on the mare’s EHV specific total IgG levels in serum at birth, the foals were grouped in two groups, high and low. There was a notable correlation between the level of maternal antibodies on the one hand and viral load and induction of endogenous antibody production in the foals on the other hand. These effects were more evident for EHV-5 as there were seven months between the viral load peaks for the high and low groups, compared to one month for EHV-2. Amplification of viral DNA was detected in some foals’ nasal swab and blood leukocyte samples taken before colostrum intake, indicating possible integration of viral DNA sequences into the host chromosome. Overall, these results provide information on how maternal antibody transfer affects EHV shedding and antibody production of the offspring and extends our knowledge on the EHV-2 and EHV-5 infection in foals during the first two years of life. An expression cassette was designed to express EGFP protein as a fusion protein with EHV-2 glycoprotein B. An infectious recombinant EHV-2 virus, rEHV2-gB-egfp, was constructed with homologous recombination. The virus was able to infect both equine and rabbit cells (RK13). The fluorescent virus could be a useful tool in EHV-2 in vitro studies. Recombinant baculoviruses carrying a mammalian cell-active expression cassette have been used for gene delivery in vitro and in vivo. We constructed and tested 6 different rBac-viral vectors in vitro and used three of them in vaccination trials in foals. Regardless of injection site, the foals did not develop measurable antibodies against the target. Overall, the study provides improved tools for EHV research and deeper understanding of the EHV status in the Icelandic horse population., The work was funded by the Icelandic Research Fund, the University of Iceland Research Fund, the Developmental Fund for Icelandic Horse Breeding, the Icelandic Horse Conservation Fund and the Agricultural Productivity Fund of Iceland.

Insect bite hypersensitivity (IBH) is a type I allergy of horses with production of IgE and release of inflammatory mediators. It is caused by bites of midges of the genus Culicoides. The disease is a recurrent dermatitis characterized by pruritic skin and hair loss, which can result in secondary infections. All breeds of horses can be affected, but horses born in Iceland and exported are more frequently affected than Icelandic horses born abroad. Allergens have been identified at the molecular level from three Culicoides spp., C. sonorensis, C. nubeculosus and C. obsoletus. They have all been produced in E. coli and some in insect cells and barley. The aim of the study was to develop immunotherapy for IBH using vaccination with purified allergens in adjuvants and a method to treat horses via the oral mucosa with transgenic barley expressing allergens. Different injection routes and adjuvants were tested for development of prophylactic immunotherapy. Healthy Icelandic horses were vaccinated intradermally or intralymphatically with a small amount of purified allergens with or without a Th1 adjuvant. The intradermal and the intralymphatic vaccinations with adjuvant resulted in significant generation of allergen specific IgG1 and IgG4/7, the highest response being observed following intralymphatic vaccination. Furthermore, the antibodies produced after intralymphatic vaccination with the allergens in an adjuvant were able to partly inhibit binding of IgE to the allergens, an important mechanism of allergen immunotherapy. The intralymphatic route was employed to compare the immune response induced after vaccination with purified allergens in Alum alone or in a mixture of Alum and Monophosphoryl lipid A (MPLA). The vaccinated horses mounted a strong IgG1 and IgG4/7 response and the antibodies had the capacity to block the binding of IgE to the allergens. The adjuvant groups only differed with regard to the cytokine response but not in the antibody response. Compared to unvaccinated control horses, the IFNγ and IL-10 induced following in vitro restimulation of peripheral blood mononuclear cells (PBMCs) with an allergen was significantly higher in the Alum/MPLA but not in the Alum group. This indicated an increased Th1/Treg response after vaccination with Alum/MPLA. A spiral bit and a barley flour mixture were developed to treat horses via the mucosa of the mouth. Healthy Icelandic horses were treated with barley expressing the Cul n 2 allergen. The horses receiving the Cul n 2 barley mounted a specific IgG1 and IgG4/7 response in the sera and saliva. The antibodies induced were able to partly inhibit binding of IgE to Cul n 2 as well as to Cul o 2, the corresponding allergen from C. obsoletus. For evaluation of the benefit of immunotherapy as well as to confirm the diagnosis of IBH it is important to have recombinant allergens with similar three-dimensional structure and post-translational modification to the native proteins which can be easily purified. Barley grains are an excellent source of proteins and ideal for stable storage. Two allergens produced in barley showed similar performance in ELISA to that of the corresponding proteins expressed in E. coli and insect cells. In conclusion, intralymphatic vaccination with purified allergens in the adjuvant mixture Alum/MPLA is a promising approach for development of a prophylactic and probably also therapeutic immunotherapy against IBH. Furthermore, a first study has demonstrated that oral immunotherapy with barley expressing allergens could be an attractive alternative to conventional allergen immunotherapy, avoiding the expensive purification of recombinant allergens and the need of injection., Sumarexem er ofnæmi af gerð I í hrossum með framleiðslu á IgE mótefnum sem leiða til losunar á bólguþáttum. Ofnæmið orsakast af biti smámýs af ættkvísl Culicoides. Sumarexem er ítrekuð húðbólga og einkennist af kláða, hárlausum svæðum, sáramyndun og jafnvel sýkingum í sárunum. Útfluttir íslenskir hestar fá sumarexem í um 50% tilfella, séu þeir óvarðir á flugusvæðum, en önnur hestakyn og íslenskir hestar fæddir á flugusvæðum fá sumarexem í tíðninni 5-10%. Ofnæmisvakar úr þremur smámýs tegundum, C. sonorensis, C. nubeculosus og C. obsoletus hafa verið skilgreindir, tjáðir í E.coli og nokkrir í skordýrafrumum og byggi. Markmiðið með verkefninu var að þróa ónæmismeðferð gegn sumarexemi; bólusetningu með hreinsuðum ofnæmisvökum í ónæmisglæðum og meðhöndlun um munnslímhúð með erfðabreyttu byggi sem tjáir ofnæmisvaka. Við þróun á ofnæmisvakasértækri ónæmismeðferð voru mismunandi sprautunarstaðir og ónæmisglæðar prófaðir. Heilbrigðir íslenskir hestar voru sprautaðir í húð og í eitla með litlu magni af ofnæmisvökum með eða án Th1 stýrandi glæðis. Bólusetning í eitla og í húð með ónæmisglæði örvaði myndun á sértækum IgG1 og IgG4/7 mótefnum. Bólusetning í eitla með glæði gaf ívíð sterkara svar og mótefnin sem mynduðust hindruðu að hluta til bindingu IgE við ofnæmisvaka. Í næstu tilraun voru íslenskir hestar sprautaðir í eitla með ofnæmisvökum í Alum eða blöndu af Alum og Monophosphoryl lipid A. Bólu-setningin örvaði sterka IgG1 og IgG4/7 mótefnasvörun sem hindruðu bindingu IgE við ofnæmisvaka. Munur á ónæmissvörun hópanna var mælanlegur í boðefnasvari í kjölfar örvunar á hvítfrumum en ekki í mótefnasvari. Marktækt meiri IFNγ og IL-10 framleiðsla var hjá Alum/MPLA hópnum samanborið við óbólusetta viðmiðunar hesta, þessi munur sást ekki hjá Alum hópnum. Þetta bendir til að Alum/MPLA blandan hvetji Th1/Tstjórnfrumu miðað svar. Þróuð voru byggmél og byggblanda til að meðhöndla hross um munnslímhúð. Heilbrigðir íslenskir hestar voru meðhöndlaðir með byggi sem tjáir ofnæmisvakann Cul n 2. Í kjölfar meðhöndlunar mældust sérvirk IgG1 og IgG4/7 mótefni í sermi og munnvatni. Þau gátu að hluta til hindrað bindingu IgE við Cul n 2 en einnig við Cul o 2 sem er sambærilegt prótein úr C. obsoletus. Mikilvægt er að hafa endurraðaða ofnæmisvaka sem auðvelt er að hreinsa, hafa svipaða þriðja stigs byggingu og eftirbreytni og náttúrulegu próteinin þegar árangur ónæmismeðferðar er metinn og klínísk greining sjúkdómsins staðfest. Byggfræ eru vel til þess fallin að framleiða prótein og fyrir langvarandi geymslu proteina á stöðugu formi. Tveir ofnæmisvakar framleiddir í byggi voru jafn vel fallnir til notkunar í ELISA mótefnaprófi og samsvarandi vakar framleiddir í E.coli og skordýrafrumum. Niðurstöðurnar sýna að bólusetning í eitla með hreinum ofnæmisvökum í blöndu af Alum/MPLA er vænleg leið í þróun á fyrirbyggjandi meðferð gegn sumarexemi og mögulega líka afnæmingu. Enn fremur fengust vísbendingar um að meðhöndlun um slímhúð munns með byggi sem tjáir ofnæmisvaka gæti verið góður valkostur. Þannig væri komist hjá vandasamri sprautun og kostnaðarsamri próteinhreinsun., Rannís, Eimskipasjóður Háskóla Íslands, Rannsóknasjóður Háskóla Íslands, Framleiðnisjóður landbúnaðarins, Hrossaræktarsamtök Suðurlands, VETSUISSE.