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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 (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).

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 detection of anti-Streptococcus equi antibodies in the blood serum of horses can assist with the identification of apparently healthy persistently infected carriers and the prevention of strangles outbreaks. The aim of the current study was to use genome sequencing data to develop an indirect enzyme linked immunosorbent assay (iELISA) that targets two S. equi-specific protein fragments. The sensitivity and specificity of the antigen A and antigen C iELISAs were compared to an SeM-based iELISA marketed by IDvet – diagnostic Vétérinaire (IDvet). Individually, each assay compromised specificity in order to achieve sufficient sensitivity (SeM iELISA had a sensitivity of 89.9%, but a specificity of only 77.0%) or sensitivity to achieve high specificity. However, combining the results of the antigen A and antigen C iELISAs permitted optimisation of both sensitivity (93.3%) and specificity (99.3%), providing a robust assay for the identification of horses exposed to S. equi.

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.

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.

Allergic diseases occur in most mammals, although some species such as humans, dogs and horses seem to be more prone to develop allergies than others. In horses, insect bite hypersensitivity (IBH), an allergic dermatitis caused by bites of midges, and recurrent airway obstruction (RAO), a hyperreactivity to stable born dust and allergens, are the two most prevalent allergic diseases. Allergic diseases involve the interaction of three major factors: (i) genetic constitution, (ii) exposure to allergens, and (iii) a dysregulation of the immune response determined by (i) and (ii). However, other environmental factors such as infectious diseases, contact with endotoxin and degree of infestation with endoparasites have been shown to influence the prevalence of allergic diseases in humans. How these factors may impact upon allergic disease in the horse is unknown at this time. The 3rd workshop on Allergic Diseases of the Horse, with major sponsorship from the Havemeyer Foundation, was held in Holar, Iceland, in June 2007 and focussed on immunological and genetic aspects of IBH and RAO. This particular venue was chosen because of the prevalence of IBH in exported Icelandic horses. The incidence of IBH is significantly different between Icelandic horses born in Europe or North America and those born in Iceland and exported as adults. Although the genetic factors and allergens are the same, exported adult horses show a greater incidence of IBH. This suggests that environmental or epigenetic factors may contribute to this response. This report summarizes the present state of knowledge and summarizes important issues discussed at the workshop.

We have investigated the role of vif in maedi-visna virus (MVV), a lentivirus of sheep, by studying in vitro replication of vif-deleted MVV in several cell types, and the effects of vif deletion on in vivo infection. By measuring RT activity, we found that in comparison to wild-type MVV, growth of vif-deleted MVV was similar in fetal ovine synovial (FOS) cells, highly attenuated in sheep choroid plexus (SCP) cells, and not detectable in macrophages, natural target cells of MVV. Productive infection by vif-deleted MVV could not be demonstrated in sheep. An increased mutation frequency was observed in DNA produced by endogenous reverse transcription of viral RNA in vif-deleted virions, indicating the existence of a factor comparable in action to human APOBEC3G. These results suggest that the vif gene of MVV is essential for infectivity and that the Vif protein protects the viral genome from enpackaged mutagenic activities.

DMV, dolphin morbillivirus, a paramyxovirus of uncertain origin recently emerged in Mediterranean dolphins. This study presents the complete nucleotide sequence of the hemagglutinin (H) gene including the gene boundaries. The single open reading frame of the DMV H gene encodes a protein of 604 residues which exhibits overall sequence characteristics similar to the H genes of other morbilliviruses. When compared to its closest homologues, measles virus (MV) and rinderpest virus (RPV), DMV has, respectively, 44 and 46% of amino acid residues in identical positions. The primary sequence of the DMV H protein is markedly less conserved than that of the fusion protein. The comparative data at the genomic level correspond with cross-neutralization studies with different morbilliviruses. Retrospective serogical studies dating back to 1983 indicate DMV-like infections in whales of the eastern Atlantic. The presented data support and extend previous studies suggesting that this novel morbillivirus is one of the phylogenetically oldest morbilliviruses known to circulate today. The relationship of DMV and established morbilliviruses to the newly emerged candidate morbillivirus infecting horse and man is discussed.

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.

Horses are hosts to 2 types of gammaherpesviruses, Equid herpesvirus 2 and 5 (EHV-2 and EHV-5, respectively). Both EHV-2 and EHV-5 are common in horses in Iceland. An Icelandic EHV-5 isolate was recovered by sequential culture in primary fetal horse kidney and rabbit kidney cells. Glycoprotein B, glycoprotein H, and DNA terminase genes of the isolate were fully sequenced, and the DNA polymerase gene was partly sequenced. To date, the glycoprotein B gene of EHV-5 was the only gene that has been reported to be completely sequenced in addition to small parts of the glycoprotein H, DNA polymerase, and DNA terminase genes. The present report, therefore, is a significant addition to previously reported EHV-5 sequences.

The immunological relationships between distemper viruses, isolated from a seal and mink in Denmark and from a dog in Greenland, were investigated with 39 previously developed monoclonal antibodies (MAbs) directed against four major structural proteins of canine distemper virus (CDV). They were also investigated with 16 newly developed MAbs directed against the fusion (F) and large glycoprotein (named H in analogy with measles virus) of phocid distemper virus (PDV) isolated from a harbour seal (Phoca vitulina). These MAbs were reacted with the three different isolated viruses and with the LEC strain of measles virus, in ELISA and immunofluorescence tests. In addition, immunoprecipitation tests were carried out with some of the cross-reacting antibodies. All 55 MAbs reacted identically with distemper virus isolated from seals or mink. When the MAbs produced against CDV were tested, 37 of 39 antibodies reacted with a virus isolated from a sled dog diseased in an outbreak of distemper in Greenland prior to the epizootic among seals in the North Sea. Of the 39 antibodies, 25 reacted with PDV and distemper virus isolated from mink. Of these antibodies, only three of the nine antibodies directed against the H protein of CDV cross-reacted with PDV and distemper virus from mink. Eleven MAbs, reacting with six epitopes of the H protein of PDV, were produced. All 11 antibodies reacted with distemper virus from mink, two of the antibodies reacted with CDV and none reacted with measles virus. All five antibodies reacting with three different epitopes of the F protein of PDV reacted with distemper virus from mink and CDV. Of these five antibodies three, directed against two epitopes, reacted with measles virus. Of the two envelope proteins, the H protein shows pronounced immunological differences between PDV and CDV. In contrast, immunologically the F protein appears to be well conserved among morbilliviruses. It is concluded that the virus causing the epizootic in seals in the North Sea in 1988 may have infected mink on land, or, alternatively, the virus in the sea may have originated from virus-infected mink.

Maedi-visna virus (MVV) is a lentivirus of sheep sharing several key features with the primate lentiviruses. The virus causes slowly progressive diseases, mainly in the lungs and the central nervous system of sheep. Here, we investigate the molecular basis for the differential growth phenotypes of two MVV isolates. One of the isolates, KV1772, replicates well in a number of cell lines and is highly pathogenic in sheep. The second isolate, KS1, no longer grows on macrophages or causes disease. The two virus isolates differ by 129 nucleotide substitutions and two deletions of 3 and 15 nucleotides in the env gene. To determine the molecular nature of the lesions responsible for the restrictive growth phenotype, chimeric viruses were constructed and used to map the phenotype. An L120R mutation in the CA domain, together with a P205S mutation in Vif (but neither alone), could fully convert KV1772 to the restrictive growth phenotype. These results suggest a functional interaction between CA and Vif in MVV replication, a property that may relate to the innate antiretroviral defense mechanisms in sheep.

Background In a stable of eight horses in Northern Iceland, six horses presented with clinical signs, such as ataxia and reduced appetite, leading to euthanasia of one severely affected horse. Serological investigations revealed no evidence of active equine herpes virus type 1 infection, a common source of central nervous system disease in horses, nor equine arteritis virus and West Nile virus. Another neurotropic virus, Borna disease virus, was therefore included in the differential diagnosis list. Findings Serological investigations revealed antibodies against Borna disease virus in four of five horses with neurological signs in the affected stable. One horse without clinical signs was seronegative. Four clinically healthy horses in the stable that arrived and were sampled one year after the outbreak were found seronegative, whereas one of four investigated healthy horses in an unaffected stable was seropositive. Conclusions This report contains the first evidence of antibodies to Borna disease virus in Iceland. Whether Borna disease virus was the cause of the neurological signs could however not be confirmed by pathology or molecular detection of the virus. As Iceland has very restricted legislation regarding animal imports, the questions of how this virus has entered the country and to what extent markers of Bornavirus infection can be found in humans and animals in Iceland remain to be answered.

Since 1988 morbilliviruses have been increasingly recognized and held responsible for mass mortality amongst harbour seals (Phoca vitulina) and other seal species. Virus isolations and characterization proved that morbilliviruses from seals in Northwest Europe were genetically distinct from other known members of this group including canine distemper virus (CDV), rinderpest virus, peste des petits ruminants virus and measles virus. An epidemic in Baikal seals in 1987 was apparently caused by a morbillivirus closely related to CDV so that two morbilliviruses have now been identified in two geographically distant seal populations, with only the group of isolates from Northwest Europe forming a new member of the genus morbillivirus: phocid distemper virus (PDV). Because of distemper-like disease, the Baikal seal morbillivirus was tentatively named PDV-2 in spite of its possible identity with CDV. The appearance of morbilliviruses in the Mediterranean Sea causing high mortality amongst dolphins should further increase the research activities on protection strategies for endangered species of marine mammals.

One of the prerequisite for developing DNA vaccines for horses are vectors that are efficiently expressed in horse cells. We have analysed the ectopic expression of the human serum albumin gene in primary horse cells from different tissues. The vectors used are of pcDNA and pUC origin and include the cytomegalovirus (CMV) promoter. The pUC vectors contain CMV intron A whereas the pcDNA vectors do not. Insertion of intron A diminished the expression from the pcDNA vectors whereas insertion of a Kozak sequence upstream of the gene in two types of pUC vectors increased significantly the in vitro expression in primary horse cells derived from skin, lung, duodenum and kidney. We report for the first time the significance of full consensus Kozak sequences for protein expression in horse cells in vitro.

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.