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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.

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/).

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.

Equine insect bite hypersensitivity (IBH) is a seasonally recurrent, pruritic skin disorder caused by an IgE-mediated reaction to salivary proteins of biting flies, predominantly of the genus Culicoides. The aim of this study was to define T cell subsets and cytokine profile in the skin of IBH-affected Icelandic horses with particular focus on the balance between T helper (Th) 1, Th2 and T regulatory (Treg) cells. Distribution and number of CD4+, CD8+ and Forkhead box P3 (FoxP3)+ T cells were characterized by immunohistochemical staining in lesional and non-lesional skin of moderately and severely IBH-affected horses (n=14) and in the skin of healthy control horses (n=10). Using real-time quantitative reverse transcription-polymerase chain reaction, mRNA expression levels of Th2 cytokines (Interleukin (IL)-4, IL-5, IL-13), Th1 cytokines (Interferon-γ), regulatory cytokines (Transforming Growth Factor β1, IL-10) and the Treg transcription factor FoxP3 were measured in skin and blood samples. Furthermore, Culicoides nubeculosus specific serum IgE levels were assessed. Lesions of IBH-affected horses contained significantly higher numbers of CD4+ cells than skin of healthy control horses. Furthermore, the total number of T cells (CD4+ and CD8+) was significantly increased in lesional compared to non-lesional skin and there was a tendency (p=0.07) for higher numbers of CD4+ cells in lesional compared to non-lesional skin. While the number of FoxP3+ T cells did not differ significantly between the groups, the ratio of Foxp3 to CD4+ cells was significantly lower in lesions of severely IBH-affected horses than in moderately affected or control horses. Interestingly, differences in FoxP3 expression were more striking at the mRNA level. FoxP3 mRNA levels were significantly reduced in lesional skin, compared both to non-lesional and to healthy skin and were also significantly lower in non-lesional compared to healthy skin. Expression levels of IL-13, but not IL-4 or IL-5, were significantly elevated in lesional and non-lesional skin of IBH-affected horses. IL-10 levels were lower in lesional compared to non-lesional skin (p=0.06) and also lower (p=0.06) in the blood of IBH-affected than of healthy horses. No significant changes were observed regarding blood expression levels of Th1 and Th2 cytokines or FoxP3. Finally, IBH-affected horses had significantly higher Culicoides nubeculosus specific serum IgE levels than control horses. The presented data suggest that an imbalance between Th2 and Treg cells is a characteristic feature in IBH. Treatment strategies for IBH should thus aim at restoring the balance between Th2 and Treg cells.

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.

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.

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.

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 two types of gammaherpesviruses, equine herpes virus (EHV) 2 and 5. While EHV-2 is ubiquitous in adult horses, EHV-5 has been less frequently described. Due to strong serological cross-reactivity, EHV-2 and -5 cannot be discriminated in broad spectrum antibody tests and are thus commonly referred to as gamma-EHV. Total IgG and IgG subclass response against gamma-EHV were determined in serum from 41 healthy Icelandic horses, thereof 20 adults, 10 foals aged 10 months, and 11 foals aged 1-4 months. Additionally, in 10 of the adult horses, interferon (IFN)-gamma and interleukin (IL)-4 expression were measured by real-time PCR in white blood cells upon in vitro stimulation with EHV-2. With the exception of one orphan foal, all tested individuals were seropositive for gamma-EHV. All but one adult had high titer of EHV-specific IgG4/7 (IgGb) in combination with much lower titer of IgG1 (IgGa) and IgG3/5 (IgG(T)), indicating a stabilized response. IgG titer and subclasses in the foals showed considerably more variation, possibly dependant on maternal antibodies and/or recent infection. In all the 10 horses tested for cytokine expression, IFN-gamma production exceeds production of IL-4. These results indicate that equine gammaherpesvirus infection is characterized by an induction of IgG1, IgG4/7 and IgG3/5 with prevailing IgG4/7 and cytokine profile dominated by IFN-gamma. To our knowledge, this is the first report on the cytokine and IgG subclass response against gamma-EHV in horses.

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.

An upsurge of canine distemper was recognized at the beginning of 1991 in the urban dog population of the Copenhagen area. The outbreak had the characteristics of a virulent morbillivirus introduction in a partly immune population, where the disease primarily was manifested in young individuals. Testing of single serum samples for the presence of canine distemper virus (CDV) IgM antibodies using an IgM ELISA confirmed current and recent CDV infections in an urban dog population, where the use of attenuated CDV vaccines was widespread. In 49 out of 66 sera from clinical cases suspected of canine distemper we detected CDV IgM antibodies, as compared to the detection of viral antigen by indirect immunofluorescence in 27 of 65 specimens of conjunctival cells. The antigenic make-up of isolates from acute and subacute clinical cases was investigated with a panel of 51 monoclonal antibodies directed against CDV and the related phocine distemper virus. The isolates exhibited an homogeneous reaction pattern and shared overall antigenic characteristics of the CDV prototype. The majority of cases were diagnosed among unvaccinated dogs and individuals with unknown or obscure vaccination record. However, severe clinical cases were also diagnosed in vaccinated individuals.