Vaccination of foals with a modified live, equid herpesvirus-1 gM deletion mutant (RacHΔgM) confers partial protection against infection

Equid herpesvirus-1 (EHV-1) causes respiratory and neurological disease and late gestation abortion in pregnant mares. Current vaccines contain either inactivated or live EHV-1, but fail to provide complete clinical or virological protection, namely prevention of nasopharyngeal shedding and cell-associated viraemia. Thus, the development of novel products, such as modified live virus (MLV) vaccines which stimulate virus-specific, humoral and cell mediated immune responses more effectively remains a priority. Two groups of weaned foals (n = 6 each group) were used in a longitudinal, prospective, experimental study to evaluate immune responses elicited by two vaccinations with a glycoprotein M (gM) deletion mutant of EHV-1 (RacHdeltagM). Following two concurrent intranasal and intramuscular inoculations six weeks apart, vaccinated (8.4 ± 0.2 months old) and control foals (6.2 ± 0.4 months) were challenge infected intranasally with EHV-1 Ab4/8 four weeks after the second vaccination and clinical signs and virological replication measured. Vaccination caused no adverse events, but did stimulate significantly higher complement fixing and virus neutralizing antibodies in serum compared with control foals at either equivalent or pre-vaccination time points. Virus-specific nasopharyngeal antibody levels and cytotoxic T lymphocyte responses were not significantly different between the groups. Following challenge infection, these immune responses were associated with a reduction in clinical signs and virological replication in the vaccinated foals, including a reduction in duration and magnitude of pyrexia, nasopharyngeal shedding and cell-associated viraemia. We conclude that the RacHΔgM MLV primed EHV-1-specific humoral immune responses in weaned foals. However, complete virological protection by vaccination against EHV-1 requires further research

Development of a comprehensive protein microarray for immunoglobulin E profiling in horses with severe asthma

Background: Severe asthma in horses, known as severe equine asthma (SEA), is a prevalent, performance-limiting disease associated with increased allergen-specific immunoglobulin E (IgE) against a range of environmental aeroallergens. Objective: To develop a protein microarray platform to profile IgE against a range of proven and novel environmental proteins in SEA-affected horses. Animals: Six SEA-affected and 6 clinically healthy Warmblood performance horses. Methods: Developed a protein microarray (n = 384) using protein extracts and purified proteins from a large number of families including pollen, bacteria, fungi, and arthropods associated with the horses, environment. Conditions were optimized and assessed for printing, incubation, immunolabeling, biological fluid source, concentration techniques, reproducibility, and specificity. Results: This method identified a number of novel allergens, while also identifying an association between SEA and pollen sensitization. Immunolabeling methods confirmed the accuracy of a commercially available mouse anti-horse IgE 3H10 source (R2 = 0.91). Biological fluid source evaluation indicated that sera and bronchoalveolar lavage fluid (BALF) yielded the same specific IgE profile (average R2 = 0.75). Amicon centrifugal filters were found to be the most efficient technique for concentrating BALF for IgE analysis at 40-fold. Overnight incubation maintained the same sensitization profile while increasing sensitivity. Reproducibility was demonstrated (R2 = 0.97), as was specificity using protein inhibition assays. Arthropods, fungi, and pollens showed the greatest discrimination for SEA. Conclusions and clinical importance: We have established that protein microarrays can be used for large-scale IgE mapping of allergens associated with the environment of horses. This technology provides a sound platform for specific diagnosis, management, and treatment of SEA

Polarisation of Major Histocompatibility Complex II Host Genotype with Pathogenesis of European Brown Hare Syndrome Virus.

A study was conducted in order to determine the occurrence of European Brown Hare Syndrome virus (EBHSV) in Denmark and possible relation between disease pathogenesis and Major Histocompatibility Complex (MHC) host genotype. Liver samples were examined from 170 brown hares (hunted, found sick or dead), collected between 2004 and 2009. Macroscopical and histopathological findings consistent with EBHS were detected in 24 (14.1%) hares; 35 (20.6%) had liver lesions not typical of the syndrome, 50 (29.4%) had lesions in other tissues and 61 (35.9%) had no lesions. Sixty five (38.2%) of 170 samples were found to be EBHSV-positive (RT-PCR, VP60 gene). In order to investigate associations between viral pathogenesis and host genotype, variation within the exon 2 DQA gene of MHC was assessed. DQA exon 2 analysis revealed the occurrence of seven different alleles in Denmark. Consistent with other populations examined so far in Europe, observed heterozygosity of DQA (H o = 0.1180) was lower than expected (H e = 0.5835). The overall variation for both nucleotide and amino acid differences (2.9% and 14.9%, respectively) were lower in Denmark than those assessed in other European countries (8.3% and 16.9%, respectively). Within the peptide binding region codons the number of nonsynonymous substitutions (dN) was much higher than synonymous substitutions (dS), which would be expected for MHC alleles under balancing selection. Allele frequencies did not significantly differ between EBHSV-positive and -negative hares. However, allele Leeu-DQA*30 was detected in significantly higher (P = 0.000006) frequency among the positive hares found dead with severe histopathological lesions than among those found sick or apparently healthy. In contrast, the latter group was characterized by a higher frequency of the allele Leeu-DQA*14 as well as the proportion of heterozygous individuals (P = 0.000006 and P = 0.027). These data reveal a polarisation between EBHSV pathogenesis and MHC class II genotype within the European brown hare in Denmark

Development of a DNA-based microarray for the detection of zoonotic pathogens in rodent species

The demand for diagnostic tools that allow simultaneous screening of samples for multiple pathogens is increasing because they overcome the limitations of other methods, which can only screen for a single or a few pathogens at a time. Microarrays offer the advantages of being capable to test a large number of samples simultaneously, screening for multiple pathogen types per sample and having comparable sensitivity to existing methods such as PCR. Array design is often considered the most important process in any microarray experiment and can be the deciding factor in the success of a study. There are currently no microarrays for simultaneous detection of rodent-borne pathogens. The aim of this report is to explicate the design, development and evaluation of a microarray platform for use as a screening tool that combines ease of use and rapid identification of a number of rodent-borne pathogens of zoonotic importance. Nucleic acid was amplified by multiplex biotinylation PCR prior to hybridisation onto microarrays. The array sensitivity was comparable to standard PCR, though less sensitive than real-time PCR. The array presented here is a prototype microarray identification system for zoonotic pathogens that can infect rodent species

Use of wild bird surveillance, human case data and GIS spatial analysis for predicting spatial distributions of West Nile Virus in Greece

West Nile Virus (WNV) is the causative agent of a vector-borne, zoonotic disease with a worldwide distribution. Recent expansion and introduction of WNV into new areas, including southern Europe, has been associated with severe disease in humans and equids, and has increased concerns regarding the need to prevent and control future WNV outbreaks. Since 2010, 524 confirmed human cases of the disease have been reported in Greece with greater than 10% mortality. Infected mosquitoes, wild birds, equids, and chickens have been detected and associated with human disease. The aim of our study was to establish a monitoring system with wild birds and reported human cases data using Geographical Information System (GIS). Potential distribution of WNV was modelled by combining wild bird serological surveillance data with environmental factors (e.g. elevation, slope, land use, vegetation density, temperature, precipitation indices, and population density). Local factors including areas of low altitude and proximity to water were important predictors of appearance of both human and wild bird cases (Odds Ratio = 1,001 95%CI = 0,723–1,386). Using GIS analysis, the identified risk factors were applied across Greece identifying the northern part of Greece (Macedonia, Thrace) western Greece and a number of Greek islands as being at highest risk of future outbreaks. The results of the analysis were evaluated and confirmed using the 161 reported human cases of the 2012 outbreak predicting correctly (Odds = 130/31 = 4,194 95%CI = 2,841–6,189) and more areas were identified for potential dispersion in the following years. Our approach verified that WNV risk can be modelled in a fast cost-effective way indicating high risk areas where prevention measures should be implemented in order to reduce the disease incidence

Detection of a Yersinia pestis gene homologue in rodent samples

A homologue to a widely used genetic marker, pla, for Yersinia pestis has been identified in tissue samples of two species of rat (Rattus rattus and Rattus norvegicus) and of mice (Mus musculus and Apodemus sylvaticus) using a microarray based platform to screen for zoonotic pathogens of interest. Samples were from urban locations in the UK (Liverpool) and Canada (Vancouver). The results indicate the presence of an unknown bacterium that shares a homologue for the pla gene of Yersinia pestis, so caution should be taken when using this gene as a diagnostic marker

Prevalence of equine herpesvirus types 2 and 5 in horse populations by using type-specific PCR assays

Equine herpesvirus types 2 and 5 (EHV-2 and EHV-5) have a rather unclear pathogenicity and distribution within the equid population. In order to gain more information on the prevalence of these two viruses, type-specific PCR assays were developed to detect viral DNA in nasal specimens and in peripheral blood leukocytes (PBLs) of adult horses and foals from various regions of Europe, i.e. Sweden, Hungary and the United Kingdom. In adult horses, the prevalence of EHV-2 in PBLs was up to 68% in Sweden and 71% in the United Kingdom. EHV-2 DNA was detected in the PBLs from all the foals tested in all countries and most (93%) of the nasal specimens also yielded positive results. The prevalence of EHV-5 DNA in the PBLs of foals in Hungary was 15 and 24% in adult horses in the United Kingdom. This observation was among the very few reports of the presence of EHV-5 in horses. In summary, the specific PCR assays revealed important data on the occurrence and distribution of EHV-2 and EHV-5 in large horse populations. The findings indicated that infection with EHV-5 occurred later than EHV-2 in foals. This study may contribute to a better understanding of the etiological role of these gammaherpesviruses in equine diseases.Prévalence des herpesvirus équins types 2 et 5 dans les populations de chevaux par l'utilisation de PCR spécifiques du type. Les herpesvirus équins de types 2 et 5 (EHV-2 et EHV-5) ont un pouvoir pathogène et une distribution mal connus parmi la population des équidés. Afin de recueillir plus d'informations sur la prévalence de ces deux virus, des tests PCR spécifiques du type ont été développés afin de détecter l'ADN viral dans les prélèvements nasaux et dans les leucocytes du sang périphérique (LSP) de chevaux adultes et de poulains provenant de diverses régions d'Europe : Suède, Hongrie et Royaume Uni. Chez les chevaux adultes, la prévalence de EHV-2 dans les LSP atteignait 68 % en Suède et 71 % au Royaume-Uni. L'ADN de EHV-2 a été détecté dans les LSP de tous les poulains testés, et la plupart (93 %) des prélèvements nasaux étaient également positifs. La prévalence de l'ADN de EHV-5 dans les LSP des poulains en Hongrie était de 15 % et de 24 % chez les chevaux adultes au Royaume-Uni. Cette observation fait partie des très rares signalements de la présence de EHV-5 chez les chevaux. En résumé, les tests PCR spécifiques ont révélé des données importantes sur la présence et la distribution de EHV-2 et EHV-5 dans d'importantes populations de chevaux. Les résultats ont montré que l'infection par le EHV-5 se produisait plus tard que celle par le EHV-2 chez le poulain. Cette étude apporte une meilleure compréhension du rôle étiologique de ces herpesvirus gamma dans les maladies équines

Diagnosis of severe asthma via protein microarray IgE profiling and mathematical modelling

International audienc

Development of a multiple protein extract microarray for profiling allergen-specific immunoglobin E in horses with severe asthma

International audienc