Detection of equine herpesvirus in horses with idiopathic keratoconjunctivitis and comparison of three sampling techniques

ObjectivesTo determine the role of equine herpesvirus (EHV) in idiopathic keratoconjunctivitis in horses and to determine whether sample collection method affects detection of EHV DNA by quantitative polymerase chain reaction (qPCR).Animals studiedTwelve horses with idiopathic keratoconjunctivitis and six horses without signs of ophthalmic disease.ProceduresConjunctival swabs, corneal scrapings, and conjunctival biopsies were collected from 18 horses: 12 clinical cases with idiopathic keratoconjunctivitis and six euthanized controls. In horses with both eyes involved, the samples were taken from the eye judged to be more severely affected. Samples were tested with qPCR for EHV-1, EHV-2, EHV-4, and EHV-5 DNA. Quantity of EHV DNA and viral replicative activity were compared between the two populations and among the different sampling techniques; relative sensitivities of the sampling techniques were determined.ResultsPrevalence of EHV DNA as assessed by qPCR did not differ significantly between control horses and those with idiopathic keratoconjunctivitis. Sampling by conjunctival swab was more likely to yield viral DNA as assessed by qPCR than was conjunctival biopsy. EHV-1 and EHV-4 DNA were not detected in either normal or IKC-affected horses; EHV-2 DNA was detected in two of 12 affected horses but not in normal horses. EHV-5 DNA was commonly found in ophthalmically normal horses and horses with idiopathic keratoconjunctivitis.ConclusionsBecause EHV-5 DNA was commonly found in control horses and in horses with idiopathic keratoconjunctivitis, qPCR was not useful for the etiological diagnosis of equine keratoconjunctivitis. Conjunctival swabs were significantly better at obtaining viral DNA samples than conjunctival biopsy in horses in which EHV-5 DNA was found

Evaluation of a blocking ELISA for the detection of antibodies against Lawsonia intracellularis in pig sera

<p>Abstract</p> <p>Background</p> <p><it>Lawsonia intracellularis </it>is a common cause of chronic diarrhoea and poor performance in young growing pigs. Diagnosis of this obligate intracellular bacterium is based on the demonstration of the microbe or microbial DNA in tissue specimens or faecal samples, or the demonstration of <it>L. intracellularis</it>-specific antibodies in sera. The aim of the present study was to evaluate a blocking ELISA in the detection of serum antibodies to <it>L. intracellularis</it>, by comparison to the previously widely used immunofluorescent antibody test (IFAT).</p> <p>Methods</p> <p>Sera were collected from 176 pigs aged 8-12 weeks originating from 24 herds with or without problems with diarrhoea and poor performance in young growing pigs. Sera were analyzed by the blocking ELISA and by IFAT. Bayesian modelling techniques were used to account for the absence of a gold standard test and the results of the blocking ELISA was modelled against the IFAT test with a "2 dependent tests, 2 populations, no gold standard" model.</p> <p>Results</p> <p>At the finally selected cut-off value of percent inhibition (PI) 35, the diagnostic sensitivity of the blocking ELISA was 72% and the diagnostic specificity was 93%. The positive predictive value was 0.82 and the negative predictive value was 0.89, at the observed prevalence of 33.5%.</p> <p>Conclusion</p> <p>The sensitivity and specificity as evaluated by Bayesian statistic techniques differed from that previously reported. Properties of diagnostic tests may well vary between countries, laboratories and among populations of animals. In the absence of a true gold standard, the importance of validating new methods by appropriate statistical methods and with respect to the target population must be emphasized.</p

Proximity assays for sensitive quantification of proteins

Proximity assays are immunohistochemical tools that utilise two or more DNA-tagged aptamers or antibodies binding in close proximity to the same protein or protein complex. Amplification by PCR or isothermal methods and hybridisation of a labelled probe to its DNA target generates a signal that enables sensitive and robust detection of proteins, protein modifications or protein–protein interactions. Assays can be carried out in homogeneous or solid phase formats and in situ assays can visualise single protein molecules or complexes with high spatial accuracy. These properties highlight the potential of proximity assays in research, diagnostic, pharmacological and many other applications that require sensitive, specific and accurate assessments of protein expression

Differential diagnosis and aspects on epidemiology and pathogenesis of equine herpesviruses

Herpesviruses constitute a family of DNA viruses found in virtually all animal species. In horse five herpesviruses have been identified namely Equine herpesvirus (EHV) type 1, 3 and 4 belonging to the subfamily alphaherpesvirinae and Equine herpesvirus type 2 and 5 belonging to the gammaherpesvirinae. EHV-1 and 4 are the major causes of abortion and respiratory disease respectively and both cause large economical losses. EHV-2 has been associated with respiratory disease, immunosuppression and is a predisposing factor for more severe secondary infections. EHV-5 is a new virus first isolated in Australia from horses suffering from upper respiratory disease. This thesis is dealing with the diagnosis, epidemiology and pathogenesis ofEHV-1,2,4 and 5.EHV-2 is a predisposing factor for bacterial infection particularly with Rhodococcus equi and this role was studied by vaccination of foals with an EHV-2 subunit (ISCOM) vaccine. Foals vaccinated twice developed neutralising antibodies and were protected against the pneumonia while the non-vaccinated control group developed respiratory disease including pneumonia with abscesses containing R.equi which in some cases was fatal. Natural infection induce neutralising EHV-2 antibody as detected by a blocking ELISA. This ELISA recorded a much higher number of seropositive horses in a stable with annual EHV-2/R.equi infections compared to a stable with no such problems. Thus, the blocking ELISA was found to be a reliable tool for detecting recent EHV-2 infections. Likewise antibodies induced by a newly acquired EHV-2 infection in foals were also detected by the blocking ELISA and all foals tested were found to have experienced an EHV-2 infection by the age of 4 to 6 months.The prevalence of EHV-2 and EHV-5 were studied by type-specific Polymerase chain reaction (PCR). Up to 68% of tested horses in Sweden were EHV-2 positive, i.e. had virus DNA in peripheral blood leukocytes, while 100% ofthe tested foals in Sweden and Hungary were positive before 2 to 8 months of age. No EHV-5 positive horses were found in Sweden while 4 out of 27 tested foals in Hungary had EHV-5 specific DNA by the age of 13 to 23 weeks showing that EHV-5 cause infections later in life.Convalescent sera from horses contain EHV-l/EHV-4 cross-reactive antibody which up to recently made differentiation virtually impossible. A new indirect ELISA that differentiate between the two virus types showed that the epidemiology between EHV-1 and EHV-4 differ considerably. While almost 100% of the horses were seropositive to EHV-4, the number of EHV-1 seropositive horses varied between 9 and 56 % in different stables. Most foals had maternally-derived antibodies to EHV-4 until 4 to 6 months of age, after which they all became infected with EHV-4 and seroconverted. Only 2 out of 48 foals had maternal antibodies to EHV-1 and very few became infected

Assessment of the First Commercial ELISA Kit for the Diagnosis of Theileria annulata

The present study assesses the efficacy of SVANOVIR Theileria annulata-Ab, the first commercial ELISA kit for the diagnosis of Theileria annulata infection in cattle based on a recombinant protein known as T. annulata surface protein (TaSp). As a reference test, a polymerase chain reaction (PCR) assay depending on T. annulata merozoite surface antigen (Tams-1) was applied. A total of 468 blood samples as well as serum samples were randomly collected from cattle and tested in the PCR as well as in the ELISA developed in this study. Moreover, all samples were also analyzed by conventional Giemsa-stained blood smear. The results of this study revealed a good correlation between the results obtained by PCR and the ELISA, whereas all PCR positive samples scored correctly positive in the ELISA and 73 of the 125 PCR negative samples scored correctly negative. Taken together, a sensitivity of 91.25% and a specificity of 78.4% were recorded, when compared to the PCR data. In conclusion, the SVANOVIR Theileria annulataAb is a suitable diagnostic assay for use in the diagnosis and epidemiological surveys of Theileria annulata infection in chronic and carrier animals

Assessment of the First Commercial ELISA Kit for the Diagnosis of Theileria annulata

The present study assesses the efficacy of SVANOVIR Theileria annulata-Ab, the first commercial ELISA kit for the diagnosis of Theileria annulata infection in cattle based on a recombinant protein known as T. annulata surface protein (TaSp). As a reference test, a polymerase chain reaction (PCR) assay depending on T. annulata merozoite surface antigen (Tams-1) was applied. A total of 468 blood samples as well as serum samples were randomly collected from cattle and tested in the PCR as well as in the ELISA developed in this study. Moreover, all samples were also analyzed by conventional Giemsa-stained blood smear. The results of this study revealed a good correlation between the results obtained by PCR and the ELISA, whereas all PCR positive samples scored correctly positive in the ELISA and 73 of the 125 PCR negative samples scored correctly negative. Taken together, a sensitivity of 91.25% and a specificity of 78.4% were recorded, when compared to the PCR data. In conclusion, the SVANOVIR Theileria annulata-Ab is a suitable diagnostic assay for use in the diagnosis and epidemiological surveys of Theileria annulata infection in chronic and carrier animals

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