Detection of equine arteritis virus in semen by reverse transcriptase polymerase chain reaction-ELISA.
Abstract: The reverse transcriptase polymerase chain reaction (RT-PCR) assay was used to detect Equine Arteritis Virus (EAV) in the semen of 88 horses and 2 donkeys, with neutralising antibodies against EAV, on the basis of the amplification of a 279 bp long fragment located in the viral polymerase gene. The RT-PCR assay revealed the virus at 4 TCID50/ml in cell culture and showed a greater sensitivity (54.4%) than cell culture isolation (33.3%). Moreover, the two samples of donkey semen were found positive. The cDNAs obtained from 14 samples of horse and 2 of donkey semen were sequenced. Comparing the sequence of reference strain Bucyrus, the analysed samples were 78-100% identical and showed a 84-97% nucleotide identity with Bucyrus isolate. The results demonstrate high levels of genomic heterogeneity among the extracted RNAs, but inside the fragment amplified a well-preserved region of 24 bp was found with only three mismatches in some samples, suggesting that this could be ideal as a probe for RT-PCR-ELISA. The RT-PCR-ELISA assay using the EAV 7 and 8 primer set, has proved to be sensitive, specific and above all directly applicable to semen. Additionally, the short time needed for the overall procedure makes this method suitable for diagnostic purposes.
Publication Date: 1999-07-03 PubMed ID: 10391506DOI: 10.1016/s0147-9571(98)00136-2Google Scholar: Lookup
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- Journal Article
- Comparative Study
- Diagnosis
- Diagnostic Technique
- Disease
- Disease Diagnosis
- Enzyme-Linked Immunosorbent Assay (ELISA)
- Epidemiology
- Equid Semen
- Equine Diseases
- Equine Health
- Equine Viral Arteritis
- Genomics
- Horses
- Infection
- Infectious Disease
- Laboratory Methods
- Polymerase Chain Reaction
- RNA
- Veterinary Medicine
- Virology
- Virus
Summary
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This research paper presents the successful use of a reverse transcriptase polymerase chain reaction (RT-PCR) test to detect the presence of Equine Arteritis Virus (EAV) in the semen of horses and donkeys, suggesting this method is potent, speedy and effective for diagnostic purposes.
Methodology and Sample
- The study deployed the RT-PCR assay to check for EAV in the semen of 90 animals, which included 88 horses and 2 donkeys. These animals had previously demonstrated neutralising antibodies against EAV.
- The test focused specifically on detecting a 279 bp long fragment that is found in the EAV’s polymerase gene.
Test Sensitivity and Comparative Analysis
- The RT-PCR assay was able to detect the presence of the virus when the concentration was as low as 4 TCID50/ml in cell culture. This indicates a high sensitivity as it succeeded in discovering a large percentage (54.4%) of positive cases.
- RT-PCR proved to be more sensitive in comparison to the cell culture isolation method, which had a sensitivity of only 33.3%.
- Most importantly, the assay was successful in identifying the virus in the semen samples of the two donkeys included in the experiment.
Genomic Divergence and Probe Identification
- On sequencing the cDNAs obtained from the semen samples of 14 horses and 2 donkeys, a high level of genomic heterogeneity was noticed among the RNAs extracted.
- However, within the amplified fragment, a consistently well-preserved region of 24 bp was identified, making it an ideal candidate for a probe in future RT-PCR-ELISA testing.
- When compared with the sequence of reference strain Bucyrus, the analysed samples showed a 78-100% identity – further suggesting this particular region’s suitability as a probe.
Feasibility and Diagnostic Application
- The study demonstrated that the RT-PCR-ELISA assay is not only sensitive and specific, but can be directly applied to semen, making it significantly useful for diagnosing EAV.
- Another notable advantage of this testing method is the short duration required for the overall procedure, enhancing its practical application in a diagnostic setting.
Cite This Article
APA
Ramina A, Dalla Valle L, De Mas S, Tisato E, Zuin A, Renier M, Cuteri V, Valente C, Cancellotti FM.
(1999).
Detection of equine arteritis virus in semen by reverse transcriptase polymerase chain reaction-ELISA.
Comp Immunol Microbiol Infect Dis, 22(3), 187-197.
https://doi.org/10.1016/s0147-9571(98)00136-2 Publication
Researcher Affiliations
- Istituto Zooprofilattico Sperimentale delle Venezie, Agripolis, Padova, Italy.
MeSH Terms
- Animals
- Antibodies, Viral / blood
- Arterivirus Infections / veterinary
- Base Sequence
- Cells, Cultured
- DNA Primers
- DNA, Viral / analysis
- Enzyme-Linked Immunosorbent Assay / veterinary
- Equartevirus / immunology
- Equartevirus / isolation & purification
- Horse Diseases / virology
- Horses
- Kidney
- Male
- Molecular Sequence Data
- RNA, Viral / isolation & purification
- RNA, Viral / metabolism
- Rabbits
- Reverse Transcriptase Polymerase Chain Reaction / veterinary
- Semen / virology
Citations
This article has been cited 2 times.- Reichel MP, Lanyon SR, Hill FI. Moving past serology: Diagnostic options without serum. Vet J 2016 Sep;215:76-81.
- Balasuriya UB, Go YY, MacLachlan NJ. Equine arteritis virus. Vet Microbiol 2013 Nov 29;167(1-2):93-122.
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