Detection of respiratory herpesviruses in foals and adult horses determined by nested multiplex PCR.

L Wang, Sharanne Raidal, A. Pizzirani, Graham Wilcox

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Abstract

A nested multiplex PCR was developed as a rapid (<12 h), sensitive test for the simultaneous identification of equine herpes viruses (EHV1, EHV4, EHV2 and EHV5) in clinical samples from horses. Peripheral blood and nasal swab (NS) samples from 205 weanling Thoroughbred foals on 6 different studs over 3 consecutive seasons and from 92 adult horses without clinical signs of respiratory disease were examined using direct multiplex PCR of clinical samples (direct PCR) and conventional cell culture with differentiation of EHV in cell cultures by multiplex PCR. Multiplex PCR proved a sensitive and specific technique for the detection of EHV in cell culture and clinical samples. The technique described appeared equally sensitive as one using a single set of primers for individual EHV but reduced labour and reagent costs. Cell cultures showing cytopathic effect (CPE) were always positive for EHV on PCR. EHV were also detected by multiplex PCR in 11 samples which failed to show CPE. By a combination of multiplex PCR and cell culture or direct multiplex PCR, the presence of up to three EHV in the same sample was detected. Overall, EHV5 was detected by direct multiplex PCR of peripheral blood mononuclear cells (PBMC) and/or NS samples from 78% of foals and 47% of adult horses. Repeated sampling or cell culture in combination with multiplex PCR and with the incorporation of IL-2 in culture medium increased the sensitivity for detection of EHV in PBMC and demonstrated that EHV5 DNA could be identified in PBMC from 89% of foals and 100% of adult horses. EHV2 was identified from approximately 30% of foals, but was more frequently identified in samples from 17 foals with mild respiratory disease and was isolated infrequently from adult horses. EHV1 and EHV4 were identified uncommonly in any population in the current study.
Original languageEnglish
Pages (from-to)18-28
Number of pages11
JournalVeterinary Microbiology
Volume121
Issue number1-2
DOIs
Publication statusPublished - Mar 2007

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Herpesviridae
Multiplex Polymerase Chain Reaction
foals
Horses
horses
Polymerase Chain Reaction
cell culture
Cell Culture Techniques
mononuclear leukocytes
sampling
Blood Cells
cytopathogenicity
Nose
respiratory tract diseases
interleukin-2
weanlings
Interleukin-2
Culture Media
labor
culture media

Cite this

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title = "Detection of respiratory herpesviruses in foals and adult horses determined by nested multiplex PCR.",
abstract = "A nested multiplex PCR was developed as a rapid (<12 h), sensitive test for the simultaneous identification of equine herpes viruses (EHV1, EHV4, EHV2 and EHV5) in clinical samples from horses. Peripheral blood and nasal swab (NS) samples from 205 weanling Thoroughbred foals on 6 different studs over 3 consecutive seasons and from 92 adult horses without clinical signs of respiratory disease were examined using direct multiplex PCR of clinical samples (direct PCR) and conventional cell culture with differentiation of EHV in cell cultures by multiplex PCR. Multiplex PCR proved a sensitive and specific technique for the detection of EHV in cell culture and clinical samples. The technique described appeared equally sensitive as one using a single set of primers for individual EHV but reduced labour and reagent costs. Cell cultures showing cytopathic effect (CPE) were always positive for EHV on PCR. EHV were also detected by multiplex PCR in 11 samples which failed to show CPE. By a combination of multiplex PCR and cell culture or direct multiplex PCR, the presence of up to three EHV in the same sample was detected. Overall, EHV5 was detected by direct multiplex PCR of peripheral blood mononuclear cells (PBMC) and/or NS samples from 78{\%} of foals and 47{\%} of adult horses. Repeated sampling or cell culture in combination with multiplex PCR and with the incorporation of IL-2 in culture medium increased the sensitivity for detection of EHV in PBMC and demonstrated that EHV5 DNA could be identified in PBMC from 89{\%} of foals and 100{\%} of adult horses. EHV2 was identified from approximately 30{\%} of foals, but was more frequently identified in samples from 17 foals with mild respiratory disease and was isolated infrequently from adult horses. EHV1 and EHV4 were identified uncommonly in any population in the current study.",
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Detection of respiratory herpesviruses in foals and adult horses determined by nested multiplex PCR. / Wang, L; Raidal, Sharanne; Pizzirani, A.; Wilcox, Graham.

In: Veterinary Microbiology, Vol. 121, No. 1-2, 03.2007, p. 18-28.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Detection of respiratory herpesviruses in foals and adult horses determined by nested multiplex PCR.

AU - Wang, L

AU - Raidal, Sharanne

AU - Pizzirani, A.

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AB - A nested multiplex PCR was developed as a rapid (<12 h), sensitive test for the simultaneous identification of equine herpes viruses (EHV1, EHV4, EHV2 and EHV5) in clinical samples from horses. Peripheral blood and nasal swab (NS) samples from 205 weanling Thoroughbred foals on 6 different studs over 3 consecutive seasons and from 92 adult horses without clinical signs of respiratory disease were examined using direct multiplex PCR of clinical samples (direct PCR) and conventional cell culture with differentiation of EHV in cell cultures by multiplex PCR. Multiplex PCR proved a sensitive and specific technique for the detection of EHV in cell culture and clinical samples. The technique described appeared equally sensitive as one using a single set of primers for individual EHV but reduced labour and reagent costs. Cell cultures showing cytopathic effect (CPE) were always positive for EHV on PCR. EHV were also detected by multiplex PCR in 11 samples which failed to show CPE. By a combination of multiplex PCR and cell culture or direct multiplex PCR, the presence of up to three EHV in the same sample was detected. Overall, EHV5 was detected by direct multiplex PCR of peripheral blood mononuclear cells (PBMC) and/or NS samples from 78% of foals and 47% of adult horses. Repeated sampling or cell culture in combination with multiplex PCR and with the incorporation of IL-2 in culture medium increased the sensitivity for detection of EHV in PBMC and demonstrated that EHV5 DNA could be identified in PBMC from 89% of foals and 100% of adult horses. EHV2 was identified from approximately 30% of foals, but was more frequently identified in samples from 17 foals with mild respiratory disease and was isolated infrequently from adult horses. EHV1 and EHV4 were identified uncommonly in any population in the current study.

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KW - Cell culture

KW - Equine herpesvirus 1

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KW - Equine herpesvirus 4

KW - Equine herpesvirus 5

KW - Multiplex PCR

KW - Peripheral blood mononuclear cells

KW - Respiratory disease

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JO - Veterinary Microbiology

JF - Veterinary Microbiology

SN - 0378-1135

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