Development and Field Testing of a Real-Time PCR Assay for Caprine Arthritis-Encephalitis-Virus (CAEV)



Giovanni Brajon*, 1, Daniela Mandas 2, Manuele Liciardi 4, Flavia Taccori 1, Mauro Meloni 2, Franco Corrias 1, Caterina Montaldo 5, Ferdinando Coghe 2, Cristina Casciari 3, Monica Giammarioli 3, Germano Orrù*, 2, 5
1 Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Sezione di Firenze, Firenze, Italy
2 DNA Sequencing Service (DSS), AOU, Cagliari, Italy
3 Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche, Perugia, Italy
4 Istituto Zooprofilattico Sperimentale della Sardegna, Dipartimento di Cagliari, Cagliari, Italy
5 OBL, Diparimento di Scienze Chirurgiche, Università di Cagliari, Cagliari, Italy


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© Brajon et al.; Licensee Bentham Open.

open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http: //creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

* Address correspondence to these authors at the DSS. Università degli Studi di Cagliari, Via Binaghi 4, 09121 Cagliari, Italy; Tel: 0039 070 537413; Fax: 0039 070 537437; E-mail: orru@unica.it and Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana. Sezione di Firenze, Via Castelpulci 43, 50018 Scandicci, Italy; Tel: 0039 055 721308; Fax: 0039 055 7311323; E-mail: giovanni.brajon@izslt.it


Abstract

Caprine arthritis/encephalitis (CAE) of goats and occasionally sheep are persistent virus infections caused by a lentivirus (CAEV). This viral infection results in arthritis in adult animals and encephalitis in kids. Prognosis for the encephalitic form is normally poor, with substantial economic loss for the farm. In this context an early/fast laboratory diagnosis for CAEV infection could be useful for effective prophylactic action. In this work we performed a quantitative real time PCR designed on the CAEV env gene to detect/quantify in goat/sheep samples, viral RNA or proviral DNA forms of CAEV. This procedure was validated in 15 sheep, experimentally infected with CAEV or with a highly correlated lentivirus (visna maedi, MVV); in addition, a total of 37 clinical goat specimens recruited in CAEV positive herds were analyzed and compared using serological analysis (Elisa and AGID). All samples infected with MVV resulted negative. In sheep experimentally infected with CAEV, proviral DNA was detectable 15 days post infection, whereas the serological methods revealed an indicative positivity after 40-60 days.This method showed a sensitivity of 102 env fragments/PCR) with a linear dynamic range of quantitation from 103 to 107env fragments/PCR; the R2 correlation coefficient was 0.98. All subjects with a clinical diagnosis for Caprine Arthritis-Encephalitis (CAE) resulted CAEV DNA positive.

Keywords: Caprine arthritis-encephalitis, CAEV, lentivirus, real time PCR, agar gel immunodiffusion (AGID), enzyme-linked immunosorbent assay (ELISA)..