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

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 107 env fragments/PCR; the R2 correlation coefficient was 0.98. All subjects with a clinical diagnosis for Caprine Arthritis-Encephalitis (CAE) resulted CAEV DNA positive.

Development of TaqMan-based qPCR method for detection of caprine arthritis–encephalitis virus (CAEV) infection

“…As reported previously [2], real-time PCR can identify an infection much earlier (15 days postinfection) than serological methods (ELISA and AGID, 40-60 days postinfection). When applied to clinical diagnosis of CAEV, this method serves as a feasible and attractive method for large-scale screening, particularly at times of CAEV outbreaks.…”

“…The control measures therefore rely heavily on accurate and reasonable laboratory diagnosis to identify and cull the CAEV-infected subjects so as to reduce economic losses [2]. …”

“…CAEV is mainly macrophage-tropic; expression of the viral genome depends on the maturation state of the cells, and viral transcripts are produced only when the cells mature into macrophages [9, 21]. Epidemiological evidence indicates that the virus is transmitted from infected does to their offspring through the consumption of virus-infected colostrum and milk [29] or through prolonged close contact with infected adult animals [2]. …”

“…PCR-based diagnostic techniques vary according to their targets, such as reverse transcription PCR for the detection of viral RNA [16], semi-nested PCR [7] or loop-mediated isothermal amplification (LAMP) of proviral DNA [12], and real-time PCR detection of the CAEV env gene. The real-time PCR gave earlier positive detection results (15 days postinfection) than serological methods(ELISA and AGID, about 40-60 days postinfection) [2]. An early/fast laboratory diagnosis for CAEV infection can be very useful for effective prophylactic action, and PCR is a useful tool for decreasing the risk of breeding AGID-false-negative animals [19].…”

Expanding Possibilities for Intervention against Small Ruminant Lentiviruses through Genetic Marker-Assisted Selective Breeding

“…The first is by direct measurement of proviral concentration [73,79,80,81,82] in peripheral blood, specific tissues, or both [83]. Such measurements have the advantage of reflecting viral replication.…”