Application of quantitative real-time polymerase chain reaction for the diagnosis of toxoplasmosis and enzootic abortion of ewes

Abstract

Toxoplasma gondii and Chlamydophila abortus are the 2 most common infectious causes of ovine abortion worldwide. These obligate intracellular pathogens are associated with severe placentitis leading to abortion or stillbirth in pregnant ewes, and resulting in significant economic losses. The objectives of the current study were the development, validation, and application of a duplex real-time polymerase chain reaction (PCR) assay capable of quantifying the burden of infection by T. gondii and C. abortus in material submitted for diagnostic purposes. The validation was carried out using samples from ewes experimentally infected with these organisms. Based on the numbers of genome copies detected, an arbitrary cutoff level was established to correlate with significant pathological changes sufficient to give rise to abortion. When the PCR assay was applied to samples from 66 Irish farms with naturally occurring outbreaks of ovine abortion, toxoplasmosis and enzootic abortion of ewes (EAE) accounted for 14% and 20% of the farms, respectively, while on 6% of the farms, there was evidence of dual infection. When standard diagnostic techniques including histopathological examination, serological analysis, chlamydial antigen detection, and bacteriological culture, were used on samples from the same farms, toxoplasmosis was diagnosed in 17% of farms, and EAE in 12%; dual infection was diagnosed on 3% of the farms. In general, good agreement was found between the PCR and the standard methods. The duplex real-time PCR assay developed in this study has proved to be a very sensitive and rapid tool that might provide a valuable addition to the methods currently available for routine diagnosis of ovine abortions.

A prospective study of sheep and goat abortion using real-time polymerase chain reaction and cut point estimation shows Coxiella burnetii and Chlamydophila abortus infection concurrently with other major pathogens

“…The study had similar findings to the present study in that C. abortus and T. gondii were commonly identified with other pathogens in the same abortion case. 12 Campylobacter spp. was only identified in sheep, and several submissions were from the same farm.…”

Influence of the gestational stage on the clinical course, lesional development and parasite distribution in experimental ovine neosporosis

“…Placenta and foetal brain and liver samples that had tested positive by nested-PCR were adjusted to 20 ng DNA/μL and the parasite load was quantified using real-time PCR. Primer pairs from the N. caninum Nc-5 sequence [ 25 ] were used for parasite quantification, and primers from the β-actin gene [ 26 ] were used for the quantification of host DNA. Amplification reactions were performed as described by Collantes-Fernández et al [ 25 ], in a final volume of 20 μL using Power SYBR® Green PCR Master Mix (Applied Biosystems, Foster City¸ CA, USA), 20 pmol of each primer and 100 ng of DNA in a ABI 7300 Real Time PCR System (Applied Biosystems).…”

Experimental ovine toxoplasmosis: influence of the gestational stage on the clinical course, lesion development and parasite distribution

“…Experimental infection of sheep with Neospora caninum has shown that there is a close relation between parasite burden and the severity of lesions identified in placenta or foetus, and how these parameters could affect the course of clinical disease [ 8 ]. The close relationship between the presence of T. gondii and the development of histological lesions has been shown through quantitative real time-PCR (qPCR), in sheep experimentally infected at mid gestation [ 9 ]. Whether there are differences in the quantity of parasite and the presence of lesions at different terms of gestation in ovine toxoplasmosis has not yet been investigated.…”