In vitro culture, PCR , and nested PCR for the detection of Theileria equi in horses submitted to exercise

Abstract

This study compared the usefulness of in vitro culture, PCR, and nested PCR for the diagnosis of Theileria equi in horses submitted to stress during exercise. Blood samples from 15 apparently healthy horses, previously conditioned to a high-speed equine treadmill, were taken prior to and after exercise. The animals were divided into two experimental groups: 30-day training schedule (G1) and 90-day training schedule (G2). Statistical analysis was performed using a chi-square test and kappa statistic was used in order to assess agreement. No significant difference was observed between samples collected at resting or after exercise. In G1, merozoites of T. equi were detected in the blood smears of four horses before in vitro culture, whereas 14 samples were positive, confirmed by culture. In G2, five and 11 horses were positive before and after culture, respectively. No PCR amplified product was observed in any of the tested animals although the PCR system based on the 16S rRNA gene of T. equi detected DNA in blood with an equivalent 8x10-5% parasitaemia. The nested PCR based on the T. equi merozoite antigen gene (EMA-1) allowed the visualization of amplified products in all the horses. Therefore, nested PCR should be considered as a means of detection of sub-clinical T. equi infections and in vitro culture could be used as a complement to other methods of diagnosis.

Serologic evidence of equine granulocytic anaplasmosis in horses from central West Brazil

“…The nPCR for the diagnosis of T. equi were performed as previously described by Baldani et al (2008).…”

Occurrence of Theileria equi in horses raised in the Jaboticabal microregion, São Paulo State, Brazil

“…DNA concentrations were determined using the NanoDrop ND-1000 spectrometer (NanoDrop Technologies, DE, USA). Nested PCR assays were performed as described previously by Nicolaiewsky et al (2001) and Baldani et al (2008). The primers sets EMAE-F (5'-CCGCCCTTCACCTCGTTCTCAA-3')/EMAE-R (5'-TCTCGGCGGCATCCTTGACCTC-3') and EMAI-F (5'-CCGTCTCCGTTGACTTGGCCG-3')/EMAI-R (5'-GGACGCGCTTGCCTGGAGCCT-3') were chosen to flank 396 and 102 bp regions of the EMA-1 gene sequence (GenBank accession number L13784), respectively.…”

“…The assays are mainly based on ribosomal 18S RNA sequence or EMA-1 gene and have showed to have high sensitivity and specificity (BALDANI et al, 2008;CACCIÒ et al, 2000;FRITZ, 2010;HEIM et al, 2007;NICOLAIEWSKY et al, 2001;RAMPERSAD et al, 2003). Therefore, this direct diagnostic method has now been incorporated to routine diagnosis, especially in association with serological assays.…”

“…Another diagnostic technique available for equine piroplasmosis is in vitro blood culture, which has been used for the detection of carrier host animals. Although considered reliable and species-specific, it is a very time-consuming technique and unsuitable for routine diagnosis (BALDANI et al, 2008;HOLMAN et al, 1997). Therefore, the advantages and disadvantages of each test should be considered while choosing the best test for the diagnosis of equine piroplasmosis.…”

Prevalence of equine Piroplasmosis and its association with tick infestation in the State of São Paulo, Brazil

“…al., 1995;HEUCHERT et al, 1999;KERBER et al, 1999, CANOLA et al, 2007HEIM et al 2007;BALDANI et al, 2008). The severity of clinical signs is variable, and in many cases spontaneous recovery may occur without obvious clinical signs.…”