Comparative evaluation of the sensitivity of LAMP, PCR and in vitro culture methods for the diagnosis of equine piroplasmosis

Abstract

The sensitivity of LAMP, PCR and in vitro culture methods for the detection of Theileria equi and Babesia caballi was evaluated using tenfold serially diluted culture parasites. On day 1 post-culture, both T. equi and B. caballi parasites could only be observed at 1% parasite dilution from the in vitro culture method, whereas LAMP could detect up to 1 x 10(-3)% of both T. equi and B. caballi parasite dilutions, whilst PCR could detect 1 x 10(-3)% T. equi and 1 x 10(-1)% B. caballi parasite dilutions. On day 7 post-culture, the detection limit for T. equi and B. caballi in the in vitro culture increased up to 1 x 10(-6)%, whereas LAMP detection limit increased to 1 x 10(-10)% for both parasites, whilst the PCR detection limit increased to 1 x 10(-10)% and 1 x 10(-6)% for T. equi and B. caballi, respectively. Furthermore, LAMP and PCR amplified the T. equi DNA extracted from the organs of an experimentally infected horse. This study further validates LAMP as an alternative molecular diagnostic tool, which can be used in the diagnosis of early infections of equine piroplasmosis and together with PCR can also be used as supplementary methods during post-mortems.

Loop-mediated isothermal amplification assay for the detection of Ehrlichia canis DNA in blood samples from dogs

“…The sensitivity of the assay, which was established using 10-fold serial dilutions of plasmid DNA containing a fragment of the groESL operon, was 1 copy of plasmid DNA. This sensitivity can be compared to that reported for other LAMP assays developed for Anaplasmataceae (Ma et al 2011, Nakao et al 2010 and other studies have demonstrated the higher sensitivity of the method when compared to PCR (Alhassan et al 2007, Cho et al 2006, Ma et al 2011, Matovu et al 2010.…”

Detection of Theileria equi in spleen and blood of asymptomatic piroplasm carrier horses

“…During the clinical phase of the disease, the parasites may reach several tissues of the host. In fact, in experimentally infected horses, T. equi DNA was detected in the liver, spleen, groin lymph node, lungs, heart, bone marrow, and brain (Alhassan et al 2007a). Recent studies have been shown that bone marrow may be a potential reservoir site of T. equi and B. caballi in clinically healthy horses (Alhassan et al 2007a;Pitel et al 2010).…”

“…In fact, in experimentally infected horses, T. equi DNA was detected in the liver, spleen, groin lymph node, lungs, heart, bone marrow, and brain (Alhassan et al 2007a). Recent studies have been shown that bone marrow may be a potential reservoir site of T. equi and B. caballi in clinically healthy horses (Alhassan et al 2007a;Pitel et al 2010). In our study, piroplasms were found in the spleen of asymptomatic horses, and this may be an important tissue supporting the persistence of the parasite in the organism.…”

“…However, because we just tested the spleen it is possible that the parasite might be present in other organs like the results of Alhassan et al (2007a) in experimentally infected horses. However, Babesia canis and Babesia bovis parasitized erythrocytes are able to escape the passage through the spleen (Allsopp et al 1994;Mehlhorn and Schein 1998;Schetters et al 1998;Botteon et al 2005;Carcy et al 2006).…”

“…It was determined that in some *Corresponding author: benito.blanco@pq.cnpq.br Theileria equi in spleen of horses 219 horses, T. equi and B. caballi can persist in bone marrow (Pitel et al 2010). Furthermore, T. equi was detected in the liver, spleen, groin lymph node, lung, heart, bone marrow and brain of experimentally infected horses (Alhassan et al 2007a). However, it has not been determined whether horses with piroplasm in tissues always present with parasitemia.…”