Analytical evaluation of an immunomagnetic separation PCR assay to detect pathogenic Leptospira in cattle urine samples obtained under field conditions

Abstract

Clinical manifestations of leptospirosis are diverse and very similar to other febrile diseases, hence early and accurate detection of subclinical infections is a key element in disease control. We evaluated immunomagnetic separation (IMS) capture technology coupled with a standard quantitative PCR (qPCR) system for the detection of pathogenic Leptospira in urine samples from 803 cows from dairy herds with a history of clinical cases of leptospirosis. The urine samples were first processed in a purification step, then subdivided into 2 subsamples, one that continued to DNA extraction and direct qPCR, and one that was pretreated by IMS before continuing to DNA extraction and qPCR. Overall, 133 of 803 (16.6%) samples were IMS-qPCR positive, whereas only 92 of 803 (11.5%) were positive when using direct qPCR. Statistically significant differences were observed between the mean estimated Leptospira load between the IMS-qPCR and the direct qPCR positive urine samples. The IMS-qPCR technology revealed a larger number of positive results and higher bacterial loads than direct qPCR. This difference is most likely the result of the high antigen-binding capacity and capture efficiency of the IMS system. The use of polyclonal antibodies produced by the inoculation of 3 synthetic peptides, which make up the extracellular regions of the LipL32 protein, provided a high detection capacity to the IMS-qPCR technique, resulting in performance superior to direct qPCR.

Cats shedding pathogenic Leptospira spp.—An underestimated zoonotic risk?

“…The urine sample aliquot was pre-treated with an IMS method published previously [ 28 ], followed by a DNA extraction purification and molecular confirmation qPCR. The IMS-qPCR consisted of 4 steps ( in silico peptide production; polyclonal antibody production; coating magnetic beads with polyclonal antibodies, and IMS–qPCR Leptospira spp.…”

“…With the reference of the molecular weight of the genome of Leptospira interrogans serovar Hardjo-prajitno (GenBank accession number EU357983.1) a standard curve for estimation of pathogenic Leptospira spp. numbers in urine using a Roche 2.0 real-time PCR, according to the following equation was established [ 30 , 28 ]: …”

Detection of pathogenic leptospira as a cause of abortion in cattle-observations on diagnosis

“…Urine samples were pretreated using an immunomagnetic separation (IMS) protocol coupled to real time PCR (qPCR), according to a published protocol (Tomckowiack et al, 2020). A 25 mL aliquot of each urine sample was centrifuged at 4,000 g for 15 min and the pellet was resuspended in 1 mL of phosphate buffered saline (PBS) [137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 (pH 7)] and then transferred to a 1.5 mL microcentrifuge tube and recentrifuged at 11,000 g for 5 min.…”

“…A 25 mL aliquot of each urine sample was centrifuged at 4,000 g for 15 min and the pellet was resuspended in 1 mL of phosphate buffered saline (PBS) [137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 (pH 7)] and then transferred to a 1.5 mL microcentrifuge tube and recentrifuged at 11,000 g for 5 min. Finally, the supernatant was discarded, the pellet was resuspended in 1 mL of PBS and a 100 μL aliquot was submitted to be used in the IMS protocol (Tomckowiack et al, 2020), before proceeding with DNA extraction by High Pure DNA Template Preparation Kit protocol (Roche, USA).…”

“…Pathogenic leptospira cell numbers (genome equivalents) detected by qPCR were estimated according to a published protocol used in Tomckowiack et al (2020), using the molecular weight of the genome of Leptospira interrogans serovar Hardjo type prajitno strain Hardjoprajitno (GenBank accession number EU357983.1) to establish a standard curve for the estimation of leptospira numbers by qPCR, according to a published algorithm (Dzieciol et al, 2010).…”