Passive immunization does not provide protection against experimental infection with Mycoplasma haemofelis

Sugiarto, Sarah; Spiri, Andrea M.; Riond, Barbara; Novacco, Marilisa; Oestmann, Angelina; de Miranda, Luisa H. Monteiro; Meli, Marina L.; Boretti, Felicitas S.; Hofmann-Lehmann, Regina; Willi, Barbara

Quantification of Mhf blood loads by qPCR was performed on day 0, twice weekly pi until week 7 and weekly thereafter up to day 100 pi. Total nucleic acid (TNA) was extracted from 100 μL of EDTA anti-coagulated blood using the MagNa Pure LC (Roche Diagnostics AG, Rotkreuz, Switzerland) and the MagNa Pure LC TNA Isolation Kit (Roche Diagnostics) following the manufacturer's instructions. TNA was eluted in 100 µL elution buffer and stored at −80 °C until use. With each batch of extraction, a negative control consisting of 200 µL of phosphate-buffered saline (PBS) was used to monitor for cross-contamination. All TNA samples were tested with a Mhf-specific qPCR assay to detect and quantify Mhf as previously described [12] . For absolute quantification, tenfold serial dilutions of a standard plasmid were used as described [12] . Positive and negative controls were included in each PCR run.