Bartonella henselae

Weber, Katie, Karnik, Dhruva & Brown, Lisa D., 2024, Transcriptional induction of the IMD signaling pathway and associated antibacterial activity in the digestive tract of cat fleas (CtenoCephalideS feliS), Parasites & Vectors (546) 17 (1), pp. 1-13 : 3

Quantification of Bartonella henselae by qPCR

Genomic DNA was extracted from bacterial cultures and individual fleas using the PureLink ™ Genomic DNA Mini Kit (Invitrogen ™) according to the manufacturer’s instructions. Bacterial cell lysates were generated from 10 μL of diluted B. henselae culture, and flea tissue lysates comprised the ground bodies of whole fleas as described above. Each gDNA extraction process utilized a negative environmental control (DNA extraction reagents without biological samples). All gDNA preparations were eluted in 50 μL of the PureLink ™ genomic elution buffer and were stored in a −20 °C freezer until use. Real-time qPCR assays were performed to quantify Bartonella gene copy numbers as described previously [ 23, 25, 26]. Briefly, serial dilutions of a plasmid containing a region of the B. henselae citrate synthase (gltA) gene were used to generate a standard curve. Each qPCR reaction included 2× PowerUp SYBR Green PCR Master Mix (Applied Biosystems ™), B. henselae gltA gene-specific primers, DNase/ RNase-free water, and either gDNA template (samples), water (negative control), or serial tenfold dilutions of the plasmid containing the B. henselae gltA gene (3.5 × 108 to 350 copies). Additionally, for flea tissue lysates, a portion of the flea 18S rDNA gene was amplified in each assay as a control to verify the integrity of the template DNA [ 27]. Te reactions were run on a QuantStudio ™ 3 real-time PCR system (Applied Biosystems ™), and quantities of the Bartonella gene copy numbers per sample were interpolated from the standard curves.