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Table 2 Experimental conditions and results of D. variabilis subolesin RNAi and CF with different pathogens.

From: Subolesin expression in response to pathogen infection in ticks

Pathogen
(isolate/strain)
Inoculum CF tickmeal Subolesin expression silencing
(%)a
Tick infection ratio
(Subolesin/Rs86)b
A. marginale
(Oklahoma, OK [33])
4.3% infected erythrocytes Blood from splenectomized calves experimentally infected with isolate stabilate 89 ± 17* 0.85 ± 0.09*
A. marginale
(Okeechobee, FL [33])
3.3% infected erythrocytes Blood from splenectomized calves experimentally infected with isolate stabilate 55 ± 32* 0.83 ± 0.10*
A. marginale
(Bison) [33]
7.4% infected erythrocytes Blood from splenectomized calves experimentally infected with isolate stabilate 86 ± 17* 0.95 ± 0.10*
A. phagocytophilum
(NY18) [34]
50% infected cells ISE6 cultured tick cells in L15B with 10% FBS 92 ± 14* 0.91 ± 0.09*
F. tularensis
(Live Vaccine Strain LVS; ATCC 29684)
107 CFU/ml DMEM with 10% FBS 99 ± 2* 1.74 ± 0.86*
E. canis
(Ebony)
2% infected cells DH82 cultured dog cells in DMEM with 10% FBS 94 ± 11* 0.89 ± 0.16*
E. coli
(JM109; Promega)
107 CFU/ml DMEM with 10% FBS 97 ± 3* 0.92 ± 0.07*
B. subtilis
(culture 125-1 kindly supplied by H. Evers)
107 CFU/ml DMEM with 10% FBS 71 ± 21* 0.65 ± 0.58
P. pastoris
(X33; Invitrogen)
106 CFU/ml YPD 80 ± 16* 0.60 ± 0.31
  1. aSubolesin mRNA levels were determined by real-time RT-PCR and normalized against tick 16S rRNA using the comparative Ct method. Percent subolesin expression silencing was calculated in subolesin dsRNA-injected ticks with respect to control ticks injected with the unrelated Rs86 dsRNA and expressed as average ± SD. Subolesin normalized Ct values were compared between subolesin dsRNA and control Rs86 dsRNA injected ticks by Student's t-test (*P < 0.05).
  2. bInfection levels were determined by real-time PCR using pathogen-specific gene sequences and normalizing against tick 16S rRNA using the comparative Ct method. Tick infection ratio was calculated as subolesin dsRNA to average control Rs86 dsRNA injected ticks normalized Ct values and expressed as average ± SD. Pathogen-specific gene normalized Ct values were compared between subolesin dsRNA and control Rs86 dsRNA injected ticks by Student's t-test (*P < 0.05).
  3. Abbreviations: CF, capillary feeding; CFU, colony forming units; L15B, modification of Leibovitz's L15 medium containing additional glucose, amino acids, vitamins and trace minerals (Sigma-Aldrich, St Louis, MO, USA); FBS, fetal bovine serum (Sigma); DMEM, Dulbecco's Modified Eagle Medium (Gibco, Invitrogen, Carlsbad, CA, USA); YPD, Yeast Extract Peptone Dextrose medium (10 g/l yeast extract, 20 g/l peptone, 20 g/l glucose) (Sigma).