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Our results were generally consistent with previously reported effects of Bcc strains on C. elegans [20], [21], [24] and were usually qualitatively consistent with assays in our liquid model (linear regression of [mean % mortality on PGS agar, 24 h] versus [mean nematode virulence index in liquid, 119 h], r2 = 0.65, F1,8 = 11.0, p = 0.016).
The virulence index was calculated as previously described [ 19] [virulence index=number of internalized parasites × number of infected macrophages/number of examined macrophages].
A similar virulence index was obtained from the weight, V W (E i H j ).
(b) Virulence index of Cavier et al.[ 23] ranges from 0 (avirulent) to 16 (maximum virulence).
Overall, the fly virulence index had the highest predictive power for mouse liver burden at day 7 p.i.
As a measure for virulence, we calculated a fly virulence index (FVI) of all mutants (supplementary material Table S1).
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This is reflected by the relative abundance of mutants with reduced virulence indices and unaltered in vitro growth.
Interestingly, the L. donovani r LdMAcP mRFP1 promastigotes generated in this study, showed significantly higher infectivity and virulence indexes than control parasites in the infection of J774 mouse macrophages highlighting thereby a role for LdMAcP in the parasite's virulence.
An ANOVA with the same factors was used to analyze the two virulence indices and the "generalist/specialist" factor did not have any significant effect neither on V S (F1,4 = 0.650, P = 0.422) nor on V W (F1,4 = 0.334, P = 0.565).
Looking at results from the mutants that showed large deviations between virulence indices in mouse and fly can give insight into differences between the models, as experienced by the fungus during the infection process.
At any given time point in any organ, the mean virulence indices of the mutants were found to be slightly lower than those of the wild type, indicating – as expected and similar to the fly model – that, whereas most individual mutants did not deviate much in fitness from the wild type, on average, gene deletions result in at least slight growth defects in the murine system (Fig. 2B).
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