Table 1.
RT-qPCR primers designed to target the Leishmania sherp gene in this study.
Fig 1.
Detection and quantification of culture-derived Leishmania metacyclic promastigotes by RT-qPCR.
(A) Ten-fold serial dilutions of cDNA prepared from purified metacyclic promastigotes of stationary phase cultures were used to generate a standard curve for each target gene. Each symbol presents a sample ranging from 106 to 1 parasite equivalents for sherp, 18S rRNA, and kDNA RT-qPCR targets. The RT-qPCR was run employing a single plate in a single RT-qPCR reaction. (B–D) Cultured-derived L. (L.) major, L. (L.) infantum, and L. (L.) donovani non-metacyclic and infectious metacyclic promastigotes (106 parasite cells per lifecycle stage) were purified and assessed for sherp transcript expression using RT-qPCR. In (B-D) n=5 pooled individual experiments. **P=0.0079 employing a Mann-Whitney test.
Fig 2.
Detection and quantification of Leishmania metacyclic promastigotes in infected Lu. longipalpis sand fly midguts employing the sherp RT-qPCR.
(A) Relative expression levels of the sherp gene determined by RT-qPCR at early (day 4-), mid (day 7-), and late (day 14-) time points post-sand fly infection (p.i.) of dissected infected Lu. longipalpis midguts normalized to Leishmania-specific 18S gene expression and expressed as a fold increase over uninfected midguts (calibrator samples). (B) Total number of parasites per infected midgut calculated employing the sherp-RT-qPCR. (C) Total number of parasites per infected midgut calculated employing the kDNA-RT-qPCR. (D) Total number of metacyclic promastigotes per infected midgut as determined by microscopic examination versus parasite number as determined by sherp-RT-qPCR from the same fly and shown in (B). (E) Calculation of a metacyclogenesis score using the log transformed number of parasites in matched samples determined by the sherp divided by the number determined by the kDNA RT-qPCR. Each symbol represents 1 dissected sand fly midgut. Asterisks indicate values that are statistically significant using a non-parametric Kruskall-Wallis test and a Dunn’s multiple comparison post-test (Panels A-C, E) or Multiple Unpaired t-tests with Welch correction (Panel D) comparing microscopy versus sherp PCR.
Fig 3.
Screening of experimentally infected ethanol-fixed whole Lu. longipalpis sand fly samples for sherp gene expression or kDNA by RT-qPCR.
(A) Expression levels of the sherp gene from ethanol-fixed sand fly samples on days 4-, 7-, 14-, and 21 p.i. The day 21 samples were flies used for experimental parasite transmission to naïve mice on day 14 p.i. (see Fig 4), as indicated by the dotted line. (B and C) The total number of parasites per infected sand fly employing the sherp-RT-qPCR (B) or kDNA-RT-qPCR (C). (D) Calculation of a metacyclogenesis score using the log transformed number of parasites in matched samples determined by the sherp divided by the number determined by the kDNA RT-qPCR. (E) Comparison of sample preservation in absolute ethanol medium at room temperature for 2- versus 14- months employing the sherp (left panel) or kDNA (right panel) RT-qPCR. Asterisks indicate values that are statistically significant using a non-parametric Kruskall-Wallis test and a Dunn’s multiple comparison post-test (Panels A-D) or Multiple Unpaired t-tests with Welch correction (Panel E) comparing 2- versus 14- months. In panels A-C, only significant differences are shown.
Table 2.
Summary of sand flies screened by sherp- and kDNA-RT-qPCR.
Fig 4.
Detection and quantification of L.
(L.) major parasites in infected mice employing sherp- or kDNA-RT-qPCR. Naïve female C57BL/6J (WT) mice were needled inoculated with 2 x 105 L. (L.) major in the ear dermis (48 hr. data) or exposed to the bites of L. major infected Lu. longipalpis sand flies (8-week data, see Materials and Methods). (A) Mean ear lesion area (mm2) following infected sand fly exposure, n=8 ears. (B and D) sherp mRNA relative gene expression levels determined by RT-qPCR in ears at 8 weeks (B) and 48 hours (D) p.i., n=8 ears/group. (C and E) The total number of parasites per infected sand fly calculated employing the sherp- or total number of parasites per infected sand fly calculated employing the kDNA- RT-qPCR at 8 wks (C) or 48 hrs. (E) p.i.. Asterisks indicate statistically significant values, *P=0.014 and ***p=0.0002, n=8, employing a Mann-Whitney test.