Leishmania macropodum

Panahi, Elina, Shivas, Martin, Hall-Mendelin, Sonja, Kurucz, Nina, Rudd, Penny A., Araujo, Rachel De, Skinner, Eloise B., Melville, Lorna & Herrero, Lara J., 2020, Utilising a novel surveillance system to investigate species of Forcipomyia (Lasiohelea) (Diptera: Ceratopogonidae) as the suspected vectors of Leishmania macropodum (Kinetoplastida: Trypanosomatidae) in the Darwin region of Australia, International Journal for Parasitology: Parasites and Wildlife 12, pp. 192-198 : 194-195

3.2. Detection of L. macropodum in F. (Lasiohelea)

To confirm ongoing Leishmania circulation at the TWP site, 123 pools (a total of 536 individuals) and 47 individual F. (Lasiohelea) were screened for L. macropodum by real-time qPCR. 14/123 pools and 1/47 individual F. (Lasiohelea) were positive for L. macropodum DNA. High parasitemia was detected in several pools and individual F. (Lasiohelea) with ≥ 5 × 10 6 parasites ( Fig. 2 ). Moreover, non-destructive DNA extractions from field-collected F. (Lasiohelea) (n = 50) species confirmed L. macropodum DNA in specimens identified as F. (Lasiohelea) sp.1 (n = 2/39; Table 1).

In addition to the steady-state detection of L. macropodum in randomly selected F. (Lasiohelea) species, we further examined the possibility of biting midges to sustain infection and support a successful development of L. macropodum . Towards this, the presence of L. macropodum DNA by qPCR was performed over an 8-day period post insect field-collection. Our data shows that parasite DNA was detected up to 7 days ( Table 2) suggesting possible parasite development beyond the blood meal stage (day 2–3).

3.3. Detection of L. macropodum on FTA Ṝ cards after exposure to field-collected F. (Lasiohelea)

Stability of L. macropodum DNA on FTAṜ cards was assessed over a 10-week time course by qPCR. Results are shown for cards inoculated with 10 6 parasites per card over the 10-week period ( Fig. 3 ). A two-way ANOVA with a post Tukey's comparison test confirmed the detected parasite load over the 10 weeks were not significantly different. Furthermore, the use of honey was not found to be associated with any interference of parasite load detection or the assay's sensitivity.

From the field experiments, 145 FTAṜ cards were screened for L. macropodum DNA by qPCR within 60 days' post-exposure to field collected F. (Lasiohelea). Due to the insect's small size, a high proportion of F. (Lasiohelea) became stuck in the coated honey, and prior to screening with qPCR 44/145 cards (30%) had insects removed. Overall, 7/145 (4.83%) of FTAṜ cards were detected positive for L. macropodum DNA when screened with (n = 4/44) and without (3/101) adhered F. (Lasiohelea) ( Fig. 4 ).

Figures

Fig. 2. Leishmania macropodum DNA detection by qPCR. Individual or pools of F. (Lasiohelea) species were assessed for the presence of L. macropodum DNA. Only positive samples are shown, with each pair of columns representing results from one sample. Black columns depict the parasitic load detected and the dashed columns show the number of insects processed in that sample. Asterisks represent groups that contained ≥ 5 × 106 F. (Lasiohelea) parasites.

Fig. 3. Evaluation of L. macropodum viability on FTAṜ cards and the effect of honey. To determine L. macropodum detection on honeycoated and plain FTAṜ cards, an experiment was designed over a 10-week time course. FTAṜ cards (2.5 × 2.5 cm) were inoculated with 10 6 L. macropodum per card in triplicates and DNA was tested at five time points (week 0, 2, 5, 8 and 10).

Fig. 4. Assessment of L. macropodum DNA using FTAṜ card technology. FTAṜ cards were exposed to field-collected F. (Lasiohelea). Cards with and without insects adhered were processed and parasite load was determined with qPCR. 4.83% (7/145) FTAṜ cards were positive for L. macropodum DNA. Black columns show parasite load detected on each positive FTAṜ card (numbered FTA.1 – FTA.7). Dashed columns show the number of insects adhered to each card. When dashed columns are absent, this signifies the absence of insects on the positive cards.