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 : 193

2.1. Cultivating L. macropodum

Leishmania macropodum parasites had previously been isolated from clinical infected macropod species at the Territory Wildlife Park (TWP). Skin lesions had been grown in biphasic Novy-MacNeal-Nicolle (NNN) medium and incubated at 26 ̊C for promastigote growth ( Dougall et al., 2009). To obtain an on-going in vitro culture, promastigotes were cultivated in Grace's Insect medium (Invitrogen, Australia), containing 20% heat-inactivated foetal bovine serum, 2 mM L-glutamine, 100 U/ mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich) at 26 ̊C.

2.2. FTA Ṝ card preparation

FTAṜ cards (2.5 cm × 2.5 cm) were coated with honey 24-48 h prior to use. In order to identify which insects had fed on the coated FTAṜ cards, blue food dye was added to the honey ( Hall-Mendelin et al., 2010). Waxed-paper containers were converted to contain field-collected biting insects and to hold a FTAṜ card ( Fig. 1 ). A total of 145 waxed-paper containers were set up, each holding one honey-coated FTAṜ card.

Figures

Fig. 1. Wax-paper cups were used to contain and maintain field-collected biting midges. (A) Honey-coated FTAṜ cards were left at room temperature for 48 h allowing even absorption of honey into the cards. (B) A 2.5 cm slit was carved into the bottom of disposable cup and sealed with adhesive tape. (C) Insects were aspirated directly into the bottom of the containers through a small perforation created before field collection. Once biting midges were collected from the macropods, the small perforation was sealed with a rubber plug. Gauze was used as a lid to seal the top of the containers and fastened securely with a rubber band. The honey-coated FTAṜ card was inserted through the bottom slit after insect collection and once again sealed with adhesive tape.