Sterilization of Chrysomya putoria (Insecta: Diptera: Calliphoridae) eggs for use in biotherapy

Abstract

Large-scale, quality-controlled laboratory production of fly larvae is needed for biotherapy. The objective of this study was to assess the action of glutaraldehyde on the sterilization of Chrysomya putoria eggs by applying pharmaceutical sterility tests. Egg masses with 0.600 g were divided into three parts of 0.200 g, the eggs were separated using sterile distilled water, and the suspensions obtained were mixed with activated 2% glutaraldehyde solution. After 15-min contact, the suspensions were filtered through Whatman filter paper, and the glutaraldehyde residue obtained in the filtrate was neutralized by rinsing with Tryptone Soy Broth. The treated eggs were placed aseptically on Petri dishes containing gauze moistened with sterile saline solution. About 10% of the sterilized mass was transferred to test tubes containing Tryptone Soy Broth and Fluid Thioglycollate Broth. The tubes were incubated, respectively, at 22.5 and 35.0°C for 14 d to verify egg mass sterility. The plates containing the rest of the eggs (90%) were sealed with plastic film and kept in a climatized chamber at 30°C/d, 28°C per night, 60 ± 10% relative humidity, and under a 12-h light period to assess insect viability and survival. Each experiment was carried out in triplicate using a biological class II safety cabinet. No change in color or turgidity was observed with the agent tested, proving the sterility of the product and that there was no trace of contamination. Forty larvae (in three replications) in the periods of 12, 24, and 48 h after sterilization, when transferred to diet, produced larvae, pupae, and total viability similar to the control (larvae without sterilization). However, for the 72-h treatment, larvae and total viability were significantly lower than for the other treatments. There was no significant difference for the pupal stage. The product tested was shown to be efficacious for use as a sterilizer of C. putoria eggs for all the parameters assessed.

Keywords: glutaraldehyde treatment; larval therapy; screw worm; sterility test; wound.

Fig. 1.

Demonstrative scheme of the sterilization process sequence: (a) egg mass on chicken gizzard protein diet; (b) egg mass transferred aseptically to a Petri dish for weighing and separating for disassociation; (c) Bactor Sterile monitor filter cup and 0.8 -µm Whatman filter paper with sterilized egg mass; (d) sample of sterilized egg mass transferred to test tube containing 10-ml TSB and FTB for the sterility test; (e) hatched larvae after 12 h transferred to chicken gizzard diet; and (f) larvae with normal development. Only the larva viability is shown in this study.

Fig. 2.

Mapa Mundi illustrating the countries where biotherapy is currently used: the United States, Canada, Mexico, Venezuela, Colombia, Chile, Norway, the United Kingdom, Israel, Germany, Sweden, Austria, Hungary, Switzerland, Belgium, the Ukraine, Australia, Thailand, Turkey, China, Russia, France, Spain, Indonesia, Egypt, South Africa, India, Poland, Estonia, Slovakia, and Belgium ( Dallavecchia et al . 2011 ).