Involvement of Insects in the Dissemination of Cryptosporidium in the Environment
Cryptosporidium is a protozoan pathogen that cause prolonged diarrhea in host mammals. In humans, children and immunocompromised people are mainly susceptible. Oocysts are the infective stages responsible for transmission of the Cryptosporidium organisms and infected hosts with active diarrhea are particularly involved in the environment contamination.
Synanthropic insects, such as houseflies are recognized as transport hosts for a variety of infectious micro-organisms or viral pathogens for humans. There is a phoretic association between some insects and Cryptosporidium [3]. Flies are thus excellent carriers of oocysts on their body surfaces as well as in their gut [2]. In our investigations, the sheep blowfly Lucilia sericata (Diptera: Calliphoridae) was chosen as a model for the transport of Cryptosporidium. The objective was to know if the larval stages of the flies breeding or having access to C. parvwm-contaminatedmeat, will mechanically carry the C. parvum oocysts in their digestive tracts.
MATERIALS AND METHODS
Experimental model. Approximately 100 eggs of the fly Lucilia sericata were placed on 200 g of minced beef meat, contaminated with 106 oocysts. First instar maggots (L1) were collected after 10 h, washed, ground and analyzed for detecting the presence of C. parvum oocysts in their gastrointestinal tracts by microscopy and nested-PCR. Individuals of other larval stages (L2 and L3 respectively at 18 h and 24 h), post-feeding larvae (48 h), pupae (5 days) and adults (10 days) were also examined.
Immunofluorescence assay (IFA) and Modified Ziehl-Neelsen staining (MZN). All samples were microscopically examined on smears of larvae or adult insect extracts, after staining with MZN or IFA by using the Crypto/Giardia-Cel Test IF kit (Cellabs, Biomedical Diagnostics, France).
DNA extraction and nested-PCR. Genomic DNA was extracted by phenol/chloroform extraction. The primers described by Xiao et al. were used to amplify a 830pb portion of the 18S rDNA gene [5].
RESULTS AND DISCUSSION
Exposure of L sericata eggs to beef meat contaminated with oocysts of C. parvum resulted in ingestion of the oocysts by the maggots. Oocysts were detected by IFA and nested-PCR in the digestive tract only in stages L2 and L3. In the other stages, we were unable to detect any oocysts nor parasite DNA in the samples.
Our results indicated that oocysts passed through the mouthparts and gastrointestinal tracts of the maggots. However, we don't know if they are viable or not. In a recent study, Escherichia coli was used to investigate the fate of bacteria in the alimentary tract of sterile grown maggots of L. sericata. It has been shown that they were mainly destroyed in the midgut of the maggots, indicating that the faeces were either sterile or contained only small numbers of bacteria [4]. It is really important to establish if Cryptosporidium can survive in the digestive tract of flies and remains infectious because the insect faeces can be deposited on human skin, food, and household surfaces. Thus, strongly immunocompromised people could be infected by a small amount of oocysts transported by flies. We know that the mean number of oocysts, which can be carried by a wild filth fly, was experimentally estimated from 4 to 131 [1]. This publication insects could carry oocysts for at least 3 weeks and these oocysts are still infectious for mice [1].
The absence of parasite DNA in stages after L3 could be in part explained by the low inoculum we used (5 × 104 oocysts/g compared to 106 oocysts/g of faecal material which is currently observed in the case of bovine cryptosporidiosis). In order to verify this hypothesis, a further experiment will be performed with 107 oocysts/g of meat. Moreover, the larval stages placed on contaminated meat will be respectively L1, L2 and L3.
The fate of C parvum oocysts ingested by dung beetles and the possible role these coprophagous insects serve in the dissemination of Cryptosporidium were also considered in the literature [3]. A prolonged presence of Cryptosporidium-infected faecal material on soil may permit the dispersion of oocysts by flies as well as by coprophagous insects. These insects could also be integrated into the food chain by insectivorous animals. In conclusion, coprophagous and necrophagous insects can acquire infectious Cryptosporidium oocysts from unsanitary sites in contact with infected water or faecal material and then deposit these oocysts on visited surfaces. Therefore, they may be involved in the dissemination of Cryptosporidium.
ACKNOWLEDGMENTS
AFD was supported by a grant (a campaign named Ensemble, bdtissons demain) from the Catholic University of Lille, France. This work was also supported by funds from VIH-PAL, an ANRS/IRD projects.
