House fly (Musca domestica) as a transport vector of Giardia lamblia

Abstract

The role of arthropods in the indirect transmission of giardiasis has been long suspected but little investigated. A polymerase chain reaction (PCR) assay specific for a heatshock protein (HSP) gene of Giardia lamblia (Lambl, 1859) was used for the direct detection of G. lamblia DNA in house flies (Musca domestica L.) captured in two towns in Aragon (Spain). Six hundred flies were captured and clustered in groups of ten, and each group was processed separately. An amplification product of the expected size (163 bp) was detected in 13 of the 60 groups (22%). Giardia lamblia (Lambl, 1859) is an enteric parasitic protozoon from humans and animals, and is a common cause of parasitic infections in humans in Spain and other countries (Jarabo M.T., Garcia-Moran N.P., Garcia-Moran J.L. 1995: Enferm. Infecc. Microbiol. Clin. 13: 464-468; RodriguezHernandez J., Canut-Blasco A., Martin-Sanchez A.M. 1996: Eur. J. Epidemiol. 12: 291-295; Del Aguila C., Navajas R., Gurbindo D., Ramos J.T., Mellado M.J., Fenoy S., Munoz Fernandez M.A., Subirats M., Ruiz J., Pieniazek N.J. 1997: J. Euk. Microbiol. 44: 84S-85S). Numerous waterborne outbreaks of giardiasis have been documented (Kirner J.C., Littler J.D., Angelo L.A. 1978: J. Am. Water Works Assoc. 70: 3540; Dykes A.C., Juranek D.D., Lorenz R.A., Sinclair S.P., Jakubowski W., Davies R.B. 1980: Ann. Intern. Med. 92: 165170; Hibler C.P. 1988: In: P.M. Wallis and B.R. Hammond (Eds.), Advances in Giardia Research, University of Calgary Press, Calgary, pp. 197-204; Kent G.P., Greenspan J.R., Herndon J.L., Mofenson L.M., Harris J.A., Eng T.R., Waskin H.A. 1988: Am. J. Public Health 78: 139-143), but the role of other indirect transmission mechanisms (for example arthropods) has not been extensively studied. Giardia cysts have been found in flies (Khan A.R., Huq F. 1978: Bangladesh Med. Res. Counc. Bull. 4: 86-93; Sterling C.R., Miranda E., Gilman R.H. 1987: Am. J. Trop. Med. Hyg. 36, Suppl.: 233) and cockroaches (Kasprzak W., Majewska A. 1981: Wiad. Parazytol. 27: 555-563). Given the low infective dose of Giardia cysts (Rendtorff R.C. 1954: Am. J. Hyg. 59: 209-220), arthropods, and especially flies, could be an important vehicle between faeces and food; promiscuouslanding synanthropic flies are recognised as transport hosts for a variety of protozoan and metazoan parasites in addition to viral and bacterial pathogens of public health importance (Graczyk T.K., Cranfield M.R., Fayer R., Bixler H. 1999: Am. J. Trop. Med. Hyg. 61: 500-504). It was the purpose of this work to detect possible Giardia lamblia cysts naturally occurring in house flies in our geographical area. In July and August, 1998, twenty adhesive fly traps were set outdoors in two towns in Aragon (north-eastern Spain) where cattle and sheep are raised. House flies (Musca domestica L.) captured in them were selected and crushed in a mortar in groups of ten; each group was resuspended separately in 4.5 ml of PBS. Approximately 200 μl of the suspension, 900 μl of L6 lysis buffer (10 M guanidinium thiocyanate, 0.1 M Tris-hydrocholoride pH 6.4, 35 mM EDTA pH 8, and 2.6% (w/v) Triton X-100) (Boom R., Sol C.J.A., Salimans M.M.M., Jansen C.L., Wertheim-van Dillen P.M.E., van der Noordaa J. 1990: J. Clin. Microbiol. 28: 495-503), 60 μl of isoamyl alcohol and 0.5 g of 0.5 mm diameter zirconia/silica beads (Biospec Products, Inc., Bartlesville, Oklahoma) were added into 1.9 ml Eppendorf tubes. The tubes were then shaken in a MiniBeadbeater (Biospec Products, Inc.) for 2 min at maximum speed, left at room temperature for 5 min and centrifuged at 12,000 g for 15 s. The supernatant was transferred to another tube, to which 40 μl of coarse activated silica suspension (Boom et al. 1990, op. cit.) were added. The suspension was then mixed in a Vortex mixer and allowed to stand at room temperature for 10 min and, after centrifugation (as above), the supernatant was discarded. The pellet was washed by centrifugation, twice with 200 μl of L2 washing buffer (10 M guanidinium thiocyanate, 0.1 M Trishydrochloride) (Boom et al. 1990, op. cit.), twice with 200 μl of ice-cold 80% ethanol, and once with 200 μl of acetone. The acetone was discarded and the pellet dried at 55oC for 5 min; 100 μl of water were added and, after vortexing briefly, the tube was incubated at 55oC for 5 min. It was then centrifuged for 2 min at 12,000 g and the supernatant was transferred to another tube. In a second phase, the nucleic acid sample was treated with polyvinyl pyrrolidone (PVP) to remove potential inhibitors of the PCR (Young C.C., Burghoff R.L., Keim L.G., MinakBernero V., Lute J.R., Hinton S.M. 1993: Appl. Environ. Microbiol. 59: 1972-1974). A 50 μl aliquot of the extracted sample was added to 150 μl of PVP-TE (10% (w/v) PVP (Sigma) in TE buffer), mixed and incubated at room temperature for 10 min. The DNA was then selectively precipitated with isopropanol (Sambrook J., Maniatis T., Fritsch E.F. 1982: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, pp. E.10-E.15), 100 μl of 2.0 M ammonium acetate and 600 μl of isopropanol were added, mixed well and held at –20oC for 30 min. The tube was centrifuged (12,000 g for 10 min) and the supernatant fluid discarded carefully with a pipette. The precipitated DNA was dried with hot air and then reconstituted in 50 μl of TE buffer and stored at –20oC (Lawson A.J., Linton D., Stanley J., Owen R.J. 1997: J. Appl. Microbiol. 83: 375-380). The primers selected for the PCR were GLC-1 (5’-AGG GCT CCG GCA TAA CTT TCC-3’) and GLC-2 (5’-GTA TCT GTG ACC CGT CCG AG-3’) (Abbaszadegan M., Huber FOLIA PARASITOLOGICA 47: 330-331, 2000