Studies on the novel detection and destruction of the enteroparasite Giardia lamblia and other veterinary problematic microorganisms examined under varying culture conditions
Aims & Rationale: This study focuses on the hypothesis that every pet entering a hospital environment runs the potential risk of contracting a nosocomial infection. Giardia lamblia (G. lamblia) is a zoonotic unicellular flagellated protozoan parasite that infects both human and non-human mammals. The presence of pathogenic organisms in veterinary settings is a public health concern in relation to human and animal exposure. Veterinary clinics represent a significant risk factor for the zoonotic transfer of pathogens especially in cases of wound infection and the shedding of faecal matter. This study aims to provide a means of detecting veterinary relevant parasite species in bacterial biofilms, and to provide a means of disinfecting these parasites and biofilms. This study aims to breach the testing void for G. lamblia that currently exists in Ireland by using a combined in vitro HCT-8 cell culture-quantitative PCR assay for evaluating the efficacy of using pulsed UV light for treating G. lamblia parasites. This study aims to provide a source of data into novel disinfection mechanisms such as Pulsed UV light technologies as a potential clinical application in veterinary practice. Methods & Results: Biofilms were grown on veterinary relevant surfaces, i.e. stainless steel and PVC coupons using a CDC biofilm reactor and treated using Pulsed UV light. Pulsed light successfully inactivated all test species (Listeria, Salmonella, Bacillus, Escherichia) in planktonic and biofilm form with an increase in inactivation for every increase in UV dose. Biofilms were also used as part of a parasite entrapment study for this project. Giardia lamblia was seeded in the biofilm reactor and disinfection studies were carried out. In order to independently define inactivation during testing of the novel disinfection methods an in vitro cell culture model was adapted and used. A real time PCR assay was utilized to detect parasite DNA in Bacillus cereus biofilms on stainless steel and PVC surfaces. Results show that Giardia attach to biofilms in large numbers (100-1000 cysts) in as little as 72 hours. Conclusion: This represents the first study on the use of a combined cell culture - real time PCR in vitro assay for the viability assessment of low-pressure and pulsed UV light treated Giardia lamblia cysts using human intestinal derived cell lines. It is envisioned that such an assay provides an alternative approach to that of in vivo testing by allowing for a rapid method of determining parasitic inactivation following UV and other disinfection processes. The observations from these findings further enhance the hypothesis that pulsed UV light would be an effective sterilization technique in a veterinary clinical setting once regular cleaning had taken place. Findings indicated that current Giardia detection methods are limited to using vital stains before and after cyst excystation are not appropriate for monitoring or evaluating cyst destruction post PUV-treatments. Use of the human ileocecal HCT-8 cell line was superior to that of the human colon Caco-2 cell line for in vitro culture and determining PUV sensitivity of treated cysts. The use of the in vitro HCT-8 cell culture assay may replace use of animal models for determining disinfection performances of PUV for treating G. lamblia. The extraction and amplification of the parasitic DNA via real time PCR provides a rapid measurement of infective parasite numbers allowing for the measurement of live or dead parasites.
National University of Ireland - Maynooth
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