Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based analysis of Giardia lamblia and Giardia muris.

IARDIA is the protozoan parasite that is the etiologic agentof giardiasis. This illness is the most common parasitic dis-ease and is estimated to infect at least 100,000 people each year inthe United States (Furness, Beach, and Roberts 2000). Symptomsof giardiasis range from asymptomatic to severe abdominal pain,chronic diarrhea, andin rarecases,death, withyoung children andimmunocompromised individuals being at the greatest risk of ser-ious illness (Adam 1991; Upcroft and Upcroft 2001). Infectiontypically occurs through the fecal-oral route and has been docu-mented to be associated with many waterborne disease outbreaksworldwide. A majority of these outbreaks occurred due to con-tamination of the drinking water supplies with untreated sewage.There are at least six species of Giardia including Giardia lam-blia (also known as Giardia duodenalis or Giardia intestinalis),which can infect a wide range of hosts including humans (Caccioetal.2005).Thecurrentdetectionmethodemployedtomonitorthepresence of Giardia cysts in surface and drinking water relies pri-marily on microscopic techniquesthat detect thepresenceof Giar-dia cysts in the sample, but the method is not species specific nordoes it determine cyst viability (U.S. Environmental ProtectionAgency 1999). More recently, a PCR-based genotyping tool hasbeen developed and used to identify the different Giardia spp.present in environmental water (Sulaiman et al. 2004). This tech-nique however can be prone to contamination and thus other al-ternative approaches are currently being explored. In particular,matrix-assisted laser desorption/ionization time-of-ﬂight massspectrometry (MALDI-TOF MS) has been used to identify andclassify bacteria (Donohue et al. 2006) and parasites (Magnuson,Owens,andKelty2000;Mouraetal.2003),althoughthisapproachhas not been applied to study Giardia spp.This manuscript describes a MALDI-TOF MS-based approachthat is used to characterize the mass spectral ﬁngerprints of intactG. lambliaandGiardiamuriscysts.Thisstudy identiﬁed commonmass spectral peaks shared by the two species as well as peaksspecific to G. lamblia and others specific to G. muris, which areuseful in differentiating the two organisms. Additional analysesrevealed that the mass spectral proﬁles of intact cysts consistedpartly of peaks representing trophozoite-derived proteins, basedon comparison with puriﬁed trophozoites. These results suggestthe potential application of intact cell MALDI-TOF MS as an al-ternative high throughput approach for species identiﬁcation ofGiardia spp.MATERIALS AND METHODSCyst propagation and puriﬁcation. Giardia lamblia (H3strain; assemblage B) and G. muris (obtained from Drs. ErikHewlett and John Andrews, Case Western Reserve School ofMedicine, Cleveland, OH) cysts were propagated using Mongo-lian gerbils or CF-1 mice, respectively (Jackson Laboratories, BarHarbor, ME). Feces from infected rodents were collected andcysts were harvested and puriﬁed using a sucrose/percoll (Sigma,St. Louis, MO) gradient (Hayes et al. 2003). Puriﬁed cysts used inall experiments were 7 days from the time of collection.In vitro excystation and trophozoite puriﬁcation. The invitro excystation of cysts was performed as described (Rice andSchaefer 1981) and efﬁciency of excystation was determinedmicroscopically to routinely be 90% excysted. Following ex-cystation, trophozoite/cyst mixtures were labeled with FITC-con-jugated anti-Giardia antibody (Giardi-a-Glo