Submitted to: American Association of Veterinary Parasitologists
Publication Type: Abstract Only
Publication Acceptance Date: May 7, 2008
Publication Date: July 19, 2008
Citation: Trout, J.M., Obrien, C.N., Miska, K.B., Schwarz, R.S., Jenkins, M.C. Cloning, expression, and characterization of Giardia duodenalis, beta-giardin. The American Association of Veterinary Parasitologists (AAVP) 53rd Annual Meeting, July 19-22, 2008, New Orleans, LA. No. 57. Technical Abstract: Giardiasis is frequently diagnosed in humans and is highly prevalent in many animal species as well. The cumulative prevalence of infection in dairy cattle frequently reaches 100%. The replicating trophozoite stage colonizes the upper small intestine and attaches to the surface epithelium via a ventral adhesive disk, whereas infective cysts are shed in the feces. A comparison of gene expression between the trophozoite and cyst stages was performed using suppressive subtractive hybridization. The cyst and trophozoite libraries generated by this protocol were used to construct cDNA libraries enriched for sequences present in cysts and trophozoites. Analysis of clones revealed the presence of a gene encoding beta-giardin, one of the components of the ventral adhesive disk. Real-Time RT-PCR showed consistently greater beta-giardin mRNA in the trophozoite stage, although there was signal in the cyst stage as well. The entire beta-giardin gene was cloned into pET28 vector as a polyHis fusion protein. A recombinant protein (rGd-bG) of approximately 36-37 kDa was expressed and purified by NiNTA affinity chromatography. Antisera was prepared by immunizing rabbits with the recombinant protein. In immunoblotting of SDS-PAGE fractionated recombinant or native protein, anti-rGd-bG sera identified both the recombinant 36-37 kDa protein and a 31-33 kDa native trophozoite protein. Immunofluorescent staining of cultured trophozoites with anti-rGd-bG sera revealed specific labeling of the ventral adhesive disc. Preliminary studies comparing the effects of anti-rGd-bG antisera on cultured trophozoites indicates that treatment with anti-Gd-bG serum alters the morphology of trophozoites compared to treatment with pre-bleed or irrelevant antisera. This could indicate that binding of anti-rGd-bG antibodies interferes with the function of the ventral adhesive disk, which could result in a potential control strategy for Giardia infections.