Giardia duodenalis 5741 Infection is initiated by the ingestion of live infective cysts from contaminated water or food or through person to person transmission. When cysts reach the stomach, gastric acidity and intestinal proteases promote excystation. Newly emerging excyzoites immediately divide into two trophozoites that migrate to the duodenum and adhere to the intestinal mucosa by virtue of their ventral discs. Giardia trophozoites reproduce asexually by binary fission and gradually populate the host's intestinal lumen. Under the influence of bile salts, a number of trophozoites undergo differentiation resulting in cyst formation. Encystation of trophozoites represents an important step in the completion of the parasite's life cycle since it leads to the formation of the cyst structure, necessary for the transmission of the parasite from host to host. Infected humans are estimated to excrete between 150 and 20,000 cysts/g feces daily, whereas infected cattle can excrete up to 10^6 Giardia cysts/g feces. The quadrinucleate cysts are resistant to the external environment and can remain viable from days to months if kept wet (Abdul-Wahid and Faubert, 2007). Giardiasis (Beaver fever) Humoral immunity is believed to be important in elimination of Giardia trophozoites from the host intestine. Raised levels of serum and mucosal antibodies are found in experimentally infected animals and infected humans during the elimination phase of the Giardia parasite, and the host produces specific serum and mucosal antibodies directed against both surface and cytosolic antigens (Olson et al., 2000). Giardia infects humans, but is also one of the most common parasites infecting cats, dogs and birds. Mammalian hosts also include cows, beavers, deer, and sheep (Wiki: Giardia lamblia). Giardia lamblia (synonymous with Lamblia intestinalis and Giardia duodenalis) is a flagellated protozoan parasite that colonises and reproduces in the small intestine, causing giardiasis. The giardia parasite attaches to the epithelium by a ventral adhesive disc, and reproduces via binary fission[1]. Giardiasis does not spread via the bloodstream, nor does it spread to other parts of the gastro-intestinal tract, but remains confined to the lumen of the small intestine. Giardia trophozoites absorb their nutrients from the lumen of the small intestine, and are anaerobes. If the organism is split and stained, it has a very characteristic pattern that resembles a familiar "smiley face" symbol. Colonization of the gut results in inflammation and villous atrophy, reducing the gut's absorptive capability. In humans, infection is symptomatic only about 50% of the time, and protocol for treating asymptomatic individuals is controversial. Symptoms of infection include (in order of frequency) diarrhea, malaise, excessive gas (often flatulence or a foul or sulphuric-tasting belch, which has been known to be so nauseating in taste that it can cause the infected person to vomit), steatorrhoea (pale, foul smelling, greasy stools), epigastric pain, bloating, nausea, diminished interest in food, possible (but rare) vomiting which is often violent, and weight loss (Wiki: Giardia lamblia). Baboon Papio cynocephalus 9556 Bank vole Clethrionomys glareolus 447135 Bear Ursus americanus 9643 Birds Passeroidea 175121 Brown Trout Salmo trutta 8032 Buffalo Bison bison 9901 Carnivores Vulpes 9625 Cat Felis catus 9685 Catfishes Siluriformes 7995 Cattle Bos taurus 9913 Chicken Gallus gallus 9031 Chimpanzee Pan troglodytes 9598 chinchillas Chinchillidae 10150 Copper Pheasant Syrmaticus soemmerringii 9067 Deer Cervus elaphus 9860 Deer mouse Peromyscus maniculatus 10042 Dog Canis familiaris 9615 Ducks Anas 8835 Ferret Mustela putorius furo 9669 Fish Hyperotreti 117565 Gerbil Gerbillina 10045 Goat Capra hircus 9925 Gray wolf Canis lupus 9612 Guinea pig Cavia porcellus 10141 Hamster Mesocricetus auratus 10036 Horse Equus caballus 9796 Human Homo sapiens 9606 Macaque Macaca fascicularis 9541 Mongolian Gerbil Meriones unguiculatus 10047 Monkey Platyrrhini 9479 Mouse Mus musculus 10090 None None Parrot Psittacidae 9224 Pig Sus scrofa 9823 Rabbit Oryctolagus cuniculus 9986 Rainbow trout Oncorhynchus mykiss 8022 Rat Rattus 10114 Raven Corvus corax 56781 sei whale Balaenoptera borealis 9768 Sheep Ovis aries 9940 Squirrel Spermophilus richardsonii 37591 Tree shrew Tupaiidae 9393 Trouts, salmons & chars Salmoninae 504568 Turkey Meleagris gallopavo 9103 Vole Microtus ochrogaster 79684 Water buffalo Bubalus bubalis 391902 G. duodenalis DNA vaccine encoding CWP2 VO_0011550 DNA vaccine Research pCDNA3 mammalian expression vector Oral Oral G. duodenalis cyst wall protein-2 (CWP2) DNA vaccine construction The cwp2 open reading frame was amplified by PCR and sub-cloned between the EcoRI and NotI restriction sites in the multiple cloning site of pCDNA3. Both the empty and recombinant pCDNA3 plasmids were purified from transformed Escherichia coli DH5α using the Qiafilter plasmid purification kit from Qiagen (Ont., Canada) according to the manufacturer's instructions. The plasmids were transformed into the SL1655 restriction modified strain of S. typhimurium (kindly provided by Dr. Ken Sanderson; Salmonella genetic stock center, Calgary, Canada) to stabilize the plasmids in STM1, and then purified and re-introduced into the STM1 strain of S. typhimurium (kindly provided by Dr. Peter Smooker; RMIT University, Melbourne, Australia). All bacterial cultures were maintained in Lauria Bertani (LB) culture medium supplemented with ampicillin (150 μg per mL) (Abdul-Wahid and Faubert, 2007). BALB/c For oral DNA immunization, mid-log phase Salmonellae were collected by centrifugation and re-suspended in ice cold PBS. Mice were orally immunized with 300 μL containing 10^9 CFUs by gavage using a blunt-ended probe. The animals were immunized every 2 weeks for a total of three immunizations. This dose and regimen were used as they were found to stimulate the highest level of CWP2-specific immune response without causing any toxic side effects to the mice (Abdul-Wahid and Faubert, 2007). The gene encoding the full length cyst wall protein-2 (CWP2) from Giardia lamblia was subcloned into the pCDNA3 mammalian expression vector and stably introduced into S. typhimurium STM1. Challenge infection with live Giardia muris cysts revealed that mice receiving the CWP2-encoding DNA vaccine were able to reduce cyst shedding by approximately 60% compared to control mice (Abdul-Wahid and Faubert, 2007). DNA immunized and control mice (n = 12 mice per group) were challenged with 10^5 G. muris cysts a week following the last immunization (Abdul-Wahid and Faubert, 2007). Giardia Lamblia Killed Protozoa Vaccine (USDA: 18G5.00) Wyeth VO_0001750 Inactivated or "killed" vaccine Licensed USA Giardiavax Giardiavax Fort Dodge VO_0001008 Subunit vaccine Licensed Intramuscular injection (i.m.) Cultured trophozoites, reduces disease and cyst shedding(Olson et al., 2000) Intramuscular injection (i.m.) licensed Giardiasis human vaccine Generic Unknown VO_0001083 Inactivated or "killed" vaccine Licensed A generic representation of vaccines developed using inactivated Giardia lamblia organisms to prevent giardiasis (Beaver fever) infection in humans. These vaccines aim to elicit protective immunity without the risk of causing disease. CWP2 Giardia lamblia ATCC 50803 VO_0011080 5703171 159120052 GL50803_5435 AACB02000001 XP_001710242 50803 1061836 1062924 - 8.13 37700.21 362 >NW_002477099.1:1061836-1062924 Giardia lamblia ATCC 50803 SC_592, whole genome shotgun sequence TTCACCTTCTGCGGACAATAGGCTTGTTCGTGCTGCGAGGCAGAGCTCTCACGGGTGCACGCTTCGATTG GGTCGGCTTGGTGATCGCACGAGCGGTGGGGCGGGCTACAGCAGAGCGGCTAGCATTGCCAGTCACAGAG CTCCTCGATTGGACCGTCTTCTTGGGAGCCGTCCTGGAGCGGGACGTTGAGGCCGTCTTGCTAGATGACC TAACCACTGTCCTGAGCGGTGTTTGGCTGCGGCTGTTTGGCATGTTGCATTTCCTCTGCGCATTGTGTGC ACTGTTGTGCATGTTGCTCTTGGCCTTGCCACAGTTGGAGGCGCTACGAGCGTTGCAGCTTGTCTTTCGT CCCGACGCATTGCGGACTACTGTCTGCCTGTAGTACGTGCATCCATCTCTCTCGAGAGTTGTGGGGCAGT TTGTCGGCGGGAGAGCAGTGCAGTAATCGCAGTCTGTTGTTCCACAGAGGTAGATTCCGGTGTAGGTGTC AGGCGCAGCTGTGCAAACGAGATCGGTGTTGCACTGGACATGAATCTCCATCATGTAAGGCAGGTCGAAG AGATCTGCCGGAACGTCGCCAGTCAGCTCGTTGCAGTCAAGGTGGAGCTCCTTGAGGAATTGGAGTTCGT TGACGCAGGTGGGGATGGGACCAGTGAGAGCATTAATACTCATGTACCAAAACATCATGTGGGTTAAATC ACAGATGCACTCGGGAATGTCACCCGTCAGCCCAGCGTTGTTAATTTGCAGGTACTGCATTGAGGTGAGG GCACAAAGATCCGTCGGGATCGGGCCCGCAAGGTCATTATTATTAAGGTACAGCGACCTCAAAAGTGGGA AGCAGCCTATGTCGGCAGGGAGGGCACCAGTGAGCCCCATGTCAGAGAGGTCGATCCCAATAACGTTATT GTTAGAGTCGCATGTGATGCCGGTCCAGCTACAGTAAGACACGTCGGGCGTAAGCCAGTTATTCGACTTC CAATTAGCACCATCCAGGGCATCGTACAAATTGGTCAGGGCCTCCTCCTCGGTGGCAGGGCAAGCGGCGC GAGCAAGACCAAGAAGTCCTAGAACAAGGGCTGCGATCA >XP_001710242.1 Cyst wall protein 2 [Giardia lamblia ATCC 50803] MIAALVLGLLGLARAACPATEEEALTNLYDALDGANWKSNNWLTPDVSYCSWTGITCDSNNNVIGIDLSD MGLTGALPADIGCFPLLRSLYLNNNDLAGPIPTDLCALTSMQYLQINNAGLTGDIPECICDLTHMMFWYM SINALTGPIPTCVNELQFLKELHLDCNELTGDVPADLFDLPYMMEIHVQCNTDLVCTAAPDTYTGIYLCG TTDCDYCTALPPTNCPTTLERDGCTYYRQTVVRNASGRKTSCNARSASNCGKAKSNMHNSAHNAQRKCNM PNSRSQTPLRTVVRSSSKTASTSRSRTAPKKTVQSRSSVTGNASRSAVARPTARAITKPTQSKRAPVRAL PRSTNKPIVRRR Protective antigen The gene encoding the full length cyst wall protein-2 (CWP2) from Giardia lamblia was subcloned into the pCDNA3 mammalian expression vector and stably introduced into S. typhimurium STM1. Challenge infection with live Giardia muris cysts revealed that mice receiving the CWP2-encoding DNA vaccine were able to reduce cyst shedding by approximately 60% compared to control mice [Ref1093:Abdul-Wahid and Faubert, 2007]. Abdul-Wahid and Faubert, 2007 journal Abdul-Wahid A, Faubert G 2007 25 50 8372-8383 Vaccine Lee et al., 2009 journal Lee P, Abdul-Wahid A, Faubert GM 2009 11 1 20-28 Microbes and infection / Institut Pasteur Olson et al., 2000 journal Olson ME, Ceri H, Morck DW 2000 16 5 213-217 Parasitology today (Personal ed.) Wiki: Giardia lamblia website http://en.wikipedia.org/wiki/Giardia_duodenalis