4 Genotyping of Giardia intestinalis Isolates from Dogs by Analysis of gdh , tpi , and bg Genes

Giardia intestinalis, a flagellated protozoan parasite, is the most prevalent human intestinal protozoan worldwide (Adam, 2001). About 200 million people in the world are infected with giardiasis and each individual eliminates up to 900 million cysts per day (Minivielle , 2008). Higher prevalence is found in tropical and subtropical areas, where Giardia affects up to 30% of the population. In epidemiological studies carried out in Mexico and other sudamerican countries, prevalence between of 10-16% has been found in urban areas and 34% in shantytowns (Gamboa “et al”, 2003; Giraldo-Gomez, 2005; Sulaiman, 2004). G. intestinalis is a cosmopolitan pathogen with a very wide host range, including humans, domestic animals, and wild animal species (Caccio, 2008; Thompson, et al 1993). The most common cause of infection with Giardia is the consumption of contaminated food or water (Ortega, 1997), although zoonotic transmission is also possible. Once a person is infected, the parasite lives in the intestines and is passed in the stool of the infected person. Animals such as cats, dogs and cattle can also be infected and spread the disease to humans.


Introduction
Giardia intestinalis, a flagellated protozoan parasite, is the most prevalent human intestinal protozoan worldwide (Adam, 2001).About 200 million people in the world are infected with giardiasis and each individual eliminates up to 900 million cysts per day (Minivielle , 2008).Higher prevalence is found in tropical and subtropical areas, where Giardia affects up to 30% of the population.In epidemiological studies carried out in Mexico and other sudamerican countries, prevalence between of 10-16% has been found in urban areas and 34% in shantytowns (Gamboa "et al", 2003;Giraldo-Gomez, 2005;Sulaiman, 2004).G. intestinalis is a cosmopolitan pathogen with a very wide host range, including humans, domestic animals, and wild animal species (Caccio, 2008;Thompson, et al 1993).The most common cause of infection with Giardia is the consumption of contaminated food or water (Ortega, 1997), although zoonotic transmission is also possible.Once a person is infected, the parasite lives in the intestines and is passed in the stool of the infected person.Animals such as cats, dogs and cattle can also be infected and spread the disease to humans.
Infections may be asymptomatic or include symptoms of chronic diarrhea, weight loss, and malabsorption.When children infected with Giardia have no symptoms of giardiasis, the parasite is present in their feces and they can pass the infection to others.Other symptoms of chronic giardiasis include: Loose, soft, greasy stools, discomfort in the abdomen, general feeling of discomfort or illness, weakness and fatigue.
The parasite has two interchangeable forms that guarantee a simple and efficient life cycle.The cyst that contaminate the environment and the trophozoite, which attach to the intestinal villi via a specialized microtubule structure, the ventral disc.
There are at least seven major genotypes referred to as assemblages (A-G) including 2 (A and B) known to infect humans (Mcpherson, 2005;Monis, 2009).
Assemblages A and B, of clinical significance to humans, differ from each other by as much as 20% at the DNA sequence level (Caccio, 2008).There is also evidence that genetic exchange has resulted in hybrids, or mixed types, based on assemblage-specific PCR of Giardia isolates from cases of human infection (Monis, 1999).Clinically, G. intestinalis assemblage A appears to be less prevalent than assemblage B and other mixed types worldwide (Tungtrongchitr, 2010;Singh A, 2009).However, infections with assemblage A appear to be more symptomatic.Although assemblages A and B are the only ones that infect humans, they also can infect other mammals (Volotao, et al. 2007).Interestingly, assemblage A is more frequently associated with animal hosts that may serve as reservoirs of infection for humans (Ballweber, 2010).
Although Giardia isolated from dogs typically belong to assemblages C or D, assemblages A and B have been identified in dogs in regions of high endemicity (Cooper, 2010).Genotypes E, F, and G have been isolated from pigs and other farm mammals (Jerlström-Hultqvist J, 2010), cats (Ballweber, 2010), and rodents (Monis, 1999), respectively.
The ability to genetically characterize Giardia strains isolated from clinical and animal samples should contribute to the understanding of the epidemiology and pathogenesis of Giardia infection and the relative contributions of distinct genotypes to the severity of clinical infection and zoonotic potential.Here we report our attempts to develop assays for the characterization of Giardia genomic DNA extracted from dog stool samples and from Giardia trophozoites Portland I, based on the Polymerase Chain Reaction amplification of multiple loci.We targeted the G. intestinalis tpi, gdh, and bg genes that encode the enzyme triose-phosphate isomerase, the enzyme glutamate dehydrogenase, and β-giardin, respectively.

Coproparasitoscopic analysis
Fecal specimens were stained with Lugol's iodine (Faust, 1938) and examined by microscopy to find cysts of Giardia intestinalis.Cysts were concentrated from dog feces by repeated washing in distilled water and stored at 4 ºC until use.

DNA extraction
DNA was extracted from the fecal samples by use of the QIAamp DNA Stool Mini Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions.All DNA concentrations were determined by using an Epoch spectrophotometer (Biotek, Winooski, VT).

PCR amplification
The gdh gene encoding glutamate dehydrogenase, the tpi gene encoding triose phosphate isomerase, and the bg gene encoding β-giardin were each amplified using the Polymerase Chain Reaction (PCR) as follows.

tpi gene amplification
The tpi gene was amplified by nested-PCR in which the first round was a duplex reaction to amplify two fragments corresponding to genotypes A and B simultaneously using four primers (TPIAF 5´-CGAGACAAGTGTTGAGATGC-3´, TPIAR 5´-GGTCAAGAGCTTACAACACG-3´ and TPIBF 5´-GTTGCTCCCTCCTTTGTGC-3´, TPIBR www.intechopen.com5´-CTCTGCTCATTGGTCTCGC-3´).PCR amplification was performed in a volume of 50-µL with 500 ng of DNA in 1X PCR buffer, 2 mM MgCl 2 , 0.25 mM of dNTP and 1 U of Taq DNA Polymerase.Amplification was achieved with 25 cycles of [94 ºC for 20 s, 50 ºC for 30 s and 72 ºC for 1 min].The second round of PCR comprised two separate hemi-nested PCRs to amplify internal fragments of 476 bp and 140 bp corresponding to the A and B genotypes respectively.
To amplify genotype A, primers TPIAIF: 5´-CCAAGAAGGCTAAGCGTGC-3´ and TPIAR were used using 3 µL of the first round amplicon as template in a 50-µL volume reaction.The amplification step used 33 cycles of [94 °C for 20 s, 56 °C for 30 s, and 72 °C for 1 min].Alternatively, the 140 bp fragment corresponding to genotype B was amplified with primers TPIBIF: 5´-GCACAGAACGTGTATCTGG-3´ and TPIBR.Amplification was performed under the same conditions used for A except that the MgCl 2 concentration in the PCR mixture was 1.5 mM.(Amar, 2003. Molina, 2005)

Restriction analysis
The amplicons generated by PCR were digested with restriction enzymes for the purpose of subtyping.The tpi gene amplicons were digested with restriction enzyme RsaI, the bg gene amplicons were digested with HaeIII, and the gdh amplicons were digested with BspHI.The products of restriction enzyme digestion were separated by 2% agarose gel electrophoresis, using 100bp DNA ladder (Promega, Madison,Wi.USA) as a size standard, and visualized by staining with ethidium bromide.

Results
We developed a molecular method to test stool samples for the presence of G. intestinalis genotypes that are of clinical significance to human infection possibly by zoonotic transmission from dogs.Giardia infection of dog stool samples was confirmed by coproparasitoscópico analysis (data not shown).Giardia cysts isolated from feces was genotyped by a combination of multi-locus (gdh, bg, tpi) PCR followed by restriction analysis of the PCR amplicons.We analyzed nine samples of dog stool, one sample of human stool, and G. intestinalis cysts from the Portland-1 control strain.The Portland-1 standard is a control for the A-I assemblage.

Figure 2
Illustrates genotyping based on amplification and subsequent BspHI enzyme digestion of the gdh locus encoding glutamate dehydrogenase.All of the dog and human samples tested in this way yielded the same 1200 bp fragment as the Portland-1 control and after digestion they yielded two fragments of 900 and 300 bp respectively indicative of assemblage A.

Figure 3
Illustrates genotyping based on amplification and HaeIII enzyme digestion of the bg locus encoding β-giardin.Three fragments ranging from 100 to 200 bp are indicative of assemblage A-I.All 9 dog samples and 1 human sample were classified as assemblage A-I, the same as the Portland-1 control.

Figures 4
Illustrate genotyping based on amplification and RsaI enzyme digestion of the tpi locus encoding triose-phosphate isomerase.Again, all samples yielded products consistent with their identification as belonging to G. intestinalis genotype AI.

Discussion
Human giardiasis is caused by two genetically distinct assemblages (A and B) of G. intestinalis.A number of molecular assays have been developed for their specific detection in stool and environmental samples (Caccio, 2008).
We have developed a method to detect Giardia based on the PCR amplification of three genes (gdh, bg, tpi) used in prior genotyping studies.
Although DNA-based methods reported in the literature have been used with success to genotype Giardia, we did not have observed differences among the analysis of different genes in studied samples.Some researchers had found frequent mismatches, intraassemblage discordances and mixed positions, in tpi and in bg sequences, especially in assemblage B (Bonhomme, 2011).
All of the fecal samples analyzed in this report (9 from dogs, 1 from humans) were determined to belong to the sub-genotype A-I assemblage.This predominance of assemblage A-I probably reflects the mechanism that led to infection of the animals from which the fecal samples came.The dogs might have drunk from water that had been