Multilocus genotyping of Giardia duodenalis in lambs from Spain reveals a high heterogeneity
Introduction
Giardia duodenalis is a ubiquitous parasite that infects humans and many other mammals. Because the parasite shows little morphologic variation, microscopically based investigation of outbreaks to determine zoonotic potential is impossible. Therefore, molecular techniques have been developed and used for identification. Seven assemblages (assemblages A–G) have been described in mammals mainly based on allozymic and genetic analysis (Monis et al., 2003). However, two new genotypes have been recently reported in mammals, genotype H and the quenda genotype (Adams et al., 2004; Lasek-Nesselquist et al., 2010; Ng et al., 2011). Most assemblages are found in closely related host species, for example, assemblage C and D are found in canids, assemblage F in felids, assemblage E in hoofed mammals and assemblage G in rodents). However, assemblages A and B, have been found in a wide variety of mammals including humans.
Three assemblages have been found in sheep, assemblage E which predominates in all the genotyping studies, and assemblages A and B which have been found mostly sporadically but are potentially zoonotic. A recent study showed that assemblage A was more prevalent in older lambs than in younger animals (Sweeny et al., 2011). In some studies sheep have not appeared to be an important potential source of infection for humans (Ryan et al., 2005, Santín et al., 2007, Yang et al., 2009, Robertson et al., 2010), whereas in other studies lambs were found be frequently infected with zoonotic assemblages. Although the percentage of sheep infected with potentially zoonotic assemblages varies greatly among studies, values approximating 40–50% have been obtained (Geurden et al., 2008, Lebbad et al., 2010) depending on the gene examined in molecular studies. Such data indicate that sheep could be an important source of infection for humans (reviewed by Robertson, 2009).
Most molecular studies involving sheep have used a single locus to assign the isolates to assemblages (Aloisio et al., 2006, Santín et al., 2007, Giangaspero et al., 2005, Castro-Hermida et al., 2006, Castro-Hermida et al., 2010, Di Giovanni et al., 2006, Ryan et al., 2005, Gómez-Muñoz et al., 2009, Nolan et al., 2010). Only a few studies have used two or more gene loci but these have used a small number of samples (Castro-Hermida et al., 2007, Yang et al., 2009, Robertson et al., 2010, Lebbad et al., 2010, Geurden et al., 2008). The use of only one locus in animal studies has recently raised concern regarding assemblage assignment of isolates, but a similar situation exists in studies involving humans (Cacciò et al., 2008). Therefore, it has been proposed that multilocus genotyping (MLG) using at least four genes could improve the assignment of each isolate to a specific assemblage and thereby better clarify the epidemiology of giardiasis (Cacciò et al., 2008).
The present study was designed to compare the utility of the four most frequently used loci for genotyping Giardia, beta giardin (bg), glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi) and small subunit ribosomal RNA (ssurRNA) genes, on a large number of samples from sheep to identify the most sensitive loci for detecting the presence of Giardia while determining if multilocus patterns provide helpful epidemiological information.
Section snippets
Faecal samples
Individual faeces were collected directly from the rectum of 1- to 3-month-old lambs on 4 farms near Valencia (Spain). On each farm, 30 lambs were randomly selected resulting in 120 samples. Three grams of faeces from each lamb were suspended in 30 ml of distilled water, filtered through a 50 μm pore size mesh sieve and centrifuged for 10 min at 1000×g. Between samples the sieves were placed in a 5% aqueous solution of sodium hypochlorite for 10 min. Each pellet was suspended in 7.5 ml of distilled
Comparison of diagnostic methods
The number of samples detected by IFA as well as each of the four nested-PCR techniques used in this study is shown in Table 1. Above all, the nested-PCR for the ssurRNA was the most sensitive of the methods employed. Of the 120 samples examined 107 were positive using this technique (89.2%). Thirty samples negative by IFA were positive by nested-PCR at the ssurRNA locus while only 7–12 samples negative by IFA were positive using nested-PCR for the gdh, tpi or bg loci. A good correlation was
Discussion
The sensitivity of the loci used for identifying assemblages of G. duodenalis isolates varies. Also, some loci represent more conserved genes, such as ssurRNA, whereas other loci such bg, gdh and tpi are much less conserved (reviewed in Cacciò and Ryan, 2008). In this study the ssurRNA locus was the most sensitive, and therefore well suited for prevalence studies. This finding is in agreement with other studies (Cacciò and Ryan, 2008). The main reason is most likely the multicopy nature of this
Acknowledgements
This work has been supported by a grant from the Spanish Ministerio de Ciencia y Tecnología (AGL2007/62435GAN).
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