Multilocus genotyping of Giardia duodenalis in lambs from Spain reveals a high heterogeneity

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Abstract

Fecal specimens from 120 lambs in Valencia (Spain) were analyzed for Giardia duodenalis by IFA and nested-PCR using the beta giardin (bg), glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi) and small subunit ribosomal RNA (ssurRNA) genes. The highest prevalence was obtained using the ssurRNA gene (89.2%), whereas values from other techniques ranged from 64.1% to 69.2%. Sequences of the ssurRNA showed a high proportion of assemblage A or mixed assemblage A/E samples (55.1% and 25.2%, respectively). When the other 3 loci were analyzed, between 6.5% and 15.4% were found to be assemblage A or A/E, respectively. Nested PCR for the tpi gene was the most variable of the targets employed. Twelve new sequences of gdh and tpi for G. duodenalis from sheep were found. Multilocus genotyping resulted in 63 patterns from the 71 samples sequenced at the four loci. This high variability among isolates possibly reflects the high frequency of mixed infections.

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).

References (41)

  • E. Lasek-Nesselquist et al.

    The identification of a new Giardia duodenalis assemblage in marine vertebrates and a preliminary analysis of G. duodenalis population biology in marine systems

    International Journal for Parasitology

    (2010)
  • M. Lebbad et al.

    From mouse to moose: Multilocus genotyping of Giardia isolates from various animal species

    Veterinary Parasitology

    (2010)
  • P.T. Monis et al.

    Genetic diversity within the morphological species Giardia intestinalis and its relationship to host origin

    Infection, Genetics and Evolution

    (2003)
  • J. Ng et al.

    Molecular characterization of Cryptosporidium and Giardia in pre-weaned calves in Western Australia and New South Wales

    Veterinary Parasitology

    (2011)
  • C.M. Read et al.

    Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP

    Infection, Genetics and Evolution

    (2004)
  • L.J. Robertson et al.

    The zoonotic potential of Giardia and Cryptosporidium in Norwegian sheep: a longitudinal investigation of 6 flocks of lambs

    Veterinary Parasitology

    (2010)
  • A. Ruiz et al.

    Ocurrence and genetic characterization of Giardia duodenalis in goat kids from the Canary Islands, Spain

    Veterinary Parasitology

    (2008)
  • M. Santín et al.

    Prevalence and molecular characterization of Cryptosporidium and Giardia species and genotypes in sheep in Maryland

    Veterinary Parasitology

    (2007)
  • J.P. Sweeny et al.

    Longitudinal investigation of protozoan parasites in meat lamb farms in southern Western Australia

    Preventive Veterinary Medicine

    (2011)
  • J.M. Trout et al.

    Prevalence of Giardia duodenalis genotypes in pre-weaned dairy calves

    Veterinary Parasitology

    (2004)
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