Introduction

Traditionally, diseases of wildlife populations have attracted attention mostly if these affect human wellbeing or because of economic implications. Moreover, the emergence of wildlife diseases has been recognized as a threat to domestic animal and human health, as well as a substantial risk to the conservation of global biodiversity (^{DASZAK et al., 2000}). However, during the past decade, an increased number of studies have pointed at the links between human activity and the emergence of wildlife diseases. These studies have assessed the potential role that the anthropogenic environmental alterations and the increasing human encroachment into wild habitats represent as causes of this disease emergence (^{THOMPSON et al., 2010}; ^{BREARLEY et al., 2013}). Recently, the investigations of wildlife diseases are recognized as a part of global health and their surveillance has become part of the activities against zoonotic emerging diseases. Therefore, integrated approaches to human and animal health, including their respective social and environmental contexts, are required (^{ZINSSTAG et al., 2011}). Furthermore, the health status of wildlife can be used as an indicator of environmental health (^{CARIGNAN & VILLARD, 2002}).

Giardia is a genus of flagellated protozoa that infect the gut of different classes of vertebrates. Currently, six species are recognized within this genus: Giardia agilis in amphibians, Giardia ardeae and Giardia psittaci in birds, Giardia muris and Giardia microti in rodents, and G. duodenalis in a wide range of mammals. G. duodenalis (syn. Giardia lamblia, Giardia intestinalis) is recognized as a complex of at least eight different assemblages with different host distribution: assemblages A and B are found in a wide range of domestic and wild mammals, including humans; assemblages C and D are specific for dogs and other canids; assemblage E is found in livestock; assemblage F in felids; assemblage G in rats and assemblage H in marine mammals (^{CACCIÒ, 2015}).

Species of this genus were first described basing on the presumed host specificity, because of the lack of differentiating morphological features. During the first half of the XX century, over 40 species of Giardia were recognized, two of them in reptiles: Giardia varani from monitor lizard (Varanus niloticus) and Giardia serpentis from Cape viper (Causus rhombeatus) (^{THOMPSON et al., 1990}). In 1952, the increasing number of Giardia species and the uncertainty regarding host specificity led to a taxonomic rationalisation. Thus, most species infecting vertebrates, including those described in reptiles, were named as G. duodenalis (^{FILICE, 1952}). Since then, only one description of a G. varani-like flagellate from a water monitor (Varanus salvator) has been reported in Malaysia (^{UPTON & ZIEN, 1997}). In the last years, few studies about the presence of Giardia in reptiles, both in wild and in captivity, were carried out, and their results did not show evidence of Giardia species in these hosts (^{LALLO et al., 2009}; ^{RINALDI et al., 2012}; ^{RAŚ-NORYŃSKA & SOKÓL, 2015}).

This work reports for the first time the presence of zoonotic and livestock-specific assemblages of G. duodenalis in several species of free-living wild lizards from Galicia (northwest Spain), demonstrating the wide environmental contamination by this protozoan parasite, possibly as a result of anthropogenic activities.

Materials and Methods

Results and Discussion

This is one of the very few investigations of Giardia in reptiles and the first report of the presence of zoonotic and livestock-specific assemblages of G. duodenalis in two species of free-living wild lizards. Thus, among the 31 samples analysed, five samples were positive (16.1%), corresponding to Iberian rock lizard (I. monticola) (4/21, 19.0%) and Bocage’s wall lizard (P. bocagei) (1/1, 100%) (Table 1). Partial nucleotide sequences of the fragment encompassing the ITS1-5.8S-ITS2 region in the ribosomal unit of Giardia were obtained from all these isolates. Four sequences were 99% similar to other deposited in GenBank, of which two corresponded to G. duodenalis assemblage A2 and the other two to G. duodenalis assemblage B (accession numbers GU126432 and GU126440, respectively). The remaining sequence was identical to the livestock-specific G. duodenalis assemblage E (GenBank® accession number GU126434). On the other hand, the results of the PCR amplification and sequencing of a fragment of the SSU-rDNA gene show the presence of G. duodenalis assemblage A in four of these samples. In two of them, the presence of this assemblage was confirmed, whereas in the two remaining samples, the results were inconsistent in relation with those obtained from the ITS1-5.8S-ITS2 region (see Table 1). These inconsistencies in the results obtained for both molecular markers can be due to the small size of the fragment of the SSU-rDNA gene sequenced (^{WIELINGA & THOMPSON, 2007}). In any case, the use of this gene allowed the confirmation of the presence of G. duodenalis in these hosts.

To the best of our knowledge, this is the first detection of G. duodenalis in free-living wild reptiles. ^{Rinaldi et al. (2012)} did not identify this parasite in a study carried out in pet reptiles (25 lizards and 125 snakes) surveyed in Italy. Likewise, Giardia was not detected in three wild lizards from Brazil (^{LALLO et al., 2009}). Nevertheless, Giardia cysts were observed in the feces of one chameleon among several pet reptile species analyzed in Poland (76 lizards, 15 turtles and 10 snakes), but the authors did not characterize this isolate at the molecular level (^{RAŚ-NORYŃSKA & SOKÓL, 2015}).

Most Giardia infections naturally occurring in wildlife are caused by zoonotic species, which are considered to have been introduced into wildlife habitats and, once established, maintained by direct contact or environmental routes (^{THOMPSON, 2013}; ^{ABEYWARDENA et al., 2015}). The study region (Galicia, northwest Spain) is characterized by an important livestock sector and extensive rural areas, where farmers usually applied slurry as manure in the grasslands. Moreover, the region has a high rainfall and fecal contamination of surface waters by runoff from manure-fertilized fields is common. Thus, assemblage E is the most frequently G. duodenalis genotype detected in surface waters followed by assemblage A (^{CASTRO-HERMIDA et al., 2015}). On the other hand, the lizard species under study feed on flies and other insects that can act as mechanical vectors of this parasite in rural areas, as previously demonstrated by several authors (^{CONN et al., 2007}; ^{ZHAO et al., 2014}). Therefore, since hoofed animals are natural hosts for G. duodenalis assemblages A, B and E (^{CACCIÒ, 2015}) and they were identified in Galician livestock (^{CASTRO-HERMIDA et al., 2007}), the detection of zoonotic and livestock-specific assemblages of G. duodenalis in free-living wild lizards indicates the importance that livestock management practices have in the transmission of this parasite.

Finally, the results of the present study increase the number of species in which the presence of Giardia was reported and suggest that this parasite is widely dispersed in wildlife. Although we cannot confirm the existence of real infection in these lizards, the detection of zoonotic and livestock-specific assemblages of G. duodenalis supports the increasing evidence that the presence of this protozoon in wildlife is the result of the environmental contamination from anthropogenic activities.