Abstract
The aim of this work was to study the epidemiological status of Salmonella spp. and Shiga toxin–producing Escherichia coli (STEC) in an ocellated lizard research center focusing on the risk and hygiene aspects. Fecal and environmental samples were collected and examined for Salmonella spp. and STEC. Isolates were detected using real-time polymerase chain reaction (RT-PCR) and characterized using serotyping and pulsed-field gel electrophoresis (PFGE). Overall, 52% of samples were positive for Salmonella spp. using RT-PCR and seven isolates were obtained from samples from ocellated lizards and their environment, whereas no samples were positive for STEC. Salmonella isolates belonged to S. enterica subsp. enterica serovar Kibusi and S. enterica subsp. salamae serovars 41:z10:z6 and 18:z10:z6, some of which have previously been isolated from human sources. Indistinguishable and closely related PFGE types were found, which supported the existence of horizontal transmission between animals due to crowding of animals and the persistence of Salmonella in the environment. The results of the current study emphasize the need for improved prevention efforts and good hygiene practices in research centers, recuperation centers, and zoos with reptiles to minimize the exposure of personnel and visitors to this pathogen.
Introduction
Shiga toxin–producing Escherichia coli (STEC) strains have recently emerged as important foodborne pathogens, but, to our knowledge, the existence of STEC in reptiles has not yet been described.
The ocellated lizard (Timon lepidus) lives in southwest Europe and it is classified as a species of special interest within the National Catalogue of Threatened Species (EFSA, 2007). In fact, in Spain it has been in danger of extinction, with a declining population in nature.
The aim of this work was to study the epidemiological status of Salmonella spp. and STEC in an ocellated lizard research centers focusing on the risk and hygiene aspects.
Materials and Methods
Sample collection, culture, and Salmonella and STEC screening
In September 2009, about 20 samples of rectal feces from apparently healthy ocellated lizards (one per animal) were collected (Table 1). Sampled animals were randomly selected from 85 reproductive adults (50 females and 35 males) from a research center in the Extremadura region of Southwest Spain. In addition, four food samples (insects and worms) and one sample from lizards' drinking water were collected before feeding the lizards (Table 1). Samples were collected using aseptic techniques, transported to the laboratory on ice, and analyzed within 24 h of collection.
Real-time PCR for the invA gene.
PCR for stx 1 and stx 2 genes.
STEC, Shiga toxin–producing Escherichia coli; PCR, polymerase chain reaction.
Each fecal sample was placed into 9 mL buffered peptone water (BPW) (Oxoid) and incubated for 16–24 h at 37°C under aerobic conditions. Also, 25 g of food samples or 25 mL of water sample was homogenized in 225 mL of BPW in a stomacher filter bag. Filtrates were transferred to sterile bottles and incubated for 16–24 h at 37°C under aerobic conditions. After incubation, enrichment cultures were subdivided into two aliquots: one aliquot was analyzed for Salmonella and the other aliquot was analyzed for E. coli O157:H7.
For detection of Salmonella spp., real-time polymerase chain reaction (RT-PCR) from each BPW aliquot was done by duplicate to detect the invA gene as previously described (Nam et al., 2005) with some modifications. For isolation of Salmonella, each aliquot was cultured onto xylose lysine desoxycholate agar (Oxoid) at 37°C under aerobic conditions. After 24–48 h of incubation, 10 presumptive Salmonella colonies were tested to detect the invA gene as previously described (Nam et al., 2005) and positive colonies were biochemically confirmed as Salmonella spp. by the API 20E system (bioMérieux). One positive colony was stored at −20°C until further characterization.
For isolation of E. coli O157:H7, each aliquot was processed as previously described (Sánchez et al., 2010), whereas for isolation of non-O157 STEC, fecal samples were analyzed as described by Rey et al. (2003).
Characterization of isolates
S. enterica isolates were serotyped at the Spanish National Reference Laboratory for Salmonella and Shigella by the slide agglutination method using commercial antisera (Bio-Rad; Statens Serum Institut; Izasa). Pulsed-field gel electrophoresis (PFGE) was performed as previously described (Sánchez et al., 2010) to establish relatedness and diversity among Salmonella isolates.
Results
Overall, 52% (13/25) of samples were RT-PCR-positive for Salmonella spp. (Table 1), with a 60% (12/20) of fecal samples detected as RT-PCR-positives and one invA PCR-positive sample obtained from lizards' drinking water. Salmonella isolates were only recovered from 53.9% (7/13) of the RT-PCR-positive samples (Table 1) and all the Salmonella isolates were biochemically identified as S. enterica. In contrast, although E. coli was found, no positive samples for STEC were detected in either fecal or environmental samples.
Two subspecies and three different serovars have been observed among the isolates (Fig. 1) and six distinct PFGE types (1–6) in three PFGE clusters were found among them (Fig. 1). One cluster was composed of PFGE types 3 to 6, all belonging to Salmonella Kibusi, with PFGE types differing only in less than or equal to two restriction fragments (90.7% similarity). Moreover, two clusters with two PFGE types belonging to S. enterica II were observed in which isolates of the serovar 41:z10:z6 were indistinguishable (PFGE type 1).
Dendrogram generated with InfoQuestFP software showing the pulsed-field gel electrophoresis (PFGE)-XbaI digestion types of Salmonella isolates recovered from reproductive adults of ocellated lizards. The bands generated were analyzed by using the Dice coefficient and the unweighted-pair group method with arithmetic averages. The scales at the top indicate the similarity indices (in percentages) and molecular sizes (in kilobases).
Discussion
The ocellated lizard is an important carrier of Salmonella spp. High prevalence values of Salmonella as in our study have also been previously observed in other different lizard species (Woodward et al., 1997; Geue and Loschner, 2002) and in ocellated lizards sampled in Spain (Briones et al., 2004). In contrast, although E. coli was found, no positive samples for STEC were detected in agreement with previous studies about E. coli in reptiles (Murinda et al., 2004; Naldo et al., 2009).
As in our study, S. enterica I and II have been routinely isolated in previous studies in reptiles (Geue and Loschner, 2002; Briones et al., 2004). Salmonella survives well in the environment and on different surfaces (Mermin et al., 2004; Bauwens et al., 2006). So, the presence of S. enterica I in the water creates enhanced opportunities for transmission, eventually to humans, and suggests a contact with a source of infection, such as wild animals, possibly birds (Katribe et al., 2009), but further epidemiological studies must be done to determine the origin of this subspecies.
Salmonella Kibusi, previously detected in lizard from a zoo in Belgium (Bauwens et al., 2006), and S.II 41:z10:z6 have been isolated from human sources causing disease (CDC, 2008). However, to our knowledge, both S. II 41:z10:z6 and S. II 18:z10:z6 found in this study have not been previously described in lizards. The high similarity among PFGE types supports the existence of horizontal transmission between animals. In our opinion, this transmission is due to crowding animals in a small enclosure and the persistence of Salmonella in the environment, which may favor the reinfection in animals and represent a potential risk for the research center personnel and visitors.
Some recommendations about prevention of reptile-associated salmonellosis in human have been made (CDC, 2003). Routine precautions with basic hygiene measures to prevent human infection, including the use of hand gloves and hand washing after handling reptiles, cages, equipment, and reptile feces, should be applied to reduce the risk of infection. Also, to reduce the exposure, it is important to feed the animals with clean water and balanced food, to reduce the stress and to clean carefully the fecal material from pens. It is recommended to avoid the contact of the ocellated lizards with children with <5 years, pregnant women, and immunocompromised persons. Finally, visitors and personnel of the research center should be informed about the risk of acquiring salmonellosis from reptiles.
In conclusion, this report describes the absence of STEC in lizards and the high prevalence of Salmonella in lizards with serovars previously isolated from human sources, representing a potential reservoir of infection for the research center personnel and visitors. These results emphasize the need for improved prevention efforts and good hygiene practices in research centers, recuperation centers, and zoos with reptiles to minimize the exposure of personnel and visitors to this pathogen.
Footnotes
Acknowledgments
Thanks to the Ocellated Lizard Research Centre Tencarral S.L. (QLK5-CT2002-70670). We thank Raquel Rubio and Ana Aladueña for their skilful technical assistance. R. Martínez acknowledges the Junta de Extremadura for his research fellowship (PRE06053). S. Sánchez acknowledges the Junta de Comunidades de Castilla-La Mancha and Fondo Social Europeo for his research fellowship (09/02-C).
Disclosure Statement
No competing financial interests exist.