Detection of Salmonellae in Captive and Free-Ranging Turtles Using Enrichment Culture and Polymerase Chain Reaction

Author(s): James P. Gaertner, Dittmar Hahn, Jacob Jackson, Michael R. J. Forstner, and Francis L. Rose Source: Journal of Herpetology, 42(2):223-231. 2008. Published By: The Society for the Study of Amphibians and Reptiles DOI: http://dx.doi.org/10.1670/07-1731.1 URL: http://www.bioone.org/doi/full/10.1670/07-1731.1

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Journal of Herpetology, Vol. 42, No. 2, pp. 223231, 2008 Copyright 2008 Society for the Study of Amphibians and Reptiles

Detection of Salmonellae in Captive and Free-Ranging Turtles Using Enrichment Culture and Polymerase Chain Reaction
JAMES P. GAERTNER, DITTMAR HAHN,1 JACOB JACKSON, MICHAEL R. J. FORSTNER, FRANCIS L. ROSE
AND

Department of Biology, Texas State University, 601 University Drive, San Marcos, Texas 78666, USA ABSTRACT.Information on the importance of captive turtles as sources of human Salmonella infections is well established; however, data on the potential of free-ranging turtles as carriers of salmonellae are scarce and contradictory. We combined traditional culture techniques and molecular tools to analyze swabs obtained from the cloacae and from different body parts of captive and free-ranging turtles for salmonellae. Salmonellae were detected in 50% of captive turtles (N = 10). A similar percentage of detection (51%) was obtained for salmonellae in free-ranging turtles from the Rio Grande (N = 80) with six sites at Big Bend National Park, Texas, and one site at Elephant Butte Reservoir, New Mexico, analyzed. Here, 46% of Trachemys gaigeae (N = 36), 56% of Apalone spinifera (N = 43), and the only individual of Chrysemys picta were positive for salmonellae. These percentages of detection of salmonellae in turtles were independent of the location of the sampling in the Rio Grande, the species and the gender of the turtles. Although individuals of captive turtles testing positive for salmonellae were generally positive for all body parts tested (i.e., the cloacae, the carapace, the ventral base of the left rear leg, underneath one or more of the claws on the front feet, and the ventral base of the tail), individuals of free-ranging turtles testing positive for salmonellae were often positive for only one or two body parts. Our results demonstrate that salmonellae are prevalent in high rates in both captive and free-ranging turtles.

A large number of studies have confirmed the presence of salmonellae in pet amphibians and reptiles such as lizards, snakes or turtles (Sanyal schner, 2002; Nakadai et al., 1997; Geue and Lo et al., 2005). Reptiles are generally asymptomatic carriers of salmonellae (Chiodini and Sundberg, 1981; Anonymous, 1995; Pasmans et al., 2002), and several taxa have been commonly identified as sources of human Salmonella infections (Anonymous, 1995, 1999; Wells et al., 2004). Pet turtles are a well-recognized source of human Salmonella infections (Johnson-Delaney, 1996), but information on the potential for free-ranging turtles to be a source of human Salmonella infections is limited. Although some studies have failed to detect salmonellae in free-ranging turtles (Brenner et al., 2002; Richards et al., 2004; Saelinger et al., 2006), others have reported significant rates of infection with 32100% of turtles analyzed harboring salmonellae (Briones et al., 2004; Chambers and Hulse 2006). In recent studies, we detected salmonellae not only in cloacal swabs, but also in samples from biofilms on the carapace of different turtle species, such as Common Musk Turtles (Sternotherus odoratus), Red-Eared Sliders (Trachemys scripta elegans), Texas River Cooters (Pseudemys texana), and Common Snapping Turtles (Chelydra serpentina
1

serpentina) in percentages between 32 and 61% of the individuals analyzed (Hahn et al., 2007; unpubl. data). These results indicated that salmonellae were established members of the microbial community in, and on, free-ranging turtles even in the supposedly clean habitat of the headwaters of the San Marcos River, Texas (Hahn et al., 2007; unpubl. data). The purpose of this study was to expand these previous analyses to habitats with higher nutrient loads including holding ponds with captive turtles and the Rio Grande/Bravo with free-ranging turtles. High nutrient loads and permanent reinoculation of salmonellae through feces in small holding ponds with captive turtles have been consistently assumed to promote growth and transmission of salmonellae; thus, infection rate of turtles, although the heavily anthropogenically impacted Rio Grande/Bravo, provided a natural but contaminated ecosystem containing fecal coliforms and salmonellae as nonsource pollutants. The study not only focused on cloacal contamination but also included analyses of salmonellae on different body parts of the turtles. MATERIALS AND METHODS Turtle Species Tested for Salmonellae.Captive turtles were obtained from the WaterLife Collection, San Marcos, Texas. Ten individuals that belonged to five different species (Callagur

Corresponding Author. E-mail: dh49@txstate.edu

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FIG. 1. Schematic presentation of seven sampling sites for free-ranging turtles in the Rio Grande (i.e., six sites within Big Bend National Park, Texas [Boquillas, Rio Grande Village, Gravel Pit, La Clocha, Solis, and Tally] and an additional site near Elephant Butte Reservoir, New Mexico).

borneoensis [N 5 3], Siebenrockiella crassicollis [N 5 2], Graptemys nigrinoda [N 5 1], Trachemys gaigeae [N 5 2], and Pseudemys gorzugi [N 5 2]) were caught by hand from two pond enclosures. All turtles had carapace length . 12.2 cm. Eighty free-ranging turtles (36 T. gaigeae, 43 Apalone spinifera, and one Chrysemys picta) were caught in baited hoop nets in the Rio Grande/ Bravo, at six sites within Big Bend National Park, Texas, and an additional site near Elephant Butte Reservoir, New Mexico (Fig. 1). All free-living turtles had a carapace length . 13.2 cm. Sampling for and Enrichment of Salmonellae. Samples were taken with sterile cotton wool swabs from the cloacae of all turtle species (Hahn et al., 2007) and from four locations on the body: the carapace, the ventral base of the left rear leg, underneath one or more of the claws on the front feet, and the ventral base of the tail. Swabs were incubated in 1 ml of

buffered peptone water (l21: 10 g peptone, 5 g NaCl, 9 g Na2HPO4, 1.5 g KH2PO4, pH 7.2) in 2ml cryotubes at 37uC for 1620 h (International Standard Organization, 1993), and 100-ml subsamples of these preenrichment cultures subsequently transferred to 2-ml cryotubes that contained 1 ml of Rappaport-Vassiliadis (RVS) Broth (l21: 4.5 g peptone (soymeal), 29 g MgCl2 3 7 H2O, 8 g NaCl, 0.4 g KH2PO4, 0.036 g malachite-green, pH 5.2; Vassiliadis et al., 1981). Tubes were incubated at 43uC for 24 h to enrich for salmonellae (Vassiliadis et al., 1981). Aliquots of preenrichment and enrichment cultures (100 ml) were subsequently processed for polymerase chain reaction (PCR)-assisted detection of salmonellae, whereas the remainder of the cultures was mixed with 600 ml of 60% glycerol and stored at 280uC until further use. PCR-Based Detection of Salmonellae.Detection of all Salmonella enterica subspecies as well as Salmonella bongori was based on the amplifica-

SALMONELLAE IN TURTLES tion of a 284-bp-fragment of the invA gene that encodes a protein of a type III secretion system, essential for the invasion of epithelial cells by rez and Ru ssmann, 1998; Khan salmonellae (Sua et al., 2000). This procedure was recently validated and proposed as the international standard diagnostic method for quality assurance laboratories in epidemiological studies on Salmonella spp. (Malorny et al., 2003). Aliquots were centrifuged at 14,000 rpm for 1 min, the bacterial pellets washed once in sterile distilled water, and the bacteria lysed in 100 ml of 50 mM NaOH by incubation at 65uC for 15 min (Hahn et al., 2007). One ml of lysate was used as template for PCR amplification with primers 139 (59-GTG AAA TTA TCG CCA CGT TCG GGC AA) and 141 (59-TCA TCG CAC CGT CAA AGG AAC C) (Rahn et al., 1992) in a final volume of 50 ml containing 10 3 PCR buffer (500 mM KCl, 25 mM MgCl2, 200 mM Tris/ HCl, pH 8.4, 0.1% Triton 100), 1 ml dNTPs (each 10 mM in 10 mM Tris/HCl, pH 7.5), 0.2 ml Taq polymerase (5 U ml21), and 1 ml of each primer (100 ng ml21). After an initial 2-min denaturation at 96uC, 35 rounds of temperature cycling were performed in a PTC-200 Thermocycler (BioRad, Hercules, CA) with denaturation at 96uC, primer annealing at 64uC, and elongation at 72uC, each for 30 sec (Malorny et al., 2003). This was followed by incubation at 72uC for 7 min (Hahn et al., 2007). Lysates of Salmonella typhimurium ATCC 14028 and Escherichia coli DH5a as well as sterilized distilled water were used as positive and negative controls, respectively. PCR products were analyzed by gel electrophoresis on 2% agarose gels in TAE buffer after staining with ethidium bromide (0.5 mg ml21) (Sambrook et al., 1989). Method Verification.Because the approach of using PCR-based analyses of salmonellae in enrichment cultures from samples obtained from turtles was proven to be reliable in previous studies (Hahn et al., 2007; unpubl. data), only a limited number of enrichments chosen at random were used for method verification. Method verification was based on preenrichment of previously frozen enrichment cultures in buffered peptone water, subsequent enrichment in RVS Broth, and isolation of salmonellae after plating on RVS solidified with agar and incubation at 43uC for 24 h as in our previous study (Hahn et al., 2007). Ten isolates per sample were identified by PCR targeting the invA gene, positive isolates characterized by rep-PCR to reduce redundancy, and representatives of unique rep-PCR patterns sent to the Texas Department of State Health Services (Austin, TX) for serotyping (Hahn et al., 2007).

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RESULTS Preenrichment and enrichment cultures of all samples (i.e., all samples from all body parts of both captive and free-ranging turtles) showed significant microbial growth as indicated by the visual change of cultures from clear to turbid. The enrichment step was a necessary prerequisite for the detection of invA gene fragments by PCR because no signals were obtained on preenrichment cultures (data not shown). Because enrichment conditions were only semiselective, the increase in turbidity in enrichment cultures did not presuppose enrichment of salmonellae only; thus, not all samples from enrichment cultures resulted in the amplification of invA gene fragments (Fig. 2). Five of the 10 captive turtles sampled in pond enclosures at the WaterLife Collection tested positive for salmonellae by PCR. All three C. borneoensis and one S. crassicollis were positive for salmonellae at all body locations sampled (Fig. 2). The second individual of S. crassicollis was positive for salmonellae in four of the five samples, whereas all other turtles, G. nigrinoda (N 5 1), T. gaigeae (N 5 2), and P. gorzugi (N 5 2), were negative for salmonellae. Thus, salmonellae were detected in 50% of the captive turtles, with individuals generally positive for salmonellae at all body parts tested (Fig. 3). Of the total of 80 turtles sampled in the Rio Grande, 41 individuals (51%) were positive for salmonellae in at least one of the five body locations subsampled. Forty-six percent of A. spinifera (N 5 43), 56% of T. gaigeae (N 5 36), and the only C. picta tested positive for salmonellae. Most of these turtles were positive at one or two locations (13 and 17 of 41 turtles), followed by those positive at three, four, or all five locations (six, three, and two of 41 turtles), which resulted in a total of 22% positive samples (87 of 400 subsamples analyzed). Within the group of turtles that had tested positive for the invA gene, amplification products were obtained with relatively similar percentages between body parts with 36% of the carapace samples, 66% of the samples from the leg, 44% of the samples from the tail, 36% of the claw samples and 44% of the samples from the cloacae being positive (Fig. 3). These percentages did not differ significantly between sex (female, male) and species (A. spinifera, T. gaigeae) (Fig. 3). The fraction of turtles testing positive for salmonellae from site to site ranged from 4075% (Fig. 4). However, differences, were not significant among sampling sites (Fig. 4), and results were potentially impacted by large differences in numbers of turtles caught and analyzed per site (Fig. 4).

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FIG. 2. Typical pattern of invA gene fragment detection by PCR with DNA from enrichment cultures obtained from individuals of captive Callagur borneoensis (A) and free-ranging Apalone spinifera (B). Lane a represents a fragment size marker (lambda HindIII), lane b a positive control using DNA of Salmonella typhimurium ATCC 14028, and lane c a negative control using DNA of Escherichia coli DH5a as template for PCR amplification. Lanes dh show results of PCR amplifications from enrichment cultures from the carapace (d), the ventral base of the left rear leg (e), underneath one or more of the claws on the front feet (f ), and the ventral base of the tail (g), and the cloacae (h).

Three enrichment cultures positive for the invA gene were subsequently selected for the isolation of salmonellae as control for and verification of the accuracy of the PCR-based analyses. All samples were obtained from individuals of A. spinifera, although from two different sites (two sites at La Clocha and one Gravel Pit) and different body locations (leg, cloacae, tail). Isolation attempts retrieved six, nine, and 10 of 10 colonies analyzed from these enrichments, respectively, that were identified as salmonellae by PCR amplification of the invA gene fragment. Rep-PCR revealed identical patterns for all positive isolates on each individual turtle (Fig. 5) but different patterns between isolates from different individuals.

Three representative isolates for the rep-PCR profiles were subsequently identified by the Texas Department of State Health Services, Austin, as salmonellae by physiological methods and further characterized as serovars Newport (leg, Gravel Pit), Assen (tail, La Clocha), and an undetermined serotype within group 2 : y : 1,7 (leg, La Clocha). DISCUSSION Similar to our previous studies (Hahn et al., 2007; unpubl. data), the combination of traditional culture techniques enriching salmonellae in semiselective media, and of molecular techniques providing sensitive and specific tools

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FIG. 3. Detection of salmonellae (% 6 95% confidence intervals) in samples from different locations on the body (carapace, leg, claw, tail, and cloacae) of captive (dark bars) and free-ranging (light bars) turtles (upper part), and of free-ranging turtles, differentiating between females (dark bars) and males (light bars; middle part), and between turtle species Trachemys gaigeae (dark bars) and Apalone spinifera (light bars; lower part). The 100% value corresponds to the number of turtles being positive for salmonellae on at least one location. Proportions with overlapping confidence intervals were assumed to be the same.

allowed us to detect salmonellae in, and on, different turtles species both in captivity and in the wild. Captive turtles have been identified as carriers for salmonellae in many studies (Sanyal schner, 2002; Nakadai et al., 1997; Geue and Lo et al., 2005). However, detection of salmonellae in free-ranging turtles is less common (Briones et al., 2004; Chambers and Hulse, 2006), and several surveys failed to detect them at all (Brenner et al., 2002; Richards et al., 2004; Saelinger et al., 2006). Although the basic

procedures in most of the earlier studies were similar to ours, those studies that did not detect salmonellae in samples from free-ranging turtles differed from ours in two aspects. We included a preenrichment in buffered peptone water that was meant to resuscitate salmonellae from environmental samples. Preenrichments were shown to increase the recovery of salmonellae in aquatic samples (Thomason et al., 1977; Anselmo et al., 1989) as well as in a variety of other settings such as orange juice (Hammack et al., 2001) and egg shells (Valentin-Bon et al., 2003). In addition, we used PCR-based analysis of enrichments instead of culture- or ELISAbased analyses with a correspondent increase in sensitivity (i.e., 98% vs. 55% and 78% detection, respectively; Mitchell et al., 2000). Thus, studies that did not detect salmonellae in samples of free-ranging turtles (Brenner et al., 2002; Richards et al., 2004; Saelinger et al., 2006) probably reflect a lower probability of detection by less sensitive methodology (Mitchell, 2006). Limited sensitivity of other methods has not been a problem for the detection of salmonellae in captive turtles, which suggests significantly higher loads of salmonellae in turtles held in small and contained systems such as ponds than in larger and potentially open systems, such as lakes and rivers. Small enclosures for captive turtles experience less frequent water turnover than open systems and, thus, enrich with organic material such as turtle food and feces. Easily available carbon resources, as well as frequent reinoculation through feces of turtles, might help sustain populations of salmonellae at densities much higher than in open systems. Under these conditions, cells of salmonellae might not be stressed and, thus, resuscitation not necessary to achieve significant growth for detection. In addition to these environmental issues, captive turtles are often analyzed in response to cases of human Salmonella infections (Anonymous, 2005), which results in highly focused analyses that further enhance the chances of detection. In contrast to captive turtles that were usually positive for salmonellae on all body locations analyzed, the vast majority of freeranging turtles were positive on only one or two of the five sample locations analyzed. This result does support speculation toward higher densities of salmonellae in the environments of captive turtles compared to those that are freeranging. A lower density of salmonellae in habitats of free-ranging turtles, including compromised habitats like that of the Rio Grande, would also explain the need of highly sensitive detection procedures for salmonellae that include resuscitation, enrichment and molecular

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FIG. 4. Detection of salmonellae (% 6 95% confidence intervals) in samples of free-ranging turtles Trachemys gaigeae (dark bars) and Apalone spinifera (light bars) from seven different sites on the Rio Grande (i.e., six sites within Big Bend National Park, Texas [Boquillas, Rio Grande Village, Gravel Pit, La Clocha, Solis, and Tally] and an additional site near Elephant Butte Reservoir, New Mexico). Numbers presented within the bars represent numbers of turtles caught at the respective site. Proportions with overlapping confidence intervals were assumed to be the same.

detection methods (Hahn et al., 2007; unpubl. data). Despite the potentially low abundance of salmonellae on free-ranging turtles, we were able to obtain isolates. One of the isolates was identified as a Salmonella spp. of an unknown serotype in group 2 : y : 1,7, whereas two other isolates were identified as serovars Newport and Assen, respectively. Serovar Newport is among the 20 most commonly isolated serovars from human sources (Anonymous, 2003) as well as one of the most common among reptiles (Anonymous, 1995; Woodward et al., 1997). Serotype Assen is also associated with human sources; however, it is uncommon and only accounted for three reported cases of salmonel-

losis in the United States from 19922002 (Anonymous, 2003). Thus, free-ranging turtles from the Rio Grande/Bravo are carriers of salmonellae with the potential to cause human-associated salmonellosis. Notably, similar results were obtained for several turtle species with different life histories within supposedly clean natural environments, the headwaters of the San Marcos River, with serovars Rubislaw, Newport, Gaminara, and Thompson representing potentially human pathogenic salmonellae (Hahn et al., 2007; unpubl. data). The presence of salmonellae in captive turtles led to regulations meant to reduce the probability of human infection with pathogenic salmonellae. Regulations were enacted that

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FIG. 5. Rep-PCR profiles of 10 salmonellae (110) isolated from enrichments from the carapace of the same turtle. Lane M represents a size marker (Lambda/HindIII).

prohibit the commercial sale or public distribution of turtle eggs and of hatchlings smaller than four inches (10.16 cm) as pets within the United States but allow selling them for educational and scientific purposes and for export (Anonymous, 1975; Cohen, 1980). Even though these regulations exclude lizards and snakes that may carry salmonellae (Anonymous, 1995, 1999), they seem to have proven a necessary and valuable public health measure to protect humans, especially small children, from salmonellosis (Cohen, 1980). However, the assumption that captive turtles are more prone to carry salmonellae than free-ranging turtles and are, therefore, more likely reservoirs for salmonellosis is not supported by our results. The perception that salmonellae are somehow a component of contaminated or filthy waters is also different from our recent results (Hahn et al., 2007; unpubl. data). The results of our study demonstrate that salmonellae can be easily detected in, and on, captive and free-ranging turtles. This suggests that the normal habitat for Salmonella spp. can no longer be seen as the gut or cloaca

of turtles but encompasses bacterial biofilms both within and upon turtles regardless of water quality. Because herpetology is a field particularly invested in the hands on study of the organisms, awareness of the potential for salmonellosis is warranted when working with living turtles regardless of origin. Simple hygiene procedures including use of gloves or of disinfecting hand washes are needed to prevent transmission of salmonellae to humans. Acknowledgments.The authors are indebted to the Austin Community Foundation and the River Systems Institute of Texas State University-San Marcos for financial support. The research was carried out in compliance to the rules overseen by the Texas State Institutional Animal Care and Use Committee (IACUC, permit 04-4D4436A6) and with sampling permits of the National Park Service (NPS, permit BIBE-2005-SCI-0027), the Texas Parks and Wildlife Department (TPWD, permit SPR-0102-191), and the New Mexico Department of Game and Fish (permit 3236).

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