Free-Living Turtles Are a Reservoir for Salmonella but Not for Campylobacter

Different studies have reported the prevalence of Salmonella in turtles and its role in reptile-associated salmonellosis in humans, but there is a lack of scientific literature related with the epidemiology of Campylobacter in turtles. The aim of this study was to evaluate the prevalence of Campylobacter and Salmonella in free-living native (Emys orbicularis, n=83) and exotic ( Trachemys scripta elegans, n=117) turtles from 11 natural ponds in Eastern Spain. In addition, different types of samples (cloacal swabs, intestinal content and water from Turtle containers) were compared. Regardless of the turtle species, natural ponds where individuals were captured and the type of sample taken, Campylobacter was not detected. Salmonella was isolated in similar proportions in native (8.0±3.1%) and exotic (15.0±3.3%) turtles (p=0.189). The prevalence of Salmonella positive turtles was associated with the natural ponds where animals were captured. Captured turtles from 8 of the 11 natural ponds were positive, ranged between 3.0±3.1% and 60.0±11.0%. Serotyping revealed 8 different serovars among four Salmonella enterica subspecies: S. enterica subsp. enterica (n = 21), S. enterica subsp. salamae (n = 2), S. enterica subsp. diarizonae (n = 3), and S. enterica subsp. houtenae (n = 1). Two serovars were predominant: S. Thompson (n=16) and S . typhimurium (n=3). In addition, there was an effect of sample type on Salmonella detection. The highest isolation of Salmonella was obtained from intestinal content samples (12.0±3.0%), while lower percentages were found for water from the containers and cloacal swabs (8.0±2.5% and 3.0±1.5%, respectively). Our results imply that free-living turtles are a risk factor for Salmonella transmission, but do not seem to be a reservoir for Campylobacter . We therefore rule out turtles as a risk factor for human campylobacteriosis. Nevertheless, further studies should be undertaken in other countries to confirm these results.


Introduction
Campylobacteriosis and salmonellosis are the two most prevalent zoonoses worldwide [1]. These zoonoses represent an important public health problem and controlling the disease has become a vital challenge in most countries [1][2][3][4][5][6][7]. Campylobacteriosis and salmonellosis were responsible respectively for 212,064 and 99,020 cases of illnesses in the EU [1]. Moreover, campylobacteriosis is the most common cause of acute bacterial gastroenteritis in the EU [1,8,9].
Apart from acute gastroenteritis, campylobacteriosis may lead to more severe, occasionally long-term sequelae such as Guillain-Barré syndrome, reactive arthritis and irritable bowel syndrome [10,11]. The high and rapidly increasing incidence and the capacity of Campylobacter to cause considerable morbidity make campylobacteriosis a public health problem of considerable magnitude [2]. However, compared to Salmonella, few outbreaks are reported, and most cases of campylobacteriosis are considered to be "sporadic" rather than a part of recognised outbreaks, with a seasonal peak during summer [12].
Campylobacter are commensally widespread in the intestines of wild and domesticated animals, resulting in contamination of the environment, including water sources [13]. Although Campylobacter is mostly perceived as a food-borne pathogen, there is evidence for other transmission pathways, including direct and indirect contact with infected animals, people and environment [2,[14][15][16][17]. In recent years, the popularity and number of exotic reptiles kept as pets has risen, leading to an increase in the number of reptile-associated zoonotic pathogens infections, especially in vulnerable patients such as infants, young children, the elderly or immunocompromised adults [18][19][20][21][22][23]. In this way, turtles represent a special risk, as they are commonly kept as pets for children [24]. Similar to Campylobacter, Salmonella infections are caused by consumption of contaminated food, person-toperson transmission, waterborne transmission and numerous environmental and animal exposures [25]. Specifically, reptiles and other coldblooded animals (often referred to as 'exotic pets') can act as reservoirs of Salmonella, and cases of infection have been associated with direct or indirect contact with these animals [3]. Trachemys scripta elegans is the most common pet turtle worldwide and has been identified as an important source of infection in human cases and outbreaks of salmonellosis since 1963 [26][27][28][29][30][31][32][33][34][35][36][37]. For this reason, the epidemiology of pathogenic microorganisms in free-living and pet turtles has been studied [7,24,[38][39][40]. In particular, results from these studies have shown that the incidence of Salmonella in pet turtles ranged from 0% to 72.2% [5,39,41,42] and from 0% to 15.4% in free-living turtles [39,[43][44][45][46][47]. To our knowledge, no prevalence studies of Campylobacter in pet and free-living turtles have been carried out.
In this context the aim of this study was to assess the prevalence of Campylobacter and Salmonella in free-living native (Emys orbicularis) and exotic turtles (Trachemys scripta elegans) located in 11 natural pond areas in Eastern Spain (Valencia Region). Additionally, we assessed the relative sensitivity of different sample types (cloacal swabs, intestinal content and water from containers) to estimate Salmonella prevalence in turtles.

Material and Methods
The Ethics and Animal Welfare Committee of the Universidad CEU Cardenal Herrera approved this study. All animals were handled according to the principles of animal care published by Spanish Royal Decree 1201/2005 (BOE, 2005; BOE = Official Spanish State Gazette). The Conselleria de Infraestructuras, Territorio y Medio ambiente (regional administration) gave permission to take samples. This project is included in the LIFE + Biodiversity section, which aims to develop innovative projects or demonstrations that contribute to the implementation of the objectives of the Commission communication (COM (2006) 216 final) "Halting the loss of Biodiversity for 2010-and beyond." During the period between July and October 2012, 200 freeliving turtles were captured from 11 natural ponds in Eastern Spain (Pego-Oliva, Almenara, Castellón, Xeraco, Peñíscola, Villanueva de Alcolea, Pobla Tornesa, Cabanes, Vaca River, Moros and La Safor). After capture, each individual was housed singly in a plastic container with 2 litres of sterile water to prevent bacterial transmission among them. As bacteria excretion is not continuous, water samples were taken after two days in captivity. This study was undertaken within the framework of an eradication programme for exotic turtles. Therefore, native turtles were returned to their habitat after sampling, while exotic turtles were euthanised by sodium pentobarbital injection before taking the samples (Dolethal, Vétoquinol, E.V.S.A).
For each individual, two cloacal samples were taken using sterile cotton swabs (Cary Blair sterile transport swabs, DELTALAB ® ). After 2 days in the container, two water samples from plastic containers were collected. Each sample was analysed for Campylobacter and for Salmonella isolation. For exotic turtles, after euthanisation 2 cm of large intestine were collected and the content was homogenised.

Detection of Campylobacter spp
The procedure was based on ISO 10272:2006 recommendations (Annex E). Intestinal content and swabs were directly streaked onto the two selective agar plates (mCCDA and Preston, AES laboratories®, Bruz Cedex, France) and incubated at 41.5±1°C for 44±4 hours. Water samples were pre-enriched in 1: 10 vol/vol Bolton Broth (OXOID, Dardilly, France) and then pre-incubated at 37±1°C for 5±1hours. Afterwards, 100 µl of the sample was cultured on the two selective agar plates as described above. All plates and broths were incubated in a micro-aerobic atmosphere (84% N 2 , 10% CO 2 and 6% O 2 ) generated in a gas charged incubator (CampyGen, Oxoid). Plates were examined for grey, flat, irregular and spreading colonies typical of Campylobacter. One putative colony was subcultured from each plate onto sheep blood agar for confirmation as Campylobacter spp. Campylobacter confirmation was performed by a mobility test using a dark field microscope, by oxidase and catalase biochemical test and by streaking at different temperatures and atmospheres on Columbia blood agar (AES Laboratories ®, Bruz Cedex, France). Finally, characterisation of the bacteria species was done with a hippurate hydrolysis test.

Statistical analyses
A generalised linear model, which assumed a binomial distribution for Salmonella shedding, was fitted to the data to determine whether there was an association with turtle species (native and exotic), natural ponds where turtles were captured (Pego-Oliva, Almenara, Castellón, Xeraco, Peñíscola, Villanueva de Alcolea, Pobla Tornesa, Cabanes, Vaca river, Moros and La Safor) and sample type (cloacal swabs, intestinal content and water from the containers). A P value of less than 0.05 was considered to indicate a statistically significant difference. Data are presented as least squares means ± standard error of the least squares means. All statistical analyses were carried out using a commercially available software program (SPSS 16.0 software package; SPSS Inc., Chicago, Illinois, USA, 2002).

Results
For Campylobacter isolation, overall 517 samples were analysed; 200 samples were from water containers (117 from exotic and 83 from native turtles), 200 from cloacal swabs (117 from exotic and 83 from native turtles) and 117 from intestinal content (only from exotic turtles). Regardless of the turtle species captured, the natural pond where animals were captured and the sample type (water from the container, cloacal swabs and intestinal content), Campylobacter was not detected.
For Salmonella isolation, overall 517 samples were examined; 200 samples were from water from the container (117 from exotic and 83 from native turtles), 200 from cloacal swab (117 from exotic and 83 from native turtles) and 117 from intestinal content (only from exotic turtles). Independently of the species of turtle analysed, 11.0±2.3% of the turtles tested positive. Moreover, of the exotic turtles sampled and the native turtles sampled, 15.0±3.3% and 8.0±3.1% were positive, respectively. No significant differences were found between the percentage of Salmonella and the turtle species studied. Salmonella was detected in exotic and native turtles from eight natural ponds investigated (Table 1). In positive natural ponds significant differences were found. The mean prevalence of Salmonella was 16.2±4.6% (ranged between 3.0±3.1% to 60.0±11.0%). In the natural ponds of Cabanes, Pobla Tornesa and Xeraco, Salmonella was not isolated.
Significant differences for Salmonella detection were found among the different type of samples collected ( Table 3). The highest isolation of Salmonella was obtained from intestinal content samples (12.0±3.0%), while for water from the containers and cloacal swabs lower percentages were found (8.0±2.5% and 3.0±1.5%, respectively).

Discussion
Symptoms of Campylobacter (fever, abdominal cramps and diarrhoea) are clinically indistinguishable from those of bacterial gastroenteritis caused by other organisms, such as Salmonella or Shigella species [48,49]. Pet turtles are considered an important reservoir for Salmonella [39,46,50] and we tested the hypothesis that free-living turtles would also   be an important reservoir for Campylobacter. Members of the Campylobacter genus naturally colonise humans, farm animals, wild mammals, birds, reptiles and shellfish [51]. However, to date only two reports identify Campylobacter foetus of turtle origin as a human pathogen [38,52]. In the present study, Campylobacter was not detected. As for Salmonella isolation, Campylobacter detection is likely to be highly dependent on the choice of an adequate sampling procedure combined with a sensitive culture method [53,54]. However, direct plating of faecal samples has been shown to yield the best isolation efficiency for detection of Campylobacter [54,55]. One possible explanation for the lack of detectable Campylobacter from cloacal swabs is a lack of appreciable faecal material. Nevertheless, in our study neither cloacal swabs nor intestinal content were positive. Although molecular methods (PCR and qPCR) have several advantages over classical bacteriology for Campylobacter detection, a high level of agreement between both methods has been reported, especially with faecal samples [54,56]. Nevertheless, if Campylobacter had been present, it seems highly unlikely that the bacteria would not have been isolated in any of the samples analysed. Thus, our results show that free-living turtles appear not to be a reservoir for Campylobacter and we therefore rule out turtles as a risk factor for human campylobacteriosis.
The prevalence of Salmonella detected in this study among free-living Valencian turtles was moderate (11.0±2.3%) and consistent with those of other studies [39,47,57]. To our best knowledge, few studies on the prevalence of Salmonella in free-living turtles have been carried out in Spain [39,46,50]. However, contradictory results are present in the literature, since some authors revealed low prevalence of Salmonella in free-living turtles [39,43,45,[57][58][59], while other authors reported a medium and high prevalence [42,50,60,61]. Freeliving turtles are believed to shed Salmonella at lower rates than captive turtles because they are less or not even exposed to stress factors that increase shedding rates, or because they are not natural carriers of the bacteria [45,58,62]. However, for other authors free-living turtles are considered an important reservoir for Salmonella [46].
The serovar most frequently identified was S. Thompson All serovars identified have previously been reported in reptiles and have been associated with human salmonellosis [63][64][65][66][67][68]. Although many of these serovars may be considered as types rarely associated with human disease, 10% or more of isolates belong to subsp. enterica, which comprises potential human pathogens, e.g. S. Typhimurium. This is, together with S. Enteritidis, one of the most frequently reported serovar involved in human salmonellosis [1,35]. The infections in reptiles are usually asymptomatic, although clinical salmonellosis in reptiles has been reported with the following symptoms: septicaemia, salpingitis, dermatitis, osteomyelitis and granulomatous disease [69]. In addition, Salmonella subsp. houtenae has been recently associated as a cause of meningitis in a child [63].
In the cloaca of turtles, the presence of Salmonella was lower than in the intestinal content. The lower recovery of Salmonella from cloacal swabs was probably due to wild turtles shedding Salmonella at lower rates because they are less stressed, as mentioned above [45,58,62]. Cloacal swabs appeared to be less sensitive than faecal samples [70]. As Salmonella excretion is not continuous [39], in our study turtles were kept for two days in water containers to increase shedding rates. As expected, analyses also indicated that stress increased shedding of Salmonella. Specifically, keeping the turtles for 48 hours in containers could increase the sensitivity of water samples, as suggested by our findings. For this reason, this sampling method may be applied in further studies to determine the prevalence of Salmonella in turtles.
This study showed free-living turtles as a risk factor for Salmonella infection, but our findings also indicate that freeliving turtles appear not to be a reservoir for Campylobacter and we therefore discard the turtles as a risk factor for human campylobacteriosis. To our best knowledge, this is the first study in which campylobacteriosis is investigated in relation to free-living turtles as a possible reservoir. Nevertheless, further studies should be undertaken in other countries to confirm these results.