Salmonella is an important zoonotic pathogen of Enterobacteriaceae, which is gram-negative, faculta-tive anaerobic bacterium. Currently Salmonella has found 2 449 serotypes in the world (Brenner et al., 2000), there are about 292 serotypes reported in China (Zhang and Zhu, 2002). As early as 1975, turtles were ascertained to carry and transmit Salmonella, red-eared sliders, as the same as many other reptiles can carry and transmit Salmonella, humans may infect Salmonella through direct or indirect contacting with red-eared sliders and other reptiles, thereby causing typhoid, paratyphoid, gastroenteritis and other diseases and even death (Ackman et al., 1995). Therefore, FDA (the Food and Drug Administration) in 1975 banned the pet turtle trade except for education purpose use in the USA.

In the United States, red-eared turtle is popular pet sliders, particularly loved by children. Each year about 1.4 million Americans infected with Salmonella. Reptile and amphibian exposure is associated with ~74,000 Salmonella infections annually in the United States (Mermin et al., 2004). Clearly, pet turtle has a serious public health impact because of carrying Salmonella. Previous studies of Salmonella focused on captive turtles (McCoy and Seidler, 1973) and commercial turtles (Siebeling et al., 1984). Under the conditions of captive wildlife, the detection of Salmonella in reptiles was higher than that in mammals and birds (Gopee et al., 2000). There are few studies of Salmonella on the turtles on the wild environment reported.

Red-eared sliders in China is a foreign invasive species, Guangdong and other southern regions of China have the habit of eating red-eared sliders, it was also the red-eared sliders as pets. So far, There is no information on wild red-eared sliders carrying salmonella reported. In recent years, red-eared sliders have intruded on Hainan Island, populations have been found in red-eared sliders across the island, in order to know the impacts of alien species, red-eared sliders, on island ecosystems, especially on potential harms of environment and human in the cayying and transmiting of Salmonella. We collected samples of red-eared sliders in Nandu river, Wanquan river on Hainan Island, Donghu Lake in downtown of Haikou city of Hainan to isolate Salmonella bacteria from cloaca of red-eared sliders, in order to make clear the status of the wild populations of red-eared sliders carrying Salmonella.

1 Results and Analysis
1.1 Red-eared sliders collection and Salmonella identification
We collected 68 of wild red-eared sliders in Nandujiang (Nandu river), Wanquanhe (Wanquan river) and Haikoudonghu (East Lake of Haikou City) during the period between Feb. to Sep. 2010 (Figure 1), of which Nandujiang was 26, Wanquanhe was 34 and Haikoudonghu was 8. Total tentative 98 Salmonella isolates were obtained from 68 samples of red-eared sliders. Further biochemical identification showed that 79 Salmonella bacteria were detected out of Total tentative 98 Salmonella isolates, of which 37 red-eared sliders carried Salmonella, Total carrying rate of Salmonella in collected red-eared sliders was 54.41%, of which Nandujiang was 53.85%, Wanquanhe was 58.82%, Haikoudonghu was 50%.
 

Figure 1 Selected photos of Red-eared sliders (Trachemys scripta elegans) collected in Hainan island

1.2 Biochemical identification of Salmonella
In this study we used Salmonella biochemical identification kit produced by Huankai Biotechnology Co., Ltd. Guangdong to determine serological reactions of Salmonella. The employed Kit contained three response categories (A1, A2 and A3) with six standard identifications of Salmonella (Table 1). The results showed that 24 individuals of red-eared sliders response to A1-1 reaction.
 

Table 1 Biochemical test response to Salmonella of red-eared sliders

According to biochemical test positive data, further statistics showed that the tested sample of 68 red-eared sliders had a total carrying rate of 54.41%, of which the red-eared sliders from Nandujiang was 53.85%, Wanquanhe was 58.82% and Haikoudonghu was 50.00%. The sample of group A from Nandujiang carries 75.00% of Salmonella, and group B 50.00% of Salmonella (Table 2). 34 of red-eared sliders from Wanquanhe in Group A carried 66.67% of Salmonella, group B carried 56.00% of Salmonella, Whereas 8 of red-eared sliders in group A carried 42.85% of Salmonella, group B was not for statistical analysis due to small sample size.
 

Table 2 Positive carrying rate of Salmonella by biochemic test

1.3 Identification of Salmonella serotypes
The employed Salmonella diagnostic serum kit (Model 60) produced by Lanzhou Institute of Biological Products can theoretically detect Salmonella OA-F serotypes, while Salmonella diagnostic kits produced by Thailand S & A company can detect OA-I serotypes. The results showed that the two sampling points of Nandujiang and Wanquanhe detected out Salmonella common serotypes as S. litchfiled, S. chailey, S. senftenberg, and S. Stanley. We were unable to detect out any Salmonella common serotypes in samples of Salmonella isolated from Haikoudonghu, which indicated that the Salmonella serotypes of isolates from red-eared sliders in Haikoudonghu should be outside of the A-F group (Table 3). 
 

Table 3 Serotypes detection of Salmonella isolates in collected red-eared sliders

2 Discussions
According to the literature, 14% of human Salmonella infections in is caused by the spread of turtles (Cohen et al., 1980), Richard et al (2004) detected 0% of wild turtles carrying Salmonella. Saelinger et al (2006) detected less than 5% of wild turtles carrying Salmonella, Vila et al (2007) detected 12% of overall detection rate of Salmonella in one kind of upland tortoises and two species of freshwater turtles, Gaertner (2008) detected Salmonella in a variety of wild freshwater turtles that the total carrier rate was 51%, of which the wild red-eared sliders carried Salmonella rate of 38%. The results of this study for the total rate of wild red-eared sliders carrying Salmonella was 54.41% (n = 68). 

There is a little difference of Salmonella carrying rate among three different sampling locations, it indicated that habitation should have no effect on the carrying rate of Salmonella, which was consistent with Gaertner' s report (Gaertner, 2007). Nandujiang is a kind of brackish water river, where Salmonella in the sample was detected out, indicating that Salmonella as less salt tolerant baterium, can be carried by amphibians reptiles living in these aquatic environment, Salmonella detection rate (50.00%) of red-eared sliders from semi-artificial Lake of Haikoudonghu was lower than that of the natural rivers, which does not match the Salmonella carring rate of turtles in the artificial environment reported in the previous studies (Keymer, 1972). The reason might be due to regularly release bleaching powder, Comparison of two groups by carapace length, Salmonella carrying rate of red-eared sliders with small carapace length was higher than that of red-eared sliders with big carapace length, in which red-eared sliders with small carapace length from Nandujiang  carried highest rate of Salmonella that was consistent with the findings of Keymer (1972).

Amphibians and reptiles carrying Salmonella usually without any symptoms (Anonymous, 1999). Mitchell (1980) and other studies have shown that S. Urbana, S. Lichfield, and S. Java were the most human infection serotypes caused by turtles in the Salmonella, especially affecting infants and young children. Other serotypes can also be detected from humans (Geue and Loschner, 2002). This study detected the S. Lichfield, which indicated the invasive red-eared sliders in china might cause potential harm to humans. Detected S. Stanley also is a common pathogens causing human food poison. In this study, we might have a preliminary suggestion that the invasive alien red-eared sliders carrying some Salmonella serotypes closely to human health, which may have a direct impact on human health (OIsen et al., 2001).

In China, red-eared sliders, albeit an exotic invasive species, but is still widely used as food turtle and pet turtle. Obviously, the red-eared sliders, as one of the main disseminators of Salmonella in reptiles, have entered the human food chains and ecosystem, human health and the environment posed by the potential risks and dangers.

3 Materials and Methods
3.1 Experimental samples collected
We collected 68 of wild red-eared sliders in the two major rivers of Hainan Island, Nandujiang and Wanquanhe, and in the downtown lake of Haikou city, Haikoudonghu during the period between February to September in 2010, of which Nandu were 26, Wanquan were 34, and Haikoudonghu were 8. In this study we have collected wild red-eared sliders divided into three groups, Nandu was as first group, Wanquan as second group, Haikoudonghu as third group. Meanwhile each group was sub-divided as subgroups, the carapace length of a red-eared sliders less than 4-inch divided into group A, greater than or equal to 4 inches into the B group. 26 samples from Nandu (First Group) were gone into Group A of 4 red-eared sliders and Group B of 22 red-eared sliders. 34 samples from Wanquan (Second Group) were gone into Group A of 9 red-eared sliders and Group B of 25 red-eared sliders. 8 red-eared sliders from Haikoudonghu were grouped into 7 of Group A and 1 of group B.

3.2 Standard strains of Salmonella
In this study, Salmonella typhimurium was used as the standard strains, the standard accession number of the Salmonella typhimurium is CMCC (B) 50071, come from the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP).

3.3 Bacterium culture medium
Nutrient broth, Salmonella chromogenic Medium, vassiliadis enrichment broth, and Salmonella biochemical identification kit, provided by HuanKai Biotechnology Co., Ltd. Guangdong; Salmonella diagnostic serum (60 reactions) were purchased from Lanzhou Institute of Biological Products; Salmonella diagnostic serum for OA-I, purchased from S & A company in Thailand.

3.4 Method for Salmonella Identification
Salmonella bacteria were identified by following the procedures of GB 4789.4-2010 established as the Chinese National Standard Food Safety Inspection.

3.4.1 Sampling and enrichment
Bacterium samples were collected with a steriled cotton swab in red-eared sliders cloaca, then add 1 mL nutrient broth, cultured 6~8 h at 37℃; then took 60 μL medium transferring into 1 mL magnesium chloride malachite green, cultured 16~18 h at 37℃.

3.4.2 Bacterial isolation of Salmonella
Took one loop of Salmonella cultured medium with the inoculation loop to inoculate by crossing the plate of Salmonella chromogenic medium and incubated 16~18 h at 37℃. If there is purple colonies appeared, the isolate will be considered as tentative Salmonella bacterium. Pick three purple single colonies ready for further purified culture. If no any colony appeared purple, then extend the incubation time to 48 hours to observe whether there is purple colonies appeared, The tentative colonies of Salmonella were conducted by the assay of Gram staining, Gram-negative bacteria were considered as Salmonella. 

3.4.3 Biochemical identification of Salmonella
Salmonella biochemical identification was carried out by using biochemical identification kit. The colonies on nutrient agar slant was inoculated in 1 mL of nutrient broth to make as bacterial suspension for incubation 4~6 h, and then took 70 μL of the liquid of enriched bacteria to inoculate in 10 reaction bottles of biochemical identification kit (of which three are in control), after incubating 18~24 hours at 37℃ by following guideline, all observation items except for lysine decarboxylase reaction can be done, in addition to lysine decarboxylase, the result of the reaction can be observed. If there was no any reaction of lysine decarboxylase observed in incubation of 18~24 h, incubation will be extended 4 days and then re-observed.

3.4.4 Serological identification of Salmonella
Positive Salmonella isolates identified by the biochemical identification was inoculated to Salmonella chromogenic medium and then the purple colonies were picked to steriled petri dishes on a clean slide for agglutination test. The first agglutination test was for OA-â…  serum agglutination, positive strains then used for OA-F slide agglutination serum test, Salmonella strains were genotyped by comparing the common Salmonella antigen.

Authors' contributions
LS is the executor of experimental research in this study, LS, DL and XPP completed data analysis and manuscript preparation; HTS and RPW are the persons who conceived the project and took responsibility to make the experimental design, data analysis, paper writing and revising. All authors have read and agreed the final text.

Acknowledgments
The study was funded by the National Natural Science Foundation of International Cooperation Project (NO: 30910103916). The authors thank to the two anonymous peer reviewers for their critical comments and revising suggestion. Regarding the reagent suppliers mentioned in this article in our experiments, this is not to provide recommend or endorsement for their products and services.

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