Prevalence of Salmonella spp in cloacal, fecal, and gastrointestinal mucosal samples from wild North American turtles

Carley A. Saelinger School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104

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Gregory A. Lewbart Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606.

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Larry S. Christian Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606.

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Carol L. Lemons Department of Molecular and Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606.

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Abstract

Objective—To determine prevalence of Salmonella spp in samples collected from wild North American turtles.

Animals—94 wild North American turtles of 6 species in 2 genera.

Design—Prospective microbiologic study.

Procedures—A convenience sample of wild North Carolina turtles admitted to a veterinary college was evaluated for Salmonella spp by use of standard techniques via microbiologic culture of cloacal swab and fecal samples. Gastrointestinal mucosa samples were also collected at necropsy from turtles that died or were euthanized. Cloacal swab samples were also collected from wild pond turtles for bacteriologic culture. Controls were established by use of wild-type Salmonella Typhimurium LT2.

Results—94 turtles were tested for Salmonella spp; Salmonella spp were not detected in any sample. By use of a pathogen-prevalence and sample-size table, the true prevalence of Salmonella spp was estimated as < 5%.

Conclusions and Clinical Relevance—Results suggested that wild turtles in central North Carolina may not be active shedders or carriers of Salmonella spp. Despite this 0% prevalence of infection, proper hygiene practices should be followed when handling wild turtles.

Abstract

Objective—To determine prevalence of Salmonella spp in samples collected from wild North American turtles.

Animals—94 wild North American turtles of 6 species in 2 genera.

Design—Prospective microbiologic study.

Procedures—A convenience sample of wild North Carolina turtles admitted to a veterinary college was evaluated for Salmonella spp by use of standard techniques via microbiologic culture of cloacal swab and fecal samples. Gastrointestinal mucosa samples were also collected at necropsy from turtles that died or were euthanized. Cloacal swab samples were also collected from wild pond turtles for bacteriologic culture. Controls were established by use of wild-type Salmonella Typhimurium LT2.

Results—94 turtles were tested for Salmonella spp; Salmonella spp were not detected in any sample. By use of a pathogen-prevalence and sample-size table, the true prevalence of Salmonella spp was estimated as < 5%.

Conclusions and Clinical Relevance—Results suggested that wild turtles in central North Carolina may not be active shedders or carriers of Salmonella spp. Despite this 0% prevalence of infection, proper hygiene practices should be followed when handling wild turtles.

Salmonellosis is an important zoonotic infection often associated with contact between pet reptiles and humans in the United States.1 The CDC estimates that the fecal carriage rate of Salmonella spp in pet reptiles is > 90% and that there are approximately 74,000 cases of reptile-associated salmonellosis in the United States annually.1 The CDC also estimates that since banning the sale of turtles < 4 inches in diameter, 100,000 cases of salmonellosis have been prevented.1 A number of studies have investigated the risk of salmonellosis associated with the international pet turtle trade.2-5 A recent study6 found 74% prevalence of Salmonella infection in pet reptiles from Japan. A 2002 study of 62 samples (28 cloacal swab specimens and 34 soil samples) from pet store turtles and privately owned turtles identified Salmonella spp in 11.3% of samples.7 This finding was consistent with other reports8-10 of Salmonella infection rates of 4.5%, 14%, and 16%, respectively, in a variety of captive chelonian species. Although the prevalence of salmonellosis contracted from pet reptiles in humans is high, it is hypothesized that the risk from wild reptiles, particularly turtles, is low because they are not active shedders or carriers of Salmonella spp, as estimated on the basis of previous studies11-13 of freeliving reptiles.This disparity in reptilian Salmonella infection rates may be attributable in part to environmental variations between wild and captive reptiles (captive reptiles may be crowded or subjected to poor hygienic protocol). Salmonella studies of wild North American reptiles are limited, compared with the numerous studies conducted on captive reptiles.3-10,14 In 2004, a Virginia study13 revealed a prevalence of 0% for Salmonella infection in 82 free-living reptiles.

The purpose of the study reported here was to determine the prevalence of Salmonella spp in samples collected from wild North American turtles.

Materials and Methods

Ninety-four wild turtles, including 46 admitted to the NCSU-CVM TRT15 and 48 captured from local ponds, were tested for Salmonella spp during May, June, and July 2005. There were 14 eastern painted turtles (Chrysemys picta picta), 5 common snapping turtles (Chelydra serpentina), 13 Florida cooters (Pseudemys floridana), 17 eastern box turtles (Terrapene carolina carolina), 10 common musk turtles (Sternotherus odoratus), and 35 yellowbelly sliders (Pseudemys concinna concinna).

Cloacal swaba samples were collected from live turtles by inserting a sterile polyester swab into the cloaca and rotating the swab against the inner cloacal wall until fecal material was obtained. Cloacal swabbings were repeated 3 weeks after admittance in 3 eastern box turtles. Cloacal swab samples were also collected from 6 turtles undergoing rehabilitation that were rechecked by the NCSU-CVM TRT. Gastrointestinal mucosa and a cloacal swab sample or fecal material were collected at necropsy from turtles that were dead or euthanized. Gastrointestinal mucosa samples were included to test for nonshedding (carrier-state) turtles.

Each sample was incubated in selenite F brothb for 18 to 24 hours at 35°C and plated on Salmonella-Shigella agarc for 18 to 24 hours at 35°C. After incubation, the agar plates were examined for colonies resembling those of Salmonella spp (clear colonies with a black center characteristic of hydrogen sulfide production). If there were no suspicious colonies, the agar plates were re-incubated for 24 hours at 35°C and rechecked for growth. Suspicious colonies were plated on Christensen urea slantsc and TSI agar slantsd for 18 to 24 hours at 35°C. Salmonella spp were suspected if there was no urease production (no color change to pink on the slant) and the TSI agar slants yielded carbon dioxide and hydrogen sulfide production and the typical reactions of alkaline slant over acidic butt. Suspicious colonies from the urea or TSI agar slants were isolated for biochemical profiling at the NCSU-CVM Clinical Microbiology Laboratory by use of a computerized automated interpretive database.e

Controls were established with saline (0.9% NaCl) solution–diluted samples and feces (equine)-diluted samples with wild-type Salmonella Typhimurium LT2. Salmonella organisms were recovered from the saline solution controls at a concentration of 15 CFU/mL and from the fecal controls at a concentration of 150 CFU/mL.

Results

Overall, 97 cloacal swab samples, 1 fecal sample, and 16 mucosal samples were tested. Prevalence of Salmonella spp was 0% for all samples from the 94 wild turtles, suggesting that the organism was not being actively shed and/or carried without clinical signs. Most samples yielded negative results on SalmonellaShigella agarf after 18 to 24 hours and 48 hours of incubation at 35°C in ambient air. In many samples, a lack of color change on Christensen urea agar slants indicated inability of bacteria to hydrolyze urea. Other samples yielded negative results on TSI agar slants because of failure to produce carbon dioxide or hydrogen sulfide and failure to ferment glucose, lactose, or sucrose. Few bacterial isolates required biochemical profiling; these were determined not to be Salmonella spp. There were 6 Citrobacter freundii isolates.

By interpolation with a pathogen-prevalence and sample-size table (Table 1) used commonly for fish pathogens,16 and assuming a sensitivity of 65% with an observed prevalence of 0%, the true prevalence of Salmonella spp in the turtles was < 5%. If the culture test was 100% sensitive and a large theoretical population was used, a 0% observed prevalence would yield 95% confidence that the true prevalence would be approximately < 3.3%. With a sensitivity of just 50%, the true prevalence would be approximately < 6.6%.

Table 1—

Sample sizes required to detect a sample with positive results, assuming test sensitivity of 100%, in populations of various sizes and at various pathogen prevalences.

Population sizePrevalence
2%5%10%
50503520
100754523
2501105025
5001305526
1,0001405527
10,0001456027
> 100,0001506030

Adapted from Ossiander FJ, Wedemeyer G. Computer program for sample size required to determine disease incidence in fish populations. J Fish Res Board Can 1973;30:1383–1384. Reprinted with permission.16

Discussion

The 0% prevalence of Salmonella carriers and shedders detected in this study among free-living North Carolina turtles was similar to results of some studies11-13 of wild reptiles in North America, but differed from other studies1,6,14 that revealed high prevalence of reptile-associated salmonellosis in humans.

There are 2 general hypotheses regarding the reason captive reptiles shed Salmonella spp at a higher rate than wild reptiles. The first hypothesis is that wild turtles are natural carriers of Salmonella spp but do not actively shed the organism unless relocated to a stressful environment, such as captivity. Our results suggested that wild turtles in North Carolina are not natural carriers of Salmonella organisms. Three turtles admitted to the NCSU-CVM TRT yielded negative results for Salmonella spp upon entrance to the facility and again 3 weeks later, after undergoing the stress of hospitalization. Six wild turtles that were admitted to the NCSU-CVM TRT, and cared for by a turtle rehabilitator, all yielded negative results for Salmonella spp. The second hypothesis is that free-living turtles are not natural carriers of Salmonella spp, but rather acquire the organism and start shedding when brought into the pet industry. The study reported here did not address that issue, but did suggest that wild turtles may not be carriers of the organisms before capture.

One possible explanation for the lack of detectable Salmonella spp from cloacal swabs is a lack of appreciable fecal material, although Salmonella organisms were detected at 150 CFU/mL in the fecal control samples. Lack of detection of Salmonella organisms that may have been in the carrier state could have resulted from poor technique in gastrointestinal scrapings. Another possible explanation is that in some cases, multiple samples may be required to detect Salmonella spp in feces via bacterial culture, perhaps because of intermittent shedding of the organism. In 1 report,17 a PCR assay was more sensitive than ELISA and bacterial culture in detecting Salmonella spp from the cloacalcolon samples of green iguanas (Iguana iguana). The authors estimated that from 30% to 45% of infected animals could go undetected with a culture-only Salmonella screening protocol based on test sensitivities of 55% and 70% in 2 populations of 120 animals each. A study18 of captive lizards revealed that use of single bacterial cultures of cloacal swab samples or feces indicated a prevalence of 62.5% for Salmonella infection in clinically normal lizards and that this method was 75.8% sensitive. Nevertheless, it seems highly unlikely that the present study would have failed to detect Salmonella spp in 94 turtles if the organisms were present.

Results of this study suggest that further investigation of the pet reptile industry and the differences in the environment between captive and wild reptiles are needed. Despite the findings reported here, proper hygiene practices should be followed when handling and maintaining all reptiles.19,20

ABBREVIATIONS

NCSU-CVM

North Carolina State University College of Veterinary Medicine

TRT

Turtle Rescue Team

TSI

Triple sugar iron

CFU

Colony-forming unit

a.

Solon 6-inch Dacron tipped plastic applicator, Solon Manufacturing Co, Solon, Me.

b.

Selenite F broth, VWR International, West Chester, Pa.

c.

Christensen's urea agar slant, Remel Inc, Lenexa, Kan.

d.

Triple sugar iron agar slant, Remel Inc, Lenexa, Kan.

e.

Vitek32, bioMérieux Inc, Hazelwood, Mo.

f.

Salmonella-Shigella agar plate, Remel Inc, Lenexa, Kan.

References

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    CDC Healthy Pets Web site. Spotlight on turtles. Available at: www.cdc.gov/healthypets/spotlight_an_turtles.htm. Accessed Sep 1, 2005.

  • 2.

    Baker EF, Anderson HW, Allard J. Epidemiological aspects of turtle-associated salmonellosis. Arch Environ Health 1972;24:19.

  • 3.

    Chassis G, Gross EM & Greenberg Z, et al. Salmonella in turtles imported to Israel from Louisiana (lett). JAMA 1986;256:1003.

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    Shane SM, Gilbert R, Huntington KS. Salmonella colonization in commercial pet turtles. Epidemiol Infect 1990;105:307316.

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    Tauxe RV, Rigau-Perez JG & Wells JG, et al. Turtle-associated salmonellosis in Puerto Rico. Hazards of the global turtle trade. JAMA 1985;254:237239.

    • Search Google Scholar
    • Export Citation
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    Nakadai A, Kuroki T & Kato Y, et al. Prevalence of Salmonella spp in pet reptiles in Japan. J Vet Med Sci 2005;1:97101.

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    Pasmans F, De Herdt P, Haesebrouck F. Presence of Salmonella infections in freshwater turtles. Vet Rec 2002;150:692693.

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    • Search Google Scholar
    • Export Citation
  • 9.

    Greenberg Z, Sechter I. Salmonella serotypes isolated from snakes and other reptiles—Israel, 1953–1989. Isr J Vet Med 1992;47:4960.

    • Search Google Scholar
    • Export Citation
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    Gopee NV, Adesyun AA, Caesar K. Retrospective and longitudinal study of salmonellosis in captive wildlife in Trinidad. JWildl Dis 2000;36:284293.

    • Search Google Scholar
    • Export Citation
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    Brenner D, Lewbart G & Stebbins M, et al. Health survey of wild and captive bog turtles (Clemmys muhlenbergiii) in North Carolina and Virginia. JZoo Wildl Med 2002;33:311316.

    • Search Google Scholar
    • Export Citation
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    Mitchell JC, McAvoy BV. Enteric bacteria in natural populations of freshwater turtles in Virginia. Va J Sci 1990;41:233242.

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    Richards JM, Brown JD & Kelly TR, et al. Absence of detectable Salmonella cloacal shedding in free-living reptiles on admission to the wildlife center of Virginia. JZoo Wildl Med 2004;35:562563.

    • Search Google Scholar
    • Export Citation
  • 14.

    Geue L, Loschner U. Salmonella enterica in reptiles of German and Austrian origin. Vet Microbiol 2002;84:7991.

  • 15.

    Lewbart GA, Kishimori J, Christian LS. The North Carolina State University College of Veterinary Medicine Turtle Rescue Team: a model for a successful wild-reptile clinic. JVet Med Educ 2005;32:377381.

    • Search Google Scholar
    • Export Citation
  • 16.

    Ossiander FJ, Wedemeyer G. Computer program for sample size required to determine disease incidence in fish populations. J Fish Res Board Can 1973;30:13831384.

    • Search Google Scholar
    • Export Citation
  • 17.

    Mitchell MA, Shane SM & Orr K, et al. Salmonella diagnostic testing in the absence of a gold standard, in Proceedings. Annu Meet Assoc Reptilian Amphib Vet 2000;143144.

    • Search Google Scholar
    • Export Citation
  • 18.

    Pasmans F, Martel A & Boyen F, et al. Characterization of Salmonella isolates from captive lizards. Vet Microbiol 2005;110:285291.

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    Johnson-Delaney CA. Reptile zoonoses and threats to public health. In: Mader DM, ed. Reptile medicine and surgery. 2nd ed. Philadelphia: WB Saunders Co, 2006;10171030.

    • Search Google Scholar
    • Export Citation
  • 1.

    CDC Healthy Pets Web site. Spotlight on turtles. Available at: www.cdc.gov/healthypets/spotlight_an_turtles.htm. Accessed Sep 1, 2005.

  • 2.

    Baker EF, Anderson HW, Allard J. Epidemiological aspects of turtle-associated salmonellosis. Arch Environ Health 1972;24:19.

  • 3.

    Chassis G, Gross EM & Greenberg Z, et al. Salmonella in turtles imported to Israel from Louisiana (lett). JAMA 1986;256:1003.

  • 4.

    Shane SM, Gilbert R, Huntington KS. Salmonella colonization in commercial pet turtles. Epidemiol Infect 1990;105:307316.

  • 5.

    Tauxe RV, Rigau-Perez JG & Wells JG, et al. Turtle-associated salmonellosis in Puerto Rico. Hazards of the global turtle trade. JAMA 1985;254:237239.

    • Search Google Scholar
    • Export Citation
  • 6.

    Nakadai A, Kuroki T & Kato Y, et al. Prevalence of Salmonella spp in pet reptiles in Japan. J Vet Med Sci 2005;1:97101.

  • 7.

    Pasmans F, De Herdt P, Haesebrouck F. Presence of Salmonella infections in freshwater turtles. Vet Rec 2002;150:692693.

  • 8.

    Cambre RC, Green DE & Smith EE, et al. Salmonellosis and arizonosis in the reptile collection at the National Zoological Park. JAm Vet Med Assoc 1980;177:800803.

    • Search Google Scholar
    • Export Citation
  • 9.

    Greenberg Z, Sechter I. Salmonella serotypes isolated from snakes and other reptiles—Israel, 1953–1989. Isr J Vet Med 1992;47:4960.

    • Search Google Scholar
    • Export Citation
  • 10.

    Gopee NV, Adesyun AA, Caesar K. Retrospective and longitudinal study of salmonellosis in captive wildlife in Trinidad. JWildl Dis 2000;36:284293.

    • Search Google Scholar
    • Export Citation
  • 11.

    Brenner D, Lewbart G & Stebbins M, et al. Health survey of wild and captive bog turtles (Clemmys muhlenbergiii) in North Carolina and Virginia. JZoo Wildl Med 2002;33:311316.

    • Search Google Scholar
    • Export Citation
  • 12.

    Mitchell JC, McAvoy BV. Enteric bacteria in natural populations of freshwater turtles in Virginia. Va J Sci 1990;41:233242.

  • 13.

    Richards JM, Brown JD & Kelly TR, et al. Absence of detectable Salmonella cloacal shedding in free-living reptiles on admission to the wildlife center of Virginia. JZoo Wildl Med 2004;35:562563.

    • Search Google Scholar
    • Export Citation
  • 14.

    Geue L, Loschner U. Salmonella enterica in reptiles of German and Austrian origin. Vet Microbiol 2002;84:7991.

  • 15.

    Lewbart GA, Kishimori J, Christian LS. The North Carolina State University College of Veterinary Medicine Turtle Rescue Team: a model for a successful wild-reptile clinic. JVet Med Educ 2005;32:377381.

    • Search Google Scholar
    • Export Citation
  • 16.

    Ossiander FJ, Wedemeyer G. Computer program for sample size required to determine disease incidence in fish populations. J Fish Res Board Can 1973;30:13831384.

    • Search Google Scholar
    • Export Citation
  • 17.

    Mitchell MA, Shane SM & Orr K, et al. Salmonella diagnostic testing in the absence of a gold standard, in Proceedings. Annu Meet Assoc Reptilian Amphib Vet 2000;143144.

    • Search Google Scholar
    • Export Citation
  • 18.

    Pasmans F, Martel A & Boyen F, et al. Characterization of Salmonella isolates from captive lizards. Vet Microbiol 2005;110:285291.

  • 19.

    Chin JC. Control of communicable diseases manual. 17th ed. Washington, DC: American Public Health Organization, 2000.

  • 20.

    Johnson-Delaney CA. Reptile zoonoses and threats to public health. In: Mader DM, ed. Reptile medicine and surgery. 2nd ed. Philadelphia: WB Saunders Co, 2006;10171030.

    • Search Google Scholar
    • Export Citation

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