As reptiles continue to gain in popularity as pets and exhibition animals there is a growing concern regarding zoonotic diseases. Reptiles have long been known to carry Salmonella spp. and reptile associated salmonellosis in humans increases relative to exposure. Hatchling aquatic turtles, Pseudemys scripta elegans, were associated with over 300,000 incident cases of salmonellosis in children annually until interstate commerce was banned in 1974. More recently, increased import and exhibition of reptiles have been correlated with an increase of human salmonellosis.2,6
Salmonella spp. has been diagnosed as a primary disease agent in reptiles, but is most often considered to be a normal component of the intestinal tract. Microbiologic techniques are used to isolate the organism, but diagnostic sensitivity can be affected by a number of factors.3 The time required to isolate and confirm the identity of Salmonella spp. is 72–96 hr with conventional microbiology. The delay makes management of cases and therapy more difficult. The application of a genus specific technique, such as the polymerase chain reaction, is rapid and sensitive and could improve the diagnostic capability and management of cases. Depending on sample quality, PCR can provide a diagnosis within 24–48 hr. The purpose of this study was to determine if available oligonucleotide primers used to diagnose Salmonella spp. in domestic species would be of value in identifying Salmonella serotypes in reptiles. Two distinct oligonucleotide sequences were evaluated in this study to determine their applicability to future studies.
Six Salmonella serotypes were isolated from green iguanas and their environment in Costa Del Sol, El Salvador, including Salmonella arizonae 48: I–Z, S. seftenberg, S. newport, S. havana, S. matadi, and S. saint-paul. Escherichia coli and Pseudomonas aeruginosa were also used to assess the specificity of the primers. Bacteria were collected from stock agar and the DNA was extracted using a commercial kit (Qiagen Inc., Valencia, CA, USA). The first oligonucleotide sequence (SGS) defined the amplified region of a 496 bp highly conserved segment of the histidine transport operon gene of S. typhimurium.4 The primer sequences for the upper and lower oligonucleotides were: lower-5’ ACT GGC GTT ATC CCT TTC TCT GGT G 3’ and upper-5’ ATG TTG TCC TGC CCC TGG TAA GAG A 3’. The second oligonucleotide sequence defined the amplified region of a 457 bp invasion protein (InvA) gene of S. typhimurium.5 The primer sequences for the upper and lower oligonucleotides were: lower-5’ TGC CTA CAA GCA TGA AAT GG 3’ and upper-5’ AAA CTG GAC CAC GGT GAC AA 3’. Polymerase chain reaction mixtures were prepared including a primer set (20 µM SGS or InvA), 6 µl MgCl, 10 µl dNTP, 1 µl Taq polymerase, 10 µl 10x buffer, and 65 µl of sterile water. Seven microliters of the DNA template extracted from each of the Salmonella samples was added to a PCR mixture. Each Salmonella serotype was tested in triplicate for each of the primer sets. The samples were placed in a 9600 thermal cycler (Perkin-Elmer, Saint Quentin, France) and run for 30 sec at 94°C, 30 sec at 60°C, and 45 sec at 72°C for 33 cycles with an additional 10-min extension cycle at 72°C. The amplified samples were subjected to electrophoresis on a 1% agarose gel containing ethidium bromide. The gel was visualized and photographed using a UV transilluminator. A positive result for the SGS amplicon was indicated by a 496 bp band on the gel and a negative result by absence of band formation. A positive result for the InvA amplicon was indicated by a 457 bp band on the gel.
All six Salmonella serotypes produced 496 bp and 457 bp bands on their respective gels. No band was observed for the E. coli and P. aeruginosa control samples. These findings suggest that these genes are conserved at the generic level and would be useful to identify common Salmonella serotypes in the green iguana.
Further studies are required to evaluate the effectiveness of the PCR technique in comparison to standard microbiologic culture to identify Salmonella spp. infection in reptiles. Currently, three successive negative fecal cultures are required to confirm the negative status of a green iguana.1 The polymerase chain reaction is more rapid and sensitive than culture and may reduce time for diagnosis.3
1. Burnham BR, DH Atchley, RP DeFusco, KE Ferris, JC Zicarelli, JH Lee, FJ Angulo. 1998. Prevalence of fecal shedding of Salmonella organisms among captive green iguanas and potential public health implications. JAVMA. 213: 48–51.
2. Centers for Disease Control. 1992. Iguana-associated salmonellosis. Morbidity and Mortality Weekly. 41: 38–39.
3. Cohen ND, LJ Martin, B Simpson, DE Wallis, HL Neibergs. 1996. Comparison of polymerase chain reaction and microbiological culture for detection of salmonellae in equine feces and environmental samples. AJVR. 57: 780–786.
4. Cohen ND, HL Neibergs, ED McGruder, HW Whitford, RW Behle, PM Ray, BM Hargis. 1993. Genus-specific detection of salmonellae using the polymerase chain reaction. J Vet Diagn Invest. 5: 368–371.
5. Galan JE, C Ginocchio, P Costeas. 1992. Molecular and functional characterization of the Salmonella invasion A gene: homology of InvA to members of a new protein. J Bacteriol. 174: 4338–4349.
6. Gangarosa EJ. 1985. Boundaries of conscience. JAMA. 254: 265–266.