Detection of Salmonellae in Turtles and Their Aquatic Habitats
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A sensitive and accurate methodology for the detection of salmonellae in wild turtles and in the environment was developed using a combination of traditional culture techniques and molecular tools. A combination of a semi-selective enrichment in growth media and subsequent detection by polymerase chain reaction (PCR) enhanced the sensitivity of detection and allowed us to detect salmonellae in sediments and wild turtles. The methodology was developed and tested on captive turtles, which are well-documented carriers of salmonellae. In order to determine the versatility and resolution of the proposed methods they were subsequently used to analyze environmental samples collected from both wild turtles and sediments from a relatively undisturbed environment (Spring Lake, San Marcos, Texas) as well as heavily impacted environments (Rio Grande River, Big Bend National Park, Texas, and near Elephant Butte Reservoir, New Mexico). Sterile swabs were used to gather samples from sediments as well as the cloacae and carapace of turtles from Spring Lake. Captive turtles and turtles from the Rio Grande River were swabbed at five different locations on the body. Swabs were collected into 2-ml-cryotubes containing 1 ml buffered peptone water (pre-enrichment cultures). Samples were enriched in Rappaport-Vassiliadis (RVS) broth, which is semi-selective for salmonellae. Cultures were then tested for salmonellae using PCR, targeting the invA gene that encodes a protein of a type III secretion system, essential for the invasion of epithelial cells by salmonellae. The presence of salmonellae was verified on selected samples using in situ hybridization with probe Sal3, which binds to the 23S rRNA of the vast majority of Salmonella enterica sub-species. Finally, individual colonies from selected turtle and sediment samples were isolated using traditional plating techniques and subjected to molecular, physiological and serological identification in order to verify the reliability of the methodology developed.