Use of a SYBR Green Real-Time Polymerase Chain Reaction and Melt Analysis to Detect Mycoplasma Species in Lymph Nodes from Elk (Cervus elaphus)
Mycoplasmas have been identified as disease agents for humans, livestock and wildlife, and mycoplasmas have caused large population declines in some animals. Detecting mycoplasmas in wildlife and identifying them as the cause of disease syndromes is complicated by the culture requirements of many mycoplasmas and the difficulty of obtaining fresh specimens from free-ranging animals.1 A real-time PCR method utilizing SYBR dye has been developed for the detection of Mycoplasma spp. in several different types of biological samples.2 With it, several species of mycoplasma can be differentiated on the basis of the melt temperature and the length and sequence of the PCR amplicon. Twenty-nine elk lymph nodes collected in 2008 by Utah Department of Wildlife Resources during annual chronic wasting disease surveillance were selected for testing. Mycoplasmas were detected in 4 of the lymph node samples. The PCR products were further analyzed with standard gel electrophoresis followed by extraction of the DNA and sequencing. The length of the amplicon was estimated to be 235 bp with a capillary electrophoresis platform (2100 Bioanalyzer, Agilent). The sequences of 2 samples were homologous and further analyzed for similarity to known bacterial sequences with BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences were most similar to M. ovipneumoniae (96%), M. hyopneumoniae (93%), and M. dispar (93%). The biological relevance of the identification of a Mycoplasma spp. in elk is unknown. Outbreaks of mycoplasmal respiratory disease have not been reported in elk. The results suggest that additional research into the occurrence of mycoplasmas in elk as well as other wildlife species is warranted.
The authors would like to thank the Utah State University Center for Integrated Biosystems and the Utah Veterinary Diagnostic Laboratory for the funding and technical support of this project.
1. Besser TE, Cassirer EF, Potter KA, VanderSchalie J, Fischer A, Knowles DP, Herndon DR, Rurangirwa FR, Weiser GC, Srikumaran S. Association of Mycoplasma ovipneumoniae infection with population-limiting respiratory disease in free-ranging Rocky Mountain bighorn sheep (Ovis canadensis canadensis). J Clin Microbiol. 2008;46:423–430.
2. Trujillo J, Justice-Allen A, Morley T, Wilson D. SYBR green real-time polymerase chain reaction detection and differentiation assay for Mycoplasma species in biological samples (abstr.). Proceedings of the American Association of Veterinary Laboratory Diagnosticians. San Diego, CA, USA; 2009.