Denaturing gradient gel electrophoresis (DGGE): A tool to assess diversity of Mycoplasma species in small ruminants

Abstract

Mycoplasma diseases of livestock such as contagious bovine pleuropneumonia, contagious caprine pleuropneumonia and contagious agalactia still represent major problems for animal health authorities worldwide. Many significant improvements have been seen as a result of research into these degenerate bacteria, mainly in the area of diagnosis, with the availability of specific, effective new molecular tools to detect small quantities of these pathogens (some of them highly fastidious, unculturable and often in mixed bacterial culture) directly from clinical samples. The objective of the present study was to identify and differentiate Mycoplasma species of sheep, goats and cattle by a new diagnostic test based on PCR of the 16S rRNA gene with specific primers and separation of the PCR product according to primary sequence using denaturing gradient gel electrophoresis (DGGE). Detection and identification results were compared with the ones obtained by sequence analysis. The development of a PCR DGGE offers advantages of a rapid identification of many Mycoplasma species for which no specific PCR is yet available, enabling the differentiation of animal Mycoplasma species. It represents a significant improvement from conventional culture and serological tests in term of specificity and time to perform. *Correspondence to: Nicholas RAJ, The Oaks, Nutshell Lane, Farnham, Surrey, UK, E-mail: robin.nicholas@btinternet.com Received: August 20, 2019; Accepted: August 28, 2019; Published: August 30, 2019 Introduction Mycoplasmas cause a wide range of disease in both humans and animals and are commonly associated with pneumonia, arthritis, conjunctivitis, infertility and abortion. Mycoplasmas are highly fastidious, typically taking weeks to culture while many serological tests are non-specific and poorly sensitive. More recently PCRs have been used to detect Mycoplasma species, but it is not feasible to develop PCR tests for each species. Consequently, there is a significant need for a single, generic test that can both detect and differentiate Mycoplasmas. Denaturing gradient gel electrophoresis (DGGE) can theoretically detect single base mutations in DNA [1-3]. Amplicons of the same length but with different nucleotide compositions are separated in a denaturing gradient gel of polyacrylamide, based on their differential denaturation profile. During denaturation, the two strands of a DNA molecule separate or melt at a specific denaturant concentration, and the DNA sequence stops its migration in the gel. PCR DGGE allows the detection and differentiation of mixed and very small amounts of Mycoplasmas showing higher sensitivity than the conventional tests. Using this method McAuliffe et al. [4,5] enabled the differentiation of 67 Mycoplasma species of human and animal origin representing a significant improvement on current tests. Diagnosis of Mycoplasma infection can be made directly from clinical samples in less than 24 h and can even detect new and unculturable Mycoplasmas. We report the application of DGGE to detect pathogenic and nonpathogenic Mycoplasmas from small ruminants.