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Separating Mycoplasma gallisepticum field strains from nonpathogenic avian mycoplasmas
Cynthia M Boettger1, John E Dohms
1Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716-2150, USA.
Abstract:
Mycoplasma gallisepticum (MG) has repeatedly emerged as a serious problem in U.S. broiler, layer, and turkey industries. Tracing the source of an outbreak is essential if MG control is to be accomplished. Amplified fragment length polymorphism (AFLP), random amplification of polymorphic DNA (RAPD), and restriction fragment length polymorphism (RFLP) are valuable tools used to study MG epidemiology, allowing diagnosticians to determine the source of MG infections. In some past outbreaks, AFLP, RAPD, and RFLP fingerprinting, which require pure MG cultures, were not successful because of contaminating nonpathogenic mycoplasmas from field samples. The objective of this research was to develop a method to separate rapidly growing nonpathogenic avian mycoplasma species from slower-growing MG field strains. Mixtures of MG and three separate nonpathogenic avian mycoplasmas were inoculated onto chick embryo fibroblasts cells (CEF) allowing MG to penetrate the CEF cells. Later, gentamicin sulphate was added to the culture, eliminating the nonpathogenic mycoplasmas and allowing MG to be isolated in pure culture. Mixtures of Mycoplasma synoviae (MS) and MG could not be separated in this assay. However, removal of nicotinamide adenine dinucleotide and cysteine hydrochloride during serial passage in Frey broth medium successfully eliminated growth of MS.
Insights
A new method effectively isolates Mycoplasma gallisepticum (MG) from avian samples by using chick embryo fibroblasts and gentamicin, overcoming contamination issues. This aids in tracing MG outbreaks and improving poultry health management.
Area of Science:
- Veterinary Microbiology
- Avian Pathology
- Molecular Epidemiology
Background:
- Mycoplasma gallisepticum (MG) outbreaks pose significant economic threats to the U.S. poultry industry (broilers, layers, turkeys).
- Accurate identification of MG sources is crucial for effective disease control and prevention strategies.
- Existing molecular techniques like AFLP, RAPD, and RFLP for MG strain differentiation are hindered by nonpathogenic mycoplasma contamination in field samples.
Purpose of the Study:
- To develop a rapid and effective method for isolating pure Mycoplasma gallisepticum (MG) field strains from samples contaminated with faster-growing, nonpathogenic avian mycoplasmas.
- To improve the reliability of epidemiological studies and outbreak source tracing for MG.
Main Methods:
- Co-cultivation of MG with nonpathogenic avian mycoplasmas on chick embryo fibroblasts (CEF) to allow selective MG penetration.
- Treatment of co-cultures with gentamicin sulphate to eliminate contaminating nonpathogenic mycoplasmas, enabling pure MG isolation.
- Modification of Frey broth medium by removing specific components (nicotinamide adenine dinucleotide, cysteine hydrochloride) to inhibit Mycoplasma synoviae (MS) growth.
Main Results:
- The developed method successfully separated and isolated pure MG cultures from mixtures containing nonpathogenic avian mycoplasmas using CEF cells and gentamicin treatment.
- The assay was unable to separate mixtures of Mycoplasma synoviae (MS) and MG using the CEF/gentamicin method.
- Serial passage in a modified Frey broth medium (lacking nicotinamide adenine dinucleotide and cysteine hydrochloride) effectively eliminated MS contamination.
Conclusions:
- A novel isolation technique using CEF cells and gentamicin provides a reliable method for obtaining pure MG cultures, essential for accurate molecular fingerprinting and epidemiological investigations.
- While the CEF/gentamicin method is effective against common nonpathogenic contaminants, a separate strategy involving modified broth medium is required to eliminate Mycoplasma synoviae.
- These advancements enhance the ability to trace MG outbreak sources, contributing to improved disease management and reduced economic losses in the poultry sector.
