Application of PCR to Diagnose a Chicken Disease Caused by Mycoplasma Gallisepticum

News source: Department of Agriculture and Forestry Extension, Ministry of Agriculture and Rural Development (MARD), Vietnam
For further information, contact Le Minh Sat, National Institute of Animal Husbandary, Vietnam: Phan Thanh Phuong, National Institute for Veterinary Research, MARD, Vietnam.
See PDF file for e-mail address., 2002-02-01

Mycoplasma Disease of Chickens

Mycoplasma disease of chickens is a major problem in poultry production. The pathogen has many strains, so that diagnosis of the disease using rapid coagulation on Lamel SPA is not very effective. The aim of this study was to find a more accurate method of diagnosing the disease.

Extraction of Dna

The extraction of nucleic acids from test samples for mycoplasma detection was undertaken, following the CIRAD-EMVT protocol. Primer pairs RP5 and FP2 for polymerase chain reaction were made from gene fragment 16s rARN of Mycoplasma gallisepticum. Two hundred and seventy-five diseased samples were used in determining the mycoplasma strains. A Promega kit was used to extract the DNA from the samples.

PCR Analysis

PCR analysis followed by electrophoresis of PCR products showed that the line of M. gallisepticum has 4 bands 401, 143, 199 and 78 base pairs. Meanwhile the line of M. gallinarum has 2 bands of 538 and 199 base pairs. This enabled us to distinguish the two strains of mycoplasma by PCR and RFLP. PCR is a highly sensitive method, giving a positive reaction at a concentration of 6.10-2 pg DNA or 50 CFU (colony forming unit). PCR analysis showed that M. gallisepticum was detected in 66.0% of samples from diseased chickens, while M. gallinarum was detected in 38.5% of samples. However, mycoplasma was detected in only 38.0% of disease samples when SPA was used.

Accuracy of PCR

In conclusion, PCR allows us to determine the percentage of infested chickens with mycoplasma more accurately than SPA. This method also allows us to detect pathogens in cattle manure and drinking water, and in chicken embryos that test negative with SPA.

M. gallisepticum and M. gallinarum can be distinguished by PCR in combination with restricted enzyme Rsal.

Download the PDF. of this document, 29,668 bytes (29.0 KB).