Posted by Dr. Steve Dudley on February 18 2004 15:51
Isolation of Mycoplasma hyopneumoniae field strains and their characterization by molecular-based technique
The objective of this study is to isolate Mycoplasma hyopneumoniae from field samples, which will be then evaluated and compared by molecular-based diagnostic techniques. Results will be analyzed using a commercial software to determine relationships and then compare them with the field epidemiological information.
Besides generating a Mycoplasma hyopneumoniae collection, results form several Polymerase Chain Reaction (PCR) based tests will be compared.
To achieve this goal, we will proceed as follows:
- Field samples will be obtained from euthanized pigs from various US states. Veterinarians will select animals based on Enzootic Pneumonia clinical sings and/or farm history; empathizing samples from animals in the early stages of infection. For example animals from groups that are just starting to cough. Samples of whole, refrigerated lungs as well as nasal swabs are needed for this study.
- A Bronchoalveolar lining fluid lavage will be performed upon sample arrival in our laboratory. The obtained liquid will be inoculated into previously prepared Friis medium, broth and agar, and incubated at 37°C, CO2 5%, for at least four weeks, while checking for growth indication. Presence of Mycoplasma hyopneumoniae will be confirmed by microscopic observation of colonies and Nested-PCR. DNA concentration (µg/mL) will be measured using a spectrophotometer.
In order to compare strains, DNA extraction will be done and several PCR’s will be performed: RAPD, ERIC, BOX and REP using the specific set of primers for each one and following its own procedure.
Resulting products will be run in an agarose gel, to obtain the sample bands distribution pattern. The information will be processed with BioNumerics software and expressed in dendrograms, where strains will be grouped by percent similarity.
Dendogram information will be contrasted with on-field epidemiological information, in order to decide which PCR gives the best and most logical discrimination.