A simple random amplified polymorphic DNA genotyping method for field isolates of Dermatophilus congolensis.
Dermatophilus congolensis is the pathogenic actinomycete that causes dermatophilosis in cattle, lumpy wool in sheep and rain scald in horses. Phenotypic variation between isolates has previously been described, but its genetic basis, extent and importance have not been investigated. Standard DNA extraction methods are not always successful for D. congolensis due to its complex life cycle, one stage of which is encapsulated. Here we describe the development of rapid and reliable DNA extraction and random amplified polymorphic DNA (RAPD) methods that can be used for genotyping D. congolensis field isolates. Our results suggest that genotypic variation between isolates correlates with host species. Several DNA extraction methods and RAPD protocols were compared. An extraction method based on incubation of the bacterium in lysozyme, sodium dodecyl sulphate (SDS) and proteinase K treatments and phenolic extraction yielded high-quality DNA, which was used to optimize RAPD-polymerase chain reaction (PCR) protocols for two random primers. An alternative rapid, non-phenolic extraction method based on proteinase K treatment and thermal shock was selected for routine RAPD typing of isolates. DNA extracted from reference strains from cattle, sheep and horse using either method gave reproducible banding patterns with different DNA batches and different thermal cyclers. The rapid DNA extraction method and RAPD-PCR were applied to 38 D. congolensis field isolates. The band patterns of the field and type isolates correlated with host species but not with geographical location.
Larrasa
J
J
Cátedra de Patologia Infecciosa, Universidad de Extremadura, Facultad de Veterinaria, Cáceres, Spain.
Garcia
A
A
Ambrose
N C
NC
Alonso
J M
JM
Parra
A
A
de Mendoza
M Hermoso
MH
Salazar
J
J
Rey
J
J
de Mendoza
J Hermoso
JH
D000192:Actinomycetales / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D000193:Actinomycetales Infections / Q000382:microbiology / Q000662:veterinary*; D000818:Animals; D002417:Cattle / Q000382:microbiology; D002418:Cattle Diseases / Q000382:microbiology; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics*; D005838:Genotype; D006734:Horse Diseases / Q000382:microbiology; D006736:Horses / Q000382:microbiology; D019105:Random Amplified Polymorphic DNA Technique / Q000592:standards / Q000662:veterinary*; D012756:Sheep / Q000382:microbiology; D012757:Sheep Diseases / Q000382:microbiology; D017192:Skin Diseases, Bacterial / Q000382:microbiology / Q000662:veterinary*
D017931:DNA Primers; D004269:DNA, Bacterial