TDK conference 2013

The disease with greatest prevalence and economic importance in dairy cows is mastitis, which is caused mostly by bacteria. By reducing the most common pathogens efficiently, the less known environmental pathogens have started to spread, like the representatives of Corynebacterinae suborder in genera Mycobacterium, Corynebacterium, Rhodococcus, Nocardia and Dietzia, as well as Gordonia paraffinovorans in genus Gordonia, whose role in mastitis has not been published in the literature, yet.

The aim of this study was to develop a diagnostic method, which can help identify these special pathogens for routine laboratories.

The bovine milk samples collected from 2006 were isolated on Columbia agar, incubated for 48 hours on 37ºC temperature and another 48 hours on room temperature, then morphology of the colonies were examined, Gram and Ziehl-Neelsen staining, catalase, oxidase and indole tests were executed. One colony from each sample was inoculated to Columbia or Middlebrook agar to make a pure culture, then urease and nitrate tests were performed. 52 strains were analysed by Biolog Microlog M system, and 62 strains were defined by multiplex polymerase chain reaction and sequencing.

The colonies evolved during the longer incubation period were not suitable to differentiation with quick tests and classical biochemical examinations, so the Microlog system capable to analyse 94 phenotypic tests was used. From the 52 analyzed strains 16 were identified at species level, however only two were proved to be accurate. The strains defined by sequencing were 34 Mycobacterium smegmatis, 1 Mycobacterium fortuitum, 22 Gordonia paraffinivorans, 2 Nocardia sp., and 1 strain of Corynebacterium bovis, Rhodococcus erythropolis and Dietzia sp.

The database of Microlog does not contain the Mycobacterium and Gordonia species identified by sequencing, but the system allows to create a custom ID, which can be made in a further study by using a larger number of samples, so this program can worked up suitable to identify these slowly growing mastitis pathogens. The sequences obtained as results of the molecular biological investigations were not completely identical for the same species, the analysis of the sections differing consistently at certain locations may be subject of a further research.