APPENDIX B
Soil samples collection
For the purpose of collecting appropriate soil samples from farms visited, the farms
were categorized as intensive, semi-intensive and extensive farms. For the intensively
managed farms, the soil samples were taken within the farm premises especially near
the sources of the animals’ feed and water. In farms practicing semi intensive and
extensive systems, soil samples were taken within the farm premises near sources of
animals’ feed and water and outside the premises where the animals graze. In each
farm, triplicate samples were taken.
The soil sampling procedure was as described by Chantratita et al., (2008).
Accordingly, a small hole was dug with a clean spade to a depth of approximately
30cm. About 500 - 700g of soil was taken from the base of the hole and placed in a
clean plastic bag. Each plastic bag was labeled with permanent marker denoting farm
code and date of collection. The plastic bags were sealed to prevent moisture loss and
stored under shade out of direct sunlight at ambient temperature inside an iceman’s
Cool Box. Triplicate samples were collected from three different spots in each farm
visited. The sampling utensils were cleaned between each use by rinsing with water to
remove soil debris and then disinfected with 70% alcohol after which were allowed to
air-dried before next usage. The samples were transported to the laboratory and
processed the same day.
Processing and culturing of soil samples in laboratory
The processing of the soil samples is known as the Simplified Method of Isolation of
the agent described by Limmathurotsakul et al., (2012). The method uses 100g of soil
and pre-enrichment of soil solution in selective broth. Accordingly, 100g of soil
samples were measured into plastic bags and 100ml of distilled water were added,
shaken manually for 1 minute and the mixtures allowed to sediment overnight. After
overnight sedimentation, 100μl of the supernatant was plated on Ashdown agar plates.
The plates were incubated in air at 37°C and observed for 4 days for growth of
presumptive isolates. Suspected isolates were screened using catalase and oxidase
tests and positive samples were preserved in glycerol/BHI media until final
confirmation using PCR amplification.
For the liquid culture enrichment of B. pseudomallei from the soil samples, 1 ml of
the same supernatants were inoculated into 9 ml of Ashdown’s selective broth
supplemented with 1g/l of polymixin B. The broths and agar plates were incubated in
air at 37°C for 48hours. The plates were inspected daily for the typical B.
pseudomallei colonies while the broths were sub-cultured on Ashdown’s agar,
incubated at 37°C in air and observed again for another 4 days for the typical B.
pseudomallei colonies. The presumptive colonies were treated the same way as in
methods were screened using biochemical tests and API 20NE KITSand stored
pending final confirmation using PCR.
References:
Chantratita, N., V. Wuthiekanun, D. Limmathurotsakul, M. Vesaratchavest, A.
Thanwisai, P. Amornchai, S. Tumapa, E. J. Feil, N. P. Day and S. J. Peacock,
2008: Genetic diversity and microevolution of Burkholderia pseudomallei in
the environment. PLoS Neglected Tropical Diseases, 2, e182
Limmathurotsakul, D., V. Wuthiekanun, P. Amornchai, G. Wongsuwan, N. P. Day
and S. J. Peacock, 2012: Effectiveness of a simplified method for isolation of
Burkholderia pseudomallei from soil. Applied and Environmental
Microbiology, 78, 876-877.