Download Investigating the Use of Multiplex PCR to Detect Pathogens

Investigating the Use of Multiplex PCR to Detect
Pathogens Causing Bovine Respiratory Disease
Ellen Graham
Supervisors: Dr. Hywell Ball and Colin Bell
Introduction
Results
Bovine respiratory disease is a major cause of pneumonia in
cattle leading to mortality and resulting in economic losses.
Symptoms of the disease include fever, coughing, discharge
from the eyes and nose. Major bacterial pathogens involved
are Mycoplasma bovis, Trueperella pyogenes, Pasteurella
multocida, Mannhiemia haemolytica and Histophilus somni
which are commonly isolated in various combinations. These
pathogens are currently detected by culture but antibiotics
administered on the farm can mask infection. Polymerase
chain reaction (PCR) procedures that have been developed
for each of these pathogens provide an alternative solution
by amplifying DNA from both viable and non viable cells.
Multiple combinations of PCR were tried together to form a
multiplex but only one gave some positive results that were
comparable to the PCR tests done individually.
The H. somni and T. pyogenes multiplex was the only one
able to detect both pathogens present in a sample. However
this multiplex is not sensitive enough to pick up both
pathogens when the DNA is present in small amounts.
Other multiplex combinations failed to detect multiple
organisms.
Figure 2 shows the results for the multiplex using 16
different field samples which all tested positive individually
for both H. somni and T. pyogenes. Only 5 samples showed
a positive multiplex.
Figure 3 shows the gels of 5 of these 16 samples that had
tested positive individually for both bacteria. The H. somni
and T. pyogenes multiplex was able to detect both
pathogens in some samples, however it was only detecting
one in other samples.
Figure 1 - Picture of livestock
Aims
This study investigated the application of various multiplex
PCR combinations of these bacteria. Detection of multiple
pathogens by this procedure could improve time and cost
efficiency.
Methods
Template DNA was isolated from lung tissue obtained from
the post-mortem of animals submitted to VSD for diagnostic
investigation. Samples were tested by multiplex PCRs and
compared to individual pathogen PCR using previously
described primers (1,2,3 & 4). PCR products were run in a
0.9% agarose gel electrophoresis.
Figure 2 - Graph showing pathogen DNA present in 16 different field
samples
T. pyogenes and M. haemolytica multiplex may have worked
but due to the comparative product size (150 bp and 143 bp)
it was impossible to distinguish between them on a gel.
Multiplexes incorporating P. multocida showed that PCR
reagents
favoured smaller product size, both
M. haemolytica (143 bp) and H. somni (400 bp) were
detected but none showed positive for P. multocida (567 bp)
however in an individual PCR there were positive results for
this bacterial pathogen.
Conclusion
Figure 3 - Gel electrophoresis showing multiplex for H. somni and
T.pyogenes respectively
The multiplex for H. somni and T. pyogenes does work but
the PCR is unable to detect all positives and therefore is not
suitable to be used as a diagnostic test.
References:
1 Angen et al (1998) Veterinary Microbiology 63:39-48.
2. Silva et al (2008) Veterinary Microbiology 132:111-118.
3. Dongyou et al (2004) Journal of Microbiological Methods 58:263-267.
4. Guenther et al (2008) Journal of Clinical Methods 75:75-80.