COMPARISON OF DIFFERENT MOLECULAR TYPING METHODS FOR CHARACTERISATION OF Riemerella anatipestifer ISOLATES
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Date
2019
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College of Veterinary and animal Science,Mannuthy
Abstract
The pathogen causing septicaemia and infectious serositis in ducks and
wide variety of wild birds, Riemerella anatipestifer has been categorised as a
member of Flavobacteriaceae by 16S rRNA sequence analysis. At present, 21
serotypes have been characterised using serum agglutination test. So far, the
molecular determinants for R. anatipestifer serotyping are not clear; moreover,
serotype analysis is time-consuming and labour-intensive. To find a better
molecular typing method for R. anatipestifer, serotype-associated genetic
variations has to be determined. Hence, the 20 field isolates (maintained as
lyophilised culture and also collected from the suspected cases reported to the
Department of Veterinary Microbiology) were subjected to different molecular
methods like Polymerase chain reaction- Restriction fragment length
polymorphism (PCR-RFLP), Repetitive-sequence based PCR (REP- PCR),
Internal transcribed spacer (ITS) nucleotide sequencing, Omp A gene sequencing
and pulsed field gel electrophoresis (PFGE) to determine the genetic variations.
The phylogenetic trees constructed based on the variations of the ITS region
grouped the organism into four clusters and the amino acid sequence variation of
OmpA region categorised it into two clusters. The Omp A sequence correlated
much better with serotyping than ITS. By comparing the pattern based methods,
REP-PCR and PCR-RFLP showed similarity with PFGE which distinguished the
isolates into three pulsotypes, Although all methods displaying broadly similar
discriminatory powers, REP-PCR subtyping proved to be a much easier, cheaper
and more rapid method. To conclude many techniques are available but each of
them has advantages and limitations which making them useful in some studies
and restrictive in others. Hence to group the local isolates with universally
categorised isolates, one or more of the different typing methods need to be
applied in conjunction with conventional methods.