Molecular studies on the fish pathogen Yersinia ruckeri
Scott, Callum James Walter
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Yersinia ruckeri is the aetiological agent of enteric redmouth (ERM), a disease of salmonids, notably rainbow trout (Oncorhynchus mykiss, Walbaum). Until the 1990s, prophylaxis was achieved using a formalin-inactivated whole-cell vaccine of a motile (= flagellin producing) Y. ruckeri strain. However, outbreaks of ERM have since occurred in vaccinated livestock which heralded the emergence of a new biogroup. In addition to giving a negative result for the Voges–Proskauer (VP) reaction and the production of an extracellular lipase, strains responsible for the majority of these new outbreaks in vaccinated stock were non-motile and unable to produce detectable flagellin. It was the aim of this study to determine what protective role flagellin may have towards Y. ruckeri infection, both as a component of the whole-cell vaccine and as a vaccine in itself (i.e. sub-unit vaccine). Results showed that protection against bacterial challenge, either with a motile or non-motile Y. ruckeri strain, was not entirely dependent on the presence of flagellin within the whole-cell vaccine. On the other hand, administering native flagellin (50 μg/fish) via intraperitoneal injection (without adjuvant) resulted in excellent levels of protection (relative percent survival = 100%) against challenge 28 days postvaccination with a flagellin-producing (YR1) or flagellin-devoid (R1) Y. ruckeri strain. Use of recombinant flagellin (r-flagellin) as a vaccine again confirmed the protective properties against challenge with both YR1 and R1 strains, even at lower concentrations i.e. 10 μg/fish. Protection was also conferred after a relatively short period of time (14 days) without any detrimental effect on health or weight of the fish. Thus flagellin has the potential to be an efficacious, non-specific sub-unit vaccine for rainbow trout. Analysis of whole cell proteins by SDS-PAGE from both motile and non-motile isolates demonstrated that highly virulent EX5 isolates which caused disease in vaccinated livestock were overexpressing a 30 to 40 kDa protein. 2D SDS-PAGE and Maldi-tof mass spectrometry identified this protein as outer membrane protein A (OmpA). However, attempts to disrupt the gene encoding the OmpA protein (ompA) using transposon mutagenesis and PCR screening failed to isolate a mutant with a transposon within the gene of interest (ompA::Tn-RL27).