Salmonella enterica subspecies enterica serotype Dublin (S. dublin) is a major cause of salmonellosis in cattle. Double antimicrobial-resistant mutants were derived from S. dublin wild strain FD436 utilising spontaneous point-chromosomal (metabolic-drift) mutations induced by high concentrations of nalidixic acid and rifampicin. The estimated frequency of
reversion to the parent strain of the mutants was calculated to be in the range of 10-20- 10-22 Eight mutants with reduced growth rates were selected for fiirther evaluation as live, attenuated vaccine candidates.
The eight mutants selected were easily distinguishable from FD436 on the basis of both genetic and phenotypic characteristics. All mutants had at least one mutation in both gyrA and rpoB genes. The mutants were less motile than FD436 and also differed from the parent strain in terms of sugar fermentation profiles, gas production during glucose fermentation, LPS profiles and protein profiles. One mutant, N-RM25, produced small flat colonies on SBA and took longer to produce hydrogen sulphide on XLD medium compared to FD436 and the other mutants. Three vaccine candidates, N-RM4, N-RM25 and R-NM29, were then selected for further study
on the basis of conventional criteria, such as stability of mutation and absence of cross-resistance to other antimicrobials.
A novel immune activation concept, based on slow "drip-feeding" stimulation (gastrointestinal immunity from a reservoir of organisms in the gall bladder), was hypothesised to define a vaccine strain that would produce long-lasting protection against S. dublin infection without deleterious side-effects on the host. This concept resulted in the creation of three additional novel selection criteria: 1) the sensitivity of the candidates to bile; 2) penetration of the gall bladder, and; 3) shedding of the candidate to the intestine, potentially promoting mucosal immunity. N-RM25 satisfied both the conventional and novel criteria. This strain grew at a markedly slower, but consistent, rate in the presence of a broad range of bile concentrations when compared with the
wild sfrain and the other mutants. In addition, despite significant attenuation (ID50: >5.1 X 108 cfu/mouse), N-RM25 still penetrated the liver, spleen and gall bladder in a mouse model following administration via the intraperitoneal route, and survived in these organs at < 102 cfu/g tissue (liver and spleen) for a prolonged period (> 24 days) post inoculation without causing clinical symptoms. The strain also reached the intestine, most likely via the biliary system, and was eliminated from the body within 2 months.
N-RM25 showed excellent promise as a vaccine candidate, as it provided 100% protection against lethal challenge, caused no disease and was not shed for prolonged periods (< 7 days) following vaccination studies in the mouse model of S. dublin infection. This protection was produced by
intraperitoneal vaccination with doses of greater than 107 cfu/mouse, irrespective of whether a single or a double dose was administered. In conclusion, N-RM25 is sufficiently attenuated and retains sufficient antigenicity to use as a vaccine candidate in further studies including vaccine trials in calves and cattle.