Mark Breslin

Diversity of Strains of Salmonella enterica Serotype Enteritidis from English Poultry Farms Assessed by Multiple Genetic Fingerprinting

ABSTRACT Reliable and sufficiently discriminative methods are needed for differentiating individual strains of Salmonella entericaserotype Enteritidis beyond the phenotypic level; however, a consensus has not been reached as to which molecular method is best suited for this purpose. In addition, data are lacking on the molecular fingerprinting of serotype Enteritidis from poultry environments in the United Kingdom. This study evaluated the combined use of classical methods (phage typing) with three well-established molecular methods (ribotyping, macrorestriction analysis of genomic DNA, and plasmid profiling) in the assessment of diversity within 104 isolates of serotype Enteritidis from eight unaffiliated poultry farms in England. The most sensitive technique for identifying polymorphism wasPstI-SphI ribotyping, distinguishing a total of 22 patterns, 10 of which were found among phage type 4 isolates. Pulsed-field gel electrophoresis of XbaI-digested genomic DNA segregated the isolates into only six types with minor differences between them. In addition, 14 plasmid profiles were found among this population. When all of the typing methods were combined, 54 types of strains were differentiated, and most of the poultry farms presented a variety of strains, which suggests that serotype Enteritidis organisms representing different genomic groups are circulating in England. In conclusion, geographical and animal origins ofSalmonella serotype Enteritidis isolates may have a considerable influence on selecting the best typing strategy for individual programs, and a single method cannot be relied on for discriminating between strains.

The efficacy of Salenvac, a Salmonella enterica subsp. Enterica serotype Enteritidis iron-restricted bacterin vaccine, in laying chickens

The protective effect of two vaccination regimes using Salenvac, a commercially available iron-restricted Salmonella enterica subsp. Enterica serotype Enteritidis PT4 bacterin vaccine, was verified in laying birds. Immunization was intramuscular at 1 day old and again at 4 weeks of age (V2), or at 1 day and 4 weeks with a third dose at 18 weeks of age (V3). Challenge S . Enteritidis (5 to 7.5) × 10 7 colony forming units) was given intravenously at 8, 17, 23, 30 and 59 weeks of age. For all age groups, both vaccination regimes reduced significantly the number of tissues and faecal samples that were culture positive for the challenge strain. For laying birds, fewer eggs ( P < 0.001) were culture positive for S . Enteritidis after challenge from vaccinated laying birds (56/439 batches of eggs) than unvaccinated birds (99/252 batches). The data give compelling evidence that the vaccine is efficacious and may contribute to the reduction of layer infection and egg contamination.

Persistence and clearance of different Salmonella serovars in buildings housing laying hens

We investigated factors associated with persistence of different Salmonella serovars in buildings housing laying hens in Great Britain using survival analysis. A total of 264 incidents of Salmonella detection occurring between July 1998 and August 2007 in 152 houses were recorded. For incidents involving Salmonella Enteritidis (SE), both the rodent score of the house and the type of house were positively associated with persistence. For non-SE serovars, only the type of house was associated with persistence. Persistence of SE in the houses was longest (>15 months) in step-cage and cage-scraper houses when high levels of rodents were present, and lowest in non-cage and cage-belt houses. We estimated that 42% (95% CI 23.3-63.1) of SE incidents may be cleared during the lay period, and this was related to elimination of rodents from the houses. From January 2009, EU legislation will ban the sale of fresh eggs from SE-positive and S. Typhimurium-positive flocks over their remaining lifespan. If infection is eliminated from such flocks, they would cease to represent a public health risk.

A longitudinal study of environmental salmonella contamination in caged and free-range layer flocks

The environmental contamination by salmonella was examined over a 12-month period in 74 commercial layer flocks from eight farms in the UK, which previously had been identified as being contaminated with salmonella. Samples of faeces, dust, litter, egg belt spillage and wildlife vectors were taken, plus swabs of cages, feeders, drinkers, floors, egg belts and boots. Some sampling was performed in each month of the year. Numerous serovars were detected but Salmonella enterica serotype Enteritidis was the only persistent serotype found among single-age flocks. There was a significant correlation between qualitative environmental samples and semi-quantitative faeces samples. The level of environmental contamination increased significantly over time. There were significant temperature and seasonal effects upon contamination. Wildlife vectors proved to be sensitive samples for the detection of salmonella. The efficacy of cleaning and disinfection upon residual salmonella contamination, and upon subsequent flock contamination, was highly variable between and within premises. The variability between detected prevalences over time and between flocks indicates a need for regular, sensitive monitoring of flocks for salmonella to permit targeting of control measures aimed at eliminating contamination of the layer environment by salmonella. There is substantial scope for improvement of cleaning and disinfection procedures.

Molecular fingerprinting evidence of the contribution of wildlife vectors in the maintenance of Salmonella Enteritidis infection in layer farms

Aims: To provide molecular fingerprinting evidence of the contribution of wildlife vectors in the on‐farm epidemiology of Salmonella Enteritidis infections.

A laboratory study of an inactivated bivalent iron restricted Salmonella enterica serovars Enteritidis and Typhimurium dual vaccine against Typhimurium challenge in chickens

A commercial inactivated iron restricted Salmonella Typhimurium and Salmonella Enteritidis vaccine was used to vaccinate chicks at 1 day and again at 4 weeks of age, with challenge by a high and a low dose of S. Typhimurium given either orally or by contact with seeder birds inoculated orally with a high dose of S. Typhimurium. In all three challenge regimes, the shedding of challenge strain was reduced significantly (p < 0.05) in vaccinated birds compared with unvaccinated controls. Vaccination reduced colonisation of internal organs after challenge by contact seeder birds. However, no effect of vaccination upon colonisation of internal organs after either high or low oral challenge was apparent. In conclusion, the data indicate that the vaccine should be a useful tool in the control of S. Typhimurium infection in chickens.

A comparison of the efficacy of cleaning and disinfection methods in eliminating Salmonella spp. from commercial egg laying houses

Effective terminal cleaning and disinfection (C&D) is regarded as a necessary step for the elimination of Salmonella spp. from laying houses. A total of 60 commercial laying houses that had housed laying flocks infected with Salmonella enterica serovar Enteritidis or Salmonella enterica serovar Typhimurium that were representative of all production systems (cage, barn, free-range) were intensively sampled immediately after C&D as well as in the follow-on flock. The procedures investigated were: (1) a compound disinfectant consisting of a mixture of formaldehyde, glutaraldehyde and quarternary ammonium applied at the recommended concentration; (2) a 10% (vol/vol) dilution of the standard 37% commercial formalin, applied by a contractor; and (3) other disinfection procedures selected and applied by the farmer. The recovery of Salmonella in the cleaned and disinfected houses was variable, with samples from floor and dropping boards/belts (cage houses) and scratching areas (non-cage houses) being the most likely to remain contaminated. In cage houses, the use of the 10% formalin dilution led to a statistically greater reduction in the sample prevalence than using any of the other C&D methods. A negative post-C&D result predicted clearance of Salmonella in 52% of cases, although the isolation of Salmonella from the houses immediately after C&D was not a perfect predictor of carry-over of infection.

Semiquantitative assessment of the distribution of Salmonella in the environment of caged layer flocks

Aims:  To evaluate a semiquantitative technique for the enumeration of Salmonella in the environment of layer flocks and to compare findings with those of a standard qualitative technique.

Evaluation of commonly-used farm disinfectants in wet and dry models of Salmonella farm contamination

Two experimental models of Salmonella contamination were used in an attempt to mimic the conditions of disinfectant use on farms. A wet model, for conditions such as boot dips, used disinfectant application to a slurry of poultry faeces inoculated with Salmonella Enteritidis or Salmonella Typhimurium. A dry model, for disinfectant application to surfaces and equipment with adherent or residual organic material, used Salmonella-inoculated poultry faeces that were air-dried onto wooden dowels, immersed in disinfectant solution then left in air at room temperature overnight. All samples were subjected to a disinfectant neutralization step and resuscitation in broth, followed by Salmonella culture on semi-solid then indicator media. Disinfectants were tested at 0.5x, 1x and 2x the concentrations specified for the general control of bacterial pathogens on livestock premises in the UK (Defra General Orders rates). Chlorocresol-based disinfectants provided consistently high rates of Salmonella killing in both wet and dry tests. Formaldehyde-containing disinfectants showed very high efficacy in the dry test but were less effective in the shorter wet test, whereas the efficacy of glutaraldehyde without formaldehyde was variable between products. Other chemical classes tested (quaternary ammonium compounds, amphoteric surfactants, iodine preparations, peroxygens and a substituted phenol blend) were only moderately effective. They often required concentrations above General Orders rates to eliminate the test salmonellas, and frequently elimination was not achieved even under maximal conditions of concentration and exposure.

Investigation of the distribution and control of Salmonella enterica serovar Enteritidis PT6 in layer breeding and egg production

Investigations were carried out in a layer breeder hatchery, a layer parent rearing farm, a layer parent farm and in a commercial pullet rearing and cage layer farm where Salmonella enterica serovar Enteritidis (S. Enteritidis) PT6 had become established. PT6 was initially found in focal points in the hatchery, such as hatcher ventilation ducting, tray wash areas and waste areas, but improved disinfection was followed by a rapid disappearance of contamination. Several different phage types of S. Enteritidis were found in the hatchery but most of these proved to be genotypically identical with PT6. Investigations of contaminated layer breeder and rearing sites showed that the terminal disinfection programmes in place were effective in that no carry-over of infection occurred and the organism was rapidly eliminated from the organization. Infection with PT6 originating from chicks was investigated on a commercial pullet rearing farm. After several rounds of treatment with a fluoroquinolone antibiotic and competitive exclusion, no Salmonella was found in faeces or cloacal swabs but was present in dust in one of six houses. Sampling carried out after cleaning and disinfection confirmed clearance of the organism from the site, but infection did become established in a commercial laying house receiving the birds.