Reliably colonising broiler chickens with Campylobacter spp. using a litter-based method.

1. Chicken-associated Campylobacter spp. are the cause of most food poisoning cases in Europe. In order to study the host-pathogen interactions, a reliable and reproducible method of colonising chickens with the bacteria is required. 2. This study aimed to identify a more appropriate and less invasive method of colonisation (cf. gavaging) by seeding bedding material (litter) that commercial chickens are kept on with a mixture of Campylobacter spp., broth and faeces. 3. The first phase of the study tested the longevity of Campylobacter spp. recovery in seeded litter over 24 h: significantly more Campylobacter spp. was recovered at 0 or 3 h post-seeding than at 6 and 24 h post-seeding, indicating that the pathogen can survive to detectable levels for at least 3 h in this environment. 4. In the second phase, three groups of 10 broiler chickens (negative for Campylobacter spp. prior to exposure) were exposed at 21 days of age to one of three different Campylobacter jejuni and C. coli mixes (A, B, C), using the method above. At 28 days of age, birds were euthanised by overdose of barbiturate or cervical dislocation, and livers and caeca removed for Campylobacter spp. assessment. 5. All liver and 28/30 caeca samples tested positive for Campylobacter spp., with mix A and C giving higher counts in the caeca than mix B. The method of euthanasia did not affect Campylobacter spp. counts. 6. In conclusion, a successful method for reliably colonising broiler chickens with Campylobacter spp. has been developed which negates the need for gavaging and is more representative of how contamination occurs in the field.

Infection of chickens with antimicrobial-resistant Salmonella enterica Typhimurium DT193 and monophasic Salmonella Typhimurium-like variants: an emerging risk to the poultry industry?

Antimicrobial-resistant Salmonella enterica poses a particular risk to public health, and in particular isolates belonging to clonal lineages such as Salmonella Typhimurium DT104 cause epidemics across species including poultry. In recent years, antimicrobial-resistant S. Typhimurium DT193 and specifically the monophasic S. Typhimurium-like variants of this phage type, serotypes 4,12:i:- and 4,5,12:i:-, have become an increasing risk to public health in Europe and the USA and now account for nearly one-half of human S. Typhimurium infections in the UK. Unlike S. Typhimurium that possesses two forms of flagella which can vary between phase 1 and phase 2 during infection, monophasic variants possess only phase 1 flagella. These monophasic antimicrobial-resistant variants have become a major problem in pig production but human cases have also been associated with poultry consumption and have been found in UK flocks through surveillance schemes since 2010. In this study we determined the ability of antimicrobial-resistant DT193 serotype 4,12:i:- and 4,5,12:i:- isolates from pigs to infect chickens. All isolates were found to colonize the caeca and liver. All but one isolate of serotype 4,5,12:i:- also infected the spleen. Levels of infection and pathology were comparable with those found with the virulent S. Typhimurium isolate 4/74. These findings indicate that both S. Typhimurium DT193 and monophasic variants of this phage type usually associated with pigs are capable of colonizing the chicken. This shows that both S. Typhimurium DT193 and monophasic variants represent a significant and potential emerging threat to poultry production from "spill-over" of these isolates from the pig industry or other sources.

Campylobacter infection has different outcomes in fast- and slow-growing broiler chickens.

Campylobacter spp. are frequently carried by poultry, but they are not believed to cause significant disease in these animals. Modern poultry breeds have been selected to grow rapidly under intensive conditions, but recently, consumers have moved toward purchasing birds produced in higher welfare, free-range or organic systems. Birds reared in these systems tend to be a slower growing breed and are fed a different diet. Birds reared in such systems are stocked at a lower density compared with the standard conventional broilers, and they have access to environmental enrichment, such as perches. In previous research, these slower growing birds have been shown to have different levels of Campylobacter carriage in commercial rearing conditions, but the reasons for, and effect of, these different levels are unknown; is it the bird breed, diet, or environmental conditions? In this study, experimental flocks of fast- and slow-growing breeds of broiler chickens were reared to a standard commercial slaughter weight, with their weight gain being measured during the growing period. At 21 days, birds were either infected with Campylobacter jejuni or given a placebo as control. Cohorts of birds were euthanatized at various intervals, and samples were taken for examination for Campylobacter. The fast-growing birds gained weight more rapidly than the slow-growing birds. By 2 days postinfection (dpi), C. jejuni was detected in the caeca and by enrichment from the liver and spleen samples from both breeds of birds. Low-level colonization persisted in the spleen and liver samples but was undetectable by 28 dpi. Fast- and slow-growing birds did not show detectably different levels of Campylobacter carriage. Infection with C. jejuni affected the incidence of hock marks and pododermatitis in both breeds of birds, but the differences were greater with the fast-growing breed compared with the uninfected control birds. In addition, the incidence of pododermatitis was significantly higher in Campylobacter-positive fast-growing birds than in their slower-growing counterparts. The results show that infection with Campylobacter can have an indirect welfare effect on birds via increased incidence of hock marks and pododermatitis.

Enrichment culture can bias the isolation of Campylobacter subtypes.

Enrichment culture is often used to isolate Campylobacter. This study compared isolation of Campylobacter spp. from 119 broiler chicken environments from two farms, using Preston and modified Exeter (mExeter) and modified Bolton (mBolton) enrichments. mExeter was significantly more effective in isolating Campylobacter spp. from the environmental samples compared to Preston (P<0.001) and mBolton (P<0.04) broths but there was no significant difference between the latter two methods (P>0.05). Enrichment broth type did not affect isolation from chicken faecal or soil and litter samples. C. jejuni was isolated from significantly more environmental samples using mExeter broth compared to Preston (P<0.01) and mBolton (P<0.003) broths; there was no difference between the latter two methods or between all methods for detection of C. coli (P>0.05). Only C. coli was isolated from the soil and litter samples and although both C. jejuni and C. coli were recovered from the faecal samples there was no effect of using different enrichment broths. The majority of samples where the same species had been isolated yielded the same or closely related genotypes as defined by pulsed-field gel electrophoresis. Isolates recovered using Preston and mBolton broths were less genetically diverse than those from mExeter broth. We conclude that the enrichment method used affects both the number and species of Campylobacter isolated from naturally contaminated samples.

SulA-induced filamentation in Salmonella enterica serovar Typhimurium: effects on SPI-1 expression and epithelial infection.

AIMS: Salmonella enterica serovar Typhimurium is capable of adopting a filamentous phenotype in response to damage. How this adaptive response affects bacterial virulence is unclear. We have examined the hypothesis that filamentation affects the ability of Salmonella to infect host cells. METHODS AND RESULTS: Expression of the cell division inhibitor SulA in Salm. Typhimurium SL1344 from an arabinose-inducible plasmid resulted in filamentation. We examined expression of the type 3 secretion system (T3SS) encoded by Salmonella pathogenicity island 1 (SPI-1) using SL1344 expressing a chromosomal PprgH-gfp reporter. Single cell analysis of SulA-induced SL1344 PprgH-gfp revealed a relationship between increasing cell length and decreasing propensity for prgH expression, but there was no evidence of a significant change in prgH expression evident at the whole population level. Filamentous Salm. Typhimurium were capable of initiating membrane ruffling on MDCK epithelial cells, but only nonfilamentous bacteria (< 6 µm) invade. CONCLUSIONS: Induction of SulA expression in Salmonella inhibits septation. Increasing filament length is associated with down-regulation of SPI-1 gene expression, but a significant proportion of filaments retain the ability to produce SPI-1 T3SS and induce membrane ruffles on epithelia. Despite an active SPI-1 T3SS, filamentous Salmonella are unable to invade epithelial cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings that filamentous Salmonella can express an invasive phenotype but fail to invade cells suggest that their presence in food does not constitute an immediate risk of infection until septation occurs. The described SulA expression model provides a convenient model for studying the impact of filamentation in the absence of additional stresses.

Influence of season and geography on Campylobacter jejuni and C. coli subtypes in housed broiler flocks reared in Great Britain.

Geographical and seasonal variation in the incidence and prevalence of Campylobacter jejuni and C. coli in housed broiler flocks reared in Great Britain in 2004 to 2006 was investigated in this study. Ceca (30) from 797 flocks, not subject to prior partial depopulation and reared on 211 farms, were examined individually for the presence of Campylobacter spp. The best-fitting climatic factors explained approximately 46% of the prevalence of Campylobacter-colonized flocks at slaughter and consisted of a combination of temperature at slaughter, number of sunshine hours in placement month, and millimeters of rainfall in placement month. Positive flocks were more likely to be slaughtered between June and November than during the rest of the year and to be reared in northern Great Britain than in central or southern Great Britain. C. jejuni was identified in approximately 90% of flocks, and C. coli was present in 10% of flocks. The most common clonal complexes identified in 226 isolates typed by multilocus sequence typing (MLST) were ST-45, ST-21, ST-574, ST-443, and ST-828. Flocks slaughtered at the same time were more likely to have similar complexes, and ST-45 had a seasonal pattern, with the highest prevalence in June, and was also more likely to be present in flocks reared in northern Great Britain.

Sub-clinical infection with Salmonella in chickens differentially affects behaviour and welfare in three inbred strains.

1. Much evidence exists detailing how animals respond to pathogen challenge, yet information explaining how the various behavioural, immunological, and physiological systems in chickens interplay during such challenges remains limited. 2. To gain an understanding of this interplay while controlling for genetic variation, the current study collected a variety of behavioural, physiological and immunological measures from three inbred lines (P, O and N) of laying hens before and after a sub-clinical infection with Salmonella enterica Typhimurium at 56 d of age. For comparison, an equal number of control birds were inoculated with a Salmonella-free broth. To identify an underlying profile, which might result in reduced susceptibility to infection, data were also collected in the pre-infection period. Post-infection blood and faeces were collected at 1-d post infection (dpi) and faeces again at 8 dpi. Animals were killed 15 d after infection and faeces, caecal contents, and spleen were examined for the presence of Salmonella. 3. Statistical analysis was performed to identify pre- and post-infection differences between genetic lines, changes in bird behavioural patterns between the two periods, and associations between a positive test for Salmonella and the various response measures. 4. Tissues from Line P birds were more often negative for Salmonella than those from birds of other lines, though this was inconsistent and tissue-dependent. The P line was also characterised by relatively greater serum concentrations of immunoglobulins at 1 dpi and α(1)-acid glycoprotein at 15 dpi. In addition, P line birds were more timid and their growth was reduced during the pre-infection period suggesting the possibility of a profile with reduced susceptibility to the bacterial challenge. 5. The current work has identified correlations between attributes of chicken strains and improved clearance. Future work using hypothesis-based testing will be required to determine whether the identified correlations are causally related.

Risk factors for Campylobacter colonisation during rearing of broiler flocks in Great Britain.

We investigated the associations between Campylobacter colonisation and management practices and farm characteristics in 603 housed broiler batches originating from 137 farms in Great Britain. All study batches were the initial batch slaughtered from the selected house on enrolled farms. Between 1 and 15 batches were sampled from each farm throughout the study. A total of 34.2% of the batches was Campylobacter positive and multivariable multilevel logistic regression revealed that the risk of Campylobacter colonisation was highest in July (OR=3.4, CI95%:1.8; 6.4), August (OR=3.4, CI95%:1.9; 6.2) and September (OR=3.7, CI95%:1.9; 7.1). Cattle on or adjacent to the farm increased the risk (OR=1.7, CI95%:1.1; 2.7), whereas chlorinated drinking water reduced it (OR=0.5, CI95%:0.2; 0.9). If the first removed batch from the previous flock in the house had been Campylobacter positive, the first batch of the following flock was also more likely to be colonised (OR=3.2, CI95%:2.1; 4.9). This association was more likely due to a persistent risk practice or source of Campylobacter on the farm than a direct carry-over from previous flock.

Campylobacter in housed broiler chickens: a longitudinal study of risk factors.

Infections by Campylobacter spp. are a major cause of gastrointestinal disease in the United Kingdom. Most cases are associated with the consumption of chicken that has become contaminated during production. We investigated the epidemiology of Campylobacter spp. in chickens in a 3-year longitudinal study of flocks reared on 30 farms in the United Kingdom. We used Generalized Linear Mixed Effect Models (GLMM) to investigate putative risk factors associated with incidence and prevalence of flock infection arising from farm and flock management and local environmental conditions during rearing. We used survival analysis to investigate infection events and associated risk factors over the course of the study using two marginal models - the independent increment approach, which assumed that individual infection events were independent; and a conditional approach, which assumed that events were conditional on those preceding. Models of flock prevalence were highly overdispersed suggesting that infection within flocks was aggregated. The key predictors of flock infection identified from the GLMM analyses were mean temperature and mean rainfall in the month of slaughter and also the presence of natural ventilation. Mean temperature in the month of slaughter was also a significant predictor of flock infection, although the analyses suggested that the risk in flocks increased in a unimodal way in relation to temperature, peaking at 12 degrees C. The extent of pad burn was also identified as a predictor in these analyses. We conclude that predicting prevalence within flocks with linear modelling approaches is likely to be difficult, but that it may be possible to predict when flocks are at risk of Campylobacter infection. This is a key first step in managing disease and reducing the risks posed to the human food chain.

Demographic determinants for Campylobacter infection in England and Wales: implications for future epidemiological studies.

Despite a significant public health burden the epidemiology of human Campylobacter infection remains blurred. The identification of demographic determinants for Campylobacter infection is therefore essential for identifying potential areas for intervention. Demographic data from an active, population-based sentinel surveillance system for Campylobacter infection (from 2000 until 2003, n=15 907) were compared with appropriate denominator data from the 2001 United Kingdom Census. Incidence was higher in males from birth until the late teens and in females from 20 to 36 years. Age- and gender-specific differences in Campylobacter incidence were observed in different ethnic and socioeconomic groups and hence are all major drivers for Campylobacter infection. Epidemiological studies on Campylobacter infection need to take these factors into consideration during design and analysis. The collation of detailed epidemiological data and its comparison with appropriate denominator data provides a valuable epidemiological tool for studying infection.

Persistence of Campylobacter species, strain types, antibiotic resistance and mechanisms of tetracycline resistance in poultry flocks treated with chlortetracycline.

OBJECTIVES: The aim of this study was to investigate the persistence of Campylobacter species, strain types, antibiotic resistance and mechanisms of tetracycline resistance in poultry flocks treated with chlortetracycline. METHODS: Three commercially reared broiler flocks, naturally colonized with Campylobacter, were treated with chlortetracycline under experimental conditions. The numbers of Campylobacter isolated, and the species, flaA short variable region allele, and antimicrobial resistance of isolates were determined. RESULTS: For two of three flocks, tetracycline-resistant strains predominated prior to chlortetracycline exposure. Presence of the antibiotic had no discernible effect on the numbers or types of Campylobacter and the tetracycline-resistant strains persisted in numbers similar to those observed before treatment. With all flocks, some faecal samples were obtained that contained no Campylobacter, irrespective of exposure to chlortetracycline; this was more common as the birds grew older. For the third flock, tetracycline-resistant Campylobacter were in the minority of samples before and during exposure to chlortetracycline, but at sampling times after this, no resistant strains were found in the treated (or untreated) birds, irrespective of exposure to the antibiotic. All tetracycline-resistant isolates (MICs 16 to >128 mg/L) contained tet(O) and, for some isolates, this was transferable to Campylobacter jejuni 81116. The efflux pump inhibitor PAbetaN reduced the MICs of tetracycline for these isolates by 4-fold, suggesting that an intact efflux pump, presumably CmeABC, is required for high-level tetracycline resistance. CONCLUSIONS: Our data indicate that chlortetracycline treatment does not eradicate tetracycline-resistant Campylobacter spp. from poultry. However, if a low number of resistant isolates are present, then the antibiotic pressure appears insufficient to select such strains as the dominant population.

Effects of repeated cycles of acid challenge and growth on the phenotype and virulence of Salmonella enterica.

AIMS: The aim of the study was to investigate how stresses like low pH, which may be encountered in farms or food preparation premises, shape populations of Salmonella enterica by the selection of stress-resistant variants. METHODS AND RESULTS: Stationary-phase cultures of S. enterica serovar Enteritidis and serovar Typhimurium (one strain of each) were exposed to pH 2.5 for up to 4 h, followed by growth at pH 7 for 48 h. This process was repeated 15 times in two separate experiments, which increased the acid resistance of the three out of four populations we obtained, by three- to fourfold. Sustainable variants derived from the populations showed changes in colony morphology, expression of SEF17 fimbriae, growth, increased heat resistance and reduced virulence. CONCLUSIONS: The study demonstrates that low pH environments can select for populations of S. enterica with persistent phenotypic changes such as increased acid resistance and occasionally increased SEF17 expression and lower virulence. SIGNIFICANCE AND IMPACT OF THE STUDY: There is a common belief that increased acid resistance coincides with increased virulence. This study demonstrates for the first time that increased acid resistance often impairs virulence and affects the general phenotype of S. enterica.

Pathotyping of Salmonella enterica by analysis of single-nucleotide polymorphisms in cyaA and flanking 23S ribosomal sequences.

The egg-contaminating phenotype of Salmonella enterica serotype Enteritidis was linked to single-nucleotide polymorphisms (SNPs) occurring in cyaA, which encodes adenylate cyclase that produces cAMP and pyrophosphate from ATP. Ribotyping indicated that SNPs in cyaA were linked to polymorphisms occurring in the rrlC and rrlA 23S ribosomal subunits. Phylogenetic analysis of cyaA discriminated between Salmonella enterica serotypes and within serotype Enteritidis. Serotypes Typhimurium, Heidelberg and Enteritidis produced one, three and six cyaA allelic variants, respectively, among the set of 56 isolates examined. Asparagine(702) of CyaA was converted to serine in a biofilm-producing isolate. Statistical analysis was applied to 42 other genes encoding proteins between 800 and 1000 amino acids (aa). Results show that the 848 aa CyaA of serovar Enteritidis evolved by nucleotide substitutions that did not significantly alter the purine-to-pyrimidine nucleotide substitution ratio, which was a characteristic of large genes that was positively correlated with increasing gene size. In summary, these analyses link SNPs occurring in the rrlC-rrlA genomic fragment of S. enterica to genetic drift within S. Enteritidis that is associated with egg contamination.

Campylobacter spp. contamination of chicken carcasses during processing in relation to flock colonisation.

The presence and numbers of campylobacters on chicken carcasses from 26 slaughter groups, originating from 22 single-house flocks and processed in four UK plants, were studied in relation to the level of flock colonisation determined by examining the caecal contents of at least ten birds per group. The prevalence of campylobacters on carcasses from five campylobacter-negative flocks processed just after other negative flocks was low (8.0 log(10) cfu) than carcasses originating from low prevalence flocks (average of 2.3 log(10) cfu; range: <1.1 to 4.1 log(10) cfu). There was a reduction in the numbers of campylobacters on carcasses between plucking and chilling in eight of ten fully colonised flocks. In another eight flocks, a significant (P<0.001) decrease (0.8 log(10) cfu) in the number of campylobacters on carcasses from just before to after chilling was detected. Campylobacter spp. could be isolated from aerosols, particles and droplets in considerable numbers in the hanging-on, defeathering and evisceration areas but not in the chillers. This was the case even when campylobacters were not isolated from the target flock. Campylobacters on carcasses from two partly colonised flocks were either the same subtype, as determined by speciation, Multi-Locus Sequence Typing (MLST) and flaA Restricted Fragment Length Polymorphism (RFLP) typing, as those in the fully colonised flocks processed previously, although not necessarily the most prevalent ones; or were the same subtypes as those found in the caeca of the flock itself. The prevalences of the different campylobacter subtypes found on carcasses from two fully colonised flocks did not closely reflect those found in the caeca. MLST combined with flaA RFLP provided a good method for ascertaining the relatedness of strains isolated from carcasses and caecal contents. This study showed that carcass contamination is related to the within-flock prevalence of campylobacter colonisation, but that contamination from previously processed flocks was also significant, especially on carcasses from low prevalence flocks. Forced dry air cooling of carcasses reduced contamination levels.

Norepinephrine increases the pathogenic potential of Campylobacter jejuni.

BACKGROUND: Campylobacter jejuni can cause a spectrum of diseases in humans, ranging from enteritis and diarrhoea to severe inflammation, profuse bloody diarrhoea and chronic relapsing infection. Norepinephrine (NE) levels in the intestine increase under conditions of stress and trauma, and are thought to result in spill over of NE into the intestinal lumen. NE is known to stimulate the growth of a range of bacterial species, and to increase the pathogenicity of Escherichia coli. AIM: To determine the effects of NE on the pathogenic potential of C jejuni in a model system. METHODS: C jejuni was grown in iron-replete and iron-limited media in the presence and absence of 100 microM NE. Several virulence-associated characteristics, including motility and cell invasion, were measured. RESULTS: When C jejuni was grown in iron-limited media in the presence of NE, growth rate, motility and invasion of cultured epithelial cells were increased compared with cultures grown in the absence of NE. Bacteria exposed to NE during growth also caused greater subsequent disruption of cultured epithelial cell monolayers, inducing widespread breakdown of tight junctions. CONCLUSION: Exposure to NE causes an increase in the virulence-associated properties of Campylobacter. Stress and concomitant infection could therefore be contributory factors to the variable presentation of this disease.

Sources of Campylobacter spp. colonizing housed broiler flocks during rearing.

The study aimed to identify sources of campylobacter in 10 housed broiler flocks from three United Kingdom poultry companies. Samples from (i) the breeder flocks, which supplied the broilers, (ii) cleaned and disinfected houses prior to chick placement, (iii) the chickens, and (iv) the environments inside and outside the broiler houses during rearing were examined. Samples were collected at frequent intervals and examined for Campylobacter spp. Characterization of the isolates using multilocus sequence typing (MLST), serotyping, phage typing, and flaA restriction fragment length polymorphism typing was performed. Seven flocks became colonized during the growing period. Campylobacter spp. were detected in the environment surrounding the broiler house, prior to as well as during flock colonization, for six of these flocks. On two occasions, isolates detected in a puddle just prior to the birds being placed were indistinguishable from those colonizing the birds. Once flocks were colonized, indistinguishable strains of campylobacter were found in the feed and water and in the air of the broiler house. Campylobacter spp. were also detected in the air up to 30 m downstream of the broiler house, which raises the issue of the role of airborne transmission in the spread of campylobacter. At any time during rearing, broiler flocks were colonized by only one or two types determined by MLST but these changed, with some strains superseding others. In conclusion, the study provided strong evidence for the environment as a source of campylobacters colonizing housed broiler flocks. It also demonstrated colonization by successive campylobacter types determined by MLST during the life of a flock.

Effect of sunlight on the survival of Salmonella on surfaces.

AIMS: To investigate the effect of simulated full-spectrum tropical sunlight on the survival of Salmonella in droplets on surfaces. MATERIALS AND RESULTS: The survival on surfaces of three Zambian strains of Salmonella enterica serovars Enteritidis and Heidelberg was compared with that of a strain of S. enterica serovar Enteritidis phage type (PT) 4 with known characteristics which had been isolated from poultry in the UK. Samples were taken from surfaces every hour for 3 h and after 24 h exposure in either dark or 12 h light/12 h dark cycle conditions. Differences were analysed for significance using a one-way analysis of variance (anova). Results show that there were a significantly higher number of cells surviving on surfaces after 24 h in the dark when compared with populations exposed to a 12 h light/12 h dark cycle. Significantly more cells also survived exposure to sunlight under dirty than clean conditions. CONCLUSIONS: Exposure to sunlight results in a significant decrease in numbers of Salmonella on surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: Under field conditions exposure of contaminated surfaces to sunlight could be used in place of chemical methods of control as a cheaper way to reduce Salmonella contamination of surfaces.

Flagella and curli fimbriae are important for the growth of Salmonella enterica serovars in hen eggs.

Salmonella enterica serovar Enteritidis is unable to multiply in the albumen of fresh eggs and must gain access to the yolk contents in order to multiply to a high level (>10(6) c.f.u. per ml egg contents). As human Salmonella infections resulting from the consumption of infected eggs more frequently involve serovar Enteritidis phage type (PT) 4 than other serovars or PTs, a number of isolates of various S. enterica serovars were examined for their ability to multiply to a high level in eggs over a period of 8 days storage at 20 degrees C. Their behaviour was compared to that of a range of defined fimbrial and flagella mutants of S. Enteritidis. Strains that did not express flagella were unable to multiply in eggs, and those deficient for curli fimbriae, including strains of S. Enteritidis PT6, displayed high-level growth in significantly fewer eggs than those able to express curli. Most S. Enteritidis strains multiplied to a high level in between 5 and 10 % of eggs during 8 days storage. One PT4 strain, though, showed high levels of growth in more than 25 % of eggs over this period, significantly higher than the other PTs or the two other isolates of PT4 tested. This ability may be important for the association of PT4 infection with the consumption of eggs.

An assessment of the microbiological risks involved with egg washing under commercial conditions.

The potential benefits of washing eggs is offset by a historical perception in the European Union that wetted eggs are prone to spoilage and water loss. This study describes the effects of spray jet washing under various processing conditions to shell surface counts of Salmonella and the presence of bacteria in egg contents. Experiments used eggs that were contaminated with Salmonella Enteritidis PT4 or Salmonella Typhimurium DT104 before cuticle hardening. Washing of contaminated eggs under optimum conditions resulted in a more than 5-log reduction of Salmonella counts from the shell surface. Salmonella was not isolated from the yolk or albumen of any egg washed by the optimal protocol, suggesting that when properly controlled, egg washing did not cause Salmonella to enter the contents. However, contamination did arise if strict control was not maintained over the wash and rinse water temperatures. Both Salmonella Enteritidis and Salmonella Typhimurium were shown to enter the egg contents when water temperatures were lowered, indicating that strict temperature control must be maintained in order to prevent the ingress of Salmonella into egg contents. Other washing machine parameters that were investigated did not significantly affect Salmonella entry into the egg contents but influenced shell surface kill levels to varying degrees.