Abstract
Egg-related outbreaks of salmonellosis are a significant health concern. Although Salmonella Enteritidis (SE) is the major egg-associated serotype, Salmonella Typhimurium (ST) can also infect the hen's reproductive tract and contaminate eggs. Recently, monophasic and aphasic variants of ST have been reported with increased frequency in Europe, and the isolation of these variants from laying flocks triggers the same legislative restrictions associated with biphasic ST strains. However, little is known about the colonization, invasiveness and persistence of monophasic and aphasic ST strains in laying hens. In this study, seven groups of 1-day-old and point-of-lay commercial Hy-line chicken layers were separately challenged with four different strains of monophasic ST, one aphasic ST, one biphasic ST and one egg-invasive SE strain. Tissue samples and cloacal swabs (point-of-lay chickens only) were collected at regular intervals post challenge in order to recover the Salmonella challenge strains. In 1-day-old chicks, only the aphasic ST strain and the SE strain were recovered after direct plating, suggesting that the number of salmonellas colonizing the tissues of the chicks infected with the other strains was likely to be low. Interestingly, all of the strains colonized well in the point-of-lay chickens, and there was no statistical difference in the overall number of positive samples or Salmonella counts between the seven strains. Salmonella was recovered from the point-of-lay birds to the end of the study (20 days after challenge). Monophasic and aphasic ST strains colonized point-of-lay birds as efficiently as biphasic ST and SE strains. Further studies are necessary to estimate the invasiveness of these strains in naturally-infected vaccinated laying hens, and to assess the impact of natural infection on egg contamination.
Introduction
Salmonella spp. are responsible for the majority of the reported food poisoning outbreaks in Europe. In the European Union, the two serotypes responsible for the majority of salmonellosis outbreaks are Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) (respectively, 44.4% and 24.9% of all reported serotypes in human cases in 2011) (EFSA, Citation2013). Consumption of contaminated eggs and egg products has been linked with 17.3% of salmonellosis outbreaks in 2009 in Europe (EFSA, Citation2011). SE is mostly associated with the contamination of eggs, whilst ST outbreaks are generally related to the consumption of contaminated pork meat or produce (EFSA, Citation2010a).
ST infects laying flocks across Europe and is able to contaminate eggs (Wales & Davies, Citation2011). Egg-associated ST outbreaks have been reported (Scuderi et al., Citation1996; Carraminana et al., Citation1997; Greig & Ravel, Citation2009; EFSA, Citation2010a; Le Hello et al., Citation2012). Recently in the UK and Republic of Ireland, several outbreaks caused by ST definitive type (DT) 8 and related to the consumption of contaminated duck eggs have been reported (Noble et al., Citation2012; Garvey et al., Citation2013). In other geographic regions, such as India and Australia, SE is rarely isolated from laying flocks or eggs, and ST is responsible for most egg contamination and egg-related outbreaks (OzFoodNet, Citation2009; Renu et al., Citation2011; Chousalkar & Roberts, Citation2012).
Most Salmonella strains are motile by means of peritrichous flagella that are encoded by the flagellin genes fliC and fljB present on the bacterial chromosome. The alternate expression of fliC or fljB is called “phase regulation”. Some strains lack genes for one of the two phases and are therefore called monophasic (Switt et al., Citation2009). In recent years an increase in the number of human outbreaks caused by monophasic variants of ST (mST) has been reported throughout the European Union, the Far East and the USA. Most commonly, the European strains lack the second-phase flagellar antigens, therefore presenting a 1,4,[5],12:i:- antigenic formula and characteristic pulsed field gel electrophoresis patterns, together with the presence of two novel genomic islands. Different clonal lines of mST can be identified across Europe, presenting different phage types, genotypes and antimicrobial resistance profiles (EFSA, Citation2010b). The most recent widely distributed clone is S. 4,5,12:i:- DT193, tetra-resistant to ampicillin, streptomycin, sulphonamides and tetracyclines (Lucarelli et al., Citation2010). In 2010, DT193 was the most common phage type for ST in Europe, representing 21% of the ST isolates (EFSA, Citation2012). Outbreaks of 4,5,12:i:- infection have been associated with the consumption of pork products in Europe (Hauser et al., Citation2010; Gossner et al., Citation2012). Here, mST was the fourth most commonly reported serotype in 2010 (1.5% of confirmed cases) (EFSA, Citation2012). In people, it is not clear whether infections caused by mST are more or less severe than those caused by ST, as there are conflicting reports in the literature (EFSA, Citation2010b). In the UK, a National Control Programme for the control of Salmonella in laying hens has been implemented since 2008. From January 2010, the eggs produced by flocks in which SE or ST have been identified cannot be sold for human consumption unless they undergo heat treatment (Defra, Citation2007). The National Control Programme implements Commission Regulation (EC) No. 1168/2006. After publication by EFSA in 2010 of the Opinion on “Salmonella Typhimurium”-like strains (EFSA, Citation2010b), the EU regulation 1168/2006 was revised (Commission Regulation 517/2011). This further regulation includes mST in Typhimurium requirements. As a consequence of this legislation, the isolation of mST strains from a commercial laying flock triggers the same restrictions as ST strains.
ST infections of laying flocks are often caused by wild-bird-related strains, and are normally short lived (Martelli & Davies, Citation2011). However, after experimental challenge, ST strains have been shown capable of causing systemic infection and egg contamination in laying hens (Wales & Davies, Citation2011). The occurrence of reproductive tract infection and egg contamination is related to the challenge dose, and this is normally low in naturally-infected laying hen flocks (Gast et al., Citation2011). Invasion can also vary depending on Salmonella serotype, phage type and genotype (Suzuki, Citation1994). Understanding the pathobiology of mST in laying hens could help to elucidate the risk posed by these strains to human health and aid in the development of novel control strategies.
The objective of this study was to undertake the first assessment of the pathogenicity of selected ST, mST and aphasic ST strains in commercial laying hens. To acquire information on the invasiveness of these strains in layer chickens, six groups of point-of-lay commercial hens were challenged separately with one ST strain, four mST strains and one aphasic ST strain. A seventh group of hens was challenged with an egg-invasive SE strain for comparison.
Materials and Methods
This study comprised two separate in vivo challenge trials, the first performed using commercial 1-day-old chicks (strain passage trial) and the second using commercial point-of-lay hens (invasiveness trial in point-of-lay hens).
Ethical statement
The project was approved by the Animal Health and Veterinary Laboratories Agency (AHVLA) ethics committee and all procedures were conducted in accordance with the UK Animals (Scientific Procedures) Act 1986 under the jurisdiction of HO project licence PPL 70_7319.
Selection of strains
Seven strains of Salmonella were selected for use in this study (). These represented currently circulating and emerging mST strains (four strains) and aphasic ST strains (one strain). One biphasic ST strain and one egg-invasive SE strain were also included in the study. The strains were selected on the basis of their epidemiological relevance, to ultimately obtain a panel representative of mST strains circulating in the UK or egg-invasive strains that could occur in the UK poultry population. Strain A is a ST DT8 originally isolated in the UK from a flock of duck layers that produced eggs for human consumption and was linked to human outbreaks (Noble et al., Citation2012). Strains B, D and E are tetra-resistant mST 4, 12:i:- and 4,5,12:i:- DT193 strains of UK origin. Strain C is a non-resistant S. 4,5,12:i:- strain. Strain F is a non-motile aphasic ST DT104b strain originating from a laying flock in France, where it was responsible for egg-related human outbreaks (Le Hello et al., Citation2012). Strain G is an egg-invasive SE phage type (PT) 14b strain that spread from continental Europe to the UK through contaminated eggs and was isolated from eggs in an outbreak investigation (Janmohamed et al., Citation2011). Prior to the commencement of the in vivo studies, all strains were subjected to confirmatory serotyping and phage typing (OIE, Citation2012). Furthermore, antimicrobial resistance patterns were determined using the AHVLA panel, which comprises 16 antimicrobial agents (amikacin amoxicillin/clavulanic acid, ampicillin, apramycin, cefotaxime, ceftazidime, chloramphenicol, tetracycline, ciprofloxacin, furazolidone, gentamicin, nalidixic acid, neomycin, streptomycin, trimethoprim/sulphamethoxazole and sulphonamide compounds) (Snow et al., Citation2008).
Table 1. Serotype, phage type and antimicrobial resistance pattern of the strains included in the study.
Strain passage trial
Prior to the start of the challenge studies with point-of-lay birds, the Salmonella strains were passaged in commercial 1-day-old layer chicks to ensure they could cause infection and to overcome any infectivity problems that may be associated with long-term storage. Three chicks per strain were challenged by oral gavage with approximately 1 × 105 colony-forming units (CFU) of the respective test Salmonella strain. Three days post challenge the chicks were euthanized and subjected to post-mortem examination. At post-mortem examination, the liver, spleen, caeca, ovary and heart blood of each bird were aseptically sampled. The tissues were homogenized and direct plated in order to recover the challenge strains as described below.
Invasiveness trial in point-of-lay hens
One hundred and forty commercial Hy-line layer chickens were sourced at 1 day old. All birds received all of the standard vaccinations routinely administered at the hatchery, but remained unvaccinated against Salmonella. The transport crates were swabbed on arrival to confirm absence of Salmonella. The birds were reared in biosecure accommodation until they reached 20 weeks of age. At this point, the birds were split into seven groups of 20 and housed in environmentally controlled rooms using heat-treated wood shavings as bedding and were fed on a poultry mash. Water was provided ad libitum. Nest boxes and perches were provided in each room. All birds were wing tagged in order to aid identification throughout the study. Prior to challenge, all birds were cloacally swabbed to confirm that they were negative for Salmonella.
The Salmonella isolates used for challenge were those recovered from the tissues of the chicks from the strain passaging trial, where possible after direct plating. When more than one tissue was positive per strain, the isolate from the liver was selected; if this was not available, isolates from the spleen or caeca (in this order of preference) were selected instead.
Before challenge, feed was withdrawn for 8 h, to enhance the susceptibility of the hens to Salmonella (Suzuki, Citation1994). Immediately prior to challenge, each bird received 2 ml of 10% sodium bicarbonate to neutralize the crop acid. Following neutralization of the crop acid, each bird received 1 ml of approximately 1 × 109 CFU/ml of the Salmonella challenge strain by oral gavage. After challenge, cloacal swabs were collected serially from all birds in each group to evaluate levels of shedding of Salmonella. The experiment continued for 20 days after challenge (d.p.c.), and post-mortem examinations were performed 3 and 7 d.p.c. (four birds per group) and 17 and 20 d.p.c. (six birds per group). At post-mortem examination, the liver, spleen, ovary and caeca of each bird were sampled aseptically. Two pooled faecal samples (approximately 25 g faeces) and one dust sample (approximately 10 g) were collected from each room on a weekly basis.
Bacteriological methods
Cloacal swabs were plated directly into Rambach agar (1.07,500.0002; Merck, Whitehouse Station, NJ, USA) and incubated at 37 ± 1°C for 24 ± 3 h. All swabs were also incubated in 18 ml buffered peptone water (10.07228.0500; Merck) at 37 ± 1°C for 16 to 20 h, subsequently inoculated into Mueller–Kauffmann tetrathionate broth at 37 ± 1°C for 24 ± 3 h, and then plated onto Rambach agar. In order to enumerate Salmonella from tissues, 1 g sub-samples were homogenized, and decimal dilutions of homogenates were made in phosphate-buffered saline (pH 7.4) and plated onto Rambach agar (Clifton-Hadley et al., Citation2002). The tissues were also enriched in buffered peptone water and any negative tissues from the necropsy were enriched in Mueller–Kauffmann tetrathionate broth and re-plated onto Rambach agar. The environmental samples (pooled faeces and dust) were pre-enriched in buffered peptone water and tested as described above. Slide agglutination tests on isolates from samples were also carried out to confirm positive results. A selection of the positive samples was subjected to serotyping and phage typing for confirmation (Anderson et al., Citation1977; Ward et al., Citation1987; Popoff et al., Citation2004).
Statistical methods
A comparison between the numbers of positive samples was carried out with a Fisher's exact test. The log10-transformed CFU/g counts for the positive birds were compared at each post-mortem examination for each organ by one-way analysis of variance using Bonferroni adjusted t tests.
Results
In the strain passage trial, only strain F (4,5,12:-:-) and strain G (SE) were recovered by direct plating. Strain F (4,5,12:-:-) was recovered by direct plating also from the heart blood. All other strains were only recovered post enrichment, with strain A (ST DT8), strain B (4, 12:i:-), strain D (4, 5, 12:i:-) and strain E (4, 5, 12:i:-) being found in the liver and/or spleen, whereas strain C (4,5,12:i:-) was recovered only from cloacal swabs and caeca ().
Table 2. Results of the strain passage trial, after direct plating and enrichment.
In the invasiveness trial in point-of-lay hens, all of the samples collected before challenge tested negative for Salmonella. All pooled faeces and dust samples collected after challenge were positive for Salmonella. All of the strains colonized well in the birds, yielding positive samples in all tissues harvested at post-mortem examination 3 and 7 d.p.c. and in most cloacal swabs taken serially during the experiment. The number of positive samples after direct plating and post enrichment for the post-mortem samples is shown in . Where available, the mean of the log10-transformed Salmonella counts for each organ positive after direct plating are also shown in . Salmonella could be recovered from cloacal swabs by direct plating only on two sampling occasions (2 and 6 d.p.c.), and the mean of the log10-transformed Salmonella counts per group, where available, is shown in .
Table 3. Number of positive tissue samples (including post-enrichment data) at post-mortem examination 3, 7, 17 and 20 d.p.c.
Table 4. Number of Salmonella-positive cloacal swabs for each strain on all post challenge sampling occasions.
The birds were still shedding Salmonella in their faeces at 16 d.p.c. in all challenge groups, apart from the group challenged with strain A. At 10 d.p.c., no Salmonella was isolated from cloacal swabs in the group challenged with strain D (4, 5, 12:i:-). Salmonella was recovered from ovaries in the first post-mortem examinations after direct plating, and after enrichment also in the third and fourth post-mortem examinations. At the fourth post-mortem examination, Salmonella was recovered only from the ovaries of birds challenged with strain D (4, 5, 12:i:-), strain F (4, 5, 12:-:-) and strain G (SE). In the spleen, 10 to 103 Salmonella CFU/g were recovered in the last post-mortem for all strains except strain B (4,12:i:-) and strain G (SE). In the caeca, Salmonella was isolated up to the last post-mortem examination in all challenge groups, apart from the group challenged with strain A (ST).
No statistically significant differences (P > 0.05, Fisher's exact test) could be detected between the recovery of the different strains after direct plating of cloacal swabs. After enrichment of the cloacal swabs, there was significantly (P < 0.001, Fisher's exact test) less recovery of strain A (ST DT8) than the other strains. After direct plating of tissues collected post-mortem, there were no significant differences (P > 0.05, Fisher's exact test) in recovery of strains. When comparing the total number of tissues positive after enrichment/tested per each strain, only two statistically significant differences (P ≤ 0.05, Fisher's exact test) could be detected, where two strains had a significantly higher number of Salmonella-positive samples when compared with other strains. A significant difference (P = 0.005) in the liver of strain G (SE, third post-mortem examination) and in the ovary (P = 0.012) of strain F (4,5,12:-:-, fourth post-mortem examination) was observed. By Bonferroni adjusted t test, at the second post-mortem examination, strain G (SE) had significantly higher counts (P < 0.05) than strain A (ST DT8), strain B (4,12:i:-), strain C (4,5,12:i:-) and strain D (4,5,12:i:-).
Statistically significant differences between strains, both in the cloacal swabs and in the post-mortem results, were only sporadic and are therefore of doubtful biological significance. Overall, all strains were readily recovered from tissue samples and cloacal swabs on most sampling occasions. No significant difference could be consistently detected between the ST, mST, aphasic ST and SE strains in the final sampling or cumulative results.
Discussion
All of the strains utilized in this study were able to readily colonize commercial birds, both in the strain passage trial in 1-day-old chicks and in the invasiveness trial in point-of-lay hens.
The number of positive tissues recovered from the chicks in the passaging study was lower than expected (). The strains might have been less virulent as a consequence of long-term storage (Matthews et al., Citation2011). The chicks used in this study were not specific pathogen free, and they could therefore have presented more significant resistance to invasion by the Salmonella strains (Roy et al., Citation2001). Also, the challenge dose (1 × 105 CFU) administered to the chicks was selected to minimize the risk of any clinical signs and thus reduce the chance of any adverse consequences for the birds. A higher dose might have led to a more extensive tissue invasion, but could have caused clinical disease (Barrow et al., Citation1987; Dhillon et al., Citation2001). Strain F (4,5,12:-:-) and strain G (SE) colonized more effectively in the chicks (the number of positive tissues recovered was higher and there were positive samples after direct plating, suggesting that a higher number of salmonellas was present in the tissues). Both of these strains were isolated from egg-related outbreaks, suggesting that they may be particularly successful in colonizing laying hens, resulting in high levels of ovary transmission (Janmohamed et al., Citation2011). Strain F (4,5,12:-:-) was isolated by direct plating from liver and heart blood, suggesting that high numbers of Salmonella were circulating in the bloodstream and invading tissues soon after challenge.
In the invasiveness trial in point-of-lay hens, all strains were recovered at high levels from cloacal swabs and tissue samples immediately after challenge (2 d.p.c. and 3 d.p.c., respectively). The number of positive samples and the Salmonella counts decreased progressively during the study, but most Salmonella strains were recovered after enrichment in most tissues at the last post-mortem examination. This suggests that although the Salmonella shedding might have progressively decreased over time, an active or latent Salmonella infection was still present 20 days post challenge. Salmonella was recovered from the ovaries only of a limited number of challenge groups—birds challenged with strain D (4, 5, 12:i:-), strain F (4, 5, 12:-:-) and strain G (SE) at the last post-mortem examination (20 d.p.c.). The results obtained for strains F and G confirm what was observed in the passaging study. The progressive decrease of the frequency of Salmonella recovery from cloacal swabs and internal organs has been observed in previous studies (Gast & Beard, Citation1990a, Citationb; Okamura et al., Citation2001). Significant differences in the number of positive samples and in the Salmonella counts between strains were only sporadic, indicating that the tissue invasion and shedding levels of monophasic/aphasic strains is comparable with those of a fully typeable ST strain and an outbreak-associated SE strain. These results are in agreement with those of previous in vivo challenge studies, which have highlighted no difference in the internal organ colonization and cloacal shedding between chickens infected with SE and ST (Shaffer et al., Citation1957; Keller et al., Citation1997). A recent study in 8-day-old specific pathogen free broiler chickens also showed that ST, 4, 5, 12:i:- and 4, 12:i:- can colonize the intestines of chickens and are able to invade systemic sites (Parsons et al., Citation2013). The findings from this study and from published in vivo studies, however, are generated by a high-dose challenge and might therefore not be comparable with natural infections, where contamination usually involves low numbers of organisms, at least in most cases (Wales & Davies, Citation2011). In previous studies, similar levels of tissue contamination were observed in hens challenged with high doses of different Salmonella serotypes (Gast et al., Citation2011). ST infections are likely to evoke a stronger immune response in the birds, and therefore can be cleared more quickly when compared with SE infections (Rabsch et al., Citation2002), but longer term studies would be needed to evaluate the persistence characteristics of the strains used in this study.
In conclusion, a high-dose mST and aphasic ST experimental challenge in point-of-lay hens resulted in tissue invasion and prolonged shedding. Field studies would be required in order to accurately assess the level of the invasiveness of mST strains in naturally-infected chickens, and to elucidate the impact of the infection on the contamination of table eggs.
Acknowledgements
The authors would like to thank Dr Sophie A. Granier from ANSES for kindly providing the aphasic ST strain used in this study and the AHVLA Animal Services Unit staff for assistance with all in vivo studies.
Funding
This study was funded by the UK Department for Environment, Food and Rural Affairs project OZ0341.
Additional information
Funding
References
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