Abstract
Research on the role of type 1 fimbriae of Salmonella enterica serotype Enteritidis in poultry to date has been focused on the intestinal phase of the infection. This study aimed to investigate the role of type 1 fimbriae in a systemic infection by intravenously inoculating chickens with a fimD mutant or its parent strain. The fimD mutant was present in the blood for 3 weeks after infection, while the wild type parent strain was cleared within the first 3 days. The fimD mutant was isolated at least as much as the parent strain from the liver and spleen for up to 3 weeks after inoculation. The wild type strain was cleared from the caeca in the second week, while the fimD mutant was isolated from the caeca for up to 3 weeks after infection. The ovaries were more heavily infected by the fimD mutant than by the wild-type strain. In the first and second week after inoculation, the oviducts were more frequently infected by the mutant strain. The eggs of birds infected with the fimD mutant were less frequently contaminated with Salmonella. The shells of the eggs were more frequently contaminated by the wild type strain than with the mutant strain. Thus, the absence of type 1 fimbriae prolongs bacteraemia, modifies reproductive tract infection and reduces egg shell contamination by Salmonella enterica serovar Enteritidis.
Introduction
Salmonella enterica serotype Enteritidis continues to be a threat to public health. Human illness caused by this serotype of Salmonella can often be traced back to egg consumption (Angulo & Swerdlow, Citation1999). Why serotype Enteritidis is specifically associated with egg contamination still is incompletely understood. The low incidence of egg contamination in an infected flock, the intermittent production of contaminated eggs by infected hens (Humphrey et al., Citation1989) and the possibility of different mechanisms of contamination (Keller et al., Citation1995) all make experimental studies of this phenomenon difficult and time consuming.
Little is known about the virulence factors that enable Salmonella Enteritidis to contaminate chicken eggs. The virulence factors that have been suggested to be associated with egg contamination are high molecular weight lipopolysaccharide and a capacity for growth to high densities (Petter, Citation1993; Guard-Petter et al., Citation1997; Guard-Petter, Citation1998; Parker et al., Citation2001). Moreover, the specific interaction of S. Enteritidis with the granulosa cells is believed to play an important role in vertical transmission (Thiagarajan et al., Citation1994, Citation1996a). We recently showed that type 1 fimbriae interact with the secretions of the oviduct that are incorporated into the forming egg (De Buck et al., Citation2003), especially with the isthmus secretions that make up the shell membranes. Analysis of the different components of fresh eggs has shown that S. Enteritidis can be associated with egg shells as well as with yolk or egg white (Humphrey et al., Citation1991b; Bichler et al., Citation1996; Miyamoto et al., Citation1997; Okamura et al., Citation2001a,Citationb). This led us to the hypothesis that persistent colonization of the oviduct, mediated by type 1 fimbriae, may be a key element in egg contamination by S. Enteritidis.
The purpose of the present study was to compare the spread of type 1 fimbriated and unfimbriated S. Enteritidis from the blood to different organs including the reproductive tract. Therefore, a fimD mutant strain of S. Enteritidis and its wild-type parent strain were used (Allen-Vercoe & Woodward, Citation1999b). FimD is involved in the export and assembly of fimA fimbrial subunits across the outer membrane (Klemm & Christiansen, Citation1990). The mutant and parent strains were inoculated intravenously, allowing investigation of the role of type 1 fimbriae in infection after the intestinal colonization and invasion phase, during which these appendages are known to be of importance (Craven et al., Citation1992; Dibb-Fuller et al., Citation1999).
Materials and Methods
Animals
Forty-eight commercial non-Salmonella-vaccinated laying hens (ISA Warren Brown) were housed in wire-bottom isolation units from the age of 19 weeks until the end of the experiment. The Salmonella status of the hens was tested by bacteriological analysis of cloacal swabs and litter samples and serological testing for anti-Salmonella antibodies using a previously described ELISA test (Desmidt et al., Citation1996). The hens had free access to drinking water and were fed ad libitum. They were subjected to a 16 h light/8 h darkness lighting scheme.
Bacterial strains and culture
A fimD insertional mutant of S. Enteritidis phage type 4, defective in the production of type 1 fimbriae, and the wild-type parent strain S1400/94 were kindly provided by Prof. M.J. Woodward, V.L.A., Weybridge, UK. The characteristics of these strains have been described previously (Allen-Vercoe & Woodward, Citation1999b). The strains were cultured overnight at 37°C on Isosensitest agar (Oxoid, Basingstoke, UK) plates. Ten colonies were transferred into Brain Heart Infusion broth (Biolife, Milano, Italy) and cultured for 20 h at 37°C with shaking. The suspension was checked for purity and the number of colony forming units (CFU) was determined by plating 10-fold dilutions on Isosensitest agar.
Experimental design
As soon as all the hens were in lay, they were all intravenously inoculated with 2×107 CFU fimD mutant of S. Enteritidis or the corresponding wild-type strain S1400/94 in 0.5 ml phosphate-buffered saline. This is a dose comparable with those used in previous reports (Miyamoto et al., Citation1997; Gast et al., Citation2002). The eggs were collected on a daily basis. Three hens from each group were euthanized at 1, 2, 4 and 8 days after inoculation. Six hens of each group were euthanized at 14 and 21 days after inoculation.
Bacteriological examination
The lumens of the five segments of the oviduct were swabbed during necropsy and the swabs used to inoculate brilliant green agar (BGA) (Oxoid) as well as buffered peptone water (BPW) pre-enrichment media (Oxoid). Enrichment was performed in tetrathionate brilliant green broth (Oxoid), followed by plating of a loopful of the bacterial suspension on BGA.
The spleen, liver, caeca, cardiac blood, ovary and the different segments of the oviduct (vagina, uterus, isthmus, magnum and infundibulum) were collected and homogenized. The concentration of Salmonella in each sample was determined by directly plating 10-fold dilutions in BPW on BGA. Samples that were negative after direct plating were pre-enriched in BPW for 20 h at 37°C and enriched in tetrathionate brilliant green broth for 20 h at 37°C, followed by plating a loopful of the bacterial suspension on BGA. Colonies thought to be Salmonella were identified by Salmonella group D-specific slide agglutination (Difco, Kansas City, MO, USA). Samples that were negative by direct plating but positive after enrichment were presumed to contain 10 CFU/g tissue. Samples that were negative after enrichment were treated as containing 1 CFU/g for statistical analysis. The mean concentration of Salmonella in tissue was calculated for three (1, 2, 4 and 8 days post-inoculation [p.i.]) or six (14 and 21 days p.i.) hens for each time point.
Bacteriology was also carried out on the eggs. Upon collection, the eggs were passed through a chloramine solution (5 g/l). Faeces on the surface of the eggs was removed and the egg surface was decontaminated by dipping in ethanol (95%) (Gast & Holt, Citation2000; Gast et al., Citation2002). The eggs were broken aseptically. The shell, yolk and white were separated and pre-enriched by incubating overnight in BPW at 37°C. The BPW for the pre-enrichment of the egg whites was supplemented with 0.005% ferric ammonium citrate. The enrichment (overnight, 37°C) was performed in tetrathionate brilliant green broth. Thereafter, a loopful of the enrichment broth was plated on BGA.
Statistical analysis
The data were analysed with SPSS 9.0 software using Student's t test to compare the mean log10 concentration of Salmonella in the organs, the chi-squared test to compare the total number of positive eggs and Fisher's Exact Test to compare the oviduct lumen swabs. To compare the infection of the oviducts, each oviduct was allocated a score from 0 to 5, corresponding to the number of oviduct segments positive after enrichment. The scores were compared by the Mann–Whitney test.
Results
Isolation of S. Enteritidis from internal organs and blood
Prior to the experiment, excretion of Salmonella was not detected and the birds were found to be serologically negative for antibodies against Salmonella. No clinical signs of infection were seen before necropsy. During necropsy, swollen spleens and pale livers were frequently found and a few birds had atrophic ovaries. Results from the attempted isolation of S. Enteritidis after inoculation are summarized in . All spleen samples of both groups (mutant and parent strain) were Salmonella-positive at all time points after inoculation. From 14 days onwards the concentration of salmonella in the spleen was significantly higher after infection with the mutant strain.
Table 1. Isolation of a S. Enteritidis fimD mutant and the corresponding wild-type strain from the organs of intravenously inoculated laying hens
Almost all liver samples were Salmonella-positive at all time points after inoculation, except for some samples from hens infected with the wild-type strain at 2 and 3 weeks after inoculation.
Both the wild type and the mutant strain were isolated from the ovaries at a high frequency.
The caeca of hens infected with the parent strain were only found Salmonella-positive after enrichment. Taken over all time points, the caecal samples were significantly (P=0.000) more frequently contaminated with the fimD mutant (21/24) than with the wild-type strain (6/24).
From the fourth day after infection the wild-type strain was cleared from the blood in all hens. During the first 2 weeks complete clearance of the mutant strain from the blood did not occur in most birds. Even at 3 weeks p.i. the mutant was still present in the blood of one out of six hens. Taken over all time points, the blood samples were significantly (P=0.003) more frequently contaminated in birds inoculated with the fimD mutant (15/24) than in birds inoculated with the wild-type strain (4/24).
Both strains were able to colonize the oviduct at 1 day p.i. After 2 days almost all segments of the oviduct of all hens in both groups were Salmonella-positive. The median oviduct colonization score of the hens infected with the fimD mutant was scored significantly higher (P=0.015) at 2 weeks p.i. than that of the hens infected with the wild-type strain. At 3 weeks p.i. with the wild-type strain, the median colonization score of the oviduct was significantly higher (P=0.015) than at 2 weeks p.i. Taken over all time points, the median colonization score of the oviduct was significantly higher (P=0.047) in hens infected with the mutant strain than in those infected with the parent strain.
Isolation of S. Enteritidis from the oviduct lumen
The results are summarized in . All but one of the swabs of the lumen of the oviduct were negative for Salmonella after infection with the wild type strain. Taken over all different time points, the swabs of the vagina and isthmus were significantly (P≤0.05) more frequently positive after infection with the mutant strain than with the parent strain.
Table 2. Isolation of a fimD mutant of S. Enteritidis and the corresponding wild-type strain from the lumen of the oviduct of intravenously inoculated laying hens
Isolation of S. Enteritidis from the eggs
The results are presented in . A total of 117 eggs were laid by all hens during the experiment, 69 of which were laid by hens infected with the wild-type strain and 48 by hens infected with the fimD mutant. Taken over the whole experiment, infection with the wild-type strain resulted in a significantly higher incidence (P=0.009) of egg contamination (31.9%) than the fimD mutant (10.4%). The shells of surface decontaminated eggs were significantly (P=0.035) more frequently contaminated after infection with the wild-type strain than with the fimD mutant.
Table 3. Isolation of a fimD mutant of S. Enteritidis and the corresponding wild-type strain from eggs of intravenously inoculated laying hens
Discussion
In this study, intravenous inoculation of laying hens with a fimD mutant of S. Enteritidis resulted in heavier and more frequently contaminated internal organs. This may be due to the effect of the afimbriate state on bacteraemia. Bacteraemia in the laying hens after infection with the fimD mutant bacteria was more extensive and longer lasting than after infection with the wild type. This is in accordance with the results of several independent studies demonstrating that when bacteria (Escherichia coli as well as Salmonella) are administered parenterally to mice or rats, the concentrations of fim+ organisms in the animals decrease more rapidly than the concentrations of fim– organisms (Leunk & Moon, Citation1982; Perry & Ofek, Citation1984; Rumelt et al., Citation1988; Saukkonen et al., Citation1988). A S. Typhimurium fim+ strain that was injected intravenously into mice was removed from the blood and retained in the liver more efficiently than a fim– mutant strain (Leunk & Moon, Citation1982). In another study the increased virulence of a fim– mutant of S. Typhimurium was associated with a greater bacteraemia after oral infection in mice (Lockman & Curtiss III, Citation1992). Avian Escherichia coli that are lacking F1 fimbriae have been shown to be resistant to phagocytosis (Pourbakhsh et al., Citation1997a,Citationb). It has been suggested that chicken macrophages may express receptors for type 1 fimbriae, resulting in phagocytosis of bacteria expressing these fimbriae (Rodrigues-Ortega et al., Citation1987; Ofek & Sharon, Citation1988). However, a type 1 fimbrial mutant and wild-type strain are phagocytosed equally well by the chicken macrophage cell lines HD11 and MQ-NCSU (Rajashekara et al., Citation2000). Earlier reports suggested selective trapping of type 1 fimbriated S. Typhimurium from the blood by the endothelial and Kupffer cells of the liver (Leunk & Moon, Citation1982).
The expression of type 1 fimbriae is known to be phase variable (Duguid et al., Citation1966; Old & Duguid, Citation1970). The type 1 fimbrial expression of S. Typhimurium is strongly regulated, in part, by the products of four genes found within the fim gene cluster (Tinker & Clegg, Citation2000, Citation2001; Tinker et al., Citation2001). The different environments inside the host that the bacteria encounter during the consecutive phases of an infection could result in switching of the expression of type 1 fimbriae during the infection. In vitro, the expression of SEF21 is influenced by environmental conditions (Walker et al., Citation1999). However, environmental conditions that might influence fimbrial variation in vivo are not known. Fimbrial expression seems to be required during the intestinal phase (Thiagarajan et al., Citation1996b). However, other studies have shown that flagella and not fimbriae are important in caecal colonization of 1-day-old chicks (Allen-Vercoe & Woodward, Citation1999a,Citationb; Allen-Vercoe et al., Citation1999). Our studies suggest that type 1 fimbrial expression may also affect colonization of the oviduct. E. coli in chickens has been shown to switch off the expression of type 1 fimbriae during invasion of the bloodstream from the respiratory tract in chickens (Pourbakhsh et al., Citation1997b), where the unfimbriated state has an advantage over the fimbriated state.
The oviduct was more frequently infected by the mutant strain. Possibly, the higher incidence of the mutant bacteria in the blood may give rise to some incidental isolation of Salmonella from the oviduct. However, the higher frequency of infection of the oviduct tissue samples by the mutant strain corresponded with the higher number of isolations of the mutant strain from the oviduct lumen swabs, indicating that the bacteria were actually colonizing the oviduct. The extent of oviduct colonization increased in hens infected with the wild-type strain in the third week p.i. A high frequency of positive samples was seen in both of the oviduct segments that have a lot of tubular gland tissue (magnum and isthmus). This is in accordance with previous studies showing that S. Enteritidis is associated with this tubular gland tissue in natural (Hoop & Pospischil, Citation1993) and experimental (Keller et al., Citation1995) infections.
In the present study, S. Enteritidis lacking type 1 fimbriae were found to be less capable of contaminating eggs, and the contamination of shell and shell membranes was significantly reduced. However, the differences in rate of egg contamination between the two experimental groups might be biased by the relative difference in egg production between the two groups. Although in this study the yolks were overall the most frequently contaminated site in the eggs, several other studies of hens experimentally infected with S. Enteritidis have indicated that the egg shell, containing the shell membranes, is often the most frequently contaminated site in eggs after the surface has been decontaminated (Bichler et al., Citation1996; Miyamoto et al., Citation1997; Okamura et al., Citation2001a). This could be explained by the binding of the type 1 fimbriae to the secretions of the glands that produce the egg shell membrane (De Buck et al., Citation2003). Horizontal contamination of the eggs by contact with litter could not be excluded. However, the egg shells are likely to have been contaminated inside the reproductive tract, as very few Salmonella were isolated from the caeca of hens. Salmonella-contaminated shells in the absence of intestinal carriage have been reported previously (Humphrey et al., Citation1991a).
In conclusion, there are indications that type 1 fimbriae are involved in clearance of S. Enteritidis from the blood and in egg contamination by S. Enteritidis in laying hens.
Acknowledgements
This work was supported by the Belgian Ministry of Agriculture (DG6-R&D), Brussels, Belgium (grant number S6035-3). The authors would like to thank P.A. De Groot for his excellent technical assistance and M.J. Woodward (Bacteriology Department, Central Veterinary Laboratory, Addlestone, Surrey, UK) for providing the fimD mutant of S. Enteritidis.
Related Research Data
References
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