Subdivision of Salmonella enterica serovar enteritidis phage types PT14b and PT21 by plasmid profiling

We have shown that plasmid profiling is a sensitive method for further identification of strains of Salmonella enterica serovar enteritidis (S. enteritidis) phage type PT21 and to a lesser extent the strains of phage type PT14b. Five and three plasmid types were identified within 33 strains of phage type PT21 and 19 strains of phage type PT14b, respectively. Plasmid types in strains of phage type PT21 showed significant correlation with geographical origin of the strain. In strains of phage type PT14b a single isolate predominated suggesting that the plasmid designated as 'C' can be directly linked with S. enteritidis PT14b strains. Application of IS200 fingerprinting did not reveal any other differences and showed just one copy of IS200 in all the 52 analysed strains. All the strains were tested for antibiotic resistance and only four strains were resistant to ampicillin, cefotaxime, cefuroxime and cotrimoxazole. This indicates that low molecular weight plasmids in Salmonella enteritidis are not responsible for the spread of antibiotic resistance.     

Pulsed-field gel electrophoresis-based subtyping of DNA degradation-sensitive Salmonella enterica subsp. enterica serovar Livingstone and serovar Cerro isolates obtained from a chicken layer farm

Salmonella enterica serovar subsp. enterica Livingstone and serovar Cerro isolates from a commercial egg-producing farm, which had previously been untypeable by pulsed-field gel electrophoresis (PFGE) because of DNA degradation during the PFGE process, successfully gave banding patterns using electrophoresis buffer supplemented with 50 microM thiourea. By PFGE in the presence of thiourea, DNA degradation-sensitive S. enterica serovar Cerro isolates from the commercial egg-producing farm were found to be genetically unrelated to S. enterica serovar Cerro isolates that gave the patterns in the absence of thiourea. Forty-five of 50 (90%) S. enterica serovar Livingstone isolates from the farm showed arbitrarily designated XbaI-digested patterns X1 and X2 that were distinguished by one-band difference and had an identical BlnI-digested pattern. In one of the two layer houses in the farm, the numbers of isolates having the pattern X2 increased from 57% in 1997 to 89% in 1998, whereas virtually all the isolates obtained from the other house in the same period showed the profile X1. This suggests that strains having the pattern X2 might have an advantage to preferentially colonize in the former house.     

Longitudinal monitoring of two commercial layer flocks and their environments for Salmonella enterica serovar enteritidis and other Salmonellae

Between August 20, 2001, and September 17, 2002, 1429 samples including drag swabs, egg belt or egg rollout swabs, fan-blade swabs, rodent organ and intestinal pools, beetle (Alphitobius diaperinus) pools, housefly (Musca domestica) pools, chicken organ and intestinal pools, and egg pools were obtained for Salmonella culture from two flocks from two different commercial layer ranches. The two ranches were purposefully selected for the study based on their previous status of Salmonella Enteritidis isolation using environmental drag swabs in cooperation with practicing veterinarians. Salmonella sp. was isolated from 337 out of 979 (34.42%) non-egg samples. No Salmonella was isolated from 450 egg pools collected from either ranch. S. enteritidis was isolated from samples obtained from ranch 1 from manure drag swabs, 4/284 (1.4%); rodent organs, 1/24 (4.2%); and housefly pool cultures 1/21 (4.8%). Salmonella Enteritidis was isolated from ranch 2 from mouse organ and intestinal pool samples, 1/24 (4.2%). Salmonella group B was isolated from all sample types except the insects. There was a statistically significant difference in isolation rates among seven serogroups of Salmonella: groups B, C1, C2, D, E, K, and untypeable (Pearson chi-square 18.96, P = 0.002). Overall, statistically significant differences were observed with respect to Salmonella isolation among the types of samples taken (Pearson chi-square 118.54, P < 0.0001). Intensive monitoring for Salmonella Enteritidis can be used to optimize a Salmonella reduction program for an individual poultry biosecurity unit.     

Effects of prior coinfection with different Salmonella serovars on the progression of a Salmonella enterica serovar enteritidis infection in hens undergoing induced molt

Four trials were conducted to evaluate whether prior infection with Salmonella enterica serovar typhimurium (S. typhimurium) or Salmonella enterica serovar muenchen (S. muenchen) would modify the severity or the transmission of Salmonella enterica serovar enteritidis (S. enteritidis) challenge in hens undergoing molt via feed withdrawal. Hens were separated into two groups where one group received a prior S. typhimurium or S. muenchen infection, whereas the other group remained untreated until S. enteritidis challenge. In trials 1 and 2, one group of hens was infected with S. typhimurium 5 days prior to feed withdrawal. Both groups of hens were then challenged with S. enteritidis on day 4 post feed withdrawal. In trials 3 and 4, one group of hens received S. typhimurium or S. muenchen, respectively, 1 day after feed was withdrawn. Transmission of S. enteritidis was evaluated by challenging the center hen in rows of 11 hens per row with S. enteritidis at 4 days post feed withdrawal and following the progression of the S. enteritidis down the row of hens over time. In trials 1 and 2, where hens received S. typhimurium 5 days prior to feed withdrawal, shedding of the S. enteritidis challenge was significantly reduced in hens on day 10 postchallenge in trial 1 and on days 3 and 10 postchallenge in trial 2 compared with the hens subjected only to the molt procedure. Significantly fewer S. enteritidis were recovered in livers and spleens at day 9 postchallenge in trial 2 from hens receiving the prior S. typhimurium infection. In trial 3, where hens received S. typhimurium 1 day after feed withdrawal, S. enteritidis transmission was significantly reduced in these hens on days 3, 10, and 24 postchallenge. In trial 4, similar in methodology to trial 3 except that, rather than S. typhimurium, hens received S. muenchen, a Salmonella organism totally lacking any antigen cross-reactive with S. enteritidis, S. enteritidis transmission was significantly reduced on days 3, 10, 17, and 24 postchallenge, suggesting that factors other than specific immunity were involved in the observed resistance to S. enteritidis infection. These results indicate that prior infection of a flock with a non-S. enteritidis paratyphoid Salmonella can reduce S. enteritidis problems that may occur during a molt.     

Eggshell characteristics and penetration by Salmonella enterica serovar Enteritidis through the production period of a layer flock

1. Egg weight, shell thickness, number of pores, cuticle deposition and ability of Salmonella enterica serovar Enteritidis (SE) to penetrate the shell were determined for eggs from one layer flock through the entire production period. 2. Penetration was assessed by filling the eggs with a selective medium that allowed visualising Salmonella growth on the inside of the shell and membrane complex. After inoculation of each shell with on average 2.59 log cfu, the eggs were stored for up to 20 d at 20 degrees C and 60% relative humidity (RH). 3. On average 38.7% of the eggshells became penetrated. Mostly penetration occurred on d 3. Although it affected all shell characteristics studied, hen age did not significantly influence eggshell penetration. 4. No correlations were observed between any of the shell characteristics studied and the ability of SE to penetrate the shell. The growth of SE on the shell is of major importance because shell contamination at 20 d of storage and SE penetration were highly correlated.     

Age at primary infection with Salmonella enterica serovar Typhimurium in the chicken influences persistence of infection and subsequent immunity to re-challenge

Salmonella enterica remains one of the most important food-borne pathogens of humans and is often acquired through consumption of infected poultry meat or eggs. Control of Salmonella infections in chicken is therefore an important public health issue. Infection with S. enterica serovar Typhimurium results in a persistent enteric infection without clinical disease in chickens of more than 3 days of age, and represents a source for contamination of carcass at slaughter and entry into the human food chain. Data presented indicate a profound effect of age at initial exposure on the persistence of infection and a lesser effect on the development of effective immunity to re-challenge. The percentage of birds positive for Salmonella was high until 8–9 weeks of age, regardless of the age at which the birds were infected (1, 3 or 6 weeks). The birds infected at 3 and 6 weeks of age produced a more rapid and higher antibody response (IgY and IgA) than those infected at 1 week of age, but in all cases infection persisted for a considerable period despite the presence of high antibody levels. Following a re-challenge infection with S. Typhimurium, all three previously-infected groups had fewer bacteria in the gut, spleen and liver compared with age-matched birds receiving a parallel primary infection. However, the birds primary infected at 3 and 6 weeks of age cleared infection more rapidly than those infected at a younger age. Interestingly older-primed birds had higher specific T lymphocyte proliferative responses and specific circulating levels of IgY antibody at time of re-challenge. Although birds initially infected at 1 week of age and those that were previously uninfected produced a stronger antibody response following re-challenge, they were slower to clear Salmonella from the gut than the older-primed groups which expressed a stronger T lymphocyte response. The data presented indicate that clearance of Salmonella from the gut is age-dependent and we propose that this relates to the increased competence of the enteric T cell response. The findings that Salmonella persists beyond 8–9 weeks, irrespective of age at exposure, has implications for the broiler sector and indicates the need to remain Salmonella free throughout the rearing period. Moreover, the re-challenge data demonstrates that infection at a young age is less effective in producing protective immunity than in older chickens. This feature of the development of protective immunity needs to be considered when developing vaccines for the broiler sector of the poultry industry.     

Distribution of Salmonella enterica serovars from humans, livestock and meat in Vietnam and the dominance of Salmonella Typhimurium phage type 90

Epidemiologically unrelated non-typhoid Salmonella isolates from humans (n = 56) and animal origin (n = 241, from faeces, carcasses and meat) in Vietnam were investigated. Salmonella Typhimurium, S. Anatum, S. Weltevreden, S. Emek, and S. Rissen were the most prevalent serovars. S. Typhimurium phage type 90 was predominant among S. Typhimurium isolates. The serotype and phage type distribution of the Salmonella isolates was different from that in Europe and America. Many sero- and phage types found in humans were also found in cattle, pigs, and poultry suggesting that food producing animals are an important source of human non-typhoid Salmonella infection in Vietnam.     

The attenuated sopB mutant of Salmonella enterica serovar Typhimurium has the same tissue distribution and host chemokine response as the wild type in bovine Peyer's patches

Salmonella enterica serovar Typhimurium is an important cause of enteric infections in farm animals and it is one of the most frequent food borne infections worldwide. Serovar Typhimurium lacking the sopB gene is attenuated for induction of host inflammatory response and fluid accumulation into the intestinal lumen, which correlates with clinical diarrhea. SopB is an inositol phosphate phosphatase, but its exact role in the pathogenesis of salmonellosis is still unclear. We employed the bovine ileal ligated loop model to compare the tissue distribution of a sopB mutant and its wild type parent serovar Typhimurium. Sections of the Peyer's patches were histologically processed and immuno-stained for detection of serovar Typhimurium. In addition, samples were processed for transmission electron microscopy, and the profile of expression of host chemokine and cytokine responses was assessed. Ultrastructurally both strains had the same ability to invade intestinal epithelial cells. No differences were detected in the tissue distribution of the sopB mutant and the wild type organism and both strains elicited the same profile of chemokines and pro-inflammatory cytokines. In conclusion, our results indicate that the attenuation of the sopB mutant is associated with pathogenic mechanisms other than invasion and distribution in host intestinal tissues.     

Observations on Salmonella contamination of commercial laying farms before and after cleaning and disinfection

Little is known about the effectiveness of the cleaning and disinfection methods in use on commercial laying farms in Great Britain. Samples were taken from poultry house structures and equipment of five cage layer flocks, five barn egg production flocks and two free-range flocks. In the free-range houses there was a decrease in Salmonella after cleaning and disinfection, although the soil in the paddocks remained contaminated. In the barn and especially the cage layer houses, significant residual contamination remained on the surfaces of buildings and equipment. Wildlife pests were also found to be carrying Salmonella in the disinfected houses and free-range paddocks.     

Deposition of phage type 4 and 13a Salmonella enteritidis strains in the yolk and albumen of eggs laid by experimentally infected hens

Because egg yolk and albumen differ substantially in their abilities to support bacterial growth, the initial level and location of Salmonella enteritidis deposition are critical for determining whether proposed standards for refrigerating eggs are likely to protect public health by preventing extensive microbial multiplication. In the present study, three groups of laying hens were infected with oral doses of approximately 10(9) cells of different S. enteritidis strains (two were phage type 4 and one was phage type 13a) in two replicate trials. For all three S. enteritidis strains, the incidence of yolk contamination (approximately 2.5% overall) was significantly greater than the incidence of albumen contamination (approximately 0.5% overall). The phage type 13a strain was less often isolated from fecal samples at 2 wk post-inoculation than were the phage type 4 strains, but no significant differences between strains were observed in the incidence of egg contamination. Most freshly laid contaminated eggs contained fewer than 1 S. enteritidis cell/ml of egg yolk or albumen, and no sample contained more than 67 S. enteritidis cells/ml.     

Effects of using a chicken-origin competitive exclusion culture and probiotic cultures on reducing Salmonella in broilers

Three commercial products, a partially defined chicken-origin competitive exclusion culture (Aviguard, 0.50 mL/chick) and 2 single-organism probiotic cultures (Saccharomyces cerevisiae, Levucell SC, 1 g/kg of feed, and Pediococcus acidilactici, Bactocell, 100 mg/kg of feed) were evaluated for their ability to reduce Salmonella in broilers and their effects on production performance. It was found that all the treatments significantly (P < 0.05) reduced Salmonella concentrations, as compared with the control, on the chicken, in the ceca, and on the chicken carcass. In addition, it was found that these treatments had no adverse effect on any of the production parameters that were measured. Finally, this study showed the importance of using these preharvest treatments as part of an integrated program to control Salmonella at the broiler farm.     

Comparison of real-time PCR with conventional PCR and culture to assess the efficacy of a live attenuated Salmonella enterica serovar Typhimurium vaccine against Salmonella enterica serovar Enteritidis in commercial leghorn chicks vaccinated under field and laboratory conditions

The efficacy of a live attenuated Salmonella Typhimurium Megan Vac 1 vaccine (MV1) was evaluated against Salmonella Enteritidis in chicken pullets with the use of PCR and culture methods. Two hundred Hyline W-32 white leghorn chicks were obtained from a local hatchery and divided into four treatment groups. Two of the groups served as positive and negative controls. The MV1 vaccine was administered to the chicks in the remaining two groups at 1 and 35 days old by either the coarse spray (field) or the oral route (laboratory) method. The chicks were challenged with a high dose of a Salmonella Enteritidis strain at 10 wk old and euthanatized 3 days postinoculation. Samples for PCR analysis were collected prior to enrichment, after pre-enrichment in buffered peptone water (BPW) and after primary enrichment from the ceca, liver, and spleen. None of the samples tested yielded positive results for the Salmonella Typhimurium vaccine strain by either the culture or PCR methods. Results from the standard culture method showed that vaccinating the birds with MV1 reduced the counts of Salmonella Enteritidis recovered from the challenged birds. In addition, fewer pre-enriched samples tested positive for Salmonella Enteritidis among the challenged groups that were vaccinated when compared to the unvaccinated challenged group. Under the conditions of this study, MV1 was unable to prevent colonization of other internal organs such as the liver and spleen. Real-time PCR was significantly more sensitive than conventional PCR (C-PCR) prior to enrichment, but after enrichment the sensitivities of the two methods were similar. Enrichment significantly increased the sensitivity of both PCR methods for the detection of Salmonella Enteritidis in cecal samples, but did not significantly increase the sensitivity for detection of Salmonella Enteritidis in liver and spleen samples that were pre-enriched in BPW. There was no significant difference between the laboratory or field vaccination methods with respect to either the prevalence of Salmonella Enteritidis isolation or the bacterial loads in culture-positive samples. Collectively, the data suggest that MV1 offered some protection against Salmonella Enteritidis in commercial layer chick pullets under the conditions of this study. Given the labor and time required to perform the C-PCR and culture methods, the real-time PCR method may prove to be a more useful method to use in diagnostics.     

Effect of a commerical competitive exclusion product on the colonization of Salmonella infantis in day-old pheasant chicks

One-day-of-age pheasant chicks were treated orally with the competitive exclusion product Broilact in three replicate trials. The following day the treated chicks and untreated control chicks were challenged likewise with approximately 10(3) colony-forming units (cfu) of Salmonella infantis. Five days after challenge the cecal contents of the birds were examined quantitatively and by enrichment for S. infantis. In all three trials Broilact effectively reduced colonization of the challenge organism, the mean infection factor (IF) value (the logarithmic number of colony forming units of salmonella organisms per gram of cecal contents) for the treated groups being 2.9 and that for the salmonella control groups 8.4. Mortality during the 1 week rearing period was 5.0% in the Broilact treated groups and 8.5% in the salmonella control groups.     

Occurrence of Salmonella serotype Typhimurium DT104 on a commercial swine farm before, during, and after depopulation and repopulation

OBJECTIVE: To determine whether depopulation-repopulation could be used to eradicate Salmonella serotype Typhimurium DT104 from a commercial swine farm in the midwestern United States. DESIGN: Observational study SAMPLE POPULATION: A commercial swine farm undergoing depopulation-repopulation to eliminate porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae. PROCEDURE: Pooled fecal samples, tissue samples, and serum samples were collected from pigs on the farm before and after depopulation-repopulation. When there were no pigs on the farm, environmental swab specimens were collected for bacterial culture. Serum was analyzed for anti-Salmonella antibodies with an indirect ELISA. Salmonella isolates obtained by bacterial culture of fecal, tissue, and environmental samples were characterized by means of serotyping, phage typing, pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing. RESULTS: 167 Salmonella isolates representing 9 serotypes were recovered from the farm. Results of PFGE and antimicrobial susceptibility testing suggested that S. Typhimurium DT104 strain was not eradicated from the farm. However, seroprevalence of anti-Salmonella antibodies and the percentage of pooled fecal samples positive for Salmonella spp were significantly decreased following repopulation. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that depopulation-repopulation in conjunction with stringent cleaning and disinfection, attention to biosecurity procedures, control of other diseases, and changes in feed management may reduce the occurrence of, but likely will not eliminate, Salmonella spp in commercial swine herds.     

Prevalence of Escherichia coli O157:H7 prophage-like sequences among German Salmonella enterica serotype Typhimurium phage types and their use in detection of phage type DT104 by the polymerase chain reaction

A 1.6kb DNA fragment identified by random amplifiable polymorphic DNA differentiation (RAPD) from a Salmonella enterica serotype Typhimurium phage type DT104 isolate was used to investigate the prevalence of the region in 160 DT104 isolates, 83 other epidemiological important S. Typhimurium phage types and 20 strains selected from 17 other Salmonella serotypes. PCR screening tests using two different primer-sets derived from the RAPD fragment's nucleotide sequence showed that 76% of the 160 DT104 isolates investigated, including subtypes DT104A, DT104B, DT104B low, DT104H and DT104L, reacted positively. High sensitivity was shown for DT104 strains expressing at least the penta-resistance pattern ACSSuT (97% of 104 strains tested). DT104 susceptible strains showed only a sensitivity of 35% (17 strains tested). In contrast, 83% of the 83 strains from the other S. Typhimurium phage types reacted negatively. Strains from five out of the 17 other serotypes showed a positive signal with one primer-set. The other primer-set exhibited only a positive reaction with one S. Dublin isolate. The analysis of a 2415bp extended sequence revealed homologies to genes encoded by Escherichia coli O157:H7 prophages, suggesting that the described region contains genes of a prophage specific for DT104 and related phage types.     

Biofilm formation in field strains of Salmonella enterica serovar Typhimurium: identification of a new colony morphology type and the role of SGI1 in biofilm formation

In this study we examined the extent of biofilm formation in field strains of Salmonella enterica serovar Typhimurium (S. Typhimurium), an important foodborne pathogen. Ninety-four field strains of S. Typhimurium were tested for their ability to form biofilm and components contributing to its formation. Most S. Typhimurium strains were highly capable of biofilm formation except for strains of phage type DT2 originating from pigeons. The most efficient biofilm forming strains were those of phage type DT104 positive for Salmonella genomic island 1 (SGI1). A comparison of SGI1 positive and negative strains indicated that the increased biofilm formation of SGI1 positive strains was associated with the presence of this genomic island. Finally, in five strains we found an alternative strategy of biofilm formation independent of curli fimbriae and cellulose production but solely dependent on an overproduction of capsular polysaccharide. Due to a mucoid and brown appearance on Congo Red agar we designated these strains as belonging to the SBAM (smooth brown and mucoid) morphotype.     

The effects of a competitive exclusion product and two probiotics on Salmonella colonization and nutrient digestibility in broiler chickens

Competitive exclusion (CE) cultures, given as a single dose on the day of hatch, together with good hygienic practices has been shown to be a novel approach to control Salmonella in poultry. The ability of the CE product Broilact and 2 probiotics, FloraMax-B11 and Colostrum, to prevent Salmonella colonization in newly hatched chickens was evaluated employing a slightly modified Mead-model chicken assay. In a parallel study the effect of the 3 treatments on the production of volatile fatty acids in the ceca were determined. In the Salmonella study 2 separate experiments were done. In the first experiment all 3 treatment materials were given as a single dose on d 1. In the second experiment, which consisted only of Broilact and FloraMax-B11, the latter was given in the drinking water during the 3 first d after hatch. In both experiments the chicks were challenged with Salmonella enterica serovar Infantis on d 2. The results of the present study show that Broilact was superior to the 2 other treatment materials in protecting the newly hatched chickens against Salmonella colonization. The parallel study showed only minor differences among the different treatments. Based on the results of the Salmonella challenge study, it was concluded that Broilact was the only treatment material that was established in the gut of the newly hatched chickens in such a way that the colonization of Salmonella was prohibited.