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OBJECTIVE: To compare the efficacy of a Salmonella bacterin and a modified live Salmonella ser. Choleraesuis vaccine on a commercial dairy. ANIMALS: 450 cows in late gestation and 80 calves. PROCEDURE: Group-1 cows (n = 150) were vaccinated once with a modified live S. Choleraesuis (serogroup C1) strain 54 (SC54) vaccine, group-2 cows (150) were vaccinated on enrollment and 30 days later with a Salmonella ser. Montevideo (serogroup C1) bacterin, and group-3 cows (150) served as unvaccinated controls. One gallon of colostrum harvested from the first 80 cows to calve was fed to each calf. Outcome assessments included fecal shedding of Salmonella spp for the first 10 days after parturition (cows) or birth (calves), milk production, involuntary culling rate, mastitis incidence, antimicrobial use, and mortality rate. RESULTS: Salmonellae were isolated from 306 of 309 (99%) cows and 64 of 74 (86.5%) calves. Shedding frequency was less in SC54-vaccinated cows and calves that received colostrum from those cows, compared with the other groups, and vaccination was specifically associated with less shedding of serogroup C1 salmonellae. Production data were similar among groups. CONCLUSIONS AND CLINICAL RELEVANCE: Vaccination of pregnant cows with an autogenous Salmonella bacterin had no effect on fecal shedding of salmonellae, whereas vaccination with a modified live S. Choleraesuis vaccine reduced the frequency of fecal shedding of serogroup C1 salmonellae during the peripartum period. A commercial S. Choleraesuis vaccine licensed for use in swine may be more efficacious than autogenous Salmonella bacterins on dairies infected with serogroup C1 salmonellae.
Loop-mediated isothermal amplification (LAMP) assay was effective in detecting Salmonella enterica in naturally contaminated liquid egg samples. Salmonella was detected in 110 samples taken from four egg-breaking plants. The egg samples were pre-enriched in buffered peptone water (BPW) at 37 degrees C for 20 h. The selective enrichment was done in Rappaport-Vassiliadis or tetrathionate broth and plated onto xylose lysine deoxycholate agar and brilliant green agar, modified. In addition, the PCR assay was used to detect Salmonella after pre-enrichment in BPW at 37 degrees C for 20 h. The culture method and PCR assay were compared to the LAMP assay, which was also performed after pre-enrichment in BPW. PCR failed to detect Salmonella in 10% of 110 samples, whereas the culture method and LAMP assay successfully identified Salmonella in all samples. However, the LAMP assay was found to be much more rapid than the culture method and as sensitive in detecting Salmonella from liquid eggs. In all of the egg-breaking plants studied, Salmonella was isolated on most tested days. The positive samples showed that more than 75% of the Salmonella strains had identical genetic patterns when analyzed by pulsed-field gel electrophoresis. This suggests that the same Salmonella strains having survived long periods of time in the plants were contaminating the production line. The LAMP assay is rapid, specific, and sensitive for Salmonella detection in liquid eggs and is able to monitor Salmonella contamination in egg-handling plants more reliably.
The effect of acid shock with hydrochloric, citric, or lactic acid on the survival and growth of Salmonella Typhi and Salmonella Typhimurium in acidified broth was evaluated. Salmonella serovars were acid shocked (1 h at 35 degrees C) in Trypticase soy broth acidified with hydrochloric, citric, or lactic acid at pH 5.5. Unshocked cells were exposed to the same media that had been neutralized before use to pH 7.0. Shocked and unshocked cells were inoculated into broth acidified with hydrochloric acid (pH 3.0), citric acid (pH 3.0), or lactic acid (pH 3.8), and growth and survival ability were evaluated. The acid shock conferred protection to Salmonella against the lethal effects of low pH and organic acids. The adaptive response was not specific to the anion used for adaptation. The biggest difference in reduction of survival between shocked and unshocked strains (approximately 2 log CFU/ml) was observed when the microorganisms were shocked with lactic acid and then challenged with citric acid. Salmonella Typhi was more tolerant of citric acid than was Salmonella Typhimurium, but Salmonella Typhimurium had higher acid tolerance in response to acid shock than did Salmonella Typhi. The acid shock decreased the extension of the lag phase and enhanced the physiological state values of Salmonella Typhi and Salmonella Typhimurium when the pH of growth was 4.5. This increased ability to tolerate acidity may have an important impact on food safety, especially in the case of Salmonella Typhi, given the very low infectious dose of this pathogen.
The goal of this study was to evaluate the suitability of the fimA gene amplification by PCR as a specific method for detection of Salmonella strains. Salmonella typhimurium and other pathogenic members of the family Enterobacteriaceae produce morphologically and antigenically related, thin, aggregative, type 1 fimbriae. A single gene, fimA, encodes the major fimbrial unit. In order to obtain higher specificity, we have selected a series of primers internal to the fimA gene sequence and have developed a PCR method for detecting Salmonella strains. A collection of 376 strains of Salmonella comprising over 80 serovars, isolated from animals and humans in Canada, have been used to evaluate this PCR method. Forty non-Salmonella strains were also tested by the same procedure. Cultures were screened by inoculating a single colony of bacteria directly into a PCR mixture containing a pair of primers specific for the fimA gene. The specific PCR product is an 85-bp fragment which was visualized by polyacrylamide gel electrophoresis and ethidium bromide staining. All Salmonella strains gave positive results by the PCR. Feed and milk samples contaminated by Salmonella strains were also detected by this procedure. The detection of all Salmonella strains tested and the failure to amplify the fragment from non-Salmonella strains confirm that the fimA gene contains sequences unique to Salmonella strains and demonstrate that this gene is a suitable PCR target for detection of Salmonella strains in food samples.
We describe broad-range salmonellae (ie, Salmonella) and Salmonella serotype Typhi-specific LightCycler (Roche Diagnostics, Indianapolis, IN) real-time polymerase chain reaction assays. We validated these with a battery of 280 bacteria, 108 of which were salmonellae representing 20 serotypes. In addition, 298 isolates from 170 clinical specimens that were suspected to possibly represent Salmonella were tested with the pan- Salmonella assay. Finally, the pan-Salmonella assay also was used to test DNA extracts from 101 archived, frozen stool specimens, 55 of which were culture-positive for salmonellae. Both assays were 100% sensitive and specific when cultured isolates of the battery were tested. The pan- Salmonella assay also characterized correctly all salmonellae on the primary isolation agar and was 96% sensitive (53/55) and 96% specific (49/51) when nucleic acid extracts from direct stool specimens were tested. These assays represent potential tools the clinical microbiologist could use to screen suspect isolates or stool specimens for Salmonella.
A stable live avirulent, genetically modified delta cya delta crp Salmonella typhimurium vaccine strain, chi 3985, was used in several vaccination strategies to evaluate its use in the control of Salmonella infection in chickens. Oral vaccination of chickens at 1 and at 14 days of age with 10(8) CFU of chi 3985 protected against invasion of spleen, ovary, and bursa of Fabricius and colonization of the ileum and cecum in chickens challenged with 10(6) CFU of virulent homologous Salmonella strains from group B. Chickens challenged with heterologous Salmonella strains from groups C, D, and E were protected against visceral invasion of spleen and ovary, while invasion of the bursa of Fabricius and colonization of ileum and cecum was reduced in vaccinated chickens. Oral vaccination at 2 and at 4 weeks of age induced an excellent protection against challenge with virulent group B Salmonella serotypes and very good protection against challenge with group D or E Salmonella serotypes, while protection against challenge with group C Salmonella serotypes was marginal but significant. Vaccination at 2 and at 4 weeks of age also protected vaccinated chickens against challenge with 10(8) CFU of highly invasive S. typhimurium or S. enteritidis strains. The protection of chickens vaccinated with chi 3985 against challenge with homologous and heterologous Salmonella serotypes is outstanding, and the complete protection against ovarian invasion in chickens challenged with 10(8) CFU of highly invasive S. typhimurium or S. enteritidis strains suggests that vaccination of chickens with chi 3985 can complement the present hygiene- and sanitation-based Salmonella control measures. This paper reports a breakthrough in the use of live avirulent vaccine to control Salmonella carriers in chickens.
In Experiment 1, mature laying hens were inoculated intravaginally with 10(6) colony-forming units of Salmonella enterica serovar enteritidis (S. enteritidis), Salmonella typhimurium, Salmonella infantis, Salmonella hadar, Salmonella heidelberg, or Salmonella montevideo to compare their abilities to colonize the reproductive organs of chickens and to contaminate eggs. Salmonella enteritidis was more frequently recovered (from 11 of 40 eggs, 27.5%) than the other serovars, and especially the inner shell was contaminated with these organisms in 10 of 40 eggs (25.0%). The contamination rates and the viable counts in cloaca were significantly (P < 0.05) higher in hens inoculated with S. enteritidis than in those inoculated with the other serovars at 4 days postinoculation (PI). In the vagina, the positive rates were 90%-100% in hens inoculated with S. enteritidis, and the viable counts of the organisms in this portion were significantly (P < 0.05) higher than those of the other serovars at 2, 4, and 7 days PI. The ceca were colonized similarly by each serovar at 7 days PI. The spleen and ovary were infected with S. enteritidis in three and one hen, respectively. No Salmonella was recovered from liver and peripheral blood in any hen. Salmonella enteritidis was recovered from other oviductal portions than the vagina (10%-20%), whereas no forming egg was contaminated in the oviduct. In Experiment 2, the in vitro adherence of these six serovars to the vaginal epithelium was compared with vaginal explants. The mean number of S. enteritidis attaching to the secondary villi in the vaginal lumen was significantly (P < 0.05) higher than those of the other serovars. These results suggest that S. enteritidis has a specific advantage over the other Salmonella serovars by its capacity to colonize the vaginal tissues of hens, and this higher affinity of S. enteritidis to the vagina may play a significant role in the production of many S. enteritidis-contaminated eggs.
Amplification of nucleotide sequences within the invA gene of Salmonella typhimurium was evaluated as a means of detecting Salmonella. A collection of 630 strains of Salmonella comprising over 100 serovars, including the 20 most prevalent serovars isolated from animals and humans in Canada, was examined. Controls consisted of 142 non-Salmonella strains comprising 21 genera of bacteria. Cultures were screened by inoculating a single colony of bacteria directly into a polymerase chain reaction (PCR) mixture which contained a pair of primers specific for the invA gene. The specific PCR product was a 284 bp DNA fragment which was visualized in 2% agarose gels. With the exception of two S. litchfield and two S. senftenberg strains, all Salmonella strains were detected. In contrast, none of the non-Salmonella strains yielded the specific amplification product. Non-specific amplification of a few non-Salmonella strains resulted in a product that was distinctly different in size from the specific 284 bp product. Specificity of amplification was further confirmed by demonstration of hybridization of a 32P-labelled invA gene fragment only to the specific 284 bp product. The detection of 99.4% of Salmonella strains tested and the failure to specifically amplify DNA from non-Salmonella strains confirm that the invA gene contains sequences unique to Salmonella and demonstrate that this gene is a suitable PCR target, with potential diagnostic applications.
The occurrence and incidence of gram-positive organisms which possess some biochemical and serological characteristics in common with salmonellae are described. These organisms were encountered as contaminants in various food products as well as in feces. Their growth in various Salmonella enrichment media, typical Salmonella-like growth on differential selective media, biochemical similarities to Salmonella, and agglutination in Salmonella polyvalent O antiserum serve as a source of possible confusion in analyses for Salmonella. Although a definitive identification was not performed, preliminary results indicate that these bacteria closely resemble members of the genus Brevibacterium. No sanitary significance could be associated with these bacteria, because they were never found to coexist with Salmonella. The occurrence of these bacteria illustrates the pitfalls of a cursory examination for Salmonella. On the premise that ultimate identification of Salmonella rests upon serological procedures, a rapid and definitive identification of suspect Salmonella isolates is proposed, based upon reactions with polyvalent group O, group O, and Spicer-Edwards H antisera.
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.
Egg contamination by Salmonella Enteritidis has remained a significant public health problem for nearly two decades, and Salmonella Heidelberg has also been recently implicated in egg-transmitted human illness. Colonization of the intestinal tract is a necessary precursor to the invasion of reproductive organs and subsequent deposition inside eggs laid by infected hens, but the relationship between the persistence of Salmonella in the intestinal tract and the likelihood of egg contamination has been uncertain. In this study, groups of laying hens were inoculated with large oral doses of strains of Salmonella Enteritidis and Salmonella Heidelberg, including variants of the original parent strains that had been reisolated from eggs laid by infected hens in a prior study. The shedding of Salmonella in voided feces was monitored for 6 wk postinoculation, and all eggs laid by infected hens between 5 and 22 days postinoculation were cultured for Salmonella in their contents. The mean duration of fecal shedding was significantly longer for the previously passaged Salmonella strains (26.7 days) than for the original parent strains (17.5 days), and the passaged strains caused a significantly higher frequency of egg contamination (6.4%) than did the parent strains (3.3%). However, the duration of fecal shedding and the frequency of egg contamination were not correlated for any of the Salmonella Enteritidis or Salmonella Heidelberg strains.
OBJECTIVE: To estimate prevalence of fecal shedding of Salmonella spp among horses in the US horse population and prevalence of Salmonella spp in grain or other concentrate used as horse feed on equine operations in the United States. DESIGN: Cross-sectional survey. SAMPLE POPULATION: Horses on 972 operations in 28 states. PROCEDURE: Fecal samples were collected from horses resident at each operation. Only a single sample was collected from any individual horse; number of horses from which samples were collected on each operation was determined on the basis of number of horses on the operation. A single sample of grain or concentrate was also collected from each operation. All samples were tested for Salmonella spp by means of bacterial culture. RESULTS: Overall, 0.8% (SE, 0.5) of resident horses shed Salmonella spp in their feces. The overall prevalence of operations positive for fecal shedding of Salmonella spp (i.e., operations with > or = 1 horse shedding Salmonella spp in its feces) was 1.8% (SE, 0.7). Prevalence of grain or other concentrate samples positive for Salmonella spp was 0.4%. Serotypes of Salmonella spp that were identified in grain or other concentrate were not those typically associated with clinical disease in horses. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the national prevalence of fecal shedding of Salmonella spp by horses in the United States was 0.8%, and that prevalence of Salmonella spp in grain or other concentrate used for horse feed was 0.4%.
OBJECTIVE: To determine sources of Salmonella organisms in a veterinary teaching hospital, compare bacterial culture with polymerase chain reaction (PCR) testing for detection of Salmonella organisms in environmental samples, and evaluate the effects of various disinfectants on detection of Salmonella organisms on surface materials. DESIGN: Prospective study. SAMPLE POPULATION: Fecal samples from 638 hospitalized horses and 783 environmental samples. PROCEDURE: Standard bacterial culture techniques were used; the PCR test amplified a segment of the Salmonella DNA. Five disinfectants were mixed with Salmonella suspensions, and bacterial culture was performed. Swab samples were collected from 7 surface materials after inoculation of the surfaces with Salmonella Typhimurium, with or without addition of a disinfectant, and submitted for bacterial culture and PCR testing. RESULTS: Salmonella organisms were detected in fecal samples from 35 (5.5%) horses. For environmental samples, the proportion of positive bacterial culture results (1/783) was significantly less than the proportion of positive PCR test results (110/783), probably because of detection of nonviable DNA by the PCR test. Detection of Salmonella organisms varied with the surface material tested, the method of detection (bacterial culture vs PCR testing), and the presence and type of disinfectant. CONCLUSIONS AND CLINICAL RELEVANCE: Results of the present study suggested that Salmonella organisms can be isolated from feces of hospitalized horses and a variety of environmental surfaces in a large animal hospital. Although recovery of Salmonella organisms was affected by surface material and disinfectant, bleach was the most effective disinfectant on the largest number of surfaces tested.
Salmonella typhimurium causes an invasive disease in mice that has similarities to human typhoid. A type III protein secretion system encoded by Salmonella pathogenicity island 2 (SPI2) is essential for virulence in mice, as well as survival and multiplication within macrophages. Reactive nitrogen intermediates (RNI) synthesized by inducible nitric oxide synthase (iNOS) are involved in the control of intracellular pathogens, including S. typhimurium. We studied the effect of Salmonella infection on iNOS activity in macrophages. Immunofluorescence microscopy demonstrated efficient colocalization of iNOS with bacteria deficient in SPI2 but not wild-type Salmonella, and suggests that the SPI2 system interferes with the localization of iNOS and Salmonella. Furthermore, localization of nitrotyrosine residues in the proximity was observed for SPI2 mutant strains but not wild-type Salmonella, indicating that peroxynitrite, a potent antimicrobial compound, is excluded from Salmonella-containing vacuoles by action of SPI2. Altered colocalization of iNOS with intracellular Salmonella required the function of the SPI2-encoded type III secretion system, but not of an individual "Salmonella translocated effector." Inhibition of iNOS increased intracellular proliferation of SPI2 mutant bacteria and, to a lesser extent, of wild-type Salmonella. The defect in systemic infection of a SPI2 mutant strain was partially restored in iNOS(-/-) mice. In addition to various strategies to detoxify RNI or repair damage due to RNI, avoidance of colocalization with RNI is important in adaptation of a pathogen to an intracellular life style.
A questionnaire-based, retrospective field study was conducted in 78 Danish broiler houses (analytical units) on 42 farms. In spring 1997, all these broiler houses had been infected with Salmonella Enteritidis, phage type 8, and/or Salmonella Typhimurium, definitive-type 66, by day-old chicks delivered from the same hatchery. Because these two salmonella types had not been detected in the Danish broiler sector before and the broiler houses were infected in a period of low salmonella prevalence, the study could focus on farm-related factors, without being distorted by other salmonella infections. The same person visited all 78 broiler houses, and farm-related factors were listed both from personal observations and by interrogating the person in charge of attending to the broilers. Additional factors (noted at the visits) were recorded in a later telephone interview. Altogether, 187 variables were analyzed.Broiler houses were divided into single-infection houses (which had one or both of the salmonella types in only one crop), and multiple-infection houses (where infection occurred in multiple crops). This was done both generally and separately for each of the salmonella types. Factors associated with house status-but that were beyond the control of the farmer-were the salmonella type and the percentage of salmonella positive samples in the first crop. Factors which could be controlled by the farmer and which were associated with reduced prevalence of multiple-infection houses included antiseptic soap and water for washing hands in the anteroom, hygiene barriers when removing dead broilers, gravel alongside the broiler house, systematic checks of indoor rodent-bait depots, and combined surface and pulse-fogging disinfection.
Salmonella enterica serovar Typhimurium is a major foodborne pathogen throughout the world. Until now, the specific target genes for the detection and identification of serovar Typhimurium have not been developed. To determine the specific probes for serovar Typhimurium, the genes of serovar Typhimurium LT2 that were expected to be unique were selected with the BLAST (Basic Local Alignment Search Tool) program within GenBank. The selected genes were compared with 11 genomic sequences of various Salmonella serovars by BLAST. Of these selected genes, 10 were expected to be specific to serovar Typhimurium and were not related to virulence factor genes of Salmonella pathogenicity island or to genes of the O and H antigens of Salmonella. Primers for the 10 selected genes were constructed, and PCRs were evaluated with various genomic DNAs of Salmonella and non-Salmonella strains for the specific identification of Salmonella serovar Typhimurium. Among all the primer sets for the 10 genes, STM4497 showed the highest degree of specificity to serovar Typhimurium. In this study, a specific primer set for Salmonella serovar Typhimurium was developed on the basis of the comparison of genomic sequences between Salmonella serovars and was validated with PCR. This method of comparative genomics to select target genes or sequences can be applied to the specific detection of microorganisms.