Outbreaks of foodborne Salmonella enteritidis in the United States between 1990 and 2015: An analysis of epidemiological and spatial-temporal trends.

OBJECTIVES: To evaluate the role of eggs and other food vehicles as risk factors associated with Salmonella enteritidis (SE) outbreaks in order to address the endemicity of SE infections in the USA. METHODS: We retrieved and analyzed data relating to all SE outbreaks reported to the Centers for Disease Control and Prevention (CDC) between 1990 and 2015. We then used descriptive and analytical statistical methods, including negative binomial regression models for the estimation of rate-ratios, to analyze the data. RESULTS: Analyses showed that egg-based dishes were the most common food vehicle associated with outbreaks of SE in the USA (273 cases [24%]); this was followed by several other food items, including meat (130 cases [11%]), vegetables (96 cases [8%]), chicken items (95 cases [8%]), dairy products (55 cases [5%]), and bakery items (8 cases [1%]). Compared to egg-based dishes, other food items such as meat (exp(ß) = 0.51, 95% CI 0.37, 0.69), chicken (exp(ß) = 0.42, 95% CI 0.30, 0.58), vegetables (exp(ß) = 0.41, 95% CI 0.29, 0.55), and dairy items (exp(ß) = 0.27, 95% CI 0.18, 0.40) were significantly associated with outbreaks of SE in the USA. Of 1144 SE outbreaks, 402 (35%) occurred in the Northeast region of the USA, followed by the South (253 [22%]), West (250 [22%]), and Midwestern regions (239 [21%]). CONCLUSIONS: Epidemiological and spatiotemporal trends analyses demonstrated that a significant proportions of Salmonella enteritidis outbreaks in the USA are attributed to food vehicles other than eggs. Our findings can be used to plan effective strategies to mitigate the increasing occurrence of foodborne SE outbreaks.

Single-nucleotide polymorphism typing analysis for molecular subtyping of Salmonella Tennessee isolates associated with the 2007 nationwide peanut butter outbreak in the United States.

BACKGROUND: In 2007, a nationwide Salmonella Tennessee outbreak occurred via contaminated peanut butter. Here, we developed a single-nucleotide polymorphism (SNP)-typing method for S. Tennessee to determine the clonal subtypes of S. Tennessee that were associated with the peanut butter outbreak. METHODS AND RESULTS: One seventy-six S. Tennessee isolates from various sources, including humans, animals, food, and the environment, were analyzed by using the SNP technique. Eighty-four representative SNP markers were selected by comparing the sequences of three representative S. Tennessee strains with different multi-locus sequence typing and variable number tandem repeats from our collection. The set of eighty-four SNP markers showed 100% typeability for the 176 strains, with the nucleotide diversity ranging from 0.011 to 0.107 (mean = 0.049 ± 0.018, median = 0.044) for each marker. Among the four clades and nine subtypes generated by the SNP typing, subtype 1, which comprised 142 S. Tennessee strains, was the most predominant. The dominance of single-strain clones in subtype 1 revealed that S. Tennessee is highly clonal regardless of outbreak-association, source, or period of isolation, suggesting the presence of an S. Tennessee strain prototype. Notably, a minimum 18 SNP set was able to determine clonal S. Tennessee strains with similar discrimination power, potentially allowing more rapid and economic strain genotyping for both outbreaks and sporadic cases. CONCLUSIONS: The SNP-typing method described here might aid the investigation of the epidemiology and microevolution of pathogenic bacteria by discriminating between outbreak-related and sporadic clinical cases. In addition, this approach enables us to understand the population structure of the bacterial subtypes involved in the outbreak.

Generation of high-titer of neutralizing polyclonal antibodies against heat-stable enterotoxin (STa) of enterotoxigenic Escherichia coli.

In this study, polyclonal antibodies with high titer and avidity to native heat-stable enterotoxin (STa) of enterotoxigenic Escherichia coli (ETEC) have been generated and evaluated for their neutralizing effect in STa-induced enterotoxic animal model. Native STa was purified to homogeneity and coupled to modified bovine serum albumin (MBSA) using dimethylformamide (DMF)-based conjugation protocol. STa conjugate was used for immunization of female New Zealand white rabbits. The humoral immune response of the rabbits against native STa was monitored and evaluated for its antibody binding and neutralization capacity by ELISA and suckling mouse assay, respectively. After three subsequent boosts by STa conjugate, the animals were capable of eliciting high levels of STa-antibody binding titer (10(6)) and STa-neutralizing antibody capacity (3×10(4) mouse units of STa/ml serum). STa antibody maturation (avidity) was improved dramatically after multiple boosters with the STa conjugate. Comparison of the avidity of STa antibodies demonstrated that the strength in the STa antibody avidity developed in time corresponding to the development of the STa-neutralizing and binding titers. High avid STa antibodies (48.21% avidity index) were demonstrated 24 weeks post immunization (PI). However, differences in the onset of STa antibody production were noticed among animals and may need further investigation.

An enhanced protocol for expression and purification of heat-stable enterotoxin of enterotoxigenic Escherichia coli.

We present an improved protocol for expression and purification of heat-stable enterotoxin (STa) of enterotoxigenic Escherichia coli (ETEC). In this protocol, controlled growth conditions at different pHs (7.4, 8.0, and 8.6) were adopted using a bioreactor. In addition, specific adsorbent resins, methacrylate, were used for STa purification. The bioreactor provided optimal ETEC growth at pH 7.4 with high STa production. Furthermore, methacrylate bounded specifically to STa and dramatically enhanced the purification process of STa. The STa-specific activity was high (8.9 × 10(6) units/mg protein), and the minimal effective dose of STa required for production of gut weight to remaining body weight ratio ≥ 0.083 was recorded as less than 0.2 ng in 2-3 days old suckling mice. The protocol presented, produces highly purified STa as documented by matrix-assisted laser desorption ionization-time of flight mass spectroscopy/. Also, as compared with the traditional methods, this procedure is trouble-free and practical for scale-up production and purification of STa peptides.

Design and characterization of highly immunogenic heat-stable enterotoxin of enterotoxigenic Escherichia coli K99(+).

In this study, STa peptide of enterotoxigenic Escherichia coli K99(+) was purified and successfully covalently cross-linked to modified bovine serum albumin after thorough evaluation of three different hapten-carrier conjugation protocols. Dimethyformamide (DMF) based STa-conjugation protocol demonstrated higher biological activity (10×10(6) STa Total Mouse Units [MU]) and 100% conjugation efficiency. A range of conjugation ratio of 4-12 STa molecules per one molecule of BSA was achieved and confirmed by matrix-assisted laser desorption ionization-time of flight/mass spectroscopy (MALDI-TOF/MS). This conjugate was used for immunization of ten rabbits for STa antibody production. A high antibody binding titer (10(6)) against STa was obtained with a neutralization capacity of 3×10(4) STa MUs/ml serum. These levels of high STa binding and neutralizing antibodies titers propose the potential use of this conjugate for the development of immunotherapeutic reagents and/or STa-based vaccine against ETEC K99(+).

Allele distribution and genetic diversity of VNTR loci in Salmonella enterica serotype Enteritidis isolates from different sources.

BACKGROUND: Salmonella enterica serotype Enteritidis (S. Enteritidis) is a zoonotic pathogen, which can be found in many sources including animals and the environment. However, little is known about the molecular relatedness among S. Enteritidis isolates from different sources. We have applied multiple-locus variable number tandem repeat analysis (MLVA) to study the genetic diversity of S. Enteritidis isolates from human and non-human sources. RESULTS: We identified 38 unique MLVA types using nine VNTR loci markers for discrimination between 145 S. Enteritidis isolates from different sources including humans (n = 41), chickens (n = 45), and eggs (n = 40). There were 20 distinct MLVA types identified from human isolates, 17 distinct MLVA types from chicken isolates, and 5 from egg isolates. We compared allele distribution and frequency for each VNTR marker and measured allelic polymorphism within each VNTR locus of S. Enteritidis isolates from the sources using Nei's diversity index (D). Differences in allele distribution and frequency were detected in most loci of study isolates. Different genetic diversity for certain loci was identified in isolates from different sources. The average of genetic diversity (D) was lower in egg isolates (0.16) compared to human (0.41) and chicken (0.30). However, for loci SE3, SE7, and SE9, human isolates showed significantly higher diversity than both chicken and egg isolates. Whereas for loci SE5 and SE10, chicken isolates had significantly higher diversity than both human and egg isolates. Minimum-spanning tree (MST) comprised one major cluster, a minor cluster, and four clonal expansions. MLVA application enabled a cluster analysis by the MST of the S. Enteritidis isolates by sources, which allows a great insight into the genetic relatedness and the possible flow of these organisms between different reservoirs and humans. CONCLUSION: Differences in allele distribution and genetic diversity of VNTR loci in S. Enteritidis isolates from different sources were found. Polymorphism in most of the VNTR loci was more frequent among human S. Enteritidis isolates than isolates from chickens or eggs. Therefore, VNTR profiles of S. Enteritidis isolates from a specific source should be further evaluated as potential markers in epidemiologic investigations to trace S. Enteritidis to their probable source.

Epidemiologic attributes of invasive non-typhoidal Salmonella infections in Michigan, 1995--2001.

OBJECTIVES: To determine: (1) the incidence of invasive salmonellosis, (2) the distribution of specific Salmonella serotypes associated with invasive disease, and (3) the role of demographic characteristics in invasive salmonellosis in Michigan. DESIGN: We conducted a cross-sectional study using laboratory-confirmed cases of salmonellosis reported to the Michigan Department of Community Health between 1995 and 2001. METHODS: Average annual incidences (AAIs) for invasive salmonellosis were computed, and Poisson regression analysis was used to model the association between demographic attributes and invasive salmonellosis. RESULTS: Of 6797 cases of salmonellosis, 347 (5.1%) were characterized as invasive having an AAI of 0.5/100000. A covariate-adjusted Poisson model showed children aged <1 year to be at higher risk for invasive salmonellosis compared to adults aged 40-49 years (rate ratio (RR) 8.98, 95% confidence interval (CI) 5.69-14.17). No significant differences were found between males and females (RR 1.02, 95% CI 0.83-1.26), African-Americans and Caucasians (RR 0.95, 95% CI 0.64-1.42), and urban and rural residents (RR 0.98, 95% CI 0.76-1.26). Among the most common serotypes associated with invasive salmonellosis (Heidelberg, Typhimurium, and Enteritidis), Salmonella serotype Heidelberg had the highest blood invasiveness ratio (15.79/100). CONCLUSIONS: Children aged <1 year, especially of minority groups, are at higher risk for invasive Salmonella infections. Invasive salmonellosis may be included in the differential diagnosis of acute bacterial infections in young children with positive stool cultures for Salmonella.

A registry-based study on the association between human salmonellosis and routinely collected parameters in Michigan, 1995-2001.

PURPOSE: Salmonella serotypes are among the most common bacterial causes of foodborne gastroenteritis in the United States, associated with approximately 1.4 million human illnesses annually. Studies on trends of the serotypes and host-related factors are necessary for the development of effective prevention plans for foodborne diseases caused by these pathogens. MATERIALS AND METHODS: To determine the epidemiologic trends of human infections with the most common Salmonella serotypes in Michigan, we analyzed cases of culture-confirmed salmonellosis at the Michigan Department of Community Health (MDCH) from 1995 to 2001. RESULTS: A total of 6797 cases were reported, with an average annual incidence per 100,000 population (AAI) of 9.9. Among cases for which information on Salmonella serotype were available (6292 cases), the most common serotypes were S. Typhimurium (1596 cases, 26%), followed by S. Enteritidis (1309, 22%), S. Heidelberg (466, 8%) and S. Newport (222, 4%). From 1998 to 2001, the incidence of S. Typhimurium and S. Enteritidis decreased significantly by 39% (95% confidence interval [CI], 49% to 26% decrease) and 32% (95% CI, 44% to 18% decrease) respectively. Whereas the incidence of S. Newport increased by 101% (95% CI, 25% to 225% increase) and S. Heidelberg remained stable. Infection with these serotypes frequently occurred in the summer months. As a group, infants had the highest AAI for all Salmonella serotypes (75.0), S. Typhimurium (21.9), S. Enteritidis (14.0), S. Heidelberg (5.4), and S. Newport (1.7). Among patients whose race was known, blacks had a significantly higher AAI compared to whites for S. Typhimurium (2.5 vs. 1.3; RR = 2.3, 95% CI, 1.6-3.3), S. Enteritidis (1.4 vs. 1.1; relative rate (RR) = 1.4; 95% CI, 1.1-1.6), S. Heidelberg (0.8 vs. 0.3; RR = 3.6; 95% CI, 2.8-4.6), and S. Newport (0.3 vs. 0.1; RR = 2.8; 95% CI, 1.9-4.2). Among patients whose ethnicity was known, Hispanics had a significantly higher AAI for S. Enteritidis compared to non-Hispanics (1.0 vs. 0.5; RR = 1.9; 95% CI, 1.2-3.0), but not different significantly for S. Typhimurium, S. Heidelberg, and S. Newport. CONCLUSION: This study revealed the emergence of S. Newport and the high incidence of the most common Salmonella serotypes among infants, people of African descent, and Hispanics. This information can be used by the state and local health departments of Michigan to enhance salmonellosis prevention efforts by rationalizing the allocation of appropriate public health resources and personnel.

Interaction of Escherichia coli heat-stable enterotoxin (STa) with its putative receptor on the intestinal tract of newborn kids.

The elaboration of heat stable enterotoxin (STa) is an important step in the pathogenesis of enterotoxigenic Escherichia coli (ETEC), which causes severe diarrhea in newborn animals. In this study, the distribution of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from the anterior jejunum, posterior jejunum, ileum and colon of newborn kids was investigated. The density of STa-receptors on enterocytes and BBMVs was higher in the posterior jejunum than that in other segments of the kids' intestines. Additionally, the affinity of the posterior jejunum STa-receptors was higher than the affinity of receptors present on the epithelium of other intestinal segments. Our findings suggest that the posterior jejunum is a major target for STa within the intestinal tract of newborn kids.

Demographic risk factors and incidence of Salmonella enteritidis infection in Michigan.

Salmonella Enteritidis is a major cause of foodborne disease worldwide. In the United States, this serotype has been associated with significant morbidity and mortality accounting for about 17% of all human Salmonella infections. Data on risk factors associated with serotype-specific Salmonella infections have not been reported in Michigan before. We carried out this study using surveillance data to identify high-risk groups and regions for S. Enteritidis infection in Michigan. Results of this study may be used to develop better control and prevention measures against this common foodborne pathogen. We analyzed data on Salmonella infections collected by the Michigan Department of Community Health's surveillance system (1995-2001). We computed age, gender, and place of residence-specific incidences of S. Enteritidis infection in Michigan using population-based denominators. We used Poisson regression analysis to study the associations between S. Enteritidis infection and certain predictor demographic variables, while controlling for confounders and covariates. We found an overall 1.87/100,000 average annual S. Enteritidis incidence with 1.91/100,000 in females and 1.81/100,000 in males. A higher incidence of S. Enteritidis infection was associated with Michigan children<5 years old when compared with average national estimates (p<0.01). The final adjusted Poisson model revealed that children<1 year of age [RR: 9.75, CI: 7.99-11.90] and children 1-4 years of age [RR: 3.37, CI: 2.83-4.02] are at higher risk for S. Enteritidis infection compared to adults aged 15-39 years. No significant differences in incidence of S. Enteritidis infections were found between urban and rural dwellers or between genders (p>0.05). The incidence rate of S. Enteritidis infection in the Michigan pediatric population (<5 years) was found to be significantly higher than average national estimates, meriting immediate public health attention. Risk factors for S. Enteritidis infection in Michigan children should be identified in order to devise effective control and prevention measures.

Clonal structure and variation in virulence of Salmonella enteritidis isolated from mice, chickens, and humans.

The clonal analysis of a diverse collection of Salmonella Enteritidis indicates that most strains belong to a single multilocus genotype (i.e., ET-3) regardless of phage type, geographic origin, or time of isolation that spanned over 2 decades (1978 to 2004). Attachment and invasion assays, however, indicate that, among ET-3 isolates, there is a distinct invasive bacterial subpopulation that is more readily recovered from eggs and clinical cases in humans than from chicken cecal samples. These observations support the hypothesis that the specialized ability of S. Enteritidis to infect the avian reproductive tract and contaminate eggs has been critical in its emergence as a frequent cause of human illness.

An outbreak of besnoitiosis in miniature donkeys.

Fourteen miniature donkeys (Equus asinus) in a mid-Michigan herd of 38 animals presented with clinical signs of besnoitiosis, including the presence of typical tissue cysts in the ocular sclera, the buccal and nasal mucosa, together with characteristic dermatitis in specific areas of the body. The common histopathological change seen was the presence of many 100-200-microm diameter, thick walled, typical Besnoitia sp. tissue cysts together with a chronic cellular response associated with degenerating cysts. Microscopy of isolated scleral cysts and skin biopsies showed the presence of protozoal organisms consistent in morphology with that of Besnoitia bennetti bradyzoites. Molecular analysis of these parasites indicates that they differ from previously described coccidia, including Besnoitia sp., from rabbits and opossums. Isolated cases of infection with this agent have been reported infrequently in equids; however, this is the first report of an outbreak in a herd of donkeys in the United States.

Sarcocystis inghami n. sp. (Sporozoa: Sarcocystidae) from the skeletal muscles of the Virginia opossum Didelphis virginiana in Michigan.

This report describes the newly identified Sarcocystis inghami n. sp. from the skeletal muscles of opossums (Mammalia: Didelphidae) that were collected from south central Michigan (42 degrees 43'-42 degrees 79'N, 84 degrees 18'-84 degrees 86'W), USA. The new species is distinguished from all species described from North and South American opossums by the distinctive morphology of the villar protrusions on the cyst wall. Sarcocysts of S. inghami are microscopic, up to 700 microm long and 110 microm wide. The sarcocyst wall is up to 7 microm thick, with long, stalked protrusions which average 5.5 x 1.2 microm. These are constricted at the base, expanded laterally, rounded off distally and occasionally bifid. The villar protrusions have numerous microtubules without electron-dense bodies that extend from the tips into the granular layer. Bradyzoites are 10.7 x 4.3 (8-12 x 4-5) microm. This is the second species of Sarcocystis sarcocyst described from the Virginia opossum in North America.

Growth and Heat Resistance of Salmonella enteritidis in Refrigerated and Abused Eggs.

The effect of storage temperature on the growth and heat resistance of Salmonella enteritidis (SE) in eggs artificially inoculated with 20 CFU per egg has been investigated. SE organisms grew to a stationary phase (1 × 109 CFU/ml of egg) within 2-3 d after storing the eggs at room temperature (23°C), while minimal or no growth occurred in similarly inoculated eggs that were refrigerated at 4°C. None of the common methods of egg cooking was found totally effective in eliminating SE organisms in massively contaminated eggs. SE organisms survived in significantly higher proportions of the eggs that were stored at 23°C than in refrigerated eggs after cooking by different methods (P < 0.05). Higher rates of Salmonella survival after cooking were detected in the eggs that were stored at room temperature for 5 d or longer than in eggs stored for shorter time (P < 0.05). Data from this study indicated: i) storage abuse of eggs can be a major factor in increasing the prevalence of eggborne SE infections by increasing the infectious load and heat resistance of the SE organisms in the contaminated eggs, and ii) that breaking and pooling of eggs immediately before cooking can minimize the substantial multiplication of SE organisms from slightly contaminated eggs. Refrigeration, therefore, can be a significant barrier against SE infections through its growth inhibitory and heat resistance-reducing effects on the SE organisms in contaminated shell eggs as well as in large volumes of liquid eggs intended for pasteurization.