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(β) = 0.51, 95% CI 0.37, 0.69), chicken (exp(β) = 0.42, 95% CI 0.30, 0.58), vegetables (exp(β) = 0.41, 95% CI 0.29, 0.55), and dairy items (exp(β) = 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.

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.

We conducted an epidemiologic study on the incidence of Salmonella infections and the associated risk factors in children aged <1 in the state of Michigan. Data on laboratory-confirmed cases of salmonellosis from 1995-2001 were abstracted from the records of Michigan Department of Community Health. We computed the incidence of the disease by sex, race, and area of residence and analyzed the relationships between selected demographic characteristics and Salmonella infection using Poisson regression analysis. Based on a total of 690 cases, an overall incidence of 73.3/100,000, with an incidence of 84.3/100,000 in male children and 64.9/100,000 in female children, were found. The final covariate-adjusted Poisson model showed that children aged 1-5 months are at higher risk for salmonellosis compared to children aged 6-11 months (RR: 1.80, CI: 1.54-2.10), African-American children are at higher risk than their Caucasian counterparts (RR: 2.63, CI: 2.16-3.19), and male children more commonly represented among cases (RR: 1.30, CI: 1.12-1.51). These findings should be considered by healthcare authorities in Michigan and in states with similar population demographics to investigate possible risk factors that may be associated with the high incidence of salmonellosis in African-American children and in children <6 months.

BACKGROUND:Acquired immunodeficiency syndrome (AIDS) is a continuing global public health threat affecting millions of individuals. In 2009, 33.3 million people worldwide were living with human immunodeficiency virus (HIV) infection. HIV-infected individuals are at an increased risk of acquiring hepatitis B and hepatitis C viral (HBV, HCV)infections because of shared transmission routes. The purpose of this study was to identify geographical clusters of HIV-(HBV/HCV) co-infection in the State of Michigan.METHODS:Retrospective cohort data on HIV-infected individuals were matched to all hepatitis B and C cases in Michigan during the period of January 1, 2006 through December 31, 2009. A prevalence map of HIV infection was created and spatial clusters of HIV-hepatitis B or C co-infection were detected using GeoDa's bivariate local Moran's I and SaTScan's discrete Poisson model.RESULTS:A bivariate cluster of high prevalence HIV and hepatitis B or C was identified in the Detroit Metropolitan Area and surrounding counties. A Poisson cluster of HIV-hepatitis B or C co-infection was identified, relative risk (RR) = 1.38 (p = 0.029) in the western and northwestern counties of Lower Michigan, controlling for sex, race, and AIDS status.CONCLUSION:This study identified elevated HIV-hepatitis B or C co-infection unexplained by sex, race or AIDS status in counties outside of the Detroit Metropolitan Area where HIV prevalence was highest in Michigan. The findings from this study may be used to target future public health policy and healthcare interventions for HIV-hepatitis co-infection in these areas.

The multi-state outbreak of Salmonella serotype Tennessee infections associated with peanut butter during 2006-2007 was the first outbreak in the United States associated with this food vehicle. We investigated whether the outbreak-related strains had any distinct virulence attributes. We have analyzed 96 representative isolates from human and non-human sources from multiple states for attachment and invasion of caco-2 cell. In logistic regression analysis, we found that Salmonella Tennessee strains associated with the peanut butter outbreak were more likely to be highly invasive than strains from non-outbreak sources, OR 4.03 (95% CI 1.42, 11.41). Results from this study suggest that peanut butter could have provided an impetus for the expression of certain sets of virulence genes leading to the observed high level of invasiveness of the Salmonella Tennessee contaminants. The occurrence of this outbreak underscores the importance of hygienic practices in peanut butter manufacturing plants for the prevention of such mass contamination. Keywords: Salmonella Tennessee; peanut butter; newly emerging food vehicles for Salmonella ; risk factors for Salmonella Tennessee

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.

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 (106) and STa-neutralizing antibody capacity (3×104 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.

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 × 106 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 (106) against STa was obtained with a neutralization capacity of 3 × 104 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+.

Background Infections with Salmonella serotypes continue to be a significant global public health problem. In addition to contaminated foods, several other sources contribute to infections with Salmonella serotypes. We have assessed the role of socioeconomic factors, exposure to food, and environmental sources in the etiology of non-typhoidal Salmonella infections in Michigan children. Findings A case-control study among Michigan children aged ≤ 10 years was conducted. A total of 123 cases of children with laboratory-confirmed Salmonella infections and 139 control children, who had not experienced symptoms of gastrointestinal illness during the month prior to the interviews, were enrolled. The cases and controls were matched on age-category (<1 year, 2-<6 years and 6-10 years). Data on socioeconomic status, food intake, and environmental exposures, were collected on the queried case and control subjects. After adjusting for race and household-income the final regression multivariable model revealed that Salmonella infections were significantly associated with attendance of a daycare center (adjusted matched odds ratio = 5.00, 95% CI: 1.51 - 16.58), contact with cats (MOR = 2.53, 95% CI: 1.14 - 5.88), and contact with reptiles (MOR = 7.90, 95% CI: 1.52 - 41.01), during the 3 days prior to the onset of child's illness. Conclusions Study results suggest that exposure to environmental sources may play an important role in sporadic infections with Salmonella serotypes in children. Additional efforts are needed to educate parents and caretakers about the risk of Salmonella transmission to children from these sources.

The emergence of Salmonella enterica serovar Enteritidis (S. Enteritidis) during the past three decades as major contaminant of eggs and other poultry products caused a surge in human infections. This could have been mediated in part by emerging S. Enteritidis strains with enhanced virulence. The overall pathogenicity of Salmonella is controlled by numerous genes. To assess the role of a few specific genes thought to contribute to the pathogenicity of S. Enteritidis in egg laying hens, we conducted an experimental infection of egg-laying hens, with a wild type (WT) S. Enteritidis phage type 4 strain and three mutants (M1, M2, M3). These mutants were produced from the wild type by the inactivation of the prgH, SEN4287, and tyrR genes, respectively. We observed that the WT and the M1 mutant had shorter durations of fecal shedding and faster clearance from internal organs of the infected hens than the M2 and M3 mutants. The isolation rates of the wild type S. Enteritidis and the mutants were highest from the ceca, moderately high from the liver and spleen, and lowest from the ovaries of the infected hens. Hep-2 cells attachment assay revealed attenuated attachment for the M1 mutant while the M3 mutant seemed to have enhanced attachment. Colonization of tissues of the infected hens by M1 mutant appears to have been attenuated.

ABSTRACT The gene for the Salmonella enterica serovar Enteritidis fimbrial protein SefA was cloned into an Escherichia coli surface expression vector and confirmed by Western blot assay. E. coli clones expressing SefA attached to avian ovary granulosa cells and HEp-2 cells, providing evidence for the involvement of SefA in the ability of Salmonella to attach to eukaryotic cells.

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.

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.We conducted a cross-sectional study using laboratory-confirmed cases of salmonellosis reported to the Michigan Department of Community Health between 1995 and 2001.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.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).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.

Simplified multiple-locus variable-number tandem repeat analysis (MLVA) was developed using one-shot multiplex PCR for seven variable-number tandem repeats (VNTR) markers with high diversity capacity. MLVA, phage typing, and PFGE methods were applied on 34 diverse Salmonella Enteritidis isolates from human and non-human sources. MLVA detected allelic variations that helped to classify the S. Enteritidis isolates into more evenly distributed subtypes than other methods. MLVA-based S. Enteritidis clonal groups were largely associated with sources of the isolates. Nei's diversity indices for polymorphism ranged from 0.25 to 0.70 for seven VNTR loci markers. Based on Simpson's and Shannon's diversity indices, MLVA had a higher discriminatory power than pulsed field gel electrophoresis (PFGE), phage typing, or multilocus enzyme electrophoresis. Therefore, MLVA may be used along with PFGE to enhance the effectiveness of the molecular epidemiologic investigation of S. Enteritidis infections.

Background: The majority of U.S. disease surveillance systems contain incomplete information regarding socioeconomic status (SES) indicators like household or family income and educational attainment in case reports, which reduces the usefulness of surveillance data for these parameters. We investigated the association between select SES attributes at the neighborhood level and Salmonella infections in the three most populated counties in Michigan using a geographic information system. Methods: We obtained data on income, education, and race from the 2000 U.S. Census, and the aggregate number of laboratory-confirmed cases of salmonellosis (1997–2006) at the block group level from the Michigan Department of Community Health. We used ArcGIS to visualize the distribution, and Poisson regression analysis to study associations between potential predictor variables and Salmonella infections. Results: Based on data from 3,419 block groups, our final multivariate model revealed that block groups with lower educational attainment were less commonly represented among cases than their counterparts with higher education levels (< high school degree vs. ≥ college degree: rate ratio (RR) = 0.79, 95% confidence interval (CI):0.63, 0.99; ≥ and high school degree, but no college degree vs. ≥ college degree: RR = 0.84, 95% CI: 0.76, 0.92). Levels of education also showed a dose-response relation with the outcome variable, i.e., decreasing years of education was associated with a decrease in Salmonella infections incidence at the block group level. Conclusion: Education plays a significant role in health-seeking behavior at the population level. It is conceivable that a reporting bias may exist due to a greater detection of Salmonella infections among high education block groups compared to low education block groups resulting from differential access to healthcare. In addition, individuals of higher education block groups who also have greater discretionary income may eat outside the home frequently and be more likely to own pets considered reservoirs of Salmonella , which increase the likelihood of contracting Salmonella infections compared to their counterparts with lower levels of education. Public health authorities should focus on improving the level of disease detection and reporting among communities with lower income and education and further evaluate the role of higher educational attainment in the predisposition for salmonellosis.

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.