Determining the DNA Fingerprinting Profiles of Salmonella Isolates from Raw Poultry Meats and Human Clinical Samples from the Same Geographic Area Using Pulsed-Field Gel Electrophoresis.

ABSTRACT: Foods of animal origin, such as poultry, eggs, and pork, are recognized sources of Salmonella infections, but determination of the proportion of foodborne infections associated with various food sources has been challenging. In the present study, 141 Salmonella isolates recovered from 1,322 poultry product samples purchased over a 1-year period from retail stores across Seattle, WA were subtyped by pulsed-field gel electrophoresis (PFGE) using restriction enzyme XbaI. The objectives of the study were (i) to analyze the longitudinal distribution of Salmonella PFGE profiles throughout the sampling period and their clonality within and between poultry processing establishments, (ii) to determine the association between PFGE profiles of Salmonella isolates from locally distributed poultry products and those of clinical isolates submitted to the Washington State Department of Health (WA-DOH) laboratories, and (iii) to compare the PFGE profiles of Salmonella isolates from the National Antimicrobial Resistance Monitoring System (NARMS) retail meats program. During the 1-year sampling period, multiple indistinguishable PFGE patterns were found across multiple poultry processing establishments. Twelve of the 30 unique PFGE profiles of Salmonella isolates from locally purchased poultry products were indistinguishable from the PFGE profiles of clinical Salmonella isolates submitted to the WA-DOH. When the PFGE profiles from the poultry samples were compared with those found in the NARMS database, eight indistinguishable PFGE matches were found with isolates recovered from chicken breasts, ground turkey, and ground beef from multiple states. Although this study revealed some association between PFGE profiles from raw poultry products and those of clinical isolates from the same geographical area, these results do not prove that all of those clinical isolates were from infections acquired through consumption or handling of poultry.

Prevalence, concentrations, and antibiotic sensitivities of Salmonella serovars in poultry from retail establishments in Seattle, Washington.

Poultry have been identified as one of the major sources of salmonellosis, with estimates ranging from 10 to 22% of total cases. Despite several advances in the industry and new performance standards, the incidence of salmonellosis in the population has not declined over the last 15 years. Salmonella is pervasive in a wide variety of foods, and thus, estimating its burden resulting from specific food categories has been challenging and plagued with uncertainty due to critical data gaps. The objective of this study was to conduct a year-long market survey (1,322 samples) to help bridge the data gaps on the contamination rates and levels of Salmonella on raw poultry by product type (i.e., breast, thighs, drums, wings, and split breast) and production method (conventional versus organic). The isolates recovered were serotyped and tested for antibiotic sensitivities. A PCR method was utilized for initial screening of samples after an overnight enrichment in tryptic soy broth. Three-tube most-probable-number (MPN) assays and anti-Salmonella immunomagnetic separation methods were utilized to determine the levels of Salmonella and aid with the recovery of Salmonella species, respectively. Eleven percent of the samples were positive for Salmonella. Significant differences in percent positive rates by product type included up to a 4-fold difference in percent positive rates between establishments, ranging from 7 to 31%. Of the samples positive for Salmonella species, 94% had <30 MPN/100 g. Production methods identified as organic or as not using antibiotics had significantly higher rates of recovery of Salmonella. On the other hand, all of the Salmonella isolates that were resistant to two or more antibiotics originated from conventional processing establishments where antibiotics were utilized. In addition, a significant proportion of isolates from conventionally processed products were serotypes clinically relevant to humans.

Vaccination with plasmid DNA encoding TSA/LmSTI1 leishmanial fusion proteins confers protection against Leishmania major infection in susceptible BALB/c mice.

We have recently shown that a cocktail containing two leishmanial recombinant antigens (LmSTI1 and TSA) and interleukin-12 (IL-12) as an adjuvant induces solid protection in both a murine and a nonhuman primate model of cutaneous leishmaniasis. However, because IL-12 is difficult to prepare, is expensive, and does not have the stability required for a vaccine product, we have investigated the possibility of using DNA as an alternative means of inducing protective immunity. Here, we present evidence that the antigens TSA and LmSTI1 delivered in a plasmid DNA format either as single genes or in a tandem digene construct induce equally solid protection against Leishmania major infection in susceptible BALB/c mice. Immunization of mice with either TSA DNA or LmSTI1 DNA induced specific CD4(+)-T-cell responses of the Th1 phenotype without a requirement for specific adjuvant. CD8 responses, as measured by cytotoxic-T-lymphocyte activity, were generated after immunization with TSA DNA but not LmSTI1 DNA. Interestingly, vaccination of mice with TSA DNA consistently induced protection to a much greater extent than LmSTI1 DNA, thus supporting the notion that CD8 responses might be an important accessory arm of the immune response for acquired resistance against leishmaniasis. Moreover, the protection induced by DNA immunization was specific for infection with Leishmania, i.e., the immunization had no effect on the course of infection of the mice challenged with an unrelated intracellular pathogen such as Mycobacterium tuberculosis. Conversely, immunization of BALB/c mice with a plasmid DNA that is protective against challenge with M. tuberculosis had no effect on the course of infection of these mice with L. major. Together, these results indicate that the protection observed with the leishmanial DNA is mediated by acquired specific immune response rather than by the activation of nonspecific innate immune mechanisms. In addition, a plasmid DNA containing a fusion construct of the two genes was also tested. Similarly to the plasmids encoding individual proteins, the fusion construct induced both specific immune responses to the individual antigens and protection against challenge with L. major. These results confirm previous observations about the possibility of DNA immunization against leishmaniasis and lend support to the idea of using a single polygenic plasmid DNA construct to achieve polyspecific immune responses to several distinct parasite antigens.

Protection against cutaneous leishmaniasis induced by recombinant antigens in murine and nonhuman primate models of the human disease.

Leishmaniasis affects approximately 2 million people each year throughout the world. This high incidence is due in part to the lack of an efficacious vaccine. We present evidence that the recombinant leishmanial antigens LmSTI1 and TSA, which we identified and characterized previously, induce excellent protection in both murine and nonhuman primate (rhesus monkey) models of human cutaneous leishmaniasis. The remarkable protection induced by LmSTI1 and TSA in an animal model that is evolutionarily close to humans qualifies this antigen combination as a promising candidate subunit vaccine against human leishmaniasis.