Prevalence and counts of Salmonella and enterohemorrhagic Escherichia coli in raw, shelled runner peanuts.

Three major outbreaks of salmonellosis linked to consumption of peanut butter during the last 6 years have underscored the need to investigate the potential sources of Salmonella contamination in the production process flow. We conducted a study to determine the prevalence and levels of Salmonella in raw peanuts. Composite samples (1,500 g, n = 8) of raw, shelled runner peanuts representing the crop years 2009, 2010, and 2011 were drawn from 10,162 retained 22-kg lot samples of raw peanuts that were negative for aflatoxin. Subsamples (350 g) were analyzed for the presence of Salmonella and enterohemorrhagic Escherichia coli. Salmonella was found in 68 (0.67%) of 10,162 samples. The highest prevalence rate (P < 0.05) was for 2009 (1.35%) compared with 2010 (0.36%) and 2011 (0.14%). Among four runner peanut market grades (Jumbo, Medium, No. 1, and Splits), Splits had the highest prevalence (1.46%; P < 0.05). There was no difference (P > 0.05) in the prevalence by region (Eastern versus Western). Salmonella counts in positive samples (most-probable-number [MPN] method) averaged 1.05 (range, 0.74 to 5.25) MPN per 350 g. Enterohemorrhagic E. coli was found in only three samples (0.030%). Typing of Salmonella isolates showed that the same strains found in Jumbo and Splits peanuts in 2009 were also isolated from Splits in 2011. Similarly, strains isolated in 2009 were also isolated in 2010 from different peanut grades. These results indicated the persistence of environmental sources throughout the years. For five samples, multiple isolates were obtained from the same sample that had different pulsed-field gel electrophoresis types. This multistrain contamination was primarily observed in Splits peanuts, in which the integrity of the kernel is usually compromised. The information from the study can be used to develop quantitative microbial risk assessments models.

Tracking the sources of salmonella in ground beef produced from nonfed cattle.

The objective of this study was to determine the source(s) of Salmonella contamination in ground beef. One hundred dairy cows were harvested in a U.S. commercial beef processing plant. Samples of hides, carcasses after hide removal and before exposure to antimicrobial intervention, carcasses after all antimicrobial interventions, superficial cervical lymph nodes from the chuck, trim, ground beef, and air were obtained. Ninety-six percent of the hide samples, 47% of the carcasses before intervention, 18% of the lymph nodes, 7.14% of the trim, and 1.67% of the ground beef samples were positive for Salmonella. None of the samples obtained from the carcasses after the full complement of interventions and none of the air samples were positive for Salmonella. All Salmonella-positive samples were subjected to pulsed-field gel electrophoresis, and eight DNA Xba I restriction patterns were identified. The majority of isolates had one of two restriction digest patterns. The strain isolated from ground beef had the same pattern as the strains isolated from hides and from carcasses immediately after hide removal. The Salmonella isolates from trim samples and lymph nodes also had the same restriction digest pattern. These results indicate that hide and lymph nodes are the most likely sources of Salmonella in ground beef. Dressing practices that effectively reduce or eliminate the transfer of bacteria from hide to carcass and elimination of lymph nodes as a component of raw ground beef should be considered as measures to reduce Salmonella contamination of ground beef. Because total elimination of lymph nodes from ground beef is not possible, other approaches should be explored. Easily accessible lymph nodes could be screened for Salmonella very early in the slaughter process. When the results are positive for Salmonella, the corresponding carcasses should be fabricated separately at the end of the production run, and the trim from these carcasses should be subjected to a treatment that destroys Salmonella.

Cloning and characterization of a gene encoding an immunoglobulin-binding receptor on the cell surface of some members of the family Trypanosomatidae.

Several members of the Trypanosomatidae family, when freshly isolated from their mammalian hosts, have immunoglobulins adsorbed to their cell surfaces. However, a significant portion of these antibody molecules is not parasite specific, i.e., the immunoglobulins are bound to the parasite's cell surface molecules via noncognitive interactions. It has been proposed that this noncognitive adsorption of immunoglobulins to the parasite is mediated by an Fc-like receptor present in several members of the Trypanosomatidae family. However, the molecular identification of this receptor has never been defined. Here, we describe the cloning of a gene encoding a protein that might represent this molecule. The gene, named Lmsp1, was cloned by screening a Leishmania major cDNA expression library using a rabbit antiserum. Lmsp1 is present in both Leishmania and Trypanosoma and is expressed in all developmental stages of these parasites. The predicted protein has a molecular mass of 16.6 kDa and contains an RGD sequence starting at residue 104 and three cysteine residues at positions 55, 74, and 116. The purified recombinant protein strongly binds to normal immunoglobulins of various animal species (humans, rabbits, sheep, goats, guinea pigs, donkeys, rats, and mice) and the binding to human immunoglobulins appears to be immunoglobulin G (IgG) and IgM isotype specific. Moreover, Lmsp1 binds to both purified Fc and Fab fragments of IgG from both humans and rabbits. The mapping of the Lmsp1 epitopes that bind human IgG revealed that different sequences of the molecule bind to Fc or Fab. In addition, fluorescence-activated cell sorter analyses with a specific rabbit anti-Lmsp1 antiserum showed that Lmsp1 is associated with the parasite's cell surface. Finally, inhibition experiments point to an active role of this molecule in the immunoglobulin-mediated attachment and penetration of Trypanosoma cruzi in its macrophage host cells, thus suggesting that Lmsp1 is a putative Trypanosomatidae immunoglobulin receptor.

Protective efficacy of a tandemly linked, multi-subunit recombinant leishmanial vaccine (Leish-111f) formulated in MPL adjuvant.

Three immunodominant leishmanial antigens (TSA, LmSTI1 and LeIF) previously identified in the context of host response to infection in infected donors and BALB/c mice, as well as their ability to elicit at least partial protection against Leishmania major infection in the BALB/c mouse model, were selected for inclusion into a subunit based vaccine. This is based on the premise that an effective vaccine against leishmaniasis (a complex parasitic infection) would require a multivalent cocktail of several antigens containing a broader range of protective epitopes that would cover a wide range of MHC types in a heterogeneous population. For practical considerations of vaccine development, we report on the generation of a single recombinant polyprotein comprising the sequences of all three open reading frames genetically linked in tandem. The resulting molecule, Leish-111f, comprises an open reading frame that codes for a 111kDa polypeptide. Evaluation of the immunogenicity and protective efficacy of Leish-111f formulated with IL-12 revealed that the immune responses to the individual components were maintained and as well, rLeish-111f protected BALB/c mice against L. major infection to a magnitude equal or superior to those seen with any of the individual components of the vaccine construct or SLA, a soluble Leishmania lysate. But because rIL-12 is expensive and difficult to manufacture and its efficacy and safety as an adjuvant for human use is questionable, we screened for other adjuvants that could potentially substitute for IL-12. We report that monophosphoryl lipid A (MPL) plus squalene (MPL-SE) formulated with rLeish-111f elicited protective immunity against L. major infection. The demonstrated feasibility to manufacture a single recombinant vaccine comprising multiple protective open reading frames and the potential use of MPL-SE as a substitute for IL-12, takes us closer to the realization of an affordable and safe Leishmania vaccine.

Immunization with a polyprotein vaccine consisting of the T-Cell antigens thiol-specific antioxidant, Leishmania major stress-inducible protein 1, and Leishmania elongation initiation factor protects against leishmaniasis.

Development of an effective vaccine against Leishmania infection is a priority of tropical disease research. We have recently demonstrated protection against Leishmania major in the murine and nonhuman primate models with individual or combinations of purified leishmanial recombinant antigens delivered as plasmid DNA constructs or formulated with recombinant interleukin-12 (IL-12) as adjuvant. In the present study, we immunized BALB/c mice with a recombinant polyprotein comprising a tandem fusion of the leishmanial antigens thiol-specific antioxidant, L. major stress-inducible protein 1 (LmSTI1), and Leishmania elongation initiation factor (LeIF) delivered with adjuvants suitable for human use. Aspects of the safety, immunogenicity, and vaccine efficacy of formulations with each individual component, as well as the polyprotein referred to as Leish-111f, were assessed by using the L. major challenge model with BALB/c mice. No adverse reactions were observed when three subcutaneous injections of the Leish-111f polyprotein formulated with either MPL-squalene (SE) or Ribi 529-SE were given to BALB/c mice. A predominant Th1 immune response characterized by in vitro lymphocyte proliferation, gamma interferon production, and immunoglobulin G2A antibodies was observed with little, if any, IL-4. Moreover, Leish-111f formulated with MPL-SE conferred immunity to leishmaniasis for at least 3 months. These data demonstrate success at designing and developing a prophylactic leishmaniasis vaccine that proved effective in a preclinical model using multiple leishmanial antigens produced as a single protein delivered with a powerful Th1 adjuvant suitable for human use.