Systematic Review: Impact of point sources on antibiotic-resistant bacteria in the natural environment.

Point sources such as wastewater treatment plants and agricultural facilities may have a role in the dissemination of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARG). To analyse the evidence for increases in ARB in the natural environment associated with these point sources of ARB and ARG, we conducted a systematic review. We evaluated 5,247 records retrieved through database searches, including both studies that ascertained ARG and ARB outcomes. All studies were subjected to a screening process to assess relevance to the question and methodology to address our review question. A risk of bias assessment was conducted upon the final pool of studies included in the review. This article summarizes the evidence only for those studies with ARB outcomes (n = 47). Thirty-five studies were at high (n = 11) or at unclear (n = 24) risk of bias in the estimation of source effects due to lack of information and/or failure to control for confounders. Statistical analysis was used in ten studies, of which one assessed the effect of multiple sources using modelling approaches; none reported effect measures. Most studies reported higher ARB prevalence or concentration downstream/near the source. However, this evidence was primarily descriptive and it could not be concluded that there is a clear impact of point sources on increases in ARB in the environment. To quantify increases in ARB in the environment due to specific point sources, there is a need for studies that stress study design, control of biases and analytical tools to provide effect measure estimates.

Vaccination as a control strategy against Salmonella infection in pigs: A systematic review and meta-analysis of the literature.

Consumption or handling of improperly processed or cooked pork is considered one of the top sources for foodborne salmonellosis, a common cause of intestinal disease worldwide. Asymptomatic carrier pigs may contaminate pork at slaughtering; therefore, pre-harvest reduction of Salmonella load can contribute to reduce public health risk. Multiple studies have evaluated the impact of vaccination on controlling Salmonella in swine farms, but results are highly variable due to the heterogeneity in vaccines and vaccination protocols. Here, we report the results of an inclusive systematic review and a meta-analysis of the peer-reviewed scientific literature to provide updated knowledge on the potential effectiveness of Salmonella vaccination. A total of 126 articles describing the use of Salmonella vaccines in swine were identified, of which 44 fulfilled the inclusion criteria. Most of the studies (36/44) used live vaccines, and S. Typhimurium and S. Choleraesuis were the predominant serotypes evaluated. Vaccine efficacy was most often measured through bacteriological isolation, and pooled estimates of vaccine efficacy were obtained as the difference in the percentage of positive animals when available. Attenuated and inactivated vaccines had similar efficacy [Risk Difference=-26.8% (-33.8, -19.71) and -29.5% (-44.4, -14.5), respectively]. No serotype effect was observed on the efficacy recorded for attenuated vaccines; however, a higher efficacy of inactivated vaccines against S. Choleraesuis was observed, though in a reduced sample. Results from the meta-analysis here demonstrate the impact that vaccination may have on the control of Salmonella in swine farms and could help in the design of programs to minimize the risk of transmission of certain serotypes through the food chain.

An increased frequency of IgE-producing B cell precursors contributes to the elevated levels of plasma IgE in atopic subjects.

BACKGROUND: The production of specific IgE, which underlies the allergic response, may be a normal correlate of the immune response to a certain class of antigen (allergens), or could represent a unique response driven by regulatory signals that are absent in non-allergic individuals. If atopic subjects do possess a regulatory environment favoring IgE production, they may display not only allergen-specific IgE, but also higher levels of total IgE and higher frequencies of IgE-producing B lymphocytes. OBJECTIVE: To address the contribution of antibody-producing cell number to the circulating IgE titre in atopic vs non-atopic subjects. METHODS: Frequency determination by limiting dilution of EBV transformants and Poisson distribution analysis. Titres of total and allergen-specific IgM, IgG, and IgE by specific ELISA. RESULTS: In contrast to findings reported by others, the atopic subjects had a significantly higher frequency of IgE-producing B cells than non-atopics (0.79% of total Ig-producing cells, as compared with 0.17% for the control group; P < 0.01), suggesting that one factor contributing to the high plasma IgE titres in atopic subjects is the high frequency of B lymphocytes with the potential to produce IgE. Although only the atopic subjects produced allergen-specific IgE, the frequency of specific IgE-producing B cells was undetectable in both groups. CONCLUSION: Atopic subjects have higher frequencies of IgE-producing B cell precursors than non-atopics. A correlation exists between IgE-producing B cell frequency and levels of circulating IgE.

Enhanced immunoreactivity and preferential heterodimer formation of reassociated Fel d I recombinant chains.

In this study we have addressed the question of whether reassociating the two recombinant protein chains that comprise the major cat dander allergen, Fel d I, would change the overall IgE and allergic patient T cell immunoreactivity compared to the native molecule. To accomplish this, the chains were combined under reducing and denaturing conditions, then allowed to reassociate by dilution and extensive dialysis against a physiological buffer. An initial examination of the reaction products using quantitative capture ELISA demonstrated comparable reactivity to Fel d I. Further analysis, using a pool of cat allergic patient plasma, showed that the products of the reassociation reaction (rFel d I) also possessed an enhanced IgE binding capacity. Depletion ELISA results gave only a 5% difference in reactivity between rFel d I and the native protein versus a 20% difference with the mixture of the two chains. Comparative secondary T cell stimulation assays were subsequently performed using cat allergic patient peripheral blood lymphocytes. Here the results demonstrated no loss of reactivity with the reassociated chains as compared to Fel d I or the two mixed recombinant chains. To biochemically characterize the products of the reassociation reaction we have performed reverse phase HPLC and then analysed the isolated fractions by mass spectrometry. It was clear from these results that like the native Fel d I, the products of the reassociation reaction favored heterodimer formation, with no homodimer being detected. This implies that the reassociated protein chains had preferentially adopted a native-like conformation.

Potential therapeutic recombinant proteins comprised of peptides containing recombined T cell epitopes.

The complete primary structure of Fel d I2 has been determined and shown to be comprised of two separate polypeptide chains (designated chain 1 and 2). Overlapping peptides covering the entire sequence of both chains of Fel d I have been used to map the major areas of human T cell reactivity. The present study describes three non-contiguous T cell reactive regions of < 30 aa in length that were assembled in all six possible configurations using PCR and recombinant DNA methods. These six recombinant proteins comprised of defined non-contiguous T cell epitope regions artificially combined into single polypeptide chains have been expressed in E. coli, highly purified, and examined for their ability to bind to human cat-allergic IgE and for human T cell reactivity. Several of these recombined T cell epitope-containing polypeptides exhibit markedly reduced IgE binding as compared to the native Fel d I. Importantly, the human T cell reactivity to individual T cell epitope-containing regions is maintained even though each was placed in an unnatural position as compared to the native molecule. In addition, T cell responses to potential junctional epitopes were not detected. It was also demonstrated in mice that s.c. injection of T cell epitope-containing polypeptides inhibits the T cell response to the individual peptides upon subsequent challenge in vitro. Thus, these recombined T cell epitope-containing polypeptides, which harbor multiple T cell reactive regions but have significantly reduced reactivity with allergic human IgE, constitute a novel potential approach for desensitization to important allergens.

Native and recombinant Fel dI as probes into the relationship of allergen structure to human IgE immunoreactivity.

To delineate the relationship between the structural conformation and the stability of an allergen and its antigenicity, we have chosen the major allergen from cat dander, Fel dI. From protein sequence analysis data we have examined the structure of the naturally occurring Fel dI and we have found it to exist as an anti-parallel heterodimer. We have used ELISA, RAST, Western blot and histamine release techniques to compare the IgE reactivity of a set of cat allergic patient samples to purified, native Fel dI and the E. coli expressed chains 1 and 2. Results from these studies demonstrate a significant level of IgE reactivity to all forms when examined for direct binding. However, both blot and ELISA competition assays show a much higher reactivity to Fel dI in solution compared to the separate recombinant chains and this is supported by the histamine release data. Although native Fel dI chain 2 contains an N-linked carbohydrate moiety, this does not seem to play a role in the reactivity of IgE to chain 2. Denaturation of Fel dI with alkali conditions leads to a dramatic decrease in IgE reactivity, even though measurable changes to the backbone structure of the protein are minimal. One proposed explanation is that both chains possess a core region determined by their primary structures and that the major IgE epitopes are dependent upon them. The relative reactivity amongst these allergen forms varied with the method of analysis, implying that the conformational requirements for IgE antibody binding are best studied by the application of more than one experimental protocol. Results from these qualitative analyses afford insight into the allergenicity of this exceptionally stable cat pelt protein.

Sequence polymorphism of Amb a I and Amb a II, the major allergens in Ambrosia artemisiifolia (short ragweed).

Two of the major allergens in the pollen of short ragweed are Amb a I and Amb a II (formerly antigen E and antigen K, respectively). The genes for Amb a I and Amb a II have recently been cloned, and it was shown that Amb a I is a family of proteins with at least three distinct polymorphic family members. This study addresses the number of individual Amb a I and Amb a II family members, the polymorphism in each family member and the expression of these genes in ragweed plants from different geographical locations. This work led to the cloning and characterization of a fourth Amb a I family member, designated Amb a I.4.

Characterization of the Escherichia coli protein-export gene secB.

The Escherichia coli secB gene product is required for normal export of envelope proteins out of the cell cytoplasm. In this report, we present the identification and nucleotide sequence of the secB coding sequence. The secB structural gene overlaps almost completely with a predicted open reading frame (ORF) that is encoded on the opposite strand. To establish the identity of the secB ORF, we characterized a secB mutation that caused total loss of secB function, based upon its phenotype. This mutation resulted from a nucleotide change that caused an ochre mutation in one ORF (the secB gene) and a silent (no amino acid change) codon change in the opposite ORF.