Antibacterial effect of Moringa oleifera on Staphylococcus aureus and Pseudomonas aeruginosa isolated from raw milk and some dairy products with special reference to biofilm gene expression.

Background: Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) are well defined as food poisoning pathogens that are highly resistant and need continuous studies. Aim: The purpose of the work was to examine phenotypic and genotypic characteristics of both P. aeruginosa and S. aureus, and treatment trials with medicinal plants. Methods: Samples were examined for isolation of P. aeruginosa and S. aureus on selective media followed by biochemical confirmation, biofilm formation, genes detection, and expression of P. aeruginosa pslA biofilm gene was performed by quantitative real-time polymerase chain reaction after treatment with 0.312 mg/ml Moringa oleifera aqueous extract as a minimum inhibitory concentration. Results: The highest isolation rate of P. aeruginosa was 20% from both raw milk and Kariesh cheese, followed by 16% and 12% from ice cream and processed cheese, respectively, while the highest isolation rate of S. aureus was 36% from raw milk followed by 28% in ice cream and 16% in both Kariesh cheese and processed cheese. 30% of P. aeruginosa isolates were biofilm producers, while only 21% of S. aureus isolates were able to produce biofilm. The P. aeruginosa isolates harbor virulence-associated genes nan1, exoS, toxA, and pslA at 100%, 80%, 40%, and 40%, respectively. Staphylococcus aureus SEs genes were examined in S. aureus strains, where SEA and SEB genes were detected with 60%, but no isolate harbored SEC, SED, or SEE. The significant fold change of P. aeruginosa pslA expression was 0.40332 after treatment with M. oleifera aqueous extract. Conclusion: Pseudomonas aeruginosa and S. aureus harbor dangerous virulence genes that cause food poisoning, but M. oleifera extract could minimize their action.

Prevalence of cpe-positive Clostridium perfringens in surface-attached soil of commercially available potatoes and its significance as a potential source of food poisoning.

OBJECTIVE: We aimed to examine the surface-attached soil of commercially available potatoes in Japan to determine the association between foodborne infection and the circulation of Clostridium perfringens through vegetables, soil, and environments. METHODS: C. perfringens spores were isolated from 30 surface-attached soil samples of potatoes obtained from six regions in Japan. We performed multiplex polymerase chain reaction (PCR) and sequencing to detect the presence of six toxin and plasmid-related genes in the isolates. RESULTS: Sulfite-reducing clostridial spores were detected in 28 (93%) of 30 potato samples, and toxin gene PCR was performed using 613 isolates. The C. perfringens α toxin gene (cpa) was detected in 288 isolates (288/613; 47%) from 25 potato samples (83%), and these isolates were presumed to be the strains of C. perfringens. The toxin types of C. perfringens were classified into type A, in which 73% of isolates had only cpa, followed by type F in 20%, type C in 6%, and type E in 0.003% (1 isolate). The enterotoxin gene (cpe) related to food poisoning was detected in 64 isolates from 9 potato samples (3%). Of these, 59 isolates had cpa and cpe, whereas five had cpa, C. perfringens ß toxin gene, and cpe. All tested cpe-positive isolates had plasmid-type cpe. CONCLUSIONS: The isolation of culturable cpe-positive C. perfringens from the surface-attached soil of commercially available potatoes indicates that potatoes are a potential source of foodborne transmission of C. perfringens.

Short Communication: Enterotoxin Genes and Antibiotic Susceptibility of Bacillus cereus Isolated from Garlic Chives and Agricultural Environment.

This study aims to investigate the enterotoxin profiles and antibiotic susceptibility of Bacillus cereus isolated from garlic chives and environmental samples. A total of 103 B. cereus isolates were used to identify enterotoxin genes, including hblA, hblC, hblD, nheA, nheB, and nheC. The hemolysin BL enterotoxin complex (hblACD) was detected in 38 isolates (36.9%), and the non-hemolytic enterotoxin complex (nheABC) was detected in 8 (7.8%) isolates. Forty-five isolates (43.7%) had hblACD and nheABC genes. B. cereus was resistant to ß-lactam antibiotics and susceptible to non-ß-lactam antibiotics. However, some B. cereus strains showed intermediate resistance to ß-lactam and non-ß-lactam antibiotics. B. cereus isolated from garlic chives showed intermediate resistance to cefotaxime (7.7%), rifampin (15.4%), clindamycin (30.8%), erythromycin (7.7%), and tetracycline (7.7%). B. cereus isolates from the agricultural environment were moderately resistant to cefotaxime (18.9%), rifampin (15.6%), clindamycin (12.2%), erythromycin (4.4%), and tetracycline (5.6%). Moreover, B. cereus isolates from garlic chives and cultivation environments could change their antibiotic resistance profile from susceptible to intermediate-resistant to rifampin, clindamycin, erythromycin, and tetracycline and exhibit multidrug resistance. These results indicate that continuous monitoring of B. cereus contamination in the produce and agricultural environment might be needed to ensure the safety of consuming fresh vegetables.

Inhibition of the sea Gene Expression in Staphylococcus aureus Using the Aqueous and Alcoholic Extracts of the Grapevine (Vitis vinifera L.) Seeds.

Staphylococcus aureus is an important etiological agent for causing food poisoning leading to high mortality in the world. The sea gene is encoded in a polymorphic family of temperate bacteriophage chromosomes and became a prophage, and the transcription of this gene is associated with the life cycle of this prophage. It has been suggested that the grape polyphenols can eradicate the enterotoxin production of food-borne bacteria. This study aimed to evaluate the activity of the aqueous and alcoholic extracts of the grape seeds in inhibiting the expression of the sea gene encoding staphylococcal enterotoxin type A in S. aureus isolated from different sources. This study used five enterotoxin A producing isolates belonging to S. aureus. The results showed that minimum inhibition concentration and sub-minimum inhibition concentration of the aqueous extract were 32 and 16 µg/mL for all isolates, respectively. However, in the case of the alcoholic extract, these concentrations were 16 and 8 µg/mL for all isolates, respectively, and the results of the chemical analysis of the aqueous and alcoholic extracts confirmed that they contain active chemical compounds, such as flavonoids, alkaloids, tannins, and glycosides; moreover, they contain many functional groups according to the analysis of the infrared spectrum. Both extracts were shown to be active in inhibiting the expression of the sea gene in the isolates under study. As the results indicated, the gene expression of these isolates was inhibited by approximately 0.31-0.63 fold, and all pathogenic and environmental isolates showed a decrease in the expression of this gene. These results practically open the door to the possibility of using these extracts to inhibit the ability of S. aureus to produce these dangerous enterotoxins; thereby decreasing or preventing their pathogenicity, especially their food poisoning infections.

Cell-Free Systems Enable the Production of AB5 Toxins for Diagnostic Applications.

Cell-free protein synthesis (CFPS) represents a versatile key technology for the production of toxic proteins. As a cell lysate, rather than viable cells, is used, the toxic effects on the host organism can be circumvented. The open nature of cell-free systems allows for the addition of supplements affecting protein concentration and folding. Here, we present the cell-free synthesis and functional characterization of two AB5 toxins, namely the cholera toxin (Ctx) and the heat-labile enterotoxin (LT), using two eukaryotic cell-free systems based on Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cells. Through an iterative optimization procedure, the synthesis of the individual AB5 toxins was established, and the formation of multimeric structures could be shown by autoradiography. A functional analysis was performed using cell-based assays, thereby demonstrating that the LT complex induced the characteristic cell elongation of target cells after 24 h. The LT complex induced cell death at higher concentrations, starting at an initial concentration of 5 nM. The initial toxic effects of the Ctx multimer could already be detected at 4 nM. The detection and characterization of such AB5 toxins is of utmost importance, and the monitoring of intracellular trafficking facilitates the further identification of the mechanism of action of these toxins. We showed that the B-subunit of LT (LTB) could be fluorescently labeled using an LTB-Strep fusion protein, which is a proof-of-concept for future Trojan horse applications. Further, we performed a mutational analysis of the CtxA subunit as its template was modified, and an amber stop codon was inserted into CtxA's active site. Subsequently, a non-canonical amino acid was site-specifically incorporated using bio-orthogonal systems. Finally, a fluorescently labeled CtxA protein was produced using copper-catalyzed click reactions as well as a Staudinger ligation. As expected, the modified Ctx multimer no longer induced toxic effects. In our study, we showed that CFPS could be used to study the active centers of toxins by inserting mutations. Additionally, this methodology can be applied for the design of Trojan horses and targeted toxins, as well as enabling the intracellular trafficking of toxins as a prerequisite for the analysis of the toxin's mechanism of action.

Further Insights into the Toxicity of Bacillus cytotoxicus Based on Toxin Gene Profiling and Vero Cell Cytotoxicity Assays.

Bacillus cytotoxicus belongs to the Bacillus cereus group that also comprises the foodborne pathogen Bacillus cereus sensu stricto, Bacillus anthracis causing anthrax, as well as the biopesticide Bacillus thuringiensis. The first B. cytotoxicus was isolated in the context of a severe food poisoning outbreak leading to fatal cases of diarrheal disease. Subsequent characterization of the outbreak strain led to the conclusion that this Bacillus strain was highly cytotoxic and eventually resulted in the description of a novel species, whose name reflects the observed toxicity: B. cytotoxicus. However, only a few isolates of this species have been characterized with regard to their cytotoxic potential and the role of B. cytotoxicus as a causative agent of food poisoning remains largely unclear. Hence, the aim of this study was to gain further insights into the toxicity of B. cytotoxicus. To this end, 19 isolates were obtained from mashed potato powders and characterized by toxin gene profiling and Vero cell cytotoxicity assays. All isolates harbored the cytK1 (cytotoxin K1) gene and species-specific variants of the nhe (non-hemolytic enterotoxin) gene. The isolates exhibited low or no toxicity towards Vero cells. Thus, this study indicates that the cytotoxic potential of B. cytotoxicus may be potentially lower than initially assumed.

Traceability of potential enterotoxigenic Bacillus cereus in bee-pollen samples from Argentina throughout the production process.

Bee-pollen is a functional food sold for human and animal consumption but also is a favorable microhabitat for many spore-forming bacteria. Among them, Bacillus cereus can produce several toxins and other virulence factors, causing an emetic or diarrheal syndrome after ingestion. The study involved 36 bee-pollen samples obtained from different sampling points throughout the production process (collecting, freezing, drying, and cleaning) in Argentina. Fifty isolates of B. cereus yielded 24 different fingerprint patterns with BOX and ERIC primers. Only three fingerprint patterns were maintained throughout the production process. In contrast, others were lost or incorporated during the different steps, suggesting that cross-contamination occurred as shown by differences in fingerprint patterns after freezing, drying, and cleaning steps compared to the initial collection step. Genes encoding for cereulide (ces), cytotoxin K (cytK), sphingomyelinase (sph), the components of hemolysin BL (hblA, hblB, hblC, hblD) and non-hemolytic complex (nheAB) were studied. All the isolates displayed one or more enterotoxin genes. The most frequent virulence genes detected belong to the HBL complex, being the most abundant hblA (98%), followed by hblD (64%), hblB (54%), and hblC (32%), respectively. Ten strains (20%), present at all sampling points, carried all the subunits of the HBL complex. The non-hemolytic enterotoxic complex (nheAB) was found in 48 strains (96%), while seven strains (14%) present at all sampling points showed the amplification product for sphingomyelinase (sph). One cereulide-producer was isolated at the cleaning step; this strain contained all the components for the hemolytic enterotoxin complex HBL, the NHE complex, and cytotoxin K related to the foodborne diarrhoeal syndrome. In total, 11 different virulence patterns were observed, and also a correlation between rep-fingerprint and virulence patterns. The results suggest that bee-pollen can be contaminated at any point in the production process with potential enterotoxic B. cereus strains, emphasizing the importance of hygienic processing.

Expression of Escherichia coli Heat-Labile Enterotoxin B Subunit in Centella (Centella asiatica (L.) Urban) via Biolistic Transformation.

BACKGROUND: Heat-Labile enterotoxin B subunit (LTB) produced by Escherichia coli, a non-toxic protein subunit with potential biological properties, is a powerful mucosal and parenteral adjuvant which can induce a strong immune response against co-administered antigens. OBJECTIVE: In the present study, LTB protein, encoded by the optimized ltb (also known synthetic ltb, s-ltb) gene in centella plant (Centella asiatica) for use as an antigen, has been discussed. METHODS: The s-ltb gene was cloned into a plant expression vector, pMYO51, adjacent to the CaMV 35S promoter and was then introduced into centella plant by biolistic transformation. PCR amplification was conducted to determine the presence of s-ltb gene in the transgenic centella plant. The expression of s-ltb gene was analyzed by immunoblotting and quantified by ELISA. In vitro activity of LTB protein was determined by GM1-ELISA. RESULTS: PCR amplification has found seven transgenic centella individuals. However, only five of them produced LTB protein. ELISA analysis showed that the highest amount of LTB protein detected in transgenic centella leaves was about 0.8% of the total soluble protein. GM1-ELISA assay indicated that plant LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits formed active pentamers. CONCLUSION: The s-ltb gene that was successfully transformed into centella plants by the biolistic method has produced a relatively high amount of plant LTB protein in the pentameric quaternary structure that has GM1-ganglioside binding affinity, a receptor on the intestinal epithelial membrane.

[The first identification of epidemic clone of enterotoxic Escherichia coli O∶6 serogroup highly associated with azithromycin resistance in Shanghai].

Objective: To investigate the molecular characterization of adult diarrhea cases caused by enterotoxic Escherichia coli (ETEC) and explore the practical model of epidemiology for laboratory technique and data needs based on the surveillance network. Methods: Epidemiological design and sampling targeted adult cases ETEC caused diarrhea in epidemic season. The enterotoxin type, serogroup, resistance, colonization factor and molecular type of ETEC were identified. Multiple dynamic phenotypic characteristics of ETEC were indicated by multidimensional and multivariable data. Results: From 2016 to 2018, 84 eligible ETEC strains were detected. The dominant serums/toxins were O∶6 (STh), O∶25 (LT), O∶159 (STh), O∶153 (STh). O∶6 (STh+CS21), which replaced O∶25 and O∶159 as the popular clones in 2018. Six cases of O∶153 (STh+CFA/I+CS8+PT34) in outbreak in 2017 were imported ones. The resistance rates of ETEC strains detected in adults to sulfasoxazole, naproxinic acid, ampicillin and azithromycin were more than 30%, multidrug resistance (MDR) reached 58.3%. Serum/toxin types suggested that attenuated strains were more likely to become MDR. Molecular typing confirmed that the genetic similarity of the dominant clone of O∶6 serogroup (PT20-24) was higher than O∶25 and O∶159. There was a high correlation between the minimal inhibitory concentration (MIC) of azithromycin and the resistant gene mphA (87.5%, 28/32). O∶6 (STh+CS21+mphA) resistant clone was first detected in 2016. Conclusion: A new epidemic clone in adult ETEC diarrhea cases in Shanghai was O∶6 (STh+CS21+mphA). For the first time the association between azithromycin resistance gene mphA and a serum group of ETEC was observed. Multidimensional and multivariate analysis techniques based on epidemiology can help reveal the potential transmission pattern of ETEC for the accurate surveillance and early warning of outbreaks.

Antimicrobial Susceptibility and Enterotoxin-Encoding Genes in Staphylococcus spp. Recovered from Kitchen Equipment from a University Hospital in Rio de Janeiro, Brazil.

This study was conducted to determine the occurrence of antimicrobial resistance and enterotoxin-encoding genes (EEGs) in Staphylococcus spp. recovered from equipment used to prepare hospital meals, in a university hospital in Rio de Janeiro, Brazil. Sixty samples were collected from semi-industrial equipment (one blender and one mixer) in the hospital's kitchen. Resistance genes and SCCmec types were detected by PCR. From the 40 isolates of Staphylococcus spp. identified, 8 were Staphylococcus aureus. Thirty-two (80%) Staphylococcus spp. isolates were resistant to at least one antimicrobial agent. Resistance genetic determinants were detected: erm gene (Staphylococcus epidermidis [n = 2]; Staphylococcus hominis [n = 1]), mecA gene (S. epidermidis [n = 2]), and aa(6')-aph(2'') gene (Staphylococcus caprae [n = 1], S. epidermidis [n = 2], S. hominis [n = 1], Staphylococcus pausteri [n = 1], Staphylococcus simulans [n = 1], and Staphylococcus warneri [n = 1]). The presence of at least one EEG in 83% (n = 33) of the isolates was identified. Two strains of S. epidermidis were methicillin-resistant S. epidermidis (MRSE) and harboring SCCmec type IV. Staphylococcus spp. contaminated some hospital kitchen's equipment, indicating that hygiene procedures should be improved. Results also indicate that meals can be a vehicle to disseminate multiresistant Staphylococcus spp., including MRSE, and Staphylococcus with EEGs.

Leaf-Encapsulated Vaccines: Agroinfiltration and Transient Expression of the Antigen Staphylococcal Endotoxin B in Radish Leaves.

Transgene introgression is a major concern associated with transgenic plant-based vaccines. Agroinfiltration can be used to selectively transform nonreproductive organs and avoid introgression. Here, we introduce a new vaccine modality in which Staphylococcal enterotoxin B (SEB) genes are agroinfiltrated into radishes (Raphanw sativus L.), resulting in transient expression and accumulation of SEB in planta. This approach can simultaneously express multiple antigens in a single leaf. Furthermore, the potential of high-throughput vaccine production was demonstrated by simultaneously agroinfiltrating multiple radish leaves using a multichannel pipette. The expression of SEB was detectable in two leaf cell types (epidermal and guard cells) in agroinfiltrated leaves. ICR mice intranasally immunized with homogenized leaves agroinfiltrated with SEB elicited detectable antibody to SEB and displayed protection against SEB-induced interferon-gamma (IFN-γ) production. The concept of encapsulating antigens in leaves rather than purifying them for immunization may facilitate rapid vaccine production during an epidemic disease.

Elucidation of enterotoxigenic Bacillus cereus outbreaks in Austria by complementary epidemiological and microbiological investigations, 2013.

Identifying Bacillus cereus as the causative agent of a foodborne outbreak still poses a challenge. We report on the epidemiological and microbiological investigation of three outbreaks of food poisoning (A, B, and C) in Austria in 2013. A total of 44% among 32 hotel guests (A), 22% among 63 employees (B) and 29% among 362 residents of a rehab clinic (C) fell sick immediately after meal consumption. B. cereus isolated from left overs or retained samples from related foods were characterized by toxin gene profiling, and molecular typing using panC sequencing and M13-PCR typing (in outbreak A and C). We identified two B. cereus strains in outbreak A, and six B. cereus strains, each in outbreak B and C; we also found Staphylococcus aureus and staphylococcal enterotoxins in outbreak A. The panC sequence based phylogenetic affiliation of the B. cereus strains, together with findings of the retrospective cohort analyses, helped determining their etiological role. Consumption of a mashed potatoes dish in outbreak A (RR: ∞), a pancake strips soup in outbreak B (RR 13.0; 95% CI 1.8-93.0) and for outbreak C of a fruit salad (RR 1.50; 95% CI 1.09-2.00), deer ragout (RR: 1.99; 95% CI 1.23-3.22) and a cranberry/pear (RR 2.46; 95% CI 1.50-4.03)were associated with increased risk of falling sick. An enterotoxigenic strain affiliated to the phylogenetic group with the highest risk of food poisoning was isolated from the crème spinach and the strawberry buttermilk, and also from the stool samples of the one B. cereus positive outbreak case-patient, who ate both. Our investigation of three food poisoning outbreaks illustrates the added value of a combined approach by using epidemiological, microbiological and genotyping methods in identifying the likely outbreak sources and the etiological B. cereus strains.

Potential pathogens, antimicrobial patterns and genotypic diversity of Escherichia coli isolates in constructed wetlands treating swine wastewater.

Escherichia coli populations originating from swine houses through constructed wetlands were analyzed for potential pathogens, antimicrobial susceptibility patterns, and genotypic diversity. Escherichia coli isolates (n = 493) were screened for the presence of the following virulence genes: stx1, stx2 and eae (Shiga toxin-producing E. coli [STEC]), heat-labile enterotoxin (LT) genes and heat stable toxin STa and STb (enterotoxigenic E. coli (ETEC), cytotoxin necrotizing factors 1 and 2 (cnf1 and cnf2 [necrotoxigenic E. coli- NTEC]), as well as O and H antigens, and the presence of the antibiotic resistance genes blaTEM, blaSHV, blaCMY-2, tet A, tet B, tet C, mph(A), aadA, StrA/B, sul1, sul2 and sul3. The commensal strains were further screened for 16 antimicrobials and characterized by BOX AIR-1 PCR for unique genotypes. The highest antibiotic resistance prevalence was for tetracycline, followed by erythromycin, ampicillin, streptomycin, sulfisoxazole and kanamycin. Our data showed that most of the isolates had high distribution of single or multidrug-resistant (MDR) genotypes. Therefore, the occurrence of MDR E. coli in the wetland is a matter of great concern due to possible transfer of resistance genes from nonpathogenic to pathogenic strains or vice versa in the environment.

Infection with Toxin A-Negative, Toxin B-Negative, Binary Toxin-Positive Clostridium difficile in a Young Patient with Ulcerative Colitis.

Large clostridial toxin-negative, binary toxin-positive (A(-) B(-) CDT(+)) strains of Clostridium difficile are almost never associated with clinically significant C. difficile infection (CDI), possibly because such strains are not detected by most diagnostic methods. We report the isolation of an A(-) B(-) CDT(+) ribotype 033 (RT033) strain of C. difficile from a young patient with ulcerative colitis and severe diarrhea.

Analysis and application of a neutralizing linear epitope on liable toxin B of enterotoxin Escherichia coli.

Heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC) is one of the major virulence factors for causing diarrhea in piglets, and LT is a strong immunogen. Thus, LT represents an important target for development of vaccines and diagnostic tests. In this study, bioinformatic tools were used to predict six antigenic B cell epitopes in the B subunit of LT protein (LTB) of ETEC strains. Then, seven antigenic B cell epitopes of LTB were identified by polyclonal antisera (polyclonal antibody (PAb)) using a set of LTB-derived peptides expressed as maltose-binding protein (MBP) fusion protein. In addition, one LTB-specific monoclonal antibody (MAb) was generated and defined its corresponding epitope as mentioned above. This MAb was able to specifically bind with native LT toxin and has no cross-reaction with LT-II (type II heat-labile enterotoxin), Stx1 (Shiga toxin I), Stx2 (Shiga toxin II), STa (heat-stable enterotoxin I), and STb (heat-stable enterotoxin II) toxins. Further, this MAb was able to interrupt LT toxin specific binding to GM1 receptor, indicating that the corresponding epitope is the specific binding region to GM1 receptor. Moreover, in vitro and in vivo assay showed that the MAb was able to neutralize the native LT toxin. Diarrheal suckling pigs challenged with LT-positive ETEC strain recovered when an enema with this purified MAb was administered. This study will provide the foundation for further studies about the interaction between LT toxin and GM1 receptor and about the developing of epitope-based vaccines and specific therapeutic agent.

Effect of maternal supplementation with seaweed extracts on growth performance and aspects of gastrointestinal health of newly weaned piglets after challenge with enterotoxigenic Escherichia coli K88.

In the present study, a 2 × 2 factorial arrangement was conducted to investigate the effect of maternal supplementation with seaweed extracts ( - SWE v. +SWE, n 20) from day 83 of gestation until weaning (day 28) on post-weaning (PW) growth performance, faecal score, faecal enterotoxigenic Escherichia coli (ETEC) toxin quantification, intestinal histology and cytokine mRNA of unchallenged and ETEC-challenged pigs. Pigs were ETEC challenged on day 9 PW. There was a maternal treatment × challenge (SWE × ETEC) interaction effect on growth performance and faecal score (P< 0.05). Pigs from SWE-supplemented sows and ETEC-challenged (SE) had higher average daily gain (ADG) during 0-13 d PW and reduced faecal score during 0-72 h post-challenge than those from basal-fed sows and ETEC-challenged (BE) (P< 0.05). However, there was no difference between unchallenged pigs from the SWE-supplemented sows (SC) and basal-fed sows (BC) (P>0.10). Pigs from the SWE-supplemented sows had reduced heat-labile enterotoxin gene copy numbers than those from the basal-fed sows (P< 0.05). Maternal SWE supplementation increased the villus height in the ileum of pigs (P< 0.05). There was a SWE × ETEC interaction effect (P< 0.05) on IL-6 mRNA and a SWE × gastrointestinal (GI) region interaction effect (P< 0.05) on transforming growth factor-ß1 (TGF-ß1) and TNF-α mRNA. IL-6 mRNA was down-regulated in SC pigs than BC pigs (P< 0.05). However, there was no difference in IL-6 mRNA between SE and BE pigs. The mRNA of TGF-ß1 and TNF-α was down-regulated in the colon of pigs from the SWE-supplemented sows compared with those from the basal-fed sows (P< 0.05). However, there was no difference in TGF-ß1 and TNF-α mRNA in the ileum between the pigs from the SWE-supplemented sows and basal-fed sows. In conclusion, maternal SWE supplementation improves ADG and the aspects of GI health of weaned pigs following an ETEC challenge.

A standardised challenge model with an enterotoxigenic F4+ Escherichia coli strain in piglets assessing clinical traits and faecal shedding of fae and est-II toxin genes.

This study evaluated the effect of five feed additives on post weaning diarrhoea (PWD) in piglets challenged 3 d after weaning with an enterotoxigenic Escherichia coli strain (ETEC). In three experimental runs, a total of 84 piglets was weaned at 21 days of age and randomly assigned to seven treatments. As dietary treatment, piglets were fed a basal diet or diets with addition of bovine colostrum (0.2%), pineapple stem extract containing bromelain (0.2%), an autolysed yeast preparation (Saccharomyces cerevisiae) (0.1%), a combination of organic acids (0.7%) and a phytogenic product with thyme essential oil (0.015%). A porcine ETEC, serotype O149:K91:K88ac was given twice via oral infection on day 3 after weaning at 10(10) colony forming units/animal. One group of piglets was fed the basal diet without ETEC challenge. Traits included clinical sores, body temperature, faecal scoring and determination of faecal dry matter and the shedding of fae and est-II ETEC toxin genes. After weaning, non-challenged control piglets did not show signs of diarrhoea or impaired health, while the majority of infected piglets had a drop in body temperature, signs of diarrhoea and impaired general health. Mortality, the decrease of faecal dry matter and shedding of the toxin genes fae and est-II were not affected by the different additives. In conclusion, the ETEC challenge model induced distinct clinical signs of PWD in piglets, but the tested feed additives had no preventive effect under these conditions.

The hybrid between the ABC domains of synapsin and the B subunit of Escherichia coli heat-labile toxin ameliorates experimental autoimmune encephalomyelitis.

The B subunit of Escherichia coli heat-labile enterotoxin (LTB) acts as efficient mucosal carrier for conjugated antigens. We expressed two heterologous proteins using E. coli as a host: a hybrid consisting of LTB and the A, B and C domain of synapsin (LTBABC) and the separated ABC peptide of this synaptic protein. Refolded LTBABC and LTB bound to the GM1 receptor and internalized into CHO-K1(GM1+) cells. LTBABC showed enhanced solubility and cell binding ability respect to the former hybrid LTBSC. Several oral doses of LTBABC were administered to rats with experimental autoimmune encephalomyelitis (EAE) from induction to the acute stage of the disease. This treatment decreased disease severity, delayed type hypersensitivity reaction and lymph node cell proliferation stimulated by myelin basic protein. Amelioration of EAE was also associated with modulation of the Th1/Th2 cytokine ratio, increased TGF-ß secretion in mesenteric lymph nodes as well as expansion of CD4(+)CD25(+)Foxp3(+) regulatory T cell population. These results indicate that the fusion protein LTBABC is suitable for further exploration of its therapeutic effect on EAE development.

Molecular and toxigenic characterization of Bacillus cereus and Bacillus thuringiensis strains isolated from commercial ground roasted coffee.

Thirty samples of roasted ground coffee beans from 10 different commercial brands were analyzed to investigate the occurrence and levels of Bacillus cereus and Bacillus thuringiensis strains. Strains were evaluated for their genetic diversity by repetitive element sequence polymorphism PCR (Rep-PCR) and for their toxigenic profiles, i.e., the presence of hblA, hblC, hblD, nheA, nheB, nheC, cytK, ces, and entFM. Survival and multiplication of B. cereus sensu lato in the ready-to-drink coffee was determined to evaluate this beverage as a possible vehicle for B. cereus infection. B. cereus was detected in 17 (56.7%) of the 30 samples, and B. thuringiensis was detected in 8 (26.7%) of the 30 samples. Five samples did not produce any characteristic growth. The most common gene, entFM, was detected in 23 strains (92%). The NHE complex (nheA, nheB, and nheC genes) was found in 19 strains (76%). The HBL complex (hblA, hblC, and hblD) was found in 16 strains (64%). All strains were negative for ces. The cytK gene was found in 16 strains (64%). The computer-assisted cluster analysis of Rep-PCR profiles using a clustering criterion of 80% similarity revealed four main clusters. Cluster 1 was the predominant and comprised three B. thuringiensis strains with 100% similarity, cluster 2 comprised two B. cereus strains (100% similarity), cluster 3 comprised two B. thuringiensis strains (90% similarity), and cluster 4 comprised one B. thuringiensis strain and one B. cereus strain (85% similarity). The cluster analysis of fingerprints generated by Rep-PCR revealed a high genetic diversity among the B. cereus strains, suggesting that the contamination could have originated from different sources. In our experiments, when sugar was added and the beverage was kept in thermic bottles there was a significant increase in B. cereus sensu lato levels, which may increase the risk of food poisoning. These results highlight the need for additional studies on this subject to better evaluate coffee as a food poisoning vehicle.

Expression of the cholera toxin B subunit (CT-B) in maize seeds and a combined mucosal treatment against cholera and traveler's diarrhea.

The non-toxic B subunit (CT-B) of cholera toxin from Vibrio cholerae is a strong immunogen and amplifies the immune reaction to conjugated antigens. In this work, a synthetic gene encoding for CT-B was expressed under control of a γ-zein promoter in maize seeds. Levels of CT-B in maize plants were determined via ganglioside dependent ELISA. The highest expression level recorded in T(1) generation seeds was 0.0014% of total aqueous soluble protein (TASP). Expression level of the same event in the T(2) generation was significantly increased to 0.0197% of TASP. Immunogenicity of maize derived CT-B was evaluated in mice with an oral immunization trial. Anti-CTB IgG and anti-CTB IgA were detected in the sera and fecal samples of the orally immunized mice, respectively. The mice were protected against holotoxin challenge with CT. An additional group of mice was administrated with an equal amount (5 µg per dose each) of mixed maize-derived CT-B and LT-B (B subunit of E. coli heat labile toxin). In the sera and fecal samples obtained from this group, the specific antibody levels were enhanced compared to either the same or a higher amount of CT-B alone. These results suggest that a synergistic action may be achieved using a CT-B and LT-B mixture that can lead to a more efficacious combined vaccine to target diarrhea induced by both cholera and enterotoxigenic strains of Escherichia coli.