Development of a Salmonella Typhimurium challenge model in weaned pigs to evaluate effects of water and feed interventions on fecal shedding and growth performance.

The aim of this study was to develop a Typhimurium (ST) challenge model in weaned pigs suitable to evaluate effects of water and feed interventions on fecal shedding and growth performance. Two studies were performed. In Exp. 1 weaned pigs were fed either a standard diet (CON) or a diet with a high buffer capacity (HB) and challenged for either 3 or 7 consecutive days in a Latin square design with 4 × 8 individually housed pigs. In Exp. 2, the CON 7-d challenge method was chosen for further model development and validation. Thirty-two individually housed weaned pigs were divided over 4 treatments: a nonchallenged control group (NCON), a challenged positive control group (PCON), a challenged intervention group with acidified water (WATER), and a challenged intervention group with acidified feed (FEED). Pigs were orally challenged once daily on d 7 to 9 or d 7 to 13 after weaning (d 0) with 1 ×10 cfu ST. From d 0 to 28, rectal temperature and occurrence of diarrhea were recorded daily, and BW and feed intake were measured weekly. Fecal samples were collected on d 0, 2, 7, 9, 13, 16, 20, 23, and 27 in Exp. 1 and d 0, 2, 7, 8, 9, 13, 15, and 27 in Exp. 2 for quantification. The results of both experiments showed quantifiable fecal shedding (average peak shedding of approximately 3.5 log and 5.5 log cfu/g, respectively), accompanied by a transient 0.5°C increase in rectal temperature and an increase in occurrence of diarrhea. In Exp. 2 during the week of challenge (i.e., d 7 to 14), a reduction in growth performance (ADG: -157 to 200 g/d and G:F: -0.22 to 0.25 g/d; < 0.01) in PCON and FEED was observed compared to NCON, with WATER showing an intermediate response. The WATER treatment also showed a numerically lower peak shedding (difference of -1.3 to 1.4 log cfu/g) compared to PCON and FEED. To conclude, we repeatedly infected weaned pigs successfully with 1 × 10 cfu of ST for 7 consecutive days, resulting in detectable and quantifiable fecal shedding. This ST challenge model may be suitable for evaluation of effects of water and feed interventions on peak fecal shedding and growth performance.

Technical note: Development of a feed matrix as inoculum in Salmonella infection studies in piglets.

Current methods used for oral administration of pathogens to piglets are stressful for the animals because of fixation and use of inoculation methods such as oral gavage. In the present study an alternative way to challenge piglets with Typhimurium (ST) is investigated. The strategy was to incorporate the in a feed matrix, which is fed to the piglets and eaten voluntary. Different types of feed matrices were tested for their absorption capacity, handling properties, and palatability to piglets. Furthermore, the viability of ST in feed matrices was studied. The ST could be incorporated in ladyfinger biscuits; a 2-cm piece absorbs 1 mL of culture media. The ladyfinger biscuits were very well accepted by the piglets. After a short training period, the piglets consumed the -incorporated biscuits out of the feeding trough. In this way the animals were infected in a more natural way and without stress. The safety of farm workers was also increased because the incorporated ST results in less spoilage and spreading of ST during the infections. Results indicated that the ST cell count was reduced by only 0.2 log unit to 8.7 log cfu per inoculum after 24 h at 4°C incorporation of ST into the biscuits, which is sufficient for an infection study and indicates excellent viability of the . The viability was also indicated by increased fecal shedding of ST. Thus, it is concluded that ladyfinger biscuits are a suitable matrix to challenge piglets with ST.

Soya bean tempe extracts show antibacterial activity against Bacillus cereus cells and spores.

AIMS: Tempe, a Rhizopus ssp.-fermented soya bean food product, was investigated for bacteriostatic and/or bactericidal effects against cells and spores of the food-borne pathogen Bacillus cereus. METHODS AND RESULTS: Tempe extract showed a high antibacterial activity against B. cereus ATCC 14579 based on optical density and viable count measurements. This growth inhibition was manifested by a 4 log CFU ml(-1) reduction, within the first 15 min of exposure. Tempe extracts also rapidly inactivated B. cereus spores upon germination. Viability and membrane permeability assessments using fluorescence probes showed rapid inactivation and permeabilization of the cytoplasmic membrane confirming the bactericidal mode of action. Cooked beans and Rhizopus grown on different media did not show antibacterial activity, indicating the unique association of the antibacterial activity with tempe. Subsequent characterization of the antibacterial activity revealed that heat treatment and protease addition nullified the bactericidal effect, indicating the proteinaceous nature of the bioactive compound. CONCLUSIONS: During fermentation of soya beans with Rhizopus, compounds are released with extensive antibacterial activity against B. cereus cells and spores. SIGNIFICANCE AND IMPACT OF STUDY: The results show the potential of producing natural antibacterial compounds that could be used as ingredients in food preservation and pathogen control.

Fermented soya bean (tempe) extracts reduce adhesion of enterotoxigenic Escherichia coli to intestinal epithelial cells.

AIMS: This study aimed to investigate the effect of processed soya bean, during the successive stages of tempe fermentation and different fermentation times, on adhesion of enterotoxigenic Escherichia coli (ETEC) K88 to intestinal brush border cells as well as Caco-2 intestinal epithelial cells; and to clarify the mechanism of action. METHODS AND RESULTS: Tempe was prepared at controlled laboratory scale using Rhizopus microsporus var. microsporus as the inoculum. Extracts of raw, soaked and cooked soya beans reduced ETEC adhesion to brush border cells by 40%. Tempe extracts reduced adhesion by 80% or more. ETEC adhesion to Caco-2 cells reduced by 50% in the presence of tempe extracts. ETEC K88 bacteria were found to interact with soya bean extracts, and this may contribute to the observed decrease of ETEC adhesion to intestinal epithelial cells. CONCLUSIONS: Fermented soya beans (tempe) reduce the adhesion of ETEC to intestinal epithelial cells of pig and human origin. This reduced adhesion is caused by an interaction between ETEC K88 bacteria and soya bean compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: The results strengthen previous observations on the anti-diarrhoeal effect of tempe. This effect indicates that soya-derived compounds may reduce adhesion of ETEC to intestinal cells in pigs as well as in humans and prevent against diarrhoeal diseases.

Validation of growth as measurand for bacterial adhesion to food and feed ingredients.

AIMS: A miniaturized adhesion test was designed to study the binding capacity of food and feed ingredients for bacterial cells. METHODS AND RESULTS: Bacteria were allowed to adhere to different fibrous materials supplied as well coatings in microtitration plates. The amount of bacteria retained on the materials was determined in an automated way as growth after addition of liquid medium. The test principle was based on an inverse relationship between initial cell densities and the appearance of growth: The higher adhering cell numbers are, the shorter are the detection times of growth. The growth curves obtained were fitted by nonlinear regression analysis employing a sigmoidal curve model. Growth parameters as (i) the time after incubation at which half of the maximum growth yield was reached; (ii) the time-coordinate of the point of inflection; (iii) the detection time calculated as x-axis intercept of the maximum specific growth rate in the point of inflection; and (iv) the time-coordinate of a growth detection threshold at OD = 0.05 were highly separating for the binding capacity of different food and feed ingredients for bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: With growth as measurand for adhesion, a simple, high-throughput method was developed for the screening of huge numbers of different binding matrices and bacteria.