SUMMARY OF TRIAL SET UP:

Location:
Belgium

Timing of trial:
2003

Investigator:
Prof. Dr. L. Ducatelle and Ir. F. Van Immerseel, RUG, University of Ghent, Belgium

Purpose of evaluation:
Study the effects of contacts of Salmonella Typhimurium with Alphamune^TM G, D-Mannose and Bio-Mos* on in-vitro invasion of an intestinal epithelial cell line.

EXPERIMENTAL SET UP:

• Intestinal epithelial cells were seeded in 96-well cell culture plates (Greiner, Fricken-hausen, Germany) at a density of 5.105 cells per ml culture medium (without antibiotics) and grown overnight.
  
  

• Bacteria were grown for 20 hours in LB-medium. Then the bacteria were 1/50 diluted in LB-media, supplemented with D-Mannose, Bio-Mos* or Alphamune^TM G (at concentrations of 0.5 and 1%) and grown for 4 hours at 37°C.
  
  

• The number of cfu/ml was determined by plating 6 x 20 ml of a dilution series of the suspensions on BGA, whereafter the plates were incubated overnight at 37°C. The suspensions were put at 4°C until they were used in the assay. The bacterial suspensions were diluted to a density of 5.106 cfu/ml. From these diluted suspensions, 200 ml was added to the cells.This was centrifuged for 10 min at 1500 rpm, whereafter the plates were incubated for 1 hour at 37°C and 5% CO2. Then cells were rinsed 3 times with Hanks’ Balanced Salt Solution (HBSS, Life Technologies, Paisley, Scotland), cell culture medium with gentamicin (50 mg/ml) was added and plates were incubated for 1 hour at 37°C and 5% CO2.
  
  

• Hereafter, cells were rinsed 3 times with PBS and lysed with 1% Triton X-100 in distilled water (Sigma, St. Louis, USA). From this lysate, a 10-fold dilution series was made.
  
  

• From each dilution, 6 x 20 ml was inoculated on BGA, to determine the number of cfu of S. Typhimurium per ml.
  
  

• The tests were performed in triplicate with three repeats in each experiment.
  
  

• Analysis of variance methods with the S-Plus software was used.
  
  

• Equal concentrations of the different products were compared (with control), as well as different concentrations of the same products.

RESULTS OF Alphamune^TM G ON S. TYPHIMURIUM INVASIVENESS

Results are shown in hereunder figure, in which the mean of the three experiments (± standard error of the mean) is shown. Standard error of the mean is shown to indicate the probable interval in which the mean is lying.

• On the vertical axis, the percent of invasion relative to the initial number of bacteria, brought on the cells, is shown.

• On the horizontal axis, the different conditions, being the different contacts with the feed supplements added at various concentrations to the growth media, are shown.

• For S. typhimurium, significant differences were detected between the Control group and the Alphamune^TM G Group at a concentration of 0,5 % (P < 0.05).

• Alphamune^TM G when used at a low concentration of 0,5% had more numerical effects on reduction of invasiveness of S. Typhimurium in intestinal cellines than Bio-Moss and even more than D-Mannose at similar concentrations.

OVERALL CONCLUSIONS

• S. Typhimurium, a zoonotic pathogen in swine shows a significant reduced level of invasiveness of intestinal cellines when placed in contact with Alphamune^TM G at 0,5%.

• This in-vitro experiment demonstrates that Alphamune^TM G has pre-biotic effects against S. Typhimurium, very likely by its mannano-oligosaccharide content.

• From these observations it could be concluded that Alphamune^TM G when supplemented in pig feeds could contribute to the reduction of invasiveness of S. Typhimurium in the swine intestinal mucosa.

• Alphamune^TM G could be a valuable tool in the reduction of S. Typhimurium shedding of pigs.

• Correct dosing of feed supplements is likely to be mandatory to get the right assumed pre-biotic effects.