Studies of the binding of swine bacterial pathogens to commercial
yeast products
R.E. Wullschleger, K. Skjolaas, and J.E. Minton
Kansas State University
Summary
Antibiotics are thought to stimulate growth in livestock by controlling the
bacterial load of the gut, thereby making nutrients needed for immune vigilance to be
available for growth. However, there is a growing concern that continued use of
antibiotics for this purpose is not a sustainable practice. There has been an active search
for alternatives to antibiotic feeding. Yeast products may be one such non-antibiotic
alternative because they are thought to stimulate growth by virtue of their ability to bind
certain bacteria. BIOSAF, a commercial yeast product, manufactured by Saf Agri and
available for incorporation into swine diets, was observed to bind important swine
pathogens under laboratory conditions of this study. The current study examined the
ability of BIOSAF and two additional yeast products from Saf Agri, Procreatin-7 and
ADY 20, to bind Salmonella enterica serovar Typhimurium and three additional swine
pathogens Haemophilus parasuis, Actinobacillus pleuropneumoniae, and Streptococcus
suis. Qualitative binding data were obtained using scanning electron microscopy to
visual bacteria binding to yeast. Binding of Salmonella was used as a positive control for
the conditions of the binding studies, and as expected, Salmonella abundantly bound all
three yeast products. Similarly, all yeast products bound to S. suis, but to a lesser
degree than Salmonella. Still fewer numbers of H. parasuis and A. pleuropneumoniae
were generally observed bound to all three yeasts. We conclude that, whereas all the
bacteria examined bound to BIOSAF, Procreatin-7, and ADY 20 under the conditions of
our study, binding was generally most abundant with Salmonella.
Introduction
For my honors project, I worked with Dr. J.E. Minton from the Animal Science
Department at Kansas State University. Dr. Minton’s research group had recently
determined that yeast products commonly sold as direct fed microbials in livestock diets
stimulated growth performance when included in the diets of nursery pigs. In vitro
experiments have further demonstrated that these same strains of yeast bind
Salmonella enterica serovar Typhimurium, which is an important swine enteric
pathogen. Data from Dr. Brad Johnson’s laboratory indicated that E. coli F88, another
important swine pathogen, was likewise bound to yeast. Therefore, the ability of yeast
to bind to potential pathogens may account for their ability to stimulate growth
performance. Yeast may have the ability to serve as a surrogate binding site for
potential pathogens, rather than the gut wall.
In my project, I evaluated the binding of additional yeast products to a broader
range of important swine pathogens. The pathogens I used, in addition to Salmonella,
were Haemophilus parasuis, Actinobacillus pleuropneumoniae, and Streptococcus suis.
H. parasuis has emerged as a pathogen in high-health status and has been associated
with septicemia, sudden death, pneumonia, and polyserositis/arthritis in pigs of all age
groups. A. pleuropneumoniae is an etiologic bacteria of severe, and often fatal, swine
pleuropneumonia. S. suis has been associated with septicemia, meningitis, endocarditis,
arthritis, and pneumonia in pigs.
Materials and Methods
Isolates of A. pleuropneumoniae, H. parasuis, and S. suis were obtained from Dr.
M.M. Chengappa (Department of Diagnostic Medicine/Pathobiology). The Salmonella
enterica serovar Typhimurium strain was used as a positive control because it was
known to bind BIOSAF from previous studies. It was obtained from Dr. Jerome
Nietfeld, who is in the Department of Diagnostic Medicine/Pathobiology. Dr. Minton’s
laboratory has used this isolate in a number of studies that have already been published.
Bacteria were cultured in accord with accepted procedures. Saf Agri of Milwaukee, WI
donated BIOSAF, Procreatin-7, and ADY 20 yeast. Bacteria and yeast were cultured
overnight and equilibrated to McFarland standards. Salmonella were grown in Luria
broth at 37C. A. pleuropneumoniae and H. parasuis were grown on chocolate agar at
37C. S. suis was grown on trypticase soy agar with sheep blood. Bacteria were washed
with phosphate buffered saline (PBS), centrifuged, then washed a second time. Yeast
were suspended in 5 ml of PBS. Equal volumes (0.2 to 0.4 ml) of bacterial and yeast
cultures were incubated in 15-ml conical tubes. Co-incubation continued for 10 min at
37C. Co-incubation was continued for 9 h. Following co-incubation, the samples were
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filtered and fixed, then placed in a dessicator overnight. Finally, samples were mounted
on aluminum stubs and sputter coated to observation with the scanning electron
microscope (SEM). The ability of individual pathogens to bind to yeast was assessed
qualitatively in randomly selected SEM fields.
Results and Discussion
Figure 1 illustrates representative binding of Salmonella enterica serovar
Typhimurium to the three yeast products evaluated. Binding of Salmonella to BIOSAF
was used as a positive control for the binding assay because this pathogen had been
observed to bind BIOSAF in previous studies. As expected, in virtually all fields in
which yeast could be found, multiple bacteria were observed bound to clumps of yeast
and bacteria were rarely seen bound to fibers from the filter. This was generally true of
the other two yeasts as well, and in at least one field, Salmonella bindging was
impressively numerous on Procreatin-7 (Figure 1, far right image).
Figure 1. Representative binding of Salmonella enterica serovar Typhimurium to BIOSAF
(left), ADY 20 (middle) and Procreatin-7 (right).
S. suis too was frequently observed bound to all of the yeast products (Figure 2),
and like Salmonella, the bacteria were rarely seen bound to the filter fibers. Even so,
Salmonella binding in a given field was always more abundant. For these two
pathogens, in fields where bacteria were observed, they were always associated with
the yeast.
In contrast, although both A. pleuropneumoniae (Figure 3) and H. parasuis
Figure 2. Representative binding of Streptococcus suis to BIOSAF (left), ADY 20 (middle)
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and Procreatin-7 (right).
(Figure 4) could be observed to bind to an occasional yeast cell, fields in which bacteria
could be observed bound to yeast were infrequent. In particular for H. parasuis, bacteria
could be observed in most fields in which yeast was present, but in those fields, bacteria
generally were more abundantly bound to filter fibers than to yeast.
Conclusion
We confirmed the binding of S. typhimurium to BIOSAF yeast, and found both
Procreatin-7 and ADY 20 to likewise readily bind Salmonella. Our qualitative assessment
was that all yeast products likewise bound to S. suis, but to a lesser degree than
Salmonella. We generally conclude that, whereas all the bacteria examined bound to
BIOSAF under the conditions of our study, SEM fields where H. parasuis and A.
pleuropneumoniae were bound yeast were more difficult to observe. Although the
Figure 3. Representative binding of Actinobacillus pleuropneumoniae to BIOSAF (left), ADY
20 (middle) and Procreatin-7 (right).
practical implications of these findings remains to be determined, the results suggest
that yeast incorporation into swine diets may stimulate growth, in part, through
providing surrogate binding surface to that of mucosal surfaces of the alimentary and
respiratory tracks of the pig. Such binding could reduce the likelihood of pathogen entry
through mucosal surfaces and perhaps spare nutrients by allowing the animal to
maintain a less defensive mucosal immune system.
Figure 4. Representative binding of Haemophilus parasuis to BIOSAF (left), ADY 20 (middle)
and Procreatin-7 (right).
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