Bacterial Analysis

To test the effect of Ration Plus™ for Horses on digestive microbial populations Cytozyme Laboratories, Inc. secured the services of the Associated Regional and University Pathologists, Inc. (A.R.U.P.) and Dr. Clay Cannon, D.V.M.. A.R.U.P. conducted a bacterial analysis of fecal specimens collected by Dr. Cannon from four healthy horses stabled at the Parker Ranch in South Jordan, Utah. Each horse had a fecal sample taken directly from the rectum, to avoid contamination by outside microorganisms, every 10 days for a 50 day period. The first 20 days (2 samples) the animals were fed their regular diet of quality alfalfa hay and oats. Horses had access to free choice mineral salt block and fresh water at all times. On the 21st day, Ration Plus™ was added to each horse's diet (4cc/day for 30 days).

One gram of horse fecal material was removed from the transport tubes and diluted in buffered saline. Appropriate dilutions were plated to selective (MAC) and non selective (SAB) agar. Cultures were incubated for 24 hours. The number of colony types were determined, corrected and the organisms were identified by standard laboratory procedures.

It was possible to isolate specific organisms when they were present in concentration of 100,000 per gram. When they were present at concentration less than that, they were often overgrown by other bacteria making their isolation impossible. The greatest number of any given organism was present in the order of 300,000,000 per gram, so our effective range of examination was about 3000 fold.

In general the following patterns seem clear:
1. The absolute number of bacteria per gram of feces increased from 105 - 106 to 107 - 108 organisms per gram.
2. The diversity of recoverable bacterial species increased from an average of 5.5 detectable species in untreated horses to 7-7.5 detectable species in horses receiving supplement.
3. The change in the absolute number of species recovered was reflected in a shift to a general increase in the number of species of gram negative bacillus.
4. Although streptococci were recovered in all specimens, they were received less often and in smaller numbers from treated horses.
5. A variety of Bacillus species were recovered from all specimens. However, in 3 of the 4 horses they were more common during supplementation than before.

6. Specific changes noted were:
a. Escherichia coli was uniformly present and changed little as a result of supplementation. In three horses, there appeared to be a slight increase in E. coli per gram of feces after supplementation.
b. Staphylococci and mirococci - remained constant in the study.
c. Streptococci and bacilli were noted in #4 and #5 above.
d. Species form Citrobacter, Acinetobacter, Enterobacter and Lactobacillus occurred intermittently but were much more likely to be recovered from horses receiving supplementation.

The data may be instructive in terms of what does not happen as what does. There is no apparent absolute change in the types of bacteria inhabiting the bowel as a function of supplementation. No species is inhibited by this procedure, although as noted earlier the variety of species increased (a subtle change) and the total number of bacteria per gram of feces increased (also a subtle change).

None of the bacteria Isolates were unexpected or unusual. The bacteria strains were representative of normal gut flora and could not be considered either unique or exciting.