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The Effect of Increasing Concentrations of Terramycin on the Growth and Intestinal Microflora of Chicks1
T h e Effect of Increasing Concentrations of Terramycin on the Growth and Intestinal Microflora of Chicks1 M . M . R O S E N B E R G , W. M O R I K A W A AND O. A. B U S H N E L L Departments of Poultry Husbandry and Bacteriology, University of Hawaii, Honolulu, Hawaii (Received for publication December 10, 1951)
UMEROUS reports have been published concerning the beneficial effects of antibiotics on poultry and swine. Smith and Robinson (1945) found that streptomycin administered orally produced a marked reduction in the coliform bacteria of the intestine of mice, and Moore et al. (1946) in a study of the use of antibacterial agents found that the inclusion of streptomycin in the diet resulted also in a significant response in growth of chicks. Stokstad et al. (1949) were the first to report that fermentation liquors of Streptomyces aureofaciens were capable of producing growth increments in chicks greater than those obtained with vitamin B12 alone. Subsequently, Matterson and Singsen (1951), working with chicks, and Carpenter (1951) with swine, observed that aureomycin, terramycin, streptomycin, penicillin, and other antibiotics stimulated the growth rates of their experimental subjects. Various theories have been discussed by Groschke (1950), Groschke and Evans (1950), and Biely and March (1951) to account for this remarkable growth stimulation in young chickens. The latter investigators suggested that "the effect of aureomycin, in lowering the dietary requirements for certain vitamins, is 1 Published with the approval of the Director of the Hawaii Agricultural Experiment Station as Technical Paper No. 246.
brought about through a reduction in the numbers of intestinal microflora which might compete with the host for members of the vitamin B complex and/or by permitting the proliferation of microorganisms which synthesize these vitamins." Furthermore, from a study of the effects of certain antibiotics on the intestinal microflora of turkeys and pigs, Sieburth et al. (1951) suggested that penicillin and terramycin promoted growth in poults, and terramycin in pigs, by inhibiting C. perfringens, thereby preventing enterotoxemia. Because the other theories proposed to account for these beneficial effects were not founded on adequate bacteriological evidence, and inasmuch as studies were already underway at this Station on the relationships between rations made up of ingredients produced in Hawaii and the intestinal microflora of chicks, a preliminary investigation was undertaken to determine what effect aureomycin and terramycin, added in the recommended amounts to chick rations, would have upon the various groups of intestinal microorganisms being assayed. These preliminary observations indicated that during the first 2 weeks of the chicks' life there were no profound changes in the groups of the intestinal microflora being assayed, although the growth rate of chicks was definitely stimulated by the two antibiotics. 708
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709
TERRAMYCIN AND INTESTINAL MICROFLORA
These observations led to an investigation of the effects of increasing concentrations of terramycin fed during a period of 4 weeks upon the growth of chicks and upon their intestinal microflora. The results of these trials are reported in detail in this paper. MATERIALS AND METHODS
TABLE 1.—Formulas of rations fed to the six experimental groups of chicks in trials 1 and 2 Rations Ingredients 1 Ground yellow corn Ground wheat Ground oats Herring meal (70% protein) Soybean oil meal (44% protein) Alfalfa leaf meal Ground oyster shell Defluorophos* Iodized salt Manganese sulfate, gm. Delsterol,f gm. Choline chloride, gm. Riboflavin, mg. Bi-Con TM-5,t gm.
2
26.5 20.0 10.0 6.0 30.0 5.0 1.0 1.0 0.5 10.0 15.0 125.0 160.0 0.0
3
5
6
480.0
960.0
4
,
Same as in ration 1
60.0
120.0
240.0
* Defluorophos: 31.0 percent calcium and 13.1 percent phosphorus, t Delsterol: 2000 AOAC units per gram. % Bi-Con TM-5: terramycin feed supplement; guaranteed equivalent 5.0 grams crystalline terramycin hydrochloride per pound. Chas. Pfizer and Co., Inc.
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Day-old New Hampshire chicks were randomized into groups containing 25 chicks. Each group was reared in a standard Oakes battery brooder. The rations devised for different groups of chicks, as shown in Table 1, were fed for 28 days in two separate trials. As may be seen, the rations differed only in the concentration of terramycin, supplied in the form of Bi-Con TM-5 (Pfizer). Ration 1, the control ration, contained no Bi-Con TM5; to ration 2, 60 grams of Bi-Con TM-5 were added per 100 pounds of feed; in ration 3, 120 grams were added. By progressive doubling of the amounts of BiCon TM-5 employed, the other rations were prepared until, in ration 6, 960
grams of this antibiotic feed supplement were used per 100 pounds of feed. Microbiological analyses of fecal samples were made every third day. The bacteriological techniques were those employed by Johansson et al. (1948) in their study of the intestinal microflora of hens. Fecal droppings were collected on sheets of clean waxed paper spread beneath the wire floors of the battery brooders. Specimens were collected at approximately 11:00 a.m. on each day of sampling during a period not exceeding 30 minutes. The pooled specimens from each group of chicks were transferred t,o separate sterile paper cups kept in sterile paper bags and were transported immediately to the laboratory for analysis. A sample of approximately 0.5 gram, measured to the nearest milligram, was used as the test specimen from each lot of chicks. From this specimen were made all the dilutions needed for inoculation into the several media utilized by Johansson et al. (1948). The media and the manner in which they were used were designed to
710
M. M. ROSENBERG, W. MORIKAWA AND O. A. BUSHNELL
detect the following groups of microorganisms: Medium (1) Thioglycollate broth (2) Tryptose-glucose-yeast extract agar ("aerobic agar") (3) Carrot-liver agar (4) S-F broth (5) (Difco) eosin-methylene blue agar (6) Potato-glucose agar
Purpose (1) for "total counts," including both aerobes and anaerobes (2) for "aerobes" (3) (4) (5) (6)
for for for for
"lactic acid-producing bacteria" "enterococci" "coliform organisms" "yeasts"
a gram of gain, the groups fed rations 2 to 6 required 2.0, 1.93, 1.83, 1.98, and 1.85 grams, respectively. The results of trial 2 confirmed the trend observed in trial 1. As may be seen in Table 2, graded increments of Bi-Con TM-5, with the exception of ration 2, stimulated growth rate and increased the efficiency of feed conversion. Upon analysis of variance, the effect of various concentrations of terramycin on growth rate was highly significant (P<0.01); but its effect on efficiency was not significant. In both analyses, the variation in results RESULTS between trials was highly significant Effect on growth rate and feed efficiency.— (P<0.01). The effects of the addition of various conEffect on number of microorganisms.— centrations of Bi-Con TM-5 to a standard A summary of the average counts obstarter ration upon the growth of chicks tained in each medium is presented in may be seen in the data summarized in Table 3 and the significant differences in Table 2. Terramycin stimulated significounts are shown in Table 4. The followcantly the growth rate of the experiing is a brief analysis of the data obtained mental chicks. In trial 1, for example, the 2.—Average body weights and efficiency of feed average chick weights among the groups TABLE utilization of groups fed various confed rations 2, 3, 4, 5, and 6 exceeded the centrations of terramycin control group, at the end of 28 days, by Average body Percentage Unit feed 9.5, 16.6, 13.7, 15.4, and 19.1 percent, weights of both Rations of controls' per unit sexes at 28 gain (groups) weight respectively. Furthermore, the efficiency days of age of feed utilization was affected beneficialTrial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2 ly: whereas the chicks fed the control 1 343.4 408.6 100.0 100.0 2.20 2.24 2 375.9 403.8 109.5 98.8 2.00 2.43 diet required 2.2 grams of feed to produce 3 400.4 444.9 116.6 108.9 1.93 2.12
* Comparison of counts obtained in earlier work had shown that there was not enough statistical difference between counts adjusted to dry-weight basis and those calculated on a wet-weight basis to warrant the added trouble involved in dry-weight determinations.
4 5 6
390.5 396.4 408.9
449.5 454.0 458.5
Among rations F = 11.76 Between trials F=86.78 L.S.D. between any two rations = 19.97
113.7 115.4 119.1
110.0 111.1 112.2
1.83 1.98 1.85
2.03 2.02 1.93
Among rations F=4.00 Between trials F=9.00 L.S.D. between any two rations = 0.0257
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All counts obtained for microorganisms growing in these media were adjusted on the basis of wet-weight determinations to give the approximate numbers of organisms per half gram of natural feces.* Each count was converted into its respective logarithm to the base 10, with the mantissa carried to the second place. All computations were based on the log numbers of the different groups of microorganisms. Data were collected also on body weight and feed consumption.
TERRAMYCIN AND INTESTINAL MICROFLORA TABLE 3.—Average log number of microorganisms per 0.5 gram of natural feces Groups of microorganisms Total count
robes
Lactic acid producers
1
1 2 3 4 5 6
10.59 10.35 10.42 10.70 10.95 11.19
8.53 8.21 8.32 8.37 8.88 8.76
8.68 8.76 8.78 8.48 9.03 9.08
8.13 8.06 8.08 8.26 8.52 8.45
5.94 6.39 6.40 6.90 7.52 7.65
2.27 2.21 2.39 2.52 2.79 2.32
2
1 2 3 4 5 6
10.81 10.47 10.19 10.44 10.58 10.23
8.34 8.19 8.04 8.33 8.19 8.03
8.76 8.67 8.58 8.65 8.55 8.27
7.84 7.91 7.84 8.11 7.87 7.88
6.17 6.52 7.00 6.98 7.46 7.84
1.78 1.92 2.08 2.10 2.10 2.09
Ae-
Con- Enter- Yeasts forms ococci
on each medium: Trial 1. (a) Thioglycollate broth: the variation in total number of microorganisms among the experimental groups was significant (P<0.05); the counts increased as the concentration of terramycin was increased. When the least significant difference (L.S.D.) was applied, it was observed that the group fed ration 6 had a significantly higher count that either group 2 or 3.
(b) Aerobic agar: there was a significant difference among the counts from the six groups, and a highly significant F value was obtained among the counts within each group. Groups 5 and 6, fed the higher levels of terramycin, had significantly higher counts than group 2; similarly, group 5 had a significantly higher count that group 3. (c) Carrot-liver agar: although there was no real difference among the six groups in their numbers of lactic acidproducing bacteria, group 6 showed a significantly greater count than group 4. (d) Eosin-methylene blue agar: the variation in counts among the six groups in the numbers of coliform organisms was highly significant. The L.S.D. showed that group 5 had a significantly higher count than did the control, and groups 5 and 6 significantly exceeded groups 2 and 3. (e) S-F broth: the variation in numbers of enterococci between rations was highly significant. Groups 4, 5, and 6 displayed
TABLE 4.—Significant differences in microbial counts as affected by the concentration of terramycin* Columns G
D Trial Rations
Total Count
Aerobes
Lactic acid producers
Coliforms
Enterococci
Yeasts
#4,5&6 #5&6
#6 #5&6
l,4,5&6
* Chicks fed rations in column A had a significantly lower count than those of the group number shown in columns B through G.
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tions
Trial
Ra-
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M. M. ROSENBERG, W. MORIKAWA AND 0. A. BUSHNELL
no significant difference in coliform organisms among the groups or between any two groups in trial 2. There was a tendency for the counts to drop during the first 2 weeks but subsequent counts increased in numbers, resulting in a significant F value for successive counts within groups. (e) S-F broth: as in trial 1, the variation in numbers of enterococci among the groups was highly significant; the groups receiving the greater concentrations of terramycin showed significantly higher counts. As may be seen in Table 4, the results of trials 1 and 2 were similar in trend. (f) Potato-dextrose agar: there was no significant difference in yeast counts between any two groups or among the six groups. DISCUSSION AND CONCLUSIONS
The chief conclusion to be drawn from these observations is the fact that, contrary to the common belief, the addition of terramycin to chick rations does not "sterilize" the intestinal tract or even significantly reduce the numbers of intestinal microorganisms that can be detected by the cultural methods that were used in this investigation. Even by increasing the concentration of terramycin to 16 times the amount recommended for commercial poultry rations, the relatively large amount of the antibiotic did not significantly depress the numbers of the several physiological groups of organisms assayed. In fact, at the higher concentrations of terramycin higher counts were obtained for both enterococci and yeasts than were found at lower concentrations, the enterococcus counts being significantly higher. Only in the case of the lactic acidproducing bacteria, in only one of the trials (trial 2), was there a significant reduction in numbers, the counts for the
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significantly greater counts than did the control group; indeed, the control group fed no terramycin had the lowest counts! The L.S.D. also showed that groups 2 and 3 had significantly lower counts than did either group 5 or 6, and group 4 had a significantly lower count than group 6. (f) Potato-dextrose agar: there was no significant difference between the groups in the numbers of yeasts. Although the L.S.D. between each experimental ration and the control showed no real differences, group 5 had a significantly greater count than group 2. Trial 2. (a) Thioglycollate broth: in this series no significant difference was observed between treatments, and none of the comparisons between the average counts of any two groups exceeded the value of the L.S.D. As in the preceding trial, although terramycin was fed at concentrations up to 16 times the level recommended in chick starter rations, these high levels did not depress the total count of the intestinal microflora. (b) Aerobic agar: the counts for aerobic organisms were not significantly different among the groups of chicks both between and within groups; nor was there any real difference between the average counts of any two of the six groups. In both trials, the groups fed terramycin did not show significantly different counts from that of the control when measured by the L.S.D. (c) Carrot-liver agar: as in trial 1, the F value measuring the magnitude of the variation in counts among the six groups was not significant. However, group 6 had a significantly lower count than either groups 1 and 2. This is a marked reversal from trial 1, where group 6 showed the highest number of "lactic acid-producers." (d) Eosin-methylene blue agar: contrary to the findings of trial 1, there was
TERRAMYCIN AND INTESTINAL MICROFLORA
tected by thioglycollate broth, tryptoseglucose-yeast extract agar, carrot-liver agar, S-F broth, Difco's modification of eosin-methylene blue agar, and potatoglucose agar. Perhaps among the microorganisms that are not detected by these techniques are "harmful" microorganisms which are susceptible to the action of terramycin and are so affected that the relieved chick shows an increase in its size and improvement in the utilization of its foodstuff. It is evident to the bacteriologist that much more fundamental work in establishing the identity of all the microorganisms found in the "normal flora" of the intestinal tracts of chicks must be done before anyone can say which organisms, if any, are so affected by antibiotics incorporated in rations that the reduction or elimination of these organisms results in increased growth of the chicks. Unpublished data suggest that the physiological response of chicks to BiCon TM-5 may depend upon the completeness of the chicks' diet and upon the quality of the ingredients incorporated in it. This evidence, combined with the impressions gained from an appreciation of the persistence of great numbers of microorganisms in the intestinal tract even in the presence of high concentrations of terramycin, leads inevitably to the rash but pertinent question whether, in the intestinal tract of chicks, terramycin acts primarily as an antibiotic or benefits the chick in some other way. Biely and March (1951) have shown that dietary levels of nicotinic acid, folic acid, or riboflavin that are suboptimal for maximum growth rate of the chick under normal conditions may be adequate when aureomycin is fed. SUMMARY
Terramycin, introduced into the diet
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group fed 960 grams of Bi-Con TM-5 per 100 pounds of feed being significantly affected. However, the counts for "lactic acid-producing bacteria" made on the groups fed from 60 to 480 grams of BiCon TM-5 per 100 pounds of feed were not reduced significantly in either trial. Counts for the different groups of organisms did exhibit variations from a neatly mannered curve and from a precise repetition between trials; but inspection of the data shows that counts made on feces from the chicks in the control groups varied in the same way. The usual explanations can be offered to account for some of the variations, but no criticism of techniques can explain away the persisting high counts during the 4 weeks of exposure to comparatively high concentrations of terramycin for all of the groups of microorganisms which were studied; Moreover, the addition of Bi-Con TM-5 to the rations did stimulate growth rate and efficiency of feed utilization of the experimental groups of chicks, indicating thereby that these observations were not confounded by a lack of the so-called "antibiotic supplement" effect. How, then, does terramycin present in Bi-Con TM-5 exert its beneficial effect upon the growing chick? Obviously these investigations have not found the answer. Perhaps the techniques employed were far from subtle enough to observe what effect terramycin would have upon many groups of organisms which might exist in the intestinal tracts of chicks and which could not be detected by the media under the conditions in which they were used. It can be concluded, however, that terramycin in the concentrations employed does not significantly reduce the numbers and kinds of microorganisms— which surely must form an important part, if not the major part, of the intestinal microflora of chicks—that are de-
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POULTRY SCIENCE TEACHING AND EXTENSION PRIZE AWARDS
REFERENCES Biely, J., and B. March, 1951. The effect of aureomycin and vitamins on the growth rate of chicks. Science 114: 330-331. Carpenter, L. E., 1951. The effect of antibiotics and vitamin B12 on the growth of swine. Arch. Biochem. Biophysics 32: 187.
Groschke, A. C , 1950. Relation of growth stimulating properties of antibiotics to unidentified growth factors required by the chick. Poultry Sci. 29: 760. Groschke, A. C , and R. J. Evans, 1950. Effect of antibiotics, synthetic vitamins, vitamin Bi 2 , and an APF supplement on chick growth. Poultry Sci. 29: 616-619. Johansson, K. R., W. B. Sarles and S. K. Shapiro, 1948. The intestinal microflora of hens as influenced by various carbohydrates in a biotindeficient ration. J. Bact. 56: 619-634. Matterson, L. A., and E. P. Singsen, 1951. A comparison of several antibiotics as growth stimulants in practical chick-starting rations. Storrs Agr. Exp. Sta. Bull. 275: 1-20. Moore, P. R., A. Evenson, T. D. Luckey, E. McCoy, C. A. Elvehjem and E. B. Hart, 1946. Use of sulfasuxidine, streptothricin and streptomycin in nutritional studies with the chick. J. Biol. Chem. 165:437-441. Sieburth, J. McN., J. Gutierrez, J. McGinnis, J. R. Stern and B. H. Schneider, 1951. Effect of antibiotics on intestinal microflora and on growth of turkeys and pigs. Proc. Soc. Exp. Biol. Med. 76: 15-18. Smith, D. G., and H. J. Robinson, 1945. The influence of streptomycin and streptothricin on the intestinal flora of mice. J. Bact. 50: 613-621. Stokstad, E. L. R., T. H. Jukes, J. Pierce, A. C. Page and A. L. Franklin, 1949. The multiple nature of the animal protein factor. J. Biol. Chem. 180: 647-654.
POULTRY SCIENCE TEACHING AND EXTENSION PRIZE AWARDS {Continued from page 707) All information shall be forwarded to the Chairman of the Award Committee not later than 60 days prior to the annual meeting. In making the award, the following points shall be considered and in addition such other information as may have a bearing on the work accomplished: (a) Organization—This shall include a digest of the plan of work. Emphasis shall be placed on cooperation with other subject matter specialists in allied fields, cooperation, utilization and development of leadership in the poultry industry organization and in the development and application of new subject matter. (b) Economic need—The economic application
of the subject matter to the poultry industry and the timeliness of the subject shall be considered. (c) Results—Results shall be measured in terms of approval of those who are associated with the nominee and in terms of the improved practices that have been adopted. Consideration shall be given as to the durability and the test of time achieved by the program. The extent to which these methods, programs and procedures have been adopted by other states shall also be considered. (d) Appeal—Consideration shall be given to the originality and appeal of the methods in use and the extent to which they have stimulated interest in the project and problem.
(Continued on page 722)
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in the form of Bi-Con TM-5, was fed at five progressively doubled concentrations, in two trials, to determine its effect on the intestinal microflora of chicks. Counts of organisms present in natural feces were made using the following media: thioglycollate broth, tryptose-glucose-yeast extract agar, corrot-liver agar, S-F broth, Difco's modification of eosin-methylene blue agar, and potato-glucose agar. Although terramycin significantly stimulated the growth rate of chicks during the two 28-day studies and increased the efficiency of feed utilization, the microflora detectable by the techniques used was not eliminated or even significantly reduced in number, even when 16 times the recommended amount of terramycin was fed. In both trials the enterococci counts were significantly greater among the groups receiving the higher concentrations of terramycin.
UMEROUS reports have been published concerning the beneficial effects of antibiotics on poultry and swine. Smith and Robinson (1945) found that streptomycin administered orally produced a marked reduction in the coliform bacteria of the intestine of mice, and Moore et al. (1946) in a study of the use of antibacterial agents found that the inclusion of streptomycin in the diet resulted also in a significant response in growth of chicks. Stokstad et al. (1949) were the first to report that fermentation liquors of Streptomyces aureofaciens were capable of producing growth increments in chicks greater than those obtained with vitamin B12 alone. Subsequently, Matterson and Singsen (1951), working with chicks, and Carpenter (1951) with swine, observed that aureomycin, terramycin, streptomycin, penicillin, and other antibiotics stimulated the growth rates of their experimental subjects. Various theories have been discussed by Groschke (1950), Groschke and Evans (1950), and Biely and March (1951) to account for this remarkable growth stimulation in young chickens. The latter investigators suggested that "the effect of aureomycin, in lowering the dietary requirements for certain vitamins, is 1 Published with the approval of the Director of the Hawaii Agricultural Experiment Station as Technical Paper No. 246.
brought about through a reduction in the numbers of intestinal microflora which might compete with the host for members of the vitamin B complex and/or by permitting the proliferation of microorganisms which synthesize these vitamins." Furthermore, from a study of the effects of certain antibiotics on the intestinal microflora of turkeys and pigs, Sieburth et al. (1951) suggested that penicillin and terramycin promoted growth in poults, and terramycin in pigs, by inhibiting C. perfringens, thereby preventing enterotoxemia. Because the other theories proposed to account for these beneficial effects were not founded on adequate bacteriological evidence, and inasmuch as studies were already underway at this Station on the relationships between rations made up of ingredients produced in Hawaii and the intestinal microflora of chicks, a preliminary investigation was undertaken to determine what effect aureomycin and terramycin, added in the recommended amounts to chick rations, would have upon the various groups of intestinal microorganisms being assayed. These preliminary observations indicated that during the first 2 weeks of the chicks' life there were no profound changes in the groups of the intestinal microflora being assayed, although the growth rate of chicks was definitely stimulated by the two antibiotics. 708
Downloaded from http://ps.oxfordjournals.org/ at Ritsumeikan University on June 8, 2015
N
709
TERRAMYCIN AND INTESTINAL MICROFLORA
These observations led to an investigation of the effects of increasing concentrations of terramycin fed during a period of 4 weeks upon the growth of chicks and upon their intestinal microflora. The results of these trials are reported in detail in this paper. MATERIALS AND METHODS
TABLE 1.—Formulas of rations fed to the six experimental groups of chicks in trials 1 and 2 Rations Ingredients 1 Ground yellow corn Ground wheat Ground oats Herring meal (70% protein) Soybean oil meal (44% protein) Alfalfa leaf meal Ground oyster shell Defluorophos* Iodized salt Manganese sulfate, gm. Delsterol,f gm. Choline chloride, gm. Riboflavin, mg. Bi-Con TM-5,t gm.
2
26.5 20.0 10.0 6.0 30.0 5.0 1.0 1.0 0.5 10.0 15.0 125.0 160.0 0.0
3
5
6
480.0
960.0
4
,
Same as in ration 1
60.0
120.0
240.0
* Defluorophos: 31.0 percent calcium and 13.1 percent phosphorus, t Delsterol: 2000 AOAC units per gram. % Bi-Con TM-5: terramycin feed supplement; guaranteed equivalent 5.0 grams crystalline terramycin hydrochloride per pound. Chas. Pfizer and Co., Inc.
Downloaded from http://ps.oxfordjournals.org/ at Ritsumeikan University on June 8, 2015
Day-old New Hampshire chicks were randomized into groups containing 25 chicks. Each group was reared in a standard Oakes battery brooder. The rations devised for different groups of chicks, as shown in Table 1, were fed for 28 days in two separate trials. As may be seen, the rations differed only in the concentration of terramycin, supplied in the form of Bi-Con TM-5 (Pfizer). Ration 1, the control ration, contained no Bi-Con TM5; to ration 2, 60 grams of Bi-Con TM-5 were added per 100 pounds of feed; in ration 3, 120 grams were added. By progressive doubling of the amounts of BiCon TM-5 employed, the other rations were prepared until, in ration 6, 960
grams of this antibiotic feed supplement were used per 100 pounds of feed. Microbiological analyses of fecal samples were made every third day. The bacteriological techniques were those employed by Johansson et al. (1948) in their study of the intestinal microflora of hens. Fecal droppings were collected on sheets of clean waxed paper spread beneath the wire floors of the battery brooders. Specimens were collected at approximately 11:00 a.m. on each day of sampling during a period not exceeding 30 minutes. The pooled specimens from each group of chicks were transferred t,o separate sterile paper cups kept in sterile paper bags and were transported immediately to the laboratory for analysis. A sample of approximately 0.5 gram, measured to the nearest milligram, was used as the test specimen from each lot of chicks. From this specimen were made all the dilutions needed for inoculation into the several media utilized by Johansson et al. (1948). The media and the manner in which they were used were designed to
710
M. M. ROSENBERG, W. MORIKAWA AND O. A. BUSHNELL
detect the following groups of microorganisms: Medium (1) Thioglycollate broth (2) Tryptose-glucose-yeast extract agar ("aerobic agar") (3) Carrot-liver agar (4) S-F broth (5) (Difco) eosin-methylene blue agar (6) Potato-glucose agar
Purpose (1) for "total counts," including both aerobes and anaerobes (2) for "aerobes" (3) (4) (5) (6)
for for for for
"lactic acid-producing bacteria" "enterococci" "coliform organisms" "yeasts"
a gram of gain, the groups fed rations 2 to 6 required 2.0, 1.93, 1.83, 1.98, and 1.85 grams, respectively. The results of trial 2 confirmed the trend observed in trial 1. As may be seen in Table 2, graded increments of Bi-Con TM-5, with the exception of ration 2, stimulated growth rate and increased the efficiency of feed conversion. Upon analysis of variance, the effect of various concentrations of terramycin on growth rate was highly significant (P<0.01); but its effect on efficiency was not significant. In both analyses, the variation in results RESULTS between trials was highly significant Effect on growth rate and feed efficiency.— (P<0.01). The effects of the addition of various conEffect on number of microorganisms.— centrations of Bi-Con TM-5 to a standard A summary of the average counts obstarter ration upon the growth of chicks tained in each medium is presented in may be seen in the data summarized in Table 3 and the significant differences in Table 2. Terramycin stimulated significounts are shown in Table 4. The followcantly the growth rate of the experiing is a brief analysis of the data obtained mental chicks. In trial 1, for example, the 2.—Average body weights and efficiency of feed average chick weights among the groups TABLE utilization of groups fed various confed rations 2, 3, 4, 5, and 6 exceeded the centrations of terramycin control group, at the end of 28 days, by Average body Percentage Unit feed 9.5, 16.6, 13.7, 15.4, and 19.1 percent, weights of both Rations of controls' per unit sexes at 28 gain (groups) weight respectively. Furthermore, the efficiency days of age of feed utilization was affected beneficialTrial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2 ly: whereas the chicks fed the control 1 343.4 408.6 100.0 100.0 2.20 2.24 2 375.9 403.8 109.5 98.8 2.00 2.43 diet required 2.2 grams of feed to produce 3 400.4 444.9 116.6 108.9 1.93 2.12
* Comparison of counts obtained in earlier work had shown that there was not enough statistical difference between counts adjusted to dry-weight basis and those calculated on a wet-weight basis to warrant the added trouble involved in dry-weight determinations.
4 5 6
390.5 396.4 408.9
449.5 454.0 458.5
Among rations F = 11.76 Between trials F=86.78 L.S.D. between any two rations = 19.97
113.7 115.4 119.1
110.0 111.1 112.2
1.83 1.98 1.85
2.03 2.02 1.93
Among rations F=4.00 Between trials F=9.00 L.S.D. between any two rations = 0.0257
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All counts obtained for microorganisms growing in these media were adjusted on the basis of wet-weight determinations to give the approximate numbers of organisms per half gram of natural feces.* Each count was converted into its respective logarithm to the base 10, with the mantissa carried to the second place. All computations were based on the log numbers of the different groups of microorganisms. Data were collected also on body weight and feed consumption.
TERRAMYCIN AND INTESTINAL MICROFLORA TABLE 3.—Average log number of microorganisms per 0.5 gram of natural feces Groups of microorganisms Total count
robes
Lactic acid producers
1
1 2 3 4 5 6
10.59 10.35 10.42 10.70 10.95 11.19
8.53 8.21 8.32 8.37 8.88 8.76
8.68 8.76 8.78 8.48 9.03 9.08
8.13 8.06 8.08 8.26 8.52 8.45
5.94 6.39 6.40 6.90 7.52 7.65
2.27 2.21 2.39 2.52 2.79 2.32
2
1 2 3 4 5 6
10.81 10.47 10.19 10.44 10.58 10.23
8.34 8.19 8.04 8.33 8.19 8.03
8.76 8.67 8.58 8.65 8.55 8.27
7.84 7.91 7.84 8.11 7.87 7.88
6.17 6.52 7.00 6.98 7.46 7.84
1.78 1.92 2.08 2.10 2.10 2.09
Ae-
Con- Enter- Yeasts forms ococci
on each medium: Trial 1. (a) Thioglycollate broth: the variation in total number of microorganisms among the experimental groups was significant (P<0.05); the counts increased as the concentration of terramycin was increased. When the least significant difference (L.S.D.) was applied, it was observed that the group fed ration 6 had a significantly higher count that either group 2 or 3.
(b) Aerobic agar: there was a significant difference among the counts from the six groups, and a highly significant F value was obtained among the counts within each group. Groups 5 and 6, fed the higher levels of terramycin, had significantly higher counts than group 2; similarly, group 5 had a significantly higher count that group 3. (c) Carrot-liver agar: although there was no real difference among the six groups in their numbers of lactic acidproducing bacteria, group 6 showed a significantly greater count than group 4. (d) Eosin-methylene blue agar: the variation in counts among the six groups in the numbers of coliform organisms was highly significant. The L.S.D. showed that group 5 had a significantly higher count than did the control, and groups 5 and 6 significantly exceeded groups 2 and 3. (e) S-F broth: the variation in numbers of enterococci between rations was highly significant. Groups 4, 5, and 6 displayed
TABLE 4.—Significant differences in microbial counts as affected by the concentration of terramycin* Columns G
D Trial Rations
Total Count
Aerobes
Lactic acid producers
Coliforms
Enterococci
Yeasts
#4,5&6 #5&6
#6 #5&6
l,4,5&6
* Chicks fed rations in column A had a significantly lower count than those of the group number shown in columns B through G.
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tions
Trial
Ra-
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no significant difference in coliform organisms among the groups or between any two groups in trial 2. There was a tendency for the counts to drop during the first 2 weeks but subsequent counts increased in numbers, resulting in a significant F value for successive counts within groups. (e) S-F broth: as in trial 1, the variation in numbers of enterococci among the groups was highly significant; the groups receiving the greater concentrations of terramycin showed significantly higher counts. As may be seen in Table 4, the results of trials 1 and 2 were similar in trend. (f) Potato-dextrose agar: there was no significant difference in yeast counts between any two groups or among the six groups. DISCUSSION AND CONCLUSIONS
The chief conclusion to be drawn from these observations is the fact that, contrary to the common belief, the addition of terramycin to chick rations does not "sterilize" the intestinal tract or even significantly reduce the numbers of intestinal microorganisms that can be detected by the cultural methods that were used in this investigation. Even by increasing the concentration of terramycin to 16 times the amount recommended for commercial poultry rations, the relatively large amount of the antibiotic did not significantly depress the numbers of the several physiological groups of organisms assayed. In fact, at the higher concentrations of terramycin higher counts were obtained for both enterococci and yeasts than were found at lower concentrations, the enterococcus counts being significantly higher. Only in the case of the lactic acidproducing bacteria, in only one of the trials (trial 2), was there a significant reduction in numbers, the counts for the
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significantly greater counts than did the control group; indeed, the control group fed no terramycin had the lowest counts! The L.S.D. also showed that groups 2 and 3 had significantly lower counts than did either group 5 or 6, and group 4 had a significantly lower count than group 6. (f) Potato-dextrose agar: there was no significant difference between the groups in the numbers of yeasts. Although the L.S.D. between each experimental ration and the control showed no real differences, group 5 had a significantly greater count than group 2. Trial 2. (a) Thioglycollate broth: in this series no significant difference was observed between treatments, and none of the comparisons between the average counts of any two groups exceeded the value of the L.S.D. As in the preceding trial, although terramycin was fed at concentrations up to 16 times the level recommended in chick starter rations, these high levels did not depress the total count of the intestinal microflora. (b) Aerobic agar: the counts for aerobic organisms were not significantly different among the groups of chicks both between and within groups; nor was there any real difference between the average counts of any two of the six groups. In both trials, the groups fed terramycin did not show significantly different counts from that of the control when measured by the L.S.D. (c) Carrot-liver agar: as in trial 1, the F value measuring the magnitude of the variation in counts among the six groups was not significant. However, group 6 had a significantly lower count than either groups 1 and 2. This is a marked reversal from trial 1, where group 6 showed the highest number of "lactic acid-producers." (d) Eosin-methylene blue agar: contrary to the findings of trial 1, there was
TERRAMYCIN AND INTESTINAL MICROFLORA
tected by thioglycollate broth, tryptoseglucose-yeast extract agar, carrot-liver agar, S-F broth, Difco's modification of eosin-methylene blue agar, and potatoglucose agar. Perhaps among the microorganisms that are not detected by these techniques are "harmful" microorganisms which are susceptible to the action of terramycin and are so affected that the relieved chick shows an increase in its size and improvement in the utilization of its foodstuff. It is evident to the bacteriologist that much more fundamental work in establishing the identity of all the microorganisms found in the "normal flora" of the intestinal tracts of chicks must be done before anyone can say which organisms, if any, are so affected by antibiotics incorporated in rations that the reduction or elimination of these organisms results in increased growth of the chicks. Unpublished data suggest that the physiological response of chicks to BiCon TM-5 may depend upon the completeness of the chicks' diet and upon the quality of the ingredients incorporated in it. This evidence, combined with the impressions gained from an appreciation of the persistence of great numbers of microorganisms in the intestinal tract even in the presence of high concentrations of terramycin, leads inevitably to the rash but pertinent question whether, in the intestinal tract of chicks, terramycin acts primarily as an antibiotic or benefits the chick in some other way. Biely and March (1951) have shown that dietary levels of nicotinic acid, folic acid, or riboflavin that are suboptimal for maximum growth rate of the chick under normal conditions may be adequate when aureomycin is fed. SUMMARY
Terramycin, introduced into the diet
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group fed 960 grams of Bi-Con TM-5 per 100 pounds of feed being significantly affected. However, the counts for "lactic acid-producing bacteria" made on the groups fed from 60 to 480 grams of BiCon TM-5 per 100 pounds of feed were not reduced significantly in either trial. Counts for the different groups of organisms did exhibit variations from a neatly mannered curve and from a precise repetition between trials; but inspection of the data shows that counts made on feces from the chicks in the control groups varied in the same way. The usual explanations can be offered to account for some of the variations, but no criticism of techniques can explain away the persisting high counts during the 4 weeks of exposure to comparatively high concentrations of terramycin for all of the groups of microorganisms which were studied; Moreover, the addition of Bi-Con TM-5 to the rations did stimulate growth rate and efficiency of feed utilization of the experimental groups of chicks, indicating thereby that these observations were not confounded by a lack of the so-called "antibiotic supplement" effect. How, then, does terramycin present in Bi-Con TM-5 exert its beneficial effect upon the growing chick? Obviously these investigations have not found the answer. Perhaps the techniques employed were far from subtle enough to observe what effect terramycin would have upon many groups of organisms which might exist in the intestinal tracts of chicks and which could not be detected by the media under the conditions in which they were used. It can be concluded, however, that terramycin in the concentrations employed does not significantly reduce the numbers and kinds of microorganisms— which surely must form an important part, if not the major part, of the intestinal microflora of chicks—that are de-
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POULTRY SCIENCE TEACHING AND EXTENSION PRIZE AWARDS
REFERENCES Biely, J., and B. March, 1951. The effect of aureomycin and vitamins on the growth rate of chicks. Science 114: 330-331. Carpenter, L. E., 1951. The effect of antibiotics and vitamin B12 on the growth of swine. Arch. Biochem. Biophysics 32: 187.
Groschke, A. C , 1950. Relation of growth stimulating properties of antibiotics to unidentified growth factors required by the chick. Poultry Sci. 29: 760. Groschke, A. C , and R. J. Evans, 1950. Effect of antibiotics, synthetic vitamins, vitamin Bi 2 , and an APF supplement on chick growth. Poultry Sci. 29: 616-619. Johansson, K. R., W. B. Sarles and S. K. Shapiro, 1948. The intestinal microflora of hens as influenced by various carbohydrates in a biotindeficient ration. J. Bact. 56: 619-634. Matterson, L. A., and E. P. Singsen, 1951. A comparison of several antibiotics as growth stimulants in practical chick-starting rations. Storrs Agr. Exp. Sta. Bull. 275: 1-20. Moore, P. R., A. Evenson, T. D. Luckey, E. McCoy, C. A. Elvehjem and E. B. Hart, 1946. Use of sulfasuxidine, streptothricin and streptomycin in nutritional studies with the chick. J. Biol. Chem. 165:437-441. Sieburth, J. McN., J. Gutierrez, J. McGinnis, J. R. Stern and B. H. Schneider, 1951. Effect of antibiotics on intestinal microflora and on growth of turkeys and pigs. Proc. Soc. Exp. Biol. Med. 76: 15-18. Smith, D. G., and H. J. Robinson, 1945. The influence of streptomycin and streptothricin on the intestinal flora of mice. J. Bact. 50: 613-621. Stokstad, E. L. R., T. H. Jukes, J. Pierce, A. C. Page and A. L. Franklin, 1949. The multiple nature of the animal protein factor. J. Biol. Chem. 180: 647-654.
POULTRY SCIENCE TEACHING AND EXTENSION PRIZE AWARDS {Continued from page 707) All information shall be forwarded to the Chairman of the Award Committee not later than 60 days prior to the annual meeting. In making the award, the following points shall be considered and in addition such other information as may have a bearing on the work accomplished: (a) Organization—This shall include a digest of the plan of work. Emphasis shall be placed on cooperation with other subject matter specialists in allied fields, cooperation, utilization and development of leadership in the poultry industry organization and in the development and application of new subject matter. (b) Economic need—The economic application
of the subject matter to the poultry industry and the timeliness of the subject shall be considered. (c) Results—Results shall be measured in terms of approval of those who are associated with the nominee and in terms of the improved practices that have been adopted. Consideration shall be given as to the durability and the test of time achieved by the program. The extent to which these methods, programs and procedures have been adopted by other states shall also be considered. (d) Appeal—Consideration shall be given to the originality and appeal of the methods in use and the extent to which they have stimulated interest in the project and problem.
(Continued on page 722)
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in the form of Bi-Con TM-5, was fed at five progressively doubled concentrations, in two trials, to determine its effect on the intestinal microflora of chicks. Counts of organisms present in natural feces were made using the following media: thioglycollate broth, tryptose-glucose-yeast extract agar, corrot-liver agar, S-F broth, Difco's modification of eosin-methylene blue agar, and potato-glucose agar. Although terramycin significantly stimulated the growth rate of chicks during the two 28-day studies and increased the efficiency of feed utilization, the microflora detectable by the techniques used was not eliminated or even significantly reduced in number, even when 16 times the recommended amount of terramycin was fed. In both trials the enterococci counts were significantly greater among the groups receiving the higher concentrations of terramycin.