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Effect of the Antibiotic Thiopeptin on Clostridium perfringens and Growth and Feed Efficiency of Broiler Chicks
Effect of the Antibiotic Thiopeptin on Clostridium perfringens and Growth and Feed Efficiency of Broiler Chicks M. W. STUTZ,1 S. L. JOHNSON,2 F. R. JUDITH, and L. A. MUIR Merck Institute for Therapeutic Research, Rahway, New Jersey 07065 (Received for publication August 9, 1982)
1983 Poultry Science 62:1633-1638
INTRODUCTION
Thiopeptin is a sulfur-containing peptide antibiotic produced in the mycelium of Streptomyces tateyamensis. The antibiotic is a mixture of thiopeptin B and small amounts of thiopeptins A (Aj to A 4 ). The components have similar physical, chemical, and antimicrobial properties. Thiopeptins are very effective against certain Gram-positive bacteria and mycoplasma, but not against Gram-negative bacteria and yeasts. Fermentation, extraction, purification, and antimicrobial activity of the thiopeptins have been described by Miyairi et al. (1970, 1972). Muir and Barreto (1979) demonstrated that thiopeptin was active against the Gram-positive organism Streptococcus bonis, an organism implicated in the development of lactic acidosis in ruminants. Although the chemical structures of the thiopeptins are not completely known, on the basis of physical and chemical studies they appear to be similar to thiostrepton, a known chemical structure (Anderson et al, 1970).
1 Diamond Shamrock Corporation, T. R. Evans Research Center, P. O. Box 348, Painesville, OH 44077. 2 Kettering Animal Hospital, Inc., Kettering, OH 45420.
In animal studies, thiopeptin appears to be a safe antibiotic when administered orally. Acute oral doses of 4 and 8 g/kg were safe in the rat and mouse, respectively, and daily oral doses of 2 g/kg for 3 months were without adverse effects in rats (Mine et al., 1972). Mine et al. (1972) also showed that thiopeptin is not absorbed and, as a result, does not produce residues in tissues. No residues were found in any tissue, organ, or biological fluid of chickens and swine. Nearly 100% of the thiopeptin was recovered in the feces of dosed animals. Thiopeptin has been shown to improve the weight gain and feed efficiency of broilers and swine and to increase the egg production of layers (Mine et al., 1972). Thiopeptin has also been shown to be effective in the prevention of induced lactic acidosis in sheep (Muir et al., 1980a,b) and cattle (Muir et al, 1981). The antibiotic is currently marketed in Japan as a feed additive antibiotic for poultry and swine. The objectives of the following experiments were to determine the potential of thiopeptin as a growth permittant for poultry and to continue studies on the mode of action of antimicrobial agents as growth permittants (Muir et al, 1977; Stutz and Judith, 1977). Investigations were conducted to determine in vitro the activity of thiopeptin against the
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ABSTRACT Experiments were conducted to determine the potential of the antibiotic thiopeptin as a growth permittant for poultry. Thiopeptin as the semipurified antibiotic and as fermentation solids demonstrated activity against Clostridium perfringens at .03 ppm based on suppression of gas production in an anaerobic tube test. Supplementing a soybean protein and sucrose-based diet with levels of 2.2, 11, and 55 ppm of thiopeptin from each of the two sources resulted in significant improvements in weight gain and feed efficiency of chicks fed the higher levels. Computed indexes for each antibiotic treatment showed that the antibiotic as fermentation solids was slightly more active than the semipurified material at the lower levels. Supplementing the soybean protein and sucrose-based diet with 1.1, 5.5, 16.5, and 55 ppm thiopeptin reduced significantly (P<.01) the numbers of C. perfringens organisms in ileal contents of chicks. Chicks fed 5.5, 16.5, and 55 ppm thiopeptin also had significantly fewer C. perfringens organisms in the intestine than those fed 1.1 ppm. A discussion is presented of the biological responses of thiopeptin relative to the antibiotics efrotomycin and bacitracin. (Key words: antibiotic, thiopeptin, weight gain, feed efficiency, Clostridium perfringens, chicks)
1634
STUTZ ET AL.
anaerobe Clostridium perfringens, to evaluate the effect of thiopeptin on growth rate and feed efficiency of the chick, and to examine in vivo the effect of the antibiotic on the intestinal population of C. perfringens. MATERIALS AND METHODS
A growth and feed efficiency experiment was conducted, using a soybean protein and sucrose-based diet, to evaluate the activity of the two sources of thiopeptin. The diet and experimental procedures were similar to those previously used and described (Stutz et al., 1983a,b). Day-old male Hubbard x Hubbard broiler chicks were started on a nonmedicated practical diet (Laboratory Broiler Feed, Pennfield Corporation, Lancaster, PA), grouped on a weight basis at 4 days of age, and fed the experimental diets for a 10-day period. Six replicate groups of 8 chicks each were fed the control diet, and three replicate groups were fed the diet supplemented with 2.2, 11, and 55 ppm of thiopeptin from each of the two
The data from the in vitro C. perfringens assays were analyzed statistically by analysis of variance and the least significant difference test (Steel and Torrie, 1960). Weight gains and feed efficiency values from the growth experiment were analyzed by analysis of variance and significant differences among treatments were determined by Duncan's new multiple range test (Duncan, 1955). The C. perfringens counts in the ileum of chicks from the second chick experiment were analyzed for significance using the Mann-Whitney U test (Campbell, 1974).
RESULTS
Table 1 shows results of the in vitro activity of thiopeptin from the two sources against C. perfringens. Based on the measurement of gas production in this anaerobic tube test, both sources of thiopeptin gave a minimum inhibitory concentration of .03 ppm. The semipurified
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 8, 2015
Three titration experiments were conducted with thiopeptin as semipurified (71% active drug) and as fermentation solids (1% active drug) to determine the minimum inhibitory concentrations against Clostridium perfringens (type A, ATCC 3624). An anaerobic tube test based on the measurement of gas production similar to that of Ceballos (1971) was utilized. The experimental procedures for the test were previously described in detail (Stutz et al., 1983a). The assay medium consisted of nutrient broth, brewer's thioglycolate media, yeast extract, dextrose, and ground limestone in distilled water. Nineteen milliliters of medium were dispensed into screw cap tubes which contained smaller inverted tubes. An inoculum was prepared and standardized by diluting with assay medium, without limestone, to give approximately 1.0 to 1.5 x 10 9 bacteria/ml. The test materials were solubilized in dimethylsulfoxide and dilutions were prepared in a final volume of 9.0 ml with sterilized water. Two milliliters of inoculum were added per tube to give a final volume per assay tube of 30 ml. The contents were incubated at 45 C and the gas displacement in the inverted tubes was measured in millimeters after approximately 6 hr or when control tubes had reached approximately 40 mm of gas production. Three control tubes and three tubes per level of test material were used in each experiment.
sources. Chicks were maintained in battery brooders with raised wire floors and subjected to continuous lighting. Feed and water were supplied ad libitum. Index scores were calculated for each thiopeptin treatment based on the average relative percent weight gain and feed efficiency compared to the controls. A second chick experiment was conducted to evaluate the effect of thiopeptin on the intestinal populations of C. perfringens. The experimental procedures were again similar to those described in experiments with the antibiotics efrotomycin and bacitracin (Stutz et al., 1983a,b). The soybean protein and sucrosebased diet was supplemented with levels of 1.1, 5.5, 16.5, and 55 ppm of thiopeptin as the semipurified antibiotic (71% active drug), and the diets were fed for 14 to 17 days. Eight chicks were sacrificed on each of the consecutive 4 days and samples of approximately .5 g of contents from the ileal section of the small intestine were collected. Serial dilutions of the intestinal contents were made in phosphate buffered saline with sodium thioglycolate added as a reducing agent. The organisms were enumerated using the TSN (tryptose-sulfiteneomycin) agar plate method of Marshall et al. (1965). Spread agar plates with TSN agar overlays were incubated anaerobically at 46 C for approximately 24 hr using the Gas Pak system (BBL). During all procedures, samples were treated in a manner to minimize exposure to aerobic conditions.
THIOPEPTIN ON CLOSTRIDIA AND BROILERS
1635
TABLE 2. Effect of level and source of thiopeptin on weight gain and feed efficiency of broiler chicks (Experiment 4) Treatment
Level
Chicks3
(ppm)
(no.)
Weight gain4
Index s
(relative %)
8 15 30
(g/g) 1.81 a 1.71*b 1.68ab 1.48 c
- 7 -18
7 11 24
14 20 32
1.69 ab 1.58 b c 1.55 b c
- 6 -13 -14
10 16 23
48
(g) 110*
(relative %)
0
Thiopeptin 1 Thiopeptin Thiopeptin
2.2 11.0 55.0
24 24 24
118ab 126 b 142cd
Thiopeptin 2 Thiopeptin Thiopeptin
2.2 11.0 55.0
23 24 24
125b 13lbc 145 d
None
Feed/gain
-
5
' ' ' Values within a column with different superscript letters are significantly different (P<.05). Semipurified antibiotic corrected for purity of 71%. 2 Fermentation solids corrected for purity of 1%. 3 Started with 8 chicks per pen. Six replicates of controls and three replicates of each level of antibiotic treat1
' Average initial weight of 68 g at 4 days of age. Final weights at 14 days of age. 5 Index = [(relative % weight gain) — (relative % feed/gain)] 12.
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TABLE 1. Effect of level and source of thiopeptin The effect of level and source of thiopeptin on in vitro activity against Clostridium perfringens, on weight gain and feed efficiency of broiler ATCC 3624 (Experiments 1, 2, and 3) chicks is shown in Table 2. The same materials were evaluated in this experiment that were Average gas assayed against C. perfringens and were likewise Treatment Concentration Tubes production corrected for their respective purities. Similar response trends in both weight gain and feed (ppm) (no.) (mm) efficiency were obtained for both sources of 0 None 39.5 a 9 the antibiotic. Levels of 11 and 55 ppm of both 1 0d Thiopeptin 1.0 3 sources of thiopeptin resulted in significant Thiopeptin .1 3 0d responses in both weight gain and feed effiThiopeptin .04 3 0d ciency. At 2.2 ppm, thiopeptin as fermentation Thiopeptin .03 3 0d Thiopeptin .02 6 6.5C solids significantly improved weight gain and a Thiopeptin .01 6 40.4 thiopeptin as the semipurified antibiotic 0d Thiopeptin 2 1.0 3 improved both parameters, but the responses Thiopeptin .1 3 0d were not statistically significant. Computed Thiopeptin .04 3 0d indexes for each antibiotic treatment, which Thiopeptin .03 3 0d represent the combined effects of both weight Thiopeptin .02 3 5.8 C Thiopeptin .01 3 31.3b gain and feed efficiency, indicate that thiopeptin in the fermentation solids was slightly more ' ' ' Values with different superscript letters are active than the semipurified antibiotic, especially significantly different (P<.05). at the lower levels. 1 Semipurified antibiotic corrected for purity of The effect of level of thiopeptin on C. 71%. perfringens counts from the small intestine of 2 Fermentation splids corrected for purity of 1%. broiler chicks is presented in Table 3. Levels of 1.1, 5.5, 16.5, and 55 ppm of thiopeptin as the semipurified antibiotic resulted in significant antibiotic was corrected for a purity of 71% reductions in the numbers of the organism in and the fermentation solids were corrected for ileal contents when compared to controls (all P<.01). Chicks fed 5.5, 16.5, and 55 ppm of a purity of 1%.
STUTZ ET AL.
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TABLE 3. Effect of level of thiopeptin on Clostridium perfringens counts from the small intestine of broiler chicks (Experiment 5) Treatment
None Thiopeptin Thiopeptin Thiopeptin Thiopeptin
Level
Chicks2
(ppm)
(r (no.) 8
0 1
1.1 5.5 16.5 55.0
Numbers in ileal contents •dog.o/g) 6.5 a b
5.0 2.9 C 2.9 C 2.5 e
6 6 6 6
(5.5--8.0) 3 (4.1- -5.6) (2.1- -3.4) (1.9--4.6) (2.1- -3.0)
Semipurified antibiotic corrected for purity of 71%.
2
Sacrificed and samples obtained at 14 to 17 days on diets.
3
Values in parentheses represent the ranges. Detection limits were 40 and 10' organisms/g.
thiopeptin also had significantly fewer C. perfringens organisms in the intestine than those fed 1.1 ppm (P<.01). The detection limits in this experiment were 40 and 10 9 organisms per gram of contents. DISCUSSION Thiopeptin demonstrated excellent activity against C. perfringens at .03 ppm based on suppression of gas production in the anaerobic tube test. The antibiotics, bacitracin and penicillin, and the antibacterial agent, nitrovin, gave minimum inhibitory concentrations of .5, .1, and .5 ppm, respectively (Stutz and Johnson, 1976), and a new antibiotic, efrotomycin, gave a minimum inhibitory concentration of .1 to .2 ppm in the same assay (Stutz etai, 1983a). Smith (1972) observed that bacitracin, virginiamycin, and nitrovin were very active against seven strains of C. perfringens. Hennessey and Edwards (1972) reported similar findings with quindoxin, nitrovin, penicillin, and bacitracin. Jeffries et al. (1977) reported the in vitro results of 17 antibiotics and synthetic antibacterial agents against 20 organisms. Eleven of the compounds including bacitracin, penicillin, virginiamycin, and nitrovin were active against C. perfringens below 1 ppm. The results presented from our studies confirm the reports of others that C. perfringens in especially sensitive to the commonly used feed additive antimicrobial agents under anaerobic conditions. Our results also show that other more recently discovered antibiotics, thiopeptin and efrotomycin, are very active against this organism.
Moore (1969) stated that the oxidationreduction potential of the gastrointestinal tract of man and animals is very low and contributes to the extremely anaerobic environment. Because C. perfringens is an anaerobe that has been shown to require a low oxidation-reduction potential for growth (Bryan, 1970) similar to that present in the intestine, in vitro procedures should be utilized that attempt to simulate those conditions. Thiopeptin improved the weight gain and feed efficiency of broiler chicks fed the soybean protein and sucrose-based diet. Increased responses were obtained in chicks fed this diet supplemented with the antibiotics efrotomycin (Stutz et al., 1983a) and bacitracin (Stutz et al., 1983b). Efrotomycin in those studies, however, appeared to be somewhat more active than thiopeptin with a maximum response near the 11 ppm level. In a dose-response experiment with bacitracin, a regression analysis of the index scores on the log of the significant dose levels gave a linear response line. Based on this equation, the ineffective level of bacitracin was determined to be 4 ppm and the maximum effective level was 31 ppm. The results of the index scores for thiopeptin presented in this paper show that the antibiotic is slightly more active than bacitracin. Our results with thiopeptin confirm the reports of Stokstad et al. (1953) and Eyssen and De Somer (1963) that the carbohydrate sucrose in a semipurified diet has a pronounced effect on the growth response of chicks to antibiotics. More recent reports show that the poorly utilized carbohydrates, pectin, lactose, and xylitol, also reduce the
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' ' Values with different superscript letters are significantly different (P<.01). 1
THIOPEPTIN ON CLOSTRIDIA AND BROILERS
growth of chicks and rats as a result of bacterial fermentation in the intestine (Wagner and Thomas, 1977; Kim et al, 1978; Wekell et al, 1980). Wagner and Thomas (1977) and Day and Thomas (1980) showed that the depressions in growth of chicks by rye, pectin, and rye extracts of pectic substances could be partially overcome by penicillin supplementation. Stokstad et al. (1953) demonstrated a similar effect with chicks fed lactose as the carbohydrate and supplemented with chlortetracycline.
A committee report published by the National Academy of Sciences (National Research Council, 1980) suggested that studies should be conducted in which short-term performance measurements are correlated with similar short-term measurements related to the microbiology and biochemistry of control animals and treated animals responding to antibiotics. The committee further stated that experiments of this type might uncover mechanisms related to the action of antibiotics in growth promotion. The objectives of our studies have been to utilize this approach to elucidate further the mode of action of antimicrobial agents as growth permittants.
ACKNOWLEDGMENTS
The authors with to thank Bridget Butler for excellent technical assistance. REFERENCES Anderson, B., D. C. Hodgkin, and M. A. Viswamitra, 1970. The structure of thiostrepton. Nature 225:233-235. Bryan, F. L., 1970. What the sanitarian should know about Clostridium perfringens foodborne illness. J. Milk Food Technol. 32.381-389. Campbell, R. C , 1974. Statistics for Biologists. Cambridge Univ. Press, London. Ceballos, E., 1971. Effect of beans, other food products and inorganic residues from foods on in vitro gas production by Clostridium perfringens and the influence of basal medium composition on the response to supplements of foods and ash residues. Ph.D. diss., Washington State Univ., Pullman, WA. Day, R. M., and O. P. Thomas, 1980. Growth depression of chicks fed a crude rye extract containing pectic substances. Poultry Sci. 59:2754-2759. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics 11:1—42. Eyssen, H., and P. De Somer, 1963. Effect of antibiotics on growth and nutrient absorption of chicks. Poultry Sci. 42:1373-1379. Hennessey, T. D., and J. R. Edwards, 1972. Antibacterial properties of quindoxin: a new growthpromoting agent. Vet. Rec. 90:187—191. Jeffries, L., K. Coleman, and J. Bunyan, 1977. Antimicrobial substances and chick growth promotion: Comparative studies on selected compounds in vitro and in vivo. Br. Poult. Sci. 18:295—308. Kim, K„ N. J. Benevenga, and R. H. Grummer, 1978. Estimation of the fraction of the lactose in a high lactose diet available for fermentation in the cecum and colon of the rat. J. Nutr. 108:79—89. Marshall, R. S., J. F. Steenbergen, and L. S. McClung, 1965. Rapid technique for the enumeration of Clostridium perfringens. Appl. Microbiol. 13: 559-563. Mine, K., N. Miyairi, N. Takano, S. Mori, and N. Watanabe, 1972. Thiopeptin, a new feed-additive antibiotic: Biological studies and field trials. Antimicrob. Agents Chemother. 1:496—503. Miyairi, N., T. Miyoshi, H. Aoki, M. Kohsaka, H. Ikushima, K. Kunugita, H. Sakai, and H. Imanaka, 1970. Studies on thiopeptin antibiotics I. Characteristics of thiopeptin B. J. Antibiot. 23:113 — 119. Miyairi, N., T. Miyoshi, H. Aoki, M. Kohsaka, H. Ikushima, K. Kunugita, H. Sakai, and H. Imanaka, 1972. Thiopeptin, a new feed additive antibiotic: Microbiological and chemical studies. Antimicrob. Agents Chemother. 1:192—196. Moore, W.E.C., 1969. Current research on the anaerobic flora of the gastrointestinal tract. Pages 107—113 in A Symposium: The Use of Drugs in Animal Feeds. Natl. Acad. Sci., Washington, DC. Muir, L. A., and A. Barreto, 1979. Sensitivity of Streptococcus bovis to various antibiotics. J. Anim. Sci. 4 8 : 4 6 8 - 4 7 3 . Muir, L. A., P. F. Duquette, E. L. Rickes, and G. E.
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Thiopeptin had a significant effect on C. perfringens counts from the small intestine of broiler chicks fed the soybean protein and sucrose-based diet. The antibiotic reduced the population of C. perfringens in the ileum of chicks as did the antibiotics efrotomycin (Stutz et al, 1983a) and bacitracin (Stutz et al, 1983b). Efrotomycin, however, reduced the population of the organism to a greater extent and increased the weight gain of chicks at lower levels than observed for both thiopeptin and bacitracin. Thiopeptin appeared to be slightly more active than bacitracin for both criteria. In terms of in vitro activity, thiopeptin was inhibitory to C. perfringens at a lower level than either efrotomycin or bacitracin. The results of the three antibiotics show that the reductions in the population of the organism in the ileum of the chick correlate more closely with the improvements in weight gain and feed efficiency than with the in vitro activities. Jeffries et al. (1977) concluded that the antibacterial activity of selected compounds in vitro did not correlate with their ability to promote growth when added to the diet of chicks.
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STUTZ ET AL. 1953. The growth-promoting effect of Aureomycin on various types of diets. Poultry Sci. 32:1054-1058. Stutz, M. W., and S. L. Johnson, 1976. In vitro and in vivo evaluations of antimicrobial agents as potential growth permittants. Poultry Sci. 55:2098. (Abstr.) Stutz, M. W., S. L. Johnson, and F. R. Judith, 1983b. Effects of diet and bacitracin on growth, feed efficiency, and populations of Clostridium perfringens in the intestine of broiler chicks. Poultry Sci. 62:1619-1625. Stutz, M. W., S. L. Johnson, F. R. Judith, and B. M. Miller, 1983a. In vitro and in vivo evaluations of the antibiotic efrotomycin. Poultry Sci. 62: 1612-1618. Stutz, M. W., and F. R. Judith, 1977. Studies on the mode of action of antimicrobials as growth permittants in the chick. Pages 12—16 in Proc. Carolina Poult. Nutr. Conf., Charlotte, NC. Wagner, D. D., and O. P. Thomas, 1977. A rye type growth depression of chicks fed pectin. Poultry Sci. 56:615-619. Wekell, M. M., W. J. Hartman, and F. M. Dong, 1980. Incidence of increased numbers of Clostridium perfringens in the intestinal tract of rats fed xylitol. J. Nutr. 110:2103-2108.
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Smith, 1980a. Thiopeptin for the prevention of ovine lactic acidosis induced by diet change. J. Anim. Sci. 51:1182-1188. Muir, L. A., E. L. Rickes, P. F. Duquette, and G. E. Smith, 1980b. Control of wheat-induced lactic acidosis in sheep by thiopeptin and related antibiotics. J. Anim. Sci. 50:547-553. Muir, L. A., E. R. Rickes, P. F. Duquette, and G. E. Smith, 1981. Prevention of induced lactic acidosis in cattle by thiopeptin. J. Anim. Sci 52:635—643. Muir, L. A., M. W. Stutz, and G. E. Smith, 1977. Feed additives. Pages 27—37 in Livestock Feeds and Feeding. D. C. Church, ed. O & B Books, Corvallis, OR. National Research Council, 1980. Antibiotics in animal feeds-void in knowledge and suggested research. Pages 368—371 in A Report: The Effects on Human Health of Subtherapeutic Use of Antimicrobials in Animal Feeds. Natl. Acad. Sci., Washington, DC. Smith, H. W., 1972. The antibacterial activity of nitrovin in vitro: the effect of this and other agents against Clostridium welcbii in the alimentary tract of chickens. Vet. Rec. 90:310—312. Steel, R.G.D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill, New York, NY. Stockstad, E.L.R., T. H. Jukes, and W. L. Williams,
1983 Poultry Science 62:1633-1638
INTRODUCTION
Thiopeptin is a sulfur-containing peptide antibiotic produced in the mycelium of Streptomyces tateyamensis. The antibiotic is a mixture of thiopeptin B and small amounts of thiopeptins A (Aj to A 4 ). The components have similar physical, chemical, and antimicrobial properties. Thiopeptins are very effective against certain Gram-positive bacteria and mycoplasma, but not against Gram-negative bacteria and yeasts. Fermentation, extraction, purification, and antimicrobial activity of the thiopeptins have been described by Miyairi et al. (1970, 1972). Muir and Barreto (1979) demonstrated that thiopeptin was active against the Gram-positive organism Streptococcus bonis, an organism implicated in the development of lactic acidosis in ruminants. Although the chemical structures of the thiopeptins are not completely known, on the basis of physical and chemical studies they appear to be similar to thiostrepton, a known chemical structure (Anderson et al, 1970).
1 Diamond Shamrock Corporation, T. R. Evans Research Center, P. O. Box 348, Painesville, OH 44077. 2 Kettering Animal Hospital, Inc., Kettering, OH 45420.
In animal studies, thiopeptin appears to be a safe antibiotic when administered orally. Acute oral doses of 4 and 8 g/kg were safe in the rat and mouse, respectively, and daily oral doses of 2 g/kg for 3 months were without adverse effects in rats (Mine et al., 1972). Mine et al. (1972) also showed that thiopeptin is not absorbed and, as a result, does not produce residues in tissues. No residues were found in any tissue, organ, or biological fluid of chickens and swine. Nearly 100% of the thiopeptin was recovered in the feces of dosed animals. Thiopeptin has been shown to improve the weight gain and feed efficiency of broilers and swine and to increase the egg production of layers (Mine et al., 1972). Thiopeptin has also been shown to be effective in the prevention of induced lactic acidosis in sheep (Muir et al., 1980a,b) and cattle (Muir et al, 1981). The antibiotic is currently marketed in Japan as a feed additive antibiotic for poultry and swine. The objectives of the following experiments were to determine the potential of thiopeptin as a growth permittant for poultry and to continue studies on the mode of action of antimicrobial agents as growth permittants (Muir et al, 1977; Stutz and Judith, 1977). Investigations were conducted to determine in vitro the activity of thiopeptin against the
1633
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 8, 2015
ABSTRACT Experiments were conducted to determine the potential of the antibiotic thiopeptin as a growth permittant for poultry. Thiopeptin as the semipurified antibiotic and as fermentation solids demonstrated activity against Clostridium perfringens at .03 ppm based on suppression of gas production in an anaerobic tube test. Supplementing a soybean protein and sucrose-based diet with levels of 2.2, 11, and 55 ppm of thiopeptin from each of the two sources resulted in significant improvements in weight gain and feed efficiency of chicks fed the higher levels. Computed indexes for each antibiotic treatment showed that the antibiotic as fermentation solids was slightly more active than the semipurified material at the lower levels. Supplementing the soybean protein and sucrose-based diet with 1.1, 5.5, 16.5, and 55 ppm thiopeptin reduced significantly (P<.01) the numbers of C. perfringens organisms in ileal contents of chicks. Chicks fed 5.5, 16.5, and 55 ppm thiopeptin also had significantly fewer C. perfringens organisms in the intestine than those fed 1.1 ppm. A discussion is presented of the biological responses of thiopeptin relative to the antibiotics efrotomycin and bacitracin. (Key words: antibiotic, thiopeptin, weight gain, feed efficiency, Clostridium perfringens, chicks)
1634
STUTZ ET AL.
anaerobe Clostridium perfringens, to evaluate the effect of thiopeptin on growth rate and feed efficiency of the chick, and to examine in vivo the effect of the antibiotic on the intestinal population of C. perfringens. MATERIALS AND METHODS
A growth and feed efficiency experiment was conducted, using a soybean protein and sucrose-based diet, to evaluate the activity of the two sources of thiopeptin. The diet and experimental procedures were similar to those previously used and described (Stutz et al., 1983a,b). Day-old male Hubbard x Hubbard broiler chicks were started on a nonmedicated practical diet (Laboratory Broiler Feed, Pennfield Corporation, Lancaster, PA), grouped on a weight basis at 4 days of age, and fed the experimental diets for a 10-day period. Six replicate groups of 8 chicks each were fed the control diet, and three replicate groups were fed the diet supplemented with 2.2, 11, and 55 ppm of thiopeptin from each of the two
The data from the in vitro C. perfringens assays were analyzed statistically by analysis of variance and the least significant difference test (Steel and Torrie, 1960). Weight gains and feed efficiency values from the growth experiment were analyzed by analysis of variance and significant differences among treatments were determined by Duncan's new multiple range test (Duncan, 1955). The C. perfringens counts in the ileum of chicks from the second chick experiment were analyzed for significance using the Mann-Whitney U test (Campbell, 1974).
RESULTS
Table 1 shows results of the in vitro activity of thiopeptin from the two sources against C. perfringens. Based on the measurement of gas production in this anaerobic tube test, both sources of thiopeptin gave a minimum inhibitory concentration of .03 ppm. The semipurified
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 8, 2015
Three titration experiments were conducted with thiopeptin as semipurified (71% active drug) and as fermentation solids (1% active drug) to determine the minimum inhibitory concentrations against Clostridium perfringens (type A, ATCC 3624). An anaerobic tube test based on the measurement of gas production similar to that of Ceballos (1971) was utilized. The experimental procedures for the test were previously described in detail (Stutz et al., 1983a). The assay medium consisted of nutrient broth, brewer's thioglycolate media, yeast extract, dextrose, and ground limestone in distilled water. Nineteen milliliters of medium were dispensed into screw cap tubes which contained smaller inverted tubes. An inoculum was prepared and standardized by diluting with assay medium, without limestone, to give approximately 1.0 to 1.5 x 10 9 bacteria/ml. The test materials were solubilized in dimethylsulfoxide and dilutions were prepared in a final volume of 9.0 ml with sterilized water. Two milliliters of inoculum were added per tube to give a final volume per assay tube of 30 ml. The contents were incubated at 45 C and the gas displacement in the inverted tubes was measured in millimeters after approximately 6 hr or when control tubes had reached approximately 40 mm of gas production. Three control tubes and three tubes per level of test material were used in each experiment.
sources. Chicks were maintained in battery brooders with raised wire floors and subjected to continuous lighting. Feed and water were supplied ad libitum. Index scores were calculated for each thiopeptin treatment based on the average relative percent weight gain and feed efficiency compared to the controls. A second chick experiment was conducted to evaluate the effect of thiopeptin on the intestinal populations of C. perfringens. The experimental procedures were again similar to those described in experiments with the antibiotics efrotomycin and bacitracin (Stutz et al., 1983a,b). The soybean protein and sucrosebased diet was supplemented with levels of 1.1, 5.5, 16.5, and 55 ppm of thiopeptin as the semipurified antibiotic (71% active drug), and the diets were fed for 14 to 17 days. Eight chicks were sacrificed on each of the consecutive 4 days and samples of approximately .5 g of contents from the ileal section of the small intestine were collected. Serial dilutions of the intestinal contents were made in phosphate buffered saline with sodium thioglycolate added as a reducing agent. The organisms were enumerated using the TSN (tryptose-sulfiteneomycin) agar plate method of Marshall et al. (1965). Spread agar plates with TSN agar overlays were incubated anaerobically at 46 C for approximately 24 hr using the Gas Pak system (BBL). During all procedures, samples were treated in a manner to minimize exposure to aerobic conditions.
THIOPEPTIN ON CLOSTRIDIA AND BROILERS
1635
TABLE 2. Effect of level and source of thiopeptin on weight gain and feed efficiency of broiler chicks (Experiment 4) Treatment
Level
Chicks3
(ppm)
(no.)
Weight gain4
Index s
(relative %)
8 15 30
(g/g) 1.81 a 1.71*b 1.68ab 1.48 c
- 7 -18
7 11 24
14 20 32
1.69 ab 1.58 b c 1.55 b c
- 6 -13 -14
10 16 23
48
(g) 110*
(relative %)
0
Thiopeptin 1 Thiopeptin Thiopeptin
2.2 11.0 55.0
24 24 24
118ab 126 b 142cd
Thiopeptin 2 Thiopeptin Thiopeptin
2.2 11.0 55.0
23 24 24
125b 13lbc 145 d
None
Feed/gain
-
5
' ' ' Values within a column with different superscript letters are significantly different (P<.05). Semipurified antibiotic corrected for purity of 71%. 2 Fermentation solids corrected for purity of 1%. 3 Started with 8 chicks per pen. Six replicates of controls and three replicates of each level of antibiotic treat1
' Average initial weight of 68 g at 4 days of age. Final weights at 14 days of age. 5 Index = [(relative % weight gain) — (relative % feed/gain)] 12.
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TABLE 1. Effect of level and source of thiopeptin The effect of level and source of thiopeptin on in vitro activity against Clostridium perfringens, on weight gain and feed efficiency of broiler ATCC 3624 (Experiments 1, 2, and 3) chicks is shown in Table 2. The same materials were evaluated in this experiment that were Average gas assayed against C. perfringens and were likewise Treatment Concentration Tubes production corrected for their respective purities. Similar response trends in both weight gain and feed (ppm) (no.) (mm) efficiency were obtained for both sources of 0 None 39.5 a 9 the antibiotic. Levels of 11 and 55 ppm of both 1 0d Thiopeptin 1.0 3 sources of thiopeptin resulted in significant Thiopeptin .1 3 0d responses in both weight gain and feed effiThiopeptin .04 3 0d ciency. At 2.2 ppm, thiopeptin as fermentation Thiopeptin .03 3 0d Thiopeptin .02 6 6.5C solids significantly improved weight gain and a Thiopeptin .01 6 40.4 thiopeptin as the semipurified antibiotic 0d Thiopeptin 2 1.0 3 improved both parameters, but the responses Thiopeptin .1 3 0d were not statistically significant. Computed Thiopeptin .04 3 0d indexes for each antibiotic treatment, which Thiopeptin .03 3 0d represent the combined effects of both weight Thiopeptin .02 3 5.8 C Thiopeptin .01 3 31.3b gain and feed efficiency, indicate that thiopeptin in the fermentation solids was slightly more ' ' ' Values with different superscript letters are active than the semipurified antibiotic, especially significantly different (P<.05). at the lower levels. 1 Semipurified antibiotic corrected for purity of The effect of level of thiopeptin on C. 71%. perfringens counts from the small intestine of 2 Fermentation splids corrected for purity of 1%. broiler chicks is presented in Table 3. Levels of 1.1, 5.5, 16.5, and 55 ppm of thiopeptin as the semipurified antibiotic resulted in significant antibiotic was corrected for a purity of 71% reductions in the numbers of the organism in and the fermentation solids were corrected for ileal contents when compared to controls (all P<.01). Chicks fed 5.5, 16.5, and 55 ppm of a purity of 1%.
STUTZ ET AL.
1636
TABLE 3. Effect of level of thiopeptin on Clostridium perfringens counts from the small intestine of broiler chicks (Experiment 5) Treatment
None Thiopeptin Thiopeptin Thiopeptin Thiopeptin
Level
Chicks2
(ppm)
(r (no.) 8
0 1
1.1 5.5 16.5 55.0
Numbers in ileal contents •dog.o/g) 6.5 a b
5.0 2.9 C 2.9 C 2.5 e
6 6 6 6
(5.5--8.0) 3 (4.1- -5.6) (2.1- -3.4) (1.9--4.6) (2.1- -3.0)
Semipurified antibiotic corrected for purity of 71%.
2
Sacrificed and samples obtained at 14 to 17 days on diets.
3
Values in parentheses represent the ranges. Detection limits were 40 and 10' organisms/g.
thiopeptin also had significantly fewer C. perfringens organisms in the intestine than those fed 1.1 ppm (P<.01). The detection limits in this experiment were 40 and 10 9 organisms per gram of contents. DISCUSSION Thiopeptin demonstrated excellent activity against C. perfringens at .03 ppm based on suppression of gas production in the anaerobic tube test. The antibiotics, bacitracin and penicillin, and the antibacterial agent, nitrovin, gave minimum inhibitory concentrations of .5, .1, and .5 ppm, respectively (Stutz and Johnson, 1976), and a new antibiotic, efrotomycin, gave a minimum inhibitory concentration of .1 to .2 ppm in the same assay (Stutz etai, 1983a). Smith (1972) observed that bacitracin, virginiamycin, and nitrovin were very active against seven strains of C. perfringens. Hennessey and Edwards (1972) reported similar findings with quindoxin, nitrovin, penicillin, and bacitracin. Jeffries et al. (1977) reported the in vitro results of 17 antibiotics and synthetic antibacterial agents against 20 organisms. Eleven of the compounds including bacitracin, penicillin, virginiamycin, and nitrovin were active against C. perfringens below 1 ppm. The results presented from our studies confirm the reports of others that C. perfringens in especially sensitive to the commonly used feed additive antimicrobial agents under anaerobic conditions. Our results also show that other more recently discovered antibiotics, thiopeptin and efrotomycin, are very active against this organism.
Moore (1969) stated that the oxidationreduction potential of the gastrointestinal tract of man and animals is very low and contributes to the extremely anaerobic environment. Because C. perfringens is an anaerobe that has been shown to require a low oxidation-reduction potential for growth (Bryan, 1970) similar to that present in the intestine, in vitro procedures should be utilized that attempt to simulate those conditions. Thiopeptin improved the weight gain and feed efficiency of broiler chicks fed the soybean protein and sucrose-based diet. Increased responses were obtained in chicks fed this diet supplemented with the antibiotics efrotomycin (Stutz et al., 1983a) and bacitracin (Stutz et al., 1983b). Efrotomycin in those studies, however, appeared to be somewhat more active than thiopeptin with a maximum response near the 11 ppm level. In a dose-response experiment with bacitracin, a regression analysis of the index scores on the log of the significant dose levels gave a linear response line. Based on this equation, the ineffective level of bacitracin was determined to be 4 ppm and the maximum effective level was 31 ppm. The results of the index scores for thiopeptin presented in this paper show that the antibiotic is slightly more active than bacitracin. Our results with thiopeptin confirm the reports of Stokstad et al. (1953) and Eyssen and De Somer (1963) that the carbohydrate sucrose in a semipurified diet has a pronounced effect on the growth response of chicks to antibiotics. More recent reports show that the poorly utilized carbohydrates, pectin, lactose, and xylitol, also reduce the
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' ' Values with different superscript letters are significantly different (P<.01). 1
THIOPEPTIN ON CLOSTRIDIA AND BROILERS
growth of chicks and rats as a result of bacterial fermentation in the intestine (Wagner and Thomas, 1977; Kim et al, 1978; Wekell et al, 1980). Wagner and Thomas (1977) and Day and Thomas (1980) showed that the depressions in growth of chicks by rye, pectin, and rye extracts of pectic substances could be partially overcome by penicillin supplementation. Stokstad et al. (1953) demonstrated a similar effect with chicks fed lactose as the carbohydrate and supplemented with chlortetracycline.
A committee report published by the National Academy of Sciences (National Research Council, 1980) suggested that studies should be conducted in which short-term performance measurements are correlated with similar short-term measurements related to the microbiology and biochemistry of control animals and treated animals responding to antibiotics. The committee further stated that experiments of this type might uncover mechanisms related to the action of antibiotics in growth promotion. The objectives of our studies have been to utilize this approach to elucidate further the mode of action of antimicrobial agents as growth permittants.
ACKNOWLEDGMENTS
The authors with to thank Bridget Butler for excellent technical assistance. REFERENCES Anderson, B., D. C. Hodgkin, and M. A. Viswamitra, 1970. The structure of thiostrepton. Nature 225:233-235. Bryan, F. L., 1970. What the sanitarian should know about Clostridium perfringens foodborne illness. J. Milk Food Technol. 32.381-389. Campbell, R. C , 1974. Statistics for Biologists. Cambridge Univ. Press, London. Ceballos, E., 1971. Effect of beans, other food products and inorganic residues from foods on in vitro gas production by Clostridium perfringens and the influence of basal medium composition on the response to supplements of foods and ash residues. Ph.D. diss., Washington State Univ., Pullman, WA. Day, R. M., and O. P. Thomas, 1980. Growth depression of chicks fed a crude rye extract containing pectic substances. Poultry Sci. 59:2754-2759. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics 11:1—42. Eyssen, H., and P. De Somer, 1963. Effect of antibiotics on growth and nutrient absorption of chicks. Poultry Sci. 42:1373-1379. Hennessey, T. D., and J. R. Edwards, 1972. Antibacterial properties of quindoxin: a new growthpromoting agent. Vet. Rec. 90:187—191. Jeffries, L., K. Coleman, and J. Bunyan, 1977. Antimicrobial substances and chick growth promotion: Comparative studies on selected compounds in vitro and in vivo. Br. Poult. Sci. 18:295—308. Kim, K„ N. J. Benevenga, and R. H. Grummer, 1978. Estimation of the fraction of the lactose in a high lactose diet available for fermentation in the cecum and colon of the rat. J. Nutr. 108:79—89. Marshall, R. S., J. F. Steenbergen, and L. S. McClung, 1965. Rapid technique for the enumeration of Clostridium perfringens. Appl. Microbiol. 13: 559-563. Mine, K., N. Miyairi, N. Takano, S. Mori, and N. Watanabe, 1972. Thiopeptin, a new feed-additive antibiotic: Biological studies and field trials. Antimicrob. Agents Chemother. 1:496—503. Miyairi, N., T. Miyoshi, H. Aoki, M. Kohsaka, H. Ikushima, K. Kunugita, H. Sakai, and H. Imanaka, 1970. Studies on thiopeptin antibiotics I. Characteristics of thiopeptin B. J. Antibiot. 23:113 — 119. Miyairi, N., T. Miyoshi, H. Aoki, M. Kohsaka, H. Ikushima, K. Kunugita, H. Sakai, and H. Imanaka, 1972. Thiopeptin, a new feed additive antibiotic: Microbiological and chemical studies. Antimicrob. Agents Chemother. 1:192—196. Moore, W.E.C., 1969. Current research on the anaerobic flora of the gastrointestinal tract. Pages 107—113 in A Symposium: The Use of Drugs in Animal Feeds. Natl. Acad. Sci., Washington, DC. Muir, L. A., and A. Barreto, 1979. Sensitivity of Streptococcus bovis to various antibiotics. J. Anim. Sci. 4 8 : 4 6 8 - 4 7 3 . Muir, L. A., P. F. Duquette, E. L. Rickes, and G. E.
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Thiopeptin had a significant effect on C. perfringens counts from the small intestine of broiler chicks fed the soybean protein and sucrose-based diet. The antibiotic reduced the population of C. perfringens in the ileum of chicks as did the antibiotics efrotomycin (Stutz et al, 1983a) and bacitracin (Stutz et al, 1983b). Efrotomycin, however, reduced the population of the organism to a greater extent and increased the weight gain of chicks at lower levels than observed for both thiopeptin and bacitracin. Thiopeptin appeared to be slightly more active than bacitracin for both criteria. In terms of in vitro activity, thiopeptin was inhibitory to C. perfringens at a lower level than either efrotomycin or bacitracin. The results of the three antibiotics show that the reductions in the population of the organism in the ileum of the chick correlate more closely with the improvements in weight gain and feed efficiency than with the in vitro activities. Jeffries et al. (1977) concluded that the antibacterial activity of selected compounds in vitro did not correlate with their ability to promote growth when added to the diet of chicks.
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STUTZ ET AL. 1953. The growth-promoting effect of Aureomycin on various types of diets. Poultry Sci. 32:1054-1058. Stutz, M. W., and S. L. Johnson, 1976. In vitro and in vivo evaluations of antimicrobial agents as potential growth permittants. Poultry Sci. 55:2098. (Abstr.) Stutz, M. W., S. L. Johnson, and F. R. Judith, 1983b. Effects of diet and bacitracin on growth, feed efficiency, and populations of Clostridium perfringens in the intestine of broiler chicks. Poultry Sci. 62:1619-1625. Stutz, M. W., S. L. Johnson, F. R. Judith, and B. M. Miller, 1983a. In vitro and in vivo evaluations of the antibiotic efrotomycin. Poultry Sci. 62: 1612-1618. Stutz, M. W., and F. R. Judith, 1977. Studies on the mode of action of antimicrobials as growth permittants in the chick. Pages 12—16 in Proc. Carolina Poult. Nutr. Conf., Charlotte, NC. Wagner, D. D., and O. P. Thomas, 1977. A rye type growth depression of chicks fed pectin. Poultry Sci. 56:615-619. Wekell, M. M., W. J. Hartman, and F. M. Dong, 1980. Incidence of increased numbers of Clostridium perfringens in the intestinal tract of rats fed xylitol. J. Nutr. 110:2103-2108.
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Smith, 1980a. Thiopeptin for the prevention of ovine lactic acidosis induced by diet change. J. Anim. Sci. 51:1182-1188. Muir, L. A., E. L. Rickes, P. F. Duquette, and G. E. Smith, 1980b. Control of wheat-induced lactic acidosis in sheep by thiopeptin and related antibiotics. J. Anim. Sci. 50:547-553. Muir, L. A., E. R. Rickes, P. F. Duquette, and G. E. Smith, 1981. Prevention of induced lactic acidosis in cattle by thiopeptin. J. Anim. Sci 52:635—643. Muir, L. A., M. W. Stutz, and G. E. Smith, 1977. Feed additives. Pages 27—37 in Livestock Feeds and Feeding. D. C. Church, ed. O & B Books, Corvallis, OR. National Research Council, 1980. Antibiotics in animal feeds-void in knowledge and suggested research. Pages 368—371 in A Report: The Effects on Human Health of Subtherapeutic Use of Antimicrobials in Animal Feeds. Natl. Acad. Sci., Washington, DC. Smith, H. W., 1972. The antibacterial activity of nitrovin in vitro: the effect of this and other agents against Clostridium welcbii in the alimentary tract of chickens. Vet. Rec. 90:310—312. Steel, R.G.D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill, New York, NY. Stockstad, E.L.R., T. H. Jukes, and W. L. Williams,