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Effect of Feeding Escherichia coli to Turkey Poults and Chicks in the Presence of Certain Antibiotics1
728
W. C. LOCKHART, R. L. BRYANT AND D. W. BOLIN
proteins in chick diets. Poultry Sci. 23: 486490. Kratzer, F. H., D. E, Williams and B. Marshall, 1952. The requirement for isoleucine and the activities of its isomers for the growth of turkey poults. J. Nutrition, 47: 631-635. Kratzer, F. H., and N. Green, 1957. The availability of lysine in blood meal for chicks and poults. Poultry Sci. 36: 562-565. Mitchell, H. H., 1924. A method of determining the biological value of protein. J. Biol. Chem. 58: 873-903. Mussehl, F. E., and C. W. Ackerson, 1931. Utilization of proteins by the growing chick. Nebraska Agric. Exp. Sta. Res. Bull. 55. Slinger, S. J., W. F. Pepper and D. C. HiU, 1955. The use of keratin products in poultry rations. Poultry Sci. 34: 919-922.
Snedecor, G. W., 1946. Statistical Methods. 4th Edition, Iowa State College Press. Ames, Iowa. Squibb, R. L., and M. K. Wyld, 1950. Further studies on the value of dismodium meal in the baby chick diet. Poultry Sci. 29: 586-589. Squibb, R. L., and J. E. Braham, 1955. Blood meal as a lysine supplement to all-vegetable protein rations for chicks. Poultry Sci. 34: 1050-1053. Titus, H. W., T. C. Byerly, N. R. Ellis and R. B. Nestler, 1936. Effects of packinghouse by-products in the diet of chickens on the production and hatchability of eggs. J. Agr. Res. 53: 453456. Titus, H. W., 1955. Energy Values of Feedstuffs for Poultry. The Interstate, Danville, Illinois. Titus, H. W., 1957. Energy values of feedstuffs for poultry. Mineo. report, Linestone Products Corporation of America, Lime Crest Res. Lab
Effect of Feeding Escherichia coli to Turkey Poults and Chicks in the Presence of Certain Antibiotics1 W. K. WARDEN 2 AND PHILIP J. SCHAIBLE Department of Poultry Science, Michigan State University, East Lansing (Received for publication September 17,1959)
D
URING the past few years, great importance has been ascribed to shifts in bacterial population of the gut as a major factor in explaining the growthstimulating action of antibiotics. Supporting this theory is the fact that zinc bacitracin, a complex polypeptide molecule, is not absorbed into the blood stream, so any improvement in growth must result from its action in the digestive tract. Tetracycline-type antibiotics, on the other hand, are absorbed into the blood stream to some extent and so could act systemically as well as enterically. Furthermore, "broad-spectrum" antibiotics, such as the tetracycline-type inhibit both gram-posi1 Journal article No. 2495 Michigan Agricultural Experiment Station. Research supported in part by a grant from Commercial Solvents Corporation. 2 Presented by the senior author in partial fulfillment of Ph.D. requirements.
tive and gram-negative organisms; whereas, "narrow-spectrum" antibiotics, such as penicillin and bacitracin, act more specifically against the common, diseaseproducing, gram-positive forms. The bacteriological approach has appealed to many workers since it was demonstrated early (Coates et al, 1951, 1952; Bird et al, 1952; Hill et al, 1953; Jacobs et al, 1953; and Waibel et al, 1954) that antibiotics are more effective in contaminated than in new quarters. Also, the bacterial population of the gut changes significantly when antibiotics are fed at levels as low as one-half gram per ton of feed. Elam et al. (1953) and March and Biely (1952) suggested that growth responses may result from decreasing the incidence of enterotoxemia—primarily by reducing the number of Clostridia and Lactobacillus-type organisms in the gut,
729
E. COLI AND ANTIBIOTICS
Before the advent of antibiotics as specific compounds King (1905) and Emmel (1930) showed that the coliform types {Escherichia coli, Aerobacter aerogenes) constitute about 65 percent of the bacterial population of the intestinal tract of chickens. These findings have been verified more recently by Schumacher and Heuser (1941) and Yacowitz and Bird (1953). Work by Couch et al. (1948), Driesens (1951) and Sieburth et al. (1952) provided evidence that coliform bacteria have the ability to synthesize folic acid, riboflavin, biotin, niacin and other nutritional factors. It is conceivable then that optimum concentrations of coliform organisms or their metabolic products may improve nutrient availability and utilization in growing birds. Competition for B-vitamins by certain normal gut microorganisms may explain, in part, the method by which antibiotics work. Hauser et al. (1956) have associated antibiotic growth response with an increased coliform count in the gut. While these findings are not conclusive, they do lend support to the theory that bacteriological modifications are an important consideration in explaining the mechanisms of antibiotic action. In a search for other gut organisms that might be involved, Cook et al. (1954) observed that turkeys fed penicillin had a lower-thannormal count of Lactobacilli-typz organisms in the gut. It was their belief that this organism competes with the host for B-vitamins and that a reduction in the number of organisms by the use of penicillin in the feed was responsible for the improved performance in their experiment with turkeys. Luckey et al. (1956) and Forbes et al. (1958) have investigated the growth effects of antibiotics on poults and chicks or poults respectively under germ-free conditions. Whereas, Luckey et al. ob-
served significant growth responses to penicillin in germ-free poults, but not in germ-free chicks, Forbes' group found no effect from feeding germ-free poults approximately the same levels of the antibiotic. Bogdonoff et al. (1957) observed a growth effect in broilers from bi-weekly crop inoculations of mixed cultures of E. coli and A. aerogenes in the presence of several antibiotics. Coliform inoculation per se appeared to be without effect on growth or feed efficiency. Recent theories concerning the method of action of antibiotics have been reviewed by researchers at Kansas State College (Anonymous, 1956). They suggest that antibiotics have their effect on the physiology of bacteria rather than on the numbers or types of intestinal forms. These workers proposed that certain physiological changes in the bacteria, which happen as a consequence of antibiotic feeding, make them respond better to defense mechanisms of the animal body. Still another proposal along the same line has been suggested by Goebel and Barry (1957). Discovery that E. coli produce colominic acid led these workers to associate it with sialic acid, since colominic acid contains a large percentage of a substance resembling sialic. It is claimed by medical researchers that sialic acid interferes with the metabolism of certain viruses which affect animal health. Romoser et al. (1953) compared the effects of adding lyophilized coliforms to chick rations in the presence or absence of penicillin. Under these treatments, antibiotic response was increased significantly in the presence of the bacterial products. EXPERIMENTAL
The possibility of an association of E. coli population with growth stimulation in poultry prompted a series of experi-
730
W.
K. W A R D E N AND P. J.
TABLE 1.—Experimental rations Ingredients
Turkey starter
SCHAIBLE
TABLE 1A.—Calculated analyses of starter rations Chick broiler starter
Yellow corn, ground Oats, pulverized Wheat standard middlings
30.96
% — 10.00
46.43 1.00 1.00
%
Soybean oil meal (50% protein) Dehydrated alfalfa meal (17% protein) Stabilized animal fat
33.18 5.00 3.79
31.32 2.00 6.94
5.00 5.00
2.00 2.50 2.00
Units Crude protein Arginine Methionine Cystine Lysine Tryptophane Crude fat
Fish meal (55% protein) M e a t and bone scraps ( 5 0 % protein) Dried corn fermentation solubles Dried whey (50% delactosed) Dicalcium phosphate Ground limestone Salt (iodized) Trace mineral mix* -Methionine Arsanilic acid mixturef Butylated hydroxy toluene (antioxidant) Vitamin mixture
— 3.00 1.38 1.79
Productive energy 2.00 0.50 1.50
0.30 0.10
0.30 0.10 0.059
— — 0.50'
0.025 0.01 .3162
100.00
Crude fiber
100.00
* Trace mineral mixture supplied the following per pound of ration: 27.2 mg. of manganese, 0.54 mg. of iodine:, 9.07 mg. of iron, 0.91 mg. of copper, 0.045 mg. of zinc and 0.09 ]mg. of cobalt. t Abbott Laboratories, Progen containing 2 0 % arsanilic acid. 1 Vitamin mixture supplied the following per pound of ration: 2,270 I.U. of vitamin A, 671 I.C.U. of vitamin Dj, 6.0 mcgm. of vitamin B12, 2.0 mg. of riboflavin, 9.0 mg. of niacin, 4.0 mg. of pantothenic acid, 112 mg. of choline, 0.5 mg. of folic acid and 10 mg. of alpha tocopherol acetate. 2 Vitamin mixture supplied the following per pound of ration: 1,818 I.U. of vitamin A, 300 I.C.U. of vitamin D 3 , 6.0 mcgm. of vitamin B12, 1.0 mg. of ribofia'vin, 8.08 mg. of niacin, 2.0 mg. of pantothenic acid and 169 mg. of choline.
% % % % % % % % Cal./lb.
Calorie-Protein ratio
Turkey starter
Chick broiler starter
28.0 1.39 .55
24.0 1.36 .50
.38 1.50 .26
.32 1.23 .25
5.9
9.7
3.8
2.5
840
1,020
30
42
% % %
0.30 2.09 1.10
0.30 1.21 0.66
Manganese Iron Copper
mg./lb. mg./lb. mg./lb.
27.00 8.08 0.91
27.00 8.08 0.91
Iodine Zinc Cobalt
mg./lb. mg./lb. mg./lb.
Vitamin A Vitamin D3 Vitamin B12
I.U./lb. I.C.U./lb. mcg./lb.
Riboflavin Niacin Pantothenic acid Choline Folic acid
mg./lb. mg./lb. mg./lb. mg./lb. mg./lb.
Arsanilic acid
mg./lb.
Vitamin E (supplemental)
mg./lb.
Salt Calcium Phosphorus
0.54 0.027 0.09 6,733 681 7.3 3.87 43.90 8.27 916 0.68
0.54 0.027 0.09 4,118 300 6.0 2.57 21.41 6.02 744 0.72 23.5
10
-
ments with turkey poults and broiler chicks. Experiment 1. I n the first experiment, six lots of one-day-old Broad Breasted Bronze turkey poults were randomly allotted into four replicate pens of ten birds each and fed an all-mash starter ration (Tables 1 and 1A) for the 28-day test period. Equal numbers of males and females were reared in wire-floored, electrically-heated battery brooders and fed 200 grams of zinc bacitracin or terramycin per ton of feed continuously throughout the test period. I n addition, trypticase soy broth or a culture of E. coli in trypticase soy broth was administered directly into the crop of the test birds at bi-weekly intervals.
of age) to four ml. a t the fourth week—by one ml. increments weekly. The broth culture population of E. coli was adjusted on the basis of light transmission as follows: a. One loopful of stock culture* of E. coli was transferred to 10 ml. of trypticase soy broth and incubated at 37°C. for 18 hours. b. By use of photoelectric meter, the cultured medium was adjusted from approximately 10 to 25 percent light transmission by addition of sterile trypticase soy broth. c. 0.2 ml. of the 18-hour culture was added to each 100 ml. of soy broth and incubated by shaking an additional 18 hours a t 37°C. to produce
The bi-weekly dosage of broth or E. coli in broth was increased from one ml. per bird the first week (starting at three days
* Zinc bacitracin and stock cultures of E. coli provided by Commercial Solvents Corporation, Terre Haute, Indiana.
731
E. COLI AND ANTIBIOTICS
approximately 6.8X1012 organisms per milliliter. A representative bird was sacrificed from each treatment 72 hours after final inoculation. Contents of each area of the digestive tract were removed aseptically and appropriately diluted in sterile saline solution. The Mallmann-Peabody (1957) drop plate method for determining E. coli was employed. Composition of the media is given in Table 2.
TABLE 2.—Mallmann-Peabody {1957) agar adjusted to pH 7.2
of bi-weekly crop inoculation of E. coli on relative concentrations of E. coli in digestive tract of turkey poults at four weeks Crop inoculation
1 2 3 4 5 6 ,
10.00 5.00 1.50 5.00 0.10 15.00 1,000.00
and 1A. Equal numbers of males and females were reared together in electricallyheated battery brooders with raised wire floors for the 28-day test period. Two hundred grams of zinc bacitracin or terramycin per ton of feed were fed continuously throughout the experiment. In addition, crop inoculations of trypticase soy broth plus coliforms approximating 6.8X1012 organisms per ml., supernatant of broth plus coliforms, or centrifuged cells triple washed in physiological saline were administered weekly. Oral dosage of broth, E. coli, supernatant, or cells was increased from one ml. per bird the first week (starting at three days of age) to four ml. at the fourth week—in one ml. per week increments.
As shown in Table 3, digestive tract E. coli population in four-week poults varied in the several areas of the tract sampled, with highest counts occurring nearest the cloaca. No particular pattern of these microorganisms occurred due to treatment based on drop plate cultures of one representative bird per lot. Significant growth responses (P<.01) were obtained from feeding both zinc bacitracin and terramycin in the presence and absence of crop induced E. coli (Table 4). Experiment 2. In this experiment 480 White Rock chicks were allocated to twelve treatments of four replicates of ten birds each. They were fed the all-mash, broiler-starter ration shown in Tables 1
Treatment No.
Amount (gm.)
* Peptone, Difco Lab. f 1 ml. per liter of a 1.6 percent brome cresol purple indicator.
RESULTS
TABLE 3.—Effect
Composition Tryptose* Lactose Bile salts #3 NaCl Laurel sulphate Agar Water (to bring to) Indicator f
Supplement Broth only None None 200 gm./ton zinc bacitracin 200 gm./ton zinc bacitracin 200 gm./ton terramycin 200 gm./ton terramycin
+ + — + —
Broth plus coliforms
+ +
E. coli count/gm. of digestive tract contents* 2"above Cropp
80. 5. TNCf 58. TNCf None
Duod.
cecum
counts (10*)
.3 270,000 10.00 12,100 TNCf TNCf TNCf 92. 97,200 140. None None
Cecum
Feces
65,400 19,800 703,000 750,000 108,000 22,900
537,000 80,070 TNCf 192,000 459,000 44,000
* One representative bird per lot, average counts calculated from best dilution 1:100, 1:1,000,1:10,000, 1:100,000, 1:1,000,000. f Too numerous to count.
732
W. K. W A R D E N AND P . J.
SCHAIBLE
TABLE 4.—Growth, feed utilization and livability of 28-day old poults Crop inoculation
Treatment No.
Supplement
Broth only
1 2 3 4 5 6
None None 200 gm./ton zinc bacitracin 200 gm./ton zinc bacitracin 200 gm./ton terramycin 200 gm./ton terramycin
+
Broth plus coliforms
+ +
At four weeks of age
Initial weights (gm.)
Weight (gm.)
Feed/ gain (gm.)
Alive of 40 started
52 50 51 51 52 51
594 573 678* 683* 665* 678*
2.15 2.19 2.12 2.02 2.07 2.02
37 34 32 35 37 37
+ + +
* Significant increase in growth over control at 1 percent level of probability, analysis of variance, Duncan's multiple range and multiple F tests (1955). Coliform counts were determined only in the gut area approximately two inches above the cecum. I t was thought t h a t this location would represent an area wh,-ch would give meaningful differences in population, if E. coli were involved in growth stimulation. Results. As shown on Table 5, significant growth increases ( P < . 0 1 ) in four-week chicks were obtained from feeding zinc bacitracin only. Crop induced E. coli had no effect on growth rate and were ineffective in mediating responses to zinc bacitracin. TABLE 5.—Effect
DISCUSSION If, in the mechanism by which antibiotics promote growth in poultry, E. coli is directly involved by providing vitamins or other nutrients, then the addition of these microorganisms as part of the diet should enhance the value of a ration containing broad-spectrum antibiotics. This follows because the latter types of antibiotics would inhibit coliforms. An indication of a depression in coliform bacteria throughout the digestive tract occurred in the terramycin-fed poults t h a t had received E. coli by crop inoculation. Since
of crop inoculation of E. coli fractions on antibiotic response in chicks (4-week results)
Treatment
E. coli count* 2" above cecum (104)
Av. wei s h t (gm-)
Feed/gain
Alive of 40 started
Control Soybroth Broth plus E. coli Supernatant of E. coli broth Washed cells
1. 420. 0.7 None
424 419 414 411
2.12 2.12 2.05 1.96
39 40 38 39
Zinc bacitracin 200 gms./ton Soy broth Broth plus E. coli Supernatant of E. coli broth Washed cells
0.8 0.8 0.4 78.0
4771 471 4601 469-f
1.84 1.76 1.88 1.79
37 39 39 38
Terramycin 200 gms./ton Soy broth Broth plus E. coli Supernatant of E. coli broth Washed cells
None 0.7 None None
441 452 452 421
1.84 1.94 1.94 1.92
40 39 39 39
* One representative bird per lot, average counts calculated from best dilution 1:100, 1:1,000, 1:10,000, 1:100,000,1:1,000,000. t Significant increase over control at one percent level of probability. Analysis of variance, Duncan's multiple range and multiple F tests.
733
E. COLI AND ANTIBIOTICS
the naturally acquired E. coli appeared in birds fed terramycin alone, it appears t h a t normal coliforms m a y differ in resistance to the antibiotic compared with cultured strains. Furthermore, since a significant growth response was observed in terramycin-fed turkeys with a relatively low concentration of E. coli, the mechanism of growth promotion from broad spectrum antibiotics must involve factors in addition to relative coliform population. Weekly crop inoculations in chicks of several fractions of coliform cultures were ineffective in changing growth rate in the presence of zinc bacitracin or terramycin. Whereas, only zinc bacitracin treatments promoted a significant growth response over the controls, no relationship between the improved growth and E. coli population was evident. I n addition, none of the various coliform-crop inoculation treatments tempered or increased antibiotic responses. While the lack of significant responses to any of the terramycin-fed chicks was associated with an absence of E. coli population, the differences in organism count compared with bacitracinfed groups were not marked. I n a preliminary study (Tables 3, 5) involving a single representative bird per treatment, E. coli population was determined in the various areas of the digestive tract of poults, and two inches above the cecum of chicks. While no specific p a t t e r n of gut flora was evident, there was a general increase in coliforms nearest the cloaca for all treatments. SUMMARY AND CONCLUSIONS Both narrow spectrum (zinc bacitracin) and broad spectrum (oxytetracycline) antibiotics promoted significant growth increases in four-week-old turkeys in the presence or absence of added coliforms. Addition of a trypticase soy broth culture of E. coli by bi-weekly crop inoculation to
Broad Breasted Bronze turkey poults, however, was ineffective in stimulating growth irrespective of antibiotic treatment. I n chicks, weekly crop inoculation of broth cultures of E. coli, supernatant of an E. coli culture or saline washed cells of E. coli were also ineffective in changing growth rate in the presence or absence of zinc bacitracin or oxytetracycline. Further, all zinc bacitracin treatments promoted significant increases in growth regardless of coliform treatment, while none of the terramycin-fed birds grew significantly faster than the control birds. Under the conditions of these experiments, the addition of pure coliform cultures into the digestive tract, via the crop inoculation route, had no effect on the antibiotic, growth-stimulating mechanism in turkey poults or broiler chicks. REFERENCES Anonymous, 1956. Kansas reports on how antibiotics function. Feedstuffs, Dec. 1:70. Bird, H. R., R. J. Lillie and J. R. Sizemore, 1952. Environment and stimulation of chick growth by antibiotics. Poultry Sci. 31: 907. Bogdonoff, P. D., J. M. Pensack, J. N. Henson and R. S. Baldwin, 1957. Effect of coliform organisms on broiler growth. Informal Poultry Nutrition Conference. Coates, M. E., C. D. Dickinson, G. F. Harrison, S. K. Kon, S. H. Cummins and W. F. J. Cuthbertson, 1951. Mode of action of antibiotics in stimulating growth of chicks. Nature, 168: 332. Coates, M. E., C. D. Dickinson, G. F. Harrison, S. K. Kon, J. W. G. Porter, S. H. Cummins and W. F. J. Cuthbertson, 1952. A mode of action of antibiotics in chick nutrition. J. Sci. Food Agric. 3: 43-48. Cook, F. D., R. M. Blakely, H. I. MacGregor and R. W. Anderson, 1954. The effect of antibiotics on the intestinal flora of turkey poults. Poultry Sci. 33:38-40. Couch, J. R., W. W. Cravens, C. A. Elvehjem and J. G. Halpin, 1948. Relation of carbohydrate to intestinal synthesis of biotin and hatchability in mature fowl. J. Nutrition, 35:57-72. Driesens, R. J., 1951. Studies on the intestinal bacterial flora of the chick. I. Studies on the effect of certain antibiotics upon bacterial populations.
734
W. K. WARDEN AND P. J. SCHAIBLE
II. Studies on the effect of penicillin on the production of certain B-vitamins. Thesis, Michigan State College. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Elam, J. F., R. J. Jacobs, W. L. Tidwell, L. L. Gee and J. R. Couch, 1953. Possible mechanism involved in the growth-promoting responses obtained from antibiotics. J. Nutrition, 49: 307317. Emmel, M. W., 1930. Bacterial flora in feces of the normal fowl. J. Infect. Dis. 46: 293-297. Forbes, M., W. C. Supplee and G. F. Combs, 1958. Response of germ-free and conventionally reared turkey poults to dietary supplementation with penicillin and oleandomycin. Proc. Soc. Exptl. Biol. Med. 99:110-113. Goebel, W. F., and G. T. Barry, 1957. Colominic acid, a substance of bacterial origin related to sialic acid. Nature, 179: 206. Hauser, M. L., G. W. Anderson, W. F. Pepper and S. J. Slinger, 1956. Further evidence on the relation of colif orms to the growth response of chicks to antibiotics. Poultry Sci. 35: 27-36. Hill, D. C , H. D. Branion, S. J. Slinger and G. W. Anderson, 1953. Influence of environment on the growth response of chicks to penicillin. Poultry Sci. 32: 462-466. Jacobs, R. L., J. F. Elam. G. W. Anderson, L. L. Gee, J. Fowler and J. R. Couch, 1953. Further evidence as to the possible mechanism involved in the growth-promoting response obtained from
antibiotics. J. Nutrition, 51: 507-513. King, W. E., 1905. The bacterial flora of the intestinal mucosa and conjunctiva of the normal chicken. J. Am. Med. 10: 400-404. Luckey, T. D., H. A. Gordon, M. Wagner and J. A. Reyniers, 1956. Growth of germ-free birds fed antibiotics. Antibiotics and Chemotherapy. Vol. 6. No. 1. Mallmann, W. A., and F. R. Peabody, 1957. Michigan State University, Unpublished data. March, B., and J. Biely, 1952. The effect of feeding aureomycin on the bacterial content of chick feces. Poultry Sci. 31: 177-178. Romoser, G. L., M. S. Shorb and G. F. Combs, 1953. Effect of orally administered penicillin resistant microorganisms on the growth of chicks. Proc. Soc. Exptl. Biol. Med. 83: 17-21. Schumacher, A. E., and G. F. Heuser, 1941. Isolation of an organism responsible for the increased riboflavin content of the feces of the fowl. Poultry Sci. 20: 272-273. Sieburth, J. McN., J. McGinnis and C. E. Skinner, 1952. The effect of terramycin on the antagonism of certain bacteria against species of Proteus. J. Bact. 64: 163-169. Waibel, P. E., O. J. Abbott, C. A. Baumann and H. R. Bird, 1954. Disappearance of the growth response of chicks to dietary antibiotics in an "old" environment. Poultry Sci. 33: 1141-1146. Yacowitz, H., and O. D. Bird, 1953. Antibiotic levels in the digestive tract of the chick. Poultry Sci. 32:966-968.
NEWS AND NOTES {Continued from page 696) BRITISH COLUMBIA NOTES Mrs. Beryl E. March, Research Associate, Department of Poultry Science, University of British Columbia, Vancouver, Canada, has been awarded a Royal Society and Nuffield Commonwealth bursary for advanced study at Cambridge University, England. She will carry out further studies on the nutritive value of fish meals under the direction of Dr. K. J. Carpenter who has designed "short cut" methods of estimating the nutritive value of different sources of proteins by chemical means. PENB NOTES D. L. Vogt has been appointed News and Information Officer for the Poultry and Egg National Board, Chicago, Illinois. Previously he was
Publicity Manager for Orange-Crush Company, and prior to that was with Needham, Louis, and Brorby, Inc., advertising agency. COLORADO NOTES The National Institutes of Health have made a research grant of $11,500 to the Colorado State University for a study of the effect of x-ray irradiation on chicken embryos. The studies will be conducted by R. E. Moreng and P. A. Thornton. P.P.I. NOTES At the annual meeting of the Poultry Products Institute (Canada) in Vancouver, the following officers were elected: President—D. McQ. Shaver, Gait, Ontario; First Vice-President—W. G. Sharpe, Regina, Saskatchewan; Second Vice-Preson page 745)
W. C. LOCKHART, R. L. BRYANT AND D. W. BOLIN
proteins in chick diets. Poultry Sci. 23: 486490. Kratzer, F. H., D. E, Williams and B. Marshall, 1952. The requirement for isoleucine and the activities of its isomers for the growth of turkey poults. J. Nutrition, 47: 631-635. Kratzer, F. H., and N. Green, 1957. The availability of lysine in blood meal for chicks and poults. Poultry Sci. 36: 562-565. Mitchell, H. H., 1924. A method of determining the biological value of protein. J. Biol. Chem. 58: 873-903. Mussehl, F. E., and C. W. Ackerson, 1931. Utilization of proteins by the growing chick. Nebraska Agric. Exp. Sta. Res. Bull. 55. Slinger, S. J., W. F. Pepper and D. C. HiU, 1955. The use of keratin products in poultry rations. Poultry Sci. 34: 919-922.
Snedecor, G. W., 1946. Statistical Methods. 4th Edition, Iowa State College Press. Ames, Iowa. Squibb, R. L., and M. K. Wyld, 1950. Further studies on the value of dismodium meal in the baby chick diet. Poultry Sci. 29: 586-589. Squibb, R. L., and J. E. Braham, 1955. Blood meal as a lysine supplement to all-vegetable protein rations for chicks. Poultry Sci. 34: 1050-1053. Titus, H. W., T. C. Byerly, N. R. Ellis and R. B. Nestler, 1936. Effects of packinghouse by-products in the diet of chickens on the production and hatchability of eggs. J. Agr. Res. 53: 453456. Titus, H. W., 1955. Energy Values of Feedstuffs for Poultry. The Interstate, Danville, Illinois. Titus, H. W., 1957. Energy values of feedstuffs for poultry. Mineo. report, Linestone Products Corporation of America, Lime Crest Res. Lab
Effect of Feeding Escherichia coli to Turkey Poults and Chicks in the Presence of Certain Antibiotics1 W. K. WARDEN 2 AND PHILIP J. SCHAIBLE Department of Poultry Science, Michigan State University, East Lansing (Received for publication September 17,1959)
D
URING the past few years, great importance has been ascribed to shifts in bacterial population of the gut as a major factor in explaining the growthstimulating action of antibiotics. Supporting this theory is the fact that zinc bacitracin, a complex polypeptide molecule, is not absorbed into the blood stream, so any improvement in growth must result from its action in the digestive tract. Tetracycline-type antibiotics, on the other hand, are absorbed into the blood stream to some extent and so could act systemically as well as enterically. Furthermore, "broad-spectrum" antibiotics, such as the tetracycline-type inhibit both gram-posi1 Journal article No. 2495 Michigan Agricultural Experiment Station. Research supported in part by a grant from Commercial Solvents Corporation. 2 Presented by the senior author in partial fulfillment of Ph.D. requirements.
tive and gram-negative organisms; whereas, "narrow-spectrum" antibiotics, such as penicillin and bacitracin, act more specifically against the common, diseaseproducing, gram-positive forms. The bacteriological approach has appealed to many workers since it was demonstrated early (Coates et al, 1951, 1952; Bird et al, 1952; Hill et al, 1953; Jacobs et al, 1953; and Waibel et al, 1954) that antibiotics are more effective in contaminated than in new quarters. Also, the bacterial population of the gut changes significantly when antibiotics are fed at levels as low as one-half gram per ton of feed. Elam et al. (1953) and March and Biely (1952) suggested that growth responses may result from decreasing the incidence of enterotoxemia—primarily by reducing the number of Clostridia and Lactobacillus-type organisms in the gut,
729
E. COLI AND ANTIBIOTICS
Before the advent of antibiotics as specific compounds King (1905) and Emmel (1930) showed that the coliform types {Escherichia coli, Aerobacter aerogenes) constitute about 65 percent of the bacterial population of the intestinal tract of chickens. These findings have been verified more recently by Schumacher and Heuser (1941) and Yacowitz and Bird (1953). Work by Couch et al. (1948), Driesens (1951) and Sieburth et al. (1952) provided evidence that coliform bacteria have the ability to synthesize folic acid, riboflavin, biotin, niacin and other nutritional factors. It is conceivable then that optimum concentrations of coliform organisms or their metabolic products may improve nutrient availability and utilization in growing birds. Competition for B-vitamins by certain normal gut microorganisms may explain, in part, the method by which antibiotics work. Hauser et al. (1956) have associated antibiotic growth response with an increased coliform count in the gut. While these findings are not conclusive, they do lend support to the theory that bacteriological modifications are an important consideration in explaining the mechanisms of antibiotic action. In a search for other gut organisms that might be involved, Cook et al. (1954) observed that turkeys fed penicillin had a lower-thannormal count of Lactobacilli-typz organisms in the gut. It was their belief that this organism competes with the host for B-vitamins and that a reduction in the number of organisms by the use of penicillin in the feed was responsible for the improved performance in their experiment with turkeys. Luckey et al. (1956) and Forbes et al. (1958) have investigated the growth effects of antibiotics on poults and chicks or poults respectively under germ-free conditions. Whereas, Luckey et al. ob-
served significant growth responses to penicillin in germ-free poults, but not in germ-free chicks, Forbes' group found no effect from feeding germ-free poults approximately the same levels of the antibiotic. Bogdonoff et al. (1957) observed a growth effect in broilers from bi-weekly crop inoculations of mixed cultures of E. coli and A. aerogenes in the presence of several antibiotics. Coliform inoculation per se appeared to be without effect on growth or feed efficiency. Recent theories concerning the method of action of antibiotics have been reviewed by researchers at Kansas State College (Anonymous, 1956). They suggest that antibiotics have their effect on the physiology of bacteria rather than on the numbers or types of intestinal forms. These workers proposed that certain physiological changes in the bacteria, which happen as a consequence of antibiotic feeding, make them respond better to defense mechanisms of the animal body. Still another proposal along the same line has been suggested by Goebel and Barry (1957). Discovery that E. coli produce colominic acid led these workers to associate it with sialic acid, since colominic acid contains a large percentage of a substance resembling sialic. It is claimed by medical researchers that sialic acid interferes with the metabolism of certain viruses which affect animal health. Romoser et al. (1953) compared the effects of adding lyophilized coliforms to chick rations in the presence or absence of penicillin. Under these treatments, antibiotic response was increased significantly in the presence of the bacterial products. EXPERIMENTAL
The possibility of an association of E. coli population with growth stimulation in poultry prompted a series of experi-
730
W.
K. W A R D E N AND P. J.
TABLE 1.—Experimental rations Ingredients
Turkey starter
SCHAIBLE
TABLE 1A.—Calculated analyses of starter rations Chick broiler starter
Yellow corn, ground Oats, pulverized Wheat standard middlings
30.96
% — 10.00
46.43 1.00 1.00
%
Soybean oil meal (50% protein) Dehydrated alfalfa meal (17% protein) Stabilized animal fat
33.18 5.00 3.79
31.32 2.00 6.94
5.00 5.00
2.00 2.50 2.00
Units Crude protein Arginine Methionine Cystine Lysine Tryptophane Crude fat
Fish meal (55% protein) M e a t and bone scraps ( 5 0 % protein) Dried corn fermentation solubles Dried whey (50% delactosed) Dicalcium phosphate Ground limestone Salt (iodized) Trace mineral mix* -Methionine Arsanilic acid mixturef Butylated hydroxy toluene (antioxidant) Vitamin mixture
— 3.00 1.38 1.79
Productive energy 2.00 0.50 1.50
0.30 0.10
0.30 0.10 0.059
— — 0.50'
0.025 0.01 .3162
100.00
Crude fiber
100.00
* Trace mineral mixture supplied the following per pound of ration: 27.2 mg. of manganese, 0.54 mg. of iodine:, 9.07 mg. of iron, 0.91 mg. of copper, 0.045 mg. of zinc and 0.09 ]mg. of cobalt. t Abbott Laboratories, Progen containing 2 0 % arsanilic acid. 1 Vitamin mixture supplied the following per pound of ration: 2,270 I.U. of vitamin A, 671 I.C.U. of vitamin Dj, 6.0 mcgm. of vitamin B12, 2.0 mg. of riboflavin, 9.0 mg. of niacin, 4.0 mg. of pantothenic acid, 112 mg. of choline, 0.5 mg. of folic acid and 10 mg. of alpha tocopherol acetate. 2 Vitamin mixture supplied the following per pound of ration: 1,818 I.U. of vitamin A, 300 I.C.U. of vitamin D 3 , 6.0 mcgm. of vitamin B12, 1.0 mg. of ribofia'vin, 8.08 mg. of niacin, 2.0 mg. of pantothenic acid and 169 mg. of choline.
% % % % % % % % Cal./lb.
Calorie-Protein ratio
Turkey starter
Chick broiler starter
28.0 1.39 .55
24.0 1.36 .50
.38 1.50 .26
.32 1.23 .25
5.9
9.7
3.8
2.5
840
1,020
30
42
% % %
0.30 2.09 1.10
0.30 1.21 0.66
Manganese Iron Copper
mg./lb. mg./lb. mg./lb.
27.00 8.08 0.91
27.00 8.08 0.91
Iodine Zinc Cobalt
mg./lb. mg./lb. mg./lb.
Vitamin A Vitamin D3 Vitamin B12
I.U./lb. I.C.U./lb. mcg./lb.
Riboflavin Niacin Pantothenic acid Choline Folic acid
mg./lb. mg./lb. mg./lb. mg./lb. mg./lb.
Arsanilic acid
mg./lb.
Vitamin E (supplemental)
mg./lb.
Salt Calcium Phosphorus
0.54 0.027 0.09 6,733 681 7.3 3.87 43.90 8.27 916 0.68
0.54 0.027 0.09 4,118 300 6.0 2.57 21.41 6.02 744 0.72 23.5
10
-
ments with turkey poults and broiler chicks. Experiment 1. I n the first experiment, six lots of one-day-old Broad Breasted Bronze turkey poults were randomly allotted into four replicate pens of ten birds each and fed an all-mash starter ration (Tables 1 and 1A) for the 28-day test period. Equal numbers of males and females were reared in wire-floored, electrically-heated battery brooders and fed 200 grams of zinc bacitracin or terramycin per ton of feed continuously throughout the test period. I n addition, trypticase soy broth or a culture of E. coli in trypticase soy broth was administered directly into the crop of the test birds at bi-weekly intervals.
of age) to four ml. a t the fourth week—by one ml. increments weekly. The broth culture population of E. coli was adjusted on the basis of light transmission as follows: a. One loopful of stock culture* of E. coli was transferred to 10 ml. of trypticase soy broth and incubated at 37°C. for 18 hours. b. By use of photoelectric meter, the cultured medium was adjusted from approximately 10 to 25 percent light transmission by addition of sterile trypticase soy broth. c. 0.2 ml. of the 18-hour culture was added to each 100 ml. of soy broth and incubated by shaking an additional 18 hours a t 37°C. to produce
The bi-weekly dosage of broth or E. coli in broth was increased from one ml. per bird the first week (starting at three days
* Zinc bacitracin and stock cultures of E. coli provided by Commercial Solvents Corporation, Terre Haute, Indiana.
731
E. COLI AND ANTIBIOTICS
approximately 6.8X1012 organisms per milliliter. A representative bird was sacrificed from each treatment 72 hours after final inoculation. Contents of each area of the digestive tract were removed aseptically and appropriately diluted in sterile saline solution. The Mallmann-Peabody (1957) drop plate method for determining E. coli was employed. Composition of the media is given in Table 2.
TABLE 2.—Mallmann-Peabody {1957) agar adjusted to pH 7.2
of bi-weekly crop inoculation of E. coli on relative concentrations of E. coli in digestive tract of turkey poults at four weeks Crop inoculation
1 2 3 4 5 6 ,
10.00 5.00 1.50 5.00 0.10 15.00 1,000.00
and 1A. Equal numbers of males and females were reared together in electricallyheated battery brooders with raised wire floors for the 28-day test period. Two hundred grams of zinc bacitracin or terramycin per ton of feed were fed continuously throughout the experiment. In addition, crop inoculations of trypticase soy broth plus coliforms approximating 6.8X1012 organisms per ml., supernatant of broth plus coliforms, or centrifuged cells triple washed in physiological saline were administered weekly. Oral dosage of broth, E. coli, supernatant, or cells was increased from one ml. per bird the first week (starting at three days of age) to four ml. at the fourth week—in one ml. per week increments.
As shown in Table 3, digestive tract E. coli population in four-week poults varied in the several areas of the tract sampled, with highest counts occurring nearest the cloaca. No particular pattern of these microorganisms occurred due to treatment based on drop plate cultures of one representative bird per lot. Significant growth responses (P<.01) were obtained from feeding both zinc bacitracin and terramycin in the presence and absence of crop induced E. coli (Table 4). Experiment 2. In this experiment 480 White Rock chicks were allocated to twelve treatments of four replicates of ten birds each. They were fed the all-mash, broiler-starter ration shown in Tables 1
Treatment No.
Amount (gm.)
* Peptone, Difco Lab. f 1 ml. per liter of a 1.6 percent brome cresol purple indicator.
RESULTS
TABLE 3.—Effect
Composition Tryptose* Lactose Bile salts #3 NaCl Laurel sulphate Agar Water (to bring to) Indicator f
Supplement Broth only None None 200 gm./ton zinc bacitracin 200 gm./ton zinc bacitracin 200 gm./ton terramycin 200 gm./ton terramycin
+ + — + —
Broth plus coliforms
+ +
E. coli count/gm. of digestive tract contents* 2"above Cropp
80. 5. TNCf 58. TNCf None
Duod.
cecum
counts (10*)
.3 270,000 10.00 12,100 TNCf TNCf TNCf 92. 97,200 140. None None
Cecum
Feces
65,400 19,800 703,000 750,000 108,000 22,900
537,000 80,070 TNCf 192,000 459,000 44,000
* One representative bird per lot, average counts calculated from best dilution 1:100, 1:1,000,1:10,000, 1:100,000, 1:1,000,000. f Too numerous to count.
732
W. K. W A R D E N AND P . J.
SCHAIBLE
TABLE 4.—Growth, feed utilization and livability of 28-day old poults Crop inoculation
Treatment No.
Supplement
Broth only
1 2 3 4 5 6
None None 200 gm./ton zinc bacitracin 200 gm./ton zinc bacitracin 200 gm./ton terramycin 200 gm./ton terramycin
+
Broth plus coliforms
+ +
At four weeks of age
Initial weights (gm.)
Weight (gm.)
Feed/ gain (gm.)
Alive of 40 started
52 50 51 51 52 51
594 573 678* 683* 665* 678*
2.15 2.19 2.12 2.02 2.07 2.02
37 34 32 35 37 37
+ + +
* Significant increase in growth over control at 1 percent level of probability, analysis of variance, Duncan's multiple range and multiple F tests (1955). Coliform counts were determined only in the gut area approximately two inches above the cecum. I t was thought t h a t this location would represent an area wh,-ch would give meaningful differences in population, if E. coli were involved in growth stimulation. Results. As shown on Table 5, significant growth increases ( P < . 0 1 ) in four-week chicks were obtained from feeding zinc bacitracin only. Crop induced E. coli had no effect on growth rate and were ineffective in mediating responses to zinc bacitracin. TABLE 5.—Effect
DISCUSSION If, in the mechanism by which antibiotics promote growth in poultry, E. coli is directly involved by providing vitamins or other nutrients, then the addition of these microorganisms as part of the diet should enhance the value of a ration containing broad-spectrum antibiotics. This follows because the latter types of antibiotics would inhibit coliforms. An indication of a depression in coliform bacteria throughout the digestive tract occurred in the terramycin-fed poults t h a t had received E. coli by crop inoculation. Since
of crop inoculation of E. coli fractions on antibiotic response in chicks (4-week results)
Treatment
E. coli count* 2" above cecum (104)
Av. wei s h t (gm-)
Feed/gain
Alive of 40 started
Control Soybroth Broth plus E. coli Supernatant of E. coli broth Washed cells
1. 420. 0.7 None
424 419 414 411
2.12 2.12 2.05 1.96
39 40 38 39
Zinc bacitracin 200 gms./ton Soy broth Broth plus E. coli Supernatant of E. coli broth Washed cells
0.8 0.8 0.4 78.0
4771 471 4601 469-f
1.84 1.76 1.88 1.79
37 39 39 38
Terramycin 200 gms./ton Soy broth Broth plus E. coli Supernatant of E. coli broth Washed cells
None 0.7 None None
441 452 452 421
1.84 1.94 1.94 1.92
40 39 39 39
* One representative bird per lot, average counts calculated from best dilution 1:100, 1:1,000, 1:10,000, 1:100,000,1:1,000,000. t Significant increase over control at one percent level of probability. Analysis of variance, Duncan's multiple range and multiple F tests.
733
E. COLI AND ANTIBIOTICS
the naturally acquired E. coli appeared in birds fed terramycin alone, it appears t h a t normal coliforms m a y differ in resistance to the antibiotic compared with cultured strains. Furthermore, since a significant growth response was observed in terramycin-fed turkeys with a relatively low concentration of E. coli, the mechanism of growth promotion from broad spectrum antibiotics must involve factors in addition to relative coliform population. Weekly crop inoculations in chicks of several fractions of coliform cultures were ineffective in changing growth rate in the presence of zinc bacitracin or terramycin. Whereas, only zinc bacitracin treatments promoted a significant growth response over the controls, no relationship between the improved growth and E. coli population was evident. I n addition, none of the various coliform-crop inoculation treatments tempered or increased antibiotic responses. While the lack of significant responses to any of the terramycin-fed chicks was associated with an absence of E. coli population, the differences in organism count compared with bacitracinfed groups were not marked. I n a preliminary study (Tables 3, 5) involving a single representative bird per treatment, E. coli population was determined in the various areas of the digestive tract of poults, and two inches above the cecum of chicks. While no specific p a t t e r n of gut flora was evident, there was a general increase in coliforms nearest the cloaca for all treatments. SUMMARY AND CONCLUSIONS Both narrow spectrum (zinc bacitracin) and broad spectrum (oxytetracycline) antibiotics promoted significant growth increases in four-week-old turkeys in the presence or absence of added coliforms. Addition of a trypticase soy broth culture of E. coli by bi-weekly crop inoculation to
Broad Breasted Bronze turkey poults, however, was ineffective in stimulating growth irrespective of antibiotic treatment. I n chicks, weekly crop inoculation of broth cultures of E. coli, supernatant of an E. coli culture or saline washed cells of E. coli were also ineffective in changing growth rate in the presence or absence of zinc bacitracin or oxytetracycline. Further, all zinc bacitracin treatments promoted significant increases in growth regardless of coliform treatment, while none of the terramycin-fed birds grew significantly faster than the control birds. Under the conditions of these experiments, the addition of pure coliform cultures into the digestive tract, via the crop inoculation route, had no effect on the antibiotic, growth-stimulating mechanism in turkey poults or broiler chicks. REFERENCES Anonymous, 1956. Kansas reports on how antibiotics function. Feedstuffs, Dec. 1:70. Bird, H. R., R. J. Lillie and J. R. Sizemore, 1952. Environment and stimulation of chick growth by antibiotics. Poultry Sci. 31: 907. Bogdonoff, P. D., J. M. Pensack, J. N. Henson and R. S. Baldwin, 1957. Effect of coliform organisms on broiler growth. Informal Poultry Nutrition Conference. Coates, M. E., C. D. Dickinson, G. F. Harrison, S. K. Kon, S. H. Cummins and W. F. J. Cuthbertson, 1951. Mode of action of antibiotics in stimulating growth of chicks. Nature, 168: 332. Coates, M. E., C. D. Dickinson, G. F. Harrison, S. K. Kon, J. W. G. Porter, S. H. Cummins and W. F. J. Cuthbertson, 1952. A mode of action of antibiotics in chick nutrition. J. Sci. Food Agric. 3: 43-48. Cook, F. D., R. M. Blakely, H. I. MacGregor and R. W. Anderson, 1954. The effect of antibiotics on the intestinal flora of turkey poults. Poultry Sci. 33:38-40. Couch, J. R., W. W. Cravens, C. A. Elvehjem and J. G. Halpin, 1948. Relation of carbohydrate to intestinal synthesis of biotin and hatchability in mature fowl. J. Nutrition, 35:57-72. Driesens, R. J., 1951. Studies on the intestinal bacterial flora of the chick. I. Studies on the effect of certain antibiotics upon bacterial populations.
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W. K. WARDEN AND P. J. SCHAIBLE
II. Studies on the effect of penicillin on the production of certain B-vitamins. Thesis, Michigan State College. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Elam, J. F., R. J. Jacobs, W. L. Tidwell, L. L. Gee and J. R. Couch, 1953. Possible mechanism involved in the growth-promoting responses obtained from antibiotics. J. Nutrition, 49: 307317. Emmel, M. W., 1930. Bacterial flora in feces of the normal fowl. J. Infect. Dis. 46: 293-297. Forbes, M., W. C. Supplee and G. F. Combs, 1958. Response of germ-free and conventionally reared turkey poults to dietary supplementation with penicillin and oleandomycin. Proc. Soc. Exptl. Biol. Med. 99:110-113. Goebel, W. F., and G. T. Barry, 1957. Colominic acid, a substance of bacterial origin related to sialic acid. Nature, 179: 206. Hauser, M. L., G. W. Anderson, W. F. Pepper and S. J. Slinger, 1956. Further evidence on the relation of colif orms to the growth response of chicks to antibiotics. Poultry Sci. 35: 27-36. Hill, D. C , H. D. Branion, S. J. Slinger and G. W. Anderson, 1953. Influence of environment on the growth response of chicks to penicillin. Poultry Sci. 32: 462-466. Jacobs, R. L., J. F. Elam. G. W. Anderson, L. L. Gee, J. Fowler and J. R. Couch, 1953. Further evidence as to the possible mechanism involved in the growth-promoting response obtained from
antibiotics. J. Nutrition, 51: 507-513. King, W. E., 1905. The bacterial flora of the intestinal mucosa and conjunctiva of the normal chicken. J. Am. Med. 10: 400-404. Luckey, T. D., H. A. Gordon, M. Wagner and J. A. Reyniers, 1956. Growth of germ-free birds fed antibiotics. Antibiotics and Chemotherapy. Vol. 6. No. 1. Mallmann, W. A., and F. R. Peabody, 1957. Michigan State University, Unpublished data. March, B., and J. Biely, 1952. The effect of feeding aureomycin on the bacterial content of chick feces. Poultry Sci. 31: 177-178. Romoser, G. L., M. S. Shorb and G. F. Combs, 1953. Effect of orally administered penicillin resistant microorganisms on the growth of chicks. Proc. Soc. Exptl. Biol. Med. 83: 17-21. Schumacher, A. E., and G. F. Heuser, 1941. Isolation of an organism responsible for the increased riboflavin content of the feces of the fowl. Poultry Sci. 20: 272-273. Sieburth, J. McN., J. McGinnis and C. E. Skinner, 1952. The effect of terramycin on the antagonism of certain bacteria against species of Proteus. J. Bact. 64: 163-169. Waibel, P. E., O. J. Abbott, C. A. Baumann and H. R. Bird, 1954. Disappearance of the growth response of chicks to dietary antibiotics in an "old" environment. Poultry Sci. 33: 1141-1146. Yacowitz, H., and O. D. Bird, 1953. Antibiotic levels in the digestive tract of the chick. Poultry Sci. 32:966-968.
NEWS AND NOTES {Continued from page 696) BRITISH COLUMBIA NOTES Mrs. Beryl E. March, Research Associate, Department of Poultry Science, University of British Columbia, Vancouver, Canada, has been awarded a Royal Society and Nuffield Commonwealth bursary for advanced study at Cambridge University, England. She will carry out further studies on the nutritive value of fish meals under the direction of Dr. K. J. Carpenter who has designed "short cut" methods of estimating the nutritive value of different sources of proteins by chemical means. PENB NOTES D. L. Vogt has been appointed News and Information Officer for the Poultry and Egg National Board, Chicago, Illinois. Previously he was
Publicity Manager for Orange-Crush Company, and prior to that was with Needham, Louis, and Brorby, Inc., advertising agency. COLORADO NOTES The National Institutes of Health have made a research grant of $11,500 to the Colorado State University for a study of the effect of x-ray irradiation on chicken embryos. The studies will be conducted by R. E. Moreng and P. A. Thornton. P.P.I. NOTES At the annual meeting of the Poultry Products Institute (Canada) in Vancouver, the following officers were elected: President—D. McQ. Shaver, Gait, Ontario; First Vice-President—W. G. Sharpe, Regina, Saskatchewan; Second Vice-Preson page 745)