2147
RACHITOGENIC EFFECTS OF GRAIN
obtained at the low level of vitamin D 3 in the first experiment. Triticale, a hybrid between wheat and rye, was found to have a rachitogenic effect similar to that of rye. Wheat did not differ from corn in its effect on chick bone ash.
Association of Official Agricultural Chemists, 1970. Official Methods of Analysis, Eleventh ed., Washington, D.C. Duncan, D. B., 1955. Multiple range and multiple F test. Biometrics, 11: 1-42. Fernandez, R., E. Lucas and J. McGinnis, 1973. Fractionation of a chick growth depressing factor from rye. Poultry Sci. 52: 2252-2259. MacAuliffe, T., A. Pietraszek and J. McGiniis, 1976. The effect of adding procaine penicillin to diets with different levels of vitamin D 3 on the performance of turkey poults. Poultry Sci. 55: 183-187. National Academy of Sciences, National Research Council, 1971. Nutrient Requirements of Poultry. Sixth revised ed., Washington, D.C. Steel, G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York, NY.
Effect of Dietary Oxy tetracycline on Microorganisms in Turkey Feces 1 B . B . BALDWIN, M. C. BROMEL, D . W . A I R D , R. L . JOHNSON AND J. L .
SELL.
Departments of Bacteriology and Animal Science, North Dakota State University, Fargo, North Dakota 58102 (Received for publication December 3, 1975)
ABSTRACT Thirty-six two-day old male turkey poults were divided into three groups and fed three levels of oxytetracycline; 0, 110 or 440 mg. per kg. of ration, respectively, for nine weeks. The poults were given an oral spectinomycin plus vitamin treatment at the hatchery but were not fed before the start of the experiment. Defecated feces from each group were sampled at weekly intervals and analyzed for bacteriological content, especially for members of the Enterobacteriaceae. Total aerobic counts of 3 x 108 and total anaerobic counts of 2 x 109 were obtained. The majority of the Enterobacteriaceae were Escherichia coli but some Klebsiella sp., the Bethesda-Ballerup group of paracolon bacteria and Arizona sp. were also isolated. Antibiograms of the enteric isolates indicated that an increase in multiple antibiotic resistance occurred when increased amounts of antibiotic were fed. POULTRY SCIENCE 55: 2147-2154, 1976
INTRODUCTION
A
NTIBIOTICS have been used by the poultry industry as feed additives (singly and in combination) for over 20 years. Good-
1. Published with the approval of the Director of the North Dakota Agricultural Experiment Station as Journal Article No. 000.
ling et al. (1972) and Lucas (1972) have shown that increased feed efficiency, better egg production and better disease control can still be demonstrated from the continued use of dietary antibiotics. However, Smith (1968), Datta (1971), Anderson (1965), and Guinee (1970) have voiced concern regarding the development of multiple antibiotic resistant bacteria in domestic animals due to the use
Downloaded from http://ps.oxfordjournals.org/ by guest on November 22, 2014
The bone ash results obtained in Experiment 3 show clearly that a factor in rye which depresses bone mineralization is extractable with water and that acid autoclaving of the rye destroys or inactivates this factor, especially when penicillin is also added to the diet. In subsequent trials not reported in this paper, significant increases in bone ash have been obtained by acid autoclaving the rye. The factor in rye that appears to interfere with vitamin D 3 utilization by chicks and turkeys is under investigation.
REFERENCES
2148
BALDWIN, BROMEL, AIRD, JOHNSON AND SELL
MATERIALS AND METHODS Test Animals. Thirty-six two-day old male turkey poults (previously treated at the hatchery with a single oral dose of spectinomycin and vitamins) were divided into twelve groups. Each group of three poults was kept in an individual metabolism cage designed to facilitate collection of excreta (Lockhart, 1963). The cages, water pans and feed pans were steam sterilized prior to the experiment. Each of three ration treatments was assigned to four groups of poults. The ration treatments consisted of: 1) Control, a standard starter ration (Table 1) with no antibiotics; 2) starter ration plus 110 mg. of oxytetracycline (OTC) per kg. and 3) starter ration plus 440 mg. OTC per kg. Sampling Procedures. The poults were weighed weekly and composite samples of defecated feces (eight hour accumulations) were collected from each of the treatment groups of three birds with a sterile spatula and transmitted to the laboratory in sterile specimen jars. At the termination of the study at nine weeks, the poults were killed and
TABLE 1.—Starter mash Ingredient Percent Ground yellow corn 23.70 Barley 10.00 Soybean meal (48% protein) 51.00 Animal tallow 5.25 Meat and bone meal (50% protein) 4.00 Dehydrated alfalfa meal (17% protein) 2.00 Dicalcium phosphate 2.00 Ground limestone 1.45 DL methionine 0.05 Vitamin premixa 0.25 Trace mineral premixb 0.30 a The vitamin premix supplied the following vitamins: (mg./kg. diet) menadione, 1; riboflavin, 6; pantothenic acid, 22; niacin, 75; choline, 750; DL methionine, 0.5 g; vitamin A, 7500 I.U.; vitamin D 3 , 1500 I.C.U.; and vitamin E, 6 I.U. b The trace mineral premix supplied the following trace minerals: (mg./kg. diet) manganese, 50; zinc, 50; iron, 30; copper, 5; iodized salt, 2600. livers examined and removed asceptically into sterile specimen jars for further study (Aird, 1973). Gut samples were taken at autopsy with a sterile swab after an incision into the mid section portion of the small intestine with a sterile scalpel. Plate Counts. Defecated feces were mixed with a sterile mortar and pestle and then homogenized in 99 ml. sterile water in a Waring blender for two minutes. Dilutions were made in sterile water and plated in triplicate for total aerobic counts on Trypticase Soy Agar (TSA) from Baltimore Biological Laboratories, (BBL) and for enteric counts on MacConkey's Agar (Mac, BBL) and Hektoen Enteric Agar (He, Difco). Plates were incubated at 37° C. for 24 hours. Plates of TSA plus 5% defibrinated sheep blood were incubated in a H 2 : C 0 2 atmosphere (Gas Pak, BBL) at 35° C. for 5 days to determine total anaerobic counts. Enrichment Procedures for Salmonella. One gram of ground composite defecated fecal
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of antibiotics for growth promotion and prevention of disease. Little data are available relative to the numbers and types of bacteria present in poultry feces and the effect thereon of dietary antibiotics. This lack of information has compounded problems involved in adequate disposal or utilization of poultry feces. The following study was designed to determine the effect of dietary oxytetracycline on the microflora in the intestinal digesta and in the defecated feces. Concurrently, the development of multiple resistant bacteria in defecated feces was also studied with special attention given to the Enterobacteriaceae because of their traditional use as indicators of potential public health hazards.
OXYTETRACYCLINE AND FECAL MICROORGANISMS
material was added to 9 ml. of Selinite Cystine Enrichment Growth (BBL). After 12 to 15 hours incubation at 41° C , cultures were streaked on Mac and incubated at 37° C. for 24 to 48 hours. Feed Analysis. Feed from each of the regimens was routinely tested for bacterial contamination following the protocol employed for fecal samples. The feed was received in paper bags and one batch of feed sufficed for the nine week experiment.
Gut Swabs. A sterile swab was inserted into the asceptically incised gut at autopsy, then immediately immersed in Trypticase Soy Broth (BBL) which served as an enrichment medium. Cultures were incubated 24 hours at 37° C. and subsequently streaked on Mac and TSA. Identification of Enterobacteriaceae. A minimum of four isolates from the composite defecated feces of each group fed the three levels of OTC were chosen weekly by colonial characteristics and fermentation reactions on Mac and He. A total of 120 gram negative glucose fermenting bacterial isolates obtained from Mac and He media, from enrichment cultures of defecated samples and from incised gut swabs were randomly chosen for identification. These representative isolates were then transferred to Kligler's Iron Agar (BBL) after which the following biochemical tests were used for the identification of the Enterobacteriaceae (Ewing and Edwards, 1972): glucose, lactose and dulcitol fermentations; motility, indole and sulfide production on Sulfide Indole Motility Medium; urease production on Urease Test Medium; lysine
decarboxylase on Lysine Iron Agar; phenylalanine deamination on Phenylalanine Agar (all BBL); citrate utilization on Simmons Citrate Agar and ornithine decarboxylation on Motility Indole Ornithine Media (both Difco). Possible Salmonella isolates were serotyped with Salmonella O antiserum polyvalent (BBL). Antibiogram Assay. Enteric isolates were assayed for drug resistance by the disc method of Bauer (1966) for their sensitivities to the following levels of antimicrobial agents: penicillin, 10 units; bacitracin, 10 units; aureomycin, 20 meg.; OTC, 30 meg.; ampicillin, 10 units; gantrisin, 300 meg.; and streptomycin, 10 units.
RESULTS AND DISCUSSION Weight Gain and Feed Efficiency. Gain in body weight was not affected significantly by including OTC in the ration. However, poults fed the ration containing 440 mg. OTC per kg. required significantly (P < 0.01) less feed per kg. of gain (1.94 kg.) than did poults fed no antibiotic (2.11 kg.). Feed efficiency was also improved slightly by 100 mg. OTC per kg. of ration but not significantly (Duncan, 1955). Total Counts. Problems encountered in feeding the two-day old poults made proper collection of feces impossible until the birds were nine days old. All counts (aerobic and anaerobic) showed considerable variation for the first four weeks. After the fifth week the total aerobic counts from TSA of the defecated feces of all three regimens stabilized at 3 x 108 organisms per gram while the gram negative counts from Mac and He stabilized at 5 x 107 cells per gram. Only in the anaerobic count (TSA plus 5% def ibrinated sheep blood) was there any variation that could possibly be attributed to the
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Water Analysis. The tap water supply and a composite sample from the watering troughs were tested weekly for bacterial contamination, employing procedures outlined by the American Public Health Association (1971).
2149
2150
BALDWIN, BROMEL, AIRD, JOHNSON AND SELL
TABLE 2.—Enterobacteriaceae isolates (From composite defecated feces or gut samples)
Organism E. coli Klebsiella Beth-Ballerup Citrobacter Arizona Enterobacter Pseudomonas
Defecated feces 54 16 16 6 5 4 2
Gut 10 2 2 3
— — —
% of 120 isolates 53 15 15 8 4 3 2
10C
80
6C
% 4C
2C
FIG. 1. Percentages of predominant microorganisms—Control Abbreviations: Citro, Citrobacter; Ariz, Arizona; Kleb, Klebsiella; Be-Ba, Bethesda-Ballerup group; E. coli, Escherichia coli; Entero, Enterobacter. All figures refer to composite gut and defecated fecal samples.
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amount of OTC fed. Both control and low level groups levelled off at 3 x 109 while the anaerobic count from the feces of birds on the high antibiotic regimen decreased to 8 x 108 organisms per gram which is consistent with one of the reported benefits of antibiotic supplementation of feed (Jacobs et al., 1955).
The bacterial counts in defecated turkey feces were in the same range as those obtained from defecated cattle feces in this laboratory (Coles, 1973) and from cattle feedlot wastes as reported by Rhodes et al. (1972). Lovett et al. (1971) reported total counts in poultry litter to be approximately 108 per gram both aerobically and anaerobically. Differences between litter and defecated feces as well as differences in media and anaerobic methodology may have contributed to the higher anaerobic counts (4 x 10 8 tol x 109) obtained in this study. In an investigation into the effect of coliform organisms on broiler growth, Bogdonoff et al. (1959) obtained aerobic coliform counts of 1.07 x 108 on Eosin Methylene Blue Agar from the defecated feces of chicks fed 200 grams per ton OTC in a modified broiler ration. These counts are similar to
2151
OXYTETRACYCLINE AND FECAL MICROORGANISMS
KX
8C
60
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2C E. K c L E B
— K A B
P S E A K U R L L) 1 E Z B 5 Weeks
E. c
K B 1 E E B A
FIG. 2. Percentages of predominant microorganisms—Low level OTC Abbreviations: Citro, Citrobacter; Ariz, Arizona; Kleb, Klebsiella; Be-Ba, Bethesda-Ballerup group, E. coli, Escherichia coli; Entero; Enterobacter. All figures refer to composite gut and defecated fecal samples. the results in this study obtained by using Mac and He selective media. Species o/Enterobacteriaceae Isolated. The percentages of isolates that belonged to various Enterobacteriaceae are shown in Table 2. Escherichia coli, comprising 54% of the isolates, appeared to be the predominant microorganism in defecated poultry feces. Significant numbers of Klebsiella and the Bethesda-Ballerup paracolon group, (15 and 16%, respectively), were also found. No salmonellae were isolated although Arizona sp. (an avian and human pathogen) was isolated from the defecated feces of all three groups but not from the intestinal samples. Analysis of the feed and water supplies showed both to be free of any enteric organisms.
Effect of OTC on Genera Isolated. The effect of OTC on the percentages of Enterobacteriaceae isolated is shown in Figs. 1, 2, and 3. In the control birds (Fig. 1) there was a marked preponderance of E. coli until the seventh week, when a mixed paracolon group appeared, including 20% Arizona at the end of the feeding trials. In the defecated feces of birds fed low levels of antibiotics, (Fig. 2) E. coli was less predominant, while the Klebsiella sp. and the paracolon group, including Arizona sp., were much more prevalent. E. coli was the predominant fecal organism obtained from birds fed high levels of OTC (Fig. 3) during the early weeks of the trial, but E. coli disappeared by the eighth week to be replaced by Klebsiella and Citrobacter. Finegold (1970) reported a similar change in
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R
B E B A
001
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40
E. c
o
E
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O
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2152
BALDWIN, BROMEL, AIRD, JOHNSON AND SELL
10C
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E. c o I i
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Weeks FIG. 3. Percentages of predominant microorganisms—High level OTC Abbreviations: Citro, Citrobacter; Ariz, Arizona; Kleb, Klebsiella; Be-Ba, Bethesda-Ballerup group, E. coli, Escherichia coli; Entero, Enterobacter. All figures refer to composite gut and defecated fecal samples.
intestinal flora in human patients treated with penicillin or the tetracyclines; namely, a shift to Klebsiella, Enterobacter and the intermediate (paracolon) coliforms. The appearance of the Klebsiella sp. may indicate a selective pressure for the more OTC resistant Klebsiella organisms by the antibiotic. Conversely, the avian pathogen Arizona appeared only during the fifth week in high level antibiotic feces and may have subsequently been eliminated by the antibiotic. Sieburth et al. (1952) compared the fecal microflora of OTC fed poults to that of control poults. They reported an increase in Proteus sp. and a decrease in coliform numbers which they attributed to the OTC.
Our data show a decrease in typical E. coli isolates, but no Proteus sp. were isolated at any time. Antibiogram Sensitivities. Fifty-four fecal E. coli isolates were tested for OTC sensitivity and sensitivity to six of the antibiotics often used by the animal industry. It is evident from Table 3 that, regardless of the level of OTC fed, 100% of the isolates tested were resistant to penicillin and bacitracin. The percentage resistance among E. coli isolates was 15 units higher in defecated feces from birds fed the high level of OTC than from the control group fed no OTC. The high levels of bacteria showing multiple antibiotic resistances in the control birds may have been
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4C
2153
OXYTETRACYCLINE AND F E C A L MICROORGANISMS
TABLE 3 . -
- Antimicrobial resistance patterns of 54 E. coli isolates
Antimicrobial agent Oxytetracycline Bacitracin Penicillin Ampicillin Gantricin Aureomycin Streptomycin
% Resistance Control— Low— High— Og./T 50g./T 200g./T 100 93 85 100 100 100 100 100 100 36 22 0 57 36 54 100 100 96 72 79 81
TABLE 4.-—Percent
g-/T OTC fed 0 50 200
#Org. isolated 36 38 29
OTC 87 89 97
antimicrobial resistance of fecal isolates
Pen 100 100 100
Antimicrobial agents Bac Aur Amp 100 92 14 100 100 37 100 97 41
Gan 56 47 55
Str 83 74 66
Abbreviations: OTC, oxytetracycline; Pen, penicillin; Bac, bacitracin; Aur, aureomycin; Amp, ampicillin; Gan, gantrisin; Str, streptomycin.
Downloaded from http://ps.oxfordjournals.org/ by guest on November 22, 2014
due to the oral medication (including spectinomycin and vitamin supplements) given to the two-day old poults before leaving the hatchery. The most interesting increase in resistance was to Ampicillin. There were no resistant orgainsms from defecated feces of control birds but 36% of the organisms from birds fed high levels of antibiotic had developed resistance. Ampicillin is not used by the poultry industry but is widely employed in human medicine. Resistance to streptomycin decreased by about 10 units while Gantrisin (sulfisoxazole) resistance was not affected by the feeding of OTC. The increasing number of disease conditions in poultry attributed to E. coli cannot be overlooked (Bentley, 1970). McCune and Russell (1972) estimated that 542,000 birds were known to be infected with E. coli manifested as enteritis, osteomyelitis, septicemia or airsacculitis. McCune and Russell also stated that antibiotic treatment with
tetracyclines, streptomycin and other drugs cleared for use by the Food and Drug Administration appear to be ineffective in over half the cases. Our data indicate not only a high percentage of naturally occurring multiple resistant strains of E. coli, but also an increase in multiple resistance in E. coli strains following the feeding of OTC. The feeding of dietary antibiotics may be selective for some of the antibiotic resistant, opportunistic E. coli strains responsible for the disease outbreaks that have been attributed to this microorganism. Table 4 shows a summary of the antibiotic resistance of all the enteric genera isolated from defecated feces in this study. The pattern is the same as for E. coli; namely, increasing OTC resistance and increasing Ampicillin resistance with increased levels of OTC fed, no change in Gantrisin resistance and decreased streptomycin resistance. The latter may be due to the influence of OTC on segregration of the streptomycin determinant (Watenabe, 1967). Insufficient numbers of gut isolates made a significant survey of their resistance patterns difficult. In general, their resistance patterns were similar to those of the defecated fecal isolates. Although the amount of OTC fed did not appear to effect any significant change in the total numbers of bacteria present in defecated turkey feces, except at high levels where it decreased the anaerobic count, the poults on high levels of OTC showed a significant increase in feed efficiency.
2154
BALDWIN, BROMEL, AIRD, JOHNSON AND SELL
REFERENCES
NEWS AND NOTES (Continued from page 2141)
reputation while at K.S.U. by discovering a method to determine the sex of baby chicks by feather characteristics, and also did pioneering work on hybrid vigor and inheritance of rate of feathering, he was the recipient of K.S.U.'s Distinguished Service Award in Agriculture in 1972, one of the few individuals to be so honored who have not received degrees from K.S.U. Warren, Scott, and others collaborated during the 1930's at K.S.U. on research into egg formation in the body of the hen. These investigations led to revision of all textbook materials on the subject, and resulted
in preparation of a motion picture film showing actual ovulation and formation of an egg as it passed through the different stages. With Warren and Scott providing the technical expertise and Floyd Hanna, K-State photographer, doing the filming, the three produced a 13-minute, 16 millimeter color film in 1939 that still is the finest film available on the subject. Warren, Scott, and other members of the K.S.U. poultry staff also collaborated with K-State scientists in such areas as biochemistry, zoology, bacteriology, and veterinary medicine to make contributions in
(Continued on page 2165)
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Aird, D., 1973. Antibiotic resistance in bacterial isolates from turkey livers and blood. M.S. Thesis, North Dakota State University Library, Fargo, North Dakota. American Public Health Association, 1971. Standard Methods for the Examination of Water and Wastewater. 13th ed. American Public Health Association, Inc., New York. Anderson, E. S., 1965. Origin of transferable drug resistance in the Enterobacteriaceae. Brit. Med. J. 2: 1289-1291. Bauer, A. W., C. E. Roberts and W. Kirby, 1966. Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Path. 45: 493-496. Bentley, O. E., 1970. E. coli—Ever present opportunist. Poultry Digest, 29: 324-327. Bogonoff, P. D., J. M. Pensack, J. N. Nelson and R. S. Baldwin, 1959. Effect of coliform organisms on broiler growth. Poultry Sci. 38: 1355-1366. Coles, F. T., 1973. Bacteriological studies of confinement animal waste and survival studies of indicator bacteria in animal waste lagoons. M.S. Thesis, North Dakota State University Library, Fargo, North Dakota. Datta, N., M. C. Fraiers, D. S. Reeves, W. Brumfitt, F. Orskov and I. Orskov, 1971. R factors in Escherichia coli in feces after oral chemotherapy in general practice. Lancet, 1: 312-315. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Edwards, P. R., and W. H. Ewing, 1972. Identification of Enterobacteriaceae. Burgess Pub. Co., Minneapolis.
Finegold, S. M., 1970. Interaction of antimicrobial therapy and intestinal flora. Am. J. CI. Nutr. 23: 1466-1471. Goodling, A. C , R. S. Woodward and A. B. Watts, 1972. Antibiotic feeding for market egg production. Louisiana State Univ. Bull. 16(2): 8-9. Guinee, P. A. M., 1970. Resistance transfer to the resident intestinal Escherichia coli of rats. J. Bacterid. 102:291-292. Jacobs, R. L., J. F. Elam and J. R. Couch., 1955. Effect of administering antibiotics upon egg production, growth and antibiotic-resistant microorganisms. Poultry Sci. 34: 1232. Lockhart, W. C , R. L. Bryant and D. W. Bolin, 1963. A shifting type metabolism pen. North Dakota Farm Res. Bull., 23(1): 16-19. Lovett, J., J. Messer and R. Read, Jr., 1971. The microflora of Southern Ohio poultry litter. Poultry Sci. 50:746-751. Lucas, I. A., 1972. Food additives and contaminants. Proc. Nutr. Soc. 31: 1-8. McCune, E. I., and W. Russell, 1972. Resistant E. coli strains . . . a major disease problem. Poultry Digest, 31: 137-138. Rhodes, R. A., and G. R. Hrubrant, 1972. Microbial population of feedlot waste and associated sites. App. Microbiol. 24: 369-377. Sieburth, J. M., J. McGinnis and C. E. Skinner, 1952. The effect of Terramycin on the antagonisms of certain bacteria against species of Proteus. J. Bacterid. 64: 163-169. Smith, H. W., 1968. Anti-microbial drugs in animal feeds. Nature 218: 728-731. Watenabe, T., 1967. Infectious drug resistance. Sc. Am. 217: 19-27.
RACHITOGENIC EFFECTS OF GRAIN
obtained at the low level of vitamin D 3 in the first experiment. Triticale, a hybrid between wheat and rye, was found to have a rachitogenic effect similar to that of rye. Wheat did not differ from corn in its effect on chick bone ash.
Association of Official Agricultural Chemists, 1970. Official Methods of Analysis, Eleventh ed., Washington, D.C. Duncan, D. B., 1955. Multiple range and multiple F test. Biometrics, 11: 1-42. Fernandez, R., E. Lucas and J. McGinnis, 1973. Fractionation of a chick growth depressing factor from rye. Poultry Sci. 52: 2252-2259. MacAuliffe, T., A. Pietraszek and J. McGiniis, 1976. The effect of adding procaine penicillin to diets with different levels of vitamin D 3 on the performance of turkey poults. Poultry Sci. 55: 183-187. National Academy of Sciences, National Research Council, 1971. Nutrient Requirements of Poultry. Sixth revised ed., Washington, D.C. Steel, G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York, NY.
Effect of Dietary Oxy tetracycline on Microorganisms in Turkey Feces 1 B . B . BALDWIN, M. C. BROMEL, D . W . A I R D , R. L . JOHNSON AND J. L .
SELL.
Departments of Bacteriology and Animal Science, North Dakota State University, Fargo, North Dakota 58102 (Received for publication December 3, 1975)
ABSTRACT Thirty-six two-day old male turkey poults were divided into three groups and fed three levels of oxytetracycline; 0, 110 or 440 mg. per kg. of ration, respectively, for nine weeks. The poults were given an oral spectinomycin plus vitamin treatment at the hatchery but were not fed before the start of the experiment. Defecated feces from each group were sampled at weekly intervals and analyzed for bacteriological content, especially for members of the Enterobacteriaceae. Total aerobic counts of 3 x 108 and total anaerobic counts of 2 x 109 were obtained. The majority of the Enterobacteriaceae were Escherichia coli but some Klebsiella sp., the Bethesda-Ballerup group of paracolon bacteria and Arizona sp. were also isolated. Antibiograms of the enteric isolates indicated that an increase in multiple antibiotic resistance occurred when increased amounts of antibiotic were fed. POULTRY SCIENCE 55: 2147-2154, 1976
INTRODUCTION
A
NTIBIOTICS have been used by the poultry industry as feed additives (singly and in combination) for over 20 years. Good-
1. Published with the approval of the Director of the North Dakota Agricultural Experiment Station as Journal Article No. 000.
ling et al. (1972) and Lucas (1972) have shown that increased feed efficiency, better egg production and better disease control can still be demonstrated from the continued use of dietary antibiotics. However, Smith (1968), Datta (1971), Anderson (1965), and Guinee (1970) have voiced concern regarding the development of multiple antibiotic resistant bacteria in domestic animals due to the use
Downloaded from http://ps.oxfordjournals.org/ by guest on November 22, 2014
The bone ash results obtained in Experiment 3 show clearly that a factor in rye which depresses bone mineralization is extractable with water and that acid autoclaving of the rye destroys or inactivates this factor, especially when penicillin is also added to the diet. In subsequent trials not reported in this paper, significant increases in bone ash have been obtained by acid autoclaving the rye. The factor in rye that appears to interfere with vitamin D 3 utilization by chicks and turkeys is under investigation.
REFERENCES
2148
BALDWIN, BROMEL, AIRD, JOHNSON AND SELL
MATERIALS AND METHODS Test Animals. Thirty-six two-day old male turkey poults (previously treated at the hatchery with a single oral dose of spectinomycin and vitamins) were divided into twelve groups. Each group of three poults was kept in an individual metabolism cage designed to facilitate collection of excreta (Lockhart, 1963). The cages, water pans and feed pans were steam sterilized prior to the experiment. Each of three ration treatments was assigned to four groups of poults. The ration treatments consisted of: 1) Control, a standard starter ration (Table 1) with no antibiotics; 2) starter ration plus 110 mg. of oxytetracycline (OTC) per kg. and 3) starter ration plus 440 mg. OTC per kg. Sampling Procedures. The poults were weighed weekly and composite samples of defecated feces (eight hour accumulations) were collected from each of the treatment groups of three birds with a sterile spatula and transmitted to the laboratory in sterile specimen jars. At the termination of the study at nine weeks, the poults were killed and
TABLE 1.—Starter mash Ingredient Percent Ground yellow corn 23.70 Barley 10.00 Soybean meal (48% protein) 51.00 Animal tallow 5.25 Meat and bone meal (50% protein) 4.00 Dehydrated alfalfa meal (17% protein) 2.00 Dicalcium phosphate 2.00 Ground limestone 1.45 DL methionine 0.05 Vitamin premixa 0.25 Trace mineral premixb 0.30 a The vitamin premix supplied the following vitamins: (mg./kg. diet) menadione, 1; riboflavin, 6; pantothenic acid, 22; niacin, 75; choline, 750; DL methionine, 0.5 g; vitamin A, 7500 I.U.; vitamin D 3 , 1500 I.C.U.; and vitamin E, 6 I.U. b The trace mineral premix supplied the following trace minerals: (mg./kg. diet) manganese, 50; zinc, 50; iron, 30; copper, 5; iodized salt, 2600. livers examined and removed asceptically into sterile specimen jars for further study (Aird, 1973). Gut samples were taken at autopsy with a sterile swab after an incision into the mid section portion of the small intestine with a sterile scalpel. Plate Counts. Defecated feces were mixed with a sterile mortar and pestle and then homogenized in 99 ml. sterile water in a Waring blender for two minutes. Dilutions were made in sterile water and plated in triplicate for total aerobic counts on Trypticase Soy Agar (TSA) from Baltimore Biological Laboratories, (BBL) and for enteric counts on MacConkey's Agar (Mac, BBL) and Hektoen Enteric Agar (He, Difco). Plates were incubated at 37° C. for 24 hours. Plates of TSA plus 5% defibrinated sheep blood were incubated in a H 2 : C 0 2 atmosphere (Gas Pak, BBL) at 35° C. for 5 days to determine total anaerobic counts. Enrichment Procedures for Salmonella. One gram of ground composite defecated fecal
Downloaded from http://ps.oxfordjournals.org/ by guest on November 22, 2014
of antibiotics for growth promotion and prevention of disease. Little data are available relative to the numbers and types of bacteria present in poultry feces and the effect thereon of dietary antibiotics. This lack of information has compounded problems involved in adequate disposal or utilization of poultry feces. The following study was designed to determine the effect of dietary oxytetracycline on the microflora in the intestinal digesta and in the defecated feces. Concurrently, the development of multiple resistant bacteria in defecated feces was also studied with special attention given to the Enterobacteriaceae because of their traditional use as indicators of potential public health hazards.
OXYTETRACYCLINE AND FECAL MICROORGANISMS
material was added to 9 ml. of Selinite Cystine Enrichment Growth (BBL). After 12 to 15 hours incubation at 41° C , cultures were streaked on Mac and incubated at 37° C. for 24 to 48 hours. Feed Analysis. Feed from each of the regimens was routinely tested for bacterial contamination following the protocol employed for fecal samples. The feed was received in paper bags and one batch of feed sufficed for the nine week experiment.
Gut Swabs. A sterile swab was inserted into the asceptically incised gut at autopsy, then immediately immersed in Trypticase Soy Broth (BBL) which served as an enrichment medium. Cultures were incubated 24 hours at 37° C. and subsequently streaked on Mac and TSA. Identification of Enterobacteriaceae. A minimum of four isolates from the composite defecated feces of each group fed the three levels of OTC were chosen weekly by colonial characteristics and fermentation reactions on Mac and He. A total of 120 gram negative glucose fermenting bacterial isolates obtained from Mac and He media, from enrichment cultures of defecated samples and from incised gut swabs were randomly chosen for identification. These representative isolates were then transferred to Kligler's Iron Agar (BBL) after which the following biochemical tests were used for the identification of the Enterobacteriaceae (Ewing and Edwards, 1972): glucose, lactose and dulcitol fermentations; motility, indole and sulfide production on Sulfide Indole Motility Medium; urease production on Urease Test Medium; lysine
decarboxylase on Lysine Iron Agar; phenylalanine deamination on Phenylalanine Agar (all BBL); citrate utilization on Simmons Citrate Agar and ornithine decarboxylation on Motility Indole Ornithine Media (both Difco). Possible Salmonella isolates were serotyped with Salmonella O antiserum polyvalent (BBL). Antibiogram Assay. Enteric isolates were assayed for drug resistance by the disc method of Bauer (1966) for their sensitivities to the following levels of antimicrobial agents: penicillin, 10 units; bacitracin, 10 units; aureomycin, 20 meg.; OTC, 30 meg.; ampicillin, 10 units; gantrisin, 300 meg.; and streptomycin, 10 units.
RESULTS AND DISCUSSION Weight Gain and Feed Efficiency. Gain in body weight was not affected significantly by including OTC in the ration. However, poults fed the ration containing 440 mg. OTC per kg. required significantly (P < 0.01) less feed per kg. of gain (1.94 kg.) than did poults fed no antibiotic (2.11 kg.). Feed efficiency was also improved slightly by 100 mg. OTC per kg. of ration but not significantly (Duncan, 1955). Total Counts. Problems encountered in feeding the two-day old poults made proper collection of feces impossible until the birds were nine days old. All counts (aerobic and anaerobic) showed considerable variation for the first four weeks. After the fifth week the total aerobic counts from TSA of the defecated feces of all three regimens stabilized at 3 x 108 organisms per gram while the gram negative counts from Mac and He stabilized at 5 x 107 cells per gram. Only in the anaerobic count (TSA plus 5% def ibrinated sheep blood) was there any variation that could possibly be attributed to the
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Water Analysis. The tap water supply and a composite sample from the watering troughs were tested weekly for bacterial contamination, employing procedures outlined by the American Public Health Association (1971).
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TABLE 2.—Enterobacteriaceae isolates (From composite defecated feces or gut samples)
Organism E. coli Klebsiella Beth-Ballerup Citrobacter Arizona Enterobacter Pseudomonas
Defecated feces 54 16 16 6 5 4 2
Gut 10 2 2 3
— — —
% of 120 isolates 53 15 15 8 4 3 2
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FIG. 1. Percentages of predominant microorganisms—Control Abbreviations: Citro, Citrobacter; Ariz, Arizona; Kleb, Klebsiella; Be-Ba, Bethesda-Ballerup group; E. coli, Escherichia coli; Entero, Enterobacter. All figures refer to composite gut and defecated fecal samples.
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amount of OTC fed. Both control and low level groups levelled off at 3 x 109 while the anaerobic count from the feces of birds on the high antibiotic regimen decreased to 8 x 108 organisms per gram which is consistent with one of the reported benefits of antibiotic supplementation of feed (Jacobs et al., 1955).
The bacterial counts in defecated turkey feces were in the same range as those obtained from defecated cattle feces in this laboratory (Coles, 1973) and from cattle feedlot wastes as reported by Rhodes et al. (1972). Lovett et al. (1971) reported total counts in poultry litter to be approximately 108 per gram both aerobically and anaerobically. Differences between litter and defecated feces as well as differences in media and anaerobic methodology may have contributed to the higher anaerobic counts (4 x 10 8 tol x 109) obtained in this study. In an investigation into the effect of coliform organisms on broiler growth, Bogdonoff et al. (1959) obtained aerobic coliform counts of 1.07 x 108 on Eosin Methylene Blue Agar from the defecated feces of chicks fed 200 grams per ton OTC in a modified broiler ration. These counts are similar to
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OXYTETRACYCLINE AND FECAL MICROORGANISMS
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FIG. 2. Percentages of predominant microorganisms—Low level OTC Abbreviations: Citro, Citrobacter; Ariz, Arizona; Kleb, Klebsiella; Be-Ba, Bethesda-Ballerup group, E. coli, Escherichia coli; Entero; Enterobacter. All figures refer to composite gut and defecated fecal samples. the results in this study obtained by using Mac and He selective media. Species o/Enterobacteriaceae Isolated. The percentages of isolates that belonged to various Enterobacteriaceae are shown in Table 2. Escherichia coli, comprising 54% of the isolates, appeared to be the predominant microorganism in defecated poultry feces. Significant numbers of Klebsiella and the Bethesda-Ballerup paracolon group, (15 and 16%, respectively), were also found. No salmonellae were isolated although Arizona sp. (an avian and human pathogen) was isolated from the defecated feces of all three groups but not from the intestinal samples. Analysis of the feed and water supplies showed both to be free of any enteric organisms.
Effect of OTC on Genera Isolated. The effect of OTC on the percentages of Enterobacteriaceae isolated is shown in Figs. 1, 2, and 3. In the control birds (Fig. 1) there was a marked preponderance of E. coli until the seventh week, when a mixed paracolon group appeared, including 20% Arizona at the end of the feeding trials. In the defecated feces of birds fed low levels of antibiotics, (Fig. 2) E. coli was less predominant, while the Klebsiella sp. and the paracolon group, including Arizona sp., were much more prevalent. E. coli was the predominant fecal organism obtained from birds fed high levels of OTC (Fig. 3) during the early weeks of the trial, but E. coli disappeared by the eighth week to be replaced by Klebsiella and Citrobacter. Finegold (1970) reported a similar change in
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BALDWIN, BROMEL, AIRD, JOHNSON AND SELL
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Weeks FIG. 3. Percentages of predominant microorganisms—High level OTC Abbreviations: Citro, Citrobacter; Ariz, Arizona; Kleb, Klebsiella; Be-Ba, Bethesda-Ballerup group, E. coli, Escherichia coli; Entero, Enterobacter. All figures refer to composite gut and defecated fecal samples.
intestinal flora in human patients treated with penicillin or the tetracyclines; namely, a shift to Klebsiella, Enterobacter and the intermediate (paracolon) coliforms. The appearance of the Klebsiella sp. may indicate a selective pressure for the more OTC resistant Klebsiella organisms by the antibiotic. Conversely, the avian pathogen Arizona appeared only during the fifth week in high level antibiotic feces and may have subsequently been eliminated by the antibiotic. Sieburth et al. (1952) compared the fecal microflora of OTC fed poults to that of control poults. They reported an increase in Proteus sp. and a decrease in coliform numbers which they attributed to the OTC.
Our data show a decrease in typical E. coli isolates, but no Proteus sp. were isolated at any time. Antibiogram Sensitivities. Fifty-four fecal E. coli isolates were tested for OTC sensitivity and sensitivity to six of the antibiotics often used by the animal industry. It is evident from Table 3 that, regardless of the level of OTC fed, 100% of the isolates tested were resistant to penicillin and bacitracin. The percentage resistance among E. coli isolates was 15 units higher in defecated feces from birds fed the high level of OTC than from the control group fed no OTC. The high levels of bacteria showing multiple antibiotic resistances in the control birds may have been
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OXYTETRACYCLINE AND F E C A L MICROORGANISMS
TABLE 3 . -
- Antimicrobial resistance patterns of 54 E. coli isolates
Antimicrobial agent Oxytetracycline Bacitracin Penicillin Ampicillin Gantricin Aureomycin Streptomycin
% Resistance Control— Low— High— Og./T 50g./T 200g./T 100 93 85 100 100 100 100 100 100 36 22 0 57 36 54 100 100 96 72 79 81
TABLE 4.-—Percent
g-/T OTC fed 0 50 200
#Org. isolated 36 38 29
OTC 87 89 97
antimicrobial resistance of fecal isolates
Pen 100 100 100
Antimicrobial agents Bac Aur Amp 100 92 14 100 100 37 100 97 41
Gan 56 47 55
Str 83 74 66
Abbreviations: OTC, oxytetracycline; Pen, penicillin; Bac, bacitracin; Aur, aureomycin; Amp, ampicillin; Gan, gantrisin; Str, streptomycin.
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due to the oral medication (including spectinomycin and vitamin supplements) given to the two-day old poults before leaving the hatchery. The most interesting increase in resistance was to Ampicillin. There were no resistant orgainsms from defecated feces of control birds but 36% of the organisms from birds fed high levels of antibiotic had developed resistance. Ampicillin is not used by the poultry industry but is widely employed in human medicine. Resistance to streptomycin decreased by about 10 units while Gantrisin (sulfisoxazole) resistance was not affected by the feeding of OTC. The increasing number of disease conditions in poultry attributed to E. coli cannot be overlooked (Bentley, 1970). McCune and Russell (1972) estimated that 542,000 birds were known to be infected with E. coli manifested as enteritis, osteomyelitis, septicemia or airsacculitis. McCune and Russell also stated that antibiotic treatment with
tetracyclines, streptomycin and other drugs cleared for use by the Food and Drug Administration appear to be ineffective in over half the cases. Our data indicate not only a high percentage of naturally occurring multiple resistant strains of E. coli, but also an increase in multiple resistance in E. coli strains following the feeding of OTC. The feeding of dietary antibiotics may be selective for some of the antibiotic resistant, opportunistic E. coli strains responsible for the disease outbreaks that have been attributed to this microorganism. Table 4 shows a summary of the antibiotic resistance of all the enteric genera isolated from defecated feces in this study. The pattern is the same as for E. coli; namely, increasing OTC resistance and increasing Ampicillin resistance with increased levels of OTC fed, no change in Gantrisin resistance and decreased streptomycin resistance. The latter may be due to the influence of OTC on segregration of the streptomycin determinant (Watenabe, 1967). Insufficient numbers of gut isolates made a significant survey of their resistance patterns difficult. In general, their resistance patterns were similar to those of the defecated fecal isolates. Although the amount of OTC fed did not appear to effect any significant change in the total numbers of bacteria present in defecated turkey feces, except at high levels where it decreased the anaerobic count, the poults on high levels of OTC showed a significant increase in feed efficiency.
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REFERENCES
NEWS AND NOTES (Continued from page 2141)
reputation while at K.S.U. by discovering a method to determine the sex of baby chicks by feather characteristics, and also did pioneering work on hybrid vigor and inheritance of rate of feathering, he was the recipient of K.S.U.'s Distinguished Service Award in Agriculture in 1972, one of the few individuals to be so honored who have not received degrees from K.S.U. Warren, Scott, and others collaborated during the 1930's at K.S.U. on research into egg formation in the body of the hen. These investigations led to revision of all textbook materials on the subject, and resulted
in preparation of a motion picture film showing actual ovulation and formation of an egg as it passed through the different stages. With Warren and Scott providing the technical expertise and Floyd Hanna, K-State photographer, doing the filming, the three produced a 13-minute, 16 millimeter color film in 1939 that still is the finest film available on the subject. Warren, Scott, and other members of the K.S.U. poultry staff also collaborated with K-State scientists in such areas as biochemistry, zoology, bacteriology, and veterinary medicine to make contributions in
(Continued on page 2165)
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Aird, D., 1973. Antibiotic resistance in bacterial isolates from turkey livers and blood. M.S. Thesis, North Dakota State University Library, Fargo, North Dakota. American Public Health Association, 1971. Standard Methods for the Examination of Water and Wastewater. 13th ed. American Public Health Association, Inc., New York. Anderson, E. S., 1965. Origin of transferable drug resistance in the Enterobacteriaceae. Brit. Med. J. 2: 1289-1291. Bauer, A. W., C. E. Roberts and W. Kirby, 1966. Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Path. 45: 493-496. Bentley, O. E., 1970. E. coli—Ever present opportunist. Poultry Digest, 29: 324-327. Bogonoff, P. D., J. M. Pensack, J. N. Nelson and R. S. Baldwin, 1959. Effect of coliform organisms on broiler growth. Poultry Sci. 38: 1355-1366. Coles, F. T., 1973. Bacteriological studies of confinement animal waste and survival studies of indicator bacteria in animal waste lagoons. M.S. Thesis, North Dakota State University Library, Fargo, North Dakota. Datta, N., M. C. Fraiers, D. S. Reeves, W. Brumfitt, F. Orskov and I. Orskov, 1971. R factors in Escherichia coli in feces after oral chemotherapy in general practice. Lancet, 1: 312-315. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Edwards, P. R., and W. H. Ewing, 1972. Identification of Enterobacteriaceae. Burgess Pub. Co., Minneapolis.
Finegold, S. M., 1970. Interaction of antimicrobial therapy and intestinal flora. Am. J. CI. Nutr. 23: 1466-1471. Goodling, A. C , R. S. Woodward and A. B. Watts, 1972. Antibiotic feeding for market egg production. Louisiana State Univ. Bull. 16(2): 8-9. Guinee, P. A. M., 1970. Resistance transfer to the resident intestinal Escherichia coli of rats. J. Bacterid. 102:291-292. Jacobs, R. L., J. F. Elam and J. R. Couch., 1955. Effect of administering antibiotics upon egg production, growth and antibiotic-resistant microorganisms. Poultry Sci. 34: 1232. Lockhart, W. C , R. L. Bryant and D. W. Bolin, 1963. A shifting type metabolism pen. North Dakota Farm Res. Bull., 23(1): 16-19. Lovett, J., J. Messer and R. Read, Jr., 1971. The microflora of Southern Ohio poultry litter. Poultry Sci. 50:746-751. Lucas, I. A., 1972. Food additives and contaminants. Proc. Nutr. Soc. 31: 1-8. McCune, E. I., and W. Russell, 1972. Resistant E. coli strains . . . a major disease problem. Poultry Digest, 31: 137-138. Rhodes, R. A., and G. R. Hrubrant, 1972. Microbial population of feedlot waste and associated sites. App. Microbiol. 24: 369-377. Sieburth, J. M., J. McGinnis and C. E. Skinner, 1952. The effect of Terramycin on the antagonisms of certain bacteria against species of Proteus. J. Bacterid. 64: 163-169. Smith, H. W., 1968. Anti-microbial drugs in animal feeds. Nature 218: 728-731. Watenabe, T., 1967. Infectious drug resistance. Sc. Am. 217: 19-27.
