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Effect of Oxytetracycline as a Turkey Feed Additive1
ENVIRONMENT AND HEALTH Effect of Oxytetracycline as a Turkey Feed Additive1 J. L. SWEZEY,2 B. B. BALDWIN, and M. C. BROMEL Department of Bacteriology, North Dakota State University, Fargo, North Dakota 58105 (Received for publication March 26, 1980)
1981 Poultry Science 60:738-743
mented feed (Sanger et al, 1956), Turkeys are routinely fed antibiotics for growth promotion and disease prevention. The impact of such practice on increased resistance in bacteria from internal organs, as well as the increased number of abnormalities in internal organs, has not been fully documented. Studies in this area were considered important in the evaluation of the use of antibiotics as feed supplements in turkey husbandry. The present study reports on the increase in drug resistant organisms in blood and liver of turkeys fed oxytetracycline-supplemented feed and documents an increase in liver abnormalities as a result of such practice.
INTRODUCTION
The use of antibiotics as a feed additive for growth promotion and disease control in domestic animals has increased over the past years. This practice has resulted in an increase in antibiotic resistance by the intestinal bacteria of domestic animals (Siegel et al, 1974). In addition to the increase in resistance, it has been demonstrated that antibiotics may have a detrimental effect on the animal body. Forsgren and Gnarpe (1973) reported that the use of tetracycline caused an inhibition of immune globulin synthesis. Administration of streptomycin to poultry has been correlated with prolongation of the carrier state following acute infections with salmonellae (Garside et al, 1960). Furthermore, as a result of chlorotetracycline feeding, therapeutic failure of this drug was documented in cases of colibacillosis in pigs (Dey et al, 1977). The feeding of chlorotetracycline resulted in increased shedding of salmonellae and enhanced the severity of disease in calves (Dey et al, 1978).
MATERIALS AND METHODS
Limited research has been conducted on the increased resistance of the bacteria isolated from internal organs of domestic animals fed antibiotic-supplemented diet. However, a higher incidence of liver abscesses was reported when chickens were given sulfaquinoxaline-supple-
1 Published with the approval of the Director of the North Dakota Agricultural Experiment Station as Journal Article No. 1078. 2 Present Address: US Department of Agriculture, Northern Regional Research Center, AR, SEA, 1815 North University Street, Peoria, IL 61604.
Housing and Feeding of Birds. The study was conducted at the Poultry Research Center of North Dakota State University. The cages and pens met the specifications of turkey husbandry. Day-old male poults (Nicholas Strain) were obtained from Swift and Co. (Detroit Lakes, MN). The birds were initially housed in 38 C incubators. The temperature of the incubators was decreased 3 C each week for 2 weeks after which the birds were transferred to starter cages at 23 C for 10 days. At 21 days the birds were transferred into i x 6 m pens and held for the remainder of the study period. The 207 birds were divided randomly into nine different cages, with three cages designated for each of the three levels of antibiotic supplement. A standard poultry feed (Gold Label Feed Co., West Fargo, ND) consisting of soybean meal, corn, and barley was fed to the poults for the 18-week experiment. The OTC (Sigma Chemi-
738
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 9, 2015
ABSTRACT Turkeys were given oxytetracycline (OTC) as a feed supplement at levels of 0 g, 50 g, and 200 g OTC per ton of feed. After 8, 16, and 18 weeks the birds were sacrificed and bacteria were isolated from heart blood and liver tissue. When these isolates were tested for resistance against eight antibiotics, a direct correlation was observed between the level of supplement and the level of antibiotic resistance in the bacterial isolates. In postmortem studies on the livers from birds consuming both levels of OTC, an increase in the occurrence of lesions was observed. The livers from control birds appeared to have more granulomas. (Key words:
OXYTETRACYCLINE AS A FEED ADDITIVE
Specimen Collection. After 8 and 16 weeks of the study, 4 birds from each pen were sacrificed; at 18 weeks, 8 birds from each pen were sacrificed. Heart punctures were performed with sterile 5 cc syringes on every other bird immediately before slaughter. Of this blood, 2 ml were cultured in Trypticase Soy Broth (TSB) (BBL) tubes for the enumeration of aerobic organisms and 2 ml aliquots were also cultured into Thioglycollate medium (Difco Laboratories, Detroit, MI) for the enumeration of anaerobic or facultative anaerobic organisms. The liver from each bird was aseptically removed and placed in sterile Whirl Pak Bags (Difco) for transportation to the laboratory where the livers were examined for gross abnormalities. Following necropsy, 10 g of liver were aseptically removed and placed into TSB for enumeration of aerobes. A separate 10 g portion of tissue was placed in Cooked Meat medium (Difco) for isolation of anaerobes. After incubation, the TSB with blood and liver tissue samples were subcultured to Trypticase
Soy Agar (TSA) (BBL) plates. Bacteria from the Thioglycollate and Cooked Meat medium were subcultured to TSA plates containing 5% defibrinated sheep blood. The latter plates were incubated anaerobically. RESULTS
The antibiotic resistance patterns of the bacteria isolated at 8, 16, and 18 weeks from the blood and liver samples of turkeys fed different levels of OTC are given in Tables 1,2, and 3. From each group of birds, the percentages of antibiotic resistance from bacterial isolates within each supplement feeding group were obtained by dividing the actual number of resistant organisms by the maximum possible resistance. The maximum resistance was obtained by multiplying the number of bacteria isolated from that group by the number of antibiotics tested (8). At 8 weeks, 6 bacteria were isolated from the liver and blood of 4 birds not receiving antibiotic supplement. These isolates were resistant to 29% (13 of a possible 48) of the antibiotics (Table 1). At 16 weeks, 4 isolates from 2 birds in the same feeding group were resistant to 44% (14 of 32) the antibiotics (Table 1). There were no bacteria isolated from the control group at 18 weeks. At 8 weeks, from birds fed a low-level of supplement, 5 bacteria isolated from 4 birds were resistant to 50% (20 of 40) of the antibiotics (Table 2). From this feeding group at 16 weeks, 5 isolates from 2 birds were resistant to 85% (34 of 40) of the antibiotics (Table 2). From this feeding group at 18 weeks, 8 bacteria isolated from 3 birds were resistant to 63% (40 of 64) of the antibiotics (Table 2). At 8 weeks, from birds fed a high-level of antibiotic supplement, 4 isolates from 3 birds were resistant to 69% (22 of 32) of the antibiotics (Table 3). At 16 weeks from this group of birds, 2 isolates from 2 birds were resistant to 88% (14 of 16) of the antibiotics (Table 3). There were no bacteria isolated from this group at 18 weeks. Most isolates from all three levels of supplement had high resistance against OTC as compared to other antibiotics. Tables 4, 5, and 6 list the types of gross liver abnormalities observed in birds fed OTC at different levels. In control birds the number of liver abnormalities were three at 8 weeks, one at 16 weeks, and none at 18 weeks (Table 4). The number of liver abnormalities observed in
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 9, 2015
cal Co., St. Louis, MO) was combined into the feed at rates of 0 g/ton (control group), 50 g/ton (low level), and 200 g/ton (high level). Bacteriological Procedures. Reagents and media were used in classifying the bacteria according to biochemical tests described in Bergey's Manual of Determinative Bacteriology (Breed etal., 1975). Commercial media were prepared according to manufacturer's directions. Aerobic organisms were incubated at 37 C for 24 hr except for the urease and coagulase biochemical tests, which were incubated in a 37 C water bath for 6 hr. The anaerobic or facultative anaerobic organisms were grown at 37 C for 48 hr in a Brewer's Anaerobic Jar with a Gas Pak (Baltimore Biological Laboratory, Cockeysville, MD) (BBL) and a redox indicator containing sodium bicarbonate and .001% methylene blue. The Kirby-Bauer method (Bauer et al, 1966) was applied to test the antibiotic sensitivity of isolated organisms against the following: ampicillin (10 Mg), penicillin (10 Mg), bacitracin (10 Mg), naladixic acid (30 Mg), streptomycin (10 Mg), gantricin (300 Mg), aureomycin (300 Mg), and OTC (30 Mg). All zones of inhibition produced by the standard discs against the growth of organisms were measured to the nearest millimeter. The results were interpreted for antibiotic sensitivity in accordance to the BBL Manual of Products and Laboratory Procedures (Rhode, 1968).
739
SWEZEYETAL. birds fed a low level supplement were four at 8 weeks, one at 16 weeks, and two at 18 weeks (Table 5). The number of abnormalities in the livers of turkeys fed a high-level supplement were three at 8 weeks, two at 16 weeks, and none at 18 weeks (Table 6).
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DISCUSSION Data on the feeding of OTC at various levels in turkeys indicate that use of an antibiotic as feed supplement increased the level of antibiotic resistance in bacteria present in the liver and blood. Furthermore, it was evident that the degree of resistance in isolates correlated directly to the level of antibiotic concentration administered to the birds. It has been shown in earlier studies (Smith and Crabb, 1957; Starr and Reynolds, 1951; Mercer et al, 1971) that OTC, used as a feed supplement, increased the level of multiple drug resistance in intestinal flora in various domestic animals. In control birds, the amount of bacterial resistance in blood and liver isolates increased from 29% at 8 weeks to 44% at 16 weeks (Table 1). This resistance found in turkeys not exposed to the antibiotic could be due to the development of naturally occurring resistant organisms. Similar increases in the resistance of bacteria living in nature has been documented (Mare, 1968; Smith and Crabb, 1957; Grabow and Prozesky, 1973). At 8 weeks, the liver and blood isolates from birds fed a low level of supplement demonstrated 50% resistance (Table 2). This indicates that development of increased resistance can occur by 8 weeks if birds are placed on feed supplement immediately after hatching. At 8 weeks, isolates from the liver and blood of birds fed a high level of supplement showed 72% resistance (Table 3). These results confirm the development of bacterial resistance within 8 weeks of placing the birds on supplemented feed. The resistance percentages received at the 8 week sampling period also demonstrate the direct correlation between the amount of resistance with the level of antibiotic supplement fed. The 16 week results indicate that the bacteria inhibiting the liver and blood of turkeys fed OTC supplement continue to develop resistance. After 16 weeks of consuming OTC supplement, the isolates from birds on OTC had significantly higher percentages of resistance when compared to birds not feeding on OTC. However, there was no significant difference in
S S R I R S S S S R S S R S S R R R
Staphylococcus epidermidis3 Propionibacterium acnes3 Peptococcus anaerobius3 Peptococcus aerogenes3 Bacillus cereus0 Staphylococcus epidermidis* Staphylococcus epidermidis3Streptococcus zooepidemicus" Lactobacillus acidophilus0 Peptostreptococcus anaerobius3 Bacillus psycbrosaccharolyticus0 Streptococcus avium0 Staphylococcus epidermidis3 Lactobacillus acidophilus0 Streptococcus faecalis3 Micrococcus spP Lactobacillus fermentum3 Staphylococcus epidermidis3
8 8 8 8 8 16 16 16 16 16 16 16 16 16 16 16 18 18
16 16 20 22 24 39 39 39 39 71 75 75 75 75 80 80 138 138
Indicates organism isolated from blood sample.
Indicates organism isolated from liver tissue.
wk wk wk wk wk wk wk wk wk wk wk wk wk wk wk wk wk wk
Ampicillin
Isolate
Age at slaughter
Tag no.
S R R R R R R R S S R S S S
S S R S
Aureomycin
S R S S
R R I R R R S R R S S
S
Bacitracin
R
R
S R R S S S R S R S
R R
R
S S R R S
Gantncin tricin
TABLE 2. Antibiotic resistance spectra of bacteria isolated from the liver an of birds fed 50 g OTC/ton of feed
d from http://ps.oxfordjournals.org/ at University of Manchester on May 9, 2015
742
SWEZEY ET AL.
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TABLE 4. Liver abnormalities in turkeys fed 0 g OTC/ton of feed as oi as as OS OS
Tag no.
Age at slaughter
Type of liver abnormality
5 10 12 50
8 8 8 16
Granuloma Lesion Granuloma Lesion
OS as as os as oS
.i.
c
as OS as as OS OS
Z =5
the percentages of resistance found in bacteria from birds receiving the two different levels of OTC. We were unable to explain the decrease in resistance observed in birds feeding on low levels of supplement at the 18 weeks sampling period. In observing turkey livers, the lesion is a common type of abnormality. In accordance with Dorland (1965), livers were classified as having lesions when gross abnormalities were observed with pathological or traumatic discontinuity of tissue. The liver abnormalities observed consisted of infarcts, enlargement of the organ with red-colored foci, and mottling. One type of liver lesion often found on the surface is a granuloma (Moore and Gross, 1968), which is described as a hard, light-colored, spheroid mass having a dense fibrinous lining surrounding a hard caseous core. Lesions were diagnosed as granulomas when the appearance was similar to the description given. Tables 4, 5, and 6 list the types of liver abnormalities found in the birds examined. Differences were observed when the types of abnormalities in livers of control group birds were compared to the livers of birds receiving supplement.
as as as as as os
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TABLE 5. Liver abnormalities in turkeys fed 50 g OTC/ton of feed
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Type of liver abnormality
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OXYTETRACYCLINE AS A FEED ADDITIVE TABLE 6. Liver abnormalities in turkeys fed 200 g OTC/ton of feed
Tag no.
Age at slaughter
Type of liver abnormality
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8 wk 8 wk 8 wk 16 wk 16 wk
Lesion Granuloma Lesion Lesion Lesion
The increase of drug-resistant organisms as a result of widespread use of an antibiotic as feed supplements to domestic animals should be reevaluated. Increased difficulty in disease treatment may result if levels of antibiotic resistance continue to increase due to such practice. The problem of increased organ abnormalities due to the presence of antibiotics in feed should also be further investigated. REFERENCES Bauer, A. W., C. E. Roberts, and W. Kirby, 1966. Antibiotic susceptibility testing by a standardi-
zed single disc method. Amer. J. Clin. Pathol. 45:493. Breed, R. S., E. G. D. Murray, and N. R. Smith, 1971. Bergey's manual of determinative bacteriology. 8th ed. Williams and Wilkins, Baltimore, MD. Dey, B. P., D. C. Blenden, B. C. Burton, H. E. Mercer, and R. D. Tsutakawa, 1977. Therapeutic responses of piglets to experimentally induced colibacillosis. Res. Vet. Sci. 23:340-343. Dey, B. P., D. C. Blenden, B. C. Burton, H. E. Mercer, and R. D. Tsutakawa, 1978. Influence of chlorotetracycline feeding in Salmonellosis in calves. Int. J. Zoon. 5:97-110. Dorland, W. A., 1965. American illustrated medical dictionary. W. B. Saunders Co., Philadelphia, PA. Forsgren, A., and H. Gnarpe, 1973. Tetracycline and host-defense mechanisms. Antimicrob. Agents Chemother. 3:711-715. Garside, J. S., R. F. Gordon, and J. F. Tucker, 1960. The emergence of resistant strains of Salmonella typhimurium in the tissues and alimentary tracts of chickens following the feeding of an antibiotic. Res. Vet. Sci. 1:184-199. Grabow, W. O. K., and O. W. Prozesky, 1973. Drug resistance of coliform bacteria in hospital and city sewage. Antimicrob. Agents Chemother. 3:175-180. Mare, J. L., 1968. Incidence of r-factor among gram negative bacteria in drug-free humans and animals. Nature 220:1046-1047. Mercer, H. D., D. Pocurull, S. Gaines, S. Wilson, and J. Bennett, 1971. Characteristics of antimicrobial resistance of E. coli from animals. Appl. Microbiol. 22:700-703. Moore, W. E. C , and W. B. Gross, 1968. Liver granuloma of turkeys causative agents and mechanisms of infection. Avian Dis. 12:417-422. Rhode, P. A., 1968. BBL Manual of products and laboratory procedures. 5th ed. Division of Beeton, Dickerson and Co., Cockeysville, MD. Sanger, V. S., J. Yacowitz, and E. N. Moore, 1956. Micro-pathological changes in experimental hemorrhagic syndrome in chickens fed sulfaquinoxaline and suggested cause of the disease. Amer. J. Vet. Res. 17:766-770. Siegel, D., W. G. Huber, and F. Enloe, 1974. Continuous non-therapeutic use of antibacterial drugs in feed and drug resistance of the gram negative enteric flora of food producing animals. Antimicrob. Agent Chemother. 6:697—701. Smith, H. S., and W. E. Crabb, 1957. The effect of continuous administration of diets containing low levels of tetracyclines on the incidence of drug-resistant bacteria in the feces of pigs and chickens. Vet. Rec. 69:24-30. Starr, M., and D. Reynolds, 1951. Streptomycin resistance of coliform bacteria from turkeys fed streptomycin. Amer. J. Public Health. 41:1375-1380.
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 9, 2015
At the 8-week sampling period, two livers from control birds contained granulomas, whereas only one liver with granulomas was found in the low level and one in the high level supplement group (Tables 4, 5, and 6). In the same sampling period, one lesion was found in the livers of control birds, while three livers from birds feeding on low level supplement and one liver from birds feeding on high level supplement were found to have lesions (Tables 4, 5, and 6). At the 16 and 18 week sampling periods, one liver with lesions was observed in the control birds but three livers from birds consuming a low level of supplement and three livers from birds consuming a high level of supplement were found to have lesions. These results support evidence that the feeding of OTC as a feed supplement may reduce the number of granulomas that appear on liver tissue in turkeys, but that the occurrence of liver lesions may be increased by the use of the supplement.
743
1981 Poultry Science 60:738-743
mented feed (Sanger et al, 1956), Turkeys are routinely fed antibiotics for growth promotion and disease prevention. The impact of such practice on increased resistance in bacteria from internal organs, as well as the increased number of abnormalities in internal organs, has not been fully documented. Studies in this area were considered important in the evaluation of the use of antibiotics as feed supplements in turkey husbandry. The present study reports on the increase in drug resistant organisms in blood and liver of turkeys fed oxytetracycline-supplemented feed and documents an increase in liver abnormalities as a result of such practice.
INTRODUCTION
The use of antibiotics as a feed additive for growth promotion and disease control in domestic animals has increased over the past years. This practice has resulted in an increase in antibiotic resistance by the intestinal bacteria of domestic animals (Siegel et al, 1974). In addition to the increase in resistance, it has been demonstrated that antibiotics may have a detrimental effect on the animal body. Forsgren and Gnarpe (1973) reported that the use of tetracycline caused an inhibition of immune globulin synthesis. Administration of streptomycin to poultry has been correlated with prolongation of the carrier state following acute infections with salmonellae (Garside et al, 1960). Furthermore, as a result of chlorotetracycline feeding, therapeutic failure of this drug was documented in cases of colibacillosis in pigs (Dey et al, 1977). The feeding of chlorotetracycline resulted in increased shedding of salmonellae and enhanced the severity of disease in calves (Dey et al, 1978).
MATERIALS AND METHODS
Limited research has been conducted on the increased resistance of the bacteria isolated from internal organs of domestic animals fed antibiotic-supplemented diet. However, a higher incidence of liver abscesses was reported when chickens were given sulfaquinoxaline-supple-
1 Published with the approval of the Director of the North Dakota Agricultural Experiment Station as Journal Article No. 1078. 2 Present Address: US Department of Agriculture, Northern Regional Research Center, AR, SEA, 1815 North University Street, Peoria, IL 61604.
Housing and Feeding of Birds. The study was conducted at the Poultry Research Center of North Dakota State University. The cages and pens met the specifications of turkey husbandry. Day-old male poults (Nicholas Strain) were obtained from Swift and Co. (Detroit Lakes, MN). The birds were initially housed in 38 C incubators. The temperature of the incubators was decreased 3 C each week for 2 weeks after which the birds were transferred to starter cages at 23 C for 10 days. At 21 days the birds were transferred into i x 6 m pens and held for the remainder of the study period. The 207 birds were divided randomly into nine different cages, with three cages designated for each of the three levels of antibiotic supplement. A standard poultry feed (Gold Label Feed Co., West Fargo, ND) consisting of soybean meal, corn, and barley was fed to the poults for the 18-week experiment. The OTC (Sigma Chemi-
738
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 9, 2015
ABSTRACT Turkeys were given oxytetracycline (OTC) as a feed supplement at levels of 0 g, 50 g, and 200 g OTC per ton of feed. After 8, 16, and 18 weeks the birds were sacrificed and bacteria were isolated from heart blood and liver tissue. When these isolates were tested for resistance against eight antibiotics, a direct correlation was observed between the level of supplement and the level of antibiotic resistance in the bacterial isolates. In postmortem studies on the livers from birds consuming both levels of OTC, an increase in the occurrence of lesions was observed. The livers from control birds appeared to have more granulomas. (Key words:
OXYTETRACYCLINE AS A FEED ADDITIVE
Specimen Collection. After 8 and 16 weeks of the study, 4 birds from each pen were sacrificed; at 18 weeks, 8 birds from each pen were sacrificed. Heart punctures were performed with sterile 5 cc syringes on every other bird immediately before slaughter. Of this blood, 2 ml were cultured in Trypticase Soy Broth (TSB) (BBL) tubes for the enumeration of aerobic organisms and 2 ml aliquots were also cultured into Thioglycollate medium (Difco Laboratories, Detroit, MI) for the enumeration of anaerobic or facultative anaerobic organisms. The liver from each bird was aseptically removed and placed in sterile Whirl Pak Bags (Difco) for transportation to the laboratory where the livers were examined for gross abnormalities. Following necropsy, 10 g of liver were aseptically removed and placed into TSB for enumeration of aerobes. A separate 10 g portion of tissue was placed in Cooked Meat medium (Difco) for isolation of anaerobes. After incubation, the TSB with blood and liver tissue samples were subcultured to Trypticase
Soy Agar (TSA) (BBL) plates. Bacteria from the Thioglycollate and Cooked Meat medium were subcultured to TSA plates containing 5% defibrinated sheep blood. The latter plates were incubated anaerobically. RESULTS
The antibiotic resistance patterns of the bacteria isolated at 8, 16, and 18 weeks from the blood and liver samples of turkeys fed different levels of OTC are given in Tables 1,2, and 3. From each group of birds, the percentages of antibiotic resistance from bacterial isolates within each supplement feeding group were obtained by dividing the actual number of resistant organisms by the maximum possible resistance. The maximum resistance was obtained by multiplying the number of bacteria isolated from that group by the number of antibiotics tested (8). At 8 weeks, 6 bacteria were isolated from the liver and blood of 4 birds not receiving antibiotic supplement. These isolates were resistant to 29% (13 of a possible 48) of the antibiotics (Table 1). At 16 weeks, 4 isolates from 2 birds in the same feeding group were resistant to 44% (14 of 32) the antibiotics (Table 1). There were no bacteria isolated from the control group at 18 weeks. At 8 weeks, from birds fed a low-level of supplement, 5 bacteria isolated from 4 birds were resistant to 50% (20 of 40) of the antibiotics (Table 2). From this feeding group at 16 weeks, 5 isolates from 2 birds were resistant to 85% (34 of 40) of the antibiotics (Table 2). From this feeding group at 18 weeks, 8 bacteria isolated from 3 birds were resistant to 63% (40 of 64) of the antibiotics (Table 2). At 8 weeks, from birds fed a high-level of antibiotic supplement, 4 isolates from 3 birds were resistant to 69% (22 of 32) of the antibiotics (Table 3). At 16 weeks from this group of birds, 2 isolates from 2 birds were resistant to 88% (14 of 16) of the antibiotics (Table 3). There were no bacteria isolated from this group at 18 weeks. Most isolates from all three levels of supplement had high resistance against OTC as compared to other antibiotics. Tables 4, 5, and 6 list the types of gross liver abnormalities observed in birds fed OTC at different levels. In control birds the number of liver abnormalities were three at 8 weeks, one at 16 weeks, and none at 18 weeks (Table 4). The number of liver abnormalities observed in
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 9, 2015
cal Co., St. Louis, MO) was combined into the feed at rates of 0 g/ton (control group), 50 g/ton (low level), and 200 g/ton (high level). Bacteriological Procedures. Reagents and media were used in classifying the bacteria according to biochemical tests described in Bergey's Manual of Determinative Bacteriology (Breed etal., 1975). Commercial media were prepared according to manufacturer's directions. Aerobic organisms were incubated at 37 C for 24 hr except for the urease and coagulase biochemical tests, which were incubated in a 37 C water bath for 6 hr. The anaerobic or facultative anaerobic organisms were grown at 37 C for 48 hr in a Brewer's Anaerobic Jar with a Gas Pak (Baltimore Biological Laboratory, Cockeysville, MD) (BBL) and a redox indicator containing sodium bicarbonate and .001% methylene blue. The Kirby-Bauer method (Bauer et al, 1966) was applied to test the antibiotic sensitivity of isolated organisms against the following: ampicillin (10 Mg), penicillin (10 Mg), bacitracin (10 Mg), naladixic acid (30 Mg), streptomycin (10 Mg), gantricin (300 Mg), aureomycin (300 Mg), and OTC (30 Mg). All zones of inhibition produced by the standard discs against the growth of organisms were measured to the nearest millimeter. The results were interpreted for antibiotic sensitivity in accordance to the BBL Manual of Products and Laboratory Procedures (Rhode, 1968).
739
SWEZEYETAL. birds fed a low level supplement were four at 8 weeks, one at 16 weeks, and two at 18 weeks (Table 5). The number of abnormalities in the livers of turkeys fed a high-level supplement were three at 8 weeks, two at 16 weeks, and none at 18 weeks (Table 6).
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DISCUSSION Data on the feeding of OTC at various levels in turkeys indicate that use of an antibiotic as feed supplement increased the level of antibiotic resistance in bacteria present in the liver and blood. Furthermore, it was evident that the degree of resistance in isolates correlated directly to the level of antibiotic concentration administered to the birds. It has been shown in earlier studies (Smith and Crabb, 1957; Starr and Reynolds, 1951; Mercer et al, 1971) that OTC, used as a feed supplement, increased the level of multiple drug resistance in intestinal flora in various domestic animals. In control birds, the amount of bacterial resistance in blood and liver isolates increased from 29% at 8 weeks to 44% at 16 weeks (Table 1). This resistance found in turkeys not exposed to the antibiotic could be due to the development of naturally occurring resistant organisms. Similar increases in the resistance of bacteria living in nature has been documented (Mare, 1968; Smith and Crabb, 1957; Grabow and Prozesky, 1973). At 8 weeks, the liver and blood isolates from birds fed a low level of supplement demonstrated 50% resistance (Table 2). This indicates that development of increased resistance can occur by 8 weeks if birds are placed on feed supplement immediately after hatching. At 8 weeks, isolates from the liver and blood of birds fed a high level of supplement showed 72% resistance (Table 3). These results confirm the development of bacterial resistance within 8 weeks of placing the birds on supplemented feed. The resistance percentages received at the 8 week sampling period also demonstrate the direct correlation between the amount of resistance with the level of antibiotic supplement fed. The 16 week results indicate that the bacteria inhibiting the liver and blood of turkeys fed OTC supplement continue to develop resistance. After 16 weeks of consuming OTC supplement, the isolates from birds on OTC had significantly higher percentages of resistance when compared to birds not feeding on OTC. However, there was no significant difference in
S S R I R S S S S R S S R S S R R R
Staphylococcus epidermidis3 Propionibacterium acnes3 Peptococcus anaerobius3 Peptococcus aerogenes3 Bacillus cereus0 Staphylococcus epidermidis* Staphylococcus epidermidis3Streptococcus zooepidemicus" Lactobacillus acidophilus0 Peptostreptococcus anaerobius3 Bacillus psycbrosaccharolyticus0 Streptococcus avium0 Staphylococcus epidermidis3 Lactobacillus acidophilus0 Streptococcus faecalis3 Micrococcus spP Lactobacillus fermentum3 Staphylococcus epidermidis3
8 8 8 8 8 16 16 16 16 16 16 16 16 16 16 16 18 18
16 16 20 22 24 39 39 39 39 71 75 75 75 75 80 80 138 138
Indicates organism isolated from blood sample.
Indicates organism isolated from liver tissue.
wk wk wk wk wk wk wk wk wk wk wk wk wk wk wk wk wk wk
Ampicillin
Isolate
Age at slaughter
Tag no.
S R R R R R R R S S R S S S
S S R S
Aureomycin
S R S S
R R I R R R S R R S S
S
Bacitracin
R
R
S R R S S S R S R S
R R
R
S S R R S
Gantncin tricin
TABLE 2. Antibiotic resistance spectra of bacteria isolated from the liver an of birds fed 50 g OTC/ton of feed
d from http://ps.oxfordjournals.org/ at University of Manchester on May 9, 2015
742
SWEZEY ET AL.
kr*
TABLE 4. Liver abnormalities in turkeys fed 0 g OTC/ton of feed as oi as as OS OS
Tag no.
Age at slaughter
Type of liver abnormality
5 10 12 50
8 8 8 16
Granuloma Lesion Granuloma Lesion
OS as as os as oS
.i.
c
as OS as as OS OS
Z =5
the percentages of resistance found in bacteria from birds receiving the two different levels of OTC. We were unable to explain the decrease in resistance observed in birds feeding on low levels of supplement at the 18 weeks sampling period. In observing turkey livers, the lesion is a common type of abnormality. In accordance with Dorland (1965), livers were classified as having lesions when gross abnormalities were observed with pathological or traumatic discontinuity of tissue. The liver abnormalities observed consisted of infarcts, enlargement of the organ with red-colored foci, and mottling. One type of liver lesion often found on the surface is a granuloma (Moore and Gross, 1968), which is described as a hard, light-colored, spheroid mass having a dense fibrinous lining surrounding a hard caseous core. Lesions were diagnosed as granulomas when the appearance was similar to the description given. Tables 4, 5, and 6 list the types of liver abnormalities found in the birds examined. Differences were observed when the types of abnormalities in livers of control group birds were compared to the livers of birds receiving supplement.
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TABLE 5. Liver abnormalities in turkeys fed 50 g OTC/ton of feed
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Age at slaughter
Type of liver abnormality
16 20 22 24 71 75 138
8 8 8 8 16 18 18
Granuloma Lesion Lesion Lesion Lesion Lesion Lesion
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OXYTETRACYCLINE AS A FEED ADDITIVE TABLE 6. Liver abnormalities in turkeys fed 200 g OTC/ton of feed
Tag no.
Age at slaughter
Type of liver abnormality
3 26 34 43 68
8 wk 8 wk 8 wk 16 wk 16 wk
Lesion Granuloma Lesion Lesion Lesion
The increase of drug-resistant organisms as a result of widespread use of an antibiotic as feed supplements to domestic animals should be reevaluated. Increased difficulty in disease treatment may result if levels of antibiotic resistance continue to increase due to such practice. The problem of increased organ abnormalities due to the presence of antibiotics in feed should also be further investigated. REFERENCES Bauer, A. W., C. E. Roberts, and W. Kirby, 1966. Antibiotic susceptibility testing by a standardi-
zed single disc method. Amer. J. Clin. Pathol. 45:493. Breed, R. S., E. G. D. Murray, and N. R. Smith, 1971. Bergey's manual of determinative bacteriology. 8th ed. Williams and Wilkins, Baltimore, MD. Dey, B. P., D. C. Blenden, B. C. Burton, H. E. Mercer, and R. D. Tsutakawa, 1977. Therapeutic responses of piglets to experimentally induced colibacillosis. Res. Vet. Sci. 23:340-343. Dey, B. P., D. C. Blenden, B. C. Burton, H. E. Mercer, and R. D. Tsutakawa, 1978. Influence of chlorotetracycline feeding in Salmonellosis in calves. Int. J. Zoon. 5:97-110. Dorland, W. A., 1965. American illustrated medical dictionary. W. B. Saunders Co., Philadelphia, PA. Forsgren, A., and H. Gnarpe, 1973. Tetracycline and host-defense mechanisms. Antimicrob. Agents Chemother. 3:711-715. Garside, J. S., R. F. Gordon, and J. F. Tucker, 1960. The emergence of resistant strains of Salmonella typhimurium in the tissues and alimentary tracts of chickens following the feeding of an antibiotic. Res. Vet. Sci. 1:184-199. Grabow, W. O. K., and O. W. Prozesky, 1973. Drug resistance of coliform bacteria in hospital and city sewage. Antimicrob. Agents Chemother. 3:175-180. Mare, J. L., 1968. Incidence of r-factor among gram negative bacteria in drug-free humans and animals. Nature 220:1046-1047. Mercer, H. D., D. Pocurull, S. Gaines, S. Wilson, and J. Bennett, 1971. Characteristics of antimicrobial resistance of E. coli from animals. Appl. Microbiol. 22:700-703. Moore, W. E. C , and W. B. Gross, 1968. Liver granuloma of turkeys causative agents and mechanisms of infection. Avian Dis. 12:417-422. Rhode, P. A., 1968. BBL Manual of products and laboratory procedures. 5th ed. Division of Beeton, Dickerson and Co., Cockeysville, MD. Sanger, V. S., J. Yacowitz, and E. N. Moore, 1956. Micro-pathological changes in experimental hemorrhagic syndrome in chickens fed sulfaquinoxaline and suggested cause of the disease. Amer. J. Vet. Res. 17:766-770. Siegel, D., W. G. Huber, and F. Enloe, 1974. Continuous non-therapeutic use of antibacterial drugs in feed and drug resistance of the gram negative enteric flora of food producing animals. Antimicrob. Agent Chemother. 6:697—701. Smith, H. S., and W. E. Crabb, 1957. The effect of continuous administration of diets containing low levels of tetracyclines on the incidence of drug-resistant bacteria in the feces of pigs and chickens. Vet. Rec. 69:24-30. Starr, M., and D. Reynolds, 1951. Streptomycin resistance of coliform bacteria from turkeys fed streptomycin. Amer. J. Public Health. 41:1375-1380.
Downloaded from http://ps.oxfordjournals.org/ at University of Manchester on May 9, 2015
At the 8-week sampling period, two livers from control birds contained granulomas, whereas only one liver with granulomas was found in the low level and one in the high level supplement group (Tables 4, 5, and 6). In the same sampling period, one lesion was found in the livers of control birds, while three livers from birds feeding on low level supplement and one liver from birds feeding on high level supplement were found to have lesions (Tables 4, 5, and 6). At the 16 and 18 week sampling periods, one liver with lesions was observed in the control birds but three livers from birds consuming a low level of supplement and three livers from birds consuming a high level of supplement were found to have lesions. These results support evidence that the feeding of OTC as a feed supplement may reduce the number of granulomas that appear on liver tissue in turkeys, but that the occurrence of liver lesions may be increased by the use of the supplement.
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