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An Experimental Study on the Control of Pullorum Disease1,2
An Experimental Study on the Control of Pullorum Disease1,2 A B E PETAL, 3 ' 4 H . J. STAFSETH 3 AND E. H . L U C A S 6
Michigan State College, East Lansing (Received for publication June 1, 1953)
A
Streptomycin is one of the few antibiotics which has been employed to any appreciable extent in the control of pullorum infection (Benson, 1947; Gwatkin, 1950). T h e results obtained with streptomycin are not too encouraging, since the carrier condition does not appear to be eliminated. I n addition, it is also known t h a t organisms rapidly develop resistance to this antibiotic. These factors m a y preclude further consideration of streptomycin as a therapeutic agent in pullorum disease control.
MATERIALS AND METHODS
Experimental data concerning the use of other antibiotics have primarily been confined to a small number of in vitro observations (Jackson et ah, 1950). T h e present study is mainly of a preliminary nature and was undertaken with a view 1
toward a more complete in vitro and in vivo evaluation of three common antibiotic agents. These include aureomycin, Chloromycetin and penicillin. 6 T h e inclusion of penicillin in this study was prompted by repeated in vitro observations on the sensitivity of various Salmonella pullorum strains to moderately low concentrations of penicillin. I n addition, previous work by P r a t t , Dufrenoy and Strait (1947, 1948a, b) had demonstrated t h a t among a number of heavy metals tested, only cobalt, in appropriate concentrations, produced a marked enhancement of penicillin activity. Enhancing effects were obtained with selected gram-negative and gram-positive organisms. T h e extension of their findings to an organism like 5 . pullorum was of theoretical as well as practical interest.
Journal Article No. 1508 of the Michigan Agricultural Experiment Station. 2 Based on a portion of a thesis submitted to the School of Graduate Studies of Michigan State College, June 1952, by the senior author in partial fulfillment of the requirements for the Ph.D. degree. 3 Department of Bacteriology and Public Health. 4 Present address: Camp Detrick, Frederick, Md. 6 Department of Horticulture.
A two-fold b r o t h dilution method was used in determining the sensitivity of 5 . pullorum' to aureomycin hydrochloride and Chloromycetin. A modification of the broth dilution method was employed with penicillin (G, buffered potassium salt). Penicillin concentrations were increased by increments of five, starting with five units/ml. as the lowest amount. Tubes were incubated for 24 hours a t 37°C. and 6
The antibiotics were supplied through the courtesy of the following companies: Aureomycin— Lederle Laboratories, Pearl River, N. Y.; Chloromycetin—Parke-Davis, Detroit, Mich.; Penicillin— The Upjohn Company, Kalamazoo, Mich. 7 Strain #89817 (Department of Bacteriology and Public Health, Michigan State College) was used in all experiments.
134
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N T I B I O T I C agents have received ^ limited application in the control of pullorum disease. This is in contrast to the sulfonamides which have been used with moderate frequency (Bottorf a n d Kiser, 1947; Anderson et al., 1948; Dickinson and Stoddard, 1949). T h e ultimate value of sulfonamides remains questionable and especially with reference to eradication of the carrier condition among infected birds.
CONTROL OF PULLORUM
these plates were examined for evidence of growth inhibition. The Barcroft-Warburg apparatus was used for determining the effect of cobalt on oxygen uptake by 5. pullorum. Comparative respiration studies were made between organisms in plain broth and in broth containing cobalt. In general, identical experimental procedures were used in testing the in vivo effectiveness of aureomycin and Chloromycetin. Each antibiotic was administered to day-old chicks by incorporation in a starter feed. A period of prophylactic feeding was allowed prior to artificial infection and was varied in several of the experimental groups. Chicks were infected per os with a 0.5 cc. saline suspension of the organism. A pooled saline suspension was prepared from actively grow" ing cultures on nutrient agar slants. The inoculum was adjusted to contain approximately 109 organisms per ml. of suspension. Following infection, the chicks were maintained on an antibiotic-starter feed for the duration of the experimental period. Chicks succumbing to infection were necropsied and an attempt was made to recover the infecting organism. The experimental procedure employed for in vivo evaluation of penicillin did not differ from that described for aureomycin and Chloromycetin. On the other hand, the combination of cobalt and penicillin necessitated a change in experimental method. Day-old chicks (White Leghorn; were allowed to drink water containing cobalt (0.2 mg./ml.) for a 24-hour period and at the same time were given a starter feed which did not contain penicillin. At the end of 24 hours the chicks were infected per os with a 0.5 ml. suspension of S. pullorum (approximately 109 organisms per ml.). Thirty minutes after infection each chick was given 0.5 ml. of cobalt water per os. Plain starter feed and
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examined for visible growth. The lowest concentration of antibiotic completely inhibiting growth of the test organism in 24 hours was regarded as the endpoint reading. The preceding method plus subculture studies were used in determining the penicillin sensitivity of 5. pullorum. Subcultures in broth were prepared from all penicillin tubes after 24, 48 and 72 hours of incubation. The cobalt enhancement (in vitro) of penicillin activity against S. pullorum was evaluated by means of the seeded plate method. Plates were prepared in the following manner: 18 ml. of Penassay base agar (Difco) was added to each Petri dish and allowed to solidify. A flask containing 200 ml. of Penassay seed agar was then inoculated with 2 ml. of a 24-hour Penassay broth culture of S. pullorum. Three to four ml. of seeded agar were added to the solidified base and the plate gently rotated to insure a uniform distribution of the seed layer. Two to three ml. of cobalt solution (0.2 mg. CoCl2-6H20 per ml. distilled water) was applied to each plate. These plates were then incubated for 30 minutes at 37°C. in the presence of cobalt. At the end of this period, the cobalt solution was decanted and penicillin (100 units/ml.) immediately added utilizing the paper disk technique of Vincent and Vincent (1944). Following the addition of penicillin, the plates were incubated for 24 hours at 37°C. and the zones of inhibition produced were compared with corresponding controls which did not have contact with cobalt. Further controls to ascertain the possible toxic effects of cobalt on the test organism were made by preparing plates in the manner previously described, but with the exception that penicillin was omitted. Also, paper disks were immersed in cobalt solution (0.2 mg./ml.) and placed on prepared plates. After a suitable incubation time,
135
136
A. PITAL, H. J. STAFSETH AND E. H. LUCAS
TABLE 1.—Evaluation of the effect of feeding aureomycin to chicks infected with S. pullorum Series 1 (White Leghorn)
3 (Rhode Island Red)
4 (New Hampshire)
5 (Barred Rock X White Rock)
Number of birds
Percent Hours of Antibiotic concentration prophylactic mortality in 21 days feeding mg./gm. feed
Non-treated infected control Aureomycin Aureomycin Aureomycin Aureomycin
24
0
0
37.5
29 29 29 29
1 1 1 1
72 72 72 72
3.4 0.0 10.3 6.9
Non-treated infected control Non-treated noninfected control Aureomycin Aureomycin
29
0
0
34.5
24
0
0
0.0
25 30
1 1
24 24
0.0 13.3
24
0
0
29.2
25
0
0
4.0
24 25
1 1
48 48
16.7 4.0
25
0
0
28.0
25
0
0
4.0
27 27
1 2
48 48
3.7 3.7
25
0
0
32.0
32
0
0
3.1
24
1
24
8.3
Non-treated infected control Non-treated noninfected control Aureomycin Aureomycin Non-treated infected control Non-treated noninfected control Aureomycin Aureomycin Non-infected infected control Non-treated noninfected control Aureomycin
cobalt water were then removed and a feed containing penicillin along with ordinary drinking water were substituted for the duration of the experiment. A noninfected group of control chicks was allowed to drink cobalt for one week in order to determine any possible toxic effects. RESULTS AND DISCUSSION The average sensitivity values of S. Pullorum to aureomycin, Chloromycetin and penicillin were as follows: 2.0 mcgm./ml. Aureomycin 1.56 mcgm./ml. Chloromycetin 15.0 units/ml. Penicillin Aureomycin and Chloromycetin appeared to exert a bacteriostatic rather than a bactericidal effect. Penicillin on the other
hand, seemed to be slowly bactericidal for S. pullorum. Subcultures in broth from all penicillin tubes revealed a slow bactericidal action within 48 hours of incubation. Subculture studies provided a more accurate evaluation of penicillin sensitivity than did turbidity tests. In vivo observations appear to indicate that both aureomycin and Chloromycetin are effective in reducing mortality resulting from artificial infection with S. pullorum. Results obtained with aureomycin and Chloromycetin are listed in Tables 1 and 2. The value of both antibiotics in preventing the development of carriers among recovered birds still remains to be determined. In vivo experiments with oral penicillin preparations have shown that this anti-
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2 (White Leghorn)
Experimental group
137
CONTROL OF PULLORUM TABLE 2.—Evaluation of the effect of feeding Chloromycetin to chicks infected with S. pullorum
Series 1 (Barred Rock X White Rock)
3 (White Leghorn)
4 (White Leghorn)
5 (White Leghorn)
Non-treated infected control Non-treated-noninfected control Chloromycetin Non-treated infected control Non-treated noninfected control Chloromycetin Chloromycetin Non-treated infected control Non-treated noninfected control Chloromycetin Non-treated infected control Non-treated noninfected control Chloromycetin Chloromycetin Non-treated infected control Non-treated noninfected control Chloromycetin
biotic must be incorporated in feed at relatively high levels in order to be therapeutically effective (Table 4). A partial explanation for this may be found in the inactivation of penicillin by the enzyme penicillinase which is elaborated by vari- ' ous organisms of the chick gut. The use of anti-penicillinase agents like sodium benzoate may eventually counteract this effect. From in vitro data, it would appear that penicillin blood levels of 15 units/ ml. are probably necessary during the acute stages of infection. This value will undoubtedly vary for different strains. Failure to maintain an adequate concentration may render penicillin therapy ineffective. Preliminary observations by Boger et al. (1951) showed that successful penicillin therapy for typhoid fever in humans is dependent upon maintaining a
Number of birds
Antibiotic Hours of concentration prophylactic mg./gm. feed feeding 0
Percent mortality in 21 days
25
0
20.0
30
0
0
0.0
25
1
48
0.0
25
0
0
32.0
32
0
0
3.1
22 25
1 1
24 24
13.6 12.0
25
0
0
24.0
35
0
25
1.5
25
0
35
0
25 25
1.5 1.5
25
0
0.0
24
0.0
0
40.0
0
2.9
24 24
12.0 12.0
0
0
32.0
35
0
0
2.9
25
1
24
4.0
penicillin blood level of 10 units/ml. during acute stages of infection and for some time thereafter. The practical utilization of penicillin in pullorum disease control appeared to depend on some method which would allow for a reduction in penicillin levels without impairing therapeutic efficiency. The use of cobalt in conjunction with penicillin appears to be a promising method. Experimental results in this study showed that cobalt in a concentration of 0.2 mg./ml. produced a marked enhancement of penicillin activity against .S. pullorum. Enhancing effects were recorded for both in vitro and in vivo experiments. This is illustrated in Tables 3 and 4. A more pronounced enhancing phenomenon became evident when the test or-
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2 (Barred Rock X White Rock)
Experimental group
138
A. PITAL, H. J. STAFSETH AND E. H. LUCAS TABLE 3.—The cobalt enhancement (in vitro) of penicillin activity against S. pullorum Penicillin—100 units/ml Series 1
Series 2
2 ml. cobalt soln. (0.2 mg./ml.)
No cobalt
3 ml. cobalt soln. (0.2 mg./ml.)
29.2
22.0
29.1
Series 3 3 ml. cobalt soln. (0.2 mg./ml.)
No cobalt
Zones of inhibition (mm.)* 22.4 29.1
No cobalt
Cobalt control (no penicillin) 0.2 mg./ml.
22.8
0.0
ganism was incubated in the presence of cobalt for a short period of time prior to contact with penicillin. For in vitro tests a period of 30 minutes was found to be optimal. The interval of time elapsing between artificial infection and resumption of normal feeding activities (with penicillin) seemed to provide the necessary incubation time for chick experiments. The particular concentration of cobalt used in this study did not produce any observable inhibitory effects on the test organism. Chicks serving as cobalt toxicity controls exhibited no ill effects from prolonged drinking of cobalt water. Respiration studies, however, conducted with a Barcroft-Warburg apparatus, revealed that cobalt by itself, in a concentration of 0.2 mg./ml., produced a marked inhibition of oxygen uptake by TABLE 4.—The therapeutic effect of penicillin and penicillin plus cobalt on chicks infected with S. pullorum
Series 1
2
Experimental group
Percent Penicillin Number morunits/g. of tality in 21 feed birds days
Non-treated infected control 0 Non-treated non-infected 0 control 2,290 Penicillin 2,290 Penicillin 3,500 Penicillin
25
40.0
35 25 25 25
2.8 28.0 24.0 20.0
Non-treated infected control Non-treated non-infected control Penicillin Penicillin Penicillin 4-cobalt PeniciUin+cobalt Penicillin+oobalt
0
25
32.0
0 5,500 5,500 2,290 2,290 2,290
35 25 25 25 25 25
2.8 8.0 16.0 12.0 0.0 4.0
TABLE 5.—Oxygen uptake by S. f the presence of cobalt
Series
Experimental group
Total oxygen uptake in 3 hours (microliters)
1
Broth control Broth control Broth+cobalt (0.001 mg./ml.) Broth+cobalt (0.2 mg./ml.)
101.8 111.5 96.9 33.3
2
Broth control Broth control Broth control Broth+cobalt (0.2 mg./ml.) Broth+cobalt (0.2 mg./ml.) Broth+cobalt (0.2 mg./ml.)
88.7 95.6 81.1 21.6 23.0 40.6
S. pullorum. A summary of oxygen uptake studies is presented in Table 5. It may be postulated that a similar early action of cobalt (applied without penicillin) might have taken place when the organism was grown on plates, but that its effect was overcome during the later part of a 24hour incubation period. At the end of this time, the organism presented abundant growth. If, however, cobalt was employed together with penicillin, the initial inhibitory effect appeared to be of sufficient magnitude to induce a synergistic phenomenon as illustrated in Table 3. Further experiments with cobalt and penicillin are being conducted in order to evaluate preliminary findings and to refine present'techniques and procedures. Optimal enhancing concentrations of cobalt may vary for different S. pullorum strains
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* Each zone reading represents the average value of eight plate determinations.
139
NEWS AND NOTES
SUMMARY In vitro tests showed that 6". pullorum #89817 is sensitive to low concentrations of aureomycin and Chloromycetin. This organism is also sensitive to moderately low concentrations of penicillin. The latter appears to exert a slow bactericidal action, while aureomycin and Chloromycetin appear to be bacteriostatic. The use of proper concentrations of cobalt greatly enhanced the antibacterial action of penicillin. This was found in both in vitro and in vivo experiments. Aureomycin and Chloromycetin were effective in reducing chick mortalities resulting from artificial infection with S. pullorum. Penicillin also reduced mortalities, but only at relatively high concentration levels. A decided reduction in penicillin levels may eventually be realized by the combined use of cobalt and penicillin. The value of all three antibiotic agents in prevention of carriers among recovered birds, still remains to be determined.
All treated birds received antibiotics for varying periods of time prior to infection. These same groups continued to receive antibiotics for the duration of the experimental period following inoculation. REFERENCES Anderson, G. W., J. B. Cooper, J. C. Jones and C. L. Morgan, 1948. Sulfonamides in the control of pullorum disease. Poultry Sci. 27: 172-175. Benson, D. V., 1947. Streptomycin in the treatment of pullorum in baby chicks. Vet. Med. 17: 72-73. Boger, W. P., N. H. Schimmel and W. V. Matteucci, 1951. Typhoid fever successfully treated with penicillin. J. Antibiotics Chemotherapy, 1: 140145. Bottorf, C. A., and J. S. Kiser, 1947. The use of sulfonamides in the control of pullorum disease. Poultry Sci. 26:335-339. Dickinson, E. M., and E. D. Stoddard, 1949. Sulfamerazine against Salmonella pullorum in adult chickens. Poultry Sci. 28: 153-155. Gwatkin, R., 1950. Observations on the use of streptomycin in Salmonella pullorum infection in chicks by the oral and intraperitoneal routes. Canadian J. Comp. Med. 14: 157. Jackson, G. G., T. M. Gocke, H. S. Collins and M. Finland, 1950. In vitro sensitivity of pathogenic enteric bacteria to various antibiotics. J. Infec. Diseases, 87:63-70. Pratt, R., and J. Dufrenoy, 1947. Comparative responses of gram-positive and gram-negative bacteria to penicillin. J. Bact. 54: 719. Pratt, R., J. Dufrenoy and L. A. Strait, 1948. The enhancement of penicillin effectiveness in vivo by traces of cobalt. J. Bact. 55: 75-77. Strait, L. A., J. Dufrenoy and R. Pratt, 1948. Enhancement of penicillin effectiveness by traces of cobalt. J. Amer. Pharm. Assoc. 37: 133. Vincent, J. G., and H. W. Vincent, 1944. Filter paper disc modification of the Oxford cup penicillin determination. Proc. Soc. Exptl. Biol. Med. 55: 162-164.
NEWS AND NOTES {Continued from page 133) and from 1914-1918 he held the same position at Barryton, Michigan. He was also the County Superintendent of Mecosta Schools during the years 1918-1920. In 1920 Professor Card was ap-
pointed to the position of the Director of the Vocational School at Michigan State College, a position which he held until 1923 when he was appointed as an Assistant Professor of Poultry Husbandry. The
(Continued on page 154)
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susceptible to penicillin. This possibility is receiving further attention as well as the role of cobalt in a practical program of penicillin therapy. The possible use of penicillin in pullorum disease control merits consideration from two important aspects: First, the slow bactericidal action of penicillin may be a factor in preventing the development of carrier birds and secondly, in comparison to other antibiotic agents, penicillin is relatively cheap and readily available.
Michigan State College, East Lansing (Received for publication June 1, 1953)
A
Streptomycin is one of the few antibiotics which has been employed to any appreciable extent in the control of pullorum infection (Benson, 1947; Gwatkin, 1950). T h e results obtained with streptomycin are not too encouraging, since the carrier condition does not appear to be eliminated. I n addition, it is also known t h a t organisms rapidly develop resistance to this antibiotic. These factors m a y preclude further consideration of streptomycin as a therapeutic agent in pullorum disease control.
MATERIALS AND METHODS
Experimental data concerning the use of other antibiotics have primarily been confined to a small number of in vitro observations (Jackson et ah, 1950). T h e present study is mainly of a preliminary nature and was undertaken with a view 1
toward a more complete in vitro and in vivo evaluation of three common antibiotic agents. These include aureomycin, Chloromycetin and penicillin. 6 T h e inclusion of penicillin in this study was prompted by repeated in vitro observations on the sensitivity of various Salmonella pullorum strains to moderately low concentrations of penicillin. I n addition, previous work by P r a t t , Dufrenoy and Strait (1947, 1948a, b) had demonstrated t h a t among a number of heavy metals tested, only cobalt, in appropriate concentrations, produced a marked enhancement of penicillin activity. Enhancing effects were obtained with selected gram-negative and gram-positive organisms. T h e extension of their findings to an organism like 5 . pullorum was of theoretical as well as practical interest.
Journal Article No. 1508 of the Michigan Agricultural Experiment Station. 2 Based on a portion of a thesis submitted to the School of Graduate Studies of Michigan State College, June 1952, by the senior author in partial fulfillment of the requirements for the Ph.D. degree. 3 Department of Bacteriology and Public Health. 4 Present address: Camp Detrick, Frederick, Md. 6 Department of Horticulture.
A two-fold b r o t h dilution method was used in determining the sensitivity of 5 . pullorum' to aureomycin hydrochloride and Chloromycetin. A modification of the broth dilution method was employed with penicillin (G, buffered potassium salt). Penicillin concentrations were increased by increments of five, starting with five units/ml. as the lowest amount. Tubes were incubated for 24 hours a t 37°C. and 6
The antibiotics were supplied through the courtesy of the following companies: Aureomycin— Lederle Laboratories, Pearl River, N. Y.; Chloromycetin—Parke-Davis, Detroit, Mich.; Penicillin— The Upjohn Company, Kalamazoo, Mich. 7 Strain #89817 (Department of Bacteriology and Public Health, Michigan State College) was used in all experiments.
134
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N T I B I O T I C agents have received ^ limited application in the control of pullorum disease. This is in contrast to the sulfonamides which have been used with moderate frequency (Bottorf a n d Kiser, 1947; Anderson et al., 1948; Dickinson and Stoddard, 1949). T h e ultimate value of sulfonamides remains questionable and especially with reference to eradication of the carrier condition among infected birds.
CONTROL OF PULLORUM
these plates were examined for evidence of growth inhibition. The Barcroft-Warburg apparatus was used for determining the effect of cobalt on oxygen uptake by 5. pullorum. Comparative respiration studies were made between organisms in plain broth and in broth containing cobalt. In general, identical experimental procedures were used in testing the in vivo effectiveness of aureomycin and Chloromycetin. Each antibiotic was administered to day-old chicks by incorporation in a starter feed. A period of prophylactic feeding was allowed prior to artificial infection and was varied in several of the experimental groups. Chicks were infected per os with a 0.5 cc. saline suspension of the organism. A pooled saline suspension was prepared from actively grow" ing cultures on nutrient agar slants. The inoculum was adjusted to contain approximately 109 organisms per ml. of suspension. Following infection, the chicks were maintained on an antibiotic-starter feed for the duration of the experimental period. Chicks succumbing to infection were necropsied and an attempt was made to recover the infecting organism. The experimental procedure employed for in vivo evaluation of penicillin did not differ from that described for aureomycin and Chloromycetin. On the other hand, the combination of cobalt and penicillin necessitated a change in experimental method. Day-old chicks (White Leghorn; were allowed to drink water containing cobalt (0.2 mg./ml.) for a 24-hour period and at the same time were given a starter feed which did not contain penicillin. At the end of 24 hours the chicks were infected per os with a 0.5 ml. suspension of S. pullorum (approximately 109 organisms per ml.). Thirty minutes after infection each chick was given 0.5 ml. of cobalt water per os. Plain starter feed and
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examined for visible growth. The lowest concentration of antibiotic completely inhibiting growth of the test organism in 24 hours was regarded as the endpoint reading. The preceding method plus subculture studies were used in determining the penicillin sensitivity of 5. pullorum. Subcultures in broth were prepared from all penicillin tubes after 24, 48 and 72 hours of incubation. The cobalt enhancement (in vitro) of penicillin activity against S. pullorum was evaluated by means of the seeded plate method. Plates were prepared in the following manner: 18 ml. of Penassay base agar (Difco) was added to each Petri dish and allowed to solidify. A flask containing 200 ml. of Penassay seed agar was then inoculated with 2 ml. of a 24-hour Penassay broth culture of S. pullorum. Three to four ml. of seeded agar were added to the solidified base and the plate gently rotated to insure a uniform distribution of the seed layer. Two to three ml. of cobalt solution (0.2 mg. CoCl2-6H20 per ml. distilled water) was applied to each plate. These plates were then incubated for 30 minutes at 37°C. in the presence of cobalt. At the end of this period, the cobalt solution was decanted and penicillin (100 units/ml.) immediately added utilizing the paper disk technique of Vincent and Vincent (1944). Following the addition of penicillin, the plates were incubated for 24 hours at 37°C. and the zones of inhibition produced were compared with corresponding controls which did not have contact with cobalt. Further controls to ascertain the possible toxic effects of cobalt on the test organism were made by preparing plates in the manner previously described, but with the exception that penicillin was omitted. Also, paper disks were immersed in cobalt solution (0.2 mg./ml.) and placed on prepared plates. After a suitable incubation time,
135
136
A. PITAL, H. J. STAFSETH AND E. H. LUCAS
TABLE 1.—Evaluation of the effect of feeding aureomycin to chicks infected with S. pullorum Series 1 (White Leghorn)
3 (Rhode Island Red)
4 (New Hampshire)
5 (Barred Rock X White Rock)
Number of birds
Percent Hours of Antibiotic concentration prophylactic mortality in 21 days feeding mg./gm. feed
Non-treated infected control Aureomycin Aureomycin Aureomycin Aureomycin
24
0
0
37.5
29 29 29 29
1 1 1 1
72 72 72 72
3.4 0.0 10.3 6.9
Non-treated infected control Non-treated noninfected control Aureomycin Aureomycin
29
0
0
34.5
24
0
0
0.0
25 30
1 1
24 24
0.0 13.3
24
0
0
29.2
25
0
0
4.0
24 25
1 1
48 48
16.7 4.0
25
0
0
28.0
25
0
0
4.0
27 27
1 2
48 48
3.7 3.7
25
0
0
32.0
32
0
0
3.1
24
1
24
8.3
Non-treated infected control Non-treated noninfected control Aureomycin Aureomycin Non-treated infected control Non-treated noninfected control Aureomycin Aureomycin Non-infected infected control Non-treated noninfected control Aureomycin
cobalt water were then removed and a feed containing penicillin along with ordinary drinking water were substituted for the duration of the experiment. A noninfected group of control chicks was allowed to drink cobalt for one week in order to determine any possible toxic effects. RESULTS AND DISCUSSION The average sensitivity values of S. Pullorum to aureomycin, Chloromycetin and penicillin were as follows: 2.0 mcgm./ml. Aureomycin 1.56 mcgm./ml. Chloromycetin 15.0 units/ml. Penicillin Aureomycin and Chloromycetin appeared to exert a bacteriostatic rather than a bactericidal effect. Penicillin on the other
hand, seemed to be slowly bactericidal for S. pullorum. Subcultures in broth from all penicillin tubes revealed a slow bactericidal action within 48 hours of incubation. Subculture studies provided a more accurate evaluation of penicillin sensitivity than did turbidity tests. In vivo observations appear to indicate that both aureomycin and Chloromycetin are effective in reducing mortality resulting from artificial infection with S. pullorum. Results obtained with aureomycin and Chloromycetin are listed in Tables 1 and 2. The value of both antibiotics in preventing the development of carriers among recovered birds still remains to be determined. In vivo experiments with oral penicillin preparations have shown that this anti-
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 23, 2015
2 (White Leghorn)
Experimental group
137
CONTROL OF PULLORUM TABLE 2.—Evaluation of the effect of feeding Chloromycetin to chicks infected with S. pullorum
Series 1 (Barred Rock X White Rock)
3 (White Leghorn)
4 (White Leghorn)
5 (White Leghorn)
Non-treated infected control Non-treated-noninfected control Chloromycetin Non-treated infected control Non-treated noninfected control Chloromycetin Chloromycetin Non-treated infected control Non-treated noninfected control Chloromycetin Non-treated infected control Non-treated noninfected control Chloromycetin Chloromycetin Non-treated infected control Non-treated noninfected control Chloromycetin
biotic must be incorporated in feed at relatively high levels in order to be therapeutically effective (Table 4). A partial explanation for this may be found in the inactivation of penicillin by the enzyme penicillinase which is elaborated by vari- ' ous organisms of the chick gut. The use of anti-penicillinase agents like sodium benzoate may eventually counteract this effect. From in vitro data, it would appear that penicillin blood levels of 15 units/ ml. are probably necessary during the acute stages of infection. This value will undoubtedly vary for different strains. Failure to maintain an adequate concentration may render penicillin therapy ineffective. Preliminary observations by Boger et al. (1951) showed that successful penicillin therapy for typhoid fever in humans is dependent upon maintaining a
Number of birds
Antibiotic Hours of concentration prophylactic mg./gm. feed feeding 0
Percent mortality in 21 days
25
0
20.0
30
0
0
0.0
25
1
48
0.0
25
0
0
32.0
32
0
0
3.1
22 25
1 1
24 24
13.6 12.0
25
0
0
24.0
35
0
25
1.5
25
0
35
0
25 25
1.5 1.5
25
0
0.0
24
0.0
0
40.0
0
2.9
24 24
12.0 12.0
0
0
32.0
35
0
0
2.9
25
1
24
4.0
penicillin blood level of 10 units/ml. during acute stages of infection and for some time thereafter. The practical utilization of penicillin in pullorum disease control appeared to depend on some method which would allow for a reduction in penicillin levels without impairing therapeutic efficiency. The use of cobalt in conjunction with penicillin appears to be a promising method. Experimental results in this study showed that cobalt in a concentration of 0.2 mg./ml. produced a marked enhancement of penicillin activity against .S. pullorum. Enhancing effects were recorded for both in vitro and in vivo experiments. This is illustrated in Tables 3 and 4. A more pronounced enhancing phenomenon became evident when the test or-
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on June 23, 2015
2 (Barred Rock X White Rock)
Experimental group
138
A. PITAL, H. J. STAFSETH AND E. H. LUCAS TABLE 3.—The cobalt enhancement (in vitro) of penicillin activity against S. pullorum Penicillin—100 units/ml Series 1
Series 2
2 ml. cobalt soln. (0.2 mg./ml.)
No cobalt
3 ml. cobalt soln. (0.2 mg./ml.)
29.2
22.0
29.1
Series 3 3 ml. cobalt soln. (0.2 mg./ml.)
No cobalt
Zones of inhibition (mm.)* 22.4 29.1
No cobalt
Cobalt control (no penicillin) 0.2 mg./ml.
22.8
0.0
ganism was incubated in the presence of cobalt for a short period of time prior to contact with penicillin. For in vitro tests a period of 30 minutes was found to be optimal. The interval of time elapsing between artificial infection and resumption of normal feeding activities (with penicillin) seemed to provide the necessary incubation time for chick experiments. The particular concentration of cobalt used in this study did not produce any observable inhibitory effects on the test organism. Chicks serving as cobalt toxicity controls exhibited no ill effects from prolonged drinking of cobalt water. Respiration studies, however, conducted with a Barcroft-Warburg apparatus, revealed that cobalt by itself, in a concentration of 0.2 mg./ml., produced a marked inhibition of oxygen uptake by TABLE 4.—The therapeutic effect of penicillin and penicillin plus cobalt on chicks infected with S. pullorum
Series 1
2
Experimental group
Percent Penicillin Number morunits/g. of tality in 21 feed birds days
Non-treated infected control 0 Non-treated non-infected 0 control 2,290 Penicillin 2,290 Penicillin 3,500 Penicillin
25
40.0
35 25 25 25
2.8 28.0 24.0 20.0
Non-treated infected control Non-treated non-infected control Penicillin Penicillin Penicillin 4-cobalt PeniciUin+cobalt Penicillin+oobalt
0
25
32.0
0 5,500 5,500 2,290 2,290 2,290
35 25 25 25 25 25
2.8 8.0 16.0 12.0 0.0 4.0
TABLE 5.—Oxygen uptake by S. f the presence of cobalt
Series
Experimental group
Total oxygen uptake in 3 hours (microliters)
1
Broth control Broth control Broth+cobalt (0.001 mg./ml.) Broth+cobalt (0.2 mg./ml.)
101.8 111.5 96.9 33.3
2
Broth control Broth control Broth control Broth+cobalt (0.2 mg./ml.) Broth+cobalt (0.2 mg./ml.) Broth+cobalt (0.2 mg./ml.)
88.7 95.6 81.1 21.6 23.0 40.6
S. pullorum. A summary of oxygen uptake studies is presented in Table 5. It may be postulated that a similar early action of cobalt (applied without penicillin) might have taken place when the organism was grown on plates, but that its effect was overcome during the later part of a 24hour incubation period. At the end of this time, the organism presented abundant growth. If, however, cobalt was employed together with penicillin, the initial inhibitory effect appeared to be of sufficient magnitude to induce a synergistic phenomenon as illustrated in Table 3. Further experiments with cobalt and penicillin are being conducted in order to evaluate preliminary findings and to refine present'techniques and procedures. Optimal enhancing concentrations of cobalt may vary for different S. pullorum strains
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* Each zone reading represents the average value of eight plate determinations.
139
NEWS AND NOTES
SUMMARY In vitro tests showed that 6". pullorum #89817 is sensitive to low concentrations of aureomycin and Chloromycetin. This organism is also sensitive to moderately low concentrations of penicillin. The latter appears to exert a slow bactericidal action, while aureomycin and Chloromycetin appear to be bacteriostatic. The use of proper concentrations of cobalt greatly enhanced the antibacterial action of penicillin. This was found in both in vitro and in vivo experiments. Aureomycin and Chloromycetin were effective in reducing chick mortalities resulting from artificial infection with S. pullorum. Penicillin also reduced mortalities, but only at relatively high concentration levels. A decided reduction in penicillin levels may eventually be realized by the combined use of cobalt and penicillin. The value of all three antibiotic agents in prevention of carriers among recovered birds, still remains to be determined.
All treated birds received antibiotics for varying periods of time prior to infection. These same groups continued to receive antibiotics for the duration of the experimental period following inoculation. REFERENCES Anderson, G. W., J. B. Cooper, J. C. Jones and C. L. Morgan, 1948. Sulfonamides in the control of pullorum disease. Poultry Sci. 27: 172-175. Benson, D. V., 1947. Streptomycin in the treatment of pullorum in baby chicks. Vet. Med. 17: 72-73. Boger, W. P., N. H. Schimmel and W. V. Matteucci, 1951. Typhoid fever successfully treated with penicillin. J. Antibiotics Chemotherapy, 1: 140145. Bottorf, C. A., and J. S. Kiser, 1947. The use of sulfonamides in the control of pullorum disease. Poultry Sci. 26:335-339. Dickinson, E. M., and E. D. Stoddard, 1949. Sulfamerazine against Salmonella pullorum in adult chickens. Poultry Sci. 28: 153-155. Gwatkin, R., 1950. Observations on the use of streptomycin in Salmonella pullorum infection in chicks by the oral and intraperitoneal routes. Canadian J. Comp. Med. 14: 157. Jackson, G. G., T. M. Gocke, H. S. Collins and M. Finland, 1950. In vitro sensitivity of pathogenic enteric bacteria to various antibiotics. J. Infec. Diseases, 87:63-70. Pratt, R., and J. Dufrenoy, 1947. Comparative responses of gram-positive and gram-negative bacteria to penicillin. J. Bact. 54: 719. Pratt, R., J. Dufrenoy and L. A. Strait, 1948. The enhancement of penicillin effectiveness in vivo by traces of cobalt. J. Bact. 55: 75-77. Strait, L. A., J. Dufrenoy and R. Pratt, 1948. Enhancement of penicillin effectiveness by traces of cobalt. J. Amer. Pharm. Assoc. 37: 133. Vincent, J. G., and H. W. Vincent, 1944. Filter paper disc modification of the Oxford cup penicillin determination. Proc. Soc. Exptl. Biol. Med. 55: 162-164.
NEWS AND NOTES {Continued from page 133) and from 1914-1918 he held the same position at Barryton, Michigan. He was also the County Superintendent of Mecosta Schools during the years 1918-1920. In 1920 Professor Card was ap-
pointed to the position of the Director of the Vocational School at Michigan State College, a position which he held until 1923 when he was appointed as an Assistant Professor of Poultry Husbandry. The
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susceptible to penicillin. This possibility is receiving further attention as well as the role of cobalt in a practical program of penicillin therapy. The possible use of penicillin in pullorum disease control merits consideration from two important aspects: First, the slow bactericidal action of penicillin may be a factor in preventing the development of carrier birds and secondly, in comparison to other antibiotic agents, penicillin is relatively cheap and readily available.