- Email: [email protected]
Anticoccidial Activity of Sulfadimethoxine1 Potentiated Mixture (Ro 5–0013)2 in Chickens
Anticoccidial Activity of Sulfadimethoxine1 Potentiated Mixture (Ro 5-0013)2 in Chickens M . MiTROVIC, E . G. SCHILDKNECHT AND G. FUSIEK Chemical Research Division, Hoffmann-La Roche Inc., Nutley, New Jersey 07110 (Received for publication June 27, 1968)
1
UX (1954) has shown that the ac- ' tivity of sulfonamides against E. tenella was enhanced when used in combination with members of a class of pyrimidine and triazine compounds. Notable among these were 2,4-diaminopyrimidines. Joyner and Kendall (1955, 1956), Horton-Smith et al. (1960), Ball (1964) and Clarke (1962, 1964) not only confirmed the above reported findings, but expanded them to include other Eimeria species. It is known today that sulfonamide-potentiator mixtures interfere with folic acid, dihydrofolic acid and tetrahydrofolic acid metabolism in sequential biogenic fashion. The resulting beneficial therapeutic responses are the enhancement in activity, lower drug concentrations, reduction in toxicity and nil drug resistance.
diamino-5-(4,5-dimethoxy-2-methylbenzyl) pyrimidine-sulfadimethoxine mixture. Further laboratory investigations have shown that Ro 5-0013 (sulfadimethoxinepotentiated mixture, ratio 5:3), at the dosage of 0.02%, was the most effective against Eimeria infections in chickens. Since the optimum dosage for sulfadimethoxine alone against Eimeria infections in chickens is 0.1% in feed, while the potentiator is ineffective at 0.0075%, the activity of the combined mixture at 0.02% (in excess of two-fold) should be considered as a true potentiation. While Marusich et al. (1969) have reported on the safety and compatibility of the sulfadimethoxine potentiated mixture (Ro 5-0013) in chickens, the present report deals with its anticoccidial activity.
Mitrovic and Bauernfeind (1967) and Mitrovic (1967, 1968) have reported on sulfadimethoxine [(N'- (2,6-dimethoxy4-pyrimidinyl) sulfanilamides-anticoccidial and antibacterial activity in fowl when used alone. In evaluating the newly synthesized 2,4-diamino-5-substituted benzylpyrimidines in combination with sulfadimethoxine against bacterial and protozoal laboratory infections, a high degree of activity was exhibited by 2,4-
MATERIALS AND METHODS
1 Agribon® is the Hoffmann-La Roche tradename for a coccidiostat and antibacterial containing sulfadimethoxine. 2 Rofenaid™ is the Hoffmann-La Roche trademark for a coccidiostat and antibacterial containing sulfadimethoxine and 2,4-diamino-5-(4,5-dimethoxy2-methylbenzyl) pyrimidine.
The chemoprophylactic efficacy studies were carried out in battery and floor pen trials. Battery trials and experimental design. Day-old Peterson Cross broiler chicks, obtained from a commercial hatchery, were kept in wire floored, electrically heated battery brooders in isolation rooms to insure complete freedom from coccidiosis until two weeks of age. At that time, they were evenly distributed by sex and body weight and placed on test. Ten birds were used per group, and all trials were repeated four times. The tests were conducted in identical rooms and batteries, under uniform temperature and light conditions.
210
ANTICOCCIDIAL ACTIVITY OF R O 5-0013
Cultures and infections. T h e coccidia suspensions used in our studies were either isolated by us or obtained from workers engaged in avian coccidiosis research. All cultures were checked for purity and virulence prior to use. T h e number of sporulated oocysts used for infection varied according to coccidial species. For single species infections, 100,000 oocysts of Eimeria tenella, E. maxima, E. brunetti; 50,000 of E. necatrix; 5,000,000 of E. acervulina and 1,000,000 of E. tnivati were used per bird. For mixed species infections, 200,000 and 150,000 oocysts were used per bird, each species being represented b y 100,000 and 50,000 oocysts. T h e sporulated oocysts were suspended in 1.0 ml of sterile distilled water and inoculated directly into the chick crop b y means of a blunt needle attached to a calibrated syringe. Medication. Purina broiler starter mash, a complete feed formula free of drugs, was used as the basal ration. T h e medicated feed was prepared by adding to the mash enough Ro 5-0013 to obtain the desired 0.02% concentration of active drug in feed. T h e drug was thoroughly mixed into the mash just prior to use to provide a uniform blend. I n all instances, the medicated feed was fed two days before infection and then for nine consecutive days thereafter. Efficacy evaluation. At the termination of the trials, the surviving birds were sacrificed, autopsied and scored for gross lesions. A special scoring system was devised for each individual species of Eimeria tested; and the lesions were graded as O-normal, 1-slight, 2-moderate, 3-severe and 4-dead. T h e readings obtained were recorded as average degree of infection (ADI). I n addition, bird weights, daily feed intake, feed efficiency and mor-
211
tality records were kept. The parameters of activity were based upon mortality, weight gain, feed efficiency and lesion score (ADI) of the medicated-infected versus the unmedicated-uninfected controls (UUC) and the infected-unmedicated controls ( I U C ) . Challenge and immunity. T h e procedures employed were identical to those described for mixed infections, except t h a t following completion of a regular medication-infection period, all the groups were placed on an unmedicated ration for two weeks. At t h a t time, all groups together with the newly introduced uninfected, unmedicated, unexposed controls ( U U U C ) , were challenged with homologous species of Eimeria—three times the amount of the first exposure. Floor pen broiler trials. Chicks of the same breed were used in these trials. T h e y were floor reared and medicated from one d a y to eight weeks of age. One hundred chicks were used per test, and each test was replicated (2 X100). At four weeks of age each bird, except the U U C , received a total of 250,000 sporulated oocysts of E. tenella, E. acervulina, E. necatrix, E. brunetti and E. maxima, each species being represented by 50,000 oocysts. T h e medicated feed was prepared b y adding enough Ro 5-0013 to Purina broiler starter and finisher mash to obtain the desired concentration of 0.02% of active drug in feed. Parallel sets of U U C and I U C were used in these trials. T h e birds were fed and watered ad libitum. Parameters of activity were the same as previously described. Floor pen replacement trials. Chicks of the same breed as previously described were used in these trials. T h e y were floor reared on litter artificially contaminated with
212
M . MlTROVIC, E . G. SCHILDKNECHT AND G. FUS1EK
E. tenella, E. necatrix, E. acervulina, E. maxima, E. brunetti and E. mivati and medicated from one day to sixteen weeks of age. One hundred birds were used per test, and each test was replicated (2X100). The medicated feed was prepared by addingTenough Ro 5-0013 to Purina broiler starter and finisher mash to obtain the desired concentrations of 0.02 and 0.01% of active drug in feed. For the first eight weeks of feeding trial, Ro 5-0013 was fed at the 0.02% dosage, which was then reduced to 0.01% for the remaining eight weeks (8 to 16 weeks). Parallel sets of UC (unmedicated controls) were used in these trials. The birds were fed and watered ad libitum. Parameters of activity were identical to those previously described.
RESULTS AND DISCUSSION Battery trials-single infections. The efficacy data on the chemoprophylactic activity of Ro 5-0013 against Eimeria species of chicks are listed in Tables 1, 2, 3, 4, 5 and 6. The results showed that Ro 5-0013, given in feed two days prior to infection and then for nine consecutive days thereafter, exhibited a high degree of activity against E. tenella (3 strains), E. necatrix, E. acervulina (2 strains), E. maxima (2 strains), E. brunetti (2 strains) and E. mivati at the dosage tested—0.02% active drug in feed. At this dosage, the Ro 5-0013 medicated chicks infected singly with the six pathogenic coccidial species had no mortality, an ADI of 0.0 to 1.0, an average weight gain of 86
TABLE 1.—Anticoccidial efficacy of Ro 5-0013 against three strains of E. tenella in two-week old chicks following infection with 100,000 oocysts r Grou
P
UUC IUC Ro 5-0013
Cone, in feed, %
No. of chicks
Average weight gain, %
Feed efficiency
Mortality %
. -p.,
None None 0.02
120 120 120
100 45 95
1.94 3.19 2.12
0 42 0
0.0 3.2 0.1
A m
UUC = Uninfected, unmedicated controls; IUC = Infected, unmedicated controls; ADI = Average degree of infection.
TABLE 2.—Anticoccidial efficacy of Ro 5-0013 against E. necatrix in
two-week old chicksfollowing infection with 50,000 oocysts Group
Cone, in feed, %
No. of chicks
Average weight gain, %
Feed efficiency
Mortality
UUC IUC Ro 5-0013
None None 0.02
40 40 40
100 44 86
1.71 2.51 1.83
0 30 0
%
ADI 0.0 2.8 1.0
TABLE 3.—Anticoccidial efficacy of Ro 5-0013 against two strains of E. acervulina in two-week old chicks following infection with 5,000,000 oocysts r W0U
P
UUC IUC Ro 5-0013
Cone, in feed, %
No. of chicks
Average weight gain, %
Feed efficiency
None None 0.02
80 80 80
100 49 100
1.92 2.81 1.91
Mortality % 0 0 0
ATYr AJJ1
0.0 2.1 0.2
ANTICOCCIDIAL ACTIVITY OF RO
5-0013
213
TABLE 4.—Anticoccidial efficacy of Ro 5-0013 against two strains of E. maxima in two-week old chicks following infection with 100,000 oocysts Group
Cone, in feed, %
No. of chicks
Average weight gain, %
Feed efficiency
Mortality
UUC IUC Ro 5-0013
None None 0.02
80 80 80
100 69 96
1.70 2.00 1.78
0 0 0
%
ADI 0.0 0.0 0.0
TABLE 5.—Anticoccidial efficacy of Ro 5-0013 against two strains of E. brunetti in two-week old chicks following infection with 100,000 oocysts Group
Cone, in feed, %
No. of chicks
Average weight gain, %
Feed efficiency
UUC IUC Ro 5-0013
None None 0.02
80 80 80
100 71 105
1.70 1.99 1.65
Mortality
% 0 6.25 0
ADI 0.0 0.3 0.0
TABLE 6.—Anticoccidial efficacy of Ro 5-0013 against E. mivati in two-week old chicks following infection with 1,000,000 oocysts Group
Cone, in feed, %
No. of chicks
Average weight gain, %
Feed efficiency
Mortality
UUC IUC Ro S-0013
None None 0.02
40 40 40
100 67 95
1.68 2.10 1.72
0 0 0
%
ADI 0.0 1.8 0.5
TABLE 7.—Anticoccidial efficacy of Ro 5-0013 against E. maxima and E. brunetti (mixed infection) in two-week old chicks following infection with 200,000 oocysts* Group
Cone, in feed, %
No. of chicks
Average weight gain, %
Feed efficiency
Mortality
UUC IUC Ro 5-0013
None None 0.02
40 40 40
100 62 97
1.73 2.03 1.77
0 18 0
%
ADI 0.0 0.7 0.0
* 100,000 oocysts of each Eimeria species per bird.
to 105% and feed efficiency of 1.65 to 2.12. This was in contrast with 0 to 42% mortality, ADI of 0.0 to 3.2, average weight gain of 44 to 7 1 % and feed emciency of 1.99 to 3.19 for the IUC. The weight gain and feed efficiency of Ro 5-0013-medicated infected chicks compared very favorably with these parameters for the UUC. Mixed infections. The efficacy data pertinent to chemoprophylactic efficacy of
Ro 5-0013 against mixed infections are listed in Tables 7 and 8. The results showed that Ro 5-0013, given in feed under the conditions previously described, exhibited a high degree of activity at the 0.02% dosage against mixed infection with E. maxima and E. brunetti. Mortality of 18% with an ADI of 0.7, weight gain of 62% and feed efficiency of 2.03 were recorded among the IUC, in contrast to no mortality, 0.0 ADI, 97% weight gain and 1.77 feed efficiency for
214
M . MlTROVIC, E . G. SCHILDKNECHT AND G. FUSIEK TABLE 8.—Anticoccidial efficacy of Ro 5-0013 against E. tenella, E. necatrix and E. acervulina {mixed infection) in two-week old chicks following infection with 150,000 oocysts* Group
Cone, in feed, %
No. of chicks
Average weight gain, %
Feed efficiency
Mortality
UUC IUC Ro 5-0013
None None 0.02
40 40 40
100 48 93
1.71 2.53 1.79
0 28 0
ADI
%
0.0 2.4 0.4
* 50,000 oocysts of each Eimeria species per bird.
birds medicated with 0.02% Ro 5-0013. The results also showed that Ro 5-0013, given in feed under identical conditions, exhibited a high degree of activity at the 0.02% dosage against mixed infection with E. tenella, E. necatrix and E. acervulina. Mortality of 28% with an ADI of 2.4 occurred among the IUC, in contrast to no mortality and a negligible ADI (0.4) for birds medicated with 0.02% Ro 5-0013. The weight gain and feed efficiency of the Ro 5-0013-medicated group was far superior to that of the IUC. In both mixed infections, the weight gain and feed efficiency of Ro 5-0013-medicated infected birds compared very favorably with those of the UUC. Challenge and immunity. The results pertinent to the chemoprophylactic activity of Ro 5-0013 (first exposure) and subsequent challenge and immunity re-
sponses (second exposure) in mixed infections are shown in Table 9. It appeared that Ro 5-0013 was highly effective in preventing mortality at the 0.02% dosage against mixed infection with E. tenella, E. necatrix and E. acervulina. On the first exposure, mortality of 20% occurred among the IUC as compared with no mortality for Ro 5-0013-medicated infected chicks. When the Ro 5-0013-medicated infected chicks were subsequently challenged with homologous species, after a two-week period without medication, they proved to be resistant. On the second exposure, mortality of 40 and 60% with an ADI of 3.0 and 3.4 occurred among each of the unmedicated control groups (UUC and UUUC), as compared with no mortality and 0.0 ADI for the previously Ro 5-0013-medicated infected chicks. Floor pen broiler trials. The results perti-
ity results of Ro 5-0013 against mixed infection in chicks
TABLE 9.—Challenge and
First exposure
Second exposure
E. tennella, E. necatrix, E. acervulina (50,000 oocysts of each per bird— birds' age, 2 weeks)
E. tenella, E. necatrix, E. acervulina (150,000 oocysts of each per bird— birds' age, 6 weeks)
Group UUC IUC Ro 5-0013 UUUC*
No. of TV/T .* r* No. of chicks chicks Mortality from first '° exposure challenged
Cone, in feed, %
No. of chicks
None None 0.02
10 10 10
0 20 0
10 8 10
—
—
—
—
* UUUC = Uninfected, unmedicated, unexposed controls.
10 8 10 10
Mortality
% 40 0 0 60
ADI
Immunity
3.0 0.0 0.0 3.4
S R R S
R=resistant S = susceptible
215
ANTICOCCIDIAL ACTIVITY OF R O 5-0013 TABLE 10.—Anticoccidial efficacy of Ro 5-0013 against mixed injection in broiler chicks reared in floor pens to 8 weeks of age and infected with 250,000 oocysts*
Group
Cone, in feed, %
No. of chicks
Average initial wt. gms./chick
UUC IUC Ro 5-0013
None None 0.02
200 200 200
37 37 37
av. final wt. gms./chick
av. wt. gain, %
Feed efficiency
Mortality due to Coccidiosis, %
1,587 1,557 1,578
100 97.7 99.1
2.22 2.92 2.26
0.0 70.5 0.0
End trial 8 weeks
* 50,000 oocysts of each Eimeria species at 4 weeks of age. Species used: E. lenella, E. necatrix, E. acervulina, E. brunetti and E. maxima.
nent to the chemoprophylactic activity of Ro 5-0013 in experimentally infected broiler chicks (5 species of Eimeria), reared in floor pens u p to eight weeks of age, are shown in Table 10. T h e results showed t h a t the chicks medicated with 0.02% Ro 5-0013 performed well. Mortality among the R o 5-0013-medicated infected birds was absent, in contrast to 7 0 . 5 % for the I U C . I n spite of the severity of exposure, the average weight gain and feed efficiency of Ro 5-0013medicated chicks was almost identical to t h a t of the U U C . Floor pen replacement trials. T h e d a t a pertinent to growth, feed efficiency and viability of chicks medicated with Ro 5-0013 (0.02 and 0.01%) and reared on contaminated litter (six Eimeria species) up to sixteen weeks of age, are listed in
Table 11. T h e results showed t h a t birds medicated with 0.02% Ro 5-0013 (up to 8 weeks) and 0 . 0 1 % Ro 5-0013 (up to 16 weeks) performed well. T h e y gained more weight and converted feed more efficiently t h a n UC (unmedicated controls). Mortality of 5 % occurred among the UC as compared with zero for the Ro 5-0013-medicated groups.
SUMMARY AND CONCLUSIONS 1. I n b a t t e r y trials, Ro 5-0013 at a dosage of 0.02% active drug in feed exhibited a high degree of chemoprophylactic activity against single and mixed infections with various strains of E. tenella, E. necatrix, E. acervulina, E. maxima, E. brunetti a n d E. mivati in chicks. 2. Infected birds medicated with 0.02% of Ro 5-0013 proved to be resistant to
TABLE 11.—Growth, feed efficiency and viability of replacement chicks medicated with Ro 5-0013 and reared in contaminated* floor pens to 16 weeks of age Group
Cone, in feed, %
No. of chicks
Av. wt. gain, %
Feed efficiency
Mort. % due to Coccidiosis
Oocysts per gram/litter
End 8-week medication period UC Ro 5-0013
None 0.02
200 200
100 101
2.49 2.33
5.0 0.0
5,000 5,000
End 16-week medication period UC Ro 5-0013
None 0.01
190 200
100 101
4.24 4.02
0.0 0.0
17,500 25,000
UC = Unmedicated controls. * Litter contaminated with E. tenella, E. necatrix, E. acervulina, E. maxima, E. brunetti and E. mivati.
216
M . MlTROVIC, E . G. SCH1LDKNECHT AND G . FUSIEK
subsequent challenge infection with multiple homologous coccidial species. 3. In floor pen broiler and replacement trials, Ro 5-0013 was highly effective at dosages of 0.02% and 0.01% in feed. 4. Under both battery and pen trials, Ro 5-0013-medicated infected birds showed no mortality, maintained either normal or close to normal body weight, converted feed efficiently and showed little or no pathology. 5. This new broad spectrum coccidiostat (Ro 5-0013), at dosages of 0.02% and 0.01% active drug in feed, is suggested for effective coccidiosis control in broiler and replacement chickens. REFERENCES Ball, S. J., 1964. Synergistic action of sulphaquinoxaline and 2-amino-4-dimethylamino-5-(4-chlorophenyl)-6 ethylpyrimidine in caecal coccidiosis in chickens. J. Comp. Path. 74: 487-499. Clarke, M. L., 1962. A mixture of diaveridine and sulphaquinoxaline as a coccidiostat for poultry. 1. Preliminary studies on efficacy against Eimeria tenetta and E. necatrix infection, and on toxicity in poultry. Vet. Rec. 74: 845-848. Clarke, M. L., 1964. A mixture of diaveridine and sulphaquinoxaline as a coccidiostat for poultry. 2. Efficacy against Eimeria acervulina, E. brunetti
and E. maxima infections together with a field survey of coccidiosis in S. E. England. Vet. Rec. 76: 818-822. Horton-Smith, C , P. L. Long and H. O. Collier, 1960. Potentiation of sulphadimidine by 2,4diamino-6,7-diisopropylpteridine and other 6,7disubstituted 2,4-diaminopteridines against Eimeria infections of chicks. Brit. J. Pharmacol. 15: 298-303. Joyner, L. P., and S. B. Kendall, 1955. Synergism in the chemotherapy of Eimeria tenetta. Nature, 176: 975. Joyner, L. P., and S. B. Kendall, 1956. The mode of action of a mixture of pyrimethamine and sulphadimidine on Eimeria tenetta. Brit. J. Pharmacol. 11:454-457. Lux, R. E., 1954. The chemotherapy of Eimeria tenetta. 1. Diaminopyrimidines and dihydrotriazines. Antibiotics and Chemotherapy, 4: 971977. Marusich, W. L., E. Ogrinz, M. Brand and M. Mitrovic, 1969. Safety and compatibility of sulfadimethoxine potentiated mixture (Ro 5 0013), a new broad spectrum coccidiostat-antibacterial, in chickens. Poultry Sci. 48:217-222. Mitrovic, M., 1967. Chemotherapeutic efficacy of sulfadimethoxine against fowl cholera and infectious coryza. Poultry Sci., 46: 1153-1158. Mitrovic, M., 1968. Sulfadimethoxine in prevention of turkey coccidiosis. Poultry Sci. 47: 314-319. Mitrovic, M., and J. C. Bauernfeind, 1967. Sulfadimethoxine therapy of avian coccidiosis. Poultry Sci. 46: 402-411.
NEWS AND NOTES U.E.P. NOTES The United Egg Producers, a federation of regional egg cooperatives, including National Egg Cooperative, New England Egg Marketing Association, Southwestern Egg Producers, and Western Egg Cooperative, elected the following officers at their inaugural meeting in Chicago: President— Maurice Stein, Maine; Vice-President—Don Nicolayson, California, and Secretary-Treasurer—Mike Rossiter, California. SOUTH DAKOTA NOTES Dedication of new facilities for poultry research was held November 7, 1968. Speaking at the program were Gordon Mydland, Attorney-General
'rom page 209) Elect, and Dr. Ted Hartung, President of the Poultry Science Association. Dr. Hartung described the extent to which research with the fowl has aided in making poultry a major agricultural industry. He reminded us that chickens are a biological converter of feed to protein food and will be of increasing importance in the 1970's when "food power will be stronger than atomic power." He pointed out that the chick has been and is a research tool for many disciplines. He commended South Dakotans for their foresight in providing facilities that will help find answers to the industry's problems. The facilities include a headquarters laboratory with chemical and physiology areas, battery brooder rooms and poultry and egg processing. A
{Continued on page 260)
1
UX (1954) has shown that the ac- ' tivity of sulfonamides against E. tenella was enhanced when used in combination with members of a class of pyrimidine and triazine compounds. Notable among these were 2,4-diaminopyrimidines. Joyner and Kendall (1955, 1956), Horton-Smith et al. (1960), Ball (1964) and Clarke (1962, 1964) not only confirmed the above reported findings, but expanded them to include other Eimeria species. It is known today that sulfonamide-potentiator mixtures interfere with folic acid, dihydrofolic acid and tetrahydrofolic acid metabolism in sequential biogenic fashion. The resulting beneficial therapeutic responses are the enhancement in activity, lower drug concentrations, reduction in toxicity and nil drug resistance.
diamino-5-(4,5-dimethoxy-2-methylbenzyl) pyrimidine-sulfadimethoxine mixture. Further laboratory investigations have shown that Ro 5-0013 (sulfadimethoxinepotentiated mixture, ratio 5:3), at the dosage of 0.02%, was the most effective against Eimeria infections in chickens. Since the optimum dosage for sulfadimethoxine alone against Eimeria infections in chickens is 0.1% in feed, while the potentiator is ineffective at 0.0075%, the activity of the combined mixture at 0.02% (in excess of two-fold) should be considered as a true potentiation. While Marusich et al. (1969) have reported on the safety and compatibility of the sulfadimethoxine potentiated mixture (Ro 5-0013) in chickens, the present report deals with its anticoccidial activity.
Mitrovic and Bauernfeind (1967) and Mitrovic (1967, 1968) have reported on sulfadimethoxine [(N'- (2,6-dimethoxy4-pyrimidinyl) sulfanilamides-anticoccidial and antibacterial activity in fowl when used alone. In evaluating the newly synthesized 2,4-diamino-5-substituted benzylpyrimidines in combination with sulfadimethoxine against bacterial and protozoal laboratory infections, a high degree of activity was exhibited by 2,4-
MATERIALS AND METHODS
1 Agribon® is the Hoffmann-La Roche tradename for a coccidiostat and antibacterial containing sulfadimethoxine. 2 Rofenaid™ is the Hoffmann-La Roche trademark for a coccidiostat and antibacterial containing sulfadimethoxine and 2,4-diamino-5-(4,5-dimethoxy2-methylbenzyl) pyrimidine.
The chemoprophylactic efficacy studies were carried out in battery and floor pen trials. Battery trials and experimental design. Day-old Peterson Cross broiler chicks, obtained from a commercial hatchery, were kept in wire floored, electrically heated battery brooders in isolation rooms to insure complete freedom from coccidiosis until two weeks of age. At that time, they were evenly distributed by sex and body weight and placed on test. Ten birds were used per group, and all trials were repeated four times. The tests were conducted in identical rooms and batteries, under uniform temperature and light conditions.
210
ANTICOCCIDIAL ACTIVITY OF R O 5-0013
Cultures and infections. T h e coccidia suspensions used in our studies were either isolated by us or obtained from workers engaged in avian coccidiosis research. All cultures were checked for purity and virulence prior to use. T h e number of sporulated oocysts used for infection varied according to coccidial species. For single species infections, 100,000 oocysts of Eimeria tenella, E. maxima, E. brunetti; 50,000 of E. necatrix; 5,000,000 of E. acervulina and 1,000,000 of E. tnivati were used per bird. For mixed species infections, 200,000 and 150,000 oocysts were used per bird, each species being represented b y 100,000 and 50,000 oocysts. T h e sporulated oocysts were suspended in 1.0 ml of sterile distilled water and inoculated directly into the chick crop b y means of a blunt needle attached to a calibrated syringe. Medication. Purina broiler starter mash, a complete feed formula free of drugs, was used as the basal ration. T h e medicated feed was prepared by adding to the mash enough Ro 5-0013 to obtain the desired 0.02% concentration of active drug in feed. T h e drug was thoroughly mixed into the mash just prior to use to provide a uniform blend. I n all instances, the medicated feed was fed two days before infection and then for nine consecutive days thereafter. Efficacy evaluation. At the termination of the trials, the surviving birds were sacrificed, autopsied and scored for gross lesions. A special scoring system was devised for each individual species of Eimeria tested; and the lesions were graded as O-normal, 1-slight, 2-moderate, 3-severe and 4-dead. T h e readings obtained were recorded as average degree of infection (ADI). I n addition, bird weights, daily feed intake, feed efficiency and mor-
211
tality records were kept. The parameters of activity were based upon mortality, weight gain, feed efficiency and lesion score (ADI) of the medicated-infected versus the unmedicated-uninfected controls (UUC) and the infected-unmedicated controls ( I U C ) . Challenge and immunity. T h e procedures employed were identical to those described for mixed infections, except t h a t following completion of a regular medication-infection period, all the groups were placed on an unmedicated ration for two weeks. At t h a t time, all groups together with the newly introduced uninfected, unmedicated, unexposed controls ( U U U C ) , were challenged with homologous species of Eimeria—three times the amount of the first exposure. Floor pen broiler trials. Chicks of the same breed were used in these trials. T h e y were floor reared and medicated from one d a y to eight weeks of age. One hundred chicks were used per test, and each test was replicated (2 X100). At four weeks of age each bird, except the U U C , received a total of 250,000 sporulated oocysts of E. tenella, E. acervulina, E. necatrix, E. brunetti and E. maxima, each species being represented by 50,000 oocysts. T h e medicated feed was prepared b y adding enough Ro 5-0013 to Purina broiler starter and finisher mash to obtain the desired concentration of 0.02% of active drug in feed. Parallel sets of U U C and I U C were used in these trials. T h e birds were fed and watered ad libitum. Parameters of activity were the same as previously described. Floor pen replacement trials. Chicks of the same breed as previously described were used in these trials. T h e y were floor reared on litter artificially contaminated with
212
M . MlTROVIC, E . G. SCHILDKNECHT AND G. FUS1EK
E. tenella, E. necatrix, E. acervulina, E. maxima, E. brunetti and E. mivati and medicated from one day to sixteen weeks of age. One hundred birds were used per test, and each test was replicated (2X100). The medicated feed was prepared by addingTenough Ro 5-0013 to Purina broiler starter and finisher mash to obtain the desired concentrations of 0.02 and 0.01% of active drug in feed. For the first eight weeks of feeding trial, Ro 5-0013 was fed at the 0.02% dosage, which was then reduced to 0.01% for the remaining eight weeks (8 to 16 weeks). Parallel sets of UC (unmedicated controls) were used in these trials. The birds were fed and watered ad libitum. Parameters of activity were identical to those previously described.
RESULTS AND DISCUSSION Battery trials-single infections. The efficacy data on the chemoprophylactic activity of Ro 5-0013 against Eimeria species of chicks are listed in Tables 1, 2, 3, 4, 5 and 6. The results showed that Ro 5-0013, given in feed two days prior to infection and then for nine consecutive days thereafter, exhibited a high degree of activity against E. tenella (3 strains), E. necatrix, E. acervulina (2 strains), E. maxima (2 strains), E. brunetti (2 strains) and E. mivati at the dosage tested—0.02% active drug in feed. At this dosage, the Ro 5-0013 medicated chicks infected singly with the six pathogenic coccidial species had no mortality, an ADI of 0.0 to 1.0, an average weight gain of 86
TABLE 1.—Anticoccidial efficacy of Ro 5-0013 against three strains of E. tenella in two-week old chicks following infection with 100,000 oocysts r Grou
P
UUC IUC Ro 5-0013
Cone, in feed, %
No. of chicks
Average weight gain, %
Feed efficiency
Mortality %
. -p.,
None None 0.02
120 120 120
100 45 95
1.94 3.19 2.12
0 42 0
0.0 3.2 0.1
A m
UUC = Uninfected, unmedicated controls; IUC = Infected, unmedicated controls; ADI = Average degree of infection.
TABLE 2.—Anticoccidial efficacy of Ro 5-0013 against E. necatrix in
two-week old chicksfollowing infection with 50,000 oocysts Group
Cone, in feed, %
No. of chicks
Average weight gain, %
Feed efficiency
Mortality
UUC IUC Ro 5-0013
None None 0.02
40 40 40
100 44 86
1.71 2.51 1.83
0 30 0
%
ADI 0.0 2.8 1.0
TABLE 3.—Anticoccidial efficacy of Ro 5-0013 against two strains of E. acervulina in two-week old chicks following infection with 5,000,000 oocysts r W0U
P
UUC IUC Ro 5-0013
Cone, in feed, %
No. of chicks
Average weight gain, %
Feed efficiency
None None 0.02
80 80 80
100 49 100
1.92 2.81 1.91
Mortality % 0 0 0
ATYr AJJ1
0.0 2.1 0.2
ANTICOCCIDIAL ACTIVITY OF RO
5-0013
213
TABLE 4.—Anticoccidial efficacy of Ro 5-0013 against two strains of E. maxima in two-week old chicks following infection with 100,000 oocysts Group
Cone, in feed, %
No. of chicks
Average weight gain, %
Feed efficiency
Mortality
UUC IUC Ro 5-0013
None None 0.02
80 80 80
100 69 96
1.70 2.00 1.78
0 0 0
%
ADI 0.0 0.0 0.0
TABLE 5.—Anticoccidial efficacy of Ro 5-0013 against two strains of E. brunetti in two-week old chicks following infection with 100,000 oocysts Group
Cone, in feed, %
No. of chicks
Average weight gain, %
Feed efficiency
UUC IUC Ro 5-0013
None None 0.02
80 80 80
100 71 105
1.70 1.99 1.65
Mortality
% 0 6.25 0
ADI 0.0 0.3 0.0
TABLE 6.—Anticoccidial efficacy of Ro 5-0013 against E. mivati in two-week old chicks following infection with 1,000,000 oocysts Group
Cone, in feed, %
No. of chicks
Average weight gain, %
Feed efficiency
Mortality
UUC IUC Ro S-0013
None None 0.02
40 40 40
100 67 95
1.68 2.10 1.72
0 0 0
%
ADI 0.0 1.8 0.5
TABLE 7.—Anticoccidial efficacy of Ro 5-0013 against E. maxima and E. brunetti (mixed infection) in two-week old chicks following infection with 200,000 oocysts* Group
Cone, in feed, %
No. of chicks
Average weight gain, %
Feed efficiency
Mortality
UUC IUC Ro 5-0013
None None 0.02
40 40 40
100 62 97
1.73 2.03 1.77
0 18 0
%
ADI 0.0 0.7 0.0
* 100,000 oocysts of each Eimeria species per bird.
to 105% and feed efficiency of 1.65 to 2.12. This was in contrast with 0 to 42% mortality, ADI of 0.0 to 3.2, average weight gain of 44 to 7 1 % and feed emciency of 1.99 to 3.19 for the IUC. The weight gain and feed efficiency of Ro 5-0013-medicated infected chicks compared very favorably with these parameters for the UUC. Mixed infections. The efficacy data pertinent to chemoprophylactic efficacy of
Ro 5-0013 against mixed infections are listed in Tables 7 and 8. The results showed that Ro 5-0013, given in feed under the conditions previously described, exhibited a high degree of activity at the 0.02% dosage against mixed infection with E. maxima and E. brunetti. Mortality of 18% with an ADI of 0.7, weight gain of 62% and feed efficiency of 2.03 were recorded among the IUC, in contrast to no mortality, 0.0 ADI, 97% weight gain and 1.77 feed efficiency for
214
M . MlTROVIC, E . G. SCHILDKNECHT AND G. FUSIEK TABLE 8.—Anticoccidial efficacy of Ro 5-0013 against E. tenella, E. necatrix and E. acervulina {mixed infection) in two-week old chicks following infection with 150,000 oocysts* Group
Cone, in feed, %
No. of chicks
Average weight gain, %
Feed efficiency
Mortality
UUC IUC Ro 5-0013
None None 0.02
40 40 40
100 48 93
1.71 2.53 1.79
0 28 0
ADI
%
0.0 2.4 0.4
* 50,000 oocysts of each Eimeria species per bird.
birds medicated with 0.02% Ro 5-0013. The results also showed that Ro 5-0013, given in feed under identical conditions, exhibited a high degree of activity at the 0.02% dosage against mixed infection with E. tenella, E. necatrix and E. acervulina. Mortality of 28% with an ADI of 2.4 occurred among the IUC, in contrast to no mortality and a negligible ADI (0.4) for birds medicated with 0.02% Ro 5-0013. The weight gain and feed efficiency of the Ro 5-0013-medicated group was far superior to that of the IUC. In both mixed infections, the weight gain and feed efficiency of Ro 5-0013-medicated infected birds compared very favorably with those of the UUC. Challenge and immunity. The results pertinent to the chemoprophylactic activity of Ro 5-0013 (first exposure) and subsequent challenge and immunity re-
sponses (second exposure) in mixed infections are shown in Table 9. It appeared that Ro 5-0013 was highly effective in preventing mortality at the 0.02% dosage against mixed infection with E. tenella, E. necatrix and E. acervulina. On the first exposure, mortality of 20% occurred among the IUC as compared with no mortality for Ro 5-0013-medicated infected chicks. When the Ro 5-0013-medicated infected chicks were subsequently challenged with homologous species, after a two-week period without medication, they proved to be resistant. On the second exposure, mortality of 40 and 60% with an ADI of 3.0 and 3.4 occurred among each of the unmedicated control groups (UUC and UUUC), as compared with no mortality and 0.0 ADI for the previously Ro 5-0013-medicated infected chicks. Floor pen broiler trials. The results perti-
ity results of Ro 5-0013 against mixed infection in chicks
TABLE 9.—Challenge and
First exposure
Second exposure
E. tennella, E. necatrix, E. acervulina (50,000 oocysts of each per bird— birds' age, 2 weeks)
E. tenella, E. necatrix, E. acervulina (150,000 oocysts of each per bird— birds' age, 6 weeks)
Group UUC IUC Ro 5-0013 UUUC*
No. of TV/T .* r* No. of chicks chicks Mortality from first '° exposure challenged
Cone, in feed, %
No. of chicks
None None 0.02
10 10 10
0 20 0
10 8 10
—
—
—
—
* UUUC = Uninfected, unmedicated, unexposed controls.
10 8 10 10
Mortality
% 40 0 0 60
ADI
Immunity
3.0 0.0 0.0 3.4
S R R S
R=resistant S = susceptible
215
ANTICOCCIDIAL ACTIVITY OF R O 5-0013 TABLE 10.—Anticoccidial efficacy of Ro 5-0013 against mixed injection in broiler chicks reared in floor pens to 8 weeks of age and infected with 250,000 oocysts*
Group
Cone, in feed, %
No. of chicks
Average initial wt. gms./chick
UUC IUC Ro 5-0013
None None 0.02
200 200 200
37 37 37
av. final wt. gms./chick
av. wt. gain, %
Feed efficiency
Mortality due to Coccidiosis, %
1,587 1,557 1,578
100 97.7 99.1
2.22 2.92 2.26
0.0 70.5 0.0
End trial 8 weeks
* 50,000 oocysts of each Eimeria species at 4 weeks of age. Species used: E. lenella, E. necatrix, E. acervulina, E. brunetti and E. maxima.
nent to the chemoprophylactic activity of Ro 5-0013 in experimentally infected broiler chicks (5 species of Eimeria), reared in floor pens u p to eight weeks of age, are shown in Table 10. T h e results showed t h a t the chicks medicated with 0.02% Ro 5-0013 performed well. Mortality among the R o 5-0013-medicated infected birds was absent, in contrast to 7 0 . 5 % for the I U C . I n spite of the severity of exposure, the average weight gain and feed efficiency of Ro 5-0013medicated chicks was almost identical to t h a t of the U U C . Floor pen replacement trials. T h e d a t a pertinent to growth, feed efficiency and viability of chicks medicated with Ro 5-0013 (0.02 and 0.01%) and reared on contaminated litter (six Eimeria species) up to sixteen weeks of age, are listed in
Table 11. T h e results showed t h a t birds medicated with 0.02% Ro 5-0013 (up to 8 weeks) and 0 . 0 1 % Ro 5-0013 (up to 16 weeks) performed well. T h e y gained more weight and converted feed more efficiently t h a n UC (unmedicated controls). Mortality of 5 % occurred among the UC as compared with zero for the Ro 5-0013-medicated groups.
SUMMARY AND CONCLUSIONS 1. I n b a t t e r y trials, Ro 5-0013 at a dosage of 0.02% active drug in feed exhibited a high degree of chemoprophylactic activity against single and mixed infections with various strains of E. tenella, E. necatrix, E. acervulina, E. maxima, E. brunetti a n d E. mivati in chicks. 2. Infected birds medicated with 0.02% of Ro 5-0013 proved to be resistant to
TABLE 11.—Growth, feed efficiency and viability of replacement chicks medicated with Ro 5-0013 and reared in contaminated* floor pens to 16 weeks of age Group
Cone, in feed, %
No. of chicks
Av. wt. gain, %
Feed efficiency
Mort. % due to Coccidiosis
Oocysts per gram/litter
End 8-week medication period UC Ro 5-0013
None 0.02
200 200
100 101
2.49 2.33
5.0 0.0
5,000 5,000
End 16-week medication period UC Ro 5-0013
None 0.01
190 200
100 101
4.24 4.02
0.0 0.0
17,500 25,000
UC = Unmedicated controls. * Litter contaminated with E. tenella, E. necatrix, E. acervulina, E. maxima, E. brunetti and E. mivati.
216
M . MlTROVIC, E . G. SCH1LDKNECHT AND G . FUSIEK
subsequent challenge infection with multiple homologous coccidial species. 3. In floor pen broiler and replacement trials, Ro 5-0013 was highly effective at dosages of 0.02% and 0.01% in feed. 4. Under both battery and pen trials, Ro 5-0013-medicated infected birds showed no mortality, maintained either normal or close to normal body weight, converted feed efficiently and showed little or no pathology. 5. This new broad spectrum coccidiostat (Ro 5-0013), at dosages of 0.02% and 0.01% active drug in feed, is suggested for effective coccidiosis control in broiler and replacement chickens. REFERENCES Ball, S. J., 1964. Synergistic action of sulphaquinoxaline and 2-amino-4-dimethylamino-5-(4-chlorophenyl)-6 ethylpyrimidine in caecal coccidiosis in chickens. J. Comp. Path. 74: 487-499. Clarke, M. L., 1962. A mixture of diaveridine and sulphaquinoxaline as a coccidiostat for poultry. 1. Preliminary studies on efficacy against Eimeria tenetta and E. necatrix infection, and on toxicity in poultry. Vet. Rec. 74: 845-848. Clarke, M. L., 1964. A mixture of diaveridine and sulphaquinoxaline as a coccidiostat for poultry. 2. Efficacy against Eimeria acervulina, E. brunetti
and E. maxima infections together with a field survey of coccidiosis in S. E. England. Vet. Rec. 76: 818-822. Horton-Smith, C , P. L. Long and H. O. Collier, 1960. Potentiation of sulphadimidine by 2,4diamino-6,7-diisopropylpteridine and other 6,7disubstituted 2,4-diaminopteridines against Eimeria infections of chicks. Brit. J. Pharmacol. 15: 298-303. Joyner, L. P., and S. B. Kendall, 1955. Synergism in the chemotherapy of Eimeria tenetta. Nature, 176: 975. Joyner, L. P., and S. B. Kendall, 1956. The mode of action of a mixture of pyrimethamine and sulphadimidine on Eimeria tenetta. Brit. J. Pharmacol. 11:454-457. Lux, R. E., 1954. The chemotherapy of Eimeria tenetta. 1. Diaminopyrimidines and dihydrotriazines. Antibiotics and Chemotherapy, 4: 971977. Marusich, W. L., E. Ogrinz, M. Brand and M. Mitrovic, 1969. Safety and compatibility of sulfadimethoxine potentiated mixture (Ro 5 0013), a new broad spectrum coccidiostat-antibacterial, in chickens. Poultry Sci. 48:217-222. Mitrovic, M., 1967. Chemotherapeutic efficacy of sulfadimethoxine against fowl cholera and infectious coryza. Poultry Sci., 46: 1153-1158. Mitrovic, M., 1968. Sulfadimethoxine in prevention of turkey coccidiosis. Poultry Sci. 47: 314-319. Mitrovic, M., and J. C. Bauernfeind, 1967. Sulfadimethoxine therapy of avian coccidiosis. Poultry Sci. 46: 402-411.
NEWS AND NOTES U.E.P. NOTES The United Egg Producers, a federation of regional egg cooperatives, including National Egg Cooperative, New England Egg Marketing Association, Southwestern Egg Producers, and Western Egg Cooperative, elected the following officers at their inaugural meeting in Chicago: President— Maurice Stein, Maine; Vice-President—Don Nicolayson, California, and Secretary-Treasurer—Mike Rossiter, California. SOUTH DAKOTA NOTES Dedication of new facilities for poultry research was held November 7, 1968. Speaking at the program were Gordon Mydland, Attorney-General
'rom page 209) Elect, and Dr. Ted Hartung, President of the Poultry Science Association. Dr. Hartung described the extent to which research with the fowl has aided in making poultry a major agricultural industry. He reminded us that chickens are a biological converter of feed to protein food and will be of increasing importance in the 1970's when "food power will be stronger than atomic power." He pointed out that the chick has been and is a research tool for many disciplines. He commended South Dakotans for their foresight in providing facilities that will help find answers to the industry's problems. The facilities include a headquarters laboratory with chemical and physiology areas, battery brooder rooms and poultry and egg processing. A
{Continued on page 260)