- Email: [email protected]
Efficacy Studies With Robenidine, A New Type of Anticoccidial, in the Diet
KUBENA, CHEN, DEATON AND REECE
978
REFERENCES Deaton, J. W., F. N. Reece, L. F. Kubena, B. D. Lott and J. D. May, 1973. The ability of the broiler chicken to compensate for early growth depression. Poultry Sci. 52: 262-265. Deaton, J. W., L. F. Kubena, T. C. Chen and F. N. Reece, 1974. Factors influencing the quantity of abdominal fat in broilers 2. Cage versus floor rearing. Poultry Sci. 53: 574-577.
Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Essary, E. O., L. E. Dawson, E. L. Wisman and C. E. Holmes, 1960. Influence of different levels of fat and protein in the diet on areas of fat deposition in fryers. Poultry Sci. 39: 1249. Haram, D., R. E. Childs and A. J. Mercuri, 1973. Relationship between fats in broiler finisher rations and fats in chiller water from broiler processing. Poultry Sci. 52: 88-92. Kubena, L. F., B. D. Lott, J. W. Deaton, F. N. Reece and J. D. May, 1972. Body composition of chicks as influenced by environmental temperature and selected dietary factors. Poultry Sci. 51: 517522. Kubena, L. F., J. W. Deaton, T. C. Chen and F. N. Reece, 1974. Factors influencing the quantity of abdominal fat in broilers. 1. Rearing temperature, sex, age or weight, and dietary choline chloride and inositol supplementation. Poultry Sci. 53: 211214. Macklin, L. J., and R. S. Gordon, 1961. Effect of dietary fatty acids and cholesterol on growth and fatty acid composition of the chicken. J. Nutr. 75: 157-164. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics, Chapter 8. McGrawHill Book Company, Inc., New York, N. Y.
Efficacy Studies With Robenidine, A New Type of Anticoccidial, in the Diet R. L . KENNETT, S. KANTOR AND A. GALLO
Agriculture Center, American Cyanamid Company, P. O. Box 400, Princeton, New Jersey 08540 (Received for publication August 20, 1973)
ABSTRACT Robenidine, l,3-bis(p-chlorobenzylideneamino)guanidine hydrochloride has broad spectrum efficacy in chickens. At recommended 30 g./ton in the diet it is completely effective in preventing clinical disease from 8 laboratory species of Eimeria administered individually or in combination; it is equally effective against at least 95 strains of 6 economically important species obtained in 1968 and 1969 from various geographical areas of the United States. Comparative floor-pen trials on litter seeded with 6 pathogenic species of coccidia indicated that robenidine at 30 g./ton overall provided better weight gains and feed efficiencies than recommended concentrations of amprolium plus ethopabate. Robenidine plus roxarsone (45 g./ton) was equivalent to recommended levels of clopidol plus roxarsone (45 g./ton) in these performance parameters. POULTRY SCIENCE 53: 978-986, 1974
INTRODUCTION OCCIDIOSIS in chickens continues to be a problem despite efforts to control the disease by chemical means and poultry house management. Many broad spectrum
C
anticoccidials have been developed and their use has greatly contributed to the growth of the poultry industry. However, since most anticoccidials fail after a period of time, because of misuse and the development of
Downloaded from http://ps.oxfordjournals.org/ at Carleton University on June 13, 2015
had a significantly smaller percentage of abdominal fat than the 9-week-old females receiving the same diet. This agrees with the previous report of Kubena et al. (1974). Within each dietary treatment females had a numerically higher percentage of abdominal fat than the males, which agrees with the report of Kubena et al. (1974). It is interesting to note that, although the females were considerably lighter in weight than the males, the absolute quantity of abdominal fat was comparable to that of the males within each dietary treatment.
EFFICACY OF ROBENIDINE
MATERIALS AND METHODS Battery Trails. Standardized methods and criteria of efficacy are those reported by Waletzky (1970). Broiler-type hybrid cockerel chicks, oneweek of age, were initially distributed into equal-weight groups. All chicks were reared on wire-floored cages in adequately heated rooms and maintained on a 12-hour alternating light-dark cycle. Normal and medicated rations were available ad libitum in a typical and nutritionally adequate basal broiler ration from 2 days before inoculation until termination of the test 14 days after inoculation. Sporulated oocysts were inoculated by gavage, with a level of infection selected from previously titrated inocula in non-medicated birds of similar age. Duplicate tests, each containing 4 replicates of 10 chicks each, were conducted utilizing ©Registered trademark of American Cyanamid Company.
a commercial mixture of 8 coccidial species (no E. mitis), as well as individual inoculations with each of the following 6 pathogenic species maintained routinely in the laboratory: Eimeria acervulina, E. brunetti, E. maxima, E. mivati, E. necatrix and E. tenella. Mixed field cultures of coccidia were also utilized to assess better the performance of robenidine in single experiments containing 2 replicates. Most cultures were obtained between May, 1968 and May, 1969 from various diagnostic laboratories located in nine states. Average weight gains and feed intakes were calculated from day of inoculation (0 day) to 6 or 7 days post-inoculation near the height of infection, and finally at 14 days post-inoculation when the experiments were terminated. Weight gains and feed conversions were compared at the 5% level of probability by analysis of variance, and the studentized range test for differences between all means (Snedecor and Cochran, 1967). Lesions were scored (Johnson and Reid, 1970, slightly modified) in selected groups sacrificed at 6 or 7 days post-inoculation in all experiments, except those receiving the 8 species combination; in these no lesion scoring was performed. Oocyst counts were made using minor modifications of standard methods (Davies et al., 1963) in Levy cell counting chambers, or after flotation of 1-2% of the sample in a saturated sugar solution when relatively few oocysts were present. Significance of differences in mortality were determined by Chi Square analysis. Floor Pen Trials. Two trials under simulated commercial conditions were conducted in the same facilities. In the first trial robenidine was compared with reference coccidiostat amprolium plus ethopabate through five consecutive flocks, and in a second trial robenidine, with and without roxarsone (45 g. /ton), was compared with reference coccidiostat
Downloaded from http://ps.oxfordjournals.org/ at Carleton University on June 13, 2015
resistance, a continuing search for novel, improved chemotherapeutic agents is necessary. Robenz® robenidine, l,3-bis(p-chlorobenzylideneamino)guanidine hydrochloride, first reported by Kantor et al. (1970) to have high broad spectrum activity and Berger et al. (1972) who proved that its margin of safety is at least sixfold, is a novel anticoccidial for use in chickens. Additional data confirming the high efficacy of robenidine in battery and floor pen studies have appeared in the literature (Badiola et al., 1970; Kantor, 1973 a,b;Reid era/., 1970; Ryley and Wilson, 1971; Waletzky et al., 1970). Efficacy against species of turkey coccidia has also been reported (Joyner and Norton, 1972). This paper describes the results of recent battery and floor-pen experiments on the prophylactic efficacy of robenidine in chickens at its recommended concentration of 30 g./ton of feed against both laboratory and field collected strains of Eimeria.
979
980
R. L. KENNETT, S. KANTOR AND A. GALLO
Examination of susceptible three-week-old chicks placed in all "seeded" pens for approximately 4 days after completion of seeding in each trial indicated the premises were successfully contaminated with coccidia. Criteria of efficacy were average terminal weights and feed conversion for all treatments and flocks in each trial. RESULTS AND DISCUSSION Battery Trials. Robenidine at its recommended concentration of 30 g./ton of feed was highly effective against the 6 major pathogenic species of chicken coccidia, tested both individually, and against a mixed challenge of 8 species. In Table 1 the average weight gains of the infected, nonmedicated control groups through 6 or 7 days, and 14 days post-inoculation were all significantly (P < 0.05) lower than the non-infected controls. No significant differences were ob* Trademark of Sterwin Laboratories, Inc., New York, N.Y., U.S.A.
served in weight gains of birds fed robenidine at 30g./ton either at 6 or 7 days post-inoculation, or at the termination of the test at 14 days. Feed efficiencies over 14 days paralleled the weight gains. The non-medicated infected treatments were inferior to the normal control feed efficiencies; those of the robenidine medicated groups were not different (P > 0.05). No feed efficiencies were calculated in the E. necatrix and E. tenella tests because high mortality in control birds distorts their value. Table 1 also summarizes robenidine efficacy based on mortality, lesion scores, and oocyst production. No mortality was observed in any robenidine treated birds, even though significant (P < 0.05) numbers of birds died in non-medicated treatments inoculated with E. necatrix and E. tenella. Lesions in the robenidine medicated groups were prevented, except in 18 of 40 birds inoculated with E. maxima. However, as compared to the non-medicated, E. maxima inoculated birds, robenidine reduced E. maxima lesions to the extent that microscopic examination of mucosal smears was necessary to determine infection in most birds. E. maxima oocysts were recovered from medicated groups inoculated with this species either in a single or mixed coccidial inoculum, but numbers were reduced by more than 99.9% in each case. No oocysts representative of the other species were recovered from birds treated with robenidine in these experiments. Against 32 different field collections containing at least 95 strains of Eimeria (Table 2), the average weight gain of robenidine treated birds, calculated from 0-6, or 7 days, was 100 percent (range 88%-112%) relative to growth in the non-medicated, non-inoculated (normal) controls. Growth of replicates in 30 of the 32 infected, non-medicated control treatments were significantly reduced (P <0.05) as compared to their non-inoculated,
Downloaded from http://ps.oxfordjournals.org/ at Carleton University on June 13, 2015
clopidol plus roxarsone (45 g./ton) through four consecutive flocks. In each trial all flocks contained 4 replicates of 500 birds per treatment and were provided with 0.85 sq. ft. of floor space. Floor litter was renewed only between the two trials after careful cleaning of the pens: no changes in floor litter were made between the series of 5 and 4 flocks, respectively. Immediately before the initial flock in each trial was introduced each pen was seeded by two consecutive groups of 100 infected non-medicated birds on litter with adequate moisture content for oocyst sporulation. In the first trial each seeder bird received 10 times the recommended immunizing dose of CocciVac* type D which contains all chicken coccidia except Eimeria mitis. A mixture of laboratory and recently collected field strains containing at least 6 economically important species of Eimeria was administered to seeder birds in the second trial (approximately 50,000 sporulated oocysts per chick administered).
56*
68*
283
282
265
302
305
261
92
92
%
118
126
113
brunetti
mivati
maxima
necatnx
tenella
8 spp. mixedb
100
99
99 74*
58*
56*
83*
102
102
101
102
97
102
102
1.51
1.55
1.49
1.54
1.67*
NC
NC
1.65*
1.70*
1.54
1.51
1.55
1.50
1.54
1.55
1.54
1.51
1.73*
1.72
1.58
1.50
IR
IC
heed efficiency (0-14 days p.i.) -
5
23*
55*
0
0
0
0
14 day IC
% Mort
* Significantly different from normal control (P < 0.05) aAverage of eight groups of 10 birds each; four groups of birds sacrificed for lesion scores at 7 days except in mixed species (p.i.) in acervulina, brunetti, and mivati experiments. b50 x CocciVac, Type D (E. mitis absent) c E. maxima only recovered NC = High mortality with E. necatrix and E. tenella prevented meaningful efficiency calculations IC = Inoculated, non-medicated controls IR = Inoculated, robenidine treated
62*
48*
24*
101
81*
97
72* 74*
97 102
ill
74*
65*
309
102
acervulina
0-14 days p.i. IC IR
% of average normal weight gain
0-7 days p.i." IC IR
0-14 days
Eimeria spp.
0-7 days
Non-inoculated, non-medicated (normal) control weight gain (g.)
TABLE 1.—Anticoccidial efficacy of robenidine at 30 g./ton against laboratory Ei
ded from http://ps.oxfordjournals.org/ at Carleton University on June 13, 2015
+ + + + +
+ + + + + +
+ + + + + +
7 8 9 10 11 12
13 14 15 16 17 18
+
•
acervulina/ mivati
Isolate no. 1 2 3 4 5 6
+ + + + + +
+ +
+ +
+ +
brunetti + +
+
+ + + +
+
+ +
+
+
tenella
+ + + + + + + + +
+
necatrix
862
103
0-6 or 7 days p.i. gms. /chick 96
Weight gain
Non-inoculated, non-medicated (Normal) control
Wei
of robenidine at 30 g./ton against 32 different Eimeria
maxima + + + + + +
Species present
TABLE 2.—Efficacy
ded from http://ps.oxfordjournals.org/ at Carleton University on June 13, 2015
+
+ +
+
23 24 25 26 27 28
29
30 31
32
+
+ +
+
+
brunetti +
+
maxima
Species present
+
+
+
+ +
+
+
+
+
tenella +
necatrix
Weig
acervulina/E
136
140
131
1122
0-6 or 7 days p.i. gms./chick 1202
Weight gain
Non-inoculated, non-medicated (Normal) control
* Significantly different from normal controls (P < .05) 1 At least 95 individual strains of Eimeria spp. 2 Weight gains calculated at 0-6 days after inoculation 3 Two replicates of 10 birds per treatment 4 E . maxima only species recovered with the exception of isolate number 31 in which E. p.i. = post inoculation
acervulina/ mivati + + + +
++++++
Isolate no. 19 20 21 22
++++++
TABLE 2.—(Cont'd)
ed from http://ps.oxfordjournals.org/ at Carleton University on June 13, 2015
3.40" 3.66" 3.75" 3.72"
Non-medicated controls Clopidol + Roxarsone 2 Robenidine + Roxarsone 3 Robenidine
3.36" 3.58" 3.50"" 3.51""
3.30" 3.29" 3.43"
2
3.60" 3.82" 3.88" 3.75""
2.95" 3.09" 3.24c
3
3.65" 3.80" 3.77" 3.70"
3.03" 3.19" 3.35c
4
—
3.22" 3.41" 3.56c
5
(3.50) (3.72) (3.72) (3.67)
(3.20) (3.32) (3.43)
Avg.
2.01" 2.01" 1.99" 2.03"
Trial 2
1.99" 2.00" 1.99"
1
1.97 1.89 1.91 1.91
1.99 1.99 1.97
2
Fe
Figures in same flock having different superscript letters are significantly different from each other ( 'Amprolium concentration in feed equal to 125 p.p.m.; ethopabate equals 4 p.p.m. 2 Clopidol concentration in feed equal to 125 p.p.m.; roxarsone equals 50 p.p.m. 'Roxarsone concentration in feed equal to 50 p.p.m.
3.50" 3.60" 3.66"
1
Non-medicated controls Amprolium + Ethopabate 1 Robenidine
Treatment
Flock number
Avg . weight/bird (lb.) 8 weeks
Trial 1
TABLE 3.—Comparative efficacy of robenidine at 30 g./ton and two reference anticoccidials in floor p hundred birds per treatment on reused litter
ded from http://ps.oxfordjournals.org/ at Carleton University on June 13, 2015
985
EFFICACY OF ROBENIDINE
Floor-Pen Trials. (Table 3) Average terminal 8-week weights of robenidine and amprolium plus ethopabate medicated birds in the first trial were significantly superior (P < 0.05) to the non-medicated controls in all but the second of 5 consecutive flocks. Robenidine weights were significantly better than amprolium + ethopabate in the third, fourth, and fifth flocks. Overall average weight improvement was 4% for amprolium plus ethopabate, and 7% for robenidine. The feed efficiencies of robenidine treated birds were significantly improved as compared to the non-medicated controls in the third, fourth and fifth flocks, and superior to amprolium + ethopabate in the third and fifth flocks. Little improvement in feed efficiencies were apparent in amprolium + ethopabate treated flocks. In the second comparison the overall improvement in bird weights at the end of eight weeks was 6% for clopidol plus roxarsone and robenidine plus roxarsone, and 5% for robenidine alone. All treatments appeared to improve weight gains within each flock, sometimes significantly, but no statistical
differences were noted among treatments. Feed efficiencies of treated groups were improved only occasionally over the untreated controls, particularly clopidol plus roxarsone in the second and fourth flocks, and robenidine, with and without roxarsone in the third flock. No significant differences were evident among the birds on the different treatments. The data reported here, and as yet unpublished data, confirm the high and broad spectrum efficacy of robenidine at 30 g./ton against chicken coccidiosis. Robenidine will be a useful addition to the commercial anticoccidial market. REFERENCES Badiola, C , I. Badiola and F. Carrasco, 1970. Robenzidene in the feed of broilers: Determination of the effect of the withdrawal of robenzidene for periods up to 48 hours on the course of experimental coccidial infection. Proc. XIV Congr. Mund. de Avicultura, Madrid, Section III: 186-187. Berger, H., A. L. Shor, G. O. Gale and K. L. Simkins, 1972. Safety evaluation of robenidine in the feed of broiler chickens. (Unpublished data). Davies, S. F. M., L. P. Joyner and S. B. Kendall, 1963. Coccidiosis. Oliver and Boyd, Ltd., London, Chapter XV. Johnson, J., and W. M. Reid, 1970. Anticoccidial drugs: Lesion scoring techniques in battery and floor-pen experiments with chickens. Experimental Parasitol. 28: 30-36. Joyner, L. P., and C. C. Norton, 1972. The drug sensitivity of recently isolated strains of Eimeria meleagrimitis and a laboratory strain of Eimeria adenoeides in turkeys to robenidine. Res. Vet. Sci. 13: 279-281. Kantor, S., 1973a. Robenidine—a new type of anticoccidial. World's Poultry Sci. J., 29: 56-57. Kantor, S., 1973b. Robenidine—a new type of anticoccidial agent. 4th Europ. Poultry Conf., London, pp. 141-146. Kantor, S., R. L. Kennett, Jr., E. Waletzky and A. S. Tomcufcik, 1970. 1,3-Bis(p-chlorobenzylideneamino)guanidine hydrochloride (Robenzidene): new poultry anticoccidial agent. Science, 168: 373-374. Reid, W. M., L. M. Kowalski, E. M. Taylor and J.Johnson, 1970. Efficacy evaluation of robenzidene for control of coccidiosis in chickens. Avian Diseases, 14: 788-796.
Downloaded from http://ps.oxfordjournals.org/ at Carleton University on June 13, 2015
non-medicated controls with an overall average growth of 57% of normal (range 19%-89%). In the robenidine treated birds, E. maxima oocysts were recovered from 19 of 24 field cultures, but average reduction was greater than 95% where oocysts were recovered. E. acervulina and/or E. mivati oocysts were recovered in 1 of 32 cultures, but reduction was greater than 99%. Representative inoculated, non-medicated controls necropsied primarily for species identification had moderate to severe coccidial lesions. No coccidiosis related mortality occurred in any groups treated with robenidine. At least 16 cultures had previous exposure to commercial anticoccidials. All 32 collections contained E. acervulina and/or E. mivati, 20 contained E. brunetti, 24 E. maxima, 6 E. necatrix, and 13 E. tenella.
986
R. L. KENNETT, S. KANTOR AND A. GALLO
Ryley, J. F., and R. G. Wilson, 1971. Studies on the mode of action of the coccidiostat robenidine. Z. Parasitenk. 37: 85-93. Snedecor, G. W., and W. G. Cochran, 1967. Statistical Methods, 6th ed., Iowa State U., Ames, p. 272-273. Waletzky, E., 1970. Coccidiosis: evaluation of tech-
niques for battery testing of field-collected Eimeria oocysts. Experimental Parasitol. 28: 37-41. Waletzky, E., S. Kantor and A. L. Shor., 1970. A new poultry feed anticoccidial, Cycostat robenidine. Proc. XIV Cong. Mund. de Avicultura, Madrid, Sect. 111:631-637.
Anti-tumor Activity in Regression Fluid N . R. G Y L E S , P . MARINI' AND A. BOWANKO
Department of Animal Sciences, University of Arkansas, Fayetteville, Arkansas 72701
ABSTRACT Portions of growing tumors were ground in a Waring blender and mixed with Hanks' balanced salt solution. This tumor homogenate was highly infectious and produced a large proportion of progressive tumors upon inoculation into the wing webs of susceptible birds. However, upon mixing fluid taken from the site of regressing tumors with tumor homogenate and holding it at refrigerator temperature, there was a slight reduction in the ability of the homogenate to produce tumors. A greater reduction of infectivity was obtained when tumor homogenate was mixed with fluid and stored in vivo at the site of regression of a tumor for 24 hours. Mixing regression fluid with Rous sarcoma virus, before inoculation into susceptible White Leghorns, greatly reduced the tumor inducing capacity of the virus. These results suggest the presence of anti-tumor activity in regression fluid. This activity was demonstrated in vitro against Rous sarcoma virus and in vivo against tumor homogenate. POULTRY SCIENCE 53: 986-989, 1974
INTRODUCTION
MATERIALS AND METHODS
PONTANEOUS regressions of Rous sarcoma tumors in chickens have been reported by Andrewes (1933) and Greenwood et al. (1948). It was determined by Gyles and Brown (1971) that the incidence of spontaneous regression in chickens may be increased by genetic selection. During the course of this work, it was observed that fluid usually develops at the site of regression of large tumors. This fluid may be withdrawn with a needle and syringe and is referred to as regression fluid. Regression usually is completed within one or two weeks in small tumors as compared with three to five weeks in large tumors, offering less opportunity to detect any occurrence of fluid in the small tumors. The purpose of this study was to examine regression fluid for the presence of anti-tumor activity.
Experiment No. 1. Regression fluid was taken from 15 different regressing tumors in a strain of chicken selected specifically for increasing incidence of regression. A composite of the fluid was made and immediately placed in an ice water bath. Tumor homogenate was made by excising a portion of a growing Rous sarcoma and grinding it in a Waring blender with sufficient Hanks' balanced salt solution (BSS) to make a sludge. This was filtered through gauze and the filtrate was mixed in equal portions with Hanks' BSS. It was immediately placed in an ice water bath. Portions of tumor homogenate and regression fluid were mixed at the rate of 0.4 ml. of homogenate to 1.0 ml. regression fluid. Each of 8 White Leghorn chickens, 10 weeks of age, were inoculated in the wing-web with 1.4 ml. of this mixture. The inoculum was delivered by inserting the needle through the
S
1. Present Address: Peterson Poultry Breeding Farm, Decatur, Arkansas 72722.
Downloaded from http://ps.oxfordjournals.org/ at Carleton University on June 13, 2015
(Received for publication August 20, 1973)
978
REFERENCES Deaton, J. W., F. N. Reece, L. F. Kubena, B. D. Lott and J. D. May, 1973. The ability of the broiler chicken to compensate for early growth depression. Poultry Sci. 52: 262-265. Deaton, J. W., L. F. Kubena, T. C. Chen and F. N. Reece, 1974. Factors influencing the quantity of abdominal fat in broilers 2. Cage versus floor rearing. Poultry Sci. 53: 574-577.
Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Essary, E. O., L. E. Dawson, E. L. Wisman and C. E. Holmes, 1960. Influence of different levels of fat and protein in the diet on areas of fat deposition in fryers. Poultry Sci. 39: 1249. Haram, D., R. E. Childs and A. J. Mercuri, 1973. Relationship between fats in broiler finisher rations and fats in chiller water from broiler processing. Poultry Sci. 52: 88-92. Kubena, L. F., B. D. Lott, J. W. Deaton, F. N. Reece and J. D. May, 1972. Body composition of chicks as influenced by environmental temperature and selected dietary factors. Poultry Sci. 51: 517522. Kubena, L. F., J. W. Deaton, T. C. Chen and F. N. Reece, 1974. Factors influencing the quantity of abdominal fat in broilers. 1. Rearing temperature, sex, age or weight, and dietary choline chloride and inositol supplementation. Poultry Sci. 53: 211214. Macklin, L. J., and R. S. Gordon, 1961. Effect of dietary fatty acids and cholesterol on growth and fatty acid composition of the chicken. J. Nutr. 75: 157-164. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics, Chapter 8. McGrawHill Book Company, Inc., New York, N. Y.
Efficacy Studies With Robenidine, A New Type of Anticoccidial, in the Diet R. L . KENNETT, S. KANTOR AND A. GALLO
Agriculture Center, American Cyanamid Company, P. O. Box 400, Princeton, New Jersey 08540 (Received for publication August 20, 1973)
ABSTRACT Robenidine, l,3-bis(p-chlorobenzylideneamino)guanidine hydrochloride has broad spectrum efficacy in chickens. At recommended 30 g./ton in the diet it is completely effective in preventing clinical disease from 8 laboratory species of Eimeria administered individually or in combination; it is equally effective against at least 95 strains of 6 economically important species obtained in 1968 and 1969 from various geographical areas of the United States. Comparative floor-pen trials on litter seeded with 6 pathogenic species of coccidia indicated that robenidine at 30 g./ton overall provided better weight gains and feed efficiencies than recommended concentrations of amprolium plus ethopabate. Robenidine plus roxarsone (45 g./ton) was equivalent to recommended levels of clopidol plus roxarsone (45 g./ton) in these performance parameters. POULTRY SCIENCE 53: 978-986, 1974
INTRODUCTION OCCIDIOSIS in chickens continues to be a problem despite efforts to control the disease by chemical means and poultry house management. Many broad spectrum
C
anticoccidials have been developed and their use has greatly contributed to the growth of the poultry industry. However, since most anticoccidials fail after a period of time, because of misuse and the development of
Downloaded from http://ps.oxfordjournals.org/ at Carleton University on June 13, 2015
had a significantly smaller percentage of abdominal fat than the 9-week-old females receiving the same diet. This agrees with the previous report of Kubena et al. (1974). Within each dietary treatment females had a numerically higher percentage of abdominal fat than the males, which agrees with the report of Kubena et al. (1974). It is interesting to note that, although the females were considerably lighter in weight than the males, the absolute quantity of abdominal fat was comparable to that of the males within each dietary treatment.
EFFICACY OF ROBENIDINE
MATERIALS AND METHODS Battery Trails. Standardized methods and criteria of efficacy are those reported by Waletzky (1970). Broiler-type hybrid cockerel chicks, oneweek of age, were initially distributed into equal-weight groups. All chicks were reared on wire-floored cages in adequately heated rooms and maintained on a 12-hour alternating light-dark cycle. Normal and medicated rations were available ad libitum in a typical and nutritionally adequate basal broiler ration from 2 days before inoculation until termination of the test 14 days after inoculation. Sporulated oocysts were inoculated by gavage, with a level of infection selected from previously titrated inocula in non-medicated birds of similar age. Duplicate tests, each containing 4 replicates of 10 chicks each, were conducted utilizing ©Registered trademark of American Cyanamid Company.
a commercial mixture of 8 coccidial species (no E. mitis), as well as individual inoculations with each of the following 6 pathogenic species maintained routinely in the laboratory: Eimeria acervulina, E. brunetti, E. maxima, E. mivati, E. necatrix and E. tenella. Mixed field cultures of coccidia were also utilized to assess better the performance of robenidine in single experiments containing 2 replicates. Most cultures were obtained between May, 1968 and May, 1969 from various diagnostic laboratories located in nine states. Average weight gains and feed intakes were calculated from day of inoculation (0 day) to 6 or 7 days post-inoculation near the height of infection, and finally at 14 days post-inoculation when the experiments were terminated. Weight gains and feed conversions were compared at the 5% level of probability by analysis of variance, and the studentized range test for differences between all means (Snedecor and Cochran, 1967). Lesions were scored (Johnson and Reid, 1970, slightly modified) in selected groups sacrificed at 6 or 7 days post-inoculation in all experiments, except those receiving the 8 species combination; in these no lesion scoring was performed. Oocyst counts were made using minor modifications of standard methods (Davies et al., 1963) in Levy cell counting chambers, or after flotation of 1-2% of the sample in a saturated sugar solution when relatively few oocysts were present. Significance of differences in mortality were determined by Chi Square analysis. Floor Pen Trials. Two trials under simulated commercial conditions were conducted in the same facilities. In the first trial robenidine was compared with reference coccidiostat amprolium plus ethopabate through five consecutive flocks, and in a second trial robenidine, with and without roxarsone (45 g. /ton), was compared with reference coccidiostat
Downloaded from http://ps.oxfordjournals.org/ at Carleton University on June 13, 2015
resistance, a continuing search for novel, improved chemotherapeutic agents is necessary. Robenz® robenidine, l,3-bis(p-chlorobenzylideneamino)guanidine hydrochloride, first reported by Kantor et al. (1970) to have high broad spectrum activity and Berger et al. (1972) who proved that its margin of safety is at least sixfold, is a novel anticoccidial for use in chickens. Additional data confirming the high efficacy of robenidine in battery and floor pen studies have appeared in the literature (Badiola et al., 1970; Kantor, 1973 a,b;Reid era/., 1970; Ryley and Wilson, 1971; Waletzky et al., 1970). Efficacy against species of turkey coccidia has also been reported (Joyner and Norton, 1972). This paper describes the results of recent battery and floor-pen experiments on the prophylactic efficacy of robenidine in chickens at its recommended concentration of 30 g./ton of feed against both laboratory and field collected strains of Eimeria.
979
980
R. L. KENNETT, S. KANTOR AND A. GALLO
Examination of susceptible three-week-old chicks placed in all "seeded" pens for approximately 4 days after completion of seeding in each trial indicated the premises were successfully contaminated with coccidia. Criteria of efficacy were average terminal weights and feed conversion for all treatments and flocks in each trial. RESULTS AND DISCUSSION Battery Trials. Robenidine at its recommended concentration of 30 g./ton of feed was highly effective against the 6 major pathogenic species of chicken coccidia, tested both individually, and against a mixed challenge of 8 species. In Table 1 the average weight gains of the infected, nonmedicated control groups through 6 or 7 days, and 14 days post-inoculation were all significantly (P < 0.05) lower than the non-infected controls. No significant differences were ob* Trademark of Sterwin Laboratories, Inc., New York, N.Y., U.S.A.
served in weight gains of birds fed robenidine at 30g./ton either at 6 or 7 days post-inoculation, or at the termination of the test at 14 days. Feed efficiencies over 14 days paralleled the weight gains. The non-medicated infected treatments were inferior to the normal control feed efficiencies; those of the robenidine medicated groups were not different (P > 0.05). No feed efficiencies were calculated in the E. necatrix and E. tenella tests because high mortality in control birds distorts their value. Table 1 also summarizes robenidine efficacy based on mortality, lesion scores, and oocyst production. No mortality was observed in any robenidine treated birds, even though significant (P < 0.05) numbers of birds died in non-medicated treatments inoculated with E. necatrix and E. tenella. Lesions in the robenidine medicated groups were prevented, except in 18 of 40 birds inoculated with E. maxima. However, as compared to the non-medicated, E. maxima inoculated birds, robenidine reduced E. maxima lesions to the extent that microscopic examination of mucosal smears was necessary to determine infection in most birds. E. maxima oocysts were recovered from medicated groups inoculated with this species either in a single or mixed coccidial inoculum, but numbers were reduced by more than 99.9% in each case. No oocysts representative of the other species were recovered from birds treated with robenidine in these experiments. Against 32 different field collections containing at least 95 strains of Eimeria (Table 2), the average weight gain of robenidine treated birds, calculated from 0-6, or 7 days, was 100 percent (range 88%-112%) relative to growth in the non-medicated, non-inoculated (normal) controls. Growth of replicates in 30 of the 32 infected, non-medicated control treatments were significantly reduced (P <0.05) as compared to their non-inoculated,
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clopidol plus roxarsone (45 g./ton) through four consecutive flocks. In each trial all flocks contained 4 replicates of 500 birds per treatment and were provided with 0.85 sq. ft. of floor space. Floor litter was renewed only between the two trials after careful cleaning of the pens: no changes in floor litter were made between the series of 5 and 4 flocks, respectively. Immediately before the initial flock in each trial was introduced each pen was seeded by two consecutive groups of 100 infected non-medicated birds on litter with adequate moisture content for oocyst sporulation. In the first trial each seeder bird received 10 times the recommended immunizing dose of CocciVac* type D which contains all chicken coccidia except Eimeria mitis. A mixture of laboratory and recently collected field strains containing at least 6 economically important species of Eimeria was administered to seeder birds in the second trial (approximately 50,000 sporulated oocysts per chick administered).
56*
68*
283
282
265
302
305
261
92
92
%
118
126
113
brunetti
mivati
maxima
necatnx
tenella
8 spp. mixedb
100
99
99 74*
58*
56*
83*
102
102
101
102
97
102
102
1.51
1.55
1.49
1.54
1.67*
NC
NC
1.65*
1.70*
1.54
1.51
1.55
1.50
1.54
1.55
1.54
1.51
1.73*
1.72
1.58
1.50
IR
IC
heed efficiency (0-14 days p.i.) -
5
23*
55*
0
0
0
0
14 day IC
% Mort
* Significantly different from normal control (P < 0.05) aAverage of eight groups of 10 birds each; four groups of birds sacrificed for lesion scores at 7 days except in mixed species (p.i.) in acervulina, brunetti, and mivati experiments. b50 x CocciVac, Type D (E. mitis absent) c E. maxima only recovered NC = High mortality with E. necatrix and E. tenella prevented meaningful efficiency calculations IC = Inoculated, non-medicated controls IR = Inoculated, robenidine treated
62*
48*
24*
101
81*
97
72* 74*
97 102
ill
74*
65*
309
102
acervulina
0-14 days p.i. IC IR
% of average normal weight gain
0-7 days p.i." IC IR
0-14 days
Eimeria spp.
0-7 days
Non-inoculated, non-medicated (normal) control weight gain (g.)
TABLE 1.—Anticoccidial efficacy of robenidine at 30 g./ton against laboratory Ei
ded from http://ps.oxfordjournals.org/ at Carleton University on June 13, 2015
+ + + + +
+ + + + + +
+ + + + + +
7 8 9 10 11 12
13 14 15 16 17 18
+
•
acervulina/ mivati
Isolate no. 1 2 3 4 5 6
+ + + + + +
+ +
+ +
+ +
brunetti + +
+
+ + + +
+
+ +
+
+
tenella
+ + + + + + + + +
+
necatrix
862
103
0-6 or 7 days p.i. gms. /chick 96
Weight gain
Non-inoculated, non-medicated (Normal) control
Wei
of robenidine at 30 g./ton against 32 different Eimeria
maxima + + + + + +
Species present
TABLE 2.—Efficacy
ded from http://ps.oxfordjournals.org/ at Carleton University on June 13, 2015
+
+ +
+
23 24 25 26 27 28
29
30 31
32
+
+ +
+
+
brunetti +
+
maxima
Species present
+
+
+
+ +
+
+
+
+
tenella +
necatrix
Weig
acervulina/E
136
140
131
1122
0-6 or 7 days p.i. gms./chick 1202
Weight gain
Non-inoculated, non-medicated (Normal) control
* Significantly different from normal controls (P < .05) 1 At least 95 individual strains of Eimeria spp. 2 Weight gains calculated at 0-6 days after inoculation 3 Two replicates of 10 birds per treatment 4 E . maxima only species recovered with the exception of isolate number 31 in which E. p.i. = post inoculation
acervulina/ mivati + + + +
++++++
Isolate no. 19 20 21 22
++++++
TABLE 2.—(Cont'd)
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3.40" 3.66" 3.75" 3.72"
Non-medicated controls Clopidol + Roxarsone 2 Robenidine + Roxarsone 3 Robenidine
3.36" 3.58" 3.50"" 3.51""
3.30" 3.29" 3.43"
2
3.60" 3.82" 3.88" 3.75""
2.95" 3.09" 3.24c
3
3.65" 3.80" 3.77" 3.70"
3.03" 3.19" 3.35c
4
—
3.22" 3.41" 3.56c
5
(3.50) (3.72) (3.72) (3.67)
(3.20) (3.32) (3.43)
Avg.
2.01" 2.01" 1.99" 2.03"
Trial 2
1.99" 2.00" 1.99"
1
1.97 1.89 1.91 1.91
1.99 1.99 1.97
2
Fe
Figures in same flock having different superscript letters are significantly different from each other ( 'Amprolium concentration in feed equal to 125 p.p.m.; ethopabate equals 4 p.p.m. 2 Clopidol concentration in feed equal to 125 p.p.m.; roxarsone equals 50 p.p.m. 'Roxarsone concentration in feed equal to 50 p.p.m.
3.50" 3.60" 3.66"
1
Non-medicated controls Amprolium + Ethopabate 1 Robenidine
Treatment
Flock number
Avg . weight/bird (lb.) 8 weeks
Trial 1
TABLE 3.—Comparative efficacy of robenidine at 30 g./ton and two reference anticoccidials in floor p hundred birds per treatment on reused litter
ded from http://ps.oxfordjournals.org/ at Carleton University on June 13, 2015
985
EFFICACY OF ROBENIDINE
Floor-Pen Trials. (Table 3) Average terminal 8-week weights of robenidine and amprolium plus ethopabate medicated birds in the first trial were significantly superior (P < 0.05) to the non-medicated controls in all but the second of 5 consecutive flocks. Robenidine weights were significantly better than amprolium + ethopabate in the third, fourth, and fifth flocks. Overall average weight improvement was 4% for amprolium plus ethopabate, and 7% for robenidine. The feed efficiencies of robenidine treated birds were significantly improved as compared to the non-medicated controls in the third, fourth and fifth flocks, and superior to amprolium + ethopabate in the third and fifth flocks. Little improvement in feed efficiencies were apparent in amprolium + ethopabate treated flocks. In the second comparison the overall improvement in bird weights at the end of eight weeks was 6% for clopidol plus roxarsone and robenidine plus roxarsone, and 5% for robenidine alone. All treatments appeared to improve weight gains within each flock, sometimes significantly, but no statistical
differences were noted among treatments. Feed efficiencies of treated groups were improved only occasionally over the untreated controls, particularly clopidol plus roxarsone in the second and fourth flocks, and robenidine, with and without roxarsone in the third flock. No significant differences were evident among the birds on the different treatments. The data reported here, and as yet unpublished data, confirm the high and broad spectrum efficacy of robenidine at 30 g./ton against chicken coccidiosis. Robenidine will be a useful addition to the commercial anticoccidial market. REFERENCES Badiola, C , I. Badiola and F. Carrasco, 1970. Robenzidene in the feed of broilers: Determination of the effect of the withdrawal of robenzidene for periods up to 48 hours on the course of experimental coccidial infection. Proc. XIV Congr. Mund. de Avicultura, Madrid, Section III: 186-187. Berger, H., A. L. Shor, G. O. Gale and K. L. Simkins, 1972. Safety evaluation of robenidine in the feed of broiler chickens. (Unpublished data). Davies, S. F. M., L. P. Joyner and S. B. Kendall, 1963. Coccidiosis. Oliver and Boyd, Ltd., London, Chapter XV. Johnson, J., and W. M. Reid, 1970. Anticoccidial drugs: Lesion scoring techniques in battery and floor-pen experiments with chickens. Experimental Parasitol. 28: 30-36. Joyner, L. P., and C. C. Norton, 1972. The drug sensitivity of recently isolated strains of Eimeria meleagrimitis and a laboratory strain of Eimeria adenoeides in turkeys to robenidine. Res. Vet. Sci. 13: 279-281. Kantor, S., 1973a. Robenidine—a new type of anticoccidial. World's Poultry Sci. J., 29: 56-57. Kantor, S., 1973b. Robenidine—a new type of anticoccidial agent. 4th Europ. Poultry Conf., London, pp. 141-146. Kantor, S., R. L. Kennett, Jr., E. Waletzky and A. S. Tomcufcik, 1970. 1,3-Bis(p-chlorobenzylideneamino)guanidine hydrochloride (Robenzidene): new poultry anticoccidial agent. Science, 168: 373-374. Reid, W. M., L. M. Kowalski, E. M. Taylor and J.Johnson, 1970. Efficacy evaluation of robenzidene for control of coccidiosis in chickens. Avian Diseases, 14: 788-796.
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non-medicated controls with an overall average growth of 57% of normal (range 19%-89%). In the robenidine treated birds, E. maxima oocysts were recovered from 19 of 24 field cultures, but average reduction was greater than 95% where oocysts were recovered. E. acervulina and/or E. mivati oocysts were recovered in 1 of 32 cultures, but reduction was greater than 99%. Representative inoculated, non-medicated controls necropsied primarily for species identification had moderate to severe coccidial lesions. No coccidiosis related mortality occurred in any groups treated with robenidine. At least 16 cultures had previous exposure to commercial anticoccidials. All 32 collections contained E. acervulina and/or E. mivati, 20 contained E. brunetti, 24 E. maxima, 6 E. necatrix, and 13 E. tenella.
986
R. L. KENNETT, S. KANTOR AND A. GALLO
Ryley, J. F., and R. G. Wilson, 1971. Studies on the mode of action of the coccidiostat robenidine. Z. Parasitenk. 37: 85-93. Snedecor, G. W., and W. G. Cochran, 1967. Statistical Methods, 6th ed., Iowa State U., Ames, p. 272-273. Waletzky, E., 1970. Coccidiosis: evaluation of tech-
niques for battery testing of field-collected Eimeria oocysts. Experimental Parasitol. 28: 37-41. Waletzky, E., S. Kantor and A. L. Shor., 1970. A new poultry feed anticoccidial, Cycostat robenidine. Proc. XIV Cong. Mund. de Avicultura, Madrid, Sect. 111:631-637.
Anti-tumor Activity in Regression Fluid N . R. G Y L E S , P . MARINI' AND A. BOWANKO
Department of Animal Sciences, University of Arkansas, Fayetteville, Arkansas 72701
ABSTRACT Portions of growing tumors were ground in a Waring blender and mixed with Hanks' balanced salt solution. This tumor homogenate was highly infectious and produced a large proportion of progressive tumors upon inoculation into the wing webs of susceptible birds. However, upon mixing fluid taken from the site of regressing tumors with tumor homogenate and holding it at refrigerator temperature, there was a slight reduction in the ability of the homogenate to produce tumors. A greater reduction of infectivity was obtained when tumor homogenate was mixed with fluid and stored in vivo at the site of regression of a tumor for 24 hours. Mixing regression fluid with Rous sarcoma virus, before inoculation into susceptible White Leghorns, greatly reduced the tumor inducing capacity of the virus. These results suggest the presence of anti-tumor activity in regression fluid. This activity was demonstrated in vitro against Rous sarcoma virus and in vivo against tumor homogenate. POULTRY SCIENCE 53: 986-989, 1974
INTRODUCTION
MATERIALS AND METHODS
PONTANEOUS regressions of Rous sarcoma tumors in chickens have been reported by Andrewes (1933) and Greenwood et al. (1948). It was determined by Gyles and Brown (1971) that the incidence of spontaneous regression in chickens may be increased by genetic selection. During the course of this work, it was observed that fluid usually develops at the site of regression of large tumors. This fluid may be withdrawn with a needle and syringe and is referred to as regression fluid. Regression usually is completed within one or two weeks in small tumors as compared with three to five weeks in large tumors, offering less opportunity to detect any occurrence of fluid in the small tumors. The purpose of this study was to examine regression fluid for the presence of anti-tumor activity.
Experiment No. 1. Regression fluid was taken from 15 different regressing tumors in a strain of chicken selected specifically for increasing incidence of regression. A composite of the fluid was made and immediately placed in an ice water bath. Tumor homogenate was made by excising a portion of a growing Rous sarcoma and grinding it in a Waring blender with sufficient Hanks' balanced salt solution (BSS) to make a sludge. This was filtered through gauze and the filtrate was mixed in equal portions with Hanks' BSS. It was immediately placed in an ice water bath. Portions of tumor homogenate and regression fluid were mixed at the rate of 0.4 ml. of homogenate to 1.0 ml. regression fluid. Each of 8 White Leghorn chickens, 10 weeks of age, were inoculated in the wing-web with 1.4 ml. of this mixture. The inoculum was delivered by inserting the needle through the
S
1. Present Address: Peterson Poultry Breeding Farm, Decatur, Arkansas 72722.
Downloaded from http://ps.oxfordjournals.org/ at Carleton University on June 13, 2015
(Received for publication August 20, 1973)