Efficacy of Carbon Disulfide Against Ascaridia Galli (Roundworm) of Chickens1,2

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YIELD OF TURKEY MEAT

of live weight) was comparable to that for turkeys fed the other rations. Some variations in the percentage of drippings, and the proportion of fat in the drippings, were noted,in torn and hen turkeys fed rations which differed in the source of protein or in the addition of fat. In general, after roasting at 325°F. or braising at 450°, about 28 percent was light meat, 18 percent dark meat, 9 percent skin and fat, and 18 percent bones, based on the weight of ready-to-cook turkeys. ACKNOWLEDGMENTS

The authors wish to express their appre-

ciation to Ruth A. Redstrom and Carroll Kitzmiller for assistance in the laboratory. REFERENCES Harkin, A. M., C. Kitzmiller, G. L. Gilpin and S. J. Marsden, 1958. Quality of the cooked meat of turkeys fed animal or vegetable protein diets with vitamin and fat supplements. Poultry Sci. 37: 1328-1339. Sizemore, J. R., S. J. Marsden and C. A. Denton, 1956. The effect of diet and supplementary fat on the performance of Beltsville Small White turkeys. Poultry Sci. 35: 1172. Swickard, M. T., and A. M. Harkin, 1954. Percentage relationships of raw carcass weights and yield of cooked edible portion for young Beltsville Small White turkeys. Poultry Sci. 33: 775779.

Efficacy of Carbon Disulfide Against Ascaridia (Roundworm) of Chickens1,2

Galli

S. E. KNAPP 3 AND M. F. HANSEN Department of Zoology, Kansas State University, Manhattan, Kansas (Received for publication November 30, 1959)

INTRODUCTION P TO now, CS2 has not been the drug of choice to remove Ascaridia galli from chickens. Its limited use against this parasite is probably related to its toxicity, difficult administration, and availability of more nearly satisfactory anthelmintics. Recently, CS2 has been combined with a piperazine compound.4 This anthelmintic was tested by Shumard (1957) and found

U

1 Contribution No. 299, Kansas Agricultural Experiment Station, Manhattan and No. 329 from the Department of Zoology. 2 A portion of a dissertation presented by S. E. Knapp in partial fulfillment of the requirements for the degree Doctor of Philosophy in Parasitology at Kansas State University of Agriculture and Applied Sciences, Manhattan. 8 Present address: Department of Veterinary Medicine, Oregon State College, Corvallis. 4 "Parvex"—The Upjohn Company (Betaine of 1-piperazine carbodithiotic acid).

to be highly efficacious for removing chicken ascarids. Use of CS2 in this form should renew interest in it as an anthelmintic. The first reported use of CS2 to remove A. galli from chickens was by Roberts (1937). His work embodied a series of studies involving numerous anthelmintics so time permitted only a cursory study of CS2. Later, Knapp and Hansen (1954),' in a preliminary report, showed that the drug could be used effectively to remove A. galli. Because of limited information, the present study was initiated to investigate the action of CS2 against A. galli and the nature of its actions on the host. MATERIALS AND METHODS Adult Worm Removal. Straight run, dayold White Rock chickens were obtained

1106

S. E. KNAPP AND M. F. HANSEN

from a local hatchery, given an intranasal inoculation of Newcastle disease vaccine,1 and placed in electrically heated incubators. At 14 days of age each chicken was infected with 100 +10 A. galli ova cultured according to the method described by Hansen et al. (1954). A period of 60 days was allowed to permit complete development of the worms prior to the administration of the drug (Tugwell and Ackert, 1952). In several experiments the antibiotic, Aureomycin,2 was included at the level of 2.15 gm./gal. of drinking water for several days to modify bird losses from an unidentified respiratory disorder. To avoid any possible anthelmintic interference, antibiotic treatment was discontinued several days prior to administration of CS2. The control birds did not lose worms following treatment with the antibiotic. All birds were maintained on a commercial feed (18 percent protein egg-layer mash). Experiment 1 was a pilot study designed to determine the approximate dosage range considered effective to remove ascarids from test birds. Four groups of 10 birds each were established by weight according to the method of Gardiner and Wehr (1950). Prior to drug administration each chicken was fasted for 12 hours. Carbon disulfide was administered in hard gelatin capsules, which were dropped directly into the crop through a hollow glass tube. The control birds, group 1, were given empty capsules. Groups 2, 3, and 4 received 0.15, 0.30, and 0.60 ml. of drug, respectively. Each bird was isolated in a separate cage and given feed immediately after treatment. Dropping trays were examined for expelled ascarids prior 1 Live Virus Bi Strain, American Cyanamid Company, New York, N. Y. 2 American Cyanamid Company, New York, N. Y.

to treatment and then hourly for 12 hours post-treatment. At 24 hours post-treatment the birds were weighed and all trays were again examined for worms. This procedure was repeated for 5 days posttreatment at which time the birds were killed and examined for ascarids. The data were analyzed statistically, using weight change and reduction in worm burden as an index of toxicity and efficacy. Since information from the preliminary experiment indicated that an effective dosage range had been reached and that weight fluctuations were, in part, an indication of drug toxicity, a second efficacy test was undertaken. Experiment 2 was designed to evaluate more precisely the dose of CS2 that would give the highest efficacy and lowest toxicity to the host. This experiment tested also whether the fasting of chickens prior to treatment conditioned the action of CS2. Sixty infected chickens were divided into 6 groups. These were subdivided into groups of 5 birds each. Six of the 12 subgroups were on full ration while the other 6 subgroups were fasted for 15 hours. The birds within each of the groups were given a specific dosage of CS2. Controls, group 1, were given empty gelatin capsules. The remaining 6 groups, 2 through 6, received 0.10, 0.20, 0.30, 0.40, and 0.50 ml. of the drug, respectively. After treatment, the subgroups were placed in separate batteries. As individual cages were not available, efficacy was computed on the basis of worms passed per subgroup versus worms retained per subgroup. There were 3 replications of this experiment. Weights of individual birds were recorded daily from the day prior to treatment until the fifth day after treatment. The experiment was terminated when the birds were killed and examined for ascarids. Efficacy results were analyzed statistically by transforming the number of

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CARBON DISULFIDE AND ROUNDWORMS

worms recovered to arc sine V % efficacy in an attempt to make their distribution more nearly normal (Bartlett, 1947). An analysis of variance was conducted on the transformed data. A final efficacy test, experiment 3, was done to determine the efficacy of CS2 using individual chickens. Fifty-four infected birds were treated. These were grouped and dosed similarly to those of experiment 2 except that each was retained in a separate cage. All chickens were weighed the day preceding treatment and at 72 hours post-treatment when they were autopsied. Immature Worm Removal. Experiment 4 was designed to test the efficacy of CS2 against the larval phase of A. galli. This stage in the nematode's life history has been reported as lasting from the 10th to the 20th day post-infection (Tugwell and Ackert, 1952). Two hundred and ten 14-day-old chickens were individually infected with 100+10 ascarid ova. Ten of these birds were given 0.35 ml. per kilo of CS2 24 hours post-infection. This procedure was

TABLE 1.—Efficacy of carbon disulfide against Ascaridia galli Treatment (ml. C & / kilo) 0.0 (control) 0.15 0.30 0.60

Number Average weight birds/ change treat. (gm.) 10 9 10 9

125 46 -29 -89

Worms passed

Worms retained

2 13 16 41

16 7 2 0

Efficacy

(%)

11* 65 89 100

* Spontaneous elimination.

repeated daily using 10 birds each day until a total of 200 birds had been treated. Ten birds, when 16 days of age, were given empty gelatin capsules and retained as infected controls. All treated groups were fed ad libitum until they were 50 days old, when they were sacrificed and examined for adult nematodes. RESULTS

Adult Worm- Removal. The data for experiment 1 indicated that although 100 percent efficacy was reached at the level of 0.6 ml. of CS2 per kilogram body weight, the weight losses for these chickens were relatively large (Table 1). At a level of 0.3 ml. per kilo, CS2 was 89 per-

TABLE 2.—Efficacy of various concentrations of carbon disulfide against Ascaridia galli in fasted and nonfasted chickens Groups

I

II

III

IV

V

VI

•

Replicates

Birds per replicate

Worms passed

0 . 0 (Control) F (fasted) Nf (non-fasted)

5 5

0 0

51 112

0 0

0 0

80 54

0 0

0 0

63 25

0 0

0 0

0.1 F Nf

5 5

1 2

48 97

2 2

25* 1

93 13

7 7

0 0

20 18

0 0

3 3

0.2 F Nf

5 5

20 0

15 33

58 0

17 0

17 67

50 0

9 0

8 25

53 0

54 0

0.3 F Nf

5 5

10 10

22 12

31 46

14 35

13 17

52 67

16 4

0 3

100 57

61 57

0.4 F Nf

5 5

63 30

12 15

84 66

43 46

12 79

78 37

10 1

0 13

100 7

87 37

0.5 F Nf

5 5

so3

0 46

100 6

25 33

24 1

51 97

15 12

1 2

94 86

82 63

Treatment (ml. C&Ailo)

Worms Efficacy retained

(%)

Worms passed

Worms Efficacy retained

(%)

Worms passed

Worms Efiicacy Efficacy retained (average)

(%)

* Eighteen worms in this group were passed prior to treatment. Efficacy was calculated on the basis of the remaining seven worms (7+93 = 100.7/100=7%).

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S. E. KNAPP AND M. F. HANSEN

DAY POST-TREATMENT

MEAN WEIGHT lor TREATMENT MEAN WEIGHT for FASTED BIRDS ft

MEAN WEIGHT lor NON-FASTED BIRDS

FIG. 1. Graph showing mean weights for chickens treated with different dosages of CS2 (Experiment 2). There were 30 birds per treatment of which 15 were fasted and IS non-fasted.

cent effective and the mean weight loss was less than that experienced by birds receiving 0.6 ml. per kilo. At 0.15 ml. per kilo, CS2 was 65 percent effective and the group gained in weight after the first day of treatment. The control group lost spontaneously 11 percent of its worms. In experiment 2, the data showed that as dosage increased from 0.1 ml. to 0.5 ml. per kilo body weight, the mean efficacy increased (Table 2). Fasting improved the efficacy percentage beginning at the level

of 0.2 ml. per kilo. An analysis of variance and F-test conducted on the transformed data revealed a highly significant variation in efficacy of the several dosages. Significant differences in drug efficacy were also recorded for fasted and nonfasted birds. Data concerning daily weight changes for birds treated in experiment 2 are shown in Figure 1. The first period of weight loss occurred on the day of treatment among the fasted birds. Twenty

1109

CARBON DISULFIDE AND ROUNDWORMS

four hours post-treatment (3rd day) birds receiving 0.2 to 0.5 ml. per kilo had marked depressions in weight compared with the controls. However, fasting of birds that received 0.3 to 0.5 ml. per kilo resulted in their making a relatively more rapid recovery in body weight (6th and 7th days) than the non-fasted birds at these drug levels. Table 3 shows the results for experiment 3. Efficacy percentages were lower in this experiment than in the previous ones, reaching only 65 percent at 0.5 ml CS2 per kilo body weight. The mean difference in weight loss between treatments 0.4 and 0.5 ml. per kilo was 303 grams; whereas, the resulting efficacies between these two groups differed by only 1 percent. A dosage of 0.3 ml. per kilo body weight was only 27 percent effective. Worm losses occurred in the control group through spontaneous elimination. The method of Kruskal and Wallis (1952) was used in statistical analyses of the data since the results did not appear to be normally distributed. This analysis led to a Chi-square value of 19.02 with 5 degrees of freedom for treatments (P<.01). Spearman's rank correlation, as presented by Fryer (1954), indicated that there was a significant linear association between efficacy and dosage (r 8 = 0.881). Immature Worm Removal. The arithmetic mean for numbers of worms found TABLE 3.—Efficacy of various doses of carbon disulfide against Ascaridia galli Average Number Number weight worms Efficacy change worms (%) passed retained (gm.)

Treatment (ml. CS2/ kilo)

Birds per treatment

0.0 (control) 0.1 0.2 0.3 0.4 0.5

9

-

8

0.1

8.3

9 9 9 9 9

+ 10

0.5 1.8 1.4 5.2 3.3

7.5 3.4 3.6 2.9 1.9

- 65 - 18 - 60 -363

' Spontaneous elimination.

1.3* 6.2 36.0 27.C 64.0 65.0

TABLE 4.—Results of testing the effect of carbon disulfide on the larval phase of Ascaridia galli

Treatment group

1 2 3 (Control) 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Number birds examined per group*

Age of birds (days)

5 10 9 9 10 9 8 9 9 10 10 9 8 10 10 10 10 10 10 10 10

15 16 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

Average number worms per bird 14.0 5.1 3.6 6.2 5.4 2.8 5.6 2.4 3.8 6.7 2.8 5.5 4.7 8.2 3.4 4.2 3.5 5.4 4.7 5.7 5.0

* Ten birds were treated for each group; however, bird losses occurred in several of the groups.

in each of the treated groups of experiment 4 was 5.2 compared with 3.6 for the controls (Table 4). Variability of worm counts was greater within treatment groups than it was among such groups. There was no linear association between the date of treatment and the number of worms found in the fowl on post-mortem. There were also no efficacy differences among the dates of treatment when they were grouped into larval stages based on the paper by Tugwell and Ackert (1952). DISCUSSION The results of this study indicate that although CS2 was not a particularly effective anthelmintic for A. galli in chickens, it could still be used against this parasite as a means of studying the mechanism of its anthelmintic action. Preliminary experiments indicated that dosages ranging between 0.1 and 0.6 ml.

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S. E. KNAPP AND M. F. HANSEN

per kilo body weight would remove the worms from adult birds with varying degrees of efficacy. Further studies revealed that higher dosages were more toxic and accounted for large weight losses in the treated birds. Dosages at the 0.3 and 0.4 ml. per kilo level were the most promising considering not only efficacy but also toxicity. The resulting efficacies of the three experiments, using fasted birds, revealed a rather inconsistent efficacy with dosages of 0.3 and 0.4 ml. per kilo. The average efficacy of 0.3 ml. per kilo was 59% with a range of 27% to 100% (Tables 1, 2, 3). The average efficacy of 0.4 ml. per kilo was higher, 81%, with a range of 64% to 100% (Tables 2, 3), but was accompanied by a relatively high toxicity to the host. From these results it was arbitrarily decided that an intermediate dosage of 0.35 ml. per kilo would be used in the study reported on the removal of immature worms. This dosage was used in a study with sulfur-35 labeled CS2 reported by Knapp et al. (1960). Fasting prior to treatment aided in raising the efficacy of the drug and reducing its toxicity to the host. Based on the amount of fecal material produced, it was observed that fasted chickens began feeding 24 to 48 hours after the administration of CS2, while only a few of the nonfasted birds fed during the 7-day experimental period. Also, at post-mortem no chyme was present in the intestines of the non-fasted birds, in contrast with the chyme-filled ones of the fasted and control chickens. The explanation for a greater efficacy and reduced toxicity in the fasted birds involves a complexity of factors. In a fasted chicken, CS2 would have a greater opportunity to pass directly to the intestinal region where the worms are located; whereas, the rate of passage of drug would

be considerably reduced in the crop and gizzard of a non-fasted bird. This delay in passage would afford an opportunity for the drug to be metabolized or absorbed into the circulatory system, thereby reducing the quantity of CS2 contacting the worms. Since a specific level of CS2 in the worms is probably necessary to affect their removal (Knapp et al., 1960), a lowering of this level through increased host metabolism would account for differences in drug efficacy between treatment groups and within treatment groups. The latter point is mentioned as it is necessary to recognize that individual metabolic differences may account for some of the variations in drug efficacy in these experiments. In the larval efficacy test, a greater variation within treatment groups than between treatment groups was obtained. No recommendations can be made for administering CS2 at any particular day during larval development. This does not mean that CS2 cannot be effective against larvae since a dose greater than 0.35 ml. per kilo body weight might be efficient. SUMMARY

In order to study the anthelmintic activity of CS2 on Ascaridia galli in the chicken, it was first necessary to determine its percent efficacy. Using weight changes in the chicken as an indication of drug toxicity and percent of worms removed after treatment, a series of experiments involving varying dosages and methods of administration was conducted. A statistical analysis of the data indicated that there was a significant linear association between efficacy and dosage. At dosage levels of 0.3 ml. per kilo of body weight and higher, the per cent efficacy was variable in different groups of birds at each dosage level tested. At dosages of 0.4 ml. per kilo body

CARBON DISULFIDE AND ROUNDWORMS

weight and higher toxicity was manifested by large weight losses. A higher efficacy was found among fasted birds than among non-fasted ones, thus emphasizing the importance of fasting chickens prior to administering CS2. The drug was not effective against any of the larval stages of A. galli at the dosage used in this study. ACKNOWLEDGMENTS The authors wish to thank Dr. Stanley Wearden, Department of Statistics, Kansas State University for his assistance with the statistical analyses, and the United States Atomic Energy Commission for funds supporting the study under Contract At(ll-l)-308. REFERENCES Bartlett, M. S., 1947. The use of transformations. Biometrics, 3: 39-51. Fryer, H. C , 1954. Elements of Statistics. John Wiley and Sons, Inc., New York, 262 pp. Gardiner, J. L., and E. E. Wehr, 1950. Selecting ex-

1111

perimental groups of chicks by weight. Proc. Helm. Soc. Wash. 17: 25-26. Hansen, M. F., L. J. Olson and J. E. Ackert, 1954. Improved techniques for culturing and administering ascarid eggs to experimental chicks. Exptl. Parasit. 3: 464-473. Knapp, S. E., and M. F. Hansen, 1954. Observations on the anthelmintic action of carbon disulfide on the fowl ascarid, Ascaridia galli. J. Parasit. 40: 17-18. Knapp, S. E., M. F. Hansen, H. C. Moser and R. H. McFarland, 1960. Uptake of sulfur-35-labeled carbon disulfide by Ascaridia galli (Roundworm) and its chicken host. Exptl. Parasit. 9: 56-62. Kruskal, W. H., and W. A. Wallis, 1952. Use of ranks in one-criterion variance analysis. J. Amer. Stat. Assoc. 47: 583-621. Roberts, F . H. S., 1937. Studies on the biology and control of the large roundworm of fowls, Ascaridia galli (Schrank, 1788) Freeborn, 1923. Bui. No. 2, Animal Health Station, Yeerongpilly, Australia. 106 pp. Shumard, R. F., 1957. The toxicity to chickens and the anthelmintic effect of two forms of a piperazine-carbon disulfide complex on Ascaridia galli and Heterakis gallinae. Poultry Sci. 36: 613-618. Tugwell, R. L., and J. E. Ackert, 1952. On the tissue phase of the life cycle of the fowl nematode Ascaridia galli (Schrank). J. Parasit. 38: 277-288.

Magnesium Requirement of the Chick* E. E. GARDINER, J. C. ROGLER AND H. E. PARKER Purdue University, Lafayette, Ind. (Received for publication December 1, 1959)

ALMQUIST (1942), working with a -**• washed casein, cerelose-type diet containing 40 p.p.m. of magnesium, reported that the addition of 350 p.p.m. of supplemental magnesium produced as good growth rates as any higher level. The author concluded that 350 p.p.m. was a barely adequate supplementary level and stated that the requirement during the first weeks of life was approximately 400 p.p.m. The deficiency symptoms that * Journal paper no. 1539 of the Purdue University Agricultural Experiment Station.

were observed included convulsions, gasping and a coma which sometimes was fatal but usually ceased in a few minutes. Bird (1946) reported a cerebellum disorder in chicks being fed a washed casein, cerelose diet. Increasing the basal dietary level of MgS04 to supply 500 p.p.m. (calculated) of elemental magnesium corrected the condition. His description of the deficiency syndrome was very similar to that of Almquist's. Scott et al. (1956) supplemented a cerelose, Drackett-protein diet with 0,50,100,