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The Anthelmintic Effect of Three Piperazine Derivatives on Ascaridia Galli (Schrank 1788)
The Anthelmintic Effect of Three Piperazine Derivatives on Ascaridia Galli (Schrank 1788) C. HORTON-SMITH AND P. L. LONG
Poultry Research Station, Houghton, Huntingdon, England (Received for publication November 21, 1955)
A
periment a comparison has been made of the anthelmintic activity of the piperazine compounds with that of other substances which are, or have been, in common use against ascaridial infestations in fowls. METHODS
Suspensions of Ascaridia ova were obtained from freshly-collected female worms by removing the uteri and grinding them up into a thin paste with a little distilled water. A 1:30 solution of "Milton" (Active ingredient: sodium hypochlorite 1% and sodium chloride 16.5%) was added and allowed to act for fifteen minutes after which the ova were washed free from the solution by repeated spinning in distilled water. The ova were embryonated by suspending them in about 100 ml. of 0.5 percent formalin solution in a 250 ml. Erlenmeyer flask and incubating at 30°C. for fourteen days. Embryonated ova were administered to fowls per os in numbers varying from approximately 75-110 to each chicken when the effect of a compound on adult worms was being studied. White Leghorn chickens of mixed sexes and aged from 3-6 weeks at time of infecting were used in this work. The chickens were maintained in wire-floor units until about the thirty fifth day when they were transferred to wire-floor battery cages. One or two chickens were housed in each cage. Faecal examinations were made and only chickens showing Ascaridia ova in their faeces were included in the tests. The chickens were distributed amongst the groups by selecting birds of closely ap-
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CONSIDERABLE literature on the anthelmintic effects of piperazine compounds has accumulated. Since this has been reviewed in several recent papers mention is only made of publications which have a direct bearing on the subject of the present paper. Guilhon (1951) discussed the activity of piperazine against Ascaridia columbae and Capillaria columbae in pigeons. Sloan, Kingsbury and Jolly (1954) were the first British workers to report results obtained with some forty piperazine compounds. These authors described the value of piperazine in full-scale trials in horses, pigs, dogs, cats and poultry. Following closely on this publication, Leiper (1954) mentions that piperazine is effective eliminating Ascaridia and, to some extent, Capillaria and Heterakis as well. Poynter (1955a) found that piperazine adipate was an efficient anthelmintic against Parascaris equorum in horses. Lee (1955) described piperazine adipate as a safe and efficient anthelmintic against Neoascaris vitulorum when administered to calves in single doses. He noted that the activity of the compound against immature worms enhances its value. Shumard and Eveleth (1955) found piperazine citrate effective in removing Ascaridia galli. The present paper sets out the results of experiments to determine the anthelmintic activity of piperazine adipate, piperazine citrate and an equimolecular complex of piperazine and carbon disulphide (hereinafter referred to as piperazine carbon disulphide) against the larval and mature forms of Ascaridia in fowls. In the preliminary ex-
ANTHELMINTIC EFFECT OF PIPERAZINE DERIVATIVES
In determining the effect of medication on the larvae, two methods were employed depending on whether searches were being made for tissue phase or lumen phase larvae. In both cases White Leghorn chicks of mixed sexes and aged from one to five weeks were each given approximately 100
to 500 embryonated ova per os. Single doses of the compounds were administered on the 14th or 17th day after infection. The chickens were sacrificed 24-48 hrs. after medication and, in the case of the search for tissue phase larvae, the intestines were removed and digested by the method described by Tugwell and Ackert (1952). The resulting suspensions were then diluted to about ten times their original volume with tap water. Using small quantities of these diluted suspensions at a time, searches for larvae were carried out until the whole suspension had been examined. The larvae were transferred by pipette to another vessel and counted. The method for the isolation of 20-21 day old larvae was that used by Ackert and Nolf (1929) in which a gentle stream of tap water was passed through the unopened intestine into a glass jar. The larvae were isolated by serial examinations of small quantities of the suspension of intestinal contents until the whole had been examined. The lumen larvae were more conspicuous than the tissue phase larvae and could usually be sexed. RESULTS
The effect of various anthelmintics on adult Ascaridia Experiment 1. Seventy-eight five week old chickens which had been reared on wire floors, were transferred to wire floor battery cages each of which contained two chickens. When the chickens were thirty-eight days old each of them was given seventy five embryonated ova per os. When the birds were seventy-five days old, and had been infected for thirtyseven days, the faeces from each cage were examined for Ascaridia ova and were found to be positive. Thirty-nine days after the initial infections with ova, twelve chickens were selected at random and weighed. The average weight proved
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proximating weights so that finally the average weight for each group within a series was approximately the same. The piperazine compounds and the phenothiazine were introduced into the crop in gelatin capsules. Carbon tetrachloride was administered into the crop by a syringe fitted with polythene tubing of about | inch diameter and 3§ inches in length; by this means the carbon tetrachloride could be passed into the crop well clear of the epiglottis. In a final experiment the piperazine compounds were incorporated in the food or in the drinking water. All the faeces from the control and treated chickens were collected at 24 hrs. and again at 48 hrs. after the administration of anthelmintic. Most of the worms were passed within 24 hours of dosing. Each faecal sample was broken up and washed through a wire mesh screen, from which the worms were collected and transferred to A dish of water. The worms were counted and sexed. All chickens were killed at 48 or 72 hrs. after medication and the intestines removed. The intestines were cut longitudinally and any remaining worms collected. The contents of the opened intestines were then washed into a bowl of warm water to remove any worms that had been overlooked in the previous operation. The efficiency of each drug in removing adult worms was determined by comparing the number of worms discharged in the faeces during the period of 0-48 hrs. after medication with the total number recovered from the intestines at examination post mortem.
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C. HORTON-SMITH AND P. L. LONG
Oil of chenopodium and phenothiazine expelled 4.5 percent and 39 percent respectively of the worms, carbon tetrachloride and the two piperazine compounds when applied at a dosage of 200 mg. or more were 100% effective. A small number of male worms remained in the intestine at the two lower dosages of the piperazine carbon disulphide. The effect of various anthelmintics on larval Ascaridia Experiment 2. Sixty-two five week old chickens each received approximately 100 embryonated ova -per os. Five groups of
five chickens each were treated with the same anthelmintics, and a sixth group remained untreated. The average weight of the chickens was only If lb. at fourteen days after initial infection with ova, but the dosages used were as follows: piperazine adipate, 300 mg. and piperazine carbon disulphide, 100 mg. The remaining compounds were dosed at levels similar to those used in experiment 1. These and seven chickens in a control group were killed two days later. The intestines were removed from each chicken and the tissue and lumen phases of the larvae recovered. The results of this experiment are given in Tables 2 and 3. The chickens of all groups were sacrificed two days after receiving the anthelmintics, i.e. on the sixteenth day after infection. Another twenty-five chickens were treated on the twentieth day after administering the ova. A study of Table 2 shows that none of the compounds tested is outstandingly effective against tissue-larvae. On the other hand, there has probably been some reduction in the numbers of larvae in chickens treated with carbon tetrachloride and the two piperazine derivatives. When the effect of the two piperazine derivatives and carbon tetrachloride on 20-day old lumen larvae are considered (Table 3) the differences in recoveries of larvae are sufficiently great to assume an effect. The
on A. galli TABLE 1 —Effects of various anthelmintics adult
Group No.
1 2 3 4 5 6 7 8 9 10 11
Anthelmintic and dosage used
Oil of Chenopodium, 4 drops Phenothiazine, 0.5 g. Carbon tetrachloride, 2 ml. Piperazine carbon disulphide, 50 mg. Piperazine carbon disulphide, 100 mg. Piperazine carbon disulphide, 200 mg. Piperazine adipate, 200 mg. Piperazine adipate, 300 mg. Piperazine adipate, 400 mg. Piperazine adipate, 500 mg. Control no treatment
N o . of chickens in group
Worms recovered from faeces 0-48 hours following dosage
Recovery of worms i t autopsy of birds 48 hours after dosing Average N o . per bird
effect of drug
17.3 13.3 0
4.58 38.93 100
%
c?
9
Total from group
0.83 8.5 19.16
46 40 0
58 40 0
104 80 0
103
17.16
10
0
10
1.6
91.15
144
24.0
6
0
6
1.0
96.0
99 112 171 82 92 0
16.5 18.6 28.5 13.6 15.3 0
0 0 0 0 0 105
0 0 0 0 0 153
0 0 0 0 0 258
0 0 0 0 0 21.5
c?
9
Total from group
6 6 6
1 21 44
4 30 71
5 51 115
6
46
57
6
61
83
6 6 6 6 6 12
45 49 64 37 43 0
54 63 107 45 49 0
Average N o . per bird.
100 100 100 100 100 0
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to be 2 lb. 5§ oz. Since it had been decided to use commonly adopted dosages of anthelmintic the piperazine compounds were administered on a mg./kg. body weight basis. Any birds registering a weight of less than 2 lb. were not medicated. The faeces were collected at 24 and 48 hrs. after dosing with anthelmintic and the worms recovered as described. The anthelmintics used in the test were oil of chenopodium (4 drops in 2 ml. liquid paraffin), phenothiazine (0.5 gm. administered in a gelatin capsule), carbon tetrachloride (2 ml.), piperazine adipate (200, 300, 400, and 500 mg.), piperazine carbon disulphide (50, 100 and 200 mg.). The results of this experiment are given in Table 1.
609
ANTHELMINTIC EFFECT OF PIPERAZINE DERIVATIVES TABLE 2.—The effect of single doses of various anthelmintics on 14 day old larvae of A. galli
Anthelmintic and dosage used
Piperazine carbon disulphide, 100 mg. Piperazine adipate, 300 mg. Phenothiazine, 500 mg. Carbon tetrachloride, 2 ml. Oil of chenopodium, 4 drops Control
No. of chickens
5 5 5 5 5 5
Number of larvae recovered From lumen of intestine
By digestion of intestine
39 36 64 15 60 51
15 12 19 6 51 26
Total larvae recovered 54 48 83 26 111 77
The chickens were sacrificed and autopsied 72 hours after medication and the intestines searched for worms. The results are summarised in Table 4. The results of this experiment also show the differences in infection rate obtained by approximately the same number of embryonated ova. The three piperazine derivatives at the higher levels of dosage are effective in removing adult worms from the intestinal tract. The male worms appear to be more resistant to the action of the compounds.
The effect of three piperazine derivatives on adult Ascaridia galli
The effects of three piperazine compounds on on 14 day old larvae of Ascaridia galli
Experiment 3. Fifty-seven six week old chickens each received 100 embryonated ova. The chickens were housed in wire floor battery cages containing one or two birds each. The faeces of these chickens were examined at 32 and 43 days after the ova were administered and were found to contain Ascaridia ova. Forty-eight days after receiving the ova the compounds were administered in capsules to each of the forty-eight chickens which were distributed among twelve groups of four chickens each. Nine chickens were kept as controls. At this time the weights of the chickens varied from 1 lb. 14 oz. to 2 lb. 4 oz. In this experiment the chickens were treated as weighing approximately one kg. and the quantities of compound were adjusted accordingly. The worms recovered from the faeces at 24 and 48 hrs. after medication were counted and sexed.
Experiment 4. Forty-two one week old chickens received 500 embryonated ova each and thirteen days later were distributed amongst six groups of six chickens each when their average weight approximated to 200 gm. The compounds were administered in gelatin capsules to the chickens of five groups on the fourteenth day after they received the ova. All the chickens from the five treated groups and the single untreated group TABLE 3.—The effect of single doses of various anthelmintics on 20 day old larvae of A. galli
Anthelmintic and dosage used
Piperazine carbon disulphide, 100 mg. Piperazine adipate, 300 mg. Phenothiazine, 500 mg. Carbon tetrachloride, 2 ml. Oil of chenopodium, 4 drops Control
Number of Number of larvae chickens recovered in group from intestine 0 2 56 0 50
53
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effects of the piperazine derivatives on tissue phase and lumen larvae receive further attention in later experiments. Since the piperazine derivatives compared favourably with carbon tetrachloride in removing larval and adult worms, and were considerably less toxic to chickens, it was decided to confine the remainder of the investigation to a study of the anthelmintic properties of piperazine carbon disulphide, piperazine adipate and piperazine citrate.
610
C. H O R T O N - S M I T H AND P . L.
TABLE 4.—Effect
Group No.
No. of chickens in group
Anthelmintic and dosage used
of three piperazine compounds on adult A. galli Worms recovered in faeces 0-48 hours after treatment ft
1 2 3 4
13
9
Worms recovered at auopsy 82 hours after treatment
Total Average from No. per group bird 3.25
ri<
9
compound
Total Average from No. per group bird
9
Both sexes
54.5
43.3
88.63
58.5
cf
4
1
12
13
20
10
30
7.5
4
9
39
48
12.0
29
5
34
8.5
23.6
4.76
4
23
33
56
14.0
14
0
14
3.5
62.1
4 4 4 4 4 4 4 4
22 24 23 14 20 15 11 24
35 51 33 21 31 17 17 41
57 75 56 35 51 32 28 65
13.75 18.75 14.0 8.75 12.75 8.0 7.0 16.25
2 15 10 3 1 4 0 0
0 2 0 0 0 0 0 0
2 17 10 3 1 4 0 0
0.5 4.25 2.5 0.75 0.25 1.0 0 0
91.6 61.5 69.6 82.3 95.2 78.9 100 100
100 96.2 100 100 100 100 100 100
96.4 75.0 84.8 92.1 98.0 88.8 100 100
4 9
32 1
41 3
73 4
18.25 0.44
0 68
0 86
0 154
100
100
100
0 17.1
100
80.0
four groups to give an average weight for three groups of 175 gm. and 154 gm. in the fourth group. The piperazine compounds were then administered in gelatin capsules to the chickens of the three groups with the higher average weight. The fourth group remained untreated. There were four chickens in each of the treated groups and six in the control group. The anthelmintics were given as single doses at the rate of 300 mg./kg. body weight, The effect of three piperazine compounds on i.e. 53 mg. per chicken. The chickens were 17 day old larvae of Ascaridia galli killed forty-eight hours after treatment and the intestines were examined in the Experiment 5. Eighteen one week old usual way. The group which received chickens were each given 500 embryonpiperazine adipate and piperazine citrate ated ova. Seventeen days later the chickens were weighed and distributed among yielded only one larva each, the group were killed on the following day. The intestines from the chickens of each group were kept separate and washed out with warm water to collect any lumen larvae present. The larvae were counted and sexed. The intestines from each group were then cut up into small pieces and digested as described. The tissue larvae were then isolated. The results are given in Table 5.
TABLE S.—The effect of three piperazine compounds on 14 day old larvae of A. galli Larvae recovered Group
1 2 3 4 5 6
No. of chickens in group 6 6 6 6 6 6
Piperazine derivative administered adipate adipate citrate citrate carbon disulphide No treatment
Dosage in mgs.
60 120 60 120 20
Equivalent dosage in mg./kilo 300 600 300 600 200
Worms marked "x" were damaged and impossible to sex.
Lumen
Tissue
&
9
d"
9
Total recovered from group
4 0 0 0 6 18
5 0 0 0
5 0
2 1 4 0 1 11
16+1 unsexed* 1 6 0 16+1 unsexed* 5 2 + 5 unsexed*
s 14
2 0 4 9
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5 6 7 8 9 10 11 12
Piperazine carbon disulphide 25 Piperazine carbon disulphide 50 Piperazine carbon disulphide 100 Piperazine carbon disulphide 200 Piperazine adipate 75 Piperazine adipate 150 Piperazine adipate 300 Piperazine adipate 600 Piperazine citrate 150 Piperazine citrate 300 Piperazine citrate 600 Piperazine citrate 1,200 Control no treatment
LONG
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ANTHELMINTIC EFFECT OF PIPERAZINE DERIVATIVES TABLE 6.—Effect
of three piperazine compounds on 21 day old larvae of A. galli
Group
No. of chickens
Piperazine compound administered
Dosage in
Equivalent dosage in mg./kilo
Total number of larvae recovered from intestine
1 2 3 4 5 6 7
4 4 4 4 4 4 4
adipate adipate citrate citrate carbon disulphide carbon disulphide No treatment
84 168 84 168 28 56
300 600 300 600 100 200
1 0 0 0 0 0 38
—•
azine citrate and piperazine carbon disulphide respectively the chicks of each group were killed, including those from the untreated group. The intestines were removed. The larvae from each group were collected, counted and sexed. The results are summarized in Table 6. When the last three experiments are compared it is evident that the three piperazine compounds under consideration are extremely effective in removing lumen larvae from the intestines. Their effect on tissue larvae is now receiving consideration in further work. The anthelmintic activity of piperazine compounds administered in the food or water
Experiment 8. A single experiment was conducted to ascertain the anthelmintic effect of administering the piperazine compounds in the food or drinking water, to fowls which had been inoculated with 110 The effect of three piperazine compounds on Ascaridia ova forty-three days previously. 21 day old larvae of Ascaridia galli Piperazine adipate or piperazine citrate Experiment 7. Twenty-eight one week were incorporated in the food at the rate old chickens were given 500 embryonated of 300 mg. in 100 gm. wet mash or in the ova. Three weeks later the chickens, now drinking water at the rate of 300 mg. of aged four weeks, were weighed and sepa- compound per 200 ml. water. The birds rated into seven groups of four birds each. were starved overnight to ensure that the The chickens in the group to be treated whole of the medicated mash was conweighed approximately 280 gm. each and sumed within the day it was presented to those in the untreated group about 281 them. There was substantial elimination gm. each. Twenty-four hours after dosing of worms and the efficiency of the two each group with piperazine adipate, piper- piperazine compounds varied from 80-
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treated with piperazine carbon disulphide yielded three and the control group thirty-five larvae. Experiment 6. A similar experiment was conducted in which thirty-two 2 week old chickens were infected with 500 embryonated ova. Groups of eight chicks were treated seventeen days later with piperazine adipate, piperazine citrate and piperazine carbon disulphide respectively. The compounds were administered at the rate of 600 mg. per kg. body weight. Since the chickens were of an average weight of 200 gm. in each group 120 mg. of compound were administered to each bird. A group of eight birds was not treated. The chickens were killed forty-eight hours after medication and the intestines were searched for larvae. A single larva was recovered from each treated group and fifty-three were recovered from the untreated group.
—
612
C. HORTON-SMITH AND P. L. LONG
compound acts as a narcotic which so reduces the activity of the worms that they are carried, almost passively, to the exterior by the ordinary peristaltic movements of the fowl intestine. Since the action of the drug was gradual and produced no irritation of the worms, the danger of rupture of the bowel wall or occlusion of the common bile duct was much reduced; such might well occur as a consequence of the increase in activity of large muscular worms endeavouring to escape from an unfavourable environment (Standen, 1955).
Action of piperazine adipate on adult Ascaridia galli
DISCUSSION
Goodwin and Standen (1954) reported that piperazine citrate in a concentration of 1:500 exerted a narcotic effect on Ascaris lumbricoides in vitro. The same conclusion was reached by Poynter (1955a) in regard to Parascaris equorum in vitro. Standen (1955) showed that piperazine citrate and piperazine phosphate produced a state of narcosis in Ascaris lumbricoides obtained from the pig and maintained in vitro. Tests on similar lines were made in the present series of experiments to determine the effect of piperazine adipate on adult Ascaridia galli in vitro, and using Standen's medium in which the compound was incorporated at 100 mg. percent. The worms maintained at 39°C. in the medium in which the piperazine adipate was incorporated were less active within 15 minutes and after 2 hours became sluggish. After 2\ hours and up to 28 hours only occasional movements were observed. When these worms were restored to a drug free medium an activity comparable with that of control worms maintained throughout in drugfree medium at 39°C. was attained within 24 hours. These findings confirm the conclusion of Standen and Poynter that the
The elimination of sexually mature worms from fowls is of value in reducing the numbers of ova passed out onto the ground and in relieving birds of massive infestations (cf. Hansen et al., 1954). Should chickens ingest large numbers of Ascaridia ova considerable damage will result from the larval penetration of the mucosa at or about the tenth day after the ingestion of ova. As pointed out by the above authors, much time will have elapsed between the penetration of the bowel wall by the larvae and the time when a definite diagnosis of helminthiasis can be made by recovery of ova in the faeces. Probably the only solution, if no anthelmintic action on tissue larvae could be expected, to the problem of depopulating the intestine of worms would be some form of preventive treatment which would eliminate newly-hatched larvae in the pretissue phase. On the other hand, if an anthelmintic could be found which was active, or even partially active, against tissue larvae as well as adult worms, a single treatment, by removing both, would have a marked effect in still further reducing the numbers of ova discharged over a longer period of time. There would, however, be no provision against reinfection
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100% as based on a comparison of the number of worms expelled and the number recovered at autopsy. There was no significant difference between the results obtained by these methods of medication and those obtained by direct oral administration of the compounds in the case of piperazine adipate in food or water and piperazine citrate in water. In this connection, Poynter (1955b) concluded that the efficiency of piperazine adipate as an anthelmintic was in no way impaired when it was administered to horses in bran mash.
ANTHELMINTIC EFFECT or PIPERAZINE DERIVATIVES
termined on the assumption which is open to doubt that larvae always pass through a tissue phase. If this assumption is correct any effect on tissue phase larvae could be detected by treating fowls between the tenth and fifteenth days of infection and sacrificing them at forty days. A comparison could then be made of the numbers of adult worms recovered from them with those recovered from a group of chickens sacrificed at the same time, which bad received a comparable number of ova at the same time, but which had received no treatment. Care must be exercised to ensure that the fowls are parasite-free before they receive the experimental doses of ova and are adequately isolated during the term of the experiment. Future work is proceeding along the lines of correlating treatment with life history, together with the isolation of tissue larvae from treated and untreated chickens by digestion techniques. SUMMARY
1. The piperazine compounds studied completely eliminated adult Ascaridia when administered in single doses varying from 100 to 500 mg./kg. body weight according to the compound used. 2. The three compounds removed a considerable number of the larvae but to what extent the removal of tissue phase larvae occurred has not yet been determined with any certainty, although some reduction in their numbers may have been effected. 3. Substantial control of Ascaridia infestations was obtained in a single experiment by administering the compounds in the food or water. 4. The anthelmintic action of the piperazine compounds depends on their capacity to produce a state of narcosis in the worms which are then carried to the exterior in the flow of intestinal contents pro-
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and subsequent build-up of ova on the ground. The accumulation of ova would, of course, be of much less importance to chickens maintained in wire floor battery cages. The piperazine compounds and carbon tetrachloride proved extremely effective in removing adult Ascaridia from the intestines and were much superior to phenothiazine and oil of chenopodium in this respect. (Experiments 1 and 3). It was noted that adult male worms appeared to be slightly more resistant to the action of these compounds (Table 4). None of the anthelmintics at the dosages used in Experiments 2, 5 and 6 was markedly effective in removing 14 and 17 day old larvae although good results were obtained with piperazine adipate and piperazine citrate at the higher dosages used, i.e. 600 mg./kg. body weight (Experiment 4). Twenty and twenty-one day old larvae were completely eliminated from the intestines of chickens receiving piperazine derivatives at the dosages used in Experiments 2 and 7. Further work is proceeding to determine the effects of selected piperazine derivatives on the larval phases of Ascaridia galli and this work must be completed before the action of these compounds on larvae, and, particularly tissue-phase larvae, can be assessed with any accuracy. If the anthelmintic action of the piperazines is dependent on their narcotic effect on the worms, as it appears to be, and their subsequent discharge to the exterior by peristalsis it seems probable that such action would affect lumen larvae as well as adults. However, it is open to speculation that such action would release all tissue phase larvae and especially those which have penetrated the mucosa at depth. Apart from digestion techniques, the question of whether the effect on tissue phase larvae is total, partial or indifferent could be de-
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C. HORTON-SMITH AND P. L. LONG
duced by normal peristaltic action. The mode of action on Ascaridia is therefore similar to that described for other nematodes. ACKNOWLEDGEMENTS
REFERENCES Ackert, J. E., and L. O. Nolf, 1929. New technique for collecting intestinal roundworms. Science, 70: 310-311. Goodwin, L. G., and O. D . Standen, 1954. Treatment of roundworm with piperazine citrate "Antepar," Brit. Med. J. 4900: 1332-1334. Guilhon, J., 1951. Un nouvel anthelmintique: le diethylene diamine. Bull, de FAcad. Vet. de France, 24:243-245. Helm. abs. (1951) 697(a).
The Specificity of Action of the K-k Alleles Affecting Feathering in the Fowl T U L S A R A M AND F . B. H U T T
Cornell University Agricultural Experiment Station, Ithaca, New York (Received for publication November 22, 1955)
T
HE sex-linked alleles, K and k, induce remarkably different rates of feathering which are most evident in young chicks. The effects of these genes and their economic importance have been discussed elsewhere (Hutt, 1949). It would not be surprising if the influence of K and k on the growth of cells in the feather follicles were merely the outwardly visible manifestations of more general influences affecting the bird's physiology,
but no such effects have yet been conclusively demonstrated. Contrary to an earlier suggestion, it has been shown by Hays (1951), Hale (1952) and Godfrey and Farnsworth (1952) that slowly feathering chicks do not differ in rate of body growth from those that feather rapidly. Since pullets of these two types do not differ in age at first egg (Hays and Spear, 1951), it seems probable that K and k do not influence the physiol-
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Our thanks are due to Boots Pure Drug Co. Ltd., Cooper McDougall and Robertson, Ltd., and Burroughs Wellcome and Co. for the supply of the compounds used in this work. We would also like to express our gratitutde to Mr. J. W. G. Leiper and Mr. J. E. N. Sloan for their helpful advice and to Dr. R. FJ Gordon for his encouragement throughout.
Hanson, M. F., B. R. B. Persaud and J. E. Ackert., 1954. Effects of certain anthelmintics and an antibiotic on lumen and tissue phase larvae of Ascaridia galli (Schrank). Poultry Sci. 33: 140-146. Lee, R. P., 1955. The anthelmintic efficiency of piperazine adipate against Neoascaris vitulorum (Goeze 1782). Vet. Rec. 67: 146-149. Leiper, J. W. G., 1954. The piperazine compound V.19 for the removal of Ascaris and Oesophagostomum from the pig. Vet. Rec. 66: 596-599. Poynter, D., 1955a. Piperazine adipate as an equine anthelmintic. Vet. Rec. 67: 159-163. Poynter, D., 1955b. The efficiency of piperazine adipate administered in bran mash to horses. Vet. Rec. 67:625-626. Shumard, R. F., and D. F. Eveleth, 1955. A preliminary report on the anthelmintic action of piperazine citrate on Ascaris galli and Heterakis gallinae in hens. Vet. Med. 50:203-205. Sloan, J. E. N., P. A. Kingsbury and D. W. J. Jolly, 1954. Preliminary trials with piperazine adipate as a veterinary anthelmintic. J. Pharm. Pharmacol. 6: 718-724. Standen, O. D., 1955. Activity of piperazine, in vitro, against Ascaris lumricoides. Brit. Med. J. 4930: 20-22. Tugwell, R. L., and J. E. Ackert, 1952. On the tissue phase of the life cycle of the fowl nematode Ascaridia gaUi (Schrank). J. Parasit. 38:277-288.
Poultry Research Station, Houghton, Huntingdon, England (Received for publication November 21, 1955)
A
periment a comparison has been made of the anthelmintic activity of the piperazine compounds with that of other substances which are, or have been, in common use against ascaridial infestations in fowls. METHODS
Suspensions of Ascaridia ova were obtained from freshly-collected female worms by removing the uteri and grinding them up into a thin paste with a little distilled water. A 1:30 solution of "Milton" (Active ingredient: sodium hypochlorite 1% and sodium chloride 16.5%) was added and allowed to act for fifteen minutes after which the ova were washed free from the solution by repeated spinning in distilled water. The ova were embryonated by suspending them in about 100 ml. of 0.5 percent formalin solution in a 250 ml. Erlenmeyer flask and incubating at 30°C. for fourteen days. Embryonated ova were administered to fowls per os in numbers varying from approximately 75-110 to each chicken when the effect of a compound on adult worms was being studied. White Leghorn chickens of mixed sexes and aged from 3-6 weeks at time of infecting were used in this work. The chickens were maintained in wire-floor units until about the thirty fifth day when they were transferred to wire-floor battery cages. One or two chickens were housed in each cage. Faecal examinations were made and only chickens showing Ascaridia ova in their faeces were included in the tests. The chickens were distributed amongst the groups by selecting birds of closely ap-
606
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CONSIDERABLE literature on the anthelmintic effects of piperazine compounds has accumulated. Since this has been reviewed in several recent papers mention is only made of publications which have a direct bearing on the subject of the present paper. Guilhon (1951) discussed the activity of piperazine against Ascaridia columbae and Capillaria columbae in pigeons. Sloan, Kingsbury and Jolly (1954) were the first British workers to report results obtained with some forty piperazine compounds. These authors described the value of piperazine in full-scale trials in horses, pigs, dogs, cats and poultry. Following closely on this publication, Leiper (1954) mentions that piperazine is effective eliminating Ascaridia and, to some extent, Capillaria and Heterakis as well. Poynter (1955a) found that piperazine adipate was an efficient anthelmintic against Parascaris equorum in horses. Lee (1955) described piperazine adipate as a safe and efficient anthelmintic against Neoascaris vitulorum when administered to calves in single doses. He noted that the activity of the compound against immature worms enhances its value. Shumard and Eveleth (1955) found piperazine citrate effective in removing Ascaridia galli. The present paper sets out the results of experiments to determine the anthelmintic activity of piperazine adipate, piperazine citrate and an equimolecular complex of piperazine and carbon disulphide (hereinafter referred to as piperazine carbon disulphide) against the larval and mature forms of Ascaridia in fowls. In the preliminary ex-
ANTHELMINTIC EFFECT OF PIPERAZINE DERIVATIVES
In determining the effect of medication on the larvae, two methods were employed depending on whether searches were being made for tissue phase or lumen phase larvae. In both cases White Leghorn chicks of mixed sexes and aged from one to five weeks were each given approximately 100
to 500 embryonated ova per os. Single doses of the compounds were administered on the 14th or 17th day after infection. The chickens were sacrificed 24-48 hrs. after medication and, in the case of the search for tissue phase larvae, the intestines were removed and digested by the method described by Tugwell and Ackert (1952). The resulting suspensions were then diluted to about ten times their original volume with tap water. Using small quantities of these diluted suspensions at a time, searches for larvae were carried out until the whole suspension had been examined. The larvae were transferred by pipette to another vessel and counted. The method for the isolation of 20-21 day old larvae was that used by Ackert and Nolf (1929) in which a gentle stream of tap water was passed through the unopened intestine into a glass jar. The larvae were isolated by serial examinations of small quantities of the suspension of intestinal contents until the whole had been examined. The lumen larvae were more conspicuous than the tissue phase larvae and could usually be sexed. RESULTS
The effect of various anthelmintics on adult Ascaridia Experiment 1. Seventy-eight five week old chickens which had been reared on wire floors, were transferred to wire floor battery cages each of which contained two chickens. When the chickens were thirty-eight days old each of them was given seventy five embryonated ova per os. When the birds were seventy-five days old, and had been infected for thirtyseven days, the faeces from each cage were examined for Ascaridia ova and were found to be positive. Thirty-nine days after the initial infections with ova, twelve chickens were selected at random and weighed. The average weight proved
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proximating weights so that finally the average weight for each group within a series was approximately the same. The piperazine compounds and the phenothiazine were introduced into the crop in gelatin capsules. Carbon tetrachloride was administered into the crop by a syringe fitted with polythene tubing of about | inch diameter and 3§ inches in length; by this means the carbon tetrachloride could be passed into the crop well clear of the epiglottis. In a final experiment the piperazine compounds were incorporated in the food or in the drinking water. All the faeces from the control and treated chickens were collected at 24 hrs. and again at 48 hrs. after the administration of anthelmintic. Most of the worms were passed within 24 hours of dosing. Each faecal sample was broken up and washed through a wire mesh screen, from which the worms were collected and transferred to A dish of water. The worms were counted and sexed. All chickens were killed at 48 or 72 hrs. after medication and the intestines removed. The intestines were cut longitudinally and any remaining worms collected. The contents of the opened intestines were then washed into a bowl of warm water to remove any worms that had been overlooked in the previous operation. The efficiency of each drug in removing adult worms was determined by comparing the number of worms discharged in the faeces during the period of 0-48 hrs. after medication with the total number recovered from the intestines at examination post mortem.
607
608
C. HORTON-SMITH AND P. L. LONG
Oil of chenopodium and phenothiazine expelled 4.5 percent and 39 percent respectively of the worms, carbon tetrachloride and the two piperazine compounds when applied at a dosage of 200 mg. or more were 100% effective. A small number of male worms remained in the intestine at the two lower dosages of the piperazine carbon disulphide. The effect of various anthelmintics on larval Ascaridia Experiment 2. Sixty-two five week old chickens each received approximately 100 embryonated ova -per os. Five groups of
five chickens each were treated with the same anthelmintics, and a sixth group remained untreated. The average weight of the chickens was only If lb. at fourteen days after initial infection with ova, but the dosages used were as follows: piperazine adipate, 300 mg. and piperazine carbon disulphide, 100 mg. The remaining compounds were dosed at levels similar to those used in experiment 1. These and seven chickens in a control group were killed two days later. The intestines were removed from each chicken and the tissue and lumen phases of the larvae recovered. The results of this experiment are given in Tables 2 and 3. The chickens of all groups were sacrificed two days after receiving the anthelmintics, i.e. on the sixteenth day after infection. Another twenty-five chickens were treated on the twentieth day after administering the ova. A study of Table 2 shows that none of the compounds tested is outstandingly effective against tissue-larvae. On the other hand, there has probably been some reduction in the numbers of larvae in chickens treated with carbon tetrachloride and the two piperazine derivatives. When the effect of the two piperazine derivatives and carbon tetrachloride on 20-day old lumen larvae are considered (Table 3) the differences in recoveries of larvae are sufficiently great to assume an effect. The
on A. galli TABLE 1 —Effects of various anthelmintics adult
Group No.
1 2 3 4 5 6 7 8 9 10 11
Anthelmintic and dosage used
Oil of Chenopodium, 4 drops Phenothiazine, 0.5 g. Carbon tetrachloride, 2 ml. Piperazine carbon disulphide, 50 mg. Piperazine carbon disulphide, 100 mg. Piperazine carbon disulphide, 200 mg. Piperazine adipate, 200 mg. Piperazine adipate, 300 mg. Piperazine adipate, 400 mg. Piperazine adipate, 500 mg. Control no treatment
N o . of chickens in group
Worms recovered from faeces 0-48 hours following dosage
Recovery of worms i t autopsy of birds 48 hours after dosing Average N o . per bird
effect of drug
17.3 13.3 0
4.58 38.93 100
%
c?
9
Total from group
0.83 8.5 19.16
46 40 0
58 40 0
104 80 0
103
17.16
10
0
10
1.6
91.15
144
24.0
6
0
6
1.0
96.0
99 112 171 82 92 0
16.5 18.6 28.5 13.6 15.3 0
0 0 0 0 0 105
0 0 0 0 0 153
0 0 0 0 0 258
0 0 0 0 0 21.5
c?
9
Total from group
6 6 6
1 21 44
4 30 71
5 51 115
6
46
57
6
61
83
6 6 6 6 6 12
45 49 64 37 43 0
54 63 107 45 49 0
Average N o . per bird.
100 100 100 100 100 0
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to be 2 lb. 5§ oz. Since it had been decided to use commonly adopted dosages of anthelmintic the piperazine compounds were administered on a mg./kg. body weight basis. Any birds registering a weight of less than 2 lb. were not medicated. The faeces were collected at 24 and 48 hrs. after dosing with anthelmintic and the worms recovered as described. The anthelmintics used in the test were oil of chenopodium (4 drops in 2 ml. liquid paraffin), phenothiazine (0.5 gm. administered in a gelatin capsule), carbon tetrachloride (2 ml.), piperazine adipate (200, 300, 400, and 500 mg.), piperazine carbon disulphide (50, 100 and 200 mg.). The results of this experiment are given in Table 1.
609
ANTHELMINTIC EFFECT OF PIPERAZINE DERIVATIVES TABLE 2.—The effect of single doses of various anthelmintics on 14 day old larvae of A. galli
Anthelmintic and dosage used
Piperazine carbon disulphide, 100 mg. Piperazine adipate, 300 mg. Phenothiazine, 500 mg. Carbon tetrachloride, 2 ml. Oil of chenopodium, 4 drops Control
No. of chickens
5 5 5 5 5 5
Number of larvae recovered From lumen of intestine
By digestion of intestine
39 36 64 15 60 51
15 12 19 6 51 26
Total larvae recovered 54 48 83 26 111 77
The chickens were sacrificed and autopsied 72 hours after medication and the intestines searched for worms. The results are summarised in Table 4. The results of this experiment also show the differences in infection rate obtained by approximately the same number of embryonated ova. The three piperazine derivatives at the higher levels of dosage are effective in removing adult worms from the intestinal tract. The male worms appear to be more resistant to the action of the compounds.
The effect of three piperazine derivatives on adult Ascaridia galli
The effects of three piperazine compounds on on 14 day old larvae of Ascaridia galli
Experiment 3. Fifty-seven six week old chickens each received 100 embryonated ova. The chickens were housed in wire floor battery cages containing one or two birds each. The faeces of these chickens were examined at 32 and 43 days after the ova were administered and were found to contain Ascaridia ova. Forty-eight days after receiving the ova the compounds were administered in capsules to each of the forty-eight chickens which were distributed among twelve groups of four chickens each. Nine chickens were kept as controls. At this time the weights of the chickens varied from 1 lb. 14 oz. to 2 lb. 4 oz. In this experiment the chickens were treated as weighing approximately one kg. and the quantities of compound were adjusted accordingly. The worms recovered from the faeces at 24 and 48 hrs. after medication were counted and sexed.
Experiment 4. Forty-two one week old chickens received 500 embryonated ova each and thirteen days later were distributed amongst six groups of six chickens each when their average weight approximated to 200 gm. The compounds were administered in gelatin capsules to the chickens of five groups on the fourteenth day after they received the ova. All the chickens from the five treated groups and the single untreated group TABLE 3.—The effect of single doses of various anthelmintics on 20 day old larvae of A. galli
Anthelmintic and dosage used
Piperazine carbon disulphide, 100 mg. Piperazine adipate, 300 mg. Phenothiazine, 500 mg. Carbon tetrachloride, 2 ml. Oil of chenopodium, 4 drops Control
Number of Number of larvae chickens recovered in group from intestine 0 2 56 0 50
53
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effects of the piperazine derivatives on tissue phase and lumen larvae receive further attention in later experiments. Since the piperazine derivatives compared favourably with carbon tetrachloride in removing larval and adult worms, and were considerably less toxic to chickens, it was decided to confine the remainder of the investigation to a study of the anthelmintic properties of piperazine carbon disulphide, piperazine adipate and piperazine citrate.
610
C. H O R T O N - S M I T H AND P . L.
TABLE 4.—Effect
Group No.
No. of chickens in group
Anthelmintic and dosage used
of three piperazine compounds on adult A. galli Worms recovered in faeces 0-48 hours after treatment ft
1 2 3 4
13
9
Worms recovered at auopsy 82 hours after treatment
Total Average from No. per group bird 3.25
ri<
9
compound
Total Average from No. per group bird
9
Both sexes
54.5
43.3
88.63
58.5
cf
4
1
12
13
20
10
30
7.5
4
9
39
48
12.0
29
5
34
8.5
23.6
4.76
4
23
33
56
14.0
14
0
14
3.5
62.1
4 4 4 4 4 4 4 4
22 24 23 14 20 15 11 24
35 51 33 21 31 17 17 41
57 75 56 35 51 32 28 65
13.75 18.75 14.0 8.75 12.75 8.0 7.0 16.25
2 15 10 3 1 4 0 0
0 2 0 0 0 0 0 0
2 17 10 3 1 4 0 0
0.5 4.25 2.5 0.75 0.25 1.0 0 0
91.6 61.5 69.6 82.3 95.2 78.9 100 100
100 96.2 100 100 100 100 100 100
96.4 75.0 84.8 92.1 98.0 88.8 100 100
4 9
32 1
41 3
73 4
18.25 0.44
0 68
0 86
0 154
100
100
100
0 17.1
100
80.0
four groups to give an average weight for three groups of 175 gm. and 154 gm. in the fourth group. The piperazine compounds were then administered in gelatin capsules to the chickens of the three groups with the higher average weight. The fourth group remained untreated. There were four chickens in each of the treated groups and six in the control group. The anthelmintics were given as single doses at the rate of 300 mg./kg. body weight, The effect of three piperazine compounds on i.e. 53 mg. per chicken. The chickens were 17 day old larvae of Ascaridia galli killed forty-eight hours after treatment and the intestines were examined in the Experiment 5. Eighteen one week old usual way. The group which received chickens were each given 500 embryonpiperazine adipate and piperazine citrate ated ova. Seventeen days later the chickens were weighed and distributed among yielded only one larva each, the group were killed on the following day. The intestines from the chickens of each group were kept separate and washed out with warm water to collect any lumen larvae present. The larvae were counted and sexed. The intestines from each group were then cut up into small pieces and digested as described. The tissue larvae were then isolated. The results are given in Table 5.
TABLE S.—The effect of three piperazine compounds on 14 day old larvae of A. galli Larvae recovered Group
1 2 3 4 5 6
No. of chickens in group 6 6 6 6 6 6
Piperazine derivative administered adipate adipate citrate citrate carbon disulphide No treatment
Dosage in mgs.
60 120 60 120 20
Equivalent dosage in mg./kilo 300 600 300 600 200
Worms marked "x" were damaged and impossible to sex.
Lumen
Tissue
&
9
d"
9
Total recovered from group
4 0 0 0 6 18
5 0 0 0
5 0
2 1 4 0 1 11
16+1 unsexed* 1 6 0 16+1 unsexed* 5 2 + 5 unsexed*
s 14
2 0 4 9
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5 6 7 8 9 10 11 12
Piperazine carbon disulphide 25 Piperazine carbon disulphide 50 Piperazine carbon disulphide 100 Piperazine carbon disulphide 200 Piperazine adipate 75 Piperazine adipate 150 Piperazine adipate 300 Piperazine adipate 600 Piperazine citrate 150 Piperazine citrate 300 Piperazine citrate 600 Piperazine citrate 1,200 Control no treatment
LONG
611
ANTHELMINTIC EFFECT OF PIPERAZINE DERIVATIVES TABLE 6.—Effect
of three piperazine compounds on 21 day old larvae of A. galli
Group
No. of chickens
Piperazine compound administered
Dosage in
Equivalent dosage in mg./kilo
Total number of larvae recovered from intestine
1 2 3 4 5 6 7
4 4 4 4 4 4 4
adipate adipate citrate citrate carbon disulphide carbon disulphide No treatment
84 168 84 168 28 56
300 600 300 600 100 200
1 0 0 0 0 0 38
—•
azine citrate and piperazine carbon disulphide respectively the chicks of each group were killed, including those from the untreated group. The intestines were removed. The larvae from each group were collected, counted and sexed. The results are summarized in Table 6. When the last three experiments are compared it is evident that the three piperazine compounds under consideration are extremely effective in removing lumen larvae from the intestines. Their effect on tissue larvae is now receiving consideration in further work. The anthelmintic activity of piperazine compounds administered in the food or water
Experiment 8. A single experiment was conducted to ascertain the anthelmintic effect of administering the piperazine compounds in the food or drinking water, to fowls which had been inoculated with 110 The effect of three piperazine compounds on Ascaridia ova forty-three days previously. 21 day old larvae of Ascaridia galli Piperazine adipate or piperazine citrate Experiment 7. Twenty-eight one week were incorporated in the food at the rate old chickens were given 500 embryonated of 300 mg. in 100 gm. wet mash or in the ova. Three weeks later the chickens, now drinking water at the rate of 300 mg. of aged four weeks, were weighed and sepa- compound per 200 ml. water. The birds rated into seven groups of four birds each. were starved overnight to ensure that the The chickens in the group to be treated whole of the medicated mash was conweighed approximately 280 gm. each and sumed within the day it was presented to those in the untreated group about 281 them. There was substantial elimination gm. each. Twenty-four hours after dosing of worms and the efficiency of the two each group with piperazine adipate, piper- piperazine compounds varied from 80-
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treated with piperazine carbon disulphide yielded three and the control group thirty-five larvae. Experiment 6. A similar experiment was conducted in which thirty-two 2 week old chickens were infected with 500 embryonated ova. Groups of eight chicks were treated seventeen days later with piperazine adipate, piperazine citrate and piperazine carbon disulphide respectively. The compounds were administered at the rate of 600 mg. per kg. body weight. Since the chickens were of an average weight of 200 gm. in each group 120 mg. of compound were administered to each bird. A group of eight birds was not treated. The chickens were killed forty-eight hours after medication and the intestines were searched for larvae. A single larva was recovered from each treated group and fifty-three were recovered from the untreated group.
—
612
C. HORTON-SMITH AND P. L. LONG
compound acts as a narcotic which so reduces the activity of the worms that they are carried, almost passively, to the exterior by the ordinary peristaltic movements of the fowl intestine. Since the action of the drug was gradual and produced no irritation of the worms, the danger of rupture of the bowel wall or occlusion of the common bile duct was much reduced; such might well occur as a consequence of the increase in activity of large muscular worms endeavouring to escape from an unfavourable environment (Standen, 1955).
Action of piperazine adipate on adult Ascaridia galli
DISCUSSION
Goodwin and Standen (1954) reported that piperazine citrate in a concentration of 1:500 exerted a narcotic effect on Ascaris lumbricoides in vitro. The same conclusion was reached by Poynter (1955a) in regard to Parascaris equorum in vitro. Standen (1955) showed that piperazine citrate and piperazine phosphate produced a state of narcosis in Ascaris lumbricoides obtained from the pig and maintained in vitro. Tests on similar lines were made in the present series of experiments to determine the effect of piperazine adipate on adult Ascaridia galli in vitro, and using Standen's medium in which the compound was incorporated at 100 mg. percent. The worms maintained at 39°C. in the medium in which the piperazine adipate was incorporated were less active within 15 minutes and after 2 hours became sluggish. After 2\ hours and up to 28 hours only occasional movements were observed. When these worms were restored to a drug free medium an activity comparable with that of control worms maintained throughout in drugfree medium at 39°C. was attained within 24 hours. These findings confirm the conclusion of Standen and Poynter that the
The elimination of sexually mature worms from fowls is of value in reducing the numbers of ova passed out onto the ground and in relieving birds of massive infestations (cf. Hansen et al., 1954). Should chickens ingest large numbers of Ascaridia ova considerable damage will result from the larval penetration of the mucosa at or about the tenth day after the ingestion of ova. As pointed out by the above authors, much time will have elapsed between the penetration of the bowel wall by the larvae and the time when a definite diagnosis of helminthiasis can be made by recovery of ova in the faeces. Probably the only solution, if no anthelmintic action on tissue larvae could be expected, to the problem of depopulating the intestine of worms would be some form of preventive treatment which would eliminate newly-hatched larvae in the pretissue phase. On the other hand, if an anthelmintic could be found which was active, or even partially active, against tissue larvae as well as adult worms, a single treatment, by removing both, would have a marked effect in still further reducing the numbers of ova discharged over a longer period of time. There would, however, be no provision against reinfection
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100% as based on a comparison of the number of worms expelled and the number recovered at autopsy. There was no significant difference between the results obtained by these methods of medication and those obtained by direct oral administration of the compounds in the case of piperazine adipate in food or water and piperazine citrate in water. In this connection, Poynter (1955b) concluded that the efficiency of piperazine adipate as an anthelmintic was in no way impaired when it was administered to horses in bran mash.
ANTHELMINTIC EFFECT or PIPERAZINE DERIVATIVES
termined on the assumption which is open to doubt that larvae always pass through a tissue phase. If this assumption is correct any effect on tissue phase larvae could be detected by treating fowls between the tenth and fifteenth days of infection and sacrificing them at forty days. A comparison could then be made of the numbers of adult worms recovered from them with those recovered from a group of chickens sacrificed at the same time, which bad received a comparable number of ova at the same time, but which had received no treatment. Care must be exercised to ensure that the fowls are parasite-free before they receive the experimental doses of ova and are adequately isolated during the term of the experiment. Future work is proceeding along the lines of correlating treatment with life history, together with the isolation of tissue larvae from treated and untreated chickens by digestion techniques. SUMMARY
1. The piperazine compounds studied completely eliminated adult Ascaridia when administered in single doses varying from 100 to 500 mg./kg. body weight according to the compound used. 2. The three compounds removed a considerable number of the larvae but to what extent the removal of tissue phase larvae occurred has not yet been determined with any certainty, although some reduction in their numbers may have been effected. 3. Substantial control of Ascaridia infestations was obtained in a single experiment by administering the compounds in the food or water. 4. The anthelmintic action of the piperazine compounds depends on their capacity to produce a state of narcosis in the worms which are then carried to the exterior in the flow of intestinal contents pro-
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and subsequent build-up of ova on the ground. The accumulation of ova would, of course, be of much less importance to chickens maintained in wire floor battery cages. The piperazine compounds and carbon tetrachloride proved extremely effective in removing adult Ascaridia from the intestines and were much superior to phenothiazine and oil of chenopodium in this respect. (Experiments 1 and 3). It was noted that adult male worms appeared to be slightly more resistant to the action of these compounds (Table 4). None of the anthelmintics at the dosages used in Experiments 2, 5 and 6 was markedly effective in removing 14 and 17 day old larvae although good results were obtained with piperazine adipate and piperazine citrate at the higher dosages used, i.e. 600 mg./kg. body weight (Experiment 4). Twenty and twenty-one day old larvae were completely eliminated from the intestines of chickens receiving piperazine derivatives at the dosages used in Experiments 2 and 7. Further work is proceeding to determine the effects of selected piperazine derivatives on the larval phases of Ascaridia galli and this work must be completed before the action of these compounds on larvae, and, particularly tissue-phase larvae, can be assessed with any accuracy. If the anthelmintic action of the piperazines is dependent on their narcotic effect on the worms, as it appears to be, and their subsequent discharge to the exterior by peristalsis it seems probable that such action would affect lumen larvae as well as adults. However, it is open to speculation that such action would release all tissue phase larvae and especially those which have penetrated the mucosa at depth. Apart from digestion techniques, the question of whether the effect on tissue phase larvae is total, partial or indifferent could be de-
613
614
C. HORTON-SMITH AND P. L. LONG
duced by normal peristaltic action. The mode of action on Ascaridia is therefore similar to that described for other nematodes. ACKNOWLEDGEMENTS
REFERENCES Ackert, J. E., and L. O. Nolf, 1929. New technique for collecting intestinal roundworms. Science, 70: 310-311. Goodwin, L. G., and O. D . Standen, 1954. Treatment of roundworm with piperazine citrate "Antepar," Brit. Med. J. 4900: 1332-1334. Guilhon, J., 1951. Un nouvel anthelmintique: le diethylene diamine. Bull, de FAcad. Vet. de France, 24:243-245. Helm. abs. (1951) 697(a).
The Specificity of Action of the K-k Alleles Affecting Feathering in the Fowl T U L S A R A M AND F . B. H U T T
Cornell University Agricultural Experiment Station, Ithaca, New York (Received for publication November 22, 1955)
T
HE sex-linked alleles, K and k, induce remarkably different rates of feathering which are most evident in young chicks. The effects of these genes and their economic importance have been discussed elsewhere (Hutt, 1949). It would not be surprising if the influence of K and k on the growth of cells in the feather follicles were merely the outwardly visible manifestations of more general influences affecting the bird's physiology,
but no such effects have yet been conclusively demonstrated. Contrary to an earlier suggestion, it has been shown by Hays (1951), Hale (1952) and Godfrey and Farnsworth (1952) that slowly feathering chicks do not differ in rate of body growth from those that feather rapidly. Since pullets of these two types do not differ in age at first egg (Hays and Spear, 1951), it seems probable that K and k do not influence the physiol-
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Our thanks are due to Boots Pure Drug Co. Ltd., Cooper McDougall and Robertson, Ltd., and Burroughs Wellcome and Co. for the supply of the compounds used in this work. We would also like to express our gratitutde to Mr. J. W. G. Leiper and Mr. J. E. N. Sloan for their helpful advice and to Dr. R. FJ Gordon for his encouragement throughout.
Hanson, M. F., B. R. B. Persaud and J. E. Ackert., 1954. Effects of certain anthelmintics and an antibiotic on lumen and tissue phase larvae of Ascaridia galli (Schrank). Poultry Sci. 33: 140-146. Lee, R. P., 1955. The anthelmintic efficiency of piperazine adipate against Neoascaris vitulorum (Goeze 1782). Vet. Rec. 67: 146-149. Leiper, J. W. G., 1954. The piperazine compound V.19 for the removal of Ascaris and Oesophagostomum from the pig. Vet. Rec. 66: 596-599. Poynter, D., 1955a. Piperazine adipate as an equine anthelmintic. Vet. Rec. 67: 159-163. Poynter, D., 1955b. The efficiency of piperazine adipate administered in bran mash to horses. Vet. Rec. 67:625-626. Shumard, R. F., and D. F. Eveleth, 1955. A preliminary report on the anthelmintic action of piperazine citrate on Ascaris galli and Heterakis gallinae in hens. Vet. Med. 50:203-205. Sloan, J. E. N., P. A. Kingsbury and D. W. J. Jolly, 1954. Preliminary trials with piperazine adipate as a veterinary anthelmintic. J. Pharm. Pharmacol. 6: 718-724. Standen, O. D., 1955. Activity of piperazine, in vitro, against Ascaris lumricoides. Brit. Med. J. 4930: 20-22. Tugwell, R. L., and J. E. Ackert, 1952. On the tissue phase of the life cycle of the fowl nematode Ascaridia gaUi (Schrank). J. Parasit. 38:277-288.