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Action of Piperazine Compounds on Lumen and Tissue Phase Larvae of Ascaridia Galli (Schrank), A Roundworm of Chickens1
Action, of Piperazine Compounds on Lumen and Tissue Phase Larvae of Ascaridia Galli (Schrank), A Roundworm of Chickens 1 D. E. WORLEY, M. F. HANSEN AND B. R. B. PERSAUD
Department of Zoology, Kansas State College, Manhattan, Kansas (Received for publication February 28, 1957)
INTRODUCTION
TNCREASED recognition of the value of •*• certain piperazine compounds as anthelmintics is indicated by their current widespread use in combating nematode parasitism in domestic animals. The first evidence of the effectiveness of a piperazine compound against intestinal helminthiasis was reported by Hewitt et al. (1948). Their work demonstrated efficacy for removal of ascarid infections in dogs. Later, Kanegis (1948) obtained comparable results in both dogs and cats treated for ascariasis. Both papers stated that the test anthelmintics, Hetrazan®* and Caricide,®* approached 100 percent efficiency in worm removal when administered at therapeutic levels. Guilhon and Groulade (1951), working with Toxocara infections in dogs and a cat, found that piperazine treatment effectively reduced fecal egg counts in these hosts. Colglazier and Enzie (1951) reported that Caricide removed 98 percent of the ascarids in a group of treated cats. It has been reported that piperazine citrate was of value for field use in the treatment of chickens infected with adult Ascaridia galli (Shumard and Eveleth, 1955; Bradley, 1955). In a preliminary study, Riedel (1950) reported erratic results with 1
Contribution No. 272 from the Kansas Agricultural Experiment Station in the Department of Zoology and No. 314 from the Department of Zoology, Kansas State College. * Diethylcarbamazine, American Cyanamid Company.
Caricide in chickens, but in a later study (1951) he obtained up to 81 percent efficacy when high concentrations of the drug were incorporated in the birds' feed. These successful trials with various piperazines against several species of adult ascarids suggested their possible potential value as anthelmintics against larval stages of nematodes. The need for such a drug for use in poultry has been pointed out by the experimental work of Ackert (1923), Guberlet (1924), Ackert and Herrick (1928), and Todd et al. (1949), among others. These studies convincingly demonstrated the pre-eminent role of the early stages of Ascaridia in causing host injury during the time when they inhabit the hosts' intestinal crypts. Hansen et al. (1954a) indicated that several compounds, including nicotine, formerly the drug of choice for removal of adult ascarids in chickens, were almost totally ineffective in combatting the larval stages of this parasite. The objective of the present study was to determine the activity of several of the more promising piperazine compounds against the immature stages of Ascaridia galli in the domestic fowl. MATERIALS AND METHODS
The five compounds tested in this study and the percentage of piperazine content were Caricide (l-diethylcarbamyl-4-methylpiperaxine dihydrogen citrate, 22%), Compound 180-C (l-carbethoxy-4-methylpiperazine hydrochloride, 41.2%), piperazine
865
866
D. E. WOELEY, M. F. HANSEN AND B. R. B. PERSAUD
dihydrochloride (52.1%), Parvex®* (betaine of 1-piperazine carbodithioic acid, 53%), and piperazine hexahydrate (44%). A total of seven tests were conducted, using White Plymouth Rock and New Hampshire chickens as experimental hosts. These were obtained as non-sexed, day-old chicks from a commercial hatchery and were fed a standard antibiotic-free commercial feed containing 18 percent protein. They were maintained in wire-floored batteries throughout the experiments. All of the birds used in the trials were infected per os at 14 days of age with either 75 ± 5 or 100 ± 10 embryonated Ascaridia galli ova. The preparation of a standardized suspension of ova for administration to the chickens was patterned after the sugar solution technique described by Hansen et al. (1954b). After infection, two types of experimental procedures were followed. In the first three tests, two treated and two untreated birds from Groups A and B, respectively, were sacrificed and examined each day for 10 days, beginning 11 days post inoculation. This procedure was designed to test the action of the anthelmintic during the progression of the tissue phase of the Ascaridia life cycle. According to Tugwell and Ackert (1952), the peak invasion of the crypts of the intestine occurs about 14 days after feeding embryonated ova, and continues until about the 21st day post-inoculation. To furnish evidence of the activity of the drug toward lumen larvae, the period of larval life following the tissue phase, an entire group of treated chicks (Group C) and an equal number of control birds (Group D) were killed and examined 21 days after inoculation. In Experiments 4, 5, 6, and 7, only Groups C and D were used. In Experiment 5-6, 2 groups received treatment with piper* The Upjohn Company.
azine dihydrochloride and Parvex, respectively, and are designated as Groups Ct and C2, respectively, in Table 1. Group D served as a common control for both medicated groups in this test. The flushing technique of Ackert and Nolf (1929) was used to recover worms present in the intestinal lumen of birds at autopsy. In Experiments 1,2, and 3 a count was also made of the tissue phase larvae. These imbedded larvae were recovered from the intestinal crypts by artificially digesting in a solution of 0.5% HC1 and 1.0% pepsin the portion of the small intestine from the gizzard to a point slightly posterior to the yolk sac diverticulum of each bird. In addition to enumerating the number of tissue phase and lumen larvae present in each bird, the length of the latter larvae from each group of birds was measured with the aid of a microprojection apparatus. All experimental results were evaluated statistically, using the t-test for group comparisons. If the value of "t" obtained by this technique was at, or close to a significant level, a modified t-test using a squareroot transformation was also computed, as an additional means of confirming the test hypothesis. RESULTS The effects of the 5 piperazine compounds on the growth of the fowl host are recorded in Table 1. A statistical comparison of the mean weights at autopsy of the treated chickens and their controls was used as a criterion of the effect of the anthelmintics on the host. Such a comparison is valid because the average weights of the two groups were equalized at the start of each experiment (Gardiner and Wehr, 1950). In neither of the first two Caricide tests, in which the drug was given by capsule,
PIPERAZINE COMPOUNDS AS ANTHELMINTICS
was any significant effect noted on the growth rates of the chickens. In Experiment 3 a continuous low-level dosage of Caricide, added as a powder to the feed, was accompanied by significantly greater weight gains in the treated group. Inasmuch as there was no significant difference between worm burdens of chicks in this experiment, some direct or indirect action of the drug as a growth stimulant may have occurred. In a replication of this experiment at a later date, comparable results were not obtained, indicating that these results should perhaps be regarded with reservation. In Experiment 4, Compound 180-C in aqueous solution had no significant effect TABLE 1.—Effect
Experiment No.
1
2
3
Av. No. lumen larvae
A. B. C. D.
Caricide 25 mgm. (Day 10 p.i.) Control Caricide 25 mgm. (Day 10 p.i.) Control
200
20 20 22 22
A. B. C. D.
Caricide 12.5 mgm. (Days 4-11 p.i.) Control Caricide 12.5 mgm. (Days 4-11 p.i.) Control
100
18
A. Caricide 12.5 mgm. (Continuous from hatching until slaughter) B. Control C. Caricide 12.5 mgm. (Continuous) D. Control
100 100
220.8* 188.0
13.1 7.3 17.1
C. Compound 180-C 25 mgm. (Days 10-17 p.i.) D. Control
200
236.1
23.5
247.8
15.1
Cj. Piperazine dihydrochloride 25 mgm. (Days 7-14 p.i.) C2. Parvex 30 mgm. (Day 11 p.i.) D. Control
200
259.5*
200
235.9 246.4
14.1 13.5
C. Piperazine hexahydrate 25 mgm. (Days 7-15 p.i.) D. Control
200
317.1
2.66*
316.2
7.59
46
52 52 54
7
Group treatment (Mgm./day)
Approx. drug dosage Av. Wt. of (mgm. chickens total at autopsy cmpd./kilo. (gms.) body wt.)
19 19 17 17
46 5-6
on the growth rates of the treated chickens. In Experiments 5-6 and 7, Parvex capsules and aqueous piperazine hexahydrate had no significant influence on the weight of the treated birds. However, in the piperazine dihydrochloride test, in which the chicks were dosed individually with an aqueous solution, significantly higher average weights were attained by the treated hosts compared with their controls. Throughout the tests the experimental data indicated the difficulty in establishing a quantitative relationship between the number of larvae present in, and the weight gains made by, infected but untreated control birds. As one of several noticeable ex-
of certain piperazine compounds on lumen larvae of A. galli and on the chicken host
No. chickens
18 14 17 4
867
50 50
* Significant, 1-5% level, p.i. = post ova inoculation.
200
100
268.5 248.6
8.6 8.2 9.0 5.7
309.8 294.1
12.2 10.3 6.3 6.1 17.0
2.2*
868
D. E. WORLEY, M. F. HANSEN AND B. R. B. PERSAUD
amples of this phenomenon, a bird ranked third in order of greatest weight gain had the heaviest infection, whereas, the bird which ranked third from the lowest in weight gain harbored only four worms. Similar observations were made by Todd and Hansen (1951), and Egerton and Hansen (19SS). The former authors theorized that chickens supporting the least number of worms failed to gain weight at a normal rate because they expended energy against the parasitic infection. The latter authors, from studies on immunity and tolerance, theorized that chickens able to produce antibodies in large enough quantity and at a rapid rate against metabolic products of parasites would provide a more suitable habitat for parasites. Consequently, these chickens would tolerate a greater number of parasites with less host damage. The effect of the anthelmintics on the numbers of lumen larvae in treated birds is also presented in Table 1. Caricide in the various dosages tested had no effect on the number of lumen larvae. Likewise, Compound 180-C used in Experiment 4 did not affect the number of lumen larvae in the treated birds. Piperazine dihydrochloride and hexahydrate, used in Experiments 5-6 and 7, respectively, significantly reduced the number of lumen larvae in treated birds. Secondary in importance to the efficacy of the piperazine compounds tested were the effects of the drugs on the growth rates of the lumen larvae remaining in the birds after treatment. Caricide at the level used in Experiment 1 had no effect on the growth of lumen larvae. However, in Experiment 2, the lumen larvae recovered from chickens given 12.5 mgm. continuously for 8 days were significantly longer than those from untreated controls. Worms from Group A hosts averaged 1.48 cm. in length, compared with 0.39 cm. in Group B birds.
Group C chickens contained worms averaging 1.0 cm. long, compared with an average of 0.29 cm. for those from Group D chickens. These differences were significant at the 1 percent level. In Experiment 3, the reverse was true. Chickens receiving 12.5 mgm. Caricide continuously in their diet contained worms significantly shorter than those from their controls. Lumen larvae from Group A birds averaged 0.40 cm. in length, compared with an average length of 0.70 cm. in Group B hosts. In Experiment 4, Compound 180-C significantly repressed growth of the larvae, since those from treated birds averaged 1.0 cm. in length, compared with 1.4 cm. in the untreated group, at 21 days post-inoculation. In these experiments the effect of the piperazine compounds on the growth of larvae was conditioned by the chemical type and dosage of the drug. An indirect effect which presumably could result from anthelmintic medication is a modification of the normal tissue stage in the Ascandia life cycle, due to a toxic chemical environment created by the drug in the host's intestine. Since the tissue stage is one of the principal times at which host injury is incurred in Ascaridia infections (Ackert and Herrick, 1928), observations were made on this phase of the host-parasite-drug interrelationship in the three Caricide experiments. The peak of the tissue phase, i.e. the day post-inoculation on which the largest number of worms was recovered from the intestinal mucosa, occurred on day 11 in Experiment 1 for both treated and control groups, on day 13 for both groups in Experiment 2. and on day 13 for both treated and control birds in Experiment 3. Likewise, the end of the tissue phase, as indicated by the day post-inoculation on which the last tissue stage worms were
PIPERAZINE COMPOUNDS AS ANTHELMINTICS
found, appeared to follow the normal time schedule for Ascaridia, as reported by Tugwell and Ackert (1952). They found that the majority of the worms were in the mucosa between the 10th and 17th days after inoculation of the host. The present data show that in Experiment 1, the last mucosa larva was recovered on day IS post-inoculation in the treated birds, and on day 18 in the controls. In Experiment 2, the last larvae were recovered on days 19 and 18, in the treated and control groups, respectively. In Experiment 3, the last tissue larvae were found on day 18 post-inoculation in the treated chicks, and in the controls on day 19. Thus, the presence of Caricide in single or extended dosages appeared to have no appreciable influence in either suppressing or accelerating the life cycle time for development of Ascaridia.
DISCUSSION
A study by Hansen et al. (1954a) revealed that nicotine, antimonyl potassium tartrate, butynorate, phenothiazine, and aureomycin given at therapeutic levels did not remove tissue phase larvae. Only butynorate and a mixture of nicotine and phenothiazine showed any efficacy against lumen larvae. The present study revealed promising action of piperazine dihydrochloride and hexahydrate against lumen larvae. That the results obtained with these two compounds were related to their higher piperazine content is not certain in view of the results obtained with Compound 180-C which has very nearly the same piperazine content as piperazine hexahydrate. Of these two compounds, piperazine dihydrochloride would be more suitable than the hexahydrate because of its non-hygroscopic nature. Its low toxicity, chemical stability, and adaptability for use either in
869
feed or in water make it a desirable anthelmintic. SUMMARY
1. None of the anthelmintics, at the dosages used, retarded the growth of the experimental chickens. In view of the experimental design used, the growth stimulation obtained with Caricide and piperazine dihydrochloride must be accepted with reservation. 2. There was no apparent relationship between host worm burden and host weight gains under the conditions of the experiments. 3. Neither Caricide, Compound 180-C (l-carbethoxy-4-methylpiperazine hydrochloride), nor Parvex significantly reduced the number of lumen larvae of Ascaridia galli. 4. The effects of Caricide and Compound 180-C on the growth of lumen larvae were conditioned by the dosage level of the drugs. 5. Caricide in single or extended dosages did not influence the life cycle time of tissue phase larvae. ACKNOWLEDGMENTS
This work was supported by a grant-inaid from the Lederle Laboratories Division, American Cyanamid Company. REFERENCES Ackert, J. E., 1923. On the habitat of Ascaridia perspicillum (Rud.). J. Parasitol. 10: 101-103. Ackert, J. E., and C. A. Herrick, 1928. Effects of the nematode Ascaridia lineata (Schneider) on growing chickens. J. Parasitol. I S : 1-13. Ackert, J. E., and L. O. Nolf, 1929. New technique for collecting intestinal roundworms. Science, 70: 310-311. Bradley, R. E., 1955. Observations on the anthelmintic effect of piperazine citrate in chickens. Vet. Med. 50: 444-446. Colglazier, M. L., and F. D. Enzie, 1951. Notes on caricide as an anthelmintic for cats and
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D. E. WOELEY, M. F. HANSEN AND B. R. B. PERSAUD
dogs. Proc. Helm. Soc. Wash. 18: 50-52. Egerton, J. R., and M. F. Hansen, 1955. Immunity and tolerance of chickens to the roundworm, Ascaridia galli (Schrank). Exper. Parasitol. 4: 335-350. Gardiner, J. L., and E. E. Wehr, 1950. Selecting experimental groups of chicks by weight. Proc. Helm. Soc. Wash. 17 : 25-26. Guberlet, J., 1924. Notes on the life history of Ascaridia perspicillum (Rud.). Trans. Am. Micro. Soc. 43 : 152-156. Guilhon, J., and P. Groulade, 1951. Action du d'ethylene-diamine sur les ascarides des carnivores. Bull, de FAcad. Veternaire de France. 24:301-303. Hansen, M. F., B. R. B. Persaud and J. E. Ackert, 1954a. Effects of certain anthelmintics and an antibiotic on lumen and tissue phase larvae of Ascaridia galli (Schrank). Poultry Sci. 33: 140-146. Hansen, M. F., L. J. Olsen and J. E. Ackert, 1954b. Improved techniques for culturing and administering ascarid eggs to experimental chicks. Exper. Parasitol. 3 : 464-473. Hewitt, R., W. Wallace, E. White and Y. Subbarow, 1948. The treatment of ascariasis in
dogs with l-diethylcarbamyl-4-methylpiperazine hydrochloride. J. Parasitol. 34: 237-239. Kanegis, L. A., 1948. A new treatment for ascariasis in dogs and cats. J. Amer. Vet. Med. Assoc. 113: 579-581. Riedel, B. B., 1950. The effect of caricide on Ascaridia galli in chickens. Poultry Sci. 29: 394-397. Riedel, B. B., 1951. Group treatment with caricide for ascariasis in poultry. J. Parasitol. 37: 318319. Shumard, R. F., and D. F. Eveleth, 1955. A preliminary report on the anthelmintic action of piperazine citrate on Ascaridia galli and Heterakis gallinae in hens. Vet. Med. 50: 203-205. Todd, A. C , W. M. Insko, Jr., G. W. Kelley and M. F. Hansen, 1949. Aggregate worm infections and growth of broilers. Trans. Am. Micro. Soc. 48: 256-260. Todd, A. C , and M. F. Hansen, 1951. The economic import of host resistance to helminth infection. Am. J. Vet. Res. 12: 58-61. Tugwell, R. L. and J. E. Ackert, 1952. On the tissue phase of the life cycle of the fowl nematode Ascaridia galli (Schrank). J. Parasitol. 38: 277-288.
NEWS AND NOTES (Continued from page 864) of Hawaii, to conduct poultry nutrition research. He graduated from the University of Arizona, and received a M.S. and a Ph.D. degree at Ohio State University in 1952 and 1955 respectively. WORLD'S POULTRY CONGRESS H. H. Alp, Commodity Director, American Farm Bureau Federation, Chicago, Illinois, has been named Chairman of the U.S. Committee for the Eleventh World's Poultry Congress to be held in Mexico City in 1958, probably in August. Other officers are: Vice-Chairman—C. D. Carpenter, President, Institute of American Poultry Industries; Secretary—E. Karpoff, Agricultural Marketing Service, U.S. Department of Agriculture; and Treasurer—G. F. Heuser, Cornell University. Sub-Committee Chairmen are: Exhibits—H. L. Shrader, Senior Extension Poultryman, U.S. Department of Agriculture; Membership—M. C. Small, Executive Secretary, National Turkey Federation; Publicity—A. N. Schwartz, Editor, The Poultryman; Papers—E. M. Funk, University of
Missouri; and Tours—J. W. Kinghorne, Washington. P.E.N.B. NOTES L. H. Geil has been appointed General Manager of the Poultry and Egg National Board, effective June 1, replacing J. M. Gwin who resigned to join the Ralston Purina Company. Geil has been Director of Public Relations of the National Dairy Council since 1948. Prior to 1948 he served for 5 years as Professor of Journalism and Advertising, and, for 6 years, as Director of Public Relations at Michigan State University. He obtained a B.A. degree at North Central College in 1927, and a M.A. degree at Northwestern University in 1934. NEBRASKA NOTES Professor Frank E. Mussehl will retire on July 1, after 40 years of service as Professor and Head of the Poultry Department of the University of Nebraska. He was born in Jefferson, Wisconsin,
(Continued on page 897)
Department of Zoology, Kansas State College, Manhattan, Kansas (Received for publication February 28, 1957)
INTRODUCTION
TNCREASED recognition of the value of •*• certain piperazine compounds as anthelmintics is indicated by their current widespread use in combating nematode parasitism in domestic animals. The first evidence of the effectiveness of a piperazine compound against intestinal helminthiasis was reported by Hewitt et al. (1948). Their work demonstrated efficacy for removal of ascarid infections in dogs. Later, Kanegis (1948) obtained comparable results in both dogs and cats treated for ascariasis. Both papers stated that the test anthelmintics, Hetrazan®* and Caricide,®* approached 100 percent efficiency in worm removal when administered at therapeutic levels. Guilhon and Groulade (1951), working with Toxocara infections in dogs and a cat, found that piperazine treatment effectively reduced fecal egg counts in these hosts. Colglazier and Enzie (1951) reported that Caricide removed 98 percent of the ascarids in a group of treated cats. It has been reported that piperazine citrate was of value for field use in the treatment of chickens infected with adult Ascaridia galli (Shumard and Eveleth, 1955; Bradley, 1955). In a preliminary study, Riedel (1950) reported erratic results with 1
Contribution No. 272 from the Kansas Agricultural Experiment Station in the Department of Zoology and No. 314 from the Department of Zoology, Kansas State College. * Diethylcarbamazine, American Cyanamid Company.
Caricide in chickens, but in a later study (1951) he obtained up to 81 percent efficacy when high concentrations of the drug were incorporated in the birds' feed. These successful trials with various piperazines against several species of adult ascarids suggested their possible potential value as anthelmintics against larval stages of nematodes. The need for such a drug for use in poultry has been pointed out by the experimental work of Ackert (1923), Guberlet (1924), Ackert and Herrick (1928), and Todd et al. (1949), among others. These studies convincingly demonstrated the pre-eminent role of the early stages of Ascaridia in causing host injury during the time when they inhabit the hosts' intestinal crypts. Hansen et al. (1954a) indicated that several compounds, including nicotine, formerly the drug of choice for removal of adult ascarids in chickens, were almost totally ineffective in combatting the larval stages of this parasite. The objective of the present study was to determine the activity of several of the more promising piperazine compounds against the immature stages of Ascaridia galli in the domestic fowl. MATERIALS AND METHODS
The five compounds tested in this study and the percentage of piperazine content were Caricide (l-diethylcarbamyl-4-methylpiperaxine dihydrogen citrate, 22%), Compound 180-C (l-carbethoxy-4-methylpiperazine hydrochloride, 41.2%), piperazine
865
866
D. E. WOELEY, M. F. HANSEN AND B. R. B. PERSAUD
dihydrochloride (52.1%), Parvex®* (betaine of 1-piperazine carbodithioic acid, 53%), and piperazine hexahydrate (44%). A total of seven tests were conducted, using White Plymouth Rock and New Hampshire chickens as experimental hosts. These were obtained as non-sexed, day-old chicks from a commercial hatchery and were fed a standard antibiotic-free commercial feed containing 18 percent protein. They were maintained in wire-floored batteries throughout the experiments. All of the birds used in the trials were infected per os at 14 days of age with either 75 ± 5 or 100 ± 10 embryonated Ascaridia galli ova. The preparation of a standardized suspension of ova for administration to the chickens was patterned after the sugar solution technique described by Hansen et al. (1954b). After infection, two types of experimental procedures were followed. In the first three tests, two treated and two untreated birds from Groups A and B, respectively, were sacrificed and examined each day for 10 days, beginning 11 days post inoculation. This procedure was designed to test the action of the anthelmintic during the progression of the tissue phase of the Ascaridia life cycle. According to Tugwell and Ackert (1952), the peak invasion of the crypts of the intestine occurs about 14 days after feeding embryonated ova, and continues until about the 21st day post-inoculation. To furnish evidence of the activity of the drug toward lumen larvae, the period of larval life following the tissue phase, an entire group of treated chicks (Group C) and an equal number of control birds (Group D) were killed and examined 21 days after inoculation. In Experiments 4, 5, 6, and 7, only Groups C and D were used. In Experiment 5-6, 2 groups received treatment with piper* The Upjohn Company.
azine dihydrochloride and Parvex, respectively, and are designated as Groups Ct and C2, respectively, in Table 1. Group D served as a common control for both medicated groups in this test. The flushing technique of Ackert and Nolf (1929) was used to recover worms present in the intestinal lumen of birds at autopsy. In Experiments 1,2, and 3 a count was also made of the tissue phase larvae. These imbedded larvae were recovered from the intestinal crypts by artificially digesting in a solution of 0.5% HC1 and 1.0% pepsin the portion of the small intestine from the gizzard to a point slightly posterior to the yolk sac diverticulum of each bird. In addition to enumerating the number of tissue phase and lumen larvae present in each bird, the length of the latter larvae from each group of birds was measured with the aid of a microprojection apparatus. All experimental results were evaluated statistically, using the t-test for group comparisons. If the value of "t" obtained by this technique was at, or close to a significant level, a modified t-test using a squareroot transformation was also computed, as an additional means of confirming the test hypothesis. RESULTS The effects of the 5 piperazine compounds on the growth of the fowl host are recorded in Table 1. A statistical comparison of the mean weights at autopsy of the treated chickens and their controls was used as a criterion of the effect of the anthelmintics on the host. Such a comparison is valid because the average weights of the two groups were equalized at the start of each experiment (Gardiner and Wehr, 1950). In neither of the first two Caricide tests, in which the drug was given by capsule,
PIPERAZINE COMPOUNDS AS ANTHELMINTICS
was any significant effect noted on the growth rates of the chickens. In Experiment 3 a continuous low-level dosage of Caricide, added as a powder to the feed, was accompanied by significantly greater weight gains in the treated group. Inasmuch as there was no significant difference between worm burdens of chicks in this experiment, some direct or indirect action of the drug as a growth stimulant may have occurred. In a replication of this experiment at a later date, comparable results were not obtained, indicating that these results should perhaps be regarded with reservation. In Experiment 4, Compound 180-C in aqueous solution had no significant effect TABLE 1.—Effect
Experiment No.
1
2
3
Av. No. lumen larvae
A. B. C. D.
Caricide 25 mgm. (Day 10 p.i.) Control Caricide 25 mgm. (Day 10 p.i.) Control
200
20 20 22 22
A. B. C. D.
Caricide 12.5 mgm. (Days 4-11 p.i.) Control Caricide 12.5 mgm. (Days 4-11 p.i.) Control
100
18
A. Caricide 12.5 mgm. (Continuous from hatching until slaughter) B. Control C. Caricide 12.5 mgm. (Continuous) D. Control
100 100
220.8* 188.0
13.1 7.3 17.1
C. Compound 180-C 25 mgm. (Days 10-17 p.i.) D. Control
200
236.1
23.5
247.8
15.1
Cj. Piperazine dihydrochloride 25 mgm. (Days 7-14 p.i.) C2. Parvex 30 mgm. (Day 11 p.i.) D. Control
200
259.5*
200
235.9 246.4
14.1 13.5
C. Piperazine hexahydrate 25 mgm. (Days 7-15 p.i.) D. Control
200
317.1
2.66*
316.2
7.59
46
52 52 54
7
Group treatment (Mgm./day)
Approx. drug dosage Av. Wt. of (mgm. chickens total at autopsy cmpd./kilo. (gms.) body wt.)
19 19 17 17
46 5-6
on the growth rates of the treated chickens. In Experiments 5-6 and 7, Parvex capsules and aqueous piperazine hexahydrate had no significant influence on the weight of the treated birds. However, in the piperazine dihydrochloride test, in which the chicks were dosed individually with an aqueous solution, significantly higher average weights were attained by the treated hosts compared with their controls. Throughout the tests the experimental data indicated the difficulty in establishing a quantitative relationship between the number of larvae present in, and the weight gains made by, infected but untreated control birds. As one of several noticeable ex-
of certain piperazine compounds on lumen larvae of A. galli and on the chicken host
No. chickens
18 14 17 4
867
50 50
* Significant, 1-5% level, p.i. = post ova inoculation.
200
100
268.5 248.6
8.6 8.2 9.0 5.7
309.8 294.1
12.2 10.3 6.3 6.1 17.0
2.2*
868
D. E. WORLEY, M. F. HANSEN AND B. R. B. PERSAUD
amples of this phenomenon, a bird ranked third in order of greatest weight gain had the heaviest infection, whereas, the bird which ranked third from the lowest in weight gain harbored only four worms. Similar observations were made by Todd and Hansen (1951), and Egerton and Hansen (19SS). The former authors theorized that chickens supporting the least number of worms failed to gain weight at a normal rate because they expended energy against the parasitic infection. The latter authors, from studies on immunity and tolerance, theorized that chickens able to produce antibodies in large enough quantity and at a rapid rate against metabolic products of parasites would provide a more suitable habitat for parasites. Consequently, these chickens would tolerate a greater number of parasites with less host damage. The effect of the anthelmintics on the numbers of lumen larvae in treated birds is also presented in Table 1. Caricide in the various dosages tested had no effect on the number of lumen larvae. Likewise, Compound 180-C used in Experiment 4 did not affect the number of lumen larvae in the treated birds. Piperazine dihydrochloride and hexahydrate, used in Experiments 5-6 and 7, respectively, significantly reduced the number of lumen larvae in treated birds. Secondary in importance to the efficacy of the piperazine compounds tested were the effects of the drugs on the growth rates of the lumen larvae remaining in the birds after treatment. Caricide at the level used in Experiment 1 had no effect on the growth of lumen larvae. However, in Experiment 2, the lumen larvae recovered from chickens given 12.5 mgm. continuously for 8 days were significantly longer than those from untreated controls. Worms from Group A hosts averaged 1.48 cm. in length, compared with 0.39 cm. in Group B birds.
Group C chickens contained worms averaging 1.0 cm. long, compared with an average of 0.29 cm. for those from Group D chickens. These differences were significant at the 1 percent level. In Experiment 3, the reverse was true. Chickens receiving 12.5 mgm. Caricide continuously in their diet contained worms significantly shorter than those from their controls. Lumen larvae from Group A birds averaged 0.40 cm. in length, compared with an average length of 0.70 cm. in Group B hosts. In Experiment 4, Compound 180-C significantly repressed growth of the larvae, since those from treated birds averaged 1.0 cm. in length, compared with 1.4 cm. in the untreated group, at 21 days post-inoculation. In these experiments the effect of the piperazine compounds on the growth of larvae was conditioned by the chemical type and dosage of the drug. An indirect effect which presumably could result from anthelmintic medication is a modification of the normal tissue stage in the Ascandia life cycle, due to a toxic chemical environment created by the drug in the host's intestine. Since the tissue stage is one of the principal times at which host injury is incurred in Ascaridia infections (Ackert and Herrick, 1928), observations were made on this phase of the host-parasite-drug interrelationship in the three Caricide experiments. The peak of the tissue phase, i.e. the day post-inoculation on which the largest number of worms was recovered from the intestinal mucosa, occurred on day 11 in Experiment 1 for both treated and control groups, on day 13 for both groups in Experiment 2. and on day 13 for both treated and control birds in Experiment 3. Likewise, the end of the tissue phase, as indicated by the day post-inoculation on which the last tissue stage worms were
PIPERAZINE COMPOUNDS AS ANTHELMINTICS
found, appeared to follow the normal time schedule for Ascaridia, as reported by Tugwell and Ackert (1952). They found that the majority of the worms were in the mucosa between the 10th and 17th days after inoculation of the host. The present data show that in Experiment 1, the last mucosa larva was recovered on day IS post-inoculation in the treated birds, and on day 18 in the controls. In Experiment 2, the last larvae were recovered on days 19 and 18, in the treated and control groups, respectively. In Experiment 3, the last tissue larvae were found on day 18 post-inoculation in the treated chicks, and in the controls on day 19. Thus, the presence of Caricide in single or extended dosages appeared to have no appreciable influence in either suppressing or accelerating the life cycle time for development of Ascaridia.
DISCUSSION
A study by Hansen et al. (1954a) revealed that nicotine, antimonyl potassium tartrate, butynorate, phenothiazine, and aureomycin given at therapeutic levels did not remove tissue phase larvae. Only butynorate and a mixture of nicotine and phenothiazine showed any efficacy against lumen larvae. The present study revealed promising action of piperazine dihydrochloride and hexahydrate against lumen larvae. That the results obtained with these two compounds were related to their higher piperazine content is not certain in view of the results obtained with Compound 180-C which has very nearly the same piperazine content as piperazine hexahydrate. Of these two compounds, piperazine dihydrochloride would be more suitable than the hexahydrate because of its non-hygroscopic nature. Its low toxicity, chemical stability, and adaptability for use either in
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feed or in water make it a desirable anthelmintic. SUMMARY
1. None of the anthelmintics, at the dosages used, retarded the growth of the experimental chickens. In view of the experimental design used, the growth stimulation obtained with Caricide and piperazine dihydrochloride must be accepted with reservation. 2. There was no apparent relationship between host worm burden and host weight gains under the conditions of the experiments. 3. Neither Caricide, Compound 180-C (l-carbethoxy-4-methylpiperazine hydrochloride), nor Parvex significantly reduced the number of lumen larvae of Ascaridia galli. 4. The effects of Caricide and Compound 180-C on the growth of lumen larvae were conditioned by the dosage level of the drugs. 5. Caricide in single or extended dosages did not influence the life cycle time of tissue phase larvae. ACKNOWLEDGMENTS
This work was supported by a grant-inaid from the Lederle Laboratories Division, American Cyanamid Company. REFERENCES Ackert, J. E., 1923. On the habitat of Ascaridia perspicillum (Rud.). J. Parasitol. 10: 101-103. Ackert, J. E., and C. A. Herrick, 1928. Effects of the nematode Ascaridia lineata (Schneider) on growing chickens. J. Parasitol. I S : 1-13. Ackert, J. E., and L. O. Nolf, 1929. New technique for collecting intestinal roundworms. Science, 70: 310-311. Bradley, R. E., 1955. Observations on the anthelmintic effect of piperazine citrate in chickens. Vet. Med. 50: 444-446. Colglazier, M. L., and F. D. Enzie, 1951. Notes on caricide as an anthelmintic for cats and
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dogs. Proc. Helm. Soc. Wash. 18: 50-52. Egerton, J. R., and M. F. Hansen, 1955. Immunity and tolerance of chickens to the roundworm, Ascaridia galli (Schrank). Exper. Parasitol. 4: 335-350. Gardiner, J. L., and E. E. Wehr, 1950. Selecting experimental groups of chicks by weight. Proc. Helm. Soc. Wash. 17 : 25-26. Guberlet, J., 1924. Notes on the life history of Ascaridia perspicillum (Rud.). Trans. Am. Micro. Soc. 43 : 152-156. Guilhon, J., and P. Groulade, 1951. Action du d'ethylene-diamine sur les ascarides des carnivores. Bull, de FAcad. Veternaire de France. 24:301-303. Hansen, M. F., B. R. B. Persaud and J. E. Ackert, 1954a. Effects of certain anthelmintics and an antibiotic on lumen and tissue phase larvae of Ascaridia galli (Schrank). Poultry Sci. 33: 140-146. Hansen, M. F., L. J. Olsen and J. E. Ackert, 1954b. Improved techniques for culturing and administering ascarid eggs to experimental chicks. Exper. Parasitol. 3 : 464-473. Hewitt, R., W. Wallace, E. White and Y. Subbarow, 1948. The treatment of ascariasis in
dogs with l-diethylcarbamyl-4-methylpiperazine hydrochloride. J. Parasitol. 34: 237-239. Kanegis, L. A., 1948. A new treatment for ascariasis in dogs and cats. J. Amer. Vet. Med. Assoc. 113: 579-581. Riedel, B. B., 1950. The effect of caricide on Ascaridia galli in chickens. Poultry Sci. 29: 394-397. Riedel, B. B., 1951. Group treatment with caricide for ascariasis in poultry. J. Parasitol. 37: 318319. Shumard, R. F., and D. F. Eveleth, 1955. A preliminary report on the anthelmintic action of piperazine citrate on Ascaridia galli and Heterakis gallinae in hens. Vet. Med. 50: 203-205. Todd, A. C , W. M. Insko, Jr., G. W. Kelley and M. F. Hansen, 1949. Aggregate worm infections and growth of broilers. Trans. Am. Micro. Soc. 48: 256-260. Todd, A. C , and M. F. Hansen, 1951. The economic import of host resistance to helminth infection. Am. J. Vet. Res. 12: 58-61. Tugwell, R. L. and J. E. Ackert, 1952. On the tissue phase of the life cycle of the fowl nematode Ascaridia galli (Schrank). J. Parasitol. 38: 277-288.
NEWS AND NOTES (Continued from page 864) of Hawaii, to conduct poultry nutrition research. He graduated from the University of Arizona, and received a M.S. and a Ph.D. degree at Ohio State University in 1952 and 1955 respectively. WORLD'S POULTRY CONGRESS H. H. Alp, Commodity Director, American Farm Bureau Federation, Chicago, Illinois, has been named Chairman of the U.S. Committee for the Eleventh World's Poultry Congress to be held in Mexico City in 1958, probably in August. Other officers are: Vice-Chairman—C. D. Carpenter, President, Institute of American Poultry Industries; Secretary—E. Karpoff, Agricultural Marketing Service, U.S. Department of Agriculture; and Treasurer—G. F. Heuser, Cornell University. Sub-Committee Chairmen are: Exhibits—H. L. Shrader, Senior Extension Poultryman, U.S. Department of Agriculture; Membership—M. C. Small, Executive Secretary, National Turkey Federation; Publicity—A. N. Schwartz, Editor, The Poultryman; Papers—E. M. Funk, University of
Missouri; and Tours—J. W. Kinghorne, Washington. P.E.N.B. NOTES L. H. Geil has been appointed General Manager of the Poultry and Egg National Board, effective June 1, replacing J. M. Gwin who resigned to join the Ralston Purina Company. Geil has been Director of Public Relations of the National Dairy Council since 1948. Prior to 1948 he served for 5 years as Professor of Journalism and Advertising, and, for 6 years, as Director of Public Relations at Michigan State University. He obtained a B.A. degree at North Central College in 1927, and a M.A. degree at Northwestern University in 1934. NEBRASKA NOTES Professor Frank E. Mussehl will retire on July 1, after 40 years of service as Professor and Head of the Poultry Department of the University of Nebraska. He was born in Jefferson, Wisconsin,
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