A study of the activity of chemotherapeutic agents on infections of Syphacia oblevata and Aspiculuris tetraptera

A study of the activity of chemotherapeutic agents on infections of Syphacia oblevata and Aspiculuris tetraptera

A Study Agents of the Activity of Chemotherapeutic on Infections of Syphacia oblevata and Aspiculuris tetrapteral H. W. Brown, Culumbia University ...

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A Study Agents

of the Activity of Chemotherapeutic on Infections of Syphacia oblevata and Aspiculuris tetrapteral

H. W. Brown, Culumbia

University

K. F. Chan, and B. D. Ferrell School of Public

(Submitted

for publication,

Health,

New York, IVew York

18 March 1953)

The search for new, effective non-toxic drugs for the treatment of human enterobiasis has led to the use of various related oxyurids as test organisms. Wells (1951) tried several antibiotics against Aspiculuris tetraptera of the mouse, which led to the discovery of the efficacy of oxytetracycline against Enterobius vermicularis in man (Wells et al., 1951; Loughlin et al., 1951). Chan (1952a) found that a number of the antibiotics and at least one poorly absorbable sulfonamide are active against Syphacia obvelata in mice. Wells (1952)) Chan (1952b) and Hsieh (1952a) worked out the details of the life cycle of these two worms, making it possible to give mice experimental infections against which various chemotherapeutic agents could be evaluated. The life cycle of Syphacia is completed in 11 to 15 days with migration of the worms out of the host. Aspiculuris reaches maturity in approximately 25 days and has a longevity of 45 to 50 days; it therefore lends itself to long continued chemotherapy much better tha,n does Syphacia. However, the technique of infecting mice with Syphacia is much simpler than that used with Aspicduris. The present study was designed to ascertain the comparative usefulness of these two parasites for screening new drugs against human enterobiasis. MATERXALS AND METHODS The mice used in this study were from a helminth-free colony. The eggs of A. telraptera were obtained from gravid 29 to 33-day-old female worms and they were incubated in Syracuse watch dishes in distilled water at 27°C for 8 days. A uniform suspension of the eggs was made by the addition of a small amount of gum traga1 The authors wish to thank Burroughs Wellcome and Co.; Commercial Solvents Co.; Dr. Hess and Clark, Inc.; Chas. Pfizer; Sharp and Dohme, for supplying chemotherapeutic agents used in this study. 45

canth to the culture liquid and the dosage was determined by a dilution counting method in which only completely embryonated eggs were counted. Three-week-old mice were infected with APJJ~‘CIIIZI~I’S by atlminister~ng approai matjely 500 fully embryonated A. tclrapt~n eggs through a slomach 1ubr. Thcrcafter, thesr mice were kept, in a. room frrc of S. ohr,c/rrln-illfcc,t(~~ianimals. Chemotherapy was instituted on the fourtcent h day of infection \vith those drugs given for 3 days, and on the fifteenth day of the infection bvit.11drugs given for 2 days. These mice were autopsied on the eighteent,h day of infection. The large intestine was opened in zinc sulfate solution (spgr. 1.18) and the worms present counted under a dissecting microscope. Control auimals similarl~~ infected were autopsied on the same day, the number of Aspiculwis aspertained and the cecum esamined to be sure no S. obvelata were present. Three-week-old mice from the helminth-free colony were infected with S. obvelata by placing them with infected mice for 7 days following the method developed by Chan. On that day the newly infected mice were removed from the coloq and treatment initiated. (With drugs given for 2 days, treatment was initiated after 8 days.) A4t autopsy 2 days after the completion of t,reatment, the cecum of each mouse was opened in a shallow dish containing the zinc sulfate solution, and the number of worms exclusive of reinfection-larvae (0.6 mm or less in length) counted under a dissecting microscope. The large intestines of these mice were examined for the presence of Aspicuhis. The mixed infections were obtained by giving 500 fully embryonated Aspiculuris eggs to 3-week-old mice and on the seventh day of this infection the animals were placed with Suphacia-infected mice for 7 days. The drugs were then tested against 13, l-1 and Sday-old Aspic?tluris and a concurrent infection of all stages of &phacia up to i, 8, and 9 days of age. Tetrachlorethyleue and bacitracin were given for only 2 days and therefore were tested against 1-l and 15-day-old Aspicdun’s and stages up to 8 and g-day-old Syphacia infections. At autopsy the cecum and large intestine from each mouse were placed in separate dishes and examined for pinworms. The average number of worms in the cont,rol mice was compared to the average number in treated mice and the reduction reported on a percentage basis. The drugs used in this study were: methylrosaniline2, tetrachlorethylene, phenothiazine, benzylphenyl carbamate 3, thym)-1 in-iso:lm~lcarbanInte4, hexylbacitracin, and a comt)inat,ion of Itacitracin and resorcinol, oxytetracycline5, sulfaguanidine. These drugs were all given by stomach tube in relatively large amounts, 5 to 15 times the human dose, for 2 or 3 consecutive days. .I11 mice were autopsied 2 days following the completion of treat,ment. The results obtained were examined for statistical significance by the use of the t-test,. Reduction in worm burden was considered significant when the probability was 1% or less, of borderline significance when the probability was 1 to 5oJ,, and not significant when the probability exceeded 5%. * Methylrosaniline chloride, gentian violet. 3 p-Benzylphenyl carbamate, diphenan, butolan. 4 Thymyl N-isoamylcarbamate, egressin. 6 Osytetracycline, terramycin.

CHEMOTHERAPEUTIC

47

AGENTS ON MOUSE PINWORMS

TABLE I The Effect of Chemotherapeutic Agents against S. obvelata and A. tetraptera Single Infections

I-

Syphacia Chemotherapeutic agent

No. of mice

AverageNo. worms

Aspiculuris

No. of AverageNo. worms mice

Treated % ReTreated ‘rated,I ” lice/Contr< % 1‘reated mice/ConControl mice 31 Ikduction ,Controi trol mice Luction -Methylrosaniline 35 mg/kg/ 3 days.................... Tetrachlorethylene 1631mg/ kg/2 days. Phenothiazine 500 mg/kg/3 days. . Benzylphenyl carbamate 500 mg/kg/3 days. Thymyl N-isoamylcarbamate 500 mg/kg/3 days. Hexylresorcinol 100 mg/kg/ 3 days.................... Oxytetracycline 1000 mg/kg/ 3 days.. Bacitrscin 50,000u/kg/2 days Bacitracin 10,000u/kg & Sulfaguanidine 1 gm/kg/3 days......................

15/11

77/133

42a

15/16

89/201

56=

15/9

49/165

70”

15/19

178/171

0

15/s

97/173

44”

14/10

139/a 1

35”

10/9

174/164

0

10/12

187/219

15

10/a

153/181

15

10/7

204/227

10

10/8

133/140

5

10/7

210/227

10/7 U/10

1.5/162 2.2/154

>9ga 99”

18/16 18/16

92/193 125/183

53” 32

IO/5


>99”

lO/ll

139/200

30

7.4

a Indicatesstatistical significance. * Indicatesborderlinestatisticalsignificance. RESULTS

The data on the effectiveness of commonly used pinworm chemotherapeutic agents are presented in Table I. Each animal in this experimental group harbored an experimental infection of either Syphacia or Aspiculurk It will be noted that relatively massive doses of oxytetracycline, bacitracin, and bacitracin-sulfaguanidine mixture were highly effective against Syphacia. Methylrosaniline, tetrachlorethylene, and phenothiazine were less effective and benzylphenyl carbamate, thymyl N-isoamylcarbamate and hexylresorcinol showed little or no activity in the dosages employed. Except for methylrosaniline, none of the compounds was as active against Aspiculuris as against Syphacia.

48

II. W. BROWN,

K. F. CH.W,

.\ND

B. D. FERRELL

Aspiculuris appears to be somewhat more difficult to remove from mice than Syphacia, which may be due to an inherent resistance of Aspiculuris or due to its habitat throughout the large intestine. Syphacia concentrated in the cecum may be exposed to higher drug concentration. Most of the mice that are found naturally infected with pinworms harbor both rlspicduris and Syphacin in varying numbers. Inasmuch as our original studies were made on naturally infected mice and since a number of investigators at present are using naturally acquired double infections, we felt that it was important to ascertain whether these mixed infections responded to chemotherapeutic agents in a similar manner to single infections. We therefore infected mice with known numbers of both Aspiculuris and Syphacia and treated them with some of the standard drugs. Table II gives the results of these studies. It will be noted that in general the results of chemotherapy against the double infections were quantitatively similar to the results obtained in the treatment of single infections. Several of the drugs seemed to be slightly less effective against Syphacia in mixed infections than in single infections. However, it is apparent from our studies that treatment of mixed infections is a satisfactory method of assessing chemotherapeutic activity. Several investigators (Thompson and Reinertson, 1952) have assessed the activity of chemotherapeutic agents against pinworm in mice by TABLE The Effect of Chemotherapeutic

II

Agents against Mixed and A. tetraptera

Chemotherapeuticagent

No. of mice treated/ I ---__

Infections

Syphacin

of S. obvelata

Aspiculuria

%

Treated Reduction ~ Contra{ Methylrosaniline 35 mg/kg/3 days. Tetrachlorethylene 1631 mg/kg/2 days. .._.......... Phenothiazine 500 mg/kg/3 days Oxytetracycline 1000 mg/kg/3 days Bacitracin 50,000 u/kg/a days Hacitracin 10,000 u/kg & Sulfaguanidine 1 gm/kg/3 days.

%

Reduction

IO/10

106/168

37”

130/262

50”

12/10 19/10 17/14 13/x

115/168 116/204 24/180 I 9/182

31 43” 86a 95”

190/206 X4/278 71/175 61/221

7.3 45” 60a 73a

58/169

66”

l&4/241

40”

li/lJ,

CHEMOTHER~4PEUTIC

AGENTS

TABLE

MOUSE

49

PIWVORMB

III

and Per cent of Mice Cleared of Syphacia and in Single Infections Compared to the Efectiveness of the Same Agents against Enterobius in Man

The Worm Burden

Aspiculuris

OX

Reduction

T Chemotherapeutic

agent

-

Methylrosaniline 35 mg/kg/3 days Tetrachlorethylene 1631mg/kg/2 days. Phenothiazine 500 mg/kg/3 days Benzylphenyl carbamate 500 mg/ kg/3 days.. Thymyl N-isoamylcarbamate 500 mg/kg/3 days. Hexylresorcinol 100 mg/kg/3 days Osytetracycline 1 gm/kg/3 days. Bacitracin 50,000u/kg/2 days.. . . Bacitracin, 10,000u/kg & Sulfaguanidine, 1 gm/kg/3 days.

Worm

WOIltl b,~~~;i t104, % ~~~

-

Aspiculuris

Syphacia

ZiZ cleared

br::::” tion, %

% of lZlice cleared

Enterobius % of Patients cured by standard course of treatment

42

6.6

-156

0

60-91

70 44

6.6 0

0 35

0 0

47-55 60-82

0

15

0

1044

15 5 >99 99

0 0 90 72.6

10 7.4 53 32

0 0 5.6 0

30-40 0 85-100 40

>99

90

30

0

0

ascertaining the number of mice that were completely cleared of their infections by treatment. Tables III and IV give our data comparing the worm burden reduction to the percentage of mice cleared of worms. It will be seen that in general, although most of the compounds effect a reduction in worm burden, relatively few of the mice are completely cleared of their pinworms. For example, methylrosaniline reduced the Syphacia worm burden 42 % and cleared completely only 6.6 % of the mice. Similarly, this drug reduced the Aspiculuris worm burden 56% but did not eliminate all of the worms from a single mouse. A study of Tables III and IV suggests that a chemotherapeutic testing method against pinworm which relies on complete cure of mice is not as satisfactory as the method depending on worm burden reduction. Such agents as oxytetracycline, which eliminates 99 y0 of the worm burden, likewise clears as high as 90 Y0 of the mice, and with compounds like this either method of assay would be useful. On the other hand, such standard compounds as methylrosaniline, tetrachlorethylene and phenothiazine would probably not be correctly assayed by the test involving complete clearance of mice of pinworms. Hsieh’s (1952b) summary of the activity

50

H. W. BRONX,

Ii.

F. CHAIY, AND B. D. FERRELL

TABLE Th.e Worm Burden

IV

and Per cent of Mice Cleurcd o.f Syphacin. nun! Aspiculuris in Mixed Injections

Reduction

worm % of burden Mice reduction, 7% cleared

Chemotherapeuticagent

Methylrosaniline 35 mg/kg/3 days. Tetrachlorethylene 1631 mg/kg/2 days. Phenothiazine 500 mg/kg/3 days.. Oxytetracycline 1 gm/kg/3 days. Bacitracin 50,000 u/kg/2 days.. Bacitracin 10,000 u/kg & Sulfaguanidine 1 gm/kg/3 days..

~

i ~ 1

37 31 13 86 95

0 0 0 59 54

50 ~ 7.’J ~ 41 60 is

0 0 0 5.!) 54

66

35 -~

40

0

of these agents against Enterobius in man indicates they are active, yet on the basis of mice cleared of all worms they appear to be relatively inactive. Our results indicate that both S~~phacia and Aspiculuris are satisfactory helminths for the screening of new chemotherapeutic agent’s. Both of these worms appear to be somewhat more resistant than Bnterobius vermicularis to massive doses of the standard pinworm chemotherapeutic agents and hence drugs found active against them would probably be likewise active against the human pinworm. SUMMARY Syphacia obvelata and Rspiculuris tetraptera, the pinworms of mice, respond to the anthelmintics commonly used against the pinworm in man. Although ~lspiculuris appears to be more resistant to several of the anthelmintics than Xyphacia, both worms appear to be useful in rhemotherapeutic tests. Our data indicate that the reduction in the worm burden of infected mice is more sensitive than tests relying on the romplete elimination of all worms from the mice. REFERENCES GHAN, K. F. 1952a. Chemotherapeutic studies on Syphacia obvelata infection in mice. Am. J. Hyg. 66, 22-30. CHAN, K. F. 1952b. Life cycle studies on the nematode Syphncia obvelata. Anz. J. Hyg. 66, 14-21.

CHEMOTHERhPEUTIC

AGENTS

ON

HSIEH, K. N. 1952a. The life cycle of Aspicuhis

MOUSE

PINWORMS

51

letraptera and its relationship to chemotherapeutic agents. Doctorate thesis, Columbia University. HSIEH, K. N. 1952b. The effect of the standard pinworm chemotherapeutic agents on the mouse-pinworm Aspiculuris tetraptera. Am. J. Hug. 66, 287-293. LOUGHLIN, E. H., RAPPAPORT, I., MULLIN, W. G., WELLS, H. S., JOSEPH, A. A., AND SHOOKIIOFF, H. B. 1951. The treatment of enterobiasis with terramycin base. Antibiotics and Chemofherapg 1, 588-593. THOUPSON, P. E., AND REINERTSON, J. W. 1952. Chemotherapeutic studies of natural pinworm infections in mice. Exptl. Parasitol. 1, 384391. WELLS, H. 1951. Studies of the effect of antibiotics on infections with the mousepinworm Aspiculuris tetraptera. I. The action of terramycin hydrochloride. J. Infectious Diseases 89, 190-192. WELLS, H. S., SHOOKHOFF, H. B., MULLIN, W. G., STERMAN, M. M., LOUGHLIN E. H., AND RAPPAPORT, I. 1951. Terramycin HCl in the treatment of human pinworm infections. Antibiotics and Chemotherapy 1, 299-304. WELLS, H. 1952. Studies on the effect of antibiotics on infection with the mouscpinworm Aspiculuris tetraptera. Doctorate thesis, Columbia University.