A comparison of the susceptibility to niridazole of two geographical strains of Schistosoma mansoni in mice with a note on the susceptibility of S. mattheei

169 TRANSACTIONSOF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. Vol. 65. No. 2. 1971.

A C O M P A R I S O N OF T H E S U S C E P T I B I L I T Y TO N I R I D A Z O L E OF T W O G E O G R A P H I C A L S T R A I N S OF SCHISTOSOMA MANSONI I N M I C E W I T H A N O T E O N T H E S U S C E P T I B I L I T Y O F S. IVIATTHEEI. M. G. TAYLOR AND G. S. NELSON Medical Helminthology Department, London School of Hygiene and Tropical Medicine Introduction There is experimental and clinical evidence that different strains of Schistosoma mansoni react differently to a variety of drugs, (GONNERT and VOGEL, 1955; TI-IOtCd'SON et al., 1965; ABDALLAHand SAIF, 1969; JORDAN, 1969). It has also been shown that the Japanese strain of S. japonicum in mice is much more resistant to antimonial drugs than the other strains (Hsu et al., 1963). In the present experimental study in mice a Tanzanian strain of S. mansoni was found to be more resistant to niridazole than a Puerto Rican strain. This is in agreement with the clinical observations and is further evidence of strain variation in S. mansoni. Gross differences in susceptibility to drugs occur between different species of schistosomes. In man, niridazole and other drugs are more effective against S. haematobium than against S. mansoni, and S. japonicum infections are particularly difficult to cure. This is so for niridazole in spite of the fact that the concentration of the unmetabolised compound (the active agent) is five times higher in the portal blood than in the peripheral c i r ~ a t i o n (FAIGLE and KEBERLE, 1969). It has been suggested that this apparent difference in the susceptibility of the parasites is really due to a "lung shift" of S. haematobium (FoRSYTH, 1965). According to this theory the worms are paralysed by the drug and swept to the lung capillaries and survivors are unable to return to the vesical veins. In S. rnansoni and S. japonicum, however, an "hepatic shift" is thought to occur; the surviving worms may regain the mesenteric veins after treatment has stopped, producing a "relapse." An alternative explanation for the difference in susceptibility is that genetic differences between the different species of worms are responsible. The present results which show that S. mattheei, which has a mesenteric site in the mouse is much more susceptible to niridazole than S. mansoni, favour the latter theory. It has often been suggested that the size of the worm burden affects the outcome of chemotherapy: it is claimed that heavily infected patients are harder to cure than lightly infected ones. (JORDAN, 1969) and GONNERT and VOGEL (1955) produced experimental evidence that lighter worm burdens of S. mansoni respond better to Miracil D. However, some clinical evidence is contradictory and in other experimental studies (LUTTERMOSER and DE WITT, 1961) the size of the worm burden did not affect the outcome of stibophen treatment of S. mansoniinfections in mice. In the present studies, the responses of light and heavy infections of the Puerto Rican strain of S. mansoni to niridazole were compared: it was found that it was the heavy infections which were slightly more responsive to the drug. M a t e r i a l s and m e t h o d s The Puerto Rican strain of S. mansoni was originally isolated from a Puerto Rican patient and since 1962 has been maintained in London in an albino strain of Biomphalaria glabrata from Puerto Rico and white mice.

170

A C O M P A R I S O N O F T H E S U S C E P T I B I L I T Y T O N I R I D A Z O L E OF T W O S T R A I N S OF S. M A N S O N I

The Tanzanian strain of S. mansoni was isolated at Mwanza in 1965 and has been maintained there subsequently in B. sudanica and white mice. Infected snails were sent to London in 1968 by Dr. F. McCullough and cercariae shed by these snails were used for the present work. Both strains of S. mansoni were thus thoroughly "mouse adapted". A South African strain of S. mattheei originating from Nelspruit has been maintained in Bulinus globosus from Nelspruit and hamsters at the School since 1965. The techniques used for mouse infection and for recovery of the adult worms by perfusion have been described by NELSON et al. (1968). The drug chosen for this work was niridazole (Ambilhar, CIBA) which was first shown to be effective against S. mansoni infections in mice by LAMBERT(1964). The drug was given orally by stomach tube to mice with mature infections on consecutive days and was prepared by homogenising the powdered compound in a suspension of 1% NaC1 with 0.1% "Tween 80" detergent, as described by LAMBERT (1964). Every mouse was weighed before each treatment and the exact amount of the drug solution was administered each time. The effectiveness of the treatment schedules was assessed at autopsy 10-13 days after the final treatment; worms which had survived treatment were recovered by perfusion, the dead worms by this time having been fixed in the liver by connective tissue sheaths (STRIEBEL,1969). To assess the effect of treatment on the eggs, the livers of all the mice in each group were pooled and an egg hatching test was done using the technique described in previous work for obtaining miracidia for snail exposure (see TAYLOR et al. 1969). S t u d i e s o n S. mansoni

Effect of strain difference Two groups of 6 weeks old male albino T.O. strain mice were exposed to cercariae of the two strains and kept on a uniform synthetic diet. T h e worm recovery using 150 cercariae of the Tanzanian strain was higher than that obtained in previous experimental work with the Puerto Rican strain. I n order that the worm burdens in the two series would be comparable mice were exposed to higher doses (265 cercariae) of the Puerto Rican strain. Treatment was begun 8 weeks after infection and continued for 10 days, using various concentrations of the drug. The results, set out in Table I, show that the Tanzanian strain was more resistant to the drug than the Puerto Rican strain: a unit dose of 50 mg./kg, did not reduce the mean worm load significantly with the Tanzanian strain (P >0.05) whereas with the Puerto Rican strain the worm burden was reduced significantly (P <0.05). Also, a unit dose of 100 mg./kg, cured none of the mice with the Tanzanian strain and a dosage of 200 mg./kg. cured only 4 out o f 15, whereas with the Puerto Rican strain a dose of 100 mg./kg, cured 7 out of i0 of the mice, and all 10 were cured at 200 mg./kg. A series oft-tests performed after converting the mean worm recoveries for the mice in each dosage group to percentages of the mean worm recovery in the untreated control group showed that the differences between the percentages of worms alive after treatment at 50, 75 and 100 mg./ kg. were significant for the two strains (P < 0 . 0 5 ; P <0.001; P <0.001).

Effect of worm load T o see if the size of the worm burden had any effect on the success of chemotherapy 6 weeks old mice were exposed to a relatively light infection of the Puerto Rican strain (150 cercariae) and treated 8 weeks later. The results (c.f. Tables I and II) show that the size of the worm burden did slightly affect the outcome of treatment. With the heavy infections, at a unit dose of 50 mg./kg. there was a significant reduction in the mean worm burden (P <0.05) but not with the light infections. Also, a unit dose of 100 mg./kg, cured only 6 out of 14 o f the mice with the light worm burdens, but 7 out of 10 of the mice with heavier worm burdens. A series of t-tests carried out after converting the worm recoveries of all the mice in each dosage

M. G. TAYLOR AND G. S. NELSON

171

group to percentages o f the mean worm burden of the untreated control mice showed that there was a significant difference between the two series in the percentages of the original worm burdens recovered alive after treatment at a unit dose o f 100 mg./kg. (P <0.05) though not with a unit dose of 50 mg./kg. TABLE I.

Comparison of the susceptibility to niridazole of a Puerto Rican and a Tanzanian strain of S. mansoni in mice ] Unit No. of No. of No. of dose mice mice mice in rag./ infected autopsied cured ! kg. ____ _ _

~

.~

Mean living worms per mouse Males

Females

10

0

34'1

26'9

61"0 ± 14.0

50

15

11

0

33'7

14'1

47"8 ±13"5

75

15

9

1

11'9

1.8

13.7 ±11'0

100

15

10

7

1.0

0

1'0 *- 2"2

200

15

10

10

0

0

0

]

0

13

11

0

31-8

20"8

52"5 ~k18 "0

I-

50

14

14

0

34"3

21.4

55"7 ~15'7

75

14

11

0

25"7

11.5

37.2 :~_13"4

100

14

13

0

18"9

6.5

25 "4 :-k15"5

200

15

15

4

1"6

0-3

1.9 2"2

u

r

T

e

.4 N

Total = S.D.

15 l

Egg hatching test

D

+

Although the light infections were thus slightly less susceptible to the drug than the heavy infections with the same strain there were still significantly lower percentages of the original w o r m b u r d e n alive after treatment in the mice with the light infections of the Puerto Rican strain than in those with the Tanzanian strain at unit dosage o f 50 mg./kg, and 100 mg./kg. (P < 0 . 0 5 and P <0"001, respectively). W i t h both strains all the eggs in the tissue were killed at the lowest unit dosage used (25 mg./kg.) and the surviving worms were sterilized, at least temporarily, since all egg hatching tests on livers o f treated mice were negative.

The susceptibility of S. mattheei Six weeks old male T.O. strain albino mice were exposed to 50 cercariae o f S. mattheei each; treatment was begun 8 weeks later. T h e results, shown in Table I I I , indicate that

172

A C O M P A R I S O N OF THE S U S C E P T I B I L I T Y TO N I R I D A Z O L E OF T W O STRAINS OF S. M A N S O N I

parasitological cure was achieved with unit doses of 100 mg./kg, and over. This species was much more susceptible to the drug than S. mansoni (cf Tables I to III). For example, a unit dosage of 50 mg./kg., which had little effect on the S. mansoni infections, killed nearly all the worms and cured 9/13 of the mice infected with S. mattheei. As before, a unit dosage of 25 mg./kg, apparently sterilized the worms and also killed the eggs already deposited in the tissues as hatching tests were negative. TABLE II.

The effect of niridazole on a "light" infection with the Puerto Rican strain of S. mansoni (24' 2 worms compared with 61-0 worms in Table I)

Unit dose No. of mice No. ofmice No. ofmice in mg.jkg. infected autopsied cured

Mean living worms per mouse - - - - 1 Males Females Total ± S.D.

0

14

12

0

11.6

12-6

24.2 ±11.5

25

14

14

0

11.9

14.9

26.7 ±7.9

50

14

14

0

9.2

10-6

19.8 ±7.4

100

14

14

6

2.5

0.7

3.2 ± 3-4

200

14

14

14

0

0

TABLE III. Unit dose inmg./ kg.

No. of daily treatmerits

Egg hatching test

+

0

The effect of niridazole on S. mattheei infections in mice No. of No. of No. of mice mice mice infected autopsied cured

Mean living worms per mouse Males

Females

Egg hatching test

Total ± S.D.

0

0

14

9

0

10.1

9.7

19.8 ± 7"0

25

10

14

14

0

7 '9

6.7

14"6 ±7"1

50

10

14

13

9

0"4

0"2

0-6 ±1-1

75

10

14

13

10

0"1

0"5

0"6 zk 1"7

100

10

14

14

14

0

0

0

200

10

14

14

14

0

0

0

100

15

14

14

14

0

0

0

+

w

M. G. TAYLOR AND G. S. NELSON

173

Discussion The finding that the Tanzanian strain of S. mansoni is more resistant to niridazole than the Puerto Rican strain may explain the different results observed in clinical trials in different geographical regions (see ABDALLAHand SAIF, 1969; JORDAN, 1969). Differences were also observed in the response of different strains of S. mansoni in mice treated with Miracil D by GONNERTand VOGEL (1955). They compared the therapeutic responses of a Liberian and an Egyptian strain in mice at four levels of dosage and found that adult worms of the Liberian strain were more susceptible at all dosage levels. Strain differences in response of S. mansoni to drugs have been reported for a third class of drugs by THOMPSON et al. (1965) who investigated the joint effect o f T A C pamoate and tartar emetic in S. mansoniinfections of mice and noted that a Liberian strain appeared to be more susceptible to TACP than their Puerto Rican strain. There is also strain variation in the response of S. japonicum to chemotherapy. The relative susceptibility of four strains of S. japonicum to antimonial therapy was investigated in mice by Hsu et al. (1963) who found that with both stibophen and tartar emetic the Japanese strain was more resistant to treatment than the Chinese, Formosan and Philippine strains, although the susceptibility of the last three strains was similar. In the present study with the Puerto Rican strain of S. mansoni there was a slightly higher worm kill with the heavier infections than with the lighter ones. Previous experimental work has produced conflicting results; GONNERT and VOGEL (1955) found that with both a Liberian and an Egyptian strain of S. mansoni that mice were more responsive to Miracil D when lighter worm burdens were being treated, but LUTTERMOSERand DE WITT (1961) found no significant differences in the percentage reduction in S. mansoni worm loads in heavily and in lightly infected mice treated with stibophen. The clinical evidence is inconclusive. JORDAN(1969) reported that cure rates are often higher in lightly infected patients than in those excreting large numbers of eggs. JORDAN(1965) found that the percentage reductions in egg load in S. haematobium patients treated with T W S b were independent of the size of the worm load. Nevertheless, the lightly infected patients were apparently more easily "cured", probably because eggs were more difficult to find in these patients. A unit dose of only 25 mg./kg, of niridazole killed no worms of either strain of S. mansoni or of S. mattheei but it killed all the eggs in the tissues and also sterilized the worms, at least temporarily. MONTEIRO et al. (1969) also noticed that niridazole damages eggs deposited in the tissues and STRIEBEL(1969) reported that a treatment of 20 mg./kg, of niridazole for ten days completely suppressed egg laying of a Liberian strain of S. mansoni in mice. STRIEBEL(personal communication) has studied the effect of niridazole on the genital cells of male and female schistosomes and has shown that "even severely damaged sex organs of the worms do regenerate and that egg laying starts anew about 4-5 weeks after the end of treatment." VOGEL and MINNING (1947) reported similar results in their experiments with S.japonicum in rabbits treated with antimony compounds. These observations emphasize the necessity for long-term follow-up studies of patients treated for schistosomiasis since the absence of living eggs in the faeces or urine may be due only to a temporary sterilization of the worms and not to a curative dose of the drug. The present study has shown that niridazole is much more effective against S. mattheei than against S. mansoni in mice. DINNIK (1968) had a similar experience with S. boris and S. rodhaini in hamsters. She found that S. boris was more susceptible than S. rodhaini to metriphonate (Neguvon, Bayer); all the hamsters infected with S. boris were cured after 4-14 oral treatments at doses of 78-130 mg./kg., but with S. rodhaini some hamsters were not cured even by 25 treatments at this dosage. It seems that S. mattheei and S. C*

174 A COMPARISONOFTHE SUSCEPTIBILITYTO NIRIDAZOLEOFTWO STRAINS OFS. MANSONI bovis, which are closely related to S. haematobium but occupy the mesenteric veins in rodents, are more susceptible to niridazole and metriphonate than S. mansoni and S. rodhaini which also inhabit the portal system of rodents. This suggests that the better therapeutic response of human infections of S. haematobium to treatment with these drugs may be due to differences in the susceptibility of the different species of worms and not to their different sites in the host as was suggested by FORSYTH(1965). Further evidence against the "lung shift" theory comes from the work of GOLDSMITH and KEAlq(1969) who were unable to obtain a single worm from heavily infected S. haematobium patients by extra-corporeai filtration of inferior vena cava blood after tartar emetic treatment. The route of migration of S. haematobium to the vesical veins of the definitive host has not been established, because of the lack of any suitable experimental host for such a study, but it is possible that S. haematobium migrate to the vesical plexus of man via the anastomoses with the inferior mesenteric vein, and not via the vena cava. I f this is so, worms subsequently dislodged by treatment might be shifted to the liver and not to the lungs.

Summary and conclusions Previous experimental studies have confirmed the clinical impressions that different strains of S. mansoni and S. japonicum have differem susceptibilities to miracil and antimonial drugs. We have now extended these observations to show that in mice a Tanzanian strain of S. mansoni is more resistant to niridazole than a Puerto Rican strain. We have also studied the effect of the intensity of infection on the success of chemotherapy, and found that heavy infections with S. mansoni were slightly more responsive to treatment than light infections. Finally, we tested the susceptibility of S. mattheei to niridazole and found that this parasite, a sibling species of S. haematobium, is much more susceptible than S. mansoni in mice. We deduce that the better therapeutic response seen in patients with S. haematobium compared with S. mansoni may be due to genetic differences in the parasites rather than to differences in their location in the hosts. REFERENCES ABDALLAH,A. & SAIF, M. (1969). Ann. N . Y . Acad. Sci., 160, 686. DINNIK, N. N. (1968). Vet. Med. Rev., 1, 30. FAIGLE, J. W. & KEBERLE,H. (1969). Ann. N . Y . Acad. Sci., 160, 544. FORSYTI-I,n . M. (1965). Lancet, 2, 354. GOLDSMITH,E. I. & KEAN, B. H. (1969). Am. J. trop. Med. Hyg., 18, 382. GONNERT, R. & VOGEL, H. (1955). Z. Tropenmed. Parasit., 6, 193. Hsu, S. Y. L., CHU, K. Y. & Hsu, H. F. (1963). Ibid., 14, 37. JORDAN, P. (1965). Bull. Wld Hlth Org., 33, 553. (1969). Ann. N . Y . Acad. Sci., 160, 602. LAMBERT, C. R. (1964). Ann. trop. ivied. Parasit., 58, 292. LUTTERMOSER,G. W. & DE WITT, W. B. (1961). Am. J. trop. Med. Hyg., 10, 541. MONTEIRO, W., PELLEGRINO,J. & DA SILVA,M. L. H. (1969). Revta Bras. Pesquisas ivied. Biol., 2, 45. NELSON, G. S., AMIN, M. A., SAOUD, M. F. A. & TEESDALE, C. (1968). Bull. Wld Hlth Org., 38, 9. STRIEBEL, H. (1969). Ann, N . Y . Acad. Sci., 160, 491. TAYLOR, M. G., AMIN, M. A. & NELSON, G. S. (1969). J. Helm., 43, 197. THOMVSON, P. E. MEISEm-mLDER,J. E., MOORE, A. K. & WAITZ, J. A. (1965). Bull. Wld I-Ilth Org., 33, 517. VOGEL, H. & MINNING, W. (1947). Acta Trop., 4, 21.