Journal for Parasitology,Vol. 10,Pp. 205-211 1980.Printedin GreatBritain. 0 AustralianSociety for Parasitology.
Pergamon Press Ltd.
THE EXPULSION OF ASPICULURIS TETRAPTERA AND SYPHACIA SPP. FROM MICE AFTER ANTHELMINTIC TREATMENT J. C. W. COMLEY* Department
of Zoology and Applied Entomology, Imperial College Field Station, Ashurst Lodge, Silwood Park, Ascot, Berkshire, England (Received 30 July 1979; in revised.form 5 November 1979)
Abstract-CoMLEY JOHN C. W. 1980. The expulsion of Aspiculuris tetraptera and Syphacia spp. from mice after anthelmintic treatment. International Journal for Parasitology 10:205-211. The effect of four benzimidazoles, piperazine and levamisole on the expulsion of adult Aspiculuris tetraptera and Syphacia spp. from mice is described on a quantitative basis. Levamisole and piperazine were found to initiate expulsion within a few hours, but this ceased by 24 h. Following benzimidazole treatment most pinworms were expelled between 24 and 48 h. Syphaciu spp. responded earlier to the benzimidazoles than did A. tetraptera, but later to levamisole; both species responded similarly to piperazine. Only levamisole .and mebendazole were completely effective against both species within 24 h, piperazine being the least effective. The in vitro effects of levamisole on the motility and the recovery from drug-induced paralysis of the same nematode species are also reported. INDEX KEY WORDS: Pinworms; Aspicuhwis tetraptera; Syphacia spp.; mice; expulsion; mintics; benzimidazoles; piperazine; levamisole; in vitro motility.
MICE NATURALLY infected with the pinworms tetraptera and Syphacia spp., and with other artificial helminth parasite infections are used as a primary screen by several drug companies for evaluating compounds with potential anthelmintic activity (Theodorides, 1976). The efficacy of many known anthelmintics against A. tetraptera and S. obvelata has been extensively studied by Brody & Elward (1971) and reviewed by Taffs (1976). However, few anthelmintic tests against A. tetraptera and S. obveIata have taken the form of the critical test (see Theodorides, 1976) and only very limited descriptions are available on the fate of anthelmintic-treated worms (Vakil & Dalal, 1975; Van den Bossche, 1972; Thomas & Got-inert, 1977). In particular no attempt has been made to quantify the elimination of helminth worms following anthelmintic treatment, although the kinetics of the natural expulsion of some equine parasites has been monitored during a critical test (Folz, 1977). The main objective of the present investigation was to examine the process of nematode expulsion following anthelmintic treatment in order to establish a temporal basis for the interpretation of the biochemical effects of anthelmintics in viva. In addition, the in vitro effects of levamisole on the
motility of A. tetraptera and Syphacia spp. are also reported.
*Present address: The University of Vermont, Department of Pharmacology, Given Building, Burlington, Vermont, 05401, U.S.A.
Laboratory mice and nematode parasites. Four-month old female mice (IC obese strain) obtained from the Biochemistry Department, Imperial College, London were used in all experiments. These mice were shown to have a natural concurrent infection of the mouse pinworms A. tetraptera and Syphaciu spp., with a mean worm burden on post mortem of 167 f 12 (S.E.M.) adult nematodes, of which 83 ‘4 were A. tetraptera. During the experiments mice were housed individually in plastic cages (type Rl _ 38 x 25 x 18 cm) fitted with metal grids (North Kent Plastics Ltd.), and with plastic trays to collect the faeces. Mice were placed in the cages at least 12 h before the administration of anthelmintics (which was always started at 09.00 h). Twelve mice could conveniently be studied concurrently and each experiment was repeated until ten mice had been examined for each treatment. Anthelmintics. The following anthelmintics were used: Piperazine hexahydrate (PIP) (Sigma Chemical CO., (Poole) Ltd.); Levamisole hydrochloride (LVZ) and Mebendazole (MBZ) (Janssen Pharmaceutics; Beerse, Belgium); Cambendazole (CBZ) (Merck, Sharp and Dohme,. Hoddesdon, Herts.) and Fenbendazole (FBZ) (Farb-Werke Hoechst AG. Hounslow, Middx.). The drugs were used in their pure form apart from FBZ which was used as a 2.5 oA (w/v) commercial suspension ([email protected]
). Single doses of anthelmintics were given per OSby stomach tube at a concentration of 100 mg/kg body weight. In addition, MBZ was also tested at 1 and 10
mg/kg. Drugs were either dissolved or prepared as a 205
JOHN C. W. COMLEY
I.J.P. VOL. 10. 1980
FIGS l-4. Anthelmintic-induced expulsion of mouse pinworms. FIGS l-2. Nematodes within and closely adhering to the surface of a faecal pellet after benzimidazole or piperazine treatment of the host, Figs 3 and 4. Levamisole-induced mucous secretion containing nematodes. suspension by sonication in distilled water and given on a volumetric basis; 0.1 cm3 anthelmintic suspension per log body weight. Panacura was diluted with distilled water to the same concentration, as thepureunformulated drugs. Control mice were dosed with a corresponding volume of distilled water. In addition, the efficacy of Thiabendazole (TBZ) formulated of 0.3% (w/wj on PRM diet (E. Dixon and Sons. Ware) suoolied ad libitum to thk mice was tested. Unmedicated fiiM diet was given to both test and control mice for 5 days before each experiment and to the control mice throughout the experiment. The TBZ-diet remaining in the test cages was weighed at 12 h intervals to estimate the amount of TBZ ingested by each mouse. Determination of nematode expulsion from mice treated with anthelmintics. Following treatment of mice with anthelmintics faecal collections were made at 12 h intervals for 48 h except for LVZ and PIP where additional collections were made at 3,6 and 9 h, and at 12 h intervals for 84 h for TBZ diet. At the end of the experiment mice were killed by cervical dislocation. The caecum and the complete length of the colon to the anus was rapidly removed, placed in Petri dishes and stored at -20°C. The faeces were collected in a Petri dish and either examined for nematodes directly or stored at -20°C. Before examination faeces were softened in an artificial sea water medium (minus Na Br. 2H,O) diluted to 35 % (v/v), which Anya (1966) found to be isotonic with A. tetraptera. Adult nematodes were transferred to a watch glass containing 35 % sea water to be identified, sexed and counted. Mouse gastrointestinal tracts were thawed and dissected in 35% sea water, then examined as above.
In vitro effects qf levamisole on the motility of A. tetraptera and Syphacia spp. Gravid female A. tetraptera and Syphacia spp. were collected from mouse guts dissected in warm saline medium. This medium was equivalent to 35 % (v/v) sea water but also contained glucose (1.0 g 1-I) and antibiotics (penicillin G, 100,000 U I-‘) and streptomycin sulphate, 0.1 g 1-l). Worms were then washed several times in fresh medium, and ten individuals were incubated in 1 cm’ of saline at 37°C in covered watch glasses which were agitated every 30 min. Worms were examined for motility under a binocular microscope with a warm stage at about 40°C at various time intervals for up to 8 h. Various concentrations of LVZ were added in 100 mm3 of saline, and all experiments were started at 09.30 h. Statistical analysis qf expulsion data. Nematode expulsion was expressed as a percent of the nematodes remaining within the host at 24 and 48 h, and all comparisons were made on angularly transformed data by one-way analysis of variance (ANOVA) between the drug treatments for each species and sex of nematode, at 24 and 48 h. In addition, planned comparisons of the ANOVA were carried out between various groups of compounds. The difference between the percent remaining at 24 and 48 h after treatment with each drug were compared using a paired t test for each sex and species of nematode. Inter-sex and inter-specific comparisons between the same treatments at either 24 or 48 h were made using unpaired t tests. In addition, nematode expulsion was expressed as the percent nematodes eliminated per 12 h as a proportion of the number of nematodes available for elimination at the beginning of that 12 h period. These so-
Expulsion of pinworms
I.J.P. VOL. 10. 1980
24 h A. t.
100 68 0 95 92 100 92 62 100 96
99 61 5 95 96 100 96 63 100 98
PIP LVZ CBZ FBZ MBZ 1 MBZ 10 MBZ 100 PBM Diet TBZ Diet
s. spp. s. spp. d
88 46 8 60 18 97 51 39 100 53
83 57 2 63 66 93 70 25 100 95
Significant i differences
68 0 42* 34* 99 68* 2* 100 39*
60 1* 39* 38’ 100 77* 3” 100 .57*
b,c,d a C
48 h s. spp. 6
Significant t differences
80 43 0 4” 0* 45 16* 0’ 100 3
59 56 0 7% 0* 78 12* 0 100 0
cd cd c,d c
? Significant differences (P c 0.05) between:
a, A. i. 3 v A. t. 0; b, S. spp. $ Y S. spp. $T;c, A. t. 9 v S. spp. 9; d, A. spp. 6; * the percent remaining at 48 h from that at 24 h.
COMPARISONS BY ANOVA
OF THE PERCENT REMAINING AT 24 TREATMENT.
48 H AFTER ANTHELMINTIC
Comparisons Between all drugs LVZ v PIP LVZ v MBZIOO PIP v MBZlOO LVZ v MBZlO PIP v MBZlO MBZIO v MBZlOO LVZ v (CBZ, FBZ, TBZ diet) PIP v (CBZ, FBZ, TBZ diet) MBZlOO v (CBZ, FBZ, TBZ diet) MBZlO v (CBZ, FBZ, TBZ diet)
A. t. $
A. t. i!
* * *
* * *
* * * + *
Significant differences (PC 0.05) are indicated by *. called ‘death’ rates were determined for each drug and species of nematode and were based on the combined number of nematodes present in the ten replicates for
each drug and no statistical analysis was thus possible. RESULTS Expelled nematodes were found either within the faecal pellet or closely adhering to its surface after treatment with the benzimidazole compounds or PIP (Figs. 1 and 2). LVZ, however, rapidly induced a mucous secretion containing the pinworms in the first three hours following drug administration, thereafter worms were recovered from the faeces (Figs. 3 and 4). Nematodes recovered from fresh faecal samples or from the LVZ-mucous secretion were found to be alive and capable of some movement when placed in warm (37°C) artificial 35% sea water. The percent A. fetraptera and Syphacia spp. remaining within the host at 24 and 48 h after anthelmintic treatment are shown in TabIe I.
Statistical analyses of the percent remaining at 24 compared with that at 48 11,and the inter-sex and interspecific comparisons for the same treatments at either 24 or 48 h are also given in Table 1. Differences between the response to all drugs at 24 and 48 h for both sexes and species were tested by ANOVA and found to be highly significant (P<@OOl) and together with the planned comparisons between specific groups of drugs these are presented in Table 2. Only LVZ and MBZ 100 were greater than 99 % effective against both species within 48 h. MBZ 1 was completely without effect on A. tetraptera and only marginally effective against Syphacia spp. Both species of nematode showed no further change between the percent remaining at 24 h compared with those at 48 h after treatment with PIP and LVZ, whilst after ~nzimidazole treatment the greatest change in the percent remaining occurred between 24 and 48 h. Syphacia spp. appeared to respond earlier to the benzimidazoles but later to
FIG. 5. The expulsion of A. tetra~tera and Syp~ae~a spp. after LVZ and PIP treatment. (A) A. tetra~tera male, (B) A. tetraptera female, (C) Syphacia spp. male, (D) Syphacia spp. female, o - Control, A - PIP, a - LVZ.
expulsion patterns after treatment
(at 0.3% w/w) in the diet of •I - Ad. 9, mean of thia~ndazole + SD.
m - A.t. 3, A - S. spp. 9, A - S. spp. 3, Q - expulsion of unmedicated PRM-diet treated worms (Control). o -The
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I.J.P. VOL. 1% 1980
FIG. 7. The death rates of female nematodes after anthelmintic treatment. A, B and C - A. tetroptera, a, b, and c Syphaciu sgp. In A and a; o - Control, A -PIP, n - LVZ. In B and b; o - Control, o - CBZ, n - FBZ, A - PRM-diet, a - TBZ-diet. In C and c; o - Control, A - MB2 1, A - MBZ 10, q - MBZ 100. TABLE &--THE
Duration of treatment 2 min 4 min 10 min 30 min lh 2h 3h 4h 5h 6h 7h 8h
252 272 4,2 4+4 4&4 4+2 222 422
OF LEVAMISOLE ON THE MOTILITY * OF
Concentration of levamisole (ppm) A. tetraptera Syphaciaspp. 10 100 0 1 1 10 2+2 4+2 612 622 14 * 12 * 642 8+4 10 + 14 + 14 +
5 4 4 8 8
18 18 26 30 40 58 70 72 80 84 82 68
rf: I F 7 + 5 + 4 & 8 _t 10 + 5 ): 6 rt 5 + 8 f: 7 2 10
7628 88k4 8825 96 + 2 100 100 98 + 2 94 * 4 92 k 6 80 * 7 76 f 5 72 f 6
-.-4+4 2E2 41-2 4t2 612 8+4
2+2 4k2 2*2 36 + 8 762 13 96 + 2 96 + 2 94 + 4 100 98 f 2 100 100
6+4 18 + 11 34 f 13 96 f 2 98 + 2 100 100 100 100 100 100 100
100 62 _e 7 100 100 100 100 98 * 2 100 98 + 2 100 98 +- 2 98 * 2 100
* Motility expressed as the mean percent of worms inactive + S.E.M. (n = 5), in saline containing varying concentrations of levamisole. LVZ than A. t~t~~pfer~, both species responded similarly to PIP. The expulsion patterns elicited by PIP and LVZ in the first few hours after anthelmintic
treatment are given in more detail in Fig. 5. With the exception of male Syphaciu spp. notable expulsion of the nematode population did not occur until at least 24 h after exposure to TBZ-diet had elapsed (Fig. 6). After 24 h it was calculated each mouse had received a mean cumulative dose (2 S.E.M.) of 490.2 + 68.2 mg TBZ/Kg body weight, nearly five times the 100 mg/Kg oral dose of TBZ that was shown to be without effect against A. tetraptera and Syphacia spp. (Comley, unpublished). After treatment with LVZ and PIP the ‘death’ rates of each nematode species rose to a peak within
24 h and declined thereafter (Fig. 7). This result contrasts with the ‘death’ rates initiated by the benzimidazole compounds where the rates only started to rise after 12 h and were still increasing after 36 and 48 h. Males and females of the same species showed similar death rates. The effects of LVZ on the in vitro motility of A. fetmpfera and Sy~hac~a spp. are presented in Table 3. From the results it is apparent that the rate of paralysis was faster in Syphacia spp., and the concentration of LVZ needed to produce 100% inhibition of motility lower for Syphacia spp. compared to A. tetraptera. In addition, only A. tetraptera showed any degree of recovery during the experiment and worms recovered motility earlier at
the higher concentrations of LVZ. In both species the rate of paralysis increased with increasing concentrations of LVZ. DISCUSSION Theonset oftheeffectsofanthelminticsonintestinal nematodes in viva has only been briefly reported in the literature. Van den Bossche (1972) noted that the MBZ-induced expulsion of Syphacia muris, Ascaridia galli and Syngamus trachea took almost 3 days, while Coles & McNeillie (1977) observed that when various benzimidazoles were supplied in the host diet worm counts of Nematospiroides dubius did not start to decrease significantly until after the third day of treatment. No mention was made in these studies of the state of the expelled nematodes. Vakil & Dalal (1975), however, observed that after LVZ therapy of a human ascarid infection all expelled worms were dead, whereas after TBZ, PIP and TBZ treatment the worms were alive following expulsion. This confirmed the earlier report of Cavier & Hawking (1973), that ascarids were alive when expelled following treatment with PIP. The hitherto unreported secretion of mucous found in the present work appears to be a response of the host’s colon to thesudden backwardmovement of large numbers of paralysed nematodes (in some instances in excess of 500 adult worms per mouse). This LVZ-induced secretion was not produced when uninfected mice were treated with LVZ. Although there are reports of comparisons between critical and controlled tests (Herlich, 1977) and on the development of statistical methods for the assessment of anthelmintic efficiency (Groeneveld & Reinecke, 1969; Clark, 1970. Abstract. Journal of Parasitology 56: (4. sect. 2), p. 57, 100) there have been few attempts previous to the present work to quantify the process of worm elimnation. Since LVZ is thought to act by rapid stimulation of ganglionic structures followed by a depolarising type of neuromuscular inhibition (Van Neuten, 1972), it is probable that the greater susceptibility of A. tetraptera to this drug is a function of the proximity of this species to the anus, the majority of Syphacia spp. being located more anteriorly in the caecum. However, the in vitro experiments on motility also suggest some intrinsic differences between A. tetraptera and Syphacia spp., since when adult female A. tetraptera were left in a solution of LVZ, neuromuscular paralysis passes off and motility resumes. The rate of paralysis and of recovery of motility occurring earlier in those worms exposed to a higher concentration of LVZ (Table 3). Coles, East & Jenkins (1974) first observed this phenomenon in three species of adult nematodes, Ascaris lumbricoides, Nippostrongylas brasiliensis and N. dubius and later suggested it might be a type of tachyphylaxis (Coles, East & Jenkins, 1975). This effect does not, however, occur in all nematodes as Coles et al. (1974
I.J.P. VOL. 10. 1980
and 1975) noted that 3rd stage infective larvae of N. brasiliensis showed no reversal of paralysis. This was also found for the adult female Syphacia spp. used in the present study. Syphacia spp. was, however, more susceptible to all of the benzimidazole compounds tested, a result which would suggest that either Syphacia spp. encounters a greater concentration of these drugs within the caecum compared to colon, or, that this species is intrinsically more susceptible to benzimidazoles than A. tetraptera. The present results do not indicate that Syphacia spp. is more susceptible to elimination during the period in which female worms migrate to perianal skin, although this might have been expected. The ecological term ‘death’ rate has not previously been used in this type of study. ‘Death’ rates clearly distinguished the effect of benzimidazole anthelmintics from those of PIP and LVZ. In hindsight more information would have been gained if the expulsion experiments had been extended for at least one more day (up to 72 h), as was the case in the TBZ diet experiment (Fig. 7). The efficacy of many of the anthelmintics tested against A. tetraptera and Syphacia spp. has already b:en reported (Lynch & Hoegl, 1959; Brody & Elward, 1971; Baeder, et al., 1974; Taffs, 1975, 1976; Sharp & Wescott, 1976). In all these studies efficacy was determined from a ‘controlled’ anthelmintic test, after a period of treatment when all susceptible nematodes would have been eliminated. In the present study efficacy was determined 24 and 48 h after anthelmintic treatment for the same nematode population using the ‘critical’ anthelmintic test. The selection of these two times highlighted the different action of PIP and LVZ compared with the benzimidazole treatments. The benzimidazoles CBZ, FBZ and TBZ-diet were similar in their effects on A. tetraptera which in all cases commenced later than their effects on Syphacia spp. If CBZ and FBZ were to cause similar patterns of expulsion as TBZ-diet over the period 48-74 h we might expect CBZ and FBZ to eliminate their entire worm burdens by 72 h. The inactivity of TBZ compared with the effect of other benzimidazoles can probably be explained by pharmacokinetic differences, affecting both the distribution and persistence of the drug within the host, and the distribution of the drug within the parasite. However, the necessary experiments to substantiate this have not yet been fully reported, although it is known that higher concentrations of MBZ are found in the intestinal cells than other parts of Ascaris suum (Van den Bossche & Nollin, 1973). It seems most likely that nematodes need to be exposed to TBZ for a considerable length of time, presumably to permit the accumulation of the drug (Coles & McNeillie, 1977), a point well illustrated after the continuous treatment of mice with TBZ diet.
I.J.P. VOL. 10. 1980
Expulsion of pinworms
The different expulsion patterns observed in the present work can therefore beattributed to differences in the mode of action and the pharamacokinetic behaviour of the anthelmintics tested. LVZ and PIP rapidly eliminated pinworms of both species within a few hours, which is consistent with their known effects on the neuromuscular system of nematodes. The benzimidazoles however, are slower in their action and generally required at least 24 h to induce expulsion. All benzimidazoles probably have a similar mode of action, which according to Coles (1978) may be on the microtubular system of helminths, although the evidence for this being the principal site of action is still at present poor.
COLES G. C., EAST J. M. & JENKINS S. N. 1975. The
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