Parasitol Res (1991) 77:517-520 004432559100080U
Parasitnlngy Research
9 Springer-Verlag1991
Drug treatment of experimental
Capillaria hepatica infection in mice
R.F. Cheetham and M.B. Markus
Department of Zoology,Universityof the Witwatersrand,Johannesburg, 2050 South Africa Accepted February t5, 1991
Abstract. This report presents the results obtained using
14 anthelmintic compounds that were tested in experimental white mice of the Swiss-Webster strain for their action against Capillaria hepatica. Four of the drugs effectively prevented deposition of C. hepatica ova in mouse liver. The doses at which these four drugs prevented >99% of egg deposition were: albendazole, 30 mg/kg; febantel, 30 mg/kg; mebendazole, 3.13 mg/kg; and oxfendazole, 12.5 mg/kg. Of these, mebendazole is the only agent for which the application of five daily doses of 3.13 mg/kg lay within the dose range recommended for man.
Capillaria hepatica is synonymous with both Trichocephalus hepaticus and Hepaticola hepatica and is in the same family as the well-known whipworm Trichuris trichiura. C. hepatica can occur in the liver of various animals but its prevalence is undoubtedly highest in rodents. Published infection rates in rodents vary from 22 % in juvenile Rattus norvegicus (see Luttermoser 1936) to 81% in adults of the same species (Conlogue et al. 1979). The prevalence of infection amongst 235 Rattus rattus from the Witwatersrand area of the Transvaal province, South Africa, has been found to be 58% (Markus and Yeo, unpublished data). The importance of rodents as the primary host and transmission of the parasite to man, is explained by the unique life cycle of C.
hepatica. The adult worms are found in the liver. After mating, the female worms produce many thousands of eggs, which are not excreted from the host's body but remain within the parenchyma of the liver. These eggs are liberated only following the death and subsequent decomposition of the host or after the ingestion of an infected animal by a carnivore, in which case the eggs pass unaltered through the digestive tract of the latter and are excreted with the faeces (Shorb 1931). Once free in the
Offprint requests to: R.F. Cheetham
soil, the eggs must embryonate to become infective. Factors that determine the rate of embryonation include oxygen and moisture availability and the ambient temperature. When ingested by a new host, embryonated ova hatch in the intestine. From approximately 6 h onwards after hatching, the larvae penetrate the intestinal mucosa, enter the hepatic portal vein and are carried to the liver (Luttermoser 1938b). Larval maturation is swift, and female worms bearing ova may be seen 18 21 days after the ingestion of embryonated eggs by the host (Luttermoser 1938 b; Wright 1961). Normal egg production increases rapidly from day 20 of the infection to its peak at day 40, with no further egg production occurring after about day 70 (L/immler et al. 1974). After their reproductive functions have been completed, C. hepatica worms die and slowly disintegrate. In all, 13 cases of human infestation with C. hepatica have been documented in the literature (Dive et al. 1924; McQuown 1950; Otto et al. 1954; Turhan et al. 1954; Ewing and Tilden 1956; Ward and Dent 1959; Cochrane and Skinstad 1960; Calle 1961; Kallichurum and Elsdon-Dew 1961 ; Romero Garcia et al. 1962; Cislaghi and Radice 1970; Silverman etal. 1973; Attah et al. 1983). Over half of the 13 cases resulted in death, with hepatic capillariasis being discovered at autopsy; even in cases in which the infection was detected at laparotomy or liver biopsy, the patient died due to lack of suitable by treatment. There is no established cure for C. hepatica infection in man. L/immler and Grfiner (1976) have examined the effect of a number of drugs on C. hepatica in the multimammate mouse Mastomys nataIensis, but we felt that there was scope for further investigations using other compounds and a different host animal.
Materials and methods The animals used for the experiments were male white mice of
the Swiss-Webster strain. The mice weighed between 20 and 35 g
518
R.F. Cheetham and M.B. Markus: Drug treatment of Capillaria hepatica infection
at the start of the drug trials, and each animal was given an individual number by means of ear punching according to a pre-determined code. The initial supply of Capillaria hepatica eggs was obtained from a wild black rat trapped in the Transvaal. Once the infection had been established in laboratory white mice, it was maintained by routine passage in these rodents. Eggs were released from an infected liver by pulverising the latter in dechlorinated tap water with a pestle and mortar to break open the fibrotic masses containing C. hepatica eggs. After sieving this material through gauze, the resulting filtrate was maintained at 4 ~ C and washed repeatedly over a period of 7 days until the supernatant became clear. Most of the supernatant was discarded, and the eggs were incubated at 27~ ~ C in a small amount of dechlorinated water in petri dishes for 6-8 weeks and were aerated two to three times per week (Shorb 1931; Luttermoser 1938a; Wright 1961; Vollerthun et al. 1974; L/immler and Grtiner 1976; Zahner et al. 1976). The percentage embryonation was then determined by microscopic examination. A modified McMaster technique was used to count the C. hepatica eggs (Ministry of Agriculture, Fisheries and Food 1971; Vollerthun et al. 1974; L/immler and Grfiner 1976; Dunn 1978). A known volume of eggs in a minimal volume of dechlorinated water was added to a known amount of zinc chloride solution with a specific gravity of approximately 1.6 g/cm 3, and a sample was placed in the counting chamber of the McMaster slide. The eggs rose to the underside of the upper surface of the chamber and could then be counted microscopically. Three counts were averaged and the total number of eggs per millilitre of egg suspension was calculated. The number of embryonated eggs per millilitre could also be determined, based on the percentage embryonation. On day 1 of each experiment, mice were inoculated p.o. with 150 embryonated C. hepatica eggs in 0.2 ml dechlorinated tap water using a blunted metal cannula attached to a 1-ml disposable syringe. The body weights of the animals were also recorded. On days 14-18 post-inoculation, each drug being tested was given to separate groups of five mice. The animals were dosed according to their daily body weights, the drugs being prepared for each dose level such that 1 ml drug solution was used per 100 g body weight. The drugs were prepared by one of the following methods: (a) solution in distilled water, (b) suspension in gum tragacanth, (c) dilution with water or (d) dilution with supplied solvent. Groups of mice serving as controls were dosed with distilled water, gum tragacanth or solvent, as appropriate. On day 29 postinoculation, each animal was weighed and anaesthetised with ether and its jugular vein was then severed for exsanguination to ensure consistent liver weights. The body cavity was opened, any gross abnormalities were noted and the liver was removed and weighed. Each liver was individually wrapped, labelled and frozen at - 70 ~ C for a minimttm of 72 h so as to render the C. hepatica eggs uninfective (Wright 1961). The livers were then processed by means of a modified liverdigestion technique (Matsusaki 1951) as follows: a solution containing 0.7 g pepsin, 6 ml molar hydrochloric acid and 94 ml distilled water was placed in a 250-ml round-bottomed flask. The liver was cut into small pieces and added to the flask, which was held in a water bath at 37.5 ~ C and stirred using a vibrating stirrer for at least 3 h or until all the fibrotic masses had disintegrated. The resulting solution was made up to 200 ml with distilled water and kept at 4 ~ until required for examination. The eggs were counted in a McMaster chamber as described above. The number of eggs per gram of liver and the percentage reduction in deposition of ova were then calculated. Pilot experiments were first performed using very high doses of each test drug. For compounds that appeared to have an advantageous effect on the infection, further experiments were carried out using a much lower range of doses. The following compounds were tested in the experiments: albendazole (Valbazen, SmithKline), amoscanate (Ciba Geigy), febantel (Rintal, Bayer), mebendazole (Vermox, Janssen Pharmaceutica), niclosamide (Lintex, Bayer), oxamniquine (Vansil, Pfizer), oxfendazole (Systemex,
Coopers), oxyclozanide (ICI Liver Fluke Remedy, ICI), piperazine adipate (Ascaradina, Panvet), piperazine citrate (Rid, WDC), piperazine dihydrochloride (Wazine, Salsbury), praziquantel (Droncit, Bayer), pyrantel (Combantrin, Pfizer) and rafoxanide (Ranide, MSD).
Results The results of the high-dose pilot experiments are pres e n t e d in T a b l e 1. O n t h e basis o f t h e s e results, t h e d r u g s albendazole, febantel, mebendazole and oxfendazole were selected for low-dose experimentation. The criterio n u s e d f o r this s e l e c t i o n w a s t h a t t h e d r u g s h o u l d h a v e Table 1. Results of pilot experiments Compound
Dose (mg/kg x 5)
Number Eggs/g Eggs/g of mice liver liver surviving (X X 10 4) (% reduction)
Control group
Albendazole
125 250 500
5 5 5
0.1 0.2 0.3
99.8 99.7 99.6
B B B
Amoscanate
125 250 500
5 5 2
163.1 150.7 255.8
20.2 26.3 0
E1 El E1
Febantel
62.5 125 250
5 5 5
0.1 0.4 0.2
99.9 99.8 99.9
C C C
5 5 5
0.1 0.1 0.1
99.9 99.9 99.9
A1 A1 A1
Mebendazole
6.25 12.5 25
Niclosamide
125 250 500
4 4 5
153.6 135.8 67.5
9.5 20 60.2
C C C
Oxamniquine
125 250 500
5 5 2
90.3 74.5 73.2
0 1.3 3.1
E2 E2 E2
Oxfendazole
56.25 112.5 225
5 5 5
0 0 0
Oxyclozanide
85 170 340
5 5 4
26.6 40.7 5.3
65.8 47.6 93.1
B B B
Piperazine adipate
125 250 500
5 5 5
123.7 124.3 175.6
0 0 0
B B B
Piperazine citrate
125 250 500
5 5 5
196 110.3 248.6
28.4 59.7 9.1
A A A
Piperazine dihydrochloride
125 250 500
2 4 4
100 133.9 157.1
0 0 0
D D D
Praziquantel
50 100
5 5
72.9 229.7
73.4 16.1
A A
100 100 100
200
0
--
Pyrantel
125 250 500
5 5 5
66.7 86.5 117
75.6 68.4 57.3
A A A
Rafoxanide
62.5 125 250
5 3 0
66.4 70.1
60.9 58.7 --
C C C
-
--
D D D
A
R.F. Cheetham and M.B. Markus: Drug treatment of CapilIaria hepatica infection Table 2. Results of low-dose experiments Compound
Dose (mg/kg x 5)
Number Eggs/g Eggs/g of mice liver liver surviving (~ • ~04) (% reduction)
Control group
Albendazole
1.88 3.75 7.5 15 30a 60 120 1.25 2.5 5 10 15 30" 6O 3.13a 6.25 12.5 1.25 2.5 5 10 12.5" 25 50
5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
F F F F H H H F F F F H H H J J J F F F F H H H
Febantel
Mebendazole
Oxfendazole
26.9 20 14.2 6.1 0.1 0 0 40.6 47.7 34.3 4.1 5.6 0.1 0 0.2 0 0 45.1 41.3 28.6 11.2 0.1 0.1 0
20.5 41.1 78.7 91.8 99.8 100 100 0 0 0 87.9 90.2 99.9 100 99.7 100 100 0 0 15.7 67 99.8 99.9 100
" Lowest dose effective against Capillaria hepatica Table 3. Results obtained in control groups Control group
Number of mice surviving
Eggs/g liver (~ x 104)
AI A B C D E1 E2 F H J
5 5 5 5 5 5 5 5 5 5
120.7 273.7 77.6 169.7 87.9 204.3 75.5 33.9 56.8 66.9
achieved a reduction of > 9 9 % in the number of C. hepatica ova deposited in the liver. The results of the low-dose experiments are shown in Table 2. Table 3 lists the results obtained in the control groups. Groups A1, A, B, C, D, El and E2 were used as control animals during the pilot experiments, whereas groups F, H and J served as control groups during the low-dose experiments.
Discussion As our criterion for the efficacy of a c o m p o u n d in preventing the deposition of Capillaria hepatica eggs was that it should prevent at least 99% of egg deposition, we rejected the following as being ineffective: amoscan-
519
ate, niclosamide, oxamniquine, oxyclozanide, piperazine adipate, piperazine citrate, piperazine dihydrochloride, praziquantel, pyrantel and rafoxanide. Four drugs were found to fulfil the above criteria for effectiveness against C. hepatica, these being albendazole, febantel, mebendazole and oxfendazole. The lowest doses at which these compounds achieved a reduction of > 99% in the deposition of C. hepatica ova in the liver of white mice are indicated in Table 2 and in the abstract of this paper. Mebendazole was the only c o m p o u n d tested for which the experimental dose fell within the manufacturer's guidelines for use in man. Mebendazole is a benzimidazole carbamate derivative that displays broad spectrum anthelmintic activity in h u m a n helminthiasis and has been recognised as being effective against C. hepatica (Reynolds 1989; L/immler and Griiner 1976). Albendazole is also a benzimidazole anthelmintic that is structurally related to mebendazole and has been registered for use in man. It is effective against most nematode and some cestode worms. Although albendazole was effective in the present study, the dose of 30 mg/kg given on 5 consecutive days lay above the manufacturer's recommended dose of 10 mg/kg daily. However, there have been reports of the use of 30 mg/kg daily (Reynolds 1989). Oxfendazole is also structurally related to mebendazole, whereas febantel is not. Both of these products have been registered for veterinary use only; in both cases the doses used in the present study were higher than those recommended by the manufacturers. It can be concluded that although the present research revealed three additional compounds that are effective in preventing the deposition of C. hepatica eggs (namely albendazole, febantel and oxfendazole), mebendazole possibly remains the drug of choice for treating h u m a n capillariasis.
Acknowledgements. The support of the South African Council for Scientific and Industrial Research, the South African Medical Research Council and the University of the Witwatersrand is gratefully acknowledged. The following pharmaceutical companies are thanked for the donation of anthelmintic drugs : Bayer, Ciba Geigy, Coopers, ICI, MSD, Panvet, Salsbury and Smith-Kline. The assistance of A.N. Markus and W. Steinke in the translation of Spanish and German publications, respectively, is acknowledged with thanks.
References Attah EB, Nagarajan S, Obineche EN, Gera SC (1983) Hepatic capillariasis. Am J Clin Pathol 79:127-130 Calle S (1961) Parasitism by Capillaria hepatica. Pediatrics 27:648655 Cislaghi F, Radice C (1970) Infection by Capillaria hepatica: first case report in Italy. Helv Paediatr Acta 25:647-654 Cochrane JC, Skinstad EE (1960) Capillaria hepatica in man: follow-up of a case. S Afr Med J 34:21-22 Conlogue G, Foreyt W, Adess M, Levine H (1979) Capillaria hepatica (Bancroft) in select rat populations of Hartford, Connecticut, with possible public health implications. J Parasitol 65:105-108 Dive GH, Lafrenais HM, MacArthur WP (I924) A case of deposition of eggs of Hepaticola hepatica in the human liver. J R Army Med Corps 43 : 1-4
520
R.F. Cheetham and M.B. Markus: Drug treatment of Capillaria hepatica infection
Dunn AM (1978) Veterinary helminthology, 2nd edn. William Heinemann, London, pp 297-298 Ewing GM, Tilden IL (1956) Capillaria hepatica: report of the fourth true case of human infestation. J Pediatr 48 : 341-348 Kallichurum S, Elsdon-Dew R (1961) Capillaria in man: a case report. S Afr Med J 35:860-861 Lfimmler G, Grfiner D (1976) Zur Wirksamkeit yon Anthelminthika gegen Capillaria hepatica. Berl Mfinch Tierfirztl Wochenschr 89:222-225, 89: 229-233 L/immler G, Zahner H, Vollerthun R, Rudolph R (1974) Egg production and host reaction in Capillaria hepatica infection of Mastomys natalensis. In: Soulsby EJL (ed) Parasitic zoonoses. Academic Press, New York San Francisco London, pp 327-341 Luttermoser GW (1936) A helminthological survey of Baltimore house rats (Rattus norvegicus). Am J Hyg 24:350-360 Luttermoser GW (1938 a) Factors influencingthe development and viability of the eggs of Capillaria hepatica. Am J Epidemiol 27: 275-289 Luttermoser GW (1938 b) An experimental study of Capillaria hepatica in the rat and the mouse. Am J Epidemiol 27:321-340 Matsusaki G (1951) Studies on the life history of the hookworm: Part VII. On the development of Ancylostoma caninurn in the abnormal host. Yokohama Med Bull 2:154-160 McQuown AL (1950) Capillaria hepatica: report of genuine and spurious cases. Am J Trop Med Hyg 30 : 761-767 Ministry of Agriculture, Fisheries and Food (1971) Manual of veterinary parasitological laboratory techniques (Technical bulletin 18). Her Majesty's Stationery Office, London, pp 5-7
Note added in proof
Additional cases of hepatic capillariasis in man have recently been reported in the following papers : Berger T, Degr~mont A, Gebbers JO, T6nz O (1990) Hepatic capillariasis in a 1-year-old child. Eur J Pediatr 149:333-336 Kokai GK, Misic S, Perisic VN, Grujovska S (1990) Capillaria hepatica infestation in a two-year-old girl. Histopathology 17: 275-278
Otto GF, Berthrong M, Appleby RE, Rawlins JC, Wilbur O (1954) Eosinophilia and hepatomegaly due to Capillaria hepatica infection. Bull Johns Hopkins Hosp 94:319-336 Reynolds JEF (ed) (1989) Martindale: the extra pharmacopoeia, 29th edn. Pharmaceutical Press, London Romero Garcia F, Mendiola J, Biaji F (1962) Eosinophilia elevada con manifestaciones viscerales. IV Primer caso de infectcion pot Capillaria hepatica en M6xico. Bot Med Hosp Infant Mex 19 :473-479 Shorb DA (1931) Experimental infection of white rats with Hepaticola hepatica. J Parasitol 17:151-154 Silverman NH, Katz JS, Levin SE (1973) Capillaria hepatica infestation in a child. S Aft Med J 47:219-221 Turhan B, Unat EK, Yenermen M, Sumer C (1954) Insan karacigerinde Capillaria hepatica (Bancroft, 1893), Travossos, 1915. Microbiol Dergisi 7:149-159 Vollerthun R, L/immler G, Schuster J (1974) Capillaria hepatica - Infektion der Mastomys natalensis: Verfinderungen der Enzymaktivit/iten im Serum. Z Parasitenkd 44:43-58 Ward RL, Dent JH (1959) Capillaria hepatica infection in a child. Bull Tulane Univ Med Fac 19:27-33 Wright KA (1961) Observations on the life cycle of Capillaria hepatica (Bancroft, 1893) with a description of the adult. Can J Zool 39:167-182 Zahner H, Bruckmann G, Schmidt H, L/immler G, Geyer E (1976) Capillaria hepatica - Infektion der Mastomys natalensis: zur Entwicklung, Eierproduktion und Wirtsreaktion. Z Parasitenkd 49: 41-61
Parasitnlngy Research
9 Springer-Verlag1991
Drug treatment of experimental
Capillaria hepatica infection in mice
R.F. Cheetham and M.B. Markus
Department of Zoology,Universityof the Witwatersrand,Johannesburg, 2050 South Africa Accepted February t5, 1991
Abstract. This report presents the results obtained using
14 anthelmintic compounds that were tested in experimental white mice of the Swiss-Webster strain for their action against Capillaria hepatica. Four of the drugs effectively prevented deposition of C. hepatica ova in mouse liver. The doses at which these four drugs prevented >99% of egg deposition were: albendazole, 30 mg/kg; febantel, 30 mg/kg; mebendazole, 3.13 mg/kg; and oxfendazole, 12.5 mg/kg. Of these, mebendazole is the only agent for which the application of five daily doses of 3.13 mg/kg lay within the dose range recommended for man.
Capillaria hepatica is synonymous with both Trichocephalus hepaticus and Hepaticola hepatica and is in the same family as the well-known whipworm Trichuris trichiura. C. hepatica can occur in the liver of various animals but its prevalence is undoubtedly highest in rodents. Published infection rates in rodents vary from 22 % in juvenile Rattus norvegicus (see Luttermoser 1936) to 81% in adults of the same species (Conlogue et al. 1979). The prevalence of infection amongst 235 Rattus rattus from the Witwatersrand area of the Transvaal province, South Africa, has been found to be 58% (Markus and Yeo, unpublished data). The importance of rodents as the primary host and transmission of the parasite to man, is explained by the unique life cycle of C.
hepatica. The adult worms are found in the liver. After mating, the female worms produce many thousands of eggs, which are not excreted from the host's body but remain within the parenchyma of the liver. These eggs are liberated only following the death and subsequent decomposition of the host or after the ingestion of an infected animal by a carnivore, in which case the eggs pass unaltered through the digestive tract of the latter and are excreted with the faeces (Shorb 1931). Once free in the
Offprint requests to: R.F. Cheetham
soil, the eggs must embryonate to become infective. Factors that determine the rate of embryonation include oxygen and moisture availability and the ambient temperature. When ingested by a new host, embryonated ova hatch in the intestine. From approximately 6 h onwards after hatching, the larvae penetrate the intestinal mucosa, enter the hepatic portal vein and are carried to the liver (Luttermoser 1938b). Larval maturation is swift, and female worms bearing ova may be seen 18 21 days after the ingestion of embryonated eggs by the host (Luttermoser 1938 b; Wright 1961). Normal egg production increases rapidly from day 20 of the infection to its peak at day 40, with no further egg production occurring after about day 70 (L/immler et al. 1974). After their reproductive functions have been completed, C. hepatica worms die and slowly disintegrate. In all, 13 cases of human infestation with C. hepatica have been documented in the literature (Dive et al. 1924; McQuown 1950; Otto et al. 1954; Turhan et al. 1954; Ewing and Tilden 1956; Ward and Dent 1959; Cochrane and Skinstad 1960; Calle 1961; Kallichurum and Elsdon-Dew 1961 ; Romero Garcia et al. 1962; Cislaghi and Radice 1970; Silverman etal. 1973; Attah et al. 1983). Over half of the 13 cases resulted in death, with hepatic capillariasis being discovered at autopsy; even in cases in which the infection was detected at laparotomy or liver biopsy, the patient died due to lack of suitable by treatment. There is no established cure for C. hepatica infection in man. L/immler and Grfiner (1976) have examined the effect of a number of drugs on C. hepatica in the multimammate mouse Mastomys nataIensis, but we felt that there was scope for further investigations using other compounds and a different host animal.
Materials and methods The animals used for the experiments were male white mice of
the Swiss-Webster strain. The mice weighed between 20 and 35 g
518
R.F. Cheetham and M.B. Markus: Drug treatment of Capillaria hepatica infection
at the start of the drug trials, and each animal was given an individual number by means of ear punching according to a pre-determined code. The initial supply of Capillaria hepatica eggs was obtained from a wild black rat trapped in the Transvaal. Once the infection had been established in laboratory white mice, it was maintained by routine passage in these rodents. Eggs were released from an infected liver by pulverising the latter in dechlorinated tap water with a pestle and mortar to break open the fibrotic masses containing C. hepatica eggs. After sieving this material through gauze, the resulting filtrate was maintained at 4 ~ C and washed repeatedly over a period of 7 days until the supernatant became clear. Most of the supernatant was discarded, and the eggs were incubated at 27~ ~ C in a small amount of dechlorinated water in petri dishes for 6-8 weeks and were aerated two to three times per week (Shorb 1931; Luttermoser 1938a; Wright 1961; Vollerthun et al. 1974; L/immler and Grtiner 1976; Zahner et al. 1976). The percentage embryonation was then determined by microscopic examination. A modified McMaster technique was used to count the C. hepatica eggs (Ministry of Agriculture, Fisheries and Food 1971; Vollerthun et al. 1974; L/immler and Grfiner 1976; Dunn 1978). A known volume of eggs in a minimal volume of dechlorinated water was added to a known amount of zinc chloride solution with a specific gravity of approximately 1.6 g/cm 3, and a sample was placed in the counting chamber of the McMaster slide. The eggs rose to the underside of the upper surface of the chamber and could then be counted microscopically. Three counts were averaged and the total number of eggs per millilitre of egg suspension was calculated. The number of embryonated eggs per millilitre could also be determined, based on the percentage embryonation. On day 1 of each experiment, mice were inoculated p.o. with 150 embryonated C. hepatica eggs in 0.2 ml dechlorinated tap water using a blunted metal cannula attached to a 1-ml disposable syringe. The body weights of the animals were also recorded. On days 14-18 post-inoculation, each drug being tested was given to separate groups of five mice. The animals were dosed according to their daily body weights, the drugs being prepared for each dose level such that 1 ml drug solution was used per 100 g body weight. The drugs were prepared by one of the following methods: (a) solution in distilled water, (b) suspension in gum tragacanth, (c) dilution with water or (d) dilution with supplied solvent. Groups of mice serving as controls were dosed with distilled water, gum tragacanth or solvent, as appropriate. On day 29 postinoculation, each animal was weighed and anaesthetised with ether and its jugular vein was then severed for exsanguination to ensure consistent liver weights. The body cavity was opened, any gross abnormalities were noted and the liver was removed and weighed. Each liver was individually wrapped, labelled and frozen at - 70 ~ C for a minimttm of 72 h so as to render the C. hepatica eggs uninfective (Wright 1961). The livers were then processed by means of a modified liverdigestion technique (Matsusaki 1951) as follows: a solution containing 0.7 g pepsin, 6 ml molar hydrochloric acid and 94 ml distilled water was placed in a 250-ml round-bottomed flask. The liver was cut into small pieces and added to the flask, which was held in a water bath at 37.5 ~ C and stirred using a vibrating stirrer for at least 3 h or until all the fibrotic masses had disintegrated. The resulting solution was made up to 200 ml with distilled water and kept at 4 ~ until required for examination. The eggs were counted in a McMaster chamber as described above. The number of eggs per gram of liver and the percentage reduction in deposition of ova were then calculated. Pilot experiments were first performed using very high doses of each test drug. For compounds that appeared to have an advantageous effect on the infection, further experiments were carried out using a much lower range of doses. The following compounds were tested in the experiments: albendazole (Valbazen, SmithKline), amoscanate (Ciba Geigy), febantel (Rintal, Bayer), mebendazole (Vermox, Janssen Pharmaceutica), niclosamide (Lintex, Bayer), oxamniquine (Vansil, Pfizer), oxfendazole (Systemex,
Coopers), oxyclozanide (ICI Liver Fluke Remedy, ICI), piperazine adipate (Ascaradina, Panvet), piperazine citrate (Rid, WDC), piperazine dihydrochloride (Wazine, Salsbury), praziquantel (Droncit, Bayer), pyrantel (Combantrin, Pfizer) and rafoxanide (Ranide, MSD).
Results The results of the high-dose pilot experiments are pres e n t e d in T a b l e 1. O n t h e basis o f t h e s e results, t h e d r u g s albendazole, febantel, mebendazole and oxfendazole were selected for low-dose experimentation. The criterio n u s e d f o r this s e l e c t i o n w a s t h a t t h e d r u g s h o u l d h a v e Table 1. Results of pilot experiments Compound
Dose (mg/kg x 5)
Number Eggs/g Eggs/g of mice liver liver surviving (X X 10 4) (% reduction)
Control group
Albendazole
125 250 500
5 5 5
0.1 0.2 0.3
99.8 99.7 99.6
B B B
Amoscanate
125 250 500
5 5 2
163.1 150.7 255.8
20.2 26.3 0
E1 El E1
Febantel
62.5 125 250
5 5 5
0.1 0.4 0.2
99.9 99.8 99.9
C C C
5 5 5
0.1 0.1 0.1
99.9 99.9 99.9
A1 A1 A1
Mebendazole
6.25 12.5 25
Niclosamide
125 250 500
4 4 5
153.6 135.8 67.5
9.5 20 60.2
C C C
Oxamniquine
125 250 500
5 5 2
90.3 74.5 73.2
0 1.3 3.1
E2 E2 E2
Oxfendazole
56.25 112.5 225
5 5 5
0 0 0
Oxyclozanide
85 170 340
5 5 4
26.6 40.7 5.3
65.8 47.6 93.1
B B B
Piperazine adipate
125 250 500
5 5 5
123.7 124.3 175.6
0 0 0
B B B
Piperazine citrate
125 250 500
5 5 5
196 110.3 248.6
28.4 59.7 9.1
A A A
Piperazine dihydrochloride
125 250 500
2 4 4
100 133.9 157.1
0 0 0
D D D
Praziquantel
50 100
5 5
72.9 229.7
73.4 16.1
A A
100 100 100
200
0
--
Pyrantel
125 250 500
5 5 5
66.7 86.5 117
75.6 68.4 57.3
A A A
Rafoxanide
62.5 125 250
5 3 0
66.4 70.1
60.9 58.7 --
C C C
-
--
D D D
A
R.F. Cheetham and M.B. Markus: Drug treatment of CapilIaria hepatica infection Table 2. Results of low-dose experiments Compound
Dose (mg/kg x 5)
Number Eggs/g Eggs/g of mice liver liver surviving (~ • ~04) (% reduction)
Control group
Albendazole
1.88 3.75 7.5 15 30a 60 120 1.25 2.5 5 10 15 30" 6O 3.13a 6.25 12.5 1.25 2.5 5 10 12.5" 25 50
5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
F F F F H H H F F F F H H H J J J F F F F H H H
Febantel
Mebendazole
Oxfendazole
26.9 20 14.2 6.1 0.1 0 0 40.6 47.7 34.3 4.1 5.6 0.1 0 0.2 0 0 45.1 41.3 28.6 11.2 0.1 0.1 0
20.5 41.1 78.7 91.8 99.8 100 100 0 0 0 87.9 90.2 99.9 100 99.7 100 100 0 0 15.7 67 99.8 99.9 100
" Lowest dose effective against Capillaria hepatica Table 3. Results obtained in control groups Control group
Number of mice surviving
Eggs/g liver (~ x 104)
AI A B C D E1 E2 F H J
5 5 5 5 5 5 5 5 5 5
120.7 273.7 77.6 169.7 87.9 204.3 75.5 33.9 56.8 66.9
achieved a reduction of > 9 9 % in the number of C. hepatica ova deposited in the liver. The results of the low-dose experiments are shown in Table 2. Table 3 lists the results obtained in the control groups. Groups A1, A, B, C, D, El and E2 were used as control animals during the pilot experiments, whereas groups F, H and J served as control groups during the low-dose experiments.
Discussion As our criterion for the efficacy of a c o m p o u n d in preventing the deposition of Capillaria hepatica eggs was that it should prevent at least 99% of egg deposition, we rejected the following as being ineffective: amoscan-
519
ate, niclosamide, oxamniquine, oxyclozanide, piperazine adipate, piperazine citrate, piperazine dihydrochloride, praziquantel, pyrantel and rafoxanide. Four drugs were found to fulfil the above criteria for effectiveness against C. hepatica, these being albendazole, febantel, mebendazole and oxfendazole. The lowest doses at which these compounds achieved a reduction of > 99% in the deposition of C. hepatica ova in the liver of white mice are indicated in Table 2 and in the abstract of this paper. Mebendazole was the only c o m p o u n d tested for which the experimental dose fell within the manufacturer's guidelines for use in man. Mebendazole is a benzimidazole carbamate derivative that displays broad spectrum anthelmintic activity in h u m a n helminthiasis and has been recognised as being effective against C. hepatica (Reynolds 1989; L/immler and Griiner 1976). Albendazole is also a benzimidazole anthelmintic that is structurally related to mebendazole and has been registered for use in man. It is effective against most nematode and some cestode worms. Although albendazole was effective in the present study, the dose of 30 mg/kg given on 5 consecutive days lay above the manufacturer's recommended dose of 10 mg/kg daily. However, there have been reports of the use of 30 mg/kg daily (Reynolds 1989). Oxfendazole is also structurally related to mebendazole, whereas febantel is not. Both of these products have been registered for veterinary use only; in both cases the doses used in the present study were higher than those recommended by the manufacturers. It can be concluded that although the present research revealed three additional compounds that are effective in preventing the deposition of C. hepatica eggs (namely albendazole, febantel and oxfendazole), mebendazole possibly remains the drug of choice for treating h u m a n capillariasis.
Acknowledgements. The support of the South African Council for Scientific and Industrial Research, the South African Medical Research Council and the University of the Witwatersrand is gratefully acknowledged. The following pharmaceutical companies are thanked for the donation of anthelmintic drugs : Bayer, Ciba Geigy, Coopers, ICI, MSD, Panvet, Salsbury and Smith-Kline. The assistance of A.N. Markus and W. Steinke in the translation of Spanish and German publications, respectively, is acknowledged with thanks.
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R.F. Cheetham and M.B. Markus: Drug treatment of Capillaria hepatica infection
Dunn AM (1978) Veterinary helminthology, 2nd edn. William Heinemann, London, pp 297-298 Ewing GM, Tilden IL (1956) Capillaria hepatica: report of the fourth true case of human infestation. J Pediatr 48 : 341-348 Kallichurum S, Elsdon-Dew R (1961) Capillaria in man: a case report. S Afr Med J 35:860-861 Lfimmler G, Grfiner D (1976) Zur Wirksamkeit yon Anthelminthika gegen Capillaria hepatica. Berl Mfinch Tierfirztl Wochenschr 89:222-225, 89: 229-233 L/immler G, Zahner H, Vollerthun R, Rudolph R (1974) Egg production and host reaction in Capillaria hepatica infection of Mastomys natalensis. In: Soulsby EJL (ed) Parasitic zoonoses. Academic Press, New York San Francisco London, pp 327-341 Luttermoser GW (1936) A helminthological survey of Baltimore house rats (Rattus norvegicus). Am J Hyg 24:350-360 Luttermoser GW (1938 a) Factors influencingthe development and viability of the eggs of Capillaria hepatica. Am J Epidemiol 27: 275-289 Luttermoser GW (1938 b) An experimental study of Capillaria hepatica in the rat and the mouse. Am J Epidemiol 27:321-340 Matsusaki G (1951) Studies on the life history of the hookworm: Part VII. On the development of Ancylostoma caninurn in the abnormal host. Yokohama Med Bull 2:154-160 McQuown AL (1950) Capillaria hepatica: report of genuine and spurious cases. Am J Trop Med Hyg 30 : 761-767 Ministry of Agriculture, Fisheries and Food (1971) Manual of veterinary parasitological laboratory techniques (Technical bulletin 18). Her Majesty's Stationery Office, London, pp 5-7
Note added in proof
Additional cases of hepatic capillariasis in man have recently been reported in the following papers : Berger T, Degr~mont A, Gebbers JO, T6nz O (1990) Hepatic capillariasis in a 1-year-old child. Eur J Pediatr 149:333-336 Kokai GK, Misic S, Perisic VN, Grujovska S (1990) Capillaria hepatica infestation in a two-year-old girl. Histopathology 17: 275-278
Otto GF, Berthrong M, Appleby RE, Rawlins JC, Wilbur O (1954) Eosinophilia and hepatomegaly due to Capillaria hepatica infection. Bull Johns Hopkins Hosp 94:319-336 Reynolds JEF (ed) (1989) Martindale: the extra pharmacopoeia, 29th edn. Pharmaceutical Press, London Romero Garcia F, Mendiola J, Biaji F (1962) Eosinophilia elevada con manifestaciones viscerales. IV Primer caso de infectcion pot Capillaria hepatica en M6xico. Bot Med Hosp Infant Mex 19 :473-479 Shorb DA (1931) Experimental infection of white rats with Hepaticola hepatica. J Parasitol 17:151-154 Silverman NH, Katz JS, Levin SE (1973) Capillaria hepatica infestation in a child. S Aft Med J 47:219-221 Turhan B, Unat EK, Yenermen M, Sumer C (1954) Insan karacigerinde Capillaria hepatica (Bancroft, 1893), Travossos, 1915. Microbiol Dergisi 7:149-159 Vollerthun R, L/immler G, Schuster J (1974) Capillaria hepatica - Infektion der Mastomys natalensis: Verfinderungen der Enzymaktivit/iten im Serum. Z Parasitenkd 44:43-58 Ward RL, Dent JH (1959) Capillaria hepatica infection in a child. Bull Tulane Univ Med Fac 19:27-33 Wright KA (1961) Observations on the life cycle of Capillaria hepatica (Bancroft, 1893) with a description of the adult. Can J Zool 39:167-182 Zahner H, Bruckmann G, Schmidt H, L/immler G, Geyer E (1976) Capillaria hepatica - Infektion der Mastomys natalensis: zur Entwicklung, Eierproduktion und Wirtsreaktion. Z Parasitenkd 49: 41-61
