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Determination of the effective dose of an experimental fasciolicide in naturally and experimentally infected cattle
Veterinary Parasitology 120 (2004) 65–74
Determination of the effective dose of an experimental fasciolicide in naturally and experimentally infected cattle Froylán Ibarra a,∗ , Yolanda Vera a , Héctor Quiroz a , Jorge Cantó b , Rafael Castillo c , Alicia Hernández c , Pedro Ochoa d a
Departamento de Parasitolog´ıa, Fac. de Med. Vet. y Zoot., UNAM. Cd. Universitaria, 04510 Mexico, DF, Mexico b Departamento de Parasitolog´ıa, Universidad de Querétaro, Qro., Mexico c Facultad de Qu´ımica, UNAM. Cd. Universitaria, 04510 Mexico, DF, Mexico d Departamento de Genética y Estad´ıstica, Fac. de Med. Vet. y Zoot., UNAM. Cd. Universitaria, 04510 Mexico, DF, Mexico
Received 28 May 2003; received in revised form 15 December 2003; accepted 15 December 2003
Abstract The aim of the present study was to determine the effective dose of an experimental fasciolicide called compound alpha or 5-chloro-2-methylthio-6-(1-napthyloxy)1H-benzimidazole in experimentally and naturally infected cattle. In the first experiment, 24 fluke-free heifers were each infected with 800 metacercariae of Fasciola hepatica and re-infected on day 45 with other 600 cysts per animal. On day 75, when the animals had 4- and 10-week-old flukes respectively, they were divided into four groups (G) of six animals each according to fluke egg counts. Groups 1–3 received compound alpha at 10, 12 and 14 mg/kg/p.o., respectively. G4 remained as an untreated control. Twenty days after treatment, the animals were sacrificed for the recovery of flukes. Efficacy was assessed as a percentage of egg or fluke reduction relative to the untreated control. In the second experiment (naturally infected cattle), 24-year-old steers positive to F. hepatica eggs were blocked into four groups of five animals each. Groups 1–3 received compound alpha at 10, 12 and 14 mg/kg/p.o., respectively. Group 4 served as a non-treated control. All procedures to determine efficacy were carried out as mentioned in the first experiment. The results in the first study showed a percentage on egg reduction of 97.3, 100 and 100 and overall fluke reduction of 94.3, 100 and 100 for Groups 1–3, respectively. In the second study, the percentage of egg reduction was of 87.5, 99.1 and 100 and overall efficacy regarding fluke reduction was of 84.2, 99.6, and 100 for
∗ Corresponding author. Tel.: +52-56-22-58-99; fax: +52-56-22-59-71. E-mail address: [email protected] (F. Ibarra). 0304-4017/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2003.12.005
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Groups 1–3, respectively. It is concluded that the effective dose selected for compound alpha was of 12 mg/kg/p.o. in cattle having an induced or natural F. hepatica infection. © 2004 Elsevier B.V. All rights reserved. Keywords: Fasciola hepatica; Experimental chemotherapy; Cattle
1. Introduction Fasciolosis is the most important hepatic parasitic disease of ruminants. For several years now in vitro and in vivo screening of several compounds to achieve fasciolicide efficacy has been carried out. From that screening the compound “Alpha” or 5-chloro-2-methylthio-6-(1napthyloxy)1H-benzimidazole (Hernández et al., 2002) has shown percentages of efficacy from 80.6 to 100 in experimentally infected sheep and cattle (Ibarra et al., 1997a,b, 2000; Vera et al., 2001, 2003, 2004; Rivera et al., 2002). 1.1. Chemical properties It is a white powder with light characteristic odor, its condensed formula is C18 H13 CIN2 OS, molecular weight of 340.6 g/mol, melting point of 171–179 ◦ C, with a pka of 2.87, insoluble in water and lightly soluble in organic solvents. It is a novel compound related to the bencimidazole derivatives but different from the bencimidazole-2-carbamates. Efficacy of pharmacokinetic data suggest that the sulfoxide metabolite is the active principle responsible for fasciolicidal activity in vivo (Vertiz, 2000). It is formulated as a 10% drench suspension w/v alpha as described by Ibarra et al. (2000). On the other hand, compound alpha has been generally tested in sheep but population of cattle in our country overtakes by far that of sheep, therefore, efforts aimed to determine activity of this drug in cattle, should be encouraged. The aim of the present study was to determine the effective dose of 5-chloro-2-methylthio6-(1-napthyloxy)1H-benzimidazole in experimentally and naturally infected cattle.
2. Materials and methods 2.1. First experiment (experimentally infected heifers) 2.1.1. Animals Twenty-four, 4-year-old male cross-bred heifers, were located in a free-fluke area at the “G.B.” ranch located at Querétaro, in the state of Querétaro, Mexico. 2.1.2. Infections On day 0, animals were each infected with 800 metacercariae of Fasciola hepatica and were re-infected on day 45 with additional 600 metacercariae per animal, given orally in
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a gelatin capsule. They were raised in Lymnaea cubensis snails experimentally infected in our laboratory with miracidia from eggs collected from gallbladders of cattle sacrificed at the local slaughterhouse. 2.1.3. Procedure of the experiment On day 75 the animals were divided into four groups (G) of six animals each, all being ranked in descending order on the basis of fluke egg counts in fecal samples taken on day 67 (MAFF, 1988). 2.1.4. Treatments On day 75, Groups 1–3 received compound alpha at 10, 12 and 14 mg/kg/p.o., formulated as a 10% suspension, respectively. G4 remained as a non-treated control. 2.1.5. Evaluations Sixteen days after treatment, the heifers were sacrificed at the local slaughterhouse, and the livers were dissected for the recovery of flukes (Wood et al., 1995). The livers were then cut into slices approximately 1 cm thick and squeezed while in the saline solution; later the liver tissue was removed and the liberated parasites were collected after 30 min of sedimentation. To estimate their ages, the intact flukes were measured and examined microscopically for the presence of eggs in the uterus. The recovered worms were measured and classified as either immature (4-week-old) or adult flukes (10-week-old). Efficacy was assessed according to the formula described by Foreyt (1988) and Eckert et al. (1984): Mean no. of eggs or flukes in the control group − Mean number of eggs or flukes in the treated groups % Efficacy = × 100 Mean number of eggs or flukes in the control group 2.1.6. Statistical analysis Comparison of means of treatment and the total numbers of F. hepatica collected were submitted to a Kruskal–Wallis test to determine possible statistical differences on the egg or fluke reduction (Zar, 1996). All analyses were carried out using a statistical package. (SAS, 1990). 2.2. Second experiment (naturally infected cattle) 2.2.1. Location The study was carried out at the ranch “El Cocal”, located at the municipality of Mart´ınez de la Torre, in the state of Veracruz, Mexico. 2.2.2. Animals Twenty, 3–4-year-old cross-bred steers naturally infected with F. hepatica and selected for inclusion in the trial were determined positive to fluke eggs by the sedimentation test and to antibodies anti-F. hepatica by the Indirect ELISA test (Ibarra et al., 1998). The animals were then blocked into four groups of five animals each, according to this ranking order.
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2.2.3. Treatments Groups 1, 2 and 3 received compound alpha given orally at the doses of 10, 12 and 14 mg/kg of body weight (bw), respectively. Group 4 served as an untreated control. 2.2.4. Evaluations Thirty days after treatment the animals were sacrificed, and all procedures were carried out as mentioned in experiment 1. 3. Results 3.1. First experiment (experimentally infected heifers) Results on the fluke egg reduction showed a high decrease of eggs on the day of slaughter. Egg reduction was at very high level, being 97.3, 100 and 100% for Groups 1, 2 and 3, respectively (Table 1). No statistical difference was observed among the treated groups. Table 1 also shows the adult and immature fluke recovery after the compound alpha treatment. The average number of flukes for Group 1 was 1.5 adult and 7.6 immature. No flukes were obtained from Groups 2 and 3. The percentage of efficacy for adult flukes was 98.8, 100 and 100, and for immature flukes 90.9, 100 and 100, for Groups 1, 2 and 3, respectively. The control group had an average of 187 flukes/animal and a mean number of 102 adults and 85 immature flukes per animal. Statistical differences were observed in Group 1 when compared with Groups 2 and 3 (P < 0.051). Fig. 1 shows the size distribution in which 312 flukes were measured in the range of 5.1–10 mm and another large group of 286 flukes in which the range was 15.1–20.0 mm. Flukes measured from 3.0 to 30 mm in length. 3.2. Second experiment (naturally infected steers) Table 2 shows the fluke egg reduction in which very few eggs were observed in Group 1, an even lower number in Group 2 and no eggs at all were observed in Group 3, when compared with day 0. The percentage of egg reduction obtained for Groups 1, 2 and 3 was 87.5, 99.1 and 100, respectively. The untreated control always remained with the highest number of fluke eggs. Statistical differences were observed in Group 1 when compared with Groups 2 and 3 (P < 0.051).
Fig. 1. Size distribution of flukes recovered from the experimentally untreated heifers.
Group (n = 5)
1 2 3 4
Dose rate (mg/kg/p.o.)
10 12 14 Control a b
Recovered flukes Adult (mean ± range)
S.E.b ,
1.5 a,b ± 0.7 (0–4) 0a±0 0a±0 102 b ± 10.7 (76–141)
Efficacy (%) Immature (mean ± S.E.b , range) 7.6 a,b ± 3.7 (0–24) 0a±0 0a±0 85 b ± 5.6 (66–106)
Different letters in the same column indicate statistical difference (P < 0.051). Standard error.
Total (x) ± S.E. (range)
8.8 a,b ± 3.8 (0–24) 0a 0a 187 b ± 13.2 (158–247)
Egg reduction
97.3 a 100 a 100 a –
Fluke reduction Adult
Immature
Overall
98.8 100 100 –
90.9 100 100 –
94.3 100 100 –
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Table 1 Efficacy of compound alpha against immature and adult F. hepatica in experimentally infected heifersa
69
70
Group (n = 6)
1 2 3 4
Dose rate mg/kg/p.o.
10 12 14 Control a b
Recovered flukes
% Efficacy
Adult (mean ± S.E.b , range)
Immature (mean ± S.E.b , range)
Total (x) ± S.E. (range)
Egg reduction
7.6 a,b ± 5.2 (0–27) 0a±0 0a±0 45.8 b ± 18.9 (14–118)
2.2 a,b ± 1.6 (0–8) 0.2 a ± 0.04 (0–1) 0a±0 16.6 b ± 4.7 (9–31)
9.8 ± 6.8 (0–35) 0 0 62.4 ± 24 (24–149)
87.5 a 99.1 a,b 100 b –
Different letters in the same column indicate statistical difference (P < 0.051). Standard error.
Fluke reduction Adult
Immature
Overall
83.4 100 100 –
86.7 98.7 100 –
84.2 99.6 100 –
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Table 2 Efficacy of compound alpha against different stages of F. hepatica in naturally infected steersa
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Fig. 2. Size distribution of flukes recovered from the control group in naturally infected steers.
Table 2 also shows a mean of 7.6 adults and 2.2 immature flukes collected from Group 1, only one fluke in Group 2 and no flukes in Group 3, corresponding to percentage of fluke reduction of 83.4, 100 and 100 for adults and 86.7, 98.7 and 100 for immature flukes, respectively. The untreated control group had an average number of 62.4 flukes/animal and a mean of 45.8 adult and 16.6 immature flukes per animal. Comparisons carried out between groups showed a statistical difference in Group 1 when compared with the other groups (P < 0.051). Fig. 2 shows the size distribution in which 85 flukes were measured in the range 15.1–20 mm and another large group of 81 flukes in which the range was 20.1–25.0 mm. Flukes measured from 1.0 to 30 mm in length. 4. Discussion 4.1. First experiment Although the performance exerted by compound alpha either in eggs or fluke reduction in both experiments was at a very high level when evaluations were carried out in Groups 2 and 3, some weakness in its efficacy was observed in the data of Group 1 (10 mg/kg). The experimental compound showed slightly better capability of removing adult flukes the activity exerted being highly acceptable when compared with other commercial fasciolicides whose range of activity is reported to be lower (Richards et al., 1990; Boray, 1986; Shin-Sung et al., 1995; Ibarra et al., 2001). 4.2. Second experiment The pattern of infections in the steers was different considering that they had a natural infection of Fasciola. The infections had all stages of the parasite including flukes younger than 6 weeks. The flukes recovered from the slaughtered animals ranged in body length from 1.0 to 30.0 mm. According to Boray et al. (1967) it is considered that flukes measuring
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between 10.2 and 13.2 mm at the time of treatment 10 weeks earlier would have been approximately only between 1 day and 3-week-old. The larger flukes cannot be categorized as mature, thus making impossible to establish how many would have been immature or adult at the time of treatment; however, against this range of flukes of different ages, compound alpha showed high efficacy. It is also important to note that compound alpha overcomes the lack of activity shown against immature stages by older fasciolicides such as meniclofolan, nitroxinil, rafoxanide, albendazole, closantel and clorsulon, among others (Cordero del Campillo and Rojo Vázquez, 1999). On the other hand, triclabendazole (TCBZ) has been widely used to control liver fluke because its excellent activity against immature stages. However, resistance in adult fluke has been confirmed in sheep of Australia (Overend and Bowen, 1995), in sheep from Scotland (Mitchell et al., 1998), in a controlled trial with an isolate form county Sligo in Ireland (Coles et al., 2000). In South Wales, Thomas et al. (2000) treated a flock of ewes with a product containing triclabendazole and levamisole and 5 out of 9 fecal samples examined at the laboratory contained fluke eggs. All animals were then treated with closantel (Flukiver; Janseen Animal Health) and, 7 days later no eggs were observed by coprological examination. Moll et al. (2000) treated sheep and cattle with TCBZ in a farm in North Holland, The Netherlands. Fecal examinations of the animals after treatment showed high number of liver fluke eggs producing very low levels of efficacy. Their results were highly indicative of the presence of TCBZ-resistant F. hepatica in sheep and cattle on this farm. Gaasenbeek et al. (2001) in a study carried out in sheep, compared the efficacy of TCBZ against a resistant liver fluke strain with a susceptible laboratory strain. Their results showed efficacies of 10.8% in the resistant strain and 99.8% in the susceptible strain. On the other hand, Fairweather et al., from Belfast University (personal communication) have carried out an initial experiment with compound alpha against triclabendazole-resistant flukes. It was an in vitro experiment to obtain some SEM data and the flukes were inactive “dead” within 24 h. By comparison, in a separate experiment involving triclabendazole sulfoxide and a triclabendazole-susceptible isolate, the flukes were still moving after 24 h. He mentioned that he is awaiting for SEM results with compound alpha with considerable interest. Therefore, further investigations will be carried out testing compound alpha against a liver fluke resistant strain. It is concluded that the effective dose selected for compound alpha was of 12 mg/kg/p.o. in cattle having an induced or natural F. hepatica infection. Acknowledgements To the Comité Estatal de Fomento y Protección Pecuaria, ranch “G.B.” and Mr. Rodrigo Grappin from ranch “El Cocal” for all given facilities. Studies supported by the projects: PAPIIT-DEGAPA, UNAM, IN227998 and CONACyT 34942-B. References Boray, J.C., Happich, F.A., Andrews, J.C., 1967. Comparative chemotherapeutical tests in sheep infected with immature and mature Fasciola hepatica. Vet. Rec. 80, 218–224.
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Boray, J.C., 1986. Trematode infections of domestic animals. In: Campbell, W.C., Rew, R.S. (Eds.), Chemotherapy of Parasitic Diseases. Plenum Press, New York, pp. 401–425. Coles, G.C., Rhodes, A.C., Stafford, K.A., 2000. Activity of closantel against triclabendazole-resistant Fasciola hepatica. Vet. Rec. 146, 504. Cordero del Campillo, M., Rojo Vázquez, F.A., 1999. Parasitolog´ıa Veterinaria. McGraw-Hill-Interamericana, Madrid, España, p. 270. Eckert, J., Schneiter, G., Wolf, K., 1984. Fasinex (Triclabendazole)-ein neues Fazciolizide. Berl. Munch. Tierarztl. Wochr. 91, 249–356. Foreyt, W.J., 1988. Efficacy of a Fenbendazole-Triclabendazole combination against Fasciola hepatica and gastrointestinal nematodes in sheep. Vet. Parasitol. 26, 265–271. Gaasenbeek, C.P.H., Mol, L., Cornelissen, P.J.B.W., Vellema, P., Borgsteede, F.H.M., 2001. An experimental study on triclabendazole resistance of Fasciola hepatica in sheep. Vet. Parasitol. 95, 37–43. Hernández, C.A., Ibarra, V.F., Vera, M.Y., Rivera, F.N., Castillo, B.R., 2002. Synthesis and Fasciolicidal activity of 5-chloro-2-methylthio-6-(1-naphtiloxy)-1H-benzimidazole. Chem. Pharm. Bull. 50, 649–652. Ibarra, V.F., Garc´ıa, S.E., Fernández, R.M., Vera, M.Y., Castillo, B.R., Hernández, C.A., 1997a. Eficacia de dos compuestos de s´ıntesis qu´ımica in vivo e in vitro en ovinos. Vet. Méx. 28, 291–296. Ibarra, V.F., Vera, M.Y., Hernández, C.A., Castillo, B.R., 1997b. Eficacia fasciolicida del compuesto ALFA contra estadios juveniles y adultos en ovinos. Vet. Méx. 28, 297–301. Ibarra, V.F., Montenegro, N., Vera, M.Y., Boulard, C., Quiroz, R.H., Flores, J., Ochoa, G.P., 1998. Comparison of three ELISA tests for seroepidemiology of bovine fasciolosis. Vet. Parasitol. 77, 229–236. Ibarra, V.F., Montenegro, C.N., Flores, C.J., Hernández, C.A., Castillo, B.R., 2000. Evaluación de cuatro veh´ıculos para formular un fasciolicida experimental. Vet. Méx. 31, 47–51. Ibarra, V.F., Vera, M.Y., Nájera Fuentes, R., Sánchez Albarrán, A., 2001. Efficacy of combined chemotherapy against gastrointestinal nematodes and Fasciola hepatica in cattle. Vet. Parasitol. 99, 199–204. Ministry of Agriculture Fisheries and Food (MAFF), 1988. Manual of Veterinary Parasitological Laboratory Techniques. Reference Book 418, 3rd ed. London. Mitchell, G.B.B., Maris, L., Bonniwell, M.A., 1998. Triclabendazole-resistance liver fluke in Scottish sheep. Vet. Rec. 143, 399–403. Moll, L., Gaasenbeek, C.P.H., Vellema, P., Borgsteede, F.H.M., 2000. Resistance of Fasciola hepatica against triclabendazole in cattle and sheep in the Netherlands. Vet. Parasitol. 91, 153–158. Overend, D.J., Bowen, F.L., 1995. Resistance of Fasciola hepatica to triclabendazole. Aust. Vet. J. 72, 275– 276. Richards, R.J., Bowen, F.I., Essenwein, F., Teiger, R.F., Buscher, G., 1990. The efficacy of triclabendazole and other anthelmintics against Fasciola hepatica in controlled studies in cattle. Vet. Rec. 126, 213– 216. Rivera, F.N., Ibarra, V.F., Olazarán, J.S., Vera, M.Y., Castillo, B.R., Hernández, C.A., 2002. Eficacia del 5-cloro-2-metiltio-6-(1-naftiloxi)-ih-bencimidazol contra diversas edades de Fasciola hepatica en ovinos pelibuey. Vet. Méx. 33, 55–61. Shin-Sung, S., Lee-Chung, G., Cho-Shinn, H., Kim-Jong, T., Wee-Sung., Shin, S., 1995. Efficacy of closantel for the treatment of naturally acquired and experimentally induced Fasciola hepatica infections in cattle. Kor. J. Vet. Res. 35, 347–352. Statistical Analysis Systems (SAS), 1990. User’s Guide, 4th ed. SAS/STAT Institute Inc., Cary, NC. Thomas, I., Coles, G.C., Duffus, K., 2000. Triclabendazole-resistant Fasciola hepatica in southwest Wales. Vet. Rec. 146, 200–203. Vera, M.Y., Ibarra, V.F., Quiroz, R.H., Rios, U.A., Castillo, B.R., Hernández, C.A., 2001. Eficacia del 6-cloro-2-metiltio-5-(1-naftiloxi)bencimidazol contra Fasciola hepatica de cuatro y diez semanas de edad en bovinos de México. Vet. Méx. 32, 77–80. Vera, M.Y., Ibarra, V.F., Quiroz, R.H., Hernández, C.A., Castillo, B.R., 2003. Field trial on the efficacy of an experimental fasciolicide compared with some commercial compounds in naturally infected cattle. Parasitol. Res. 91, 1–4. Vera Montenegro, Y., Ibarra Verlarde, F., Liébano Hernández, E., Quiroz Romero, H., Castillo Bocanegra, R., Hernández Campos, A., Ochoa Galván, P., 2004. Efficacy of an experimental fasciolicide against immature and mature Fasciola hepatica in artificially infected. Parasitol. Res. 92, 211–214.
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Vertiz, S.G., 2000. Evaluación farmacocinética de ␣-biof10 en ganado vacuno. Tesis de Maestr´ıa en Farmacia. Facultad de Qu´ımica, Universidad Nacional Autónoma de México. Wood, I.B., Amaral, N.K., Bairden, K., Duncan, J.L., Kassai, J., Malone, J.B., Pankavich, J.A., Reinecke, R.K., Slocombe, O., 1995. World Association for the Advancement of Veterinary Parasitology (WAAVP), second edition of guidelines for evaluating the efficacy of anthelmintics in ruminants. Vet. Parasitol. 58, 181–213. Zar, J.H. (Ed.), 1996. Biostatistical Analysis. Prentice-Hall, NJ, 620 pp.
Determination of the effective dose of an experimental fasciolicide in naturally and experimentally infected cattle Froylán Ibarra a,∗ , Yolanda Vera a , Héctor Quiroz a , Jorge Cantó b , Rafael Castillo c , Alicia Hernández c , Pedro Ochoa d a
Departamento de Parasitolog´ıa, Fac. de Med. Vet. y Zoot., UNAM. Cd. Universitaria, 04510 Mexico, DF, Mexico b Departamento de Parasitolog´ıa, Universidad de Querétaro, Qro., Mexico c Facultad de Qu´ımica, UNAM. Cd. Universitaria, 04510 Mexico, DF, Mexico d Departamento de Genética y Estad´ıstica, Fac. de Med. Vet. y Zoot., UNAM. Cd. Universitaria, 04510 Mexico, DF, Mexico
Received 28 May 2003; received in revised form 15 December 2003; accepted 15 December 2003
Abstract The aim of the present study was to determine the effective dose of an experimental fasciolicide called compound alpha or 5-chloro-2-methylthio-6-(1-napthyloxy)1H-benzimidazole in experimentally and naturally infected cattle. In the first experiment, 24 fluke-free heifers were each infected with 800 metacercariae of Fasciola hepatica and re-infected on day 45 with other 600 cysts per animal. On day 75, when the animals had 4- and 10-week-old flukes respectively, they were divided into four groups (G) of six animals each according to fluke egg counts. Groups 1–3 received compound alpha at 10, 12 and 14 mg/kg/p.o., respectively. G4 remained as an untreated control. Twenty days after treatment, the animals were sacrificed for the recovery of flukes. Efficacy was assessed as a percentage of egg or fluke reduction relative to the untreated control. In the second experiment (naturally infected cattle), 24-year-old steers positive to F. hepatica eggs were blocked into four groups of five animals each. Groups 1–3 received compound alpha at 10, 12 and 14 mg/kg/p.o., respectively. Group 4 served as a non-treated control. All procedures to determine efficacy were carried out as mentioned in the first experiment. The results in the first study showed a percentage on egg reduction of 97.3, 100 and 100 and overall fluke reduction of 94.3, 100 and 100 for Groups 1–3, respectively. In the second study, the percentage of egg reduction was of 87.5, 99.1 and 100 and overall efficacy regarding fluke reduction was of 84.2, 99.6, and 100 for
∗ Corresponding author. Tel.: +52-56-22-58-99; fax: +52-56-22-59-71. E-mail address: [email protected] (F. Ibarra). 0304-4017/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2003.12.005
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Groups 1–3, respectively. It is concluded that the effective dose selected for compound alpha was of 12 mg/kg/p.o. in cattle having an induced or natural F. hepatica infection. © 2004 Elsevier B.V. All rights reserved. Keywords: Fasciola hepatica; Experimental chemotherapy; Cattle
1. Introduction Fasciolosis is the most important hepatic parasitic disease of ruminants. For several years now in vitro and in vivo screening of several compounds to achieve fasciolicide efficacy has been carried out. From that screening the compound “Alpha” or 5-chloro-2-methylthio-6-(1napthyloxy)1H-benzimidazole (Hernández et al., 2002) has shown percentages of efficacy from 80.6 to 100 in experimentally infected sheep and cattle (Ibarra et al., 1997a,b, 2000; Vera et al., 2001, 2003, 2004; Rivera et al., 2002). 1.1. Chemical properties It is a white powder with light characteristic odor, its condensed formula is C18 H13 CIN2 OS, molecular weight of 340.6 g/mol, melting point of 171–179 ◦ C, with a pka of 2.87, insoluble in water and lightly soluble in organic solvents. It is a novel compound related to the bencimidazole derivatives but different from the bencimidazole-2-carbamates. Efficacy of pharmacokinetic data suggest that the sulfoxide metabolite is the active principle responsible for fasciolicidal activity in vivo (Vertiz, 2000). It is formulated as a 10% drench suspension w/v alpha as described by Ibarra et al. (2000). On the other hand, compound alpha has been generally tested in sheep but population of cattle in our country overtakes by far that of sheep, therefore, efforts aimed to determine activity of this drug in cattle, should be encouraged. The aim of the present study was to determine the effective dose of 5-chloro-2-methylthio6-(1-napthyloxy)1H-benzimidazole in experimentally and naturally infected cattle.
2. Materials and methods 2.1. First experiment (experimentally infected heifers) 2.1.1. Animals Twenty-four, 4-year-old male cross-bred heifers, were located in a free-fluke area at the “G.B.” ranch located at Querétaro, in the state of Querétaro, Mexico. 2.1.2. Infections On day 0, animals were each infected with 800 metacercariae of Fasciola hepatica and were re-infected on day 45 with additional 600 metacercariae per animal, given orally in
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a gelatin capsule. They were raised in Lymnaea cubensis snails experimentally infected in our laboratory with miracidia from eggs collected from gallbladders of cattle sacrificed at the local slaughterhouse. 2.1.3. Procedure of the experiment On day 75 the animals were divided into four groups (G) of six animals each, all being ranked in descending order on the basis of fluke egg counts in fecal samples taken on day 67 (MAFF, 1988). 2.1.4. Treatments On day 75, Groups 1–3 received compound alpha at 10, 12 and 14 mg/kg/p.o., formulated as a 10% suspension, respectively. G4 remained as a non-treated control. 2.1.5. Evaluations Sixteen days after treatment, the heifers were sacrificed at the local slaughterhouse, and the livers were dissected for the recovery of flukes (Wood et al., 1995). The livers were then cut into slices approximately 1 cm thick and squeezed while in the saline solution; later the liver tissue was removed and the liberated parasites were collected after 30 min of sedimentation. To estimate their ages, the intact flukes were measured and examined microscopically for the presence of eggs in the uterus. The recovered worms were measured and classified as either immature (4-week-old) or adult flukes (10-week-old). Efficacy was assessed according to the formula described by Foreyt (1988) and Eckert et al. (1984): Mean no. of eggs or flukes in the control group − Mean number of eggs or flukes in the treated groups % Efficacy = × 100 Mean number of eggs or flukes in the control group 2.1.6. Statistical analysis Comparison of means of treatment and the total numbers of F. hepatica collected were submitted to a Kruskal–Wallis test to determine possible statistical differences on the egg or fluke reduction (Zar, 1996). All analyses were carried out using a statistical package. (SAS, 1990). 2.2. Second experiment (naturally infected cattle) 2.2.1. Location The study was carried out at the ranch “El Cocal”, located at the municipality of Mart´ınez de la Torre, in the state of Veracruz, Mexico. 2.2.2. Animals Twenty, 3–4-year-old cross-bred steers naturally infected with F. hepatica and selected for inclusion in the trial were determined positive to fluke eggs by the sedimentation test and to antibodies anti-F. hepatica by the Indirect ELISA test (Ibarra et al., 1998). The animals were then blocked into four groups of five animals each, according to this ranking order.
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2.2.3. Treatments Groups 1, 2 and 3 received compound alpha given orally at the doses of 10, 12 and 14 mg/kg of body weight (bw), respectively. Group 4 served as an untreated control. 2.2.4. Evaluations Thirty days after treatment the animals were sacrificed, and all procedures were carried out as mentioned in experiment 1. 3. Results 3.1. First experiment (experimentally infected heifers) Results on the fluke egg reduction showed a high decrease of eggs on the day of slaughter. Egg reduction was at very high level, being 97.3, 100 and 100% for Groups 1, 2 and 3, respectively (Table 1). No statistical difference was observed among the treated groups. Table 1 also shows the adult and immature fluke recovery after the compound alpha treatment. The average number of flukes for Group 1 was 1.5 adult and 7.6 immature. No flukes were obtained from Groups 2 and 3. The percentage of efficacy for adult flukes was 98.8, 100 and 100, and for immature flukes 90.9, 100 and 100, for Groups 1, 2 and 3, respectively. The control group had an average of 187 flukes/animal and a mean number of 102 adults and 85 immature flukes per animal. Statistical differences were observed in Group 1 when compared with Groups 2 and 3 (P < 0.051). Fig. 1 shows the size distribution in which 312 flukes were measured in the range of 5.1–10 mm and another large group of 286 flukes in which the range was 15.1–20.0 mm. Flukes measured from 3.0 to 30 mm in length. 3.2. Second experiment (naturally infected steers) Table 2 shows the fluke egg reduction in which very few eggs were observed in Group 1, an even lower number in Group 2 and no eggs at all were observed in Group 3, when compared with day 0. The percentage of egg reduction obtained for Groups 1, 2 and 3 was 87.5, 99.1 and 100, respectively. The untreated control always remained with the highest number of fluke eggs. Statistical differences were observed in Group 1 when compared with Groups 2 and 3 (P < 0.051).
Fig. 1. Size distribution of flukes recovered from the experimentally untreated heifers.
Group (n = 5)
1 2 3 4
Dose rate (mg/kg/p.o.)
10 12 14 Control a b
Recovered flukes Adult (mean ± range)
S.E.b ,
1.5 a,b ± 0.7 (0–4) 0a±0 0a±0 102 b ± 10.7 (76–141)
Efficacy (%) Immature (mean ± S.E.b , range) 7.6 a,b ± 3.7 (0–24) 0a±0 0a±0 85 b ± 5.6 (66–106)
Different letters in the same column indicate statistical difference (P < 0.051). Standard error.
Total (x) ± S.E. (range)
8.8 a,b ± 3.8 (0–24) 0a 0a 187 b ± 13.2 (158–247)
Egg reduction
97.3 a 100 a 100 a –
Fluke reduction Adult
Immature
Overall
98.8 100 100 –
90.9 100 100 –
94.3 100 100 –
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Table 1 Efficacy of compound alpha against immature and adult F. hepatica in experimentally infected heifersa
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70
Group (n = 6)
1 2 3 4
Dose rate mg/kg/p.o.
10 12 14 Control a b
Recovered flukes
% Efficacy
Adult (mean ± S.E.b , range)
Immature (mean ± S.E.b , range)
Total (x) ± S.E. (range)
Egg reduction
7.6 a,b ± 5.2 (0–27) 0a±0 0a±0 45.8 b ± 18.9 (14–118)
2.2 a,b ± 1.6 (0–8) 0.2 a ± 0.04 (0–1) 0a±0 16.6 b ± 4.7 (9–31)
9.8 ± 6.8 (0–35) 0 0 62.4 ± 24 (24–149)
87.5 a 99.1 a,b 100 b –
Different letters in the same column indicate statistical difference (P < 0.051). Standard error.
Fluke reduction Adult
Immature
Overall
83.4 100 100 –
86.7 98.7 100 –
84.2 99.6 100 –
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Table 2 Efficacy of compound alpha against different stages of F. hepatica in naturally infected steersa
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Fig. 2. Size distribution of flukes recovered from the control group in naturally infected steers.
Table 2 also shows a mean of 7.6 adults and 2.2 immature flukes collected from Group 1, only one fluke in Group 2 and no flukes in Group 3, corresponding to percentage of fluke reduction of 83.4, 100 and 100 for adults and 86.7, 98.7 and 100 for immature flukes, respectively. The untreated control group had an average number of 62.4 flukes/animal and a mean of 45.8 adult and 16.6 immature flukes per animal. Comparisons carried out between groups showed a statistical difference in Group 1 when compared with the other groups (P < 0.051). Fig. 2 shows the size distribution in which 85 flukes were measured in the range 15.1–20 mm and another large group of 81 flukes in which the range was 20.1–25.0 mm. Flukes measured from 1.0 to 30 mm in length. 4. Discussion 4.1. First experiment Although the performance exerted by compound alpha either in eggs or fluke reduction in both experiments was at a very high level when evaluations were carried out in Groups 2 and 3, some weakness in its efficacy was observed in the data of Group 1 (10 mg/kg). The experimental compound showed slightly better capability of removing adult flukes the activity exerted being highly acceptable when compared with other commercial fasciolicides whose range of activity is reported to be lower (Richards et al., 1990; Boray, 1986; Shin-Sung et al., 1995; Ibarra et al., 2001). 4.2. Second experiment The pattern of infections in the steers was different considering that they had a natural infection of Fasciola. The infections had all stages of the parasite including flukes younger than 6 weeks. The flukes recovered from the slaughtered animals ranged in body length from 1.0 to 30.0 mm. According to Boray et al. (1967) it is considered that flukes measuring
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between 10.2 and 13.2 mm at the time of treatment 10 weeks earlier would have been approximately only between 1 day and 3-week-old. The larger flukes cannot be categorized as mature, thus making impossible to establish how many would have been immature or adult at the time of treatment; however, against this range of flukes of different ages, compound alpha showed high efficacy. It is also important to note that compound alpha overcomes the lack of activity shown against immature stages by older fasciolicides such as meniclofolan, nitroxinil, rafoxanide, albendazole, closantel and clorsulon, among others (Cordero del Campillo and Rojo Vázquez, 1999). On the other hand, triclabendazole (TCBZ) has been widely used to control liver fluke because its excellent activity against immature stages. However, resistance in adult fluke has been confirmed in sheep of Australia (Overend and Bowen, 1995), in sheep from Scotland (Mitchell et al., 1998), in a controlled trial with an isolate form county Sligo in Ireland (Coles et al., 2000). In South Wales, Thomas et al. (2000) treated a flock of ewes with a product containing triclabendazole and levamisole and 5 out of 9 fecal samples examined at the laboratory contained fluke eggs. All animals were then treated with closantel (Flukiver; Janseen Animal Health) and, 7 days later no eggs were observed by coprological examination. Moll et al. (2000) treated sheep and cattle with TCBZ in a farm in North Holland, The Netherlands. Fecal examinations of the animals after treatment showed high number of liver fluke eggs producing very low levels of efficacy. Their results were highly indicative of the presence of TCBZ-resistant F. hepatica in sheep and cattle on this farm. Gaasenbeek et al. (2001) in a study carried out in sheep, compared the efficacy of TCBZ against a resistant liver fluke strain with a susceptible laboratory strain. Their results showed efficacies of 10.8% in the resistant strain and 99.8% in the susceptible strain. On the other hand, Fairweather et al., from Belfast University (personal communication) have carried out an initial experiment with compound alpha against triclabendazole-resistant flukes. It was an in vitro experiment to obtain some SEM data and the flukes were inactive “dead” within 24 h. By comparison, in a separate experiment involving triclabendazole sulfoxide and a triclabendazole-susceptible isolate, the flukes were still moving after 24 h. He mentioned that he is awaiting for SEM results with compound alpha with considerable interest. Therefore, further investigations will be carried out testing compound alpha against a liver fluke resistant strain. It is concluded that the effective dose selected for compound alpha was of 12 mg/kg/p.o. in cattle having an induced or natural F. hepatica infection. Acknowledgements To the Comité Estatal de Fomento y Protección Pecuaria, ranch “G.B.” and Mr. Rodrigo Grappin from ranch “El Cocal” for all given facilities. Studies supported by the projects: PAPIIT-DEGAPA, UNAM, IN227998 and CONACyT 34942-B. References Boray, J.C., Happich, F.A., Andrews, J.C., 1967. Comparative chemotherapeutical tests in sheep infected with immature and mature Fasciola hepatica. Vet. Rec. 80, 218–224.
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