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Larvicidal effects of several chemicals on Strongyloides infective larvae
Veterinary Parasitology 118 (2003) 165–168
Short communication
Larvicidal effects of several chemicals on Strongyloides infective larvae Carlos D.E. Rebollo a , N. Taira b,∗ , S. Ura c , J.C. Williams d a
d
Parasitology Laboratory, Faculty of Veterinary Medicine, University of Asuncion, C.O. 1061, Asuncion, Paraguay b Kyushu Research Station, National Institute of Animal Health, 2702 Chuzan-cho, Kagoshima-shi 891-0105, Japan c Kyodoken Institute, 585 Shimoitabashi, Fushimi-ku, Kyoto 612-8073, Japan Department of Veterinary Science, Louisiana State University AgCenter, Baton Rouge, LA 70803-6002, USA Received 4 March 2003; received in revised form 5 August 2003; accepted 26 August 2003
Abstract The larvicidal effects of 11 anthelmintics, 7 pesticides and 4 disinfectants were evaluated with infective larvae of Strongyloides papillosus (SPL) and Strongyloides venezuelensis (SVZ). The lethal concentrations against SPL and SVZ were found to be similar. Three chemicals (dichlorvos, levamisole and trichlorfon) showed highest larvicidal effects. The 50% lethal concentration (LC50 ) values for the three compounds against SPL larvae were 0.08, 0.24, and 0.59 ppm, respectively. © 2003 Elsevier B.V. All rights reserved. Keywords: Strongyloides papillosus; Strongyloides venezuelensis; Larvicidal effect; Anthelmintics; Pesticides; Disinfectants
1. Introduction Strongyloidosis that resulted in sudden death in calves as described by Taira and Ura (1991), occurred with calves maintained on sawdust litter confinement pens during the late summer to early autumn in Japan. On some of the farms where death occurred, severe itching was observed in some of the calves due to Strongyloides papillosus (SPL) infection. The sawdust litter in the pens was likely contaminated with large numbers of SPL larvae. Methods of killing the larvae in the sawdust litter would be most useful for prevention of infection. The purpose of this study was to evaluate cidal effects of selected anthelmintics, pesticides and disinfectants against both SPL and Strongyloides venezuelensis (SVZ) infective larvae. ∗ Corresponding author. Tel.: +81-99-268-2078; fax: +81-99-268-3088. E-mail address: [email protected] (N. Taira).
0304-4017/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2003.08.009
166
C.D.E. Rebollo et al. / Veterinary Parasitology 118 (2003) 165–168
2. Materials and methods 2.1. Infective larvae used S. papillosus used in the study was the Himeji strain (Taira et al., 1991), and SVZ was the Kagoshima strain (Taira et al., 1994). The fresh faeces of calves infected with SPL and of rats infected with SVZ were cultured by the “polyethylene tube faecal culture for Strongyloides L3 ” reported by Taira et al. (1991) at 25 ◦ C for 3–4 days. The larvae, while migrating up the inside wall of a polyethylene bag, were collected in a glass pipette with a small amount of tap water and transferred to a glass tube. The larval suspension was adjusted to a population range of 20–40 larvae per 0.25 ml. Only motile or obviously living larvae were counted. 2.2. Chemicals and dilution Eleven anthelmintics, seven pesticides and four disinfectants were evaluated (Table 1). The anthelmintics were levamisole, trichlorfon, morantel, methyuridine, piperazine citrate, diethylcarbamazine, flubendazole, ivermectin, thiabendazole, parbendazole and praziquantel. The pesticides were dichlorvos (emulsifiable concentrate form; Rabbiphos® ec veterinary use, Sankyo Co. Ltd., Tokyo), MEP (dimethyl 4-nitro-m-tolylphosphorothionate), disulfoton, BPMC (o-sec-butylphenyl methylcarbamate), cartap, PCP (pentachlorophenol) and propoxur. The disinfectants were triethylammonium chloride, iodine, o-dichlorobenzene and phenol. All chemicals were made up to 2000 ppm as the active constituent concentration with tap water (the stock solution). Serial dilution was performed according to Taira et al. (1977), in that the 2000 ppm stock solution was diluted to 632, 200, 64, 20, 6.4 and 2 ppm for each chemical. 2.3. Exposure and evaluation of larvae The 0.25 ml larval suspensions were placed in wells in glass microscope slides and numbers of larvae in each well were counted. Subsequently, 0.25 ml of a diluted chemical was placed in a well with larvae. Therefore, actual serial concentrations of chemicals to which the larvae were exposed were 1000, 316, 100, 32, 10, 3.2 and 1 ppm in the experiment. The microscope slides with wells containing larvae and the various chemical dilutions were placed in a moister-maintained chamber and kept at 25 ◦ C for 18–24 h. Following the exposure period, larval movement were examined microscopically (100×); during 30 s of observation, determination was made of larval death or survival by the movement. 2.4. LC 50 and LC100 determination The 50% lethal concentration (LC50 ) was calculated according to Reed and Muench (1938). That is, the formula for the proportionate distance of the endpoint above the dilution giving next below 50% mortality is: 50% − (mortality at dilution next below) = proportional distance (mortality next above) − (mortality next below)
C.D.E. Rebollo et al. / Veterinary Parasitology 118 (2003) 165–168
167
Since dilutions are increasing on a logarithmic scale, it is necessary to obtain the final reading as follows: (logarithm of the lower dilution factor) + (proportional distance) × log 10 The LC100 was determined by the dilution in which all of the larvae were dead.
3. Results LC50 and LC100 values for SPL and SVZ larvae gave similar values (Table 1). The most effective chemicals, i.e. those active at less than 6 ppm LC50 for SPL, were levamisole and trichlorfon (anthelmintics), dichlorvos and MEP (pesticides), and trimethylammonium Table 1 Larvicidal effects of chemicals to S. papillosus and S. venezuelensis Chemicals Category
Infective larvae of Strongyloides Generic name
p/s/ca
S. papillosus LC50
(ppm)b
S. venezuelensis LC100 (ppm)
LC50 (ppm)
LC100 (ppm)
Anthelmintics 1 Levamisole 2 Trichlorfon 3 Morantel 4 Methyuridine 5 Piperazine citrate 6 Ivermectin 7 Diethylcarbamazine 8 Flubendazole 9 Thiabendazole 10 Parbendazole 11 Praziquantel
s s p s p s p p p p p
0.24 0.59 37.76 76.12 177.80 390.80 110.20 268.00 >1000 >1000 >1000
1.00 3.16 100.00 316.00 316.00 1000 >1000 >1000 >1000 >1000 >1000
0.30 0.17 28.18 177.80 177.80 2.92 644.80 >1000 613.00 >1000 >1000
1.00 1.00 100.00 316.00 316.00 316.00 >1000 >1000 >1000 >1000 >1000
Pesticides 1 2 3 4 5 6 7
s s p s p p p
0.08 0.56 16.92 17.78 17.78 133.40 143.30
0.32 1.00 31.60 31.60 31.60 316.00 316.00
0.04 0.17 5.28 17.78 17.78 280.00 141.20
0.10 0.32 10.00 31.60 31.60 1000.00 316.00
s
5.53
10.00
5.37
1.00
s s c
19.50 87.39 562.30
100.00 1000.00 1000.00
48.70 82.54 163.80
100.00 316.00 316.00
Dichlorvos MEP Disulfoton BPMC Cartap PCP Propoxur
Disinfectants 1 Trimethylammonium chloride 2 Iodine 3 Orthodichlorobenzene 4 Phenol a b
p: powder/tablet/granule; s: solution; c: crystal. Active constituent concentration.
168
C.D.E. Rebollo et al. / Veterinary Parasitology 118 (2003) 165–168
chloride (disinfectant). The most effective chemical was dichlorvos. The values of LC50 and LC100 were 0.08 and 0.32 ppm for SPL, and 0.04 and 0.10 ppm for SVZ larvae, respectively. 4. Discussion The LC50 and LC100 values against SPL and SVZ larvae were quite similar with all chemicals. The use of SVZ larvae in such screening tests of chemicals, provides for much easier handling than with SPL. Additionally, patent infections of SVZ in Mongolian gerbils persist very well (Tsuji et al., 1993). Of the anthelmintics tested, levamisole and trichlorfon showed the highest larvicidal effect. Levamisole may be useful as a disinfectant for human hands if individuals are exposed dermally to Strongyloides larvae. In an experimental infection of calves with SPL larvae and following accidental dermal infection on the hand of a person, intense itching of the hand ceased after a hand rinse with 2.5% levamisole in water (Taira, unpublished data). Of the disinfectants evaluated, trimethylammonium chloride had a LC50 of 5.53 ppm against SPL larvae and 5.37 ppm against SVZ larvae. Such a disinfectant might be useful as a foot or boot dip placed at the entrance to animal pens. Of the pesticides tested, dichlorvos had considerable effect on larvae. Dichlorvos is used not only as a pesticide, but also as an anthelmintic, synonyms or trade name: Dichlorphos, Atgard, Equigard, Equigel and Task, etc. (Campbell and Rew, 1986). The LC50 of dichlorvos was 0.08 ppm against SPL larvae and 0.04 ppm against SVZ larvae and were the lowest values in the experiment. Dichlorvos could be a useful drug for field application to infected premise against SPL infective larvae. Acknowledgements We are grateful to Dr. K. Kusano of Sankyo Co. Ltd., Tokyo, Dr. M. Motokawa of Kuji Animal Hygiene Service Center of Iwate Prefecture, and Mr. H. Hazeyama of Kyushu Research Station of National Institute of Animal Health for their considerable assistance. References Campbell, W.C., Rew, R.S., 1986. Chemistry of antinematodal agents. In: Chemotherapy of Parasitic Diseases. Plenum Press, New York, pp. 239–266. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty per cent endpoints. Am. J. Hyg. 27, 493–497. Taira, N., Ura, S., 1991. Sudden death in calves associated with Strongyloides papillosus infection. Vet. Parasitol. 39, 313–319. Taira, N., Yoshihara, S., Ikeda, I., Ueno, H., 1977. A simple method for screening molluscicidal activity of pesticides using Lymnaea ollula egg. Bull. Natl. Inst. Anim. Health 75, 16–22 (in Japanese, English summary in Natl. Inst. Anim. Health Q. 17, 185 (1977)). Taira, N., Minami, T., Smitanon, J., 1991. Dynamics of faecal egg counts in rabbits experimentally infected with Strongyloides papillosus. Vet. Parasitol. 39, 333–336. Taira, N., Hirooka, M., Saeki, H., 1994. Isolation of Strongyloides venezuelensis from Rattus norvegicus in Kagoshima Prefecture. J. Vet. Med. Sci. 56, 255–258. Tsuji, N., Nakamura, Y., Taira, N., 1993. Long-lasting parasitism of Strongyloides venezuelensis in Mongolian gerbils (Meriones unguiculatus). J. Parasitol. 79, 305–607.
Short communication
Larvicidal effects of several chemicals on Strongyloides infective larvae Carlos D.E. Rebollo a , N. Taira b,∗ , S. Ura c , J.C. Williams d a
d
Parasitology Laboratory, Faculty of Veterinary Medicine, University of Asuncion, C.O. 1061, Asuncion, Paraguay b Kyushu Research Station, National Institute of Animal Health, 2702 Chuzan-cho, Kagoshima-shi 891-0105, Japan c Kyodoken Institute, 585 Shimoitabashi, Fushimi-ku, Kyoto 612-8073, Japan Department of Veterinary Science, Louisiana State University AgCenter, Baton Rouge, LA 70803-6002, USA Received 4 March 2003; received in revised form 5 August 2003; accepted 26 August 2003
Abstract The larvicidal effects of 11 anthelmintics, 7 pesticides and 4 disinfectants were evaluated with infective larvae of Strongyloides papillosus (SPL) and Strongyloides venezuelensis (SVZ). The lethal concentrations against SPL and SVZ were found to be similar. Three chemicals (dichlorvos, levamisole and trichlorfon) showed highest larvicidal effects. The 50% lethal concentration (LC50 ) values for the three compounds against SPL larvae were 0.08, 0.24, and 0.59 ppm, respectively. © 2003 Elsevier B.V. All rights reserved. Keywords: Strongyloides papillosus; Strongyloides venezuelensis; Larvicidal effect; Anthelmintics; Pesticides; Disinfectants
1. Introduction Strongyloidosis that resulted in sudden death in calves as described by Taira and Ura (1991), occurred with calves maintained on sawdust litter confinement pens during the late summer to early autumn in Japan. On some of the farms where death occurred, severe itching was observed in some of the calves due to Strongyloides papillosus (SPL) infection. The sawdust litter in the pens was likely contaminated with large numbers of SPL larvae. Methods of killing the larvae in the sawdust litter would be most useful for prevention of infection. The purpose of this study was to evaluate cidal effects of selected anthelmintics, pesticides and disinfectants against both SPL and Strongyloides venezuelensis (SVZ) infective larvae. ∗ Corresponding author. Tel.: +81-99-268-2078; fax: +81-99-268-3088. E-mail address: [email protected] (N. Taira).
0304-4017/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2003.08.009
166
C.D.E. Rebollo et al. / Veterinary Parasitology 118 (2003) 165–168
2. Materials and methods 2.1. Infective larvae used S. papillosus used in the study was the Himeji strain (Taira et al., 1991), and SVZ was the Kagoshima strain (Taira et al., 1994). The fresh faeces of calves infected with SPL and of rats infected with SVZ were cultured by the “polyethylene tube faecal culture for Strongyloides L3 ” reported by Taira et al. (1991) at 25 ◦ C for 3–4 days. The larvae, while migrating up the inside wall of a polyethylene bag, were collected in a glass pipette with a small amount of tap water and transferred to a glass tube. The larval suspension was adjusted to a population range of 20–40 larvae per 0.25 ml. Only motile or obviously living larvae were counted. 2.2. Chemicals and dilution Eleven anthelmintics, seven pesticides and four disinfectants were evaluated (Table 1). The anthelmintics were levamisole, trichlorfon, morantel, methyuridine, piperazine citrate, diethylcarbamazine, flubendazole, ivermectin, thiabendazole, parbendazole and praziquantel. The pesticides were dichlorvos (emulsifiable concentrate form; Rabbiphos® ec veterinary use, Sankyo Co. Ltd., Tokyo), MEP (dimethyl 4-nitro-m-tolylphosphorothionate), disulfoton, BPMC (o-sec-butylphenyl methylcarbamate), cartap, PCP (pentachlorophenol) and propoxur. The disinfectants were triethylammonium chloride, iodine, o-dichlorobenzene and phenol. All chemicals were made up to 2000 ppm as the active constituent concentration with tap water (the stock solution). Serial dilution was performed according to Taira et al. (1977), in that the 2000 ppm stock solution was diluted to 632, 200, 64, 20, 6.4 and 2 ppm for each chemical. 2.3. Exposure and evaluation of larvae The 0.25 ml larval suspensions were placed in wells in glass microscope slides and numbers of larvae in each well were counted. Subsequently, 0.25 ml of a diluted chemical was placed in a well with larvae. Therefore, actual serial concentrations of chemicals to which the larvae were exposed were 1000, 316, 100, 32, 10, 3.2 and 1 ppm in the experiment. The microscope slides with wells containing larvae and the various chemical dilutions were placed in a moister-maintained chamber and kept at 25 ◦ C for 18–24 h. Following the exposure period, larval movement were examined microscopically (100×); during 30 s of observation, determination was made of larval death or survival by the movement. 2.4. LC 50 and LC100 determination The 50% lethal concentration (LC50 ) was calculated according to Reed and Muench (1938). That is, the formula for the proportionate distance of the endpoint above the dilution giving next below 50% mortality is: 50% − (mortality at dilution next below) = proportional distance (mortality next above) − (mortality next below)
C.D.E. Rebollo et al. / Veterinary Parasitology 118 (2003) 165–168
167
Since dilutions are increasing on a logarithmic scale, it is necessary to obtain the final reading as follows: (logarithm of the lower dilution factor) + (proportional distance) × log 10 The LC100 was determined by the dilution in which all of the larvae were dead.
3. Results LC50 and LC100 values for SPL and SVZ larvae gave similar values (Table 1). The most effective chemicals, i.e. those active at less than 6 ppm LC50 for SPL, were levamisole and trichlorfon (anthelmintics), dichlorvos and MEP (pesticides), and trimethylammonium Table 1 Larvicidal effects of chemicals to S. papillosus and S. venezuelensis Chemicals Category
Infective larvae of Strongyloides Generic name
p/s/ca
S. papillosus LC50
(ppm)b
S. venezuelensis LC100 (ppm)
LC50 (ppm)
LC100 (ppm)
Anthelmintics 1 Levamisole 2 Trichlorfon 3 Morantel 4 Methyuridine 5 Piperazine citrate 6 Ivermectin 7 Diethylcarbamazine 8 Flubendazole 9 Thiabendazole 10 Parbendazole 11 Praziquantel
s s p s p s p p p p p
0.24 0.59 37.76 76.12 177.80 390.80 110.20 268.00 >1000 >1000 >1000
1.00 3.16 100.00 316.00 316.00 1000 >1000 >1000 >1000 >1000 >1000
0.30 0.17 28.18 177.80 177.80 2.92 644.80 >1000 613.00 >1000 >1000
1.00 1.00 100.00 316.00 316.00 316.00 >1000 >1000 >1000 >1000 >1000
Pesticides 1 2 3 4 5 6 7
s s p s p p p
0.08 0.56 16.92 17.78 17.78 133.40 143.30
0.32 1.00 31.60 31.60 31.60 316.00 316.00
0.04 0.17 5.28 17.78 17.78 280.00 141.20
0.10 0.32 10.00 31.60 31.60 1000.00 316.00
s
5.53
10.00
5.37
1.00
s s c
19.50 87.39 562.30
100.00 1000.00 1000.00
48.70 82.54 163.80
100.00 316.00 316.00
Dichlorvos MEP Disulfoton BPMC Cartap PCP Propoxur
Disinfectants 1 Trimethylammonium chloride 2 Iodine 3 Orthodichlorobenzene 4 Phenol a b
p: powder/tablet/granule; s: solution; c: crystal. Active constituent concentration.
168
C.D.E. Rebollo et al. / Veterinary Parasitology 118 (2003) 165–168
chloride (disinfectant). The most effective chemical was dichlorvos. The values of LC50 and LC100 were 0.08 and 0.32 ppm for SPL, and 0.04 and 0.10 ppm for SVZ larvae, respectively. 4. Discussion The LC50 and LC100 values against SPL and SVZ larvae were quite similar with all chemicals. The use of SVZ larvae in such screening tests of chemicals, provides for much easier handling than with SPL. Additionally, patent infections of SVZ in Mongolian gerbils persist very well (Tsuji et al., 1993). Of the anthelmintics tested, levamisole and trichlorfon showed the highest larvicidal effect. Levamisole may be useful as a disinfectant for human hands if individuals are exposed dermally to Strongyloides larvae. In an experimental infection of calves with SPL larvae and following accidental dermal infection on the hand of a person, intense itching of the hand ceased after a hand rinse with 2.5% levamisole in water (Taira, unpublished data). Of the disinfectants evaluated, trimethylammonium chloride had a LC50 of 5.53 ppm against SPL larvae and 5.37 ppm against SVZ larvae. Such a disinfectant might be useful as a foot or boot dip placed at the entrance to animal pens. Of the pesticides tested, dichlorvos had considerable effect on larvae. Dichlorvos is used not only as a pesticide, but also as an anthelmintic, synonyms or trade name: Dichlorphos, Atgard, Equigard, Equigel and Task, etc. (Campbell and Rew, 1986). The LC50 of dichlorvos was 0.08 ppm against SPL larvae and 0.04 ppm against SVZ larvae and were the lowest values in the experiment. Dichlorvos could be a useful drug for field application to infected premise against SPL infective larvae. Acknowledgements We are grateful to Dr. K. Kusano of Sankyo Co. Ltd., Tokyo, Dr. M. Motokawa of Kuji Animal Hygiene Service Center of Iwate Prefecture, and Mr. H. Hazeyama of Kyushu Research Station of National Institute of Animal Health for their considerable assistance. References Campbell, W.C., Rew, R.S., 1986. Chemistry of antinematodal agents. In: Chemotherapy of Parasitic Diseases. Plenum Press, New York, pp. 239–266. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty per cent endpoints. Am. J. Hyg. 27, 493–497. Taira, N., Ura, S., 1991. Sudden death in calves associated with Strongyloides papillosus infection. Vet. Parasitol. 39, 313–319. Taira, N., Yoshihara, S., Ikeda, I., Ueno, H., 1977. A simple method for screening molluscicidal activity of pesticides using Lymnaea ollula egg. Bull. Natl. Inst. Anim. Health 75, 16–22 (in Japanese, English summary in Natl. Inst. Anim. Health Q. 17, 185 (1977)). Taira, N., Minami, T., Smitanon, J., 1991. Dynamics of faecal egg counts in rabbits experimentally infected with Strongyloides papillosus. Vet. Parasitol. 39, 333–336. Taira, N., Hirooka, M., Saeki, H., 1994. Isolation of Strongyloides venezuelensis from Rattus norvegicus in Kagoshima Prefecture. J. Vet. Med. Sci. 56, 255–258. Tsuji, N., Nakamura, Y., Taira, N., 1993. Long-lasting parasitism of Strongyloides venezuelensis in Mongolian gerbils (Meriones unguiculatus). J. Parasitol. 79, 305–607.