Year : 2007 | Volume
: 52 | Issue : 1 | Page : 43--47
Larvicidal efficacy of essential oil of betel leaf ( Piper betle ) on the larvae of the old World screwworm fly, Chrysomya bezziana in vitro
April H Wardhana1, Sujith Prasad W Kumarasinghe2, LDAM Arawwawala3, Lakshmi SR Arambewela3,
1 Research Institute for Veterinary Science, Bogor, Indonesia
2 Colombo North Teaching Hospital, Ragama, Sri Lanka (Currently at National Skin Centre, Singapore)
3 Industrial Technology Institute, Colombo, Sri Lanka
Sujith Prasad W Kumarasinghe
National Skin Centre, No. 1, Mandalay Road, Singapore - 308205
Larvae of Chrysomya bezziana are the commonest cause of wound myiasis in some parts of the world. This obligatory parasite is important in humans and in commercial livestock. Kumarasinghe et al have reported that essential oil of betel leaf (EOBL) is larvicidal to C. megacephala but there are no publications on its effect on C. bezziana . This study was done to evaluate the efficacy of essential oil of betel leaf ( Piper betle ) against the larvae of C. bezziana in vitro . EOBL was prepared at the Industrial Technology Institute Colombo, Sri Lanka, according to a standard protocol. The experiment on larvae was carried out at the Research Institute for Veterinary Sciences in Bogor, Indonesia. EOBL concentrations of 2%, 3% and 4% were prepared with Tween 80. Two ml of 4%, 3% and 2% EOBL in 1% Tween 80 (v/v/aq) were poured into separate Petri dishes. Ten 1st and 2nd instar larvae were placed in each Petri dish. Asuntol (Chaumaphos) 1% was used as positive control and distilled water with 1% tween 80 was the negative control. Larval mortality was assessed half-hourly. The experiment was repeated five times and averages were compared. Sustained immobility of the larvae, after exposure to the relevant substances was considered as death. The efficacy of EOBL depended on, the stages of C. bezziana larvae and the concentration. With 4% EOBL, all first instar larvae were killed within two hours and the second instar larvae were killed by four hours. The positive control showed no mortality until four hours but all larvae were weak, from the first 30 minutes. In the negative control, larvae were mobile and active. EOBL 3% killed all the first instar larvae by 150 minutes and 74% of the second instar at four hours. By 210 minutes, 2% preparation had killed 100% of the first instars. EOBL is an effective larvicidal for C. bezziana first and second instar larvae in vitro . This natural product has a great potential to be developed as a novel larvicide against this parasite.
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Wardhana AH, Kumarasinghe SW, Arawwawala L, Arambewela LS. Larvicidal efficacy of essential oil of betel leaf ( Piper betle ) on the larvae of the old World screwworm fly, Chrysomya bezziana in vitro.Indian J Dermatol 2007;52:43-47
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Wardhana AH, Kumarasinghe SW, Arawwawala L, Arambewela LS. Larvicidal efficacy of essential oil of betel leaf ( Piper betle ) on the larvae of the old World screwworm fly, Chrysomya bezziana in vitro. Indian J Dermatol [serial online] 2007 [cited 2020 Aug 10 ];52:43-47
Available from: http://www.e-ijd.org/text.asp?2007/52/1/43/31924
Larvae of Chrysomya bezziana are the commonest cause of wound myiasis in some parts of the world., This obligatory parasite is not only important in humans but also in commercial livestock.,, Chrysomya bezziana larvae are not suitable for maggot debridment therapy as the larvae destroy the normal healthy tissue as well. Development of a nontoxic natural product as a treatment would be of immense value as there is no uniformity effective safe product for cutaneous myiasis. Kumarasinghe et al have reported that essential oil of betel leaf is effective in killing larvae of the facultative parasite C. megacephala , but there have been no published reports on its effects in C. bezziana , which is an obligatory parasite.
The objective of this study was to assess the in vitro efficacy of essential oil of the betel leaf ( Piper betle ) in killing C. bezziana larvae.
Materials and Methods
A Chrysomya bezziana culture was maintained at the Department of Parasitology, Research Institute for Veterinary Science (Balitvet), Bogor Indonesia. The establisment and maintenance of the colony has been previously described by Sukarsih.
Essential oil of betel
The betel leaves were collected from Kalutara area, 22 miles South of Colombo, in Sri Lanka. Essential oil of betel was prepared at the Industrial Technology Institute (ITI) Colombo, Sri Lanka, using a standard distillation process. Briefly; crushed betel leaves were steam-distilled for five hours, using a Clevenger arm, to obtain the volatile essential oil of betel leaf. This oil was used for the study carried out at the laboratory in the Research Institute of Veterinary Sciences in Bogor Indonesia.
In vitro assay
The procedure used was similar to that described for C. megachepala larvae with a little modification. Betel essential oil concentrations of 2%, 3% and 4% were prepared with Tween 80. Two hundred (200) microlitres each of 4%, 3% and 2% of essential oil of betel leaf (EOBL) in 1% Tween 80 (v/v/aq) were poured into separate Petri dishes (35x10 mm, FalconŽ). Ten first and second-instar larvae were placed in each Petri dish. One percent Asuntol (Coumaphos); a known active agent against fly larvae, was used as the positive control and distilled water with 1% Tween 80 solvent was used as the negative control. Larval mortality was assessed half-hourly for four hours, as before. The experiment was repeated five times and arithmetic means were taken for assessment of larvicidal effect. Sustained immobility of the larvae, after exposure to the relevant substances, was considered as death of the larvae.
The efficacy of betel essential oil depended on; both the stage of C. bezziana larvae and the concentration. All the first instar larvae that were exposed to 4% of betel essential oil were killed within two hours [Table 1] and [Figure 1][Figure 2] and the second instar larvae were killed by four hours [Table 2]. The positive control showed no mortality until four hours but all the larvae were weak from the first 30 minutes onwards. In the negative control, the larvae were mobile and active. EOBL 3% preparation killed all the first instar larvae by 150 minutes and 74% of the second instar at four hours. By 210 minutes, 2% preparation killed 100% of the first instar larvae.
The old World screw-worm fly, Chrysomya bezziana , is an obligate parasite of all warm-blooded animals including humans. The infestation of this larvae on the host tissue is called myiasis. It is common in many tropical countries throughout Africa, South East Asia and the Indian Subcontinent.,,,,, Poor hygiene, working in contaminated areas, particularly during warm seasons and inability to prevent files from depositing ova on open wounds etc. provide a setting for infestation with this parasite. Clinical manifestations of myiasis are not specific and vary according to the involved area of the body and the extent of infestation. Fever, chills, pain, bleeding from infested site, secondary infection, neutrophil leukocytosis or hypereosinophilia may be seen in more severe cases. While wound myiasis in humans is not a major medical problem in many countries, animals myiasis especially on sheep and cattle is of major economic importance. In tropical countries, human myiasis is not uncommon. As serious complications are possible if human myiasis is neglected,, it deserves more medical and research attention. Also there is a potential danger of further spread of Chrysomya beyond the traditional regions where Chrysomya species has been found, considering the recordings of this fly in various countries of the world.
Many methods have been employed for myiasis treatment with variable success.,, Turpentine has been used in some countries and a topical formulation containing 1% ivermectin in propylene glycol is reported to effective. Therapeutic modalities including sterile oil and 15% chloroform have also used. Surgical removal of larvae is another method employed where larvae cannot be extracted out of the existing opening of the wound. Kumarasinghe et al have reported on the in vitro larvicidal efficacy of low aromatic white spirits (LAWS), which is the main ingredient of mineral turpentine, against C. megacephala .
Much of research on larvicides of Chrysomya larvae have been carried out on animals such as sheep and cattle.,,,,,,,, In veterinary studies, several products have been tried against C. bezziana larvae. Spradbery et al have reported that organophosphates coumaphos, fenchlorphos, diazinon, chlorfenvinphos and fenthion methyl are useful. Eprinomectin and doramectin are some of the macrolcyclic lactones that have proven some efficacy against C. bezziana larvae. Perkins has reported that ivermectin, 1% diclofenthion plus gention violet and 3% lindane plus pine oil smear were all effective in preventing wound infection by C. bezziana . Subcutaneous ivermectin has also been used experimentally in cattle. Immunological control of the ectoparasite has received much attention in the recent past. However, there have been no breakthroughs with regards to vaccines against C. bezziana . A study done in collaboration with the Commonwealth Scientific and Industrial Research Organization (CSIRO), Australia and Research Institute for Veterinary Science (Balitvet), in Bogor Indonesia to develop a vaccine against C. bezziana showed that a recombinant vaccine was not protective in sheep, against infestation with C. bezziana larvae., Many products, such as organophosphates, used in veterinary studies would not be acceptable in humans. Therefore, our attention was drawn to finding a safe natural product for treatment of myiasis.
In our study, the efficacy of betel essential oil depended on; both stages of C. bezziana larvae and the concentration. All the first instar larvae exposed to 4% EOBL were killed within two hours and the second instar larvae were killed by four hours. The positive control showed no mortality until four hours, but all larvae were weak from first 30 minutes. In the negative control, the larvae were unaffected. EOBL 3% preparation killed all the first instar larvae by 150 minutes and 74% of the second instar larvae at four hours. By 210 minutes, 2% preparation had killed 100% of the first instar larvae. The low efficacy of the positive control coumaphos is probably due to the low concentration (1%) used. A higher percentage(2%) of coumaphos was larvicidal to C. bezziana in a subsequent experiment(Hariwardhana and Kumarasinghe unpublished data).
Piper betle leaf is one of the most commonly used plants as a masticate in Asia. It is claimed to have antibacterial, fungicidal, as well as deodorant properties due to its essential oils. Essential oil of betel leaf also has been reported to have insecticide properties.,,, Its larvicidal effects on mosquitoes and C. megacephala have been investigated., Kumarasinghe et al reported that essential oil of Piper betle is effective in vitro against the larvae of the facultative parasite C. megacephala . But this paper is the first report on its efficacy on the obligatory parasite C. bezziana larvae in vitro .
There are no definite detrimental effects attributable to betel leaf in humans or other mammals, although the arecanut, which is consumed with betel leaf in some communities have been reported to be carcinogenic. Recently there have been reports of antioxidative protective effects of betel leaf extract in in vitro and in vivo experiments in rats. These studies suggest that betel leaf derivatives would be good alternatives to be used as a safe product on wounds infested by larvae of Chrysomya in higher animals and humans. An in vivo experiment on Chrysomya infested sheep is underway. The fact that betel leaf is a harmless natural product which has been used by humans for millennia of years, makes it more promising compared to other products. Further large-scale studies would be needed before recommending it for use in humans.
Efficacy of betel essential oil depends on the concentration and the stage of C. bezziana larvae. The 4% concentration killed all the first instar larvae by two hours and the second instar larvae by four hours.. The potential of betel essential oil for treatment of wound myiasis should be harnessed. This will not only benefit treatment of human myiasis, but also myiasis of the cattle and sheep, which is of agro-economic importance. We have planned an in-vivo study on sheep in this respect.
The authors would like to thank Sandhy Santosa and Eko Prasetyo for their assistance.
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