J Arthropod-Borne Dis, December 2013, 7(2): 188–193 A Veysi et al.: Laboratory Evaluation of … http://jad.tums.ac.ir Published Online: August 31, 2013 Short Communication Laboratory Evaluation of a Rodenticide-insecticide, Coumavec®, against Rhombomys opimus, the Main Reservoir Host of Zoonotic Cutaneouse Leishmaniasis in Iran Arshad Veysi 1, Hassan Vatandoost 1, Mohammad Hossein Arandian 2, Reza Jafari 2, Mohammad Reza Yaghoobi-Ershadi 1, Yavar Rassi 1, *Amir Ahmad Akhavan 3,1 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Esfahan Health Research Station, National Institute of Health Research, Tehran University of Medical Sciences, Esfahan, Iran 3Institute for Environmental Research (IER), Tehran University of Medical Sciences, Tehran, Iran (Received 11 Feb 2013; accepted 22 Apr 2013) Abstract Background: Zoonotic cutaneous leishmaniasis is a growing health problem in many rural areas of Iran. Rhombo- mys opimus, the great gerbil, is the main animal reservoir of ZCL in the northeast and central part of Iran. The aim of the current study was to evaluate the rodenticidal effect of Coumavec® (a mixture of Coumatetralyl 0.5% and Etofenprox 0.5%) on R. opimus under laboratory condition. Methods: Great gerbils were collected from Sejzi rural district, Esfahan Province, Iran. Four groups of 19 great ger- bils were treated with the poisoned baits of different concentrations and one group was considered as control. The bating procedure was conducted in three stages: first, second (a week after first) and third (a month after first stage), in each stage baits were offered in 1 day, based on national protocol for rodent control operation in purpose of ZCL control. Results: The mortality rate for 0.03, 0.0625, 0.125 and 0.25% concentrations in the first stage of baiting were ob- tained 36.8%, 31.5%, 52.6% and 36.8%, in the second stage 47.3%, 52.6%, 68.4% and 52.6%, and in the third stage 52.6%, 63.1%, 68.4% and 57.8% respectively. The maximum and minimum mortality has occurred in 5-6 days and 31-40 days intervals consequently. Conclusion: The results of this study showed that, Coumavec® has some rodenticidal effects on R. opimus in labo- ratory condition. For the appropriate rodenticide-insecticide contamination of the rodent body and also considering to the economic issues, we suggest the use of 0.125% concentration for rodent control operation in the field condition. Keywords: Rhombomys opimus, Rodenticide, Coumatetralyl, Zoonotic cutaneous leishmaniasis, Iran Introduction Zoonotic cutaneous leishmaniasis (ZCL) is a growing health problem in many rural areas of Iran, which involves 17 out of 31 provinces of the country (Yaghoobi-Ershadi 2012). Ro- dents, especially those belonging to gerbilli- nae subfamily have significance role as res- ervoir hosts of zoonotic cutaneous leishma- niasis (Yaghoobi-Ershadi 2008, Yaghoobi- Ershadi and Javadian 1996). In addition, different kinds of pathogens like bacteria, rickettsia, viruses, protozoa and hel- minthes can be transmited by rodents to human and animals (Bell et al. 1988). Phle- botomus papatasi is the main vector of ZCL and Leishmania major is the causative agent of the disease in the most parts of the country (Yaghoobi-Ershadi 2012). Rhombomys opimus, the great gerbil, is the main animal reservoir *Corresponding author: Dr Amir Ahmad Akhavan, E-mail: aaakhavan@tums.ac.ir 188 J Arthropod-Borne Dis, December 2013, 7(2): 188–193 A Veysi et al.: Laboratory Evaluation of … http://jad.tums.ac.ir Published Online: August 31, 2013 of ZCL in the northeast and central part of Iran (Yaghoobi-Ershadi and Javadian 1996). This gerbil is relatively large and has diurnal activity whereas the most other species have nocturnal activity (Dubrovskiy 1979). A karyo- type study revealed that diploid chromosome number for R. opimus was 2n=40 (Akhavan et al. 2004). Rhombomys opimus is an herbi- vores rodent and feeds on green dried leaves as well as stems of plants. This species has social life and lives with other members of the colony in their burrows. This species has 1 to 2 and sometimes 3 generations in spring and 1 more generation in summer or fall. They have 5–14 offsprings in each genera- tion. The longevity of this species reaches to 3 years. This long longevity provides appro- priate condition for the maintenance of Leishmania parasites in the rodent popula- tion while sand flies are inactive. Many eco- logical characters of great gerbils and pres- ence of sand flies, directly or indirectly, ef- fect the leishmaniasis cycle in the nature (Bell et al. 1988). Attempts to control of leishma- niasis in the field condition by rodent control operation have been conducted in some coun- tries including Iran. In central Asia, a large- scale operation against great gerbils through poisoned baits successfully eliminated the rodents (Dergacheva and Zherikhina 1980). Along April to January 1997, a field trial car- ried out to control ZCL by destruction rodent burrows and using zinc phosphide 2.5% baits in radius of 500 meters from houses in cen- tral Iran. The results showed that the control program reduced the incidence of ZCL 12- folds at the end of the first year of the opera- tion and 5-folds at the end of the second year (Yaghoobi-Ershadi et al. 2000, Yaghoobi- Ershadi et al. 2005). Recently some behav- ioural resistance and/or bait shyness against the conventional rodenticide, zinc phosphide, among the population of great gerbils has been reported from some endemic areas of the dis- ease (unpublished data, Esfahan Health Re- search Station, Iran). It is necessary to intro- duce some new, safer and more effective al- ternative rodenticides to control the reservoir hosts and subsequently the disease in en- demic areas of ZCL in Iran. Furthermore, Coumavec® has insecticidal ef- fect on the ectoparasites and other blood feed- ing insects of the gerbils but the aim of the current study was the evaluation of roden- ticidal effect of Coumavec® (a mixture of Coumatetralyl 0.5% and Etofenprox 0.5%) on R. opimus in laboratory condition. Materials and Methods Rodent collection Active colonies of gerbils were identified in Sejzi rural district (32˚39˙54.84”N/ 52˚08˙07.38”E), Esfahan Province, Iran. Sherman live traps were placed near the rodent holes from January to February 2010. Around 80 to 100 live traps baited with cu- cumber and sometimes carrot were used in each day. They were set up daily in the early morning to evening in winter. The collected gerbils were transferred to the animal house of Esfahan Health Research Station. Differ- ent morphologic criteria of rodents were used for R. opimus identification. Captured ro- dents were identified by valid identification keys (Etemad 1978). Just R. opimus species were included in the study. All the animals, which were used in this study, had no ex- perience of exposing to the rodenticides. Laboratory tests All experiments were conducted with ani- mals maintained individually in metal cages. Five groups of 19 great gerbils per group were sexed and selected, four groups were treated with the poisoned baits of each con- centration and one group was as control. The commercial formulation of Coumavec®, a mix- ture of Coumatetralyl 0.5% and Etofenprox 0.5% (Levant Overseas Development Ltd, Argenteul, France) which was evaluated in 189 J Arthropod-Borne Dis, December 2013, 7(2): 188–193 A Veysi et al.: Laboratory Evaluation of … http://jad.tums.ac.ir Published Online: August 31, 2013 these tests, recommended for direct applica- tion as a rodenticide dust or for dilution in bait. Poisoned baits were prepared using a mixture of grain and four concentration of Coumavec® (0.03, 0.0625, 0.125 and 0.25%). Sexually mature animals were acclimatized to the laboratory condition at least 7 days. The bating procedure was conducted in three stages: first, second (a week after first) and third (a month after first stage), in each stage baits were offered in 1 day, based on na- tional protocol for rodent control operation in purpose of ZCL control. Along the keep- ing animals in the animal house, before the tests, they were fed on the standard animal diet (pellet). For providing required water, they were offered carrot. In each stage, the diets were withdrawn and a fresh container, holding 15 gr of poised bait for each rodent, was placed in the cages. The ordinary diet was not resumed until complete consump- tion of poison bait. The gerbils were then fed on ordinary laboratory diet until the next stage of baiting. Dead animals were exam- ined for internal bleeding and other symptoms of anticoagulant poisoning. Gerbils, which survived during the test period, were main- tained on laboratory diet, and those still alive 30 days after third offered poisoned bait, were considered as survived individuals. Animal ethics consideration Animal experiments were approved by the Ethical Committee of Tehran University of Medical Sciences, Tehran, Iran. Data analysing The data were analyzed using SPSS 11.5 and graphs were prepared using Excel. The cumulative mortality rate of gerbils among the treated and control groups was compared using Chi-squared test. Results Totally 95 healthy-mature rodents were used in this investigation. All dead animals were examined for internal bleeding and other symptoms of anticoagulant poisoning (Fig. 1). The results of toxicity test based on con- centration in each stage using Coumavec® is shown in Fig. 2. The mortality rate for 0.03, 0.0625, 0.125 and 0.25% concentrations in the first stage of baiting were obtained 36.8%, 31.5%, 52.6% and 36.8%, in the second stage 47.3%, 52.6%, 68.4 and 52.6%, and in the third stage 52.6, 63.1, 68.4 and 57.8% respectively. The maximum and minimum mortality has occurred in 5–6 days and 31–40 days intervals consequently. The maximum rate of mortality for female and male was ob- served in 0.0625 and 0.125% concentrations consequently (Fig. 3). In spite of more mor- tality rate in 0.125% concentration, statis- tical differences between the concentrations have not seen. The number of dead rodents in the intervals between 1–5, 6–10, 11–15, 16–30 and 31–40 were summarised in Table 1. As the table shows, the maximum and minimum mortal- ity has occurred in 5–6 and 31–40 interval consequently. The statistical analyses showed that there were no significant differences between four selected concentrations of Coumavec® on the rodent’s mortality rate. Fig. 1. Internal bleeding of a Rhombomys opimus treated by Coumavec® 190 http://jad.tums.ac.ir Published Online: August 31, 2013 J Arthropod-Borne Dis, December 2013, 7(2): 188–193 A Veysi et al.: Laboratory Evaluation of … http://jad.tums.ac.ir Published Online: August 31, 2013 Table 1. Mortality time of the tested rodents with Coumavec® under laboratory condition in each stage of the baiting Concentration Total 1–5 6–10 11–15 16–30 31–40 0.03 19 8 (41.1%) 0 (0%) 1 (5.2%) 1 (5.2%) 0 (0%) 0.625 19 6 (31.5%) 1 (5.2%) 2 (10.5%) 3 (15.78%) 0 (0%) 0.125 19 6 (31.5%) 4 (21%) 2 (10.5%) 0 (0%) 1 (5.2%) 0.25 19 6 (31.5%) 3 (15.7%) 0 (0%) 1 (5.2%) 1 (5.2%) Control 19 0 0 0 0 0 Fig. 2. Mortality rate of the tested rodents with Coumavec® in laboratory condition in each stage of the baiting Fig. 3. Mortality rate of the tested rodents with Coumavec® in laboratory condition based on sex Discussion The results showed that maximum mortal- ity occurred along 1–5 days after first bait- ing, slightly more than 40 percent for 0.25, 0.125, and 0.0625% and over 40 percent for 0.03% concentrations. It is suggested that the later baiting stages did not have dramatic effects on the mortality. The complete mor- tality was not obtained for all concentration as well. The maximum mortality was slightly more than 68% and the minimum was al- most 52% occurred in 0.125 and 0.03% con- centration, consequently. In addition Gill and Redfern (Gill and Redfern 1983) showed that complete mortality with Coumatetralyl (15 days feeding, in 0.0375% concentration) against Meriones shawi were not obtained. 191 J Arthropod-Borne Dis, December 2013, 7(2): 188–193 A Veysi et al.: Laboratory Evaluation of … http://jad.tums.ac.ir Published Online: August 31, 2013 All concentrations have an increasing mor- tality trend, as prepared baits offered in each stage, the mortality increased consequently. In dissected dead animals, the expected or- gans were checked. Bleeding mostly occurred in stomach and after that extent to the intes- tine, spleen and liver. Therefore, all concentrations can be used in the field operations but in view of the fact that this pesticide is an anticoagulant-insec- ticide mixture, for the appropriate rodenti- cide-insecticide contamination of the rodents’ body and also considering to the economic issues, this article suggests 0.125% concen- tration for rodent control operation in the field condition. Acknowledgements Authors would like to express their appre- ciation to the staff of Esfahan Health Station, National Institute of Health Research, Teh- ran University of Medical Sciences (TUMS) for their collaboration along the study. In ad- dition, the authors are grateful to Levant Over- seas Development (LOD), Ltd, France for providing and sending Coumavec® to con- duct this project. This research was finan- cially supported by School of Public Health, TUMS, Institute for Environmental Research (IER), TUMS and Department of Zoonosis, CDC, Ministry of Health and Medical Edu- cation, Islamic Republic of Iran. 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