J Arthropod-Borne Dis, June 2019, 13(2): 214–223 S Madreseh-Ghahfarokhi et al.: Zingiber officinalis and … 214 http://jad.tums.ac.ir Published Online: June 24, 2019 Original Article Zingiber officinalis and Eucalyptus globulus, Potent Lethal/Repellent Agents against Rhipicephalus bursa, Probable Carrier for Zoonosis Samin Madreseh-Ghahfarokhi1; *Amir Dehghani-Samani2,3; Yaser Pirali4; Azam Dehghani-Samani5 1Department of Clinical Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran 2Faculty of Medicine, Birjand University of Medical Sciences, Birjand, Iran 3Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran 4Department of Pathobiology, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran 5Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran (Received 20 Jun 2017; accepted 17 June 2019) Abstract Background: Rhipicephalus bursa is a hard tick with importance in transmission of tick-borne diseases and zoono- sis. Natural products are excellent alternative to pesticides. In this study for the first time, lethal and repellent activity of Zingiber officinalis and Eucalyptus globulus against Rh. bursa were studied. Methods: In July till September of 2017, essential oils were extracted in Shahrekord University, Iran from fresh plant ma- terials and engorged mature ticks were collected from infested sheep herd located in Saman, Iran. Ticks were chal- lenged by different concentrations of essential oils including 300, 500 and 800µl/ml and 1 (pure) singly and/or in combination. Percentages of killed and repellent ticks as efficacy of acaricidal and repellent activity of essential oils against Rh. bursa were calculated and analyzed statistically. Results: Efficacy of eucalyptus essential oils was more in whole concentrations and its highest performance was observed in concentration 1 (pure). Efficacies of ginger and combined essential oils were different for each concen- tration but their highest efficacies were observed in concentration 1 (pure), too. Conclusion: This study showed considerable values of acaricidal and repellent activity against Rh. bursa for both essential oils singly and in combination, so they can be considered as potent lethal/repellent agents for control of ticks, but more studies need for this purpose, study on lethal/repellent activity of these essential oils and/or other plants against other important arthropods can be considered as subjects for next experiments. Keywords: Acaricidal activity; Essential oil; Eucalyptus; Ginger; Repellent activity Introduction Ticks can transfer some of important dis- eases that named tick-borne diseases (1). Rhip- icephalus bursa is a hard tick from the genus Rhipicephalus which is the fourth largest in the Family Ixodidae and has an important role in transferring of different diseases (2). Sheep, goats, cattle, horses are the preferred hosts of Rh. bursa, it is a two-host species, usually has a mono-tropic type of behavior, with immature stages feeding on the same host species as the adult tick stages. However, the immature- stages can also feed on rodents and rabbits (3). Rhipicephalus bursa is an important mem- ber of common tick fauna in different part of Iran (4–6) and several important microorgan- isms including: Crimean Congo hemorrhagic fever (CCHF) virus (7), Ehrlichiosis bacteria (8), Anaplasma phagocytophilum (9), Rickett- sia aeschlimannii (10) and other bacteria (11) were isolated repeatedly from Rh. bursa. Use of chemical pesticide agents is in- creasing every day in order to control ticks populations. About 2.5 million tons of pesti- cides are used on crops each year and the *Corresponding author: Dr Amir Dehghani-Samani, E-mail: amirds2008@gmail.com mailto:amirds2008@gmail.com J Arthropod-Borne Dis, June 2019, 13(2): 214–223 S Madreseh-Ghahfarokhi et al.: Zingiber officinalis and … 215 http://jad.tums.ac.ir Published Online: June 24, 2019 worldwide damage caused by pesticides reach- es $100 billion annually. The reasons for this are: (a) the high toxicity and non-biodegrada- ble properties of pesticides and (b) the resi- dues in soil, water resources and crops that affect public health (12). Addition to environmental concerns, there are several reports about insecticide-resistant against chemical pesticides like Pyrethroid and Propetamphos in ticks including Rh. bursa (13, 14). Thus, searching for replace of chemical pesticides is necessary in order to firstly solve the problem of long term toxicity to mammals and secondly to find environmental friendly pesticides and develop techniques used to re- duce pesticide use while maintaining crop yields. Natural products are an excellent al- ternative to chemical insecticides. Several plants contain compounds that they use in preventing attack from arthropods and/ or insects. These chemicals fall into several categories, including repellents, feeding de- terrents, toxins, and growth regulators (15). Ginger (rhizome of Zingiber officinale Ros- coe) is one of the most widely used herbal med- ications in oriental medicine against pain, in- flammation, stomach problems, nausea, vom- iting, epilepsy, sore throat, cough, common cold, bruises, wounds, liver complaints, rheu- matism, muscular pains, atherosclerosis, mi- graine headaches, high cholesterol, ulcers, and etc. (16). Ginger essential oil can produce from fresh rhizomes and it has many efficient ef- fects like antibacterial, antiviral, antifungal and other properties (17, 18). Moreover, the genus Eucalyptus that knows by over 700 species distributed throughout the world (19), provides variety of components extracted from its essential oil known as in- secticide and repellent agent (20). Antimicro- bial and antioxidant activity of eucalyptus es- sential oil were reported (21, 22), also effect of eucalyptus essential oil on respiratory bac- teria and viruses, traditional use of eucalyptus in treatment of rhino-sinusitis and anti-diabetic effect of eucalyptus were identified (23–25). There is no study on lethal and repellent ac- tivity of Z. officinalis and E. globulus essen- tial oils against Rh. bursa, and this is the first survey in this way. In this study acaricidal and repellent activity of essential oils of gin- ger and eucalyptus against Rh. bursa as a wide- distributed species of ticks in different parts of Iran with an important role in transmission of zoonosis were studied singly and in combi- nation with together. Materials and Methods Extraction of essential oils Current study were done from July till Sep- tember of 2017, fresh rhizomes of Z. offici- nalis and leafs of E. globulus were used to essential oil extraction. Fresh rhizomes of Z. officinalis were prepared from the shops and fresh leafs of E. globulus were collected in summer (July 2017) from grown trees located in Najafabad City, Isfahan Province in center of Iran (32°50′43″N 51°36′00″E). Essential oils extractions were done separately for each sample via conventional hydrodistillation meth- od. Hydrodistillation in a Clevenger-type ap- paratus consists of immersing the ground plant material directly in a flask filled with water that is then brought to the boil. Vapors carry volatile compounds and the condensate drops on the pentane/ether trap in the inner tube of the apparatus where the volatiles are retained. In current study, 200gr of each sample were crushed and added to 800ml distilled water in a round bottom flask. The flask was heated and the Clevenger apparatus was attached. The mixture was boiled at 100 °C and then the tem- perature was reduced to 60 °C and kept for 3 h, the recovered mixture was allowed to settle and finally essential oil was withdrawn for each sample separately (26). Collection of ticks Mature engorged male and/or female ticks J Arthropod-Borne Dis, June 2019, 13(2): 214–223 S Madreseh-Ghahfarokhi et al.: Zingiber officinalis and … 216 http://jad.tums.ac.ir Published Online: June 24, 2019 (disregard of their sex) were collected from a highly infested sheep herd located in the Sa- man City, Chaharmahal and Bakhtiari Prov- ince, southwest of Iran (32°27′06″N 50°54′ 38″E). Infestation was so high in the herd and ticks were mostly presented around or inside of outer ear, perineal area and around the vent and under the groin area, ticks dis-attaching was done via sterile forceps, without any palpation and with the low pressure of forceps and their rotation. Guard glass, disposable dress, and multi-layer disposable gloves were used dur- ing the work and whole the health concerns were considered. In order to species-specific collecting, morphological aspects of Rh. bur- sa species like color were considered during the collecting procedure. Collected ticks were immediately transferred to specific glass box- es, kept in cool place away from any chemi- cal agents and sunlight, transferred to labora- tory for experiment. Note that dis-attaching of ticks was a difficult procedure and some ticks were injured during the collecting or transfer- ring, that they were removed for experiment. Totally, about 700 ticks were collected. Con- firmed species identification was done under the laboratory optic loop via identification keys for tick's species (3), also health of ticks was controlled again under the loop and probable injured ticks were removed. Differentially, 93 % of collected ticks were identified as Rh. bur- sa and about 7% were identified as other spe- cies of hard ticks. Finally, 640 engorged health ticks were prepared for experiment, in order to deletion of biases in experiment, collected ticks were immediately examined after col- lection and till the experiment, ticks were in- cubated in 25 °C and 80% humidity, in com- mon glass box with air circulation. Treatments preparation Concentrations of 300, 500 and 800µl/ml and 1 (pure) were prepared from essential oils of E. globulus and Z. officinalis separately via combination by different amount of normal saline, also mixed concentrations of 150µl/ ml Eucalyptus plus 150µl/ml ginger, 250µl/ ml Eucalyptus plus 250µl/ml ginger, 400µl /ml Eucalyptus plus 400µl/ml ginger and 500 µl/ml Eucalyptus plus 500µl/ml ginger were prepared with the same method. In order to sure of tests validity, a positive control group with concentration of 100mg/ml was prepared via dilution of 25ml of Ripcord ® (Cyperme- thrin 40%, Spiagri Company, Tehran, Iran) in 75ml normal saline, also a negative control group was prepared from pure normal saline without any additive components. Evaluation of acaricidal activity of essential oils Three replication tests were done for every treatment (including positive and negative con- trol groups) and for each replication 13 health ticks were transferred to plastic nets and dipped in different concentrations of essential oils, and/or positive and negative control solution, with same temperature (25 °C) for 30sec, then they were transferred to specific glass dishes with same condition including humidity, light and air circulation for all of treatments and then were incubated in 25 °C and 80% humidity for two hours. Note that all the methods were same for every groups and replications. After the two hours, percentage of died ticks as ac- aricidal efficacy of each treatment were count- ed and mean of efficacy for each treatment were calculated and analyzed statistically. For sure whole of died ticks were carefully ob- served under the laboratory optic loop. Evaluation of the repellent activity of essen- tial oils Repellent activity of each essential oils against Rh. bursa in concentration of 1 (pure) singly and 500µl/ml eucalyptus plus 500µl/ ml ginger in combination were studied by Y- tube olfactometer bioassay. Y-tube olfactom- eter consists of a glass Y-tube with the main arm (the stem) and 2 arms containing one re- J Arthropod-Borne Dis, June 2019, 13(2): 214–223 S Madreseh-Ghahfarokhi et al.: Zingiber officinalis and … 217 http://jad.tums.ac.ir Published Online: June 24, 2019 pellent and control in another one, where a low rate air movement is created by sucking the air in the two arms of the Y-tube with a pump connected to the stem. The essential oil sample and control are applied on a paper attached to the arms of the tube. Arthropods are introduced into the tube by a hole located at the center (the joint point of the three tubes). After introduction, the hole is closed with a rubber stopper and the pump is oper- ated. After specific time of exposition, the number of arthropods on each of the 2 tubes (treated and control) are scored to assess the percentage of repellency (27, 28). In this study, triplicate tests were done and in each replica- tion 13 ticks were put into every arm and 10 ml of each mentioned treatments was added into treated tube and nothing into control tube. Hard ticks are slow, so more time considered and five hours after pumping, the number of repellent ticks was reported as repellent per- centage of treatment and control groups. Note that after each application of Y-tube olfac- tometer, it was cleaned, washed and dried for next examination. Statistical analysis The analyzed data were expressed as the Mean±standard error of the mean (SEM) us- ing Sigma stat (ver. 3.1) software. Groups were compared using one-way ANOVA for repeat- ed measurements. A value of (P≤ 0.05) was considered significant. Results Acaricidal activity of essential oils Results of current study showed different amounts of efficacy (lethal effect) for differ- ent concentration of essential oils, singly and in combination. Highest acaricidal activities against Rh. bursa were observed for concen- tration 1 (pure) of eucalyptus and gingers es- sential oils singly. The most potent treatment (about 54%) was eucalyptus essential oil in concentration 1 (pure) and the lowest activi- ty (about 8.5%) was observed for combined essential oils in concentration 150+150 µl/ml of essential oils. Significant differences (P≤ 0.05) between different concentrations of eu- calyptus essential oil were also observed and the highest increase in its acaricidal activity was observed between concentration 800µl/ ml and 1 (pure) of this essential oil. Ginger es- sential oil also had different values o acari- cidal activity in different concentrations, its highest activity was observed in concentra- tion 1 (pure) too, but its activity had equal in- crease between different concentrations and the highest increase in its acaricidal activity of combined essential oils was observed be- tween concentrations mixed 150+150µl/ml and mixed 250+250µl/ml of essential oils. Table 1 shows different values of acaricidal activi- ty in different concentrations of essential oils singly and in combination. Comparison between efficacies of different essential oil treatments in each concentration shows that eucalyptus essential oils were the most potent agents than others in whole con- centrations. Efficacies of ginger essential oils in different concentration were between the efficacies of eucalyptus and combined essen- tial oils except for concentration 500µl/ml that efficacy of combined essential oils was inter- mediate. Performances of different treatments in each concentration are compared in Fig. 1. Different values of repellent activity were observed for different essential oils group ver- sus control group. Eucalyptus essential oil had the highest repellent activity significantly (P≤ 0.05) against Rh. bursa and other groups had different efficacies. Comparison between re- pellent activities of examined groups is shown in Fig. 2. J Arthropod-Borne Dis, June 2019, 13(2): 214–223 S Madreseh-Ghahfarokhi et al.: Zingiber officinalis and … 218 http://jad.tums.ac.ir Published Online: June 24, 2019 Table 1. Acaricidal activity (Mean±SEM)% of different concentrations for each treatment (essential oils) versus negative and positive control groups Groups Z. officinalis Combined E. globulus Negative Control 0*a 0a 0a 300µl/ml singly or mixed 150µl/ml 11.47±0.29b 8.53±0.86b 15.21±1.09b 500µl/ml singly or mixed 250µl/ml 16.73±1.54c 21.38±2.92c 24.38±0.42c 800µl/ml singly or mixed 400µl/ml 28.36±2.09d 25.62±1.37c 37.65±2.64d 1 (pure) singly or mixed 500 µl/ml 39.08±1.29e 33.75±2.83d 53.83±3.14e Positive Control 100f 100e 100f *Presence of different superscript lowercase letters (a-f) shows the significant differences (P≤0.05) between different concentrations (rows) of each essential oil treatment (column). Fig. 1. Comparison between acaricidal activities of different treatments in each concentration. Presence of different lowercase letters (a–f) in each concentration shows the significant differences (P≤ 0.05) between different essential oils (lines). Fig. 2. Repellent activities of different essential oils versus control group. Presence of different uppercase letters (A– D) shows the significant differences (P≤ 0.05) between groups. J Arthropod-Borne Dis, June 2019, 13(2): 214–223 S Madreseh-Ghahfarokhi et al.: Zingiber officinalis and … 219 http://jad.tums.ac.ir Published Online: June 24, 2019 Discussion Role of Rh. bursa in transmission of many important zoonosis microorganisms (7-11), failure of chemical pesticides and presence of pesticides-resistant ticks (13, 14) and lack of studies on acaricidal and repellent natural prod- ucts against Rh. bursa were the reasons for design of current study. Natural products, such as essential oils produced by the secondary metabolism of herbs and are used in human consumption as functional food, food addi- tives, medicines, nutritional supplements and the manufacture of cosmetics due to their prop- erties (29) are good candidates as replace for chemical products. There is no similar study about acaricidal and/or repellent activity of essential oil of nat- ural plants like Z. officinalis and E. globulus against Rh. bursa. In this study for the first time acaricidal and repellent activity of es- sential oils of Z. officinalis and E. globulus against Rh. bursa as a wide-distributed spe- cies of ticks in different parts of Iran with an important role in transmission of zoonosis were studied singly and in combination with together. Acaricidal and repellent activities of eu- calyptus essential oil were studied against the poultry red mite, Dermanyssus gallinae (Ac- ari: Mesostigmata) and its efficacies were re- ported about 90% and 94% for acaricidal and repellent activity respectively (20), in current study acaricidal and repellent activities of eu- calyptus essential oil against Rh. bursa (Aca- ri, Ixodidae) were determined about 54% and 76% respectively, that maybe its lowest per- formances occurred due to different resistance between these ticks species. Insecticidal and repellent activity of es- sential oil of Z. officinalis and E. globulus against Culex theileri Theobald were report- ed in another study, the highest value of in- secticidal activity of eucalyptus essential oil was 66% and its highest repellent activity against Culex theileri Theobald was 74% and for ginger essential oil insecticidal and repel- lent activities was 39% and 62%, respective- ly (30). Results of current study agreed with them. In current study eucalyptus, essential oil had the highest acaricidal (54%) and repellent (76%) activates against Rh. bursa. Ginger es- sential oil had intermediate effects between treatments and its acaricidal and repellent ac- tivities was 39% and 62% respectively and values of 34% and 42% were observed as the lowest results for acaricidal and repellent activities of combined essential oils respec- tively. Acaricidal effect of Pelargonium roseum and eucalyptus essential oils against adult stage of Rhipicephalus annulatus were also studied and dose-dependent effects were reported, high- est acaricidal effects were observed for ticks treated with 5% dilutions of Pelargonium roseum (79.2%) and eucalyptus (16.7%) af- ter one day incubation (31), that higher value of acaricidal activity of eucalyptus essential oil in current study versus their results may- be was observed due to higher concentration of this essential oil in current study. Pesticide and/or repellent activity of eu- calyptus essential oil against Acanthoscelides obtectus (Say) (32), Culex quinquefasciatus (33), housefly, Musca domestica (34), Pedic- ulus humanus capitis (Anoplura:  Pediculidae) (35) and Lutzomyia longipalpis (36) were re- ported. Result of current study agreed with them and shows acaricidal/repellent activity for eucalyptus essential oil due to presence of components such as 1, 8-cineole, citron- ellal, citronellol, citronellyl acetate, p-cymene, eucamalol, limonene, linalool, a-pinene, g- terpinene, a-terpineol, alloocimene, andaro- madendrene in eucalyptus essential oil (37). There is a little data about insecticidal and/ or repellent activity of ginger essential oil. In- secticidal, repellent and oviposition-deterrent activity of ginger essential oil against Anoph- eles stephensi and Aedes aegypti (38) and lar- J Arthropod-Borne Dis, June 2019, 13(2): 214–223 S Madreseh-Ghahfarokhi et al.: Zingiber officinalis and … 220 http://jad.tums.ac.ir Published Online: June 24, 2019 vae of Spodoptera littoralis (39) were re- ported. Result of current study was agrees with them and showed acaricidal/repellent activi- ty of essential oil of ginger due to presence of presence of bioactive compounds such as gingerols, shogaols, diarylheptanoids, phenyl- butenoids, flavanoids, diterpenoids and ses- quiterpenoids in it (40). Moreover, briefly, insecticidal and/or ac- aricidal effect of Micromeria fruticosa, Nep- eta racemosa and Origanum vulgare (Lami- aceae) essential oils against Tetranychus ur- ticae and Bemisia tabaci (41) were reported in another study, also essential oils of Cum- inum cyminum, Pimenta dioica and Ocimum basilicum had different values of acaricidal effects against the cattle tick Rhipicephalus (Boophilus) microplus (42). In another study, acaricidal activity of essential oils of Lippia graveolens, Rosmarinus officinalis, and Alli- um sativum against Rhipicephalus microplus were identified (43). Acaricidal properties of Artemisia absinthium and Tanacetum vul- gare essential oils against Tetranychus urti- cae were also studied (44). Conclusion Briefly, eucalyptus essential oil had the highest acaricidal (54%) and repellent (76%) activates against Rh. bursa. Ginger essential oil had intermediate effects between treat- ments and its acaricidal and repellent activi- ties were 39% and 62% respectively and val- ues of 34% and 42% were observed as the lowest results for acaricidal and repellent ac- tivities of combined essential oils respective- ly. These agents can be considered as potent lethal/repellent agents for control of ticks, but more studies need for this purpose, study on lethal/repellent activity of these essential oils and/or other plants essential oils against other/this important arthropods can be con- sidered as subjects for next experiments. Acknowledgements Authors are grateful to Prof Yaser Pirali (Designer and adviser during the study), Dr Azam Dehghani Samani: Chief Technology Officer (CTO) of Tolou Tejarat Khavar Com- pany, Mr Ayat Dehghani Samani (Owner of farm and animals) and all of the persons that helped them to do this research. 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