Iranian Journal of Pharmaceutical Research (2017), 16 (2): 653-660 Copyright © 2016 by School of Pharmacy Received: Jun. 2015 Shaheed Beheshti University of Medical Sciences and Health Services Accepted: Dec. 2015 Original Article Neutralization Capacity of Monovalant Antivenom Against Existing Lethal Scorpions in the Turkish Scorpiofauna Ozcan Ozkana* and Ersen Aydın Yağmurb aDepartment of Biology, Çankırı Karatekin University, Çankırı, Turkey. bAlaşehir Vocational School, Celal Bayar University, Alaşehir, Manisa, Turkey. Abstract In this study, Mesobuthus gibbosus and Mesobuthus eupeus eupeus venom samples were compared for lethality, in-vivo effects and proteins. Neutralization capacity of monovalent Androctonus crassicauda antivenom (RSHA anti-Ac) was tested against the lethal effects of the venoms. Venom was obtained from mature scorpions by electrical stimulation of the telson. The lethality of the venom and potency of Horse RSHA anti-Ac were determined in Swiss mice. The protein profiles of the scorpion venoms were analysed by NuPAGE® 4–12% gradient Bis- Tris gel followed by Coomassie blue staining. Western blotting was performed to determine immunogenic compounds in the venom samples. The median lethal doses of M. e. eupeus, M.gibbosus scorpion and A.crassicauda venoms were determined to be 1.92 mg/kg by i.v. injection route, 0.67 mg/kg and 0.24 mg/kg by s.c. injection route, respectively. A.crassicauda (Olivier, 1807) venom was used as control. One millilitre of the RSHA anti-Ac neutralises 23 LD50 of M. e. eupeus, 32 LD50 of M.gibbosus and 42 LD50 of A. crassicauda venom in mice. Analysis of electrophoresis indicates that three scorpion venoms posses low molecular weight proteins. Immunoblotting indicated that RSHA anti-Ac strongly reacted with both the specific venom and Mesobuthus species venoms which have antigenic similarity. The result of our study showed that M.e. eupeus and M.gibbosus could be medically important scorpions for humans, particullary children. The RSHA anti-Ac can be used in the treatment of envenomation by M. e.eupeus and M.gibbosus scorpion stings. Key words: Turkish Scorpiofauna; lethality; Androctonus crassicauda antivenom. Introduction species of the genera Androctonus, Leiurus, and Mesobuthus are mainly responsible for Envenomation by arachnids causes significant envenomation in Turkey (4). injuries all over the world (1). Scorpion Although Mesobuthus eupeus has been sting is the most important type of arachnid considered to be a single species for many envenomation resulting in adult morbidity and years, two subspecies of M. eupeus, which pediatric mortality (2). Lethal scorpions mostly have been identified as M. eupeus phillipsii belong to the Buthidae family (3), among which, and M. eupeus eupeus have been recorded in the Turkish scorpiofauna recently by Kovařík * Corresponding author: et al. (5). Subsequently, Mirshamsi et al. (6) E-mail: [email protected] revised subspecies of M. eupeus existing in Iran Ozkan O et al. / IJPR (2017), 16 (2): 653-660 that include the subspecies M. eupeus phillipsii profiles (molecular weight), minimal lethal and M. eupeus eupeus, and M. eupeus phillipsii dose and also to show in-vivo effects in mice elevated to species level as M. phillipsii. to improve quality and paraspecific activity of Mesobuthus gibbosus occurs throughout national antivenom. Anatolia, except for the Black Sea coast in northern Turkey, European Turkey, Eastern and Experimental South-eatern Anatolia, and is the most abundant scorpion in this region (7-10). Mesobuthus is Scorpions one of the most widely distributed genera of M.gibbosus was collected from Aydın, the family Buthidae, with species being present M.e.eupeus from Karacadağ Mountain throughout Turkey. Stings by Mesobuthus (Şanlıurfa, 37°44› 45»N 39°49›50»E, 1752 m) species are more frequent than those by other and A.crassicauda from Şanlıurfa province at scorpion species (4, 11-13). The previous reports night, by using a UV lamp (Figure 1). Avoiding also indicated that those Mesobuthus species cannibalism, captive scorpions were housed are involved in numerous serious and deadly in individual boxes at the Department of envenomations, especially in children (14–18). Entomology, Faculty of Veterinary Medicine, Scorpion anti-venom is the only specific Ankara University, Turkey. The scorpions were treatment for scorpion envenomation and is still fed with crickets or cockroaches and received widely used in many countries (19, 20) as there water daily. are no vaccines or other effective agents against animal venoms (21). However, anti-venoms are Venoms still administered empirically and consequently Venom was obtained from mature M. eupeus their efficacy is controversial, particularly eupeus, A. crassicauda scorpions (Figure 1) from in the case of mild or moderate scorpion Şanlıurfa province and M. gibbosus scorpions envenomations (22). As venom is a complex from Aydın province by electrical stimulation of mixture of antigens wherein not all components the telson (26). The venoms were dissolved in are equally important for the production of sterile double-distilled water and centrifuged at neutralizing antibodies, the identification of 14,000 rpm for 15 min at 4 ºC. The supernatant immunogenic protein(s) and/or their neutralizing was stored at -20oC. The protein content of the epitopes may lead to the use of more clearly venoms was determined by the Bradford method defined substances as immunogens to develop using BSA as the standard (27). efficient anti-venoms. RSHA anti-Ac has been produced at the Refik Saydam Public Health Antivenom Agency (RSHA), Ankara in Turkey since 1942 Horse RSHA anti-Ac, produced by the Refik by venom of A. crassicauda and administering Saydam Public Health Agency is the unique to horses (23, 24). Consequently, identification antivenom available in the country and has been of the factors that lead to effective antivenom used to treat all scorpion stings in the country generation is important. With the median lethal since 1942 (4). Dose is normalized to neutralize doses of M.gibbosus (12), M.eupeus (25) two minimal lethal doses (MLD) in rats when scorpion venom and the potency of RSHA they are tested subcutaneously (4). antivenom previously determined, there was no confirmed cross-reactivity of the three venoms Experimental animals with the horse anti-venom produced by RSHA, Experimental protocols followed the Turkey. We studied venom samples obtained guidelines of the Bioethics Commission, the from two different Mesobuthus which were RSHA, and were approved by this Commission. Mesobuthus gibbosus from the West of Turkey Swiss mice of both sexes were employed and M.eupeus eupeus from Southeastern Turkey. to determine the median lethal dose (LD50) The present study is aimed to determine the and the median effective dose (ED50) by paraspecific effects and potency of RSHA anti- intravenous (IV) and subcutaneous (SC) route Ac against the venom and determine the protein of administration. They were breed in animal 654 Turkish Scorpiofauna, lethality, Androctonus crassicauda antivenom Figure 1. Map of distributionFigure of1. MesobuthusMap of speciesindistribution Turkey of (Blue Mesobuthus line; Mesobuthus speciesin gibbosus, Turkey red line;(Blue Mesobuthus line; Mesobuthus eupeus eupeus, gibbosus, red line; orange line: M. e. phillipsii,Mesobuthus brown line: eupeus M. caucasicus, eupeus, orgreenange line: line: M. M.nigrocinctus e. phillipsii ). The). Thevenom venom of A. crassicauda of A. crassicauda (A) has been(A )used has been used in the in the production of antivenom. M. e. phillipsii was commonly found in the Southeast Anatolia Region, but M.eupeus eupeus (B) was reported only in the Karacadagproduction Mountain of antivenom. (Red circle) M.within e. thephillipsii region of wasTurkey commonly (Kovařík, etfound al., 2011) in .the Mesobuthus Southeast gibbosus Anatolia (C) isRegion, the but M.eupeus source of many scorpioneupeus envenomation (B) was cases reported in the Aegean, only in Mediterranean, the Karacadag Marmara Mountain and Central within Anatolia the regionregions of Turkey.Turkey (Kovařík, et al., 2011). Mesobuthus gibbosus (C) is the source of many scorpion envenomation cases in the Aegean, Mediterranean, Marmara and Central Anatolia regions of Turkey. facility of the RSPHA. The animals were housed and A. crassicauda venom (SC injection; 3.15, under controlled temperature (20±2°C), with 3.49, 3.89, and 4.14µg/ 20g mouse [n: 20 mice]) a 12:12 light/dark schedule and were fed with diluted in physiological saline solution (PSS: commercial rodentLethal pellets potency and water of the ad scorpion libitum venom0.85% NaCl). An equivalent volume of PSS was throughout the experiment. injected into five mice as negative control group. Protein content of M. e. eupeus, M.gibbosusDeaths occurring and A. aftercrassicauda 24 h werescorpion recorded venom in was found to Lethal potency of the venoms order to determine the median lethal dose. The The lethality ofbe the3.72, venom 1.15 wasand determined1.54 µg protein/ lethalty µl, respectively. was expressed The as themedian LD50 .lethal dose for M. eupeus as described by Behrens and Karber (28) using Swiss mice. For pereupeus, each M.gibbosusdose group, andfiveA. micecrassicauda Antivenomscorpion potency venoms assay was determined in mice. The LD50 of were injected with increasing volume doses of Neutralization studies were carried out by M. eupeus eupeusM venoms. eupeus (IVeupeus injection: was determined 28.6, mixingas 1.92 amg/kg constant by i.vfixed injection amount route. of TheRSHA LD anti-50 of M.gibbosus 30.1, 33.2, 35.4 µg / 16g mouse [n: 20 mice]), Ac (1 mL) with various dilutions of M. eupeus M. gibbosus venomsand A.(SC crassicauda injection: scorpion7.6, 11.3, venoms eupeus were venomsfound as (IV 0.67 injection: mg/kg and 10, 0.24 14, mg/kg 18, 25 by s.c injection 14.9, 18.5 and 22.1 µg / 20g mouse [n: 25 mice]) and 28LD50 [n: 30 mice]), M. gibbosus venoms route, respectively. 655 7 Ozkan O et al. / IJPR (2017), 16 (2): 653-660 (SC injection; 29, 31, 33, 35, 38 and 40LD50 buffered saline (TBS, pH:7.5) containing 0.1% [n: 36 mice]) and A.