J Arthropod-Borne Dis, March 2019, 13(1): 104–115 O Ozkan and ME Alcigir: A Comparative … 104 http://jad.tums.ac.ir Published Online: April 27, 2019 Original Article A Comparative Pathomorphological Findings Between Leiurus abdullahbayrami and Androctonus crassicauda (Scorpion: Buthidae) Envenomation in Rabbit Animal Model *Ozcan Ozkan1; Mehmet Eray Alcigir2 1Çankırı Karatekin University, Çankırı, Turkey 2Department of Pathology, Kirikkale University, Faculty of Veterinary Medicine, Kirikkale, Turkey (Received 15 May 2017; accepted 23 Dec 2018) Abstract Background: The aim of study was to compare macroscopical and histopathological findings between venoms be- longing to two scorpion species, Androdoctonus crassicauda, and the newly discovered Leirus abdullahbayrami. Methods: The animals used in this experimental study were fifteen New Zealand bred rabbits. Three groups were constituted as group I (L. abdullahbayrami group, n= 6), group II (A. crassicauda group, n= 6) and group III (control group, n= 3). The animals in the L. abdullahbayrami group and the A. crassicauda group were envenomed through an intravenous route. The rabbits were monitored for the first 24h following the envenomation. The animals dead within that time period were examined and all animals were sacrificed and standard necropsy process was performed at 24h. Results: The pathomorphological findings from group I were found to be more severe than those observed in group II. The venom from the newly identified L. abdullahbayrami has a greater effect than the venom from the A. crassicauda. Moreover, as this was a rabbit modeling study, the L. abdullahbayrami might pose the most serious health threat to infants in particular due to their smaller body weight. Conclusion: These findings will provide a better understanding of envenomation of human beings in terms of the possible consequences of scorpion toxication on the organs. Keywords: Leirus abdullahbayrami; Androctonus crassicauda; Venom; Pathomorphology; Rabbit Introduction In many parts of the world, millions of people are stung by various scorpion species yearly. These stings can result in death, par- ticularly in children (1, 2). This is because of the serious health problems that result, such as cardiovascular, respiratory, and/or neurologic complications. Nowadays, despite advances in medical science, scorpion envenomation cas- es still continue to be a current public health problem all over the world, including in Tur- key. Scorpions are considered to be life-threat- ening venomous animals. Arguably, the med- ical knowledge of the scorpion's species is most critical for the scorpion species that are typi- cally found in Mexico, the middle and northern regions of South America, North and South Africa, India, and the Middle Eastern countries because those areas have scorpion species with the most potent venoms as they are classified as neurotoxic, hemotoxic, cardiotoxic, nephrotox- ic, and myotoxic (3-5). In Turkey, the most hazardous scorpions are the yellow scorpion, Leiurus abdul- lahbayrami, which is endemic in Southeast- ern Anatolia, and a black scorpion, Androcto- nus crassicauda, commonly found in South- eastern Anatolia and in a part of Eastern Ana- tolia (Fig. 1) and neighbouring countries Iran, Iraq and Syria (2, 5-7). Furthermore, in these regions, the species are responsible for the most deadly cases, par- ticularly those involving children. According- *Corresponding author: Dr Ozcan Ozkan, E-mail: ozcanozkan@karatekin.edu.tr J Arthropod-Borne Dis, March 2019, 13(1): 104–115 O Ozkan and ME Alcigir: A Comparative … 105 http://jad.tums.ac.ir Published Online: April 27, 2019 ly, these scorpion species are described as med- ically significant to both the world and to Tur- key (1, 3, 5, 7). In the toxicokinetic studies, radiolabeled venom from the L. quinquestriatus and venom from the A. crassicauda scorpion have reached the maximum value of total plasma radioac- tivity in less than two minutes after a subcu- taneous injection and begin distribution with- in four to seven minutes after the injection with a half-life of between 4–21h. At that time, the degree of the clinical symptoms depends on the amount of venom circulating in the body (7-9). A low venom dose triggers an adrenergic effect while high scorpion venom concentra- tions result in venom cholinergic symptoms. The venom results in an excessive acetylcho- line (Ach) release or a decrease in the destruc- tion of Ach, which is acting on the postgan- glionic nerve endings. Thus, cholinergic activ- ity begins. On the other side, catecholamine is released into the peripheral sympathetic nerve endings from the adrenal medulla in response to the venom. Then, an autonomic storm begins because of the renin secretion by the alpha ad- renergic receptor. The receptor stimulation plays a significant role because it increases blood pressure and ultimately results in the pathogenesis of pulmonary edema. Addition- ally, it causes an inflammatory reaction in the vital organs (8, 10-13). Scorpion envenomation has been reported to mainly culminate in a syndrome of fuel-en- ergy deficiency. This situation develops as a deficiency during the usage of existing meta- bolic substrates. As an aftermath, a failure oc- curs within vital organs including a multi-or- gan system deficiency that can lead to death (14, 15). The main reason for this failure is the triggering of an inflammatory response cascade that results in a release of several mediators, such as prostaglandins, cytokines, and nitric ox- ide, and an infiltration of inflammatory cells (16-19). The highest venom concentrations can be found in the kidneys, liver, heart, and lungs following a venom injection (20-22). In enven- omations from different scorpion species, the lungs, heart, liver, intestines, and pancreas were documented as the most affected organs in ad- dition to the recently described effects on the brain following a Leiurus envenomation (23- 27). As a result of the effect of scorpion venom, different clinical tables have reported varying effects from localized signs to more serious au- tonomic and neurologic findings as well as fatal cases due to multisystem organ failure. Ac- cording to medical records from Turkey, most deaths were caused by the results of cardiopul- monary complications, such as myocarditis and acute pulmonary edema following a scorpion’s sting (1, 2, 7). In animal models, scorpion envenomation symptoms were similar to those described by the clinical findings in humans. Therefore, to mimic human victims, especially children, rab- bits were chosen as the animal model for ex- perimental scorpion envenomation to mimic a natural sting accident. Thus, in this study, different pathomorphological findings devel- oped by two scorpion venoms were revealed out L. abdullahbayrami and A. crassicauda, which species were most encountered in south- east region of Turkey and also were showed L. abdullahbayrami having unusual harmful effects onto several organs during long term. Materials and Methods Scorpions Origin Scorpions were collected using an ultra- violet lamp at night in the Sanliurfa and Ga- ziantep Provinces, southeastern part of Tur- key. The animals were kept in plastic boxes at the Department of Entomology, Faculty of Veterinary Medicine, Ankara. The scorpions were received fresh water daily and fed crick- ets or cockroaches weekly. J Arthropod-Borne Dis, March 2019, 13(1): 104–115 O Ozkan and ME Alcigir: A Comparative … 106 http://jad.tums.ac.ir Published Online: April 27, 2019 Scorpion venom and the median lethal dose (LD50) The venom of scorpions was milked by electrical stimulation of 24 Volt. The venoms were added sterile double-distilled water, dis- solved and centrifuged at 14000rpm for 15 min at 4 °C. Supernatant was dissolved in PSS. The median lethal doses (LD50) of the scor- pions were determined as in previous studies (28, 29). Animals This study was conducted on 15 of healthy New Zealand rabbits 12 months old of both sexes and between 2.4±0.1kg body weight for this experiment. The rabbits were fed with a special rabbit pellet diet ad libitum until en- venomation. The animals were kept in room temperature set to 19±1 °C and with 12h light/ 12h dark schedule. The experimental protocol was approved by the local Laboratory Animal Ethics Committee. After 24h all animals were subjected to euthanasia for macroscopical and histopathological findings. Experimental envenomation of the animals The animals were envenomed by the ven- om of L. abdullahbayrami in group I (GI, n: 6), and the venom of A. crassicauda in group II (GII, n: 6). Finally, three rabbits were se- lected as a control group and will be referred to as GIII. The venoms (2xLD50 for both) were injected into the marginal ear vein by intrave- nous (IV) route after dissolving the venoms within 0.5mL PSS. For control group, any en- venomation was not performed. 0.9% Physi- ological saline solution only was administrated to rabbits of this group. The animals were mon- itored and kept in individual cages up to 24h following the injections because the deaths were not shown during first 24h of critical period. Histopathological Examination After providing sedation by xylazine (10 mg/kg Intramuscular route) and ketamine hy- drochloride (5mg/kg intramuscular route), So- dium Pentobarbital injection (100mg/kg Intra- venous route) were done and then cervical dis- location were performed for each one. Necrop- sy process was done systematically and all or- gans and tissues were examined routinely. Le- sions were photographed. For histopathologi- cal examination, tissue samples were taken sys- tematically from each organ in 10% buffered formalin. After fixation for 48h, tissue samples were processed routinely through alcohol and xylol series and mounted in paraffin. Processed tissues were embedded in paraffin wax. Five- micron thickness-sections were cut from par- affin blocks and Haematoxylin-Eosin (H and E) staining method. Results Macroscopical Findings In all groups, cadavers were well-fed, and rigor mortis did not happen. However, the blood in group I was lately clotted in contrast to the other groups. Conjunctiva was hyper- emic in two of the animals from group I. The gut walls were thickened and filled with a yel- lowish content in all of the members of group I and II. The liver, kidneys, and lungs were con- gested in all members of both groups I and II. However, there was haemorrhagia that hap- pened in the kidneys in the members of group II, and also atrophia and capsular contraction in the spleen was noticed in all animals within group II. In group I, a meningeal vessel was congested in one animal. In contrast, none of these macroscopical findings were evident in the animals of group III. Histopathological Findings Heart Myocardiocytes in some areas were degen- erated or partly necrotic in the members of group I. There was a hyalinosis in appearance in the degenerated myocardiocytes in the group I. Haemorrhagia between cardiomyocytes was formed in two animals from group I and four J Arthropod-Borne Dis, March 2019, 13(1): 104–115 O Ozkan and ME Alcigir: A Comparative … 107 http://jad.tums.ac.ir Published Online: April 27, 2019 animals from group II. However, there was fo- cal mononuclear cell infiltration in one animal from group II. There were no changes in con- trol group. Lungs All veins and vessels were hyperemic in members from group I. Furthermore, there was edema in all of the animals in group I. Micro- haemorrhagies were noticed in three of the members of group I and all of the members of group II. BALT hyperplasia was noted in one member of group I and all of the mem- bers of group II. It was not encountered with any findings in control group. Kidneys In the kidneys, there was hyperemia in all vessels and glomerules in all animals from group I, there were no such findings in the group II animals. On the other hand, vacuolar and hydropic degeneration in tubular epitheli- um were common findings for members of the first group except for one of group I animals. There is no any changes in control group. Liver Hepatocytes contained vacuoles in different sizes in their cytoplasm and degenerated nuclei in especially the periphery of lobules for all an- imals from the first two groups. In some an- imals of group II, reticular degeneration was also noted in addition to those findings. Ad- ditionally, mononuclear cell infiltrations in the portal field were evident in the group I find- ings and in all animals of group II. Finding in control group in terms of this organs were un- remarkable. Spleen Although no prominent findings were observed in animals of group II, focal follic- ular necrosis and haemorrhagia were noted in each animal of group I. Finding in control group in terms of this organ were unremark- able. Central Nervous System Lesions on the central nervous system were predominantly seen in group I. Menin- geal and parenchymal vessels were severely hyperemic in one animal of group I. Stratum pyramidal cells of the brain often seemed to have lost their nuclear chromatin in almost all foci. Perivascular mononuclear cell infil- tration was observed in substantia alba of the cerebrum within one of the animals in group I. In the cerebellum, cytoplasms and nuclei of Purkinje cells generally degenerated in both groups of animals. Moreover, some of the cells were necrotic in group I animals, seen in Fig. 2 and Fig. 3A–F. However, there were no conspicuous findings in the control group in terms of any of these organ. Gastrointestinal system Regarding the intestinal lesions, in group I, lymphocyte-plasma cell infiltration was noted in three animals, and neutrophil leuco- cyte infiltration was evident in one animal in the duodenum. In the group II, lymphocyte and macrophage infiltrations were seen in the jejunum as well as the duodenum in two of the animals. In addition, aggregate lym- phoid follicles were hyperplasic in three an- imals from group I. There were no findings in the stomach of any of the members of group I in contrast to the focal periarteriolar haemorrhagia noted in one animal from group II. In control group, there were no any pathologies. J Arthropod-Borne Dis, March 2019, 13(1): 104–115 O Ozkan and ME Alcigir: A Comparative … 108 http://jad.tums.ac.ir Published Online: April 27, 2019 Fig. 1. The distribution of the black colored scorpion is the Androdoctonus crassicauda (black dots), and a yellow colored scorpion the Leiurus abdullahbayrami species (red dots) are found in Hatay, Kilis, Sanliurfa, Mardin, Gaziantep provinces of the Southeastern Anatolia as shown map of Turkey. The scorpions were captured in Gaziantep and Sanliurfa Fig. 2. A. Vacuolar degeneration (arrows), periphery of lobules, liver, x100, H and E. B. Hyperemia (asterisks), glomerules and capillary vessels, kidney, x100, H and E. C. Vacuolar degeneration (arrows), kidney, x400, H and E. D. Haemorrhagia (arrows), lung, x100, H and E. E. Necrosis in myocardiocytes (arrows), x100, H and E. F. Degeneration (arrows) and necrosis (arrowhead) in Purkinje cells x100, H and E J Arthropod-Borne Dis, March 2019, 13(1): 104–115 O Ozkan and ME Alcigir: A Comparative … 109 http://jad.tums.ac.ir Published Online: April 27, 2019 Fig. 3. A. Focal lymphocytes in propria mucosa (arrows), duodenum, x100, H and E. B. Focal mononüklear cell infiltration (asteriks) in portal region and vacuolar degeneration in hepatocytes (oklar), liver, x100, H and E. C. Hydropic degeneration in tubul epitheliums (arrows), kidney, x100, H and E. D. Haemorrhagia (arrows), lung, x100, H and E. E. Focal lymphocyte infiltration (arrow), heart, x100, H and E. F. Degenerative and necrotic changes (arrows), cerebellum, x100, H and E Discussion Scorpion envenomation continues to be a global problem despite some national measures taken by authorities in various countries. Re- garding the the clinical symptoms, the degree of the envenomation depends on some factors related to the scorpion such as the species and the size of the scorpion as well as the number of stings, the content of the venom, and the amount of venom injected. Another factor as- sociated with a patient’s prognosis include the patient’s health status, the part of the body that has been stung, the patient’s body mass, and the patient’s age. After the sting, the symptoms of envenomation can be evident within a few minutes, and maximum severity is usually achieved within five hours. Consequently, the clinical table has shown localized symptoms at the sting site up to a significantly severe generalized envenomation (7, 11, 28). In Turkey, the scorpion sting is very com- mon in the provinces of Southeastern Anato- lia and Eastern Anatolia due to social and ge- ographical factors. In these areas, the scorpion sting cases have been reported as coming from both the yellow colored scorpion, which is the L. abdullahbayrami, and a black colored scor- pion, which is the A. crassicauda species. In addition, scorpions have been the main animal responsible for medically important enven- omation cases (5, 11-13). In the Kilis Province, southeastern Turkey, recently, acute pulmonary edema and cardio J Arthropod-Borne Dis, March 2019, 13(1): 104–115 O Ozkan and ME Alcigir: A Comparative … 110 http://jad.tums.ac.ir Published Online: April 27, 2019 myopathy were due to the excessive catechol- amine release that resulted from a sympathetic overstimulation in an 11-yr-old male child fol- lowing an L. abdullahbayrami stung (7). An- other study conducted at the Sanliurfa Chil- dren’s Hospital reported on a patient with symp- toms that included fever, hypersalivation, my- driasis, tachycardia, lethargic, and respiratory distress during admission time. Later, this pa- tient had to be intubated because of pulmonary edema and myocarditis. Another girl had neu- rological signs similar to the first victim as well as anisocoria and seizure. Two girls, ages 2-yr- old and 4-yr-old, had pulseless ventricular tach- ycardia and died (13). In the province of Hatay in the Southeast Anatolia region, the patients were categorized according to the color of the scorpion that had stung them as yellow (54.2%) and black (28.7%). Two children passed away due to cardiac and respiratory complications resulting from a scorpion sting (30). In this same province, a 4-yr-old boy died because of pulmonary edema (31). Furthermore, in Adıya- man Province, the patients were categorized by the color of the scorpion as well with black consisting of 40% of the cases and yellow re- sulting in 60% of the cases (12). One of these patients died due to cardiac and respiratory ar- rest. 50.8% of their scorpion cases were A. crassicauda stings (5). Additionally, four of the deaths in children were caused by the A. crassicauda species, and only one was caused by an L. abdullahbayrami (32). Especially in children, similar findings have been shown in previous studies concerning these two important scorpion species. These scorpions were respon- sible for serious medical cases and deaths in Turkey as well. Therefore, our study aimed to determine and compare the pathological ef- fects of the venom based on an animal model. The L. abdullahbayrami scorpion venom- induced potassium and sodium voltage-gated ion channels and then led to excessive cate- cholamine release due to the over stimulation of the sodium and potassium ion channels. As a result of the catecholamines storm, car- diomyopathy and acute pulmonary edema were caused by severe envenomation, especially among small children or infants because of their smaller body weight (7). Namely, following envenomation, an acute failure leads to multi- organ system deficiency among the vital or- gans, which leads to death (14, 15), especially within the heart and lungs. After acute enven- omation, a cardiac dysfunction occurred, and as a result of this cardiac failure, the formation of edema and hemorrhages happened in the lungs (8, 9, 33). Thrombosis of cardiac vessels, endothelial cells of those vessels, edema, and necrosis in cardiac muscle cells were all ob- served during the 24h following envenomation (33). In another acute envenomation study with A. australis hector, LD50 of the venom resulted in myocardial necrosis and degeneration. Neu- trophil leucocyte and mononuclear cell infil- tration in the interstitium of myocardium were noted within the first 24h period in mice as well. Suppurative bronchopneumonia, focal haemorrhagia, and fibrin deposits are found in the lungs of the mice. Cardiac edema, myocar- dial haemorrhagia, degeneration and necrosis were also detected with envenomation attribut- ed to Centuroides sculpturatus venom (34). In another study that experimented with LD50 doses of Mesobuthus eupeus envenomation on rabbits, several important consequences pro- gressed including myocardial necrosis, focal haemorrhage, thrombus formation and inflam- mation in myocardium and the endocardium surfaces of the heart and lungs (35). In our study, more serious fatal findings, such as hy- peremia, edema, and haemorrhage, were evi- dent in the lungs. Degeneration and necrosis in the myocardiocytes occurred within the rab- bits in group I envenomated with L. abdul- lahbayrami. However, BALT hyperplasia and microhemorrhage were encountered in the lungs and focal mononuclear cell infiltration in heart in the rabbits envenomated with A. crassicauda. Leiurus abdullahbayrami trig- gered a more acute reaction especially in re- gards to the cardiopulmonary system within the J Arthropod-Borne Dis, March 2019, 13(1): 104–115 O Ozkan and ME Alcigir: A Comparative … 111 http://jad.tums.ac.ir Published Online: April 27, 2019 first 24h as compared to another type of ven- oms documented in several previous studies. Therefore, the results obtained from these study groups were found to parallel the findings of previous studies which also examined the ef- fects that occur within the first 24h following a scorpion sting. Some deaths in humans and animals were related to the effect of the venom on the kid- neys. The scorpion toxin caused hyperemia and degenerative-necrotic changes in the tubules and glomeruli. This situation resulted in acute renal failure (2, 33, 34, 36, 37). Even an enven- omation attributed to a Hemiscorpius lepturus scorpion can sometimes result in a uremic syn- drome in a child (38). In an acute envenomation from M. eupeus, congestion in the vessels and glomeruli of the envenomated animals has been reported. In our study, group I animals en- venomated with L. abdullahbayrami showed similar important damages, including vascular congestion and degenerative changes in tubules, as compared to group II animals envenomat- ed by the venom of the A. crassicauda. On the other hand, certain studies conduct- ed in association with particularly both acute as well as sub-acute envenomation have prov- en that venom of the M. eupeus species can promptly effect animals within 30min up to three to five hours later by resulting in con- gestion within the central and portal vein of the liver and in interstitial vessels (35). In sub- acute envenomation caused by the C. sculp- turatus’s venom, the liver of rats was affect- ed within two hours and up to five days after the envenomation. In the livers of envenomat- ed animals, congestion of central veins, hy- dropic degeneration, and single cell necrosis of hepatocytes was also observed (34, 38). In our study, the findings were observed in both experimental groups. However, mononuclear cell infiltrations in the portal field were also noted in all of the animals of group II. The last finding is a different finding as compared to previous reports. Apart from the findings with- in the vital organs, milder findings such as con- gestion were encountered in both the spleen and the pancreas (36). In our study, no other prom- inent findings were evident. However, focal follicular necrosis and haemorrhagia were both noted in all of the animals in group I. The ven- om of the L. abdullahbayrami might be more effective since it triggered an inflammatory re- action. In relation to the gastrointestinal system, edema and mildly necrosis have occurred in the guts of these envenomed animals. On the other hand, no inflammatory changes were ev- ident even though there were severe microscop- ical changes noted, such as gastrointestinal haemorrhagia, necrosis, and inflammation (33, 36). In our study, in group I, inflammatory re- actions was evident in some of the animals from both groups. Additionally, aggregate lym- phoid follicles were found hyperplasic in three animals of group I. There were no findings in the stomach of any of the members from group I, which was in contrast to the focal periarte- riolar haemorrhagia found in one of the animals from group II. These findings correlated with the previous findings (36). In our study, the most significant findings in almost all organs were mostly hyperemia, focal haemorrhagia, and mononuclear cell infiltrations as was in- dicated in several previous studies. Such results have been reported as a result of inflammatory responses under influence of the variety of cy- tokines as reported in previous reports (18, 24, 25). In our study, the acute findings belonging to L. abdullahbayrami envenomation were found to be more severe than the A. crassi- cauda envenomation because it caused much more of an inflammatory reaction within the first 24h. In this sense, the newly identified L. abdullahbayrami venom had more of an ef- fect than the A. crassicauda venom. Additionally, many studies have reported on this inflammatory responses following en- venomation. Among them, leucocytosis has been mentioned as occurring in the hours fol- lowing envenomation (14, 18, 25, 39, 40). In the present study, mononuclear cell infiltrations J Arthropod-Borne Dis, March 2019, 13(1): 104–115 O Ozkan and ME Alcigir: A Comparative … 112 http://jad.tums.ac.ir Published Online: April 27, 2019 were more evident in several organs, especial- ly among the Leirus sp. group. The Leirus envenomation resulted in an ongoing or final stage of acute inflammatory reaction. On the other hand, the main affected organs included the lungs, liver, intestine, and pancreas, respec- tively (22-26). However, some important find- ings in the kidneys and the central nervous sys- tems were also evident in our study, which was less likely to be mentioned in prior documents except for the most recent document (27). The inflammation might be a long-term effect from the two types of venom. Particularly, another reason for multiple organ disfailure was the hy- dropic-vacuolar degeneration in parenchyma- tous organs. It has been previously associated with the loss of function of the Na-K ATPase pump (15, 27, 41, 42). This situation could be further clarified by examining several fractions of these venoms that target one or more vital cells such as those from the liver, kidneys and also the cerebellum rather than simply focus- ing on the most prominent vital organs of the heart and the lungs. Hence, we think that the hepatocytes, tubular epitheliums, and Purkinje cells were more affected based on the findings of this current study. In our study, the main acute findings were mainly hyperemia, haem- orrhagia and degenerative-necrotic changes in the aforementioned vital organs. The period of experimentation for this study was longer as compared to other envenomation studies. Find- ings from this study related to the other organs besides just the two vital organs have been made more evident due to the longer time that allowed these effects from both types of ven- oms to be noticed. In addition, histopatholog- ical findings might change according to the type of scorpion venom and the envenomation meth- od for an organism. Conclusion Leirus abdullahbayrami and A. crassicauda venoms cause to more slight findings beyond simply resulting in death for rabbits. However, L. abdullahbayrami has resulted in more severe lesions when compared to the A. crassicauda. Especially, findings were evident in both the vital parenchymatous organs, such as the heart, lungs, liver, kidneys, as well as other organs, such as the brain, spleen, gastrointestinal sys- tem, during the first 24h. In addition, the lesions were more related to vascular changes and de- generative-necrotic changes in envenomated an- imals with L. abdullahbayrami. Thus, some cy- tokines might have change the immune re- sponse of the animals against the preservation of body homeostasis. This situation might be more related to the degrees of toxic components of mentioned scorpion species. In this regard, therapeutic strategy against envenomation by several scorpion species and course of toxi- cation might be changed during critical first 24h-period. Acknowledgements No financial support was received for this study. The authors declare that there is no conflict of interests. References 1. 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