Recombinant expression and antigenicity of two peptide families of neurotoxins from Androctonus sp

Background: Scorpion neurotoxins such as those that modify the mammalian voltagegated sodium ion channels (Nav) are the main responsible for scorpion envenomation. Their neutralization is crucial in the production of antivenoms against scorpion stings. Methods: In the present study, two in silico designed genes ­ one that codes for a native neurotoxin from the venom of the Anatolian scorpion Androctonus crassicauda, named Acra 4 ­ and another non-native toxin ­ named consensus scorpion toxin (SccTx) obtained from the alignment of the primary structures of the most toxic neurotoxins from the Middle Eastern and North African scorpions ­ were recombinantly expressed in E. coli Origami. Results: Following bacterial expression, the two expressed neurotoxins, hereafter named HisrAcra4 and HisrSccTx, were obtained from inclusion bodies. Both recombinant neurotoxins were obtained in multiple Cys-Cys isoforms. After refolding, the active protein fractions were identified with molecular masses of 8,947.6 and 9,989.1 Da for HisrAcra4 and HisrSccTx, respectively, which agreed with their expected theoretical masses. HisrAcra4 and HisrSccTx were used as antigens to immunize two groups of rabbits, to produce either anti-HisrAcra4 or anti-HisrSccTx serum antibodies, which in turn could recognize and neutralize neurotoxins from venoms of scorpion species from the Middle East and North Africa. The antibodies obtained from rabbits neutralized the 3LD50 of Androctonus australis, Leiurus quinquestriatus hebraeus and Buthus occitanus venoms, but they did not neutralize A. crassicauda and A. mauritanicus venoms. In addition, the anti-HisrAcra4 antibodies did not neutralize any of the five scorpion venoms tested. However, an antibody blend of anti-HisrAcra4 and anti-HisrSccTx was able to neutralize A. crassicauda and A. mauritanicus venoms. Conclusions: Two recombinant Nav neurotoxins, from different peptide families, were used as antigens to generate IgGs for neutralizing scorpion venoms of species from the Middle East and North Africa.(AU)

A study on the genetic diversity of Androctonus crassicauda (Olivier, 1807; Scorpiones: Buthidae) from Turkey

Mitochondrial DNA (mtDNA) has been widely employed in phylogeographic and phylogenetic studies. In the present study, the genetic identification of the scorpion Androctonus crassicauda (Olivier, 1807) was carried out by using the 16S mitochondrial gene, since this scorpion represents the most important species in Turkey regarding scorpionism and antivenom production. Two genetic groups were found according to the sequence analysis results, while five different loci at the nucleotide level presented genetic variation in the 16S region when compared to a known A. crassicauda sequence data (GenBank, AJ277598). Nucleotide variations found in the current work constitute the first descriptive report for A. crassicauda. Moreover, future studies may enlighten the genetic and venom composition variations for this scorpion species.(AU)

A study on the genetic diversity of Androctonus crassicauda (Olivier, 1807; Scorpiones: Buthidae) from Turkey

Mitochondrial DNA (mtDNA) has been widely employed in phylogeographic and phylogenetic studies. In the present study, the genetic identification of the scorpion Androctonus crassicauda (Olivier, 1807) was carried out by using the 16S mitochondrial gene, since this scorpion represents the most important species in Turkey regarding scorpionism and antivenom production. Two genetic groups were found according to the sequence analysis results, while five different loci at the nucleotide level presented genetic variation in the 16S region when compared to a known A. crassicauda sequence data (GenBank, AJ277598). Nucleotide variations found in the current work constitute the first descriptive report for A. crassicauda. Moreover, future studies may enlighten the genetic and venom composition variations for this scorpion species.(AU)

In vivo and in vitro effects of scorpion venoms in Turkey: a mini-review

Scorpion stings constitute a common type of accident in Turkey due to the geographic location, climate and socioeconomic structure of this country. Moreover, envenomation cases are considered a public health problem throughout Turkey. Based on data in the literature, the main clinical features of scorpion envenomations are defined as systemic manifestations including sweating, hypertension and vomiting, and among prominent causes of death are cardiorespiratory manifestations, toxic myocarditis and pulmonary edema. The most important health-threatening scorpions found in Turkey are: Androctonus crassicauda, Leiurus quinquestriatus, Mesobuthus gibbosus and Mesobuthus eupeus, all of which belong to the Buthidae family. This study indicates that there is no data related to venom toxicity and in vivo effects of other scorpions found in Turkey - such as Mesobuthus caucasicus, Mesobuthus nigrocinctus and Hottentotta saulcyi - and that further research should be performed to determine their health effects.(AU)

In vivo and in vitro effects of scorpion venoms in Turkey: a mini-review

Scorpion stings constitute a common type of accident in Turkey due to the geographic location, climate and socioeconomic structure of this country. Moreover, envenomation cases are considered a public health problem throughout Turkey. Based on data in the literature, the main clinical features of scorpion envenomations are defined as systemic manifestations including sweating, hypertension and vomiting, and among prominent causes of death are cardiorespiratory manifestations, toxic myocarditis and pulmonary edema. The most important health-threatening scorpions found in Turkey are: Androctonus crassicauda, Leiurus quinquestriatus, Mesobuthus gibbosus and Mesobuthus eupeus, all of which belong to the Buthidae family. This study indicates that there is no data related to venom toxicity and in vivo effects of other scorpions found in Turkey - such as Mesobuthus caucasicus, Mesobuthus nigrocinctus and Hottentotta saulcyi - and that further research should be performed to determine their health effects.(AU)

New additions to the scorpion fauna of Riyadh region, Saudi Arabia

In this work, we present additional morphological data about the scorpion fauna of Saudi Arabia. This investigation was carried out in the central region (Riyadh) of the country and identified existing and newly discovered scorpion species for taxonomic documentation while determining the medically important ones. The survey covered the entire Riyadh region, including all major districts, and collected a total of 4,164 specimens. Morphological identification of collected animals was based on identification keys. There were two species (one with a subspecies) that belonged to the family Scorpionidae, namely Scorpio maurus kruglovi (0.02 percent) and Hemiscorpius arabicus (0.05 percent). The latter, currently, is part of the Hemiscorpiidae family that had been upgraded from a subfamily. Eight more species from the Buthidae family were found: Leiurus quinquestriatus (7.20 percent), Androctonus crassicauda (17.24 percent), Androctonus bicolor (64.60 percent), Compsobuthus arabicus (3.84 percent), Compsobuthus werneri (0.94 percent), Buthacusyotvatensis nigroaculeatus (2.31 percent), Buthacusleptochelys (3.24 percent) and Orthochirus innesi (0.55 percent). The major locations of collection were the outskirts of Riyadh city and the airport vicinity. The specimens were transported from all central region areas in 124 short trips.(AU)

New additions to the scorpion fauna of Riyadh region, Saudi Arabia

In this work, we present additional morphological data about the scorpion fauna of Saudi Arabia. This investigation was carried out in the central region (Riyadh) of the country and identified existing and newly discovered scorpion species for taxonomic documentation while determining the medically important ones. The survey covered the entire Riyadh region, including all major districts, and collected a total of 4,164 specimens. Morphological identification of collected animals was based on identification keys. There were two species (one with a subspecies) that belonged to the family Scorpionidae, namely Scorpio maurus kruglovi (0.02 percent) and Hemiscorpius arabicus (0.05 percent). The latter, currently, is part of the Hemiscorpiidae family that had been upgraded from a subfamily. Eight more species from the Buthidae family were found: Leiurus quinquestriatus (7.20 percent), Androctonus crassicauda (17.24 percent), Androctonus bicolor (64.60 percent), Compsobuthus arabicus (3.84 percent), Compsobuthus werneri (0.94 percent), Buthacusyotvatensis nigroaculeatus (2.31 percent), Buthacusleptochelys (3.24 percent) and Orthochirus innesi (0.55 percent). The major locations of collection were the outskirts of Riyadh city and the airport vicinity. The specimens were transported from all central region areas in 124 short trips.(AU)

Evaluation of the neutralizing capacity of Androctonus crassicauda (Olivier, 1807) antivenom against Leiurus quinquestriatus (Ehrenberg, 1928) venom (Scorpiones: Buthidae)

The two most venomous species of the family Buthidae, Leiurus quinquestriatus and Androctonus crassicauda, are found in Africa and in the Middle East. Potency and paraspecific activities of A. crassicauda antivenom (RSHC anti-Ac) were tested against L. quinquestriatus venom. The sera produced by Refik Saydam Hygiene Center (RSHC) showed strong reactivity against the venoms of A. crassicauda and L. quinquestriatus in western blotting and dot-blot analysis. RSHC anti-Ac presents immunoactivity and neutralizing potential against Leiurus quinquestriatus venom. Neutralization capacity of antivenom was found to be 400 µL against 40 minimum lethal doses (MLD) of A. crassicauda scorpion venom and 10 MLD of L. quinquestriatus venom. This study indicates that the RSHC anti-Ac could be used for treating L. quinquestriatus stings.

Effects of Androctonus crassicauda (Olivier, 1807) (Scorpiones: Buthidae) venom on rat metabolism

Scorpions are venomous arthropods of the Arachnida class and are considered relatives of spiders, ticks and mites. There is not any study about the biochemical effects of Androctonus crassicauda (Olivier, 1807) venom. Therefore, in the present study, we aimed at evaluating the toxicity of the venom from A. crassicauda, which is responsible for a number of deaths of infants, children and adults in tropical and subtropical countries. For this purpose, rats (n=35) were divided into seven groups of five animals each; venom solutions (250µg/kg) were subcutaneously injected into rats; blood samples were taken from each animal at various times; and serum biochemical parameters were measured (levels of total proteins, total bilirubin, albumin, globulin, urea, creatinine, uric acid, glucose, cholesterol, triglycerides, sodium, chlorine, potassium and calcium, and the activity of the enzymes alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, lactate dehydrogenase). Serum levels of glucose, cholesterol, aspartate aminotransferase, alanine aminotransferase and uric acid increased in envenomed animals, compared to controls. There was a statistically positive correlation between Na+ and Cl- ions.

Evaluation of the neutralizing capacity of Androctonus crassicauda (Olivier, 1807) antivenom against Leiurus quinquestriatus (Ehrenberg, 1928) venom (Scorpiones: Buthidae)

The two most venomous species of the family Buthidae, Leiurus quinquestriatus and Androctonus crassicauda, are found in Africa and in the Middle East. Potency and paraspecific activities of A. crassicauda antivenom (RSHC anti-Ac) were tested against L. quinquestriatus venom. The sera produced by Refik Saydam Hygiene Center (RSHC) showed strong reactivity against the venoms of A. crassicauda and L. quinquestriatus in western blotting and dot-blot analysis. RSHC anti-Ac presents immunoactivity and neutralizing potential against Leiurus quinquestriatus venom. Neutralization capacity of antivenom was found to be 400 µL against 40 minimum lethal doses (MLD) of A. crassicauda scorpion venom and 10 MLD of L. quinquestriatus venom. This study indicates that the RSHC anti-Ac could be used for treating L. quinquestriatus stings.(AU)

Histology and histochemistry of the venom apparatus of the black scorpion Androctonus crassicauda (Olivier, 1807) (Scorpiones: Buthidae)

The venom apparatus of the black scorpion Androctonus crassicauda has been characterized histologically and histochemically in the present study. The results showed that this apparatus consists of paired venom glands, each of which initially presents its own canal and posteriorily both fuse into a single common one. Each gland is covered by a sheath of striated muscle and is lined with extensively folded secretory epithelium (formed of non-secretory and secretory venom-producing cells). The outcomes also revealed that the venom-producing cells of both glands produce neutral mucosubstances, sialomucins, sulfomucins and proteins, but are devoid of glycogen. Cysteine, tyrosine, tryptophan and arginine were also detected along with activities of acid and alkaline phosphatases, mitochondrial adenosine triphosphatase, aminopeptidase, cholinesterase and non-specific esterases. Structure and secretion of scorpion venom glands are discussed within the context of the present results.

Effects of Androctonus crassicauda (Olivier, 1807) (Scorpiones: Buthidae) venom on rat metabolism

Scorpions are venomous arthropods of the Arachnida class and are considered relatives of spiders, ticks and mites. There is not any study about the biochemical effects of Androctonus crassicauda (Olivier, 1807) venom. Therefore, in the present study, we aimed at evaluating the toxicity of the venom from A. crassicauda, which is responsible for a number of deaths of infants, children and adults in tropical and subtropical countries. For this purpose, rats (n=35) were divided into seven groups of five animals each; venom solutions (250µg/kg) were subcutaneously injected into rats; blood samples were taken from each animal at various times; and serum biochemical parameters were measured (levels of total proteins, total bilirubin, albumin, globulin, urea, creatinine, uric acid, glucose, cholesterol, triglycerides, sodium, chlorine, potassium and calcium, and the activity of the enzymes alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, lactate dehydrogenase). Serum levels of glucose, cholesterol, aspartate aminotransferase, alanine aminotransferase and uric acid increased in envenomed animals, compared to controls. There was a statistically positive correlation between Na+ and Cl- ions.(AU)

Evaluation of scorpion sting incidence in turkey

Scorpion stings are common in Turkey due to its geographical location, climate and socioeconomic structure. Scorpion envenomation cases are a considerable public health problem in all regions of the country. Important health-threatening scorpions in Turkey are Androctonus crassicauda, Leiurus quinquestriatus, Mesobuthus gibbosus and M. eupeus, all of which belong to the Buthidae family. They are described to be potentially dangerous to humans. So far, there is no study about scorpion sting incidence covering all geographical regions of Turkey; therefore, in this study, we analyzed the frequency of scorpion sting cases in the country. A total of 24,261 scorpion sting cases were reported during 2005. Most of which (about 80%) occurred in the Southeastern Anatolia (30.4%), Mediterranean Sea (24.9%) and Aegean regions (23.5%), where medically important scorpion species are found. The sting cases mostly occurred during the summer period. Our results suggest that scorpion stings are an important problem in Turkey. This simple descriptive study will help develop interventions to prevent scorpion stings, which should take local sting cases into consideration.

Evaluation of scorpion sting incidence in turkey

Scorpion stings are common in Turkey due to its geographical location, climate and socioeconomic structure. Scorpion envenomation cases are a considerable public health problem in all regions of the country. Important health-threatening scorpions in Turkey are Androctonus crassicauda, Leiurus quinquestriatus, Mesobuthus gibbosus and M. eupeus, all of which belong to the Buthidae family. They are described to be potentially dangerous to humans. So far, there is no study about scorpion sting incidence covering all geographical regions of Turkey; therefore, in this study, we analyzed the frequency of scorpion sting cases in the country. A total of 24,261 scorpion sting cases were reported during 2005. Most of which (about 80%) occurred in the Southeastern Anatolia (30.4%), Mediterranean Sea (24.9%) and Aegean regions (23.5%), where medically important scorpion species are found. The sting cases mostly occurred during the summer period. Our results suggest that scorpion stings are an important problem in Turkey. This simple descriptive study will help develop interventions to prevent scorpion stings, which should take local sting cases into consideration.

Histology and histochemistry of the venom apparatus of the black scorpion Androctonus crassicauda (Olivier, 1807) (Scorpiones: Buthidae)

The venom apparatus of the black scorpion Androctonus crassicauda has been characterized histologically and histochemically in the present study. The results showed that this apparatus consists of paired venom glands, each of which initially presents its own canal and posteriorily both fuse into a single common one. Each gland is covered by a sheath of striated muscle and is lined with extensively folded secretory epithelium (formed of non-secretory and secretory venom-producing cells). The outcomes also revealed that the venom-producing cells of both glands produce neutral mucosubstances, sialomucins, sulfomucins and proteins, but are devoid of glycogen. Cysteine, tyrosine, tryptophan and arginine were also detected along with activities of acid and alkaline phosphatases, mitochondrial adenosine triphosphatase, aminopeptidase, cholinesterase and non-specific esterases. Structure and secretion of scorpion venom glands are discussed within the context of the present results.(AU)

Evaluation of scorpion sting incidence in turkey

Scorpion stings are common in Turkey due to its geographical location, climate and socioeconomic structure. Scorpion envenomation cases are a considerable public health problem in all regions of the country. Important health-threatening scorpions in Turkey are Androctonus crassicauda, Leiurus quinquestriatus, Mesobuthus gibbosus and M. eupeus, all of which belong to the Buthidae family. They are described to be potentially dangerous to humans. So far, there is no study about scorpion sting incidence covering all geographical regions of Turkey; therefore, in this study, we analyzed the frequency of scorpion sting cases in the country. A total of 24,261 scorpion sting cases were reported during 2005. Most of which (about 80 percent) occurred in the Southeastern Anatolia (30.4 percent), Mediterranean Sea (24.9 percent) and Aegean regions (23.5 percent), where medically important scorpion species are found. The sting cases mostly occurred during the summer period. Our results suggest that scorpion stings are an important problem in Turkey. This simple descriptive study will help develop interventions to prevent scorpion stings, which should take local sting cases into consideration.(AU)

Determination of potency and paraspecific effects of Androctonus crassicauda (Olivier, 1807) antivenom against Mesobuthus gibbosus (Brullé, 1832) venom (Scorpiones: Buthidae)

Scorpion envenomation remains a real health problem in many countries. In scorpionism cases, it is often recommended that patients be treated with species-specific antivenom. Androctonus crassicauda venom has been used as antigen for antivenom production in Turkey, where this antivenom, called Turkish antivenom, has also been effective in the treatment of envenomation caused by species other than A. crassicauda. The present study aimed at determining the paraspecific effects and potency of the Turkish antivenom against Mesobuthus gibbosus (Brullé, 1832) venom. To assess the venom toxicity and the antivenom efficacy, we determined the Minimum Lethal Dose (MLD) and the Minimum Effective Dose (MED) instead of LD50 and ED50, respectively. Androctonus crassicauda antivenom was capable of neutralizing M. gibbosus venom (20 MLD). This was the first study indicating that A. crassicauda antivenom can be used for the treatment of Mesobuthus gibbosus stings, especially in Aegean Region, Turkey.

Determination of potency and paraspecific effects of Androctonus crassicauda (Olivier, 1807) antivenom against Mesobuthus gibbosus (Brullé, 1832) venom (Scorpiones: Buthidae)

Scorpion envenomation remains a real health problem in many countries. In scorpionism cases, it is often recommended that patients be treated with species-specific antivenom. Androctonus crassicauda venom has been used as antigen for antivenom production in Turkey, where this antivenom, called Turkish antivenom, has also been effective in the treatment of envenomation caused by species other than A. crassicauda. The present study aimed at determining the paraspecific effects and potency of the Turkish antivenom against Mesobuthus gibbosus (Brullé, 1832) venom. To assess the venom toxicity and the antivenom efficacy, we determined the Minimum Lethal Dose (MLD) and the Minimum Effective Dose (MED) instead of LD50 and ED50, respectively. Androctonus crassicauda antivenom was capable of neutralizing M. gibbosus venom (20 MLD). This was the first study indicating that A. crassicauda antivenom can be used for the treatment of Mesobuthus gibbosus stings, especially in Aegean Region, Turkey.(AU)

Effects of Androctonus crassicauda (Olivier, 1807) (scorpiones: buthidae) venom on rats: correlation among acetylcholinesterase activities and electrolytes levels

Scorpions can be considered living fossils because they have changed so little during the last 400 million years. They are venomous arthropods of the Arachnida class and regarded as relatives of spiders, ticks and mites. The aim of the present study was to evaluate the toxicity of Androctonus crassicauda (Olivier, 1807) venom and its effects on the acetylcholinesterase (AchE) activity and on electrolytes levels in rats. Animals were divided into seven groups of five rats each. Test groups received 250µg/kg of venom solution while control group was treated with 200µl of physiological saline solution (PSS). Blood samples were collected from the animals on the 1st, 2nd 4th, 8th, 12th, and 24th hours after subcutaneous injection of venom. Animals were monitored for 24 hours. Androctonus crassicauda venom significantly reduced AchE activity on the 12th hour when compared with control group. A statistically negative correlation between Na+ and K+ (p 0.05) and a positive correlation between Na+ and CL- (p 0.001) ions levels were observed after the administration of A. crassiccauda venom to rats. We can conclude that the differences in the electrolytes levels are due to acute renal failure, since elimination of toxin occurs primarily via the kidney.

Comparison of proteins, lethality and immunogenic compounds of Androctonus crassicauda (Olivier, 1807) (Scorpiones: Buthidae) venom obtained by different methods

Scorpions are venomous arthropods of the class Arachnida and are considered relatives of spiders, ticks and mites. There are approximately 1,500 species of scorpions worldwide, which are characterized by an elongated body and a segmented tail that ends in a venomous stinger. No specific treatment is available for scorpion envenomation, except for the use of antivenom. The current study aimed at comparing protein content and lethality of Androctonus crassicauda venom extracted by two different methods (electric stimulation and maceration of telsons). The LD50 calculated by probit analysis was 1.1mg/kg for venom obtained by electric stimulation and 39.19mg/kg for venom obtained by maceration of telsons. In the electrophoretic analysis, protein bands of the venom sample obtained by electric stimulation were between 12 and 53kDa (total: five bands), and those of venom extracted by maceration appeared as multiple protein bands, relative to the other venom sample. Low-molecular-weight proteins, revealed by western blotting, played an important immunogenic role in the production of antivenom. Lethality and protein levels varied according to the extraction method; venom obtained by the maceration technique showed lower toxicity than that obtained by electric stimulation.