read more can occur, particularly in snake handlers who have been previously sensitized. If envenomation has occurred, edema and erythema at the bite site and in adjacent tissues occur, usually within 30 to 60 minutes. Oozing from the wound suggests envenomation. Edema can progress rapidly and may involve the entire extremity within hours.

Lymphangitis and enlarged, tender regional lymph nodes may develop; temperature increases over the bite area. At least one read more , ecchymosis is common and may appear at and around the bite site within 3 to 6 hours.

Ecchymosis is most severe after bites by. The skin around the bite may appear tense and discolored. Bullae—serous, hemorrhagic, or both—usually appear at the bite site within 8 hours.

Edema resulting from North American rattlesnake envenomations may be severe but is usually limited to dermal and subcutaneous tissues, although severe envenomation rarely causes edema in subfascial tissue, causing compartment syndrome Compartment Syndrome Compartment syndrome is increased tissue pressure within a closed fascial space, resulting in tissue ischemia.

The earliest symptom is pain out of proportion to the severity of injury. Necrosis around the bite site is common after rattlesnake envenomations. Most venom effects on soft tissues peak within 2 to 4 days.

Systemic manifestations of envenomation can include nausea, vomiting, diarrhea, diaphoresis, anxiety, confusion, spontaneous bleeding, fever, chest pain, difficulty breathing, paresthesias, hypotension, and shock.

Some patients with rattlesnake bites experience a rubbery, minty, or metallic taste in their mouth. The venom of most North American pit vipers causes minor neuromuscular conduction changes, including generalized weakness and paresthesias and muscle fasciculations.

Some patients have alterations in mental status. Venom of Mojave and eastern diamondback rattlesnakes may cause serious neurologic deficits, including respiratory depression. Rattlesnake envenomations may induce various coagulation abnormalities, including thrombocytopenia, prolongation of prothrombin time PT, measured by the INR [international normalized ratio] or activated partial thromboplastin time PTT , hypofibrinogenemia, elevated fibrin degradation products, or a combination of these disorders, resembling a disseminated intravascular coagulation DIC -like syndrome.

Thrombocytopenia is usually the first manifestation and may be asymptomatic or, in the presence of a multicomponent coagulopathy, cause spontaneous bleeding. Patients with coagulopathy typically hemorrhage from the bite site or from venipuncture sites or mucous membranes, with epistaxis, gingival bleeding, hematemesis, hematochezia, hematuria, or a combination.

A rise in hematocrit Hct is an early finding secondary to edema and hemoconcentration. Later, Hct may fall as a result of fluid replacement and blood loss due to DIC-like syndrome.

In severe cases, hemolysis may cause a rapid fall in Hct. Pain and swelling may be minimal or absent and are often transitory. The absence of local symptoms and signs may erroneously suggest a dry bite, producing a false sense of security for both patient and clinician.

Suspect envenomation with all bites caused by venomous snakes, even if there are no signs of envenomation soon after the bite. Weakness of the bitten extremity may become evident within several hours.

Systemic neuromuscular manifestations may be delayed for 12 hours and include weakness and lethargy; altered sensorium eg, euphoria, drowsiness ; cranial nerve palsies causing ptosis, diplopia, blurred vision, dysarthria, and dysphagia; increased salivation; muscle flaccidity; and respiratory distress or failure.

Once the neurotoxic venom effects manifest, they are difficult to reverse and may last 3 to 6 days. Untreated, respiratory muscle paralysis may be fatal.

Definitive diagnosis of a snakebite is aided by positive identification of the snake and clinical manifestations of envenomation. History should include the time of bite, description of the snake, type of field therapy, underlying medical conditions, allergy to horse or sheep products, and history of previous venomous snakebites and therapy.

A complete physical examination should be done. A marker should be used to indicate the leading edge of edema on the affected limb or area, and the time the mark was made should be recorded.

Snakebites should be assumed to be venomous until proved otherwise by clear identification of the species or by a period of observation. Pit vipers and nonvenomous snakes can be distinguished by some physical features see figure Identifying pit vipers Identifying pit vipers.

Consultation with a zoo, an aquarium, or a poison center can help in the identification of snake species. Coral snakes in the US have round pupils and black snouts but lack facial pits. They have blunt or cigar-shaped heads and alternating bands of red, yellow cream , and black, often causing them to be mistaken for the common nonvenomous scarlet king snake, which has alternating bands of red, black, and yellow.

Coral snakes have short, fixed fangs and inject venom through successive chewing movements. Fang marks are suggestive but not conclusive; rattlesnakes may leave single or double fang marks or other teeth marks, whereas bites by nonvenomous snakes usually leave multiple superficial teeth marks.

However, the number of teeth marks and bite sites may vary because snakes may strike and bite multiple times. A dry pit viper bite is diagnosed when no symptoms or signs of envenomation appear within 8 hours after the bite. Location and depth of the bite eg, envenomation in bites to the head and trunk tends to be more severe than in bites to the extremities.

Severity of envenomation can be graded as minimal, moderate, or severe based on local findings, systemic symptoms and signs, coagulation parameters, and laboratory results see table. Grading should be determined by the most severe symptom, sign, or laboratory finding.

If systemic symptoms begin immediately, anaphylaxis Anaphylaxis Anaphylaxis is an acute, potentially life-threatening, IgE-mediated allergic reaction that occurs in previously sensitized people when they are reexposed to the sensitizing antigen.

read more should be assumed. They should avoid exertion and be reassured, kept warm, and transported rapidly to the nearest medical facility. A bitten extremity should be wrapped loosely and immobilized in a functional position at about heart level, and all rings, watches, and constrictive clothing should be removed.

Pressure immobilization to delay systemic absorption of venom eg, by wrapping wide crepe or other fabric bandages around the limb may be appropriate for coral snake bites but is not recommended in the US, where most bites are from pit vipers; pressure immobilization may cause arterial insufficiency and necrosis.

First responders should support airway and breathing, administer oxygen, and establish IV access in an unaffected extremity while transporting patients. All other out-of-hospital interventions eg, tourniquets, topical preparations, any form of wound suction with or without incision, cryotherapy, electrical shock are of no proven benefit, may be harmful, and may delay appropriate treatment.

However, tourniquets that are already placed, unless causing limb-threatening ischemia, should remain in place until patients are transported to the hospital and envenomation is excluded or definitive treatment is initiated.

Serial assessment and testing begin in the emergency department. Outlining the leading margin of local edema with an indelible marker every 15 to 30 minutes can help clinicians assess progression of local envenomation.

Extremity circumference should also be measured on arrival and at regular intervals until local progression subsides. All but trivial pit viper bites require. Measurement of serum electrolytes, blood urea nitrogen, and creatinine. In the management of patients with coral snake bites, neurotoxic venom effects necessitate monitoring of oxygen saturation Pulse Oximetry Gas exchange is measured through several means, including Diffusing capacity for carbon monoxide Pulse oximetry Arterial blood gas sampling The diffusing capacity for carbon monoxide DLCO read more and baseline and serial pulmonary function tests ie, peak flow, vital capacity.

Duration of close observation for all patients with pit viper bites should be at least 8 hours. Patients without evidence of envenomation after 8 hours may be sent home after adequate wound care Adjunctive measures. Patients with coral snake bites should be monitored closely for at least 12 hours in case respiratory paralysis develops.

Envenomation initially assessed as mild may progress to severe within several hours. Supportive care may include respiratory support, benzodiazepines for anxiety and sedation, opioids for pain, and fluid replacement and vasopressor support for shock. Transfusions eg, packed red blood cells, fresh frozen plasma, cryoprecipitate, platelets may be required but should not be given before patients have received adequate quantities of neutralizing antivenom because most coagulopathies respond to sufficient quantities of neutralizing antivenom.

Suspected anaphylaxis eg, with immediate onset of systemic symptoms is treated with standard measures, including epinephrine. Tracheostomy Tracheostomy If the upper airway is obstructed because of a foreign body or massive facial trauma or if ventilation cannot be accomplished by other means, surgical entry into the trachea is required.

read more may be needed if trismus, laryngeal spasm, or excessive salivation is present. Along with aggressive supportive care, antivenom is the mainstay of treatment for patients with anything more than the mildest envenomation grade. For pit viper envenomation , the mainstay of treatment in the US is an ovine-derived Crotalidae polyvalent immune FAb antivenom purified FAb fragments of IgG harvested from pit viper venom—immunized sheep.

The effectiveness of this antivenom is time and dose related; it is most effective in preventing venom-induced tissue damage when given early. It is less effective if delayed but can reverse coagulopathies and be effective even when started 24 hours after envenomation.

Crotalidae polyvalent immune FAb is very safe, although it can still cause acute cutaneous or anaphylactic reactions and delayed hypersensitivity reactions serum sickness.

The same dose can be repeated 2 times as needed to achieve initial control of symptoms, reverse coagulopathies, and correct physiologic parameters.

In children, the dose is not decreased eg, based on weight or size. Measuring the circumference of the involved extremity at 3 points proximal to the bite and measuring the advancing border of edema every 15 to 30 minutes can guide decisions about the need for additional doses.

Once control is achieved, a 2-vial dose in mL saline is given at 6, 12, and 18 hours to prevent recurrence of limb swelling and other venom effects. Crotalidae immune F ab' 2 equine is a newly available horse-derived antivenom consisting of reconstituted Crotalidae immune Fab2 fragments and is used to treat North American rattlesnake bites in adults and children.

This initial dose may be repeated every hour if needed to arrest the progression of symptoms. Late or reemerging symptoms may be treated with additional 4 vial doses. Pit viper species may affect dose. Cottonmouth, copperhead, and pygmy rattlesnake envenomations may require smaller doses of antivenom.

However, antivenom should not be withheld based on the species of snake and should be given based on envenomation grading regardless of the species. Special attention is warranted for children, older patients, and patients with medical conditions eg, diabetes mellitus, coronary artery disease who may be more susceptible to venom effects.

For coral snake envenomation , equine-derived polyvalent coral snake antivenom is given at a dose of 5 vials for suspected envenomation and an additional 10 to 15 vials if symptoms develop.

Dose is similar for adults and children. This dosing recommendation may be reduced during national shortages of coral snake antivenom. Antivenom pretreatment precautions should be considered for patients with known hypersensitivity to the specific antivenom being considered, horse or sheep serum, and those with a history of asthma or multiple allergies.

In such patients, if the envenomation is considered life or limb threatening, H1 and H2 blockers should be given before antivenom in a critical care setting equipped to treat anaphylaxis Anaphylaxis Anaphylaxis is an acute, potentially life-threatening, IgE-mediated allergic reaction that occurs in previously sensitized people when they are reexposed to the sensitizing antigen.

This should reduce the mortality resulting from neurotoxic snakebite envenomation. Snakebite envenomation causes significant morbidity and mortality in the world, particularly in sub-Saharan Africa, Asia and Latin America, with about 2. This is primarily due to a limited production and inadequate supply of effective and affordable antivenoms 4.

Snake antivenoms are specific against venoms used as immunogens, and those of closely related species; cross reaction or cross neutralization with venoms from other phylogenetically distant species is not often observed 5 , 6 , 7 , 8.

Thus, antivenoms are mainly used to treat envenomations by snakes that are native to a particular country or region, and generally cannot be used on a larger geographical scale, in contrast to immunoglobulins for rabies or tetanus toxin.

Consequently, antivenoms are produced in relatively small volumes for local or regional use and, as a result, the cost of the product is high. One way to overcome these problems is to produce pan-specific antivenoms that can neutralize large numbers of venoms from snakes inhabiting wide geographic areas 4.

Such antivenoms could save lives of victims where no locally made antivenom is available. These lifesaving products would then be more affordable to poor people and health authorities in developing countries where the highest incidences of snakebites occur 9 , Furthermore, pan-specific antivenoms with wide para specificity can be useful in cases where the culprit snake is not identified or captured, and consequently species identification of the snake cannot be made.

In this context, we have previously produced an experimental pan-specific equine antiserum that is capable of neutralizing 27 neurotoxic venoms from homologous and heterologous snake species inhabiting Asia and Africa.

This should result in the production of antibodies with a variety of paratopes against the diverse toxin epitopes, and consequently, exhibit wide para-specificity.

These toxin fractions contained all the toxic components of the venoms, mostly presynaptic and postsynaptic neurotoxins and cytotoxins, but were devoid of the high molecular mass, highly immunogenic non-toxic proteins 11 , In the present study, we demonstrated that this pan-specific antiserum also neutralized nine additional neurotoxic venoms of elapids from Central America, Africa, and Australia, including sea snakes and sea kraits.

Altogether, 36 neurotoxic venoms from 4 continents have been shown to be neutralized by the antiserum. The 10 neurotoxic venoms hereby tested are shown in Table 1. The list includes venoms of the coral snake Micrurus nigrocinctus , the most medically important elapid in Central America, the yellow-lipped sea krait Laticauda colubrina , and the beaked sea snake Hydrophis schistosus distributed from Australian waters to the Arabian Sea.

Other venoms tested include those of the tiger snake Notechis scutatus , the king brown snake Pseudechis australis and the coastal taipan Oxyuranus scutellatus , which are classified within WHO Category 1 most medically important snakes from Australia and Papua New Guinea. In addition, neutralization of venoms of the African species black mamba Dendroaspis polylepis , the green mamba Dendroaspis angusticeps , the western green mamba Dendroaspis viridis , and the Senegalese cobra Naja senegalensis was assessed.

All these snakes, except Micrurus nigrocinctus , are within WHO Category 1 species, i. nigrocinctus is in WHO Category 2 snakes, i. From the median lethal dose LD 50 results, the coastal taipan O. angusticeps venom Tanzania with LD 50 of 1. Of the ten venoms studied, nine of them, including those from the two sea snakes, the Central American coral snake and the Australian snakes were cross-neutralized, and so were those of two African mambas D.

viridis and D. polylepis and one African cobra N. Only the green mamba D. angusticeps venom was not neutralized by the pan-specific antiserum.

The antiserum most effectively neutralized the venom of N. senegalensis with a Potency P of 0. nigrocinctus , the two African mambas and the three Australian elapids.

The P value of antiserum against the sea krait L. colubrina venom underscored that it was still capable of neutralization. Thus the results showed that 9 out of ten neurotoxic venoms were neutralized by the pan-specific antiserum; only the venom of D.

It is relevant to analyse the neutralization results vis-à-vis the proteomic profiles previously reported for these venoms. Table 2 depicts the major toxic components described for these venoms, with the exception of N. senegalensis , whose venom proteome has not been characterized.

The proteomics toxin profiles show the major toxic lethal components of each of these 10 venoms. Seven of them contain short Type I and long Type II α-neurotoxins, which belong to the three-finger toxin family 3FTx. The α-neurotoxins in these seven venoms are highly toxic and could lead to neuromuscular paralysis and death.

Neurotoxicity caused by P. australis venom has been demonstrated and it was more potent to diapsids than to synapsids Moreover, a short- and a long α-neurotoxins had been reported from P. australis venom UniProt database entries: P and P, respectively.

The toxins were present at very low level that probably explained its non-detection in the proteomic study 15 , and in our in vitro assay based on T. californica nAChR binding Fig. Each points was the mean ± SEM of 3 separate determinations. All nine venoms with available proteomic information contained phospholipases A 2 PLA 2 s , some of which are basic PLA 2 that contribute to presynaptic neurotoxicity in O.

scutellatus known as taipoxin 17 and N. scutatus known as notexin 18 venoms, and hemolytic, anticoagulant as well as myotoxic activities in P. australis venom Besides, the myotoxic PLA 2 was also found abundantly in the sea snake H.

schistosus venom, which causes rhabdomyolysis and nephrotoxicity in envenomation Both lethal α-neurotoxins and presynaptic PLA 2 s are present in the venoms of H. schistosus 20 and N. scutatus 21 , In addition to α- and β-neurotoxins, procoagulant serine proteases in the venoms of Australian elapids O.

scutellatus and N. scutatus can cause hemostatic alterations which may lead to bleeding or thrombosis 17 , 23 , although it is likely that neurotoxins are the main culprits of lethality in these venoms. The lethal toxins present in the nine venoms were presumably neutralized by the pan-specific antiserum, as evidenced by neutralization results.

It should be mentioned that the pan-specific antivenom was prepared with the aim of neutralizing lethal neurotoxins mainly α- and β-neurotoxins and not against the high molecular weight enzymes e. prothrombin activators which were filtered out during the preparation of immunogens. As such, the pan specific antiserum was not tested for neutralization of these activities associated with high molecular mass components.

Regarding the three mamba venoms, α-neurotoxins are present in D. polylepis 24 , In addition, fasciculins, members of the 3FTx family which induce fasciculations by inhibiting acetylcholinesterase, were found in D.

angusticeps venoms Dendrotoxins, which have homology to Kunitz-type proteinase inhibitors and block voltage-dependent potassium channels, are typical of mamba venoms, with highest concentration in the venom of D. polylepis Both dendrotoxins and fasciculins were probably not neutralized by the pan-specific antiserum since these toxins are not present in the immunogen mix.

α-Neurotoxins are the most lethal toxins of D. polylepis venom, with dendrotoxins playing a minor role in lethality This explains why the lethality of this venom was neutralized by the pan-specific antiserum even though the dendrotoxins were unlikely neutralized. angusticeps venom was the only one not neutralized by the pan-specific antiserum.

From its proteome, fourty-two different proteins were detected, among which 3FTxs were the most abundant, followed by the Kunitz-type proteinase inhibitor family. However, no α-neurotoxin was identified in the venom 23 which is in agreement with an in vitro potency assay based on nAChR binding 26 Fig.

None of the venom HPLC fractions was lethal to mice at the doses tested. Thus, it was proposed that the lethality of the venom was due to the synergistic action of various components, such as fasciculins and dendrotoxins, and probably other synergistically-acting toxins It is not surprising that the pan-specific antiserum did not neutralize the lethal effects of the venom since the toxins of the venom were not present in the immunogen mix, and simultaneous neutralization of various synergistic acting toxins are required in order to neutralize the lethality of the venom.

Proteomics data indicate that α-neurotoxins in N. nigricollis venom was only 0. The antiserum most likely contains antibodies against these components in this spitting cobra venom probably due to the presence of similar toxins in the venoms used in the immunizing mix.

In the case of N. senegalensis venom, no proteomics data are available. However, clinical cases are associated with neuromuscular paralysis and respiratory failure 28 , suggesting that α- neurotoxins are likely to play a key role in the overall toxicity.

This venom was effectively neutralized by the pan-specific antiserum, underscoring that these lethal toxins were immunorecognized by the antibodies.

Nevertheless, caution should be exerted when extrapolating data from mouse experiments to the human situation when studying venom-induced neurotoxicity Table 3 shows the proteomics toxin profiles of the 16 heterologous venoms previously shown to be neutralized by the pan-specific antiserum.

Ten of them are from species of the genus Naja , characterized by the presence of short Type I and long Type II α-neurotoxins and cytotoxins. Venoms from species of Bungarus , e. candidus and B. multicinctus , contain both pre-synaptic β- and post-synaptic α-neurotoxins 30 , In the case of Ophiophagus hannah , its venom contains α-neurotoxins but not β-neurotoxins These toxins, except for cytotoxins, are highly lethal in mice and are known to be the cause of death in elapid envenomations.

On the basis of our observations, they were likely neutralized by the antibodies in the pan-specific antiserum. Thus, the pan-specific antiserum neutralized most, if not all, the potentially lethal toxins in the 25 heterologous neurotoxic venoms tested, hence stressing the value of using fractions enriched with various lethal toxins of several venoms in the immunization process.

Another alternative proposed to raise an antiserum of high neutralizing coverage against neurotoxic venoms includes the use of recombinant consensus short α-neurotoxins as immunogen In this case, however, the antiserum is not effective in the neutralization of presynaptic β-neurotoxins, and recombinant consensus PLA 2 neurotoxins would need to be added to the immunizing mix to neutralize venoms whose toxicity is driven by β-neurotoxins.

and kraits Bungarus spp. inhabiting various countries of Asia. Table 4 presents the toxin profiles of these 12 venoms. These elapid venoms contain a diverse set of toxins, such as short and long α-neurotoxins, β-neurotoxins and cytotoxins as the major lethal toxins.

Table 4 shows the number of isoforms of each type of these toxins in these 12 venoms. Altogether, there are about 23 isoforms of short Type I α-neurotoxins, 17 isoforms of long Type II α-neurotoxins and about 15 isoforms of PLA 2 β-neurotoxins which are exposed to the horses.

These large numbers of toxin isoforms are likely to contain numerous epitopes of these lethal toxins. It is therefore conceivable that the immune system of the horses generated a diverse set of paratopes against all the isoforms of these lethal toxins, hence explaining the ability of the antiserum to neutralize the lethal activity of 36 neurotoxic elapid venoms 25 heterologous and 11 homologous venoms from 10 snake genera.

It is evident that the experimental antiserum showed very wide para-specificity against numerous neurotoxic venoms. The following considerations may form the bases for explaining this phenomenon:.

The most lethal components in the majority of these neurotoxic venoms are the post synaptically-acting α—neurotoxins Tables 2 , 3 and 4. For some venoms, e. Bungarus spp. and O. scutellatus , highly lethal β-neurotoxins are also relevant to human envenomation 22 , 34 , 35 , 36 , 37 , For simplicity, this discussion will be confined to the case of α—neurotoxins, which constitute the main lethal factors of many, but not all, elapid venoms α—neurotoxins are small polypeptides of about 61—62 amino acids with 4 disulfide linkages short α-neurotoxins or 66—75 amino acids with 5 disulfide linkages long α-neurotoxins They adopt a planar structure similar to a 3-finger configuration and are referred to as three-finger toxins 3FTxs The amino acid sequences of the over α—neurotoxins from snakes have been described; the top of these sequences share about All of these α—neurotoxins bind specifically and strongly to the α—subunits of nAchR at the motor endplate in the neuromuscular junction 40 , 42 , Thus all these toxins share structural and functional homology.

Each elapid venom may contain several α—neurotoxins short, long, dimers, precursors, etc. that show sequence variations Most of the venoms contain 1—5 α-neurotoxin isoforms Table 4 ; therefore, the total number of α-neurotoxin isoforms in the immunogen mixture may reach several dozens.

These numerous α-neurotoxin isoforms together contain a large number of toxin epitopes to which the horses were exposed to, with a vast number of antibody paratopes generated against these epitopes. Hence, the diversity of α-neurotoxin isoforms is the strength in this immunization strategy, as it contributes to generate a wide repertoire of antibodies.

Given their small molecular size and constraint to form a biologically active conformation, it is likely that each α—neurotoxin contains a relatively small number of dominant epitopes on its surface, as shown for venoms of Dendroaspis spp. using a high-throughput microarray analysis 45 , with each epitope covering an area of 6—7 amino acid residues Some of the epitopes from homologous toxins are conserved for structural and functional reasons.

Because of the high sequence identity, some of these epitopes are expected to be structurally similar, though not identical, and thus explain some degree of immunochemical cross reactivity of antisera 47 , 48 , In the case of monospecific polyclonal antisera raised against a single neurotoxic venom, they are likely to contain high affinity antibodies against the α—neurotoxins of the homologous venom.

Some of these antibodies may cross react with heterologous α—neurotoxins due to epitope structural similarity. This could be a reason for the low cross-neutralization of monospecific antivenoms usually observed 5. Moreover, it has been shown that a single antibody could adopt different conformations of its paratope to bind different epitopes, thus enhancing its antigenic coverage The more diverse the paratopes of the antisera antibodies, the better chance for some of them to interact with the epitopes of the heterologous α-neurotoxins.

These interactions, albeit with lower affinity should, through cross-linking and lattice formation 52 , result in antisera with higher avidity leading to more effective neutralization of diverse heterologous neurotoxic venoms Similar situations may occur with the highly lethal presynaptic β-neurotoxins 37 and other types of venom toxins when using a variety of venoms in the immunizing mixture.

The large pool of diverse antibody paratopes makes it likely that high affinity antibodies are present to interact effectively with a variety of α-neurotoxins.

This is crucial because, due to steric hindrance, no more than two antibody molecules can interact simultaneously with one toxin molecule Supplementary Fig. Whether or not the proposed bases for the wide para-specificity of our antiserum are correct, the results of these studies show that this is the widest cross-neutralizing antiserum ever reported against neurotoxic snake venoms from wide geographical distribution.

Our results represent a proof of concept that an antiserum with wide spectrum of cross-neutralization against elapid venoms can be raised. The genus-wide analysis of venom composition and toxicity of these venoms to identify the lethal toxins 24 followed by use of the combined toxin fractions to immunize horses, is likely to result in widely para-specific antiserum against these snake venoms.

As shown in Tables 1 , 2 and 3 , the antiserum could neutralize lethality of 25 heterologous venoms, but its neutralizing potency against some of them is rather low. This becomes a problem especially when dealing with species that inject a large volume of venom in a bite.

However, the potency of the antiserum can be improved by a concentration process during plasma fractionation.

Since horse hyperimmune sera have an average protein concentration at After such concentration process, the present horse pan-specific antiserum could have higher neutralizing activity against the lethality of many neurotoxic venoms.

This may not only increase the potency against the venoms tested, but also provide neutralization of additional elapid venoms. There is a growing interest in the development of recombinant antivenoms 55 , This involves, for example, the preparation of animal- or human-derived monoclonal antibodies against the lethal components of venoms.

Proofs of concept of this strategy have been published 57 , One major requirement of this approach is that the major lethal toxin s of the venom must be identified and used for antibody selection. When more than one toxin is relevant in a particular venom, there is a need to generate additional antibodies for a successful neutralization.

Since these antibodies are produced against one or few toxins, a challenging issue for this strategy is to ensure the neutralization of heterologous toxins present in other venoms. It should be possible to further increase the para-specificity of the antiserum by including additional venom toxin fractions in the immunization mix.

For example, inclusion of toxin fractions of some African mamba venoms D. angusticeps and D. viridis and some American coral snakes e.

could increase the neutralizing scope of the antiserum. By carefully selecting the venoms and fractions to be added to the immunizing mix it should be possible to expand the scope of coverage of neurotoxic venoms, ideally to neutralize the most important elapid venoms in the world.

Snakebite envenomation is a WHO classified category A Neglected Tropical Diseases, i. a disease of highest importance. One of the four pillars of this strategy is to ensure safe and effective treatments, particularly referring to antivenoms, which represent the only scientifically-validated therapy for these envenomings.

As shown in this work, a pan-specific antivenom against neurotoxic venoms would be a powerful therapeutic tool to save lives of people suffering these envenomings in different parts of the world, by neutralizing a wide spectrum of neurotoxic snake venoms which otherwise require region- or species-specific antivenoms for treatment.

The antiserum exhibited a wide para-specificity by neutralizing at least 36 neurotoxic venoms of snakes of 10 genera from four continents. The pool of diverse toxin antigens in the immunogen mix enabled the production of diverse antibody paratopes, which facilitate the interaction of the antibodies with the epitopes of various neurotoxins from homologous as well as heterologous snake venoms.

Dendroaspis polylepis, D. angusticeps, D. viridis and Naja senegalensis venoms were obtained from Latoxan Valence, France ; Laticauda colubrina, Pseudechis australis, Oxyuranus scutellatus and Notechis scutatus venoms were obtained from Venom Supplies Pty Ltd Australia.

Hydrophis schistosus venom was provided by Dr. CH Tan, and Micrurus nigrocinctus venom was provided by Prof. José María Gutiérrez. These two venoms were obtained from several specimens kept in captivity M.

nigrocinctus or captured wild H. Naja kaouthia Thailand principal post-synaptic neurotoxin 3 NK3 was purified as described by Karlsson et al. The pan-specific antiserum used in the present study was from the same batch as that obtained from horses immunized with mixtures of venoms and venom fractions 11 using the protocol briefly described below.

All chemicals and biochemical were from Sigma Chemical Co. St Louis, Missouri, USA unless otherwise stated. Experiments carried out in horses regarding care, bleeding and immunization were approved by the Animal Care and Use Committee of the Faculty of Veterinary Science, Mahidol University, Protocol and clearance no.

MUVS in accordance with the Guidelines of the National Research Council of Thailand. Preparation of the pan-specific antiserum was described previously and Bungarus spp.

inhabiting different geographical locations of Asia The toxin fractions of Naja spp. The presence of α-neurotoxins in venoms was estimated by the venom-mediated inhibition of the binding of purified nAChR to immobilized elapid post-synaptic neurotoxins, as described previously californica electroplax.

The amount of nAChR bound to the immobilized NK3 in the wells was estimated by adding rat anti-nAChR serum at dilution followed by a dilution of goat anti-rat IgG-enzyme conjugated HRP and enzyme substrate. If the tested neurotoxic venom contained α-neurotoxin which could specifically interact with nAChR, the percent binding of the receptor to the NK3 immobilized plate is reduced and can be calculated using the following formula:.

Venom lethality median lethal dose, LD 50 and the median effective doses ED 50 of the pan-specific antiserum against the venoms tested were determined and analyzed as previously reported 11 and are briefly described below. The median lethal dose LD 50 of a venom was determined by i. In all experiments, the control groups of mice, regardless of whether 5x, 2.

The neutralization potency P of the antiserum, defined as the amount of venom completely neutralized per unit volume of antiserum, was expressed as previously described Chippaux, J. Snake-bites: appraisal of the global situation.

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