Analysis of the Necrosis-Inducing Components of the Venom of Naja atra and Assessment of the Neutralization Ability of Freeze-Dried Antivenom.

Cheng-Hsuan Ho,Yan-Chiao Mao,Chin-Sheng Lin,Kuang-Ting Chen,Wei-Hsuan Fang,Yu-Jen Shih,Dapi Meng-Lin Chiang,Chi-Hsin Lee,Yuan-Sheng Tzeng,Wen-Loung Lin,Shing-Hwa Liu,Shih-Hung Tsai,Kuo-Cheng Lan,Liao-Chun Chiang

doi:10.3390/toxins13090619

Abstract

Patients bitten by Naja atra who are treated with bivalent freeze-dried neurotoxic antivenom in Taiwan have an improved survival rate but develop necrotic wound changes. The World Health Organization (WHO) has suggested using the minimum necrotizing dose (MND) of venom as a method of evaluating the neutralization effect of antivenom. The aim of this study was to evaluate the effectiveness of antivenom for the prevention of necrosis based on the MND and clarify which component of the venom of N. atra induces necrosis. The neurotoxins (NTXs) were removed from the crude venom (deNTXs), and different concentrations of deNTXs were injected intradermally into the dorsal skin of mice. After three days, the necrotic lesion diameter was found to be approximately 5 mm, and the MND was calculated. A reduction in the necrotic diameter of 50% was used to identify the MND50. Furthermore, both phospholipase A2 (PLA2) and cytotoxins (CTXs) were separately removed from the deNTXs to identify the major necrosis-inducing factor, and the necrotic lesions were scored. All mice injected with deNTXs survived for three days and developed necrotic wounds. The MND of the deNTXs for mice was 0.494 ± 0.029 µg/g, that of the deNTXs-dePLA2 (major component retained: CTXs) was 0.294 ± 0.05 µg/g, and that of the deNTX-deCTX (major component retained: PLA2) venom was greater than 1.25 µg/g. These values show that CTX is the major factor inducing necrosis. These results suggest that the use of the deNTXs is necessary to enable the mice to survive long enough to develop venom-induced cytolytic effects. CTXs play a major role in N. atra-related necrosis. However, the MND50 could not be identified in this study, which meant that the antivenom did not neutralize venom-induced necrosis.

Highlights

Naja atra, a member of the Elapidae family, is a medically significant venomous snake that is common in Central Taiwan [1]
The aim of this study was to evaluate the effectiveness of antivenom with regard to the prevention of necrosis based on the minimum necrotizing dose (MND)/MND50 and to identify which component of the venom of N. atra leads to necrosis
N. atra, atra, which which was was identified identified by the the patient, patient, received one vial of the bivalent antivenom against atra and develreceived of the bivalent antivenom oped progressive necrosis without any neurologic symptoms two days later

Summary

Introduction

A member of the Elapidae family, is a medically significant venomous snake that is common in Central Taiwan [1]. Patients bitten by N. atra are treated with bivalent antivenom-freeze-dried neurotoxic antivenom in Taiwan [2]. Local injuries are more common than neurologic toxicity after bites from many Naja species, including Naja nigricollis, Naja mossambica, Naja nigricincta, Naja pallida, Naja nubiae, and Naja katiensis [3,4,5]. Among patients bitten by N. atra, 65.6% progress to skin necrosis, and 42.1% develop necrotizing soft tissue infections [1,6]. Even when patients receive antivenom, skin necrosis still occurs, and debridement surgery is usually suggested 3.5 days after snake bite [6]. Studies have investigated the mechanism for N. nigricollis [7,8] and N. atra [9], there has been less research on the latter species

Objectives

Methods

Conclusion