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    Анализ ядовитых свойств секрета Azemiops feae

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    Toxicon, Vol. 24, No.5, pp. 510-513, 1986.
    Printed in Great Britain.

    0041-0101186 $3.00+ .00

    Pergamon Journals Ltd.



    Department of Zoology, Washington State University, Pullman, WA 99164-4220, U.S.A.
    (Accepted jor publication 11 December 1985)

    D. K. VEST. Preliminary studies on the venom of the Chinese snake Azemiops jeae, Boulenger
    (Fea's viper). Toxicon 24, 510- 513, 1986. - Fea's viper (Azemiops jeae) produces a venom
    which is highly toxic to mice when injected by the s.c. or Lv. routes. The Lv. LDso of Azemiops
    venom for Swiss-Webster laboratory mice is 0.52 mg/kg. Azemiops venom produces no
    hemorrhagic activity in mice or rabbits. Immunodiffusion indicates that some fractions of
    Azemiops venom are antigenically related to viperid, elapid and crotalid venoms. SDS-PAGE
    electrophoresis reveals that this venom contains as many as 22 proteinaceous components.

    THE FEA'S VIPER (Azemiops jeae) is a small viperine snake distributed in the Chinese
    provinces of Yunan, Guizhou, Sichuan, Guangxi, Fujian, Jiangxi and Zhejiang (ZHAO
    and ZHAO, 1981) and also occurs in Burma. Due to the rarity of this serpent, virtually
    nothing is known concerning its venom. Histological investigation of Azemiops venom
    glands by KOCHVA and GANS (1965, 1966) and KOCHVA et ale (1967) show that Azemiops
    venom glands exhibit the general pattern of taxonomic characteristics of all viperine
    Seven adult specimens of Azemiopsjeae were obtained from a commercial source (J. J.
    Vandenbrink, Jabria B.V.) and were subjected to venom extraction. The venom was
    collected in a snlall plastic 'auto-analysis' cup (Sardstedt no. 73.641), immediately frozen,
    then lyophilized and weighed. Lyophilized venom was reconstituted in 0.9070 physiological
    saline. Twenty-five healthy Swiss-Webster laboratory mice weighing 18 - 22 g were used
    for the lethality screen. The Lv. LD so was calculated according to the method of
    LITCHFIELD and WILCOXON (1949). All mice were closely observed for 12 hr post­
    challenge. Respiratory rates were measured by counting respirations against a lap timer.
    Thirteen Swiss-Webster mice were depilated and challenged s.c. with venom
    concentrations of 5 - 50 JAg in 100 JAI 0.9070 physiological saline. Additionally, a California
    giant laboratory rabbit was dorsally depilated and challenged intracutaneously with six
    concentrations of (5 - 50 JAg) Azemiops venom reconstituted as described above. Mice
    surviving s.c. challenge, as well as the rabbit, were killed at 24 hr post-injection and
    examined for hemorrhagic and local responses. Immunodiffusion of Azemiops venom
    against several commercial monovalent and polyvalent antivenoms, as well as active
    monovalent antisera of rabbit origin (Arizona State University), was performed on
    Ouchterlony plates and micro-immunodiffusion slides using 2070 agar noble in the

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    diffusion medium. Antigen wells were loaded with venom solutions containing 5 mg/ml
    Azemiops venom, and were developed at room temperature. SDS-Polyacrylamide gel
    electrophoresis (slab format) was performed at pH 6.8 according to the method of
    LAEMMLI (1970). Wells were loaded in duplicate with venom quantities of 50, 75, 100, 125
    and 150 IJg. All samples were reduced with (3-mercaptoethanol and run at 10 rnA per gel
    through a 3 mm thick, 5070 stacking gel, then a 15070 separating gel.
    Five of the Azemiops specimens subjected to extraction delivered only trace amounts of
    venom, despite vigorous biting. The other two specimens delivered significant,
    approximately equivalent volumes of venom ( rv l.75 mg/snake). The venom appeared as
    a clear, golden-yellow liquid, similar to that of many other viperine snakes. Its viscosity,
    likewise, was similar to that delivered by most viperines.
    The i.v. LD so of Azemiops feae venom in Swiss-Webster mice was 0.52 mg/kg. Mice
    challenged with doses of 0.50 - 0.60 mg/kg usually exhibited a transitory, minor-to­
    moderate vasodilatation of ear vessels within 10 min of injection. These mice became
    torpid within 30 ± 10 min and respirations began to decrease, generally followed by clonic
    convulsions. Paralysis became virtually complete 90 - 110 min post-injection, although
    mice retained a very slight ability to move the legs and were able to right themselves until
    the very terminal stage of poisoning. A paroxysm occurred 86 -150 min post-injection, in
    which respirations reached a critical minimum (less than 35 per min). This paroxysm
    terminated in death for mice challenged by a lethal dose; all mice that tolerated
    envenomation for 180 min survived. All mice receiving s.c. challenges of between 0.50
    and 0.60 mg/kg died, while those receiving 0.40 mg/kg or less survived.
    Local tissue responses and hemorrhagic activity in laboratory mice were only slight. No
    indications of frank hemorrhagic activity nor local tissue degradation were evident.
    Intracutaneous injection of Azemiops venom into rabbit skin produced no local
    manifestations or evidence of hemorrhagic phenomenon.
    Ouchterlony and micro-immunodiffusion of Azemiops venom vs. commercial
    antivenins resulted in the formation of precipitation lines between wells containing
    Azemiops venom and several commercial antivenins (Fig. 1). Significant precipitation
    lines developed against the antisera to tiger-snake, death adder, mamba and Iran cobra,
    while weak reactions were seen against Thai cobra, and no reaction developed against
    king cobra. Strong, multiple precipitation lines developed against Serum Europe, Serum
    North Africa, Fitzsimmon's Polyvalent and Wyeth Polyvalent, with weaker lines visible
    against Haffkine Polyvalent and Serum Near and Middle East. No precipitation reaction
    was observed against monovalent Agkistrodon acutus antivenene or Malayan pit viper
    antivenene, nor against monovalent Agkistrodon bilineatus, Agkistrodon piscivorus,
    Crotalus atrox, Crotalus scutulatus or Heloderma suspectum antisera.
    SDS-PAGE of fJ-mercaptoethanol-reduced Azemiops venom revealed the presence of
    22 visible proteinaceous bands with molecular weights ranging from approximately 10,000
    to 80,000. When 100 - 150 IJg of venom were applied, 20 - 22 bands were visible,
    including one smaller component (mol. wt 9000 - 11,000) not well visualized when lesser
    amounts of venom were applied.
    Fea's viper (Azemiops feae) produces a venom which causes marked flaccid paralysis,
    respiratory depression and clonic convulsions in mice, while eliciting only negligable local
    tissue responses. There was no apparent hemorrhagic phenomenon, either local or
    systemic. Generally, convulsions signaled the onset of a paroxysm in which respiratory
    rates fell below 30 per min and finally ceased altogether, terminating in the death of the
    mouse. Superficially, the progressive respiratory depression, which closely paralleled the


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    progression of flaccid paralysis, resembles poisoning by those elapid snakes whose
    venoms contain a post-synaptic, peptide neurotoxin.
    The present study using mice indicates that Azemiops envenomation may be expected to
    produce visual, respiratory and paralytic effects in humans, but does not suggest that this
    venom is capable of inducing local or hemorrhagic phenomenon, as reported for humans
    (ZHAO and ZHAO, 1981).
    Immunodiffusion of Azemiops venom against multi-genus polyvalent antivenins
    demonstrate the presence of antigenically related components between Azemiops and all
    major groups of terrestrial venomous snakes. Precipitation lines between Azemiops
    venom and monovalent Australian antivenenes, as well as genus-specific mamba
    antivenene, indicate a definite antigenic relationship between these venoms and
    Azemiops, but it should be noted that these sera react with an exceptionally large number
    of heterologous venoms, including colubrid Duvernoy's secretions (MINTON, 1979;
    Minton, personal communication). Wyeth Polyvalent Antivenin proved to be the only
    crotalid preparation reacting against Azemiops venom, with no reaction seen against
    monovalent Agkistrodon or Crotalus antisera.
    Azemiops jeae may be considered a member of the Viperidae which possesses a venom
    with immunological and electrophoretic characteristics similar to those of other viperine


    .-- • .~ •


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    Azemiops /eae


    Photographed 72 hr post-loading: (A) Azemiops /eae venom, 5 mg/ml; (D) death adder

    antivenom; (F) Fitzsimmon's Polyvalent; (H) Haffkine Polyvalent; (I) Iran cobra antivenom; (M)

    mamba Polyvalent; (T) tiger snake antivenom; (NA) Serum North Africa; (NM) Serum Near and

    Middle East; (TC) Thai cobra antivenom. Comparable, but more sharply defined precipitations

    were observed using micro-immunodiffusion slides.

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    snakes. Its toxicity characteristics, however, are not typical of the majority of the better
    known, clinically significant Viperidae, whose venoms induce changes in local tissues,
    hemorrhage and hematological discrasias. Azemiops venom appears to be one of the
    'atypical' viperid venoms, which, like the berg adder Bitis atropos (CHRISTENSEN, 1955,
    1968), elicits pronounced paralytic phenomenon in mice, without evidence of local and/or
    hemorrhagic syndromes.
    Acknowledgement - For comments on the manuscript, I thank SHERMAN A. MINTON, EDWARD K. JOHNSON
    and K. V. KARDONG. For technical assistance, I thank TERRY ELTON, K. GASSER, H. L. HOSICK, J. D. HUBER, R.
    REEVES, P. C. SCHROEDER and L. KIRSCHNER. This work was supported in part by grant BNS 7817465,
    Psychobiology Program, National Science Foundation, to K. V. KARDONG.

    CHRISTENSEN, P. A. (1955) South African Snake Venoms and Antivenoms. Johannesburg: South African
    Institute of Medical Research.
    CHRISTENSEN, P. A. (1968) Venoms of Central and South African snakes. In: Venomous Animals and their
    Venoms, Vol. 1, p. 447 (HOCHERL, W., BUCKLEY, E. and DEULOFEU, V., Eds). New York: Academic Press.
    KOCHVA, E. and GANS, C. (1965) The venom gland of Vipera palaestinae with comments on the glands of some
    other Viperinae. Acta Anat. 62, 365.
    KOCHVA, E. and GANS, C. (1966) Histology and histochemistry of the venom gland of some crotaline snakes.

    Copeia 3, 506.
    KOCHVA, E., SHAYER-WOLLBERG, M. and SOBOL, .R. (1967) The special pattern of the venom gland in
    Atractaspis and its bearing on the taxonomic status of the genus. Copeia 4,·763.
    LAEMMLI, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.

    Nature 227,680.
    LITCHFIELD, J. T. JR and WILCOXON, I. (1949) A simplified method of evaluating dose-effect experiments. J.
    Pharmac. expo Ther. 96, 99.
    MINTON, S. A. (1979) Common antigens in snake venoms. In: Handbook ofExperimental Pharmacology, Vol.
    52, Snake Venoms, p. 847 (Lee, C.-Y., Ed.). New York: Springer-Verlag.
    ZHAO, E. and ZHAO, G. (1981) Notes on Fea's viper (Azemiopsfeae Boulenger) from China. Acta herpetol. sin.