Snake Sera Research Articles

Identification of Antibacterial Molecule(s) from Animals Living in Polluted Environments.

Snakes feed on germ-infested rodents, while water monitor lizards thrive on rotten matter in unhygienic conditions. We hypothesize that such creatures survive the assault of superbugs and are able to fend off disease by producing antimicrobial substances. In this study, we investigated the potential antibacterial activity of sera/lysates of animals living in polluted environments. Snake (Reticulatus malayanus), rats (Rattus rattus), water monitor lizard (Varanus salvator), frog (Lithobates catesbeianus), fish (Oreochromis mossambicus), chicken (Gallus gallus domesticus), and pigeon (Columba livia) were dissected and their organ lysates/sera were collected. Crude extracts were tested for bactericidal effects against neuropathogenic E. coli K1, methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Pseudomonas aeruginosa, Bacillus cereus and Klebsiella pneumoniae. To determine whether lysates/sera protect human cells against bacterialmediated damage, cytotoxicity assays were performed by measuring lactate dehydrogenase release as an indicator of cell death. Lysates/sera were partially characterized using heat-treatment and pronasetreatment and peptide sequences were determined using the Liquid Chromatography Mass Spectrometry (LC-MS). Snake and water monitor lizard sera exhibited potent broad-spectrum bactericidal effects against all bacteria tested. Heat inactivation and pronase-treatment inhibited bactericidal effects indicating that activity is heat-labile and pronase-sensitive suggesting that active molecules are proteinaceous in nature. LCMS analyses revealed the molecular identities of peptides. The results revealed that python that feeds on germ-infested rodents and water monitor lizards that feed on rotten organic waste possess antibacterial activity in a heat-sensitive manner and several peptides were identified. We hope that the discovery of antibacterial activity in the sera of animals living in polluted environments will stimulate research in finding antibacterial agents from unusual sources as this has the potential for the development of novel strategies in the control of infectious diseases.

Generation of Anti-Boa Immunoglobulin Antibodies for Serodiagnostic Applications, and Their Use to Detect Anti-Reptarenavirus Antibodies in Boa Constrictor.

Immunoglobulins (Igs), the key effectors of the adaptive immune system, mediate the specific recognition of foreign structures, i.e. antigens. In mammals, IgM production commonly precedes the production of IgG in the response to an infection. The reptilian counterpart of IgG is IgY, but the exact kinetics of the reptilian immune response are less well known. Boid inclusion body disease (BIBD), an often fatal disease of captive boas and pythons has been linked to reptarenavirus infection, and BIBD is believed to be immunosuppressive. However, so far, the study of the serological response towards reptarenaviruses in BIBD has been hampered by the lack of reagents. Thus we set up a purification protocol for boa constrictor IgY and IgM, which should also be applicable for other snake species. We used centrifugal filter units, poly ethylene glycol precipitation and gel permeation chromatography to purify and separate the IgM and IgY fractions from boa constrictor serum, which we further used to immunise rabbits. We affinity purified IgM and IgY specific reagents from the produced antiserum, and labelled the reagents with horseradish peroxidase. Finally, using the sera of snakes with known exposure to reptarenaviruses we demonstrated that the newly generated reagents can be utilised for serodiagnostic purposes, such as immunoblotting and immunofluorescent staining. To our knowledge, this is the first report to show reptarenavirus-specific antibodies in boa constrictors.

Prospection, structural analysis and phylogenetic relationships of endogenous γ-phospholipase A 2 inhibitors in Brazilian Bothrops snakes (Viperidae, Crotalinae)

During the last 20 years, there have been an increasing number of reports on endogenous phospholipase A 2 inhibitors (PLIs) in the sera of snakes. These studies have demonstrated the existence of three different structural classes of PLIs (α, β and γ). The γ class members are potent inhibitors of phospholipases A 2 (PLA 2) from the venom of Viperidae snakes. These enzymes, together with the mammalian pro-inflammatory PLA 2, belong to the IIA class of the PLA 2-superfamily. Although coming from distinct sources, these phospholipases A 2 share main structural features. For this reason, γPLIs have been considered as potential models for the development of selective inhibitors of pro-inflammatory PLA 2 in humans. In spite of the rich diversity of the ophidian fauna in Brazil, only two γPLI representatives, from Crotalus durissus terrificus and Lachesis muta, have been described in Brazilian snakes so far. Here we investigated the presence of transcripts of novel γPLIs in six Bothrops species (Viperidae, Crotalinae) commonly found in our country: Bothrops alternatus, Bothrops erythromelas, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni and Bothrops neuwiedi. γPLI transcripts were present in every species analysed. The deduced mature proteins possessed 181 amino acid residues following a 19-residue signal peptide, similar to the γPLIs from C. d. terrificus taken as our model, with the exception of the deduced proteins from B. erythromelas and B. neuwiedi snakes. In these particular cases, an insertion of 4-amino acid residues was consistently present. A Bayesian tree was obtained for the Brazilian Bothrops γPLIs, showing four clusters: (1) L. muta and B. jararacussu, (2) B. alternatus, (3) B. erythromelas and B. neuwiedi, (4) B. jararaca and B. moojeni. Detailed structural analysis and further comparisons of these novel Bothrops inhibitors with γPLIs from New and Old World snakes are provided.

Inhibitory effects of snake sera on several venom-induced myonecrosis, haemorrhage and odema activities in mice

Inhibitory effects of snake sera on several venom-induced myonecrosis, haemorrhage and odema activities in mice

Sphero-echinocytosis of human red blood cells caused by snake, red-back spider, bee and blue-ringed octopus venoms and its inhibition by snake sera

It was found that bee (Apis mellifera) venom, red-back spider (Latrodectus mactans) venom, blue-ringed octopus (Hapalochlaena maculosa) venom, ten different snake venoms, phospholipase A2 and four snake toxins caused sphero-echinocytosis of human red blood cells at 200 ng/ml. Most venoms and toxins lost the ability to deform human red blood cells when their components of less than mol. wt 10,000 were applied. In a number of cases the sphero-echinocytotic effect was also inhibited by blood sera of Notechis scutatus and Pseudonaja textilis.

Appendix: Specimens of snake sera tested

Appendix: Specimens of snake sera tested

Natural hemolytic activity of snake serum II. Heterogeneity of putative natural antibody and levels of hemolytic activity

The erythrocytes of various vertebrates, such as mice, rabbits, sheep, chickens, bullfrogs, and toads are lysed by normal snake sera. However, snake erythrocytes were not lysed by serum from different snake species. Putative natural antibody seems with different specificities to comprise heterogeneous antibodies. Thus, absorption of snake serum with mouse erythrocytes, for example, abrogated hemolytic activity for mice but not for rabbit or sheep erythrocytes. We observed no significant intraspecies individual differences in serum hemolytic titer, but interspecies differences were obvious. Immunization of snakes with sheep erythrocytes caused no further elevation of hemolytic activity, though high titer antibody was produced in response to certain bacterial antigens. Even the sera of newly-hatched snakes showed hemolytic activity at modestly high levels. No seasonal change in hemolytic activity was observed.

Neutralization of crotalid venoms by fractions from snake sera

Neutralization of crotalid venoms by fractions from snake sera. Toxicon 16, 603–609, 1978.—A protein fraction is present in the sera of snakes belonging to Crotalidae and Colubridae. which protects mice from the lethal effect of 5 ld 50 of crotalid venoms injected i.p. When the sera were subjected to ammonium sulfate fractionation, the 0–45% fraction afforded little or no protection. The 45–90% precipitate on the other hand contained the neutralizing agent(s). Molecular sieving of this fraction on Sephadex G-200 gave two distinct protein bands. The neutralizing agent(s) was recovered in the second band containing proteins with mol. wt. of 50,000–100,000. The protective effect of this fraction was completely destroyed by pronase treatment. The most pronounced effect of the purified serum factor(s) was its ability to inhibit venom protease activity. The venom esterase activity and many other venom enzyme activities were not affected by the serum factor(s). It has been observed that venom protease in the presence of plasma produces large amounts of bradykinin. The serum fraction which protects mice from the lethal action of the venoms also blocks the release of bradykinin. It is proposed that the lethal action of venoms is related to the action of unique proteases which release bradykinin from plasma proteins and destroy fibrinogen. This combined action sets the stage for extensive hemorrhage. The neutralizing effect of crotalid and colubrid sera is due in part to a powerful antiprotease activity.

Natural hemolytic activity of snake serum I. Natural antibody and complement

Normal snake sera from five species were highly active in lysing sheep erythrocytes (SRBC). No significant hemolytic activity, however, was found in the sera of tortoises, lizards, or bullfrogs. Natural hemolytic activity of snake sera was dependent upon natural antibody and complement. The antibody was heat-labile and inactivated by treatment at 55° for 15 min. A component (C S), probably the first component, of snake complement may be snake-specific; thus, sera from guinea pigs, bullfrogs, tortoises, chickens, and rabbits were ineffective in lysing SRBC sensitized with snake anti-SRBC natural antibody. Components (C G) of snake complement other than C S could be replaced by those of guinea pig complement. The activity of the snake complement system was inactivated by treatment of sera at 55° for 15 min. The titer of complement exceeded that of natural antibody, indicating that the limiting factor in the natural hemolytic activity of snake sera depended upon the level of natural antibody.

Neutralization of Viperidae and Elapidae snake venoms by sera of different animals

The venoms of three vipers, Vipera palaestinae, Echis colorata and Pseudocerastes fieldi, and the elapid Walterinnesia aegyptia, all from Israel, show a similar ld 50 of 250–300 μg/kg body weight when injected intravenously into mice. The venoms of the viperid Aspis cerastes from Israel and the elapid Naja nigricollis from East Africa are less toxic and have an ld 50 of 600 and 1200 μg/kg respectively. Compared with mice on a weight basis, V. palaestinae, Natrix tessellata (a non-venomous water snake) and the mammal Herpestes ichneumon (mongoose) are highly resistant to V. palaestinae venom; Mesocricetus auratus (hamster) also shows some degree of tolerance. The sera from these and other animals were tested for their capacity to neutralize this venom in vitro. The snake sera, which represented the families Elapidae, Viperidae, Crotalidae and Colubridae, all neutralized V. palaestinae venom, whereas sera from two lizards ( Uromastix aegyptius and Agama stellio) failed to do so. Of the mammalian sera tested, neutralization was clearly demonstrated with hamster serum and to a lesser degree with hedgehog ( Erinaceus europeus) serum, but not with human serum, cat serum or rabbit serum, and even serum from the highly resistant mongoose failed to show any neutralization. In tests involving these sera and the 6 venoms listed above, the Viperidae venoms were neutralized by sera from snakes of the same family, by W. aegyptia serum, and, with the exception of E. colorata venom, by N. nigricollis serum. The neutralization by sera from non-venomous snakes was slightly less complete, and sera from the mongoose and hamster failed to neutralize in some cases. N. nigricollis venom could be neutralized by the homologous serum and by W. aegyptia serum, but not by any others. All test sera, including the homologous serum, were without effect on W. aegyptia venom. Despite the lack of neutralizing capacity of its serum, W. aegyptia is highly resistant to its own venom and the mongoose to both of the elapid venoms tested. This resistance is apparently not due to humoral factors.

Antivenin activity of some snake sera

Studies were done to determine the effects of certain snake sera on snake venoms, to observe the tryptic inhibitor activity of these sera, and to determine their effects on erythrocytes in the presence of venom.

Serological Studies of the Reptilia

Summary This paper had for its primary purpose, a study of the hemagglutinins and hemagglutinogens of snake blood and their similarity to human hemagglutinins and hemagglutinogens. By an appropriate technic it was found that isohemagglutination did not occur with the samples of snake blood examined. Heterohemagglutination and heterohemolysis did occur between genera and between families of the Serpentes. This heterohemagglutination showed no correlation to zoological grouping. From a comparative study of snake and human bloods it was observed that the snake erythrocytes examined did not contain agglutinogens for human Alpha and Beta agglutinins. Snake serum, however, may or may not contain agglutinins which act similarly to the Alpha and Beta agglutinins of human serum. In addition to these 2 specifically acting agglutinins, it was found that some snake sera contain an agglutinin which can be absorbed by human cells of any group. The specific agglutinins of snake serum and the Alpha and Beta agglutinins of human sera are equally thermolabile. Appropriately selected and absorbed snake sera can be used to determine the blood grouping of unknown human cells.

Societies and Academies

PARIS Academy of Sciences, July 8 (CR., 201, 105–180). RICHARD FOSSE, PAUL EMLLE THOMAS and PAUL DE GKAEVE: The estimation by weight and the identification by elementary analysis of small quantities of formol in very dilute solutions. The method is based on the reaction of formol with fj-naphtbol, and determines formol at concentrations botween 30 and 200 parts per million, with an acciiracy of one per cent or less. HENRI DEVAUX; The insolubility of thin plates of albumen. H. GRUYELLE and CHARLES POISSON: The magnetic anomaly of Tsiafajavona. MARC KRASNMR: The theory of the ramification of ideals. H. AUEEBACH and S. XJLAM: The number of generators of a serni-simple group. PAUL MONTEL: Positive harmonic functions. OCTAVE ONICESCU: Holotope functions. ANDRE AURJC: An empirical formula giving the distances at which the successive rings of the nebular hypothesis are formed. MLLE. MARGUERITE ROUMENS: The systematic inclination to the west of the equatorial solar prominences of mean latitudes. DANIEL BARBIBR, DANIEL CHALOXCE and ETIENNE VASSY: The continuous spectrum of Nova Herculis. DIKRAN G. DERVICHIAN: The interpretation of the Eotvos constant and of its different values. NICOLAS STOYKO and RAYMOND JOUAUST: The propagation of short radio-electric waves in the region of the polar aurora. LOTTIS NEEL: The number electrons which contribute to the paramagiictism of nickel. The study of nickel alloys shows that an electron of the alloy metal annuls the magnetic contribution of a nickel electron. There is complete continuity between ferro- and para-magnctism, and the same electrons intervene in both cases. LEON and EUGENE BLOCK: The spectra of zinc, cadmium and mercury in the extreme ultra-violet. GEORGES DECHENE: The influence of the passage of an electric current on the phosphorescence of zinc sulphide, MAURICE CURIE: The hyperbolic law of the decline of phosphorescence. MAURICE PROST: The radiation accompanying the dehydration of quinine sulphate. The ease with which the radiation can be deflected shows that it is not due to large ions, thus differentiating this emission from that arising from the oxidation of phosphorus, HENRI MOUREU and “PAUL ROCQUET. The struoture of phosphorus ponta-chloride and of phosphorus pentamide P(NHS)E. PIERRE CARRE and DAVID LIBERMANN: The reaction of thionyl chloride with m- and p-amino-benzoic acids. ROBERT LANTZ: Study of the mechanism of the monoaulphonation of naphthalene. GEORGES DARZENS and ANDRE LEVY: The action of organomagnesium compounds on methylnaphtha-lenie and phenanthrenic esters. JACQUES DE LAPPARENT: The stages of the metamorphism of the Samoa emeries. PIERRE DRACH: The modifications undergone by the skeleton in the decapod Crustacea before casting the shell. GEORGES A. NADSON and MME. CATHERINE STERN: The action of metals at a distance on germinating seeds. The inhibiting effect on growth produced by plates of various metals is shown to depend on the radioactivity of the surrounding air. MME. VERA DANTCHAKOFF: The differences of sensibility of tissue receivers towards folliculin at various embryonic stages. MME. MARIE PHISALIX and FELIX PASTEUR: The action of short waves on antivenom snake sera as well as on their neutral mixtures with the corresponding poisons. THKOPHILE CAHN and JACQUES HOUGET: The transport of lipids in the animal organism. AZSTDRE BOIVIN and MME. LYDIA MESROBEANU: The presence of a ‘complete'antigen and of a ‘residual’ antigen in various bacteria. A. BESREDKA, I. MAGATandP. BESNARD: The importance of the mode of entrance in the evolution of the Pearce-Brown epitholiorna. Testioular epithelioma of the rabbit, resulting in fatal generalised cancer, changes its character when it is inoculated under the skin. The tumour on the skin, although epithelio-matous in structure, is benign and is resorbed. PIERRE LEPINE: The comparative action of immersion in glycerol and of freezing on the preservation of the virulence of rabic marrows. Tho usual method of preservation in glycerol at about 0° C, leads to a gradual loss in activity, due to the action of the glycerol. Freezing at 10° C. leads to no loss of activity for at least 70 days. CONSTANTLY LEVADITI, RENE MARTIN, ANTOINE BONNEFOI and JILLE. RACHEL SCHEN: The etiology of mumps. RAYMOND-HAMET: Some pharmacological effects of ergometrine, a now alkaloid from ergot of rye. JEAN CUILLE, CHELLE and BERLUREAIT: The identity of French and Algerian ovine anaplasmosis.

The constitution of snake venom and snake sera

The constitution of snake venom and snake sera