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Mercury (Hg) is a heavy metal that enters the environment through natural and anthropogenic means. Once in the environment, Hg can biomagnify in food webs and is known to cause immunotoxic effects to wildlife. Compared with other vertebrates, knowledge of the reptilian immune system is lacking, especially in snakes. Further, even less is known about the impact of environmental contaminants on snake immunity. This gap in knowledge is largely due to an absence of established immune-based assays or specific reagents for these species. In this study, brown watersnakes (Nerodia taxispilota; n = 23) were captured on the Savannah River (Augusta, Georgia, USA), weighed, measured, bled, and released. Peripheral blood leukocytes (24 h old) were enriched and evaluated with an established mammalian in vitro lymphocyte proliferation assay. Enriched leukocytes were then exposed to mercury chloride (HgCl 2 ) at 3.75, 37.5, and 75 μM. Total mercury (THg) in whole blood was also quantified. Snake peripheral blood leukocyte enrichment yielded >90% lymphocytes with viabilities averaging >70%. Exposure to HgCl 2 resulted in significant dose-dependent suppression of proliferative responses relative to spontaneous proliferation at 37.5 and 75 μM (both p ≤ 0.01) but not 3.75 μM (p = 0.99). Mean ± 1 SE concentration of THg in whole blood was 0.127 ± 0.027 mg/kg (wet weight). Based on the in vitro findings with HgCl 2 , snakes in systems with heavy Hg pollution may be at risk of immunosuppression, but N. taxispilota at the site in this study appear to be at low risk., (© 2021 John Wiley & Sons, Ltd.)

Snakebite envenoming is predominantly an occupational disease of the rural tropics, causing death or permanent disability to hundreds of thousands of victims annually. The diagnosis of snakebite envenoming is commonly based on a combination of patient history and a syndromic approach. However, the availability of auxiliary diagnostic tests at the disposal of the clinicians vary from country to country, and the level of experience within snakebite diagnosis and intervention may be quite different for clinicians from different hospitals. As such, achieving timely diagnosis, and thus treatment, is a challenge faced by treating personnel around the globe. For years, much effort has gone into developing novel diagnostics to support diagnosis of snakebite victims, especially in rural areas of the tropics. Gaining access to affordable and rapid diagnostics could potentially facilitate more favorable patient outcomes due to early and appropriate treatment. This review aims to highlight regional differences in epidemiology and clinical snakebite management on a global scale, including an overview of the past and ongoing research efforts within snakebite diagnostics. Finally, the review is rounded off with a discussion on design considerations and potential benefits of novel snakebite diagnostics., Competing Interests: CK, JJ, AH, RF, SD, and AL are shareholders in VenomAid Diagnostics ApS. CK is enrolled in an industrial PhD programme sponsored partially by the company BioPorto Diagnostics A/S, and is therefore employed by this company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with several of the authors, CK, JJ, and AL., (Copyright © 2021 Knudsen, Jürgensen, Føns, Haack, Friis, Dam, Bush, White and Laustsen.)

Snake envenomation is still a serious threat to many countries in the world. The only mainstay treatment depends on the administration of animal derived immunoglobulin based antivenom. Significant limitations to these antivenoms are a challenge in the treatment of snake envenomation. Many alternate approaches have been explored to overcome the limitations of antivenom. Exploring alternate approaches like use of bioactive components from plant sources, use of peptide and small molecule inhibitors are some aspects taken towards improving the current limitations of antivenom therapy. However, all these alternate approaches also have many drawbacks which should be improved by more in vitro and in vivo experiments. Here, we review some of the limitations of current antivenom therapy and developments as well as drawbacks of these alternate treatment strategies., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The immunocompetence of a community of free-living animals can be affected by seasonality, sex, and parasite burden. However, each of these factors is often examined independently. Recent studies have also found that dehydration can enhance aspects of immunocompetence in drought-adapted species. To explore how all of these factors interact, and their effect on the immune system in mesic-adapted species, we collected blood samples from a community of free-ranging snakes in coastal South Carolina, United States, across 2 years. We specifically examined (a) how sex and seasonality influence humoral and cellular immunocompetence and parasite burden, (b) the dynamics among hydration state, parasite burden, and immunocompetence, and (c) whether mesic-adapted species also show enhanced innate immunity with dehydration. Consistent with previous work on drought-adapted species, we found that dehydration enhances multiple aspects of humoral immunity in mesic species, and we are the first to report that dehydration also enhances aspects of cellular immunocompetence. Contrary to previous results in other squamates, sex and season did not impact immunocompetence or parasite prevalence. Our results also reveal complex interactions among parasite prevalence, immunocompetence, and hydration state demonstrating that hydration state and parasitism are two ubiquitous factors that should continue to be considered in future studies examining ecoimmunological variation., (© 2020 Wiley Periodicals, Inc.)

Boid Inclusion Body Disease (BIBD) is a potentially fatal disease reported in captive boid snakes worldwide that is caused by reptarenavirus infection. Although the detection of intracytoplasmic inclusion bodies (IB) in blood cells serves as the gold standard for the ante mortem diagnosis of BIBD, the mechanisms underlying IB formation and the pathogenesis of BIBD are unknown. Knowledge on the reptile immune system is sparse compared to the mammalian counterpart, and in particular the response towards reptarenavirus infection is practically unknown. Herein, we investigated a breeding collection of 70 Boa constrictor snakes for BIBD, reptarenavirus viraemia, anti-reptarenavirus IgM and IgY antibodies, and population parameters. Using NGS and RT-PCR on pooled blood samples of snakes with and without BIBD, we could identify three different reptarenavirus S segments in the collection. The examination of individual samples by RT-PCR indicated that the presence of University of Giessen virus (UGV)-like S segment strongly correlates with IB formation. We could also demonstrate a negative correlation between BIBD and the presence of anti-UGV NP IgY antibodies. Further evidence of an association between antibody response and BIBD is the finding that the level of anti-reptarenavirus antibodies measured by ELISA was lower in snakes with BIBD. Furthermore, female snakes had a significantly lower body weight when they had BIBD. Taken together our findings suggest that the detection of the UGV-/S6-like S segment and the presence of anti-reptarenavirus IgY antibodies might serve as a prognostic tool for predicting the development of BIBD., Competing Interests: The authors have declared that no competing interests exist.

Each year, millions of humans fall victim to animal envenomings, which may either be deadly or cause permanent disability to the effected individuals. The Nobel Prize-winning discovery of serum therapy for the treatment of bacterial infections (tetanus and diphtheria) paved the way for the introduction of antivenom therapies for envenomings caused by venomous animals. These antivenoms are based on polyclonal antibodies derived from the plasma of hyperimmunized animals and remain the only specific treatment against animal envenomings. Following the initial development of serum therapy for snakebite envenoming by French scientists in 1894, other countries with high incidences of animal envenomings, including Brazil, Australia, South Africa, Costa Rica, and Mexico, started taking up antivenom production against local venomous animals over the course of the twentieth century. These undertakings revolutionized envenoming therapy and have saved innumerous patients worldwide during the last 100 years. This review describes in detail the above-mentioned historical events surrounding the discovery and the application of serum therapy for envenomings, as well as it provides an overview of important developments and scientific breakthroughs that were of importance for antibody-based therapies in general. This begins with discoveries concerning the characterization of antibodies, including the events leading up to the elucidation of the antibody structure. These discoveries further paved the way for other milestones in antibody-based therapies, such as the introduction of hybridoma technology in 1975. Hybridoma technology enabled the expression and isolation of monoclonal antibodies, which in turn formed the basis for the development of phage display technology and transgenic mice, which can be harnessed to directly obtain fully human monoclonal antibodies. These developments were driven by the ultimate goal of producing potent neutralizing monoclonal antibodies with optimal pharmacokinetic properties and low immunogenicity. This review then provides an outline of the most recent achievements in antivenom research, which include the application of new biotechnologies, the development of the first human monoclonal antibodies that can neutralize animal toxins, and efforts toward creating fully recombinant antivenoms. Lastly, future perspectives in the field of envenoming therapies are discussed, including rational engineering of antibody cross-reactivity and the use of oligoclonal antibody mixtures.

Several snake species that feed infrequently in nature have evolved the ability to massively upregulate intestinal form and function with each meal. While fasting, these snakes downregulate intestinal form and function, and upon feeding restore intestinal structure and function through major increases in cell growth and proliferation, metabolism and upregulation of digestive function. Previous studies have identified changes in gene expression that underlie this regenerative growth of the python intestine, but the unique features that differentiate this extreme regenerative growth from non-regenerative post-feeding responses exhibited by snakes that feed more frequently remain unclear. Here, we leveraged variation in regenerative capacity across three snake species-two distantly related lineages ( Crotalus and Python) that experience regenerative growth, and one ( Nerodia) that does not-to infer molecular mechanisms underlying intestinal regeneration using transcriptomic and proteomic approaches. Using a comparative approach, we identify a suite of growth, stress response and DNA damage response signalling pathways with inferred activity specifically in regenerating species, and propose a hypothesis model of interactivity between these pathways that may drive regenerative intestinal growth in snakes.

Taiwan is an island located in the south Pacific, a subtropical region that is home to 61 species of snakes. Of these snakes, four species-Trimeresurus stejnegeri, Protobothrops mucrosquamatus, Bungarus multicinctus and Naja atra-account for more than 90% of clinical envenomation cases. Currently, there are two types of bivalent antivenom: hemorrhagic antivenom against the venom of T. stejnegeri and P. mucrosquamatus, and neurotoxic antivenom for treatment of envenomation by B. multicinctus and N. atra. However, no suitable detection kits are available to precisely guide physicians in the use of antivenoms. Here, we sought to develop diagnostic assays for improving the clinical management of snakebite in Taiwan. A two-step affinity purification procedure was used to generate neurotoxic species-specific antibodies (NSS-Abs) and hemorrhagic species-specific antibodies (HSS-Abs) from antivenoms. These two SSAbs were then used to develop a sandwich ELISA (enzyme-linked immunosorbent assay) and a lateral flow assay comprising two test lines. The resulting ELISAs and lateral flow strip assays could successfully discriminate between neurotoxic and hemorrhagic venoms. The limits of quantification (LOQ) of the ELISA for neurotoxic venoms and hemorrhagic venoms were determined to be 0.39 and 0.78 ng/ml, respectively, and the lateral flow strips were capable of detecting neurotoxic and hemorrhagic venoms at concentrations lower than 5 and 50 ng/ml, respectively, in 10-15 min. Tests of lateral flow strips in 21 clinical snakebite cases showed 100% specificity and 100% sensitivity for neurotoxic envenomation, whereas the sensitivity for detecting hemorrhagic envenomation samples was 36.4%. We herein presented a feasible strategy for developing a sensitive sandwich ELISA and lateral flow strip assay for detecting and differentiating venom proteins from hemorrhagic and neurotoxic snakes. A useful snakebite diagnostic guideline according to the lateral flow strip results and clinical symptoms was proposed to help physicians to use antivenoms appropriately. The two-test-line lateral flow strip assay could potentially be applied in an emergency room setting to help physicians diagnose and manage snakebite victims., Competing Interests: The authors have declared that no competing interests exist.

Reptiles are the unique ectothermic amniotes, providing the key link between ectothermic anamniotes fish and amphibians, and endothermic birds and mammals; becoming an important group to study with the aim of providing significant knowledge into the evolutionary history of vertebrate immunity. Classification systems for reptiles' leukocytes have been described by their appearance rather than function, being still inconsistent. With the advent of modern techniques and the establishment of analytical protocols for snakes' blood by flow cytometry, we bring a qualitative and quantitative assessment of innate activities presented by snakes' peripheral blood leukocytes, thereby linking flow cytometric features with fluorescent and light microscopy images. Moreover, since corticosterone is an important immunomodulator in reptiles, hormone levels of all blood samples were measured. We provide novel and additional information which should contribute to better understanding of the development of the immune system of reptiles and vertebrates., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Animals will continue to encounter increasingly warm environments, including more frequent and intense heat waves. Yet the physiological consequences of heat waves remain equivocal, potentially because of variation in adaptive plasticity (reversible acclimation) and/or aspects of experimental design. Thus, we measured a suite of physiological variables in the corn snake (Pantherophis guttatus) after exposure to field-parameterized, fluctuating temperature regimes (moderate temperature and heat wave treatments) to address two hypotheses: (1) a heat wave causes physiological stress, and (2) thermal performance of immune function exhibits adaptive plasticity in response to a heat wave. We found little support for our first hypothesis because a simulated heat wave had a negative effect on body mass, but it also reduced oxidative damage and did not affect peak performance of three immune metrics. Likewise, we found only partial support for our second hypothesis. After exposure to a simulated heat wave, P. guttatus exhibited greater performance breadth and reduced temperature specialization (the standardized difference between peak performance and performance breadth) for only one of three immune metrics and did so in a sex-dependent manner. Further, a simulated heat wave did not elicit greater performance of any immune metric at higher temperatures. Yet a heat wave likely reduced innate immune function in P. guttatus because each metric of innate immune performance in this species (as in most vertebrates) was lower at elevated temperatures. Together with previous research, our study indicates that a heat wave may have complex, modest, and even positive physiological effects in some taxa.

Background: Non-front-fanged colubroid snakes comprise about two-thirds of extant ophidian species. The medical significance of the majority of these snakes is unknown, but at least five species have caused life-threatening or fatal human envenomings. However, the venoms of only a small number of species have been explored., Methods: A combined venomic and venom gland transcriptomic approach was employed to characterise of venom of Dispholidus typus (boomslang), the snake that caused the tragic death of Professor Karl Patterson Schmidt. The ability of CroFab™ antivenom to immunocapture boomslang venom proteins was investigated using antivenomics., Results: Transcriptomic-assisted proteomic analysis identified venom proteins belonging to seven protein families: three-finger toxin (3FTx); phospholipase A 2 (PLA 2 ); cysteine-rich secretory proteins (CRISP); snake venom (SV) serine proteinase (SP); C-type lectin-like (CTL); SV metalloproteinases (SVMPs); and disintegrin-like/cysteine-rich (DC) proteolytic fragments. CroFab™ antivenom efficiently immunodepleted some boomslang SVMPs., Conclusions: The present work is the first to address the overall proteomic profile of D. typus venom. This study allowed us to correlate the toxin composition with the toxic activities of the venom. The antivenomic analysis suggested that the antivenom available at the time of the unfortunate accident could have exhibited at least some immunoreactivity against the boomslang SVMPs responsible for the disseminated intravascular coagulation syndrome that caused K.P. Schmidt's fatal outcome., General Significance: This study may stimulate further research on other non-front-fanged colubroid snake venoms capable of causing life-threatening envenomings to humans, which in turn should contribute to prevent fatal human accidents, such as that unfortunately suffered by K.P. Schmidt., (Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.)

Present study was designed to examine daily and seasonal variability in the innate immune responses of splenocytes in the fresh water snake, Natrix piscator. Animals were mildly anesthetized and spleen was aseptically isolated and processed for macrophage phagocytosis, NBT reduction, nitrite production, splenocyte proliferation and serum lysozyme activity. Samples were collected at seven time points, viz., 0000, 0400, 0800, 1200, 1600, 2000 and 0000 h during three different seasons, namely summer, winter and spring. Cosinor analysis revealed that percent phagocytosis had a significant 24-h rhythm during summer and spring seasons. The peaks of rhythms in NBT reduction and nitrite release occurred in the morning hours at 10.88 h and 8.31 h, respectively, in winter. A significant 24-h rhythm was also observed in lysozyme concentration and splenocyte proliferation (both Basal and Concanavalin A stimulated) in all three seasons. A significant phase shift in splenocyte proliferation was obtained with a trend of delayed phase shift from winter to spring and from spring to summer. Of the nine variables, significant annual (seasonal) rhythms were detected in almost all variables, excluding phagocytic and splenosomatic indices. All rhythmic variables, except spleen cellularity, exhibited tightly synchronized peaks coinciding with the progressive and recrudescence phases of annual reproductive cycle. It is concluded that the snake synchronizes its daily and seasonal immune activity with the corresponding external time cues. The enhancement of immune function coinciding with one of its crucial reproductive phases might be helping it to cope with the seasonal stressors, including abundance of pathogens, which would otherwise jeopardize the successful reproduction and eventual survival of the species.

The immunocompetence "pace-of-life" hypothesis proposes that fast-living organisms should invest more in innate immune defenses and less in adaptive defenses compared to slow-living ones. We found some support for this hypothesis in two life-history ecotypes of the snake Thamnophis elegans; fast-living individuals show higher levels of innate immunity compared to slow-living ones. Here, we optimized a lymphocyte proliferation assay to assess the complementary prediction that slow-living snakes should in turn show stronger adaptive defenses. We also assessed the "environmental" hypothesis that predicts that slow-living snakes should show lower levels of immune defenses (both innate and adaptive) given the harsher environment they live in. Proliferation of B- and T-lymphocytes of free-living individuals was on average higher in fast-living than slow-living snakes, opposing the pace-of-life hypothesis and supporting the environmental hypothesis. Bactericidal capacity of plasma, an index of innate immunity, did not differ between fast-living and slow-living snakes in this study, contrasting the previously documented pattern and highlighting the importance of annual environmental conditions as determinants of immune profiles of free-living animals. Our results do not negate a link between life history and immunity, as indicated by ecotype-specific relationships between lymphocyte proliferation and body condition, but suggest more subtle nuances than those currently proposed.

The ability of lizards to withstand infections after wounding or amputation of the tail or limbs has suggested the presence of antimicrobial peptides in their tissues. Previous studies on the lizard Anolis carolinensis have identified several beta-defensin-like peptides that may potentially be involved in protection from infections. The present ultrastructural immunocytochemical study has analyzed tissues in different reptilian species in order to localize the cellular source of one of the more expressed beta-defensins previously sequenced in lizard indicated as AcBD15. Beta-defensin-like immunoreactivity is present in some of the larger, nonspecific granules of granulocytes in two lizard species, a snake, the tuatara, and a turtle. The ultrastructural study indicates that only heterophilic and basophilic granulocytes contain this defensin while other cell types from the epidermis, mesenchyme, and dermis, muscles, nerves, cartilage or bone are immunonegative. The study further indicates that not all granules in reptilian granulocytes contain the beta-defensin peptide, suggesting the presence of granules with different content as previously indicated for mammalian neutrophilic leucocytes. No immunolabeling was instead observed in granulocytes of the alligator and chick using this antibody. The present immunocytochemical observations suggest a broad cross-reactivity and conservation of beta-defensin-like sequence or steric motif across lepidosaurians and likely in turtles while archosaurian granulocytes may contain different beta-defensin-like or other peptides., (Copyright © 2013 Wiley Periodicals, Inc.)

Purity is a characteristic that, together with effectiveness and safety, must be tested to determine the quality of biopharmaceutical products. In therapeutic immunoglobulins, such as human intravenous immunoglobulin (IVIG), purity is evaluated on the basis of physicochemical properties, and is usually assessed by chromatography and electrophoresis. However, in the case of antivenoms these methods fail to discriminate between antibodies towards venom antigens, which constitute the active substance, and antibodies towards non-venom antigens, which are the major impurities in most of the current formulations. The assessment of this aspect of purity requires the use of the immunochemical methods. In this study, it was demonstrated that antivenoms showing physicochemical purity higher than 90% might present immunochemical purity lower than 40%. It is proposed that a comprehensive analysis of antivenom purity should combine physicochemical and immunochemical parameters. In addition, these results are crucial to decide the more appropriate strategies to improve antivenom purity. Taking into account that the current methods of antivenom purification remove most non-antibodies proteins, we propose that efforts must be primarily directed to the improvement of immunization protocols to enhance the antibody response towards venom components in hyperimmunized animals, and secondarily, in the realm of immunoglobulin purification technology., (Copyright © 2012 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

We studied the immunoreactivity between venoms and commercial antiserums in four Chinese venomous snakes, Bungarus multicinctus, Naja atra, Deinagkistrodon acutus and Gloydius brevicaudus. Venoms from the four snakes shared common antigenic components, and most venom components expressed antigenicity in the immunological reaction between venoms and antiserums. Antiserums cross-reacted with heterologous venoms. Homologous venom and antiserum expressed the highest reaction activity in all cross-reactions. Species-specific antibodies (SSAbs) were obtained from four antiserums by immunoaffinity chromatography: the whole antiserum against each species was gradually passed through a medium system coated with heterologous venoms, and the cross-reacting components in antiserum were immunoabsorbed by the common antigens in heterologous venoms; the unbound components (i.e., SSAbs) were collected, and passed through Hitrap G protein column and concentrated. The SSAbs were found to have high specificity by western blot and enzyme-linked immunosorbent assay (ELISA). A 6-well ELISA strip coated with SSAbs was used to assign a venom sample and blood and urine samples from the envenomed rats to a given snake species. Our detections could differentiate positive and negative samples, and identify venoms of a snake species in about 35 min. The ELISA strips developed in this study are clinically useful in rapid and reliable identification of venoms from the above four snake species., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Immunoglobulins are basic molecules of the immune system of vertebrates. In previous studies we described the immunoglobulins found in two squamata reptiles, Anolis carolinensis and Eublepharis macularius. Snakes are squamata reptiles too but they have undergone an extreme evolutionary process. We therefore wanted to know how these changes affected their immunoglobulin coding genes. To perform this analysis we studied five snake transcriptomes and two genome draft sequences. Sequences coding for immunoglobulin M (IgM), immunoglobulin D (IgD) and two classes of immunoglobulin Y (IgY - named IgYa and IgYb-) were found in all of them. Moreover, the Thamnophis elegans transcriptome and Python molurus genome draft sequences showed a third class of IgY, the IgYc, whose constant region only presents three domains and lacks the CH2. All data suggest that the IgYb is the evolutionary origin of this IgYc. An exhaustive search of the light chains were carried out, being lambda the only light chain found in snakes. The results provide a clear picture of the immunoglobulins present in the suborder Serpentes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Squamates (lizards and snakes) have independently evolved viviparity over 100 times, and exhibit a wide range of maternal investment in developing embryos from the extremes of lecithotrophic oviparity to matrotrophic viviparity. This group therefore provides excellent comparative opportunities for studying endocrine and immune involvement during pregnancy, and their possible interactions. We studied the cottonmouth (Agkistrodon piscivorus), since they exhibit limited placentation (e.g., ovoviviparity), allowing comparison with squamate species hypothesized to require considerable maternal immune modulation due to the presence of a more extensive placental connection. Furthermore, the cottonmouth's biennial reproductive cycle provides an opportunity for simultaneously comparing pregnant and non-pregnant females in the wild. We document significantly elevated concentrations of progesterone (P4) and significantly lower concentrations of estradiol (E2) in pregnant females relative to non-pregnant females. Pregnant females had lower plasma bacteria lysis capacity relative to non-pregnant females. This functional measure of innate immunity is a proxy for complement performance, and we also determined significant correlations between P4 and decreased complement performance in pregnant females. These findings are consistent with studies that have determined P4's role in complement modulation during pregnancy in mammals, and thus this study joins a growing number of studies that have demonstrated convergent and/or conserved physiological mechanisms regulating viviparous reproduction in vertebrates., (Copyright © 2011. Published by Elsevier Inc.)

The parenteral administration of antivenoms is the cornerstone of snakebite envenoming therapy. Efforts are made to ensure that antivenoms of adequate efficacy and safety are available world-wide. We address the main issues to be considered for the development and manufacture of improved antivenoms. Those include: (a) A knowledge-based composition design of venom mixtures used for immunization, based on biochemical, immunological, toxicological, taxonomic, clinical and epidemiological data; (b) a careful selection and adequate management of animals used for immunization; (c) well-designed immunization protocols; (d) sound innovations in plasma fractionation protocols to improve recovery, tolerability and stability of antivenoms; (e) the use of recombinant toxins as immunogens to generate antivenoms and the synthesis of engineered antibodies to substitute for animal-derived antivenoms; (f) scientific studies of the contribution of existing manufacturing steps to the inactivation or removal of viruses and other zoonotic pathogens; (g) the introduction of novel quality control tests; (h) the development of in vitro assays in substitution of in vivo tests to assess antivenom potency; and (i) scientifically-sound pre-clinical and clinical assessments of antivenoms. These tasks demand cooperative efforts at all main stages of antivenom development and production, and need concerted international partnerships between key stakeholders., (Copyright © 2011 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.)

Vipera ammodytes is the most venomous European snake, whose venom has been used as antigen for immunization of antivenom-producing animals. Same as venom of any other snake, it is a complex mixture of proteins, peptides and other compounds which biochemical and pharmacological variability has been demonstrated at interspecies and intraspecies level. In this work we demonstrated intraspecific variability between 8 venom production batches using both the conventional and the new methodology. Moreover, in contrast to the literature on different venoms' variability, for the first time we were able to select those biochemical differences that are related to and give information on the venom's toxicity and immunogenicity. We have shown that methods quantifying ammodytoxin (the most toxic compound identified so far in the Vipera ammodytes ammodytes venom) content of the venom clearly distinguish between high and low immunogenic venoms., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Passive immunotherapy was discovered in 1894. It is the only etiological treatment of envenomations by snakes or scorpions. Immunotherapy is based on administration of antibodies produced by an animal hyperimmunised against venom. Improvement of whole antivenomous sera was obtained, first by separating the antibodies from other components of blood plasma, then by using enzyme digestion of immunoglobulins G and, finally, by purifying the final product. Efficacy and also tolerance were significantly increased. Antivenom administration should be performed through the intravascular route to ensure better diffusion and to facilitate the complexion with the venom. The sale of antivenoms, in particular in Africa, is considerably reduced since about thirty years and is not in adequacy with the epidemiologic needs. In addition to the high cost of the current products, the difficulties of supplying, storage and use seem at the origin of this rejection by both the health staff and the victims who resort massively to traditional medicine. Whereas WHO reminds the rules of production and use of the antivenoms to producers and authorities in charge of drug regulations, it appears that the accessibility of the antivenoms depends on a concerted effort from all the actors, coordinated by health authorities: producers, distributers, manufacturers and public. Beyond technological improvements necessary in antivenom production, the strategy to be implemented must include the strict evaluation of the needs based on epidemiologic studies, the share of cost recovery between all the actors, and the optimization of the therapeutic protocol to make it applicable in remote health centres. Once confidence towards immunotherapy will be restored, mortality will be significantly reduced., (© Société de Biologie, 2010.)

The venom proteomes of Bothrops atrox from Colombia, Brazil, Ecuador, and Perú were characterized using venomic and antivenomic strategies. Our results evidence the existence of two geographically differentiated venom phenotypes. The venom from Colombia comprises at least 26 different proteins belonging to 9 different groups of toxins. PI-metalloproteinases and K49-PLA(2) molecules represent the most abundant toxins. On the other hand, the venoms from Brazilian, Ecuadorian, and Peruvian B. atrox contain predominantly PIII-metalloproteinases. These toxin profiles correlate with the venom phenotypes of adult and juvenile B. asper from Costa Rica, respectively, suggesting that paedomorphism represented a selective trend during the trans-Amazonian southward expansion of B. atrox through the Andean Corridor. The high degree of crossreactivity of a Costa Rican polyvalent (Bothrops asper, Lachesis stenophrys, Crotalus simus) antivenom against B. atrox venoms further evidenced the close evolutionary kinship between B. asper and B. atrox. This antivenom was more efficient immunodepleting proteins from the venoms of B. atrox from Brazil, Ecuador, and Perú than from Colombia. Such behaviour may be rationalized taking into account the lower content of poorly immunogenic toxins, such as PLA(2) molecules and PI-SVMPs in the paedomorphic venoms. The immunological profile of the Costa Rican antivenom strongly suggests the possibility of using this antivenom for the management of snakebites by B. atrox in Colombia and the Amazon regions of Ecuador, Perú and Brazil.

A 55-year-old herpetologist developed rhinitis, asthma, urticaria and anaphylaxis when handling 4 different viper snake venoms. Allergen characterizations were done using SDS-PAGE, IgE immunoblotting and IgE inhibition experiments. The most prominent immunoreactive proteins were analyzed by MALDI-TOF mass spectrometry, and peptide identity was demonstrated by homology with known peptide sequences. SDS-PAGE showed several protein bands ranging from 5 to 99 kDa in each of the 4 snake venoms. Immunoblotting demonstrated 4 IgE-binding bands in the Bothrops extract of about 60, 28, 14 and 7 kDa. The bands of 28 and 14 kDa were also present in Lachesis muta. Two IgE-binding proteins of about 50 and 35 kDa were found in Bothrops atrox and L. muta, respectively. A strong inhibition of IgE binding to immobilize Bothrops asper proteins was observed after preabsorption of sera with B. asper, B. atrox,Bothrops xanthograma and L. muta extracts. MALDI-TOF analysis showed a 14-kDa phospholipase and the 60- and 28-kDa proteins showed significant similarity with metalloproteinases. In this report we have characterized the snake venom allergens that can elicit IgE-mediated symptoms., (Copyright 2009 S. Karger AG, Basel.)

Snakebites in rural areas of tropical and subtropical regions are commonly treated with medicinal plants. In this report, we have studied the ability of Anacardium occidentale bark extract to neutralize enzymatic as well as pharmacological effects induced by Vipera russelii venom. The extract neutralized the viper venom hydrolytic enzymes such as phospholipase, protease, and hyaluronidase in a dose dependent manner. These enzymes are responsible for both local effects of envenomation such as local tissue damage, inflammation and myonecrosis, and systemic effects including dysfunction of vital organs and alteration in the coagulation components. In addition, extract neutralized the pharmacological effects such as edema, hemorrhage, and myotoxic effects including lethality, induced by venom. Since, it inhibits both hydrolytic enzymes and pharmacological effects; it may be used as an alternative treatment to serum therapy and, in addition, as a rich source of potential inhibitors of hydrolytic enzymes involved in several physiopathological diseases.

Little is known about specific proteins involved in keratinization of the epidermis of snakes, which is composed of alternating beta- and alpha-keratin layers. Using immunological techniques (immunocytochemistry and immunoblotting), the present study reports the presence in snake epidermis of proteins with epitopes that cross-react with certain mammalian cornification proteins (loricrin, filaggrin, sciellin, transglutaminase) and chick beta-keratin. alpha-keratins were found in all epidermal layers except in the hard beta- and alpha-layers. beta-keratins were exclusively present in the oberhautchen and beta-layer. After extraction and electrophoresis, alpha-keratins of 40-67 kDa in molecular weights were found. Loricrin-like proteins recorded molecular weights of 33, 50, and 58 kDa; sciellin, 55 and 62 kDa; filaggrin-like, 52 and 65 kDa; and transglutaminase, 45, 50, and 56 kDa. These results suggest that alpha-layers of snake epidermis utilize proteins with common epitopes to those present during cornification of mammalian epidermis. The beta-keratin antibody on extracts from whole snake epidermis showed a strong cross-reactive band at 13-16 kDa. No cross-reactivity was seen using an antibody against feather beta-keratin, indicating absence of a common epitope between snake and feather keratins.

We have demonstrated previously that antisera from mice immunised with DNA encoding the carboxy-terminal domain (JD9) of a potent haemorrhagic metalloproteinase, jararhagin, neutralised over 70% of the haemorrhagic activity of the whole Bothrops jararaca venom. Here, we demonstrate that the JD9-specific antibody possesses extensive immunological reactivity to venom components in snakes of distinct species and genera. The polyspecific immunological reactivity of the antibody showed a correlation with amino acid sequence identity and with predicted antigenic domains of JD9-analogues in venoms of snakes with closest phylogenetic links to B. jararaca. This study further promotes the potential of DNA immunisation to generate toxin-specific antibodies with polyspecific cover. An analysis of the reactivity of the JD9-specific antisera to B. atrox complex venoms that exhibited intraspecific variation in the venom proteome revealed, however, that the toxin-specific approach to antivenom development requires a more in-depth knowledge of the target molecules than is required for conventional antivenoms.

The sera of 16 species of wild animals representing 5 classes of vertebrates were assayed for amebicidal activity against species of Naegleria. The greatest activity was observed for sera of bullfrogs, muskrats, and raccoons, all of which are animals associated with water. In contrast, the sera from animals such as toads, box turtles, sparrows, and squirrels exhibited minimal or no amebicidal activity. In general, pathogenic Naegleria tended to be less susceptible than nonpathogenic Naegleria to the lytic effect of raccoon serum. Heat-inactivated serum was not amebicidal, suggesting that perhaps complement may be involved in the serum-mediated lysis of amebas.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of systemic Cryptosporidium-specific antibodies (Ab) in the serum of captive snakes. An optimal concentration of Cryptosporidium soluble protein-cocktail antigen was 20 micrograms/mL. In a direct ELISA, alkaline phosphatase-labeled rabbit-anti-snake polyvalent Ab detected snake immunoglobulins at a dilution of 1:12800. It was possible to detect Cryptosporidium-specific Ab at a serum dilution of 1:1600. Nineteen of 26 (73%) captive snakes (eight species) were Cryptosporidium-seroconverted; however, the differences in Ab titer among species were not significant. The distribution of absorbance values did not conform to a normal distribution (P < 0.05); mean Ab titer of nine snakes with cryptosporidiosis was significantly higher than mean Ab titer of the remaining ten snakes (P < 0.01). The monoclonal Ab test showed that the stools of 17 (89%) of 19 seropositive snakes contained Cryptosporidium oocysts; nine of those 19 (47%) stools were positive by acid-fast-stained (AFS) direct wet smear (DWS). A significant positive relationship between Cryptosporidium-seroconversion and snake age was observed (P < 0.03); the mean age of seropositive snakes was significantly higher than the mean age of seronegative animals (P < 0.01). The proposed assay is fast, reproducible, could be used for retrospective and prospective seroepizootiological studies, and assist ophidian centers in establishing Cryptosporidium-free collections. After standardization, the assay can be used for large-scale screening of captive snakes.

Crotaline snakes, which have infrared-sensitive pit organs, provide a good model for linking neuron morphology with sensory modality. In the trigeminal ganglion of the habu, Trimeresurus flavoviridis, cells positive for calcitonin gene-related peptide-like (CGRP) immunoreactivity were found to be of two types, darkly stained and lightly stained. They were pseudo-unipolar, having an axon divided into stem, peripheral branch, and central branch, all of which were 1 micron or less in diameter. Other, CGRP-negative cells in the ganglion were also pseudo-unipolar, but much larger. In configuration, some of the positive cells were similar to the neurons with A-delta fibers, and others to the neurons with C fibers that have been reported by other workers. On the basis of their distribution and density, and physiological studies by other workers, the CGRP-positive cells were judged to be not part of the infrared-receptive system, but to be involved in the transmission of nociception in small fibers.

Thymocytes, splenocytes and peripheral blood mononuclear cells (PBMC) of the snake Psammophis sibilans consistently killed the human erythroleukemic cells K562 in a 4 h assay as judged by lactate dehydrogenase enzyme release. PBMC and splenocyte natural cytotoxicity (NC) increased proportionally with increase in the effector/target cell ratio. Spontaneous killer cell activity was consistently 2-3 times higher in peripheral blood (PB) than in spleen. On the other hand, thymocytes displayed low, yet detectable, NC. In an attempt to define the cell subpopulation responsible for natural killer (NK) activity, PBMC were depleted of macrophages or B lymphocytes before use in NK cell assays against K562 cells. Depletion of macrophages did not impair NK activity thus suggesting that macrophages do not mediate spontaneous lysis in the present 4 h assay. Conversely, removal of B lymphocytes by panning onto dishes coated with monoclonal antibody against snake Ig significantly reduced, but did not eliminate, PBMC spontaneous cytotoxicity. These data suggest that T, B and perhaps distinct NK cells participate in spontaneous lysis. This suggestion was confirmed by studies of NC in thymus, spleen and PB the year round. Strong NC was detected during spring and autumn when high numbers of leukocytes including T and B cells can be recovered from spleen and PB. Negligible spontaneous cytotoxicity was observed during early and mid-summer and in winter, periods of the year when snakes are thymus-less and contain few T and B cells in peripheral lymphoid organs. These findings, the first to document natural cytotoxic activity in snakes, were discussed in relation to the issue of NK cell identity in vertebrates.

1. Two sets of polyclonal antibodies to two highly purified prey-derived snake-attractive proteins, a low molecular weight (3000) protein and a 20,000 mol. wt protein, were generated in rabbits. 2. They are immunospecific for their respective purified immunogens and do not cross-react with each other. 3. Eight prey-derived proteins that elicit attack by garter snakes (Thamnophis sirtalis) from earthworms (Lumbricus terrestris) were analyzed with these antibodies, and can be assigned to three distinct groups on the basis of their antigenic properties. 4. Unfolding or denaturation of the low molecular weight protein did not alter its antigenic activity to its polyclonal antibodies, suggesting the antigenic epitopes contain contiguous amino acid sequences. 5. In contrast, unfolding of the 20,000 mol. wt protein resulted in a loss of its binding with antibodies, suggesting that the epitope of this protein contains noncontiguous amino acid sequences. 6. The snake-attractivity of the 20,000 mol. wt protein could not be neutralized by reacting it with its antiserum, suggesting that the antigenic determinant (the epitope) of the antigen is not an integral part of the attractive domain of the ES20 protein. 7. In contrast, the attractivity of the purified low molecular weight protein could be neutralized by the polyclonal antibodies.

A mouse monoclonal antibody (mAb) raised against serum immunoglobulins (Ig) of the snake. Psammophis sibilans stained in indirect immunofluorescence a proportion of snake splenic and peripheral blood lymphocytes, whereas it did not react with thymocytes, erythrocytes, brain, heart, lung, liver or kidney cells. The mAb, designated SR-2, combined in enzyme-linked immunosorbent assay (ELISA) with serum proteins of each of 20 individual P. sibilans tested. On Western blots of P. sibilans reduced whole serum proteins, purified Ig, or anti-rat erythrocyte (RRBC) antibodies eluted from glutaraldehyde-fixed RRBC, mAb SR-2 identified two bands of apparent molecular weight (m.w.) of 60,000 and 51,000 daltons. These bands were due to distinct polypeptides and not resulting from heterogeneous glycosylation of a single polypeptide, as they both were readily detected after periodate oxidation or endoglycosidase-F treatment of serum proteins and isolated Ig. MAb SR-2 bound to CNBr-activated Sepharose 4B precipitated from P. sibilans 125I-labeled serum proteins under non-reducing conditions a band that did not enter 7.5 or 9-16% gel and one of about 150,000 daltons. Under reducing conditions, two heavy bands of approximately 63,000 and 50,000 daltons and two light chains of apparent mass 23,000 and 20,000 bands were precipitated. The data presented provide, for the first time, substantial information on the molecular characteristics of snake Ig.

A phospholipase myotoxin (MOO-1) and a non-phospholipase myotoxin (JSU-5) were studied for their antigenic cross-reactivity and neutralization by different antisera. Antisera against JSU-5 and MOO-1 reacted equally with both myotoxins in ELISA assays. The specificity of these antisera was also similar, recognizing the same 14,000-18,000 mol. wt components in the venoms of Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi and Bothrops pradoi. Using creatine kinase assays, JSU-5 myotoxicity was completely neutralized by B. jararacussu antivenom or anti-JSU-5 antibodies and partially neutralized by B. moojeni antivenom or anti-MOO-1 antibodies. MOO-1 myotoxicity was completely neutralized by antisera against JSU-5 and MOO-1 and B. jararacussu antivenom, and only partially neutralized by B. moojeni antivenom. B. jararacussu venom induced high titres of antibodies against purified myotoxins. This antiserum completely inhibited the myotoxicity of the homologous venom and significantly reduced the myotoxicity of the remaining myotoxin-containing venoms. It is suggested that B. jararacussu venom is a good immunogen to induce antibodies against myotoxins present in the venoms of the different species of Bothrops.

In optimal seasonal conditions, outbred adult snakes Psammophis sibilans displayed the major immunological functions related, in mammals, to the presence of the major histocompatibility gene complex (MHC). Thus, out of 30 snake random pairs that exchanged skin transplants 72.9% rejected their allograft in an acute or subacute manner. Strongly significant proliferative response was recorded in 67.3% of 168 separate one-way mixed leukocyte reaction (MLR) cultures. Lymphocytes from 6/11 snakes immunized by skin allografting displayed, after secondary stimulation in vitro, cell-mediated lympholysis (CML) in vitro of 51Cr-labeled lymphoblasts derived from the donor snake. Finally, cytotoxic alloantibodies were readily generated after snake priming with skin allograft and blood cells. Snakes did not only exhibit the major cell- and humoral-mediated immune functions, but these functions appeared to be linked with the degree of MLR disparity. Thus, animals with different MLR rejected skin allografts acutely and produced cytotoxic effector cells. In contrast, MLR-identical animals rejected the skin allograft of their partner chronically and failed to produce killer cells in CML. This significant positive correlation between MLR disparity, graft rejection and CML suggests that the responsible antigens are encoded, as in other vertebrates, by the same genetic system, the MHC.

Monovalent antivenoms were raised in mice against the venoms of Causus maculatus, Vipera ammodytes, Echis carinatus, Cerastes cerastes, Bitis arietans, Agkistrodon rhodostoma and Bothrops atrox. These antivenoms as well as four commercially available antivenoms were tested against the venoms of 15 viperid species by means of immunoelectrophoresis and/or ELISA. Cross-reactive protein bands were determined by immunoblot. ELISA cross-reactions of C. maculatus antivenom were low with all heterologous venoms. When investigating the other viperine antivenoms in ELISA stronger cross-reactions were observed with several heterologous venoms. In immunoblot, two heterologous antivenoms cross-reacted with one or two protein bands of C. maculatus venom whereas there were at least four heterologous antivenoms cross-reacting with each of the other venoms. The findings indicate that there is little antigenic affinity between C. maculatus venom and the other venoms investigated. Broad in vitro cross-reactions between viperine antivenoms and Causus venom which were reported in literature seem to be attributable to the use of antivenoms of commercial grade. Specificity of commercially produced, mono- or polyvalent antivenoms may not be strictly limited to those venoms, against which potency is claimed on the label of the product.

The sera of T. flavoviridis (Habu), H. edwardsii (mongoose) and D. semicarinatus (Akamata, non-venomous snake) were tested for their capacity to neutralize 28 species of hemorrhagic snake venoms in vitro. The sera of these animals neutralized a variety of hemorrhagic venoms, suggesting a common structure for a hemorrhagic factor and a similar mechanism of neutralization for the antihemorrhagic factor in the sera. The serum of T. flavoviridis neutralized the lethal toxicity of the T. flavoviridis venom but could not neutralize those of the other hemorrhagic venoms at all. The sera of H. edwardsii and D. semicarinatus did not inhibit the activity of all hemorrhagic venoms.

A comparative study was performed on some enzymatic and toxic activities of venoms collected from newborn, one-year old, two-years old and adult (more than five-years old) specimens of Lachesis muta stenophrys. There was an increase in lethal, hemorrhagic, edema-forming, myotoxic, proteolytic and phospholipase A2 activities of venoms as snakes aged. The venom of newborn specimens was almost devoid of toxicity. On the other hand, venom from newborn specimens showed the highest coagulant effect of human plasma. Electrophoretic and immunochemical results demonstrated conspicuous differences between venoms of different ages. Observations on the feeding behavior indicated that specimens of L. muta of different ages displayed a similar pattern, characterized by rapid strike and bite, holding the prey until they stopped their movements and swallowing them afterwards. It is concluded that venom of newborn L. muta has very low toxic and proteolytic activities and that it undergoes conspicuous changes during the first year of life.

We studied aerosol deposition and the response to inhaled particles and irritants in lungs of Boa constrictor. Snakes which breathed submicrometric particles radiolabeled with 99mTc retained 41.4 +/- 9.9% of the aerosol in the trachea, 42.5 +/- 8.8% in the anterior faveolar regions, and 8.7 +/- 4.1% in the posterior saccular regions of the lungs. Low activity recovered in the gastrointestinal tract over a 5-h period following aerosol exposure indicated slow clearance of inhaled particles. In contrast to mammalian lungs, there are no macrophages resident on the surface of boa lungs, and uningested particles persist for up to 4 days without being phagocytized. Particles and irritant stimuli (Fe2O3, endotoxin, and N-formylmethionylphenylalanine) elicited only eosinophilic granulocytes that were not phagocytic. The numbers of these cells peaked at 24 h following exposure and declined gradually over the next 7 days. Lavage fluid from stimulated snake lungs contained many large lamellar figures continuous with tubular myelin, a form of surfactant. Very little of this material was recovered from control lungs. Response to inhaled particles and lung injury in boas increased surfactant release, elicited eosinophilic granulocytes, but did not recruit phagocytic mononuclear cells.

A specific antiserum was raised in rabbits against thymocytes from snakes, Spalerosophis diadema, and was absorbed repeatedly with snake erythrocytes and kidney cells. In complement-dependent cytotoxicity assays, the absorbed anti-thymocyte serum (ATS) was, at any given dilution, cytotoxic to Sp. diadema thymocytes > peripheral blood lymphocytes (PBL) > spleen cells and could be titrated to a plateau defining a population of about 98% of thymocytes, 80% of PBL and 72% of spleen cells. Antiserum directed against snake immunoglobulins was obtained by injecting rabbits with gamma-globulins separated from snake serum by DEAE-cellulose filtration. the anti-gamma globulin serum was absorbed with snake erythrocytes, and in indirect membrane immunofluorescence stained no thymocytes while reacted with about 15% of PBL and 29% of spleen lymphocytes up to a 1:8 dilution. Fluorescence of positive cells was distributed in spots, patches or caps; cap formation could be inhibited by maintaining the immunofluorescence test at +4 degrees. In each of six separate experiments performed during spring, the percentage of lymphocytes which reacted with anti-snake gamma-globulin serum complemented the percentage of cells recognized by ATS. It was shown, furthermore, that about 3%, 8% and 21% of lymphocytes from thymus, peripheral blood and spleen, respectively, possess a receptor for 2-mercaptoethanol-insensitive antibody-sheep erythrocyte complexes. The results indicate that lymphocyte structural heterogeneity exists in reptiles.

Proteins antigenically related to CoF, the anticomplementary protein of the venom of the Indian hooded cobra (Naja naja), were found in a variety of elapid and viper venoms but not in the venom of Brazilian crotalids . In keeping with this finding was the weak ability of Brazilian snake venom to convert C3 in human serum. All snake serums tested, including Brazilian crotalids , contained a beta-globulin antigenically related to CoF. This serum protein in Brazilian snake serum had a number of characteristics of mammalian C3, including conversion on storage or on incubation of this serum with endotoxin, zymosan or mammalian antigen-antibody precipitates. The serum protein did not, however, convert on incubation with hydrazine. Brazilian crotalid serum did not, as did cobra serum, have the ability to inactivate CoF's ability to activate complement in normal human serum. The crotalid serum had hemolytic activity for rabbit antibody-sensitized and unsensitized sheep red blood cells that was active in the presence of Ca++ and Mg++ or Mg++ alone but greater with Ca++ present, suggesting the presence of both classical and alternative pathways of complement activation. This activity was maximal at 37 degrees C, but was destroyed or inactive after heating at 50 degrees C for 1 hr, incubation with hydrazine or by addition of EDTA. A marked reduction of hemolytic activity of Bothrops serum occurred after removal of the CoF-like protein. These findings suggest that Brazilian snake venom has little CoF-like material, but its serum contains a CoF-like protein with many characteristics of C3.(ABSTRACT TRUNCATED AT 250 WORDS)

The presence of a factor immunologically related to cobra venom factor (CVF) was demonstrated in serum and plasma from the Indian cobra (Naja naja kaoutia). The factor was purified from cobra plasma by affinity chromatography on an anti-CVF gel and was found to consist of a protein composed of two polypeptide chains similar in size to those of human C3. With use of immunoblotting technique, common antigenic determinants were found in the smaller chain of the prepared material and the beta-chain of human C3; the larger chain may display antigenic determinants present in the alpha-chain of human C3. These findings suggest that this molecule represents the C3 of the cobra complement system. Common antigenic determinants were also demonstrated in the alpha-chain of CVF and the beta-chains of human and cobra C3. No reactions were observed between the beta- and gamma-chains of CVF and any antiserum against human C3 or its subunits. Upon immunodiffusion analysis, cobra serum was found to contain a factor besides C3 sharing antigens specific for CVF, while cobra C3 was antigenically deficient compared to CVF. This suggests that cobra C3 physiologically is degraded to a molecule very similar to or identical with CVF.

The fixation of pemphigus antibodies was revealed by indirect immunofluorescence in shedding grass snake epidermis. The antibodies of patients with pemphigus vulgaris (PV) were specifically binding to the antigenic substance which appears in the snake epidermis at the initial stage of integument change. At the peak of shedding, the fixation of PV antibodies was observed only in the upper layers of the 'old' epidermis, although the intensity of fluorescent staining was considerably reduced. At the same stage of shedding, there was marked fixation of antibodies of patients suffering from pemphigus foliaceus (PF). The specific binding of PF antibodies was noted predominantly on the cell membranes of the 'old' keratinocytes, localized in the upper layers of the epidermis. In contrast to bullous pemphigoid antibodies, PV and PF antibodies failed to fix in the skin of the snake prior to or after physiologic shedding. The data obtained suggest that pemphigus may have an atavistic origin.

The spleen of the snake Python reticulatus, was investigated as to its general histology as well as the presence of immune complex trapping cells both at the light and electron microscopical level. Histological examination revealed that the spleen of this reptile was encapsulated and contained some trabeculae. In the splenic parenchyma two different regions could be distinguished: viz. red and white pulp. The white pulp appeared to be arranged around "central arterioles" and their smaller branches extending towards the periphery of the white pulp. The red pulp was composed of blood sinusoids and cell cords. Electron microscopy revealed at least three types of non-lymphoid cells in the white pulp of the spleen of python: reticulum cells, forming the framework; some macrophages and dendritic cells predominantly located in the periphery of the white pulp. Of these types of non-lymphoid cells, only dendritic cells were able to trap and to retain intravenously injected horseradish peroxidase (HRP)-rabbit-anti-HRP immune complexes on their cell surface as determined by enzymehistochemistry at the light and electron microscopical level. These dendritic cells were frequently found in association with collagen fibres and did not engulf large quantities of carbon particles. These data suggest that dendritic cells in the spleen of the python might be the phylogenetic precursors of the mammalian follicular dendritic cells.

Garter snakes (Thamnophis ordinoides) were immunized with hen egg albumin, human gamma-globulin and Keyhole limpet haemocyanin in Freund's adjuvant. Antibody was consistently detected by radioimmunoelectrophoresis and in three different gamma- and beta-globulin precipitin lines called IgM (approximately or equal to 20S), Ig-1 (approximately or equal to 9S) and Ig-2 (approximately or equal to 8-5S). Early antibody (day 31 after immunization) was frequently Ig-M whereas Ig-2 and especially Ig-1 were detectable for the longest duration (992 days). After immunization with antigen in Freund's adjuvant, Ig-1 serum concentration showed the greatest increase, from almost undetectable levels to the most prominent immunoglobulin in immune serum.

In spring, spleen cells (SC) derived from outbred adult snakes, Psammophis sibilans, proliferated vigorously in mixed leucocyte cultures (MLC), thus indicating that reptiles are endowed with the subset(s) of lymphocytes capable of recognizing and responding to alloantigens. Sephadex G-10- or nylon wool-adherent latex-ingesting cells provided accessory, rather than stimulatory, function in MLC. The data, based on numerous one- and two-way MLR studies, showed that the snake P. sibilans possesses strong lymphocyte-activating determinants (LAD) which, however, appeared not to be extensively diverse, as mutually or unilaterally unresponsive SC combinations were quite often observed. Furthermore, seasonal investigations revealed that the lymphoproliferative response in MLR is considerable only in some months of spring and autumn, being abrogated during the rest of the year. These findings suggest that antigen sharing and environmental conditions greatly restrict the cell-mediated immune potential of snakes.

Rabbit erythrocytes are lysed by cobra plasma. The membranes of the lysed rabbit erythrocytes exhibited typical ultrastructural complement lesions with an inner diameter of 72 A. Structures similar to human C5b-8 complexes were also visualized. The proteins found in membranes lysed by cobra plasma resembled closely the proteins of the human membrane attack complex in number and molecular weight. Cobra poly C9 that is resistant to reduction and boiling in SDS could also be demonstrated. These results indicate that the membrane attack complex of cobra complement is very similar to its mammalian counterpart.