Kathleen Fernandes Grego

Semen Collection and Evaluation in Free-Ranging Brazilian Rattlesnakes (Crotalus durissus terrificus)

Two hundred-ninety species of reptiles are estimated to need urgent action for conservation, with at least 113 threatened species worldwide. The International Union for Conservation of Nature and Natural Resources (IUCN) Red List of Threatened Species includes 80 species of snakes, with six native Brazilian species, a number likely to be an underestimation. Some authors believe that assisted reproduction would be an important tool to improve reproduction in captivity of some reptiles. An efficient technique for semen collection and evaluation is an important step in development of protocols for cryopreservation of semen or artificial insemination in snakes, contributing to the conservation of endangered species. Although these techniques are important, some basic semen parameters are described for four of the approximately 2,900 snake species in the world. The Brazilian rattlesnake (Crotalus durissus terrificus) was chosen as a model for semen collection in snakes because it is found quite often in Sao Paulo State. Semen was collected once from each animal by the same investigator during the mating season of this species in Brazil. After antiseptic cleansing of the skin around the cloaca, the snakes were injected subcutaneously with a dose of 15 mg/kg of 1% solution of lidocaine around the cloaca. Semen was collected with ventral massages after cloacal relaxation and directly from genital papilla inside the cloaca. A total of 28 ejaculates from 39 animals were obtained, representing collection efficiency of 71.80%. Semen volume and concentration in Brazilian rattlesnakes ranged from 3-70 microl and from 0.94-2.23 x 10(9) spermatozoa/ml, respectively. Zoo Biol 0:1-6, 2007. (c) 2007 Wiley-Liss, Inc.

Clinical, serological, and parasitological analysis of snakes naturally infected with Cryptosporidium serpentis

Infection by Cryptosporidium serpentis is one of the most important diseases in reptiles and is characterized by chronic clinical or subclinical infection and the presence of hypertrophic gastritis, food regurgitation, progressive weight loss, mortality, and intermittent or continuous shedding of oocysts in the feces. The objectives of this study were to standardize an indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against C. serpentis and to evaluate the clinical, parasitological, and humoral immune response in snakes naturally infected with C. serpentis. Twenty-one snakes naturally infected with C. serpentis and housed at the Butantan Institute, São Paulo, Brazil, underwent clinical and parasitological analyses for C. serpentis infection through daily records of clinical signs and a monthly survey of fecal shedding of oocysts using the Kinyouns acid-fast staining. The serological evaluation was performed monthly by indirect ELISA using crude total antigen from oocysts of C. serpentis to detect anti-C. serpentis antibodies. Clinical symptoms consisted of food regurgitation, inappetence, and progressive weight loss. The parasitological analysis revealed intermittent fecal shedding of a variable number of oocysts in all snakes, with positivity in 85.32% (157/184) of the samples. The indirect ELISA was positive in 68.25% (86/126) of the samples. A humoral immune response was observed in most animals; however, fluctuating antibodies levels, leading to alternating positive and negative results, were observed in most snakes.

Proteomic Analysis of the Ontogenetic Variability in Plasma Composition of Juvenile and Adult Bothrops jararaca Snakes.

The ontogenetic variability in venom composition of some snake genera, including Bothrops, as well as the biological implications of such variability and the search of new molecules that can neutralize the toxic components of these venoms have been the subject of many studies. Thus, considering the resistance of Bothrops jararaca to the toxic action of its own venom and the ontogenetic variability in venom composition described in this species, a comparative study of the plasma composition of juvenile and adult B. jararaca snakes was performed through a proteomic approach based on 2D electrophoresis and mass spectrometry, which allowed the identification of proteins that might be present at different levels during ontogenetic development. Among the proteins identified by mass spectrometry, antihemorrhagic factor Bj46a was found only in adult plasma. Moreover, two spots identified as phospholipase A2 inhibitors were significantly increased in juvenile plasma, which can be related to the higher catalytic PLA2 activity shown by juvenile venom in comparison to that of adult snakes. This work shows the ontogenetic variability of B. jararaca plasma, and that these changes can be related to the ontogenetic variability described in its venom.

Ontogenetic Variation in Biological Activities of Venoms from Hybrids between Bothrops erythromelas and Bothrops neuwiedi Snakes.

Lance-headed snakes are found in Central and South America, and they account for most snakebites in Brazil. The phylogeny of South American pitvipers has been reviewed, and the presence of natural and non-natural hybrids between different species of Bothrops snakes demonstrates that reproductive isolation of several species is still incomplete. The present study aimed to analyze the biological features, particularly the thrombin-like activity, of venoms from hybrids born in captivity, from the mating of a female Bothrops erythromelas and a male Bothrops neuwiedi, two species whose venoms are known to display ontogenetic variation. Proteolytic activity on azocoll and amidolytic activity on N-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPNA) were lowest when hybrids were 3 months old, and increased over body growth, reaching values similar to those of the father when hybrids were 12 months old. The clotting activity on plasma diminished as hybrids grew; venoms from 3- and 6-months old hybrids showed low clotting activity on fibrinogen (i.e., thrombin-like activity), like the mother venom, and such activity was detected only when hybrids were older than 1 year of age. Altogether, these results point out that venom features in hybrid snakes are genetically controlled during the ontogenetic development. Despite the presence of the thrombin-like enzyme gene(s) in hybrid snakes, they are silenced during the first six months of life.

Examination of biochemical and biological activities of Bothrops jararaca (Serpentes: Viperidae; Wied-Neuwied 1824) snake venom after up to 54 years of storage

ABSTRACT The number of snakes donated to the Brazilian Instituto Butantan has been decreasing in the past 10 years. This circumstance motivated us to compare the properties of five venom pools of Bothrops jararaca snake stored for up to 54 years. Results showed differences among venom pools regarding enzymatic and other biological activities, such as caseinolytic, phospholipase A2, hemorrhagic and coagulant activities, as well as antigenicity. Protein content, reverse‐phase chromatographic profile, and immunorecognition by commercial Bothrops antivenom were comparable for all venom pools, although lethality of the most recent preparations was higher. Since the lowest functional activities did not always correspond to older venoms, differences among venom pools used for antivenom production during the period 1963–2008 may correlate with the different proportions of venoms from different localities used in their generation, rather than to long‐term storage. We conclude that B. jararaca venoms properly stored for long periods of time retain their structural and pharmacological activities, thus representing useful materials for scientific research and antivenom production. Graphical abstract Figure. No Caption available. HighlightsBioactivities of Bothrops jararaca venom pools stored for up to 54 years were analyzed.Protein content, chromatographic and immunorecognition profile were comparable for all venom pools.Lethality of lyophilized venom pools (1997 and 2008) was higher than that of venom pools dried at RT (1963, 1973, 1977‐88).Vintage venom collections represent valuable materials for research purposes and possibly for the production of antivenom.

Viperid venom glands with defective venom production. Morphological study

The venom of viperid snakes is collected monthly at Butantan Institute for research purposes and production of antivenoms. Here we describe histological and ultrastructural changes on Crotalus durissus terrificus and Bothrops sp. venom glands with defective venom production. Secretory tubules commonly showed partial or total obliteration of their lumina by masses of necrotic cells and cellular debris. Secretory cells showed varying degrees of degenerative and/or metaplastic alterations seriously affecting the structures responsible for the synthesis and secretion of venom. The intertubular connective tissue presented fibroblast hyperplasia, inflammatory cells infiltration, vacuolated cells and blood vessels alterations. In two venom glands out of nineteen snakes examined, virus-like particles were found. The alterations observed in most of the glands could have been caused by excessive manual pressure, during venom extraction routine, causing disruption of the secretory tubules and leakage of venom to the intertubular connective tissue.

Functional and proteomic comparison of Bothrops jararaca venom from captive specimens and the Brazilian Bothropic Reference Venom

Snake venom is a variable phenotypic trait, whose plasticity and evolution are critical for effective antivenom production. A significant reduction of the number of snake donations to Butantan Institute (São Paulo, Brazil) occurred in recent years, and this fact may impair the production of the Brazilian Bothropic Reference Venom (BBRV). Nevertheless, in the last decades a high number of Bothrops jararaca specimens have been raised in captivity in the Laboratory of Herpetology of Butantan Institute. Considering these facts, we compared the biochemical and biological profiles of B. jararaca venom from captive specimens and BBRV in order to understand the potential effects of snake captivity upon the venom composition. Electrophoretic analysis and proteomic profiling revealed few differences in venom protein bands and some differentially abundant toxins. Comparison of enzymatic activities showed minor differences between the two venoms. Similar cross-reactivity recognition pattern of both venoms by the antibothropic antivenom produced by Butantan Institute was observed. Lethality and neutralization of lethality for B. jararaca venom from captive specimens and BBRV showed similar values. Considering these results we suggest that the inclusion of B. jararaca venom from captive specimens in the composition of BBRV would not interfere with the quality of this reference venom. BIOLOGICAL SIGNIFICANCE Snakebite envenomation is a neglected tropical pathology whose treatment is based on the use of specific antivenoms. Bothrops jararaca is responsible for the majority of snakebites in South and Southeastern Brazil. Its venom shows individual, sexual, and ontogenetic variability, however, the effect of animal captivity upon venom composition is unknown. Considering the reduced number of wild-caught snakes donated to Butantan Institute in the last decades, and the increased life expectancy of the snakes raised in captivity in the Laboratory of Herpetology, this work focused on the comparative profiling of B. jararaca venom from captive snakes and the Brazilian Bothropic Reference Venom (BBRV). BBRV is composed of venom obtained upon the first milking of wild-caught B. jararaca specimens, and used to assess the potency of all bothropic antivenoms produced by Brazilian suppliers. The use of proteomic strategies, added to biochemical and neutralization tests, allowed to conclude that, despite some subtle differences detected between these two venoms, venom from captive specimens could be used in the BBRV composition without affecting its quality in antivenom potency assays.

The detection of Cryptosporidium serpentis in snake fecal samples by real-time PCR.

Infection by Cryptosporidium serpentis occurs in reptiles, particularly in snakes. This disease is characterized by chronic infection with the presence of hypertrophic gastritis. The objectives of this study were to use real-time polymerase chain reaction (PCR) targeting the heat shock protein 70 (Hsp70) gene for the detection of C. serpentis in fecal samples from snakes and to determine the analytical and epidemiological specificity and sensitivity of this approach relative to the gold standard of nested PCR for the amplification of a fragment of the 18S subunit of the ribosomal RNA (18S rRNA) gene followed by the sequencing of amplified fragments (nPCR/S). Individual fecal samples were collected on a single occasion from 503 asymptomatic adult snakes housed in the serpentarium of the Butantan Institute in São Paulo, Brazil. The nested PCR revealed that 60 samples (11.98%) were positive for Cryptosporidium sp. The sequencing of amplified fragments, which was possible for 38 samples, resulted in the identification of Cryptosporidium tyzzeri (7), Cryptosporidium muris (4), Cryptosporidium varanii (12) and C. serpentis (15) in fecal samples from several snake species. The real-time PCR approach indicated that 17 samples (3.37%) were positive for C. serpentis, whereas the nPCR/S indicated that 15 samples (2.98%) were positive for C. serpentis. The epidemiological sensitivity and specificity of real-time PCR were 93.8% and 99.5%, respectively. Thus, we conclude that real-time PCR targeting the Hsp70 gene is a sensitive and specific method for the detection of C. serpentis in snake fecal samples.

Does the administration of pilocarpine prior to venom milking influence the composition of Micrurus corallinus venom

Considering that the scarcity of venom represents a huge challenge for biochemical and functional studies of Micrurus species (coral snakes), in this report we describe for the first time the influence of pilocarpine administration prior to venom milking on the yield and protein composition of Micrurus corallinus venom. The administration of pilocarpine resulted in an increase of about 127% in the volume of venom milked, with similar protein content. Venoms showed similar protein bands distribution and intensity by SDS-PAGE and equivalents RP-HPLC profiles. Our proteomic analysis showed that venoms milked in the presence and absence of pilocarpine presented comparable protein profiles, in terms of protein composition and relative abundance. The toxins identified were assigned to 13 protein families and represent the most complete M. corallinus venom proteome described so far, in terms of number of protein families identified. Our data indicate that the administration of pilocarpine prior to venom milking increases the venom yield and does not change significantly the venom composition of M. corallinus. The employment of pilocarpine represents a useful approach to increase the yield of venom not only for Micrurus species, but also for other genera of snakes with limitations regarding the amount of venom available. SIGNIFICANCE In this report, we evaluated the influence of pilocarpine administration prior to venom milking in the overall composition of M. corallinus venom. We showed that the use of pilocarpine 10min before M. corallinus venom milking increases venom yield by ~127%. Not only the volume of venom obtained is higher, but also the protein concentration of both venoms is similar, opposing the idea that a more diluted venom is obtained as a result of pilocarpine administration, observed in non-front-fanged snakes. Shotgun proteomics analysis revealed that venom milked with and without the use of this drug showed similar overall protein composition and relative abundances. In addition, our proteomic approach allowed the identification of 13 toxin families in M. corallinus venom, representing the most complete M. corallinus venom proteome described so far. Moreover, two of these toxin families were identified for the first time in the venom of this species. Thus, considering the scarcity of Micrurus venom for biochemical and functional studies, we highlighted the usefulness of pilocarpine administration prior to venom milking to increase the venom yield of these snakes.

Phylogenetic analysis of standard metabolic rate of snakes: a new proposal for the understanding of interspecific variation in feeding behavior

The current proposal about the variation of standard metabolic rates (SMR) in snakes predicts that SMR is influenced by the feeding frequency (frequent or infrequent feeders). However, feeding frequency in snakes is poorly studied and hard to quantify under natural conditions. Alternatively, foraging strategy was studied for a large number of species and is usually related to the feeding frequency. In this work, we performed a meta-analysis on the SMR of compiled data from 74 species of snakes obtained from the literature and five more different species of lanceheads (genus Bothrops), after categorization according to the foraging mode (ambush or active foraging) and regarding their phylogenetic history. We tested the hypothesis that foraging mode (FM) is a determinant factor on the interspecific variation of SMR despite the phylogenetic relationship among species. We demonstrated that FM predicted SMR, but there is also a partial phylogenetic structuration of SMR in snakes. We also detected that evolution rates of SMR in active foragers seem to be higher than ambush-hunting snakes. We suggested that foraging mode has a major effect over the evolution of SMR in snakes, which could represent an ecophysiological co-adaptation, since ambush hunters (with low feeding rates) present a lower maintenance energetic cost (SMR) when compared to active foragers. The higher SMR evolution rates for active foraging snakes could be related to a higher heterogeny in the degree of activity during hunting by active foragers when compared to ambush-hunting snakes.