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N Altman - One of the best experts on this subject based on the ideXlab platform.
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molecular analysis of the 18s rrna gene of cryptosporidium serpentis in a wild caught Corn Snake elaphe guttata guttata and a five species restriction fragment length polymorphism based assay that can additionally discern c parvum from c wrairi
Applied and Environmental Microbiology, 1999Co-Authors: Lewis M Kimbell, Debra L Miller, W Chavez, N AltmanAbstract:An adult wild-caught Corn Snake (Elaphe guttata guttata) was presented for humane euthanasia and necropsy because of severe cryptosporidiosis. The animal was lethargic and >5% dehydrated but in good flesh. Gastric lavage was performed prior to euthanasia. Histopathologic findings included gastric mucosal hypertrophy and a hemorrhagic erosive gastritis. Numerous 5- to 7-μm-diameter round extracellular organisms were associated with the mucosal hypertrophy. A PCR, acid-fast stains, Giemsa stains, and an enzyme immunoassay were all positive for Cryptosporidiumspp. PCR and restriction fragment length polymorphism (RFLP) analysis on gastric lavage and gastric mucosal specimens, and subsequent sequencing of the 18S rRNA gene, enabled a distinct molecular characterization of the infecting organism as Cryptosporidium serpentis. Until recently, studies on SnakeCryptosporidium have relied on host specificity and gross and histopathologic observations to identify the infecting species. A multiple alignment of our sequence against recently published sequences of the 18S rRNA gene of C. serpentis (GenBank accession no.AF093499, AF093500, and AF093501 [L. Xiao et al., unpublished data, 1998]) revealed 100% homology with the C. serpentis(Snake) sequence (AF093499) previously described by Xiao et al. An RFLP method to differentiate the five presently sequenced strains of Cryptosporidium at this locus was developed. This assay, which uses SpeI andSspI, complements a previously reported assay by additionally distinguishing the bovine strain ofCryptosporidium from Cryptosporidium wrairi.
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molecular analysis of the 18s rrna gene of cryptosporidium serpentis in a wild caught Corn Snake elaphe guttata guttata and a five species restriction fragment length polymorphism based assay that can additionally discern c parvum from c wrairi
Applied and Environmental Microbiology, 1999Co-Authors: Lewis M Kimbell, Debra L Miller, W Chavez, N AltmanAbstract:Cryptosporidium is a 5- to 10-μm round protozoal parasite that affects the digestive epithelium of a variety of animal species. More than 20 species of Cryptosporidium have been identified with questionable host specificity (8, 17). Recently, questions regarding identification of the organism to species level have been brought to the forefront for five reasons, as follows. (i) The organism has been identified as causing a fatal diarrhea in immune-compromised humans (6). (ii) There have been numerous water- and food-borne outbreaks of the disease in humans (22). (iii) There is no effective treatment for the disease. (iv) Water treatment protocols lack effective disinfection methods to rid drinking water of viable organisms (22, 27). (v) A variety of environmental sources may be contaminated with oocysts, and therefore, they may harbor a variety of different species of Cryptosporidium (11, 23). Identification of Cryptosporidium organisms to species level is important because species-specific pathogenicity to humans has not yet been determined. Until we are able to determine the specificity of the different species of Cryptosporidium, we cannot determine the epidemiological significance of its presence within environmental samples (i.e., understanding its importance and implementing control methods). In Snakes, the absence of an effective treatment for cryptosporidiosis necessitates euthanasia to eliminate patient suffering and prevent further spread of the parasite. Historically, oocyst size and location in the host have been used in identification to species level (1a, 8, 16, 17, 21). However, a recent, sequence-specific PCR has been used to distinguish Cryptosporidium parvum from other species (4). Leng et al. (15) have described a PCR and restriction fragment length polymorphism (RFLP) method to analyze sequence variation of the 18S rRNA gene of Cryptosporidium spp. The high degree of sequence homology present in the 18S rRNA genes among different isolates and species of Cryptosporidium (3, 13, 14) allows the use of specific primers to amplify a wide range of Cryptosporidium species. Concomitant RFLP analysis is capable of exploiting any subtle differences found in these highly homologous nucleotide areas, provided restriction sites which enable differentiation exist. In this study, two oligonucleotide primers, sequences 18SFwd (5′-AACCTggTTgATCCTgCCAg-3′) and 18SRev (5′-TgATCCTTCTgCAggTTCACCTA-3′) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"L16997","term_id":"290016"}}L16997) (2a, 17a) were used to amplify ca. 1,750-bp fragment of the 18S rRNA gene of Cryptosporidium. These primers target the 18S rRNA gene of all presently sequenced species of Cryptosporidium. This fragment was analyzed by RFLP and sequenced. We give the resulting complete nucleotide sequence of the 18S rRNA gene of Cryptosporidium serpentis (1,743 bp; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF151376","term_id":"5870846","term_text":"AF151376"}}AF151376) from Elaphe guttata guttata and align the sequence against other known C. serpentis sequences at this locus. Based on our results we suggest an RFLP-based assay capable of distinguishing five species of Cryptosporidium and of discerning bovine C. parvum from Cryptosporidium wrairi.
David D. Pollock - One of the best experts on this subject based on the ideXlab platform.
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Comparative mitochondrial genomics of Snakes: extraordinary substitution rate dynamics and functionality of the duplicate control region.
BMC Evolutionary Biology, 2007Co-Authors: Zhi J. Jiang, Christopher L Parkinson, Matthew D. Herron, Christopher C. Austin, Todd A. Castoe, Jimmy A Mcguire, Frank T Burbrink, David D. PollockAbstract:Background The mitochondrial genomes of Snakes are characterized by an overall evolutionary rate that appears to be one of the most accelerated among vertebrates. They also possess other unusual features, including short tRNAs and other genes, and a duplicated control region that has been stably maintained since it originated more than 70 million years ago. Here, we provide a detailed analysis of evolutionary dynamics in Snake mitochondrial genomes to better understand the basis of these extreme characteristics, and to explore the relationship between mitochondrial genome molecular evolution, genome architecture, and molecular function. We sequenced complete mitochondrial genomes from Slowinski's Corn Snake (Pantherophis slowinskii) and two cottonmouths (Agkistrodon piscivorus) to complement previously existing mitochondrial genomes, and to provide an improved comparative view of how genome architecture affects molecular evolution at contrasting levels of divergence.
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Comparative mitochondrial genomics of Snakes: extraordinary substitution rate dynamics and functionality of the duplicate control region
BMC Evolutionary Biology, 2007Co-Authors: Zhi J. Jiang, Christopher L Parkinson, Matthew D. Herron, Christopher C. Austin, Todd A. Castoe, Jimmy A Mcguire, Frank T Burbrink, David D. PollockAbstract:Background The mitochondrial genomes of Snakes are characterized by an overall evolutionary rate that appears to be one of the most accelerated among vertebrates. They also possess other unusual features, including short tRNAs and other genes, and a duplicated control region that has been stably maintained since it originated more than 70 million years ago. Here, we provide a detailed analysis of evolutionary dynamics in Snake mitochondrial genomes to better understand the basis of these extreme characteristics, and to explore the relationship between mitochondrial genome molecular evolution, genome architecture, and molecular function. We sequenced complete mitochondrial genomes from Slowinski's Corn Snake ( Pantherophis slowinskii ) and two cottonmouths ( Agkistrodon piscivorus ) to complement previously existing mitochondrial genomes, and to provide an improved comparative view of how genome architecture affects molecular evolution at contrasting levels of divergence. Results We present a Bayesian genetic approach that suggests that the duplicated control region can function as an additional origin of heavy strand replication. The two control regions also appear to have different intra-specific versus inter-specific evolutionary dynamics that may be associated with complex modes of concerted evolution. We find that different genomic regions have experienced substantial accelerated evolution along early branches in Snakes, with different genes having experienced dramatic accelerations along specific branches. Some of these accelerations appear to coincide with, or subsequent to, the shortening of various mitochondrial genes and the duplication of the control region and flanking tRNAs. Conclusion Fluctuations in the strength and pattern of selection during Snake evolution have had widely varying gene-specific effects on substitution rates, and these rate accelerations may have been functionally related to unusual changes in genomic architecture. The among-lineage and among-gene variation in rate dynamics observed in Snakes is the most extreme thus far observed in animal genomes, and provides an important study system for further evaluating the biochemical and physiological basis of evolutionary pressures in vertebrate mitochondria.
N M Loskutoff - One of the best experts on this subject based on the ideXlab platform.
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161 cryopreservation of Corn Snake elaphe gutatta semen
Reproduction Fertility and Development, 2009Co-Authors: K M Mattson, A T Devries, J Krebs, N M LoskutoffAbstract:The purpose of this investigation was to develop a protocol for cryopreserving Snake semen using the Corn Snake, Elaphe gutatta, as the model species. This experiment is part of a five year investigation where the influences of diluents, cryoprotectants, cooling and thawing rates on sperm survival were studied. This report presents one protocol found to be effective for cryopreserving Corn Snake semen as determined by post-thaw motility parameters in vitro. Semen was collected by applying pressure to the lower abdomen and continuing distally towards the cloaca to remove any feces or urates. The cloaca was washed using PBS, then a more local pressure was applied to each side of the vent to cause the hemipenes to evert and subsequently ejaculate. The semen (approximately 5 μL) was then collected using a sterile transfer pipette, placed in 120 μL Biladyl A containing 20% egg yolk (Minitube, 13502/0501), and analyzed for motility, rate of forward progression (RFP; 0–5), and concentration. The semen was further diluted at room temperature at 1:1 v/v with Biladyl A containing 20% egg yolk and 34% Glycerol (Sigma, G2025), yielding a final concentration of 17% Glycerol. The diluted semen was then loaded into 250-μL straws and slowly cooled for 1 hour. The straws were then placed 1 inch above a liquid nitrogen bath for ten minutes and finally plunged into the nitrogen where it remained frozen. The cryopreserved semen was thawed by placing the straws into a 50°C water bath for 8 s, then emptied into microcentrifuge tubes and the sperm were evaluated for motility and RFP. The mean motility of the fresh samples was 72.5% (66.4–77.7%). The mean post-thaw motility of sperm over six trials was 27.1% (17.8–50.2%). The mean RFP was 0.75 (0.5–1.0). The differences between fresh and post-thawed mean motilities were shown to be significant using a chi-square analysis (P < 0.0001). Density gradient centrifugation (DGC) was applied in one trial where the semen had an initial post-thaw motility of 50.2% with an RFP of 0.5. After the centrifugation treatment, the motility increased to 64.8% with an RFP of 3. The DGC media was composed of 400 μL 45% Percoll (Sigma, P4937) layered over 400 μL 90% Percoll. The density gradients were centrifuged at 700g for 30 min after which time the pellets were washed in 500 μL pre-warmed TL Hepes Solution (Lonza, 04-616F) and centrifuged at 300g for 10 min to remove the Percoll. The resulting sperm pellets were then resuspended in a small volume of the pre-warmed Hepes. Thus far, the protocol using 17% Glycerol in Biladyl A with 20% egg yolk has proven to be the most successful for cryopreserving Corn Snake semen. The use of DGC enhanced the number of usable sperm leaving sperm of higher motility and RFP possibly due to the absence of seminal plasma or cryoprotective agents that may detrimentally affect sperm quality. There are no known reports of the use of DCG with Snake semen. Further studies are underway to improve these results and successfully use cryopreserved Snake semen for artificial insemination and cryobanking for the long-term genetic management of endangered Snake species.
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247 successful artificial insemination in the Corn Snake elaphe gutatta using fresh and cooled semen
Reproduction Fertility and Development, 2007Co-Authors: J K Mattson, Susie M. Mcguire, Edward E. Louis, A T Devries, J Krebs, N M LoskutoffAbstract:The purpose of this investigation was to develop a non-invasive technique to artificially inseminate Snakes using the Corn Snake, Elaphe gutatta, as the model representative for this taxon. Semen was collected by first applying pressure to the lower abdomen in a continuous distal motion toward the cloaca to remove any feces or urates. The cloaca was then gently washed using phosphate-buffered saline, and a more localized pressure was applied to each side of the vent to evert the hemipenes and, subsequently, the ejaculate. The semen was collected using a sterile transfer pipette and placed into 70 to 90 µL of medium (TL-HEPES solution; Cambrex Bio Science, Inc., Baltimore, MD, USA04–616F) in a sterile microcentrifuge tube, and then analyzed for overall motility, rate of forward progression (RFP, 0–5), and concentration. Based on a previously reported procedure, 10 females were inseminated with either fresh (n = 5) or cooled semen (n = 5; refrigerated for 3 days) one week after recovering from a hibernation period required to stimulate reproduction in this species. The overall sperm motility and concentration for females inseminated with fresh or cooled semen was 92%, 9.6 million sperm mL-1; and 85%, 6.1 million sperm mL-1, respectively. Immediately prior to insemination, the same method for expressing feces and urates in the males was applied to the females. The insemination dose (50 µL semen per oviduct) was drawn into a 1-mL latex- and silicone-free tuberculin syringe (Norm-Ject; VWR, Batavia, IL, USA) that was connected to a feeding/dosing needle (EJAY International, Issaquah, WA, USA) with a ball tip to prevent any potential damage during the insemination. The tip of the needle was then moved around the inner tissue of the vent to relax the cloaca, and the insertion continued until resistance was found indicating the vicinity of the oviducts. The extended semen was carefully deposited on both sides, and then the needle was slowly withdrawn. The offspring were tested for parentage to verify the success of the insemination. Blood was collected from the dorsal aorta posterior to the cloaca and stored in 10 mM Tris at 4°C. The DNA was extracted using a phenyl : chloroform : isoamyl alcohol (PCI) extraction method. Eight microsatellite loci were used for the paternity exclusion analysis: Eobµ1, Eobµ3, Eobµ10, Eobµ13, Eobµ16, Eobµ34, Eobµ366, and Eobµ373 (IDT, Coraville, IA, USA). All males and females in the collection were tested, and parental candidates were excluded if 2 or more allele mismatches occurred. From the total number of females inseminated, 3 females laid 51 eggs. Two females inseminated with cooled semen laid 36 eggs, of which 5 eggs were hatched, and the remaining were either unfertilized (n = 25) or non-viable (n = 6). All 5 hatched eggs were laid by one of the females. The third female inseminated with fresh semen laid 15 eggs, resulting in 5 hatching and 10 unfertilized eggs. The parentage test validated the AI a success as the alleles correlated between the adults and the offspring. In conclusion, artificial insemination was successful using both freshly collected and cooled (3 days) semen. Further studies are underway to improve the success rate in order to maximize the efficiency of this technology, and thus assist in the genetic preservation of endangered Snake species.
Michel C. Milinkovitch - One of the best experts on this subject based on the ideXlab platform.
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genome mapping of a lyst mutation in Corn Snakes indicates that vertebrate chromatophore vesicles are lysosome related organelles
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Asier Ullateagote, Ingrid Burgelin, Adrien Debry, Carine Langrez, Florent Montange, Rodrigue Peraldi, Jean Daraspe, Henrik Kaessmann, Michel C. MilinkovitchAbstract:Reptiles exhibit a spectacular diversity of skin colors and patterns brought about by the interactions among three chromatophore types: black melanophores with melanin-packed melanosomes, red and yellow xanthophores with pteridine- and/or carotenoid-containing vesicles, and iridophores filled with light-reflecting platelets generating structural colors. Whereas the melanosome, the only color-producing endosome in mammals and birds, has been documented as a lysosome-related organelle, the maturation paths of xanthosomes and iridosomes are unknown. Here, we first use 10x Genomics linked-reads and optical mapping to assemble and annotate a nearly chromosome-quality genome of the Corn Snake Pantherophis guttatus The assembly is 1.71 Gb long, with an N50 of 16.8 Mb and L50 of 24. Second, we perform mapping-by-sequencing analyses and identify a 3.9-Mb genomic interval where the lavender variant resides. The lavender color morph in Corn Snakes is characterized by gray, rather than red, blotches on a pink, instead of orange, background. Third, our sequencing analyses reveal a single nucleotide polymorphism introducing a premature stop codon in the lysosomal trafficking regulator gene (LYST) that shortens the corresponding protein by 603 amino acids and removes evolutionary-conserved domains. Fourth, we use light and transmission electron microscopy comparative analyses of wild type versus lavender Corn Snakes and show that the color-producing endosomes of all chromatophores are substantially affected in the LYST mutant. Our work provides evidence characterizing xanthosomes in xanthophores and iridosomes in iridophores as lysosome-related organelles.
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Amelanism in the Corn Snake is associated with the insertion of an LTR-retrotransposon in the OCA2 gene
Scientific Reports, 2015Co-Authors: Suzanne V. Saenko, Alvaro Martinez Barrio, Nima Rafati, Sangeet Lamichhaney, Leif Andersson, Michel C. MilinkovitchAbstract:The Corn Snake ( Pantherophis guttatus ) is a new model species particularly appropriate for investigating the processes generating colours in reptiles because numerous colour and pattern mutants have been isolated in the last five decades. Using our captive-bred colony of Corn Snakes, transcriptomic and genomic next-generation sequencing, exome assembly and genotyping of SNPs in multiple families, we delimit the genomic interval bearing the causal mutation of amelanism, the oldest colour variant observed in that species. Proceeding with sequencing the candidate gene OCA2 in the uncovered genomic interval, we identify that the insertion of an LTR-retrotransposon in its 11^th intron results in a considerable truncation of the p protein and likely constitutes the causal mutation of amelanism in Corn Snakes. As amelanistic Snakes exhibit white, instead of black, borders around an otherwise normal pattern of dorsal orange saddles and lateral blotches, our results indicate that melanocytes lacking melanin are able to participate to the normal patterning of other colours in the skin. In combination with research in the zebrafish, this work opens the perspective of using Corn Snake colour and pattern variants to investigate the generative processes of skin colour patterning shared among major vertebrate lineages.
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Multiple applications of R2OBBIE.
2015Co-Authors: António F. Martins, Michel Bessant, Liana Manukyan, Michel C. MilinkovitchAbstract:A) PMVS reconstruction (~24 hours of reconstruction time) of a 155–161 million-year-old marine crocodylian fossil (Metriorhynchus superciliosus; specimen PIMUZ A/III 14 from the Paläontologisches Institut und Museum, Universität Zürich, Switzerland). Multiple scans in the ‘static support’ and ‘turn-table’ configurations were combined for a total of 746 pictures; linear distance between the two arrows = 21.4cm. B) Geometry of a 0.5 Swiss-franc coin scanned in the ‘static support’ configuration (30 pictures, ~20 seconds of scanning time) and reconstructed with PS (~30 minutes of reconstruction time); Scale bar: 2mm. C) and D) Details of B; Scale bars: 200 μm. E) Corn Snake (Pantherophis guttatus) geometry (and colour texture on bottom half of the mesh) scanned in the ‘hanging anaesthetised Snake’ configuration (100 SFM pictures + 30 PS pictures, ~7 minutes of scanning time) and reconstructed with visual hull (VH, ~2 hours of reconstruction time), structure-from-motion (PMVS, ~6 hours of reconstruction time), photometric stereo (PS, ~15 minutes of reconstruction time), and the hybrid mode (PMVS + PS, ~7 hours of total reconstruction time, i.e., including the PMVS and PS reconstruction times and the combination step). Scale bar: 10 mm. Insets: zoom on geometry.
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Animal scans performed with R2OBBIE.
2015Co-Authors: António F. Martins, Michel Bessant, Liana Manukyan, Michel C. MilinkovitchAbstract:A) An adult day gecko (Phelsuma grandis) of 20.5cm (total length) scanned in the ‘static support’ configuration (74 pictures, ~5 minutes of scanning time) and reconstructed with PMVS (~6 hours of reconstruction time); upper panel, overall view; mid panel, close-up with shaded geometry and colour texture (double-headed arrow: 1mm); lower panel, same closeup with geometry only. B) Corn Snake (Pantherophis guttatus) scanned in the ‘hanging anaesthetised Snake’ configuration (424 pictures, ~25 minutes of scanning time) and reconstructed with PMVS (~24 hours of reconstruction time); upper panel, overall view with shaded geometry and colour texture; lower panel, close-up of the shaded geometry with and without colour texture. Scale bars: 40mm (upper panel) and 5mm (lower panel). C) Sea urchin’s (Echinometra mathaei) skeleton scanned in the ‘static support’ configuration (30 pictures, ~20 seconds of scanning time) and reconstructed with PS (~20 minutes of reconstruction time); upper-left, photography under a stereoscopic microscope (scale bar: 1mm) without white-balance correction; lower left, reconstructed geometry with and without colour texture (with calibrate white-balance, i.e., the colour texture is realistic); upper-right and lower right panels, stereoscope image and reconstructed geometry of two small features (arrows) of the specimen (Scale bar: 100μm).
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the genome sequence of the Corn Snake pantherophis guttatus a valuable resource for evodevo studies in squamates
The International Journal of Developmental Biology, 2014Co-Authors: Asier Ullateagote, Michel C. Milinkovitch, Athanasia C TzikaAbstract:Squamates (Snakes and lizards) exhibit a striking variety of phenotypes, with little known on their generative mechanisms. Studies aiming to understand the genetic basis of this wide diversity in morphology, physiology and ecology will greatly benefit from whole genome sequencing initiatives, as they provide the foundation for comparative analyses and improve our understanding of the evolution, development and diversification of traits. Here, we present the first draft genome of the Corn Snake Pantherophis guttatus, an oviparous Snake that we promote as a particularly appropriate model species for evolutionary developmental studies in squamates. We sequenced 100-base paired-end reads from multiple individuals of a single family (parents and offspring) that produced a genome assembly of 1.53 gigabases (Gb), roughly covering 75% of the expected total genome size, and 297,768 scaffolds >1 Kb. We were able to fully retrieve 86, and partially another 106, of the 248 CEGMA core genes, indicating that a high genome completeness was achieved, even though the assembly is fragmented. Using MAKER2, we annotated 10,917 genes with high confidence (Annotation Edit Distance (AED)
Lewis M Kimbell - One of the best experts on this subject based on the ideXlab platform.
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molecular analysis of the 18s rrna gene of cryptosporidium serpentis in a wild caught Corn Snake elaphe guttata guttata and a five species restriction fragment length polymorphism based assay that can additionally discern c parvum from c wrairi
Applied and Environmental Microbiology, 1999Co-Authors: Lewis M Kimbell, Debra L Miller, W Chavez, N AltmanAbstract:An adult wild-caught Corn Snake (Elaphe guttata guttata) was presented for humane euthanasia and necropsy because of severe cryptosporidiosis. The animal was lethargic and >5% dehydrated but in good flesh. Gastric lavage was performed prior to euthanasia. Histopathologic findings included gastric mucosal hypertrophy and a hemorrhagic erosive gastritis. Numerous 5- to 7-μm-diameter round extracellular organisms were associated with the mucosal hypertrophy. A PCR, acid-fast stains, Giemsa stains, and an enzyme immunoassay were all positive for Cryptosporidiumspp. PCR and restriction fragment length polymorphism (RFLP) analysis on gastric lavage and gastric mucosal specimens, and subsequent sequencing of the 18S rRNA gene, enabled a distinct molecular characterization of the infecting organism as Cryptosporidium serpentis. Until recently, studies on SnakeCryptosporidium have relied on host specificity and gross and histopathologic observations to identify the infecting species. A multiple alignment of our sequence against recently published sequences of the 18S rRNA gene of C. serpentis (GenBank accession no.AF093499, AF093500, and AF093501 [L. Xiao et al., unpublished data, 1998]) revealed 100% homology with the C. serpentis(Snake) sequence (AF093499) previously described by Xiao et al. An RFLP method to differentiate the five presently sequenced strains of Cryptosporidium at this locus was developed. This assay, which uses SpeI andSspI, complements a previously reported assay by additionally distinguishing the bovine strain ofCryptosporidium from Cryptosporidium wrairi.
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molecular analysis of the 18s rrna gene of cryptosporidium serpentis in a wild caught Corn Snake elaphe guttata guttata and a five species restriction fragment length polymorphism based assay that can additionally discern c parvum from c wrairi
Applied and Environmental Microbiology, 1999Co-Authors: Lewis M Kimbell, Debra L Miller, W Chavez, N AltmanAbstract:Cryptosporidium is a 5- to 10-μm round protozoal parasite that affects the digestive epithelium of a variety of animal species. More than 20 species of Cryptosporidium have been identified with questionable host specificity (8, 17). Recently, questions regarding identification of the organism to species level have been brought to the forefront for five reasons, as follows. (i) The organism has been identified as causing a fatal diarrhea in immune-compromised humans (6). (ii) There have been numerous water- and food-borne outbreaks of the disease in humans (22). (iii) There is no effective treatment for the disease. (iv) Water treatment protocols lack effective disinfection methods to rid drinking water of viable organisms (22, 27). (v) A variety of environmental sources may be contaminated with oocysts, and therefore, they may harbor a variety of different species of Cryptosporidium (11, 23). Identification of Cryptosporidium organisms to species level is important because species-specific pathogenicity to humans has not yet been determined. Until we are able to determine the specificity of the different species of Cryptosporidium, we cannot determine the epidemiological significance of its presence within environmental samples (i.e., understanding its importance and implementing control methods). In Snakes, the absence of an effective treatment for cryptosporidiosis necessitates euthanasia to eliminate patient suffering and prevent further spread of the parasite. Historically, oocyst size and location in the host have been used in identification to species level (1a, 8, 16, 17, 21). However, a recent, sequence-specific PCR has been used to distinguish Cryptosporidium parvum from other species (4). Leng et al. (15) have described a PCR and restriction fragment length polymorphism (RFLP) method to analyze sequence variation of the 18S rRNA gene of Cryptosporidium spp. The high degree of sequence homology present in the 18S rRNA genes among different isolates and species of Cryptosporidium (3, 13, 14) allows the use of specific primers to amplify a wide range of Cryptosporidium species. Concomitant RFLP analysis is capable of exploiting any subtle differences found in these highly homologous nucleotide areas, provided restriction sites which enable differentiation exist. In this study, two oligonucleotide primers, sequences 18SFwd (5′-AACCTggTTgATCCTgCCAg-3′) and 18SRev (5′-TgATCCTTCTgCAggTTCACCTA-3′) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"L16997","term_id":"290016"}}L16997) (2a, 17a) were used to amplify ca. 1,750-bp fragment of the 18S rRNA gene of Cryptosporidium. These primers target the 18S rRNA gene of all presently sequenced species of Cryptosporidium. This fragment was analyzed by RFLP and sequenced. We give the resulting complete nucleotide sequence of the 18S rRNA gene of Cryptosporidium serpentis (1,743 bp; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF151376","term_id":"5870846","term_text":"AF151376"}}AF151376) from Elaphe guttata guttata and align the sequence against other known C. serpentis sequences at this locus. Based on our results we suggest an RFLP-based assay capable of distinguishing five species of Cryptosporidium and of discerning bovine C. parvum from Cryptosporidium wrairi.