Exposure to fluctuating temperatures leads to reduced immunity and to stress response in rattlesnakes.

Ectothermic organisms often experience considerable variation in their body temperature throughout the circadian cycle. However, studies focusing on the measurement of physiological traits are usually performed under constant temperature regimes. This mismatch between thermal exposure in the field and experimental conditions could act as a stressor agent, as physiological functions are strongly influenced by temperature. Herein, we asked the question whether constant thermal regimes would cause a stress response and impact the immunity of the South American rattlesnake (Crotalus durissus) when compared with a fluctuating thermal regime. We addressed this question by determining heterophil:lymphocyte (H:L) ratio, plasma bacteria-killing ability (BKA) and corticosterone (CORT) levels in snakes kept under a constant temperature regime at 30°C, and under a fluctuating regime that oscillated between 25°C at night and 35°C during the day. The experiments had a mirrored design, in which half of the snakes were subjected to a fluctuating-to-constant treatment, while the other half was exposed to a constant-to-fluctuating treatment. The shift from constant to fluctuating thermal regime was accompanied by an increase in plasma CORT levels, indicating the activation of a stress response. Exposure to a fluctuating thermal regime at the onset of the experiments induced a decrease in the BKA of rattlesnakes. H:L ratio was not affected by treatments and, therefore, the shift between thermal regimes seems to have acted as a low-intensity stressor. Our results suggest that removal from temperatures close to the snake's preferred body temperature triggers a stress response in rattlesnakes.

Snake fungal disease alters skin bacterial and fungal diversity in an endangered rattlesnake.

Snake Fungal Disease (SFD), caused by Ophidiomyces ophiodiicola, is the most recently described fungal disease afflicting wildlife populations across North America and Europe. It has been proposed as a significant conservation threat yielding high mortality and yet much its ecology is unknown. We collected 144 skin swabs from Eastern Massasaugas (Sistrurus catenatus) in 2015 and 2016 to determine document ongoing prevalence and assess differences in microbial assemblages between positive and negative individuals. Alpha diversity of fungi was reduced in SFD positive animals, while beta diversity identified distinct assemblages of microbes between SFD-positive and -negative samples. Ophidiomyces was present on the skin of affected animals, even on body sites distant to lesions indicating that the microbiome on entire surface of the skin is altered. Ophidiomyces was not detected in any non-SFD snake. There were smaller, but significant, influences of year sampled. Bacterial genera Janthinobacterium and Serratia were significantly increased in SFD snakes, while Xylanimicrobium, Cellulosimicrobium, and Rhodococcus were the only bacterial taxa significantly reduced. The relative abundance of fungi within the orders Pleosporales and Canopdiales was reduced in SFD-positive samples, though Pyrenochaetopsis pratorum was the only species found to differ significantly. This is the first study to determine the impact that this fungal pathogen has on the skin microbiome.

Enterococcus crotali sp. nov., isolated from faecal material of a timber rattlesnake.

A facultatively anaerobic, Gram-stain-positive bacterium, designated ETRF1T, was found in faecal material of a timber rattlesnake (Crotalus horridus). Based on a comparative 16S rRNA gene sequence analysis, the isolate was assigned to the genus Enterococcus. The 16S rRNA gene sequence of strain ETRF1T showed >97 % similarity to that of the type strains of Enterococcus rotai, E. caccae, E. silesiacus, E haemoperoxidus, E. ureasiticus, E. moraviensis, E. plantarum, E. quebecensis, E. ureilyticus, E. termitis, E. rivorum and E. faecalis. The organism could be distinguished from these 12 phylogenetically related enterococci using conventional biochemical testing, the Rapid ID32 Strep system, comparative pheS and rpoA gene sequence analysis, and comparative whole genome sequence analysis. The estimated in silico DNA-DNA hybridization values were <70 %, and average nucleotide identity values were <96 %, when compared to these 12 species, further validating that ETRF1T represents a unique species within the genus Enterococcus. On the basis of these analyses, strain ETRF1T (=CCUG 65857T=LMG 28312T) is proposed as the type strain of a novel species, Enterococcus crotali sp. nov.

Thrombelastographic characterization of the thrombin-like activity of Crotalus simus and Bothrops asper venoms.

: Annually, thousands suffer venomous snake-bite from Crotalus simus and Bothrops asper vipers in central and South America. The goals of the present study were to generally characterize the thrombin-like effects of venom from these snakes in human plasma with viscoelastic methods. Human plasma was exposed to the venom of three different C. simus subspecies and venoms obtained from B. asper vipers located in three different locations in Mexico. To characterize the factor X-activating and thrombin-like activity of these venoms, plasma (normal or factor XIII deficient) was pretreated with a variety of additives (e.g., heparin) in the absence or presence of calcium prior to exposure to 2.0 µg/ml of each viper's venom. These profiles were compared with plasma without venom that had contact activation of coagulation. Coagulation kinetics were determined with thrombelastography. All venoms had thrombin-like activity, with C. s. simus creating a slow growing, weak clot that was likely mediated by metalloproteinases. In contrast, B. asper venoms had rapid onset of coagulation and a high velocity of thrombus growth. Further, B. asper venom activity was calcium-independent, activated prothrombin, activated factor XIII, and independently polymerized fibrinogen. The viscoelastic methods used were able to differentiate subspecies of C. simus and specimens of B. asper, and provide insight into the mechanisms by which the venoms acted on plasma. These methods may be useful in the profiling of similar venoms and perhaps can assist in the assessment of interventions designed to treat envenomation (e.g., antivenom).

Anti-Leptospira spp. antibodies in Crotalus durissus collilineatus kept in captivity and its zoonotic relevance.

Leptospirosis is a worldwide spread zoonosis that can affect all groups of vertebrates, including reptiles. Because it has been little studied in snakes, this study focused on determining the occurrence of anti-Leptospira spp. antibodies in 64 Crotalus durissus collilineatus kept in captivity and on identifying the most common serovars in these animals, using the microscopic agglutination test. Of these, almost 90% were positive and there were reactions to the 22 serovars used in the study. The most common serovar in these snakes was Javanica, Andamana and Patoc. Most frequent titers were 25 and 50, although high titers (such as 1600) were also recorded, despite the absence of clinical symptoms. The possibility should be considered of captive snakes serving as a serious source of leptospiral infection in humans, which is why it is essential to study, prevent and control the disease in breeding centers and serpentariums.

Ophidiomyces ophiodiicola dermatitis in eight free-ranging timber rattlesnakes (Crotalus horridus) from Massachusetts.

Eight free-ranging timber rattlesnakes (Crotalus horridus) from two geographically isolated Massachusetts populations were observed with skin lesions located primarily on the head but occasionally also on the lateral and ventral surfaces of the body. The snakes underwent health assessments that included physical examination, clinical pathology, full body radiographs, and full thickness biopsies of skin lesions. Each snake had fungal elements present histologically in tissue sections from skin lesions. Ophidiomyces ophiodiicola was identified from skin lesions using polymerase chain reaction in all eight snakes.

Metagenomic analysis of the gut microbiota of the Timber Rattlesnake, Crotalus horridus.

Snakes are capable of surviving long periods without food. In this study we characterized the microbiota of a Timber Rattlesnake (Crotalus horridus), devoid of digesta, living in the wild. Pyrosequencing-based metagenomics were used to analyze phylogenetic and metabolic profiles with the aid of the MG-RAST server. Pyrosequencing of samples taken from the stomach, small intestine and colon yielded 691696, 957756 and 700419 high quality sequence reads. Taxonomic analysis of metagenomic reads indicated Eukarya was the most predominant domain, followed by bacteria and then viruses, for all three tissues. The most predominant phylum in the domain Bacteria was Proteobacteria for the tissues examined. Functional classifications by the subsystem database showed cluster-based subsystems were most predominant (10-15 %). Almost equally predominant (10-13 %) was carbohydrate metabolism. To identify bacteria in the colon at a finer taxonomic resolution, a 16S rRNA gene clone library was created. Proteobacteria was again found to be the most predominant phylum. The present study provides a baseline for understanding the microbial ecology of snakes living in the wild.

Isolation of Lactococcus garvieae strain TRF1 from the fecal material of a Timber Rattlesnake.

In certain species of fish, such as rainbow trout, infection by the Firmicutes Lactococcus garvieae is problematic. This organism is the causative agent of lactococcosis disease in fish, and it is also considered a potential zoonotic bacterium, since it can cause several opportunistic infections in humans. In this study, L. garvieae strain TRF1 was grown and isolated in pure culture from the fecal material of a Timber Rattlesnake (Crotalus horridus), living in the wild. The presence/absence of several putative virulence factors was identified using staining, PCR amplification, and the construction of a draft genome. Strain TRF1 shared several putative virulence factors with strain Lg2, a known fish pathogen. However, the capsule gene cluster, found in strain Lg2, was not found in strain TRF1. Since this gene cluster is absent in several non-pathogenic strains of L. garvieae, it suggests strain TRF1 may not be pathogenic. However, this hypothesis will have to be tested in an animal model.

Additional observations and notes on the natural history of the prairie rattlesnake (Crotalus viridis) in Colorado.

On account of their unique anatomy, physiology, natural history, ecology, and behavior, rattlesnakes make ideal subjects for a variety of different scientific disciplines. The prairie rattlesnake (Crotalus viridis) in Colorado was selected for investigation of its relationship to colonies of black-tailed prairie dogs (Cynomys ludovicianus) with regard to spatial ecology. A total of 31 snakes were anesthetized and had radiotransmitters surgically implanted. In addition, at the time of their capture, all snakes underwent the following: (1) they had bacterial culture taken from their mouths for potential isolation of pathogenic bacteria; (2) similarly, they had cloacal bacterial cultures taken to assess potentially harmful bacteria passed in the feces; and (3) they had blood samples drawn to investigate the presence of any zoonotic agents in the serum of the snakes. The results of the study and their implications are discussed here. Traditionally, a low incidence of bacterial wound infection has been reported following snakebite. Nevertheless, the oral cavity of snakes has long been known to house a wide variety of bacterial flora. In our study, 10 different bacterial species were isolated from the mouths of the rattlesnakes, 6 of which are capable of being zoonotic pathogens and inducing human disease. More studies are necessary to see why more rattlesnake bites do not become infected despite the presence of such pathogenic bacteria. The results of fecal bacteria isolated revealed 13 bacterial species, 12 of which can cause disease in humans. Of the snakes whose samples were cultured, 26% were positive for the presence of the pathogen Salmonella arizonae, one of the causative agents of reptile-related salmonellosis in humans. It has long been reported that captive reptiles have a much higher incidence than wild, free-ranging species. This study shows the incidence of Salmonella in a wild, free-ranging population of rattlesnakes. In addition, Stenotrophomonas maltophilia was isolated. This bacterium is associated with wound and soft tissue infections that can lead to sepsis, endocarditis, meningitis, and peritonitis. In addition, this bacterium has been increasingly implicated as an opportunistic pathogen to humans during pregnancies, hospitalizations, malignancies and chemotherapy, chronic respiratory diseases, and presurgical endotracheal intubation. Furthermore, S. maltophilia has an intense resistance to broad-spectrum antibiotics, the results of our study showed the bacterium was resistant to multiple antibiotics. Our results indicate that anyone working with snake feces, dead skin, or their carcasses must follow reasonable hygiene protocols. Rattlesnakes tested for West Nile antibodies had positive results but these were invalidated owing to possible cross-reactivity with other unknown viruses, interference with snake serum proteins, and the fact that the test was not calibrated for rattlesnake serum. Still, the interesting implication remains, should we be regularly testing these animals as sentinels against potentially zoonotic diseases. The results of this study clearly show the value of veterinarians in a multidisciplinary study of this sort and the particular skill set they can offer. Veterinarians must get involved in conservation studies if the biodiversity of the planet is to be preserved.

Comparison of wildlife and captivity rattlesnakes (Crotalus durissus terrificus) microbiota / Comparação da microbiota de cascavéis (Crotalus durissus terrificus) de vida-livre e cativeiro

The study evaluated and compared the aerobic microbiota from the oral cavity, cloaca and venom of Crotalus durissus terrificus snakes, recently caught from the wild and kept under quarantine (WQ), individual captivity (IC) and collective captivity (CC). Antimicrobial drug effectiveness on isolated agents also was assayed. From group I, II and III were isolated, respectively, 29 (63.04 percent), 38 (90.48 percent) and 21 (42.86 percent) microorganisms from the cloaca; 15 (32.61 percent), 3 (7.14 percent) and 25 (51.02 percent) microorganisms from the oral cavity; and, 2 (4.35 percent), 1 (2.38 percent) and 3 (6.12 percent) microorganisms from venom. The most frequent bacteria were Pseudomonas aeruginosa, Proteus vulgaris and Morganella morganii, with sensitivity to amikacin, gentamicin, norfloxacin, sulfazotrin and tobramycin. Snakes kept in semi-open captivity exhibited the fewest microorganisms in oral cavities, perhaps due to the environment in captivity, with different temperature gradients, running water, absence of daily handling, circulating air, possibility of moving around, daily cleaning, and sunlight access.

Este estudo avaliou e comparou a microflora aeróbica da cavidade oral, cloaca e veneno de serpentes Crotalus durissus terrificus recém-capturadas da natureza e mantidas sob quarentena (WQ), mantidas em cativeiro coletivo (CC) e em cativeiro individual (IC). A eficácia de drogas antimicrobianas de agentes isolados foi também avaliada. Foram isolados microorganismos dos grupos I, II e III respectivamente: 29 (63.04 por cento), 38 (90.48 por cento) e 21 (42.86 por cento) da cloaca; 15 (32.61 por cento), 3 (7.14 por cento) e 25 (51.02 por cento) da cavidade oral, e finalmente 2 (4.35 por cento), 1 (2.38 por cento) e 3 (6.12 por cento) do veneno. As bactérias mais frequentes foram Pseudomonas aeruginosa, Proteus vulgaris e Morganella morganii, com sensibilidade para amikacina, gentamicina, norfloxacina, sulfazotrina e tobramicina. Serpentes mantidas no cativeiro semi-aberto mostraram menor número de agentes infecciosos em cavidade oral, talvez devido ao ambiente de cativeiro com diferentes gradientes de temperatura, água corrente, ausência de manejo diário, ampla circulação de ar, possibilidade de movimentação pelos animais, limpeza diária e acesso ao Sol.

Salmonella surveillance in a collection of rattlesnakes (Crotalus spp.).

Over the past 15 yr, Salmonella enterica ssp. arizonae (IIIa) 56:z4,z23:- has repeatedly been isolated from individual Crotalus willardi rattlesnakes with progressively debilitating osteomyelitis at the Knoxville Zoological Gardens. In April 2004, the serotype was linked with a fatal case of septicemia in another Crotalus species in this collection. Although the association of IIIa 56:z4,z23:- with disease in this colony of C. willardi is well established, prior disease or isolation of this serotype outside of the C. willardi colony had not been documented previously, and the serotype's distribution throughout the remainder of the Crotalus collection had yet to be determined. Forty-one fecal samples were obtained from each individual (n = 36) or exhibit group (n = 5) of crotalid snakes, representing nine species, housed at the zoo. Salmonella spp. were isolated from every sample, with 21 different serotypes. The 21 serotypes were distributed among S. enterica ssp. I (24%), IIIa (9%), and IIIb (67%). Although not recovered in the primary study, S. arizonae 56:z4,z23:- was recovered from additional samples taken from two C. willardi willardi. Although the overall recovery rate of this serotype from feces has been low, it seems that its distribution among the Crotalus collection at Knoxville Zoological Gardens remains largely restricted to the C. willardi species.

Comparison of phenotypic traits and genetic relatedness of Salmonella enterica subspecies arizonae isolates from a colony of ridgenose rattlesnakes with osteomyelitis.

Reptiles are well-known sources of human Salmonella infections; however, little is known about the ability of Salmonella to cause disease in reptiles. Thirty-seven isolates of Salmonella enterica subspecies arizonae (S. arizonae) were obtained from retrospective and prospective studies of a closed colony of ridgenose rattlesnakes (Crotalus willardi) with osteomyelitis. All isolates (N = 7) from bone lesions were of a single serotype, 56:z4,z23, and this serotype was found on only 1 occasion among 8 other serotypes isolated from 21 cloacal and intestinal samples. The remainder (N = 7) of serotype 56:z4,z23 isolates were from other extraintestinal sites, including liver, ovary, blood, and testis. S. arizonae isolates were susceptible to most antimicrobials, and plasmid profiles did not correlate with serotype or antimicrobial resistance. Isolates of the 56:z4,z23 serotype (N = 14) formed a tight cluster with 95% similarity by XbaI macrorestriction analysis. Individual isolates of serotypes, 56:z4,z23, 38:(k)-z35, and 48:i-z invaded HeLa cells but an isolate of serotype 50:r-z did not. The same individual isolates of serotype 56:z4,z23 and 48:i-z also invaded viper heart cells. The Salmonella InvA gene was detected by polymerase chain reaction (PCR) in all S. arizonae serotypes tested, including 5 serotype 56:z4,z23 isolates and individual isolates of serotypes 48:i-z and 50:r-z. A source or possible explanation for increased virulence of S. arizonae serotype 56:z4,z23 in this unique host has not been found.

A new serovar and a new serological variant belonging to Salmonella enterica subspecies diarizonae.

Description of a new serovar (S. IIIb 16:k:e,n,x,z15) and a new serological variant (S. IIIb 42:z10:e,n,x,z15:z60 ) belonging to the genus Salmonella isolated from stool specimens of Brazilian snakes (Crotalus durissus).