Studies on Trueperella pyogenes isolated from an okapi (Okapia johnstoni) and a royal python (Python regius).

BACKGROUND: The present study was designed to characterize phenotypically and genotypically two Trueperella pyogenes strains isolated from an okapi (Okapia johnstoni) and a royal python (Python regius). CASE PRESENTATION: The species identity could be confirmed by phenotypic properties, by MALDI-TOF MS analysis and by detection of T. pyogenes chaperonin-encoding gene cpn60 with a previously developed loop-mediated isothermal amplification (LAMP) assay. Furthermore, sequencing of the 16S ribosomal RNA (rRNA) gene, the 16S-23S rDNA intergenic spacer region (ISR), the target genes rpoB encoding the ß-subunit of bacterial RNA polymerase, tuf encoding elongation factor tu and plo encoding the putative virulence factor pyolysin allowed the identification of both T. pyogenes isolates at species level. CONCLUSIONS: Both strains could be clearly identified as T. pyogenes. The T. pyogenes strain isolated in high number from the vaginal discharge of an okapi seems to be of importance for the infectious process; the T. pyogenes strain from the royal python could be isolated from an apparently non-infectious process. However, both strains represent the first isolation of T. pyogenes from these animal species.

Leptospira interrogans in wild Boa constrictor snakes from Northeast Brazil peri­urban rainforest fragments.

Leptospirosis, a disease that occurs worldwide, especially in tropical regions, is caused by bacteria of the genus Leptospira and affects mammals, amphibians, and reptiles. Boa constrictor snakes are commonly found in Atlantic rainforest fragments in peri­urban areas, which indicates a greater possibility of the contact of these animals with humans residing there. Therefore, the aim of this work was to detect Leptospira spp infection through molecular assays in wild B. constrictor snakes rescued in peri­urban areas and verify seroreactivity, by the microscopic agglutination test (MAT), as well as the most common serogroups. Among the 46 samples tested, 7 (15.21%) were positive according to PCR and confirmed as Leptospira interrogans through secY gene sequencing. In MAT, 37 (80.43%) of the 46 samples were classified as reactive. Panama was the serogroup with the highest occurrence. The results showed the presence of Leptospira spp DNA in asymptomatic snakes rescued in rainforest fragments located in peri­urban areas and support further investigations on the influence of these animals in the epidemiology of leptospirosis in tropical peri­urban areas.

Molecular detection of reptile-associated Borrelia in Boa constrictor (Squamata: Boidae) from Veracruz, Mexico.

The reptile-associated Borrelia represent a monophyletic group of bacteria transmitted by several species of hard ticks, which has been reported to only infect amphibians and reptiles in Eurasia and Middle East, however, this bacterial group has not been studied in North America. The aim of this study was to assess the presence of Borrelia spirochetes in blood samples of native reptiles of Mexico. Blood samples were directly obtained from individuals, DNA extractions were performed using Chelex-100. The Borrelia detection was performed by conventional PCR. From 102 reptiles tested, only five individuals of Boa constrictor were positive for the presence of DNA of the reptile-associated Borrelia group. Supported by phylogenetic analysis, this study presents the first record of these spirochetes group in Mexico, and initial evidence of B. constrictor as a host of this group.

Cutaneous mycobiota of boid snakes kept in captivity / Microbiota cutânea de serpentes Boidae mantidas em cativeiro

Boids are large, constrictor snakes that feed mostly on mammals, reptiles, and birds. These animals are commonly raised as pets, and their improper handling can favor the emergence of fungal infections, which can lead to dermatological diseases that are undiagnosed in nature. Here, we isolate and identify the filamentous fungi that compose the mycobiota of the scales of boid snakes kept in captivity at the Biological Museum of the Butantan Institute. Thirty individuals of four species were evaluated: four Eunectes murinus, twelve Boa constrictor constrictor, seven Corallus hortulanus, and seven Epicrates crassus. Microbiological samples were collected by rubbing small square carpets on the snake scales. We isolated five genera of fungi: Penicillium sp. (30%), Aspergillus sp. (25%), Mucor sp. (25%), Acremonium sp. (10%), and Scopulariopsis sp. (10%). Approximately half of the snakes evaluated had filamentous fungi on the scales, but only 12% of the individuals were colonized by more than one fungal genus. We found no dermatophytes in the evaluated species. Our results provide an overview of the fungal mycobiota of the population of boids kept in the Biological Museum, allowing the identification of possible pathogens.(AU)

Boidae são serpentes grandes e constritoras, que comem normalmente mamíferos, répteis e aves. São comumente criadas como animais de estimação, e seu manuseio inadequado pode favorecer infecções fúngicas emergentes, as quais podem causar doenças dermatológicas que não são diagnosticadas na natureza. Foram isolados e identificados os fungos filamentosos da microbiota das escamas das serpentes Boidae mantidas em cativeiro no Museu Biológico do Instituto Butantan. Trinta indivíduos de quatro espécies foram avaliados: quatro Eunectes murinus, 12 Boa constrictor constrictor, sete Corallus hortulanus e sete Epicrates crassus. As amostras microbiológicas foram coletadas por fricção, em pequenos quadrados de carpete das escamas das serpentes. Isolaram-se cinco gêneros de fungos: Penicillium sp. (30%), Aspergillus sp. (25%), Mucor sp. (25%), Acremonium sp. (10%) e Scopulariopsis sp. (10%). Aproximadamente metade das serpentes avaliadas tinham fungos filamentosos nas escamas e apenas 12% dos indivíduos foram colonizados por mais de um gênero. Não foram encontrados dermatófitos nas espécies avaliadas. Os resultados mostraram uma visão geral da microbiota fúngica da população de boídeos mantidos no Museu Biológico, o que permitiu a identificação de possíveis patógenos.(AU)

Proposal of Lysobacter pythonis sp. nov. isolated from royal pythons (Python regius).

The bacterial strains 4284/11T and 812/17 isolated from the respiratory tract of two royal pythons in 2011 and 2017, respectively were subjected to taxonomic characterization. The 16S rRNA gene sequences of the two strains were identical and showed highest sequence similarities to Lysobacter tolerans UM1T (97.2%) and Luteimonas aestuarii DSM 19680T (96.7 %). The two strains were identical in the sequences of the 16S-23S rRNA internal transcribed spacer (ITS) and partial groEL gene sequences and almost identical in genomic fingerprints. In the ITS sequence Ly. tolerans DSM 28473T and in the groEL nucleotide sequence Luteimonas mephitis DSM 12574T showed the highest similarity. In silico DDH analyses using genome sequence based ANIb and gANI similarity coefficients demonstrated that strain 4284/11T represents a novel species and revealed Ly. tolerans UM1T as the next relative (ANIb = 76.2 %, gANI = 78.0 %). Based on the topology of a core gene phylogeny strain 4284/11T could be assigned to the genus Lysobacter. Chemotaxonomic characteristics including polyamine pattern, quinone system, polar lipid profile and fatty acid profile were in accordance with the characteristics of the genera Lysobacter and Luteimonas. Strains 4284/11T and 812/17 could be differentiated from the type strains of the most closely related species by several physiological tests. In conclusion we are here proposing the novel species Lysobacter pythonis sp. nov. The type strain is 4284/11T (= CCM 8829T = CCUG 72164T = LMG 30630T) and strain 812/17 (CCM 8830) is a second strain of this species.

Pulmonary fungal granulomas and fibrinous pneumonia caused by different hypocrealean fungi in reptiles.

In contrast to fungal dermatitis, fungal glossitis and disseminated visceral mycosis, fungal infection of the lung has so far rarely been described in reptiles. Pulmonary fungal granulomas were diagnosed histopathologically within the scope of post mortem examinations. Fragments of the 18S-internal transcribed spacer1-5.8S rDNA (SSU-ITS1-5.8S) and 28S rDNA (LSU), including domains (D)1 and D2 as well as the protein coding gene translation elongation factor 1 alpha (TEF) were used for phylogenetical analysis after isolation of the fungal pathogen by culturing. Ten reptiles, including lizards (n = 6), snakes (n = 1), crocodilians (n = 2) and tortoises (n = 1) presented with pulmonary fungal granulomas (n = 8) and fibrinous pneumonia (n = 2) caused by different non-clavicipitaceous and clavicipitaceous species of the order Hypocreales. Purpureocillium lavendulum (n = 2) and Metarhizium robertsii (n = 1) as the etiologic agents of pneumonia in reptile species are described for the first time. Fungal pulmonary granulomas caused by clavicipitaceous fungi (n = 6) were all associated with disseminated visceral mycosis as well as oral fungal granulomas (n = 4) and/or fungal dermatitis (n = 1). Differing infection routes being likely for clavicipitaceous and non-clavicipitaceous fungal pathogens. A potential zoonotic health risk should be taken into account during necropsy or lung sampling in live reptiles with pulmonary fungal granulomas, since human infections, mainly keratitis and sclerokeratitis, caused by Beauveria bassiana, Metarhizium robertsii and Purpureocillium lilacinum, have occasionally been described.

[Successful treatment of a necrotizing, multi-resistant bacterial pyoderma in a python with cold plasma therapy]. / Erfolgreiche Behandlung einer nekrotisierenden, multiresistenten bakteriellen Pyodermie bei einem Python mittels Kaltplasmatherapie.

A 4-year-old ball python was presented 3 weeks after multiple bite wounds from a prey rat with large skin lesions, a concurrent deep bacterial pyoderma and clinical signs for septicemia, including neurolo -gical symptoms. Affected tissue separated from the underlying muscular layer revealing parts of the muscles. Clinical examination and cyto -logy was consistent with bacterial pyoderma; septicemia was an additional tentative clinical diagnosis. Empirical lincomycin and marbo -floxacin (bacterial culture revealed a multi-resistant Stenotrophomonas maltophilia susceptible to fluoroquinolones) treatment improved the patient's general condition but skin wounds deteriorated to multifocal eschars with intracellular rods. Further diagnostics were limited for financial reasons, euthanasia was considered. Cold atmospheric pressure plasma (CAPP) therapy was performed six times in 4 weeks. Within 1 week, inflammatory symptoms resolved. Re-epithelialization was completed few weeks later. In the following year, the snake sloughed three times without any signs of dysecdysis. CAPP therapy may offer a viable treatment option for bacterial (especially multiresistant) pyoderma and necrotizing dermatitis in snakes.

Isolation and identification of bacterial populations of zoonotic importance from captive non-venomous snakes in Malaysia.

AIM: Captivity of non-venomous snakes such as python and boa are common in zoos, aquariums and as pets in households. Poor captivity conditions expose these reptiles to numerous pathogens which may result in disease conditions. The purpose of this study was to investigate the common bacteria isolated from necropsied captive snakes in Malaysia over a five year period. MATERIALS AND METHODS: A total of 27 snake carcasses presented for necropsy at the Universiti Putra Malaysia (UPM) were used in this survey. Samples were aseptically obtained at necropsy from different organs/tissues (lung, liver, heart, kindey, oesophagus, lymph node, stomach, spinal cord, spleen, intestine) and cultured onto 5% blood and McConkey agar, respectively. Gram staining, morphological evaluation and biochemical test such as oxidase, catalase and coagulase were used to tentatively identify the presumptive bacterial isolates. RESULTS: Pythons had the highest number of cases (81.3%) followed by anaconda (14.8%) and boa (3.7%). Mixed infection accounted for 81.5% in all snakes and was highest in pythons (63%). However, single infection was only observed in pythons (18.5%). A total of 82.7%, 95.4% and 100% of the bacterial isolates from python, anaconda and boa, respectively were gram negative. Aeromonas spp was the most frequently isolated bacteria in pythons and anaconda with incidences of 25 (18%) and 8 (36.6%) with no difference (p > 0.05) in incidence, respectively, while Salmonella spp was the most frequently isolated in boa and significantly higher (p < 0.05) than in python and anaconda. Bacteria species were most frequently isolated from the kidney of pythons 35 (25.2%), intestines of anacondas 11 (50%) and stomach of boa 3 (30%). CONCLUSION: This study showed that captive pythons harbored more bacterial species than anaconda or boa. Most of the bacterial species isolated from these snakes have public health importance and have been incriminated in human infections worldwide.

Akkermansia glycaniphila sp. nov., an anaerobic mucin-degrading bacterium isolated from reticulated python faeces.

A Gram-stain-negative, non-motile, strictly anaerobic, oval-shaped, non-spore-forming bacterium (strain PytT) was isolated from reticulated python faeces. Strain PytT was capable of using mucin as sole carbon, energy and nitrogen source. Cells could grow singly, in pairs, and were also found to aggregate. Scanning electron microscopy revealed the presence of filamentous structures connecting individual bacterial cells. Strain PytT could grow on a limited number of single sugars, including N-acetylglucosamine, N-acetylgalactosamine, glucose, lactose and galactose, but only when a plentiful protein source was provided. Phylogenetic analysis based on 16S rRNA gene sequencing showed strain PytT to belong to the Verrucomicrobiae class I, family Akkermansiaceae, genus Akkermansia, with Akkermansia muciniphila MucT as the closest relative (94.4 % sequence similarity). DNA-DNA hybridization revealed low relatedness of 28.3 % with A. muciniphila MucT. The G+C content of DNA from strain PytT was 58.2 mol%. The average nucleotide identity (ANI) of the genome of strain PytT compared to the genome of strain MucT was 79.7 %. Chemotaxonomic data supported the affiliation of strain PytT to the genus Akkermansia. Based on phenotypic, phylogenetic and genetic characteristics, strain PytT represents a novel species of the genus Akkermansia, for which the name Akkermansia glycaniphila sp. nov. is proposed. The type strain is PytT (=DSM 100705T=CIP 110913T).

Molecular evidence of Chlamydophila pneumoniae infection in reptiles in Argentina.

In the central area of Argentina, the epidemiological and molecular characteristics of Chlamydophila pneumoniae infections in reptiles are still unknown. A nested polymerase chain reaction of the rpoB gene was used to detect C. pneumoniae in cloacal swab samples from 19 reptiles at a recreational area. Eleven (57.89%) reptiles were positive; the sequencing and phylogenetic analysis confirmed the presence of this bacterium. Neither C. pneumoniae DNA in the caregivers pharynges nor IgM antibodies anti-C. pneumoniae in their serum samples were detected; however, caregivers presented very high titers of IgG anti-C. pneumoniae. The detection of C. pneumoniae DNA in reptiles demonstrated the circulation of this agent in the recreational area and could be responsible for the exacerbated immune response of the personnel handling the reptiles, which suggests a potential zoonotic cycle. This is the first report of the detection of C. pneumoniae in reptiles in Argentina.

Oral flora of Python regius kept as pets.

UNLABELLED: This study was aimed at evaluating the oral bacterial flora of 60 Python regius kept as pets by culture and biochemical methods. All isolates were also submitted to antimicrobial susceptibility testing using the disc diffusion method. The oral cavity of snakes sampled harboured a wide range of Gram-negative bacteria mainly constituted by Pseudomonas spp., Morganella morganii, Acinetobacter calcoaceticus, Aeromonas hydrophila, but also by Salmonella spp. Staphylococcus spp. was the commonest Gram-positive isolates, and various anaerobic Clostridium species were also found. The most effective antimicrobial agents were enrofloxacin and ciprofloxacin, followed by doxycycline and gentamicin. SIGNIFICANCE AND IMPACT OF THE STUDY: The oral cavity of snakes sampled harboured a wide range of bacteria. Our results suggest that people who come in contact with snakes could be at risk of infection and should follow proper hygiene practices when handling these reptiles.

Molecular evidence of Chlamydophila pneumoniae infection in reptiles in Argentina. / Molecular evidence of Chlamydophila pneumoniae infection in reptiles in Argentina.

In the central area of Argentina, the epidemiological and molecular characteristics of Chlamydophila pneumoniae infections in reptiles are still unknown. A nested polymerase chain reaction of the rpoB gene was used to detect C. pneumoniae in cloacal swab samples from 19 reptiles at a recreational area. Eleven (57.89

) reptiles were positive; the sequencing and phylogenetic analysis confirmed the presence of this bacterium. Neither C. pneumoniae DNA in the caregivers pharynges nor IgM antibodies anti-C. pneumoniae in their serum samples were detected; however, caregivers presented very high titers of IgG anti-C. pneumoniae. The detection of C. pneumoniae DNA in reptiles demonstrated the circulation of this agent in the recreational area and could be responsible for the exacerbated immune response of the personnel handling the reptiles, which suggests a potential zoonotic cycle. This is the first report of the detection of C. pneumoniae in reptiles in Argentina.

Myiasis by Megaselia scalaris (Diptera: Phoridae) in a python affected by pulmonitis.

Myiases are caused by the presence of maggots in vertebrate tissues and organs. Myiases have been studied widely in humans, farm animals, and pets, whereas reports of myiasis in reptiles are scarce. We describe a case of myiasis caused by the Megaselia scalaris (Loew) in an Indian python (Python molurus bivittatus, Kuhl) (Ophida: Boidae). The python, 15 yr old, born and reared in a terrarium in the mainland of Venice (Italy), was affected by diffuse, purulent pneumonia caused by Burkholderia cepacia. The severe infestation of maggots found in the lungs during an autopsy indicated at a myiasis.

Detection of pathogens in Boidae and Pythonidae with and without respiratory disease.

Respiratory diseases in boid snakes are common in captivity, but little information is available on their aetiology. This study was carried out to determine the occurrence of lung associated pathogens in boid snakes with and without respiratory signs and/or pneumonia. In total, 80 boid snakes of the families Boidae (n = 30) and Pythonidae (n = 50) from 48 private and zoo collections were included in this survey. Husbandry conditions were evaluated using a detailed questionnaire. All snakes were examined clinically and grouped into snakes with or without respiratory signs. Tracheal wash samples from all snakes were examined bacteriologically as well as virologically. All snakes were euthanased, and a complete pathological examination was performed. Respiratory signs and pneumonia were detected more often in pythons than in boas. An acute catarrhal pneumonia was diagnosed more often in snakes without respiratory signs than in snakes with respiratory signs, which revealed fibrinous and fibrous pneumonia. Poor husbandry conditions are an important trigger for the development of respiratory signs and pneumonia. Different bacterial pathogens were isolated in almost all snakes with pneumonia, with Salmonella species being the most common. Ferlavirus (formerly known as ophidian paramyxovirus)-RNA was detected only in pythons. Inclusion body disease was rarely seen in pythons but often in boas. Adenovirus and Mycoplasma were other pathogens that were diagnosed in single snakes with pneumonia. In living boid snakes with respiratory signs, tracheal wash samples were found to be a useful diagnostic tool for the detection of viral and bacterial pathogens.

Postprandial remodeling of the gut microbiota in Burmese pythons.

The vertebrate gut microbiota evolved in an environment typified by periodic fluctuations in nutrient availability, yet little is known about its responses to host feeding and fasting. As many model species (for example, mice) are adapted to lifestyles of frequent small meals, we turned to the Burmese python, a sit-and-wait foraging snake that consumes large prey at long intervals (>1 month), to examine the effects of a dynamic nutrient milieu on the gut microbiota. We used multiplexed 16S rRNA gene pyrosequencing to characterize bacterial communities harvested from the intestines of fasted and digesting snakes, and from their rodent meal. In this unprecedented survey of a reptilian host, we found that Bacteroidetes and Firmicutes numerically dominated the python gut. In the large intestine, fasting was associated with increased abundances of the genera Bacteroides, Rikenella, Synergistes and Akkermansia, and with reduced overall diversity. A marked postprandial shift in bacterial community configuration occurred. Between 12 h and 3 days after feeding, Firmicutes, including the taxa Clostridium, Lactobacillus and Peptostreptococcaceae, gradually outnumbered the fasting-dominant Bacteroidetes, and overall 'species'-level diversity increased significantly. Most lineages seemed to be indigenous to the python rather than ingested with the meal, but a dietary source of Lactobacillus could not be ruled out. Thus, the observed large-scale alterations of the gut microbiota that accompany the Burmese python's own dramatic physiological and morphological changes during feeding and fasting emphasize the need to consider both microbial and host cellular responses to nutrient flux. The Burmese python may provide a unique model for dissecting these interrelationships.

Septicaemia secondary to infection by Corynebacterium macginleyi in an Indian python (Python molurus).

A seven-year-old female Indian python (Python molurus) weighing about 35kg was euthanased after several clinical episodes of stomatitis, pneumonia, ophthalmitis and dystocia over a period of four years. The animal had been maintained in a terrarium in a circus truck at an adequate temperature. During shows, however, the snake was considered to be exposed to stressful conditions for several hours at a time at low temperatures and with noise and bright lights. A post-mortem examination indicated ulcerative stomatitis, osteomyelitis, severe pneumonia and numerous granulomata and multifocal necrosis in stomach and spleen. Corynebacterium macginleyi was isolated in pure culture from the ulcerative stomatitis, and mixed with Stenotrophomonas maltophilia from the lungs and spleen. The findings indicated that the snake had died from a septicaemic process caused by C. macginleyi, probably originating from the stomatitis. The role of S. maltophilia as a secondary agent is discussed. The stress of the circus show and poor husbandry may have predisposed the animal to infection and septicaemia. This is the first report of C. macginleyi causing disease in a snake.

An ecological risk assessment of nonnative boas and pythons as potentially invasive species in the United States.

The growing international trade in live wildlife has the potential to result in continuing establishment of nonnative animal populations in the United States. Snakes may pose particularly high risks as potentially invasive species, as exemplified by the decimation of Guam's vertebrate fauna by the accidentally introduced brown tree snake. Herein, ecological and commercial predictors of the likelihood of establishment of invasive populations were used to model risk associated with legal commercial imports of 23 species of boas, pythons, and relatives into the United States during the period 1989-2000. Data on ecological variables were collected from multiple sources, while data on commercial variables were collated from import records maintained by the U.S. Fish and Wildlife Service. Results of the risk-assessment models indicate that species including boa constrictors (Boa constrictor), ball pythons (Python regius), and reticulated pythons (P. reticulatus) may pose particularly high risks as potentially invasive species. Recommendations for reducing risk of establishment of invasive populations of snakes and/or pathogens include temporary quarantine of imports to increase detection rates of nonnative pathogens, increasing research attention to reptile pathogens, reducing the risk that nonnative snakes will reach certain areas with high numbers of federally listed species (such as the Florida Keys), and attempting to better educate individuals purchasing reptiles.