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.

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).

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.

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.

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.

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.

[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.

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.

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.

Identification of Chlamydophila pneumoniae in an emerald tree boa, Corallus caninus.

Tissues were evaluated from emerald tree boas, Corallus caninus, from a collection in which chlamydiosis was diagnosed. To determine the strain of chlamydia infecting these snakes, tissue samples from 5 frozen snakes were tested by a quantitative TaqMan polymerase chain reaction (PCR) test and a PCR sequence analysis test. Of the 22 samples tested, 9 were categorized as either positive or weakly positive with the TaqMan test, and 6 yielded an amplicon using a serial PCR test that amplified a portion of the 23S ribosomal RNA gene. A PCR product suitable for sequencing was obtained from the heart of one of the snakes. Sequence analysis showed that the snake had been infected with Chlamydophila pneumoniae. These findings show that C. pneumoniae can infect emerald tree boas, broadening the range of reptiles known to be infected by this primarily human pathogen.

Immunohistochemical staining of chlamydial antigen in emerald tree boas (Corallus caninus).

Of 120 privately owned captive-bred and wild-collected emerald tree boas (ETBs) (Corallus caninus), 97 died or were euthanatized. Eighteen snakes were necropsied, and tissues were collected from all major organs and processed for light microscopy. Histologic examination demonstrated histiocytic granulomas in the small intestine, heart, and esophageal tonsils of one ETB, small intestine of a second ETB, and in an esophageal tonsil of a third ETB. Within the center of these granulomas, small, basophilic, punctate organisms were demonstrated using hematoxylin and eosin staining. Transmission electron microscopic examination of an intestinal granuloma demonstrated developmental stages of organisms consistent with members of the family Chlamydiaceae. An immunoperoxidase staining technique and 2 different commercially available monoclonal antibodies against chlamydial lipopolysaccharide antigen was used to identify chlamydial antigen in these lesions. Liver of a puff adder (Bitis arietans) with previously reported systemic chlamydiosis served as the positive control. Both monoclonal antibodies stained antigen in these granulomas. Additionally, macrophages within aggregates of lymphoplasmacytic cells in the colon, small intestine, and esophageal tonsils of 3 other ETBs contained antigen. Although both antibodies labeled antigen in serial sections of tissue, a difference in staining intensity was noted.

A novel Mycoplasma sp. associated with proliferative tracheitis and pneumonia in a Burmese python (Python molurus bivittatus).

Proliferative lymphocytic tracheitis and pneumonia were observed histologically in the respiratory tract of a captive Burmese python (Python molurus bivittatus). A mycoplasma species was isolated from the respiratory tissue. Polymerase chain reaction analysis of the 16S rRNA gene sequence of the isolate showed 0.90 similarity to Mycoplasma agassizii, an organism previously shown to cause respiratory disease in reptiles. Based on these findings, a novel Mycoplasma species was suspected to be the causative agent of respiratory disease in this snake.

Laboratory reference values for a group of captive Ball Pythons (Python regius)

OBJECTIVE: Laboratory reference values, including hematologic and serum biochemical variables, and oropharyngeal bacteria flora, were determined in a group of captive Ball Pythons (Python regius). ANIMALS: 20 adult Ball Pythons, weighing between 700 and 1,510 g, were allowed to acclimate at the recommended temperature range for the species (25 C night-time, up to 30 C daytime), then were evaluated for internal parasites and treated with appropriate medication prior to the start of the study. PROCEDURE: Hematologic values determined included WBC, hemoglobin, hematocrit, plasma protein, and differential cell count. Clinical biochemical analysis included determination of glucose, uric acid, calcium, phosphorus, total protein, alanine transaminase, alkaline phosphatase, and aspartate transaminase values. In addition to blood values, oropharyngeal swab specimens of the mouth were submitted for culture to determine the species of bacteria found in this population. Descriptive statistics were calculated for each hematologic and clinical biochemical value. Mean, SEM, and ranges were calculated. RESULTS: Hematologic values were similar to those reported in other snake species, except the hematocrit, which was lower. Clinical biochemical values different from those of other species were alkaline phosphatase activity, which was lower, and calcium and phosphorus concentrations, which were lower than values in other species. Bacteria isolated from the oropharynx were principally gram-negative organisms. CONCLUSION: Reference intervals reported in this study are important for establishing a database for comparative studies of Ball Pythons in other locations and under different husbandry conditions. CLINICAL RELEVANCE: Accumulated laboratory reference values will assist veterinarians in assessing the health status of Ball Pythons.

Pulmonary mycobacteriosis caused by Mycobacterium haemophilum and M. marinum in a royal python.

An adult female royal python was referred with an 18-month history of chronic respiratory tract disease. Anemia and moderate leukocytosis with heterophilia and monocytosis were detected and interpreted as evidence of a chronic inflammatory condition. Evaluation of lateral and dorsoventral radiographic views revealed multiple soft-tissue opacities within the cranial lung fields. Endoscopic evaluation revealed that the normal reticulated pattern on the surface of the lung had been largely replaced by diffuse, granulomatous tissue. Histologic examination of biopsy specimens revealed classic pyogranulomas. Ziehl-Neelsen stains revealed numerous acid-fast bacilli consistent with Mycobacterium spp. Molecular methods including polymerase chain reaction restriction assays and DNA sequencing confirmed the identification of M. haemophilum and M. marinum. The snake was euthanatized. Mycobacteriosis is an uncommon and sporadic pyogranulomatous disease of reptiles. In most cases of reptile mycobacteriosis, treatment is not advised because of the chronic nature and often advanced stage of the disease, long-term and expensive nature of potential treatment regimens, and the risk of spread to other animals, including humans.

An epizootic of chronic regurgitation associated with chlamydophilosis in recently imported emerald tree boas (Corallus caninus).

One hundred and five wild-caught emerald tree boas (Corallus caninus) were added to a collection of 15 others. in Central Florida, during a 4-mo period. Eighty-one boas (67%) developed repetitive regurgitation during the 23-mo period after the initial introduction, and 61 (75%) of these died. Regurgitation occurred 3-4 days after feeding. Prevalence of regurgitation in this population of snakes was 25%/mo (range 0-42%), and incidence was 3.52/mo (range 0-13/mo). The cumulative mortality for those boas developing repetitive regurgitation (61 of 120) during the 23-mo epizootic was 51%. Hematologic findings included anemia and leukocytosis, with lymphocytosis, monocytosis, and azurophilia. Histologic evaluation of the gastrointestinal tract showed multifocal to diffuse lymphoplasmacytic inflammation with granuloma formation and positive immunohistochemical staining for chlamydial antigen. Electron microscopic evaluation of granulomas showed organisms consistent with Chlamydophila sp.

Cutaneous and pulmonary mycosis in green anacondas (Euncectes murinus).

Two dead, captive green anacondas (Eunectes murinus), including one male and one female, submitted for necropsy were in poor body condition, having multiple, scattered, dark red foci on the scales and mottled lungs. Both snakes had severe mycotic dermatitis. In addition, the male snake had mycotic stomatitis, and the female snake had mycotic pneumonia. Trichophyton sp., Verticillium sp., and Alternaria sp. were isolated from the dermal lesions. The pulmonary lesions were morphologically consistent with Aspergillus sp. Bacterial organisms isolated from skin and internal organs included Chryseobacterium meningosepticum, Stenotrophomonas maltophilia, Aeromonas hydrophila, and Providencia rettgeri. Mycotic diseases can be devastating to reptiles, and suboptimal husbandry and captivity were likely the predisposing factors that led to opportunistic invasion in these snakes.