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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 (nâ[euro][per thousand]=â[euro][per thousand]30) and Pythonidae (nâ[euro][per thousand]=â[euro][per thousand]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.
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Introduction
Respiratory diseases in boid snakes are common in captivity, and often take a long time to develop before clinical signs are evident ( Chitty 2004 ). Deficits in husbandry conditions or primary viral infections especially with ferlaviruses (formerly known as ophidian paramyxoviruses) or inclusion body disease (IBD) are common triggers of bacterially induced pneumonia in boid snakes (Kurath and others 2004, Marschang and others 2009, Pees and others 2010). Gram-negative aerobic bacteria appear to be more commonly associated with pneumonia than other bacteria ( Mayer and Frank 1974 , Jacobsen 1984 ). Species relating to Pseudomonas , Acinetobacter , Aeromonas , Alcaligenes , Bacillus , Bordetella , Citrobacter freundii , Corynebacterium , Enterobacter , Escherichia coli , Flavobacterium , Hafnia alvei , Klebsiella , Micrococcus , Proteus , Providencia , Salmonella , Shigella , Stenotrophomonas and coagulase-negative Staphylococcus , have frequently been isolated from the oral cavities and from glottal swabs of healthy snakes (Goldstein and others 1979, Goldstein and others 1981, Hilf and others 1990, Johnson and others 1996, Miller and others 2004, Hejnar and others 2007, Pees and others 2010). Reports of Mycobacterium , Mycoplasma and Chlamydia species, or fungi-induced pneumonia of boid snakes are rare ( Olson and Woodard 1974 , Penner and others 1997, Hernandez-Divers and Shearer 2002 , Miller and others 2004, Soldati and others 2004). The aim of this study was to compare the prevalence of infectious agents isolated from a tracheal wash sample in living boid snakes suffering from respiratory disease, and that which revealed pneumonia postmortem, with that found in boid snakes without respiratory signs. Furthermore, findings should help to clarify the pathogenic relevance of bacterial and virological findings from tracheal wash samples, as one-third of the snakes in a previous study revealed remarkable bacterial findings in these samples without a significant correlation to respiratory signs.
Material and methods
Snakes
In total, 80 boid snakes of seven different species including the family Boidae (n = 30), as well as the family Pythonidae (n = 50) were examined (Table 1 ). The snakes originated from 48 private and zoo collections throughout Germany in order to minimise the influence of regional differences and connections between the collections. Most of the collections consisted of one species within one family, whereas in one collection, species of both families were kept together. Owners agreed to complete a detailed questionnaire, which had been previously established (Pees and others 2010). In 20 of 48 collections, the local conditions and the health status of the collection, especially the occurrence of viral pathogens, was known from previous examination (Pees and others 2010). In those cases, snakes were submitted for follow-up examinations, and four owners decided to cull their collections, as they had recurrent respiratory or central nervous disease problems in their collections caused by IBD or ferlavirus infections. The remaining snakes were submitted alive for pathological examination to determine the health status of the collections.
Species | Numbers of individuals | Clinical signs | ||
Boa constrictor | 30 | 13 (2/2)* | 14 (3/3) | 3 (1/3) |
Ball python (Python regius ) | 12 | 0 | 10 (4/2) | 2 (1/2) |
Burmese python (Python molurus ) | 26 | 8 (0/1) | 5 (1/3) | 13 (9/10) |
Carpet python (Morelia spilota ) | 4 | 0 | 3 (1/1) | 1 (1/1) |
Green tree python (Morelia viridis ) | 6 | 0 | 1 (0/1) | 5 (3/5) |
Python brongersmai | 1 | 0 | 1 (0/1) | 0 |
Reticulated python (Python reticulates) | 1 | 0 | 0 | 1 (0/1) |
All | 80 | 21 (2/3) | 34 (9/11) | 25 (15[dagger]/22[double dagger]) |
*Number of snakes with remarkable husbandry conditions/number of snakes with pneumonia
[dagger]P < 0.001
[double dagger]P < 0.001
A thorough clinical examination was conducted on each individual snake (Pees and others 2010). Respiratory signs were defined as gasping and cloudy tracheal wash samples with white to yellow clumps. According to the clinical findings, snakes were grouped in three categories: snakes with no clinical signs, snakes with clinical signs other than respiratory signs and snakes with respiratory signs (Table 1 ). Snakes with different health status from inside one collection were found in five collections.
Snakes were euthanased by decapitation after initial anaesthesia by intramuscular injection with ketamine (80 mg/kg body weight) and diazepam (10 mg/kg body weight). Necropsy was performed according to standard procedures ( Garner 2006 ). Samples of the contents of the lung, small and large intestines were collected immediately after opening of the carcase and examined microscopically for parasites at 200x and 400x magnification. Stained impression smears (DiffQuik; Dade Behring, Marburg, Germany) prepared from liver, spleen, pancreas, lung and small and large intestines were examined microscopically at 1000x magnification. Tissue samples were fixed in 4.5 per cent phosphate-buffered formalin, embedded in paraffin, sectioned at 4 [proportional, variant]m and stained with haematoxylin and eosin.
Bacteriological and mycological examination
A tracheal wash sample was collected before the snakes were euthanased (Pees and others 2010). During necropsy, swabs for bacteriological and mycological culture were taken from liver, lung, heart, kidney and small intestine. Bacteriological and mycological culture were performed according to standard protocols (Hilf and others 1990, Pees and others 2007). Differentiation of bacteria was carried out using Gram staining, Crystal Tube (BD Biosciences, Heidelberg, Germany) and MALDI-TOF mass spectrometry (Bruker microflex LT mass spectrometer, Bruker Daltonik GmbH, Leipzig, Germany) (Grosse-Herrenthey and others 2008). The spectra were analysed using Bruker BioTyper 1.1 software (Bruker Daltonik GmbH, Leipzig, Germany) (Grosse-Herrenthey and others 2008). For detection of Salmonella species, samples of lung were inoculated into Rappaport Vassiliadis enrichment broth (Oxoid) and Selenit enrichment broth (Oxoid), incubated at 41 °C for 48 hours followed by culture on XLT4 agar (Oxoid) and brilliant green agar (Oxoid) at 41°C for 24 hours.
Serotyping of Salmonella species
All strains were serotyped at the German National Salmonella Reference Laboratory in Berlin. The classification of Salmonella subspecies and below the subspecies level was carried out according to the White-Kauffmann-Le Minor Scheme ( Grimont and Weill 2007 ) by agglutination with O- and H-antigen-specific sera.
Molecular detection of Mycoplasma
Pieces of trachea were collected in 2SP-medium (48.46 g sucrose, 2.088 g K2 HPO4 , 1.088 g KH2 PO4 ad 1000 ml distilled water, supplemented with 120 ml fetal calf serum) and stored at -80 °C. The samples were transported via aeroplane, cooled on ice in a cool bag, and were stored again at -80 °C immediately after arrival until further analysis.
DNA was extracted from swabs and tissue samples using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich Handels GmbH, Vienna, 1120, Austria) following the manufacturer's instructions. Molecular detection of Mycoplasma was carried out by genus-specific amplification (Vojdani and others 1998). Amplicons were analysed by gel electrophoresis and ethidium bromide staining (Sigma-Aldrich Handels GmbH, Vienna, 1120, Austria). In addition, samples were cultivated for Mycoplasma using SP4, Friis and Frey medium as described previously ( Kleven 2008 ). Mycoplasma isolates were identified by 16S rRNA gene sequencing and serotyping.
Virological examination
A swab taken from the choana and the cloaca, as well as fluid from the tracheal wash sample and samples from lung, kidney, gut, pancreas, liver and/or brain from dead snakes were collected in 3 ml Dulbecco's modified Eagle's medium (Biochrom AG, Berlin, Germany) supplemented with antibiotics. Samples were shipped cooled to the laboratory and stored at 4°C until processing. RNA was prepared from 300 [proportional, variant]l of sample as described by Boom and others (1990). PCR detection of a portion of the L gene of ferlaviruses was carried out as described previously (Papp and others 2010). Samples were examined for the presence of adenoviruses (AdV) by nested PCR targeting the DNA polymerase gene (Wellehan and others 2004, Papp and others 2009). Isolation of viruses was attempted from all samples on Russell's viper heart cells (VH-2, ATCC: CCL-140) (Abbas and others 2011). For the verification of reovirus isolation, a nested RT-PCR targeting a portion of the RNA-dependent RNA polymerase gene was carried out as described previously (Wellehan and others 2004, Abbas and others 2011).
Statistical analysis
Statistical analysis was performed using the program SPSS, V.17.0 (SPSS, Chicago, Illinois, USA). Differences between snakes with pneumonia and snakes without pneumonia were examined for significance at a 0.05 significance level using contingency tables, Pearson's χ2 and Fisher's exact test.
Results
Origin of the snakes and clinical signs
In total, 40 males and 40 females including 44 immature snakes were examined. Most of them were captive bred in Germany (n = 68). The others (n = 12) were bred in captivity in the country of origin depending on the snake species. The majority of snakes had been kept in the same collection for more than four months (n = 50). Body condition ranged from poor (n = 22), through reduced body condition (n = 22), moderate body condition (n = 23), good body condition (n = 12), to obese (n = 1). Unremarkable husbandry quality (all climatic, feeding and technical parameters within the ranges recommended for the respective species) was determined for 56 snakes of 31 collections and remarkable husbandry quality (deficits in the husbandry conditions) for 24 snakes of 17 collections (Table 1 ).
According to the findings of the clinical examination, snakes were grouped in three categories: snakes with no clinical signs (21 snakes from four collections), snakes with clinical signs other than respiratory signs (34 snakes from 30 collections) and snakes with respiratory signs (25 snakes from 20 collections) (Table 1 ). Snakes with clinical signs other than respiratory signs included snakes with central nervous signs (n = 21), anorexia (n = 8), vomiting (n = 3), stomatitis (n = 3), dermatitis (n = 2) and diarrhoea (n = 1).
Pythons (22 of 25 snakes with respiratory signs; P < 0.001), adult snakes (17 of 25 snakes with respiratory signs; P < 0.008), snakes with remarkable husbandry conditions (15 of 25 snakes with respiratory signs; P < 0.001) and snakes with stomatitis (14 of 25 snakes with respiratory signs; P < 0.001) showed respiratory signs significantly more often.
Pathological findings
In total, pneumonia was diagnosed in 36 of 80 (45 per cent) snakes from 29 of 48 (60 per cent) collections. Pneumonia was detected significantly more often in snakes with respiratory signs (22 of 25 snakes with respiratory signs, 88 per cent; P < 0.001) as well as pythons (28 of 50 pythons, 56 per cent; P = 0.012) (Table 1 ). Aside from one green tree python (Morelia viridis ) with formation of gout tophi in the lung, pneumonias were characterised by an inflammatory reaction of different stages and, in most cases, by detectable bacterial foci (34 of 36 snakes with pneumonia). An acute catarrhal pneumonia characterised by cuboidal metaplasia of the respiratory epithelial cells, multifocal infiltration of lymphocytes, plasma cells, histiocytes, heterophiles and mild oedema of the interstitial tissue was diagnosed in nine snakes, more often in snakes without respiratory signs (six of 14 snakes with pneumonia and absence of respiratory signs, 43 per cent) compared with snakes with pneumonia and respiratory signs (three of 22 examined snakes with pneumonia and respiratory signs, 14 per cent). Subacute fibrinous pneumonia characterised by fibrin and desquamated respiratory epithelial cell deposition in the lumen of the faveoli, complete atrophy of the cilia and severe inflammatory reaction in the interstitium consisting of heterophiles, histiocytes, lymphocytes, plasma cells and fibrin deposition were the most common forms (n = 19). A chronic pneumonia characterised by fibrous thickening of the interstitial tissue with mixed cell inflammatory reaction, as well as fibrin deposition in the faveoli was detected in six pythons with respiratory signs. In one case of a carpet python (Morelia spilota ), infection was associated with a granulomatous inflammatory reaction with intralesional mycotic hyphae. Stomatitis was detected significantly more often in snakes with pneumonia (14 of 36 snakes with pneumonia, 39 per cent; P < 0.001) than in snakes without pneumonia (two of 44 snakes without pneumonia, five per cent). Furthermore, pneumonia was diagnosed significantly (P = 0.035) more often in snakes from collections with husbandry deficits (15 of 24 snakes from collections with husbandry deficits) than in snakes from collections with unremarkable husbandry conditions (21 of 56 snakes).
Microbiological findings
Tracheal wash samples from 28 snakes of 19 collections contained bacteria (28 of 80 snakes, 35 per cent). Pythons (25 of 50 pythons, 50 per cent; P < 0.0001), snakes with respiratory signs (22 of 25 snakes with respiratory signs, 88 per cent, P < 0.001), snakes with stomatitis (15 of 16 snakes with stomatitis, 94 per cent, P < 0.001) and snakes with pneumonia (22 of 36 snakes with pneumonia, 61 per cent, P < 0.001) revealed significantly more often bacteria in the tracheal wash sample than boas (three of 30 boas, 10 per cent), snakes without respiratory signs (six of 55 snakes without respiratory signs, 11 per cent), snakes without stomatitis (13 of 64 snakes without stomatitis, 20 per cent) or snakes without pneumonia (six of 44 snakes without pneumonia, 14 per cent). Bacterial isolation from lung tissue was positive in 44 of 80 (55 per cent) snakes. Snakes with pneumonia (34 of 36 snakes with pneumonia, 94 per cent, P < 0.001) and with stomatitis (14 of 16 snakes with stomatitis, 88 per cent, P = 0.004) revealed significantly more often bacteria in the lung than snakes without pneumonia (10 of 44 snakes without pneumonia, 23 per cent) or without stomatitis (30 of 64 snakes without stomatitis, 47 per cent). Twenty-five of 80 snakes (31 per cent) revealed bacteria in the tracheal wash sample as well as in swabs taken from the lung, and 22 of them revealed pneumonia (22 of 36 snakes with pneumonia, 61 per cent). A mixed bacterial infection was detected in three lungs. Different bacteria from tracheal wash samples compared with the lung isolates were cultured in five snakes. Isolation of bacteria only from the lung was successful in 19 of 80 (24 per cent) snakes. Pneumonia was diagnosed in 12 of these 19 snakes and most of them (11 of 12 snakes) showed no respiratory signs, and were from collections with husbandry deficits (12 of 12 snakes, P = 0.018). Isolation only from the tracheal wash sample was possible in three snakes without pneumonia, but two of them had respiratory signs.
In total, 12 different bacterial species from six families were cultured (Tables 2 , 3). Enterobacteriaceae were the most commonly isolated bacteria from the tracheal wash samples (13 of 80 snakes, 16 per cent) as well as from the lungs (32 of 80 snakes, 40 per cent). Enterobacteriaceae were isolated from tracheal wash samples irrespective of clinical status. They caused inflammatory reactions in the lungs of 23 (64 per cent) snakes with pneumonia. Fibrinous pneumonias were detected in 12 cases, followed by catarrhal and fibrous (n = 5, each) inflammation of the lungs.
Bacteria and viruses | Clinical signs | Pneumonia (n = 8) | ||||||
Absent (n = 13) | Other than respiratory signs (n = 14) | Respiratory signs (n = 3) | ||||||
Salmonella species[dagger] | 1 ( 2 )[double dagger] | 1 ( 8 ) | 0 | 3 ( 21 ) | 0 | 0 | 0 | 3 (38) |
Salmonella subspecies IIIb | 0 | 2 ( 15 ) | 0 | 0 | 0 | 0 | 0 | 1 ( 13 ) |
Salmonella Muenchen | 0 | 1 ( 8 ) | 0 | 0 | 0 | 0 | 0 | 0 |
Klebsiella pneumoniae | 0 | 0 | 0 | 0 | 1 ( 33 ) | 1 ( 33 ) | 1 ( 13 ) | 1 ( 13 ) |
Pseudomonas aeruginosa | 0 | 0 | 0 | 1 ( 7 ) | 1 ( 33 ) | 1 ( 33 ) | 1 ( 13 ) | 2 ( 25 ) |
Stenotrophomonas maltophilia | 0 | 0 | 0 | 0 | 1 ( 33 ) | 1 ( 33 ) | 1 ( 13 ) | 1 ( 13 ) |
Adenovirus | 0 | 0 | 0 | 1 ( 7 ) | 0 | 0 | 0 | 0 |
*Tracheal wash sample
[dagger]Not serotyped
[double dagger]Total number of animals (percentage)
Salmonella isolation
With a total of 20 isolates from 20 snakes of 12 collections, Salmonella were the most commonly isolated bacterial species. Isolation of Salmonella species from tracheal wash samples and lung tissue was possible in four of 80 (five per cent) snakes. Isolation of Salmonella species only from the lung was successful in 16 of 80 (20 per cent) snakes. Salmonella was isolated significantly more often from the lungs of snakes without respiratory signs (16 of 55 snakes without respiratory signs, 29 per cent, P < 0.001) than from snakes with respiratory signs (four of 25 snakes with respiratory signs, 16 per cent). In snakes with pneumonia, Salmonella was the most commonly isolated bacterium (12 of 36 snakes with pneumonia, 33 per cent) (Tables 2 , 3). Fibrinous pneumonias were detected in eight Salmonella-positive snakes, followed by catarrhal (n = 2) and fibrous (n = 1) inflammation of the lungs. The remaining one snake had a granulomatous pneumonia caused by urate tophi.
Detection of Mycoplasma and fungi
In one snake with respiratory signs, and in one snake with clinical signs other than respiratory signs, Mycoplasma was detected by PCR and cultivation procedures. Pneumonia was diagnosed in both cases at necropsy. One had no respiratory signs, a catarrhal pneumonia and no other bacteria were isolated; 16S rRNA gene analysis revealed identical sequences for both isolates with highest sequence similarity values to Mycoplasma caviae and Mycoplasma fermentans , respectively (95 per cent, accession number FR869692 ).
Fungi belonging to Paecilomyces variotii were isolated from the lung of a Morelia spilota with respiratory signs and a granulomatous pneumonia without isolation of bacterial agents.
Detection of IBD
IBD was diagnosed in 28 of 80 (35 per cent) snakes. Nineteen of these snakes were from previously known IBD-positive collections (n = 6). In total, 15 of 48 (31 per cent) collections were affected. Boas had IBD significantly more often IBD (26 of 30 boas, 87 per cent; P < 0.001). Pneumonia was diagnosed in six of 28 (21 per cent) snakes with IBD. On the other hand, six of 36 (17 per cent) snakes with pneumonia had IBD. Two IBD-positive snakes had a catarrhal pneumonia and four snakes had a fibrinous pneumonia. Bacterial isolation from tracheal wash samples was positive in four (13 per cent) snakes with IBD and from lungs in nine (32 per cent) snakes with IBD, while Salmonella secies was detected in six of these nine snakes. Inclusion bodies in the two pythons were only detected in the brain. IBD was detected irrespective of body condition, quality of the husbandry conditions, age and/or origin of the snakes.
Detection of ferlavirus-specific RNA
Ferlavirus-specific RNA was detected and sequenced from a total of eight out of 80 (10 per cent) snakes, four of which were from previously known ferlavirus-positive collections (n = 2). Tracheal wash samples as well as lung samples were positive in seven of these eight snakes (Table 3 , Fig 1 ). In one snake, ferlavirus-specific RNA was only amplified from the kidney and gut. In total, six of 48 (13 per cent) collections were affected. Ferlavirus-specific RNA was only detected in samples from pythons (eight of 50 Pythonidae, 16 per cent). Positive amplification of ferlavirus-specific RNA was irrespective of husbandry conditions or age of the snakes. No clinical signs were seen in three of eight ferlavirus-specific RNA-positive snakes. Respiratory signs were seen in three of eight ferlavirus-specific RNA positive snakes. Four of eight (50 per cent) postmortem-examined snakes with ferlavirus-specific RNA had pneumonia. On the other hand, four of 36 (11 per cent) examined snakes with pneumonia were positive for ferlavirus-specific RNA. One ferlavirus-specific RNA positive snake had a catarrhal pneumonia, and three snakes had a fibrinous pneumonia. Bacterial isolation from lungs was successful in six of eight ferlavirus-specific RNA-positive snakes, and in three of eight tracheal wash samples from ferlavirus-specific RNA-positive snakes.
FIG 1:Phylogenetic tree of the ferlaviruses, including the ferlaviruses detected in this study based on 443bp of the large RNA-dependent RNA polymerase (L) gene. Sequences were analysed using the Mega 5 program using the Neighbour Joining method (NJ). Bootstrap values over 60 (500 replications) of the NJ are indicated beside the nodes. Branches with lower values are drawn with checkerboard lines. All diagnostic samples sited on the tree are 100% similar to the previously described paramyxovirus (PMV) and shaded with grey colour and indicated with arrows. Light shaded subgroups 'a' and 'b' and the proposed 'c' (Pangut GER 09) correspond to those described already in an earlier publication (Abbas and others 2011). Corresponding sequences (GenBank accession number) from Newcastle disease virus/NDV/( AF375823 ), Sendai ( NC_001552 ), and Human parainfluenza type 1/HPIV-1/( AF457102 ) were used as outgroups. GenBank accession numbers for the reptilian PMV: Bush viper virus/ATCC-VR-1409/( AF286043 ), CeraCe-98 ( AF351137 ), Crot-GER03 ( GQ277611 ), CrotX1-96 ( AF349405 ), Dasy-GER00 ( GQ277613 ), ElaGut-91 ( AF349408 ), Fer-de-Lance virus ( NC_005084 ), GonoGER-85 ( AF349404 ), Igu-GER00+ ( GQ277617 ), Neotropical rattlesnake virus/ATCC-VR-1408/( AF286045 ), Pyth-GER01 ( GQ277612 ), TORTOISE-GER99( GQ277615 ), Xeno-USA99+ ( GQ277614 ), Pangut GER09 ( HQ148084 ), Pyt-6 intestine ( GU393348 ). The following sequences (Ahne and others 1999) were kindly provided by Dr Gael Kurath and Dr William N Batts: Crot1-VA95, Ela-FL93, More-GER86, Pyth-GER88, Trim-MD97
Bacteria and viruses | Clinical symptoms | Pneumonia (n = 28) | ||||||
Absent (n = 8) | other than respiratory signs (n = 20) | respiratory signs (n = 21) | ||||||
Salmonella species[dagger] | 0 | 0 | 0 | 2 ( 10 ) | 1 ( 5 ) | 2 ( 10 ) | 1 ( 4 ) | 3 ( 11 ) |
Salmonella subspecies IIIb | 0 | 2 ( 25 ) | 0 | 1 ( 5 ) | 0 | 0 | 0 | 2 ( 7 ) |
Salmonella subspecies IV | 0 | 2 ( 25 ) | 1 ( 5 ) | 1 ( 5 ) | 1 ( 5 ) | 2 ( 10 ) | 2 ( 7 ) | 1 ( 11 ) |
Salmonella Paratyphi B-enteric pathovar | 0 | 0 | 1 ( 5 ) | 1 ( 5 ) | 0 | 0 | 0 | 0 |
Enterobacter cloacae | 0 | 1 ( 13 ) | 1 ( 5 ) | 2 ( 10 ) | 1 ( 5 ) | 1 ( 5 ) | 2 ( 7 ) | 3 ( 11 ) |
Citrobacter freundii | 0 | 0 | 0 | 0 | 3 ( 14 ) | 4 ( 19 ) | 3 ( 11 ) | 4 ( 14 ) |
Klebsiella pneumoniae | 0 | 0 | 0 | 1 ( 5 ) | 0 | 1 ( 5 ) | 0 | 2 ( 7 ) |
Klebsiella oxytoca | 0 | 0 | 0 | 0 | 1 ( 5 ) | 1 ( 5 ) | 1 ( 4 ) | 1 ( 4 ) |
Proteus vulgaris | 0 | 0 | 0 | 0 | 2 ( 10 ) | 0 | 1 ( 4 ) | 0 |
Pseudomonas aeruginosa | 0 | 0 | 0 | 0 | 3 ( 14 ) | 4 ( 19 ) | 0 | 4 ( 14 ) |
Pseudomonas floureszens | 0 | 0 | 0 | 0 | 1 ( 5 ) | 0 | 1 ( 4 ) | 0 |
Stenotrophomonas maltophilia | 0 | 0 | 0 | 0 | 5 ( 24 ) | 6 ( 29 ) | 5 ( 18 ) | 6 ( 21 ) |
Elizabethkingia meningoseptica | 0 | 0 | 0 | 0 | 1 ( 5 ) | 1 ( 5 ) | 1 ( 4 ) | 1 ( 4 ) |
Bordetella hinzii | 3 (38)[double dagger] | 1 ( 13 ) | 0 | 0 | 0 | 0 | 0 | 0 |
Stapylococcus species | 0 | 0 | 0 | 1 ( 5 ) | 1 ( 5 ) | 0 | 0 | 1 ( 4 ) |
Ferlavirus | 3 (38) | 3 (38) | 2 ( 10 ) | 2 ( 10 ) | 2 ( 10 ) | 2 ( 10 ) | 3 ( 11 ) | 3 ( 11 ) |
*Tracheal wash sample
[dagger]Not serotyped
[double dagger]Total number of animals (percentage)
Detection of adenovirus-specific DNA and virus isolation
AdV-specific DNA was amplified and sequenced from various organs (lung, kidney, gut) from one imported immature anorectic boa constrictor without pneumonia (Fig 2 ).
![View Image - FIG 2:Phylogenetic tree of the adenoviruses including the atadenoviruses detected in this study (shaded with grey colour) based on 247bp of the adenovirus DNA-polymerase nucleotide gene. Sequences were analysed with the Mega 5 program using the Neighbour Joining method (NJ). Bootstrap values over 50 from 500 replications of the NJ are indicated beside the nodes. Branches with lower values are drawn with checkerboard lines. GenBank accession numbers for sequences included in the analysis are as follows: A (AAS89694), A1 (ACI28425), A2 (ACI28426), A4 (ACI28429), A5 (ACI28430), A6 (ACI28431), B1 ( YP_094032 ), B3 ( AP_000026 ), B4 (AAC41020), Chameleonid (AAS89695), C2 (AAB38716), C1 (AAB05434), Duck 1 ( AP_000088 ), Eublepharid (AAF13265), F1 ( AP_000410 ), F9 ( AP_000385 ), Frog 1 (AAF86924), Gekkonid (AAS89697), H40 (AAC13953), H52 (ABK35035), OAV 287 (AAA84979), Raptor (YP_004414800), Snake1 (AAL92452), Snake 2 (ACH91014), Snake 3 (ACH91015), Simian1 (AAX19399), Scincid (AAS89698), T3 ( AP_000478 ), Viperid (ACH86254). Aâ[euro][per thousand]=â[euro][per thousand]Agamid, Bâ[euro][per thousand]=â[euro][per thousand]Bovine, Câ[euro][per thousand]=â[euro][per thousand]Canine, Fâ[euro][per thousand]=â[euro][per thousand]Fowl, Hâ[euro][per thousand]=â[euro][per thousand]Human, Oâ[euro][per thousand]=â[euro][per thousand]Ovine, Tâ[euro][per thousand]=â[euro][per thousand]Turkey](https://media.proquest.com/media/hms/THUM/3/Zfj4S?_a=ChgyMDI2MDEyMTExMDU0OTY2NTozMTk2OTYSBzE0MTM1MzcaCk9ORV9TRUFSQ0giDjIxNi43My4yMTYuMTE0KgcyMDQxMDI3MgoxNzc5ODYxODEwOgtJbmxpbmVJbWFnZUIBMFIGT25saW5lWgNJTEliBFRIVU1qCjIwMTMvMDMvMDJyCjIwMTMvMDMvMDJ6AIIBPFAtMTAwNzA2Ny0yMDg2MTEtQ1VTVE9NRVItMTAwMDAzNDgvMTAwMDAyNTUvMTAwMDAxNTUtNjY5MzE2NJIBBk9ubGluZcoBDlNlYXJjaFJldHJpZXZl0gESU2Nob2xhcmx5IEpvdXJuYWxzmgIHUHJlUGFpZKoCLE9TOkVNUy1UZXh0UGx1c0dyYXBoaWNzQ29udGFpbmVyLXByZXBhcmVCb2R5ygIPQXJ0aWNsZXxGZWF0dXJl4gIA8gIA%2BgIBToIDA1dlYooDHENJRDoyMDI2MDEyMTExMDUwMzQyNjo1OTE3MzY%3D&_s=OvETg5bw3bb%2BdQk5pI3JglgGEMY%3D "View Image - FIG 2:Phylogenetic tree of the adenoviruses including the atadenoviruses detected in this study (shaded with grey colour) based on 247bp of the adenovirus DNA-polymerase nucleotide gene. Sequences were analysed with the Mega 5 program using the Neighbour Joining method (NJ). Bootstrap values over 50 from 500 replications of the NJ are indicated beside the nodes. Branches with lower values are drawn with checkerboard lines. GenBank accession numbers for sequences included in the analysis are as follows: A (AAS89694), A1 (ACI28425), A2 (ACI28426), A4 (ACI28429), A5 (ACI28430), A6 (ACI28431), B1 ( YP_094032 ), B3 ( AP_000026 ), B4 (AAC41020), Chameleonid (AAS89695), C2 (AAB38716), C1 (AAB05434), Duck 1 ( AP_000088 ), Eublepharid (AAF13265), F1 ( AP_000410 ), F9 ( AP_000385 ), Frog 1 (AAF86924), Gekkonid (AAS89697), H40 (AAC13953), H52 (ABK35035), OAV 287 (AAA84979), Raptor (YP_004414800), Snake1 (AAL92452), Snake 2 (ACH91014), Snake 3 (ACH91015), Simian1 (AAX19399), Scincid (AAS89698), T3 ( AP_000478 ), Viperid (ACH86254). Aâ[euro][per thousand]=â[euro][per thousand]Agamid, Bâ[euro][per thousand]=â[euro][per thousand]Bovine, Câ[euro][per thousand]=â[euro][per thousand]Canine, Fâ[euro][per thousand]=â[euro][per thousand]Fowl, Hâ[euro][per thousand]=â[euro][per thousand]Human, Oâ[euro][per thousand]=â[euro][per thousand]Ovine, Tâ[euro][per thousand]=â[euro][per thousand]Turkey")
FIG 2:Phylogenetic tree of the adenoviruses including the atadenoviruses detected in this study (shaded with grey colour) based on 247bp of the adenovirus DNA-polymerase nucleotide gene. Sequences were analysed with the Mega 5 program using the Neighbour Joining method (NJ). Bootstrap values over 50 from 500 replications of the NJ are indicated beside the nodes. Branches with lower values are drawn with checkerboard lines. GenBank accession numbers for sequences included in the analysis are as follows: A (AAS89694), A1 (ACI28425), A2 (ACI28426), A4 (ACI28429), A5 (ACI28430), A6 (ACI28431), B1 ( YP_094032 ), B3 ( AP_000026 ), B4 (AAC41020), Chameleonid (AAS89695), C2 (AAB38716), C1 (AAB05434), Duck 1 ( AP_000088 ), Eublepharid (AAF13265), F1 ( AP_000410 ), F9 ( AP_000385 ), Frog 1 (AAF86924), Gekkonid (AAS89697), H40 (AAC13953), H52 (ABK35035), OAV 287 (AAA84979), Raptor (YP_004414800), Snake1 (AAL92452), Snake 2 (ACH91014), Snake 3 (ACH91015), Simian1 (AAX19399), Scincid (AAS89698), T3 ( AP_000478 ), Viperid (ACH86254). Aâ[euro][per thousand]=â[euro][per thousand]Agamid, Bâ[euro][per thousand]=â[euro][per thousand]Bovine, Câ[euro][per thousand]=â[euro][per thousand]Canine, Fâ[euro][per thousand]=â[euro][per thousand]Fowl, Hâ[euro][per thousand]=â[euro][per thousand]Human, Oâ[euro][per thousand]=â[euro][per thousand]Ovine, Tâ[euro][per thousand]=â[euro][per thousand]Turkey
Cultivation on VH-2 resulted in the isolation of a reovirus from the gut of one Python regius without pneumonia or respiratory signs.
Discussion
Respiratory diseases in snakes often take a long time to develop before clinical signs are evident ( Chitty 2004 ). Therefore, it is not surprising that pneumonia was diagnosed in 45 per cent of the snakes from 60 per cent of the involved collections, though more than one-third (39 per cent) of the snakes with pneumonia showed no respiratory signs. Pneumonia was detected in pythons significantly more often compared with boas. The cause of this could be that ferlavirus-specific RNA was detected in pythons only. It has been experimentally proven that respiratory epithelial cells are one of the primary target cells for the replication of ferlaviruses in vipers (Jacobson and others 1997). This may be true for pythons, as well, as 50 per cent of the postmortem-examined pythons with ferlavirus-specific RNA had pneumonia. However, ferlaviruses were detected only in 11 per cent of the snakes with pneumonia, and other triggers, like deficits in husbandry conditions clearly influence the health status of reptiles as ectothermic organisms (Murray 2006).
Pneumonia in snakes can be induced by inhalation of bacteria following a bacterial stomatitis, or as a result of a bacterial septicaemia ( Soveri 1984 ). Stomatitis was a common finding (59 per cent) in snakes with pneumonia. Isolation of bacteria in those snakes was successful in tracheal wash samples and lungs, so that bacterial infection of the lung was most likely caused via inhalation of bacteria in those cases. Bacteria isolated in this study have been described as a part of the healthy oral cavity of snakes (Goldstein and others 1979, Goldstein and others 1981, Hilf and others 1990, Johnson and others 1996, Hejnar and others 2007), as well as in diseased snakes (Miller and others 2004, Pees and others 2010). On the other hand, one-third of the tracheal wash samples revealed a negative bacteriological result despite isolation of bacteria from lungs with different inflammatory stages. Furthermore, these snakes revealed fibrinous splenitis and hepatitis with isolation of the same bacteria (data not shown) as in the lungs, so that a bacterial septicaemia was most likely the cause of the pneumonia in these cases. Statistical analysis revealed that snakes with pneumonia and isolation of bacteria only from the lungs were significant more often from collections with deficits in husbandry conditions.
Salmonella were the most frequently isolated bacteria from lungs without inflammatory reaction (15 per cent), as well as from lungs with pneumonia (33 per cent). This bacterium is common in snakes and appears to be a component of the normal gastrointestinal flora ( Chiodini and Sundberg 1981 ). The German National Salmonella Reference Laboratory in Berlin reports increasing numbers of Salmonella isolates in snakes, and also generally in reptiles in the last years (Friedrich and others 2011). In the study presented here, Salmonella was isolated significantly more often from snakes without clinical signs, but on the other hand it was the most common bacteria associated with pneumonia. Most of the human pathogenic Salmonella are in the subgenus I (Aleksic and others 1996), whereas Salmonella Muenchen and Salmonella Paratyphi B (d-tatrate +)-enteric pathovar were isolated from one snake each in the present study. Neither snake had respiratory signs or pneumonia. Both Salmonella serovars are known pathogens for foodborne salmonellosis in humans, so that an infection of the snakes via chickens used as food is possible ( Nicklas 1987 ), especially as both snakes were fed with day-old-chicks. Salmonella enterica subspecies IV and Salmonella enterica subspecies IIIb were the two subspecies isolated from boid snakes with pneumonia.
Mycoplasma was isolated from two pythons from two collections. One revealed a catarrhal pneumonia without isolation of other bacterial or viral pathogens. A causative role of Mycoplasma in the pathogenesis of pneumonia in at least this snake cannot be excluded with certainty, as a catarrhal pneumonia induced by an unidentified Mycoplasma species has been described in a Burmese python (Penner and others 1997). However, both isolates most likely represent a new species within the class of Mollicutes, as the serotyping with antisera directed against Mycoplasma caviae and Mycoplasma fermentans was negative.
A fungal infection caused by Paecilomyces variotii was diagnosed in one python. Paecilomyces variotii is a ubiquitous saprophyte found in such diverse substrates as soil, a variety of moist or viscous substances, and water ( Summerbell 2003 ).
Conclusion
Pneumonia is an important differential diagnosis in snakes with respiratory signs or stomatitis, as well as in snakes from collections with deficits in husbandry conditions, especially when Salmonella can be isolated from the faeces or cloacal swabs. In two-thirds of the snakes with pneumonia, bacteria were isolated from the tracheal wash sample as well as from swabs taken from the lung. Furthermore, a ferlavirus was detected in tracheal wash samples in almost all ferlavirus-positive animals. Therefore, at least in snakes with respiratory signs, a bacteriological and virological examination of tracheal wash samples seems to be a reliable diagnostic tool. On the other hand, snakes can suffer from pneumonia due to bacterial septicaemia not associated with respiratory signs, which could result in false negative findings in tracheal wash samples. Salmonella species were isolated more often from affected lungs without successful isolation from the tracheal wash samples, so that the use of a Salmonella enrichment broth prior to cultivation from a tracheal wash sample could help to avoid these false negative results.
Acknowledgement: The present study was passed by the ethical committee of animal welfare and experimental studies of the University of Leipzig and was funded by the German Research Foundation (DFG Pe 877/2-1). This sponsor had to no role in the collection, analysis and interpretation of data, nor in the writing and the decision to submit this manuscript.
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