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Rachel E. Marschang - One of the best experts on this subject based on the ideXlab platform.
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Comparison of three different PCR protocols for the detection of Ferlaviruses
BMC Veterinary Research, 2019Co-Authors: Ekaterina Kolesnik, Timothy H Hyndman, Michael Pees, Elisabeth Müller, Rachel E. MarschangAbstract:BACKGROUND: Ferlaviruses are important pathogens in snakes often associated with respiratory and neurological disease. The detection of ferlaviral RNA by PCR is considered to be the most reliable method for the diagnosis of infection. The PCRs that have been used most commonly for this purpose have not been properly assessed to determine their sensitivity, specificity and ability to detect the known genetic diversity of this group of viruses. The aim of this study was to compare three published PCR protocols so that a single method could be recommended to laboratories that perform this testing. RESULTS: Comparisons were carried out using cell culture isolates and tissues from snakes infected with specific virus genotypes. A single round PCR targeting a short segment of the viral polymerase (L) gene provided the highest sensitivity and specificity, and detected isolated Ferlaviruses from all four described genogroups, as well as from tissues of infected snakes. CONCLUSION: A broadly-reactive PCR for the detection of all known Ferlaviruses was found to provide a good combination of detection limit, specificity and speed. Based on these criteria, this method is recommended for the diagnosis of Ferlavirus infections.
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Comparison of three different PCR protocols for the detection of Ferlaviruses
BMC Veterinary Research, 2019Co-Authors: Ekaterina Kolesnik, Timothy H Hyndman, Michael Pees, Elisabeth Müller, Rachel E. MarschangAbstract:Background Ferlaviruses are important pathogens in snakes often associated with respiratory and neurological disease. The detection of ferlaviral RNA by PCR is considered to be the most reliable method for the diagnosis of infection. The PCRs that have been used most commonly for this purpose have not been properly assessed to determine their sensitivity, specificity and ability to detect the known genetic diversity of this group of viruses. The aim of this study was to compare three published PCR protocols so that a single method could be recommended to laboratories that perform this testing. Results Comparisons were carried out using cell culture isolates and tissues from snakes infected with specific virus genotypes. A single round PCR targeting a short segment of the viral polymerase (L) gene provided the highest sensitivity and specificity, and detected isolated Ferlaviruses from all four described genogroups, as well as from tissues of infected snakes. Conclusion A broadly-reactive PCR for the detection of all known Ferlaviruses was found to provide a good combination of detection limit, specificity and speed. Based on these criteria, this method is recommended for the diagnosis of Ferlavirus infections.
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Three genetically distinct Ferlaviruses have varying effects on infected corn snakes (Pantherophis guttatus).
PLOS ONE, 2019Co-Authors: Michael Pees, Annkatrin Neul, Volker Schmidt, Tibor Papp, Ákos Gellért, Maha Diekan Abbas, J. Matthias Starck, Rachel E. MarschangAbstract:Ferlaviruses are important pathogens in snakes and other reptiles. They cause respiratory and neurological disease in infected animals and can cause severe disease outbreaks. Isolates from this genus can be divided into four genogroups-A, B, and C, as well as a more distantly related sister group, "tortoise". Sequences from large portions (5.3 kb) of the genomes of a variety of Ferlavirus isolates from genogroups A, B, and C, including the genes coding the surface glycoproteins F and HN as well as the L protein were determined and compared. In silico analyses of the glycoproteins of genogroup A, B, and C isolates were carried out. Three isolates representing these three genogroups were used in transmission studies with corn snakes (Pantherophis guttatus), and clinical signs, gross and histopathology, electronmicroscopic changes in the lungs, and isolation of bacteria from the lungs were evaluated. Analysis of the sequences supported the previous categorization of Ferlaviruses into four genogroups, and criteria for definition of Ferlavirus genogroups and species were established based on sequence identities (80% resp. 90%). Analysis of the Ferlavirus glycoprotein models showed parallels to corresponding regions of other paramyxoviruses. The transmission studies showed clear differences in the pathogenicities of the three virus isolates used. The genogroup B isolate was the most and the group A virus the least pathogenic. Reasons for these differences were not clear based on the differences in the putative structures of their respective glycoproteins, although e.g. residue and consequential structure variation of an extended cleavage site or changes in electrostatic charges at enzyme binding sites could play a role. The presence of bacteria in the lungs of the infected animals also clearly corresponded to increased pathogenicity. This study contributes to knowledge about the structure and phylogeny of Ferlaviruses and lucidly demonstrates differences in pathogenicity between strains of different genogroups.
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Ferlavirus related deaths in a collection of viperid snakes
Journal of Zoo and Wildlife Medicine, 2018Co-Authors: E J Flach, Rachel E. Marschang, Mark P Dagleish, Yedra Feltrer, Iri Gill, Nic Masters, Jorge Oros, Ann Pocknell, Matthew Rendle, Taina StrikeAbstract:Abstract Between June and October 2013, 26 snakes of six viperid species kept in two adjoining rooms died (n = 16) or were euthanized on medical (1) or welfare grounds (9). Two were from the main zoo collection, but the other 24 had been imported and quarantined for a minimum of 6 mo. Four of those that died and the single snake euthanized on medical grounds showed minor signs of respiratory disease prior to death, and five were weak, lethargic, and/or poor feeders. Frequent postmortem findings among all snakes were poor body condition (18) and respiratory disease (13). Seventeen cases were examined histologically, and pneumonia, sometimes with air sacculitis and/or tracheitis, was present in 15 individuals. Lung samples from 24 snakes were Ferlavirus polymerase chain reaction (PCR) positive, and one of the two snakes for which only liver was available was also positive. The negative liver sample was from a snake that died of sepsis following anesthesia for surgical removal of a spindle cell sarcoma. Corr...
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Immunologic responses in corn snakes (Pantherophis guttatus) after experimentally induced infection with Ferlaviruses
American Journal of Veterinary Research, 2017Co-Authors: Annkatrin Neul, Wieland Schrödl, Rachel E. Marschang, Tina Bjick, Uwe Truyen, Heiner Von Buttlar, Michael PeesAbstract:OBJECTIVE To measure immunologic responses of snakes after experimentally induced infection with Ferlaviruses. ANIMALS 42 adult corn snakes (Pantherophis guttatus) of both sexes. PROCEDURES Snakes were inoculated intratracheally with genogroup A (n = 12), B (12), or C (12) Ferlavirus (infected groups) or cell-culture supernatant (6; control group) on day 0. Three snakes from each infected group were euthanized on days 4, 16, 28, and 49, and 3 snakes from the control group were euthanized on day 49. Blood samples were collected from live snakes on days −6 (baseline), 4, 16, 28, and 49. Hematologic tests were performed and humoral responses assessed via hemagglutination-inhibition assays and ELISAs. Following euthanasia, gross pathological and histologic evaluations and virus detection were performed. RESULTS Severity of clinical signs of and immunologic responses to Ferlavirus infection differed among snake groups. Hematologic values, particularly WBC and monocyte counts, increased between days 4 and 16 af...
M Pees - One of the best experts on this subject based on the ideXlab platform.
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morphology and morphometry of the lung in corn snakes pantherophis guttatus infected with three different strains of Ferlavirus
Journal of Comparative Pathology, 2017Co-Authors: J M Starck, Annkatrin Neul, Volker Schmidt, T Kolb, S Franzguess, D Balcecean, M PeesAbstract:Summary Ophidian paramyxovirus (Ferlavirus) is a global threat to reptilian sauropsids in herpetological collections, with occasional but fatal effects. This study characterizes the effects of three different genetic strains of Ferlavirus on the dynamic changes of histology and morphometry of the lung of corn snakes (Pantherophis guttatus). Lungs from 42 corn snakes were either sham-infected or infected experimentally under standardized conditions. From 4 to 49 days after intratracheal inoculation, the lungs were examined qualitatively and quantitatively. Progressive microscopical changes were seen in the lung. Initially, increased numbers of heterophils were observed in the interstitium followed by proliferation and vacuolation of epithelial cells lining faveoli. Electron microscopy revealed loss of type-I pneumocytes, hyperplasia of type-II pneumocytes, and interstitial infiltrates of heterophils and mononuclear cells. With progression of disease the respiratory epithelium was initially overgrown by transformed type-II pneumocytes and later became multilayered. The results of the study suggest that the respiratory capacity of the lungs declines with disease development. The dynamics of disease development and histopathology differed in snakes infected with different Ferlavirus genogroups. Animals infected with virus genogroup B developed histopathological changes and morphometric changes more rapidly and of greater intensity than snakes infected with viruses from genogroups A or C.
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virus distribution and detection in corn snakes pantherophis guttatus after experimental infection with three different Ferlavirus strains
Veterinary Microbiology, 2016Co-Authors: M Pees, Annkatrin Neul, Volker Schmidt, Uwe Truyen, K Muller, Nadja Leinecker, Rachel E. MarschangAbstract:Ferlaviruses are important pathogens of snakes. However, factors influencing the pathogenicity of individual isolates as well as optimal protocols for virus detection in tissues of infected snakes have been insufficiently studied. The objectives of this study were to compare virus detection using previously described PCR and cell culture protocols following infection with three genetically distinct Ferlaviruses in corn snakes (Pantherophis guttatus) as a model species. Groups of 12 corn snakes were each inoculated intratracheally with a genogroup A, B, or C Ferlavirus. Tracheal washes and cloacal swabs were tested for virus shedding on days 16 and 28. Three animals were each euthanized on days 4, 16, 28, and 49. Beside immunohistochemistry of lung tissue, several organs (lung, intestine, pancreas, kidney, brain) were tested for the presence of Ferlavirus. Distinct differences were noted in the pathogenicity of the three viruses, with a genotype B isolate causing the greatest pathology. PCR was more sensitive in comparison to cell culture, but results varied depending on the tissues. Ferlaviruses spread rapidly into the tissues, including the brain. Overall average detection rate was 72%, and was highest on day 16. There were differences between the groups, with the most virulent strain causing 100% positive samples at the end of the study. Some snakes were able to clear the infection. Shedding via cloaca was seen only on day 28. For ante-mortem sampling, a tracheal wash sample is recommended, for post mortem diagnosis, a pooled organ sample should be tested.
Tibor Papp - One of the best experts on this subject based on the ideXlab platform.
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Three genetically distinct Ferlaviruses have varying effects on infected corn snakes (Pantherophis guttatus).
PLOS ONE, 2019Co-Authors: Michael Pees, Annkatrin Neul, Volker Schmidt, Tibor Papp, Ákos Gellért, Maha Diekan Abbas, J. Matthias Starck, Rachel E. MarschangAbstract:Ferlaviruses are important pathogens in snakes and other reptiles. They cause respiratory and neurological disease in infected animals and can cause severe disease outbreaks. Isolates from this genus can be divided into four genogroups-A, B, and C, as well as a more distantly related sister group, "tortoise". Sequences from large portions (5.3 kb) of the genomes of a variety of Ferlavirus isolates from genogroups A, B, and C, including the genes coding the surface glycoproteins F and HN as well as the L protein were determined and compared. In silico analyses of the glycoproteins of genogroup A, B, and C isolates were carried out. Three isolates representing these three genogroups were used in transmission studies with corn snakes (Pantherophis guttatus), and clinical signs, gross and histopathology, electronmicroscopic changes in the lungs, and isolation of bacteria from the lungs were evaluated. Analysis of the sequences supported the previous categorization of Ferlaviruses into four genogroups, and criteria for definition of Ferlavirus genogroups and species were established based on sequence identities (80% resp. 90%). Analysis of the Ferlavirus glycoprotein models showed parallels to corresponding regions of other paramyxoviruses. The transmission studies showed clear differences in the pathogenicities of the three virus isolates used. The genogroup B isolate was the most and the group A virus the least pathogenic. Reasons for these differences were not clear based on the differences in the putative structures of their respective glycoproteins, although e.g. residue and consequential structure variation of an extended cleavage site or changes in electrostatic charges at enzyme binding sites could play a role. The presence of bacteria in the lungs of the infected animals also clearly corresponded to increased pathogenicity. This study contributes to knowledge about the structure and phylogeny of Ferlaviruses and lucidly demonstrates differences in pathogenicity between strains of different genogroups.
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DETECTION OF ANTIBODIES AGAINST PARAMYXOVIRUSES IN TORTOISES
Journal of Zoo and Wildlife Medicine, 2013Co-Authors: Reinhild Rösler, Rachel E. Marschang, Tibor Papp, Maha Diekan Abbas, Ftä Mikrobiologie, Zb ReptilienAbstract:Abstract: Sera from a total of 202 tortoises from six countries and nine species were tested for antibodies against four different reptilian paramyxoviruses (Ferlaviruses, ferlaVs) by hemagglutination inhibition (HI) test. The viruses used were a tortoise PMV (tPMV) and three squamatid PMV isolates, each belonging to a different subgroup of ferlaV within the genus Ferlavirus. HI tests revealed that antibodies against ferlaVs occurred regularly in the tested samples (5.5%). One and a half percent of the tested samples have measurable antibody titers against the group A isolate, 3% had antibodies against the group B isolate, and 1% had antibodies against the group C isolate. The significantly highest number of positive reactions was detected against the tortoise isolate (5%). Most of the animals that tested positive for one of the snake isolates also tested positive in HI assays with the tortoise isolate. Of the samples from different origins, the sera from Great Britain showed the highest percentage of po...
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a novel type of paramyxovirus found in hungary in a masked water snake homalopsis buccata with pneumonia supports the suggested new taxonomy within the Ferlavirus genus
Veterinary Microbiology, 2013Co-Authors: Tibor Papp, Rachel E. Marschang, Maha Diekan Abbas, Szilvia L FarkasAbstract:Abstract During the course of a longitudinal survey on the occurrence of viruses in Hungarian exotic reptile collections a dead masked water snake ( Homalopsis buccata ) was submitted for virologic examination in September 2009. Based on history, gross pathological and histopathological findings paramyxovirus infection was suspected and later confirmed by RT-PCR and sequencing of the RNA dependent RNA polymerase (L), the hemaggluitinin–neuraminidase (HN) and the unknown (U) genes. Sequence analyses revealed that the detected virus, HoBuc-HUN09, belongs to the recently described “group C” within the genus Ferlavirus . Our paper presents the first description of this novel reptilian paramyxovirus from a homalopsid snake with mucopurulent pneumonia in Hungary.
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A unique novel reptilian paramyxovirus, four atadenovirus types and a reovirus identified in a concurrent infection of a corn snake (Pantherophis guttatus) collection in Germany.
Veterinary microbiology, 2011Co-Authors: Maha Diekan Abbas, Volker Schmidt, Rachel E. Marschang, Astrid Kasper, Tibor PappAbstract:In 2009, 26 clinical samples (organs and oral/cloacal swabs) from a total of 24 corn snakes (Pantherophis guttatus) from a single owner were sent to our laboratory to be tested for the presence of viruses. Paramyxoviruses (PMV), adenoviruses (AdV) and reoviruses were detected by RT-PCR, PCR and virus isolation methods. Three snakes were infected with all three viruses at the same time, while two other snakes had a double infection (PMV and reo, AdV and reo) and nine other snakes had a single infection with any of the three viruses. No viruses were detected in 10 animals. All isolated reoviruses were identical to one another and to the reptilian orthoreovirus isolate 55-02 in the partial RNA dependent RNA polymerase (RDRP) gene sequence. AdV partial polymerase sequences represented four different types, one of which was first described here: most similar to SnAdV-1, while the other three were identical to known types: SnAV-1, -2 and -3. However, the detected single PMV differed distinctly from described reptile PMV and was a new type. According to partial L gene, HN gene and U gene sequences it may be the first described representative of a third squamatid PMV cluster: "group C" within the proposed reptilian PMV genus "Ferlavirus". Nucleotide identity values for the L gene of the new PMV compared to group A viruses range between 76.5 and 80.3%, and between 80.5 and 81.2% compared to group B viruses. For the HN gene, these values were similar: 78.2-80% (A) and 79.9-80.5% (B) and somewhat lower for the U gene: 72.7-75.4% (A) and 69.7-70% (B). No reports on the prevalence of concurrent viral infection in captive snake populations have been published so far. The possibility of concurrent infection with several different viruses and subsequent consequences for animal health should be kept in mind when testing reptile samples for viruses.
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Comparison of paramyxovirus isolates from snakes, lizards and a tortoise.
Virus research, 2009Co-Authors: Rachel E. Marschang, Tibor Papp, Jens W FrostAbstract:Previously uncharacterized paramyxovirus (PMV) isolates from four snakes, three lizards and a tortoise were compared based on partial sequences of the L, HN, and U genes. Analysis of the sequences supported the classification of all reptilian PMVs in a separate genus (Ferlavirus) in the subfamily Paramyxovirinae. Within each of the gene segments, the squamatid isolates could be divided into two groups with a sequence divergence of 0.3-15.6% nt (0-6.8% aa) within the groups and 19.5-22.3% nt (5-7.4% aa) between the groups for the L gene, and 0.9-15.4% nt (0-6.9% aa) within the groups and 18.2-22.5% nt (4.4-9.5% aa) between the groups for the HN gene while higher values of 0.4-17.1% nt (0-13.3% aa) within the groups and 28.9-31.3% nt (25.5-27.8% aa) between the groups were found for the U gene. Isolates from lizards were found in both groups. There was no host species specificity in the grouping of the isolates from snakes and lizards. However, the L gene sequence obtained from the tortoise isolate differed significantly from the sequences obtained from the snake and lizard isolates. This isolate showed divergence values of 24.2-27% nt (18.5-20.9% aa) compared to the squamatid sequences. The tortoise isolate clustered together with the other reptilian PMVs, but not into any of the squamatid groups on the phylogenetic tree. It is hypothesized that this chelonian PMV has a more unique genome sequence as neither HN nor U gene parts could be amplified using newly designed consensus nested PCRs.
Volker Schmidt - One of the best experts on this subject based on the ideXlab platform.
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Three genetically distinct Ferlaviruses have varying effects on infected corn snakes (Pantherophis guttatus).
PLOS ONE, 2019Co-Authors: Michael Pees, Annkatrin Neul, Volker Schmidt, Tibor Papp, Ákos Gellért, Maha Diekan Abbas, J. Matthias Starck, Rachel E. MarschangAbstract:Ferlaviruses are important pathogens in snakes and other reptiles. They cause respiratory and neurological disease in infected animals and can cause severe disease outbreaks. Isolates from this genus can be divided into four genogroups-A, B, and C, as well as a more distantly related sister group, "tortoise". Sequences from large portions (5.3 kb) of the genomes of a variety of Ferlavirus isolates from genogroups A, B, and C, including the genes coding the surface glycoproteins F and HN as well as the L protein were determined and compared. In silico analyses of the glycoproteins of genogroup A, B, and C isolates were carried out. Three isolates representing these three genogroups were used in transmission studies with corn snakes (Pantherophis guttatus), and clinical signs, gross and histopathology, electronmicroscopic changes in the lungs, and isolation of bacteria from the lungs were evaluated. Analysis of the sequences supported the previous categorization of Ferlaviruses into four genogroups, and criteria for definition of Ferlavirus genogroups and species were established based on sequence identities (80% resp. 90%). Analysis of the Ferlavirus glycoprotein models showed parallels to corresponding regions of other paramyxoviruses. The transmission studies showed clear differences in the pathogenicities of the three virus isolates used. The genogroup B isolate was the most and the group A virus the least pathogenic. Reasons for these differences were not clear based on the differences in the putative structures of their respective glycoproteins, although e.g. residue and consequential structure variation of an extended cleavage site or changes in electrostatic charges at enzyme binding sites could play a role. The presence of bacteria in the lungs of the infected animals also clearly corresponded to increased pathogenicity. This study contributes to knowledge about the structure and phylogeny of Ferlaviruses and lucidly demonstrates differences in pathogenicity between strains of different genogroups.
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morphology and morphometry of the lung in corn snakes pantherophis guttatus infected with three different strains of Ferlavirus
Journal of Comparative Pathology, 2017Co-Authors: J M Starck, Annkatrin Neul, Volker Schmidt, T Kolb, S Franzguess, D Balcecean, M PeesAbstract:Summary Ophidian paramyxovirus (Ferlavirus) is a global threat to reptilian sauropsids in herpetological collections, with occasional but fatal effects. This study characterizes the effects of three different genetic strains of Ferlavirus on the dynamic changes of histology and morphometry of the lung of corn snakes (Pantherophis guttatus). Lungs from 42 corn snakes were either sham-infected or infected experimentally under standardized conditions. From 4 to 49 days after intratracheal inoculation, the lungs were examined qualitatively and quantitatively. Progressive microscopical changes were seen in the lung. Initially, increased numbers of heterophils were observed in the interstitium followed by proliferation and vacuolation of epithelial cells lining faveoli. Electron microscopy revealed loss of type-I pneumocytes, hyperplasia of type-II pneumocytes, and interstitial infiltrates of heterophils and mononuclear cells. With progression of disease the respiratory epithelium was initially overgrown by transformed type-II pneumocytes and later became multilayered. The results of the study suggest that the respiratory capacity of the lungs declines with disease development. The dynamics of disease development and histopathology differed in snakes infected with different Ferlavirus genogroups. Animals infected with virus genogroup B developed histopathological changes and morphometric changes more rapidly and of greater intensity than snakes infected with viruses from genogroups A or C.
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virus distribution and detection in corn snakes pantherophis guttatus after experimental infection with three different Ferlavirus strains
Veterinary Microbiology, 2016Co-Authors: M Pees, Annkatrin Neul, Volker Schmidt, Uwe Truyen, K Muller, Nadja Leinecker, Rachel E. MarschangAbstract:Ferlaviruses are important pathogens of snakes. However, factors influencing the pathogenicity of individual isolates as well as optimal protocols for virus detection in tissues of infected snakes have been insufficiently studied. The objectives of this study were to compare virus detection using previously described PCR and cell culture protocols following infection with three genetically distinct Ferlaviruses in corn snakes (Pantherophis guttatus) as a model species. Groups of 12 corn snakes were each inoculated intratracheally with a genogroup A, B, or C Ferlavirus. Tracheal washes and cloacal swabs were tested for virus shedding on days 16 and 28. Three animals were each euthanized on days 4, 16, 28, and 49. Beside immunohistochemistry of lung tissue, several organs (lung, intestine, pancreas, kidney, brain) were tested for the presence of Ferlavirus. Distinct differences were noted in the pathogenicity of the three viruses, with a genotype B isolate causing the greatest pathology. PCR was more sensitive in comparison to cell culture, but results varied depending on the tissues. Ferlaviruses spread rapidly into the tissues, including the brain. Overall average detection rate was 72%, and was highest on day 16. There were differences between the groups, with the most virulent strain causing 100% positive samples at the end of the study. Some snakes were able to clear the infection. Shedding via cloaca was seen only on day 28. For ante-mortem sampling, a tracheal wash sample is recommended, for post mortem diagnosis, a pooled organ sample should be tested.
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Detection of pathogens in Boidae and Pythonidae with and without respiratory disease.
Veterinary Record, 2013Co-Authors: Volker Schmidt, Rachel E. Marschang, Maha Diekan Abbas, Inna Ball, Istvan Szabo, R. Helmuth, Bastian Plenz, Joachim Spergser, Michael PeesAbstract: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.
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A unique novel reptilian paramyxovirus, four atadenovirus types and a reovirus identified in a concurrent infection of a corn snake (Pantherophis guttatus) collection in Germany.
Veterinary microbiology, 2011Co-Authors: Maha Diekan Abbas, Volker Schmidt, Rachel E. Marschang, Astrid Kasper, Tibor PappAbstract:In 2009, 26 clinical samples (organs and oral/cloacal swabs) from a total of 24 corn snakes (Pantherophis guttatus) from a single owner were sent to our laboratory to be tested for the presence of viruses. Paramyxoviruses (PMV), adenoviruses (AdV) and reoviruses were detected by RT-PCR, PCR and virus isolation methods. Three snakes were infected with all three viruses at the same time, while two other snakes had a double infection (PMV and reo, AdV and reo) and nine other snakes had a single infection with any of the three viruses. No viruses were detected in 10 animals. All isolated reoviruses were identical to one another and to the reptilian orthoreovirus isolate 55-02 in the partial RNA dependent RNA polymerase (RDRP) gene sequence. AdV partial polymerase sequences represented four different types, one of which was first described here: most similar to SnAdV-1, while the other three were identical to known types: SnAV-1, -2 and -3. However, the detected single PMV differed distinctly from described reptile PMV and was a new type. According to partial L gene, HN gene and U gene sequences it may be the first described representative of a third squamatid PMV cluster: "group C" within the proposed reptilian PMV genus "Ferlavirus". Nucleotide identity values for the L gene of the new PMV compared to group A viruses range between 76.5 and 80.3%, and between 80.5 and 81.2% compared to group B viruses. For the HN gene, these values were similar: 78.2-80% (A) and 79.9-80.5% (B) and somewhat lower for the U gene: 72.7-75.4% (A) and 69.7-70% (B). No reports on the prevalence of concurrent viral infection in captive snake populations have been published so far. The possibility of concurrent infection with several different viruses and subsequent consequences for animal health should be kept in mind when testing reptile samples for viruses.
Maha Diekan Abbas - One of the best experts on this subject based on the ideXlab platform.
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Three genetically distinct Ferlaviruses have varying effects on infected corn snakes (Pantherophis guttatus).
PLOS ONE, 2019Co-Authors: Michael Pees, Annkatrin Neul, Volker Schmidt, Tibor Papp, Ákos Gellért, Maha Diekan Abbas, J. Matthias Starck, Rachel E. MarschangAbstract:Ferlaviruses are important pathogens in snakes and other reptiles. They cause respiratory and neurological disease in infected animals and can cause severe disease outbreaks. Isolates from this genus can be divided into four genogroups-A, B, and C, as well as a more distantly related sister group, "tortoise". Sequences from large portions (5.3 kb) of the genomes of a variety of Ferlavirus isolates from genogroups A, B, and C, including the genes coding the surface glycoproteins F and HN as well as the L protein were determined and compared. In silico analyses of the glycoproteins of genogroup A, B, and C isolates were carried out. Three isolates representing these three genogroups were used in transmission studies with corn snakes (Pantherophis guttatus), and clinical signs, gross and histopathology, electronmicroscopic changes in the lungs, and isolation of bacteria from the lungs were evaluated. Analysis of the sequences supported the previous categorization of Ferlaviruses into four genogroups, and criteria for definition of Ferlavirus genogroups and species were established based on sequence identities (80% resp. 90%). Analysis of the Ferlavirus glycoprotein models showed parallels to corresponding regions of other paramyxoviruses. The transmission studies showed clear differences in the pathogenicities of the three virus isolates used. The genogroup B isolate was the most and the group A virus the least pathogenic. Reasons for these differences were not clear based on the differences in the putative structures of their respective glycoproteins, although e.g. residue and consequential structure variation of an extended cleavage site or changes in electrostatic charges at enzyme binding sites could play a role. The presence of bacteria in the lungs of the infected animals also clearly corresponded to increased pathogenicity. This study contributes to knowledge about the structure and phylogeny of Ferlaviruses and lucidly demonstrates differences in pathogenicity between strains of different genogroups.
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Diagnosis of Ferlaviruses in snakes and characterization of isolates based on gene sequences
2014Co-Authors: Maha Diekan AbbasAbstract:PMV are important pathogens for reptiles especially snakes and have been isolated from wild and private collections. During a period (2009-2011), a total of 495 clinical samples originating from 251 snakes of several families including Boidae, Pythonidae, and Colubridae were screened for the presence of PMV by RT-PCR described by Ahne et al. (1999) targeting a partial sequence of the L gene and virus isolation on the reptilian cell line viper heart cells (VH2). Samples with positive amplicons (566 bp, L gene) were subjected to RT-PCR targeting partial sequence of HN as described by Ahne et al. (1999) and Marschang et al. (2009) and U gene as described by Marschang et al. (2009). All RT-PCR positive amplicons were subjected to sequencing in order to exclude false positive results. Phylogenetic analyses using several programs (Phylip 3.36 and Mega 5.05) were carried out to explore the associations between the viruses detected and to broaden our understanding of their taxonomic relationships. Unspecific size products and specific size products with non specific amplicons were repeatedly obtained using the previously published protocol. Several trials were therefore carried out in an attempt to increase the specificity of the original RT-PCR protocol (Ahne et al., 1999) including optimization and sensitivity tests. Several concentrations of MgCl2 (1, 1.5, 2, 2.5) mM and different annealing temperatures (45, 48 and 51) Co were used in order to eliminate the unspecific size products. Sensitivity tests using several Ferlavirus isolates were conducted using new degenerate primers targeting the conserved L gene. Changes in annealing temperature and MgCl2 concentration did not decrease the number of unspecific reactions detected. Sensitivity tests showed that the RT-PCR protocol described by Ahne et al. (1999) has the highest sensitivity. However, this protocol has been shown to be highly unspecific. Sequencing of RT-PCR products is therefore necessary to ensure specific results. Ferlaviruses were detected in 5.97% of the snakes tested (15 of the 251 snakes screened). All Ferlavirus positive snakes were from the families Colubridae and Pythonidae. The low infection rate might indicate a fluctuation in the infection rate. A total of six different partial L gene sequences were obtained from 19 RT-PCR products using RT-PCR (L gene) and verified by sequencing. Three of these products clustered within subgroup B isolates. The one detected in an Indian python was 97% similar to FDLV (AY141760.2) (Subgroup A). Two (Pangut GER09 and Hobuc HUN09) were not assigned to subgroup A or B. However, they clustered together forming the first two representatives of the novel subgroup C within the Ferlavirus genus extending its classification into three squamate subgroups; A, B and C. Concurrent viral infections (PMV, reo and AdV) were detected in a group of corn snakes in Germany which highlight the significance for testing for different pathogens and different organs and tissues. In order to assess the degree of genetic diversity within this group of viruses, complete CDS regions of the F and HN genes from nine Ferlaviruses were sequenced and compared (on both nt and deduced aa sequence levels) with each other and with the corresponding sequences of other genera of the Paramyxovirinae. Phylogenetic analyses were conducted for each gene separately and for the concatenated sequences of F, HN and the extended portion (1544 nts) of L gene. On a genomic level, squamate Ferlaviruses are closely related; however they are distributed into three different genogroups (A, B and C). Deduced animo acid sequences of both F and HN genes of all Ferlavirus isolates revealed conserved domains corresponding to those described for other members of the Paramyxovirinae. However, the chelonid Ferlavirus showed for both genes and for some motifs some differences to the squamate (snake and lizard) group. PMV sind bedeutende Krankheitserreger bei Reptilien und konnten sowohl aus wildlebenden als auch in Gefangenschaft gehaltenen Populationen isoliert werden. Uber einen Zeitraum von drei Jahren (2009-2011) wurden insgesamt 495 klinische Proben von 251 Schlangen verschiedener Familien wie Boidae, Phytonidae und Colubridae auf PMV untersucht, dabei wurde das Virus mittels RT-PCR nach Ahne et al. (1999), die auf das L-Gen des Virus abzielt nachgewiesen und Virusisolation durch Vermehrung in VH2 (Viper Herz Zellen) durchgefuhrt. Proben mit positivem PCR-Ergebnis (566 bp, L-Gen) wurden weiterhin in RT-PCRs, welche auf die Gene HN und U abzielen, getestet. Alle positiven PCR-Ergebnisse wurden sequenziert um falsch-positive Resultate auszuschliesen. Die erhaltenen Sequenzen wurden des Weiteren phylogenetische Analysen mit unterschiedlichen Programmen (Phylip 3.36 und Mega 5.05) unterzogen um die genetischen Relationen zwischen den Subtypen weiter zu erforschen und das Verstandnis der taxonomischen Zusammenhange zu erweitern. Produkte mit unspezifischer Lange und Produkte mit spezifischer Lange aber unspezifischen Amplicons Sequencen wurden wiederholt getestet. Dafur wurden verschiedene Anpassungen des ursprunglichen Protokolls (Ahne et al, 1999) vorgenommen. Auserdem wurden Sensitivitatstests anhand neuer Primer, welche auf das konservierte L-Gen abzielten, durchgefuhrt. Veranderungen der Annealing Temperatur und der MgCl2 Konzentration hatten keinen Effekt auf die Anzahl unspezifischer Reaktionen. Sensitivitatstests zeigten, dass das RT-PCR Protokoll nach Ahne et al. (1999) die hochste Sensitivitat aufweist. Da die PCR nach diesem Protokoll jedoch einen hohen Anteil falsch positiver Resultate aufweist, sollten, um spezifische Ergebnisse zu erhalten, die Produkte stets sequenziert werden. Insgesamt wurden in 5.97% der getesteten Schlangen (15 von 251 Tieren) Ferlaviren nachgewiesen, dabei gehorten alle positiv getesteten Tiere den Familien Colubridae und Phytonidae an. Die in der Studie gemessene geringe Infektionsrate konnte durch eine Schwankung in der Gesamtinfektionsrate zustande gekommen sein. Aus den 19 RT-PCR Produkten wurden sechs unterschiedliche partielle L-Gen Sequenzen nachgewiesen. Drei dieser Produkte liesen sich dem Subtyp B zuordenen, eines aus einer indischen Phyton wies 97% Homologie zum FDLV (AY141760.2), (Subgruppe A), auf. Zwei Isolate (Pangut GER09 and Hobuc HUN09) konnten in keines der bestehenden Cluster der Subtypen A oder B eingeordnet werden. Sie bildeten zusammen eine eigene Gruppe und sind damit die ersten Mitglieder des neuen Subtyps C innerhalb des Genus Ferlavirus, welches nun erweitert und in die drei squamaten Subgrupen A, B und C unterteilt werden kann. Es wurden multiple Virusinfektionen (PMV, reo und AdV) in einer Gruppe von Kornnattern/Kornschlangen in Deutschland entdeckt, was die Bedeutung des Testens verschiedener Pathogene und unterschiedliche Organe und Geweben hervorhebt. Um den Grad der genetischen Diversitat innerhalb dieser neuen Virusgruppe einzuschatzen, wurden die kompletten CDS Regionen der Gene F und HN von neun Ferlaviren sequenziert und untereinander und mit entsprechenden Sequenzen anderer Mitglieder der Paramyxovirinae verglichen (sowohl auf der Ebene der Nukleotidsequenzen als auch der abgeleiteten Aminosaure-sequenz). Die phylogenetischen Analysen wurden dabei zum einen fur jedes Gen separat und des weiteren anhand der aneinandergereiten Sequenzen der Gene F, HN und einem erweiterten Anteil des L-Gens (1544 Nuckleotide) durchgefuhrt. Dabei konnte gezeigt werden, dass die squamaten Ferlaviren auf genomischer Ebene eng miteinander verwandt sind, sich jedoch in drei verschiedene Genogruppen (A, B und C) aufspalten. Hergeleitete Aminosauresequenzen der F und HN Gene aller Ferlavirusisolate in dieser Studie zeigten konservierten Domanen ubereinstimmend mit denen anderer Mitglieder der Paramyxovirinae, allerdings konnten in beiden Genen und einigen Motiven Unterschiede zwischen dem cheloniden Ferlavirus und den squamaten Isolaten (Schlangen und Echsen) nachgewiesen warden.
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DETECTION OF ANTIBODIES AGAINST PARAMYXOVIRUSES IN TORTOISES
Journal of Zoo and Wildlife Medicine, 2013Co-Authors: Reinhild Rösler, Rachel E. Marschang, Tibor Papp, Maha Diekan Abbas, Ftä Mikrobiologie, Zb ReptilienAbstract:Abstract: Sera from a total of 202 tortoises from six countries and nine species were tested for antibodies against four different reptilian paramyxoviruses (Ferlaviruses, ferlaVs) by hemagglutination inhibition (HI) test. The viruses used were a tortoise PMV (tPMV) and three squamatid PMV isolates, each belonging to a different subgroup of ferlaV within the genus Ferlavirus. HI tests revealed that antibodies against ferlaVs occurred regularly in the tested samples (5.5%). One and a half percent of the tested samples have measurable antibody titers against the group A isolate, 3% had antibodies against the group B isolate, and 1% had antibodies against the group C isolate. The significantly highest number of positive reactions was detected against the tortoise isolate (5%). Most of the animals that tested positive for one of the snake isolates also tested positive in HI assays with the tortoise isolate. Of the samples from different origins, the sera from Great Britain showed the highest percentage of po...
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a novel type of paramyxovirus found in hungary in a masked water snake homalopsis buccata with pneumonia supports the suggested new taxonomy within the Ferlavirus genus
Veterinary Microbiology, 2013Co-Authors: Tibor Papp, Rachel E. Marschang, Maha Diekan Abbas, Szilvia L FarkasAbstract:Abstract During the course of a longitudinal survey on the occurrence of viruses in Hungarian exotic reptile collections a dead masked water snake ( Homalopsis buccata ) was submitted for virologic examination in September 2009. Based on history, gross pathological and histopathological findings paramyxovirus infection was suspected and later confirmed by RT-PCR and sequencing of the RNA dependent RNA polymerase (L), the hemaggluitinin–neuraminidase (HN) and the unknown (U) genes. Sequence analyses revealed that the detected virus, HoBuc-HUN09, belongs to the recently described “group C” within the genus Ferlavirus . Our paper presents the first description of this novel reptilian paramyxovirus from a homalopsid snake with mucopurulent pneumonia in Hungary.
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Detection of pathogens in Boidae and Pythonidae with and without respiratory disease.
Veterinary Record, 2013Co-Authors: Volker Schmidt, Rachel E. Marschang, Maha Diekan Abbas, Inna Ball, Istvan Szabo, R. Helmuth, Bastian Plenz, Joachim Spergser, Michael PeesAbstract: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.