First recorded case of paramyxovirus infection introduced into a healthy snake collection in Croatia
© The Author(s). 2017
Received: 5 November 2016
Accepted: 30 March 2017
Published: 8 April 2017
In the present study, we describe the first paramyxovirus infection in a snake collection in Croatia caused by an introduction of new snakes that were not previously tested and didn’t show any signs of disease.
In less than a month after introduction into a healthy colony, new snakes began to show respiratory symptoms (i.e. mouth opening, wheezing, etc.) and died within a month and a half after antibiotic therapy was applied. The same symptoms and a high mortality rate were then observed in in-contact snakes from other collections belonging to different snake families.
Two entries of new snakes in different time periods were recorded and recognized as possible sources of infection. We stress the need for veterinary health control and monitoring of snakes prior to transportation as well as implementing obligatory quarantine measures to minimize the risk of infection among newly established snake groups.
KeywordsSnake Paramyxovirus Nested RT-PCR Phylogenetic characterization Infection Croatia
Paramyxoviruses (PMV) are negative sensed single stranded RNA viruses with a helical nucleocapsid packaged in a pleomorphic envelope and are classified as members of the Paramyxoviridae family, genus Ferlavirus [5, 6, 11]. According to studies based on partial sequences of L, HN, and U genes [1, 11, 3], members of the genus Ferlavirus are grouped into three subgroups, subgroups A, B and C. There are numerous reports of Ferlavirus isolation worldwide from zoological and private collections. Up to now, the occurrence of PMVs has been detected in several snake (sub) families: Colubridae, Elapidae, Viperidae, Crotalidae, Boidae and Pythonidae [4, 7, 9, 11]. The purchase and introduction of new reptiles into an established collection is of high risk in regards to disease spread and manifestation as well as a probable cause of a higher mortality rate. It has been demonstrated that viral infections such as inclusion body disease (IBD) and PMV , as well as some bacterial infections, represent a significant risk when latently infected snakes are introduced into healthy snake collections .
The objective of this study was to detect PMV RNA in samples derived during 2015 from a collection of exotic snakes showing signs of disease. Furthermore, sequencing of a highly conserved fragment within PMV L-gene region was performed to define the genetic heterogeneity of the virus isolates and to compare the sequences with previously described ones from the GenBank. The aim was to retroactively define the probable sources of infection. In consensus with earlier publications [8, 10] we used a nested RT-PCR protocol targeting the L-gene  as the most sensitive method currently available for basic diagnostics of PMV infection.
In less than a month after introduction of Dendroaspis jamesoni jamesoni (purchased from a private keeper from the Czech Republic in September 2014) into a healthy colony, the mamba began to show respiratory symptoms (i.e. mouth opening, wheezing, etc.) and died within a month and a half after antibiotic therapy (Marbocyl for 10 days) was applied. The same symptoms and a high mortality rate were then observed in other snakes from collections belonging to different snake families. In February 2015 the breeder purchased new snakes and introduced them into the collection. One pair (male and female) of Dendroaspis viridis (caught in the wild in Ghana) was purchased from a private keeper from the Czech Republic, whereas snakes Bitis nasicornis were purchased from Poland. Dendroaspis viridis didn’t show any respiratory symptoms but was in a poor condition and died within a month and a half. The snake owner noticed the same clinical signs and a high mortality rate in other snakes from his collection belonging to different snake families. Autopsy showed signs of fibrotic pneumonia.
Animals used in the study
Origin of snakes
Sample/time of death
Crotalus m. molossus
Imported in September 2014 from Germany
Crotalus m. molossus
Imported in September 2014 from Germany
Crotalus m. pyrrus
Imported in 2013 from Germany
Imported in February 2015 from Czech Republic
Dendroaspis j. jamesoni
Imported in September 2014 from Czech Republic
Imported in 2012 from South Africa
Imported in February 2015 from Poland
Imported in 2009 from Croatia
Tracheal/ cloacal swab
Crotalus l. klauberi
Imported in 2014 from Austria
Tracheal/ cloacal swab
Imported in 2011 from Germany
The amplification of PMV L-gene fragments was adopted from previously published methodology . One-step reverse transcription PCR and nested PCR assays for the amplification of 627-bp and 566-bp long products of L-gene, respectively, were performed using SuperScript III One-Step RT-PCR with PlatinumTaq (Invitrogen, USA) and PlatinumBlue PCR SuperMix (Invitrogen, USA) using primer pairs and the amplification procedure described by Ahne et al. . PCR products were separated by agarose gel electrophoresis in a 1.5% agarose gel stained with ethidium bromide and visualized by UV transillumination. Twelve gel purified PCR amplicons (Wizard SV gel and PCR Clean up system, Promega, USA) derived from 10 snakes were sequenced directly (Macrogen Inc., Amsterdam, The Netherlands). To determine the phylogenetic grouping of the obtained sequences (GenBank KU207735 to KU207746) the prototype PMV sequences  were retrieved from GenBank. Also, similar sequences obtained using BLAST algorithm (http://www.ncbi.nlm.nih.gov) were included in the study. Sequences were aligned using ClustalX, version 2.0  and analyzed by MEGA 5 .
All Croatian PMV isolates showed high nucleotide identity with sequences of PMV isolates from Germany (GQ277611, AF351137, GQ277612, GU393348 and GQ277617) and Austria (AF286043).
According to the data gained, there are two possible sources of infection. The first source of infection was Dendroaspis jamesoni jamesoni (CRO-PMV-4) imported from the Czech Republic in September 2014. The disease spread to Crotalus molosus molosus (CRO-PMV 1). Virus sequences isolated from these snakes are mutually identical. The second sources of infection were Dendroaspis viridis (CRO-PMV-3) and Bitis nasicornis (CRO-PMV-12) that were introduced into the collection in February 2015. We can presume that the disease spread from Bitis nasicornis (CRO-PMV-12) to Crotalus molosus molosus (CRO-PMV-2) since the virus sequences isolated from these snakes are mutually identical. However, the source of infection for the strain isolated from Crotalus molosus Pyrrus (CRO-PMV-5) could not be identified. We presume that point mutations could be initiated by multiple passages in vivo originating from previously introduced clusters since this snake was in the colony since 2013. The passages in Vero cells couldn’t be responsible for the observed mutations, since the sequences from swab samples and cell culture supernatants were 100% identical.
Our results imply that the infection was introduced into the healthy collection by new snakes that were not previously tested or showing signs of disease. Since no vaccine is presently commercially available, basic veterinary health checks and quarantine measures are of great importance to minimize the risk of infection among newly established snake groups.
The authors are grateful to Mr. Neven Vrbanic for providing the experimental samples and for excellent technical assistance in handling snakes.
Availability of data and materials
All data generated or analysed during this study are included in this manuscript.
JP made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data. TK, DF, MLB, LJ have been involved in drafting the manuscript or revising it critically for important intellectual content. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Taking swabs from live snake and sampling tissue of dead animals is done in accordance with good veterinary practice. On the other side, Croatian legislation is harmonized with the Code of good veterinary practice. (https://ec.europa.eu/digital-agenda/en/content/federation-veterinarians-europe-code-good-veterinary-practice)(http://www.fve.org/news/publications/pdf/gvp.pdf).
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