Detection of nidoviruses in live pythons and boas

Tierarztl Prax, Kleintiere Heimtiere. 44(6): PDF   doi: 10.15654/TPK-151067

(Marschang RE, Kolesnik E.)


Nidoviruses have recently been described as a putative cause of severe respiratory disease in pythons in the USA and Europe. The objective of this study was to establish the use of a conventional PCR for the detection of nidoviruses in samples from live animals and to extend the list of susceptible species.


A PCR targeting a portion of ORF1a of python nidoviruses was used to detect nidoviruses in diagnostic samples from live boas and pythons. A total of 95 pythons, 84 boas and 22 snakes of unknown species were included in the study. Samples tested included oral swabs and whole blood.


Nidoviruses were detected in 27.4% of the pythons and 2.4% of the boas tested. They were most commonly detected in ball pythons (Python [P.] regius) and Indian rock pythons (P. molurus), but were also detected for the first time in other python species, including Morelia spp. and Boa constrictor. Oral swabs were most commonly tested positive.


The PCR described here can be used for the detection of nidoviruses in oral swabs from live snakes. These viruses appear to be relatively common among snakes in captivity in Europe and screening for these viruses should be considered in the clinical work-up.


Nidoviruses are believed to be an important cause of respiratory disease in pythons, but can also infect boas. Detection of these viruses in live animals is now possible and can be of interest both in diseased animals as well as in quarantine situations.

Repeated detection of an invertebrate iridovirus (IIV) in amphibians

Journal of Herpetological Medicine and Surgery 26:P.54-58. 

(Stöhr AC, Papp T, Marschang RE)


Invertebrate iridoviruses (IIVs) (family: Iridoviridae) are known pathogens for invertebrates causing high mortality and reduced fertility in affected insects. During the last years, IIVs have also been increasingly found in lizards in association with nonspecific clinical signs. It has been hypothesized that IIVs from insects are able to infect reptiles.

In the years 2010-2011, IIVs were repeatedly detected via PCR and virus isolation methods in routine diagnostic samples from different amphibians: three blue poison dart frogs (Dendrobates tinctorius azureus), four edible frogs (Pelophylax kl. esculentus), a giant ditch frog (Leptodactylus fallax), an Amazon milk frog (Trachycephalus resinifictrix), mixed organs from agile frogs (Rana dalmatina), a black-spined toad (Bufo melanostictus), and one Lake Urmia newt (Neurergus crocatus). IIVs were found in skin swabs from apparently healthy animals, as well as in multiple organs of frogs that died of unknown causes. Prey insects (crickets) from one owner also tested positive for the presence of IIV. The obtained partial sequences from the major capsid protein (MCP) gene (222nt) from each of these were 100% identical to each other and 98% identical to IIV-6, the type species of the genus Iridovirus. Although the pathogenicity of IIV in amphibians remains unclear, these findings provide further evidence that IIVs may be able to infect vertebrates under some conditions and underline the importance of the genus Iridovirus in vertebrates.

High prevalence of intestinal adenocarcinoma in a captive population of Amazon milk frog (Trachycephalus resinifictrix)

Journal of Zoo and Wildlife Medicine 2016 Dec;47(4):1061-1068.  doi: 10.1638/2016-0037.1

(López J, Barbón AR, Smithyman J, Goetz M, Marschang RE, Dastjerdi A, Stidworthy MF)


A series of eight cases of intestinal adenocarcinoma in Amazon milk frog (Trachycephalus resinifictrix) is described. All cases presented with signs of inappetence and weight loss, and evidence of large intestinal distention on gross postmortem, with six of the eight cases showing a grossly visible large intestinal mass. Histologic examination identified the mass as an intestinal adenocarcinoma in all cases. No specific etiologic agent could be identified. This is the first report of neoplasia in the Amazon milk frog, and the first reported series of amphibian gastrointestinal neoplasia.

Detection of testudinid herpesvirus type 4 in a leopard tortoise (Stigmochelys pardalis)

Tierarztl Prax, Kleintiere Heimtiere. 2016 Aug 17;44(4):p. 283-6. doi: 10.15654/TPK-150843

(Kolesnik E, Mittenzwei F, Marschang RE)


Several animals from a mixed species collection of tortoises in Germany died unexpectedly. Some of the affected leopard tortoises (Stigmochelys pardalis) from this group showed respiratory signs. Samples were collected from one of the ill tortoises, and a Mycoplasma spp. and a herpesvirus were detected by PCR. Sequencing of a portion of the DNA polymerase gene of the herpesvirus showed 99% identity with testudinid herpesvirus 4, previously described only once in a bowsprit tortoise (Chersina angulata) in the United States.

Viral diseases of reptiles

In Practice. 38(6):p. 275–385, JUN 2016   DOI: 10.1177/1040638716628584

(Rachel E. Marschang)


As reptile medicine has grown and more is known about various diseases in these animals, the importance of some viruses in clinical reptile practice has become more widely understood. More and more studies have become available on the prevalence of infections with specific viruses in different reptiles, as well as clinical signs associated with these infections. An increasing body of knowledge on the genomes of specific viruses is available, which has also led to the development of more sensitive and more specific testing methods. The availability of new, high-throughput sequencing methods has allowed the detection of previously unknown viruses that are associated with specific disease syndromes in the tissues of reptiles. All of this has led to a much expanded pallet of diagnostic tests available to practitioners, while knowledge of the interpretation of results may, in many cases, have lagged behind. This article describes some important viruses in reptiles that may be commonly seen in private practice, and how to diagnose them.

Development of a consensus reverse transcription polymerase chain reaction assay for the specific detection of tortoise picornaviruses

J Vet Diagn Invest. 28: P.309-314.

(Marschang RE)


Picornaviruses (PVs) of different terrestrial tortoise species, previously designated as Virus "X," have been frequently detected from various tissues by virus isolation in Terrapene heart cell culture as the preferred laboratory method for diagnosis. Here, we describe the development of 2 diagnostic reverse transcription (RT)-PCR-based assays for the identification and characterization of tortoise PVs belonging to the tentative genus Topivirus To test the novel diagnostic systems, PVs were isolated from swab and tissue samples collected in Germany, Italy, and Hungary between 2000 and 2013. All 25 tested isolates gave positive results with both novel consensus primer sets. Sequencing of the amplified products confirmed that all studied viruses were members of the new proposed genus Topivirus Phylogenetic analyses clearly distinguished 2 lineages within the genus. Based on sequence analysis, no association was observed between the geographic distribution and genetic relatedness. Furthermore, no strict host specificity was indicated. The PCR-based diagnosis may provide a time-saving and sensitive method to detect tortoise PVs, and evaluation of PV presence in these animals may help control virus spread.

Usefulness of serum cardiac troponin I concentration as a marker of survival of harbor seal (Phoca vitulina) pups during rehabilitation

(Sonja Fonfara, Janne Sundermeyer, Domingo Casamian Sorrosal, Corinna Weber, Tanja Rosenberger)


To measure serum cardiac troponin I (cTnI) concentrations in orphaned harbor seal (Phoca vitulina) pups at various points during rehabilitation in a seal rescue center and determine whether cTnI concentration was associated with survival during rehabilitation and duration of rehabilitation. 


Serial cross-sectional study. 


Fifty-five 2- to 9-day-old harbor seal pups. 


Blood samples for serum cTnI concentration measurement, CBC, and serum biochemical analysis were obtained from seal pups at admission into a seal rescue center, after 2 weeks of rehabilitation at the center, and prior to release. Serum cTnI concentrations were compared between seals that did or did not survive rehabilitation. 


Median serum cTnI concentration was highest at admission (0.03 ng/mL). After 2 weeks, the median value was 0.01 ng/mL; prior to release, it was 0.01 ng/mL. Seal pups that were found to have died during or after rehabilitation (n = 7) had a significantly higher median serum cTnI concentration at admission (0.06 ng/mL) than did seal pups that survived rehabilitation (and for which the postrelease fate was unknown; 48; 0.03 ng/mL). No correlation was identified between serum cTnI concentration and duration of rehabilitation. 


The results of this study suggested some degree of myocardial injury was present in most of the orphaned seal pups admitted for rehabilitation. Measurement of serum cTnI concentration in seal pups at admission might provide prognostic information about their likelihood of survival during or after rehabilitation.

Herd-level seroprevalence of Neospora caninum infection in dairy cattle in central and northeastern Poland

Acta Parasitol. 2016 Jan;61(1):p. 63-65

J. Kowalczyk, Michał Czopowicz, Corinna N. Weber, Elisabeth Müller, Lucjan Witkowski and Jarosław Kaba(Sławomir)


A serosurvey was carried out to estimate the herd-level seroprevalence of Neospora caninum infection in cattle in central and northeastern Poland. Ninety seven dairy cattle herds from 2 provinces of Poland (Podlaskie, 47 herds and Łodzkie, 50 herds) were randomly enrolled in the study using two-stage cluster method. A simple random selection was applied within each herd to select a sample of adult cows (≥18 month-old). A total number of 734 cows were enrolled in the study. The animals were screened with a commercial competitive ELISA (Bio-X Diagnostics, Belgium). To calculate true herd-level seroprevalence test sensitivity and specificity were adjusted from an individual- to a herd-level using FreeCalc method. The true overall herd-level seroprevalence of N. caninum infection was 56.7% (95% CI: 47.5%, 65.9%). The true herd-level seroprevalence in Podlaskie was 63.3% (95% CI: 43.0%, 83.6%) and 50.5% (95% CI: 32.8%, 68.2%) in Łodzkie province and these figures did not differ significantly between the two provinces (chi2 test p = 0.238). One hundred forty three of 734 cows (19.5%) were seropositive which gave the true overall individual-level seroprevalence of 20.1% (95% CI: 17.4%, 23.2%). Percentage of seropositive cows in each herd varied from 6% to 80%. This study is the first epidemiological investigation of herd-level seroprevalence of N. caninum infection in Polish dairy cattle population. In conclusion, the result of the study confirmed previous data that N. caninum infection is widespread in the Polish cattle population and thus should be considered as a potential cause of spontaneous abortions.

Polypoid hypertrophic gastropathia similar to human Morbus Ménétrier-Disease in the pars pylorica of a warmblood-mixgelding

Pferdeheilkunde 32 (2016) p. 606-610

(Rapp S.; Aupperle H.)

Language: German


A 12-year-old warmblood-mix gelding showed clinical signs of poor feed intake and decreased performance. Additionally cough was des-cribed. Therefore the gelding was first treated against chronic bronchitis. The cough disappeared but clinical signs of weight loss and poorfeed intake prolonged. That was the reason why an endoscopic examination of the stomach was performed. First endoscopy showed a largeconcretion of feed material. A visualization of the pars glandularis and the pars non-glandularis as well as the pylorus was not possible. Therefore a laxative therapy with paraffin and Glauber solution over a nasogastric tube was initiated. The endoscopic examination of thestomach could be done on the third day. Abnormal radially arranged mucosal growths on the pars pylorica ensured that polyps were seen.The margo plicatus also showed ulcera (ESGD 3/4). The horse was treated with omeprazole and metoclopramide for six weeks. After that,a second endoscopic examination of the stomach was performed. As well as the ulcera on the margo plicatus as the gastric polyps of thepars pylorica showed no improvement. Another period of treatment should start for about two months of duration. In that time, the geldingshould be kept on pasture during the day and get on shavings over the night. Next to gras, soaked cobs of hay should be offered for feed.Ten days later, the gelding showed acute signs of colic at home. The condition of the horse was getting bad so the decision for surgery wasmade. During the laparotomy under general anesthesia some incarcerated small intestine due to a hernia foraminis omentalis was detected.The owner did not give the permission for resection (3m), so the gelding was euthanized during surgery. After that a transmural biopsy wastaken from the region of the pars pylorica due to scientific interests. A post-mortem examination was not desired by the owner. The tissuesample was fixed in 4% formalin and sent for histological examination. Here a diffuse polypoid hypertrophic gastropathy (Morbus Ménétrier)was diagnosed. Main characteristics were the markedly elongated foveolae gastricae of the hyperplastic gastric mucosa without any signs ofmalignancy. Helicobacter sp. was not found in the pathological examination. So far the Morbus Ménétrier was not described in a horse yet.

Triple infection with agamid adenovirus 1, Encephaliton cuniculi-like microsporidium and enteric coccidia in a bearded dragon (Pogona vitticeps)

Tierärztliche Praxis Kleintiere 2016: Heft 5, S.355-358 

(Lionel Schilliger; Véronique Mentré; Rachel E. Marschang; Alexandra Nicolier; Barbara Richter)


A 2-month-old juvenile central bearded dragon was presented for anorexia and cachexia. Another specimen from the same cage had died suddenly 2 weeks prior. Fecal analysis revealed a high quantity of Isospora amphiboluri and a few pinworm eggs. Other examinations were not performed and the animal died a few days later despite supportive care. A third individual from the same cage presented with anorexia and a distended coelom and was euthanized. In this third dragon, histological examination revealed intestinal coccidiosis, basophilic intranuclear inclusions compatible with adenovirus infection, acute hepatic necrosis with intrahepatocytic and intraenteritic organisms typical of microsporidia and renal gout. 

A PCR confirmed the diagnosis of adenovirosis. Sequencing showed that the PCR product was 100% identical to the corresponding portion of the agamid adenovirus 1 genome. A PCR for the detection of Encephalitozoon (E.) cuniculi was positive. Partial sequencing revealed 100% identity to an E. cuniculi-like organism previously found in bearded dragons. In cases where environmental factors such as poor hygiene or stress can be excluded, the presence of opportunistic pathogens in high numbers can be due to a systemic (viral) infection with temporary immunosuppression.

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What do the values really mean? - A comparison of an enzyme-linked fluorescent assay vs chemiluminescent immunoassay for measuring serum progesterone in bitches

Proceedings of the 8th International Symposium on Canine and Feline Reproduction ISCFR 2016, Paris, P.223

(Jenny Uhlmann, Corinna Weber, Uwe Kuechenmeister, Andrea Muennich)

Cutaneous and Subcutaneous Soft Tissue Tumours in Snakes: A Retrospective Study of 33 Cases

Journal of Comparative Pathology, 155 (2016) 76-87.  doi:10.1016/j.jcpa.2016.05.009

(J. Dietz, K. O. Heckers, H. Aupperle and M. Pees)


Cutaneous and subcutaneous soft tissue tumours have been rarely described in detail in snakes. Several malignant entities show strikingly similar histological patterns and therefore the term soft tissue sarcoma (STS) has become a standard histopathological diagnosis. The present study characterizes soft tissue tumours in 33 snakes. Samples included 29 surgically excised masses and four carcasses. Additionally, six animals were humanely destroyed and submitted for necropsy examination following tumour recurrence. Benign neoplasms (n = 8) were described as lipomas of varying differentiation. Recurrence was observed in two of five snakes in which the clinical course was recorded. Malignant neoplasms (n = 25) were diagnosed as STS and graded according to a three-point system previously applied to canine STS. Five (20%) of the primary tumours were classified as grade 1, eleven (44%) as grade 2 and nine (36%) as grade 3 sarcomas. Clinically, recurrence of STS was observed in 11 of 17 cases with available follow-up information. Pathologically, multiple cutaneous metastases were found in one grass snake (Natrix natrix), while visceral metastases were observed in one carpet python (Morelia spilota) and two corn snakes (Pantherophis guttatus). Metastatic risk appears to increase with histological grade. Surgical excision generally represents the current therapy of choice for STS. This study includes the first reports of conventional lipomas in a ribbon snake (Thamnophis radix), angiolipomas in a black-headed python (Aspidites melanocephalus) and a corn snake as well as of STS in a Jamaican boa (Epicrates subflavus), emerald tree boa (Corallus caninus), grass snake (N. natrix), African house snake (Lamprophis fuliginosus), California kingsnake (Lampropeltis getula californiae) and common garter snake (Thamnophis sirtalis).

Whole genome characterization of a chelonian orthoreovirus strain identifies significant genetic diversity and may classify reptile orthoreoviruses into distinct species

Virus Research, Volume 215, 2 April 2016, Pages 94–98. ELSEVIER

(Kugler R, Marschang RE, Ihász K, Lengyel G, Jakab F, Bányai K, Farkas S.)


In this study we report the sequence and phylogenetic characterization of an orthoreovirus strain, CH1197/96, isolated from a spur-thighed tortoise (Testudo graeca) on chicken embryo fibroblast cells. The 23,957bp long genome sequence was obtained by combined use of semiconductor and capillary sequencing. Although the genomic characterization showed that the virus was most similar to the bush viper reovirus strain, 47/02, and in phylogenies performed with all segments the two strains formed a monophyletic group, the nucleotide (48.4-70.3%) and amino acid (39.2-80.7%) sequence identity values were moderate between the two reptile origin reoviruses. Based on our results and existing classification criteria for the genus Orthoreovirus, the tortoise reovirus strain CH1197/96 might be the first representative of a novel reptilian origin Orthoreovirus species.

Virus distribution and detection in corn snakes (Pantherophis guttatus) after experimental infection with three different ferlavirus strains

Vet Mircrobiol 182, 2016: p.213-222.   doi:10.1016/j.vetmic.2015.11.024.

(Pees M, Neul A, Müller K, Schmidt V, Truyen U, Leineceker N, Marschang RE)


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.