Reptarenaviruses in apparently healthy snakes in an Australian zoological collection.

BACKGROUND: Inclusion body disease (IBD) is a disease of snakes with a global distribution and has recently been shown to be caused by reptarenaviruses. Testing for this group of viruses in asymptomatic snakes allows the association between infection and disease to be further elucidated. METHODS: A reptarenavirus was detected by RT-PCR in a reticulated python (Malayopython reticulatus) from an Australian zoological collection that was open-mouth breathing and had erythematous oral mucosa. Another 27 pythons, 4 elapids, 2 colubrids and 2 boas from this collection were then screened. From these animals, swabs, whole blood and/or tissue were tested for reptarenaviruses by RT-PCR. Additionally, blood films from 10 snakes were examined by light microscopy for the presence of inclusion bodies. The majority of samples were collected over a 484-day period. RESULTS: A total of 8 animals were RT-PCR-positive (8/36 = 22.2%): 6 were pythons, 1 was a corn snake (Pantherophis guttatus) and 1 was a Madagascar tree boa (Sanzinia madagascariensis). From them, 57 samples were collected, but only one from each animal was RT-PCR-positive (8/57 = 14.0%). From all 36 animals in this study, 8/182 samples were RT-PCR-positive (4.4%). Inclusion bodies were not recognised in any of the blood films. Only the reticulated python showed signs of illness, which improved without any further intervention. All other RT-PCR-positive snakes were apparently healthy throughout the duration of the study. CONCLUSION: This study showed a weak association between the presence of reptarenaviruses and disease. Testing serially collected swab and whole-blood samples increased the number of animals in which reptarenaviruses were detected.

Morphological Pulmonary Diffusion Capacity for Oxygen of Burmese Pythons (Python molurus): a Comparison of Animals in Healthy Condition and with Different Pulmonary Infections.

A qualitative and quantitative morphological study of the pulmonary exchange capacity of healthy and diseased Burmese pythons (Python molurus) was carried out in order to test the hypothesis that the high morphological excess capacity for oxygen exchange in the lungs of these snakes is one of the reasons why pathological processes extend throughout the lung parenchyma and impair major parts of the lungs before clinical signs of respiratory disease become apparent. Twenty-four Burmese pythons (12 healthy and 12 diseased) were included in the study. A stereology-based approach was used to quantify the lung parenchyma using computed tomography. Light microscopy was used to quantify tissue compartments and the respiratory exchange surface, and transmission electron microscopy was used to measure the thickness of the diffusion barrier. The morphological diffusion capacity for oxygen of the lungs and the anatomical diffusion factor were calculated. The calculated anatomical diffusion capacity was compared with published values for oxygen consumption of healthy snakes, and the degree to which the exchange capacity can be obstructed before normal physiological function is impaired was estimated. Heterogeneous pulmonary infections result in graded morphological transformations of pulmonary parenchyma involving lymphocyte migration into the connective tissue and thickening of the septal connective tissue, increasing thickness of the diffusion barrier and increasing transformation of the pulmonary epithelium into a columnar pseudostratified or stratified epithelium. The transformed epithelium developed by hyperplasia of ciliated cells arising from the tip of the faveolar septa and by hyperplasia of type II pneumocytes. These results support the idea that the lungs have a remarkable overcapacity for oxygen consumption and that the development of pulmonary disease continuously reduces the capacity for oxygen consumption. However, due to the overcapacity of the lungs, this reduction does not result in clinical signs and disease can progress unrecognized for an extended period.

Identification of snake arenaviruses in live boas and pythons in a zoo in Germany.

OBJECTIVE: Recent studies have described the detection and characterisation of new, snake specific arenaviruses in boas and pythons with inclusion body disease (IBD). The objective of this study was to detect arenaviral RNA in live snakes and to determine if these were associated with IBD in all cases. Samples for arenavirus detection in live animals were compared. Detected viruses were compared in order to understand their genetic variability. MATERIALS AND METHODS: Esophageal swabs and whole blood was collected from a total of 28 boas and pythons. Samples were tested for arenaviral RNA by RT-PCR. Blood smears from all animals were examined for the presence of inclusion bodies. Internal tissues from animals that died or were euthanized during the study were examined for inclusions and via RT-PCR for arenaviral RNA. All PCR products were sequenced and the genomic sequences phylogenetically analysed. RESULTS: Nine live animals were found to be arenavirus-positive. Two additional snakes tested positive following necropsy. Five new arenaviruses were detected and identified. The detected viruses were named "Boa Arenavirus Deutschland (Boa Av DE) numbers 1-4" and one virus detected in a python (Morelia viridis) was named "Python Av DE1". Results from sequence analyses revealed considerable similarities to a portion of the glycoprotein genes of recently identified boid snake arenaviruses. CONCLUSIONS: Both oral swabs and whole blood can be used for the detection of arenaviruses in snakes. In most cases, but not in all, the presence of arenaviral RNA correlated with the presence of inclusions in the tissues of infected animals. There was evidence that some animals may be able to clear arenavirus infection without development of IBD. This is the first detection of arenaviruses in live snakes. CLINICAL RELEVANCE: The detection of arenaviruses in live snakes is of importance for both disease detection and prevention and for use in quarantine situations. The findings in this study support the theory that arenaviruses are the cause of IBD, but indicate that in some cases it may be possible for animals to clear arenavirus infections without developing IBD.

[Herpesvirus detection in clinically healthy West African mud turtles (Pelusioscastaneus)]. / Nachweis eines Herpesvirus bei klinisch gesunden Klappbrust-Pelomedusenschildkröten (Pelusios castaneus).

OBJECTIVE: First description of a herpesvirus in West African mud turtles. MATERIALS AND METHODS: A herpesvirus was detected in two clinically healthy West African mud turtles (Pelusios castaneus) by PCR during a quarantine exam. The animals had been imported from Togo, West Africa to Germany for the pet trade. RESULTS: Analysis of a portion of the genome of the detected virus showed that it is a previously unknown virus related to other chelonid herpesviruses. The virus was named pelomedusid herpesvirus 1. DISCUSSION: This case highlights the importance of testing for infectious agents during quarantine, even in clinically healthy animals.

Mass-mortality in green striped tree dragons (Japalura splendida) associated with multiple viral infections.

In spring 2011, high mortality in association with skin lesions, systemic haemorrhages and necrosis occurred in a group of green striped tree dragons (Japalura splendida) which were imported from southwestern China via Florida to Germany. Infections with various endoparasites were diagnosed in coprological examinations. Different antiparasitic and antibiotic treatments over a period of three months did not reduce the mortality rate. The remaining animals were therefore euthanased and submitted for additional testing. Predominant findings in pathological examination were granulomatous and necrotising inflammation of the skin, vacuolar tubulonephrosis of the distal renal tubules, hyperaemia and liver necrosis. Eosinophilic intranuclear and basophilic intracytoplasmic inclusion bodies were detected in the liver. Virological testing (PCR and virus isolation methods) demonstrated the presence of ranavirus, adenovirus and invertebrate iridovirus.

Detection of pathogens in Boidae and Pythonidae with and without respiratory disease.

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.

Environmental persistence of amphibian and reptilian ranaviruses.

Ranaviruses infect fish, amphibians, and reptiles. The present study was conducted to compare the persistence of amphibian and reptilian ranaviruses in a pond habitat. The 4 viruses used in this study included 2 amphibian ranaviruses, Frog virus 3 (FV3, the type species of the genus Ranavirus) and an isolate from a frog, and 2 ranaviruses of reptilian origin (from a tortoise and from a gecko). A sandwich germ-carrier technique was used to study the persistence of these viruses in sterile and unsterile pond water (PW) and soil obtained from the bank of a pond. For each virus, virus-loaded carriers were placed in each of the 3 substrates, incubated at 4 and 20°C, and titrated at regular intervals. Serial data were analyzed using a linear regression model to calculate T-90 values (time required for 90% reduction in the virus titer). Resistance of the viruses to drying was also studied. All 4 viruses were resistant to drying. At 20°C, T-90 values of the viruses were 22 to 31 d in sterile PW and 22 to 34 d in unsterile PW. Inactivation of all 4 viruses in soil at this temperature appeared to be non-linear. T-90 values at 4°C were 102 to 182 d in sterile PW, 58 to 72 d in unsterile PW, and 30 to 48 d in soil. Viral persistence was highest in the sterile PW, followed by the unsterile PW, and was lowest in soil. There were no significant differences in the survival times between the amphibian and reptilian viruses. The results of the present study suggest that ranaviruses can survive for long periods of time in pond habitats at low temperatures.

Prevalence of viral infections in captive collections of boid snakes in Germany.

Data on viral infections in apparently healthy snake collections in Germany were obtained with respect to husbandry conditions and health status. Samples from 100 boid snakes (from 14 collections) were examined microbiologically and for the presence of paramyxoviruses (PMVs) using RT-PCR. Blood was tested for the presence of antibodies against PMV, adenovirus and reovirus and for inclusion bodies indicative of inclusion body disease. Nine snakes tested positive for PMV, and inclusion bodies were detected in six snakes. Antibodies against PMV were found in one snake, and two snakes had antibodies against an adenovirus. A significant correlation was found between the origin of the snake and the presence of PMV, and between the presence of remarkable microbiological findings and husbandry conditions.

Identification of bovine enteric Caliciviruses (BEC) from cattle in Baden-Württemberg.

Caliciviruses are known to cause different diseases in many animal species. The bovine enteric caliciviruses (BEC) are associated with diarrhoea in cattle. These viruses have been classified in the genus Norovirus and are closely related to human noroviruses, the leading cause of gastroenteritis in humans. This has raised questions about zoonotic transmission and an animal reservoir for the human viruses. Two samples from 41 stool samples collected for diagnostic purposes from diarrheic cattle in Aulendorf, Germany tested positive for BEC. The samples were amplified with new degenerate BEC specific primers, which amplify a 263 bp portion of the RNA polymerase region. Analysis of the nucleotide sequences showed that these viruses are most closely related to the Norovirus genogroup III/2 (Bo/NLV/Newbury-2/76/UK) viruses.

Virus isolation and vaccination of Mediterranean tortoises against a chelonid herpesvirus in a chronically infected population in Italy.

A chelonid herpesvirus was isolated from a group of tortoises in Italy with a history of increased mortality and upper digestive and respiratory tract disease. The isolated virus was inactivated with formalin and used to prepare a nonadjuvanted vaccine and a vaccine adjuvanted with aluminum hydroxide. 57 tortoises, 26 Testudo hermanni, 25 T. graeca, and 6 T. marginata, were included in the study. The animals were vaccinated 3 times at 45 day intervals. Blood was collected from the animals 14 days prior to the first vaccination, and on day 0, 25, 45, 90, 113 and 369 after the first vaccination. Plasma antibody titers to the homologous chelonid herpesvirus were determined using a virus neutralization test (VNT). No significant rise in antibody titer was noted in the vaccinated animals. Antibody titers measured dropped below the cutoff-level sporadically in all positive animals. Repeat serological testing may therefore be necessary in order to detect seropositive animals.

Enzyme-linked immunosorbent assay for detecting herpesvirus exposure in Mediterranean tortoises (spur-thighed tortoise [Testudo graeca] and Hermann's tortoise [Testudo hermanni]).

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies to a herpesvirus associated with an upper respiratory tract disease in Mediterranean tortoises [spur-thighed tortoise (Testudo graeca) and Hermann's tortoise (Testudo hermanni)]. This serodiagnostic test was validated through a hyperimmunization study. The mean of the A(405) readings of the plasma samples collected at time zero of the hyperimmunization study plus three times the standard deviation was used as the cutoff for seropositivity in tortoises. ELISA results were compared to serum neutralization (SN) values for the same samples by using the McNemar test. The results obtained by SN and ELISA were not significantly different (P > 0.05). This new ELISA could be used as an important diagnostic tool for screening wild populations and private and zoo collections of Mediterranean tortoises.

Comparison of 16 chelonid herpesviruses by virus neutralization tests and restriction endonuclease digestion of viral DNA.

A total of 16 chelonid herpesviruses that were isolated between 1992 and 1998 were compared with one another on the basis of serology and restriction enzyme digestion patterns of viral DNA. The viruses stem from tortoises of three different species in four different European countries and the United States of America. The majority of the isolates were similar to one another. One isolate, however, differed strongly from all others both serologically and in the restriction cleavage pattern of its DNA, showing that there are at least two different sero- and genotypes of herpesviruscs that infect tortoises.

Differentiation of psittacine herpesvirus field isolates by restriction endonuclease analysis.

Thirty-one viruses were isolated from 15 different psittacine species. They were all identified as herpesviruses on the basis of chloroform sensitivity, inhibition of replication in cell cultures by 5-iodo-2'-deoxyuridine and morphology by electron microscopy. DNA from all 31 field isolates of psittacine herpesviruses (PsHV) was analysed by cleavage with restriction endonucleases EcoRI, PstI, and BglII. Using this technique, 12 different restriction patterns were recognizable. In previous work, we have differentiated PsHV strains according to their serological interrelationships. Restriction endonuclease profiles confirmed the results of conventional serogrouping but additional differences were observed among the five established serological subtypes. Using this method, it was possible to demonstrate a spontaneous reinfection of parrots in an aviary with a serologically and genetically different PsHV strain 4 years after the first outbreak of Pacheco's parrot disease (PPD). The results of this study reaffirm that antigenetically and genetically different PsHV exist and can cause PPD.

Detection of chelonid herpesvirus DNA by nonradioactive in situ hybridization in tissues from tortoises suffering from stomatitis-rhinitis complex in Europe and North America.

Chelonid herpesvirus (ChHV) infection in tortoises associated with stomatitis-rhinitis complex is a severe, mostly epizootic disease characterized by proliferative and diphtheroid-necrotizing glossitis, pharyngitis, rhinitis, and tracheitis, often occurring with pneumonia and encephalitis. The UL5 gene from a German ChHV isolate was used to generate a digoxigenin-labeled 307-base-pair DNA probe by polymerase chain reaction (PCR). ChHV DNA was detected in paraffin-embedded tissues of five naturally infected tortoises (two Afghan tortoises [Testudo horsfieldii], USA; two Hermann's tortoises [Testudo hermanni], Switzerland; one T. hermanni, Germany) by means of in situ hybridization (ISH) and PCR. Distribution of ChHV DNA exhibits many characteristics of alphaherpesvirus but also some characteristics of betaherpesvirus infections. The amino acid sequence of a portion of the ChHV UL5 homolog exhibited more than 50% similarity to alphaherpesvirus UL5 proteins. Nuclear hybridization signals were detected in epithelial cells of the lingual mucosa and glands. Furthermore, ChHV DNA was observed in tracheal epithelium, pneumocytes, hepatocytes, the renal tubular epithelium, cerebral glia cells and neurons, and intramural intestinal ganglia. ChHV DNA in endothelial cells of many organs underlines the systemic character of the disease. Importantly, ChHV DNA was detected by ISH in multiple tissues of tortoises originating from different geographic provenances. This indicates a high degree of conservation of the UL5 gene fragment among viruses prevalent in tortoises on different continents. With the described ISH, a molecular biological tool is available for rapid and specific diagnosis of ChHV infections and, more importantly, comparative pathogenetic studies of ChHV isolates from geographically unrelated regions.

Isolation and characterization of an iridovirus from Hermann's tortoises (Testudo hermanni).

A virus was isolated from tissues of 2 diseased Hermann's tortoises (Testudo hermanni) and preliminarily characterized as an iridovirus. This conclusion was based on the presence of inclusion bodies in the cytoplasm of infected cells, sensitivity to chloroform, inhibition of virus replication by 5-iodo-2'-desoxyuridine and the size and icosahedral morphology of viral particles. The virus was able to replicate in several reptilian, avian and mammalian cell lines at 28 degrees C, but not at 37 degrees C. Restriction enzyme analysis showed resistance of the ral DNA to digestion with HpaII due to methylation of the internal cytosine at CCGG sequences. Part of the genomic region encoding the major capsid protein was amplified by PCR and subjected to sequence analysis. Comparative analysis of the obtained nucleotide sequence revealed that the isolate is closely related to frog virus 3, the type species of the genus Ranavirus.

Paramyxoviral and reoviral infections of iguanas on Honduran Islands.

Thirty-five free-ranging healthy spiny-tailed iguanas (31 Ctenosaura bakeri, 4 C. similis) and 14 green iguanas (Iguana iguana rhinolopha) were caught and held in captivity for 2 days. Blood was collected from all animals and their sera were evaluated for antibody titres against reptilian reoviruses, reptilian paramyxoviruses, and avian paramyxovirus-1 (PMV-1). Cloacal and pharyngeal swabs also were collected and examined for viral content by incubation on chicken embryo fibroblasts (CEF) and terrapene heart cells (TH-1). No virus was isolated from the pharyngeal and cloacal swabs on CEF and TH-1. Twenty-three (47%) of 49 sera samples tested positive for reptilian reoviruses by virus neutralization tests. Twenty (41%) of 49 samples had antibodies against one reptilian PMV isolate by virus neutralization tests and 3 (9%) of 34 by hemagglutination inhibition tests. No antibodies were detected against the other PMV isolate of reptilian origin nor against avian PMV-1. This is the first description of serum antibodies against reptilian reoviruses and PMV in wild iguanas.

Quantification of the herpesvirus content in various tissues and organs, and associated post mortem lesions of psittacine birds which died during an epornithic of pacheco's parrot disease (PPD).

This paper reports on the viral content of up to 52 tissue and organ samples of 18 individual large psittacines which died during an epornithic of Pacheco's parrot disease (PPD) caused by psittacid herpesvirus 1 (PsHV1). Associated clinical signs and pathological lesions are described. The large spectrum of samples found to be positive for PsHVl suggests that birds succumb to PPD during viraemia. Tissues and organs from which the virus could be isolated included the integument and associated structures, the muscular, respiratory and circulatory system, bone marrow, the nervous system, thyroid and adrenal glands, spleen and liver, the urogenital tract and the gastro-intestinal tract. Nevertheless, individual and organ (but not species)-specific variation does occur. Virus isolation appears to be most promising from the respiratory, vascular and nervous system and the liver. Highest titres were obtained from heart blood and liver (up to 7.6 log(10)/g tissue), airsac, Nervus vagus and pulp and quill of pin feathers. Pin feathers may therefore be suitable for in-vivo diagnosis. In contrast, HV could not be isolated from any of the feather vanes examined. For the most part, post mortem lesions do not reflect the organ pattern found to be most permissive for virus replication as judged by the success of virus isolation and virus titres. A closer quantitative correlation is indicated for the lungs, spleen and liver, only. Corresponding findings as to frequency of gross pathological lesions and virus quantification appear to be restricted to the liver. In accordance with clinical observations and experimental findings, tissue virus content indicates that horizontal spread of herpesviruses is mediated by cloacal contents or secretions from the respiratory system.

Herpesviruses in tortoises: investigations into virus isolation and the treatment of viral stomatitis in Testudo hermanni and T. graeca.

Various studies were done during a spontaneous outbreak of stomatitis-rhinitis-complex (mouth rot) in a collection of Mediterranean land tortoises (21 Testudo hermanni, Hermann's tortoises, and three Testudo graeca, spur-thighed tortoises) in southern Germany. These studies were intended to help diagnose the causative agent, establish a possible diagnostic method in vivo and provide information on the efficacy of aciclovir and ganciclovir against chelonian herpesviruses. Thirteen T. hermanni and no T. graeca died within a period of 6 weeks following the introduction of one apparently healthy T. graeca. Two of the dead Testudo hermanni were submitted for post-mortem examination. In addition, blood samples from 11 of the 12 tortoises still surviving at the beginning of this study were cultured for virus content and for the presence of neutralizing antibodies to chelonian herpesviruses and swabs from conjunctiva, pharynx and cloaca were cultured for the presence of viruses. Herpesviruses were isolated from tissues of the two dead Testudo hermanni (tongue, intestine, trachea, lung, spleen, heart and brain). Peripheral leukocytes from one of 11 blood samples were positive for herpesvirus isolation, indicating viremia in at least one animal. Nine of 11 pharyngeal swabs but none of the conjunctival and cloacal swabs yielded herpesviruses. Circulating neutralizing antibodies were present in two of two tested T. graeca, but absent in all of the nine samples from T. hermanni. Aciclovir and ganciclovir were effective when tested in vitro against one of the herpesvirus isolates.