Prevalence of Mycoplasma agassizii and Chelonian herpesvirus in captive tortoises (Testudo sp.) in the United Kingdom.

During the months of April to August in 1999 and 2002, oral swabs were collected from 146 tortoises (Testudo sp.) in private collections in the United Kingdom and tested by polymerase chain reaction (PCR) for the presence of Mycoplasma agassizii and Chelonian herpesvirus (ChHV). The presence of M. agassizii was confirmed by restriction digestion of the PCR product. A 307-bp fragment of the ChHV UL5 homologue gene was sequenced and found to show most similarity to equine herpesvirus type 1. A prevalence of 15.8 and 8.2% was found for M. agassizii and ChHV, respectively. Comparison of the carriage of both M. agassizii and ChHV in different species of tortoises correlated the presence of M. agassizii with Testudo horsfieldii and ChHV with Testudo marginata and Testudo graeca iberia. An association of ChHV with stomatitis was also found. Mixed infections with both agents were detected. The findings further demonstrate this pathogen-tortoise association and the cross transmission of these infections if different tortoise species are housed together.     

Herpesvirus outbreak in a group of mediterranean tortoises (Testudo spp).

ChHV and Mycoplasma agassizii infections in tortoises share similar clinical signs of lethargy, anorexia, rhinitis, and conjunctivitis. In addition, ChHV infection is associated with glossitis and stomatitis and often causes high morbidity and mortality. As was seen in this case, ChHV infection tends to cause higher mortality in T hermanni compared with T graeca and T marginata. T horsfieldi is also considered highly susceptible to ChHV but appeared unaffected in this outbreak.     

Seasonal variations in haematological data from Mediterranean tortoises (Testudo graeca and Testudo hermanni) in captivity

Reference values for a variety of haematological parameters in 18 Mediterranean tortoises of two species (Testudo graeca and Testudo hermanni) were determined on six occasions during the year. Statistically significant seasonal variations were demonstrated in all parameters.     

Plasma concentrations of 25-hydroxycholecalciferol in 22 captive tortoises (Testudo species)

The plasma concentration of 25-hydroxycholecalciferol was measured in 13 adult Hermann's tortoises (Testudo hermanni), seven adult spur-thighed tortoises (Testudo graeca) and two adult marginated tortoises (Testudo marginata) during 2004. They were healthy, of both sexes, and kept in captivity under natural unfiltered sunlight in southern England with no dietary sources of cholecalciferol. Blood samples were taken in March, June and August, and the concentration of 25-hydroxycholecalciferol did not vary significantly with the seasons. However, the concentrations in the female tortoises were always significantly lower than in the males.     

Obstetrical problems in two tortoises

The management of two tortoises ( Testudo hermanni and Testudo graeca ) with retained ova is described and discussed. Caesarean delivery of the ova was successful in one case but the other tortoise died post-operatively. Pseudomonas and Proteus spp. were cultured from an ovum in the latter case and the same Pseudomonas was recovered post-mortem from a heart blood sample.     

Calcium and phosphorus values and their derivatives in captive tortoises (Testudo species)

Objective : To evaluate the relationships between total calcium and phosphorus and ionised calcium and phosphorus values in clinically healthy tortoises. Methods : Jugular blood samples were obtained from 25 tortoises, as part of a health screen of the population. These comprised Hermann’s tortoises, Testudo hermanni boettgeri, spur-thighed tortoises, Testudo graeca ibera, marginated tortoises, Testudo marginata, and horsfield tortoises, Testudo horsfieldi. Plasma from these samples were analysed for total calcium, ionised calcium and phosphorus levels. These samples were taken in the immediate posthibernation period, before the onset of reproductive activity. Results : Females exhibited statistically significantly higher levels of phosphorus. Ionised calcium and total calcium levels were elevated in females compared with males, but this was not statistically significant. Females did have statistically significantly higher calculated solubility indexes and statistically significantly lower ratios compared with males. Clinical Significance : This study has provided an insight into the ratios and solubility indexes in tortoises, and this information may lead to further understanding of the significance of these parameters in chelonians.     

Comparison of 11 herpesvirus isolates from tortoises using partial sequences from three conserved genes

Herpesviruses are an important cause of epidemic disease in tortoises. There are at least two serologically distinct herpesviruses capable of infecting tortoises. Methods for the diagnosis of herpesvirus infections in tortoises include virus isolation and a number of different PCRs. We have compared 11 virus isolates collected from various species in different countries over several years using sequences from three different viral genes. During this study we used four different PCR protocols described for the diagnosis of herpesvirus infections in tortoises. The protocols used included two based on portions of the DNA polymerase gene, one targeting the UL5 homologue, and one targeting the UL39 homologue. Comparison of the methods showed that the tortoise herpesvirus-specific protocols were all serotype specific. Sequences of the obtained amplicons were compared with one another and with sequences of herpesviruses available in GenBank. The sequence alignments showed that the tortoise herpesviruses were most closely related to members of the subfamily Alphaherpesvirinae. They also showed that the tortoise isolates could be clearly divided into two genogroups.     

Phylogenetic Variation of Chelonid Alphaherpesvirus 5 (ChHV5) in Populations of Green Turtles Chelonia mydas along the Queensland Coast,Australia

Abstract

Sea turtle fibropapillomatosis (FP) is a disease marked by the proliferation of benign but debilitating cutaneous and occasional visceral tumors, likely to be caused by chelonid alphaherpesvirus 5 (ChHV5). This study presents a phylogeny of ChHV5 strains found on the east coast of Queensland, Australia, and a validation for previously unused primers. Two different primer sets (gB-1534 and gB-813) were designed to target a region including part of the UL27 glycoprotein B (gB) gene and part of UL28 of ChHV5. Sequences obtained from FP tumors found on juvenile green turtles Chelonia mydas (<65 cm curved carapace length) had substantial homology with published ChHV5 sequences, while a skin biopsy from a turtle without FP failed to react in the PCRs used in this study. The resulting sequences were used to generate a neighbor-joining tree from which three clusters of ChHV5 from Australian waters were identified: north Australian, north Queensland, and Queensland clusters. The clusters reflect the collection sites on the east coast of Queensland with a definitive north–south trend.

Received October 22, 2016; accepted May 7, 2017 Published online July 26, 2017     

Potential Noncutaneous Sites of Chelonid Herpesvirus 5 Persistence and Shedding in Green Sea Turtles Chelonia mydas

Abstract

Chelonid herpesvirus 5 (ChHV5), the likely etiologic agent of sea turtle fibropapillomatosis (FP), is predicted to be unevenly distributed within an infected turtle, in which productive virus replication and virion shedding occurs in cutaneous tumor keratinocytes. In this study, we measured and compared ChHV5 DNA quantities in tumors, skin, urine, major organs, and nervous tissue samples from green turtles Chelonia mydas. These samples were taken from the carcasses of 10 juvenile green turtles with and without clinical signs of FP that stranded in Florida during 2014. Quantitative PCR for ChHV5 UL30 was used to identify ChHV5 DNA in tumors, skin, heart, kidney, nerves, and urine sampled from five out of five FP-positive and three out of five FP-free turtles. The most frequently co-occurring sites were cutaneous tumor and kidney (n = 4). Novel data presented here include the identification of ChHV5 DNA in kidney, heart, and nerve samples from three FP-free turtles. These data support candidate nontumored anatomic sites of ChHV5 DNA localization and mobilization during two different disease states that may be involved in the ChHV5 infection cycle.

Received September 8, 2016; accepted April 17, 2017 Published online July 26, 2017     

Feasibility and variability of corneal esthesiometry in Hermann's tortoises (Testudo hermanni) depending on age and body weight

BackgroundHermann's tortoise is one of the most popular reptiles kept as pet which underlines the importance to reinforce the information needed to provide advanced and adequate veterinary care in exotic animal species. Therefore, the purpose of this study, performed in Testudo hermanni, was to evaluate corneal touch threshold (CTT) and its feasibility according to age and body weight.MethodsFifty-one healthy tortoises were classified in 2 groups (≤2 years [young; n = 25] and 8–10 years [subadult; n = 26]). Central CTT was measured by means of a Cochet-Bonnet esthesiometer and defined as the filament length required to elicit a blink in at least 3/5 applications. CTT feasibility according to body weight was also evaluated by diving the individuals in weight groups consisting of “lighter” (<400 g [n = 31]) and “heavier” (≥400 g [n = 20]) animals.ResultsMean CTT was 5.99 cm for the whole population (90% CI: 5.87–6.11), being 5.98 cm for young (90% CI: 5.81–6.15) and 6 cm for all subadult (90% CI: 6.00–6.00) tortoises, and 5.98 cm for lighter (90% CI: 5.84–6.13) and 6 cm for all heavier (90% CI: 6.00–6.00) tortoises. No statistical differences were detected between age and weight groups (P = 0.159 and P = 0.159, respectively). Three animals presented unilateral faint fluorescein uptake postesthesiometry (3/51; 2.9%) that resolved spontaneously within 48 hours.Conclusions and clinical relevanceCochet-Bonnet esthesiometer was a safe means of confirming high corneal sensitivity in all tortoises, which was high regardless of age. Increasing filament lengths would ultimately be required to determine the true corneal sensitivity scope of Testudo hermanni.     

Evaluation of pulmonary function in European land tortoises using whole-body plethysmography

The aim of this study was to evaluate the use of whole-body plethysmography as a non-invasive method to determine the respiratory parameters and profiles in two tortoise species belonging to the genus Testudo. Pulmonary functions and volumetric parameters were determined in 10 adults of Testudo hermanni and in seven Testudo marginata animals, using whole-body plethysmography. A profile pattern was regularly observed: an inspiratory flow peak, an expiratory peak, an apnoea phase and a second expiratory peak, previous to the beginning of the next respiratory cycle. Positive and significant correlation was observed between the inspiratory time, weight and length of the tortoises. Larger tortoises showed a higher time of inhalation. The peak of inspiratory flow was correlated with the sex, being longer in the females. T. marginata had an inspiratory time longer than that of T. hermanii. In T. hermanii, differences related to the sex were observed in the tidal volume, peak inspiratory flow, peak expiratory flow, expiratory flow of 50 per cent and enhanced pause, which could be related to the smaller size of males. The results suggest that additional information on new technologies currently used in pet medicine or even in human medicine should be developed and adjusted as alternative ways to support the rehabilitation of turtles and tortoises.     

Hematologic and plasma biochemical changes associated with fenbendazole administration in Hermann's tortoises (testudo hermanni).

Toxicosis associated with benzimidazole anthelmintics has been reported with increasing frequency in zoologic collections. Clinical signs, clinicopathologic abnormalities, and gross and histologic lesions are primarily the result of damage to the gastrointestinal and hematopoietic systems. Profound leukopenia, especially granulocytopenia, is the most common and severe clinicopathologic change associated with benzimidazole administration. Death usually occurs from overwhelming systemic bacterial and/or fungal infections secondary to severe immunosuppression. In this 125-day study, six male Hermann's tortoises (Testudo hermanni) were treated orally with two 5-day courses of fenbendazole 2 wk apart at a dosage of 50 mg/kg. Serial blood samples were used to assess hematologic and plasma biochemical changes before, during, and following the treatment period. Although the tortoises remained healthy, blood sampling indicated an extended heteropenia with transient hypoglycemia, hyperuricemia, hyperphosphatemia, and equivocal hyperproteinemia/hyperglobulinemia, which were considered to be in response to fenbendazole administration. Changes in several other clinicopathologic parameters appeared to correlate with fenbendazole administration. The hematologic and biochemical changes seen in the healthy animals in this study should be considered when treating compromised tortoises with fenbendazole. Hematologic and plasma biochemical status of tortoises/reptiles should be determined before treatment and monitored during the treatment period. The risk of mortality of an individual from nematode infection should be assessed relative to the potential for metabolic alteration and secondary septicemia following damage to hematopoietic and gastrointestinal systems by fenbendazole.     

Polymerase chain reaction (PCR) for the detection of herpesvirus in tortoises

A polymerase chain reaction (PCR)-based method using a herpesvirus consensus primer was assessed for the identification of herpesviral infections in tortoises. A single band of about 230 bp was detected in PCR products from two out of twenty swabs taken from the oral cavity, three out of three paraffin-embedded tissue sections from the liver (two cases) and oral mucosa (one case), and one out of two fresh tissue samples from the oral mucosa. Nucleotide sequencing of these PCR products indicated that the herpesvirus present in these tortoises might belong to the alphaherpesvirinae. PCR using swabs and biopsy tissues was a sensitive and highly specific method for the diagnosis of herpesviral infections in tortoises.     

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.     

Development of species-specific PCR techniques for the detection of tortoise herpesvirus.

Previously, a nested polymerase chain reaction (PCR) was employed with consensus degenerate primers targeting highly conserved motifs within herpesviral DNA polymerase genes to detect a newly described tortoise herpesvirus. However, nucleotide sequence information obtained from the final amplified fragment was restricted to a small region of 181 bp. In the present study, additional sequences flanking this segment were determined from a PCR product successfully amplified using a set of known degenerate primers, which covered a 692-bp region within the tortoise herpesviral DNA polymerase gene. Polymerase chain reaction primers for specific amplification of the tortoise herpesviral DNA were designed on the basis of these nucleotide sequences and successfully amplified tortoise herpesviral DNA from the tissues of tortoises that were well characterized histopathologically with herpesviral infection. The lower limit of detection was 1,000 herpesviral DNA equivalents in the presence of normal tortoise genomic DNA. Furthermore, a more sensitive and specific PCR technique for the identification of herpesviral infections in tortoises was developed employing a heminested form, which will enable the detection of latent infections or herpesvirus carriers in tortoises.     

Seasonal variation in blood biochemistry of long-term captive Mediterranean tortoises (Testudo graeca and T hermanni)

Reference values for five blood chemistry parameters in 18 Mediterranean tortoises of two species, Testudo graeca and T hermanni, were determined on up to 10 occasions during the year. Statistically significant seasonal variations were demonstrated in blood urea and blood glucose. Seasonal variations were demonstrated in blood urea and blood glucose. Seasonal variations in total plasma proteins, lipids and cholesterol, however, were limited to gravid females. The study also suggested that three energy sources were available to the tortoise during hibernation, lipids stored in the fat body, endogenous protein degradation and glucose derived from hepatic glycogen.     

Evidence of infection in tortoises by Chlamydia-like organisms that are genetically distinct from known Chlamydiaceae species

Nasal lavage fluid was collected from 155 tortoises, mostly Testudo spp., that were kept as companion animals and suffered from nasal discharge. Examination for chlamydial DNA by PCR assays targeting the ompA, ompB, and groESL genes, as well as the 16S rRNA signature region and the 16S-23S intergenic spacer, respectively, revealed 16 (10.3%) positive animals. Sequence analysis of PCR products indicated high homology to the family Chlamydiaceae. Phylogenetic trees constructed from partial sequences of the ompA and 16S rRNA genes showed that the present samples clustered outside the nine species of Chlamydia and Chlamydophila. Sequences of the nearest relative, Chlamydophila pecorum, were still clearly distinct from those of the positive tortoise samples. This suggests that the tortoises had been infected by Chlamydia-like agents, the taxonomic identity and pathogenic importance of which has yet to be established.     

Development of in situ nest PCR and comparison of five molecular biological diagnostic methods for the detection of intracellular viral DNAs in paraffin sections

Nest polymerase chain reaction (PCR), in situ hybridization (ISH), in situ PCR, in situ PCR/hybridization (PCR-ISH) and in situ nest PCR were compared for the detection and localization of intracellular viral DNAs in paraffin sections. MDBK cells were infected with alcelapine herpesvirus 1 ranging from 10(1) to 10(5) 50% tissue culture infected doses (TCID(50)), incubated 18 hr, then fixed and processed into paraffin blocks. Sections of the cell preparation were subjected to nest PCR, ISH, in situ PCR, PCR-ISH and in situ nest PCR using specific oligonucleotide primers or probes directed against the viral open reading frame 50. In situ nest PCR and nest PCR were found to be capable of detecting the viral DNA in the cells infected with the lowest virus titer. As compared with other molecular biological methods for the detection of the virus, in situ nest PCR was found to be more sensitive than ISH, in situ PCR and PCR-ISH. In situ nest PCR has wide applications for sensitive localization of low copy viral sequences within cells to investigate the role of viruses in a variety of clinical conditions.     

Atypical systemic mycobacteriosis in Hermann's tortoises (Testudo hermanni)

Background: Hermann's tortoise (Testudo hermanni) is a near threatened species found throughout southern Europe. Captive breeding of endangered species is subject to health risks, such as the stress of captivity and ambient conditions that may favor the emergence of infectious diseases. An episode of systemic atypical mycobacteriosis was investigated in six Hermann's tortoises from a captive population. The symptoms appeared in the form of anorexia, weakness, and lethargy, with inflammatory and edematous lesions of the hind limbs and tail. Four of the affected tortoises were euthanized and necropsied. Methods: Blood samples and joint aspirates were obtained for assessment. Euthanasia of four affected animals was performed. Postmortem examination included necropsy, histopathology, and mycobacterium detection by culture and polymerase chain reaction (PCR). In the other two animals, only hematology and blood biochemistry was carried out. Results: Hematological and blood biochemistry analyses gave inconsistent results. In joint aspirates, mononuclear cells with phagocytozed bacillary structures were observed. Ziehl-Neelsen stain revealed the presence of acid-fast bacteria. Granulomatous lesions were observed in liver, lungs, spleen, heart, muscle, kidney, and ovaries. Culture and identification were positive for mycobacteria and molecular sequencing led to the identification of Mycobacterium nonchromogenicum. Conclusions and clinical relevance: Although mycobacteriosis is rare in reptile collections, strict hygienic and prophylactic measures in species of high ecological value must be performed, especially if the animals are to be used for reintroduction projects.     

Detection of circovirus infection in pigeons by in situ hybridization using cloned DNA probes.

Degenerate primers were designed based on known sequence information for the circoviruses psittacine beak and feather disease virus and porcine circovirus and applied by polymerase chain reaction (PCR) to known virus-infected bursa of Fabricius (BF) from a pigeon. A 548-bp DNA fragment was amplified and shown to be specific to a novel circovirus, named pigeon circovirus (PiCV), and was used to produce sensitive and specific probes for detection of circovirus DNA by in situ hybridization (ISH). Using ISH on BF from 107 pigeons submitted for necropsy, infection was detected in 89%, compared with a histologic detection rate of 66%. Using the ISH technique, infected cells were also found in liver, kidney, trachea, lung, brain, crop, intestine, spleen, bone marrow, and heart of some birds. Large quantities of DNA were present in some of these tissues, and in the absence of BF, liver in particular is identified as a potentially useful organ to examine for presence of PiCV. This high prevalence of infection in diseased birds is noteworthy, emphasizing the need for studies to determine the precise role of this virus as a disease-producing agent.