The role of Virus "X" (Tortoise Picornavirus) in kidney disease and shell weakness syndrome in European tortoise species determined by experimental infection

Tortoise Picornavirus (ToPV) commonly known as Virus "X" was recently discovered in juvenile European tortoises suffering from soft carapace and plastron as well as kidney disease. Therefore, this virus was a potential candidate to be a causative agent for these disease patterns. Spur thighed tortoises (Testudo graeca) seemed to be more susceptible to establish clinical symptoms than other European species like T. hermanni. Thus this trial investigated the role of ToPV in the described syndrome. Two groups of juvenile European tortoises (T. graeca and T.hermanni) each of 10 animals, were cloacally, oronasally and intracoelomically inoculated with an infectious dose (~ 2000 TICD) of a ToPV strain isolated from a diseased T. graeca. A control group of two animals of each species received non-infected cell culture supernatant. The tortoises were examined daily and pharyngeal and cloacal swabs for detection of ToPV-RNA by RT-PCR were taken from each animal every six days for a period of 6 months. At the end of the study the remaining animals were euthanised and dissected. Bacteriological and parasitological tests were performed and organ samples of all tortoises were investigated by RT-PCR for the presence of ToPV and histopathology. Animals that were euthanised at the end of the experiment, were examined for presence of specific anti-ToPV antibodies. Several animals in both inoculated groups showed retarded growth and a light shell weakness, in comparison to the control animals. Three animals were euthanised during the trial, showing reduced weight gain, retarded growth, severe shell weakness and apathy, in parallel to clinical observations in naturally infected animals. In all inoculated animals of both species an intermittent virus shedding, starting from 18 days post inoculation (d.p.i.), till 164 d.p.i. was detected, while the control animals remained negative. The virus was successfully reisolated in terrapene heart cell culture in 16 of 20 inoculated animals of both species. Histopathology of most inoculated animals revealed a lack of bone remodeling and vacuolisation in kidney tubuli which supports the described pathogenesis of nephropathy and osteodystrophy. Anti- ToPV antibody titres ranged from 1:2 to >1:256 in 13 of 20 animals, whereas all control animals were seronegative. The study proofed the Henle Koch`s postulates of ToPV as causative agent for shell dystrophy and kidney disease in both testudo species. The proposed species specific sensitivity towards clinical disease was not observed

High prevalence of Sarcocystis calchasi in racing pigeon flocks in Germany

The apicomplexan parasite Sarcocystis calchasi (Coccidia: Eimeriorina: Sarcocystidae) is the causative agent of Pigeon Protozoal Encephalitis (PPE) and infects birds of the orders Columbiformes, Piciformes and Psittaciformes. Accipiter hawks (Aves: Accipitriformes) are the definitive hosts of this parasite. Infections of S. calchasi have been detected in Germany, the United States and Japan. However, the prevalence of the parasite in racing pigeon flocks has not yet been determined. Here, the first cross-sectional prevalence study to investigate S. calchasi in pigeon racing flocks was accomplished including 245 pigeon flocks across Germany. A total of 1,225 muscle biopsies, were taken between 2012 and 2016 and examined by semi-nested PCR for S. calchasi DNA targeting the ITS gene. Additionally, a questionnaire on construction of the aviary as well as management and health status of the flock was conducted. In 27.8% (95% C.I. = 22.3–33.8%) of the flocks, S. calchasi DNA was detected in at least one pigeon. Positive flocks were located in 15 out of 16 federal states. A significant increase of infected racing pigeons was seen in spring. Half-covered or open aviary constructions showed a trend of increase of the prevalence rate, while anti-coccidian treatment and acidified drinking water had no effects. The high prevalence and the geographical distribution of S. calchasi suggest a long-standing occurrence of the parasite in the German racing pigeon population. For pigeons presented with neurological signs or other symptoms possibly related to PPE, S. calchasi should be considered as a potential cause throughout Germany

Behaviour of adipose-derived canine mesenchymal stem cells after superparamagnetic iron oxide nanoparticles labelling for magnetic resonance imaging

Background: Therapy with mesenchymal stem cells (MSCs) has been reported to provide beneficial effects in the treatment of neurological and orthopaedic disorders in dogs. The exact mechanism of action is poorly understood. Magnetic resonance imaging (MRI) gives the opportunity to observe MSCs after clinical administration. To visualise MSCs with the help of MRI, labelling with an MRI contrast agent is necessary. However, it must be clarified whether there is any negative influence on cell function and viability after labelling prior to clinical administration. Results: For the purpose of the study, seven samples with canine adipose-derived stem cells were incubated with superparamagnetic iron oxide nanoparticles (SPIO: 319.2 µg/mL Fe) for 24 h. The internalisation of the iron particles occurred via endocytosis. SPIO particles were localized as free clusters in the cytoplasm or within lysosomes depending on the time of investigation. The efficiency of the labelling was investigated using Prussian blue staining and MACS assay. After 3 weeks the percentage of SPIO labelled canine stem cells decreased. Phalloidin staining showed no negative effect on the cytoskeleton. Labelled cells underwent osteogenic and adipogenic differentiation. Chondrogenic differentiation occurred to a lesser extent compared with a control sample. MTT-Test and wound healing assay showed no influence of labelling on the proliferation. The duration of SPIO labelling was assessed using a 1 Tesla clinical MRI scanner and T2 weighted turbo spin echo and T2 weighted gradient echo MRI sequences 1, 2 and 3 weeks after labelling. The hypointensity caused by SPIO lasted for 3 weeks in both sequences. Conclusions: An Endorem labelling concentration of 319.2 µg/mL Fe (448 µg/mL SPIO) had no adverse effects on the viability of canine ASCs. Therefore, this contrast agent could be used as a model for iron oxide labelling agents. However, the tracking ability in vivo has to be evaluated in further studies

Natural killer (NK) and lymphokine-activated killer (LAK) cell functions from healthy dogs and 29 dogs with a variety of spontaneous neoplasms

To investigate natural killer (NK) and lymphokine-activated killer (LAK) cell functions from 10 healthy dogs and 29 dogs with a variety of spontaneous neoplasms, large granular lymphocytes (LGLs) from blood samples were separated by a 58.5% Percoll density gradient. LGLs were stimulated with a low dose of recombinant human interleukin 2 (rhIL-2) for 7days. Cytotoxicity of effector cells against the susceptible CTAC cell line was measured before and after stimulation. Compared with those before stimulation, the percentage of LGLs after stimulation with rhIL-2 was found to be significantly increased (P<0.01) in both dogs with tumors and controls. However, the increase was significantly higher in control animals, indicating a defect in proliferation ability of NK cells in canine tumor patients. After stimulation with rhIL-2, lymphokine-activated killer (LAK) cell activity in dogs with tumors was significantly lower (P<0.01) when compared with controls. Reduced cytotoxicity of rhIL-2-activated NK cells in dogs with tumors seems to be attributable to the presence of a diminished proliferative capacity of NK cells and a decreased ability of LAK cells to lyse target cells. Further knowledge of the precise function of IL-2-activated NK cells in dogs with tumors may help to optimize new and therapeutically beneficial treatment strategies in canine and human cancer patients. Our findings suggest that the dog could also serve as a relevant large animal model for cancer immunotherapy with IL-

Besnoitia besnoiti infection alters both endogenous cholesterol de novo synthesis and exogenous LDL uptake in host endothelial cells

Besnoitia besnoiti, an apicomplexan parasite of cattle being considered as emergent in Europe, replicates fast in host endothelial cells during acute infection and is in considerable need for energy, lipids and other building blocks for offspring formation. Apicomplexa are generally considered as defective in cholesterol synthesis and have to scavenge cholesterol from their host cells for successful replication. Therefore, we here analysed the influence of B. besnoiti on host cellular endogenous cholesterol synthesis and on sterol uptake from exogenous sources. GC-MS-based profiling of cholesterol-related sterols revealed enhanced cholesterol synthesis rates in B. besnoiti-infected cells. Accordingly, lovastatin and zaragozic acid treatments diminished tachyzoite production. Moreover, increased lipid droplet contents and enhanced cholesterol esterification was detected and inhibition of the latter significantly blocked parasite proliferation. Furthermore, artificial increase of host cellular lipid droplet disposability boosted parasite proliferation. Interestingly, lectin-like oxidized low density lipoprotein receptor 1 expression was upregulated in infected endothelial hostcells, whilst low density lipoproteins (LDL) receptor was not affected by parasite infection. However, exogenous supplementations with non-modified and acetylated LDL both boosted B. besnoiti proliferation. Overall, current data show that B. besnoiti simultaneously exploits both, endogenous cholesterol biosynthesis and cholesterol uptake from exogenous sources, during asexual replication

Besnoitia besnoiti infection alters both endogenous cholesterol de novo synthesis and exogenous LDL uptake in host endothelial cells

Besnoitia besnoiti, an apicomplexan parasite of cattle being considered as emergent in Europe, replicates fast in host endothelial cells during acute infection and is in considerable need for energy, lipids and other building blocks for offspring formation. Apicomplexa are generally considered as defective in cholesterol synthesis and have to scavenge cholesterol from their host cells for successful replication. Therefore, we here analysed the influence of B. besnoiti on host cellular endogenous cholesterol synthesis and on sterol uptake from exogenous sources. GC-MS-based profiling of cholesterol-related sterols revealed enhanced cholesterol synthesis rates in B. besnoiti-infected cells. Accordingly, lovastatin and zaragozic acid treatments diminished tachyzoite production. Moreover, increased lipid droplet contents and enhanced cholesterol esterification was detected and inhibition of the latter significantly blocked parasite proliferation. Furthermore, artificial increase of host cellular lipid droplet disposability boosted parasite proliferation. Interestingly, lectin-like oxidized low density lipoprotein receptor 1 expression was upregulated in infected endothelial hostcells, whilst low density lipoproteins (LDL) receptor was not affected by parasite infection. However, exogenous supplementations with non-modified and acetylated LDL both boosted B. besnoiti proliferation. Overall, current data show that B. besnoiti simultaneously exploits both, endogenous cholesterol biosynthesis and cholesterol uptake from exogenous sources, during asexual replication

Diagnosis of gastrointestinal parasites in reptiles: comparison of two coprological methods

BACKGROUND: Exotic reptiles have become increasingly common domestic pets worldwide and are well known to be carriers of different parasites including some with zoonotic potential. The need of accurate diagnosis of gastrointestinal endoparasite infections in domestic reptiles is therefore essential, not only for the well-being of captive reptiles but also for the owners. Here, two different approaches for the detection of parasite stages in reptile faeces were compared: a combination of native and iodine stained direct smears together with a flotation technique (CNF) versus the standard SAF-method. RESULTS: A total of 59 different reptile faeces (20 lizards, 22 snakes, 17 tortoises) were coprologically analyzed by the two methods for the presence of endoparasites. Analyzed reptile faecal samples contained a broad spectrum of parasites (total occurence 93.2%, n=55) including different species of nematodes (55.9%, n=33), trematodes (15.3%, n=9), pentastomids (3.4%, n=2) and protozoans (47.5%, n=28). Associations between the performances of both methods to detect selected single parasite stages or groups of such were evaluated by Fishers exact test and marginal homogeneity was tested by the McNemar test. In 88.1% of all examined samples (n=52, 95% confidence interval [CI]=77.1 - 95.1%) the two diagnostic methods rendered differing results, and the McNemar test for paired observations showed highly significant differences of the detection frequency (P<0.0001). CONCLUSION: The combination of direct smears/flotation proved superior in the detection of flagellates trophozoites, coccidian oocysts and nematode eggs, especially those of oxyurids. SAF-technique was superior in detecting larval stages and trematode eggs, but this advantage failed to be statistically significant (P=0.13). Therefore, CNF is the recommended method for routine faecal examination of captive reptiles while the SAF-technique is advisable as additional measure particularly for wild caught animals and individuals which are to be introduced into captive collections

Comparison of the endocranial- and brain volumes in brachycephalic dogs, mesaticephalic dogs and Cavalier King Charles spaniels in relation to their body weight

BACKGROUND: A number of studies have attempted to quantify the relative volumes of the endocranial volume and brain parenchyma in association with the pathogenesis of the Chiari-like malformation (CLM) in the Cavalier King Charles spaniel (CKCS). In our study we examine the influence of allometric scaling of the brain and cranial cavity volume on morphological parameters in different dog breeds. MRI scans of 110 dogs (35 mesaticephalic dogs, 35 brachycephalic dogs, 20 CKCSs with SM, and 20 CKCSs without SM) have been used to create 3-dimensional volumetric models of skull and brain parts. Volumes were related to body weight calculating the adjusted means for different breeds. RESULTS: There was a strong global dependency of all volumes to body weight (P<0.0001). The adjusted means of the absolute and relative volumes of brain parenchyma and cranial compartments are not significantly larger in CKCSs in comparison to brachycephalic and mesaticephalic dogs. A difference in absolute or relative volumes between CKCSs with and without SM after relating these values to body weight could not be identified. The relative volume of the hindbrain parenchyma (caudal fossa parenchyma percentage) was larger in brachycephalic dogs than in CKCSs, without causing herniation or SM. CONCLUSION: An influence of body weight exist in dogs, which can be sufficiently large to render conclusions on the difference in volumes of the brain and skull unsafe unless some account of the body weight is taken in the analysis. The results of this study challenge the role of overcrowding for the development of SM in dogs

Accipiter hawks and Common Woodpigeon in Germany

The apicomplexan parasite Sarcocystis calchasi (S. calchasi) triggers pigeon protozoal encephalitis, a neurologic disease in columbids. Accipiter hawks have been identified as the final host, and Columbidae and Psittaciformes as intermediate hosts. In this study, 368 free-ranging Accipiter hawks and 647 free-ranging common woodpigeons were sampled in a country-wide study in order to identify the prevalence of S. calchasi in these populations. A semi-nested PCR specific for S. calchasi tested positive in 7.3% (4.9–10.5) of submitted samples from Accipiter hawks. Juvenile Accipiter hawks (13.7%; 7.7–22.0) had a significantly higher infection rate with S. calchasi than adult Accipiter hawks (5.8%; 2.7–9.3). The prevalence of S. calchasi in common woodpigeons was 3.3% (5.4–9.7). Positive pigeons were identified in 14/16 federal states, and a region-dependency was detected, with higher rates of infection in the eastern parts of Germany. The results of this study suggest that the common woodpigeon is a natural reservoir for S. calchasi. In a study of one region for four consecutive years, an increase in prevalence was not detected. Findings indicate that the parasite is not newly introduced to Germany, but rather long established. The prevalence suggests that there is a substantial risk of S. calchasi infections in other free-ranging as well as captive host species

Single tracheal inoculation of Aspergillus fumigatus conidia induced aspergillosis in juvenile falcons (Falco spp.)

Aspergillosis is a common and life-threatening respiratory disease in raptors with acute and chronic courses. Among raptors, gyrfalcons (Falco rusticolus) and their hybrids are often declared to be highly susceptible with juvenile individuals being the most susceptible. However, species-and age-specific experimental studies are lacking and minimal infective doses (IDs) for Aspergillus spp. conidia are unknown. Therefore, 8-week-old, healthy gyr-hybrid falcons (F. rusticolus X F. cherrug) (N = 18) were experimentally infected with Aspergillus fumigatus using a single intratracheal inoculation with varying dosages of conidia (10(2) to 10(7) conidia). Over 28 days, clinical signs were monitored as well as haematological and serological parameters. Following euthanasia, necropsy, histopathology, bacteriology, and mycology were performed. Re-isolated fungi were compared to the inoculum using microsatellite length polymorphisms. During the trial, clinical signs and dyspnoea correlated significantly with the ID. Necropsy revealed fungal lesions in the upper and lower airways of 10/18 inoculated falcons, but not in the control birds. In 9/18 inoculated falcons, fungal granulomas were confirmed in histopathology and A. fumigatus was re-isolated from these granulomas. Except one nasal isolate all re-isolated fungal strains were identical to the inoculum strain. Based on mycology and histopathology a minimal ID of 50% was calculated to be MID50% (+/- S.E.) = 10(4.52 +/- 0.67) for a single tracheal inoculation of A. fumigatus conidia. This study demonstrates for the first time that a single exposure is able to cause acute aspergillosis in juvenile falcons