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

Modulation of the host Th1 immune response in pigeon protozoal encephalitis caused by Sarcocystis calchasi

Pigeon protozoal encephalitis (PPE) is an emerging central-nervous disease of domestic pigeons (Columba livia f. domestica) reported in Germany and the United States. It is caused by the apicomplexan parasite Sarcocystis calchasi which is transmitted by Accipter hawks. In contrast to other members of the Apicomplexa such as Toxoplasma and Plasmodium, the knowledge about the pathophysiology and host manipulation of Sarcocystis is scarce and almost nothing is known about PPE. Here we show by mRNA expression profiling a significant down-modulation of the interleukin (IL)-12/IL-18/interferon (IFN)-γ axis in the brains of experimentally infected pigeons during the schizogonic phase of disease. Concomitantly, no cellular immune response was observed in histopathology while immunohistochemistry and nested PCR detected S. calchasi. In contrast, in the late central-nervous phase, IFN-γ and tumor necrosis factor (TNF) α-related cytokines were significantly up-modulated, which correlated with a prominent MHC-II protein expression in areas of mononuclear cell infiltration and necrosis. The mononuclear cell fraction was mainly composed of T-lymphocytes, fewer macrophages and B-lymphocytes. Surprisingly, the severity and composition of the immune cell response appears unrelated to the infectious dose, although the severity and onset of the central nervous signs clearly was dose-dependent. We identified no or only very few tissue cysts by immunohistochemistry in pigeons with severe encephalitis of which one pigeon repeatedly remained negative by PCR despite severe lesions. Taken together, these observations may suggest an immune evasion strategy of S. calchasi during the early phase and a delayed-type hypersensitivity reaction as cause of the extensive cerebral lesions during the late neurological phase of disease

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

Repatriation of an old fish host as an opportunity for myxozoan parasite diversity: The example of the allis shad, Alosa alosa (Clupeidae), in the Rhine

Background: Wildlife repatriation represents an opportunity for parasites. Reintroduced hosts are expected to accumulate generalist parasites via spillover from reservoir hosts, whereas colonization with specialist parasites is unlikely. We address the question of how myxozoan parasites, which are characterized by a complex life-cycle alternating between annelids and fish, can invade a reintroduced fish species and determine the impact of a de novo invasion on parasite diversity. We investigated the case of the anadromous allis shad, Alosa alosa (L.), which was reintroduced into the Rhine approximately 70 years after its extinction in this river system. Methods: We studied parasites belonging to the Myxozoa (Cnidaria) in 196 allis shad from (i) established populations in the French rivers Garonne and Dordogne and (ii) repatriated populations in the Rhine, by screening the first adults returning to spawn in 2014. Following microscopical detection of myxozoan infections general myxozoan primers were used for SSU rDNA amplification and sequencing. Phylogenetic analyses were performed and cloned sequences were analyzed from individuals of different water sources to better understand the diversity and population structure of myxozoan isolates in long-term coexisting vs recently established host-parasite systems. Results: We describe Hoferellus alosae n. sp. from the renal tubules of allis shad by use of morphological and molecular methods. A species-specific PCR assay determined that the prevalence of H. alosae n. sp. is 100 % in sexually mature fish in the Garonne/Dordogne river systems and 22 % in the first mature shad returning to spawn in the Rhine. The diversity of SSU rDNA clones of the parasite was up to four times higher in the Rhine and lacked a site-specific signature of SNPs such as in the French rivers. A second myxozoan, Ortholinea sp., was detected exclusively in allis shad from the Rhine. Conclusions: Our data demonstrate that the de novo establishment of myxozoan infections in rivers is slow but of great genetic diversity, which can only be explained by the introduction of spores from genetically diverse sources, predominantly via straying fish or by migratory piscivorous birds. Long-term studies will show if and how the high diversity of a de novo introduction of host-specific myxozoans succeeds into the establishment of a local successful strain in vertebrate and invertebrate hosts

Process development of health benefits from corn in pet diets that enhance dog utilization

Master of ScienceDepartment of Grain Science and IndustryMajor Professor Not ListedThe methods of manufacturing pet diets have evolved over the past 160 years. Starting with a baked biscuit in 1860 to the widely used extrusion technology today. Formulas have also changed with once popular corn now being criticized as inferior and indigestible. Different processing methods can change starch digestion. Less processing can lead to more indigestible starch (resistant starch; RS) within corn diets. This RS may benefit gastrointestinal health. Therefore, the objective of the first study was to determine the effect of process on dietary utilization of corn-based diets, and changes to starch utilization. Experimental diets containing 56% corn as the sole starch source were produced through pelleting, baking, and extrusion and compared to a baked control diet in which the corn was replaced with dextrose. The pelleted diet had the highest (P < 0.05) differential scanning calorimetry (DSC) residual gelatinization peak (3.5 J/g) followed by the baked diet (1.4 J/g), and then similar to the extruded diet the control had the lowest result (0.1 J/g). The extruded diet resulted in the highest (P < 0.05) level of gelatinized starch (34.2%) by the glucoamylase procedure followed by the baked treatment (14.7%) and pelleted had the lowest (P < 0.05) gelatinized starch (8.4%). As a percentage of total starch, the extruded diet had the highest (P < 0.05) level of rapidly digestible starch (90.0%), the baked treatment was intermediate (P < 0.05, 60.9%) and pelleted the lowest (P < 0.05, 34.8%). The pelleted diet had the highest (P < 0.05) amount of slowly digestible starch (SDS, 42.6%), with the baked treatment having an intermediate (P < 0.05) amount of SDS (27.1%), and the extruded diet had the lowest (P < 0.05) amount of SDS (7.1%) Among corn treatments the extruded diet had the lowest (P < 0.05) level of RS (1.3%). The baked diet was intermediate (P < 0.05, 3.6%) with the highest RS (P < 0.05) in the pelleted diet. To evaluate the in vivo effects of these process treatments, twelve Beagle dogs were fed the experimental diets for 9 d adaptation and 5 d of collection in a replicated 4 x 4 Latin square designed study. The data were analyzed using the general linear mixed model with statistical analysis software (GLIMMIX proc; SAS v9.4, Cary NC) and means were considered different at ∝=5%. The experimental diet was the fixed effect with period and dog as random effects. Dogs were fed to maintain body weight, with food intake similar among corn treatments and lower (P < 0.05) for those fed the control diet. Fecal scores (1: soft-liquid to 5: hard-dry) were slightly different across the three corn treatments, but each exceeded 3.5 which was considered ideal. However, dogs fed the control diet (dextrose) had soft-runny stools (average score of 1). Feces from dogs fed the baked treatment had a lower fecal pH (5.37; P < 0.05) than the other corn treatments (average 5.71); whereas the pH of feces from the dogs fed the control (dextrose) diet were the highest (P < 0.05) at 6.87. The dry matter apparent total tract digestibility (ATTD) was greater (P < 0.05) for the dogs fed the extruded corn-based diet and the control diet (average 75.75%) versus the baked and pelleted diets (average 69.49%). In conclusion, the corn diets had different levels of gelatinized starch thereby influencing the in vivo digestibility, Resistant starch was higher in treatments of lower cooking intensity including lower fecal pH which may indicate improved colonic fermentation leading to positive impacts on gut health. To assess the level of RS in commercial products for comparison purposes, 30 baked dog treats were evaluated in a 2 x 3 factorial design. Samples were purchased and separated into main effects of size (small, medium, large), and presence or absence of wheat. Samples were analyzed for their resistant starch concentration. It was determined that the size and presence of wheat did not impact the total starch (average 40.7%), digestible starch (average 39.5%), or resistant starch (average 1.8%) concentration. However, a post-hoc analysis did indicate the few (n=5) grain-free products that were selected had a higher (P < 0.05) concentration of resistant starch (average 4.9%). In conclusion, among treatments like extrusion that had more energy inputs there was a lower concentration of RS while the inverse was observed in processes with fewer energy inputs or lower cooking intensity. This also impacted stool quality and the digestion of some nutrients but significantly increased starch digestion. Treatments with lower levels of RS are common to commercially available foods. So, using different processing methods can change the digestible and indigestible starch ratio, which can have promote several health benefits

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

Detection of hepatitis E virus in wild boars of rural and urban regions in Germany and whole genome characterization of an endemic strain

<p>Abstract</p> <p>Background</p> <p>Hepatitis E is an increasingly diagnosed human disease in Central Europe. Besides domestic pigs, in which hepatitis E virus (HEV) infection is highly prevalent, wild boars have been identified as a possible source of human infection. In order to assess the distribution of HEV in the wild boar population of Germany, we tested liver samples originating from different geographical regions for the presence of the HEV genome and compared the detected sequences to animal and human HEV strains.</p> <p>Results</p> <p>A total of 148 wild boar liver samples were tested using real-time RT-PCR resulting in an average HEV detection rate of 14.9% (95% CI 9.6–21.6). HEV was detected in all age classes and all geographical regions. However, the prevalence of HEV infection was significantly higher in rural as compared to urban regions (p < 0.001). Sequencing of the PCR products indicated a high degree of heterogenicity of the detected viruses within genotype 3 and a grouping according to their geographical origin. The whole genome sequence of an HEV isolate (wbGER27) detected in many wild boars in the federal state of Brandenburg was determined. It belongs to genotype 3i and shows 97.9% nucleotide sequence identity to a partial sequence derived from a human hepatitis E patient from Germany.</p> <p>Conclusion</p> <p>The results indicate that wild boars have to be considered as a reservoir for HEV in Germany and that a risk of HEV transmission to humans is present in rural as well as urban regions.</p

Modulation of the host Th1 immune response in pigeon protozoal encephalitis caused by Sarcocystis calchasi

Pigeon protozoal encephalitis (PPE) is an emerging central-nervous disease of domestic pigeons (Columba livia f. domestica) reported in Germany and the United States. It is caused by the apicomplexan parasite Sarcocystis calchasi which is transmitted by Accipter hawks. In contrast to other members of the Apicomplexa such as Toxoplasma and Plasmodium, the knowledge about the pathophysiology and host manipulation of Sarcocystis is scarce and almost nothing is known about PPE. Here we show by mRNA expression profiling a significant down-modulation of the interleukin (IL)-12/IL-18/interferon (IFN)-γ axis in the brains of experimentally infected pigeons during the schizogonic phase of disease. Concomitantly, no cellular immune response was observed in histopathology while immunohistochemistry and nested PCR detected S. calchasi. In contrast, in the late central-nervous phase, IFN-γ and tumor necrosis factor (TNF) α-related cytokines were significantly up-modulated, which correlated with a prominent MHC-II protein expression in areas of mononuclear cell infiltration and necrosis. The mononuclear cell fraction was mainly composed of T-lymphocytes, fewer macrophages and B-lymphocytes. Surprisingly, the severity and composition of the immune cell response appears unrelated to the infectious dose, although the severity and onset of the central nervous signs clearly was dose-dependent. We identified no or only very few tissue cysts by immunohistochemistry in pigeons with severe encephalitis of which one pigeon repeatedly remained negative by PCR despite severe lesions. Taken together, these observations may suggest an immune evasion strategy of S. calchasi during the early phase and a delayed-type hypersensitivity reaction as cause of the extensive cerebral lesions during the late neurological phase of disease

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