Flow-cytometric analysis of circulating leukocyte populations in turkeys: Establishment of a whole blood analysis approach and investigations on possible influencing factors.

The blood cell counting methods used for diagnostic and research purposes in turkeys are, up to date, inferior to the techniques established for mammals and chickens. While microscopic counts are time consuming, previous flow cytometric approaches did not cover all blood cell types of interest due to the lack of turkey-specific markers for the different cell populations. Moreover, it is unknown to what extent the different leukocyte populations are affected by host and environmental factors including age, breed of the bird, and housing environment, respectively. In this study we established a whole blood based flow cytometric analysis method for turkeys. This method was used to determine baseline values depending on the age of the birds as well as under consideration of variations between trials and animal room effects. During three trials whole blood samples of B.U.T. 6 female turkeys were collected to analyze different leukocyte concentrations (cells/µl whole blood). In the first trial one group and in the second and third trial two groups with 22 birds/group were housed. Blood samples were collected at days one, 23, 43, 60, and 88 post hatch and concentrations of monocytes, MHC class II-positive, CD4+, and CD8+ lymphocytes, as well as thrombocytes and granulocytes were determined by flow cytometric analysis. Concentrations of all identified populations were not only influenced by the bird's age (p < 0.05), they varied also among trials (p < 0.05) and even for some of the populations between animal rooms within the same trial despite comparable housing and management conditions. Therefore, for the establishment of baseline values for leucocyte concentrations in whole blood effects of age and housing have to be considered. In addition, our data emphasize the importance of the establishment of baseline values for different age groups, as age had the strongest effect on the blood cell numbers in this study.

H/L ratio as a measurement of stress in laying hens - methodology and reliability.

Measuring the ratio of heterophils and lymphocytes (H/L) in response to different stressors is a standard tool for assessing long-term stress in laying hens but detailed information on the reliability of measurements, measurement techniques and methods, and absolute cell counts is often lacking. Laying hens offered different sites of the nest boxes at different ages were compared in a two-treatment crossover experiment to provide detailed information on the procedure for measuring and the difficulties in the interpretation of H/L ratios in commercial conditions. H/L ratios were pen-specific and depended on the age and aviary system. There was no effect for the position of the nest. Heterophiles and lymphocytes were not correlated within individuals. Absolute cell counts differed in the number of heterophiles and lymphocytes and H/L ratios, whereas absolute leucocyte counts between individuals were similar. The reliability of the method using relative cell counts was good, yielding a correlation coefficient between double counts of r > 0.9. It was concluded that population-based reference values may not be sensitive enough to detect individual stress reactions and that the H/L ratio as an indicator of stress under commercial conditions may not be useful because of confounding factors and that other, non-invasive, measurements should be adopted.

Soft plastron, soft carapace with skeletal abnormality in juvenile tortoises. Histopathology and isolation of a novel picornavirus from Testudo graeca and Geochelone elegans.

OBJECTIVE: A disease is described in juvenile tortoises (Testudo graeca and Geochelone elegans) consisting mainly of a soft carapace, soft plastron and deformed skeleton. The aim of this study was to determine histopathological lesions and the biological properties of the isolated viruses. MATERIALS AND METHODS: Clinical signs and gross pathology were determined on diseased and healthy appearing tortoises. Paraffin sections were stained with HE, PAS and Prussian Blue and histologically examined. Terrapene heart (TH-1) cell cultures served for virus isolations from 64 tissues and 104 swabs. One isolate (isolate 1243/37 tongue) was used in neutralization tests on 19 sera. RESULTS: Retarded growth and increasingly soft plastron and carapace were the prominent signs in diseased tortoises. Pathological lesions consisted of dilated urinary sac, enlarged kidneys and livers. Histopathologically, hepatic hemosiderosis, hypoplastic anaemia, congestive glomerulonephrosis and osteodystrophy were seen. A novel vi- rus ("virus X") was isolated from 64 organs and 79 of 104 swabs. The isolated viruses were identified as a novel chelonid picornavirus based on cytopathic effect, resistance to chloroform and stability at low pH. Co-cultivation with 5-iodo-2'-deoxyuridine and actinomycin D did not reduce virus titres. Electron microscopically, round, non-enveloped particles (25-30 nm) were detected. Neutralizing antibodies to the isolate 1243/37tongue were present in 17 of 19 sera from seven species of tortoises. CONCLUSION AND CLINICAL RELEVANCE: Nephropathy, osteodystrophy and virus isolations suggest a viral aetiology. Metabolic bone disease is the major differential diagnosis. Further investigations in vivo are needed to evaluate the likely effects of the picornavirus on tortoises.