Development of a framework for genotyping bovine-derived Cryptosporidium parvum, using a multilocus fragment typing tool

Background: There is a need for an integrated genotyping approach for C. parvum; no sufficiently discriminatory scheme to date has been fully validated or widely adopted by veterinary or public health researchers. Multilocus fragment typing (MLFT) can provide good differentiation and is relatively quick and cheap to perform. A MLFT tool was assessed in terms of its typeability, specificity, precision (repeatability and reproducibility), accuracy and ability to genotypically discriminate bovine-derived Cryptosporidium parvum. Methods: With the aim of working towards a consensus, six markers were selected for inclusion based on their successful application in previous studies: MM5, MM18, MM19, TP14, MS1 and MS9. Alleles were assigned according to the fragment sizes of repeat regions amplified, as determined by capillary electrophoresis. In addition, a region of the GP60 gene was amplified and sequenced to determine gp60 subtype and this was added to the allelic profiles of the 6 markers to determine the multilocus genotype (MLG). The MLFT tool was applied to 140 C. parvum samples collected in two cross-sectional studies of UK calves, conducted in Cheshire in 2004 (principally dairy animals) and Aberdeenshire/Caithness in 2011 (beef animals). Results: Typeability was 84 %. The primers did not amplify tested non-parvum species frequently detected in cattle. In terms of repeatability, within- and between-run fragment sizes showed little variability. Between laboratories, fragment sizes differed but allele calling was reproducible. The MLFT had good discriminatory ability (Simpson’s Index of Diversity, SID, was 0.92), compared to gp60 sequencing alone (SID 0.44). Some markers were more informative than others, with MS1 and MS9 proving monoallelic in tested samples. Conclusions: Further inter-laboratory trials are now warranted with the inclusion of human-derived C. parvum samples, allowing progress towards an integrated, standardised typing scheme to enable source attribution and to determine the role of livestock in future outbreaks of human C. parvum

Manipulation of the Spin Memory of Electrons in n-GaAs

We report on the optical manipulation of the electron spin relaxation time in a GaAs based heterostructure. Experimental and theoretical study shows that the average electron spin relaxes through hyperfine interaction with the lattice nuclei, and that the rate can be controlled by the electron-electron interactions. This time has been changed from 300 ns down to 5 ns by variation of the laser frequency. This modification originates in the optically induced depletion of n-GaAs layer

Phenotypic characterisation of the cellular immune infiltrate in placentas of cattle following experimental inoculation with Neospora caninum in late gestation

Abstract Despite Neospora caninum being a major cause of bovine abortion worldwide, its pathogenesis is not completely understood. Neospora infection stimulates host cell-mediated immune responses, which may be responsible for the placental damage leading to abortion. The aim of the current study was to characterize the placental immune response following an experimental inoculation of pregnant cattle with N. caninum tachyzoites at day 210 of gestation. Cows were culled at 14, 28, 42 and 56 days post inoculation (dpi). Placentomes were examined by immunohistochemistry using antibodies against macrophages, T-cell subsets (CD4, CD8 and γδ), NK cells and B cells. Macrophages were detected mainly at 14 days post inoculation. Inflammation was generally mild and mainly characterized by CD3+, CD4+ and γδ T-cells; whereas CD8+ and NK cells were less numerous. The immune cell repertoire observed in this study was similar to those seen in pregnant cattle challenged with N. caninum at early gestation. However, cellular infiltrates were less severe than those seen during first trimester Neospora infections. This may explain the milder clinical outcome observed when animals are infected late in gestation.The authors acknowledge the Scottish Government’s Rural and Environment Science and Analytical Services Division (RESAS), UK, and Instituto Nacional de Tecnología Agropecuaria (INTA), Argentina, for funding this study and Dr Alex Schock from Animal Health and Veterinary Laboratories Agency and Prof. Gary Entrican from Moredun Research Institute for useful and constructive discussion.Peer Reviewe

Shedding of Cryptosporidium in calves and dams – evidence of re-infection and shedding of different gp60 subtypes

One of the most common causes of calf diarrhoea is the parasite Cryptosporidium parvum. Two longitudinal studies were carried out on a dairy farm Scotland to determine the prevalence of Cryptosporidium species and subtypes in a group of calves and to determine whether dams were a possible source of calfhood infection. Fecal samples were collected from 25 calves from birth to 12 months in the first year. In the second year, fecal samples were collected from pregnant cows (n = 29) and their calves (n = 30) from birth to 6 months. The samples were tested for Cryptosporidium and speciated. Cryptosporidium parvum-positive samples were subtyped by GP60 fragment analysis. All calves in both studies shed Cryptosporidium during the study period. Cryptosporidium parvum was the predominant species detected in calves ⩽6 weeks of age and at 6 months of age, C. bovis and C. ryanae were detected in calves older than 4 weeks of age but ⩽6 months of age. The prevalence of Cryptosporidium was higher in younger animals than in older animals. GP60 subtyping revealed two subtypes in calves on this farm (IIaA15G2R1 and IIaA19G2R1) that differed in frequency by age. Adult cattle also shed C. parvum, of four gp60 genotypes

Visualizing exertional dyspnea in a post-COVID patient using electrical impedance tomography

Purpose and method Many post-COVID patients suffer from dyspnea on exertion. To visualize exercise-induced dyspnea, a post-COVID patient and a healthy volunteer underwent an exercise test on a treadmill under stress relevant to everyday life monitored by electrical impedance tomography (EIT). Results The lung-healthy volunteer showed an even ventilation distribution throughout the assessment, a large ventilated area, and a butterfly-like lung shape with a convex lung rim. The post-COVID patient showed clear differences in the ventilated area compared to the control subject. During exercise, a constantly changing picture of differently ventilated areas is shown. However, especially the anterior regions were under-ventilated and larger areas were partially absent from ventilation. Overall, uncoordinated breathing and an uneven distribution of ventilation dominated the findings. Conclusion EIT is suitable for visualizing disturbed ventilation of the lungs, both at rest and under stress. The potential as a diagnostic tool in dyspnea assessment should be investigated

Selection of Neospora caninum antigens stimulating bovine CD4+ve T cell responses through immuno-potency screening and proteomic approaches

Neospora caninum is recognised worldwide as a major cause of bovine infectious abortion. There is a real need to develop effective strategies to control infection during pregnancy which may lead to either abortion or congenital transmission. Due to the intracellular nature of the parasite, cell-mediated immune (CMI) responses involving CD4+ve, CD8+ve, γ/δ TCR+ve T cells and NK cells, as well as production of IFN-γ, are thought to be important for protective immunity. In this study we applied a combination of proteomic and immunological approaches to identify antigens of N. caninum that are recognized by CD4+ve T cell lines derived from infected cattle. Initially, N. caninum tachyzoite Water Soluble Antigens (NcWSA) were fractionated by size-exclusion HPLC and then screened for immune-potency using CD4+ve T cell lines. LC-ESI-MS/MS (liquid chromatography electrospray ionisation tandem mass spectrometry) was employed to catalogue and identify the proteins comprising three immunologically selected fractions and led to the identification of six N. caninum target proteins as well as sixteen functional orthologues of Toxoplasma gondii. This approach allows the screening of biologically reactive antigenic fractions by the immune cells responsible for protection (such as bovine CD4+ve cells) and the subsequent identification of the stimulating components using tandem mass spectrometry