Study of the economic impact of cryptosporidiosis in calves after implementing good practices to manage the disease on dairy farms in Belgium, France, and the Netherlands.

Cryptosporidium spp. are widespread parasitic protozoans causing enteric infections in humans and animals. The parasites cause neonatal diarrhoea in calves, leading to a high mortality rate in the first three weeks. Losses are significant for farmers, but the cost of cryptosporidiosis remains poorly documented. In the absence of a vaccine, only preventive measures are available to farmers to combat the infection. This study, conducted between 2018 and 2021, aimed to evaluate the economic impact of Cryptosporidium spp. on European dairy farms and monitor changes in costs after implementing disease management measures. First, a field survey was carried out and questionnaires administered to 57 farmers in Belgium, France, and the Netherlands. The aim of the survey was to assess the losses associated with the occurrence of diarrhoea in calves aged between 3 days and 3 weeks. The economic impact of diarrhoea was calculated based on mortality losses, health expenditures, and additional labour costs. To refine the cost estimation specifically for Cryptosporidium spp., stool samples were collected from 10 calves per farm. The prevalence of Cryptosporidium spp. was determined, and the economic impact of diarrhoea was adjusted accordingly. The assumption was made that a certain percentage of costs was attributed to cryptosporidiosis based on the prevalence. These protocols were repeated at the end of the study to observe changes in costs. In the three years, the cost of diarrhoea for the 28 farms that stayed in the panel all along the study improved from €140 in 2018 to €106 on average per diarrhoeic calf in 2021. With a stable prevalence at 40%, the cost of cryptosporidiosis per infected calf decreased from €60.62 to €45.91 in Belgium, from €43.83 to €32.14 in France, and from €58.24 to €39.48 in the Netherlands. This represented an average of €15 saved per infected calf. The methodology employed in this study did not allow us to conclude that the improvement is strictly due to the implementation of preventive measures. However, with 11 million calves raised in the Interreg 2 Seas area covered by the study, it provided valuable insights into the economic burden of Cryptosporidium spp.

The control of microtubule stability in vitro and in transfected cells by MAP1B and SCG10.

In neurons, the regulation of microtubules plays an important role for neurite outgrowth, axonal elongation, and growth cone steering. SCG10 family proteins are the only known neuronal proteins that have a strong destabilizing effect, are highly enriched in growth cones and are thought to play an important role during axonal elongation. MAP1B, a microtubule-stabilizing protein, is found in growth cones as well, therefore it was important to test their effect on microtubules in the presence of both proteins. We used recombinant proteins in microtubule assembly assays and in transfected COS-7 cells to analyze their combined effects in vitro and in living cells, respectively. Individually, both proteins showed their expected activities in microtubule stabilization and destruction respectively. In MAP1B/SCG10 double-transfected cells, MAP1B could not protect microtubules from SCG10-induced disassembly in most cells, in particular not in cells that contained high levels of SCG10. This suggests that SCG10 is more potent to destabilize microtubules than MAP1B to rescue them. In microtubule assembly assays, MAP1B promoted microtubule formation at a ratio of 1 MAP1B per 70 tubulin dimers while a ratio of 1 SCG10 per two tubulin dimers was needed to destroy microtubules. In addition to its known binding to tubulin dimers, SCG10 binds also to purified microtubules in growth cones of dorsal root ganglion neurons in culture. In conclusion, neuronal microtubules are regulated by antagonistic effects of MAP1B and SCG10 and a fine tuning of the balance of these proteins may be critical for the regulation of microtubule dynamics in growth cones.

The structure of human apolipoprotein E2, E3 and E4 in solution 1. Tertiary and quaternary structure.

Three recombinant apoE isoforms fused with an amino-terminal extension of 43 amino acids were produced in a heterologous expression system in E. coli. Their state of association in aqueous phase was analyzed by size-exclusion liquid chromatography, sedimentation velocity and sedimentation equilibrium experiments. By liquid chromatography, all three isoforms consisted of three major species with Stokes radii of 4.0, 5.0 and 6.6 nm. Sedimentation velocity confirmed the presence of monomers, dimers and tetramers as major species of each isoform. The association schemes established by sedimentation equilibrium experiments corresponded to monomer-dimer-tetramer-octamer for apoE2, monomer-dimer-tetramer for apoE3 and monomer-dimer-tetramer-octamer for apoE4. Each of the three isoforms exhibits a distinct self-association pattern. The apolipoprotein multi-domain structure was mapped by limited proteolysis with trypsin, chymotrypsin, elastase, subtilisin and Staphylococcus aureus V8 protease. All five enzymes produced stable intermediates during the degradation of the three apoE isoforms, as described for plasma apoE3. The recombinant apoE isoforms, thus, consist of N- and C-terminal domains. The presence of the fusion peptide did not appear to alter the apolipoprotein tertiary organization. However, a 30 kDa amino-terminal fragment appeared during the degradation of the recombinant apoE isoforms resulting from cleavage in the 273-278 region. This region, not accessible in plasma apoE3, results from a different conformation of the C-terminal domain in the recombinant isoforms. A specific pattern for the apoE4 C-terminal domain was observed during the proteolysis. The region 230-260 in apoE4, in contrast to that of apoE3 and apoE2, was not accessible to proteases, probably due to the existence of a longer helix in this region of apoE4 stabilized by an interdomain interaction.

The structure of human apolipoprotein E2, E3 and E4 in solution. 2. Multidomain organization correlates with the stability of apoE structure.

The stabilities toward thermal and chemical denaturation of three recombinant isoforms of human apolipoprotein E (r-apoE2, r-apoE3 and r-apoE4), human plasma apoE3, the recombinant amino-terminal (NT) and the carboxyl-terminal (CT) domains of plasma apoE3 at pH 7 were studied using near and far ultraviolet circular dichroism (UV CD), fluorescence and size-exclusion chromatography. By far UV CD, thermal unfolding was irreversible for the intact apoE isoforms and consisted of a single transition. The r-apoE3 was found to be less stable as compared to the plasma protein and the stability of recombinant isoforms was r-apoE4

Early-glycation of apolipoprotein E: effect on its binding to LDL receptor, scavenger receptor A and heparan sulfates.

Glycation is responsible for disruption of lipoprotein functions leading to the development of atherosclerosis in diabetes. The effects of apolipoprotein E (apoE) glycation were investigated with respect to its interaction with receptors. The interaction of apoE with the low density lipoprotein receptor (LDL-R) and scavenger receptor A (SR-A) was measured by competition experiments performed using, respectively, on a human fibroblast cell line 125I-LDL, and on a murine macrophage cell line (J774) 125I-acetylated LDL, and unlabeled apoE/phospholipid complexes. Glycated apoE binding to heparin and heparan sulfates (HS) was assessed by surface plasmon resonance (SPR) technology. Site-directed mutagenesis was then performed on Lys-75, the major glycation site of the protein. The prepared mutant protein proved to be useful as a tool to study the role of Lys-75 in apoE glycation. The findings showed that, although glycation has no effect on apoE binding either to the LDL-R or to SR-A, it impairs its binding to immobilized heparin and HS. The glycation of Lys-75 was found to be proceed rapidly and contributed significantly to total protein glycation. We propose that, in the case of diabetes, glycation may lead to the atherogenicity of apoE-containing lipoproteins disturbing their uptake via the HS proteoglycan pathway.