Diagnosis of sheep fasciolosis caused by Fasciola hepatica using cathepsin L enzyme-linked immunosorbent assays (ELISA)

Publication history: Accepted - 3 July 2021; Published online - 6 July 2021.Fasciolosis, a global parasitic disease of agricultural livestock, is caused by the liver fluke Fasciola hepatica. Management and strategic control of fasciolosis on farms depends on early assessment of the extent of disease so that control measures can be implemented quickly. Traditionally, this has relied on the detection of eggs in the faeces of animals, a laborious method that lacks sensitivity, especially for sub-clinical infections, and identifies chronic infections only. Enzyme linked immunosorbent assays (ELISA) offer a quicker and more sensitive serological means of diagnosis that could detect early acute infection before significant liver damage occurs. The performance of three functionally-active recombinant forms of the major F. hepatica secreted cathepsins L, rFhCL1, rFhCL2, rFhCL3, and a cathepsin B, rFhCB3, were evaluated as antigens in an indirect ELISA to serologically diagnose liver fluke infection in experimentally and naturally infected sheep. rFhCL1 and rFhCL3 were the most effective of the four antigens detecting fasciolosis in sheep as early as three weeks after experimental infection, at least five weeks earlier than both coproantigen and faecal egg tests. In addition, the rFhCL1 and rFhCL3 ELISAs had a very low detection limit for liver fluke in lambs exposed to natural infection on pastures and thus could play a major role in the surveillance of farms and a ‘test and treat’ approach to disease management. Finally, antibodies to all three cathepsin L proteases remain high throughout chronic infection but decline rapidly after drug treatment with the flukicide, triclabendazole, implying that the test may be adapted to trace the effectiveness of drug treatment.This work was supported by a European Research Council Advanced Grant (HELIVAC, 322725) and Science Foundation Ireland (SFI) Professorship grant (17/RP/5368) awarded to J.P. Dalton

Molecular investigation into the roles of Sigma B and the Stressosome in the response of Listeria monocytogenes to environmental stress

The alternative sigma factor, SigB, has been studied extensively in Listeria monocytogenes, Bacillus subtilis and Staphylococcus aureus, and is known to regulate the general stress response in these bacteria. The activation of SigB by a stress occurs via a signalling cascade, upstream of which is the stressosome complex. The stressosome is a large protein complex composed of RsbS, RsbT and RsbR proteins, with the RsbR proteins hypothesised to function as stress sensors. RsbR has four paralogues, Lmo0161, Lm0799, Lmo1642 and Lmo1842, of which only Lmo0799 has been established to act as a stress sensor. Lmo0799 is a blue light sensor, but somewhat surprisingly inactivation of the corresponding gene does not result in sensitivity to blue light. In the current study, environmental factors including growth phase and temperature, along with exposure to ethanol or alternative carbon sources, were identified to alter the sensitivity of L. monocytogenes to killing by blue light. Whole transcriptomic analysis of the wild-type, SigB deletion mutant and a mutant for which the conserved cysteine residue critical for blue light sensing in Lmo0799, C56, has been changed to an alanine, designated lmo0799 C56A, in the presence of blue light identified a key role for SigB in altering gene transcription in response to blue light exposure. In addition, it confirmed the ability of the C56A mutant to upregulate genes under the control of SigB in response to blue light exposure, although to a lesser extent than the wild-type. These data provide further evidence that L. monocytogenes is able to sense and respond to secondary stresses associated with blue light exposure, such as ROS. Finally, the reconstruction of Dlmo1642 and Dlmo1842 deletion mutants enabled the confirmation that there is a redundancy between the stress sensing abilities of these proteins and the other RsbR paralogues that has previously been associated with Lmo0161

Molecular investigation into the roles of Sigma B and the Stressosome in the response of Listeria monocytogenes to environmental stress

The alternative sigma factor, SigB, has been studied extensively in Listeria monocytogenes, Bacillus subtilis and Staphylococcus aureus, and is known to regulate the general stress response in these bacteria. The activation of SigB by a stress occurs via a signalling cascade, upstream of which is the stressosome complex. The stressosome is a large protein complex composed of RsbS, RsbT and RsbR proteins, with the RsbR proteins hypothesised to function as stress sensors. RsbR has four paralogues, Lmo0161, Lm0799, Lmo1642 and Lmo1842, of which only Lmo0799 has been established to act as a stress sensor. Lmo0799 is a blue light sensor, but somewhat surprisingly inactivation of the corresponding gene does not result in sensitivity to blue light. In the current study, environmental factors including growth phase and temperature, along with exposure to ethanol or alternative carbon sources, were identified to alter the sensitivity of L. monocytogenes to killing by blue light. Whole transcriptomic analysis of the wild-type, SigB deletion mutant and a mutant for which the conserved cysteine residue critical for blue light sensing in Lmo0799, C56, has been changed to an alanine, designated lmo0799 C56A, in the presence of blue light identified a key role for SigB in altering gene transcription in response to blue light exposure. In addition, it confirmed the ability of the C56A mutant to upregulate genes under the control of SigB in response to blue light exposure, although to a lesser extent than the wild-type. These data provide further evidence that L. monocytogenes is able to sense and respond to secondary stresses associated with blue light exposure, such as ROS. Finally, the reconstruction of Dlmo1642 and Dlmo1842 deletion mutants enabled the confirmation that there is a redundancy between the stress sensing abilities of these proteins and the other RsbR paralogues that has previously been associated with Lmo0161

Empagliflozin in Patients with Chronic Kidney Disease

Background The effects of empagliflozin in patients with chronic kidney disease who are at risk for disease progression are not well understood. The EMPA-KIDNEY trial was designed to assess the effects of treatment with empagliflozin in a broad range of such patients. Methods We enrolled patients with chronic kidney disease who had an estimated glomerular filtration rate (eGFR) of at least 20 but less than 45 ml per minute per 1.73 m(2) of body-surface area, or who had an eGFR of at least 45 but less than 90 ml per minute per 1.73 m(2) with a urinary albumin-to-creatinine ratio (with albumin measured in milligrams and creatinine measured in grams) of at least 200. Patients were randomly assigned to receive empagliflozin (10 mg once daily) or matching placebo. The primary outcome was a composite of progression of kidney disease (defined as end-stage kidney disease, a sustained decrease in eGFR to < 10 ml per minute per 1.73 m(2), a sustained decrease in eGFR of & GE;40% from baseline, or death from renal causes) or death from cardiovascular causes. Results A total of 6609 patients underwent randomization. During a median of 2.0 years of follow-up, progression of kidney disease or death from cardiovascular causes occurred in 432 of 3304 patients (13.1%) in the empagliflozin group and in 558 of 3305 patients (16.9%) in the placebo group (hazard ratio, 0.72; 95% confidence interval [CI], 0.64 to 0.82; P < 0.001). Results were consistent among patients with or without diabetes and across subgroups defined according to eGFR ranges. The rate of hospitalization from any cause was lower in the empagliflozin group than in the placebo group (hazard ratio, 0.86; 95% CI, 0.78 to 0.95; P=0.003), but there were no significant between-group differences with respect to the composite outcome of hospitalization for heart failure or death from cardiovascular causes (which occurred in 4.0% in the empagliflozin group and 4.6% in the placebo group) or death from any cause (in 4.5% and 5.1%, respectively). The rates of serious adverse events were similar in the two groups. Conclusions Among a wide range of patients with chronic kidney disease who were at risk for disease progression, empagliflozin therapy led to a lower risk of progression of kidney disease or death from cardiovascular causes than placebo