Validation of a real-time polymerase chain reaction for the detection and quantification of the nucleic acid of Histoplasma from equine clinical samples.

Histoplasma capsulatum var. farciminosum (HCF) is a dimorphic fungus that causes epizootic lymphangitis in equids. Current diagnostic approaches, including culture, microscopy, and clinical presentation, lack speed, sensitivity, and specificity when diagnosing clinical cases. In this study, equine blood and pus samples on Whatman FTA cards from Senegal (n = 3), The Gambia (n = 19), Ethiopia (n = 16), and Mali (n = 13) were tested using a real-time PCR (qPCR) protocol. The assay was optimized and tested for its suitability to detect and quantify HCF in blood and pus loaded onto Whatman FTA cards at sampling. Whatman FTA cards were tested for their suitability for use with qPCR and were found to recover DNA more efficiently than from direct extraction. Using TaqMan fluorescent probes and specific primers, the assay demonstrated 100% analytical specificity when detecting multiple strains of Histoplasma and no false positives with off-target organisms. The assay's diagnostic performance was measured against an existing nested internal transcribed spacer PCR protocol using a receiver operating characteristic curve. The test was found to have a diagnostic specificity and sensitivity of 100% and 71.4%, respectively, when analyzing pus samples using a cycle threshold (Ct) cutoff determined by Youden's index (27.75). Blood sample cutoff Ct value was proposed at 34.55. Further optimization is required to improve the performance of the protocol when applied to blood samples. This study has, for the first time, demonstrated the ability to detect and quantify the DNA of Histoplasma spp. in equine blood and pus samples with a high degree of accuracy, providing a platform to further investigate the pathogenesis and epidemiology of this disease. IMPORTANCE: Histoplasmosis is a neglected yet major cause of morbidity and mortality in both equids and people in resource-scarce settings. One of the major hindrances to the control of histoplasmosis is a lack of readily available diagnostic tests. Tests are needed to support clinical decision-making and to be applied in population-based research to further understand this disease in situ. This paper reports, for the first time, the validation and application of a qPCR to detect Histoplasma directly from equine clinical samples, bypassing the need to culture this notoriously difficult organism. We report and comment on the performance of the qPCR in comparison with our previously developed nested PCR.

Genotypic Variation in Grain P Loading across Diverse Rice Growing Environments and Implications for Field P Balances.

More than 60% of phosphorus (P) taken up by rice (Oryza spp.) is accumulated in the grains at harvest and hence exported from fields, leading to a continuous removal of P. If P removed from fields is not replaced by P inputs then soil P stocks decline, with consequences for subsequent crops. Breeding rice genotypes with a low concentration of P in the grains could be a strategy to reduce maintenance fertilizer needs and slow soil P depletion in low input systems. This study aimed to assess variation in grain P concentrations among rice genotypes across diverse environments and evaluate the implications for field P balances at various grain yield levels. Multi-location screening experiments were conducted at different sites across Africa and Asia and yield components and grain P concentrations were determined at harvest. Genotypic variation in grain P concentration was evaluated while considering differences in P supply and grain yield using cluster analysis to group environments and boundary line analysis to determine minimum grain P concentrations at various yield levels. Average grain P concentrations across genotypes varied almost 3-fold among environments, from 1.4 to 3.9 mg g-1. Minimum grain P concentrations associated with grain yields of 150, 300, and 500 g m-2 varied between 1.2 and 1.7, 1.3 and 1.8, and 1.7 and 2.2 mg g-1 among genotypes respectively. Two genotypes, Santhi Sufaid and DJ123, were identified as potential donors for breeding for low grain P concentration. Improvements in P balances that could be achieved by exploiting this genotypic variation are in the range of less than 0.10 g P m-2 (1 kg P ha-1) in low yielding systems, and 0.15-0.50 g P m-2 (1.5-5.0 kg P ha-1) in higher yielding systems. Improved crop management and alternative breeding approaches may be required to achieve larger reductions in grain P concentrations in rice.

Effect of sodium bicarbonate supplementation on carcass characteristics of lambs fed concentrate diets at different ambient temperature levels.

The objective of this study was to investigate the influence of ambient temperatures on carcass characteristics of lambs fed concentrate diets with or without NaHCO3 supplementation. A slaughter study was carried on 12 male Black Belly Barbados lambs randomly drawn from a growth trial (35 weeks). The lambs were divided into four equal groups and allotted in a 2×2 factorial design. The lambs were allotted at random to two dietary treatments of a basal diet (35:65 roughage:concentrate) or basal diet supplemented with 4% NaHCO3 at different ambient temperatures (20°C and 30°C) in an environment controlled chamber for 10 days. Lambs were slaughtered for carcass evaluation at about 262 days of age (245 days of growth trial, 7 days adaptation and 10 days of experimental period). Ambient temperature had significant (p<0.05, p<0.05, p<0.01, and p<0.001) effects on meat color from the ribeye area (REA), fat, leg and longissimus dorsi muscles with higher values recorded for lambs in the lower temperature group than those from the higher ambient temperature group. Significant differences (p<0.05) in shear force value (kg/cm(2)) recorded on the leg muscles showed higher values (5.32 vs 4.16) in lambs under the lower ambient temperature group compared to the other group. Dietary treatments had significant (p<0.01, p<0.01, and p<0.05) effects on meat color from the REA, fat, and REA fat depth (cm(2)) with higher values recorded for lambs in the NaHCO3 supplementation group than the non supplemented group. Similarly, dietary treatments had significant differences (p<0.05) in shear force value (kg/cm(2)) of the leg muscles with the NaHCO3 groups recording higher (5.30 vs 4.60) values than those from the other group. Neither ambient temperature nor dietary treatments had any significant (p>0.05) effects on pH, and water holding capacity on both muscles. These results indicated that NaHCO3 supplementation at low ambient temperatures had caused an increase in carcass characteristics leading to significant effect on meat quality.