Short communication: Utilisation of n-alkanes to estimate feed intake in horses fed known amounts of a labelled concentrate supplement.

The assessment of feed intake in stabled horses is a difficult task to accomplish. Faecal markers, namely n-alkanes, have been used successfully for the estimation of this important nutritional parameter. This usually involves the dosing of synthetic n-alkanes via different matrices, a laborious task that may also influence the animal normal foraging behaviour. An experiment was conducted to evaluate a relative simple methodology to quantify feed intake in horses, based on the provision of measured amounts of a concentrate supplement labelled with beeswax and the utilisation of n-alkanes as faecal markers. Four Lusitano horses were used in three consecutive experimental periods. Animals were fed on cereal straw and different proportions of a previously prepared beeswax-labelled concentrate supplement (BLCS; 0.05, 0.10 and 0.20, DM basis). Beeswax labelling was performed to provide a distinct n-alkane profile for the concentrate feed. Prior to feed intake calculations, proportions of labelled concentrate supplement in the diets were estimated using n-alkanes C25 to C33 by least-square optimisation procedures. Results showed that the beeswax labelling resulted in high n-alkane concentrations in the concentrate feed, especially for the odd-chain n-alkanes. Estimates of diet composition did not differ from the measured values, except for the diet with highest BLCS incorporation, with an underestimation of 10%. DM intake was accurately estimated by the "labelled supplement method" in all diets. However, for the lowest BLCS incorporation, DM intake was underestimated by 16% whereas for the higher levels of BLCS in the diet, measured and estimated DM intake values were almost identical with a slight overestimation of only 0.7 and 0.2% (10 and 20% of BLCS, respectively). Results indicate that both diet composition and feed intake can be accurately estimated in horses using the "labelled supplement method", even when very low levels of the labelled concentrate supplement are included in the animals' diet. This method eliminates the need for daily dosing with external synthetic markers, providing advantages in terms of minimising animal management and interference with their normal foraging behaviour.

Predictive value of faecal calprotectin in ulcerative colitis - single centre experience.

OBJECTIVES: Faecal calprotectin is an important biomarker used in the evaluation of inflammatory bowel disease. The aim of this study was to establish the value of faecal calprotectin concentration as a predictor of remission in ulcerative colitis and its correlation with laboratory, endoscopic and clinical findings. METHODS: The single centre study included 126 adult patients with established diagnosis of ulcerative colitis consecutively visiting our Day clinic from March 2017 to March 2019. We measured serum biomarkers- CRP, haemoglobin, leukocytes and platelets. Faecal calprotectin was determined from stool, and endoscopy was performed with calculation of MAYO endoscopic subscore system (MES 0-1: remission, and MES 2-3: active disease). Clinical assessment was done by using Mayo score for ulcerative colitis (clinical Mayo score <2:remission, >5: active disease).The statistical analysis was performed using an univariate and multivariate model of disease remission prediction using logistic regression. RESULTS: According to univariate analysis the increase of faecal calprotectin concentration by 10 ug/g is associated with an 8% decrease in probability of disease remission (OR 0.9921, p < .05). In the multivariate analysis, faecal calprotectin remained a significant predictor of disease remission (OR 0.9948, 95% CI 0.9914-0.9982, p = .0028), however, with a significant contribution of C-reactive protein (OR 0.8340, 95% CI 0.7085-0.9818, p = .0292). According to our model the cut off value for faecal calprotectin was 154 ug/g. CONCLUSION: Our results have shown that faecal calprotectin is an independent predictor of remission in UC patients. The results of our study represent real-life data from a single university centre dealing with FC as a prognostic marker in patients with UC. KEY MESSAGESFaecal calprotectin is an independent predictor of remission in UC patients.Recent studies have suggested that calprotectin correlates well with endoscopic activity of inflammation but correlation of faecal calprotectin in a phase of remission hasn't been evaluated yet.We have found that other inflammatory biomarkers do not correlate well with either endoscopic or clinical activity in ulcerative colitis.

Genetic and shared environmental risk factors do not lead to eosinophil activation in healthy twins of IBD patients.

OBJECTIVE: To examine the role of eosinophils in the pre-diagnostic phase of inflammatory bowel disease (IBD), we studied the influence of genetic and shared environmental risk factors in a twin cohort of IBD. MATERIAL AND METHODS: We analysed eosinophil derived neurotoxin (EDN) and eosinophil cationic protein (ECP) in faecal samples from twin pairs with Crohn's disease (n = 37) or ulcerative colitis (n = 21) and from external healthy controls (n = 44). Eosinophils stained with eosinophil peroxidase (EPO) were quantified in rectal biopsies. Ratios with 95% confidence intervals were calculated. RESULTS: Twins with Crohn' disease displayed higher levels of EDN (Ratio = 2.98, 1.65-5.37) and ECP (Ratio 1.83, 1.24-2.70) than their healthy siblings. Levels did not differ between healthy twin-siblings and external controls (EDN, Ratio = 1.52, 0.79-2.94 and ECP, Ratio = 0.93, 0.56-1.54). Higher levels of EDN (Ratio = 2.43, 1.13-5.24) and ECP (Ratio = 1.53, 0.92-2.53) were observed among twins with ulcerative colitis vs their healthy siblings. Levels did not differ between healthy twin-siblings and external controls (EDN, Ratio = 1.08, 0.51-2.25 and ECP, Ratio = 1.29, 0.74-2.26). Using intra-class correlation coefficient (ICC), we found no agreement in levels of EDN or ECP in discordant pairs, except for ECP in monozygotic Crohn's disease pairs (ICC = 0.63). In contrast, agreement was observed in monozygotic pairs concordant for Crohn's disease (EDN, ICC = 0.67 and ECP, ICC = 0.66). The number of eosinophils in rectum was increased in twins with ulcerative colitis vs their healthy sibling (Ratio = 2.22, 1.50-3.27). CONCLUSIONS: Activation of eosinophils in IBD seems to be a consequence of inflammation rather than an effect of genetic and shared environmental risk factors alone.

Seasonal influences on the use of genetic markers as performance indicators for small wastewater treatment plants.

The development of microbial source tracking methods has resulted in an array of genetic faecal markers for assessing human health risks posed from surface water pollution. However, their use as performance metrics at wastewater treatment plants (WWTPs) has not been explored extensively. Here we compared three Bacteroides (HF183, HumM2, AllBac) and two E. coli (H8, RodA) genetic markers for summer and winter performance monitoring at twelve small rural (<250 PE) and three larger WWTPs in NE England. Small WWTPs are of interest because they are poorly understood and their impact on surface water quality may be underestimated. Overall, genetic marker data showed significant differences in treatment performance at smaller versus larger WWTPs. For example, effluent abundances of HF183 and HumM2 were significantly higher in smaller systems (p = 0.003 for HumM2; p = 0.02 for HF183). Genetic markers also showed significant differences in performance between seasons (p < 0.01, n = 120), with human-specific markers (i.e., HF183, HumM2, H8) being generally better for summer WWTP monitoring. In contrast, Bacteroides markers were much more suitable for winter monitoring, possibly because the E. coli markers are less sensitive to differences in temperature and sunlight conditions. Overall, Bacteroides markers best described WWTP treatment performance across all samples, although seasonal differences suggest caution is needed when markers are used for performance monitoring. Genetic markers definitely provide rapid and new information about WWTP performance, but more spatially diverse studies are needed to refine their use for routine WWTP monitoring.

Multiparametric monitoring of microbial faecal pollution reveals the dominance of human contamination along the whole Danube River.

The microbial faecal pollution of rivers has wide-ranging impacts on a variety of human activities that rely on appropriate river water quality. Thus, detailed knowledge of the extent and origin of microbial faecal pollution is crucial for watershed management activities to maintain safe water use. In this study, the microbial faecal pollution levels were monitored by standard faecal indicator bacteria (SFIB) along a 2580 km stretch of the Danube, the world's most international river, as well as the Danube's most important tributaries. To track the origin of faecal pollution, host-associated Bacteroidetes genetic faecal marker qPCR assays for different host groups were applied in concert with SFIB. The spatial resolution analysis was followed by a time resolution analysis of faecal pollution patterns over 1 year at three selected sites. In this way, a comprehensive faecal pollution map of the total length of the Danube was created, combining substantiated information on both the extent and origin of microbial faecal pollution. Within the environmental data matrix for the river, microbial faecal pollution constituted an independent component and did not cluster with any other measured environmental parameters. Generally, midstream samples representatively depicted the microbial pollution levels at the respective river sites. However, at a few, somewhat unexpected sites, high pollution levels occurred in the lateral zones of the river while the midstream zone had good water quality. Human faecal pollution was demonstrated as the primary pollution source along the whole river, while animal faecal pollution was of minor importance. This study demonstrates that the application of host-associated genetic microbial source tracking markers in concert with the traditional concept of microbial faecal pollution monitoring based on SFIB significantly enhances the knowledge of the extent and origin of microbial faecal pollution patterns in large rivers. It constitutes a powerful tool to guide target-oriented water quality management in large river basins.

Effect of faecal calprotectin assay variability on the management of inflammatory bowel disease and potential role of faecal S100A12.

AIMS: To prospectively evaluate whether between-assay variability of different faecal calprotectin (f-Cp) assays influences diagnostic accuracy for inflammatory bowel disease (IBD) in a cohort of patients with confirmed IBD and irritable bowel syndrome (IBS). To also evaluate the diagnostic accuracy of faecal S100A12 (f-S100A12) against f-Cp in the same patient cohort and assess whether f-S100A12 offers additional diagnostic value. METHODS: F-Cp using four commercially available f-Cp assays, f-S100A12 and blood biomarkers were measured in patients, recruited from the local IBD clinic, who had established IBS or active ulcerative colitis (UC) and Crohn's disease (CD). Diagnostic sensitivities and specificities for each assay and biomarker were calculated and compared. RESULTS: Median f-Cp levels in all assays were significantly higher in UC (347-884 µg/g; n=28) and CD (377-838 µg/g; n=15) compared with IBS (6-27 µg/g; n=17). Sensitivities and specificities at 50 µg/g were 94%-100% and 82%-100%, respectively. Median f-S100A12 levels were significantly higher in UC (81.0 µg/g; IQR 38.3-159.8) and CD (47.2 µg/g; IQR 5.3-108.9) compared with IBS (0.7 µg/g; IQR 0.5-0.8). At 2.8 µg/g, f-S100A12 had a sensitivity of 97% and specificity of 94%. The blood biomarkers demonstrated sensitivities and specificities of 44%-63% and 80%-92%, respectively. CONCLUSIONS: The diagnostic sensitivity of the calprotectin assays was similar despite inter-kit variability in absolute values. There is a need for f-Cp assay standardisation, but in its absence assay-specific cut-off values may optimise their diagnostic performance. F-S100A12 demonstrated comparable sensitivity and specificity to f-Cp and although a research tool at present, may have a future role to play in the diagnosis and management of these patients.

Accuracy of Faecal Calprotectin and Neutrophil Gelatinase B-associated Lipocalin in Evaluating Subclinical Inflammation in UlceRaTIVE Colitis-the ACERTIVE study.

BACKGROUND AND AIMS: Mucosal healing and histological remission are different targets for patients with ulcerative colitis, but both rely on an invasive endoscopic procedure. This study aimed to assess faecal calprotectin and neutrophil gelatinase B-associated lipocalin as biomarkers for disease activity in asymptomatic ulcerative colitis patients. METHODS: This was a multicentric cross-sectional study including 371 patients, who were classified according to their endoscopic and histological scores. These results were evaluated alongside the faecal levels of both biomarkers. RESULTS: Macroscopic lesions [i.e. endoscopic Mayo score ≥1] were present in 28% of the patients, and 9% had active disease according to fht Ulcerative Colitis Endoscopic Index of Severity. Moreover, 21% presented with histological inflammation according to the Geboes index, whereas 15% and 5% presented with focal and diffuse basal plasmacytosis, respectively. The faecal levels of calprotectin and neutrophil gelatinase B-associated lipocalin were statistically higher for patients with endoscopic lesions and histological activity. A receiver operating characteristic-based analysis revealed that both biomarkers were able to indicate mucosal healing and histological remission with an acceptable probability, and cut-off levels of 150-250 µg/g for faecal calprotectin and 12 µg/g for neutrophil gelatinase B-associated lipocalin were proposed. CONCLUSIONS: Faecal calprotectin and neutrophil gelatinase B-associated lipocalin levels are a valuable addition for assessment of disease activity in asymptomatic ulcerative colitis patients. Biological levels of the analysed biomarkers below the proposed thresholds can rule out the presence of macroscopic and microscopic lesions with a probability of 75-93%. However, caution should be applied whenever interpreting positive results, as these biomarkers present consistently low positive predictive values.

Diagnostic importance of faecal markers in long-term monitoring of anti-TNF-α therapy in primary responders with Crohn's disease.

INTRODUCTION: Monitoring the response to biological treatment in Crohn's disease (CD) is a very important element of the therapeutic optimisation. AIM: To evaluate the usefulness of measuring calprotectin, lactoferrin, and myeloperoxidase in stool as markers of long-term clinical and endoscopic response to anti-tumour necrosis factor α (anti-TNF) treatment in CD. MATERIAL AND METHODS: The studied group consisted of 35 CD patients treated with anti-TNF-α antibodies. Clinical activity was evaluated using Crohn's Disease Activity Index (CDAI), and the exacerbation of endoscopic changes was evaluated using a Simple Endoscopic Score for Crohn's Disease (SES-CD). The concentration of calprotectin, lactoferrin, and myeloperoxidase was measured using the ELISA method. All measurements were performed three times - before, after 3 months, and after a year of therapy. RESULTS: During anti-TNF treatment the concentrations of all measured faecal markers decreased significantly in relation to baseline values. We observed a significant correlation at all time-points: before the therapy, after 3 months, and 12 months after starting the therapy, between the concentration of calprotectin and SES-CD, calprotectin and CDAI, as well as between lactoferrin and SES-CD, and lactoferrin and CDAI. Myeloperoxidase correlated with both SES-CD and CDAI only after 1 year of treatment. CONCLUSIONS: Faecal calprotectin and lactoferrin are valuable markers of clinical and endoscopic activity of CD in patients treated with anti-TNF antibodies. They are useful in monitoring the response to treatment. The usefulness of myeloperoxidase in this respect remains controversial.

Application of long-chain alcohols as faecal markers to estimate diet composition of horses and cattle fed with herbaceous and woody species.

Utilization of long-chain alcohols (LCOH) as diet composition markers in horses and cattle was assessed in a study conducted with 12 mature crossbreed mares (385±47 kg BW) and six adult non-lactating cows (499±36 kg BW) of Asturiana de los Valles breed. The LCOH data were combined with alkane and long-chain fatty acid (LCFA) data to test the applicability of combining these markers to estimate diet composition. Animals were randomly divided into groups of three animals and received a daily total amount of 1.0 kg dry matter/100 kg BW of diets composed of different proportions of ryegrass (Lolium perenne) and woody species (Ulex gallii and heather). Diet composition was estimated from even-chain LCOH (C(20)-OH to C(30)-OH) combined or not with alkane (C(25)-C(31) and C(33)) and/or LCFA (C(22)-FA to C(28)-FA, C(30)-FA, C(32)-FA and C(34)-FA) concentrations in diet components and faeces by least-squares procedures, using marker faecal concentrations uncorrected for incomplete faecal recovery (FR0) or corrected using mean recoveries across diets within animal species (FR1). Results showed large differences between plant species in their LCOH profiles, and that these markers offered additional discriminatory information to that provided by alkanes and LCFA. The LCOH markers were incompletely recovered in the faeces of both animal species. In cattle, LCOH FR tended to increase with carbon-chain length in a linear manner in both diets (P < 0.001), whereas in horses overall data showed a curvilinear relationship between these variables. Combination of LCOH, LCFA and alkanes resulted in more accurate diet estimates. Correction of faecal LCOH concentrations to incomplete FR led to more accurate diet composition estimates in both animal species. Results obtained in this study suggest the usefulness of LCOH markers combined with alkanes and LCFA to estimate diet composition of horses and cattle grazing mixed grassy-woody plant communities.