Tributyrin

The study was approved by the Ethics Committee at the London School of Hygiene & Tropical Medicine and the relevant ethical committees at each of the seven participating African centers (Comité National d'Ethique pour la Recherche en Santé (CNERS) in Senegal; Research & Ethical Committee of the University of Maiduguri Teaching Hospital in Nigeria; Comité Consultatif National d'Ethique in Niger; Comité d'Ethique de la Faculté de Médecine et Pharmacie D’Odonto-Stomatologie de l’Université de Bamako in Mali; Navrongo Health Research Center Institutional Review Board in Ghana; Armauer Hansen Research Institute/All Africa Leprosy Rehabilitation and Training Center (AHRI/ALERT) Ethical Review Committee in Ethiopia, and in Chad approval was granted by a committee established to oversee MenAfriCar studies by the Ministry of Health since no formal ethical committee was in place in the country at the time). During enrollment, staff explained the purpose and nature of the study, and a parent or guardian provided written, informed consent. Older children provided written assent (age determined by local practices) and younger children provided oral assent.
During 2009 and early 2010, cross-sectional surveys were conducted in schoolchildren at five urban sites (N'Djamena, Chad; Navrongo, Ghana; Butajira, Ethiopia; Bamako, Mali; Maiduguri, Nigeria;) and two rural sites (Say, Niger; Niakhar, Senegal) in seven countries of the African meningitis belt to compare methods for collecting pharyngeal swabs. Standard field and laboratory operating procedures were implemented in all centers; these have been described previously [18 (link)]. The samples collected in Chad, Ghana, and Nigeria had to be discarded following technical difficulties maintaining proper storage temperatures; results from the surveys in Ethiopia, Mali, Niger, and Senegal are reported in this analysis.
Each center chose one or more schools in an area where no meningitis vaccination campaign had occurred in the previous two years (or previous six months in Niger) and recruited a convenience sample until the target sample size of 250 children in each center was reached. Children were eligible if they were 5–15 years of age, had no severe acute or long-term illness, and if they had not received a meningitis vaccine during the specified time period.
Two pharyngeal samples were collected from each child using sterile, dacron-tipped swabs with plastic shafts. Two techniques for swabbing the pharynx through the mouth were compared; one method involved swabbing the posterior pharynx behind the uvula (hereafter referred to as method “U”) and the other method involved swabbing both the posterior pharynx behind the uvula and one tonsillar fossa (hereafter referred to as method “T”). The order of the swabbing method was alternated every 25 participants in Ethiopia, and every 20 participants in Mali; in Niger and Senegal the U swab was collected followed by the T swab throughout. The sample labels did not indicate which method was used for sample collection so that the laboratory personnel could not distinguish between the T and U swabs during processing.
Modified Thayer-Martin (TM) selective agar plates were prepare locally using Gonococci agar base (Oxoid CM0367B), hemoglobin powder (Oxoid LP053B) containing 3mg/liter vancomycin, 7.5mg/liter colistin, 12.5 U/liter nystatin, 5mg/liter trimetropin lactate (Oxoid SR00991E) and Vitox enrichment supplement (Oxoid SR0090A) (Thermo Scientific, UK) [19 (link)]. Swabs were plated immediately in the field onto TM plates, returned to the laboratory within six hours, and incubated in 5% CO₂ at 35-37°C for 24-48 hours to determine growth. A single colony with morphology typical of N. meningitidis (large or medium size, blue-grey color, and mucoid in appearance) was selected, sub-cultured on a blood agar plate (BAP), streaked, and incubated in 5% CO₂ at 35-37°C for an additional 18-24 hours. BAPs were prepared locally with blood agar base number 2 (Oxoid, CM0271, Thermo Scientific, UK) supplemented with 5% defibrinated sheep’s blood. The colonies remaining on the TM selective agar plate were collected with a sterile plastic loop, suspended in a cryotube containing 1mL of Brain heart infusion (BHI) broth supplemented with 15% glycerol and stored at -80°C. The remaining colonies from the BAP were emulsified in 0.5 mL phosphate buffered saline (PBS) in microcentrifuge tubes, boiled for 20 minutes to release DNA, cooled, divided into four aliquots in 250μL tubes and stored at -20°C for future molecular testing.
During the pilot survey in Mali, the selected colonies sub-cultured onto BAP underwent oxidase testing and Gram staining to ensure that they were Gram-negative diplococci. However, DNA samples prepared from the selected colonies sent from Mali to the University of Oxford for molecular testing showed that these samples contained DNA from many organisms that were not Gram-negative diplococci. This prompted the inclusion of three additional biochemical tests in a new protocol circulated to all the sites: γ-glutamyl transferase activity (GGT) (Rosco Diagnostica, Denmark) for identification of presumptive N. meningitidis, β-galactosidase activity with ortho-nitrophenyl-β-D-galactopyranoside (ONPG) (Rosco Diagnostica, Denmark) for identification of Neisseria lactamica, and butyrate esterase activity (Tributyrin) (Rosco Diagnostica, Denmark) to further distinguish Moraxella species from Neisseria species which was the main cause of the initial misidentification [18 (link)]. The new protocol piloted in Mali in June 2010 indicated that the introduction of the biochemical tests improved species identification. The new protocol incorporating the biochemical tests was introduced in the rest of the sites two months later using samples from the original pilot study that had been stored in BHI broth supplemented with glycerol at -80°C. These samples were thawed at room temperature, vortex mixed briefly, and plated on TM plates followed by BAPs as described above. Growth from all oxidase positive, Gram negative diplococcic samples were harvested into microcentrifuge tubes containing 1 mL PBS, placed in a boiling water bath for 20 minutes to release DNA and inactivate nucleases, cooled, divided into lots and stored at -20°C for molecular testing. Heat killed cell suspensions, prepared from all oxidase-positive, Gram-negative diplococci from each site (49 from Ethiopia, 188 from Mali, 23 from Niger, 95 from Senegal) were sent to the University of Oxford for molecular characterization [18 (link)].
A swab was positive for N. meningitidis if the rplF sequence-based assay, described previously in [18 (link)], identified N. meningitidis. A participant was classified as a positive carrier if at least one of the samples provided met this definition. Data for each center were collected and managed locally using Microsoft Excel; data were cleaned and merged centrally using STATA for Mac version 12.1 (StataCorp LP 2012). Data were analyzed to determine the overall prevalence of N. meningitidis among the swabs collected and among the participants overall and at each center using standard statistical measures and calculating the exact binomial 95% confidence intervals (CIs) (also known as the Clopper-Pearson CIs) [20 ] for the point prevalence estimates. The two methods for collecting pharyngeal swabs were compared by calculating the concordance, kappa, and applying McNemar’s test for paired samples to test the null hypothesis that there was no difference between the proportion of positive samples observed using one method compared to the other. Analyses were performed combined (pooling all samples) and by center. Data were analyzed using STATA for Mac version 12.1 (StataCorp LP 2012).

C57BL/6 female mice (10–11 weeks) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Mice were randomized to receive a chronic–binge ethanol Lieber DeCarli feeding model consisting of 10 days chronic ethanol (5% v/v) exposure and ±daily supplementation with saline or 5 mM tributyrin by oral gavage. Control animals were pair-fed an isocaloric diet with maltose dextrin substituted for ethanol ± 5 mM tributyrin or saline supplementation by oral gavage daily. On the 11th day, mice were administered an oral binge gavage of either 5 g/kg ethanol or maltose with either 2.5 mM tributyrin or saline as previously randomized. There were four groups in total: pair-fed saline (PF-S), pair-fed tributyrin (PF-TB), ethanol-fed saline (EF-S), and ethanol-fed tributyrin (EF-TB). Mice were anesthetized 6 h post-gavage, blood was collected from the inferior vena cava, and tissue (small intestine, cecum, lung) was dissected and stored for future analyses.

In the experimental feeding trial, each tank was randomly assigned to one of the dietary treatments as follows: basal control diet (Cont), diet supplemented with a tributyrin product (TBT, supplied by Lucta S.A., Barcelona, Spain) at 0.1% (m/m) (TBT1), 0.2% (TBT2), and 0.4% (TBT4) (triplicate tanks per diet). This product consisted of 98% purity tributyrin (55%) and silica carrier (45%). A detailed description of the formulation of each diet can be found in Supplementary Table S1. Despite using 60% plant-based meals, it is worth noting that this was not a fishmeal-free diet, and 10% fishmeal was included. This formulation was developed to be nutritionally challenging but with a realistic application in the commercial context. Fish were hand-fed twice a day to apparent satiety at 9:00 h and 16:00 h during 50 days. Rainbow trout had an initial body weight (IBW) of 18.99 ± 0.19 g (mean ± SE) and a final body weight (FBW) of 63.23 ± 1.02 g at the end of the trial. No significant differences were found in the IBW (P = 0.478).