<b>Background and Objective:</b> The presence of Asian small-clawed otters (<i>Aonyx cinereus</i>) in West Sumatra has been reported from ecological data in the form of footprints and feces, while its genetic information has not been reported yet. This genetic information needs to be reported along with the determination of <i>A. cinereus</i> as a vulnerable species and is experiencing population decline by the International Union for Conservation of Nature (IUCN). This study aimed to determine the phylogenetic relationship of <i>A. cinereus</i> found in West Sumatra with other regions. <b>Materials and Methods:</b> The samples used were <i>A. cinereus</i> stool collected from several wetland locations in West Sumatra. <i>Aonyx cinereus</i> DNA was extracted from stool samples following the QIAamp Fast DNA Stool Mini Kit protocol (Qiagen). Amplification was performed using the CO1 gene. The IQTree was used to provide phylogenetic information on <i>A. cinereus</i> and MEGA 7 was used to determine the uncorrected genetic distance of <i>A. cinereus</i>. <b>Results:</b> <i>Aonyx cinereus</i> clustered to form three sub-clusters namely <i>A. cinereus</i> Sundaland, Laos lineage and unknown lineage. <i>Aonyx cinereus</i> Sundaland consists of <i>A. cinereus</i> West Sumatra and <i>A. cinereus</i> Sarawak, Malaysia which are closely related with a genetic distance of 0.68%. Moreover, compared to <i>A. cinereus</i> from unknown lineage (including Captive Copenhagen Zoo) and Laos lineage, <i>A. cinereus</i> of West Sumatra had a genetic distance of 0.68-1.20 and 4.18%, respectively. <b>Conclusion:</b> Wetland conversion and the role of humans have influenced the obstacle to connectivity among populations that cause genetic variation.
Sewage contamination is a principal concern in water quality management as pathogens in sewage can cause diseases and lead to detrimental health effects in humans. This study examines the distribution of seven sterol compounds, namely coprostanol, epi-coprostanol, cholesterol, cholestanol, stigmasterol, campesterol, and β-sitosterol in filtered and particulate phases of sewage treatment plants (STPs), groundwater, and river water. For filtered samples, solid-phase extraction (SPE) was employed while for particulate samples were sonicated. Quantification was done by using gas chromatography-mass spectrometer (GC-MS). Faecal stanols (coprostanol and epi-coprostanol) and β-sitosterol were dominant in most STP samples. Groundwater samples were influenced by natural/biogenic sterol, while river water samples were characterized by a mixture of sources. Factor loadings from principal component analysis (PCA) defined fresh input of biogenic sterol and vascular plants (positive varimax factor (VF)1), aged/treated sewage sources (negative VF1), fresh- and less-treated sewage and domestic sources (positive VF2), biological sewage effluents (negative VF2), and fresh-treated sewage sources (VF3) in the samples. Association of VF loadings and factor score values illustrated the correlation of STP effluents and the input of biogenic and plant sterol sources in river and groundwater samples of Linggi. This study focuses on sterol distribution and its potential sources; these findings will aid in sewage assessment in the aquatic environment.
The Langat River in Malaysia has been experiencing anthropogenic input from urban, rural and industrial activities for many years. Sewage contamination, possibly originating from the greater than three million inhabitants of the Langat River Basin, were examined. Sediment samples from 22 stations (SL01-SL22) along the Langat River were collected, extracted and analysed by GC-MS. Six different sterols were identified and quantified. The highest sterol concentration was found at station SL02 (618.29 ng/g dry weight), which situated in the Balak River whereas the other sediment samples ranged between 11.60 and 446.52 ng/g dry weight. Sterol ratios were used to identify sources, occurrence and partitioning of faecal matter in sediments and majority of the ratios clearly demonstrated that sewage contamination was occurring at most stations in the Langat River. A multivariate statistical analysis was used in conjunction with a combination of biomarkers to better understand the data that clearly separated the compounds. Most sediments of the Langat River were found to contain low to mid-range sewage contamination with some containing 'significant' levels of contamination. This is the first report on sewage pollution in the Langat River based on a combination of biomarker and multivariate statistical approaches that will establish a new standard for sewage detection using faecal sterols.
This is the first report on fecal pollution using molecular markers in Southeast Asia where serious sewage pollution has occurred. A simple and sensitive analytical method using gas chromatography-mass spectrometry for 10 sterols in various environmental samples was developed to monitor extensive areas of tropical Asia. First, the method was applied to wastewater to confirm that >95% of sterols existed in the particulate phase. Then the approach was applied to a tropical Asian region, Malaysia and Vietnam, with a selection of 59 sampling stations in total. River water and sediment samples were collected and analyzed for chemical markers (coprostanol and other sterols) and microbiological markers (fecal coliforms and fecal streptococci). Particulate coprostanol concentrations ranged from <0.0001 to 13.47 microg/L in tropical river and estuarine waters, indicating severe fecal pollution in populous areas. Coprostanol concentrations in the sediments ranged from 0.005 to 15.5 microg/g-dry. The sedimentary coprostanol concentrations were lower than those reported in some urban areas of industrialized countries. This is probably because frequent heavy rain induces intensive input of eroded soil, which dilutes fecal material in river sediments. The relationship between the concentrations of fecal sterols and bacterial indicators was examined in an attempt to develop public health criteria for coprostanol levels applicable to the tropical region. Coprostanol concentrations of 30-100 ng/L or percent coprostanol levels of 2% corresponded to approximately 1000 fecal coliforms per 100 mL, which is set for secondary contact limit in many countries. These coprostanol concentrations were lower than those proposed as criteria in temperate countries, probably owing to greater survival of bacteria in warmer tropical waters. On the basis of these criteria, extensive monitoring of sediments suggests that poor sanitary conditions exist in most of the urbanized area of Malaysia and in several urban and rural sites in Vietnam.
Gas-Liquid chromatography (GLC) was used to detect the presence of isocaproic acid produced by Clostridium difficile from 54 stool samples grown in cycloserine-cefoxitin broth. Isocaproic acid was detected in 12 samples of which 5 were confirmed to be Clostridium difficile by culture and biochemical tests. The detection of isocaproic acid by GLC together with the presence of presumptive colonies on primary selective culture media provides a more rapid laboratory diagnosis for Clostridium difficile.
In this study, we sequenced a partial segment of the mitochondrial control region from 21 proboscis monkeys of the Klias peninsula, the last large population remaining on the west coast of Sabah, Malaysia. Our results showed that this population retains substantial genetic variation, and subpopulations from different river systems in the central and southern portions of the Klias share multiple haplotypes. We also compared our data with previously generated sequences from 2 eastern populations of proboscis monkeys in Sabah and found little evidence of regional genetic structure. Based on these results, we argue that conservation efforts should focus on restoring connectivity between central and southern Klias peninsula proboscis monkeys and discuss future analyses needed to better understand the mitochondrial structure of proboscis monkeys in Sabah.
Fecal estradiol concentrations were measured in three captive unmated female sun bears (Helarctos malayanus) from August 1998 to July 1999 in Sarawak, Malaysia and vaginal smears from one of the females was observed in August 1998 and March 1999. A single peak in fecal estradiol concentration was obvious for each bear in August or September 1998, and there was a much higher percentage of superficial vaginal anuclear cells in August 1998 than in March 1999. These results suggest that sun bears in Sarawak are likely to be a seasonal breeder associated with a peak of estrogen production in August or September.
In the present study, the influences of diets (i.e. chow and AIN-93 diets) on the interpretation of various fecal parameters including viable microbiota, moisture, weight, and short-chain fatty acids in rats fed different amounts of inulin (0.5-2 g/kg). Eight groups of rats (n = 8/group) were fed, for 4 weeks, chow or AIN-93 diets with or without inulin supplementation. Fecal samples were analyzed for different fecal parameters. After a 2-week adaptation, apparent differences in some fecal parameters were observed between the chow and AIN-93 diet groups. Throughout the 4-week intervention period, significantly (p
The gut microbiome is involved in the pathogenesis of many diseases including polycystic ovarian syndrome (PCOS). Modulating the gut microbiome can lead to eubiosis and treatment of various metabolic conditions. However, there is no proper study assessing the delivery of microbial technology for the treatment of such conditions. The present study involves the development of guar gum-pectin-based solid self-nanoemulsifying drug delivery system (S-SNEDDS) containing curcumin (CCM) and fecal microbiota extract (FME) for the treatment of PCOS. The optimized S-SNEDDS containing FME and CCM was prepared by dissolving CCM (25 mg) in an isotropic mixture consisting of Labrafil M 1944 CS, Transcutol P, and Tween-80 and solidified using lactose monohydrate, aerosil-200, guar gum, and pectin (colon-targeted CCM solid self-nanoemulsifying drug delivery system [CCM-CT-S-SNEDDS]). Pharmacokinetic and pharmacodynamic evaluation was carried out on letrozole-induced female Wistar rats. The results of pharmacokinetic studies indicated about 13.11 and 23.48-fold increase in AUC of CCM-loaded colon-targeted S-SNEDDS without FME (CCM-CT-S-SNEDDS (WFME)) and CCM-loaded colon-targeted S-SNEDDS with FME [(CCM-CT-S-SNEDDS (FME)) as compared to unprocessed CCM. The pharmacodynamic study indicated excellent recovery/reversal in the rats treated with CCM-CT-S-SNEDDS low and high dose containing FME (group 13 and group 14) in a dose-dependent manner. The developed formulation showcasing its improved bioavailability, targeted action, and therapeutic activity in ameliorating PCOS can be utilized as an adjuvant therapy for developing a dosage form, scale-up, and technology transfer.
The purpose of this paper is to investigate the potential prebiotic properties and proliferation mechanism of fermented milk-derived peptides. In this study, fermented milk-derived polypeptides were obtained by extraction, separation, and purification. The purified peptides were used to culture fecal flora in vitro, and the relative abundance and composition of the flora were analyzed by high-throughput 16S rRNA sequencing technology. The results showed that peptides can promote the proliferation of beneficial bacteria Lactococcus in the intestine and inhibit the proliferation of harmful bacteria Escherichia coli-Shigella. The amino acid sequence of polypeptide components was determined and synthesized in vitro to verify the proliferation of intestinal flora; the proliferation mechanism of peptides on Lactococcus lactis was studied using non-targeted LC-MS metabolomics technology. Five important peptides with molecular weights of 1000-2000 Da were identified by LC-MS: GRP1 (LTEEEK), GRP2 (ENDAPSPVM*K), GRP3 (ITVDDK), GRP4 (EAM*APK) and GRP5 (LPPPEK). The results showed that the peptides could affect the arginine biosynthesis pathway and the amino sugar and nucleotide sugar metabolism of Lactococcus lactis. In addition, the peptides increased the expression of organic acids and their derivatives in Lactococcus lactis. This study provides a research basis for expanding the potential sources of new prebiotics and also opens up a new idea for discovering new prebiotics in vitro.
Understanding the basal gut bacterial community structure and the host metabolic composition is pivotal for the interpretation of laboratory treatments designed to answer questions pertinent to host-microbe interactions. In this study, we report for the first time the underlying gut microbiota and systemic metabolic composition in BALB/c mice during the acclimatisation period. Our results showed that stress levels were reduced in the first three days of the study when the animals were subjected to repetitive handling daily but the stress levels were increased when handling was carried out at lower frequencies (weekly). We also observed a strong influence of stress on the host metabolism and commensal compositional variability. In addition, temporal biological compartmental variations in the responses were observed. Based on these results, we suggest that consistency in the frequency and duration of laboratory handling is crucial in murine models to minimise the impact of stress levels on the commensal and host metabolism dynamics. Furthermore, caution is advised in consideration of the temporal delay effect when integrating metagenomics and metabonomics data across different biological matrices (i.e. faeces and urine).
We investigated the genetic structure within and among Bornean orang-utans (Pongo pygmaeus) in forest fragments of the Lower Kinabatangan flood plain in Sabah, Malaysia. DNA was extracted from hair and faecal samples for 200 wild individuals collected during boat surveys on the Kinabatangan River. Fourteen microsatellite loci were used to characterize patterns of genetic diversity. We found that genetic diversity was high in the set of samples (mean H(E) = 0.74) and that genetic differentiation was significant between the samples (average F(ST) = 0.04, P < 0.001) with F(ST) values ranging from low (0.01) to moderately large (0.12) values. Pairwise F(ST) values were significantly higher across the Kinabatangan River than between samples from the same river side, thereby confirming the role of the river as a natural barrier to gene flow. The correlation between genetic and geographical distance was tested by means of a series of Mantel tests based on different measures of geographical distance. We used a Bayesian method to estimate immigration rates. The results indicate that migration is unlikely across the river but cannot be completely ruled out because of the limited F(ST) values. Assignment tests confirm the overall picture that gene flow is limited across the river. We found that migration between samples from the same side of the river had a high probability indicating that orang-utans used to move relatively freely between neighbouring areas. This strongly suggests that there is a need to maintain migration between isolated forest fragments. This could be done by restoring forest corridors alongside the river banks and between patches.
Voluntary food intake, digestibility and water turnover were determined in adult Malaysian lesser mouse-deer (Tragulus javanicus) given unlimited access to lundai foliage (Sapium baccatum). Daily dry matter (DM) intake was 42.4 g/kg metabolic live mass (M0.73) or 3.7% M. Digestible energy intake was 853 kJ/day (571 kJ metabolisable energy per M0.73), calculated to be used with 79% efficiency. Apparent digestibility (%) of organic matter was 83.8, crude fibre 63.7, acid detergent fibre 60.5, neutral detergent fibre 72.1 and crude protein 65.0. Urinary excretion of the purine derivative, allantoin, was 0.05 mg/g digestible DM intake suggesting rumen microbial yield efficiency may be lower than in other ruminant species. Total water intake was 182 ml/M0.82. The body-water content of the fed mouse-deer, from tritiated water dilution, was 77% M, consistent with a very lean carcass. Turnover of body water was 17% per day. The mouse-deer produced relatively dry, well-defined faecal pellets.
1. Various dosages of metabolite combinations of the Lactobacillus plantarum RI11, RG14 and RG11 strains (COM456) were used to study the egg production, faecal microflora population, faecal pH, small intestine morphology, and plasma and egg yolk cholesterol in laying hens. 2. A total of 500 Lohmann Brown hens were raised from 19 weeks to 31 weeks of age. The birds were randomly divided into 5 groups and fed on various treatment diets: (i) basal diet without supplementation of metabolites (control); (ii) basal diet supplemented with 0·3% COM456 metabolites; (iii) basal diet supplemented with 0·6% COM456 metabolites; (iv) basal diet supplemented with 0·9% COM456 metabolites; and (v) basal diet supplemented with 1·2% COM456 metabolites. 3. The inclusion of 0·6% liquid metabolite combinations, produced from three L. plantarum strains, demonstrated the best effect in improving the hens' egg production, faecal lactic acid bacteria population, and small intestine villus height, and reducing faecal pH and Enterobacteriaceae population, and plasma and yolk cholesterol concentrations. 4. The metabolites from locally isolated L. plantarum are a possible alternative feed additive in poultry production.
1. Four combinations of metabolites produced from strains of Lactobacillus plantarum were used to study the performance of broiler chickens. 2. A total of 432 male Ross broilers were raised from one-day-old to 42 d of age in deep litter pens (12 birds/pen). These birds were divided into 6 groups and fed on different diets: (i) standard maize-soybean-based diet (negative control); (ii) standard maize-soybean-based diet + Neomycin and Oxytetracycline (positive control); (iii) standard maize-soybean-based diet + 0.3% metabolite combination of Lactobacillus plantarum RS5, RI11, RG14 and RG11 strains (com3456); (iv) standard maize-soybean-based diet + 0.3% metabolite combination of L. plantarum TL1, RI11 and RG11 (Com246); (v) standard maize-soybean-based diet + 0.3% metabolite combination of L. plantarum TL1, RG14 and RG11 (Com256) and (vi) standard maize-soybean-based diet + 0.3% metabolite combination of L. plantarum TL1, RS5, RG14 and RG11 (Com2356). 3. Higher final body weight, weight gain, average daily gain and lower feed conversion ratio were found in all 4 treated groups. 4. The addition of a metabolite combination supplementation also increased faecal lactic acid bacteria population, small intestine villus height and faecal volatile fatty acids and faecal Enterobacteriaceae population.
Fecal progesterone assays were conducted in 3 captive female sun bears (Helarctos malayanus) segregated from males to characterize the species-specific reproductive pattern in their original distribution area in Sarawak, Malaysia. Peaks of fecal progesterone concentrations were observed once annually, and lactation was observed after increasing progesterone concentrations in all females without mating stimulus. These results suggest that sun bears in Sarawak, Malaysia, may have a seasonal reproductive pattern and ovulation was noted to occur spontaneously, followed by pseudopregnancy.
A molecular sexing method by polymerase chain reaction (PCR) amplification of a portion of the sex-determining region Y (SRY) and the zinc finger (ZF) gene, as well as six equine Y-chromosome-specific microsatellite markers, were tested in the Malayan tapir (Tapirus indicus). While the microsatellite markers did not yield any male-specific amplicons for sex-typing, the SRY/ZF marker system produced reliable molecular sexing results by accurately sex-typing 31 reference Malayan tapirs, using whole blood, dried blood spot (DBS), or tissue samples as materials for DNA extraction. The marker system was also tested on 16 faecal samples, and the results were in general consistent with the pre-determined sexes of the animals, despite some amplification failures. A preliminary estimation of wild Malayan tapir population sex ratio was estimated from the Wildlife Genomic Resource Bank (WGRB) database of the Malaysian Department of Wildlife and National Parks (PERHILITAN), zoos, and the Sungai Dusun Wildlife Conservation Centre (WCC), as well as from the results of molecular sexing 12 samples of unknown sex. The overall sex ratio favoured females, but the deviation from parity was statistically not significant when tested using the binomial test (p > 0.05), which may be due to reduced statistical power caused by small sample sizes.
We use individual-based information on the behavior of wild female Japanese macaques in two consecutive years with different food availability (nut-rich vs. nut-poor) to test effects of dominance rank and nut fruiting on seed dispersal parameters. We predicted that social rank would affect dispersal (1) quantity, (2) quality, (3) species richness, and (4) percentage of berries in the diet in the nut-poor year, while these differences would disappear in the nut-rich year. We found seeds of nine fleshy-fruited plant species in the feces of the monkeys. The frequency of seed occurrence for two plant species (Viburnum dilatatum and Rosa multiflora) showed an interaction between dominance ranks and years; in the nut-poor year V. dilatatum seeds were more abundant among dominant females and R. multiflora among subordinates, while such inter-rank differences disappeared in the nut-rich year. Similarly, the intact ratio of V. dilatatum seeds was lower for dominants in the nut-poor year, while inter-rank variations disappeared in the nut-rich year. Finally, percentage of berries in diet and seed richness showed no inter-annual nor inter-rank variations. Our study highlights that differences in individuals' social rank lead to within-group variation in seed dispersal services and that these differences are dependent on nut availability.
Bacterial polyhydroxyalkanoates (PHA) are expensive partly due to the recovery and purification processes. Thus, many studies have been carried out in order to minimize the cost. Here we report on the use of mealworm, which is the larva of mealworm beetle (Tenebrio molitor) to recover PHA granules from Cupriavidus necator. Mealworms were shown to readily consume the freeze-dried C. necator cells and excrete the PHA granules in the form of whitish feces. Further purification using water, detergent and heat resulted in almost 100% pure PHA granules. Comparison with chloroform extraction showed no signs of reduction in the molecular weight and dispersion of the PHA molecules. Scanning electron microscopy and dynamic light scattering measurements revealed that the biologically recovered PHA granules retained their native spherical morphology. The PHA granules were subjected to a battery of tests to determine their purity and properties in comparison to the chloroform extracted PHA. This study has demonstrated the possibility of using mealworms as a biological agent to partially purify the PHA granules.
Analysis on fly artifacts produced by forensically important blowfly, Chrysomya megacephala (Fabricius) (Diptera:Calliphoridae), revealed several unique patterns. They can be divided into fecal spots, regurgitation spots and swiping stains. The characteristics of fecal spots are round with three distinct levels of pigmentation; creamy, brownish and darkly pigmented. Matrix of the spots appears cloudy. The round spots are symmetrical and non-symmetrical, delineated by irregular and darker perimeter which only visible in fairly colored fecal spots. Diameter of these artifacts ranged from 0.5 mm to 4 mm. Vomit or regurgitation spots are determined by the presence of craters due to sucking activity of blowflies and surrounded by thickly raised and darker colored perimeter. The size of these specks ranged from 1 mm to 2 mm. Matrix of the spots displays irregular surface and reflective under auxiliary microscope light. Swiping stains due to defecation by flies consists of two distinguishable segments, the body and tail. It can be seen as a tear drop-like, sperm-like, snake-like and irregular tadpole-like stain. The direction of body and tail is inconsistent and length ranged between 4.8 mm to 9.2 mm. A finding that should be highlighted in this observation is the presence of crater on tadpole-like swiping stain which is apparent by its raised border characteristic and reflective under auxiliary microscope light. The directionality of this darkly brown stain is random. This unique mix of regurgitation and swiping stain has never been reported before. Highlighting the features of artifacts produced by flies would hopefully add our understanding in differentiating them from blood spatters produced from victims at crime scenes.