Fecal Water Samples Research Articles

Evaluation of crAssphages as a potential marker of human viral contamination in environmental water and fresh leafy greens.

CrAssphages are human gut bacteriophages with potential use as an indicator of human fecal contamination in water and other environmental systems. We determined the prevalence and abundance of crAssphages in water, food, and fecal samples and compared these estimates with the prevalence of norovirus. Samples were tested using two crAssphage-specific qPCR assays (CPQ056 and TN201-203) and for norovirus using TaqMan realtime RT-PCR. CrAssphage was detected in 40% of human fecal specimens, 61% of irrigation water samples, 58.5% of stream water samples, and 68.5% of fresh leafy greens samples. Interestingly, across all sample categories, crAssphage concentrations were 2-3 log10 higher than norovirus concentrations. The correlation of detection of crAssphage and norovirus was significant for the irrigation water samples (r = 0.74, p = 7.4e-06). Sequences obtained from crAssphage positive samples from human fecal and stream water samples phylogenetically clustered with genotype I crAssphages, whereas sequences derived from irrigation water samples clustered differently from other genotypes. Our data show that crAssphages were prevalent in norovirus-positive water samples and in fresh leafy green samples, there was a strong correlation between the presence of crAssphage and norovirus. CrAssphage genomic copies were consistently higher than norovirus copies in all sample types. Overall, our findings suggest that crAssphages could be used as reliable indicators to monitor fecal-borne virus contamination within the food safety chain.

Campylobacter jejuni Outbreak Linked to Raw Oysters in Rhode Island, 2021

Oysters and other shellfish are not a food that is commonly highlighted as high risk for Campylobacter contamination. The Rhode Island Department of Health (RIDOH) conducted a multiagency investigation of a Campylobacter jejuni outbreak that was linked to the consumption of raw oysters; the first such outbreak was detected in Rhode Island. The environmental investigation identified birds as the likely source of contamination of the aquacultured oysters. As a result of this outbreak response, several investigative processes and best practice recommendations are offered. 1) RIDOH will be including exposure to raw shellfish as a question on their case report forms to better identify future oyster-related Campylobacter clusters. 2) It is important that shellfish aquaculture farms be aware of the risks of using floating gear to hold oyster cages and of the importance of using bird abatement to keep birds off floating aquaculture gear to prevent contamination of oysters from bird feces. 3) It should be recognized that fecal coliform water samples collected near an oyster aquaculture farm may not act as an adequate indicator for the presence of Campylobacter. 4) For the first time, Rhode Island has developed guidelines for the closure and reopening of oyster harvest areas due to contamination with Campylobacter. It is hoped that increased awareness and mitigation of the described risk factors will help prevent future similar outbreaks of illness.

1H NMR fecal metabolic phenotyping of periductal fibrosis- and cholangiocarcinoma-specific metabotypes defining perturbation in gut microbial-host co-metabolism.

The liver fluke Opisthorchis viverrini (OV), which subsequently inhabits the biliary system and results in periductal fibrosis (PDF), is one of the primarily causes of cholangiocarcinoma (CCA), a bile duct cancer with an exceptionally high incidence in the northeast of Thailand and other Greater Mekong Subregion (GMS) countries. Insights in fecal metabolic changes associated with PDF and CCA are required for further molecular research related to gut health and potential diagnostic biological marker development. In this study, nuclear magnetic resonance (NMR) metabolomics was applied for fecal metabolic phenotyping from 55 fecal water samples across different study groups including normal bile duct, PDF and CCA groups. By using NMR spectroscopy-based metabolomics, fecal metabolic profiles of patients with CCA or PDF and of individuals with normal bile duct have been established with a total of 40 identified metabolites. Further multivariate statistical analysis and hierarchical clustering heat map have demonstrated the PDF- and CCA-specific metabotypes through various altered metabolite groups including amino acids, alcohols, amines, anaerobic glycolytic metabolites, fatty acids, microbial metabolites, sugar, TCA cycle intermediates, tryptophan catabolism substrates, and pyrimidine metabolites. Compared to the normal bile duct group, PDF individuals showed the significantly elevated relative concentrations of fecal ethanol, glycine, tyrosine, and N-acetylglucosamine whereas CCA patients exhibited the remarkable fecal metabolic changes that can be evident through the increased relative concentrations of fecal uracil, succinate, and 5-aminopentanoate. The prominent fecal metabolic alterations between CCA and PDF were displayed by the reduction of relative concentration of methanol observed in CCA. The metabolic alterations associated with PDF and CCA progression have been proposed with the involvement of various metabolic pathways including TCA cycle, ethanol biogenesis, hexamine pathway, methanol biogenesis, pyrimidine metabolism, and lysine metabolism. Among them, ethanol, methanol, and lysine metabolism strongly reflect the association of gut-microbial host metabolic crosstalk in PDF and/or CCA patients. The PDF- and CCA-associated metabotypes have been investigated displaying their distinct fecal metabolic patterns compared to that of normal bile duct group. Our study also demonstrated that the perturbation in co-metabolism of host and gut bacteria has been involved from the early step since OV infection to CCA tumorigenesis.

Isolation and characterization of esbl producing Escherichia coli from healthy Goats and from their environment in west coastal India

The emergence of extended spectrum ß-lactamases (ESBL) producing Escherichia coli in food animals is a serious public health concern globally as it causes severe infections in humans. A study was conducted to describe the occurrence, antibiogram profile and plasmid-mediated genetic determinants and virulence factors of ESBL producing E. coli in healthy goats and in their environment. A total of 168 samples of faecal swabs, soil and water were collected from healthy goats and from the environment of Goa and coastal Karnataka. A total of 111 E. coli isolates were retrieved from 118 faecal swabs and 50 environment samples, with overall prevalence of 66.07% (111/168). From faecal samples, 89 isolates (89/118, 75.42%), soil, 15 (15/29, 51.7%) and water samples, 07 (7/21, 33.3%) isolates were recovered. Multidrug resistance was detected in 19 isolates. Multiplex-PCR detected blaCTX (67.56%), blaSHV (12.61%), blaTEM (14.41%), stx1 (6.30%), eaeA (5.40%) and hylA (0.90) in isolates. Stx2 was absent in all the isolates. The current study implies that goats are a potential reservoir of non-virulent multi-resistant ESBL producing E. coli which harbours resistant genes and may expose to humans via different transmission routes.

Decline in Cryptosporidium Infection in Free-Ranging Rhesus Monkeys in a Park After Public Health Interventions.

Nonhuman primates (NHPs) are considered an important source of parasitic zoonoses. A study in 2010 revealed high prevalence of Cryptosporidium spp. in free-ranging rhesus monkeys (Macaca mulatta) in a public park in Guiyang, southwestern China, which called for the control of disease in animals and long-term epidemiological tracking of Cryptosporidium spp. After the initiation of a series of public health interventions, we collected 2,402 fecal samples from monkeys and 123 water samples from lakes in the park on six occasions during 2013-2019. They were analyzed and genotyped for Cryptosporidium spp. using PCR and sequence analyses of the small subunit rRNA gene. The C. hominis and C. parvum identified were further subtyped by sequence analysis of the 60 kDa glycoprotein gene. Compared with the high prevalence of Cryptosporidium spp. in fecal samples (10.9% or 45/411) and water samples (47.8% or 11/23) in 2010, only 18 (0.7%) fecal samples and 3 (2.4%) water samples collected in the present study were positive for Cryptosporidium spp., including C. hominis (n = 9) and C. parvum (n = 12). The former belonged to the NHP-adapted IfA17G2R3 subtype, while the latter mostly belonged to rodent-adapted IIpA9. Therefore, the detection rate and genetic diversity of Cryptosporidium spp. during this study period were much lower than those before the public health interventions, and there was a switch from common occurrence of anthroponotic C. hominis subtypes to sporadic occurrence of NHP-adapted C. hominis and rodent-adapted C. parvum subtypes.

Prevalence, Characterization, and Pathogenicity of Salmonella enterica Subspecies enterica Serovar Derby from Yaks in the Aba Tibetan Autonomous Prefecture, China.

Simple SummarySalmonella spp. is a very important pathogen in the livestock industry and public health, which poses a major threat to global public health. Yaks and their by-products have a significant economic status in the Qinghai–Tibetan Plateau. The aim of this study was to investigate the prevalence of salmonella in yak farms and to conduct a molecular characterization and tests on its pathogenicity in mice with the use of salmonella isolated from yaks with diarrhea as well as from drinking water samples. The prevalence of salmonella was 19.75% of 162 samples collected from yak farms, and all isolates were found to belong to the serovar of Salmonella Derby and ST40. All Salmonella Derby isolates from both fecal and drinking water samples from 13 farms were clonally related based on SNP alignment. Salmonella Derby was still detected positively in the feces of model mice on day 24 post-injection. This study reports the prevalence of Salmonella Derby in yaks with diarrhea and in their drinking water. In addition, the pathogenicity of the S. Derby in mice was investigated. Findings suggest that Salmonella Derby excreted by diarrheic yaks is a source of contamination for other yaks and the environment and is highly pathogenic to mice. Seeing that Salmonella Derby has become one of the most common Salmonella serovars, this situation gives rise to further risk from the potential spread of food-borne diseases.Salmonella enterica subsp. enterica serovar Derby (S. Derby) is one of the numerous non-typhoidal Salmonella serovars and has been recognized as a food-borne pathogen. In 2019, outbreaks of salmonellosis were reported in 13 yak farms in the Aba Tibetan Autonomous Prefecture, China. A total of 32 salmonella strains were isolated from 162 fecal samples of yaks with diarrhea as well as from drinking water samples. The isolates were subjected to serovar identification, animal experiments, and whole-genome sequencing (WGS) analyses. The serovar of all the isolates was S. Derby, and the sequence types (STs) were ST40. The analysis of the differences of single-nucleotide polymorphisms (SNPs) showed that the salmonella strains isolated from 13 farms were clonally related. Animal experiments showed that the lethal dose (LD50) was 4.57 × 107 CFU (colony-forming units); the shedding time of S. Derby in mice was 24 days; the bacterial loads in spleen were higher than those in other organs (ileum, liver, and cecum). Pathological analyses by hematoxylin and eosin (H&E) staining revealed obvious damage in the spleen, liver, and intestine. These results indicate that the S. Derby from yaks can cause infection in mice.

Isolation of Protease Producing Strains and Evaluation of Protease Assay from Fecal Water and Curd Samples

Isolation of Protease Producing Strains and Evaluation of Protease Assay from Fecal Water and Curd Samples

The occurrence of multidrug-resistant Salmonella nagoya and other serovars in healthy commercial layers in Ilorin, Nigeria

The occurrence of drug-resistant non-typhoidal Salmonella (NTS) in poultry has serious economic implications for the poultry industry and has the potential to cause human Salmonellosis. This study, therefore, aimed to determine the circulating serovars of NTS and their antibiotic susceptibility patterns in poultry in Ilorin. This cross-sectional study was conducted from January to March 2015. A total of 420 samples (cloacal, n=140; fecal, n=140; feed, n=70, and water, n=70) were aseptically collected from live adult birds from 14 farms using a systematic random sampling technique. Salmonella was isolated using the ISO 6579 method. Antibiotic sensitivity testing of NTS serovars was performed using the Kirby Bauer disc – diffusion method and interpreted using the epidemiological cut-off (ECOFF) values. The prevalence of NTS in poultry was 7.4% (n=31). Feed samples were the most contaminated samples (42%, n=13/31). Faecal sample (32%, n=10/31), cloacal swabs (19.5%, n= 6/31), and water samples (6.5%, n=2/31) also contained NTS. There was a significant difference between NTS isolation rates between farms (p<0.05). Only 21 isolates purposively selected across farms and sample types were serotyped. Salmonella nagoya was the most prevalent (52%, n=11/21). Other serovars were Salmonella brijbhumi (5%, n=1/21); Salmonella enteritidis (5%, n=1/21); and Salmonella enterica subsp. enterica serovar 6, 8: z4 (19%, n=4/21). Four isolates (19%) were untypable. All isolates showed multidrug resistance. Most of the isolates were resistant to ampicillin (82.3%) and tetracycline (76.5%). Some isolates were resistant to cefotaxime (23.5%) and ciprofloxacin (29.4%). The occurrence of multidrug-resistant salmonella isolates is considered a critical public health threat that requires urgent global action. There is a need for a coordinated national salmonella surveillance program in Nigeria.

Investigation of Shiga Toxin-Producing Escherichia coli in Groundwater, River Water, and Fecal Sources in the Kathmandu Valley, Nepal

Shiga toxin-producing Escherichia coli (STEC) is one of the most commonly heard sources of foodborne outbreaks but presently lacks studies of in the Kathmandu Valley. This study explored the presence of STEC in river water (n = 17), groundwater (n = 83), feces (n = 50), and manure (n = 20) in the Kathmandu Valley, Nepal. Samples that were confirmed to be E. coli–positive using the Colilert assay in previous studies were selected to examine the presence of the sfmD, stx1, and stx2 genes using quantitative polymerase chain reaction. Based on Colilert/sfmD gene ratios, the percentage of viable cells of E. coli in the fecal (2.0% ± 2.5%), manure (0.1% ± 0.1%), river water (3.4% ± 3.1%), and groundwater samples (3.9% ± 5.8%) were determined. The presence of the stx1 gene was observed in the fecal (6%), river water (53%), and groundwater (2%) samples, whereas the stx2 gene was detected in the fecal (8%), manure (5%), and river water (71%) samples. Interestingly, the stx/sfmD gene ratios in the groundwater samples were greater than 100%, suggesting the presence of other stx1/stx2-harboring microorganisms. These findings indicated the importance of continuing investigation into waterborne sources of STEC contamination within the Kathmandu Valley.

Antibacterial Effectiveness of Fecal Water and In Vitro Activity of a Multi-Strain Probiotic Formulation against Multi-Drug Resistant Microorganisms

Introduction: Intestinal colonization with multi-drug resistant (MDR) microorganisms is a consequence of antimicrobial-induced gut dysbiosis. Given the effect of probiotics in modulating gut microbiota, the aim of the study was to investigate whether the ingestion of high concentration multi-strain probiotic formulation would change the antibacterial activity of the feces against clinical strains of MDR microorganisms. The corresponding in vitro antibacterial activity was also investigated. Materials/Methods: The feces of healthy donors (n = 6) were analyzed before and after a 7-day dietary supplementation with a multi-strain probiotic formulation and tested against MDR microorganisms of clinical concern (carbapenem-resistant (CR), Klebsiella pneumoniae (CR-Kp), CR-Acinetobacter baumannii (CR-Ab), CR-Pseudomonas aeruginosa (CR-Pa), and methicillin-resistant Staphylococcus aureus (MRSA)). The tested MDR pathogens were cultured with decreasing concentrations of fecal water obtained before and after the treatment period. Furthermore, to corroborate the results obtained from the feces of healthy donors, the in vitro antibacterial activity of probiotic formulation (both whole probiotic (WP) and probiotic surnatant (PS)) against the same collection of MDR microorganisms was evaluated at different incubation times throughout the minimum bactericidal dilution and the corresponding serial silution number. Results: While before probiotic administration, the fecal water samples did not inhibit MDR microorganism growth, after supplementation, a reduced bacterial growth was shown. Accordingly, a noticeable in vitro activity of WP and PS was observed. Conclusions: Although preliminary, these experiments demonstrated that a specific multi-strain probiotic formulation exhibits in vitro antibacterial activity against MDR microorganisms of clinical concern. If confirmed, these results may justify the administration of probiotics as a decolonization strategy against MDR microorganisms.

Antiviral Activity of Fecal Water Samples from HIV-1 Infected Subjects Treated with a Specific Probiotic Formulation.

The aim of the study was to investigate if the supplementation with multistrain probiotics may be able to modulate T cell response in HIV-1 infected patients and to evaluate the anti-HIV activity of probiotic by studying fecal water (FW) samples. Three HIV-1-positive patients (Pt1, Pt2 and Pt3) on long-term suppressive combined antiretroviral therapy (cART) received a specific multi-strain probiotic supplementation (Vivomixx ®), for six months (T6). Levels of T cell subsets were evaluated by flow cytometry. Anti- HIV activity of FW samples was evaluated in vitro. CD4+ T cells levels increased in all HIV-1 infected patients whereas activation markers (CD38 and HLA-DR) were decreased both on CD4+ and CD8+ T cells. FW samples presented an increased inhibitory activity against HIV-1 compared to T0 (FW-Pt1: T0 =40%, T6 = 65% of reduction; FW Pt2: T0 = 26%, T6 = 46% of reduction; FW Pt3: T0 = 47%, T6 = 94% of reduction). Our data suggest that the administration of the specific probiotic formulation improves the antiviral status of people living with HIV-1 under cART, also modulating T cell response. Anti-HIV activity of FW may have several public health and social implications for sexually transmitted diseases that need to be further explored.

Chronic exposure to sub-lethal concentrations of antibiotics in rural population and consequent health risks

TPS 751: Farms, antibiotics, infections, Exhibition Hall, Ground floor, August 26, 2019, 3:00 PM - 4:30 PM Background: Chronic exposure to sub-lethal concentrations of antibiotic is mostly ignored. The rural environment receives antibiotics and their residues from agriculture, livestock and human that may persist for long-time. Hence public exposure in rural world to antibiotics is multifaceted and need health risk assessment. Aims: This study aimed to (i) quantify ethical and unethical antibiotic usage, their concentrations and residues in rural environment (ii) assess chronic exposure of rural population to antibiotics and consequent health risks among rural population. Methods: Field soil, fecal and irrigation water samples were analyzed for fluoroquinolones and quinolone antibiotics. Target rural population (n=234) were surveyed which includes livestock handlers, agricultural farmers, field workers, female households and children, to assess extent of exposure to antibiotic persist in their immediate environment and epidemiological symptoms. Results: Significant use of ciprofloxacin as self-medication (unethical) was strongly correlated with flu and gastrointestinal infections. Among females gastro (OR=1.51; 95% CI=1.16–1.95) whereas in livestock workers both flu (OR=1.39; 95% CI=1.07–1.80) and gastro (OR=2.11; 95% CI=1.62–2.74) incidences were recorded as consequent impact of chronic exposure to antibiotics. In feces wide range of ciprofloxacin was detected (97.5–188.2 mg kg–1) whereas amount of levofloxacin and ofloxacin were within the WHO regulatory limits. Wastewater channels from rural dwellings and livestock farms were identified as potential hotspots for chronic exposure. Indiscriminate use of antibiotics to enhance dairy produce has forced rural people to indulge in unethical drug administration which has increased their susceptibility to further exposure to antibiotics and their residues in rural environment. Conclusions: We conclude that chronic exposure of rural population to antibiotics and their residues provoke health risks and this exposure is on rise due to continuous add-up of sub-lethal levels linked with self-administrated usage which must be strictly checked to minimize their release in environment.

Prevalence of antimicrobial resistance and potential pathogenicity, and possible spread of third generation cephalosporin resistance, in Escherichia coli isolated from healthy chicken farms in the region of Dakar, Senegal.

Escherichia coli is a normal inhabitant of the intestinal microbiota of chickens, a small proportion of which may be avian pathogenic E. coli (APEC) or potential extraintestinal pathogenic E. coli (ExPEC), capable of causing disease in humans. These E. coli may also be resistant to antimicrobials of critical importance in human or veterinary health. This study aims to 1) determine the prevalence of antimicrobial resistance (AMR) and resistance genes, multidrug resistance (MDR), chromosomal mechanisms of quinolone-resistance and virulence profiles of E. coli isolated from healthy chicken farms in the region of Dakar, Senegal, 2) investigate the spread of third-generation cephalosporins (3GC) resistance in E. coli isolated from healthy chicken farms with respect to virulence and resistance genes, serogroups, Pulsed-Field Gel Electrophoresis (PFGE), phylogenetic groups, plasmid types and transferability and 3) determine whether nonsusceptibility against 3GC on farms could be linked to risk factors. More than 68% of isolates from environmental faecal and drinking water samples, carcasses and carcass washes collected on 32 healthy chicken farms were multidrug resistant (MDR), resistance to antimicrobials critical in human health (3GC or ciprofloxacin) being found in all types of samples. Ciprofloxacin resistance was due to mutations in the gyrA and parC genes, 95% of tested farms harboring isolates carrying three mutations, in gyrA (Ser83Ile and Asp87Asn) and parC (Ser80Ile). Nine of the 32 farms (28.1%) demonstrated the presence of one or more 3GC-nonsusceptible indicator isolates but none of the potential risk factors were significantly associated with this presence on farms. Following ceftriaxone enrichment, presumptive extended-spectrum beta-lactamase/AmpC-beta-lactamase (ESBL/AmpC)-producer isolates were found in 17 of the 32 farms. 3GC resistance was mediated by blaCMY-2 or blaCTX-M genes, blaCTX-M being of genotypes blaCTX-M-1, blaCTX-M-8 and for the first time in chickens in Senegal, the genotype blaCTX-M-15. Clonally related ESBL/AmpC-producer isolates were found on different farms. In addition, blaCTX-M genes were identified on replicon plasmids I1 and K/B and blaCMY-2 on K/B, I1 and B/O. These plasmids were found in isolates of different clusters. In addition, 18 isolates, some of which were ESBL/AmpC-producers, were defined as potential human ExPEC. In conclusion, E. coli isolates potentially pathogenic for humans and demonstrating MDR, with resistance expressed against antimicrobials of critical importance in human health were found in healthy chickens in Senegal. Our results suggest that both clonal spreading and horizontal gene transfer play a role in the spread of 3GC-resistance and that chickens in Senegal could be a reservoir for AMR and ExPEC for humans. These results highlight the importance of raising awareness about compliance with biosecurity measures and prudent use of antimicrobials.

Broadly reactive real-time reverse transcription-polymerase chain reaction assay for the detection of human sapovirus genotypes.

Sapoviruses are associated with acute gastroenteritis. Human sapoviruses are classified into four distinct genogroups (GI, GII, GIV, and GV) based on their capsid gene sequences. A TaqMan probe-based real-time reverse transcription-polymerase chain reaction (RT-PCR) assay that detects the representative strains of these four genogroups is widely used for screening fecal specimens, shellfish, and environmental water samples. However, since the development of this test, more genetically diverse sapovirus strains have been reported, which are not detectable by the previously established assays. In this study, we report the development of a broader-range sapovirus real-time RT-PCR assay. The assay can detect 2.5 × 107 and 2.5 × 10 1 copies of sapovirusand therefore is as sensitive as the previous test. Analysis using clinical stool specimens or synthetic DNA revealed that the new system detected strains representative of all the 18 human sapovirus genotypes: GI.1-7, GII.1-8, GIV.1, and GV.1, 2. No cross-reactivity was observed against other representative common enteric viruses (norovirus, rotavirus, astrovirus, and adenovirus). This new assay will be useful as an improved, broadly reactive, and specific screening tool for human sapoviruses.

Exosomes and Metabolic Function in Mice Exposed to Alternating Dark-Light Cycles Mimicking Night Shift Work Schedules.

Sleep is an important modulator of metabolic function. Disruptions of sleep in circadian rhythm are common in modern societies and are associated with increased risk of developing cardiometabolic disorders. Exosomes are ubiquitous extracellular vesicles that may play a mechanistic role in metabolic derangements. We hypothesized that alternating dark-light cycles mimicking shift work in mice would alter fecal microbiota and colonic epithelium permeability and alter plasma exosome cargo and metabolic function. C57BL/6 mice were randomly assigned to (i) control day light (CL), or (ii) inverted dark-light every 2 weeks for 8 weeks (IN). Body weight, fat mass and HOMA-IR were measured, along with Tregs, metabolic, and resident macrophages in visceral white adipose tissue (vWAT). Fecal water samples were incubated with confluent colonic epithelium cell cultures in electric cell-substrate impedance sensing (ECIS) arrays, and plasma exosomes were added to differentiated adipocytes and insulin-induced pAKT/AKT expression changes were assessed by western blots. Mice exposed to IN showed elevated HOMA-IR, and their fecal samples showed altered microbiota which promote increased permeability of the colonic epithelial cell barrier. Plasma exosomes decreased pAKT/AKT responses to exogenous insulin compared to CL, and altered expression of circadian clock genes. Inflammatory macrophages (Ly-6chigh) were increased in IN-exposed vWAT, while Tregs were decreased. Thus, gut microbiota and the cargo of plasma exosomes are altered by periodic shifts in environmental lighting, and effectively alter metabolic function, possibly via induction of systemic inflammation and altered clock expression in target tissues. Further exploration of exosomal miRNA signatures in shift workers and their putative metabolic organ cell targets appears warranted.

Identifying Key Transmission Route of Avian Influenza A(H9N2) in Live Poultry Markets

ObjectiveThis study assessed the transmission of low pathogenic avianinfluenza in live poultry market setting, using paired fecal anddrinking water samples from a longitudinal surveillance program.The relative contribution of transmission via direct fecal-oral routeversus drinking water will be determined.IntroductionLive poultry markets (LPMs) continue to operate in many Asiancountries. Low pathogenic avian influenza (LPAI) viruses areoften endemic in the poultry, and LPM presents the opportunity forhuman-poultry interactions and potential human infections with avianinfluenza viruses.As a series of interventions to control avian influenza transmissionin Hong Kong LPMs, local health authority implemented marketrest days once every month since mid-2001, and an additional restday every month since 2003, during which all unsold poultry wereslaughtered and the stalls cleaned and disinfected. Rest days werefound to effectively reduce avian influenza A(H9N2) isolation rateto baseline level for a few days following the rest days. However,H9N2 isolation rate was still observed to be increasing between therest days, indicating the existence of efficient transmission in spite ofrapid turnover of poultry.In LPMs, poultry are usually stored in cages where drinkingwater is shared among poultry. This is analogous to environmentalcontamination in the wild, but transmissibility may even be higherdue to the dense environment. The use of drinking water for avianinfluenza surveillance in LPM setting was suggested to be moresensitive than fecal samples (1). However, the relative contributionof direct fecal-oral versus water transmission routes in the LPMsetting was not yet understood. This study aimed to determine theirrole, which will have implications in the control of avian influenzatransmission.MethodsWe analyzed 7,321 paired fecal and drinking water samplesfrom a longitudinal surveillance programme during the period with2 monthly rest days in the LPMs. Samples were collected fromchicken cages and subsequently cultured. Positive isolates weresubtyped by hemagglutination-inhibition tests and neuraminidaseinhibition test. Data were aggregated by sampling occasion and daysafter the rest days.A compartmental transmission model which incorporated turnoverand overnight stay of poultry, virus contamination and decay indrinking water was fitted to the data (Figure 1). A 12-hour tradingday was assumed. Based on the parameterized model, we simulatedthe scenario that water transmission was prohibited to assess the roleof transmission via drinking water.ResultsH9N2 isolation rates ranged from 0-25% for fecal samples and0-56% for drinking water samples. A clear increasing trend can beseen over days after the rest days (Figure 2). The estimated parameterfor water transmission is higher than the parameter for direct fecal-oraltransmission. Simulation results show that transmission via drinkingwater plays a major role in the amplification of LPAI in the LPMsetting (Figure 2).ConclusionsOur study showed that drinking water has a major role in thetransmission and amplification of LPAI H9N2 in LPMs, comparingto direct fecal-oral transmission route. Given the relatively lowprevalence of H9N2 in chicken, direct transmission is governed bychance events, while chickens are consistently exposed to viruses indrinking water if contaminated. Drinking water could be targeted forintervention to control LPAI transmission in LPM. The use of drinkingfountain or frequent disinfection of drinking water may be considered.Avian influenza viruses (e.g. H5N1) may differ in their pattern ofvirus shedding via oral versus fecal routes and thus extrapolation ofthese results to other viruses needs to be done with caution. However,H7N9 viruses are similar to H9N2 viruses by being shed primarilyvia the respiratory / oral route (2) and it is reasonable to assume thatthese conclusions would apply to H7N9 virus which is of major publichealth concern. However, our model could not differentiate the effectof indirect fecal-oral transmission through contamination of drinkingwater by droppings versus contamination through drinking.

NMR-Based Metabonomic Analysis of Physiological Responses to Starvation and Refeeding in the Rat.

Starvation is a postabsorptive condition derived from a limitation on food resources by external factors. Energy homeostasis is maintained under this condition by using sources other than glucose via adaptive mechanisms. After refeeding, when food is available, other adaptive processes are linked to energy balance. However, less has been reported about the physiological mechanisms present as a result of these conditions, considering the rat as a supraorganism. Metabolic profiling using (1)H nuclear magnetic resonance spectroscopy was used to characterize the physiological metabolic differences in urine specimens collected under starved, refed, and recovered conditions. In addition, because starvation induced lack of faecal production and not all animals produced faeces during refeeding, 24 h pooled faecal water samples were also analyzed. Urinary metabolites upregulated by starvation included 2-butanamidoacetate, 3-hydroxyisovalerate, ketoleucine, methylmalonate, p-cresyl glucuronide, p-cresyl sulfate, phenylacetylglycine, pseudouridine, creatinine, taurine, and N-acetyl glycoprotein, which were related to renal and skeletal muscle function, β-oxidation, turnover of proteins and RNA, and host-microbial interactions. Food-derived metabolites, including gut microbial cometabolites, and tricarboxylic acid cycle intermediates were upregulated under refed and recovered conditions, which characterized anabolic urinary metabotypes. The upregulation of creatine and pantothenate indicated an absorptive state after refeeding. Fecal short chain fatty acids, 3-(3-hydroxyphenyl)propionate, lactate, and acetoin provided additional information about the combinatorial metabolism between the host and gut microbiota. This investigation contributes to allow a deeper understanding of physiological responses associated with starvation and refeeding.

Estimating the contribution of nitrogen and phosphorus to waterbodies by colonial nesting waterbirds

Fecal deposition by colonial nesting waterbirds is a potential source of nutrient enrichment and pollution of nearby waterways. Excess concentrations of nitrogen (N) and phosphorus (P) can cause water quality concerns or impairment. We estimated concentrations of N and P deposited to waterbodies by birds nesting in four colonies in east-central Texas during 2011–2013, and developed an age-structured compartment model to estimate the amount of fecal N and P material deposited by birds during the entire breeding season. There was a seasonal variation in the accumulation of N and P in waterbodies where heronries were located with a significant increase from June to July at the peak of the breeding season. Also, there was a significant positive correlation in concentrations of N (P=0.023) in fecal material and water samples from one of the colonies (Murphy Park), suggesting a significant contribution of N from feces to water. Concentrations of N in water near the colonies were also significantly correlated (P=0.004, R2=0.22) with the average amount of precipitation recorded for each region. The simulation model results indicated that total N and P deposition increased proportionally with heronry size, and daily deposition rates varied due to the shifting daily activity budgets of the birds as the breeding season progressed. The total estimated loads from the model reached a maximum of 2170kg N and 240kg P for a given colony, with daily deposition estimated at 22.8kg N, and 2.5kg P. Based on the model, one bird can release about 1mg of N and 0.115mg of P on a daily basis to the soil substrate or directly over water. Results from this study could be useful to estimate the contribution of N and P from wildlife to waterbodies, and for watershed management plans.

Optimized Sample Handling Strategy for Metabolic Profiling of Human Feces.

Fecal metabolites are being increasingly studied to unravel the host-gut microbial metabolic interactions. However, there are currently no guidelines for fecal sample collection and storage based on a systematic evaluation of the effect of time, storage temperature, storage duration, and sampling strategy. Here we derive an optimized protocol for fecal sample handling with the aim of maximizing metabolic stability and minimizing sample degradation. Samples obtained from five healthy individuals were analyzed to assess topographical homogeneity of feces and to evaluate storage duration-, temperature-, and freeze-thaw cycle-induced metabolic changes in crude stool and fecal water using a (1)H NMR spectroscopy-based metabolic profiling approach. Interindividual variation was much greater than that attributable to storage conditions. Individual stool samples were found to be heterogeneous and spot sampling resulted in a high degree of metabolic variation. Crude fecal samples were remarkably unstable over time and exhibited distinct metabolic profiles at different storage temperatures. Microbial fermentation was the dominant driver in time-related changes observed in fecal samples stored at room temperature and this fermentative process was reduced when stored at 4 °C. Crude fecal samples frozen at -20 °C manifested elevated amino acids and nicotinate and depleted short chain fatty acids compared to crude fecal control samples. The relative concentrations of branched-chain and aromatic amino acids significantly increased in the freeze-thawed crude fecal samples, suggesting a release of microbial intracellular contents. The metabolic profiles of fecal water samples were more stable compared to crude samples. Our recommendation is that intact fecal samples should be collected, kept at 4 °C or on ice during transportation, and extracted ideally within 1 h of collection, or a maximum of 24 h. Fecal water samples should be extracted from a representative amount (∼15 g) of homogenized stool sample, aliquoted, and stored at <-20 °C, avoiding further freeze-thaw cycles.

Impact of β-glucan on the Fecal Water Genotoxicity of Polypectomized Patients

ABSTRACTThe aim of the study was to determine the effect of β-glucan on the cytotoxicity and genotoxicity of polypectomized patient's fecal water (FW). Polypectomized volunteers (n = 69) were randomly assigned to consume bread with or without β-glucan, for 3 months. FW was collected at the beginning (t = 0), the 30th and 90th day and 2 wk after the intervention. Cytotoxicity and genotoxicity were estimated on Caco-2 cells, using trypan blue exclusion test and comet assay, respectively. Gastrointestinal symptoms were recorded and subjects kept a 3-day food diary at baseline and after completion. Trypan blue exclusion test revealed cell survival of approximately 87% after incubation with FW. The FW samples showed 49% genotoxicity at the baseline. Genotoxicity in the intervention group decreased during the trial reaching statistical significance on the 90th day compared to control. An increase was noticed 2 wk after the trial, but it still remained significantly lower compared to control. Group-specific analysis for β-glucan also revealed significant decrease in the genotoxicity on the 90th day compared to baseline. β-glucan ingestion in polypectomized patients significantly decreased the genotoxicity of their FW. Our findings suggest that β-glucan consumption could possibly provide protection against colon cancer development.