Evidence for Extraintestinal Growth of Bacteroidales Originating from Poultry Litter

Water quality monitoring techniques that target microorganisms in the order Bacteroidales are potential alternatives to conventional methods for detection of fecal indicator bacteria. Bacteroidales and members of the genus Bacteroides have been the focus of microbial source tracking (MST) investigations for discriminating sources of fecal pollution (e.g., human or cattle feces) in environmental waters. For accurate source apportionment to occur, one needs to understand both the abundance of Bacteroides in host feces and the survival of these host-associated microbial markers after deposition in the environment. Studies were undertaken to evaluate the abundance, persistence, and potential for growth of Bacteroidales originating from poultry litter under oxic and anoxic environmental conditions. Bacteroidales abundance, as determined by quantitative PCR (qPCR) with GenBac primers and probe, increased 2 to 5 log gene copies ml⁻¹ and 2 log gene copies g litter⁻¹ under most conditions during incubation of poultry litter in a variety of laboratory microcosm and field mesocosm studies. DNA sequencing of the Bacteroidales organisms in the litter identified taxa with sequences corresponding exactly to the GenBac primer and probe sequences and that were closely related to Bacteroides uniformis, B. ovatus, and B. vulgatus. These results suggest that MST studies using qPCR methods targeting Bacteroidales in watersheds that are affected by poultry litter should be interpreted cautiously. Growth of Bacteroidales originating from poultry litter in environmental waters may occur while Bacteroidales growth from other fecal sources declines, thus confounding the interpretation of MST results.     

Long-Term Survival of Pathogenic and Sanitation Indicator Bacteria in Experimental Biowaste Composts

For economic, agricultural, and environmental reasons, composting is frequently used for organic waste recycling. One approach to limiting the potential risk from bacterial food-borne illnesses is to ensure that soil amendments and organic fertilizers are disinfected. However, more knowledge concerning the microbiological safety of composted substrates other than sludge and manure is necessary. Experimental in-vessel biowaste composts were used to study the survival of seeded Listeria monocytogenes, Salmonella enterica subsp. enterica serotype Enteritidis, and Escherichia coli. Four organic waste mixtures, containing various proportions of paper and cardboard, fruits and vegetables, and green waste, were composted in laboratory reactors with forced aeration. The physicochemical and microbiological parameters were monitored for 12 weeks during composting. The survival of bacteria over a 3-month period at 25°C was assessed with samples collected after different experimental composting times. Strain survival was also monitored in mature sterilized composts. Nonsterile composts did not support pathogen growth, but survival of seeded pathogens was observed. Salmonella serovar Enteritidis survived in all composts, and longer survival (3 months) was observed in mature composts (8 and 12 weeks of composting). Mature biowaste composts may support long-term survival of Salmonella serovar Enteritidis during storage at room temperature. E. coli and L. monocytogenes survival was observed only in 4-week-old composts and never in older composts. Proper composting may prevent long-term survival of E. coli and L. monocytogenes. These results suggest that like composted sewage sludge or manure, domestic waste composts may support pathogen survival. Survival was not related to the physicochemical characteristics of the composts.     

Second Survey of Market Poultry for Salmonella Infection

On 34 days over a period of 30 months, samplings were made of randomly selected birds slaughtered in randomly chosen processing plants. Methods were designed so that Salmonella and paracolon bacteria isolated were indigenous to the intestinal tract of the selected bird. Incidence rates were 5.25% for turkeys, 0.54% for chicken hens, and 1.05% for chicken fryers. Infected birds, providing the potential for contamination, were processed on 58% (turkeys), 25% (chicken hens), and 43% (chicken fryers) of the days. Combined data from the present and previous surveys represent a total of 9,851 carcasses sampled on 94 separate days over a 4.5-year period; these data provide a more accurate analysis of incidence rates and days that positive birds were being slaughtered. For turkeys, chicken hens, and chicken fryers, incidence rates were 4.16, 0.65, and 1.42%, respectively, and days positive were 56, 14, and 32%.     

Direct Measurement Versus Surrogate Indicator Species for Evaluating Environmental Change and Biodiversity Loss

The enormity and complexity of problems like environmental degradation and biodiversity loss have led to the development of indicator species and other surrogate approaches to track changes in environments and/or in biodiversity. Under these approaches particular species or groups of species are used as proxies for other biota, particular environmental conditions, or for environmental change. The indicator species approach contrasts with a direct measurement approach in which the focus is on a single entity or a highly targeted subset of entities in a given ecosystem but no surrogacy relationships with unmeasured entities are assumed. Here, we present a broad philosophical discussion of the indicator species and direct measurement approaches because their relative advantages and disadvantages are not well understood by many researchers, resource managers and policy makers. A goal of the direct measurement approach is to demonstrate a causal relationship between key attributes of the target ecosystem system (for example, particular environmental conditions) and the entities selected for measurement. The key steps in the approach are based on the fundamental scientific principles of hypothesis testing and associated direct measurement that drive research activities, management activities and monitoring programs. The direct measurement approach is based on four critical assumptions:(1) the ‘right’ entities to measure have been selected, (2) these entities are well known, (3) there is sufficient understanding about key ecological processes and (4) the entities selected can be accurately measured. The direct measurement approach is reductionist and many elements of the biota, many biotic processes and environmental factors must be ignored because of practical considerations. The steps in applying the indicator species approach are broadly similar to the direct measurement approach, except surrogacy relationships also must be quantified between a supposed indicator species or indicator group and the factors for which it is purported to be a proxy. Such quantification needs to occur via: (1) determining the taxonomic, spatial and temporal bounds for which a surrogacy relationship does and does not hold. That is, the extent of transferability of a given surrogate such as an indicator species to other biotic groups, to landscapes, ecosystems, environmental circumstances or over time in the same location can be determined; and (2) determining the ecological mechanisms underpinning a surrogacy relationship (for example, through fundamental studies of community structure). Very few studies have rigorously addressed these two tasks, despite the extremely widespread use of the indicator species approach and similar kinds of surrogate schemes in virtually all fields of environmental, resource and conservation management. We argue that this has the potential to create significant problems; thus, the use of an indicator species approach needs to be better justified. Attempts to quantify surrogacy relationships may reveal that, in some circumstances, the alternative of direct measurement of particular entities of environmental or conservation interest will be the best option.     

Molecular Epidemiology of Nontyphoidal Salmonella in Poultry and Poultry Products in India: Implications for Human Health

Human infections with non-typhoidal Salmonella (NTS) serovars are increasingly becoming a threat to human health globally. While all motile Salmonellae have zoonotic potential, Salmonella Enteritidis and Salmonella Typhimurium are most commonly associated with human disease, for which poultry are a major source. Despite the increasing number of human NTS infections, the epidemiology of NTS in poultry in India has not been fully understood. Hence, as a first step, we carried out epidemiological analysis to establish the incidence of NTS in poultry to evaluate the risk to human health. A total of 1215 samples (including poultry meat, tissues, egg and environmental samples) were collected from 154 commercial layer farms from southern India and screened for NTS. Following identification by cultural and biochemical methods, Salmonella isolates were further characterized by multiplex PCR, allele-specific PCR, enterobacterial repetitive intergenic consensus (ERIC) PCR and pulse field gel electrophoresis (PFGE). In the present study, 21/1215 (1.73 %) samples tested positive for NTS. We found 12/392 (3.06 %) of tissue samples, 7/460 (1.52 %) of poultry products, and 2/363 (0.55 %) of environmental samples tested positive for NTS. All the Salmonella isolates were resistant to oxytetracycline, which is routinely used as poultry feed additive. The multiplex PCR results allowed 16/21 isolates to be classified as S. Typhimurium, and five isolates as S. Enteritidis. Of the five S. Enteritidis isolates, four were identified as group D Salmonella by allele-specific PCR. All of the isolates produced different banding patterns in ERIC PCR. Of the thirteen macro restriction profiles (MRPs) obtained by PFGE, MRP 6 was predominant which included 6 (21 %) isolates. In conclusion, the findings of the study revealed higher incidence of contamination of NTS Salmonella in poultry tissue and animal protein sources used for poultry. The results of the study warrants further investigation on different type of animal feed sources, food market chains, processing plants, live bird markets etc., to evaluate the risk factors, transmission and effective control measures of human Salmonella infection from poultry products.     

Species Diversity and Substrate Utilization Patterns of Thermophilic Bacterial Communities in Hot Aerobic Poultry and Cattle Manure Composts

This study investigated the species diversity and substrate utilization patterns of culturable thermophilic bacterial communities in hot aerobic poultry and cattle manure composts by coupling 16S rDNA analysis with Biolog data. Based on the phylogenetic relationships of 16S rDNA sequences, 34 thermophilic (grown at 60 degrees C) bacteria isolated during aerobic composting of poultry manure and cattle manure were classified as Bacillus licheniformis, B. atrophaeus, Geobacillus stearothermophilus, G. thermodenitrificans, Brevibacillus thermoruber, Ureibacillus terrenus, U. thermosphaericus, and Paenibacillus cookii. In this study, B. atrophaeus, Br. thermoruber, and P. cookii were recorded for the first time in hot compost. Physiological profiles of these bacteria, obtained from the Biolog Gram-positive (GP) microplate system, were subjected to principal component analysis (PCA). All isolates were categorized into eight different PCA groups based on their substrate utilization patterns. The bacterial community from poultry manure compost comprised more divergent species (21 isolates, seven species) and utilized more diverse substrates (eight PCA groups) than that from cattle manure compost (13 isolates, five species, and four PCA groups). Many thermophilic bacteria isolated in this study could use a variety of carboxylic acids. Isolate B110 (from poultry manure compost), which is 97.6% similar to U. terrenus in its 16S rDNA sequence, possesses particularly high activity in utilizing a broad spectrum of substrates. This isolate may have potential applications in industry.     

Purification of Concentrated Hemoglobin Using Organic Solvent and Heat Treatment

A simple method for obtaining a purified and concentrated hemoglobin (Hb) solution (25 g/100 ml) from human red blood cells has been established. To prevent MetHb formation during the purification procedure, Hb in red blood cells was carbonylated in advance, and then washed red blood cells were mixed with organic solvents such as diethyl ether or dichloromethane for hemolysis and removal of stroma. The Hb solution was isolated by centrifugation (1 900g) with the high removal efficiency of phospholipid (>99.8%). After the solution was heated (60°C, 1 h), the precipitates were removed by centrifugation. The purity of Hb was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing and oxygen-binding properties of the obtained Hb solution demonstrated its purity and showed no denaturation of globin. This purification procedure is applicable to large-scale production of the purified Hb.     

Breeding for Biomass Yield in Switchgrass Using Surrogate Measures of Yield

Development of switchgrass (Panicum virgatum L.) as a dedicated biomass crop for conversion to energy requires substantial increases in biomass yield. Most efforts to breed for increased biomass yield are based on some form of indirect selection. The objective of this paper is to evaluate and compare the expected efficiency of several indirect measures of breeding value for improving sward-plot biomass yield of switchgrass. Sward-plot biomass yield, row-plot biomass, and spaced-plant biomass were measured on 144 half-sib families or their maternal parents from the WS4U-C2 breeding population of upland switchgrass. Heading date was also scored on row plots and anthesis date was scored on spaced plants. Use of any of these indirect selection criteria was expected to be less efficient than direct selection for biomass yield measured on sward plots, when expressed as genetic gain per year. Combining any of these indirect selection criteria with half-sib family selection for biomass yield resulted in increases in efficiency of 14 to 36%, but this could only be achieved at a very large cost of measuring phenotype on literally thousands of plants that would eventually have no chance of being selected because they were derived from inferior families. Genomic prediction methods offered the best solution to increase breeding efficiency by reducing average cycle time, increasing selection intensity, and placing selection pressure on all additive genetic variance within the population. Use of genomic selection methods is expected to double or triple genetic gains over field-based half-sib family selection.     

Compositional and Thermal Evaluation of Lignocellulosic and Poultry Litter Chars via High and Low Temperature Pyrolysis

Inorganic elements in biomass feedstocks can influence thermochemical reactions as well as the resultant char’s elemental, compositional, and thermal characteristics. Chars were produced using slow pyrolysis under low (≤400°C) and high (≥500°C) temperature regimes from sugarcane bagasse, peanut hulls, pecan shell, pine chips, poultry litter, and switchgrass. The chars and raw feedstocks were characterized for their elemental, structural, and thermal properties to ascertain the implications of feedstock selection and pyrolysis temperatures on these properties. Char mass yields from the six feedstocks ranged between 28% and 78% by weight while carbon yields ranged between 44% and 89%. In both instances, lower yields were obtained with increasing pyrolysis temperature. Higher pyrolysis temperatures (≥500°C) resulted in more neutral to alkaline chars possessing greater ash contents and increased aromatic character with narrow O/C and H/C ratios. A significant exponential curve response (r ²?=?0.87, P?<?0.001) was revealed between char mass yields vs. pyrolysis temperature. All raw feedstocks and chars contained mixed amounts of macro-, micro-, and trace element concentrations. The higher heating values (HHV) tended to increase with heightened pyrolysis temperature with some chars producing >30 MJ kg^?1. The chars’ HHV values inversely correlated to their total ash and Cl content. Lignocelluloses chars had better thermal characteristics and lower ash quality concerns implying suitable service in thermal energy production. In contrast, poultry litter char had greater ash contents, medium HHV values, and contained corrosive inorganic elements, which rendered it problematic as a feedstock for thermal energy generation.     

Safety of the Surrogate Microorganism Enterococcus faecium NRRL B-2354 for Use in Thermal Process Validation

Enterococcus faecium NRRL B-2354 is a surrogate microorganism used in place of pathogens for validation of thermal processing technologies and systems. We evaluated the safety of strain NRRL B-2354 based on its genomic and functional characteristics. The genome of E. faecium NRRL B-2354 was sequenced and found to comprise a 2,635,572-bp chromosome and a 214,319-bp megaplasmid. A total of 2,639 coding sequences were identified, including 45 genes unique to this strain. Hierarchical clustering of the NRRL B-2354 genome with 126 other E. faecium genomes as well as pbp5 locus comparisons and multilocus sequence typing (MLST) showed that the genotype of this strain is most similar to commensal, or community-associated, strains of this species. E. faecium NRRL B-2354 lacks antibiotic resistance genes, and both NRRL B-2354 and its clonal relative ATCC 8459 are sensitive to clinically relevant antibiotics. This organism also lacks, or contains nonfunctional copies of, enterococcal virulence genes including acm, cyl, the ebp operon, esp, gelE, hyl, IS16, and associated phenotypes. It does contain scm, sagA, efaA, and pilA, although either these genes were not expressed or their roles in enterococcal virulence are not well understood. Compared with the clinical strains TX0082 and 1,231,502, E. faecium NRRL B-2354 was more resistant to acidic conditions (pH 2.4) and high temperatures (60°C) and was able to grow in 8% ethanol. These findings support the continued use of E. faecium NRRL B-2354 in thermal process validation of food products.     

Litter and aerosol sampling of chicken houses for rapid detection of Salmonella typhimurium contamination using gene amplification

Rapid screening of poultry houses for contamination is critical for Salmonella control. Use of air filter sampling has great potential for efficient and reliable monitoring of Salmonella spp., as it could represent an entire poultry house and solve sample-size problems. Two sampling methods (litter and air filter) were compared for detection in four chicken pens inoculated with a S. typhimurium antibiotic resistant strain. Salmonella levels in both litter and air filter samples were determined by PCR amplification and by conventional enrichment. Although amplified DNA was not directly detected, amplified DNA could be detected using a dual probe hybridization sensor. The ratio of the positive samples to total samples determined by gene amplification was much lower than that obtained by conventional enrichments (29/128 versus 102/128 samples). However, the ratio obtained by gene amplification with air filter samples was greater than that with litter samples (26/64 versus 3/64). These results demonstrate that the air filter sampling method is an alternative method of Salmonella detection in poultry house using PCR gene amplification protocol. Journal of Industrial Microbiology & Biotechnology (2000) 24, 379–382. Received 12 August 1999/ Accepted in revised form 17 February 2000     

Rapid Detection of Campylobacter coli, C. jejuni, and Salmonella enterica on Poultry Carcasses by Using PCR-Enzyme-Linked Immunosorbent Assay

Contamination of retail poultry by Campylobacter spp. and Salmonella enterica is a significant source of human diarrheal disease. Isolation and identification of these microorganisms require a series of biochemical and serological tests. In this study, Campylobacter ceuE and Salmonella invA genes were used to design probes in PCR-enzyme-linked immunosorbent assay (ELISA), as an alternative to conventional bacteriological methodology, for the rapid detection of Campylobacter jejuni, Campylobacter coli, and S. enterica from poultry samples. With PCR-ELISA (40 cycles), the detection limits for Salmonella and Campylobacter were 2 × 10² and 4 × 10¹ CFU/ml, respectively. ELISA increased the sensitivity of the conventional PCR method by 100- to 1,000-fold. DNA was extracted from carcass rinses and tetrathionate enrichments and used in PCR-ELISA for the detection of Campylobacter and S. enterica, respectively. With PCR-ELISA, Salmonella was detected in 20 of 120 (17%) chicken carcass rinses examined, without the inclusion of an enrichment step. Significant correlation was observed between PCR-ELISA and cultural methods (kappa = 0.83; chi-square test, P < 0.001) with only one false negative (1.67%) and four false positives (6.67%) when PCR-ELISA was used to screen 60 tetrathionate enrichment cultures for Salmonella. With PCR-ELISA, we observed a positive correlation between the ELISA absorbance (optical density at 405 nm) and the campylobacter cell number in carcass rinse, as determined by standard culture methods. Overall, PCR-ELISA is a rapid and cost-effective approach for the detection and enumeration of Salmonella and Campylobacter bacteria on poultry.     

Microorganisms in Heat Supply Systems and Internal Corrosion of Steel Pipelines

In laboratory experiments with batch cultures of thermophilic microorganisms isolated from urban heat supply systems, the growth of sulfate-reducing, iron-oxidizing, and iron-reducing bacteria was found to accelerate the corrosion rate of the steel-3 plates used in pipelines. In the absence of bacteria and dissolved oxygen, minimal corrosion was determined. The aforementioned microorganisms, as well as sulfur-oxidizing bacteria, were found to be widespread in water and corrosion deposits in low-alloy steel pipelines (both delivery and return) of the Moscow heat networks, as well as in the corrosion deposits on the steel-3 plates in a testing unit supplied with the network water. The microorganisms were found in samples with a water pH ranging from 8.1 to 9.6 and a temperature lower than 90°C. Magnetite, lepidocrocite, goethite, and X-ray amorphous ferric oxide were the corrosion products identified on the steel-3 plates, as well as siderite, aragonite, and S⁰. The accumulation of corrosion deposits and variation in the total and local corrosion of the steel plates in a testing unit were considered in terms of the influence of microbial processes.     

干热灭菌温度指示剂的筛选和效果验证

利用结晶物质在一定温度下熔融的特性,通过对四种化学试剂进行熔点测定。选出试剂Ａ和试剂Ｂ作为干热灭菌温度指示剂和参考指示剂。试验结果表明,这两种温度指示剂终点明确,简便快速,能满足１８０℃干热灭菌的温度要求,可以对干热灭菌的温度验证提供客观证据     

Salmonella in Broiler Litter and Properties of Soil at Farm Location

Contamination of litter in a broiler grow-out house with Salmonella prior to placement of a new flock has been shown to be a precursor of the flock''s Salmonella contamination further down the production continuum. In the southern USA, broiler grow-out houses are primarily built on dirt pad foundations that are placed directly on top of the native soil surface. Broiler litter is placed directly on the dirt pad. Multiple grow-out flocks are reared on a single litter batch, and the litter is kept in the houses during downtime between flocks. The effects of environmental determinants on conditions in broiler litter, hence Salmonella ecology within it, has received limited attention. In a field study that included broiler farms in the states of Alabama, Mississippi and Texas we assessed Salmonella in broiler litter at the end of downtime between flocks, i.e. at the time of placement of a new flock for rearing. Here we utilized these results and the U.S. General Soil Map (STATSGO) data to test if properties of soil at farm location impacted the probability of Salmonella detection in the litter. The significance of soil properties as risk factors was tested in multilevel regression models after accounting for possible confounding differences among the farms, the participating broiler complexes and companies, and the farms'' geographical positioning. Significant associations were observed between infiltration and drainage capabilities of soil at farm location and probability of Salmonella detection in the litter.     

Leaching of Zinc,Cadmium, and Lead in a Sandy Soil Due to Application of Poultry Litter

Dissolved organic matter in poultry litter could contribute organic ligands to form complexes with heavy metals in soil. The soluble complexes with heavy metals can be transported downward and possibly deteriorate groundwater quality. To better understand metal mobilization by soluble organic ligands in poultry litter, soil columns were employed to investigate the movement of zinc (Zn), cadmium (Cd), and lead (Pb). Uncontaminated soil was amended with Zn, Cd, and Pb at rates of 400, 8, and 200 mg kg ^{? 1} soil, respectively. Glass tubes, 4.9-cm-diameter and 40-cm-long, were packed with either natural or metal-amended soil. The resulting 20-cm-long column of soils had bulk density of about 1.58 g cm ^{? 3} . Columns repacked with natural or amended soil were leached with distilled water, 0.01 M EDTA, 0.01 M CaCl ₂ , or poultry litter extract (PLE) solutions. Low amounts of Zn, Cd, and Pb were leached from natural soil with the solutions. Leaching of Zn, Cd, or Pb was negligible with distilled water. In the metal-amended soil, EDTA solubilized more Zn, Cd, and Pb than CaCl ₂ and PLE. The breakthrough curves of Zn and Pb in the PLE and CaCl ₂ were similar, indicating they have similar ability to displace Zn and Pb from soils. Compared with Zn and Cd the PLE had a small ability to solubilize Pb from metal-amended soil. Thus, the application of poultry litter on metal-contaminated soils might enhance the mobility of Zn and Cd.