Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth.

This study tested the hypothesis that a host mucogenic response to an intestinal coccidial infection promotes the onset of necrotic enteritis (NE). A chick NE model was used in which birds were inoculated with Eimeria acervulina and E. maxima and subsequently with Clostridium perfringens (EAM/CP). A second group of EAM/CP-infected birds was treated with the ionophore narasin (NAR/EAM/CP). These groups were compared to birds that were either non-infected (NIF), or infected only with E. acervulina and E. maxima (EAM), or C. perfringens (CP). The impact of intestinal coccidial infection and anti-coccidial treatment on host immune responses and microbial community structure were evaluated with histochemical-, cultivation- and molecular-based techniques. Barrier function was compromised in EAM/CP-infected birds as indicated by elevated CFUs for anaerobic bacteria and C. perfringens in the spleen when compared to NIF controls at day 20, with a subsequent increase in intestinal NE lesions and mortality at day 22. These results correlate positively with a host inflammatory response as evidenced by increased ileal interleukin (IL)-4, IL-10 and IFN-gamma RNA expression. Concurrent increases in chicken intestinal mucin RNA expression, and goblet cell number and theca size indicate that EAM/CP induced an intestinal mucogenic response. Correspondingly, the growth of mucolytic bacteria and C. perfringens as well as alpha toxin production was greatest in EAM/CP-infected birds. The ionophore narasin, which directly eliminates coccidia, reduced goblet cell theca size, IL-10 and IFN-gamma expression, the growth of mucolytic bacteria including C. perfringens, coccidial and NE lesions and mortality in birds that were co-infected with coccidia and C. perfringens. Collectively the data support the hypothesis that coccidial infection induces a host mucogenic response providing a growth advantage to C. perfringens, the causative agent of NE.

"Candidatus Paraporphyromonas polyenzymogenes" encodes multi-modular cellulases linked to the type IX secretion system.

BACKGROUND: In nature, obligate herbivorous ruminants have a close symbiotic relationship with their gastrointestinal microbiome, which proficiently deconstructs plant biomass. Despite decades of research, lignocellulose degradation in the rumen has thus far been attributed to a limited number of culturable microorganisms. Here, we combine meta-omics and enzymology to identify and describe a novel Bacteroidetes family ("Candidatus MH11") composed entirely of uncultivated strains that are predominant in ruminants and only distantly related to previously characterized taxa. RESULTS: The first metabolic reconstruction of Ca. MH11-affiliated genome bins, with a particular focus on the provisionally named "Candidatus Paraporphyromonas polyenzymogenes", illustrated their capacity to degrade various lignocellulosic substrates via comprehensive inventories of singular and multi-modular carbohydrate active enzymes (CAZymes). Closer examination revealed an absence of archetypical polysaccharide utilization loci found in human gut microbiota. Instead, we identified many multi-modular CAZymes putatively secreted via the Bacteroidetes-specific type IX secretion system (T9SS). This included cellulases with two or more catalytic domains, which are modular arrangements that are unique to Bacteroidetes species studied to date. Core metabolic proteins from Ca. P. polyenzymogenes were detected in metaproteomic data and were enriched in rumen-incubated plant biomass, indicating that active saccharification and fermentation of complex carbohydrates could be assigned to members of this novel family. Biochemical analysis of selected Ca. P. polyenzymogenes CAZymes further iterated the cellulolytic activity of this hitherto uncultured bacterium towards linear polymers, such as amorphous and crystalline cellulose as well as mixed linkage ß-glucans. CONCLUSION: We propose that Ca. P. polyenzymogene genotypes and other Ca. MH11 members actively degrade plant biomass in the rumen of cows, sheep and most likely other ruminants, utilizing singular and multi-domain catalytic CAZymes secreted through the T9SS. The discovery of a prominent role of multi-modular cellulases in the Gram-negative Bacteroidetes, together with similar findings for Gram-positive cellulosomal bacteria (Ruminococcus flavefaciens) and anaerobic fungi (Orpinomyces sp.), suggests that complex enzymes are essential and have evolved within all major cellulolytic dominions inherent to the rumen.

Partial purification and characterization of Ral8I, a class-IIS restriction endonuclease from Ruminococcus albus 8 which recognizes 5'-GGATC.

Heparin-agarose chromatography was used to isolate a restriction endonuclease (ENase) from the cellulolytic Gram+ anaerobe, Ruminococcus albus 8. The enzyme, Ral8I, was eluted from the column using 230-310 mM Na+. However, the preparation was active only with DNA substrates that were not Dam-methylated. Moreover, the restriction fragment pattern generated from simian virus 40 (SV40) DNA was not consistent with the expected number of Dam-methylation sites. Alignment of the Dam-methylation sites in SV40 DNA indicated that Ral8I may actually recognize the asymmetric sequence, GGATC. This was confirmed by nucleotide (nt) sequence analysis and, further, Ral8I was found to cause cleavage of the DNA approx. 5 nt downstream from the recognition sequence. Ral8I can therefore be classified as a type-IIS restriction endonuclease and is an isoschizomer of AlwI, BinI and BthII.

Developmental microbial ecology of the neonatal gastrointestinal tract.

The gastrointestinal tract of a normal fetus is sterile. During the birth process and rapidly thereafter, microbes from the mother and surrounding environment colonize the gastrointestinal tract of the infant until a dense, complex microbiota develops. The succession of microbes colonizing the intestinal tract is most marked in early development, during which the feeding mode shifts from breast-feeding to formula feeding to weaning to the introduction of solid food. Dynamic balances exist between the gastrointestinal microbiota, host physiology, and diet that directly influence the initial acquisition, developmental succession, and eventual stability of the gut ecosystem. In this review, the development of the intestinal microbiota is discussed in terms of initial acquisition and subsequent succession of bacteria in human infants. Intrinsic and extrinsic factors influencing succession and their health significance are discussed. The advantages of modern molecular ecology techniques that provide sensitive and specific, culture-independent evaluation of the gastrointestinal ecosystem are introduced and discussed briefly. Further advances in our understanding of developmental microbial ecology in the neonatal gastrointestinal tract are dependent on the application of these modern molecular techniques.

The restriction endonuclease RflFII, isolated from Ruminococcus flavefaciens FD-1, recognizes the sequence 5'-AGTACT-3', and is inhibited by site-specific adenine methylation.

Molecular studies of the rumen bacterium Ruminococcus flavefaciens are constrained by the lack of stable gene transfer systems. We report here on the characterization of RflFII, a restriction endonuclease isolated from R. flavefaciens FD-1. The enzyme is an isoschizomer of ScaI, and cleavage of the DNA is blunt-ended, between the internal TA dinucleotide sequence of 5'-AGTACT-3'. Chromosomal DNA preparations were used to demonstrate that adenine methylation of DNA within the sequence 5'-GTAC-3' inhibits both RflFII and the restriction endonucleases RsaI and ScaI. Chromosomal DNA from R. flavefaciens FD-1 is also host modified to protect against cleavage by ScaI.

Complete nucleotide sequence of a cryptic plasmid, pBAW301, from the ruminal anaerobe Ruminococcus flavefaciens R13e2.

The complete nucleotide sequence of a cryptic plasmid designated pBAW301, from the Gram-positive ruminal bacterium Ruminococcus flavefaciens R13e2, has been determined. This plasmid is 1768 bp in size and has an overall G+C content of 43.5%. Computer analysis of the sequence data revealed an open reading frame, ORF1 (256 amino acids), which is similar to the Rep protein of the Bacillus borstelensis plasmid pHT926. ORF1 is preceded by Shine-Dalgarno and Escherichia coli-10 and -35 like sequences. Nine smaller open reading frames showed no significant homologies to known protein sequences. Analysis of replication intermediates and the nucleotide sequence indicate that the plasmid does not replicate by a rolling-circle mode of replication similar to other plasmids from Gram-positive bacteria. Moreover, sequences typical of theta replication origins were not found in the nucleotide sequence of pBAW301. These data suggest that this plasmid either replicates by an as yet undescribed mechanism, or represents a new class of theta replicating plasmids.

Application of denaturant gradient gel electrophoresis for the analysis of the porcine gastrointestinal microbiota.

The porcine gastrointestinal tract (GIT) microbiota has been studied to increase production efficiency, improve product quality, and help attempt to reduce disease. During the developmental period from birth through weaning, the intestinal microbiota undergoes a rapid ecological succession. There is interest in developing a monitoring technique that allows for analysis of bacterial population levels and shifts within the pig intestine. The objective of this study was to determine if denaturant gradient gel electrophoresis (DGGE) could be effectively applied to measure changes in bacterial populations of the pig GIT, as influenced by age, diet or compartment. Bacterial genetic diversity was determined using DGGE analysis of the V3 region of 16S rDNA PCR products (approximately 200 bp) obtained from primers specific for the domain Bacteria. Protocol development included optimization of: DNA extraction procedures, PCR amplification, removal of PCR artifacts, and optimization of gel preparation and image capture. DGGE analysis revealed diverse bacterial populations between pigs of different ages and among individual gut compartments. Comparison of fecal DNA from different aged pigs revealed several unique PCR product bands indicating the presence of unique bacterial populations. Comparison of different gut compartments demonstrated that bacterial populations were most similar (C, value > 50%) within a single compartment and between adjacent ones. Thus, DGGE can be used to examine bacterial diversity and population shifts in the pig GIT.

Design and evaluation of a Lactobacillus group-specific ribosomal RNA-targeted hybridization probe and its application to the study of intestinal microecology in pigs.

The potential importance of anaerobic bacteria belonging to the Lactobacillus group has been well documented. Appropriate methods to rapidly evaluate species diversity and fluctuations in their population levels within the Lactobacillus group are being developed. Molecular tools such as hybridization probes based on rRNA sequences are well suited to these studies. The work reported here was undertaken to test the specificity of an hybridization probe to specifically recognize microorganisms of the Lactobacillus group and assess its usefulness as a quantitative tool to study fluctuations of the Lactobacillus population relative to the total bacterial population in gastrointestinal contents of pigs. We have designed a 25-mer oligonucleotide that targets a region common to and specific for the Lactobacillus group 16S rRNA sequences within the available database. The optimal wash temperature of the probe was experimentally determined to be 54 degrees C. The results obtained using the Lactobacillus group-specific probe (LGP) shows that Lactobacillus populations vary along the different segments of the gastrointestinal tract (GIT). In weaning piglets, the relative Lactobacillus signal intensity obtained constituted 100% of the relative RNA index in the stomach contents as determined by a bacterial domain probe (BDP), and between 90 to 100% in the duodenum. The signal of the Lactobacillus population decreased and reached its minimum in the distal part of the GIT. The same trend was observed in adult pigs, but in the stomach they constituted no more than 30% as determined by the BDP, and were present at lower levels in the other parts of the GIT. These studies document the quantitative importance of the lactobacilli in the stomach and small intestine of pigs. Further studies to investigate the role of lactobacilli in promoting the ecological balance of gut bacteria for probiotic therapy are being undertaken.

Anaerobic codigestion of municipal solid waste and biosolids under various mixing conditions--I. Digester performance.

The feasibility of codigestion of the organic fraction of municipal solid waste, primary sludge, and waste activated sludge was evaluated in mesophilic (37 degrees C), laboratory-scale digesters. In a first experiment, different startup strategies were compared using four digesters, operated under continuously mixed conditions. After two weeks, the experiment was continued under minimally mixed conditions. Results demonstrated that reducing the level of mixing improved digester performance. Therefore, in a second experiment, six digesters were operated to compare performance under continuous mixing and reduced mixing levels at various loading rates and solids levels. The continuously mixed digesters exhibited unstable performance at the higher loading rates, while the minimally mixed digesters performed well for all loading rates evaluated. In a third experiment, it was demonstrated that an unstable, continuously mixed digester was quickly stabilized by reducing the mixing level. These experiments confirmed that continuous mixing was not necessary for good performance and was inhibitory at higher loading rates. In addition, reduction of mixing levels may be used as an operational tool to stabilize unstable digesters.

Anaerobic codigestion of municipal solid waste and biosolids under various mixing conditions--II: Microbial population dynamics.

Microbial population dynamics were evaluated in anaerobic codigesters treating municipal solid waste and sewage sludge. Ribosomal RNA based oligonucleotide probes were used to characterize changes in population abundance of syntrophic volatile fatty acid degrading bacteria and methanogens. Changes in community structure were linked to traditional performance parameters during the recovery of previously unstable codigesters induced by a reduction in mixing levels. Methanosarcina spp. were the most abundant aceticlastic methanogens in unstable codigesters with high acetate concentrations, while Methanosaeta concilii was dominant in stable systems with low levels of acetate. Growth of Syntrophobacter wolinii was enhanced during stabilization of a codigester with a well-developed population of Methanobacteriaceae, possibly because the presence of adequate numbers of these hydrogenotrophic methanogens encouraged the syntrophic oxidation of propionate. Mesophilic saturated fatty acid beta-oxidizing syntrophs were most abundant in previously unstable codigesters. One minimally mixed reactor became unstable after switching to continuously mixed conditions. After the switch, total archaeal abundance decreased sharply, though Methanobacteriaceae and Methanosarcina spp. levels increased as the fermentation became unbalanced. Based on the results presented here, mixing appears to inhibit the syntrophic oxidation of volatile fatty acids, possibly by disrupting the spatial juxtaposition of syntrophic bacteria and their methanogenic partners.

Biochemical identification and biological origin of key odor components in livestock waste.

Animal production results in conversion of feeds into valuable products such as meat, milk, eggs, and wool as well as into unavoidable and less desirable waste products. Intensification of animal numbers and increasing urbanization has resulted in considerable attention to odorous gases produced from animal wastes. It is clear that animal manure was, and still is, a valuable resource. However, it may be a major obstacle to future development of the animal industry if its impact on the environment is not properly controlled. Poor odor prevention and control from animal wastes is related to a lack of knowledge of the fundamental nature of odor and its production by farm animals. Odor, like noise, is a nuisance or disturbance and there is no universally accepted definition of an objectionable odor. Thus, regulation and control of odors in the environment is difficult because of the technical difficulties of defining odor limits and their measurement and evaluation. A variety of direct (sensory) and indirect (analytical instruments) methods for measuring odor intensity and determination of individual or key odor components are discussed. The biological origins of the four principal classes of odor compounds, namely branched- and straight-chain VFA, ammonia and volatile amines, indoles and phenols, and the volatile sulfur-containing compounds, are reviewed. Because more than 50% of N from animals is excreted as urea, one strategy to conserve N in waste is to inhibit the urease enzyme that converts urea to ammonia. Laboratory studies to evaluate di- and triamide compounds to control urea hydrolysis in slurries of cattle and swine wastes are presented. Finally, a brief overview of various intervention strategies is provided. Multiple combinations of nutritional management, housing systems, treatment options as well as storage and disposal of animal wastes will be required to reduce environmental pollution and provide for long-term sustainable growth.

Degradation of wheat straw and alkaline hydrogen peroxide-treated wheat straw by Ruminococcus albus 8 and Ruminococcus flavefaciens FD-1.

Degradation of wheat straw (WS) and alkaline hydrogen peroxide (AHP)-treated wheat straw (AHPWS) by Ruminococcus albus 8 and Ruminococcus flavefaciens FD-1 was determined by measuring the growth (OD600) of each bacterium and determining DM disappearance (DMD) of the substrate. Complex medium and defined medium with or without the addition of phenylpropanoic acid (PPA) and phenylacetic acid (PAA) were used. Tubes were incubated at 39 degrees C for 8 d. Both OD600 and DMD indicated that AHPWS was degraded to a much greater extent by either bacterium (R. flavefaciens FD-1, 60.8 +/- 1.8% and R. albus 8, 42.3 +/- 3.5%) vs untreated WS (R. flavefaciens FD-1, 16.5 +/- 1.8% and R. albus 8, 8.6 +/- 6%) in the complex medium. Most degradation occurred between d 1 and 4. With the complex medium, addition of PPA and PAA did not stimulate degradation by either bacterium. When the defined medium was used, the addition of PPA and PAA enhanced (P less than .05) degradation of AHPWS (39.6 +/- 2.6%) vs AHPWS with no added PPA and PAA (24.9 +/- 7.6%) by R. albus 8. There was no synergistic effect on degradation when the two species were co-cultured with either WS or AHPWS as the substrate. No effect of PPA and PAA on disappearance of AHPWS was observed for R. flavefaciens FD-1 or when the two bacteria were grown together. Dry matter disappearance analysis showed that R. flavefaciens FD-1 degraded AHPWS more rapidly (6.1 mg/d) than R. albus 8 did (4.2 mg/d) in complex medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of defaunation and various nitrogen supplementation regimens on microbial numbers and activity in the rumen of sheep.

Five sheep (average BW 62 kg) were fed 65% roughage: 35% concentrate diets (CP = 15%) in a 5 x 5 Latin square design to study the effects of combinations of defaunation and N supplements (soybean meal [SBM], corn gluten meal [CGM], blood meal [BM], urea, and casein) differing in ruminal degradation on ruminal microbial numbers and activity. Diets were fed twice daily (DM intake 1,759 g/d). Defaunation was accomplished with doses of 30 ml of alkanate 3SL3.sheep-1.d-1 for 3 d with 2 d of fasting. Treatment 1 (control) involved feeding faunated sheep a diet in which the supplemental N was 67% SBM N and 33% urea N. Treatment 2 involved feeding defaunated sheep the same diet as the control. Treatments 3, 4, and 5 involved feeding defaunated sheep diets in which the supplemental N source was either 67% CGM-BM N (CGM and BM combined on a 1:1 N ratio): 33% urea N, or 33% CGM-BM N:67% urea N or 33% CGM-BM N:33% urea N:33% casein N, respectively. Compared with the faunated control, defaunation (Treatments 2, 3, 4, and 5) increased (P less than .05) total direct counts of ruminal bacteria (2.7 vs 1.3 x 10(11)/ml), fungal zoospores (2.8 vs 1.4 x 10(5)/ml), and ruminal microbial protease activity (1.4 vs 1.0 mg azocasein/[ml ruminal fluid.h]). Defaunation did not have a consistent effect on ruminal microbial deaminase activity. Compared with the control, defaunation resulted in lower (P less than .05) total perchloric acid-soluble amino N in ruminal fluid at 4 and 10 h after the morning feeding. Defaunation did not decrease (P greater than .05) total free amino acid concentrations in ruminal fluid, but it altered the profile of free amino acids. Although defaunation increased (P less than .05) ruminal bacterial numbers, no increases in total microbial CP or OM concentrations in ruminal contents were observed.

Manipulation of nitrogen digestion by sheep using defaunation and various nitrogen supplementation regimens.

Five ruminally, duodenally, and ileally cannulated sheep (average BW 62 kg) were fed 65% roughage: 35% concentrate diets (CP = 15%) in a 5 x 5 Latin square design to study the applicability of using a combination of defaunation with N supplements (soybean meal [SBM], corn gluten meal [CGM], blood meal [BM], urea, and casein) with different extents of ruminal degradation to manipulate microbial protein synthesis and amount of ruminal escape protein. Diets were fed twice daily (1,759 g DM/d). Defaunation was accomplished with 30-ml doses of alkanate 3SL3 (active ingredient: sodium lauryl diethoxy sulfate)/sheep daily for 3 d with 2 d of fasting. Treatment 1 (control) involved feeding faunated sheep a diet in which the supplemental N (45% of total dietary N) was 67% SBM N and 33% urea N. Treatment 2 involved feeding defaunated sheep the same diet as the control. Treatments 3, 4, and 5 involved feeding defaunated sheep diets in which the supplemental N source was either 67% CGM-BM (1:1 N ratio) N:33% urea N, or 33% CGM-BM N:67% urea N or 33% CGM-BM N:33% urea N:33% casein N, respectively. Compared with the faunated control, defaunation decreased (P less than .05) ruminal ammonia concentration (19 vs 26 mg/dl) and increased (P less than .05) CP flow to the duodenum (253 vs 214 g/d) due to a trend for increases in both bacterial (BCP) and nonbacterial (NBCP) CP flows.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of weaning diet on the ecology of adherent lactobacilli in the gastrointestinal tract of the pig.

Sixteen pigs were included in an investigation of the effects of weaning and weaning diet on the ecology of adherent Lactobacillus species in the gastrointestinal tract. At 28 d of age four pigs were killed and were designated as the preweaning control (PW). Four pigs remained on the sow (Sow), four pigs were fed a corn-soy-lactose (CSL) diet, and the remaining four pigs were fed a corn-soy (CS) diet. Pigs from the latter three treatments were killed at 38 d of age. Tissue samples were taken from the pars esophagus, ileum, and cecum and the adherent lactobacilli were enumerated using Rogosa SL agar. Bacterial colonies were randomly selected from Rogosa SL agar plates and speciated using cell type, morphology, and substrate fermentation tests. The species data were used to calculate the Shannon, Simpson, and evenness diversity indices. Shannon and Simpson diversity index values when averaged across tissues were lower (P < .05) for PW than for postweaning treatments (Sow, CSL, and CS) and lower (P < .05) when pigs receiving sow's milk (PW and Sow) were compared with pigs receiving the dry diets (CSL and CS). The diversity of adherent Lactobacillus is altered by the form of the diet fed to weanling pigs, and statistical ecological methods provide a powerful way of analyzing the ecology of the gastrointestinal tract.

Effects of dietary energy level and protein source on nutrient digestion and ruminal nitrogen metabolism in steers.

Four Simmental steers with ruminal, duodenal, and ileal cannulas were used to examine effects of dietary forage: concentrate ratio and supply of ruminally degradable true protein on site of nutrient digestion and net ruminal microbial protein synthesis. Steers (345 kg) were fed ammoniated corn cob (high forage; HF)- or corn cob/ground corn/cornstarch (low forage; LF)-based diets supplemented with soybean meal (SBM) or a combination of corn gluten meal and blood meal (CB). Diets were fed at 2-h intervals with average DM intake equal to 2.2% of BW. Feeding LF vs HF increased (P less than .05) OM digestion (percentage of intake) in the stomach, small intestine, and total tract. Efficiency of microbial CP synthesis (EMCP; g of N/kg of OM truly fermented) decreased (P less than .05) for LF vs HF (24.1 vs 26.8), but microbial N and total N flows to the small intestine were similar (P greater than .05) between energy levels (average 112 and 209 g/d, respectively). Total N flows to the small intestine were 13.1% greater (P less than .05) for CB than for SBM because of increased (P less than .05) passage of nonmicrobial N. Feeding SBM vs CB increased (P less than .05) EMCP (27.3 vs 23.3) and microbial N flow to the small intestine (127.5 vs 112.5 g/d), but these increases were not likely due to increased ruminal concentrations of ammonia N (NH3 N). Decreased (P less than .05) incorporation of NH3 N into bacterial N and slower turnover rates of ruminal NH3 N for SBM vs CB suggest that direct incorporation of preformed diet components into cell mass increased when SBM was fed. Results of this study suggest that the inclusion of ruminally degradable protein in the diet may increase the supply of products from proteolysis and that this can increase EMCP and microbial protein flow to the small intestine.

Development of a signature probe targeting the 16S-23S rRNA internal transcribed spacer of a ruminal Ruminococcus flavefaciens isolate from reindeer.

The cellulolytic Ruminococcus flavefaciens has previously been introduced into the ruminant rumen to increase microbial degradation of plant cell wall carbohydrates. The functional effect of an introduced bacterium depends on its ability to establish in the digestive tract, and signature probes can be used as a tool to track and quantify introduced strains. The purpose of this current study was to develop an oligonucleotide signature probe targeting the 16S-23S rRNA internal transcribed spacer (ITS) of a putative probiotic cellulolytic isolate (R. flavefaciens strain 8/94-32) from the rumen of reindeer (Rangifer tarandus tarandus). The 16S-23S rRNA gene ITS of three Ruminococcus strains; R. flavefaciens strain 8/94-32, R. flavefaciens FD-1 and Ruminococcus albus Ra-8, was investigated. The ITS region has been reported to vary more between closely related bacteria compared to the widely used 16S rRNA gene, and a high degree of sequence polymorphism was indeed detected between the three Ruminococcus strains studied. Based on observed sequence differences, two oligonucloetide probes, ITSRumi1 and ITSRumi2, targeting the ITS region of the R. flavefaciens isolate 8/94-32 were developed. Probe specificity was evaluated in dot blot hybridisations with R. flavefaciens isolate 8/94-32 and four other Ruminococcus-strains tested. The probe ITSRumi1 gave positive signals for the R. flavefaciens isolate 8/94-32 only, while probe ITSRumi2 gave positive signals for R. flavefaciens isolate 8/94-32 as well as for R. albus Ra-8. The result of hybridisations with the probe ITSRumi1 indicates that the probe is specific for the R. flavefaciens strain 8/94-32 amongst the four Ruminococcus-strains tested, and is promising for further studies using it as a signature probe for tracking this strain when re-introduced to the reindeer rumen.

Digestive challenges for vertebrate animals: microbial diversity, cardiorespiratory coupling, and dietary specialization.

The digestive system is the interface between the supply of food for an animal and the demand for energy and nutrients to maintain the body, to grow, and to reproduce. Digestive systems are not morphologically static but rather dynamically respond to changes in the physical and chemical characteristics of the diet and the level of food intake. In this article, we discuss three themes that affect the ability of an animal to alter digestive function in relation to novel substrates and changing food supply: (1) the fermentative digestion in herbivores, (2) the integration of cardiopulmonary and digestive functions, and (3) the evolution of dietary specialization. Herbivores consume, digest, and detoxify complex diets by using a wide variety of enzymes expressed by bacteria, predominantly in the phyla Firmicutes and Bacteroidetes. Carnivores, such as snakes that feed intermittently, sometimes process very large meals that require compensatory adjustments in blood flow, acid secretion, and regulation of acid-base homeostasis. Snakes and birds that specialize in simple diets of prey or nectar retain their ability to digest a wider selection of prey. The digestive system continues to be of interest to comparative physiologists because of its plasticity, both phenotypic and evolutionary, and because of its widespread integration with other physiological systems, including thermoregulation, circulation, ventilation, homeostasis, immunity, and reproduction.

Monitoring and source tracking of tetracycline resistance genes in lagoons and groundwater adjacent to swine production facilities over a 3-year period.

To monitor the dissemination of resistance genes into the environment, we determined the occurrence of tetracycline resistance (Tc(r)) genes in groundwater underlying two swine confinement operations. Monitoring well networks (16 wells at site A and 6 wells at site C) were established around the lagoons at each facility. Groundwater (n = 124) and lagoon (n = 12) samples were collected from the two sites at six sampling times from 2000 through 2003. Total DNA was extracted, and PCR was used to detect seven Tc(r) genes [tet(M), tet(O), tet(Q), tet(W), tet(C), tet(H), and tet(Z)]. The concentration of Tc(r) genes was quantified by real-time quantitative PCR. To confirm the Tc(r) gene source in groundwater, comparative analysis of tet(W) gene sequences was performed on groundwater and lagoon samples. All seven Tc(r) genes were continually detected in groundwater during the 3-year monitoring period at both sites. At site A, elevated detection frequency and concentration of Tc(r) genes were observed in the wells located down-gradient of the lagoon. Comparative analysis of tet(W) sequences revealed that the impacted groundwater contained gene sequences almost identical (99.8% identity) to those in the lagoon, but these genes were not found in background libraries. Novel sequence clusters and unique indigenous resistance gene pools were also found in the groundwater. Thus, antibiotic resistance genes in groundwater are affected by swine manure, but they are also part of the indigenous gene pool.

Effect of insertional mutations in the pueA and pueB genes encoding two polyurethanases in Pseudomonas chlororaphis contained within a gene cluster.

AIMS: To better understand the role of PueA and PueB from Pseudomonas chlororaphis in polyurethane degradation, the present study was conducted to create insertional mutants in their respective genes. METHODS AND RESULTS: Growth kinetic studies showed that the pueA knockout mutant had a greater effect than the pueB knockout mutant. The pueA mutant had an 80% decrease in cell density from that of the wild type, while the pueB mutant had an 18% decrease in cell density. Polyurethane utilization followed Michaelis-Menten kinetics. The pueA and pueB mutants exhibited a 17% and 10% decrease respectively in growth rate using polyurethane when compared with the wild type. CONCLUSIONS: In this present study, pueA and pueB, are shown to be part of an ABC transporter gene cluster that consists of seven open reading frames. Mutational analysis results suggest that PueA may play a more major role in polyurethane degradation than PueB based on cell density and growth rates. SIGNIFICANCE AND IMPACT OF THE STUDY: The results from this study provide a starting point for the eventual enhancement and bioremediation of polyurethane waste. Understanding the role of polyurethane-degrading enzymes is useful for the creation of strains for this purpose.