Succession in the intestinal microbiota of preadolescent turkeys

In the present study, automated ribosomal intergenic spacer analysis (ARISA), library sequence analysis, real-time PCR detection of Bacteroides uniformis and Campylobacter coli and dot-blot hybridizations of Clostridiaceae were used to identify trends in microbial colonization of the ceca of male turkeys. Two separate trials were performed with six and five birds, respectively. ARISA community profiles identified a period of community transition at week 12 of age in both trials. A significant increase of Ca. coli was also detected at week 12 in one trial, suggesting a possible correlation between microbiota destabilization and pathogen prevalence. Libraries of ribosomal small subunit 16S genes representing weeks 9, 11, 12 and 14 of both trials were sequenced. Whereas fingerprint and sequence analyses indicated significant differences in the species composition between the two trials, in general sequence library and dot-blot analyses indicated that Clostridia-like species decreased in prevalence over time. While B. uniformis prevalence in the two trials rose from 7% and 0% of the library clones at week 9 to 84% and 79% at week 11, real-time PCR did not support these results, with only approximately twofold and sixfold increases in internal transcribed spacer copy numbers observed.     

Comparison of the Cecal Microbiota of Domestic and Wild Turkeys

The extent to which production methods alter intestinal microbial communities of livestock is currently unknown. As the intestinal microbiota may affect animal health, nutrition, and food safety, a baseline comparison of the cecal communities of domestic and wild turkeys was performed. Oligonucleotide fingerprinting of ribosomal RNA (rRNA) genes (OFRG) of 2,990 16S rRNA clones and dot blot quantification of dominant populations were used to identify the dominant bacterial taxa. Seventy-three percent of all the clones belonged to as yet uncultured genera. However, at a higher phylogenetic level, the OFRG library was composed of 54% Bacteroidetes clones (52% of the domestic library clones, 56% of the wild library clones), 30% Firmicutes clones (33% of the domestic library clones, 32% of the wild library clones), 3% Proteobacteria clones (5% domestic, 2% wild), and 3% Deferribacteres clones (4% domestic, 1% wild). Seven percent of the clones were unidentifiable (6% domestic, 9% wild). Bacteroidetes clones included the genera Alistipes, Prevotella, Megamonas, and Bacteroides. Of the Clostridiales clones, groups IV, IX, and XIV including genera Faecalibacterium, Megasphaera, Phascolarctobacterium, and Papillibacter were predominant. Lactobacillus, Enterococcus, and Streptococcus bacilli were also identified. beta- delta- and gamma-proteobacterial genera included Acinetobacter, Sutterella, and Escherichia. Deferribacteres clones showed high similarity to Mucispirillum schaedleri. Statistical comparison of the domestic and wild turkey clone libraries indicated similar levels of community richness and evenness despite the fact that the two libraries shared only 30% of the total clone operational taxonomic units. Together these results indicate that although high level taxonomic community structure is similar, high-density turkey production causes considerable divergence of the genera found in the ceca of commercial birds from those of their wild counterparts.     

Interaction of Sindbis virus glycoproteins during morphogenesis.

In cells infected with the Sindbis temperature-sensitive mutants ts-23 and ts-10 (complementation group D), which contain a defect in the envelope glycoprotein E1, the precursor polypeptide PE2 is not cleaved to the envelope glycoprotein E2 at the nonpermissive temperature. This defect is phenotypically identical to the defect observed in the complementation group E mutant, ts-20. The lesion in ts-23 is reversible upon shift to permissive temperature, whereas that of ts-10 is not. Antiserum against whole virus, E1, or E2 also prevents the cleavage of PE2 in cells infected with wild-type Sindbis virus. Because the cleavage of PE2 is inhibited by the lesion in mutants that are genotypically distinct and by anti-E1 or -E2 serum, it appears that PE2 and E1 exist as a complex in the membrane of the infected cell.     

Identifying microorganisms which fill a niche similar to that of the pathogen: a new investigative approach for discovering biological control organisms

Understanding the mechanisms of suppressive soils should lead to the development of new strategies to manage pests and diseases. For suppressive soils that have a biological nature, one of the first steps in understanding them is to identify the organisms contributing to this phenomenon. Here we present a new approach for identifying microorganisms involved in soil suppressiveness. This strategy identifies microorganisms that fill a niche similar to that of the pathogen by utilizing substrate utilization assays in soil. To demonstrate this approach, we examined an avocado grove where a Phytophthora cinnamomi epidemic created soils in which the pathogen could not be detected with baiting techniques, a characteristic common to many soils with suppressiveness against P. cinnamomi. Substrate utilization assays were used to identify rRNA genes (rDNA) from bacteria that rapidly grew in response to amino acids known to attract P. cinnamomi zoospores. Six bacterial rDNA intergenic sequences were prevalent in the epidemic soils but uncommon in the non-epidemic soils. These sequences belonged to bacteria related to Bacillus mycoides, Renibacterium salmoninarum, and Streptococcus pneumoniae. We hypothesize that bacteria such as these, which respond to the same environmental cues that trigger root infection by the pathogen, will occupy a niche similar to that of the pathogen and contribute to suppressiveness through mechanisms such as nutrient competition and antibiosis.     

Abundant and Diverse Fungal Microbiota in the Murine Intestine

Enteric microbiota play a variety of roles in intestinal health and disease. While bacteria in the intestine have been broadly characterized, little is known about the abundance or diversity of enteric fungi. This study utilized a culture-independent method termed oligonucleotide fingerprinting of rRNA genes (OFRG) to describe the compositions of fungal and bacterial rRNA genes from small and large intestines (tissue and luminal contents) of restricted-flora and specific-pathogen-free mice. OFRG analysis identified rRNA genes from all four major fungal phyla: Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota. The largest assemblages of fungal rRNA sequences were related to the genera Acremonium, Monilinia, Fusarium, Cryptococcus/Filobasidium, Scleroderma, Catenomyces, Spizellomyces, Neocallimastix, Powellomyces, Entophlyctis, Mortierella, and Smittium and the order Mucorales. The majority of bacterial rRNA gene clones were affiliated with the taxa Bacteroidetes, Firmicutes, Acinetobacter, and Lactobacillus. Sequence-selective PCR analyses also detected several of these bacterial and fungal rRNA genes in the mouse chow. Fluorescence in situ hybridization analysis with a fungal small-subunit rRNA probe revealed morphologically diverse microorganisms resident in the mucus biofilm adjacent to the cecal and proximal colonic epithelium. Hybridizing organisms comprised about 2% of the DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride)-positive organisms in the mucus biofilm, but their abundance in fecal material may be much lower. These data indicate that diverse fungal taxa are present in the intestinal microbial community. Their abundance suggests that they may play significant roles in enteric microbial functions.     

Examination of the microbial ecology of the avian intestine in vivo using bromodeoxyuridine

Bromodeoxyuridine, a thymidine analogue that can be incorporated into the DNA of actively dividing cells, has been used in vivo to identify intestinal bacteria that are metabolically active in 3-week-old turkey poults during an acute period of feed withdrawal. Automated ribosomal intergenic spacer analysis was used to identify amplicons unique to animals subjected to feed withdrawal. One amplicon was unique to fasted birds while two amplicons were present in 60% of fasted birds and absent in all fed birds. Sequence analysis of 16S ribosomal genes indicated the caecal communities of all birds were dominated by Clostridiaceae while also harbouring low levels of metabolically active gamma-proteobacteria and Bacteroides. Twenty per cent of clones from the fasted animals were identified as belonging to the genus Papillibacter, suggesting these microbes may be specifically dividing in response to environmental conditions present in the caeca of fasted birds.     

Comparison of DNA extraction methods for analysis of turkey cecal microbiota

AIM: As a prelude to long-term studies to characterize the microbiota of the turkey ceca, 14 DNA isolation protocols were evaluated for their ability to reproducibly characterize microbial diversity. METHODS AND RESULTS: Eight commercially available DNA extraction kits were assessed. DNA quantity and quality were assessed and competitive PCR was used to quantify the 16S bacterial rRNA genes. The Invitrogen Easy-DNA Kit extraction method for large samples yielded over eight times more DNA than any other method (3144 +/- 873 microg g(-1) of sample, P < 0.05). Bacterial and fungal species richness was estimated by Automated Ribosomal Intergenic Spacer Analysis. The Invitrogen Easy-DNA Kit generated the greatest bacterial species richness (46 +/- 7 peaks) while Bio-Rad Aquapure yielded the highest fungal species richness (71 +/- 9.5 peaks). CONCLUSION: Cluster analysis indicated different DNA extraction methods generated different microbial community compositions using the same cecal matrix from a single donor bird. SIGNIFICANCE AND IMPACT OF THE STUDY: Optimized DNA extraction protocols Invitrogen Easy-DNA Kit extraction method for large samples and Bio-Rad Aquapure outperform other methods for extraction of DNA from poultry fecal samples, although these methods do not necessarily recover all available DNA. They will be used in future studies to monitor the dynamics of microbial communities of the avian ceca.     

Abundant and diverse fungal microbiota in the murine intestine

Enteric microbiota play a variety of roles in intestinal health and disease. While bacteria in the intestine have been broadly characterized, little is known about the abundance or diversity of enteric fungi. This study utilized a culture-independent method termed oligonucleotide fingerprinting of rRNA genes (OFRG) to describe the compositions of fungal and bacterial rRNA genes from small and large intestines (tissue and luminal contents) of restricted-flora and specific-pathogen-free mice. OFRG analysis identified rRNA genes from all four major fungal phyla: Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota. The largest assemblages of fungal rRNA sequences were related to the genera Acremonium, Monilinia, Fusarium, Cryptococcus/Filobasidium, Scleroderma, Catenomyces, Spizellomyces, Neocallimastix, Powellomyces, Entophlyctis, Mortierella, and Smittium and the order Mucorales. The majority of bacterial rRNA gene clones were affiliated with the taxa Bacteroidetes, Firmicutes, Acinetobacter, and Lactobacillus. Sequence-selective PCR analyses also detected several of these bacterial and fungal rRNA genes in the mouse chow. Fluorescence in situ hybridization analysis with a fungal small-subunit rRNA probe revealed morphologically diverse microorganisms resident in the mucus biofilm adjacent to the cecal and proximal colonic epithelium. Hybridizing organisms comprised about 2% of the DAPI (4',6-diamidino-2-phenylindole, dihydrochloride)-positive organisms in the mucus biofilm, but their abundance in fecal material may be much lower. These data indicate that diverse fungal taxa are present in the intestinal microbial community. Their abundance suggests that they may play significant roles in enteric microbial functions.