Genealogy of an in-vivo passaged isolate of western Canadian bovine respiratory syncytial virus.

Bovine respiratory syncytial virus (BRSV) is a primary respiratory pathogen in calves. Clinical infection with this pathogen has been experimentally modelled to assess vaccine efficacy using a field isolate (Asquith) of BRSV that has been sequentially passaged in vivo in neonatal calves to maintain virulence. The objective of this retrospective cumulative analysis of passages over approximately 20 years was to determine if there have been any changes in the viral genome of this isolate because of this process. Sequence analyses indicated that the Asquith isolate placed genetically in a clade comprising US and some European isolates and a recently described Chinese BRSV isolate (DQ). Furthermore, there were rare changes in bases over time in the N, G, and F gene segments examined when comparing among different passages ranging from 1996 to 2019. These results indicated the absence of significant mutations in the absence of significant adaptive immunological pressure.

Le virus respiratoire syncitial bovin (BRSV) est un agent pathogène respiratoire primaire chez les veaux. Une infection clinique avec cet agent pathogène a été expérimentalement modélisée pour évaluer l'efficacité vaccinale en utilisant un isolat de champ (Asquith) de BRSV qui a été passé séquentiellement in vivo chez des veaux nouveau-nés pour maintenir sa virulence. L'objectif de cette analyse rétrospective cumulative des passages sur une période d'approximativement 20 ans était de déterminer s'il y avait eu des changements dans le génome viral de cet isolat à cause de ce processus. L'analyse des séquences indiquaient que l'isolat Asquith se positionnait génétiquement dans un clade comprenant des isolats américains et quelques isolats européens et un isolat chinois de BRSV récemment décrit (DQ). Également, il y avait de rares changements de bases dans le temps dans les segments de gènes N, G et F examinés lors de la comparaison parmi les différents passages allant de 1996 à 2019. Ces résultats indiquent l'absence de mutation significative en absence de pression immunologique adaptative significative.(Traduit par Docteur Serge Messier).

Sarcolipin Exhibits Abundant RNA Transcription and Minimal Protein Expression in Horse Gluteal Muscle.

Ca2+ regulation in equine muscle is important for horse performance, yet little is known about this species-specific regulation. We reported recently that horse encode unique gene and protein sequences for the sarcoplasmic reticulum (SR) Ca2+-transporting ATPase (SERCA) and the regulatory subunit sarcolipin (SLN). Here we quantified gene transcription and protein expression of SERCA and its inhibitory peptides in horse gluteus, as compared to commonly-studied rabbit skeletal muscle. RNA sequencing and protein immunoblotting determined that horse gluteus expresses the ATP2A1 gene (SERCA1) as the predominant SR Ca2+-ATPase isoform and the SLN gene as the most-abundant SERCA inhibitory peptide, as also found in rabbit skeletal muscle. Equine muscle expresses an insignificant level of phospholamban (PLN), another key SERCA inhibitory peptide expressed commonly in a variety of mammalian striated muscles. Surprisingly in horse, the RNA transcript ratio of SLN-to-ATP2A1 is an order of magnitude higher than in rabbit, while the corresponding protein expression ratio is an order of magnitude lower than in rabbit. Thus, SLN is not efficiently translated or maintained as a stable protein in horse muscle, suggesting a non-coding role for supra-abundant SLN mRNA. We propose that the lack of SLN and PLN inhibition of SERCA activity in equine muscle is an evolutionary adaptation that potentiates Ca2+ cycling and muscle contractility in a prey species domestically selected for speed.

Coding sequences of sarcoplasmic reticulum calcium ATPase regulatory peptides and expression of calcium regulatory genes in recurrent exertional rhabdomyolysis.

BACKGROUND: Sarcolipin (SLN), myoregulin (MRLN), and dwarf open reading frame (DWORF) are transmembrane regulators of the sarcoplasmic reticulum calcium transporting ATPase (SERCA) that we hypothesized played a role in recurrent exertional rhabdomyolysis (RER). OBJECTIVES: Compare coding sequences of SLN, MRLN, DWORF across species and between RER and control horses. Compare expression of muscle Ca2+ regulatory genes between RER and control horses. ANIMALS: Twenty Thoroughbreds (TB), 5 Standardbreds (STD), 6 Quarter Horses (QH) with RER and 39 breed-matched controls. METHODS: Sanger sequencing of SERCA regulatory genes with comparison of amino acid (AA) sequences among control, RER horses, human, mouse, and rabbit reference genomes. In RER and control gluteal muscle, quantitative real-time polymerase chain reaction of SERCA regulatory peptides, the calcium release channel (RYR1), and its accessory proteins calsequestrin (CASQ1), and calstabin (FKBP1A). RESULTS: The SLN gene was the highest expressed horse SERCA regulatory gene with a uniquely truncated AA sequence (29 versus 31) versus other species. Coding sequences of SLN, MRLN, and DWORF were identical in RER and control horses. A sex-by-phenotype effect occurred with lower CASQ1 expression in RER males versus control males (P < .001) and RER females (P = .05) and higher FKBP1A (P = .01) expression in RER males versus control males. CONCLUSIONS AND CLINICAL IMPORTANCE: The SLN gene encodes a uniquely truncated peptide in the horse versus other species. Variants in the coding sequence of SLN, MLRN, or DWORF were not associated with RER. Males with RER have differential gene expression that could reflect adaptations to stabilize RYR1.

Prevalence of the E321G MYH1 variant for immune-mediated myositis and nonexertional rhabdomyolysis in performance subgroups of American Quarter Horses.

BACKGROUND: Immune-mediated myositis (IMM) in American Quarter Horses (QHs) causes acute muscle atrophy and lymphocytic infiltration of myofibers. Recently, an E321G mutation in a highly conserved region of the myosin heavy chain 1 (MYH1) gene was associated with susceptibility to IMM and nonexertional rhabdomyolysis. OBJECTIVES: To estimate prevalence of the E321G MYH1 variant in the QH breed and performance subgroups. ANIMALS: Three-hundred seven elite performance QHs and 146 random registered QH controls. METHODS: Prospective genetic survey. Elite QHs from barrel racing, cutting, halter, racing, reining, Western Pleasure, and working cow disciplines and randomly selected registered QHs were genotyped for the E321G MYH1 variant and allele frequencies were calculated. RESULTS: The E321G MYH1 variant allele frequency was 0.034 ± 0.011 in the general QH population (6.8% of individuals in the breed) and the highest among the reining (0.135 ± 0.040; 24.3% of reiners), working cow (0.085 ± 0.031), and halter (0.080 ± 0.027) performance subgroups. The E321G MYH1 variant was present in cutting (0.044 ± 0.022) and Western Pleasure (0.021 ± 0.015) QHs at lower frequency and was not observed in barrel racing or racing QHs. CONCLUSIONS AND CLINICAL IMPORTANCE: Knowing that reining and working cow QHs have the highest prevalence of the E321G MYH1 variant and that the variant is more prevalent than the alleles for hereditary equine regional dermal asthenia and hyperkalemic periodic paralysis in the general QH population will guide the use of genetic testing for diagnostic and breeding purposes.

Proteome and transcriptome profiling of equine myofibrillar myopathy identifies diminished peroxiredoxin 6 and altered cysteine metabolic pathways.

Equine myofibrillar myopathy (MFM) causes exertional muscle pain and is characterized by myofibrillar disarray and ectopic desmin aggregates of unknown origin. To investigate the pathophysiology of MFM, we compared resting and 3 h postexercise transcriptomes of gluteal muscle and the resting skeletal muscle proteome of MFM and control Arabian horses with RNA sequencing and isobaric tags for relative and absolute quantitation analyses. Three hours after exercise, 191 genes were identified as differentially expressed (DE) in MFM vs. control muscle with >1 log2 fold change (FC) in genes involved in sulfur compound/cysteine metabolism such as cystathionine-beta-synthase ( CBS, ↓4.51), a cysteine and neutral amino acid membrane transporter ( SLC7A10, ↓1.80 MFM), and a cationic transporter (SLC24A1, ↓1.11 MFM). In MFM vs. control at rest, 284 genes were DE with >1 log2 FC in pathways for structure morphogenesis, fiber organization, tissue development, and cell differentiation including > 1 log2 FC in cardiac alpha actin ( ACTC1 ↑2.5 MFM), cytoskeletal desmoplakin ( DSP ↑2.4 MFM), and basement membrane usherin ( USH2A ↓2.9 MFM). Proteome analysis revealed significantly lower antioxidant peroxiredoxin 6 content (PRDX6, ↓4.14 log2 FC MFM), higher fatty acid transport enzyme carnitine palmitoyl transferase (CPT1B, ↑3.49 MFM), and lower sarcomere protein tropomyosin (TPM2, ↓3.24 MFM) in MFM vs. control muscle at rest. We propose that in MFM horses, altered cysteine metabolism and a deficiency of cysteine-containing antioxidants combined with a high capacity to oxidize fatty acids and generate ROS during aerobic exercise causes chronic oxidation and aggregation of key proteins such as desmin.

An E321G MYH1 mutation is strongly associated with nonexertional rhabdomyolysis in Quarter Horses.

BACKGROUND: An E321G mutation in MYH1 was recently identified in Quarter Horses (QH) with immune-mediated myositis (IMM) defined by a phenotype of gross muscle atrophy and myofiber lymphocytic infiltrates. HYPOTHESIS/OBJECTIVES: We hypothesized that the MYH1 mutation also was associated with a phenotype of nonexertional rhabdomyolysis. The objective of this study was to determine the prevalence of the MYH1 mutation in QH with exertional (ER) and nonexertional (nonER) rhabdomyolysis. ANIMALS: Quarter Horses: 72 healthy controls, 85 ER-no atrophy, 56 ER-atrophy, 167 nonER horses selected regardless of muscle atrophy. METHODS: Clinical and histopathologic information and DNA was obtained from a database for (1) ER > 2 years of age, with or without atrophy and (2) nonER creatine kinase (CK) ≥ 5000 U/L, <5 years of age. Horses were genotyped for E321G MYH1 by pyrosequencing. RESULTS: The MYH1 mutation was present in a similar proportion of ER-no atrophy (1/56; 2%) and in a higher proportion of ER-atrophy (25/85; 29%) versus controls (4/72; 5%). The MYH1 mutation was present in a significantly higher proportion of nonER (113/165; 68%) than controls either in the presence (39/42; 93%) or in absence (72/123; 59%) of gross atrophy. Lymphocytes were present in <18% of muscle samples with the MYH1 mutation. CONCLUSIONS AND CLINICAL IMPORTANCE: Although not associated with ER, the MYH1 mutation is associated with atrophy after ER. The MYH1 mutation is highly associated with nonER regardless of whether muscle atrophy or lymphocytic infiltrates are present. Genetic testing will enhance the ability to diagnose MYH1 myopathies (MYHM) in QH.

A missense mutation in MYH1 is associated with susceptibility to immune-mediated myositis in Quarter Horses.

BACKGROUND: The cause of immune-mediated myositis (IMM), characterized by recurrent, rapid-onset muscle atrophy in Quarter Horses (QH), is unknown. The histopathologic hallmark of IMM is lymphocytic infiltration of myofibers. The purpose of this study was to identify putative functional variants associated with equine IMM. METHODS: A genome-wide association (GWA) study was performed on 36 IMM QHs and 54 breed matched unaffected QHs from the same environment using the Equine SNP50 and SNP70 genotyping arrays. RESULTS: A mixed model analysis identified nine SNPs within a ~ 2.87 Mb region on chr11 that were significantly (Punadjusted < 1.4 × 10- 6) associated with the IMM phenotype. Associated haplotypes within this region encompassed 38 annotated genes, including four myosin genes (MYH1, MYH2, MYH3, and MYH13). Whole genome sequencing of four IMM and four unaffected QHs identified a single segregating nonsynonymous E321G mutation in MYH1 encoding myosin heavy chain 2X. Genotyping of additional 35 IMM and 22 unaffected QHs confirmed an association (P = 2.9 × 10- 5), and the putative mutation was absent in 175 horses from 21 non-QH breeds. Lymphocytic infiltrates occurred in type 2X myofibers and the proportion of 2X fibers was decreased in the presence of inflammation. Protein modeling and contact/stability analysis identified 14 residues affected by the mutation which significantly decreased stability. CONCLUSIONS: We conclude that a mutation in MYH1 is highly associated with susceptibility to the IMM phenotype in QH-related breeds. This is the first report of a mutation in MYH1 and the first link between a skeletal muscle myosin mutation and autoimmune disease.

Differences in CD8αα and cecal microbiome community during proliferation and late cytolytic phases of Marek's disease virus infection are associated with genetic resistance to Marek's disease.

Marek's disease (MD) is an important neoplastic disease of chickens caused by Marek ': s disease virus (MDV), a highly oncogenic alphaherpesvirus. In this study using two chicken lines, one resistant and another susceptible to MD, splenic T cells and cecal microbiome were profiled to gain a better understanding of primary differences in these lines. The percent of splenic CD4+ T cells were similar regardless of MDV challenge status in both bird lines. In contrast, CD8αα profiles were different (P < 0.005) between chicken lines under naïve status and under MDV challenge, suggesting that CD8αα T cells play a key role in mediating MDV infection. Microbiome composition was different between naïve resistant (Blautia spp.) and susceptible birds (Streptococcus spp.) (P < 0.05) during initial colonization. With MDV challenge, both chicken lines showed lower numbers of beneficial Faecalibacterium spp. and increased number of Lactobacillus spp. Metabolic profiles between naïve chicken types were similar but with MDV challenge, there were differences in metabolism in both chicken lines, with amino acid metabolism impacted in resistant birds and lipid metabolism in susceptible birds. These results provide insights into immune response and potential interplay with the microbiome during infection with an oncogenic virus.

Fine mapping of QTL and genomic prediction using allele-specific expression SNPs demonstrates that the complex trait of genetic resistance to Marek's disease is predominantly determined by transcriptional regulation.

BACKGROUND: Marek's disease (MD) is a lymphoproliferative disease of poultry induced by Marek's disease virus (MDV), a highly oncogenic alphaherpesvirus. Identifying the underlying genes conferring MD genetic resistance is desired for more efficacious control measures including genomic selection, which requires accurately identified genetic markers throughout the chicken genome. METHODS: Hypothesizing that variants located in transcriptional regulatory regions are the main mechanism underlying this complex trait, a genome-wide association study was conducted by genotyping a ~1,000 bird MD resource population derived from experimental inbred layers with SNPs containing 1,824 previously identified allele-specific expression (ASE) SNPs in response to MDV infection as well as 3,097 random SNPs equally spaced throughout the chicken genome. Based on the calculated associations, genomic predictions were determined for 200 roosters and selected sires had their progeny tested for Marek's disease incidence. RESULTS: Our analyses indicate that these ASE SNPs account for more than 83 % of the genetic variance and exhibit nearly all the highest associations. To validate these findings, 200 roosters had their genetic merit predicted from the ASE SNPs only, and the top 30 and bottom 30 ranked roosters were reciprocally mated to random hens. The resulting progeny showed that after only one generation of bidirectional selection, there was a 22 % difference in MD incidence and this approach gave a 125 % increase in accuracy compared to current pedigree-based estimates. CONCLUSIONS: We conclude that variation in transcriptional regulation is the major driving cause for genetic resistance to MD, and ASE SNPs identify the underlying genes and are sufficiently linked to the causative polymorphisms that they can be used for accurate genomic prediction as well as help define the underlying molecular basis. Furthermore, this approach should be applicable to other complex traits.

Marek's disease virus influences the core gut microbiome of the chicken during the early and late phases of viral replication.

Marek's disease (MD) is an important neoplastic disease of chickens caused by the Marek's disease virus (MDV), an oncogenic alphaherpesvirus. In this study, dysbiosis induced by MDV on the core gut flora of chicken was assessed using next generation sequence (NGS) analysis. Total fecal and cecum-derived samples from individual birds were used to estimate the influence of MDV infection on the gut microbiome of chicken. Our analysis shows that MDV infection alters the core gut flora in the total fecal samples relatively early after infection (2-7 days) and in the late phase of viral infection (28-35 days) in cecal samples, corresponding well with the life cycle of MDV. Principle component analyses of total fecal and cecal samples showed clustering at the early and late time points, respectively. The genus Lactobacillus was exclusively present in the infected samples in both total fecal and cecal bird samples. The community colonization of core gut flora was altered by viral infection, which manifested in the enrichment of several genera during the early and late phases of MDV replication. The results suggest a relationship between viral infection and microbial composition of the intestinal tract that may influence inflammation and immunosuppression of T and B cells in the host.

Characterizing the molecular basis of attenuation of Marek's disease virus via in vitro serial passage identifies de novo mutations in the helicase-primase subunit gene UL5 and other candidates associated with reduced virulence.

UNLABELLED: Marek's disease (MD) is a lymphoproliferative disease of chickens caused by the oncogenic Gallid herpesvirus 2, commonly known as Marek's disease virus (MDV). MD vaccines, the primary control method, are often generated by repeated in vitro serial passage of this highly cell-associated virus to attenuate virulent MDV strains. To understand the genetic basis of attenuation, we used experimental evolution by serially passing three virulent MDV replicates generated from an infectious bacterial artificial chromosome (BAC) clone. All replicates became completely or highly attenuated, indicating that de novo mutation, and not selection among quasispecies existing in a strain, is the primary driving force for the reduction in virulence. Sequence analysis of the attenuated replicates revealed 41 to 95 single-nucleotide variants (SNVs) at 2% or higher frequency in each population and several candidate genes containing high-frequency, nonsynonymous mutations. Five candidate mutations were incorporated into recombinant viruses to determine their in vivo effect. SNVs within UL42 (DNA polymerase auxiliary subunit) and UL46 (tegument) had no measurable influence, while two independent mutations in LORF2 (a gene of unknown function) improved survival time of birds but did not alter disease incidence. A fifth SNV located within UL5 (helicase-primase subunit) greatly reduced in vivo viral replication, increased survival time of birds, and resulted in only 0 to 11% disease incidence. This study shows that multiple genes, often within pathways involving DNA replication and transcriptional regulation, are involved in de novo attenuation of MDV and provides targets for the rational design of future MD vaccines. IMPORTANCE: Marek's disease virus (MDV) is a very important pathogen in chickens that costs the worldwide poultry industry $1 billion to $2 billion annually. Marek's disease (MD) vaccines, the primary control method, are often produced by passing virulent strains in cell culture until attenuated. To understand this process, we identified all the changes in the viral genome that occurred during repeated cell passage. We find that a single mutation in the UL5 gene, which encodes a viral protein necessary for DNA replication, reduces disease incidence by 90% or more. In addition, other candidate genes were identified. This information should lead to the development of more effective and rationally designed MD vaccines leading to improved animal health and welfare and lower costs to consumers.

Comparison and contrast of genes and biological pathways responding to Marek's disease virus infection using allele-specific expression and differential expression in broiler and layer chickens.

BACKGROUND: Marek's disease (MD) is a commercially important neoplastic disease of chickens caused by the Marek's disease virus (MDV), a naturally occurring oncogenic alphaherpesvirus. Enhancing MD genetic resistance is desirable to augment current vaccines and other MD control measures. High throughput sequencing was used to profile splenic transcriptomes from individual F1 progeny infected with MDV at 4 days of age from both outbred broilers (meat-type) and inbred layer (egg-type) chicken lines that differed in MD genetic resistance. The resulting information was used to identify SNPs, genes, and biological pathways exhibiting allele-specific expression (ASE) in response to MDV infection in each type of chicken. In addition, we compared and contrasted the results of pathway analyses (ASE and differential expression (DE)) between chicken types to help inform on the biological response to MDV infection. RESULTS: With 7 individuals per line and treatment group providing high power, we identified 6,132 single nucleotide polymorphisms (SNPs) in 4,768 genes and 4,528 SNPs in 3,718 genes in broilers and layers, respectively, that exhibited ASE in response to MDV infection. Furthermore, 548 and 434 genes in broilers and layers, respectively, were found to show DE following MDV infection. Comparing the datasets, only 72 SNPs and 850 genes for ASE and 20 genes for DE were common between the two bird types. Although the chicken types used in this study were genetically different, at the pathway level, both TLR receptor and JAK/STAT signaling pathways were enriched as well as exhibiting a high proportion of ASE genes, especially at the beginning of both above mentioned regulatory pathways. CONCLUSIONS: RNA sequencing with adequate biological replicates is a powerful approach to identify high confidence SNPs, genes, and pathways that are associated with transcriptional response to MDV infection. In addition, the SNPs exhibiting ASE in response to MDV infection provide a strong foundation for determining the extent to which variation in expression influences MD incidence plus yield genetic markers for genomic selection. However, given the paucity of overlap among ASE SNP sets (broilers vs. layers), it is likely that separate screens need to be incorporated for each population. Finally, comparison of gene lists obtained between these two diverse chicken types indicate the TLR and JAK/STAT signaling are conserved when responding to MDV infection and may be altered by selection of genes exhibiting ASE found at the start of each pathway.

Anaerobic transformation of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) by ovine rumen microorganisms.

Explosives such as octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) provide a challenge in terms of bioremediation. In the present study, sheep rumen was studied for its potential to detoxify HMX using analytical chemistry and molecular microbial ecology tools. Results indicated significant loss (p < 0.05) of HMX at 8 h post-incubation and complete disappearance of the parent molecule after 16 h. Qualitative LC-MS/MS analysis provided evidence for the formation of 1-NO-HMX and MEDINA metabolites. A total of 1006 16S rRNA-V3 clones were sequenced and the Classifier tool of the RDPII database was used to sort the sequences at their phylum level. Most sequences were associated with either the phylum Bacteroidetes or Firmicutes. Significant differences at the phylum level (p < 0.001) were found between 0 h and 8 h HMX treatments. Using LibCompare analysis, 8 h HMX treatment showed enrichment of clones (p < 0.01) belonging to the genus Prevotella. From these results, it could be concluded that members of the genus Prevotella are enriched in the rumen and are capable of detoxifying HMX.

Biochemical and microbial analysis of ovine rumen fluid incubated with 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX).

In this study, the rumen was assessed for its potential to detoxify RDX using molecular microbial ecology as well as analytical chemistry techniques. Results indicated significant loss (P < 0.05) of RDX in <8-h post incubation, and qualitative LC-MS/MS analysis showed evidence for the formation of 1-NO-RDX (M-O + HCOO) and methylenedinitramine metabolites. A total of 1106 16S rRNA-V3 clones were sequenced, and most sequences associated with either the phyla Bacteroidetes or Firmicutes. A LibCompare analysis for the RDX treatment showed an enrichment (P < 0.01) of the genus Prevotella. From these results, it can be concluded that the rumen is capable of detoxifying RDX, and the members of the genus Prevotella are linked to this detoxification.

Design and in vitro evaluation of new rpoB-DGGE primers for ruminants.

Two new primer sets based on the rpoB gene were designed and evaluated with bovine and ovine rumen samples. The newly developed rpoB-DGGE primer set was used along with the 16S rRNA gene-V3, and another (old) rpoB-DGGE-based primer set from a previous study to in vitro compare the bovine and ovine rumen ecosystems. The results indicate a significant (P<0.001) difference in the microbial population between the two ruminants irrespective of the primers used in the analysis. Qualitative comparison of the data provides evidence for the presence of similar phyla profiles between the 16S rRNA gene and the newly developed rpoB primers. A comparison between the two rpoB-based primer sets (old and new) showed that the old rpoB-based primers failed to amplify phylum Bacteroidetes (a common phylum in the rumen) in both bovine and ovine rumen samples. The old and new rpoB-DGGE-based primers amplified a large number of clones belonging to phylum Proteobacteria, providing a useful insight into the microbial structure of the rumen. ChaoI, ACE, Simpson, and Shannon-Weaver index analysis estimated the bovine rumen to be more diverse than the ovine rumen for all three primer sets. These results provide a new insight into the community structure among ruminants using the newly developed primers in this study.

Changes to the rumen bacterial population of sheep with the addition of 2,4,6-trinitrotoluene to their diet.

Previous work has shown that bacterial isolates from the sheep rumen are capable of detoxifying 2,4,6-trinitrotoluene (TNT) into polar constituents. In this study, the dietary effects of TNT on the sheep rumen microbial community were evaluated using molecular microbiology ecology tools. Rumen samples were collected from sheep fed with and without TNT added to their diet, genomic DNA was extracted, and the 16S rRNA-V3 gene marker was used to quantify changes in the microbial population in the rumen. Control and treatment samples yielded 533 sequences. Phylogenetic analyses were performed to determine the microbial changes between the two conditions. Results indicated the predominant bacterial populations present in the rumen were comprised of the phyla Firmicutes and Bacteroidetes, irrespective of presence/absence of TNT in the diet. Significant differences (P < 0.001) were found between the community structure of the bacteria under TNT (-) and TNT (+) diets. Examination of the TNT (+) diet showed an increase in the clones belonging to family Ruminococcaceae, which have previously been shown to degrade TNT in pure culture experiments.

Microbiomic comparison of the intestine of the earthworm Eisenia fetida fed ergovaline.

Tall fescue toxicosis and ergot alkaloids cost U.S. livestock producers approximately one billion dollars in annual livestock production loss annually. Ergovaline (EV) is the tall fescue alkaloid primarily responsible for clinical disease in livestock. Since native ruminal microorganisms have not been attributed to the detoxification of EV, finding detoxifying microbes from other environments is desirable. One possible source for potential microorganisms that can degrade EV is the anaerobic gut of the earthworm, Eisenia fetida. This study describes a comparative microbial analysis of earthworm digestive tracts receiving 10,000 ppb EV (E+ treatment) when compared with a control treatment with no detectable amounts of EV (E- treatment). An HPLC assay determined a 25% loss of EV from the E+ treatment was microbial in nature. A community microbiomic approach of constructing 16S-rRNA gene clone libraries was used to compare the microbes affected by the two treatments. RDPII tools such as Classifier and Libcompare were used in the analysis of 16S sequences. DOTUR analysis was used to examine the richness and diversity of the two microbial populations in these experiments. The results indicate there are few significant differences in the microbial community structure between the two microbiomes.

A bioremediation approach using natural transformation in pure-culture and mixed-population biofilms.

Bacterial transformation by naked DNA is thought to contribute to gene transfer and microbial evolution within natural environments. In nature many microbial communities exist as complex assemblages known as biofilms where genetic exchange is facilitated. It may be possible to take advantage of natural transformation processes to modify the phenotypes of biofilm communities giving them specific and desirable functions. Work described here shows that biofilms composed of either pure cultures or mixed populations can be transformed with specific catabolic genes such that the communities acquire the ability to degrade a particular xenobiotic compound. Biofilms were transformed by plasmids bearing genes encoding green fluorescent protein (mut2) and/or atrazine chlorohydrolase (atzA). Confocal microscopy was used to quantify the number of transformants expressing mut2 in the biofilms. Degradation of atrazine by expressed atzA was quantified by tandem mass spectrometry. PCR analysis was performed to confirm the presence of atzA in transformed biofilms. These results indicate that it should be possible to use natural transformation to enhance bioremediation processes performed by biofilms.