Streptococcus zooepidemicus in dogs: Exploring a canine pathogen through multilocus sequence typing.

Streptococcus equi. subsp. zooepidemicus (S. zooepidemicus) associated diseases in dogs have emerged as a significant concern over recent decades. S. zooepidemicus occurs sporadically in dog populations globally, with increased prevalence in shelters/kennels. This study used multilocus sequence typing (MLST) of 149 independent canine S. zooepidemicus isolates to assess associations between sequence type and breed, country of origin, disease severity, sampling type, year, and behaviour within an outbreak. No clear associations for breed, country, sampling type and year were determined in this study. ST-10 and 123 strains were present within all disease categories, from no clinical signs to severe disease. Assessment of S. zooepidemicus infection in 3 UK outbreaks at the same location found ST-10, 18, 123 strains, and a ST-173 strain in a US outbreak, were associated with haemorrhagic pneumonia and persisted in kennelled populations over time. The ST-173 clonal complex has been noted to have severe virulence capabilities in dogs and other species. S. zooepidemicus seems to thrive in environments with a high risk of transmissibility, overcrowding, stress and naïve populations, particularly for those in shelters/kennels. MLST alone cannot determine the virulence phenotype of S. zooepidemicus in dogs. However, a level of conservancy and diversity within ST allelic loci aids the opportunity to cause severe disease in dogs. Thus, further research into whole genome sequencing and characterising the virulence factors of S. zooepidemicus is warranted in dogs.

Evaluation of subchondral bone cysts in canine elbows with radiographic osteoarthritis secondary to elbow dysplasia.

OBJECTIVE: To investigate whether subchondral bone cysts (SBCs) were present in dogs with radiographic elbow osteoarthritis (OA) and to investigate their relationship with radiographic OA severity. STUDY DESIGN: Retrospective cross-sectional study. SAMPLE POPULATION: Thirty-eight Labrador retrievers (total of 76 elbows). METHODS: Elbow computed tomography (CT) images of 18 young (≤2 years old) and 20 old (>2 years old) Labrador retrievers, which presented for elbow-associated lameness, were reviewed. Radiographic elbow OA was graded into four groups based on the largest osteophyte size on CT. The presence, number, and maximum diameter of SBCs were determined. RESULTS: Subchondral bone cysts were only identified in elbows with osteophytic new bone formation. The number and size of SBCs were associated with radiographic OA severity (p < .001 and p = .041 respectively). Specifically, the rate at which SBCs were present increased for both moderate and severe OA in comparison with the mild OA (moderate OA RR = 2.46, 95% CI 2.08-2.92, p < .001; severe OA RR = 5.60, 95% CI 4.79-6.55, p < .001). For dogs with severe OA, there was an increased likelihood that their SBCs were larger than SBCs from dogs with mild OA (OR = 1.056, 95% CI 1.012-1.101, p = .012). No SBCs were observed in elbows without radiographic evidence of OA. CONCLUSION: Subchondral bone cysts were identified as a feature of radiographic elbow osteoarthritis in Labrador retrievers, and their number and size were indicative of the presence and severity of radiographic elbow OA. CLINICAL SIGNIFICANCE: Subchondral bone cysts are a potential imaging biomarker for quantitative assessment for canine OA.

Moving Beyond the Limits of Detection: The Past, the Present, and the Future of Diagnostic Imaging in Canine Osteoarthritis.

Osteoarthritis (OA) is the most common orthopedic condition in dogs, characterized as the chronic, painful end-point of a synovial joint with limited therapeutic options other than palliative pain control or surgical salvage. Since the 1970s, radiography has been the standard-of-care for the imaging diagnosis of OA, despite its known limitations. As newer technologies have been developed, the limits of detection have lowered, allowing for the identification of earlier stages of OA. Identification of OA at a stage where it is potentially reversible still remains elusive, however, yet there is hope that newer technologies may be able to close this gap. In this article, we review the changes in the imaging of canine OA over the past 50 years and give a speculative view on future innovations which may provide for earlier identification, with the ultimate goal of repositioning the limit of detection to cross the threshold of this potentially reversible disease.

Troubled spots: Human impacts constrain the density of an apex predator inside protected areas.

Effective conservation requires understanding the processes that determine population outcomes. Too often, we assume that protected areas conserve wild populations despite evidence that they frequently fail to do so. Without large-scale studies, however, we cannot determine what relationships are the product of localized conditions versus general patterns that inform conservation more broadly. Leopards' (Panthera pardus) basic ecology is well studied but little research has investigated anthropogenic effects on leopard density at broad scales. We investigated the drivers of leopard density among 27 diverse protected areas in northeastern South Africa to understand what conditions facilitate abundant populations. We formulated 10 working hypotheses that considered the relative influence of bottom-up biological factors and top-down anthropogenic factors on leopard density. Using camera-trap survey data, we fit a multi-session spatial capture-recapture model with inhomogenous density for each hypothesis and evaluated support using an information theoretic approach. The four supported hypotheses indicated that leopard density is primarily limited by human impacts, but that habitat suitability and management conditions also matter. The proportion of camera stations that recorded domestic animals, a proxy for the extent of human impacts and protected area effectiveness, was the only predictor variable present in all four supported models. Protected areas are the cornerstone of large felid conservation, but only when the human-wildlife interface is well managed and protected areas shelter wildlife populations from anthropogenic impacts. To ensure the long-term abundance of large carnivore populations, reserve managers should recognize the ineffectiveness of "paper parks" and promote contiguous networks of protected areas that offer leopards and other large mammal populations greater space and reduced human impacts.

Ecological and genomic analyses of candidate phylum WPS-2 bacteria in an unvegetated soil.

Members of the bacterial candidate phylum WPS-2 (or Eremiobacterota) are abundant in several dry, bare soil environments. In a bare soil deposited by an extinct iron-sulfur spring, we found that WPS-2 comprised up to 24% of the bacterial community and up to 108 cells per g of soil based on 16S rRNA gene sequencing and quantification. A single genus-level cluster (Ca. Rubrimentiphilum) predominated in bare soils but was less abundant in adjacent forest. Nearly complete genomes of Ca. Rubrimentiphilum were recovered as single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs). Surprisingly, given the abundance of WPS-2 in bare soils, the genomes did not indicate any capacity for autotrophy, phototrophy, or trace gas metabolism. Instead, they suggest a predominantly aerobic organoheterotrophic lifestyle, perhaps based on scavenging amino acids, nucleotides, and complex oligopeptides, along with lithotrophic capacity on thiosulfate. Network analyses of the entire community showed that some species of Chloroflexi, Actinobacteria, and candidate phylum AD3 (or Dormibacterota) co-occurred with Ca. Rubrimentiphilum and may represent ecological or metabolic partners. We propose that Ca. Rubrimentiphilum act as efficient heterotrophic scavengers. Combined with previous studies, these data suggest that the phylum WPS-2 includes bacteria with diverse metabolic capabilities.

Stable Isotope and Metagenomic Profiling of a Methanogenic Naphthalene-Degrading Enrichment Culture.

Polycyclic aromatic hydrocarbons (PAH) such as naphthalene are widespread, recalcitrant pollutants in anoxic and methanogenic environments. A mechanism catalyzing PAH activation under methanogenic conditions has yet to be discovered, and the microbial communities coordinating their metabolism are largely unknown. This is primarily due to the difficulty of cultivating PAH degraders, requiring lengthy incubations to yield sufficient biomass for biochemical analysis. Here, we sought to characterize a new methanogenic naphthalene-degrading enrichment culture using DNA-based stable isotope probing (SIP) and metagenomic analyses. 16S rRNA gene sequencing of fractionated DNA pinpointed an unclassified Clostridiaceae species as a putative naphthalene degrader after two months of SIP incubation. This finding was supported by metabolite and metagenomic evidence of genes predicted to encode for enzymes facilitating naphthalene carboxylic acid CoA-thioesterification and degradation of an unknown arylcarboxyl-CoA structure. Our findings also suggest a possible but unknown role for Desulfuromonadales in naphthalene degradation. This is the first reported functional evidence of PAH biodegradation by a methanogenic consortium, and we envision that this approach could be used to assess carbon flow through other slow growing enrichment cultures and environmental samples.

Identification of a gene involved in the biosynthesis pathway of the terminal sugar of the archaellin N-linked tetrasaccharide in Methanococcus maripaludis.

In Methanococcus maripaludis, the three archaellins which comprise the archaellum are modified at multiple sites with an N-linked tetrasaccharide with the structure of Sug-4-ß-ManNAc3NAmA6Thr-4-ß-GlcNAc3NAcA-3-ß-GalNAc, where Sug is a unique sugar (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-L-erythro-hexos-5-ulo-1,5-pyranose, so far found exclusively in this species. In this study, a six-gene cluster mmp1089-1094, neighboring one of the genomic regions already known to contain genes involved with the archaellin N-glycosylation pathway, was examined for its potential involvement in the archaellin N-glycosylation or sugar biosynthesis pathway. The co-transcription of these six genes was demonstrated by RT-PCR. Mutants carrying an in-frame deletion in mmp1090, mmp1091 or mmp1092 were successfully generated. The Δmmp1090 deletion mutant was archaellated when examined by electron microscopy and mass spectrometry analysis of purified archaella showed that the archaellins were modified with a truncated N-glycan in which the terminal sugar residue and the threonine linked to the third sugar residue were missing. Both gene annotation and bioinformatic analyses indicate that MMP1090 is a UDP-glucose 4-epimerase, suggesting that the unique terminal sugar of the archaellin N-glycan might be synthesised from UDP-glucose or UDP-N-acetylglucosamine with an essential early step in synthesis catalysed by MMP1090. In contrast, no detectable phenotype related to archaellin glycosylation was observed in mutants deleted for either mmp1091 or mmp1092 while attempts to delete mmp1089, mmp1093 and mmp1094 were unsuccessful. Based on its demonstrated involvement in the archaellin N-glycosylation pathway, we designated mmp1090 as aglW.

Stable-Isotope Probing Identifies Uncultured Planctomycetes as Primary Degraders of a Complex Heteropolysaccharide in Soil.

The exopolysaccharides (EPSs) produced by some bacteria are potential growth substrates for other bacteria in soil. We used stable-isotope probing (SIP) to identify aerobic soil bacteria that assimilated the cellulose produced by Gluconacetobacter xylinus or the EPS produced by Beijerinckia indica. The latter is a heteropolysaccharide comprised primarily of l-guluronic acid, d-glucose, and d-glycero-d-mannoheptose. (13)C-labeled EPS and (13)C-labeled cellulose were purified from bacterial cultures grown on [(13)C]glucose. Two soils were incubated with these substrates, and bacteria actively assimilating them were identified via pyrosequencing of 16S rRNA genes recovered from (13)C-labeled DNA. Cellulose C was assimilated primarily by soil bacteria closely related (93 to 100% 16S rRNA gene sequence identities) to known cellulose-degrading bacteria. However, B. indica EPS was assimilated primarily by bacteria with low identities (80 to 95%) to known species, particularly by different members of the phylum Planctomycetes. In one incubation, members of the Planctomycetes made up >60% of all reads in the labeled DNA and were only distantly related (<85% identity) to any described species. Although it is impossible with SIP to completely distinguish primary polysaccharide hydrolyzers from bacteria growing on produced oligo- or monosaccharides, the predominance of Planctomycetes suggested that they were primary degraders of EPS. Other bacteria assimilating B. indica EPS included members of the Verrucomicrobia, candidate division OD1, and the Armatimonadetes. The results indicate that some uncultured bacteria in soils may be adapted to using complex heteropolysaccharides for growth and suggest that the use of these substrates may provide a means for culturing new species.

Identification of genes involved in the biosynthesis of the third and fourth sugars of the Methanococcus maripaludis archaellin N-linked tetrasaccharide.

N-glycosylation is a protein posttranslational modification found in all three domains of life. Many surface proteins in Archaea, including S-layer proteins, pilins, and archaellins (archaeal flagellins) are known to contain N-linked glycans. In Methanococcus maripaludis, the archaellins are modified at multiple sites with an N-linked tetrasaccharide with the structure Sug-1,4-ß-ManNAc3NAmA6Thr-1,4-ß-GlcNAc3NAcA-1,3-ß-GalNAc, where Sug is the unique sugar (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-l-erythro-hexos-5-ulo-1,5-pyranose. In this study, four genes--mmp1084, mmp1085, mmp1086, and mmp1087--were targeted to determine their potential involvement of the biosynthesis of the sugar components in the N-glycan, based on bioinformatics analysis and proximity to a number of genes which have been previously demonstrated to be involved in the N-glycosylation pathway. The genes mmp1084 to mmp1087 were shown to be cotranscribed, and in-frame deletions of each gene as well as a Δmmp1086Δmmp1087 double mutant were successfully generated. All mutants were archaellated and motile. Mass spectrometry examination of purified archaella revealed that in Δmmp1084 mutant cells, the threonine linked to the third sugar of the glycan was missing, indicating a putative threonine transferase function of MMP1084. Similar analysis of the archaella of the Δmmp1085 mutant cells demonstrated that the glycan lacked the methyl group at the C-5 position of the terminal sugar, indicating that MMP1085 is a methyltransferase involved in the biosynthesis of this unique sugar. Deletion of the remaining two genes, mmp1086 and mmp1087, either singularly or together, had no effect on the structure of the archaellin N-glycan. Because of their demonstrated involvement in the N-glycosylation pathway, we designated mmp1084 as aglU and mmp1085 as aglV.

Identification of genes involved in the acetamidino group modification of the flagellin N-linked glycan of Methanococcus maripaludis.

N-linked glycosylation of protein is a posttranslational modification found in all three domains of life. The flagellin proteins of the archaeon Methanococcus maripaludis are known to be modified with an N-linked tetrasaccharide consisting of N-acetylgalactosamine (GalNAc), a diacetylated glucuronic acid (GlcNAc3NAc), an acetylated and acetamidino-modified mannuronic acid with a substituted threonine group (ManNAc3NAmA6Thr), and a novel terminal sugar residue [(5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-L-erythro-hexos-5-ulo-1,5-pyranose]. To identify genes involved in biosynthesis of the component sugars of this glycan, three genes, mmp1081, mmp1082, and mmp1083, were targeted for in-frame deletion, based on their annotation and proximity to glycosyltransferase genes known to be involved in assembly of the glycan. Mutants carrying a deletion in any of these three genes remained flagellated and motile. A strain with a deletion of mmp1081 had lower-molecular-mass flagellins in Western blots. Mass spectrometry of purified flagella revealed a truncated glycan with the terminal sugar absent and the threonine residue and the acetamidino group missing from the third sugar. No glycan modification was seen in either the Δmmp1082 or Δmmp1083 mutant grown in complex Balch III medium. However, a glycan identical to the Δmmp1081 glycan was observed when the Δmmp1082 or Δmmp1083 mutant was grown under ammonia-limited conditions. We hypothesize that MMP1082 generates ammonia and tunnels it through MMP1083 to MMP1081, which acts as the amidotransferase, modifying the third sugar residue of the M. maripaludis glycan with the acetamidino group.

Biosynthesis and role of N-linked glycosylation in cell surface structures of archaea with a focus on flagella and s layers.

The genetics and biochemistry of the N-linked glycosylation system of Archaea have been investigated over the past 5 years using flagellins and S layers as reporter proteins in the model organisms, Methanococcus voltae, Methanococcus maripaludis, and Haloferax volcanii. Structures of archaeal N-linked glycans have indicated a variety of linking sugars as well as unique sugar components. In M. voltae, M. maripaludis, and H. volcanii, a number of archaeal glycosylation genes (agl) have been identified by deletion and complementation studies. These include many of the glycosyltransferases and the oligosaccharyltransferase needed to assemble the glycans as well as some of the genes encoding enzymes required for the biosynthesis of the sugars themselves. The N-linked glycosylation system is not essential for any of M. voltae, M. maripaludis, or H. volcanii, as demonstrated by the successful isolation of mutants carrying deletions in the oligosaccharyltransferase gene aglB (a homologue of the eukaryotic Stt3 subunit of the oligosaccharyltransferase complex). However, mutations that affect the glycan structure have serious effects on both flagellation and S layer function.

S-layer glycoproteins and flagellins: reporters of archaeal posttranslational modifications.

Many archaeal proteins undergo posttranslational modifications. S-layer proteins and flagellins have been used successfully to study a variety of these modifications, including N-linked glycosylation, signal peptide removal and lipid modification. Use of these well-characterized reporter proteins in the genetically tractable model organisms, Haloferax volcanii, Methanococcus voltae and Methanococcus maripaludis, has allowed dissection of the pathways and characterization of many of the enzymes responsible for these modifications. Such studies have identified archaeal-specific variations in signal peptidase activity not found in the other domains of life, as well as the enzymes responsible for assembly and biosynthesis of novel N-linked glycans. In vitro assays for some of these enzymes have already been developed. N-linked glycosylation is not essential for either Hfx. volcanii or the Methanococcus species, an observation that allowed researchers to analyze the role played by glycosylation in the function of both S-layers and flagellins, by generating mutants possessing these reporters with only partial attached glycans or lacking glycan altogether. In future studies, it will be possible to consider questions related to the heterogeneity associated with given modifications, such as differential or modulated glycosylation.