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Microbiology and Immunology Jun 2010Bovine digital epidermitis involves different pathologies, including PDD, interdigital dermatitis, and foot rot. Bacteriological and molecular biological studies suggest...
Bovine digital epidermitis involves different pathologies, including PDD, interdigital dermatitis, and foot rot. Bacteriological and molecular biological studies suggest that these are multimicrobial infections. During our study on the isolation of treponemes from biopsies of PDD, colonies producing black pigment were isolated frequently from the primary cultures, suggesting that Porphyromonas species were present. Moreover, 16S rRNA genes of Fusobacterium necrophorum and Porphyromonas levii-like species were detected in the lesions. We therefore determined whether an immunological response could be elicited by a P. levii-like organism isolated from a PDD lesion, as well as two subspecies of F. necrophorum in the sera from cows with and without PDD. A total of 151 serum samples were collected from 85 cows with PDD lesions and 33 cows without lesions on 12 PDD-positive farms and from 33 cows on two PDD-free farms. ELISA data showed that IgG antibody levels against antigens of P. levii-like species and F. necrophorum subsp. necrophorum were significantly higher in cows on PDD-positive farms than in cows on PDD-free farms, regardless of the presence of PDD lesions in the cows on the PDD-positive farms. However, F. necrophorum subsp. funduliforme was present at low levels in both groups. The ELISA results were confirmed by western blot analysis. Furthermore, antigens of these bacteria were detected in PDD-biopsy sections examined by immunohistochemical staining. F. necrophorum subsp. necrophorum and P. levii-like species may be involved in the pathogenesis of PDD.
Topics: Animals; Antibodies, Bacterial; Antigens, Bacterial; Cattle; Cattle Diseases; Dermatitis; Enzyme-Linked Immunosorbent Assay; Female; Foot Dermatoses; Fusobacterium necrophorum; Immunohistochemistry; Porphyromonas; Rabbits
PubMed: 20536732
DOI: 10.1111/j.1348-0421.2010.00220.x -
Journal of Dairy Science Dec 2019Until 2010, our knowledge of the uterine microbiome in cows that developed uterine disease relied almost exclusively on culture-dependent studies and mostly included... (Review)
Review
Until 2010, our knowledge of the uterine microbiome in cows that developed uterine disease relied almost exclusively on culture-dependent studies and mostly included cows with clinical endometritis (i.e., with purulent uterine discharge). Those studies consistently found a strong positive correlation between Trueperella pyogenes and clinical endometritis, whereas other pathogens such as Escherichia coli, Fusobacterium necrophorum, Prevotella melaninogenica, and Bacteroides spp. were also commonly cocultured. In contrast, Streptococcus spp., Staphylococcus spp., and Bacillus spp. were usually isolated from healthy cows. Starting in 2010, culture-independent studies using PCR explored the microbiome of cows with metritis and clinical endometritis, and observed that E. coli was a pioneer pathogen that predisposed cows to infection with F. necrophorum, which was strongly associated with metritis, and to infection with T. pyogenes, which was strongly associated with clinical endometritis. Starting in 2011, culture-independent studies using metagenomic sequencing expanded our knowledge of the uterine microbiome. It has been shown that cows have bacteria in the uterus even before calving, they have an established uterine microbiome within 20 min of calving, and that the microbiome structure is identical between cows that develop metritis and healthy cows until 2 d postpartum, after which the bacterial structure of cows that developed metritis deviates in favor of greater relative abundance of Bacteroidetes and Fusobacteria and lesser relative abundance of Proteobacteria and Tenericutes. The shift in the uterine microbiome in cows that develop metritis is characterized by a loss of heterogeneity and a decrease in bacterial richness. At the genus level, Bacteroides, Porphyromonas, and Fusobacterium have the strongest association with metritis. At the species level, we observed that Bacteroides pyogenes, Porphyromonas levii, and Helcococcus ovis were potential emerging uterine pathogens. Finally, we have shown that the hematogenous route is a viable route of uterine infection with uterine pathogens. Herein, we propose that metritis is associated with a dysbiosis of the uterine microbiota characterized by decreased richness, and an increase in Bacteroidetes and Fusobacteria, particularly Bacteroides, Porphyromonas, and Fusobacterium.
Topics: Animals; Bacteria; Bacteroidetes; Cattle; Cattle Diseases; Dysbiosis; Endometritis; Female; Fusobacteria; Microbiota; Polymerase Chain Reaction; Postpartum Period; Uterine Diseases; Uterus
PubMed: 31587913
DOI: 10.3168/jds.2019-17106 -
Journal of Clinical Microbiology Sep 2005Porphyromonas levii is an anaerobic, pigmented gram-negative bacillus originally isolated from bovine rumen. We describe 58 human clinical strains of P. levii-like...
Porphyromonas levii is an anaerobic, pigmented gram-negative bacillus originally isolated from bovine rumen. We describe 58 human clinical strains of P. levii-like organisms, isolated from various human clinical specimens that are phenotypically similar to the type strain of P. levii, a rumen isolate (ATCC 29147). Our biochemical, comparative 16S rRNA sequence analyses, and DNAlpha-DNA relatedness studies indicate that the human P. levii-like organisms are similar to each other but genetically different from the P. levii type strain isolated from bovine rumen. We therefore propose the name Porphyromonas somerae to encompass the human P. levii-like organisms. P. somerae was predominantly isolated from patients with chronic skin and soft tissue or bone infections, especially in the lower extremities.
Topics: Animals; Bacterial Typing Techniques; Bacteroidaceae Infections; DNA, Bacterial; DNA, Ribosomal; Humans; Molecular Sequence Data; Nucleic Acid Hybridization; Phenotype; Porphyromonas; RNA, Ribosomal, 16S; Sequence Analysis, DNA
PubMed: 16145091
DOI: 10.1128/JCM.43.9.4455-4459.2005 -
Biofilm Dec 2022Host immune cells and clinical interventions often fail to eradicate biofilm-mediated infections, resulting in chronic inflammation. The role of the biofilm...
Host immune cells and clinical interventions often fail to eradicate biofilm-mediated infections, resulting in chronic inflammation. The role of the biofilm three-dimensional structure in this tolerant phenotype has been studied extensively; however, the impact of small molecules released from biofilm-bacteria in modulating host immune function is less well understood. A model of mixed-species biofilms composed of and was developed to evaluate bovine neutrophil responses to bioactive molecules released from either biofilm or planktonic bacteria. We hypothesized that different soluble extracellular factors (ECFs) would be released from planktonic and biofilm bacteria, resulting in altered neutrophil function. Neutrophils exposed to ECFs from planktonic bacteria showed significantly elevated levels of reactive oxygen species (ROS). In contrast, biofilm components from these same species of bacteria failed to induce such a response. Size-exclusion filtration of ECFs revealed that the bioactive molecule causing neutrophil ROS responses was below 3 kDa. Intensive heat, nuclease, lipase, or protease treatments of the <3 kDa fractions did not alter neutrophil functional responses. Protoporphyrin IX (PPIX) is an important heme precursor and growth requirement for many anaerobes. species can accumulate environmental PPIX at the cell surface as a strategy to protect the bacteria from oxidative stress and we investigated the direct interaction of bovine neutrophils with PPIX. In the present study, evidence suggests that the accumulation of protoporphyrin in these dual-species biofilm ECFs attenuates neutrophil ROS production and chemotaxis. The diminished neutrophil response to biofilm ECFs via the action of PPIX may represent a biofilm immune-evasion strategy that could assist in explaining the ineffectual host clearance of biofilm-mediated infections involving these bacteria.
PubMed: 36478961
DOI: 10.1016/j.bioflm.2022.100095 -
Canadian Journal of Veterinary Research... Apr 1999Acute interdigital phlegmon (AIP) is a commonly occurring anaerobic bacterial infection in cattle. This study examined in vitro the interaction of bovine...
Acute interdigital phlegmon (AIP) is a commonly occurring anaerobic bacterial infection in cattle. This study examined in vitro the interaction of bovine polymorphonuclear granulocytic neutrophils (PMN) from blood with bacterial species involved in AIP. Polymorphonuclear neutrophils were purified from whole bovine blood, exposed to one of the three putative etiologic agents of AIP and comparatively assessed for phagocytosis using light microscopy. Fusobacterium necrophorum and Prevotella intermedia were effectively phagocytosed by PMN, but Porphyromonas levii was phagocytosed significantly less effectively by PMN. The effect of high titre anti-P. levii bovine serum on antibody-mediated phagocytosis by PMN was also evaluated. High titre serum increased the efficiency of phagocytosis of P. levii by bovine PMN. This was independent of heat labile complement factors. Antibodies specific for P. levii were assessed for protease activity capable of cleaving bovine immunoglobulins (IgG, IgG1, IgG2, and IgM). Partially purified supernatant from broth cultures of P. levii were incubated with biotinylated immunoglobulins (Igs). Samples were taken from times 0 to 72 h and examined using SDS-PAGE followed by Western blot analysis. Streptavidin-alkaline phosphatase and NBT-BCIP were used to visualize the Igs for heavy and light chains as well as lower molecular weight fragments of these glycoproteins. Porphyromonas levii produced an immunoglobulin protease which readily cleaved bovine IgG into fragments, but did not act against IgM. Specifically, the enzyme may be a significant virulence factor as it may act to neutralize the antibodies demonstrated necessary for effective PMN-mediated phagocytosis.
Topics: Animals; Bacterial Proteins; Biotinylation; Cattle; Fusobacterium; Immunoglobulin G; Immunoglobulin M; Male; Metalloendopeptidases; Neutrophils; Phagocytosis; Porphyromonas; Prevotella; Substrate Specificity
PubMed: 10369568
DOI: No ID Found -
American Journal of Veterinary Research May 2002To examine the host response toward Porphyromonas levii, by evaluating chemotaxis, phagocytosis, and oxidative burst of bovine macrophages in vitro.
OBJECTIVE
To examine the host response toward Porphyromonas levii, by evaluating chemotaxis, phagocytosis, and oxidative burst of bovine macrophages in vitro.
SAMPLE POPULATION
Cultured bovine macrophages obtained from monocytes harvested from blood samples of 15 Holstein steers. Porphyromonas levii was isolated from the foot rot lesion of an acutely affected feedlot steer.
PROCEDURE
Monocytes were cultured for macrophage differentiation over 7 days. Porphyromonas levii was cultured in strict anaerobic conditions for experimentation. Chemotaxis was evaluated by quantifying macrophage migration toward P. levii in Boyden chambers. Phagocytosis was assessed by quantification of macrophages engulfing P. levii following incubation with or without anti-P. levii serum or purified IgG. Oxidative burst was measured by use of the nitroblue tetrazolium reduction assay.
RESULTS
Chemotaxis toward P. levii was not significantly different from control values at any of the tested bacterial concentrations. Phagocytosis of P. levii was approximately 10% at a 10:1 bacterium to macrophage ratio and did not change significantly over time. When higher proportions of P. levii were tested for phagocytosis, the 1,000:1 bacterium to macrophage ratio had a significant increase, compared with the 10:1 test group. Opsonization of P. levii with high-titeranti-P. levii serum or anti-P. levii IgG produced a significant increase in macrophage phagocytosis. Oxidative production significantly increased compared with control in the 1,000:1 test group only.
CONCLUSIONS AND CLINICAL RELEVANCE
Porphyromonas levii may evade host detection by decreased chemotaxis, phagocytosis, and oxidative burst by macrophages. Acquired immunity may be beneficial for clearance of P. levii in foot rot lesions in cattle.
Topics: Animals; Antibodies, Bacterial; Cattle; Cattle Diseases; Chemotaxis; Foot Rot; Indicators and Reagents; Macrophages; Male; Microscopy, Electron; N-Formylmethionine Leucyl-Phenylalanine; Nitroblue Tetrazolium; Phagocytosis; Pseudomonas; Pseudomonas Infections; Respiratory Burst
PubMed: 12013480
DOI: 10.2460/ajvr.2002.63.757 -
MSystems Aug 2021Bovine digital dermatitis (DD) is a skin disorder that is a significant cause of infectious lameness in cattle around the world. However, very little is known about the...
Bovine digital dermatitis (DD) is a skin disorder that is a significant cause of infectious lameness in cattle around the world. However, very little is known about the etiopathogenesis of the disease and the microbiota associated with DD in beef cattle. In this study, we provide a comprehensive characterization of DD and healthy skin microbiota of feedlot beef cattle. We also developed and validated a novel multiplex quantitative PCR (qPCR) assay to quantify the distribution of DD-associated bacterial species across DD lesion stages. We determined the DD-associated microbiota with deep amplicon sequencing of the V3-V4 hypervariable region of the 16S rRNA gene, followed by the application of novel and existing qPCR assays to quantify species distributions of Treponema, , , and across lesion stages. Deep amplicon sequencing revealed that Treponema, , , and were associated with DD lesions. Culturing of DD biopsy specimens identified Porphyromonas levii, Bacteroides pyogenes, and two spp. within DD lesions. Using species-specific qPCR on DD lesion DNA, we identified P. levii in 100% of active lesion stages. Early-stage lesions were particularly associated with Treponema medium, T. phagedenis, and . This study suggests a core DD microbial group consisting of species of Treponema, , , and , which may be closely tied with the etiopathogenesis of DD. Further characterizations of these species and spp. are necessary to understand the microbial factors involved in DD pathogenesis, which will help elucidate DD etiology and facilitate more targeted and effective mitigation and treatment strategies. Previous work, primarily in dairy cattle, has identified various taxa associated with digital dermatitis (DD) lesions. However, there is a significant gap in our knowledge of DD microbiology in beef cattle. In addition, characterization of bacteria at the species level in DD lesions is limited. In this study, we provide a framework for the accurate and reproducible quantification of major DD-associated bacterial species from DNA samples. Our findings support DD as a polymicrobial infection, and we identified a variety of bacterial species spanning multiple genera that are consistently associated with DD lesions. The DD-associated microbiota identified in this study may be capable of inducing the formation and progression of DD lesions and thus should be primary targets in future DD pathogenesis studies.
PubMed: 34313462
DOI: 10.1128/mSystems.00708-21 -
Infection and Immunity Sep 2016Periodontitis is a significant problem in companion animals, and yet little is known about the disease-associated microbiota. A major virulence factor for the human...
Periodontitis is a significant problem in companion animals, and yet little is known about the disease-associated microbiota. A major virulence factor for the human periodontal pathogen Porphyromonas gingivalis is the lysyl- and arginyl-specific proteolytic activity of the gingipains. We screened several Porphyromonas species isolated from companion animals-P. asaccharolytica, P. circumdentaria, P. endodontalis, P. levii, P. gulae, P. macacae, P. catoniae, and P. salivosa-for Lys- and Arg-specific proteolytic activity and compared the epithelial and macrophage responses and induction of alveolar bone resorption of the protease active species to that of Porphyromonas gingivalis Only P. gulae exhibited Lys-and Arg-specific proteolytic activity. The genes encoding the gingipains (RgpA/B and Kgp) were identified in the P. gulae strain ATCC 51700 and all publicly available 12 draft genomes of P. gulae strains. P. gulae ATCC 51700 induced levels of alveolar bone resorption in an animal model of periodontitis similar to those in P. gingivalis W50 and exhibited a higher capacity for autoaggregation and binding to oral epithelial cells with induction of apoptosis. Macrophages (RAW 264.7) were found to phagocytose P. gulae ATCC 51700 and the fimbriated P. gingivalis ATCC 33277 at similar levels. In response to P. gulae ATCC 51700, macrophages secreted higher levels of cytokines than those induced by P. gingivalis ATCC 33277 but lower than those induced by P. gingivalis W50, except for the interleukin-6 response. Our results indicate that P. gulae exhibits virulence characteristics similar to those of the human periodontal pathogen P. gingivalis and therefore may play a key role in the development of periodontitis in companion animals.
Topics: Alveolar Bone Loss; Animals; Bacteroidaceae Infections; Cell Line; Disease Models, Animal; Epithelial Cells; Female; Humans; Interleukin-6; Macrophages; Mice; Mice, Inbred BALB C; Periodontitis; Porphyromonas; Porphyromonas gingivalis; Virulence; Virulence Factors
PubMed: 27354442
DOI: 10.1128/IAI.01500-15 -
Canadian Journal of Veterinary Research... Apr 2002The objective was to evaluate the pro-inflammatory response of bovine macrophages towards Porphyromonas levii, an etiologic agent of acute interdigital phlegmon, by...
The objective was to evaluate the pro-inflammatory response of bovine macrophages towards Porphyromonas levii, an etiologic agent of acute interdigital phlegmon, by evaluating the mRNA expression of tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), and interleukin 8 (IL-8). Bovine macrophages detect the presence of bacteria, such as P. levii, and respond by upregulating transcription of the genes for pro-inflammatory cytokines TNF-alpha and IL-1beta in addition to the neutrophil chemoattractant IL-8. Monocytes were isolated from blood obtained from Holstein steers. These cells were cultured and differentiated into macrophages over 7 d, followed by exposure to P. levii, Escherichia coli lipopolysaccharide (LPS), or tissue culture medium for 1, 1.5, 2, 4, 6, 8,12, or 24 h. Total RNA was extracted, and reverse transcriptase polymerase chain reaction was conducted to examine the presence of TNF-alpha, IL-1beta, or IL-8 mRNA. Products were visualized on agarose gels to determine the presence or absence of cytokine mRNA amplified DNA. Bovine macrophages, when exposed to P. levii or E. coli LPS, produced mRNA for TNF-alpha, IL-1beta, and IL-8. Expression of all 3 cytokines was higher in the P. levii and LPS-exposed macrophages at all time points examined, compared with tissue culture medium-treated cells. Expression of these cytokines by macrophages is likely directly involved in orchestration of the immune response, and particularly in neutrophil recruitment to affected tissues in acute interdigital phlegmon.
Topics: Animals; Bacteroidaceae Infections; Cattle; Cattle Diseases; Cells, Cultured; Cytokines; Interleukin-1; Interleukin-8; Lipopolysaccharides; Macrophage Activation; Macrophages; Male; Porphyromonas; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Tumor Necrosis Factor-alpha
PubMed: 11989740
DOI: No ID Found -
Journal of Bacteriology Feb 1994The phylogenetic structure of the bacteroides subgroup of the cytophaga-flavobacter-bacteroides (CFB) phylum was examined by 16S rRNA sequence comparative analysis.... (Comparative Study)
Comparative Study
The phylogenetic structure of the bacteroides subgroup of the cytophaga-flavobacter-bacteroides (CFB) phylum was examined by 16S rRNA sequence comparative analysis. Approximately 95% of the 16S rRNA sequence was determined for 36 representative strains of species of Prevotella, Bacteroides, and Porphyromonas and related species by a modified Sanger sequencing method. A phylogenetic tree was constructed from a corrected distance matrix by the neighbor-joining method, and the reliability of tree branching was established by bootstrap analysis. The bacteroides subgroup was divided primarily into three major phylogenetic clusters which contained most of the species examined. The first cluster, termed the prevotella cluster, was composed of 16 species of Prevotella, including P. melaninogenica, P. intermedia, P. nigrescens, and the ruminal species P. ruminicola. Two oral species, P. zoogleoformans and P. heparinolytica, which had been recently placed in the genus Prevotella, did not fall within the prevotella cluster. These two species and six species of Bacteroides, including the type species B. fragilis, formed the second cluster, termed the bacteroides cluster. The third cluster, termed the porphyromonas cluster, was divided into two subclusters. The first contained Porphyromonas gingivalis, P. endodontalis, P. asaccharolytica, P. circumdentaria, P. salivosa, [Bacteroides] levii (the brackets around genus are used to indicate that the species does not belong to the genus by the sensu stricto definition), and [Bacteroides] macacae, and the second subcluster contained [Bacteroides] forsythus and [Bacteroides] distasonis. [Bacteroides] splanchnicus fell just outside the three major clusters but still belonged within the bacteroides subgroup. With few exceptions, the 16 S rRNA data were in overall agreement with previously proposed reclassifications of species of Bacteroides, Prevotella, and Porphyromonas. Suggestions are made to accommodate those species which do not fit previous reclassification schemes.
Topics: Bacteroides; Base Sequence; DNA Primers; Molecular Sequence Data; Phylogeny; Porphyromonas; RNA, Ribosomal, 16S; Sequence Alignment; Sequence Homology, Nucleic Acid
PubMed: 8300528
DOI: 10.1128/jb.176.3.725-732.1994