Fusobacterium Necrophorum Research Articles

ETIOLOGICAL STRUCTURE OF POSTPARTUM METRITIS IN DAIRY COWS UNDER VARIOUS TECHNOLOGIES OF THEIR MANAGEMENT

The article presents the data on the frequency of isolation of postpartum metritis pathogens in dairy cows. The microflora of cervical-vaginal mucus and exudate (n=150) was isolated in the form of monocultures in 44.7% of cases, in the form of associations - in 55.3% of cases. E. coli was most often isolated, which allows us to consider it one of the main causative agents of this disease. At enterprises with loose keeping, the microbial landscape of the genital tract of cows with postpartum metritis had a wider range of pathogens, which was represented by numerous associations. Fusobacterium necrophorum and Actinomyces pyogenes were also isolated from these animals, contributing to a more severe course of the inflammatory process with its manifestation.

Monitoramento de lesões periodontais e seus efeitos na gestação: aspectos microbiológicos da cavidade bucal e líquido amniótico de ovelhas gestantes

ABSTRACT: Periodontitis affects the teeth supporting tissues, leading to tooth loss and damage to animal health. Evidence in humans suggests that oral microorganisms spread systemically, increasing the risk of pregnancy disorders such as miscarriage, prematurity, and low birth weight. This study aimed to verify whether periodontopathogenic microorganisms reach the transplacental unit, culminating in problems in pregnant ewes. After analyzing the oral cavity, 10 clinically healthy pregnant ewes (OGCH group) and 10 pregnant ewes with periodontitis (OGP group) were selected. The subgingival biofilm was collected for the polymerase chain reaction (PCR) test and amniotic fluid for both the PCR and interleukin (IL) analysis. Peripheral blood was collected for complete blood count, and analyses of IL-6, IL1-β, and tumor necrosis factor-α were performed. Placental fragments were collected to assess the inflammatory changes using optical microscopy. After giving birth, both the ewes and their lambs were weighed. On clinical examination, a positive correlation between bleeding and suppuration (correlation index - CI=0.54), suppuration and marginal gingivitis (CI=0.34), and marginal gingivitis and edema (CI=0.54) was observed. The weights of the ewes (p=0.013) and their respective lambs (p=0.04) in the OGP group were lower than those of their OGCH group counterparts. The hematological analysis revealed that the OGP group ewes showed a slight increase in the mean corpuscular volume (p=0.2447), segmented cells (p=0.3375), and eosinophils (p=0.3823) when compared with the OGCH group ewes, without a statistical difference. Regarding the microorganisms detected in the oral cavity, there was a significant difference between the occurrence of periodontal pockets and the presence of Fusobacterium necrophorum (p=0.0328), Porphyromonas asaccharolytica (p=0.0392), and the Mollicutes class (p=0.0352). Staphylococcus genus (p=0.9107) and Archaea domain (p=0.7245) were detected in the amniotic samples of both groups, without a significant difference, whereas P. asaccharolytica (p=0.2685) was only detected in one sample in the OGCH group. The expression of cytokine IL-6 in the OGP group differed significantly between the prepartum and postpartum periods (p=0.0039); moreover, it differed significantly in the postpartum period between the OGCH and OGP groups (p=0.0198). Histological examination showed a higher percentage of placental changes in the OGP group (70%) than in the OGCH group, such as the presence of macrophages, neutrophils, plasma cells, and multifocal areas of calcification. These results do not corroborate the hypothesis of dissemination of oral microorganisms to the placental unit, suggesting that it constitutes placental isolation in sheep.

Dynamics and Diversity of Intrauterine Anaerobic Microbiota in Dairy Cows with Clinical and Subclinical Endometritis

The aim of the study was to characterize the dynamics of anaerobic cultivable postpartum microbiota in the uterus of dairy cows. In total, 122 dairy cows were enrolled and sampled on day 0 (day of calving) and on days 3, 9, 15, 21, and 28 postpartum (pp). Samples were cultivated anaerobically and analyzed by MALDI-TOF MS. In total, 1858 isolates were recovered. The most prevalent facultative anaerobic genera were Trueperella (27.8%), Streptococcus (25.4%), and Escherichia (13.1%). The most prevalent obligate anaerobes were Peptoniphilus (9.3%), Bacteroides (3.3%), and Clostridium (2.4%). The microbial communities were highly dynamic and diverse. On the animal level, Trueperella pyogenes on day 21 and 28 pp was associated with clinical endometritis, and E. coli on day 21 pp was associated with subclinical endometritis. The occurrence of Streptococcus pluranimalium on day 28 was related to uterine health. The presence of T. pyogenes, Streptococcus, and Peptoniphilus was significantly associated with an increased risk for purulent vaginal discharge. Primiparous cows showed a higher prevalence of T. pyogenes, Fusobacterium necrophorum, Porphyromonas levii, and Peptoniphilus spp. than multiparous cows but were not more susceptible to uterine diseases. This study might provide a suitable basis for future co-cultivation studies to elucidate potential synergistic interactions between microbiota.

Lemierre’s syndrome: The “forgotten disease” that should not be forgotten

Introduction: Lemierre’s syndrome (LS) is often misdiagnosed as a common cold or viral infection. It is characterized by sepsis often evolving after a sore throat or tonsillitis and then complicated by various septic emboli and thrombosis of the internal jugular vein. Fusobacterium necrophorum is the most common causative organism. The recommended management is antibiotic treatment. Case Report: We present a case of LS with positive blood cultures for F. necrophorum and pulmonary septic emboli without internal jugular vein thrombosis. The patient was successfully treated with prolonged antibiotic management. Conclusion: Once observed, LS is unlikely to be forgotten by the spectator. Early antibiotic initiation of septic young patients with tonsillitis or neck infections is essential.

Two Streptococcus pyogenes emm types and several anaerobic bacterial species are associated with idiopathic cutaneous ulcers in children after community-based mass treatment with azithromycin.

In yaws-endemic areas, two-thirds of exudative cutaneous ulcers (CU) are associated with Treponema pallidum subsp. pertenue (TP) and Haemophilus ducreyi (HD); one-third are classified as idiopathic ulcers (IU). A yaws eradication campaign on Lihir Island in Papua New Guinea utilizing mass drug administration (MDA) of azithromycin initially reduced but failed to eradicate yaws; IU rates remained constant throughout the study. Using 16S rRNA gene sequencing, we previously determined that Streptococcus pyogenes was associated with some cases of IU. Here, we applied shotgun metagenomics to the same samples we analyzed previously by 16S rRNA sequencing to verify this result, identify additional IU-associated microorganisms, and determine why S. pyogenes-associated IU might have persisted after MDA of azithromycin. We sequenced DNA extracted from 244 CU specimens separated into four groups based upon microorganism-specific PCR results (HD+, TP+, TP+HD+, and TP-HD- or IU). S. pyogenes was enriched in IU (24.71% relative abundance [RA]) specimens compared to other ulcer sub-groups, confirming our prior results. We bioinformatically identified the emm (M protein gene) types found in the S. pyogenes IU specimens and found matches to emm156 and emm166. Only ~39% of IU specimens contained detectable S. pyogenes, suggesting that additional organisms could be associated with IU. In the sub-set of S. pyogenes-negative IU specimens, Criibacterium bergeronii, a member of the Peptostreptococcaceae, and Fusobacterium necrophorum (7.07% versus 0.00% RA and 2.18% versus 0.00% RA, respectively), were enriched compared to the S. pyogenes-positive sub-set. Although a broad range of viruses were detected in the CU specimens, none were specifically associated with IU. Our observations confirm the association of S. pyogenes with IU in yaws-endemic areas, and suggest that additional anaerobic bacteria, but not other microorganisms, may be associated with this syndrome. Our results should aid in the design of diagnostic tests and selective therapies for CU.

Comprehensive Analyses of the Bacterial Population in Non-Healing Claw Lesions of Dairy Cattle.

Non-healing claw lesions (NHCLs) are a newly characterized disorder affecting the deep dermis of the hoof in dairy cattle. Although NHCLs are thought to be associated with bovine digital dermatitis (BDD), their precise etiology is not yet understood. To investigate the bacterial populations present in each type of NHCL (toe necrosis: TN, non-healing white line disease: nhWLD, and a non-healing sole ulcer: nhSU), and the newly added entity non-healing verrucous-like lesions (nhVLL), 16S rRNA-based metagenomic analysis with next-generation sequencing (NGS) was employed. Twelve cases of NHCLs (3 TN, 3 nhWLD, 4 nhSU, and 2 nhVLL) were collected from five dairy farms in two prefectures in Japan. Three samples of healthy hoof dermis collected from two farms and a slaughterhouse were used as controls. Furthermore, culture-dependent and -independent approaches were conducted for detecting Treponema species and Fusobacterium necrophorum. As reported in BDD, Treponema species and F. necrophorum were detected frequently from NHCLs by PCR and immunohistochemistry, but NGS showed that these bacterial genera were not predominant in NHCLs. The predominant bacterial genera in NHCLs differed among the lesions examined, suggesting that Treponema species present predominantly in BDD were not predominant in NHCLs and that the bacterial population in NHCLs may vary among individual cattle and/or farms.

An In Vitro Model Using TRIS-Buffered Plasma-Activated Water to Reduce Pathogenic Microorganisms Involved in Digital Dermatitis Infection in Cattle

Bovine digital dermatitis is an important infectious claw disease caused by multimicrobial infections with bacteria such as Fusobacterium (F.) necrophorum or Porphyromonas (P.) levii. To analyze the antibacterial effects of a TRIS-buffered plasma-activated water (Tb-PAW) on the bacterial number of F. necrophorum, P. levii, Escherichia (E.) coli, Staphylococcus (S.) aureus and Clostridium (C.) sporogenes 1 mL of each bacterial solution (106–108 CFU/mL) was incubated with 9 mL Tb-PAW up to 15 min. E. coli, F. necrophorum and P. levii were significantly reduced by 5.0 log after 1 min of treatment, while S. aureus and C. sporogenes required 15 min to reach a 3.0 log reduction. The addition of bovine serum albumin did not negatively affect the bactericidal effect. Tb-PAW storage at 7 °C and 21 °C is possible for up to 24 h without any change in the bactericidal effect, while Tb-PAW stored at 30 °C can only be used for a period of 12 h. The present data indicate that Tb-PAW can be used to reduce various bacteria even under the influence of different parameters. However, due to the complexity of Tb-PAW, further studies are required to ensure its microbicidal activity before practical application.

Treatment of digital dermatitis using salicylic acid in European bison (Bison bonasus) reveals promising results.

Digital dermatitis (DD) associated with the presence of multiple Treponema spp. was recently described for the first time in European bison (Bison bonasus). DD is characterized by skin inflammation in the distal foot area in various ungulates. The objective of this proof of concept study was to test a treatment protocol adopted from cattle for its applicability in this wildlife species using five animals. Keratolytic salicylic acid paste was administered topically under bandages for seven days to enable removal of the affected skin. All interventions were performed under general anesthesia. To evaluate the treatment efficacy, photographs and biopsies were taken pre- and post-treatment. The biopsies were examined histologically, by PCR for the presence of different bacterial species, by Treponema-specific fluorescent in situ hybridization (FISH), and by transmission electron microscopy. Based on photographs, complete clinical healing of the 15 feet with macroscopical DD lesions was achieved. Histological examination showed mild to moderate dermatitis in 17/20 feet before, and in 12/20 feet after treatment. 17/20 feet were Treponema spp. PCR positive before, and none was positive after treatment. Dichelobacter nodosus, Fusobacterium necrophorum, and Porphyromonas levii could not be detected in any of the samples. By FISH and electron microscopy, Treponema spp. could be visualized in the stratum corneum before, but not after treatment. These results suggest that this treatment method can be applied as standard practice prior to transporting DD affected European bison to prevent the spread of this contagious disease.

Protoporphyrin IX derived from dual-species anaerobic biofilms of Fusobacterium necrophorum and Porphyromonas levii attenuates bovine neutrophil function

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 Fusobacterium necrophorum and Porphyromonas levii 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. Porphyromonas 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.

Natural Mycoplasma Infection Reduces Expression of Pro-Inflammatory Cytokines in Response to Ovine Footrot Pathogens.

Ovine footrot is a complex multifactorial infectious disease, causing lameness in sheep with major welfare and economic consequences. Dichelobacter nodosus is the main causative bacterium; however, footrot is a polymicrobial disease with Fusobacterium necrophorum, Mycoplasma fermentans and Porphyromonas asaccharolytica also associated. There is limited understanding of the host response involved. The proinflammatory mediators, interleukin (IL)-1β and C-X-C Motif Chemokine Ligand 8 (CXCL8), have been shown to play a role in the early response to D. nodosus in dermal fibroblasts and interdigital skin explant models. To further understand the response of ovine skin to bacterial stimulation, and to build an understanding of the role of the cytokines and chemokines identified, primary ovine interdigital fibroblasts and keratinocytes were isolated, cultured and stimulated. The expression of mRNA and protein release of CXCL8 and IL-1β were measured after stimulation with LPS, D. nodosus or F. necrophorum, which resulted in increased transcript levels of IL-1β and CXCL8 in the M. fermentans-free cells. However, only an increase in the CXCL8 protein release was observed. No IL-1β protein release was detected, despite increases in IL-1β mRNA, suggesting the signal for intracellular pre-IL-1β processing may be lacking when culturing primary cells in isolation. The keratinocytes and fibroblasts naturally infected with M. fermentans showed little response to the LPS, a range of D. nodosus preparations or heat-inactivated F. necrophorum. Primary single cell culture models complement ex vivo organ culture models to study different aspects of the host response to D. nodosus. The ovine keratinocytes and fibroblasts infected with M. fermentans had a reduced response to the experimental bacterial stimulation. However, in the case of footrot where Mycoplasma spp. are associated with diseased feet, this natural infection gives important insights into the impact of multiple pathogens on the host response.

Detection and quantification of bacterial species DNA in bovine digital dermatitis lesions in swabs and fine-needle aspiration versus biopsies.

Digital Dermatitis (DD) is a polymicrobial disease characterized by ulcerative lesions on the heel bulb of cattle and for which, despite being reported almost 50 years ago, information on the causative agent is still lacking. Tissue biopsies are regularly collected to identify bacterial presence-absence and their relative abundance in the microbiome, with sufficient evidence for the high abundance of species of Treponema spp. and other anaerobes in lesions. However, it is unclear what the potential of less-invasive sampling methods is for bacterial detection and quantification. This study aimed to test whether less-invasive sampling techniques, such as swabs and fine-needle aspiration (FNA), can be a convenient alternative to tissue biopsies in detecting and quantifying seven DD-associated bacteria in active, ulcerative DD lesions by qPCR. Twenty-two M2 DD lesions were collected using corresponding swabs, aspirates, and biopsies from dairy cows. Presence/absence and quantities of Treponema phagedenis, Treponema medium, Treponema pedis, Porphryromonas levii, Bacteroides pyogenes, Fusobacterium necrophorum, and Fusobacterium mortiferum were correlated, and Bland-Altman plot, McNemar's test, and Cohen's kappa coefficient were used to calculate the agreement among the methods. The quantities of all species were larger in swabs and smaller in aspirates compared to biopsies; however, the differences in bacterial enumeration observed between biopsies and swabs were smaller than in biopsies and aspirates. A strong correlation was observed between the quantity of T. pedis, T. medium, P. levii, and F. mortiferum in biopsies, swabs, and FNA. Yet, T. phagedenis presented the smallest difference between biopsies and swabs, followed by T. pedis and T. medium. In conclusion, swabs, aspirates, and biopsies were equal in their capacity to detect Treponema species based on the good agreement for bacteria presence/absence, with a more limited agreement for the other anaerobes, which were more often present in M2 lesions swabs by qPCR. Bacterial numbers were higher in swabs and lower in aspirates compared to biopsies, with the amounts of treponemes in swabs being closer to biopsies than in aspirates to biopsies. Therefore, aspirates were less suitable for bacterial quantification in DD lesions compared to the other methods.

Immunological response of lame sheep to clinical interdigital dermatitis and footrot: Procalcitonin, acute phase proteins, and pro-inflammatory cytokines

In many countries, sheep lameness is a cause of economic concern and a contributing factor to a declining economy. This study aimed to investigate changes in procalcitonin (PCT), acute phase proteins (APPs), and cytokines (CYTs) in response to interdigital dermatitis and footrot in sheep under field conditions, to emphasize their role in the disease pathogenesis, diagnosis, as well as monitoring treatment response. Fifty-three sheep with foot diseases (26 clinical cases with interdigital dermatitis and 27 clinical cases with footrot) and 20 clinically healthy naemi sheep were used in this study. Real time PCR for detection of Fusobacterium necrophorum (F. necrophorum) and Dichelobacter nodosus (D. nodosus) revealed that, all samples collected from lame sheep (N = 53) were positive for D. nodosus (100 %), whereas F. necrophorum was detected in 19 out of 53 samples (35.84 %). The virulent D. nodosus was detected in 48 lameness cases where non-virulent D. nodosus were identified in 5 cases (in concurrent with F. necrophorum). The mean serum levels of PCT, C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (HP), fibrinogen (Fg) and CYTs (IL1-β, IL-1α, IFN-γ, IL-6 and TNF-α) in sheep with clinical interdigital dermatitis and footrot were remarkably higher than those detected in control healthy sheep. The serum levels of PCT, CRP, SAA, HP, Fg, and CYTs markers in lame sheep pre- and post-treatment were measured. A substantial decline was detected in serum levels of tested biomarkers of lame sheep after 14 days of treatment. The ROC curves were created. The AUC was assessed to evaluate the accuracy of each variable in distinguishing diseased and healthy sheep. Based on the ROC curves and AUCs; PCT, CRP, SAA, HP, and CYTs were highly diagnostic and predictive for the treatment response of sheep with clinical interdigital dermatitis and footrot. Moreover, all tested biomarkers had a noteworthy role in disease immuno-pathogenesis. Nevertheless, PCT and CRP are better than other tested APPs and CYTs as diagnostic markers for interdigital dermatitis and footrot. However, PCT only has the ability to differentiate sheep with different lameness score.

“Non-healing” claw horn lesions in dairy cows: Clinical, histopathological and molecular biological characterization of four cases

The increasing prevalence of bovine digital dermatitis (BDD) contributes to a higher occurrence of secondary infections of exposed corium with Treponema spp. in bovine claws. "Non-healing" claw horn lesions (NHL) clinically resemble BDD lesions. They are severe, cause chronic lameness, and may persist for several months. They poorly respond to standard treatments of BDD and represent a serious welfare issue. In this study, four cases of NHL were classified clinically either as BDD-associated axial horn fissures (BDD-HFA; n = 3) or BDD-associated sole ulcer (BDD-SU; n = 1). In all four cases, pronounced multifocal keratinolysis of the stratum corneum, ulceration, and severe chronic lymphoplasmacytic perivascular to interstitial dermatitis were observed. All lesional samples tested positive for Treponema spp., Fusobacterium (F.) necrophorum, and Porphyromonas (P.) levii by PCRs. BDD-HFA lesions contained Treponema pedis as revealed by genetic identities of 93, 99, and 100%. Treponemes in the BDD-SU lesion were 94% homologous to Treponema phylotype PT3. Fluorescent in situ hybridization (FISH) revealed extensive epidermal infiltration by treponemes that made up > 90% of the total bacterial population in all four lesions. FISH also tested positive for P. levii and negative for F. necrophorum in all four cases, whilst only one BDD-HFA contained Dichelobacter nodosus. Our data point to BDD-associated treponemes and P. levii constituting potential etiological agents in the development of "non-healing" claw horn lesions in cattle.

Comparative Genomic Analysis of Fusobacterium necrophorum Provides Insights into Conserved Virulence Genes.

Fusobacterium necrophorum is a Gram-negative, filamentous anaerobe prevalent in the mucosal flora of animals and humans. It causes necrotic infections in cattle, resulting in a substantial economic impact on the cattle industry. Although infection severity and management differ within F. necrophorum species, little is known about F. necrophorum speciation and the genetic virulence determinants between strains. To characterize the clinical isolates, we performed whole-genome sequencing of four bovine isolates (8L1, 212, B17, and SM1216) and one human isolate (MK12). To determine the phylogenetic relationship and evolution pattern and investigate the presence of antimicrobial resistance genes (ARGs) and potential virulence genes of F. necrophorum, we also performed comparative genomics with publicly available Fusobacterium genomes. Using up-to-date bacterial core gene (UBCG) set analysis, we uncovered distinct Fusobacterium species and F. necrophorum subspecies clades. Pangenome analyses revealed a high level of diversity among Fusobacterium strains down to species levels. The output also identified 14 and 26 genes specific to F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme, respectively, which could be essential for bacterial survival under different environmental conditions. ClonalFrameML-based recombination analysis suggested that extensive recombination among accessory genes led to species divergence. Furthermore, the only strain of F. necrophorum with ARGs was F. necrophorum subsp. funduliforme B35, with acquired macrolide and tetracycline resistance genes. Our custom search revealed common virulence genes, including toxins, adhesion proteins, outer membrane proteins, cell envelope, type IV secretion system, ABC (ATP-binding cassette) transporters, and transporter proteins. A focused study on these genes could help identify major virulence genes and inform effective vaccination strategies against fusobacterial infections. IMPORTANCE Fusobacterium necrophorum is an anaerobic bacterium that causes liver abscesses in cattle with an annual incidence rate of 10% to 20%, resulting in a substantial economic impact on the cattle industry. The lack of definite biochemical tests makes it difficult to distinguish F. necrophorum subspecies phenotypically, where genomic characterization plays a significant role. However, due to the lack of a good reference genome for comparison, F. necrophorum subspecies-level identification represents a significant challenge. To overcome this challenge, we used comparative genomics to validate clinical test strains for subspecies-level identification. The findings of our study help predict specific clades of previously uncharacterized strains of F. necrophorum. Our study identifies both general and subspecies-specific virulence genes through a custom search-based analysis. The virulence genes identified in this study can be the focus of future studies aimed at evaluating their potential as vaccine targets to prevent fusobacterial infections in cattle.

Lemierre's Syndrome: A Lethal Complication of Acute Tonsillitis.

Tonsillitis is a condition involving the infection of the lymphoid tissue of the tonsils. This pathology of the tonsils is frequently reported as a childhood illness in children of school-going age. The leading causative agent which is associated with tonsillitis is Group A and B Haemolytic Streptococcus and Staphylococcus and Haemophilus influenzae. With rapid and correct treatment and management with antibiotics and analgesics, it can be resolved, and the patient can be free of the symptoms such as sore throat, dysphagia, pain over the throat, and fever. Though in the minority of cases the diseases can progress and can result in multiple complications which sometimes can be lethal and extremely serious. These can be rheumatic fever, acute glomerulonephritis, or tonsillar cyst. One of the sporadic but equally important and grave syndromes is Lemierre's syndrome, which unfortunately has been labelled as a forgotten disease due to the development of antibiotic therapy and management of the disease. The mortality of the disease was extremely high in the pre-antibiotic era. This disease presents the following findings, such as thrombophlebitis of the internal jugular vein, which usually occurs after the presentation of pharyngeal infection. Which in later stages also gives rise to thrombi that advance and extend throughout the body, in the form of septic emboli. The important microorganism which is isolated and associated with the Lemierre's is Fusobacterium necrophorum, a strict gram-negative anaerobe. This article emphasizes and discusses the pathophysiology, and microbiology of Lemierre's syndrome. It also focuses on the clinical symptoms that include the appropriate and timely diagnosis and treatment of this deadly and fatal syndrome, together with the complications that arise with Lemierre's syndrome as the presenting problem.

A RARE CASE OF ACTINOMYCES EMPYEMA

A RARE CASE OF ACTINOMYCES EMPYEMA

Effect of live yeast supplementation on energy partitioning and ruminal fermentation characteristics of steers fed a grower-type diet in heat-stress conditions.

The objective of this trial was to determine the influence of live yeast supplementation (LY), environmental condition (ENV), and their interaction (TRT) on energy partitioning, nitrogen metabolism, and ruminal fermentation dynamics of steers receiving a grower-type diet. The effects of LY and ENV were investigated using a 2 × 2 crossover design that spanned five periods. Eight Angus-crossbred steers were randomly split into pairs and housed in four outdoor pens outfitted with an individualized feeding system. Animals were limit-fed a grower diet (DIET) at 1.2% shrunk body weight (SBW) with no live yeast supplementation (NOY) or a grower diet top-dressed with 10 g LY/d for 14 d (1.2 × 1012 CFU/d). On days 13 and 14, animals were subjected to one of two ENV conditions, thermoneutral (TN; 18.4 ± 1.1 °C, 57.6 ± 2.8% relative humidity [RH]) or heat stress (HS; 33.8 ± 0.6 °C, 55.7 ± 2.7% RH), in two side-by-side, single-stall open-circuit, indirect respiration calorimetry chambers. Data were analyzed using a random coefficients model. Carryover effects were examined and removed from the model if not significant. Gross (GE), digestible, metabolizable, heat, and retained energies were not influenced by DIET, ENV, or TRT (P ≥ 0.202). Gaseous energy, as a percentage of GE, tended to increase during HS (P = 0.097). The only carryover effect in the study was for oxygen consumption (P = 0.031), which could be attributed to the tendency of NOY (P = 0.068) to have greater oxygen consumption. DIET, ENV, or TRT (P ≥ 0.154) had no effects on total animal methane or carbon dioxide emissions. Similarly, DIET, ENV, or TRT (P ≥ 0.157) did not affect ruminal pH, redox, protozoa enumeration, ruminal ammonia concentrations, and acetate-to-propionate ratio. Propionate concentrations were the greatest in animals in TN conditions receiving LY (P = 0.034) compared to the other TRT. This effect is mirrored by TN-LY tending to have greater acetate concentrations (P = 0.076) and total VFA concentrations (P = 0.065). Butyrate concentrations tended to be greater for animals fed LY (P = 0.09). There was a tendency for LY to have elevated numbers of Fusobacterium necrophorum (P = 0.053). Although this study lacked effects of LY on energy partitioning, nitrogen metabolism, and some ruminal parameters during HS, further research should be completed to understand if LY is a plausible mitigation technique to enhance beef animals' performance in tropical and sub-tropical regions of the world.

333 Development of a 4-Plex Quantitative PCR Assay for the Detection and Quantification of Species and Subspecies of Fusobacterium Necrophorum and Fusobacterium Varium in Bovine Rumen Fluid

Abstract The Fusobacterium genus contains Gram-negative, rod-shaped anaerobic bacteria. Fusobacterium necrophorum and its two subspecies, necrophorum and funduliforme, are normal ruminal inhabitants of cattle. Subsp. necrophorum is the primary causative agent of liver abscesses in feedlot cattle. Fusobacterium varium is also a normal rumen inhabitant, but its role in ruminal fermentation and involvement in liver abscesses is unknown. Currently, no PCR assays exist to quantify F. varium and both subspecies of F. necrophorum. Therefore, our objective was to develop, validate, and determine the applicability of a quantitative PCR assay for quantification of F. varium and the two subspecies of F. necrophorum. Assay targets were the species-specific hgdA gene, encoding for (R)-2-hydroxyglutarylCoA dehydratase subunit alpha, and the subspecies-specific promoter sequence of the leukotoxin gene, lktA. Assay specificity was validated with different species and strains of Fusobacterium, and other bacterial species. Assay sensitivity was determined using pure cultures. Applicability of the assay for quantification of F. necrophorum and F. varium was determined using ruminal fluid from slaughtered cattle. Species and subspecies-level specificity was confirmed. Pure culture quantification limits (CFU/ml) were 2.1x103 for the F. necrophorum species, 5.6x102 for subsp. necrophorum, 1.5x103 for subsp. funduliforme, and 1.8x103 for F. varium. Analysis of ruminal fluid collected from cattle with healthy livers (n=83) and abscessed livers (n=68) indicated that 97% of samples contained F. funduliforme, with mean concentrations of 7.8 x103, and 8.1x103, respectively. Additionally, subsp. necrophorum and F. varium were more prevalent in ruminal fluid from cattle with abscessed livers (41% and 59%) compared with samples from cattle with healthy livers (31% and 41%). In conclusion, subsp. funduliforme appears to be the normal inhabitant of the rumen, while subsp. necrophorum and F. varium are more prevalent in the rumen of cattle with abscessed livers than those with healthy livers.

PSXIV-11 Microbial Characterization of Digital Dermatitis and Foot rot in Feedlot Cattle

Abstract Lameness is a significant health issue in feedlot cattle, having major economic, labor, and welfare implications. Digital dermatitis (DD), a lesion of the plantar surface of the foot, and foot rot (FR), which affects the interdigital cleft, are common infectious causes of feedlot lameness with bacterial origins. These hoof lesions can occur alone or in combination in the same hoof (DD+FR). Over a one-year period (2019-2020), 208 hoof swabs were obtained from cattle with FR (n = 55), DD (n = 18), and DD+FR (n = 29), and 5 completely healthy cattle (controls) from three southern Alberta commercial feedlots. 16S rRNA gene sequencing was used to characterize the bacterial microbiota of DD, FR, and DD+FR lesions relative to corresponding healthy skin controls found within the same affected animals. DD and FR lesion microbiotas showed reduced diversity compared with corresponding control skin. Multiple Treponema (members of Spirochaetaceae Family) species were differentially abundant in DD lesions compared with contralateral control skin. FR lesions are characterized by an abundance of Fusobacterium necrophorum and Porphyromonas levii, compared with contralateral control skin. The microbiome of DD+FR lesions was similar to FR lesions, where F. necrophorum and P. levii were increased compared with contralateral control skin. Other differentially abundant taxa (Helcococcus, Succiniclasticum spp.) found in FR and DD+FR lesions had been previously associated with DD, suggesting there may be an overlap of bacteria that have a role in both types of hoof lesions. The findings support previous literature showing that FR and DD are polybacterial in nature. The identification of novel bacterial species of DD and FR lesions could contribute information for the development of more targeted and effective prevention and treatment strategies. A novel finding of the sequencing analysis for hooves with concurrent DD and FR infection was characterized.

Associations Between Findings of Fusobacterium necrophorum or β-Hemolytic Streptococci and Complications in Pharyngotonsillitis-A Registry-Based Study in Southern Sweden.

Most pharyngotonsillitis guidelines focus on the identification of group A streptococci (GAS), guided by clinical scores determining whom to test with a rapid antigen detection test. Nevertheless, many patients testing negative with this test are evaluated for group C/G streptococci (GCS/GGS) and Fusobacterium necrophorum, yet their importance remains debated. Our primary aim was to evaluate associations between complications and findings of F. necrophorum, GAS, or GCS/GGS in pharyngotonsillitis. This was a retrospective, registry-based study of pharyngotonsillitis cases tested for F. necrophorum (polymerase chain reaction) and β-hemolytic streptococci (culture) in the Skåne Region, Sweden, in 2013-2020. Patients with prior complications or antibiotics (within 30 days) were excluded. Data were retrieved from registries and electronic charts. Logistic regression analyses were performed with a dichotomous composite outcome of complications as primary outcome, based on International Classification of Diseases, Tenth Revision, codes. Cases with negative results (polymerase chain reaction and culture) were set as reference category. Complications within 30 days were defined as peritonsillar or pharyngeal abscess, otitis, sinusitis, sepsis or septic complications, recurrence of pharyngotonsillitis (after 15-30 days) or hospitalization. Of 3700 registered cases, 28% had F. necrophorum, 13% had GCS/GGS, 10% had GAS, and 54% had negative results. The 30-day complication rates were high (20%). F. necrophorum (odds ratio, 1.8; 95% confidence interval, 1.5-2.1) and GAS (1.9; 1.5-2.5) were positively associated with complications, whereas GCS/GGS were negatively associated (0.7; 0.4-0.98). Our results indicate that F. necrophorum is a relevant pathogen in pharyngotonsillitis, whereas the relevance of testing for GCS/GGS is questioned. However, which patient to test and treat for F. necrophorum remains to be defined.