Genomics and virulence factors of Fusobacterium necrophorum

Fusobacterium necrophorum, a Gram negative, anaerobic bacterium, is a common cause of acute pharyngitis and tonsillitis and a rare cause of more severe infections of the head and neck. At the beginning of the project, there was no available genome sequence for F. necrophorum. The aim of this project was to sequence the F. necrophorum genome and identify and study its putative virulence factors contained using in silico and in vitro analysis. Type strains JCM 3718 and JCM 3724,F. necrophorum subspecies necrophorum (Fnn) and funduliforme (Fnf), respectively, and strain ARU 01 (Fnf), isolated from a patient with LS, were commercially sequenced by Roche 454 GS-FLX+ next generation sequencing and assembled into contigs using Roche GS Assembler. Sequence data was annotated semi-automatically, using the xBASE pipeline, BLASTp and Pfam. The F. necrophorum genome was determined to be approximately 2.1 – 2.3 Mb in size, with an estimated 1,950 ORFs and includes genes for a leukotoxin, ecotin, haemolysin, haemagglutinin, haemin receptor, adhesin and type Vb and Vc secretion systems. The prevalence of the leukotoxin gene was investigated in strains JCM 3718, JCM 3724 and ARU 01, as well as a clinical collection of 25 Fnf strains, identified using biochemical and molecular tests. The leukotoxin operon was found to be universal within the strain collection by PCR. HL-60 cells subjected to aliquots of concentrated high molecular weight culture supernatant, predicted to contain the secreted leukotoxins of strains JCM 3718, JCM 3724 and ARU 01, were killed in a dose-dependent manner. The cytotoxic effect of the leukotoxin against human donor white blood cells was also tested to validate the HL-60 assay. The differences in the results between the two assays were not statistically significant. Ecotin, a serine protease inhibitor, was found to be present in 100 % of the strain collection and had a highly conserved sequence with primary and secondary binding sites exposed on opposing sides of the protein. During enzyme inhibition studies, a purified recombinant F. necrophorum ecotin protein inhibited human neutrophil elastase, a protease that degrades bacteria at inflammation sites, and human plasma kallikrein, a component of the host clotting cascade. The recombinant ecotin also prolonged human plasma clotting times by up to 7-fold for the extrinsic pathway, and up to 40-fold for the intrinsic pathway. The genome sequence data provides important information about F. necrophorum type strains and enables comparative study between strains and subspecies. Results from the leukotoxin and ecotin assays can be used to build up an understanding of how the organism behaves during infection.

Presence and characterisation of ecotin in F. necrophorum

Fusobacterium necrophorum is a causative agent of Lemierre’s syndrome (LS) in humans. LS is characterised by thrombophlebitis of the jugular vein and bacteraemia. Disseminated intravascular coagulation is also a documented symptom. F. necrophorum is a Gram-negative, anaerobic bacterium known to possess virulence genes such as a haemolysin, filamentous haemagglutinin and leukotoxin, which target host blood components. Ecotin is a serine protease inhibitor that has not previously been characterised in F. necrophorum, but in E.coli has been shown to have a potent anticoagulant effect. Next generation and Sanger sequencing were used to confirm the presence of the ecotin gene in the genomes of a collection of F. necrophorum clinical and reference strains. When translated, it was found to be a highly conserved protein made up of159 amino acids. Enzyme/substrate inhibition assays demonstrated that F. necrophorum ecotin inhibits human plasma kallikrein and human neutrophil elastase in a dose-dependent manner. Data will also be presented on the anticoagulant effects of ecotin during activated partial thromboplastin time, thrombin time and prothrombin time tests on human donor blood. The mechanisms for how this organism reaches the bloodstream and the significance of this serine protease inhibitor during F. necrophorum infections remain to be elucidated

Comparison of virulence genes found in draft genomes of F. necrophorum

Fusobacterium necrophorum is a causative agent of persistent sore throat syndrome, tonsillar abscesses and Lemierre’s syndrome (LS) in humans. LS is characterised by thrombophlebitis of the jugular vein and bacteraemia. It is a Gram-negative, anaerobic bacterium which to date has no available reference genome. Draft genomes suggest it to be a single circular chromosome of approximately 2.2Mb. A reference strain of each of the two F. necrophorum subspecies and a clinical isolate from a LS patient were sequenced on a Roche 454 GS-FLX+. Sequence data was assembled using Roche GS Assembler and the resulting contigs annotated using xBASE, Pfam and BLAST. The annotation data was mined for gene products associated with virulence revealing a leukotoxin, haemolysin, filamentous haemagglutinnin, adhesin, hemin receptor, phage genes, CRISPR-associated proteins, ecotin and a putative type V secretion system. Data will be presented on comparative genomics of the three strains, with a focus on putative virulence genes. Tools such as Artemis Comparison Tool and ClustalO were used for sequence alignments and PhyML was used to generate phylogenetic trees. Conserved motifs associated with virulence were also located. Understanding variations at the genomic level may help to explain the increased virulence of some F. necrophorum strains.

Effects of adding polyclonal antibody preparations on ruminal fermentation patterns and digestibility of cows fed different energy sources

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito da suplementação com anticorpos policlonais e/ou monensina sódica sobre a saúde ruminal de bovinos jovens confinados

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Características físicas e químicas da carcaça de bovinos jovens suplementados com monensina sódica ou anticorpos policlonais aviários

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Monensina sódica ou anticorpos policlonais contra bactérias precursoras de distúrbios nutricionais em bovinos induzidos à acidose ruminal

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeito do preparado de anticorpos policlonais sobre o consumo alimentar, fermentação ruminal e digestibilidade in vivo de bovinos suplementados com três fontes energéticas

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Advanced Lemierre syndrome requiring surgery

A 38-year-old homeless man was admitted with a 2-week history of a sore throat, increasing shortness of breath, and high fever. Clinical examination showed enlarged and tender submandibular and anterior cervical lymph nodes and a pronounced enlargement of the left peritonsillar region (Figure 1a). CT scan of the throat and the chest showed left peritonsillar abscess formation, occlusion of the left internal jugular vein with inflammatory wall thickening and perijugular soft tissue infiltration, pulmonary abscesses, and bilateral pleural effusions (Figures 1b-e, arrowed). Anaerobe blood cultures grew Fusobacterium necrophorum, leading to the diagnosis of Lemierre's syndrome. Treatment with high-dose amoxicillin and clavulanic acid improved the oropharyngeal condition, but the patient's general status declined further, marked by dyspnea and tachypnea. Repeated CT scans showed progressive lung abscesses and bilateral pleural empyema. Bilateral tonsillectomy, ligation of the left internal jugular vein, and staged decortication of bilateral empyema were performed. Total antibiotic therapy duration was 9 weeks, including a change to peroral clindamycin. Clinical and laboratory findings had returned to normal 12 weeks after surgery.The patient's history and the clinical and radiological findings are characteristic for Lemierre's syndrome. CT scans of the neck and the chest are the diagnostic methods of choice. F. necrophorum is found in over 80% of cases of Lemierre's syndrome and confirms the diagnosis. Prolonged antibiotic therapy is usually sufficient, but in selected patients, a surgical intervention may be necessary. Reported mortality rates are high, but in surviving patients, the recovery of pulmonary function is usually good.

Clínica e cirurgia de espécies pecuárias

O presente relatório, inerente ao estágio curricular do Mestrado Integrado em Medicina Veterinária da Universidade de Évora, tem como objetivo descrever as atividades desenvolvidas durante o estágio na área da clínica e cirurgia de espécies pecuárias. A primeira parte do relatório consiste na descrição do local de estágio e na distribuição casuística das atividades acompanhadas. A segunda parte consiste numa revisão bibliográfica e no desenvolvimento de dois casos clínicos ocorridos em bovinos, relacionados com o tema, “necrobacilose hepática bovina” (NHB). A NHB é uma doença infecciosa de grande impacto económico, principalmente em explorações de bovinos de engorda intensiva. Carateriza-se pela formação de abcessos hepáticos, cujo agente etiológico primário é Fusobacterium necrophorum. É de difícil diagnóstico em vida, raramente demonstra sinais clínicos e o seu tratamento é pouco eficaz. A prevenção baseia-se na adição de antimicrobianos na alimentação, num correto maneio alimentar e eventualmente vacinação; Abstract: Medical and Surgical Pathology of livestock species The present report, inherent of the master in Veterinary Medicine from the University of Évora, aims to describe the activities carried out during the internship in the field of clinic and surgery of livestock species. The first part of the report is the description of the local of internship and the statistical distribution of activities. The second part consists of a literature review and the development of two clinical cases in cattle, related to the theme, “hepatic bovine necrobacillosis "(HBN). The HBN is an infectious disease of great economic impact, especially in feedlot cattle. It is characterized by the formation of liver abscesses, whose primary etiological agent is Fusobacterium necrophorum. It is difficult to diagnose in life, rarely show clinical signs and their treatment is ineffective. Prevention is based on the addition of antimicrobials in food, a correct feed management and eventually vaccination.

Antimicrobial effect of human serum on oral Fusobacterium nucleatum isolates from humans and monkeys

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Plasmid profile in oral Fusobacterium nucleatum from humans and Cebus apella monkeys

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)