Spotlight on avian pathology: Campylobacter hepaticus, the cause of Spotty Liver Disease in layers.

Campylobacter hepaticus was recently identified as the aetiological agent of Spotty Liver Disease (SLD). SLD causes significant health and productivity losses in the Australian egg industry and the disease is present in other countries. Following the isolation and characterization of C. hepaticus, molecular tools and refined culturing methods have been developed to identify the pathogen. It is suspected that the application of these tools will lead to identification of the pathogen in many poultry production systems throughout the world. As C. hepaticus has only recently been identified, little is known about the mechanisms of pathogenesis and, hence, new research needs to be directed towards understanding SLD epidemiology and C. hepaticus virulence mechanisms to inform efforts to develop intervention strategies.

Evolution of higher torque in Campylobacter-type bacterial flagellar motors.

Understanding the evolution of molecular machines underpins our understanding of the development of life on earth. A well-studied case are bacterial flagellar motors that spin helical propellers for bacterial motility. Diverse motors produce different torques, but how this diversity evolved remains unknown. To gain insights into evolution of the high-torque ε-proteobacterial motor exemplified by the Campylobacter jejuni motor, we inferred ancestral states by combining phylogenetics, electron cryotomography, and motility assays to characterize motors from Wolinella succinogenes, Arcobacter butzleri and Bdellovibrio bacteriovorus. Observation of ~12 stator complexes in many proteobacteria, yet ~17 in ε-proteobacteria suggest a "quantum leap" evolutionary event. Campylobacter-type motors have high stator occupancy in wider rings of additional stator complexes that are scaffolded by large proteinaceous periplasmic rings. We propose a model for motor evolution wherein independent inner- and outer-membrane structures fused to form a scaffold for additional stator complexes. Significantly, inner- and outer-membrane associated structures have evolved independently multiple times, suggesting that evolution of such structures is facile and poised the ε-proteobacteria to fuse them to form the high-torque Campylobacter-type motor.

Detecção de resistência às fluoroquinolonas em Campylobacter isolados de frangos de criação orgânica / Detection of fluoroquinolone resistance in Campylobacter strains from organic poultry

Estudos têm revelado que a resistência às quinolonas em cepas de Campylobacter está relacionada à presença da mutação Treonina-86 para Isoleucina. Com o objetivo de investigar a presença dessa mutação em cepas de Campylobacter sensíveis e resistentes à ciprofloxacina e enrofloxacina, o conteúdo cecal de 80 frangos de corte de criação orgânica, abatidos sob Serviço de Inspeção Estadual (S.I.E.) do Estado do Rio de Janeiro, foram coletados e investigados para a presença de Campylobacter. A determinação da resistência à ciprofloxacina e enrofloxacina foi feita pela técnica de difusão em disco e de diluição em ágar para determinação da Concentração Inibitória Mínima (CIM). A detecção da mutação na Região Determinante de Resistencia às Quinolonas (RDRQ) no gene gyrA foi realizada através de sequenciamento. Campylobacter foi isolado a partir de 100% das amostras avaliadas, sendo 68,75% correspondente à C. jejuni e 31,25% à C. coli. No teste de difusão em disco, 100% das cepas foram resistentes à ciprofloxacina e 56,25% das cepas foram resistentes à enrofloxacina. No teste de diluição em ágar, todas as cepas foram resistentes à ciprofloxacina apresentando CIM variando de ≥ 16-64μg/mL, e resistência ou resistência intermediaria à enrofloxacina foi detectada em 42,50% (CIM ≥ 4-32μg/mL) e 38,75% (CIM = 2μg/mL) das cepas, respectivamente. A mutação Tre-86-Ile, foi observada em 100% das cepas analisadas. Além dessa mutação, foram observadas outras mutações não silenciosas (Val-73-Glu, Ser-114-Leu, Val-88-Asp, Ala-75-Asp, Ser-119-Gli, Arg-79-Lis) e mutações silenciosas (His-81-His, Ser-119-Ser, Ala-120-Ala, Fen-99-Fen, Ala-122-Ala, Gli-74-Gli, Ile-77-Ile, Ala-91-Ala, Leu-92-Leu, Val-93-Val, Ile-106-Ile, Tre-107-Tre, Gli-113-Gli, Ile-115-Ile, Gli-110-Gli). A observação de que cepas sensíveis à enrofloxacina pelos testes fenotípicos apresentavam a substituição Tre-86 para Ile sugere que outros mecanismos podem contribuir para a resistência à enrofloxacina em Campylobacter...

Studies have shown that resistance to quinolones in Campylobacter strains is related with Threonine-86-Isoleucine mutation. In order to investigate the presence of this mutation in sensitive and resistant Campylobacter strains to ciprofloxacin and enrofloxacin, the cecal contents of 80 broilers from organic raising chickens, slaughtered under State Inspection Service (S.I.S) of the State of Rio de Janeiro, were collected and tested for the presence of Campylobacter. The determination of ciprofloxacin and enrofloxacin susceptibility was done by disk diffusion and agar dilution methods for determining the Minimum Inhibitory Concentration (MIC). The detection of mutation in Quinolone Resistance Determinant Region (QRDR) in gyrA gene was done by sequencing. Campylobacter was isolated from 100% of the samples, being 68.75% C. jejuni and 31.25% C. coli. By the disk diffusion method, resistance to ciprofloxacin was observed in all isolates and 56.25% of the strains were resistant to enrofloxacin. By agar dilution method, all strains were resistant to ciprofloxacin (MIC ≥ 16μg/mL to ≥ 64μg/mL) and full and intermediate resistance to enrofloxacin was detected in 42.50% (MIC ≥ 4-32μg/mL) and 38.75% (MIC =2μg/mL) of the strains, respectively. Mutation Thr-86-Ile was observed in 100% of the isolates investigated. In addition to this mutation, others no silent mutations (Val-73-Glu, Ser-114-Leu, Val-88-Asp, Ala-75-Asp, Gly-119-Ser, Arg-79-Lys) and silent mutations (His-81-His, Ser-119-Ser, Ala-120-Ala, Phe-99-Phe, Ala-122-Ala, Gly-74-Gly, Ile-77-Ile, Ala-91-Ala, Leu-92-Leu, Val-93-Val, Ile-106-Ile, Thr-107-Thr, Gly-113-Gly, Ile-115-Ile, Gly-110-Gly) were detected. All the enrofloxacin-sensitive strains by the phenotypic methods had the Thr-86 to Ile substitution, which suggests other mechanisms contributing to enrofloxacin resistance in Campylobacter...

Obturation Over an S1 ProTaper Instrument Fragment in a Mandibular Molar with Three Years of Follow-up

This case report describes root canal filling performed over a large S1 ProTaper file fragment in a second mandibular molar with irreversible pulpitis. An S1 ProTaper file was fractured during the instrumentation of the mesiobuccal canal. Approximately 10 mm of file fragment remained in the apical and middle thirds of the canal. The obturation was performed over this fragment using thermomechanically compacted gutta-percha and sealer. Radiographic findings and the absence of clinical signs and symptoms at 3-year follow up indicated successful treatment. Cone-beam computed tomography images revealed absence of periapical lesion and details of intracanal file fragment related to root fillings and apex morphology. In this case, the presence of a large intracanal fractured instrument did not have a negative impact on the endodontic prognosis during the follow up evaluation period.

Este relato de caso descreve a obturação do canal radicular realizada sobre um grande fragmento da lima ProTaper S1 em um segundo molar inferior com pulpite irreversível. Uma lima ProTaper S1 fraturou durante a instrumentação do canal mésio-vestibular. Aproximadamente 10 mm de remanescente do fragmento da lima permaneceu nos terços apical e médio do canal. A obturação foi realizada sobre este fragmento usando guta-percha compactada termomecanicamente e cimento endodôntico. Achados radiográficos e ausência de sinais e sintomas clínicos após 3 anos de acompanhamento indicaram o sucesso do tratamento. Imagens de tomografia computadorizada de feixes cônicos revelaram a ausência de lesão periapical e detalhes do fragmento da lima intracanal relacionados à obturação do canal radicular e à morfologia do ápice. Neste caso, a presença de grande instrumento fraturado intracanal não teve impacto negativo no prognóstico endodôntico durante o período de acompanhamento.

Unique features of the motility and structures in the flagellate polar region of Campylobacter jejuni and other species: an electron microscopic study.

Similarly to Helicobacter pylori but unlike Vibrio cholerae O1/O139, Campylobacter jejuni is non-motile at 20°C but highly motile at ≥37°C. The bacterium C. jejuni has one of the highest swimming speeds reported (>100 µm/s), especially at 42°C. Straight and spiral bacterial shapes share the same motility. C. jejuni has a unique structure in the flagellate polar region, which is characterized by a cup-like structure (beneath the inner membrane), a funnel shape (opening onto the polar surface) and less dense space (cytoplasm). Other Campylobacter species (coli, fetus, and lari) have similar motility and flagellate polar structures, albeit with slight differences. This is especially true for Campylobacter fetus, which has a flagellum only at one pole and a cup-like structure composed of two membranes.

The pathogenic potential of Campylobacter concisus strains associated with chronic intestinal diseases.

Campylobacter concisus has garnered increasing attention due to its association with intestinal disease, thus, the pathogenic potential of strains isolated from different intestinal diseases was investigated. A method to isolate C. concisus was developed and the ability of eight strains from chronic and acute intestinal diseases to adhere to and invade intestinal epithelial cells was determined. Features associated with bacterial invasion were investigated using comparative genomic analyses and the effect of C. concisus on host protein expression was examined using proteomics. Our isolation method from intestinal biopsies resulted in the isolation of three C. concisus strains from children with Crohn's disease or chronic gastroenteritis. Four C. concisus strains from patients with chronic intestinal diseases can attach to and invade host cells using mechanisms such as chemoattraction to mucin, aggregation, flagellum-mediated attachment, "membrane ruffling", cell penetration and damage. C. concisus strains isolated from patients with chronic intestinal diseases have significantly higher invasive potential than those from acute intestinal diseases. Investigation of the cause of this increased pathogenic potential revealed a plasmid to be responsible. 78 and 47 proteins were upregulated and downregulated in cells infected with C. concisus, respectively. Functional analysis of these proteins showed that C. concisus infection regulated processes related to interleukin-12 production, proteasome activation and NF-κB activation. Infection with all eight C. concisus strains resulted in host cells producing high levels of interleukin-12, however, only strains capable of invading host cells resulted in interferon-γ production as confirmed by ELISA. These findings considerably support the emergence of C. concisus as an intestinal pathogen, but more significantly, provide novel insights into the host immune response and an explanation for the heterogeneity observed in the outcome of C. concisus infection. Moreover, response to infection with invasive strains has substantial similarities to that observed in the inflamed mucosa of Crohn's disease patients.

The biofilm forming potential of bacterial species in the genus Campylobacter.

The biofilm forming abilities of 16 strains representative of 14 of the 16 species comprising the genus Campylobacter were determined on glass, stainless steel, and polystyrene plastic. The formation of biofilms has been suggested as a means by which Campylobacter is able to persist within an inhospitable environment. Of the eight microaerophilic Campylobacter species, including two strains each of Campylobacter jejuni and Campylobacter fetus, only C. jejuni strain 81-176 reliably produced a visible biofilm on multiple surfaces. Alternately, all six strains of the anaerobic Campylobacter species reliably produced visible biofilms on multiple surfaces. Electron micrographs of the individual biofilms showed relatively homogeneous biofilms produced by the anaerobic strains, while the microaerophilic C. jejuni strain 81-176 produced a biofilm containing similar quantities of both the spiral and coccoid forms. This survey suggests a difference in the biofilm forming potentials and the morphologies of the bacteria comprising the biofilms between anaerobic and microaerophilic species of Campylobacter. Additionally, differences observed in the biofilm forming ability of two strains of C. jejuni suggest the need for a further investigation of the biofilm forming potential of this species using a larger number of strains.

Campylobacter flagella: not just for motility.

Campylobacter jejuni and Campylobacter coli are among the major causes of diarrheal disease worldwide. The motility imparted by the polar flagella of these pathogens is required for colonization of the mucus lining of the gastrointestinal tract. However, recent studies have revealed a more complex role for flagella in Campylobacter pathogenesis that includes the ability to secrete non-flagellar proteins that modulate virulence and the co-regulation of secreted and non-secreted virulence factors with the flagella regulon. Campylobacter flagellins are heavily glycosylated and changes in glycan composition affect autoagglutination and microcolony formation on intestinal epithelial cells; these traits are associated with disease in an animal model. Here, these recent advances in our understanding of the multifaceted role of flagella in Campylobacter virulence are summarized.

Apparent attachment of Campylobacter and Salmonella to broiler breeder rooster spermatozoa.

It has been demonstrated that horizontal and vertical transmission of Salmonella and Campylobacter can occur in broiler breeder flocks. The mechanism of this transmission is still unclear. Previously negative broiler breeder flocks have been reported to become positive with Salmonella, Campylobacter, or both after the introduction of "spike" roosters at 45 wk of age. To determine whether the rooster semen is a possible source of transmission to hens for colonization, we evaluated the association of both Salmonella and Campylobacter spp. to segments (head, midpiece, and tail) of individual spermatozoa after artificial inoculation. Salmonella typhimurium, Salmonella heidelberg, and Salmonella montevideo, or Campylobacter jejuni (in 0.85% saline) was added to a freshly collected (by abdominal massage) aliquot of pooled semen from roosters housed in individual cages. The semen and bacteria solutions were incubated 1 h at room temperature. Samples were fixed using Karnosvsky and Zamboni fixatives for 24 h prior to centrifuging and rinsing in 0.1 M cacodylate x HCl buffer. Individual aliquot samples were then subjected to both scanning (JSM-5800) and transmission (JEM-1210) electron microscopy. The scanning electron microscopy showed that Salmonella was associated with all 3 segments (head, midpiece, and tail) of the spermatozoa and apparently equally distributed. Campylobacter was mainly associated with the midpiece and tail segments; few isolates were located on the head segment. The transmission electron microscopy showed apparent attachment of Salmonella and Campylobacter to the spermatozoa.

Phenotypic characterisation of flagellin and flagella of urease-positive thermophilic campylobacters.

In this study, flagellin is purified biochemically from eight urease-positive thermophilic camplylobacters (UPTC) isolated from river water, sea water and mussels, and purified also from two isolates of Campylobacter jejuni and C. coli and fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Results showed that no flagellin components were detected in the two Japanese UPTC isolates (CF89-12 and CF89-14) and the two UPTC NCTC strains (NCTC12893 and NCTC12894). Flagellin components, each consisting of a single peptide, with a heterogeneous molecular mass of approximately 52-63 kDa were demonstrated in the other four UPTC isolates (NCTC12892, NCTC12895, NCTC12896 and NI15F [from Northern Ireland]) and the two Japanese isolates of C. jejuni (JCM2013 and C. coli 27). The approximate molecular mass of flagellin from the flagellin-positive UPTC isolates was smaller than those of C. jejuni and C. coli. Flagella were not detected by electron microscopy in the four flagellin-negative UPTC isolates but they were detected in the four flagellin-positive UPTC isolates and the two isolates of C. jejuni and C. coli. Thus, significant phenotypic diversity for flagellin, which must be due to genotypic variations, was demonstrated among the UPTC isolates.

A novel approach for the construction of a Campylobacter mutant library.

Given the lack of functional transposons for use in Campylobacter spp., an alternative method of insertional mutagenesis using natural transformation was developed. High efficiencies of transformation were only obtained with species-specific DNA. This feature was a key element in the construction of mutant libraries of this bacterium. A chromosomal library of Campylobacter jejuni 81116 DNA was made in shuttle vector pUOA18. Next, a kanamycin-resistance (KmR) cassette was ligated into the inserts of the plasmids. C. jejuni 81116 was then transformed with the resulting products to allow homologous recombination between genomic fragments present in the shuttle vector and the chromosome. Transformants were pooled and chromosomal DNA from these transformants was used to retransform C. jejuni 81116. This resulted in transformants containing the KmR cassette in the chromosome but lacking the vector. In order to evaluate this approach for the construction of a mutant bank, the KmR insertional mutants were screened for loss of motility. Partial characterization of 11 non-motile mutants indicated that the inserted genes are involved in motility. Four mutants had the KmR cassette inserted in genes involved in flagella biosynthesis, namely flaA/B, neuB and flgK, and produced incomplete or no flagella. Four mutants had the KmR cassette inserted in genes possibly involved in flagella motor function: pflA, fliM and orf1 downstream of the fliN gene. Three mutants had the KmR cassette inserted in genes that are homologous to genes encoding hypothetical proteins of Helicobacter pylori.

Ultrastructural characterization of Anaerobiospirillum succiniciproducens and its differentiation from Campylobacter species.

The anaerobic spiral-shaped bacterium Anaerobiospirillum succiniciproducens was isolated from the blood of an AIDS patient for the first time in Europe. Electron-microscopical methods, especially negative staining, allowed rapid morphological classification and differentiation from Campylobacter species. While A. succiniciproducens revealed lophotriche flagellation all the investigated Campylobacter species showed monotriche flagellation. The cell diameter of A. succiniciproducens was at least double that of the investigated Campylobacter species. Other ultrastructural features, such as a ring-like structure underneath the flagellar area and fibrils arranged parallel along the axis, were also specific to A. succiniciproducens.

An environmentally regulated pilus-like appendage involved in Campylobacter pathogenesis.

Examination of strains of Campylobacter jejuni, Campylobacter coli, and Campylobacter fetus by electron microscopy revealed that they produced peritrichous pilus-like appendages when the bacteria were grown in the presence of bile salts. Various bile-salt supplements were used and it was found that deoxycholate and chenodeoxycholic acid caused a significant enhancement of pilus production and resulted in a highly aggregative phenotype. Morphologically, the pili were between 4 and 7 nm in width and were greater than 1 micron in length. A gene, termed pspA, which encodes a predicted protein resembling protease IV of Escherichia coli, was identified in C. jejuni strain 81-176. A site-specific insertional mutation within this gene resulted in the loss of pilus synthesis as determined by electron microscopy. Insertions upstream and downstream of the gene had no effect on pilus production. The non-piliated mutant of strain 81-176 showed no reduction in adherence to or invasion of INT 407 cells in vitro. However, this mutant, while still possessing the ability to colonize ferrets, caused significantly reduced disease symptoms in this animal model.

Relationship between Ileal symbiont intracellularis and porcine proliferative enteritis.

The relationship between Ileal symbiont (IS) intracellularis, formerly known as a Campylobacter-like organism, and porcine proliferative enteritis (PE) was studied by use of pigs with experimentally transmitted PE. Twenty one pigs were experimentally inoculated with homogenized ileal mucosa from a pig that died with PE, and 7 were maintained as uninoculated controls. Fecal samples were collected, and pigs were necropsied weekly postinoculation. Light microscopy and electron microscopy were used to examine tissues for lesions of PE and infectious agents. DNA was extracted from the fecal samples and assayed for the presence of sequences specific for IS intracellularis by dot blot hybridization and polymerase chain reaction amplification. IS intracellularis was detected by the polymerase chain reaction in the feces of 20 of 21 inoculated pigs but not in the feces of uninoculated pigs. Seven inoculated pigs but no uninoculated pigs were detected shedding IS intracellularis by dot blot hybridization. Shedding was detected 1 to 5 weeks after inoculation, and clinical signs were seen in the second to fifth weeks after inoculation. Few pigs without lesions of PE were found to shed IS intracellularis. There was a highly significant association between the presence of IS intracellularis in feces or tissue and the presence of microscopic proliferative lesions and between the severity of the lesions of PE and the percentage of IS intracellularis-infected intestinal crypts. Pigs that ceased shedding IS intracellularis were significantly less likely to have proliferative lesions. These and previous reports are consistent with the hypothesis that IS intracellularis is a necessary causative agent of PE.

Campylobacter showae sp. nov., isolated from the human oral cavity.

Nine Campylobacter-like strains were isolated from human gingival crevices and characterized. These strains were gram-negative, straight rods that were motile by means of multiple unipolar flagella. They were asaccharolytic and preferred an anaerobic atmosphere rather than a microaerophilic atmosphere for growth, and their growth was stimulated by formate and fumarate. These strains were biochemically similar to Campylobacter curvus and Campylobacter rectus, but were clearly distinguishable from these organisms by the number of flagella (two to five flagella at one end of the cell), by being catalase positive, by their whole-cell protein profiles, by their Western blot (immunoblot) patterns, and on the basis of DNA-DNA homology data. They could also be differentiated from the other species of the genus Campylobacter. The nine Campylobacter-like strains were compared with two strains (FDC 286 and VPI 10279) representing a previously described but unnamed Wolinella sp. The nine isolates and strains FDC 286 and VPI 10279 were found to be members of a single species. The 16S rRNA sequences of two strains of the newly identified species were compared with the rRNA sequences of 21 reference Campylobacter, Wolinella, and Helicobacter species in order to generate a phylogenetic tree. We propose the name Campylobacter showae for the newly identified strains; strain SU A4 (= ATCC 51146) is the type strain of this new species.

[Current concepts of the biology of bacteria in genus Campylobacter and their role in human pathology]. / Sovremennye predstavleniia o biologii bakterii roda Campylobacter i ikh roli v patologii cheloveka.

An analytical review of literature on the problem of biological properties of campylobacteria is presented. Taxonomic characteristics of campylobacteriosis agents are given. Morphological and cultural peculiarities of campylobacteria are considered, their ability to form coccal forms is emphasized. Peculiar attention is paid to serotype and biotype characteristics of bacteria of the Campylobacter genus. It is shown expedient to develop the home schemes of sero- and biotypization. Data on biological properties of "new" agents of human campylobacteriosis (Campylobacter cinaedi, C. hypointestinalis, C. upsaliensis) are generalized for the first time in home literature. A conclusion is made that the study of biological properties of campylobacteria strains circulating in the Ukrainian territory as well as the development of efficient prophylactic and antiepidemic measures on this basis are urgent now.

Antigenic properties of Campylobacter rectus (Wolinella recta) major S-layer proteins.

The antigenic properties of the surface layer (S-layer) proteins of various Campylobacter rectus strains including 24 clinical isolates and the type strain ATCC 33238 were examined. S-layer proteins were extracted from whole cells by acid treatment according to the method of McCoy et al. (Infect. Immun. 11, 517-525, 1975). The acid extracts from 23 of the isolates and ATCC 33238 contained two major proteins with molecular masses of 130 kDa and 150 kDa, both of which were identified as subunits of the S-layer after comparison with the protein profiles of acid-treated (S-layer-deficient) cells. An S-layer protein from one isolate (CI-808) demonstrated a different molecular mass (160 kDa). Both the 150-kDa proteins of ATCC 33238 and isolate CI-306 and the 160-kDa protein of CI-808 were purified by ion-exchange chromatography in the presence of urea. In Ouchterlony immunodiffusion experiments with these purified proteins and rabbit antiserum raised to each purified protein, both common and strain-specific antigenic determinants were identified in the C. rectus S-layer proteins.

Reversible expression of flagella in Campylobacter spp.

The in vitro phase variation of flagella and the transition rates between flagellate and aflagellate phenotypes in Campylobacter species including C. jejuni, C. coli, C. lari (thermophilic campylobacters), C. fetus subsp. fetus, C. fetus subsp. venerealis and C. hyointestinalis were investigated. The change from the flagellate to aflagellate phenotype was detected in all of the 12 Campylobacter strains studied. When measured in a motility medium, flagellate to aflagellate transition in thermophilic campylobacters, C. fetus and C. hyointestinalis strains occurred at a rate of 1.8 x 10(-3) to 7.5 x 10(-3), 3.0 x 10(-4) to 7.8 x 10(-4) and 1.8 x 10(-5) to 7.7 x 10(-6) per cell per generation, respectively. Transition from aflagellate to flagellate phenotype occurred at a rate of 5.8 x 10(-6) to 9.3 x 10(-6) per cell per generation in thermophilic campylobacters and 1.0 x 10(-6) to 1.5 x 10(-6) in C. fetus strains. No reversion from aflagellate to flagellate phenotype could be detected in C. hyointestinalis strains. It was concluded that the ability to reversibly express flagella was inherent in the wild-type strains and the transition rates for both directions were consistent for each strain.