Molecular interaction of Ornithobacterium rhinotracheale with eukaryotic cells

Ornithobacterium rhinotracheale (ORT) is a emerging gram negative bacterial pathogen in poultry. To facilitate the development of novel infection intervention and prevention strategies, the pathogenesis of ORT infection was investigated. In vitro infection assays demonstrated that ORT adhered to epithelial cells in a dose-dependent fashion. Comparison of the adhesive properties of different strains indicated that strain 41 lacked the ability to infect epithelial cells. This strain gained an adhesive phenotype when it was grown in suspension rather than on a solid phase medium. Bacterial adherence was inhibited in the presence of purified lipopolysaccharide (LPS). ORT also adhered to and became ingested by chicken and murine macrophages. This interaction was not blocked by LPS. The macrophage response to ORT was investigated using the production of nitric oxide (NO) as a marker. ORT induced the production of NO by macrophages. This stimulation did not require but was enhanced by rChIFN-gamma. The NO response stimulated by ORT differed from that induced by the Gramnegative bacteria E. coli and S.Enteritidis. The ORT response was slower and was not inhibited by the lipid A neutralizing agent polymixin B. LPS derived from ORT did stimulate NO production. The stimulation of NO production by ORT was specific for chicken macrophages. Both bovine and chicken serum inhibited ORT infection of epithelial cells. The inhibition was caused via an effect of serum factors on the adhesive properties of the bacteria. Biotinylated isolated serum glycoproteins showed direct binding of multiple serum glycoproteins to the bacteria. Serum inhibition was not observed in the interaction of ORT with macrophages. Competitive binding assays, in which the bacteria were first incubated with LPS or serum and subsequently with biotinylated isolated serum glycoproteins showed that LPS and serum components inhibited cellular infection via different mechanisms. Haemagglutination assays indicated variation among ORT strains to agglutinate erythrocytes. Strain 41 was unable to adhere to epithelial cells but did cause haemagglutination when the bacteria were grown onto solid phase media. When the bacteria were grown in suspension, opposite binding characteristics were observed. The apparent transition between phenotypes with different cell-type specificities (erythrocytes versus epithelial cells) thus varied with media growth conditions. To enable the use of molecualr biology in studying ORT pathogenesis, a transformation system was developed that was based on a cryptic plasmid isolated from ORT. This plasmid was completely sequenced and characterized and contained up to 14 different open reading frames of which some appear to encode the proteins involved the transport of heavy metals across the membrane. Introduction of the origin of replication and several regulatory sequences of this plasmid (designated pOR1) into the vector pGEM7, yielded a shuttle vector that replicated as an independent unit in both E. coli and ORT. An antibiotic resistance gene cfxA had been introduced into the plasmid and the transfer of this plasmid to confirm that the plasmid can be used as a genetic tool to identify novel bacterial virulence determinants and candidate vaccine antigens

Detection and molecular characterization of infectious bronchitis virus isolated from recent outbreaks in broiler flocks in Thailand

Thirteen field isolates of infectious bronchitis virus (IBV) were isolated from broiler flocks in Thailand between January and June 2008. The 878-bp of the S1 gene covering a hypervariable region was amplified and sequenced. Phylogenetic analysis based on that region revealed that these viruses were separated into two groups (I and II). IBV isolates in group I were not related to other IBV strains published in the GenBank database. Group 1 nucleotide sequence identities were less than 85% and amino acid sequence identities less than 84% in common with IBVs published in the GenBank database. This group likely represents the strains indigenous to Thailand. The isolates in group II showed a close relationship with Chinese IBVs. They had nucleotide sequence identities of 97-98% and amino acid sequence identities 96-98% in common with Chinese IBVs (strain A2, SH and QXIBV). This finding indicated that the recent Thai IBVs evolved separately and at least two groups of viruses are circulating in Thailand

Molecular interaction of Ornithobacterium rhinotracheale with eukaryotic cells

Ornithobacterium rhinotracheale (ORT) is a emerging gram negative bacterial pathogen in poultry. To facilitate the development of novel infection intervention and prevention strategies, the pathogenesis of ORT infection was investigated. In vitro infection assays demonstrated that ORT adhered to epithelial cells in a dose-dependent fashion. Comparison of the adhesive properties of different strains indicated that strain 41 lacked the ability to infect epithelial cells. This strain gained an adhesive phenotype when it was grown in suspension rather than on a solid phase medium. Bacterial adherence was inhibited in the presence of purified lipopolysaccharide (LPS). ORT also adhered to and became ingested by chicken and murine macrophages. This interaction was not blocked by LPS. The macrophage response to ORT was investigated using the production of nitric oxide (NO) as a marker. ORT induced the production of NO by macrophages. This stimulation did not require but was enhanced by rChIFN-gamma. The NO response stimulated by ORT differed from that induced by the Gramnegative bacteria E. coli and S.Enteritidis. The ORT response was slower and was not inhibited by the lipid A neutralizing agent polymixin B. LPS derived from ORT did stimulate NO production. The stimulation of NO production by ORT was specific for chicken macrophages. Both bovine and chicken serum inhibited ORT infection of epithelial cells. The inhibition was caused via an effect of serum factors on the adhesive properties of the bacteria. Biotinylated isolated serum glycoproteins showed direct binding of multiple serum glycoproteins to the bacteria. Serum inhibition was not observed in the interaction of ORT with macrophages. Competitive binding assays, in which the bacteria were first incubated with LPS or serum and subsequently with biotinylated isolated serum glycoproteins showed that LPS and serum components inhibited cellular infection via different mechanisms. Haemagglutination assays indicated variation among ORT strains to agglutinate erythrocytes. Strain 41 was unable to adhere to epithelial cells but did cause haemagglutination when the bacteria were grown onto solid phase media. When the bacteria were grown in suspension, opposite binding characteristics were observed. The apparent transition between phenotypes with different cell-type specificities (erythrocytes versus epithelial cells) thus varied with media growth conditions. To enable the use of molecualr biology in studying ORT pathogenesis, a transformation system was developed that was based on a cryptic plasmid isolated from ORT. This plasmid was completely sequenced and characterized and contained up to 14 different open reading frames of which some appear to encode the proteins involved the transport of heavy metals across the membrane. Introduction of the origin of replication and several regulatory sequences of this plasmid (designated pOR1) into the vector pGEM7, yielded a shuttle vector that replicated as an independent unit in both E. coli and ORT. An antibiotic resistance gene cfxA had been introduced into the plasmid and the transfer of this plasmid to confirm that the plasmid can be used as a genetic tool to identify novel bacterial virulence determinants and candidate vaccine antigens

A study on Campylobacter jejuni and Campylobacter coli through commercial broiler production chains in Thailand: antimicrobial resistance, the characterization of gyrA mutation, and genetic diversity by flaA-RFLP

Contaminated poultry meat is regarded as the main source of human campylobacteriosis. During September 2014 and February 2015, breeder flocks, hatcheries, and broiler farms from two chicken production chains were investigated chronologically. Five commercial breeder flocks (Breeder Flocks 1-5), two hatcheries (Hatcheries A and B), and five broiler flocks (Broiler Flocks 1-5) were sampled in this study. Campylobacter colonization of both breeder and broiler flocks was determined from cloacal swabs and environmental samples (pan feeders, footwear, darkling beetles, flies, feed, and water). The eggs from the breeder flocks were followed to hatcheries. At the hatcheries, early embryonic deaths, egg trays, eggshells, hatchers, and water were investigated. Cloacal swabs were taken from broilers at Days 1, 14, and 28 (all broiler flocks), and either 35 (Broiler Flocks 1 and 2) or 43 (Broiler Flocks 3-5). Thirty-six Campylobacter jejuni and 94 Campylobacter coli isolates collected through two broiler production chains were tested by twofold agar dilution for their susceptibility to antimicrobial agents. Most Campylobacter isolates were multidrug resistant (MDR), defined as being resistant to three or more antimicrobial classes ( C. jejuni : 100%; C. coli : 98.9%), and exhibited high resistance to enrofloxacin ( C. jejuni : 100%; C. coli : 98.9%). The vast majority of C. coli were resistant to tetracycline (97.9%), trimethoprim-sulfamethoxazole (81.9%), and doxycycline (79.8%), but only 55.6%, 36.1%, and 50% of C. jejuni isolates revealed resistance to these antimicrobial agents, respectively. A selected subset of 24 C. jejuni and 24 C. coli were characterized for their mutations in the quinolone resistance determining region of the DNA gyrase subunit A gene by nucleotide sequence analysis. The Thr-86-Ile substitution (ACA-ATA in C. jejuni or ACT-ATT in C. coli ) was found in all isolates. Moreover, a total of 130 Campylobacter isolates were typed with the use of polymerase chain reaction-restriction fragment length polymorphism of the flagellin A gene (flaA-RFLP) to determine their genetic relationships. Ten distinct clusters were recognized by flaA-RFLP typing. The results showed that horizontal transmission was the major route of Campylobacter transmission in this study. In conclusion, the emergence of MDR and high resistance rates to several antimicrobials are major concerns identified in this study. The prudent use of these agents and active surveillance of resistance at the farm level are essential steps to reduce the public health risks identified in this work

Comparison of two attenuated infectious bursal disease vaccine strains focused on safety and antibody response in commercial broilers

Background and Aim: Infectious bursal disease (IBD) or Gumboro disease is one of the most detrimental diseases in the poultry industry worldwide. Previous scientific studies have shown that live IBD vaccination might induce transient immunosuppression, leading to suboptimal vaccine responses, and therefore lack of protection against other infectious diseases; therefore, selecting an IBD vaccine in commercial farms is a concern. This study aims to compare two commercially attenuated IBD vaccines (intermediate and intermediate-plus strains) in terms of safety and antibody response to IBD and Newcastle disease viruses (NDV) in commercial broilers. Materials and Methods: Overall, 216 Cobb broiler chickens were divided into three groups based on the IBD vaccine strain administered: V217 strain (Group 1), M.B. strain (Group 2), and an unvaccinated group (Group 3). Groups 1 and 2 were orally vaccinated with Hitchner B1 NDV vaccine strain 7 days after IBD vaccination. Blood samples were collected at IBD vaccination day (15 days of age) and at 7, 14, 21, and 28 days post-IBD vaccination. The immunosuppressive effects of the IBD vaccination were determined by NDV antibody response, the bursa:body weight (B:BW) ratio, and the histopathological lesion scores of the bursa of Fabricius. Phylogenetic analysis was also performed. Results: Phylogenetic analysis revealed that the M.B. strain belonged to a very virulent IBD strain, whereas the V217 strain belonged to a classical IBD virus strain. NDV antibody titers of the two vaccinated groups increased after ND vaccination, reaching their maximum at 14 days post-ND vaccination and decreasing thereafter. The V217 group presented the highest NDV humoral response from 7 days post-vaccination (dpv) to the end of the study. The mean NDV antibody titer of the V217 group was significantly (p<0.05) higher than that of the M.B. group at 14 dpv. In addition, the V217 strain-induced lower bursal lesions post-IBD vaccination and a higher B:BW ratio at 7 and 21 dpv compared to the M.B. group. The higher B:BW ratio, lower bursal lesions, and higher ND antibody response present in the V217 group indicate that the V217 strain induces lower immunosuppressive effects compared to the M.B. strain. Conclusion: The results of this study indicate that IBD vaccine selection merits consideration, as avoiding the immunosuppressive effects induced by live IBD vaccination and the consequent impact on response to other vaccines is important

Characterization of avian influenza H5N1 virosome

The purpose of this study was to prepare and characterize virosome containing envelope proteins of the avian influenza (H5N1) virus. The virosome was prepared by the solubilization of virus with octaethyleneglycol mono (n-dodecyl) ether (C12E8) followed by detergent removal with SM2 Bio-Beads. Biochemical analysis by SDS-PAGE and western blotting, indicated that avian influenza H5N1 virosome had similar characteristics to the parent virus and contained both the hemagglutinin (HA, 60-75 kDa) and neuraminidase (NA, 220 kDa) protein, with preserved biological activity, such as hemagglutination activity. The virosome structure was analyzed by negative stained transmission electron microscope (TEM) demonstrated that the spherical shapes of vesicles with surface glycoprotein spikes were harbored. In conclusion, the biophysical properties of the virosome were similar to the parent virus, and the use of octaethyleneglycol mono (n-dodecyl) ether to solubilize viral membrane, followed by removal of detergent using polymer beads adsorption (Bio-Beads SM2) was the preferable method for obtaining avian influenza virosome. The outcome of this study might be useful for further development veterinary virus vaccines

Antimicrobial Resistance in Bacterial Poultry Pathogens: A Review

Antimicrobial resistance (AMR) is a global health threat, and antimicrobial usage and AMR in animal production is one of its contributing sources. Poultry is one of the most widespread types of meat consumed worldwide. Poultry flocks are often raised under intensive conditions using large amounts of antimicrobials to prevent and to treat disease, as well as for growth promotion. Antimicrobial resistant poultry pathogens may result in treatment failure, leading to economic losses, but also be a source of resistant bacteria/genes (including zoonotic bacteria) that may represent a risk to human health. Here we reviewed data on AMR in 12 poultry pathogens, including avian pathogenic Escherichia coli (APEC), Salmonella Pullorum/Gallinarum, Pasteurella multocida, Avibacterium paragallinarum, Gallibacterium anatis, Ornitobacterium rhinotracheale (ORT), Bordetella avium, Clostridium perfringens, Mycoplasma spp., Erysipelothrix rhusiopathiae, and Riemerella anatipestifer. A number of studies have demonstrated increases in resistance over time for S. Pullorum/Gallinarum, M. gallisepticum, and G. anatis. Among Enterobacteriaceae, APEC isolates displayed considerably higher levels of AMR compared with S. Pullorum/Gallinarum, with prevalence of resistance over >80% for ampicillin, amoxicillin, tetracycline across studies. Among the Gram-negative, non-Enterobacteriaceae pathogens, ORT had the highest levels of phenotypic resistance with median levels of AMR against co-trimoxazole, enrofloxacin, gentamicin, amoxicillin, and ceftiofur all exceeding 50%. In contrast, levels of resistance among P. multocida isolates were less than 20% for all antimicrobials. The study highlights considerable disparities in methodologies, as well as in criteria for phenotypic antimicrobial susceptibility testing and result interpretation. It is necessary to increase efforts to harmonize testing practices, and to promote free access to data on AMR in order to improve treatment guidelines as well as to monitor the evolution of AMR in poultry bacterial pathogens

Characterization of Plasmid pOR1 from Ornithobacterium rhinotracheale and Construction of a Shuttle Plasmid

The bacterium Ornithobacterium rhinotracheale has been recognized as an emerging pathogen in poultry since about 10 years ago. Knowledge of this bacterium and its mechanisms of virulence is still very limited. Here we report the development of a transformation system that enables genetic modification of O. rhinotracheale. The system is based on a cryptic plasmid, pOR1, that was derived from an O. rhinotracheale strain of serotype K. Sequencing indicated that the plasmid consisted of 14,787 nucleotides. Sequence analysis revealed one replication origin and several rep genes that control plasmid replication and copy number, respectively. In addition, pOR1 contains genes with similarity to a heavy-metal-transporting ATPase, a TonB-linked siderophore receptor, and a laccase. Reverse transcription-PCR demonstrated that these genes were transcribed. Other putative open reading frames exhibited similarities with a virulence-associated protein in Actinobacillus actinomycetemcomitans and a number of genes coding for proteins with unknown function. An Escherichia coli-O. rhinotracheale shuttle plasmid (pOREC1) was constructed by cloning the replication origin and rep genes from pOR1 and the cfxA gene from Bacteroides vulgatus, which codes for resistance to the antibiotic cefoxitin, into plasmid pGEM7 by using E. coli as a host. pOREC1 was electroporated into O. rhinotracheale and yielded cefoxitin-resistant transformants. The pOREC1 isolated from these transformants was reintroduced into E. coli, demonstrating that pOREC1 acts as an independent replicon in both E. coli and O. rhinotracheale, fulfilling the criteria for a shuttle plasmid that can be used for transformation, targeted mutagenesis, and the construction of defined attenuated vaccine strains