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

Random amplified polymorphic DNA (RAPD) analysis of Mycobacterium tuberculosis strains in India

The usefulness of random amplification of polymorphic DNA (RAPD) analysis for typing Indian strains of M. tuberculosis was investigated. M. tuberculosis H37Rv, M. tuberculosis DT and 42 clinical isolates of M. tuberculosis were subjected to RAPD-PCR using 7 random decamer primers. All 7 primers were found to be differentiated and produced specific RAPD profiles. The polymorphic amplicons served as RAPD markers for M. tuberculosis. The dendrograms, obtained by different primers, showed the discriminatory ability of the primers. RAPD analysis provided a rapid and easy means of identifying polymorphism in M. tuberculosis isolates, and it was found to be a valuable alternative epidemiological tool. In addition, the results of the present study showed heterogeneity in the M. tuberculosis strains in the population studied

Efficacy of live B1 or Ulster 2C Newcastle disease vaccines simultaneously vaccinated with inactivated oil adjuvant vaccine for protection of Newcastle disease virus in broiler chickens

Two hundred, one-day-old broiler chicks were divided into groups 1, 2 and 3 containing 60, 70 and 70 chicks, respectively. The groups were divided into subgroups of 10 chicks that were vaccinated according to the following scheme: group 1 unvaccinated control, group 2 vaccinated subcutaneously at 1 day old with inactivated oil adjuvant vaccine (IOAV) in combination with live B1 vaccine. Group 3 was vaccinated in the same mode as group 2 with IOAV and live Ulster 2C vaccine. All birds were challenged when they were 28 days old. Mortality rate, body weight gain and feed conversion ratio (FCR) were monitored before and after challenge. All the chickens in group 1 died, indicating that there was no disease resistance of this unvaccinated control group of chickens. Conversely, the monitored disease resistance of chickens in groups 2 and 3 was 68.57% ± 18.64 and 88.57% ± 9.00, respectively (P < 0.05). The morbidity of chickens in groups 2 and 3 was 37.89% ± 14.36 and 14.76% ± 12.40, respectively (P < 0.05). The body weight gain, feed intake and FCR of group 3 were significantly better than those of group 2 (P < 0.05) during 1–42 days old. The simultaneous vaccination with B1 or Ulster 2C and IOAV of 1-day-old chicks gave some protection of 28-day-old broilers without a booster vaccination

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

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 &gt;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

A comparison of virulence genes, antimicrobial resistance profiles and genetic diversity of avian pathogenic Escherichia coli (APEC) isolates from broilers and broiler breeders in Thailand and Australia.

ABSTRACT Avian pathogenic Escherichia coli (APEC) is the causative agent of colibacillosis resulting in economic losses in the poultry industry worldwide. A total of 168 APEC isolates, equal numbers from Australian and Thai broilers/broiler breeders, were identified and tested for their susceptibility to ten antimicrobial agents. Most of the Thai APEC isolates were multidrug-resistant (MDR) (60.7%) whilst Australian APEC isolates showed a MDR rate of just 10.7%. The Thai APEC isolates exhibited high resistance to tetracycline (TET) (84.5%), amoxicillin (AMX) (70.2%) and trimethoprim-sulfamethoxazole (SXT) (51.2%) whilst the Australian APEC isolates showed lower levels of resistance (TET 36.9%, AMX 29.8%, SXT 17.86%). The 34 Thai APEC and four Australian APEC isolates which were resistant to nalidixic acid were characterized for their carriage of mutations in the quinolone resistance determining region of gyrA, gyrB, parC and parE. While no mutations were detected in gyrB in the Thai isolates, the Ser83Leu and Asp87Asn substitutions in gyrA and Ser80Ile in parC were common (n = 9/34). In regard to the Australian isolates, the Ser83Leu and Asp678Glu substitution in gyrA, Pro385Ala and Ser492Asn in gyrB and Met241Ile and Asp475Glu in parC were identified (n = 3/4). Rep-PCR analysis of the 84 Thai and 84 Australian APEC isolates showed 16 main clusters that mostly contained isolates from both countries. Our results suggest that the emergence of MDR is a major concern for the Thai APEC isolates and that more prudent use of antimicrobial agents in Thai poultry production is required