Disclosing respiratory co-infections: a broad-range panel assay for avian respiratory pathogens on a nanofluidic PCR platform

<p>Respiratory syndromes (RS) are among the most significant pathological conditions in edible birds and are caused by complex coactions of pathogens and environmental factors. In poultry, low pathogenic avian influenza A viruses, metapneumoviruses, infectious bronchitis virus, infectious laryngotracheitis virus, <i>Mycoplasma</i> spp. <i>Escherichia coli</i> and/or <i>Ornithobacterium rhinotracheale</i> in turkeys are considered as key co-infectious agents of RS. <i>Aspergillus</i> sp., <i>Pasteurella multocida, Avibacterium paragallinarum</i> or <i>Chlamydia psittaci</i> may also be involved in respiratory outbreaks. An innovative quantitative PCR method, based on a nanofluidic technology, has the ability to screen up to 96 samples with 96 pathogen-specific PCR primers, at the same time, in one run of real-time quantitative PCR. This platform was used for the screening of avian respiratory pathogens: 15 respiratory agents, including viruses, bacteria and fungi potentially associated with respiratory infections of poultry, were targeted. Primers were designed and validated for SYBR green real-time quantitative PCR and subsequently validated on the Biomark high throughput PCR nanofluidic platform (Fluidigm©, San Francisco, CA, USA). As a clinical assessment, tracheal swabs were sampled from turkeys showing RS and submitted to this panel assay. Beside systematic detection of <i>E. coli</i>, avian metapneumovirus, <i>Mycoplasma gallisepticum</i> and <i>Mycoplasma synoviae</i> were frequently detected, with distinctive co-infection patterns between French and Moroccan flocks. This proof-of-concept study illustrates the potential of such panel assays for unveiling respiratory co-infection profiles in poultry.</p

Number of human H5N1 influenza virus infections in Egypt by Governorate.

<p>Number of human H5N1 influenza virus infections in Egypt by Governorate.</p

Symptoms of influenza H5N1 virus infection in poultry, 2006–2010.

<p>Symptoms of influenza H5N1 virus infection in poultry, 2006–2010.</p

Case-fatality among human H5N1 influenza cases in Egypt by age group, sex, year, and the number of days between onset of symptoms and hospitalization.

<p>*p<0.001, Fisher's exact test.</p>†<p>p≤0.005, Pearson's chi-square test.</p

Oligonucleotide primers for detection and subtyping of avian H5N1 influenza viruses.

<p>Oligonucleotide primers for detection and subtyping of avian H5N1 influenza viruses.</p

Number of human H5N1 influenza virus infections in Egypt by age and sex.

<p>Number of human H5N1 influenza virus infections in Egypt by age and sex.</p

Co-infection of turkeys with <i>Escherichia coli</i> (O78) and H6N1 avian influenza virus

<p>Respiratory diseases are responsible for major economic losses in poultry farms. While in most cases a single pathogen is not alone responsible for the clinical outcome, the impact of co-infections is not well known, especially in turkeys. The purpose of this study was to assess the possible synergism between <i>Escherichia coli</i> (O78) and low pathogenic avian influenza virus (LPAIV, H6N1), in the turkey model. Four-week-old commercial turkeys were inoculated with either H6N1, O78 or both agents simultaneously or three days apart. We have established an experimental infection model of turkeys using aerosolization that better mimics field infections. Birds were observed clinically and swabbed on a daily basis. Necropsies were performed at 4 and 14 days post single or dual inoculation and followed by histological and immunohistochemical analyses. Combined LPAIV/<i>E. coli</i> infections resulted in more severe clinical signs, were associated with higher mortality and respiratory organ lesions (mucous or fibrinous exudative material in lungs and air sacs), in comparison with the groups given single infections (<i>P</i> < 0.05). The time interval or the sequence between H6N1 and <i>E. coli</i> inoculation (none or three days) did not have a significant effect on the outcome of the dual infection and disease although slightly greater (<i>P</i> > 0.05) respiratory signs were observed in turkeys of the <i>E. coli</i> followed by H6N1 inoculated group. Microscopic lesions and immunohistochemical staining supported clinical and macroscopic findings. Efficient virus and bacteria replication was observed in all inoculated groups. <i>E. coli</i> and H6N1 thus exercise an additive or synergistic pathogenic effect in the reproduction of respiratory disease.</p