Propagation of avian influenza virus in embryonated ostrich eggs.

Influenza A viruses (IAVs) are typically isolated and cultured by successive passages using 9- to 11-day-old embryonated chicken eggs (ECEs) and in 14-day old ECEs for virus mutational studies. Real-time reverse transcription-polymerase chain reaction tests (RT-PCRs) are commonly used for IAV diagnosis, but virus isolation remains invaluable in terms of its high sensitivity, providing viable isolates for further studies and the ability to distinguish between viable and nonviable virus. Efforts at isolating ostrich-origin IAVs from RT-PCR positive specimens using ECEs have often been unsuccessful, raising the possibility of a species bottleneck, whereby ostrich-adapted IAVs may not readily infect and replicate in ECEs, yet the capacity of an ostrich embryo to support the replication of influenza viruses has not been previously demonstrated. This study describes an optimised method for H5 and H7 subtype IAV isolation and propagation in 28-day old embryonated ostrich eggs (EOEs), the biological equivalent of 14-day old ECEs. The viability of EOEs transported from breeding sites could be maximised by pre-incubating the eggs for 12 to 14 days prior to long-distance transportation. This method applied to studies for ostrich-adapted virus isolation and in ovo studies will enable better understanding of the virus-host interaction in ostriches and the emergence of potentially zoonotic diseases.

Efficacy of a plant-produced virus-like particle vaccine in chickens challenged with Influenza A H6N2 virus.

The efficacy, safety, speed, scalability and cost-effectiveness of producing hemagglutinin-based virus-like particle (VLP) vaccines in plants are well-established for human influenza, but untested for the massive poultry influenza vaccine market that remains dominated by traditional egg-grown oil-emulsion whole inactivated virus vaccines. For optimal efficacy, a vaccine should be closely antigenically matched to the field strain, requiring that influenza A vaccines be updated regularly. In this study, an H6 subtype VLP transiently expressed in Nicotiana benthamiana was formulated into a vaccine and evaluated for efficacy in chickens against challenge with a heterologous H6N2 virus. A single dose of the plant-produced H6 VLP vaccine elicited an immune response comparable to two doses of a commercial inactivated H6N2 vaccine, with mean hemagglutination inhibition titres of 9.3 log2 and 8.8 log2 , respectively. Compared to the non-vaccinated control, the H6 VLP vaccine significantly reduced the proportion of shedders and the magnitude of viral shedding by >100-fold in the oropharynx and >6-fold in the cloaca, and shortened oropharyngeal viral shedding by at least a week. Despite its potency, the cost of the antigenic mismatch between the inactivated H6N2 vaccine and challenge strain was evident not only in this vaccine's failure to reduce viral shedding compared to the non-vaccinated group, but its apparent exacerbation of oropharyngeal viral shedding until 21 days post-challenge. We estimate that a kilogram of plant leaf material can produce H6 VLP vaccines sufficient for between 5000 and 30 000 chickens, depending on the effective dose and whether one or two immunizations are administered.

Comparative Evaluation of the Pathogenicity of Mycoplasma gallinaceum in Chickens.

Mycoplasma gallinaceum is not among the most pathogenic mycoplasmas affecting poultry, but its continuous re-isolation from flocks in South Africa displaying typical signs of mycoplasmosis prompted us to revisit its role in respiratory disease. Specific-pathogen-free white leghorn chickens were co-challenged with either M. gallinaceum (MGC) and QX-like infectious bronchitis virus (IBV), or the more virulent Mycoplasm gallisepticum (MG) and IBV. No clinical signs were observed apart from sneezing in chickens challenged with IBV, MGC + IBV, and MG + IBV. On postmortem examination, one bird each in the MGC + IBV and IBV groups developed peritonitis or airsacculitis, respectively. In the tracheas, the MG + IBV group showed the most severe ciliary damage with a mean ciliostatic score of 32.40 compared to scores of 26.83 and 20.4 for the MGC + IBV and IBV groups, respectively. Corresponding tracheal lesions were recorded. Quantitation of the challenge pathogens by quantitative real-time PCR and real-time reverse transcriptase-PCR determined that MGC was shed in much higher titers from the trachea than MG, when co-infected with IBV. Interestingly, the presence of both MG and MGC appeared to enhance IBV replication in the tracheas of infected chickens, whereas the presence of IBV suppressed MG and MGC proliferation in the trachea. In general, the nonpathogenicity of M. gallinaceum in chickens was confirmed, but it was able to aggravate respiratory disease and pathogen proliferation with virulent QX-like IBV.